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...

<ol>
	<li>Saini, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+and+functionalization+of+graphene+and+application+in+electrochemical+biosensing.">Synthesis and functionalization of graphene and application in electrochemical biosensing.</a> Nanotechnology Reviews. 5 (4), 393-416 (2016).</li><li>Emtsev, K. V., Bostwick, A., Horn, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Towards+wafer-size+graphene+layers+by+atmospheric+pressure+graphitization+of+silicon+carbide.">Towards wafer-size graphene layers by atmospheric pressure graphitization of silicon carbide.</a> Nature Materials. 8 (3), 203-207 (2009).</li><li>Wang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrochemical+delamination+of+CVD-grown+graphene+film:+Toward+the+recyclable+use+of+copper+catalyst.">Electrochemical delamination of CVD-grown graphene film: Toward the recyclable use of copper catalyst.</a> ACS Nano. 5 (12), 9927-9933 (2011).</li><li>Carvalho Fernandes, D. C., Lynch, D., Berry, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D-printed+graphene/polymer+structures+for+electron-tunneling+based+devices.">3D-printed graphene/polymer structures for electron-tunneling based devices.</a> Scientific Reports. 10 (1), 1-8 (2020).</li><li>Gao, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Repeated+growth+and+bubbling+transfer+of+graphene+with+millimetre-size+single-crystal+grains+using+platinum.">Repeated growth and bubbling transfer of graphene with millimetre-size single-crystal grains using platinum.</a> Nature Communications. 3, 699 (2012).</li><li>Singh, J., Rathi, A., Rawat, M., Gupta, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Graphene:+From+synthesis+to+engineering+to+biosensor+applications.">Graphene: From synthesis to engineering to biosensor applications.</a> Frontiers of Materials Science. 12 (1), 1-20 (2018).</li><li>Randviir, E. P., Brownson, D. A. C., Banks, C. 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P., Pei, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Graphene+segregated+on+Ni+surfaces+and+transferred+to+insulators.">Graphene segregated on Ni surfaces and transferred to insulators.</a> Applied Physics Letters. 93 (11), 113103 (2008).</li><li>Xu, S. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+synthesis+of+graphene+on+SiO2+substrates+by+chemical+vapor+deposition.">Direct synthesis of graphene on SiO2 substrates by chemical vapor deposition.</a> CrystEngComm. 15 (10), 1840-1844 (2013).</li><li>Zhang, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Facile+synthesis+of+graphene+on+dielectric+surfaces+using+a+two-temperature+reactor+CVD+system.">Facile synthesis of graphene on dielectric surfaces using a two-temperature reactor CVD system.</a> Nanotechnology. 24 (39), 395603 (2013).</li><li>Zhang, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+formation+of+graphene-carbon+nanotubes+hybrid+on+SiO2+substrate+via+chemical+vapor+deposition.">Direct formation of graphene-carbon nanotubes hybrid on SiO2 substrate via chemical vapor deposition.</a> Science of Advanced Materials. 6 (2), 399-404 (2014).</li><li>Sun, J., Finklea, H. 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Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Label-free+detection+of+DNA+hybridization+using+transistors+based+on+CVD+grown+graphene.">Label-free detection of DNA hybridization using transistors based on CVD grown graphene.</a> Biosensors and Bioelectronics. 41 (1), 103-109 (2013).</li><li>Xu, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+growth+of+graphene+on+quartz+substrates+for+label-free+detection+of+adenosine+triphosphate.">Direct growth of graphene on quartz substrates for label-free detection of adenosine triphosphate.</a> Nanotechnology. 25 (16), 165702 (2014).</li><li>Dan, Y., Lu, Y., Kybert, N. J., Luo, Z., Johnson, A. T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intrinsic+response+of+graphene+vapor+sensors.">Intrinsic response of graphene vapor sensors.</a> Nano Letters. 9 (4), 1472-1475 (2009).</li><li>Zhang, A., Lieber, C. M. -. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nano-Bioelectronics.">Nano-Bioelectronics.</a> Chemical Reviews. 116 (1), 215-257 (2015).</li><li>Forsyth, R., Devadoss, A., Guy, O. 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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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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&#34; x 2&#34; 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

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...

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

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, ...

The method effectively removes PMMA residue while preserving the underlying graphene lattice. The functional device shows consistent results in detecting IgG antibodies in blood serum. Also, the protocol ensures the implementation of CVD graphene in a realtime, label-free biosensing device.The steps are pretty simple and can be done with minimal training. The device offers high selectivity, high sensitivity, and realtime detection over other biosensing devices Begin by cutting the graphene sheet on a copper substrate in half using a scalpel. Apply heat-resistant tape to fix the four corners of the graphene square on a spinner gasket.Spin coat the square sheet of the graphene with a thin layer of 100 to 200 nanometers of PMMA 495K A4, spinning at 500 rotations per minute for 10 seconds, and then 2, 000 rotations per minute for 50 seconds. Then, bake the sample at 150 degrees Celsius for five minutes. Remove the backside of the graphene with oxygen plasma at 30 watt using a rate flow of 15 standard cubic centimeters per minute for five minutes.Cut the plasma-treated graphene square into a dimension of one-centimeter width and two-centimeter height for device fabrication. Cut the pre-cleaned silica substrate into small pieces with an approximate four-centimeter width and two-centimeter height. Etch the copper off using the graphene etchant ferric chloride without dilution.Float the sample with the copper side down and the PMMA side up on the liquid etchant. After copper etching, lift the graphene film slowly using the plasma-treated substrate. Air dry the transferred graphene film for two hours, then bake on a hot plate.To remove PMMA, start by warming up the sample with acetone vapor at 70 degrees Celsius by keeping the sample approximately two centimeters above acetone vapor for four minutes with the PMMA side facing down. Then, immerse the sample in acetone for five minutes. Wash the sample with deionized water cautiously.Finally, gently blow dry the sample with nitrogen. Wash the substrate with the transferred graphene using acetone, isopropyl alcohol, and deionized water. Then, bake the substrate on a hot plate at 75 degrees Celsius for 30 minutes.Using an electron beam evaporator, deposit nickel and gold of 5 and 45 nanometers thickness, respectively, on the graphene sample. Apply the first photolithography process using mask A for the patterning of the electrodes. Spin AZ 5214E positive photoresist on the sample at 2, 000 rotations per minute for 45 seconds and cure the sample at 120 degrees Celsius for one minute.Place the sample in the ultraviolet flood exposure system and expose it for approximately 10 seconds under 200 millijoules per square centimeter. Develop the sample with a photoresist developer AZ 300 MIF for approximately two minutes and then rinse with deionized water. Immerse the sample in a gold etchant to etch the gold layer for 10 seconds, rinse with deionized water, and remove the remaining photoresist layer by immersing in acetone for 10 minutes.Using acetone, isopropyl alcohol, and deionized water, wash the sample followed by baking on a hot plate at 75 degrees Celsius for 30 minutes. Then, apply the second photolithography process using mask B to pattern the graphene channels. Immerse the sample in nickel etchant at 60 degrees Celsius to etch the nickel layer for 10 seconds.Rinse with deionized water and blow dry using nitrogen. Place the sample in the plasma asher and remove the exposed graphene using oxygen plasma. Later, remove the photoresist layer by immersing in acetone for 10 minutes.Wash the sample using acetone, IPA, and deionized water and bake on a hot plate at 75 degrees Celsius for 30 minutes. Apply the third photolithography process using mask C to pattern the passivation photoresist layer to protect the underlying graphene on the substrate. Use the same process parameters as previously mentioned, including spinning with positive photoresist, curing the sample, and developing with photoresist developer.Later, immerse the sample in nickel etchant at 60 degrees Celsius for 10 seconds to remove the remaining nickel layer, and then rinse with deionized water and blow dry using nitrogen. Finally, bake the sample on a hot plate at 120 degrees Celsius for 30 minutes. The representative results show the transferred CVD graphene characterized by Raman and atomic force microscopy.The G peak and the two-dimensional peaks of the Raman image give comprehensive information regarding the existence and the quality of the transferred monolayer graphene. The figure shows EEG FET biosensor integrated with a standard silver in silver chloride reference electrode and a polydimethyl siloxane well for containing the sample. Further, the enlarged view of the graphene channel demonstrates the connection to the source electrode to the ground, the drain, and the gate electrodes to the source.PBASE, a widely used functionalization reagent for graphene, can be absorbed on graphene surface through a pi-pi interaction without damaging graphene's electrical properties. A 5-prime amino-modified IgG aptamer is conjugated with PBASE by the amide bond linkages between the reactive and hydroxy-6 cinnamide ester in PBASE, and the amine group on the 5-prime end of the IgG aptamer. Bovine serum albumin incubation was used to block the remaining unconjugated sites after rinsing the device with single-strength PBS The figure shows the IgG detection under different electrolyte conditions.The quality of the graphene is the key to the best performance of this device. So during plasma etching, it needs to be ensured that the plasma does not harm the useful regions of the graphene. Also, the PMMA residues need to be cleaned altogether to get a clean graphene surface.The functional device shows consistent results in detecting human IgG antibody, so the procedure can be used as a reference to build devices with other nanomaterials to study interface interactions and biosensing.

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3.1K Görüntüleme.  West Virginia University. Yöntem, altta yatan grafen kafesi korurken PMMA kalıntılarını etkili bir şekilde giderir. Fonksiyonel cihaz, kan serumundaki IgG antikorlarının saptanmasında tutarlı sonuçlar göstermektedir. Ayrıca, protokol CVD grafeninin gerçek zamanlı, etiketsiz bir biyo-algılama cihazında uygulanmasını sağlar.Adımlar oldukça basittir ve minimum eğitimle yapılabilir. Cihaz, diğer biyolojik algılama cihazlarına göre yüksek seçicilik, yüksek hassasiyet ve gerçek zamanl?...