Enzyme-Linked Amplified Electrochemical Sensing of Oligonucleotide

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Langmuir 1999, 15, 3703-3706

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Enzyme-Linked Amplified Electrochemical Sensing of Oligonucleotide-DNA Interactions by Means of the Precipitation of an Insoluble Product and Using Impedance Spectroscopy Fernando Patolsky, Eugenii Katz, Amos Bardea, and Itamar Willner* Institute of Chemistry, The Hebrew University of Jerusalem, Jerusalem 91904, Israel Received December 4, 1998. In Final Form: March 19, 1999 A novel method for the sensitive and specific electrochemical analysis of DNA is described using Faradaic impedance spectroscopy. A thiol-thymine-tagged oligonucleotide (1) capable of forming only one doublestranded turn with the target DNA analyte (2) is assembled on a Au electrode and acts as the sensing interface. The resulting functionalized electrode is reacted with a complex between the target DNA (2) and a biotinylated oligonucleotide (3) to yield a bifunctional double-stranded assembly on the electrode support. The Faradaic impedance spectra, using Fe(CN)63- as redox probe, reveal an increase in the electrontransfer resistance at the electrode surface upon the construction of the double-stranded assembly. This is attributed to the electrostatic repulsion of Fe(CN)63- upon formation of the negatively charged doublestranded superstructure. Binding of an avidin-HRP conjugate to the oligonucleotide-DNA assembly further insulates the electrode and increases the interfacial electron-transfer resistance. The HRP-mediated biocatalyzed oxidation of 4-chloro-1-naphthol (4) by H2O2 yields a precipitate (5) on the conductive support and stimulates a very high barrier for interfacial electron transfer, Ret ) 14.7 kΩ. Thus, the precipitation of 5 confirms and amplifies the sensing process of the target DNA (2). The analyte DNA (2) corresponds to the mutated gene fragment characteristic of the Tay-Sachs genetic disorder. The normal gene (2a) is easily discriminated by the sensing interface. The sensor device enables detection of the target DNA (2) with a sensitivity of at least 20 × 10-9 g‚mL-1. Cyclic voltammetry experiments further confirm the formation of barriers for the interfacial electron transfer upon the buildup of the double-stranded oligonucleotide-DNA structure and upon the biocatalytic deposition of 5 on the electrode surface.

The development of DNA sensor devices has attracted substantial recent research efforts directed to gene analysis, detection of genetic disorders, tissue matching, and forensic applications.1,2 Optical detection of DNA was accomplished by the application of fluorescence-labeled oligonucleotides3 or by the use of surface plasmon resonance.4 Electronic transduction of the formation of oligonucleotide complexes with a target DNA and, particularly, in the quantitative assay of DNA is a major challenge of bioelectronics.5 The organization of DNA sensors requires the assembly of the sensing interface on a transducer and the design of the appropriate electronic output that signals the formation of the recognition complex with the target DNA analyte on the transducer element. Electrochemical DNA sensors based on the amperometric transduction6 of the formation of doublestranded (ds)-oligonucleotide-DNA assemblies or the electrostatic attraction of electroactive transition metal complexes or organic dyes to oligonucleotide-DNA ds* Corresponding author. Telephone: 972-2-6585272. Fax: 9722-6527715. E-mail: [email protected]. (1) (a) Bard, A. J.; Carter, M. T. J. Am. Chem. Soc. 1987, 109, 75287530. (b) Bard, A. J.; Rodriguez, M. Anal. Chem. 1990, 62, 2658-2662. (c) Wilson, E. K. Chem. Eng. News 1998, 76, 47-49. (2) (a) Millan, K. M.; Mikkelsen, S. R. Anal. Chem. 1993, 65, 23172323. (b) Hashimoto, K.; Ito, K.; Ishimori, Y. Anal. Chem. 1994, 66, 3830-3833. (c) Yang, M.; McGovern, M. E.; Thompson, M. Anal. Chim. Acta 1997, 346, 259-275. (3) (a) Piunno, P. A. E.; Krull, V. J.; Hudson, R. H. E.; Damha, M. J.; Cohen, H. Anal. Chim. Acta 1994, 288, 205-209. (b) Mandenius, C. F.; Chollet, A.; Lenburg, M. M.; Lundstro¨m, I. Anal. Lett. 1989, 22, 2961-2964. (4) (a) Lidberg, B.; Nylander, C.; Lundstro¨m, I. Sens. Actuators 1983, 4, 299-302. (b) Jonsson, V. Biotechniques 1991, 11, 620-624. (5) Mikkelsen, S. R., Electroanalysis 1996, 8, 15-23. (6) (a) Korri-Youssoufi, H.; Garnier, F.; Srivastava, P.; Godillot, P.; Yassar, A. J. Am. Chem. Soc. 1997, 119, 7388-7389. (b) Livache, T.; Roget, A.; Dejean, E.; Barthet, C.; Bidan, G.; Teoule, R. Synth. Met. 1995, 71, 2143-2146.

complexes,7,8 for example, Co(bpy)33+, acridin, or Hoechst 33258, were reported. Microgravimetric quartz-crystalmicrobalance, QCM,9 analyses were also applied to sense the formation of complementary oligonucleotide-DNA complexes. Two major difficulties are still encountered in the development of DNA sensors and relate to the sensitivity and specificity of the resulting sensing systems. The biocatalyzed precipitation of insoluble products on solid supports was employed as a means to assay biorecognition events.10 Within our broad activities in the organization of layered enzyme electrodes,11,12 we have recently assembled monolayer enzyme electrodes, for example peroxidase, that stimulate the biocatalytic precipitation of a product upon sensing an analyte.13 Here we wish to report on the development of a novel oligonucleotide monolayer interface for the specific and sensitive (7) (a) Millan, K. M.; Sanauloo, A.; Mikkelsen, S. R. Anal. Chem. 1994, 66, 3830-3833. (b) Hashimoto, K.; Ito, K.; Ishimori, Y. Anal. Chem. 1994, 66, 1236-1241. (8) (a) Hashimoto, K.; Ito, K.; Ishimori, Y. Anal. Chim. Acta 1994, 286, 219-224. (b) Wang, J.; Palecek, E.; Nielson, P. E. J. Am. Chem. Soc. 1996, 118, 7667-7670. (c) Ihara, T.; Nakayama, M.; Murata, M.; Nakano, K.; Maeda, M. Chem. Commun. 1997, 1069-1070. (9) Bardea, A.; Dagan, A.; Ben-Dov, I.; Amit, B.; Willner, I. Chem. Commun. 1998, 839-840. (10) (a) Bobrow, M. N.; Harris, T. D.; Shaughnessy, K. J.; Litt, G. J. J. Immunol. Methods 1989, 125, 279-285. (b) Bobrow, M. N.; Shaughnessy, K. J.; Litt, G. J. J. Immunol. Methods 1991, 137, 103-112. (c) Bobrow, M. N.; Litt, G. J.; Shaughnessy, K. J.; Mayer, P. C.; Conlon, J. J. Immunol. Methods 1992, 150, 145-149. (11) (a) Willner, I.; Katz, E.; Riklin, A.; Kasher, R. J. Am. Chem. Soc. 1992, 114, 10965-10966. (b) Willner, I.; Katz, E.; Willner, B. Electroanalysis 1997, 13, 965-977. (c) Riklin, A.; Willner, I. Anal. Chem. 1995, 67, 4118-4126. (12) (a) Willner, I.; Heleg-Shabtai, V.; Blonder, R.; Katz, E.; Tao, G.; Bu¨ckmann, A. F.; Heller, A. J. Am. Chem. Soc. 1996, 118, 1032110322. (b) Willner, I.; Riklin, A.; Shoham, B.; Rivenzon, D.; Katz, E. Adv. Mater. 1993, 5, 912-915. (13) Patolsky, F.; Zayats, M.; Katz, E.; Willner, I. Anal. Chem., in press.

10.1021/la981682v CCC: $18.00 © 1999 American Chemical Society Published on Web 04/29/1999

3704 Langmuir, Vol. 15, No. 11, 1999

Letters

Scheme 1. Stepwise Assembly of a DNA-sensing Electrode. Electrochemical Sensing and Amplification of the Target DNA (2) Using an Oligonucleotide Functionalized Electrode and a Labeled Oligonucleotide Probe

analysis of a DNA fragment (the gene fragment responsible for the Tay-Sachs genetic disorder). An enzyme-avidin conjugate, associated with the biotinylated oligonucleotide probe, linked to the target DNA, leads to the biocatalytic precipitation of a product. This process is used to sense and amplify the formation of the ds-recognition complex with the target DNA analyte. Impedance spectroscopy is used to follow the interfacial properties (capacitance, electron-transfer resistance) of the layered electrode upon

formation of the oligonucleotide complexes and precipitation of the enzyme product.14 The methods to analyze the target DNA analyte and to assemble the DNA sensor bioelectronic system are depicted in Scheme 1. The 18-mer oligonucleotide 1 includes (14) (a) Bard, A. J.; Faulkner, L. R. Electrochemical Methods: Fundamentals and Applications; Wiley: New York, 1980. (b) Stoynov, Z. B.; Grafov, B. M.; Savova-Stoynov, B. S.; Elkin, V. V. Electrochemical Impedance; Nauka Publisher: Moscow, 1991.

Letters

a 12-base sequence that is complementary to a part of the analyte, the Tay-Sachs (TS) mutant (2). In addition, it includes a 5-base thiophosphate thymine-TS tag for its assembly on the Au electrode, and a single T-base separating the tag from the sensing oligonucleotide sequence. A disk Au electrode, 0.05 cm2, was interacted with (1) (20 µM, 10 h), resulting in the assembly of the sensing interface on the gold support15 (step x in Scheme 1). The resulting 1-functionalized electrode was interacted with a solution that included the target analyte, the TS mutant (2) (5.8 × 10-7 g‚mL-1, 4 h), and the biotinylated oligonucleotide (3) (2 × 10-5 g‚mL-1) (step y in Scheme 1). The latter DNA-labeled oligonucleotide is complementary to the sensing interface assembled on the electrode. The resulting bifunctional double-stranded DNA-oligonucleotide assembly is subsequently treated with avidinlabeled-horseradish peroxidase (HRP) (1 × 10-8 g‚mL-1, 3 h) (step z in Scheme 1). Association of the enzyme label to the assembly enables the biocatalytic oxidation of 4-chloro-1-naphthol (4) by H2O2 and the precipitation of the product (5) onto the electrode. As the oligonucleotide and oligonucleotide-DNA layered assemblies are negatively charged, the electrostatic repulsion of a negatively charged redox probe, for example Fe(CN)63-/4-, from the electrode support is anticipated to perturb the interfacial electron transfer. This is expected to introduce an electron-transfer resistance that can be detected by Faradaic impedance spectroscopy or other electrochemical means. The biocatalytic precipitation of the product (5) on the electrode is expected to further insulate the conductive support and to lead to a high interfacial electron-transfer resistance. Figure 1A shows the impedance features, using Fe(CN)63-/4- as redox probe, presented as Nyquist plots (Zim versus Zre), of the bare electrode (curve a), the 1-functionalized electrode (curve b), and the layered bifunctional double-stranded oligonucleotide-analyte DNA and biotinylated oligonucleotide assembly (curve c). The respective semicircle diameters correspond to the interfacial electron-transfer resistances Ret. It can be seen that the electron-transfer resistance increases upon the buildup of the biotinylated oligonucleotide-DNA assembly. For example, for the 1-functionalized electrode Ret ) 1.1 kΩ whereas Ret increases to approximately 2 kΩ upon the association of the complex between 2 and the biotinylated oligonucleotide 3. These results are consistent with the fact that the negative charge associated with the phosphate groups of the different oligonucleotides increases upon the two-step organization of the assembly. This results in the enhanced electrostatic repulsion of the redox probe and introduces higher interfacial electron-transfer resistances. Figure 1B shows the impedance spectra of the bifunctional double-stranded assembly consisting of the target DNA linked to the sensing interface and the biotinylated oligonucleotide, before (curve c) and after (curve d) interaction with the avidin-HRP conjugate. Upon the association of the avidin-HRP biocatalytic conjugate to the layer, a considerable increase in the electron-transfer resistance is observed due to the partial insulation of the electrode by the proteins. In the presence of H2O2 and the substrate 4, biocatalytic precipitation of the product onto the electrode occurs. This insulates the conductive support, resulting in a very high increase in the electron-transfer resistance (curve e; Ret ) 17 kΩ). Note the difference in the scales of the Zre axes of parts A and B of Figure 1. The association of the avidin-HRP conjugate to the oligo(15) A detailed analysis of the effect of the time interval of precipitation of 5 on the sensitivity limits of the analysis of 2 will be reported in a forthcoming comprehensive report.

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Figure 1. Nyquist diagram (Zim versus Zre) for the Faradaic impedance measurement. (A) stepwise assembly of the bifunctional double-stranded oligonucleotides-DNA assembly: (a) bare electrode; (b) step x, after deposition of the sensing oligonucleotide 1 onto the interface; (c) step y, after formation of the complex between the sensing interface and the target DNA analyte (2) complexed with the biotinylated oligonucleotide (3). Concentration of 2 ) 5.8 × 10-7 g‚mL-1 and that of 3 ) 2.6 × 10-6 g‚mL-1; interaction time ) 2 h. (B) Stepwise deposition of the avidin-HRP conjugate and precipitation of 5 onto the electrode: (c) step y, bifunctional double-stranded assembly formed between the complex of 2 with 3 and the sensing interface; (d) step z, after deposition of avidin-HRP, 1 × 10-8 g‚mL-1, 3 h; (e) after deposition of the precipitate (5) onto the electrode by incubation of the superstructured electrode in a phosphate buffer solution, 0.1 M, pH ) 7.3, which includes 4, 5 × 10-3 M, and H2O2, 5 × 10-3 M, for 20 min. All measurements were recorded in a phosphate buffer solution, 0.1 M, pH ) 7.3, using Fe(CN)3-/4-, 5 × 10-3 M, as redox probe. The impedance spectra were recorded within the frequency range 0.1 Hz to 10 kHz at the formal potential of the [Fe(CN)6]3-/4- redox couple. The amplitude of the alternating voltage was 5 mV. Inset: Cyclic voltammograms of the superstructured electrode in the presence of Fe(CN)63-/4-, 5 × 10-3 M; parts a-e correspond to the same electrode structures described for the impedance measurements.

nucleotide-DNA assembly and the precipitation of the product induce an approximately 10-fold increase in the interfacial electron-transfer resistance as compared to that for the changes that occurred upon the formation of the ds-assembly between the sensing interface and the complex between the DNA analyte (2) and the biotinlabeled oligonucleotide (3). It should be noted that the two parameters controlling the sensitivity of the DNAsensing devices are the time of incubation of the 1-functionalized monolayer electrode with the complex between the analyte DNA and the biotinylated oligonucleotide, and, more important, the time interval used to precipitate the product by the avidin-HRP biocatalytic conjugate. Figure 2 shows the electron-transfer resistance at the sensing interface upon precipitation of the insoluble product at different concentrations of the analyte DNA (2). It is evident that as the bulk concentration of the DNA is

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Figure 2. Electron-transfer resistance observed at the sensing electrode upon the precipitation of the insoluble product (5) at different bulk concentrations of the DNA analyte (2). For all experiments, the DNA analyte was interacted with 3, 2.6 × 10-6 g‚mL-1, for 2 h. The resulting ds-oligonucleotide DNAanalyte assembly was interacted with avidin-HRP, 1 × 10-8 g‚mL-1, for 3 h. Precipitation of the insoluble product (5) was conducted in the presence of 4, 5 × 10-3 M, and H2O2, 5 × 10-3 M, for 40 min. Impedance spectra were recorded in a phosphate buffer solution, 0.1 M, pH ) 7.3, using Fe(CN)63-/4-, 5 × 10-3 M, as redox probe. The impedance spectra were recorded as described in the caption of Figure 1.

lowered, the observed electron-transfer resistance decreases as a result of the precipitation of the insoluble product. This is consistent with the fact that lower bulk concentrations of 2 yield a lower coverage of the 2 and biotin-labeled oligonucleotide complex on the sensing interface. This results in lower coverage of the interface with avidin-HRP, and consequently, a decreased efficiency in the deposition of the insoluble product is observed. Using this configuration, and upon precipitation of 5 for 40 min, we were able to sense 2 at a concentration of 20 × 10-9 g‚mL-1, Ret ) 7.9 kΩ. It should be noted that, upon the application of longer precipitation time intervals, the sensitivity of the analysis could be enhanced.15 Control experiments reveal that the oligonucleotidesensing assembly reveals high specificity and selectivity. Treatment of the 1-functionalized electrode with the biotinylated oligonucleotide, and then with the avidinHRP conjugate, but without the interaction with the DNA analyte, yields only a minute change in the electrontransfer resistance. Also, the sensing interface, for example, the 1-functionalized monolayer electrode, was interacted with a solution that included the DNA fragment (2a) and the biotin-labeled oligonucleotide (3). The oligonucleotide (2a) corresponds to the normal gene sequence in which the 7-base mutation leads to the TS genetic disorder. After treatment of the sensing interface with the complex between 2a and 3, the system was subjected to the biocatalytic precipitation process using the avidinHRP conjugate. No noticeable changes in the electrontransfer resistances at the electrode were observed, implying that the lack of formation of the ds-oligonucleotide-DNA (analyte) complex with the sensing interface prevented the subsequent formation of the precipitate layer on the electrode. Furthermore, interaction of the electrode consisting of the ds-sensing oligonucleotide, the DNA analyte (2), and the biotin-labeled oligonucleotide (3) with avidin (rather than avidin-HRP) and incubation of the resulting system with 5 and H2O2 do not yield any significant change in the interfacial electron-transfer resistance. This implies that 5 by itself (or in the presence of H2O2) does not precipitate on the ds-oligonucleotideDNA-avidin assembly.

Letters

Cyclic voltammetry experiments further confirm that the stepwise organization of the bifunctional doublestranded oligonucleotide DNA-biotinylated oligonucleotide array and the precipitation of the insulating layer of 5 on the electrode gradually perturb the electrontransfer kinetics of Fe(CN)63-. Figure 1B, inset, shows the cyclic voltammograms of Fe(CN)63- at a bare Au electrode, curve a, upon the assembly of the oligonucleotide-sensing layer of 1, curve b, and upon the formation of the double-stranded assembly with the complex between the target DNA analyte (2) and the biotinylated oligonucleotide (3), curve c. The stepwise assembly of the layers is accompanied by a decrease in the amperometric response of the electrode and an increase in the peakto-peak separation between the cathodic and anodic waves of the redox probe. This is consistent with the enhanced electron-transfer barriers introduced upon the assembly of the negatively charged oligonucleotide assembly. Association of the avidin-HRP conjugate onto the layer, curve d, further separates the redox waves of Fe(CN)63-, implying that binding of the proteins insulates the electrode and perturbs the interfacial electron transfer. Biocatalytic precipitation of 5 onto the electrode insulates the conductive support, and the electrical response of the redox probe is almost entirely blocked, curve e. It should be noted, however, that formation of the double-stranded assembly between the sensing interface and the complex between the analyte DNA (3) and the biotin-labeled oligonucleotide yields only a minor change in the cyclic voltammogram of Fe(CN)63-/4-, whereas the binding of the avidin-HRP conjugate and the subsequent precipitation of the product (5) significantly perturb the resulting cyclic voltammogram of the redox probe. This is in agreement with the partial coverage of the Au surface by the sensing oligonucleotide (1).16 The resulting free surface domains, presumably originating from the electrostatic repulsion of internucleotide strands, enable the direct electrical contact between the redox probe and the electrode. The high-molecular-weight avidin-HRP and specifically the precipitation of the insoluble product (5) block these free surface-domains and prohibit the electron transfer between Fe(CN)63-/4- and the electrode support. In conclusion, we have demonstrated a novel method for the sensitive analysis of DNA using Faradaic impedance spectroscopy. The use of a biotinylated oligonucleotide and an avidin-HRP conjugate for the precipitation of the insulating layer on the electrode provides a means to confirm and amplify the analysis of the target DNA. Preliminary studies reveal that other transduction means, such as microgravimetric quartz-crystal-microbalance, QCM, or surface plasmon resonance, SPR, enable probing the formation of the insoluble product on the DNA layer. Also, other avidin-enzyme conjugates, for example alkaline phosphatase, can be used as biocatalysts for the precipitation of insoluble products that amplify the formation of the ds-complex between the analyte DNA and the sensing interface. Acknowledgment. This research is supported by the Infrastructure Project on Biomicroelectronics, The Israel Ministry of Science. LA981682V (16) Microgravimetric quartz-crystal-microbalance experiments on a Au quartz crystal (9 MHz) indicate a surface coverage of 1 on the Au support corresponding to 4.1 × 10-11 mol‚cm-2.