Beyond the Gold-Thiol Paradigm: Exploring Alternative Interfaces for Electrochemical Nucleic Acid-Based Sensing.

Nucleic acid-based electrochemical sensors (NBEs) use oligonucleotides as affinity reagents for the detection of a variety of targets, ranging from small-molecule therapeutics to whole viruses. Because of their versatility in molecular sensing, NBEs are being developed broadly for diagnostic and biomedical research applications. Benchmark NBEs are fabricated via self-assembly of thiol-based monolayers on gold. Although robust for rapid prototyping, thiol monolayers suffer from limitations in terms of stability under voltage modulation and in the face of competitive ligands such as thiolated molecules naturally occurring in biofluids. Additionally, gold cannot be deployed as an NBE substrate for all biomedical applications, such as in cases where molecular measurements coupled to real-time, under-the-sensor tissue imaging is needed. Seeking to overcome these limitations, the field of NBEs is pursuing alternative ligands and electrode surfaces. In this perspective, I discuss new interface fabrication strategies that have successfully achieved NBE sensing, or that have the potential to allow NBE sensing on conductive surfaces other than gold. I hope this perspective will provide the reader with a fresh view of how future NBE interfaces could be constructed and will serve as inspiration for the pursuit of collaborative developments in the field of NBEs.

Erratum: Survey of Conductive Polymers for the Fabrication of Conformation Switching Nucleic Acid-Based Electrochemical Biosensors.

[This corrects the article DOI: 10.1021/acsapm.3c02206.].

Label-Free, Real-Time Monitoring of Cytochrome C Responses to Drugs in Microdissected Tumor Biopsies with a Multi-Well Aptasensor Platform.

Functional assays on intact tumor biopsies can potentially complement and extend genomics-based approaches for precision oncology, drug testing, and organs-on-chips cancer disease models by capturing key determinants of therapeutic response, such as tissue architecture, tumor heterogeneity, and the tumor microenvironment. Currently, most of these assays rely on fluorescent labeling, a semi-quantitative method best suited to be a single-time-point terminal assay or labor-intensive terminal immunostaining analysis. Here, we report integrated aptamer electrochemical sensors for on-chip, real-time monitoring of increases of cytochrome C, a cell death indicator, from intact microdissected tissues with high affinity and specificity. The platform features a multi-well sensor layout and a multiplexed electronic setup. The aptasensors measure increases in cytochrome C in the supernatant of mouse or human microdissected tumors after exposure to various drug treatments. Since the aptamer probe can be easily exchanged to recognize different targets, the platform could be adapted for multiplexed monitoring of various biomarkers, providing critical information on the tumor and its microenvironment. This approach could not only help develop more advanced cancer disease models but also apply to other complex in vitro disease models, such as organs-on-chips and organoids.

Nucleic Acid-based Electrochemical Sensors Facilitate the Study of DNA Binding by Platinum (II)-based Antineoplastics.

DNA crosslinking agents such as cisplatin and related platinum(II) analogs are effective drugs to treat solid tumors. However, these therapeutics can cause high toxicity in the body, and tumors can develop resistance to them. To develop less toxic and more effective DNA crosslinkers, medicinal chemists have focused on tuning the ligands in square planar platinum(II) complexes to modulate their bioavailability, targeted cell penetration, and DNA binding rates. Unfortunately, linking in vitro DNA binding capacity of DNA crosslinkers with their in vivo efficacy has proven challenging. Here we report an electrochemical biosensor strategy that allows the study of platinum(II)-DNA binding in real time. Our biosensors contain a purine-rich deoxynucleotide sequence, T6 (AG)10 , modified with a 5' hexylthiol linker for easy self-assembly onto gold electrodes. The 3' terminus is functionalized with the redox reporter methylene blue. Electron transfer from methylene blue to the sensor is a function of platinum(II) compound concentration and reaction time. Using these biosensors, we resolve DNA binding mechanisms including monovalent and bivalent binding, as well as base stacking. Our approach can measure DNA binding kinetics in buffers and in 50 % serum, offering a single-step, real-time approach to screen therapeutic compounds during drug development.

Rational Approach to Optimizing Conformation-Switching Aptamers for Biosensing Applications.

The utilization of structure-switching aptamers (SSAs) has enabled the development of novel sensing platforms for the sensitive and continuous detection of molecules. De novo development of SSAs, however, is complex and laborious. Here we describe a rational approach to SSA optimization that simultaneously improves aptamer binding affinity and introduces target-dependent conformation-switching for compatibility with real-world biosensor applications. Key structural features identified from NMR and computational modeling were used to optimize conformational switching in the presence of target, while large-scale, microarray-based mutation analysis was used to map regions of the aptamer permissive to mutation and identify combinations of mutations with stronger binding affinity. Optimizations were carried out in a relevant biofluid to ensure a seamless transition of the aptamer to a biosensing platform. Initial proof-of-concept for this approach is demonstrated with a cortisol binding aptamer but can easily be translated to other relevant aptamers. Cortisol is a hormone correlated with the stress response that has been associated with various medical conditions and is present at quantifiable levels in accessible biofluids. The ability to continuously track levels of stress in real-time via cortisol monitoring, which can be enabled by the aptamers reported here, is crucial for assessing human health and performance.

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.

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.

Stability of N-Heterocyclic Carbene Monolayers under Continuous Voltammetric Interrogation.

N-Heterocyclic carbenes (NHCs) are promising monolayer-forming ligands that can overcome limitations of thiol-based monolayers in terms of stability, surface functionality, and reactivity across a variety of transition-metal surfaces. Recent publications have reported the ability of NHCs to support biomolecular receptors on gold substrates for sensing applications and improved tolerance to prolonged biofluid exposure relative to thiols. However, important questions remain regarding the stability of these monolayers when subjected to voltage perturbations, which is needed for applications with electrochemical platforms. Here, we investigate the ability of two NHCs, 1,3-diisopropylbenzimidazole and 5-(ethoxycarbonyl)-1,3-diisopropylbenzimidazole, to form monolayers via self-assembly from methanolic solutions of their trifluoromethanesulfonate salts. We compare the electrochemical behavior of the resulting monolayers relative to that of benchmark mercaptohexanol monolayers in phosphate-buffered saline. Within the -0.15 to 0.25 V vs Ag|AgCl voltage window, NHC monolayers are stable on gold surfaces, wherein they electrochemically perform like thiol-based monolayers and undergo similar reorganization kinetics, displaying long-term stability under incubation in buffered media and under continuous voltammetric interrogation. At negative voltages, NHC monolayers cathodically desorb from the electrode surface at lower bias (-0.1 V) than thiol-based monolayers (-0.5 V). At voltages more positive than 0.25 V, NHC monolayers anodically desorb from electrode surfaces at similar voltages to thiol-based monolayers. These results highlight new limitations to NHC monolayer stability imposed by electrochemical interrogation of the underlying gold electrodes. Our results serve as a framework for future optimization of NHC monolayers on gold for electrochemical applications, as well as structure-functionality studies of NHCs on gold.

Continuous Molecular Monitoring in the Body via Nucleic Acid-based Electrochemical Sensors: The Need for Statistically-powered Validation.

Nucleic acid-based electrochemical (NBE) sensors offer real-time and reagent-free sensing capabilities that overcome limitations of target-specific reactivity via affinity-based molecular detection. By leveraging affinity probes, NBE sensors become modular and versatile, allowing the monitoring of a variety of molecular targets by simply swapping the recognition probe without the need to change their sensor architecture. However, NBE sensors have not been rigorously validated in vivo in terms of analytical performance and clinical agreement relative to benchmark methods. In this article, we highlight reports from the past three years that evaluate NBE sensors performance in vivo. We hope this discussion will inspire future translational efforts with statistically robust experimental design, thus enabling real-world clinical applications and commercial development of NBE sensors.

High-precision monitoring of and feedback control over drug concentrations in the brains of freely moving rats.

Knowledge of drug concentrations in the brains of behaving subjects remains constrained on a number of dimensions, including poor temporal resolution and lack of real-time data. Here, however, we demonstrate the ability of electrochemical aptamer-based sensors to support seconds-resolved, real-time measurements of drug concentrations in the brains of freely moving rats. Specifically, using such sensors, we achieve <4 µM limits of detection and 10-s resolution in the measurement of procaine in the brains of freely moving rats, permitting the determination of the pharmacokinetics and concentration-behavior relations of the drug with high precision for individual subjects. In parallel, we have used closed-loop feedback-controlled drug delivery to hold intracranial procaine levels constant (±10%) for >1.5 hours. These results demonstrate the utility of such sensors in (i) the determination of the site-specific, seconds-resolved neuropharmacokinetics, (ii) enabling the study of individual subject neuropharmacokinetics and concentration-response relations, and (iii) performing high-precision control over intracranial drug levels.

Perspective-The Feasibility of Continuous Protein Monitoring in Interstitial Fluid.

Real-time continuous monitoring of proteins in-vivo holds great potential for personalized medical applications. Unfortunately, a prominent knowledge gap exists in the fundamental biology regarding protein transfer and correlation between interstitial fluid and blood. Additionally, technological sensing will require affinity-based platforms that cannot be robustly protected in-vivo and will therefore be challenged in sensitivity, longevity, and fouling over multi-day to week timelines. Here we use electrochemical aptamer sensors as a model system to discuss further research necessary to achieve continuous protein sensing.

Expanding the Monolayer Scope for Nucleic Acid-Based Electrochemical Sensors Beyond Thiols on Gold: Alkylphosphonic Acids on ITO.

Electrochemical biosensors are a powerful and rapidly evolving molecular monitoring technology. Evidenced by the success of the continuous glucose monitor in managing Type 1 Diabetes, these sensors are capable of precise, accurate measurements in unprocessed biological environments. Nucleic acid-based electrochemical sensors (NBEs) are a specific type of biosensor that employs the target binding and conformational dynamics of nucleic acids for signal transduction. Currently, the vast majority of NBEs are fabricated via self-assembly of alkylthiols on Au electrodes. However, this architecture is limited in scope, as Au electrodes are not universally deployable for all potential NBE applications. Here, to expand the repertoire of materials on which NBEs can be made, we describe the multistep procedure for creating sensing monolayers of alkylphosphonic acids on a conductive oxide surface. Using such monolayers on indium tin oxide (ITO)-coated glass slides, we couple redox reporter-modified nucleic acids and demonstrate signaling of procaine-binding NBE sensors in buffer and human serum. We investigate the operational stability of these NBE sensors to reveal faster signal loss relative to benchmark thiol-on-gold sensing layers, a result that arises due to poor stability of the underlying ITO. Finally, we discuss future directions to continue expansion of NBE sensor materials and applications.

Explaining the Decay of Nucleic Acid-Based Sensors under Continuous Voltammetric Interrogation.

Nucleic acid-based electrochemical sensors (NBEs) can support continuous and highly selective molecular monitoring in biological fluids, both in vitro and in vivo, via affinity-based interactions. Such interactions afford a sensing versatility that is not supported by strategies that depend on target-specific reactivity. Thus, NBEs have significantly expanded the scope of molecules that can be monitored continuously in biological systems. However, the technology is limited by the lability of the thiol-based monolayers employed for sensor fabrication. Seeking to understand the main drivers of monolayer degradation, we studied four possible mechanisms of NBE decay: (i) passive desorption of monolayer elements in undisturbed sensors, (ii) voltage-induced desorption under continuous voltammetric interrogation, (iii) competitive displacement by thiolated molecules naturally present in biofluids like serum, and (iv) protein binding. Our results indicate that voltage-induced desorption of monolayer elements is the main mechanism by which NBEs decay in phosphate-buffered saline. This degradation can be overcome by using a voltage window contained between -0.2 and 0.2 V vs Ag|AgCl, reported for the first time in this work, where electrochemical oxygen reduction and surface gold oxidation cannot occur. This result underscores the need for chemically stable redox reporters with more positive reduction potentials than the benchmark methylene blue and the ability to cycle thousands of times between redox states to support continuous sensing for long periods. Additionally, in biofluids, the rate of sensor decay is further accelerated by the presence of thiolated small molecules like cysteine and glutathione, which can competitively displace monolayer elements even in the absence of voltage-induced damage. We hope that this work will serve as a framework to inspire future development of novel sensor interfaces aiming to eliminate the mechanisms of signal decay in NBEs.

Os(II/III) complex supports pH-insensitive electrochemical DNA-based sensing with superior operational stability than the benchmark methylene blue reporter.

DNA-based electrochemical sensors use redox reporters to transduce affinity events into electrical currents. Ideally, such reporters must be electrochemically reversible, chemically stable for thousands of redox cycles, and tolerant to changing chemical environments. Here we report the first use of an Os(II/III) complex in DNA-based sensors, which undergoes pH-insensitive electron transfer with 35% better operational stability relative to the benchmark methylene blue, making it a promising reporter for continuous molecular monitoring applications where pH fluctuates with time.

Optimization of Vancomycin Aptamer Sequence Length Increases the Sensitivity of Electrochemical, Aptamer-Based Sensors In Vivo.

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.

The challenge of long-term stability for nucleic acid-based electrochemical sensors.

Nucleic acid-based electrochemical sensors are a versatile technology enabling affinity-based detection of a great variety of molecular targets, regardless of inherent electrochemical activity or enzymatic reactivity. Additionally, their modular interface and ease of fabrication enable rapid prototyping and sensor development. However, the technology has inhibiting limitations in terms of long-term stability that have precluded translation into clinically valuable platforms like continuous molecular monitors. In this opinion, we discuss published methods to address various aspects of sensor stability, including thiol-based monolayers and anti-biofouling capabilities. We hope the highlighted works will motivate the field to develop innovative strategies for extending the long-term operational life of nucleic acid-based electrochemical sensors.

Human Cyclophilin B Nuclease Activity Revealed via Nucleic Acid-Based Electrochemical Sensors.

Human cyclophilin B (CypB) is oversecreted by pancreatic cancer cells, making it a potential biomarker for early-stage disease diagnosis. Our group is motivated to develop aptamer-based assays to measure CypB levels in biofluids. However, human cyclophilins have been postulated to have collateral nuclease activity, which could impede the use of aptamers for CypB detection. To establish if CypB can hydrolyze electrode-bound nucleic acids, we used ultrasensitive electrochemical sensors to measure CypB's hydrolytic activity. Our sensors use ssDNA and dsDNA in the biologically predominant d-DNA form, and in the nuclease resistant l-DNA form. Challenging such sensors with CypB and control proteins, we unequivocally demonstrate that CypB can cleave nucleic acids. To our knowledge, this is the first study to use electrochemical biosensors to reveal the hydrolytic activity of a protein that is not known to be a nuclease. Future development of CypB bioassays will require the use of nuclease-resistant aptamer sequences.

Microneedle Aptamer-Based Sensors for Continuous, Real-Time Therapeutic Drug Monitoring.

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) was 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. In the past, continuous monitoring in ISF was enabled by microneedle sensor arrays. Yet, benchmark microneedle sensors can only detect molecules 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 electrochemical, aptamer-based (E-AB) sensors. This platform achieves molecular recognition based on affinity interactions, vastly expanding the scope of molecules that can be sensed. We report the fabrication of microneedle E-AB sensor arrays and a method to regenerate them for multiple uses. In addition, we demonstrate continuous molecular 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 preclinical animal models.

Antibody-Invertase Fusion Protein Enables Quantitative Detection of SARS-CoV-2 Antibodies Using Widely Available Glucometers.

Rapid diagnostics that can accurately inform patients of disease risk and protection are critical to mitigating the spread of the current COVID-19 pandemic and future infectious disease outbreaks. To be effective, such diagnostics must rely on simple, cost-effective, and widely available equipment and should be compatible with existing telehealth infrastructure to facilitate data access and remote care. Commercial glucometers are an established detection technology that can overcome the cost, time, and trained personnel requirements of current benchtop-based antibody serology assays when paired with reporter molecules that catalyze glucose conversion. To this end, we developed an enzymatic reporter that, when bound to disease-specific patient antibodies, produces glucose in proportion to the level of antibodies present in the patient sample. Although a straightforward concept, the coupling of enzymatic reporters to secondary antibodies or antigens often results in low yields, indeterminant stoichiometry, reduced target binding, and poor catalytic efficiency. Our enzymatic reporter is a novel fusion protein that comprises an antihuman immunoglobulin G (IgG) antibody genetically fused to two invertase molecules. The resulting fusion protein retains the binding affinity and catalytic activity of the constituent proteins and serves as an accurate reporter for immunoassays. Using this fusion, we demonstrate quantitative glucometer-based measurement of anti-SARS-CoV-2 spike protein antibodies in blinded clinical sample training sets. Our results demonstrate the ability to detect SARS-CoV-2-specific IgGs in patient serum with precise agreement to benchmark commercial immunoassays. Because our fusion protein binds all human IgG isotypes, it represents a versatile tool for detection of disease-specific antibodies in a broad range of biomedical applications.