Electrochemical, aptamer-based (E-AB) sensors are analytical platforms that achieve continuous monitoring of specific molecular targets in vivo. E-AB sensors present an architecture typically consisting of three elements (
E-AB sensors can be successfully interrogated via chronoamperometry, differential pulse techniques such as square-wave voltammetry and differential pulse voltammetry, alternating current voltammetry, and electrochemical impedance spectroscopy. Ultimately, the choice of technique is typically determined by the final intended application of the E-AB sensor. For example, the simplicity of the voltage program in chronoamperometry is ideal for drift-free measurements at sub-second interrogation frequencies, which may be needed for the study of fast biological processes like neurotransmitter modulation in the brain. Electrochemical impedance, in contrast, offers the convenience of interrogating E-AB sensors in a label-free manner, without using a redox reporter. However, the vast majority of reported E-AB sensors have been interrogated by pulse techniques and, in particular, by square wave voltammetry. This widespread use likely arose because pulsed techniques differentially remove currents originating from charging the electrode-electrolyte double layer, significantly improving the signal-to-noise ratio of E-AB measurements. Yet, pulsed techniques also remove valuable electrochemical information regarding sensor stability (e.g., the capacitive current reports on monolayer stability) and differential voltage pulsing also strains the E-AB interface causing faster loss of signal.
Cyclic voltammetry (CV) is frequently used for the surface characterization of E-AB sensors, as this technique provides valuable information regarding monolayer stability (by proxy of double layer capacitance) and surface coverage of the redox reporter-modified aptamer (from faradaic peak areas). However, CV is not commonly used for the direct interrogation of E-AB sensors, in part because sensors with defective blocking monolayers or redox reporter-modified aptamers with slow electron transfer kinetics present large capacitive currents that can hide the faradaic waves of methylene blue, resulting in low signal-to-noise E-AB measurements. Moreover, for many E-AB sensors, CV peak currents do not change significantly with increasing target concentrations.
Accordingly, there is a need for additional methods, and related aspects, of interrogating electrochemical sensors using cyclic voltammetry.
The present disclosure relates, in certain aspects, to methods, systems, and computer readable media of use in detecting target molecules using cyclic voltammetry (CV). Some aspects, for example, include a general CV-based interrogation approach that employs voltammogram peak-to-peak separation (ΔEP) to directly measure binding-induced changes in the apparent electron transfer kinetics of E-AB sensors. Certain embodiments demonstrate that voltage programs used in CV are less damaging to E-AB interfaces over time, and that ΔEP-based interrogation can achieve E-AB measurements with significantly reduced batch-to-batch and day-to-day variability relative to the benchmark square wave voltammetry. These and other aspects will be apparent upon a complete review of the present disclosure, including the accompanying figures.
In one aspect, the present disclosure provides a method of detecting a target molecule using an electrochemical sensor comprising biomolecular receptor-bound redox reporters. The method includes contacting the electrochemical sensor with at least one sample that comprises the target molecule such that one or more of the biomolecular receptors undergo conformational changes when the biomolecular receptors bind the target molecule. The method also includes generating one or more cyclic voltammograms from the electrochemical sensor using cyclic voltammetry (CV). In addition, the method also includes determining a change in a target peak-to-peak separation, ΔEP,T, from the cyclic voltammograms generated from the electrochemical sensor, thereby detecting the target molecule using the electrochemical sensor. In some embodiments, electrochemical sensors are configured to be worn by users as wearable devices (e.g., wearable microneedle sensor arrays, etc.) that continuously detect and monitor target molecule concentrations in and/or on the users.
In some embodiments, the determining step comprises comparing the ΔEP,T to a no target peak-to-peak separation, ΔEP,NT, determined from one or more cyclic voltammograms generated from the electrochemical sensor in the absence of the target molecule. In some embodiments, the methods disclosed herein include determining a concentration of the target molecule in the sample by comparing the ΔEP,T to a standard curve. In some embodiments, the methods disclosed herein include determining the ΔEP,T from at least a first cyclic voltammogram and at least a second cyclic voltammogram generated from the electrochemical sensor. In some embodiments, the determining step comprises correlating at least two currents with corresponding peak potentials and calculating a separation between the peak potentials.
In some embodiments, the methods disclosed herein include determining a concentration of the target molecule in the sample via the change in the target peak-to-peak separation, ΔEP,T. In some embodiments, the electrochemical sensor is substantially resistant to drift. In some embodiments, the methods disclosed herein include determining the change in the target peak-to-peak separation, ΔEP,T, from the cyclic voltammograms with about 900 milliseconds, about 800 milliseconds, about 700 milliseconds, about 600 milliseconds, about 500 milliseconds, about 400 milliseconds, about 300 milliseconds, about 200 milliseconds, about 100 milliseconds, or less of contacting the electrochemical sensor with the sample. In some embodiments, the methods disclosed herein include generating the cyclic voltammograms from the electrochemical sensor using a voltage scanning rate of about 5 V s−1 or more. In some embodiments, the voltage scanning rate is between about 5 V s−1 and about 10 V s−1. In some embodiments, the methods disclosed herein include continuously monitoring the change in the target peak-to-peak separation, ΔEP,T over time from multiple cyclic voltammograms generated from the electrochemical sensor.
In some embodiments, the biomolecular receptor comprises an aptamer. In some embodiments, the biomolecular receptor comprises a deoxyribonucleic acid (DNA) molecule. In some embodiments, the redox reporters comprise methylene blue (MB).
In some embodiments, the sample is substantially unprocessed. In some embodiments, the sample comprises an environmental sample. In some embodiments, the target molecule comprises a therapeutic agent. In some embodiments, the methods disclosed herein include generating a dose-response curve for the therapeutic agent. In some embodiments, the target molecule comprises a metabolite. In some embodiments, the target molecule comprises a biomarker (e.g., a biomolecule or the like).
In some embodiments, the sample comprises a biological sample. In some embodiments, the biological sample is obtained from a subject. In some embodiments, the biological sample is selected from the group consisting of: serum, plasma, blood, saliva, interstitial fluid, urine, feces, semen, and cerebrospinal fluid.
In another aspect, the present disclosure provides a system that includes at least one electrochemical sensor comprising biomolecular receptor-bound redox reporters. The system also includes at least one controller operably connected to the electrochemical sensor. The controller comprises, or is capable of accessing, computer readable media comprising non-transitory computer executable instructions which, when executed by at least one electronic processor, perform at least: generating one or more cyclic voltammograms from the electrochemical sensor using cyclic voltammetry (CV) when the electrochemical sensor is contacted with at least one sample that comprises the target molecule such that one or more of the biomolecular receptors undergo conformational changes when the biomolecular receptors bind the target molecule; and determining a change in a target peak-to-peak separation, ΔEP,T, from the cyclic voltammograms generated from the electrochemical sensor to detect the target molecule in the sample.
In another aspect, the present disclosure provides computer readable media comprising non-transitory computer executable instructions which, when executed by at least electronic processor, perform at least: generating one or more cyclic voltammograms from an electrochemical sensor comprising biomolecular receptor-bound redox reporters using cyclic voltammetry (CV) when the electrochemical sensor is contacted with at least one sample that comprises the target molecule such that one or more of the biomolecular receptors undergo conformational changes when the biomolecular receptors bind the target molecule; and determining a change in a target peak-to-peak separation, ΔEP,T, from the cyclic voltammograms generated from the electrochemical sensor to detect the target molecule in the sample.
In some embodiments of the system or computer readable media disclosed herein, the instructions further perform at least: comparing the ΔEP,T to a no target peak-to-peak separation, ΔEP,NT, determined from one or more cyclic voltammograms generated from the electrochemical sensor in the absence of the target molecule. In some embodiments of the system or computer readable media disclosed herein, the instructions further perform at least: determining a concentration of the target molecule in the sample by comparing the ΔEP,T to a standard curve. In some embodiments of the system or computer readable media disclosed herein, the instructions further perform at least: determining the ΔEP,T from at least a first cyclic voltammogram and at least a second cyclic voltammogram generated from the electrochemical sensor. In some embodiments of the system or computer readable media disclosed herein, the instructions further perform at least: determining a concentration of the target molecule in the sample via the change in the target peak-to-peak separation, ΔEP,T. In some embodiments of the system or computer readable media disclosed herein, the electrochemical sensor comprises a wearable device that is worn by the subject.
In some embodiments of the system or computer readable media disclosed herein, the electrochemical sensor is substantially resistant to drift. In some embodiments of the system or computer readable media disclosed herein, the cyclic voltammograms are determined from the electrochemical sensor using a voltage scanning rate of about 5 V s−1 or more. In some embodiments of the system or computer readable media disclosed herein, the target molecule comprises a therapeutic agent and wherein the instructions further perform at least: generating a dose-response curve for the therapeutic agent. In some embodiments of the system or computer readable media disclosed herein, the instructions further perform at least: continuously monitoring the change in the target peak-to-peak separation, ΔEP,T over time from multiple cyclic voltammograms generated from the electrochemical sensor. In some embodiments of the system or computer readable media disclosed herein, the biomolecular receptor comprises an aptamer. In some embodiments of the system or computer readable media disclosed herein, the biomolecular receptor comprises a deoxyribonucleic acid (DNA) molecule. In some embodiments of the system or computer readable media disclosed herein, the redox reporters comprise methylene blue (MB).
In some embodiments of the system or computer readable media disclosed herein, the biomolecular receptor comprises an aptamer. In some embodiments of the system or computer readable media disclosed herein, the biomolecular receptor comprises a deoxyribonucleic acid (DNA) molecule. In some embodiments of the system or computer readable media disclosed herein, the DNA molecule is single-stranded. In some embodiments of the system or computer readable media disclosed herein, the DNA molecule is double-stranded. In some embodiments of the system or computer readable media disclosed herein, the redox reporters comprise methylene blue (MB).
The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate certain embodiments, and together with the written description, serve to explain certain principles of the methods, systems, and related computer readable media disclosed herein. The description provided herein is better understood when read in conjunction with the accompanying drawings which are included by way of example and not by way of limitation. It will be understood that like reference numerals identify like components throughout the drawings, unless the context indicates otherwise. It will also be understood that some or all of the figures may be schematic representations for purposes of illustration and do not necessarily depict the actual relative sizes or locations of the elements shown.
In order for the present disclosure to be more readily understood, certain terms are first defined below. Additional definitions for the following terms and other terms may be set forth through the specification. If a definition of a term set forth below is inconsistent with a definition in an application or patent that is incorporated by reference, the definition set forth in this application should be used to understand the meaning of the term.
As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. Thus, for example, a reference to “a method” includes one or more methods, and/or steps of the type described herein and/or which will become apparent to those persons skilled in the art upon reading this disclosure and so forth.
It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. Further, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. In describing and claiming the methods, systems, and component parts, the following terminology, and grammatical variants thereof, will be used in accordance with the definitions set forth below.
About: As used herein, “about” or “approximately” or “substantially” as applied to one or more values or elements of interest, refers to a value or element that is similar to a stated reference value or element. In certain embodiments, the term “about” or “approximately” or “substantially” refers to a range of values or elements that falls within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value or element unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value or element).
Bind: As used herein, “bind,” in the context of pathogen detection, refers to a state in which a first chemical structure (e.g., a therapeutic agent) is sufficiently associated a second chemical structure (e.g., a bioreceptor) such that the association between the first and second chemical structures can be detected.
Detecting: As used herein, “detecting,” “detect,” or “detection” refers to an act of determining the existence or presence of one or more target analytes in a sample.
Biomolecule: As used herein, “biomolecule” refers to an organic molecule produced by a living organism. Examples of biomolecules, include macromolecules, such as nucleic acids, proteins, carbohydrates, and lipids.
Bioreceptor: As used herein, “bioreceptor” refers to a biochemical structure that receives or binds other chemical structures (e.g., therapeutic agents, nucleic acids, proteins, metabolites, and the like).
Sample: As used herein, “sample” means anything capable of being analyzed using a device or system disclosed herein. Exemplary sample types include environmental samples and biological samples. In some embodiments, subjects exhale, spit, sneeze, cough, and/or the like to produce aerosolized samples.
Specifically Bind: As used herein, “specifically bind,” in the context of pathogen detection, refers to a state in which substantially only target chemical structures (e.g., biomolecules) are sufficiently associated with a corresponding or cognate binding agent, to the exclusion of non-target chemical structures, such that the association between the target chemical structures and the binding agent can be detected.
System: As used herein, “system” in the context of analytical instrumentation refers a group of objects and/or devices that form a network for performing a desired objective.
Subject: As used herein, “subject” refers to an animal, such as a mammalian species (e.g., human) or avian (e.g., bird) species. More specifically, a subject can be a vertebrate, e.g., a mammal such as a mouse, a primate, a simian or a human. Animals include farm animals (e.g., production cattle, dairy cattle, poultry, horses, pigs, and the like), sport animals, and companion animals (e.g., pets or support animals). A subject can be a healthy individual, an individual that has or is suspected of having a disease or a predisposition to the disease, or an individual that is in need of therapy or suspected of needing therapy. The terms “individual” or “patient” are intended to be interchangeable with “subject.”
Electrochemical, aptamer-based (E-AB) sensors support continuous, real-time measurements of specific molecular targets in complex fluids such as undiluted serum, among many other sample types. They typically achieve these measurements by using redox-reporter-modified, electrode-attached aptamers that undergo target binding-induced conformational changes which, in turn, change electron transfer between the reporter and the sensor surface. Traditionally, E-AB sensors are interrogated via pulse voltammetry to monitor binding-induced changes in transfer kinetics. While these pulse techniques are sensitive to changes in electron transfer, they also respond to progressive changes in the sensor surface driven by biofouling or monolayer desorption and, consequently, present significant drift. Moreover, as described herein, it has been empirically observed that differential voltage pulsing can accelerate monolayer desorption from the sensor surface, presumably via field-induced actuation of aptamers. The present disclosure, in contrast, demonstrates the advantages of employing cyclic voltammetry to measure electron transfer changes directly in some embodiments. In certain embodiments, target concentration is reported via changes in the peak-to-peak separation, ΔEP, of cyclic voltammograms. Because the magnitude of ΔEP is insensitive to variations in the number of aptamer probes on the electrode, ΔEP-interrogated E-AB sensors are resistant to drift and show decreased batch-to-batch and day-to-day variability in sensor performance. Moreover, ΔEP-based measurements can also be performed in a few hundred milliseconds and are, thus, competitive with other sub-second interrogation strategies such as chronoamperometry, but with the added benefit of retaining sensor capacitance information that can report on monolayer stability over time.
To illustrate some of these aspects,
In some embodiments, method 300 includes comparing the ΔEP,T to a no target peak-to-peak separation, ΔEP,NT, determined from one or more cyclic voltammograms generated from the electrochemical sensor in the absence of the target molecule. In some embodiments, method 300 includes determining a concentration of the target molecule in the sample by comparing the ΔEP,T to a standard curve. In some embodiments, method 300 includes determining the ΔEP,T from at least a first cyclic voltammogram and at least a second cyclic voltammogram generated from the electrochemical sensor. In some embodiments, method 300 includes correlating at least two currents with corresponding peak potentials and calculating a separation between the peak potentials.
In some embodiments, method 300 includes determining a concentration of the target molecule in the sample via the change in the target peak-to-peak separation, ΔEP,T. In some embodiments, the electrochemical sensor is substantially resistant to drift. In some embodiments, method 300 includes determining the change in the target peak-to-peak separation, ΔEP,T, from the cyclic voltammograms with about 900 milliseconds, about 800 milliseconds, about 700 milliseconds, about 600 milliseconds, about 500 milliseconds, about 400 milliseconds, about 300 milliseconds, about 200 milliseconds, about 100 milliseconds, or less of contacting the electrochemical sensor with the sample. In some embodiments, method 300 includes generating the cyclic voltammograms from the electrochemical sensor using a voltage scanning rate of about 5 V s−1 or more. In some embodiments, the voltage scanning rate is between about 5 V s−1 and about 10 V s−1. In some embodiments, method 300 includes continuously monitoring the change in the target peak-to-peak separation, ΔEP,T over time from multiple cyclic voltammograms generated from the electrochemical sensor.
In some embodiments, the sample is substantially unprocessed. In some embodiments, the sample comprises an environmental sample. In some embodiments, the target molecule comprises a therapeutic agent. In some embodiments, the methods disclosed herein include generating a dose-response curve for the therapeutic agent. In some embodiments, the target molecule comprises a metabolite. In some embodiments, the target molecule comprises a biomolecule. In some embodiments, the sample comprises a biological sample. In some embodiments, the biological sample is obtained from a subject. In some embodiments, the biological sample is selected from the group consisting of, for example, serum, plasma, blood, urine, feces, semen, and cerebrospinal fluid, among other sample types.
The present disclosure also provides various systems and computer program products or machine-readable media. In some aspects, for example, the methods described herein are optionally performed or facilitated at least in part using systems, distributed computing hardware and applications (e.g., cloud computing services), electronic communication networks, communication interfaces, computer program products, machine readable media, electronic storage media, software (e.g., machine-executable code or logic instructions) and/or the like. To illustrate,
As understood by those of ordinary skill in the art, memory 606 of the server 602 optionally includes volatile and/or nonvolatile memory including, for example, RAM, ROM, and magnetic or optical disks, among others. It is also understood by those of ordinary skill in the art that although illustrated as a single server, the illustrated configuration of server 602 is given only by way of example and that other types of servers or computers configured according to various other methodologies or architectures can also be used. Server 602 shown schematically in
As further understood by those of ordinary skill in the art, exemplary program product or machine readable medium 608 is optionally in the form of microcode, programs, cloud computing format, routines, and/or symbolic languages that provide one or more sets of ordered operations that control the functioning of the hardware and direct its operation. Program product 608, according to an exemplary aspect, also need not reside in its entirety in volatile memory, but can be selectively loaded, as necessary, according to various methodologies as known and understood by those of ordinary skill in the art.
As further understood by those of ordinary skill in the art, the term “computer-readable medium” or “machine-readable medium” refers to any medium that participates in providing instructions to a processor for execution. To illustrate, the term “computer-readable medium” or “machine-readable medium” encompasses distribution media, cloud computing formats, intermediate storage media, execution memory of a computer, and any other medium or device capable of storing program product 608 implementing the functionality or processes of various aspects of the present disclosure, for example, for reading by a computer. A “computer-readable medium” or “machine-readable medium” may take many forms, including but not limited to, non-volatile media, volatile media, and transmission media. Non-volatile media includes, for example, optical or magnetic disks. Volatile media includes dynamic memory, such as the main memory of a given system. Transmission media includes coaxial cables, copper wire and fiber optics, including the wires that comprise a bus. Transmission media can also take the form of acoustic or light waves, such as those generated during radio wave and infrared data communications, among others. Exemplary forms of computer-readable media include a floppy disk, a flexible disk, hard disk, magnetic tape, a flash drive, or any other magnetic medium, a CD-ROM, any other optical medium, punch cards, paper tape, any other physical medium with patterns of holes, a RAM, a PROM, and EPROM, a FLASH-EPROM, any other memory chip or cartridge, a carrier wave, or any other medium from which a computer can read.
Program product 608 is optionally copied from the computer-readable medium to a hard disk or a similar intermediate storage medium. When program product 608, or portions thereof, are to be run, it is optionally loaded from their distribution medium, their intermediate storage medium, or the like into the execution memory of one or more computers, configuring the computer(s) to act in accordance with the functionality or method of various aspects. All such operations are well known to those of ordinary skill in the art of, for example, computer systems.
To further illustrate, in certain aspects, this application provides systems that include one or more processors, and one or more memory components in communication with the processor. The memory component typically includes one or more instructions that, when executed, cause the processor to provide information that causes at least one result, data, and/or the like to be displayed or otherwise indicated (e.g., via a result indicator of electrochemical sensor 618 and/or via communication devices 614, 616 or the like) and/or receive information from other system components and/or from a system user (e.g., via communication devices 614, 616, or the like).
In some aspects, program product 608 includes non-transitory computer-executable instructions which, when executed by electronic processor 604 perform at least: generating one or more cyclic voltammograms from the electrochemical sensor using cyclic voltammetry (CV) when the electrochemical sensor is contacted with at least one sample that comprises the target molecule such that one or more of the biomolecular receptors undergo conformational changes when the biomolecular receptors bind the target molecule; and determining a change in a target peak-to-peak separation, ΔEP,T, from the cyclic voltammograms generated from the electrochemical sensor to detect the target molecule in the sample.
Additional exemplary aspects of the present disclosure also shown in the following slides:
The magnitude of ΔEP in cyclic voltammetry can be used to interrogate changes in the apparent electron transfer kinetics of E-AB sensors. ΔEP is affected by a mass transfer (diffusional) component related to the conformation switching behavior of aptamers; thus, measured changes in ΔEP reflect target-binding induced changes in aptamer conformation. We first illustrate this effect employing tobramycin-detecting E-AB sensors (
The magnitude of ΔEP in cyclic voltammograms of E-AB sensors can be a strong function of the voltage scanning rate. To investigate this effect, we interrogated a fresh batch of tobramycin-detecting E-AB sensors at scanning rates ranging from 0.5 to 100 V·s−1, in the presence and absence of saturating tobramycin concentrations (
As with all E-AB interrogation methods, there is a tradeoff between the maximum signal gain observed by ΔEP and the apparent dissociation constant of the sensors. To illustrate this, we measured dose-response curves as shown in
E-AB interrogation via ΔEP achieves larger signal gains relative to interrogation based on CV peak heights. To illustrate this point, we measured dose-response curves at the optimal voltage scanning rate of 5 V·s−1 and compared the response based on voltammogram peak currents, IP, at varying tobramycin concentrations relative to the proposed ΔEP-based method (
The enhanced signal gain seen with ΔEP-based interrogation can be explained by its strong correlation to electron transfer kinetics. For diffusion-less, quasi-reversible systems, Laviron established that ΔEP can be accurately used to determine electron transfer rate constants (Laviron, E. J. Electroanal. Chem. Interf. Electrochem. 1979, 101, 19). Although, technically, the E-AB system is not entirely diffusion-less—previous works have demonstrated a diffusional contribution to the electrochemical response for DNA sequences longer that 10 nucleotides—ΔEP does directly reflect binding-induced changes in apparent electron transfer kinetics. Voltammetric peak currents, in contrast, are more reflective of the total number of electrons being transferred in the voltammetric sweep (i.e., total moles of reporter-modified aptamer), and not of electron transfer rate. We experimentally demonstrate this for tobramycin-binding E-AB sensors by comparing side-by-side the behavior of ΔEP and IP in the absence and presence of target and at various scanning rates (
ΔEP-based interrogation of E-AB sensors supports molecular measurements in complex media such as undiluted serum. Moreover, the approach can be used to interrogate E-AB sensors detecting different molecular targets. We demonstrate these points here by interrogating sensors employing aptamers that bind to 3 structurally different therapeutics: the aminoglycoside tobramycin, the glycopeptide vancomycin, and the amino ester procaine (
Because ΔEP can rapidly respond to fluctuating target levels, our approach supports continuous molecular monitoring in complex fluids. We evaluate this by serially interrogating E-AB sensors by CV in undiluted serum in the presence and absence of saturating target concentrations (
A critical point to consider when selecting the most suitable electrochemical interrogation technique for E-AB sensors is how the voltage program of that technique can affect long-term sensor stability. The added value of CV over, for example, square-wave voltammetry, is that its linear voltage sweeps can be gentler on biosensor interfaces than differential voltage pulsing. To illustrate this beneficial effect, we compared the signal stability of tobramycin-detecting E-ABs under continuous electrochemical interrogation when using square-wave voltammetry vs CV (
The similar drift resistance of CV IP and ΔEP seen in phosphate-buffered saline does not translate to unprocessed biological fluids, where ΔEP outperforms IP in long-term stability. This difference is revealed by repeating the CV experiment from
One important advantage of ΔEP-based E-AB interrogation over square-wave voltammetry is that ΔEP presents less batch-to-batch and day-to-day variations in sensing performance in unprocessed serum. To illustrate this effect, we serially interrogated three independent batches of six E-AB sensors each by square-wave voltammetry (at the same square-wave frequency and amplitude) in undiluted serum, every 5 s for 24 h on separate days, observing significant variability in peak currents (
The work presented here is focused on the use of ΔEP to interrogate E-AB sensors for the detection of small-molecule targets in biological media. However, we note the same interrogation approach can be readily applied to DNA-based sensors used in the detection of other target types and in different media, as long as the sensing mechanism involves binding-induced changes in reporter electron transfer kinetics. For example, previous works have shown changes in ΔEP upon hybridization of surface-bound, MB-modified DNA with complementary strands. In these cases, DNA hybridization moves MB further away from the electrode surface, leading to an increase in ΔEP (i.e. signal-ON sensors). Similarly, the approach can be used to interrogate E-DNA sensors containing an antibody-binding epitope, aptamer-based sensors binding to protein targets, or DNA-origami based sensors binding to single-entity, mesoscale targets.
Method selection for the interrogation of E-AB sensors typically requires careful consideration of the intended application, the time resolution needed, and the importance given to electrochemical information offered by different methods. We have previously discussed the advantages and disadvantages of many electrochemical methods, mentioning in passing that cyclic voltammetry is most often used for sensor characterization and not for interrogation. Here, however, we describe an approach to use cyclic voltammetry for the direct interrogation of E-AB sensors, with the added benefit of retaining the rich electrochemical information provided by this technique. We have also adapted SACMES, a software we previously reported, to enable real-time, CV-based interrogation of E-AB sensors. The new version of the software allows tracking of voltammetric peak currents and voltages, ΔEP, and arbitrary currents at user specified voltages, continuously and with millisecond processing speeds. Using this platform, we demonstrate that the peak-to-peak separation in cyclic voltammograms of E-AB sensors can be used to directly, rapidly and reversibly interrogate this class of sensors. The method works across sensors binding to structurally different molecular targets and in complex media such as undiluted whole serum. Moreover, because ΔEP is not a function of the moles of aptamer bound to the electrode surface but of the fractional populations of bound vs unbound aptamers, this parameter is drift resistant and expands the operational lifetime of E-AB sensors relative to interrogation based on voltammetric peak currents. This approach supports fast, sub-second voltammetric measurements that could be valuable for the study of aptamer-target binding and dissociation kinetics, or for the time resolved study of dynamic processes in biological systems.
Chemicals and materials. Phosphate buffered saline (PBS; 11.9 mM HPO32-, 137 mM NaCl, 2.7 mM KCl; pH=7.4), sulfuric acid, sodium hydroxide, and procaine hydrochloride were purchased from Fisher Scientific (Waltham, MA). 6-mercaptohexanol, tris(2-carboxyethyl)phosphine hydrochloride (TCEP), and the three nucleic acid aptamers were purchased from Sigma-Aldrich (St. Louis, MO). Human serum was purchased from BioIVT (Washington, D.C.). Tobramycin sulfate was purchased from GoldBio (St. Louis, MO). Vancomycin hydrochloride was purchased from Alfa-Aesar (Ward Hill, MA). All solutions were prepared using ultrapure Milli-Q water with 18 Ω resistance.
The nucleic acid aptamer sequences used in this work were obtained from previous works:
These sequences were purchased modified on the 5′end with hexanethiol and on the 3′end with MB, and double HPLC purified. To prepare aptamer solutions for electrode modification, 1 μL of 100 μM of the modified sequences was incubated with 2 μL of 5 mM TCEP solution in water for 1 hour to reduce the disulfide bonds. Then, we diluted the aptamer solutions in 1 mM 6-mercaptohexanol aqueous solution to a final aptamer concentration of 500 nM.
Electrochemical measurements. Gold working (PN 002314, d=1.6 mm) and coiled platinum wire counter electrodes (PN 012961) were purchased from ALS Inc. (Japan). Ag/AgCl (1M KCl) reference electrodes (PN CHI111) were purchased from CH Instruments (USA). All the electrochemical measurements were done using a multichannel potentiostat CHI 1040C (CH Instruments). Cyclic voltammograms were recorded at different scan rates in the potential window from −0.5 V to +0.1 V vs Ag/AgCl (1M KCl). Square-wave voltammograms were recorded from 0 V to −0.5 V vs Ag/AgCl (1M KCl) with an amplitude of 50 mV, a step size of 1 mV, and at 300 Hz. When specified, the temperature of the electrochemical cell was held constant by using a Huber Microprocessor Control water recirculation bath obtained from Huber (USA).
Electrode modification. Gold electrodes were polished on a 1200/P2500 silicon carbide grinding paper (PN 36-08-1200, Buehler, USA), and on a cloth pad and alumina slurry (PN CF-1050, BASi, USA). After rinsing and sonicating them with water for 1 min to remove polishing debris, the were electrochemically activated by continuous cycles of CV from −0.3 V to −1.6 V in 0.5 M NaOH, and from 0 V to 1.6 V in 0.5 M H2SO4, 250 times in each solution at a scan rate of 0.5 V/s. After this, electrodes were rinsed with water and dipped in the aptamer-thiol mixture for 15 h at room temperature. After rinsing with water, electrodes were ready for use. For the long-term stability experiments of
Data analysis. To process the data obtained from the stability experiments where the sensors were continuously interrogated for long periods of time, we used a previously reported, open-source Python script called SACMES.30 This software supports the real-time analysis, visualization, and control of electrochemical data with millisecond resolution, providing users the ability to extract peak currents (SWV, CV), peak-to-peak separation (CV), half-lives (Chronoamperometry), and area under the curve (SWV, CV). Furthermore, this software provides the user with dynamic control over smoothing and regression algorithms used to filter and fit the raw data.
The data obtained from all experiments were processed using data analysis software Origin Pro v8.5. Multi-panel figures were assembled using Adobe Creative Cloud 2021.
While the foregoing disclosure has been described in some detail by way of illustration and example for purposes of clarity and understanding, it will be clear to one of ordinary skill in the art from a reading of this disclosure that various changes in form and detail can be made without departing from the true scope of the disclosure and may be practiced within the scope of the appended claims. For example, all the methods, devices, systems, computer readable media, and/or component parts or other aspects thereof can be used in various combinations. All patents, patent applications, websites, other publications or documents, and the like cited herein are incorporated by reference in their entirety for all purposes to the same extent as if each individual item were specifically and individually indicated to be so incorporated by reference.
This application is the national stage entry of International Patent Application No. PCT/US2022/017102, filed on Feb. 18, 2022, and published as WO 2022/178327 A1 on Aug. 25, 2022, which claims the benefit of U.S. Provisional Patent Application Ser. No. 63/151,478, filed Feb. 19, 2021, both of which are hereby incorporated by reference herein in their entireties. The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Feb. 17, 2022, is named 0184_0125-PCT_SL.txt and is 1,012 bytes in size.
Filing Document | Filing Date | Country | Kind |
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PCT/US2022/017102 | 2/18/2022 | WO |
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63151478 | Feb 2021 | US |