Patent Application
20210285936
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. Several challenges, however, have 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 biosensing moves towards detection of small proteins, and large, low concentration analytes (nM to pM and lower).
In particular, many known sensor technologies for detecting small molecules are ill-suited for use in wearable biofluid 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 many biofluid sensing applications. 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 biofluid, while being capable of multiple analyte capture events during the sensor lifespan. However, a chief obstacle to the development of such sensors is the ability to select suitable aptamers capable of capturing, and by extension allowing the sensor to detect, target analytes.
The state of the art technology for aptamer selection relies on techniques, such as systematic evolution of ligands by exponential enrichment (“SELEX”) processes, that iteratively select for aptamers having the desired capture properties for the target analyte. One such SELEX process works by tethering target molecules to a substrate, and then washing the tethered analytes with a library of about 1014 different aptamer sequences. The non-binding aptamers are removed, and the aptamers that successfully bonded to the target analyte are polymerase chain reaction (“PCR”) amplified and reintroduced to the target analyte. After several iterations, candidate aptamers that preferentially bind to the target analyte will emerge.
These candidate aptamers are then functionalized into prior art multi-capture aptamer sensing elements, as depicted in
With reference to
Unfortunately, most SELEX processes only identify candidate aptamers that preferentially bind to the target analyte. They do not select for aptamers that display a conformational change sufficient to produce an analyte capture signal that is distinguishable from the unbound signal. Such selection instead is done though an intensive trial and error process that involves functionalizing the candidate aptamers and testing their performance empirically. Not only is this process time-consuming, but in the end, it may not produce a suitable aptamer. Clearly, aptamer selection and EAB sensor configuration requires improvement if EAB sensing is to become practical for wearable biofluid sensing. Accordingly, there is a need for new EAB sensor configurations and methods of detecting analyte capture with selected aptamers. In particular, there is a need for sensing devices having selected aptamers that not only preferentially bind to a target analyte, but also produce a reliably detectable signal upon analyte capture. Such devices and methods are the subject of the present disclosure.
Many of the other challenges to successful biofluid 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 biofluid to sensors and sample preparing or concentrating subsystems.
Electrochemical aptamer-based biosensing devices and methods are described herein that are configured to produce a detectible signal upon target analyte interaction with reduced reliance on a conformational change by the aptamer. In the disclosed devices, aptamers can be selected for preferential binding with a target analyte, with reduced reliance on the further step of selecting for a detectable conformation change in the presence of the analyte. Embodiments are disclosed herein for an EAB sensor with docked aptamers for measuring the presence of a target analyte in a biofluid sample. In the disclosed embodiments, the sensor includes an electrode capable of sensing redox events, and a plurality of aptamer sensing elements with aptamers selected to interact with a target analyte. Each aptamer sensing element includes a molecular docking structure attached to the electrode, and an analyte capture complex that includes an aptamer releasably bound to the docking structure, and an electroactive redox moiety. Upon the aptamer binding with a target analyte, the analyte capture complex separates from the docking structure. The separation of the analyte capture complex from the docking structure produces a positional change in the redox moiety that is detectable by the sensing device on interrogation of the electrode.
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 multiple aptamer sensing elements that, in the presence of a target analyte in a biofluid 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 of the target analyte.
“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 fractionalization may include tagging the aptamer with a redox moiety, or attaching thiol binding molecules, docking structures, or other components to the aptamer or capture complex. Multiple aptamer sensing elements functionalized on an electrode comprise an EAB sensor.
“Multi-capture Aptamer Sensor” means an EAB sensor capable of a plurality of analyte capture interactions, as disclosed in U.S. Pat. Nos. 7,803,542 and 8,003,374, which are hereby incorporated herein in their entirety.
“Docked aptamer EAB sensor” means an EAB sensor that employs docking strategies to connect analyte capture complexes with the sensor electrode, and wherein such analyte capture complexes are configured for one analyte capture interaction.
“Reference EAB sensor” means a reference sensor that comprises aptamer sensing elements functionalized on an electrode base, where the aptamers have been modified to not interact with target analyte molecules. A reference EAB sensor is configured to perform substantially identically to a comparable active EAB biosensor but will not bind to a target analyte.
“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.
“Time-to-threshold” means the amount of time required for an 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.
The embodiments described herein solve a shortcoming of SELEX processes with respect to biofluid sensor development through the use of docked aptamer EAB sensors. The disclosed sensors reduce the requirement of identifying aptamers that not only preferentially bind to the target analyte, but also display a conformational change sufficient to produce an analyte capture signal that is distinguishable from the unbound signal. Rather, the docked aptamer sensors use a change in position of a redox moiety, caused by detachment of a bound aptamer from a docking structure, to detect analyte capture. As described herein, a docked aptamer EAB sensor includes an aptamer initially bound to a docking structure. While bound to the docking structure, a redox moiety associated with the aptamer produces a first redox signal measurable by the sensing device. Upon interaction with the analyte, the aptamer changes shape to bind with the analyte, causing the aptamer to break away from the docking structure. The separation of the aptamer from the docking structure produces a change in the location of the associated redox moiety. The change in redox moiety location produces a second redox signal that is measurably different from the first redox signal. The difference between the first and second measured redox signals can be correlated and compared to a threshold to detect analyte capture.
Turning now to
The aptamer sensing element 210 also includes an analyte capture complex 212 for binding to a target analyte 260. The analyte capture complex includes an aptamer 240 selected to bind to a target analyte, and may also include one or more linker nucleotide sections, here depicted as a complementary pair of linkers 242, 244. The complementary linkers 242, 244 may be of differing lengths, specifically, the first linker 242 may include more nucleotide bases than the second linker 244. Such an arrangement makes the second linker competitive to the binding between the dock 220 and first linker. In this embodiment, a redox chemical moiety 250 such as, for example, a methylene blue group, a viologen, or a ferrocene group, is attached to the free end of the first linker 242.
In the initial arrangement, shown in
With reference to
Because this embodiment is a signal-off sensor, it can be vulnerable to false positives caused by physical degradation of the individual aptamer sensing elements, or changes in conditions. Over time, aptamer sensing elements within an EAB sensor will physically degrade, meaning docks will release from the electrode and the sensing elements will become unattached to the electrode surface independent of target analyte concentration in the biofluid. Further, docked aptamer sensors have a second source of degradation since the analyte capture complexes will gradually detach from their respective docks independent of target analyte concentration. Similarly, changes in external or internal temperature, humidity, non-specific binding factors, and biofluid sample pH and salinity can affect the rate at which the sensors degrade. Therefore, some embodiments of the disclosed invention further include a reference EAB sensor to provide drift correction and calibration for a companion active EAB sensor. While reference EAB sensors are discussed here in the context of signal-off and docked aptamer EAB sensors, their use is not so limited, and other types of active EAB sensors, including multi-capture EAB sensors, can benefit from appropriately configured companion reference sensors.
An embodiment of the disclosed reference EAB sensor is configured to be substantially identical to its companion active EAB sensor, however, the aptamer is altered so that it will no longer interact with the target molecule. By adhering as closely as possible to the companion active sensor's physical characteristics, the reference sensor will mirror the drift or physical degradation experienced by the active sensor due to time or conditions. Like its companion sensor, the reference sensor will include an electrode, and a plurality of aptamer sensing elements, each including a deactivated aptamer, and a redox moiety. A deactivated aptamer may be produced by, e.g., switching out one of more nucleotide bases in the aptamer to render it incapable of interacting with the target analyte without substantially altering the aptamer's structural configuration. In use, the reference EAB sensor will allow the biofluid sensing device to chart the drift or physical degradation of its companion active sensor. The reference EAB sensor can also perform an initial diagnostic test of a device by providing a measurement of physical degradation within the active sensor that has occurred up until the time of use.
In operation, the EAB sensor is exposed to a biofluid sample containing a concentration of the target analyte 360. On interaction with the target analyte, the aptamer changes shape to bind with the analyte, causing the linker 342 to break free from the dock 320, and the complex moves away from the dock, as shown in
Once the dock 320 is free from the linker 342B, the dock becomes more flexible, and begins to move freely about its point of attachment to the electrode. As the attached redox moiety 350 moves about the dock attachment point, the redox moiety moves sufficiently close to the electrode to promote a detectable electron transduction, eTB. Interrogation of the electrode 330 following analyte capture, therefore, will return a detectable signal due to movement of the redox moiety 350 closer to the electrode. In this embodiment, the EAB sensor has a signal-off condition prior to analyte capture and a signal-on condition after analyte capture, enabling a positive detected signal to provide confirmation of analyte capture.
In operation, the EAB sensor is exposed to a biofluid sample containing a concentration of the target analyte 460. On interaction with the target analyte, the aptamer changes shape to bind with the analyte, causing the second linker 444 to move into physical proximity to the first linker 442. The physical proximity of the complementary linkers causes the first linker to break free from the dock 420 and bind to the second linker 444, and the complex is carried away from the docking structure 420, as shown in
Once the dock 420 is unbound from the first linker 442B, the dock becomes more flexible, and the complementary sections 422B, 424B bind together. The folding of dock 420 caused by the sections binding locks the attached redox moiety 450 in a position close to the electrode 430, thereby promoting a detectable electron transduction, eTB. Interrogation of the electrode 430 following analyte capture, therefore, will return a detectable signal due to the proximity of the redox moiety to the electrode. In this embodiment, the EAB sensor has a signal-off condition prior to analyte capture and a signal-on condition after analyte capture, enabling a positive detected signal to provide confirmation of analyte capture. Relative to the embodiment depicted in
In an initial arrangement, the first electrode 530A is configured with a multitude of aptamer sensing elements, each of which includes a docking structure 520 immobilized on the electrode, an analyte capture complex 512, and a redox moiety 550, here shown bonded to the free end of a first linker 542. The dock 520 may be attached to the electrode 530A by covalently bonding a first end to a thiol, which is then in turn covalently bonded to the electrode, which may be comprised of gold or another suitable conductive material. The aptamer sensing elements may be arranged in any manner described in the previous embodiments, and here they are depicted as similar to those described in
In operation, the sensor 500 is exposed to a biofluid sample containing a concentration of target analytes 560. As the biofluid sample flows through the channel 580 in the direction of the arrow 16, target analyte molecules interact with the aptamers 540, causing the second linker 544 to move into physical proximity to the first linker 542. The first linker then breaks free from the dock 520, and binds to the second linker. The analyte capture complex 512 then moves away from the dock, causing the attached redox moiety 550 to also move away from the first electrode. As the redox moiety 550 moves away from the first electrode, the electron transfer between the redox moiety and the first electrode is blocked due to the distance between the two, creating a decreasing signal condition at the first electrode.
After separation, the freed analyte capture complex 512 and captured analyte 560 are carried as a unit by the biofluid through the fluid channel 580 in the sample flow direction 16. As the analyte capture complexes 512 move away from the first electrode 530A and toward the second electrode 530B, a number of the complexes will approach the second electrode 530B so that the second electrode registers a signal. As individual complexes approach the second electrode, the proximity of the redox moiety 550 to the second electrode will enable redox of the redox moiety in response to potentials applied via the second electrode. The increase from no redox signal to a measurable signal will be detected through the second electrode as an indication of analyte capture. The decreasing signal at the first electrode 530A, combined with increasing signal at the second electrode 530B will provide an indication of the concentration or presence of target analyte in the sample.
With each of the embodiments depicted above, the interactions among the dock and the one or more linker sections may prove critical to the performance of the docked aptamer sensors. Therefore, linker and dock length and composition may be adjusted to improve or allow sensor function. Relative bond strength can be adjusted by varying the nucleotide bases (adenine (A), thymine (T), guanine (G), cytosine (C), uracil (U)) in the linkers and dock, to include adding non-native or unnatural bases. For example, an A-T bond is weaker than a G-C bond. By creating relatively more G-C complementary pairs between a linker and a dock, a stronger bond can be created. Similarly, placing a G-C pair at the end of the linker-dock complex creates a stronger bond. Conversely, the inclusion of more A-T complementary pairs produces a relatively weaker bond between dock and linker. Bond strength can also be adjusted by making the length of a component longer or shorter. For example, two complementary linkers that are 9 bases each would have a stronger bond relative to two complementary 3 base linkers.
Adjusting these parameters will allow adjustment of EAB sensitivity and drift. For example, a strong bond between a dock and its associated analyte capture complex may increase sensor lifespan (reduce drift) by reducing sensor degradation over time, i.e., by slowing analyte capture complex detachment from its dock. However, stronger bonds between docks and analyte capture complexes could also reduce sensitivity, e.g., analyte capture produces an insufficient conformation change to disrupt the bond with the dock, and no signal is produced.
In addition to the description above, sensing devices may be further configured for improved performance in low-concentration detection. For example, one or more filtering membranes can be placed before and after the electrodes, or the sensors may be electromagnetically shielded to reduce the effects of electrical noise, thereby improving sensitivity. Similarly, an EAB sensing element may be surrounded by neutral pH fluid to improve sensitivity.
While several exemplary embodiments have been described with reference to a molecular docking structure, it is anticipated that other types of docking structures may also be used, provided the docking structure is designed to release an aptamer upon binding of the aptamer to a target 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 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/US18/39274, filed Jun. 25, 2018, and U.S. Provisional Application Ser. No. 62/523,835, filed Jun. 23, 2017, and has specification that builds upon PCT/US17/23399, filed Mar. 21, 2017, the disclosures of which are hereby incorporated herein by reference in their entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US18/39274 | 6/25/2018 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62523835 | Jun 2017 | US |
