A highly sensitive nanosensing method for the combined selective capture and SERS detection of Microcystin-LR (MC-LR) in blood plasma has been developed. The new method utilizes gold coated magnetic nanoparticles that are functionalized with anti MC-LR antibody Fab' fragments for the selective capture of MC-LR from aqueous media and blood plasma. Using an oriented immobilization approach, the Fab' fragments are covalently attached to gold surface to form a monolayer with high capture efficiency towards the toxin. After the selective capture, the purified MC-LR molecules were released from the extractor nanoparticles within 5min by manipulating the pH environment of the nanoparticles. The regenerated extractor nanoparticles maintained their capture efficiency and, therefore, were re-used to capture of MC-LR from successive samples. The released purified toxin was screened within 10min on gold coated silicon nanopillars and a new paper-based SERS substrate by handheld Raman spectrometer. The SERS enhancement factors of the nanopillars and the new paper-based substrate were 2.5×10 and 3×10 respectively. The lower limit of quantification (LOQ) of MC-LR by SERS on the nanopillar substrate was 10fM (R=0.9975) which is well below the clinically required detection limit of the toxin. The SERS determination of MC-LR was cross validated against ELISA. By using antibody fragments that are specific to the target biomolecule, the new methodology can be extended to the rapid extraction and detection of other toxins and proteins.

The detection of protein biomarkers for the clinical diagnosis of diseases requires selective and sensitive methodologies and biosensors that can be easily used at pathology laboratories and points of care. An ideal methodology would be able to conduct multimode screening of low and high concentrations of proteins in biological fluids using recyclable platforms. In this work, we demonstrate a novel nanosensing methodology for the dual detection of cystatin C (CST-C), as a protein biomarker model, in blood plasma by surface-enhanced Raman spectroscopy and electrochemistry. The new methodology utilizes the thiol chemistry of biomolecules to develop a target-specific and recyclable extractor chip for the rapid isolation of protein biomarkers from blood plasma. This is followed by the rapid reduction of the disulfide bonds within the isolated protein to influence its oriented immobilization onto a conductive gold coated silicon nanopillar substrate via stable gold-sulfur (Au-S) bonds. The oriented immobilization led to reproducible surface-enhanced Raman spectroscopy (SERS) measurements of the reduced protein (RSD = 3.8%) and allowed for its direct electrochemical determination. After the SERS measurement, differential pulse voltammetry (DPV) was used to desorb the analyte from the substrate and generate a reduction current that is proportional to its concentration. CST-C was determined down to 1 pM and 62.5 nM by SERS and DPV, respectively, which satisfies the requirements for monitoring Alzheimer's and kidney failure diseases. The new dual nanosensing methodology has strong potential for miniaturization in a lab-on-a-chip platform for the screening of many protein biomarkers that have a disulfide bond structure.

Clostridium difficile (C. diff) infection is one of the most contagious diseases associated with high morbidity and mortality rates in hospitalised patients. Accurate diagnosis can slow its spread by determining...

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