Name: Development and Functionalization of Electrolyte-Gated Graphene Field-Effect Transistor for Biomarker Detection (Video) | JoVE Journal
Uploaded: 2022-02-01T14:20:00.000+00:00
Duration: 7 min 51 s
Description: Learn to develop an electrolyte-gated graphene field-effect transistor biosensor for IgG biomarker detection using CVD graphene. This protocol enables real-time, label-free biosensing with high sensitivity and selectivity.

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5 minutes

The experiments were conducted at West Virginia University. We acknowledge the Shared Research Facilities at West Virginia University for device fabrication and material characterization. This work was supported by the US National Science Foundation under Grant No. NSF1916894.

1. Transferring chemical vapor deposition of graphene
<ol>
	<li>Cut the graphene sheet on a copper substrate in half (2.5 cm x 5 cm) using scissors. Apply heat resistive tape to fix the four corners of the graphene square on a spinner gasket (see Table of Materials). 
	NOTE: The purchased graphene has a dimension of 5 cm x 5 cm (see Table of Materials).</li>
	<li>Spin-coat the sheet of the graphene with a thin layer (100-200 nm) of PMMA 495K A4 spinning at 500 rpm for 10 s and then 2000 rpm for 50 s. Then bake the sample at 150 &#176;C for 5 min.</li>
	<li>Remove the backside of the graphene with oxygen plasma (see Table of Materials) at 30 W, 15 sccm for 5 min.</li>
	<li>Cut the plasma-treated graphene square into smaller dimensions (1 cm x 2 cm) for device fabrication.</li>
	<li>Cut the pre-cleaned substrate (SiO2) into small pieces with an approximate dimension of 2.5 cm x 2 cm.</li>
	<li>Etch the copper off using the graphene commercial etchant (Ferric chloride) (see Table of Materials). Do not dilute the etchant. Float the sample with the copper side down and the PMMA side up on the liquid etchant.</li>
	<li>After copper etching, lift the graphene film slowly using the plasma-treated substrate.</li>
	<li>Air-dry the transferred graphene for 2 h and then bake at 80 &#176;C for 15 min.</li>
	<li>Remove the PMMA following the steps below.
	<ol>
		<li>Warm up the sample with acetone vapor at 70 &#176;C. Keep the sample at ~2 cm above acetone vapor for 4 min with the PMMA side facing down. Then immerse the sample in acetone for 5 min.</li>
		<li>Wash the sample with DI water cautiously and observe the transferred graphene under a microscope. Finally, gently blow-dry the sample with N2.</li>
		<li>Perform Atomic force microscopy (AFM) observation to ensure PMMA residue-free graphene. If PMMA residue is visible in the image, perform the acetone vapor cleaning and immersion once again.</li>
	</ol>
	</li>
	<li>Perform Raman and AFM characterization to confirm the monolayer of graphene transferring and observe the surface properties (Figure 1A,B).</li>
</ol>
2. Fabrication of Graphene Field Effect Transistor (GFET)
<ol>
	<li>Wash the substrate with the transferred graphene using acetone, IPA, and DI water; then bake the substrate on a hot plate at 75 &#176;C for 30 min (Figure 2A).</li>
	<li>Using the E-beam evaporator30 (see Table of Materials), deposit 5 nm nickel and 45 nm gold on the graphene sample (Figure 2B).</li>
	<li>Apply the first photolithography30 process using mask A (Supplementary Figure 1) for the patterning of the electrodes (Figure 2C).</li>
	<li>Spin a positive photoresist (AZ 5214E, see Table of Materials) on the sample (2000 rpm for 45 s) and cure the sample at 120 &#176;C for 1 min.</li>
	<li>Place the sample in the UV flood exposure system and expose it for ~10 s under 200 mJ/cm2.</li>
	<li>Develop the sample with a photoresist developer (AZ300 MIF, see Table of Materials) for ~2 min, and then rinse with DI water.</li>
	<li>Immerse the sample in a gold etchant to etch the gold layer for 10 s; rinse with DI water and remove the remaining photoresist layer by immersing in acetone for 10 min (Figure 2C).</li>
	<li>Using acetone, IPA, and DI water, wash the sample; bake on a hot plate at 75 &#176;C for 30 min. Then apply the second photolithography process using mask B (Supplementary Figure 1) to pattern the graphene channels. 
	NOTE: Use the same process parameters as the first one (step 2.4-2.6), except the UV exposure system in the mask aligner (Figure 2D).</li>
	<li>Immerse the sample in nickel etchant at 60 &#176;C to etch the nickel layer for 10 s; rinse with DI water; blow dry using N2&#160;(Figure 2D).</li>
	<li>Place the sample in the plasma asher and remove the exposed graphene using oxygen plasma (100 W for 90 s with oxygen flow at 49 sccm); after that, remove the photoresist layer by immersing in acetone for 10 min (Figure 2E).</li>
	<li>Wash the sample using acetone, IPA, and DI water; bake on a hot plate at 75 &#176;C for 30 min and apply the third photolithography process using mask C (Supplementary Figure 1) for the patterning of the passivation photoresist layer to protect the underlying graphene on the substrate. Use the same process parameters as the first one (step 2.4-2.6), except the UV exposure system in the mask aligner (Figure 2F).</li>
	<li>After the third photolithography process, immerse the sample in nickel etchant at 60 &#176;C for 10 s to remove the remaining nickel layer; then rinse with DI water and blow dry using N2 (Figure 2G). Finally, bake the sample on a hotplate at 120 &#176;C for 30 min (Figure 2H).</li>
</ol>
3. Functionalization of GFET for IgG Detection
<ol>
	<li>Assemble the sample-delivery channel.
	<ol>
		<li>Fabricate the sample-delivery channel in PDMS using soft lithography techniques31.</li>
		<li>Immerse the graphene device in 0.1 M of NaOH solution for 30 s; rinse with DI water and leave a thin water layer on the device surface to assist PDMS well&#39;s alignment and bonding. Then activate the PDMS well&#39;s surface using oxygen plasma.</li>
		<li>Align the sample delivery channel and the graphene device under a microscope; place the aligned device in a 60 &#176;C oven for 3 h to allow the bonding. The assembled device is shown in Figure 3A.</li>
	</ol>
	</li>
	<li>Functionalize the GFET.
	<ol>
		<li>Functionalize the graphene surface with IgG aptamer (see Table of Materials). Use pipettes to load and remove each reagent or buffer from the PDMS well. The schematic process is shown in Figure 4. 
		NOTE: The following steps were operated at room temperature.</li>
		<li>After rinsing the graphene surface with DMSO three times, apply 1-pyrene butyric acid N-hydroxysuccinimide ester (PBASE, 10 mM dissolved in DMSO, see Table of Materials) and keep for 2 h.</li>
		<li>After rinsing with DMSO, apply 5&#39;amino-modified IgG aptamer (20 &#181;M in 1x PBS), incubate for 3 h, and rinse with 1x PBS three times.</li>
		<li>Apply bovine serum albumin (BSA, 10% w/v in 1x PBS) on graphene for 1 h and rinse with 1x PBS three times.</li>
	</ol>
	</li>
</ol>
4. IgG detection
<ol>
	<li>Rinse the device with 0.01x PBS three times. Fill the PDMS well with 0.01x PBS (detection buffer) (Figure 3A,B).</li>
	<li>Connect the electrodes with a high-performance parameter analyzer (see Table of Materials). Connect the source electrode to the ground, the drain, and the gate electrodes to Source Measurement Units (SMU 1 and SMU 2) equipped with the parameter analyzer, respectively (Figure 3C).</li>
	<li>Set up the measurement parameters and turn on the sampling process.</li>
	<li>Test the response of the EGGFET to IgG by continuously monitoring the drain current. Dissolve IgG in 0.01x PBS with different concentrations, add the solution into the detection chamber, and monitor the drain current continuously. Save the data.</li>
</ol>

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The authors have no competing interests or conflicting interests to disclose.

The purchased CVD graphene on copper film needs to be trimmed to the right size for the following fabrication steps. Cutting of the films can cause wrinkling, which needs to be prevented. The parameters provided in the fabrication step can be referred to for plasma etching of graphene, and these numbers could be varied when using different instruments. The etched sample must be closely monitored and inspected to ensure complete graphene etching. Multiple pre-cleaning methods can be applied to clean the substrates, such a...

Graphene and its derivatives have been investigated and used for many applications, including electronics1,2, sensing3,4,5, energy storage6,7, and photocatalysis1,6,8. Synthesis and fabrication of high quality, good uniformity, and low defects graphene are critical for high-performance and highly sensitive devices. Since the development of Chemical vapor deposition (CVD) in 2009, it has shown colossal promise and set its place as an essential member of the graphene family9,10,11,12,13. It is grown on a metal substrate and, later for practical uses, is transferred onto insulating substrates14. Several transferring methods have been used to transfer CVD graphene recently. The poly (methyl methacrylate) (PMMA) assisted method is the most used among the different techniques. This method is particularly well-suited for industrial usage because of its large-scale capability, lower cost, and high quality of the transferred graphene14,15. The critical aspect of this method is getting rid of the PMMA residue for CVD graphene&#39;s applications because the residues can cause declination of the electronic properties of graphene14,15,16, cause an effect on biosensors&#39; sensitivity and performance17,18, and create significant device-to-device variations19.
Nanomaterials-based biosensors have been significantly investigated over the past decades, including silicon nanowire (SiNW), carbon nanotube (CNT), and graphene20. Because of its single-atom-layer structure and distinctive properties, graphene demonstrates superior electronic characteristics, good biocompatibility, and facile functionalization, making it an attractive material for developing biosensors14,21,22,23. Due to field-effect transistors (FET) characteristics such as high sensitivity, standard configuration, and cost-effective mass producibility21,24, FET is more preferred in portable and point-of-care implementations than other electronics-based biosensing devices. The electrolyte-gated graphene field-effect transistor (EGGFET) biosensors are examples of the stated FETs21,24. EGGFET can detect various targeting analytes such as nucleic acids25, proteins24,26, metabolites27, and other biologically relevant analytes28. The technique mentioned here ensures the implementation of CVD graphene in a label-free biosensing nanoelectronics device which offers higher sensitivity and accurate time detection over other biosensing devices29.
In this work, an overall process for developing an EGGFET biosensor and functionalizing it for biomarker detection, including transferring CVD graphene onto an insulating substrate, Raman, and AFM characterizations of the transferred graphene, are demonstrated. Furthermore, fabrication of EGGFET and integration with a polydimethylsiloxane (PDMS) sample delivery well, bioreceptor functionalization, and successful detection of human immunoglobulin G (IgG) from serum by spike-and-recovery experiments are also discussed here.

<table><tbody><tr><td>1-pyreneutyric acid N- hydroxysuccinimide ester</td><td>Sigma Aldrich</td><td>457078-1G</td><td>functionalization</td></tr><tr><td>Asylum MFP-3D Atomic Force Microscope</td><td>Oxford Instruments</td><td></td><td>graphene characterization</td></tr><tr><td>AZ 300 MIF</td><td>MicroChemicals</td><td>AZ 300 MIF</td><td>photoresist developer</td></tr><tr><td>AZ 300 MIF</td><td>MicroChemicals</td><td>AZ 300 MIF</td><td>photoresist</td></tr><tr><td>Bovine Serum Albumin</td><td>Sigma Aldrich</td><td>810014</td><td>blocking</td></tr><tr><td>Branson 1210 Sonicator</td><td>SONITEK</td><td></td><td>sample cleaning</td></tr><tr><td>Copper Etchant</td><td>Sigma Aldrich</td><td>667528-500ML</td><td>removing copper film to release graphene</td></tr><tr><td>Dimethyl Sulfoxide (DMSO)</td><td>VWR</td><td>97063-136</td><td>functionalization</td></tr><tr><td>Disposable Biopsy Punches, Integra Miltex</td><td>VWR</td><td>21909-144</td><td>create well in PDMS</td></tr><tr><td>Gold etchant</td><td>Gold Etch, TFA, Transene</td><td>658148</td><td>enchant</td></tr><tr><td>Graphene</td><td>Graphene supermarket</td><td>2&quot; x 2&quot; sheet</td><td>biosensing element of the device</td></tr><tr><td>IgG aptamer</td><td>Base Pair Biotechnologies</td><td>customized</td><td>bioreceptor</td></tr><tr><td>Keithley 4200A-SCS Parameter Analyzer</td><td>Tektronix</td><td></td><td>measurement and detection</td></tr><tr><td>KMG CR-6</td><td>KMG chemicals</td><td>64216</td><td>Chromium etchant</td></tr><tr><td>Kurt J. Lesker E-beam Evaporator</td><td>Kurt J. Lesker</td><td></td><td>metal deposition</td></tr><tr><td>Laurell Technologies 400 Spinners</td><td>Laurell Technologies</td><td>WS-400BZ-6NPP/LITE</td><td>thin film coating</td></tr><tr><td>March PX-250 Plasma Asher</td><td>March Instruments</td><td></td><td>sample cleaning</td></tr><tr><td>Nickel etchant</td><td>Nickel Etchant, TFB, Transene</td><td>600016000</td><td>etchant</td></tr><tr><td>OAI Flood Exposure</td><td>OAI</td><td></td><td>photolithography</td></tr><tr><td>Phosphate Buffered Saline (PBS)</td><td>Sigma Aldrich</td><td>806552-500ML</td><td>buffer</td></tr><tr><td>PMMA 495K A4</td><td>MicroChemicals</td><td>PMMA 495K A4</td><td>Photoresist for assisting graphene transferring</td></tr><tr><td>Polydimethylsiloxane (PDMS)</td><td>Sigma Aldrich</td><td>Sylgard 184</td><td>sample delivery well</td></tr><tr><td>Renishaw InVia Raman Microscope</td><td>Renishaw</td><td></td><td>graphene characterization</td></tr><tr><td>Sodium Hydroxide (NaOH)</td><td>Sigma Aldrich</td><td>221465-25G</td><td>functionalization</td></tr><tr><td>Suss Microtech MA6 Mask Aligner</td><td>Suss MicroTec</td><td></td><td>photolithography</td></tr><tr><td>Thermo Scientific Cimarec Hotplate</td><td>Thermo Scientific</td><td>SP131635</td><td>sample and device Baking</td></tr></tbody></table>

development and functionalization of electrolyte-gated graphene field-effect transistor for biomarker detection

Development and Functionalization of Electrolyte-Gated Graphene Field-Effect Transistor for Biomarker Detection

In the current study, graphene and its derivatives have been investigated and used for many applications, including electronics, sensing, energy storage, and photocatalysis. Synthesis and fabrication of high quality, good uniformity, and low defects graphene are critical for high-performance and highly sensitive devices. Among many synthesis methods, chemical vapor deposition (CVD), considered a leading approach to manufacture graphene, can control the number of graphene layers and yield high-quality graphene. CVD graphene needs to be transferred from the metal substrates on which it is grown onto&#160;insulating substrates for practical applications. However, separation and transferring of graphene onto new substrates are challenging for a uniform layer without damaging or affecting graphene&#39;s structures and properties. Additionally, electrolyte-gated graphene field-effect transistor (EGGFET) has been demonstrated for its wide applications in various biomolecular detections because of its high sensitivity and standard device configuration. In this article, poly (methyl methacrylate) (PMMA)-assisted graphene transferring approach, fabrication of graphene field-effect transistor (GFET), and biomarker immunoglobulin G (IgG) detection are demonstrated. Raman spectroscopy and atomic force microscopy were applied to characterize the transferred graphene. The method is shown to be a practical approach for transferring clean and residue-free graphene while preserving the underlying graphene lattice onto an insulating substrate for electronics or biosensing applications.

The representative results show the transferred CVD graphene characterized by Raman and AFM, respectively. The G peak and the 2D peaks of the Raman image give comprehensive information regarding the existence and the quality of the transferred monolayer graphene32 (Figure 1). Standard lithography processes30,31 were applied for fabricating the GFET device, as shown in Figure 2. <s...

The present protocol demonstrates the development of electrolyte-gated graphene field-effect transistor (EGGFET) biosensor and its application in biomarker immunoglobulin G (IgG) detection.

In the current study, graphene and its derivatives have been investigated and used for many applications, including electronics, sensing, energy storage, ...

development-functionalization-electrolyte-gated-graphene-field-effect

Research

JoVE Journal

Engineering

This article describes a biofabrication approach:  deposition of stimuli-responsive polysaccharides in the presence of biased electrodes to create biocompatible films which can be functionalized with cells or proteins. We demonstrate a bench-top strategy for the generation of the films as well as their basic uses for creating interactive biofunctionalized surfaces for lab-on-a-chip applications.

Bridging the Bio-Electronic Interface with Biofabrication

In this video, we describe the field-effect biosensing (FEB) technology to detect and quantify protein-protein interactions. The target proteins immobilized on the graphene biosensor interact with analyte proteins in solution, resulting in electrical current alterations in the FEB system. This change in the electrical response indicates the binding affinity between the proteins.

Graphene Biosensor-Based Field-Effect Biosensing to Detect Protein-Protein Interactions

We describe the device fabrication and measurement protocol for carbon nanotube based high frequency biosensors. The high frequency sensing technique mitigates the fundamental ionic (Debye) screening effect and allows nanotube biosensor to be operated in high ionic strength solutions where conventional electronic biosensors fail. Our technology provides a unique platform for point-of-care (POC) electronic biosensors operating in physiologically relevant conditions.

Fabrication of Carbon Nanotube High-Frequency Nanoelectronic Biosensor for Sensing in High Ionic Strength Solutions

The Organic Electrochemical Transistor is integrated with live cells and used to monitor ion flux across the gastrointestinal epithelial barrier. In this study, an increase in ion flux, related to disruption of tight junctions, induced by the presence of the calcium chelator EGTA (ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetra acetic acid), is measured.

Sensing of Barrier Tissue Disruption with an Organic Electrochemical Transistor

A microdevice with high throughput potential is used to demonstrate three-dimensional (3D) dielectrophoresis (DEP) with novel materials. Graphene nanoplatelet paper and double sided tape were alternately stacked; a 700 μm micro-well was drilled transverse to the layers. DEP behavior of polystyrene beads was demonstrated in the micro-well.

Development of a 3D Graphene Electrode Dielectrophoretic Device

We describe key steps for biosensing by using polysilicon nanowire field-effect transistors, including the preparation of the device and the immobilization and confirmation of a DNA molecular probe on the nanowire surface, as well as conditions for DNA sensing.

Preparation of Silicon Nanowire Field-effect Transistor for Chemical and Biosensing Applications

A microfluidic biosensor platform was designed and fabricated using low-cost dry film photoresist technology for the rapid and sensitive quantification of various analytes. This single-use system allows for the electrochemical readout of on-chip-immobilized enzyme-linked assays by means of the stop-flow technique.

Dry Film Photoresist-based Electrochemical Microfluidic Biosensor Platform: Device Fabrication, On-chip Assay Preparation, and System Operation

Here, we present the fabrication protocol of an organic charge-modulated field-effect transistor (OCMFET)-based device for in vitro cellular interfacing. The device, called a micro OCMFET array, is a flexible, low-cost, and reference-less device, which will enable the monitoring of the electrical and metabolic activities of electroactive cell cultures.

In Vitro Multiparametric Cellular Analysis by Micro Organic Charge-modulated Field-effect Transistor Arrays

Field-effect biosensing (FEB) is a label-free technique for detecting biomolecular interactions. It measures the electric current through the graphene biosensor to which the binding targets are immobilized. The FEB technology was used to evaluate biomolecular interactions between Hsp90 and Cdc37 and a strong interaction between the two proteins was detected.

Exploring Biomolecular Interaction Between the Molecular Chaperone Hsp90 and Its Client Protein Kinase Cdc37 using Field-Effect Biosensing Technology

This protocol describes the fabrication of a one-piece indium-tin-oxide (ITO)-based ion-sensitive field-effect transistor (ISFET), which can be constructed as a solution-gated FET sensor (e.g., pH sensor) using a short and simple process (approximately half a day). This one-piece ITO-ISFET can also be applied to biosensing.

Fabrication of a Solution-gated Indium-Tin-Oxide-based One-piece Transistor Enabling Sensitive Biosensing

Learn to develop an electrolyte-gated graphene field-effect transistor biosensor for IgG biomarker detection using CVD graphene. This protocol enables real-time, label-free biosensing with high sensitivity and selectivity.

Development and Functionalization of Electrolyte-Gated Graphene Field-Effect Transistor for Biomarker Detection

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