Surface modification and conjugation strategies for bioassay/biomaterial applications

The aims of this research were to develop novel surface modification strategies that can be used on a range of solid supports including polymeric and metallic matrices, as these could have a significant impact on the performance of bio/sensors employing these surfaces. These approaches were used to develop methods for immobilizing biomolecular recognition elements, such as antibodies, on modified matrices, and to exploit these approaches for the generation of high sensitivity bio-assays. Human fetuin A, mouse immunoglobulin G, and horseradish peroxidase were employed as model analytes. A silane-based surface modification strategy was designed and optimized for planar or flat surfaces such as polymeric sheets, chips or microtitre plates. These polymeric surfaces were activated prior to silane-functionalization using potassium hydroxide (aq.)-mediated mild oxidation (wet method) and oxygen-plasma etching. This novel surface activation strategy was further optimized in combination with surface functionalization and covalent immobilization of antibodies, for enhancing immunoassay sensitivities. Sensitivities obtained for immunoassays using antibodies immobilized with the developed and adsorption-based conventional strategies were 39 and 625 pg/mL, respectively, for human fetuin A. The strategy was demonstrated to be generic in nature and could be employed to activate a wide range of polymeric and metallic surfaces. In addition, highly sensitive detection of human fetuin A was achieved with antibodies captured in an oriented manner on covalently immobilized protein A (EC50 3.7 ng/mL) in comparison to randomly captured antibodies (EC50 5.8 ng/mL). High-brightness NIR664 dye-doped silica nanoparticles were employed to probe various activation states of platelets. These NPs were functionalized with silanes (viz. amine and carboxy-terminal) followed by conjugation to a platelet surface biomarker-specific antibody (anti-CD41) and successfully employed for probing platelet activation. The antibody-NP conjugates were found to be highly sensitive (>95%) and specific (≈100%). In addition, aggregation of NPs was minimized by controlling their surrounding chemical environment and their stability after antibody conjugation

A facile molecularly imprinted polymer-based fluorometric assay for detection of histamine

Histamine is a biogenic amine naturally present in many body cells. It is also a contaminant that is mostly found in spoiled food. The consumption of foods containing high levels of histamine may lead to an allergy-like food poisoning. Analytical methods that can routinely screen histamine are thus urgently needed. In this paper, we developed a facile and cost-effective molecularly imprinted polymer (MIP)-based fluorometric assay to directly quantify histamine. Histamine-specific MIP nanoparticles (nanoMIPs) were synthesized using a modified solid-phase synthesis method. They were then immobilized in the wells of a microplate to bind the histamine in aqueous samples. After binding, o-phthaldialdehyde (OPA) was used to label the bound histamine, which converted the binding events into fluorescent signals. The obtained calibration curve of histamine showed a linear correlation ranging from 1.80 to 44.98 μM with the limit of detection of 1.80 μM. This method was successfully used to detect histamine in spiked diary milk with a recovery rate of more than 85%

Identification and Biosensing Application of Molecular Recognition Elements

Molecular recognition elements (MREs) are biomolecules such as single-stranded DNA (ssDNA), RNA, small peptides and antibody fragments that can bind to user defined targets with high affinities and specificities. This binding property allows MREs to have a wide range of applications, including therapeutic, diagnostic, and biosensor applications. The identification of MREs can be achieved by using the process called Systematic Evolution of Ligands by Exponential Enrichment (SELEX). This process begins with a large library of 109 to 1015 different random molecules, molecules that bind to the user defined target or positive target are enriched in the process. Subsequently, this process can be modified and tailored to direct the enriched library away from binding to related targets or negative targets, and thus increasing the specificity. Single-stranded DNA (ssDNA) MREs are particularly favorable for biosening applications due to their relative stability, reusability and low cost in production. This work investigated the identification and application of ssDNA MREs to detect different bacterial toxins and pesticide.;In Chapter 1, it begins by reviewing recent discovery and advancement in the SELEX technique for the identification and biosensing application of ssDNA MREs specific for bacteria, viruses, their related biomolecules, and selected environmental toxins. It is then followed by a brief discussion on major biosensing principles based upon ssDNA MREs. In Chapter 2, the pilot project of this work, ssDNA MRE specific for Pseudomonas aeruginosa exotoxin A was identified. In this chapter, a novel variation of SELEX called Decoy-SELEX, previously developed by our laboratory is described in greater detail. Additionally, the development of a ssDNA MRE modified enzyme-linked immunosorbent assay (ELISA) for the exotoxin A detection is also discussed. In Chapter 3, similar methodology was applied to identify a ssDNA MRE specific for the second target, Clostridium difficile toxin B. Subsequently, similar ssDNA MRE modified ELISA was developed for target detection in clinically relevant samples. In Chapter 4, ssDNA MRE specific for alpha toxin of Staphylococcus aureus was identified, and it was applied for sensitive detection of the target in clinically relevant samples. In Chapter 5, the overall conclusion and potential future studies as a result from this work is discussed. Lastly, in Appendix, the project of identifying and potential future application of ssDNA MREs specific for a pesticide, Fipronil is described.;Overall, this work has shown the proof-of-principle of using ssDNA MREs in biosensing application for target detections in clinically relevant samples. The work will be useful in the development of potential point-of-care diagnostic tools for rapid diagnosis of bacterial infections

Optical sensor for the analysis of biomarkers

The continuous measurement of biomarkers such as proteins and hormones in blood and serum are the cornerstone of healthcare monitoring and optimising treatment plans. Often such biomarkers are present in minute concentrations. Conventional analysis methods involve assays with costly labelled antibodies and lengthy protocols on microtitre well plates. For example fluorescence detection is often favoured for antibody based assays as it is very sensitive, however considerable sample clean-up is required (they will contains interfering materials such as other proteins, salts, etc.) and a protocol is required that requires fluorescence labels. Label-free biosensors are therefore an attractive alternative to improve biomarker analysis. In this work, a label-free optical biosensor was developed based on dye-doped leaky waveguides (DDLW). Chitosan was selected as the porous waveguide material because it has a large surface area, offering the probability of immobilizing a large number of biomolecules, which significantly increases the possibility of capturing target species.The DDLW biosensor consisted of a 1% of chitosan layer, deposited by spin coating onto a glass substrate from a solution of 0.1 M acetic acid. Reactive Blue 4 dye 100 mM was then incorporated in the chitosan waveguide via reaction with the free amines of the chitosan. The chitosan waveguide on the glass substrate was then sealed within a microfluidics flow cell to allow controlled flushing with reagents. The Reactive Blue 4 dye absorbs light most strongly at the resonance angle and thus generates a dip in reflectivity at that angle which could be used to make measurement.The DDLW sensing mechanism is based on shifts in resonance angle as a result of changes in refractive index in the waveguide, which may change when molecules bind to antibodies immobilised in the waveguide. The surface flatness and thickness of the spin-coated chitosan layer was characterised via white light interferometer and a Dektak profiler. The porosity of the waveguide layer is the main factor determining the maximum number of antibodies that can be immobilized in its entire volume. The pore size of the waveguide film needed to be large enough to allow the antibodies to enter the waveguide layer. The pore size of the waveguide layer was tailored using different methodologies. Silica nanoparticles were investigated as well as porogens, but the chemicals required to dissolve the silica nanoparticles and the porogens used were found to damage the chitosan waveguide. Hence, we have developed a facile method of tailoring the porosity of the waveguide layer by controlling a dry time of chitosan to some extend that can provide a waveguide mode with an enhancement in the porosity of has achieved and shown fascinating results where the pore size was being large enough to different molecular weight of biomolecules. Where we optimised the drying time following different concentrations of chitosan film fabrication by spin coating. The detection method was initially tested using the binding of rabbit IgG to anti-rabbit IgG as a model example. Initially, carboxylic group of the antibody can bind to the amino groups of chitosan film through an activation process using different ratio of EDC-Sulfo-NHS linkage chemistry; the efficiency of immobilisation was investigated with (confocal) fluorescence microscopy, but it was found that the amount of attachment was insufficient. To increase the sensitivity an alternative approach was used in which the antibodies were immobilised into the waveguide layer by first attaching Streptavidin to the amine groups of the chitosan waveguide using glutaraldehyde. Biotinylated anti-rabbit IgG was then immobilised by binding between streptavidin and biotin. Finally, the binding of rabbit-IgG and the immobilised anti-rabbit IgG was studied.Once the immobilisation chemistry is successfully developed, the waveguide system was intended and used to measure the release of tissue factor (TF) that expressed on pancreatic cell lines. Two types of pancreatic cells (Aspc-1 and Miapaca-2 cells) were used to investigate the concentration of TF in a real-time. There were a strong correlations between the level of TF antigen and activity in these cells, showing that the TF present on the cancer cells was active. The calibration curve of Tissue factor (TF) has been created by preparing a range of TF standard solutions (7.825, 15.65, 31.3, 62.5, 125, 250 pg mL-1) and recorded by ELISA plate reader at 450 nm to inspect the concentration of TF that expressed from unknown sample solutions. The highest expression of tissue factor (107.82 pg mL-1) was recorded on the unknown sample solution of Aspc-1, whereas, TF concentration was very low (57.27 pg mL-1) on unknown sample solution of Miapaca-2 cell lines. A leaky waveguide system was also used to investigate the lowest concentration of TF that released from pancreatic cell lines (7.86±1 pg mL-1) that was expressed on unknown sample solution of Aspc-1 cell lines and (6.89±1 pg mL-1) on unknown sample solution of Miapaca-2 cell lines. Different concentrations of TF standard solutions (0.003, 0.03, 0.3, 3, and 30 pg mL-1) were applied using dye-doped leaky waveguide sensor. Then, the results were compared to the conventional microwell-based ELISA system. In conclusion, when applying lowest concentrations of TF that were expressed on unknown sample solutions of (Aspc-1 and Miapaca-2 cell lines) the degrees of shifting in angles were very close to the shifting in angles of standard solutions TF that were prepared to investigate the detection limit