Methods and related aspects for monitoring antiretroviral drug therapy

Abstract

Provided herein are methods of detecting an antiretroviral therapeutic (ART) agent in a sample from a subject. In some embodiments, the methods include contacting the sample that comprises the ART agent with a plurality of biomolecular receptors such that one or more of the biomolecular receptors undergo conformational changes when the biomolecular receptors bind the ART agent, and detecting one or more detectable signals produced when the biomolecular receptors undergo the conformational changes using a detector. Related devices, kits, systems, computer readable media, and additional methods are also provided.

Description

METHODS AND RELATED ASPECTS FOR MONITORING ANTIRETROVIRALDRUG THERAPYCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to, and the benefit of, U.S. Provisional Patent Application Ser. No. 63 / 633,108, filed April 12, 2024, the disclosure of which is incorporated herein by reference.STATEMENT OF GOVERNMENT SUPPORT

[0002] This invention was made with Government support under grant numbers GM140143 and GM124974, awarded by the National Institutes of Health. The Government has certain rights in the invention.SEQUENCE LISTING

[0003] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on April 10, 2025, is named 0184_0305- PCT_SL.xml and is 49,438 bytes in size.BACKGROUND

[0004] Human immunodeficiency virus (HIV) infection continues to be a global public health concern with an estimated 39 million people living with HIV worldwide and 1.5 million new infections each year. Antiretroviral (ARV) drug therapy has been identified as a key pillar to end the HIV epidemic in the United States. Daily oral dosing of ARV combinations that directly inhibit HIV replication allows people living with HIV to maintain suppressed viral loads, improve clinical outcomes, hinder emergence of drug-resistant virus, and prevent subsequent transmission to uninfected individuals. Additionally, daily oral dosing with the ARVs tenofovir disoproxil fumarate (TDF) or tenofovir alafenamide (TAF) in combination with emtricitabine (FTC, FIG. 1 ) is highly effective at preventing HIV infection amongst healthy men and women. However, ARV effectiveness in HIV treatment and prevention is tightly associated with adherence to daily oral dosing. In response, long-acting injectable ARV formulations have been developed to reduce reliance on daily regimens. Nonetheless, irrespective of dosing schedule, adherence to ARV therapy remains important to sustaining viral suppression and prevent infection.

[0005] Because adherence to HIV treatment is so critical to therapeutic efficacy, monitoring ARV concentrations in biofluids is necessary for the development and implementation of HIV treatment and prevention strategies. Preclinical and clinical evaluation of ARVs and novel dosing strategies requires pharmacokinetic assessment to define subject-specific concentrations associated with viral load suppression or protection from infection. ARV concentration monitoring is increasingly being used to provide objective measures of adherence to HIV treatment, and for the establishment of prevention strategies. This contrasts with previously used self-reporting methods which were unreliable. Additionally, screening samples for ARVs is important to the identification of unreported ARV use in cases of people canceling their HIV status during behavioral surveillance studies and at blood donation centers.

[0006] Accordingly, there exists a growing need to support ARV monitoring at the point of care and specimen collection venues at a larger scale than currently being conducted.SUMMARY

[0007] The present disclosure relates, in certain aspects, to methods, systems, kits, and computer readable media of use in detecting antiretroviral therapeutic (ART) agents in samples obtained from subjects to monitor therapy adherence, among other applications. Monitoring the concentration of antiretroviral drugs is important to ensuring patient adherence to HIV treatment, which is necessary to effectively manage infection status, prevent infection via prophylaxis therapy, and support sample screening at blood donation banks to avert infection spread. The current benchmark method to support antiretroviral drug monitoring, liquid chromatography coupled to mass spectrometry (LC-MS), typically utilizes centralized facilities with costly instrumentation, and has blood-to-result turnaround times of days, making it impractical for effective drug monitoring. Seeking to overcome this issue, in some embodiments, the present disclosure provides aptamers for the antiretroviral drug emtricitabine, which is present in most antiretroviral combination therapies in the market and used for both infection management and prevention. The aptamers have clinically relevant sensitivity in biofluids and are highly selective relative to close analogs such as cytosine, cytidine, and fluorocytidine. Using these aptamers, the present disclosure provides twoanalytical assays, one for continuous, in-vivo emtricitabine monitoring in rodent research models, and one for rapid and high-throughput screening of emtricitabine levels in human plasma. Through a blinded analytical validation, our clinical assay achieved 100% negative correlation and 95% positive correlation relative to the benchmark LC-MS method used at the Centers of Disease Control and Prevention. These and other aspects will be apparent upon a complete review of the present disclosure, including the accompanying figures.

[0008] In one aspect, the present disclosure provides a method of detecting an antiretroviral therapeutic (ART) agent in a sample from a subject. The method comprises: contacting the sample that comprises the ART agent with a plurality of biomolecular receptors such that one or more of the biomolecular receptors undergo conformational changes when the biomolecular receptors bind the ART agent; and, detecting one or more detectable signals produced when the biomolecular receptors undergo the conformational changes using a detector, thereby detecting the ART agent in the sample from the subject.

[0009] In some embodiments, biomolecular receptor-bound redox reporters comprise the biomolecular receptors, wherein the biomolecular receptor-bound redox reporters are attached to a surface of an electrochemical sensor, and wherein when the biomolecular receptors undergo the conformational changes to produce one or more electrochemical signals that are detected using the detector. In some embodiments, the electrochemical sensor comprises a wearable device that is worn by the subject. In some embodiments, the detector comprises an electrochemical analyzer. In some embodiments, the redox reporters comprise methylene blue (MB) or an osmium-based complex. In some embodiments, the electrochemical sensor is configured to generate one or more square wave or cyclic voltammograms using square wave voltammetry (SWV) and / or cyclic voltammetry (CV) and determine a change in peak-to-peak separation, EP.T, from the voltammograms to detect the ART agent when the biomolecular receptors bind the ART agent. In some embodiments, the biomolecular receptor-bound redox reporters are attached to the surface of the electrochemical sensor via an electrically conductive layer disposed on the surface. In some embodiments, a thickness of the electrically conductive layer is less than about 1 pm. In some embodiments, the plurality of biomolecular receptor-bound redox reporters operably attached to the electrically conductive layercomprise one or more self-assembling biosensing monolayers that comprise one or more electrode-blocking alkanethiols, one or more alkanethiols, and one or more redox reporter-tagged aptamers.

[0010] In some embodiments, the biomolecular receptors comprise fluorescent reporter and quencher labeling moieties, wherein the quencher labeling moieties substantially quench fluorescent signals emitted from the fluorescent reporter labeling moieties when the biomolecular receptors are not bound to the ART agent, wherein the fluorescent reporter and quencher labeling moieties separate from one another a sufficient distance when the biomolecular receptors bind to the ART agent such that the fluorescent signals emitted from the fluorescent reporter labeling moieties are not substantially quenched by the quencher labeling moieties and are detected using the detector.

[0011] In some embodiments, the method comprises repeating the contacting and detecting steps one or more times using samples obtained from the subject at different time points to monitor levels of the ART agent in the subject over time. In some embodiments, the sample is substantially unprocessed prior to the contacting step. In some embodiments, the sample comprises whole blood, serum, or plasma. In some embodiments, the sample comprises urine.

[0012] In some embodiments, the subject comprises a human immunodeficiency virus (HIV) infection. In some embodiments, the subject is a human subject. In some embodiments, the ART agent comprises emtricitabine. In some embodiments, the ART agent comprises elvitegravir. In some embodiments, the biomolecular receptor comprises a nucleic acid molecule. In some embodiments, the biomolecular receptor comprises an aptamer. In some embodiments, the aptamer comprises a 5’- thiol modification and a 3’ methylene blue modification. In some embodiments, the aptamer comprises a nucleotide sequence selected from SEQ ID NOS: 1 -10 and 14-30 or a nucleotide sequence having sufficient sequence identity thereto so that the aptamer binds (e.g., specifically binds) the ART agent. In some embodiments, the aptamer can, for instance, be at least about 30%, 50%, 75%, 80%, 90%, 98% or more identical in nucleotide sequence to a nucleotide sequence selected from SEQ ID NOS: 1-10 and 14-30.

[0013] In another aspect, the present disclosure provides a system that comprises: a substrate comprising a plurality of biomolecular receptors; a detector configured to detect one or more detectable signals produced when a sample that comprises an antiretroviral therapeutic (ART) agent is contacted with the plurality of biomolecular receptors such that one or more of the biomolecular receptors undergo conformational changes when the biomolecular receptors bind the ART agent; and, at least one controller operably connected to the detector, which 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: detecting the one or more detectable signals produced when the biomolecular receptors undergo the conformational changes using a detector.

[0014] In some embodiments, biomolecular receptor-bound redox reporters comprise the biomolecular receptors, wherein an electrochemical sensor comprises the substrate, wherein the biomolecular receptor-bound redox reporters are attached to a surface of the electrochemical sensor, and wherein the biomolecular receptors undergo the conformational changes to produce one or more electrochemical signals that are detected using the detector. In some embodiments, the electrochemical sensor comprises a wearable device that is worn by the subject. In some embodiments, the detector comprises an electrochemical analyzer. In some embodiments, the redox reporters comprise methylene blue (MB) or an osmium- based complex. In some embodiments, the electrochemical sensor is configured to generate one or more square wave or cyclic voltammograms using square wave voltammetry (SWV) and / or cyclic voltammetry (CV) and determine a change in peak- to-peak separation, EP.T, from the voltammograms to detect the ART agent when the biomolecular receptors bind the ART agent. In some embodiments, the biomolecular receptor-bound redox reporters are attached to the surface of the electrochemical sensor via an electrically conductive layer disposed on the surface. In some embodiments, a thickness of the electrically conductive layer is less than about 1 pm. In some embodiments, the plurality of biomolecular receptor-bound redox reporters operably attached to the electrically conductive layer comprise one or more self-assembling biosensing monolayers that comprise one or moreelectrode-blocking alkanethiols, one or more alkanethiols, and one or more redox reporter-tagged aptamers.

[0015] In some embodiments, the biomolecular receptors comprise fluorescent reporter and quencher labeling moieties, wherein the quencher labeling moieties substantially quench fluorescent signals emitted from the fluorescent reporter labeling moieties when the biomolecular receptors are not bound to the ART agent, wherein the fluorescent reporter and quencher labeling moieties separate from one another a sufficient distance when the biomolecular receptors bind to the ART agent such that the fluorescent signals emitted from the fluorescent reporter labeling moieties are not substantially quenched by the quencher labeling moieties and are detected using the detector. In some embodiments, wherein the non- transitory computer executable instructions which, when executed by the electronic processor, further perform at least: repeating the detecting step one or more times using samples obtained from the subject at different time points to monitor levels of the ART agent in the subject over time.

[0016] In some embodiments, the sample is substantially unprocessed. In some embodiments, the sample comprises whole blood, serum, or plasma. In some embodiments, the sample comprises urine. In some embodiments, the subject comprises a human immunodeficiency virus (HIV) infection. In some embodiments, the subject is a human subject. In some embodiments, the ART agent comprises emtricitabine. In some embodiments, the ART agent comprises elvitegravir.

[0017] In some embodiments, wherein the biomolecular receptor comprises a nucleic acid molecule. In some embodiments, the biomolecular receptor comprises an aptamer. In some embodiments, the aptamer comprises a 5’- thiol modification and a 3’ methylene blue modification. In some embodiments, the aptamer comprises a nucleotide sequence selected from SEQ ID NOS: 1-10 and 14-30 or a nucleotide sequence having sufficient sequence identity thereto so that the aptamer binds (e.g., specifically binds) the ART agent. In some embodiments, the aptamer can, for instance, be at least about 30%, 50%, 75%, 80%, 90%, 98% or more identical in nucleotide sequence to a nucleotide sequence selected from SEQ ID NOS: 1-10 and 14-30.

[0018] In another aspect, the present disclosure provides a computer readable media comprising non-transitory computer executable instructions which, when executed by at least electronic processor, perform at least: detecting one or more detectable signals produced when a plurality of biomolecular receptors undergo conformational changes using a detector, which detector is configured to detect the one or more detectable signals produced when a sample that comprises an antiretroviral therapeutic (ART) agent is contacted with the plurality of biomolecular receptors such that one or more of the biomolecular receptors undergo the conformational changes when the biomolecular receptors bind the ART agent.BRIEF DESCRIPTION OF THE DRAWINGS

[0019] 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.

[0020] FIG. 1. The therapeutic emtricitabine (FTC, circled) is found in two of the most used antiretroviral (ARV) therapies, making it an ideal target for drug adherence studies.

[0021] FIG. 2 schematically depicts an E-AB biosensor for the point-of-care monitoring of antiretroviral treatment adherence via the measurement of drug levels in unprocessed human fluids according to an exemplary embodiment.

[0022] FIG. 3 is a flow chart that schematically depicts steps in a method of detecting an antiretroviral therapeutic (ART) agent in a sample from a subject according to an exemplary embodiment.

[0023] FIG. 4 schematically depicts a system according to an exemplary embodiment.

[0024] FIGS. 5A-5C. Selection of DNA aptamers against FTC. (A) The capture-SELEX procedure: (1 ) The ssDNA library (Lib_1 ) is immobilized to streptavidin-coated magnetic beads via a biotinylated capture strand (Bio-Cap_1 ); (2) The beads are washed to remove weakly bound ssDNA; (3) A counter selections are carried out against deoxycytidine (dC, from round 9 onward); (4) The beads are washed to remove unbound ssDNA from the counter selection; (5) The positive selection step is carried out in the presence of FTC; (6) ssDNA eluted during the positive selection step is amplified by PCR and made single stranded for the next SELEX round. (B) Sequence of Bio-Cap_1 and sequence and secondary structure of Lib_1. FIG. 5B discloses SEQ ID NOS 11 and 31 , respectively, in order of appearance. (C) Parent sequences for the top-5 clusters identified after 11 rounds of capture-SELEX. Sequences and structures of all oligonucleotides used in this work are listed in Table 1 .

[0025] FIGS. 6A-6E. Characterization of FTC_1_Full binding affinity and selectivity. (A) Sequence and Nupack-predicted secondary structure of FTC_1_Full. FIG. 6A discloses SEQ ID NO: 1. (B) FTC affinity measurement via isothermal titration calorimetry (ITC) displaying a dissociation constant of 150 nM ± 40 nM. Challenging the FTC_1_Full sequence with (C) cytosine, (D) 5-fluorocytidine, or (E) cytidine resulted in no heat exchange, underscoring the high selectivity of this aptamer for FTC relative to close structural analogs. The solid line in panel B represents a non-linear regression to the Hill Isotherm.

[0026] FIGS. 7A-7D. Development of electrochemical, aptamer-based (E- AB) sensors for FTC. (A) The E-AB sensing mechanism leverages changes in electron transfer rates ( eT,i vs eT,2) driven by target binding. In the bound state, the aptamer is favored to form a secondary structure that places the redox reporter (blue circle) closer to the electrode surface, increasing electron transfer kinetics, eT,2 » eT,i. To enable this mechanism, we truncated FTC_1_Full from 72 nt to 28 nt to destabilize stem S1 , resulting in sequence FTC_1_EAB. FIG. 7A discloses SEQ ID NOS 19 and 19. (B) This truncation retained FTC binding as determined via ITC measurements, albeit with a modest loss in affinity. (C) The FTC_1_EAB sequence was modified with hexanethiol at the 5’ end and methylene blue (reporter) at the 3’ end and immobilized on gold electrodes as shown schematically in panel A. Interrogating this modified aptamer in the absence (black) and presence (red) of 10pM FTC via square wave voltammetry resulted in frequency maps that shifted maxima to higher frequencies (i.e., higher electron transfer rate) in the presence of FTC. (D) Challenging FTC_1_EAB with increasing concentration of FTC in phosphate-buffered saline produced dose response curves covering the clinical range for FTC.

[0027] FIGS. 8A-8I. Pharmacokinetic measurements of FTC in preclinical animal models. (A) Schematic of sensor coordinates for mouse in-brain measurements. (B) Protocol timeline. (C) Photograph of brain E-AB probe. (D) Confirmation of sensor placement in the cortex via histology. The red circle represents the average of 3 independent sensor placements, errors show the standard deviation. A representative brain scan post implantation is shown in FIG. 17. (E) In-cortex E-AB measurements show an immediate increase in FTC concentration following intravenous dosing (absorption rate, ko = 19.6 pM’1h’1) as the drug crosses the blood-brain barrier, followed by steady state kinetics that eventually lead to excretion after ~1 h (excretion rate, k = 0.3 pM’1h’1). Circles represent the average to two measurements; errors show the dispersion across these two measurements. (F) Schematic of sensor insertion into rat jugular vein. (G) Protocol timeline of in-vein measurements. (H) Vein E-AB probe. (I) In-vein E-AB measurements show an instantaneous increase in FTC, as expected given the intravenous bolus, followed by first-order excretion kinetics, k = 1 .4 pM’1h’1.

[0028] FIG. 9. Monitoring capture-SELEX progress by elution profiles. Several eluents, including the library input (Input), 1stand 10thwashing step prior to selection (W1 and W10, respectively), three counter selection washes with deoxycytidine (C1 - 3), and three positive selection washes with FTC (Pos1 - 3), were collected during the selection, amplified via small scale PCR, and analyzed by agarose gel. Round number (R) is indicated next to each row. The counter selection was introduced during round 9 and onward.

[0029] FIG. 10. Clone SELEX elution profiles of 10 candidates. The elution profile of clone F20 (FTC_1_Full) is the best.

[0030] FIG. 11. Sequencing results of round 11 pool from aptamer selection. The parent sequence from each cluster is shown.

[0031] FIGS. 12A-12E. Binding of FTC to top cluster sequences via ITC. Clusters 1 (A) and 2 (B) display strong binding to FTC via isothermal titration calorimetry. Top sequences from clusters 3 (C) and 4 (D) display no apparent binding, but do display some interaction with the molecule, as shown by a negative heat exchange upon addition of target. (E) The top sequence form cluster 5 displays strong binding to FTC.

[0032] FIGS. 13A-13G. Serial truncations of FTC_1_Full maintain FTC binding. ITC thermograms showing affinity-based interaction between various truncations of FTC_1_Full and FTC. FIG. 13A discloses SEQ ID NO: 32.

[0033] FIGS. 14A-14C. ITC thermograms of FTC_1_EAB against close analogs of FTC. When challenged with either cytosine (A), cytidine (B), or 5- Fluorocytidine (C), FTC_1_EAB displays no binding.

[0034] FIG. 15. A scrambled version of FTC_1_EAB displays no binding to FTC. Scrambled sequence FTC_1_EAB_Scramble found in Table 1.

[0035] FIGS. 16A-16D. Frequency maps of FTC_1 truncations. FTC_1 truncations were modified with a hexanethiol linker at the 5’ end with a methylene blue. Sensors are interrogated in the absence (black trace) and presence (red trace) of 10 pM FTC resulting in frequency maps displaying relative charge at a given square wave frequency. The presence of a lateral shift along the x-axis signifies conformational switching of the surface bound aptamer similar to Fig. 7A.

[0036] FIG. 17. Representative histology image from in vivo brain experiments. Prior to animal euthanasia a 300 pA current was passed through the sensor for 2 s to locally bum the tissue, highlighting sensor placement in the circle.

[0037] FIGS. 18A-18F. Pharmacokinetic measurements of FTC in preclinical animal models. (A) Schematic of sensor coordinates for mouse in-brain measurements. FIG. 18A discloses SEQ ID NOS 33, 20, and 21 , respectively, in order of appearance. (B) Protocol timeline. (C) Photograph of brain E-AB probe. (D) Confirmation of sensor placement in the cortex via histology. The red circle represents the average of 3 independent sensor placements, errors show the standard deviation. A representative brain scan post implantation is shown in FIG. 17. (E) In-cortex E-AB measurements show an immediate increase in FTC concentration following intravenous dosing (absorption rate, ko = 19.6 pM’1h’1) as thedrug crosses the blood-brain barrier, followed by steady state kinetics that eventually lead to excretion after ~1 h (excretion rate, k = 0.3 pM’1h’1). Circles represent the average to two measurements; errors show the dispersion across these two measurements. (F) Schematic of sensor insertion into rat jugular vein. (G) Protocol timeline of in-vein measurements. (H) Vein E-AB probe. (I) In-vein E-AB measurements show an instantaneous increase in FTC, as expected given the intravenous bolus, followed by first-order excretion kinetics, k = 1 .4 pM’1h’1.

[0038] FIGS. 19A-19E. ITC thermograms of FTC binding aptamers against BSA. All sequences are found in Table 1 .

[0039] FIG. 20. Mechanism of action of FTC_1_Fluor. Upper panels represent mechanism of action for FTC_1_5’trunc3. Lower panels represent mechanism of action of FTC_1_Fluor. FIG. 20A discloses SEQ ID NOS 34, 21 , 35, and 21 , respectively, in order of appearance. FIG. 20B discloses SEQ ID NOS 20, 21 , 36, and 21 , respectively, in order of appearance.

[0040] FIG. 21. Normalized fluorescence of modified constructs. Truncation of 14 nucleotides from the 3’terminus of the FTC_1_5’trunc3 to eliminate unpaired nucleotides extending from stem S1 , resulting in a rapid aptamer-based optical sensor. This truncation achieved optimal FTC detection across the clinically relevant range of 100 nM to 10 pM.

[0041] FIG. 22. ITC Thermogram of FTC_1_Fluor against FTC. The truncated construct maintains similar binding affinity to the full-length construct (Fig. 6B).

[0042] FIG. 23. Specificity of FTC_1_Fluor against dC. No fluorescence signal was observed in the presence of excess dC, confirming the molecular specificity of the sensor for FTC.

[0043] FIG. 24. Stability in human plasma. The FTC_1_Fluor / Fluor- Capture probe is stable in 50% human plasma for up to 24 hours, as monitored via 20% denaturing polyacrylamide gel.

[0044] FIG. 25. Effect of human plasma on sensor performance. The addition of up to 50% human plasma had little effect on the performance of the sensor.

[0045] FIG. 26. Kinetic analysis of measurement in 50% human plasma. The maximum fluorescent signal was obtained within 1 min following FTC addition (arrow) and maximum fluorescence signal was stable at least 40 minutes.

[0046] FIG. 27. Receiver Operating Characteristic curve (ROC curve) of the clinical sample measurement with fluorescence assay. ROC curve was generated using R package PROC.

[0047] FIGS. 28A and 28B. ESI-MS spectra of FTC_1_Fluor components. (A) Q-Capture. Mass calculated: 5110.5 Da; Mass found: 5109.5 Da. (B) FTC_1_Fluor. Mass calculated: 10906.9 Da; Mass found: 17907.1 Da.

[0048] FIG. 29. ESI-MS spectra of FTC_1_EAB + Surface Modifiers. Mass calculated: 10069.6 Da; Mass found: 10069.5 Da.DEFINITIONS

[0049] 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.

[0050] 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.

[0051] 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.

[0052] 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).

[0053] Bind. As used herein, “bind,” in the context of ART drug 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.

[0054] 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.

[0055] 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).

[0056] 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.

[0057] Specifically Bind: As used herein, "specifically bind,” in the context of ART drug detection, refers to a state in which substantially only target chemical structures (e.g., the antiretroviral drug emtricitabine or another ART agent) 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.

[0058] 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.

[0059] 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.”DETAILED DESCRIPTION

[0060] Infection with human immunodeficiency virus (HIV) continues to be a major global health issue. The World Health Organization estimates that about 37 million people were living with HIV at the end of 2017 and 1.8 million became newly infected the same year. Although large-scale prevention, diagnosis and treatment programs have decreased the incidence rate of HIV infection by almost 40% over the past two decades - a significant achievement - eradication of the disease remains a serious challenge. To date, no cure exists for those already infected. And, while available treatments based on antiretroviral therapy (ART) allow infected patients to live a high-quality life, such treatments currently depend on dosing regimens requiring daily adherence, a feat difficult to enforce and monitor. Poor adherence to ART regimens can produce drug-resistance which, in turn, can lead to treatment failure. Thus, there is a need for technologies enabling the measurement of ART drugs in biological fluids as a means to monitor therapy adherence at the point-of- care.

[0061] The current gold standard to measuring ART drug levels in body fluids - liquid chromatography coupled to mass spectrometry detetion (LC-MS) - relies on expensive benchtop laboratory equipment needing air-conditioned facilities and scientifically-trained personnel to operate. These requirements are unrealistic for many developing nations and for poor areas in developed nations where the majority of all HIV-infected humans live. Instead, to achieve broad adoption in these regions, an ideal ART drug-monitoring platform should be portable, self-powered, convenient, and easy to use. Accordingly, in some embodiments, the present disclosure provides electrochemical and optically biosensing platforms that enable the measurement ofspecific ART drug levels in blood and urine or other sample types at the point of care (FIG. 1 ). In some embodiments, the sensors of the present disclosure employ nucleic acid aptamers as molecular recognition elements against two ART drugs: emtricitabine and elvitegravir. Both of these drugs are administered to infected patients as part of combination therapies; thus, their point-of-care monitoring can directly report on treatment adherence for a significant fraction of the infected population. Moreover, urine emtricitabine levels (not elvitegravir) correlate well with blood drug levels, thus allowing the monitoring of adherence from less invasive samples (relative to blood obtained from finger pricks). The methods and related aspects of the present disclosure create an unprecedented clinical tool to identify individuals struggling with adherence and provide the behavioral interventions they need to achieve sustained viral suppression.

[0062] The platforms of the present include numerous attributes, including, for example: 1 ) E-AB sensors have been demonstrated to support precise and highly specific drug measurements in vitro and in vivo in biological fluids, including unprocessed whole blood; 2) the considered ART drugs reach hundred-nanomolar to micromolar concentrations in plasma, which are easily detectable via E-AB sensors; and 3) electrochemical and optical detection can be easily adapted to achieve low- cost, point-of-care drug measurements. Moreover, the approaches disclosed herein are agnostic to sensor architecture, meaning that it can be adapted to work on test strips or, for example, in wearable devices. Thus, in some aspects, the present disclosure provides an ART adherence monitoring tool that is highly adaptable to real-life design restrictions.

[0063] The technology disclosed herein moves away from the status quo of indirect, self-reporting-based approaches to monitoring adherence and, instead, produces a platform achieving the chemically specific, direct measurement of ART drug levels in human samples. Such platforms are superior to already available adherence monitors which track patterns of pill intake instead of tracking in-fluid drug levels. For example, electronic monitors like the Medication Event Monitoring System (MEMS) bottle cap - which contains a microcontroller recording the opening and closing of a pill bottle with a time / date stamp - can be easily fooled by openings that do not lead to pill taking. Similarly, ingestible pill monitors depend on user adherence. In contrast, an electrochemical or optical biosensor enabling the point-of-care measurement of blood or urine ART drug levels does not involve patient action. Furthermore, as required to achieve high adoptability, the chemistry of the platforms disclosed herein is compatible with affordable (disposable) materials, portable, have low-energy requirements and easily interface with globally accessible electronic devices like cell phones.

[0064] Exemplary Methods

[0065] To illustrate some of these aspects, FIG. 3 is a flow chart that schematically depicts steps in a method of detecting an antiretroviral therapeutic (ART) agent in a sample from a subject, for example, as part of a process of monitoring therapy adherence. As shown, method 300 includes contacting the sample that comprises the ART agent with a plurality of biomolecular receptors such that one or more of the biomolecular receptors undergo conformational changes when the biomolecular receptors bind the ART agent (step 302). Method 300 also includes detecting one or more detectable signals produced when the biomolecular receptors undergo the conformational changes using a detector (step 304).

[0066] In some embodiments, biomolecular receptor-bound redox reporters comprise the biomolecular receptors. In some embodiments, the biomolecular receptor-bound redox reporters are attached to a surface of an electrochemical sensor. The biomolecular receptors undergo conformational changes to produce electrochemical signals that are detected using the detector. In some embodiments, the electrochemical sensor comprises a wearable device that is worn by the subject. In some embodiments, the detector comprises an electrochemical analyzer. In some embodiments, the redox reporters comprise methylene blue (MB) or an osmium- based complex. In some embodiments, the electrochemical sensor is configured to generate one or more square wave or cyclic voltammograms using square wave voltammetry (SWV) and / or cyclic voltammetry (CV) and determine a change in peak- to-peak separation, EP.T, from the voltammograms to detect the ART agent when the biomolecular receptors bind the ART agent.

[0067] In some embodiments, the biomolecular receptor-bound redox reporters are attached to the surface of the electrochemical sensor via an electrically conductive layer disposed on the surface. In some embodiments, a thickness of the electrically conductive layer is less than about 1 pm. In some embodiments, theplurality of biomolecular receptor-bound redox reporters operably attached to the electrically conductive layer comprise one or more self-assembling biosensing monolayers that comprise one or more electrode-blocking alkanethiols, one or more alkanethiols, and one or more redox reporter-tagged aptamers.

[0068] In some embodiments, the biomolecular receptors comprise fluorescent reporter and quencher labeling moieties, in which the quencher labeling moieties substantially quench fluorescent signals emitted from the fluorescent reporter labeling moieties when the biomolecular receptors are not bound to the ART agent. The fluorescent reporter and quencher labeling moieties separate from one another a sufficient distance when the biomolecular receptors bind to the ART agent such that the fluorescent signals emitted from the fluorescent reporter labeling moieties are not substantially quenched by the quencher labeling moieties and are detected using the detector.

[0069] In some embodiments, the method comprises repeating the contacting and detecting steps one or more times using samples obtained from the subject at different time points to monitor levels of the ART agent in the subject over time (e.g., to monitor therapy adherence). In some embodiments, the sample is substantially unprocessed prior to the contacting step. In some embodiments, the sample comprises whole blood, serum, or plasma. In some embodiments, the sample comprises urine.

[0070] In some embodiments, the subject comprises a human immunodeficiency virus (HIV) infection. In some embodiments, the subject is a human subject. In some embodiments, the ART agent comprises emtricitabine (Emtriva). In some embodiments, the ART agent comprises elvitegravir (Vitekta). In some embodiments, the biomolecular receptor comprises a nucleic acid molecule. In some embodiments, the biomolecular receptor comprises an aptamer. In some embodiments, the aptamer comprises a 5’- thiol modification and a 3’ methylene blue modification. In some embodiments, the aptamer comprises a nucleotide sequence selected from SEQ ID NOS: 1 -10 and 14-30 or a nucleotide sequence having sufficient sequence identity thereto so that the aptamer binds (e.g., specifically binds) the ART agent. In some embodiments, the aptamer can, for instance, be at least about 30%, 50%, 75%, 80%, 90%, 98% or more identical in nucleotide sequence to a nucleotide sequence selected from SEQ ID NOS: 1-10 and 14-30.

[0071] Exemplary Systems and Computer Readable Media

[0072] 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, FIG. 4 provides a schematic diagram of an exemplary system suitable for use with implementing at least aspects of the methods disclosed in this application. As shown, system 600 includes at least one controller or computer, e.g., server 602 (e.g., a search engine server), which includes processor 604 and memory, storage device, or memory component 606, and one or more other communication devices 614, 616, (e.g., client-side computer terminals, telephones, tablets, laptops, other mobile devices, etc. (e.g., for receiving data for further analysis, etc.)) positioned remote from electrochemical sensor device 618, and in communication with the remote server 602, through electronic communication network 612, such as the Internet or other internetwork. Communication devices 614, 616 typically include an electronic display (e.g., an internet enabled computer or the like) in communication with, e.g., server 602 computer over network 612 in which the electronic display comprises a user interface (e.g., a graphical user interface (GUI), a web-based user interface, and / or the like) for displaying results upon implementing the methods described herein. In certain aspects, communication networks also encompass the physical transfer of data from one location to another, for example, using a hard drive, thumb drive, or other data storage mechanism. System 600 also includes program product 608 stored on a computer or machine readable medium, such as, for example, one or more of various types of memory, such as memory 606 of server 602, that is readable by the server 602, to facilitate, for example, a guided search application or other executable by one or more other communication devices, such as 614 (schematically shown as a desktop or personal computer). In some aspects, system 600 optionally also includes at least one database server, such as, for example, server 610 associated with an online website having data stored thereon searchable either directly or through search engine server 602. System 600optionally also includes one or more other servers positioned remotely from server 602, each of which are optionally associated with one or more database servers 610 located remotely or located local to each of the other servers. The other servers can beneficially provide service to geographically remote users and enhance geographically distributed operations.

[0073] 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 FIG. 4, represents a server or server cluster or server farm and is not limited to any individual physical server. The server site may be deployed as a server farm or server cluster managed by a server hosting provider. The number of servers and their architecture and configuration may be increased based on usage, demand and capacity requirements for the system 600. As also understood by those of ordinary skill in the art, other user communication devices 614, 616 in these aspects, for example, can be a laptop, desktop, tablet, personal digital assistant (PDA), cell phone, server, or other types of computers. As known and understood by those of ordinary skill in the art, network 612 can include an internet, intranet, a telecommunication network, an extranet, or world wide web of a plurality of computers / servers in communication with one or more other computers through a communication network, and / or portions of a local or other area network.

[0074] 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.

[0075] 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.

[0076] 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.

[0077] 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 or optical sensor device 618and / 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).

[0078] EXAMPLE: Monitoring HIV Antiretroviral Therapy via Aptamer- Based Measurements in Preclinical Animal Models and in Patient Plasma

[0079] INTRODUCTION

[0080] This example reports the development of a nucleic acid aptamer binding the ARV drug FTC, which is a component of the standard-of-care combination therapy to both treat HIV and prevent infection (prophylaxis). The aptamer is completely selective against close analogs of FTC, including cytosine, cytidine, deoxycytidine, and fluorocytidine. We integrated this aptamer into two sensing modalities: an electrochemical approach that allows real-time monitoring of FTC pharmacokinetics in live rodents for biomedical research applications, and an optical approach that enables, for the first time in high-throughput format, the monitoring of ARV concentrations in patient plasma. In a blinded comparative validation against the current benchmark LC-MS method, our optical diagnostic assay achieves 100% correlation in negative samples and >95% correlation with positive samples, paving the way for high-throughput ARV monitoring.

[0081] RESULTS AND DISCUSSION

[0082] Nucleic acid aptamers are synthetic biorecognition elements that bind molecular targets with high specificity. Aptamers that bind small molecules often have affinities in the hundred nanomolar to few micromolar range. Their affinity is limited by the low surface area of small molecule targets and the few functional groups they typically display. However, aptamers are ideal for ARV monitoring because these drugs are administered at high doses that result in pharmacokinetic profiles with hundred nanomolar to micromolar concentrations in blood. Nucleic acids can be easily modified to be nuclease resistant and functionalized with reporters to support signal transduction. Here, we specifically designed conformation-switching aptamers that either transfer electrons at a faster rate, or emit fluorescence when in the presence of FTC, enabling preclinical and clinical measurements of FTC concentrations in unprocessed biofluids in vitro and in vivo.

[0083] Selection of Conformation Switching Aptamers for FTC. Toisolate aptamers with selective binding affinity for FTC, we employed a modified version of capture-SELEX (Systematic Evolution of Ligands by Exponential Enrichment) (FIG. 5A). The single-stranded DNA (ssDNA) library (Lib_1 ) contained a 30-nucleotide random domain flanked on both sides by fixed sequences with partial complementarity, resulting in the formation of a stem-loop structure (FIG. 5B). To begin the selection, the library (~1014individual molecules) was immobilized onto streptavidin-coated magnetic beads via a biotinylated capture strand (Bio-Cap_1 in FIG. 5B) that also disrupts formation of the stem-loop. After extensive washing to remove unbound sequences, the immobilized library was exposed to 100 pM FTC in PBS buffer (pH = 7.2) containing 2 mM Mg2+(i.e., the positive selection step). Aptamer sequences that bound FTC and consequently underwent a conformational change caused by stabilization of the stem-loop were released from the capture strand on the beads and collected for PCR amplification. The double-stranded DNA (dsDNA) resulting from PCR amplification was used to generate the corresponding pool of ssDNA for the next selection round.

[0084] The selection pressure was gradually increased during successive SELEX rounds by decreasing the concentration of FTC and the duration of the positive selection step. Table 2 summarizes selection conditions for each round. In rounds 9 and onward, we incorporated a counter-selection step wherein the immobilized DNA library was first exposed to deoxycytidine (dC), which is structurally similar to FTC. Aptamer sequences that bound dC were displaced from the beads and discarded; aptamer sequences that remained immobilized were advanced into the positive selection step against FTC. The progress of the selection was monitored by comparing the amount of DNA eluted by FTC relative to the amount eluted during the washing and counter-selection steps (FIG. 9). After 11 rounds, this analysis revealed that more DNA was being eluted by FTC (FIG. 10) compared to the washing steps and in much less time compared to the counter-target dC, and thus, the selection was halted at this point.

[0085] Candidate aptamers from the eleventh round of selection were identified using next-generation sequencing (NGS) and bioinformatics analysis using the FASTAptamer toolkit. The 4,620 unique sequences identified by this analysis were clustered using a Levenshtein edit distance of 4 and the clusters ranked based on the abundance of the parent sequence (FIG. 11 ). Parent sequences from the topfive clusters (FIG. 5C) were synthesized and screened for affinity (FIG. 12). The parent sequence from cluster 1 (FIG. 6A) showed affinity for FTC in the few hundred nM range (FIG. 6B) and was coincidentally the most abundant sequence in the enriched pool. Additionally, this sequence underwent target-induced conformation switching as evaluated via electrochemistry (FIG. 7C). This sequence, here named FTC_1_Full (see Table 1 ), was carried forward for further biophysical characterizations (FIG. 6).Table 1 : DNA sequences used in this example.

[0086] All sequences were synthesized following the above protocol. Allsurface constructs were modified on their 5’ end with hexanethiol and on their 3’ end with methylene blue.Table 2: Detailed conditions used for FTC aptamer in vitro selection. Q = quick wash; R = repeat times.Rounds 1 2 3 4 5 6 7 8 9 10 11Cub (pM) 4 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.4 nub(pmol) 1000 100 100 100 100 100 100 100 100 100 100CBio_c (pM) 20 2 2 2 2 2 2 2 2 2 2 nBio_c(pmol) 5000 500 500 500 500 500 500 500 500 500 500Vbb8Bl0.c(pL) 250 250 250 250 250 250 250 250 250 250 250Vwash(mL) 0.25 0.25 1 1 1 1 1 1 1 1 1Twash(mins) Q Q 1 1 1 1 1 1 1 1 1Rwash(times) 10 10 10 10 10 10 10 10 10 10 10Cdc (pM) - - - - - - - - 50 50 50Vdc(mL) . . . . . . . . 1 1 1Tdc(mins) - - - - - - - - 1 30 30 dc (times) - - - - - - - - 3 3 3VNsw(mL) . . . . . . . . 1 1 1TNsw(mins) - - - - - - - - 1 1 1 Nsw(times) - - - - - - - . 3 3 3CFTC (pM) 100 100 100 50 50 25 25 10 10 1 1VF-rc(mL) 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25TF-rc(mins) Q Q 1 1 1 1 1 1 1 1 1(tRimftecs.) 3 3 3 3 3 3 3 3 3 3 3Temp. (°C) 23 23 23 23 23 23 23 23 23 23 23CMg2+(mM) 2 2 2 2 2 2 2 2 2 2 2Notes: a. The 1stblock (light orange) shows the library and Bio_cap strand mixture conditions from round 1 to 11. b. The 2ndblock (light gold) shows the wash conditions from round 1 to 11. c. The 3rdblock (light blue) shows the negative / counter selection conditions from round 1 to 11. d. The 4thblock (light green) shows the conditions of extra wash after negative selection from round 1 to 11. e. The 5thblock (light blue-grey) shows the positive selection conditions from round 1 to 11. f. The 6thblock (light grey) shows other selection conditions from round 1 to 11.

[0087] Characterization of Binding Affinity and Selectivity. We characterized the binding affinity of FTC_1_Full against FTC using isothermal titration calorimetry (ITC) [see Methods and reference (16)]. For these measurements, we employed the aptamer sequence as the receptor (at aconcentration of 20 pM) and titrated FTC from a stock solution (at 200 pM). The resulting ITC thermogram (FIG. 6B) revealed a dissociation constant of 150 nM ± 40 nM, which matches clinically relevant concentrations of FTC (> 160 nM at steadystate dosing)(T7 Next, we investigated the selectivity of the aptamer against three structurally related compounds, cytosine (FIG. 6C), 5-fluorocytidine (FIG. 6D), and cytidine (FIG. 6E). Remarkably, FTC_1_Full displayed no heat exchange in the presence of the structural analogs. Because both 5-fluorocytidine and FTC contain a 5-fluorocytosine base, these data suggest that FTC_1_Full interacts with the FTC nucleoside through both its base and sugar moieties. Given this specificity, we continued developing the aptamer for diagnostic assays.

[0088] Analysis of Truncations for Conformation Switching-based Sensing. We set out to develop electrochemical and fluorescence-based sensors for two independent FTC measurement applications by exploiting the aptamer conformation-switching mechanism instilled through the selection process. First, we developed electrochemical aptamer-based sensors (E-ABs) to allow continuous FTC monitoring in vivo, as a tool for the study of FTC disposition in preclinical animal models(T8). To create the E-ABs, we required FTC binding to induce conformationswitching in the aptamer. This is because in E-ABs, target binding-induced conformation switching is used to modulate electron transfer between an aptamerbound redox reporter and the electrode surface (FIG. 7A), which is then measured via electrochemistry (typically square wave voltammetry). Because this sensing mechanism does not employ a complementary oligonucleotide like in the capture- SELEX strategy used to select the aptamer (Bio-Capture strand in Table 1 ), it was critical to optimize the length of stem S1 in FTC_1_Full so that the aptamer remained unfolded in the absence of FTC, but the stem would be rapidly stabilized upon FTC binding.

[0089] Accordingly, we investigated the effect of serial truncations to stem S1 of FTC_1_Full (FIG. 6A) on its FTC binding affinity, shortening it from 13 base pairs (bps) to 2 bps (FIG. 13). Remarkably, hundred nanomolar affinity for FTC was maintained across all truncations tested, indicating that a stem with as little as two bps (FIG. 7A) was sufficient to allow proper folding of the aptamer and binding to FTC (FIG. 7B). Similarly, aptamer selectivity was not affected by the truncations relative to cytosine, cytidine, and 5-fluorocytidine (FIG. 14). Additionally, a scrambledversion of the aptamer showed no binding to FTC, further demonstrating high selectivity (FIG. 15).

[0090] To determine which aptamer truncation generated the largest effect in E-AB sensor signaling output (i.e., the largest conformational change), we synthesized all truncated variants with a 5' alkylthiol linker and a 3' methylene blue reporter modification as indicated in the Methods. Using these modified aptamers, we built E-AB sensors and interrogated them via square wave voltammetry to create frequency maps (FIG. 16), in the presence and absence of FTC. These measurements generate volcano plots with maxima that reflects the average rate of electron transfer of a given E-AB sensor. If FTC binding induces structure switching in the aptamer, the maxima should shift to higher square wave frequency values. Based on these measurements, we determined that the aptamer sequence with only two base pairs in the stem (FIG. 7A) achieved the largest change in electron transfer upon FTC addition (FIG. 7C vs. FIG. 16A-16D). This sequence had 19 nucleotide truncations from the 5' terminus and 25 truncations from the 3' terminus, expressed as FTC_1_5' truncl 9_3'trunc25 in Table 1 , but hereby referred to as FTC_1_EAB for simplicity. By fabricating E-AB sensors with sequence FTC_1_EAB and challenging them with an increasing concentration of FTC in phosphate-buffered saline, we built dose-response curves (FIG. 7D) that successfully covered the clinically relevant range of FTC concentrations, 100 nM to 10 pM, with a sensor gain at the high concentration of ~200%.

[0091] FTC Pharmacokinetic Measurements in Live Preclinical Animal Models. To demonstrate the ability of FTC_1_EAB to support E-AB sensing in vivo in continuous mode, we used this aptamer to fabricate brain probes as previously reported by our group (21). We wanted to demonstrate the E-AB measurement of FTC transport from blood to the brain across the blood brain barrier because this drug and its metabolites may have implications in neurodegenerative processes in HIV patients undergoing FTC therapy.

[0092] For this proof-of-concept demonstration, we placed the E-AB sensors in the cortex of mice (FIG. 8A) and dosed FTC at 75 mg / kg via an intravenous bolus. Our measurement protocol (FIG. 8B) consisted of placing the FTC E-AB sensor in the brain and starting the recording of a sensor baseline for 1 h, interrogating thesensor every 83 s. After this period, we dosed FTC via the tail vein and continued performing FTC measurements for a total of 4 h. An optical photograph of the E-AB brain probes is shown in FIG. 8C. To confirm correct sensor placement in the brain, at the end of the measurements and prior to animal euthanasia we passed a current of 300 pA through the sensor for 2 s to locally bum the tissue, allowing us to determine sensor placement location via histology (FIG. 8B and FIG. 17). The resulting measurements (FIG. 8E, black data) revealed a quick uptake of FTC into the brain cortex immediately after intravenous dosing of FTC. As a negative control, we dosed the rodents with the bolus vehicle, phosphate-buffered saline, without FTC and observed a flat baseline with minimal drift (FIG. 8E, blue data). The cortex FTC pharmacokinetic profile showed transport saturation with a plateau at 2 pM, with excretion beginning ~2.5 h after dosing. The red lines in FIG. 8E illustrate non-linear regression of the data to a model of continuous infusion and first order excretion kinetics. At this stage we do not know if the shape of the profile is dose dependent, but we plan to conduct such experiments in future work. However, these in vivo results confirm the ability of our aptamer to support E-AB sensing in vivo in the brain of mice with high selectivity for FTC.

[0093] As an additional demonstration, we performed measurements in the vein of rats (FIG. 8F) following previously published methods (FIG. 8F, see Methods). The measurement protocol (FIG. 8G) in this case was shorter because the overall drug pharmacokinetics were faster than those observed in the brain. We recorded a 30 min baseline followed by an intravenous FTC bolus, and continued monitoring for 1 h post dosing. For these measurements, we fabricated E-AB sensors in wire bundle format and encased them into 22G medical-grade catheters, as previously reported (FIG. 8H). We then performed surgery in the rats to symmetrically dissect the external jugular veins. The right vein was used for sensor placement and the left vein for FTC dosing (FIG. 8F). In contrast to the profiles measured in the brain, which plateaued at ~2 pM, the intravenous dose achieved a much higher concentration, CMAX of ~20 pM, followed by rapid, first order excretion kinetics, with a half-life of ti / 2 ~ 50 min (FIG. 8I). These results reflect expected differences in drug levels between compartments because the blood-brain-barrier should limit absorption of FTC into the brain. The results also confirm different pharmacokinetics between compartments. Future work beyond the scope of thisstudy will seek out quantitative validation of our in vivo measurements relative to benchmark methods such as immunoassays. We also seek to apply these in vivo measurements to the study of drug-drug interactions and FTC metabolism in the brain.

[0094] The discussed E-AB sensors are not without limitations. Because no proteins were included in the selection buffer during FTC aptamer enrichment, the FTC aptamer binds serum albumin. To illustrate this, we performed ITC measurements using bovine serum albumin (BSA) as the ligand; these measurements revealed a reproducible heat change between FTC_1 and BSA at mole ratios <0.1 (FIG. 19A). However, we overcame this limitation in our in-vein in vivo measurements by encasing the E-AB probes in medical grade catheters filled with phosphate-buffered solution, which allowed the rapid exchange of FTC during our measurement period (FIG. 8I) while filtering proteins via diffusion across the buffer blank, without significant sensor fouling by albumin. The in-brain measurements successfully employed bare sensors without buffer filtering, a potential indication that albumin levels in brain tissue are negligible. Alternative sequences from the enriched aptamer pool that showed less albumin binding (FIGS. 19A-19D), could not be successfully reengineered to undergo FTC-binding induced structure switching that was functional in the E-AB platform.

[0095] Fluorescence-based FTC Measurements in Human Plasma. Similar to E-AB sensing, the reporting mechanism for our fluorescence-based sensor relied on FTC-induced structure switching (FIG. 18A). In this case, the capture strand (Bio-Cap_1 ) originally employed during in vitro selection was repurposed to quench fluorescence upon binding to the dye-labeled aptamer. When challenged with FTC, formation of the aptamer-FTC complex was expected to promote displacement of the quencher-labeled capture strand (Q-Cap_1 ) and closure of the aptamer stem S1 , leading to increased fluorescence emission. Additionally, we truncated 14 nucleotides from the 3'terminus of the FTC_1_Full aptamer to eliminate unpaired nucleotides extending from stem S1 upon FTC binding (see FIGS. 6A and 18A). These truncations also destabilized stem S2 in the unfolded aptamer that could impair structure switching (FIGS. 20 and 21 ). The resulting aptamer-based fluorescent sensor is hereby referred to as FTC_1_Fluor (FTC_1_5'trunc3_3'trunc14 in Table 1 ).

[0096] We showed that affinity and selectivity of FTC_1_Fluor for FTC was comparable to the parent aptamer FTC_1_Full (FIGS. 22 and 23). Importantly, FTC_1_Fluor was stable in 50% human plasma for up to 24 hours (FIG. 24) and the addition of up to 50% human plasma had little effect on sensor performance (FIG. 25), demonstrating the ability of FTC_1_Fluor to support FTC sensing in human biofluids. A kinetic analysis revealed that the maximum fluorescent signal was obtained within 1 minute following exposure of FTC_1_Fluor to FTC in 50% human plasma, highlighting the rapid operation of this sensing platform (FIG. 26). Finally, we obtained a standard curve for FTC detection in 50% human plasma, revealing a limit of detection (LOD) of 0.046 ± 0.001 pM, a limit of quantification (LOQ) of 0.156 ± 0.005 pM, and a half maximal effective concentration (ECso) of 3.536 ± 0.069 pM (R2= 0.99) (FIG. 18B). Unlike Full_1_EAB, the FTC_1_Fluor sequence hybridized with Bio-Cap_1 showed no albumin binding. We confirmed this property via ITC (FIG. 19E).

[0097] To validate the precision of our high-throughput, FTC_1_Fluor for clinical use, we obtained 162 deidentified, FTC-concentration-blinded patient plasma samples from the Centers of Disease Control (CDC) of the United States. We followed the validation study protocol shown in FIG. 19C. First, the samples were deidentified and rescreened for FTC levels at the CDC labs, using the benchmark standard LC-MS assay. Then, the samples were blinded and shipped to the Arroyo lab at the Johns Hopkins University School of Medicine. We screened these samples for FTC concentration using our optimized assay conditions (see Methods). For these measurements, we employed 384-well black plates. We added 20 pL plasma volume from each of the 162 specimens into individual wells. We then diluted with a 20 pL buffer solution of the FTC_1_Fluor / Q-Capture probe to a final concentration of 100 nM / 150 nM (2-fold dilution factor). After 5 min, the plates were interrogated using a benchtop plate reader. Each plate contained an internal calibration curve, shown in FIG. 19B for the data reported in this work. The resulting fluorescence emission intensities were normalized and converted to FTC concentration as discussed in the Methods. The aptamer-measured concentrations were then mailed to the CDC for differential comparison against LC-MS measurements. FTC concentrations measured by our fluorescence assay were in excellent agreement with benchmark LC-MS measurements (R2of 0.85, FIG. 19D). Additionally, ourfluorescent assay achieved 100% correlation in reporting true negatives (i.e., it reported no false positives, FIG. 19E). To stratify the patient sample into FTC positive vs FTC negative groups, we empirically set an FTC threshold at 96 ng / mL, based on current CDC standards (24). This exercise resulted in positive percent (PPA) and negative percent agreements (NPA) of 86.9% and 100%, respectively, relative to LC / MS measurements (FIG. 19F). The overall rate of agreement (ORA) was 95%. To further demonstrate the specificity of our fluorescence assay, we built a receiver operating characteristic curve (ROC curve), which gave a 94.2% area under the curve (AUC) score (FIG. 27). This ROC analysis also confirmed that the threshold for a positive sample was set properly for our assay. Taken together, the data demonstrate that our fluorescent assay achieves similar performance relative to benchmark LC / MS methods currently employed by the CDC, but in a fraction of the time and in high throughput format.

[0098] CONCLUSIONS

[0099] This example shows a complete translational workflow of two aptamer-based sensors. We started by identifying a valuable clinical application, in this case, monitoring adherence to ARV therapy. Then, we carried out aptamer development, biophysical characterizations, and translation to two biosensing platforms: one for in vivo molecular monitoring, and a second one for single-point ARV concentration measurements. The results of this work highlight the current maturity of aptamer technology and the high value it can bring to clinical practice, which by currently being limited to analysis via costly, benchtop instrumentation at centralized facilities, lacks the flexibility to support population-level screening of ARV. Given the advanced stage of the reported FTC sensors, future efforts from our laboratories will focus on continuing clinical translation via formal pilot trials with larger patient cohorts. We will also seek to establish our detection methods at the CDC as routine protocols for convenient ARV monitoring at the point of blood collection and evaluate the applicability of our FTC_1_Fluor to other sample matrices, including urine, saliva, and blood spots.

[0100] METHODS

[0101] Materials. Oligonucleotides were either purchased from Integrated DNA Technologies (Coralville, IA) or prepared by solid-phase synthesis on anExpedite 8909 DNA / RNA Synthesizer. Solid-phase oligonucleotide synthesis reagents, including nucleoside phosphoramidites, 3'-PT-Amino-Modifier C6 CPG, thiol-modifier C6 S-S phosphoram idite, MB NHS ester, and Glen-Pak DNA purification cartridges, were purchased from Glen Research (Sterling, Va). All oligonucleotides were purified by denatured polyacrylamide gel electrophoresis (PAGE), desalted using an Amicon Ultra-15 Centrifugal Filtration Unit (3kDa MWCO), and ethanol-precipitated prior to use. Taq DNA polymerase was expressed and purified according to literature procedures (25). MyOne™ Streptavidin C1 magnetic Dynabeads, high-capacity streptavidin agarose beads (50% slurry), and SuperScript II Reverse Transcriptase were purchased from Thermo Fischer Scientific (Waltham, MA). Deoxynucleotide triphosphates (dNTPs), deoxycytidine, and FTC (PHR2120- 500MG) were purchased from Sigma Aldrich. All the other chemicals were purchased from either Sigma-Aldrich or Alfa Aesar and used as received. Phosphate buffered saline (PBS) (46-013-CM) was purchased from Coming Inc (Corning, NY). All other buffers were prepared in house using Milli-Q purified water (18.2 MQ) and filtered using a 0.22 pm membrane prior to use.

[0102] Capture SELEX. 200 picomoles of a single-stranded DNA library (All sequences used during capture SELEX are listed in Table S1 ) was amplified by PCR in a 10 mL reaction volume containing 0.5 pM of each primer (Fwd_1 and Rev_1 ), 50 mM KCI, 1.5 mM MgCI2, 0.1% TRITON-X, 10 mM Tris (pH 9.0), 0.5 mM each of the four dNTPs, and 0.05 U / pL Taq DNA Polymerases. The PCR reaction was carried out using the following temperature cycling: i) 95° C for 3 min, (ii) 95° C for 30 s, (iii) 59° C for 30 s, (iv) 72° C for 1 min, and (v) 72° C for 5 min, with repeating steps (ii) to (iv) for 5 total cycles. The resulting dsDNA products were precipitated by ethanol, redispersed in 100 pL TE buffer (10 mM Tris (pH 7.6), 1 mM EDTA), and then added directly to 500 pL of settled high-capacity streptavidin coated agarose beads in wash buffer (WB; 100 mM NaCI, 50 mM Tris, pH 7.6). After incubating at room temperature for 30 min, the beads were washed twice with 500 pL WB buffer and once with 500 pL water. The nonbiotinylated strands were then eluted from the beads using 2 x 175 pL of ice-cold elution buffer (EB, 50 mM NaOH, 1 mM EDTA). The eluent was immediately neutralized by the addition of 35 pL 1 M Tris (pH 7.6) and 35 pL 3 M NaOAc, and the resulting mixture was precipitated by ethanol. The obtained ssDNA library was further purified by denaturing PAGE (10%, 19:1acrylamide:bis-acrylamide) prior to use.

[0103] Meanwhile, 200 pL streptavidin coated magnetic beads (slurry) were washed following manufacturer’s instructions and the non-specific sited were blocked using 500 pL of SELEX buffer (SB; 1x PBS, 2 mM MgCl2) containing 1 mg / mL yeast tRNA. The ssDNA library prepared above (Lib_1 ) and a capture oligonucleotide (Bio-Cap_1 ) were mixed at a 1 :5 molar ratio in 250 pL of SB buffer. The DNAs were heated to 98° C for 5 min and allowed slowly cool to room temperature. The library / capture oligonucleotide mixture was then added to the prepared magnetic beads and the slurry was allowed to incubate at room temperature for 30 minutes with gentle shaking. The beads were then washed 10 times with 1 mL SB buffer to remove unbound or weakly bound sequences. The length of each washing step is listed in Table 2 and varied per round. After washing, functional aptamers were eluted from the beads using 3 x 250 pL washes with SB buffer containing FTC (this is referred to as the positive selection step). Again, the length of each washing step and the FTC concentration used is listed in Table 2 and varied per round. The eluent from each of the three positive selection washes were combined, precipitated by ethanol, and the resulting pellet was dissolved in 100 pL water. Half of this DNA was used as template for PCR to generate the enriched ssDNA library for the next round selection (as described above). For rounds 9 and onward, a negative selection step was introduced following the initial 10 x 1 mL SB buffer washes. Here, non-selective aptamers were eluted from the beads using 3 x 1 mL washes with SB buffer containing 50 pM deoxycytidine (see Table 2). The washed beads were then progressed directly into the positive selection step. To track the selection process, a small-scale PCR was run during each selection round to generate an elution profile using the eluant from (1 ) the first and last washes from the initial 10 x 1 mL SB washing step, (2) the final wash of the negative selection step (if applicable), and (3) all three washes from the positive selection step. Representative data is shown in FIG. 9.

[0104] DNA Sequencing. The ssDNA eluted from SELEX round 11 was amplified as described above and purified in a 2% agarose gel. The purified DNA was subjected to a second PCR round using sequencing primers (Table 1 ). Nextgeneration sequencing was carried out at the Johns Hopkins School of Medicine Genetic Resources Core Facility using an Illumina MiSeq System. Sequence readswere analyzed and clustered using the FASTAptamer toolkit (Levenshtein edit distance of 4 nucleotides).

[0105] Isothermal Titration Calorimetry (ITC). All measurements were performed using an Affinity ITC (TA Instruments, New Castle, DE) coupled to an autosampler and associated software. All target and aptamer solutions were first prepared in PBS + 2 mM MgCl2 followed by 10 min of degassing prior to ITC measurements. Before each titration, the system was cleaned three times with deionized water, followed by a 300 s equilibration in the same buffer. The cell was loaded with 20 pM of the respective aptamer, and the syringe was loaded with 200 pM of the appropriate target. Heat-exchange was recorded after a 1 pL injection of target, over 35 injection points, with 150 - 180 s of equilibration time between each injection. All titrations were carried out at 25° C. Experimental and cleaning protocols may be found in the data repository associated with this publication.

[0106] Synthesis of Fluorescent Sensor FTC_1_Fluor. The truncated aptamer FTC_1_Fluor was purchased from Integrated DNA Technologies (Coralville, IA) with a 5' amino modifier (5' Amino Modifier C6). The quencher strand Q_Cap_1 was prepared by solid-phase synthesis on an Expedite 8909 DNA / RNA Synthesizer using a 3'-Black Hole Quencher 2 (BHQ2)-CPG according to manufacturer protocol. Sulfo-Cy5 N-hydroxysuccinimide (NHS) ester (Lumiprobe Corp., Hunt Valley, MD) was conjugated to the 5' end of FTC_1_Fluor. Conjugation reactions were performed by combining the 5' amino modified oligonucleotide (100 pM) with the dye NHS ester (5 mM final concentration) in 0.1 M sodium bicarbonate buffer (pH 8.5). The reaction was vortexed intermittently over a 16-hour period at 23°C. Samples were then ethanol precipitated and the labeled oligonucleotide was purified by 20% denaturing PAGE (19:1 acrylamide: bisacrylamide). Purified oligonucleotides were excised from the gel and eluted overnight at 23 °C in elution buffer (200 mM NaCI, 10 mM EDTA, and 10 mM Tris (pH 7.6)). The solution was then filtered to remove gel fragments, and eluted oligonucleotides were desalted by ethanol precipitation and an Amicon Ultra Centrifugal Filter 3KDa (MilliporeSigma, Burlington, MA). The concentration was determined by absorbance at 260 nm on a NanoDrop 2000c (ThermoFisher, Waltham, MA). The identity of the modified aptamer was confirmed by mass spectrometry (FIG. 28).

[0107] Fluorescent Biosensor Measurements. We first prepared a 4X assay master mix and a 4X control master mix containing all required components. The 4X assay master mix was composed of 400 nM Cy5-aptamer FTC_1_Fluor and 600 nM Q_Cap_1 in buffer F (4x PBS and 4 mM MgCl2). The 4X control master mix was identical but lacked Q_Cap_1. Prior to use, both the assay mix and control mix were heated to 95°C for 5 minutes, and slowly cooled to 22° C to anneal the aptamer and capture-strand components. A standard curve was generated by conducting the fluorescence assay using a two-fold dilution of FTC in human plasma. In brief, a solution of 320 pM FTC in plasma was first prepared, followed by two-fold serial dilution with plasma to generate a set of standard samples for calibration. 10 pL of either the 4X assay or control master mix was diluted with 10 pL H2O and transferred to a black 384-well plate (Corning #3575). 20 pL of the indicated FTC standard plasma sample was added to the assay mixtures. Plasma without FTC was added to the control assay mixtures. After 5 min, the fluorescence was measured on a GloMax Discover multi-well plate reader (Ex: 627 nm; Em: 660-720 nm) or a Molecular Devices SpectraMax ID5 (Ex: 627 nm; Em: 670 nm). All fluorescence was normalized to the control assay.

[0108] The standard curve was generated by curve fitting in GraphPad Prism (v.9.3.1 ) using one-phase exponential model, according to eq. 1 :where Y represents the normalized fluorescence of measurement, YMax represents the maximum normalized fluorescence, Ysiank represents the normalized background fluorescence, K represents the concentration scaling factor. Limit of detection (LOD) and limit of quantification (LOQ) were determined by performing fluorescence assays with 15 blank plasma samples. LOD was defined as the average fluorescence of blank plus 3 standard deviations of the blank and LOQ was determined as the average fluorescence of blank plus 10 standard deviations of the blank measurement. The calculated fluorescence value for LOD and LOQ was converted into concentration using the above-described standard curve. Kinetic assays (FIG. 24) were carried out similarly and were monitored for 40 minutes at 22° C.

[0109] Plasma Stability Assay. A 40 pL reaction mixture was prepared as described above containing 50% human plasma with or without 10 pM FTC. At theindicated times, a 3 pL aliquot was taken and quenched with 12 pL denaturing PAGE loading buffer (90% formamide, 10 mM EDTA). The quenched solution was then resolved by 20% denaturing PAGE (19:1 acrylamide: bisacrylamide) and imaged by a Typhoon FLA9500 Multimode Imager (Cy5; excitation / emission: 649 nm / 670 nm).

[0110] Clinical Samples. Peripheral blood was collected from HIV-negative male participants in two clinical studies registered at clinicaltrials.gov and conducted at the Emory Hope Clinic in Atlanta, Georgia. (27, 28) Both trials were funded by the US Centers for Disease Control and Prevention (CDC) and approved by Emory University and CDC Institutional Review Boards. All study participants gave written informed consent, and the trials conform to the US Federal Policy for the Protection of Human Subjects. In one study, 40 male participants provided 108 blood specimens after receiving a single oral dose formulation containing 200 mg emtricitabine (FTC), 10 mg tenofovir alafenamide (TAF), 150 mg elvitegravir (EVG) and 150 mg cobicistat (COBI) as well as a single 800 mg dose of darunavir (DRV) (NCT03472963). In the second study, 31 male participants provided 54 blood specimens after receiving two doses of a single formulation containing 200 mg FTC, 10 mg TAF, 150 mg EVG and 150 mg COBI given 24 hours apart (NCT03976752). Peripheral blood specimens were collected in sodium citrate cell preparation tubes (CPT) (Becton Dickinson, Franklin Lakes, New Jersey, USA). Plasma aliquots were collected from CPT following centrifugation. Concentrations of FTC were measured in plasma specimens using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) (Sciex, Foster City, CA, Shimadzu Scientific Instruments, Durham, NC) as previously described.

[0111] Aptamers for Electrochemical Sensors. Aptamers for electrochemical sensing were functionalized with a 6-carbon disulfide group (thiol- modifier C6 S-S, Glen Research) at their 5' terminus and two methylene blue (MB) units at their 3' terminus. Solid-phase synthesis was initiated using 3'-PT-Amino- Modifier C3 CPG (Glen Research) followed by coupling of a TFA-Amino C7 Multiaddition CE-phosphoramidite (BioSearch Technologies), allowing for the eventual conjunction of two MB molecules to the 3' terminus. Following synthesis of the aptamer sequence (Table 1 ), a 6-carbon disulfide group (thiol-modifier C6 S-S, Glen Research) was added to the 5' terminus. The terminal 4,4'-dimethoxytrityl (DMT) group was retained for downstream purification. The synthesized aptamerswere deprotected using AMA (ammonium hydroxide / 40% aqueous methylamine, 1 :1 v / v) and concentrated using a Glen-Pak DNA purification cartridge following the manufactures instructions. The deprotected aptamers were then purified by 20% denaturing PAGE (19:1 acrylamide / bisacrylamide). Purified aptamers were excised from the gel and eluted overnight at 23 °C in a buffer consisting of 200 mM NaCI, 10 mM EDTA, and 10 mM Tris (pH 7.6). The suspension was then filtered to remove gel fragments, and eluted aptamers were concentrated using an a 3K Am icon® filter device (Sigma Aldrich, St. Louis, MO) and desalted by ethanol precipitation.

[0112] MB conjugation reactions were performed by combining the amino modified aptamer (100 pM) with the MB NHS ester (2 mM final concentration) in 20 pL of 0.1 M bicarbonate buffer (pH 9) containing 10% DMSO. The reaction was vortexed intermittently over a 16 hour period at 23 °C. The reaction mixture was then passed through a NAP-5 Sephadex G-25 Column (GE Healthcare, Chicago, IL) to remove excess MB and the modified aptamer was further purified by 20% denaturing PAGE as before. The identity of all MB-modified aptamers was confirmed by mass spectrometry (FIG. 29).

[0113] Fabrication of In-Vivo Electrochemical Probes. We fabricated two sensor form factors, one for deployment in the jugular vein of rats (i.e. , for blood drug measurements) and one for in-brain measurements. For blood measurements, E- ABs were fabricated as described in previous reports(22, 29). Segments of pure gold (7.75 cm in length), platinum (7.25 cm), and silver (6.75 cm) wire, were cut to make the sensor body. The insulation at both ends of these wires, about 1 cm, was removed using a surgical blade to allow electrical contact. These were then soldered each to one of the three ends of a connector cable using 60% tin / 40% lead rosincore solder (0.8 mm diameter) and then attached together by applying heat to shrinkable tubing around the body of the wires, except for a small window of about 5 mm at the edge of each wire. The wires were stacked staggered, with the gold wire being insulated alone first, then both gold and platinum wires together, and finally all three wires together. To prevent electrical shorts between wires, different lengths were used for each wire as described above. The sensor window (i.e., the region devoid of insulation) in the gold wire was cut to approximately 3 mm in length. For brain measurements, we fabricated probes by adapting the carbon-fiber electrode fabrication protocol from the field of fast-scan cyclic voltammetry to create gold-based microprobes for in-brain measurements (30). Briefly, Au wire (50 pm in diameter, GoodFellow, Huntington, Eng) was inserted into a 75 pm ID / 150 pm OD fused silica capillary (Molex Incorporated, Lisle, IL) and sealed on one end using two-part 3-5 min curing Double / Bubble epoxy (McMaster-Carr, Elmhurst, IL), with ~ 0.5 cm of Au wire protruding from each end of the capillary. This capillary / Au wire was then inserted inside a guide cannula (18GA cut 1 mm below and 10 mm above pedestal, PlasticsOne Technologies, Roanoke, VA) and glued on both end using the same epoxy, leaving 1 cm of the capillary / Au wire exposed on the long end of the guide cannula (side to be functionalized with the FTC aptamer) and ~300 pm on the other end. Electrical connection to the latter was achieved by soldering the exposed Au wire on the short end to a 30 AWG Insulated Kynar Copper Wire Roll cable (Amazon) which had been previously pealed on both ends to expose the conductive material (named hereafter as Cu-cable). The soldering process was aided with 280 pM in diameter shrink tubing (Zeus, Orangeburg, SC) to hold the capillary / Au wire protruding from the short end to the Cu-wire. The Cu-wire’s end opposite to the Au wire-connection was previously soldered to an Au pin. Finally, 28-26 AWG shrink tube (FP-301 thin wall, 3M, Saint Paul, MN) was used to protect and reinforce all soldered areas. The active sensor windows of the probes were approximately 500 pm-1 mm long and 50 pm in diameter. The reference electrodes were fabricated by soldering one extreme end of a Cu-cable to a Au pin and the other extreme end to a Ag wire. We added adhesive to the soldered area to provide more support. Before implantation in the brain, we immersed ~2 mm of the Ag wire in a solution of 0.3 M FeCh in 0.1 M HCI for 30 s to produce the Ag / AgCI reference electrode. For the counter electrodes, we soldered a stainless-steel screw to a Cu-cable.

[0114] Preparation of Electrochemical, Aptamer-Based Sensors (E-ABs). Regardless of form factor, all electrochemical probes were prepared as follows: we electrochemically cleaned the in vivo probes using a Gamry Reference 600+ potentiostat (Warminster, PA) by performing 200 cycles using cyclic voltammetry first from -0.3 to -1.6 V (vs Ag|AgCI) in 0.5 M NaOH at a scan rate of 0.5 V / s, and then from 0 to 1.55 V (vs Ag|AgCI) in 0.5 M H2SO4 also at a scan rate of 0.5 V / s. We then increased the electroactive surface area of the electrodes by roughening their surface via chronoamperometry. For this, we performed 300 pulses (0.01 s pulse width and lowest sensitivity) from 0 to 2 V (vs Ag|AgCI). This was repeated 100 timesusing a macro (for a total of 30,000 pulses). Finally, we performed an additional 20 cycles in H2SO4 (same conditions as during electrochemical cleaning) to ensure the stability of the roughened surface. While the electrodes were being cleaned and roughened, a 1 pL aliquot of 100 pM FTC aptamer solution was reduced with 2 pL of 5 mM TCEP for 1 h and then diluted to 500 nM using PBS. The probes were incubated in the diluted aptamer solution, immediately after electrochemical roughening, for 2 h at 22° C (manually stirred the electrode in the solution to homogenize the solution / electrode interface). Finally, the electrodes were removed from the aptamer solution and immediately transferred into 1 mM MCH at room temperature overnight. During both incubation periods, we protected the solutions from evaporation by covering the electrodes / solution with a petri dish that was taped to the bench to minimize air flow.

[0115] The next day, we removed the electrodes from the MCH solution and thoroughly rinsed with deionized water before placing them in a beaker with PBS solution. We then checked the quality of the sensors by performing square wave voltammograms from 0 to 0.5 V (vs Ag|AgCI) at 30 and 300 Hz (50 mV amplitude and 2 mV step size). At this point, we ran 100 consecutive square wave voltammograms at 300 Hz to “pretreat” the sensor and remove any nonspecifically bound aptamer from the electrode surface. We kept the sensors in this PBS solution until ready to implant in the mice brain.

[0116] Rodent Surgical Procedures. In-blood FTC measurements were performed in male Sprague-Dawley rats purchased from Charles River Laboratories (Code: 400, Severn, MD), weighing between 300 and 350 g. All animals were pair housed in a standard light cycle room (08:00 on, 20:00 off) and allowed ad libitum access to food and water. The rats were induced under 4% isoflurane anesthesia in a Plexiglas anesthesia chamber. The rats were then maintained at 2-3% isoflurane gas for the duration of the experiment. While anesthetized, surgery was performed to emplace the E- AB sensor within the jugular veins. Briefly, the area above the left jugular vein was shaved and cleaned with betadine and 70% ethanol. A small incision was made above the vein, then the vein was isolated. A small hole was cut into the vein with spring-loaded micro scissors. We then inserted the E-ABs and tied them into place sterile silk suture (Fine Science Tools, Foster City, CA). Once E-AB measurements were concluded, the rats were overdosed on 5% isoflurane gas untilbreathing and heartbeat ceased and then decapitated using a guillotine. For in-brain measurements, we used male C57BL / 6J mice (Strain #: 000664; The Jackson Laboratory, Bar Harbor, ME). Before surgery, we removed the mice from their home cage and placed them in an anesthesia box where we flowed O2 at 1 L / min and 5% isoflurane until the mouse is under the surgical place of anesthesia (confirmed by lack of toe-pinch reaction). We shaved the hair off the head from eyes to ears level and, once we immobilized the head in the stereotaxic frame (Stoelting, Wood Dale, IL), we lowered the isoflurane level to 3% (always checking for lack of toe-pinch reaction). We applied eye lube to the eyes and betadine to the shaved area using a cotton-tip applicator before making an incision using a scalpel. We removed the connective tissue from the exposed skull using a cotton-tip applicator and added 3% hydrogen peroxide solution to aid in the visualization of the sagittal, bregma, and lambda suture lines (for alignment of the head with the stereotaxic frame). Next, we drilled three holes in the skull with the following coordinates:1. Reference electrodes: +4.80 AP (from bregma), -1.00 ML (from bregma), and -2.00 DV (from brain surface)2. E-ABs electrodes: -1.70 AP (from bregma), +0.50 ML (from bregma), and -1.00 DV (from brain surface)3. Counter-electrode screws: -2.90 AP (from bregma), -2.30 ML (from bregma), and 3-4 full rotations inside the hole.To find the brain surface with the E-ABs, we connected the Gamry Reference 600+ potentiostat to all three electrodes but kept the E-ABs near the entrance of its hole. We initiated 10 consecutive cyclic voltammograms from 0 to 0.5 V at 0.01 V / s and slowly lowered the sensor tip toward the brain. When the sensor is not touching the brain, the cyclic voltammogram shows just noise because the electrical circuit is open. Once the tip of the sensor touches the brain, a spike in the cyclic voltammogram current is observed, indicating the circuit is closed. At this point, we zeroed the DV coordinate of the digital stereotaxic arm reader and lowered the electrode to its final depth inside the brain. Finally, we run another “pretreatment” session of 60 square wave voltammograms from 0 to 0.5 V at a frequency of 300 Hz (2 mV step, 50 mV amplitude) to ensure the sensor is homogenized with the brain tissue before starting the experiment. All procedures were approved by the JohnsHopkins University Animal Care and Use Committee (Protocols: MO19L258, MO22M234, RA22M242) and in accordance with the Guide for the Care and Use of Laboratory Animals.

[0117] In vivo FTC measurements. For all in vivo measurements a 60 min sensor baseline was established before drug infusions. After establishing a stable baseline, FTC was infused at a dose of 75 mg / kg via tail-vein intravenous administration. E-AB recordings continued for up to 6 h before experiment termination and euthanasia. In-brain measurements were performed at 10 and 550 Hz, 50 mV amplitude and 2 mV potential step. The real-time plotting and analysis of voltammetric data were carried out via SACMES, an open access script previously reported by our group.

[0118] Additional exemplary aspects of the present disclosure are provided in the accompanying APPENDICES A and B, which are incorporated by reference in their entirety.

[0119] Some further aspects are defined in the following clauses:

[0120] Clause 1 : A method of detecting an antiretroviral therapeutic (ART) agent in a sample from a subject, the method comprising: contacting the sample that comprises the ART agent with a plurality of biomolecular receptors such that one or more of the biomolecular receptors undergo conformational changes when the biomolecular receptors bind the ART agent; and, detecting one or more detectable signals produced when the biomolecular receptors undergo the conformational changes using a detector, thereby detecting the ART agent in the sample from the subject.

[0121] Clause 2: The method of Clause 1 , wherein biomolecular receptorbound redox reporters comprise the biomolecular receptors, wherein the biomolecular receptor-bound redox reporters are attached to a surface of an electrochemical sensor, and wherein when the biomolecular receptors undergo the conformational changes to produce one or more electrochemical signals that are detected using the detector.

[0122] Clause 3: The method of Clause 1 or Clause 2, wherein the electrochemical sensor comprises a wearable device that is worn by the subject.

[0123] Clause 4: The method of any one of the preceding Clauses 1-3,wherein the detector comprises an electrochemical analyzer.

[0124] Clause 5: The method of any one of the preceding Clauses 1-4, wherein the redox reporters comprise methylene blue (MB) or an osmium-based complex.

[0125] Clause 6: The method of any one of the preceding Clauses 1-5, wherein the electrochemical sensor is configured to generate one or more square wave or cyclic voltammograms using square wave voltammetry (SWV) and / or cyclic voltammetry (CV) and determine a change in peak-to-peak separation, EP.T, from the voltammograms to detect the ART agent when the biomolecular receptors bind the ART agent.

[0126] Clause 7: The method of any one of the preceding Clauses 1-6, wherein the biomolecular receptor-bound redox reporters are attached to the surface of the electrochemical sensor via an electrically conductive layer disposed on the surface.

[0127] Clause 8: The method of any one of the preceding Clauses 1-7, wherein a thickness of the electrically conductive layer is less than about 1 pm.

[0128] Clause 9: The method of any one of the preceding Clauses 1-8, wherein the plurality of biomolecular receptor-bound redox reporters operably attached to the electrically conductive layer comprise one or more self-assembling biosensing monolayers that comprise one or more electrode-blocking alkanethiols, one or more alkanethiols, and one or more redox reporter-tagged aptamers.

[0129] Clause 10: The method of any one of the preceding Clauses 1-9, wherein the biomolecular receptors comprise fluorescent reporter and quencher labeling moieties, wherein the quencher labeling moieties substantially quench fluorescent signals emitted from the fluorescent reporter labeling moieties when the biomolecular receptors are not bound to the ART agent, wherein the fluorescent reporter and quencher labeling moieties separate from one another a sufficient distance when the biomolecular receptors bind to the ART agent such that the fluorescent signals emitted from the fluorescent reporter labeling moieties are not substantially quenched by the quencher labeling moieties and are detected using the detector.

[0130] Clause 11 : The method of any one of the preceding Clauses 1-10,comprising repeating the contacting and detecting steps one or more times using samples obtained from the subject at different time points to monitor levels of the ART agent in the subject over time.

[0131] Clause 12: The method of any one of the preceding Clauses 1-11 , wherein the sample is substantially unprocessed prior to the contacting step.

[0132] Clause 13: The method of any one of the preceding Clauses 1-12, wherein the sample comprises whole blood, serum, or plasma.

[0133] Clause 14: The method of any one of the preceding Clauses 1-13, wherein the sample comprises urine.

[0134] Clause 15: The method of any one of the preceding Clauses 1-14, wherein the subject comprises a human immunodeficiency virus (HIV) infection.

[0135] Clause 16: The method of any one of the preceding Clauses -15, wherein the subject is a human subject.

[0136] Clause 17: The method of any one of the preceding Clauses -16, wherein the ART agent comprises emtricitabine.

[0137] Clause 18: The method of any one of the preceding Clauses -17, wherein the ART agent comprises elvitegravir.

[0138] Clause 19: The method of any one of the preceding Clauses -18, wherein the biomolecular receptor comprises a nucleic acid molecule.

[0139] Clause 20: The method of any one of the preceding Clauses 1-19, wherein the biomolecular receptor comprises an aptamer.

[0140] Clause 21 : The method of any one of the preceding Clauses 1-20, wherein the aptamer comprises a 5’- thiol modification and a 3’ methylene blue modification.

[0141] Clause 22: The electrochemical sensor device of any one of the preceding Clauses 1-21 , wherein the aptamer comprises a nucleotide sequence selected from SEQ ID NOS: 1 -10 and 14-30 or a nucleotide sequence having sufficient sequence identity thereto so that the aptamer binds (e.g., specifically binds) the ART agent.

[0142] Clause 23: A system, comprising: a substrate comprising a plurality ofbiomolecular receptors; a detector configured to detect one or more detectable signals produced when a sample that comprises an antiretroviral therapeutic (ART) agent is contacted with the plurality of biomolecular receptors such that one or more of the biomolecular receptors undergo conformational changes when the biomolecular receptors bind the ART agent; and, at least one controller operably connected to the detector, which 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: detecting the one or more detectable signals produced when the biomolecular receptors undergo the conformational changes using a detector.

[0143] Clause 24: The system of Clause 23, wherein biomolecular receptorbound redox reporters comprise the biomolecular receptors, wherein an electrochemical sensor comprises the substrate, wherein the biomolecular receptorbound redox reporters are attached to a surface of the electrochemical sensor, and wherein the biomolecular receptors undergo the conformational changes to produce one or more electrochemical signals that are detected using the detector.

[0144] Clause 25: The system of Clause 23 or Clause 24, wherein the electrochemical sensor comprises a wearable device that is worn by the subject.

[0145] Clause 26: The system of any one of the preceding Clauses 23-25, wherein the detector comprises an electrochemical analyzer.

[0146] Clause 27: The system of any one of the preceding Clauses 23-26, wherein the redox reporters comprise methylene blue (MB) or an osmium-based complex.

[0147] Clause 28: The system of any one of the preceding Clauses 23-27, wherein the electrochemical sensor is configured to generate one or more square wave or cyclic voltammograms using square wave voltammetry (SWV) and / or cyclic voltammetry (CV) and determine a change in peak-to-peak separation, EP.T, from the voltammograms to detect the ART agent when the biomolecular receptors bind the ART agent.

[0148] Clause 29: The system of any one of the preceding Clauses 23-28, wherein the biomolecular receptor-bound redox reporters are attached to the surface of the electrochemical sensor via an electrically conductive layer disposed on thesurface..

[0149] Clause 30: The system of any one of the preceding Clauses 23-29, wherein a thickness of the electrically conductive layer is less than about 1 pm.

[0150] Clause 31 : The system of any one of the preceding Clauses 23-30, wherein the plurality of biomolecular receptor-bound redox reporters operably attached to the electrically conductive layer comprise one or more self-assembling biosensing monolayers that comprise one or more electrode-blocking alkanethiols, one or more alkanethiols, and one or more redox reporter-tagged aptamers.

[0151] Clause 32: The system of any one of the preceding Clauses 23-31 , wherein the biomolecular receptors comprise fluorescent reporter and quencher labeling moieties, wherein the quencher labeling moieties substantially quench fluorescent signals emitted from the fluorescent reporter labeling moieties when the biomolecular receptors are not bound to the ART agent, wherein the fluorescent reporter and quencher labeling moieties separate from one another a sufficient distance when the biomolecular receptors bind to the ART agent such that the fluorescent signals emitted from the fluorescent reporter labeling moieties are not substantially quenched by the quencher labeling moieties and are detected using the detector.

[0152] Clause 33: The system of any one of the preceding Clauses 23-32, wherein the non-transitory computer executable instructions which, when executed by the electronic processor, further perform at least: repeating the detecting step one or more times using samples obtained from the subject at different time points to monitor levels of the ART agent in the subject over time.

[0153] Clause 34: The system of any one of the preceding Clauses 23-33, wherein the sample is substantially unprocessed.

[0154] Clause 35: The system of any one of the preceding Clauses 23-34, wherein the sample comprises whole blood, serum, or plasma.

[0155] Clause 36: The system of any one of the preceding Clauses 23-35, wherein the sample comprises urine.

[0156] Clause 37: The system of any one of the preceding Clauses 23-36, wherein the subject comprises a human immunodeficiency virus (HIV) infection.

[0157] Clause 38: The system of any one of the preceding Clauses 23-37, wherein the subject is a human subject.

[0158] Clause 39: The system of any one of the preceding Clauses 23-38, wherein the ART agent comprises emtricitabine.

[0159] Clause 40: The system of any one of the preceding Clauses 23-39, wherein the ART agent comprises elvitegravir.

[0160] Clause 41 : The system of any one of the preceding Clauses 23-40, wherein the biomolecular receptor comprises a nucleic acid molecule.

[0161] Clause 42: The system of any one of the preceding Clauses 23-41 , wherein the biomolecular receptor comprises an aptamer.

[0162] Clause 43: The system of any one of the preceding Clauses 23-42, wherein the aptamer comprises a 5’- thiol modification and a 3’ methylene blue modification.

[0163] Clause 44: The electrochemical sensor device of any one of the preceding Clauses 23-43, wherein the aptamer comprises a nucleotide sequence selected from SEQ ID NOS: 1 -10 and 14-30 or a nucleotide sequence having sufficient sequence identity thereto so that the aptamer binds (e.g., specifically binds) the ART agent.

[0164] Clause 45: A computer readable media comprising non-transitory computer executable instructions which, when executed by at least electronic processor, perform at least: detecting one or more detectable signals produced when a plurality of biomolecular receptors undergo conformational changes using a detector, which detector is configured to detect the one or more detectable signals produced when a sample that comprises an antiretroviral therapeutic (ART) agent is contacted with the plurality of biomolecular receptors such that one or more of the biomolecular receptors undergo the conformational changes when the biomolecular receptors bind the ART agent.

[0165] 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 thedisclosure 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.

Claims

WHAT IS CLAIMED IS:1 . A method of detecting an antiretroviral therapeutic (ART) agent in a sample from a subject, the method comprising: contacting the sample that comprises the ART agent with a plurality of biomolecular receptors such that one or more of the biomolecular receptors undergo conformational changes when the biomolecular receptors bind the ART agent; and, detecting one or more detectable signals produced when the biomolecular receptors undergo the conformational changes using a detector, thereby detecting the ART agent in the sample from the subject.

2. The method of claim 1 , wherein biomolecular receptor-bound redox reporters comprise the biomolecular receptors, wherein the biomolecular receptorbound redox reporters are attached to a surface of an electrochemical sensor, and wherein when the biomolecular receptors undergo the conformational changes to produce one or more electrochemical signals that are detected using the detector.

3. The method of claim 2, wherein the electrochemical sensor comprises a wearable device that is worn by the subject.

4. The method of claim 2, wherein the detector comprises an electrochemical analyzer.

5. The method of claim 2, wherein the redox reporters comprise methylene blue (MB) or an osmium-based complex.

6. The method of claim 2, wherein the electrochemical sensor is configured to generate one or more square wave or cyclic voltammograms using square wave voltammetry (SWV) and / or cyclic voltammetry (CV) and determine a change in peak-to-peak separation, EP.T, from the voltammograms to detect the ART agent when the biomolecular receptors bind the ART agent.

7. The method of claim 2, wherein the biomolecular receptor-bound redox reporters are attached to the surface of the electrochemical sensor via an electrically conductive layer disposed on the surface.

8. The method of claim 7, wherein a thickness of the electrically conductive layer is less than about 1 pm.

9. The method of claim 7, wherein the plurality of biomolecular receptorbound redox reporters operably attached to the electrically conductive layer comprise one or more self-assembling biosensing monolayers that comprise one or more electrode-blocking alkanethiols, one or more alkanethiols, and one or more redox reporter-tagged aptamers.

10. The method of claim 1 , wherein the biomolecular receptors comprise fluorescent reporter and quencher labeling moieties, wherein the quencher labeling moieties substantially quench fluorescent signals emitted from the fluorescent reporter labeling moieties when the biomolecular receptors are not bound to the ART agent, wherein the fluorescent reporter and quencher labeling moieties separate from one another a sufficient distance when the biomolecular receptors bind to the ART agent such that the fluorescent signals emitted from the fluorescent reporter labeling moieties are not substantially quenched by the quencher labeling moieties and are detected using the detector.11 . The method of claim 1 , comprising repeating the contacting and detecting steps one or more times using samples obtained from the subject at different time points to monitor levels of the ART agent in the subject over time.

12. The method of claim 1 , wherein the sample is substantially unprocessed prior to the contacting step.

13. The method of claim 1 , wherein the sample comprises whole blood, serum, or plasma.

14. The method of claim 1 , wherein the sample comprises urine.

15. The method of claim 1 , wherein the subject comprises a human immunodeficiency virus (HIV) infection.

16. The method of claim 1 , wherein the subject is a human subject.

17. The method of claim 1 , wherein the ART agent comprises emtricitabine.

18. The method of claim 1 , wherein the ART agent comprises elvitegravir.

19. The method of claim 1 , wherein the biomolecular receptor comprises a nucleic acid molecule.

20. The method of claim 1 , wherein the biomolecular receptor comprises an aptamer.21 . The method of claim 20, wherein the aptamer comprises a 5’- thiol modification and a 3’ methylene blue modification.

22. The method of claim 20, wherein the aptamer comprises a nucleotide sequence selected from SEQ ID NOS: 1-10 and 14-30 or a nucleotide sequence having sufficient sequence identity thereto so that the aptamer binds the ART agent.

23. A system, comprising: a substrate comprising a plurality of biomolecular receptors; a detector configured to detect one or more detectable signals produced when a sample that comprises an antiretroviral therapeutic (ART) agent is contacted with the plurality of biomolecular receptors such that one or more of the biomolecular receptors undergo conformational changes when the biomolecular receptors bind the ART agent; and, at least one controller operably connected to the detector, which 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: detecting the one or more detectable signals produced when the biomolecular receptors undergo the conformational changes using a detector.

24. The system of claim 23, wherein biomolecular receptor-bound redox reporters comprise the biomolecular receptors, wherein an electrochemical sensor comprises the substrate, wherein the biomolecular receptor-bound redox reporters are attached to a surface of the electrochemical sensor, and wherein the biomolecular receptors undergo the conformational changes to produce one or more electrochemical signals that are detected using the detector.

25. The system of claim 24, wherein the electrochemical sensor comprises a wearable device that is worn by the subject.

26. The system of claim 24, wherein the detector comprises an electrochemical analyzer.

27. The system of claim 24, wherein the redox reporters comprise methylene blue (MB) or an osmium-based complex.

28. The system of claim 24, wherein the electrochemical sensor is configured to generate one or more square wave or cyclic voltammograms using square wave voltammetry (SWV) and / or cyclic voltammetry (CV) and determine a change in peak-to-peak separation, EP.T, from the voltammograms to detect the ART agent when the biomolecular receptors bind the ART agent.

29. The system of claim 24, wherein the biomolecular receptor-bound redox reporters are attached to the surface of the electrochemical sensor via an electrically conductive layer disposed on the surface.

30. The system of claim 29, wherein a thickness of the electrically conductive layer is less than about 1 pm.31 . The system of claim 29, wherein the plurality of biomolecular receptorbound redox reporters operably attached to the electrically conductive layer comprise one or more self-assembling biosensing monolayers that comprise one or more electrode-blocking alkanethiols, one or more alkanethiols, and one or more redox reporter-tagged aptamers.

32. The system of claim 23, wherein the biomolecular receptors comprise fluorescent reporter and quencher labeling moieties, wherein the quencher labeling moieties substantially quench fluorescent signals emitted from the fluorescent reporter labeling moieties when the biomolecular receptors are not bound to the ART agent, wherein the fluorescent reporter and quencher labeling moieties separate from one another a sufficient distance when the biomolecular receptors bind to the ART agent such that the fluorescent signals emitted from the fluorescent reporter labeling moieties are not substantially quenched by the quencher labeling moieties and are detected using the detector.

33. The system of claim 23, wherein the non-transitory computer executable instructions which, when executed by the electronic processor, further perform at least: repeating the detecting step one or more times using samples obtained from the subject at different time points to monitor levels of the ART agent in the subject over time.

34. The system of claim 23, wherein the sample is substantially unprocessed.

35. The system of claim 23, wherein the sample comprises whole blood, serum, or plasma.

36. The system of claim 23, wherein the sample comprises urine.

37. The system of claim 23, wherein the subject comprises a human immunodeficiency virus (HIV) infection.

38. The system of claim 23, wherein the subject is a human subject.

39. The system of claim 23, wherein the ART agent comprises emtricitabine.

40. The system of claim 23, wherein the ART agent comprises elvitegravir.41 . The system of claim 23, wherein the biomolecular receptor comprises a nucleic acid molecule.

42. The system of claim 23, wherein the biomolecular receptor comprises an aptamer.

43. The system of claim 42, wherein the aptamer comprises a 5’- thiol modification and a 3’ methylene blue modification.

44. The system of claim 42, wherein the aptamer comprises a nucleotide sequence selected from SEQ ID NOS: 1-10 and 14-30 or a nucleotide sequence having sufficient sequence identity thereto so that the aptamer binds the ART agent.

45. A computer readable media comprising non-transitory computer executable instructions which, when executed by at least electronic processor, perform at least: detecting one or more detectable signals produced when a plurality of biomolecular receptors undergo conformational changes using a detector, which detector is configured to detect the one or more detectable signals produced when a sample that comprises an antiretroviral therapeutic (ART) agent is contacted with the plurality of biomolecular receptors such that one or more of the biomolecular receptors undergo the conformational changes when the biomolecular receptors bind the ART agent.

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