Graphene Oxide Signal Reporter Based Multifunctional Immunosensing Platform for Amperometric Profiling of Multiple Cytokines in Serum

Cytokines are small proteins and form complicated cytokine networks to report the status of our health. Thus, accurate profiling and sensitive quantification of multiple cytokines is essential to have a comprehensive and accurate understanding of the complex physiological and pathological conditions in the body. In this study, we demonstrated a robust electrochemical immunosensor for the simultaneous detection of three cytokines IL-6, IL-1β, and TNF-α. First, graphene oxides (GO) were loaded with redox probes nile blue (NB), methyl blue (MB), and ferrocene (Fc), followed by covalent attachment of anti-cytokine antibodies for IL-6, IL-1β, and TNF-α, respectively, to obtain Ab₂-GO-NB, Ab₂-GO-MB, and Ab₂-GO-Fc, acting as the signal reporters. The sensing interface was fabricated by attachment of mixed layers of 4-carboxylic phenyl and 4-aminophenyl phosphorylcholine (PPC) to glassy carbon surfaces. After that, the capture monoclonal antibody for IL-6, IL-1β, and TNF-α was modified to the carboxylic acid terminated sensing interface. And finally a sandwich assay was developed. The quantitative detection of three cytokines was achieved by observing the change in electrochemical signal from signal reporters Ab₂-GO-NB, Ab₂-GO-MB, and Ab₂-GO-Fc. The designed system has been successfully used for detection of three cytokines (IL-6, IL-1β, and TNF-α) simultaneously with desirable performance in sensitivity, selectivity, and stability, and recovery of 93.6%–105.5% was achieved for determining cytokines spiked in the whole mouse serum

Decoration of Reduced Graphene Oxide Nanosheets with Aryldiazonium Salts and Gold Nanoparticles toward a Label-Free Amperometric Immunosensor for Detecting Cytokine Tumor Necrosis Factor‑α in Live Cells

In this study, a label-free electrochemical immunosensor was developed for detection of cytokine tumor necrosis factor-alpha (TNF-α). First, AuNPs loaded reduced graphene oxides nanocomposites (RGO-ph-AuNP) were prepared, and then, a mixed layer of 4-carbxyphenyl and 4-aminophenyl phosphorylcholine (PPC) was modified to the surface of AuNPs for the subsequent modification of anti-TNF-α capture antibody (Ab₁) to form the capture surface (Au-RGO-ph-AuNP-ph-PPC­(-ph-COOH)) for the analyte TNF-α with the antifouling property. For reporting the presence of analyte, the anti-TNF-α detection antibody (Ab₂) was modified to the graphene oxides which have been modified with the 4-ferrocenylaniline through diazonium chemistry to form Ab₂-GO-ph-Fc. Then, a sandwich assay was formed on gold surfaces for the quantitative detection of TNF-α based on the electrochemical signal of ferrocene. X-ray photoelectron spectra (XPS), transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FT-IR), UV–vis, and electrochemistry were used for characterization of the stepwise fabrications on the interface. The prepared electrochemical immunosensor was successfully used for the detection of TNF-α over the range of 0.1–150 pg mL^–1. The lowest detection limit of this immunosensor is 0.1 pg mL^–1 TNF-α in 50 mM phosphate buffer at pH 7.0. The fabricated immunosensor provided high selectivity and stability and can be used to detect TNF-α secreted by live BV-2 cells with comparable accuracy to enzyme-linked immunosorbent assay (ELISA) but with lower limit of detection

Graphene Oxide Thin Film with Dual Function Integrated into a Nanosandwich Device for in Vivo Monitoring of Interleukin‑6

Graphene oxide (GO), with its exceptional physical and chemical properties and biocompatibility, holds a tremendous potential for sensing applications. In this study, GO, acting both as the electron-transfer bridge and the signal reporter, was attached on the interface to develop a label-free electrochemical nanosandwich device for detection of interleukin-6 (IL-6). First, a single layer of GO was covalently modified on gold electrodes, followed by attachment of anti-IL-6 capture antibody to form the sensing interface. The 4-aminophenyl phosphorylcholine was further attached to the surface of GO to minimize nonspecific protein adsorption. For reporting the presence of analyte, the anti-IL-6 detection antibody was covalently modified to the GO, which has been integrated with the redox probe Nile blue (NB). Finally, a nanosandwich assay was fabricated on gold surfaces for detection of IL-6 on the basis of the electrochemical signal of NB. The prepared nanosandwiches demonstrated high selectivity and stability for detection of IL-6 over the range of 1–300 pg mL^–1 with the lowest detectable concentration of 1 pg mL^–1. The device was successfully used for monitoring of IL-6 secretion in RAW cells and live mice. By tailoring the GO surface with functional components, such devices were able to detect the analyte in vivo without causing inflammatory response