One particular aspect of the present invention is directed to a device for detecting the presence of, or measuring the concentration or amount of, at least one analyte in a sample fluid. The device includes a sensor fluid, a plurality of adapter molecules in the sensor fluid that are capable of binding to an analyte, a plurality of aptamers in the sensor fluid that are capable of binding to the analyte when analyte is bound to one or more adapter molecules of the plurality of adapter molecules, at least one membrane in communication with the sensor fluid, and at least one detection component that is capable of detecting the binding of analyte by one or more of the plurality of aptamers. The membrane is permeable to the analyte but impermeable to the adapter molecule. 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 bringing a sample fluid into contact with at least one adapter molecule of a plurality of adapter molecules that are present in a sensor fluid. This includes passing the sample fluid through a membrane that is permeable to analyte but impermeable to adapter molecules of the plurality of adapter molecules. Thereafter, the binding of analyte by one or more adapter molecules of the plurality of adapter molecules is detected or measured.

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. SA, 5B, and SC). 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. SA, 5B, SC, and SD). 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∝D C/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 ~0.6th 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 10s to 100s 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