Chromogenic detection of dipeptidyl peptidase IV (DPP-IV) activity using peptide-functionalized gold nanoparticles

Metal nanoparticles offer a useful platform for a wide range of biological applications especially for biosensing, bioimaging and drug delivery. This thesis presents a body of original research describing the synthesis, characterisation and development of a novel and convenient biosensing assay for detection of dipeptidyl peptidase IV (DPP-IV) enzyme activity using peptide functionalized gold nanoparticles. The distinctive optical and physical properties of gold nanoparticles (Au NP) were harnessed for the development of a colorimetric assay for rapid sensing of DPP-IV activities and screening DPP-IV inhbitors. The citrate reduction method for Au NPs synthesis was optimised and several potential peptide substrates (GPDC, VP-EN-DC, C/G dipeptide, GPG-EN-PEG4-LA, GPDCALNNC) were designed to provide substrates that mimic the DPP-IV natural substrates. The performances of the substrate functionalized Au NPs were assessed for their appropriateness for the detection of the enzyme activity. Addition of DPP-IV to the solutions containing the functionalized Au NPs resulted in cleavage of the substrate and thus causing the aggregation of the Au NPs which in turn led to a shift of the surface plasmon peak toward longer wavelengths, and a change of the colour of the colloidal suspension from red to blue. Overall, real-time detection of DPP-IV activity over a broader range (0-40 U/L) with high selectivity and stability was obtained, thus providing a method that can be used to determine the levels of DPP-IV/CD26 in biological fluids such as serum and plasma. Further assay developments were conducted to overcome limitations encountered with the original Au NP assay, especially the narrow dynamic linear range and stability in high ionic strength solutions. Validation and comparison of the Au NP assay developed has revealed that this method is highly correlated to the gold standard chromogenic Gly-Pro-pNA method for detection of enzyme activity in biological samples. Very good recoveries (in the range 83.6 –114.9%) were obtained in spiked serum samples, which indicate that this assay could provide a suitable alternative for enzyme activity detection with the naked eye and without the need for sophisticated instruments. Investigations into the effects of incorporating different stabilizers in order to improve the stability of the peptide functionalized Au NP in high ionic strength solutions were also investigated. Gold nanoparticles have different shapes and structures and an alternative approach for detection of DPP-IV activity using gold nanorods due to their higher refractive index sensitivities was explored. As a conclusion, three out of five approaches, all utilising Au NP-ligand conjugates were demonstrated useful for the detection of the DPP-IV activity. The system developed here is portable and would permit on-site analysis of samples, which offers a real alternative approach from traditional assays and reduces the need for laboratory testing. The logical next step in this research would be the continuation of experiments to transform this test into a point of care testing device that could offer an early detection tool for disease management.