Detection of∼ 103 Copies of DNA by an Electrochemical Enzyme

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Detection of ∼103 Copies of DNA by an Electrochemical Enzyme-Amplified Sandwich Assay with Ambient O2 as the Substrate Yongchao Zhang,†,‡ Arti Pothukuchy,†,‡ Woonsup Shin,§ Yousung Kim,§ and Adam Heller*,†

Department of Chemical Engineering, University of Texas, Austin, Texas 78712, and Department of Chemistry, Sogang University, Seoul, 121-742, Korea

The electrochemical sandwich-type, enzyme-amplified assay of Zhang, Kim, and Heller (Anal. Chem. 2003, 75, 3267-3269) was simplified by replacing the amplifying horseradish peroxidase with bilirubin oxidase (BOD). BOD catalyzes the reduction of ambient O2 to water and obviates the need for adding H2O2. Femtomolar (10-15 M) concentrations of DNA were detected at a 10-µmdiameter tip of a carbon fiber electrode. Correspondingly, a few thousand copies of DNA were detected in ∼5-µL samples. The sandwich is formed in an electron-conducting redox hydrogel, to the polymer of which a DNA capture sequence is bound. Capture of the analyte DNA and its hybridization with a BOD-labeled complementary DNA sequence, electrically connects the BOD label to the electron-conducting redox polymer, which is in electrical contact with the electrode. Placing the BOD in contact with the redox polymer thus converts the noncatalytic base layer into a catalyst for the electroreduction of O2 to water at +0.12 V (vs Ag/AgCl) (Figure 1). In an exemplary assay, ∼3000 copies of the iron transporting sequence of the sit gene of Shigella flexneri were detected without PCR amplification. We reported recently the enzyme-amplified amperometric DNA assay detection of as few as ∼3000 copies of DNA in 10-µL samples at 10-15 M concentration.1 The assays were performed using 10µm-diameter carbon fiber electrode tips, on which a redox polymer and a capture DNA sequence were electrodeposited. When present, the target DNA was captured by the tip. Further hybridization of the target with a horseradish peroxidase (HRP)labeled oligonucleotide made the tip catalytic for the electroreduction of H2O2 to water, the electrons flowing through the redox polymer, via the HRP, to H2O2. Preceding our work, Ghindilis, Yaropolov, and their colleagues2-4 used laccase, an O2 reduction catalyzing enzyme for electrochemical immunoassays, * Corresponding author. E-mail: [email protected]. † University of Texas. ‡ These authors contributed equally to this work. § Sogang University. (1) Zhang, Y.; Kim, H.-H.; Heller, A. Anal. Chem. 2003, 75, 3267-3269. (2) Ghindilis, A. Biochem. Soc. Trans. 2000, 28, 84-89. (3) Kuznetsov, B. A.; Shumakovich, G. P.; Koroleva, O. V.; Yaropolov, A. I. Biosens. Bioelectron. 2001, 16, 425. (4) Milligan, C.; Ghindilis, A. Electroanalysis 2002, 14, 415-419. 10.1021/ac0495034 CCC: $27.50 Published on Web 06/08/2004

© 2004 American Chemical Society

including sandwich-type assays. Though laccase is an excellent O2 reduction catalyst at pH 5 and in the absence of halides, it is a poor catalyst at neutral pH and in chloride-containing solutions.5,6 Because DNA or RNA hybridization requires high ionic strength, which is usually maintained by high NaCl or MgCl2 concentration, laccase was not used as a label in affinity assays of nucleic acids and its reported use was limited to assays of antigens and antibodies. Because equilibria of antigen-antibody pairings do not favor product formation nearly as much as pairings of long complementary DNA or RNA sequences, the sensitivities realized in affinity assays with laccase labels were those typical for immunoassays, in which antigens or antibodies are detected at concentrations higher than ∼0.1 nM. Like laccase, bilirubin oxidase (BOD) also catalyzes the reduction of O2 to water, but unlike laccase, it effectively catalyzes the reduction at neural pH and at high chloride concentrations.7-10 In a study aimed at establishing that BOD can be used as a label in a sandwich-type amperometric DNA assay, but not at probing the detection limits reached with this label,11 we used printed carbon macroelectrodes and bound the BOD to the sandwich after it was formed, not to the detection sequence itself. To bind the BOD to the completed sandwich, we linked it with glutaraldehyde, through forming a pair of Schiff bases, an amine incorporated in the detection sequence with one of the amines of BOD. The assay detected DNA only at concentrations above 0.1 nM. Here we show that detection at 10-15 M (1 fM) concentration, the detection limit for the HRP-labeled sandwich,1 and for a glucose oxidase-labeled sandwich12 is reached also with the BODcontaining sandwich when a microelectrode is used and when the detection probe is prelabeled with BOD before its hybridization. We also show that ∼3000 copies of Shigella DNA can be selectively detected in the BOD-amplified amperometric assay with ambient O2 as the electroreduced substrate. (5) Naqui, A.; Varfolomeev, S. D. FEBS Lett. 1980, 113, 157-160. (6) Xu, F. Biochemistry 1996, 35, 7608-7614. (7) Hirose, J.; Inoue, K.; Sakuragi, H.; Kikkawa, M.; Minakami, M.; Morikawa, T.; Iwamoto, H.; Hiromi, K. Inorg. Chim. Acta 1998, 273, 204-212. (8) Mano, N.; Mao, F.; Heller, A. J. Am. Chem. Soc. 2002, 124, 12962-12963. (9) Mano, N.; Kim, H.-H.; Zhang, Y.; Heller, A. J. Am. Chem. Soc. 2002, 124, 6480-6486. (10) Mano, N.; Mao, F.; Heller, A. J. Am. Chem. Soc. 2003, 125, 6588-6594. (11) Kim, H.-H.; Zhang, Y.; Heller, A. Anal. Chem. 2004, 76, 2411-2414. (12) Xie, H.; Zhang, C.; Gao, Z. Anal. Chem. 2004, 76, 1611-1617.

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EXPERIMENTAL SECTION Materials and Reagents. BOD was purchased from Amano Enzyme Inc. Poly Pak II cartridges, Thiol-Modifier C6S-S, and 3′ Amino-Modifier C7 were obtained from Glen Research (Sterling, VA). -N-Maleimidocaproic acid (2-nitro-4-sulfo)phenyl ester was purchased from Bachem Biosciences (King of Prussia, PA). NAP columns (prepacked disposable columns containing Sephadex G-25 medium) used in the oligonucleotide-BOD conjugate synthesis and DEAE Sepharose FF for purification of the oligo-BOD conjugate were from Amersham Pharmacia Biotech (Piscataway, NJ). The buffering salts and other chemicals were purchased from Sigma (St. Louis, MO), Aldrich (Milwaukee, WI), or Fisher Scientific (Pittsburgh, PA) and were used as received. The phosphate-buffered saline solution (PBS: 4.3 mM NaH2PO4, 15.1 mM Na2HPO4, 140 mM NaCl), the hybridization buffer (pH 7, 10 mM HEPES, 1 M NaCl, 1 mM EDTA), the washing buffer (4.3 mM NaH2PO4, 15.1 mM Na2HPO4, 500 mM NaCl, 0.5% Tween 20), and the other solutions were prepared using deionized water (Barnstead, Nanopure II, Van Nuys, CA). The electron-conducting redox polymer, polyacrylamide (PAA)poly(4-vinylpyridine) (PVP)-[Os(bpy)2Cl]+/2+, a copolymer of PAA and PVP complexed with [Os(2,2′-bipyridine)2Cl2]2+/3+, was synthesized as previously described.13 Instruments and Electrodes. The oligonucleotides were synthesized on a Perseptive Biosystems 8909 DNA synthesizer. Oligo-BOD conjugate was concentrated in a Speedvac concentrator SPD121P (Savant, Holbrook, NY). The BioLogic LP lowpressure chromatography system (Bio-Rad, Hercules, CA) was used to purify the oligo-BOD conjugate. The electrochemical measurements were carried out in a Faraday cage with a CH Instruments (Austin, TX) model 832A electrochemical detector, interfaced to a computer (Dell Dimension 8100, Austin, TX). The DNA solutions were prepared, and the hybridizations were carried out, in 0.5-mL polypropylene PCR tubes (Catalog No. PCR06) and 1.5-mL polypropylene microcentrifuge tubes (Catalog No. MCT-149) from United Scientific Products (San Leandro, CA). The three electrodes of the electrochemical cells were a 10µm-diameter glassy carbon microelectrode (Catalog No. EE017), a 0.5-mm-diameter platinum wire counter electrode, and a miniature Ag/AgCl reference electrode (Catalog No. EE008), all purchased from Cypress Systems (Lawrence, KS). The microelectrodes were polished sequentially with 1.0-, 0.3-, and 0.05-µm alumina pastes with sonication between each polishing step. The polished electrodes were rinsed extensively with water and were stored in deionized water. Preparation of the Capture and the Target Sequences. The capture sequence was modified at its 3′ end with a 12-T spacer, to which a seven-carbon spacer terminated with an amine was attached. The capture probe, the target oligonucleotides, and the detection sequence were synthesized with retained dimethoxytrityl protective groups, in a DNA synthesizer. The capture probe and the target oligonucleotides were purified using Poly Pak II cartridges, per instructions provided by the manufacturer. The product of the synthesis and the success of its purification were confirmed by mass spectrometry. The detection sequence was stored at -20 °C until it was used in making the oligo-BOD conjugate. (13) Zhang, Y.; Kim, H.-H.; Mano, N.; Dequaire, M.; Heller, A. Anal. Bioanal. Chem. 2002, 374, 1050-1055.

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Scheme 1. Linking the Detection Oligonucleotide to BOD, Utilizing E-N-Maleimidocaproic Acid (2-Nitro-4-sulfo)phenyl Ester as the Linker

Synthesis of the BOD-Conjugated Detection Sequence. Enzyme-Linker Coupling. 40-mg of BOD was dissolved in 1.5 mL of 0.1 M sodium phosphate buffer (pH 7.5). After adding 3 mg of -N-maleimidocaproic acid (2-nitro-4-sulfo)phenyl ester, the mixture was incubated for 30 min at room temperature in the dark. After desalting using a pre-packed G-25 Sephadex (NAP-25) column, equilibrated with 0.1 M, pH 6.0 sodium phosphate buffer, the enzyme solution was collected in 2.0 mL of buffer. Reduction of the Disulfide with Dithiothreitol. Approximately 75 nmol of the detection oligonucleotide was incubated at room temperature for 30 min in 0.1 mL of 50 mM, pH 8.5 sodium phosphate buffer containing 100 mM dithiothreitol. The solution was desalted by loading on prepacked G-25 Sephadex (NAP-5) column, equilibrated with 0.1 M, pH 6.0 sodium phosphate buffer. The desalted and detritylated oligonucleotides were eluted with 0.5 mL of the buffer. Binding of BOD to the Detection Sequence. Approximately 6075 nmol of the detritylated oligonucleotide (0.5-1.0 mL) was combined with 0.5 mL of the activated enzyme solution (∼10 mg). The mixture was concentrated to ∼1 mL using a Speedvac concentrator and was incubated at 4 °C for 18-24 h. The reactions are summarized in Scheme 1. Purification of the BOD-Labeled Detection Sequence. The detection sequence-BOD conjugate was separated from the unconjugated detection sequence and from the non-DNA bound enzyme by gradient elution from a DEAE-Sepharose FF column using pH 4.5, 20 mM citrate buffer and 1 M NaCl in 20 mM pH 4.5 citrate buffer solutions. The elution sequence was (1) unmodified enzyme, (2) detection sequence-BOD, and (3) unmodified detection sequence. To eliminate the unmodified BOD, the column was washed with 10 column bed volumes of citrate buffer (20 mM pH 4.5) containing 0.15 M NaCl. The BOD-oligo conjugate was eluted by raising the gradient’s upper NaCl concentration in the

Figure 1. Electrocatalysis of the reduction of O2 to water when the bilirubin oxidase (BOD) label is “wired” by the Os2+/3+ complex containing redox polymer gel.

Figure 2. DEAE-Sepharose FF column profile of unreacted BOD and of the BOD-oligo conjugate after gradient elution.

Figure 3. SDS-PAGE of the oligo-BOD conjugate. The 10% gels used were run for 1 h at a fixed 120-V potential gradient. M indicates the molecular weight marker in the gel. Lane 1, unreacted BOD; lane 2, the BOD-oligo conjugate.

pH 4.5, 20 mM citrate buffer (Figure 2). Elution of the conjugate started at 0.4 M NaCl. The absorbances of the eluted fractions at 280 nm were tracked, and the fractions absorbing at 280 nm were pooled and concentrated using Centricon centrifugal filter units (Millipore, Billerica, MA). Fractions containing protein were checked for purity as well as molecular weight. The bilirubin oxidase purchased from Amano contained biuret-positive protein impurities, seen in SDS-PAGE. The molecular weight of the oligo-BOD conjugate was, as expected, >65 000, above the 62 000 molecular weight of the unmodified BOD (Figure 3). Even after purification, two or more bands were observed on the gel, a possible impurity being the BOD modified with the linker, but not with the DNA.

Figure 4. Native polyacrylamide gel electrophoresis of unbound BOD and of the BOD-oligonucleotide conjugate. Lane 1, unreacted BOD; lane 2, BOD-oligo conjugate.

The protein was assayed with a Bio-Rad (Hercules, CA, Catalog No. 500-0006) protein assay kit. The activity of the detection sequence-bound BOD was determined according to the protocol of Shimizu et al.14 The purity of the detection sequence-BOD conjugate and their molecular weights were confirmed by SDSPAGE and native PAGE following the protocol of Sambrook et al.15 The specific activity of oligo-BOD conjugate was 0.70 unit/ mg of protein, close to that of the unmodified BOD (1.1 units/ mg of protein). Electrodeposition of the Redox Polymer and the Capture Sequence. The redox polymer, and then the 13-base-long capture sequence, were electrodeposited on 10-µm-diameter carbon fiber tips in a miniature three-electrode cell. The polymer was electrodeposited from 200 µL of a 1 mg mL-1 PAA-PVP-Os solution in PBS (20 mM phosphate, 100 mM NaCl, pH 7.4), with the microelectrode poised at -1.4 V versus Ag/AgCl for 2 min. After washing the electrode thoroughly with deionized water to remove all soluble redox polymer, the presence of the deposited redox polymer film was confirmed by cyclic voltammetry. The capture sequence was then electrodeposited by placing the microelectrode in 200 µL of the 1 µM capture sequence solution in PBS and poising the electrode at -1.4 V (Ag/AgCl) for 20 min. The electrodes were rinsed and stored in PBS at 4 °C until use. Assay. The 5-µL droplet to be tested for the presence of the Shigella DNA (pH 7, 10 mM HEPES, 1 M NaCl, 1 mM EDTA) was placed in a 0.5-mL polypropylene test tube (United Scientific Products, San Leandro, CA). The tip of the microelectrode was exposed to the tested liquid for 1 h at 43 °C, exposed for 20 min to 400 µL of the 10 nM BOD-labeled 14-base-long detection sequence solution (20 mM phosphate, 0.1 M NaCl, pH 7.4, 32 °C, magnetic stirring), and rinsed with the same buffer for 5 min. The O2 electroreduction current was then measured with the microelectrode poised at +0.12 V (Ag/AgCl) in 200 µL of airsaturated, pH 7.4, 20 mM phosphate, 0.1 M NaCl solution. RESULTS AND DISCUSSION Simplicity of the Assay. Figure 5 illustrates the scheme of the assay. The part of the assay to be performed not by the user but by the supplier of the target-specific microelectrode is shown (14) Shimizu, A.; Kwon, J.-H.; Sasaki, T.; Satoh, T.; Sakurai, N.; Sakurai, T.; Yamaguchi, S.; Samejima, T. Biochemistry 1999, 38, 3034-3042. (15) Sambrook, J.; Fritsch, E. F.; Maniatis, T. Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, NY, 1989.

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Figure 5. Scheme for the ambient O2-utilizing BOD-amplified amperometric detection of DNA.

at the top of the figure. The supplier-performed operations would be the preparation of the microelectrode and electrodepositions of the redox polymer and of the capture sequence. The userperformed operations are shown at the bottom of Figure 5. These are exposure of the tip to the tested solution, rinsing, and exposure to the detection sequence containing solution and rinsing. Overall, the assay is simpler than any of the earlier reported assays whereby DNA was detected at 1 fM concentration. A related assay, where the enzyme label was HRP,1 required use of an additional solution, 1 mM H2O2, and the additional step of placing the sandwich-carrying microelectrode in this solution. The recently reported assay of Xie et al.,12 requires four user-performed steps (including measurement) other than rinses, while the present assay requires only three user-performed operations (hybridization of the capture probe with the target, hybridization of the target with the detection probe, and measurement of the current). Additionally, the number of solutions required is reduced from four to three. Rapid Equilibration. The microelectrode, modified with the completed sandwich, reaches its steady-state O2 electroreduction current in less than 1 min. (Figure 6). Components of the Sandwich. The synthesized components of the sandwich are listed in Table 1. Note the modifying functions of the capture and the detection sequences. 4096 Analytical Chemistry, Vol. 76, No. 14, July 15, 2004

Figure 6. Time dependence of the O2 reduction current: +0.12 V (Ag/AgCl); 200 µL of air-saturated, pH 7.4, 0.1 M NaCl, 20 mM phosphate buffer solution. (a) No Shigella DNA; (b) [Shigella DNA] ) 1.0 fM; (c) [Shigella DNA] ) 10.0 fM. The hybridization volumes were 5 µL.

BOD Labeling of the Detection Sequence. The core of the novel assay is the BOD labeling of the detection sequence. The synthetic strategy for this labeling was derived from protocols of Nakagami et al.16 and Ghosh et al.17 for binding of alkaline phosphatase and horseradish peroxidase to DNA through a

Figure 7. Dependence of the O2 electroreduction current on the Shigella DNA concentration. Ta, current in the absence of any DNA; Tb, current in the presence of 10 pM noncomplementary DNA. Table 1. The NH2-Modified Capture, the Capture, and the BOD-Modified detection Sequences of the Sandwich

maleimide linker. Their protocols were substantially simplified by adding a dithiol function onto the oligonucleotide. Reductive cleavage of the dithiol by dithiothreitol yielded the thiol-terminted oligonucleotide. One or more of the surface amines of the BOD were reacted with -N-maleimidocaproic acid (2-nitro-4-sulfo)phenyl ester. In the final step, the thiol was condensed with the double bond of the maleic anhydride. Because the conjugate had one or more oligonucleotides, its negative charge was increased and it migrated more rapidly on native PAGE in its purification (Figure 4). Shigella DNA Assay. The microelectrodes were stored in buffer solution at 4 °C up to 6 months without significant loss of the deposited redox polymer. When present, the target DNA hybridized through part of its sequence to the immobilized capture sequence. A different part of the target was complementary to and hybridized with the BOD-labeled detection sequence, completing the formation of the “sandwich”. Formation of the sandwich brought the BOD label into contact with the redox polymermodified microelectrode. The contacted (“wired”) BOD catalyzed the electroreduction of O2, the observed current scaling with the concentration of the target DNA (Figure 7). As seen in Figure 7, the current increased with the concentration of the target DNA in the range between 0.5 and 20 fM. Above 20 fM, the current did not increase further, apparently because of saturation of the

DNA-capturing sites of the redox polymer film. The sensitivity of the assay was similar to that of an earlier reported H2O2-requiring assay,1 where the label of the detection sequence was HRP. More concentrated solutions would be assayed after dilution, conventional dilution providing the desired upper limit of the dynamic range. In actual assays in the field, endonucleases would be added to the bacterial lysate to hydrolyze the DNA to well-defined segments. Control Experiments. Two sets of control experiments were performed. In the first, no target DNA was added. In the second, a large excess of a noncomplementary ss-DNA was added, to bring DNA concentration to 10 pM, 104 times above the detectable concentration of the true target. No change in the O2 reduction current was observed in either experiment (Figure 7), showing that the assay was highly selective.

(16) Nakagami, S.; Matsunaga, H.; Oka, N.; Yamane, A. Anal. Biochem. 1991, 198, 75-79. (17) Ghosh, S. S.; Kao, P. M.; McCue, A. W.; Chapplelle, H. L. Bioconjugate Chem. 1990, 1, 71-76.

Received for review March 29, 2004. Accepted April 28, 2004.

ACKNOWLEDGMENT The research reported in this document/presentation was performed in connection with Contract DAAD13-02-C-0079 with the U.S. Edgewood Biological Chemical Command. A.P. thanks Dr. Sean M. Kerwin and Dr. Walter Fast, College of Pharmacy, University of Texas at Austin, for providing facilities for making the BOD-labeled detection sequence.

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