Development of robust and low-cost biopolymer-based surface plasmon resonance nano immunosensor for the detection of ochratoxin a in coffee | |
| Author | Rehmat, Zainia |
| Call Number | AIT Diss. no.FB-18-02 |
| Subject(s) | Ochratoxins Mycotoxins--Analysis |
| Note | A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Food Engineering and Bioprocess Technology, School of Environment, Resources and Development |
| Publisher | Asian Institute of Technology |
| Series Statement | Dissertation ; no. FB-18-02 |
| Abstract | Ochratoxin A (OTA) is a secondary toxic metabolite produced by several fungi and considered as pot entially carcinogenic molecule . It is the most abundant contaminant in various food commodities especially wine, cereals, coffee and dried food s . Coffee beverages, highly consumed worldwide are often contaminated with OTA and required continues monitoring of OTA level during all stages of processing. Most of t he techniques and sensors used for the detection of OTA are of high cost, time - consuming and required trained personnel. Therefore, the development of robust, rapid and low - cost system is required for the analysis of OTA in complex food system . The purpose of the current study was to develop and fabricate surface plasmon resonance (SPR) immunosensor for the analysis of OTA level in coffee. Chitosan - based SPR nanostructure thin films intended on a compact 3D printed system were examined and used for the immobilization of conjugate (OTA - BSA) through activation, coupling, and deactivation of the surface. Surface plasmon resonance (SPR) has been commonly used as an optical transducer in many sensing applications. It consists of an optical transducer and a se lective detection mechanism for the targeted substance. The optical transducer responses to the changes of the surrounding effective complex refractive index (phase and absorption), corresponding to a measurable change in the light intensity. SPR has high sensitivity to small index change and relative simplicity in realization, which allows it to be widely used for real - time analysis in food, biomedi cal and environmental sciences. C ompact SPR system along microfluidi c channel was developed with 3D printing technology in which a gold - coated surface was used as a slab waveguide. The developed system consis ted of glass slide coated with 50 nm gold, light emitting diode (LED) and detector, connected with a spectrometer. The external coupling elements were elimin ated because the excitation wa s done at the edge of glass slide . L arge core fiber was placed at proper angl e near the glass slide edge to detect the light from system. The sensitivity and limit of detection of the device were measured with various concentration of glycerol (5%, 10%, 15% v/v ) and the limit of detection o f the system ( LOD) was found to be 5.28×10 - 4 refractive index unit ( RIU ) . P oly - dimethylsiloxane (PDMS) was used for the fabrication of flow channels on compact SPR system. The PDMS microfluid ic flow cells consisted of two channels (Length 10 mm × Width 1 mm × depth 100 μm) having an inlet (1 mm diameter) and outlet (1 mm diameter). Channels were made through photolithography method in which resins were synthesized to get the photomask of the m icrofluidic channels. It was observed that sensitivity of the system was increased with microfluidic channels. Chitosan and carboxymethyl chitosan were used as biopolymer s for the binding of biological molecu les on the SPR sensor surface. Chitosan was chemically modified by carboxymethylation as a result, some of the hydroxyl groups of chitosan were replaced by carboxymethyl groups and cross - linked with glutaraldehyde prior to the coating on SPR chips. The characterization and the presence of functional groups on chitosan and carboxymet hyl chitosan were confirmed by F ourier transform infrared spectroscopy (FTIR). Furthermore, the experiments were conducted to measure the adsorption of the OTA with chitosan in a solution form. The results indicated that t he concentration s of the OTA in the solution decreased with the increase of incubation time. At the end of the incubation, almost 85% of the OTA was found adsorbed. The OTA was conjugated with the most common carrier protein bovine serum albumin (BSA) and were used as a ligand for the immobilization. The absorption spectra were analyzed on UV spectroscopy (250 - 500 nm wavelength range) for OTA, BSA and OTA - BSA conjugates. The maximum adsorption wavelength (nm) observed for BSA, OTA and OTA - BSA conjugates wer e 285 nm, 310 nm, and 330 nm respectively. A rapid and sensitive competitive inhibition immunoassay was developed on 3D printed compact SPR system. Chitosan - based nanostructure thin films were fur ther examined for the analysis of ochratoxin A in buffer an d coffee. Various analytical methods were optimized including regeneration of the substrate, immobilization of conjugate and reduction of non - specific binding , c alibration curves were established with the SPR bioassay for spiked buffer samples and coffee s tandards in the biologically releva nt concentration range between 0 to 10 ng/ mL . The limit of detection (LOD) for OTA on carboxyme thyl chitosan surface was lower as compared to chitosan both in buffer and coffee. On chitosan, coated surface the LOD for buf fer and coffee was found to be 3.2 and 5.2 ng/ mL respectively, while for carboxymethyl chitosan it was 2.5 and 3.8 ng/ mL . This study described the use of compact surface plasmon resonance with biopolymer chitosan for the detection of OTA in coffee with hig h sensitivity, accuracy, ease - of - use and cost - effective. |
| Year | 2018 |
| Corresponding Series Added Entry | Asian Institute of Technology. Dissertation ; no. FB-18-02 |
| Type | Dissertation |
| School | School of Environment, Resources, and Development (SERD) |
| Department | Department of Food, Agriculture and Natural Resources (Former title: Department of Food Agriculture, and BioResources (DFAB)) |
| Academic Program/FoS | Food Engineering and Bioprocess Technology (FB) |
| Chairperson(s) | Anal, Anil Kumar |
| Examination Committee(s) | Loc Thai Nguyen;Shipin, Oleg;Mohammed, Waleed;Adhikari, Benu P. |
| Scholarship Donor(s) | Sardar Bahadu r Khan Women University, Pakistan;AIT Fellowship |
| Degree | Thesis (Ph.D.) - Asian Institute of Technology, 2018 |