- Email: [email protected]
Reagentless detection of DNA sequences on chemically modified electrodes
Update
522
TRENDS in Biotechnology
Vol.21 No.12 December 2003
Reagentless detection of DNA sequences on chemically modified electrodes H. Holden Thorp Department of Chemistry, University of North Carolina, Chapel Hill, North Carolina, 27599-3290, USA
Microarray analysis requires complex optical instruments and numerous reagents. Several new electrochemical methods for creating sequence-selective microlocations are emerging; such approaches potentially facilitate electrochemical (electronic) readouts of microarrays. These developments parallel the migration of glucose sensing in diabetes monitoring from optical to electrochemical methods. Recent work provides a strategy that minimizes the use of added reagents and potentially produces a reusable sensor that can be applied to continuous monitoring applications. The electrochemical detection of specific DNA sequences on spatially resolved microlocations potentially enables microarray analysis of complicated mixtures of nucleic acids without the use of optical equipment such as lasers and charge-coupled device (CCD) cameras [1– 6]. These advances would vastly reduce the cost and complexity of array technology. Electrochemistry ultimately facilitates much smaller microlocations (or nanolocations) than those allowed by optical methods [1,7], which are constrained to the micrometer-scale spatial resolution of optical fabrication and detection techniques. Because of these potential advantages, there has been considerable effort directed toward the development of chemically modified electrodes that can be used to detect specific DNA or RNA sequences [1,8 – 11]. Such a device consists of a surface suitable for electrochemical analysis, such as carbon, gold or metal oxide, which is modified with a single-stranded probe nucleic acid that is complementary to a desired target. When the desired target hybridizes to the probe attached to the electrode, an electrochemical signal is produced. Desirable systems are those that are highly sequence-specific, are reusable, require as few added reagents as possible and show very low limits of detection. Recently, Fan et al. [12] reported the realization of an electrochemical sensor involving ‘molecular beacon’ technology that is reagentless and partially reusable. This in-progress report demonstrates significant technical advances and provides evidence of the continued enthusiasm for developing electrochemical DNA analysis. In 2000, Kuhr wrote a commentary on a similar theme entitled ‘Electrochemical DNA analysis comes of age’, suggesting that this area of biotechnology was here to stay [4]. Indeed, despite great changes in the economic environment, the pursuit of electrochemical DNA analysis Corresponding author: H. Holden Thorp ([email protected]). http://tibtec.trends.com
continues unabated in both academia and industry [1,8 –11]. In this report, I describe the approach of Fan et al. [12] and how it fits into the larger picture of the field. Electrochemical detection using the E-DNA sensor The design of the sensor developed by Fan et al. [12] is shown in Figure 1. The general strategy involves an E-DNA sensor – a hairpin-forming oligonucleotide immobilized to a conductive electrode. The distal end of the oligonucleotide is labeled with a redox-active moiety that can be oxidized or reduced by the electrode. The hairpin-forming oligonucleotide belongs to a family known as ‘molecular beacons’ [13 – 15], in which the relatively large loop region of the hairpin is complementary to a target sequence. In the hairpin form, the redox-active moiety is close to the electrode and undergoes oxidation or reduction. Hybridization of the loop region to the target disrupts the hairpin, increasing the distance from the redox-active moiety to the electrode and thereby lowering the electrochemical signal. Such a configuration has been described conceptually in the patent literature [16], and Mao et al. have recently described elements of a similar system [17]. Here, I discuss the system of Fan et al., which involves a gold electrode and a thiol-modified oligonucleotide with ferrocene as the redox-active moiety. The electrochemical response of the E-DNA sensor is shown in Figure 2, which plots background-subtracted anodic scans from cyclic voltammetry measurements obtained in the presence of increasing quantities of the target sequence. The probable reason for the decrease in electrochemical signal at the sensor is the increase in charge-transfer distance for the extended duplex as compared with the stem-loop; such an assignment is consistent with that deduced from recent solution studies involving an osmium –nucleobase donor– acceptor pair situated similarly on a hairpin oligonucleotide [18]. The impressive characteristic of the response is the dynamic range, which spans six orders of magnitude of DNA concentration (Figure 2). Although this broad dynamic range is useful in some applications, the sensitivity of the method (i.e. the percentage change per increment of input concentration) is correspondingly less marked, making E-DNA more suitable for applications such as the analysis of single nucleotide polymorphisms or the detection of pathogens, in which reading small changes in input concentration is less important (than it is in expression analysis). The limit of detection, 5 fmol (10 pM), is excellent given the reagentless and potentially reusable configuration, but
Update
TRENDS in Biotechnology
523
Vol.21 No.12 December 2003
Iron
+cDNA
e T
Denaturation and re-annealing
eT
Fc
Gold electrode TRENDS in Biotechnology
Figure 1. Representation of the E-DNA strategy. cDNA is the sequence complementary to the loop region, Fc is ferrocene. Picture reproduced with permission from [12].
it is not as low as that of other electrochemical methods that use catalytic cycles or other amplification schemes [1,10,19]. When the hybridized electrode is heatdenatured and re-challenged with the target sequence, about 80% of the signal is recovered. The 20% loss is attributed to the instability of ferrocene, and this problem must be addressed if the reagentless, reusable nature of the sensor is to be properly exploited for the continuous monitoring of pathogens. Broader context for electrochemical sensing As stated above, the E-DNA sensor has added to a growing number of electrochemical detection schemes under development [1,8 – 11]. Each presents a slightly different
45
30
Peak current (nA)
Peak current (nA)
45
30
15
0 10–11 10–10 10–9 10–8 10–7 10–6 10–5 Concentration of cDNA (M)
15
0 0.1
0.2
0.3
0.4 0.5 E (V vs. NHE)
0.6
0.7
TRENDS in Biotechnology
Figure 2. Plots of background-subtracted anodic scans from the cyclic voltammograms of the E-DNA sensor with 0 M, 30 pM, 500 pM, 30 nM, 800 nM and 5 mM input target concentrations. Inset shows peak currents plotted against target concentrations. Picture reproduced with permission from [12]. http://tibtec.trends.com
set of advantages and disadvantages. In addition to the attractive features noted above, the E-DNA sensor has two distinct disadvantages that must be overcome. First, Fan et al. [12] note that the E-DNA sensor is a ‘signal-off ’ sensor, in which the binding of the target produces a decrease in electrochemical signal; signal decreases are difficult to measure because of the high background in the absence of analyte. Second, in its current configuration, sensing involves the transfer of only one electron from the ferrocene moiety to the electrode; therefore, the limit of detection is controlled by the current generated from a single electron transfer and thus the change in signal owing to only one electron transfer event per hybridization. This trade-off is inherent in the ‘reagentless’ set-up, because methods that produce catalytic cycles or multiple labels naturally require more reagents [1,10,19]. The authors point out that miniaturization of the electrode should improve the limit of detection, and numerous studies suggest that such efforts should be successful [1,10,11,20]. The work of Fan et al. confirms that electrochemical DNA detection has indeed ‘come of age’ [4]. The method joins several systems under development, including the catalytic oxidation of nucleobases [1], sandwich assays involving ferrocene-labeled signal probes [8,21], intercalator-mediated ferricyanide electrocatalysis [9], nanoparticle assays [10] and the detection of enzyme-labeled sandwich-type configurations [11]. Each of these methods presents a distinct set of advantages in terms of manufacturability, sensitivity, limits of detection, reusability, reagent requirements and reliability. The E-DNA approach makes significant advances towards a reagentless, reusable sensor and provides further encouragement that, as has occurred with glucose monitoring [22], detection of nucleic acids on spatially resolved microlocations will ultimately migrate from optical to electrochemical methods.
Update
524
TRENDS in Biotechnology
References 1 Popovich, N. and Thorp, H.H. (2002) New strategies for electrochemical detection of nucleic acids. Interface 11, 30 – 34 2 Tarlov, M.J. and Steel, A.B. (2003) DNA-based sensors. In Biomolecular Films: Design, Function, and Applications (Rusling, J.F., ed.), pp. 545 – 608, Marcel Dekker 3 Palecek, E. (2002) Past, present and future of nucleic acids electrochemistry. Talanta 56, 809– 819 4 Kuhr, W.G. (2000) Electrochemical DNA analysis comes of age. Nat. Biotechnol. 18, 1042– 1043 5 Heller, M.J. (2002) DNA microarray technology: devices, systems, and applications. Annu. Rev. Biomed. Eng. 4, 129 – 153 6 Willner, I. (2002) Biomaterials for sensors, fuel cells, and circuitry. Science 298, 2407 – 2408 7 Fan, F.R.F. et al. (1996) Single molecule electrochemistry. J. Am. Chem. Soc. 118, 9669– 9675 8 Umek, R.M. et al. (2001) Electronic detection of nucleic acids – a versatile platform for molecular diagnostics. J. Mol. Diagn. 3, 74 – 84 9 Boon, E.M. et al. (2000) Mutation detection by electrocatalysis at DNA-modified electrodes. Nat. Biotechnol. 18, 1096– 1100 10 Park, S.J. et al. (2002) Array-based electrical detection of DNA with nanoparticle probes. Science 295, 1503 – 1506 11 Oleinikov, A.V. et al. (2003) Self-assembling protein arrays using electronic semiconductor microchips and in vitro translation. J. Proteome Res. 2, 313 – 319 12 Fan, C. et al. (2003) Electrochemical interrogation of conformational changes as a reagentless method for the sequence-specific detection of DNA. Proc. Natl. Acad. Sci. U. S. A. 100, 9134– 9137
Vol.21 No.12 December 2003
13 Fang, X.H. et al. (1999) Designing a novel molecular beacon for surface-immobilized DNA hybridization studies. J. Am. Chem. Soc. 121, 2921– 2922 14 Hui, D. et al. (2003) Hybridization-based unquenching of DNA hairpins on Au surfaces: prototypical “molecular beacon” biosensors. J. Am. Chem. Soc. 125, 4012– 4013 15 Broude, N.E. (2002) Stem-loop oligonucleotides: a robust tool for molecular biology and biotechnology. Trends Biotechnol. 20, 249– 256 16 Bamdad, C. and Yu, C. (1999) Electronic methods for the detection of analytes using monolayers. European Patent Number WO99/57317 17 Mao, Y. et al. (2003) Studies of temperature-dependent electronic transduction on DNA hairpin loop sensor. Nucleic Acids Res. 31, e108 18 Holmberg, R.C. et al. Intramolecular electrocatalysis of 8-oxo-guanine oxidation: secondary structure control of electron transfer in osmiumlabeled oligonucleotides. Inorg. Chem. (in press) 19 Armistead, P.M. and Thorp, H.H. (2000) modification of metal oxides with nucleic acids: detection of attomole quantities of immobilized DNA by electrocatalysis. Anal. Chem. 72, 3764– 3770 20 Szalai, V.A. and Thorp, H.H. (2000) Electrocatalysis of guanine electron transfer: new insights from submillimeter carbon electrodes. J. Phys. Chem. B. 104, 6851 – 6859 21 Yu, C.J. et al. (2001) Electronic detection of single-base mismatches in DNA with ferrocene-modified probes. J. Am. Chem. Soc. 123, 11155– 11161 22 Henry, C.M. (2003) Taking the plunge. Chem. Eng. News 81, 37 – 38
0167-7799/$ - see front matter q 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.tibtech.2003.10.003
Could you name the most significant papers published in life sciences this month? Updated daily, Research Update presents short, easy-to-read commentary on the latest hot papers, enabling you to keep abreast with advances across the life sciences. Written by laboratory scientists with a keen understanding of their field, Research Update will clarify the significance and future impact of this research. Our experienced in-house team is under the guidance of a panel of experts from across the life sciences who offer suggestions and advice to ensure that we have high calibre authors and have spotted the ‘hot’ papers. Visit the Research Update daily at http://update.bmn.com and sign up for email alerts to make sure you don’t miss a thing. This is your chance to have your opinion read by the life science community, if you would like to contribute, contact us at [email protected] http://tibtec.trends.com
522
TRENDS in Biotechnology
Vol.21 No.12 December 2003
Reagentless detection of DNA sequences on chemically modified electrodes H. Holden Thorp Department of Chemistry, University of North Carolina, Chapel Hill, North Carolina, 27599-3290, USA
Microarray analysis requires complex optical instruments and numerous reagents. Several new electrochemical methods for creating sequence-selective microlocations are emerging; such approaches potentially facilitate electrochemical (electronic) readouts of microarrays. These developments parallel the migration of glucose sensing in diabetes monitoring from optical to electrochemical methods. Recent work provides a strategy that minimizes the use of added reagents and potentially produces a reusable sensor that can be applied to continuous monitoring applications. The electrochemical detection of specific DNA sequences on spatially resolved microlocations potentially enables microarray analysis of complicated mixtures of nucleic acids without the use of optical equipment such as lasers and charge-coupled device (CCD) cameras [1– 6]. These advances would vastly reduce the cost and complexity of array technology. Electrochemistry ultimately facilitates much smaller microlocations (or nanolocations) than those allowed by optical methods [1,7], which are constrained to the micrometer-scale spatial resolution of optical fabrication and detection techniques. Because of these potential advantages, there has been considerable effort directed toward the development of chemically modified electrodes that can be used to detect specific DNA or RNA sequences [1,8 – 11]. Such a device consists of a surface suitable for electrochemical analysis, such as carbon, gold or metal oxide, which is modified with a single-stranded probe nucleic acid that is complementary to a desired target. When the desired target hybridizes to the probe attached to the electrode, an electrochemical signal is produced. Desirable systems are those that are highly sequence-specific, are reusable, require as few added reagents as possible and show very low limits of detection. Recently, Fan et al. [12] reported the realization of an electrochemical sensor involving ‘molecular beacon’ technology that is reagentless and partially reusable. This in-progress report demonstrates significant technical advances and provides evidence of the continued enthusiasm for developing electrochemical DNA analysis. In 2000, Kuhr wrote a commentary on a similar theme entitled ‘Electrochemical DNA analysis comes of age’, suggesting that this area of biotechnology was here to stay [4]. Indeed, despite great changes in the economic environment, the pursuit of electrochemical DNA analysis Corresponding author: H. Holden Thorp ([email protected]). http://tibtec.trends.com
continues unabated in both academia and industry [1,8 –11]. In this report, I describe the approach of Fan et al. [12] and how it fits into the larger picture of the field. Electrochemical detection using the E-DNA sensor The design of the sensor developed by Fan et al. [12] is shown in Figure 1. The general strategy involves an E-DNA sensor – a hairpin-forming oligonucleotide immobilized to a conductive electrode. The distal end of the oligonucleotide is labeled with a redox-active moiety that can be oxidized or reduced by the electrode. The hairpin-forming oligonucleotide belongs to a family known as ‘molecular beacons’ [13 – 15], in which the relatively large loop region of the hairpin is complementary to a target sequence. In the hairpin form, the redox-active moiety is close to the electrode and undergoes oxidation or reduction. Hybridization of the loop region to the target disrupts the hairpin, increasing the distance from the redox-active moiety to the electrode and thereby lowering the electrochemical signal. Such a configuration has been described conceptually in the patent literature [16], and Mao et al. have recently described elements of a similar system [17]. Here, I discuss the system of Fan et al., which involves a gold electrode and a thiol-modified oligonucleotide with ferrocene as the redox-active moiety. The electrochemical response of the E-DNA sensor is shown in Figure 2, which plots background-subtracted anodic scans from cyclic voltammetry measurements obtained in the presence of increasing quantities of the target sequence. The probable reason for the decrease in electrochemical signal at the sensor is the increase in charge-transfer distance for the extended duplex as compared with the stem-loop; such an assignment is consistent with that deduced from recent solution studies involving an osmium –nucleobase donor– acceptor pair situated similarly on a hairpin oligonucleotide [18]. The impressive characteristic of the response is the dynamic range, which spans six orders of magnitude of DNA concentration (Figure 2). Although this broad dynamic range is useful in some applications, the sensitivity of the method (i.e. the percentage change per increment of input concentration) is correspondingly less marked, making E-DNA more suitable for applications such as the analysis of single nucleotide polymorphisms or the detection of pathogens, in which reading small changes in input concentration is less important (than it is in expression analysis). The limit of detection, 5 fmol (10 pM), is excellent given the reagentless and potentially reusable configuration, but
Update
TRENDS in Biotechnology
523
Vol.21 No.12 December 2003
Iron
+cDNA
e T
Denaturation and re-annealing
eT
Fc
Gold electrode TRENDS in Biotechnology
Figure 1. Representation of the E-DNA strategy. cDNA is the sequence complementary to the loop region, Fc is ferrocene. Picture reproduced with permission from [12].
it is not as low as that of other electrochemical methods that use catalytic cycles or other amplification schemes [1,10,19]. When the hybridized electrode is heatdenatured and re-challenged with the target sequence, about 80% of the signal is recovered. The 20% loss is attributed to the instability of ferrocene, and this problem must be addressed if the reagentless, reusable nature of the sensor is to be properly exploited for the continuous monitoring of pathogens. Broader context for electrochemical sensing As stated above, the E-DNA sensor has added to a growing number of electrochemical detection schemes under development [1,8 – 11]. Each presents a slightly different
45
30
Peak current (nA)
Peak current (nA)
45
30
15
0 10–11 10–10 10–9 10–8 10–7 10–6 10–5 Concentration of cDNA (M)
15
0 0.1
0.2
0.3
0.4 0.5 E (V vs. NHE)
0.6
0.7
TRENDS in Biotechnology
Figure 2. Plots of background-subtracted anodic scans from the cyclic voltammograms of the E-DNA sensor with 0 M, 30 pM, 500 pM, 30 nM, 800 nM and 5 mM input target concentrations. Inset shows peak currents plotted against target concentrations. Picture reproduced with permission from [12]. http://tibtec.trends.com
set of advantages and disadvantages. In addition to the attractive features noted above, the E-DNA sensor has two distinct disadvantages that must be overcome. First, Fan et al. [12] note that the E-DNA sensor is a ‘signal-off ’ sensor, in which the binding of the target produces a decrease in electrochemical signal; signal decreases are difficult to measure because of the high background in the absence of analyte. Second, in its current configuration, sensing involves the transfer of only one electron from the ferrocene moiety to the electrode; therefore, the limit of detection is controlled by the current generated from a single electron transfer and thus the change in signal owing to only one electron transfer event per hybridization. This trade-off is inherent in the ‘reagentless’ set-up, because methods that produce catalytic cycles or multiple labels naturally require more reagents [1,10,19]. The authors point out that miniaturization of the electrode should improve the limit of detection, and numerous studies suggest that such efforts should be successful [1,10,11,20]. The work of Fan et al. confirms that electrochemical DNA detection has indeed ‘come of age’ [4]. The method joins several systems under development, including the catalytic oxidation of nucleobases [1], sandwich assays involving ferrocene-labeled signal probes [8,21], intercalator-mediated ferricyanide electrocatalysis [9], nanoparticle assays [10] and the detection of enzyme-labeled sandwich-type configurations [11]. Each of these methods presents a distinct set of advantages in terms of manufacturability, sensitivity, limits of detection, reusability, reagent requirements and reliability. The E-DNA approach makes significant advances towards a reagentless, reusable sensor and provides further encouragement that, as has occurred with glucose monitoring [22], detection of nucleic acids on spatially resolved microlocations will ultimately migrate from optical to electrochemical methods.
Update
524
TRENDS in Biotechnology
References 1 Popovich, N. and Thorp, H.H. (2002) New strategies for electrochemical detection of nucleic acids. Interface 11, 30 – 34 2 Tarlov, M.J. and Steel, A.B. (2003) DNA-based sensors. In Biomolecular Films: Design, Function, and Applications (Rusling, J.F., ed.), pp. 545 – 608, Marcel Dekker 3 Palecek, E. (2002) Past, present and future of nucleic acids electrochemistry. Talanta 56, 809– 819 4 Kuhr, W.G. (2000) Electrochemical DNA analysis comes of age. Nat. Biotechnol. 18, 1042– 1043 5 Heller, M.J. (2002) DNA microarray technology: devices, systems, and applications. Annu. Rev. Biomed. Eng. 4, 129 – 153 6 Willner, I. (2002) Biomaterials for sensors, fuel cells, and circuitry. Science 298, 2407 – 2408 7 Fan, F.R.F. et al. (1996) Single molecule electrochemistry. J. Am. Chem. Soc. 118, 9669– 9675 8 Umek, R.M. et al. (2001) Electronic detection of nucleic acids – a versatile platform for molecular diagnostics. J. Mol. Diagn. 3, 74 – 84 9 Boon, E.M. et al. (2000) Mutation detection by electrocatalysis at DNA-modified electrodes. Nat. Biotechnol. 18, 1096– 1100 10 Park, S.J. et al. (2002) Array-based electrical detection of DNA with nanoparticle probes. Science 295, 1503 – 1506 11 Oleinikov, A.V. et al. (2003) Self-assembling protein arrays using electronic semiconductor microchips and in vitro translation. J. Proteome Res. 2, 313 – 319 12 Fan, C. et al. (2003) Electrochemical interrogation of conformational changes as a reagentless method for the sequence-specific detection of DNA. Proc. Natl. Acad. Sci. U. S. A. 100, 9134– 9137
Vol.21 No.12 December 2003
13 Fang, X.H. et al. (1999) Designing a novel molecular beacon for surface-immobilized DNA hybridization studies. J. Am. Chem. Soc. 121, 2921– 2922 14 Hui, D. et al. (2003) Hybridization-based unquenching of DNA hairpins on Au surfaces: prototypical “molecular beacon” biosensors. J. Am. Chem. Soc. 125, 4012– 4013 15 Broude, N.E. (2002) Stem-loop oligonucleotides: a robust tool for molecular biology and biotechnology. Trends Biotechnol. 20, 249– 256 16 Bamdad, C. and Yu, C. (1999) Electronic methods for the detection of analytes using monolayers. European Patent Number WO99/57317 17 Mao, Y. et al. (2003) Studies of temperature-dependent electronic transduction on DNA hairpin loop sensor. Nucleic Acids Res. 31, e108 18 Holmberg, R.C. et al. Intramolecular electrocatalysis of 8-oxo-guanine oxidation: secondary structure control of electron transfer in osmiumlabeled oligonucleotides. Inorg. Chem. (in press) 19 Armistead, P.M. and Thorp, H.H. (2000) modification of metal oxides with nucleic acids: detection of attomole quantities of immobilized DNA by electrocatalysis. Anal. Chem. 72, 3764– 3770 20 Szalai, V.A. and Thorp, H.H. (2000) Electrocatalysis of guanine electron transfer: new insights from submillimeter carbon electrodes. J. Phys. Chem. B. 104, 6851 – 6859 21 Yu, C.J. et al. (2001) Electronic detection of single-base mismatches in DNA with ferrocene-modified probes. J. Am. Chem. Soc. 123, 11155– 11161 22 Henry, C.M. (2003) Taking the plunge. Chem. Eng. News 81, 37 – 38
0167-7799/$ - see front matter q 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.tibtech.2003.10.003
Could you name the most significant papers published in life sciences this month? Updated daily, Research Update presents short, easy-to-read commentary on the latest hot papers, enabling you to keep abreast with advances across the life sciences. Written by laboratory scientists with a keen understanding of their field, Research Update will clarify the significance and future impact of this research. Our experienced in-house team is under the guidance of a panel of experts from across the life sciences who offer suggestions and advice to ensure that we have high calibre authors and have spotted the ‘hot’ papers. Visit the Research Update daily at http://update.bmn.com and sign up for email alerts to make sure you don’t miss a thing. This is your chance to have your opinion read by the life science community, if you would like to contribute, contact us at [email protected] http://tibtec.trends.com