A simultaneous electrochemical multianalyte immunoassay of high sensitivity C-reactive protein and soluble CD40 ligand based on reduced graphene oxide-tetraethylene pentamine that directly adsorb metal ions as labels

• The detection of hsCRP and sCD40L by an immunosensor in a single run.
• rGO-TEPA as an ideal template for the adsorption of metal ions.
• The use of rGO-TEPA simplified the previous modification steps.
• The combined measurement provides new cardiac markers for the clinical diagnosis and risk evaluation of cardiovascular events.

A simplified electrochemical multianalyte immunosensor for the simultaneous detection of high sensitivity C-reactive protein (hsCRP) and soluble CD40 ligand (sCD40L) that uses reduced graphene oxide-tetraethylene pentamine (rGO-TEPA) that directly adsorbs metal ions as labels is reported. rGO-TEPA contains a large number of amino groups and has excellent conductivity, making it an ideal template for the loading of Pb2+ and Cu2+, which greatly amplifies the detection signals. 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 peak reflects the identity and the level of the corresponding antigen. Primarily designed for an application in a sandwich-type immunoassay based on Pb2+ and Cu2+ labels, two main challenges are accomplished with the herein presented nanosheets: fabrication of the template and the amination process for Pb2+ and Cu2+ adsorption. 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 sCD40L 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 (S/N=3), respectively. The assay results on clinical serum samples with the proposed immunosensor were in acceptable agreement with those using the standard single-analyte test of the enzyme-linked immunosorbent assay (ELISA). This novel immunosensing system provides a simple, sensitive and low-cost approach for a multianalyte immunoassay.