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Sub-Femtomolar Electrochemical Detection of DNA Hybridization Based on Latex/Gold Nanoparticle-Assisted Signal Amplification Suttiporn Pinijsuwan,† Patsamon Rijiravanich,‡ Mithran Somasundrum,*,‡ and Werasak Surareungchai*,† School of Bioresources and Technology and National Center for Genetic Engineering and Biotechnology, King Mongkut’s University of Technology Thonburi, Bangkhuntien, Bangkok 10150, Thailand We report a relatively simple electrostatic method for modifying submicrometer-size latex spheres with gold nanoparticles (AuNPs) based on layer-by-layer modification of the latex by polyelectrolytes. The AuNP coverages for 343- and 501-nm-diameter spheres were 4.0 × 1010 ( 1.3 × 1010 and 8.2 × 1010 ( 2.7 × 1010 particles cm-2, respectively, which is an increase of 1 order of magnitude on the previously reported coverage at latexAuNPs using streptavidin-biotin binding (Kawde, A.N.; Wang, J. Electroanalysis 2004, 16, 101-107). Due to the fact that the AuNPs used here are also of a larger size (mean diameter 15.5 ( 1.6 nm, cf. 5 nm), this represents an increase of 2 orders of magnitude in the number of Au atoms delivered per sphere. The spheres were attached to DNA probes specific to E. coli and used to detect probe hybridization by dissolution of the AuNPs, followed by measurement of Au3+ ions by anodic stripping voltammetry (ASV). Use of differential pulse voltammetry for the stripping step, along with optimization of the ASV conditions, enabled a detection limit of 0.5 fM, which is, to the best of our knowledge, equal or lower than previous voltammetric nanoparticle methods for detection of DNA hybridization. The detection of specific sequences of DNA is of use for the sensing or diagnosis of viruses, pathogenic microorganisms, human genetic diseases, food-borne bacteria, and biological warfare agents. Methods of detection that have been used include optical,1–4 chemiluminescence,5 surface plasmon resonance,6,7 * To whom correspondence should be addressed. E-mail: s_mithran@ yahoo.co.uk;
[email protected]. Fax: +66-2-452-3455. Tel: +66-2-470-7474. † School of Bioresources and Technology. ‡ National Center for Genetic Engineering and Biotechnology. (1) Pavlov, V.; Shlyahovsky, B.; Willner, I. J. Am. Chem. Soc. 2005, 127, 6522– 6523. (2) Knermeyer, J.-P.; Marme, N.; Sauer, M. Anal. Chem. 2000, 72, 3717–3724. (3) Ho, H. A.; Dore, K.; Boissinot, M.; Bergeron, M. G.; Tanguay, R. M.; Boudreau, D.; Leclerc, M. J. Am. Chem. Soc. 2005, 127, 12673–12676. (4) Peng, H.; Zhang, L.; Kjallman, T. H. M.; Soeller, C.; Travas-Sejdic, J. J. Am. Chem. Soc. 2007, 129, 3048–3049. (5) Miao, W.; Bard, A. J. Anal. Chem. 2004, 76, 5379–5386. (6) Fang, S.; Lee, H. J.; Wark, A. W.; Corn, R. M. J. Am. Chem. Soc. 2006, 128, 14044–14046. (7) Lee, C.-Y.; Nguyen, P.-C. T.; Grainger, D. W.; Gamble, L. J.; Castner, D. G. Anal. Chem. 2007, 79, 4390–4400. 10.1021/ac800566d CCC: $40.75 2008 American Chemical Society Published on Web 07/30/2008
quartz crystal microbalance,8–10 and electrochemical techniques.11–21 The latter methods are of interest because they have the potential for providing sensors of high sensitivity and low cost, suitable for on-site, decentralized testing. Additionally, electrochemical methods can be coupled with currently available miniaturization technologies to produce, for example, microchip-based DNA detection.22 The electrochemical label can be a redox compound, such as ferrocene.23 Alternatively, the use of metal/semiconductor nanoparticle labels has become attractive.24–28 These labels can be deposited in micrometer-scale gaps between microelectrodes, providing a measureable conductivity change on hybridization.29 Otherwise, they can be oxidized at a planar electrode,11 or, more usefully, can be first dissolved and then measured by anodic stripping voltammetry (ASV), a technique that imparts high sensitivity due to the preconcentration step. Gold nanoparticles (AuNPs) have been attached to DNA probes by steptavidin-biotin binding12,13 and by Au-thiol (8) Yao, C.; Zhu, T.; Tang, J.; Wu, R.; Chen, Q.; Chen, M.; Zhang, B.; Huang, J.; Fu, W. Biosens. Bioelectron. 2008, 23, 879–885. (9) Wu, V. C. H.; Chen, S.-H.; Lin, C.-S. Biosens. Bioelectron. 2007, 22, 2967– 2975. (10) Lu, W.; Jiang, L. Biosens. Bioelectron. 2007, 22, 1101–1105. (11) Ozsoz, M.; Erdem, A.; Kerman, K.; Ozkan, D.; Tugrul, B.; Topcuoglu, N.; Ekren, H.; Taylan, M. Anal. Chem. 2003, 75, 2181–2187. (12) Wang, J.; Xu, D.; Kawde, A.-N.; Polsky, R. Anal. Chem. 2001, 73, 5576– 5581. (13) Wang, J.; Polsky, R.; Xu, D. Langmuir 2001, 17, 5739–5741. (14) Authier, L.; Grossiord, C.; Brossier, P.; Limoges, B. Anal. Chem. 2001, 73, 4450–4456. (15) Rochelet-Dequaire, M.; Limoges, B.; Brossier, P. Analyst 2006, 131, 923– 929. (16) Lee, T. M.-H.; Li, L.-L.; Hsing, I.-M. Langmuir 2003, 19, 4338–4343. (17) Kawde, A.-N.; Wang, J. Electroanalysis 2004, 16, 101–107. (18) Wang, J.; Rincon, O.; Polsky, R.; Dominguez, E. Electrochem. Commun. 2003, 5, 83–86. (19) Wang, J.; Liu, G.; Zhu, Q. Anal. Chem. 2003, 75, 6218–6222. (20) Wang, J.; Liu, G.; Jan, M. R.; Zhu, Q. Electrochem. Commun. 2003, 5, 1000– 1004. (21) Rijiravanich, P.; Somasundrum, M.; Surareungchai, W. Anal. Chem. 2008, 80, 3904–3909. (22) Shiddiky, M. J. A.; Shim, Y.-B. Anal. Chem. 2007, 79, 3724–3733. (23) Yu, C. J.; Wan, Y.; Yowanto, H.; Li, J.; Tao, C.; James, M. D.; Tan, C. L.; Blackburn, G. F.; Meade, T. J. J. Am. Chem. Soc. 2001, 123, 11155–11161. (24) Katz, E.; Willner, I.; Wang, J. Electroanalysis 2004, 16, 19–44. (25) Willner, I.; Katz, E. Angew. Chem., Int. Ed. 2004, 43, 6064–6108. (26) Wong, E. L. S. Biophys. Rev. Lett. 2007, 2, 167–189. (27) Drummond, T. G.; Hill, M. G.; Barton, J. K. Nat. Biotechnol. 2003, 21, 1192–1199. (28) Rosi, N. L.; Mirkin, C. A. Chem. Rev. 2005, 105, 1547–1562. (29) Park, S.-J.; Taton, T. A.; Mirkin, C. A. Science 2002, 295, 1503–1506.
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binding.14–16 Since the analytical signal is a faradic current, the sensitivity can be increased by increasing the number AuNPs attached to each DNA probe. This can be done by the autocatalytic deposition of Au15 or Ag13,16 onto the existing AuNPs. Nonspecific Au deposition has been avoided by using polyelectrolyte films as blocking agents16 or by adding PEG/NaCl to the Au enhancer solution.15 Another method to increase the quantity of AuNPs, as described by Kawde and Wang, is to load streptavidin-coated latex particles with biotin-coated AuNPs and then attach the latex to a DNA probe.17 In this note, we report an alternative, relatively simple electrostatic method of loading, which produced AuNP coverages ∼1 order of magnitude greater than the streptavidinbiotin method. As an example of analytical utility, the latex particles were attached to DNA probes for Escherichia coli and detected by ASV, with the stripping step performed by differential pulse voltammetry (DPASV). Optimization of the DPASV conditions enabled a limit of detection that is, to the best of our knowledge, equal or lower than previous voltammetric nanoparticle methods for detection of DNA hybridization11–21 and competitive with many of the other methods of DNA measurement.1,2,4,5,8–10 EXPERIMENTAL SECTION Materials. Styrene (St) and acrylic acid (AA) were purchased from Fluka. Ammonium persulfate (APS) was from Riedel-De Hae¨n. Poly(allylamine) hydrochloride (PAA, MW ∼70000), poly (sodium 4-styrene) sulfonate (PSS, MW ∼70 000), HAuCl4 · 3H2O, trisodium citrate dehydrate, sodium dodecyl sulfate (SDS), and avidin from egg white were from Sigma-Aldrich. Hydrobromic acid was from Ajax Finechem, and bromine was from Panreac. All solutions were prepared with deionized water. The 30-base synthetic target oligonucleotides were purchased from the Bio Service Unit (NSTDA). An oligonucleotide probe with a biotin-modified 5′-position was acquired from Bioneer. The DNA strands had the following sequences: DNA probe: biotin-5′CTT CCT GAG TAA TAA3′ target: 5′TAT TCA CTC AGG AAG TTA TTA CTC AGG AAG3′ single mismatch: 5′TAT TCA CTC AGG AAG TTA TTA CTC ACG AAG3′ noncomplementary strand: 5′CTT CCT GAG TAA TAA CTT CCT GAG TGA ATA3′ Oligonucleotide stock solutions were prepared at 1 mg mL-1 and kept frozen. Target oligonucleotide solutions were diluted with 0.1 M phosphate buffer (pH 7). Capture probe solutions were diluted with hybridization buffer (5× SSPE, pH 7.4, containing 0.2% SDS). All solutions were prepared with deionized water. Apparatus. Transmission electron microscopy (TEM) was carried out with a JEOL model JM-2100. UV-visible spectra were recorded using a Beckman model DU-7000 spectrophotometer. Electrochemical experiments were performed using an Autolab PGSTAT 10 computer-controlled potentiostat (Eco Chemie) with GPES software. The working electrodes were screen-printed carbon tracks, used for DNA immobilization with a screen-printed Ag/AgCl track as combined referenced and counter and used for DPASV with a Pt disk counter and Ag/AgCl (3 M NaCl) reference. Electrode Preparation. Screen-printed electrodes (SPEs) were fabricated using a semiautomatic screen printer (model 248, DEK-S). The conductive carbon ink (type 145, MCA Services) and 6780
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silver/silver chloride ink (type C2DR15, MCA Services) were printed onto PVC sheets (150 × 200 mm) through a patterned stencil to give a group of 24 SPEs (each consisted of a carbon working electrode and Ag/AgCl combined reference and counter electrode). Following each print, the electrodes were cured overnight at 55 °C and then allowed to cool at room temperature. A layer of insulating tape (Permacel model p221) was then placed over a position of the conducting lines, exposing a 1.5 mm × 3.5 mm working electrode and a 2 mm × 3.5 mm reference/counter electrode. Preparation of Polystyrene-co-Acrylic Acid Particles (PSA). PSA particles were prepared as described in the literature30 with some modifications. Microspheres of 338-nm diameter were prepared as follows: 19 g of deionized water was charged into a three-necked flask submerged in a water bath. After purging with nitrogen for ∼1 h under stirring at 350 rpm, 20 g of St and 4 g of AA were added and then washed (at 70 °C) under stirring at 350 rpm while purging with N2. The polymerization was started by adding 0.2 g of APS into 10 mL of deionized water; the polymerization time was 8 h. For microspheres of 493-nm diameter, the same procedure was used, but with 0.5 g of AA. In both cases, the resulting PSA latex suspensions were centrifuged with distilled water twice at 13 000 rpm for 20 min. Preparation of Gold/Avidin Conjugate. AuNPs were prepared by sodium citrate reduction,31 following which gold/avidin conjugates were formed by the method of Mao et al.32 Briefly, 100 µL of avidin solution (3 mg mL-1) was added to 0.5 mL of colloidal gold suspension; the mixture adjusted to pH 10 and then incubated at room temperature for 15 min under stirring. After being kept at
