Label-Free Dual Sensing of DNA Molecules Using GaN Nanowires

Dec 1, 2008 - Yu-Kuei Hsu,‡ Ying-Chih Chang,§ Kuei-Hsien Chen,*,†,‡ and Li-Chyong Chen*,† ... We demonstrate a rationale for using GaN nanowi...
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Anal. Chem. 2009, 81, 36–42

Label-Free Dual Sensing of DNA Molecules Using GaN Nanowires Chin-Pei Chen,†,† Abhijit Ganguly,†,† Chen-Hao Wang,† Chih-Wei Hsu,‡ Surojit Chattopadhyay,| Yu-Kuei Hsu,‡ Ying-Chih Chang,§ Kuei-Hsien Chen,*,†,‡ and Li-Chyong Chen*,† Center for Condensed Matter Sciences, National Taiwan University, Taipei 10617, Taiwan, Institute of Atomic and Molecular Sciences, Academia Sinica, Taipei 10617, Taiwan, Genomics Research Center, Academia Sinica, Taipei 11529, Taiwan, and Institute of Biophotonics, National Yang Ming University, Taipei 112, Taiwan We demonstrate a rationale for using GaN nanowires (GaNNWs) in label-free DNA-sensing using dual routes of electrochemical impedance spectroscopy (EIS) and photoluminescence (PL) measurements, employing a popular target DNA with anthrax lethal factor (LF) sequence. The in situ EIS reveals that both high surface area and surface band-bending in the nanowires, providing more binding sites and surface-enhanced charge transfer, respectively, are responsible for the enhanced sensitivity to surface-immobilized DNA molecules. The net electrontransfer resistance can be readily deconvoluted into two components because of the coexistence of two interfaces, GaN/DNA and DNA/electrolyte interfaces, in series. Interestingly, the former, decreasing with LF concentration (CLF), serves as a signature for the extent of hybridization, while the latter as a fingerprint for DNA modification. For PL-sensing, the band-edge emission of GaNNWs serves as a parameter for DNA modification, which quenches exponentially with CLF as the incident light is increasingly blocked from reaching the core nanowire by rapidly developing a UV-absorbing DNA sheath at high CLF. Furthermore, successful application for detection of “hotspot” mutations, related to the human p53 tumor-suppressor gene, revealed excellent selectivity and specificity, down to picomolar concentration, even in the current unoptimized sensor design/condition, and in the presence of mutations and noncomplementary strands, suggesting the potential pragmatic application in complex clinical samples.

high surface-to-volume ratio offers the opportunity for efficient biobinding and high sensitivity in detecting biomolecules.1-6 A wide range of nanomaterials and sensing techniques, including optical absorbance or Raman scattering (via surface plasmon),5-11 electrochemical or electrical,3,12-14 colorimetry,1,2,15 photoluminescence (PL),16-18 and chemoluminescence,19 have been explored. These techniques employed mostly metals,2,7,8 silicon,2,3,12,13 II-VI semiconductors,2,5-9,16,17 and magnetic materials,1,20,21 and the use of III-V semiconductors have been scarce.22-26 (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) (14) (15) (16) (17)

Biosensing is an important area for clinical diagnosis, medicine, and bioengineering. Sensing single or minute amount of biomolecules requires integration of the highly selective recognition of biomaterials with the unique electronic, photonic, and catalytic features of nanomaterials. Proteins, nucleic acid fragments and their biomolecular complexes have nanometric dimensions comparable to the inorganic nanomaterials, of which the inherently * To whom correspondence should be addressed. E-mail: chenkh@ pub.iams.sinica.edu.tw (K.-H.C.) and [email protected] (L.-C.C.). † National Taiwan University. ‡ Institute of Atomic and Molecular Sciences, Academia Sinica. | National Yang Ming University. § Genomics Research Center, Academia Sinica.

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Katz, E.; Willner, I. Angew. Chem., Int. Ed. 2004, 43, 6042–6108. Alivisatos, P. Nat. Biotechnol. 2004, 22, 47–52. Cui, Y.; Wei, Q.; Park, H.; Lieber, C. M. Science 2001, 293, 1289–1292. Nguyen, C. V.; Delzeit, L.; Cassell, A. M.; Li, J.; Han, J.; Meyyappan, M. Nano Lett. 2002, 2, 1079–1081. Bruchez, M., Jr.; Moronne, M.; Gin, P.; Weiss, S.; Alivisatos, A. P. Science 1998, 281, 2013–2016. Chan, W. C. W.; Nie, S. Science 1998, 281, 2016–2018. Cao, Y. C.; Jin, R.; Nam, J.-M.; Thaxton, C. S.; Mirkin, C. A. J. Am. Chem. Soc. 2003, 125, 14676–14677. Grubisha, D. S.; Lipert, R. J.; Park, H.-Y.; Driskell, J.; Porter, M. D. Anal. Chem. 2003, 75, 5936–5943. Patolsky, F.; Gill, R.; Weizmann, Y.; Mokari, T.; Banin, U.; Willner, I. J. Am. Chem. Soc. 2003, 125, 13918–13919. Chattopadhyay, S.; Lo, H.-C.; Hsu, C.-H.; Chen, L.-C.; Chen, K.-H. Chem. Mater. 2005, 17, 553–559. Chattopadhyay, S.; Shi, S. C.; Lan, Z. H.; Chen, C. F.; Chen, K.-H.; Chen, L.-C. J. Am. Chem. Soc. 2005, 127, 2820–2821. Hahm, J.; Lieber, C. M. Nano Lett. 2004, 4, 51–54. Li, Z.; Chen, Y.; Li, X.; Kamins, T. I.; Nauka, K.; Williams, R. S. Nano Lett. 2004, 4, 245–247. Katz, E.; Willner, I. Electroanalysis 2003, 15, 913–947. Elghanian, R.; Storhoff, J. J.; Mucic, R. C.; Letsinger, R. L.; Mirkin, C. A. Science 1997, 277, 1078–1081. Mahtab, R.; Rogers, J. P.; Murphy, C. J. J. Am. Chem. Soc. 1995, 117, 9099–9100. Mahtab, R.; Rogers, J. P.; Singleton, C. P.; Murphy, C. J. J. Am. Chem. Soc. 1996, 118, 7028–7032. Mahtab, R.; Harden, H. H.; Murphy, C. J. J. Am. Chem. Soc. 2000, 122, 14–17. Patolsky, F.; Weizmann, Y.; Katz, E.; Willner, I. Angew. Chem., Int. Ed. 2003, 42, 2372–2376. Uhlen, M. Nature 1989, 340, 733–734. Gruttner, C.; Teller, J. J. Magn. Magn. Mater. 1999, 194, 8–15. Steinhoff, G.; Purrucker, O.; Tanaka, M.; Stutzmann, M.; Eickhoff, M. Adv. Funct. Mater. 2003, 13, 841–846. Kang, B. S.; Ren, F.; Wang, L.; Lofton, C.; Tan, W. W.; Pearton, S. J.; Dabiran, A.; Osinsky, A.; Chow, P. P. Appl. Phys. Lett. 2005, 87, 023508. Kang, B. S.; Wang, H. T.; Lele, T. P.; Tseng, Y.; Ren, F.; Pearton, S. J.; Johnson, J. W.; Rajagopal, P.; Roberts, J. C.; Piner, E. L.; Linthicum, K. J. Appl. Phys. Lett. 2007, 91, 112106. Wang, H. T.; Kang, B. S.; Ren, F.; Pearton, S. J.; Johnson, J. W.; Rajagopal, P.; Roberts, J. C.; Piner, E. L.; Linthicum, K. J. Appl. Phys. Lett. 2007, 91, 222101. 10.1021/ac800986q CCC: $40.75  2009 American Chemical Society Published on Web 12/01/2008

GaN,27 as a material, is known to be chemically robust, nontoxic, and biocompatible for long periods of time, and its potential application in biotechnology is reported only very recently.22-26,28 Initial studies22,28 revealed that GaN surface could promote reasonably well neuronal cell attachment, differentiation, and neuritic growth, without a least requirement of any surface modification, and significantly improved the neuronal survival compared to Si, a common template for biochip application.28 Recently, it has been shown by a few groups, namely, M. Eickhoff and F. Ren et al., that GaN thin film can be successfully applied in a biosensing application, by using AlGaN/GaN field effect transistors, dealing with electrical detection of protein adsorption,23 binding of antigens with immobilized antibodies,24,25 or catalytic activity of immobilized enzymes.26 Meanwhile, one-dimensional (1D) nanostructures, such as nanowires (NWs),27,29 with the advantages of large surface to volume ratio and direct electrical path, could possess some novel properties unmatched by their thin film counterparts. Our recent study30 exhibited a higher surface conductivity in GaNNWs, compared to its planar film counterparts, which naturally possess high resistivity, as a consequence of strong spatial charge separation between the surface and core. These surface-dominated properties of NWs should be highly sensitive to the local environment3,12,13 where neighboring charges could alter the overall behavior of NWs. A strong interaction between GaN and DNA, known to be negatively charged in aqueous solution, is foreseen prompting us to explore the sensing capabilities. Although initial studies of biosensing using GaN are trickling in, the exact rationale for its use is still unknown. Moreover, our GaNNWs, having a reasonable conductivity and, simultaneously, being an inheritor of optoelectronic nature of its bulk counterpart, are supposed to be an attractive material for dual-sensing (optical and electrical-based) application, which can provide more/complete information related to the sensing analytes compared to the sensors based on a single technique. In addition to making probe or target modification redundant in the sensing process, we attempt to establish a clear basis for the use of GaNNWs in label-free DNA-sensing through dual routes of electrochemical impedance spectroscopy (EIS) corroborated by photoluminescence (PL) measurements. Here, we report the feasibility of GaNNWs for DNA sensor applications via a dual and label-free approach for the specific capture and detection of Bacillus anthracis lethal factor sequence. Anthrax lethal factor has been identified as a potential target for antianthrax drug discovery programs.31 A synthetic 12-base oligonucleotide single-strand (LF) with sequence coding for the anthrax lethal factor was selected as a target. The 12-base probe (26) Baur, B.; Howgate, J.; von Ribbeck, H.-G.; Gawlina, Y.; Bandalo, V.; Steinhoff, G.; Stutzmann, M.; Eickhoff, M. Appl. Phys. Lett. 2006, 89, 183901. (27) Chen, L. C.; Chen, K. H.; Chen, C. C. In Nanowires and Nanobelts: Materials, Properties and Devices; Wang, Z. L., Ed.; Kluwer Academic Publisher: Boston, MA, 2003; Vol. 1 (Metal and Semiconductor Nanowires), Chapter 9 (Group III- and Group IV-Nitride Nanorods and Nanowires), pp 257309. (28) Young, T.-H.; Chen, C.-R. Biomaterials 2006, 27, 3361–3367. (29) Chen, C.-C.; Yeh, C.-C.; Chen, C.-H.; Yu, M.-Y.; Liu, H.-L.; Wu, J.-J.; Chen, K.-H.; Chen, L.-C.; Peng, J.-Y.; Chen, Y.-F. J. Am. Chem. Soc. 2001, 123, 2791–2798. (30) Chen, R.-S.; Chen, H.-Y.; Lu, C.-Y.; Chen, K.-H.; Chen, C.-P.; Chen, L.-C.; Yang, Y.-J. Appl. Phys. Lett. 2007, 91, 223106. (31) Taton, T. A.; Mirkin, C. A.; Letsinger, R. L. Science 2000, 289, 1757–1760.

Figure 1. Schematic diagram of immobilization and hybridization of DNA on GaNNWs: (a) as-grown nanowire, (b) after hydroxylation, (c) after MPTS modification, (d) after immobilization of probe ssDNA, (e) after hybridization, dsDNA-modified NWs. Inset: typical scanning electron microscopic image of as-grown GaNNWs, with lengths up to several micrometers and diameters about 40-70 nm.

strand (pLF), bearing a sequence complementary to LF, adapted from the 141 base pair anthrax protective antigen gene. EXPERIMENTAL SECTION The complete experimental procedure is presented in the schematic diagram (Figure 1). In short, the surfaces of the NWs were chemically functionalized by organosilane linker MPTS (3-mercaptopropyl trimethoxysilane), which in turns binds the probe pLF on the NW surface (ssDNA immobilization). The pLF molecules, immobilized on GaNNWs, were subjected to hybridization with their target LF (dsDNA modification). Materials. In the present study, except for the Cleland’s reductacryl reagents, which were purchased from Merck, all the chemicals and solvents were purchased from Fluka and SigmaAldrich. The DNA molecules were purchased from Integrated DNA Technologies, Inc. The GaNNWs (25-100 nm) were grown on silicon substrate coated with Au catalyst, using Ga as source material and NH3 (10 standard cubic centimeters per minute (sccm)) as reactant gas, in a tubular furnace (substrate temperature 900 °C) by the air pressure chemical vapor deposition technique.29 Typical GaNNWs exhibit nearly defectfree single-crystalline quality of wurtzite structures with growth directions of [110] or [100] (m or a axis, respectively) in parallel to the long axis of the nanowires.29,30 The carrier density of the GaNNWs, grown by thermal CVD process, is usually at the levels of 1018-1020 cm-3,30,32 which are reportedly 1∼2 orders higher than that (1017-1018 cm-3) of the MBE-grown GaNNWs.33 Surface Modification of Nanowires. Prior to the immobilization of probe DNA strands on GaNNWs (Figure 1), NWs were hydroxylated in acidic solution (0.12 M H2SO4 + 0.52 M H2NO3) at room temperature (RT) for 1 h and the NW surface would be terminated by hydroxyl groups. Then hydroxylated NWs were treated with a solution of MPTS in methanol (volume ratio 1:15), at RT for 1 h. The MPTS-modified GaNNWs was allowed to be incubated with pLF strands for 24 h at 4 °C for pLF immobilization on the NW surface. Before the immobilization, thiol-modified pLF, 5′-DMT-(CH2)6-S-S-(CH2)6-CCTAATAACAAT(32) Stern, E.; Cheng, G.; Cimpoiasu, E.; Klie, R.; Guthrie, S.; Klemic, J.; Kretzschmar, I.; Steinlauf, E.; Turner-Evans, D.; Broomfield, E.; Hyland, J.; Koudelka, R.; Boone, T.; Young, M.; Sanders, A.; Munden, R.; Lee, T.; Routenberg, D.; Reed, M. A. Nanotechnology 2005, 16, 2941–2953. (33) Calarco, R.; Marso, M.; Richter, T.; Aykanat, A. I.; Meijers, R.; Hart, A. v. d.; Stoica, T.; Luth, H. Nano Lett. 2005, 5, 981–984.

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Figure 2. Verification for surface modification of GaNNWs: (a) Typical bright-field and dark-field (inset) TEM images showing the binding of Au NPs on MPTS-modified NWs. (b) The corresponding HR-TEM image and SAD pattern (inset) confirming the identity of Au NPs on the NWs. The spacing of the (111) plane of Au is also indicated. (c-e) Verification of DNA hybridization: fluorescence images of pLF-immobilized GaNNWs, after incubation with FAM-labeled LF, (c) fluorescent image, (d) corresponding transmitted image, and (e) the resultant overlapped image; fluorescence from FAM indicates the presence of FAM-labeled LF, which hybridized with nonlabeled pLF, immobilized on GaNNWs.

3′, were diluted to 10 µM with 2× saline sodium citrate (SSC) buffer. For hybridization, the pLF-modified NWs were incubated at 4 °C for 12 h in target solution, which diluted the LF of 5′-ATTGTTATTAGG-3′ in a buffer solution (consisting of 6× SSC pH 7, 5× Denhardt’s solution) to a final concentration of 10 µM. After each abovementioned step, the sample was washed carefully in deionized water, followed by drying in nitrogen flow. For selectivity detection, a specific sequence p53 of SH-5′-ATGGGCCTCCGGTTC-3′ has been chosen as a probe strand, along with LF, Arg248 (5′-GAACCAGAGGCCCAT-3′), Arg249 (5′-GAACCGGAGTCCCAT-3′), and WT (5′-GAACCGGAGGCCCAT-3′) as the target DNA strands. Instrumentation. TEM experiments were performed by dispersing the nanowires on lacey carbon covered Cu grid, using JEOL, JEM-4000EX. A confocal spectral microscope imaging system (Leica TCS SP2) was used for fluorescence studies, using FAM compatible laser and filter settings (excitation at 495 nm, emission at 520 nm). The nanowires, modified with FAM-labeled or nonlabeled pLF or LF, were collected into water, followed by ultrasonication and washing. Finally, 10 µL of each dilution was spotted onto a clean microscope slide. The fluorescent images reveal features as small as 1 µm, which can be attributed to a small bundle and possibly a single NW. EC impedance measurements were carried out with a Solartron analytical 1470E CellTest system using GaNNWs sample as a working electrode (WE), with Ag/AgCl (saturated KCl) and platinum wire as reference and counter electrode, respectively. The in situ impedance study of DNA hybridization was performed at every hour, by varying the concentration (C) of target strands (e.g., LF) at every second hour, using a duplex buffer solution (30 mM Hepes, pH 7.5, 100 mM potassium acetate) of 3 mL 38

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volume as the background electrolyte. All the impedance spectra were measured at a 0.3 V (vs RE), from 10 MHz down to 0.1 Hz (ac amplitude 10 mV). A low positive potential was chosen to prevent the oxidation of the oligonucleotides; on the other hand, a negative potential, which may cause etching effects on GaNNWs, was avoided. Room temperature PL studies were performed by using a Fluorolog Tau-3 spectrometer (a 450 W xenon source). All PL spectra were collected under the excitation at 268 nm (PL excitation peak for the as-grown GaNNWs), and PL intensity was calibrated with respect to a reference spectrum from epitaxial GaN film purchased from South Epitaxy Corporation, Taiwan. It is worth mentioning that the wavelength scan was optimized to a faster rate in order to avoid the unwanted damage of DNA due to prolonged UV exposure. The experimental errors were estimated from a number of measurements corresponding to each concentration of target DNA. RESULTS AND DISCUSSION Surface Modification of GaNNWs. The MPTS modification of GaNNWs is a crucial step prior to the DNA immobilization. To verify and ensure covalent bonding formation, the MPTS-modified GaNNWs were further treated with Au nanoparticles (NPs). The size of Au NPs was comparable to that of pLF molecules used in these experiments. X-ray photoelectron spectroscopic (XPS) studies (shown in the Supporting Information, Figure S-1) of S2p and Au4f could reveal the existence of thiolate and Au+ species, proving the binding of Au onto the thiol-terminated surface. However, the transmission electron microscopy (TEM) image (Figure 2a) and its corresponding dark-field image (Figure 2a,

Figure 3. Electrochemical impedance spectroscopy based “in situ” DNA-sensing: (a) Nyquist plots and (b) corresponding Bode plots of asgrown, pLF-modified, and dsDNA-modified GaNNWs (at different concentrations of LF targets, in situ DNA hybridization detection). (c) The equivalent circuit model used for the as-grown GaNNWs system and (d) the same for the DNA-modified GaNNWs system. (e-g) Proposed schematic illustration of lowering in surface band bending and enhanced charge transport at the GD interface: (e) maximum band bending for as-grown GaNNWs, (f) lowering of band bending with MPTS and pLF modification, and (g) further lowering of band bending with pLF-LF hybridization. CB and VB denote conduction and valence band, respectively, while E represents the electric field direction. Other symbols are explained in the text.

inset) provided a visual evidence of a distribution of Au NPs on the surface of MPTS-modified GaNNWs (also seen in the Supporting Information, Figure S-2). Figure 2b shows the high resolution TEM image, and the corresponding selected-area diffraction (SAD) pattern revealed the Au reflection superposed with a typical hexagonal structure of GaN (Figure 2b, inset). For visualization of DNA immobilization and its hybridization, the fluorescence technique was employed (see Figure 2 and also see the Supporting Information, Figure S-3, for details) using FAMlabeled LF, subjected to hybridization with the pLF (without any label), immobilized on NWs. Observation of fluorescence from FAM, labeled on LF, (Figure 2c-e) clearly suggests that the pLF, immobilized on NWs, has been hybridized efficiently with its complementary strands. The possibility of physisorption of DNA molecules (or Au NPs) on the NW surface can be ruled out, since the sample was subjected to multiple cleaning and rinsing. For comparison, control experiments have been performed in detail (see the Supporting Information, Figures S-2 and S-3). Electrochemical Impedance Spectroscopy Based “in situ” DNA-Sensing. In situ sensing of the DNA-hybridization phenomena was verified by EIS studies on the pLF-GaNNWs system without the use of any redox marker. EIS is very sensitive to the changes in interfacial impedance of the electrodes (conductor or semiconductor) upon biorecognition events occurring at the surface/electrolyte interface.14 EIS results were presented by the Nyquist plots, imaginary impedance Z′′(ω) vs real impedance Z′(ω) (Figure 3a), of as-grown pristine GaNNWs, pLF-immobilized NWs,

and dsDNA-hybridized NWs sample. Nyquist plot for as-grown GaNNWs exhibits a typical shape of a Faradaic impedance spectrum, having a semicircle along with a straight line, implying finite impedance and suppressed diffusion-limited electrochemical behavior. Note that the conventional use of metallic electrodes for EIS does not exhibit this Faradaic nature unless redox markers are used.14 This feature distinguishes the existing techniques with the current one, enabling us to quantify the individual charge transfer resistances across the DNA/electrolyte and GaN/DNA interfaces, separately. On immobilization of pLF, there appears an additional semicircle region at the higher Z′ range. The presence of this double feature is found to be more pronounced in the Bode plot, phase angle vs frequency, as shown in Figure 3b. This additional impedimetric element (P2 in Figure 3b), appearing only after pLF immobilization (for the in situ DNA immobilization study, see the Supporting Information, Figure S-4), is a fingerprint for DNA modification of GaNNWs. Subsequent DNA hybridization led to a stronger evolution of P2 and a regression of the original peak at higher frequency (P1). Confirming the DNA modification of GaNNWs by P2, the rate of decrease of the integrated intensity of P1, with increasing concentration (CLF), is now a measure of the sensitivity of detection. Analyses of EIS Results. The physical origin of the observed response can be understood by introducing the following model of the electrode/electrolyte interfaces. For the as-grown GaN, a standard equivalent circuit model (Figure 3c), which provides the best fit to the data, is constructed from the following parameters: Analytical Chemistry, Vol. 81, No. 1, January 1, 2009

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Figure 4. Analyses of EIS results. (a) Variation of the electron transfer resistances RGD and RDS, of the interfaces GD and DS, respectively, with the concentration (CLF) of the target LF strand; lines joining the data points denote fitting according to exponential decay for RGD and linear for RDS. (b) EIS-based in situ DNA-selectivity detection: variation of the same RGD with CLF; inset: Nyquist plots of p53-modified GaNNWs before and after the addition of target DNA strands, in the order as follows, LF, Arg248, Arg249, and WT.

the ohmic resistance of the electrolyte solution, RS, Warburg impedance, W (resulting from the diffusion of ions from bulk electrolyte to electrode interface), double layer capacitance, CGS, of the GaN/electrolyte interface (GS), and electron transfer resistance, RGS, through the GS. On the other hand, Figure 3d represents the model that provides the best fit to the data for DNA-modified GaN (Figures 3a,b), supporting the existence of two interfaces, the GaN/DNA interface (GD) and the DNA/ electrolyte interface (DS), in series. The circuit includes the double layer capacitances CGD and CDS and electron transfer resistances RGD and RDS through the respective interfaces. The immobilization of pLF molecules on the GaNNW surface provides a layer of negative charges,14 creating an additional capacitive element in series with GaNNWs, and exhibiting its own individual electrical characteristics with an impedance comparable to GaN. Consequently, the electron would face double interfaces, DS and GD, in series, while transferring from electrolyte to electrode (Figure 3f,g). This leads to the formation of two individual but mutually dependent impedimetric elements represented by P1 and P2 (Figure 3d,f). With the capture of target LF (increasing CLF), as more negative charges accumulate on the GaN surface (Figure 3g), the impedance (RDS) of the DS interface increase marginally (Figure 4a) due to electrostatic repulsion (Figure 3g). However, the impedance (RGD) of the GD interface decreases. Meanwhile, it should be mentioned that the “hydroxylation” process might result in the formation of a thin oxide layer on the NWs surface, which was however practically difficult to be evident experimentally. The only characteristic that could be observed was that the “hydroxylated” NWs surface showed more hydrophilic nature compared to the as-grown GaNNWs surface. The role of the surface oxide and its effect on the surface electronic properties has not been clarified yet. Nevertheless, as observed from the EIS study (see the Supporting Information, Figure S-5), neither the “hydroxylated” nor the MPTS-modified GaNNWs showed any considerable feature, other than the increase in the electron-transfer resistance (Ret) at the interface between GaN and the electrolyte/DNA. Moreover, in situ hybridization phenomena (Figure 4a) revealed a sharp and considerably large decrease in Ret at the GaNNWs surface, which contradicts the effect of the oxide/MPTS layer. Hence, it can be rationalized to assume that DNA-GaNNWs correlation play a crucial role in the EC response of DNA-modified GaNNWs. However, our 40

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extended studies, we hope, may clarify the exact role of the surface-oxide/MPTS and their contribution in the ultrasensitive nature of GaNNWs, toward immobilized DNA molecules, more explicitly, in the near future. The explanation of the anomalous behavior of RGD (Figure 4a) is GaNNWs specific. As-grown GaNNWs, reportedly, have strong band bending and trap states at the surface30 (Figure 3e). Bending up of the bands at the surface can create a depletion or barrier against the surrounding solution, imposing a resistance to the electron transfer from the neighborhood. Here, it is worth mentioning that the width of the surface-depletion region [w ∝ n-1/2] would be much narrower30 for our CVD-grown GaNNWs, which possess relatively higher intrinsic carrier concentration (n)32 compared to the MBE-grown NWs.33 Hence, we believe that, not unreasonably, for our sample, most of the NWs having considerably larger diameter (. w) would adequately experience the strong spatial separation of charge carriers and consequently would contribute to the sensing mechanism. As evident, with subsequent MPTS/DNA modification and hybridization (increasing CLF) of the GaNNWs, the bands flatten up (Figure 3f,g) as a result of trap state passivation and charge redistribution, decreasing the barrier or resistance (Figure 4a). Additionally, the relatively poor electrical contact of the GaNNWs with pLF improves with increasing CLF, as one would expect for a thin film sheath on a core nanowire. In this report, we can provide only a possible hypothesis, based on direct experimental evidence, as a most possible rationale for the unique characteristics of the GaNNWs/DNA construct. Since the original Ret at the GaNNWs surface arises due to the surface band-bending, as per conventional understanding. Hence, the prominent and consistent decrease in Ret (here, RGD) would definitely lead to the most possible (speculative) explanation of the band-flattening phenomena (Figure 3f,g). At this stage, it is difficult to clarify the exact reasons for this band-flattening. Several factors, like passivation of surface defects or charge redistribution, can contribute inseparably from each other, especially for the nanometric system, where the surface dominates. Nevertheless, it is definite that a biocompatible material whose interfacial (with the biomolecule) electron transfer resistance can be modulated and measured will serve the purpose of biosensing. GaNNW transducers and the EIS measurement technique precisely does this (also see the Supporting Information, Figure S-6).

EIS-Based “in situ” DNA-Selectivity Detection. To evaluate the selectivity of our NWs system, the MPTS-modified GaNNWs were subjected to immobilization of a different probe (p53), with the sequence adapted from the human p53 gene, which is noncomplimentary to the LF target. The p53 tumor suppressor gene encodes a nuclear phosphoprotein with cancer-inhibiting properties and well-known subject for the “hotspot” mutation studies. Two important “hotspot” residues34 Arginine 248 (Arg248) and Arginine 248 (Arg249), found in lung cancer (caused by smoking) and liver cancer (hepatocellular carcinomas), respectively, have a single mismatch with p53 and have been employed as targets for selectivity studies, along with a fully noncomplimentary LF and a fully complimentary wild type (WT) sequence of p53. Figure 4b exhibits the in situ selectivity experiment, in which the targets were added in the following order: LF, Arg248, Arg249, and WT. The p53-GaNNWs showed electrochemical indifference to all molecules except the WT targets, even in picomolar concentrations, that trigger a decrease in RGD, even in the presence of noncomplementary and mismatched molecules, without any appreciable optimization for sensor design or detection condition. Hence, the decrease in RGD is definitively attributed to DNA hybridization on the GaNNWs. Photoluminescence Spectroscopy Based DNA-Sensing. Luminescence quenching in semiconductor nanoparticles is a useful signal of binding (or adsorption) of molecular species to the particle surface.16-18 The origin of such quenching, however, is yet to be clarified. Application of the pLF-GaNNWs system, as a probe in the LF recognition process, has been illustrated via ex situ room temperature PL with 268 nm excitation (Figure 5a). The unmodified pristine GaNNWs showed an excitonic emission peak around 365 nm and a blue emission around 425 nm. With subsequent MPTS modification, the PL signal intensifies and a previously unknown shoulder at 330 nm appears (Figure 5a), implying a flattening of the band bending35 at the surface of the NWs as proposed for the explanation of the EIS results. However, the PL features were quenched post pLF immobilization (Figure 5a). The quenching behavior (Figure 5a) is clearly correlated to the DNA induced surface modification,16-18 since the phenomenon is more pronounced with increasing concentration of pLF. Apart from the quenching behavior, the other changes in the PL spectra can be an artifact of the MPTS modification on the NW surface (see also the Supporting Information, Figure S-7). The integrated PL intensity, IUV, (corresponding to band-edge emission from the GaNNWs) exhibits a nonlinear quenching behavior with the CLF (Figure 5b), which resembles the variation of RGD with CLF as shown in Figure 4a. At higher CLF (>1 µM), the quenching factor (∝ IUV-1) tends to saturate limited by the number of probes available on the NW surface. An increasing surface coverage (as CLF increases) on the GaNNWs would expectedly result in an improved contact resistance and hence lower RGD (Figure 4a). However, then the quenching of the PL signal must be attributed to the increasing surface coverage of the GaNNW with the DNA strands, forming a sheath (inset, Figure 5b), both having absorption in the ultraviolet.1 (34) Hainaut, P.; Hernandez, T.; Robinson, A.; Rodriguez-Tome, P.; Flores, T.; Hollstein, M.; Harris, C. C.; Montesano, R. Nucleic Acids Res. 1998, 26, 205–213. (35) Nakagawa, O. S.; Ashok, S.; Sheen, C. W.; Ma¨rtensson, J.; Allara, D. L. Jpn. J. Appl. Phys. 1991, 30, 3759–3762.

Figure 5. Photoluminescence spectroscopy based DNA-sensing. (a) PL spectra of as-grown, MPTS-modified, pLF-modified, and dsDNA-modified GaNNWs. (b) Variation of IUV with the target concentration CLF; the line joining the data points represents a fit according to exponential decay. Inset: a schematic illustrating the formation of a UV absorbing DNA sheath on the core GaNNW at high surface coverage (CLF > few micromolar).

Here, the surface-limited nature of PL-quenching suggests that the DNA molecules act as static quenchers,36 suppressing the density of available radiative states of GaNNWs. However, the other possible origins for PL-quenching, corresponding to DNA modification, cannot be ruled out also. Immobilization of DNA may create an alternative nonradiative path, affecting the recombination rates and hence the PL lifetimes (dynamic quenching behavior36), as observed for the gas detection using metal oxide NWs.37 The results encourage further investigations needed to clarify this point. CONCLUSION In summary, the MPTS-modified GaNNWs offer an excellent transducer for label-free electrochemical and optical sensors. Interestingly, GaN in the nanowires form, with the advantages of enhanced binding sites and surface-enhanced charge transfer, is found to be highly sensitive to the surface-immobilized biomolecules in EIS studies. DNA hybridization on this special transducer exhibits remarkably distinct Faradaic characteristics, compared to the as-grown nanowires, in electrochemical impedance spectroscopy. Electrical modeling of the results convey a rapid change in the electron transfer resistance at the GaN/DNA interface with changing target concentrations between pico to micromolar levels that serve as the sensing parameter. On the other hand, quenching (36) Lettieri, S.; Setaro, A.; Baratto, C.; Comini, E.; Faglia, G.; Sberveglieri, G.; Maddalena, P. New J. Phys. 2008, 10, 043013. (37) Faglia, G.; Baratto, C.; Sberveglieri, G.; Zha, M.; Zappettini, A. Appl. Phys. Lett. 2005, 86, 011923.

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of the GaN band edge photoluminescence intensity with probe and target modifications of the nanowire signals immobilization and hybridization at nanomolar concentrations of the target. In addition, the same NWs sensor showed an in situ selective detection of “hotspot” mutations in the p53 genome, from a mixed system of noncomplimentary, single mismatch, and fully complementary wild type targets. We believe these results would stimulate further studies on the charge transfer phenomenon between MPTS/DNA and GaNNWs, and the determinations of the molecular orbital energy levels in MPTS or DNA as well as their carrier dynamics will supplement the information contained herein. ACKNOWLEDGMENT C. P. Chen and A. Ganguly contribute equally to the present work. This research was financially supported by the Ministry of

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Education and National Science Council in Taiwan. Technical support provided by the Core Facilities for Nano Science and Technology in Academia Sinica and National Taiwan University are acknowledged. Fruitful discussions with Prof. C.-C. Chen were also appreciated.

SUPPORTING INFORMATION AVAILABLE Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

Received for review May 14, 2008. Accepted October 29, 2008. AC800986Q