A Study On Electrochemical Sensor Using Metal Decorated RGO-TEPA As Nanotracer For The Detection Of Biomarkers

Electrochemical immunosensors based on metal ions functionalized rGO-TEPA as signal amplification protocol, which combine the electro activity of metal ions and bulk properties of rGO-TEPA, have shown great application prospective in the fields of clinical cardiovascular disease diagnosis. rGO-TEPA contains a large number of amino groups and has excellent conductivity, which makes metal ions adsorbed on its surface directly for developing highly sensitive electrochemical immunoassay. Compared with the usual metal ion functionalized nanomaterial probes, metal ion functionalized rGO-TEPA simplified the modification steps. Meanwhile, the metal ions in the bioconjugates can be detected by differential pulse voltammetry without the acid dissolution and preconcentration, which greatly simplifies the detection steps. This thesis focused on the preparation of metal ion functionalized rGO-TEPA nanoprobes, the fabrication of immunosensor and the detection of biomarkers for cardiovascular disease, our specific work are as follows: 1. Ultrasensitive electrochemical detection of secretoneurin based on Pb²⁺-decorated reduced graphene oxide-tetraethylene pentamine as a labelrGO-TEPA contains a large number of amino groups, which makes it an ideal templet for the loading of metal ions. Secondary secretoneurin（SN） antibody（Ab2） was immobilised on rGO-TEPA-Pb²⁺ through glutaraldehyde, and the resulting nanocomposite(Ab2-rGO-TEPA-Pb²⁺) was used as a trace tag for signal amplification. A modified electrode consisting of functionalised graphene nanosheets（Au@GS） was used as a substrate to immobilise the antibodies. Under the optimal conditions, the immunoassay exhibited high sensitivity, acceptable stability and reproducibility with a wide linear range from 0.001 to 100 ng mL-1, and an ultra-low detection limit of 0.33 pg mL-1. Furthermore, the immunosensor could be employed to detect SN in clinical serum samples.2. A simultaneous electrochemical multianalyte immunoassay of high sensitivity C-reactive protein and soluble CD40 ligand based on reduced graphene oxide-tetraethylene pentamineThrough the abundant amino groups of rGO-TEPA, Pb²⁺ and Cu²⁺ were separately absorbed on its surface directly. The high sensitivity C-reactive protein（hsCRP） and soluble CD40 ligand（sCD40L） antibodies were conjugated with the rGO-TEPA-Pb²⁺ and rGO-TEPA-Cu²⁺ hybrids through glutaraldehyde for amplifying the detection signals, respectively. The signals could be directly detected in a single run through differential pulse voltammetry（DPV）, and each biorecognition event produces a distinct voltammetric peak. The position and size of each peaks reflects the identity and the level of the corresponding antigen. To further improve the analytical performance of the immunosensor, Au@bovine serum albumin（BSA） nanospheres synthesized through a “green” synthesis route were used as a sensor platform, which not only provides a biocompatible microenvironment for the immobilization of antibodies but also amplifies the electrochemical signals. Under optimal conditions, hsCRP and sCD40 L could be assayed in the range of 0.05 to 100 ng mL-1 with detection limits of 16.7 and 13.1 pg mL-1, respectively. The assay results on clinical serum samples with the proposed immunosensor were in acceptable agreement with the enzyme-linked immunosorbent assay. This novel immunosensing system provides a simple, sensitive and low-cost approach for a multianalyte immunoassay.