Electrochemical Genosensor Based on Colloidal Gold Nanoparticles

Ege University, 35100, Bornova, Izmir, Turkey, and INOVA Biotechnology Inc., Gaziemir, Izmir, Turkey. Electrochemical genosensors for the detection of...
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Anal. Chem. 2003, 75, 2181-2187

Electrochemical Genosensor Based on Colloidal Gold Nanoparticles for the Detection of Factor V Leiden Mutation Using Disposable Pencil Graphite Electrodes Mehmet Ozsoz,*,† Arzum Erdem,† Kagan Kerman,† Dilsat Ozkan,† Berrin Tugrul,‡ Nejat Topcuoglu,‡ Hayati Ekren,§ and Muzaffer Taylan§

Department of Analytical Chemistry, Faculty of Pharmacy, and Department of Medicinal Biology, Faculty of Medicine, Ege University, 35100, Bornova, Izmir, Turkey, and INOVA Biotechnology Inc., Gaziemir, Izmir, Turkey

Electrochemical genosensors for the detection of the Factor V Leiden mutation from polymerase chain reaction (PCR) amplicons using the oxidation signal of colloidal gold (Au) is described. A pencil graphite electrode (PGE) modified with target DNA, when hybridized with complementary probes conjugated to Au nanoparticles, responded with the appearance of a Au oxide wave at ∼+1.20 V. Specific probes were immobilized onto the Au nanoparticles in two different modes: (a) Inosine-substituted probes were covalently attached from their amino groups at the 5′ end using N-(3-dimethylamino)propyl)N′-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysulfosuccinimide (NHS) as a coupling agent onto a carboxylate-terminated L-cysteine self-assembled monolayer (SAM) preformed on the Au nanoparticles, and (b) probes with a hexanethiol group at their 5′ phosphate end formed a SAM on Au nanoparticles. The genosensor relies on the hybridization of the probes with their complementary targets, which are covalently immobilized at the PGE surface. Au-tagged 23-mer capture probes were challenged with the synthetic 23-mer target, 131-base singlestranded DNA or denatured 256-base polymerase chain reaction (PCR) amplicon. The appearance of the Au oxidation signal shortened the assay time and simplified the detection of the Factor V Leiden mutation from PCR amplified real samples. The discrimination between the homozygous and heterozygous mutations was also established by comparing the peak currents of the Au signals. Numerous factors affecting the hybridization and nonspecific binding events were optimized. The detection limit for the PCR amplicons was found to be as low as 0.78 fmol; thus, it is suitable for point-of-care applications. Even one base alteration in a DNA sequence can lead to mutations causing many human diseases. The main conventional methods for detecting these point mutations are polymerase chain reaction (PCR) followed by restriction enzyme analysis,1 ligase * To whom correspondence should be addressed. E-mail: ozsozs@pharmacy. ege.edu.tr. † Department of Analytical Chemistry, Ege University. ‡ Department of Medicinal Biology, Ege University. § INOVA Biotechnology Inc. 10.1021/ac026212r CCC: $25.00 Published on Web 04/03/2003

© 2003 American Chemical Society

chain reaction,2 and nick translation PCR with fluorogenic DNA probes.3 These detection schemes are mostly based on the coupling of gel electrophoresis and radioisotopic labeling and are thus not suitable for automation. One of the most sought-after mutations in routine analysis is the Factor V Leiden mutation. The Factor V Leiden mutation in the coagulation factor V gene results in the resistance of Factor V to inactivation by activated protein C (APC).4 To overcome the need for simple and rapid Factor V Leiden mutation analysis, several optical genosensors have been developed. Potter et al.5 have reported a time-resolved fluorescence-based genosensor using europium- and samariumlabeled oligonucleotide probes for the detection of wild-type (WT) and mutant (MT) Factor V Leiden DNA sequences. The optical array analysis of real samples prepared by strand displacement amplification has also been described for the detection of Factor V mutation.6-8 The reporter oligonucleotides modified with either Cy3 or Cy5 were introduced onto the microarray, and fluorescence at each location was quantified. Genosensors have recently been the subject of extensive research activities.9-15 Colloidal gold (Au) nanoparticles are promising nanomaterials that play an important role in the design of genosensors.16 Optical DNA detection schemes based on the (1) Mullis, K. B.; Faloora, F. A. Methods Enzymol. 1987, 155, 335-350. (2) Landegran, U.; Kaiser, R.; Sanders, J.; Hood, L. Science 1988, 241, 10771080. (3) Lee, L. G.; Connel, C. R.; Bloch, W. Nucleic Acids Res. 1993, 21, 37613766. (4) Bertina, R. M.; Koeleman, B. P. C.; Koster, T.; Rosendaal, F. R.; Dirven, R. J.; Deronde, H.; Vandervelden, P. A.; Reitsma, P. H. Nature 1994, 369, 64-67. (5) Potter, C. G.; Liu, Y. T.; Rees, D. C. Genet. Test. 2001, 5, 291-297. (6) Westin, L.; Xu, X.; Miller, C.; Wang, L.; Edman, C. F.; Nerenberg, M. Nat. Biotechnol. 2000, 18, 199-204. (7) Edman, C. F.; Mehta, P.; Press, R.; Spargo, C. A.; Walker, G. T.; Nerenberg, M. J. Invest. Med. 2000, 48, 93-101. (8) Westin, L.; Miller, C.; Vollmer, D.; Canter, D.; Radtkey, R.; Nerenberg, M.; O’Connel, J. J. Clin. Microbiol. 2001, 39, 1097-1104. (9) Thorp, H. H. Trends Biotechnol. 1998, 16, 117-121. (10) Palecek, E.; Jelen, F. Crit. Rev. Anal. Chem. 2002, 32, 261-270. (11) Wang, J. Anal. Chim. Acta 2002, 469, 63-71. (12) Erdem, A.; Ozsoz, M. Electroanalysis 2002, 14, 965-974. (13) Gooding, J. J. Electroanalysis 2002, 14, 1149-1156. (14) Marrazza, G.; Chiti, G.; Mascini, M.; Anichini, M. Clin. Chem. 2000, 46, 31-37. (15) Kobayashi, M.; Mizukami, T.; Morita, Y.; Murakami, Y.; Yokoyama, K.; Tamiya, E. Electrochemistry 2001, 69, 1013-1016. (16) Shipway, A. N.; Katz, E.; Willner, I. ChemPhysChem 2000, 1, 18-52.

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hybridization of target DNA to Au nanoparticles with alkanethiolcapped probes have been reported.17-22 These reports describe a colorimetric change associated with particle aggregation caused by oligonucleotide hybridization. Demers et al.21 reported a fluorescence-based scheme for determining the surface coverage of hexanethiol-modified probes on Au nanoparticles and the accessibility of these immobilized oligonucleotides for hybridization with complementary targets in solution. In this report, the hexanethiol modified capture probes were also used as described by Demers et al.,21 but in an electrochemical detection scheme. Disulfide-coupling chemistry was used by Hilliard et al.22 for the immobilization of oligonucleotides onto silica nanoparticles in a fluorescence-based genosensor. Probe-modified silica nanoparticles were able to detect a complementary target concentration in the nanomolar range. Microgravimetric quartz crystal microbalance studies have also been reported in connection with Au nanoparticles. Willner et al.23 hybridized a biotinylated nucleotide, complementary to the mutation site, in the presence of polymerase. Subsequent binding of the Au nanoparticle with an avidin conjugate and the biocatalyzed deposition of gold on the nanoparticles increased the mass and thus changed the resonance frequency of the crystal. Willner et al.23 compared three different schemes in their report and found that a Au nanoparticle-based scheme is the most sensitive one, with a detection limit of 3 fM. Au nanoparticles were also used in many electrochemical schemes. Wang et al.24 described a colloidal Au tag for electrochemical detection and amplification of DNA hybridization. Biotinylated target oligonucleotides were bound to the streptavidincoated Au nanoparticles. The acid dissolution of Au tags was monitored by chronopotentiometric stripping analysis at bare carbon strip electrodes. Authier et al.25 described the detection of human cytomegalovirus from PCR amplicons by using Autagged probes. After the release of Au atoms by oxidative metal dissolution using acidic bromine-bromide solution, the Au signal was monitored in connection with anodic stripping voltammetry. A similar procedure was reported by Dequaire et al.26 for a noncompetitive heterogeneous immunoassay of immunoglobulin G. Cai et al.27 described the self-assembly of Au nanoparticles onto a cysteamine-modified Au electrode. Their procedure resulted in a larger surface area for DNA hybridization. Hu et al.28 studied the adsorption kinetics of [Fe(CN)6]3-/4- on the self-assembled (17) Elghanian, R.; Storhoff, J. J.; Mucic, R. C.; Letsinger, R. L.; Mirkin, C. A. Science 1997, 277, 1078-1081. (18) Storhoff, J. J.; Elghanian, R.; Mucic, R. C.; Mirkin, C. A.; Letsinger, R. L. J. Am. Chem. Soc. 1998, 120, 1959-1964. (19) Taton, T. A.; Lu, G.; Mirkin, C. A. J. Am. Chem. Soc. 2001, 123, 51645165. (20) Reynolds, R. A.; Mirkin, C. A.; Letsinger, R. L. J. Am. Chem. Soc. 2000, 122, 3795-3796. (21) Demers, L. M.; Mirkin, C. A.; Mucic, R. C.; Reynolds, R. A.; Letsinger, R. L.; Elghanian, R.; Viswanadham, G. Anal. Chem. 2000, 72, 5535-5541. (22) Hilliard, L. R.; Zhao, X.; Tan, W. Anal. Chim. Acta 2002, 470, 51-56. (23) Willner, I.; Patolsky, F.; Weizmann, Y.; Willner, B. Talanta 2002, 56, 847856. (24) Wang, J.; Xu, D.; Kawde, A.-N.; Polsky, R. Anal. Chem. 2001, 73, 55765581. (25) Authier, L.; Grossiord, C.; Brossier, P.; Limoges, B. Anal. Chem. 2001, 73, 4450-4456. (26) Dequaire, M.; Degrand, C.; Limoges, B. Anal. Chem. 2000, 72, 55215528. (27) Cai, H.; Xu, C.; He, P.; Fang, Y. J. Electroanal. Chem. 2001, 510, 78-85. (28) Hu, X.-Y.; Xiao, Y.; Chen, H.-Y. J. Electroanal. Chem. 1999, 466, 26-30.

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monolayer (SAM) of Au nanoparticles attached to cysteaminemodified Au electrodes. Nam et al. 29 recently utilized oligonucleotide-modified Au nanoparticles as biobarcodes for the simultaneous screening of multiple target polyvalent proteins. Au nanoparticle-catalyzed silver deposition-based electrochemical detection of DNA hybridization has recently been reported by several groups. Park et al.30 localized Au nanoparticle-tagged probes in an electrode gap. Silver deposition catalyzed by Au nanoparticles bridged the gap and led to measurable conductivity changes. Wang et al.31 precipitated silver on Au nanoparticles and subsequently determined silver at a bare carbon strip electrode. Cai et al.32 electrostatically adsorbed target oligonucleotides onto glassy carbon electrodes and hybridized them with Au nanoparticle-tagged probes. After silver deposition onto Au nanoparticles, hybridization was monitored using the silver oxidation signal. Wang et al.33 recently formed a silver cluster on the DNA phosphate backbone and detected the silver oxidation by using bare carbon strip electrodes. Wang et al.34,35 attached biotinylated inosine substituted oligonucleotides onto streptavidin-coated magnetic beads. The electrochemical detection was based on the guanine oxidation signal. Ozkan et al.36 also described a guanine oxidation signal based the detection scheme for the Factor V Leiden mutation by using carbon paste electrodes (CPE) with a detection limit of ∼50 fmol. In the current paper, an electrochemical genosensor is introduced for the detection of the same mutation by using the oxidation signal of Au anchored within the hybrid at the singleuse pencil graphite electrode (PGE) surface in connection with differential pulse voltammetry (DPV). The electrochemistry of Au nanoparticles was first reported in connection with CPE by Gonzales-Garcia et al.37 The electroactivity of Au nanoparticles is used for the detection of hybridization for the first time without any external indicators. The specific sequences of DNA are detected electrochemically by tagging a probe strand with Au colloid and immobilizing the target onto an electrode. Hybridization of the Au-tagged probe to the adsorbed target allows the Au to be detected by anodic stripping analysis of the Au colloid. There have yet to be any literature reports about the discrimination of heterozygous and homozygous mutations from PCR-amplified real samples which use the Au signal without the need for acidic dissolution of Au tag. Furthermore, the method of transduction by adsorbing the target onto the detecting electrode first represents a different approach. The work also has a realistic potential application, since the experiments were carried out using real PCR amplicons. This scheme can be applied for the detection of many different diseases using different PCR amplicons. The limit of detection is lower than that previously reported by Authier et al.;25 (29) Nam, J.-M.; Park, S.-J.; Mirkin, C. A. J. Am. Chem. Soc. 2002, 124, 38203821. (30) Park, S.-J.; Taton, A.; Mirkin, C. A. Science 2002, 295, 1503-1506. (31) Wang, J.; Polsky, R.; Xu, D. Langmuir 2001, 17, 5739-5741. (32) Cai, H.; Wang, Y.; He, P.; Fang, Y. Anal. Chim. Acta 2002, 469, 165-172. (33) Wang, J.; Rincon, O.; Polsky, R.; Dominguez, E. Electrochem. Commun. 2003, 5, 83-86. (34) Wang, J.; Kawde, A.-N.; Erdem, A.; Salazar, M. Analyst 2001, 126, 20202024. (35) Wang, J.; Xu, D. K.; Erdem, A.; Polsky, R.; Salazar, M. A. Talanta 2002, 56, 931-938. (36) Ozkan, D.; Erdem, A.; Kara, P.; Kerman, K.; Meric, B.; Hassmann, J.; Ozsoz, M. Anal. Chem. 2002, 74, 5931-5936. (37) Gonzales-Garcia, M. B.; Costa-Garcia, A. Bioelectrochem. Bioenerg. 1995, 38, 389-395.

thus, it is suitable for the needs of genetic diagnosis. The represented work is also cost-effective, because a disposable PGE was used as the detecting electrode. The modification of DNA probes with it is simple and rapid. In the following sections, the features of the protocol are discussed, and results are compared with the other protocols previously reported. EXPERIMENTAL SECTION Apparatus. Differential pulse voltammetry (DPV) was performed with an Autolab PGSTAT 30 electrochemical analysis system (Eco Chemie, The Netherlands). The UV-vis spectrophotometer (Schimadzu, Japan) was used with quartz cuvettes of 1 mL and 10 mm path length (Starna, England). The threeelectrode system consisted of a pencil graphite electrode (PGE) as the working electrode, the reference electrode (Ag/AgCl), and a platinum wire as the auxiliary electrode. A Noki pencil model 2000 (Japan) was used as a holder for the graphite lead (Tombo, Japan). Electrical contact with the lead was obtained by soldering a metallic wire to the metallic part. The pencil lead was held vertically with 12 mm of the lead extruded outside (10 mm of which was immersed into the solution). A magnetic stirrer provided the convective transport. Chemicals. The synthetic PCR product and its complementary thiol-capped probe and also the oligonucleotides of the WT and MT capture probes and their complementary targets were purchased (as lyophilized powder) from Thermo Hybaid (Erlangen, Germany). The base sequences used were as follows. Synthetic PCR product: 5′-CCT GCC CCA ATC CCT TTA TTA CCC CCT CCT TCA GAC ACC TCT AAC CTC TTC TGG CTC AAA AAG AGA ATT GGG GGC TTA GGG TCG GAA CCC AAG CTT AGA ACT TTA AGC AAC AAG ACC ACC ACT TCG AAA CC-3′ Thiol-Capped Probe: 5′-SH-C6-GGT TTC GAA GTG GTG GTC TTG-3′ Factor V-Wild-Type (WT) Capture Probe: 5′-NH2-AAT ACC TIT ATT CCT CIC CTI TC-3′ Factor V-Wild-Type Target: 5′-GAC AGG CGA GGA ATA CAG GTA TT-3′ Factor V MutantType (MT) Capture Probe: 5′-NH2-AAT ACC TIT ATT CCT TIC CTI TC-3′ Factor V MutantType Target: 5′-GAC AGG CAA GGA ATA CAG GTA TT-3′ The oligonucleotide stock solutions (100 mg/L) were prepared with 10 mM Tris-HCl, 1 mM EDTA (pH 8.00, TE) and kept frozen. More dilute solutions of capture probes were prepared using 0.5 M acetate buffer containing 20 mM NaCl (pH 4.80, ABS). More dilute solutions of targets were prepared using 20 mM TrisHCl buffer solution containing 20 mM NaCl (pH 7.00, TBS). The inosine (I)-labeled capture probes had the same sequence as a region of the gene for Factor V. This sequence was included in the DNA fragment amplified by PCR. Factor V Leiden mutation is caused by a point mutation from G to A at the 1691st position of the human genome. A 23-mer inosine-substituted oligonucleotide collinear with the portion of the human genome for Factor V was designed to serve as a wild-type (WT) capture probe.8 A second probe containing a single G-to-A transition at position 16 from the 5′ end of the oligonucleotide was used as the mutanttype (MT) capture probe.8 Capture probes were perfect complements to WT or MT Factor V alleles. Amplicons harboring the

point mutation were prepared by B. Tugrul and N. Topcuoglu in the Department of Medicinal Biology in the Faculty of Medicine, Ege University, using a restriction fragment length polymorphism (RFLP)-PCR technique from DNA samples. The amplicons were characterized as homozygous WT, homozygous MT, or heterozygous by using 1.5% agarose gel electrophoresis. The amplicons were 256 bp DNA fragments. Totally 30 patient samples of each genotype were studied. The concentrations of all oligonucleotides and PCR products were determined by following a spectrophotometric method in which 1 A260 unit of double-stranded DNA represented 50 µg/mL and 1 A260 unit of single-stranded DNA represented 33 µg/mL.38 N-Hydroxysulfosuccinimide (NHS) and N-(3-dimethylamino)propyl)-N′-ethylcarbodiimide hydrochloride (EDC) were purchased from Aldrich. Other chemicals were of analytical reagent grade. In-house sterilized and deionized water was used in all solutions. All experiments were conducted at room temperature (25 ( 0.5 °C). Methods. The procedure consists of the following steps and is illustrated in Scheme 1. Target Immobilization onto PGE Surface. The target was immobilized onto the desired length of PGE by dipping it into the covalent agent solution, which contained 2 mM EDC and 5 mM NHS in PBS, for 1 h. After covalent activation of the surface, the PGE was rinsed with blank PBS and then dipped into the ABS solution, which contained 15 µg/mL of synthetic target sample. The target immobilization continued for 1 h at room temperature. The amplicons were analyzed independently by using both WT and MT capture probes. The real sample obtained from the PCR amplification was diluted with TBS; the diluted sample was then placed in a vial and denatured by heating in a water bath at 95 °C for 6 min and then freezing the sample in an ice bath for 2 min. When the denatured real PCR amplicons were used as target, the electrodes were kept at 8 °C for 1 h. During this period, the renaturation process slowed, and more single-stranded target molecules were available for hybridization. Preparation of Au Nanoparticle-Tagged Capture Probes. Au nanoparticles were prepared by H. Ekren and M. Taylan as reported in the literature.21 The average diameter of a typical particle preparation was 5 ( 1.3 nm. The modification of Au nanoparticles with capture probes is illustrated in Scheme 2. For the modification of the L-cysteine self-assembled monolayer (SAM) on Au nanoparticles, the Au nanoparticles were kept overnight in 10 mM L-cysteine in PBS at room temperature in a manner similar to previous reports.39 Covalent activation was performed as reported by Millan et al.40 and others.41 Solutions of 2 mM EDC and 5 mM NHS were added onto the SAM-modified Au nanoparticles and kept for 1 h. Afterward, capture probes at a final concentration of 10 µg/mL were added to the solution and kept for 1 h. By modifying the colloids with L-cysteine, the surfaces were covered with both carboxylic acid and amine groups. The activation with EDC/NHS converted the carboxylic acids to succinimide esters, which allowed the formation of a peptide bond (38) http://biochem.roche.com/labfaqs/index.html. (39) Yang, W.; Hibbert, D. B.; Gooding, J. J. J. Electroanal. Chem. 2001, 516, 10-16. (40) Millan, K. M.; Spurmanis, A. J.; Mikkelsen, S. R. Electroanalysis 1992, 4, 929-932. (41) Gooding, J. J.; Praig, V. G.; Hall, E. A. H. Anal. Chem. 1998, 70, 23962402.

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Scheme 1. Detection of Hybridization Using Au Nanoparticle-Tagged Capture Probes

with amines. To prevent the colloids from covalently attaching to each other rather than oligonucleotides, standing time was limited to 1 h. Allowing the Au colloidal solution to stand for more than 24 h resulted in the aggregation of the amine-covered nanoparticles. The Au nanoparticles were also modified with hexanethioltagged capture oligonucleotides, as described by Demers et al. 21 by adding these oligonucleotides to the aqueous nanoparticle solution (particle concentration ∼10 nM) to a final oligonucleotide concentration of 10 µg/mL in TBS and kept for 1 h. Hybridization with Au Nanoparticle-Tagged Probes. The target-immobilized PGEs were dipped into the Au nanoparticletagged capture probe solution. The hybridization was allowed to proceed for 1 h. Nonspecific adsorption effects onto PGE were suppressed with the following washing step. The hybrid modified PGE was dipped into the 1% sodium dodecylsulfate solution dissolved in TBS (SDS) for 3 s and then immediately dipped into blank TBS for 3 s. Voltammetric Transduction. The oxidation signal of Au was measured by using differential pulse voltammetry (DPV) in blank TBS by scanning from +0.85 to +1.40 V with an amplitude of 10 mV at 20 mV/s scan rate. The raw voltammograms were treated by using the Savitzky and Golay filter (level 2) included in the General Purpose Electrochemical Software (GPES) of Eco Chemie (The Netherlands) with moving average baseline correction, as described in the literature,36,42 using a “peak width” of 0.01 V. Control experiments were performed with a real sample, which contained a PCR product that had a sequence noncomplementary 2184

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to either probe. Noncomplementary PCR samples were also prepared by B. Tugrul and N. Topcuoglu in the Department of Medicinal Biology in the Faculty of Medicine, Ege University. The WT probe was also challenged with the one-base mismatched real sample that contained a sequence complementarity to the MT probe, and the MT probe was challenged with the one-base mismatched real sample that contained a sequence complementarity to the WT probe. The MT and WT probes were also challenged with the PCR blank solution, which contained the primers and polymerase without the target amplified DNA. RESULTS AND DISCUSSION For the detection of hybridization between the Factor V Leiden WT or MT capture probe-immobilized Au nanoparticles and target DNA, an aliquot of the probe modified Au nanoparticles was simply introduced onto the target immobilized electrode. The appearance of the Au oxidation signal confirmed the presence of the sought-after DNA sequence. All voltammograms obtained from the anodic stripping scan, after moving average baseline correction, was applied to the raw voltammograms treated by using the Savitzky and Golay filter (level 2) included in the GPES program, using a peak width of 0.01 V. Figure 1 represents the Au signals obtained when the WT capture probe-immobilized Au nanoparticle was hybridized with the target oligonucleotide on the PGE surface. The WT capture probe was covalently immobilized onto the L-cysteine SAM on the (42) Palecek, E.; Kizek, R.; Havran, L.; Billova, S.; Fojta, M. Anal. Chim. Acta 2002, 496, 73-83.

Scheme 2. DNA Modification of Au Nanoparticles

Au nanoparticle. Target oligonucleotide was covalently attached onto the PGE surface. The activation with covalent agents converted the carboxylic acid moieties on the carbon surface to succinimide esters, which allowed the formation of a peptide bond with the amines of the PCR amplified sample. In Figure 1, the Au

signals obtained when the WT capture probe immobilized Au nanoparticles were used in the hybridization. Increases in the Au signal indicated the formation of a hybrid (Figure 1a). When the Au nanoparticles modified with MT capture probes were used, the hybrid signal was approximately one-half of the completely Analytical Chemistry, Vol. 75, No. 9, May 1, 2003

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Figure 1. Differential pulse voltammograms for the oxidation signals of Au at PGE in TBS at (a) wild-type (WT) capture probe-modified Au nanoparticle after hybridization with WT synthetic target, (b) mutant (MT) capture probe-modified Au nanoparticle after hybridization WT with synthetic target, (c) WT capture probe-modified Au nanoparticle after hybridization with no target on PGE surface, (d) WT capture probe-modified Au nanoparticle after hybridization with synthetic noncomplementary DNA sequence, and (e) the same experimental procedure performed with no probe-modified Au nanoparticle and no target, only TBS at a bare PGE.

complementary sample. The difference in current signal is attributed to the presence of a point mutation (Figure 1b). When Au nanoparticules with no capture oligonucleotides immobilized on the surface were used as the target, only minor penetration of Au nanoparticles onto PGE was observed (Figure 1c). A low Au signal was observed when a noncomplementary oligonucleotide was immobilized on the PGE (Figure 1d). No electrochemical signals were observed at +1.20 V when only TBS buffer was used (Figure 1e). Covalent agents (EDC and NHS) gave a high signal at ∼+1 V as a result of the oxidation to their respective carboxylic forms. Thus, the detection scheme could not be based on the oxidation signal of guanine, which was also observed at exactly the same potential (not shown). The peaks at ∼1 V do not belong to guanine because guanine does not give such high oxidation peaks in a neutral buffer, such as TBS (pH 7.00). The optimum pH medium for such high guanine oxidation peaks would be ABS. To further verify the peaks at 1 V were due to the coupling agents, only EDC/NHS were applied to the PGE surface, as described in the Experimental Section, without any probe or target. Again, the oxidation peaks at ∼1 V were clearly observed (not shown). The Au signal obtained from the WT probe-modified Au nanoparticles after hybridization with WT target at PGE gave a RSD value of 7.64% (n ) 5). The Au signal obtained from the hybridization of the MT probe with the MT target at PGE gave a RSD value of 7.42% (n ) 5). The detection limits, estimated from S/N ) 3, correspond to 0.78 fmol/mL target with WT probe-modified gold nanoparticles and 0.83 fmol/mL target with MT probe-modified gold nanoparticles. The limit of detection in this study, based on gold nanoparticle assay and PGE, is 50-60 times lower than the previous report, in which CPE was used for the discrimination of Factor V Leiden mutation.36 The crucial difference between the CPE and PGE, and hence, the success of the latter approach, relied on the activation step of the PGE followed by covalent attachment of the target. Chu et al.43 have reported that graphitic materials have at least two distinct types of surface sites, namely, the basal plane and edge plane sites. It is generally regarded that 2186 Analytical Chemistry, Vol. 75, No. 9, May 1, 2003

Figure 2. Histogram for the oxidation signal of Au at PGE in TBS at (A) hexanethiol-capped probe-modified Au nanoparticle after hybridization with synthetic PCR product, (B) hexanethiol-capped probe-modified Au nanoparticle at only covalent agent-modified PGE without any target, and (C) Au nanoparticle with no probe at covalent agent-modified PGE.

the active sites for electrochemical reactions are associated with the edge plane sites, while the basal plane is inactive. The electrochemical pretreatment was found to improve the electrochemical behavior by introducing more edge sites on the carbon surface. The effect of oxidation on the electrochemical behavior and the chemical composition of the electrode was also reported by Regisser et al.44 It was reported that the concentration of strong and weak acidic groups increased upon electrochemical oxidation of the graphite electrode. The weight increase after electrochemical pretreatment was attributed to the formation of the oxidized graphite and the intercalation of solvent molecules and anions into graphitic material. Thus, changing the ketone and aldehyde groups on the PGE into their respective acidic groups provided a surface available for the covalent attachment. Then the covalent agents converted the carboxylic acid moieties on the carbon surface to succinimide esters. DNA was immobilized onto covalently modified PGE, as first described by Millan et al.,40 which allowed the formation of a peptide bond with the amines of the PCR-amplified sample. Thus, target DNA could be strongly attached to the electrode surface. Figure 2 represents the hybridization detection with the synthetic PCR product immobilized on PGE. The appearance of the Au signal obtained with the PCR product, which contained the sought-after DNA sequence, confirmed hybridization. When the SAM hexanethiol-capped probe was formed on the Au nanoparticles, the high Au oxidation signal (Figure 2A) showed that the PCR product contained target DNA. Only a low Au signal was obtained when thiol-capped oligonucleotides were used, where there was only covalent modification but no synthetic PCR product on the PGE surface (Figure 2B). A low voltammetric signal (Figure 2C) was obtained when only the Au nanoparticles without any capture probe modification, were used for hybridization with PCR product-modified electrode. Data are given in vertical bar charts in Figure 2A, where each bar shows the mean and the standard deviation of the signal values obtained with five different replicates of the genosensor. The detection limits, estimated from S/N ) 3, correspond to 0.81 fmol/mL synthetic target with thiolcapped probes immobilized on the Au nanoparticles. (43) Chu, X.; Kinoshita, K. Mater. Sci. Eng. B 1997, 49, 53. (44) Regisser, F.; Lavoi, M.-A.; Champagne, G. Y.; Belange, D. J. Electroanal. Chem. 1996, 415, 47-54.

Figure 3. Histogram for the oxidation signal of Au at PGE in TBS at wild-type (WT) capture probe-modified Au nanoparticle (A) after hybridization with homozygous WT PCR real sample, (B) after hybridization with heterozygous PCR real sample, (C) after hybridization with homozygous MT PCR real sample, (D) mutant (MT) capture probe-modified Au nanoparticle after hybridization with homozygous MT PCR real sample, (E) after hybridization with heterozygous PCR real sample, (F) after hybridization with homozygous WT PCR real sample, (G) WT capture probe-modified Au nanoparticle after hybridization with PCR blank solution, and (H) WT capture probemodified Au nanoparticle after hybridization with noncomplementary PCR real sample.

Figure 3 represents the hybridization detection studies with the real PCR samples. When the amino-capped inosine-substituted WT probe was immobilized on the L-cysteine SAM and covalent agent-modified Au nanoparticles, the high Au oxidation signal (Figure 3A) showed that the PCR product contained WT DNA. It is a genetic fact that the alleles of the heterozygous patients contain DNA sequences that are complementary to both WT and MT capture probes. Therefore, about one-half of the heterozygous samples had complementary to the WT probe on the PGE surface, so only one-half of the signal was obtained from the heterozygous patients (Figure 3B). Both of the alleles of the homozygous mutant patient contained point mutation. Thus, none of the strands of the PCR product was complementary to the WT probe and nearly no Au signals appeared. A low voltammetric signal (Figure 3C) was obtained when homozygous MT PCR product was immobilized on the PGE. When the MT probe was immobilized on PGE, a high Au oxidation signal (Figure 3D) showed that the PCR product was homozygous MT DNA. Only one-half of the signal was obtained from the heterozygous patients (Figure 3E). A low Au signal was obtained when homozygous WT PCR product was immobilized on the surface and hybridized with MT capture probe-modified Au nanoparticle (Figure 3F). A very low Au signal was obtained when WT or MT capture probe oligonucleotides were used as the target solution at PGE with only PCR blank solutions immobilized on the surface (Figure 3G). When a noncomplementary PCR product was immobilized on the PGE, a low Au signal appeared, because nonspecific binding effects were suppressed by the washing step, as described in the Experimental Section (Figure 3H). The five subsequent experiments for the detection of hybridization between the WT and MT capture probes and the target DNA from six real samples gave reproducible results. The Au signal obtained from the WT probe-modified PGE after hybridization with homozygous WT and MT real samples gave RSD values of 7.82 and 7.63%, respectively. The Au signal obtained from the hybridization of the WT probe with the heterozygous real samples yielded a RSD value of 8.14%. The Au

signal obtained from the MT probe-modified PGE after hybridization with homozygous WT and MT real samples gave RSD values of 7.56 and 7.48%, respectively. The Au signal obtained from the hybridization of the MT probe with the heterozygous real samples yielded a RSD value of 8.43%. The results obtained from the real PCR samples are given in vertical bar charts in Figure 3, where each bar shows the mean and the standard deviation of the signal values obtained with five replicates of the genosensor. In the meantime, an electrophoresis of the amplified samples was performed in the Department of Medicinal Biology to check the compatibility of the electrochemical test results for the amplified samples (not shown). Electrochemical results were in good agreement with the test results obtained from the conventional detection method. Further work is in progress in our laboratory to achieve 100% accuracy with the results obtained from the reference method. CONCLUSIONS The objective in DNA genosensor research is to make them cheaper and easier to use. In this report, the appearance of the Au signal enables the monitoring of hybridization at a disposable PGE in a simple way in a short time. Both the SAM of hexanethiolcapped probes and also the SAM of covalently attached inosinesubstituted probes on L-cysteine were found successful in the hybridization detection scheme. The genosensor is able to detect the complementary sequence in the PCR amplified real samples by using the appearance of the Au oxidation signal. The main advantage of this new protocol is its cost-effectiveness because of its lack of need for external hybridization indicators, such as carcinogenic antitumor drugs, metal complexes, and organic dyes. The PGE genosensor scheme is easy to use and portable, attributes that are the crucial properties of devices for point-ofcare tasks. The success of PGE over existing carbon electrodes is its commercial availability. Carbon strip electrodes are suitable for mass production, but expensive instrumentation with special training is required. While the preparation of the carbon strip electrodes is time-consuming, pencil graphite leads can be obtained from a local store. Several different types of pencil graphite leads are on trial in our laboratory in an effort to find the lead with the most suitable chemical surface composition. The developed method also has a sufficient detection limit for realworld analysis in regard to diagnosis. This procedure also eliminates the use of toxic chemicals such as ethidium bromide, which is commonly used in the gel electrophoresis step of the reference methods in mutation analysis. ACKNOWLEDGMENT This work has been supported by the Turkish Academy of Sciences in the framework of the Young Scientist Award Program (KAE/TUBA-GEBIP/2001-2-8). Authors acknowledge the financial support from TUBITAK (Project no. TBAG-2161) and Department of State Planning of Turkish Republic, Project No. 2002K120220.

Received for review October 8, 2002. Accepted February 28, 2003. AC026212R Analytical Chemistry, Vol. 75, No. 9, May 1, 2003

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