Development of Molecularly Imprinted Polymer Nanoparticles Based Assay and Sensor for Fumonisin B1
thesisposted on 17.09.2018, 08:26 authored by Hasim Munawar
Fumonisin B1 (FB1) is categorised as a human and animal carcinogen produced by Fusarium moulds which occur mainly in corn, wheat and other cereals. For decades, immunoassay (ELISA) has been developed as the reference established method for FB1 determination in food and animal feed. Unfortunately, the current assays are inefficient due to factors such as temperature instability of the antibody (recognition element) and enzyme elements in the immunoassay, the presence of natural inhibitors in the samples tested and high levels of non-specific protein binding. Other important factors are the time when results are needed rapidly and the cost of use. This work aims to develop an assay and sensor for FB1 using molecularly imprinted polymer nanoparticles (nanoMIPs) to overcome these limitations. Firstly, computational modelling was used to identify the best functional monomers that form a complex with FB1. These results were verified by solid phase extraction (SPE) experiments. Ethylene glycol methacrylate phosphate (EGMP) was identified as a suitable functional monomer for FB1. The nanoMIPs for FB1 have been synthesised by solid phase synthesis using the composition based on EGMP. From hot water fraction, the nanoMIPs were collected with concentration 0.06 mg mL-1 and particle size 249 ± 29 nm. The image of nanoMIPs for FB1 was taken by TEM. The phosphate (PO43-) and carbonyl (C=O) as functional groups of nanoMIPs were identified by FT-IR Spectrometer. The dissociation constant of nanoMIPs is 0.2 μM by SPR (Chapter 3). Development of molecularly imprinted polymer nanoparticle-based assay (MINA) was started by producing complex conjugate based on horseradish peroxidase (HRP). HRP was complexed with FB1 by carbodiimide reaction using EDC and NHS. After optimisation of the concentration of nanoMIPs (0.06 mg mL-1) and HRP-FB1 conjugate (1:400), MINA was capable producing satisfactory detection of FB1 in concentration range 10 pM – 10 nM. The selectivity and cross-reactivity have been tested. The response from commercial monoclonal antibody (mAb) and non-specific imprinted polymer nanoparticles (nanoNIPs) have been lower with the same concentration range (10 pM – 10 nM). Also, the interaction between nanoMIPs and other mycotoxins such as aflatoxin B1 (AFB1), citrinin (CTT), deoxynivalenol (DON), fumonisin B2 (FB2), and zearalenone (ZEA) were shown to be negligible. The application of MINA has been tested in real samples. A total of 18 corn samples has been contaminated by fumonisins with a range from 0.02 – 1.29 ppm. From these results, all samples are safe because the concentration is lower than maximum residue limit of fumonisins (2 ppm). All samples have been further analysed for comparative study with a commercial ELISA kit and HPLC. Statistically, t-test has shown that there is significant similarity of the results obtained by MINA and commercial ELISA kit (AgraQuant, Romer Lab) (Chapter 4). Development of a molecularly imprinted nanoparticle-based electrochemical sensor (MINES) was fabricated with two types, ferrocyanide-ferricyanide ([Fe(CN)6]4-/3-) labelled MINES and label free MINES. To gain this technology, the platinum working electrode was electropolymerised by a Zinc(II)porphyrin (ZnP) and pyrrole (Pyr) and immobilised by nanoMIPs using carbodiimide chemicals. The final electrode is nanoMIPs/ZnP/Pyr/Pt. The electrode was tested by differential pulse voltammetry (DPV) using [Fe(CN)6]4-/3- for generating the label based MINES and by electrochemical impedance sensor (EIS) for producing label free MINES. In concentration range of FB1 at 1fM to 10 pM, the linearity and limit detection from EIS (R2 = 0.98, LoD = 0.7 fM) and DPV (R2 = 0.96, LoD = 0.03 fM) show the excellent performance of both methods. The EIS (0.442 kΩ/M) is two times more sensitive than DPV (0.281 μA/M) (Chapter 5). In conclusion, the nanoMIPs based assay (MINA) and electrochemical sensor (MINES) are a very promising method for the detection of FB1 in food and animal feed at very low concentrations with no cross-reactivity offering a fast, cost-effective and reliable technique.