Interrogation of Electrochemical Aptamer-Based Sensors via Peak-to-Peak Separation in Cyclic Voltammetry Improves the Temporal Stability and Batch-to-Batch Variability in Biological FluidsClick to copy article linkArticle link copied!
- Miguel Aller PelliteroMiguel Aller PelliteroDepartment of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, Maryland 21202, United StatesMore by Miguel Aller Pellitero
- Samuel D. CurtisSamuel D. CurtisDepartment of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, Maryland 21202, United StatesMore by Samuel D. Curtis
- Netzahualcóyotl Arroyo-Currás*Netzahualcóyotl Arroyo-Currás*Email: [email protected]. Phone: 443-287-4798.Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, Maryland 21202, United StatesDepartment of Chemical and Biomolecular Engineering, Whiting School of Engineering, Johns Hopkins University, Baltimore, Maryland 21218, United StatesInstitute for Nanobiotechnology, Johns Hopkins University, Baltimore, Maryland 21218, United StatesMore by Netzahualcóyotl Arroyo-Currás
Abstract
Electrochemical, aptamer-based (E-AB) sensors support continuous, real-time measurements of specific molecular targets in complex fluids such as undiluted serum. They achieve these measurements by using redox-reporter-modified, electrode-attached aptamers that undergo target binding-induced conformational changes which, in turn, change electron transfer between the reporter and the sensor surface. Traditionally, E-AB sensors are interrogated via pulse voltammetry to monitor binding-induced changes in transfer kinetics. While these pulse techniques are sensitive to changes in electron transfer, they also respond to progressive changes in the sensor surface driven by biofouling or monolayer desorption and, consequently, present a significant drift. Moreover, we have empirically observed that differential voltage pulsing can accelerate monolayer desorption from the sensor surface, presumably via field-induced actuation of aptamers. Here, in contrast, we demonstrate the potential advantages of employing cyclic voltammetry to measure electron-transfer changes directly. In our approach, the target concentration is reported via changes in the peak-to-peak separation, ΔEP, of cyclic voltammograms. Because the magnitude of ΔEP is insensitive to variations in the number of aptamer probes on the electrode, ΔEP-interrogated E-AB sensors are resistant to drift and show decreased batch-to-batch and day-to-day variability in sensor performance. Moreover, ΔEP-based measurements can also be performed in a few hundred milliseconds and are, thus, competitive with other subsecond interrogation strategies such as chronoamperometry but with the added benefit of retaining sensor capacitance information that can report on monolayer stability over time.
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Figure 1
Figure 1. E-AB sensors undergo target binding-induced changes in electron-transfer kinetics of the redox reporter that can be monitored in real time via electrochemical interrogation. (A) In this work, we employed three different DNA aptamers modified at the 5′ terminus with alkanethiol linkers and at the 3′ terminus with the redox reporter MB. We codeposited these modified oligonucleotides with 6-mercapto-1-hexanol on the surface of gold electrodes via self-assembly. (B) In the presence of their target molecule, the aptamers undergo a conformational change that, presumably, brings the redox reporter closer to the electrode surface, increasing the electron-transfer rate.
Results and Discussion
Figure 2
Figure 2. Target binding-induced changes in apparent electron-transfer rates of E-AB sensors can be monitored via ΔEP. (A) When we interrogate tobramycin-detecting E-AB sensors using CV (at 5 V·s–1), we observe a decrease in ΔEP upon the addition of tobramycin (10 mM). (B) Same voltammograms as in (A) but zoomed-in relative to the x-axis show more clearly the change in electron-transfer rate (ket) from ΔEP,1 to ΔEP,2, with ΔEP,2 < ΔEP,1. (C) The magnitude of ΔEP decreases monotonically with increasing target concentration. The solid line is a nonlinear fit to the Hill equation, resulting in a Hill coefficient n = 1 and KD = 80 ± 7 μM. Error bars represent the standard deviation between five electrodes. All measurements performed in 1× phosphate-buffered saline (PBS).
Figure 3
Figure 3. E-AB sensor gain and sensitivity based on ΔEP are a strong function of the voltage scanning rate. (A) We illustrate this effect by showing ΔEP values in the absence (black circles, ΔEP,1) and presence (red circles, ΔEP,2) of saturating concentrations of tobramycin (10 mM) at increasing voltage scanning rates. Note that, although ΔEP increases with the increasing scanning rate in both cases, it does not do so with the same correlation function. Thus, CV scanning rates ranging between 5 and 10 V s–1 produce the largest signal change based on ΔEP. (B) Dose–response curves built from ΔEP measurements result in apparent aptamer dissociation constants that strongly depend on the CV scanning rate. Interrogating tobramycin-detecting E-AB sensors at CV scanning rates between 5 and 10 V s–1 achieve the most sensitive measurements (lowest apparent KD) with the largest overall signal gain. (C) Side-by-side comparison of sensors interrogated by CV using voltammetric peak heights, IP (blue circles) vs ΔEP (black circles) to illustrate that ΔEP achieves a 3-fold improvement in signal gain relative to IP, albeit with the opposite sign. Error bars represent the standard deviation between the five electrodes. All measurements were performed in 1× PBS.
Figure 4
Figure 4. ΔEP-based interrogation supports E-AB measurements irrespective of the aptamer used. We evaluated the general suitability of our method for E-AB sensing in undiluted serum by fabricating sensors using (A) tobramycin-, (B) vancomycin-, and (C) procaine-binding aptamers. Here, we show the performance of these sensors in undiluted serum when interrogated by CV. Left. Cyclic voltammograms measured in the absence (black trace) and presence (red trace) of a saturating target show significant differences in ΔEP. Note that for tobramycin and vancomycin, we observed minimal change in IP. However, voltammograms measured with the procaine sensor did show a significant change in IP matching a broadening of the faradaic waves. Center. Scan rate dependency of each sensor to determine the region of maximum signal decrease between the unbound and bound states. Right. We use the scanning rate giving the best compromise between maximum signal change and low apparent KD to build calibration curves for each analyte. Error bars represent the standard deviation between five electrodes. The calculated detection limit was 117 ± 4, 45 ± 6, and 60 ± 10 μM for the tobramycin, vancomycin, and procaine sensors, respectively. The precision of each sensor observed by this method can be found in Figure S5.
Figure 5
Figure 5. Serially interrogating E-AB sensors via ΔEP supports second to subsecond monitoring of fluctuating target concentrations in real time. Here, we show serial ΔEP measurements (black traces) recorded at (A) 5 V s–1 for tobramycin, (B) 1 V s–1 for vancomycin, and (C) 1 V s–1 for procaine. Using a voltage window of 600 mV (e.g., see Figure 2A), these scanning rates achieve measurements every 0.24, 1.2, and 1.2 s, respectively. To demonstrate the E-AB performance over time, in these panels we recorded measurements for 7 h alternating between 100% serum, and serum + target at a saturating concentration. To reveal the percentage contribution of drift to our measurements, we present the data as the relative change in signal with respect to the signal measured at the start of each experiment. We eliminated the effect of temperature fluctuations by maintaining the electrochemical cell at 25 °C using a water jacket and a temperature-controlled recirculation bath. We also continuously stirred the serum at ∼100 rpms to avoid precipitation of solids from the serum. Red traces represent the expected relative signal change in the absence or presence of a target as given by our calibration curves from Figure 4. Error bars represent the standard deviation between the five electrodes.
Figure 6
Figure 6. Long-term stability of E-AB sensors under different interrogation methods. (A) To investigate the extent to which continuously interrogating the sensors affects their operational stability, we interrogated E-AB sensors in PBS for 72 h by square wave voltammetry, either in continuous (5 × 104 scans) or single-point (10 scans) regimes, and by CV (7 × 104 scans) monitoring both peak-to-peak separation and the oxidation peak current. We observe that the continuous square wave voltammetry-based interrogation contributes to a fast loss in the signal (red trace), which dramatically decreases with less total measurements (at equal total experiment time, green circles). However, continuous CV-based interrogation (7 × 104 scans) by monitoring either ΔEP or IP does not contribute to the sensor signal loss. (B) When we switched to a more complex matrix such as undiluted serum, the CV-based interrogation presents an initial loss of signal (∼15%) during the first 10 h likely due to monolayer reorganization, desorption, and the nonspecific binding of proteins. While CV peak currents continue to drop linearly after this initial decay (blue trace), ΔEP remains constant for ∼50 h, the point at which our software is no longer able to resolve voltammetric peaks from the charging current. These measurements were performed using tobramycin-binding E-AB sensors at controlled 25 °C and under continuous stirring. Shaded areas or error bars represent the standard deviation between the five electrodes.
Figure 7
Figure 7. The batch-to-batch and day-to-day performance of E-AB sensors in undiluted serum is affected by the sensitivity of the interrogation method to changing sensor interfaces. (A) Square wave voltammetry-based measurements of E-AB peak current performed continuously every 5 s for 24 h. These measurements present significant variability in signal output between three batches of six sensors each (different colored traces), measured on three separate days. (B) The same variability is not seen for the ΔEP-based interrogation, which produces indistinguishable traces between electrode batches and measurement days. Specifically, the initial decay in signal driven by monolayer reorganization and the nonspecific protein binding is identical between batches and, in all cases, it stabilizes at ∼75% of its initial value. These measurements were performed using tobramycin-binding E-AB sensors at controlled 25 °C and under continuous stirring. Shaded areas represent the standard deviation between the five electrodes.
Conclusions
Methods
Chemicals and Materials
Electrochemical Measurements
Electrode Modification
Data Analysis
Supporting Information
The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acssensors.0c02455.
Study of the effect of the CV scan rate on dose–response curves, evaluation of peak broadening in CV with increasing scan rates, comparison of IP and ΔEP change for tobramycin sensors, evaluation of the precision of the CV-based method, calibration curve for procaine-binding E-AB sensors by SWV, evaluation of the response of sensors with different surface coverages, comparison of the electrochemical response of sensors prepared by a coimmobilization or a backfilling protocol, and Python script used for real-time data analysis (PDF)
Terms & Conditions
Most electronic Supporting Information files are available without a subscription to ACS Web Editions. Such files may be downloaded by article for research use (if there is a public use license linked to the relevant article, that license may permit other uses). Permission may be obtained from ACS for other uses through requests via the RightsLink permission system: http://pubs.acs.org/page/copyright/permissions.html.
Acknowledgments
N.A.-C. thanks the Oak Ridge Associated Universities for granting a 2019 Ralph E. Powe Junior Faculty Enhancement Award to support this work.
References
This article references 39 other publications.
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- 2Xiao, Y.; Lubin, A. A.; Heeger, A. J.; Plaxco, K. W. Label-Free Electronic Detection of Thrombin in Blood Serum by Using an Aptamer-Based Sensor. Angew. Chem., Int. Ed. 2005, 44, 5456– 5459, DOI: 10.1002/anie.200500989Google Scholar2https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXpvF2ksLg%253D&md5=03fba0f9b28e0c7c8375e8fef4a5446fLabel-free electronic detection of thrombin in blood serum by using an aptamer-based sensorXiao, Yi; Lubin, Arica A.; Heeger, Alan J.; Plaxco, Kevin W.Angewandte Chemie, International Edition (2005), 44 (34), 5456-5459CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)A methylene blue (MB) tagged, thrombin-binding DNA aptamer immobilized on a gold surface undergoes a large conformational change upon target binding and inhibits electron transfer. This folding produces a large, readily measurable change in redox current and allows the electrochem. detection of thrombin in blood serum.
- 3Arroyo-Currás, N.; Somerson, J.; Vieira, P. A.; Ploense, K. L.; Kippin, T. E.; Plaxco, K. W. Real-time measurement of small molecules directly in awake, ambulatory animals. Proc. Natl. Acad. Sci. U.S.A. 2017, 114, 645– 650, DOI: 10.1073/pnas.1613458114Google Scholar3https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXmslWktA%253D%253D&md5=04842755081ae2272a933ce3461cd953Real-time measurement of small molecules directly in awake, ambulatory animalsArroyo-Curras, Netzahualcoyotl; Somerson, Jacob; Vieira, Philip A.; Ploense, Kyle L.; Kippin, Tod E.; Plaxco, Kevin W.Proceedings of the National Academy of Sciences of the United States of America (2017), 114 (4), 645-650CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)The development of a technol. capable of tracking the levels of drugs, metabolites, and biomarkers in the body continuously and in real time would advance our understanding of health and our ability to detect and treat disease. It would, for example, enable therapies guided by high-resoln., patient-specific pharmacokinetics (including feedback-controlled drug delivery), opening new dimensions in personalized medicine. In response, we demonstrate here the ability of electrochem. aptamer-based (E-AB) sensors to support continuous, real-time, multihour measurements when emplaced directly in the circulatory systems of living animals. Specifically, we have used E-AB sensors to perform the multihour, real-time measurement of four drugs in the bloodstream of even awake, ambulatory rats, achieving precise mol. measurements at clin. relevant detection limits and high (3 s) temporal resoln., attributes suggesting that the approach could provide an important window into the study of physiol. and pharmacokinetics.
- 4Schoukroun-Barnes, L. R.; Macazo, F. C.; Gutierrez, B.; Lottermoser, J.; Liu, J.; White, R. J. Reagentless, Structure-Switching, Electrochemical Aptamer-Based Sensors. Annu. Rev. Anal. Chem. 2016, 9, 163– 181, DOI: 10.1146/annurev-anchem-071015-041446Google Scholar4https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XmtVOntL0%253D&md5=32b573c81f86a07a525f4682802ae6a1Reagentless, Structure-Switching, Electrochemical Aptamer-Based SensorsSchoukroun-Barnes, Lauren R.; Macazo, Florika C.; Gutierrez, Brenda; Lottermoser, Justine; Liu, Juan; White, Ryan J.Annual Review of Analytical Chemistry (2016), 9 (), 163-181CODEN: ARACFU; ISSN:1936-1327. (Annual Reviews)The development of structure-switching, electrochem., aptamer-based sensors over the past ∼10 years has led to a variety of reagentless sensors capable of anal. detection in a range of sample matrixes. The crux of this methodol. is the coupling of target-induced conformation changes of a redox-labeled aptamer with electrochem. detection of the resulting altered charge transfer rate between the redox mol. and electrode surface. Using aptamer recognition expands the highly sensitive detection ability of electrochem. to a range of previously inaccessible analytes. In this review, we focus on the methods of sensor fabrication and how sensor signaling is affected by fabrication parameters. We then discuss recent studies addressing the fundamentals of sensor signaling as well as quant. characterization of the anal. performance of electrochem. aptamer-based sensors. Although the limits of detection of reported electrochem. aptamer-based sensors do not often reach that of gold-std. methods such as enzyme-linked immunosorbent assays, the operational convenience of the sensor platform enables exciting anal. applications that we address. Using illustrative examples, we highlight recent advances in the field that impact important areas of anal. chem. Finally, we discuss the challenges and prospects for this class of sensors.
- 5Arroyo-Currás, N.; Dauphin-Ducharme, P.; Scida, K.; Chávez, J. L. From the beaker to the body: translational challenges for electrochemical, aptamer-based sensors. Anal. Methods 2020, 12, 1288– 1310, DOI: 10.1039/d0ay00026dGoogle ScholarThere is no corresponding record for this reference.
- 6Kang, D.; Zuo, X.; Yang, R.; Xia, F.; Plaxco, K. W.; White, R. J. Comparing the properties of electrochemical-based DNA sensors employing different redox tags. Anal. Chem. 2009, 81, 9109– 9113, DOI: 10.1021/ac901811nGoogle Scholar6https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXht1eltrfI&md5=2c9656095c0b3a872e207465445ddff7Comparing the properties of electrochemical-based DNA sensors employing different redox tagsKang, Di; Zuo, Xiaolei; Yang, Renqiang; Xia, Fan; Plaxco, Kevin W.; White, Ryan J.Analytical Chemistry (Washington, DC, United States) (2009), 81 (21), 9109-9113CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)Many electrochem. biosensor approaches developed in recent years utilize redox-labeled (most commonly methylene blue or ferrocene) oligonucleotide probes site-specifically attached to an interrogating electrode. Sensors in this class have been reported that employ a range of probe architectures, including single- and double-stranded DNA, more complex DNA structures, DNA and RNA aptamers, and, most recently, DNA-small mol. chimeras. Signaling in this class of sensors is generally predicated on binding-induced changes in the efficiency with which the covalently attached redox label transfers electrons with the interrogating electrode. Here we have investigated how the properties of the redox tag affect the performance of such sensors. Specifically, we compare the differences in signaling and stability of electrochem. DNA sensors (E-DNA sensors) fabricated using either ferrocene or methylene blue as the signaling redox moiety. We find that while both tags support efficient E-DNA signaling, ferrocene produces slightly improved signal gain and target affinity. These small advantages, however, come at a potentially significant price: the ferrocene-based sensors are far less stable than their methylene blue counterparts, particularly with regards to stability to long-term storage, repeated electrochem. interrogations, repeated sensing/regeneration iterations, and employment in complex sample matrixes such as blood serum.
- 7Kang, D.; Ricci, F.; White, R. J.; Plaxco, K. W. Survey of Redox-Active Moieties for Application in Multiplexed Electrochemical Biosensors. Anal. Chem. 2016, 88, 10452– 10458, DOI: 10.1021/acs.analchem.6b02376Google Scholar7https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhsFGrs7jL&md5=3a25d233ddfb2b611131f474ad18d4dbSurvey of Redox-Active Moieties for Application in Multiplexed Electrochemical BiosensorsKang, Di; Ricci, Francesco; White, Ryan J.; Plaxco, Kevin W.Analytical Chemistry (Washington, DC, United States) (2016), 88 (21), 10452-10458CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)Recent years have seen the development of a large no. of electrochem. sandwich assays and reagentless biosensor architectures employing biomols. modified via the attachment of a redox-active "reporter.". Here the authors survey a large set of potential redox reporters to det. which exhibits the best long-duration stability in thiol-on-gold monolayer-based sensors and to identify reporter "sets" signaling at distinct, non-overlapping redox potentials in support of multiplexing and error correcting ratiometric or differential measurement approaches. Specifically, the authors have characterized the performance of more than a dozen potential reporters that are, first, redox active within the potential window over which thiol-on-gold monolayers are reasonably stable and, second, are available com. in forms that are readily conjugated to biomols. or can be converted into such forms in one or two simple synthetic steps. To test each of these reporters the authors conjugated it to one terminus of a single-stranded DNA "probe" that was attached by its other terminus via a six-carbon thiol to a gold electrode to form an "E-DNA" sensor responsive to its complementary DNA target. The authors then measured the signaling properties of each sensor as well as its stability against repeated voltammetric scans and against deployment in and reuse from blood serum. Doing so the performance of methylene blue-based, thiol-on-gold sensors is unmatched; the near-quant. stability of such sensors against repeated scanning in even very complex sample matrixes is unparalleled. While more modest, the stability of sensors employing a handful of other reporters, including anthraquinone, Nile blue, and ferrocene, is reasonable. The authors' work thus serves as both to highlight the exceptional properties of methylene blue as a redox reporter in such applications and as a cautionary tale; the authors wish to help other researchers avoid fruitless efforts to employ the many, seemingly promising and yet ultimately inadequate reporters the authors have studied. Finally, the authors hope that their work also serves as an illustration of the pressing need for the further development of useful redox reporters.
- 8Lubin, A. A.; Plaxco, K. W. Folding-Based Electrochemical Biosensors: The Case for Responsive Nucleic Acid Architectures. Acc. Chem. Res. 2010, 43, 496– 505, DOI: 10.1021/ar900165xGoogle Scholar8https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXislyktb0%253D&md5=fc266597938a204ca094b771e7ae30d2Folding-Based Electrochemical Biosensors: The Case for Responsive Nucleic Acid ArchitecturesLubin, Arica A.; Plaxco, Kevin W.Accounts of Chemical Research (2010), 43 (4), 496-505CODEN: ACHRE4; ISSN:0001-4842. (American Chemical Society)A review. Biomol. recognition is versatile, specific, and high affinity, qualities that have motivated decades of research aimed at adapting biomols. into a general platform for mol. sensing. Despite significant effort, however, so-called "biosensors" have almost entirely failed to achieve their potential as reagentless, real-time anal. devices; the only quant., reagentless biosensor to achieve com. 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 biomols. 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 biomol. 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 biol. samples. Anal. approaches based on biomol. recognition are therefore mostly cumbersome, multistep processes relying on analyte sepn. and isolation (such as Western blots, ELISA, and other immunochem. methods); these techniques have proven enormously useful, but are limited almost exclusively to lab. settings. In this Account, we describe how we have refined a potentially general soln. 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 electrochem. 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-mol. targets, are rapid (responding in seconds to minutes), sensitive (detecting sub-picomolar to micromolar concns.), 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 exts., cell lysates, and other grossly contaminated clin. and environmental samples. Indeed, we have recently demonstrated the ability to quant. monitor a specific small mol. 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.
- 9Schoen, I.; Krammer, H.; Braun, D. Hybridization kinetics is different inside cells. Proc. Natl. Acad. Sci. U.S.A. 2009, 106, 21649– 21654, DOI: 10.1073/pnas.0901313106Google Scholar9https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXltlKjtg%253D%253D&md5=7332fdfffcbdb0aabdd12e916c3023bfHybridization kinetics is different inside cellsSchoen, Ingmar; Krammer, Hubert; Braun, DieterProceedings of the National Academy of Sciences of the United States of America (2009), 106 (51), 21649-21654, S21649/1-S21649/12CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)It is generally expected that the kinetics of reactions inside living cells differs from the situation in bulk solns. Macromol. crowding and specific binding interactions could change the diffusion properties and the availability of free mols. Their impact on reaction kinetics in the relevant context of living cells is still elusive, mainly because the difficulty of capturing fast kinetics in vivo. This article shows spatially resolved measurements of DNA hybridization kinetics in single living cells. HeLa cells were transfected with a FRET-labeled dsDNA probe by lipofection. We characterized the hybridization reaction kinetics with a kinetic range of 10 μs to 1 s by a combination of laser-driven temp. oscillations and stroboscopic fluorescence imaging. The time const. of the hybridization depended on DNA concn. within individual cells and between cells. A quant. anal. of the concn. dependence revealed several-fold accelerated kinetics as compared with free soln. for a 16-bp probe and decelerated kinetics for a 12-bp probe. We did not find significant effects of crowding agents on the hybridization kinetics in vitro. Our results suggest that the reaction rates in vivo are specifically modulated by binding interactions for the two probes, possibly triggered by their different lengths. In general, the presented imaging modality of temp. oscillation optical lock-in microscopy allows to probe biomol. interactions in different cell compartments in living cells for systems biol.
- 10Vallée-Bélisle, A.; Plaxco, K. W. Structure-switching biosensors: inspired by Nature. Curr. Opin. Struct. Biol. 2010, 20, 518– 526, DOI: 10.1016/j.sbi.2010.05.001Google Scholar10https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXhtVKhtLjN&md5=1057758a3ce05e727a47c4c5e89b6895Structure-switching biosensors: inspired by NatureVallee-Belisle, Alexis; Plaxco, Kevin W.Current Opinion in Structural Biology (2010), 20 (4), 518-526CODEN: COSBEF; ISSN:0959-440X. (Elsevier Ltd.)A review. Chemosensing in nature relies on biomol. switches, biomols. that undergo binding-induced changes in conformation or oligomerization to transduce chem. information into specific biochem. outputs. Motivated by the impressive performance of these natural 'biosensors', which support continuous, real-time detection in highly complex environments, significant efforts have gone into the adaptation of such switches into artificial chem. sensors. Ongoing advances in the fields of protein and nucleic acid engineering (e.g. computational protein design, directed evolution, selection strategies and labeling chemistries) have greatly enhanced the ability to design new structure-switching sensors. Coupled with the development of advanced optical readout mechanisms, including genetically encoded fluorophores, and electrochem. readouts supporting detection directly in highly complex sample matrixes, switch-based sensors have already seen deployment in applications ranging from real time, in vivo imaging to the continuous monitoring of drugs in blood serum.
- 11Ferapontova, E. E.; Olsen, E. M.; Gothelf, K. V. An RNA Aptamer-Based Electrochemical Biosensor for Detection of Theophylline in Serum. J. Am. Chem. Soc. 2008, 130, 4256– 4258, DOI: 10.1021/ja711326bGoogle Scholar11https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXivV2gu7c%253D&md5=99abb5e0434e5620fdcf35432c509ea7An RNA Aptamer-Based Electrochemical Biosensor for Detection of Theophylline in SerumFerapontova, Elena E.; Olsen, Eva M.; Gothelf, Kurt V.Journal of the American Chemical Society (2008), 130 (13), 4256-4258CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)An electrochem. RNA aptamer-based biosensor for rapid and label-free detection of the bronchodilator theophylline was developed. The 5'-disulfide-functionalized end of the RNA aptamer sequence was immobilized on a gold electrode, and the 3'-amino-functionalized end was conjugated with a ferrocene (Fc) redox probe. Upon binding of theophylline the aptamer switches conformation from an open unfolded state to a closed hairpin-type conformation, resulting in the increased electron-transfer efficiency between Fc and the electrode. The electrochem. response, which was measured by differential pulse voltammetry, reaches satn. within a few minutes after addn. of theophylline, and the dynamic range for detecting theophylline is 0.2-10 μM. The electrode displays an inhibited response when applied directly in serum samples treated with RNase inhibitors; however a full response to the theophylline serum concn. was obtained by transferring the electrode to blank serum-free buffer solns. It was demonstrated that theophylline is detected with high selectivity in the presence of caffeine and theobromine.
- 12Somerson, J.; Plaxco, K. Electrochemical aptamer-based sensors for rapid point-of-use monitoring of the mycotoxin ochratoxin a directly in a food stream. Molecules 2018, 23, 912– 919, DOI: 10.3390/molecules23040912Google Scholar12https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhslGmsLfO&md5=08d42c284a1fdb130fd753b2a4eac12bElectrochemical aptamer-based sensors for rapid point-of-use monitoring of the mycotoxin ochratoxin A directly in a food streamSomerson, Jacob; Plaxco, Kevin W.Molecules (2018), 23 (4), 912/1-912/7CODEN: MOLEFW; ISSN:1420-3049. (MDPI AG)The ability to measure the concn. of specific small mols. continuously and in real-time in complex sample streams would impact many areas of agriculture, food safety, and food prodn. Monitoring for mycotoxin taint in real time during food processing, for example, could improve public health. Towards this end, we describe here an inexpensive electrochem. DNA-based sensor that supports real-time monitor of the mycotoxin ochratoxin A in a flowing stream of foodstuffs.
- 13Parolo, C.; Idili, A.; Ortega, G.; Csordas, A.; Hsu, A.; Arroyo-Currás, N.; Yang, Q.; Ferguson, B. S.; Wang, J.; Plaxco, K. W. Real-Time Monitoring of a Protein Biomarker. ACS Sens. 2020, 5, 1877– 1881, DOI: 10.1021/acssensors.0c01085Google Scholar13https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhtlWqsLzO&md5=0f21fed8f246326266979277a7bb67f3Real-Time Monitoring of a Protein BiomarkerParolo, Claudio; Idili, Andrea; Ortega, Gabriel; Csordas, Andrew; Hsu, Alex; Arroyo-Curras, Netzahualcoyotl; Yang, Qin; Ferguson, Brian Scott; Wang, Jinpeng; Plaxco, Kevin W.ACS Sensors (2020), 5 (7), 1877-1881CODEN: ASCEFJ; ISSN:2379-3694. (American Chemical Society)The ability to monitor protein biomarkers continuously and in real-time would significantly advance the precision of medicine. Current protein-detection techniques, however, including ELISA and lateral flow assays, provide only time-delayed, single-time-point measurements, limiting their ability to guide prompt responses to rapidly evolving, life-threatening conditions. In response, here the authors present an electrochem. aptamer-based sensor (EAB) that supports high-frequency, real-time biomarker measurements. Specifically, the authors have developed an electrochem., aptamer-based (EAB) sensor against Neutrophil Gelatinase-Assocd. Lipocalin (NGAL), a protein that, if present in urine at levels above a threshold value, is indicative of acute renal/kidney injury (AKI). When deployed inside a urinary catheter, the resulting reagentless, wash-free sensor supports real-time, high-frequency monitoring of clin. relevant NGAL concns. over the course of hours. By providing an "early warning system", the ability to measure levels of diagnostically relevant proteins such as NGAL in real-time could fundamentally change how the authors detect, monitor, and treat many important diseases.
- 14Arroyo-Currás, N.; Dauphin-Ducharme, P.; Ortega, G.; Ploense, K. L.; Kippin, T. E.; Plaxco, K. W. Subsecond-Resolved Molecular Measurements in the Living Body Using Chronoamperometrically Interrogated Aptamer-Based Sensors. ACS Sens. 2018, 3, 360– 366, DOI: 10.1021/acssensors.7b00787Google Scholar14https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhslynu7nL&md5=71345b6d63ba81e4474706be1d55091dSubsecond-Resolved Molecular Measurements in the Living Body Using Chronoamperometrically Interrogated Aptamer-Based SensorsArroyo-Curras, Netzahualcoyotl; Dauphin-Ducharme, Philippe; Ortega, Gabriel; Ploense, Kyle L.; Kippin, Tod E.; Plaxco, Kevin W.ACS Sensors (2018), 3 (2), 360-366CODEN: ASCEFJ; ISSN:2379-3694. (American Chemical Society)Electrochem., aptamer-based (E-AB) sensors support the continuous, real-time measurement of specific small mols. directly in situ in the living body over the course of many hours. They achieve this by employing binding-induced conformational changes to alter electron transfer from a redox-reporter-modified, electrode-attached aptamer. Previously the authors have used voltammetry (cyclic, a.c., and square wave) to monitor this binding-induced change in transfer kinetics indirectly. Here, however, the authors demonstrate the potential advantages of employing chronoamperometry to measure the change in kinetics directly. In this approach target concn. is reported via changes in the lifetime of the exponential current decay seen when the sensor is subjected to a potential step. Because the lifetime of this decay is independent of its amplitude (e.g., insensitive to variations in the no. of aptamer probes on the electrode), chronoamperometrically interrogated E-AB sensors are calibration-free and resistant to drift. Chronoamperometric measurements can also be performed in a few hundred milliseconds, improving the previous few-second time resoln. of E-AB sensing by an order of magnitude. To illustrate the potential value of the approach the authors demonstrate here the calibration-free measurement of the drug tobramycin in situ in the living body with 300 ms time resoln. and unprecedented, few-percent precision in the detn. of its pharmacokinetic phases.
- 15Radi, A.-E.; Acero Sánchez, J. L.; Baldrich, E.; O’Sullivan, C. K. Reagentless, Reusable, Ultrasensitive Electrochemical Molecular Beacon Aptasensor. J. Am. Chem. Soc. 2006, 128, 117– 124, DOI: 10.1021/ja053121dGoogle Scholar15https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXhtlShtLfI&md5=c61b8799326b09d8f8eb54c7a2c299fbReagentless, Reusable, Ultrasensitive Electrochemical Molecular Beacon AptasensorRadi, Abd-Elgawad; Acero Sanchez, Josep Lluis; Baldrich, Eva; O'Sullivan, Ciara K.Journal of the American Chemical Society (2006), 128 (1), 117-124CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)A bifunctional deriv. of the thrombin-binding aptamer with a redox-active ferrocene (Fc) moiety and a thiol group at the termini of the aptamer strand was synthesized. The ferrocene-labeled aptamer thiol was self-assembled through S-Au bonding on a polycryst. gold electrode surface and the surface was blocked with 2-mercaptoethanol to form a mixed monolayer. By use of a fluorescent mol. beacon, the effect of counterions on quadruplex formation was established. The aptamer-modified electrode was characterized electrochem. by cyclic voltammetry (CV), differential pulse voltammetry (DPV), and electrochem. impedance spectroscopy (EIS). The modified electrode showed a voltammetric signal due to a one-step redox reaction of the surface-confined ferrocenyl moiety of the aptamer immobilized on the electrode surface in 10 mM N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonic acid (HEPES) buffer of pH 8.0. An increase in the DPV current signal was evident after blocking with 2-mercaptoethanol, effectively removing aptamer nonspecifically absorbed rather than bound to electrode surface or due to the formation of the aptamer-thrombin affinity interaction. The impedance measurement, in agreement with the differential pulse voltammetry (DPV), showed decreased Faradaic resistances in the same sequence. The "signal-on" upon thrombin assocn. could be attributed to a change in conformation from random coil-like configuration on the probe-modified film to the quadruplex structure. The DPV of the modified electrode showed a linear response of the Fc oxidn. signal to the increase in the thrombin concn. in the range between 5.0 and 35.0 nM with a linear correlation of r = 0.9988 and a detection limit of 0.5 nM. The mol. beacon aptasensor was amenable to full regeneration by simply unfolding the aptamer in 1.0 M HCl, and could be regenerated 25 times with no loss in electrochem. signal upon subsequent thrombin binding.
- 16Zhao, S.; Yang, W.; Lai, R. Y. A folding-based electrochemical aptasensor for detection of vascular endothelial growth factor in human whole blood. Biosens. Bioelectron. 2011, 26, 2442– 2447, DOI: 10.1016/j.bios.2010.10.029Google Scholar16https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXhvVeqsw%253D%253D&md5=1362bef49e8b2c8c18e6b835a9862147A folding-based electrochemical aptasensor for detection of vascular endothelial growth factor in human whole bloodZhao, Shuang; Yang, Weiwei; Lai, Rebecca Y.Biosensors & Bioelectronics (2011), 26 (5), 2442-2447CODEN: BBIOE4; ISSN:0956-5663. (Elsevier B.V.)We herein report a folding-based electrochem. DNA aptasensor for the detection of vascular endothelial growth factor (VEGF) directly in complex biol. samples, including blood serum and whole blood. The electrochem. signal generation is coupled to a large, target-induced conformational change in a methylene blue-modified and surface immobilized anti-VEGF aptamer. The sensor is sensitive, selective and essentially reagentless: we can readily detect VEGF down to 5 pM (190 pg/mL) directly in 50% blood serum. Similar to other aptasensors of this class, the VEGF sensor is also regenerable and reusable. In addn., the sensor performs comparably well even when fabricated on a gold-plated screen-printed carbon electrode and can potentially be implemented as a cost-effective, single-use biosensor for diseases diagnosis and therapy monitoring. The exceptional sensitivity, selectivity, and reusability of this electrochem. aptasensor platform suggest it may be a promising strategy for a wide variety of sensing applications.
- 17Downs, A. M.; Gerson, J.; Ploense, K. L.; Plaxco, K. W.; Dauphin-Ducharme, P. Sub-second-resolved Molecular Measurements Using Electrochemical Phase Interrogation of Aptamer-Based Sensors. Anal. Chem. 2020, 92, 14063– 14068, DOI: 10.1021/acs.analchem.0c03109Google Scholar17https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhvVygtbfE&md5=b32ad23d59d4f00652afc24725ec1181Subsecond-Resolved Molecular Measurements Using Electrochemical Phase Interrogation of Aptamer-Based SensorsDowns, Alex M.; Gerson, Julian; Ploense, Kyle L.; Plaxco, Kevin W.; Dauphin-Ducharme, PhilippeAnalytical Chemistry (Washington, DC, United States) (2020), 92 (20), 14063-14068CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)Recent years have seen the development of a no. of biosensor architectures that rely on target binding-induced changes in the rate of electron transfer from an electrode-bound receptor. Most often, the interrogation of these sensors has relied on voltammetric methods, such as square-wave voltammetry, which limit their time resoln. to a few seconds. Here, we describe the use of an impedance-based approach, which we have termed electrochem. phase interrogation, as a means of collecting high time resoln. measurements with sensors in this class. Specifically, using changes in the electrochem. phase to monitor target binding in an electrochem.-aptamer based (EAB) sensor, we achieve subsecond temporal resoln. and multihour stability in measurements performed directly in undiluted whole blood. Electrochem. phase interrogation also offers improved insights into EAB sensors' signaling mechanism. By modeling the interfacial resistance and capacitance using equiv. circuits, we find that the only parameter that is altered by target binding is the charge-transfer resistance. This confirms previous claims that binding-induced changes in electron-transfer kinetics drive signaling in this class of sensors. Considering that a wide range of electrochem. biosensor architectures rely on this signaling mechanism, we believe that electrochem. phase interrogation may prove generalizable toward subsecond measurements of mol. targets.
- 18Li, F.; Yu, Z.; Han, X.; Lai, R. Y. Electrochemical aptamer-based sensors for food and water analysis: A review. Anal. Chim. Acta 2019, 1051, 1– 23, DOI: 10.1016/j.aca.2018.10.058Google Scholar18https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXitFSqur7O&md5=c39c9f6456fdef2b38b1efa4c041bb3cElectrochemical aptamer-based sensors for food and water analysis: A reviewLi, Fengqin; Yu, Zhigang; Han, Xianda; Lai, Rebecca Y.Analytica Chimica Acta (2019), 1051 (), 1-23CODEN: ACACAM; ISSN:0003-2670. (Elsevier B.V.)A review. Global food and water safety issues have prompted the development of highly sensitive, specific, and fast anal. techniques for food and water anal. The electrochem. aptamer-based detection platform (E-aptasensor) is one of the more promising detection techniques because of its unique combination of advantages that renders these sensors ideal for detection of a wide range of target analytes. Recent research results have further demonstrated that this technique has potential for real world anal. of food and water contaminants. This review summaries the recently developed E-aptasensors for detection of analytes related to food and water safety, including bacteria, mycotoxins, algal toxins, viruses, drugs, pesticides, and metal ions. Ten different electroanal. techniques and one opto-electroanal. technique commonly employed with these sensors are also described. In addn. to highlighting several novel sensor designs, this review also describes the strengths, limitations, and current challenges this technol. faces, and future development trend.
- 19Pellitero, M. A.; Shaver, A.; Arroyo-Currás, N. Critical Review─Approaches for the Electrochemical Interrogation of DNA-Based Sensors: A Critical Review. J. Electrochem. Soc. 2020, 167, 037529, DOI: 10.1149/2.0292003jesGoogle Scholar19https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXjt1agtrY%253D&md5=e78a4f721400cb76bde262181801b32bCritical review-approaches for the electrochemical interrogation of DNA-based sensors: a critical reviewPellitero, Miguel Aller; Shaver, Alexander; Arroyo-Curras, NetzahualcoyotlJournal of the Electrochemical Society (2020), 167 (3), 037529CODEN: JESOAN; ISSN:0013-4651. (Electrochemical Society)A review. The desire to improve and decentralize diagnostic platforms to facilitate highly precise and personalized medicine has motivated the development of a large no. of electrochem. sensing technologies. Such a development has been facilitated by electrochem.'s unparalleled ability to achieve highly specific mol. measurements in complex biol. fluids, without the need for expensive instrumentation. However, for decades, progress in the field had been constrained to systems that depended on the chem. reactivity of the analyte, obstructing the generalizability of such platforms beyond redox- or enzymically active clin. targets. Thus, the pursuit of alternative, more general strategies, coupled to the timely technol. advances in DNA sequencing, led to the development of DNA-based electrochem. sensors. The anal. value of these arises from the structural customizability of DNA and its ability to bind analytes ranging from ions and small mols. to whole proteins and cells. This versatility extends to interrogation methods, as DNA-based sensors work through a variety of detection schemes that can be probed via many electroanal. techniques. As a ref. for those experienced in the field, and to guide the unexperienced scientist, here the authors review the specific advantages of the electroanal. methods most commonly used for the interrogation of DNA-based sensors.
- 20Santos-Cancel, M.; Lazenby, R. A.; White, R. J. Rapid two-millisecond interrogation of electrochemical, aptamer-based sensor response using intermittent pulse amperometry. ACS Sens. 2018, 3, 1203– 1209, DOI: 10.1021/acssensors.8b00278Google Scholar20https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXpsVCitL8%253D&md5=120fb76b9377014f03b08c9604220fa6Rapid Two-Millisecond Interrogation of Electrochemical, Aptamer-Based Sensor Response Using Intermittent Pulse AmperometrySantos-Cancel, Mirelis; Lazenby, Robert A.; White, Ryan J.ACS Sensors (2018), 3 (6), 1203-1209CODEN: ASCEFJ; ISSN:2379-3694. (American Chemical Society)In this manuscript, the authors employ the technique intermittent pulse amperometry (IPA) to interrogate equil. and kinetic target binding to the surface of electrochem., aptamer-based (E-AB) sensors, achieving as fast as 2 ms time resoln. E-AB sensors comprise an electrode surface modified with a flexible nucleic acid aptamer tethered at the 3'-terminus with a redox-active mol. The introduction of a target changes the conformation and flexibility of the nucleic acid, which alters the charge transfer rate of the appended redox mol. Typically, changes in charge transfer rate within this class of sensor were monitored via voltammetric methods. Here, the use of IPA enables the detection of changes in charge transfer rates (i.e., current) at times <100 μs after the application of a potential pulse. Changes in sensor current are quant. related to target analyte concn. and can be used to create binding isotherms. Furthermore, the application of IPA enables rapid probing of the electrochem. surface with a time resoln. equiv. to as low as twice the applied potential pulse width, not previously demonstrated with traditional voltammetric techniques employed with E-AB sensors (a.c., square wave, cyclic). To visualize binding, the authors developed false-color plots analogous to those used in the field of fast-scan cyclic voltammetry. The use of IPA is universal, as demonstrated with two representative small mol. E-AB sensors directed against the aminoglycoside antibiotic tobramycin and ATP. Intermittent pulse amperometry exhibits an unprecedented sub-microsecond temporal response and is a general method for measuring rapid sensor performance.
- 21Arya, S. K.; Zhurauski, P.; Jolly, P.; Batistuti, M. R.; Mulato, M.; Estrela, P. Capacitive aptasensor based on interdigitated electrode for breast cancer detection in undiluted human serum. Biosens. Bioelectron. 2018, 102, 106– 112, DOI: 10.1016/j.bios.2017.11.013Google Scholar21https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhslyksbzN&md5=de062d9272f1afdaf736904e0dc19962Capacitive aptasensor based on interdigitated electrode for breast cancer detection in undiluted human serumArya, Sunil K.; Zhurauski, Pavel; Jolly, Pawan; Batistuti, Marina R.; Mulato, Marcelo; Estrela, PedroBiosensors & Bioelectronics (2018), 102 (), 106-112CODEN: BBIOE4; ISSN:0956-5663. (Elsevier B.V.)We report the development of a simple and powerful capacitive aptasensor for the detection and estn. of human epidermal growth factor receptor 2 (HER2), a biomarker for breast cancer, in undiluted serum. The study involves the incorporation of interdigitated gold electrodes, which were used to prep. the electrochem. platform. A thiol terminated DNA aptamer with affinity for HER2 was used to prep. the bio-recognition layer via self-assembly on interdigitated gold surfaces. Non-specific binding was prevented by blocking free spaces on surface via starting block phosphate buffer saline-tween20 blocker. The sensor was characterized using cyclic voltammetry, impedance electrochem. spectroscopy (EIS), at. force microscopy and contact angle studies. Non-Faradic EIS measurements were utilized to investigate the sensor performance via monitoring of the changes in capacitance. The aptasensor exhibited logarithmically detection of HER2 from 1 pM to 100 nM in both buffer and undiluted serum with limits of detection lower than 1 pM. The results pave the way to develop other aptamer-based biosensors for protein biomarkers detection in undiluted serum.
- 22Shaver, A.; Curtis, S. D.; Arroyo-Currás, N. Alkanethiol Monolayer End Groups Affect the Long-Term Operational Stability and Signaling of Electrochemical, Aptamer-Based Sensors in Biological Fluids. ACS Appl. Mater. Interfaces 2020, 12, 11214– 11223, DOI: 10.1021/acsami.9b22385Google Scholar22https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXisFKqsro%253D&md5=5f10c2efa8960f892202a6184bf9d63aAlkanethiol Monolayer End Groups Affect the Long-Term Operational Stability and Signaling of Electrochemical, Aptamer-Based Sensors in Biological FluidsShaver, Alexander; Curtis, Samuel D.; Arroyo-Curras, NetzahualcoyotlACS Applied Materials & Interfaces (2020), 12 (9), 11214-11223CODEN: AAMICK; ISSN:1944-8244. (American Chemical Society)Electrochem. aptamer-based (E-AB) sensors achieve highly precise measurements of specific mol. targets in untreated biol. fluids. This unique ability, together with their measurement frequency of seconds or faster, has enabled the real-time monitoring of drug pharmacokinetics in live animals with unprecedented temporal resoln. However, one important weakness of E-AB sensors is that their bioelectronic interface degrades upon continuous electrochem. interrogation-a process typically seen as a drop in faradaic and an increase in charging currents over time. This progressive degrdn. limits their in vivo operational life to 12 h at best, a period that is much shorter than the elimination half-life of the vast majority of drugs in humans. Thus, there is a crit. need to develop novel E-AB interfaces that resist continuous electrochem. interrogation in biol. fluids for prolonged periods. In response, our group is pursuing the development of better packed, more stable self-assembled monolayers (SAMs) to improve the signaling and extend the operational life of in vivo E-AB sensors from hours to days. By invoking hydrophobicity arguments, we have created SAMs that do not desorb from the electrode surface in aq. physiol. solns. and biol. fluids. These SAMs, formed from 1-hexanethiol solns., decrease the voltammetric charging currents of E-AB sensors by 3-fold relative to std. monolayers of 6-mercapto-1-hexanol, increase the total faradaic current, and alter the electron transfer kinetics of the platform. Moreover, the stability of our new SAMs enables uninterrupted, continuous E-AB interrogation for several days in biol. fluids, like undiluted serum, at a physiol. temp. of 37°C.
- 23Zhang, S.; Hu, R.; Hu, P.; Wu, Z.-S.; Shen, G.-L.; Yu, R.-Q. Blank peak current-suppressed electrochemical aptameric sensing platform for highly sensitive signal-on detection of small molecule. Nucleic Acids Res. 2010, 38, e185 DOI: 10.1093/nar/gkq728Google Scholar23https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXhsVektrzJ&md5=1177becb4b82947c02aa5fabf7ff52a5Blank peak current-suppressed electrochemical aptameric sensing platform for highly sensitive signal-on detection of small moleculeZhang, Songbai; Hu, Rong; Hu, Peng; Wu, Zai-Sheng; Shen, Guo-Li; Yu, Ru-QinNucleic Acids Research (2010), 38 (20), e185/1-e185/8CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)In this contribution, an electrochem. aptameric sensing scheme for the sensitive detection of small mols. is proposed using adenosine as a target model. A ferrocene (Fc)-functionalized thiolated aptamer probe is adapted and immobilized onto an electrode surface. Introducing a recognition site for EcoRI into the aptamer sequence not only suppresses the peak current corresponding to blank sample but also provides a signal-on response mechanism. In the absence of adenosine, the aptamer can fold into a hairpin structure and form a cleavable double-stranded region. Fc is capable of being removed from electrode surface by treatment with endonuclease, and almost no peak current is obsd. The adenosine/aptamer binding induces the conformational transition of designed aptamer, dissocg. the cleavable double-stranded segment. Therefore, the integrated aptamer sequence is maintained when exposing to endonuclease, generating a peak current of Fc. Utilizing the present sensing scheme, adenosine even at a low concn. can give a detectable current signal. Thus, a detection limit of 10-10 M and a linear response range from 3.74 × 10-9 to 3.74 × 10-5 M are achieved. The proposed proof-of-principle of a novel electrochem. sensing is expected to extend to establish various aptameric platforms for the anal. of a broad range of target mols. of interest.
- 24Huang, K.-C.; White, R. J. Random Walk on a Leash: A Simple Single-Molecule Diffusion Model for Surface-Tethered Redox Molecules with Flexible Linkers. J. Am. Chem. Soc. 2013, 135, 12808– 12817, DOI: 10.1021/ja4060788Google Scholar24https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXht1amtrvL&md5=6e0fd31ad02778e9b0ea33933aad17c4Random Walk on a Leash: A Simple Single-Molecule Diffusion Model for Surface-Tethered Redox Molecules with Flexible LinkersHuang, Kuan-Chun; White, Ryan J.Journal of the American Chemical Society (2013), 135 (34), 12808-12817CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)The authors develop a random walk model to simulate the Brownian motion and the electrochem. response of a single mol. confined to an electrode surface via a flexible mol. tether. The authors use the authors' simple model, which requires no prior knowledge of the physics of the mol. tether, to predict and better understand the voltammetric response of surface-confined redox mols. when motion of the redox mol. becomes important. The single mol. is confined to a hemispherical vol. with a max. radius detd. by the flexible mol. tether (5-20 nm) and is allowed to undergo true three-dimensional diffusion. Distance- and potential-dependent electron transfer probabilities are evaluated throughout the simulations to generate cyclic voltammograms of the model system. At sufficiently slow cyclic voltammetric scan rates the electrochem. reaction behaves like an adsorbed redox mol. with no mass transfer limitation; thus, the peak current is proportional to the scan rate. Conversely, at faster scan rates the diffusional motion of the mol. limits the simulated peak current, which exhibits a linear dependence on the square root of the scan rate. The switch between these two limiting regimes occurs when the diffusion layer thickness, (2Dt)1/2, is ∼10 times the tether length. Finally, the authors' model predicts the voltammetric behavior of a redox-active methylene blue tethered to an electrode surface via short flexible single-stranded, polythymine DNAs, allowing the estn. of diffusion coeffs. for the end-tethered mol.
- 25Rowe, A. A.; Miller, E. A.; Plaxco, K. W. Reagentless Measurement of Aminoglycoside Antibiotics in Blood Serum via an Electrochemical, Ribonucleic Acid Aptamer-Based Biosensor. Anal. Chem. 2010, 82, 7090– 7095, DOI: 10.1021/ac101491dGoogle Scholar25https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXpvVGmsLw%253D&md5=7f05a835e365d5460cdd7141d3c946f6Reagentless Measurement of Aminoglycoside Antibiotics in Blood Serum via an Electrochemical, Ribonucleic Acid Aptamer-Based BiosensorRowe, Aaron A.; Miller, Erin A.; Plaxco, Kevin W.Analytical Chemistry (Washington, DC, United States) (2010), 82 (17), 7090-7095CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)Biosensors built using RNA aptamers show promise as tools for point-of-care medical diagnostics, but they remain vulnerable to nuclease degrdn. when deployed in clin. samples. To explore methods for protecting RNA-based biosensors from such degrdn. we have constructed and characterized an electrochem., aptamer-based sensor for the detection of aminoglycosidic antibiotics. We find that while this sensor achieves low micromolar detection limits and subminute equilibration times when challenged in buffer, it deteriorates rapidly when immersed directly in blood serum. In order to circumvent this problem, we have developed and tested sensors employing modified versions of the same aptamer. Our first effort to this end entailed the methylation of all of the 2'-hydroxyl groups outside of the aptamer's antibiotic binding pocket. However, while devices employing this modified aptamer are as sensitive as those employing an unmodified parent, the modification fails to confer greater stability when the sensor is challenged directly in blood serum. As a second potentially naive alternative, we replaced the RNA bases in the aptamer with their more degrdn.-resistant DNA equiv. Surprisingly and unlike control DNA-stem loops employing other sequences, this DNA aptamer retains the ability to bind aminoglycosides, albeit with poorer affinity than the parent RNA aptamer. Unfortunately, however, while sensors fabricated using this DNA aptamer are stable in blood serum, its lower affinity pushes their detection limits above the therapeutically relevant range. Finally, we find that ultrafiltration through a low-mol.-wt.-cutoff spin column rapidly and efficiently removes the relevant nucleases from serum samples spiked with gentamicin, allowing the convenient detection of this aminoglycoside at clin. relevant concns. using the original RNA-based sensor.
- 26White, R. J.; Phares, N.; Lubin, A. A.; Xiao, Y.; Plaxco, K. W. Optimization of Electrochemical Aptamer-Based Sensors via Optimization of Probe Packing Density and Surface Chemistry. Langmuir 2008, 24, 10513– 10518, DOI: 10.1021/la800801vGoogle Scholar26https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXps1Citb8%253D&md5=c568824267126eb2d8f9cb8b7cd3c7b4Optimization of Electrochemical Aptamer-Based Sensors via Optimization of Probe Packing Density and Surface ChemistryWhite, Ryan J.; Phares, Noelle; Lubin, Arica A.; Xiao, Yi; Plaxco, Kevin W.Langmuir (2008), 24 (18), 10513-10518CODEN: LANGD5; ISSN:0743-7463. (American Chemical Society)Electrochem., 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. To further improve this technol. the authors have systematically studied the effects of probe (aptamer) packing d., 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 mol. cocaine and the protein thrombin. The authors find that, by controlling the concn. of aptamer employed during sensor fabrication, the authors can control the d. of probe DNA mols. 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 max. gain obsd. 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 obsd. at intermediate densities. Above cut-offs at low hertz frequencies, neither sensor displays any significant dependence on the frequency of the alternating potential employed in their interrogation. Finally, the authors find that E-AB signal gain is sensitive to the nature of the alkanethiol SAM employed to passivate the interrogating electrode; while thinner SAMs lead to higher abs. sensor currents, reducing the length of the SAM from 6-carbons to 2-carbons reduces the obsd. signal gain of the authors' cocaine sensor 10-fold. The authors demonstrate that fabrication and operational parameters can be varied to achieve optimal sensor performance and that these can serve as a basic outline for future sensor fabrication.
- 27Liu, Y.; Canoura, J.; Alkhamis, O.; Xiao, Y. Immobilization Strategies for Enhancing Sensitivity of Electrochemical Aptamer-Based Sensors. ACS Appl. Mater. Interfaces 2021.Google ScholarThere is no corresponding record for this reference.
- 28Campos, R.; Kotlyar, A.; Ferapontova, E. E. DNA-Mediated Electron Transfer in DNA Duplexes Tethered to Gold Electrodes via Phosphorothioated dA Tags. Langmuir 2014, 30, 11853– 11857, DOI: 10.1021/la502766gGoogle Scholar28https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhs1CiurzM&md5=8ac830b30c846608a8529aa68006fc89DNA-Mediated Electron Transfer in DNA Duplexes Tethered to Gold Electrodes via Phosphorothioated dA TagsCampos, Rui; Kotlyar, Alexander; Ferapontova, Elena E.Langmuir (2014), 30 (40), 11853-11857CODEN: LANGD5; ISSN:0743-7463. (American Chemical Society)The efficiency of DNA-based bioelectronic devices strongly depends on the way DNA mols. are linked to the electronic component. Commonly, DNA is tethered to metal electrodes via an alkanethiol linker representing an addnl. barrier for electron transport. The replacement of the alkanethiol linker for a phosphorothioated adenosine tag increases the rate of DNA-mediated electron transfer (ET) up to 259 s-1, representing the highest hitherto reported rate of electrochem.-modulated ET, and improves the stability of DNA-electrode surface binding. Both results offer pronounced technol. and scientific benefits for DNA-based electronics.
- 29Campos, R.; Kékedy-Nagy, L.; She, Z.; Sodhi, R.; Kraatz, H.-B.; Ferapontova, E. E. Electron Transfer in Spacer-Free DNA Duplexes Tethered to Gold via dA10 Tags. Langmuir 2018, 34, 8472– 8479, DOI: 10.1021/acs.langmuir.8b01412Google Scholar29https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhtFyqtLzK&md5=b9aa2e07002cb454597a0a5665bc1824Electron Transfer in Spacer-Free DNA Duplexes Tethered to Gold via dA10 TagsCampos, Rui; Kekedy-Nagy, Laszlo; She, Zhe; Sodhi, Rana; Kraatz, Heinz-Bernhard; Ferapontova, Elena E.Langmuir (2018), 34 (29), 8472-8479CODEN: LANGD5; ISSN:0743-7463. (American Chemical Society)Elec. properties of DNA critically depend on the way DNA mols. are integrated within the electronics, particularly on DNA-electrode immobilization strategies. Here, the rate of electron transport in DNA duplexes spacer-free tethered to gold via the adenosine terminal region (a dA10 tag) is enhanced compared to the hitherto reported DNA-metal electrode tethering chemistries. The rate of DNA-mediated electron transfer (ET) between the electrode and methylene blue intercalated into the dA10-tagged DNA duplex approached 361 s-1 at a ca. half-monolayer DNA surface coverage ΓDNA (with a linear regression limit of 670 s-1 at ΓDNA → 0), being 2.7-fold enhanced compared to phosphorothioated dA5* tethering (6-fold for the C6-alkanethiol linker representing an addnl. ET barrier). XPS evidenced dA10 binding to the Au surface via the purine N, whereas dA5* predominantly coordinated to the surface via sulfur atoms of phosphothioates. The latter apparently induces the DNA strand twist in the point of surface attachment affecting the local DNA conformation and, as a result, decreasing the ET rates through the duplex. Thus, a spacer-free DNA coupling to electrodes via dA10 tags thus allows a perspective design of DNA electronic circuits and sensors with advanced electronic properties and no implication from more expensive, synthetic linkers.
- 30Laviron, E. General expression of the linear potential sweep voltammogram in the case of diffusionless electrochemical systems. J. Electroanal. Chem. Interfacial Electrochem. 1979, 101, 19– 28, DOI: 10.1016/s0022-0728(79)80075-3Google Scholar30https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaE1MXltFCnt7k%253D&md5=9d280d1e17f00b28bd56b6cade497eddGeneral expression of the linear potential sweep voltammogram in the case of diffusionless electrochemical systemsLaviron, E.Journal of Electroanalytical Chemistry and Interfacial Electrochemistry (1979), 101 (1), 19-28CODEN: JEIEBC; ISSN:0022-0728.The equation of a linear potential sweep voltammogram is derived for any degree of reversibility of the electrochem. reaction for the following methods:surface voltammetry when both the oxidized and the reduced froms are strongly adsorbed, and a Langmuir isotherm is obeyed, thin-layer voltammetry, and linear potential sweep coulometry. The results are expressed in a math. form valid for the 3 cases. The transfer coeff. and the rate const. of the electrochem. reaction can be deduced from an exptl. study of the variations of the peak potentials as a function of the sweep rate.
- 31Dauphin-Ducharme, P.; Arroyo-Currás, N.; Adhikari, R.; Somerson, J.; Ortega, G.; Makarov, D. E.; Plaxco, K. W. Chain Dynamics Limit Electron Transfer from Electrode-Bound, Single-Stranded Oligonucleotides. J. Phys. Chem. C 2018, 122, 21441– 21448, DOI: 10.1021/acs.jpcc.8b06111Google Scholar31https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhsFOlsbnJ&md5=5f587654c6194f591b42c4b1a9dc014fChain Dynamics Limit Electron Transfer from Electrode-Bound, Single-Stranded OligonucleotidesDauphin-Ducharme, Philippe; Arroyo-Curras, Netzahualcoyotl; Adhikari, Ramesh; Somerson, Jacob; Ortega, Gabriel; Makarov, Dmitrii E.; Plaxco, Kevin W.Journal of Physical Chemistry C (2018), 122 (37), 21441-21448CODEN: JPCCCK; ISSN:1932-7447. (American Chemical Society)A wide range of new devices aimed at in vivo mol. detection and point-of-care diagnostics rely on binding-induced changes in electron-transfer kinetics from an electrode-attached, redox-reporter-modified oligonucleotide as their signaling mechanism. In an effort to better characterize the mechanisms underlying these sensors, we have measured the electron-transfer kinetics assocd. with surface-attached, single-stranded DNAs modified with a methylene blue redox reporter either at the chain's distal end or at an internal chain position. We find that although the rate of electron transfer from a reporter placed either terminally or internally is independent of chain length for chains shorter than the length scale of methylene blue (and its linker), for longer chains it follows a power-law dependence on length of exponent approx. -2.2. Such behavior is consistent with a diffusion-controlled mechanism in which the diffusion of the DNA-bound reporter to the surface controls the rate of electron transfer. This said, the obsd. rates are, at 5-400 s-1, orders of magnitude slower than the intramol. dynamics of single-stranded oligonucleotides when free in soln. Likewise, the rates of transfer from reporters placed internally are several-fold slower than those seen for the equiv. terminally modified construct. We attribute these effects to electrostatic repulsion between the oligonucleotide and the electrode surface, which is neg. charged at the redox potential of methylene blue. Consistent with this, changing monolayer compn. so as to increase the neg. charge of the surface reduces the transfer rate still more without significantly altering its power-law chain length dependence. Simple theor. models and computer simulations performed in support of our exptl. studies find similar power-law dependencies, similar electrostatic slowing of the transfer rate, and similar rate differences between terminally an internally modified constructs.
- 32Dauphin-Ducharme, P.; Yang, K.; Arroyo-Currás, N.; Ploense, K. L.; Zhang, Y.; Gerson, J.; Kurnik, M.; Kippin, T. E.; Stojanovic, M. N.; Plaxco, K. W. Electrochemical Aptamer-Based Sensors for Improved Therapeutic Drug Monitoring and High-Precision, Feedback-Controlled Drug Delivery. ACS Sens. 2019, 4, 2832– 2837, DOI: 10.1021/acssensors.9b01616Google Scholar32https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXhvVCnt7vM&md5=67cd35b6a26e364ac78a9c4339925c3bElectrochemical Aptamer-Based Sensors for Improved Therapeutic Drug Monitoring and High-Precision, Feedback-Controlled Drug DeliveryDauphin-Ducharme, Philippe; Yang, Kyungae; Arroyo-Curras, Netzahualcoyotl; Ploense, Kyle L.; Zhang, Yameng; Gerson, Julian; Kurnik, Martin; Kippin, Tod E.; Stojanovic, Milan N.; Plaxco, Kevin W.ACS Sensors (2019), 4 (10), 2832-2837CODEN: ASCEFJ; ISSN:2379-3694. (American Chemical Society)The Electrochem. Aptamer-Based (E-AB) sensing platform appears a convenient (rapid, single-step, calibration-free), modular approach to measure concns. of specific mols. (irresp. of their chem. reactivity) directly in blood and even in situ in the living body. Given these attributes, the platform may thus provide significant opportunities to render therapeutic drug monitoring (the clin. practice in which dosing is adjusted in response to plasma drug measurements) as frequent and convenient as the measurement of blood sugar has become for diabetics. The ability to measure arbitrary mols. in the body in real time could even enable closed-loop feedback control over plasma drug levels in a manner analogous to the recently commercialized controlled blood sugar systems. As initial exploration of this, the authors describe here the selection of an aptamer against vancomycin, a narrow therapeutic window antibiotic for which therapeutic monitoring is a crit. part of the std. of care, and its adaptation into an electrochem. aptamer-based (E-AB) sensor. Using this sensor the authors then demonstrate: (1) rapid (seconds), convenient (single-step, calibration-free) measurement of plasma vancomycin in finger-prick-scale samples of whole blood, (2) high-precision measurement of subject-specific vancomycin pharmacokinetics (in a rat animal model), and (3) high precision, closed-loop feedback control over plasma levels of the drug (in a rat animal model). The ability to not only track (with continuous-glucose-monitor-like measurement frequency and convenience), but also actively control plasma drug levels provides an unprecedented route towards improving therapeutic drug monitoring and, more generally, the personalized, high-precision delivery of pharmacol. interventions.
- 33Curtis, S. D.; Ploense, K. L.; Kurnik, M.; Ortega, G.; Parolo, C.; Kippin, T. E.; Plaxco, K. W.; Arroyo-Currás, N. Open Source Software for the Real-Time Control, Processing, and Visualization of High-Volume Electrochemical Data. Anal. Chem. 2019, 91, 12321– 12328, DOI: 10.1021/acs.analchem.9b02553Google Scholar33https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXhs1KksL%252FI&md5=8a2fb45b4fd84748c8c38c8754c1db12Open Source Software for the Real-Time Control, Processing, and Visualization of High-Volume Electrochemical DataCurtis, Samuel D.; Ploense, Kyle L.; Kurnik, Martin; Ortega, Gabriel; Parolo, Claudio; Kippin, Tod E.; Plaxco, Kevin W.; Arroyo-Curras, NetzahualcoyotlAnalytical Chemistry (Washington, DC, United States) (2019), 91 (19), 12321-12328CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)Electrochem. sensors are major players in the race for improved mol. diagnostics due to their convenience, temporal resoln., manufg. scalability, and their ability to support real-time measurements. This is evident in the ever-increasing no. of health-related electrochem. sensing platforms, ranging from single-measurement point-of-care devices to wearable devices supporting immediate and continuous monitoring. In support of the need for such systems to rapidly process large data vols. the authors describe here an open-source, easily customizable, multi-platform compatible program for the real-time control, processing and visualization of electrochem. data. The software's architecture is modular and fully documented, allowing the easy customization of the code to support the processing of voltammetric (e.g., square-wave and cyclic) and chronoamperometric data. The program, which the authors have called Software for the Anal. and Continuous Monitoring of Electrochem. Systems (SACMES), also includes a graphical interface allowing the user to easily change anal. parameters (e.g., signal/noise processing, baseline correction) in real-time. To demonstrate the versatility of SACMES the authors use it here to analyze the real-time data output by: (1) the electrochem., aptamer-based measurement of a specific small-mol. target, (2) a monoclonal antibody-detecting DNA-scaffold sensor, and (3) the detn. of the folding thermodn. of an electrode-attached, redox-reporter-modified protein.
- 34Rant, U.; Arinaga, K.; Scherer, S.; Pringsheim, E.; Fujita, S.; Yokoyama, N.; Tornow, M.; Abstreiter, G. Switchable DNA interfaces for the highly sensitive detection of label-free DNA targets. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 17364– 17369, DOI: 10.1073/pnas.0703974104Google Scholar34https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXht1ymtrjL&md5=df81bafa5a13f7ba4612578d19fc7f7aSwitchable DNA interfaces for the highly sensitive detection of label-free DNA targetsRant, Ulrich; Arinaga, Kenji; Scherer, Simon; Pringsheim, Erika; Fujita, Shozo; Yokoyama, Naoki; Tornow, Marc; Abstreiter, GerhardProceedings of the National Academy of Sciences of the United States of America (2007), 104 (44), 17364-17369CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)We report a method to detect label-free oligonucleotide targets. The conformation of surface-tethered probe nucleic acids is modulated by alternating elec. fields, which cause the mols. to extend away from or fold onto the biased surface. Binding (hybridization) of targets to the single-stranded probes results in a pronounced enhancement of the layer-height modulation amplitude, monitored optically in real time. The method features an exceptional detection limit of <3 × 108 bound targets per cm2 sensor area. Single base-pair mismatches in the sequences of DNA complements may readily be identified; moreover, binding kinetics and binding affinities can be detd. with high accuracy. When driving the DNA to oscillate at frequencies in the kHz regime, distinct switching kinetics are revealed for single- and double-stranded DNA. Mol. dynamics are used to identify the binding state of mols. according to their characteristic kinetic fingerprints by using a chip-compatible detection format.
- 35Rant, U.; Arinaga, K.; Tornow, M.; Kim, Y. W.; Netz, R. R.; Fujita, S.; Yokoyama, N.; Abstreiter, G. Dissimilar Kinetic Behavior of Electrically Manipulated Single- and Double-Stranded DNA Tethered to a Gold Surface. Biophys. J. 2006, 90, 3666– 3671, DOI: 10.1529/biophysj.105.078857Google Scholar35https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28Xkt1OktLY%253D&md5=50245d6b79f863cc0d45ea955cf9a796Dissimilar kinetic behavior of electrically manipulated single- and double-stranded DNA tethered to a gold surfaceRant, Ulrich; Arinaga, Kenji; Tornow, Marc; Kim, Yong Woon; Netz, Roland R.; Fujita, Shozo; Yokoyama, Naoki; Abstreiter, GerhardBiophysical Journal (2006), 90 (10), 3666-3671CODEN: BIOJAU; ISSN:0006-3495. (Biophysical Society)We report on the elec. manipulation of single- and double-stranded oligodeoxynucleotides that are end tethered to gold surfaces in electrolyte soln. The response to alternating repulsive and attractive elec. surface fields is studied by time-resolved fluorescence measurements, revealing markedly distinct dynamics for the flexible single-stranded and stiff double-stranded DNA, resp. Hydrodynamic simulations rationalize this finding and disclose two different kinetic mechanisms: stiff polymers undergo rotation around the anchoring pivot point; flexible polymers, on the other hand, are pulled onto the attracting surface segment by segment.
- 36Yang, W.; Lai, R. Y. Comparison of the Stem-Loop and Linear Probe-Based Electrochemical DNA Sensors by Alternating Current Voltammetry and Cyclic Voltammetry. Langmuir 2011, 27, 14669– 14677, DOI: 10.1021/la203015vGoogle Scholar36https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXhtlOqu7fP&md5=8a9bfecb8d26b6ac349fe681bab6265dComparison of the stem-loop and linear probe-based electrochemical DNA sensors by alternating current voltammetry and cyclic voltammetryYang, Weiwei; Lai, Rebecca Y.Langmuir (2011), 27 (23), 14669-14677CODEN: LANGD5; ISSN:0743-7463. (American Chemical Society)Here we systematically characterized the sensor performance of the stem-loop probe (SLP) and linear probe (LP) electrochem. DNA sensors using a.c. voltammetry (ACV) and cyclic voltammetry (CV), with the goal of generating the set of operational criteria that best suits each sensor architecture, in addn. to elucidating the signaling mechanism behind these sensors. Although the LP sensor shows slightly better % signal suppression (SS) upon hybridization with the perfect match target at 10 Hz, our frequency-dependent study suggests that it shows optimal % SS only in a very limited AC frequency range. Similar results are obsd. in CV studies in which the LP sensor, when compared to the SLP sensor, displays a narrower range of voltammetric scan rates where the optimal % SS can be achieved. More importantly, the difference between the two sensors' performance is particularly pronounced if the change in integrated charge (Q) upon target hybridization, rather than the peak current (I), is measured in CV. The temp.-dependent study further highlights the differences between the two sensors, where the LP sensor, owing to the flexible linear probe architecture, is more readily perturbed by temp. changes. Both SLP and LP sensors, however, show a loss of % SS when operated at elevated temps., despite the significant improvement in the hybridization kinetics. In conjunction with the ACV, CV, and temp.-dependent studies, the electron-transfer kinetics study provides further evidence in support of the proposed signaling mechanism of these two sensors, in which the SLP sensor's signaling efficiency and sensor performance is directly linked to the hybridization-induced conformational change in the redox-labeled probe, whereas the performance of the LP sensor relies on the hybridization-induced change in probe dynamics.
- 37Lai, R. Y.; Walker, B.; Stormberg, K.; Zaitouna, A. J.; Yang, W. Electrochemical techniques for characterization of stem-loop probe and linear probe-based DNA sensors. Methods 2013, 64, 267– 275, DOI: 10.1016/j.ymeth.2013.07.041Google Scholar37https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhtlCnsbjF&md5=449c8f20d0cfb0263d7d541c26721423Electrochemical techniques for characterization of stem-loop probe and linear probe-based DNA sensorsLai, Rebecca Y.; Walker, Bryce; Stormberg, Kent; Zaitouna, Anita J.; Yang, WeiweiMethods (Amsterdam, Netherlands) (2013), 64 (3), 267-275CODEN: MTHDE9; ISSN:1046-2023. (Elsevier B.V.)Here we present a summary of the sensor performance of the stem-loop probe (SLP) and linear probe (LP) electrochem. DNA sensors when interrogated using a.c. voltammetry (ACV), cyclic voltammetry (CV), and differential pulse voltammetry (DPV). Specifically, we identified one crit. parameter for each voltammetric technique that can be adjusted for optimal sensor performance. Overall, the SLP sensor displayed good sensor performance (i.e., 60 + % signal attenuation in the presence of the target) over a wider range of exptl. conditions when compared to the LP sensor. When used with ACV, the optimal frequency range was found to be between 5 and 5000 Hz, larger than the 5-100 Hz range obsd. with the LP sensor. A similar trend was obsd. for the two sensors in CV; the LP sensor was operational only at scan rates between 30 and 100 V/s, whereas the SLP sensor performed well at scan rates between 1 and 1000 V/s. Unlike ACV and CV, DPV has demonstrated to be a more versatile sensor interrogation technique for this class of sensors. Despite the minor differences in total signal attenuation upon hybridization to the target DNA, both SLP and LP sensors performed optimally under most pulse widths used in this study. More importantly, when used with longer pulse widths, both sensors showed "signal-on" behavior, which is generally more desirable for sensor applications.
- 38Kang, D.; Parolo, C.; Sun, S.; Ogden, N. E.; Dahlquist, F. W.; Plaxco, K. W. Expanding the Scope of Protein-Detecting Electrochemical DNA “Scaffold” Sensors. ACS Sens. 2018, 3, 1271– 1275, DOI: 10.1021/acssensors.8b00311Google Scholar38https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhtFGju7fL&md5=1136584de066e909565c10aa3aca91c1Expanding the Scope of Protein-Detecting Electrochemical DNA "Scaffold" SensorsKang, Di; Parolo, Claudio; Sun, Sheng; Ogden, Nathan E.; Dahlquist, Frederick W.; Plaxco, Kevin W.ACS Sensors (2018), 3 (7), 1271-1275CODEN: ASCEFJ; ISSN:2379-3694. (American Chemical Society)The ability to measure the levels of diagnostically relevant proteins, such as antibodies, directly at the point of care could significantly impact healthcare. Thus motivated, we explore here the E-DNA "scaffold" sensing platform, a rapid, convenient, single-step means to this end. These sensors comprise a rigid nucleic acid "scaffold" attached via a flexible linker to an electrode and modified on its distal end with a redox reporter and a protein binding "recognition element". The binding of a targeted protein reduces the efficiency with which the redox reporter approaches the electrode, resulting in an easily measured signal change when the sensor is interrogated voltammetrically. Previously we have demonstrated scaffold sensors employing a range of low mol. wt. haptens and linear peptides as their recognition elements. Expanding on this here we have characterized sensors employing much larger recognition elements (up to and including full length proteins) in order to (1) define the range of recognition elements suitable for use in the platform; (2) better characterize the platform's signaling mechanism to aid its design and optimization; and (3) demonstrate the anal. performance of sensors employing full-length proteins as recognition elements. In doing so we have enlarged the range of mol. targets amenable to this rapid and convenient sensing platform.
- 39Arroyo-Currás, N.; Sadeia, M.; Ng, A. K.; Fyodorova, Y.; Williams, N.; Afif, T.; Huang, C.-M.; Ogden, N.; Andresen Eguiluz, R. C.; Su, H.-J.; Castro, C. E.; Plaxco, K. W.; Lukeman, P. S. An electrochemical biosensor exploiting binding-induced changes in electron transfer of electrode-attached DNA origami to detect hundred nanometer-scale targets. Nanoscale 2020, 12, 13907– 13911, DOI: 10.1039/d0nr00952kGoogle Scholar39https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhtF2isLzK&md5=fdd1e2f538ebe9f782c8b2124370ed8aAn electrochemical biosensor exploiting binding-induced changes in electron transfer of electrode-attached DNA origami to detect hundred nanometer-scale targetsArroyo-Curras, Netzahualcoyotl; Sadeia, Muaz; Ng, Alexander K.; Fyodorova, Yekaterina; Williams, Natalie; Afif, Tammy; Huang, Chao-Min; Ogden, Nathan; Andresen Eguiluz, Roberto C.; Su, Hai-Jun; Castro, Carlos E.; Plaxco, Kevin W.; Lukeman, Philip S.Nanoscale (2020), 12 (26), 13907-13911CODEN: NANOHL; ISSN:2040-3372. (Royal Society of Chemistry)The specific detection in clin. samples of analytes with dimensions in the tens to hundreds of nanometers, such as viruses and large proteins, would improve disease diagnosis. Detection of these "mesoscale" analytes (as opposed to their nanoscale components), however, is challenging as it requires the simultaneous binding of multiple recognition sites often spaced over tens of nanometers. In response, we have adapted DNA origami, with its unparalleled customizability to precisely display multiple target-binding sites over the relevant length scale, to an electrochem. biosensor platform. Our proof-of-concept employs triangular origami covalently attached to a gold electrode and functionalized with redox reporters. Electrochem. interrogation of this platform successfully monitors mesoscale, target-binding-induced changes in electron transfer in a manner consistent with coarse-grained mol. dynamics simulations. Our approach enables the specific detection of analytes displaying recognition sites that are sepd. by ~ 40 nm, a spacing significantly greater than that achieved in similar sensor architectures employing either antibodies or aptamers.
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Abstract
Figure 1
Figure 1. E-AB sensors undergo target binding-induced changes in electron-transfer kinetics of the redox reporter that can be monitored in real time via electrochemical interrogation. (A) In this work, we employed three different DNA aptamers modified at the 5′ terminus with alkanethiol linkers and at the 3′ terminus with the redox reporter MB. We codeposited these modified oligonucleotides with 6-mercapto-1-hexanol on the surface of gold electrodes via self-assembly. (B) In the presence of their target molecule, the aptamers undergo a conformational change that, presumably, brings the redox reporter closer to the electrode surface, increasing the electron-transfer rate.
Figure 2
Figure 2. Target binding-induced changes in apparent electron-transfer rates of E-AB sensors can be monitored via ΔEP. (A) When we interrogate tobramycin-detecting E-AB sensors using CV (at 5 V·s–1), we observe a decrease in ΔEP upon the addition of tobramycin (10 mM). (B) Same voltammograms as in (A) but zoomed-in relative to the x-axis show more clearly the change in electron-transfer rate (ket) from ΔEP,1 to ΔEP,2, with ΔEP,2 < ΔEP,1. (C) The magnitude of ΔEP decreases monotonically with increasing target concentration. The solid line is a nonlinear fit to the Hill equation, resulting in a Hill coefficient n = 1 and KD = 80 ± 7 μM. Error bars represent the standard deviation between five electrodes. All measurements performed in 1× phosphate-buffered saline (PBS).
Figure 3
Figure 3. E-AB sensor gain and sensitivity based on ΔEP are a strong function of the voltage scanning rate. (A) We illustrate this effect by showing ΔEP values in the absence (black circles, ΔEP,1) and presence (red circles, ΔEP,2) of saturating concentrations of tobramycin (10 mM) at increasing voltage scanning rates. Note that, although ΔEP increases with the increasing scanning rate in both cases, it does not do so with the same correlation function. Thus, CV scanning rates ranging between 5 and 10 V s–1 produce the largest signal change based on ΔEP. (B) Dose–response curves built from ΔEP measurements result in apparent aptamer dissociation constants that strongly depend on the CV scanning rate. Interrogating tobramycin-detecting E-AB sensors at CV scanning rates between 5 and 10 V s–1 achieve the most sensitive measurements (lowest apparent KD) with the largest overall signal gain. (C) Side-by-side comparison of sensors interrogated by CV using voltammetric peak heights, IP (blue circles) vs ΔEP (black circles) to illustrate that ΔEP achieves a 3-fold improvement in signal gain relative to IP, albeit with the opposite sign. Error bars represent the standard deviation between the five electrodes. All measurements were performed in 1× PBS.
Figure 4
Figure 4. ΔEP-based interrogation supports E-AB measurements irrespective of the aptamer used. We evaluated the general suitability of our method for E-AB sensing in undiluted serum by fabricating sensors using (A) tobramycin-, (B) vancomycin-, and (C) procaine-binding aptamers. Here, we show the performance of these sensors in undiluted serum when interrogated by CV. Left. Cyclic voltammograms measured in the absence (black trace) and presence (red trace) of a saturating target show significant differences in ΔEP. Note that for tobramycin and vancomycin, we observed minimal change in IP. However, voltammograms measured with the procaine sensor did show a significant change in IP matching a broadening of the faradaic waves. Center. Scan rate dependency of each sensor to determine the region of maximum signal decrease between the unbound and bound states. Right. We use the scanning rate giving the best compromise between maximum signal change and low apparent KD to build calibration curves for each analyte. Error bars represent the standard deviation between five electrodes. The calculated detection limit was 117 ± 4, 45 ± 6, and 60 ± 10 μM for the tobramycin, vancomycin, and procaine sensors, respectively. The precision of each sensor observed by this method can be found in Figure S5.
Figure 5
Figure 5. Serially interrogating E-AB sensors via ΔEP supports second to subsecond monitoring of fluctuating target concentrations in real time. Here, we show serial ΔEP measurements (black traces) recorded at (A) 5 V s–1 for tobramycin, (B) 1 V s–1 for vancomycin, and (C) 1 V s–1 for procaine. Using a voltage window of 600 mV (e.g., see Figure 2A), these scanning rates achieve measurements every 0.24, 1.2, and 1.2 s, respectively. To demonstrate the E-AB performance over time, in these panels we recorded measurements for 7 h alternating between 100% serum, and serum + target at a saturating concentration. To reveal the percentage contribution of drift to our measurements, we present the data as the relative change in signal with respect to the signal measured at the start of each experiment. We eliminated the effect of temperature fluctuations by maintaining the electrochemical cell at 25 °C using a water jacket and a temperature-controlled recirculation bath. We also continuously stirred the serum at ∼100 rpms to avoid precipitation of solids from the serum. Red traces represent the expected relative signal change in the absence or presence of a target as given by our calibration curves from Figure 4. Error bars represent the standard deviation between the five electrodes.
Figure 6
Figure 6. Long-term stability of E-AB sensors under different interrogation methods. (A) To investigate the extent to which continuously interrogating the sensors affects their operational stability, we interrogated E-AB sensors in PBS for 72 h by square wave voltammetry, either in continuous (5 × 104 scans) or single-point (10 scans) regimes, and by CV (7 × 104 scans) monitoring both peak-to-peak separation and the oxidation peak current. We observe that the continuous square wave voltammetry-based interrogation contributes to a fast loss in the signal (red trace), which dramatically decreases with less total measurements (at equal total experiment time, green circles). However, continuous CV-based interrogation (7 × 104 scans) by monitoring either ΔEP or IP does not contribute to the sensor signal loss. (B) When we switched to a more complex matrix such as undiluted serum, the CV-based interrogation presents an initial loss of signal (∼15%) during the first 10 h likely due to monolayer reorganization, desorption, and the nonspecific binding of proteins. While CV peak currents continue to drop linearly after this initial decay (blue trace), ΔEP remains constant for ∼50 h, the point at which our software is no longer able to resolve voltammetric peaks from the charging current. These measurements were performed using tobramycin-binding E-AB sensors at controlled 25 °C and under continuous stirring. Shaded areas or error bars represent the standard deviation between the five electrodes.
Figure 7
Figure 7. The batch-to-batch and day-to-day performance of E-AB sensors in undiluted serum is affected by the sensitivity of the interrogation method to changing sensor interfaces. (A) Square wave voltammetry-based measurements of E-AB peak current performed continuously every 5 s for 24 h. These measurements present significant variability in signal output between three batches of six sensors each (different colored traces), measured on three separate days. (B) The same variability is not seen for the ΔEP-based interrogation, which produces indistinguishable traces between electrode batches and measurement days. Specifically, the initial decay in signal driven by monolayer reorganization and the nonspecific protein binding is identical between batches and, in all cases, it stabilizes at ∼75% of its initial value. These measurements were performed using tobramycin-binding E-AB sensors at controlled 25 °C and under continuous stirring. Shaded areas represent the standard deviation between the five electrodes.
References
This article references 39 other publications.
- 1Fan, C.; Plaxco, K. W.; Heeger, A. J. Electrochemical interrogation of conformational changes as a reagentless method for the sequence-specific detection of DNA. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 9134– 9137, DOI: 10.1073/pnas.16335151001https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3sXmtlyksLk%253D&md5=85001b6478051243f8e5b3d70d5cb032Electrochemical interrogation of conformational changes as a reagentless method for the sequence-specific detection of DNAFan, Chunhai; Plaxco, Kevin W.; Heeger, Alan J.Proceedings of the National Academy of Sciences of the United States of America (2003), 100 (16), 9134-9137CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)We report a strategy for the reagentless transduction of DNA hybridization into a readily detectable electrochem. signal by means of a conformational change analogous to the optical mol. beacon approach. The strategy involves an electroactive, ferrocene-tagged DNA stem-loop structure that self-assembles onto a gold electrode by means of facile gold-thiol chem. Hybridization induces a large conformational change in this surface-confined DNA structure, which in turn significantly alters the electron-transfer tunneling distance between the electrode and the redoxable label. The resulting change in electron transfer efficiency is readily measured by cyclic voltammetry at target DNA concns. as low as 10 pM. In contrast to existing optical approaches, an electrochem. DNA (E-DNA) sensor built on this strategy can detect femtomoles of target DNA without employing cumbersome and expensive optics, light sources, or photodetectors. In contrast to previously reported electrochem. approaches, the E-DNA sensor achieves this impressive sensitivity without the use of exogenous reagents and without sacrificing selectivity or reusability. The E-DNA sensor thus offers the promise of convenient, reusable detection of picomolar DNA.
- 2Xiao, Y.; Lubin, A. A.; Heeger, A. J.; Plaxco, K. W. Label-Free Electronic Detection of Thrombin in Blood Serum by Using an Aptamer-Based Sensor. Angew. Chem., Int. Ed. 2005, 44, 5456– 5459, DOI: 10.1002/anie.2005009892https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXpvF2ksLg%253D&md5=03fba0f9b28e0c7c8375e8fef4a5446fLabel-free electronic detection of thrombin in blood serum by using an aptamer-based sensorXiao, Yi; Lubin, Arica A.; Heeger, Alan J.; Plaxco, Kevin W.Angewandte Chemie, International Edition (2005), 44 (34), 5456-5459CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)A methylene blue (MB) tagged, thrombin-binding DNA aptamer immobilized on a gold surface undergoes a large conformational change upon target binding and inhibits electron transfer. This folding produces a large, readily measurable change in redox current and allows the electrochem. detection of thrombin in blood serum.
- 3Arroyo-Currás, N.; Somerson, J.; Vieira, P. A.; Ploense, K. L.; Kippin, T. E.; Plaxco, K. W. Real-time measurement of small molecules directly in awake, ambulatory animals. Proc. Natl. Acad. Sci. U.S.A. 2017, 114, 645– 650, DOI: 10.1073/pnas.16134581143https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXmslWktA%253D%253D&md5=04842755081ae2272a933ce3461cd953Real-time measurement of small molecules directly in awake, ambulatory animalsArroyo-Curras, Netzahualcoyotl; Somerson, Jacob; Vieira, Philip A.; Ploense, Kyle L.; Kippin, Tod E.; Plaxco, Kevin W.Proceedings of the National Academy of Sciences of the United States of America (2017), 114 (4), 645-650CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)The development of a technol. capable of tracking the levels of drugs, metabolites, and biomarkers in the body continuously and in real time would advance our understanding of health and our ability to detect and treat disease. It would, for example, enable therapies guided by high-resoln., patient-specific pharmacokinetics (including feedback-controlled drug delivery), opening new dimensions in personalized medicine. In response, we demonstrate here the ability of electrochem. aptamer-based (E-AB) sensors to support continuous, real-time, multihour measurements when emplaced directly in the circulatory systems of living animals. Specifically, we have used E-AB sensors to perform the multihour, real-time measurement of four drugs in the bloodstream of even awake, ambulatory rats, achieving precise mol. measurements at clin. relevant detection limits and high (3 s) temporal resoln., attributes suggesting that the approach could provide an important window into the study of physiol. and pharmacokinetics.
- 4Schoukroun-Barnes, L. R.; Macazo, F. C.; Gutierrez, B.; Lottermoser, J.; Liu, J.; White, R. J. Reagentless, Structure-Switching, Electrochemical Aptamer-Based Sensors. Annu. Rev. Anal. Chem. 2016, 9, 163– 181, DOI: 10.1146/annurev-anchem-071015-0414464https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XmtVOntL0%253D&md5=32b573c81f86a07a525f4682802ae6a1Reagentless, Structure-Switching, Electrochemical Aptamer-Based SensorsSchoukroun-Barnes, Lauren R.; Macazo, Florika C.; Gutierrez, Brenda; Lottermoser, Justine; Liu, Juan; White, Ryan J.Annual Review of Analytical Chemistry (2016), 9 (), 163-181CODEN: ARACFU; ISSN:1936-1327. (Annual Reviews)The development of structure-switching, electrochem., aptamer-based sensors over the past ∼10 years has led to a variety of reagentless sensors capable of anal. detection in a range of sample matrixes. The crux of this methodol. is the coupling of target-induced conformation changes of a redox-labeled aptamer with electrochem. detection of the resulting altered charge transfer rate between the redox mol. and electrode surface. Using aptamer recognition expands the highly sensitive detection ability of electrochem. to a range of previously inaccessible analytes. In this review, we focus on the methods of sensor fabrication and how sensor signaling is affected by fabrication parameters. We then discuss recent studies addressing the fundamentals of sensor signaling as well as quant. characterization of the anal. performance of electrochem. aptamer-based sensors. Although the limits of detection of reported electrochem. aptamer-based sensors do not often reach that of gold-std. methods such as enzyme-linked immunosorbent assays, the operational convenience of the sensor platform enables exciting anal. applications that we address. Using illustrative examples, we highlight recent advances in the field that impact important areas of anal. chem. Finally, we discuss the challenges and prospects for this class of sensors.
- 5Arroyo-Currás, N.; Dauphin-Ducharme, P.; Scida, K.; Chávez, J. L. From the beaker to the body: translational challenges for electrochemical, aptamer-based sensors. Anal. Methods 2020, 12, 1288– 1310, DOI: 10.1039/d0ay00026dThere is no corresponding record for this reference.
- 6Kang, D.; Zuo, X.; Yang, R.; Xia, F.; Plaxco, K. W.; White, R. J. Comparing the properties of electrochemical-based DNA sensors employing different redox tags. Anal. Chem. 2009, 81, 9109– 9113, DOI: 10.1021/ac901811n6https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXht1eltrfI&md5=2c9656095c0b3a872e207465445ddff7Comparing the properties of electrochemical-based DNA sensors employing different redox tagsKang, Di; Zuo, Xiaolei; Yang, Renqiang; Xia, Fan; Plaxco, Kevin W.; White, Ryan J.Analytical Chemistry (Washington, DC, United States) (2009), 81 (21), 9109-9113CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)Many electrochem. biosensor approaches developed in recent years utilize redox-labeled (most commonly methylene blue or ferrocene) oligonucleotide probes site-specifically attached to an interrogating electrode. Sensors in this class have been reported that employ a range of probe architectures, including single- and double-stranded DNA, more complex DNA structures, DNA and RNA aptamers, and, most recently, DNA-small mol. chimeras. Signaling in this class of sensors is generally predicated on binding-induced changes in the efficiency with which the covalently attached redox label transfers electrons with the interrogating electrode. Here we have investigated how the properties of the redox tag affect the performance of such sensors. Specifically, we compare the differences in signaling and stability of electrochem. DNA sensors (E-DNA sensors) fabricated using either ferrocene or methylene blue as the signaling redox moiety. We find that while both tags support efficient E-DNA signaling, ferrocene produces slightly improved signal gain and target affinity. These small advantages, however, come at a potentially significant price: the ferrocene-based sensors are far less stable than their methylene blue counterparts, particularly with regards to stability to long-term storage, repeated electrochem. interrogations, repeated sensing/regeneration iterations, and employment in complex sample matrixes such as blood serum.
- 7Kang, D.; Ricci, F.; White, R. J.; Plaxco, K. W. Survey of Redox-Active Moieties for Application in Multiplexed Electrochemical Biosensors. Anal. Chem. 2016, 88, 10452– 10458, DOI: 10.1021/acs.analchem.6b023767https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhsFGrs7jL&md5=3a25d233ddfb2b611131f474ad18d4dbSurvey of Redox-Active Moieties for Application in Multiplexed Electrochemical BiosensorsKang, Di; Ricci, Francesco; White, Ryan J.; Plaxco, Kevin W.Analytical Chemistry (Washington, DC, United States) (2016), 88 (21), 10452-10458CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)Recent years have seen the development of a large no. of electrochem. sandwich assays and reagentless biosensor architectures employing biomols. modified via the attachment of a redox-active "reporter.". Here the authors survey a large set of potential redox reporters to det. which exhibits the best long-duration stability in thiol-on-gold monolayer-based sensors and to identify reporter "sets" signaling at distinct, non-overlapping redox potentials in support of multiplexing and error correcting ratiometric or differential measurement approaches. Specifically, the authors have characterized the performance of more than a dozen potential reporters that are, first, redox active within the potential window over which thiol-on-gold monolayers are reasonably stable and, second, are available com. in forms that are readily conjugated to biomols. or can be converted into such forms in one or two simple synthetic steps. To test each of these reporters the authors conjugated it to one terminus of a single-stranded DNA "probe" that was attached by its other terminus via a six-carbon thiol to a gold electrode to form an "E-DNA" sensor responsive to its complementary DNA target. The authors then measured the signaling properties of each sensor as well as its stability against repeated voltammetric scans and against deployment in and reuse from blood serum. Doing so the performance of methylene blue-based, thiol-on-gold sensors is unmatched; the near-quant. stability of such sensors against repeated scanning in even very complex sample matrixes is unparalleled. While more modest, the stability of sensors employing a handful of other reporters, including anthraquinone, Nile blue, and ferrocene, is reasonable. The authors' work thus serves as both to highlight the exceptional properties of methylene blue as a redox reporter in such applications and as a cautionary tale; the authors wish to help other researchers avoid fruitless efforts to employ the many, seemingly promising and yet ultimately inadequate reporters the authors have studied. Finally, the authors hope that their work also serves as an illustration of the pressing need for the further development of useful redox reporters.
- 8Lubin, A. A.; Plaxco, K. W. Folding-Based Electrochemical Biosensors: The Case for Responsive Nucleic Acid Architectures. Acc. Chem. Res. 2010, 43, 496– 505, DOI: 10.1021/ar900165x8https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXislyktb0%253D&md5=fc266597938a204ca094b771e7ae30d2Folding-Based Electrochemical Biosensors: The Case for Responsive Nucleic Acid ArchitecturesLubin, Arica A.; Plaxco, Kevin W.Accounts of Chemical Research (2010), 43 (4), 496-505CODEN: ACHRE4; ISSN:0001-4842. (American Chemical Society)A review. Biomol. recognition is versatile, specific, and high affinity, qualities that have motivated decades of research aimed at adapting biomols. into a general platform for mol. sensing. Despite significant effort, however, so-called "biosensors" have almost entirely failed to achieve their potential as reagentless, real-time anal. devices; the only quant., reagentless biosensor to achieve com. 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 biomols. 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 biomol. 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 biol. samples. Anal. approaches based on biomol. recognition are therefore mostly cumbersome, multistep processes relying on analyte sepn. and isolation (such as Western blots, ELISA, and other immunochem. methods); these techniques have proven enormously useful, but are limited almost exclusively to lab. settings. In this Account, we describe how we have refined a potentially general soln. 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 electrochem. 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-mol. targets, are rapid (responding in seconds to minutes), sensitive (detecting sub-picomolar to micromolar concns.), 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 exts., cell lysates, and other grossly contaminated clin. and environmental samples. Indeed, we have recently demonstrated the ability to quant. monitor a specific small mol. 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.
- 9Schoen, I.; Krammer, H.; Braun, D. Hybridization kinetics is different inside cells. Proc. Natl. Acad. Sci. U.S.A. 2009, 106, 21649– 21654, DOI: 10.1073/pnas.09013131069https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXltlKjtg%253D%253D&md5=7332fdfffcbdb0aabdd12e916c3023bfHybridization kinetics is different inside cellsSchoen, Ingmar; Krammer, Hubert; Braun, DieterProceedings of the National Academy of Sciences of the United States of America (2009), 106 (51), 21649-21654, S21649/1-S21649/12CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)It is generally expected that the kinetics of reactions inside living cells differs from the situation in bulk solns. Macromol. crowding and specific binding interactions could change the diffusion properties and the availability of free mols. Their impact on reaction kinetics in the relevant context of living cells is still elusive, mainly because the difficulty of capturing fast kinetics in vivo. This article shows spatially resolved measurements of DNA hybridization kinetics in single living cells. HeLa cells were transfected with a FRET-labeled dsDNA probe by lipofection. We characterized the hybridization reaction kinetics with a kinetic range of 10 μs to 1 s by a combination of laser-driven temp. oscillations and stroboscopic fluorescence imaging. The time const. of the hybridization depended on DNA concn. within individual cells and between cells. A quant. anal. of the concn. dependence revealed several-fold accelerated kinetics as compared with free soln. for a 16-bp probe and decelerated kinetics for a 12-bp probe. We did not find significant effects of crowding agents on the hybridization kinetics in vitro. Our results suggest that the reaction rates in vivo are specifically modulated by binding interactions for the two probes, possibly triggered by their different lengths. In general, the presented imaging modality of temp. oscillation optical lock-in microscopy allows to probe biomol. interactions in different cell compartments in living cells for systems biol.
- 10Vallée-Bélisle, A.; Plaxco, K. W. Structure-switching biosensors: inspired by Nature. Curr. Opin. Struct. Biol. 2010, 20, 518– 526, DOI: 10.1016/j.sbi.2010.05.00110https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXhtVKhtLjN&md5=1057758a3ce05e727a47c4c5e89b6895Structure-switching biosensors: inspired by NatureVallee-Belisle, Alexis; Plaxco, Kevin W.Current Opinion in Structural Biology (2010), 20 (4), 518-526CODEN: COSBEF; ISSN:0959-440X. (Elsevier Ltd.)A review. Chemosensing in nature relies on biomol. switches, biomols. that undergo binding-induced changes in conformation or oligomerization to transduce chem. information into specific biochem. outputs. Motivated by the impressive performance of these natural 'biosensors', which support continuous, real-time detection in highly complex environments, significant efforts have gone into the adaptation of such switches into artificial chem. sensors. Ongoing advances in the fields of protein and nucleic acid engineering (e.g. computational protein design, directed evolution, selection strategies and labeling chemistries) have greatly enhanced the ability to design new structure-switching sensors. Coupled with the development of advanced optical readout mechanisms, including genetically encoded fluorophores, and electrochem. readouts supporting detection directly in highly complex sample matrixes, switch-based sensors have already seen deployment in applications ranging from real time, in vivo imaging to the continuous monitoring of drugs in blood serum.
- 11Ferapontova, E. E.; Olsen, E. M.; Gothelf, K. V. An RNA Aptamer-Based Electrochemical Biosensor for Detection of Theophylline in Serum. J. Am. Chem. Soc. 2008, 130, 4256– 4258, DOI: 10.1021/ja711326b11https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXivV2gu7c%253D&md5=99abb5e0434e5620fdcf35432c509ea7An RNA Aptamer-Based Electrochemical Biosensor for Detection of Theophylline in SerumFerapontova, Elena E.; Olsen, Eva M.; Gothelf, Kurt V.Journal of the American Chemical Society (2008), 130 (13), 4256-4258CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)An electrochem. RNA aptamer-based biosensor for rapid and label-free detection of the bronchodilator theophylline was developed. The 5'-disulfide-functionalized end of the RNA aptamer sequence was immobilized on a gold electrode, and the 3'-amino-functionalized end was conjugated with a ferrocene (Fc) redox probe. Upon binding of theophylline the aptamer switches conformation from an open unfolded state to a closed hairpin-type conformation, resulting in the increased electron-transfer efficiency between Fc and the electrode. The electrochem. response, which was measured by differential pulse voltammetry, reaches satn. within a few minutes after addn. of theophylline, and the dynamic range for detecting theophylline is 0.2-10 μM. The electrode displays an inhibited response when applied directly in serum samples treated with RNase inhibitors; however a full response to the theophylline serum concn. was obtained by transferring the electrode to blank serum-free buffer solns. It was demonstrated that theophylline is detected with high selectivity in the presence of caffeine and theobromine.
- 12Somerson, J.; Plaxco, K. Electrochemical aptamer-based sensors for rapid point-of-use monitoring of the mycotoxin ochratoxin a directly in a food stream. Molecules 2018, 23, 912– 919, DOI: 10.3390/molecules2304091212https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhslGmsLfO&md5=08d42c284a1fdb130fd753b2a4eac12bElectrochemical aptamer-based sensors for rapid point-of-use monitoring of the mycotoxin ochratoxin A directly in a food streamSomerson, Jacob; Plaxco, Kevin W.Molecules (2018), 23 (4), 912/1-912/7CODEN: MOLEFW; ISSN:1420-3049. (MDPI AG)The ability to measure the concn. of specific small mols. continuously and in real-time in complex sample streams would impact many areas of agriculture, food safety, and food prodn. Monitoring for mycotoxin taint in real time during food processing, for example, could improve public health. Towards this end, we describe here an inexpensive electrochem. DNA-based sensor that supports real-time monitor of the mycotoxin ochratoxin A in a flowing stream of foodstuffs.
- 13Parolo, C.; Idili, A.; Ortega, G.; Csordas, A.; Hsu, A.; Arroyo-Currás, N.; Yang, Q.; Ferguson, B. S.; Wang, J.; Plaxco, K. W. Real-Time Monitoring of a Protein Biomarker. ACS Sens. 2020, 5, 1877– 1881, DOI: 10.1021/acssensors.0c0108513https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhtlWqsLzO&md5=0f21fed8f246326266979277a7bb67f3Real-Time Monitoring of a Protein BiomarkerParolo, Claudio; Idili, Andrea; Ortega, Gabriel; Csordas, Andrew; Hsu, Alex; Arroyo-Curras, Netzahualcoyotl; Yang, Qin; Ferguson, Brian Scott; Wang, Jinpeng; Plaxco, Kevin W.ACS Sensors (2020), 5 (7), 1877-1881CODEN: ASCEFJ; ISSN:2379-3694. (American Chemical Society)The ability to monitor protein biomarkers continuously and in real-time would significantly advance the precision of medicine. Current protein-detection techniques, however, including ELISA and lateral flow assays, provide only time-delayed, single-time-point measurements, limiting their ability to guide prompt responses to rapidly evolving, life-threatening conditions. In response, here the authors present an electrochem. aptamer-based sensor (EAB) that supports high-frequency, real-time biomarker measurements. Specifically, the authors have developed an electrochem., aptamer-based (EAB) sensor against Neutrophil Gelatinase-Assocd. Lipocalin (NGAL), a protein that, if present in urine at levels above a threshold value, is indicative of acute renal/kidney injury (AKI). When deployed inside a urinary catheter, the resulting reagentless, wash-free sensor supports real-time, high-frequency monitoring of clin. relevant NGAL concns. over the course of hours. By providing an "early warning system", the ability to measure levels of diagnostically relevant proteins such as NGAL in real-time could fundamentally change how the authors detect, monitor, and treat many important diseases.
- 14Arroyo-Currás, N.; Dauphin-Ducharme, P.; Ortega, G.; Ploense, K. L.; Kippin, T. E.; Plaxco, K. W. Subsecond-Resolved Molecular Measurements in the Living Body Using Chronoamperometrically Interrogated Aptamer-Based Sensors. ACS Sens. 2018, 3, 360– 366, DOI: 10.1021/acssensors.7b0078714https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhslynu7nL&md5=71345b6d63ba81e4474706be1d55091dSubsecond-Resolved Molecular Measurements in the Living Body Using Chronoamperometrically Interrogated Aptamer-Based SensorsArroyo-Curras, Netzahualcoyotl; Dauphin-Ducharme, Philippe; Ortega, Gabriel; Ploense, Kyle L.; Kippin, Tod E.; Plaxco, Kevin W.ACS Sensors (2018), 3 (2), 360-366CODEN: ASCEFJ; ISSN:2379-3694. (American Chemical Society)Electrochem., aptamer-based (E-AB) sensors support the continuous, real-time measurement of specific small mols. directly in situ in the living body over the course of many hours. They achieve this by employing binding-induced conformational changes to alter electron transfer from a redox-reporter-modified, electrode-attached aptamer. Previously the authors have used voltammetry (cyclic, a.c., and square wave) to monitor this binding-induced change in transfer kinetics indirectly. Here, however, the authors demonstrate the potential advantages of employing chronoamperometry to measure the change in kinetics directly. In this approach target concn. is reported via changes in the lifetime of the exponential current decay seen when the sensor is subjected to a potential step. Because the lifetime of this decay is independent of its amplitude (e.g., insensitive to variations in the no. of aptamer probes on the electrode), chronoamperometrically interrogated E-AB sensors are calibration-free and resistant to drift. Chronoamperometric measurements can also be performed in a few hundred milliseconds, improving the previous few-second time resoln. of E-AB sensing by an order of magnitude. To illustrate the potential value of the approach the authors demonstrate here the calibration-free measurement of the drug tobramycin in situ in the living body with 300 ms time resoln. and unprecedented, few-percent precision in the detn. of its pharmacokinetic phases.
- 15Radi, A.-E.; Acero Sánchez, J. L.; Baldrich, E.; O’Sullivan, C. K. Reagentless, Reusable, Ultrasensitive Electrochemical Molecular Beacon Aptasensor. J. Am. Chem. Soc. 2006, 128, 117– 124, DOI: 10.1021/ja053121d15https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXhtlShtLfI&md5=c61b8799326b09d8f8eb54c7a2c299fbReagentless, Reusable, Ultrasensitive Electrochemical Molecular Beacon AptasensorRadi, Abd-Elgawad; Acero Sanchez, Josep Lluis; Baldrich, Eva; O'Sullivan, Ciara K.Journal of the American Chemical Society (2006), 128 (1), 117-124CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)A bifunctional deriv. of the thrombin-binding aptamer with a redox-active ferrocene (Fc) moiety and a thiol group at the termini of the aptamer strand was synthesized. The ferrocene-labeled aptamer thiol was self-assembled through S-Au bonding on a polycryst. gold electrode surface and the surface was blocked with 2-mercaptoethanol to form a mixed monolayer. By use of a fluorescent mol. beacon, the effect of counterions on quadruplex formation was established. The aptamer-modified electrode was characterized electrochem. by cyclic voltammetry (CV), differential pulse voltammetry (DPV), and electrochem. impedance spectroscopy (EIS). The modified electrode showed a voltammetric signal due to a one-step redox reaction of the surface-confined ferrocenyl moiety of the aptamer immobilized on the electrode surface in 10 mM N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonic acid (HEPES) buffer of pH 8.0. An increase in the DPV current signal was evident after blocking with 2-mercaptoethanol, effectively removing aptamer nonspecifically absorbed rather than bound to electrode surface or due to the formation of the aptamer-thrombin affinity interaction. The impedance measurement, in agreement with the differential pulse voltammetry (DPV), showed decreased Faradaic resistances in the same sequence. The "signal-on" upon thrombin assocn. could be attributed to a change in conformation from random coil-like configuration on the probe-modified film to the quadruplex structure. The DPV of the modified electrode showed a linear response of the Fc oxidn. signal to the increase in the thrombin concn. in the range between 5.0 and 35.0 nM with a linear correlation of r = 0.9988 and a detection limit of 0.5 nM. The mol. beacon aptasensor was amenable to full regeneration by simply unfolding the aptamer in 1.0 M HCl, and could be regenerated 25 times with no loss in electrochem. signal upon subsequent thrombin binding.
- 16Zhao, S.; Yang, W.; Lai, R. Y. A folding-based electrochemical aptasensor for detection of vascular endothelial growth factor in human whole blood. Biosens. Bioelectron. 2011, 26, 2442– 2447, DOI: 10.1016/j.bios.2010.10.02916https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXhvVeqsw%253D%253D&md5=1362bef49e8b2c8c18e6b835a9862147A folding-based electrochemical aptasensor for detection of vascular endothelial growth factor in human whole bloodZhao, Shuang; Yang, Weiwei; Lai, Rebecca Y.Biosensors & Bioelectronics (2011), 26 (5), 2442-2447CODEN: BBIOE4; ISSN:0956-5663. (Elsevier B.V.)We herein report a folding-based electrochem. DNA aptasensor for the detection of vascular endothelial growth factor (VEGF) directly in complex biol. samples, including blood serum and whole blood. The electrochem. signal generation is coupled to a large, target-induced conformational change in a methylene blue-modified and surface immobilized anti-VEGF aptamer. The sensor is sensitive, selective and essentially reagentless: we can readily detect VEGF down to 5 pM (190 pg/mL) directly in 50% blood serum. Similar to other aptasensors of this class, the VEGF sensor is also regenerable and reusable. In addn., the sensor performs comparably well even when fabricated on a gold-plated screen-printed carbon electrode and can potentially be implemented as a cost-effective, single-use biosensor for diseases diagnosis and therapy monitoring. The exceptional sensitivity, selectivity, and reusability of this electrochem. aptasensor platform suggest it may be a promising strategy for a wide variety of sensing applications.
- 17Downs, A. M.; Gerson, J.; Ploense, K. L.; Plaxco, K. W.; Dauphin-Ducharme, P. Sub-second-resolved Molecular Measurements Using Electrochemical Phase Interrogation of Aptamer-Based Sensors. Anal. Chem. 2020, 92, 14063– 14068, DOI: 10.1021/acs.analchem.0c0310917https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhvVygtbfE&md5=b32ad23d59d4f00652afc24725ec1181Subsecond-Resolved Molecular Measurements Using Electrochemical Phase Interrogation of Aptamer-Based SensorsDowns, Alex M.; Gerson, Julian; Ploense, Kyle L.; Plaxco, Kevin W.; Dauphin-Ducharme, PhilippeAnalytical Chemistry (Washington, DC, United States) (2020), 92 (20), 14063-14068CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)Recent years have seen the development of a no. of biosensor architectures that rely on target binding-induced changes in the rate of electron transfer from an electrode-bound receptor. Most often, the interrogation of these sensors has relied on voltammetric methods, such as square-wave voltammetry, which limit their time resoln. to a few seconds. Here, we describe the use of an impedance-based approach, which we have termed electrochem. phase interrogation, as a means of collecting high time resoln. measurements with sensors in this class. Specifically, using changes in the electrochem. phase to monitor target binding in an electrochem.-aptamer based (EAB) sensor, we achieve subsecond temporal resoln. and multihour stability in measurements performed directly in undiluted whole blood. Electrochem. phase interrogation also offers improved insights into EAB sensors' signaling mechanism. By modeling the interfacial resistance and capacitance using equiv. circuits, we find that the only parameter that is altered by target binding is the charge-transfer resistance. This confirms previous claims that binding-induced changes in electron-transfer kinetics drive signaling in this class of sensors. Considering that a wide range of electrochem. biosensor architectures rely on this signaling mechanism, we believe that electrochem. phase interrogation may prove generalizable toward subsecond measurements of mol. targets.
- 18Li, F.; Yu, Z.; Han, X.; Lai, R. Y. Electrochemical aptamer-based sensors for food and water analysis: A review. Anal. Chim. Acta 2019, 1051, 1– 23, DOI: 10.1016/j.aca.2018.10.05818https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXitFSqur7O&md5=c39c9f6456fdef2b38b1efa4c041bb3cElectrochemical aptamer-based sensors for food and water analysis: A reviewLi, Fengqin; Yu, Zhigang; Han, Xianda; Lai, Rebecca Y.Analytica Chimica Acta (2019), 1051 (), 1-23CODEN: ACACAM; ISSN:0003-2670. (Elsevier B.V.)A review. Global food and water safety issues have prompted the development of highly sensitive, specific, and fast anal. techniques for food and water anal. The electrochem. aptamer-based detection platform (E-aptasensor) is one of the more promising detection techniques because of its unique combination of advantages that renders these sensors ideal for detection of a wide range of target analytes. Recent research results have further demonstrated that this technique has potential for real world anal. of food and water contaminants. This review summaries the recently developed E-aptasensors for detection of analytes related to food and water safety, including bacteria, mycotoxins, algal toxins, viruses, drugs, pesticides, and metal ions. Ten different electroanal. techniques and one opto-electroanal. technique commonly employed with these sensors are also described. In addn. to highlighting several novel sensor designs, this review also describes the strengths, limitations, and current challenges this technol. faces, and future development trend.
- 19Pellitero, M. A.; Shaver, A.; Arroyo-Currás, N. Critical Review─Approaches for the Electrochemical Interrogation of DNA-Based Sensors: A Critical Review. J. Electrochem. Soc. 2020, 167, 037529, DOI: 10.1149/2.0292003jes19https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXjt1agtrY%253D&md5=e78a4f721400cb76bde262181801b32bCritical review-approaches for the electrochemical interrogation of DNA-based sensors: a critical reviewPellitero, Miguel Aller; Shaver, Alexander; Arroyo-Curras, NetzahualcoyotlJournal of the Electrochemical Society (2020), 167 (3), 037529CODEN: JESOAN; ISSN:0013-4651. (Electrochemical Society)A review. The desire to improve and decentralize diagnostic platforms to facilitate highly precise and personalized medicine has motivated the development of a large no. of electrochem. sensing technologies. Such a development has been facilitated by electrochem.'s unparalleled ability to achieve highly specific mol. measurements in complex biol. fluids, without the need for expensive instrumentation. However, for decades, progress in the field had been constrained to systems that depended on the chem. reactivity of the analyte, obstructing the generalizability of such platforms beyond redox- or enzymically active clin. targets. Thus, the pursuit of alternative, more general strategies, coupled to the timely technol. advances in DNA sequencing, led to the development of DNA-based electrochem. sensors. The anal. value of these arises from the structural customizability of DNA and its ability to bind analytes ranging from ions and small mols. to whole proteins and cells. This versatility extends to interrogation methods, as DNA-based sensors work through a variety of detection schemes that can be probed via many electroanal. techniques. As a ref. for those experienced in the field, and to guide the unexperienced scientist, here the authors review the specific advantages of the electroanal. methods most commonly used for the interrogation of DNA-based sensors.
- 20Santos-Cancel, M.; Lazenby, R. A.; White, R. J. Rapid two-millisecond interrogation of electrochemical, aptamer-based sensor response using intermittent pulse amperometry. ACS Sens. 2018, 3, 1203– 1209, DOI: 10.1021/acssensors.8b0027820https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXpsVCitL8%253D&md5=120fb76b9377014f03b08c9604220fa6Rapid Two-Millisecond Interrogation of Electrochemical, Aptamer-Based Sensor Response Using Intermittent Pulse AmperometrySantos-Cancel, Mirelis; Lazenby, Robert A.; White, Ryan J.ACS Sensors (2018), 3 (6), 1203-1209CODEN: ASCEFJ; ISSN:2379-3694. (American Chemical Society)In this manuscript, the authors employ the technique intermittent pulse amperometry (IPA) to interrogate equil. and kinetic target binding to the surface of electrochem., aptamer-based (E-AB) sensors, achieving as fast as 2 ms time resoln. E-AB sensors comprise an electrode surface modified with a flexible nucleic acid aptamer tethered at the 3'-terminus with a redox-active mol. The introduction of a target changes the conformation and flexibility of the nucleic acid, which alters the charge transfer rate of the appended redox mol. Typically, changes in charge transfer rate within this class of sensor were monitored via voltammetric methods. Here, the use of IPA enables the detection of changes in charge transfer rates (i.e., current) at times <100 μs after the application of a potential pulse. Changes in sensor current are quant. related to target analyte concn. and can be used to create binding isotherms. Furthermore, the application of IPA enables rapid probing of the electrochem. surface with a time resoln. equiv. to as low as twice the applied potential pulse width, not previously demonstrated with traditional voltammetric techniques employed with E-AB sensors (a.c., square wave, cyclic). To visualize binding, the authors developed false-color plots analogous to those used in the field of fast-scan cyclic voltammetry. The use of IPA is universal, as demonstrated with two representative small mol. E-AB sensors directed against the aminoglycoside antibiotic tobramycin and ATP. Intermittent pulse amperometry exhibits an unprecedented sub-microsecond temporal response and is a general method for measuring rapid sensor performance.
- 21Arya, S. K.; Zhurauski, P.; Jolly, P.; Batistuti, M. R.; Mulato, M.; Estrela, P. Capacitive aptasensor based on interdigitated electrode for breast cancer detection in undiluted human serum. Biosens. Bioelectron. 2018, 102, 106– 112, DOI: 10.1016/j.bios.2017.11.01321https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhslyksbzN&md5=de062d9272f1afdaf736904e0dc19962Capacitive aptasensor based on interdigitated electrode for breast cancer detection in undiluted human serumArya, Sunil K.; Zhurauski, Pavel; Jolly, Pawan; Batistuti, Marina R.; Mulato, Marcelo; Estrela, PedroBiosensors & Bioelectronics (2018), 102 (), 106-112CODEN: BBIOE4; ISSN:0956-5663. (Elsevier B.V.)We report the development of a simple and powerful capacitive aptasensor for the detection and estn. of human epidermal growth factor receptor 2 (HER2), a biomarker for breast cancer, in undiluted serum. The study involves the incorporation of interdigitated gold electrodes, which were used to prep. the electrochem. platform. A thiol terminated DNA aptamer with affinity for HER2 was used to prep. the bio-recognition layer via self-assembly on interdigitated gold surfaces. Non-specific binding was prevented by blocking free spaces on surface via starting block phosphate buffer saline-tween20 blocker. The sensor was characterized using cyclic voltammetry, impedance electrochem. spectroscopy (EIS), at. force microscopy and contact angle studies. Non-Faradic EIS measurements were utilized to investigate the sensor performance via monitoring of the changes in capacitance. The aptasensor exhibited logarithmically detection of HER2 from 1 pM to 100 nM in both buffer and undiluted serum with limits of detection lower than 1 pM. The results pave the way to develop other aptamer-based biosensors for protein biomarkers detection in undiluted serum.
- 22Shaver, A.; Curtis, S. D.; Arroyo-Currás, N. Alkanethiol Monolayer End Groups Affect the Long-Term Operational Stability and Signaling of Electrochemical, Aptamer-Based Sensors in Biological Fluids. ACS Appl. Mater. Interfaces 2020, 12, 11214– 11223, DOI: 10.1021/acsami.9b2238522https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXisFKqsro%253D&md5=5f10c2efa8960f892202a6184bf9d63aAlkanethiol Monolayer End Groups Affect the Long-Term Operational Stability and Signaling of Electrochemical, Aptamer-Based Sensors in Biological FluidsShaver, Alexander; Curtis, Samuel D.; Arroyo-Curras, NetzahualcoyotlACS Applied Materials & Interfaces (2020), 12 (9), 11214-11223CODEN: AAMICK; ISSN:1944-8244. (American Chemical Society)Electrochem. aptamer-based (E-AB) sensors achieve highly precise measurements of specific mol. targets in untreated biol. fluids. This unique ability, together with their measurement frequency of seconds or faster, has enabled the real-time monitoring of drug pharmacokinetics in live animals with unprecedented temporal resoln. However, one important weakness of E-AB sensors is that their bioelectronic interface degrades upon continuous electrochem. interrogation-a process typically seen as a drop in faradaic and an increase in charging currents over time. This progressive degrdn. limits their in vivo operational life to 12 h at best, a period that is much shorter than the elimination half-life of the vast majority of drugs in humans. Thus, there is a crit. need to develop novel E-AB interfaces that resist continuous electrochem. interrogation in biol. fluids for prolonged periods. In response, our group is pursuing the development of better packed, more stable self-assembled monolayers (SAMs) to improve the signaling and extend the operational life of in vivo E-AB sensors from hours to days. By invoking hydrophobicity arguments, we have created SAMs that do not desorb from the electrode surface in aq. physiol. solns. and biol. fluids. These SAMs, formed from 1-hexanethiol solns., decrease the voltammetric charging currents of E-AB sensors by 3-fold relative to std. monolayers of 6-mercapto-1-hexanol, increase the total faradaic current, and alter the electron transfer kinetics of the platform. Moreover, the stability of our new SAMs enables uninterrupted, continuous E-AB interrogation for several days in biol. fluids, like undiluted serum, at a physiol. temp. of 37°C.
- 23Zhang, S.; Hu, R.; Hu, P.; Wu, Z.-S.; Shen, G.-L.; Yu, R.-Q. Blank peak current-suppressed electrochemical aptameric sensing platform for highly sensitive signal-on detection of small molecule. Nucleic Acids Res. 2010, 38, e185 DOI: 10.1093/nar/gkq72823https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXhsVektrzJ&md5=1177becb4b82947c02aa5fabf7ff52a5Blank peak current-suppressed electrochemical aptameric sensing platform for highly sensitive signal-on detection of small moleculeZhang, Songbai; Hu, Rong; Hu, Peng; Wu, Zai-Sheng; Shen, Guo-Li; Yu, Ru-QinNucleic Acids Research (2010), 38 (20), e185/1-e185/8CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)In this contribution, an electrochem. aptameric sensing scheme for the sensitive detection of small mols. is proposed using adenosine as a target model. A ferrocene (Fc)-functionalized thiolated aptamer probe is adapted and immobilized onto an electrode surface. Introducing a recognition site for EcoRI into the aptamer sequence not only suppresses the peak current corresponding to blank sample but also provides a signal-on response mechanism. In the absence of adenosine, the aptamer can fold into a hairpin structure and form a cleavable double-stranded region. Fc is capable of being removed from electrode surface by treatment with endonuclease, and almost no peak current is obsd. The adenosine/aptamer binding induces the conformational transition of designed aptamer, dissocg. the cleavable double-stranded segment. Therefore, the integrated aptamer sequence is maintained when exposing to endonuclease, generating a peak current of Fc. Utilizing the present sensing scheme, adenosine even at a low concn. can give a detectable current signal. Thus, a detection limit of 10-10 M and a linear response range from 3.74 × 10-9 to 3.74 × 10-5 M are achieved. The proposed proof-of-principle of a novel electrochem. sensing is expected to extend to establish various aptameric platforms for the anal. of a broad range of target mols. of interest.
- 24Huang, K.-C.; White, R. J. Random Walk on a Leash: A Simple Single-Molecule Diffusion Model for Surface-Tethered Redox Molecules with Flexible Linkers. J. Am. Chem. Soc. 2013, 135, 12808– 12817, DOI: 10.1021/ja406078824https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXht1amtrvL&md5=6e0fd31ad02778e9b0ea33933aad17c4Random Walk on a Leash: A Simple Single-Molecule Diffusion Model for Surface-Tethered Redox Molecules with Flexible LinkersHuang, Kuan-Chun; White, Ryan J.Journal of the American Chemical Society (2013), 135 (34), 12808-12817CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)The authors develop a random walk model to simulate the Brownian motion and the electrochem. response of a single mol. confined to an electrode surface via a flexible mol. tether. The authors use the authors' simple model, which requires no prior knowledge of the physics of the mol. tether, to predict and better understand the voltammetric response of surface-confined redox mols. when motion of the redox mol. becomes important. The single mol. is confined to a hemispherical vol. with a max. radius detd. by the flexible mol. tether (5-20 nm) and is allowed to undergo true three-dimensional diffusion. Distance- and potential-dependent electron transfer probabilities are evaluated throughout the simulations to generate cyclic voltammograms of the model system. At sufficiently slow cyclic voltammetric scan rates the electrochem. reaction behaves like an adsorbed redox mol. with no mass transfer limitation; thus, the peak current is proportional to the scan rate. Conversely, at faster scan rates the diffusional motion of the mol. limits the simulated peak current, which exhibits a linear dependence on the square root of the scan rate. The switch between these two limiting regimes occurs when the diffusion layer thickness, (2Dt)1/2, is ∼10 times the tether length. Finally, the authors' model predicts the voltammetric behavior of a redox-active methylene blue tethered to an electrode surface via short flexible single-stranded, polythymine DNAs, allowing the estn. of diffusion coeffs. for the end-tethered mol.
- 25Rowe, A. A.; Miller, E. A.; Plaxco, K. W. Reagentless Measurement of Aminoglycoside Antibiotics in Blood Serum via an Electrochemical, Ribonucleic Acid Aptamer-Based Biosensor. Anal. Chem. 2010, 82, 7090– 7095, DOI: 10.1021/ac101491d25https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXpvVGmsLw%253D&md5=7f05a835e365d5460cdd7141d3c946f6Reagentless Measurement of Aminoglycoside Antibiotics in Blood Serum via an Electrochemical, Ribonucleic Acid Aptamer-Based BiosensorRowe, Aaron A.; Miller, Erin A.; Plaxco, Kevin W.Analytical Chemistry (Washington, DC, United States) (2010), 82 (17), 7090-7095CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)Biosensors built using RNA aptamers show promise as tools for point-of-care medical diagnostics, but they remain vulnerable to nuclease degrdn. when deployed in clin. samples. To explore methods for protecting RNA-based biosensors from such degrdn. we have constructed and characterized an electrochem., aptamer-based sensor for the detection of aminoglycosidic antibiotics. We find that while this sensor achieves low micromolar detection limits and subminute equilibration times when challenged in buffer, it deteriorates rapidly when immersed directly in blood serum. In order to circumvent this problem, we have developed and tested sensors employing modified versions of the same aptamer. Our first effort to this end entailed the methylation of all of the 2'-hydroxyl groups outside of the aptamer's antibiotic binding pocket. However, while devices employing this modified aptamer are as sensitive as those employing an unmodified parent, the modification fails to confer greater stability when the sensor is challenged directly in blood serum. As a second potentially naive alternative, we replaced the RNA bases in the aptamer with their more degrdn.-resistant DNA equiv. Surprisingly and unlike control DNA-stem loops employing other sequences, this DNA aptamer retains the ability to bind aminoglycosides, albeit with poorer affinity than the parent RNA aptamer. Unfortunately, however, while sensors fabricated using this DNA aptamer are stable in blood serum, its lower affinity pushes their detection limits above the therapeutically relevant range. Finally, we find that ultrafiltration through a low-mol.-wt.-cutoff spin column rapidly and efficiently removes the relevant nucleases from serum samples spiked with gentamicin, allowing the convenient detection of this aminoglycoside at clin. relevant concns. using the original RNA-based sensor.
- 26White, R. J.; Phares, N.; Lubin, A. A.; Xiao, Y.; Plaxco, K. W. Optimization of Electrochemical Aptamer-Based Sensors via Optimization of Probe Packing Density and Surface Chemistry. Langmuir 2008, 24, 10513– 10518, DOI: 10.1021/la800801v26https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXps1Citb8%253D&md5=c568824267126eb2d8f9cb8b7cd3c7b4Optimization of Electrochemical Aptamer-Based Sensors via Optimization of Probe Packing Density and Surface ChemistryWhite, Ryan J.; Phares, Noelle; Lubin, Arica A.; Xiao, Yi; Plaxco, Kevin W.Langmuir (2008), 24 (18), 10513-10518CODEN: LANGD5; ISSN:0743-7463. (American Chemical Society)Electrochem., 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. To further improve this technol. the authors have systematically studied the effects of probe (aptamer) packing d., 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 mol. cocaine and the protein thrombin. The authors find that, by controlling the concn. of aptamer employed during sensor fabrication, the authors can control the d. of probe DNA mols. 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 max. gain obsd. 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 obsd. at intermediate densities. Above cut-offs at low hertz frequencies, neither sensor displays any significant dependence on the frequency of the alternating potential employed in their interrogation. Finally, the authors find that E-AB signal gain is sensitive to the nature of the alkanethiol SAM employed to passivate the interrogating electrode; while thinner SAMs lead to higher abs. sensor currents, reducing the length of the SAM from 6-carbons to 2-carbons reduces the obsd. signal gain of the authors' cocaine sensor 10-fold. The authors demonstrate that fabrication and operational parameters can be varied to achieve optimal sensor performance and that these can serve as a basic outline for future sensor fabrication.
- 27Liu, Y.; Canoura, J.; Alkhamis, O.; Xiao, Y. Immobilization Strategies for Enhancing Sensitivity of Electrochemical Aptamer-Based Sensors. ACS Appl. Mater. Interfaces 2021.There is no corresponding record for this reference.
- 28Campos, R.; Kotlyar, A.; Ferapontova, E. E. DNA-Mediated Electron Transfer in DNA Duplexes Tethered to Gold Electrodes via Phosphorothioated dA Tags. Langmuir 2014, 30, 11853– 11857, DOI: 10.1021/la502766g28https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhs1CiurzM&md5=8ac830b30c846608a8529aa68006fc89DNA-Mediated Electron Transfer in DNA Duplexes Tethered to Gold Electrodes via Phosphorothioated dA TagsCampos, Rui; Kotlyar, Alexander; Ferapontova, Elena E.Langmuir (2014), 30 (40), 11853-11857CODEN: LANGD5; ISSN:0743-7463. (American Chemical Society)The efficiency of DNA-based bioelectronic devices strongly depends on the way DNA mols. are linked to the electronic component. Commonly, DNA is tethered to metal electrodes via an alkanethiol linker representing an addnl. barrier for electron transport. The replacement of the alkanethiol linker for a phosphorothioated adenosine tag increases the rate of DNA-mediated electron transfer (ET) up to 259 s-1, representing the highest hitherto reported rate of electrochem.-modulated ET, and improves the stability of DNA-electrode surface binding. Both results offer pronounced technol. and scientific benefits for DNA-based electronics.
- 29Campos, R.; Kékedy-Nagy, L.; She, Z.; Sodhi, R.; Kraatz, H.-B.; Ferapontova, E. E. Electron Transfer in Spacer-Free DNA Duplexes Tethered to Gold via dA10 Tags. Langmuir 2018, 34, 8472– 8479, DOI: 10.1021/acs.langmuir.8b0141229https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhtFyqtLzK&md5=b9aa2e07002cb454597a0a5665bc1824Electron Transfer in Spacer-Free DNA Duplexes Tethered to Gold via dA10 TagsCampos, Rui; Kekedy-Nagy, Laszlo; She, Zhe; Sodhi, Rana; Kraatz, Heinz-Bernhard; Ferapontova, Elena E.Langmuir (2018), 34 (29), 8472-8479CODEN: LANGD5; ISSN:0743-7463. (American Chemical Society)Elec. properties of DNA critically depend on the way DNA mols. are integrated within the electronics, particularly on DNA-electrode immobilization strategies. Here, the rate of electron transport in DNA duplexes spacer-free tethered to gold via the adenosine terminal region (a dA10 tag) is enhanced compared to the hitherto reported DNA-metal electrode tethering chemistries. The rate of DNA-mediated electron transfer (ET) between the electrode and methylene blue intercalated into the dA10-tagged DNA duplex approached 361 s-1 at a ca. half-monolayer DNA surface coverage ΓDNA (with a linear regression limit of 670 s-1 at ΓDNA → 0), being 2.7-fold enhanced compared to phosphorothioated dA5* tethering (6-fold for the C6-alkanethiol linker representing an addnl. ET barrier). XPS evidenced dA10 binding to the Au surface via the purine N, whereas dA5* predominantly coordinated to the surface via sulfur atoms of phosphothioates. The latter apparently induces the DNA strand twist in the point of surface attachment affecting the local DNA conformation and, as a result, decreasing the ET rates through the duplex. Thus, a spacer-free DNA coupling to electrodes via dA10 tags thus allows a perspective design of DNA electronic circuits and sensors with advanced electronic properties and no implication from more expensive, synthetic linkers.
- 30Laviron, E. General expression of the linear potential sweep voltammogram in the case of diffusionless electrochemical systems. J. Electroanal. Chem. Interfacial Electrochem. 1979, 101, 19– 28, DOI: 10.1016/s0022-0728(79)80075-330https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaE1MXltFCnt7k%253D&md5=9d280d1e17f00b28bd56b6cade497eddGeneral expression of the linear potential sweep voltammogram in the case of diffusionless electrochemical systemsLaviron, E.Journal of Electroanalytical Chemistry and Interfacial Electrochemistry (1979), 101 (1), 19-28CODEN: JEIEBC; ISSN:0022-0728.The equation of a linear potential sweep voltammogram is derived for any degree of reversibility of the electrochem. reaction for the following methods:surface voltammetry when both the oxidized and the reduced froms are strongly adsorbed, and a Langmuir isotherm is obeyed, thin-layer voltammetry, and linear potential sweep coulometry. The results are expressed in a math. form valid for the 3 cases. The transfer coeff. and the rate const. of the electrochem. reaction can be deduced from an exptl. study of the variations of the peak potentials as a function of the sweep rate.
- 31Dauphin-Ducharme, P.; Arroyo-Currás, N.; Adhikari, R.; Somerson, J.; Ortega, G.; Makarov, D. E.; Plaxco, K. W. Chain Dynamics Limit Electron Transfer from Electrode-Bound, Single-Stranded Oligonucleotides. J. Phys. Chem. C 2018, 122, 21441– 21448, DOI: 10.1021/acs.jpcc.8b0611131https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhsFOlsbnJ&md5=5f587654c6194f591b42c4b1a9dc014fChain Dynamics Limit Electron Transfer from Electrode-Bound, Single-Stranded OligonucleotidesDauphin-Ducharme, Philippe; Arroyo-Curras, Netzahualcoyotl; Adhikari, Ramesh; Somerson, Jacob; Ortega, Gabriel; Makarov, Dmitrii E.; Plaxco, Kevin W.Journal of Physical Chemistry C (2018), 122 (37), 21441-21448CODEN: JPCCCK; ISSN:1932-7447. (American Chemical Society)A wide range of new devices aimed at in vivo mol. detection and point-of-care diagnostics rely on binding-induced changes in electron-transfer kinetics from an electrode-attached, redox-reporter-modified oligonucleotide as their signaling mechanism. In an effort to better characterize the mechanisms underlying these sensors, we have measured the electron-transfer kinetics assocd. with surface-attached, single-stranded DNAs modified with a methylene blue redox reporter either at the chain's distal end or at an internal chain position. We find that although the rate of electron transfer from a reporter placed either terminally or internally is independent of chain length for chains shorter than the length scale of methylene blue (and its linker), for longer chains it follows a power-law dependence on length of exponent approx. -2.2. Such behavior is consistent with a diffusion-controlled mechanism in which the diffusion of the DNA-bound reporter to the surface controls the rate of electron transfer. This said, the obsd. rates are, at 5-400 s-1, orders of magnitude slower than the intramol. dynamics of single-stranded oligonucleotides when free in soln. Likewise, the rates of transfer from reporters placed internally are several-fold slower than those seen for the equiv. terminally modified construct. We attribute these effects to electrostatic repulsion between the oligonucleotide and the electrode surface, which is neg. charged at the redox potential of methylene blue. Consistent with this, changing monolayer compn. so as to increase the neg. charge of the surface reduces the transfer rate still more without significantly altering its power-law chain length dependence. Simple theor. models and computer simulations performed in support of our exptl. studies find similar power-law dependencies, similar electrostatic slowing of the transfer rate, and similar rate differences between terminally an internally modified constructs.
- 32Dauphin-Ducharme, P.; Yang, K.; Arroyo-Currás, N.; Ploense, K. L.; Zhang, Y.; Gerson, J.; Kurnik, M.; Kippin, T. E.; Stojanovic, M. N.; Plaxco, K. W. Electrochemical Aptamer-Based Sensors for Improved Therapeutic Drug Monitoring and High-Precision, Feedback-Controlled Drug Delivery. ACS Sens. 2019, 4, 2832– 2837, DOI: 10.1021/acssensors.9b0161632https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXhvVCnt7vM&md5=67cd35b6a26e364ac78a9c4339925c3bElectrochemical Aptamer-Based Sensors for Improved Therapeutic Drug Monitoring and High-Precision, Feedback-Controlled Drug DeliveryDauphin-Ducharme, Philippe; Yang, Kyungae; Arroyo-Curras, Netzahualcoyotl; Ploense, Kyle L.; Zhang, Yameng; Gerson, Julian; Kurnik, Martin; Kippin, Tod E.; Stojanovic, Milan N.; Plaxco, Kevin W.ACS Sensors (2019), 4 (10), 2832-2837CODEN: ASCEFJ; ISSN:2379-3694. (American Chemical Society)The Electrochem. Aptamer-Based (E-AB) sensing platform appears a convenient (rapid, single-step, calibration-free), modular approach to measure concns. of specific mols. (irresp. of their chem. reactivity) directly in blood and even in situ in the living body. Given these attributes, the platform may thus provide significant opportunities to render therapeutic drug monitoring (the clin. practice in which dosing is adjusted in response to plasma drug measurements) as frequent and convenient as the measurement of blood sugar has become for diabetics. The ability to measure arbitrary mols. in the body in real time could even enable closed-loop feedback control over plasma drug levels in a manner analogous to the recently commercialized controlled blood sugar systems. As initial exploration of this, the authors describe here the selection of an aptamer against vancomycin, a narrow therapeutic window antibiotic for which therapeutic monitoring is a crit. part of the std. of care, and its adaptation into an electrochem. aptamer-based (E-AB) sensor. Using this sensor the authors then demonstrate: (1) rapid (seconds), convenient (single-step, calibration-free) measurement of plasma vancomycin in finger-prick-scale samples of whole blood, (2) high-precision measurement of subject-specific vancomycin pharmacokinetics (in a rat animal model), and (3) high precision, closed-loop feedback control over plasma levels of the drug (in a rat animal model). The ability to not only track (with continuous-glucose-monitor-like measurement frequency and convenience), but also actively control plasma drug levels provides an unprecedented route towards improving therapeutic drug monitoring and, more generally, the personalized, high-precision delivery of pharmacol. interventions.
- 33Curtis, S. D.; Ploense, K. L.; Kurnik, M.; Ortega, G.; Parolo, C.; Kippin, T. E.; Plaxco, K. W.; Arroyo-Currás, N. Open Source Software for the Real-Time Control, Processing, and Visualization of High-Volume Electrochemical Data. Anal. Chem. 2019, 91, 12321– 12328, DOI: 10.1021/acs.analchem.9b0255333https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXhs1KksL%252FI&md5=8a2fb45b4fd84748c8c38c8754c1db12Open Source Software for the Real-Time Control, Processing, and Visualization of High-Volume Electrochemical DataCurtis, Samuel D.; Ploense, Kyle L.; Kurnik, Martin; Ortega, Gabriel; Parolo, Claudio; Kippin, Tod E.; Plaxco, Kevin W.; Arroyo-Curras, NetzahualcoyotlAnalytical Chemistry (Washington, DC, United States) (2019), 91 (19), 12321-12328CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)Electrochem. sensors are major players in the race for improved mol. diagnostics due to their convenience, temporal resoln., manufg. scalability, and their ability to support real-time measurements. This is evident in the ever-increasing no. of health-related electrochem. sensing platforms, ranging from single-measurement point-of-care devices to wearable devices supporting immediate and continuous monitoring. In support of the need for such systems to rapidly process large data vols. the authors describe here an open-source, easily customizable, multi-platform compatible program for the real-time control, processing and visualization of electrochem. data. The software's architecture is modular and fully documented, allowing the easy customization of the code to support the processing of voltammetric (e.g., square-wave and cyclic) and chronoamperometric data. The program, which the authors have called Software for the Anal. and Continuous Monitoring of Electrochem. Systems (SACMES), also includes a graphical interface allowing the user to easily change anal. parameters (e.g., signal/noise processing, baseline correction) in real-time. To demonstrate the versatility of SACMES the authors use it here to analyze the real-time data output by: (1) the electrochem., aptamer-based measurement of a specific small-mol. target, (2) a monoclonal antibody-detecting DNA-scaffold sensor, and (3) the detn. of the folding thermodn. of an electrode-attached, redox-reporter-modified protein.
- 34Rant, U.; Arinaga, K.; Scherer, S.; Pringsheim, E.; Fujita, S.; Yokoyama, N.; Tornow, M.; Abstreiter, G. Switchable DNA interfaces for the highly sensitive detection of label-free DNA targets. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 17364– 17369, DOI: 10.1073/pnas.070397410434https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXht1ymtrjL&md5=df81bafa5a13f7ba4612578d19fc7f7aSwitchable DNA interfaces for the highly sensitive detection of label-free DNA targetsRant, Ulrich; Arinaga, Kenji; Scherer, Simon; Pringsheim, Erika; Fujita, Shozo; Yokoyama, Naoki; Tornow, Marc; Abstreiter, GerhardProceedings of the National Academy of Sciences of the United States of America (2007), 104 (44), 17364-17369CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)We report a method to detect label-free oligonucleotide targets. The conformation of surface-tethered probe nucleic acids is modulated by alternating elec. fields, which cause the mols. to extend away from or fold onto the biased surface. Binding (hybridization) of targets to the single-stranded probes results in a pronounced enhancement of the layer-height modulation amplitude, monitored optically in real time. The method features an exceptional detection limit of <3 × 108 bound targets per cm2 sensor area. Single base-pair mismatches in the sequences of DNA complements may readily be identified; moreover, binding kinetics and binding affinities can be detd. with high accuracy. When driving the DNA to oscillate at frequencies in the kHz regime, distinct switching kinetics are revealed for single- and double-stranded DNA. Mol. dynamics are used to identify the binding state of mols. according to their characteristic kinetic fingerprints by using a chip-compatible detection format.
- 35Rant, U.; Arinaga, K.; Tornow, M.; Kim, Y. W.; Netz, R. R.; Fujita, S.; Yokoyama, N.; Abstreiter, G. Dissimilar Kinetic Behavior of Electrically Manipulated Single- and Double-Stranded DNA Tethered to a Gold Surface. Biophys. J. 2006, 90, 3666– 3671, DOI: 10.1529/biophysj.105.07885735https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28Xkt1OktLY%253D&md5=50245d6b79f863cc0d45ea955cf9a796Dissimilar kinetic behavior of electrically manipulated single- and double-stranded DNA tethered to a gold surfaceRant, Ulrich; Arinaga, Kenji; Tornow, Marc; Kim, Yong Woon; Netz, Roland R.; Fujita, Shozo; Yokoyama, Naoki; Abstreiter, GerhardBiophysical Journal (2006), 90 (10), 3666-3671CODEN: BIOJAU; ISSN:0006-3495. (Biophysical Society)We report on the elec. manipulation of single- and double-stranded oligodeoxynucleotides that are end tethered to gold surfaces in electrolyte soln. The response to alternating repulsive and attractive elec. surface fields is studied by time-resolved fluorescence measurements, revealing markedly distinct dynamics for the flexible single-stranded and stiff double-stranded DNA, resp. Hydrodynamic simulations rationalize this finding and disclose two different kinetic mechanisms: stiff polymers undergo rotation around the anchoring pivot point; flexible polymers, on the other hand, are pulled onto the attracting surface segment by segment.
- 36Yang, W.; Lai, R. Y. Comparison of the Stem-Loop and Linear Probe-Based Electrochemical DNA Sensors by Alternating Current Voltammetry and Cyclic Voltammetry. Langmuir 2011, 27, 14669– 14677, DOI: 10.1021/la203015v36https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXhtlOqu7fP&md5=8a9bfecb8d26b6ac349fe681bab6265dComparison of the stem-loop and linear probe-based electrochemical DNA sensors by alternating current voltammetry and cyclic voltammetryYang, Weiwei; Lai, Rebecca Y.Langmuir (2011), 27 (23), 14669-14677CODEN: LANGD5; ISSN:0743-7463. (American Chemical Society)Here we systematically characterized the sensor performance of the stem-loop probe (SLP) and linear probe (LP) electrochem. DNA sensors using a.c. voltammetry (ACV) and cyclic voltammetry (CV), with the goal of generating the set of operational criteria that best suits each sensor architecture, in addn. to elucidating the signaling mechanism behind these sensors. Although the LP sensor shows slightly better % signal suppression (SS) upon hybridization with the perfect match target at 10 Hz, our frequency-dependent study suggests that it shows optimal % SS only in a very limited AC frequency range. Similar results are obsd. in CV studies in which the LP sensor, when compared to the SLP sensor, displays a narrower range of voltammetric scan rates where the optimal % SS can be achieved. More importantly, the difference between the two sensors' performance is particularly pronounced if the change in integrated charge (Q) upon target hybridization, rather than the peak current (I), is measured in CV. The temp.-dependent study further highlights the differences between the two sensors, where the LP sensor, owing to the flexible linear probe architecture, is more readily perturbed by temp. changes. Both SLP and LP sensors, however, show a loss of % SS when operated at elevated temps., despite the significant improvement in the hybridization kinetics. In conjunction with the ACV, CV, and temp.-dependent studies, the electron-transfer kinetics study provides further evidence in support of the proposed signaling mechanism of these two sensors, in which the SLP sensor's signaling efficiency and sensor performance is directly linked to the hybridization-induced conformational change in the redox-labeled probe, whereas the performance of the LP sensor relies on the hybridization-induced change in probe dynamics.
- 37Lai, R. Y.; Walker, B.; Stormberg, K.; Zaitouna, A. J.; Yang, W. Electrochemical techniques for characterization of stem-loop probe and linear probe-based DNA sensors. Methods 2013, 64, 267– 275, DOI: 10.1016/j.ymeth.2013.07.04137https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhtlCnsbjF&md5=449c8f20d0cfb0263d7d541c26721423Electrochemical techniques for characterization of stem-loop probe and linear probe-based DNA sensorsLai, Rebecca Y.; Walker, Bryce; Stormberg, Kent; Zaitouna, Anita J.; Yang, WeiweiMethods (Amsterdam, Netherlands) (2013), 64 (3), 267-275CODEN: MTHDE9; ISSN:1046-2023. (Elsevier B.V.)Here we present a summary of the sensor performance of the stem-loop probe (SLP) and linear probe (LP) electrochem. DNA sensors when interrogated using a.c. voltammetry (ACV), cyclic voltammetry (CV), and differential pulse voltammetry (DPV). Specifically, we identified one crit. parameter for each voltammetric technique that can be adjusted for optimal sensor performance. Overall, the SLP sensor displayed good sensor performance (i.e., 60 + % signal attenuation in the presence of the target) over a wider range of exptl. conditions when compared to the LP sensor. When used with ACV, the optimal frequency range was found to be between 5 and 5000 Hz, larger than the 5-100 Hz range obsd. with the LP sensor. A similar trend was obsd. for the two sensors in CV; the LP sensor was operational only at scan rates between 30 and 100 V/s, whereas the SLP sensor performed well at scan rates between 1 and 1000 V/s. Unlike ACV and CV, DPV has demonstrated to be a more versatile sensor interrogation technique for this class of sensors. Despite the minor differences in total signal attenuation upon hybridization to the target DNA, both SLP and LP sensors performed optimally under most pulse widths used in this study. More importantly, when used with longer pulse widths, both sensors showed "signal-on" behavior, which is generally more desirable for sensor applications.
- 38Kang, D.; Parolo, C.; Sun, S.; Ogden, N. E.; Dahlquist, F. W.; Plaxco, K. W. Expanding the Scope of Protein-Detecting Electrochemical DNA “Scaffold” Sensors. ACS Sens. 2018, 3, 1271– 1275, DOI: 10.1021/acssensors.8b0031138https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhtFGju7fL&md5=1136584de066e909565c10aa3aca91c1Expanding the Scope of Protein-Detecting Electrochemical DNA "Scaffold" SensorsKang, Di; Parolo, Claudio; Sun, Sheng; Ogden, Nathan E.; Dahlquist, Frederick W.; Plaxco, Kevin W.ACS Sensors (2018), 3 (7), 1271-1275CODEN: ASCEFJ; ISSN:2379-3694. (American Chemical Society)The ability to measure the levels of diagnostically relevant proteins, such as antibodies, directly at the point of care could significantly impact healthcare. Thus motivated, we explore here the E-DNA "scaffold" sensing platform, a rapid, convenient, single-step means to this end. These sensors comprise a rigid nucleic acid "scaffold" attached via a flexible linker to an electrode and modified on its distal end with a redox reporter and a protein binding "recognition element". The binding of a targeted protein reduces the efficiency with which the redox reporter approaches the electrode, resulting in an easily measured signal change when the sensor is interrogated voltammetrically. Previously we have demonstrated scaffold sensors employing a range of low mol. wt. haptens and linear peptides as their recognition elements. Expanding on this here we have characterized sensors employing much larger recognition elements (up to and including full length proteins) in order to (1) define the range of recognition elements suitable for use in the platform; (2) better characterize the platform's signaling mechanism to aid its design and optimization; and (3) demonstrate the anal. performance of sensors employing full-length proteins as recognition elements. In doing so we have enlarged the range of mol. targets amenable to this rapid and convenient sensing platform.
- 39Arroyo-Currás, N.; Sadeia, M.; Ng, A. K.; Fyodorova, Y.; Williams, N.; Afif, T.; Huang, C.-M.; Ogden, N.; Andresen Eguiluz, R. C.; Su, H.-J.; Castro, C. E.; Plaxco, K. W.; Lukeman, P. S. An electrochemical biosensor exploiting binding-induced changes in electron transfer of electrode-attached DNA origami to detect hundred nanometer-scale targets. Nanoscale 2020, 12, 13907– 13911, DOI: 10.1039/d0nr00952k39https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhtF2isLzK&md5=fdd1e2f538ebe9f782c8b2124370ed8aAn electrochemical biosensor exploiting binding-induced changes in electron transfer of electrode-attached DNA origami to detect hundred nanometer-scale targetsArroyo-Curras, Netzahualcoyotl; Sadeia, Muaz; Ng, Alexander K.; Fyodorova, Yekaterina; Williams, Natalie; Afif, Tammy; Huang, Chao-Min; Ogden, Nathan; Andresen Eguiluz, Roberto C.; Su, Hai-Jun; Castro, Carlos E.; Plaxco, Kevin W.; Lukeman, Philip S.Nanoscale (2020), 12 (26), 13907-13911CODEN: NANOHL; ISSN:2040-3372. (Royal Society of Chemistry)The specific detection in clin. samples of analytes with dimensions in the tens to hundreds of nanometers, such as viruses and large proteins, would improve disease diagnosis. Detection of these "mesoscale" analytes (as opposed to their nanoscale components), however, is challenging as it requires the simultaneous binding of multiple recognition sites often spaced over tens of nanometers. In response, we have adapted DNA origami, with its unparalleled customizability to precisely display multiple target-binding sites over the relevant length scale, to an electrochem. biosensor platform. Our proof-of-concept employs triangular origami covalently attached to a gold electrode and functionalized with redox reporters. Electrochem. interrogation of this platform successfully monitors mesoscale, target-binding-induced changes in electron transfer in a manner consistent with coarse-grained mol. dynamics simulations. Our approach enables the specific detection of analytes displaying recognition sites that are sepd. by ~ 40 nm, a spacing significantly greater than that achieved in similar sensor architectures employing either antibodies or aptamers.
Supporting Information
Supporting Information
The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acssensors.0c02455.
Study of the effect of the CV scan rate on dose–response curves, evaluation of peak broadening in CV with increasing scan rates, comparison of IP and ΔEP change for tobramycin sensors, evaluation of the precision of the CV-based method, calibration curve for procaine-binding E-AB sensors by SWV, evaluation of the response of sensors with different surface coverages, comparison of the electrochemical response of sensors prepared by a coimmobilization or a backfilling protocol, and Python script used for real-time data analysis (PDF)
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