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UBC Theses and Dissertations

Development of innovative biosensors for the determination of melamine in milk Hu, Yaxi


After discovering the potential detriment of melamine to humans, the society calls for novel techniques to accomplish accurate, rapid, high-throughput, and on-line or in-field detection of melamine in foods, as required by the food industry and government laboratories. The aim of this study was to investigate different innovative biosensors combining antibodies or molecularly imprinted polymers (MIPs) with surface enhanced Raman spectroscopy (SERS) to determine melamine in a representative dairy product (i.e. milk). A “two-step” antibody-SERS biosensor was developed to detect melamine in whole milk. The anti-melamine antibody, produced by immunizing New Zealand white rabbits with melamine hapten-ovalbumin immunogen, was used to extract melamine from whole milk exclusively. After releasing melamine from the antibody, the eluents were deposited onto silver dendrite SERS-active substrate for SERS spectral collection. The limit of detection (LOD) calculated by the principal component analysis (PCA) model was lower than 0.79×10-³ mmol/L. The overall analysis was completed in 20 min. The MIP for the “two-step” MIPs-SERS biosensor was synthesized by bulk polymerization of melamine, methacrylic acid, ethylene glycol dimethacrylate and 2,2’-azobisisobutyronitrile. After confirming the specific affinity of the MIP towards melamine by adsorption capacity tests, MIP was used as sorbent for solid phase extraction (SPE) to extract melamine from whole milk. SERS spectra were collected by depositing the eluents from MISPE onto silver dendrite. The LOD and limit of quantification (LOQ) calculated by the linear regression model correlating relative intensity of melamine SERS band at 703 cm-¹ and melamine concentration in whole milk were 0.012 mmol L-¹ and 0.039 mmol L-¹, respectively, and the full analysis was accomplished in 18 min. “One-step” MIPs-SERS biosensor incorporated silver nanoparticles (AgNPs) into MIPs synthesized by bulk polymerization. Adsorption capacity tests verified the specific affinity of MIPs-AgNPs to melamine, and PCA model resulted in the LOD between 0.01 and 0.017 mmol L-¹ melamine in skim milk. The time required to detect melamine in skim milk was 25 min. The low LOD and LOQ, as well as rapid detection confirm the potential of applying these three types of biosensors for accurate and high-throughput detection of melamine in dairy products.

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