Electrochemical affinity biosensors with sensor response dependent on proximity of two or more tags

Abstract

A device for detecting or measuring at least one large analyte in a sample fluid. The device includes at least one electrode; a sample fluid; and a plurality of aptamers capable of binding to the analyte, wherein the aptamers are physically bound to the device. A majority of the aptamers comprise a first molecule and a second molecule. The first molecule is a redox tag or redox molecule. The second molecule, when it is brought into proximity of the first molecule, results in decreased redox electron transfer with an electrode at a given voltage.

Description

ELECTROCHEMICAL AFFINITY BIOSENSORS WITH SENSOR RESPONSE DEPENDENT ON PROXIMITY OF TWO OR MORE TAGSCROSS-REFERENCE TO RELATED APPLICATIONS100011 This application claims priority to, and the benefit of, U.S. Provisional Application No. 63 / 539,258, filed September 19, 2023, U.S. Provisional Application No. 63 / 607,024, filed December 6, 2023, U.S. Provisional Application No. 63 / 624,041, filed January 23, 2024, and U.S. Provisional Application No. 63 / 568,203, filed March 21, 2024 - the disclosures of each of which are hereby incorporated by reference herein in their entireties.FIELD OF THE INVENTION

[0002] This invention relates generally to electrochemical sensors.BACKGROUND OF THE INVENTION

[0003] 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.

[0004] Affinity-based electrochemical sensors, such as 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 associated with an electrode (such as by being directly or indirectly attached thereto), wherein each of the aptamers has a redox active molecule (often referred to as a redox tag) attached thereto. The redox tag can transfer electrical charge to or from the electrode. When binding occurs between an analyte and the aptamer, the aptamer changes shape, moving the redox tag closer to or further from, on average, the electrode. This results in a measurable change in electrical current that can be read as a detection of the presence of analyte in a sample, or can be translated to a measure of concentration of the analyte in a sample. Aptamers are an example of an affinitybased molecule, and so aptamer sensors are an example of an affinity-based biosensor.

[0005] A major unresolved challenge for aptamer sensors, and other affinity-based biosensors, is the limit of detection and magnitude of sensor response, especially for measuring largeranalytes such a peptide hormones and proteins. Existing electrochemical sensors for proteins are often limited to <30% change in sensor response (measured as the percentage change in the sensor response in response to increasing analyte concentration compared to sensor response if no analyte were present). This sensor response is far less than the 100-200% sensor responses that can be achieved for small molecule aptamer-based sensors (e.g. see White et al. Langmuir 2008, 24, 18, 10513-10518, or Parolo et al. ACS Sens. 2020, 5, 7, 1877-1881). Novel approaches for electrochemical aptamer sensors which eliminate the drawbacks of limited sensor response are therefore required. Furthermore, once an aptamer is found that binds to an analyte it needs to be developed into an aptamer sensor, and aptamer sensor development can be time-consuming and challenging from a predictability perspective. For example, once one has an aptamer that can bind analyte (and wishes to use same in an aptamer sensor), that aptamer needs to be able to (1) bind the target analyte, and (2) change the distance of the redox tag from the electrode in a predictable manner in order for the sensor to be effectively used and be accurate. It is not always a simple matter to design sensors where this distance changes predictably upon binding. And so, novel approaches which simplify aptamer sensor development are needed.SUMMARY OF THE INVENTION

[0006] 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.

[0007] 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.100071 One aspect of the present invention is directed to a device for detecting or measuring at least one analyte in a sample fluid, wherein the device includes at least one electrode, and a plurality of aptamers capable of binding to the analyte. The aptamers are associated with the at least one electrode (such as by being bound either directly or indirectly to the electrode). Further, at least some aptamers of the plurality of aptamers have a first molecule and a second molecule associated therewith, wherein the first molecule is a redox tag, and wherein the firstmolecule and second molecule move either closer to or further from one another when their associated aptamer binds analyte (resulting in a change in electron transfer between the first molecule and the electrode at a given voltage)

[0008] Another aspect of the present invention is directed to a device for detecting or measuring at least a first analyte and a second analyte in a sample fluid. The device of this aspect of the invention includes at least one electrode, a first plurality of aptamers capable of binding to the first analyte, and a second plurality of aptamers capable of binding to the second analyte. The aptamers of both the first plurality and second plurality of aptamers are bound to the at least one electrode (either directly or indirectly). At least some aptamers of the first plurality of aptamers have a first molecule and a second molecule associated therewith, wherein the first molecule is a redox tag, and wherein the first molecule and second molecule move either closer to or further from one another when their associated aptamer binds analyte, which results in a change in electron transfer between the first molecule and the electrode at a given voltage. Further, at least some aptamers of the second plurality of aptamers have a third molecule associated therewith, wherein the third molecule is a redox tag, and wherein the third molecule moves either closer to or further from the electrode which results in a change in electron transfer between the third molecule and the electrode at a given voltage. And the third molecule is redox active at a potential that is measurably distinct from that of the first molecule and / or second molecule.

[0009] Another aspect of the present invention is directed to a device for detecting or measuring at least one analyte in a sample fluid, wherein the device includes at least one electrode, a first aptamer capable of binding to the analyte, and a second aptamer at least partially complementary to the first aptamer. The first and second aptamers are proximal to one another and bound to the at least one electrode. Due to their at least partially complementary nature, the first and second aptamers may be hybridized to one another (such as when no analyte is present). The first aptamer has a first molecule associated therewith, and the second aptamer has a second molecule associated therewith, and the first molecule may be a redox tag. Alternatively, the second molecule may be a redox tag, or both the first and second molecules may be redox tags. The first molecule and second molecule move either closer to or further from one another when their associated aptamer binds analyte, which results in a change in electron transfer between the first molecule and the electrode at a given voltage - thereby indicating the presence of analyte, or which can be used to measure concentration of analyte in sample.

[0010] Another aspect of the present invention is directed to a device for detecting or measuring at least one analyte in a sample fluid, the device including at least one electrode; a plurality of affinity probes bound to the electrode, and a plurality of flexibly-linked bait elements bound to the electrode. At least some affinity probes of the plurality of affinity probes have a first molecule associated therewith, and at least some flexibly-linked bait elements of the plurality of flexibly-linked bait elements have a second molecule associated therewith. One of the first molecule and second molecule is a redox tag. Further, the flexibly-linked bait elements are bound by the affinity probes in the absence of analyte such that the first molecule and second molecule are proximal to one another. And the first molecule and second molecule move further from one another when the affinity probe binds analyte, which results in a change in electron transfer between the redox tag and the electrode at a given voltage.

[0011] Another aspect of the present invention is directed to a device for detecting or measuring at least one analyte in a sample fluid, wherein the device includes at least one electrode, a first aptamer, and a second aptamer at least partially complementary to the first aptamer. The first and second aptamers are proximal to one another and bound to the at least one electrode. Due to their at least partially complementary nature, the first and second aptamers may be hybridized to one another (such as when no analyte is present). The first aptamer has an affinity -based probe and a first molecule associated therewith, and the second aptamer has a second molecule associated therewith. The affinity-based probe is capable of binding to the analyte, and at least the first molecule or the second molecule is a redox tag. The first molecule and second molecule move either closer to or further from one another when the affinity-based probe binds analyte, which results in a change in electron transfer between the redox tag and the electrode at a given voltage - thereby indicating the presence of analyte, or which can be used to measure concentration of analyte in sample.BRIEF DESCRIPTION OF THE DRAWINGS

[0012] The objects and advantages of the disclosed invention will be further appreciated in light of the following detailed descriptions and drawings in which:

[0013] FIG. 1 is a schematic of a conventional prior art sensor device.

[0014] FIG. 2 is a schematic of one embodiment of a device in accordance with principles of the present invention.

[0015] FIG. 3 is a schematic of another embodiment of a device in accordance with principles of the present invention.

[0016] FIG. 4 is a schematic of another embodiment of a device in accordance with principles of the present invention.

[0017] FIG. 5 is a schematic of another embodiment of a device in accordance with principles of the present invention.

[0018] FIG. 6 is a schematic of another embodiment of a device in accordance with principles of the present invention.

[0019] FIG. 7 is specific embodiment of a device in accordance with principles of the present invention, related to the embodiment shown in the schematic of FIG. 6.

[0020] FIG. 8 is data from a specific example of an embodiment of the present invention.

[0021] FIG. 9 is a specific example and data of an embodiment of the present invention.

[0022] FIG. 10A is a schematic of a device of an embodiment of the present invention.

[0023] FIG. 10B is a schematic of a device of an embodiment of the present invention.DEFINITIONS

[0024] 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.

[0025] 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.

[0026] 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.

[0027] As used herein, the term “blocking layer” means a homogeneous or heterogeneous layer of material or of one or more types of molecules on an electrode which reduce electrochemical background current and / or current due to electrochemical interference, and which may promote proper freedom of movement for the aptamer which is required for creating a measurable response to analyte concentration.

[0028] 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.

[0029] As used herein, the term “large analyte” means an analyte with >3 kDa of molecular weight and in most cases with be >4kDa such as insulin, BNP or even >10 kDa such as C- reactive protein, IL-6 or other suitable analytes.

[0030] 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. Redox tags can be tagged at the end of an aptamer or internally along the aptamer using for example thymine base modification, referred to as ‘distal tagging’ and ‘internal tagging”, respectively.

[0031] As used herein, the term “redox quenching” refers to an effect caused by at least one first molecule that is a redox tag or redox molecule, and at least a second molecule that, when brough into proximity of the first molecule, results in decreased redox electron transfer with an electrode at a given voltage. For example, if carminic acid is utilized, the redox activity of two carminic acid molecules when adjacent to each other is largely or fully neutralized. Alternately, the redox peak voltage could simply be shifted such that measuring at fixed voltage will cause observation of an increase or decrease of redox current as the redox peak is shifted. Alternately, a redox active molecule and a second quenching molecule could be used such that, when brought into proximity, redox quenching occurs, more generally involving energy or charge transfer from the redox active molecule to the second quenching molecule.

[0032] As used herein, the term “change in electron transfer” means a redox molecule 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 molecule, or any other embodiment as taught herein thatresults in a measurable change in electron transfer between the redox molecule and the electrode.

[0033] As used herein, “redox tag current” is the amplitude of the faradaic redox tag peak current minus the background current amplitude outside the redox peak in a given voltammetric scan.

[0034] As used herein, “normalized redox-tag current” is the redox-tag current normalized to the first measurement taken.

[0035] As used herein, “background current” is the voltammetric current that would be measured if the aptamer molecules were not tagged with a redox reporter including, for example, capacitive currents and competing redox processes such as oxygen reduction.

[0036] As used herein, “adjusted current” is the combined redox tag current and background current of a square-wave voltammogram adjusted such that the minimum current is set to 0 A in the presentation of the voltammogram such that voltammograms can be plotted side by side and compared with greater ease.

[0037] As used herein, “sensor response” is the change in redox tag current due to binding of the target analyte to the aptamer, also known as signal gain, which can either increase or decrease based on the aptamer and the voltammetric time scale. Sensor response may also be applied to alternative measures such as amperometry or chronoamperometry, or other approaches which do not measure a voltammogram. As used herein “change in sensor response” is the percentage change in the sensor response in response to increasing analyte concentration compared to sensor response if no analyte were present. For example, if the peak redox tag current was 1 p A with no analyte present and adding analyte caused the peak redox tag current to be 0.5 or 1.5 |iA then the change in sensor response would be -50% sensor off response or +50% sensor on response respectively.

[0038] As used herein, the term “sensing monolayer” means at least a plurality of aptamers on a working electrode, which may also include a plurality of molecules or mixtures of molecules that form a blocking layer or an anti-fouling layer.

[0039] 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.

[0040] As used herein, the term “continuous sensing” simply means the device records a plurality of readings over time. Even a point-of-care testing device which provides a single data point can be considered a continuous sensing device if, for example, it is a 15 minute test,that operates by taking multiple data points over 15 minutes and averaging them to provide a single data measure.

[0041] As used herein, a “device” comprises at least one sensor based on at least one aptamer. 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

[0042] 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.

[0043] 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 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.

[0044] With reference to FIG. 1, a conventional prior art sensor device 100, as placed initially in a sample fluid 130, such as interstitial fluid, is shown. The device 100 includes at least one working electrode 120, which may comprise gold, carbon, or other suitable electrodematerial; at least one blocking or protective layer 122 of a plurality of molecules such as mercaptohexanol, mercaptooctanol, or other suitable chemistry that are thiol bonded to the electrode, or a plurality of natural solutes in blood that can act as a blocking layer, or other suitable molecules depending on application and on the choice of electrode 120 material; and at least one aptamer 124 that is linked to the electrode 120 via a thiol bond or other suitable bond, or to the blocking layer 122. The aptamer 124 is capable of binding to an analyte 180. A redox tag 170 (such as methylene blue) is associated with the aptamer 124, such as by being bound to the aptamer. In the generic example taught for FIG. 1 , the aptamer 124 is a simple stem-loop (hairpin) aptamer where binding of analyte 180 to aptamer 124 causes the stem- loop to form (as seen at the left side of FIG. 1), which brings the redox tag 170 closer to the electrode 120. This causes the redox tag current measured from the redox tag 170 to increase, as measured using square wave voltammetry or other suitable technique. The sensor can also have more than one stem-loop, for example such as the cocaine aptamer which has >100% sensor response to cocaine as taught in White et al. Langmuir 2008, 24, 18, 10513-10518. In the absence of analyte 180 binding to the aptamer 124, the stem- loop is not formed and the redox current would lower than when analyte binding occurs (due to the redox tag being positioned farther away from the electrode (as seen at the right side of FIG. 1). Thus, a measurement of electrical current can be used to interpret presence of, or changes in the concentration of, the analyte 180.

[0045] The majority of electrochemical aptamer sensors use ‘signal ON’ motifs like that illustrated in FIG. 1 which produce large sensor responses of >100% by leveraging a stem-loop or other stable configuration which brings the redox tag 170 close to the electrode 120 in a stable geometry with significantly increased redox tag current. These ‘signal ON’ geometries use a single binding domain of the aptamer 124 to the analyte 180 because the analyte is a small molecule and because the small molecule is at high concentrations such that single binding domains can be sufficient. Making a similar sensor for a larger peptide or protein or other analyte such as insulin, BNP, c-reactive protein, IL-6 or other analytes is more difficult because they are at much lower concentrations (e.g., in the pM to nM range) and because they are so large that a single binding domain will have difficulty in many cases binding to the analyte with a binding affinity that is close to the physiologically expected concentrations in a biofluid such as interstitial fluid. For larger analytes it is also difficult to create aptamers where binding would cause stem-loop formation, because the analyte is often so large and the concentration of the large analyte so low that the aptamer largely binds to and conforms to the shape of the larger analyte. And so, while the ‘signal ON’ motif shown in FIG. 1, is able to bind smallmolecule analytes while predictably changing the distance between redox tag and electrode, such designs are not very useful for large analytes. Furthermore, promoting instead a ‘signal OFF’ approach, where the binding of the large analyte breaks the stem-loop may increase response, but may make the sensor less sensitive because the binding of the analyte to the aptamer has to overcome the energetic stability created by the stem-loop structure. As a result, aptamer sensor development for larger analytes is more complicated.

[0046] Various aspects of the present invention provide aptamer sensors that eliminate these drawbacks of limited sensor response in current aptamer sensors. Various aspects of the present invention provide aptamer sensors that eliminate these drawbacks of having more complicated aptamer development for larger analytes. To that end, one aspect of the present invention is directed to a device for detecting or measuring at least one analyte in a sample fluid, wherein the device includes at least one electrode, and a plurality of aptamers capable of binding to the analyte. The aptamers are associated with the at least one electrode (such as by being bound either directly or indirectly to the electrode). Further, at least some aptamers of the plurality of aptamers have a first molecule and a second molecule associated therewith, wherein the first molecule is a redox tag, and wherein the first molecule and second molecule move either closer to or further from one another when their associated aptamer binds analyte (resulting in a change in electron transfer between the first molecule and the electrode at a given voltage)

[0047] With reference to FIG. 2, where like numerals refer to like features, one embodiment of the present invention includes a device 200 having an electrode 220, which can operate as a sensor to detect and / or measure concentration of an analyte 282 in a sample fluid 230 that is introduced to the device 200. For example, such an analyte 282 may be NT-proBNP in a fluid such as interstitial fluid 230. To detect the analyte, the device 200 also includes at least one aptamer 224 (and generally a plurality of aptamers 224) associated with the electrode 220, such as by being directly or indirectly bound thereto. In the embodiment shown in FIG. 2, the aptamers 224 are directly bound to a surface of the electrode 220. The aptamer 224 may be bound to the electrode 220 via a thiol bond or other suitable bond. The device 200 also includes a blocking layer 222 associated with the surface of the electrode 220. The blocking layer is formed from one or more molecules bound to electrode in those areas where aptamer is not bound. Non-limiting examples of molecules that can be used in the blocking layer 222 of this embodiment (and of the other embodiments described below) include mercaptohexanol, mercaptooctanol, or a plurality of natural solutes in blood that can act as a blocking layer.

[0048] The aptamer 224 has at least a first molecule 272 (which may be a first redox tag) and at least a second molecule 274 (which may be a second redox tag) associated therewith (such as by being bound to the aptamer). In FIG. 2 the aptamer 224 is distally tagged (e.g., at the 3’ or 5’ end of the aptamer) with first redox tag 272, and internally tagged along the aptamer with second redox tag 274. The first and second redox tags 272, 274 are positioned on the aptamer 224 such that they are brought into proximity with one another in the stem portion of a stem-loop structure when analyte 282 is not present. As a result, the first and second redox tags 272, 274 provide “redox quenching” in the absence of analyte 282, and little or no redox quenching when analyte 282 binds to aptamer 224. For example, if carminic acid is utilized as the first and second redox tags 272, 274, the redox activity of the two carminic acid molecules when adjacent to each other is largely or fully neutralized (as seen at the left side of FIG. 2, where the aptamer is in its stem-loop conformation). But when moved away from one another (as seen once aptamer binding occurs at the right side of FIG. 2 and the stem-loop conformation is broken), the first and second redox tags 272, 274 are highly active. Alternately, the redox peak voltage could simply be shifted such that measuring at fixed voltage will cause observation of an increase or decrease of redox current as the redox peak is shifted. The present invention includes other alternate embodiments, so long as a change in proximity between the two redox tags 272, 274 results in a measurable change of the redox electron transfer from one of the redox tags. As a result, the device 200 is able to provide at least one of >50%, >100%, >200%, >500% change in sensor response, measured as a change (increase or decrease) in electrochemical current over the full titration of the analyte 282 (from zero concentration to a concentration where all the aptamers 224 are bound with analyte 282).

[0049] With reference to FIG. 3, where like numerals refer to like features, in an embodiment of the present invention a device 300 achieves similar operating principles of FIG. 2 but the aptamer 324 may associate the first and second redox tags 372, 374 in an alternate manner to achieve redox quenching. Thus, the device 300 of the embodiment of FIG. 3 includes an electrode 320, which can operate as a sensor to detect and / or measure concentration of an analyte 382 in a sample fluid 330 that is introduced to the device 300. To detect the analyte, the device 300 also includes at least one aptamer 324 (and generally a plurality of aptamers 324) associated with the electrode 320, such as by being directly or indirectly bound thereto. In the embodiment shown in FIG. 3, the aptamers 324 are directly bound to a surface of the electrode 320. The aptamer 324 may be bound to the electrode 320 via a thiol bond or other suitable bond. The device 300 also includes a blocking layer 322 associated with the surface of the electrode 320. The blocking layer is formed from one or more molecules boundto electrode in those areas where aptamer is not bound. The aptamer 324 has at least a first molecule 372 (which may be a first redox tag) and at least a second molecule 374 (which may be a second redox tag) associated therewith (such as by being bound to the aptamer). In FIG. 3 the aptamer 324 is distally tagged (e.g., at the 3’ or 5’ end of the aptamer) with first redox tag 372, and internally tagged along the aptamer with second redox tag 374. In FIG. 3 the aptamer 324 is internally tagged for both the first redox tag 372 and the second redox tag 374. The first and second redox tags 372, 374 are positioned on the aptamer 324 such that they are brought into proximity with one another in the stem portion of a stem-loop structure when analyte 382 is not present. As a result, the first and second redox tags 372, 374 provide “redox quenching” in the absence of analyte 382 (as seen at the left side of FIG. 3), and little or no redox quenching when analyte 382 binds to aptamer 324, and the distance between first redox tag 372 and second redox tag 374 is increased (as seen at the right side of FIG. 3).

[0050] Like FIG. 2, FIG. 3 shows an embodiment where a stem-loop structure is formed by a portion of aptamer 324 in the absence of analyte 382. However, further examples are possible, and do not require stem-loop formation. Other examples may not require analyte binding to move first and second redox tags away from one another. For example, redox tags could initially be positioned along aptamer in locations that are distant from one another to a degree that quenching is reduced or does not occur in the absence of analyte, only to be brought close together (for quenching to occur or increase) upon binding of analyte to aptamer. For example, when aptamers bind with large analytes like proteins there are often multiple internal or external tagging sites for a first redox tag and a second redox tag that can be changed in their proximity to each other as the aptamer and analyte bind to each other and therefore provide an alternate embodiment of the present invention.

[0051] With reference to FIG. 4, where like numerals refer to like features, in an embodiment of the present invention a device 400 can provide multiplex measurement of two or more analytes 480, 482 on the same electrode. For example, the device 400 could include (1) a conventional aptamer 424 such as FIG. 1 with a methylene blue tag 470 (e.g., a small molecule detecting aptamer such as for potassium, cortisol, or phenylalanine), and (2) an aptamer 426 with redox quencher tagging (provided by redox tags 472, 474). In this embodiment, the redox tag 470 has a different redox potential than redox tag 474 or 472. [While the device 400 is shown in FIG. 4, and initially described here as including (1) a conventional aptamer or aptamers directed to a small molecule analyte and (2) an aptamer or aptamers with redox quenching tagging directed to a larger analyte, alternate embodiments may include first redox quenching tagging aptamers for a first analyte, and a separate population ofredox quenching tagging aptamers for a second analyte. Further, a device of this aspect of the invention is not limited to two populations of aptamers directed to two analytes. Rather, the device may include populations of aptamers to detect, or measure concentration of, any number of different analytes via different aptamers and different redox tags, provided that overlapping redox potentials of the various redox tags being used are avoided.]

[0052] Thus, the device 400 of the embodiment of FIG. 4 includes an electrode 420, which can operate as a sensor to detect and / or measure concentration of two or more analytes 480, 482 in a sample fluid 430 that is introduced to the device 400. To detect a first analyte 480, the device 400 also includes a first aptamer 424 (and generally a plurality of first aptamers 424) that is associated with to the electrode 420 via a thiol bond or other suitable bond. The first aptamer 424 is capable of binding to a first analyte 480 and a redox tag 470 (such as methylene blue) is associated with the aptamer 424, such as by being bound to the aptamer. In the particular example illustrated in FIG. 4, the first aptamer 424 is a simple stem-loop (hairpin) aptamer where the binding of first analyte 480 thereto causes the stem-loop to form, which brings the redox tag 470 closer to the electrode 420 (thereby causing the redox tag current measured from the redox tag 470 to increase.

[0053] The second aptamer 426 (and generally a plurality of second aptamers 426) is associated with the electrode 420, such as by being directly or indirectly bound thereto. In the embodiment shown in FIG. 4, the second aptamers 426 are directly bound to a surface of the electrode 420 (such as via a thiol bond or other suitable bond). The second aptamer 426 has at least a first molecule 472 (which may be a first redox tag) and at least a second molecule 474 (which may be a second redox tag) associated therewith (such as by being bound to the aptamer). In the embodiment shown in FIG. 4, the first and second redox tags 472, 474 may operate similarly to those shown in FIG.3: by providing “redox quenching’’ in the absence of second analyte 482, and little or no redox quenching when second analyte 482 binds to second aptamer 424, and the distance between first redox tag 472 and second redox tag 474 is increased.

[0054] The device 400 also includes a blocking layer 422 associated with the surface of the electrode 420. The blocking layer is formed from one or more molecules bound to electrode in those areas where aptamer is not bound.

[0055] With reference to FIG. 5, another embodiment of a device 500 exhibiting aspects of the present invention is shown. The device 500 includes an electrode 520, which can operate as a sensor to detect and / or measure concentration of an analyte 582 in a sample fluid 530 that is introduced to the device 500. To detect the analyte, the device 500 also includes at least oneaptamer 524 (and generally a plurality of aptamers 524) associated with the electrode 520, such as by being directly or indirectly bound thereto. In the embodiment shown in FIG. 5, the aptamers 524 are indirectly bound to a surface of the electrode 520. In particular, a substrate linker 528 is used to hold the aptamer 524 to the substrate such that the aptamer first and second molecules 572, 574 (such as first and second redox tags) may be distally tagged to the aptamer 524. The device 500 also includes a blocking layer 522 associated with the surface of the electrode 520. The blocking layer is formed from one or more molecules bound to electrode in those areas where aptamer is not bound. The substrate linker 528 and blocking layer molecules 522 may be different or may be similar in nature (e.g. both have an 8 carbon chain like mercatooctanol). In the embodiment shown in FIG. 5, the first and second redox tags 572, 574 provide “redox quenching’" when analyte 582 binds to aptamer 524 (as seen at the left side of FIG. 5), and little or no redox quenching when analyte 582 is absent, and the distance between first redox tag 572 and second redox tag 574 is increased (as seen at the right side of FIG. 5).

[0056] With reference to FIG. 6, yet another embodiment of a device 600 exhibiting aspects of the present invention is shown. This device 600 includes at least one electrode 620, a first aptamer 625 capable of binding to the analyte, and a second aptamer 624 at least partially complementary to the first aptamer 625. The first and second aptamers are proximal to one another and bound to the at least one electrode. The binding of the aptamers to the first electrode may use individual linkers 628 for each aptamer or for example may use a common linker as taught in FIG. 5 for linker 528. Due to their at least partially complementary nature, the first and second aptamers 625, 624 may be at least partially hybridized to one another (such as when no analyte is present - see the left side of FIG. 6). The first aptamer 625 has a first molecule 672 associated therewith, and the second aptamer 624 has a second molecule 674 associated therewith, and the first molecule may be a redox tag. Alternatively, the second molecule may be a redox tag, or both the first and second molecules may be redox tags. The first molecule 672 and second molecule 674 move either closer to or further from one another when their associated aptamer binds analyte 682 (see right side of FIG. 6), which results in a change in electron transfer between the first molecule and the electrode at a given voltage.

[0057] In the illustrated embodiment of FIG. 6, the first and second redox tags 672, 674 are distally tagged to the first and second aptamers 625, 624, respectively. In the absence of analyte 682 redox current is quenched, whereas in the presence of analyte 682 binding, the first and second redox tags 672, 674 are moved away from one another and the redox current increases. This specific embodiment will be described in greater detail in the Examples section(below). While the particular embodiment of FIG. 6 shows the first and second redox tags 672, 674 as being distally tagged to the first and second aptamers 625, 624, the embodiment of FIG. 6 could also have the first and second redox tags 672, 674 tagged to the first and second aptamers 625, 624 internally, such as near the substrate linkers 628, which may provide less increase in redox current as analyte binds (less separation between redox tags) but may have greater accuracy because the variability for the distance between the redox tags and the electrode 620 may be less due to the closer distance from the electrode 620. An additional advantage of FIG. 6 is that any analyte binding aptamer can be made into a redox-quenching switch like that shown in FIG. 6 by leveraging the aptamer’ s complimentary DNA strand (and thus, secondary structure switching optimization such as stem-loop formation is not required). In the approach shown in FIG. 6, one can also see a design that allows for greater predictability in change of redox tag distance to electrode. In the embodiment of FIG. 6, binding of analyte 682 to first aptamer 625 causes the hybridized first and second aptamers 625, 624 to separate. Tus, this allows aptamer to (1) bind the target analyte, and (2) change the distance of the redox tag from the electrode in a predictable manner.|OO58| With further reference to FIG. 6, in yet another embodiment of a device 600 the first redox tag 672 and second redox tag 674 may be internally tagged on the first aptamer 625 or second aptamer 624 (not shown). Distal tagging as illustrated in FIG. 6 places the redox tags 672, 674 at a greater distance from the electrode 620 which can reduce signal strength or for very long aptamers reduce their accuracy (increase standard deviation of measurement). Therefore, in some cases internal tagging may be preferred over distal tagging. The aptamers 624 and 625 also not need be the same length (same number of nucleotides). For example, if first aptamer 625 is the aptamer that binds to analyte 682 and it is very long (e.g. 60 nucleotides) then the second aptamer 624 could be 8, 12, 18, 24 or some other number of nucleotides long and, for example, the second aptamer 624 could be redox tagged distally with redox tag 674 and the first aptamer 625 could be tagged internally with redox tag 672 at a location such that when first aptamer 625 hybridizes with second aptamer 624 redox tags 672 and 674 are brought adjacent to one another to cause redox quenching (not shown).

[0059] With reference to FIGS. 10A and 10B, where like numerals refer to like features, this embodiment of the present invention includes an alternate switching mechanism. In this embodiment, a device 1000 includes an electrode 1020, which can operate as a sensor to detect and / or measure concentration of an analyte in a sample fluid that is introduced to the device 1000. To detect the analyte, the device 1000 also includes at least one affinity-based probe 1029, such as an antibody, designed to capture a target analyte 1082 (target analyte shown onlyin FIG. 10B). In the embodiment shown in FIGS 10A and 10B, the affinity -based probes 1029 are indirectly bound to a surface of the electrode 1020. In particular, a substrate linker 1028 is used to hold the affinity-based probe 1029 to the substrate A first molecule 1072 (which may be a first redox tag) is associated with the affinity-based probe 1029, such as by being bound thereto. Apart from the affinity -based probe 1029, the device also includes an element 1025, which has a bait target 1085 and a second redox tag 1074 associated therewith, (herein referred to as flexibly linked bait element). Element 1025 may be a single or double stranded aptamer, or other flexible structure such as a polyethylene glycol. As can be seen in FIG. 10A, in the absence of target analyte, the bait target 1085 is bound to affinity-based probe 1029, and this causes first and second redox tags 1072, 1074 to be positioned proximal to one another. Thus, the first and second redox tags 1072, 1074 experience redox quenching, or at least one change in redox behavior, when brought into close proximity (as taught similar to previous embodiments). Referring then to FIG. 10B, as analyte concentration increases, the bait 1085 is displaced by the target analyte 1082 and the redox tags are separated, resulting in a redox signal change as taught for other embodiments of the present invention.|0060| With further reference to FIGS. 10A and 10B, in an embodiment of the present invention affinity probe 1029 could also be a probe that is not an antibody, for example such as an aptamer, a peptide, or an analyte binding protein such as transcortin, which has a molecular weight of 52 kDa which is smaller than the molecular weight of an antibody and which may allow for greater stability, accuracy, or longevity, or other performance advantage compared to a probe 1072 that is an antibody.

[0061] Referring now to FIG. 11, where like numerals refer to like features, this embodiment of the present invention includes an alternate switching mechanism. In this embodiment, a device 1100 includes an electrode 1120, which can operate as a sensor to detect and / or measure concentration of an analyte in a sample fluid that is introduced to the device 1100. As illustrated in FIG. 11, an affinity -based probe 1185 such as small molecule, single domain antibody, nanobody, or other suitable probe will bind to an analyte 1182 and, when doing so, release a hybridized strand of aptamers 1124, 1125, resulting in a signal change with at least two redox molecules 1172, 1174 to enable redox quenching. In particular, prior to binding of analyte 1182, the hybridized strand of aptamers includes first and second aptamers 1124, 1125 that are partially or fully complimentary to one another. A first redox tag 1174 is associated with the fist aptamer 1124 (such as by being bound thereto), and a second redox tag 1172 is associated with the second aptamer 1125 (such as by being bound thereto). In the illustrated embodiment of FIG. 11 , the first redox tags 1174 is internally tagged to the first aptamer 1124(with the affinity-based probe 1185 being attached to the distal end of the first aptamer 1124). The second redox tag 1172 is shown as being distally tagged to the second aptamer 1125. In the absence of analyte 1182 redox current is quenched (see first and second aptamers 1124, 1125 at the left side of FIG. 11), whereas in the presence of analyte 1182 binding, the first and second redox tags 1174, 1172 are moved away from one another and the redox current increases (see the first and second aptamers 1124, 1125 at the right side of FIG. 11).

[0062] As an example, the probe molecule 1185 in the embodiment of FIG. 11 could be cortisol as taught herein and the analyte 1 182 could be transcortin which binds cortisol with high binding affinity. Other non-limiting examples for probe and analyte targets may include, for example, one of many small molecules drugs 1185 such as dabigatran, bivalirudin, desirudin, which are commercially available and can be used to bind to thrombin (where thrombin is the analyte 1182) or small molecules 1185 that are known to bind to albumin. Generally, for the embodiment of FIG. 11 to function properly, the analyte 1182 when binding to the probe 1185 must occupy space otherwise occupied by second aptamer 1125 when it hybridizes with first aptamer 1124 such that analyte 1182 binding to probe 1185 disrupts this hybridization. Therefore, probes 1185 may be selected to be smaller in size than antibodies, and in addition to small molecules may include nanobodies, single-domain antibodies, peptides, or other suitable probes that are smaller than antibodies in molecular weight. Examples may include Caplacizumab which targets von Willebrand factor, one of numerous nanobodies developed for targeting coronaviruses, l,6-bis(phosphocholine)-hexane for targeting C-reactive protein, or other suitable probe 1185 and analyte 1182 matches.

[0063] With further reference to FIGS. 10A, 10B, and 11, the first redox tag 1074 may be methylene blue (which is not redox quenching) and second redox tag 1072 may not be required or may be another redox tag with a different redox potential than redox tag 1074 to provide a reference redox signal that is unchanging. A non-redox quenching tag 1072 such as methylene blue can still provide robust changes in electrochemical redox current with analyte 1082 binding because in the state of FIG. 10A the redox tag 1074 can be forced far away from the electrode 1020 by virtue of the large vertical height of affinity probe 1029 if, for example, affinity probe 1028 is an antibody.

[0064] In the state of FIG. 10B, element 1025 can be flexible enough that it allows tag 1074 to come much closer to electrode 1020, or for example element 1025 could be a molecular pendulum type aptamer sensor that only actuates via positive voltage to attract a negatively charged aptamer 1025, tag 1074, and element 1085 closer to the electrode 1020 when aptamer 1025 is free to move as shown in FIG. 10B but not in FIG. 10A. This is a non-limiting example,and other constructs are possible, such as having greater redox current in the state of FIG. 10B than in FIG. 10A by having an antibody anchored, for example, horizontally to the electrode 1020 instead of vertically as shown which would bring redox tag 1074 closer the electrode 1020 in the state shown in FIG. 10B versus that shown in FIG. 10A. In this example, element 1029 is an affinity probe, element 1025 is a flexible tether for tethering bait 1085 to electrode 1020, and tag 1074 is a redox tag carried by flexible tether 1025. Furthermore, a single aptamer 1124 with methylene blue 1174 could alone provide a signal response with analyte 1182 binding. For example, the analyte 1182 binding to probe 1 185 may disrupt the secondary structure of aptamer 1124 in a manner that causes a change in electron transfer rates between tag 1174 and electrode 1120, or it may simply change the kinetics of motion for aptamer 1124 (due to mass, charge, or other properties of analyte 1182) which changes electron transfer rates between tag 1174 and electrode 1120.EXAMPLESExample 1

[0065] The following example is based on the embodiment shown in FIG. 6, and described above, although it is extendable to other embodiments as taught herein by use of the proper aptamer sequence for each target analyte and by relocating the linking and tagging chemistries.

[0066] Materials and Methods

[0067] Materials - Tris-2-carboxyethyl-phosphine (TCEP), 6-mercapto-l -hexanol (99%), l-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), carminic acid, trizmabase (tris base), sodium chloride (NaCl), magnesium chloride (MgCh), hydrochloric acid (HC1), potassium chloride (KC1), disodium phosphate (Na2HPO4), and monopotassium phosphate (KH2PO4) were all used as received from Sigma Aldrich. Destabilized ATP DNA sequence (Integrated DNA Technologies, Coralville, IA, U.S.A.) were purified using dual-HPLC.

[0068] Solution preparation - All buffer solutions were prepared using Milli-Q Ultrapure Water (Milli-Q UltraPure Water Purification, Millipore, Billercia, MA, U.S.A., 18 MO • cm) and a constant ionic strength with 20 mM trizma base, 100 mM NaCl, 5 mM MgCL, adjusted to a pH of -7.45. lx phosphate buffered saline (PBS) solution was prepared by diluting lOx PBS solution. The lOx PBS solution was prepared with 1.36 M NaCl, 26.8 mM KC1, 101 mM Na2HPO4, and 17.6 mM KH2PO4.

[0069] Electrode Preparation - 2 mm polycrystalline gold electrodes (CH Instruments, Austin, TX, U.S.A.) were polished by hand in a figure eight motion on a micro cloth (Buehler) with a 0.05 pm alumina oxide slurry for 2 minutes. The electrodes were then polished in the same manner on a micro cloth with ultrapure water. The electrodes were then cleanedelectrochemically by a series of cyclic voltametric scans in various concentrations of NaOH and H2SO4 to remove any remaining contaminants.

[0070] DNA sequences - For all experiments, the aptamer used was a Destabilized ATP aptamer, an adapted sequence for optimal sensor signaling adapted from the ATP binding aptamer sequence first reported by Szostak, et al., with a C6 thiol modification at the 5’ end and a C6 primary amine at the 3’ end. The sequence of the Destabilized ATP aptamer is as follows: 5’ CTG GGG GAG TAT TGC GGA GGA AA 3’ [SEQ. ID. NO. 1],

[0071] To promote dimerization of carminic acid labels, the destabilized ATP aptamer was hybridized with its partial complement. For this complement, several of the bases were non- complimentary so that the hybidrization was not so strong as to not allow ATP binding. This complement was modified with a C6 primary amine at the 5’ end and C6 thiol modification at the 3’ end. The sequence of the partial complement is as follows:5’ TTT CCT CCG CAA TAC TCC CCC TT 3’ [SEQ. ID. NO. 2]

[0072] Aptamer Modification - Carminic acid was covalently coupled to amine-terminated aptamer and the complementary strand as follows: After being reduced for 1 hour with 100 mM TCEP, equimolar amounts of the aptamer and its complement were mixed and allowed to hybridize for 30 minutes. Afterwards, the solution of hybridized probes was diluted to a concentration of 200 nM and clean gold electrodes were incubated in the solution for 1 hour. After incubation, the electrodes were thoroughly rinsed with distilled water and then placed in a solution of 30 mM 6-mercapto-l -hexanol for one hour. After an hour of passivation, the electrodes were thoroughly rinsed with distilled water and then placed into a solution of lx PBS buffer with 100 mM EDC and 2 mM carminic acid for a minimum of 3 hours. Finally, the electrodes were placed in tris buffer for one hour for equilibration before testing.

[0073] Electrochemical measurements - All electrochemical measurements were done on a CHI 1040C (CH Instruments, Austin, TX, U.S.A.) using a 3-electrode setup with a Ag / AgCl reference electrode (3 mM NaCl), a platinum counter electrode, and either a planar or NPGL- modified polycrystalline gold working electrode. The working electrodes were modified with DNA. For square wave measurements, the potential window is from 0.6V - 0V with a sample interval of 0.001 V at a frequency of 200 Hz. A quiet time of 2 seconds was used for all measurements.

[0074] Baseline reduction with hybridized probes - With reference to FIGS. 6 and 7, to promote dimerization of carminic acid redox tags on surface-bound aptamers, the Aptamer Modification method described above was followed: An amine-terminated aptamer was hybridized with an amine-terminated partial complement. The terminal amines were positionedat the 3’ and 5’ terminus respectively. The aptamer and partial complement were mixed in equimolar amounts for 30 minutes prior to the disulfide bond reduction step with 100 mM TCEP. This step ensured that the two strands would immobilize in proximity as they were immobilized on the gold electrode surface, thereby keeping the amine-labeled termini, and eventually carminic acid labels, in close proximity to one another. After the hybridized probes had been placed on the electrode surface, the electrodes were then passivated with 30 mM 6- mercaptohexanol for 1 hour. After passivation, the electrodes were placed in a solution containing 100 mM EDC and 2 mM carminic acid in l x PBS buffer for at least 3 hours, after which the electrodes were rinsed with distilled water and placed in tris buffer for 1 hour for equilibration.

[0075] Results

[0076] With reference to FIG. 8, with interrogation in the absence of target (ATP), the sensors showed no measurable peak in the potential window of 0.6 V to 0 V vs. Ag / AgCl. From there, ATP was titrated in starting at 0. 1 pM and ending at 1 mM. As concentration of ATP increases, so too does the peak current at approximately 0.32 V (vs Ag / AgCl), which agrees well with the measured potential for carminic acid from other reports. Typically, aptamer sensors employing other redox labels, such as methylene blue, ferrocene, etc., report percent signal change when interrogating with square wave voltammetry, however, percent signal change requires a non- zero initial point and therefore will not work with this type of sensor as the dimerization of carminic acid leads to an initial peak current of zero. In order to normalize the signal obtained from multiple sensors, the peak current at 0.32 V was divided by electroactive surface area to obtain peak current density. At 1 mM ATP concentration, the peak current density reaches an average of 90 ± 13 pA / cnr. Fitting the titration curve, the present inventors find the observed KD value to be 140 ± 30 pM.

[0077] To support the proposed dimerized signaling mechanism, additional sensors were prepared using the amine-terminated destabilized ATP aptamer without the partial complement. As such, the sensor fabrication process was nearly identical, apart from the hybridization of the aptamer to the partial complement being removed. Using a single stranded aptamer, the dimerization no longer occurs, and a measurable current is obtained in the absence of target, much like aptamer sensors using more typical redox labels, such as methylene blue or ferrocene. As seen with methylene blue-labeled destabilized ATP aptamers, the sensor saturates at -500 - 750 pM ATP, though unlike previous reports, the maximum percent signal change is much greater at 990% ± 92%. This significant increase may be due to some signal suppression that occurs at due to some dimerization at lower target concentrations where theaptamers are more flexible and in an unfolded conformation, thereby making the redox labels more likely to collide and dimerize. Therefore, this also represents an embodiment of the present invention, where there is only one redox quenching tag per aptamer.

[0078] Discussion

[0079] One of the challenges with any aptamer sensor is longevity. For this Example (covering the embodiment of FIG. 6 and covering FIGS. 7-9), the positive voltage utilized could be detrimental to sensor longevity by promoting thiol oxidation or monolayer desorption. As demonstrated by Watkins et al., continuous or near-continuous negative scanning or potential can actually stabilize an aptamer sensing monolayer (Watkins Z, Karajic A, Young T, White R, Heikenfeld J., Week-Long Operation of Electrochemical Aptamer Sensors: New Insights into Self-Assembled Monolayer Degradation Mechanisms and Solutions for Stability in Serum at Body Temperature. ACS Sens. 2023 Mar 24;8(3): 1119- 1131. doi: 10.1021 / acssensors.2c02403. Epub 2023 Mar 8. PMID: 36884003; PMCID: PMC 10443649). Therefore, the present invention may include a device where negative potential scanning is utilized for stabilization (e.g., out to -0.4V) most of time during operation, and then briefly alternated with positive potential scanning for measurement (e.g. out to +0.35V). Similarly, such scanning also supports operation of a device as taught in FIG. 4, where, for example, first and second redox tags 472, 474 could be carminic acid and positive voltage scanned for measurement, and redox tag 470 could be methylene blue and negative voltage scanned for measurement.

[0080] With further respect to longevity, maintaining a calibration free operation of a redox quenching device will be more achievable if: (1) the redox tags remain reversible (stable); (2) the aptamer monolayer does not desorb; (3) fouling which would impede aptamer movement is prevented. Techniques used by Watkins et. al. (incorporated by reference herein) can then be used to ensure longevity of the device with respect to preventing desorption and fouling. The device may experience changes in electron transfer rates as the device ages which could result in false changes in measurements. To correct for such changes, low frequency square wave voltammetry, cyclic voltammetry, or other measures may occasionally be performed to collect most or all available redox transfer events from the redox tags, therefore providing an in- variate measure of the amount of binding occurring between analytes and the aptamers. For example, a cyclic voltammogram can be scanned at 50 mV / sec or slower. In another embodiment, a square wave voltammetry can be scanned at a square wave frequency of less than 20 Hz, or alternatively, less than 5Hz can be used. As a result, at least one of >50, >80, >90, >95% of the possible redox transfer events from the redox tags can be collected(measured) from the redox tags. As a result, a device is at least partially able to self-calibrate, because the slow or low frequency measures are less dependent on electron transfer rates and therefore less dependent on changes in the monolayer, fouling, or other aspects of the sensor that may change electron transfer rates.

[0081] Alternately, carminic acid may have a negative potential window for measurementbased scanning based on its electrochemical profile seen in solution. Alternately, the redox tags which provide redox quenching need not be carminic acid and can be other tags which have a near zero potential or negative potential window for scanning, which may be achievable, for example, with other redox active anthraquinone based molecules. Beyond the possibility of substituted anthraquinone derivatives, it is known, for example, that the iron-containing porphyrin hemin is reduced at approximately -0.3 V (vs Ag / AgCl) and undergoes redox quenching due to pi-stacking interactions and therefore could also be used as a negative measurement window redox reporter whose electron transfer to an electrode is dependent on the proximity to another hemin redox tag. Hemin can be synthesized into hemin active esters or other suitable formats for attachment to aptamers or other elements as taught herein for embodiments of the present invention. For example, amine terminated aptamers can enable distal tagging of redox tags such as hemin and modified thymine bases can be used for internal tatting of redox tags such as hemin. Alternately, the redox tags need not be identical molecules as long as they provide redox quenching as taught herein.

[0082] Example 2

[0083] The following example is directed to an embodiment including a bait element, such as the embodiment seen in FIGS. 10A and 10B, and draws from: Thompson, et al., “An Antibody-Based Molecular Switch for Continuous Biosensing”, doi: https: / / doi.Org / 10.l 101 / 2023.03.07.531602 (incorporated by reference herein). For this example, the DNA sequences to be used may include:

[0084] Affinity Probes (1029, 1072).

[0085] For measuring cortisol as the target analyte 1082, cortisol monoclonal antibody (XM210) can be obtained from Abeam for affinity probe 1029 for cortisol sensing. For measuring digoxigenin as the target analyte 1082, digoxigenin polyclonal antibody (Sheep, SKU: 11333089001) can be obtained from Sigma Aldrich and digoxigenin monoclonal antibody (Clone # 611621) can be obtained from R&D Systems 1029.

[0086] Flexibly-linked bait elements (1025, 1085, 1072).

[0087] Oligonucleotide sequences can be purchased HPLC-purified from Integrated DNA Technologies or Biosearch Technologies with various internal and terminal modifications including thiol linkage to the gold electrode 1020 and including redox tags 1072, 1074 such as hemin or carminic acid, and including bait molecules 1085. Digoxigenin bait-DNA conjugates 1025 1085 can be prepared by labeling the amine in the bait-DNA sequence with DIG-NHS. Cortisol bait-DNA conjugates 1025 1085 can be prepared by coupling cortisol-3CMO 1085 to the terminal amine on the bait-DNA sequence 1025 using EDC / NHS chemistry. Alternately, corticosterone bait-DNA conjugates 1025 1085 can use a carboxyl-group conjugated onto corticosterone 1085 and then conjugated onto the bait-DNA sequence 1025 using the EDC / NHS reaction. For the affinity probe 1029, if an antibody is used for affinity probe 1029 there are a diverse number of suppliers and chemistries used for tagging of antibodies that can be used to attach redox tag 1072. For bait-DNA conjugates 1085 1025 redox tags such as 1074 can be attached using a variety of tagging chemistries including attachment hemin-active esters. One advantage of using a DNA duplex (double stranded DNA) for element 1025 is that the terminal ends (5 ’ or 3 ’ ends depending on orientation) can be used for tagging with redox tag 1074 and / or bait 1085.

[0088] 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

WHAT IS CLAIMED IS:

1. A device for detecting or measuring at least one analyte in a sample fluid, the device comprising: at least one electrode; and a plurality of aptamers capable of binding to the analyte, wherein the aptamers are bound to the at least one electrode; wherein at least some aptamers of the plurality of aptamers have a first molecule and a second molecule associated therewith; wherein the first molecule is a redox tag; wherein the first molecule and second molecule move either closer to or further from one another when their associated aptamer binds analyte, which results in a change in electron transfer between the first molecule and the electrode at a given voltage.

2. The device of claim 1, wherein the first molecule is distally tagged and the second molecule is internally tagged onto the aptamer.

3. The device of claim 1, wherein either the second molecule is distally tagged and the first molecule is internally tagged onto the aptamer.

4. The device of claim 1, wherein both the first molecule and the second molecule are internally tagged onto the aptamer.

5. The device of claim 1, wherein both the first molecule and the second molecule are distally tagged onto the aptamer.

6. The device of claim 1 , wherein at least one of the first molecule and second molecule moves either closer to or further from the electrode when their associated aptamer binds analyte.

7. The device of claim 1, wherein the analyte has a molecular weight chosen from greater than 3 kDa, greater than 4kDa, or greater than 10 kDa.

8. The device of claim 1, wherein the plurality of aptamers are directly bound to theelectrode.

9. The device of claim 1, wherein the plurality of aptamers are indirectly bound to the electrode via a substrate linker.

10. The device of claim 1, wherein the first molecule and the second molecule are the same molecule.

11. The device of claim 10, wherein the first molecule and the second molecule are carminic acid.

12. The device of claim 10, wherein the first molecule and the second molecule are hemin.

13. The device of claim 1, wherein the device is capable of providing a percentage change in sensor response selected from the group consisting of greater than 50%, greater than 100%, greater than 200%, and greater than 500%.

14. The device of claim 1, further comprising an instrument for measurement scanning, wherein the measurement scanning includes periods of both negative and positive potential scanning for measurement.

15. A device for detecting or measuring at least a first analyte and a second analyte in a sample fluid, the device comprising: at least one electrode; a first plurality of aptamers capable of binding to the first analyte, wherein the aptamers are bound to the at least one electrode, wherein at least some aptamers of the first plurality of aptamers have a first molecule and a second molecule associated therewith, wherein the first molecule is a redox tag, and wherein the first molecule and second molecule move either closer to or further from one another when their associated aptamer binds analyte, which results in a change in electron transfer between the first molecule and the electrode at a given voltage; and a second plurality of aptamers capable of binding to the second analyte, wherein the aptamers are bound to the at least one electrode, wherein at least some aptamers have a third molecule associated therewith, wherein the third molecule is a redox tag, and wherein the third molecule moves either closer to or further from the electrode when the associated aptamerbinds analyte which results in a change in electron transfer between the third molecule and the electrode at a given voltage; wherein the third molecule is redox active at a potential that is measurably distinct from the first molecule or second molecule.

16. The device of claim 15, wherein the first molecule is distally tagged and the second molecule is internally tagged onto the aptamer.

17. The device of claim 15, wherein either the second molecule is distally tagged and the first molecule is internally tagged onto the aptamer.

18. The device of claim 15, wherein both the first molecule and the second molecule are internally tagged onto the aptamer.

19. The device of claim 15, wherein both the first molecule and the second molecule are distally tagged onto the aptamer.

20. The device of claim 15, wherein at least one of the first molecule and second molecule moves either closer to or further from the electrode when their associated aptamer binds analyte.

21. The device of claim 15, wherein the first analyte has a molecular weight chosen from greater than 3 kDa, greater than 4kDa, or greater than 10 kDa.

22. The device of claim 15, wherein the first plurality of aptamers and / or the second plurality of aptamers are directly bound to the electrode.

23. The device of claim 15, wherein the first plurality of aptamers and / or the second plurality of aptamers are indirectly bound to the electrode via a substrate linker.

24. The device of claim 15, wherein the first molecule and the second molecule are the same molecule.

25. The device of claim 24, wherein the first molecule and the second molecule are selectedfrom the group consisting of carminic acid and hemin.

26. The device of claim 15, wherein the device is capable of providing a percentage change in sensor response relative to the first molecule selected from the group consisting of greater than 50%, greater than 100%, greater than 200%, and greater than 500%.

27. A device for detecting or measuring at least one analyte in a sample fluid, the device comprising: at least one electrode; and a first aptamer capable of binding to the analyte, and a second aptamer at least partially complementary to the first aptamer, wherein the first and second aptamers are proximal to one another and bound to the at least one electrode; wherein the first aptamer has a first molecule associated therewith, and the second aptamer has a second molecule associated therewith; wherein the first molecule is a redox tag; wherein the first molecule and second molecule move either closer to or further from one another when their associated aptamer binds analyte, which results in a change in electron transfer between the first molecule and the electrode at a given voltage.

28. The device of claim 27, wherein the first aptamer and second aptamer are the same number of nucleotides in length.

29. The device of claim 27, wherein the first aptamer and second aptamer have different lengths in number of nucleotides.

30. The device of claim 27, wherein the first molecule is distally tagged to the first aptamer, and the second molecule is distally tagged to the second aptamer.

31. The device of claim 27, wherein the first molecule is internally tagged to the first aptamer, and the second molecule is internally tagged to the second aptamer.

32. The device of claim 27, wherein the first molecule is distally tagged to the first aptamer, and the second molecule is internally tagged to the second aptamer.

33. The device of claim 27, wherein the first molecule is internally tagged to the first aptamer, and the second molecule is distally tagged to the second aptamer.

34. The device of claim 27, wherein at least one of the first molecule and second molecule moves either closer to or further from the electrode when either the first or the second aptamer binds analyte.

35. The device of claim 27, wherein the analyte has a molecular weight chosen from greater than 3 kDa, greater than 4kDa, or greater than 10 kDa.

36. The device of claim 27, wherein the first aptamer and / or the second aptamer is directly bound to the electrode.

37. The device of claim 27, wherein the first aptamer and / or the second aptamer is indirectly bound to the electrode via a substrate linker.

38. The device of claim 27, wherein the device is capable of providing a percentage change in sensor response relative to the first molecule selected from the group consisting of greater than 50%, greater than 100%, greater than 200%, and greater than 500%.

39. A device for detecting or measuring at least one analyte in a sample fluid, the device comprising: at least one electrode; a plurality of affinity probes, wherein the affinity probes are bound to the electrode, wherein at least some affinity probes of the plurality of affinity probes have a first molecule associated therewith; a plurality of flexibly-linked bait elements, wherein the flexibly-linked bait elements are bound to the electrode, wherein at least some flexibly-linked bait elements of the plurality of flexibly-linked bait elements have a second molecule associated therewith; wherein one of the first molecule and second molecule is a redox tag; and wherein the flexibly-linked bait elements are bound by the affinity probes in the absence of analyte such that the first molecule and second molecule are proximal to one another; and wherein the first molecule and second molecule move further from one another when the affinity probe binds analyte, which results in a change in electron transfer between the redox tag and the electrode at a given voltage.

40. The device of claim 39 where the first molecule and the second molecule are the same molecule.

41. The device of claim 39 wherein the first molecule and the second molecule are selected from the group consisting of carminic acid and hemin.

42. The device of claim 39, wherein said affinity probe is selected from the group consisting of an antibody and a protein.

43. The device of claim 39, wherein at least one of the first molecule and second molecule moves either closer to or further from the electrode when either the first or the second aptamer binds analyte.

44. The device of claim 39, wherein the analyte has a molecular weight chosen from greater than 3 kDa, greater than 4kDa, or greater than 10 kDa.

45. The device of claim 39, wherein the affinity probe and / or the flexibly-linked bait element is directly bound to the electrode.

46. The device of claim 39, wherein the affinity probe and / or the flexibly-linked bait element is indirectly bound to the electrode via a substrate linker.

47. The device of claim 39, wherein the device is capable of providing a percentage change in sensor response relative to the first molecule selected from the group consisting of greater than 50%, greater than 100%, greater than 200%, and greater than 500%.

48. The device of claim 39 further comprising an instrument for measurement scanning, wherein the measurement scanning includes periods of both negative and positive potential scanning for measurement.

49. A device for detecting or measuring at least one analyte in a sample fluid, the device comprising: at least one electrode; anda first aptamer and a second aptamer at least partially complementary to the first aptamer, wherein the first and second aptamers are proximal to one another and bound to the at least one electrode; wherein the first aptamer has an affinity-based probe and a first molecule associated therewith, and the second aptamer has a second molecule associated therewith; wherein the affinity-based probe is capable of binding to the analyte; wherein at least the first molecule or the second molecule is a redox tag; wherein the first molecule and second molecule move either closer to or further from one another when the affinity -based probe binds analyte, which results in a change in electron transfer between the redox tag and the electrode at a given voltage.

50. The device of claim 49, wherein the first and second aptamers are at least partially hybridized to one another in the absence of binding of affinity-based probe to analyte.

51. The device of claim 50, wherein the first and second hybridized aptamers release from one another when the affinity-based probe binds analyte.

52. The device of claim 49, wherein the first and second molecules are first and second redox tags.

53. The device of claim 52, wherein the first redox tag and the second redox tag are redox quenching when brought into close proximity to each other.

54. The device of claim 49, wherein said affinity probe is selected from the group consisting of an antibody and a protein.

55. The device of claim 49, wherein the first aptamer and second aptamer are the same number of nucleotides in length.

56. The device of claim 49, wherein the first aptamer and second aptamer have different lengths in number of nucleotides.

57. The device of claim 49, wherein at least one of the first redox tag and second redox tag moves either closer to or further from the electrode when either the first or the second aptamerbinds analyte.

58. The device of claim 49, wherein the analyte has a molecular weight chosen from greater than 3 kDa, greater than 4kDa, or greater than 10 kDa.

59. The device of claim 49, wherein the first aptamer and / or the second aptamer is directly bound to the electrode.

60. The device of claim 49, wherein the first aptamer and / or the second aptamer is indirectly bound to the electrode via a substrate linker.

61. The device of claim 49, wherein the device is capable of providing a percentage change in sensor response relative to the first molecule selected from the group consisting of greater than 50%, greater than 100%, greater than 200%, and greater than 500%.

62. A method for detecting or measuring at least one analyte in a sample fluid comprising: Bringing the sample fluid into proximity with at least a portion of the device of claim1, claim 15, claim 27, claim 39, or claim 49; and measuring a change in electrical current between the first molecule and the electrode.

63. The method of claim 62, further comprising performing one or more measurements selected from the group consisting of low frequency square wave voltammetry, and cyclic voltammetry.

64. The method of claim 63, wherein the measurements may occasionally be performed to collect most or all available redox transfer events from the redox tags, therefore providing an in- variate measure of the amount of binding occurring between analytes and the aptamers.

65. The method of claim 64, wherein a percentage of the possible redox transfer events from the redox tags is measured from the redox tags, the percentage selected from the group consisting of greater than 50%, greater than 80%, greater than 90%, and greater than 95%.

66. The method of claim 62, the method further comprising measuring an initial electrical current between the electrode and the first molecule.

67. The method of claim 66, further comprising detecting and / or measuring a change from the initial electrical current between the electrode and the first molecule following bringing the sample fluid into proximity with the device.

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