Gold Nanoparticle-Based Quantitative Electrochemical Detection of

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Anal. Chem. 2001, 73, 4450-4456

Gold Nanoparticle-Based Quantitative Electrochemical Detection of Amplified Human Cytomegalovirus DNA Using Disposable Microband Electrodes Laurent Authier, Ce´line Grossiord, and Pierre Brossier*

Laboratoire de Microbiologie Me´ dicale et Mole´ culaire, Faculte´ de Me´ decine et de Pharmacie, 7 Boulevard Jeanne d’Arc, 21033 Dijon, France Benoıˆt Limoges*

Laboratoire d’Electrochimie Mole´ culaire, UMR CNRS 7591, Universite´ Paris 7-Denis Diderot, 2 place Jussieu, 75251 Paris Cedex 05, France

An electrochemical DNA detection method has been developed for the sensitive quantification of an amplified 406-base pair human cytomegalovirus DNA sequence (HCMV DNA). The assay relies on (i) the hybridization of the single-stranded target HCMV DNA with an oligonucleotide-modified Au nanoparticle probe, (ii) followed by the release of the gold metal atoms anchored on the hybrids by oxidative metal dissolution, and (iii) the indirect determination of the solubilized AuIII ions by anodic stripping voltammetry at a sandwich-type screen-printed microband electrode (SPMBE). Due to the enhancement of the AuIII mass transfer by nonlinear diffusion during the electrodeposition time, the SPMBE allows the sensitive determination of AuIII in a small volume of quiescent solution. The combination of the sensitive AuIII determination at a SPMBE with the large number of AuIII released from each gold nanoparticle probe allows detection of as low as 5 pM amplified HCMV DNA fragment. Polymerase chain reaction (PCR) followed by hybridization of the target PCR-amplified product with a single-stranded oligonucleotide-labeled probe is one of the most widely used methods of sequence-specific DNA detection.1 It has opened new avenues in the diagnosis of pathogenic diseases, often replacing the traditional laborious method in the field of virology.2 Many detection techniques have been developed, and their sensitivities depend mainly on the specific activity of the label linked to the oligonucleotide probe. Labels that provide a radioactive, fluorescent, chemiluminescent, or colorimetric signal are the most popular.1a While many DNA hybridization assays are suitable for diagnosis, faster, lower cost, easier-to-use, and more sensitive approaches are highly desired, especially in the case of decentral* To whom correspondence should be addressed. E-mail: limoges@ paris7.jussieu.fr; [email protected]. (1) (a) Kricka, L. J. In Nonisotopic DNA Probe Techniques; Kricka, L. J., Ed.; Academic Press: New York, 1992. (b) Christopoulos, T. K. Anal. Chem. 1999, 71, 425R. (2) Tang, Y.-W.; Procop, G. W.; Persing, D. H. Clin. Chem. 1997, 43, 2021.

4450 Analytical Chemistry, Vol. 73, No. 18, September 15, 2001

ized screening of infectious diseases. In this context, electrochemical detection of DNA hybridization events offers innovative routes,3 and several DNA hybridizations based on electroactive groove binders have been described.4 However, the small signal variations lead to a low precision and the selectivity remains poor. Alternatively, indirect electrochemical detection of hybridization can be accomplished using enzyme labels that generate an amplified signal via the production of an electroactive compound5 or via a bioelectrocatalytic process.6 Although large signal amplification can be achieved, labeling of an oligonucleotide by an enzyme is not a straightforward route, and once prepared, the activity of the conjugate must be periodically controlled owing to the inherent poor stability of enzymes. Recently, we investigated a novel electrochemical amplification strategy based on a colloidal gold nanoparticle label that, after oxidative gold metal dissolution in an acidic solution, was indirectly quantified by anodic stripping voltammetry (ASV).7 Since thousand AuIII ions can be released from each Au nanoparticle label (e.g., 2.3 × 105 gold atoms are theoretically contained in a 20-nm spherical gold particle), it was thus possible, in the context of a sandwich immunoassay, to achieve picomolar detection of an immunoglobulin G.7 Herein, we further extend the scope of the sensitive Au nanoparticle-based electrochemical detection to analysis of DNA hybridization. For the purpose, sandwich-type screen-printed microband electrodes (SPMBEs) of low cost were developed in order to improve the sensitivity of AuIII detection in (3) (a) Mikkelsen, S. R. Electroanalysis 1996, 8, 15. (b) Wang, J. Chem. Eur. J. 1999, 5, 1681. (4) (a) Millan, K. M.; Saraullo, A.; Mikkelsen, S. R. Anal. Chem. 1994, 66, 2943. (b) Wang, J.; Cai, X.; Rivas, G.; Shiraishi, H.; Farias, P. A. M.; Dontha, N. Anal. Chem. 1996, 68, 2629. (c) Hashimoto, K.; Ito, K.; Ishimori, Y. Anal. Chem. 1994, 66, 3830. (d) Takenaka, S.; Yamashita, K.; Takagi, M.; Uto, Y.; Kondo, H. Anal. Chem. 2000, 72, 1334. (5) Azek, F.; Grossiord, C.; Joannes, M.; Limoges, B.; Brossier, P. Anal. Biochem. 2000, 284, 107. (6) (a) de Lumley-Woodyear, T.; Caruana, D. J.; Campell, C. N.; Heller, A. Anal. Chem. 1999, 71, 394. (b) Caruana, D. J.; Heller, A. J. Am. Chem. Soc. 1999, 121, 769. (7) Dequaire, M.; Degrand, C.; Limoges, B. Anal. Chem. 2000, 72, 5521. 10.1021/ac0103221 CCC: $20.00

© 2001 American Chemical Society Published on Web 08/10/2001

small volumes of nonagitated solution and to offer the opportunity to develop an inexpensive, simple, compact, and hand-held electrochemical device. To demonstrate the feasibility of the approach, the method was applied to the determination of an amplified 406-base pair human cytomegalovirus DNA sequence (HCMV DNA). This herpes virus is an important pathogen in transplant recipients and immunosuppressed individuals, and it is responsible the most common cause of congenital infections throughout the world.8 EXPERIMENTAL SECTION Screen-Printed Microband Electrode Preparation and Apparatus. High-impact polystyrene (HIPS) substrate was purchased from Sericol (Vaulx-en-Velin, France) as 1-m-long, 70-cmwide, and 1-mm-thick plates. Small rectangles of HIPS (8 cm long and 4.5 cm wide) were cut, cleaned with ethanol, and left to dry at room temperature before use. Their mat side was coated with an Electrodag PF 407A conductive carbon-based ink from Acheson Colloids Co. (Port Huron, MI), using a manual screen-printer (Circuit Imprime´ Franc¸ ais, Bagneux, France). An array of eight electrodes was produced by forcing the carbon ink to penetrate through the mesh of a screen stencil with a squeegee (120 threads cm-1) and then to deposit onto the HIPS sheet (the pattern of the electrodes is shown in Supporting Information). Each electrode consisted of an electrical contact prolonged by a conductive track (30 mm × 1.5 mm). The resulting electrodes were left to dry for 1 h at room temperature and then were cured for 1 h in an oven at 65 °C. The conductive track was then coated manually (or by screen-printing) with a thick layer of viscous solution of HIPS dissolved in mesitylene (saturated solution). The insulator layer was then dried for 72 h in a ventilated box oven at room temperature. An individual electrode was cut from the polystyrene plate and a band approximately 1.5 mm long and 10 µm wide was exposed by cutting the plate perpendicularly to the direction of the conductive track, with a scalpel (see Supporting Information). The microband electrode is then ready for measurement. Unless otherwise stated, before each measurement, a small slice of the microband electrode was cut in order to produce another fresh carbon band surface. The macroscopic (disk of 3.50-mm diameter) screen-printed electrodes (SPEs) employed in a few experiments were prepared as previously.7 An Autolab potentiostat (EcoChemie) interfaced to a PC system with GPSE version 4.4 software was used for cyclic voltammetry (CV) and ASV. A small homemade saturated Ag/AgBr reference electrode and a wire platinum counter electrode were used. Reagents and Solutions. A solution of 20-nm colloidal gold particles was supplied by Sigma Chemical Co. and 3-phenoxypropionic acid by Lancaster. The hexafluorophosphate salt of (ferrocenylmethyl)trimethylammonium (FcTMA+) was obtained by metathesis of the corresponding iodide salt purchased from Lancaster. Chloroauric acid (HAuCl4) and concentrated bromine (CAUTION: bromine is a toxic and harmful reagent) were purchased from Aldrich Chemical Co. Sodium salt of bromide and hydrobromic acid (47%) were supplied by Merck as Suprapure grade reagents. Bromine was eliminated from the commercialized HBr as previously mentioned.7 The hybridization procedure was an adaptation of the Hybridowell kit protocol developed by Arge`ne S.A. (http://www.argenbiosoft.com), and consequently, its re(8) Myers, J. B.; Amsterdam, D. Immunol. Invest. 1997, 26, 383.

agents (coating solution, hybridization and washing buffers, denaturation solutions, and negative control) were used. The amplified human ETS2 gene DNA and the 5′-hexanethiol 22-base oligonucleotide (AC3) were gifts from Arge`ne S.A. The phosphate buffer (PB, 4.3 mM NaH2PO4, 15.1 mM Na2HPO4, and 50 mM NaCl, pH 7.4) and all of the solutions were prepared with Milli-Q 18-MΩ water (Millipore purification system). Preparation of the Oligonucleotide-Colloidal Gold Conjugate. The gold nanoparticle probe was synthesized by derivatizing 0.5 mL of an aqueous 20-nm-diameter Au nanoparticle solution (∼1 nM) with 20 µL of an aqueous solution of 5′hexanethiol 22-base oligonucleotide (22.7 µM). After standing for 19 h, the solution was brought to the pH and ionic strength of the phosphate buffer (0.08 M, pH 7.4) and allowed to stand for 6 h. Then, aqueous 2 M NaCl was added to the solution to bring the total NaCl concentration of the probe solution to 0.075 M. This was repeated 2 h after to adjust the NaCl concentration to 0.1 M. Upon standing an additional 90 h, the nanoparticles were isolated by centrifugation (14 000 rpm for 1 h), washed with PB, and recentrifuged (14 000 rpm for 1 h). The red oily precipitate was mixed with an equivalent volume of glycerol and stored at -20 °C. This concentrated probe solution was 750-fold diluted with the hybridization buffer prior to the hybridization assay. Procedure of the Electrochemical HCMV DNA Hybridization Assay in Microwells. HCMV DNA was extracted from cell culture, amplified by PCR, and then quantified by agarose gel electrophoresis as previously described.5 A standard concentration range of HCMV-amplified DNA (5-5000 pM) was prepared by serial dilution of the concentrated solution (5000 pM) in the negative control buffer. The HCMV DNA assay basically consisted of four steps: (i) Target DNA Immobilization. In a propylene tube, 5 µL of standard solutions of HCMV-amplified DNA was denatured in alkaline media for 10 min at room temperature, by adding 10 µL of each of the two denaturation solutions of the Hybridowell kit. Then, 200 µL of coating solution was added, and 100 µL of the resulting solution was transferred in a polystyrene microwell (Maxisorp) and incubated overnight at room temperature. (ii) Probe Hybridization. The microwells were drained and filled with 100 µL of the colloidal gold labeled probe diluted in the hybridization buffer. After incubation at 37 °C for 30 min, they were subjected to a washing cycle consisting of five washes for 1 min with 300 µL of fresh 1× washing solution, followed by two rinses for 1 min with 300 µL of PB. (iii) Gold Metal Dissolution. After carefully removing the rinsing solution, 100 µL of acidic bromine-bromide solution (0.1 M HBr containing 10-4 M Br2) was pipeted into the microwell for 20 min, and then 90 µL was transferred into a new microwell containing 10 µL of a 3-phenoxypropionic acid solution (2 × 10-3 M in 0.1 M HBr). After waiting 5 min, a SPMBE was immersed into the solution and the AuIII ions were then quantified by ASV. (iv) ASV Detection. The following instrumental conditions were used for ASV measurements: 5-min electrodeposition at -0.3 V, immediately followed by a positive potential scan at 50 mV s-1. Between each measurement, the microband electrode surface was regenerated by cutting a small slice of its extremity. The peak current ip located at ∼0.95 V or its integration Qp was taken as the analytical response. Analytical Chemistry, Vol. 73, No. 18, September 15, 2001

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Procedure of the Electrochemical HCMV DNA Hybridization Assay Performed Directly on the Surface of a Macroscopic SPE. Immobilization of the target HCMV DNA on a macroscopic SPE (sensing area of 9.62 mm2) was performed as previously described.5 Briefly, 5 µL of amplified HCMV DNA (5000 pM) was denatured in alkaline media for 10 min at room temperature by adding 10 µL of each of the two denaturation solutions. The resulting mixture was 12-fold diluted with the coating solution, and the sensing disk area of a SPE was immersed in 150 µL of this solution and incubated to dryness at 37 °C, overnight. Once the target HCMV DNA was immobilized, the SPE was rinsed with distilled water and immersed in a polypropylene tube containing 150 µL of the gold nanoparticle probe (750-fold diluted in the hybridization buffer) for 30 min at 37 °C. After hybridization, the SPE was washed five times with 300 µL of fresh 1× washing solution for 1 min, followed by two rinses in 300 µL of PB for 1 min, and finally immersed in a microwell containing 100 µL of 0.1 M HBr. The gold nanoparticles linked to the hybrids formed on the SPE surface were then electrochemically dissolved in solution by polarizing the SPE at +1.25 V for 30 s. Then, the SPE was replaced by a SPMBE and the released AuIII ions were quantified by ASV as mentioned above. RESULTS AND DISCUSSION Fabrication of Disposable SPMBEs. In our previous work,7 ASV measurements of AuIII were performed in a 35-µL droplet of solution deposited on the macroscopic sensing disk area (geometric area of 9.62 mm2) of a carbon-based SPE. However, the sensitivity was observed to be limited by the mass transport efficiency in the quiescent droplet solution.7 With the aim to favor the mass transfer of AuIII, the replacement of the macroscopic electrode by a microelectrode was envisioned and the construction of disposable sandwich-type SPMBEs was then investigated. Among the different types of microband electrodes reported in the literature, there are two major designs of fabrications: (i) a thin conductive film is sealed between two insulators and the edge is exposed (sandwich-type microband electrodes);9,10 (ii) a thin narrow conductive line is deposited photolithographically on an insulating plane.11 In a few cases, both designs were combined.12 Although lithography is well adapted for the large-scale production of any desired configuration of microband length and width, it requires expensive equipment and a special facility for production. Consequently, the fabrication of sandwich-type microband electrodes was preferentially investigated. Microbands (9) (a) Kovach, P. M.; Caudill, W. L.; Peters, D. G.; Wightman, R. M. J. Electroanal. Chem. 1985, 185, 285. (b) Wehmeyer, K. R.; Deakin, M. R.; Wightman, R. M. Anal. Chem. 1985, 57, 1913. (c) Bartelt, J. E.; Deakin, M. R.; Amatore, C.; Wightman, R. M. Anal. Chem. 1988, 60, 2167. (d) Bowyer, W. J.; Clark, M. E.; Ingram, J. L. Anal. Chem. 1992, 64, 459. (e) Varco Shea, T.; Bard, A. J. Anal. Chem. 1987, 59, 2101. (f) Hill, H. A. O.; Klein, N. A.; Psalti, I. S. M.; Walton, N. J. Anal. Chem. 1989, 61, 2200. (10) (a) Bond, A. M.; Henderson, T. L. E.; Thormann, W. J. Phys. Chem. 1986, 90, 2911. (b) Craston, D. H.; Jones, C. P.; Williams, D. E.; El Murr, N. Talanta 1991, 38, 17. (c) Bartlett, P. N.; Birkin, P. R. Anal. Chem. 1993, 65, 1118. (d) Ball, J. C.; Scott, D. L.; Lumpp, J. K.; Daunert, S.; Wang, J.; Bachas, L. G. Anal. Chem. 2000, 72, 497. (11) (a) Morita, M.; Longmire, M. L.; Murray, R. W. Anal. Chem. 1988, 60, 2770. (b) Kittlesen, G. P.; White, H. S.; Wrighton, M. S. J. Am. Chem. Soc. 1984, 106, 7389. (c) Takahashi, M.; Morita, M.; Niwa, O.; Tabei, H. J. Electroanal. Chem. 1992, 335, 253. (12) (a) Samuelsson, M.; Armgarth, M.; Nylander, C. Anal. Chem. 1991, 63, 931. (b) Nagale, M. P.; Fritsch, I. Anal. Chem. 1998, 70, 2902-7. (c) Nagale, M. P.; Fritsch, I. Anal. Chem. 1998, 70, 2908.

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prepared from microfoils or evaporated/sputtered thin films of noble metal (Pt, Au) sealed between two insulators (glass, epoxy, polyester, mica, Teflon, Tefzel) were the first developed.9 These approaches allow the construction of microbands with thicknesses ranging from few tens of micrometers to submicrometers. However, the assembling process is generally slow, tedious, and not adapted to large-scale production of disposable microbands. As an alternative approach, the construction of sandwich-type microbands by screen-printing successive layers of conductive (carbon- or metal-based inks) and dielectric inks on an insulating substrate (glass, alumina, ceramic, PVC) was proposed.10 Although the microband thickness is limited to few micrometers (except for metallic ink fired at high temperature10a), screen-printing technology possesses the advantages of being inexpensive, simple, rapid, and versatile and it is well adapted to mass production. However, the reproducible fabrication of disposable sandwichtype SPMBEs has not yet been demonstrated, probably because they are subject to delamination defects during the polishing or cutting step required to expose the edge,13 thus leading to a poor reproducibility. With the aim to mass produce reproducible sandwich-type microbands of low cost, we investigated the printing of a polymeric carbon-based ink onto a soft polymeric substrate. Our choice was focused on the HIPS, an impact-resistant polystyrene (copolymer of polystyrene and polybutadiene) having a higher elastic modulus than polystyrene and which is currently used for screen-printing (widely used for the fabrication of credit cards). Moreover, to avoid delamination along the insulator-conductor interface by differential stress during exposition of the microband edge, the same HIPS polymer was used to insulate the carbon layer (providing an equal hardness between the insulating and conductive materials). The optical and scanning electron micrographs (a representative view is given in Figure 1) of an array of height SPMBEs allowed the measurement of an average microband length of 1.42 ( 0.02 mm and the estimation of an average thickness of 6 ( 2 µm (geometric area of 0.0085 ( 0.0028 mm2). Micrographs show an intimate contact between the insulating HIPS phases and the carbon layer, without any apparent delamination defects, and clearly indicate that the insulating layer has penetrated into the thickness of the HIPS plate (the mesitylene solvent contained in the insulating layer is able to partially dissolve the HIPS plate surface), thus reinforcing the good adhesion between insulator and carbon film. Electrochemical Characterization of SPMBEs. The CV curves recorded at a SPMBE immersed in PB (pH 7.4) containing 2 mM FcTMA+ were of sigmoidal shape at slow scan rates (Figure 2), indicating a significant contribution from nonlinear diffusion. Under these conditions, the interelectrode reproducibility of the eight SPMBEs coming from the array previously examined by microscopy was evaluated by measuring the limiting peak-shaped current (ip) at a scan rate of 10 mV s-1. The intraelectrode reproducibility was also examined by renewing the microband edge with a scalpel between each successive measurement. From a series of eight measurements performed for each of the eight SPMBEs (see Supporting Information), the statistical analysis indicates a very good intraelectrode reproducibility with an (13) Porat, Z.; Crooker, J. C.; Zhang, Y.; Le Mest, Y.; Murray, R. W. Anal. Chem. 1997, 69, 5073.

Table 1. Average Values and Reproducibility of the ASV Responses Recorded at SPMBEs Immersed in 0.1 µM AuIII (See Text for Details) entry

Qp ( SD (nC)a

RSD (%)

no. of measurements

1 2 3 4b

2.43 ( 0.57 2.90 ( 0.42 2.84 ( 0.20 2.74 ( 0.25

23.5 14.5 7.0 9.1

8 8 7 10

a Integration of the stripping peak current (Q ) was observed to be p more reproducible than the anodic peak current response. b The microband electrode surface was just clean with an anodic polarization for 60 s between each measurement.

Figure 1. Scanning electron micrograph of the edge of a SPMBE. The microband thickness (4.76 µm) is marked off by two crosses on the picture.

Figure 2. CV curve (scan rate 0.01 V s-1) obtained at a SPMBE immersed in PB (pH 7.4) containing 2 mM FcTMA+.

average relative standard deviation (RSD) of 2%, which was 2.5fold better than the interelectrode reproductibility (average RSD of 5%). Such a discrepancy was explained by systematic higher average current responses of the SPMBEs located at both extremities of the array, suggesting a slightly higher microband thickness for the lateral electrodes. A last test of reproducibility was performed for eight SPMBEs selected from eight different batches arrays, and an average current response of 115.1 ( 9.3 nA (RSD value of 8.1%) was obtained. Using the relatively confident value of the microband length (L ) 1.42 ( 0.02 mm) measured by optical microscopy, the microband width (w) was evaluated from the average ip value and the theoretical equation14 giving the relationships between ip and w, at slow scan rate:

ip )

πnFCDL ln[8x(DRT/nFv)/w]

(1)

where n is the number of electron per mole of redox species (n ) 1 for FcTMA+ ), F is the Faraday constant, C is the concentration of redox species in bulk solution (2 mM FcTMA+), (14) Amatore, C. A.; Fosset, B.; Deakin, M. R.; Wightman, R. W. J. Electroanal. Chem. 1987, 225, 33.

D is the diffusion coefficient (6.3 × 10-6 cm2 s-1 for FcTMA+ at 20 °C), R is the gas constant, T the temperature (20 °C), and v is the scan rate (0.01 V s-1). Taking the preceding average ip value of 115.1 ( 9.3 nA, a band with of w ) 4.5 ( 1.5 µm (RSD of 33.3%) was calculated, which is close to the value estimated with the optical microscope 6 ( 2 µm. The resulting high RSD value of w (33.3%) confirms a nonnegligible variation of the microband thickness from batches to batches arrays. Tests of reproducibility were also performed for the ASV determination of AuIII (10-7 M in 0.1 M HBr), and the results are summarized in Table 1. Entry 1 corresponds to the average response obtained at eight SPMBEs selected from eight random arrays, and it shows again a relatively poor interarray electrode reproducibility. Although the intra-array RSD was 2-fold better (entry 2), the ASV reproducibility was, here also, considerably improved when the same electrode was repetitively used by cutting a small slice of its extremity between each ASV experiments (entry 3), and it was even slightly better than for a nonregenerated microband surface (entry 4). It is worth noting that the overall reproducibility of the ASV measurements of AuIII was significantly lower than that of the CV current responses of FcTMA+ at slow scan rate. This can be explained by the relative insensitivity of the radial diffusion current (i.e., of the CV current response) to the microband width (see eq 1), contrary to ASV, which is a surface area-dependent method and which can be strongly influenced by fluctuations of the real microband area. In summary, the overall tests of reproducibility show that the scalpel cutting is a simple and efficient method for the generation of a reproducible microband, thus allowing one to perform a large number of measurements with the same electrode. The good intraelectrode reproducibility and the very stable CV response upon immersion of a SPMBE for a prolonged period in an aqueous solution of FcTMA+, suggest a good adhesion of the insulating materials around the carbon film and the absence of delamination defects. It demonstrates that, by appropriate selection of materials, the screen-printing of successive layers is a useful method for the production of reproducible sandwich-type SPMBEs. The main source of irreproducibility observed in the case of interbatches SPMBEs, is strongly related to the use of a manual screen-printer. The further experiments were performed with SPMBEs renewed by microband edge cutting owing to the improve reproducibility. Enhanced Sensitivity of AuIII Determination at a SPMBE. Parameters such as accumulation time and electrodeposition potential were first investigated in order to establish optimal conditions for AuIII determination at SPMBEs, and the results were Analytical Chemistry, Vol. 73, No. 18, September 15, 2001

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Table 2. Comparison of Sensitivity for DNA Assay Methods Based on the Detection of a Gold Nanoparticle Label

a

analytical method

length of the target DNA/no. of bases

homogeneous colorimetric detection heterogeneous colorimetric detection flatbed scanner surface plasmon resonance quartz crystal microbalance quartz crystal microbalance ASV

30 24 27 24 17 24 406

detection limit

ref

50 pM 500 pM 50 fM 10 pM 0.35 mg L-1 32 pM 5 pM

19 20 21 22 23 24 a

This work.

Figure 3. Log-log ASV calibration plots of AuBr4- recorded (a) at a SPMBE (0.0085 mm2) in 50 µL of 0.1 M HBr solution and (b) at a macroscopic SPE (9.62 mm2) in a 35-µL droplet of 0.1 M HBr solution deposited on the SPE surface. Electrodeposition at -0.3 V vs saturated Ag/AgBr for 5 min, immediately followed by a positive potential scan at 0.05 V s-1. Error bars represent the standard deviation of two measurements.

consistent with those previously obtained at macroscopic SPEs.7 Consequently, an electrodeposition time of 5 min at -0.3 V versus Ag/AgBr was selected. Figure 3 compares the ASV calibration plots of AuBr4- in 0.1 M HBr (anodic stripping peak current normalized to the geometric area of electrodes versus AuIII concentration) for a SPMBE (curve a) and for a macroscopic SPE (curve b) in a small volume of quiescent solution. The 10-fold increase of sensitivity obtained at the SPMBE is due to the enhancement of the AuIII mass transfer by nonlinear diffusion during the electrodeposition period, thus allowing one to reach a detection limit as low as 3 nM AuIII. Such a performance is competitive with the lowest detection limit previously reported for ASV of AuIII,15 with the further advantages that no electrode pretreatment before the measurement and no agitation of the solution during the electrodeposition time were required. Electrochemical HCMV DNA Hybridization Assay Involving a Gold Nanoparticle Label. The principle, depicted in Figure 4, consisted of four steps: (a) passive adsorption of the amplified target DNA on the walls of a polystyrene microwell, (b) hybridization with an oligonucleotide probe conjugated to a colloidal gold (15) (a) Jacobs, E. S. Anal. Chem. 1963, 35, 2112. (b) Alarnes-Varela, G.; CostaGarcia, A. Electroanalysis 1997, 9, 1262. (c) Turyan, I.; Mandler, D. Anal. Chem. 1993, 65, 2089. (d) Lintern, M.; Mann, A.; Longman, D. Anal. Chim. Acta 1988, 209, 193.

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nanoparticle, (c) oxidative gold metal dissolution in an acidic bromine-bromide solution, and (d) ASV detection of the released AuIII ions at a SPMBE immersed in the microwell. A 5′-hexanethiol 22-base oligonucleotide probe was attached to a 20-nm gold particle via thiol chemisorption, using a procedure similar to the one described by Mirkin and co-workers.16 The modified nanoparticles were stabilized by addition of glycerol at the end of their preparation. Under these conditions, they can be stored at -20 °C for two months without any apparent drop of activity. The sensitivity of the nanoparticle-based electrochemical hybridization assay was investigated by varying the concentration of the target 406-bp HCMV-amplified DNA over the 5-5000 pM range. The corresponding logarithmic standard plot is shown in Figure 5. The dynamic range for the assay extended between 5 and 500 pM. The signal saturated above 1000 pM, owing to the limited amount of DNA fragment on the surface of microwells. It is important to note the small baseline signal recorded in the absence of HCMV DNA (inset of Figure 5), indicating a very low nonspecific binding. The selectivity of the response was controlled by replacing the HCMV DNA with a noncomplementary DNAamplified sequence, a human ETS2 DNA gene. The resulting signal (open circle symbol in Figure 5) was very close to that obtained in the absence of HCMV DNA. A detection limit of 5 pM (∼10 amol or 6 × 106 copies of 406-bp HCMV DNA per microwell) can be estimated using a signal-to-noise ratio of 3. The sensitivity of the method is better than other Au nanoparticlebased hybridization assays recently reported for shorter DNA sequences (Table 2). Moreover, the HCMV DNA detection limit (DL) obtained in the present work is competitive with previously reported HCMV DNA hybridization assays based on an enzyme label,5,17 and even superior to the a recent electrochemiluminescent HCMV DNA method involving a ruthenium(II) chelatelabeled probe (DL of 3 × 108 copies of a 578-bp HCMV DNA fragment).18 (16) Storhoff, J. J.; Elghanian, R.; Mucic, R. C.; Mirkin, C. A.; Letsinger, R. L. J. Am. Chem. Soc. 1997, 120, 11959. (17) Bagel, O.; Degrand, C.; Limoges, B.; Joannes, M.; Azek, F.; Brossier, P. Electroanalysis 2000, 12, 1447. (18) Boom, R.; Sol, C.; Weel, J.; Gerrits, Y.; de Boer, M.; Dillen, P. W. J. Clin. Microbiol. 1999, 37, 1489. (19) Reynolds, R. A.; Mirkin, C. A.; Letsinger, R. L. J. Am. Chem. Soc. 2000, 122, 3795. (20) Reynolds, R. A., III; Mirkin, C. A.; Letsinger, R. L. Pure Appl. Chem. 2000, 72, 229. (21) Taton, T. A.; Mirkin, C. A.; Letsinger, R. L. Science 2000, 289, 1757. (22) He, L.; Musick M. D.; Nicewarner S. R.; Salinas F. G.; Benkovic S. J.; Natan M. J.; Keating C. D. J. Am. Chem. Soc. 2000, 122, 9071. (23) Lin, L.; Zhao, H.; Li, J.; Tang, J.; Duan, M.; Jiang, L. Biochem. Biophys. Res. Commun. 2000, 274, 817.

Figure 4. Schematic representation of the Au nanoparticle-based electrochemical detection of HCMV-amplified DNA.

Figure 6. ASV curves (v ) 50 mV s-1) recorded at a SPMBE immersed in a solution in which (a) a gold probe/HCMV DNA-coated SPE was polarized at +1.25 V for 30 s. (b) The dashed line corresponds to the negative control; i.e., the target HCMV DNA was omitted on the SPE.

Figure 5. Log-log plot of integrated anodic stripping peak current vs HCMV DNA concentration. Open circle symbol: control with a noncomplementary human ETS2 DNA sequence. Inset: set of ASV curves recorded (v ) 50 mV s-1) at a SPMBE (between each measurement the microband electrode surface was regenerated by cutting a small slice of its extremity). Error bars represent the standard deviation of two measurements.

Electrochemical Dissolution of the Gold Label Hybridized on a Macroscopic SPE. To shorten the DNA assay procedure and to avoid the use of toxic bromine solutions, the electrochemical oxidation of the colloidal gold label was investigated. For this purpose, the HCMV DNA was adsorbed passively on the sensing area of a macroscopic SPE (geometric area of 9.62 mm2). Once the target DNA was hybridized with the gold nanoparticle probe, the SPE was immersed in 0.1 M HBr and polarized at +1.25 V for 30 s. During the anodic polarization, bromide was oxidized into bromine which in turn served as oxidant for the dissolution of the gold metal label anchored onto the SPE. After the AuIII ions are released in solution, they can then be quantified by ASV at a SPMBE. Figure 6 displays the ASV responses obtained after the gold released from SPEs coated with (curve a) and without (curve b) the gold probe/HCMV DNA hybrids. After the anodic polarization of the gold probe/HCMV DNA-coated SPE, a large anodic stripping peak current was measured at the SPMBE (Figure 6a), whereas in the absence of adsorbed target DNA (24) Zhou, X. C.; O’Shea, S. J.; Li, S. F. Y. Chem. Commun. 2000, 953.

(Figure 6b) or in the presence of noncomplementary ETS2 DNA (not shown) onto the SPE, no signal was recorded. Moreover, no stripping current was observed when no anodic pretreatment was applied to the gold probe/HCMV DNA-coated SPE. These results demonstrate that the gold probe was present on the SPE only when the target DNA was first immobilized and that the gold label dissolution was effective only when bromine was generated at the SPE surface by anodic polarization. Finally, the direct visualization of the gold nanoparticles hybridized on the HCMV DNA-coated SPE was tested by cycling the SPE between 0 and 1.2 V. However, in contrast to the previous direct voltammetric detection of unmodified colloidal gold particles adsorbed on a SPE,7 no current response was observed for gold probe/HCMV DNA-coated SPE, suggesting that oligonucleotides surrounding gold act as insulating shells, thus impeding the direct electrochemical oxidation of gold particles. The above experiments clearly show that the anodic generation of a high local concentration of bromine at the surface of the gold probe/HCMV DNA-coated SPE leads to a fast and efficient dissolution of Au. They also indicate that the amount of bromine generated during the short anodic polarization is sufficiently low that, once diluted in the bulk volume of the solution, it cannot interfere on the ASV measurement of AuIII, as was previously observed.7 Consequently, the addition of a bromine-trapping reagent is not required. CONCLUSION The novel approach described in this work is promising for several reasons. (1) The successful sensitive and simple quantification of large target HCMV-amplified DNA adsorbed on the Analytical Chemistry, Vol. 73, No. 18, September 15, 2001

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bottom of microwells establishes a general detection methodology that can be extend to a variety of DNA diagnostic formats including various immobilization supports. (2) The use of SPMBEs for the sensitive AuIII detection that can be easily and reproducibly mass produced with inexpensive materials is particularly attractive for large-scale DNA testing in very small volumes. (3) The gold nanoparticle label has the advantages over radioisotopic or enzyme label of being stable, and the labeling procedure is very simple. (4) Further improvement in the detection limit by dissolution of gold in a smaller volume of liquid and/or by using DNA probes modified with much larger Au nanoparticles can be readily envisaged. For example, assuming a 5-fold increase of the gold size particles (i.e., 100-nm diameter) and their electrochemical dissolution in a volume of liquid as small as 10 µL (i.e., 10-fold smaller than the present 100 µL of 0.1 M HBr), a 1250-fold enhancement of sensitivity can be expected (assuming that the sensitivity is proportional to the cube of the particle diameter, that

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the AuIII determination is not affected by scaling down the volume, and that the physical effect of larger gold particles is insignificant). ACKNOWLEDGMENT The authors acknowledge Argene S.A. and Re´gion Bourgogne for financial support. SUPPORTING INFORMATION AVAILABLE A scheme of the SPMBEs and a table giving the 64 current responses of 8 SPMBEs repetitively used, including their statistical analysis. This material is available free of charge via the Internet at http://pubs.acs.org.

Received for review March 19, 2001. Accepted June 29, 2001. AC0103221