Graphene-Based Materials for Biosensors: A Review <s> Engineering of Biomolecules-Functionalized Graphene Based Biosensors <s> The development of sensing interfaces can significantly improve the performance of biological sensors. Graphene oxide provides a remarkable immobilization platform for surface plasmon resonance (SPR) biosensors due to its excellent optical and biochemical properties. Here, we describe a novel sensor chip for SPR biosensors based on graphene-oxide linking layers. The biosensing assay model was based on a graphene oxide film containing streptavidin. The proposed sensor chip has three times higher sensitivity than the carboxymethylated dextran surface of a commercial sensor chip. Moreover, the demonstrated sensor chips are bioselective with more than 25 times reduced binding for nonspecific interaction and can be used multiple times. We consider the results presented here of importance for any future applications of highly sensitive SPR biosensing. <s> BIB001 </s> Graphene-Based Materials for Biosensors: A Review <s> Engineering of Biomolecules-Functionalized Graphene Based Biosensors <s> Most of the reported field effect transistors (FETs) fall short of a general method to uniquely specify and detect a target analyte. For this reason, we propose a pentacene-based FET with a graphene oxide support system (GOSS), composed of functionalized graphene oxide (GO) ink. The GOSS with a specific moiety group to capture the biomaterial of interest was inkjet printed on the pentacene FET. It provided modular receptor sites on the surface of pentacene, without alteration of the device. To evaluate the performance of a GOSS-pentacene FET biosensor, we detected the artificial DNA and circulating tumor cells as a proof-of-concept. The mobility of the FET dramatically changed upon capturing the target biomolecule on the GOSS. The FET exhibited high selectivity with 0.1 pmoles of the target DNA and a few cancer cells per detection volume. This study suggests a valuable sensor for medical diagnosis that can be mass produced effortlessly at low-cost. <s> BIB002 </s> Graphene-Based Materials for Biosensors: A Review <s> Engineering of Biomolecules-Functionalized Graphene Based Biosensors <s> We present the construction of layer-by-layer (LbL) assemblies of polyethylenimine and urease onto reduced-graphene-oxide based field-effect transistors (rGO FETs) for the detection of urea. This versatile biosensor platform simultaneously exploits the pH dependency of liquid-gated graphene-based transistors and the change in the local pH produced by the catalyzed hydrolysis of urea. The use of an interdigitated microchannel resulted in transistors displaying low noise, high pH sensitivity (20.3µA/pH) and transconductance values up to 800 µS. The modification of rGO FETs with a weak polyelectrolyte improved the pH response because of its transducing properties by electrostatic gating effects. In the presence of urea, the urease-modified rGO FETs showed a shift in the Dirac point due to the change in the local pH close to the graphene surface. Markedly, these devices operated at very low voltages (less than 500mV) and were able to monitor urea in the range of 1-1000µm, with a limit of detection (LOD) down to 1µm, fast response and good long-term stability. The urea-response of the transistors was enhanced by increasing the number of bilayers due to the increment of the enzyme surface coverage onto the channel. Moreover, quantification of the heavy metal Cu2+(with a LOD down to 10nM) was performed in aqueous solution by taking advantage of the urease specific inhibition. <s> BIB003 </s> Graphene-Based Materials for Biosensors: A Review <s> Engineering of Biomolecules-Functionalized Graphene Based Biosensors <s> Since brain natriuretic peptide (BNP) has become internationally recognized biomarkers in the diagnosis and prognosis of heart failure (HF), it is highly desirable to search for a novel sensing tool for detecting the patient's BNP level at the early stage. Here we report a platinum nanoparticles (PtNPs)-decorated reduced graphene oxide (rGO) field effect transistor (FET) biosensor coupled with a microfilter system for label-free and highly sensitive detection of BNP in whole blood. The PtNPs-decorated rGO FET sensor was obtained by drop-casting rGO onto the pre-fabricated FET chip and subsequently assembling PtNPs on the graphene surface. After anti-BNP was bound to the PtNPs surface, BNP was successfully detected by the anti-BNP immobilized FET biosensor. It was found that the developed FET biosensor was able to achieve a low detection limitation of 100fM. Moreover, BNP was successfully detected in human whole blood sample treated by a custom-made microfilter, suggesting the sensor's capability of working in a complex sample matrix. The developed FET biosensor provides a new sensing platform for protein detection, showing its potential applications in clinic sample. <s> BIB004 </s> Graphene-Based Materials for Biosensors: A Review <s> Engineering of Biomolecules-Functionalized Graphene Based Biosensors <s> Abstract This work demonstrates the excellent potential of carboxyl-functionalized graphene oxide (GO–COOH) composites to form biocompatible surfaces on sensing films for use in surface plasmon resonance (SPR)-based immunoaffinity biosensors. Carboxyl-functionalization of graphene carbon can modulate its visible spectrum, and can therefore be used to improve and control the plasmonic coupling mechanism. The binding properties of the molecules between a sensing film and a protein were elucidated at various flow rates of those molecules. The bio-specific binding interaction among the molecules was investigated by performing an antigen and antibody affinity immunoassay. The results thus obtained revealed that the overall affinity binding value, K A , of the Au/GO–COOH chip can be significantly enhanced by up to ∼5.15 times that of the Au/GO chip. With respect to the shifts of the SPR angles of the chips, the affinity immunoassay interaction at a BSA concentration of 1 μg/ml for an Au/GO-COOH chip, an Au/GO chip and a traditional SPR chip are 35.5 m°, 9.128 m° and 8.816 m°, respectively. The enhancement of the antigen-antibody interaction of the Au/GO–COOH chip cause this chip to become four times as sensitive to the SPR angle shift and to have the lowest antibody detection limit of 0.01 pg/ml. These results indicate the potential of the chip in detecting specific proteins, and the development of real-time in vivo blood analysis and diagnosis based on cancer tumor markers. <s> BIB005 </s> Graphene-Based Materials for Biosensors: A Review <s> Engineering of Biomolecules-Functionalized Graphene Based Biosensors <s> Abstract A sensitive and selective optical biosensor based on reduced graphene oxide (RGO), which uses the polarization-dependent absorption effect of graphene under total internal reflection, is reported for determination of rabbit IgG. RGO sheets with the thickness of about 8.1 nm are fabricated by high temperature reduction and used as a sensing film, because of its strong polarization dependent absorption. This RGO-based optical sensor shows a satisfactory response to rabbit IgG with a minimum concentration of 0.0625 μg/ml. As a contrast, commercial SPR apparatus is used to investigate rabbit IgG with a minimum concentration of 0.3125 μg/ml. Moreover, with antigen–antibody binding, this sensor can also achieve label-free and real-time detection. Taking into account these factors, the RGO-based optical sensor may be a potential candidate for biosensing. <s> BIB006 </s> Graphene-Based Materials for Biosensors: A Review <s> Engineering of Biomolecules-Functionalized Graphene Based Biosensors <s> Abstract This paper presents a “turn-on” fluorescence biosensor based on graphene quantum dots (GQDs) and molybdenum disulfide (MoS 2 ) nanosheets for rapid and sensitive detection of epithelial cell adhesion molecule (EpCAM). PEGylated GQDs were used as donor molecules, which could not only largely increase emission intensity but also prevent non-specific adsorption of PEGylated GQD on MoS 2 surface. The sensing platform was realized by adsorption of PEGylated GQD labelled EpCAM aptamer onto MoS 2 surface via van der Waals force. The fluorescence signal of GQD was then quenched by MoS 2 nanosheets via fluorescence resonance energy transfer (FRET) mechanism. In the presence of EpCAM protein, the stronger specific affinity interaction between aptamer and EpCAM protein could detach GQD labelled EpCAM aptamer from MoS 2 nanosheets, leading to the restoration of fluorescence intensity. By monitoring the change of fluorescence signal, the target EpCAM protein could be detected sensitively and selectively with a linear detection range from 3 nM to 54 nM and limit of detection (LOD) around 450 pM. In addition, this nanobiosensor has been successfully used for EpCAM-expressed breast cancer MCF-7 cell detection. <s> BIB007
Biosensors based on the subtype of graphene materials or functionalized graphene GO, RGO, and GQD are widely used in medicine, biomedical, and bioimaging regimes. This is due to their extremely large surface area and ability to interact with various types of molecules. In addition, the outstanding properties of solubility, biocompatibility, and functionalization play an important role in sensing mechanisms. Currently, as shown in Figure 8 , the GO-based FET has simply demonstrated glucose detection without an enzymatic glucose solution  . In this device, GO is used as the selective material to glucose while the sensitivity of the sensor is enhanced down to 1 µM by adding CuNPS and AgNPs. Interestingly, functionalized graphene oxide (GO) ink has been printed on a pentacene FET for detecting artificial DNA and circulating tumor cells BIB002 . Upon capturing the DNA by its phosphate group, the negative charge attracts holes at the grain-boundary of the pentacene layer and induces the collision or scattering in the region of the pentacene layer. Therefore, the mobility of the FET changes extremely achieving a high sensitivity of 0.1 pM and this could be improved for mass-scale production of printed biosensors. In a platform for ultra-sensitive urea detection, the RGO surface is decorated by the new construction of layer-by-layer assemblies of polyethylenimine (PEI) and urease BIB003 . The RGO FET of urea detection can be analyzed as the change of pH in liquid gate, by which shifting the Dirac point at the minimum voltage of <500 mV. The limit of urea detection is down to 1 µM with very fast response and good long-term stability. In addition, the introduction the Cu 2+ improves the LOD down to 0.01 µM. In recent complex platforms, FET biosensors using RGO combined with PtNPS and anti-BNP have been explored as a brain natriuretic peptide (BNP) detector at the early stage level BIB004 . The BNP is a recognized biomarker and it is very important in heart failure diagnosis and prognosis. The RGO FET achieves the lowest of detection at 100 fM in a human whole blood sample. Surface plasmon resonance (SPR) is a widely used technique to investigate biochemical reactions in scientific research and medical diagnosis  . In particular, SPR provides label-free biosensing and real-time monitoring of biomolecule interactions. However, for small molecules or at low concentrations of the targets, the SPR signal is not sufficient to be analyzed. To improve the SPR signal and biosensing performance, linking layers of GO have been introduced into the SPR sensor system BIB001 . The sensor chip consists of gold sensor chips using PMMA as an intermediate membrane, s monolayer of CVD graphene on top, and the biotin-SA conjugate, respectively. The SA molecules allow the biosensor to select the immobilized biomolecules containing biotin. The linking layers of GO in the system provides a number of binding sites for biomolecules due to the large surface area. However, a GO thickness of more than 10 nm strongly limited the optical absorption leading to a sensitivity reduction. Recently, there has been a demonstration showing the improvement and control of the plasmonic coupling mechanism in GO SPR-based immunoaffinity biosensors by adding carboxyl groups BIB005 . The GO-COOH SPR chip can be improved four times over the SPR angle shift and achieved the lowest antibody detection limit of 0.01 pg/mL. Other work has reported the reduction of GO-based SPR fabricated by thermal reduction at high temperature, the so-called RGO SPR, with a thickness of 8.1 nm BIB006 . The performance of the RGO SPR biosensor shows a response to rabbit immunoglobulin G (rabbit IgG) with a LOD of 0.0625 µg/mL. Currently, fluorescence biosensors based on the GQDs have gained much attention as an alternative choice due to their ease of the synthesis, good stability, fast tissue internalization, and biocompatibility. The fluorescence biosensor relies on the energy transfer between the electron donor and acceptor which is powerful for drug delivery and biomolecular interactions at the nanoscale. Fluorescence resonance energy transfer (FRET) is a mechanism to describe the energy transfer between two fluorescent molecules, where one is a donor being in an excited state and ready to transfer to the other one or an acceptor via a non-radiative dipole-dipole coupling  . the so-called RGO SPR, with a thickness of 8.1 nm BIB006 . The performance of the RGO SPR biosensor shows a response to rabbit immunoglobulin G (rabbit IgG) with a LOD of 0.0625 μg/mL. Currently, fluorescence biosensors based on the GQDs have gained much attention as an alternative choice due to their ease of the synthesis, good stability, fast tissue internalization, and biocompatibility. The fluorescence biosensor relies on the energy transfer between the electron donor and acceptor which is powerful for drug delivery and biomolecular interactions at the nanoscale. Fluorescence resonance energy transfer (FRET) is a mechanism to describe the energy transfer between two fluorescent molecules, where one is a donor being in an excited state and ready to transfer to the other one or an acceptor via a non-radiative dipole-dipole coupling  . This simple technique has been reported in a novel FRET based on GQD-PEG aptamer/MoS2 for the detection of epithelial cell adhesion molecule (EpCAM), which is a glycosylated membrane protein expressed on the surface of circulating tumor cells (CTCs) BIB007 . In the mechanism, GQD is used as the FRET donor that emits fluorescence at 466 nm under an excitation of 360 nm. MoS2 having a good quenching ability is the acceptor. When the PEG is conjugated onto GQD, the PEGylated/GQD exhibits a stronger fluorescence emission because of the quantum confinement. Then the PEGylated/GQD is conjugated with the aptamer via the van der Waals binding causing a proximity BIB004 ); (e) Biotin-SA/GO SPR chip (reprinted with permission from BIB001 ); (f) BSA biosensor based on GO-COOH enhanced SPR (reprinted with permission from BIB005 ); (g) rabbit IgG detection based on RGO SPR (reprinted with permission from BIB006 ); and (h) FRET biosensor based on GQD-PEG aptamer/MoS 2 (reprinted with permission from BIB007 ). This simple technique has been reported in a novel FRET based on GQD-PEG aptamer/MoS 2 for the detection of epithelial cell adhesion molecule (EpCAM), which is a glycosylated membrane protein expressed on the surface of circulating tumor cells (CTCs) BIB007 . In the mechanism, GQD is used as the FRET donor that emits fluorescence at 466 nm under an excitation of 360 nm. MoS 2 having a good quenching ability is the acceptor. When the PEG is conjugated onto GQD, the PEGylated/GQD exhibits a stronger fluorescence emission because of the quantum confinement. Then the PEGylated/GQD is conjugated with the aptamer via the van der Waals binding causing a proximity of GQD and MoS 2 and quenching GQD. When the EpCAM protein with a strong binding affinity is introduced, the GQD could label on EpCAM aptamer and restores its fluorescence. Therefore, the EpCAM target protein can be monitored by the fluorescence emission.