SOLUTE-PHASE ELECTROCHEMICAL APTAMER SENSORS FOR IMPROVED LONGEVITY AND SENSITIVITY

Abstract

A device and method for detecting the presence of, or measuring the concentration or amount of, at least one analyte in a sample fluid. The device 100 includes at least one electrode 150, a sensor fluid 18, a plurality of aptamers freely diffusing in the sensor fluid, and a plurality of redox tags associated with at least a subset of aptamers of the plurality of aptamers. The sensor fluid is capable of having a sample fluid 14 introduced thereinto, and the detection or measurement of any analyte may occur through a change in electron transfer from at least one redox tag of the plurality of redox tags.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of the filing date of U.S. Patent Application Ser. No. 63/082,834, filed on Sep. 24, 2020; claims the benefit of the filing date of U.S. Patent Application Ser. No. 63/082,999, filed on Sep. 24, 2020; claims the benefit of the filing date of U.S. Patent Application Ser. No. 63/083,029, filed on Sep. 24, 2020; claims the benefit of the filing date of U.S. Patent Application Ser. No. 63/085,484, filed on Sep. 30, 2020; claims the benefit of the filing date of U.S. Patent Application Ser. No. 63/122,071, filed on Dec. 7, 2020; claims the benefit of the filing date of U.S. Patent Application Ser. No. 63/122,076, filed on Dec. 7, 2020; claims the benefit of the filing date of U.S. Patent Application Ser. No. 63/136,262, filed on Jan. 12, 2021; claims the benefit of the filing date of U.S. Patent Application Ser. No. 63/150,667, filed on Feb. 18, 2021; claims the benefit of the filing date of U.S. Patent Application Ser. No. 63/150,677, filed on Feb. 18, 2021; claims the benefit of the filing date of U.S. Patent Application Ser. No. 63/150,712, filed on Feb. 18, 2021; claims the benefit of the filing date of U.S. Patent Application Ser. No. 63/150,856, filed on Feb. 18, 2021; claims the benefit of the filing date of U.S. Patent Application Ser. No. 63/150,865, filed on Feb. 18, 2021; claims the benefit of the filing date of U.S. Patent Application Ser. No. 63/150,894, filed on Feb. 18, 2021; claims the benefit of the filing date of U.S. Patent Application Ser. No. 63/150,944, filed on Feb. 18, 2021; claims the benefit of the filing date of U.S. Patent Application Ser. No. 63/150,953, filed on Feb. 18, 2021; claims the benefit of the filing date of U.S. Patent Application Ser. No. 63/150,986, filed on Feb. 18, 2021; and claims the benefit of the filing date of U.S. Patent Application Ser. No. 63/197,674, filed on Jun. 7, 2021, the disclosures of each of which are incorporated by reference herein in their entireties.

FIELD OF THE INVENTION

This invention relates generally to aptamer sensors.

BACKGROUND OF THE INVENTION

This section is intended to introduce the reader to various aspects of art that may be related to various aspects of the present invention, which are described and/or claimed below. This discussion is believed to be helpful in providing the reader with background information to facilitate a better understanding of various aspects of the present invention. Accordingly, it should be understood that these statements are to be read in this light, and not as admissions of prior art.

Electrochemical aptamer sensors can identify the presence and/or concentration of an analyte of interest via the use of an aptamer sequence that specifically binds to the analyte of interest. These sensors include aptamers attached to an electrode, wherein each of the aptamers has a redox active molecule (redox couple) attached thereto. The redox couple can transfer electrical charge to or from the electrode. When an analyte binds to the aptamer, the aptamer changes shape, bringing the redox couple closer to or further from, on average, the electrode. This results in a measurable change in electrical current that can be translated to a measure of concentration of the analyte.

A major unresolved challenge for aptamer sensors (particularly those where the aptamers are bonded to the working electrode) is the lifetime of the sensors, especially for applications where continuous operation is required (“continuous” referring to multiple measurements over time by the same device). Such aptamer sensors are susceptible to degradation due to, among other things, solutes in a fluid sample that are potentially harmful to the sensor (such as nucleases that can degrade the aptamers, or fouling proteins such as albumin). Harmful solutes such as these can reduce the operational life of the sensor, and thus any device including an aptamer sensor. Thus, to date, it has been difficult to provide electrochemical aptamer sensors with a lifetime that allows continuous sensing to take place over an extended period of time. Furthermore, for aptamer sensors where the aptamer is bonded to the electrode, the flexibility of design is limited and often the sensitivity of the aptamer therefore suffers as a consequence. Devices and methods that resolve these challenges for aptamer sensors are needed.

SUMMARY OF THE INVENTION

Certain exemplary aspects of the invention are set forth below. It should be understood that these aspects are presented merely to provide the reader with a brief summary of certain forms the invention might take and that these aspects are not intended to limit the scope of the invention. Indeed, the invention may encompass a variety of aspects that may not be explicitly set forth below.

Many of the drawbacks and limitations stated above 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 sensing technology into proximity with biofluid and analytes.

One aspect of the present invention is directed to device for detecting the presence of, or measuring the concentration or amount of, at least one analyte in a sample fluid. The device includes at least one electrode, a sensor fluid, a plurality of aptamers freely diffusing in the sensor fluid, and a plurality of redox tags associated with at least a subset of aptamers of the plurality of aptamers. The sensor fluid is capable of having a sample fluid introduced thereinto, and the detection or measurement of any analyte may occur through a change in electron transfer from at least one redox tag of the plurality of redox tags.

Another aspect of the present invention is directed to a method for detecting the presence of, or measuring the concentration or amount of, at least one analyte in a sample fluid. The method includes (1) bringing a sample fluid into contact with at least one aptamer of a plurality of aptamers that are freely diffusing in a sensor fluid, wherein a plurality of redox tags are associated with at least a subset of aptamers of the plurality of aptamers; and (2) detecting or measuring a change in electron transfer from at least one redox tag of the plurality of redox tags.

BRIEF DESCRIPTION OF THE DRAWINGS

The objects and advantages of the disclosed invention will be further appreciated in light of the following detailed descriptions and drawings in which:

FIG. 1A is a cross-sectional view of a device in accordance with principles of the disclosed invention.

FIG. 1B is a cross-sectional view of an alternate embodiment of a device in accordance with principles of the disclosed invention.

FIG. 2A is a schematic showing a prior art portion of an aptamer sensor device having a passivating layer and an aptamer attached to an electrode.

FIG. 2B is a schematic showing the aptamer and passivating layer portions of the aptamer sensor device of FIG. 2A degrading over time.

FIG. 3 is a schematic of yet another embodiment of a device in accordance with principles of the present invention.

FIG. 4 is a graph showing the effect of a membrane in an aptamer sensor device on percentage of solute retention versus molecular weight of the solute.

FIG. 5A is a schematic of yet another embodiment of a device in accordance with principles of the present invention.

FIG. 5B is a schematic showing an alternate embodiment of an aptamer with attached redox tag that can be used with devices in accordance with principles of the disclosed invention.

FIG. 5C is a schematic showing yet another alternate embodiment of an aptamer with attached redox tag that can be used with devices in accordance with principles of the disclosed invention.

FIG. 5D is a schematic showing yet another alternate embodiment of an aptamer with attached redox tag that can be used with devices in accordance with principles of the disclosed invention.

FIG. 5E is a schematic showing yet another alternate embodiment of an aptamer with attached redox tag that can be used with devices in accordance with principles of the disclosed invention.

FIG. 6A is a schematic of yet another embodiment of a device in accordance with principles of the present invention.

FIG. 6B is a schematic showing an alternate embodiment of an aptamer with attached redox tag that can be used with devices in accordance with principles of the disclosed invention.

FIG. 6C is a schematic showing yet another alternate embodiment of an aptamer with attached redox tag that can be used with devices in accordance with principles of the disclosed invention.

FIG. 7 is a schematic of yet another embodiment of a device in accordance with principles of the present invention.

FIG. 8 is a schematic of yet another embodiment of a device in accordance with principles of the present invention.

FIG. 9 is a schematic of yet another embodiment of a device in accordance with principles of the present invention.

FIG. 10A is a graph showing raw chronoamperometric scans of current versus time for cortisol in an exemplary device.

FIG. 10B is a graph showing normalized current gain for three sensors versus concentration of cortisol.

DEFINITIONS

As used herein, “continuous sensing” with a “continuous sensor” means a sensor that changes in response to changing concentration of at least one solute in a solution such as an analyte. Similarly, as used herein, “continuous monitoring” means the capability of a device to provide multiple measurements of an analyte over time.

As used herein, the term “about,” when referring to a value or to an amount of mass, weight, time, volume, pH, size, concentration or percentage is meant to encompass variations of ±20% in some embodiments, ±10% in some embodiments, ±5% in some embodiments, ±1% in some embodiments, ±0.5% in some embodiments, and ±0.1% in some embodiments from the specified amount, as such variations are appropriate to perform the disclosed method.

As used herein, the term “electrode” means any material that is electrically conductive such as gold, platinum, nickel, silicon, conductive liquid infused materials such as ionic liquids, PEDOT:PSS, conductive oxides, carbon, boron-doped diamond, nanotubes or nanowire meshes, or other suitable electrically conducting materials.

As used herein, the term “blocking layer” or “passivating layer” means a homogeneous or heterogeneous layer of molecules on an electrode which alter the electrochemical behavior of the electrode. Examples include a monolayer of mercaptohexanol on a gold electrode or as another example natural small-molecule solutes in serum that form a layer on a carbon electrode.

As used herein, the term “aptamer” means a molecule that undergoes a conformation or binding change as an analyte binds to the molecule, and which satisfies the general operating principles of the sensing method as described herein. Such molecules are, e.g., natural or modified DNA, RNA, or XNA oligonucleotide sequences, spiegelmers, peptide aptamers, and affimers. Modifications may include substituting unnatural nucleic acid bases for natural bases within the aptamer sequence, replacing natural sequences with unnatural sequences, or other suitable modifications that improve sensor function, but which behave analogous to traditional aptamers. Two or more aptamers bound together can also be referred to as an aptamer (i.e., not separated in solution). Aptamers can have molecular weights of at least 1 kDa, 10 kDa, or 100 kDa.

As used herein, the term “redox tag” or “redox molecule” means any species such as small or large molecules with a redox active portion that when brought adjacent to an electrode can reversibly transfer at least one electron with the electrode. Redox tag or molecule examples include methylene blue, ferrocene, quinones, or other suitable species that satisfy the definition of a redox tag or molecule. In some cases, a redox tag or molecule is referred to as a redox mediator. Redox tags or molecules may also exchange electrons or change in behavior when brought into proximity with other redox tags or molecules.

As used herein, the term “change in electron transfer” means a redox tag whose electron transfer with an electrode has changed in a measurable manner. This change in electron transfer can, for example, originate from availability for electron transfer, distance from an electrode, diffusion rate to or from an electrode, a shift or increase or decrease in electrochemical activity of the redox tag, or any other embodiment as taught herein that results in a measurable change in electron transfer between the redox tag and the electrode.

As used herein, the term “optical tag” or “fluorescent tag” means any species that fluoresces in response to an optical source such as LED and whose fluorescence is detectable by a photodetector such as a photodiode. Example fluorescent tags include fluorescein and may be used in combination with other fluorescent tags or optical quenchers such a black-hole quencher dyes to change the fluorescence of the optical tag.

As used herein, the term “signaling aptamer” means an aptamer that is tagged with a redox active molecule or tag and/or contains a redox active portion itself and which provides a change in electrochemical signal when it is released from an anchor aptamer.

As used herein, the term “anchor aptamer” means an aptamer that that can bind to a signaling aptamer, and when bound to the signaling aptamer changes at least one property of the bound vs. unbound signaling aptamer such as molecular weight, diffusion coefficient, charge state, being floating in solution vs. being immobilized, or some other property which causes a change in electron transfer with an electrode. The binding of the anchor aptamer with the signaling aptamer can be dependent on concentration of the analyte to be measured.

As used herein, the term “folded aptamer” means an aptamer that along its length associates with itself in one or more locations creating a two or three-dimensional structure for the aptamer that is distinct from an “unfolded aptamer” that is a freely floating and oscillating strand of aptamer. Aptamers can also be partially folded or partially unfolded in structure or in time spent in the folded vs. unfolded states. Multiple folding configurations are also possible.

As used herein, the term “analyte” means any solute in a solution or fluid which can be measured using a sensor. Analytes can be small molecules, proteins, peptides, electrolytes, acids, bases, antibodies, molecules with small molecules bound to them, DNA, RNA, drugs, chemicals, pollutants, or other solutes in a solution or fluid.

As used herein, the term “membrane” means a polymer film, plug of hydrogel, liquid-infused film, tiny pore, or other suitable material which is permiselective to transport of a solute through the membrane by solute parameters such as size, charge state, hydrophobicity, physical structure, or other solute parameters than can enable permiselectivity. For example, a dialysis membrane is permselective by passing small solutes but not large solutes such as proteins. Membranes as understood herein need not be multiporous, for example a nanotube or nanopore can act as a permiselective filter and is therefore considered part of a membrane as understood for the present invention. Permiselectivity can scale with the analyte, for example a membrane with a molecular weight cut-off of 50 kDa could be used to measure a 20-30 kDa protein but could still keep out cellular or other large content (globulins, fibrogen, etc.) and retain in aptamer that adequately large or physically structured such that permeability through the membrane is slow or nil.

As used herein, the term “sample fluid” means any solution or fluid that contains at least one analyte to be measured.

As used herein, the term “sensor fluid” means a solution or fluid that differs from a sample solution by at least one property, and through which the sensor solution and the sample solution are therefore separated but are in fluidic connection through at least one pathway such as a membrane. The sensor solution comprises at least one aptamer as a solute.

As used herein, the term “reservoir fluid” means a solution or fluid that differs from a sample solution by at least one property, and through which the sensor solution and the reservoir solution are in fluidic connection through at least one pathway such as a membrane or a pin-hole connection. A reservoir fluid may have multiple functions in a device, for example, by introducing a solute continuously or as needed by diffusion equilibrium into the sensor fluid, or for example removing unwanted solutes from a sensor fluid and acting as a “waste removal element”.

As used herein, a “device” comprises at least one sensor based on at least one aptamer, at least one sensor solution, and at least one sample solution. Devices can sense multiple samples and be in multiple configurations such as a device to measure a pin-prick of blood, or a microneedle or in-dwelling sensor needle to measure interstitial fluid, or a device to measure saliva, tears, sweat, or urine sensor, or a device to measure water pollutants or food processing solutes, or other devices which measure at least one analyte found in a sample solution.

DETAILED DESCRIPTION OF THE INVENTION

One or more specific embodiments of the present invention will be described below. In an effort to provide a concise description of these embodiments, all features of an actual implementation may not be described in the specification. It should be appreciated that in the development of any such actual implementation, as in any engineering or design project, numerous implementation-specific decisions must be made to achieve the developers' specific goals, such as compliance with system-related and business-related constraints, which may vary from one implementation to another. Moreover, it should be appreciated that such a development effort might be complex and time consuming, but would nevertheless be a routine undertaking of design, fabrication, and manufacture for those of ordinary skill having the benefit of this disclosure.

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 such as a LED or laser excitation source and a photodetector, chemical, mechanical, or other known biosensing mechanisms. Sensors can be in duplicate, triplicate, or more, to provide improved data and readings. Sensors may provide continuous or discrete data and/or readings. 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 known (e.g., a reference or counter electrode, a battery, antenna, adhesive), and for purposes of brevity and focus on inventive aspects, such components may not be explicitly shown in the diagrams or described in the embodiments of the disclosed invention. All ranges of parameters disclosed herein include the endpoints of the ranges.

As described above, one aspect of the present invention is directed to device for detecting the presence of, or measuring the concentration or amount of, at least one analyte in a sample fluid. The device includes at least one electrode, a sensor fluid, a plurality of aptamers freely diffusing in the sensor fluid, and a plurality of redox tags associated with at least a subset of aptamers of the plurality of aptamers. The sensor fluid is capable of having a sample fluid introduced thereinto, and the detection or measurement of any analyte may occur through a change in electron transfer from at least one redox tag of the plurality of redox tags.

Another aspect of the present invention is directed to a method for detecting the presence of, or measuring the concentration or amount of, at least one analyte in a sample fluid. The method includes (1) bringing a sample fluid into contact with at least one aptamer of a plurality of aptamers that are freely diffusing in a sensor fluid, wherein a plurality of redox tags are associated with at least a subset of aptamers of the plurality of aptamers; and (2) detecting or measuring a change in electron transfer from at least one redox tag of the plurality of redox tags.

With reference to FIGS. 1A and 1B, exemplary embodiments of devices in accordance with principles of the disclosed invention are shown. Referring first to FIG. 1A, a device 100 is shown as being placed partially in-vivo into the skin 12 of a subject. Skin 12 includes the epidermis 12a, the dermis 12b, and the subcutaneous or hypodermis 12c. The device 100 includes a feature 112 that allows for access to sample fluids from the subject. Such sample fluids may include interstitial fluid (from the dermis 12b) and/or blood (from a capillary 12d). In the embodiment shown in FIG. 1A, the feature 112 includes a plurality of microneedles (which may be formed of metal, polymer, semiconductor, glass, or other suitable material). Each of the microneedles 112 projects from a first substrate 108. And each microneedle 112 may include a hollow lumen 132. The device 100 also includes a second substrate 110 (which may be a material such as polymer or glass) having an electrode 150 adjacent thereto. An optional passivating layer 120 may be adjacent to electrode 150, such that electrode 150 is positioned between passivating layer 120 and second substrate 110. Passivating layer 120 includes a compound such as mercaptohexanol or may comprise natural solutes that have diffused into the device 100 from the dermis 12b.

As can be seen in FIG. 1A, a defined volume 130 is present between first substrate 108 and passivating layer 120. It will be recognized by those of ordinary skill in the art that defined volume 130 does not necessarily have to be defined by first substrate 108 and passivating layer 120—and in embodiments where passivating layer is absent, volume 130 may be defined by first substrate 108 and electrode 150; or, alternatively, may be defined by first substrate 108 and second substrate 110. A sensor fluid 18 may be present within volume 130 (as shown in FIG. 1A). Further, as can be seen in the embodiment of FIG. 1A, at least one membrane 136 is present between first substrate 108 and passivating layer 120, and is positioned adjacent first substrate 108. The at least one membrane 136 may be of various materials or substances—such as a dialysis membrane or hydrogel, for example. In the particular embodiment shown in FIG. 1A, portions of the membrane 136 overlie the boundary between volume 130 and lumens 132 of each microneedle 112. Due to this positioning of membrane 136, volume 130 includes sensor fluid 18, and lumens 132 include sample fluid 14—such as interstitial fluid from dermis 12b or blood from capillary 12d. Together the total volume provided by volume 130 and lumens 132 can be a microfluidic component such as channels, a hydrogel, or other suitable material. A diffusion or other fluidic pathway exists from the sample fluid 14, such as interstitial fluid or blood, into volumes 132, 130.

Another embodiment of a device 100 is shown in FIG. 1B. This embodiment also includes first and second substrates 108, 110, microneedles 112 having lumens 132, electrode 150, passivating layer 120, defined volume 130 and at least one membrane 136. As can be seen from FIG. 1B, in this embodiment, electrode 150 and passivating layer 120 are recessed in second substrate 110 (as opposed to the configuration shown in FIG. 1A). Thus, volume 130 is defined by first substrate 108 and combination of second substrate 110 and passivating layer 120. Further, the embodiment as shown in FIG. 1B includes a plurality of membranes 136, with each membrane 136 positioned in a distal end of each lumen 132 of each microneedle 112. Due to this positioning of membranes 136, both volume 130 and lumens 132 include a sensor fluid 18 (and sample fluid is present in, and obtainable from, dermis 12b and capillary 12d, for example).

Alternative arrangements and materials to those discussed above with respect to FIGS. 1A and 1B are possible, such as using a single needle or hydrogel polymer microneedles. In addition, one or more of the features of device 100 or the entire device 100 could be implanted into the body and perform similarly as described herein. Furthermore, a device 100 could be fully outside the body, if for example sampling a fluid such as sweat or tears.

Turning now to FIGS. 2A and 2B, where like numerals refer to like features, a portion of a prior art device 200 is shown. Referring to FIG. 2A, an aptamer sensor includes a passivating or blocking layer 248 (including a compound such as mercaptohexanol) attached to an electrode 250 (made from a material such as gold), and having at least one aptamer 270 that is attached to the electrode 250, such as by being thiol-bonded to the electrode 250. The aptamer 270 has at least one redox tag or molecule 240, such as methylene blue, associated therewith. The device 200 is shown as being positioned in a sample fluid 14, such as blood or interstitial fluid (for example). This prior art device 200 may have an analyte (not shown) that binds with the aptamer 270, thereby changing the availability of the redox tag 240 to the electrode 250, such as by bringing it closer to, or further from, the electrode 250. Conventional aptamer sensors can be limited in performance because an aptamer that is bound to an electrode often has a weaker binding affinity to an analyte than an aptamer that is free in solution. In addition, as shown in FIG. 2B, the sensors can degrade as the aptamer 270 and/or blocking layer 248 degrades over time (e.g., chemical degradation, or detaching from the electrode 250). Also, because such prior art devices 200 have relied on exogenous molecules (e.g., mercaptohexanol) for passivation, the passivation layer 248 can also become thicker with fouling from solutes (such as albumin) in the sample fluid 14.

Thus, and with reference now to FIG. 3, where like numerals refer to like features, an embodiment of the disclosed invention that improves on the prior art devices and reduces or eliminates drawbacks with such devices is shown. To that end, FIG. 3 shows a device 300 (or at least a portion thereof) that includes an electrode 350 and at least one membrane 336 which separates a sample fluid 14 from a sensor fluid 18. The sensor fluid 18 contains a plurality of aptamers 370 having redox tags 340. The electrode 350 may include a passivating layer 348. The passivating layer 348 may comprise one or more endogeneous solutes 16 from the sample fluid 14 itself (or, as initially prepared, the passivating layer 348 may be prepared from molecules that are known to be endogenous to the sample fluid to be tested). Examples of such endogenous molecules 16 include small molecules such as amino acids, hormones, metabolites, or peptides. (Thus, the device 300 shown in FIG. 3 differs from that described above in prior art FIGS. 2A and 2B in that the prior art device described above included an aptamer and an exogenous molecule, such as mercaptohexanol. Similarly, electrode 350 could also contain a passivation layer 348 comprised, at least in part, by an exogenous molecule such as hexanethiol or mercaptohexanol. But, even in that case, the passivation layer could detatch from the electrode 350 and be in need of replacement.) By including endogeneous molecules 16 in the passivation layer 348, longer lifetime of the device 300 is achieved because endogenous molecules 16 can leave the electrode 350 as shown by arrow 392 and another endogenous molecule 16 can replace that now-missing molecule as shown by arrow 390. Thus, in a sense, the very molecules in the sample fluid 14 can be used to “repair” the passivation layer as it degrades, thereby extending the life of the device. (As mentioned above, these endogenous molecules can originate from the sample fluid itself, be already present as a deliberate component of the sensor fluid, or could be a mix of the two.) As a non-limiting example, membrane 336 is able to pass in small solutes (e.g., <1 kDa)—for example, an analyte such as cortisol—and passivating solutes 16, such as amino-acids and peptides, but retains the aptamer 370 (with redox tag 340) which is often >10 kDa in molecular weight. If the aptamer 370 with redox tag 340 were not retained by the membrane 336, then aptamer 370 with redox tag 340 could be lost into the body and no longer able be able to provide a measurement of the analyte.

An example of the analysis of the use of a membrane to pass small solutes (small target analyte) while retaining aptamers within device is shown with reference to FIG. 4, which shows an illustrative plot of solute retention for a membrane such as membrane 336. This is an example only, and shows that if measuring a small analyte such as cortisol (<400 Da) and using a large aptamer (>10 kDa or even >50 kDa) a membrane could be highly permeable to the analyte and poorly permeable to the aptamer. Thus, for example, in various embodiments, membranes of the present invention may have molecular weight cutoffs (i.e., the molecular weight above which a molecule will not easily pass through the membrane) that are at least one of <300 Da, <1000 Da, <3 kDa, <10 kDa, <30 kDa, <100 kDa, <300 kDa. Larger molecular weight cut-off membranes will require larger sized aptamers to prevent the aptamers from potentially escaping the device.

Several additional embodiments will be discussed below. In these additional embodiments, an increase in availability of the redox tag to the electrode can occur as a result of aptamer binding analyte, or, alternatively, without aptamer binding to an analyte. And even though each of the embodiments discussed below (and their respective figures) may show one specific example, the other the embodiments of the invention are not so limited (e.g., the various aptamer/redox tag types can be used across the various embodiments of devices disclosed herein, and vice versa.

Turning now to FIGS. 5A-5E, where like numerals refer to like features: FIG. 5A shows a portion of a device 500 including a substrate 510, a sensor fluid 18, a plurality of aptamers 570 with redox tags 540 free in the sensor fluid 18, a passivation layer 548 of endogenous solutes 16, and an electrode adjacent the substrate 510. Though not part of the device, analytes 19 are also depicted as present in the sensor fluid of the device. The schematic shown in FIG. 5A also depicts an electron transfer event that occurs between a redox tag 540 and the electrode 550. This is shown generally at reference numeral 598, and is a non-limiting example depicting that electron transfer 598 from a redox tag 540 occurs in an increased amount, or frequency, or rate, when aptamer 570 binding to analyte 19 occurs (e.g., as shown in the figure, when analyte is not bound to aptamer, the redox tag is not available—or is less available—to the electrode, due to, for example, a conformation of the aptamer that hinders or prevents such transfer when not bound to analyte; conversely, when aptamer binds analyte, the conformation of aptamer may change in a manner that positions the redox tag for electron transfer). In various embodiments, aptamer binding to analyte can provide changes in electron transfer and redox current (compared to baseline transfer and current—i.e., transfer/current in the absence of analyte binding) of greater than 5%, greater than 10%, greater than 20%, greater than 50%, greater than 100%, or greater than 200%. For the embodiments illustrated herein, non-limiting examples of electrical measurement techniques may include voltammetry, square wave voltammetry, amperometry, chronoamperometry, coulometry, chronocoulometry, with a preferred embodiment being square wave voltammetry.

FIG. 5B schematically depicts another example of an aptamer 570 with attached redox tag 540 (that differs from the aptamer 570/redox tag 540 schematically shown in FIG. 5A). The embodiment of the aptamer 570 in FIG. 5B is designed such that the redox tag 540 is more available for electron transfer with the electrode 550 in the absence of any analyte 19 binding to the aptamer 570 (high electron transfer—or high ET). Conversely, when analyte 19 binds to the aptamer 570 of FIG. 5B, the redox tag 540 is less available for electron transfer with the electrode 550 (e.g., the redox tag 540 is less exposed—low ET).

FIG. 5C schematically depicts yet another example of an aptamer with attached redox tag 540 (that differs from the aptamer 570/redox tag 540 schematically shown in FIGS. 5A and 5B). The embodiment shown in FIG. 5C is designed with two aptamer portions: a signaling aptamer 572 and an anchor aptamer 574. A redox tag 540 is associated with (such as by being attached to) the signaling aptamer 572. The anchor aptamer 574 includes a portion that has affinity for, and thus can bind, analyte 19. When analyte 19 is not bound to the anchor aptamer 574 (left side of FIG. 5C), the signaling aptamer 572 remains associated with the anchor aptamer 574, and so the redox tag 540 on signaling aptamer 572 is less available for electron transfer with the electrode 550 (low ET). However, once the anchor aptamer 574 binds to analyte 19 (right side of FIG. 5C), signaling aptamer 572 is released from anchor aptamer 574, and the redox tag 540 becomes more available for electron transfer with the electrode 550 (high ET). It will be recognized that the device of the embodiment of FIG. 5C has a plurality of aptamers—and thus includes a plurality of signaling aptamers 572, and a plurality of anchor aptamers 574. As described above, each anchor aptamer of the plurality of anchor aptamers is adapted to bind to analyte. In one embodiment, each signaling aptamer of a majority of the plurality of signaling aptamers is bound to a respective anchor aptamer when a majority of anchor aptamers are not bound to any analyte. (This may occur, for example, prior to the introduction of any analyte.) Once analyte is introduced (such as when a sample fluid is introduced into the device—e.g., by being introduced into the sensor fluid of the device), at least a subset of anchor aptamers from the plurality of anchor aptamers then binds to analyte. When this occurs, a subset of signaling aptamers (from the total plurality of aptamers) dissociates from the anchor aptamers—and the redox tag becomes more available for electron transfer with the electrode.

Further, while the embodiment shown in FIG. 5C depicts analyte 19 binding to anchor aptamer 574 and redox tag 540 on signaling aptamer 572, in an alternate embodiment analyte binding may occur with signaling aptamer (signaling aptamer having redox tag), and binding of analyte to signaling aptamer may serve to release signaling aptamer from anchor aptamer (such as by change in conformation of signaling aptamer). Even further configurations as possible, as understood by those skilled in the art of aptamers. To maximize the signal gain (change in signal) signaling aptamer 572 concentration will typically be less than or equal to the anchor aptamer 574 concentration else the signaling aptamer can cause increased background signal with or without the presence of analyte.

FIG. 5D schematically depicts yet another example of an aptamer with attached redox tag 540 (that differs from the aptamers/redox tags schematically shown in FIGS. 5A, 5B, and 5C). The embodiment of the aptamer 570 in FIG. 5D has both a redox tag 544 and a redox quencher 542 associated therewith (such as by being bound to the aptamer 570). When analyte 19 is not bound to the aptamer 570, the redox tag 544 and redox quencher 542 are spatially separated (left side of FIG. 5D) thereby allowing for greater electron transfer between redox tag 544 and electrode 550 (high ET). However, once the aptamer 570 binds to analyte 19 (right side of FIG. 5D), the redox tag 544 and redox quencher 542 are brought into closer proximity with one another, thereby causing less electron transfer between redox tag 544 and electrode 550 (low ET). Numerous quenchers are possible, including anthraquinone-based redox molecules that can be self-quenching when two of such identical molecules are brought close together (monomer vs. dimer).

FIG. 5E schematically depicts yet another example of an aptamer with attached redox tag 540 (that differs from the aptamers/redox tags schematically shown in FIGS. 5A, 5B, 5C and 5D). The embodiment of the aptamer 570 in FIG. 5E has both a first redox tag 546 and a second redox tag 548 associated therewith (such as by being bound to the aptamer 570). When analyte 19 is not bound to the aptamer 570, the first and second redox tags 546, 548 are spatially separated (left side of FIG. 5E) thereby allowing for greater electron transfer between first and second redox tags 546, 548 and electrode 550 (high ET). However, once the aptamer 570 binds to analyte 19 (right side of FIG. 5E), the first and second redox tags 546, 548 are brought closer together and the electron transfer from one of the redox tags 546, 548 to the electrode 550 is altered due to a two-step mediated electron transfer process, or other effect, for two redox tags brought into close proximity. These changes in electron transfer are depicted in the voltammograms as shown as 546a and 548a. A non-limiting example of redox tags that enable the embodiment of FIG. 5E include methylene blue and ferricyanide.

Turning now to FIGS. 6A-6C, where like numerals refer to like features: FIG. 6A shows a portion of a device 600 that includes a substrate 610, at least first and second electrodes 650, 652, a passivation layer 648 including endogenous solutes 16, a sensor fluid 18 (which, in the embodiment illustrated in FIG. 6A is inside an optional hydrogel 638), a plurality of aptamers 670 having redox tags 640 (free in solution), and a diffusion or iontophoretic pathway 694. Though not part of the device, analytes 19 are also depicted as present in the sensor fluid of the device. FIG. 6A also schematically depicts electron transfer that can occur between redox tags 640 and the first and second electrodes 650, 652. As can be seen in FIG. 6A, as a non-limiting example, electron transfer 698 from the redox tags 640 in an increased amount, or frequency, or rate, when analyte 19 is bound to the aptamer 670. For example, when analyte is bound to aptamer, the hydrodynamic radius or size of the aptamer is smaller and therefore providing a faster diffusion coefficient, which results in the redox tag being more available for electron transfer with the electrodes (such a version will be discussed in greater detail below with respect to FIG. 6B); or, for example, when analyte is not bound to aptamer, the redox tag is not available—or is less available—to the electrode, due to, for example, a conformation of the aptamer that hinders or prevents such transfer when not bound to analyte; conversely, when aptamer binds analyte, the conformation of aptamer may change in a manner that positions the redox tag for electron transfer. In various embodiments, aptamer binding to analyte can provide changes in electron transfer and redox current (compared to baseline transfer and current—i.e., transfer/current in the absence of analyte binding) of greater than 5%, greater than 10%, greater than 20%, greater than 50%, greater than 100%, or greater than 200%. For the embodiments illustrated in FIGS. 6A-6C, non-limiting examples of electrical measurement techniques may include voltammetry, square wave voltammetry, amperometry, chronoamperometry, coulometry, chronocoulometry, with a preferred embodiment being amperometry.

FIG. 6B schematically depicts another example of an aptamer 670 with attached redox tag 640 (that differs from the aptamer 670/redox tag 640 schematically shown in FIG. 6A). The embodiment of the aptamer in FIG. 6B is designed such that the redox tag 640 is less available for electron transfer with the electrodes 650, 652 in the absence of analyte binding to aptamer (left side of FIG. 6B), because of a longer diffusion time between the first and second electrodes 650, 652 where the analyte can undergo redox recycling (e.g. one electrode is a reducing electrode, one electrode is an oxidizing electrode). However, when analyte 19 binds to the aptamer 670 (right side of FIG. 6B), the hydrodynamic radius or size of the aptamer is smaller and therefore providing a faster diffusion coefficient, and therefore redox tag 640 is more available for electron transfer with the first and second electrodes 650, 652. The binding of analyte 19 transforms the aptamer 670 between a long unfolded aptamer 670 (in the absence of analyte 19 binding) and an aptamer 670 with three stems when analyte 19 binds to aptamer 670.

As described above, with respect to FIG. 6A, a non-limiting example of an environment within a device 600 may include an optional hydrogel. In such an embodiment, the hydrogel 638 (such as agar or polyacrylamide) is added to further distinguish diffusion times between aptamers 670 bound to analyte 19 and aptamers 670 not bound to analyte. This is because the hydrogel 638 creates a more tortuous and size-selective diffusion pathway than a pure fluid would by itself. For example, an aptamer 670 that fully dissociates could be modified to have a significant change in hydrodynamic radius (R), which changes its diffusion coefficient (D) according to D=kT/(6πηR). This equation is for diffusion in pure solution; a dense hydrogel 638 can be added to further distinguish the diffusion of the unfolded aptamer vs. the folded aptamer. The resulting current between the redox recycling electrodes is proportional as I∞DC/z, where C is the concentration of the aptamer 670 and z the electrode-to-electrode distance. With respect to changes in signal gain, the diffusion length of oglionucleotides (aptamers) varies with length to the ˜0.6^th power, and a 15 kDa protein that is globular/unfolded can have a change in R of 2.15/3.65.

FIG. 6C schematically depicts yet another example of an aptamer 670 with attached redox tag 640 (that differs from the aptamer 670/redox tag 640 schematically shown in FIGS. 6A and 6B). The embodiment of the aptamer in FIG. 6C is designed with two aptamer portions: a signaling aptamer 672 and an anchor aptamer 674. A redox tag 640 is associated with (such as by being attached to) the signaling aptamer 672. The anchor aptamer 674 includes a portion that has affinity for, and thus can bind, analyte 19. When analyte 19 is not bound to the anchor aptamer 674 (left side of FIG. 6C), the signaling aptamer 672 remains associated with the anchor aptamer 674, and so the redox tag 640 on signaling aptamer 672 is less available for electron transfer with the first and second electrode 650, 652 (as the combined signaling and anchor aptamers 672, 674 will exhibit slower diffusion in sensor solution and hydrogel). However, once the anchor aptamer 674 binds to analyte 19 (right side of FIG. 6C), signaling aptamer 672 is released from anchor aptamer 674, and the redox tag 640 becomes more available for electron transfer with the first and second electrodes 650, 652 (as the liberated signaling aptamer 672 will exhibit more rapid diffusion in sensor solution and hydrogel). Further, while the embodiment shown in FIG. 6C depicts analyte 19 binding to anchor aptamer 674 and redox tag 640 on signaling aptamer 672, in an alternate embodiment analyte binding may occur with signaling aptamer (signaling aptamer having redox tag), and binding of analyte to signaling aptamer may serve to release signaling aptamer from anchor aptamer (such as by change in conformation of signaling aptamer).

It will be recognized that when the device shown in FIG. 6A uses the embodiment of aptamers of FIG. 6C, it will include a plurality of signaling aptamers 672, and a plurality of anchor aptamers 674. As described above, each anchor aptamer of the plurality of anchor aptamers is adapted to bind to analyte. In one embodiment, each signaling aptamer of a majority of the plurality of signaling aptamers is bound to a respective anchor aptamer when a majority of anchor aptamers are not bound to any analyte. (This may occur, for example, prior to the introduction of any analyte.) Once analyte is introduced (such as when a sample fluid is introduced into the device—e.g., by being introduced into the sensor fluid of the device), at least a subset of anchor aptamers from the plurality of anchor aptamers then binds to analyte. When this occurs, a subset of signaling aptamers (from the total plurality of aptamers) dissociates from the anchor aptamers—and the redox tag becomes more available for electron transfer with the electrode.

With further reference to FIG. 6C, in addition to changes in diffusion coefficient, the larger the effective sphere for the aptamer the less likely it will experience electron transfer with an electrode (with a first principles estimation based on the inverse of sphere area, proportional to 1/R{circumflex over ( )}2). This example is simply to show that two factors can be at play for embodiments of the present invention, both distance of the redox tag to the electrode and diffusion time to/from the electrode. This diffusion time to an electrode applies other embodiments as well, where for example with a chronoamperometric response for an aptamer the total current baseline could remain higher or reach baseline more quickly as diffusion coefficient for the aptamers increases. This diffusion time to an electrode may also impact interrogation methods such as square wave voltammetry, as aptamer that is near the electrode can contribute to the signal as well if it is able to diffuse to the electrode during each square window (during each voltage pulse that is applied). The first and second electrodes 650 and 652 can be closely spaced via interdigitation or other suitable technique, and, in such an embodiment, may be within less than 50 μm, less than 10 μm, less than 2 μm, or less than 0.4 μm distant of each other.

With reference to FIG. 7 where like numerals refer to like features, another embodiment in accordance with aspects of the present invention is shown. As can be seen in FIG. 7, a portion of a device 700 is shown, and includes a substrate 710, at least one electrode 750, a passivation layer 748 including endogenous solutes 16, a sensor fluid 18, a plurality of aptamers having redox tags 740 (free in the sensor solution), and a poorly-mobile or non-mobile material 738 in the sensor fluid 18. Though not part of the device, analytes 19 are also depicted as present in the sensor fluid of the device.

The aptamers/redox tags component of the embodiment of FIG. 7 is similar to that shown in FIGS. 5C and 6C, and includes two aptamer portions: a signaling aptamer 772 and an anchor aptamer 774. A redox tag 740 is associated with (such as by being attached to) the signaling aptamer 772. The anchor aptamer 774 includes a portion that has affinity for, and thus can bind, analyte 19. As can be seen in FIG. 7, the anchor aptamer 774 is immobilized via linkage 739 to the poorly or non-mobile material 738. The poorly-mobile or non-mobile material 738 may comprise various materials, such as a hydrogel. In one non-limiting example, the material 738 could be a hydrogel such as polyacrylamide and the linker be a molecule such as acrydite that is attached to the anchor aptamer at a terminal end or other location. In an alternate embodiment, the anchor aptamer could be cross-linked with other anchor aptamers or the anchor aptamer made so large (e.g., >100 kDa) such that it is effectively immobile in a dense hydrogel 738.

Still referring to FIG. 7, when analyte 19 is not bound to the anchor aptamer 774, the signaling aptamer 772 remains associated with the anchor aptamer 774, and so the redox tag 740 on signaling aptamer 772 is less available for electron transfer with the electrode 750 (because the combined signaling and anchor aptamers 772, 774 will be poorly-mobile or non-mobile in the sensor fluid due to anchor aptamer 774 being linked to material 738). However, once the anchor aptamer 774 binds to analyte 19, the signaling aptamer 772 is released from anchor aptamer 774 (as indicated by arrow 796), and the redox tag 740 becomes more available for electron transfer with the electrode 750 (because the liberated signaling aptamer 772 will exhibit more rapid diffusion in sensor solution as it is no longer complexed with the anchor aptamer 774 that is linked to poorly-mobile or non-mobile material 738). Further, while the embodiment shown in FIG. 7 depicts analyte 19 binding to anchor aptamer 774 and redox tag 740 on signaling aptamer 772, in an alternate embodiment analyte binding may occur with signaling aptamer (signaling aptamer having redox tag), and binding of analyte to signaling aptamer may serve to release signaling aptamer from anchor aptamer (such as by change in conformation of signaling aptamer).

It will be recognized that the device of the embodiment of FIG. 7 has a plurality of aptamers—and thus includes a plurality of signaling aptamers 772, and a plurality of anchor aptamers 774. As described above, each anchor aptamer of the plurality of anchor aptamers is adapted to bind to analyte. In one embodiment, each signaling aptamer of a majority of the plurality of signaling aptamers is bound to a respective anchor aptamer when a majority of anchor aptamers are not bound to any analyte. (This may occur, for example, prior to the introduction of any analyte.) Once analyte is introduced (such as when a sample fluid is introduced into the device—e.g., by being introduced into the sensor fluid of the device), at least a subset of anchor aptamers from the plurality of anchor aptamers then binds to analyte. When this occurs, a subset of signaling aptamers (from the total plurality of aptamers) dissociates from the anchor aptamers—and the redox tag becomes more available for electron transfer with the electrode.

With reference to FIG. 8, where like numerals refer to like features, another embodiment in accordance with aspects of the present invention is shown. As can be seen in FIG. 8, a portion of a device 800 is shown, and includes a substrate 810, at least one electrode 850, a membrane 838, a sensor fluid 18, a plurality of aptamers having redox tags 740 (free in the sensor fluid). Though not part of the device, analytes 19 are also depicted as present in the sensor fluid of the device. The aptamers/redox tags component of the embodiment of FIG. 8 is similar to that shown in FIGS. 5C, 6C, and 7, and includes two aptamer portions: a signaling aptamer 882 and an anchor aptamer 884. A redox tag 840 is associated with (such as by being attached to) the signaling aptamer 882. The anchor aptamer 884 includes a portion that has affinity for, and thus can bind, analyte 19. The membrane 838 exhibits selective permeability based on size, charge, or at least one solute property, and is able to pass a signaling aptamer 882 but not a signaling aptamer that is attached to a larger anchor aptamer 884. Thus, the membrane 838 impacts the availability of the redox couple 840 to the electrode 850. For example, a signaling aptamer could have a radius of 3 nm/2 nm in folded/unfolded states and an anchor aptamer have 27/7 nm in folded/unfolded state, creating a difference in size of ˜3-10× when a signaling aptamer is freed from an anchor aptamer. Nanofiltration membranes can provide is nM pore sizes, and ultrafiltration 10 s to 100 s nm pore sizes (PES, track-etch, and other materials), resulting in size selective permeability that would enable mainly only the signaling aptamer 882 to penetrate the hydrogel or membrane 838.

And so, still referring to FIG. 8, when analyte 19 is not bound to the anchor aptamer 884, the signaling aptamer 882 remains associated with the anchor aptamer 884, and so the redox tag 840 on signaling aptamer 882 is less available (or not available) for electron transfer with the electrode 850 (because the signaling aptamer 882 will be unable to cross membrane 838 due to being complexed with anchor aptamer 884). However, once the anchor aptamer 884 binds to analyte 19, the signaling aptamer 882 is released from anchor aptamer 884 and is able to pass through membrane 838, resulting in the redox tag 840 becoming available for electron transfer with the electrode 850. Further, while the embodiment shown in FIG. 8 depicts analyte 19 binding to anchor aptamer 884 and redox tag 840 on signaling aptamer 882, in an alternate embodiment analyte binding may occur with signaling aptamer (signaling aptamer having redox tag), and binding of analyte to signaling aptamer may serve to release signaling aptamer from anchor aptamer (such as by change in conformation of signaling aptamer).

With reference to FIG. 9, where like numerals refer to like features, another embodiment in accordance with principles of the present invention is shown. In certain of the various embodiments discussed herein, a membrane is used to selectively allow passage of certain molecules and not of others. However, as no membrane is perfectly size selective, and as aptamers and redox tags can degrade over time, it may be advantageous to continually introduce a fresh supply of aptamers, signaling aptamers, and/or anchor aptamers or other solutes that increase performance of the sensor or improve longevity of the sensor (e.g. nuclease inhibitors, for example). Thus, as shown in FIG. 9, a portion of a device 900 includes substrates 910, at least one electrode 950, a membrane 936, a sample fluid 14, a sensor fluid 18, and a reservoir fluid 17. The membrane 936 exhibits mass flow represented at reference numeral 991, and the device also includes a diffusion restrictive feature 935 (such as a pinhole or membrane) with a mass flow represented at reference numeral 993.

As a nonlimiting example of that shown in FIG. 9, consider a 0.2 kDa dialysis membrane for membrane 936 and assume the aptamers are 10-100× larger than the solute to be detected (e.g. phenylalanine, cortisol, etc.). Assume the system is designed such that the volume of reservoir fluid 17 is at least one of 2×, 10×, 50×, or 250× greater than volume of sensor fluid 18 and that the mass flow 991 of aptamer is at least 2×, 10×, 50×, or 250× less than mass flow of aptamer 993, while the mass flow 991 of the analyte is at least 2×, 10×, 50×, or 250× greater than the mass flow of the analyte 993. As a result, the concentrations of analyte will be within at least 50%, 10%, 2%, or 0.4% of each other when comparing sample fluid 14 with sensor fluid 18, and the concentrations of aptamer will be within at least 50%, 10%, 2%, or 0.4% of each other when comparing sensor fluid 18 and reservoir fluid 17.

As a geometrical example, consider a membrane 936 with 0.2 cm² area and 10% porosity to the analyte, and a diffusion restrictive feature 935 that is a pinhole in materials 910 and 950 0.001 cm² in area and 0.001 cm in length. The mass transport for a small analyte through the membrane will be equivalent to 0.02 cm² area and the mass transport through the feature 935 0.001 cm², which is 20× different, satisfying the above stated criteria for design as shown in FIG. 9. As a result, both analyte and aptamer concentrations can be maintained for prolonged periods of times (days, weeks, months) even if aptamer is lost from the device or degraded over time. Aptamers could also degrade over time and their presence in the device and the presence of other contaminants such as nucleases or proteins could be problematic. For example, if signaling aptamers became cleaved and their molecular weight decreased, they could give a false higher reading of signal in embodiments of the present invention. With membrane protection of the sensor fluid from the sample fluid, most degradation or contamination modes will be very slow, such that the reservoir may also act as a waste removal element.

The various embodiments disclosed herein can be enabled to be user-calibrated, factory-calibrated, or calibration-free. User-calibration could for example require a pin-prick blood draw and running of a conventional assay to measure analyte concentration, and that concentration data entered into the software that runs the sensing device.

Factory-calibrated implies that the device requires calibration, but that the calibration is shelf-stable and stable for at least a portion of the use period of the device. Embodiments, such as those shown in FIG. 5B and FIG. 5C, could benefit from factory calibration if they are interrogated by square wave voltammetry, and if the passivation layer 548 thickness is kept fairly constant (e.g. using a mercaptohexanol passivation layer 548 or polyethylene glycol terminated passivation layer 548 that is resistant to fouling). In factory calibration, the device is tested with a sample fluid with a known concentration of analyte, and that information is then shipped along with the product in order to enable it to start its use with proper calibration.

Calibration-free operation is possible if one could eliminate the factors that could cause a sensor signal to drift or change. Considering the embodiments of FIGS. 6 and 9, the aptamer concentrations can be kept constant, and with a chronoamperometric measurement response the change in current vs. time will be dependent on diffusion coefficient of the signaling aptamer 672 vs. the anchor aptamer 674 and signaling aptamer 672 bound together. The diffusion coefficient will not change in a sample fluid such as interstitial fluid, and an overvoltage can be supplied to measure the chronoamperometric response even if thickness of the passivating layer 648 changes slightly. Simply, the chronamperometric response will measure the percentage of signaling aptamer 672 that is free from the anchor aptamer 674, which is directly related to the binding affinity of the analyte to the aptamers, hence enabling calibration-free operation because the concentrations of the signaling aptamers 672 and anchor aptamers 674 are known. Calibration-free operation is also possible using the constructs of FIG. 5D or FIG. 5E by measuring changes in electron transfer rates, peak position shifts, or ratios (not individual magnitudes) of two or more redox peaks from different redox tags 546, 548 (FIG. 5E).

Although not described in detail herein, the embodiments of the present invention may also be applied to a simple single-use device. For example, device 100 of FIG. 1 could be applied to the skin 12, take a single measurement of an analyte within 15 minutes of application of the device 100, and then remove the device 100 from the skin 12 surface. Alternately (not shown) a device format similar to a glucose test-strip or other device with a microfluidic channel or a wicking channel (like a lateral flow assay) can be used to transport a sample fluid to one or more sensors and embodiments as taught herein for the present invention. For example, the aptamers and redox tags could be dry and placed near the inlet of such a test strip and dissolved into the sample fluid and then transported to a working electrode where measurement of the analyte is performed.

Although not illustrated in detail herein, the embodiments of the present invention may also be applied to a device with more than one type of aptamer within a single device. For example, and referring to FIGS. 5A-5E, if an analyte had a very large dynamic range such as cortisol which can ranges from 1 s to 100 s of nM in free concentration in blood or even higher in those with Cushing's syndrome, the range of cortisol levels could be beyond the dynamic range of the device 500 if only a single type of aptamer was used for 570, 572, or 574. Instead, for example, half of the aptamers 570, 572, or 574 could have 100× weaker binding affinity for cortisol be used for a dynamic range of 0.5 to 50 nM and the other half for 50 nM to 5000 nM. Together in a single device they could provide a dynamic range for sensing cortisol that is >100× and as large as 10,000×.

A single device 500 could sense two or more analytes as well even using one electrode. If square wave voltammetry is utilized to interrogate the aptamers it is well known that there is an optimum square wave scanning frequency (typically between 10 and 1000 Hz) for each aptamer, and often a square wave scanning frequency that results in zero signal gain as analytes bind with aptamers. This same frequency dependence could be used, for example, to have a first set of aptamers 570, 572, or 574 in a device 500 (FIGS. 5A-5E) that respond to phenylalanine with a strongest response frequency at 30 Hz, and second set of aptamers 570, 572, or 574 in a device 500 that respond to cortisol with a strongest response frequency at 300 Hz, and the electrode 550 simply modulated in frequency to measure phenylalanine or cortisol. As a result, a single device can have two or more aptamer types that enable it to sense two or more analytes. Similarly, subsets of aptamers can have different redox tags with different redox peak potentials, to use applied potential to discriminate between two or more analytes. Other ways to discriminate aptamer types include electron transfer rates (time), and other variables in aptamer design that effect their signal gains.

Further, although not described in detail herein, the embodiments of the present invention may also be applied to a device that has a plurality of electrodes used at different times in order to prolong device longevity. As described above, electrodes can be passivated with passivating materials that are exogenous or endogenous, and exogenous passivation such as polyethyleglycol-terminated passivation can have a very long-lasting resistance to fouling. However, during operation of the electrode 250, 450 the passivation can degrade or the electrode itself could degrade (e.g. become pitted, delaminate, be etched entirely away, etc.). Therefore, in embodiments of the present invention a plurality of electrodes can be used at different times inorder to prolong device longevity. For example, one electrode could be used for 3 days until it degrades, then a second electrode for 3 days, then a 3rd electrode for 3 days, for a total device longevity of >1 week.

Although not described in detail herein, other steps which are readily interpreted from or incorporated along with the disclosed embodiments shall be included as part of the invention. The embodiments that have been described herein provide specific examples to portray inventive elements, but will not necessarily cover all possible embodiments commonly known to those skilled in the art.

EXAMPLES

Example 1

With reference to FIGS. 10A and 10B a cortisol binding aptamer was utilized in a manner similar to that taught in FIG. 6C, where the signaling aptamer 672 was tagged with methylene blue as a redox tag 640 with an aptamer sequence of GTCGTCCCGAGAG [SEQ ID NO.1] and where the anchor aptamer 674 with a sequence of ctctcgggacgacGCCCGCATGTTCCATGGATAGTCTTGACTAgtcgtccc [SEQ ID NO. 2]. Electrodes 650, 652 were gold interdigitated electrodes with a 5 μm spacing in between them. The gold electrodes were passivated with an exogenous molecule of mercaptohexanol. No hydrogel 638 was utilized in this experiment. The sensor solution was buffer solution with 5 μM of the aptamers 650, 652 in solution, and a reference electrode of platinum was used. The device 600 was measured amperometrically vs. a titration curve of cortisol as the analyte 19. The results are shown in FIG. 10A and FIG. 10B (open circles, open diamonds, and solid diamonds), and a control experiment with titration of simply adding more cortisol but without aptamer in solution is also shown in FIG. 10B (solid circles). The signal gain in Example 1 is as much as 70%, and if the anchor aptamer was made even larger or smaller the signal gain could be tuned to be as much as 200% or more as little as 5% based on the change in diffusion rate of the signaling aptamer to the electrode compared to the signaling aptamer when it is bound to the anchor aptamer. Signal gain is also measured above a baseline signal, and changing signaling aptamer concentration can therefore be used to tune the signal gain.

Example 2

The experiment of Example 1 was repeated but instead of using mercaptohexanol passivation of the gold electrodes 650, 652, endogenous small molecule solutes found in blood or interstitial fluid were allowed to passivate the gold electrode 650, 652. It was found that without passivation background current was very high, but that both mercaptohexanol and endogeneous solutes were able to adequately reduce background current and enable sensor operation.

Example 3

Sensors were tested with square wave voltammetry, and redox peaks via voltammetry were observable with 100 nM of aptamer. Higher aptamer concentrations only increase the amount of signal and 1 μM, and 10 μM and 100 μM of aptamer were tested as well. Generally, lower aptamer concentrations were preferred as they reduce device lag times as they require less concentration of analyte to create a change in sensor signal.

Example 4

Assume a signaling aptamer 672 of 5 kDa and an anchor aptamer 674 of 100 kDa, and assume the signaling aptamer when freed from the anchor aptamer has a diffusion coefficient increase of 4×. Assume the signaling aptamer is 98% of the concentration of the anchor aptamer. Assume a simple gold rod electrode 650 that does not necessarily rely on redox recycling as illustrated in FIG. 6A.

According to the Cotrell equation chronoamperometric current is proportional to (D/t){circumflex over ( )}½. Further assume a measurement of the slope or change in slope of a chronamperometric response at 50 ms or greater, such as between 50 ms and 100 ms, which is after charging current durations and well past the electron transfer time scales of any aptamers that are stuck on or near the electrode 650. Said in another way, chronoamperometric measurement is performed and has a data measurement window within the chronoamperometric measurement curve that is after charging currents have dissipated to less than 10% of the current value provided by electron transfer from the aptamers. This particular embodiment brings several potential advantages. First, it produces a 2× (100% signal gain) with binding/unbinding of the analyte. Secondly, this approach is calibration free. The signal gain (or decrease) due to analyte binding is proportional to the percentage of signaling aptamer that is free from the anchor aptamer. The signal gain can even be invariant to changes in passivation layer 16 thickness by simply using a small over voltage (for example −0.5V instead of −0.3V if the redox tag 640 had a peak at −0.3V). Consider an intereferent such as NADH/NAD+ (663 Da) and which is −20 nM each in serum and therefore 50 nM total concentration. The 5 kDa signaling aptamer would have D that is 3× greater than NADH, and with the Cotrell equation the current difference between NADH and the signaling aptamer would be 3{circumflex over ( )}(½) or 1.73×. So, to have 10× more current from signaling aptamer than NADH/NAD+ it would require 50 nM*10*1.73=865 nM or >100 nM of aptamer, and preferably >1 μM of aptamer. Embodiments can be enabled to be user calibrated, factory calibrated, or calibration free. User calibration could for example require a pin-prick blood draw and running of a conventional assay to measure analyte concentration, and that concentration data entered into the software that runs the sensing device. Factory calibrated implies that the device requires calibration, but that the calibration is shelf-stable and stable for at least a portion of the use period of the device. Embodiments such as FIG. 6 could benefit from factory calibration if calibration free operation is not possible, and benefit if the passivation layer 648 thickness is kept fairly constant (e.g., using a mercaptohexanol passivation layer 648 or polyethylene glycol terminated passivation layer 648 that is resistant to fouling).

Although not described in detail herein, other steps which are readily interpreted from or incorporated along with the disclosed embodiments shall be included as part of the invention. The embodiments that have been described herein provide specific examples to portray inventive elements, but will not necessarily cover all possible embodiments commonly known to those skilled in the art.

Claims

1. A device for detecting the presence of, or measuring the concentration or amount of, at least one analyte in a sample fluid, the device comprising:

at least one electrode;

a sensor fluid;

a plurality of aptamers freely diffusing in the sensor fluid; and

a plurality of redox tags associated with at least a subset of aptamers of the plurality of aptamers;

wherein the sensor fluid is capable of fluidic connection with a sample fluid introduced thereinto; and

wherein the detection or measurement of any analyte may occur through a change in electron transfer from at least one redox tag of the plurality of redox tags.

2. The device of claim 1, wherein the device is a continuous sensing device.

3. The device of claim 1, wherein the device is a single-use device.

4. The device of claim 1, further comprising a passivating layer on the at least one electrode.

5. The device of claim 4, wherein the passivating layer includes exogenous molecules.

6. The device of claim 4, wherein the passivating layer includes molecules that are endogenous to the sample fluid.

7. The device of claim 6, wherein a source of the endogenous molecules is the sample fluid.

8. The device of claim 6, wherein a source of the endogenous molecules is the sensor fluid.

9. The device of claim 1, further comprising at least one membrane, wherein the membrane separates the sample fluid and the sensor fluid and the membrane retains at least a portion of the aptamers in the sensor fluid.

10. The device of claim 9, wherein the at least one membrane has a molecular weight cutoff that is chosen from less than 300 Da, less than 1000 Da, less than 3 kDa, less than 10 kDa, less than 30 kDa, less than 100 kDa, and less than 300 kDa.

11. The device of claim 9, wherein the change in electron transfer is chosen from greater than 5%, greater than 10%, greater than 20%, greater than 50%, greater than 100%, and greater than 200%.

12. The device of claim 1, wherein the measurement of change in electron transfer is performed using voltammetry.

13. The device of claim 1, wherein the plurality of aptamers comprise a plurality of signaling aptamers and a plurality of anchor aptamers.

14. The device of claim 13, wherein the plurality of redox tags are bound to the signaling aptamers, but are not bound to the anchor aptamers.

15. The device of claim 14, wherein the plurality of anchor aptamers are adapted to bind the analyte, and wherein each signaling aptamer of a majority of the plurality of signaling aptamers is bound to a respective anchor aptamer when a majority of anchor aptamers are not bound to any analyte.

16. The device of claim 14, wherein the plurality of anchor aptamers are adapted to bind the analyte, and wherein a subset of signaling aptamers dissociates from the anchor aptamers when at least a subset of anchor aptamers bind to any analyte.

17. The device of claim 13, wherein the concentration of signaling aptamer is less than the concentration of anchor aptamer.

18. The device of claim 13, wherein the plurality of anchor aptamers are immobilized to a first material.

19. The device of claim 18, wherein the first material is not freely diffusing in fluid.

20. The device of claim 1, wherein the plurality of redox tags comprises two or more redox tags per each aptamer of the plurality of aptamers, wherein the distance between the two or more redox tags alters depending on the presence of analyte.

21. The device of claim 20, wherein the two or more redox tags are comprised of identical molecules.

22. The device of claim 20, wherein the two or more redox tags are comprised of different molecules.

23. The device of claim 1, wherein the electrode is coated with at least one membrane.

24. The device of claim 1, wherein the concentration of the plurality of aptamers is chosen from less than 500 nM, less than 5 μM, and less than 50 μM.

25. The device of claim 1, where each aptamer of the plurality of aptamers has a molecular weight chosen from at least 1 kDa, at least 10 kDa, and at least 100 kDa.

26. The device of claim 1, wherein the change in electron transfer is associated with a condition chosen from a change in folding pattern of the aptamer, a changing in binding between two or more aptamers, a change in distance between the redox tag and the electrode, a change in rate of diffusion for the redox tag to the electrode, a change in electrochemical behavior of the redox tag, a change in hydrodynamic radius of the aptamer, a change in diffusion coefficient of the aptamer, a change in redox potential of the redox tag, a change in redox current magnitude of the redox tag, and a change in electron transfer rate.

27. The device of claim 1, wherein the device is factory-calibrated.

28. The device of claim 1, wherein the device is calibration-free.

29. The device of claim 1, wherein the device contains two or more types of aptamers for measurement of two or more analytes.

30. The device of claim 29, wherein distinguishing between measurement of two or more analytes is accomplished via use of one or more of frequency, potential, and time.

31. The device of claim 1, wherein the at least one electrode further comprises a plurality of electrodes, wherein each electrode of the plurality of electrodes is are used at a different time than each of the other electrodes of the plurality of electrodes.

32. The device of claim 1, wherein the measurement of change in electron transfer is amperometry.

33. The device of claim 1, wherein the in electron transfer is due to a change in diffusion coefficient for the aptamers.

34. The device of claim 1, wherein the at least one electrode is paired with at least a second electrode and the distance between the electrodes is chosen from less than 50 μm, less than 10 μm, less than 2 μm, and less than 0.4 μm.

35. The device of claim 1, wherein the measurement of change in electron transfer is chronoamperometry or chronocoulometry.

36. The device of claim 35, wherein the chronoamperometric or chronocoulometric measurement is performed and has a data measurement window within the measurement curve that is after charging currents have dissipated to less than 10% of the current value provided by electron transfer from the aptamers.

37. The device of claim 36, wherein the chronoamperometric or chronocoulometric measurement has a slope and a change in slope and the slope or change in slope is the measurement of analyte through a change in electron transfer.

38. A method for detecting the presence of, or measuring the concentration or amount of, at least one analyte in a sample fluid, the method comprising:

bringing a sample fluid into contact with at least one aptamer of a plurality of aptamers that are freely diffusing in a sensor fluid, wherein a plurality of redox tags are associated with at least a subset of aptamers of the plurality of aptamers; and

detecting or measuring a change in electron transfer from at least one redox tag of the plurality of redox tags.

39. The method of claim 38, wherein bringing the sample fluid into contact with at least one aptamer further comprises introducing solutes in the sample fluid into the sensor fluid.

40. The method of claim 38, wherein the change in electron transfer results from a condition chosen from a change in folding pattern of the aptamer, a changing in binding between two or more aptamers, a change in distance between the redox tag and the electrode, a change in rate of diffusion for the redox tag to the electrode, a change in electrochemical behavior of the redox tag, a change in hydrodynamic radius of the aptamer, a change in diffusion coefficient of the aptamer, a change in redox potential of the redox tag, a change in redox current magnitude of the redox tag, and a change in electron transfer rate.

41. The method of claim 38, wherein detecting or measuring a change in electron transfer from at least one redox tag comprises the use of an electrical measurement technique.

42. The method of claim 41, wherein the electrical measurement technique is chosen from voltammetry, square wave voltammetry, amperometry, chronoamperometry, coulometry, and chronocoulometry.

43. The method of claim 42, wherein the electrical measurement technique is square wave voltammetry.

44. The method of claim 38, wherein detecting or measuring a change in electron transfer further comprises taking only one measurement.

45. The method of claim 38, wherein detecting or measuring a change in electron transfer further comprises taking multiple separate measurements over a defined time period.