Optimization of Vancomycin Aptamer Sequence Length Increases the Sensitivity of Electrochemical, Aptamer-Based Sensors In VivoClick to copy article linkArticle link copied!
- Alexander ShaverAlexander ShaverDepartment of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, United StatesMore by Alexander Shaver
- J.D. MahlumJ.D. MahlumChemistry-Biology Interface Program, Zanvyl Krieger School of Arts & Sciences, Johns Hopkins University, Baltimore, Maryland 21218, United StatesMore by J.D. Mahlum
- Karen ScidaKaren ScidaLieber Institute for Brain Development, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, United StatesMore by Karen Scida
- Melanie L. JohnstonMelanie L. JohnstonDepartment of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, United StatesBiochemistry, Cellular and Molecular Biology, Johns Hopkins University School of Medicine, Baltimore, Maryland 21287, United StatesMore by Melanie L. Johnston
- Miguel Aller PelliteroMiguel Aller PelliteroDepartment of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, United StatesMore by Miguel Aller Pellitero
- Yao WuYao WuDepartment of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, United StatesMore by Yao Wu
- Gregory V. CarrGregory V. CarrDepartment of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, United StatesLieber Institute for Brain Development, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, United StatesMore by Gregory V. Carr
- 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 21205, United StatesChemistry-Biology Interface Program, Zanvyl Krieger School of Arts & Sciences, Johns Hopkins University, Baltimore, Maryland 21218, United StatesBiochemistry, Cellular and Molecular Biology, Johns Hopkins University School of Medicine, Baltimore, Maryland 21287, United StatesMore by Netzahualcóyotl Arroyo-Currás
Abstract
The measurement of serum vancomycin levels at the clinic is critical to optimizing dosing given the narrow therapeutic window of this antibiotic. Current approaches to quantitate serum vancomycin levels are based on immunoassays, which are multistep methods requiring extensive processing of patient samples. As an alternative, vancomycin-binding electrochemical, aptamer-based sensors (E-ABs) were developed to simplify the workflow of vancomycin monitoring. E-ABs enable the instantaneous measurement of serum vancomycin concentrations without the need for sample dilution or other processing steps. However, the originally reported vancomycin-binding E-ABs had a dissociation constant of 45 μM, which is approximately 1 order of magnitude higher than the recommended trough concentrations of vancomycin measured in patients. This limited sensitivity hinders the ability of E-ABs to accurately support vancomycin monitoring. To overcome this problem, here we sought to optimize the length of the vancomycin-binding aptamer sequence to enable a broader dynamic range in the E-AB platform. Our results demonstrate, via isothermal calorimetry and E-AB calibrations in undiluted serum, that superior affinity and near-equal sensor gain in vitro can be achieved using a one-base-pair-longer aptamer than the truncated sequence originally reported. We validate the impact of the improved binding affinity in vivo by monitoring vancomycin levels in the brain cortex of live mice following intravenous administration. While the original sequence fails to resolve vancomycin concentrations from baseline noise (SNR = 1.03), our newly reported sequence provides an SNR of 1.62 at the same dose.
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Figure 1
Figure 1. Vancomycin binding to E-ABs results in an electrochemical signal change. (A) E-ABs consist of several parts: (i) the electrode, (ii) the self-assembled monolayer, (iii) the SAM-bound aptamer (which binds to the target, vancomycin), and (iv) the redox reporter. (B) The redox reporter exchanges electrons at rates dependent on the reporter–electrode distance, keT1 and keT2. Because target binding induces conformational changes in the aptamer that affect the reporter–electrode distance, such changes result in observable changes in voltammetric current. These voltammograms were measured via square wave voltammetry at a frequency of 250 Hz, amplitude of 25 mV, and step size of 1 mV in phosphate-buffered saline (PBS) with 2 mM MgCl2. (C) Change in voltammetric peak current (red markers) or area under the curve (blue markers) can then be plotted to build dose–response curves from which thermodynamic parameters can be extracted. Here, for example, we performed nonlinear regression of the curve to a Hill model (red trace), obtaining a dissociation constant of 11.7 ± 0.7 μM and a Hill coefficient of 0.66 ± 0.02. The clinically relevant range for serum vancomycin is 6–30 μM as indicated in Table 1. (2,6)
Figure 2
Figure 2. ITC measurements reveal worsening of vancomycin affinity with increasing aptamer truncations. (A) The modeled secondary structure of the aptamers with symmetric truncations considered in this work. From left to right: no truncations, one (1trunc), two (2trunc), three (3trunc), and four (4trunc) base-pair truncations. This modeling was done using Nupack (14) assuming 140 mM Na+ and 2 mM Mg2+ at a temperature of 25 °C. (B) An example of two raw ITC graphs. Negative is exothermic. (C) We calculated the areas for each titration point and plotted them against the vancomycin to aptamer mole ratio in the ITC cell. We then fit the curves to a binding polynomial (red, black, and blue traces). KD for the full aptamer is 0.14 ± 0.02 μM, KD for 3trunc is 3.92 ± 0.90 μM, and KD for 4trunc is 52 ± 20 μM. Error bars represent the standard deviation of the heat and mole ratio. Errors are calculated from three replicates. Shading represents the propagation of error of the 95% confidence interval for each regression based on three replicates. (D) Red markers represent the KD, and blue markers represent the n of each symmetrical truncation. Dashed lines are point-to-point connections. Error bars are based on three replicates. All measurements were done in PBS solution with 2 mM MgCl2 added.
Results and Discussion
Affinity of Truncated Vancomycin Aptamers
Conclusions from the Affinity Measurements
Circular Dichroism Spectroscopy of Truncated Vancomycin Aptamers
Figure 3
Figure 3. CD spectroscopy illustrates the change in aptamer conformation upon vancomycin binding. CD spectra for each construct with no vancomycin (red trace) and 100 μM vancomycin (black trace). Red and gray shaded areas represent the standard deviation of three measurements without and with vancomycin, respectively. Measurements were done at a concentration of 5 μM aptamer in PBS solution with 2 mM Mg2+. Insets are expanded regions to highlight band shift upon vancomycin addition. For the 4trunc sequence (C), minimal vancomycin binding-induced conformational change is seen compared to full (A) or 3trunc (B). Measurements were performed at 25 °C, with an averaging time of 2 seconds, and a 1 mm path length. Data were background-subtracted ([aptamer ± vancomycin] – [buffer ± vancomycin]).
Conclusions from CD Measurements
Performance of Truncated Aptamers in E-AB Format
Figure 4
Figure 4. Performance of truncated aptamers in E-AB format. Here, we show dose–response curves for E-ABs functionalized with a 200 nM deposition concentration of (A) 3trunc (KD = 19.5 ± 1.6 μM) and 4trunc (KD = 70.7 ± 6 μM) aptamers in PBS with 2 mM Mg2+ or (B) 3trunc (KD = 18.1 ± 1.6 μM) and 4trunc (KD = 87.3 ± 9.1 μM) E-ABs in whole serum. The y-axis in these titrations represents signal gain, computed as the change in signal relative to measurements in the absence of vancomycin. Error bars are calculated based on eight sensors representing one standard deviation for each point. All measurements were performed via square wave voltammetry with an amplitude of 25 mV, step size of 1 mV, and frequency of either 80 Hz (PBS) or 250 Hz (serum).
Conclusions from E-AB Testing of Truncated Aptamers
Calibration Considerations for Vancomycin Sensing in Static Solutions
Figure 5
Figure 5. Single-point serum vancomycin measurements in static solution exhibit worse gain than a full titration. (A) Illustration of this effect by showing a 3trunc full titration curve (gold) side-by-side with single-point additions of vancomycin into unmixed (red) and mixed (black) serum. The full vancomycin titration shown here includes 35 measurements each performed at increasing vancomycin concentrations, converted to time scale using the time stamp on the files generated after each measurement. The two single-addition traces display the same number of E-AB measurements up until the addition of 100 μM vancomycin. The blue squares indicate the E-AB signal gain measured at 100 μM vancomycin concentration 1 min after addition, either via a progressive titration or via a single concentrated spike. (B, C) When comparing the E-AB gain between three points from a full vancomycin titration and single additions to reach equivalent vancomycin concentrations in (B) 100% serum or (C) 50% serum, the titration always achieved higher gains than measurements performed after single additions, for both 3trunc- (B) and 4trunc-functionalized sensors (C), irrespective of the number of measurements or sample mixing.
Conclusions from E-AB Calibrations in Serum
Reversibility of 3trunc-Functionalized E-ABs
Figure 6
Figure 6. Reversibility and reusability of 3trunc-functionalized E-AB sensors for vancomycin detection in vitro. (A) 3D-printed flow cell to evaluate the reversibility of 3trunc-functionalized E-ABs when exposed to changing vancomycin concentrations. Two pumps, a peristaltic pump for continuous solution flow and a syringe pump for drug additions, are suspended above a plate containing gold microelectrodes. The microelectrodes are functionalized with vancomycin-binding 3trunc aptamer to make E-AB sensors, and interrogated via a handheld potentiostat. Reprinted with permission from Anal. Chem. 2022, 94, 23, 8335–8345. Copyright 2022, Analytical Chemistry. (B) Demonstration of continuous, real-time monitoring of vancomycin in the flow system following three injections of 100 μM vancomycin. Voltammetric measurements were performed every 12 s in PBS containing 2 mM MgCl2. (C) Kinetic differential measurements (KDM) obtained after subtracting data collected at 10 Hz (signal-OFF output) from data collected at 80 Hz (signal-ON output).
3trunc-Functionalized E-ABs Support Continuous Vancomycin Monitoring In Vivo
Figure 7
Figure 7. 3trunc-Functionalized E-ABs support continuous vancomycin monitoring in vivo. (A) Electrode fabrication protocols from the field of fast-scan cyclic voltammetry to make gold-based E-AB probes. The probes are 50 μm in diameter and ∼500 μm in length. (B) Placing the probes on the right hemisphere of mice brains, within the brain cortex (sagittal view). (C) In-brain placement is confirmed via histology post in vivo measurements (coronal view). (D) 3trunc-Based E-AB measurements performed every 2 min revealing an immediate increase of cortex vancomycin levels following IV dosing relative to vehicle boluses. (E) Identical experiments performed using 4trunc-based E-ABs cannot confidently resolve cortex vancomycin levels from background noise. Solid red markers represent the average of three measurements performed on n = 3 independent mice. Shaded areas represent the standard deviation. Solid black markers indicate control measurements (n = 1 for each sensor type) following vehicle dosing.
Conclusions
Methods
Chemicals and Materials

CD Measurements
ITC Measurements
Fabrication of Electrodes for In Vivo Measurements
Sensor Preparation for In Vitro Beaker Applications
Microneedle Sensor Preparation for In Vitro Flow Cell Applications
Sensor Preparation for In Vivo Applications
Surgical Procedures for Electrodes Implantation
1. | Reference electrodes: +4.80 AP (from bregma), −1.00 ML (from bregma), and −2.00 DV (from brain surface) | ||||
2. | E-ABs electrodes: −1.70 AP (from bregma), +0.50 ML (from bregma), and −1.00 DV (from brain surface) | ||||
3. | Counter-electrode screws: −2.90 AP (from bregma), −2.30 ML (from bregma), and 3–4 full rotations inside the hole. |
Electrochemical Measurements In Vivo
Electrochemical Measurements In Vitro
Data Fitting
Time Course Experiments
Flow Cell Measurements
Brain Tissue Staining and Histology
Supporting Information
The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acssensors.2c01910.
Aptamer variant sequences, measured ITC parameters, and Nupack-predicted secondary structures (Table S1)
and additional ITC, CD, and electrochemical measurements (Figures S1–S7) (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
The authors thank Zio Health for providing research-sponsored funds in support of this work. They also thank Dr. Katie Tripp and the JHU Center for Molecular Biophysics for providing access to CD spectrometers and technical assistance. The in vivo work reported here was supported by the National Institute of General Medical Sciences of the National Institutes of Health under Award Number R01GM140143. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health
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- 4Elbarbry, F. Vancomycin Dosing and Monitoring: Critical Evaluation of the Current Practice. Eur. J. Drug Metab. Pharmacokinet. 2018, 43, 259– 268, DOI: 10.1007/s13318-017-0456-4Google Scholar4https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXitVShsLzI&md5=da2e22e7c51e0cf3c82e57a43d1ba46eVancomycin Dosing and Monitoring: Critical Evaluation of the Current PracticeElbarbry, FawzyEuropean Journal of Drug Metabolism and Pharmacokinetics (2018), 43 (3), 259-268CODEN: EJDPD2; ISSN:0378-7966. (Springer France)After more than six decades of its use as the mainstay antibiotic for the treatment of multidrug-resistant Gram-pos. bacterial infections, dosing and monitoring of vancomycin therapy have not been optimized. The current vancomycin therapeutic guidelines recommend empiric doses of 15-20 mg/kg administered by intermittent infusion every 8-12 h in patients with normal kidney function. Addnl., the guidelines recommend trough concn. of 15-20 mg/L as a therapeutic goal for adult patients with severe infections. This review critically discusses the current guidelines considering the basic pharmacokinetics and pharmacodynamics of vancomycin and the recent published reports from clin. studies. More in-depth discussion will be focused on (1) providing evidence of advantages of administering vancomycin by continuous infusion compared to intermittent infusion; (2) revising the current practice of trough-only monitoring vs. the area under concn.-time curve (AUC); and (3) assessing the current practice of wt.-based dosing vs. AUC-based dosing. Using the gathered information presented in this paper, two user-friendly and scientifically based dosing strategies are proposed to improve the efficiency of vancomycin dosing while avoiding the risk of nephrotoxicity and minimizing the cost of therapeutic drug monitoring.
- 5Chen, C. Y.; Li, M. Y.; Ma, L. Y.; Zhai, X. Y.; Luo, D. H.; Zhou, Y.; Liu, Z. M.; Cui, Y. M. Precision and Accuracy of Commercial Assays for Vancomycin Therapeutic Drug Monitoring: Evaluation Based on External Quality Assessment Scheme. J. Antimicrob. Chemother. 2020, 75, 2110– 2119, DOI: 10.1093/jac/dkaa150Google Scholar5https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXitlyqtLk%253D&md5=915ae6c327141c60dc2e9b2f55c3db50Precision and accuracy of commercial assays for vancomycin therapeutic drug monitoring: evaluation based on external quality assessment schemeChen, Chao-Yang; Li, Meng-Ya; Ma, Ling-Yun; Zhai, Xing-Yu; Luo, Dao-Huang; Zhou, Ying; Liu, Zhen-Ming; Cui, Yi-MinJournal of Antimicrobial Chemotherapy (2020), 75 (8), 2110-2119CODEN: JACHDX; ISSN:1460-2091. (Oxford University Press)Vancomycin remains a mainstay of the treatment of Gram-pos. bacterial infections. It is crucial to accurately det. vancomycin serum concn. for adequate dose adjustment. To evaluate the precision and accuracy of com. assay techniques for vancomycin concn. and to assess the comparability of vancomycin detection methods in Chinese labs. Human serum samples spiked with known concns. of vancomycin were provided to labs. participating in the external quality assessment scheme (EQAS). Assay methods included chemiluminescence, enzyme immunoassay (EIA) and so on. The dispersion of the measurements was analyzed and the robust coeff. of variation (rCV), relative percentage difference (RPD) and satisfactory rate for method groups were calcd. Moreover, performance of the Chinese labs. was assessed. A total of 657 results from 75 labs. were collected, including 84 samples from 10 Chinese labs. The median rCV, median RPD and satisfactory rates classified by methods ranged from 1.85% to 15.87%, -14.75% to 13.34% and 94.59% to 100.00%, resp. Significant differences were seen in precision, between kinetic interaction of microparticles in soln. (KIMS) and other methods, and in accuracy, between enzyme-multiplied immunoassay technique (EMIT), fluorescence polarization immunoassay (FPIA) and other techniques. Vancomycin detection in China mainly depended on the chemiluminescence and EMIT methods, which tended to result in lower measurements. Although almost all assays in this study achieved an acceptable performance for vancomycin serum concn. monitoring, obvious inconsistencies between methods were still obsd. Chinese labs. were more likely to underestimate vancomycin concns. Thus, recognizing inconsistencies between methods and regular participation in vancomycin EQAS are essential.
- 6Dauphin-Ducharme, P.; Zhang, Y.; Gerson, J.; Kurnik, M.; Kippin, T. E.; Stojanovic, M. N. 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 Scholar6https://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.
- 7Li, H.; Dauphin-ducharme, P.; Ortega, G.; Plaxco, K. W. Calibration-Free Electrochemical Biosensors Supporting Accurate Molecular Measurements Directly in Undiluted Whole Blood. J. Am. Chem. Soc. 2017, 139, 11207– 11213, DOI: 10.1021/jacs.7b05412Google Scholar7https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhtFOmtLrL&md5=ab88a3fba1bac8fdc60d009b5af2a601Calibration-Free Electrochemical Biosensors Supporting Accurate Molecular Measurements Directly in Undiluted Whole BloodLi, Hui; Dauphin-Ducharme, Philippe; Ortega, Gabriel; Plaxco, Kevin W.Journal of the American Chemical Society (2017), 139 (32), 11207-11213CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)The need to calibrate to correct for sensor-to-sensor fabrication variation and sensor drift has proven a significant hurdle in the widespread use of biosensors. To maintain clin. relevant (±20% for this application) accuracy, for example, com. continuous glucose monitors require recalibration several times a day, decreasing convenience and increasing the chance of user errors. Here, however, the authors demonstrate a "dual-frequency" approach for achieving the calibration-free operation of electrochem. biosensors that generate an output by using square-wave voltammetry to monitor binding-induced changes in electron transfer kinetics. Specifically, the authors use the square-wave frequency dependence of their response to produce a ratiometric signal, the ratio of peak currents collected at responsive and non- (or low) responsive square-wave frequencies, which is largely insensitive to drift and sensor-to-sensor fabrication variations. Using electrochem. aptamer-based (E-AB) biosensors as the test bed, the authors demonstrate the accurate and precise operation of sensors against multiple drugs, achieving accuracy in the measurement of their targets of within better than 20% across dynamic ranges of up to 2 orders of magnitude without the need to calibrate each individual sensor.
- 8Arroyo-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 Scholar8https://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.
- 9Parolo, 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 Scholar9https://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.
- 10Idili, A.; Gerson, J.; Parolo, C.; Kippin, T.; Plaxco, K. W. An Electrochemical Aptamer-Based Sensor for the Rapid and Convenient Measurement of l-Tryptophan. Anal. Bioanal. Chem. 2019, 411, 4629– 4635, DOI: 10.1007/s00216-019-01645-0Google Scholar10https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXms1ansL0%253D&md5=479436452e3a26dd45ad0a08a81e5ebfAn electrochemical aptamer-based sensor for the rapid and convenient measurement of L-tryptophanIdili, Andrea; Gerson, Julian; Parolo, Claudio; Kippin, Tod; Plaxco, Kevin W.Analytical and Bioanalytical Chemistry (2019), 411 (19), 4629-4635CODEN: ABCNBP; ISSN:1618-2642. (Springer)The field of precision medicine-the possibility to accurately tailor pharmacol. treatments to each specific patient-would be significantly advanced by the ability to rapidly, conveniently, and cost-effectively measure biomarkers directly at the point of care. Electrochem. aptamer-based (E-AB) sensors appear a promising approach to this end due to their low cost, ease of use, and good anal. performance in complex clin. samples. Thus motivated, we present here the development of an E-AB sensor for the measurement of the amino acid L-tryptophan, a diagnostic marker indicative of a no. of metabolic and mental health disorders, in urine. The sensor employs a previously reported DNA aptamer able to recognize the complex formed between tryptophan and a rhodium-based receptor. We adopted the aptamer to the E-AB sensing platform by truncating it, causing it to undergo a binding-induced conformational change, modifying it with a redox-reporting methylene blue, and attaching it to an interrogating electrode. The resulting sensor is able to measure tryptophan concns. in the micromolar range in minutes and readily discriminates between its target and other arom. and non-arom. amino acids. Using it, we demonstrate the measurement of clin. relevant tryptophan levels in synthetic urine in a process requiring only a single diln. step. The speed and convenience with which this is achieved suggest that the E-AB platform could significantly improve the ease and frequency with which metabolic diseases are monitored. [Figure not available: see fulltext.].
- 11Zaric, R. Z.; Milovanovic, J.; Rosic, N.; Milovanovic, D.; Zecevic, D. R.; Folic, M.; Jankovic, S. Pharmacokinetics of Vancomycin in Patients with Different Renal Function Levels. Open Med. 2018, 13, 512– 519, DOI: 10.1515/med-2018-0068Google Scholar11https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXitVCisLzF&md5=97dc74ebbace1349b387d8d95d5a0a0bPharmacokinetics of vancomycin in patients with different renal function levelsZaric, Radica Zivkovic; Milovanovic, Jasmina; Rosic, Nikola; Milovanovic, Dragan; Zecevic, Dejana Ruzic; Folic, Marko; Jankovic, SlobodanOpen Medicine (2018), 13 (1), 512-519CODEN: OMPEAL; ISSN:2391-5463. (De Gruyter Open Ltd.)There are many determinants of vancomycin clearance, but these have not been analyzed sep. in populations with different levels of renal function, which could be why some important factors have been missed. The aim of our study was to compare the pharmacokinetic parameters and factors that may affect vancomycin pharmacokinetics in groups of patients with normal renal function and in those with chronic kidney failure. The study used a population pharmacokinetic modeling approach, based on plasma vancomycin concns. and other data from 78 patients with chronic kidney failure and 32 patients with normal renal function. The model was developed using NONMEM software and validated by bootstrapping. The final model for patients with impaired kidney function was described by the following equation: CL (L/h) = 0.284 + 0.000596 x DD + 0.00194 x AST, and that for the patients with normal kidney function by: CL (L/h) = 0.0727 + 0.205 x FIB. If our results are confirmed by new studies on two similar populations, these factors could be considered when dosing vancomycin in patients with chronically damaged kidneys, as well as in patients with normal kidneys who frequently require high doses of vancomycin.
- 12Suzuki, Y.; Kawasaki, K.; Sato, Y.; Tokimatsu, I.; Itoh, H.; Hiramatsu, K.; Takeyama, M.; Kadota, J. I. Is Peak Concentration Needed in Therapeutic Drug Monitoring of Vancomycin? A Pharmacokinetic-Pharmacodynamic Analysis in Patients with Methicillin- Resistant Staphylococcus Aureus Pneumonia. Chemotherapy 2012, 58, 308– 312, DOI: 10.1159/000343162Google Scholar12https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XhslyjsLrE&md5=e10a1ad993f3e4642c02b3699e7bd0bdIs Peak Concentration Needed in Therapeutic Drug Monitoring of Vancomycin? A Pharmacokinetic-Pharmacodynamic Analysis in Patients with Methicillin-Resistant Staphylococcus aureus PneumoniaSuzuki, Yosuke; Kawasaki, Kanako; Sato, Yuhki; Tokimatsu, Issei; Itoh, Hiroki; Hiramatsu, Kazufumi; Takeyama, Masaharu; Kadota, Jun-ichiChemotherapy (Basel, Switzerland) (2012), 58 (4), 308-312CODEN: CHTHBK; ISSN:0009-3157. (S. Karger AG)Background: We analyzed the pharmacokinetic-pharmacodynamic relationship of vancomycin to det. the drug exposure parameters that correlate with the efficacy and nephrotoxicity of vancomycin in patients with methicillin-resistant Staphylococcus aureus pneumonia and evaluated the need to use peak concn. in therapeutic drug monitoring (TDM). Methods: Serum drug concns. of 31 hospitalized patients treated with vancomycin for methicillin-resistant S. aureus pneumonia were collected. Results: Significant differences in trough concn. (Cmin)/min. inhibitory concn. (MIC) and area under the serum concn.-time curve (AUC0-24)/MIC were obsd. between the response and non-response groups. Significant differences in Cmin and AUC0-24 were obsd. between the nephrotoxicity and non-nephrotoxicity groups. Receiver operating characteristic curves revealed high predictive values of Cmin/MIC and AUC0-24/MIC for efficacy and of Cmin and AUC0-24 for safety of vancomycin. Conclusions: These results suggest little need to use peak concn. in vancomycin TDM because Cmin/MIC and Cmin are sufficient to predict the efficacy and safety of vancomycin.
- 13Turnbull, W. B.; Daranas, A. H. On the Value of c: Can Low Affiniity Systems Be Studied by Isothermal Titration Calorimetry?. J. Am. Chem. Soc. 2003, 125, 14859– 14866, DOI: 10.1002/asi.4630310417Google Scholar13https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3sXovVSiu7w%253D&md5=088d351b0836433e55b46e16189271e7On the Value of c: Can Low Affinity Systems Be Studied by Isothermal Titration Calorimetry?Turnbull, W. Bruce; Daranas, Antonio H.Journal of the American Chemical Society (2003), 125 (48), 14859-14866CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Isothermal titrn. calorimetry (ITC) allows the detn. of ΔG°, ΔH°, and ΔS° from a single expt. and is thus widely used for studying binding thermodn. in both biol. and synthetic supramol. systems. However, it is widely believed that it is not possible to derive accurate thermodn. information from ITC expts. in which the Wiseman "c" parameter (which is the product of the receptor concn. and the binding const., Ka) is less than ca. 10, constraining its use to high affinity systems. Herein, exptl. titrns. and simulated data are used to demonstrate that this dogma is false, esp. for low affinity systems, assuming that (1) a sufficient portion of the binding isotherm is used for anal., (2) the binding stoichiometry is known, (3) the concns. of both ligand and receptor are known with accuracy, and (4) there is an adequate level of signal-to-noise in the data. This study supports the validity of ITC for detg. the value of Ka and, hence, ΔG° from expts. conducted under low c conditions but advocates greater caution in the interpretation of values for ΔH°. Therefore, isothermal titrn. calorimetry is a valid and useful technique for studying biol. and synthetically important low affinity systems.
- 14Zadeh, J. N.; Steenberg, C. D.; Bois, J. S.; Wolfe, B. R.; Pierce, M. B.; Khan, A. R.; Dirks, R. M.; Pierce, N. A. NUPACK: Analysis and Design of Nucleic Acid Systems. J. Comput. Chem. 2011, 32, 170– 173, DOI: 10.1002/jcc.21596Google Scholar14https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXhsVCjsLzP&md5=9c60f0134ea55d9335301e2efe1602e8NUPACK: analysis and design of nucleic acid systemsZadeh, Joseph N.; Steenberg, Conrad D.; Bois, Justin S.; Wolfe, Brian R.; Pierce, Marshall B.; Khan, Asif R.; Dirks, Robert M.; Pierce, Niles A.Journal of Computational Chemistry (2011), 32 (1), 170-173CODEN: JCCHDD; ISSN:0192-8651. (John Wiley & Sons, Inc.)The Nucleic Acid Package (NUPACK) is a growing software suite for the anal. and design of nucleic acid systems. The NUPACK web server (http://www.nupack.org) currently enables: NUPACK algorithms are formulated in terms of nucleic acid secondary structure. In most cases, pseudoknots are excluded from the structural ensemble. © 2010 Wiley Periodicals, Inc. J Comput Chem, 2010.
- 15Chamorro-Garcia, A.; Ortega, G.; Mariottini, D.; Green, J.; Ricci, F.; Plaxco, K. W. Switching the Aptamer Attachment Geometry Can Dramatically Alter the Signalling and Performance of Electrochemical Aptamer-Based Sensors. Chem. Commun. 2021, 57, 11693– 11696, DOI: 10.1039/d1cc04557aGoogle Scholar15https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXit1Wku77E&md5=e72fd6ba0bb829251dcf92478b487837Switching the aptamer attachment geometry can dramatically alter the signalling and performance of electrochemical aptamer-based sensorsChamorro-Garcia, Alejandro; Ortega, Gabriel; Mariottini, Davide; Green, Joshua; Ricci, Francesco; Plaxco, Kevin W.Chemical Communications (Cambridge, United Kingdom) (2021), 57 (88), 11693-11696CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)Electrochem. aptamer-based (EAB) sensors, composed of an electrode-bound DNA aptamer with a redox reporter on the distal end, offer the promise of high-frequency, real-time mol. measurements in complex sample matrixes and even in vivo. Here we assess the extent to which switching the aptamer terminus that is electrode-bound and the one that is redox-reporter-modified affects the performance of these sensors. Using sensors against doxorubicin, cocaine, and vancomycin as our test beds, we find that both signal gain (the relative signal change seen in the presence of a satg. target) and the frequency dependence of gain depend strongly on the attachment orientation, suggesting that this easily investigated variable is a worthwhile parameter to optimize in the design of new EAB sensors.
- 16Shaver, 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 Scholar16https://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.
- 17White, 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 Scholar17https://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.
- 18Liu, Y.; Canoura, J.; Alkhamis, O.; Xiao, Y. Immobilization Strategies for Enhancing Sensitivity of Electrochemical Aptamer-Based Sensors. ACS Appl. Mater. Interfaces 2021, 13, 9491– 9499, DOI: 10.1021/acsami.0c20707Google Scholar18https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXht1Wjtrw%253D&md5=ba2749b65de53c20271e7e034b2cdabbImmobilization Strategies for Enhancing Sensitivity of Electrochemical Aptamer-Based SensorsLiu, Yingzhu; Canoura, Juan; Alkhamis, Obtin; Xiao, YiACS Applied Materials & Interfaces (2021), 13 (8), 9491-9499CODEN: AAMICK; ISSN:1944-8244. (American Chemical Society)Electrochem. aptamer-based (E-AB) sensors are a versatile sensing platform that can achieve rapid and robust target detection in complex matrixes. However, the limited sensitivity of these sensors has impeded their translation from proof-of-concept to com. products. Surface-bound aptamers must be sufficiently spaced to bind targets and subsequently fold for signal transduction. We hypothesized that electrodes fabricated using conventional methods result in sensing surfaces where only a fraction of aptamers are appropriately spaced to actively respond to the target. As an alternative, we presented a novel aptamer immobilization approach that favors sufficient spacing between aptamers at the microscale to achieve optimal target binding, folding, and signal transduction. We first demonstrated that immobilizing aptamers in their target-bound, folded state on gold electrode surfaces yields an aptamer monolayer that supports greater sensitivity and higher signal-to-noise ratio than traditionally prepd. E-AB sensors. We also showed that performing aptamer immobilization under low ionic strength conditions rather than conventional high ionic strength buffer greatly improves E-AB sensor performance. We successfully tested our approach with three different small-mol.-binding aptamers, demonstrating its generalizability. On the basis of these results, we believe our electrode fabrication approach will accelerate development of high-performance sensors with the sensitivity required for real-world anal. applications.
- 19Wu, Y.; Tehrani, F.; Teymourian, H.; Mack, J.; Shaver, A.; Reynoso, M.; Kavner, J.; Huang, N.; Furmidge, A.; Duvvuri, A. Microneedle Aptamer-Based Sensors for Continuous, Real-Time Therapeutic Drug Monitoring. Anal. Chem. 2022, 94, 8335– 8345, DOI: 10.1021/acs.analchem.2c00829Google Scholar19https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38XhsVGnt7rF&md5=7ec79273b3414d7e179173460bdc565cMicroneedle Aptamer-Based Sensors for Continuous, Real-Time Therapeutic Drug MonitoringWu, Yao; Tehrani, Farshad; Teymourian, Hazhir; Mack, John; Shaver, Alexander; Reynoso, Maria; Kavner, Jonathan; Huang, Nickey; Furmidge, Allison; Duvvuri, Andres; Nie, Yuhang; Laffel, Lori M.; Doyle, Francis J.; Patti, Mary-Elizabeth; Dassau, Eyal; Wang, Joseph; Arroyo-Curras, NetzahualcoyotlAnalytical Chemistry (Washington, DC, United States) (2022), 94 (23), 8335-8345CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)The ability to continuously monitor the concn. of specific mols. in the body is a long-sought goal of biomedical research. For this purpose, interstitial fluid (ISF) was proposed as the ideal target biofluid because its compn. can rapidly equilibrate with that of systemic blood, allowing the assessment of mol. concns. that reflect full-body physiol. In the past, continuous monitoring in ISF was enabled by microneedle sensor arrays. Yet, benchmark microneedle sensors can only detect mols. that undergo redox reactions, which limits the ability to sense metabolites, biomarkers, and therapeutics that are not redox-active. To overcome this barrier, here, we expand the scope of these devices by demonstrating the first use of microneedle-supported electrochem., aptamer-based (E-AB) sensors. This platform achieves mol. recognition based on affinity interactions, vastly expanding the scope of mols. that can be sensed. We report the fabrication of microneedle E-AB sensor arrays and a method to regenerate them for multiple uses. In addn., we demonstrate continuous mol. measurements using these sensors in flow systems in vitro using single and multiplexed microneedle array configurations. Translation of the platform to in vivo measurements is possible as we demonstrate with a first E-AB measurement in the ISF of a rodent. The encouraging results reported in this work should serve as the basis for future translation of microneedle E-AB sensor arrays to biomedical research in preclin. animal models.
- 20Arroyo-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 Scholar20https://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.
- 21Curtis, 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 Scholar21https://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.
- 22Miura, Y.; Fuchigami, Y.; Nomura, S.; Nishimura, K.; Hagimori, M.; Kawakami, S. Brain Microdialysis Study of Vancomycin in the Cerebrospinal Fluid After Intracerebroventricular Administration in Mice. AAPS PharmSciTech 2019, 20, 5 DOI: 10.1208/s12249-018-1232-8Google ScholarThere is no corresponding record for this reference.
- 23Nau, R.; Sörgel, F.; Eiffert, H. Penetration of Drugs through the Blood-Cerebrospinal Fluid/Blood-Brain Barrier for Treatment of Central Nervous System Infections. Clin. Microbiol. Rev. 2010, 23, 858– 883, DOI: 10.1128/CMR.00007-10Google Scholar23https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXhtlOntbg%253D&md5=470fffb97c0373a2dccafe3dbd021345Penetration of drugs through the blood-cerebrospinal fluid/blood-brain barrier for treatment of central nervous system infectionsNau, Roland; Soergel, Fritz; Eiffert, HelmutClinical Microbiology Reviews (2010), 23 (4), 858-883CODEN: CMIREX; ISSN:0893-8512. (American Society for Microbiology)A review. Central nervous system (CNS) infections caused by pathogens with a reduced sensitivity to drugs are a therapeutic challenge. This is particularly true for infections caused by penicillin- resistant pneumococci, methicillin-resistant staphylococci, multiresistant Gram-neg. aerobic bacilli, or several other organisms (including Aspergillus spp., Scedosporium apiospermum, and Nocardia asteroides) that affect primarily the CNS in immunocompromised patients. This review aims to increase the awareness of the peculiarities of the pharmacokinetics of anti-infectives within the CNS.
- 24Meunier, C. J.; Sombers, L. A. Fast-Scan Voltammetry for In Vivo Measurements of Neurochemical Dynamics. In The Brain Reward System; Springer, 2021.Google ScholarThere is no corresponding record for this reference.
- 25Arroyo-Currás, N.; Scida, K.; Ploense, K. L.; Kippin, T. E.; Plaxco, K. W. High Surface Area Electrodes Generated via Electrochemical Roughening Improve the Signaling of Electrochemical Aptamer-Based Biosensors. Anal. Chem. 2017, 89, 12185– 12191, DOI: 10.1021/acs.analchem.7b02830Google Scholar25https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhslagsr%252FO&md5=c40a96ca49d03b35bb7eaceb0ab47462High Surface Area Electrodes Generated via Electrochemical Roughening Improve the Signaling of Electrochemical Aptamer-Based BiosensorsArroyo-Curras, Netzahualcoyotl; Scida, Karen; Ploense, Kyle L.; Kippin, Tod E.; Plaxco, Kevin W.Analytical Chemistry (Washington, DC, United States) (2017), 89 (22), 12185-12191CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)The electrochem., aptamer-based (E-AB) sensor platform provides a modular approach to the continuous, real-time measurement of specific mol. targets (irresp. of their chem. reactivity) in situ in the living body. To achieve this, however, requires the fabrication of sensors small enough to insert into a vein, which, for the rat animal model the authors employ, entails devices less than 200 μm in diam. The limited surface area of these small devices leads, in turn, to low faradaic currents and poor signal-to-noise ratios when deployed in the complex, fluctuating environments found in vivo. In response the authors have developed an electrochem. roughening approach that enhances the signaling of small electrochem. sensors by increasing the microscopic surface area of gold electrodes, allowing in this case more redox-reporter-modified aptamers to be packed onto the surface, thus producing significantly improved signal-to-noise ratios. Unlike previous approaches to achieving microscopically rough gold surfaces, the method employs chronoamperometric pulsing in a 5 min etching process easily compatible with batch manufg. Using these high surface area electrodes, the authors demonstrate the ability of E-AB sensors to measure complete drug pharmacokinetic profiles in live rats with precision of better than 10% in the detn. of drug disposition parameters.
- 26Franklin, K.; Paxinos, G. The Mouse Brain in Stereotaxic Coordinates, 3rd ed.; Elsevier Academic Press: San Diego, 2007.Google ScholarThere is no corresponding record for this reference.
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Abstract
Figure 1
Figure 1. Vancomycin binding to E-ABs results in an electrochemical signal change. (A) E-ABs consist of several parts: (i) the electrode, (ii) the self-assembled monolayer, (iii) the SAM-bound aptamer (which binds to the target, vancomycin), and (iv) the redox reporter. (B) The redox reporter exchanges electrons at rates dependent on the reporter–electrode distance, keT1 and keT2. Because target binding induces conformational changes in the aptamer that affect the reporter–electrode distance, such changes result in observable changes in voltammetric current. These voltammograms were measured via square wave voltammetry at a frequency of 250 Hz, amplitude of 25 mV, and step size of 1 mV in phosphate-buffered saline (PBS) with 2 mM MgCl2. (C) Change in voltammetric peak current (red markers) or area under the curve (blue markers) can then be plotted to build dose–response curves from which thermodynamic parameters can be extracted. Here, for example, we performed nonlinear regression of the curve to a Hill model (red trace), obtaining a dissociation constant of 11.7 ± 0.7 μM and a Hill coefficient of 0.66 ± 0.02. The clinically relevant range for serum vancomycin is 6–30 μM as indicated in Table 1. (2,6)
Figure 2
Figure 2. ITC measurements reveal worsening of vancomycin affinity with increasing aptamer truncations. (A) The modeled secondary structure of the aptamers with symmetric truncations considered in this work. From left to right: no truncations, one (1trunc), two (2trunc), three (3trunc), and four (4trunc) base-pair truncations. This modeling was done using Nupack (14) assuming 140 mM Na+ and 2 mM Mg2+ at a temperature of 25 °C. (B) An example of two raw ITC graphs. Negative is exothermic. (C) We calculated the areas for each titration point and plotted them against the vancomycin to aptamer mole ratio in the ITC cell. We then fit the curves to a binding polynomial (red, black, and blue traces). KD for the full aptamer is 0.14 ± 0.02 μM, KD for 3trunc is 3.92 ± 0.90 μM, and KD for 4trunc is 52 ± 20 μM. Error bars represent the standard deviation of the heat and mole ratio. Errors are calculated from three replicates. Shading represents the propagation of error of the 95% confidence interval for each regression based on three replicates. (D) Red markers represent the KD, and blue markers represent the n of each symmetrical truncation. Dashed lines are point-to-point connections. Error bars are based on three replicates. All measurements were done in PBS solution with 2 mM MgCl2 added.
Figure 3
Figure 3. CD spectroscopy illustrates the change in aptamer conformation upon vancomycin binding. CD spectra for each construct with no vancomycin (red trace) and 100 μM vancomycin (black trace). Red and gray shaded areas represent the standard deviation of three measurements without and with vancomycin, respectively. Measurements were done at a concentration of 5 μM aptamer in PBS solution with 2 mM Mg2+. Insets are expanded regions to highlight band shift upon vancomycin addition. For the 4trunc sequence (C), minimal vancomycin binding-induced conformational change is seen compared to full (A) or 3trunc (B). Measurements were performed at 25 °C, with an averaging time of 2 seconds, and a 1 mm path length. Data were background-subtracted ([aptamer ± vancomycin] – [buffer ± vancomycin]).
Figure 4
Figure 4. Performance of truncated aptamers in E-AB format. Here, we show dose–response curves for E-ABs functionalized with a 200 nM deposition concentration of (A) 3trunc (KD = 19.5 ± 1.6 μM) and 4trunc (KD = 70.7 ± 6 μM) aptamers in PBS with 2 mM Mg2+ or (B) 3trunc (KD = 18.1 ± 1.6 μM) and 4trunc (KD = 87.3 ± 9.1 μM) E-ABs in whole serum. The y-axis in these titrations represents signal gain, computed as the change in signal relative to measurements in the absence of vancomycin. Error bars are calculated based on eight sensors representing one standard deviation for each point. All measurements were performed via square wave voltammetry with an amplitude of 25 mV, step size of 1 mV, and frequency of either 80 Hz (PBS) or 250 Hz (serum).
Figure 5
Figure 5. Single-point serum vancomycin measurements in static solution exhibit worse gain than a full titration. (A) Illustration of this effect by showing a 3trunc full titration curve (gold) side-by-side with single-point additions of vancomycin into unmixed (red) and mixed (black) serum. The full vancomycin titration shown here includes 35 measurements each performed at increasing vancomycin concentrations, converted to time scale using the time stamp on the files generated after each measurement. The two single-addition traces display the same number of E-AB measurements up until the addition of 100 μM vancomycin. The blue squares indicate the E-AB signal gain measured at 100 μM vancomycin concentration 1 min after addition, either via a progressive titration or via a single concentrated spike. (B, C) When comparing the E-AB gain between three points from a full vancomycin titration and single additions to reach equivalent vancomycin concentrations in (B) 100% serum or (C) 50% serum, the titration always achieved higher gains than measurements performed after single additions, for both 3trunc- (B) and 4trunc-functionalized sensors (C), irrespective of the number of measurements or sample mixing.
Figure 6
Figure 6. Reversibility and reusability of 3trunc-functionalized E-AB sensors for vancomycin detection in vitro. (A) 3D-printed flow cell to evaluate the reversibility of 3trunc-functionalized E-ABs when exposed to changing vancomycin concentrations. Two pumps, a peristaltic pump for continuous solution flow and a syringe pump for drug additions, are suspended above a plate containing gold microelectrodes. The microelectrodes are functionalized with vancomycin-binding 3trunc aptamer to make E-AB sensors, and interrogated via a handheld potentiostat. Reprinted with permission from Anal. Chem. 2022, 94, 23, 8335–8345. Copyright 2022, Analytical Chemistry. (B) Demonstration of continuous, real-time monitoring of vancomycin in the flow system following three injections of 100 μM vancomycin. Voltammetric measurements were performed every 12 s in PBS containing 2 mM MgCl2. (C) Kinetic differential measurements (KDM) obtained after subtracting data collected at 10 Hz (signal-OFF output) from data collected at 80 Hz (signal-ON output).
Figure 7
Figure 7. 3trunc-Functionalized E-ABs support continuous vancomycin monitoring in vivo. (A) Electrode fabrication protocols from the field of fast-scan cyclic voltammetry to make gold-based E-AB probes. The probes are 50 μm in diameter and ∼500 μm in length. (B) Placing the probes on the right hemisphere of mice brains, within the brain cortex (sagittal view). (C) In-brain placement is confirmed via histology post in vivo measurements (coronal view). (D) 3trunc-Based E-AB measurements performed every 2 min revealing an immediate increase of cortex vancomycin levels following IV dosing relative to vehicle boluses. (E) Identical experiments performed using 4trunc-based E-ABs cannot confidently resolve cortex vancomycin levels from background noise. Solid red markers represent the average of three measurements performed on n = 3 independent mice. Shaded areas represent the standard deviation. Solid black markers indicate control measurements (n = 1 for each sensor type) following vehicle dosing.
References
This article references 26 other publications.
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- 2Rybak, M. J.; Le, J.; Lodise, T. P.; Levine, D. P.; Bradley, J. S.; Liu, C.; Mueller, B. A.; Pai, M. P.; Wong-Beringer, A.; Rotschafer, J. C. Therapeutic Monitoring of Vancomycin for Serious Methicillin-Resistant Staphylococcus Aureus Infections: A Revised Consensus Guideline and Review by the American Society of Health-System Pharmacists, the Infectious Diseases Society of America, the Pediatric Infectious Diseases Society, and the Society of Infectious Diseases Pharmacists. Am. J. Heal. Pharm. 2020, 77, 835– 863, DOI: 10.1093/ajhp/zxaa0362https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BB383lsVSiug%253D%253D&md5=3e67ea42b02c2fd9fc12564374f9591dTherapeutic monitoring of vancomycin for serious methicillin-resistant Staphylococcus aureus infections: A revised consensus guideline and review by the American Society of Health-System Pharmacists, the Infectious Diseases Society of America, the Pediatric Infectious Diseases Society, and the Society of Infectious Diseases PharmacistsRybak Michael J; Le Jennifer; Lodise Thomas P; Levine Donald P; Bradley John S; Liu Catherine; Mueller Bruce A; Pai Manjunath P; Wong-Beringer Annie; Rotschafer John C; Rodvold Keith A; Maples Holly D; Lomaestro Benjamin MAmerican journal of health-system pharmacy : AJHP : official journal of the American Society of Health-System Pharmacists (2020), 77 (11), 835-864 ISSN:.There is no expanded citation for this reference.
- 3Dilworth, T. J.; Schulz, L. T.; Rose, W. E. Vancomycin Advanced Therapeutic Drug Monitoring: Exercise in Futility or Virtuous Endeavor to Improve Drug Efficacy and Safety?. Clin. Infect. Dis. 2021, 72, E675– E681, DOI: 10.1093/cid/ciaa13543https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXhtFyitrrM&md5=2f3c0a2778f415e29fe7469b17e551acVancomycin advanced therapeutic drug monitoring: exercise in futility or virtuous endeavor to improve drug efficacy and safety?Dilworth, Thomas J.; Schulz, Lucas T.; Rose, Warren E.Clinical Infectious Diseases (2021), 72 (10), e675-e681CODEN: CIDIEL; ISSN:1537-6591. (Oxford University Press)A review. Vancomycin is commonly prescribed to hospitalized patients. Decades of pharmacokinetic/pharmacodynamic research culminated in recommendations to monitor the ratio of the area under the concn.-time curve (AUC) to the min. inhibitory concn. in order to optimize vancomycin exposure and minimize toxicity in the revised 2020 guidelines. These guideline recommendations are based on limited data without high-quality evidence and limitations in strength. Despite considerable effort placed on vancomycin therapeutic drug monitoring (TDM), clinicians should recognize that the majority of vancomycin use is empiric. Most patients prescribed empiric vancomycin do not require it beyond a few days. For these patients, AUC detns. during the initial days of vancomycin exposure are futile. This added workload may detract from high-level patient care activities. Loading doses likely achieve AUC targets, so AUC monitoring after a loading dose is largely unnecessary for broad application. The excessive vancomycin TDM for decades has been propagated with limitations in evidence, and it should raise caution on contemporary vancomycin TDM recommendations.
- 4Elbarbry, F. Vancomycin Dosing and Monitoring: Critical Evaluation of the Current Practice. Eur. J. Drug Metab. Pharmacokinet. 2018, 43, 259– 268, DOI: 10.1007/s13318-017-0456-44https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXitVShsLzI&md5=da2e22e7c51e0cf3c82e57a43d1ba46eVancomycin Dosing and Monitoring: Critical Evaluation of the Current PracticeElbarbry, FawzyEuropean Journal of Drug Metabolism and Pharmacokinetics (2018), 43 (3), 259-268CODEN: EJDPD2; ISSN:0378-7966. (Springer France)After more than six decades of its use as the mainstay antibiotic for the treatment of multidrug-resistant Gram-pos. bacterial infections, dosing and monitoring of vancomycin therapy have not been optimized. The current vancomycin therapeutic guidelines recommend empiric doses of 15-20 mg/kg administered by intermittent infusion every 8-12 h in patients with normal kidney function. Addnl., the guidelines recommend trough concn. of 15-20 mg/L as a therapeutic goal for adult patients with severe infections. This review critically discusses the current guidelines considering the basic pharmacokinetics and pharmacodynamics of vancomycin and the recent published reports from clin. studies. More in-depth discussion will be focused on (1) providing evidence of advantages of administering vancomycin by continuous infusion compared to intermittent infusion; (2) revising the current practice of trough-only monitoring vs. the area under concn.-time curve (AUC); and (3) assessing the current practice of wt.-based dosing vs. AUC-based dosing. Using the gathered information presented in this paper, two user-friendly and scientifically based dosing strategies are proposed to improve the efficiency of vancomycin dosing while avoiding the risk of nephrotoxicity and minimizing the cost of therapeutic drug monitoring.
- 5Chen, C. Y.; Li, M. Y.; Ma, L. Y.; Zhai, X. Y.; Luo, D. H.; Zhou, Y.; Liu, Z. M.; Cui, Y. M. Precision and Accuracy of Commercial Assays for Vancomycin Therapeutic Drug Monitoring: Evaluation Based on External Quality Assessment Scheme. J. Antimicrob. Chemother. 2020, 75, 2110– 2119, DOI: 10.1093/jac/dkaa1505https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXitlyqtLk%253D&md5=915ae6c327141c60dc2e9b2f55c3db50Precision and accuracy of commercial assays for vancomycin therapeutic drug monitoring: evaluation based on external quality assessment schemeChen, Chao-Yang; Li, Meng-Ya; Ma, Ling-Yun; Zhai, Xing-Yu; Luo, Dao-Huang; Zhou, Ying; Liu, Zhen-Ming; Cui, Yi-MinJournal of Antimicrobial Chemotherapy (2020), 75 (8), 2110-2119CODEN: JACHDX; ISSN:1460-2091. (Oxford University Press)Vancomycin remains a mainstay of the treatment of Gram-pos. bacterial infections. It is crucial to accurately det. vancomycin serum concn. for adequate dose adjustment. To evaluate the precision and accuracy of com. assay techniques for vancomycin concn. and to assess the comparability of vancomycin detection methods in Chinese labs. Human serum samples spiked with known concns. of vancomycin were provided to labs. participating in the external quality assessment scheme (EQAS). Assay methods included chemiluminescence, enzyme immunoassay (EIA) and so on. The dispersion of the measurements was analyzed and the robust coeff. of variation (rCV), relative percentage difference (RPD) and satisfactory rate for method groups were calcd. Moreover, performance of the Chinese labs. was assessed. A total of 657 results from 75 labs. were collected, including 84 samples from 10 Chinese labs. The median rCV, median RPD and satisfactory rates classified by methods ranged from 1.85% to 15.87%, -14.75% to 13.34% and 94.59% to 100.00%, resp. Significant differences were seen in precision, between kinetic interaction of microparticles in soln. (KIMS) and other methods, and in accuracy, between enzyme-multiplied immunoassay technique (EMIT), fluorescence polarization immunoassay (FPIA) and other techniques. Vancomycin detection in China mainly depended on the chemiluminescence and EMIT methods, which tended to result in lower measurements. Although almost all assays in this study achieved an acceptable performance for vancomycin serum concn. monitoring, obvious inconsistencies between methods were still obsd. Chinese labs. were more likely to underestimate vancomycin concns. Thus, recognizing inconsistencies between methods and regular participation in vancomycin EQAS are essential.
- 6Dauphin-Ducharme, P.; Zhang, Y.; Gerson, J.; Kurnik, M.; Kippin, T. E.; Stojanovic, M. N. 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.9b016166https://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.
- 7Li, H.; Dauphin-ducharme, P.; Ortega, G.; Plaxco, K. W. Calibration-Free Electrochemical Biosensors Supporting Accurate Molecular Measurements Directly in Undiluted Whole Blood. J. Am. Chem. Soc. 2017, 139, 11207– 11213, DOI: 10.1021/jacs.7b054127https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhtFOmtLrL&md5=ab88a3fba1bac8fdc60d009b5af2a601Calibration-Free Electrochemical Biosensors Supporting Accurate Molecular Measurements Directly in Undiluted Whole BloodLi, Hui; Dauphin-Ducharme, Philippe; Ortega, Gabriel; Plaxco, Kevin W.Journal of the American Chemical Society (2017), 139 (32), 11207-11213CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)The need to calibrate to correct for sensor-to-sensor fabrication variation and sensor drift has proven a significant hurdle in the widespread use of biosensors. To maintain clin. relevant (±20% for this application) accuracy, for example, com. continuous glucose monitors require recalibration several times a day, decreasing convenience and increasing the chance of user errors. Here, however, the authors demonstrate a "dual-frequency" approach for achieving the calibration-free operation of electrochem. biosensors that generate an output by using square-wave voltammetry to monitor binding-induced changes in electron transfer kinetics. Specifically, the authors use the square-wave frequency dependence of their response to produce a ratiometric signal, the ratio of peak currents collected at responsive and non- (or low) responsive square-wave frequencies, which is largely insensitive to drift and sensor-to-sensor fabrication variations. Using electrochem. aptamer-based (E-AB) biosensors as the test bed, the authors demonstrate the accurate and precise operation of sensors against multiple drugs, achieving accuracy in the measurement of their targets of within better than 20% across dynamic ranges of up to 2 orders of magnitude without the need to calibrate each individual sensor.
- 8Arroyo-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.16134581148https://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.
- 9Parolo, 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.0c010859https://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.
- 10Idili, A.; Gerson, J.; Parolo, C.; Kippin, T.; Plaxco, K. W. An Electrochemical Aptamer-Based Sensor for the Rapid and Convenient Measurement of l-Tryptophan. Anal. Bioanal. Chem. 2019, 411, 4629– 4635, DOI: 10.1007/s00216-019-01645-010https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXms1ansL0%253D&md5=479436452e3a26dd45ad0a08a81e5ebfAn electrochemical aptamer-based sensor for the rapid and convenient measurement of L-tryptophanIdili, Andrea; Gerson, Julian; Parolo, Claudio; Kippin, Tod; Plaxco, Kevin W.Analytical and Bioanalytical Chemistry (2019), 411 (19), 4629-4635CODEN: ABCNBP; ISSN:1618-2642. (Springer)The field of precision medicine-the possibility to accurately tailor pharmacol. treatments to each specific patient-would be significantly advanced by the ability to rapidly, conveniently, and cost-effectively measure biomarkers directly at the point of care. Electrochem. aptamer-based (E-AB) sensors appear a promising approach to this end due to their low cost, ease of use, and good anal. performance in complex clin. samples. Thus motivated, we present here the development of an E-AB sensor for the measurement of the amino acid L-tryptophan, a diagnostic marker indicative of a no. of metabolic and mental health disorders, in urine. The sensor employs a previously reported DNA aptamer able to recognize the complex formed between tryptophan and a rhodium-based receptor. We adopted the aptamer to the E-AB sensing platform by truncating it, causing it to undergo a binding-induced conformational change, modifying it with a redox-reporting methylene blue, and attaching it to an interrogating electrode. The resulting sensor is able to measure tryptophan concns. in the micromolar range in minutes and readily discriminates between its target and other arom. and non-arom. amino acids. Using it, we demonstrate the measurement of clin. relevant tryptophan levels in synthetic urine in a process requiring only a single diln. step. The speed and convenience with which this is achieved suggest that the E-AB platform could significantly improve the ease and frequency with which metabolic diseases are monitored. [Figure not available: see fulltext.].
- 11Zaric, R. Z.; Milovanovic, J.; Rosic, N.; Milovanovic, D.; Zecevic, D. R.; Folic, M.; Jankovic, S. Pharmacokinetics of Vancomycin in Patients with Different Renal Function Levels. Open Med. 2018, 13, 512– 519, DOI: 10.1515/med-2018-006811https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXitVCisLzF&md5=97dc74ebbace1349b387d8d95d5a0a0bPharmacokinetics of vancomycin in patients with different renal function levelsZaric, Radica Zivkovic; Milovanovic, Jasmina; Rosic, Nikola; Milovanovic, Dragan; Zecevic, Dejana Ruzic; Folic, Marko; Jankovic, SlobodanOpen Medicine (2018), 13 (1), 512-519CODEN: OMPEAL; ISSN:2391-5463. (De Gruyter Open Ltd.)There are many determinants of vancomycin clearance, but these have not been analyzed sep. in populations with different levels of renal function, which could be why some important factors have been missed. The aim of our study was to compare the pharmacokinetic parameters and factors that may affect vancomycin pharmacokinetics in groups of patients with normal renal function and in those with chronic kidney failure. The study used a population pharmacokinetic modeling approach, based on plasma vancomycin concns. and other data from 78 patients with chronic kidney failure and 32 patients with normal renal function. The model was developed using NONMEM software and validated by bootstrapping. The final model for patients with impaired kidney function was described by the following equation: CL (L/h) = 0.284 + 0.000596 x DD + 0.00194 x AST, and that for the patients with normal kidney function by: CL (L/h) = 0.0727 + 0.205 x FIB. If our results are confirmed by new studies on two similar populations, these factors could be considered when dosing vancomycin in patients with chronically damaged kidneys, as well as in patients with normal kidneys who frequently require high doses of vancomycin.
- 12Suzuki, Y.; Kawasaki, K.; Sato, Y.; Tokimatsu, I.; Itoh, H.; Hiramatsu, K.; Takeyama, M.; Kadota, J. I. Is Peak Concentration Needed in Therapeutic Drug Monitoring of Vancomycin? A Pharmacokinetic-Pharmacodynamic Analysis in Patients with Methicillin- Resistant Staphylococcus Aureus Pneumonia. Chemotherapy 2012, 58, 308– 312, DOI: 10.1159/00034316212https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XhslyjsLrE&md5=e10a1ad993f3e4642c02b3699e7bd0bdIs Peak Concentration Needed in Therapeutic Drug Monitoring of Vancomycin? A Pharmacokinetic-Pharmacodynamic Analysis in Patients with Methicillin-Resistant Staphylococcus aureus PneumoniaSuzuki, Yosuke; Kawasaki, Kanako; Sato, Yuhki; Tokimatsu, Issei; Itoh, Hiroki; Hiramatsu, Kazufumi; Takeyama, Masaharu; Kadota, Jun-ichiChemotherapy (Basel, Switzerland) (2012), 58 (4), 308-312CODEN: CHTHBK; ISSN:0009-3157. (S. Karger AG)Background: We analyzed the pharmacokinetic-pharmacodynamic relationship of vancomycin to det. the drug exposure parameters that correlate with the efficacy and nephrotoxicity of vancomycin in patients with methicillin-resistant Staphylococcus aureus pneumonia and evaluated the need to use peak concn. in therapeutic drug monitoring (TDM). Methods: Serum drug concns. of 31 hospitalized patients treated with vancomycin for methicillin-resistant S. aureus pneumonia were collected. Results: Significant differences in trough concn. (Cmin)/min. inhibitory concn. (MIC) and area under the serum concn.-time curve (AUC0-24)/MIC were obsd. between the response and non-response groups. Significant differences in Cmin and AUC0-24 were obsd. between the nephrotoxicity and non-nephrotoxicity groups. Receiver operating characteristic curves revealed high predictive values of Cmin/MIC and AUC0-24/MIC for efficacy and of Cmin and AUC0-24 for safety of vancomycin. Conclusions: These results suggest little need to use peak concn. in vancomycin TDM because Cmin/MIC and Cmin are sufficient to predict the efficacy and safety of vancomycin.
- 13Turnbull, W. B.; Daranas, A. H. On the Value of c: Can Low Affiniity Systems Be Studied by Isothermal Titration Calorimetry?. J. Am. Chem. Soc. 2003, 125, 14859– 14866, DOI: 10.1002/asi.463031041713https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3sXovVSiu7w%253D&md5=088d351b0836433e55b46e16189271e7On the Value of c: Can Low Affinity Systems Be Studied by Isothermal Titration Calorimetry?Turnbull, W. Bruce; Daranas, Antonio H.Journal of the American Chemical Society (2003), 125 (48), 14859-14866CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Isothermal titrn. calorimetry (ITC) allows the detn. of ΔG°, ΔH°, and ΔS° from a single expt. and is thus widely used for studying binding thermodn. in both biol. and synthetic supramol. systems. However, it is widely believed that it is not possible to derive accurate thermodn. information from ITC expts. in which the Wiseman "c" parameter (which is the product of the receptor concn. and the binding const., Ka) is less than ca. 10, constraining its use to high affinity systems. Herein, exptl. titrns. and simulated data are used to demonstrate that this dogma is false, esp. for low affinity systems, assuming that (1) a sufficient portion of the binding isotherm is used for anal., (2) the binding stoichiometry is known, (3) the concns. of both ligand and receptor are known with accuracy, and (4) there is an adequate level of signal-to-noise in the data. This study supports the validity of ITC for detg. the value of Ka and, hence, ΔG° from expts. conducted under low c conditions but advocates greater caution in the interpretation of values for ΔH°. Therefore, isothermal titrn. calorimetry is a valid and useful technique for studying biol. and synthetically important low affinity systems.
- 14Zadeh, J. N.; Steenberg, C. D.; Bois, J. S.; Wolfe, B. R.; Pierce, M. B.; Khan, A. R.; Dirks, R. M.; Pierce, N. A. NUPACK: Analysis and Design of Nucleic Acid Systems. J. Comput. Chem. 2011, 32, 170– 173, DOI: 10.1002/jcc.2159614https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXhsVCjsLzP&md5=9c60f0134ea55d9335301e2efe1602e8NUPACK: analysis and design of nucleic acid systemsZadeh, Joseph N.; Steenberg, Conrad D.; Bois, Justin S.; Wolfe, Brian R.; Pierce, Marshall B.; Khan, Asif R.; Dirks, Robert M.; Pierce, Niles A.Journal of Computational Chemistry (2011), 32 (1), 170-173CODEN: JCCHDD; ISSN:0192-8651. (John Wiley & Sons, Inc.)The Nucleic Acid Package (NUPACK) is a growing software suite for the anal. and design of nucleic acid systems. The NUPACK web server (http://www.nupack.org) currently enables: NUPACK algorithms are formulated in terms of nucleic acid secondary structure. In most cases, pseudoknots are excluded from the structural ensemble. © 2010 Wiley Periodicals, Inc. J Comput Chem, 2010.
- 15Chamorro-Garcia, A.; Ortega, G.; Mariottini, D.; Green, J.; Ricci, F.; Plaxco, K. W. Switching the Aptamer Attachment Geometry Can Dramatically Alter the Signalling and Performance of Electrochemical Aptamer-Based Sensors. Chem. Commun. 2021, 57, 11693– 11696, DOI: 10.1039/d1cc04557a15https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXit1Wku77E&md5=e72fd6ba0bb829251dcf92478b487837Switching the aptamer attachment geometry can dramatically alter the signalling and performance of electrochemical aptamer-based sensorsChamorro-Garcia, Alejandro; Ortega, Gabriel; Mariottini, Davide; Green, Joshua; Ricci, Francesco; Plaxco, Kevin W.Chemical Communications (Cambridge, United Kingdom) (2021), 57 (88), 11693-11696CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)Electrochem. aptamer-based (EAB) sensors, composed of an electrode-bound DNA aptamer with a redox reporter on the distal end, offer the promise of high-frequency, real-time mol. measurements in complex sample matrixes and even in vivo. Here we assess the extent to which switching the aptamer terminus that is electrode-bound and the one that is redox-reporter-modified affects the performance of these sensors. Using sensors against doxorubicin, cocaine, and vancomycin as our test beds, we find that both signal gain (the relative signal change seen in the presence of a satg. target) and the frequency dependence of gain depend strongly on the attachment orientation, suggesting that this easily investigated variable is a worthwhile parameter to optimize in the design of new EAB sensors.
- 16Shaver, 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.9b2238516https://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.
- 17White, 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/la800801v17https://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.
- 18Liu, Y.; Canoura, J.; Alkhamis, O.; Xiao, Y. Immobilization Strategies for Enhancing Sensitivity of Electrochemical Aptamer-Based Sensors. ACS Appl. Mater. Interfaces 2021, 13, 9491– 9499, DOI: 10.1021/acsami.0c2070718https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXht1Wjtrw%253D&md5=ba2749b65de53c20271e7e034b2cdabbImmobilization Strategies for Enhancing Sensitivity of Electrochemical Aptamer-Based SensorsLiu, Yingzhu; Canoura, Juan; Alkhamis, Obtin; Xiao, YiACS Applied Materials & Interfaces (2021), 13 (8), 9491-9499CODEN: AAMICK; ISSN:1944-8244. (American Chemical Society)Electrochem. aptamer-based (E-AB) sensors are a versatile sensing platform that can achieve rapid and robust target detection in complex matrixes. However, the limited sensitivity of these sensors has impeded their translation from proof-of-concept to com. products. Surface-bound aptamers must be sufficiently spaced to bind targets and subsequently fold for signal transduction. We hypothesized that electrodes fabricated using conventional methods result in sensing surfaces where only a fraction of aptamers are appropriately spaced to actively respond to the target. As an alternative, we presented a novel aptamer immobilization approach that favors sufficient spacing between aptamers at the microscale to achieve optimal target binding, folding, and signal transduction. We first demonstrated that immobilizing aptamers in their target-bound, folded state on gold electrode surfaces yields an aptamer monolayer that supports greater sensitivity and higher signal-to-noise ratio than traditionally prepd. E-AB sensors. We also showed that performing aptamer immobilization under low ionic strength conditions rather than conventional high ionic strength buffer greatly improves E-AB sensor performance. We successfully tested our approach with three different small-mol.-binding aptamers, demonstrating its generalizability. On the basis of these results, we believe our electrode fabrication approach will accelerate development of high-performance sensors with the sensitivity required for real-world anal. applications.
- 19Wu, Y.; Tehrani, F.; Teymourian, H.; Mack, J.; Shaver, A.; Reynoso, M.; Kavner, J.; Huang, N.; Furmidge, A.; Duvvuri, A. Microneedle Aptamer-Based Sensors for Continuous, Real-Time Therapeutic Drug Monitoring. Anal. Chem. 2022, 94, 8335– 8345, DOI: 10.1021/acs.analchem.2c0082919https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38XhsVGnt7rF&md5=7ec79273b3414d7e179173460bdc565cMicroneedle Aptamer-Based Sensors for Continuous, Real-Time Therapeutic Drug MonitoringWu, Yao; Tehrani, Farshad; Teymourian, Hazhir; Mack, John; Shaver, Alexander; Reynoso, Maria; Kavner, Jonathan; Huang, Nickey; Furmidge, Allison; Duvvuri, Andres; Nie, Yuhang; Laffel, Lori M.; Doyle, Francis J.; Patti, Mary-Elizabeth; Dassau, Eyal; Wang, Joseph; Arroyo-Curras, NetzahualcoyotlAnalytical Chemistry (Washington, DC, United States) (2022), 94 (23), 8335-8345CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)The ability to continuously monitor the concn. of specific mols. in the body is a long-sought goal of biomedical research. For this purpose, interstitial fluid (ISF) was proposed as the ideal target biofluid because its compn. can rapidly equilibrate with that of systemic blood, allowing the assessment of mol. concns. that reflect full-body physiol. In the past, continuous monitoring in ISF was enabled by microneedle sensor arrays. Yet, benchmark microneedle sensors can only detect mols. that undergo redox reactions, which limits the ability to sense metabolites, biomarkers, and therapeutics that are not redox-active. To overcome this barrier, here, we expand the scope of these devices by demonstrating the first use of microneedle-supported electrochem., aptamer-based (E-AB) sensors. This platform achieves mol. recognition based on affinity interactions, vastly expanding the scope of mols. that can be sensed. We report the fabrication of microneedle E-AB sensor arrays and a method to regenerate them for multiple uses. In addn., we demonstrate continuous mol. measurements using these sensors in flow systems in vitro using single and multiplexed microneedle array configurations. Translation of the platform to in vivo measurements is possible as we demonstrate with a first E-AB measurement in the ISF of a rodent. The encouraging results reported in this work should serve as the basis for future translation of microneedle E-AB sensor arrays to biomedical research in preclin. animal models.
- 20Arroyo-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.7b0078720https://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.
- 21Curtis, 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.9b0255321https://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.
- 22Miura, Y.; Fuchigami, Y.; Nomura, S.; Nishimura, K.; Hagimori, M.; Kawakami, S. Brain Microdialysis Study of Vancomycin in the Cerebrospinal Fluid After Intracerebroventricular Administration in Mice. AAPS PharmSciTech 2019, 20, 5 DOI: 10.1208/s12249-018-1232-8There is no corresponding record for this reference.
- 23Nau, R.; Sörgel, F.; Eiffert, H. Penetration of Drugs through the Blood-Cerebrospinal Fluid/Blood-Brain Barrier for Treatment of Central Nervous System Infections. Clin. Microbiol. Rev. 2010, 23, 858– 883, DOI: 10.1128/CMR.00007-1023https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXhtlOntbg%253D&md5=470fffb97c0373a2dccafe3dbd021345Penetration of drugs through the blood-cerebrospinal fluid/blood-brain barrier for treatment of central nervous system infectionsNau, Roland; Soergel, Fritz; Eiffert, HelmutClinical Microbiology Reviews (2010), 23 (4), 858-883CODEN: CMIREX; ISSN:0893-8512. (American Society for Microbiology)A review. Central nervous system (CNS) infections caused by pathogens with a reduced sensitivity to drugs are a therapeutic challenge. This is particularly true for infections caused by penicillin- resistant pneumococci, methicillin-resistant staphylococci, multiresistant Gram-neg. aerobic bacilli, or several other organisms (including Aspergillus spp., Scedosporium apiospermum, and Nocardia asteroides) that affect primarily the CNS in immunocompromised patients. This review aims to increase the awareness of the peculiarities of the pharmacokinetics of anti-infectives within the CNS.
- 24Meunier, C. J.; Sombers, L. A. Fast-Scan Voltammetry for In Vivo Measurements of Neurochemical Dynamics. In The Brain Reward System; Springer, 2021.There is no corresponding record for this reference.
- 25Arroyo-Currás, N.; Scida, K.; Ploense, K. L.; Kippin, T. E.; Plaxco, K. W. High Surface Area Electrodes Generated via Electrochemical Roughening Improve the Signaling of Electrochemical Aptamer-Based Biosensors. Anal. Chem. 2017, 89, 12185– 12191, DOI: 10.1021/acs.analchem.7b0283025https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhslagsr%252FO&md5=c40a96ca49d03b35bb7eaceb0ab47462High Surface Area Electrodes Generated via Electrochemical Roughening Improve the Signaling of Electrochemical Aptamer-Based BiosensorsArroyo-Curras, Netzahualcoyotl; Scida, Karen; Ploense, Kyle L.; Kippin, Tod E.; Plaxco, Kevin W.Analytical Chemistry (Washington, DC, United States) (2017), 89 (22), 12185-12191CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)The electrochem., aptamer-based (E-AB) sensor platform provides a modular approach to the continuous, real-time measurement of specific mol. targets (irresp. of their chem. reactivity) in situ in the living body. To achieve this, however, requires the fabrication of sensors small enough to insert into a vein, which, for the rat animal model the authors employ, entails devices less than 200 μm in diam. The limited surface area of these small devices leads, in turn, to low faradaic currents and poor signal-to-noise ratios when deployed in the complex, fluctuating environments found in vivo. In response the authors have developed an electrochem. roughening approach that enhances the signaling of small electrochem. sensors by increasing the microscopic surface area of gold electrodes, allowing in this case more redox-reporter-modified aptamers to be packed onto the surface, thus producing significantly improved signal-to-noise ratios. Unlike previous approaches to achieving microscopically rough gold surfaces, the method employs chronoamperometric pulsing in a 5 min etching process easily compatible with batch manufg. Using these high surface area electrodes, the authors demonstrate the ability of E-AB sensors to measure complete drug pharmacokinetic profiles in live rats with precision of better than 10% in the detn. of drug disposition parameters.
- 26Franklin, K.; Paxinos, G. The Mouse Brain in Stereotaxic Coordinates, 3rd ed.; Elsevier Academic Press: San Diego, 2007.There is no corresponding record for this reference.
Supporting Information
Supporting Information
The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acssensors.2c01910.
Aptamer variant sequences, measured ITC parameters, and Nupack-predicted secondary structures (Table S1)
and additional ITC, CD, and electrochemical measurements (Figures S1–S7) (PDF)
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