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Silicon Nanowire Arrays for Label-Free Detection of DNA Zhiqiang Gao,* Ajay Agarwal, Alastair D. Trigg, Navab Singh, Cheng Fang, Chih-Hang Tung, Yi Fan, Kavitha D. Buddharaju, and Jinming Kong
Institute of Microelectronics, 11 Science Park Road, Singapore 117685, Republic of Singapore
Arrays of highly ordered n-type silicon nanowires (SiNW) are fabricated using complementary metal-oxide semiconductor (CMOS) compatible technology, and their applications in biosensors are investigated. Peptide nucleic acid (PNA) capture probe-functionalized SiNW arrays show a concentration-dependent resistance change upon hybridization to complementary target DNA that is linear over a large dynamic range with a detection limit of 10 fM. As with other SiNW biosensing devices, the sensing mechanism can be understood in terms of the change in charge density at the SiNW surface after hybridization, the so-called “field effect”. The SiNW array biosensor discriminates satisfactorily against mismatched target DNA. It is also able to monitor directly the DNA hybridization event in situ and in real time. The SiNW array biosensor described here is ultrasensitive, non-radioactive, and more importantly, label-free, and is of particular importance to the development of gene expression profiling tools and point-of-care applications.
INTRODUCTION In recent years intensive efforts have been focused on the development of ultrasensitive DNA biosensors/arrays capable of quantitative gene expression analysis. These biosensors have a wide variety of potential applications that range from genotyping to molecular diagnostics.1-3 For example, abnormalities in the expression of specific genes have been linked to a large and increasing number of diseases. Quantification of gene expression is a promising basis for early diagnosis. Among various gene expression profiling tools developed, polymerase chain reaction (PCR)-based fluorescent microarrays are the most widely studied systems since they offer the highest degree of sensitivity, the highest multiplexing capability, and the widest dynamic range. Arrays containing tens of thousands of unique probe sequences have been constructed that enable simultaneous assessment of tens of thousands genes down to a few copies.4 They have been * Author to whom correspondence should be addressed. Tel: +65-67705928, Fax: +65-67731914. E-mail:
[email protected]. (1) Lu, J.; Getz, G.; Miska, E. A.; Alvarez-Saavedra, E.; Lamb, J.; Peck, D.; SweetCordero, A.; Ebert, B. L.; Mak, R. H.; Ferrando, A. A.; Downing, J. R.; Jacks, T.; Horvitz, H. R.; Golub, T. R. Nature 2005, 435, 834-838. (2) Hutvagner, G.; Zamore, P. D. Science 2001, 293, 834-838. (3) Lee, Y.; Ahn, C.; Han, J.; Choi, H.; Kim, J.; Yim, J.; Lee, J.; Provost, P.; Radmark, O.; Kim, S. Nature 2003, 425, 415-419. (4) Noordewier, M. O.; Warren, P. V. Trends Biotechnol. 2001, 19, 412-415. 10.1021/ac061808q CCC: $37.00 Published on Web 04/04/2007
© 2007 American Chemical Society
used to identify gene expression patterns associated with specific biological functions on a global scale. However, there are several technical limitations that prevent using the PCR-based microarrays as a clinical tool because of the difficulty to reliably profile low abundance genes.5 Consequently, much effort has been put into realizing accurate, sensitive, selective, robust, and portable biosensing devices for both laboratory and point-of-care applications. The use of electrical techniques instead of fluorescence in principle allows for simple, rapid, and portable DNA detection platforms. Recent advances in nanotechnology open up new opportunities for electrical detection system for biomolecules.6,7 Semiconductor nanowires, in particular, have attracted substantial research efforts because of the extremely high surface-to-volume ratio and the extreme sensitivity of the carrier mobility to the variations in the electric field (charge density) at their surface.8 Working as the gate in a field-effect transistor (FET), the essentially one-dimensional morphology of the nanowires offers a unique advantage over macroplanar FETs and overcomes the sensitivity limitations of the latter. Binding of biomolecules onto the nanowire surface leads to depletion or accumulation of carriers in the “bulk” of the nanowire, rather than only in the surface region of a macro FET. Therefore, semiconductor nanowire-based devices have been proposed to probe bioaffinitive events at ultralow concentrations.9 Furthermore, theoretical calculations suggest that nanowire sensors are significantly more sensitive than their macroplanar counterparts and have much shorter response time.10 Among different nanowire biosensing devices investigated, silicon nanowires (SiNWs) appear particularly attractive, because they can be readily prepared using both “bottom-up” and “topdown” approaches.11 The former starts with nanocomponents/ molecules to biuld up more complex assemblies and devices, utilizing the concept of self-orgnization and molecular recognition to arrange the compoments/molecules into some useful conformation, while the latter begins with bulk materials to reduce their lateral dimensions before forming nanodevices, often involving microfabrication methods where externally controlled tools are (5) Draghici, S.; Khatri, P.; Eklund, A. C.; Szallasi, Z. Trends Genet. 2006, 22, 101-109. (6) Rosi, N. L.; Mirkin, C. A. Chem. Rev. 2005, 105, 1647-1562. (7) Patolsky, F.; Zhang, G.; Lieber, C. M. Anal. Chem. 2006, 78, 4261-4269. (8) Star, A.; Tu, E.; Niemann, J.; Gabriel, J. P.; Joiner, C. S.; Valcke, C. Proc. Natl. Acad. Sci. U.S.A. 2006, 104, 921-926. (9) Cui, Y.; Wei, Q.; Park, H.; Leiber, C. M. Science 2001, 293, 1289-1292. (10) Nair, P. R.; Alam, M. A. Appl. Phys. Lett. 2006, 88, 233120. (11) Li, Z.; Chen. Y.; Li, X.; Kamins, T. I.; Williams, R. S. Nano Lett. 2004, 4, 245-247.
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used to cut, pattern, etch, and shape them into the desired shape and order. Utilization of SiNWs has translated into new assays that improve on current methods of DNA and protein detection. The simplest of such assays would be direct (label-free) electrical detection of biomolecules using miniaturized bioelectronic devices modified with bioaffinitive agents that produce measurable electrical signals upon interacting with target biomolecules. Indeed, Leiber has demonstrated that SiNW biosensors are potentially powerful platforms for ultrasensitive label-free detection of biological species, such as DNA,12 proteins,9,13 and viruses.14 Most of the studies involving “pick and place” fabrication techniques, however, suffer from certain limitations such as device-to-device uniformity, reflecting the variations in the device fabrication processes. Poor device uniformity, yield, and scalability hinder further development of these biosensors into practical systems. Large scale fabrication of SiNW devices requires precise control at nanoscales where positioning, circuiting, and integrating individual nanodevices are some of the technical challenges. Developing a reliable and scalable fabrication technique for producing uniform and wellaligned SiNWs, integrating individual SiNWs into functional devices with high yields, is one of the great challenges facing development of the next generation SiNW biosensors. One solution is to utilize semiconductor processing techniques (top-down) to fabricate SiNW devices. Toward this path, Li et al. demonstrated that electron beam lithography can be used to fabricate SiNW devices in a precisely controlled manner.11 Although it has been elegantly demonstrated that well-defined SiNW detection devices can be fabricated in the previously proposed top-down approach, three fundamental issues that remain unanswered are the scalability, multiplexing capacity, and cost. We believe that conventional optical lithography in combination with a size reduction strategy may provide a simple and yet economic solution for the above-mentioned technical challenges, promising the realization of SiNWs in commercial devices. In this paper we present a CMOS compatible SiNW array fabrication process involving deep ultraviolet lithography and selflimiting oxidation. The feasibility of utilizing the SiNW array for direct electrical detection of DNA at femtomolar levels has been demonstrated. The electrical DNA assay described here is rapid, ultrasensitive and label-free, and is able to monitor directly DNA hybridization process in situ and in real time. Specific DNAs were detected electrically at femtomolar levels with high specificity by simply measuring the resistance changes. The label-free electrical detection demonstrated in this work greatly simplifies DNA assays, removing all the biological/chemical ligation steps and bulky optical readout equipment. By integrating the SiNW arrays into a fully automated microelectromechanical system, from sample digestion, DNA isolation and purification, to quantification, it will provide a generic platform for both laboratory and pointof-care applications. EXPERIMENTAL SECTION Materials. Butylamine (99.5%) and sodium borohydride (>99%) were purchased from Sigma-Aldrich (St Louis, MO). Trimethy(12) Hahm, J.; Lieber, C. M. Nano Lett. 2004, 4, 51-54. (13) Zheng, G. F.; Patolsky, F.; Cui, Y.; Wang, W. U.; Lieber, C. M. Nat. Biotechnol. 2005, 23, 1294-1301. (14) Patolsky, F.; Zheng, G. F.; Hayden, O.; Lakadamyali, M.; Zhuang, X. W.; Lieber, C. M. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 14017-14022.
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loxysilane aldehyde (90%) was from United Chemical Technologies (Bristol, PA). Amino-terminated peptide nucleic acid (PNA) capture probes (N f C: NH2-AAC CAC ACA ACC TAC TAC CTC A) used in this work were custom-made by Eurogentec (Herstal, Belgium), and all other oligonucleotides of PCR purity were from 1st Base Pte Ltd (Singapore). The sequences of sample oligonucleotides used in this work are as follows: 5′-TGA GGT AGT AGG TTG TGT GGT T-3′ (fully complementary), 5′-TGA GGT AGT AGG ATG TGT GGT T-3′ (one-base mismatched), 5′TGA GCT AGT AGG TTG TGA GGT T-3′ (two-base mismatched), and 5′-CAA AAC AAA GAT CTA CAT GGA T-3′ (control). All other reagents of certified analytical grade were obtained from SigmaAldrich and used without further purification. A pH 8.5 10 mM Tris-HCl-1.0 mM EDTA-30 mM NaCl buffer solution (TE) was used as the hybridization, washing, and working buffer. Apparatus. Electrical measurements were performed with an Advantest R8340A Ultra High Resistance Meter (Advantest Corp., Tokyo, Japan.). The biosensor consists of an array of 100 nanowires embedded in a microfluidic channel (Figure 1). To avoid any possible electrochemical reaction, electrical measurements were conducted at an applied voltage of
