Arica Lubin - Academia.edu (original) (raw)

Papers by Arica Lubin

There is considerable interest in the development of second and third generation photovoltaics wi... more There is considerable interest in the development of second and third generation photovoltaics with higher efficiency and lower cost of production. Dye-sensitized solar cells (DSSCs) use organic species as the absorptive AND conductive species in the photovoltaic device, where molecules enter the excited state upon photon absorption and create a current based on the excited electrons. We have used a biomimetic approach to previously develop a self-assembling peptide-porphyrin aggregate system which maintains conductivity at pH values well above the peptide-free system. This system uses a scaffold peptide to bind and orient m-Tetrakis(4-sulfonatophenyl)porphine (TPPS) molecules into conductive ''J-aggregates''. In this work we characterized a number of second generation peptide designs with the goal of better understanding the chemical and structural relationships in formation of J-aggregated species. Early studies showed an inverse relationship between alpha helix formation in the scaffold peptide and J-aggregate formation. using circular dichroism and absorbance spectroscopy we have investigated this structural relationship and found that peptide secondary structure alone is insufficient to promote J-aggregate formation in solutions above pH 3.6. Additionally, spectroscopic investigations of the binding affinity of peptide for porphyrin as a function of ionic strength confirmed that the primary driving force in complex formation is electrostatic interactions between the anionic TPPS and the cationic peptide. using this knowledge we designed a 3rd generation peptide scaffold with intent on increasing overall aggregate size and stability.

We report a signal-on, electronic DNA (E-DNA) sensor that is label-free and achieves a subpicomol... more We report a signal-on, electronic DNA (E-DNA) sensor that is label-free and achieves a subpicomolar detection limit. The sensor, which is based on a target-induced strand displacement mechanism, is composed of a ''capture probe'' attached by its 5 terminus to a gold electrode and a 5 methylene blue-modified ''signaling probe'' that is complementary at both its 3 and 5 termini to the capture probe. In the absence of target, hybridization between the capture and signaling probes minimizes contact between the methylene blue and electrode surface, limiting the observed redox current. Target hybridization displaces the 5 end of the signaling probe, generating a short, flexible single-stranded DNA element and producing up to a 7-fold increase in redox current. The observed signal gain is sufficient to achieve a demonstrated (not extrapolated) detection limit of 400 fM, which is among the best reported for single-step electronic DNA detection. Moreover, because sensor fabrication is straightforward, the approach appears to provide a ready alternative to the more cumbersome femtomolar electrochemical assays described to date.

Proceedings of the National Academy of Sciences, 2006

We report a signal-on, electronic DNA (E-DNA) sensor that is label-free and achieves a subpicomol... more We report a signal-on, electronic DNA (E-DNA) sensor that is label-free and achieves a subpicomolar detection limit. The sensor, which is based on a target-induced strand displacement mechanism, is composed of a ''capture probe'' attached by its 5 terminus to a gold electrode and a 5 methylene blue-modified ''signaling probe'' that is complementary at both its 3 and 5 termini to the capture probe. In the absence of target, hybridization between the capture and signaling probes minimizes contact between the methylene blue and electrode surface, limiting the observed redox current. Target hybridization displaces the 5 end of the signaling probe, generating a short, flexible single-stranded DNA element and producing up to a 7-fold increase in redox current. The observed signal gain is sufficient to achieve a demonstrated (not extrapolated) detection limit of 400 fM, which is among the best reported for single-step electronic DNA detection. Moreover, because sensor fabrication is straightforward, the approach appears to provide a ready alternative to the more cumbersome femtomolar electrochemical assays described to date.

Langmuir, 2008

Electrochemical, aptamer-based (E-AB) sensors, which are comprised of an electrode modified with ... more Electrochemical, aptamer-based (E-AB) sensors, which are comprised of an electrode modified with surface immobilized, redox-tagged DNA aptamers, have emerged as a promising new biosensor platform. In order to further improve this technology we have systematically studied the effects of probe (aptamer) packing density, the AC frequency used to interrogate the sensor, and the nature of the self-assembled monolayer (SAM) used to passivate the electrode on the performance of representative E-AB sensors directed against the small molecule cocaine and the protein thrombin. We find that, by controlling the concentration of aptamer employed during sensor fabrication, we can control the density of probe DNA molecules on the electrode surface over an order of magnitude range. Over this range, the gain of the cocaine sensor varies from 60% to 200%, with maximum gain observed near the lowest probe densities. In contrast, over a similar range, the signal change of the thrombin sensor varies from 16% to 42% and optimal signaling is observed at intermediate densities.

Journal of the American Chemical Society, 2010

An &a... more An "XOR" gate built using label-free, dual-analyte electrochemical sensors and the activation of this logic gate via changing concentrations of cocaine and the relevant cDNA as inputs are described.

Journal of the American Chemical Society, 2009

The development of a biosensor system capable of continuous, real-time measurement of small-molec... more The development of a biosensor system capable of continuous, real-time measurement of small-molecule analytes directly in complex, unprocessed aqueous samples has been a significant challenge, and successful implementation has been achieved for only a limited number of targets. Toward a general solution to this problem, we report here the Microfluidic Electrochemical Aptamer-based Sensor (MECAS) chip wherein we integrate target-specific DNA aptamers that fold, and thus generate an electrochemical signal, in response to the analyte with a microfluidic detection system. As a model, we demonstrate the continuous, real-time (approximately 1 min time resolution) detection of the small-molecule drug cocaine at near physiological, low micromolar concentrations directly in undiluted, otherwise unmodified blood serum. We believe our approach of integrating folding-based electrochemical sensors with miniaturized detection systems may lay the groundwork for the real-time, point-of-care detection of a wide variety of molecular targets.

Angewandte Chemie International Edition, 2005

The E-AB sensor is analogous to the E-DNA sensor, a previously described, reagentless, electronic... more The E-AB sensor is analogous to the E-DNA sensor, a previously described, reagentless, electronic DNA sensing technology.21 The E-AB sensor is constructed by covalently attaching a methylene blue (MB) labeled, thrombin-binding DNA aptamer (oligomer 1) to a gold electrode by ...

Analytical Chemistry, 2009

Previous work has described several reagentless, electrochemical DNA (E-DNA) sensing architecture... more Previous work has described several reagentless, electrochemical DNA (E-DNA) sensing architectures comprised of an electrode-immobilized, redox-tagged probe oligonucleotide. Recent studies suggest that E-DNA signaling is predicated on hybridization-linked changes in probe flexibility, which will alter the efficiency with which the terminal redox tag strikes the electrode. This, in turn, suggests that probe length, probe geometry, and redox-tag placement will affect E-DNA signaling. To test this we have characterized E-DNA sensors comprised of linear or stem-loop probes of various lengths and with redox tags placed either distal to the electrode or internally within the probe sequence (proximal). We find that linear probes produce larger signal changes upon target binding than equivalent stem-loop probes. Likewise, long probes exhibit greater signal changes than short probes provided that the redox tag is placed proximal to the electrode surface. In contrast to their improved signaling, the specificity of long probes is poorer than that of short probes, suggesting that sensor optimization represents a trade off between sensitivity and specificity. Finally, we find that sensor response time and selectivity are only minimally affected by probe geometry or length. The results of this comparative study will help guide future designs and applications of these sensors.

Analytical Chemistry, 2006

The ability to detect specific oligonucleotides in complex, contaminant-ridden samples, without t... more The ability to detect specific oligonucleotides in complex, contaminant-ridden samples, without the use of exogenous reagents and using a reusable, fully electronic platform could revolutionize the detection of pathogens in the clinic and in the field. Here, we characterize a labelfree, electronic sensor, termed E-DNA, for its ability to simultaneously meet these challenging demands. We find that because signal generation is coupled to a hybridization-linked conformational change, rather than to only adsorption to the sensor surface, E-DNA is selective enough to detect oligonucleotides in complex, multicomponent samples, such as blood serum and soil. Moreover, E-DNA signaling is monotonically related to target complementarity, allowing the sensor to discriminate between mismatched targets: we readily detect the complementary 17-base target against a 50 000-fold excess of genomic DNA, can distinguish a three-base mismatch from perfect target directly in blood serum, and under ideal conditions, observe statistically significant differences between singlebase mismatches. Finally, because the sensing components are linked to the electrode surface, E-DNA is reusable: a 30-s room temperature wash recovers >99% of the sensor signal. This work further supports the utility of E-DNA as a rapid, specific, and convenient method for the detection of DNA and RNA sequences.

Analytical Chemistry, 2011

b S Supporting Information E lectrochemical DNA biosensors (E-DNA) appear to be a promising alter... more b S Supporting Information E lectrochemical DNA biosensors (E-DNA) appear to be a promising alternative to optical sensors for the specific detection of oligonucleotide sequences. 1À3 These devices are composed of a redox-reporter-modified DNA probe immobilized on a gold electrode. Hybridization-linked changes in the flexibility of this probe (due to specific conformational changes 1,4,5 or an increase in the amount of double-helix 6 ) alter the rate with which electrons are transferred from the redox reporter, leading to a readily detectable change in Faradaic current upon voltammetric interrogation. 7,8 Because E-DNA sensors are driven by electrochemistry, rather than optical detection methods, they can be integrated into microfluidic devices, powered by inexpensive, hand-held electronics, and easily multiplexed. 9À11 Moreover, because their signaling is predicated on a binding-induced change in the physical properties of the probe DNA, rather than to adsorption of analytes to the sensor surface, E-DNA sensors are relatively insensitive to the nonspecific adsorption of interferants and are selective enough to deploy directly in complex clinical and environmental samples, such as blood or soil extracts. 12,13 E-DNA sensors thus appear well suited for point of care medical diagnostics, as well as portable analysis systems for forensics and food quality control. Although E-DNA sensors have a wide variety of positive attributes, many of the diverse E-DNA probe architectures described to date operate in a signal-off fashion, meaning that the measured current decreases as the concentration of analyte DNA increases. 14 For example, first generation E-DNA sensors employ a stem-loop architecture 1,14 such that, when a complementary target oligonucleotide is introduced, hybridization causes the stem to open. This moves the redox reporter further from the electrode surface, reducing electron transfer. This signal-off mechanism significantly limits the gain of the sensor: the maximum possible ABSTRACT: Electrochemical DNA (E-DNA) sensors, which are rapid, reagentless, and readily integrated into microelectronics and microfluidics, appear to be a promising alternative to optical methods for the detection of specific nucleic acid sequences. Keeping with this, a large number of distinct E-DNA architectures have been reported to date. Most, however, suffer from one or more drawbacks, including low signal gain (the relative signal change in the presence of complementary target), signal-off behavior (target binding reduces the signaling current, leading to poor gain and raising the possibility that sensor fouling or degradation can lead to false positives), or instability (degradation of the sensor during regeneration or storage). To remedy these problems, we report here the development of a signal-on E-DNA architecture that achieves both high signal gain and good stability. This new sensor employs a commercially synthesized, asymmetric hairpin DNA as its recognition and signaling probe, the shorter arm of which is labeled with a redox reporting methylene blue at its free end. Unlike all prior E-DNA architectures, in which the recognition probe is attached via a terminal functional group to its underlying electrode, the probe employed here is affixed using a thiol group located internally, in the turn region of the hairpin. Hybridization of a target DNA to the longer arm of the hairpin displaces the shorter arm, allowing the reporter to approach the electrode surface and transfer electrons. The resulting device achieves signal increases of ∼800% at saturating target, a detection limit of just 50 pM, and ready discrimination between perfectly matched sequences and those with single nucleotide polymorphisms. Moreover, because the hairpin probe is a single, fully covalent strand of DNA, it is robust to the high stringency washes necessary to remove the target, and thus, these devices are fully reusable.

Accounts of Chemical Research, 2010

Biomolecular recognition is versatile, specific, and high affinity, qualities that have motivated... more Biomolecular recognition is versatile, specific, and high affinity, qualities that have motivated decades of research aimed at adapting biomolecules into a general platform for molecular sensing. Despite significant effort, however, so-called "biosensors" have almost entirely failed to achieve their potential as reagentless, real-time analytical devices; the only quantitative, reagentless biosensor to achieve commercial success so far is the home glucose monitor, employed by millions of diabetics. The fundamental stumbling block that has precluded more widespread success of biosensors is the failure of most biomolecules to produce an easily measured signal upon target binding. Antibodies, for example, do not change their shape or dynamics when they bind their recognition partners, nor do they emit light or electrons upon binding. It has thus proven difficult to transduce biomolecular binding events into a measurable output signal, particularly one that is not readily spoofed by the binding of any of the many potentially interfering species in typical biological samples. Analytical approaches based on biomolecular recognition are therefore mostly cumbersome, multistep processes relying on analyte separation and isolation (such as Western blots, ELISA, and other immunochemical methods); these techniques have proven enormously useful, but are limited almost exclusively to laboratory settings. In this Account, we describe how we have refined a potentially general solution to the problem of signal detection in biosensors, one that is based on the binding-induced "folding" of electrode-bound DNA probes. That is, we have developed a broad new class of biosensors that employ electrochemistry to monitor binding-induced changes in the rigidity of a redox-tagged probe DNA that has been site-specifically attached to an interrogating electrode. These folding-based sensors, which have been generalized to a wide range of specific protein, nucleic acid, and small-molecule targets, are rapid (responding in seconds to minutes), sensitive (detecting sub-picomolar to micromolar concentrations), and reagentless. They are also greater than 99% reusable, are supported on micrometer-scale electrodes, and are readily fabricated into densely packed sensor arrays. Finally, and critically, their signaling is linked to a binding-specific change in the physics of the probe DNA, and not simply to adsorption of the target onto the sensor head. Accordingly, they are selective enough to be employed directly in blood, crude soil extracts, cell lysates, and other grossly contaminated clinical and environmental samples. Indeed, we have recently demonstrated the ability to quantitatively monitor a specific small molecule in real-time directly in microliters of flowing, unmodified blood serum. Because of their sensitivity, substantial background suppression, and operational convenience, these folding-based biosensors appear potentially well suited for electronic, on-chip applications in pathogen detection, proteomics, metabolomics, and drug discovery.

Chemical Communications, 2007

We have developed a new biosensor architecture, which is comprised of a polypeptide-peptide nucle... more We have developed a new biosensor architecture, which is comprised of a polypeptide-peptide nucleic acid tri-block copolymer and which we have termed chimeric peptide beacons (CPB), that generates an optical output via a mechanism analogous to that employed in DNA-based molecular beacons. † Electronic supplementary information (ESI) available: Thermal melt of the 2bp CPB. Saturable binding of the 3bp CPB. See

There is considerable interest in the development of second and third generation photovoltaics wi... more There is considerable interest in the development of second and third generation photovoltaics with higher efficiency and lower cost of production. Dye-sensitized solar cells (DSSCs) use organic species as the absorptive AND conductive species in the photovoltaic device, where molecules enter the excited state upon photon absorption and create a current based on the excited electrons. We have used a biomimetic approach to previously develop a self-assembling peptide-porphyrin aggregate system which maintains conductivity at pH values well above the peptide-free system. This system uses a scaffold peptide to bind and orient m-Tetrakis(4-sulfonatophenyl)porphine (TPPS) molecules into conductive ''J-aggregates''. In this work we characterized a number of second generation peptide designs with the goal of better understanding the chemical and structural relationships in formation of J-aggregated species. Early studies showed an inverse relationship between alpha helix formation in the scaffold peptide and J-aggregate formation. using circular dichroism and absorbance spectroscopy we have investigated this structural relationship and found that peptide secondary structure alone is insufficient to promote J-aggregate formation in solutions above pH 3.6. Additionally, spectroscopic investigations of the binding affinity of peptide for porphyrin as a function of ionic strength confirmed that the primary driving force in complex formation is electrostatic interactions between the anionic TPPS and the cationic peptide. using this knowledge we designed a 3rd generation peptide scaffold with intent on increasing overall aggregate size and stability.

We report a signal-on, electronic DNA (E-DNA) sensor that is label-free and achieves a subpicomol... more We report a signal-on, electronic DNA (E-DNA) sensor that is label-free and achieves a subpicomolar detection limit. The sensor, which is based on a target-induced strand displacement mechanism, is composed of a ''capture probe'' attached by its 5 terminus to a gold electrode and a 5 methylene blue-modified ''signaling probe'' that is complementary at both its 3 and 5 termini to the capture probe. In the absence of target, hybridization between the capture and signaling probes minimizes contact between the methylene blue and electrode surface, limiting the observed redox current. Target hybridization displaces the 5 end of the signaling probe, generating a short, flexible single-stranded DNA element and producing up to a 7-fold increase in redox current. The observed signal gain is sufficient to achieve a demonstrated (not extrapolated) detection limit of 400 fM, which is among the best reported for single-step electronic DNA detection. Moreover, because sensor fabrication is straightforward, the approach appears to provide a ready alternative to the more cumbersome femtomolar electrochemical assays described to date.

Proceedings of the National Academy of Sciences, 2006

We report a signal-on, electronic DNA (E-DNA) sensor that is label-free and achieves a subpicomol... more We report a signal-on, electronic DNA (E-DNA) sensor that is label-free and achieves a subpicomolar detection limit. The sensor, which is based on a target-induced strand displacement mechanism, is composed of a ''capture probe'' attached by its 5 terminus to a gold electrode and a 5 methylene blue-modified ''signaling probe'' that is complementary at both its 3 and 5 termini to the capture probe. In the absence of target, hybridization between the capture and signaling probes minimizes contact between the methylene blue and electrode surface, limiting the observed redox current. Target hybridization displaces the 5 end of the signaling probe, generating a short, flexible single-stranded DNA element and producing up to a 7-fold increase in redox current. The observed signal gain is sufficient to achieve a demonstrated (not extrapolated) detection limit of 400 fM, which is among the best reported for single-step electronic DNA detection. Moreover, because sensor fabrication is straightforward, the approach appears to provide a ready alternative to the more cumbersome femtomolar electrochemical assays described to date.

Langmuir, 2008

Electrochemical, aptamer-based (E-AB) sensors, which are comprised of an electrode modified with ... more Electrochemical, aptamer-based (E-AB) sensors, which are comprised of an electrode modified with surface immobilized, redox-tagged DNA aptamers, have emerged as a promising new biosensor platform. In order to further improve this technology we have systematically studied the effects of probe (aptamer) packing density, the AC frequency used to interrogate the sensor, and the nature of the self-assembled monolayer (SAM) used to passivate the electrode on the performance of representative E-AB sensors directed against the small molecule cocaine and the protein thrombin. We find that, by controlling the concentration of aptamer employed during sensor fabrication, we can control the density of probe DNA molecules on the electrode surface over an order of magnitude range. Over this range, the gain of the cocaine sensor varies from 60% to 200%, with maximum gain observed near the lowest probe densities. In contrast, over a similar range, the signal change of the thrombin sensor varies from 16% to 42% and optimal signaling is observed at intermediate densities.

Journal of the American Chemical Society, 2010

An &a... more An "XOR" gate built using label-free, dual-analyte electrochemical sensors and the activation of this logic gate via changing concentrations of cocaine and the relevant cDNA as inputs are described.

Journal of the American Chemical Society, 2009

The development of a biosensor system capable of continuous, real-time measurement of small-molec... more The development of a biosensor system capable of continuous, real-time measurement of small-molecule analytes directly in complex, unprocessed aqueous samples has been a significant challenge, and successful implementation has been achieved for only a limited number of targets. Toward a general solution to this problem, we report here the Microfluidic Electrochemical Aptamer-based Sensor (MECAS) chip wherein we integrate target-specific DNA aptamers that fold, and thus generate an electrochemical signal, in response to the analyte with a microfluidic detection system. As a model, we demonstrate the continuous, real-time (approximately 1 min time resolution) detection of the small-molecule drug cocaine at near physiological, low micromolar concentrations directly in undiluted, otherwise unmodified blood serum. We believe our approach of integrating folding-based electrochemical sensors with miniaturized detection systems may lay the groundwork for the real-time, point-of-care detection of a wide variety of molecular targets.

Angewandte Chemie International Edition, 2005

The E-AB sensor is analogous to the E-DNA sensor, a previously described, reagentless, electronic... more The E-AB sensor is analogous to the E-DNA sensor, a previously described, reagentless, electronic DNA sensing technology.21 The E-AB sensor is constructed by covalently attaching a methylene blue (MB) labeled, thrombin-binding DNA aptamer (oligomer 1) to a gold electrode by ...

Analytical Chemistry, 2009

Previous work has described several reagentless, electrochemical DNA (E-DNA) sensing architecture... more Previous work has described several reagentless, electrochemical DNA (E-DNA) sensing architectures comprised of an electrode-immobilized, redox-tagged probe oligonucleotide. Recent studies suggest that E-DNA signaling is predicated on hybridization-linked changes in probe flexibility, which will alter the efficiency with which the terminal redox tag strikes the electrode. This, in turn, suggests that probe length, probe geometry, and redox-tag placement will affect E-DNA signaling. To test this we have characterized E-DNA sensors comprised of linear or stem-loop probes of various lengths and with redox tags placed either distal to the electrode or internally within the probe sequence (proximal). We find that linear probes produce larger signal changes upon target binding than equivalent stem-loop probes. Likewise, long probes exhibit greater signal changes than short probes provided that the redox tag is placed proximal to the electrode surface. In contrast to their improved signaling, the specificity of long probes is poorer than that of short probes, suggesting that sensor optimization represents a trade off between sensitivity and specificity. Finally, we find that sensor response time and selectivity are only minimally affected by probe geometry or length. The results of this comparative study will help guide future designs and applications of these sensors.

Analytical Chemistry, 2006

The ability to detect specific oligonucleotides in complex, contaminant-ridden samples, without t... more The ability to detect specific oligonucleotides in complex, contaminant-ridden samples, without the use of exogenous reagents and using a reusable, fully electronic platform could revolutionize the detection of pathogens in the clinic and in the field. Here, we characterize a labelfree, electronic sensor, termed E-DNA, for its ability to simultaneously meet these challenging demands. We find that because signal generation is coupled to a hybridization-linked conformational change, rather than to only adsorption to the sensor surface, E-DNA is selective enough to detect oligonucleotides in complex, multicomponent samples, such as blood serum and soil. Moreover, E-DNA signaling is monotonically related to target complementarity, allowing the sensor to discriminate between mismatched targets: we readily detect the complementary 17-base target against a 50 000-fold excess of genomic DNA, can distinguish a three-base mismatch from perfect target directly in blood serum, and under ideal conditions, observe statistically significant differences between singlebase mismatches. Finally, because the sensing components are linked to the electrode surface, E-DNA is reusable: a 30-s room temperature wash recovers >99% of the sensor signal. This work further supports the utility of E-DNA as a rapid, specific, and convenient method for the detection of DNA and RNA sequences.

Analytical Chemistry, 2011

b S Supporting Information E lectrochemical DNA biosensors (E-DNA) appear to be a promising alter... more b S Supporting Information E lectrochemical DNA biosensors (E-DNA) appear to be a promising alternative to optical sensors for the specific detection of oligonucleotide sequences. 1À3 These devices are composed of a redox-reporter-modified DNA probe immobilized on a gold electrode. Hybridization-linked changes in the flexibility of this probe (due to specific conformational changes 1,4,5 or an increase in the amount of double-helix 6 ) alter the rate with which electrons are transferred from the redox reporter, leading to a readily detectable change in Faradaic current upon voltammetric interrogation. 7,8 Because E-DNA sensors are driven by electrochemistry, rather than optical detection methods, they can be integrated into microfluidic devices, powered by inexpensive, hand-held electronics, and easily multiplexed. 9À11 Moreover, because their signaling is predicated on a binding-induced change in the physical properties of the probe DNA, rather than to adsorption of analytes to the sensor surface, E-DNA sensors are relatively insensitive to the nonspecific adsorption of interferants and are selective enough to deploy directly in complex clinical and environmental samples, such as blood or soil extracts. 12,13 E-DNA sensors thus appear well suited for point of care medical diagnostics, as well as portable analysis systems for forensics and food quality control. Although E-DNA sensors have a wide variety of positive attributes, many of the diverse E-DNA probe architectures described to date operate in a signal-off fashion, meaning that the measured current decreases as the concentration of analyte DNA increases. 14 For example, first generation E-DNA sensors employ a stem-loop architecture 1,14 such that, when a complementary target oligonucleotide is introduced, hybridization causes the stem to open. This moves the redox reporter further from the electrode surface, reducing electron transfer. This signal-off mechanism significantly limits the gain of the sensor: the maximum possible ABSTRACT: Electrochemical DNA (E-DNA) sensors, which are rapid, reagentless, and readily integrated into microelectronics and microfluidics, appear to be a promising alternative to optical methods for the detection of specific nucleic acid sequences. Keeping with this, a large number of distinct E-DNA architectures have been reported to date. Most, however, suffer from one or more drawbacks, including low signal gain (the relative signal change in the presence of complementary target), signal-off behavior (target binding reduces the signaling current, leading to poor gain and raising the possibility that sensor fouling or degradation can lead to false positives), or instability (degradation of the sensor during regeneration or storage). To remedy these problems, we report here the development of a signal-on E-DNA architecture that achieves both high signal gain and good stability. This new sensor employs a commercially synthesized, asymmetric hairpin DNA as its recognition and signaling probe, the shorter arm of which is labeled with a redox reporting methylene blue at its free end. Unlike all prior E-DNA architectures, in which the recognition probe is attached via a terminal functional group to its underlying electrode, the probe employed here is affixed using a thiol group located internally, in the turn region of the hairpin. Hybridization of a target DNA to the longer arm of the hairpin displaces the shorter arm, allowing the reporter to approach the electrode surface and transfer electrons. The resulting device achieves signal increases of ∼800% at saturating target, a detection limit of just 50 pM, and ready discrimination between perfectly matched sequences and those with single nucleotide polymorphisms. Moreover, because the hairpin probe is a single, fully covalent strand of DNA, it is robust to the high stringency washes necessary to remove the target, and thus, these devices are fully reusable.

Accounts of Chemical Research, 2010

Biomolecular recognition is versatile, specific, and high affinity, qualities that have motivated... more Biomolecular recognition is versatile, specific, and high affinity, qualities that have motivated decades of research aimed at adapting biomolecules into a general platform for molecular sensing. Despite significant effort, however, so-called "biosensors" have almost entirely failed to achieve their potential as reagentless, real-time analytical devices; the only quantitative, reagentless biosensor to achieve commercial success so far is the home glucose monitor, employed by millions of diabetics. The fundamental stumbling block that has precluded more widespread success of biosensors is the failure of most biomolecules to produce an easily measured signal upon target binding. Antibodies, for example, do not change their shape or dynamics when they bind their recognition partners, nor do they emit light or electrons upon binding. It has thus proven difficult to transduce biomolecular binding events into a measurable output signal, particularly one that is not readily spoofed by the binding of any of the many potentially interfering species in typical biological samples. Analytical approaches based on biomolecular recognition are therefore mostly cumbersome, multistep processes relying on analyte separation and isolation (such as Western blots, ELISA, and other immunochemical methods); these techniques have proven enormously useful, but are limited almost exclusively to laboratory settings. In this Account, we describe how we have refined a potentially general solution to the problem of signal detection in biosensors, one that is based on the binding-induced "folding" of electrode-bound DNA probes. That is, we have developed a broad new class of biosensors that employ electrochemistry to monitor binding-induced changes in the rigidity of a redox-tagged probe DNA that has been site-specifically attached to an interrogating electrode. These folding-based sensors, which have been generalized to a wide range of specific protein, nucleic acid, and small-molecule targets, are rapid (responding in seconds to minutes), sensitive (detecting sub-picomolar to micromolar concentrations), and reagentless. They are also greater than 99% reusable, are supported on micrometer-scale electrodes, and are readily fabricated into densely packed sensor arrays. Finally, and critically, their signaling is linked to a binding-specific change in the physics of the probe DNA, and not simply to adsorption of the target onto the sensor head. Accordingly, they are selective enough to be employed directly in blood, crude soil extracts, cell lysates, and other grossly contaminated clinical and environmental samples. Indeed, we have recently demonstrated the ability to quantitatively monitor a specific small molecule in real-time directly in microliters of flowing, unmodified blood serum. Because of their sensitivity, substantial background suppression, and operational convenience, these folding-based biosensors appear potentially well suited for electronic, on-chip applications in pathogen detection, proteomics, metabolomics, and drug discovery.

Chemical Communications, 2007

We have developed a new biosensor architecture, which is comprised of a polypeptide-peptide nucle... more We have developed a new biosensor architecture, which is comprised of a polypeptide-peptide nucleic acid tri-block copolymer and which we have termed chimeric peptide beacons (CPB), that generates an optical output via a mechanism analogous to that employed in DNA-based molecular beacons. † Electronic supplementary information (ESI) available: Thermal melt of the 2bp CPB. Saturable binding of the 3bp CPB. See