Graphene based electrochemical sensors have also been developed to detect various protein biomarkers. A label-free immuno-sensor to specifically detect cancer marker alpha fetoprotein (AFP) using layer-by-layer construction with Electropolymerized Thionine (TH) film, GO–chitosan composite, AuNPs, and conjugates of horseradish peroxidase (HRP) and anti-AFP antibody has been fabricated and reported in the literature.
Binding of AFP molecules to the antibodies partially blocks the active center of HRP and consequently decreases the catalytic reduction of H2O2 by HRP (thus a decrease in the electrochemical signal). The electroactive TH acts synergistically with HRP to mediate the electron transfer from H2O2 to the electrode. The achieved LOD (0.7 ng ml-1) is much better than the conventional Enzyme-Linked Immuno-Sorbent Assays (ELISA). This sensor was challenged with clinical human serum samples and the negative/positive samples were correctly identified in accordance with the results from a commercial clinical device.
A simpler AFP sensor was made by incorporating TH with RGO film through p–p interaction followed by covalent cross linking of AFP antibodies with TH. Binding of AFP molecules blocks the electron-transfer and mass-transfer, leading to a decrease of electrochemical signal originated from the redox reactions of TH.
In comparison with other sensors, such as carbon nanotube or nanoparticle derived AFP sensors, a much lower LOD (5.77 pg ml-1) was achieved, due to the high electron transfer rate between the intimately interacted RGO and TH, and high loading of TH molecules and AFP-antibodies because of the large surface area provided by the RGO film. The sensor was successfully used to determine AFP in serum samples.
A different strategy to detect AFP was also demonstrated. AFP molecules bound to the primary-antibody functionalized RGO electrode complex again with carbon nanospheres (CNS) tagged with the secondary antibodies and HRP molecules, leading to an increased electrochemical signal from redox reaction of H2O2. The use of RGO and CNS gave a 7-fold increase in the detection sensitivity, because of the superior electrochemical and electrical properties of RGO and the ability of CNS to carry multiple HRP molecules.
A 20 pg ml-1 LOD was demonstrated. A similar sensor to detect prostate-specific antigen (PSA) (marker for prostate cancer) based on sandwich immuno-reactions on top of RGO modified electrode has been reported. In comparison, the CNS were replaced by small RGO flakes, because RGO flakes can carry more secondary antibodies and more HRP molecules due to their extremely large surface-to volume ratio. Dual functionalities of RGO were utilized, i.e., first as the electrode material and second as the enzyme carrier. An impressive detection limit of 1 pg ml-1 was demonstrated, superior to other PSA sensors including a sensor using carbon nanotube–HRP conjugates.
A sandwich-like immuno-detection of carcino-embryonic antigen (CEA) which is a marker for colorectal cancer was developed and reported. In their work, a nanocomposite of gold nanoparticles (AuNPs), RGO and chitosan was used to carry multi-copies of the HRP-conjugated CEA-specific secondary antibody onto a glassy carbon electrode modified with Prussian blue and AuNP. 10 pg ml-1 CEA can be detected.
In another demonstration, a RGO modified electrode for sandwich-like immuno-detection of immuno-globulin G (IgG) in human serum was developed.
Figure: Schematic illustration of an electrochemical immunosensor for detection of prostate specific antigen (PSA). GS = reduced graphene oxide sheet; TH = thionine; HRP = horseradish peroxidase; Ab2 = secondary anti-PSA antibody; Ab1 = primary anti-PSA antibody; GC = glassy carbon electrode.
Extracted and edited from “Biological and chemical sensors based on graphene materials by Yuxin Liu, Xiaochen Dong and Peng Chen in Chemical Society Reviews, 2012″