Nanostructured imaging surface plasmon resonance biosensing

Abstract

The testing and further development of a prototype nanostructured imaging surface plasmon resonance (iSPR) biosensor, with a focus on surface modification and detailed characterization of the biosensor chip and in-field and at-line applicability in the food industry is described. Furthermore, a simplified coupling of SPR and MS is described that allows identification of the mycotoxins of interest along with any other cross-reacting analytes. Chapter 1 describes general information about SPR, SPR instruments along with their components, development of a multiplex SPR biosensor and coupling of SPR to mass spectrometry. In Chapter 2, the surface modification, in-depth characterization and the antifouling performance of the nanostructured iSPR chip is described. Different types of polyethylene glycol (PEG) and zwitterionic polymers were chosen as antifouling chemistries. Various surface characterization techniques such as atomic force microscopy, scanning electron microscopy, water contact angle, X-ray photoelectron spectroscopy and direct analysis in real time high resolution mass spectrometry provided complementary information about the chip before and after the modification. Antifouling chemistry, an essential first step in the development of an SPR biosensor, prevents false positive results arising from non-specific binding of sample components to the SPR chip. Upon comparison of the surface modification and antifouling behavior with conventional flat SPR chips, the latter were only slightly better. Zwitterionic polymers and long chain PEG had the best antifouling performance. A proof-of-principle experiment was done to demonstrate the selective detection of streptavidin binding to a surface partially modified with biotin. A 6-plex SPR assay for the detection of mycotoxins in barley was developed in Chapter 3. A benchmark double 3-plex assay was developed for the detection of deoxynivalenol (DON), zearalenone (ZEA), T-2 toxin (T-2), ochratoxin A (OTA), fumonisin B1 (FB1) and aflatoxin B1 (AFB1) using benchtop SPR instrument (Biacore). Preliminary in-house validation of the competitive inhibition assay developed using ovalbumin conjugates of the mycotoxins showed that the method is suitable for detection of DON, ZEA, T-2 and FB1 whereas further improvement is required for OTA and AFB1. The method was then transferred to the nanostructured iSPR, which although less sensitive than the benchtop SPR, was able to detect DON, T-2, ZEA and FB1 at the relevant levels. In Chapter 4, the assay developed in Chapter 3 was further optimized and an entire assay along with in-house validation and measurement of naturally contaminated was developed using the nanostructured iSPR. The antifouling chemistry used in Chapter 3, PEG, was replaced by carboxymethylated dextran (CMD) that not only allowed direct immobilization of toxins but also helped to improve the stability of the chip whereby the chip could be used for more than 450 cycles. DON could be detected at the relevant levels in beer with minimal sample preparation whereas for OTA an enrichment step using solid phase extraction was required. As demonstrated in Chapter 3 and 4, the nanostructured iSPR instrument can be used for screening of different mycotoxins in beer and related ingredients. However, SPR is not able to provide chemical information of the binding analyte especially in cases where the antibodies have cross-reactivity towards conjugates of the analyte. Therefore, a simplified coupling for SPR with ambient mass spectrometry was developed in Chapter 5. The method allowed identification of DON as well as its cross-reacting conjugates such as deoxynivalenol-3-glucoside and 3-acetyl DON. The research presented in this thesis is an important step towards the use of the nanostructured iSPR instrument for label free in-field and at-line detection of various analytes. In Chapter 6, discussion of the main achievements of this thesis, challenges and future perspectives of the technology is described.