Development of amperometric immunosensors for protein biomarkers using single wall carbon nanotube forest and electrochemical detection of enzyme labels
Date of Completion
Single-walled carbon nanotube forest based electrochemical immunosensors for rapid, cost-effective, selective and highly sensitive detection of proteins and cancer biomarker proteins in various matrix including human serum and tumor tissue have been developed. The immunosensor has strong potential of being applied in large scale clinical disease screening and point-of-care diagnostics. ^ The first generation of such immunosensors was developed by conjugating capture antibodies to the carboxylated ends of vertically aligned arrays of single-wall carbon nanotubes (SWNT forests) on pyrolytic graphite (PG) surfaces. Using amperometric detection of enzyme horseradish peroxidase (HRP) labels on the tracer antibodies in a sandwich immunoassay format, as low as 75 pmol mL-1 human serum albumin (HSA) was reproducibly detected. ^ The sensitivity of this detection method was significantly improved in the second generation immunosensor with the use of water soluble mediator hydroquinone (HQ) to improve electron transfer between the HRP labels and electrode. Detection limits 0.01 pmol mL-1 Prostate Specific Antigen (PSA) was achieved. The DL is similar to clinical ELISA kit, but the sensor developed herein only requires 10 times less patient sample. An excellent match between the detection of PSA in patient samples using the methods developed and clinical ELISA kit was also observed. ^ A tremendous improvement was accomplished with the third generation immunosensors, which utilize covalent loading of multiple enzyme (HRP) labels and anti-PSA antibodies to the side wall of highly functionalized MWCNTs. Multiple enzyme labels per binding event great increased signal and lowered the detection limit. A detection limit of 0.1 fmol mL-1 PSA in merely 10 μL of undiluted serum was achieved. The mass detection limit of this method is only 40 fg, ∼100 times lower than all commercially available techniques. Using this method, PSA in as low as 1000 tumor cells from patients was reproducibly detected. ^ Also described in this thesis is the much enhanced catalytic response to H2O2 resulted from highly efficient wiring of enzymes (Mb and HRP) to electrode by using ultra-thin conductive sulfonated polyaniline (SPANI) underlayer, which behaved like molecular wires and significantly improved the efficiency of electron transfer. ^
Yu, Xin, "Development of amperometric immunosensors for protein biomarkers using single wall carbon nanotube forest and electrochemical detection of enzyme labels" (2006). Doctoral Dissertations. AAI3205763.