key: cord-1003878-lp6pwpe4
authors: Wang, Shiyu; Hossain, Md Zakir; Shinozuka, Kazuo; Shimizu, Natsuhiko; Kitada, Shunya; Suzuki, Takaaki; Ichige, Ryo; Kuwana, Anna; Kobayashi, Haruo
title: Graphene field-effect transistor biosensor for detection of biotin with ultrahigh sensitivity and specificity
date: 2020-06-04
journal: Biosens Bioelectron
DOI: 10.1016/j.bios.2020.112363
sha: 2fc4134eee7d705343f0d0fbf0b9df4094a868b5
doc_id: 1003878
cord_uid: lp6pwpe4

Because avidin and biotin molecules exhibit the most specific and strongest non-covalent interaction, avidin-biotin technology is widely used in ELISA (enzyme-linked immunosorbent assay) kits for the detection of different bio-macromolecules linked to different diseases including cancer and influenza. Combining the outstanding electrical conductivity (200,000 cm(2)V(-1)s(-1)) of graphene with the unique avidin and biotin interaction, we demonstrate a novel graphene field-effect transistor (GFET) biosensor for the quantitative detection of bio-macromolecules. The GFET consists of six pairs of interdigital Cr/Au electrodes supported on Si/SiO(2) substrate with an avidin immobilized single layer graphene channel as the sensing platform. By monitoring the real time current change upon the addition of biotin solution in bovine serum albumin (BSA) in the silicone pool preformed onto the GFET, the lowest detectable biotin concentration is estimated to be 90 fg/ml (0.37 pM). The specificity of the GFET is confirmed both by controlled and real sample measurements From the magnitude of current change upon the addition of different concentrations of biotin solutions, the dissociation constant K(d) is estimated to be 1.6 × 10(-11) M. Since biotin is capable of conjugating with proteins, nucleotides and other bio-macromolecules without altering their properties, the present GFET sensor with its ultra-high sensitivity (0.37 pM) and specificity can be tailored to the rapid point-of-care detection of different types of desired biomolecules at very low concentration level through biotinylation as well as the exogenous biotin in blood serum.

Since biotin is capable of conjugating with proteins, nucleotides and other bio-macromolecules 28 without altering their properties, the present GFET sensor with its ultra-high sensitivity (0.37 pM) 29 and specificity can be tailored to the rapid point-of-care detection of different types of desired 30 biomolecules at very low concentration level through biotinylation as well as the exogenous biotin 31 in blood serum. 32 33 34 Keywords: Graphene, Field-Effect Transistor, Biosensor, Avidin, Biotin, Clinical diagnosis. The strong interaction between avidin and biotin has been widely exploited in many 3 applications such as protein and nucleic acid detection, immobilization, and purification methods 4 (Guesdon et al., 1979; Hsu et al., 1981a Hsu et al., , 1981b . Avidin is a tetrameric biotin-binding protein 5 produced in the oviducts of birds, reptiles, and amphibians and is deposited in the whites of their 6

eggs. Dimeric members of the avidin family are also found in some bacteria (Helppolainen et al., 7 2007 ). In chicken egg white, avidin makes up approximately 0.05 % of total protein 8 (approximately 1800 µg per egg). The tetrameric protein contains four identical subunits 9

(homotetramer), each of which can bind to biotin (vitamin B7, vitamin H) with a high degree of 10 affinity and specificity (Green, 1963) . Biotin is involved in a wide range of metabolic processes, 11 both in humans and other organisms, primarily related to the utilization of fats, carbohydrates, and 12

amino acids (Zempleni et al., 2009 ). Because of its small size, biotin is a useful label for many 13 proteins and nucleotides as it does not change the original properties of the proteins or nucleotides. 14 The process of labeling a protein or nucleotide with biotin is known as biotinylation. It is also 15 important that biotin protein ligases can attach biotin to specific lysine residues in vitro or in living 16 cells (Chivers et al., 2011) . The interaction between avidin and biotin is known to be the most 17

specific and strongest non-covalent interaction (Kd = 10 -15 M) between a protein and ligand 18 (Green, 1963) . The exceptionally strong affinity of avidin for biotin arises from the hydrophobic 19 interactions of biotin and aromatic amino acids arranged in the binding pocket of avidin and 20 multiple hydrogen bonding between heteroatoms in the ureido ring of biotin and asparagine, serine, 21 tyrosine, and threonine residues in avidin (Chivers et al., 2011) . Due to the strong interaction, the 22

avidin-biotin complex is robust and stable against temperature, pH, harsh organic solvents and 23 denaturing reagents. Given the unique properties of the avidin-biotin system, it is used often in 24 enzyme-linked immunosorbent assay ( and biomedicine (Yang et al., 2013) . Because of its extremely high electrical conductivity 7

(200,000 cm 2 V -1 s -1 ) and ambipolar transfer characteristics (Geim and Novoselov, 2009; 8 Novoselov et al., 2004) , graphene is considered as the most promising material for different types 9

of ultra-sensitive graphene field-effect transistor (GFET) sensors such as chemical and biosensor. 10

The extreme sensitivity of GFET relates to the Fermi energy shift induced by the adsorption of 11

any molecules involving charge transfer between substrate and molecules, which ultimately 12 changes the conductivity of graphene (Alberto, 2020). Even without any traceable change of 13

Fermi level, any localized orbital distortion induced by the adsorbed molecule can also change the 14 conductivity of graphene (Alberto, 2020). Hence, the carrier density of graphene is expected to be 15 altered even by the non-covalent binding of biomolecules onto the surface, i.e., the electrical 16 conductivity of graphene is expected to be altered by the non-covalent binding of a very small 17 concentration of biomolecules onto the graphene surface.

The field-effect transistor (FET) is a type of transistor that uses an electric field to control studied the effect of pH on the interaction between avidin and iminobiotin using epitaxial 44 graphene on SiC based FET and emphasized the need for concentration dependent measurements 1 (Taniguchi et al., 2019) . Here, we report the development of a novel GFET biosensor consisting of 2 six pairs of interdigital electrodes covered by single layer graphene for detecting biotin molecules 3

with ultrahigh sensitivity and specificity. Real time measurements are realized by monitoring the 4 drain-source current changes upon the entry of biotin solution onto the avidin immobilized 5 graphene channel. The detection limit of biotin is estimated to be 90 fg/ml (0.37 pM). Since the 6 biotin can conjugate with a variety of proteins and nucleotides through biotinylation without 7

changing their original properties, the proposed GFET, which integrates the graphene's extreme 8 electrical conductivity with the unique avidin-biotin technology, is expected to be a breakthrough 9

in the development of GFET biosensors for the rapid and point of care diagnosis of infectious 10 diseases and different biomarkers with ultrahigh sensitivity and specificity. 11 12

2. Material and methods 13

2

A 4-inch 285 nm SiO 2 /Si wafer was purchased from ALLIANCE Biosystems (Osaka, Japan).

The chemical vapor deposition (CVD) graphene on copper foil was purchased from Chongqing 16

Graphene Technology Co., Ltd (Chongqing, China). Poly (methyl methacrylate) (PMMA) was 17

purchased from Sigma-Aldrich (Tokyo, Japan). Ammonium peroxodisulfate was purchased from 18

Kanto Chemical Co., Inc. (Tokyo, Japan). PBASE was synthesized. Neutravidin protein and 19

phosphate-buffered saline (PBS) were purchased from Thermo Fisher (Massachusetts, USA). 20

Bovine serum albumin (BSA), Lactoferrin, Vitamin C, Vitamin B3 were purchased from 21 FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan). Biotin was purchased from Tokyo 22

Chemical Industry Co., Ltd (Tokyo, Japan). Ultra-pure water was obtained from water purifier 23 WL220 (Tokyo, Japan).

3D height graphing of the interdigital electrode and substrate was carried out by 3D laser 25 microscopy OLS-4000. A 405 nm laser source is used. Raman measurements were carried out on a 26

Nicolet Almega XR Raman spectroscope with a 532 nm laser. The Raman spectra of the samples 27

were acquired with a 100X objective, which reduced the spatial resolution to 1 µm. XPS 28 measurements were performed on an AXIS-NOVA XPS system using an AlKα x-ray source. The 29

incident and emission angles were 60 and 0° to the surface normal, respectively. The analyzer pass 30 energies for the wide range and high-resolution measurements were set at 160 and 20 meV, 31

respectively. The analyzer slit was set at 110 µm. A Keysight 4155B semiconductor parameter 32

analyzer was used for the real-time GFET current measurements. 33 34 2.2 Device fabrication 35

The 4-inch 285 nm SiO 2 /Si wafer was cut into the typical size of 1 cm x 1 cm by a dicing saw 36

(DISCO Corporation, Japan). Each 1 cm x 1 cm cut SiO 2 /Si was used as platform on which the 37 interdigital electrode was fabricated. The interdigital shadow mask was carefully placed on the 38

SiO 2 /Si surface. Metal chromium (Cr) / gold (Au) was used as the deposition material. The metal 39 deposition with desired thickness was performed by the electron beam vacuum evaporation 40 deposition (EIKO Engineering, Japan) technique. The thickness of the deposited Cr/Au was about 41 50 nm / 50 nm.

In the transfer process, the graphene on the copper foil was coated with a thin layer of PMMA 43 using a spin coater (Mikasa Corporation, Japan) followed by 5 min of heating at 90 o C to 44 evaporate the acetone solvent. The PMMA/graphene on the copper foil was cut into small pieces 1 of 5 mm x 5 mm to suit the interdigital electrodes. Subsequent etching of the copper foil using 2 ammonium peroxodisulfate aqueous solution left the PMMA/graphene film floating in the 3 solution. After complete etching of copper foil, the PMMA/graphene film was transferred on the 4 ultra-pure water, held for 30 min, then carefully transferred onto the surface of the interdigitated 5 electrode substrate. The transferred PMMA/graphene film was then dried in air for 30 min at room 6 temperature and heated at 60 o C for 30 min. Finally, the removal of PMMA with boiling acetone 7

and isopropyl alcohol (IPA) left the graphene attached to the interdigital electrodes (Fei et al 2017). 8

The cleanliness of the transferred graphene was confirmed by optical images (figure S1 in 9

supporting information, SI). Note that single and bilayer graphene transferred on SiO 2 substrate 10 can be clearly visualized by optical microscope. There might have some undetected small 11

patches of PMMA on the graphene, which we think does not invalidate the principle of the present 12

GFET biosensing platform utilizing the avidin-biotin technology. 13 14 2

Initially, the transferred graphene on interdigital electrodes was modified with PBASE, which is 16

used as the linker for the non-covalent binding of the biomolecule with graphene. The surface 17 modification with PBASE is very significant in maximizing device performances. Hence to find 18 the best modification condition, the surface modification with PBASE was optimized by 19

incubating the graphene on different interdigital electrodes samples in a dry dimethylformamide 20

(DMF) solution of 50 mM PBASE for 1, 2, 4, 6 and 8 h at room temperature individually. The 21

PBASE modified graphene was washed with methanol three times and dried with rotary pump. 22

Then a pre-designed liquid pool made with silicone sheet was fixed on the top of the PBASE 23 modified graphene so that the bio-solution to be tested can be injected into this test pool. 24

To capture the avidin molecules on the surface of PBASE modified graphene, 100 µl of 1 25 mg/ml avidin-PBS solution is injected into the pool, and held for 1 h at room temperature. Then 26 the device is washed with 100 µl PBS three times. To prevent the non-specific binding of the 27 residual surface regions (regions unmodified by PBASE) to other molecules, which may affect the 28 signal during actual testing of the device, the remaining unmodified surface regions of the 29 graphene were blocked by injecting 100 µl of 0.01 mg/ml BSA-PBS solution into the pool and 30

holding for 1 h at room temperature followed by washing with 100 µl PBS three times. 31 32 2

Biotin buffer solutions with different concentrations were injected into the liquid pool while the 34 real time current was measured to achieve the quantitative detection of biotin in PBS solution at 35 pH 7.4. The source-drain voltage was maintained at 0.1 V while the gate (Si/SiO 2 ) was grounded. 36

The Keysight 4155B semiconductor parameter analyzer was used in the real-time current 37

measurements. The specificity of the graphene biosensor was investigated by two sets of 38 experiments. In the first set, the same amount of PBS, BSA, biotin, BSA solutions were injected 39

into the liquid pool separately while the current was monitored. Similarly, the same amount of 40

Vitamin C, Vitamin B3, and lactoferrin were injected separately while monitoring the current. Graphene was carefully transferred to cover all six pairs of interdigital electrodes. (b) The typical 2

Raman spectrum of the graphene transferred onto the interdigital electrodes.

The modification of graphene with the linker is an important part of designing a functional 5

GFET biosensor system, because it realizes the immobilization of a receptor biomolecule (i.e., 6 avidin) on the graphene which can specifically bind to a target ligand molecule (i.e., biotin). The 7

modification steps are shown in Figure 3 . A conventional non-covalent linker used in the case of 8

graphene, and the carbon nanotube-based device is PBASE, which is normally used to handle 9

molecules containing lysine residue (Chen et al., 2001) . The van der Waals force between the 10 graphene and the pyrene backbone of PBASE molecule ensure their tight binding, this binding is 11 also called π-p stacking. To the best of our knowledge, in spite of its importance, no detailed study 12

on the coverage dependent modification and its effect on graphene's properties has been reported. 13 Hence, to optimize the parameters for optimum coverage, we performed the time-dependent 14 measurements. Figure 4a shows the Raman spectra of transferred graphene after modification with 15 PBASE for the different times indicated (1 h, 2 h, 4 h, 6 h, and 8 h). We can see that defect 16 induced D (1350 cm -1 ) and D' band (1620 cm -1 ) appear and the intensities of both bands increase 17

with modification time. The D band is related to the disorder in the honeycomb structure of 18 sp 2 -hybridized carbon and arises from double resonances involving the nearest neighbour sites.

The mechanism that gives rise to the D' band is similar to that of the D band. Since the PBASE 20 molecule is expected to be immobilized onto the graphene through non-covalent interaction, it is 21

unlikely that the defects in graphene arise from sp 3 carbons in the graphene's structure. The most 22

likely reason for the appearance of the D and D' bands is that the localized vibrational modes of 23 the PBASE interact with the extended phonon modes of graphene. These results suggest that the 24 degree of disorder of the sp 2 hybridized carbon system gradually increases as the 25 26 Figure 3 . The schematic illustration of the stepwise modification of graphene with avidin 27 molecules followed by the binding of biotin molecules. Figure 4b shows the ratios of 2D and G bands (2D/G) at different 9 modification times. The 2D/G ratio gradually decreases and approaches 1 at around the 10 modification time of 4 h. At 6 h, the 2D/G ratio still remains close to the value of 4 h but it further 11 decreases at 8 h. Ratio of 1 is equivalent to that of the bilayer graphene. This indicates that the 12 aggregation of pyrenyl groups onto the monolayer graphene forms a structure similar to that of 13 bilayer graphene. Hence, to avoid any uncovered regions and excessive defects, the 4 h 14 modification time seems optimal given the use of the 50 mM PBASE solution in dry 15 dimethylformamide (DMF).

The modification of graphene with PBASE was also characterized by I-V and XPS 17 measurements. Typical I-V curves for clean and PBASE modified (modified for 4 h) graphene are 18

shown in Figure 4c . A large shift of the Dirac point towards higher voltages is observed. This 19

shifts in Dirac point indicates that the PBASE modification gives the graphene P-doping 20 characteristics. The XPS spectrum shown in figure 4d shows a small N 1s peak around 400 eV 21

following the PBASE modification, which ensures the presence of PBASE molecules on the 22

graphene surface. Avidin is immobilized on the PBASE modified graphene through the interaction 23 between lysine residue of avidin and N-hydroxysuccinimide ester group of PBASE. After avidin 24 modification, a strong N 1s peak is observed, which indicates the increased number of nitrogen 25

atoms from the avidin protein on the surface. No significant difference in the survey and C 1s 26 spectra acquired before and after the PBASE modification of the bare graphene is observed ( figure  1 S3 and S4 in SI), which is in agreement with previous reports . Thus the Raman, 2 XPS and I-V measurements ensure the perfect PBASE modification of the graphene and 3

immobilization of avidin onto the graphene channel of the GFET device 4 5 3.3 Quantitative detection of biotin 6

Quantitative detection of biotin was performed by adding the biotin solutions to the test pool as 7

shown in figure 5a . The 2-terminal measurement system is used while the gate-terminal was 8 value when the concentration of the biotin is close to 3.96 ng/ml. These results indicate that the 22 present GFET biosensor can detect biotin levels as low as 90 fg/ml. When the concentration of the 23 biotin is higher than 3.96 ng/ml, the graphene biosensor capability approaches saturated 24 adsorption, i.e., the detection range of the present graphene biosensor is between 90 fg/ml and 25

3.96 ng/ml. Real-time Ids measurements were also performed using the biotin concentrations 26 below the detection limit (i.e., 0.18 fg/ml, 1.8 fg/ml, 18 fg/ml), but no traceable change in stable 27

Ids values were observed (figure S5 in SI). Note that reproducibility of biotin detection was 28 confirmed in at least five independent measurements. 29 30 3

Based 3.5 The specificity of detection 8

In real applications, the sample to be tested contains not only the target molecule but also other 9

molecules. Therefore, it is necessary to investigate sensor specificity. To confirm the specificity of 10 the present GFET sensor, real time measurements with both the controlled and real samples were in figure S6 in SI. The results suggest that the current signal remains almost unchanged before and 20

after the addition of the solution. These results indicate that the immobilized avidin does not 21

interact with PBS, BSA, vitamin C, vitamin B3 and lactoferrin but readily interacted with biotin, 22

i.e., the present GFET is very specific to the biotin molecules. For testing the specificity with a 23 real sample, a preliminary experiment with chicken egg whites and yolk was performed (egg was 24 purchased from supermarket). As expected, injection of 50 µl of egg yolk solution in PBS into the 25 test pool of GFET induces the large drop of stable Ids current in real time measurements, which 1

indicates the presence of free biotin or biotinylated compound. In contrast, injection of egg white 2 solution in PBS does not induce any significant change in stable Ids current, which implies that no 3 chemical components of egg white can non-specifically bind to the immobilized avidin on GFET. 4

It is well known that the chicken egg yolk contains significant amount of biotin (13-25 µg/yolk), 5

which is normally conjugated with specific biotin binding protein (BBP) (Meslar 1978) . These 6

observations further support the specificity and sensing ability of the present GFET sensor in the 7

case of a real sample. The detailed results are shown in SI. Hence, for the analysis of blood sample using the present GFET sensor, the pre-removal of biotin 20 content or the pre-estimation of the exogenous biotin interferenceshould be considered. If the 21 target sample is other than blood such as swab or exhaust gas, then the pre-removal of biotin will 22

not be needed. immobilized graphene channel, influenza can be diagnosed with ultrahigh sensitivity even before 10 any symptom appears. Thus, because of the wide scope of avidin-biotin system, the present 11

GFET is expected to be a breakthrough in the development of various ultrasensitive GFET 12 biosensors for rapid and point-of-care low cost medical diagnosis. In addition, the ultra-high 13 sensitivity of the present GFET is expected to allow quantitative detection of the exogenous biotin 14

level in blood serum. 15 16 4. Conclusions 17

By combining the extreme electrical conductivity of graphene with the unique interaction 18 between avidin and biotin molecules, we have realized a novel two terminal GFET biosensor for 19

the detection of biotin with ultrahigh sensitivity and specificity. The GFET consists of six pairs of 20

Cr/Au electrodes and an avidin immobilized single layer graphene channel supported on the 21

Si/SiO 2 substrate. By monitoring the source-drain current change upon addition of biotin solution 22

in BSA, biotin can be detected to levels as low as 90 fg/ml (0.37 pM), and so is the most sensitive 23

GFET for biomolecule detection. The detection range of the present GFET biosensor is estimated 24

to lie between 90 fg/ml and 3.96 ng/ml. The high specificity of the GFET was confirmed both by 25 can be employed for the detection of various biomarkers and bio-macromolecules. In addition, it 29 may also be used for the rapid quantitative detection of exogenous biotin. Because of its real-time 30 rapid detection, ultra-low detection limit, and high specificity, the present GFET biosensor is 31 expected to be a breakthrough in medical devices offering real-time point-of-care clinical 32 diagnosis. This work was conducted at Nano-Processing Facility, National Institute of Advanced Industrial 3

Science and Technology (AIST), Japan. Thanks are due to Professor Hayato Sone, Professor 4

Kenta Miura for guiding the equipment operation, as well as Professor Noriyuki Koibuchi for his 5 helpful comments and suggestions. Agnolon, V., Contato, A., Meneghello, A., Tagliabue, E., Toffoli, G., Gion, M., Polo, F., Fabricio, 16

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Ballesta-Claver, J., Ametis-Cabello, J., Morales-Sanfrutos, J., Megía-Fernández, A., 20

Valencia-Mirón, M.C., Santoyo-González, F., Capitán-Vallvey, L.F., 2012.

Electrochemiluminescent disposable cholesterol biosensor based on avidin-biotin assembling 22

with the electroformed luminescent conducting polymer poly(luminol-biotinylated pyrrole). 

Noncovalent Sidewall Functionalization of 1

Single-Walled Carbon Nanotubes for Protein Immobilization

How the biotin-streptavidin 4 interaction was made even stronger: investigation via crystallography and a chimaeric 5 tetramer

Biotin Interference in Clinical Immunoassays

The biotin-(strept)avidin system: principles and 9 applications in biotechnology

Chemically Functionalized Graphene FET Biosensor for the Label-free Sensing of 13 exosomes

Utility of circulating tumor DNA in cancer diagnostics with 15 emphasis on early detection

Graphene Coated 17 Nanoprobes: A review

Graphene Transistors

The rise of graphene

Studies on the biotin-binding site of avidin. Lysine 24 residues involved in the active site

Avidin. 1. The use of [14C]biotin for kinetic studies and for assay

The use of avidin-biotin interaction in 29 immunoenzymatic techniques

Sensitivity improvement of a 32 sandwich-type ELISA immunosensor for the detection of different prostate-specific antigen 33 isoforms in human serum using electrochemical impedance spectroscopy and an ordered and 34 hierarchically organized interfacial supramolecular architecture

Probing Layer Number and Stacking Order of Few-Layer Graphene by Raman 38

Rhizavidin from Rhizobium etli: the first 42 natural dimer in the avidin protein family

Using Liposomal Fluorescent Biolabels To Develop an

The Use of Antiavidin Antibody and 4

Peroxidase Complex in Immunoperoxidase Technics

Use of avidin-biotin-peroxidase complex (ABC) in 7 immunoperoxidase techniques: a comparison between ABC and unlabeled antibody (PAP) 8 procedures

Reduced-Graphene-Oxide Hybrid Nested Hierarchical Networks for High-Performance Li-S 11

Biotin determination in food supplements by an electrochemical magneto 14 biosensor

Sensitive Detection of Biomolecules in Enzyme Linked Immunosorbent Assay

A competitive immunoassay 19 for biotin detection using magnetic beads and gold nanoparticle probes

Graphene Annealing: 22 How Clean Can It Be?

Biotin interference in immunoassays based on 24 biotin-strept(avidin) chemistry: An emerging threat

Frontiers of Graphene and Carbon Nanotubes: Devices and Applications

Biotin-binding protein from egg yolk. A protein 29 distinct from egg white avidin

Reversibility of biotin-binding by selective 31 modification of tyrosine in avidin

Direct visualization of 34 ligand-protein interactions using atomic force microscopy

Antibody Binding to Biotinylated Lipid Bilayer 38 Examined by Second-Harmonic Generation

Electric Field Effect in Atomically Thin Carbon Films

Ethanol 1 Detection at the Parts per Billion Level with Single-Stranded-DNA-Modified Graphene 2 Field-Effect Transistors. physica status solidi (b) 257

Label-Free Biosensors Based on

Aptamer-Modified Graphene Field-Effect Transistors

Electrolyte-Gated Graphene

Transistors for Detecting pH and Protein Adsorption

An 11 electrochemical biosensor to simultaneously detect VEGF and PSA for early prostate cancer 12 diagnosis based on graphene oxide/ssDNA/PLLA nanoparticles

Evaluation of a rapid immunochromatographic test for the detection 17 of OXA-48 carbapenemase

Situ Electrochemical ELISA 20 for Specific Identification of Captured Cancer Cells

Protecting copper from 24 electrochemical degradation by graphene coating

Observation of 27 the interaction between avidin and iminobiotin using a graphene FET on a SiC substrate

TGFβ drives immune evasion in genetically reconstituted colon cancer metastasis

Unique epidemiological and clinical features 35 of the emerging 2019 novel coronavirus pneumonia (COVID-19) implicate special control 36 measures

Free radical 38 sensors based on inner-cutting graphene field-effect transistors

Electrochemical Energy Storage: from Materials to Devices

Methods in Enzymology, Avidin-Biotin Technology

Graphene/metal oxide 7 composite electrode materials for energy storage

One-Step Device Fabrication of Phosphorene and Graphene Interdigital 11

Micro-Supercapacitors with High Energy Density

Real-time reliable determination of binding kinetics of DNA hybridization using a 15 multi-channel graphene biosensor

Nano-graphene in biomedicine: theranostic applications

Ru(bpy)32+-doped silica 23 nanoparticles labeling for a sandwich-type electrochemiluminescence immunosensor

Ultrafine Graphene Nanomesh with Large On/Off Ratio for High-Performance Flexible 27 Biosensors

1,4-Benzenediboronic-Acid-Induced Aggregation of Gold 30 Nanoparticles: Application to Hydrogen Peroxide Detection and Biotin-Avidin-Mediated 31 Immunoassay with Naked-Eye Detection

Electrical and Label-Free Quantification of Exosomes with a Reduced Graphene Oxide 35 Field Effect Transistor Biosensor

2D Nanomaterials: Beyond Graphene and Transition 40 Metal Dichalcogenides

Chemical vapour deposition growth of large single crystals of monolayer and 2 bilayer graphene

Label-free graphene biosensor targeting cancer molecules based on 5 non-covalent modification

Highlights:1. Combining the outstanding electrical conductivity of graphene with the unique interaction between avidin and biotin, a novel graphene field-effect transistor (GFET) biosensor for quantitative detection of bio-macromolecules is demonstrated.

The present biosensor is capable of detecting the biotin with the sensitivity of 90 fg/ml (~0.37 pM) and high specificity.

Since the biotin is capable of conjugating with protein, nucleotide and other bio-macromolecules without affecting their properties, the present GFET sensor can be tailored to various medical applications.

☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: