Hybridization Assay at a Disposable Electrochemical ... - ScienceDirect

Analytical Biochemistry 284, 107–113 (2000) doi:10.1006/abio.2000.4692, available online at http://www.idealibrary.com on

Hybridization Assay at a Disposable Electrochemical Biosensor for the Attomole Detection of Amplified Human Cytomegalovirus DNA F. Azek,* C. Grossiord,* M. Joannes,† B. Limoges,‡ ,1 and P. Brossier* ,1 *Laboratoire de Microbiologie Me´dicale et Mole´culaire, Faculte´ de Me´decine et de Pharmacie, 7 Boulevard Jeanne d’Arc, 21033 Dijon, France; †Arge`ne-Biosoft, Parc Technologique Delta-Sud, 09120 Varilhes, France; and ‡Equipe Electrosynthe`se et Electroanalyse Bioorganique, UMR CNRS 6504, Universite´ Blaise Pascal de Clermont-Ferrand, 24 Avenue des Landais, 63177 Aubie`re, France

Received March 21, 2000

A disposable electrochemical biosensor for the detection of DNA sequences related to the human cytomegalovirus (HCMV) is described. The sensor relies on the adsorption of an amplified human cytomegalovirus DNA strand onto the sensing surface of a screen-printed carbon electrode, and to its hybridization to a complementary single-stranded biotinylated DNA probe. The extent of hybrids formed was determined with streptavidin conjugated to horseradish peroxidase. The peroxidase label was indirectly quantified by measuring the amount of the chromophore and electroactive product 2,2ⴕ-diaminoazobenzene generated from the ophenylenediamine substrate. The intensity of differential pulse voltammetric peak currents resulting from the reduction of the enzyme-generated product was related to the number of target HCMV-amplified DNA molecules present in the sample, and the results were compared to those obtained with standard methods, i.e., agarose gel electrophoresis quantification and colorimetric hybridization assay in a microtiter plate. A detection limit of 0.6 amol/ml of HCMV-amplified DNA fragment was obtained with the electrochemical DNA biosensor. The electrochemical method was 23,000-fold more sensitive than the gel electrophoresis technique and 83-fold more sensitive than the colorimetric hybridization assay in a microtiter plate. © 2000 Academic Press

Nucleic acid amplification techniques dramatically increase sensitivity while still retaining a high specificity. 1

To whom correspondence should be addressed.

0003-2697/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.

The polymerase chain reaction (PCR)2 is the best-developed and most widely used method of nucleic acid amplification (1–3). It has opened new avenues of pathogen detection, often supporting or replacing the traditional laborious methods in the field of virology (4 – 6). The amplified products (amplicons) can be characterized and quantified by various methods, including electrophoretic separation of the amplified DNA, capillary electrophoresis, and nucleic acid hybridization with a labeled probe. The latter method is widely used in medical diagnostics and it usually relies on heterogeneous hybridization assays based on radioactive, fluorescent, chemiluminescent, or enzyme labels (3, 7). More recently, kinetic PCR was proposed as a homogeneous real-time hybridization technique where quantification is done without opening the reaction tube by monitoring a fluorescent signal generated during the PCR (8 –10). The main advantage of this approach is that it does not require an additional quantification step after amplification. However, special fluorescent-labeled probes combined with expensive equipment are needed, and a high number of PCR cycles is generally required to obtain a sufficient amount of detectable product. While many DNA hybridization assays are suitable for diagnostic laboratories, faster, lower cost, easier-touse, and more sensitive approaches are highly desired, especially in the case of decentralized screening of infectious diseases. In this context, DNA electrochemical sensors with the nucleic acids directly immobilized on 2

Abbreviations used: PCR, polymerase chain reaction; SPEs, screenprinted carbon electrodes; OPD, o-phenylenediamine; DPV, differential pulse voltammetry; HCMV DNA, human cytomegalovirus DNA; EBV, Epstein-Barr virus; HCV, hepatitis C virus; PBS, phosphate-buffered saline; HRP, horseradish peroxidase; DAA, 2,2⬘-diaminoazobenzene. 107

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an electrode surface for hybridization offer innovative routes (11–13). Various electrochemical detection approaches were studied. For example, DNA electrochemical sensors based on redox intercalators and electroactive groove binders have been developed (14 –17). However, these markers do not fully discriminate between single- and double-stranded DNA, and the variations of the electrochemical signals are low. A more selective response can be reached with a ferrocenelabeled oligonucleotide probe (18 –20) but the sensitivity remains low. The detection limit can be improved with the use of an enzyme label involved in a bioelectrocatalytic reaction (21–23). Using such an approach, Heller and colleagues were able to determine amperometrically the hybridization of 10 5 copies of a simple 25–30 base single-stranded DNA with a 7-␮m-diameter microelectrode, using a peroxidase-labeled oligonucleotide probe (22). The main problem associated with the electrochemical DNA sensors mentioned so far is their high-cost compared to the traditional spectrophotometric measurements in microtiter plates. Consequently, there is a growing need for developing inexpensive miniaturized sensing devices for electrochemical nucleic acid analysis. Both photolithography and screen-printing technology, adapted from the microelectronics industry, represent attractive approaches, since they allow mass production of low-cost electrochemical sensors (24 –27). A disposable gold electrode obtained by photolithography and coated by a self-assembled monolayer of DNA was recently investigated for the determination of hepatitis B virus (24). The response increased with the concentration over the 10 4–10 6 copies/ml range, using a redox-active intercalator. In this paper, disposable screen-printed carbon electrodes (SPEs) were investigated as a low-cost DNA sensor on which the target DNA could be adsorbed and hybridized with a biotinylated DNA probe. Hybrids were then indirectly determined using a streptavidinperoxidase label that converted the o-phenylenediamine (OPD) substrate into the chromophore and electroactive product 2,2⬘-diaminoazobenzene, which could be thus determined by absorption spectrophotometry and differential pulse voltammetry (DPV). The hydridization assay was applied for the determination of amplified human cytomegalovirus DNA sequences (HCMV DNA), and the analytical performance of the electrochemical DNA sensor was discussed and compared to the classical spectrophotometric measurements in microtiter plates. MATERIALS AND METHODS

Equipment A Labsystem Multiscan spectrophotometer was employed to determine optical densities. Polypropylene

and polystyrene microtiter plates were obtained from Nunc. Screen-printed carbon electrodes were prepared from a home-made polystyrene-based carbon ink (2:3 mixture of polystyrene and graphite particle in mesitylene) printed on a polyester film as previously described (28). Each SPE consisted of a disk area (9.6 mm 2 diameter), a conductive track (20 ⫻ 1 mm), and a square extremity (25 mm 2) for the electrical contact. An insulator layer was spread manually over the conductive track, leaving the sensing disk area ready for DNA coating. A platinum and a Ag/AgCl wire were used as counterelectrode and pseudoreference electrode. A ␮-Autolab potentiostat (EcoChemie) interfaced to a IBM 330 PC with a GPSE version 3 software (EcoChemie) was used for DPV, which involved a pulse height of 25 mV, a step potential of 5 mV, a pulse width of 0.05 s, and an interval time between two pulses of 0.5 s. Reagents and Buffers Oligonucleotide primers (AC1 and AC2) and a biotinylated target-specific probe (AC3) were obtained from Arge`ne-Biosoft (Varilhes, France). Taq polymerase, Taq polymerase buffer (10 mM Tris, 50 mM KCl, 1.5 mM MgCl 2, pH 8.3), and the four nucleic acid bases (dNTPs) were purchased from Pharmacia-Amersham. DNA ladder for quantification was obtained from Gibco-BRL-Life technology. The Hybridowell kit and the controls consisting of amplified Epstein-Barr virus DNA (EBV), hepatitis C virus DNA (HCV), and human ETS2 gene DNA were provided by Arge`ne-Biosoft. The solutions in the Hybridowell kit and the coating solution used for the hybridization assay are a proprietary of Arge`ne-Biosoft. Phosphate-buffered saline (PBS, 5 mM Na 2HPO 4, 2 H 2O adjusted to pH 8.0 with HCl) and citrate buffer (CB, 90 mM trisodium citrate containing 0.9 M NaCl and adjusted to pH 5.0 with HCl) were prepared using deionized and doubly distilled water. The substrate solution containing o-phenylenediamine was prepared by dissolving a 3-mg OPD tablet (Arge`ne-Biosoft) in 10 ml buffer containing 40 mM citric acid, 150 mM Na 2HPO 4, 0.02% H 2O 2, and 5 mM NaCl (pH 5.0). HCMV DNA Extraction HCMV DNA was extracted from human embryogenic lung fibroblastic (MRC5) line cells infected by the AD169 strain, with an Invisorb DNA extraction kit purchased from Invitek. Following the manufacturer’s protocol, 10 5 to 10 6 infected cells were washed and centrifuged twice with PBS buffer, and then 500 ␮l of lysis solution was added to the cell pellets, vortexed, and left at room temperature for 5 min. Then 15 ␮l of carrier was added, vortexed briefly, and incubated on

ELECTROCHEMICAL DETECTION OF CYTOMEGALOVIRUS DNA

ice for 5 min. After 10,000 rpm centrifugation, the supernatant was carefully discarded, and the pellet was washed three times with 1 ml of washing buffer and centrifuged. The pellet was dried in a vacuum desiccator to eliminate residual ethanol, resuspended into 80 ␮l elution buffer, and incubated 5 min at 60°C. The tubes were then full-speed centrifuged for 2 min and the DNA-containing supernatant was transferred into a fresh tube and stored at ⫺20°C until PCR amplification. PCR Amplification of HCMV DNA The AC1 and AC2 primers (25 nucleotides length) were selected to amplify a 406-bp HCMV DNA fragment from a highly conserved region located in the HindIII “X” region of the cytomegalovirus genome US fragment (29). PCRs performed in 100 ␮l of Taq polymerase buffer (1⫻) containing 200 ␮M of each of the four dNTPs, 400 nM of each oligonucleotide primers, and 10 units/ml of Taq polymerase. Reaction mixtures were overlaid with 100 ␮l of paraffin oil to prevent evaporation, and 10 ␮l of the target DNA extracted from cells or clinical samples was added through paraffin. The tubes were placed in the thermocycler (Genetech, Model SPCR 1) and subjected to the following thermal cycling: 92°C denaturation for 7 min in the first cycle, 92°C denaturation for 15 s, 55°C anneal for 30 s, and 72°C extension for 30 s for all subsequent 34 cycles, followed by a 72°C extension for 2 min in the last cycle. A negative control that contains all of the reagents except the DNA matrix was included in each series. After cycling, 2 ␮l of 10 mM EDTA was added and the PCR-amplified products were stored at ⫺20°C until their analysis was carried out by hybridization assays or gel electrophoresis using the protocols described below. Agarose Gel Electrophoresis Quantification The concentrated solution of amplified HCMV DNA previously extracted from cells and amplified by PCR as described above was serial and separated by gel electrophoresis on an agarose gel stained with ethidium bromide. The DNA bands were visualized by UV fluorescence (Biocom) and quantified by densitometry (a standardized DNA ladder was used for calibration). The concentration of the concentrated solution of amplified HCMV DNA was 10.5 pmol/ml corresponding to 6.3 ⫻ 10 12 copies/ml. Preparation of a Concentration Range of Amplified HCMV DNA A concentration range of amplified HCMV DNA (6.3 ⫻ 10 4– 6.3 ⫻ 10 12 copies/ml) was prepared by serial dilution of the concentrated solution of amplified

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HCMV DNA (6.3 ⫻ 10 12 copies/ml) in the negative control of the PCR. The same concentrated solution of amplified HCMV DNA was used to prepare all of the standard concentrations employed in this work. SPE-Based Electrochemical and Colorimetric Hybridization Assays The HCMV DNA sequence detection basically consisted of four steps: target DNA immobilization, probe hybridization, enzyme label binding, and voltammetric or colorimetric detection of the enzymegenerated product. The procedure was an adaptation of the Hybridowell kit protocol, and consequently its reagents (denaturation solutions, hybridization medium, streptavidine-peroxidase conjugate (50⫻)) and buffers (washing solution (10⫻), negative control, conjugate diluent) were used. In a propylene tube, 2 ␮l of amplified HCMV DNA was denatured in alkaline media for 10 min at room temperature by the addition of 30 ␮l of each of the two denaturation solutions. The resulting mixture was 10fold diluted with the coating solution, and the sensing disk area of a SPE was immersed in 100 ␮l of this solution. After incubation at 37°C overnight, the electrodes were removed, rinsed with distilled water, and incubated 30 min at 37°C in the hybridization medium containing 100 ng/ml of AC3 biotinylated probe. Next, they were subjected to a washing cycle consisting of five incubations for 1 min in 500 ␮l of washing solution (1⫻). Subsequently, SPEs were incubated in 100 ␮l of streptavidin-peroxidase conjugate (2⫻, i.e., the 50⫻ streptavidin-peroxidase conjugate solution was 25-fold diluted with the conjugate diluent) for 15 min at room temperature and immediately followed by a washing cycle as described above. The sensing disk area of the SPE was then immersed in 50 ␮l of OPD substrate solution unless otherwise stated, and incubated for 30 min at room temperature in the dark. The water-soluble and colored electroactive product 2,2⬘-diaminoazobenzene generated within the solution was then determined by both DPV and absorption spectrophotometry. In the latter case, the electrodes were removed and the wells were read at 492 nm. Each series of experiments included two SPEs coated with a PCR-amplified negative control (containing all of the reagents, except DNA) and two SPEs coated with the negative control of the Hybridowell kit. With the aim of a comparative study, ratios of the current or optical density values to the blank response were calculated, and samples were considered positive for a signal to blank response ratio ⬎2. The standard deviation calculated from duplicate negative control measurements was selected as the blank response.

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FIG. 1. Scheme of the electrochemical DNA hybridization assay on a screen-printed electrode.

Microtiter Plate-Based Colorimetric Hybridization Assay A Hybridowell kit was used for the colorimetric hybridization assay of the amplified target DNA in microtiter plates. The assay was performed following the manufacturer’s procedure, which is similar to the protocol described for the SPE-based hybridization assays. Determination of the Specificity The specificity of the hybridization assay at the SPE surface was evaluated by replacing the adsorbed HCMV DNA by three other amplified DNA fragments, i.e., a Epstein-Barr virus DNA, a hepatitis C virus DNA, and a human gene DNA (ETS2). Clinical Samples HCMV DNA extracted from four negative and six positive clinical human serum samples containing various initial amounts of HCMV DNA ranging from 2 ⫻ 10 3 to 10 5 copies/ml were amplified using the DNA PCR-amplification method described above. The amplified products were finally determined with both the SPE-based electrochemical hybridization assay and the microtiter plate-based colorimetric hybridization assay.

technique. This approach was recently investigated in sensitive electrochemical enzyme immunoassays at a dropping mercury (30, 31) and gold electrode (32). The electrochemical detection at the SPE consists of reducing the enzyme DAA product generated in the vicinity of the electrode surface (Fig. 1). Two electrons and two protons are involved in the formation of the hydrazobenzene derivative. The DPV technique was selected in this work, since it was observed to give well-defined peaks compared to other electroanalytical techniques. Figure 2 shows representative DPV curves obtained at SPEs coated with single strands of ETS2 DNA (control assay, curve b) and target HCMV DNA (curve a). After successive hybridization with a short HCMV oligonucleotide probe, enzyme label binding, and enzyme incubation for 30 min in 50 ␮l substrate solution, a significant cathodic peak located at ⫺0.154 V was obtained for the electrode coated with the target DNA (Fig. 2a), whereas only a small residual signal was observed for the control hybridization assay (Fig. 2b). The cathodic peak of Fig. 2a corresponds to the reduction of DAA, and its magnitude reflects indirectly the amount of streptavidin-peroxidase conjugate anchored to the hybrids formed on the electrode surface. It was verified that DNA cannot be adsorbed during the immobilization step on the polyester film on which the electrode is printed. Factors affecting immobilization, probe hybridization, and enzyme-conjugate reactions were optimized in order to maximize the sensitivity and to shorten the assay time. The DPV curves shown in Fig. 2 and recorded under the optimized conditions were repeated at 30 DNA-coated SPEs for assessing the sensor-to-sensor reproducibility. An average cathodic peak current i p of 3300 ⫾ 100 nA (relative standard deviation, RSD ⫽ 3%) was measured for the HCMV DNA-

RESULTS AND DISCUSSION

The electrochemical DNA hybridization assay is schematically depicted in Fig. 1. It is based on the use of o-phenylenediamine as the enzyme substrate for the measurement of the activity of peroxidase label in the DNA hybrid complex formed on the electrode surface. In the presence of hydrogen peroxide and horseradish peroxidase (HRP), OPD is very efficiently transformed into yellow 2,2⬘-diaminoazobenzene (DAA). The amount of DAA generated is usually determined by absorption spectrophotometry, but DAA is also electroactive owing to its diazo function, and it can thus be detected using an electrochemical

FIG. 2. Differential pulse voltammograms obtained at SPEs coated with (a) HCMV-amplified DNA fragment (6.3 ⫻ 10 9 copies/ml in the coating solution) and (b) ETS2-amplified DNA, and determined as depicted in Fig. 1.


coated SPE, whereas an average i p value of 61 ⫾ 12 nA (RSD ⫽ 19%) was measured for the control ETS2 DNA sensor. These results indicate the good reproducibility of the DNA sensor and confirm that the nature of electrode surface (graphite particles embedded in a polystyrene binder) is a suitable solid phase for the reproducible adsorption of DNA. Moreover, this solid phase leads to a negligible nonspecific response when the oligonucleotide probe is not complementary of the adsorbed DNA strand (Fig. 2b). The use of the HCMV DNA sensor to detect the amplified DNA product was investigated by varying the concentration of the target HCMV-amplified DNA fragment in the coating solution over the 0.1–10 6 amol/ml range (6.3 ⫻ 10 4 to 6.3 ⫻ 10 11 amplified copies/ ml). Since the enzyme-generated product was colored and electroactive, spectrophotometric and electrochemical detections could be performed to determine the amount of DAA generated in the 50 ␮l solution bulk, as sketched in Fig. 1. With the aim of comparing different analytical approaches, the signal/blank ratios (S/B) of the current or the optical density values to the standard deviation of the blank responses (samples which did not contain HCMV-amplified DNA fragment) were plotted versus the logarithmic amount of HCMV-amplified DNA fragment initially present in the coating solution (Fig. 3). Figures 3a and 3b show the calibration plots obtained for the SPE-based hybridization assay with spectrophotometric and electrochemical detection, respectively. The calibration plot obtained with the electrochemical detection (curve b) was linear over ca. two decades (50 –2000 amol/ml), and a detection limit of 50 amol/ml (3 ⫻ 10 7 copies/ml of HCMV-amplified DNA fragments) could be estimated. The plateau observed at concentrations higher than 2000 amol/ml suggests a saturation of the electrode surface by the absorbed HCMV-amplified DNA strand. Comparison of the calibration plots a and b in Fig. 3 shows that the sensitivity is ca. 10-fold better when the enzyme product generated from the DNA sensor is measured by DPV instead of absorption spectrophotometry. This result clearly indicates that under the same experimental conditions the electrochemical approach is inherently more sensitive than the spectrophotometric method. The sensitivity of the electrochemical method could be improved by introducing a lower volume of substrate solution as indicated by the calibration plot c obtained in the presence of a 5-fold lower volume of substrate solution (10 ␮l was deposited on the electrode), and an amount as low as 0.6 amol/ml could thus be measured (3.6 ⫻ 10 5 copies/ml of HCMVamplified DNA). Two other conventional methods, i.e., colorimetric enzyme hybridization assay in a microtiter plate (Hybridowell kit) (Fig. 3d) and agarose gel electrophoresis with fluorescence densitometry (Fig. 3e), were also tested in the same range of HCMV-

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FIG. 3. Log-log calibration plots (S/B: signals/blank ratios) of HCMV-amplified DNA using different detection methods. Curves a– c: SPE-based hybridization assays with spectrophotometric (a) or electrochemical (b, c) detection; the revelation volume was 50 ␮l (a, b) or 10 ␮l (c). Curve d: microtiter plate-based hybridization assay. Curve e: agarose gel electrophoresis quantification. Inset: picture of the agarose gel, where L is the DNA ladder and 1– 6 the various HCMV DNA concentrations indicated in curve e.

amplified DNA concentrations. The sensitivities of the conventional hybridization assay in microtiter plate (Fig. 3d) and of the electrochemical method (Fig. 3b) were similar, with a detection limit of 50 amol/ml of HCMV-amplified DNA in each case. The calibration plot obtained by gel electrophoresis quantification (Fig. 3e) shows a relatively poor sensitivity compared to the three preceding approaches, and a detection limit of 14 fmol was estimated. The spectrophotometric sensitivity obtained at a HCMV DNA-coated SPE (Fig. 3a) was worse than in the case of a microtiter plate (Fig. 3d), which can be explained by a difference in the area of DNA coating. Indeed, DNA is absorbed on a 10-fold smaller area in the case of the DNA-coated SPE (9.6 mm 2, area of the electrode surface) than for the assay in a microtiter well (95 mm 2). Consequently, the amount of DAA enzymatically generated from the adsorbed DNA hybrids should be ca. 10-fold higher in the 100 ␮l of substrate solution introduced in the microtiter well than in the 50 ␮l of substrate solution deposited over the DNA sensor. Clearly, the electrochemical method performed with 10 ␮l (curve c) was 23,000 times more sensitive than the agarose gel electrophoresis quantification (curve e) and ca. 83 times more sensitive than the microtiter plate-based spectrophotometric hybridization assay (curve d). However, it

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FIG. 4. Comparative study of the specificity of SPE-based hybridization assay with electrochemical (dark histograms) or spectrophotometric detection (white histograms), and microtiter plate-based hybridization assay (gray histograms). The study includes four amplified DNA fragments, i.e., an amplified human gene DNA (ETS2), and two amplified viruses DNA (EBV and HCV). One negative and one positive HCMV controls were tested in parallel.

requires some skillfulness to introduce three electrodes (SPE, reference, and counterelectrode) in 10 ␮l. The further results were obtained under the previous conditions, i.e., in 50 ␮l. Figure 4 assesses the selectivity of the sensor. The sensor was tested with three noncomplementary amplified DNA sequences, i.e., ETS2, EBV (a member of the herpes virus family like HCMV), and HCV DNAamplified products (33), and a complementary HCMVamplified DNA sequence. As expected only the complementary HCMV DNA gave a significant response. The absence of responses obtained with all of the noncomplementary DNAs show the high selectivity of the hybridization assay. The electrochemical result (dark histogram) is comparable to that obtained by absorption spectrophotometry in microtiter plates (gray histogram). The SPE-based electrochemical hybridization assay was applied to 10 amplified human serum samples (4 negative and 6 positive samples previously quantified by competitive PCR and ranging from 2 ⫻ 10 3 to 10 5 initial copies/ml before amplification of HCMV genomic DNA), and the results were compared to those obtained using the conventional hybridization assay in microtiter plate (Fig. 5). Responses below the detection limit were obtained for the 4 negative samples, whereas clear signals were measured for the 6 positive samples whatever the method, thus indicating a good reliability for each approach. Positive samples 1, 2, and 4 exhibited the same signal/blank ratio with both methods, whereas for the other positive samples 3, 5, and 6, a higher signal/blank ratio for the DNA sensor than for the microtiter plate assay was observed. CONCLUSION

The convenient use of a SPE for the adsorption of a target DNA coupled with the sensitivity of the electro-

chemical detection of HRP enzyme label has proved to be a valid tool for the sensitive detection of HCMVamplified DNA. The device is simple and cost effective, since it involves the low-cost mass production of disposable sensors (screen-printing of the reference electrode can be easily envisaged). Moreover, the electrochemical detection in a few microliters was shown to compete advantageously with the colorimetric hybridization assay in a microtiter plate. The colorimetric signal reflects the amount n (mole) of enzyme-generated product DAA, whatever the revelation volume V, whereas the electrochemical signal is proportional to the DAA concentration, i.e., the ratio n/V, making the electrochemical method very sensitive when V is as low as possible. The detection limit obtained at a DNAcoated SPE (3.6 ⫻ 10 5 copies/ml of HCMV-amplified DNA) is equivalent or superior to other electrochemical approaches using an ultramicroelectrode (10 5 copies per electrode of a simple 25–30 base single-stranded DNA) (21–23), gold electrodes obtained by photolithography (10 4–10 6 copies/ml of an amplified DNA fragment of the hepatitis B virus) (24), or using an electrochemiluminescent method (3 ⫻ 10 8 copies of an amplified 578-bp HCMV DNA fragment) (34). While this method holds great promise for screening HCMV DNA in plasma or serum, the concept of HCMV DNA electrochemical biosensing is still in the research stage. Further improvement is desired such as the development of compact, user-friendly, hand-held multichannel device (35), or the miniaturization of the sensor (i.e., microelectrodes) with the aim of working in smaller assay volumes for increasing the sensitivity. We have recently shown that a monolayer of biotin can be electrochemically grafted on the surface of carbonbased SPE, thus offering new opportunity for the immobilization of oligonucleotides on small electrode surfaces (36).

FIG. 5. Determination of 10 clinical samples by microtiter platebased hybridization assay (white histograms) and SPE-based hybridization assay (striped histograms). Samples 1 to 6 were HCMVamplified DNA from clinical samples exhibiting respectively 10 5, 7.8 ⫻ 10 4, 3.1 ⫻ 10 4, 1.7 ⫻ 10 4, 9 ⫻ 10 3, and 2 ⫻ 10 3 initial copies/ml of HCMV genomic DNA. Samples 7 to 10 were negatives. Two negative controls (⫺) and one positive control (⫹) were tested in parallel.


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