Immunosensors for the detection of endocrine disrupting chemicals in environment and foods: designing a new biorecognition interface

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Abstract
In this thesis, for fabricating immmunosensors for bisphenol-A (BPA) and 4-tert-octylphenol (4-tert-OP), the polyclonal antibodies as recognition elements and the BPA epitope functionalised gold nanoparticles as a labelled antigen were synthesised and characterised using enzyme-linked immunosorbent assay (ELISA). BPA antibodies were generated in vivo using BPA-butyrate-protein and BPA-valerate-protein conjugates. Their binding characteristics were evaluated via the competitive assays with five competing haptens (BPA-butyrate, BPA-valerate, BPA-crotonate, BPA-acetate and BPA-2-valerate). Two indirect ELISAs and one direct ELISA were developed. The 50 % inhibition of antibody binding (IC50) values were 0.78 ± 0.01 1.50 ± 0.30 µg L-1 and the limits of detection (LOD) as measured by the IC20 values were 0.10 ± 0.03 0.20 ± 0.04 µg L-1. The assays were optimised for the analysis of BPA in, bottled water, carbonated drinks and canned vegetables. The LOD for these three evaluated sample matrices were 0.10 ± 0.03, 0.60 ± 0.10 and 8.4 ± 2.2 µg L-1 respectively. The pilot survey of the three types of food and beverage products for the presence of BPA residues was conducted using three developed ELISAs. For generating 4-tert-OP antibodies, 6 haptens based on OP and NP structures (OPA, OPB, OP1, NPA, NPB and NP1) were synthesised. Specific antibodies against OPA-protein conjugate, OPB-protein conjugate and OP1-protein conjugate were generated and characterised using the direct and indirect ELISAs in 32 antibody/enzyme conjugate combinations. The most sensitive assay for 4-tert-OP was based on the antibody against OP1 hapten with the NP1-BSA conjugate (AbOP1#3;/NP1-BSA). The assay exhibited an IC50 of 100 µg L-1, and a LOD of 10 µg L-1. In the end, thiolated BPA modified spherical gold nanoparticles with different epitope coverage were synthesised and tested in competitive and displacement ELISA to measure functionality. The minimum epitope coverage required for antibody binding in this study was determined to be 4.4 × 10-10 mol cm-2. Comparing to hapten-protein conjugates in a conventional ELISA, in which epitope coverage is difficult to control, nanoparticles offer greater control of nanoparticle coverage and hence valency. Multivalent conjugation of small molecule epitope on the surface of nanoparticles can act as protein conjugate to bind to the specific antibodies.
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Author(s)
Lu, Yang
Supervisor(s)
Nanju Alice, Lee
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Publication Year
2013
Resource Type
Thesis
Degree Type
PhD Doctorate
UNSW Faculty
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