Novel Nanoscale Platforms for the Isolation and Ultra-trace Detection of Bioactive Molecules

This research presents novel functionalized nanomaterials and nanosensors for the selective and ultra-sensitive bio-analysis of different bioactive molecules using surface enhanced Raman spectroscopy (SERS) and electrochemical methods. Disposable paper SERS substrate was synthesized for the detection of sofosbuvir metabolite after its chromatographic separation from blood plasma. The developed substrate showed a good sensitivity with a quantification limit of 50 pM. The cost-effectiveness, ease of manufacture and good sensitivity of the new paper SERS substrate encouraged its utilization for the detection of the environmental toxin, microcystin – LR. A recyclable gold nanomaterial was first fabricated to selectively isolate the toxin from biological fluids. The fabricated nanomaterial was developed by decorating magnetic gold nanoparticles with target specific antibody fragments. The developed nanomaterial showed an excellent selectivity towards the isolation of the toxin from the biological fluids. After its isolation, the toxin was detected by using gold coated silicon nanopillar and paper SERS substrates. The toxin was quantified down to 10 fM using the gold coated nanopillar substrate and a handheld Raman spectrometer. The developed method demonstrated the potential for rapid protein nanosensing in biomedical and environmental applications.

The metallic nature of the gold coated nanopillar substrate promoted its utilization as a dual nanosensor for the combined SERS and electrochemical detection of bioactive compounds. Therefore, it was used for the detection of the protein biomarker, cystatin C, in human blood by both SERS and differential pulse voltammetry (DPV). To selectively extract the protein from the plasma, a recyclable 2D extractor chip was functionalized with cystatin C antibody fragments. For the first time, the protein’s own disulphide bond structure was utilized for it dual nanosensing by SERS and DPV. The molecular structure of the isolated protein was chemically reduced to break its disulfide bonds and allow its unified orientation onto the conductive SERS substrate via formation of gold – sulphur bonds. Using this approach, cystatin C was quantified by SERS and DPV down to 1 pM and 62.5 nM, respectively.

The thiol chemistry of biomolecules was also utilized for the detection of recombinant human erythropoietin (rhuEPO) in blood plasma by simple and direct electrochemical method. For the label-free electrochemical quantification of the protein, it was first extracted using antibody-functionalized magnetic beads. The isolated protein was electrochemically reduced by chronoamperometry and assembled onto the nanostructured gold electrode via gold – sulphur bonds. The protein was then quantified by reductive desorption using DPV. This electrochemical reduction allowed for the detection of rhuEPO down to 1 pM without signal amplification.

Chief Investigators