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WO2025096893A1 - Réseaux de capteurs à micro-aiguilles magnétiques et aspects associés pour la surveillance moléculaire - Google Patents

Réseaux de capteurs à micro-aiguilles magnétiques et aspects associés pour la surveillance moléculaire Download PDF

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Publication number
WO2025096893A1
WO2025096893A1 PCT/US2024/054054 US2024054054W WO2025096893A1 WO 2025096893 A1 WO2025096893 A1 WO 2025096893A1 US 2024054054 W US2024054054 W US 2024054054W WO 2025096893 A1 WO2025096893 A1 WO 2025096893A1
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Prior art keywords
sensor device
electrochemical sensor
microneedles
subject
electrochemical
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Inventor
Netzahualcoyotl ARROYO
Joseph Wang
Karen SCIDA
Yao Wu
Maria REYNOSO
An-yi CHANG
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Johns Hopkins University
University of California San Diego UCSD
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Johns Hopkins University
University of California San Diego UCSD
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    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16H—HEALTHCARE INFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR THE HANDLING OR PROCESSING OF MEDICAL OR HEALTHCARE DATA
    • G16H10/00—ICT specially adapted for the handling or processing of patient-related medical or healthcare data
    • G16H10/40—ICT specially adapted for the handling or processing of patient-related medical or healthcare data for data related to laboratory analysis, e.g. patient specimen analysis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00—Measuring for diagnostic purposes; Identification of persons
    • A61B5/145—Measuring characteristics of blood in vivo, e.g. gas concentration or pH-value ; Measuring characteristics of body fluids or tissues, e.g. interstitial fluid or cerebral tissue
    • A61B5/14507—Measuring characteristics of blood in vivo, e.g. gas concentration or pH-value ; Measuring characteristics of body fluids or tissues, e.g. interstitial fluid or cerebral tissue specially adapted for measuring characteristics of body fluids other than blood
    • A61B5/1451—Measuring characteristics of blood in vivo, e.g. gas concentration or pH-value ; Measuring characteristics of body fluids or tissues, e.g. interstitial fluid or cerebral tissue specially adapted for measuring characteristics of body fluids other than blood for interstitial fluid
    • A61B5/14514—Measuring characteristics of blood in vivo, e.g. gas concentration or pH-value ; Measuring characteristics of body fluids or tissues, e.g. interstitial fluid or cerebral tissue specially adapted for measuring characteristics of body fluids other than blood for interstitial fluid using means for aiding extraction of interstitial fluid, e.g. microneedles or suction
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00—Measuring for diagnostic purposes; Identification of persons
    • A61B5/145—Measuring characteristics of blood in vivo, e.g. gas concentration or pH-value ; Measuring characteristics of body fluids or tissues, e.g. interstitial fluid or cerebral tissue
    • A61B5/14546—Measuring characteristics of blood in vivo, e.g. gas concentration or pH-value ; Measuring characteristics of body fluids or tissues, e.g. interstitial fluid or cerebral tissue for measuring analytes not otherwise provided for, e.g. ions, cytochromes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00—Measuring for diagnostic purposes; Identification of persons
    • A61B5/68—Arrangements of detecting, measuring or recording means, e.g. sensors, in relation to patient
    • A61B5/6846—Arrangements of detecting, measuring or recording means, e.g. sensors, in relation to patient specially adapted to be brought in contact with an internal body part, i.e. invasive
    • A61B5/6847—Arrangements of detecting, measuring or recording means, e.g. sensors, in relation to patient specially adapted to be brought in contact with an internal body part, i.e. invasive mounted on an invasive device
    • A61B5/685—Microneedles
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16H—HEALTHCARE INFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR THE HANDLING OR PROCESSING OF MEDICAL OR HEALTHCARE DATA
    • G16H20/00—ICT specially adapted for therapies or health-improving plans, e.g. for handling prescriptions, for steering therapy or for monitoring patient compliance
    • G16H20/10—ICT specially adapted for therapies or health-improving plans, e.g. for handling prescriptions, for steering therapy or for monitoring patient compliance relating to drugs or medications, e.g. for ensuring correct administration to patients
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16H—HEALTHCARE INFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR THE HANDLING OR PROCESSING OF MEDICAL OR HEALTHCARE DATA
    • G16H40/00—ICT specially adapted for the management or administration of healthcare resources or facilities; ICT specially adapted for the management or operation of medical equipment or devices
    • G16H40/60—ICT specially adapted for the management or administration of healthcare resources or facilities; ICT specially adapted for the management or operation of medical equipment or devices for the operation of medical equipment or devices
    • G16H40/63—ICT specially adapted for the management or administration of healthcare resources or facilities; ICT specially adapted for the management or operation of medical equipment or devices for the operation of medical equipment or devices for local operation
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16H—HEALTHCARE INFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR THE HANDLING OR PROCESSING OF MEDICAL OR HEALTHCARE DATA
    • G16H40/00—ICT specially adapted for the management or administration of healthcare resources or facilities; ICT specially adapted for the management or operation of medical equipment or devices
    • G16H40/60—ICT specially adapted for the management or administration of healthcare resources or facilities; ICT specially adapted for the management or operation of medical equipment or devices for the operation of medical equipment or devices
    • G16H40/67—ICT specially adapted for the management or administration of healthcare resources or facilities; ICT specially adapted for the management or operation of medical equipment or devices for the operation of medical equipment or devices for remote operation

Definitions

  • Electrochemical, aptamer-based (E-AB) sensors are analytical platforms that achieve continuous monitoring of specific molecular targets in vivo.
  • E- AB sensors present an architecture typically consisting of three elements (FIG. 1A): 1 ) a self-assembled monolayer (SAM) of target-binding, alkanethiol-functionalized nucleic-acid aptamers or other bioreceptor, 2) an electrode-blocking SAM of alkanethiols to prevent undesired electrochemical reactions and confer biocompatibility to the electrode surface, and 3) a redox reporter sensitive to targetbinding events.
  • SAM self-assembled monolayer
  • the redox reporter typically methylene blue (MB)
  • MB methylene blue
  • aptamer molecules reversibly undergo binding-induced conformational changes that presumably bring the reporter closer to the electrode surface, causing a change in the electron transfer rate between the reporter and the electrode (FIG. 1B), which can be easily measured electrochemically.
  • Aptamer binding in E-AB sensors is at dynamic equilibrium, reversibly switching between bound and unbound states at rates of milliseconds. This behavior makes E-AB sensors ideal for continuous monitoring applications.
  • E-AB sensors tolerate prolonged measurements in complex matrices such as unprocessed biological fluids.
  • E-AB sensors can be successfully interrogated via chronoamperometry, differential pulse techniques such as square-wave voltammetry and differential pulse voltammetry, alternating current voltammetry, and electrochemical impedance spectroscopy.
  • the choice of technique is typically determined by the final intended application of the E-AB sensor.
  • the simplicity of the voltage program in chronoamperometry is ideal for drift-free measurements at sub-second interrogation frequencies, which may be needed for the study of fast biological processes like neurotransmitter modulation in the brain.
  • Electrochemical impedance offers the convenience of interrogating E-AB sensors in a label-free manner, without using a redox reporter.
  • E-AB sensors have been interrogated by pulse techniques and, in particular, by square wave voltammetry. This widespread use likely arose because pulsed techniques differentially remove currents originating from charging the electrode-electrolyte double layer, significantly improving the signal-to-noise ratio of E-AB measurements. Yet, pulsed techniques also remove valuable electrochemical information regarding sensor stability (e.g., the capacitive current reports on monolayer stability) and differential voltage pulsing also strains the E-AB interface causing faster loss of signal.
  • sensor stability e.g., the capacitive current reports on monolayer stability
  • differential voltage pulsing also strains the E-AB interface causing faster loss of signal.
  • Cyclic voltammetry is frequently used for the surface characterization of E-AB sensors, as this technique provides valuable information regarding monolayer stability (by proxy of double layer capacitance) and surface coverage of the redox reporter-modified aptamer (from faradaic peak areas).
  • CV is not commonly used for the direct interrogation of E-AB sensors, in part because sensors with defective blocking monolayers or redox reporter-modified aptamers with slow electron transfer kinetics present large capacitive currents that can hide the faradaic waves of methylene blue, resulting in low signal-to-noise E-AB measurements.
  • CV peak currents do not change significantly with increasing target concentrations.
  • the present disclosure relates, in certain aspects, to methods, systems, kits, and computer readable media of use in detecting target molecules in interstitial fluid (ISF) of or in subjects.
  • ISF interstitial fluid
  • Some aspects for example, provide magnetic wearable microneedle sensor arrays that enable the continuous, minimally invasive sensing of molecules in vivo via the dermal ISF, both in animal models and in humans.
  • Interstitial fluid is a body compartment that rapidly equilibrates with blood, allowing the monitoring of systemic biomarkers, among other target molecules.
  • the dermis ISF is not directly irrigated by capillaries nor innervated, allowing for painless, minimally invasive sensing.
  • Some embodiments include the use of the technology in toxicology and pharmacokinetic (PK)/pharmacodynamic (PD) labs for drug development applications.
  • the approaches disclosed herein are translated into wearable sensor devices to be placed, for example, on the ear lobe of customers for continuous health status monitoring.
  • an electrochemical sensor device includes a body structure comprising first and second surfaces, and a plurality of microneedles extending from the first surface of the body structure, wherein the plurality of microneedles comprises at least one microneedle configured as a working electrode, at least one microneedle configured as a reference electrode, and at least one microneedle configured as a counter electrode.
  • the electrochemical sensor device also includes at least one electrically conductive layer disposed on at least a portion of the plurality of microneedles, wherein the electrically conductive layer is operably connected, or connectable, to a detector, and a plurality of biomolecular receptor-bound redox reporters operably attached to the electrically conductive layer, wherein biomolecular receptors of the plurality of biomolecular receptor-bound redox reporters undergo conformational changes when the biomolecular receptors bind a target molecule to produce one or more electrochemical signals that are detected by the detector when the detector is operably connected to the electrically conductive layer.
  • the electrochemical sensor device also includes a magnet connected, or connectable, to the second surface of the body structure, wherein the magnet is configured to magnetically attach the electrochemical sensor device to a metallic structure disposed in and/or on a body of a subject such that the electrochemical sensor device is reversibly retained in position on a surface of the body of the subject at least proximal to the metallic structure and a least some of the plurality of microneedles are disposed in contact with a dermal interstitial fluid (ISF) of or in the subject, which dermal ISF comprises the target molecule.
  • ISF dermal interstitial fluid
  • the electrochemical sensor device 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 target molecule when the biomolecular receptors bind the target molecule.
  • the electrochemical sensor device is configured to perform drift correction. The drift correction is performed using kinetic differential measurements (KDM).
  • KDM kinetic differential measurements
  • the electrochemical sensor device is configured to generate substantially stable baseline electrochemical signals. A thickness of the electrically conductive layer is less than about 1 pm.
  • the body structure comprises a substantially circular cross-sectional shape, a substantially square cross-sectional shape, or a substantially rectangular cross-sectional shape.
  • the plurality of microneedles is arrayed in a substantially circular format, a substantially square format, or a substantially rectangular format. At least one of the microneedles in the plurality of microneedles has a substantially circular, a substantially square, or a substantially rectangular cross-sectional shape. At least of the microneedles in the plurality of microneedles has a substantially conical shape, a substantially pyramidal shape, or a substantially tapered shape. Edges of at least one pair of microneedles in the plurality of microneedles are separated from one another by about 5 mm or less.
  • the detector comprises an electrochemical analyzer.
  • the electrochemical analyzer comprises a potentiostat.
  • the electrically conductive layer is configured to wirelessly connect to the detector.
  • the detector is configured to measure square wave voltammograms.
  • An electrically insulating cover structure is attached to the first surface of the body structure, and wherein the plurality of microneedles extends through the electrically insulating cover structure.
  • the body structure and the plurality of microneedles are fabricated using a stereolithography (SLA) three- dimensional (3D) printing technique.
  • the plurality of microneedles comprises twelve microneedles, wherein seven of the microneedles are working electrodes, wherein two of the microneedles are reference electrodes, and wherein three of the microneedles are counter electrodes.
  • the body structure comprises a cross-sectional dimension of about 30 mm or less.
  • the magnet comprises a permanent magnet.
  • the magnet comprises a neodymium ring magnet.
  • the magnet comprises a cross-sectional dimension of about 25 mm or less.
  • the magnet comprises a thickness of about 5 mm or less.
  • 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.
  • the subject comprises a mammalian subject.
  • the mammalian subject comprises a human subject.
  • the mammalian subject is from an order Rodentia.
  • the metallic structure is configured to maintain a substantially constant and substantially homogeneous pressure across the plurality of microneedles when the plurality of microneedles is disposed in contact with the dermal ISF of the subject.
  • the metallic structure is configured to maintain a substantially constant penetration depth of the plurality of microneedles when the plurality of microneedles is disposed in contact with the dermal ISF of the subject.
  • the metallic structure is disposed at least proximal to an ear of the subject.
  • a metallic plate comprises the metallic structure.
  • the metallic structure is implanted in the subject.
  • the metallic structure comprises a biocompatible plate fabricated from a medical grade, ferromagnetic steel.
  • the plurality of microneedles is fabricated integral with the body structure.
  • the electrically conductive layer comprises a chromium layer and/or a gold layer.
  • the electrically conductive layer of the reference electrode further comprises at least one silver layer.
  • the electrically conductive layer of the counter electrode further comprises at least one platinum layer.
  • the biomolecular receptor comprises a nucleic acid molecule (e.g., RNA, DNA, PNA, LNA, L-DNA, etc.).
  • the redox reporters comprise methylene blue (MB) or an osmium-based complex.
  • the target molecule comprises a therapeutic agent.
  • the therapeutic agent comprises an antibiotic.
  • the target molecule comprises a metabolite and/or an electrolyte.
  • the target molecule comprises a biomolecule.
  • the electrochemical sensor device is a wearable device. A kit comprising the electrochemical sensor device.
  • a method of detecting a target molecule in a subject includes positioning a plurality of microneedles of an electrochemical sensor device in contact with a dermal interstitial fluid (ISF) of or in the subject, wherein the electrochemical sensor device comprises: a body structure comprising first and second surfaces; the plurality of microneedles extending from the first surface of the body structure, wherein the plurality of microneedles comprises at least one microneedle configured as a working electrode, at least one microneedle configured as a reference electrode, and at least one microneedle configured as a counter electrode; at least one electrically conductive layer disposed on at least a portion of the plurality of microneedles, wherein the electrically conductive layer is operably connected to a detector; a plurality of biomolecular receptor-bound redox reporters operably attached to the electrically conductive layer, wherein biomolecular receptors of the plurality of biomolecular receptor-bound redox reporters
  • the method further comprises implanting the metallic structure in the body of the subject prior to the positioning step.
  • the method comprises implanting the metallic structure in an ear lobe of the subject prior to the positioning step.
  • the method comprises substantially continuously monitoring the target molecule in the subject during a selected duration of time.
  • the method comprises intermittently monitoring the target molecule in the subject during a selected duration of time.
  • the method comprises performing a pharmacokinetic, pharmacodynamic, and/or toxicology assessment of the target molecule in the subject.
  • the method comprises detecting the electrochemical signals in substantially real-time.
  • the method further comprises performing drift correction of the electrochemical sensor device using one or more kinetic differential measurements (KDM).
  • KDM kinetic differential measurements
  • the electrochemical sensor device further comprises an electrically insulating cover structure attached to the first surface of the body structure, and wherein the plurality of microneedles extends through the electrically insulating cover structure.
  • 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 nucleic acids.
  • the redox reporters comprise methylene blue (MB).
  • a method of producing an electrochemical sensor device includes forming a body structure comprising first and second surfaces, and a plurality of microneedles extending from the first surface of the body structure, and disposing at least one electrically conductive layer on at least a portion of the plurality of microneedles, wherein the electrically conductive layer is operably connectable to a detector.
  • the method also includes attaching a plurality of biomolecular receptor-bound redox reporters to the electrically conductive layer, wherein biomolecular receptors of the plurality of biomolecular receptor-bound redox reporters are configured to undergo conformational changes when the biomolecular receptors bind a target molecule to produce one or more electrochemical signals that are detected by the detector when the detector is operably connected to the electrically conductive layer, and connecting a magnet to the second surface of the body structure, wherein the magnet is configured to magnetically attach the electrochemical sensor device to a metallic structure disposed in and/or on a body of a subject such that the electrochemical sensor device is reversibly retained in position on a surface of the body of the subject at least proximal to the metallic structure and a least some of the plurality of microneedles are disposed in contact with a dermal interstitial fluid (ISF) of or in the subject, which dermal ISF comprises the target molecule, thereby producing the electrochemical sensor device.
  • ISF dermal inter
  • the method further comprises attaching an electrically insulating cover structure to the first surface of the body structure such that the plurality of microneedles extends through the electrically insulating cover structure.
  • a system for detecting a target molecule in a subject includes a detector, and an electrochemical sensor device, comprising: a body structure comprising first and second surfaces; a plurality of microneedles extending from the first surface of the body structure, wherein the plurality of microneedles comprises at least one microneedle configured as a working electrode, at least one microneedle configured as a reference electrode, and at least one microneedle configured as a counter electrode; at least one electrically conductive layer disposed on at least a portion of the plurality of microneedles, wherein the electrically conductive layer is operably connected to the detector; a plurality of biomolecular receptor-bound redox reporters operably attached to the electrically conductive layer, wherein biomolecular receptors of the plurality of biomolecular receptor-bound redox reporters undergo conformational changes when the biomolecular receptors bind the target molecule to produce one or more electrochemical signals that are detected by the
  • FIGS. 1A and 1B schematically show that E-AB sensors undergo target binding-induced changes in electron transfer kinetics of the redox reporter that can be monitored in real time via electrochemical interrogation according to one exemplary embodiment.
  • A In this work we employed three different DNA aptamers modified at the 5’ terminus with alkanethiol linkers and at the 3’ terminus with the redox reporter methylene blue. We co-deposited these modified oligonucleotides with 6-mercapto-1 -hexanol on the surface of gold electrodes via self-assembly.
  • B In the presence of their target molecule, the aptamers undergo a conformational change that, presumably, brings the redox reporter closer to the electrode surface, increasing the electron transfer rate.
  • FIG. 2A is a flow chart that schematically depicts steps in a method of detecting a target molecule in a subject according to an exemplary embodiment.
  • FIG. 2B is a flow chart that schematically depicts steps in a method of producing an electrochemical sensor device according to an exemplary embodiment.
  • FIG. 3 shows an algorithm according to an exemplary embodiment.
  • FIG. 4 schematically depicts a system according to an exemplary embodiment.
  • FIGS. 5A-5D 3D-Printed, aptamer-based microneedle sensor arrays using magnetic skin placement.
  • A Schematic representation of a fully assembled sensor patch for skin placement. Unmodified microneedle arrays were 3D-printed via stereolithography. To enable magnetic placement of sensor patches on the skin of live rodents, we encased a ring-shaped permanent magnet at the bottom of the patches.
  • B Gold plated MNs allow self-assembly of biosensing monolayers, consisting of electrode-blocking alkanethiols and thiolated and redox- labelled aptamers.
  • C Rapid loss of MN sensor signals upon deployment on the skin of rodents, caused by contraction of the animal skin after the device placement.
  • D Addressing this problem by using magnetic attachment of MN sensor arrays which leads to stable sensor response during continuous pharmacokinetic measurements.
  • FIGS. 6A-6C SLA 3D printing and assembly of microneedle sensor arrays.
  • A SEM micrograph of freshly 3D-printed MNs before assembly.
  • B Device fabrication steps: (Bi) UV cured and washed MNs; (Bn) MNs with kapton tape mask, ready for sputtering; (Bm) MNs coated with a chromium layer followed by a gold film; (Biv) reference electrode coating with silver ink; (Bv) electrode connections to Dupont pins; and (Bvi) final assembled device, including neodymium magnetic ring at the back and insulating 3D-printed cover at the top.
  • C Front and back photographs of the final assembly.
  • FIGS. 7A and 7B Skin penetration tests using tapered vs conical microneedle arrays.
  • A Photograph of tapered MN sensor array. We tested three tapered microneedle array designs that differed in needle-to-needle lateral spacing: (Ai, Aiv) 2.2 mm; (AH, AV) 2mm; and (Am, Avi) 2.5mm.
  • B Photograph of conical microneedle sensor array. We tested three conical microneedle array designs that differed in the needle-to-needle lateral spacing: (Bi, Biv) 0.8 mm; (Bn, Bv) 0.5 mm; and (Biii, Bvi) 1 mm. Colors, contrast and brightness were adjusted by the iPhone used for taking the photographs without further editing.
  • FIGS. 8A-8C Comparative in-vivo performance of skin taped, glued, and magnetically placed MNs.
  • A Securing the devices via medical tape, we observed rapid skin disinsertion of MN devices over a period of 1 h.
  • B Using super glue under and around the devices improves on-skin retention relative to using medical tape, but ultimately we observed ⁇ 80% signal loss.
  • C In contrast, magnetic placement of the MN sensor arrays via a subcutaneous metallic plate leads to robust retention of sensor signals over the entire measurement period.
  • Center panels show the output of the MN sensors when interrogated via square wave voltammetry at various square wave frequencies (e.g., 80, 150, 240, 300, 600 Hz).
  • FIGS. 9A-9F Pharmacokinetics of tobramycin in ISF via magnetically placed MNs. These [tobramycin]isF vs t plots were measured following intravenous dosing of 20 mg/kg tobramycin via tail vein boluses.
  • Panel (A) illustrates the raw data obtained at signal-on frequency, 300 Hz, and signal-off frequency, 30 Hz, as well as the resulting KDM trace obtained from their difference.
  • Panel (B) depicts the calibration curve utilized for conversion of signal gain to tobramycin concentration.
  • Panels (C), (D) and (E) show the response of three individual MN arrays placed on three independent animals. Colored dashed lines show non-linear regressions to a model of first order absorption and excretion pharmacokinetics, described in detail within the main text.
  • Panel (F) shows the fits from all datasets with x-axes corrected to 0 to remove the offset from baseline measurements. The solid black line represents the mean response, with average time to plateau ⁇ 46 min post intravenous bolus. Intravenous boluses were dosed in phosphate-buffered saline with full dose delivery within 10 s.
  • FIGS. 10A-10E Monitoring therapeutic transport from blood to the dermis interstitial fluid (ISF) via microneedle sensor arrays.
  • A Top view of the sensor array employed in this study.
  • the platform features five working electrode (WE) channels for real-time drug monitoring, one counter electrode (CE) channel, and one Ag
  • WE working electrode
  • CE counter electrode
  • RE Ag
  • Each WE and CE is made of four acupuncture microneedles coated with gold nanoparticles (AuNPs).
  • the reference electrode uses five microneedles coated with silver paint, later treated in bleach to form the AgCI film.
  • Inset Scanning electron microscopy (SEM) micrograph showing an example microneedle coated with AuNPs.
  • a subcutaneously implanted magnetic plate holds the devices in place, as previously reported by our group.
  • E Using SACMES, a real-time data processing and visualization software, we can monitor ISF drug uptake and excretion in real time.
  • the innovative multichannel platforms allows redundant measurements in a single rodent, drastically decreasing the number of animals needed for statistical validation.
  • FIGS. 11A-11C Setup of microneedle sensor array in vivo and measurement protocol.
  • A Sprague-Dawley rats (300 g) were anesthetized under isoflurane. A 1 cm-wide skin cut was made on the left side of their abdomen to subcutaneously (sub-Q) slide a magnetic plate. Additionaly, their right jugular vein was cathetherized for intravenous (i.V.) dosing. Vital signs (heart rate, temperature, oxygen) were monitored throughout the 3-hour measurement periods using an oximeter.
  • the microneedle platform contains a back-mounted magnet for magnetic attahcement of the devices on the animal skin. Electrodes were wired to a potentiostat for external data collection.
  • FIGS. 12A-12F Vancomycin pharmacokinetics in the dermal ISF.
  • A We first titrated vancomycin into three independent rats, at I.V. doses of 20 mg/kg (black), 40 mg/kg (red), and 83 mg/kg (blue). Solid symbols show the statistical mean, shaded areas the standard deviation across four WE channel measurements. To assess animal-to-animal variability, we repeated the 40 mg/kg dose with a fourth rat (red triangles vs squares).
  • B Sensor calibration in 50% rat serum in phosphate- buffered saline, as a proxy for ISF. The y-axis corresponds to the gain of kinetic differential measurements.
  • the higher vancomycin doses (40 mg/kg and 83 mg/kg) caused sensor signal saturation (i.e., measurements much above the ECso), resulting in noisy plateau concentrations (D, E) or oversaturation (F).
  • the solid black lines are point connectors to illustrate trends.
  • FIGS. 13A-13D Tobramycin pharmacokinetics in the dermal ISF.
  • FIGS. 14A-14E Microneedle patch monitors hydrophobic molecules in the dermal ISF.
  • A We tested a hydrophobic amino acid phenylalanine with two doses (30 mg/kg and 60 mg/kg) injected intravenously. Sensor response over time for each dose is shown in the graph.
  • B We converted these sensor readings into actual analyte concentrations with the assistance of an ex vivo calibration curve obtained in 50% rat serum solution.
  • C Using a one-compartment pharmacokinetic model, we analyzed the analyte concentration profiles for both phenylalanine doses.
  • “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.
  • the term “about” or “approximately” or “substantially” refers to a range of values or elements that falls within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11 %, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1 %, or less in either direction (greater than or less than) of the stated reference value or element unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value or element).
  • Bind in the context of pathogen detection, refers to a state in which a first chemical structure (e.g., a therapeutic agent) is sufficiently associated a second chemical structure (e.g., a bioreceptor) such that the association between the first and second chemical structures can be detected.
  • a first chemical structure e.g., a therapeutic agent
  • a second chemical structure e.g., a bioreceptor
  • Biomolecule refers to an organic molecule produced by a living organism. Examples of biomolecules, include macromolecules, such as nucleic acids, proteins, carbohydrates, and lipids.
  • Bioreceptor refers to a biochemical structure that receives or binds other chemical structures (e.g., therapeutic agents, nucleic acids, proteins, metabolites, and the like).
  • sample means anything capable of being analyzed using a device or system disclosed herein.
  • sample types include environmental samples and biological samples.
  • bind in the context of pathogen detection, refers to a state in which substantially only target chemical structures (e.g., biomolecules) are sufficiently associated with a corresponding or cognate binding agent, to the exclusion of non-target chemical structures, such that the association between the target chemical structures and the binding agent can be detected.
  • target chemical structures e.g., biomolecules
  • system in the context of analytical instrumentation refers a group of objects and/or devices that form a network for performing a desired objective.
  • Subject 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).
  • farm animals e.g., production cattle, dairy cattle, poultry, horses, pigs, and the like
  • 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.”
  • ISF dermal interstitial fluid
  • One promising strategy for accessing ISF involves the use of wearable patches containing microneedle sensor arrays.
  • microneedle sensors have been fabricated via various manufacturing strategies based on injection molding, machining, and advanced lithography to name a few.
  • 3D-printed microneedles have previously been reported as a convenient and scalable approach to sensor fabrication that, when combined with aptamer-based molecular measurements, can support continuous molecular monitoring in ISF.
  • the inventors have identified that this problem was due to the rheological properties of, for example, the test animal (e.g., rodent) skin, which can contract post microneedle placement, physically pushing the microneedles out of the skin.
  • This sensor retraction caused a loss of electrical contact between working and reference needles, irreversibly damaging the sensors.
  • we provide innovative approaches that allow magnetic placement of microneedle sensor arrays on the skin of live rodents or other subjects, affixing the patches under light pressure that prevents needle retraction. Using this strategy, we achieved sensor signaling baselines that drift at rates comparable to those seen with other in vivo deployments of electrochemical, aptamer-based sensors.
  • An electrochemical sensor device typically includes a body structure comprising first and second surfaces, and a plurality of microneedles extending from the first surface of the body structure.
  • the plurality of microneedles includes at least one microneedle configured as a working electrode, at least one microneedle configured as a reference electrode, and at least one microneedle configured as a counter electrode.
  • the electrochemical sensor device also includes at least one electrically conductive layer disposed on at least a portion of the plurality of microneedles in which the electrically conductive layer is operably connected, or connectable, to a detector.
  • the electrochemical sensor device also includes a plurality of biomolecular receptor-bound redox reporters operably attached to the electrically conductive layer in which biomolecular receptors of the plurality of biomolecular receptor-bound redox reporters undergo conformational changes when the biomolecular receptors bind a target molecule to produce one or more electrochemical signals that are detected by the detector when the detector is operably connected to the electrically conductive layer.
  • the electrochemical sensor device also includes a magnet connected, or connectable, to the second surface of the body structure, in which the magnet is configured to magnetically attach the electrochemical sensor device to a metallic structure disposed in and/or on a body of a subject such that the electrochemical sensor device is reversibly retained in position on a surface of the body of the subject at least proximal to the metallic structure and a least some of the plurality of microneedles are disposed in contact with a dermal interstitial fluid (ISF) of or in the subject, which dermal ISF comprises the target molecule.
  • ISF dermal interstitial fluid
  • the electrochemical sensor device is a wearable device.
  • the wearable device may be one wearable device or a plurality of wearable devices (e.g., comprising electrochemical sensor device that detect/monitor the same or different target molecules).
  • the wearable device is typically configured to be wearable on or in proximity to the patient’s or subject’s body.
  • the wearable device may be attached by a strap or other means to a portion of the patient’s body such as to an arm, a leg, a waist, a neck, groin, etc.
  • the wearable device may be configured to be attached in proximity to a particular portion of the patient’s body.
  • the wearable device may be configured to be attached to clothes worn by a person.
  • the wearable device may also be integrated into or attached to another device worn by a person.
  • the wearable device can be configured to attach to a watch, to a belt, to jewelry, to glasses, to undergarment, etc.
  • the electrochemical sensor device e.g., configured as a wearable device, etc.
  • the kit is packaged as a component of a kit.
  • the wearable device comprises a metallic structure.
  • the metallic structure is disposed at least proximal to an ear of the subject.
  • a metallic plate comprises the metallic structure, the metallic structure is implanted in the subject.
  • the metallic structure comprises a biocompatible plate fabricated from a medical grade, ferromagnetic steel.
  • the metallic structure is configured to maintain a substantially constant and substantially homogeneous pressure across the plurality of microneedles when the plurality of microneedles is disposed in contact with the dermal ISF of the subject.
  • the metallic structure is configured to maintain a substantially constant penetration depth of the plurality of microneedles when the plurality of microneedles is disposed in contact with the dermal ISF of the subject.
  • the magnet comprises a permanent magnet. In some embodiments, wherein the magnet comprises a neodymium ring magnet. In some embodiments, the magnet comprises a cross- sectional dimension of about 25 mm or less. In some embodiments, the magnet comprises a thickness of about 5 mm or less.
  • the electrochemical sensor device 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 target molecule when the biomolecular receptors bind the target molecule.
  • the electrochemical sensor device is configured to perform drift correction.
  • the drift correction is performed using kinetic differential measurements (KDM).
  • the electrochemical sensor device is configured to generate substantially stable baseline electrochemical signals.
  • a thickness of the electrically conductive layer is less than about 1 pm.
  • the body structure comprises a substantially circular cross-sectional shape, a substantially square cross-sectional shape, or a substantially rectangular cross-sectional shape.
  • the plurality of microneedles is arrayed in a substantially circular format, a substantially square format, or a substantially rectangular format.
  • at least one of the microneedles in the plurality of microneedles has a substantially circular, a substantially square, or a substantially rectangular cross-sectional shape.
  • at least of the microneedles in the plurality of microneedles has a substantially conical shape, a substantially pyramidal shape, or a substantially tapered shape.
  • edges of at least one pair of microneedles in the plurality of microneedles are separated from one another by about 5 mm or less.
  • the plurality of microneedles is fabricated integral with the body structure.
  • the electrically conductive layer comprises a chromium layer and/or a gold layer.
  • the electrically conductive layer of the reference electrode further comprises at least one silver layer.
  • the electrically conductive layer of the counter electrode further comprises at least one platinum layer.
  • at least one microneedle of the plurality of microneedles extends from the surface of the body structure by about 0.5 mm to about 2 mm.
  • a base portion of at least one microneedle of the plurality of microneedles has a diameter of less than about 400 pm.
  • a tip portion of at least one microneedle of the plurality of microneedles has a diameter of less than about 100 pm.
  • the detector comprises an electrochemical analyzer.
  • the electrochemical analyzer comprises a potentiostat.
  • the electrically conductive layer is configured to wirelessly connect to the detector.
  • the detector is configured to measure square wave voltammograms.
  • an electrically insulating cover structure is attached to the first surface of the body structure, and wherein the plurality of microneedles extends through the electrically insulating cover structure.
  • the body structure and the plurality of microneedles are fabricated using a stereolithography (SLA) three-dimensional (3D) printing technique.
  • the plurality of microneedles comprises twelve microneedles, wherein seven of the microneedles are working electrodes in which two of the microneedles are reference electrodes, and in which three of the microneedles are counter electrodes.
  • the body structure comprises a cross-sectional dimension of about 30 mm or less.
  • 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.
  • the biomolecular receptor comprises an aptamer.
  • the aptamer comprises a 5’- thiol modification and a 3’ methylene blue modification.
  • the biomolecular receptor comprises a nucleic acid molecule.
  • the redox reporters comprise methylene blue (MB) or an osmium-based complex.
  • the target molecule comprises a therapeutic agent.
  • the therapeutic agent comprises an antibiotic.
  • the target molecule comprises a metabolite and/or an electrolyte.
  • the target molecule comprises a biomolecule.
  • the subject comprises a mammalian subject.
  • the mammalian subject comprises a human subject.
  • the mammalian subject is from an order Rodentia.
  • FIG. 2A is a flow chart that schematically depicts steps in a method of detecting a target molecule in a subject according to an exemplary embodiment.
  • method 200 includes positioning a plurality of microneedles of an electrochemical sensor device (as described further herein) in contact with a dermal interstitial fluid (ISF) of or in the subject (e.g., piercing the skin of an ear lobe of a human subject, etc.) (step 202).
  • ISF dermal interstitial fluid
  • the biomolecular receptor comprises an aptamer.
  • Method 200 also includes detecting the electrochemical signals using the detector, thereby detecting the target molecule in the subject (step 204).
  • method 200 further includes implanting the metallic structure in the body of the subject prior to the positioning step (i.e., step 202). In some embodiments, method 200 includes implanting the metallic structure in an ear lobe of the subject prior to the positioning step (i.e., step 202). In some embodiments, method 200 includes substantially continuously monitoring the target molecule in the subject during a selected duration of time. In some embodiments, method 200 includes intermittently monitoring the target molecule in the subject during a selected duration of time. In some embodiments, method 200 includes performing a pharmacokinetic, pharmacodynamic, and/or toxicology assessment of the target molecule in the subject. In some embodiments, method 200 includes detecting the electrochemical signals in substantially real-time.
  • method 200 further includes performing drift correction of the electrochemical sensor device using one or more kinetic differential measurements (KDM).
  • the electrochemical sensor device further includes an electrically insulating cover structure attached to the first surface of the body structure.
  • the plurality of microneedles typically extends through the electrically insulating cover structure.
  • 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 nucleic acids.
  • the redox reporters comprise methylene blue (MB).
  • FIG. 3 shows algorithm 400 that can be used to implement aspects of method 200 according to an exemplary embodiment.
  • method 200 includes comparing the AEP.T to a no target peak-to-peak separation, AEP.NT, determined from one or more cyclic voltammograms generated from the electrochemical sensor in the absence of the target molecule. In some embodiments, method 200 includes determining a concentration of the target molecule in the sample by comparing the AEP.T to a standard curve. In some embodiments, method 200 includes determining the AEP.T from at least a first cyclic voltammogram and at least a second cyclic voltammogram generated from the electrochemical sensor. In some embodiments, method 200 includes correlating at least two currents with corresponding peak potentials and calculating a separation between the peak potentials.
  • AEP.NT a no target peak-to-peak separation
  • method 200 includes determining a concentration of the target molecule in the sample via the change in the target peak- to-peak separation, AEP.T.
  • the electrochemical sensor is substantially resistant to drift.
  • method 200 includes determining the change in the target peak-to-peak separation, AEP.T, from the cyclic voltammograms with about 900 milliseconds, about 800 milliseconds, about 700 milliseconds, about 600 milliseconds, about 500 milliseconds, about 400 milliseconds, about 300 milliseconds, about 200 milliseconds, about 100 milliseconds, or less of contacting the electrochemical sensor with the sample.
  • method 200 includes generating the cyclic voltammograms from the electrochemical sensor using a voltage scanning rate of about 5 V s’ 1 or more. In some embodiments, the voltage scanning rate is between about 5 V s’ 1 and about 10 V s’ 1 . In some embodiments, method 200 includes continuously monitoring the change in the target peak-to-peak separation, EP.T over time from multiple cyclic voltammograms generated from the electrochemical sensor.
  • FIG. 2B is a flow chart that schematically depicts steps in a method of method of producing an electrochemical sensor device according to an exemplary embodiment.
  • method 210 includes forming a body structure comprising first and second surfaces, and a plurality of microneedles extending from the first surface of the body structure (step 212) and disposing at least one electrically conductive layer on at least a portion of the plurality of microneedles, wherein electrically conductive layer is operably connectable to a detector (step 214).
  • Method 210 also includes attaching a plurality of biomolecular receptor-bound redox reporters to the electrically conductive layer in which biomolecular receptors of the plurality of biomolecular receptor-bound redox reporters are configured to undergo conformational changes when the biomolecular receptors bind a target molecule to produce one or more electrochemical signals that are detected by the detector when the detector is operably connected to the electrically conductive layer (step 216).
  • method 210 also includes connecting a magnet to the second surface of the body structure in which the magnet is configured to magnetically attach the electrochemical sensor device to a metallic structure disposed in and/or on a body of a subject such that the electrochemical sensor device is reversibly retained in position on a surface of the body of the subject at least proximal to the metallic structure and a least some of the plurality of microneedles are disposed in contact with a dermal interstitial fluid (ISF) of or in the subject, which dermal ISF comprises the target molecule (step 218).
  • method 210 further includes attaching an electrically insulating cover structure to the first surface of the body structure such that the plurality of microneedles extends through the electrically insulating cover structure.
  • the present disclosure also provides various systems and computer program products or machine-readable media.
  • 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.
  • 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.
  • 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.
  • server 602 e.g., a search engine server
  • server 602 e.g., a search engine server
  • 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.
  • a user interface e.g., a graphical user interface (GUI), a web-based user interface, and/or the like
  • 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).
  • 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 600 optionally 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.
  • 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.
  • 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.
  • 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.
  • computer-readable medium refers to any medium that participates in providing instructions to a processor for execution.
  • computer-readable medium encompasses distribution media, cloud computing formats, intermediate storage media, execution memory of a computer, and any other medium or device capable of storing program product 608 implementing the functionality or processes of various aspects of the present disclosure, for example, for reading by a computer.
  • a "computer-readable medium” or “machine-readable medium” may take many forms, including but not limited to, non-volatile media, volatile media, and transmission media.
  • Non-volatile media includes, for example, optical or magnetic disks.
  • Volatile media includes dynamic memory, such as the main memory of a given system.
  • Transmission media includes coaxial cables, copper wire and fiber optics, including the wires that comprise a bus. Transmission media can also take the form of acoustic or light waves, such as those generated during radio wave and infrared data communications, among others.
  • Exemplary forms of computer-readable media include a floppy disk, a flexible disk, hard disk, magnetic tape, a flash drive, or any other magnetic medium, a CD-ROM, any other optical medium, punch cards, paper tape, any other physical medium with patterns of holes, a RAM, a PROM, and EPROM, a FLASH-EPROM, any other memory chip or cartridge, a carrier wave, or any other medium from which a computer can read.
  • Program product 608 is optionally copied from the computer-readable medium to a hard disk or a similar intermediate storage medium.
  • 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.
  • this application provides systems that include one or more processors, and one or more memory components in communication with the processor.
  • the memory component typically includes one or more instructions that, when executed, cause the processor to provide information that causes at least one result, data, and/or the like to be displayed or otherwise indicated (e.g., via a result indicator of electrochemical sensor device 618 and/or via communication devices 614, 616 or the like) and/or receive information from other system components and/or from a system user (e.g., via communication devices 614, 616, or the like).
  • EXAMPLE 1 3D-Printed, Aptamer-based Microneedle Sensor Arrays Using Magnetic Placement on Live Rats for Pharmacokinetic Measurements in Interstitial Fluid
  • MN-based electrochemical sensors are emerging as promising platforms for monitoring, minimally invasively, systemic biomarkers in the dermal interstitial fluid (ISF). With sharp tips down to few micrometers and length below one millimeter, MN-supported electrochemical sensors painlessly penetrate the epidermis to tap into the dermal ISF. Because this biofluid is highly irrigated by capillary plexuses and drained by lymphatic capillaries within the dermis, the dermal ISF represents a body compartment that dynamically equilibrates with, and therefore reflects, systemic molecular concentrations.
  • ISF dermal interstitial fluid
  • dermis-implanted MN sensors represent an ideal tool for real-time, on-body biomarker monitoring. While early MN biosensing applications have focused on the continuous monitoring of metabolites and electrolytes, the recent use of bioaffinity receptors, particularly aptamers, has expanded the scope of such MN sensors towards a broader range of important target analytes.
  • Such new magnetically supported devices may offer significant benefits to biomedical research: (1 ) pre-measurement implantation of the subcutaneous plate would allow the preparation of large animals cohorts for molecular measurements in ISF; (2) after recovery from surgery, the microneedle devices could be placed and removed as needed, to allow multiday measurements in the same animals; (3) the magnetic attachment should be sturdy enough to allow continuous measurements in awake animals; and finally, (4) the magnetic skin attachment could facilitate translation to other model systems, and even to humans, if placement is intended in discrete skin regions, such as the ear lobes.
  • Gold cleaning solution (PN: 667978), 6- mercapto-1 -hexanol (MCH), and Tris(2-carboxyethyl) phosphine hydrochloride (TCEP) were purchased from Sigma-Aldrich (St. Louis, MO). 200 proof ethanol, sodium chloride (NaCI), sodium hydroxide (NaOH), and trace-metal grade sulfuric acid (H2SO4) were purchased from Fisher Scientific (Waltham, MA). Tobramycin sulfate was ordered from Spectrum Pharmacy Products (New Brunswick, NJ). N42 neodymium ring magnets (42NEG554012-NI) were purchased from Integrated Technologies Group (Culver City, CA).
  • Neodynium magnetic discs (20 mm diameter, 3 mm thickness) were ordered from the MIN Cl store and Silver conductive epoxy adhesive was ordered from MG Chemicals store (Amazon). All aqueous solutions were prepared using deionized water from a Milli-Q® Direct purification system, with a resistivity of 18 MW.
  • Photocurable resin (clear resin) was purchased from Formlabs (Somerville, MA), and (3D printing UV sensitive resin) from Anycubic (Brighton, Co).
  • Silver ink was purchased from Ercon Inc (Wareham, MA).
  • Deidentified human serum was purchased from BiolVT (Washington, D.C.).
  • the oligonucleotide sequence for tobramycin measurements had dual modifications (5’ hexanethiol and 3’ methylene blue) and was purchased HPLC-purified from Sigma- Aldrich (Houston, TX).
  • Form3+ printer FormlabsTM
  • connection channels As shown in Figure 6B V , and lined over the straight angle header of Dupont pins. We cured the silver epoxy at 80°C for 30 min.
  • To create a pseudo-reference electrode we added a layer of silver ink only on the 2 MNs located at the center of each MN array and cured it at 80°C for 30 min. SEM micrographs of the silver-coated pseudo-reference microneedles reveal a slight increase in microneedle body and tip diameter of 40 pm caused by the coating process.
  • For final patch assembly we pipetted in a thin layer of SLA resin at the base of the MNs, then inserted the cover piece to control microneedle height and achieve electrical insulation. We cured the resin in this last step using a UV-lamp (Peopoly 450 nm UV) for 2 h.
  • Microneedle Cleaning Protocol All MN arrays were treated prior to sensing monolayer depositions following a three-stage procedure. Stage 1: First, the MNs were incubated in pure ethanol for 10 min. Stage 2: An additional layer of gold was electroplated onto their surface from a gold chloride solution consisting of: 1 .2 mg/mL HAuCk, 1.5 wt% HCI, and 0.1 M NaCI, at 50° C. We electroplated via amperometry at -0.4 V with a pseudo-reference AgCI wire for 300 s.
  • Stage 3 The electroplated devices were electrochemically cleaned via cyclic voltammetry in (I) 0.5 M NaOH and (II) 0.5 M H2SO4, following previously reported protocols (Arroyo- Curras et al., 2017a). Briefly, we first voltammetrically interrogated the MNs in 0.5 M NaOH, from -0.4 to -1.4 V vs Ag/AgCI, for 100 cycles at 4 V/s. Next, we voltammetrically interrogated the MNs in 0.5 M H2SO4, from 0.2 to -1.6 V vs Ag/AgCI at 4 V/s.
  • the MN sensor arrays were immersed in aptamer solution for 1 h in the incubation chamber, then incubated in a solution of 30 mM mercaptohexanol at 25° C for 3 h to form the final sensing monolayer.
  • Surface concentration of aptamers was determined via the AUC of cyclic voltammograms performed at 100 mV/s.
  • Mechanical Durability of 3D-Printed MNs The durability of gold- sputtered MNs was evaluated via full penetration of dissected porcine skin: 100, 500, and 1000 penetrations. We purchased porcine skin from a local supermarket and cleaned it by washing with deionized water. The porcine skin was fixed on a benchtop.
  • Electrochemical Measurements For benchtop electrochemical treatments and measurements, we employed 8-Channel Electrochemical Analyzers (CHI 1040C, Austin, TX). For in vivo measurements, we employed a hand-held potentiostat (CHI 1242, Austin, TX). All raw measurements were controlled via commercial CHI software. All in vitro measurements were carried out in a three- electrode cell configuration consisting of gold working, platinum wire counter, and Ag/AgCI (saturated KCI) reference electrodes. Cyclic voltammogram was recorded at a scan rate of 100 mV/s for evaluating the cleanliness of the gold surface of microneedles. Square wave voltammetry was performed using a square-wave amplitude of 25mV, step size of 1 mV, and various frequencies.
  • the magnetic pressure between the plate and microneedle array was 50 g/cm 2 (0.71 PSI) as measured via a flexible pressure sensor from Qinlorgo (purchased via Amazon).
  • Qinlorgo purchased via Amazon.
  • After obtaining a one to two hour-long baseline (waiting for sensor baseline to reach a steady state), we performed an intravenous bolus of 20 mg/kg tobramycin via the tail vein.
  • AVMA American Veterinary Medical Association
  • the second design was rectangular, with conical MNs located at the center of the patch (Figure 7B), with needle-to-needle lateral spacings ⁇ 0.8 mm (Figure 7Bi, iv), ⁇ 0.5 mm ( Figure 7BH, v ), and ⁇ 1.0 mm ( Figure 7Bm, v .
  • These devices had the counter and reference electrodes separated from the working electrode array.
  • the rectangular arrays performed better on the abdomen skin relative to the back.
  • the conical microneedles had inferior skin penetration relative to the tapered microneedles. We believe this inferior performance originated because it is easier to apply equal pressure across circular devices vs rectangular ones, which tend to tilt sideways upon skin placement. Given these observations, we decided to continue to use the tapered MNs with the circular base, as shown in Figures 6C and 7A. All subsequent measurements were performed on the abdomen skin of rats.
  • Microtip Stability as a Function of Chromium and Gold Layer Thicknesses To support electrochemical aptamer-based (E-AB) sensing, the MNs must be coated with a homogeneous gold film. Additionally, the film must have high purity to allow dense packing of aptamer and blocking alkylthiol monolayer elements. Finally, the film must be robust enough to tolerate chemical cleaning in ethanol and electrochemical cleaning in dilute sodium hydroxide and sulfuric acid, as required prior to monolayer deposition (see Methods section). Thus, a critical aspect of MN patch prototyping was tuning the chromium adhesion layer and gold layer thicknesses to tolerate cleaning steps prior to sensor fabrication.
  • the second strategy we tested involved magnetic placement of the microneedle sensor arrays via a subcutaneous metallic plate.
  • flat sensor responses Figure 8C, center
  • the low frequency measurements are subtracted in real time from the high frequency data, correcting the drift while simultaneously amplifying the sensor signals.
  • This approach has been discussed once for microneedle aptamer-based sensors in the context of vancomycin ISF measurements ex vivo.
  • This example reports the development of MN aptamer-based sensor arrays that were directly printed via SLA, metal coated via sputtering and electroplating, functionalized with alkylthiol- and redox reported-modified aptamers, and deployed in vivo on the skin of rodents via an innovative magnetic approach.
  • the magnetic approach consists of subcutaneously implanting a metallic plate to allow stable affixing of a magnet-containing MN sensor patch on the skin of rodents.
  • the magnetic force applied between the plate and the MN sensor patch can be adjusted by tuning the thickness of the patch, with thicker patches decreasing magnetic attraction and, therefore, pressure on the skin.
  • This innovative sensor deployment approach paves the way for future measurements in awake animals using chronic paradigms, in which measurements could be repetitively performed on the same animals by placing and removing the microneedles on demand.
  • the microneedle sensors tolerate repeated skin reinsertions without significant loss of aptamers or general damage to the biosensor interface.
  • the magnetic strategy could enable sensing applications in humans when discreet molecular monitoring is warranted such as, for example, on the ear lobes.
  • the wearable MN patches reported here achieved two critical innovations relative to previously published in-vivo MN-based platforms.
  • KDM kinetic differential measurements
  • EXAMPLE 2 Aptamer-Based Therapeutic Drug Monitoring in the Dermis Interstitial Fluid
  • the dermal interstitium is a fluid-filled elastic space that has been highlighted as an ideal body compartment for diagnostic development. This is because the interstitial fluid (ISF) contained within the interstitium is rich in molecular diversity, reflecting both skin-local and systemic compounds that could be leveraged as health and disease biomarkers.
  • ISF interstitial fluid
  • the potential value of molecules in ISF for diagnostic applications has sparked intense biosensor development efforts focused not just on sampling ISF, but also on directly interrogating it via wearable biosensor platforms.
  • ISF-probing sensors have been developed for the tracking of metabolites such as lactate, glucose and ketone bodies. Additionally, our group and others have highlighted the potential value of ISF-based measurements for therapeutic drug monitoring (TDM).
  • TDM therapeutic dosing is based on serially measured plasma drug concentrations, typically performed via blood draws followed by in vitro quantification.
  • the availability of skin-interfaced wearable monitors could significantly shorten the turnaround time of TDM, from hours and sometimes days to instantaneous, real-time drug tracking in ISF. This ability, in turn, should improve patient outcomes by precisely modulating therapeutic dosing to achieve the right in vivo concentration at the right time, while simultaneously minimizing drug toxicity.
  • microneedle sensor patches with built-in electrochemical, aptamer-based (E-AB) sensors have been previously reported for continuous molecular monitoring in ISF.
  • E-AB electrochemical, aptamer-based
  • These platforms consist of microneedle or microtip electrode arrays that can pierce the epidermis to then sit in the dermis, where they can interact with ISF.
  • the electrodes can be fabricated at scale via various methods such as 3D printing, polymer casting, and micromolding, metal- coated via sputtering, and later integrated with wearable electronic boards for wireless interrogation and data transmission.
  • the electrodes are functionalized with structure-switching nucleic acid aptamers, which can reversibly bind and dissociate from their targets at sub-second scales.
  • Target interactions with the aptamers change the electron transfer rate of aptamer-bound redox reporters, a phenomenon that can be probed in real time via electrochemistry.
  • microneedle sensor arrays have been limited to the sensing of only two therapeutic agents, tobramycin and vancomycin.
  • both of these targets are soluble in aqueous media at the doses typically delivered in vivo [their predicted solubilities are 1000 g/L ( ⁇ 2.14 M) for tobramycin and 0.4 g L’ 1 ( ⁇ 276 pM) for vancomycin]. Additionally, tobramycin has negligible protein binding in plasma while vancomycin is ⁇ 50% protein bound, leaving a significant protein-free fraction that can diffusively cross capillary barriers to go from blood to ISF. However, an open question remains as to whether microneedle sensors could be used for ISF-based TDM of more hydrophobic targets such as chemotherapeutics used in cancer treatment, which tend to be >60% protein bound in plasma.
  • the targets irinotecan and doxorubicin [predicted solubilities of 0.1 g L’ 1 ( ⁇ 170 pM) and 0.41 g L- 1 ( ⁇ 750 pM)], which are >60% protein-bound in plasma, fall below the limit of detection of current aptamer-based sensors in ISF, even following high intravenous doses.
  • phenylalanine predicted solubility for phenylalanine is 41 g L’ 1 ( ⁇ 248 mM)]
  • the microneedle sensor platform we employed in this study ( Figure 10) consisted of two main components: a stereolithographically printed shell containing microneedle insertion holes, and commercially purchased acupuncture microneedles.
  • the shell included twenty four concentric holes (700 pm in diameter) dedicated to five working electrode channels and one counter electrode. At the center, the shell also included five holes for the reference electrode (800 pm in diameter).
  • Each of the working and counter electrode channels consisted of four gold microneedles electrochemically coated with gold nanoparticles (AuNPs, Figure 10A).
  • the reference electrode consisted of five silver paint-coated microneedles, which were bleached post assembly to form a Ag
  • the four microneedles of each channel were shorted together and connected to electrically insulated copper wires, which were linked to a potentiostat for electrochemical interrogation.
  • the four shorted microneedles per channel allowed the measurement of larger baseline currents without compromising the microscopic nature of the electrodes, significantly improving signal-to-noise in our in vivo measurements.
  • the five working electrode channels provided rigorous statistical redundancy during each in vivo measurement, eliminating the need to interrogate a large number of animals.
  • microneedle electrodes were electrochemically coated with AuNPs to minimize exposure of their nickel core to buffer or biofluids, and to increase the loading capacity of aptamers on their surface.
  • Scanning electron micrographs (SEM) confirmed the presence of uniform AuNPs coatings (inset in Figure 10A). Relative to bare electrodes, the AuNPs-coated microneedles exhibited a significant increase in gold abundance under energy-dispersive spectroscopy (EDS) and in microscopic surface area as measured via cyclic voltammetry. Additionally, square wave voltammograms of the aptamer-functionalized surfaces revealed a 10-fold increase in reporter reduction currents and lower capacitive currents.
  • EDS energy-dispersive spectroscopy
  • Vancomycin is a glycopeptide antibiotic with a narrow therapeutic window and for which TDM is standard of care.
  • vancomycin-binding aptamer sequence previously optimized by our laboratory.
  • optimal square wave voltammetry parameters for sensor interrogation by building square wave frequency maps in 50% rat serum in phosphate-buffered saline. Measurements at square wave frequencies of 150 Hz and 15 Hz provided the largest ON and OFF sensor responses, respectively, while also allowing the in vivo implementation of kinetic differential measurements (KDM)- a drift correction and signal amplification strategy.
  • KDM kinetic differential measurements
  • the vancomycin concentration reached in ISF is so high the sensor signal saturates.
  • the signal scatter observed in Figure 12E, D reflects concentrations above 90% sensor gain based on our dose-response curve ( Figure 12B).
  • the curves smoothen out at the end of the measurement period because ISF vancomycin starts to decrease via excretion, bringing the measurements back to below sensor saturating concentrations.
  • a dose of 83 mg/kg grossly saturates the sensor; thus, the flat data in Figure 12F reflects our inability to quantify vancomycin concentrations above 4 pM.
  • Microneedle Patch Monitors Hydrophobic Analytes in Rodent Dermal ISF Having successfully demonstrated in vivo measurements of hydrophilic small molecules with our microneedle E-AB sensors, we next investigated the detection of a hydrophobic molecule: phenylalanine. This essential amino acid is found in many foods, but its safe intake is restricted for individuals with phenylketonuria (PKU), a rare genetic disorder causing phenylalanine buildup in the body.
  • PKU phenylketonuria
  • Hydrophobic chemotherapeutic drugs are often administered to cancer patients through intravenous infusions to maintain stable drug levels in the blood.
  • current dosing strategies based on body surface area (BSA) don't account for the complex ways these drugs are eliminated.
  • Advanced tools for precise, real-time drug level measurement would provide doctors with a better way to prescribe effective chemotherapeutic dosages.
  • the E-AB sensors immobilized on the AuNPs-coated microneedles surface are not only reproducible but also robust, allowing multiple skin reinsertions while retaining the immobilized aptamers on the surface.
  • the technological microneedle E-AB sensors enable continuous, real-time, and multiredout monitoring of diverse drugs, laying the groundwork for personalized treatment strategies and improved patient outcomes.
  • Vancomycin a hydrophilic antibiotic (20 mg/kg, I.V. bolus)
  • vancomycin a hydrophilic antibiotic (20 mg/kg, I.V. bolus)
  • tobramycin 28 mg/kg, I.V. bolus
  • another hydrophilic antibiotic displayed a typical two-compartment model with rapid, first-order drug absorption.
  • the materials and methods included chemicals and materials, microneedle patch fabrication, electroplating and e-cleaning of the microneedles, E-AB sensor fabrication and calibration, electrochemical methods and data analysis software, and regression analysis of pharmacokinetic data.
  • An electrochemical sensor device comprising: a body structure comprising first and second surfaces; a plurality of microneedles extending from the first surface of the body structure, wherein the plurality of microneedles comprises at least one microneedle configured as a working electrode, at least one microneedle configured as a reference electrode, and at least one microneedle configured as a counter electrode; at least one electrically conductive layer disposed on at least a portion of the plurality of microneedles, wherein the electrically conductive layer is operably connected, or connectable, to a detector; a plurality of biomolecular receptor-bound redox reporters operably attached to the electrically conductive layer, wherein biomolecular receptors of the plurality of biomolecular receptor-bound redox reporters undergo conformational changes when the biomolecular receptors bind a target molecule to produce one or more electrochemical signals that are detected by the detector when the detector is operably connected to the electrically conductive layer; and, a
  • Clause 2 The electrochemical sensor device of Clause 1 , wherein the electrochemical sensor device 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 target molecule when the biomolecular receptors bind the target molecule.
  • SWV square wave voltammetry
  • CV cyclic voltammetry
  • Clause 3 The electrochemical sensor device of Clause 1 or Clause 2, wherein the electrochemical sensor device is configured to perform drift correction.
  • Clause 4 The electrochemical sensor device of any one of the preceding Clauses 1-3, wherein the drift correction is performed using kinetic differential measurements (KDM).
  • KDM kinetic differential measurements
  • Clause 5 The electrochemical sensor device of any one of the preceding Clauses 1-4, wherein the electrochemical sensor device is configured to generate substantially stable baseline electrochemical signals.
  • Clause 6 The electrochemical sensor device of any one of the preceding Clauses 1-5, wherein a thickness of the electrically conductive layer is less than about 1 pm.
  • Clause 7 The electrochemical sensor device of any one of the preceding Clauses 1-6, wherein the body structure comprises a substantially circular cross-sectional shape, a substantially square cross-sectional shape, or a substantially rectangular cross-sectional shape.
  • Clause 8 The electrochemical sensor device of any one of the preceding Clauses 1-7, wherein the plurality of microneedles is arrayed in a substantially circular format, a substantially square format, or a substantially rectangular format.
  • Clause 9 The electrochemical sensor device of any one of the preceding Clauses 1-8, wherein at least one of the microneedles in the plurality of microneedles has a substantially circular, a substantially square, or a substantially rectangular cross-sectional shape.
  • Clause 10 The electrochemical sensor device of any one of the preceding Clauses 1-9, wherein at least of the microneedles in the plurality of microneedles has a substantially conical shape, a substantially pyramidal shape, or a substantially tapered shape.
  • Clause 11 The electrochemical sensor device of any one of the preceding Clauses 1-10, wherein edges of at least one pair of microneedles in the plurality of microneedles are separated from one another by about 5 mm or less.
  • Clause 12 The electrochemical sensor device of any one of the preceding Clauses 1-11 , wherein the detector comprises an electrochemical analyzer.
  • Clause 13 The electrochemical sensor device of any one of the preceding Clauses 1-12, wherein the electrochemical analyzer comprises a potentiostat.
  • Clause 14 The electrochemical sensor device of any one of the preceding Clauses 1-13, wherein the electrically conductive layer is configured to wirelessly connect to the detector.
  • Clause 15 The electrochemical sensor device of any one of the preceding Clauses 1-14, wherein the detector is configured to measure square wave voltammograms.
  • Clause 16 The electrochemical sensor device of any one of the preceding Clauses 1-15, wherein an electrically insulating cover structure is attached to the first surface of the body structure, and wherein the plurality of microneedles extends through the electrically insulating cover structure.
  • Clause 17 The electrochemical sensor device of any one of the preceding Clauses 1-16, wherein the body structure and the plurality of microneedles are fabricated using a stereolithography (SLA) three-dimensional (3D) printing technique.
  • SLA stereolithography
  • 3D three-dimensional
  • Clause 18 The electrochemical sensor device of any one of the preceding Clauses 1-17, wherein the plurality of microneedles comprises twelve microneedles, wherein seven of the microneedles are working electrodes, wherein two of the microneedles are reference electrodes, and wherein three of the microneedles are counter electrodes.
  • Clause 19 The electrochemical sensor device of any one of the preceding Clauses 1-18, wherein the body structure comprises a cross-sectional dimension of about 30 mm or less.
  • Clause 20 The electrochemical sensor device of any one of the preceding Clauses 1-19, wherein the magnet comprises a permanent magnet.
  • Clause 21 The electrochemical sensor device of any one of the preceding Clauses 1-20, wherein the magnet comprises a neodymium ring magnet.
  • Clause 22 The electrochemical sensor device of any one of the preceding Clauses 1-21 , wherein the magnet comprises a cross-sectional dimension of about 25 mm or less.
  • Clause 23 The electrochemical sensor device of any one of the preceding Clauses 1-22, wherein the magnet comprises a thickness of about 5 mm or less.
  • Clause 24 The electrochemical sensor device of any one of the preceding Clauses 1-23, 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 electrodeblocking alkanethiols, one or more alkanethiols, and one or more redox reporter- tagged aptamers.
  • Clause 25 The electrochemical sensor device of any one of the preceding Clauses 1-24, wherein the subject comprises a mammalian subject.
  • Clause 26 The electrochemical sensor device of any one of the preceding Clauses 1-25, wherein the mammalian subject comprises a human subject.
  • Clause 27 The electrochemical sensor device of any one of the preceding Clauses 1-26, wherein the mammalian subject is from an order Rodentia.
  • Clause 28 The electrochemical sensor device of any one of the preceding Clauses 1-27, wherein the metallic structure is configured to maintain a substantially constant and substantially homogeneous pressure across the plurality of microneedles when the plurality of microneedles is disposed in contact with the dermal ISF of the subject.
  • Clause 29 The electrochemical sensor device of any one of the preceding Clauses 1-28, wherein the metallic structure is configured to maintain a substantially constant penetration depth of the plurality of microneedles when the plurality of microneedles is disposed in contact with the dermal ISF of the subject.
  • Clause 30 The electrochemical sensor device of any one of the preceding Clauses 1-29, wherein the metallic structure is disposed at least proximal to an ear of the subject.
  • Clause 31 The electrochemical sensor device of any one of the preceding Clauses 1-30, wherein a metallic plate comprises the metallic structure.
  • Clause 32 The electrochemical sensor device of any one of the preceding Clauses 1-31 , wherein the metallic structure is implanted in the subject.
  • Clause 33 The electrochemical sensor device of any one of the preceding Clauses 1-32, wherein the metallic structure comprises a biocompatible plate fabricated from a medical grade, ferromagnetic steel.
  • Clause 34 The electrochemical sensor device of any one of the preceding Clauses 1-33, wherein the plurality of microneedles is fabricated integral with the body structure.
  • Clause 35 The electrochemical sensor device of any one of the preceding Clauses 1-34, wherein the electrically conductive layer comprises a chromium layer and/or a gold layer.
  • Clause 36 The electrochemical sensor device of any one of the preceding Clauses 1-35, wherein the electrically conductive layer of the reference electrode further comprises at least one silver layer.
  • Clause 37 The electrochemical sensor device of any one of the preceding Clauses 1-36, wherein the electrically conductive layer of the counter electrode further comprises at least one platinum layer.
  • Clause 38 The electrochemical sensor device of any one of the preceding Clauses 1-37, wherein at least one microneedle of the plurality of microneedles extends from the surface of the body structure by about 0.5 mm to about 2 mm.
  • Clause 39 The electrochemical sensor device of any one of the preceding Clauses 1-38, wherein a base portion of at least one microneedle of the plurality of microneedles has a diameter of less than about 400 pm.
  • Clause 40 The electrochemical sensor device of any one of the preceding Clauses 1-39, wherein a tip portion of at least one microneedle of the plurality of microneedles has a diameter of less than about 100 pm.
  • Clause 41 The electrochemical sensor device of any one of the preceding Clauses 1-40, wherein the biomolecular receptor comprises an aptamer.
  • Clause 42 The electrochemical sensor device of any one of the preceding Clauses 1-41 , wherein the aptamer comprises a 5’- thiol modification and a 3’ methylene blue modification.
  • Clause 43 The electrochemical sensor device of any one of the preceding Clauses 1-42, wherein the biomolecular receptor comprises a nucleic acid molecule.
  • Clause 44 The electrochemical sensor device of any one of the preceding Clauses 1-43, wherein the redox reporters comprise methylene blue (MB) or an osmium-based complex.
  • MB methylene blue
  • Clause 45 The electrochemical sensor device of any one of the preceding Clauses 1-44, wherein the target molecule comprises a therapeutic agent.
  • Clause 46 The electrochemical sensor device of any one of the preceding Clauses 1-45, wherein the therapeutic agent comprises an antibiotic.
  • Clause 47 The electrochemical sensor device of any one of the preceding Clauses 1-46, wherein the target molecule comprises a metabolite and/or an electrolyte.
  • Clause 48 The electrochemical sensor device of any one of the preceding Clauses 1-47, wherein the target molecule comprises a biomolecule.
  • Clause 49 The electrochemical sensor device of any one of the preceding Clauses 1-48, wherein the electrochemical sensor device is a wearable device.
  • Clause 50 A kit comprising the electrochemical sensor device of any one of the preceding Clauses 1-49.
  • a method of detecting a target molecule in a subject comprising: positioning a plurality of microneedles of an electrochemical sensor device in contact with a dermal interstitial fluid (ISF) of or in the subject, wherein the electrochemical sensor device comprises: a body structure comprising first and second surfaces; the plurality of microneedles extending from the first surface of the body structure, wherein the plurality of microneedles comprises at least one microneedle configured as a working electrode, at least one microneedle configured as a reference electrode, and at least one microneedle configured as a counter electrode; at least one electrically conductive layer disposed on at least a portion of the plurality of microneedles, wherein the electrically conductive layer is operably connected to a detector; a plurality of biomolecular receptor-bound redox reporters operably attached to the electrically conductive layer, wherein biomolecular receptors of the plurality of biomolecular receptor-bound redox reporters undergo conform
  • ISF dermal interstitial fluid
  • Clause 52 The method of Clause 51 , further comprising implanting the metallic structure in the body of the subject prior to the positioning step.
  • Clause 53 The method of Clause 51 or Clause 52, comprising implanting the metallic structure in an ear lobe of the subject prior to the positioning step.
  • Clause 54 The method of any one of the preceding Clauses 51-53, comprising substantially continuously monitoring the target molecule in the subject during a selected duration of time.
  • Clause 55 The method of any one of the preceding Clauses 51-54, comprising intermittently monitoring the target molecule in the subject during a selected duration of time.
  • Clause 56 The method of any one of the preceding Clauses 51-55, comprising performing a pharmacokinetic, pharmacodynamic, and/or toxicology assessment of the target molecule in the subject.
  • Clause 57 The method of any one of the preceding Clauses 51-56, comprising detecting the electrochemical signals in substantially real-time.
  • Clause 58 The method of any one of the preceding Clauses 51-57, further comprising performing drift correction of the electrochemical sensor device using one or more kinetic differential measurements (KDM).
  • KDM kinetic differential measurements
  • Clause 59 The method of any one of the preceding Clauses 51-58, wherein the electrochemical sensor device further comprises an electrically insulating cover structure attached to the first surface of the body structure, and wherein the plurality of microneedles extends through the electrically insulating cover structure.
  • Clause 60 The method of any one of the preceding Clauses 51-59, 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 nucleic acids.
  • Clause 61 The method of any one of the preceding Clauses 51-60, wherein the redox reporters comprise methylene blue (MB).
  • MB methylene blue
  • a method of producing an electrochemical sensor device comprising: forming a body structure comprising first and second surfaces, and a plurality of microneedles extending from the first surface of the body structure; disposing at least one electrically conductive layer on at least a portion of the plurality of microneedles, wherein electrically conductive layer is operably connectable to a detector; attaching a plurality of biomolecular receptor-bound redox reporters to the electrically conductive layer, wherein biomolecular receptors of the plurality of biomolecular receptor-bound redox reporters are configured to undergo conformational changes when the biomolecular receptors bind a target molecule to produce one or more electrochemical signals that are detected by the detector when the detector is operably connected to the electrically conductive layer; and connecting a magnet to the second surface of the body structure, wherein the magnet is configured to magnetically attach the electrochemical sensor device to a metallic structure disposed in and/or on a body of a subject such that the electrochemical sensor device.
  • Clause 63 The method of Clause 62, further comprising attaching an electrically insulating cover structure to the first surface of the body structure such that the plurality of microneedles extends through the electrically insulating cover structure.
  • a system for detecting a target molecule in a subject comprising: a detector; an electrochemical sensor device, comprising: a body structure comprising first and second surfaces; a plurality of microneedles extending from the first surface of the body structure, wherein the plurality of microneedles comprises at least one microneedle configured as a working electrode, at least one microneedle configured as a reference electrode, and at least one microneedle configured as a counter electrode; at least one electrically conductive layer disposed on at least a portion of the plurality of microneedles, wherein electrically conductive layer is operably connected to the detector; a plurality of biomolecular receptorbound redox reporters operably attached to the electrically conductive layer, wherein biomolecular receptors of the plurality of biomolecular receptor-bound redox reporters undergo conformational changes when the biomolecular receptors bind the target molecule to produce one or more electrochemical signals that are detected by the detector; and, a

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Abstract

L'invention concerne des procédés de détection de molécules cibles à l'aide de capteurs électrochimiques qui comprennent des rapporteurs redox liés au récepteur biomoléculaire. L'invention concerne également des dispositifs de capteur électrochimique, des kits, des systèmes, des supports lisibles par ordinateur associés et des procédés supplémentaires.
PCT/US2024/054054 2023-11-03 2024-11-01 Réseaux de capteurs à micro-aiguilles magnétiques et aspects associés pour la surveillance moléculaire Pending WO2025096893A1 (fr)

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US20210379370A1 (en) * 2016-05-15 2021-12-09 Biolinq, Inc. Devices And Methods For The Mitigation Of Non-Analyte Signal Perturbations Incident Upon Analyte-Selective Sensor
US20230003725A1 (en) * 2019-12-12 2023-01-05 Joseph Wang Devices and methods for aptamer-assisted microneedle-based monitoring of biomarkers
US20230012662A1 (en) * 2021-07-07 2023-01-19 The Regents Of The University Of California Wearable, non-intrusive microneedle sensor

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US20210379370A1 (en) * 2016-05-15 2021-12-09 Biolinq, Inc. Devices And Methods For The Mitigation Of Non-Analyte Signal Perturbations Incident Upon Analyte-Selective Sensor
US20190099129A1 (en) * 2017-10-04 2019-04-04 Align Technology, Inc. Intraoral appliances for sampling soft-tissue
US20190247650A1 (en) * 2018-02-14 2019-08-15 Bao Tran Systems and methods for augmenting human muscle controls
US20230003725A1 (en) * 2019-12-12 2023-01-05 Joseph Wang Devices and methods for aptamer-assisted microneedle-based monitoring of biomarkers
US20230012662A1 (en) * 2021-07-07 2023-01-19 The Regents Of The University Of California Wearable, non-intrusive microneedle sensor

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