Patent Application
20210140956
Sweat contains many of the same biomarkers, chemicals, or solutes that are carried in blood and can provide significant information enabling one to diagnose illness, health status, exposure to toxins, performance, and other physiological attributes even in advance of any physical sign. Sweat itself, the action of sweating, and other parameters, attributes, solutes, or features on, near, or beneath the skin, or within interstitial fluid, also can be measured to further reveal physiological information. Recent progress in the development of wearable sweat sensing devices has been limited to high concentration analytes (μM to mM) sampled at high sweat rates (>1 nL/min/gland) found in, for example, athletic applications. However, progress will be much more challenging as wearable biosensing moves towards detection of large, low concentration analytes (nM to pM and lower).
In particular, many known sensor technologies for detecting biofluid solutes are ill-suited for use in wearable biofluid sensing, which requires sensors that permit continuous or extended use on a wearer's skin. This means that sensor modalities that require complex microfluidic manipulation, the addition of reagents, or the use of limited shelf-life components, such as antibodies, will not be sufficient for wearable sensing. What is needed is a stable, reliable, reagentless sensor that is sensitive to target analytes in biofluid, while providing the level of specificity necessary to produce high predictive values during the lifespan of the sensor. One solution to this problem is the use of electrochemical aptamer-based (“EAB”) sensor technology, such as the multiple-capture EAB biosensors (“MCAS”) disclosed in U.S. Pat. Nos. 7,803,542 and 8,003,374, or the docked aptamer EAB biosensors (DAS) disclosed in U.S. Provisional Application No. 62/523,835, filed Jun. 23, 2017, each of which is hereby incorporated by reference herein in its entirety.
Unfortunately, both types of EAB sensors can be vulnerable to errors caused by physical degradation of the individual aptamer sensing elements, changes in sensor responses that are due to fouling, or changes in environmental conditions, rather than changes in target analyte concentrations. Over time, aptamer sensing elements within an EAB sensor will physically degrade, meaning the sensing elements will become unattached to the electrode surface, or that parts of the sensing elements will disassociate from the sensing element structure. For example, analyte capture complexes can gradually detach from their respective docks, the docks themselves can detach from the electrode surface, or redox moieties can detach from the aptamer. Another source of error is non-specific binding to the aptamer sensing element or the electrode surface. Biofluids such as sweat, blood, saliva, or interstitial fluid contain numerous solutes, including large proteins. These can bind randomly to aptamer sensing elements or the electrode surface, altering or hindering sensor response to an analyte. Similarly, changes in external weather, internal temperature, and biofluid sample potential of hydrogen (pH) and salinity can affect the rate at which the sensors degrade and can affect the sensor response to target analyte concentrations. Others in the field of EAB sensing have posed solutions to similar problems, see, e.g., Li, H., et al., “Dual-reporter drift correction to enhance the performance of electrochemical aptamer-based sensors in whole blood,” J. Am. Chem. Soc., 10.1021/jacs.6b08671, 2016 (disclosing an EAB sensor with a pair of redox moieties attached to the aptamer, each having non-overlapping potential ranges, one of which moves in response to analyte capture and the other which serves as a reference); see also PCT/US18/58020, filed Oct. 29, 2018 (disclosing chronoamperometric EAB interrogation, which provides a signal that is independent of the number of aptamer sensing elements on the electrode surface).
Therefore, some embodiments of the disclosed invention include a reference EAB sensor to provide drift correction and calibration for a companion active EAB sensor. Other embodiments include reference sensor elements incorporated alongside active sensing elements within the same sensor to provide similar drift correction and calibration. Such devices and methods are the subject of the present disclosure.
Electrochemical aptamer-based (EAB) biosensing devices are described that provide drift correction and calibration to EAB sensor measurements of biofluid analyte concentrations by disclosing reference sensors that are configured to not interact with a target analyte, but otherwise mirror the performance of active EAB sensors within the expected application parameters of the device. Such reference sensors are configured to allow comparisons with their companion active sensors to track aptamer sensing element dissociation from an electrode surface, temperature-induced effects, redox moiety dissociation, and/or the effects of surface fouling. Some embodiments include separate electrodes for active and reference aptamer sensing elements. Other embodiments include a single electrode for both active and reference aptamer sensing elements. Single electrode embodiments include two or more distinct redox moieties.
The present disclosure will be further appreciated in light of the following detailed descriptions and drawings in which:
Before continuing with a detailed description of the exemplary embodiments, a variety of definitions should be made, these definitions gaining further appreciation and scope in the detailed description and embodiments of the present disclosure.
As used herein, “sweat” means a biofluid that is primarily sweat, such as eccrine or apocrine sweat, and may also include mixtures of biofluids such as sweat and blood, or sweat and interstitial fluid, so long as advective transport of the biofluid mixtures (e.g., flow) is primarily driven by sweat.
As used herein, “biofluid” may mean any human biofluid, including, without limitation, sweat, interstitial fluid, blood, plasma, serum, tears, and saliva.
“Biofluid sensor” means any type of sensor that measures a state, presence, flow rate, solute concentration, solute presence, in absolute, relative, trending, or other ways in a biofluid. Biofluid sensors can include, for example, potentiometric, amperometric, impedance, optical, mechanical, antibody, peptide, aptamer, or other means known by those skilled in the art of sensing or biosensing.
“Analyte” means a substance, molecule, ion, or other material that is measured by a biofluid sensing device.
“Measured” can imply an exact or precise quantitative measurement and can include broader meanings such as, for example, measuring a relative amount of change of something. Measured can also imply a binary or qualitative measurement, such as ‘yes’ or ‘no’ type measurements.
“Chronological assurance” means the sampling rate or sampling interval that assures measurement(s) of analytes in biofluid in terms of the rate at which measurements can be made of new biofluid analytes emerging from the body. Chronological assurance may also include a determination of the effect of sensor function, potential contamination with previously generated analytes, other fluids, or other measurement contamination sources for the measurement(s). Chronological assurance may have an offset for time delays in the body (e.g., a well-known 5- to 30-minute lag time between analytes in blood emerging in interstitial fluid), but the resulting sampling interval is independent of lag time, and furthermore, this lag time is inside the body, and therefore, for chronological assurance as defined above and interpreted herein, this lag time does not apply.
“EAB sensor” means an electrochemical aptamer-based biosensor that is configured with a plurality of aptamer sensing elements that, in the presence of a target analyte in a fluid sample, produce a signal indicating analyte capture, and which signal can be added to the signals of other such sensing elements, so that a signal threshold may be reached that indicates the presence or concentration of the target analyte. Such sensors can be in the forms disclosed in U.S. Pat. Nos. 7,803,542 and 8,003,374 (the “Multi-capture Aptamer Sensor” (MCAS)), or in U.S. Provisional Application No. 62/523,835 (the “Docked Aptamer Sensor” (DAS)).
“Analyte capture complex” means an aptamer, or other suitable molecules or complexes, such as proteins, polymers, molecularly imprinted polymers, polypeptides, and glycans, that experience a conformation change in the presence of a target analyte, and are capable of being used in an EAB sensor. Such molecules or complexes can be modified by the addition of one or more linker sections comprised of nucleotide bases.
“Aptamer sensing element” means an analyte capture complex that is functionalized to operate in conjunction with an electrode to detect the presence of a target analyte. Such functionalization may include tagging the aptamer with a redox moiety, or attaching thiol binding molecules, docking structures, or other components to the aptamer. Multiple aptamer sensing elements functionalized on an electrode comprise an EAB sensor.
“Reference EAB sensor” means a reference sensor that comprises aptamer sensing elements functionalized on an electrode base, where the aptamers do not interact with target analyte molecules, or have reduced interaction with target analyte molecules. A reference EAB sensor is configured to perform similarly to a comparable active EAB biosensor to facilitate calibration for one of more sources of drift or error.
“Reference aptamer sensing element” means an individual aptamer sensing element that is configured to have no or reduced interaction with target analyte molecules, but otherwise performs similarly to a comparable active aptamer sensing element. A plurality of reference aptamer sensing elements may be incorporated with active sensing elements to comprise an EAB sensor with built-in reference capabilities.
“Sensitivity” means the change in output of the sensor per unit change in the parameter being measured. The change may be constant over the range of the sensor (linear), or it may vary (nonlinear).
“Signal threshold” means the combined strength of signal-on indications produced by a plurality of aptamer sensing elements that indicates the presence of a target analyte.
One skilled in the art will recognize that the various embodiments may be practiced without one or more of the specific details described herein, or with other replacement and/or additional methods, materials, or components. In other instances, well-known structures, materials, or operations are not shown or described in detail herein to avoid obscuring aspects of various embodiments of the invention. Similarly, for purposes of explanation, specific numbers, materials, and configurations are set forth herein in order to provide a thorough understanding of the invention. Furthermore, it is understood that the various embodiments shown in the figures are illustrative representations and are not necessarily drawn to scale.
Reference throughout this specification to “one embodiment” or “an embodiment” means that a particular feature, structure, material, or characteristic described in connection with the embodiment is included in at least one embodiment of the invention, but does not denote that they are present in every embodiment. Thus, the appearances of the phrases “in an embodiment” or “in another embodiment” in various places throughout this specification are not necessarily referring to the same embodiment of the invention. Further, “a component” may be representative of one or more components and, thus, may be used herein to mean “at least one.”
Certain embodiments of the disclosed invention show sensors as simple individual elements. It is understood that many sensors require two or more electrodes, reference electrodes, or additional supporting technology or features which are not captured in the description herein. Sensors are preferably electrical in nature, but may also include optical, chemical, mechanical, or other known biosensing mechanisms. Sensors can be in duplicate, triplicate, or more, to provide improved data and readings. Sensors may be referred to by what the sensor is sensing, for example: a biofluid sensor; an impedance sensor; a sample volume sensor; a sample generation rate sensor; and a solute generation rate sensor. Certain embodiments of the disclosed invention show sub-components of what would be sensing devices with more sub-components needed for use of the device in various applications, which are obvious (such as a battery), and for purposes of brevity and focus on inventive aspects, such components are not explicitly shown in the diagrams or described in the embodiments of the disclosed invention. As a further example, many embodiments of the disclosed invention could benefit from mechanical or other means known to those skilled in wearable devices, patches, bandages, and other technologies or materials affixed to skin, to keep the devices or sub-components of the skin firmly affixed to skin or with pressure favoring constant contact with skin or conformal contact with even ridges or grooves in skin, and are included within the scope of the disclosed invention.
The detailed description of the present invention will be primarily, but not entirely, limited to devices, methods and sub-methods using wearable biofluid sensing devices. Therefore, although not described in detail here, other essential steps which are readily interpreted from or incorporated along with the present invention shall be included as part of the disclosed invention. The disclosure provides specific examples to portray inventive steps, but which will not necessarily cover all possible embodiments commonly known to those skilled in the art. For example, the specific invention will not necessarily include all obvious features needed for operation. Several specific, but non-limiting, examples can be provided as follows. The invention includes reference to the article in press for publication in the journal IEEE Transactions on Biomedical Engineering, titled “Adhesive RFID Sensor Patch for Monitoring of Sweat Electrolytes”; the article published in the journal AIP Biomicrofluidics, 9 031301 (2015), titled “The Microfluidics of the Eccrine Sweat Gland, Including Biomarker Partitioning, Transport, and Biosensing Implications”; as well as PCT/US16/36038, and U.S. Provisional Application No. 62/327,408, each of which is included herein by reference in their entirety. Techniques for concentrating a biofluid sample are disclosed in PCT/US16/58356, and U.S. Provisional Application No. 62/457,604, which are also hereby incorporated herein by reference in their entirety.
The embodiments described herein address a need to provide drift correction and calibration for electrochemical aptamer-based sensors through the use of reference sensors or reference sensing elements. For example, by including reference EAB sensors, a biofluid sensing device can compare the behavior of the reference sensor to that of the active sensor during the sensors' exposure to target analyte molecules. There are a number of data points obtainable through such a comparison. For example, both MCAS and DAS sensors can degrade over time through dissociation of components from the aptamer sensing elements or the sensing elements from the electrode. In addition, normal use of DAS sensors results in the loss of aptamer sensing elements, since analyte capture causes irreversible detachment of the aptamer from the electrode. A contemporaneous comparison of the reference EAB's signal to that of the active sensor can therefore reveal the number of functional sensor elements at the time of sensor use, so that signal strength can be more accurately correlated with analyte concentration. Such corrective inputs may take the form of electronic corrections to output signals, or may be applied via algorithm.
Similarly, non-specific binding, both to the aptamer sensing elements and to the electrode surface, can alter the signal produced by EAB sensors in the presence of target analyte molecules. Such fouling can cause steric hinderance or change an aptamer's secondary structure, flexibility, or other property. Large proteins can settle onto aptamer sensing elements, physically hindering target analyte interaction. All of these effects change the aptamer's reaction to target analyte molecules, and alter the signals that would result from such interaction. In addition, non-specific binding to the electrode surface can interfere with redox proximity to the electrode, hindering electrical response to target analyte, for example, by preventing the redox from coming close enough to the electrode to allow electron exchange.
As a specific example of a non-specific binding effect, pH variability, which reflects H+ ion concentrations in the biofluid, can alter sensing element behavior by providing different binding opportunities between the sensing elements and H+ ions. The number and location of H+ ions that bind to the sensing elements can significantly alter their folding characteristics, which again affects signal strength in the event of analyte binding. Biofluid salinity acts similarly to pH, in that different levels of ions in the biofluid present different probabilities of binding between the sensing elements and ions, which translates to altered signal output from the sensor. Together, these characteristics can significantly influence EAB sensor signals, and the reference capability disclosed herein will allow biofluid sensing devices to account for such influences and better isolate sensor signal due to analyte capture.
Another point of comparison between active and reference sensors is the effect of the biofluid sensing environment on EAB sensor signal response. Environmental factors such as outside weather and internal ambient temperature can alter the behavior of aptamer sensing elements, changing how they physically present themselves in relation to the electrode, both prior to and after analyte capture. For instance, the temperature of the sensor environment or of the biofluid itself can cause aptamer sensing elements to change their physical conformations. These conformation changes may bring the sensing elements' redox moieties closer to or further away from the sensor electrode, resulting in a change to the background signal produced by the sensor in the absence of target analyte molecules. Such factors can therefore alter the signal produced by the EAB sensor and affect how the signal is translated into an analyte concentration value.
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In order to function as both an active and a reference sensor, the MCAS sensor described is configured so that the device can readily distinguish the active signals from the reference signals. In the depicted embodiment, the active sensing elements 110 are attached to a first electrode 130, while the reference sensing elements are attached to a second electrode 132. Other embodiments use a first redox moiety for the active sensing elements, and a second redox moiety for the reference sensing elements (not shown). Upon interrogation by the device, the active and reference sensing elements produce a first electrical signal, eTC.
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Tracking an EAB sensor's complement of functional sensing elements could prove especially useful for monitoring DAS function, since the normal use of the sensor causes the loss of analyte capture complexes. The loss of analyte capture complexes will necessarily reduce the amount of signal change available to the sensor when exposed to target analyte in the biofluid, and therefore affects the sensitivity of the sensor. A reference sensor could therefore calibrate the sensor by establishing a baseline signal that reflects the operational age of the active sensor (by accounting for time-based or use-based sensing element degradation), and changes due to biofluid characteristics. Such a baseline signal can then be compared to the active DAS signal to isolate the contribution of target analyte capture to the active DAS sensor signal. On subsequent uses, the baseline signal can also isolate the decrease in active sensor signal strength due to normal sensing element loss, i.e., analyte capture complexes that capture analytes and detach from their respective docks. If the background decay of both the reference and the sensing element are equivalent, then the difference of the two can be used to approximate the total accumulation of analyte over time.
Reference sensors or sensing elements may have a number of modifications with respect to their active sensor counterparts that allow the reference sensors to perform their desired calibration function. One such category of modifications includes the replacement or alteration of the randomized aptamer sequence that is selected to interact with the target analyte. For instance, at least a portion of the aptamer sequence used in the active sensing elements may be rearranged or randomized, so that the reference aptamer will not bind with the target analyte. Typically, aptamer sequences that interact strongly with a target analyte will have active binding sites that interact with the analyte interspersed along the sequence. One or more of these active binding sites can be disrupted by replacing the nucleotide base(s) at the active site with different bases, by moving the active site to another location on the aptamer, or by other suitable method. Alternatively, causing point mutations in portions of the aptamer not actively involved with binding the analyte may also disrupt the secondary structure enough to prevent binding with the analyte. Similarly, nucleotide bases may be replaced by non-native bases, or aptamers with different chirality, e.g., spiegelmers or left-handed ribonucleic acid (L-RNA) aptamers, may be used to reduce interaction with the analyte. In addition, entirely different aptamers may be used for the reference aptamer, for example, an aptamer selected to interact with a target unlikely to be present in the biofluid. Or the reference aptamer may be selected to have behavioral traits similar to the active aptamer without interacting with the target analyte, for example, aptamers having similar secondary structures to the active aptamer sequence. Such aptamers can be identified and selected through the use of isothermal titration calorimetry (“ITC”), nuclear magnetic resonance spectroscopy, x-ray crystallography, differential scanning calorimetry, or other suitable techniques which reveal the aptamer secondary structure.
Reference sensing elements or reference sensors may be configured to track specific sources of drift or error, or may be configured to track drift generally, which represents a composite of specific sources of error. For example, one source of error is dissociation of aptamer sensing elements from the electrode surface. Dissociation has many potential causes, including excessive temperatures, exposure to light and other radiation, oxidation, exposure to biofluid solutes, and other pathways known in the art. A simplified “dummy” reference sensor or sensing element may be configured to track aptamer dissociation by replacing the active aptamer with a non-nucleotide sequence, such as a simple carbon chain, or by bonding the redox moiety directly to a thiol or a docking structure without including an aptamer or aptamer substitute.
Another variable affecting EAB sensor error or drift is temperature. As a first order consideration, temperature affects aptamer conformation response to analyte capture. A temperature sensor may be used to measure the ambient temperature in the vicinity of the aptamer, and provide a benchmark that may be used with a look up table to calibrate sensor response at a given temperature. As a second order consideration, higher ambient and/or biofluid temperatures, e.g., above 70° C., also cause or accelerate aptamer sensing element dissociation from the electrode surface. To track temperature-induced dissociation, a reference sensor may be functionalized with elements known to have a specific decay rate due to temperature effects. For example, the electrode surface may be affixed with a SAM, molecule, or polymer with known temperature-induced decay rates, or an aptamer sensing element, or dummy element, may be attached to redox moiety having a known temperature-induced dissociation profile. This temperature reference sensor would provide a temperature hysteresis correction factor for the active sensor. A reference may also provide a measure of the remaining lifetime of the active sensor or sensing elements by facilitating an estimate of remaining active sensing elements on a sensor.
Another source of error is dissociation of the redox moiety from the aptamer sensing element. Some embodiments accordingly will track such error by using a plurality of different redox moieties on active sensor elements or companion reference sensing elements. Each type of redox moiety would have a distinct electrochemical and/or chemical behavior profile. For example, the redox moieties can vary based on the numbers of exchanged electrons, reversibility of redox reaction, reaction speed, pH dependence, protonation constant, redox equilibrium constant, redox potential, susceptibility to electrochemically induced degradation, oxidation, relaxation mechanism, fluorescence, hydrophilicity, and amphiphilicity. The changes in signal responses between the redox moieties can provide corrective inputs as to sensor activity relevant to analyte concentration, or can provide inputs tracking sensor lifetime.
Another specific source of drift is the tendency of EABs to experience surface fouling due to solutes settling on, or bonding to, the aptamer sensing elements, the self-assembled monolayer, or the electrode surface. In embodiments configured to track surface fouling error, a filter or selectively permeable membrane may be placed upstream of a reference sensor to isolate the influence of larger proteins and other solutes on sensor response. Such a reference sensor would see less change due to large molecule non-specific binding, or aptamer and electrode surface fouling, which is compared to readings from unfiltered reference or active sensors. Alternatively, a plurality of depletion surfaces functionalized to covalently bond potentially interfering solutes can be placed upstream of reference or active sensors. Like filters, these depletion surfaces remove potentially fouling solutes from the biofluid, allowing for measurement and comparison to sensors measuring untreated biofluid. Some embodiments may include depletion zones between two active or reference sensors to compare measurements with and without fouling species present.
In addition to the proactive reference sensors and sensor elements described, some embodiments may be configured as passive reference sensors. One such passive reference sensor uses fluorescent tags that can be read to determine the amount of sensor dissociation over time. For example, an EAB sensor includes a plurality of reference aptamer sensing elements that have a fluorescent tag affixed to their redox moieties, to their docking structures, or elsewhere. As the EAB sensor degrades over time, the amount of fluorescence remaining on the EAB sensor is measured, e.g., with an optical sensor such as a photodiode, and the degree of dissociation determined.
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As the sweat sample moves through the channel, it becomes increasingly concentrated, and interacts with at least one active EAB sensor 422, 424 and at least one reference EAB sensor 421, 423. In an alternate embodiment, instead of using separate active and reference EAB sensors, some devices will have one or more EAB sensors that include both active and reference aptamer sensing elements (not shown). Some embodiments also include one or more secondary sensors (not shown), which are one of the following: a micro-thermal flow rate sensor, one or more ISEs for measuring electrolytes (H+, Na+, Cl−, K+ Mg2+, etc.), a sweat conductivity sensor, a temperature sensor, or other sensor. Some embodiments also include a sweat stimulant gel 440 composed of sweat stimulant such as carbachol or pilocarpine, and agar, and an iontophoresis electrode 450. The electrode 450 can also be used to measure skin impedance or galvanic skin response (“GSR”), which indicates sweat onset or sweat cessation timing. In use, such a device 400 takes measurements produced by the active EAB sensors 422, 424, and compares them to measurements from the reference EAB sensors 421, 423, allowing signal output due to captured analyte molecules to be isolated from signal caused by other factors.
While several exemplary embodiments have been described herein, it is anticipated that other types configurations may also be used. Various modifications, alterations, and adaptations to the embodiments described herein may occur to persons skilled in the art with attainment of at least some of the advantages. The disclosed embodiments are therefore intended to include all such modifications, alterations, and adaptations without departing from the scope of the embodiments as set forth herein.
This has been a description of the disclosed invention along with a preferred method of practicing the disclosed invention, however the invention itself should only be defined by the appended claims.
The present application claims priority to PCT/US18/61557, filed Nov. 16, 2018, and U.S. Provisional Application No. 62/587,829, filed Nov. 17, 2017, and has specification that builds upon U.S. Provisional Application No. 62/523,835, filed Jun. 23, 2017, the disclosures of which are hereby incorporated herein by reference in their entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US18/61557 | 11/16/2018 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62587829 | Nov 2017 | US |
