Land and Food Systems, Faculty of
DSpace
UBCV
Hua, Zhong
Master of Science - MSc
University of British Columbia
Food safety remains one of the most challenging issues worldwide. To ensure food safety, regulatory authorities monitor the agri-food chain by detecting chemical hazards in foods. However, the current standard methods cannot handle large numbers of food commodities due to high cost, long analyzing time, and equipment limitations. The demand for rapid, portable, easy-to-use, and low-cost devices draws attention to the microfluidic paper-based analytical device (μPAD), a promising candidate to provide alternative solutions to the current commercial products. Previous studies of μPAD are limited to the detection of simple chemicals or the analysis of simple, lacking applications on real food samples. In the current study, a μPAD adopting the format of the conventional lateral flow immunoassay (LFI) was developed for rapid detection of chemical hazards in food. The major egg white protein, ovalbumin, was selected since egg is a common food ingredient listed in Canada’s eleven priority allergens. The patterning condition was first optimized for wax-printing and wax-stamping techniques on Grade 1 and Grade 42 filter papers. Immunological conjugates were prepared, and antibody pairs were screened for sandwiching the antigen. The surface modification solution and the geometry of patterns were optimized to functionalize the single piece of cellulose paper as an LFI-format μPAD. The developed μPAD could detect as low as 1 ppm ovalbumin solution, 0.01% (w/w) egg white protein in cake mix, and egg content in commercial food products within 15 min including sample preparation. The single-piece μPAD requires simpler fabrication procedures and much lower material cost, compared with that of the conventional LFI. With more optimizations, the developed μPAD can serve as the prototype for mass production and compete with the mature commercial products, providing an alternative food monitoring option to many sectors throughout the agri-food chain and contributing to improving food safety and public health.
DEVELOPMENT OF MICROFLUIDIC PAPER-BASED ANALYTICAL DEVICE TO DETECT CHEMICAL HAZARDS IN FOOD by Zhong Hua B.Sc., The University of British Columbia, 2017 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Food Science) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) December 2019 © Zhong Hua, 2019 The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, a thesis/dissertation entitled: Development of microfluidic paper-based analytical device to detect chemical hazards in food submitted by Zhong Hua in partial fulfillment of the requirements for the degree of Master of Science in Food Science Examining Committee: Dr. Xiaonan Lu Supervisor Dr. Simone Castellarin Supervisory Committee Member Dr. Ronaldo Cerri Additional Examiner ii Abstract Food safety remains one of the most challenging issues worldwide. To ensure food safety, regulatory authorities monitor the agri-food chain by detecting chemical hazards in foods. However, the current standard methods cannot handle large numbers of food commodities due to high cost, long analyzing time, and equipment limitations. The demand for rapid, portable, easy-to-use, and low-cost devices draws attention to the microfluidic paper-based analytical device (μPAD), a promising candidate to provide alternative solutions to the current commercial products. Previous studies of μPAD are limited to the detection of simple chemicals or the analysis of simple, lacking applications on real food samples. In the current study, a μPAD adopting the format of the conventional lateral flow immunoassay (LFI) was developed for rapid detection of chemical hazards in food. The major egg white protein, ovalbumin, was selected since egg is a common food ingredient listed in Canada’s eleven priority allergens. The patterning condition was first optimized for wax-printing and wax-stamping techniques on Grade 1 and Grade 42 filter papers. Immunological conjugates were prepared, and antibody pairs were screened for sandwiching the antigen. The surface modification solution and the geometry of patterns were optimized to functionalize the single piece of cellulose paper as an LFI-format μPAD. The developed μPAD could detect as low as 1 ppm ovalbumin solution, 0.01% (w/w) egg white protein in cake mix, and egg content in commercial food products within 15 min including sample preparation. The single-piece μPAD requires simpler fabrication procedures and much lower material cost, compared with that of the conventional LFI. With more optimizations, the developed μPAD can serve as the prototype for mass production and compete with the mature commercial products, providing an alternative food monitoring option to many sectors throughout the agri-food chain and contributing to improving food safety and public health. iii Lay Summary Food safety is critical to everyone’s life, and it is also a very challenging issue worldwide. To ensure food safety, governments regulate potential contaminants by testing the food. However, many current methods are slow and expensive, so the governments require better tools to handle large numbers of food samples. This study reported the development of a low-cost tool, made of a single piece of filter paper, to detect chemical contaminants in foods. This tool can detect a tiny amount of egg, a common food ingredient causing allergy to many Canadians, especially to children. This developed tool is the first of its kind that can detect large and complicated chemicals, such as protein, in very complex food samples. The study may aid in developing more practical tools for commercialization to test food samples and eventually contribute to the improvement of food safety. iv Preface A part of Chapter 1 has been published as a short review by MDPI: Hua, M.Z., Li, S, Wang, S, and Lu, X. Detecting chemical hazards in foods using microfluidic paper-based analytical devices (μPADs): the real-world application. Micromachines 2018, 9(1), 32. I wrote the entire manuscript, and my supervisor Dr. Lu made the editorial changes. Shenmiao Li and Shuo Wang contributed to collecting material and information during manuscript preparation. Dr. Lu proposed the thesis project. I designed and conducted all experiments, analyzed all data and draft the entire thesis, with two exceptions. The wax-stamping idea and experimental design (Section 2.3) was proposed by postdoc fellow Dr. Azadeh Nilghaz in Dr. Lu’s lab. The experiment in Section 2.4.2 was conducted by Anna Tse, an undergraduate research assistant in Dr. Lu’s lab. The thesis was edited by Dr. Lu, and the supervisory/examining committee provided critical and helpful comments. v Table of Contents Abstract ......................................................................................................................................... iii Lay Summary ............................................................................................................................... iv Preface .............................................................................................................................................v Table of Contents ......................................................................................................................... vi List of Tables ..................................................................................................................................x List of Figures ............................................................................................................................... xi List of Abbreviations ...................................................................................................................xv Acknowledgements .................................................................................................................... xvi Dedication .................................................................................................................................. xvii Chapter 1: Introduction ................................................................................................................1 1.1 Food Safety and Chemical Hazards ................................................................................ 1 1.2 Detection Tools and Challenges in Real-World Practice ............................................... 3 1.3 Microfluidic Paper-based Analytical Device .................................................................. 6 1.3.1 Re-introduction of Cellulose as a Platform ................................................................. 6 1.3.2 Fabrication Techniques ............................................................................................... 8 1.3.3 Current State of Applications in Detecting Chemical Hazards in Food ..................... 9 1.3.3.1 The Gap ............................................................................................................... 9 1.3.3.2 Detecting Pesticides .......................................................................................... 13 1.3.3.3 Detecting Inorganic Ions ................................................................................... 18 1.3.3.4 Detecting Other Organic Compounds ............................................................... 19 1.3.4 Issues and Concerns in Real-world Applications ..................................................... 20 vi 1.3.4.1 Challenges in Sample Preparation .................................................................... 20 1.3.4.2 Rapidness .......................................................................................................... 21 1.3.4.3 Aqueous Solution Limited ................................................................................ 21 1.3.4.4 Limited Understanding of Interactions between Paper-Analyte/Interference .. 22 1.3.4.5 Market Orientation ............................................................................................ 23 1.4 Research Goals, Rationale, and Challenges .................................................................. 25 Chapter 2: Methods and Materials ............................................................................................29 2.1 Overall Experimental Design ........................................................................................ 29 2.2 Filter paper .................................................................................................................... 29 2.3 Wax-stamping ............................................................................................................... 29 2.3.1 Fabrication of Stamps ............................................................................................... 29 2.3.2 Optimization of Patterning ........................................................................................ 30 2.4 Wax-Printing ................................................................................................................. 30 2.4.1 Optimization of Printing Settings ............................................................................. 30 2.4.2 Optimization of Heating Settings.............................................................................. 30 2.5 Preparation of Immunological Reagents ....................................................................... 31 2.5.1 Synthesis and Characterization of Gold Nanoparticles ............................................ 31 2.5.2 Optimization of Antibody-Gold Nanoparticle Conjugation ..................................... 31 2.5.3 Screening of Antibody Pairs on Nitrocellulose ........................................................ 32 2.6 Surface Modification of Paper Substrate for Lateral Flow ........................................... 33 2.7 Optimization of Patterns and Modification Recipes ..................................................... 33 2.7.1 Designs of Test and Control Lines ............................................................................ 33 2.7.2 Optimization of Pattern Geometry for Flow Improving ........................................... 33 vii 2.7.3 Surface Modification of Paper Substrate with Recipe 33-42 .................................... 35 2.8 Optimization of Antibody Deposition .......................................................................... 35 2.9 Detection of Ovalbumin ................................................................................................ 35 2.9.1 Food Sample Preparation .......................................................................................... 35 2.9.2 Detection ................................................................................................................... 36 Chapter 3: Results and Discussion .............................................................................................37 3.1 Filter Paper Substrate .................................................................................................... 37 3.2 Wax-stamping ............................................................................................................... 37 3.2.1 Materials of Stamps .................................................................................................. 37 3.2.2 Patterning Settings .................................................................................................... 38 3.3 Wax-printing ................................................................................................................. 41 3.3.1 Printing Settings ........................................................................................................ 41 3.3.2 Heating Settings ........................................................................................................ 43 3.4 Immunological reagents ................................................................................................ 46 3.4.1 Gold Nanoparticles ................................................................................................... 46 3.4.2 Ab-AuNPs Conjugate ............................................................................................... 46 3.4.3 Screening of Antibody Pairs on Nitrocellulose ........................................................ 47 3.5 Surface Modification of Paper Substrate ...................................................................... 48 3.6 Optimization of Patterns and Modification Recipes ..................................................... 50 3.6.1 Designs of Test and Control Lines ............................................................................ 50 3.6.2 Increasing Flow Rate by Revising the Patterns ........................................................ 51 3.6.3 Elimination of False Negative with New Recipes .................................................... 53 3.7 Optimization of Antibody Deposition .......................................................................... 55 viii 3.7.1 Test Line ................................................................................................................... 55 3.7.2 Control line ............................................................................................................... 55 3.8 Detection of Ovalbumin ................................................................................................ 56 3.9 Detection Limit and Issues in Extraction ...................................................................... 58 Chapter 4: Conclusion .................................................................................................................62 References .....................................................................................................................................63 Appendix .......................................................................................................................................70 ix List of Tables Table 1.1 Chemical hazards in foods .............................................................................................. 3 Table 1.2 Summary of microfluidic paper-based analytical device (μPAD) applications in detecting chemical hazards in food matrices ................................................................................ 12 Table 2.1 Recipes of surface modification solutions .................................................................... 34 Table 2.2 Recipes of the revised surface modification solutions .................................................. 35 Table 3.1 Minimum thickness (unit: mm) of printed shapes (before heating) required to pass the barrier test for Grade 42 and Grade 1 filter paper under various heating conditions, namely the combination of heating time (3-5 min) and temperature (120-150°C). A darker background indicates a thinner barrier required. .............................................................................................. 45 Table 3.2 pH change of 1 mL AuNPs in response to the addition of 0.2 M K2CO3. .................... 47 Table 3.3 Comparison of the claimed performance of commercial LFI products and the developed μPAD. .......................................................................................................................... 59 x List of Figures Figure 1.1 Representative μPAD applications. (A) Bidirectional lateral flow bioactive paper sensor. Adapted with permission from Reference [49]. Copyright 2009 American Chemical Society. (B) Scanning electron microscope photos of native chromatography paper (top) and molecularly imprinted polymer-coated chemiluminescence device. Adapted with permission from Reference [51]. (C) Alkylsilane self-assembling and UV/O3-patterning fabricated nitrite assay based on Griess reaction. Reprinted with permission from reference [54]. Copyright 2013 American Chemical Society. (D) Electrochemical and colorimetric dual sensor for simultaneous determination of lead, cadmium and copper. Reprinted with permission from reference [57]. ... 14 Figure 1.2 Scheme of a conventional lateral flow immunoassay (LFI) ........................................ 26 Figure 1.3 Scheme of a tentative LFI-format μPAD that integrates all functions in one piece of paper. ............................................................................................................................................. 27 Figure 3.1 Patterns generated using a PMMA stamp (A), with examples: (B) under-heating (110°C, 2 min), (C) preferred heating (130°C, 3 min) for a channel width of 2 mm, and (D) over-heating (150°C for 3 min). ............................................................................................................ 40 Figure 3.2 Example of a PMMA stamp (left) patterned LFI-format μPAD design (middle) for subsequent flow test (right). .......................................................................................................... 40 Figure 3.3 Scan (1200 dpi) of wax-printed Grade 42 filter paper, printed with lines of 0.10, 0.15, 0.20, …, 0.95, and 1.00 mm in width. The magnified images of both ends of 0.1 mm lines printed horizontally and vertically were magnified and highlighted in the red and further in the blue boxes to demonstrate the inconsistency in printing quality. ................................................. 42 xi Figure 3.4 Photos of both sides of Grade 42 filter paper and the magnified scans of 0.1 mm lines printed on both sides to demonstrate the difference in printing quality related to the surface geometry. ...................................................................................................................................... 43 Figure 3.5 An example of the barrier test, including the scan of the front side (left) and back side (right) of the patterned paper. The criteria for evaluating the functionality of the formed barrier (middle) were based on the back side. .......................................................................................... 44 Figure 3.6 UV-Vis spectrum of the synthesized gold nanoparticles (surface plasma resonance peak at 523.7 nm). ......................................................................................................................... 46 Figure 3.7 Result of antibody pair screening. Au-M291 x M293 was selected due to high signal intensity (darker dot) and lower background. ............................................................................... 48 Figure 3.8 Flow test of Recipe 1-18, LFI recipe (ϴ), and No-treated (0). Recipe 12 and 15 were selected for the next batch of flow tests due to their low background and relatively high flow rate........................................................................................................................................................ 49 Figure 3.9 Flow test of Recipe 12, 15, and 19-32. Recipe 27 was selected for further tests due to the efficient release of the conjugate, low background, and relatively high flow rate. ................ 49 Figure 3.10 Example of “bridging” (A-C) and “cutting” (D-F) methods for assembling the T/C lines: (A) front view, (B) back view, (C) a negative test; (D) front view, (E) back view, and (F) a positive test (note the high background signal when the testing time was extended for a higher T-line signal). .................................................................................................................................... 51 Figure 3.11 Effective volumes related to the geometry of different patterns. The strategy that resulted in the largest area was adopted. Total area beyond the assigned point reflected the effective volume passing the location of T/C lines. The last pattern was for reference. .............. 52 xii Figure 3.12 T line tests with Recipe 33-42 on Grade 42 filter paper. Recipe 33 was selected for further tests as the best compromise between T line signal intensity and background colour even with a longer wicking time. .......................................................................................................... 53 Figure 3.13 Comparison between modified (left pair) and unmodified (right pair) μPADs with negative and positive results tested using PBS or 50 ppm ovalbumin in PBS. The blue arrows at the front line of the sample liquid indicate the failed transportation of the conjugate due to strong non-specific binding onto the unmodified μPADs. ...................................................................... 54 Figure 3.14 Colour intensity on the C lines with various amounts of the secondary antibody. ... 56 Figure 3.15 Detection of ovalbumin in the standard solutions of 0 – 100 ppm (n ≥ 3). As low as 1 ppm of ovalbumin was visible to naked eyes. ........................................................................... 56 Figure 3.16 Detection of ovalbumin in cake mix spiked with 0 - 2% egg white powder (n ≥ 3)........................................................................................................................................................ 57 Figure 3.17 Detection of ovalbumin in muffin mix, baked muffin, mayonnaise, and skim milk powder (n ≥ 3). ............................................................................................................................ 58 Figure A1 Settings for the determination of the effect of patterning geometry on flow rate. ...... 70 Figure A2 Manufacturing direction in pre-packed sheet (left), revealed via the free spreading of aqueous food dye in both smaller cut of Grade 42 (middle) and common copy paper (right). .... 70 Figure A3 (A)Triplicate of laser-cut PMMA stamps before (protecting cover not removed), with the width pre-set as 0.2, 0.4, 0.6, …, 2.8, and 3.0 mm; (B) handle attached and cover removed transparent PMMA stamp; (C) an example of stamped pattern (0.2, 0.4, 0.6, …, 1.4, and 1.6 mm, 110°C, 3 min heating, m.p. 65°C, Grade 42). ............................................................................... 71 xiii Figure A4 Synthesis of gold nanoparticles via a modified citrate reduction method (left) and synthesized gold nanoparticles stored for 10 months (right). ....................................................... 71 Figure A5 NaCl induced aggregation test for determining the minimum ratio of antibody (μg) to AuNPs (1 mL); for M293, the least ratio without colour change from pink to blue/colourless (8 μg Ab/ 1mL AuNPs). .................................................................................................................... 72 Figure A6 Effect of various channel widths on flow rate (as the reference to tune effective volume, Fig. 3.11). ........................................................................................................................ 72 Figure A7 The increase of flow rate increased at different stages (in 600 s) due to the cuttings of various lengths in the middle of channels ..................................................................................... 73 Figure A8 Detection of ovalbumin in muffin mix, baked muffin, mayonnaise, and skim milk powder (n = 3) ............................................................................................................................... 74 xiv List of Abbreviations 2-ME 2-mercaptoethanol Ab antibody ABS acrylonitrile butadiene styrene AChE acetylcholinesterase AuNPs gold nanoparticles BSA bovine serum albumin CFIA Canadian Food Inspection Agency DMSO dimethyl sulfoxide ELISA enzyme-linked immunosorbent assay LFI lateral flow immunoassay LOD limit of detection PBS phosphate-buffered saline PDMS polydimethylsiloxane PEG polyethylene glycol PMMA poly(methyl methacrylate ppb part per billion ppm part per million PVA polyvinyl alcohol PVP polyvinylpyrrolidone SDS sodium dodecyl sulphate T/C line Test/Control line μPAD microfluidic paper-based analytical device xv Acknowledgements I would like to express my deep and sincere gratitude to my supervisor Dr. Lu for offering the opportunity to join the group as well as all the mentorship, trust and support. I would like to express my appreciation to Dr. Simone Castellarin, Dr. Paul Li, and Dr. Ronaldo Cerri for serving my supervisory and examining committees and for all the help on my thesis. I am very grateful to Dr. Hongyan Zhang (visiting professor in Lu Group) and Dr. Daniel Citterio (supervisor of my visiting research at Keio University) for saving me from hard times. Dr. Nilghaz was just as helpful as LFS 250. I would like to thank Shaolong Feng, Dr.Yaxi Hu, and Dr. Jinsong Feng for the research instructions and the survival guide. My thanks also go to labmates in Citterio Group, my undergraduate research assistant Anna Tse. I would like to acknowledge all funding agencies that make the project possible and my graduate life easier, including but not limited to NSERC, Mitacs, JSPS, and scholarship donors. Last, I would like to thank my parents and my wife for their love and support. xvi Dedication To Xerox solid ink printer. R.I.P.xvii Chapter 1: Introduction 1.1 Food Safety and Chemical Hazards Consuming safe and nutritious foods is a fundamental need and a prerequisite for maintaining physical and mental health for human beings [1]. Food safety remains one of the most important issues worldwide partially because of the ever-increasing complexity of the food systems that exacerbate the challenges in ensuring food safety. In Canada and the United States, approximately 1 in 8 people could be affected by a food-borne illness every year, which is a total of 4 and 48 million populations that lead to 11,600 and 128,000 hospitalizations as well as 238 and 3,000 deaths, respectively [2,3]. Nevertheless, considerable efforts have been made to improve the systematic approach to ensuring food safety. The monitoring of the agri-food products through the entire food chain, including the detection of potential food hazards, is adopted as a vital aspect of this approach. Food hazards refer to any agents with the potential to cause adverse health consequences to the consumers [4]. These agents can be mainly classified into three categories: physical, chemical, and biological hazards. Different from physical hazards that rarely cause major food safety issues and biological hazards that can be mostly inactivated by sufficient thermal processing, numerous chemical hazards remain stable and toxic through food processing [5,6]. Some chemical hazards are induced by inappropriate food processing, including carcinogens in the overheated oil [7] and residues of sanitation chemicals in food plants. The resulted adverse health effects have been widely reported, including the melamine contamination in infant formula powder causing kidney stones and deaths [8] and heavy metal residues in fish products. One of the recent outbreaks is the egg contamination 1 by fipronil, which is a broad-spectrum insecticide actively used to control fleas. The outbreak involved numerous countries and resulted in the recall of millions of eggs and lots of egg products (e.g., cake mix), while hundreds of thousands of the contaminated eggs had already been consumed [9-11]. Table 1.1 summarizes the classification of chemical hazards derived from the food safety guidance in the US and Canada [4], including examples of the source, specific substance, and representative food matrix for each (sub-)category. Based upon their origins and natures, chemical hazards can be sorted as follows: (1) natural toxins derived from mold, plant, and marine organisms (e.g., aflatoxin in moldy peanuts); (2) environmental contaminants including heavy metals and persistent organic pollutants (e.g., mercury in fish, polychlorinated biphenyls in milk); (3) unapproved food additives or abuse (e.g., Sudan dyes in paprika powder, nitrite in cured meat); (4) processing-induced chemicals (e.g., bisphenol A in infant formula); (5) pesticides (e.g., glyphosate in various types of foods); (6) veterinary drugs (e.g., clenbuterol in meat); (7) food sensitivities including allergens, intolerance, and sensitivities (e.g., nuts, lactose, and glutamate); (8) biochemistry and molecular biology-related food products (e.g., genetically modified food); etc. Ranging from relatively simple inorganic ions and diverse organic compounds to macromolecules such as proteins, various types of hazards can be identified in specific types of foods. 2 Table 1.1 Chemical hazards in foods Category Sub-Category Source Example Substance Example Food Matrix Natural toxins Mycotoxin Mould Aflatoxin Peanut Plant toxin Plant in response to stress Glycoalkaloids Potato tuber Marine toxin Fish decomposing Histamine Tuna Bioaccumulation from algae Saxitoxin Clam Environmental contaminants Inorganic, heavy metals Industrial manufacture, mining, pesticide degradation Arsenic, lead, mercury Seafood Persistent organic pollutants polychlorinated biphenyl Fish, milk Unapproved food additives - Adulteration, importation Sudan dye Paprika Processing-induced chemicals - Surfactant, antimicrobial, undesired reaction, migrate from container Nitrosamines, melamine, bisphenol A Processed foods Pesticides/agricultural product Herbicide, insecticide, fungicide Agricultural practice, sanitation misconduct Azoxystrobin Peach Veterinary drugs Animal disease control Clenbuterol Meat Food sensitivity Food allergens Cross-contamination, improper labelling Ovalbumin, gliadin, parvalbumin Egg, wheat, fish Food intolerances Lactose Milk Chemical sensitivity Monosodium glutamate Mushroom Biochemistry-related - Genetically modified food, cross-contamination, improper labelling, adulteration Adulterant Meat, flour, corn Novel foods and others - New formulation or processing procedure Unapproved genetically modified corn/rice for feed Corn/rice for human consumption 1.2 Detection Tools and Challenges in Real-World Practice While numerous novel sensors and innovated methods have been extensively studied at the laboratory level to provide better options to detect chemical hazards in food, rarely do researchers take the potential of mass production, broad application, and official certifications into account throughout the development. The nature of the agri-food industry 3 decides the scenario of the end-use of commercial testing products. Methods with unique requirements and higher initial investment, e.g. complicated analysis based on users’ database and mathematic models, are very unlikely to be commercialized. Consequently, in real-world food production and sample testing, the choice for routine monitoring and analysis seems quite conservative. The food industry and regulatory agencies heavily rely on column chromatography (e.g., gas chromatography, high-performance liquid chromatography), mass spectrometry, and immunoassays for accurate quantification [12]. These associated experiments typically accompanied with high costs, long turnaround time, and the demand of expertise. Furthermore, many of these gold-standard methods, such as chromatography coupled with mass spectrometry used to determine trace level of pesticide residues in fresh produce, require labour-intensive sample preparation prior to the actual running of analysis. As compensation, analytical tools for rapid screening of chemical hazards are widely adopted to test an enormous number of agri-food products, especially the varieties of imported goods. Imported fresh produce and food products, of which the production is more challenging to regulate, are more susceptible to chemical contamination and associated adulteration. In the food industry, immunoassays, particularly the enzyme-linked immunosorbent assay (ELISA) and lateral flow immunoassay (LFI), could be the favourite screening methods. With more or less compromise in accuracy and sensitivity, commercial ELISA kit usually shortens the turnaround time to 1-2 hours with decent working ranges for detection and quantification. Moreover, “test strips” (i.e. LFI) often answer the “Yes/No” question within a few and up to 30 min and thus are able to serve well for rapid screening of the target substance with a clear threshold at a more affordable price. Applications in agri-food industry represent the second-largest portion of the $4.86 billion 4 global LFI market [13], which mostly owes to the user-friendly design of LFI, including the simplified operation procedure that enables on-site analysis and the straightforward result interpretation that eliminates the barrier of experimental expertise. Despite the great efforts on regularizing the entire food chain, preventing chemical contaminated food being served to our dining table is still beyond the regulatory agencies’ capacity. As concluded by the European Court of Auditors, the EU food safety system has been overstretched and is not able to guarantee the high level of food safety respected worldwide [14]. It has been clearly determined that the tests carried by public bodies only account for a very small proportion, and private sectors are definitely needed to protect consumers from chemical hazards that may present in food [14]. Another perfect example of the government facing challenges would be the marine toxin monitoring issue in our province, British Columbia. Marine toxins are chemicals produced by marine algae and accumulated in shellfish. With a long history as early as in 1793, reported by Captain Vancouver in British Columbia, consuming shellfish contaminated with marine toxins cause diarrhetic illness, severe neurological effects and fatality to Canadian residences from coast to coast [15-17]. Marine toxins become a high threat to the First Nation community because shellfish is an important food source and shellfish harvesting is practised as an irreplaceable cultural tradition. With no method to detoxify, the provincial and federal governments have made great efforts to reduce shellfish poisoning incidents, including monitoring programs [16,18]. However, sending sample to a Canadian Food Inspection Agency (CFIA) laboratory is the only existing option for marine toxin testing in Canada currently [19]. This sole option relies on transportation from remote areas and the time-consuming gas/liquid chromatography methods along with laborious sample preparations. The 2011 shellfish 5 poisoning outbreak in BC were traced to bivalve shellfish harvested in approved open areas, and 13 out of 17 CFIA recalls directed to commercial retailers that under extensive, well-managed shellfish control program [20]. At the federal level, CFIA is currently seeking robust detecting devices that can achieve on-site detection under the field conditions in order to provide marine toxin contamination warnings in a timely manner [19,21]. As aforementioned, the lack of lab-based applications towards real-world scenarios may limit not only the improvement of food safety but also the significance of the scientific studies themselves. Essentially, the market should play the most crucial role in driving scientific studies that focus on the application side [22]. Since there is an apparent demand for rapid, portable, easy-to-use, robust, and low-cost devices to enhance the monitoring of chemical hazards in food, innovative prototypes are highly expected to be developed and optimized to be mass-producible commercial products that could comply with the government regulations. 1.3 Microfluidic Paper-based Analytical Device 1.3.1 Re-introduction of Cellulose as a Platform Ever since the re-introduction of paper (cellulose) as a platform for analysis in 2007 [23], the concept of a microfluidic paper-based analytical device (μPAD) has been spread broadly. With the initial goal to help with the limited healthcare settings by improving the clinical diagnosis tools in underdeveloped countries, the research in the Whitesides Group has inspired numerous studies on developing μPADs [24]. A well-developed μPAD is typically a small piece of paper with 2D or 3D patterns that enables the test of a few to a few tens of litres of a liquid sample within a relatively short period of time. 6 As the most abundant organic matter on earth, cellulose is the main constituent of plant fibre, with an average of 33% and as high as 90% in cotton [25]. The typical definition of “paper” refers to the thin material mostly made of fibres of cellulose. Cellulose is a liner chain homopolymer of ringed anhydro-D-glucose monomers β(1→4) linked by glycosidic bonds [26]. The triple (or less, in some cases) free hydroxyl groups endue paper’s unique structures at different levels. The hydrogen bonds between adjacent monomers stabilize the linear-chain configuration, while the inter-chain hydrogen bonds and van der Waals force stack the chains into so-called “cellulose fibrils”. The fibrils further aggregate to form cellulose fibres where tightly packed crystalline regions are spaced by less orderly oriented amorphous regions. The cellulose fibres interlace randomly and generate more irregular porous structure during a series of papermaking processes in which the hydrophobic lignin is removed from the pulp. The abundant hydroxyl group exposed on the surface of cellulose fibres leads to the high hydrophilicity of paper. The porous structure, together with the hydrophilic surface of cellulose, enables the capillary flow of polar liquid within the space of the paper. In comparison to polydimethylsiloxane (PDMS)-based microfluidic devices that require external pumps, the power-free feature of μPADs essentially won them the title of “Microfluidic 2.0” [27] and became the most important basis of paper-based microfluidics. The fluidic dynamic of capillary flow, or “wicking”, in porous media has been extensively studied, ranging from the classic simplified Lucas-Washburn model [28] to more complex mathematical simulation taking geometrical and evaporation effects into consideration [29]. With these theoretical tools and, probably more importantly, the experimental practice, one can achieve the “programming” of the fluid movement within the paper matrix by changing the surrounding 7 conditions. In this case, more complex on-paper operations and reactions can be realized instead of performing the experiment manually in the tradition analysis. 1.3.2 Fabrication Techniques The primary and most straightforward method to change the path of flow in the paper device is to pattern the paper. “Patterning” refers to the use of hydrophobic material to prevent the fluid from flowing to undesired space in order to regulate the flow direction. To date, quite many different patterning techniques have been reported, which can be classified according to the principle of material deposition. The first group of techniques involves printing (e.g., wax printing [30], inkjet printing [31], flexographic printing [32]) in which the hydrophobic material is directly deposited onto the paper, or the hydrophilic area is itched out of a piece of paper fully coated with the hydrophobic material. Note that wax-printing requires a secondary heating to further melt the heat-printed wax into the space within the paper matrix. The second group of techniques require masks with the shape of the desired patterns to turn only masked or only unmasked area hydrophobic, including photolithography [23], plasma itching [33], wax screening [34], etc. The third group uses stamps [35,36] or pen [37] to either directly transfer the hydrophobic liquid for solidification (similar to printing) or use as heating source [38] to relocate hydrophobic material to the paper substrate from a fully coated piece stacked on it. The fourth group can be understood as taking the air as the hydrophobic barrier by using a programmed laser [39] or digital blade to cut the paper substrate. Beyond basic patterning that simply changes the direction and width of the hydrophilic channel, many creative designs improve the way of manipulating the flow in μPADs, such as carved microgrooves to increase the flow rate [40] and the dissolvable bridge for preventing back-flow or for terminating the entire test [41]. 8 1.3.3 Current State of Applications in Detecting Chemical Hazards in Food 1.3.3.1 The Gap Although the clinical diagnostics-related topics are still the dominant research direction in the μPAD field, the application of μPAD on food safety-related analysis draws attention from analytical chemists and chemical engineers with less food science background. Many publications aim at detecting food hazards or include a proof-of-concept section involving the detection of chemical contaminants that may be present in foods. A recent review paper of the advance in μPAD for food and water analysis [42] summarized about 40 publications from the perspective of chemical engineering, the majority of analytes being chemicals. However, if one takes a closer look, only a few of them were able to validate the potential of using μPADs in detecting the target chemicals within real food samples or at least spiked food matrices. There seems to be a gap in understanding the detection of food hazards for researchers without an adequate food science background, which could possibly have increased the challenges in translating the device fabrication techniques to real-world applications and eventually towards the final commercial products. Detecting chemical hazards in foods is vastly different from detecting those substances in pure organic/aqueous solutions, environmental/drinking water samples, or clinical samples. In most cases, food samples are much more complex than saliva or urine—the sample liquids used in point-of-care diagnostics on which many μPAD studies have focused. For instance, in a veterinary residue test [5], the food sample could be a piece of medium-rare beef loin steak comprised of muscle (high in proteins, raw and denatured), tallow (lipids, fat-soluble compounds), meat juice and solutes (e.g., salts, amino acid), everything added during cooking (e.g., sauces, cooking oil, spices), thousands of compounds 9 generated in the Maillard reaction, etc. Due to the diversity of chemical hazards and foods, the interaction between the analyte and the matrix can be highly complicated. Depending on the nature of both analyte and matrix, strong physical and chemical interactions (e.g., physically trapped in milk emulsion, chemically bound to mono/oligosaccharides) may significantly interfere with the availability of the analyte to the detecting agent. Therefore, appropriate sample pre-treatment/preparation compatible with the selected detection method is usually required to enable the function and/or to improve the performance of such a method. In the case of using μPAD, at least the sample has to be transformed into liquid form. Typically, sample preparation involves, firstly an extraction step to transfer and/or free the analyte from the food matrix; secondly, a clean-up step to remove the co-extracted interfering substances as much as possible; and lastly, ended up with a (pre-)concentration (in a few cases, dilution) step if applicable. Bearing all the information above in mind, the gap between “being able to detect chemical hazards” and “being able to detect chemical hazards in foods” seems to be quite underestimated. It makes little sense to claim that a μPAD can be applied to the detection of chemical hazards in foods without presenting the detailed result of testing real food samples [43]. At least, it should be clear that “being able to detect chemical hazards in foods” means “the chemical currently contained in a food sample can be detected” rather than “the chemical can be detected in its pure solution or a drinking water sample, and it may also exist in a food sample” [43]. Table 1.2 summarizes the publications reporting the application of μPADs to test chemical hazards in real food samples. Although many more publications were included as applications in ensuring food safety in reviews [24,42,44] and book chapters [45], those that 10 do not present the result of testing real food samples were excluded. Meanwhile, those fabricating paper-PDMS hybrid devices [46], those only with a subtle link to typical μPADs [47,48], and those being misclassified [42] (e.g., glucose as a food additive) were excluded as well. Several special cases [49-51] will be discussed in this section. Studies utilizing nitrocellulose membranes while branded as paper-based devices are excluded.11 Table 1.2 Summary of microfluidic paper-based analytical device (μPAD) applications in detecting chemical hazards in food matrices Analyte Food Matrix Sample Preparation Liquid Phase Principle and Format Paper Type Fabrication Method Barrier Material Note Ref. Paraoxon (organophosphate pesticide) Milk, apple juice, head lettuce, apple Adjust apple juice pH, swab surface of lettuce and apple into water Aqueous Acetylcholinesterase (AChE) inhibition, colorimetric bidirectional lateral flow strip Whatman No. 1 Paper-cutting, inkjet-printing of reagents - Silica assisted reagent trapping, sampling method issue [49] Pirimicarb (carbamate pesticide) Lettuce, brown rice PBS buffer extraction (10 min) or QuEChERS method Aqueous, acetonitrile AChE inhibition, colorimetric Whatman No. 1 Cutting - Sample preparation details not reported [52] Methyl-paraoxon (organophosphate pesticide) Cabbage, green mussel QuEChERS method Aqueous (4% methanol) AChE inhibition, colorimetric Whatman No. 4 Polymer screen-printing method Polystyrene Nanoceria (CeO2) coated μPAD [53] Dichlorvos (organophosphate pesticide) Cucumber, tomato, cabbage Water eluting & filtration Aqueous Chemiluminescence, lateral flow Whatman Grade 3MM CHR chromatography paper Cutting, home-made reagent dispensing equipment - Incorrect sampling [50] Tomato skin, cabbage leaf Chemiluminescence, molecularly imprinted polymers [51] Nitrite Red cubilose 75°C water extraction 5 min, centrifugation 30 min Aqueous Griess-color nitrite assay Whatman No. 1 UV-lithography Octadecyltrichlorosi-lane Sample preparation details not specified [54] Nitrite Ham, sausage 100°C water extraction (1 h) Aqueous Griess-color nitrite assay JProlab JP40 filter paper Stamping Paraffin - [55] Cu(II) Tomato juice, rice Centrifuge & filtration (tomato juice), 4 h acid digestion & oxidation (rice) Aqueous Catalytic etching of silver nanoplates by thiosulfate in presence of Cu2+, colorimetric Whatman No. 1 Wax printing Xerox Color Qube printing wax - [56] Cu(II), Pb(II), Cd(II) Rice, fish 4 h acid digestion, pH adjustment, filtration Aqueous Electrochemistry for Pb(II), Cd(II) Whatman No. 1 Wax printing (pattern), screening-printing (electrochemical ink) Xerox Color Qube printing wax Cu(II)detection[56] incorporated [57] Clenbuterol (veterinary drug) Milk No Aqueous Competitive ELISA, HRP labelled Chromatography paper Wax printing, screening-patterning Xerox Color Qube printing wax, paraffin - [58] Formaldehyde (illegal preservative) Dried goods Micro-distillation Aqueous Hantzsch reaction, fluorescent formaldehyde-Acetoacetanilide complex Advantec No. 1 Wax printing Xerox Color Qube printing wax - [59,60] L-glutamate Instant soup No Aqueous Dehydrogenase catalyzed color change Chromatography paper No pattern - - [61] “Genotoxic activity of pollutants” Grilled chicken Dimethyl sulfoxide (DMSO) extraction DMSO Electrochemiluminescence Whatman No. 1 Screening-printing Wax from commercial wax paper Large variation, analyte not specified [62] 12 1.3.3.2 Detecting Pesticides Three studies reported their μPADs for testing several pesticides based upon a common principle, namely acetylcholinesterase (AChE) inhibition [49,52,53]. In brief, some acetylcholine substrates can be hydrolyzed by the enzyme AChE and change their colours, while the presence of AChE inhibitors (e.g., organophosphate and carbamate pesticides) interferes with such an enzymatic reaction to different extents, and therefore affect the degree of colour change. In one study, Hossain et al. [49] fabricated a bidirectional lateral flow bioactive paper sensor to detect paraoxon, an organophosphate pesticide, in liquid (i.e., milk and apple juice) and on the surface of solid foods (i.e., apple and head lettuce). For the fabrication of the device, Whatman No. 1 paper was cut into 1 × 10 cm strips and labelled at every centimetre. The substrate region (at 3 cm) and the sensing region (at 5 cm) were functionalized through printing silica/substrate/silica and polyvinylamine/silica/AChE/silica layers of reagent inks, respectively, using a piezoelectric inkjet printer (DMP-2800, Fujifilm Dimatix Inc., Tokyo, Japan). After air-drying for 1 h at room temperature, both the substrate and the enzyme were trapped within the layers of silica that fixed onto the filter paper, and the device was ready to be used. Two operation procedures were described in this study, and the bidirectional “inverted” flow system was reported to have a 10-fold better limit of detection (LOD). Briefly, the 10th cm end of the strip was first dipped into the sample liquid to be tested and immediately removed once the sample reached the sensing region (5th cm), followed by 5-min incubation in the air at room temperature for better AChE inhibition. Then, the 1st cm end of the strip was immersed into ddH2O to bring trapped substrate (3rd cm) to the sensing region (5th cm) for the enzymatic reaction, during which the red-yellow coloured substrate, indophenyl acetate, was hydrolyzed to the blue-purple indophenoxide anion (Figure 1.1A). 13 Figure 1.1 Representative μPAD applications. (A) Bidirectional lateral flow bioactive paper sensor. Adapted with permission from Reference [49]. Copyright 2009 American Chemical Society. (B) Scanning electron microscope photos of native chromatography paper (top) and molecularly imprinted polymer-coated chemiluminescence device. Adapted with permission from Reference [51]. (C) Alkylsilane self-assembling and UV/O3-patterning fabricated nitrite assay based on Griess reaction. Reprinted with permission from reference [54]. Copyright 2013 American Chemical Society. (D) Electrochemical and colorimetric dual sensor for simultaneous determination of lead, cadmium and copper. Reprinted with permission from reference [57]. 14 Photos of the developed colour were taken and analyzed by ImageJ for quantification of the concentration of pesticide, referring to a built standard curve. Regarding the liquid food samples, the pesticide standard solution was mixed into the sample (incubation time not reported). No pretreatment was performed to the milk (2%, pH 7.2) while the pH of the apple juice (pH 3.6) was adjusted to 7–8 with 1 M NaOH. This is because the optimum pH for AChE to hydrolyze indophenyl acetate was 8.0. For solid food samples, the pesticide was sprayed onto the skin of apple and surface of head lettuce for 48 h incubation. The sample liquid was prepared by swabbing the surface of the contaminated food and dissolved in water. Such sampling method is typically used in environmental monitoring on flat, non-porous surfaces (stainless steel) for pesticide residues. In the regulations of analyzing pesticide residues in foods, the entire edible part should be included as the sampling candidate with a minimum requirement of sampling mass to accurately quantify the potential hazards. This study avoided a laborious sample preparation for solid food samples but, at the same time, brought the real food analysis back to the pure solution testing and decreased the value of the method validation using mass spectrometry. Nevertheless, the matrix effect of milk and apple juice was not significant. Besides, an encouraging aspect is that the strips retained >90% of their initial change in signal in a month when stored at 4°C, which is acceptable for a commercial test strip product. In another study, Apilux et al. [52] developed a μPAD to detect pirimicarb, a carbamate pesticide, in lettuce and brown rice with the same principle but a different design. In this study, the AChE was directly dropped onto a punched-out Whatman No. 1 filter paper (6 mm diameter), followed by a blocking step with casein in phosphate-buffered saline (PBS) buffer, a washing step, and air dry. The methanol solution of indoxyl acetate (substrate) was spotted onto a square 15 filter paper (10 × 10 mm). The detection procedure includes two steps as well: 1) applying the liquid sample onto the square enzyme paper for incubation, and 2) transporting the substrate to the enzyme paper by stacking the substrate paper and adding the buffer. Regarding the sample preparation, the authors compared three methods. Immersion with 20 mM PBS buffer or 20 mM PBS buffer containing 20% methanol for 10 min only provided ca. 35% and ca. 65% recovery, respectively. However, a more laborious but efficient QuEChERS method provided excellent recovery (89–110%), although the detailed procedure was not specified. The QuEChERS (i.e., quick, easy, cheap, effective, rugged, and safe) method is a very popular sample preparation method in polar pesticide analysis for various food commodities, and the procedure and recipe can be modified to facilitate better extraction and clean-up according to the compositions. A typical QuEChERS method starts from homogenizing the food sample, followed by the addition of acetonitrile with immediate vigorous shaking. Next, MgSO4 (or Na2SO4) and NaCl are added, mixed and centrifuged, and then the supernatant (acetonitrile layer) will be cleaned up by solid-phase extraction. Lastly, the eluate is evaporated and reconstituted to the desired solution for further detection. In this study, the appropriate sampling followed by QuEChERS sample preparation significantly improved the performance of the μPAD. The overall time for testing each sample was at least 45 min and up to 3 h. Interestingly, to achieve a better LOD (0.2 ppm, part per million), the study reported that the QuEChERS pre-treated sample liquid was applied onto the μPAD for 20 times with 5-min intervals for drying, which accounted for quite a part of the overall testing time. It was not discussed why the liquid sample was not pre-concentrated in a more time-efficient way such as classical evaporation. Recently, Nouanthavong et al. [53] developed a nanoceria-coated μPAD to detect methyl-paraoxon, an organophosphate pesticide, in cabbage and green mussel. The μPAD was built on 16 Whatman No. 4 filter paper, and the 5-mm diameter circle pattern was generated by screen printing of polystyrene (25% w/v in toluene). Clear packing tape was adhered to the back of the device to prevent leaking. In the detection zone, polyethylene glycol adjusted colloidal nanoceria solution was deposited, and the device was ready to use after drying. The detection principle was partially different from the two studies summarized above, although AChE hydrolysis was still the first reaction. In this study, acetylcholine was used as the substrate, and the generated choline reacted with O2 to produce H2O2, which then oxidized nanoceria (colourless/white) to CeO2 (yellow). The detection procedure was reported as adding sample liquid and incubating 15 min, followed by adding mixture (pre-incubated 15 min) of acetylcholine and choline oxidase to develop the colour for 20 min. The sample preparation was similar to QuEChERS, and the pre-treated sample was reconstituted in 4% methanol. The 0.2 ppm methyl-paraoxon spiked into the cabbage and dried green mussel was identified to be 0.19 ± 0.02 ppm (94.8% recovery) and 0.19 ± 0.05 μg·mL−1 (95.1% recovery), respectively. For all the three studies utilizing AChE inhibition, sufficient sample preparation was necessary for solid food samples, and the colorimetric method was able to quantify the target pesticides in a decent range. Besides, Liu et al. [50,51] developed two types of chemiluminescence μPADs separately to detect dichlorvos, an organophosphate pesticide, in food samples. Both μPADs rely on the luminescence-generating oxidation of luminol by H2O2 that can be negatively affected by dichlorvos. Paper chromatography [50] and incorporated molecularly imprinted polymers [51] (Figure 1.1B) on Whatman Grade 3MM CHR chromatography paper were utilized to differentiate and to extract dichlorvos from sample liquid, respectively. However, in spiked sample analysis (tomato, cabbage, etc.), an incorrect surface-spray-and-elute sampling and 17 preparation were used, considering that dichlorvos is a systemic pesticide that can circulate through the plant’s tissues once absorbed. 1.3.3.3 Detecting Inorganic Ions Two studies [54,55] selected nitrite, a food additive possibly abused in meat products or exist in so-claimed nitrite-free products, as the analyte. Griess reaction was selected as the colorimetric principle for their μPADs in both studies. Griess reaction is a two-step diazotization reaction in which the nitrite reacts with sulphanilamide, and then the formed diazonium salt reacted with naphthylethylenediamine dihydrochloride to produce strong pink azo dye. He et al. [54] fabricated the μPAD by coupling hydrophobic octadecyltrichlorosilane to the fibres of Whatman No. 1 filter paper, followed by UV-lithography of the octadecyltrichlorosilane coating (Figure 1.1C). In another study, Cardoso et al. [55] used a pre-heated metal stamp to transfer paraffin wax from a waxed filter paper (JP 40, JProlab, São José dos Pinhais, Brazil) to another stacked native filter paper. Both fabricated μPADs were patterned as an eight-channel-circle piece for multiple tests. Although with different major compositions, both red cubilose [54] and ham/sausage [55] were extracted with hot water due to the nature of nitrite ions with no significant matrix effect. Chaiyo et al. developed two μPADs for detecting Cu(II) [56,57], Pb(II) and Cd(II) [57] in different food commodities. They firstly reported a colorimetric μPAD based upon the catalytic etching of silver nanoplates by thiosulfate in the presence of Cu(II) using wax printing [56]. Later, the authors incorporated the Cu(II)-detecting μPAD to another electrochemical μPAD that could detect both Pb(II) and Cd(II), forming a dual-sensor [57] (Figure 1.1D). Regarding sample preparation, tomato juice was simply centrifuged and filtered, while the rice was carefully treated, involving digestion with concentrated nitric acid and concentrated perchloric acid (1:1, v/v) at 18 150°C for 4 h, evaporation, titration with concentrated H2O2, and membrane filtration [56]. The procedure was slightly modified to detect all three ions together in rice and fish when electrochemistry was involved [57], including temperature change and more fine adjustment of pH (6.0). The LOD for all ions was below 10 ppb in food samples. 1.3.3.4 Detecting Other Organic Compounds Ma et al. [58] achieved the detection of clenbuterol, a veterinary drug, in milk using a paper-based ELISA. The device was fabricated by wax printing or screening patterning (with a customized mesh) on chromatography paper. A competitive ELISA with the classical horseradish peroxidase labelling format was utilized. No pre-treatment was required for milk, and the LOD was 0.2 ppb. Compared to the conventional 96-well ELISA, less antibody-antigen was required, and the overall detection time was reduced to about 1 h. More accurate tests for other foods may require more pre-treatment to cleave the conjugate clenbuterol [63]. In addition, Liu et al. [59] and Guzman et al. [60] from the same research group developed fluorescence μPADs for detecting formaldehyde, an illegal food preserving agent, in dried goods (e.g., ginseng). With distillation (time not reported), formaldehyde in food samples was extracted and concentrated. Through the Hantzsch reaction, formaldehyde reacts with ammonium and acetoacetanilide, forming fluorescent dihydropyridine derivative, which can be quantified by fluorescence detecting system demonstrated in their studies. Monosk et al. [61] applied L-glutamate, a food sensitivity causing agent for some people in western countries (typically as mono-sodium glutamate), detection to instant soup sample using chromatography paper based on the dehydrogenase catalyzed colour change. However, the pH of the sample had to be adjusted, and the interference from L-ascorbic acid could not be avoided. 19 Mani et al. [62] proposed an electrochemiluminescent μPAD to screen potential genotoxic activity in the environment and foods, targeting at unspecified chemicals with genotoxicity. Whatman No. 1 filter paper was patterned by manual screen-waxing with commercially available wax paper. Different reagents (e.g., electrodes, DNA, and enzymes) were deposited in the desired structure onto two pieces of filter paper, which were then bound together using double-sided tape. In brief, when sample liquid containing genotoxic compounds applied to the μPAD, the DNA is damaged, catalyzed by cytochrome P450s (oxidative metabolic enzymes). The guanines in the damaged DNA are more accessible than intact guanine to be oxidized by RuIIIPVP (electrically converted from RuIIPVP) to generate excited state *RuIIPVP which in turn decays to generate electrochemiluminescence at 610 nm. In this study, an over-grilled chicken was extracted with dimethyl sulfoxide and tested by using this μPAD. However, a very large variation in the result was observed, possibly due to the interferers extracted from the food matrix. 1.3.4 Issues and Concerns in Real-world Applications 1.3.4.1 Challenges in Sample Preparation It is not difficult to notice that the current studies have focused on pesticides, inorganic ions and simple organic compounds, which are small molecules without overly complex or subtle structures (Table 1.2). No macromolecules have been involved, such as proteins in the only two categories (i.e., food sensitivity and biochemistry-related). Precise and efficient separation of proteins from the food matrix is still technically challenging, especially in the case of solid food and/or cooked food. Researchers may select compounds that can be easily extracted from foods as the start or as the model analyte in fabrication-emphasizing studies. On the contrary, many small molecules are homogeneously distributed in the food sample and only 20 weakly interact with the food matrix. Therefore, solvent extraction should provide a decent recovery. Depending upon the detection principle, the analyte may not have to be present in the free form completely (e.g., when the sensitivity of the μPAD was very satisfying [58] with repeatable recovery for calibration), which may reduce the technical challenges in sample preparation or be totally exempted. 1.3.4.2 Rapidness Due to the technical challenges in sample preparation, the laborious and tedious work prior to detection reveals the time issue. It is acceptable to describe the detection of a pesticide in the environment water sample within 10 min as a rapid test. However, applying exactly the same μPAD to a food sample, it is no more a rapid test if a two-hour sample preparation time is required. For example, in the manuals of Rapid Test Kit Evaluation Program for detecting mycotoxins in grains, the length of time defined for a “rapid test” is 30 min, including sample preparation [64]. To address the time issue, μPADs with a higher interfering tolerance may simplify the prerequisite sample pre-treatment greatly. For instance, the clean-up step in the QuEChERS method may not be strictly required for some food commodities (e.g., milk). In this case, the analytes end up in acetonitrile (or other organic phases) containing a very small amount of water, and the co-extracted interferers vary in their composition. This leads to the next two issues regarding solvent: hydrophobic barrier and the paper substrate. 1.3.4.3 Aqueous Solution Limited One of the two issues is the compatibility of the sample preparation method with the μPAD, particularly the compatibility of ending liquid with the hydrophobic barrier and the paper substrate. In the aforementioned case, the ending solvent cannot be switched from acetonitrile/methanol to water or a mixture of water/alcohol due to the intrinsic reasons (e.g., free 21 pesticide may re-conjugate to co-extract matrix when the solvent is changed from acidified acetonitrile to a water/ethanol 1:1 blend) or simply because of the concern of time and labor cost. However, some fabrication techniques may not support those organic solvents perfectly, as methanol breaking through the wax-printed barrier has been observed. Dipping and efficient stamping techniques can hold methanol better as the paper substrate is more saturated with pure paraffin wax and PDMS. In most studies, the fundamentals and applications were studied using an aqueous phase [29,65], such as coloured water and PBS buffer. The wicking of organic solvents in different conditions (e.g., significantly higher evaporating effect, different solutes) may have to be further studied to promote more applications in food analysis. Special attention should be paid to acetonitrile and methanol, especially acetonitrile, since these two solvents could be the most frequently used ones in the extraction step. 1.3.4.4 Limited Understanding of Interactions between Paper-Analyte/Interference The other one of the two issues is related to the limited understanding of the interaction among all participants within a wicking system of the μPAD. For those designs involving liquid wicking within the paper substrate, the interactions between analyte/interferer and paper/barrier are observed, predicted, but not well understood. A direct question regarding the interaction between the analyte and the paper is how a large percent of the analyte can finally reach the designed sensing zone. Ota et al. [66] quantitatively evaluated the transportation of the analyte in μPAD (Advantec No. 5C filter paper patterned by wax-printing) using Ni2+ as a model metal cation and bovine serum albumin (BSA) as a model protein, which provided insightful conclusions for the design of a μPAD. Different regions of a protein molecule interact with the cellulose fibre (mainly the hydrophilic hydroxyl groups) and other components (e.g., lignin) to different extents, and the interaction may change with the composition of the solvent system and 22 the resulted change in protein folding. Moreover, the working reagent conjugated to the analyte could also be retarded by the hydrophobic barrier at the interface and thus cause the variation in the transportation of the analyte [67]. Regarding the interaction between the interferers in the sample fluid and paper substrate, the original function of the chromatography paper cannot be ignored. Such separation ability based upon polarity, along with the particle trapping ability of the space among randomly oriented fibres, can be helpful when dealing with extracted but non-cleaned samples. Particularly, all these interactions within the system of acetonitrile (with up to 10% water) and methanol (containing 0–50% water) should be further investigated as they are quite common in food sample preparation. 1.3.4.5 Market Orientation Although there seems to be a long way to go from the design of a μPAD prototype to the large-scale manufacturing of a final commercial product, it is necessary to keep the target market niche in mind from the very beginning of a study. One should consider not only the nature of the target chemical hazard and possible food matrix but also the specific needs of the end-users, so as to clarify the plan for the research emphasis. For an ideal design, the “ASSURED” criteria (i.e., Affordable, Sensitive, Specific, User-friendly, Rapid and robust, Equipment-free, Deliver to the users who need them) of point-of-care diagnostic devices with resource-limited settings could be introduced and compared [68]. Agri-food business generally has low unit value and profit, while strict regulations have to be met as the food quality and safety is directly related to consumer health. The most important benefit that a μPAD can bring to the food industry is the affordability. Essentially, the overall cost per test is what a business cares about most, and what covers most of the criteria aforementioned. Different from the estimated cost per μPAD mentioned in the publications, 23 many other factors affect the calculation to a great extent. To start with, the cost of the required equipment per test is a concern, related to the initial investment, maintenance, and the actual total test can be done on the equipment (i.e., the frequency of use, the designed time in warranty, and the time before a required update). Thus, “equipment-free” for a food plant is perhaps more a matter of cost rather than of convenience. Then, the labour cost per test, involving the overall turnaround per test and the operation capacity per person, is another factor to include. Considering the huge number of fresh produce and food products, especially imported goods that have a higher risk of chemical contamination [69], using μPAD as a fast screening method in a high-throughput manner is preferred, even though the sensitivity, accuracy and precision have to be compromised to some extent. In turn, the false positive/negative rate largely decides the extra cost of validation by typically sending out samples with questionable results to external analytical services at high prices. In the worst scenario, the failure of the μPAD may result in a costly recall and huge loss of brand value. Besides, some indirect costs could have a high impact on whether a method and associated testing products should be adopted, such as whether all data can be kept internal. The hidden risk borne in the incomplete control of the information channel, such as cloud server-based data processing or third-party certification, could bring an extra cost to a business. In brief, these real-world considerations in the food industry may question the “low-cost” tag of μPADs. “Equipment-free” and “user-friendly” become increasingly important for the applications targeting at the consumer’s level. For example, a home-use food allergen testing μPAD will undoubtedly find its market niche if it requires no equipment (at most a smartphone), very simple or no sample preparation, and an easy-to-interpret indicator. The potential risk of severe consequences due to food allergy may diminish customers’ concerns on the cost of test. For governmental inspectors with duties of on-site testing at farms, 24 slaughterhouses and food plants may prefer portable readers with recording functions to obtain relatively accurate results and to reduce sampler’s bias. Therefore, the corresponding μPAD product does not have to be equipment-free, even though the test is just qualitative or semi-quantitative and can be interpreted by the naked eyes. If the application targets the completely banned chemicals (e.g., clenbuterol), one may not have to make too much effort on the linearity of working range of the μPAD but need to emphasize the specificity and LOD. In summary, considering the real-world scenario of a specific application of μPAD in the detection of chemical hazards in food commodities from the very start of a μPAD project could be very helpful, because it may clarify the research emphasis and reduce the developing time period for researchers of both chemical engineering and food science. 1.4 Research Goals, Rationale, and Challenges The overall goal of the thesis project is to develop an LFI-format μPAD (fully paper-based) to achieve the detection of a large-molecule chemical hazard in food. As aforementioned in Section 1.2, conventional lateral flow immunoassay has achieved great success in the market of point-of-care diagnostics and also has become one of the favourite types of tools to detect chemical hazards for food safety. The user-friendly design might be the most important reason for the popularity, which will be adopted in this project. However, conventional LFI (Figure 1.2) requires relatively complicated fabrication procedures. Each part of the LFI is made of a different material along with corresponding pre-treatments and modifications. For example, the conjugate pad, typically made of glass fibre, needs blocking and pre-coating before the loading of the conjugate by either soaking–rolling–drying or spraying–drying. The prepared parts, including sample pad, conjugate pad, nitrocellulose membrane, 25 absorbent pad, supporting card, and sometimes a cassette/chamber, are then assembled to be packed. Usually more variables and uncertainties are involved when there are more parts within a system. For LFI, the deformation around the interface of different parts is a typical issue found in manufacturing troubleshooting. The most troublesome part or material is the nitrocellulose membrane where the test line (T line) and the control line (C line) locate. Nitrocellulose is very fragile and needs extraordinary caution to handle. It is very easy to have defects on this most critical and most expensive part of the entire device. Figure 1.2 Scheme of a conventional lateral flow immunoassay (LFI) On the other hand, the mechanical and chemical properties of paper (cellulose) provide much more potential beyond wicking. The decent dry and wet strengths of paper greatly reduce the design and fabrication complexity and thus the cost. At the same time, cellulose (e.g., Whatman Grade 1, $0.05 per 100 cm2) is much cheaper than nitrocellulose membrane (e.g., Millipore HF135, $3.25 per100 cm2). Besides, paper is relatively eco-friendly as it takes 3-6 months to decompose in the natural environment. However, the plastics used in LFI need tens to hundreds of years to degrade and then cause another round of microplastic contamination, a hot research topic currently in both food safety and environmental health. 26 To build a fully paper-based lateral flow immunoassay, the key and the most challenging part are to enable the desired flow of sample liquid and the transportation of analyte and reagents by only changing the patterns and modification of the surface of cellulose on a single piece of paper. This single piece of paper needs to function as the sample pad for filtering out the interfering substances, as the conjugate pad to store the labelled antibody, the nitrocellulose membrane to immobilize the capturing antibodies and enable the specific interaction, the absorbent pad to drive the liquid forward, and lastly the backing card to physically support the entire device during the test (Figure 1.2). Figure 1.3 Scheme of a tentative LFI-format μPAD that integrates all functions in one piece of paper. To the best of our knowledge, there has been only one group who reported the attempt to fabricate an LFI-format μPAD [67]. However, the authors were not able to achieve a repeatable release of the conjugate nor to eliminate the background due to non-specific binding. Besides, only the mixture of pure analyte solution was tested as a proof-of-concept for their inkjet-printing technique. Therefore, in this project, I aim to design and fabricate a fully functional μPAD with controlled reagent transportation, a negligible background signal, and the ability to detect chemical hazards in real food samples. 27 Food allergies are commonly observed, estimated as about 10% of the population [70,71], and can cause moderate, severe, or even fatal symptoms. Since there is no therapy for food allergies, the most effective way to reduce the risk is to avoid exposure to food allergens. The need for user-friendly allergen testing tools has promoted both academic studies [72] and commercial products [73] for home use based on LFI. Further, undeclared allergen leads to the most number of food recall incidents by all hazards in Canada [74] and the US [75]. Becoming more and more important, the allergic hazards are listed as an individual category out of chemical hazards sometimes, although the eleven priority allergens in Canada include a non-protein, namely sulphite [76]. In the eleven priority allergens, hen’s egg is one of the most widely used ingredients in food and one of the most common allergens in children [77]. As mentioned in Section 1.3.4.4, previous studies on μPADs have not reported any application to the detection of large molecules as chemical hazards in real food. Therefore, ovalbumin (~ 45kDa) is selected as the target analyte in this project since it is the major protein (54-58%, w/w) in the egg white. 28 Chapter 2: Methods and Materials 2.1 Overall Experimental Design The experiment can be approximately summarized in three phases, while the experiment in later phases may require further optimization from the earlier phase(s). The first phase focuses on determining the suitable patterning technique and detailed procedure to establish the paper platform. The second phase involves extensive trials to determine the recipe and procedure to achieve the desired transport of conjugate. The last stage finalizes the patterns and modification procedures to achieve the detection, followed by the optimization for testing real food samples. 2.2 Filter paper Whatman Grade 1 filter paper sheets (GE Healthcare, US) and Whatman Grade 42 filter paper sheets (GE Healthcare, US) were tested for “manufacturing direction” by pipetting the blue food dye (Club House, US) diluted 50 times with deionized water. All experiments were carried out at room temperature (20-24°C) unless otherwise stated. Replicates were tested under similar relative humidity (i.e. difference within 20%). 2.3 Wax-stamping 2.3.1 Fabrication of Stamps Stamps of three different materials were prepared for the preliminary tests, including a low carbon steel stamp faced with a milling machine, an acrylonitrile butadiene styrene (ABS) one printed by 3D printing, and a poly(methyl methacrylate) (PMMA) one by laser-cutting. The design was drawn using AutoCAD 2018 (Autodesk, Inc., USA), and the processing was operated with the assistance of external collaboration or commercial services. The first batch of stamps 29 was subject to heating test by soaking in paraffin wax (m.p. ≥ 65°C, Sigma-Aldrich; m.p. 53-57°C, Sigma-Aldrich) contained in a glass petri dish on a hot plate (C-MAG HS 10, IKA, Germany) at 80-150°C for 10 min. Note that the heating temperatures reported in wax-stamping refers to the reading on the hotplate. PMMA was used for further tests. 2.3.2 Optimization of Patterning Several parameters were compared using Grade 42 filter paper, including paraffin wax melting points (m.p. 53-57°C vs. m.p. ≥ 65°C), depth of melted wax (3, 5, and 7 mm), heating temperature (80, 110, 130, and 150°C), heating time (1, 2, and 3 min), stamp shape (round curve vs. right angle). Note that the heating temperature refers to the reading on the hotplate. 2.4 Wax-Printing 2.4.1 Optimization of Printing Settings The Grade 1 and Grade 42 filter paper sheets were separately cut into 150×150 mm and 140×140 mm pieces. The ColorQube 8580DN was set as the black colour mode, 600 dpi, and the highest printing quality to print the designs drawn in AutoCAD 2018. Lines of 0.10-1.00 mm thickness were printed at different locations with both directions on each side of the paper. The printed paper was scanned (1200 dpi, HP 3050A Printer, US) for quality examination. 2.4.2 Optimization of Heating Settings Lines of different angles (i.e., 0°, 30°, 45°, 60°, and 90°) to the paper-feeding direction in the form of (tilted) squares were printed along with circles in different line thicknesses (i.e., 0.10 - 0.65 mm). The printed paper (Grade 1, Grade 42) was heated on the top rack in a 66-L forced air oven (VWR, US) at different temperatures (i.e., 120, 130, 140, and 150°C) for various 30 periods of time (3.0, 3.5, 4.0, 4.5, and 5.0 min). After air cooling to room temperature, the food dye was then pipetted into the area within the shape to examine the integrity of the wax barrier. 2.5 Preparation of Immunological Reagents 2.5.1 Synthesis and Characterization of Gold Nanoparticles The modified citrated reduction method was adopted from a previous publication [78]. In an aqua regia-washed 500-mL round bottom flask, 2 mL of 1% (w/w) HAuCl4 (99.999%, Sigma-Aldrich, US) was added into 200 mL of ultrapure water (18.2 MΩ.cm-1 at 25°C) (Direct-Q, Milli-Q, US). With magnetic stirring, ca. 150 µL of 1 M NaOH (Sigma-Aldrich, US) was added to reach a final pH of ~4. The solution was heated to 100-105°C via an oil bath (Corning crystallizing dish on Corning PC420 Stirring Hot Plate, US) with stirring. Then, 1 mL of 500 mM sodium citrate (Sigma-Aldrich, US) was quickly added and mixed, and the reaction was kept at 100-105°C for 2 h. The resulted colloid was then naturally cooled down to room temperature and stored in the dark. The absorbance spectrum (300-700 nm) was collected by using a UV spectrophotometer (UV-1800, Shimadzu, Japan) to determine the quality and size of the synthesized gold nanoparticles. 2.5.2 Optimization of Antibody-Gold Nanoparticle Conjugation Anti-ovalbumin antibodies were purchased from Abcam (US), namely mouse monoclonal ab17291 (M291), mouse monoclonal ab17292 (M292), mouse monoclonal ab17293 (M293), and rabbit polyclonal ab181688 (P). NaCl-induced aggregation test was used to determine the minimum amount of antibody required. Briefly, the pH of AuNPs was first adjusted to 7.5 for polyclonal Ab or 8.2 for monoclonal Ab with 0.2 M K2CO3 (Fisher Sci., US), determined by precise pH-indicator strips (pH range = 6.5-10.0, resolution = 0.2-0.5) (Millipore, 31 US). Then, 25 μL of AuNPs and 25 μL of antibody (containing 0-20 μg per 1 mL AuNPs) were mixed and incubated at room temperature for 15 min, followed by the addition of 50 μL of 10% NaCl (Fisher Sci, US). The tube containing the lowest amount of antibody with no colour change indicated the minimum ratio of antibody to AuNPs. The typical conjugation was conducted as follows. In a clean 13-mL glass bottle, 32 μL of 0.2 M K2CO3 was added into 4 mL of AuNPs with magnetic stirring. Then, 20 μL of 1 μg/μL antibody was added very slowly, followed by stirring at room temperature for 1 h. Next, 400 μL of 5% BSA and 200 μL of 1% polyethylene glycol (PEG, Mw~20k) (Sigma-Aldrich, US) were added gradually, followed by stirring for another 30 min. The resulted mixture was centrifuged (Eppendorf 5424 R, Germany) at 11,000 ×g at 4°C for 30 min. The supernatant was discarded, and the precipitate was resuspended to 100 μL with the storage solution containing 1% BSA, 5% sucrose (Fisher, US), 2% trehalose (Fisher, US), 0.1% Tween-20, and 0.1% NaN3. Ab-AuNPs conjugate was stored at 4°C upon use. Note that the amount of Ab was reduced to 70% of minimum ratio (see discussion). 2.5.3 Screening of Antibody Pairs on Nitrocellulose On a sheet of nitrocellulose membrane (HF135, Millipore, US), 0.5 µL of each antibody (i.e., M291, M292, M293, and Poly; 1 μg/μL) was spotted 8 times in a row. The membrane was dried in a gravity convection oven (GCA/Precision 18EG, US) at 37°C for 15 min. The membrane was then blocked with 1% BSA (Goldbio, US) at room temperature for 10 min, followed by a quick rinse with PBST, namely 0.1% Tween 20 (Fisher, US) in PBS (Fisher, US). The excess amount of PBST was removed by gently absorbing with Kimwipes (Kimtech, US), and the membrane was dried in the oven at 37°C for 30 min. The dried membrane was then 32 attached to an absorbent pad (H5072, Jieyi Bio, China) via a backing card (DB-7, Jieyi Bio, China), and then cut into strips as appropriate. In a 96-well plate (Costar TC-treated, Corning, US), 100 µL of PBS or 100 ppm ovalbumin (Sigma-Aldrich, US) in PBS were mixed with 1 μL of each conjugate (i.e., Au-M291, Au-M292, Au-M293, and Au-Poly). The fabricated simplified strips were placed in the wells for an orthogonal pairing to seek the pair with the darkest red spot. 2.6 Surface Modification of Paper Substrate for Lateral Flow Recipes 1-18 were tested as the first batch, and the promising candidates (i.e., 12 and 15) were then tested together with Recipes 19-32 (Table 2.1) as follows. The paper strip was incubated in the solution of each recipe for 20 min, followed by removing excess liquid with Kimwipe. The paper was then oven-dried at 37°C for 30 min, followed by spotting 0.5 μL of conjugate and oven-drying at 37°C for another 15 min. In the lateral flow test, each strip was placed in a well containing 200 μL of PBS and ran for 10 min. 2.7 Optimization of Patterns and Modification Recipes 2.7.1 Designs of Test and Control Lines Two designs suitable for wax-printing were tested, namely bridging and cutting. These two ways of handling T/C-lines were used throughout the experiment, and the details were discussed in Section 3.6. 2.7.2 Optimization of Pattern Geometry for Flow Improving To increase the effective volume of sample liquid passing the T line, a series of patterns were tested, focusing on the effect of pre- and post-T line geometry to tune the flow rate (Figure A1). Details are discussed in Section 3.6. 33 Table 2.1 Recipes of surface modification solutions # BSA PVA PVP PEG Tween 20 Triton X-100 Buffer 1 0.5% 2 1% 3 3% 4 5% 5 0.5% 6 1% 7 3% 8 5% 9 3% 1% 10 3% 1% NaPB,10 mM, pH 7.5 11 3% 1% PBS 12 3% 0.1% PBS 13 0.1% 1% 14 1% 1% 15 3% 0.1% 16 1% PBS 17 1% PBS 18 1% 19 3% 0.1% KPB (10 mM, pH 7.5) 20 3% 0.1% KPB (30 mM, pH 7.5) 21 3% 0.1% KPB (100 mM, pH 7.5) 22 3% 0.1% Tris-HCl (10 mM, pH 7.5) 23 5% 0.1% 24 2% 0.1% PBS 25 3% 0.5% 0.1% 26 3% 0.5% 0.1% 27 3% 0.1% 28 3% 0.5% 29 3% 0.5% 30 3% 0.5% 31 0.5% 0.1% 32 0.5% 0.1% Note: PVA = polyvinyl alcohol (Mw 30-70kDa, Sigma, US); PVP = polyvinylpyrrolidone (Mw ~55kDa, Sigma, US); PEG = polyethylene glycol (Mw 20kDa, Sigma, US); NaPB/KPB = sodium/potassium phosphate buffer. 34 2.7.3 Surface Modification of Paper Substrate with Recipe 33-42 Recipe 33-42 (Table 2.2) were used in the revised pattern for improving the sandwich formation on the T line. Table 2.2 Recipes of the revised surface modification solutions # BSA Casein Gelatin PVA Tween 20 Triton X-100 Buffer Remark 33 5% 0.5% PBS 34 5% 0.1% PBS Dipping 35 5% 0.1% PBS 36 5% 0.5% PBS 37 5% 0.5% PBS Dipping 38 3% PBS 39 3% 1% PBS Dipping 40 3% 0.5% 1% PBS 41 2.7% 1% PBS PBST rinsing 42 3% 0.1% PBS 40°C incubation 2.8 Optimization of Antibody Deposition The original concentration of M293 was used for the T line. The rabbit anti-mouse antibody (i.e., secondary antibody) for the C line was diluted (i.e., 0.8, 0.6, 0.4, and 0.2 μg/μL; applied 0.3 μL) to reach a similar colour intensity to the maximum of that for the T line. The concentration and sugar content of the conjugate was adjusted to reduce the reagent cost and improved the performance. 2.9 Detection of Ovalbumin 2.9.1 Food Sample Preparation Cake mix (French Vanilla Cake Mix, Duncan Hines Canada) was used as the main egg-free food model. Dried egg white (Sigma-Aldrich, US) was used to spike the cake mix powder. In a 50-mL centrifuge tube (Corning, US), 20 g cake mix was spiked with 0, 4, 40, 400, and 800 35 mg egg white powder (equivalent to 0%, 0.01%, 0.1%, 1%, and 2% ovalbumin) and mixed thoroughly. Note that the recommended recipe by the manufacturer contains 3 large eggs for 432 g cake mix, which is approximately equivalent to 18 g egg proteins or 10 g (2.1%) ovalbumin. Sample pre-treatment was developed as follows. In a 50-mL centrifuge tube, 1 g of sample was mixed with 19 mL (9 mL for 0.01% spike) of extraction solution containing 1% sodium dodecyl sulphate (SDS) (Fisher, US) and 7% 2-mercaptoethanol (2-ME) (Alfa Aesar, US) in PBS [79]. The tube was shaken for 30 s and heated at 90°C for 10 min, followed by diluting with PBS for 5 times (0.01% spiked), 10 times (0.1% spiked), or 20 times (1% and 2% spiked). The volume of extraction solution was 9 mL, followed by a 10-time dilution for all other commercial products, including muffin mix (Perfect Size for 1 Blueberry Muffin Mix, Duncan Hines Canada), baked muffin (65 g mix + 45 mL water, microwave 110 s, according to instructions), mayonnaise (Hellmann's, US), and skim milk powder (Sigma-Aldrich, US). 2.9.2 Detection The fabricated μPAD was vertically placed in the 96-well plate containing 200 μL of ovalbumin standard solution (i.e., 0, 1, 5, 20, 50, and 100 ppm in PBS) or food sample extracts for 5 min. All tests were at least conducted in triplicate. The detection could also be done by allowing the triple-folded μPAD to stand freely on more than 50 μL of liquid upon test. 36 Chapter 3: Results and Discussion 3.1 Filter Paper Substrate Whatman Grade 1 filter paper is the type that has been most widely used (more than 50% of studies) [80], possibly due to the relatively low cost, compared with the ashless series, as well as decent physical properties. Whatman Grade 42 is ashless quantitative filter paper with a higher thickness and a smaller effective pore size. Grade 42 is about 3-6 times the price of Grade 1, but it is still much cheaper than nitrocellulose membrane which is over 60 times more expensive. Grade 42 was selected since the equivalent model, Advantec No. 5C (Toyo Roshi, Japan), has been well studied for analyte transportation [66] and adopted in the only study [67] as mentioned in Section 1.4. The rectangular sheets were used instead of typical circles to reduce the variation in flow rate due to the orientation of cellulose fibre [66]. The direction with a higher flow rate was defined as the manufacturing direction, along which more fibres are oriented possibly due to the stretching and rolling process during the manufacture (Figure A2). For consistency, all flow rate tests were along the manufacturing direction unless otherwise stated. 3.2 Wax-stamping 3.2.1 Materials of Stamps The melting point range of paraffin wax was only around 50-70°C which is lower than the glass transition temperature of the ABS (~105°C). In practice, maintaining the liquid phase of paraffin for stamping requires over 100°C at which the 3D-printed ABS was partially deformed. The low carbon steel stamp had no problem to bear with a high temperature. However, the lateral spreading of melted paraffin wax on paper was too high compared with the rate of penetration, 37 possibly due to the higher thermal conductivity and lower specific heat of steel compared with PMMA (i.e., 54 vs. 0.2 W/m·K, 0.45 vs. 1.47 kJ/kg·K [81-83]) and filter paper. The lateral spreading was not quickly terminated as the heating source (i.e., the metal stamp) was too close, which resulted in a very thick barrier (e.g., the 0.8-mm line stamped out 2.5 mm barrier at 130°C). In addition, machining the steel stamps of a 10-15 mm depth was expensive and difficult to revise the shape manually, compared with that for PMMA. Due to these limitations, the metal and ABS stamps were not used in the following experiments. The PMMA stamps were rather easy to fabricate by digital laser-cutting and had no problem with being kept at 150°C for at least 15 min. However, rigid and properly formed lines below 0.8 mm for the pattern on stamps with a 12-mm depth may not be easily repeated (Figure A3A). The protecting cover was removed, and a pair of handles were attached using PMMA glue (Figure A3B). However, the handles were detached at 150°C due to the possible degradation of the glue. Thinner lines of the PMMA stamp were able to hold a decent amount of melted wax, but the wax solidified quickly without enough further heat from the PMMA (Figure A3C). 3.2.2 Patterning Settings For the parameters in wax-stamping, the melting points of paraffin wax were first examined. The paraffin wax with a higher melting point range (i.e., m.p. ≥ 65°C) may stand a higher heating temperature without generating unpleasant smoke. However, the one with a lower melting point range (m.p. 53-57°C) solidified more slowly under the same heating setting, resulting in better penetration through the filter paper. Therefore, the product with melting point of 53-57°C was used for further tests. The depth of the melted-wax pool in the glass petri dish decided the amount of melted paraffin wax attached onto the sidewall of the stamp. This parameter was of less concern, and a roughly 5(±0.5) mm should work well. The heating time 38 and heating temperature largely affected the exact amount of attached liquid wax being transferred onto the paper as well as the penetration and lateral spreading rate during which the paraffin wax transform to its solid phase. Heating at 80°C was not sufficient, as aforementioned in Section 3.2.1. A combination of 110°C of at least 3 min would barely work for lines thicker than 1.4 mm. Heating at 130°C for 3 min yielded the desired patterning. However, 150°C would over-heat the paraffin wax that the unpleasant smoke could be smelt. As a result, for Grade 42 filter paper, the preferred patterning condition was determined as using a PMMA stamp to heat in a 5-mm depth of paraffin wax (m.p. 53-57°C) at 130°C for 3 min. Figure 3.1 shows the three typical patterning results. Although wax-stamping technique explored in this thesis project was successfully carried beyond the second phase of the project (Figure 3.2, an example of wax-stamped LFI-format μPAD), further experiments were continued without wax-stamping due to its limitations. The main reason was that the manual operation was low efficient in stamping compared with a possible stamping machine, which would provide consistent force, pace and angle to contact the paper substrate. The paraffin wax completely filled into space within the paper matrix, and the barrier was able to resist to many organic solvents (e.g., methanol), but the solidified barrier is subject to cracks upon bending and non-specific rendering of the conjugate. 39 Figure 3.1 Patterns generated using a PMMA stamp (A), with examples: (B) under-heating (110°C, 2 min), (C) preferred heating (130°C, 3 min) for a channel width of 2 mm, and (D) over-heating (150°C for 3 min). Figure 3.2 Example of a PMMA stamp (left) patterned LFI-format μPAD design (middle) for subsequent flow test (right). 40 3.3 Wax-printing Compared with wax-stamping, the wax-printing technique was endowed with much better flexibility to design and revise the patterns. Although the interface of hydrophobic barrier and hydrophilic cellulose was less smooth than in wax-stamping due to the secondary wicking of melted solid ink along the randomly oriented cellulose fibres, the non-specific binding within 20 min was negligible. 3.3.1 Printing Settings The result of the printing quality check was unexpected to some extent. The magnified 1200 dpi scan showed the inconsistency of wax-printing at different locations of the sheet (Figure 3.3). The variations in the printed ink could be observed by comparing the top two blue-boxed ends of the horizontal line (0.1 mm) in the red box (Figure 3.3). The magnified image of the left end of the line revealed a more split distribution of dots, while the right end aggregated more tightly. In comparison, the dot distribution for the vertical line (0.1 mm) was more even and consistent, as revealed in the magnified image of its two ends on the right of Figure 3.3. Also, the printed wax of vertical lines was found with smaller variations in general (image not shown). Without a confirmation whether this issue was only for the printer used in this thesis project or an issue commonly found in all other wax-printers, the orientation of feeding the paper sheet was fixed, and appropriate locations on the paper were carefully picked to minimize the potential variations in the following experiments. In addition, a difference in printing quality was also observed between the same location of the same lines printed on the two sides of the paper (Figure 3.4). The smoothness and geometry on the two sides were clearly different, which could be caused by the manufacturing procedures, such as rolling and drying. Therefore, the same side of the paper was used for printing throughout the experiment. 41 Figure 3.3 Scan (1200 dpi) of wax-printed Grade 42 filter paper, printed with lines of 0.10, 0.15, 0.20, …, 0.95, and 1.00 mm in width. The magnified images of both ends of 0.1 mm lines printed horizontally and vertically were magnified and highlighted in the red and further in the blue boxes to demonstrate the inconsistency in printing quality. 42 Figure 3.4 Photos of both sides of Grade 42 filter paper and the magnified scans of 0.1 mm lines printed on both sides to demonstrate the difference in printing quality related to the surface geometry. 3.3.2 Heating Settings The barrier test was conducted to determine whether the orientation or the shape of the printed pattern would affect the functionality of the formed barrier, as well as the requirement for corresponding heating conditions. Both Grade 1 and Grade 42 filter papers were evaluated under each heating setting, namely the combination of time (3-5 min) and temperature (110-150°C). The criteria for evaluating the functionality of the barrier were demonstrated in Figure 3.5. The “fail” was defined as the food dye completely penetrated the barrier and leaked out. The “half-fail” was defined as the food dye penetrated to about half on more than one direction or penetrated to more than half of the thickness of the barrier. Otherwise, the test was defined as “pass”. Only if a barrier received “pass” in all replicates, the corresponding thickness thereof was recorded. The backside of heated paper was referred to for determining the minimum thickness of the line before heating (Table 3.1). The variations related to printing quality, heating operation, and randomness of cellulose fibres were all taken into consideration. 43 Figure 3.5 An example of the barrier test, including the scan of the front side (left) and back side (right) of the patterned paper. The criteria for evaluating the functionality of the formed barrier (middle) were based on the back side. A few trends were observed and were useful for further design of patterns (Table 3.1). First, as expected, the higher the temperature and the longer the heating time, the printed wax penetrated the paper substrate better, resulting in a thinner barrier to be functional. Second, the regular squares, representing the lines to the paper feeding direction at 0° and 90°, generally required a thicker barrier to hold the liquid than all the other shapes did. This could relate to the observation that the print quality is related to the feeding direction, as aforementioned in Section 3.3.1. Third, more intense heating and thicker printed lines were required to form functional barriers for Grade 42 filter paper due to its higher thickness and smaller effective pore size, compared with Grade 1 filter paper. 44 Table 3.1 Minimum thickness (unit: mm) of printed shapes (before heating) required to pass the barrier test for Grade 42 and Grade 1 filter paper under various heating conditions, namely the combination of heating time (3-5 min) and temperature (120-150°C). A darker background indicates a thinner barrier required. 45 3.4 Immunological reagents 3.4.1 Gold Nanoparticles The synthesized gold nanoparticles (AuNPs) were clear with no aggregation observed within the following 10 months stored in dark at room temperature (Figure A4). The sharp surface plasmon resonance peak (523.7 nm) in the UV-Vis spectrum indicated a low polydispersity and narrow distribution of the particle size (Figure 3.6). The average particle size was estimated as ca. 31 nm [84]. Figure 3.6 UV-Vis spectrum of the synthesized gold nanoparticles (surface plasma resonance peak at 523.7 nm). 3.4.2 Ab-AuNPs Conjugate The pH of AuNPs should be adjusted prior to the conjugation to the antibody. Since pH meter is not suitable for measuring the pH of AuNPs due to the potential probe-clogging, non-46 bleeding precise pH-indicator strips were used to determine the pH change of AuNPs with the addition of 0.2 M K2CO3 (Table 3.2). Table 3.2 pH change of 1 mL AuNPs in response to the addition of 0.2 M K2CO3. μL of 0.2 M K2CO3 0 8 9 10 11 12 13 14 15 pH of 1 mL AuNPs <<6.5 7.4-7.7 7.7-7.9 7.9-9.1 8.1-8.3 8.3-8.5 8.5-8.7 ~8.7 ~9.0 The minimum amount for M291, M292, M293, and Poly were determined as 9, 10, 8, and 8 μg Ab per 1 mL AuNPs using the NaCl-induced aggregation test (Figure A5). However, the final ratio used was 5-6 μg Ab / 1 mL AuNPs (in experiment after antibody pair screening) for a few reasons. First, although less frequently occurred in monoclonal antibody products, batch to batch variation was observed for all of M291, M292, and M293 in terms of protein concentration and binding activity (data not shown). Second, the BSA and PEG blocking during the conjugation stabilized the conjugate, which addressed the issue of ion-induced aggregation [85]. Besides, the sub-minimum amount of antibodies potentially improved the antibody alignment and specific binding [85]. Lastly, the cost of antibodies (and overall device) would be reduced. 3.4.3 Screening of Antibody Pairs on Nitrocellulose A total of 16 pairs were tested using the four available antibodies (i.e., M291, M292, M293, and Poly) and their conjugates to AuNPs (i.e., Au-M291, Au-M292, Au-M293, and Au-Poly). The strongest signals were generated between Au-M291 x M293 and Au-M293 x M293 (Figure 3.7). Au-M291 x M293 was selected for further experiments due to its lower background signal. 47 Figure 3.7 Result of antibody pair screening. Au-M291 x M293 was selected due to high signal intensity (darker dot) and lower background. 3.5 Surface Modification of Paper Substrate The first batch of the flow test revealed two promising candidates, namely #12 and #15 (Figure 3.8). The LFI recipe (#ϴ) was the blocking buffer applied to the nitrocellulose membrane (Section 2.5.3), and the non-treated paper (#0) was for comparison (Figure 3.8). Other recipes were excluded for further test because of unsuccessful or incomplete release of the conjugate (e.g., #3 and #4), strong nonspecific binding during wicking (e.g., #1 and 5), lower flow rate (e.g., #10), wicking out onto the wax region (e.g., #14, marked by the red curve), or other reasons. The second batch revealed the best candidate so far, namely #27 (Figure 3.9). Recipe 27 resulted in a flow rate comparable to #15, a background slightly lower than #15, and more stable transportation of the conjugate than that of #24 (i.e., highest flow rate but colour partially changed from red to blue due to AuNPs aggregation). Although there was still a little overflow to 48 the hydrophobic region with #27, the conjugate zone was above the liquid frontline without being affected. Figure 3.8 Flow test of Recipe 1-18, LFI recipe (ϴ), and No-treated (0). Recipe 12 and 15 were selected for the next batch of flow tests due to their low background and relatively high flow rate. Figure 3.9 Flow test of Recipe 12, 15, and 19-32. Recipe 27 was selected for further tests due to the efficient release of the conjugate, low background, and relatively high flow rate. 49 3.6 Optimization of Patterns and Modification Recipes 3.6.1 Designs of Test and Control Lines Two designs for the T and C lines were tested using #27 as the paper modification method. In the “bridging” design (Figure 3.10 A-C), a 1-mm long segment was cut from the 2-mm wide channel manually, with ~0.5 mm more cut on both sides towards the direction of the channel. Then, the “ribbons” of the T/C lines were able to be inserted in between the space. The ribbons were immobilized with M293 and rabbit anti-mouse antibody separately. This design was highly flexible with reagent immobilization and treatment since there would be no concern about undesired spreading. Multiple ribbons of the same type were fabricated together simply as a channel, followed by cutting into individuals to be assembled. However, sometimes the ribbons would need to be fixed more tightly by sticking a small piece of clear tape on both wax sides. In the “cutting” design (Figure 3.10 D-F), tiny gaps were generated by cutting with a thin blade. A strip of thin water-repelling film was used to segregate the T/C lines (i.e., the area between each two of the four cuts) and the remaining of the channel without breaking the whole piece. Then, antibodies were loaded onto the two defined areas without spreading to other parts of the hydrophilic channel. After drying, the thin film was removed, and the segregated T/C lines recovered to the same planar during the subsequent incubation and drying process. This design utilized the elastoplasticity of paper as well as the fluid’s merit of wicking across the thin gaps. The last drying step was critical to avoid misalignment in the case of over-cutting due to the variation in manual operations. For automated cutting (e.g., Cameo cutting machine, Silhouette, US) or even potentially customized machine that integrated the segregation function, there would not be concerns of misalignment. 50 Figure 3.10 Example of “bridging” (A-C) and “cutting” (D-F) methods for assembling the T/C lines: (A) front view, (B) back view, (C) a negative test; (D) front view, (E) back view, and (F) a positive test (note the high background signal when the testing time was extended for a higher T-line signal). 3.6.2 Increasing Flow Rate by Revising the Patterns The binding between the conjugate and the secondary antibody immobilized on the C line was easily achieved. However, false-negative results on the T line were frequently experienced, which might be caused by the relatively short interaction time for the T line as well as the low total volume of sample (i.e., amount of analyte) passing the T line. According to the mathematic models and observation in the experiment, the flow rate of wicking liquid on paper of constant channel width will decrease with time [28,29]. To reduce the release rate of the conjugate, the sugar content in the storage solution was increased to 5% sucrose and 2% trehalose from the initial trial of only 2% sucrose. However, the overall flow rate of the test was also reduced. As a result, the increased testing time led to insufficient release of the conjugate and increased background signal due to non-specific binding of the conjugate or conjugate-ovalbumin complex to the paper (Figure 3.10F). To address this issue, various shapes were tested to tune the flow 51 rate (Figure 3.11, Figure A6). The desired pattern should maintain the initial flow rate when the liquid passed the T line and also accelerate the flow in order to transport more conjugate and analyte, leading to the increase of effective volume within the testing time. The total area of food dye beyond the location of the T/C lines indicated the effective volume approximately. Therefore, the strategy of the pattern with the highest volume (i.e., the largest area, ca. 28.7 mm2) was adopted (Figure 3.11). It combined a wide sample area to increase liquid supply, a narrow binding channel to decrease antibody amount and uneven flow rate, and an extremely wide absorbing area to maximize the flow rate of the latter half. The last pattern was for reference. Another interesting observation was that cutting a line within the channel would speed up the flow, and the acceleration was positively related to the length of the line (Figure A7). This phenomenon could share some similarities with the previous study in which open channels were engraved on one side of the paper substrate to accelerate or delay the flow of the fluid [40]. Figure 3.11 Effective volumes related to the geometry of different patterns. The strategy that resulted in the largest area was adopted. Total area beyond the assigned point reflected the effective volume passing the location of T/C lines. The last pattern was for reference. 52 3.6.3 Elimination of False Negative with New Recipes A few new recipes (i.e., #33-42) for surface modification were then tested on the revised pattern, which enabled a higher effective volume to eliminate false-negative results. Among #33-42 and #27, #33 resulted in the best compromise between a higher signal intensity on the T line and a low background colour elsewhere even with an extended period of wicking time (Figure 3.12, Figure 3.10F). Therefore, Recipe 33 was selected to fabricate the final prototype. Note that #37 was to demonstrate the unsuccessful treatment when a relatively high concentration (0.5%) of Triton X-100 was involved. Figure 3.12 T line tests with Recipe 33-42 on Grade 42 filter paper. Recipe 33 was selected for further tests as the best compromise between T line signal intensity and background colour even with a longer wicking time. 53 A comparison between #33-treated μPAD and bare paper (i.e., #0) verified the successful modification of the paper substrate on Grade 1 as well (Figure 3.13). In the unmodified μPAD, the strong interactions between the naked cellulose fibre surface and the conjugate led to strong non-specific binding, and no conjugate could reach the front line of the sample liquid (Figure 3.13). The modified μPAD resulted in ignorable background colour as well as the T line signal visible to naked eyes. The “flower shape” indicated the successful transportation of excessive conjugate along the absorbent area where the evenly coated modification reagents were partially re-dissolved. The stronger signal of both T and C lines on the unmodified paper was not only contributed by the specific binding between the capturing antibody but also by non-specific interaction between AuNPs to cellulose, conjugate to cellulose, conjugate-ovalbumin to cellulose, etc. For real food samples, more interfering substances in the sample extract could affect the signal intensity in more complicated ways, leading to much more unpredictable variations. Therefore, reducing the background signals as much as possible is a higher priority. Figure 3.13 Comparison between modified (left pair) and unmodified (right pair) μPADs with negative and positive results tested using PBS or 50 ppm ovalbumin in PBS. The blue arrows at the front line of the sample liquid indicate the failed transportation of the conjugate due to strong non-specific binding onto the unmodified μPADs. 54 3.7 Optimization of Antibody Deposition The colour intensity of both T and C lines required further adjustment for easier observation by naked eyes. For example, a too dark C line could diminish the perception of a relatively light pink colour on the T line despite the low background signal (i.e., optical illusion). 3.7.1 Test Line It was not feasible to further concentrate the T line capturing antibody with a limited amount in total, therefore, the final concentration was unchanged from the original 1 μg/μL. At the same time, it was also difficult to increase the volume of antibody being deposited within the defined area on the T line. On the one hand, the maximum liquid holding capacity of the filter paper had been reached, which was ~0.4 μL for the T line. On the other hand, multiple cycles of loading and drying led to observable reagent bleeding, which might relate to the hindrance from the initially immobilized antibodies to the subsequent proteins. Therefore, a total of 0.4 μg of M293 was used as the capturing antibody for the T line. 3.7.2 Control line The secondary antibody was diluted to 0.2, 0.4, 0.6, and 0.8 μg/μL, and then 0.3 μL of the diluted antibody was deposited (i.e., 0.06, 0.12, 0.18, and 0.24 μg) onto the C line. The colour intensity was as saturated beyond 0.24 μg, and that of 0.18 μg was still too high. Therefore, the concentration of 0.8 μg/μL (i.e., 0.12 μg of secondary antibody) was selected (Figure 3.14). 55 Figure 3.14 Colour intensity on the C lines with various amounts of the secondary antibody. 3.8 Detection of Ovalbumin For standard ovalbumin solutions, as low as 1 ppm could still develop a pinkish colour on the T line that was visible to naked eyes (Figure 3.15). However, the difference in the colour intensity among 5, 20, and 50 ppm was less noticeable. Figure 3.15 Detection of ovalbumin in the standard solutions of 0 – 100 ppm (n ≥ 3). As low as 1 ppm of ovalbumin was visible to naked eyes. 56 Due to the 50-200 times dilution during the sample extraction for the spiked cake mix (Figure 3.16) and other food samples (Figure 3.17), the overall sensitivity was generally lower than that for the standard solution. As low as 0.01% egg white (i.e., ~ 50 ppm ovalbumin) spiked in cake mix could be detected (less than 2 ppm in diluted extract, i.e., 0.01% with a dilution factor of 50), which was 100-fold less than the common baking recipes (~1%) (Figure 3.16). Egg white content was also detected in muffin mix, baked muffin, and mayonnaise (Figure 3.17, Figure A8), in which whole eggs were included in the ingredient lists (est. 0.1-1% egg white protein). Besides, cross-reaction to milk proteins was not directly observed (Figure 3.17, Figure A8). However, casein might interfere with the specific binding between M291/M293 and ovalbumin. This was indicated by the observation that the T line signal intensity was lower than the background in a few surface modification recipes (#38-41) containing high content of casein (Figure 3.12). The cooking process (i.e., dilution due to the addition of water for making the pastry, denaturation by microwave heating) could be the main reason for the lower colour intensity for baked muffin compared to that for the muffin mix (Figure 3.17, Figure A8). The high content of lipids in mayonnaise could also affect the performance of the μPAD. Figure 3.16 Detection of ovalbumin in cake mix spiked with 0 - 2% egg white powder (n ≥ 3). 57 Figure 3.17 Detection of ovalbumin in muffin mix, baked muffin, mayonnaise, and skim milk powder (n ≥ 3). 3.9 Detection Limit and Issues in Extraction The developed μPAD successfully detected the main protein of the allergic egg white in spiked food models and real food samples. The limit of detection by naked eyes was 1 ppm for ovalbumin solution and 0.01% egg white in the cake mix. In Canada, there is no regulatory threshold of the allergen residue level for a food product to claim “free of” a particular allergen. The only exception is gluten. Health Canada considers residue levels lower than 20 ppm gluten protein generally do not pose health risks to consumers with celiac disease, which is enforced by CFIA in “gluten-free” labelling [86]. Meanwhile, for all other allergens, CFIA relies on commercial ELISA kits with the detection limits generally at 1-5 ppm [87] which are actually 2-20 times higher than the claimed limit of detection (e.g. claimed 0.31 ppm [88] vs. evaluated 1.17 ppm whole egg [89], claimed 0.03 ppm [90] vs evaluated 0.68 ppm egg white protein [89]). 58 As for commercial LFI products for the detection of egg content, the claimed LODs are mostly within 1-10 ppm egg proteins (Table 3.3), which is about 2-10 times higher than that of commercial ELISA kits. Currently, there are no LFI products targeting at egg proteins that can generate quantitative result; only the presence or absence of egg content can be determined. Table 3.3 Comparison of the claimed performance of commercial LFI products and the developed μPAD. Brand Product Claimed LOD Cite Romer Labs AgraStrip® Egg 2 ppm dried whole egg 2 µg/25 cm2 dried whole egg [91] Neogen Reveal® 3-D for Egg 5 ppm egg 10 µg/100 cm2 egg [92] R-Biopharm Egg Lateral Flow Allergen Test Kit sprayed egg powder down to 1 ppm [93] Morinaga Rapid Test Pro II for Egg 5 ppm (μg protein /g food) 1 μg protein /swab [94] Current study μPAD 1 ppm ovalbumin (<2 ppm egg white) 0.01% egg white in cake mix The two most important factors deciding the detection limit for real food samples are the quality of antibody pair and the extraction procedure. The quality of the antibody pair involves but is not limited to the affinity, specificity, and stability. The affinity refers to the strength of the interaction between the targeted epitope of antigen and the antigen-binding site of antibody, and it is affected by many external conditions such as the pH of the sample liquid. Cross-reaction could occur when the antibody has unsatisfying specificity, and another antigen may interfere with the specific interaction between the antibody with the targeted antigen. The casein-related T line failure (Figure 3.12) could possibly be an example. Further, the stability of antibody protein on cellulose fibre under the surface modification procedure (i.e., treatment with polymers and salts, drying) and various relative humidity (from <25% to 90%) is unknown. In this thesis project, there were not many 59 choices among the commercially available antibody products. Although not as heat stable and allergenic as ovomucoid (28 kDa, 11%), ovalbumin (45kDa, 56-58%) is more abundant and potentially has more epitopes to develop different monoclonal antibodies. Consequently, antibody pairs that bind to different epitopes with least space hindrance can be prepared for the sandwich structure more easily. Most commercial products of LFI and ELISA kit are still based on anti-ovalbumin antibody pairs. The extraction procedure is more important to a great extent when there is a lack of choices of antibodies. For decades, there has been no established standard of the extraction process. The overall efficiency of the extraction process is also specific to allergen, sample matrix, and analytical tool to be applied to. Regarding the extraction of ovalbumin from food samples to be analyzed by LFI and ELISA, each company has its own patented or confidential formula of the extraction buffer incorporated in each extraction procedure. A typical extraction process includes 1) the homogenization of food sample to achieve an even distribution of antigen for better accuracy, 2) the release/dissolving of antigen into the extraction buffer from the food matrix, and 3) the reduction of co-extracted impurities to reduce interference. The effect on extraction efficiency from the component in many formulae, such as ionic strength, pH value, surfactant and denaturing reagent, has been extensively investigated [79,95]. The recipes of extraction buffer vary dramatically, ranging from simple buffer (PBS [96]) to a high content of protein (Tris-HCl, pH 8.6,10% gelatin [97]), from high content of surfactant (4% SDS, pH 8.5 [98]) to the addition of strong denaturing reagent (1% SDS, 7% 2-ME [89]). Sonication, long-time shaking, dialysis, and heating are also involved in many matrix-specific possesses [91-94]. The sacrificing proteins, such as skim milk powder and gelatin, were 60 sometimes utilized to remove the co-extracted interference (e.g., tannin and polyphenol in chocolate) [99]. In the current study, the core reagents in the extraction buffer from Morinaga (1% SDS, 7% 2-ME) were added into PBS buffer. The Morinaga’s buffer had been well recognized as the most efficient and consistent extraction buffer, especially for heat-processed food samples with relatively low allergen residue levels [79,89]. However, it was still not easy to resolve the dilemma. On the one hand, the extraction solution denatures proteins and assists the release and dissolving of ovalbumin, which can ensure decent extraction efficiency and a high antigen recovery. On the other hand, the extract had to be at least 10 times diluted due to the high content of SDS (1%) and 2-ME (7%), leading to the decrease of overall sensitivity of the detection method. Not merely the concern of its very unpleasant smell, 2-ME of a relatively high concentration could denature the antibodies by cleaving the disulphide bond [79]. In addition, the high content of surfactant (1% SDS) could also significantly alter the fluid dynamics that might cause more problems. As a result, the second round of dilution in the current study limited the performance of the developed μPADs in analyzing the model sample spiked with a low amount of allergen (<0.01%). Besides, although two recipes without reducing agent had been reported for their high extract efficiency in cake mix, the spike level was rather high (3%) that the performance of a few other mild recipes was also acceptable [98]. The extraction solutions from companies other than Morinaga either failed, or the performance was highly dependent on the specific heating process to food samples. For consistency, the Morinaga recipe was preferred. The successful detection of egg protein in microwave-baked muffin confirmed the efficiency as well. 61 Chapter 4: Conclusion In the current study, the prototype of an LFI-format microfluidic paper-based analytical device has been developed for the detection of chemical hazards in food. Being optimized in the paper platform, surface modification and geometric patterns, the developed μPAD was able to detect as low as 1 ppm ovalbumin, 0.01% egg white protein in cake mix as the food model, and egg content in samples of commercial food products in 15 min, including sample preparation. To the best of our knowledge, this study for the first time achieved the transformation of LFI to a fully cellulose-based analytical device that could detect large size of hazardous molecules in real food samples. This study expanded the potential of hydrophilic cellulose filter paper as a novel platform for the detection of chemical hazards in food, and the device may serve as a template for the development of μPADs targeting other chemical hazards. With more optimizations in the detection limit, the developed μPAD may also serve as the prototype for mass production, providing an alternative food monitoring option to many sectors throughout the food system and contributing to improving food safety and public health. In future works, more efforts could be focused on the following directions. Pulp and paper chemists and engineers could further optimize the cellulose selection and processing towards consistent quality without a significantly increased cost. Thus analytical chemists could test more paper candidates to improve the density and the orientation of immobilized antibodies. In addition, instead of AuNPs, the compatibility of alternative reporting agents (e.g., latex beads, quantum dots) with higher optical signal intensities on μPADs could be investigated so as to reduce the detection limit without relying on high-quality antibodies. 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Figure A4 Synthesis of gold nanoparticles via a modified citrate reduction method (left) and synthesized gold nanoparticles stored for 10 months (right). 71 Figure A5 NaCl induced aggregation test for determining the minimum ratio of antibody (μg) to AuNPs (1 mL); for M293, the least ratio without colour change from pink to blue/colourless (8 μg Ab/ 1mL AuNPs). Figure A6 Effect of various channel widths on flow rate (as the reference to tune effective volume, Fig. 3.11).72 Figure A7 The increase of flow rate increased at different stages (in 600 s) due to the cuttings of various lengths in the middle of channels73 Figure A8 Detection of ovalbumin in muffin mix, baked muffin, mayonnaise, and skim milk powder (n = 3) 74
Thesis/Dissertation
eng
Food Science
Vancouver : University of British Columbia Library
Attribution-NonCommercial-NoDerivatives 4.0 International
Graduate
Development of microfluidic paper-based analytical device to detect chemical hazards in food
Text