RNA Detection Based on Graphene Field-Effect Transistor Biosensor

Graphene has attracted much attention in biosensing applications due to its unique properties. In this paper, the monolayer graphene was grown by chemical vapor deposition (CVD) method. Using the graphene as the electric channel, we have fabricated a graphene field-effect transistor (G-FET) biosensor that can be used for label-free detection of RNA. Compared with conventional method, the G-FET RNA biosensor can be run in low cost, be time-saving, and beminiaturized for RNAmeasurement.The sensors show high performance and achieve the RNA detection sensitivity as low as 0.1 fM, which is two orders of magnitude lower than the previously reports. Moreover, the G-FET biosensor can readily distinguish target RNA from noncomplementary RNA, showing high selectivity for RNA detection. The developed G-FET RNA biosensor with high sensitivity, fast analysis speed, and simple operation may provide a new feasible direction for RNA research and biosensing.


Introduction
The detection of RNA can provide very useful information for molecular biology research [1], genetic disease diagnosis [2], and environmental monitoring [3], which is of great significance in life science.To date, many methods for RNA detection have been developed, such as northern blotting [4], gel electrophoresis [5], and real-time quantitative polymerase chain reaction (QRT-PCR) [6].However, these methods suffer from many limitations, such as low throughput analysis, expensive reagent, complex operation, time consuming, and poor sensitivity.Therefore, it is necessary to develop a highly efficient, sensitive, fast, and low-cost platform for RNA detection.
In recent years, the significance of biological sensors using nanomaterials, such as silicon nanowires [7,8], carbon nanotubes [9,10], and graphene [11,12], has drawn significant attention to scientific research owing to its unique physical and chemical properties, such as surface effect and microsize effect.It is expected that these biosensors can be used as nextgeneration diagnostic chips for the future, which will play an increasingly important role in life science research and medical testing.Among the nanomaterials, graphene is a twodimensional planar film and has many unique properties, such as high carrier mobility and ease of functionalization [13,14].These unique properties of graphene have unique electrical advantages in building field-effect transistor biosensors for detecting biomolecules.
In this paper, we developed a graphene field-effect transistor (G-FET) biosensor using graphene as an electric channel.The single strand DNA as probe was immobilized on the surface of graphene by using 1-pyrenebutanoic acid succinimidyl ester (PBASE) as a linker.The target RNA is introduced onto the biosensor sample cell to hybridize with probe DNA.The sensor shows high sensitivity for RNA detection.Significant sensor response was achieved for detecting trace amounts of RNA in range from 0.1 fM to 1 pM.Moreover, the sensing strategy also shows a high selectivity by performing the contrast experiment.Compared with the traditional RNA detection technology, the G-FET RNA biosensor not only provides fast analysis, simple operation and low cost, but also shows 2 Advances in Condensed Matter Physics high sensitivity and selectivity.This kind of biosensor may provide a new way for RNA biosensing applications.

Preparation of Graphene.
The graphene films were grown by chemical vapor deposition (CVD) on a copper foil substrate at 1050 ∘ C. Prior to growth, the copper foil was cleaned successively with acetone, ethanol, and deionized water for 20 min in a KQ5200 ultrasonic cleaner.Then, the copper foil was annealed at 1050 ∘ C for 10 min with flowing H 2 of 15 sccm at 90 mtorr for improving the quality of graphene [15,16].For graphene growth, the gas mixture of CH 4 and H 2 is then flowed at 460 mtorr with rates of 16 and 30 sccm for 30 min.Finally, the sample was rapidly cooled to room temperature with flowing H 2 of 15 sccm at 90 mtorr.

Fabrication of G-FETs.
In this paper, poly(methyl methacrylate) (PMMA) was used as the intermediate carrier to transfer graphene films [17,18].The PMMA acetone solution was coated on graphene/copper foils with low-speed suspension 9 s (1600 r/min) and high-speed spin-coating 60 s (3000 r/min), followed by baking at 150 ∘ C for 30 min.The copper was etched with FeCl 3 solution over a period of 10 h to release PMMA/graphene films into the solution (Figure 1(a)), after washing the PMMA/graphene film with deionized water, which was transferred onto a glass substrate with two ITO electrodes (Figures 1(b)-1(e)).Here, the ITO electrode was used as source and drain electrode, respectively.Then, the substrate/PMMA/graphene was baked on a hot plate at 180 ∘ C for 30 minutes to remove the moisture and to make the graphene more flat.After that, the substrate/PMMA/graphene was soaked in acetone solution for 24 hours to remove PMMA to leave graphene on the substrate cross source and drain electrode as the electric channel.A sample cell ( = 0.5 mm) was mounted on graphene for RNA solution addition (Figure 1(f)).An Ag/AgCl electrode as reference electrode was inserted into the sample cell to provide gate voltage ( gs ).

Immobilization of DNA Probes on Graphene.
In order to immobilize the DNA probes on graphene, PBASE is used as an intermediate linker to modify graphene due to its high chemical stability and being nonhydrophilic and insoluble in organic solvents.The pyrene group of PBASE bonds to the six-membered ring structure on the surface of graphene by the - stacking force, and the succinimide can covalently bond with the amino group on the probe DNA.PBASE solution at 1 mM in N,N-dimethylformamide (DMF) was added to the sample cell at room temperature for 1 h to make PBASE attach on graphene.Then, the G-FET device was washed with DMF and 0.1x PBS buffer sequentially.After that, 100 nM DNA was introduced to the sample cell at room temperature for 18 h to serve as probe for RNA detection.Finally, the excess unreacted DNA was rinsed with a 0.1x PBS buffer.

Detection of RNAs.
In order to reduce the degradation of RNAs, all the experiments involved in RNA were carried out in RNase-free environment.All buffers and deionized water were disposed with 0.1% DEPC overnight and autoclaved.G-FET device was treated with RNase Zap reagent before adding RNAs.The target RNAs with different concentrations were added to the sample cell to hybridize with probe DNA at room temperature for 5 h.The target RNAs were rinsed with 0.1x PBS buffer to avoid nonspecific binding.The transfer curve of G-FET was measured in a semiconductor parameter analyzer (PDA FS360) coupled with a probe station (PEH-4).In the measurement, the G-FET device was provided with a varying gate voltage  gs from −0.6 to 0.6 V with the scanning step of 0.025 V by an Ag/AgCl electrode and a constant voltage of 0.01 V was provided between drain and source electrodes.

2.6.
Characterizations.The transferred graphene on glass and the functionalization step were characterized by Raman spectra.The Raman spectra of the graphene were performed using a Raman spectrometer (Horiba HR-800) with laser excitation at 532 nm (2.33 eV).The excitation laser spot is about 0.5 m.A field emission-scanning electron microscope (FE-SEM) (ZEISS, SUPRATM-55) with accelerating voltage 5 kV was used to analyze the morphology of the as-grown graphene on Cu foil and the transferred graphene on glass.

Results and Discussion
Figure 2(a) shows SEM image of as-grown graphene film on copper foil.There are a few light ridges observed on the surface of the sample.The ridges were caused by the difference of thermal expansion coefficient between Cu and graphene [19].After the graphene was transferred onto the glass substrate, no ridges were observed as shown in Figure 2(b).It could be that the graphene film is spread out in the transfer process.The films are found to be flat and continuous without any holes and tears, indicating the well transfer of graphene.Figure 2(c) shows the Raman spectrum of graphene films after transferred on glass substrate.The D, G, and 2D bands associated with graphene are clearly seen at ∼1348 cm −1 , ∼ 1585 cm −1 , and ∼2750 cm −1 , respectively.Among them, the G band is caused by the first-order scattering of the in-plane optical phonon  2g mode, and the 2D band is the result of a second-order process involving two phonons with opposite momentum, which are regarded as characteristic bands of the graphene structure [20].The ratio of the intensity 2D band and G band ( 2D / G ) is about 2-3, indicating the grown graphene is monolayer.The D band is due to the out-ofplane breathing mode of the sp 2 atoms and is related to the structural defects [21].Raman spectra record the functionalization step of graphene as shown in Figure 2(d).After introduction PBASE molecules into graphene, several new bands were observed.The bands at 1332.7 and 1383.6 cm −1 can be assigned to the introduction of disorder arising from orbital hybridization of the molecule with the graphene plane, and the bands at 1611.3 cm −1 are due to the pyrene group resonance [22].After the probe DNA was further immobilized on the graphene/PBASE layer, more bands appeared at 1476.4,1502.5, and 1655.8 cm −1 which can be assigned to the vibrational modes of DNA molecules [23].
Figure 3 shows the transfer characteristics (drain-source current ( ds ) versus gate voltage ( gs )) of the PBASE modified G-FET (black curve) and after probe DNA functionalization (red curve).Here, the drain-source voltage was set to be a small value of 0.01 V to reduce the electrical damage to probe DNA.After the probe DNA was modified on the G-FET by PBASE, the charge neutrality point voltages ( cnp ) were shifted to the positive gate voltage direction.The change of electric potential of graphene can be explained by the negative electrostatic gating effect.As DNA has a negatively charged triphosphate group, it can modulate the Fermi level of graphene by inducing excess hole carriers and in turn shift  cnp to the positive direction [24,25].
Figure 4(a) shows the transfer characteristics of DNA probe-modified G-FETs before and after addition of target RNAs (full-complementary) with different concentration from 0.1 fM to 1 pM.With the concentration increase of the target RNAs,  cnp successively shifted to the negative direction.The shift can be explained as an electron transfer effect caused by nonelectrostatic stacking interaction between RNAs and graphene layer [26].Figure 4(b) shows the  cnp shift as a function of the concentration of target RNAs.The G-FET sensor shows a very high sensitivity.The electrical potential of graphene potential was significantly decreased when the RNA concentration was increased from 0.1 fM to 1 pM.It is noted that the detection limit of our G-FET device for RNA is as low as 0.1 fM.The detection limit is two orders of magnitude lower than that reported in the literature where the reduced graphene oxide is used as electric channel [14].Here, the high sensitivity of our G-FET device can be attributed to the excellent transfer properties of CVD graphene.To represent the capability of the quantitative detection of RNA and its reproducibility, the linear fit calibration curve ( 2 = 0.970) with error bars was also shown in Figure 4(b).The reasonable linear response of G-FET is observed from 0.1 fM to 1 pM.However, as indicated by error bars, the fluctuation of sensor response was relatively large, especially for lower concentration detection.
Besides sensitivity, selectivity is also a critical parameter to assess the performance of a sensor.In order to verify the selectivity of the G-FET device for RNA detection, identical concentrations of noncomplementary RNA (NC-RNA) and full-complementary RNA (FC-RNA) were introduced into the sample cell, respectively.As shown in Figure 5, a significant shift of  cnp occurs when detecting FC-RNA.In contrast, for NC-RNA detection, the shift of  cnp is negligible.This contrast indicates that only a small amount of NC-RNA binds to the probe DNA by nonspecific adsorption.Therefore, the G-FET biosensor makes a good distinction between target RNA and noncomplementary RNA, indicating the high selectivity of our G-FET RNA sensor.

Conclusions
In this work, we developed a G-FET biosensor for detecting trace amounts of RNA.The addition of target RNA affects the electrical potential of graphene and allows label-free detection of RNA by readout of the shift of  cnp .Benefiting from the excellent transfer properties of the CVD graphene, the sensing sensitivity of the sensor was significantly enhanced.The detection limit of G-FET biosensor for sensing RNA is as low as 0.1 fM, which is two orders of magnitude lower than the previously reports.Moreover, the G-FET biosensor can readily distinguish target RNA from NC-RNA, showing high selectivity for RNA detection.The developed G-FET provides a possible way for label-free and ultrasensitive RNA detection, suggesting a promising future for biosensing applications.

Figure 1 :Figure 2 :Figure 3 :
Figure 1: Schematic of the graphene transfer and G-FET fabrication.(a)-(e) Graphene film transferred onto the glass substrate with two ITO electrodes.(f) Photo of the fabricated G-FET.

Figure 4 :Figure 5 :
Figure 4: (a) Transfer characteristics of G-FET with addition of different concentrations of target RNA from 0.1 fM to 1 pM.(b) The change of Δ cnp of the G-FET with addition of the target RNA from 0.1 fM to 1 pM.