Aptamer functionalized addressable SWNT-film arrays between cantilever electrodes were successfully developed for biosensor applications. Dielectrophoretically aligned SWNT suspended films made possible highly specific and rapid detection of target proteins with a large binding surface area. Thrombin aptamer immobilized SWNT-film FET biosensor resulted in a real-time, label-free, and electrical detection of thrombin molecules down to a concentration of ca. 7 pM with a step-wise rapid response time of several seconds.X113338sciescopu

An, T

HAHN, SEI KWANG

Kim, KS

Lim, G

Lab on a Chip

English

포항공과대학교

COMMUNICATION www.rsc.org/loc | Lab on a ChipPublished on 09 July 2010. Downloaded by Pohang University of Science and Technology on 28/04/2015 05:50:06. View Article Online / Journal Homepage / Table of Contents for this issueReal-time, step-wise, electrical detection of protein molecules usingdielectrophoretically aligned SWNT-film FET aptasensorsTaechang An,a Ki Su Kim,b Sei Kwang Hahn*b and Geunbae Lim*acReceived 30th April 2010, Accepted 18th June 2010DOI: 10.1039/c005276kAptamer functionalized addressable SWNT-film arrays betweencantilever electrodes were successfully developed for biosensorapplications. Dielectrophoretically aligned SWNT suspended filmsmade possible highly specific and rapid detection of target proteinswith a large binding surface area. Thrombin aptamer immobilizedSWNT-film FET biosensor resulted in a real-time, label-free,and electrical detection of thrombin molecules down to a concen-tration of ca. 7 pM with a step-wise rapid response time of severalseconds.Aptamers are oligonucleotides which can specifically bind to targetmolecules such as small molecules, peptides, proteins, cells, and otherbiomolecules. Aptamers can be prepared by systematic evolution ofligands by exponential enrichment (SELEX) process.1–3Owing to thehigh binding affinity and selectivity to target biomolecules, aptamershave been used for various biosensor applications.4–7 Aptasensor isthe term for biosensors using aptamers for the detection of biomol-ecules. With the development of nanotechnology, a real-time, label-free, and electrical detection of biomolecules was successfully carriedout with the aptasensors. They have been developed using variousnano-materials such as single-walled carbon nanotube (SWNT),8conducting polymers,9 graphenes,10 and silicone nanowires (Si-NW)11,12 for real-time electrical detection of biomolecules. However,the aptasensors still had several problems such as difficulties infabrication and spatial control, reproducibility, a long detection time,insignificant electrical signals, and an unproportional sensitivity to theconcentration of target molecules. To circumvent these technicalhurdles, SWNT has been widely investigated for various biosensorapplications.SWNT-based biosensors have been initially developed using indi-vidual nanotubes13–15 resulting in many problems described above.These problems could be alleviated using SWNT-film based biosen-sors for the detection of DNA,16 proteins,17 and bacteria.18 Inparticular, it was made possible to minimize device variation, andgenerate a large active area and a high current output with a relativeinsensitivity to the spatial position.19 SWNT-film based biosensorsresulted in an improved signal-to-noise ratio and an enhanceddetection limit in comparison to the individual nanotube systems.20Furthermore, the sensitivity of SWNT-film based biosensors could beaDepartment of Mechanical Engineering, Pohang University of Science andTechnology (POSTECH), San 31, Hyoja-dong, Nam-gu, Pohang,Kyungbuk, 790-784, Korea. E-mail: limmems@postech.ac.krbDepartment of Material Science and Engineering, Pohang University ofScience and Technology (POSTECH), San 31, Hyoja-dong, Nam-gu,Pohang, Kyungbuk, 790-784, Korea. E-mail: skhanb@postech.ac.krcDepartment of Integrative Bioscience and Biotechnology, PohangUniversity of Science and Technology (POSTECH), San 31, Hyoja-dong, Nam-gu, Pohang, Kyungbuk, 790-784, Korea2052 | Lab Chip, 2010, 10, 2052–2056improved by suspending the SWNT-film away from the floorproviding more than twice the binding surface area with increasedconvectional and diffusional fluxes.21 In this work, we developeda new one-step method for the fabrication of dielectrophoreticallyaligned addressable SWNT-films and demonstrated a real-time, step-wise, rapid, electrical detection of thrombin molecules using theSWNT-film FET aptasensors.Fig. 1 shows a schematic representation for the fabrication ofthrombin aptamer functionalized SWNT-film FET biosensors.Cantilever electrode arrays were fabricated by a general MEMSprocess. A low stress silicon nitride (1 mm) was deposited on a Siwafer using a low pressure chemical vapor deposition (LPCVD) forinsulation and a cantilever structure. Gold electrode arrays (Cr/Au20/200 nm) were patterned on the SiN surface through a lift-offprocess. Then, cantilever arrays were patterned by SiN reactive ionetching (RIE). The Si below the cantilever electrodes was etched byisotropic wet etching to form a suspended cantilever structure.Finally, SU8 was coated on the electrodes for passivation except forthe cantilever surface exposing the SWNT-films to target molecules(Fig. 1a). On the other hand, a suspended SWNT-COOH film wasprepared by dielectrophoresis and the surface tension of watermeniscus. The suspension was placed between the patterned canti-lever electrodes.When anAC voltage of 1MHz frequency and 5 Vp-pamplitude was applied, SWNTs were aligned between cantileverelectrodes by dielectrophoretic force. The suspension was thenpartially removed and the remaining suspension formed a watermeniscus between the electrodes. The collected SWNTs werecompressed by the surface tension of water meniscus and attached tothe electrodes by Van der Waals force, forming a suspended SWNT-film between the electrodes (Fig. 1b). The SWNT density in the filmwas ca. 46 bundles per mm2 having ca. 10 SWNTs in each bundle,which could be controlled by changing the amplitude and frequencyofAC electric field,22 the concentration of SWNT suspension, and thecollection time.23 The suspended SWNT-films were addressedprecisely to electrodes, since they were attached only to the electrodeswhere an electric field was applied. In addition, the fabrication ofmulti-functional SWNT-film arrays was made possible by the site-specific positioning of SWNT-films with same and different compo-sitions on the same chip. It took less than 10 min to prepare theSWNT-film and 2 h to conjugate aptamers to the SWNT-film.Moreover, the aptasensor could be exploited repeatedly for thedetection of thrombin.Fig. 2a shows a SEM image of suspended SWNT-film arraysbetween electrodes. The SWNT-films constructed with severalSWNT bundle layers were well aligned and suspended between twocantilever electrodes (Fig. 2b). The large contact area betweenSWNT-films and the electrodes provided a low contact resistance,a robust mechanical contact for long-term stability, and a largebinding region for the biosensor by Schottky barrier modulationThis journal is ª The Royal Society of Chemistry 2010Fig. 1 Schematic representations for (a) the fabrication of thrombin aptamer functionalized SWNT-film field effect transistor (FET) biosensors, (b) thepreparation of suspended SWNT-films by dielectrophoresis and surface tension, and (c) the surface modification of SWNT in a strong acid to introducecarboxyl groups and the following covalent immobilization of thrombin aptamers by amide bond formation using 1-ethyl-3-[3-(dimethylamino)pro-pyl]carbodiimide (EDC) and N-hydroxysulfosuccinimide (sulfo-NHS).Published on 09 July 2010. Downloaded by Pohang University of Science and Technology on 28/04/2015 05:50:06. View Article Onlinebetween SWNT and the contact electrodes.24 The geometricaldimension of SWNT-films was controlled by the width and gap ofcantilever electrodes (Fig. 2c). We used cantilever electrodes witha width of 20 mm and a gap of 4 mm. After that, thrombin DNAaptamers were covalently immobilized on suspended SWNT-filmsfor specific binding tests to target proteins. Fig. 1c shows a schematicfor the surface modification of SWNT with thrombin aptamers.SWNT-COOH was prepared by the oxidation of carbon nanotubesin a strong acid solution with sonication.25 SWNT (5 mg) was sus-pended in 50 mL of concentrated H2SO4/HNO3 (3/1, v/v) and soni-cated in an ultrasonication bath for 3 h. The nanotube suspensionwas diluted with DI water and then centrifuged at 12 000 rpm for10min. The remaining acidwas completely removed by decanting thesupernatant and washing with DI water, repeatedly. When the pH ofThis journal is ª The Royal Society of Chemistry 2010the suspension was 7.4, the carbon nanotubes were re-dispersed inDIwater by ultrasonication. Thrombin DNA aptamers were immobi-lized as described elsewhere.12 Briefly, thrombin aptamer with anamine terminal group of 50-NH2-d(GGTTGGTGTGGTTGG)-30was synthesized by using an oligonucleotide synthesizer. Thrombinaptamers were dissolved in phosphate buffer (PB, 200mmol, pH8) toprepare an aptamer solution at a concentration of 20 mmol. Forcovalent surface immobilization, the SWNT-COOH films weretreated with 10 mL of PB and surface-activated by the addition of1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC, 3.0 mg) andN-hydroxysulfosuccinimide (sulfo-NHS, 1.7 mg). Then, the aptamersolution was added and incubated at room temperature for amidebond formation between the amine group of the aptamer and thecarboxyl group of SWNT-COOH film. The resulting aptasensorLab Chip, 2010, 10, 2052–2056 | 2053Fig. 2 SEM images of suspended SWNT-film arrays at the magnifica-tions of (a) 400 (scale bar: 50 mm) and (b) 5,000 (scale bar: 5 mm). (c)SEM image of suspended SWNT-films with a different channel length(scale bar: 20 mm). SEM images of aptamer functionalized SWNT-films(d) before and (e) after thrombin binding (scale bars: 5 mm).Published on 09 July 2010. Downloaded by Pohang University of Science and Technology on 28/04/2015 05:50:06. View Article Onlinewas rinsed extensively with PB to remove the remaining EDC andsulfo-NHS.25 In order to prevent the nonspecific binding, Tween 20was coated on the unoccupied sites of SWNT.26 As shown in Fig. 2band 2d, the thickness of SWNT-film increased after the covalentimmobilization of aptamers.The electrical properties of the SWNT-films were analyzed in realtime at room temperature using a precision semiconductor parameteranalyzer (HP 4156A). Fig. 3 shows the I–V characteristics of sus-pended SWNT-films in a buffer solution at pH 5.6. The source–draincurrent decreased when the electrolyte gate bias increased by thereference electrode. In other words, the suspended SWNT-filmsexhibited p-type characteristics in the buffer solution.22 For a realtime electrical detection of protein, nonspecific and specific bindingFig. 3 Electrical characteristics of suspended SWNT-films at variouselectrolyte gate voltages from 0.3 V to 0.3 V.2054 | Lab Chip, 2010, 10, 2052–2056tests were carried out using SWNT-COOH films without and withaptamers. Thrombin and VEGF were used as model proteins for thebinding tests. In the case of nonspecific binding tests as a controlexperiment, a 10 mL drop of the buffer and protein solutions at pH5.6 and specified concentrations were placed on the suspendedSWNT-films, and then the conductance of the suspended SWNT-films was monitored with a 10 mV bias voltage. Fig. 4a and 4b showthe current change with increasing concentrations of thrombin andVEGF, respectively. The aptasensor showed a meaningful currentchange at a concentration of 70 pMwith a response time less than 10s. In addition, the conductance decreased proportionally upon step-wise exposure to both thrombin and VEGF solution at concentra-tions from 70 pM to 700 nM. Proteins can bind to the surface ofSWNT-COOH film nonspecifically by electrostatic and hydrophobicinteractions, and p–p stacking between SWNTs.24 In order forspecific binding tests, thrombin aptamer-functionalized SWNT-filmbiosensor was surface modified with Tween 20 and assessed in thesame manner with the nonspecific binding tests. Each 3 mL of thebuffer and protein solutions with specified concentrations was addedsequentially to the aptasensor using a micropipette. As shown inFig. 4c, electric current dropped rapidly in a step-wise manner afterthe injection of thrombin, whereas no current change was observedfor the case of VEGF addition. The results confirmed the specificbinding of thrombin molecules to the SWNT-film aptasensor. Asreported elsewhere,24 the treatment with Tween 20 effectively mini-mized the non-specific binding. Finally, the sensitivity of SWNT-filmthrombin aptasensors was investigated with increasing thrombinconcentrations (Fig. 4d). The current dropped sharply in a step-wisemanner when thrombin samples were added sequentially at theconcentrations from 7 pM to 70 nM to the buffer solution. Thestandard deviation of the noise signal was estimated to be ca. 0.059.The signal detected at the concentration of 7 pMwas ca. 0.24, whichwas four times higher than the noise level and regarded as a signifi-cantly meaningful change. The variation of current after the additionof thrombin molecules can be explained by the electrostatic gatingeffect by the positive charge of thrombin molecules in the buffer atpH 5.6. In addition, the modulated Schottky barrier between elec-trodes and dielectrophoretically aligned SWNTs in the SWNT-filmdecreased the local work function, contributing for an extraordinaryrapid response time of several seconds.24 As shown in Fig. 2e, theSWNT-film surface was completely covered by thrombin moleculesafter the binding tests.In direct comparison with previous reports,10,24,26,27 the dielec-trophoretically aligned SWNT-film based aptasensor made possiblea real-time, step-wise, and electrical detection of proteins with a rapidresponse time and a high sensitivity proportional to the proteinconcentration. The response time of nanotube sensors placed on thefloor of the substrate was reported to be in the range of tens ofseconds to a few min.8,17,26–28 In contrast, the dielectrophoreticallyaligned SWNT-film based aptasensor had a very short response timeof a few seconds. In addition, the dielectrophoretically alignedSWNT-film based aptasensor had a lower detection limit of ca. 7 pMthan that of the single SWNT biosensors placed on the floor of thesubstrate (about 10 nM).8 Finally, the SWNT-film based aptasensorprovided a proportional sensitivity to the concentration of proteins.Most of the previous SWNT sensors did not exhibit a proportionalsensitivity, mostly converging with increasing protein concentra-tions.8,17,26 These patterns might be difficult to be applied in realsystems. As shown in the inset of Fig. 4d, however, the sensitivityThis journal is ª The Royal Society of Chemistry 2010Fig. 4 Real-time electrical detection of proteins using thrombin DNA aptamer-functionalized SWNT-film FET biosensors: Real-time electricaldetection for the nonspecific binding of (a) thrombin and (b) VEGF to the SWNT-COOH films. (c) Specific binding tests of thrombin molecules to theSWNT-film aptasensor after treatment with Tween 20. (d) Real-time electrical detection for the specific binding of thrombin molecules with increasingconcentrations to the SWNT-film aptasensor. Insets show the sensitivity of the SWNT-film aptasensor with increasing protein concentrations on a log–log scale (n ¼ 4).Published on 09 July 2010. Downloaded by Pohang University of Science and Technology on 28/04/2015 05:50:06. View Article Onlineincreased linearly in the protein concentrations from 70 pM to 70 nMon a log–log scale (n ¼ 4).ConclusionsDielectrophoretically aligned SWNT-films between patterned canti-lever electrodes were successfully exploited for the fabrication ofaptamer functionalized SWNT-film FET biosensors. The suspendedSWNT-film aptasensor exhibited the p-type characteristics in thebuffer solution. According to nonspecific binding tests as a controlexperiment, the aptasensor using SWNT-COOH film withoutaptamers showed a meaningful current change by the addition ofthrombin and VEGF at a concentration of 70 pM with a responsetime less than 10 s. In addition, after surface treatment with Tween20, the SWNT-film FET aptasensor demonstrated a real-time, label-free, step-wise, target-specific, and electrical detection of thrombinmolecules down to a concentration of 7 pM with an extraordinaryrapid response time of a few seconds and a proportional sensitivity.The novel addressable dielectrophoretically aligned SWNT-film FETThis journal is ª The Royal Society of Chemistry 2010aptasensor will be investigated further for various diagnostic appli-cations with biomarker-specific aptamers.AcknowledgementsThis work was supported by Mid-career Researcher Programthrough NRF grant funded by the MEST (No. 2009-0085377). Thiswork was supported by the Korea Research Foundation Grantfunded by the Korean Government (MOEHRD, Basic ResearchPromotion Fund, KRF-2008-331-D00753).Notes and references1 M. P. Robertson and A. D. Ellington,Nat. Biotechnol., 2001, 19, 650–655.2 J. Zhang, H. P. Lang, F. Huber, A. Bietsch, W. Grange, U. Certa,R. McKendry, H. J. Guntgerodt, M. Hegner and C. Gerber, Nat.Nanotechnol., 2006, 1, 214–220.3 H. A. Heus, Nat. Struct. Biol., 1997, 4, 597–600.4 P. Yin, H. M. T. Choi, C. R. Calvert and N. A. Pierce, Nature, 2008,451, 318–322.Lab Chip, 2010, 10, 2052–2056 | 2055Published on 09 July 2010. Downloaded by Pohang University of Science and Technology on 28/04/2015 05:50:06. View Article Online5 Y. Xiao, R. Y. Lai and K. W. Plaxco, Nat. Protoc., 2007, 2, 2875–2880.6 J. Bath and A. J. Turberfield, Nat. Nanotechnol., 2007, 2, 275–284.7 T. Nguyen, J. P. Hilton and Q. Lin, Microfluid. Nanofluid., 2009, 6,347–362.8 H. M. So, K. Won, Y. H. Kim, B. K. Kim, B. H. Ryu, P. S. Na,H. Kim and J. O. Lee, J. Am. Chem. Soc., 2005, 127, 11906–11907.9 O. S. Kwon, S. J. Park and J. Jang, Biomaterials, 2010, 31, 4740–4747.10 H. X. Chang, L. H. Tang, Y. Wang, J. H. Jiang and J. H. Li, Anal.Chem., 2010, 82, 2341–2346.11 H. S. Lee, K. S. Kim, C. J. Kim, S. K. Hahn and M. H. Jo, Biosens.Bioelectron., 2009, 24, 1801–1805.12 K. S. Kim, H. S. Lee, J. A. Yang, M. H. Jo and S. K. Hahn,Nanotechnology, 2009, 20, 235501.13 S. N. Kim, J. F. Rusling and F. Papadimitrakopoulos, Adv. Mater.,2007, 19, 3214–3228.14 S. S. Wong, E. Joselevich, A. T. Woolley, C. L. Cheung andC. M. Lieber, Nature, 1998, 394, 52–55.15 R. H. Baughman, A. A. Zakhidov and W. A. de Heer, Science, 2002,297, 787–792.16 A. Star, E. Tu, J. Niemann, J. C. P. Gabriel, C. S. Joiner andC. Valcke, Proc. Natl. Acad. Sci. U. S. A., 2006, 103, 921–926.2056 | Lab Chip, 2010, 10, 2052–205617 H. R. Byon and H. C. Choi, J. Am. Chem. Soc., 2006, 128, 2188–2189.18 G. A. Zelada-Guillen, J. Riu, A. Duzgun and F. X. Rius, Angew.Chem., Int. Ed., 2009, 48, 7334–7337.19 D. R. Kim and X. L. Zheng, Nano Lett., 2008, 8, 3233–3237.20 X. Q. Chen, T. Saito, H. Yamada and K. Matsushige, Appl. Phys.Lett., 2001, 78, 3714–3716.21 Q. Cao and J. A. Rogers, Adv. Mater., 2009, 21, 29–53.22 E. S. Snow, J. P. Novak, M. D. Lay and F. K. Perkins, Appl. Phys.Lett., 2004, 85, 4172–4174.23 M. Lu, M. W. Jang, G. Haugstad, S. A. Campbell and T. H. Cui,Appl. Phys. Lett., 2009, 94, 261903.24 Heller, A. M. Janssens, J. Mannik, E. D. Minot, S. G. Lemay andC. Dekker, Nano Lett., 2008, 8, 591–595.25 T. Cang-Rong and G. Pastorin, Nanotechnology, 2009, 20, 255102.26 Maehashi, T. Katsura, K. Kerman, Y. Takamura, K.Matsumoto andE. Tamiya, Anal. Chem., 2007, 79, 782–787.27 R. J. Chen, S. Bangsaruntip, K. A. Drouvalakis, N. W. S. Kam,M. Shim, Y. M. Li, W. Kim, P. J. Utz and H. J. Dai, Proc. Natl.Acad. Sci. U. S. A., 2003, 100, 4984–4989.28 C. Li, M. Curreli, H. Lin, B. Lei, F. N. Ishikawa, R. Datar, R. J. Cote,M. E. Thompson and C. W. Zhou, J. Am. Chem. Soc., 2005, 127,12484–12485.This journal is ª The Royal Society of Chemistry 2010

Real-time, step-wise, electrical detection of protein molecules using dielectrophoretically aligned SWNT-film FET aptasensors

http://oasis.postech.ac.kr/bitstream/2014.oak/25854/1/OAIR002083.pdf

Real-time, step-wise, electrical detection of protein molecules using dielectrophoretically aligned SWNT-film FET aptasensors

Abstract

Similar works

Full text

Available Versions

포항공과대학교