Electrical Detection of Viral DNA Using Ultramicroelectrode Arrays

μL/min) and current signals versus time were recorded. (23) Pozo, F.; Tenorio, A. J. ..... Taking a 5-s period after stopping the flow for getting th...
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Anal. Chem. 2004, 76, 689-696

Electrical Detection of Viral DNA Using Ultramicroelectrode Arrays Eric Nebling,*,† Thomas Grunwald,‡ Jo 1 rg Albers,† Peter Scha 1 fer,§ and Rainer Hintsche†

Fraunhofer Institute for Silicon Technology (ISIT), Fraunhoferstrasse 1, D-25524 Itzehoe, Germany, eBiochip Systems GmbH, Fraunhoferstrasse 1, D-25524 Itzehoe, Germany, and Institute for Medical Microbiology and Immunology, University of Hamburg, Martinistrasse 52, D-20246 Hamburg

A fully electrical array for voltammetric detection of redox molecules produced by enzyme-labeled affinity binding complexes is shown. The electronic detection is based on ultramicroelectrode arrays manufactured in silicon technology. The 200-µm circular array positions have 800nm-wide interdigitated gold ultramicroelectrodes embedded in silicon dioxide. Immobilization of oligonucleotide capture probes onto the gold electrodes surfaces is accomplished via thiol-gold self-assembling. Spatial separation of probes at different array positions is controlled by polymeric rings around each array position. The affinity bound complexes are labeled with alkaline phosphatase, which converts the electrochemically inactive substrate 4-aminophenyl phosphate into the active 4-hydroxyaniline (HA). The nanoscaled electrodes are used to perform a sensitive detection of enzyme activity by signal enhancing redox recycling of HA resulting in local and positionspecific current signals. Multiplexing and serial readout is realized using a CMOS ASIC module and a computercontrolled multichannel potentiostat. The principle of the silicon-based electrical biochip array is shown for different experimental setups and for the detection of virus DNA in real unpurified multiplex PCR samples. The fast and quantitative electronic multicomponent analysis for all kinds of affinity assays is robust and particle tolerant. DNA analytics using biochip technology has become an increasingly implemented method in medical sciences. Today the main field of applications focuses on genomics,1 e.g., the genetic predisposition or evaluation of pharmaceutical targets.2,3 One of the future uses of biochips will be the routine clinical diagnosis of patients for personalised medical care. The key principle of DNA biochips is the detection and quantification of hybridizationsthe interaction of complementary DNA strands to form double-stranded hybrids. At present, optical detection is used for the evaluation of fluorescence-labeled probe binding.4,5 * To whom correspondence should be addressed. E-mail: [email protected]. † Fraunhofer Institute for Silicon Technology (ISIT). ‡ eBiochip Systems GmbH. § University of Hamburg. (1) Harris, T. Med. Res. Rev. 2000, 20, 203-211. (2) Risch, N. J. Nature 2000, 405, 847-856. (3) Roses, A. D. Nature 2000, 405, 857-865. 10.1021/ac0348773 CCC: $27.50 Published on Web 12/30/2003

© 2004 American Chemical Society

An alternative way is the electrochemical detection of hybridization with direct signal coupling of the biosensing element and the transducer.6,7 These sensors utilize the intrinsic electrochemical properties of DNA8,9 or labeling methods.10-12 Some others are based on physical properties of DNA such as conductivity differences due to hybridization and base stacking or changes in sterical intercalation properties.13-15 Here we present silicon-based DNA chips using an amperometric redox-recycling reaction on interdigitated ultramicroelectrodes,16 which have been arranged in array formats. The effort of a simultaneous measuring multichannel potentiostat17 was reduced by an optimized multiplexing 16-channel potentiostat.18 The common method of self-assembling capture oligonucleotides via thiol-gold coupling19-22 was used to construct the array chipDNA interface. The advantages of this chip-based assay technique are as follows: (i) a cost-effective alternative to expensive optical devices; (4) Caillat, P.; David, D.; Belleville, M.; Clerc, F.; Massit, C.; Revol-Cavalier, F.; Peltie, P.; Livache, T.; Bidan, G.; Roget, A.; Crapez, E. Sens. Actuators, B 1999, 61, 154-162. (5) Heller, M. J.; Forster, A. H.; Tu, E. Electrophoresis 2000, 21, 157-164. (6) Scheller, F. W.; Wollenberger, U.; Warsinke, A.; Lisdat, F. Curr. Opin. Biotechnol. 2001, 1, 35-40. (7) Pividri, M. I.; Merkoci, A.; Alegret, S. Biosens. Bioelectron. 2000, 15, 291303. (8) Wang, J. Nucleic Acids Res. 2000, 28, 3011-3016. (9) Palecek, E.; Fojta, M.; Jelen, F. Bioelectrochemistry 2002, 56 (1-2), 8590. (10) Willner, I.; Willner, B.; Katz, E. J. Biotechnol. 2002, 82, 325-355. (11) Patolsky, F.; Weizmann, Y.; Willner, I. J. Am. Chem. Soc. 2002, 124, 770772. (12) Patolsky, F.; Lichtenstein, A.; Kotler, M.; Willner, I. Angew. Chem., Int. Ed. 2001, 40, 2261-2265. (13) Kelley, S. O.; Boon, E. M.; Barton, J. K.; Jackson, N. M.; Hill, M. G. Nucleic Acids Res. 1999, 27, 4830-4837. (14) Berggren, C.; Stalhandske, P.; Brundell, J.; Johansson, G. Electroanalysis 1999, 11, 156-160. (15) Alfonta, L.; Singh, A. K.; Willner, I. Anal. Chem. 2001, 73, 91-102. (16) Niwa, O.; Xu, Y.; Halsall, H. B.; Heineman, W. R. Anal. Chem. 1993, 65, 1559-1563. (17) Paeschke, M.; Dietrich, F.; Uhlig, A.; Hintsche, R. Electroanalysis 1996, 8, 891-898. (18) Hintsche, R.; Albers, J.; Bernt, H.; Eder A. Electroanalysis 2000, 12, 660665. (19) Bain, C. D.; Whitesides, G. M. J. Am. Chem. Soc. 1989, 111, 7164-7175. (20) Xu, D.; Huang, K.; Liu, Z.; Liu, Y.; Ma, L. Electroanalysis 2001, 13, 882887. (21) Steel, A. B.; Levicky, R. L.; Herne, T. M.; Tarlov, M. J. Biophys. J. 2000, 79, 975-981. (22) Edelstein, R. L.; Tamanaha, C. R.; Sheehan, P. E.; Miller, M. M.; Baselt, D. R.; Whitman, L. J.; Colton, R. J. Biosens. Bioelectron. 2000, 14, 805-813.

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(ii) small, robust, and easy to handle electrical components usable for in field measurements; (iii) direct and particle-tolerant detection in unpurified liquids; and (iv) position-specific and parallel quantitative signal readout. The detection of viral infections plays an important role in clinical diagnosis and monitoring of drug therapy. Herpes virus diagnostics for example can be applied in the fields of transplant patient monitoring, encephalitis, neurological syndromes, opportunistic infections in immune-suppressed patients, or screenings of blood donations.23-26 The molecular diagnosis of these viruses is commonly based on PCR due to the low abundance of viral DNA in biological samples. A multiplexed PCR format enabled the amplifying of multiple target sequences simultaneously in one reaction.23,24,26 The analysis of the resulting amplicon mixtures is based on hybridization of target DNA employing an enzyme label as redox molecule producing molecular species.27,28 The application of these enzyme-linked immunosorbent assay (ELISA) formats to fully electrical multiposition arrays is shown. EXPERIMENTAL SECTION Materials. Bovine serum albumin (BSA) (30% solution), Tween 20, ExtrAvidin alkaline phosphatase conjugate, phosphatebuffered saline (PBS), 4-hydroxyaniline (HA), Trizma base (Tris), and sodium citrate were purchased from Sigma (Deisenhofen, Germany). Sulfuric acid was purchased from Merck (Darmstadt, Germany) and sodium chloride from Fluka (Buchs, Switzerland). Oligonucleotides were synthesized by Interactiva (Ulm, Germany), and 4-aminophenyl phosphate (p-APP) was purchased from ICN Biomedicals Inc. (Aurora, IL). Buffers. The following buffers were used. PBS: 120 mM sodium chloride, 2,7 mM potassium chloride, 10 mM phosphate, pH 7.4. 2× SSC: 300 mM sodium chloride, 30 mM sodium citrate, pH 7.0. TBS: 100 mM sodium chloride, 30 mM Tris, pH 8,0. Oligonucleotides. The following oligonucleotides were used. HSV capture: 5′-mercaptohexyl-TTTTTGTGCGCCACTGCGTCGGCCCTCAGGGAGAG-3′. EBV capture: 5′-mercaptohexyl-TTTTTCGGGCGCAGGCCGGCTAGCCTGTGCTCTTC-3′. CMV capture: 5′-mercaptohexyl-TTTTTGGGTCCACAGGGTACTCGCCACCCGGCACC-3′. Negative control capture: 5′-mercaptohexylCGATCTGTTTTATGTAGGGTTAGGTCA-3′. Random capture (bio): 5′-mercaptohexyl-CGATAGTGTGTAAGAGATGCAAAT-biotin-3′. CMV sequence: 5′-biotin-GGTGCCGGGTGGCGAGTACCCTGTGGACCC-3′. HSV sequence: 5′-biotin-CTCTCCCTGAGGGCCGACGCAGTGGCGCAC-3′. Multiplex primer 1: 5′-biotinGACTTTGCCAGCCTSTACCC-3′ (S: G or C). Multiplex primer 2: 5′-GTCCGTGTCCCCGTAGATGA-3′. Chip Construction. Thin-film gold microelectrode arrays were manufactured in standard silicon technology on 6-in. thermally oxidized silicon wafers in the ISIT’s clean room environment. The 120-nm-thick gold electrodes were deposited onto a 20-nm-thick (23) Pozo, F.; Tenorio, A. J. Virol. Methods 1999, 79, 9-19 (24) Yamamoto, T.; Nakamura, Y. J. NeuroVir. 2000, 6, 410-417. (25) Scha¨fer, P.; Tenschert, W.; Schro¨ter, M.; Gutensohn, K.; Laufs, R. J. Clin. Microbiol. 2000, 38, 3249-3253. (26) Defoort, J.-P.; Martin, M.; Casano, B.; Prato, S.; Camilla, C.; Fert, V. J. Clin. Microbiol. 2000, 38, 1066-1071. (27) Heineman, W. R.; Halsall, H. B.; Wehmeyer, K. R.; Doyle M. J.; Wright, D. S. Methods Biochem. Anal. 1987, 32, 345-393. (28) Hintsche, R.; Paeschke, M.; Wollenberger, U.; Schnackenberg, U.; Wagner, B.; Lisec, T. Biosens. Bioelectron. 1994, 9, 697-705.

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titanium adhesion layer by chemical vapor deposition. The electrodes were patterned using the liftoff technique to generate interdigitated structures with 1-µm-wide fingers and gaps of 0.8 µm. The counter electrode, which was also integrated onto the chip was a thin-film gold pad of 0.8 mm2. The metal on-chip was encapsulated by 500-nm-thick chemical vapor deposited silicon oxinitride. The active electrode areas and connecting pads were opened by reactive ion etching. Each chip (6.4 × 4.5 mm) consisted of 28 individual positions of 200-µm diameter with spaces of 250 µm to the next positions (Figure 1). Each 200-µm position with interdigitated array structure (IDA) exhibited a gold surface of 17.000 µm2. Polymeric SU8 ring structures of 10-µm height, 15-µm width, and 10-µm distance were built up around each array position by photolithography. The array chips were mounted onto printed circuits boards of 13 mm × 20 mm (Britze Elektronik (Berlin, Germany)). Eight out of the 28 IDA positions were electrically connected by Al wire bonding (two connections per position), and the bonded areas were protected with 3140 silicon rubber (Dow Corning Corp., Midland, MI). For measuring, the printed circuit boards were connected to the potentiostat. Before use, the electrode’s surfaces were treated by an oxygen plasma for 3 min and subsequently with concentrated sulfuric acid (2-3 µL) for 1 min. The chip was finally washed with deionized water and dried in air. Instrumentation. For electrochemical measurements, a 16channel multipotentiostat (model CIPo, eBiochip Systems GmbH, Itzehoe, Germany) equipped with two multiplexers was used. The multiplexing rates were 120 ms. The resolution of the potentiostat was 2.5 pA using a current range of 100 nA. The device was connected to a commercially available personal computer by the RS232 serial port for data transfer. The amperometric response plots were presented by using the Microcal Origin 5.0 program. Measurements were taken versus an Ag/AgCl reference microelectrode (Microelectrodes Inc.). A polycarbonate flow-through cell with an internal volume of 4 µL was sealed by an O-ring onto the silicon chip and connected to silicon tubes. A peristaltic pump from Ismatec (GlattbruggZu¨rich, Switzerland) was used for moving the various fluids. For the DNA spotting procedure, we used a 10-µL Hamilton syringe with a special shaped stainless steel cannula (300 µm in diameter; flat), which was positioned by a micromanipulator 5171 system from Eppendorf AG (Hamburg, Germany). Optical control was done with a Stemi 2000-C microscope from Zeiss (Oberkochen, Germany). Test Procedures. Functionality Test by Redox Recycling. For evaluation of the gold electrode surfaces of each position, redox measurements were taken. Redox recycling is the reversible anodic oxidation and cathodic reduction of electroactive molecules as described previously16 (Scheme 1). The array positions were tested with HA and its oxidized form quinoneimine (QI). The printed circuit board with chip and the flow-through cell were plugged to the 16-channel multipotentiostat using a standard industrial connector (Figure 1). The eight anodes of the eight array positions were set to +350 mV and the eight cathodes to -50 mV versus the Ag/AgCl reference electrode. During the electrochemical measurement, a solution of 10 µM HA in PBS (pH 7.4) was pumped through the chip flow cell (100 µL/min) and current signals versus time were recorded.

Scheme 1. Illustration of the Redox Recycling Processa

a

HA, 4-hydroxyaniline; QI, quinoneimine.

Enzyme Activity of the Label Enzyme. The enzyme activity is important for calculating the density of immobilized capture DNA probes onto the electrodes and the hybridization effectiveness. For evaluating the activity of the label enzyme alkaline phosphatase, 10 µM HA in TBS (pH 8.0) was measured by redox recycling at eight array positions at room temperature (25 °C) to get a standard current signal. This values were compared with 50 fmol of ExtrAvidin alkaline phosphatase conjugate in 1 µL of TBS (pH 8.0), which was added to a solution of 500 µL of TBS (pH 8.0) with 2 mM p-APP (25 °C). Redox-recycling current signals of increasing HA concentration dependent on the enzymatic substrate hydrolysis were measured at eight positions. The resulting current signals with a linear slope were used to calculate the enzyme activity, where 1 unit of enzyme was assumed to hydrolyze 1.0 µmol of substrate/min at 37 °C at a pH of 9.8. The molecular weight of the dimeric alkaline phosphatase was assumed to be 114 000 and of the ExtrAvidin conjugate 182 000. Self-Assembling of Capture Oligonucleotides. For immobilization of capture oligonucleotides, the self-assembling of thiolated molecules at gold surfaces was used (Figure 3a). To measure the surface coverage of the gold electrodes, 5′-mercaptohexyl-labeled and 3′-biotinylated 24-mer oligonucleotides (random capture (bio)) with bound ExtrAvidin alkaline phosphatase conjugate were used (Figure 3b). After adding p-APP, the current slopes of redox recycling in stopped-flow mode were measured. The coverage density was calculated by using the enzyme activity (see above) and the electrode surface of 17.000 µm2 of one position. It was assumed that the current signals slope was proportional to the oligonucleotide density on the measured positions. Those measurements were carried out at all eight array positions. The mercaptohexyl-random-biotin capture 24-mer oligonucleotide (1 mM in water) was spotted in amounts of 6 nL onto four array positions of one chip. On the other four array positions, a negative control 27-mer capture (1 mM in water) was spotted by the same procedure for negative control. Spotting was done in a humidity chamber to minimize evaporation. After 2 h of incubation at room temperature, the whole chip was washed with deionized water and dried in air. The spotted mercaptohexyllabeled oligonucleotides were now fixed onto the gold surface by thiol-gold coupling. The chip was then incubated with 10 µL of a 3% BSA solution in PBS (pH 7.4) for 10 min at room temperature to block free areas of the gold electrode surfaces. After washing the chip 6 times with 500 µL of TBS (pH 8.0), all array positions were covered with 3 µL of TBS (pH 8.0) containing 300 fmol of the ExtrAvidin alkaline phosphatase conjugate and the resultant mixture incubated for 15 min at room temperature. The biotinylated capture probes were now enzyme labeled. After that, the chip was washed 6 times with 500 µL of TBS (pH 8.0) and

connected to the fluidic system. Detection occurred with 2 mM p-APP in TBS (pH 8.0) by redox-recycling measurements. Timedependent current signals were measured, and then the surface accessibility of each position was checked by redox-recycling measurements of HA. DNA Array Test. For testing our procedure, a hybridization assay with synthesized biotinylated target oligonucleotides was performed. This assay was done on eight array positions following the surface construction as shown in Figure 3c. Onto eight positions of a new chip, five different mercaptohexyl-labeled capture oligonucleotides were immobilized by the procedure described above. The array was loaded as follows: position 1, random capture (bio) for positive control; position 2, negative control capture DNA; positions 3 and 4, HSV capture DNA; positions 5 and 6, EBV capture DNA; positions 7 and 8, CMV capture DNA (each 1 mM in water). For evaluation of the DNA assay, 6 µL of 4× SSC with 200 fmol of HSV sequence and 200 fmol of CMV sequence (each 33 nM) was directly dropped onto all eight array positions. The chip was stored for 20 min at room temperature in a humidity chamber for hybridization and was washed 6 times with 500 µL of PBS (pH 7.4). The blocking, washing, enzyme labeling, and measurement procedures were the same as described above. Multiplex PCR and Virus DNA Detection. To determine whether our chip-based assay was able to detect target DNA directly out of a multiplexed PCR, the following procedure was used. With a QIAamp Blood Kit from Qiagen, 200 µL of a clinical blood sample was prepared according to the manufacturer’s instructions. Elution was done with 200 µL of water, and 5 µL of this eluate was used for PCR. The PCR was carried out with a DyNAzyme DNA Polymerase Kit from Finnzymes and a Primus 96 plus thermocycler from MWG-Biotech. The Mastermix contained 1 µM of each multiplex primer, 250 µM of each dNTP, 10 mM Tris-HCl (pH 8,8), 50 mM potassium chloride, 1,5 mM magnesium chloride, 0,1% Triton X-100, and 1,5 units of DNA polymerase in a 50-µL sample volume. For amplification, 40 cycles were used with 1-min denaturation at 94 °C, 1-min annealing at 55 °C, and 1-min extension at 72 °C. A final extension step was done for 5 min at 72 °C. Biotinylation of virus DNA was done during the PCR by the biotinylated multiplex primer 1. The PCR was checked by agarose gel electrophoresis, and the concentration was determined. From the PCR, a 10-µL sample was denatured for 2 min and was then directly applied onto the eight chip positions with previously spotted virus capture DNA (see above). Hybridization took place for 20 min at room temperature in a humidity chamber. The subsequent procedures and the measurement were done as described above. RESULTS AND DISCUSSION Design and Construction. A platform of silicon-based ultramicroelectrodes arranged as low-density arrays has been developed with the aim of compatibility with the common semiconductor technology and the available automated mounting and packaging techniques (Figure 1). Common printed circuits boards have been used as chip carriers. These boards are connected via a commercial plug in unit to the microprocessor-controlled Analytical Chemistry, Vol. 76, No. 3, February 1, 2004

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Figure 1. Photograph of the electrical biochip and a scheme of the measuring system: (a) chip on printed circuit board; (b) 16-channel multipotentiostat with chip and flow-through cell connected to the reference electrode, pump, and computer; (c) details of the eight used chip positions; (d) details of the submicrometer interdigitated electrode fingers and the three-dimensional polymeric ring structures.

multichannel potentiostat (Figure 1b). The fully electrical readout procedure enabled us to construct a portable and robust device. Multichannel Redox Recycling. This measuring principle excludes electrochemical interferences at the electrodes of molecular species, which undergo nonreversible redox processes. As shown by Paeschke et al., the resulting redox recycling amplification factor at the given gap/width ratio of the electrodes is eight.17 The array IDA electrodes were used for simultaneous redox recycling at eight chip positions. The electrical multichannel read out of 10 µM HA in PBS during liquid flow is shown in Figure 2. Polarizing each single anode with a potential of +350 mV and each single cathode with -50 mV and measuring the voltammetric responses were done independently and simultaneously by the multipotentiostat. In Figure 2a, the current responses of the eight anodes (channels 1, 3, 5, 7, 9, 11, 13, 15) and the corresponding cathodes (channels 2, 4, 6, 8, 10, 12, 14, 16) are presented. The sums of these currents of paired electrodes of each of the eight positions are plotted in Figure 2b. At the submicrometer IDA electrodes, an effective redox recycling takes place16 where HA is oxidized at the anode and the yielded QI is reduced at the cathode back to HA. The shape of the responses of ∼30 nA was similar for all positions, indicating identical gold electrode squares and clean electrical active surfaces. Enzyme Activity and Surface Chemistry. The enzyme activity was needed to calculate the amount of immobilized captures 692

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per square micrometer of gold surface to estimate the layer density. The redox response of 10 µM HA in TBS was 5 nA on each position, and the current slope of enzyme generated HA was 0,3 nA/min at each position (data not shown; for details, see the Experimental Section). Taking a flow cell volume of 4 µL, electrode surface of 17.000 µm2, and a flowing 500-µL buffer volume into consideration, the enzyme activity resulted in a substrate hydrolysis of 300 pmol/min (300 pmol/500 µL ) 0.6 µM). The added 50 fmol of enzyme conjugate showed an activity of 300 µunits, which resulted in a calculated activity of 53 units/mg of enzyme and 33 units/mg of ExtrAvidin enzyme conjugate. For a calculation of labeled capture molecules, the assumption was made that the measured activity of the free enzyme is equivalent to the apparent activity of the bound enzyme. The surface density of bound capture oligonucleotides was determined by using the enzyme conjugate activity. The use of electrical arrays requires highly reproducible layers of molecular capture probes as oligonucleotides at each position. The self-assembly of thiolated capture monolayers19,29 was performed by spotting the different 5′-hexanethiol-modified oligonucleotides at the different array positions (Figure 3a). The SU8 ring structures prevented leakage of the spotted droplets successfully. The layer formation could be controlled by measuring (29) Brockman, J.; Frutos, A. G.; Corn, R. M. J. Am. Chem. Soc. 1999, 121, 8044-8051.

Figure 2. Simultaneous redox-recycling responses of a portion of HA in PBS flowing through the measuring cell: (a) time-dependent anodic and cathodic current signals at eight positions; (b) summarized current signals. The anodes were set to +350 mV and the cathodes to -50 mV versus an external Ag/AgCl reference electrode. The flow was set to 100 µL/min.

the decreasing capacity of the IDA as described earlier.30 Here the decreasing access to the metallic electrode surface during layer formation was measured by redox recycling of HA, and the layer density was measured by enzymatically generated HA. Therefore, 3′-biotinylated capture oligonucleotides were spotted onto four positions of an array (Figure 3b) and the other four positions were covered with an unbiotinylated capture oligonucleotide as in Figure 3a. The directly attached ExtrAvidin alkaline phosphatase conjugate (Ex-aP) was used as an amplifier by converting its electrochemical inactive substrate p-APP into the active form HA (Figure 3b). The redox recycling was performed in TBS buffer at pH 8.0. This pH was chosen as a compromise between the optimal pH of 9.3 for the alkaline phosphatase and pH 6.5, which is optimal for redox recycling. The total current responses of the labeled and the unlabeled positions are shown in Figure 4, at first during the continuous flow of p-APP and then in stopped-flow mode. During the flow,

small constant currents are generated at the labeled positions 1, 3, 5, and 7, indicating the enzyme function. At the four negative positions, the substrate p-APP itself generated a small background current signal because it contained HA in low concentration as an impurity. After stopped flow, a significant slope of ∼60 nA/ min (at 20 s) at the labeled positions was observed, where the negative positions showed only 7 nA/min caused by small crosstalk reactions. The rapid increase of the concentration of enzymatically generated HA in the solution near to the electrodes’ surface resulted in rapidly increasing current signals of the positive positions in stopped-flow mode. After a few seconds, the substrate concentration in the reaction chamber decreased, because of the enzymatic consumption and the diffusion of products from the surface region into the bulk solution. Electrode access was estimated by redox-recycling measurements of 10 µM HA and resulted in a signal of 15 nA for each of the eight positions (data not shown). The redox current of an unmodified position was 30 nA (Figure 2b). The 50% decrease of the redox current is due to a coverage with capture oligonucleotides. From a slope of 10 nA at 80 s at each position (complete cross-talk reaction), a coverage density of capture oligonucleotides of ∼200.000 enzyme-labeled oligonucleotides/µm2 was determined. The calculations were based on a measured redox current of 2.5 nA of 10 µM HA (data not shown). An average linear slope of 10 nA/min then is equal to a HA increase of 40 µM/min (10 nA/2.5 nA × 10 µM ) 40 µM). Therefore, in the cell volume of 4 µL, the enzyme conjugate at the four positive positions produced 160 pmol of HA/min by the conversion of 160 pmol of p-APP/ min. This is equal to 160 µunits of enzyme conjugate. With an activity of 33 units/mg, the coupled amount was 4.84 ng. Enzyme conjugate was only bound to the immobilized oligonucleotides on the 4 × 17.000 µm2 gold electrode surface. With a molecular weight of 182 000, a density of 235.000 enzyme conjugate molecules/µm2 resulted. This calculation is valid if one enzyme conjugate molecule is linked to one immobilized capture molecule. In the case where the binding effectiveness is less than 100% and ExtrAvidin conjugates are binding more than one biotinylated component, this calculation shows some error. With this calculation including the error assumption the number of immobilized oligonucleotides is expected to be in the range of 100.000-300.000/µm2. This amount of thiol-gold bound capture molecules corresponds with the maximum density value of 3 × 1013 probes/cm2 (300.000/µm2) described by Steel et al.21 DNA Array. To validate the electrical biochip technology, a low-density DNA array and an assay procedure for recognizing different DNA sequences of human viruses have been carried out. Onto the eight-position chip array as shown in Figure 1c capture oligonucleotides for cytomegalovirus (CMV), Epstein-Barr virus (EMV), Herpes simplex virus (HSV), a nonbinding nucleotide for negative control, and a biotinylated oligonucleotide for 100% positive control have been immobilized onto two positions each except for the control positions. Then a hybridization reaction with a mixture of synthesized oligonucleotide sequences of HSV and CMV was performed. In Figure 5a, the redox currents of the eight positions after hybridization and enzyme labeling are shown. The absolute currents due to the redox recycling of the label enzyme product HA 4 s after stopped flow are presented in Figure (30) Paeschke, M. Dissertation, Christian-Albrechts-Universitaet zu Kiel, 1998.

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Figure 3. Schemes of capture immobilization and assay construction (a) immobilization of thiol-linked capture probe. (b) Enzyme label of biotinylated capture probe by ExtrAvidin alkaline phosphatase conjugate and redox recycling of converted substrate. (c) Affinity binding of biotinylated targets and enzyme labeling. (d) Affinity bound PCR product with label enzyme ExtrAvidin alkaline phosphatase conjugate.

Figure 4. Simultaneous redox-recycling measurements in stoppedflow mode of four positive and four negative chip positions with a portion of the enzyme substrate p-APP. Summarized current signals are shown.

5b. Recording of the current slopes during 2-7 s after stopped flow are presented in Figure 5c. The signals at the two positions with HSV captures and the two positions with CMV captures reveal that here hybridization and enzyme labeling occurred. At the positive control position, which have been labeled directly with the enzyme via the 3′-biotin residue, a significant current signal 694

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was also obtained. The nonhybridized EBV positions and the negative control position showed negative signals. The similar shape of identical positions indicates a good reproducibility of the assay and the electrical readout. The very fast readout time becomes possible because the redox recycling only measures the increase of the concentration of HA near to the electrode surface. Therefore, a linear slope can be only observed during this short time period. Both factors, the HA diffusion and the decreasing concentration of the enzyme’s substrate, cause the nonlinearity of the current responses. The signal-to-noise ratio of the current values was observed to be 4:1 (Figure 5b). Taking a 5-s period after stopping the flow for getting the current slopes resulted in a signal-to-noise ratio of 100:1 and more (Figure 5c). Comparing the current slopes at the negative positions after stopped flow, it is obvious that electrochemical cross talk due to diffusion of HA in the bulk between different positions contributes to the current values. A calculation of the number of observed hybridized and enzyme-labeled complexes has been done following the principles as discussed above. The observed current slope corresponded to 112 µunits of enzyme, which is 3.4 ng. Amounts of 3.4 ng corresponded to 1.12 × 1010 coupled enzyme conjugate molecules onto 85.000 µm2 of electrode surface (5 positive positions). This resulted in 132.000 hybridized targets/µm2 with the expectation that one enzyme conjugate molecule bound to one hybridized target oligonucleotide. The calculated amount of immobilized capture oligonucleotides was 235.000/µm2 (see above). An incubation time of 20 min at room temperature with a 33 nM target concentration showed a hybridization effectiveness onto the chip of 56%. Overall, the used electrical DNA array chip together with the evolved assay procedure was able to detect synthesized single-

Figure 6. Simultaneous redox-recycling measurements of a DNA assay at eight positions done with a multiplexed virus PCR sample. (a) Summarized currents of position-specific redox-recycling signals. (b) Column diagram of slope values as indicated in (a). For assay details, see under Experimental Section, Multiplex PCR and Virus DNA Detection.

Figure 5. Simultaneous redox-recycling measurements of a DNA assay at eight positions done with synthesized ss virus target oligonucleotides. (a) Summarized currents of position-specific redoxrecycling signals. (b) Column diagram of current values as indicated in (a). (c) Column diagram of slope values as indicated in (a). For assay details, see under Experimental Section, DNA Assay Procedure.

stranded virus target oligonucleotides with high selectivity and sensitivity. Multiplex PCR and Virus DNA Detection. For practical application of this technology, detection of virus DNA directly out of a multiplex PCR of a clinical blood sample was investigated. For this purpose, HSV-, EBV-, and CMV-DNA were amplified in one mixed PCR using biotinylated primers. The DNA concen-

tration of the multiplex PCR that was detected by the array chip was ∼2 nM, and the kind of target hybridization of this assay is shown in Figure 3d. The experimental setup was identical with that described above. Again the assay results are shown as current sum curves of the eight positions (Figure 6a). Due the better signal-to-noise ratio, only the signal slopes were used for assay evaluation (Figure 6b). The electrical biochip responses allow the diagnosis that the patient from whom the blood sample was taken was infected with CMV whereas HSV and EBV were absent (Figure 6a and b). Taking the current signal slopes from 2 to 7 s after stopping the flow resulted in an optimal signal-to-noise ratio of 100:1 or better between the three positive chip positions (positive control and 2× CMV) and the five negative positions (negative control, 2× HSV and 2× EBV). The slope of the positive control was 1,7 nA/ s, whereas the two slopes belonging to the CMV positions reached 0,6 nA/s (Figure 6b). Analytical Chemistry, Vol. 76, No. 3, February 1, 2004

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It should be mentioned that chip detection took place directly out of a PCR without any additional cleanup steps. No unspecific binding of the primer residues was observed. As expected, the electrical biochip is robust and particle tolerant. With a 20-min hybridization step after PCR denaturation, the whole assay including the measurement could be done in 50 min. CONCLUSIONS The presented study employed a silicon-based DNA array chip for fully electrical readout and showed its usability for multichannel electrochemical detection. An assay procedure of the ELISA type allows a direct virus DNA detection from a PCR-amplified clinical probe. In this study, concentrations of ∼2 nM virus target DNA could be selectively detected by the chip directly out of a multipexed PCR even in the presence of PCR primer residues. The application is robust and particle tolerant. The manufacturing is highly compatible with common semiconductor technology and the spotting procedures used in printers. The capture oligonucleotides can be immobilized fast and conveniently via thiol-gold linkage in highly ordered and stable monolayers. The electrical readout procedure is very fast

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without cross talk during the measurement interval. These electrical biochips offer a high potential for integrating as well electronic measurement functions as microchemical and microfluidic components into a real “lab on a chip”. With electrical field-supported hybridization and stringency treatment, the running time should be reduced to a few minutes. This kind of DNA chip could be used for point-of-care diagnostics. ACKNOWLEDGMENT This study was done in the scope of the SiBANAT project and supported by the Deutsche Forschungsgesellschaft (DFG) and the Bundesministerium fu¨r Bildung und Forschung (BMBF) (FKZ 01 GS 0005 and FKZ 01 M 3102 D). We thank the following SiBANAT project partners: Siemens AG, Infineon AG, November AG, and Eppendorf AG (all in Germany). The authors thank C. G. J. Schabmueller for proofreading the manuscript.

Received for review July 30, 2003. Accepted November 24, 2003. AC0348773