Graphene-Based Materials for Biosensors: A Review <s> Engineering of Pristine Graphene−Biomolecule-based Biosensors <s> The present work describes the investigations of electrochemical interactions of cholera toxin with stabilized lipid films supported on a methacrylate polymer on a glass fiber filter with incorporated ganglioside GM1 for the development of a biosensor for cholera toxin. The analyte was injected into the flowing streams of a carrier electrolyte solution, the flow of the solution stops for 5 min and an ion current transient was obtained, the magnitude of which is correlated to the toxin concentration with detection limits of 0.06 µM. The method was applied in blood serum samples. Further work is directed to the rapid detection of other toxins used in bioterrorism using this novel ultrathin film technology. <s> BIB001 </s> Graphene-Based Materials for Biosensors: A Review <s> Engineering of Pristine Graphene−Biomolecule-based Biosensors <s> The present article describes a miniaturized potentiometric urea lipid film based biosensor on graphene nanosheets. Structural characterization of graphene nanosheets for miniaturization of potentiometric urea lipid film based biosensors have been studied through atomic force microscopy (AFM) and transmission electron microscopy (TEM) measurements. UV-Vis and Fourrier transform IR (FTIR) spectroscopy have been utilized to study the pre- and postconjugated surfaces of graphene nanosheets. The presented potentiometric urea biosensor exhibits good reproducibility, reusability, selectivity, rapid response times (similar to 4 s), long shelf life and high sensitivity of ca. 70 mV/decade over the urea logarithmic concentration range from 1 x 10(-6) M to 1 x 10(-3) M. <s> BIB002 </s> Graphene-Based Materials for Biosensors: A Review <s> Engineering of Pristine Graphene−Biomolecule-based Biosensors <s> The present article describes a miniaturized potentiometric D-dimer biosensor on graphene nanosheets with incorporated lipid films. The graphene electrode was used for the development of a very selective and sensitive immunosensor for the detection of D-dimer by immobilizing the mouse anti human D-dimer antibody on stabilized lipid films. The immunosensor responded for the wide range of D-dimer concentrations with fast response time of ca. 15 s. The presented potentiometric D-dimer biosensor is easy to construct and exhibits good reproducibility, reusability, selectivity, rapid response times, long shelf life and high sensitivity of ca. 59 mV/decade over the D-dimer logarithmic concentration range from 10−6 μg/L to 10−3 μg/L. <s> BIB003 </s> Graphene-Based Materials for Biosensors: A Review <s> Engineering of Pristine Graphene−Biomolecule-based Biosensors <s> The present article describes a miniaturized potentiometric carbofuran chemical sensor on graphene nanosheets with incorporated lipid films. The graphene electrode was used for the development of a very selective and sensitive chemical sensor for the detection of carbofuran by immobilizing an artificial selective receptor on stabilized lipid films. The artificial receptor was synthesized by transformation of the hydroxyl groups of resorcin[4]arene receptor into phosphoryl groups. This chemical sensor responded for the wide range of carbofuran concentrations with fast response time of ca. 20 s. The presented potentiometric carbofuran chemical sensor is easy to construct and exhibits good reproducibility, reusability, selectivity, rapid response times, long shelf life and high sensitivity of ca. 59 mV/decade over the carbofuran logarithmic concentration range from 10−6 to 10−3 M. <s> BIB004 </s> Graphene-Based Materials for Biosensors: A Review <s> Engineering of Pristine Graphene−Biomolecule-based Biosensors <s> Lead is a cumulative toxicant, which can induce severe health issues, especially in children’s case due to their immature nervous system. While realizing large-scale monitoring of children blood lead remains challenging by utilizing traditional methods, it is highly desirable to search for alternative techniques or novel sensing materials. Here we report a label-free and portable aptasensor based on graphene field effect transistor (FET) for effective children blood lead detection. With standard solutions of different Pb2+ concentrations, we obtained a dose-response curve and a detection limitation below 37.5 ng/L, which is three orders lower than the safe blood lead level (100 μg/L). The devices also showed excellent selectivity over other metal cations such as, Na+, K+, Mg2+, and Ca2+, suggesting the capability of working in a complex sample matrix. We further successfully demonstrated the detection of Pb2+ ions in real blood samples from children by using our aptasensors, and explored their potential applications for quantification. Our results underscore such graphene FET aptasensors for future applications on fast detection of heavy metal ions for health monitoring and disease diagnostics. <s> BIB005 </s> Graphene-Based Materials for Biosensors: A Review <s> Engineering of Pristine Graphene−Biomolecule-based Biosensors <s> This work presents a fully integrated graphene field-effect transistor (GFET) biosensor for the label-free detection of lead ions (Pb2+) in aqueous-media, which first implements the G-quadruplex structure-switching biosensing principle in graphene nanoelectronics. We experimentally illustrate the biomolecular interplay that G-rich DNA single-strands with one-end confined on graphene surface can specifically interact with Pb2+ ions and switch into G-quadruplex structures. Since the structure-switching of electrically charged DNA strands can disrupt the charge distribution in the vicinity of graphene surface, the carrier equilibrium in graphene sheet might be altered, and manifested by the conductivity variation of GFET. The experimental data and theoretical analysis show that our devices are capable of the label-free and specific quantification of Pb2+ with a detection limit down to 163.7ng/L. These results first verify the signaling principle competency of G-quadruplex structure-switching in graphene electronic biosensors. Combining with the advantages of the compact device structure and convenient electrical signal, a label-free GFET biosensor for Pb2+ monitoring is enabled with promising application potential. <s> BIB006 </s> Graphene-Based Materials for Biosensors: A Review <s> Engineering of Pristine Graphene−Biomolecule-based Biosensors <s> Abstract A label-free immunosensor based on antibody-modified graphene field effect transistor (GFET) was presented. Antibodies targeting carcinoembryonic antigen (Anti-CEA) were immobilized to the graphene surface via non-covalent modification. The bifunctional molecule, 1-pyrenebutanoic acid succinimidyl ester, which is composed of a pyrene and a reactive succinimide ester group, interacts with graphene non-covalently via π-stacking. The succinimide ester group reacts with the amine group to initiate antibody surface immobilization, which was confirmed by X-ray Photoelectron Spectroscopy, Atomic Force Microscopy and Electrochemical Impedance Spectroscopy. The resulting anti-CEA modified GFET sufficiently monitored the reaction between CEA protein and anti-CEA in real-time with high specificity, which revealed selective electrical detection of CEA with a limit of detection (LOD) of less than 100 pg/ml. The dissociation constant between CEA protein and anti-CEA was estimated to be 6.35×10 −11 M, indicating the high affinity and sensitivity of anti-CEA-GFET. Taken together, the graphene biosensors provide an effective tool for clinical application and point-of-care medical diagnostics. <s> BIB007 </s> Graphene-Based Materials for Biosensors: A Review <s> Engineering of Pristine Graphene−Biomolecule-based Biosensors <s> Reliable determination of binding kinetics and affinity of DNA hybridization and single-base mismatches plays an essential role in systems biology, personalized and precision medicine. The standard tools are optical-based sensors that are difficult to operate in low cost and to miniaturize for high-throughput measurement. Biosensors based on nanowire field-effect transistors have been developed, but reliable and cost-effective fabrication remains a challenge. Here, we demonstrate that a graphene single-crystal domain patterned into multiple channels can measure time- and concentration-dependent DNA hybridization kinetics and affinity reliably and sensitively, with a detection limit of 10 pM for DNA. It can distinguish single-base mutations quantitatively in real time. An analytical model is developed to estimate probe density, efficiency of hybridization and the maximum sensor response. The results suggest a promising future for cost-effective, high-throughput screening of drug candidates, genetic variations and disease biomarkers by using an integrated, miniaturized, all-electrical multiplexed, graphene-based DNA array. <s> BIB008 </s> Graphene-Based Materials for Biosensors: A Review <s> Engineering of Pristine Graphene−Biomolecule-based Biosensors <s> A novel paper-based electrochemical biosensor was developed using an anthraquinone-labeled pyrrolidinyl peptide nucleic acid (acpcPNA) probe (AQ-PNA) and graphene-polyaniline (G-PANI) modified electrode to detect human papillomavirus (HPV). An inkjet printing technique was employed to prepare the paper-based G-PANI-modified working electrode. The AQ-PNA probe baring a negatively charged amino acid at the N-terminus was immobilized onto the electrode surface through electrostatic attraction. Electrochemical impedance spectroscopy (EIS) was used to verify the AQ-PNA immobilization. The paper-based electrochemical DNA biosensor was used to detect a synthetic 14-base oligonucleotide target with a sequence corresponding to human papillomavirus (HPV) type 16 DNA by measuring the electrochemical signal response of the AQ label using square-wave voltammetry before and after hybridization. It was determined that the current signal significantly decreased after the addition of target DNA. This phenomenon is explained by the rigidity of PNA-DNA duplexes, which obstructs the accessibility of electron transfer from the AQ label to the electrode surface. Under optimal conditions, the detection limit of HPV type 16 DNA was found to be 2.3 nM with a linear range of 10-200 nM. The performance of this biosensor on real DNA samples was tested with the detection of PCR-amplified DNA samples from the SiHa cell line. The new method employs an inexpensive and disposable device, which easily incinerated after use and is promising for the screening and monitoring of the amount of HPV-DNA type 16 to identify the primary stages of cervical cancer. <s> BIB009 </s> Graphene-Based Materials for Biosensors: A Review <s> Engineering of Pristine Graphene−Biomolecule-based Biosensors <s> Graphene is one of the new materials which has shown a large impact on the electronic industry due to its versatile properties, such as high specific surface area, high electrical conductivity, chemical stability, and large spectrum of electrochemical properties. The graphene material-based electronic industry has provided flexible devices which are inexpensive, simple and low power-consuming sensor tools, therefore opening an outstanding new door in the field of portable electronic devices. All these attractive advantages of graphene give a platform for the development of a new generation of devices in both food and environmental applications. Lipid-based sensors have proven to be a good route to the construction of novel devices with improved characteristics, such as fast response times, increased sensitivity and selectivity, and the possibility of miniaturization for the construction of portable biosensors. Therefore, the incorporation of a lipid substrate on graphene electrodes has provided a route to the construction of a highly sensitive and selective class of biosensors with fast response times and portability of field applications for the rapid detection of toxicants in the environment and food products. <s> BIB010 </s> Graphene-Based Materials for Biosensors: A Review <s> Engineering of Pristine Graphene−Biomolecule-based Biosensors <s> A miniaturized potentiometric saxitoxin sensor on graphene nanosheets with incorporated lipid films and Anti-STX, the natural saxitoxin receptor, immobilized on the stabilized lipid films is described in the present paper. An adequate selectivity for detection over a wide range of toxin concentrations, fast response time of ca. 5–20 min, and detection limit of 1 nM have been achieved. The proposed sensor is easy to construct and exhibits good reproducibility, reusability, selectivity, long shelf life and high sensitivity of ca. 60 mV/decade of toxin concentration. The method was implemented and evaluated in lake water and shellfish samples. This novel ultrathin film technology is currently adapted to the rapid detection of other toxins that could be used in bioterrorism. <s> BIB011
In the graphene-based biosensors, graphene is able to enhance the sensitivity and LOD as well as the performance of the biosensor device by improving the charge or electron transfer between graphene and the biomolecules due to its extraordinary properties. For example, as seen in Figure 7 , a label-free and portable aptasensor utilizes pristine graphene as the electrode in a field-effect transistor device BIB005 . The GFET biosensor is used to detect the Pb 2+ ions in children's blood, in which the blood matrix is very complicated. The mechanism to distinguish Pb 2+ ions from common ions in the blood, including Na + , K + , Mg 2+ and Ca 2+ at lower 0.1 M/L, is intrinsically p-doping on the CVD graphene and the surface engineering by G-quadruplex, Thrombin binding aptamer (TBA), and 8-17 DNAzyme. The lowest concentration of GFET aptasensor is at 37.5 ng/L, which was approximately one thousandth of the safety limit (100 µg/L) for Pb 2+ in blood. A GFET construction shows a similar Pb 2+ detecting platform but using G-quadruplex as the receptor BIB006 . The mechanism is due to the electrostatic potential change after the lead combines to the double layer of DNA/CVD graphene electrodes leading to the shift in Dirac point in the band structure of graphene. For this device, the LOD is only 163.7 ng/L for the first signal verification of DNA/GFET. Carcinoembryonic antigen (CEA) is a protein that can be measured in the blood of a cancer patient. Recently, a label-free immunosensor based on the antibody-modified graphene FET was reported BIB007 . The surface modification is applied via a non-covalent functionalization and π-stacking using a pyrene and a reactive succinimide ester group to interact with graphene. The GFET biosensor shows the specific monitoring of the CEA protein in real-time with high sensitivity of <100 pg/mL. In the precise quantitative measurement of DNA concentrations as well as binding affinities and kinetics of DNA hybridization, an array of six CVD graphene-based FETs was fabricated in a single multiplexed sensor for DNA analysis BIB008 . The concentration of oligonucleotides can be measured as low as 10 pM in which the single-single-base mutations can be analyzed in real time.  In the graphene-based biosensors, graphene is able to enhance the sensitivity and LOD as well as the performance of the biosensor device by improving the charge or electron transfer between graphene and the biomolecules due to its extraordinary properties. For example, as seen in Figure 7 , a label-free and portable aptasensor utilizes pristine graphene as the electrode in a field-effect transistor device BIB005 . The GFET biosensor is used to detect the Pb 2+ ions in children's blood, in which the blood matrix is very complicated. The mechanism to distinguish Pb 2+ ions from common ions in the blood, including Na + , K + , Mg 2+ and Ca 2+ at lower 0.1 M/L, is intrinsically p-doping on the CVD graphene and the surface engineering by G-quadruplex, Thrombin binding aptamer (TBA), and 8-17 DNAzyme. The lowest concentration of GFET aptasensor is at 37.5 ng/L, which was approximately one thousandth of the safety limit (100 μg/L) for Pb 2+ in blood. A GFET construction shows a similar Pb 2+ detecting platform but using G-quadruplex as the receptor BIB006 . The mechanism is due to the electrostatic potential change after the lead combines to the double layer of DNA/CVD graphene electrodes leading to the shift in Dirac point in the band structure of graphene. For this device, the LOD is only 163.7 ng/L for the first signal verification of DNA/GFET. Carcinoembryonic antigen (CEA) is a protein that can be measured in the blood of a cancer patient. Recently, a label-free immunosensor based on the antibody-modified graphene FET was reported BIB007 . The surface modification is applied via a non-covalent functionalization and π-stacking using a pyrene and a reactive succinimide ester group to interact with graphene. The GFET biosensor shows the specific monitoring of the CEA protein in real-time with high sensitivity of <100 pg/mL. In the precise quantitative measurement of DNA concentrations as well as binding affinities and kinetics of DNA hybridization, an array of six CVD graphene-based FETs was fabricated in a single multiplexed sensor for DNA analysis BIB008 . The concentration of oligonucleotides can be measured as low as 10 pM in which the single-single-base mutations can be analyzed in real time. BIB008 ); (e) paper-based biosensor for human papillomavirus (HPV) detection (reprinted with permission from BIB009 ); and (f) a lipid-based modified graphene electrochemical biosensor (reprinted with permission from BIB010 ). In the use of electrochemical properties of graphene material, a novel paper-based biosensor for human papillomavirus (HPV) detection was reported BIB009 . The graphene-polyaniline (G-PANI) BIB008 ); (e) paper-based biosensor for human papillomavirus (HPV) detection (reprinted with permission from BIB009 ); and (f) a lipid-based modified graphene electrochemical biosensor (reprinted with permission from BIB010 ). In the use of electrochemical properties of graphene material, a novel paper-based biosensor for human papillomavirus (HPV) detection was reported BIB009 . The graphene-polyaniline (G-PANI) electrode is modified using an anthraquinone-labeled pyrrolidinyl peptide nucleic acid (acpcPNA) probe (AQ-PNA) and printed by inkjet printing method. In a presence of surface engineering of a negatively charged amino acid on graphene electrode through the electrostatic attraction, a synthetic 14-base oligonucleotide target with a sequence corresponding to human papillomavirus (HPV) type 16 DNA is measured the electrochemical signal response of the AQ label to identify the primary stages of cervical cancer. On the development of electrochemical technology, graphene microelectrodes integrated with bilayer lipid membranes (BLMs) have shown promising results in both static and stirred experiments BIB010 . Moreover, due to the support made of lipid film, the biosensor achieves a good reproducibility, reusability, high selectivity, rapid response times, long-shelf life, and high sensitivity. This enables a direct potentiometric measurement. Nikolelis et al. have also reported the use of the graphene microelectrodes in detecting toxicants, i.e., carbofuran in fruit BIB004 , saxitoxin BIB011 , cholera toxin BIB001 and for diagnosis of D-dimers BIB003 , urea BIB002 and cholesterol as seen in Figure 7f .