Electrochemical Aptamer-based Research Articles

Single Voltammetric Sweep Calibration-Free Interrogation of Electrochemical Aptamer-Based Sensors Employing Continuous Square Wave Voltammetry.

Continuous square wave voltammetry (cSWV) is a technique that enables the continuous collection of current data (at 100 kHz) to maximize the information content obtainable from a single voltammetric sweep. This data collection procedure results in the generation of multiple voltammograms corresponding to different effective square wave frequencies. The application of cSWV brings significant benefits to electrochemical aptamer-based (E-AB) sensors. The E-AB sensor platform permits continuous real-time monitoring of small biological molecules. Traditionally, E-AB sensors report only on changes in analyte concentration rather than absolute quantification in matrices when basal concentrations are not known a priori. This is because they exhibit a voltammetric peak current even in the absence of a target. However, using a dual-frequency approach, calibration-free sensing can be performed effectively, eliminating the sensor-to-sensor variation by taking ratiometric current responses obtained at two different frequencies from two different voltammetric sweeps. In employing our approach, cSWV provides a great advantage over the conventionally used square wave voltammetry since the required voltammograms are collected with a single sweep, which improves the temporal resolution of the measurement when considering the current at multiple frequencies for improved accuracy and reduced surface interrogation. Moreover, we show here that using cSWV provides significantly improved concentration predictions. E-AB sensors sensitive to ATP and tobramycin were interrogated across a wide range of concentrations. With this approach, cSWV allowed us to estimate the target concentration, retaining up to an ±5% error of the expected concentration when tested in buffer and complex media.

Codeposition Enhances the Performance of Electrochemical Aptamer-Based Sensors.

Electrochemical aptamer-based (EAB) sensors, a minimally invasive means of performing high-frequency, real-time measurement of drugs and biomarkers in situ in the body, have traditionally been fabricated by depositing their target-recognizing aptamer onto an interrogating gold electrode using a "sequential" two-step method involving deposition of the thiol-modified oligonucleotide (typically for 1 h) followed by incubation in mercaptohexanol solution (typically overnight) to complete the formation of a stable, self-assembled monolayer. Here we use EAB sensors targeting vancomycin, tryptophan, and phenylalanine to show that "codeposition", a less commonly employed EAB fabrication method in which the thiol-modified aptamer and the mercaptohexanol diluent are deposited on the electrode simultaneously and for as little as 1 h, improves the signal gain (relative change in signal upon the addition of high concentrations of the target) of the vancomycin and tryptophan sensors without significantly reducing their stability. In contrast, the gain of the phenylalanine sensor is effectively identical irrespective of the fabrication approach employed. This sensor, however, appears to employ binding-induced displacement of the redox reporter rather than binding-induced folding as its signal transduction mechanism, suggesting in turn a mechanism for the improvement observed for the other two sensors. Codeposition thus not only provides a more convenient means of fabricating EAB sensors but also can improve their performance.

Chronoamperometric interrogation of an electrochemical aptamer-based sensor with tetrahedral DNA nanostructure pendulums for continuous biomarker measurements

Tetrahedral DNA nanostructure (TDN) is highly promising in developing electrochemical aptamer-based (E-AB) sensors for biomolecular detection, owing to its inherit programmability, spatial orientation and structural robustness. However, current interrogation strategies applied for TDN-based E-AB sensors, including enzyme-based amperometry, voltammetry, and electrochemical impedance spectroscopy, either require complicated probe design or suffer from limited applicability or selectivity. In this study, a TDN pendulum-empowered E-AB sensor interrogated by chronoamperometry for reagent-free and continuous monitoring of a blood clotting enzyme, thrombin, was developed. TDN pendulums with extended aptamer sequences at three vertices were immobilized on a gold electrode via a thiolated double-stranded DNA (dsDNA) at the fourth vertex, and their motion is modulated by the bonding of target thrombin to aptamers. We observed a significantly amplified signalling output on our sensor based on the TDN pendulum compared to E-AB sensors modified with linear pendulums. Moreover, our sensor achieved highly selective and rapidly responsive measurement of thrombin in both PBS and artificial urine, with a wide dynamic range from 1 pM to 10 nM. This study shows chronoamperometry-enabled continuous biomarker monitoring on a sub-second timescale with a drift-free baseline, demonstrating a novel approach to accurately detect molecular dynamics in real time.

Controlling Gold Morphology Using Electrodeposition for the Preparation of Electrochemical Aptamer-Based Sensors.

Nanostructuring of gold surfaces to enhance electroactive surface area has proven to significantly enhance the performance of electrochemical aptamer-based (E-AB) sensors, particularly for electrodes on the microscale. Unlike for sensors fabricated on polished gold surfaces, predicting the behavior of E-AB sensors on surfaces with varied gold morphologies becomes more intricate due to the effects of surface roughness and the shapes and sizes of surface features on supporting a self-assembled monolayer. In this study, we explored the impact of gold morphology characteristics on sensor performance, evaluating parameters such as signal change in response to the addition of the target analyte, aptamer probe packing density, and continuous sensing ability. Our findings reveal that surface area enhancement can either enhance or diminish sensor performance for gold nanostructured E-AB sensors, contingent upon the surface morphology. In particular, our results indicate that the aptamer packing density and target analyte signal change results are heavily dependent on gold nanostructure size and features. Sensing surfaces with larger nanoparticle diameters, which were prepared using electrodeposition at a constant potential, had a reduced aptamer packing density and exhibited diminished sensor performance. However, the equivalent packing density of polished electrodes did not yield the equivalent signal change. Other surfaces that were prepared using pulsed waveform electrodeposition achieved optimal signal change with a deposition time, tdep, of 120 s, and increased deposition time with enhanced electroactive surface area resulted in minimized signal changes and more rapid sensor degradation. By investigating sensing surfaces with varied morphologies, we have demonstrated that enhancing the electroactive surface does not always enhance the signal change of the sensor, and aptamer packing density alone does not dictate observed signal change trends. We anticipate that understanding how electrodeposition techniques enhance or diminish sensor performance will pave the way for further exploration of nanostructure-aptamer relationships, contributing to the future development of optimized, miniaturized electrochemical aptamer-based sensors for continuous, in vivo sensing.

Comparison of voltammetric methods used in the interrogation of electrochemical aptamer-based sensors

Electrochemical aptamer-based sensors support real-time, in vivo molecular measurements. Here we compare the relative merits of square-wave, alternating current, and differential pulse voltammetry in the interrogation of such sensors.

Selection, characterization, and biosensing applications of DNA aptamers targeting cyanotoxin BMAA.

Scientists have established a connection between environmental exposure to toxins like β-N-methylamino-l-alanine (BMAA) and a heightened risk of neurodegenerative disorders. BMAA is a byproduct from certain strains of cyanobacteria that are present in ecosystems worldwide and is renowned for its bioaccumulation and biomagnification in seafood. The sensitivity, selectivity, and reproducibility of the current analytical techniques are insufficient to support efforts regarding food safety and environment monitoring adequately. This work outlines the in vitro selection of BMAA-specific DNA aptamers via the systematic evolution of ligands through exponential enrichment (SELEX). Screening and characterization of the full-length aptamers was achieved using the SYBR Green (SG) fluorescence displacement assay. Aptamers BMAA_159 and BMAA_165 showed the highest binding affinities, with dissociation constants (Kd) of 2.2 ± 0.1 μM and 0.32 ± 0.02 μM, respectively. After truncation, the binding affinity was confirmed using a BMAA-conjugated fluorescence assay. The Kd values for BMAA_159_min and BMAA_165_min were 6 ± 1 μM and 0.63 ± 0.02 μM, respectively. Alterations in the amino proton region studied using solution nuclear magnetic resonance (NMR) provided further evidence of aptamer-target binding. Additionally, circular dichroism (CD) spectroscopy revealed that BMAA_165_min forms hybrid G-quadruplex (G4) structures. Finally, BMAA_165_min was used in the development of an electrochemical aptamer-based (EAB) sensor that accomplished sensitive and selective detection of BMAA with a limit of detection (LOD) of 1.13 ± 0.02 pM.

Can Electrochemical Aptasensors Achieve the Commercial Success of Glucose Biosensors?

AbstractEnzymes and antibodies are widely available biorecognition elements in bioanalytical tools such as personal glucose monitoring (PGM) devices and lateral flow assays (LFA). Meanwhile, electrochemical aptamer‐based (EAB) sensors are promising affinity‐based bioanalytical tools with potential advantages over such conventional bioassays. However, several critical factors affect the stability of EAB sensors, pivotal for their commercialization including 1) electrode defects due to surface treatment methods, 2) hampering effects of redox molecules, 3) electrical potential‐induced aptamer detachment, 4) thermal‐induced monolayer solubilization, 5) biochemical/enzymatic degradation, 6) biofouling, and 7) inadequate statistical design and analysis in EAB sensor fabrication. Herein, antidotes for the obstacles are proposed by applying novel surface treatment methods, adapting redox molecule, tuning electrochemical tests, tweaking backfilling agents, and anti‐bio‐fouling coatings. Nonetheless, the obstacles are a driving force to clear pathways toward bringing EAB sensors to the market for therapeutic drug and metabolite monitoring, point of care sensors, macromolecule detection, and pathogen diagnostics.

(Invited) Continuous Molecular Monitoring in Interstitial Fluid Via Wearable Microneedle Aptamer-Based Sensors

The ability to continuously monitor the concentration of specific molecules in the body is a long-sought goal of biomedical research. For this purpose, interstitial fluid (ISF) has been proposed as the ideal target biofluid because its composition can rapidly equilibrate with that of systemic blood, allowing the assessment of molecular concentrations that reflect full-body physiology. To tap into this measurement compartment, it is possible to use microneedle sensor arrays for continuous molecular monitoring. In this presentation, I will discuss our efforts to develop microneedle-supported electrochemical, aptamer-based (E-AB) sensors. This platform allows affinity-based monitoring of target concentrations in ISF, and takes advantage of the versatility of aptamers to selectively bind a large variety of molecular targets. I will discuss our current state of platform development, including fabrication challenges, in-vivo deployment in rodent models, and variability of pharmacokineitc profiles as a function of microneedle penetration depth in the skin.

A Guide for the Electrochemical Stability of Thiolate Self-Assembled Monolayers on Gold in Various Electrolytes

Self-assembled monolayers (SAMs) are widely used in surface chemistry to modify surfaces for various applications such as biosensing1, electronics2, and electrocatalysis3. SAMs are attractive due to their high surface coverage and stability. Although studies have been conducted to test the effects of temperature, air exposure, and solvent degradation on SAMs stability 4,5, the electrochemical stability of SAMs has not been as extensively studied. Existing literature on this topic mainly focuses on the reductive desorption limits of thiolate SAMs in alkaline conditions6,7. Recent work by Ramos et al. demonstrated pH-dependent stable potential windows in which SAMs remained intact on electrode surfaces of different metals8. However, there is still a need to verify and expand on this work.To address this need, we aim to create a comprehensive guide on the electrochemical stability of thiolate SAMs on gold electrodes. We will use cyclic voltammograms (CV), electrochemical impedance spectroscopy (EIS), and electrochemical surface plasmon resonance (EC-SPR) to probe and verify the stable electrochemical potential windows in both aqueous and non-aqueous electrolytes. We have conducted studies on three aqueous solutions at three distinct pH, perchloric acid, potassium hydroxide, and sodium chloride. For non-aqueous solvents we tested a wide range of different organic solvents including acetonitrile, dimethylformamide, propylene carbonate, and various alcohols. Our findings demonstrate that the main factor for SAM stability in aqueous and non-aqueous electrolytes is the pH and solubility of the SAM, respectively. Our poster will present our current work and outlook on this topic.(1) Dauphin-Ducharme, P.; Ploense, K. L.; Arroyo-Currás, N.; Kippin, T. E.; Plaxco, K. W. Electrochemical Aptamer-Based Sensors: A Platform Approach to High-Frequency Molecular Monitoring In Situ in the Living Body. In Biomedical Engineering Technologies: Volume 1; Ossandon, M. R., Baker, H., Rasooly, A., Eds.; Methods in Molecular Biology; Springer US: New York, NY, 2022; pp 479–492. https://doi.org/10.1007/978-1-0716-1803-5_25.(2) Hatton, R. A.; Willis, M. R.; Chesters, M. A.; Rutten, F. J. M.; Briggs, D. Enhanced Hole Injection in Organic Light-Emitting Diodes Using a SAM-Derivatised Ultra-Thin Gold Anode Supported on ITO Glass. J. Mater. Chem. 2003, 13 (1), 38–43. https://doi.org/10.1039/B208169P.(3) Shang, H.; Wallentine, S. K.; Hofmann, D. M.; Zhu, Q.; Murphy, C. J.; Baker, L. R. Effect of Surface Ligands on Gold Nanocatalysts for CO2 Reduction. Chem. Sci. 2020, 11 (45), 12298–12306. https://doi.org/10.1039/D0SC05089J.(4) Schlenoff, J. B.; Li, M.; Ly, H. Stability and Self-Exchange in Alkanethiol Monolayers. J. Am. Chem. Soc. 1995, 117 (50), 12528–12536. https://doi.org/10.1021/ja00155a016.(5) Laibinis, P. E.; Whitesides, G. M. Self-Assembled Monolayers of n-Alkanethiolates on Copper Are Barrier Films That Protect the Metal against Oxidation by Air. J. Am. Chem. Soc. 1992, 114 (23), 9022–9028. https://doi.org/10.1021/ja00049a038.(6) Salvarezza, R. C.; Carro, P. The Electrochemical Stability of Thiols on Gold Surfaces. J. Electroanal. Chem. 2018, 819, 234–239. https://doi.org/10.1016/j.jelechem.2017.10.046.(7) Beulen, M. W. J.; Kastenberg, M. I.; van Veggel, F. C. J. M.; Reinhoudt, D. N. Electrochemical Stability of Self-Assembled Monolayers on Gold. Langmuir 1998, 14 (26), 7463–7467. https://doi.org/10.1021/la981031z.(8) Ramos, N.; Medlin, J. W.; Holewinski, A. Electrochemical Stability of Thiolate Self-Assembled Monolayers on Au, Pt, and Cu; preprint; Chemistry, 2022. https://doi.org/10.26434/chemrxiv-2022-dt7dk. Figure 1

Analytical Validation of Aptamer-Based Serum Vancomycin Monitoring Relative to Automated Immunoassays.

The practice of monitoring therapeutic drug concentrations in patient biofluids can significantly improve clinical outcomes while simultaneously minimizing adverse side effects. A model example of this practice is vancomycin dosing in intensive care units. If dosed correctly, vancomycin can effectively treat methicillin-resistant streptococcus aureus (MRSA) infections. However, it can also induce nephrotoxicity or fail to kill the bacteria if dosed too high or too low, respectively. Although undeniably important to achieve effectiveness, therapeutic drug monitoring remains inconvenient in practice due primarily to the lengthy process of sample collection, transport to a centralized facility, and analysis using costly instrumentation. Adding to this workflow is the possibility of backlogs at centralized clinical laboratories, which is not uncommon and may result in additional delays between biofluid sampling and concentration measurement, which can negatively affect clinical outcomes. Here, we explore the possibility of using point-of-care electrochemical aptamer-based (E-AB) sensors to minimize the time delay between biofluid sampling and drug measurement. Specifically, we conducted a clinical agreement study comparing the measurement outcomes of E-AB sensors to the benchmark automated competitive immunoassays for vancomycin monitoring in serum. Our results demonstrate that E-ABs are selective for free vancomycin─the active form of the drug, over total vancomycin. In contrast, competitive immunoassays measure total vancomycin, including both protein-bound and free drug. Accounting for these differences in a pilot study consisting of 85 clinical samples, we demonstrate that the E-AB vancomycin measurement achieved a 95% positive correlation rate with the benchmark immunoassays. Therefore, we conclude that E-AB sensors could provide clinically useful stratification of patient samples at trough sampling to guide effective vancomycin dose recommendations.

Enhancing the sensitivity and stability of electrochemical aptamer-based sensors by AuNPs@MXene nanocomposite for continuous monitoring of biomarkers

Electrochemical aptamer-based (E-AB) sensors offer exciting potential for real-time tracking of various biomarkers, such as proteins and small molecules, due to their exceptional selectivity and adaptability. However, most E-AB sensors rely on planar gold structures, which inherently limit their sensitivity and operational stability for continuous monitoring of biomarkers. Although gold nanostructures have recently enhanced E-AB sensor performance, no studies have explored the combination of gold nanostructure with other types of nanomaterials for continuous molecular monitoring. To fill this gap, we employed gold nanoparticles and MXene Ti3C2 (AuNPs@MXene), a versatile nanocomposite, in designing an E-AB sensor targeted at vascular endothelial growth factor (VEGF), a crucial human signaling protein. Remarkably, the AuNPs@MXene nanocomposite achieved over thirty-fold and half-fold increases in active surface area compared to bare and AuNPs-modified gold electrodes, respectively, significantly elevating the analytical capabilities of E-AB sensors during continuous operation. After a systematic optimization and characterization process, the newly developed E-AB sensor, powered by AuNPs@MXene nanocomposite, demonstrated both enhanced stability and heightened sensitivity. Overall, our findings open new avenues for the incorporation of nanocomposites in E-AB sensor design, enabling the creation of more sensitive and durable real-time monitoring systems.

Development of an Electrochemical, Aptamer-Based Sensor for Dynamic Detection of Neuropeptide Y.

The ability to monitor dynamic changes in neuropeptide Y (NPY) levels in complex environments can have an impact on many fields, including neuroscience and immunology. Here, we describe the development of an electrochemical, aptamer-based (E-AB) sensor for the dynamic (reversible) measurement of physiologically relevant (nanomolar) concentrations of neuropeptide Y. The E-AB sensors are fabricated using a previously described 80 nucleotide aptamer1 reported to specifically bind NPY with a binding affinity Kd = 0.3 ± 0.2 uM. We investigated two redox tag placement locations on the aptamer sequence (terminal vs internal) and various sensor fabrication and interrogation parameters to tune the performance of the resulting sensor. The best-performing sensor architecture displayed a physiologically relevant dynamic range (nM) and low limit of detection and is selective among competitors and similar molecules. The development of this sensor accomplishes two breakthroughs: first, the development of a nonmicrofluidic aptamer-based electrochemical sensor that can detect NPY on a physiologically relevant (seconds to minutes) time scale and across a relevant concentration range; second, the expansion of the range of molecules for which an electrochemical, aptamer-based sensor can be used.

Microscale, Electrochemical, Aptamer-Based Sensors for Enhanced Small-Molecule Detection at Millisecond Time Scales.

Microscale electrodes offer the advantages of increased mass transport rates, high sensitivity, and rapid measurement capabilities. Fabricating electrochemical aptamer-based (E-AB) sensors on these electrode platforms opens new applications to chemical and biological sensing but has remained challenging due to low signal-to-noise ratios and monolayer instability. In this article, we report the development and characterization of E-AB sensors on a gold microelectrode platform (∼500 nm radius). To overcome the small current response, we modified the electrodes by growing nanostructures via electrodeposition. We interrogated the sensors with two different electroanalytical techniques, square wave voltammetry (SWV) and intermittent pulse voltammetry (IPA), to measure the representative response of an ATP sensor and determine aptamer-target binding and dissociation time scales. We find robust and stable sensor performance with an increased response rate over sensors fabricated on macroscale electrodes. These results demonstrate that sensors developed on this microelectrode platform can be employed for enhanced spatiotemporal resolution measurements in chemical and biological environments.

B-301 In-vivo Continuous Therapeutic Drug Monitoring With Electrochemical Aptamer-based Sensors

Abstract Therapeutic drug monitoring (TDM) is a clinical practice involving quantification of plasma drug concentrations to adjust treatment efficacy and minimize toxicity in patient care. TDM is relevant for drugs with a narrow therapeutic index, defined as the plasma concentration range over which a molecule is therapeutically effective without causing significant adverse effects. For narrow therapeutic index drugs, indirect predictors of a patient’s metabolism, such as age, body mass, or genotype, are insufficient to ensure effective dosing, and metabolic variability across different individuals needs to be considered. The importance of TDM is illustrated by the difficulty in treating a patient with vancomycin, an essential antibiotic used to combat gram-positive bacterial infections. The practice typically involves determining trough concentration, peak concentration, or exposure to the vancomycin area under the curve, necessitating blood sampling. After sampling, the current practices require patient blood to be sent to a laboratory, which can only provide an answer from hours after sample collection. Electrochemical aptamer-based (E-AB) sensors provide significant opportunities to make TDM a practice as frequent and convenient as the measurement of blood sugar has become for people with diabetes. This is because E-AB sensing is a molecular approach to continuously measuring concentrations of specific molecules. We present the selection of an aptamer against vancomycin and its adaptation into an E-AB sensor. Using this sensor, we then demonstrate the opportunities for rapid (seconds) measurement of plasma vancomycin and the real-time measurement of subject-specific vancomycin pharmacokinetics on rats (Fig. 1) [3]. Such capability provides a route toward improving TDM and the personalized delivery of pharmacological interventions.Fig. 1.Real-time continuous monitoring of subject-specific vancomycin pharmacokinetics with electrochemical aptamer-based sensors. Reprinted with permission from the American Chemical Society.

Implantable Hydrogel-Protective DNA Aptamer-Based Sensor Supports Accurate, Continuous Electrochemical Analysis of Drugs at Multiple Sites in Living Rats.

The ability to track the levels of specific molecules, such as drugs, metabolites, and biomarkers, in the living body, in real time and for long durations, would improve our understanding of health and our ability to diagnose, treat, and monitor disease. To this end, we are developing electrochemical aptamer-based (EAB) biosensors, a general platform supporting high-frequency, real-time molecular measurements in the living body. Here we report that the use of an agarose hydrogel protective layer for EAB sensors significantly improves their signaling stability when deployed in the complex, highly time-varying environments found in vivo. The improved stability is sufficient that these hydrogel-protected sensors achieved good baseline stability and precision when deployed in situ in the veins, muscles, bladder, or tumors of living rats without the use of the drift correction approaches traditionally required in such placements. Finally, our implantable gel-protective EAB sensors achieved good biocompatibility when deployed in vivo in the living rats without causing any severe inflammation.

Dual-Aptamer Drift Canceling Techniques to Improve Long-Term Stability of Real-Time Structure-Switching Aptasensors.

This paper presents a dual-aptamer scheme to mitigate signal drifts caused by structure-switching aptamers during long-term monitoring. Electrochemical aptamer-based (E-AB) biosensors have recently shown great potential for continuous in vivo monitoring. However, the accuracy of detection is often limited by signaling drifts. Traditional approaches rely on kinetic differential measurements (KDM) coupled with square-wave voltammetry to eliminate these drifts. Yet, we have discovered that KDM does not apply universally to all aptamers, as their responses at different SWV frequencies heavily rely on their structure-switching characteristics and the electron transfer (ET) kinetics of the redox reporters. In light of this, we propose a "dual-aptamer" scheme that utilizes two aptamers, each responding differently to the same target molecule to cancel out drift. These paired aptamers are identified through (1) screening from an existing pool of aptamers and (2) engineering the signaling behavior of the redox reporters. We demonstrate the differential signaling of the aptamer pair in the presence of ampicillin and ATP molecules and show that the pair exhibits similar drifts in undiluted goat serum. By implementing drift cancelation, sensor drift is reduced by a factor of 370. Additionally, the differential signaling enables an increased recording throughput by leveraging differential readout electronics. The authors believe that the proposed technique holds significant benefits for long-term in vivo monitoring.

Microneedle Enabled Electrochemical Aptamer-Based Sensing: Real Time Measurements Using Stainless Steel Microneedles

Electrochemical aptamer-based (EAB) sensors are among the only generalizable sensing platforms that resist fouling enough to function directly in bodily fluids and in situ in the living body. Already, these sensors have enabled the real-time measurement of a range of small molecule targets in vivo, including pharmaceuticals and drugs of abuse. EAB sensors consist of an electrode-attached, redox reporter-labeled aptamer that binds to its target species. Target binding causes a shift in electron-transfer rate between the redox reporter and electrode surface, easily detected by a range of electrochemical techniques. Here, we explore the application of EAB sensors to minimally-invasive molecular monitoring. Specifically, we adapt the platform to a microneedle device format. Microneedles can provide direct access to the skin’s interstitial space – a bodily compartment that holds valuable information ranging from drug distribution and immune status. And because microneedles can penetrate into the top layers of the skin with minimal nerve endings, they can avoid eliciting notable pain responses.In this work, we demonstrate the substantial miniaturization of the sensing platform into electrodes thinner than a human hair, and then integrate them with ultra-fine, hollow microneedles. To ensure needle sterilizability and sharpness while avoiding complex fabrication, we utilize commercial off the shelf stainless steel microneedles. Using an EAB sensor responding to the high-toxicity small molecule drug, vancomycin, as a model system, we achieve quantitative measurements directly in body temperature, undiluted whole blood. Building on this milestone, we explore the application of the sensors when inserted into porcine skin. In porcine skin (a common proxy for human skin), we observe facile microneedle penetration and stable electrochemical signaling. Because of the individual addressability of the sensing electrodes and the generalizability of the aptamer platform, this technology is capable of multiplexed, simultaneous detection of many different molecular targets. Thus, microneedle-enabled aptamer-based sensors offer a promising route forward for non-invasive physiological monitoring – applicable to therapeutic drug monitoring, pain-free biomarker detection, and disease management.

Calibration-Free, Seconds-Resolved In Vivo Molecular Measurements using Fourier-Transform Impedance Spectroscopy Interrogation of Electrochemical Aptamer Sensors.

Electrochemical aptamer-based (EAB) sensors are capable of measuring the concentrations of specific molecules in vivo, in real time, and with a few-second time resolution. For their signal transduction mechanism, these sensors utilize a binding-induced conformational change in their target-recognizing, redox-reporter-modified aptamer to alter the rate of electron transfer between the reporter and the supporting electrode. While a variety of voltammetric techniques have been used to monitor this change in kinetics, they suffer from various drawbacks, including time resolution limited to several seconds and sensor-to-sensor variation that requires calibration to remove. Here, however, we show that the use of fast Fourier transform electrochemical impedance spectroscopy (FFT-EIS) to interrogate EAB sensors leads to improved (here better than 2 s) time resolution and calibration-free operation, even when such sensors are deployed in vivo. To showcase these benefits, we demonstrate the approach's ability to perform real-time molecular measurements in the veins of living rats.

A pendulum-type electrochemical aptamer-based sensor for continuous, real-time and stable detection of proteins

Continuous detection of proteins is crucial for health management and biomedical research. Electrochemical aptamer-based (E-AB) sensor that relies on binding affinity between a recognition oligonucleotide and its specific target is a versatile platform to fulfill this purpose. Yet, the vast majority of E-AB sensors are characterized by voltammetric methods, which suffer from signal drifts and low-frequency data acquisition during continuous operations. To overcome these limitations, we developed a novel E-AB sensor empowered by Gold nanoparticle-DNA Pendulum (GDP). Using chronoamperometric interrogation, the developed sensor enabled drift-resistant, high-frequency, and real-time monitoring of vascular endothelial growth factor (VEGF), a vital signaling protein that regulates angiogenesis, endothelial cell proliferation and vasculogenesis. We assembled VEGF aptamer-anchored GDP probes to a reduced graphene modified electrode, where a fast chronoamperometric current transient occurs as the GDP rapidly transport to the electrode surface. In the presence of target molecules, longer and concentration-dependent time decays were observed because of slower motion of the GDP in its bound state. After optimizing several decisive parameters, including composition ratios of GDP, probe density, and incubation time, the GDP empowered E-AB sensor achieves continuous, selective, and reversible monitoring of VEGF in both phosphate buffered saline (PBS) solutions and artificial urine with a wide detection range from 13 fM to 130 nM. Moreover, the developed sensor acquires signals on a millisecond timescale, and remains resistant to signal degradation during operation. This study offers a new approach to designing E-AB architectures for continuous biomolecular monitoring.

Using seconds-resolved pharmacokinetic datasets to assess pharmacokinetic models encompassing time-varying physiology.

Pharmacokinetics have historically been assessed using drug concentration data obtained via blood draws and bench-top analysis. The cumbersome nature of these typically constrains studies to at most a dozen concentration measurements per dosing event. This, in turn, limits our statistical power in the detection of hours-scale, time-varying physiological processes. Given the recent advent of in vivo electrochemical aptamer-based (EAB) sensors, however, we can now obtain hundreds of concentration measurements per administration. Our aim in this paper was to assess the ability of these time-dense datasets to describe time-varying pharmacokinetic models with good statistical significance. We used seconds-resolved measurements of plasma tobramycin concentrations in rats to statistically compare traditional one- and two-compartmental pharmacokinetic models to new models in which the proportional relationship between a drug's plasma concentration and its elimination rate varies in response to changing kidney function. We found that a modified one-compartment model in which the proportionality between the plasma concentration of tobramycin and its elimination rate falls reciprocally with time either meets or is preferred over the standard two-compartment pharmacokinetic model for half of the datasets characterized. When we reduced the impact of the drug's rapid distribution phase on the model, this one-compartment, time-varying model was statistically preferred over the standard one-compartment model for 80% of our datasets. Our results highlight both the impact that simple physiological changes (such as varying kidney function) can have on drug pharmacokinetics and the ability of high-time resolution EAB sensor measurements to identify such impacts.