Enzyme-Linked Amperometric Electrochemical Genosensor Assay for

Universiti Sains Malaysia, Malaysia, Department of Analytical Chemistry, Faculty of Pharmacy, Ege University, Izmir, ... Publication Date (Web): M...
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Anal. Chem. 2008, 80, 2774-2779

Enzyme-Linked Amperometric Electrochemical Genosensor Assay for the Detection of PCR Amplicons on a Streptavidin-Treated Screen-Printed Carbon Electrode Chan Yean Yean,† Balqis Kamarudin,† Dilsat Ariksoysal Ozkan,‡ Lee Su Yin,§ Pattabhiraman Lalitha,§ Asma Ismail,⊥ Mehmet Ozsoz,*,‡ and Manickam Ravichandran*,†

Department of Medical Microbiology and Parasitology, School of Medical Sciences, Universiti Sains Malaysia, Malaysia, Department of Analytical Chemistry, Faculty of Pharmacy, Ege University, Izmir, Turkey, School of Health Sciences, Universiti Sains Malaysia, Malaysia, and Institute for Research in Molecular Medicine (INFORMM), Universiti Sains Malaysia, Malaysia

A general purpose enzyme-based amperometric electrochemical genosensor assay was developed wherein polymerase chain reaction (PCR) amplicons labeled with both biotin and fluorescein were detected with peroxidaseconjugated antifluorescein antibody on a screen-printed carbon electrode (SPCE). As a proof of principle, the response selectivity of the genosensor was evaluated using PCR amplicons derived from lolB gene of Vibrio cholerae. Factors affecting immobilization, hybridization, and nonspecific binding were optimized to maximize sensitivity and reduce assay time. On the basis of the background amperometry signals obtained from nonspecific organisms and positive signals obtained from known V. cholerae, a threshold point of 4.20 µA signal was determined as positive. Under the optimum conditions, the limit of detection (LOD) of the assay was 10 CFU/mL of V. cholerae. The overall precision of this assay was good, with the coefficient of variation (CV) being 3.7% using SPCE and intermittent pulse amperometry (IPA) as an electrochemical technique. The assay is sensitive, safe, and cost-effective when compared to conventional agarose gel electrophoresis, real-time PCR, and other enzymelinked assays for the detection of PCR amplicons. Furthermore, the use of a hand-held portable reader makes it suitable for use in the field. Over the past decade, polymerase chain reaction (PCR) has been used extensively as a diagnostic tool in various fields, such as genetic screening, infectious disease diagnosis, forensics, environmental monitoring, and veterinary science. PCR is an enzymatic process for amplifying specific regions of DNA in vitro. * To whom correspondence should be addressed. Phone: +609-7664592 (M.R.), +90 232 388 40 00 ext 1353 (M.O.). Fax: +609-7641615 (M.R.), +90 232 388 (M.O.). E-mail: [email protected] (M.R.), [email protected] (M.O.). † School of Medical Sciences, Universiti Sains Malaysia. ‡ Ege University. § School of Health Sciences, Universiti Sains Malaysia. ⊥ Institute for Research in Molecular Medicine (INFORMM), Universiti Sains Malaysia.

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This process amplifies the target DNA exponentially to generate billions of copies of target DNA from a single copy in less than 1 h.1 Conventional detection of PCR amplicons by agarose gel electrophoresis exposes the users to hazardous elements such as ethidium bromide and ultraviolet light. Other safer detection techniques, such as capillary blotting and enzyme-linked immunoassays, require specialized equipment and multiple hybridization and washing steps, which are labor-intensive and time-consuming. Newer methods such as real-time PCR and Taqman probe are faster; however, these methods are homogeneous wherein specialized stains, probes, and specialized expensive equipment are required for the detection of PCR amplicons.2 The introduction of electrochemical DNA biosensors made it possible for researchers to develop new methods for detecting nucleic acids in an inexpensive, faster, and safer way.3-6 In addition, electrochemical biosensors are highly sensitive, specific, versatile, and compatible with microfabrication technology.3,5 DNA biosensors work by converting hybridization events to analytical signals through the use of a transducer. Electrochemical DNA biosensors (or genosensors) offer greater promise for obtaining information through a variety of signal detection techniques. These can involve direct electrochemical DNA analysis based on a guanine signal (label-free)7-11 or an electrocatalytic mechanism (label-based) employing methylene (1) Saiki, R. K.; Gelfand, D. H.; Stoffel, S.; Scharf, S. J.; Higuchi, R.; Horn, G. T.; Mullis, K. B.; Erlich, H. A. Science 1988, 239, 487-491. (2) Hayden, R. T. Molecular Microbiology: Diagnostic Principles and Practice (In Vitro Nucleic Acid Amplification Techniques); ASM Press: Washington, DC, 2004. (3) Huang, T. J.; Liu, M.; Knight, L. D.; Grody, W. W.; Miller, J. F.; Ho, C. M. Nucleic Acids Res. 2002, 30, e55. (4) Aitichou, M.; Henkens, R.; Sultana, A. M.; Ulrich, R. G.; Sofi Ibrahim, M. Mol. Cell. Probes 2004, 18, 373-377. (5) Sun, C. P.; Liao, J. C.; Zhang, Y. H.; Gau, V.; Mastali, M.; Babbitt, J. T.; Grundfest, W. S.; Churchill, B. M.; McCabe, E. R.; Haake, D. A. Mol. Genet. Metab. 2005, 84, 90-99. (6) Liao, J. C.; Mastali, M.; Gau, V.; Suchard, M. A.; Moller, A. K.; Bruckner, D. A.; Babbitt, J. T.; Li, Y.; Gornbein, J.; Landaw, E. M.; McCabe, E. R.; Churchill, B. M.; Haake, D. A. J. Clin. Microbiol. 2006, 44, 561-570. (7) Ozkan, D.; Erdem, A.; Kara, P.; Kerman, K.; Meric, B.; Hassmann, J.; Ozsoz, M. Anal. Chem. 2002, 74, 5931-5936. (8) Ariksoysal, D. O.; Karadeniz, H.; Erdem, A.; Sengonul, A.; Sayiner, A. A.; Ozsoz, M. Anal. Chem. 2005, 77, 4908-4917. 10.1021/ac702333x CCC: $40.75

© 2008 American Chemical Society Published on Web 03/01/2008

blue (MB) as an intercalator;12 meldola blue (MDB), an intercalator that is an effective electron acceptor for the enzyme-based biosensor;13 and alkaline phosphatase14-16 or horseradish peroxidase (HRP)17,18 as an enzyme catalyzing oxidation signal amplification.4,14,19,20 In this paper, we describe, for the first time, the development of a sensitive, cost-effective, fast, and accurate enzyme-based electrochemical genosensor using a screen-printed carbon electrode (SPCE) and a portable reader for the detection of the PCR amplicons. As a proof of principle, the response selectivity of the genosensor was evaluated using PCR amplicons derived from lolB gene of Vibrio cholerae. PCR amplicons labeled with both biotin and fluorescein via modified primers were bound with HRPconjugated antifluorescein antibodies. The oxidation signal of the hydrogen peroxide (H2O2) substrate was detected using intermittent pulse amperometry (IPA) on a streptavidin-modified SPCE. Features of this optimized protocol for the detection of PCR amplicons obtained from lolB gene of V. cholerae are discussed. MATERIALS AND METHODS Apparatus. Intermittent pulse amperometry assays were performed with a portable pulse amperometric reader (AndCare, Durham, NC). SPCEs were designed in our laboratory and fabricated by a local company (ScrintTechnology, Penang, Malaysia). The disposable miniature SPCE consisted of a threeelectrode configuration (15 mm × 30 mm), which comprised a round-ended working electrode (4 mm in diameter), counter electrode, and silver pseudoreference electrode printed on a polycarbonate support. A ring-shaped insulating layer around the round-ended working electrode (8 mm × 8 mm) with a capacity of 100 µL was incorporated onto the SPCE as an electrochemical cell (reservoir). The preliminary electrochemical performance of the newly designed SPCE was first tested using cyclic voltammetry (staircase) with 1 mM potassium ferricyanide, K3Fe(CN)6, and the signal was measured with a µAutolab PGSTAT III potentiostat/ galvanostat (Eco Chemie, The Netherlands) interfaced to GPES 4.5 software. The reproducibility of the SPCE results was determined by calculating the relative standard deviation (RSD) of the measurements (n ) 3). Materials. The chemicals used in the study were sodium dodecyl sulfate (SDS), 1× salt sodium citrate (SSC) (150 mM NaCl (9) Kara, P.; Ozkan, D.; Erdem, A.; Kerman, K.; Pehlivan, S.; Ozkinay, F.; Unuvar, D.; Itirli, G.; Ozsoz, M. Clin. Chim. Acta 2003, 336, 57-64. (10) Ozkan, D.; Karadeniz, H.; Erdem, A.; Mascini, M.; Ozsoz, M. J. Pharm. Biomed. Anal. 2004, 35, 905-912. (11) Eskiocak, U.; Ozkan-Ariksoysal, D.; Ozsoz, M.; Oktem, H. A. Anal. Chem. 2007, 79, 8807-8811. (12) Ozkan, D.; Kara, P.; Kerman, K.; Meric, B.; Erdem, A.; Jelen, F.; Nielsen, P. E.; Ozsoz, M. Bioelectrochemistry 2002, 58, 119-126. (13) Kara, P. M. B.; Zeytinoglu, A.; Ozsoz, M. Anal. Chim. Acta 2004, 518, 6976. (14) Diaz-Gonzalez, M.; Gonzalez-Garcia, M. B.; Costa-Garcia, A. Biosens. Bioelectron. 2005, 20, 2035-2043. (15) Das, J.; Jo, K.; Lee, J. W.; Yang, H. Anal. Chem. 2007, 79, 2790-2796. (16) Miroslav Fojta, P. B.; Cahova, K.; Peinka, P. Electroanalysis 2006, 18, 141151. (17) Williams, E.; Pividori, M. I.; Merkoci, A.; Forster, R. J.; Alegret, S. Biosens. Bioelectron. 2003, 19, 165-175. (18) Azek, F.; Grossiord, C.; Joannes, M.; Limoges, B.; Brossier, P. Anal. Biochem. 2000, 284, 107-113. (19) Carpini, G.; Lucarelli, F.; Marrazza, G.; Mascini, M. Biosens. Bioelectron. 2004, 20, 167-175. (20) Caruana, D. J.; Heller, A. J. Am. Chem. Soc. 1999, 121, 769-774.

and 15 mM C3H5Na3O7; pH 7.4), ethanolamine chloride, streptavidin, bovine serum albumin (BSA), N-hydroxy succinimide (NHS), and 1-ethyl-3-(3-dimethyl-aminopropyl) carbodiimide hydrochloride (EDC), all which were purchased from Sigma (Saint Louis, MO); 3,3′5,5′-tetramethylbenzidine (TMB) and hydrogen peroxide (H2O2) were purchased from BioRad (Hercules, CA), and HRP-conjugated antifluorescein monoclonal antibodies were purchased from Roche (Basel, Switzerland). The washing buffer used was 0.05 M phosphate buffer (0.04 M K2HPO4, 0.01 M KH2PO4, 20 mM NaCl, pH 7.4) containing 0.5% (w/v) SDS. Deionized water was used for preparation of all solutions. Modified primers were custom-synthesized by First Base (Shah Alam, Malaysia), and the sequences are given below. (i) 5′ Biotin-modified primer, VHMFP (B): biotin-5′-GTT GAA CGA TTC TCG CTG ATC-3′. (ii) 5′ Fluorescein-modified primer, VHA-AS5 (F): fluorescein5′-CAA TCA CAC CAA GTC ACT C-3′. Bacterial Strains. Analytical sensitivity and specificity of the assay were tested using a panel of 10 positive controls consisting of V. cholerae strains and negative controls consisting of V. mimicus, V. parahaemolyticus, V. fluvialis, V. furnissii, V. vulnificus, Streptococcus sp. group A, Streptococcus sp. group B, Streptococcus sp. group G, Enterococcus faecium, Staphylococcus aureus, Shigella flexneri, Shigella boydii, Shigella sonnei, Salmonella typhi, Salmonella enteritidis, Escherichia coli enteroinvasive (EIEC), Escherichia coli enterotoxigenic (ETEC), Klebsiella pneumoniae, Pseudomonas aeruginosa, and Proteus mirabilis. These bacterial strains were obtained from the culture collections of the Department of Medical Microbiology and Parasitology, Universiti Sains Malaysia, Malaysia; Institute of Medical Research, Malaysia; Communicable Diseases Hospital, Thandiarpettai, Chennai, India; and Centre for Southeast Asia Studies, Kyoto University, Japan. Methods. Preparation of Bacterial Strains for PCR Amplification. DNA sample from bacterial strains were prepared by the boiling method. A single colony of each bacterial strain was inoculated into 5 mL of alkaline peptone water (APW) selectiveenrichment medium (1% (w/v) peptone, 1% (w/v) NaCl, pH 8.6) and incubated for 6 h at 37 °C. One milliliter of the culture was then harvested by centrifugation at 10 000g for 3 min at room temperature. The supernatant was discarded, and the pellet was washed once with saline solution. Next, the pellet was resuspended in 200 µL of saline solution and boiled for 10 min. After boiling, the lysate was centrifuged at 10 000g for 3 min and 2 µL of the supernatant was used for PCR amplification. For determination of the limit of detection (LOD), a single colony of V. cholerae O1 serogroup was inoculated into a LuriaBertani (LB) broth and incubated at 37 °C for overnight. The concentration was determined by measuring the optical density at a wavelength of 600 nm. The culture concentration (OD ∼ 2.0) was adjusted to 109 CFU/mL in LB broth. From this culture, 10fold serial dilutions were made, starting from 106 to 10 CFU/mL concentrations. From each dilution, 200 µL was used for preparation of the DNA template by the boiling method, whereas 100 µL was used for determination of viable count by plating on LB agar. PCR Amplification. PCR amplification was performed using VHMFP (B) as a forward primer and VHA-AS5 (F) a reverse primer. The primers were designed based on the lolB gene of V. cholerae (GenBank accession no. AF227752). The lolB gene PCR Analytical Chemistry, Vol. 80, No. 8, April 15, 2008

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Figure 1. Schematic representation of steps involved in (A) immobilization of streptavidin on SPCE and (B) the amperometric detection of biotin- and fluorescein-labeled PCR amplicons on a streptavidin-modified SPCE.

assay was carried out in 20 µL reaction volume containing 1× PCR buffer (Fermentas, Lithuania), 2.5 mM MgCl2 (Fermentas, Lithuania), 160 µM dNTPs (Fermentas, Lithuania), 1.0 pmol/µL of each forward and reverse modified primer, 0.75 U of Taq DNA polymerase (Fermentas, Lithuania), and 2 µL of DNA template. Thermal cycling consisted of one cycle at 95 °C for 5 min, 30 cycles at 95 °C for 30 s, 60 °C for 30 s, and 72 °C for 30 s, followed by one final extension cycle at 72 °C for 5 min on a thermal cycler (PTC-200, MJ Research, Waltham, MA). Agarose Gel Electrophoresis Detection of PCR Amplicons. A volume of 10 µL of the PCR amplicons was resolved by 2% (w/v) agarose gel containing ethidium bromide and was electrophorized at 80 V for 30 min. The results were compared with those obtained with the electrochemical genosensor assay. Amperometric Detection of PCR Amplicons. Pretreatment of the Electrode. Electrodes were pretreated with streptavidin before each experiment. First, 2.5 µL of covalent agent (200 mM EDC and 50 mM NHS prepared in 0.05 M phosphate buffer) was added onto the working electrode of the SPCE and incubated for 10 min at room temperature. The electrodes were washed by dipping once in 5 mL of deionized water. Then, 5 µL of 0.05 mg/ mL streptavidin was pipetted onto the working electrode again to form a meniscus and incubated at room temperature for 10 min. The electrodes were washed by dipping once in 5 mL of deionized water (schematic diagram is shown in Figure 1A). The unbound area on the streptavidin-treated SPCE reservoir area was blocked with 100 µL of 1 M ethanolamine chloride and incubated for 10 min in the dark. The electrodes were washed by dipping once in 5 mL of deionized water. Next, 100 µL of 4% (w/ v) BSA was added to the reservoir area to prevent nonspecific binding of antifluorescein-HRP and incubated for 10 min. Then, the electrodes were washed by dipping once in 5 mL of deionized water. 2776

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Capture of PCR Amplicons onto Electrodes. All experiments were carried out at room temperature and in duplicate. The capture of PCR amplicons onto electrodes and its detection was carried out as described by AndCare company protocol with some modifications. The schematic diagram of detection is shown in Figure 1B. Briefly, the biotin- and fluorescein-labeled PCR amplicons were diluted with an equal volume of 0.05 M phosphate buffer, and 5 µL of the diluted PCR amplicons was applied onto the surface of the working electrode for 5 min. During the incubation step, the biotin-labeled strands of the PCR amplicons were specifically captured onto the streptavidin-precoated working electrode. The excess PCR amplicons were removed from the electrode by absorbing it with a tissue paper and was washed by dipping 10 times in 5 mL of 0.1× SSC containing 0.5% SDS. After the washing step, the SPCE was incubated for 5 min with 5 µL of 1:200 diluted HRP-conjugated antifluorescein antibody. During this step, the antibody binds to the fluorescein-labeled strand of the PCR amplicons. Next, the SPCE was washed by dipping 10 times in 5 mL of 0.1× SSC containing 0.5% SDS. Signal Detection Using the Portable Reader. HRP substrate containing a mixture of TMB and H2O2 in a 1:10 ratio was prepared, and 100 µL of this substrate mixture was applied onto the SPCE reservoir area to cover the working, counter, and reference electrodes. The enzymatic reaction occurring on the working electrode was detected using a portable pulse amperometric reader. The reader used IPA, in which there is a 15 s incubation period followed by an applied potential of -0.1 V (vs a silver pseudoreference electrode) with a measurement time of 10 s and pulse time of 10 s, at a frequency of 5 Hz and a current range of 10 µA. Data Analysis. Precision or the RSD of the genosensor assay was calculated using the current response obtained from the serially diluted bacterial culture (n ) 4). A threshold value was

Figure 2. Analytical specificity evaluation using positive and negative reference strains. (A) Agarose gel electrophoresis detection of biotinand fluorescein-labeled V. cholerae lolB gene PCR amplicons. (B) Amperometric detection of biotin- and fluorescein-labeled V. cholerae lolB gene PCR amplicons using the portable reader.

calculated from the electrochemical signals of the positive and negative reference strains, (1 SD, to determine positivity and negativity. Regression analysis was used to evaluate the relation of CFU and amperometric signal. RESULTS AND DISCUSSION In the present study a novel SPCE for the detection of PCR product by rapid electrochemical method was developed as an alternative method to agarose gel analysis. The efficiency and specificity of SPCE-based electrochemical assay was studied using V. cholerae as a model organism. The PCR was carried out using primers specific for V. cholerae lolB gene. The electrochemical performance of the newly designed SPCE without streptavidin coating was evaluated based on the ferricyanide redox activity, using cyclic voltammetry with a µAutolab PGSTAT III potentiostat/galvanostat. Cyclic voltammogram data showed that the RSD for triplicate measurements was 3.3%, indicating that the SPCE was able to give reproducible results. The PCR assay for V. cholerae lolB gene was carried out using the modified primers. The assay samples included 10 positive reference V. cholerae strains comprised of three O1 and seven O139 serogroups, while 21 other Vibrio sp. and enteric pathogens were also included as negative controls. All the 10 V. cholerae positive reference strains yielded an expected PCR amplicons of 95 bp in size when analyzed by agarose gel electrophoresis (Figure 2A). Seven of the 21 negative control samples showed high molecular weight nonspecific PCR product by agarose gel electrophoresis (Figure 2A). For the genosensor detection of PCR product, the labeled amplicons were captured on a streptavidincoated SPCE surface and detected using H2O2 substrate and TMB as mediator. The amperometric signal generated was measured using a portable hand-held digital device (AndCare, Durham, NC). The electrochemical genosensor results were compared with agarose gel results. We found that all the 10 positive controls gave

an amperometric signal above 4.20 µA, whereas all the negative controls showed an amperometric signal below 4.20 µA as shown in Figure 2B. On the basis of these results, a threshold value for positive result was set as 4.20 ( 0.17 µA. The amperometric signal with or above this threshold value was interpreted as positive, whereas any value less than the threshold value was interpreted as negative. Analytical specificity is defined as the method’s ability to obtain negative results in concordance with negative results obtained by the reference method.21 With the use of 4.20 ( 0.17 µA as a threshold point for interpretation of results, the analytical specificity of the electrochemical genosensor assay was 100%. Determination of a threshold value facilitates the objective discrimination of positive and negative results, thus enhancing the specificity of the genosensor assay. Interpretation of the PCR result by agarose gel electrophoresis may be misleading in the presence of nonspecific bands with molecular weight close to the expected amplicon size. In this study, high molecular weight nonspecific bands were observed in seven of the negative samples. These nonspecific amplicons gave a background signal below 4.20 µA, which could be due to inefficient capture of larger amplicons on the SPCE due to its secondary structures or due to false priming of two biotin-modified forward primers or two fluoresceinmodified reverse primers. The LOD of electrochemical assay was evaluated using PCR amplicon obtained by varying dilution of V. cholerae culture (serially diluted 106 to 1 CFU/mL). The amperometric signals were greater than 4.20 µA for all dilution of V. cholerae from 106 to 10 CFU/mL, whereas it was less than 4.20 µA for 1 CFU/mL and PCR negative sample (Figure 3B). Thus the LOD for electrochemical genosensor was 10 CFU/mL compared to 100 CFU/mL by agarose gel electrophoresis. These data clearly show (21) Molecular Diagnostic Methods for Infectious Diseases; Approved Guideline; CLSI MM3-A2, 2nd ed.; Clinical and Laboratory Standards Institute: Wayne, PA, 2006.

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Figure 3. Comparison of detection limit of V. cholerae lolB gene PCR assay using varying dilutions of V. cholerae cultures. (A) Agarose gel electrophoresis detection of biotin- and fluorescein-labeled V. cholerae lolB gene PCR amplicons. (B) Amperometric detection of biotin- and fluorescein-labeled V. cholerae lolB gene PCR amplicons using the portable reader. (C) Regression analysis of current measurements (n ) 4) at different concentrations (CFU/mL) of V. cholerae with the RSD value of 1.8-3.7%.

that the genosensor method of detection is more sensitive than agarose gel electrophoresis (Figure 3A). The precision of the V. cholerae lolB genosensor assay was determined by calculating the RSD value of the replicates (n ) 4) for each CFU/mL concentration. An overall RSD value of less than 3.7% for each concentration showed that the assay results are consistent and reproducible (Figure 3C). The regression analysis showed a linear correlation between the number of cells and amperometric signal (r2 ) 0.86) as shown in Figure 3C. Conventionally, PCR amplicons are detected by agarose gel electrophoresis which takes 45 min to 1 h and utilizes harmful agents such as UV light and ethidium bromide. PCR amplicons are also detected in the presence of expensive chemicals such as SYBR green dye, Taqman, or molecular beacons using specialized instruments (real-time PCR machine). As an alternate method for PCR amplicon detection, many enzyme-based electrochemical genosensor assays have been developed and have shown promising results.4,6,14,19 But most of these electrochemical genosensors have a drawback in the sense that it requires an extra hybridization step with the probe before the detection of PCR amplicon signal. A powerful alternative technique has been designed in our present study. We have eliminated the hybridization step by labeling the PCR amplicon with both biotin and fluorescein via modified primers. The PCR amplicon is directly applied on the modified SPCE, and the HRP-enzyme reaction is read within 15 s. Among the chemicals used in this genosensor assay, only EDC is known to be an irritant to the respiratory system and skin. The cost of this genosensor assay is comparatively cheaper than conventional agarose gel electrophoresis and optical methods. 2778 Analytical Chemistry, Vol. 80, No. 8, April 15, 2008

Hence, in the present assay we have eliminated the two steps that are normally carried out on a conventional electrochemical genosensor assay, which are denaturation of PCR amplicon and hybridization. Here, we only immobilize the biotin- and fluoresceinlabeled PCR amplicon on the streptavidin-modified carbon working electrode of SPCE for 5 min, followed by addition of HRPconjugated antifluorescein antibody for 5 min, and then detect the amperometric signal directly. Electrochemical enzyme-based biosensor techniques can be employed for DNA and immunoassays (antigen-antibody) based on amperometry methods.4,6,14,22 Although the SPCE was designed for DNA detection, it can also be used for detection of any bacteria using antigen-antibody interaction. Rao et al.22 reported an antibody-based V. cholerae electrochemical biosensor assay using alkaline phosphatase (AP). But the lowest detection limit was around 105 CFU/mL, which is much less sensitive than the DNA genosensor. More over, this assay uses AP enzyme system for detection which takes more time (10 min) to read the oxidation signals. Specific primer sequences are the critical factors that need to be changed in this genosensor to detect other organisms. The optimized genosensor procedure used in this study is unique and universal in that it can detect biotin- and fluorescein-labeled PCR amplicon derived from any organisms or mutant gene to allow early and precise diagnosis. Although the use of a portable reader made this genosensor field adaptable, the PCR process needs to be carried out in a (22) Rao, V. K.; Sharma, M. K.; Goel, A. K.; Singh, L.; Sekhar, K. Anal. Sci. 2006, 22, 1207-1211.

refrigerated condition in a laboratory and the multiple washing steps also require time and reagents. Currently, we are in the process of utilizing our dry-reagent-based reaction formulation23 and lateral flow technology to overcome the refrigeration requirement and tedious washing steps. CONCLUSION The newly modified electrochemical genosensor assay reported in this study is highly sensitive, specific, and gave reproducible results. In addition, the assay is fast and user-friendly, making it a good alternative to agarose gel analysis and enzymelinked assay. The use of a portable reader makes it suitable not only for laboratories but also for use in point-of-care hospital settings, remote settings, or in fields. Besides V. cholerae, this (23) Aziah, I.; Ravichandran, M.; Ismail, A. Diagn. Microbiol. Infect. Dis. 2007, 59, 373-377.

assay methodology can be adopted for the detection of any organism or mutant genes. ACKNOWLEDGMENT This work was funded by the National Biotechnology Directorate (NBD) Top down Project of the Ministry of Science, Technology and Innovation (MOSTI), Malaysia. D.A.O. and M.O. acknowledge financial support from TUBITAK (Projects SBAG105S484 and SBAG-107S163) and the European Project under the Sixth Framework Programme Prioty 5 Food Quality and Safety (Contract No. Detectox, 514055).

Received for review November 13, 2007. Accepted January 14, 2008. AC702333X

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