Applied Science, Faculty of
Engineering, School of (Okanagan)
DSpace
UBCO
Chhajer Jain, Mandeep
Master of Applied Science - MASc
University of British Columbia
This thesis presents an innovative approach using microwave technology for contactless monitoring of microbial growth on solid growth matrices. Microwave sensors are widely employed in various biomedical applications owing to their portable, ease of fabrication, low cost, label-free, and contactless sensing capabilities. The unique ability of the microwave sensor to detect variations in the conductivity in the surrounding medium related to microbial growth makes it a promising candidate for microbial growth monitoring and related applications. This thesis presents an in-depth analysis of the design, fabrication, and testing of one-port planar microwave sensors. The designed sensors allow microbial growth monitoring capabilities in both temperature-controlled and uncontrolled environments. Microbial growth largely depends on environmental factors that include growth temperature, nutrient availability, spatial position, and presence of growth-inhibiting compounds such as antibiotics. Through this research, the potential of designed microwave sensors to investigate the influence of these factors was explored. The first study utilized a microwave sensor to detect microbial growth on a solid matrix provided with different glucose concentrations (0- 10 % w/v). Experimental results demonstrated a positive correlation between microbial growth at different nutrient concentrations with the microwave sensor’s response. In the second study, the microwave sensor successfully monitored the subsurface growth of microorganisms, confined between two layers of solid growth matrices. In the third study, the potential of the designed microwave sensor was investigated and was successfully demonstrated in monitoring the effect of antibiotics on microbial growth. The data from all these experiments and supporting data from microbial growth imaging demonstrates the high efficiency of microwave sensors in fast and contactless monitoring of microbial growth and its effectiveness in biomedical applications. Through this work, the ability of microwave resonator sensor to rapidly detect bacterial growth before any visual indicators was demonstrated. These initial studies lay the foundation and prove the microwave sensor as a prospective tool to detect and monitor microbial growth in food and pharmaceutical industries. The sensor resolution and sensitivity can be further improved by utilizing active resonators. Moreover, the sensor design can be modified and enhanced to operate wirelessly for long-range measurement systems.
Planar microwave resonator sensortechnology for real-time andcontactless monitoring of microbialgrowth on solid mediabyMandeep Chhajer JainB.Tech, The Jawaharlal Nehru Technological University, 2017A THESIS SUBMITTED IN PARTIAL FULFILLMENT OFTHE REQUIREMENTS FOR THE DEGREE OFMASTER OF APPLIED SCIENCEinTHE COLLEGE OF GRADUATE STUDIES(Electrical Engineering)THE UNIVERSITY OF BRITISH COLUMBIA(Okanagan)August 2020© Mandeep Chhajer Jain, 2020The following individuals certify that they have read, and recommendto the College of Graduate Studies for acceptance, a thesis/dissertation en-titled:Planar microwave resonator sensor technology for real-time and contactless monitoring of microbial growth onsolid mediasubmitted by Mandeep Chhajer Jain in partial fulfilment of the require-ments of the degree of Master of Applied ScienceDr. Mohammad Hossein Zarifi, School of EngineeringSupervisorDr. Ian Foulds, School of EngineeringSupervisory Committee MemberDr. Thomas Johnson, School of EngineeringSupervisory Committee MemberDr. Abbas Milani, School of EngineeringUniversity ExamineriiAbstractThis thesis presents an innovative approach using microwave technologyfor contactless monitoring of microbial growth on solid growth matrices.Microwave sensors are widely employed in various biomedical applicationsowing to their portable, ease of fabrication, low cost, label-free, and con-tactless sensing capabilities. The unique ability of the microwave sensor todetect variations in the conductivity in the surrounding medium related tomicrobial growth makes it a promising candidate for microbial growth mon-itoring and related applications. This thesis presents an in-depth analysisof the design, fabrication, and testing of one-port planar microwave sensors.The designed sensors allow microbial growth monitoring capabilities in bothtemperature-controlled and uncontrolled environments.Microbial growth largely depends on environmental factors that includegrowth temperature, nutrient availability, spatial position, and presence ofgrowth-inhibiting compounds such as antibiotics. Through this research, thepotential of designed microwave sensors to investigate the influence of thesefactors was explored. The first study utilized a microwave sensor to detectmicrobial growth on a solid matrix provided with different glucose concen-trations (0- 10 % w/v). Experimental results demonstrated a positive cor-relation between microbial growth at different nutrient concentrations withthe microwave sensor’s response. In the second study, the microwave sensorsuccessfully monitored the subsurface growth of microorganisms, confinedbetween two layers of solid growth matrices. In the third study, the poten-tial of the designed microwave sensor was investigated and was successfullydemonstrated in monitoring the effect of antibiotics on microbial growth.The data from all these experiments and supporting data from micro-bial growth imaging demonstrates the high efficiency of microwave sensorsin fast and contactless monitoring of microbial growth and its effectivenessin biomedical applications. Through this work, the ability of microwaveresonator sensor to rapidly detect bacterial growth before any visual indica-tors was demonstrated. These initial studies lay the foundation and provethe microwave sensor as a prospective tool to detect and monitor microbialgrowth in food and pharmaceutical industries. The sensor resolution andiiiAbstractsensitivity can be further improved by utilizing active resonators. More-over, the sensor design can be modified and enhanced to operate wirelesslyfor long-range measurement systems.ivLay SummaryThis thesis presents an innovative approach using microwave sensors forcontactless monitoring of microbial growth on solid growth matrices. Thedesigned sensors were employed in three independent studies to monitor mi-crobial growth under different nutrient conditions, the position of microor-ganisms, and the presence of antibiotics. The first study utilized a microwavesensor to monitor the impact of the availability of nutrients on microbialgrowth. The second study demonstrated the microwave sensor’s ability tosuccessfully monitor the subsurface growth of microorganisms. Moreover, inthe third application study, the potential of the designed microwave sensorin monitoring the effect of antibiotics on microbial growth was successfullydemonstrated. These studies conclude that microwave sensors were ableto detect microbial growth before any visual indicators demonstrating therapid detection capabilities of the microwave sensor. These initial studiesprove microwave sensors as a prospective tool to monitor microbial growthin the food and pharmaceutical industries.vPrefaceThis work was under the guidance and supervision of Prof. MohammadHossein Zarifi at the School of Engineering at The University of BritishColumbia. All copyrighted figures presented in chapters 1 and 2 werereprinted with permission, as indicated in the caption of the figures.Portions of chapter 3 have been published in the following publication:− M. C. Jain, A. V. Nadaraja, B. M. Vizcaino, D. J. Roberts, and M.H.Zarifi, “Differential Microwave Resonator Sensor Reveals Glucose-Dependent Growth Profile of E. coli on Solid Agar,”IEEE Microwaveand Wireless Components Letters, pp. 1–4, 2020viTable of ContentsAbstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . iiiLay Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vPreface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . viTable of Contents . . . . . . . . . . . . . . . . . . . . . . . . . . . viiList of Tables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xList of Figures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xiAcknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . xivDedication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xvChapter 1: Introduction . . . . . . . . . . . . . . . . . . . . . . . 11.1 Motivation . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.2 Outline . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21.3 Introduction to microwave technology . . . . . . . . . . . . . 21.4 Principle of operation . . . . . . . . . . . . . . . . . . . . . . 31.5 Microwave technologies for complex permittivity extractions 51.5.1 Free-space method . . . . . . . . . . . . . . . . . . . . 51.5.2 Open-ended coaxial probe method . . . . . . . . . . . 61.5.3 Transmission line method . . . . . . . . . . . . . . . . 81.5.4 Resonance based method . . . . . . . . . . . . . . . . 91.6 Thesis objective . . . . . . . . . . . . . . . . . . . . . . . . . . 111.7 Thesis outline . . . . . . . . . . . . . . . . . . . . . . . . . . . 12Chapter 2: Principle of operation, design, and optimizationof planar microstrip resonator . . . . . . . . . . . . 14viiTABLE OF CONTENTS2.1 Introduction to planar microwave resonator . . . . . . . . . . 142.2 Investigation on the various types of planar microwave sensors 162.2.1 Frequency variation sensor . . . . . . . . . . . . . . . 162.2.2 Coupling modulation sensors . . . . . . . . . . . . . . 172.2.3 Frequency splitting sensors . . . . . . . . . . . . . . . 182.2.4 Differential-mode sensor . . . . . . . . . . . . . . . . . 192.3 FEM analysis of a planar microstrip resonator . . . . . . . . . 202.3.1 Impact of substrate on the microstrip resonator design 222.3.2 Microstrip transmission line design and matching . . . 232.3.3 Split ring resonator design . . . . . . . . . . . . . . . . 262.3.4 Differential mode resonator design . . . . . . . . . . . 372.4 Chapter conclusion . . . . . . . . . . . . . . . . . . . . . . . . 40Chapter 3: Fabrication and evaluation of the designed sensor 413.1 Sensor fabrication . . . . . . . . . . . . . . . . . . . . . . . . 413.2 Evaluation of Frequency variation sensor . . . . . . . . . . . . 433.2.1 Impact of SUT on the sensitive region of the sensoron the electrical characteristic of the sensors . . . . . . 433.3 Evaluation of differential mode resonator . . . . . . . . . . . 443.3.1 Impact of SUT on the sensitive region of the sensoron the electrical characteristic of the sensors . . . . . . 443.3.2 Conversion of two- port to one- port microstrip deviceusing a power splitter . . . . . . . . . . . . . . . . . . 453.4 Chapter conclusion . . . . . . . . . . . . . . . . . . . . . . . . 47Chapter 4: Application of custom-designed microstrip res-onators for real-time and contactless monitoringof bacterial growth on solid media . . . . . . . . . 484.1 Microwave resonator reveal the influence of glucose on E. coligrowth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 494.1.1 Background . . . . . . . . . . . . . . . . . . . . . . . . 494.1.2 Material and method . . . . . . . . . . . . . . . . . . . 504.1.3 Measurement result and discussion . . . . . . . . . . . 524.2 Microwave resonator to monitor subsurface growth of E. coli 574.2.1 Background . . . . . . . . . . . . . . . . . . . . . . . . 574.2.2 Material and method . . . . . . . . . . . . . . . . . . . 594.2.3 Measurement result and discussion . . . . . . . . . . . 614.3 Microwave resonator to monitor the impact of erythromycinon E. coli . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 664.3.1 Background . . . . . . . . . . . . . . . . . . . . . . . . 66viiiTABLE OF CONTENTS4.3.2 Material and method . . . . . . . . . . . . . . . . . . . 674.3.3 Measurement result and discussion . . . . . . . . . . . 684.4 Chapter conclusion . . . . . . . . . . . . . . . . . . . . . . . . 72Chapter 5: Conclusion . . . . . . . . . . . . . . . . . . . . . . . . 755.1 Thesis conclusions . . . . . . . . . . . . . . . . . . . . . . . . 755.2 Contributions . . . . . . . . . . . . . . . . . . . . . . . . . . . 765.3 Future works . . . . . . . . . . . . . . . . . . . . . . . . . . . 77Bibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79Appendix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97Appendix A: . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97A.1 Measurement of effective dielectric properties of solid agar . 97ixList of TablesTable 1.1 Summary on the techniques to sense and measure di-electric properties using microwave technology . . . . 10Table 2.1 Electrical and mechanical properties of Rogers 5880,6006 and 6010 substrates . . . . . . . . . . . . . . . . 23Table 4.1 Measured growth phase and rate of change of resonantamplitude for E. coli at various concentrations of glu-cose in LB agar . . . . . . . . . . . . . . . . . . . . . 56Table 4.2 Measured growth phase and rate of change of resonantamplitude for E. coli at various concentrations of glu-cose in LB agar . . . . . . . . . . . . . . . . . . . . . 65Table 4.3 Measured zone of inhibition formed by 0 µg, 7.5 µg,30 µg and 45 µg of erythromycin. . . . . . . . . . . . 69xList of FiguresFigure 1.1 The electromagnetic spectrum. . . . . . . . . . . . . . 3Figure 1.2 A dipole in the presence of an external electric field. . 4Figure 1.3 Measurement of sample using free space method. . . . 6Figure 1.4 Measurement of tissue sample using open coaxial probe. 7Figure 1.5 Measurement using TR method with a waveguide. . . 8Figure 1.6 Measurement of thin film using cavity resonator. . . . 9Figure 2.1 Lumped element model of a series RLC resonator. . . 15Figure 2.2 Principle of operation of frequency variation sensor. . 17Figure 2.3 Principle of operation of coupling modulation sensor. 18Figure 2.4 Principle of operation of frequency splitting sensor. . 19Figure 2.5 Principle of operation of differential mode sensor. . . 20Figure 2.6 Planar microstrip resonator. Top view (a) and ex-ploded view (b). . . . . . . . . . . . . . . . . . . . . . 21Figure 2.7 Cross-section of a microstrip transmission line . . . . 23Figure 2.8 Electric field (E- field) and magnetic field (H-field)distribution across a microstrip transmission line . . . 24Figure 2.9 Plot between the input impedance and the width ofthe transmission line . . . . . . . . . . . . . . . . . . 25Figure 2.10 Microstrip feedline design and simulation . . . . . . . 26Figure 2.11 A simple resonator model . . . . . . . . . . . . . . . . 27Figure 2.12 Optimization of coupling gap between feed line andsplit ring resonator . . . . . . . . . . . . . . . . . . . 30Figure 2.13 Plot of gap distance vs capacitance . . . . . . . . . . 31Figure 2.14 Modified resonator design . . . . . . . . . . . . . . . . 32Figure 2.15 H- Field and E- Field analysis of a microstrip resonator 33Figure 2.16 Sensitivity test of a simple microstrip resonator . . . 34Figure 2.17 Impact of SUT on the response of the sensor . . . . . 35Figure 2.18 Lumped element model of a frequency variation res-onator . . . . . . . . . . . . . . . . . . . . . . . . . . . 36Figure 2.19 Differential microstrip resonator . . . . . . . . . . . . 37xiLIST OF FIGURESFigure 2.20 Field analysis of the proposed differential microstripresonator . . . . . . . . . . . . . . . . . . . . . . . . . 38Figure 2.21 Test to determine sensitive region of the proposeddifferential microstrip resonator . . . . . . . . . . . . 39Figure 3.1 Fabricated frequency variation sensor . . . . . . . . . 42Figure 3.2 Fabricated frequency variation sensor with ground . . 43Figure 3.3 Measured response of the sensor in the presence oftest sample on the ring gap and coupling gap. . . . . 44Figure 3.4 Fabricated differential- mode resonator . . . . . . . . 46Figure 3.5 Differential mode resonator with power splitter . . . . 47Figure 4.1 Cross-section of a solid agar plate. . . . . . . . . . . . 50Figure 4.2 solid agar plate contaminated with airborne fungi andprecipitation. . . . . . . . . . . . . . . . . . . . . . . . 51Figure 4.3 Placement of the inoculated plate on the microstripresonator. . . . . . . . . . . . . . . . . . . . . . . . . . 52Figure 4.4 Experimental setup to monitor bacterial growth un-der various nutrient conditions. . . . . . . . . . . . . . 53Figure 4.5 Measured ∆ Resonant amplitude (dB), ∆ Resonantfrequency (GHz), recorded temperature (◦C), and mi-croscopic images of E. coli captured at constant timeintervals for various levels of glucose in LB agar withmicroscopic images captured at constant time intervals. 55Figure 4.6 Cross section of a solid agar with a coverslip coveredwith agar. . . . . . . . . . . . . . . . . . . . . . . . . 59Figure 4.7 Solid media plate on a microstrip resonator with theinoculated E. coli aligned with the sensitive region ofthe resonator. . . . . . . . . . . . . . . . . . . . . . . 60Figure 4.8 Experimental setup to functionally verify the oper-ation of the sensor and monitor subsurface bacteriagrown on E. coli. . . . . . . . . . . . . . . . . . . . . . 61Figure 4.9 Three-dimensional growth of E. coli on solid mediawith and without the presence of cover slip. . . . . . 62Figure 4.10 Measured resonant amplitude and frequency varia-tion for 3 µL and 9 µL of E. coli on soft LB agarwith the fitted Gompertz model curve. . . . . . . . . 63Figure 4.11 Microscopic images depicting the growth of 3 µL and9 µL captured at constant time intervals. . . . . . . 65xiiLIST OF FIGURESFigure 4.12 Zone of inhibition formed by various concentrationsof antibiotics. . . . . . . . . . . . . . . . . . . . . . . . 69Figure 4.13 Measured resonant amplitude variation (∆ Resonantamplitude (dB)) (a), resonant frequency variation(∆Resonant frequency (MHz)) (b), and recorded tem-perature (c) for bacterial growth in the presence ofvarious concentrations of erythromycin. . . . . . . . . 70Figure 4.14 Microscope images captured at constant time inter-vals depicting bacterial growth in the presence of 0µg, 7.5 µg, 30 µg and 45 µg of erythromycin. . . . . . 71Figure A.1 Experimental setup for complex permittivity mea-surement. . . . . . . . . . . . . . . . . . . . . . . . . . 97Figure A.2 Solid agar plate in conjunction with the N1501A di-electric probe. . . . . . . . . . . . . . . . . . . . . . . 98Figure A.3 Measured effective permittivity and loss tangent ofLB agar and MH agar. . . . . . . . . . . . . . . . . . 99xiiiAcknowledgementsI would like to thank the Syilx Okanagan Nation for the use of theirunceded territory, the land on which the research was conducted. I alsoacknowledge the support of the Natural Sciences and Engineering ResearchCouncil of Canada (NSERC) Discovery Grant Program, Canada Foundationfor Innovation program for partial financial assistance, and CMC Microsys-tems for the use of their software and equipment.I would like to express my gratitude towards my supervisor, Prof. Mo-hammad Hossein Zarifi, for his immense support and motivation throughoutmy research. Working under his guidance makes me competent to addresschallenging research questions and find solutions for them. I truly appreci-ate the support, help, and for all the advice that will always help me in myfuture endeavors. I would also like to thank the members of my committee,Prof. Thomas Johnson, Prof. Ian Foulds, and Prof. Abbas Milani for theirwonderful guidance throughout the Master’s program.A special thanks to Dr. Anupama Pillai, for mentoring me with micro-biology experiments and constant motivation. I would also like to thank theOMEGA lab team for the support and making my research days fun andmemorable. I would like to extend my thanks to members of Prof. John-son’s lab members with whom we shared lab space. Special thanks to AaronClements. I like to thank Prof. Deborah Roberts for granting me access toher research laboratories to perform microbiology experiments.Thanks to my friends in India for supporting and believing me in thisjourney. Last but not least, I sincerely thank my parents, Madhu ChhajerJain and Lalit Kumar Chhajer Jain, my brother, Sandeep Chhajer Jain, mycousin Prerna Baid and, my grandparents for being on my side every singleday and moment.xivDedicationTo my father Lalit Kumar Chhajer Jain,whose hardwork and sacrifices are the reason I am where I am.To my Mother Madhu Chhajer Jain,whose strength, kindness and support pushes me to my very best ever day.To my brother Sandeep Chhajer Jain,my strength and my partner in crime.To my sister Prerna Baid, who supports me in my every endeavourxvChapter 1Introduction1.1 MotivationInfections are an inevitable fact of life. They pose a serious challenge topublic health and the global economy. This is evident from the current globalpandemic caused by a novel coronavirus. As per the recent report publishedby the World Health Organization (WHO), the aforementioned virus spreadrapidly across the globe affecting 188 countries with over 5 million cases andover 300 thousand deaths as of May 27, 2020 [1–6]. Statistics and futurepredictions have shown that deaths due to infection account for nearly 13-15 million deaths annually and is projected to remain constant or increaseby 2050 [7]. Although mortality due to infections is projected to rise bythe year 2050, early detection of infections and infection-causing bacteriacan reduce the number of deaths per year significantly. Early detection ofsuch disease-causing organisms can help in developing effective therapy andpostulate various disease control measures against the pathogens [8]. Thus,biologists and other researchers are constantly in search of new technologiescapable of detecting infections in earlier stages and to help develop vaccinesagainst life-threatening infections.The current technologies capable of detecting pathogens rely on eitherconventional techniques such as spread or pour plate techniques and molec-ular methods such as polymerase chain reaction (PCR) and enzyme-linkedimmunosorbent assays (ELISA) [9–11]. Although these techniques are re-liable, conventional techniques are not real-time and difficult to automatewhile molecular methods require extensive sample preparation and expen-sive instruments [12–14]. To overcome the challenges presented by existingtechnologies, rapid and portable biosensors to detect bacterial growth havebeen developed [15–20]. Among all the available biosensors, optical and elec-trochemical biosensors were extensively used for biomedical applications dueto their real-time, non-contact, and label-free detection capabilities [21–24].However, optical biosensors suffer from high cost and quick degradation ofreagents whereas electrochemical biosensors suffer from low selectivity, lowlimit of detection (LoD) and are single-use devices due to direct contact of11.2. Outlinethe sensor with the bacteria [20].Recently, microwave sensors have emerged as a promising tool for mate-rial detection and characterization [25–29], liquids characterization [30–39],gas characterization [27, 40–43], ice detection [44], light and UV detection[45–47], and wearable RFID and biomedical technology [46, 48, 49], due totheir low cost, real-time, label-free, and non-contact-based detection mech-anisms. Interestingly, bacterial growth monitoring in liquid matrices usingmicrowave technology has gained significant attention in recent years [50–53]. Liquid growth media is used extensively by microbiologists for bacterialgrowth monitoring. This is due to the availability of spectrophotometersor other sophisticated instruments capable of detecting bacterial growth inreal-time [54, 55]. Also, the volume of the liquid bacteria culture can besignificantly reduced by employing microfluidic channels [56].Solid growth media, on the other hand, is primarily used to isolate bac-teria to obtain pure bacterial cultures [54]. They are also extensively usedto perform antibiotic susceptibility tests (AST) such as disk diffusion testsand epsilometer tests [9]. However, the complex nature of solid agar coupledwith the limitations of conventional plating techniques holds back biologistsand researchers to automate the bacterial detection process. Therefore,through this work, a proof of concept employing the microwave technologyto monitor bacterial growth on solid agar media will be presented.1.2 OutlineThis chapter begins with an introduction to microwave technology, fol-lowed by the principles of operation of microwave sensors to sense and char-acterize materials. The discussion will then focus on the currently availablemethods for material sensing and characterization. Based on the meritsand demerits of each sensing method, a sensing technology suitable for thecurrent application will be chosen.1.3 Introduction to microwave technologyThe term microwave is associated with electromagnetic waves at fre-quencies in the range of 300 MHz to 300 GHz with a corresponding electricwavelength between 1m (for a frequency of 300 MHz) to 1 mm (for a fre-quency 300 GHz) respectively as shown in Figure 1.1.The theoretical foundation of microwave technology was laid by ClarkMaxwell in the early 1900s but it wasn’t until the development of Radio21.4. Principle of operationFigure 1.1: The electromagnetic spectrum. Copyright © 2005 John Wiley& Sons, Inc. All rights reserved [57].Detection and Ranging, or RADAR in World War II that engineers and sci-entists began to show substantial interest. Since then, microwave technologyhas undergone significant development in terms of power consumption, size,and cost due to the development of solid-state devices, printed circuit boardtechnology, and integrated circuits technology. Today, microwave technol-ogy finds applications in communication [58, 59], processing industry forsintering, drying and curing [60–64], healthcare [65–67] and food industry[16]. Microwave technology is also being extensively used in biomedical andpharmaceutical applications such as detecting and monitoring food spoilage,drug research, and manufacturing, human tissue characterization for cancerand tumor detection, fermentation and biomass system design, and bio-implant research [48, 68]. This is attributed to its low cost, robust, compact,non-contact, and label-free detection properties.1.4 Principle of operationA significant aspect of microwave technology is its ability to characterizeand sense materials by measuring their complex dielectric properties suchas permittivity and conductivity. Dielectric permittivity is a dimensionlessquantity that gauges the degree of polarization in a material due to thepresence of an external electric field [69]. In the presence of an electricfield, charges in the atoms and molecules of a dielectric material undergodisplacement. This results in the formation of dipoles along the direction ofthe applied electric field as shown in Figure 1.2 [69]. Dielectric permittivity31.4. Principle of operationis thus, a measure of how effectively the dipoles are formed and aligned inthe direction of electric field.Relative permittivity or dielectric constant of the dielectric (r) is rep-resented as the ratio of the permittivity of the material to that of the freespace (shown in equation 1.1) = r0 (1.1)Where  is the permittivity of the material (F/m), r is the relative permit-tivity and 0 is the permittivity of free space (8.85 x 10−12 F/m).Figure 1.2: A dipole in the presence of an external electric field. All rightsreserved [69].The complex permittivity of the medium() consists of real part knownas the dielectric constant (′) and an imaginary part which accounts for thelosses in material (′′) as shown in equation 1.2 [70]. = ′ − j.′′ (1.2)The dielectric constant (′) is a measure of the amount of energy from anexternal field stored in the material whereas loss factor(′′) is a associated todielectric loss. Loss in dielectric materials are mainly caused by (i) dielectricdamping, and (ii) conduction loss. Dielectric damping occurs due to thedisplacement of the centers of the charge carriers under the influence of theelectric field whereas conductivity loss arises due to the movement of chargecarriers in the dielectric material [71]. Thus, an important property of adielectric material i.e. the loss tangent is defined as the ratio of imaginarypart to real part of the complex permittivity and is presented in equation41.5. Microwave technologies for complex permittivity extractions1.3.tan δ =ω′′+ σω′(1.3)where, σ represents the conductive loss and ω′′represents loss due to di-electric damping [70], [57].Measuring the dielectric properties of materials plays an important rolein food and pharmaceutical industries to detect tumours and monitor fooddecay by microorganisms [16, 30, 58–64, 72]. Various standard methodshave been postulated and successfully applied to measure and determinethe complex permittivity and loss factor of materials. These methods arethoroughly discussed in the next section.1.5 Microwave technologies for complexpermittivity extractionsMeasurement of dielectric properties using microwave technology in-volves measuring the scattering parameters( S- parameters) using a vectornetwork analyzer equipped with post-processing tools capable of extractingcomplex permittivity and loss tangent of the sample under test (SUT) [73].Various methods have been developed to measure the dielectric propertiesof materials. In this section, four different permittivity characterizationtechniques will be discussed.1.5.1 Free-space methodThe Free-space method is widely used to determine the dielectric proper-ties of a sample under test at high temperatures or in hostile environments.The measurements are carried out using two spot- focusing horn lens an-tennas facing each other as shown in Figure 1.3. The antenna is connectedto a network analyzer loaded with a post-processing program capable of de-termining dielectric properties from the measured response. Prior to anymeasurements, the VNA is calibrated using a line- reflect- line calibrationmethod. The response of the sensor in the presence of a sample holder withand without a sample is recorded. By using the de-embedding function pro-vided in the VNA, the effect of the sample holder on the measured responseis canceled [73].For instance, Hakansson et al. [74], measured the dielectric propertiesof polymerized thin polypyrrole (PPy) films on synthetic textile substratesin the frequency range of 1− 18 GHz using the free-space method. Various51.5. Microwave technologies for complex permittivity extractionsFigure 1.3: Measurement of complex permittivity of a sample using freespace method. All rights reserved [73].strategies such as mathematical diffraction removal program, time–gatingtools, and high gain horns were used to improve the permittivity measure-ments. A stable real part of the permittivity was observed above a dopantconcentration of 0.018 mol/1 p-toluene sulfonic acid (pTSA). Similarly, anincrease in the imaginary part of the permittivity was observed as the dopantconcentration was increased to 0.018 mol/1 pTSA. However, a further in-crease in the dopant concentration had a negligible effect on the imaginarypart of permittivity.The Free-space method is widely used to determine the dielectric prop-erties of in-homogeneous material at high temperatures or in hostile envi-ronments [75–79]. This technique is fast and reliable but is not portable andrequires a custom fabricated sample holder for each sample. Also, a largesample is needed for permittivity measurements to avoid multiple reflectionswithin the sample and diffraction at the edge of the sample.1.5.2 Open-ended coaxial probe methodThe Open-ended coaxial probe method is a non- destructive testingmethod for complex permittivity characterization. In this method, the coax-61.5. Microwave technologies for complex permittivity extractionsial probe is placed against the specimen or immersed in the sample liquidas shown in Figure 1.4. The coaxial probe is connected to a vector networkanalyzer (VNA) and calibrated using a calibration standard (open, short,and a standard reference liquids such as water, saline, and methanol). Thecalibration program utilizes time gating function available in the VNA todecrease the reflections from the connector [80]. The calibration improvesthe ability of the network analyzer to accurately measure the dielectric prop-erties of the sample under test. Complex permittivity of the sample undertest (SUT) is assessed by analyzing the reflection coefficient of the coaxialprobe using post-processing software embedded in the VNA [73]. Shim et al.Figure 1.4: Measurement of tissue sample using open coaxial probe. Allrights reserved [80].[81], for instance, presented a complex permittivity measurement techniquefor artificial tissue emulating materials (ATEs). ATEs are used for in-vitrotesting of implantable biomedical devices. The coaxial probe was calibratedbetween the frequency range 0.5- 6 GHz. The reported results were in goodagreement with the results obtained by commercially available open-endedcoaxial probe kits.Open-ended coaxial probe method is extensively used for near field sens-ing applications and is portable and robust [82–86]. However, the device isexpensive and requires a large and custom machined sample. The measure-ment is also sensitive to air gaps between the sample and the probe.71.5. Microwave technologies for complex permittivity extractions1.5.3 Transmission line methodIn this method, a sample under test is used as a filling material of awaveguide or coaxial probe as shown in Figure 1.5. The sensor is connectedto the vector network analyzer and calibrated to accurately measure thecomplex permittivity of the sample under test. The S- parameter responseof the sensor is measured and processed using various conversion methods[73].Figure 1.5: Measurement using TR method with a waveguide. All rightsreserved [73].For instance, Bogle et al. [87], presented a method for complex per-mittivity characterization of materials using a partially filled rectangularwaveguide. The device was calibrated to operate between the frequencyrange 8- 12 GHz. Mode-matching technique was developed to accommodatethe resulting waveguide discontinuity and a Newton root search method wasutilized to subsequently extract the electromagnetic properties of the testsample.Transmission line method is widely used for complex permittivity ex-traction for medium to high loss materials [73, 88, 89]. However, the mea-surement accuracy is limited by the air- gap effects and the samples may81.5. Microwave technologies for complex permittivity extractionsneed to be machined specifically.1.5.4 Resonance based methodResonant measurements are the widely used for obtaining complex di-electric properties such as permittivity of a given sample under test due toits non-contact, portable, and label-free sensing capabilities. There are twotypes of resonant measurements commonly used: perturbation and low lossmethod. Low loss measurement methods are suitable for measurement onlow loss materials using larger samples. Perturbation methods are suitablefor permittivity measurements of medium to high loss material [73, 90].The experimental setup to measure complex permittivity of a thin filmusing a transverse magnetic (TM) cavity geometry employing perturbationmethod is shown in Figure 1.6. With resonance characteristics depending onFigure 1.6: Measurement of thin film using cavity resonator. All rightsreserved [73].91.5. Microwave technologies for complex permittivity extractionsthe material under test (MUT) in a cavity, its quality factor and resonantfrequency can be monitored to determine the dielectric parameters. Theresonant frequency and Q-factor of an empty cavity and a cavity with thematerial under test are measured and processed to determine the permittiv-ity of the material.Saeed et al. [91], presented a novel planar substrate integrated waveguidecavity resonator technique to determine and extract complex permittivityof the sample under test (SUT). The resonator was designed to resonate at8 GHz and had a high Q- factor. The proposed design had low fabricationcost, is easy to fabricate, and provided contact-free detection capabilities.Resonance-based method is used for measuring the complex dielectricproperties of small sample under tests. However, there exist limitations onthe frequencies and loss characteristics of the materials that can be measuredwith the method.Table 1.1: Summary on the techniques to sense and measure dielectric prop-erties using microwave technologyMethod Advantages DisadvantagesFree-space Contactless, Requires custommethod robust sample holders,largesampleCoaxial probe Portable, robust large sample size,method and contactless complex data analysisTransmission line Portable, Requires custom samplesmethod small sample size, for cavity resonatorsnon- contact,label- freeResonance-based Portable, non-contact Sensor responsemethod based sensing depends on the lossand label- free characteristicsof the MUTCompared to free- space and open-ended transmission line methods, theresonance-based method uses inexpensive devices [92]. However, machiningthe sample to fit in the cavity of the waveguide can be challenging. To over-come this, a microstrip line sensors can be used. Microstrip line sensors offer101.6. Thesis objectivenumerous advantages such as low-cost fabrication, small sample size, porta-bility, robust, non-contact, and label-free detection and ease of integrationwith microfluidic chips [27, 37–39, 56, 91, 93, 94].The summary of the above discussed microwave technologies for complexpermittivity extractions is presented in Table 1.1.Among the available techniques for material sensing and characterizationusing microwave sensors, microstrip line sensors offer numerous advantagessuch as low cost, contactless, ease of fabrication, and label-free detectionand will be selected to monitor bacterial growth on solid media.1.6 Thesis objectiveThis thesis aims to design a custom- designed microwave resonator sensorto monitor the influence of nutrients, subsurface position, and presence ofinhibitory compounds on the growth of microorganisms on solid growthmedia. The designed sensor should be able to detect microbial growth beforeany visual indicators.The following objectives have been set to achieve the aim:1. Employing the designed microwave sensor resonator to detect andmonitor the impact of variations in the glucose concentration in thesolid media on the bacterial growthHypothesis: The designed sensor will monitor the growth of bacteriain response to varying glucose concentration in the media.The tasks required to achieve this objective are:− Prepare LB agar medium with varying concentrations of glucose(0, 2, 5, and 10 %)− Monitoring variation in the bacterial growth at tested glucoseconcentrations using microwave sensor resonator− Analyze the data and plot the measured results. Compare themeasured results with microscopic images taken at regular timeintervals to validate the efficacy of the designed sensor to monitorbacterial growth.2. Utilizing the designed microwave sensor to detect and monitor sub-surface growth of bacteria confined between two layers of solid growthmediaHypothesis: The designed sensor will monitor the transition of bac-terial growth from 2-D surface growth to a 3-D subsurface growth.The tasks required to achieve this objective are:111.7. Thesis outline− Prepare inoculated media plates with microorganisms confinedbetween two layers of solid LB agar− Measuring conductivity changes during the transition of surfaceto subsurface growth of bacteria using microwave sensor resonator− Analyze, plot and compare the measured results with the cap-tured microscopic images as mentioned earlier3. Applying the designed microwave resonator sensor to monitor anddifferentiate the impact of various concentrations of antibiotic, ery-thromycin on the growth of BacteriaHypothesis: The microwave sensor will monitor the dielectric prop-erties of the (Mueller-Hinton) MH agar and differentiate the impactof various antibiotic concentrations on the bacterial growth.The tasks required to achieve the proposed objective are:− Prepare sample MH agar plates with varying concentrations oferythromycin (0, 7.5, 30, and 45 µg) using disk diffusion tech-niques− Measuring conductivity changes during bacterial growth inhibi-tion at tested antibiotic concentrations using microwave sensorresonator− Analyze, distinguish, and compare the measured results with thecaptured microscopic images as mentioned earlier. Based on themeasured results, determine the antibiotic concentrations capableof successfully inhibiting microbial growth1.7 Thesis outlineThis thesis is divided into four chapters. A brief summary of each chaptercontent is as follows:− Chapter 2 focuses on the theory of operation of a microstrip resonator.An investigation of the existing planar microwave resonator sensorsbased on their geometry, robustness, and efficiency will be performed.This investigation will empower the choice of the appropriate sensordesign for the proper environmental conditions. A critical review ofvarious microwave resonators schemes available is presented, and thelimitations associated with them will be discussed. Based on the draw-backs of the discussed resonator schemes and the nature of the study, a121.7. Thesis outlinethorough design procedure of the selected microwave resonator schemewill be presented. The design procedure will encompass the Finite El-ement Method (FEM) analysis of complex planar resonator structuresaimed at determining the sensitive regions, impact on the sample un-der test, and the electrical characteristics of the resonator.− Chapter 3 is devoted to the fabrication and functional verificationof the sensor designed in Chapter 2. This chapter begins with thestep by step in- house fabrication procedure followed to precisely andaccurately fabricate the sensor. The fabricated sensor will then besubjected to various functional verification using standard samples toverify the sensor’s operation as per the designed parameters.− Chapter 4 presents the capability of the designed and fabricated sensordiscussed in chapter 3 to detect and monitor the impact on bacterialgrowth on solid media subject to three mutually exclusive conditions:varying nutrient concentration, spatial position, and the presence ofinhibitory compounds such as antibiotics.− Finally, Chapter 5 concludes and summarizes the work presented inthe thesis, and discusses ideas for future work13Chapter 2Principle of operation,design, and optimization ofplanar microstrip resonatorThis chapter aims to introduce a planar microwave resonator and explorevarious factors that dictate the operation of the resonator such as resonantfrequency, resonant amplitude, and quality factor. It is followed by a thor-ough discussion of different types of planar microstrip resonator technolo-gies. An in-depth analysis of frequency variation resonator and differentialmodel resonator will be undertaken. In addition to this, the steps followedto design and study the resonator will be discussed. Various studies such asEM field analysis, sensitive region analysis, and the impact of permittivityand loss tangent of a sample under test on the response of the modelledsensors will be performed in this chapter. The sensor design modelled inthis chapter is the result of weeks and months of numerous changes in thesensor design and optimizations to improve the sensitivity of the resonator.2.1 Introduction to planar microwave resonatorA resonator is a device that stores energy at the resonant frequency [57].Consider a series RLC resonance circuit as shown in the Figure 2.1.where V is the voltage of the input electromagnetic signal(Volt), R is theresistance (Ω), L is the inductance(Henry), C is the capacitance (Farad),I is the net current (Ampere) flowing through these elements, and Zin isthe input impedance. The input impedance Zin can be calculated usingequation 2.1 [57].Zin =2 ∗ Pin|I|2 =Ploss + 2jω(Wm −We)0.5 ∗ |I|2 (2.1)where Pin is the power delivered to the series RLC resonator at the input,Ploss is the power dissipated by the resistor R, ω is the angular frequency142.1. Introduction to planar microwave resonatorFigure 2.1: Lumped element model of a series RLC resonator. All rightsreserved [57].(Radian), Wm is the average magnetic energy stored in the inductor andWe is the average electrical energy stored in the capacitor. The series RLCcircuit is said to be at resonance when the average stored magnetic energyin the circuit is equal to the average stored electrical energy. At, Wm = We,the input impedance is purely real as shown in equation 2.2.Zin =Ploss0.5 ∗ |I|2 = RΩ (2.2)The frequency at which the input impedance is purely real is known as theresonant frequency. The resonant frequency can be calculated using theequation 2.3.f =12pi√LC=cλ√effHz (2.3)Where f is the resonant frequency in Hz, c is the speed of light inmillimeter per second and λ is the wavelength in millimeter of the EMwave, eff is the effective permittivity.An important parameter associated with a resonator is the quality fac-tor. It is used to represent the EM loss in the resonator. A lower lossimplies a higher Q-factor. The relation between Q and loss tangent tan δ, adimensionless quantity to determine the amount of electromagnetic energyabsorbed within the material and dissipated as heat, is given by the equation2.4.Q =1tanδ=ωσ= − ′′′(2.4)Where σ is conductivity in siemens/m, ω is the angular frequency in152.2. Investigation on the various types of planar microwave sensorsrad, ′of the real part of the permittivity, ′′is the imaginary part of thepermittivity.2.2 Investigation on the various types of planarmicrowave sensorsAmong the available techniques for dielectric characterization using mi-crowave sensors, microstrip line sensors offer numerous advantages such aslow cost, contactless, ease of fabrication, and label-free detection capabili-ties. There are two versions of microstrip planar sensors, resonant based andnon- resonant based. Resonant based planar sensors offer better sensitivityand are compact as compared to non- resonant based planar sensors andtherefore were selected for this study. Based on the principle of operation,planar stripline resonators can be broadly grouped into four categories:− Frequency variation sensors− Coupling modulation sensors− Frequency splitting sensors− Differential- mode sensor2.2.1 Frequency variation sensorA frequency variation sensor consists of a transmission line loaded witha resonant element as shown in Figure 2.2. Material characteristics aredetermined by measuring the variation in the resonant frequency, qualityfactor, or resonant amplitude in the presence of a sample under test on theresonant element. These sensors can be used to characterize and determinethe dielectric properties of liquids and solids in environments where ambientfactor changes such as temperature and humidity have a negligible effect onthe electrical properties of the sample under test. Mohammadi et al. [31]presented a low- cost and hazard free 3D printed microfluidic microwave-based method for water quality monitoring. They employed a 2- port double-ring resonator operating at 4.5 to 4.6 GHz with a quality factor of 120.The study was able to achieve real-time monitoring of water contaminantscomparing to standard chemical-based methods. In another study, Wiltshireet al. [44] demonstrated the capabilities of a 2- port microwave resonatorto detect ice and frost. The designed sensor operated between 3.5 GHz to 5162.2. Investigation on the various types of planar microwave sensorsFigure 2.2: Principle of operation of frequency variation sensor. All rightsreserved [95].GHz with a quality factor of 250. The sensor was successfully able to detectthe phase transition between ice to water and vice- versa.Frequency variation resonator sensors suffer significant degradation ofthe response due to cross-sensitivity caused by variations in ambient factorssuch as temperature and humidity.2.2.2 Coupling modulation sensorsThe impact of the cross-sensitivity of a microstrip sensor to ambientchanges can be eliminated using symmetry-based sensors such as couplingmodulation sensors. In such sensors, the transmission line is arbitrarilyloaded using a symmetrical resonator. (Figure 2.3). In the case of perfectsymmetric loading of the transmission line by the sensor, no resonance oc-curs, and the frequency response does not exhibit a notch. However, assymmetry of the resonator is disturbed due to the dielectric loading or dis-placement of the resonator, the transmission line couples to the resonator,and the resonant profile appear. The magnitude of the resonant profile(typically a notch) is determined by the degree of coupling which is closelyrelated to the level of the asymmetry [96–99].172.2. Investigation on the various types of planar microwave sensorsFigure 2.3: Principle of operation of coupling modulation sensor. All rightsreserved [95].The sensor is easy to design and fabricate. However, the sensitivity ofthe sensor’s notch amplitude to displacement due to external factors limitsthe operation of the sensor in external environments.2.2.3 Frequency splitting sensorsThe next scheme of microstrip resonator is the frequency splitting sen-sors. In these sensors, transmission line-based structures are symmetricallyloaded with a pair of (not necessarily symmetric) resonators (Figure 2.4)leveraging the advantages of both frequency variation and coupling mod-ulation sensor. In the absence of any sample on the sensor, the sensorresponse is unchanged and a single resonant notch is observed. However, inthe presence of test material, the symmetry of the sensor is disturbed andthe resonant profile splits into two notches, one for each resonator. Thus,differences in the frequency and amplitude between the two notches canbe associated with the level of asymmetry caused by a dielectric samplethrough which dielectric properties can be inferred [100–103]. The sensor182.2. Investigation on the various types of planar microwave sensorsconfiguration is robust and easy to fabricate and operate but suffers fromlimited sensitivity and resolution as the frequency overlap of two resonatorscan affect the quality factor of the notch.Figure 2.4: Principle of operation of frequency splitting sensor. All rightsreserved [95].2.2.4 Differential-mode sensorDifferential-mode sensors comprise two sensors, one sensitive to the sam-ple whose dielectric properties need to be determined and the other sensi-tive to reference material. The output of these sensors is the differencebetween the output of the two sensors as shown in Figure 2.5. Thus, thecross-sensitivity of the sensor to ambient factors is eliminated making thesensor robust to external factor variations. Various works using differen-tial sensors to detect and characterize liquid samples have been reported[30, 33, 101, 103, 104]. These devices can be used as a four-port device orbe coupled with additional microwave circuitry to reduce the ports to twoor one.Therefore, based on the investigation of different schemes of microwaveresonators, frequency variation and differential-mode resonator schemes arechosen for microbial growth monitoring due to ease of design, and fabrica-tion. The selected designs also aid in performing complex dielectric mea-surements in both temperature-controlled and uncontrolled environments.192.3. FEM analysis of a planar microstrip resonatorFigure 2.5: Principle of operation of differential mode sensor. All rightsreserved [95].2.3 FEM analysis of a planar microstripresonatorFrom the discussion presented in the previous sections, the frequencyvariation sensor and a differential- mode resonator design were selected forfurther analysis owing to their ease of design, operation, simplicity, androbustness. In this section, the selected microstrip resonator models willbe analyzed using sophisticated Computer-Aided Design (CAD) tools ca-pable of performing complex electromagnetic simulations on the designedresonator structures. To achieve accurate simulation results, the dimensionof the mesh selected for the simulation must be less than or equal to theminimum feature size of the designed sensor. The dimension of the meshdepends upon the available Random Access Memory, or RAM on the sys-tem hosting the CAD software.Therefore, based on the available systemshosting the CAD tool and the minimum feature size of the sensor, 0.8 mmand 0.4 mm mesh sizes were selected. With a 0.8 mm mesh size, a baresensor modelled in High Frequency Structure Simulator, or HFSS took ap-proximately 20 minutes to complete simulation as opposed to 40 minutesusing 0.4 mm mesh size. In addition to this, the difference between theresonant amplitude of the simulated sensor at 0.8 mm and 0.4 mm meshsize was -0.3 dB and 0.7 MHz respectively. Therefore, as the output resultsof the simulation with 0.8 mm and 0.4 mm mesh sizes were similar, meshsize of 0.8 mm was chosen to achieve faster and accurate simulation resultsof the designed sensors. Section 2.1 presented the lumped element model ofa simple series resonance circuit. They are extensively used to describe anetwork at low frequencies. However, at microwave frequencies, the wave-202.3. FEM analysis of a planar microstrip resonatorlength of the signal is comparable to the dimension of lumped elements suchas resistors, capacitors, and inductors leading to variation in the current andvoltage across the device. Thus, at such frequencies, distributed elementsconsisting of lengths of transmission lines are widely used. A distributedelement design of a microstrip resonator is shown in Figure 2.6.Figure 2.6: Planar microstrip resonator. Top view (a) and exploded view(b).The main components of a resonator are feed-line, split ring resonator,substrate, and ground plane as shown in Figure 2.6. The feed-line couplesthe input electromagnetic waves to the split-ring resonator. The substrateon which the resonator is fabricated should be selected carefully as the elec-trical and physical characteristics of the substrate can affect the resonantfrequency of the device. The split ring resonator is the key component andneeds to be carefully designed based on the required application. The lengthof the split ring, the width of the coupling gap between the resonator andthe feed-line, and the width of the ring gap play an important role in de-termining the resonant frequency, resonant amplitude and quality factor.In this section, the impact of substrate properties such as permittivity andloss tangent on the dimensions of the microstrip resonator will be discussed.This will be followed by the design of the microstrip feed line and the splitring resonator.212.3. FEM analysis of a planar microstrip resonator2.3.1 Impact of substrate on the microstrip resonatordesignA substrate can be defined as a structural material to develop RF orMicrowave Printed circuit boards. Soft substrates such as FR- 4, Duroids,and polytetrafluoroethylene (PTFE) are used to design microwave PCBs asthey are easy to handle and readily available. Microwave substrates are oftenreferred to as “laminates” due to the presence of copper cladding attachedto either one or both sides of the dielectric material. Copper cladding canbe of various thicknesses and aid in improving the thermal characteristicsof the laminate in addition to providing weight to the board. A substrateis defined by several electrical and mechanical properties such as dielectricconstant, dissipation factor, coefficient of thermal expansion (CTE), thermalconductivity, and dielectric constant. These parameters can influence theworking of the device and must be carefully selected while designing.− Dielectric permittivity: The dielectric permittivity of the substrate canimpact the dimension of the microstrip conductors fabricated on it.For high-frequency applications, a low dielectric constant substrate ispreferred as it minimizes the parasitic capacitance developed betweentraces and conductive structures.− Dissipation factor : Substrates with low loss tangents are desirable forhigh performance and high-power applications, as it would minimizethe amount of loss within a transmission line or along the structure.− Surface roughness : The surface roughness is a measure of the smooth-ness of the surface of the copper cladding. Conductor surface rough-ness can substantially affect the loss of the conductor. Therefore,based on the reported surface roughness by Rogers Corp, laminateswith small surface roughness was selected [105].− Dielectric strength : This parameter is a measure of the intensity of theelectric field strength required to strip electronics from the dielectricmaterial [106]. This parameter has a negligible impact on the materialat low voltage and power but for application involving high voltage andhigh-power, this could be a limiting factor in a device’s operation.Therefore, based on the above factors/parameters that could influencethe operation of the sensing device, a substrate with low dielectric permittiv-ity, low loss tangent, and low surface roughness was considered by referring222.3. FEM analysis of a planar microstrip resonatorthe data sheets of the available substrates to design and fabricate the res-onator [105]. The surface roughness of the copper was not measured as itwas out of scope of the current work. However, through normalization andother data analysis techniques performed on the response of the resonatorduring contactless bacterial growth monitoring, the effect of any microscopicimperfections and ambient factors was minimized.Therefore, among the laminates available at our disposal, Rogers 5880was selected to fabricate our resonators. The electrical and mechanical prop-erties of the available laminates from Rogers is given in Table 2.1.Table 2.1: Electrical and mechanical properties of Rogers 5880, 6006 and6010 substratesLaminate name Permittivity Loss tangentRogers 5880 2.2 0.0009Rogers 6006 6.15 0.0027Rogers 6010 10.2 0.00232.3.2 Microstrip transmission line design and matchingA microstrip transmission line consists of a conductor attached to adielectric substrate with a grounded plane as shown in Figure 2.7.Figure 2.7: Cross-section of a microstrip transmission line.They are planar, easy to fabricate, portable, and support other deviceintegration such as microfluidic or passive microwave devices such as powersplitter, couplers, and mixers.Various parameters influence the signal transmission through a microstripline such as physical dimensions of the transmission line, substrate thickness,permittivity, and loss tangent. The physical dimensions of the transmission232.3. FEM analysis of a planar microstrip resonatorline and substrate such as width (w) of the stripline and the height (h) ofthe substrate define the ratio of the voltage and current signals travelingthrough the microstrip structure. This ratio is the characteristic impedance(Z0) of the line and is kept constant for reliable signal transmission.Theoretical analysis of a microstrip feed lineA time-varying electromagnetic signal traveling through a transmissionline generates electric and magnetic fields around it. The electric field isuniformly distributed across the transmission line structure. However, inthe presence of a dielectric (commonly thin with d << λ), the electric fieldlines are concentrated in the region between the conductor and the groundplane, with some fraction being in the air region above the conductor asshown in the Figure 2.8.The non-uniformity in the electric field is due to(small) longitudinal electric and magnetic field components generated atthe air-substrate interface. These components arise due to the change inthe dielectric properties at the air-substrate interface [107].Figure 2.8: Electric field (E- field) and magnetic field (H-field) distributionacross a microstrip transmission line. All rights reserved [107].As electric field propagates in more than one medium, the effective per-mittivity is calculated using equation 2.5 to 2.8.when (wh ≥ 1)eff =r + 12+r − 12[(1 + 12(hw))−0.5](2.5)Z0 =120pi√eff ∗[wh + 1.393 +23 ln(wh + 1.444)]Ω (2.6)when (wh < 1)eff =r + 12+r − 12[(1 + 12(hw))−0.5+ 0.04(1−(wh))2](2.7)242.3. FEM analysis of a planar microstrip resonatorZ0 =60√effln(8hw+ 0.25wh)Ω (2.8)Where eff is effective relative dielectric constant, w is width of the mi-crostrip line, h is the height of the microstrip line, Z0 is the characteristicimpedance of the line, r is the relative permittivity of the substrate. There-fore, to achieve an input impedance of 50 Ω, the width of the microstrip linewas calculated to be 2.48 mm as shown in the Figure 2.9.Figure 2.9: Width of the transmission line (mm) versus input impedance(Ω) for a constant dielectric height of 0.79 mm and permittivity of 2.2.Note that the thickness of the copper cladding and the effect of frequencywas omitted while calculating the width of the transmission line. A moreaccurate value for the width can be extracted using the Keysight’s LineCalctool.Microstrip feedline design and simulationUpon determining the optimum microstrip width to height ratio to achievea characteristic impedance of 50 Ω, a simple two-port model of a microstripline was simulated in High-Frequency Structure Simulator (HFSS). One portwas connected to a source and the other was connected to a 50 Ω load as252.3. FEM analysis of a planar microstrip resonatorshown in Figure 2.10. The reflection coefficient S11 was measured and plot-ted as shown in Figure 2.10.Figure 2.10: HFSS model of a matched transmission line connected toa 50 Ω load (a). Measured transmission coefficient (b), and transmissioncoefficient plotted on a smith chart (c).The feedline was matched to the characteristic impedance i.e. 50 Ω.Therefore, the EM signal was transferred from the input to output throughcharacteristic impedance i.e. 50 Ω load with little to no reflection. This isreflected in the low magnitude of the reflection coefficient from 1 GHz to 2GHz. Also, to confirm the matching, the reflection coefficient was plottedon a smith chart. As the load was matched to the feed line, the measuredresult was close to the center of the smith chart.2.3.3 Split ring resonator designA ring resonator is a transmission line formed in a closed loop. For aresonator to operate at a certain resonant frequency, the circumference of262.3. FEM analysis of a planar microstrip resonatorthe resonator must be equal to a multiple of half of the wavelength calculatedat that frequency. Equation 2.9 calculates the length of the ring resonatorrequired to achieve resonance at the desired frequency (f GHz).l =c2f√effcm (2.9)Where f is the resonant frequency in GHz, c is the speed of light in millimeterper second and l is the length of the resonator in millimeter, eff is theeffective permittivity of the medium surrounding the resonator. A resonatorin its simple form consists of a split ring coupled to a feed line as shown inFigure 2.11.Figure 2.11: A Model of a split ring resonator (a) and the S11(dB) responseof the model resonator (b).The circumference of the resonator calculated to resonate at 1.76 GHzwas found to be 57.4 mm. The resonant frequency was selected to accom-modate the loss characteristics of the solid agar. A major portion of solidagar constitutes water trapped between agar matrix. Based on the measuredeffective loss characteristics of solid agar at lower frequencies using N1501Adielectric probe from keysight (Appendix A.1), the resonant frequency wasconsidered from the lower spectrum of microwave frequencies. However, atlower frequencies, the size of resonator increases. Therefore, to design acompact and portable resonator while considering the low loss characteris-tics of solid agar at low frequencies, the resonant frequency was chosen tobe in the L- band (1 GHz– 2 GHz) of the microwave spectrum.Based on the application, it can be designed as a one-port device ortwo-port devices. Several sophisticated and complex resonator designs can272.3. FEM analysis of a planar microstrip resonatorbe formed by cascading two resonators together or integrating active andpassive devices such as power splitters and transistors. In this section, wedesign a single-port device as it requires less electronic circuitry to measurethe response and occupies less substrate space.Study on the impact of coupling gap width on the resonatorTwo transmission lines are said to be coupled when power is transferreddue to electromagnetic field interaction between the lines [57]. There are twomain modes of propagation of the signal through a coupled transmission linepair: even mode and odd mode. Even mode occurs when the currents in thetransmission lines are in equal in amplitude and in the same direction. Oddmode on the other hand occurs when the current in the coupled lines areequal in amplitude but in opposite directions. In a coupled transmission line,the amplitude of the signal can be treated as a superposition of appropriateamplitudes of even and odd modes [57, 108].The coupling gap (CG) length between the feed line and the split ringresonator impacts the resonant frequency and quality factor of the resonator.Coupling between the feed-line and resonator can be either loose or tightcoupling. The coupling between the resonator and the feed-line is consideredas loose coupling if the distance between the resonator and the feed line islarge. Loose coupling is an indication of the presence of a small capacitancebetween the coupling gaps. Tight coupling on the other hand arises when thefeed line and resonator are closer to each other leading to a large capacitancebetween the transmission line. Therefore, to determine the optimal couplingdistance between the feed line and the split ring such that the capacitancebetween the feed line and split-ring resonator is minimum, a simulation studywas performed where the response of the sensor at different coupling gapdistance was performed. The split ring was positioned at an initial distanceof 0.4 mm and gradually increased to 1.4 mm in steps of 0.2 mm. Theresponse of the study is shown in Figure 2.12 (b). To understand the impactof coupling gap variation on the electric and magnetic field distributionacross the split ring resonator, an EM field simulation was performed. Theresults of the EM field simulation is presented in Figure 2.12 (c) and (d).As per the simulated results shown in Figure 2.12 (b), the response of thesensor improved progressively with each increment in the width coupling gapup to 1.2 mm. Further increase in the width of coupling gap from 1.2 mmto 1.4 mm resulted in a decrease in the quality factor of the resonant profile.However, observing the electric and magnetic field distribution across thesplit ring resonator at various coupling gap widths, the electric field strength282.3. FEM analysis of a planar microstrip resonatoracross the coupling gap decreases as the coupling gap width increases.From Figure 2.12 (d), feed line and split ring resonator is magneticallycoupled with each other. The magnetic coupling between the two transmis-sion line is observed to be strongest at a coupling gap width of = 0.8 mmand 1 mm. Further, the coupling capacitance between the feed line and split292.3. FEM analysis of a planar microstrip resonatorFigure 2.12: A simple split ring resonator with ring gap (RG) and couplinggap (CG) (a). The coupling gap was varied from 0.4 mm to 1.4 mm insteps of 0.2 mm. The S11 (dB) vs Frequency (GHz) response of the sensorfor different coupling gap widths (b). Electric field distribution (c) andmagnetic field distribution (d) across the split ring resonator for variouscoupling gap widths.ring resonator at various gap distances was calculated using the equations2.10 to 2.12 for parallel plate capacitors [109].∆t = d+0.0885 ∗ 10 ∗ d ∗ ln(Lw + 1)pi(2.10)C =0.0885 ∗ r ∗ 10 ∗ (L+ ∆t) ∗ (w + ∆t)d(2.11)Cnorm =0.0885 ∗ r ∗ L ∗ wd(2.12)Where L is the length of the capacitor plates in mm, W is the widthof the capacitor plates in mm, d is the distance between the feed line and302.3. FEM analysis of a planar microstrip resonatorsplit ring resonator in mm, r is the relative permittivity of the substrate,C is the total capacitance including fringing capacitance in pF , ∆t is theincrease in the length and width of the capacitor due to fringing effect.Figure 2.13: Gap distance (mm) versus capacitance (pF) for a parallel platecapacitance.The effect of the gap distance on the coupling capacitance is presented inFigure 2.13. From the results, it can be observed that as the coupling gapwidth increases, the coupling capacitance decreases and remains constantafter a gap distance of 0.8 mm. Therefore, considering the electric field,magnetic field distribution, and coupling capacitance variation with respectto gap distance, 0.8 mm was selected as the optimum coupling gap widthbetween the feed line and split-ring resonator.Previous studies on bacterial growth on solid agar such as the one con-ducted by Mohammadi et al. [30] using microwave resonators highlightedthe importance of the sensing area to detect bacterial growth. The exper-iments performed by the team utilized a resonator whose sensitive regioncovered an area of 2 mm2( L=2 mm and W= 1 mm). The area covered wasable to monitor 1.5 µL of E. coli. Thus, to measure a significant area ofbacteria using our designed sensor, the ring gap was elongated. The pro-posed design is shown in Figure 2.14. An alignment marker was added tothe design to align the petri dish between the resonator and the test sample.312.3. FEM analysis of a planar microstrip resonatorFigure 2.14: Planar split ring resonator modelled in HFSS (a) and thesimulated response of the microstrip resonator (b).Sensitive region determinationA resonator detects changes in the dielectric properties in the vicinityof the sensor by measuring the interaction between the material and theelectric field around the resonator. These interactions alter the electricalcharacteristics of the resonator such as resonant amplitude, resonant fre-quency, and quality factor through which dielectric properties of the testsample can be interpreted. Thus, it is important to determine the regionsacross the resonator where electric field intensity is maximized. To deter-mine the electric field distribution across the resonator, the sensor is simu-lated in High-Frequency Structure Simulator (HFSS) and the magnitude ofelectric field (E-field) and magnetic field (H-field) across the sensor at theresonant frequency is plotted. The field distributions are shown in Figure2.15. According to Figure 2.15 (a), a strong magnetic field intensity acrossthe coupling gap of the resonator was observed. The presence of a strongmagnetic field indicates strong inductive coupling between the feed line andsplit ring resonator.According to the distribution of electric field intensity shown in Figure2.15 (b), the maximum E-field intensity was observed at the resonant gapindicating high sensitivity to variations of the dielectric properties in its nearmedium. The presence of a strong electric field intensity indicates a strongcapacitance between the ring gap. The electric field intensity is weaker nearthe coupling gap indicating the presence of a weak capacitance between thefeed line and the resonator.322.3. FEM analysis of a planar microstrip resonatorFigure 2.15: H- Field (a) and E- Field (b) analysis of a microstrip resonator.To further study the impact of the coupling gap and ring gap on theresonator, a test sample is placed on the ring gap and the coupling gap asshown in Figure 2.16 (a)- (c). The test material is a standard sample withpermittivity 2.2, loss tangent 0.0009, and dimension 6 x 6 x 1.57 mm. Thesample is placed at a height of 0.1 mm above the surface of the sensor. Thematerial characteristics and distance from the substrate were held constantfor all the simulations. The simulation results are shown in the Figure 2.16(b) .The resonant frequency of the simulated bare sensor was 1.76 GHz. Inthe presence of a test material covering the entire ring gap, a shift in theresonant frequency was observed from 1.76 GHz to 1.722 GHz with a drop inresonant amplitude from -11.57 dB to -14.36 dB. On the other hand, whenthe test material was placed on the coupling gap as shown in Figure 2.16332.3. FEM analysis of a planar microstrip resonatorFigure 2.16: Simulation results (d) of a planar microwave resonator withtest sample on the ring gap (RG) (a), coupling gap (CG) (b), and on theentire CG (c).(b), a negligible change in the resonant amplitude and resonant frequencywas observed. Therefore, based on the response of the sensor to a testsample placed on the ring and coupling gap, the ring gap was selected asthe sensitive region to detect bacterial growth.Study on the response of the sensor to changes in permittivityand loss tangent at the ring gapIn the previous section, the sensitive region of the sensor was determinedby studying the electric field distribution and the impact of test material onthe resonant frequency and amplitude of the resonator. In this section, theresponse of the sensor to changes in the permittivity and loss tangent of atest material on the ring gap will be analyzed and interpreted.A test sample is placed on the ring gap of the split ring resonator asshown in Figure 2.17 (a). The dimension of the sample was 6 x 6 x 1.57mmand was placed at a height of 0.1 mm from the surface of the substrate.The attributes of the test sample such as orientation, dimension, and thegap between the substrate and the sample were held constant throughoutthe study. According to the simulation results shown in Figure 2.17 (b)and (c), the resonant frequency and resonant amplitude decreased as thepermittivity of the sample increased from 2 to 4 in steps of 0.5 with a342.3. FEM analysis of a planar microstrip resonatorFigure 2.17: A HFSS simulated model of the microstrip resonator with aSUT on the ring gap (a). Effect on the resonant profile (resonant amplitudeand frequency) due to variation of permittivity (b) and loss tangent (c) ofa sample under test on the ring gap.constant loss tangent of 0.0009. However, as the loss tangent of the samplewas increased from 0.0001 to 0.0017 in steps of 0.0004 while maintaininga constant permittivity, the resonant amplitude decreased with no changein the resonant frequency. Thus, the resonant amplitude was found to beresponsive to changes in loss tangent or conductivity of the SUT whereasresonant frequency was found to be responsive to changes in permittivity ofthe SUT.Lumped parameter modelPrevious sections on the microstrip resonator aimed to understand thedistributed model of the resonator utilizing the transmission line. The dis-tributed model enabled us to use complex CAD tools such as HFSS to352.3. FEM analysis of a planar microstrip resonatorunderstand the interaction between the electric field and the dielectric ma-terial under test. However, to understand the behavior of the resonator,a lumped parameter model is used. Figure 2.18 (a) shows the lumped pa-rameter model of the designed resonator. The behavior of the structure ismodeled using lumped elements such as resistors (Ohm), inductors (Henry),and capacitors (Farad). In this work, the lumped-element model of the res-Figure 2.18: Lumped-element model of a frequency variation resonator (a)and the recorded S11(dB) response (b).onator was designed and simulated in Advanced Design Systems (ADS) 2020by Keysight Technologies. Based on the resonant frequency, the inductance(L) and capacitance (Cgap) of the network were calculated using equation2.13.L ∗ Cgap = (2pifres)−2 (2.13)The series resistance (R Ω) of the network was kept to a minimum to362.3. FEM analysis of a planar microstrip resonatoravoid power loss and the turn’s ratio (T = 11.08:1) of the transformer cou-pling the magnetic field from the feed line to the resonator was adjusted toimprove the quality factor of the S11 notch. The inductance (L nH) andcapacitance (CgappF ) was calculated and found to be 5 nH and 1.63 pF,respectively. The coupling capacitance (Cc = 0.0001pF ) was negligible andhad no impact on the result. On further analysis, it was observed that bymaintaining a low coupling capacitance (CcpF ) and low series resistance (RΩ), a desired S11 response at the desired resonant frequency and qualityfactor was obtained by modeling the sensor structure. The result of thesimulated lumped-element model is presented in Figure 2.18 (b). Accordingto the simulated results, the S11 response of the Lumped- element model isin good agreement with the simulated bare result of HFSS.Challenges to frequency variation sensorA frequency mode sensor is effective in determining the characteristicsof a material such as permittivity and conductivity in environments whereambient fluctuations have a negligible effect on the output of the sensor.However, they lose their advantage to cross-sensitivity caused by variationsin ambient factors such as temperature and humidity. Therefore, to over-come this limitation, a differential mode resonator is used. The next sectionis thus dedicated to the design and analysis of differential mode resonators.2.3.4 Differential mode resonator designFigure 2.19: Proposed sensor design and its simulated S11 (dB) in HFSS.A differential mode resonator consists of two resonators, sensing, anda reference resonator. The sensing resonator is sensitive to changes in the372.3. FEM analysis of a planar microstrip resonatordielectric properties of the material and the surrounding environment. Thereference resonator, on the other hand, is only sensitive to changes in thesurrounding environment and so acts as a control. Therefore, a differencebetween the sensing and reference resonator outputs eliminates the cross-sensitivity of the ambiance on the measured result.The proposed differential resonator is shown in Figure 2.19. The tworesonators: sensing resonator operating at 1.514 GHz and a reference res-onator operating at 1.797 GHz were designed. The sensor was designed onrogers 5880 with permittivity 2.2, loss tangent 0.0009, and thickness 0.79mm. Based on the resonant frequencies and permittivity of the substrate,the length of the sensing and reference resonators was calculated as 67 mmand 56.3 mm respectively. However, as a capacitive arm of 5 mm was re-quired to detect and monitor bacteria growth, the lengths of the resonatorwas adjusted to achieve the selected resonant frequencies. The width of themicrostrip transmission line was calculated to be 2.48 mm. The ring gap andcoupling gap for both the sensing and reference resonator was selected as 1mm and 0.8 mm respectively. The dimensions of the resonator are shown inFigure 2.19.Sensitive region determinationFigure 2.20: H- Field distribution (a) and E field distribution (b) of thedesigned differential resonator at 1.502 GHz.382.3. FEM analysis of a planar microstrip resonatorTo determine the sensitive region of the resonator, an electric field andmagnetic field simulation was performed in HFSS. Figure 2.20 presents theEM field distribution at 1.509 GHZ. According to the simulated results, aregion of strong magnetic field was observed at the coupling gap as comparedto the ring gap indicating a strong electromagnetic coupling between feedline and the resonator. However, figure 2.20 (b) shows strong electric fieldintensity concentrated at the ring gap of the sensing resonator indicatinghigh sensitivity to variations in the dielectric properties in its vicinity.However, to validate the sensing region of the resonator, an analysisin HFSS has been performed by placing a test material at two differentlocations as shown in 2.21.Figure 2.21: Simulation results of a differential microwave resonator withtest sample on the coupling gap (CG) (a) and ring gap (RG) (b) of thesensing resonator.The test material simulated was a standard sample with a permittivityof 2.2, loss tangent of 0.0009, and the dimension of 6 x 6 x 1.79 mm. The pa-rameters of the test sample such as orientation, dimension, and the distancebetween the substrate and the test material were kept constant. Accordingto the simulated results shown in Figure 2.21, a frequency shift of 50 MHzwas observed when the material was placed at the ring gap while the ref-erence output was unaffected. However, as the material was placed on thecoupling gap, a negligible change in the resonant amplitude and resonantfrequency of both the sensing and the reference resonator was observed.Therefore, the ring gap was found to be more responsive to the presenceof a dielectric material as opposed to the coupling gap and was chosen formeasurements.392.4. Chapter conclusion2.4 Chapter conclusionIn this chapter, two different schemes of microstrip resonators: frequencyvariation resonators and differential mode resonators have been discussed.The proposed resonator designs were thoroughly analyzed and studies to de-termine the sensitive regions of the resonator. Based on the study, frequencyvariation resonators found applications where temperature and other envi-ronmental factors were constant and had minimal impact on the resonatoroutputs. However, in the case of significant variation in the ambient fac-tors, differential mode resonators were used. The next chapter will discussthe steps followed to fabricate the resonator and also discuss the variousfunctional tests performed to validate the operation of the sensor.40Chapter 3Fabrication and evaluation ofthe designed sensorThis chapter focuses on the fabrication and functional verification ofthe sensor. This chapter begins with the step by step in- house fabricationprocedure followed to precisely and accurately fabricate the sensor. Theoperation of the fabricated sensor will then be investigated using a standarddielectric test material. This chapter presents the best outcomes of theseveral tested sensors. The final sensor design of the microwave sensors wasachieved after weeks and months of numerous sensor design optimizations,and an in-depth analysis of the measured results.3.1 Sensor fabricationTo test the viability of the simulated designs, the sensors need to befabricated. Fabrication is a crucial step and involves various chemicals andstrong etchants. If performed incorrectly, errors in this step can damage thefabricated sensor. The steps followed to fabricate the resonator using in-house fabrication equipment is described below. The finalized layout of theresonator was first transferred to laminated paper. To preserve the intricatefeatures of the resonator, a high-resolution laser printer was used to transferthe design to a photocopy paper. The printed design was then patternedon the selected substrate using a swingline thermal laminator. Under theapplication of heat, the pattern from the photo paper was transferred to theselected laminate i.e. Rogers 5880 with permittivity of 2.2 and dielectricthickness of 0.79 mm. The patterned substrate was then subjected to anetching process using a bath containing 750 gm ammonium persulphate in 5liters of water. Ammonium persulphate is an excellent copper etchant andis widely used for PCB fabrication. The ammonium persulphate reacts withthe exposed unwanted copper forming copper sulfate that dissolves in thebath. The bath was constantly aerated and warmed to increase the etchingrate. The etching was thoroughly monitored as over-etching could result in413.1. Sensor fabricationan irreversible damage to the designed sensor.Once, the unwanted copperwas etched from the substrate, the etched substrate was washed using 95%acetone. This substrate was rinsed until the ink from the patterned designwas completely removed revealing the copper traces. The sensor was thenwashed in RO water to remove traces of acetone from the substrate. ASubMiniature version A or SMA connector was soldered to the sensor. Thefabricated sensor is shown in Figure 3.1 and was functionally verified beforeusing it for its intended application.To verify the working of the developed sensor, the fabricated sensorwas connected to a vector network analyzer (VNA) ZNB20 from Rhodeand Schwarz. The fabricated sensor was mechanically stabilized on thetable to avoid measurement errors due to mechanical vibrations from theenvironment. The VNA was calibrated from 1 GHz to 2 GHz with anintermediate frequency bandwidth of 300 Hz and an input power of 0 dBm.The resolution or number of points was set to 6401 before measurements. Anautomated program was coded in LabVIEW to extract the resonant profiledetails such as resonant amplitude and the resonant frequency at a rate ofevery 2 minutes.Figure 3.1: Fabricated planar microstrip resonator.While performing a time-based measurement of the fabricated sensor, itwas observed that the response of the sensor was degrading over time. Uponinvestigation, this phenomenon was linked to the accumulation of staticcharge on the surface of the resonator. Therefore, to discharge the static, theresonator had to be connected to ground without impacting the operationof the designed sensor. Thus, the resonator was redesigned to incorporatea quarter wavelength microstrip short stub connected to a ground plane.The microstrip stub acts as open circuit to the electromagnetic signals at423.2. Evaluation of Frequency variation sensorthe resonant frequency and short circuit to the static charges. The modifiedresonator design is shown in Figure 3.2.Figure 3.2: Fabricated planar microstrip resonator with ground.3.2 Evaluation of Frequency variation sensor3.2.1 Impact of SUT on the sensitive region of the sensoron the electrical characteristic of the sensorsTo study the impact on the response of the resonator in the presence of asample under test, the reflection coefficient or S11 response of the fabricatedsensor was measured. The response of the sensor in the absence of anytest material is shown in Figure 3.3. The resonant frequency and resonantamplitude of the fabricated sensor in the absence of any test material weremeasured to be 1.76 GHz and -13.52 dB respectively.To determine the sensitive region of the resonator, a test sample whosepermittivity was close to the effective permittivity of solid agar in a petridish was chosen. The steps followed to measure the effective dielectric prop-erties of solid agar is presented in Appendix A.1. Among the available testsubstrates, a 6 x 9 mm test sample machined from a standard Rogers 5880substrate with permittivity of 2.2, loss tangent of 0.0009 and thickness 1.79mm was chosen to study the effect of test material on the response of thesensor. The sample was placed on the ring gap and the coupling gap andthe response of the sensor was measured as shown in Figure 3.3.As evident from the measured results, a resonant frequency shift of 50MHz is observed when the test sample is placed on the ring gap whereas anegligible resonant frequency shift of 1 MHz was observed when the mate-433.3. Evaluation of differential mode resonatorFigure 3.3: Measured response of the sensor in the presence of test sampleon the ring gap and coupling gap.rial was placed on the coupling gap. Thus, the ring gap was found to beresponsive to the presence of a test material as opposed to the coupling gapand is in good agreement with the simulated results.3.3 Evaluation of differential mode resonator3.3.1 Impact of SUT on the sensitive region of the sensoron the electrical characteristic of the sensorsThe fabricated differential-mode resonator is shown in Figure 3.4 (a).The sensor consists of two simple resonators: one acts as a sensing res-onator and the other as a reference resonator. The resonators are groundedusing microstrip stubs as shown in Figure 3.4 (a). The response of the bareresonator in the absence of a sample under test is shown in Figure 3.4 (b).To study the impact on the response of the resonator in the presence of asample under test, the reflection coefficient, or S11 response of the fabricatedsensor was measured. The resonant amplitude and resonant frequency of thesensing resonator and the reference resonator in the absence of any test ma-terial were measured to be -10.38 dB at 1.508 GHz and -11.56 at 1.7946443.3. Evaluation of differential mode resonatorGHz respectively. To determine the sensitive region of the resonator, a 6 x 6mm test sample machined from a standard Rogers 5880 substrate with per-mittivity of 2.2, loss tangent of 0.0009 and thickness 1.79 mm was obtained.The response of the sensing and reference resonator due to the presence oftest material on the ring and coupling gap of the sensing resonator is shownin Figure 3.4 (b).A shift in the resonant profile of the sensing resonator was observed from-10.38 dB at 1.508 GHz to -11.51 dB at 1.465 GHz due to the presence ofthe test sample with minimum to no impact on the output of the referenceresonator. However, the presence of the test sample at the coupling gap(CG) had no impact on the output of the sensing and reference resonatoras seen in Figure 3.4. Thus, the ring gap was found to be responsive to thematerial as opposed to the coupling gap and is in good agreement with thesimulated results.3.3.2 Conversion of two- port to one- port microstripdevice using a power splitterThe differential resonator fabricated in the previous section was a two-port device operating at 1.509 GHz and 1.798 GHz respectively. However,to reduce the electronics required to perform measurements, the device wastransformed into a one-port device using microwave power splitters or powerdividers. Among the available power splitters such as Wilkinson, T- Junc-tion, and Rat- race junction power dividers, the Wilkinson power divider waswidely used due to its ability to efficiently split the input power between itsoutputs while maintaining a matched condition on all ports. However, con-ventional Wilkinson power splitters operate in a narrow range of frequenciesand fail to efficiently divide input power at frequencies outside their op-erational bandwidth. Thus, to overcome this limitation, an off the shelfwideband power splitter was used.The advantage of using a wideband power splitter lies in its ability toprovide stable power output at its output ports for a wide range of frequen-cies. The power splitter also provides a high degree of isolation betweenits output ports. Therefore, a wide band power splitter ZAPD-21-S+ fromMini circuits capable of providing a stable power output between 0.5 and 2GHz was used with the sensor as shown in the Figure 3.5 (a).To study the effect of the power divider on the response of the res-onator, the transmission coefficient of the sensing and reference resonatorwas measured individually and compared with the transmission coefficientof the sensor combined with the power divider (shown in Figure 3.5 (b)).453.3. Evaluation of differential mode resonatorFigure 3.4: Fabricated resonator on Rogers 5880 (a) and measured resultswith test sample on the ring gap (RG) and coupling gap (CG) of the sensingresonator (b).According to the measured results, due to the integration of a power dividerwith the fabricated resonator, a −3 dB drop in the resonant amplitude isobserved with minimum impact on the resonant frequency. The drop in463.4. Chapter conclusionFigure 3.5: Fabricated differential resonator with a wide band power split-ter (a) and measured S11(dB) response of the fabricated sensor with andwithout the power splitter (b).the amplitude can be attributed to the insertion loss of the power splitter.Thus, integration of power divider had a minimum impact on the electricalcharacteristic of the resonator and thus was used to convert the two-portdifferential sensor to a one-port device.3.4 Chapter conclusionIn this chapter, the steps followed to fabricate two different designs ofmicrostrip resonators such as frequency variation resonators and differen-tial mode resonators have been discussed. The fabricated sensor was thensubjected to a test to determine the sensitive region of the sensor. Deter-mining the sensitive region of the sensor enables us to proceed further andinvestigate and monitor bacterial growth on solid media subjected to var-ious external factors such as nutrient availability, subsurface position, andpresence of antibiotics.47Chapter 4Application ofcustom-designed microstripresonators for real-time andcontactless monitoring ofbacterial growth on solidmediaThis chapter includes the experiments conducted to assess the ability ofthe designed planar microwave resonators for real-time and contactless bac-terial growth monitoring on solid agar medium. The growth of bacteria isinfluenced by several factors including temperature, growth media composi-tion, nutrients availability, and presence/absence of inhibitory compounds.Precisely, through this chapter, we demonstrate the ability of microwave de-vice to detect and characterize the impact of external factors on the growthprofile of bacteria (E. coli).The first study presented in this chapter is a proof of concept and exam-ines the potential of a microwave sensor to detect and monitor the influenceof various amounts of glucose (0, 2, 5, and 10 %) in Luria-Bertani (LB) agaron the growth profile of E. coli. Glucose supplements at varying concen-trations to growth media can accelerate or inhibit the growth of bacteria.Therefore, through this study, the ability of the microwave resonator to ac-curately and swiftly differentiate the impact of glucose concentration on thegrowth of bacteria is also presented. The measured results were comparedwith the microscope images captured at constant intervals of time to verifythe sensor’s competency as a bacterial growth monitoring tool. The secondstudy investigates the capability of the sensor resonator to monitor subsur-face growth of E. coli confined between two layers of LB agar. The growthof bacteria confined between two layers of solid growth media progresses by484.1. Microwave resonator reveal the influence of glucose on E. coli growthtransitioning from a 2-Dimensional growth to 3-Dimensional growth. Thevariations in the dielectric properties of the growth medium due to lateraland subsurface growth of E. coli were monitored by the sensor and presentedhere. A third novel study demonstrates the potential of the designed mi-crowave sensor to monitor the effect of antibiotics, erythromycin (0, 7.5, 30,and 45 µg) on the growth of E. coli. It is well known that various concentra-tions of antibiotics have a different impact on bacterial growth. Therefore,early detection and monitoring of the growth of bacteria in the presence ofantibiotics using sensor technology can contribute to antibiotic treatment.The studies presented in this chapter demanded a year of continuousmeasurements and sample preparations which includes the failed attemptsto prepare an agar plate for bacterial growth monitoring growth. Moreover,throughout the course of this work, a considerable amount of time was spenton researching and optimizing the sample preparation technique, analyzingthe measured results, and upgrading the experimental setup to avoid anyinterference of external factor (e.g. temperature) on sensor measurements.4.1 Microwave resonator reveals glucosedependent growth profile of E. coli on solidagar4.1.1 BackgroundBacteria require nutrients during metabolism to sustain growth. Theavailability of crucial nutrients such as carbon can modify the morphologyand impact the growth rate of microorganisms such as E. coli [110, 111].Traditional plating methods such as spread and pour plate techniques havebeen widely employed to monitor and characterize the impact of nutrientavailability on bacterial growth [112]. However, these methods were notreal-time, require a longer incubation period to detect noticeable bacterialgrowth, and were more prone to contamination. Recently, non-contact mi-crowave sensors have proved to be a viable tool to measure bacterial growthin liquid matrices [50, 113, 114]. A recent study by Mohammadi et al. [30]demonstrated the capability of planar microwave sensors to monitor bacte-rial growth on solid media. This research further investigates the capabilityof a microwave sensor to study the impact of nutritional parameters on thegrowth profile of E. coli. Their work was able to successfully demonstratethe capability of a microstrip resonator to monitor bacteria growth on thesurface of solid agar. The bacterial growth was monitored by gauging the494.1. Microwave resonator reveal the influence of glucose on E. coli growthchange in the resonant amplitude due to variation of the conductivity ofgrowth medium due to bacteria metabolism.4.1.2 Material and methodSample preparationTo prepare solid LB agar media with 2%, 5%, and 10% glucose concen-tration, 1 gm of LB broth and 0.75 gm of LB agar was mixed with 50 mlRO water in a flask. The resultant solution was then mixed with 1 gm, 2.5gm, and 5 gm of powdered glucose to achieve 2%, 5%, and 10% of glucoseconcentration in the media respectively. The media was placed in an auto-clave for 15 minutes at 121 ◦C to completely sterilize and kill any bacteriaspores present in the growth media. The sterile media was cooled down to50 ◦C. To achieve a uniform thickness of 1 mm across the petri dish, 3 ml ofthe sterilized media was poured in the petri dish. The cross-section of theagar plate is shown in Figure 4.1.Figure 4.1: Cross-section of a solid agar plate.The media preparation was performed in a fume hood or in the presenceof a bunsen burner to avoid contamination of the sample with unwantedand airborne fungi. Fungi contamination leads to degradation of the plateand results in false positives. The fungi-contaminated agar plate is shown inFigure 4.2 (a). The plate was carefully sealed using a parafilm after the solidmedia reached room temperature. Sealing the media plate while the agaris still under the process of solidification leads to precipitation which altersthe water content in the petri dish and thereby render the plate unusablefor future experiments as shown in Figure 4.2 (b).The above steps outline the general steps followed to prepare a controlplate i.e. with no bacteria inoculated on the surface of the agar. To prepare504.1. Microwave resonator reveal the influence of glucose on E. coli growthFigure 4.2: solid agar plate contaminated with airborne fungi (a) and solidagar plate with precipitation (b).inoculated agar plates, 3 µL of E. coli sample is spotted onto the agar plateusing a sterilized loop or pipette at the desired location and dried. In thisstudy, the HB101 strain of E. coli was used. The E. coli strain is non-pathogenic and has a rapid growth rate at optimal growth conditions. Theoptical density at wavelength of 600 nm (OD600nm) measured using a spec-trophotometer was found to be 1.5. The plate is then sealed using parafilmto avoid cross-contamination and preserve the nature of the contents of thepetri dish.Experimental setup and procedureTo monitor the influence of 0 % (no glucose), 2 %, 5 % and 10 % glucoseon the growth profile of E. coli, a differential microstrip resonator was used.The inoculated solid agar plate was placed on the sensor covering the sensingresonator and the reference resonator. The inoculated bacterial spot wasaligned directly above the sensitive region of the sensing resonator as shownin Figure 4.3.To measure the response of the fabricated resonator, the sensor was con-nected to a vector network analyzer, ZNB20 from Rhode and Schwarz. TheVNA was calibrated from 1 GHz to 2 GHz with an Intermediate frequencybandwidths of 300 Hz and an input power of 0 dBm. The resolution or num-ber of points was set to 6401 before measurements. An automated program514.1. Microwave resonator reveal the influence of glucose on E. coli growthFigure 4.3: Placement of agar plate on the differential resonator (a). In-oculated bacteria aligned with the sensing region of the sensing resonator(b).was coded in LabVIEW to extract the resonant profile details such as theresonant amplitude and the resonant frequency at a rate of every 2 minutes.The experimental setup is shown in Figure 4.4.4.1.3 Measurement result and discussionTo monitor the growth of E. coli in solid media, an experiment wasconducted using an inoculated agar plate with no added glucose (controlplate). The aim of this experiment was to test the working of the microwaveresonator as a device capable of monitoring bacteria growth. The sens-ing resonator was employed to monitor the media surrounding the E. coli.The reference resonator was used to monitor variations in the media dueto temperature fluctuations. The measured resonant amplitude of sensingand reference resonator was normalized by subtracting with the initial valueof their respective resonant amplitudes to diminish the impact of externalfactors. Further, the difference between the normalized sensing and refer-ence resonators’ amplitudes (∆ Resonant Amplitude (dB)) was calculatedto nullify the impact of ambient factors such as temperature and humidityvariations on the measured results. Analysis of the measured results andthe ambient temperature data revealed that the measured results were in-fluenced by external factors such as temperature and external vibrations.Considering this influence of the external factors on the sensor’s response,the sensing apparatus was modified. Permanent fixtures and foams wereincorporated into the sensing apparatus to reduce external vibrations. The524.1. Microwave resonator reveal the influence of glucose on E. coli growthFigure 4.4: Experimental setup to monitor bacterial growth under variousnutrient conditions. The setup consists of a container with sensor enclosedwithin it, a temperature probe, a microscope, VNA and LabVIEW to controlthe VNA.first 60 minutes of recorded data were excluded from the experimental mea-surements accounting stabilization of the sensing apparatus and the VNAafter calibration. The residual impact of temperature on the differential re-sponse due to non-uniformity of the agar plate was identified and eliminatedby normalizing (∆ Resonant Amplitude (dB)) with the recorded tempera-ture. The measured resonant amplitude variation and resonant frequencyvariation versus time representing growth phases of E. coli on LB agar withvarying glucose concentrations (0%, 2%, 5% and 10%) is shown in Figure4.5. From the measured results, variation in the resonant amplitude( ∆ res-onant amplitude) resembles with the growth profile of E. coli as opposed tovariation in the resonant frequency (∆ Resonant frequency) and was thusselected for further analysis.Studies have reported that high concentrations of solutes such as sugar orglucose in the environment surrounding the bacteria alters the water levelssurrounding the bacteria resulting in a high osmotic pressure in the vicinity534.1. Microwave resonator reveal the influence of glucose on E. coli growth(a) LB agar with 0% glucose(b) LB agar with 2% glucose544.1. Microwave resonator reveal the influence of glucose on E. coli growth(c) LB agar with 5% glucose(d) LB agar with 10% glucoseFigure 4.5: Measured ∆ Resonant amplitude (dB), ∆ Resonant frequency(GHz), recorded temperature (◦C), and microscopic images of E. coli cap-tured at constant time intervals for 0% (a), 2% (b), 5% (c), and 10% (d) ofglucose in LB agar.554.1. Microwave resonator reveal the influence of glucose on E. coli growthof the bacteria [115]. High osmotic pressures exerts stress on the bacteriawhich suppresses bacterial growth [115]. Therefore, a complete inhibition ofbacterial growth was witnessed at 10 % glucose. The signal equivalent to nogrowth was derived from this (0.52 dB) and was selected as a threshold todifferentiate between no growth phase and growth phase essential for growthmeasurements. The region of the measured response below the threshold i.e0.52 dB was classified as no growth region. As E. coli is introduced into thegrowth medium through inoculation, the bacteria gradually adapts to thenew environment, consumes nutrients and prepares for cell division. Variousfactors such as availability of nutrients, environment temperature, culturemedium, pH, inoculum size, the physiological history of the cells, and theprecise physiochemical environment of both the original and the new growthmedium can impact the generation time of E. coli during no growth or lagphase [116–118]. Therefore, due to various ambiguities in the no- growthphase, this study focused on detection and monitoring of changes in thegrowth phase of bacteria under various nutrient conditions. The growthphase is the region where rapid multiplication of E. coli occurs. Based onthe threshold value i.e. 0.52 dB, the onset of the growth phase for 0%, 2%,and, 5 % glucose was noted at 17, 17.03, 15.6 hr. respectively.The slope of the linear growth phase representing the rate of change ofthe resonant amplitude due to change in the growth rate was calculatedand found to be 0.10913 dB/hr, 0.23646 dB/hr and 0.21410 dB/hr witha coefficient of determination 0.9004, 0.98545 and 0.99752, respectively aspresented in Table 4.1.Table 4.1: Measured growth phase and rate of change of resonant amplitudefor E. coli at various concentrations of glucose in LB agarGlucose Onset Rate of change CoefficientPercentage of of ofgrowth phase amplitude variation determination% hr (dB/hr) R20 17 0.10913 0.900422 17.03 0.23646 0.985455 15.6 0.21410 0.99752The data shows that the rate of change in resonant amplitude was highestat 2 % glucose (0.23646 dB/hr) compared to 0 % (0.10913 dB/hr.). How-ever, a slight decrease of the rate was observed as the glucose concentrationreached 5 % (0.2141 dB/hr.) and a complete inhibition of bacterial growth564.2. Microwave resonator to monitor subsurface growth of E. coliwas observed at a glucose concentration of 10%. The accuracy and perfor-mance of the designed sensor to monitor bacterial growth on various LBagar- glucose media was further backed by microscope images as shown inFigure 4.5. This further confirms the effectiveness of the designed microwaveresonator to study factors influencing bacterial growth in solid agar therebyovercoming the shortcomings of conventional bacterial growth monitoringmethods.4.2 Microwave resonators to detect and monitorsubsurface growth of E. coli on solid agar.4.2.1 BackgroundInfectious diseases caused by biofilm forming bacteria such as periodon-titis and cystic fibrosis pneumonia pose serious challenges to public healthand global economy [119–123]. Most biofilm related infections are difficultto treat as they tend to resist various antibiotics and biocides [124–126].Moreover, contamination of medical devices and implants by biofilm form-ing bacteria can limit the usability of the device and could also lead to lifethreatening infections[121, 127–130]. The U.S Department of Health andHuman Services have reported that roughly one among 24 infection cases inUSA are related to contaminated medical devices such as catheters, heartvalves, and prosthesis [127, 131]. Therefore, it is of paramount importanceto detect and monitor subsurface bacteria biofilm growth on tissues due tocontaminated implants to prevent life threatening infections.Prior to detecting and monitoring subsurface bacterial growth, it is nec-essary to understand the growth mechanism of bacteria biofilms. A bacterialcolony in a biofilm initially grows on the surface of the medium in 2D andlater progresses into 3D [132, 133]. In the absence of any obstruction, thetransition from 2D to 3D occurs by bacteria expanding in the space above thesurface of the growth medium. However, in the presence of an obstruction,the bacteria tend to proliferate and expand into the medium [132]. Studieson the behavior of such transitions suggested that various mechanical forceson the bacteria played a vital role in the transition of microcolonies from 2-Dto 3-D [129, 134]. Various forces act on the bacterium such as i) repulsiveforces between the bacterium ii) frictional force between the bacterium andiii) elastic forces of the surrounding media [129, 132]. Such forces can strainthe bacteria to a transition point known as “buckling transition” at whichbacteria begin to invade its surrounding media.574.2. Microwave resonator to monitor subsurface growth of E. coliThe current technologies to detect these pathogens rely on either con-ventional culture–based assays or molecular methods such as polymerasechain reaction (PCR), enzyme-linked immunosorbent assays (ELISA) andmass spectroscopy. However, these are often limited by longer detectiontime as in the case of culture based assays or requires extensive samplepreparation and expensive instrumentation in the case of molecular tools[12–14]. To overcome these limitations, researchers developed biosensors forrapid detection and monitoring of microbial growth that can be used inpathogenic detection [15–20]. Of these techniques, optical and electrochem-ical biosensors were widely used owing to their real-time, automated andlabel-free detection. However optical biosensors suffer from high cost andquick degradation of reagents whereas electrochemical biosensors suffer froma low selectivity and limit of detection (LoD) [20]. Bio-FET are another classof biosensors which are gaining popularity due to their label-free, ultra-highsensitivity, scalability, low cost of fabrication and minimum power consump-tion [20, 135–137]. Although, the benefits of bio-FET make it a promisingcandidate for detection and monitoring of bacteria, these sensors cannot beused in contactless applications. Recently, microwave sensors have emergedas a potential detection method for various applications including dielectricspectroscopy, monitoring cancer, detection of glucose and electrolyte levelin aqueous solution, and classification and monitoring of bacteria in liquidmedium owing to their numerous advantages such as low cost, real time,label-free and non-contact monitoring [31, 50, 68, 138]. Interestingly, bacte-rial monitoring using microwave sensors is gaining more attention in recentyears.Through this current research, the capability of the microwave sensor tomonitor subsurface bacterial growth was explored. To the best of our knowl-edge, this is a maiden attempt in this direction. The experiment was carriedout at two different initial volumes of E. coli i.e. 3 µL and 9 µL sandwichedbetween two layers of solid LB-agar using microwave resonator owing totheir robustness, low fabrication cost, contactless and label-free properties.The changes in the characteristics of the medium due to the lateral andsubsurface growth of the bacteria are studied by measuring the variation inthe resonant amplitude of the microstrip resonator at two different volumesof bacteria.584.2. Microwave resonator to monitor subsurface growth of E. coli4.2.2 Material and methodSample preparationTo measure subsurface bacteria growth on solid agar media, a 1 mmthick sterilized soft agar media sample(1 gm of LB broth and 0.5 gm of LBagar) was prepared.Three µL of E. coli was then spotted at the designated position on theagar plate. The inoculated bacteria were then covered by a sterilized 18 x18 mm glass cover slip coated with 8 ml of soft agar as shown in Figure 4.6.The agar was cooled to room temperature to avoid precipitation and sealedusing a parafilm. The process of encapsulating the E. coli was crucial andrequired a month of practice prior to performing the study.Figure 4.6: Cross section of a solid agar with a coverslip covered with agar.Experimental setup and procedureIn this study, a thermally insulated Styrofoam enclosure was used tostabilize the variation in the external ambient factors such as temperatureand humidity. Therefore, as the ambient temperature fluctuations wereminimized, the differential-mode resonator was replaced with a simple yetefficient frequency variation resonator to detect subsurface bacteria growth.The frequency variation resonator was compact, required no additional com-ponents such as power splitter and was cost efficient. The sensor was securedto the base of the Styrofoam container and the sample E. coli plate wasfirmly placed on the sensor. The E. coli spot was aligned with the sensingregion as shown in the Figure 4.7.594.2. Microwave resonator to monitor subsurface growth of E. coliFigure 4.7: Solid media plate on a microstrip resonator with the inoculatedE. coli aligned with the sensitive region of the resonator.The Styrofoam enclosure was equipped with an LED light source, a tem-perature probe to monitor temperature in the vicinity of the sensor and amicroscope to capture images of test sample at constant time intervals. Tomeasure the response of the fabricated resonator, the sensor was connectedto a vector network analyzer, ZNB20 from Rhode and Schwarz. The VNAwas calibrated from 1 GHz to 2 GHz with an Intermediate frequency band-widths of 300 Hz and an input power of 0 dBm. The resolution or numberof points was set to 6401 prior to measurements. An automated programwas coded in LabVIEW to extract the resonant profile details such as res-onant amplitude and resonant frequency at a rate of every 2 minutes. Theexperimental setup is shown in the Figure 4.8.The measured resonant amplitude for various volumes of E. coli wasnormalized by subtracting it from the initial measured amplitude. Themeasured data pertained to the initial 90 minutes of the experiment wasexcluded accounting time for stabilization of the apparatus.604.2. Microwave resonator to monitor subsurface growth of E. coliFigure 4.8: Experimental setup to functionally verify the operation of thesensor and monitor bacteria growth. The setup consists of a thermallyinsulated Styrofoam container with sensor enclosed within it, a temperatureprobe, a microscope, VNA and LabVIEW to control the VNA.4.2.3 Measurement result and discussionPrior to subsurface bacteria detection using microwave sensor, a prelim-inary test was conducted with two solid agar plates with and without thepresence of a cover-slip to study the bacterial penetration in the medium.Both plates were incubated for 48 hours and later visually observed to de-termine the nature of bacteria growth on solid agar with and without thepresence of a cover slip. The E. coli grown on control plate exhibited three-dimensional growth on the surface of agar. However, in the presence of acover slip, a lateral surface growth was observed followed by a penetratedgrowth into the subsurface agar medium as shown in Figure 4.9.Previous studies on the growth of microorganisms have shown that acrucial stage in the subsurface bacterial growth involves the transition oflateral or 2-Dimensional growth into 3-Dimensional growth into the mediumdue to mechanical forces acting on the bacteria [132]. Various studies haveinvestigated the effects of mechanical forces on the planar to bulk transitionof bacteria sandwiched between two mediums [129, 134]. Bacterial growthand its correlation with dielectric variations in the surrounding environmenthas been successfully demonstrated [139–142]. A similar observation was614.2. Microwave resonator to monitor subsurface growth of E. coliFigure 4.9: Three-dimensional growth of E. coli on solid media with andwithout the presence of cover slip.found in this study where dielectric properties of the medium changed asbacterial subsurface growth proceeded. A significant variation in resonantamplitude was noticed compared to resonant frequency which is reasonableas bacterial metabolism alters the conductivity of the growth medium andresonant frequency is dependent on permittivity and conductivity [143].In another study, a planar microstrip resonator was used to measureresonant amplitude variation representing growth phases of E. coli confinedbetween two layers of agar. The measured results are presented in Figure4.10.From the measured ∆ Resonant amplitude (dB), a clear distinction be-tween the measured E. coli growths can be drawn between 0 µL (control),3 µL and 9 µL volume of the bacteria. A maximum amplitude of 0.037dB was measured for control i.e. with no bacteria. Considering this as athreshold value to distinguish between growth and no growth, the rate ofchange of the measured amplitude correlating to bacterial growth was foundby fitting the measured results with Type I Gompertz growth model. Thiswas obtained by plotting change in resonant amplitude (Y axis) and bac-terial growth time (X axis). Type I Gompertz growth model is a sigmoidmodel often used to describe growth of animals, plants and various bacteriaand cancer cells [144].Although Gompertz model requires an upper asymptote or adult valueto determine the growth rate of bacteria, due to the constant availabilityof nutrients in the vicinity of E. coli, it would take weeks or months ofcontinuous monitoring of bacterial growth until stationary phase is achievedand adult value is extracted. Stationary phase is defined as the period during624.2. Microwave resonator to monitor subsurface growth of E. coliFigure 4.10: Measured amplitude variation (∆ resonant amplitude (dB))with the fitted Gompertz model curve (a), measured frequency variation (∆resonant frequency (MHz)) (b), and the recorded temperature ( Tempera-ture ◦C) (c) for 3 µL and 9 µL of E. coli on soft LB agar.the life cycle of a microorganisms when the growth rate of bacteria equalsthe death rate of bacteria. Therefore,in this work, the Gompertz model wasutilized to fit the initial linear growth phases of the selected volumes of E.coli and determine the rate of change of resonant amplitude variation whichis related to the growth rate of bacteria.The equation governing the Type I Gompertz model is given by equation4.1.y(t) = A ∗ exp(−exp(−kG ∗ (t− Ti))) (4.1)Where y(t) is the expected variation in the resonant amplitude as a func-tion of time (t) due to bacteria growth, A represents the upper asymptote(adult value) (dB), kG is the growth rate coefficient (dB/hr.) (which af-634.2. Microwave resonator to monitor subsurface growth of E. coli(a) Control- no bacteria(b) E. coli- 3 µ L644.2. Microwave resonator to monitor subsurface growth of E. coli(c) E. coli- 9 µ LFigure 4.11: Microscopic images depicting the growth of 3 µL and 9 µLcaptured at constant time intervals.fects the slope), and Ti represents time at inflection (hr.). The Ti parametershifts the growth curve horizontally without changing it shape and is there-fore what is often termed a location parameter (whereas A and kG are shapeparameters). Table 4.2 presents the calculated Gompertz model parametersfor 3 µL, and 9 µL of E. coli.The fitted Gompertz growth curve demonstrated a good agreement withthe measured microwave response. The performance and accuracy of thesensor was further validated by microscope images captured at constanttime intervals as shown in Figure 4.11. The microscope images are in goodagreement with the measured results.Table 4.2: Measured growth phase and rate of change of resonant amplitudefor E. coli at various concentrations of glucose in LB agarE. coli volume (µL) A KG Ti R23 0.24182 0.08173 19.88572 0.997539 0.12456 0.139543 25.59338 0.96736654.3. Microwave resonator to monitor the impact of erythromycin on E. coliThis work exhibits a unique approach of a real-time, contactless and,label-free microwave resonator to detect subsurface growth of E. coli con-fined between two layers of solid LB-agar. The fitting of bacterial growthmeasurement to the Gompertz growth model with a coefficient of determi-nation above 96 % shows the potential of the currently used sensing tool forbacterial monitoring. A positive correlation between the measured resultsand microscopic images further strengthened the application of microwavesensors to measure subsurface bacteria growth.4.3 Microwave resonator to monitor the impactof erythromycin on E. coli growth.4.3.1 BackgroundAntibiotics have played a pivotal role in treating and killing pathogenicstrains of bacteria thereby improving the health of patients suffering frominfections caused by bacteria [145–147]. However, due to industrializationand overuse of antibiotics, microorganisms, as a mechanism of defense, haveevolved strategies capable of resisting the antibiotics [148]. These microor-ganisms are known as multidrug-resistant (MDR) bacteria or “super-bugs”and pose a serious threat to humans, animals, and the health of the environ-ment. It is predicted that causalities related to antibiotic resistance couldbe 10 million lives per year by 2050 [145]. Thus, it is essential to manufac-ture new antibiotics and determine the appropriate dose required to treatlife-threatening infections [146–149].To determine the antibiotic appropriate to treat a certain kind of in-fection, an antibiotic susceptibility test (AST) is performed. AST is a lab-oratory procedure performed by medical technologists (clinical laboratoryscientists) to determine the minimum inhibitory concentration (MIC) andidentify which antimicrobial regimen is specifically effective for individualpatients against deadly infections [9, 150]. Various AST techniques suchas Disk diffusion, dilution, Epsilometer test, PCR, DNA microarrays/chipshave been studied and developed to determine the minimum inhibitory con-centration [9, 151]. These techniques are simple and effective in determiningthe minimum inhibitory concentration for a certain type of infection but aretime-consuming and require expensive equipment. With the recent devel-opments in the field of Lab-on-a-chip devices, a network of micro-channels,sensors, and electrical circuits can be incorporated and fabricated on smallchips. This offers numerous advantages such as non-contact interface, porta-664.3. Microwave resonator to monitor the impact of erythromycin on E. colibility, quick reaction, and analysis time. Biosensors based on lab on a chiptechnology have revolutionized the field of a clinical study and pharmaceuti-cal sciences [17, 136]. They are also a promising tool for rapid and accurateASTs. Recently, microwave sensors are being widely used in pharmaceu-tical, biological and industrial applications owing to low cost, non-contact,label-free detection, high sensitivity, and ease of integration with other Lab-on-Chip (LoC) devices [48, 50, 53]. Thus, in this study, a microwave sensorcapable of monitoring bacteria growth in the presence of 7.5 µg, 30 µg, and45 µg of Erythromycin is presented. Based on the variation of growth rate,the concentrations of erythromycin achieving total inhibition of E. coli willbe determined.4.3.2 Material and methodSample preparationTo measure the impact on the growth-inhibiting substance, such as an-tibiotics, on the growth profile of bacteria, a Mueller Hinton (MH) agarmedia was prepared. Due to low levels of thymidine and thymine (excessamounts can have adverse effects on the performance of various antibiotics),MH agar is widely used for antibiotic susceptibility tests by the disk diffusionmethod.To prepare MH agar media 1.91 gm of MH agar was mixed with 50 mlof RO water in a flask. The mixture was sterilized in an autoclave at 121◦C for 15 minutes. The mixture was cooled to 50 ◦C prior to plating. Threeml of the MH agar was poured into a Petri dish to achieve a uniform agarthickness of 1 mm across the petri dish. The process was performed in afume hood or under a Bunsen burner flame to avoid contamination of agarsamples by fungi and other microorganisms. Three ml of E. coli was thenspotted at the designated position on the agar plate.Prior to preparing antibiotic paper disks, sample solutions containingvarious concentrations of erythromycin was prepared. Erythromycin, inpowdered form, is freely soluble in alcohol, acetone, and other organic sol-vents. However, in water, the solubility of erythromycin is limited to 2mg/ml. Thus, a concentration of 7.5 µg and 30 µg per 30 µL was achievedby series dilution of the parent erythromycin solution containing 2 mg/ml.To prepare antibiotic paper disks, sterile blank paper disks and E- 15µg paper disks procured by Fisher Scientific were used. Each paper diskwas hydrated with 30 µL of the prepared stock solution to maintain con-sistency among the antibiotic paper disks. A control disk containing 0 µg674.3. Microwave resonator to monitor the impact of erythromycin on E. coliof erythromycin was prepared by hydrating a blank paper disk with 30 µLof RO water. E- 45 µg paper disk was prepared by pouring 30 µL of E-30µg solution to an E-15 µg paper disk. The hydrated antibiotic paper diskswith 0 µg, 7.5 µg, 30 µg, and 45 µg erythromycin was tapped on the solidagar 5 mm from the inoculated E. coli. The plate was sealed using parafilmat room temperature to avoid precipitation and fungal contamination.Experimental setup and procedureThe fabricated frequency variation sensor was mechanically stabilizedand enclosed in a thermally insulated Styrofoam enclosure to avoid the im-pact of ambient factors such as external vibrations, temperature, and humid-ity on the response of the resonator. The Styrofoam enclosure was equippedwith an LED light source, a temperature probe to monitor the temperaturein the vicinity of the sensor, and a microscope to capture images of testsamples at constant time intervals. The sensor was connected to the VNAusing SMA cables. The experimental setup and the VNA calibration detailsare discussed in section 4.2.2 ( experimental setup and procedure).The measured resonant amplitude for various concentrations of antibi-otics was normalized by subtracting it from the initially measured amplitude.The measured data pertained to the initial 90 minutes of the experimentwere excluded accounting for mechanical stabilization of the apparatus.4.3.3 Measurement result and discussionTo study the impact of a dry paper disk (with no antibiotics) on an agarplate with no bacteria, a series of measurements were performed using a mi-crowave resonator. Analysis of the measured results revealed that dry paperdisks tend to dehydrate the growth media in their vicinity. The dehydrationof the agar media alters the electrical characteristics of the growth mediaand result in false measurements. Therefore, to avoid dehydration of thegrowth media in the vicinity of the sensor’s sensitive region, the blank diskswere hydrated with an equal volume of RO water similar to antibiotic vol-ume and used in all the further experiments. The steps followed to hydratethe paper disks are presented in section 4.3.2 (sample preparation).Another study on antibiotic disk diffusion assay showed a differential re-sponse against tested concentrations with an increase in an inhibition zonecorresponding to antibiotic concentration. The zone of inhibition was mea-sured as 1.7 cm and 2.8 cm for 30 and 45 µg respectively (Figure 4.12).684.3. Microwave resonator to monitor the impact of erythromycin on E. coliFigure 4.12: Zone of inhibition formed by 0 µg, 7.5 µg, 30 µg and 45 µg oferythromycin.This was compared with a control (plain disk) and 7.5 µg where noinhibition of E. coli was observed. The preliminary zone of inhibition studywas performed at room temperature which was measured to be 22 ◦C. Thezone of inhibition is the diameter of the region where no bacteria growthwas measured and shown in Figure 4.12. Therefore, based on the resultspresented in Figure 4.12, it can be concluded that 45 µg and 30 µg oferythromycin was able to effectively inhibit E. coli growth on solid MH agarin contrast to 7.5 µg and 0 µg.Table 4.3: Measured zone of inhibition formed by 0 µg, 7.5 µg, 30 µg and45 µg of erythromycin.Erythromycin 0.0 7.5 30 45concentration (µg)Diameter of 0 0 1.7 2.8zone of inhibition (cm)694.3. Microwave resonator to monitor the impact of erythromycin on E. coliAs bacteria grows, the interaction between the nutrients present in themedium and the bacteria such as transfer of protein to amino acids, lipidsto acetate, or carbohydrates to lactate results in the creation of new chargedend products that are expelled into the surrounding media. However, thepresence of an antibiotic can severely affect the growth of bacteria. At lowlethal concentrations of antibiotics, bacteria growth is observed as shownin Figure 4.13. Low non-lethal concentration of erythromycin triggers adifferent cellular response against the antibiotic. The low concentration ofantibiotics can stress the bacteria, but the bacteria continues to multiplyand alter the conductivity of the surrounding growth media [152, 153].Figure 4.13: Measured resonant amplitude variation (∆ Resonant amplitude(dB)) (a), resonant frequency variation(∆ Resonant frequency (MHz)) (b),and recorded temperature (Temperature ◦C) (c) for bacterial growth in thepresence of 0 µg, 7.5 µg, 30 µg, and 45 µg of erythromycin.However, at high concentrations, bacterial growth is inhibited as observedin the measured results ( Figure 4.13). At lethal or high concentrationsof erythromycin, inhibition of protein slows down or suppresses bacterial704.3. Microwave resonator to monitor the impact of erythromycin on E. coli(a) E-0 µg(b) E-7.5 µg(c) E-30 µg(d) E-45 µgFigure 4.14: Microscope images captured at constant time intervals depict-ing bacteria growth in the presence of 0 µg (a), 7.5 µg (b), 30 µg (c), and45 µg (d) of erythromycin.growth [152, 153] This change in the growth rate of bacteria is reflected by achange in the conductivity of the medium. The microwave sensor measures714.4. Chapter conclusionthe change in the conductivity of the medium surrounding the bacteria whichis interpreted by analyzing the change in the resonant amplitude which isshown in Figure 4.13.It is known from the previous literature that the zone of inhibition isproportional to the strength of the antibiotics [154]. Thus, an increase inthe zone of inhibition prevents bacterial growth in the sensitive region ofthe resonator. As evident from the bacterial growth images of E- 30 µg, asmall portion of bacteria grow within the sensitive region of the resonator.This small growth was monitored by the microwave sensor and was visibledistinctively in the response of the sensor (Figure 4.13).However, the zone of inhibition for E- 45 µg covered the entire sensitiveregion. Thus, the growth of E. coli was inhibited by the antibiotic concen-tration and the resonant amplitude variations recorded was minimal (0.01dB). Based on the microwave resonator response, E- 30 µg and E- 45 µg wereable to resist the growth of E. coli with OD600 = 1.5 which matches withthe preliminary test conducted. Therefore, at optimum growth conditions,the microwave resonator successfully distinguished the impact of differentconcentrations of erythromycin on the growth of E. coli within 18 hours asopposed to the conventional disk diffusion technique that takes around 24to 36 hours to produce visual results. Therefore, the microwave resonator’sability to rapidly detect microbial growth as compared to conventional ASTtechniques was successfully demonstrated through this study. Moreover,a positive correlation between the measured results with the preliminaryzone-of-inhibition study and the microscope images captured at constanttime intervals further strengthen the potency of the sensor to monitor theinfluence of bacterial growth due to antibiotics.4.4 Chapter conclusionIn this chapter, we have demonstrated the application of microwave res-onator for real-time and contactless monitoring of bacterial growth. Fur-thermore, the microwave sensor’s ability to detect microbial growth beforeany visual indications was demonstrated. The designed sensors were ableto successfully sense the growth of bacteria in both temperature-controlledand temperature-uncontrolled environments.From these studies and the simulation results presented in chapter 2, it isevident that resonant amplitude was sensitive to changes in the conductivityor loss tangent of the growth medium. As bacterial metabolism alters theconductivity of the growth medium, the resonant amplitude was selected724.4. Chapter conclusionover resonant frequency to monitor bacterial growth.The first study established the efficacy of the microwave resonator todetect and differentiate the impact of glucose concentration on the growthprofile of E. coli. The rate of change of amplitude that can be associatedwith the growth rate of bacteria was calculated for E. coli at various glucoseconcentrations. The presence of carbon sources (0- 2 % w/v) in LB agarenhances the growth of E. coli. Therefore, based on the measured results,the highest rate of change in resonant amplitude was observed at 2% glu-cose (0.23646 dB/hr) compared to 0% (0.10913 dB/hr). However, at higherconcentrations (10 % w/v), carbon sources inhibited bacterial growth as ob-served from the measured results. The measured results were supported byprevious microbiology studies. In addition to this, a positive correlation ofthe measured results with the microscope images captured at constant in-tervals further established the sensor’s ability to monitor bacterial growth.The results of this study are explained thoroughly in a paper entitled “Dif-ferential Microwave Resonator Sensor Reveals Glucose Dependent GrowthProfile of E. coli on Solid Agar” [53]In the second study, we demonstrated the ability of microwave sensors todetect and monitor subsurface E. coli growth. The changes in the dielectricproperties of growth media during 2D and 3D growth in the solid growthmedia were measured using microwave resonators. The successful fitting ofbacterial growth measurement to the Gompertz growth model with a co-efficient of determination above 96% shows the potential of the currentlyused sensing tool for bacterial monitoring. A positive correlation betweenthe measured results and microscopic images further strengthened the ap-plication of microwave sensors to measure subsurface bacterial growth. Thefindings of this study are submitted as a research paper and is under review.The third study successfully demonstrated the potential of the designedmicrowave resonator to differentiate the impact of antibiotics, erythromycin(0, 7.5, 30, 45 µg) on the growth profile of E. coli. From the measuredresponse of the sensor, it was observed that the growth of microorganismswas inversely proportional to the concentration of antibiotics. Among thetested concentrations of erythromycin (0, 7.5, 30 and, 45 µg), E-30 µg andE-45 µg were able to successfully inhibit bacterial growth. The microwaveresonator successfully distinguished the impact of different concentrationsof erythromycin on the growth of E. coli within 18 hours as opposed tothe conventional disk diffusion technique that takes around 24 to 36 hoursto produce visual results. The positive correlation between the measuredresults with the preliminary zone-of-inhibition study and the microscopeimages captured at constant time intervals further strengthen the potency734.4. Chapter conclusionof the sensor to monitor bacterial growth. The findings of this study aresubmitted as a research paper and is under review.74Chapter 5ConclusionIn this chapter, the main findings and contributions of the thesis aresummarized and the areas of possible improvements and research are pre-sented.5.1 Thesis conclusionsThe thesis begins with an introduction to microwave technology and itsapplications in industrial, food, biomedical, and pharmaceutical industries.Among the available microwave sensing technologies, planar microstrip res-onators were chosen for the current research due to its advantages suchas portability, ease of fabrication, label-free, and contactless sensing capa-bilities. The unique ability of microwave sensors to detect variations inthe conductivity in the surrounding medium related to microbial growthmakes it a promising candidate for microbial growth monitoring and relatedapplications. In this work, microstrip resonators were employed to moni-tor bacteria growth due to numerous advantages which include contactless,label-free monitoring, portable, cost effective, and ease of development andfabrication. The proposed planar microwave resonators were designed intwo arrangements: frequency variation resonator and differential-mode res-onator. They were designed to operate as a one-port device with their oper-ating frequency confined to the L-band. The sensor’s response to dielectricmaterial was studied through HFSS simulations. The simulation results arepresented in chapter 2. The sensors were fabricated using in-house fabrica-tion techniques, and the details of sensor operation are explained in chapter3.The fabricated sensors were later employed to detect and monitor mi-crobial growth on solid growth matrices. The sensors were used to monitorthe impact on the growth of microorganisms subjected to various factorssuch as nutrient availability, bacterial position, and presence of inhibitingcompounds such as antibiotics were conducted as three independent stud-ies. The conclusions and contributions from these studies are summarizedas follows:755.2. Contributions1. The first study demonstrated the ability of the designed sensor to suc-cessfully monitor and distinguish the impact of nutrient availability onmicrobial growth. The impact of temperature on the response of thesensor was observed and corrected by redesigning the microstrip res-onator. The measured results obtained after eliminating the influenceof ambient temperature and mechanical stabilization was presented inChapter 4. The measured data was supported by microscopic imagesrevealing changes in microbial growth at different nutrient concentra-tions. The results of this study are explained thoroughly in a paperentitled “Differential Microwave Resonator Sensor Reveals Glucose De-pendent Growth Profile of E. coli on Solid Agar” [53].2. The second study investigated the potential of the sensors to monitorsubsurface bacterial growth between two layers of solid growth matri-ces. Through the measured results and microscope images of variousexperiments, the impact of the relaxation of the solid-solid interface ofthe medium and stabilization of electronic components and VNA aftercalibration was identified and eliminated from the measured results.Additional experimental results further demonstrated a positive corre-lation between microbial growth and microwave sensor’s response andfurther demonstrated the extended capacity of the sensor to monitorsubsurface bacterial growth unlike previous studies confined to surfacegrowth. The findings of this study are submitted and under review.3. The third study investigated the effect of various concentrations ofantibiotics on microbial growth. The effect of the dry antibiotic pa-per disks on the solid agar matrices was identified through numerousfutile attempts. By using an equal volume of antibiotic solution to hy-drate the paper disks, the impact of dry paper disks was eliminated.Additional experiments confirmed a positive relationship between thezone of inhibition, measured result, and the microscopic images. Thisfurther strengthened the use of the sensor as a tool to determine theminimum inhibitory concentration of antibiotics and can be applied forantibiotic sensitivity assays. The results of this study are submittedand under review.5.2 Contributions− Resonators with various configurations were designed and simulated toachieve a highly sensitive device exclusively for monitoring microbial765.3. Future worksgrowth. Numerous futile attempts, design modifications, and opti-mization led to the final working model of a microwave resonator.− Contributed to the current state of the art sensor technology aimed forcontactless and label-free bacterial growth monitoring on solid growthmedium.− Designed a one-port sensing system by utilizing a wide band powersplitter.− Analyzed the impact of nutrient availability, subsurface growth andpresence of antibiotics on growth of bacteria using contactless mi-crowave resonator sensor.− Observed the impact of ambient factors such as temperature, stabi-lization of sensing apparatus and implemented corrective measures tominimize or diminish the impact of the above mentioned factors onthe response of the sensor.− Optimized solid agar plating techniques to achieve successful bacterialgrowth monitoring using microwave resonator sensor technology.− Designed series of experiments to understand various aspects of bac-terial growth on solid agar.5.3 Future worksThis research successfully demonstrated the potential of a prototype mi-crowave sensor for contactless real-time monitoring of microbial growth onsolid growth matrices. However, based on the current research outcomes,we identified the following research ideas could scale up the designed sensorto industrial scale:− Sensor design: Modification of sensor design by incorporating activeresonators can improve the sensitivity of the resonator. Using an ar-ray of active resonators multiple bacteria can be tested in a singleassay. Similarly, array of resonators can be used to monitor the ef-fect of multiple antibiotics on a microbial specimen and determine theappropriate antibiotics for treatment. There is a prospective to im-plement wireless resonators which can eliminate microwave cables andsoldered SMA connectors for ease in installation.775.3. Future works− Sample preparation: Using microfluidic devices to inject varying con-centrations of antibiotics can expand the application range of the cur-rent sensor. It also facilitates the reusability of the device by followingsimple cleaning procedures.− Sensor readout circuit: Including a customizable readout circuit/deviceto measure the reflected power from the sensor can eliminate the sys-tem’s dependence on the vector network analyzers. By developingsuch a self-reliant system, the cost of the sensor can also be reducedsignificantly.− Sensing apparatus: Appending an incubator for bacterial growth canenhance the growth rate and will facilitate early detection of bacterialgrowth which can be monitored with precision using a wireless sensor.78Bibliography[1] N. Zhu, D. Zhang, W. Wang, X. Li, B. Yang, J. Song, X. Zhao,B. Huang, W. Shi, R. Lu, P. Niu, F. Zhan, X. Ma, D. Wang, W. Xu,G. Wu, G. F. Gao, and W. Tan, “A Novel Coronavirus from Patientswith Pneumonia in China, 2019,” New England Journal of Medicine,vol. 382, pp. 727–733, feb 2020. → pages 1[2] E. K. Stevenson, A. R. Rubenstein, G. T. Radin, R. S. Wiener, andA. J. Walkey, “Two decades of mortality trends among patients withsevere sepsis: a comparative meta-analysis*.,” Critical care medicine,vol. 42, pp. 625–31, mar 2014. → pages[3] H.-U. Meier-Kriesche, A. O. Ojo, J. A. Hanson, and B. Kaplan, “Ex-ponentially increased risk of infectious death in older renal transplantrecipients,” Kidney International, vol. 59, pp. 1539–1543, apr 2001. →pages[4] L. van der Hoek, K. Pyrc, M. F. Jebbink, W. Vermeulen-Oost, R. J. M.Berkhout, K. C. Wolthers, P. M. E. Wertheim-van Dillen, J. Kaan-dorp, J. Spaargaren, and B. Berkhout, “Identification of a new humancoronavirus,” Nature Medicine, vol. 10, pp. 368–373, apr 2004. →pages[5] J. M. O’Brien, N. A. Ali, S. K. Aberegg, and E. Abraham, “Sepsis,”The American Journal of Medicine, vol. 120, pp. 1012–1022, dec 2007.→ pages[6] B. K. Romanov, “Coronavirus disease COVID-2019,” Safety and Riskof Pharmacotherapy, vol. 8, pp. 3–8, mar 2020. → pages 1[7] S. Wong and B. Tan, “Megatrends in Infectious Diseases: The Next10 to 15 Years,” Ann Acad Med Singapore, vol. 48, no. 6, pp. 188–194,2019. → pages 1[8] A. L. Schaefer, N. Cook, S. V. Tessaro, D. Deregt, G. Desroches, P. L.Dubeski, A. K. Tong, and D. L. Godson, “Early detection and predic-79Bibliographytion of infection using infrared thermography,” Canadian Journal ofAnimal Science, vol. 84, pp. 73–80, mar 2004. → pages 1[9] Z. A. Khan, M. F. Siddiqui, and S. Park, “Current and EmergingMethods of Antibiotic Susceptibility Testing.,” Diagnostics (Basel,Switzerland), vol. 9, may 2019. → pages 1, 2, 66[10] E. Engvall, “[28] Enzyme immunoassay ELISA and EMIT,” in Meth-ods in Enzymology, ch. 28, pp. 419–439, Elsevier Inc., 1980. → pages[11] S. Henry, E. Baudoin, J. C. Lo´pez-Gutie´rrez, F. Martin-Laurent,A. Brauman, and L. Philippot, “Quantification of denitrifying bac-teria in soils by nirK gene targeted real-time PCR,” Journal of Micro-biological Methods, vol. 59, pp. 327–335, dec 2004. → pages 1[12] S. A. Barghouthi, “A Universal Method for the Identification of Bacte-ria Based on General PCR Primers,” Indian Journal of Microbiology,vol. 51, pp. 430–444, oct 2011. → pages 1, 58[13] J.-C. Lagier, S. Edouard, I. Pagnier, O. Mediannikov, M. Dran-court, and D. Raoult, “Current and Past Strategies for Bacterial Cul-ture in Clinical Microbiology,” Clinical Microbiology Reviews, vol. 28,pp. 208–236, jan 2015. → pages[14] R. Franco-Duarte, L. Cˇerna´kova´, S. Kadam, K. S. Kaushik, B. Salehi,A. Bevilacqua, M. R. Corbo, H. Antolak, K. Dybka-Ste¸pien´,M. Leszczewicz, S. Relison Tintino, V. C. Alexandrino de Souza,J. Sharifi-Rad, H. D. Melo Coutinho, N. Martins, and C. F. Rodrigues,“Advances in Chemical and Biological Methods to Identify Microor-ganisms—From Past to Present,” Microorganisms, vol. 7, p. 130, may2019. → pages 1, 58[15] P. Cheng, Z.-G. Huang, Y. Zhuang, L.-C. Fang, H. Huang, J. Deng, L.-L. Jiang, K.-K. Yu, Y. Li, and J.-S. Zheng, “A novel regeneration-freeE. coli O157:H7 amperometric immunosensor based on functionalisedfour-layer magnetic nanoparticles,” Sensors and Actuators B: Chemi-cal, vol. 204, pp. 561–567, dec 2014. → pages 1, 58[16] Y. Wang, Z. Ye, C. Si, and Y. Ying, “Monitoring of Escherichia coliO157:H7 in food samples using lectin based surface plasmon resonancebiosensor,” Food Chemistry, vol. 136, pp. 1303–1308, feb 2013. →pages 3, 580Bibliography[17] A.-R. H. A.-A. Hassan, A. de la Escosura-Mun˜iz, and A. Merkoc¸i,“Highly sensitive and rapid determination of Escherichia coli O157:H7in minced beef and water using electrocatalytic gold nanoparticletags,” Biosensors and Bioelectronics, vol. 67, pp. 511–515, may 2015.→ pages 67[18] M. Pohanka, “The Piezoelectric Biosensors: Principles and Applica-tions, a Review,” International Journal of Electrochemical Science,pp. 496–506, jan 2017. → pages[19] L. Yao, P. Lamarche, N. Tawil, R. Khan, A. M. Aliakbar, M. H. Has-san, V. P. Chodavarapu, and R. Mandeville, “CMOS ConductometricSystem for Growth Monitoring and Sensing of Bacteria,” IEEE Trans-actions on Biomedical Circuits and Systems, vol. 5, pp. 223–230, jun2011. → pages[20] M. Salami, A. Mhs, M. Sawan, and A. Nsk, “Biosensors & Bioelec-tronics BioFET-based Integrated Platform for Accurate and RapidDetection of E . coli Bacteria : A Review,” Journal of Biosensors &Bioelectronics, vol. 10, no. 1, pp. 1–11, 2019. → pages 1, 2, 58[21] P. Damborsky´, J. Sˇvitel, and J. Katrl´ık, “Optical biosensors,” Essaysin Biochemistry, vol. 60, pp. 91–100, jun 2016. → pages 1[22] F. S. Ligler, “Perspective on Optical Biosensors and Integrated SensorSystems,” Analytical Chemistry, vol. 81, pp. 519–526, jan 2009. →pages[23] S. Zhang, “Materials and techniques for electrochemical biosensordesign and construction,” Biosensors and Bioelectronics, vol. 15,pp. 273–282, aug 2000. → pages[24] D. Ivnitski, “Electrochemical biosensor based on supported planarlipid bilayers for fast detection of pathogenic bacteria,” Electrochem-istry Communications, vol. 2, pp. 457–460, jul 2000. → pages 1[25] M. H. Zarifi and M. Daneshmand, “High-Resolution RFID LiquidSensing Using a Chipless Tag,” IEEE Microwave and Wireless Com-ponents Letters, vol. 27, pp. 311–313, mar 2017. → pages 2[26] M. Fayaz, M. H. Zarifi, M. Abdolrazzaghi, P. Shariaty, Z. Hashisho,and M. Daneshmand, “A Novel Technique for Determining the Ad-sorption Capacity and Breakthrough Time of Adsorbents Using a Non-81Bibliographycontact High-Resolution Microwave Resonator Sensor,” Environmen-tal Science & Technology, vol. 51, pp. 427–435, jan 2017. → pages[27] M. H. Zarifi, S. Deif, M. Abdolrazzaghi, B. Chen, D. Ramsawak,M. Amyotte, N. Vahabisani, Z. Hashisho, W. Chen, and M. Danesh-mand, “A Microwave Ring Resonator Sensor for Early Detection ofBreaches in Pipeline Coatings,” IEEE Transactions on Industrial Elec-tronics, vol. 65, pp. 1626–1635, feb 2018. → pages 2, 11[28] M. H. Zarifi, A. Gholidoust, M. Abdolrazzaghi, P. Shariaty,Z. Hashisho, and M. Daneshmand, “Sensitivity enhancement in planarmicrowave active-resonator using metal organic framework for CO2 de-tection,” Sensors and Actuators B: Chemical, vol. 255, pp. 1561–1568,feb 2018. → pages[29] M. A. Rafi, B. D. Wiltshire, and M. H. Zarifi, “Wideband TunableModified Split Ring Resonator Structure Using Liquid Metal and 3-DPrinting,” IEEE Microwave and Wireless Components Letters, pp. 1–4, 2020. → pages 2[30] S. Mohammadi, A. V. Nadaraja, K. Luckasavitch, M. C. Jain, D. JuneRoberts, and M. H. Zarifi, “A Label-Free, Non-Intrusive, and RapidMonitoring of Bacterial Growth on Solid Medium Using MicrowaveBiosensor,” IEEE Transactions on Biomedical Circuits and Systems,vol. 14, pp. 2–11, feb 2020. → pages 2, 5, 19, 31, 49[31] S. Mohammadi, A. V. Nadaraja, D. J. Roberts, and M. H. Zarifi,“Real-time and hazard-free water quality monitoring based on mi-crowave planar resonator sensor,” Sensors and Actuators A: Physical,vol. 303, p. 111663, mar 2020. → pages 16, 58[32] B. D. Wiltshire, T. Zarifi, and M. H. Zarifi, “Passive Split Ring Res-onator Tag Configuration for RFID-Based Wireless Permittivity Sens-ing,” IEEE Sensors Journal, vol. 20, pp. 1904–1911, feb 2020.→ pages[33] M. Amirian, G. Karimi, B. D. Wiltshire, and M. H. Zarifi, “DifferentialNarrow Bandpass Microstrip Filter Design for Material and Liquid Pu-rity Interrogation,” IEEE Sensors Journal, vol. 19, pp. 10545–10553,nov 2019. → pages 19[34] B. D. Wiltshire and M. H. Zarifi, “3-D Printing Microfluidic Chan-nels With Embedded Planar Microwave Resonators for RFID and Liq-82Bibliographyuid Detection,” IEEE Microwave and Wireless Components Letters,vol. 29, no. 1, pp. 65–67, 2019. → pages[35] B. D. Wiltshire, S. Mohammadi, and M. H. Zarifi, “Integrating 3DPrinted Microfluidic Channels With Planar Resonator Sensors for LowCost and Sensitive Liquid Detection,” in 2018 18th International Sym-posium on Antenna Technology and Applied Electromagnetics (AN-TEM), pp. 1–2, 2018. → pages[36] M. Abdolrazzaghi, M. H. Zarifi, C. F. A. Floquet, and M. Danesh-mand, “Contactless Asphaltene Detection Using an Active Planar Mi-crowave Resonator Sensor,” Energy & Fuels, vol. 31, pp. 8784–8791,aug 2017. → pages[37] M. Abdolrazzaghi, M. H. Zarifi, and M. Daneshmand, “Sensitivityenhancement of split ring resonator based liquid sensors,” in 2016IEEE SENSORS, pp. 1–3, IEEE, oct 2016. → pages 11[38] M. H. Zarifi, P. Shariaty, M. Abdolrazzaghi, Z. Hashisho, andM. Daneshmand, “Particle size characterization using a high resolutionplanar resonator sensor in a lossy medium,” Sensors and Actuators B:Chemical, vol. 234, pp. 332–337, oct 2016. → pages[39] M. H. Zarifi, M. Rahimi, M. Daneshmand, and T. Thundat, “Mi-crowave ring resonator-based non-contact interface sensor for oil sandsapplications,” Sensors and Actuators, B: Chemical, vol. 224, pp. 632–639, 2015. → pages 2, 11[40] N. R. Tanguy, B. Wiltshire, M. Arjmand, M. H. Zarifi, and N. Yan,“Highly Sensitive and Contactless Ammonia Detection Based onNanocomposites of Phosphate-Functionalized Reduced Graphene Ox-ide/Polyaniline Immobilized on Microstrip Resonators,” ACS AppliedMaterials & Interfaces, vol. 12, pp. 9746–9754, feb 2020. → pages 2[41] M. Fayaz, M. Jahandar Lashaki, M. Abdolrazzaghi, M. H. Zarifi,Z. Hashisho, M. Daneshmand, J. E. Anderson, and M. Nichols, “Mon-itoring the residual capacity of activated carbon in an emission abate-ment system using a non-contact, high resolution microwave resonatorsensor,” Sensors and Actuators B: Chemical, vol. 282, pp. 218–224,mar 2019. → pages[42] Z. Abbasi, M. H. Zarifi, P. Shariati, Z. Hashisho, and M. Daneshmand,“Flexible coupled microwave ring resonators for contactless microbead83Bibliographyassisted volatile organic compound detection,” in 2017 IEEE MTT-S International Microwave Symposium (IMS), vol. 2, pp. 1228–1231,IEEE, jun 2017. → pages[43] M. H. Zarifi, P. Shariaty, Z. Hashisho, and M. Daneshmand, “A non-contact microwave sensor for monitoring the interaction of zeolite 13Xwith CO2 and CH4 in gaseous streams,” Sensors and Actuators B:Chemical, vol. 238, pp. 1240–1247, jan 2017. → pages 2[44] B. Wiltshire, K. Mirshahidi, K. Golovin, and M. H. Zarifi, “Robustand sensitive frost and ice detection via planar microwave resonatorsensor,” Sensors and Actuators B: Chemical, vol. 301, p. 126881, dec2019. → pages 2, 16[45] M. Benlamri, S. Deif, N. Mahdi, M. Baghelani, M. H. Zarifi, D. W.Barlage, K. Shankar, and M. Daneshmand, “Planar microwave res-onator with electrodeposited ZnO thin film for ultraviolet detection,”Semiconductor Science and Technology, vol. 35, p. 025003, feb 2020.→ pages 2[46] M. H. Zarifi, H. Sadabadi, S. H. Hejazi, M. Daneshmand,and A. Sanati-Nezhad, “Noncontact and Nonintrusive Microwave-Microfluidic Flow Sensor for Energy and Biomedical Engineering,”Scientific Reports, vol. 8, p. 139, dec 2018. → pages 2[47] M. H. Zarifi, B. Wiltshire, N. Mahdi, P. Kar, K. Shankar, andM. Daneshmand, “Ultraviolet sensing using a TiO 2 nanotube inte-grated high resolution planar microwave resonator device,” Nanoscale,vol. 10, no. 10, pp. 4882–4889, 2018. → pages 2[48] N. M. Ceron Hurtado, M. H. Zarifi, M. Daneshmand, and J. AguiloLlobet, “Flexible Microdisplacement Sensor for Wearable/ Im-plantable Biomedical Applications,” IEEE Sensors Journal, vol. 17,pp. 3873–3883, jun 2017. → pages 2, 3, 67[49] M. H. Zarifi and M. Daneshmand, “Monitoring Solid Particle Deposi-tion in Lossy Medium Using Planar Resonator Sensor,” IEEE SensorsJournal, vol. 17, pp. 7981–7989, dec 2017. → pages 2[50] R. Narang, S. Mohammadi, M. M. Ashani, H. Sadabadi, H. Hejazi,M. H. Zarifi, and A. Sanati-Nezhad, “Sensitive, Real-time and Non-Intrusive Detection of Concentration and Growth of Pathogenic Bac-84Bibliographyteria using Microfluidic-Microwave Ring Resonator Biosensor,” Scien-tific Reports, vol. 8, p. 15807, dec 2018. → pages 2, 49, 58, 67[51] S. Mohammadi, R. Narang, M. Mohammadi Ashani, H. Sadabadi,A. Sanati-Nezhad, and M. H. Zarifi, “Real-time monitoring of Es-cherichia coli concentration with planar microwave resonator sensor,”Microwave and Optical Technology Letters, vol. 61, pp. 2534–2539, nov2019. → pages[52] C.-F. Liu, M.-H. Wang, and L.-S. Jang, “Microfluidics-based hairpinresonator biosensor for biological cell detection,” Sensors and Actua-tors B: Chemical, vol. 263, pp. 129–136, jun 2018. → pages[53] M. C. Jain, A. V. Nadaraja, B. M. Vizcaino, D. J. Roberts, and M. H.Zarifi, “Differential Microwave Resonator Sensor Reveals Glucose-Dependent Growth Profile of E. coli on Solid Agar,” IEEE Microwaveand Wireless Components Letters, pp. 1–4, 2020. → pages 2, 67, 73,76[54] F. BAKER and R. SILVERTON, “The Use of Culture Media,” inIntroduction to Medical Laboratory Technology, pp. 476–480, Elsevier,1976. → pages 2[55] I. De Leersnyder, L. De Gelder, I. Van Driessche, and P. Vermeir,“Influence of growth media components on the antibacterial effect ofsilver ions on Bacillus subtilis in a liquid growth medium,” ScientificReports, vol. 8, p. 9325, dec 2018. → pages 2[56] K. Grenier, D. Dubuc, P.-E. Poleni, M. Kumemura, H. Toshiyoshi,T. Fujii, and H. Fujita, “Integrated Broadband Microwave and Mi-crofluidic Sensor Dedicated to Bioengineering,” IEEE Transactions onMicrowave Theory and Techniques, vol. 57, pp. 3246–3253, dec 2009.→ pages 2, 11[57] D. M. Pozar, Microwave engineering; 3rd ed. Hoboken, NJ: Wiley,third ed., 2005. → pages 3, 5, 14, 15, 28[58] S. Ullah, C. Ruan, M. S. Sadiq, T. U. Haq, and W. He, “High Efficientand Ultra Wide Band Monopole Antenna for Microwave Imaging andCommunication Applications,” Sensors, vol. 20, p. 115, dec 2019. →pages 3, 585Bibliography[59] Y. Sun, F. Dang, C. Yuan, J. He, Q. Zhang, and X. Zhao, “A Beam-steerable Lens Antenna for Ku-band High-Power Microwave Appli-cations,” IEEE Transactions on Antennas and Propagation, pp. 1–1,2020. → pages 3[60] L. Gil-Flores, M. Salvador, F. Penaranda-Foix, A. Dalmau,A. Ferna´ndez, and A. Borrell, “Tribological and wear behaviour ofalumina toughened zirconia nanocomposites obtained by pressurelessrapid microwave sintering,” Journal of the Mechanical Behavior ofBiomedical Materials, vol. 101, p. 103415, jan 2020. → pages 3[61] D. Belamri, A. Harabi, N. Karbouaa, and N. Benyahia, “The effect ofKF on the structural evolution of natural hydroxyapatite during con-ventional and microwave sintering,” Ceramics International, vol. 46,pp. 1189–1194, jan 2020. → pages[62] E. A. Anli, “Possibilities for using microwave-vacuum drying in Lorcheese production,” International Dairy Journal, vol. 102, p. 104618,mar 2020. → pages[63] M. Nadeem, E. Ul Haq, F. Ahmed, M. Asif Rafiq, G. Hameed Awan,and M. Zain-ul Abdein, “Effect of microwave curing on the construc-tion properties of natural soil based geopolymer foam,” Constructionand Building Materials, vol. 230, p. 117074, jan 2020. → pages[64] D. Agrawal, “Microwave sintering of metal powders,” in Advances inPowder Metallurgy, pp. 361–379, Elsevier, 2013. → pages 3, 5[65] X. Lin, Y. Chen, Z. Gong, B.-C. Seet, L. Huang, and Y. Lu, “Ultra-Wideband Textile Antenna for Wearable Microwave Medical Imag-ing Applications,” IEEE Transactions on Antennas and Propagation,pp. 1–1, 2020. → pages 3[66] F. Fiore, V. Stoia, and F. Somma, “Surgical recurrence of solitaryfibrous tumor of the pleura treated with microwave (MW) thermoab-lation: A case report,” Thoracic Cancer, vol. 11, pp. 443–446, feb2020. → pages[67] K. Imajo, Y. Ogawa, M. Yoneda, S. Saito, and A. Nakajima, “A reviewof conventional and newer generation microwave ablation systems forhepatocellular carcinoma,” Journal of Medical Ultrasonics, vol. 47,pp. 265–277, apr 2020. → pages 386Bibliography[68] L. Wang, “Microwave Sensors for Breast Cancer Detection,” Sensors,vol. 18, p. 655, feb 2018. → pages 3, 58[69] S. J. Ling, J. Sanny, and W. Moebs, University Physics Volume 2.Houston, Texas: OpenStax University Physics, 2016. → pages 3, 4[70] I. Zivkovic and A. Murk, “Free-Space Transmission Method for theCharacterization of Dielectric and Magnetic Materials at MicrowaveFrequencies,” in Microwave Materials Characterization, InTech, nov2012. → pages 4, 5[71] S. Y. Huang, “Loss tangent,” feb 2012. → pages 4[72] D. Markl, M. Warman, M. Dumarey, E.-L. Bergman, S. Folestad,Z. Shi, L. F. Manley, D. J. Goodwin, and J. A. Zeitler, “Review ofreal-time release testing of pharmaceutical tablets: State-of-the art,challenges and future perspective,” International Journal of Pharma-ceutics, vol. 582, p. 119353, may 2020. → pages 5[73] K. Chee Yaw, “Measurement of Dielectric Material Properties,” 2012.→ pages 5, 6, 7, 8, 9[74] E. H˚akansson, A. Amiet, and A. Kaynak, “Dielectric characterizationof conducting textiles using free space transmission measurements:Accuracy and methods for improvement,” Synthetic Metals, vol. 157,pp. 1054–1063, dec 2007. → pages 5[75] N. Vohra, L. R. Rodriguez-Aguilar, J. S. Batista, and M. El-Shenawee,“Free-Space Characterization of Radar Absorbing Non-Magnetic Ma-terials in the W-Band,” in 2020 94th ARFTG Microwave MeasurementSymposium (ARFTG), pp. 1–4, IEEE, jan 2020. → pages 6[76] D. Ghodgaonkar, V. Varadan, and V. Varadan, “A free-space methodfor measurement of dielectric constants and loss tangents at microwavefrequencies,” IEEE Transactions on Instrumentation and Measure-ment, vol. 38, pp. 789–793, jun 1989. → pages[77] D. Ghodgaonkar, V. Varadan, and V. Varadan, “Free-space measure-ment of complex permittivity and complex permeability of magneticmaterials at microwave frequencies,” IEEE Transactions on Instru-mentation and Measurement, vol. 39, pp. 387–394, apr 1990. → pages87Bibliography[78] S. Trabelsi and S. O. Nelson, “Free-space measurement of dielectricproperties of cereal grain and oilseed at microwave frequencies,” Mea-surement Science and Technology, vol. 14, pp. 589–600, may 2003. →pages[79] F. Wee, P. Soh, A. Suhaizal, H. Nornikman, and A. Ezanuddin,“Free space measurement technique on dielectric properties of agricul-tural residues at microwave frequencies,” in 2009 SBMO/IEEE MTT-S International Microwave and Optoelectronics Conference (IMOC),pp. 183–187, IEEE, nov 2009. → pages 6[80] D. Popovic, L. McCartney, C. Beasley, M. Lazebnik, M. Okoniewski,S. Hagness, and J. Booske, “Precision open-ended coaxial probes forin vivo and ex vivo dielectric spectroscopy of biological tissues at mi-crowave frequencies,” IEEE Transactions on Microwave Theory andTechniques, vol. 53, pp. 1713–1722, may 2005. → pages 7[81] J.-Y. Shim and J.-Y. Chung, “Complex Permittivity Measurementof Artificial Tissue Emulating Material Using Open-Ended CoaxialProbe,” IEEE Sensors Journal, vol. 20, pp. 4688–4693, may 2020. →pages 7[82] T. Marsland and S. Evans, “Dielectric measurements with an open-ended coaxial probe,” IEE Proceedings H Microwaves, Antennas andPropagation, vol. 134, no. 4, p. 341, 1987. → pages 7[83] A. Mavrovic, J.-B. Madore, A. Langlois, A. Royer, and A. Roy, “Snowliquid water content measurement using an open-ended coaxial probe(OECP),” Cold Regions Science and Technology, vol. 171, p. 102958,mar 2020. → pages[84] S. K. Lau, D. Dag, S. Ozturk, F. Kong, and J. Subbiah, “A comparisonbetween the open-ended coaxial probe method and the parallel platemethod for measuring the dielectric properties of low-moisture foods,”LWT, p. 109719, jun 2020. → pages[85] V. Guihard, F. Taillade, J.-P. Balayssac, B. Steck, J. Sanahuja, andF. Deby, “Permittivity measurement of cementitious materials withan open-ended coaxial probe,” Construction and Building Materials,vol. 230, p. 116946, jan 2020. → pages88Bibliography[86] G. Maenhout, T. Markovic, I. Ocket, and B. Nauwelaers, “Effect ofOpen-Ended Coaxial Probe-to-Tissue Contact Pressure on DielectricMeasurements,” Sensors, vol. 20, p. 2060, apr 2020. → pages 7[87] A. Bogle, M. Havrilla, D. Nyquis, L. Kempel, and E. Rothwell, “Elec-tromagnetic Material Characterization using a Partially-Filled Rect-angular Waveguide,” Journal of Electromagnetic Waves and Applica-tions, vol. 19, pp. 1291–1306, jan 2005. → pages 8[88] A. Tamer, F. Karadag˘, E. U¨nal, Y. I. Abdulkarim, L. Deng, O. Al-tintas, M. Bakır, and M. Karaaslan, “Metamaterial based sensor in-tegrating transmission line for detection of branded and unbrandeddiesel fuel,” Chemical Physics Letters, vol. 742, p. 137169, mar 2020.→ pages 8[89] C.-K. Lee, S. Zhang, S. S. Bukhari, D. Cadman, J. C. Vardaxoglou,and W. G. Whittow, “Complex Permittivity Measurement System forSolid Materials Using Complementary Frequency Selective Surfaces,”IEEE Access, vol. 8, pp. 7628–7640, 2020. → pages 8[90] T. R. Jones, M. H. Zarifi, and M. Daneshmand, “MiniaturizedQuarter-Mode Substrate Integrated Cavity Resonators for HumiditySensing,” IEEE Microwave and Wireless Components Letters, vol. 27,pp. 612–614, jul 2017. → pages 9[91] K. Saeed, R. D. Pollard, and I. C. Hunter, “Substrate integratedwaveguide cavity resonators for complex permittivity characterizationof materials,” IEEE Transactions on Microwave Theory and Tech-niques, vol. 56, no. 10, pp. 2340–2347, 2008. → pages 10, 11[92] J. Baker-Jarvis, R. Geyer, J. Grosvenor, M. Janezic, C. Jones, B. Rid-dle, C. Weil, and J. Krupka, “Dielectric characterization of low-lossmaterials a comparison of techniques,” IEEE Transactions on Di-electrics and Electrical Insulation, vol. 5, no. 4, pp. 571–577, 1998.→ pages 10[93] M. H. Zarifi, T. Thundat, and M. Daneshmand, “High resolution mi-crowave microstrip resonator for sensing applications,” Sensors andActuators, A: Physical, vol. 233, pp. 224–230, 2015. → pages 11[94] M. H. Zarifi, M. Rahimi, M. Daneshmand, and T. Thundat, “Mi-crowave ring resonator-based non-contact interface sensor for oil sands89Bibliographyapplications,” Sensors and Actuators B: Chemical, vol. 224, pp. 632–639, mar 2016. → pages 11[95] J. Mun˜oz-Enano, P. Ve´lez, M. Gil, and F. Mart´ın, “Planar MicrowaveResonant Sensors: A Review and Recent Developments,” Applied Sci-ences, vol. 10, p. 2615, apr 2020. → pages 17, 18, 19, 20[96] J. Naqui, J. Coromina, A. Karami-Horestani, C. Fumeaux, andF. Mart´ın, “Angular Displacement and Velocity Sensors Based onCoplanar Waveguides (CPWs) Loaded with S-Shaped Split Ring Res-onators (S-SRR),” Sensors, vol. 15, pp. 9628–9650, apr 2015. → pages17[97] J. Naqui, A. Horestani, F. Mart´ın, C. Fumeaux, and D. Abbott, “Two-dimensional displacement and alignment sensor based on reflectioncoefficients of open microstrip lines loaded with split ring resonators,”Electronics Letters, vol. 50, pp. 620–622, apr 2014. → pages[98] J. Naqui, M. Dura´n-Sindreu, and F. Mart´ın, “Novel Sensors Based onthe Symmetry Properties of Split Ring Resonators (SRRs),” Sensors,vol. 11, pp. 7545–7553, jul 2011. → pages[99] J. Naqui, M. Dura´n-Sindreu, and F. Mart´ın, “Alignment and PositionSensors Based on Split Ring Resonators,” Sensors, vol. 12, pp. 11790–11797, aug 2012. → pages 17[100] A. K. Horestani, J. Naqui, Z. Shaterian, D. Abbott, C. Fumeaux,and F. Mart´ın, “Two-dimensional alignment and displacement sensorbased on movable broadside-coupled split ring resonators,” Sensorsand Actuators A: Physical, vol. 210, pp. 18–24, apr 2014. → pages 18[101] A. Ebrahimi, J. Scott, and K. Ghorbani, “Differential Sensors Us-ing Microstrip Lines Loaded With Two Split-Ring Resonators,” IEEESensors Journal, vol. 18, pp. 5786–5793, jul 2018. → pages 19[102] P. Velez, L. Su, K. Grenier, J. Mata-Contreras, D. Dubuc, and F. Mar-tin, “Microwave Microfluidic Sensor Based on a Microstrip Split-ter/Combiner Configuration and Split Ring Resonators (SRRs) for Di-electric Characterization of Liquids,” IEEE Sensors Journal, vol. 17,pp. 6589–6598, oct 2017. → pages[103] L. Su, J. Mata-Contreras, P. Velez, and F. Martin, “Splitter/CombinerMicrostrip Sections Loaded With Pairs of Complementary Split Ring90BibliographyResonators (CSRRs): Modeling and Optimization for DifferentialSensing Applications,” IEEE Transactions on Microwave Theory andTechniques, vol. 64, pp. 4362–4370, dec 2016. → pages 18, 19[104] A. Ebrahimi, J. Scott, and K. Ghorbani, “Transmission Lines Ter-minated With LC Resonators for Differential Permittivity Sensing,”IEEE Microwave and Wireless Components Letters, vol. 28, pp. 1149–1151, dec 2018. → pages 19[105] R. Corporation, “Product Selector Guide,” 2013. → pages 22, 23[106] A. Shrivastava, “Plastic Properties and Testing,” in Introduction toPlastics Engineering, pp. 49–110, Elsevier, 2018. → pages 22[107] F. Gustrau, RF and microwave engineering: fundamentals of wirelesscommunications. John Wiley & Sons, 2012. → pages 24[108] K. Chang and L.-H. Hsieh, Microwave Ring Circuits and RelatedStructures. Hoboken, NJ, USA: John Wiley & Sons, Inc., apr 2004.→ pages 28[109] W. J Highton, “Capacitance of a rectangular capacitor, with edgeeffect, calculator,” 2009. → pages 30[110] C. S. Westfall and P. A. Levin, “Comprehensive analysis of central car-bon metabolism illuminates connections between nutrient availability,growth rate, and cell morphology in Escherichia coli,” PLOS Genetics,vol. 14, p. e1007205, feb 2018. → pages 49[111] A. Bren, Y. Hart, E. Dekel, D. Koster, and U. Alon, “The last gener-ation of bacterial growth in limiting nutrient,” BMC Systems Biology,vol. 7, no. 1, p. 27, 2013. → pages 49[112] E. R. Sanders, “Aseptic Laboratory Techniques: Plating Methods,”Journal of Visualized Experiments, may 2012. → pages 49[113] S. Oberoi, K. S. Daya, and P. S. Tirumalai, “Microwave sensor fordetection of E. coli in water,” in 2012 Sixth International Conferenceon Sensing Technology (ICST), pp. 614–617, IEEE, dec 2012. → pages49[114] I. Piekarz, S. Go´rska, S. Odrobina, M. Drab, K. Wincza, A. Gamian,and S. Gruszczynski, “A microwave matrix sensor for multipointlabel-free Escherichia coli detection,” Biosensors and Bioelectronics,vol. 147, p. 111784, jan 2020. → pages 4991Bibliography[115] A. Tholakalabavi, J. J. Zwiazek, and T. A. Thorpe, “Effect of manni-tol and glucose-induced osmotic stress on growth, water relations, andsolute composition of cell suspension cultures of poplar (Populus del-toides var.Occidentalis) in relation to anthocyanin accumulation,” InVitro Cellular & Developmental Biology - Plant, vol. 30, pp. 164–170,jul 1994. → pages 56[116] B. Mackey and C. M. Derrick, “Conductance measurements of the lagphase of injured Salmonella typhimurium,” Journal of Applied Bacte-riology, vol. 57, pp. 299–308, oct 1984. → pages 56[117] L. Machlis, “FACTORS AFFECTING THE LAG PHASE OFGROWTH OF THE FILAMENTOUS FUNGUS, ALLOMYCESMACROGYNUS,” American Journal of Botany, vol. 44, pp. 113–119,feb 1957. → pages[118] M. D. Rolfe, C. J. Rice, S. Lucchini, C. Pin, A. Thompson, A. D. S.Cameron, M. Alston, M. F. Stringer, R. P. Betts, J. Baranyi, M. W.Peck, and J. C. D. Hinton, “Lag Phase Is a Distinct Growth PhaseThat Prepares Bacteria for Exponential Growth and Involves Tran-sient Metal Accumulation,” Journal of Bacteriology, vol. 194, pp. 686–701, feb 2012. → pages 56[119] P.-Y. Litzler, L. Benard, N. Barbier-Frebourg, S. Vilain, T. Jouenne,E. Beucher, C. Bunel, J.-F. Lemeland, and J.-P. Bessou, “Biofilmformation on pyrolytic carbon heart valves: Influence of surface freeenergy, roughness, and bacterial species,” The Journal of Thoracicand Cardiovascular Surgery, vol. 134, pp. 1025–1032, oct 2007. →pages 57[120] R. Donlan, “Biofilms and Device-Associated Infections,” Emerging In-fectious Diseases, vol. 7, pp. 277–281, apr 2001. → pages[121] Z. Khatoon, C. D. McTiernan, E. J. Suuronen, T.-F. Mah, and E. I.Alarcon, “Bacterial biofilm formation on implantable devices and ap-proaches to its treatment and prevention,” Heliyon, vol. 4, p. e01067,dec 2018. → pages 57[122] M. A. Nazir, “Prevalence of periodontal disease, its association withsystemic diseases and prevention.,” International journal of health sci-ences, vol. 11, no. 2, pp. 72–80, 2017. → pages92Bibliography[123] A. Arigbede, B. Babatope, and M. Bamidele, “Periodontitis and sys-temic diseases: A literature review,” Journal of Indian Society of Pe-riodontology, vol. 16, no. 4, p. 487, 2012. → pages 57[124] C. de la Fuente-Nu´n˜ez, F. Reffuveille, L. Ferna´ndez, and R. E. Han-cock, “Bacterial biofilm development as a multicellular adaptation:antibiotic resistance and new therapeutic strategies,” Current Opin-ion in Microbiology, vol. 16, pp. 580–589, oct 2013. → pages 57[125] A. K. Epstein, B. Pokroy, A. Seminara, and J. Aizenberg, “Bacterialbiofilm shows persistent resistance to liquid wetting and gas pene-tration,” Proceedings of the National Academy of Sciences, vol. 108,pp. 995–1000, jan 2011. → pages[126] P. S. Stewart, “Mechanisms of antibiotic resistance in bacterialbiofilms,” International Journal of Medical Microbiology, vol. 292,pp. 107–113, jan 2002. → pages 57[127] M. Hassan, E. Gonzalez, V. Hitchins, and I. Ilev, “Detecting bac-teria contamination on medical device surfaces using an integratedfiber-optic mid-infrared spectroscopy sensing method,” Sensors andActuators B: Chemical, vol. 231, pp. 646–654, aug 2016. → pages 57[128] T. Javanbakht, B. Ghane-Motlagh, and M. Sawan, “Comparativestudy of antibiofilm activity and physicochemical properties of micro-electrode arrays,” Microelectronic Engineering, p. 111305, mar 2020.→ pages[129] M. A. A. Grant, B. Wac law, R. J. Allen, and P. Cicuta, “The roleof mechanical forces in the planar-to-bulk transition in growing Es-cherichia coli microcolonies,” Journal of The Royal Society Interface,vol. 11, p. 20140400, aug 2014. → pages 57, 61[130] D. Lebeaux, J.-M. Ghigo, and C. Beloin, “Biofilm-Related Infections:Bridging the Gap between Clinical Management and Fundamental As-pects of Recalcitrance toward Antibiotics,” Microbiology and Molecu-lar Biology Reviews, vol. 78, pp. 510–543, sep 2014. → pages 57[131] S. S. Magill, J. R. Edwards, W. Bamberg, Z. G. Beldavs, G. Dumyati,M. A. Kainer, R. Lynfield, M. Maloney, L. McAllister-Hollod, J. Na-dle, S. M. Ray, D. L. Thompson, L. E. Wilson, and S. K. Fridkin,93Bibliography“Multistate Point-Prevalence Survey of Health Care–Associated In-fections,” New England Journal of Medicine, vol. 370, pp. 1198–1208,mar 2014. → pages 57[132] R. J. Allen and B. Waclaw, “Bacterial growth: a statistical physicist’sguide,” Reports on Progress in Physics, vol. 82, p. 016601, jan 2019.→ pages 57, 61[133] P.-T. Su, C.-T. Liao, J.-R. Roan, S.-H. Wang, A. Chiou, and W.-J. Syu, “Bacterial Colony from Two-Dimensional Division to Three-Dimensional Development,” PLoS ONE, vol. 7, p. e48098, nov 2012.→ pages 57[134] F. Si, B. Li, W. Margolin, and S. X. Sun, “Bacterial growth and formunder mechanical compression,” Scientific Reports, vol. 5, p. 11367,sep 2015. → pages 57, 61[135] G. Nabovati, E. Ghafar-Zadeh, A. Letourneau, and M. Sawan, “To-wards High Throughput Cell Growth Screening: A New CMOS 8 ×8 Biosensor Array for Life Science Applications,” IEEE Transactionson Biomedical Circuits and Systems, vol. 11, pp. 380–391, apr 2017.→ pages 58[136] G. Nabovati, E. Ghafar-Zadeh, and M. Sawan, “A 64 pixel ISFET-based biosensor for extracellular pH gradient monitoring,” in 2015IEEE International Symposium on Circuits and Systems (ISCAS),pp. 1762–1765, IEEE, may 2015. → pages 67[137] E. Ghafar-Zadeh, M. Sawan, V. P. Chodavarapu, and T. Hosseini-Nia,“Bacteria Growth Monitoring Through a Differential CMOS Capaci-tive Sensor,” IEEE Transactions on Biomedical Circuits and Systems,vol. 4, pp. 232–238, aug 2010. → pages 58[138] L. Y. Zhang, C. Bounaix Morand du Puch, C. Dalmay, A. Lacroix,A. Landoulsi, J. Leroy, C. Me´lin, F. Lalloue´, S. Battu, C. Lautrette,S. Giraud, A. Bessaudou, P. Blondy, M. O. Jauberteau, and A. Poth-ier, “Discrimination of colorectal cancer cell lines using microwavebiosensors,” Sensors and Actuators A: Physical, vol. 216, pp. 405–416,sep 2014. → pages 58[139] A. Sanchis, A. P. Brown, M. Sancho, G. Mart´ınez, J. L. Sebastia´n,S. Mun˜oz, and J. M. Miranda, “Dielectric characterization of bacterial94Bibliographycells using dielectrophoresis,” Bioelectromagnetics, vol. 28, pp. 393–401, jul 2007. → pages 61[140] W. Bai, K. Zhao, and K. Asami, “Dielectric properties of E. coli cell assimulated by the three-shell spheroidal model,” Biophysical Chemistry,vol. 122, pp. 136–142, jul 2006. → pages[141] B. Zavizion, Z. Zhao, A. Nittayajarn, and R. J. Rieder, “Rapid micro-biological testing: monitoring the development of bacterial stress.,”PloS one, vol. 5, p. e13374, oct 2010. → pages[142] M. Schaechter, “A brief history of bacterial growth physiology,” Fron-tiers in Microbiology, vol. 6, apr 2015. → pages 61[143] P. Jurtshuk, Bacterial Metabolism. University of Texas MedicalBranch at Galveston, 1996. → pages 62[144] K. M. C. Tjørve and E. Tjørve, “The use of Gompertz models ingrowth analyses, and new Gompertz-model approach: An addition tothe Unified-Richards family,” PLOS ONE, vol. 12, p. e0178691, jun2017. → pages 62[145] D. M. Brogan and E. Mossialos, “A critical analysis of the reviewon antimicrobial resistance report and the infectious disease financingfacility.,” Globalization and health, vol. 12, p. 8, mar 2016. → pages66[146] K. I. Mohr, History of Antibiotics Research, vol. 398. Springer, Cham,2016. → pages 66[147] S. Simoens, “Health Economics of Antibiotics.,” Pharmaceuticals(Basel, Switzerland), vol. 3, pp. 1348–1359, apr 2010. → pages 66[148] B. Aslam, W. Wang, M. I. Arshad, M. Khurshid, S. Muzammil, M. H.Rasool, M. A. Nisar, R. F. Alvi, M. A. Aslam, M. U. Qamar, M. K. F.Salamat, and Z. Baloch, “Antibiotic resistance: a rundown of a globalcrisis,” Infection and Drug Resistance, vol. Volume 11, pp. 1645–1658,oct 2018. → pages 66[149] G. D. Wright, “The antibiotic resistome: the nexus of chemical andgenetic diversity,” Nature Reviews Microbiology, vol. 5, pp. 175–186,mar 2007. → pages 6695Bibliography[150] M. L. Bayot and B. N. Bragg, Antimicrobial Susceptibility Testing.StatPearls Publishing, 2019. → pages 66[151] I. Galani, F. Kontopidou, M. Souli, P.-D. Rekatsina, E. Koratzanis,J. Deliolanis, and H. Giamarellou, “Colistin susceptibility testing byEtest and disk diffusion methods,” International Journal of Antimi-crobial Agents, vol. 31, pp. 434–439, may 2008. → pages 66[152] S. P. Bernier and M. G. Surette, “Concentration-dependent activity ofantibiotics in natural environments.,” Frontiers in microbiology, vol. 4,p. 20, 2013. → pages 70, 71[153] G. Kapoor, S. Saigal, and A. Elongavan, “Action and resistance mech-anisms of antibiotics: A guide for clinicians.,” Journal of anaesthesi-ology, clinical pharmacology, vol. 33, no. 3, pp. 300–305. → pages 70,71[154] H. Bhargav, S. D. Shastri, S. Poornav, K. Darshan, and M. M. Nayak,“Measurement of the Zone of Inhibition of an Antibiotic,” in 2016IEEE 6th International Conference on Advanced Computing (IACC),pp. 409–414, IEEE, feb 2016. → pages 7296Appendix AA.1 Measurement of effective dielectricproperties of solid agarTo measure the dielectric properties or complex permittivity of materials,the N1501 dielectric probe kit by Keysight Technologies, Inc. was used. Theprobe setup is shown in Figure A.1.Figure A.1: Experimental setup to measure the dielectric properties of sam-ple under test.It consists of an N1501A dielectric probe connected to a VNA throughan SMA cable. The probe is immersed into a test liquid or placed in closecontact with the solid material under test to perform the dielectric measure-ment.To measuring the effective dielectric properties of solid agar, the solidagar sample was enclosed in a plastic petri dish. The combination was thenplaced in proximity of the dielectric probe such that the face of the dielectricprobe is completely covered by the sample as shown in Figure A.2.97A.1. Measurement of effective dielectric properties of solid agarFigure A.2: Solid agar plate in conjunction with the N1501A dielectricprobe.The probe was calibrated from 200 MHZ to 20 GHz with a resolutionof 1001 and intermediate frequency bandwidth of 300 Hz prior to measure-ments. Sixteen measurements were taken and an average of the measuredresults were calculated and recorded using N1500A Materials ManagementSuite provided by Keysight Technologies, Inc. The measured results for MHagar and LB agar is shown in Figure A.3 (a) and (b) respectively.According to the measured result, the effective permittivity of LB agarand MH agar at frequency 1.76 GHz is 2.156 and 2.258 respectively. Theloss tangent however is measured to be 0.077 and 0.08 for LB and MH agarrespectively.98A.1. Measurement of effective dielectric properties of solid agar(a) MH agar(b) LB agarFigure A.3: Measured effective permittivity and loss tangent of MH agar(a) and LB agar (b).99
Thesis/Dissertation
eng
Electrical Engineering
Vancouver : University of British Columbia Library
Attribution-NonCommercial-NoDerivatives 4.0 International
Graduate
Planar microwave resonator sensor technology for real-time and contactless monitoring of microbial growth on solid media
Text