그래핀기반의 FET 바이오센서연구동향 성균관대학교나노소재기반휴먼인터페이스융합연구센터선임연구원윤옥자

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1 그래핀기반의 FET 바이오센서연구동향 성균관대학교나노소재기반휴먼인터페이스융합연구센터선임연구원윤옥자

2 1. 서론 Ø 바이오센서는 NT-BT-IT 의융합기술을기반으로현장진단을통한질병예방및실시간건강상태모니터링에의한종합적의료서비스가가능한종합적유비쿼터스헬스케어에목적을두고있으며나노기술의발전에힘입어급격한발전을하고있다. 1 Ø 바이오센서는의료용뿐만아니라반도체, 군사, 재료공학, 생명공학분야등의적용및응용성이매우크고거대한잠재력을가지고있는기술집약적인원천기술이다. 2 Ø 바이오센서는생물감지물질 (bioreceptor) 과신호변환기 (signal transducer) 로구성되어분석하고자하는물질을선택적으로감지할수있도록되어있고신호변환방법으로는전기화학적 (electrochemical), 형광, 발색, SPR (surface plasmon resonance), FET (field-effect transistor), QCM (quartz crystal microbalance), 열센서등다양한물리화학적방법을사용하고있다. 3 ØFET 바이오센서개발은저가, 고민감도, 선택성, 소형화, 안정성및신뢰성확보에있으며이를극복하기위하여나노선, 나노입자, 탄소나노튜브, 그래핀등의나노물질를센서에이용하는연구가보고되고있다. 2 Ø 본정보는그래핀기반 FET 바이오센서연구에대한최근연구동향에대해기술하고자한다.

3 2. 그래핀기반 FET 바이오센서의 glucose 와 glutamate 의실시간검출 ØNanoelectronic biosensors based on CVD grown graphene 4 - 그래핀은전기적, 물리적, 광학적성질이뛰어나탄소나노튜브와같이바이오센서의전극재료로써적합한나노물질이므로, field-effect transistors (FETs) 에그래핀 transistor 로사용하기위하여 CVD 합성방법으로큰사이즈의그래핀필름을제조하여생체분자를실시간센싱 (Real-time biomolecular sensing ) 하여측정함. - 그래핀필름표면을 specific redox enzyme (glucose oxidase or glutamic dehydrogenase) 으로산화시켜 doping 효과로인한 glucose 또는 glutamate 분자들의감지시그래핀 transistor 의전기적변화에의해검출하였음. - 이연구는그래핀이 real-time nanoelectronic biosensing 하기위한촉망받는잠재성을보여주었고, 그래핀의 2D 구조, 뛰어난전기적성질, 세포의부착성, 친화성등의성질때문에그래핀소자에살아있는세포 (living cells) 와인터페이스하거나, dynamic bimolecular secretion 등의응용성을입증하는연구임.

4 Figure 2. (a) Transfer curves before and after adding glucose (10 mm) to the GOD functionalized graphene FET. (b) Transfer curves before and after adding glutamate (1 mm) to the GluD functionalized graphene FET in the presence of 5 mm b-nad. Figure 1. (a) Schematic illustration of GOD functionalized graphene FET. (b) Current responses to the addition of glucose to various concentrations. The upper inset shows that GOD free graphene FET is not responsive to 10mMglucose. The lower inset shows the response curve of the graphene FET to glucose fitted by an exponential function. Figure 3 (a) Current responses of graphene FET to addition of products from glucose oxidation: H2O2 and D-glucono-1,5-lactone (1 mm). (b)current responses of graphene FET to addition of products of glutamate oxidation: NH4OH and a-ketoglutarate (1 mm).

5 3. 그래핀기반 FET 바이오센서의 ph 및단백질흡착의모니터링 ØElectrolyte-gated graphene field-effect transistors for detecting ph and protein adsorption 5 - 기계적박리 (mechanical exfoliation) 방법에의하여준비한단층그래핀으로액상에담겨져있는 GFET (electrolyte-gated graphene field-effect transistors) 를제조하여 transport behaviors 를조사하였으며액상에서 ph 와단백질 (bovine serum albumin, BSA) 흡착을전기적으로검출함. -BSA 흡착에의한그래핀 doping 에 FET capacitance 에변화를일으켜전기적특성이달라지며 ph 변화또한 H +, OH - 양에의한 charge 변화에의한전기전도도변화를입증함. - 그래핀을채널로사용하였으며용액상에서소자의성능이크게향상되었고 ph 에따른전기전도도와단백질흡착을검출함. ph 와 Dirac point 의게이트전압은선형관계를이루며 BSA 와같은단백질흡착에따른 BSA 의농도증가에의한전기전도도와 mobilty 변화역시증가함을증명함. -GFETs 는실시간측정이가능한화학적, 생물학적센서로써 ph 나생체분자농도를검출하기위한고감도전기적센서임을입증함.

6 Figure 4. (a) Optical micrograph of a typical GFET. (b) Schematic illustration of the experimental setup for electrolyte-gated GFETs. Figure 5. (a) Conductance as a function of back-gate voltage at 10-3 Pa and top-gate voltage in an electrolyte at ph 5.8. (b) Enlarged view of conductance as a function of gate voltage. Figure 6 (a) Conductance as a function of top-gate voltage of a GFET at ph 4.0, 4.8, 5.8, and 7.8. (b) Conductance versus time data of a GFET for ph values from 4.0 to 8.2. (c) Top-gate voltage at the Dirac point as a function of ph. The dashed line is a linear fit to the data points. Figure 7. (a) Conductance versus time for electrical monitoring of exposure to various BSA concentrations. Dashed lines indicate the average conductance. The inset shows the time dependence of the conductance at adding the 120 nm BSA. (b) Plot of the conductance change of a GFET versus BSA concentration. (c) BSA concentration per conductance change (CBSA/ΔG) as a function of BSA concentration.

7 4. 그래핀기반 Label-Free FET 바이오센서 ØLabel-free biosensors based on aptamer-modified graphene field-effect transistors 6 -Label-free immunosensor 기반 aptamer modified graphene field-effect transistor (G-FET) 에대한연구임. -Immunoglobulin E (IgE) aptamers ( 높이 :~3nm) 를단층그래핀채널위에고정시켰고드레인전류 (drain current) 가 IgE aptamers 의기능화에따라증가되었음을확인함. -Aptamer-modified G-FET 는 IgE 단백질를선택적으로검출하였고다른다른종류의단백질들은검출되지않았음을입증함. - 이연구의결과는 dissociation constant 는 47nM 이였고 G-FETs 가전기적으로생체분자를검출할수있는 label-free biological sensors 로써생물학적센서로이용하기에적합한뛰어난소자임을보임.

8 Figure 9. Time course of ID for an aptamer-modified G-FET. At 10, 30, and 50 min, respectively, BSA and SA (nontarget proteins) and IgE (the target protein) were injected into the aptamer-modified graphene channel. Figure 8. (a) AFM image of a G-FET with a bare graphene channel. (b) AFM image of the G-FET with an aptamermodified graphene channel. Figure 10. (a) Time course of ID for an aptamer-modified G-FET. At 10 min intervals, various concentrations of IgE were injected. (b) Change in drain current vs IgE concentration. The red dashed curve shows a fit to the Langmuir adsorption isotherm with KD ) 47 nm.

9 5. 그래핀기반 FET 바이오센서의화학적, 생물학적센싱 Ø Chemical and biosensing applications based on graphene fieldeffect transistors 7 - 그래핀은 micro-mechanical 절단방법으로단층그래핀을제조하여 graphene Field-Effect Transistors(G-FETs) 기반화학적, 생물학적센서로의응용을보고함. - 액상 ph 의변화는 의최소검출한계 (lowest detection limit (signal/noise=3)) 를가지고검출되었고 I D 는 protein 농도에의존함을보였으며 BSA농도에따른 I D 변화는 Langmuir 흡착등온식에잘맞았음을입증함. - 또한 G-FETs 의등전점 (isoelectric point) 때문에생체분자의다른전하형태를분명하게검출하였음. -G-FETs 는고감도화학적, 생물학적센서로써뛰어난소자임을입증하였고앞으로선택적으로 protein sensing 을시도할예정임.

10 Figure 11. Schematic illustration of experimental setup with G-FETs. Figure 13. ID as a function of top-gate voltage of a G-FET at ph 4.04, 4.94, 6.57 and Figure 12. Photograph of experimental setup with a G-FET. Figure 14. Time course of normalized ID for G-FETs at VD and VTGS of 0.1 and 0.1 V, respectively, in 10-mM phthalate and phosphate buffer solution. Red (blue) lines indicate that 80 (100) nm BSA was added at 10 min.

11 6. 그래핀기반 FET 바이오센서의 Sensor Arrays ØResearch article cell proliferation tracking using graphene sensor arrays 8 - 이연구는독창적인 label-free graphene sensor array 기술개발로검출방법은그래핀 FET devices 를변형하여세포배양용액의영향구성성분변화를모니터링함. - 배양용액안에있는 Escherichia coli 의 microdispensing 을통하여세포가증식하면서각각의 sensor arrays 위에정확하게위치하도록하는것이가능함. - 박테리아증식을촉진하는데사용되는영양액의농도변화를모니터링하기위하여 label-free 하면서재사용이가능한 sensor array 의가능성을보여줬으며농도변화는전기화학적센싱방법에의하여분석되었고전기전도도의변화는용액환경에서검출됨. -Graphene Sensor Arrays 는그래핀 FET device, sample dosing, initial electrical characterisation 으로구성되어있고초소형화, 진단과 drug development protocols 의소요시간단축등뛰어난성능을보여주고있음.

12 Figure 15. Production and microfabrication of graphene. (a) CVD growth of graphene on copper foil in a tube furnace, (b) graphene is attached to SiO2/Si by attaching to a support layer of PMMA and thermal-release tape and transferring by a combination of heat and pressure, (c) after transfer to SiO2/Si substrate, ani protection layer is patterned on the graphene, (d) oxygen plasma removes the unprotected graphene and the Nickel is subsequently removed by HCl etching. (e) shows the contacted graphene strips and (f) shows integration of the sensor into a chip carrier. Figure 16. (a) Example of the Raman spectrum of CVD-grown graphene after transfer. (b) Controlled deposition of small volumes of LB/glycerolmedium is shown to be accurate by opticalmicroscopy. (c) The p-type behaviour of the pristine graphene is observed, and a clear increase in hysteresis is noted upon measurement of LB. A shift in gate dependency with LB concentration is also noted. The glycerol is added to reduce droplet evaporation and is maintained at the same concentration in each case. (d) A change in resistance with LB concentration is noted using two-probe measurements on the graphene FET. Figure 17. Microdispensing of 50% LB/25% glycerol medium containing E. coli shows it is feasible to guide proliferation to the graphene sensor region. (a) An SEM image shows the graphene sensor and contacts at 80 tilt with a dense circular pattern of adhered bacteria fixed using a dehydration protocol and shown in (b) at a higher magnification.

13 7. Microfluidic Channel 에서의그래핀기반 FET 바이오 ØFlow sensing of single cell by graphene transistor in a microfluidic channel 9 - 그래핀의전기적인성질은국소적인유전체환경에의해서나 long-range charge scatterers 로부터일어나는정전기적힘 (electrostatic forces) 에영향을받아세포의표면전하밀도에민감함. -세포하나수준의말라리아에감염된적혈구를 flow catch-release 감지를위하여 microfluidic 유동세포계측방법을이용한직접화된그래핀 transistor array 를개발함. - 말라리아에감염된적혈구세포는세포막표면에감염된기생충에의해단백질이생성되고그결과 membrane 'knobs' 이발생하여 endothelial CD36 receptors 로기능화한그래핀에 membrane 'knobs' 이감지되므로써말라리아에감염된적혈구세포를선택적으로잡아냄. - 말라리아에감염된적혈구세포는그래핀의전도도에서고감도전기용량변화 ( high sensitive capacitively coupled changes) 에의하여그래핀의전도도변화를유도함. -Microfluidic channel 안에있는그래핀 transistors 를통한혈액흐름속에서감염된세포를통계적으로계산할수있어임상에서진단응용으로큰기대가됨.

14 Figure 18. Graphene-based detection of single Plasmodium falciparum-infected erythrocyte (PE). (a) (Left) Schematic illustration of an array of graphene transistors on quartz. The electrodes are protected by a SU-8 photoresist that conveniently acts as the side wall for the microfluidic channel through which cells flow. (Right) Specific binding between ligands located on positively charged membrane knobs of parasitized erythrocyte and CD36 receptors on graphene channel produces a distinct conductance change. Conductance returns to baseline value when parasitized erythrocyte exits the graphene channel. (b) DIC image of independent graphene transistors with SU-8/PDMS microfluidic channel. Inset shows the etched graphene strip between source and drain electrodes. Scale bar is 30 μm. (c) Three-dimensional AFM images of (left) parasitized erythrocyte14 (scale bar is 1 μm) and (right) 3D height plot of the surface of parasitized erythrocyte revealing protruding knobs which overlaid with adhesion maps of knob ligands (PfEMP1) using CD36- functionalized AFM tip (yellow regions).

15 Figure 19. Characterization and PE detection of CD36-functionalized graphene transistor. (a) CD36 coupled with FITC-conjugated CD36 antibody on graphene (inset) shows significant fluorescence quenching as compared to that on quartz. Scale bar is 100 μm. (b) AFM image and height profile of CD36 coupled with FITC-conjugated CD36 antibody on graphene shows a combined protein height of 3 nm. (c) Device performance for graphene and CD36-functionalized graphene gated in culture medium. Inset shows the corresponding capacitancevoltage measurements. Device channel length and width are 10 and 20 μm, respectively. (d) IdsVg curves shows distinct shifts in charge neutrality point when PE adhered onto CD36-functionalized graphene channel. The increase in the width of the minimum conductivity plateau upon PE adhesion is also evident. Figure 20. Parasite differentiation. (a) Conductance-time plots for (early to mid) trophozoite-pe and schizont-pe measured at Vg = 0.1 V and corresponding DIC images on the right. Device channel length and width is 8 and 15 μm, respectively. (b) Box plots of percentage conductance changes for trophozoite-pe and schizont-pe. The top and bottom of the box denote 75th and 25th percentiles of the population, respectively, while the top and bottom whiskers denote 90th and 10th percentiles, respectively. Maximum and minimum values are denoted by open squares. Gaussian distribution of the raw data points is shown. (c) Conductance-time plot for the occupation of 2 schizont-pe shows distinct conductance rise which corresponds to single schizont-pe.

16 4. 결론 Ø 그래핀은같은탄소나노물질인탄소나노튜브와비교하여저가비용, 높은표면적, 고순도 (Hummers method 에의하여제조된그래핀은촉매나노입자가없음 ), 분산성, 두께및크기제어, 투명성제어, safety 및제조과정이쉽다는장점을가지고있어탄소물질의 electrocatalytic effects 를연구하기에훌륭하다 10. Ø그래핀과같은나노물질을이용한 FET 바이오센서는저가제작비용, 고감도, 생체분자와결합가능한넓은표면적, 실시간측정, 소형화, 측정시간단축, 안정성및신뢰성확보등많은장점이있고앞으로 flexible FET based biosensor device를제작하기에뛰어난소재임을확인할수있다. Ø 그래핀의 biocompatibility, stability, p-p 결합에의한표면기능화등에의한선택적생체분자인식이뛰어나그래핀기반 FET 바이오센서개발은고부가가치를창출하는기술이며화학적, 생물학적바이오센서로의다양한응용이기대된다.

17 5. 참고문헌 1. 이정오, 앱타머및 FET 기바의 multiplexing 진단, NEWS & INFORMATION FOR CHEMICAL ENGINEERS, 26 (2006). 2. 최철종, 나노바이오센서연구동향, 나노세라믹스연구및산업화현황, 14 (2011) KT 종합기술원기술전략실기술전략담당, U-헬스케어확산의도화선바이오센서 (Bio Sensor), Y. Huang, X. Dong, Y. Shi, C. M. Li, L.-J. Li, P. Chen, Nanoelectronic biosensors based on CVD grown graphene Nanoscale 2 (2010) Y. Ohno, K. Maehashi, Y. Yamashiro, K. Matsumoto, Electrolyte-gated graphene field-effect transistors for detecting ph and protein adsorption Nano Lett. 9(2009) Y. Ohno, K. Maehashi, K. Matsumoto, Label-free biosensors based on aptamer-modified graphene fieldeffect transistors J. AM. CHEM. SOC. 132 (2010) Y. Ohno, K. Maehashi, K. Matsumoto, Chemical and biosensing applications based on graphene field-effect transistors (physics and applications of graphene experiments) Sergey Mikhailov (Ed.), ISBN: , InTech, R. Daly, S. Kumar, G. Lukacs, K. Lee, A.Weidlich,M. Hegner, G. S. Duesberg, Cell proliferation tracking using graphene sensor arrays Journal of Sensors, 2012 (2011) P. K. Ang, A.Li, M. Jaiswal, Y. Wang, H. W. Hou, J. T. L. Thong, C. T. Lim, K. P. Loh, Flow sensing of single cell by graphene transistor in a microfluidic channel Nano Lett. 11(2011) Y. Shao, J. Wang, H. Wu, J. Liu, I. A. Aksay, Y. Lin, Graphene based electrochemical sensors and biosensors: A review Electroanalysis 22 (2010)

[ 화학 ] 과학고 R&E 결과보고서 나노입자의표면증강을이용한 태양전지의효율증가 연구기간 : ~ 연구책임자 : 김주래 ( 서울과학고물리화학과 ) 지도교사 : 참여학생 : 원승환 ( 서울과학고 2학년 ) 이윤재 ( 서울과학고 2학년 ) 임종

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