The Integration of Reference Electrode for ISFET Ion Sensors Using Fluorothiophenol-Treated rGO.

Ion-sensitive field-effect transistors (ISFETs) detect specific ions in solutions that enable straightforward, fast, and inexpensive sensors compared to other benchtop equipment. However, a conventional reference electrode (RE) such as Ag/AgCl is limited on the miniaturization of the sensor. We introduce reduced graphene oxide (rGO), which serves as a new RE, when fluorinated (F-rGO) using fluorothiophenol through the π-π interaction. The circular RE is integrated between a fabricated microscale two-channel ISFET, which is capable of detecting two kinds of ions on an indium tin oxide (ITO) thin-film substrate, using the photolithography process. F-rGO bound to this circular region to function as an RE in the ISFETs sensor, which operated stably in solution and showed a relatively high transconductance (gm) value (1.27 mS), low drift characteristic (3.2 mV), and low hysteresis voltage (±0.05 mV). It detected proton (H+) ions in a buffer solution with high sensitivity (67.1 mV/pH). We successfully detected Na+ (62.1 mV/dec) and K+ (57.6 mV/dec) ions in human patient urine using a two-channel ISFET with the F-rGO RE. The F-rGO RE will be a suitable component in the fabrication of low-cost, mass-produced, and disposable ISFETs sensors.

Two-Dimensional Disposable Graphene Sensor to Detect Na+ Ions.

The monitoring of Na+ ions distributed in the body has been indirectly calculated by the detection of Na+ ions in urine. We fabricated a two-dimensional (2D) Na+ ion sensor using a graphene ion-sensitive field-effect transistor (G-ISFET) and used fluorinated graphene as a reference electrode (FG-RE). We integrated G-ISFET and FG on a printed circuit board (PCB) designed in the form of a secure digital (SD) card to fabricate a disposable Na+ ion sensor. The sensitivity of the PCB tip to Na+ ions was determined to be -55.4 mV/dec. The sensor exhibited good linearity despite the presence of interfering ions in the buffer solution. We expanded the evaluation of the PCB tip to real human patient urine samples. The PCB tip exhibited a sensitivity of -0.36 mV/mM and linearly detected Na+ ions in human patient urine without any dilution process. We expect that G-ISFET with FG-RE can be used to realize a disposable Na+ ion sensor by serving as an alternative to Ag/AgCl reference electrodes.

Two-Channel Graphene pH Sensor Using Semi-Ionic Fluorinated Graphene Reference Electrode.

A reference electrode is necessary for the working of ion-sensitive field-effect transistor (ISFET)-type sensors in electrolyte solutions. The Ag/AgCl electrode is normally used as a reference electrode. However, the Ag/AgCl reference electrode limits the advantages of the ISFET sensor. In this work, we fabricated a two-channel graphene solution gate field-effect transistor (G-SGFET) to detect pH without an Ag/AgCl reference electrode in the electrolyte solution. One channel is the sensing channel for detecting the pH and the other channel is the reference channel that serves as the reference electrode. The sensing channel was oxygenated, and the reference channel was fluorinated partially. Both the channels were directly exposed to the electrolyte solution without sensing membranes or passivation layers. The transfer characteristics of the two-channel G-SGFET showed ambipolar field-effect transistor (FET) behavior (p-channel and n-channel), which is a typical characteristic curve for the graphene ISFET, and the value of VDirac was shifted by 18.2 mV/pH in the positive direction over the range of pH values from 4 to 10. The leakage current of the reference channel was 16.48 nA. We detected the real-time pH value for the two-channel G-SGFET, which operated stably for 60 min in the buffer solution.

Artificial Differentiation of Hippocampal Neurons by Electrical Stimulation on Graphene Electrode.

Electrical stimulation therapy is a promising method for treating neurological diseases. This method induces the activity and differentiation of nerve cells by the direct or indirect transmission of an electrical signal through biomedical electrodes. We demonstrated the efficacy of a graphene sheet as a bioelectrode to differentiate neurites from hippocampal neuron, through electrical stimulation. In order to the artificially induce the differentiation of hippocampal neurons, we directly transmitted electrical signals of square pulse through the graphene electrode to directly stimulate neurons cultured onto graphene surface. Compared to cell culture plates, the average length of differentiated neurites increased 111.1% on pristine graphene with electrical stimulation. And the average number of differentiated neurites on a single cell increased to 281.9% on oxygenated graphene with electrical stimulation. Electrical stimulation with graphene electrodes promoted the differentiation of neurites and activated the production of intercellular networks of hippocampal neurons.

Detection of Alpha-Fetoprotein in Hepatocellular Carcinoma Patient Plasma with Graphene Field-Effect Transistor.

The detection of alpha-fetoprotein (AFP) in plasma is important in the diagnosis of hepatocellular carcinoma (HCC) in humans. We developed a biosensor to detect AFP in HCC patient plasma and in a phosphate buffer saline (PBS) solution using a graphene field-effect transistor (G-FET). The G-FET was functionalized with 1-pyrenebutyric acid N-hydroxysuccinimide ester (PBASE) for immobilization of an anti-AFP antibody. AFP was detected by assessing the shift in the voltage of the Dirac point (ΔVDirac) after binding of AFP to the anti-AFP-immobilized G-FET channel surface. This anti-AFP-immobilized G-FET biosensor was able to detect AFP at a concentration of 0.1 ng mL-1 in PBS, and the detection sensitivity was 16.91 mV. In HCC patient plasma, the biosensor was able to detect AFP at a concentration of 12.9 ng mL-1, with a detection sensitivity of 5.68 mV. The sensitivity (ΔVDirac) depended on the concentration of AFP in either PBS or HCC patient plasma. These data suggest that G-FET biosensors could have practical applications in diagnostics.

Effect of graphene on growth of neuroblastoma cells.

The unique properties of graphene have earned much interest in the fields of materials science and condensed-matter physics in recent years. However, the biological applications of graphene remain largely unexplored. In this study, we investigated the conditions and viability of a cell culture exposed to graphene onto glass and SiO2/Si, using a human nerve cell line, SH-SY5Y. Cell viability was 84% when cultured on glass and SiO2/Si coated with graphene as compared with culturing on polystyrene surface. Fluorescence data showed that the presence of graphene did not influence cell morphology. These findings suggest that graphene may be used for biological applications.