Patent Application
20190200892
Despite the many ergonomic advantages of perspiration (sweat) compared to other biofluids (particularly in “wearable” devices), sweat remains an underutilized source of biomarker analytes compared to the established biofluids: blood, urine, and saliva. Upon closer comparison to other non-invasive biofluids, the advantages may even extend beyond ergonomics: sweat might provide superior analyte information. A number of challenges, however, have historically kept sweat from occupying its place among the preferred clinical biofluids. These challenges include very low sample volumes (nL to μL), unknown concentration due to evaporation, filtration and dilution of large analytes, mixing of old and new sweat, and the potential for contamination from the skin surface. More recently, rapid progress in “wearable” sweat sampling and sensing devices has resolved several of the historical challenges. However, this recent progress has also been limited to high concentration analytes (μM to mM) sampled at high sweat rates (>1 nL/min/gland) found in, for example athletic applications. Progress will be much more challenging as sweat biosensing moves towards detection of large, low concentration analytes (nM to pM and lower).
In particular, many known sensor technologies for detecting larger molecules are ill-suited for use in wearable sweat sensing, which requires sensors that permit continuous use on a wearer's skin. This means that sensor modalities that require complex microfluidic manipulation, the addition of reagents, the use of limited shelf-life components, such as antibodies, or sensors that are designed for a single use will not be sufficient for sweat sensing. Electrochemical aptamer-based (“EAB”) sensor technology, such as is disclosed in U.S. Pat. Nos. 7,803,542 and 8,003,374, presents a stable, reliable bioelectric sensor that is sensitive to the target analyte in sweat, while being capable of multiple analyte capture events during the sensor lifespan.
As disclosed in PCT/US17/23399, EAB sensors for use in continuous sweat sensing are configured to provide stable sensor responses with a life cycle extensive enough for multiple analyte binding and release cycles. Such sensors include a plurality of individual aptamer sensing elements 110, as depicted in
With reference to
EAB sensors, as depicted in
Additionally, EAB sensors that target small analytes are inherently less stable and, thereby, produce less reliable signals, than sensors targeting larger analytes. Target analytes for sweat sensor applications as contemplated herein may range in size from ˜300 Da for hormones to ˜15 kDa for microRNA molecules to ˜600 kDa for larger proteins to ˜1000 kDa for the largest proteins. Other factors being equal, aptamers will generally develop stronger bonds to larger molecules because of the greater number of bonding sites available on such molecules. Furthermore, sweat sample composition variabilities that tend to alter bonding strength (such as pH and salinity) generally have a greater effect on smaller molecule sensors than on larger molecule sensors. Aptamer sensing elements configured to detect small molecules will tend to have shorter recovery intervals (i.e. seconds or faster), due to the low number of binding sites. The instability created by small-sized analytes is compounded as the analyte concentration in a biofluid sample decreases.
Therefore, for smaller analytes, or analytes appearing in low concentrations in a biofluid such as sweat, an EAB sensor as described above may be unable to accurately perform continuous sensing. Too few target analytes may be present in a sweat sample, such that during any given recovery interval, not enough analytes will bond with an aptamer to meet the signal threshold. Following the recovery interval, bound analytes will release back into solution before another chronologically assured sweat sample can be measured. In such a scenario, while the target analyte is present in the sweat sample, the sensing device will not be able to provide an accurate measurement of the analyte's presence, much less a reliable concentration value.
Accordingly, for small analyte and low analyte concentration applications, it is desirable to have an EAB sensor that can provide a qualitative “yes/no” measurement for the presence or absence of a target analyte. In particular, it is desirable to have EAB sensing devices and methods that can accurately assess a small or low concentration analyte's presence in a sweat sample by aggregating analyte captures over an extended period of time.
The disclosed invention can improve its performance for biofluid sensing by contextualizing data generated by the device with relevant external information. Such contextualization may include collecting the biofluid sensor data generated by biofluid sensing devices and correlating that data with relevant outside information, such as the time, date, medications, medical condition, the proximity to significant health events or stressors, age, sex, health history, or other relevant information. The sweat sensor data monitored by the user includes real-time data, trend data, or may also include aggregated sweat sensor data drawn from the system database and correlated to a particular user, a user profile (such as age, sex or fitness level), weather condition, activity, combined analyte profile, or other relevant metric. Such predictive capability can be enhanced by using correlated aggregated data, which would allow the user to compare an individual's historical analyte and external data profiles to a real-time situation as it progresses, or even to compare thousands of similar analyte and external data profiles from other individuals to the real-time situation. Biofluid sensor data may also be used to identify wearers that are in need of additional monitoring or screening.
Many of the other challenges to successful sweat sensor development can be resolved by creating novel and advanced interplays of chemicals, materials, sensors, electronics, microfluidics, algorithms, computing, software, systems, and other features or designs, in a manner that affordably, effectively, conveniently, intelligently, or reliably brings sweat to sensors and sample preparing or concentrating subsystems.
The disclosed invention includes integrative electrochemical aptamer based sensors for use in wearable biofluid sensing devices. The disclosed integrative EAB sensors are configured to detect very low concentrations of target analytes in a sweat or biofluid sample by aggregating signals from individual sensing elements over time until a signal threshold is reached. Signal aggregation is accomplished through various retention structures that extend the time sensing elements retain target analyte molecules. Embodiments include attaching complementary primers and functional groups to the aptamer, covering such retention structures with blockers until analyte capture, and coating the sensor electrode with a hydrophilic and hydrophobic monolayer. The invention also includes methods of using the disclosed integrative sensors. Some embodiments of the disclosed method include tracking time to signal threshold to develop an analyte concentration estimate.
The objects and advantages of the present invention will be further appreciated in light of the following detailed descriptions and drawings in which:
“Continuous monitoring” means the capability of a device to provide at least one measurement of sweat determined by a continuous or multiple collection and sensing of that measurement or to provide a plurality of measurements of sweat over time.
“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 (defined below) 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.
As used herein, “biofluid” may mean any human biofluid, including, without limitation, sweat, interstitial fluid, blood, plasma, serum, tears, and saliva. A biofluid may be diluted with water or other solvents inside a device because the term biofluid refers to the state of the fluid as it emerges from the body.
“Sweat” or “sweat biofluid” means a fluid that is comprised mainly of interstitial fluid or sweat as it emerges from the skin. For example, a fluid that is 45% interstitial fluid, 45% sweat, and 10% blood is a sweat biofluid as used herein. For example, a fluid that is 20% interstitial fluid, 20% sweat, and 60% blood is not a sweat biofluid as used herein.
“Sweat sampling rate” is the effective rate at which new sweat or sweat solutes, originating from the sweat gland or from skin or tissue, reaches a sensor which measures a property of sweat or its solutes. Sweat sampling rate, in some cases, can be far more complex than just sweat generation rate.
“Sweat generation rate” is the rate at which sweat is generated by the sweat glands themselves. Sweat generation rate is typically measured by the flow rate from each gland in nL/min/gland. In some cases, the measurement is then multiplied by the number of sweat glands from which the sweat is being sampled.
“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 measurement, such as ‘yes’ or ‘no’ type measurements.
“Microfluidic components” are channels in polymer, textiles, paper, or other components known in the art of microfluidics for guiding fluid movement or at least partial containment of a fluid.
“Flow rate sensing component”, is any component or components which measure the flow rate of sweat in at least one portion of a sweat sensing or collecting device.
“Analyte” means a substance, molecule, ion, or other material that is measured by a sweat sensing device.
“Biofluid sensor data” means all information collected by biofluid sensing device sensor(s) and communicated to a user or a data aggregation location.
“Correlated aggregated biofluid sensor data” means biofluid sensor data that has been collected in a data aggregation location and correlated with outside information such as time, temperature, weather, location, user profile, other biofluid sensor data, or any other relevant data.
“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 primers 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.
“Docked aptamer EAB sensor” means an EAB sensor that employs docking strategies to connect analyte capture complexes with the sensor electrode, as disclosed in U.S. Provisional No. 62/523,835, filed Jun. 23, 2017, which is hereby incorporated by reference in its entirety herein.
“Integrative EAB sensor” means an aptamer-based biosensor that is configured with multiple aptamer sensing elements that produce a sustained signal indicating target analyte capture, and which signal can be added to the signals of other such sensing elements, so that over time a signal threshold may be reached that indicates the presence of the target analyte.
“Recovery interval” means the time required for an aptamer sensing element to release a target analyte back into solution and return to its signal-off position.
“Signal threshold” means the combined strength of signal-on indications produced by a plurality of integrative aptamer sensing elements that indicates the presence of a target analyte.
“Time-to-threshold” means the amount of time required for an integrative EAB sensor to reach signal threshold. Such time may be calculated from the initiation of device use, the initiation of sweating, a sensor regeneration time, or other suitable starting point.
“Preferential energy state” means a relatively stable configuration of an analyte capture complex, typically requiring an energy input to allow the complex to change to a different configuration.
Integrative EAB sensors are described herein for use within a biofluid sensing device. The EAB sensors include a plurality of aptamer sensing elements that are selected to capture a target analyte in a biofluid, such as sweat. Rather than releasing the analyte and returning to a “signal-off” configuration following the typical aptamer recovery interval, as with the aptamer sensing elements described above, the aptamer sensing elements in the disclosed invention remain in a “signal-on” configuration for an extended period of time (e.g., several minutes, or hours), while sweat samples continuously or periodically flow past the sensors. Retaining the aptamer sensing elements in an analyte capture configuration for an extended period of time enables the “capture” signals from the sensing elements to be aggregated over the extended time period. The extended period enables sufficient analyte captures to occur for the aggregated signal to reach the signal threshold indicative of the target analyte's presence in the sweat samples.
To select suitable aptamers for the integrative EAB sensors, various methods known in the art of aptamer selection may be used, including, for example, Systematic Evolution of Ligands by Exponential Enrichment (“SELEX”) techniques. Using such techniques, an aptamer for binding a target analyte is selected to reliably detect the analyte at very low concentration levels. The aptamer is also chosen for its affinity to the target molecule, such that the analyte will be reliably captured, giving the integrative EAB sensor the desired selectivity and specificity. For many target analytes, however, the selected aptamer will not possess a recovery interval that is long enough to allow for a detectable capture signal from the electrode. For such analytes, therefore, other structural elements may be incorporated into an EAB sensor to retain an aptamer sensing element in a preferential energy state for an extended time period upon analyte capture.
For example, with reference to
With reference to
In an alternative embodiment, depicted in
When the aptamer 340 captures a target analyte 360, the first configuration is disrupted, and a second configuration is formed, causing the blocker 346 to break free from the analyte capture complex 312 and move away. The formation of the second configuration also moves the redox moiety 350 into a second position relative to the electrode 330. This change in redox position causes the electrode 330 to produce a second electrical signal eTB, that is distinguishable from the first electrical signal eTA, when the electrode is interrogated. When the aptamer 340 moves into the second configuration, functional groups 341 on opposite ends of the aptamer are drawn into close proximity with each other. This close proximity between functional groups 341 creates a favorable energy state for the analyte capture complex 312 in the second configuration, retaining the aptamer in the second configuration, and allowing the aptamer sensing element 310 to generate capture signal eTB for a sustained period of time. As with the previous embodiment, the separation of the blocker 346 from aptamer sensing element 310 on capture of analyte 360, and the subsequent bond between functional groups 341, prevents the aptamer 340 from returning to the first configuration. Accordingly, sensing devices of this embodiment are one-time use sensors. Some embodiments may include functional groups attached to the redox moiety (not shown).
In another alternative embodiment, depicted in
However, when aptamer 440 captures a target analyte 460, the first configuration is disrupted, and a second configuration, depicted in
In another alternative embodiment, depicted in
In operation, the aptamer sensing element 510 is exposed to a biofluid sample containing a concentration of the target analyte 560. With reference to
Each of the embodiments described above allows an aptamer sensing element to capture and retain a target analyte beyond a typical recovery interval for the selected aptamer. In each of the embodiments, a capture signal eTB is produced for a sustained period of time, through multiple sweat sampling intervals. The sustained signal period allows for additional analyte captures by other aptamer sensing elements in the sensor, and the aggregation of all the analyte capture signals. The aggregated signal can be compared to the sensor signal threshold to measure the presence of the target analyte. Accordingly, the devices described herein allow a greater time-to-threshold for an EAB sensor to provide increased measurement accuracy, particularly with small and/or low concentration analytes.
Turning now to
With reference to
For example, with regard to required certainty, EAB sensing devices as described herein may be designed to produce a predictive value for the desired application, which balances false positive indications and false negative indications. Some applications, such as a sweat sensing device used to screen the general population for a heart condition, may require very low false positive indications, and therefore would need to have a higher signal threshold, representing greater certainty of analyte presence. Other applications, such as preliminary screening for lead exposure in an at-risk population, may not require such high certainty, and may use a lower signal threshold. In other cases, an aptamer sensing element may have an aptamer that relatively weakly binds the target analyte, or the particular sweat sample may have challenging pH or salinity characteristics, or the target analyte may be very small. In each of these cases, the signal threshold would need to be relatively higher than in the converse case, all other factors being equal.
In other embodiments, the disclosed invention may also be configured to derive a quantitative measurement of target analytes at low concentrations. In one embodiment, the device may simply track the time required to reach the signal threshold, or time-to-threshold. If the device is placed on skin and subsequently detects the signal threshold within a few minutes, the device can infer that the analyte exists in higher sweat concentrations than if the time-to-threshold were several hours. For example, if a device configured to determine the presence of inflammation by detecting cytokines takes 5 hours to reach signal threshold, the device may recommend that no action be taken. However, if the device reaches signal threshold after only 2 hours, the device may recommend further necessary action. Such time-to-threshold calculations would not be relevant for applications requiring only qualitative measurements, such as a device monitoring for the presence of Ebolavirus or other exogenous molecules that under normal conditions would not be present in sweat. Similarly, sweat rate contributes to concentration estimates based on time-to-threshold, since an analyte that reaches signal threshold at a lower sweat rate may be interpreted as having a higher concentration than an analyte that reaches signal threshold at the same time-to-threshold, but at a higher sweat rate, other factors being equal.
By their potential to recover, or to register trend information, integrative EAB sensors represent a significant improvement over current lateral flow assay technologies. For some applications, an integrative EAB sensor can provide trend information by examining time-to-threshold for a follow-on signal threshold. For example, an integrative sensor has a first signal threshold, and is configured with a second, higher, signal threshold. The time required for the sensor to reach this second threshold could be used to indicate trend information. MCAS embodiments could also return to a signal-off state, which would indicate that the concentration trend was generally downward, and could indicate that remediation techniques were effective, for instance that water intake relieved an indicated state of dehydration.
In some embodiments, a biofluid sensing device for use with the disclosed integrative EAB sensor is configured to estimate a biofluid concentration of a target analyte.
Biofluid sensing devices with integrative EAB sensors, as described above, may be further configured for improved low analyte concentration detection. For example, a sensor may be electromagnetically shielded to reduce the effects of electrical noise, thereby improving sensor sensitivity. Alternately, an EAB sensor may be placed downstream of a sample pre-concentration channel, which would remove water and saline ions or molecules from the sweat sample, to buffer the sample, and increase relative analyte concentration. Similarly, an aptamer sensing element may be surrounded by neutral pH fluid to improve sensitivity for small and low concentration analytes.
Some embodiments benefit from additional techniques to extend the recovery interval for aptamer sensing elements. For example, aptamer sensing elements could be periodically exposed to a light source that polymerizes the sensing elements and captured analytes in their bound state, thus extending the recovery interval. Accordingly, the structures described herein allow a greater time period for creation of analyte capture signals to account for low concentration analytes. Also, the extended capture period enables aptamers to retain small molecules longer than the typical recovery interval, allowing a combined signal to be created from a higher number of captured analytes.
In some sensing device applications, it may desirable to have an EAB sensor in which the aptamer sensing elements can be regenerated after a signal threshold is reached, to allow for multiple capture and release cycles. Regeneration of the aptamer sensing elements may be accomplished, for example, by placing a heating component in proximity to the sensor, which would cause the target analytes to detach from the sensing elements and return to solution. Regeneration of the sensing elements may also be accomplished by introducing a buffering fluid to the EAB sensor such as, for example, by means of a small-volume fluid reservoir containing neutral pH fluid. The buffering fluid would allow the analytes to be released from the aptamers and returned to the solution. Various other solvents (such as alcohols, ethers, aldehydes, halogenate molecules) as known in the art, may also be used in conjunction with, or instead of, water. Other solutions may include molecules (such as a surfactant or a detergent) that cause the analytes to release and return to solution.
While several exemplary embodiments have been described herein for enabling a greater time period for creation of analyte capture signals for measuring low concentration analytes, it is anticipated that other materials, elements and configurations may also be used, provided the alternative materials, elements and/or configurations provide chronological assurance and accurate measurements of the analyte. 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 application claims priority to PCT/US17/45926, filed Aug. 8, 2017, and U.S. Provisional Application No. 62/371,902, filed Aug. 8, 2016, and has specification that builds upon PCT/US17/23399, filed Apr. 21, 2017, the disclosures of which are hereby incorporated herein by reference in their entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US17/45926 | 8/8/2017 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62371902 | Aug 2016 | US |
