Electrochemical paper-based analytical devices

Abstract

Articles and methods related to fluidic devices are generally described. In some embodiments, the articles and methods described herein allow for a liquid to flow along one or more channels positioned between two or more electrodes.

Description

[0001] ELECTROCHEMICAL PAPER-BASED ANALYTICAL DEVICES

[0002] RELATED APPLICATIONS

[0003] This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No. 63 / 729,236, filed December 6, 2024, and entitled “Electrochemical Paper-Based Analytical Devices,” which is incorporated herein by reference in its entirety for all purposes.

[0004] GOVERNMENT SPONSORSHIP

[0005] This invention was made with government support under Contract No. CBET- 1846846 awarded by the National Science Foundation. The government has certain rights in the invention.

[0006] TECHNICAL FIELD

[0007] Articles and methods related to fluidic devices are generally described.

[0008] BACKGROUND

[0009] Fluidic devices having coplanar electrodes present a number of challenges when used to detect analytes. For example, common problems include less than desirable reproducibility and / or limits of detection for analytes stemming, at least in part, from the relatively large and nonuniform distance between coplanar electrodes. Such distances and nonuniformities may be related to fabrication processes and / or patterning technologies. Accordingly, there is a need for fluidic devices having a relatively low and uniform distance between electrodes.

[0010] SUMMARY

[0011] Articles and methods related to fluidic devices are generally described. The subject matter of the present disclosure involves, in some cases, interrelated products, alternative solutions to a particular problem, and / or a plurality of different uses of one or more systems and / or articles.

[0012] In one aspect, a fluidic device is provided. According to some embodiments, the fluidic device comprises: a first layer comprising a first channel; a second layer comprising a second channel; a first electrode; a second electrode; and a third electrode, wherein: the first layer is disposed on the second layer; the first layer and the second layer each comprise a porous, absorbent material; the first channel is positioned between the first electrode and the second electrode; the second channel is positioned between the second electrode and the third electrode;

[0013] #14670283vl and the first channel comprises a first vertical flow region, the second channel comprises a second vertical flow region, and the first and second vertical flow regions are in fluidic communication.

[0014] In another aspect, a method is provided. According to some embodiments, the method comprises: flowing a liquid through a first layer comprising a first channel, and a second layer comprising a second channel, wherein: the first layer is disposed on the second layer; the first layer and the second layer comprise a porous, absorbent material; the first channel is positioned between a first electrode and a second electrode; the second channel is positioned between the second electrode and a third electrode; and the first channel comprises a first vertical flow region, the second channel comprises a second vertical flow region, and the first and second vertical flow regions are in fluidic communication.

[0015] Other advantages and novel features of the present disclosure will become apparent from the following detailed description of various non-limiting embodiments of the disclosure when considered in conjunction with the accompanying figures. In cases where the present specification and a document incorporated by reference include conflicting and / or inconsistent disclosure, the present specification shall control.

[0016] BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Non-limiting embodiments of the present disclosure will be described by way of example with reference to the accompanying figures, which are schematic and are not intended to be drawn to scale unless otherwise indicated. In the figures, each identical or nearly identical component illustrated is typically represented by a single numeral. For purposes of clarity, not every component is labeled in every figure, nor is every component of each embodiment of the disclosure shown where illustration is not necessary to allow those of ordinary skill in the art to understand the disclosure. In the figures:

[0018] FIG. 1A presents a cross-sectional schematic illustration of a fluidic device, according to some embodiments;

[0019] FIG. IB presents a cross-sectional schematic illustration of a fluidic device, according to some embodiments;

[0020] FIG. 1C presents a cross-sectional schematic illustration of a fluidic device, according to some embodiments;

[0021] FIG. ID presents a cross-sectional schematic illustration of a fluidic device, according to some embodiments;

[0022] #14670283vl FIG. 2 presents a top-view schematic illustration of a fluidic device, according to some embodiments;

[0023] FIG. 3A presents a cross-sectional schematic illustration of a fluidic device, according to some embodiments;

[0024] FIG. 3B presents a top-view schematic illustration of a fluidic device, according to some embodiments;

[0025] FIG. 4A presents a cross-sectional schematic illustration of a fluidic device, according to some embodiments;

[0026] FIG. 4B presents a cross-sectional schematic illustration of a fluidic device, according to some embodiments;

[0027] FIG. 4C presents a top-view photograph of a fluidic device, according to some embodiments;

[0028] FIG. 5 presents an exploded perspective schematic illustration of a fluidic device, according to some embodiments;

[0029] FIGS. 6A-6B show the anticipated linear relationship between the square root of CV scan rate applied and the resulting current values for various non-limiting fluidic devices, according to some embodiments;

[0030] FIG. 7 presents the difference between measured currents with respect to parameters of non-limiting fluidic devices, such as paper grade, according to some embodiments;

[0031] FIGS. 8 A presents the difference between measured currents with respect to parameters of non-limiting fluidic devices, according to some embodiments;

[0032] FIGS. 8B shows the anticipated linear relationship between the square root of CV scan rate applied and the resulting current values for various non-limiting fluidic devices, according to some embodiments;

[0033] FIG. 9 presents current decays to a steady state Faradaic current within after applying step potential, according to some embodiments;

[0034] FIGS. 10A-10B shows the calibration plots, LODs, and sensitivities produced by various non-limiting fluidic devices, according to some embodiments;

[0035] FIG. 11 presents non-limiting correlations between current signal and Pb(II) concentration measured using non-limiting fluidic devices, according to some embodiments;

[0036] FIG. 12 presents non-limiting current magnitudes of non-limiting fluidic devices in the WCR and CWR configuration, according to some embodiments;

[0037] FIGS. 13A-13B present SWASV measurements of non-limiting fluidic devices, according to some embodiments;

[0038] #14670283vl FIG. 14A presents an exploded perspective schematic illustration of a fluidic device, according to some embodiments;

[0039] FIG. 14B presents a cross-sectional schematic illustration of a fluidic device, according to some embodiments;

[0040] FIG. 14C presents a top- view photograph of a fluidic device, according to some embodiments;

[0041] FIG. 15 presents a top-view schematic of a fluidic device, according to some embodiments;

[0042] FIG. 16A presents a cross-sectional schematic illustration of a fluidic device, according to some embodiments;

[0043] FIG. 16B presents a cross-sectional schematic illustration of a fluidic device, according to some embodiments;

[0044] FIG. 17 shows cross sectional schematic and images of the ink penetration associated with printing (A) top and (B) both sides of different papers with carbon ink of non-limiting fluidic device layers, according to some embodiments;

[0045] FIG. 18A presents top-view photographs of sample flow after being introduced to paper layers from the circular zone of non-limiting fluidic devices, according to some embodiments;

[0046] FIG. 18B presents an exploded perspective schematic illustration of a non-limiting fluidic device, according to some embodiments;

[0047] FIG. 18C presents table showing the variability in peak current and potential produced by running linear sweep voltammetry (LSV) using the top-sided printed TFN-based devices, according to some embodiments;

[0048] FIG. 19 shows cross sectional schematic and actual images of the ink penetration associated with printing both faces of a non-limiting layer with carbon ink, according to some embodiments;

[0049] FIG. 20A shows top-view images of the ink penetration associated with printing both faces of a non-limiting layer with carbon ink, according to some embodiments;

[0050] FIG. 20B presents a table showing the variability in LSV peak current and potential produced using non-limited top-sided and double-sided printed fluidic devices, according to some embodiments;

[0051] FIGS. 21A-21B present the measured relationship between the thicknesses of electrodehosting and the flow layers based on the resulting LSV current, according to some embodiments;

[0052] FIGS. 22A-22B show non-limiting cyclic voltammetry using non-limiting CWR configuration fluidic devices, according to some embodiments;

[0053] #14670283vl FIGS. 23A-23B show non-limiting cyclic voltammetry of non-limiting fluidic devices using SPEs, according to some embodiments;

[0054] FIGS. 24A-24B show chronoamperometric detection of glucose using non-limiting fluidic devices, according to some embodiments;

[0055] FIGS. 25A-25B show SWASV for detection of Pb(II) using non-limiting fluidic devices, according to some embodiments;

[0056] FIG. 26 shows an exploded-perspective schematic illustration of a non-limiting fluidic device, according to some embodiments;

[0057] FIG. 27 shows an exploded-perspective schematic illustration of a non-limiting fluidic device, according to some embodiments;

[0058] FIG. 28A shows a cross-sectional schematic illustration of a non-limiting fluidic device, according to some embodiments;

[0059] FIG. 28B shows a cross-sectional schematic illustration of a non-limiting fluidic device, according to some embodiments;

[0060] FIG. 29 shows an exploded-perspective schematic illustration of a non-limiting fluidic device, according to some embodiments;

[0061] FIG. 30A shows a cross-sectional schematic illustration of a non-limiting fluidic device, according to some embodiments;

[0062] FIG. 30B shows a cross-sectional schematic illustration of a non-limiting fluidic device, according to some embodiments;

[0063] FIGS. 31A-31B present chronoamperometric detection of both glucose (FIG. 31 A) and lactate (FIG. 3 IB) using non-limiting fluidic devices, according to some embodiments;

[0064] FIGS. 32A-32B present chronoamperometric detection of both glucose and lactate in non-limiting fluidic devices, according to some embodiments;

[0065] FIGS. 32C-32D present calibration curves for detection of both glucose and lactate in non-limiting fluidic devices, according to some embodiments;

[0066] FIG. 33 presents a stability assessment of the chronoamperometric current response for both glucose and lactate using non-limiting fluidic devices, according to some embodiments;

[0067] FIGS. 34A-34B present extended analysis of chronoamperometric current stability across three measurement cycles for glucose (FIG. 34A) and lactate (FIG. 34B) using non-limiting fluidic devices, according to some embodiments;

[0068] FIG. 35 shows an exploded-perspective schematic illustration of a non-limiting fluidic device, according to some embodiments;

[0069] #14670283vl FIG. 36 shows an exploded-perspective schematic illustration of a non-limiting fluidic device, according to some embodiments;

[0070] FIG. 37 A presents voltammograms of non-limiting fluidic devices, according to some embodiments;

[0071] FIG. 37B presents the linear relationships between square root of scan rates and their measured anodic and cathodic current of non-limiting fluidic devices, according to some embodiments;

[0072] FIGS. 38A-38B present exemplary Nyquist plots of the EIS measurements of nonlimiting fluidic devices in WCR (FIG. 38A) and CWR (38B) configurations, according to some embodiments; and

[0073] FIGS. 39A-39C present quantitative comparison of ElS-derived parameters for WCR and CWR configurations of non-limiting fluidic devices, according to some embodiments.

[0074] DETAILED DESCRIPTION

[0075] Articles and methods related to fluidic devices comprising electrodes are generally provided. In some embodiments, the articles and methods described herein relate to a fluidic device having electrodes that can facilitate the detection of any of a variety of substances in at least a portion of the liquid.

[0076] In some embodiments, a fluidic device described herein comprises one or more layers and two or more electrodes such that the electrodes are separated by one or more layers of the fluidic device. In some embodiments, the electrodes are arranged such that two or more electrodes are not coplanar with each other and / or each electrode is not coplanar with another electrode. The performance of fluidic devices having coplanar electrodes are typically limited by fabrication processes used to create a separation between electrodes (e.g., screen printing). Such processes can be difficult to control when the distance between surfaces of electrodes become relatively low. Accordingly, fluidic devices having electrodes that are not coplanar may surprisingly allow the fluidic device to have a relatively low distance between the surfaces of any two electrodes. Additionally, fluidic devices having electrodes that are not coplanar may allow for the fluidic device to be used on nonplanar substrates, as the distance between surfaces of the electrodes may not significantly change when on a nonplanar substrate.

[0077] In some embodiments, a fluidic device described herein comprises two or more electrodes separated by a relatively low distance. Relatively low distances between two or more electrodes in the fluidic device can be advantageous as relatively low distances between electrodes may facilitate relatively low limits of detection (LOD). Without wishing to be bound

[0078] #14670283vl - 1 - by any particular theory, relatively low distances between electrodes may allow for increased electrochemical conductivity between electrodes and provide surprisingly low LODs. Relatively low LODs may be particularly advantageous when detecting substances (e.g., glucose and / or Pb(II)) that are present in relatively low concentrations, such as in bodily fluids (e.g., blood, saliva).

[0079] In some embodiments, a fluidic device can be fabricated such that the distance between surfaces of any two electrodes is relatively uniform. In some embodiments, the distance between the surfaces of any two electrodes can be selected by tuning the thickness of one or more layers of the fluidic device separating the two electrodes. That is, the thickness of any one of the layers of the fluidic device may be chosen to facilitate an advantageous, relatively uniform distance between the surfaces of any two electrodes. Relatively uniform distances between the surfaces of any two electrodes may promote reproducible and reliable detection of any of a variety of substances. Nonuniform distances between the surfaces of any two electrodes, on the other hand, may lead to unreliable and / or imprecise detection of various substances. Accordingly, since the distance between the surfaces of any two electrodes may be controlled by varying the thickness of any one of the layers of the fluidic device separating the two electrodes, the fluidic device may be fabricated with relatively uniform distances between the surfaces of any two electrodes such that the LOD of the fluidic device is particularly beneficial.

[0080] In some embodiments, a fluidic device comprises a first layer, a first electrode, and a second electrode. For example, as shown in FIG. 1A, fluidic device 100 comprises first layer 110, first electrode 120A, and second electrode 120B. First layer 110 comprises first channel 115 which is positioned between first electrode 120A and second electrode 120B. Fluidic device 100 further comprises inlet 105 in fluidic communication with first channel 115. When a liquid is provided to inlet 105, at least a portion of the liquid may flow along first channel 115 between first electrode 120 A and 120B.

[0081] In some embodiments, a fluidic device comprises a first layer, a second layer, a first electrode, a second electrode, and / or a third electrode. For example, as shown in FIG. IB, fluidic device 100 comprises first layer 110, second layer 130, first electrode 120A, second electrode 120B, and third electrode 120C. First layer 110 comprises first channel 115 positioned between first electrode 120A and second electrode 120B. Second layer 130 comprises second channel 150 positioned between second electrode 120B and third electrode 120C. In some embodiments, the first channel and the second channel are in fluidic communication. As an example, as shown in FIG. IB, first channel 115 comprises first vertical flow region 135 and second channel 150 comprises second vertical flow region 140. Since first vertical flow region 135 is in fluidic

[0082] #14670283vl communication with second vertical flow region 140, first channel 115 is in fluidic communication with second channel 150.

[0083] In some embodiments, the fluidic device allows at least a portion of the liquid to flow through one or more layers thereof. For example, as shown in FIG. 1C, fluidic device 100 comprises first layer 110 disposed on second layer 130. First layer 110 comprises first channel 115, having length LI, positioned between first electrode 120A and second electrode 120B. Second layer 130 comprises second channel 150, having length L2, positioned between second electrode 120B and third electrode 120C. First channel 115 comprises first vertical flow region 135 and second channel comprise second vertical flow region 140 such that first vertical flow region 135 is in fluidic communication with second vertical flow region 140.

[0084] As used herein, when a layer is referred to as being ”on” or “disposed on” another layer, it can be directly disposed on the layer, or an intervening layer also may be present. A layer that is “directly on” or “directly disposed on” another layer is positioned with respect to the layer such that no intervening layer is present.

[0085] As used herein, when a first element is positioned “between” a second element and a third element, the second and third elements are positioned on opposite sides of the first element and first and second geometric lines can be drawn as follows: a first geometric line can be drawn perpendicular to a surface on the second element most proximate to the first element and a second geometric line can be drawn perpendicular to a surface on the third element most proximate to the first element such the first element intersects both the first line and the second line. In some embodiments, the first geometric line also intersects the surface on the third element most proximate to the first element and / or the second geometric line also intersects the surface on the second element most proximate to the first element.

[0086] When a first element is positioned “between” a second element and a third element, at least some portion of the first element must be positioned between the second and third elements, but it is also possible for the first element to further comprise one or more portions that are not positioned between the second element and the third element. That is, for the first element to be “between” the second element and the third element, all portions of the first element do not need to be positioned between the second and third elements. In some cases, the first element can be positioned “between” the second and third elements despite the first element extending laterally beyond the surfaces of the second and / or third elements in one or more directions. For instance, with reference to FIG. 1A, channel 115 is between electrodes 120A and 120B even though it extends laterally beyond both of their surfaces.

[0087] #14670283vl In certain cases, the first element is positioned “between” the second and third elements despite the first element not extending fully across the surfaces of the second and / or third elements in one or more directions. For instance, and with reference to FIG. IB, electrode 120B is between channels 115 and 150 despite not extending fully across the surfaces of these channels.

[0088] As described above, the distance between the surfaces of any two electrodes of the fluid device, in accordance with certain embodiments, is relatively low. For example, as shown in FIG. 1C, distance DI between the surfaces of first electrode 120A and second electrode 120B and / or distance D2 between second electrode 120B and third electrode 120C may be relatively small. In some embodiments, the distances DI and / or D2 may be at least partially controlled by varying the thickness of one or more layers of the fluidic device. For example, turning again to FIG. 1C, fluidic device 100 comprises first layer 110 and second layer 130, wherein first layer 110 has thickness T1 and second layer 130 has thickness T2. Thickness T1 may at least partially control distance DI while thickness T2 may at least partially control distance D2. The distance between electrodes may be further controlled by the extent of which any one of the electrodes extends into one or more layers of the fluidic device. For example, as shown in FIG. 1C, first electrode 120A extends through at least a portion of thickness T1 of first layer 110 while second electrode 120B extends through at least a portion of thickness T2 of second layer 130. In some embodiments, two or more electrodes can extend into the same layer of the fluidic device. For example, as shown in FIG. 1C, first electrode 120 A and second electrode 120B extends into a portion of first layer 110. As another example, second electrode 120B and third electrode 120C extend into a portion of second layer 130. In some embodiments, one or more electrodes can extend into one or more layers of the fluidic device. As an example, as shown in FIG. 1C, second electrode 120B extends through a portion of first layer 110 and second layer 130. In some embodiments, one or more electrodes do not extend into any layers of the fluidic device. For example, in FIG. ID, first electrode 120A and second electrode 120B do not extend into first layer 110.

[0089] A top-view schematic diagram of fluidic devices shown in FIGS. 1A-1D, in accordance with certain embodiments, is shown in FIG. 2. Fluidic device 100 comprises first layer 110 comprising inlet 105 in fluidic communication with first channel 115. First channel 115 has width Wl. First electrode 120A is positioned such that first channel 115 is between first electrode 120 A and second electrode 120B (not shown in FIG. 2). When a liquid is flowed through fluidic device 100, at least a portion of the liquid may flow along first channel 115 taking up at least a portion of width Wl.

[0090] #14670283vl In some embodiments, a fluidic device comprises three or more layers. As an example, as shown in FIG. 3A, fluidic device 100 comprises first layer 110, second layer 130, and third layer 305 such that first layer 110 is disposed on second layer 130 and second layer 130 is disposed on third layer 305. Two or more electrodes may be positioned therebetween. For example, in FIG. 3A, second electrode 120B is positioned between first layer 110 and second layer 130, while third electrode 120C is positioned between second layer 130 and third layer 305.

[0091] In some embodiments, the electrodes of the fluidic device may be arranged such that accessibility to one or more lead portions of each electrode may be promoted. For example, as shown in FIG. 3B, first electrode 120 A is in electrical communication with and / or in contact with first lead portion 310A disposed on first layer 110 such that first electrode 120A is separated from another electrode (e.g., second electrode 120B and / or third electrode 120C) by at least first layer 110. Second electrode 120B is in electrical communication with and / or in contact with second lead portion 310B disposed between first layer 110 and second layer 130 such that at least first layer 110 and / or second layer 130 separates second electrode 120B from another electrode (e.g., first electrode 120A and / or third electrode 120C). Third electrode 120C is in electrical communication with and / or in contact with 310C disposed on second layer 130 such that third electrode 120C is separated from another electrode (e.g., first electrode 120A and / or second electrode 120B) by at least second layer 130. .In some embodiments, the lead portions may provide an electrically conductive path to one or more electrodes of the fluidic device. For example, as shown in FIG. 3B, first lead portion 310A provides an electrically conductive path to first electrode 120A. In some embodiments, the lead portions can be electrically isolated from each other such that the lead portions and / or the electrodes are not electrical communication with each other. Lead portions 310A-310C may be arranged such they are electrically isolated from each other. Second lead portion 310B may be disposed on a portion of the side of second layer 130 adjacent to first layer 110 (see FIG. 1) wherein the portion of second layer 130 is not covered by first layer 110. Third lead portion 310C may be disposed on a portion of the side of third layer 305 adjacent to second layer 130 (see FIG. 1) wherein the portion of third layer 305 is not covered by second layer 130.

[0092] Methods related to fluidic devices are generally described. In some embodiments, the method comprises flowing a liquid through one or more layers of the fluidic device. For example, as shown in FIG. 1C, a liquid may be provided to inlet 105 such that at least a portion of the liquid flows through first layer 110 along first channel 115. Since first channel 115 is positioned between first electrode 120A and second electrode 120B, at least a portion of the liquid, when present, may flow between first electrode 120 A and second electrode 120B before

[0093] #14670283vl flowing to second layer 130 via first vertical flow region 135 in fluidic communication with second vertical flow region 140. At least a portion of the liquid then may flow between second electrode 120B and third electrode 120C through second layer 130 and along second channel 150.

[0094] In some embodiments, the method may be suitable for the detection of a target analyte in the liquid. For example, in FIG. 1C, target analytes in the liquid may interact with first electrode 120A, second electrode 120B, and / or third electrode 120C such that the presence of the target analyte, as described elsewhere in this disclosure, may be detected. The presence of the target analyte may be detected by variations in the measured current and / or voltage between any of first electrode 120A, second electrode 120B, and third electrode 120C when an electrical signal is applied across any two electrodes 120A-120C. In some embodiments, the current between any two electrodes of the fluidic device can be altered when in the presence of the target analyte. In some embodiments, an electrical potential can be applied across any two electrodes of the fluidic device to facilitate the detection of the target analyte.

[0095] In some embodiments, a fluidic device comprises electrodes. In some embodiments, the fluidic device comprises a first electrode, a second electrode, and a third electrode. When the liquid is flowed through the fluidic device, the electrodes may be in electrical communication with each other such that, the current between any of the electrodes in the fluidic device may be altered. In some embodiments, an electrical potential can be applied across any two electrodes of the fluidic device. In some embodiments, any of the electrodes of the fluidic device (e.g., the first electrode, the second electrode, or the third electrode) can serve as a working electrode, a counter electrode, and / or a reference electrode. In some embodiments, any of the electrodes of the fluidic device can switch between serving as a working electrode, a counter electrode, or a reference electrode without altering the overall structure of the fluidic device. As an example, as shown in FIG. 1, first electrode 120 A may serve as a working electrode, a reference electrode, or a counter electrode while maintaining its position relative to second electrode 120B and third electrode 120C. Similarly, second electrode 120B may serve as a working electrode, a reference electrode, or a counter electrode while maintaining its position relative to first electrode 120A and third electrode 120C. Third electrode 120C may serve as a working electrode, a reference electrode, or a counter electrode while maintaining its position relative to first electrode 120A and second electrode 120B. Accordingly, in some embodiments, the fluidic device may be advantageously used for a variety applications (e.g., detecting any of a variety of target analytes) that may necessitate one or more electrode configurations (e.g., the first electrode may be the

[0096] #14670283vl working electrode for a first application and the counter electrode for a second application) without having to undergo any structural modifications.

[0097] Electrodes of the fluidic device (e.g., the first electrode, the second electrode, and / or the third electrode) may comprise any of a variety of suitable materials. In some embodiments, one or more of the electrodes of the fluidic device comprises a conductive material. In some embodiments, the conductive material comprises a metal (e.g., gold, silver, copper). In some embodiments, the conductive material comprises carbon (e.g., graphite, graphene, carbon nanotubes). In some embodiments, the conductive material comprises AgCl, HgSCE, CuSCE, and / or Hg2Ch.

[0098] In some embodiments, one or more electrodes of fluidic devices described herein is disposed on a substrate (e.g., a plastic film). In some embodiments, the conductive material, as described elsewhere in this disclosure, may be applied to the substrate to form the electrode. Such substrates may be positioned between layers in which channels are positioned. Accordingly, the one or more electrodes described herein (e.g., the first electrode, the second electrode, and / or the third electrode) may be disposed on and / or positioned between one or more layers of the fluidic device (e.g., one or more layers comprising channels). Without wishing to be bound by any particular theory, by applying the conductive material to the substrate to form the electrode, the distance between the surfaces of two electrodes may be advantageously controlled by selecting the thickness of each layer and / or the substrate and may not be altered by the electrode extending through a portion of any one of the layers (e.g., via absorption).

[0099] In some embodiments, a first electrode extends through a portion of the total thickness of the first layer. As described above, in FIG. 1C, first electrode 120A extends through a portion of thickness T1 of first layer 110. In some embodiments, the first layer comprises an upper surface that the first electrode may be disposed onto such that the first electrode extends through at least a portion of the first layer towards a lower surface of the first layer. In some embodiments, the first electrode extends through greater than or equal to 5%, greater than or equal to 10%, greater than or equal to 20%, greater than or equal to 30%, greater than or equal to 40%, greater than or equal to 50%, greater than or equal to 60%, greater than or equal to 70%, greater than or equal to 80%, greater than or equal to 90%, or greater than or equal to 95% of the total thickness of the first layer. In some embodiments, the first electrode extends through less than or equal to 95%, less than or equal to 90%, less than or equal to 80%, less than or equal to 70%, less than or equal to 60%, less than or equal to 50%, less than or equal to 40%, less than or equal to 30%, less than or equal to 20%, less than or equal to 10%, or less than or equal to 5% of the total thickness of the first layer. Combinations of these ranges are possible (e.g., greater than or equal to 5% and

[0100] #14670283vl less than or equal to 95%). Other ranges are also possible. In some embodiments, the first electrode does not extend through any portion of the first layer.

[0101] In some embodiments, a distance between a first electrode and a second electrode can be relatively low. In some embodiments, the distance between the first electrode and the second electrode is less than or equal to 1 millimeter, less than or equal to 750 micrometers, less than or equal to 500 micrometers, less than or equal to 200 micrometers, less than or equal to 100 micrometers, or less than or equal to 75 micrometers. In some embodiments, the distance between the first electrode and the second electrode is greater than or equal to 50 micrometers, greater than or equal to 75 micrometers, greater than or equal to 100 micrometers, greater than or equal to 200 micrometers, greater than or equal to 500 micrometers, or greater than or equal to 750 micrometers. Combinations of these ranges are also possible (e.g., less than or equal to 1 millimeter and greater than or equal to 50 micrometers). Other ranges are also possible.

[0102] In some embodiments, a second electrode extends through a portion of the total thickness of the first layer. As described above, in FIG. 1C, second electrode 120B extends through a portion of thickness T1 of first layer 110. In some embodiments, the first layer comprises a lower surface that the second electrode may be disposed onto such that the second electrode extends through at least a portion of the first layer towards an upper surface of the first layer. In some embodiments, the second electrode extends through greater than or equal to 5%, greater than or equal to 10%, greater than or equal to 20%, greater than or equal to 30%, greater than or equal to 40%, greater than or equal to 50%, greater than or equal to 60%, greater than or equal to 70%, greater than or equal to 80%, greater than or equal to 90%, or greater than or equal to 95% of the total thickness of the first layer. In some embodiments, the second electrode extends through less than or equal to 95%, less than or equal to 90%, less than or equal to 80%, less than or equal to 70%, less than or equal to 60%, less than or equal to 50%, less than or equal to 40%, less than or equal to 30%, less than or equal to 20%, less than or equal to 10%, or less than or equal to 5% of the total thickness of the first layer. Combinations of these ranges are possible (e.g., greater than or equal to 5% and less than or equal to 95%). Other ranges are also possible. In some embodiments, the second electrode does not extend through any portion of the first layer.

[0103] In some embodiments, a second electrode extends through a portion of the total thickness of the second layer. As described above, in FIG. 1C, second electrode 120B extends through a portion of thickness T2 of second layer 130. In some embodiments, the second layer comprises an upper surface that the second electrode may be disposed onto such that the second electrode extends through at least a portion of the second layer towards a lower surface of the second

[0104] #14670283vl layer. In some embodiments, the second electrode extends through greater than or equal to 5%, greater than or equal to 10%, greater than or equal to 20%, greater than or equal to 30%, greater than or equal to 40%, greater than or equal to 50%, greater than or equal to 60%, greater than or equal to 70%, greater than or equal to 80%, greater than or equal to 90%, or greater than or equal to 95% of the total thickness of the second layer. In some embodiments, the second electrode extends through less than or equal to 95%, less than or equal to 90%, less than or equal to 80%, less than or equal to 70%, less than or equal to 60%, less than or equal to 50%, less than or equal to 40%, less than or equal to 30%, less than or equal to 20%, less than or equal to 10%, or less than or equal to 5% of the total thickness of the second layer. Combinations of these ranges are possible (e.g., greater than or equal to 5% and less than or equal to 95%). Other ranges are also possible. In some embodiments, the second electrode does not extend through any portion of the second layer.

[0105] In some embodiments, the second electrode is positioned at least partially between the first and the third electrodes. That is, the second electrode may not fully extend laterally across the surfaces of the first and / or third electrode. In other words, the surfaces of the electrodes that are substantially parallel to the one or more layers of the fluidic device can each define a geometric area that, while on different planes, do not fully overlap.

[0106] In some embodiments, a third electrode extends through a portion of the total thickness of a second layer. As described above, in FIG. 1C, third electrode 120C extends through a portion of thickness T2 of second layer 130. In some embodiments, the second layer comprises an lower surface that the third electrode may be disposed onto such that the third electrode extends through at least a portion of the second layer towards a upper surface of the second layer. In some embodiments, the third electrode extends through greater than or equal to 5%, greater than or equal to 10%, greater than or equal to 20%, greater than or equal to 30%, greater than or equal to 40%, greater than or equal to 50%, greater than or equal to 60%, greater than or equal to 70%, greater than or equal to 80%, greater than or equal to 90%, or greater than or equal to 95% of the total thickness of the second layer. In some embodiments, the third electrode extends through less than or equal to 95%, less than or equal to 90%, less than or equal to 80%, less than or equal to 70%, less than or equal to 60%, less than or equal to 50%, less than or equal to 40%, less than or equal to 30%, less than or equal to 20%, less than or equal to 10%, or less than or equal to 5% of the total thickness of the second layer. Combinations of these ranges are possible (e.g., greater than or equal to 5% and less than or equal to 95%). Other ranges are also possible. In some embodiments, the third electrode does not extend through any portion of the second layer.

[0107] #14670283vl In some embodiments, the distance between a second electrode and a third electrode can be relatively low. In some embodiments, the distance between the second electrode and the third electrode is less than or equal to 1 millimeter, less than or equal to 750 micrometers, less than or equal to 500 micrometers, less than or equal to 200 micrometers, less than or equal to 100 micrometers, or less than or equal to 75 micrometers. In some embodiments, the distance between the second electrode and the third electrode is greater than or equal to 50 micrometers, greater than or equal to 75 micrometers, greater than or equal to 100 micrometers, greater than or equal to 200 micrometers, greater than or equal to 500 micrometers, or greater than or equal to 750 micrometers. Combinations of these ranges are also possible (e.g., less than or equal to 1 millimeter and greater than or equal to 50 micrometers). Other ranges are also possible..

[0108] In some embodiments, electrodes of a fluidic device comprises one or more lead portions. In some embodiments, the first electrode is in electrical communication with and / or in contact with the first lead portion. In some embodiments, the second electrode is in electrical communication with and / or in contact with the second lead portion. In some embodiments, the third electrode is in electrical communication with and / or in contact with the third lead portion. As described above, in FIG. 3B, first electrode 120 A is in electrical communication with and / or in contact with first lead portion 310A, second electrode 120B is in electrical communication with and / or in contact with second lead portion 310B, and third electrode 120C is in electrical communication with and / or in contact with third lead portion 310C. In some embodiments, the one or more lead portions serve facilitate electrical communication with one or more electrodes.

[0109] In some embodiments, the lead portions (e.g., the first lead portion, the second lead portion, and / or the third lead portion) are electrically isolated from each other through the device. Electrical isolation of each lead portion may prevent short-circuiting and / or crosstalk between each of the electrodes (e.g., the first electrode, the second electrode, and the third electrode). As each lead portion is separated by one of the layers of the fluidic device, in accordance with certain embodiments, the lead portions may not be in electrical communication with each other. However, when a liquid is present and is flowed through the fluidic device, in some embodiments, the electrodes (e.g., the first electrode, the second electrode, and / or the third electrode) can be in electrical communication with each other (e.g., through the liquid), and accordingly, the lead portions of the electrodes may no longer be electrically isolated. In some instances, despite the presence of a liquid in the fluidic device, the lead portions of the electrodes can be electrically isolated from each other.

[0110] In some embodiments, lead portions of a one or more electrodes of fluidic devices described herein can be arranged to promote accessibility such that electrical communication can

[0111] #14670283vl be established from a point external to the fluidic device to the electrodes of the fluidic device. For example, as shown in FIG. 3B, first lead portion 310A, second lead portion 31 OB, and third lead portion 3 IOC are arranged such that each lead may be accessed individually without interacting with other lead portions. In some embodiments, the first lead portion is disposed on the first layer, the second lead portion is disposed on the portion of the side of the second layer adjacent the first layer (e.g., adjacent an opposite side of the side of the first layer on which the first lead portion is disposed) that is not covered by the first layer, and the third lead portion is disposed on the portion of the side of the third layer adjacent the second layer (e.g., adjacent an opposite side of the side of the second layer on which the second lead portion is disposed) that is not covered by the second layer.

[0112] In some embodiments, a surface on one or more electrodes of the fluidic device may be treated with any of a variety of compounds. In some embodiments, compounds may be deposited onto the surface of one or more electrodes to facilitate in the detection of particular target analytes. For example, an enzyme may be deposited (e.g., glucose oxidase) on a surface of one or more electrodes (e.g., the working electrode) to facilitate in the detection of glucose.

[0113] As described above, fluidic devices described herein may comprise one or more layers. In some embodiments, the fluidic device comprises a first layer. In some embodiments, the fluidic device comprises a second layer. In some embodiments, the first layer is disposed on the second layer. As shown in FIG. IB, fluidic device 100 comprises first layer 110 disposed on second layer 130. In some embodiments, the first layer and the second layer are coupled together. In some embodiments, the first layer and the second layer are adhered (e.g., by the presence of an adhesive) together. In some embodiments, the first layer and the second layer are separated by one or more intervening layers (e.g., an adhesive layer, and / or a layer comprising one or more electrodes). In some embodiments, a portion of a side of the second layer adjacent the first layer is not covered by the first layer.

[0114] In some embodiments, one or more layers of a fluidic device described herein have any of a variety of suitable thicknesses. In some embodiments, the one or more layers of the fluidic device have a thickness of less than or equal to 1 millimeter, less than or equal to 750 micrometers, less than or equal to 500 micrometers, less than or equal to 200 micrometers, less than or equal to 100 micrometers, or less than or equal to 75 micrometers. In some embodiments, the one or more layers of the fluidic device have a thickness of greater than or equal to 50 micrometers, greater than or equal to 75 micrometers, greater than or equal to 100 micrometers, greater than or equal to 200 micrometers, greater than or equal to 500 micrometers, or greater than or equal to 750 micrometers. Combinations of these ranges are also possible (e.g.,

[0115] #14670283vl less than or equal to 1 millimeter and greater than or equal to 50 micrometers). Other ranges are also possible.

[0116] In some embodiments, fluidic devices described herein comprise a third layer. In some embodiments, the second layer is disposed on the third layer. For example, as shown in FIG. 3A, fluidic device 100 comprises first layer 110, second layer 130, and third layer 305 such that first layer 110 is disposed on second layer 130 while second layer 130 is disposed on third layer 305. In some embodiments, a portion of a side of the third layer adjacent the second layer is not covered by the second layer.

[0117] As described above, one or more layers of fluidic devices describe herein comprise one or more channels. In some embodiments, a channel may fluidically connect portions of an fluidic device. For instance, a channel may connect the inlet to one or more vertical flow regions. Thus, in some embodiments, an article is configured such that fluid may be transmitted through the channel. For example, in some embodiments the first layer comprising the porous, absorbent material is configured to transport a liquid through the fluidic device via the first channel.

[0118] In some embodiments, one or more channels of the fluidic device may be positioned between one or more electrodes of the fluidic device. For example, as shown in FIG. 1C, first channel 115 is positioned between first electrode 120A and second electrode 120B while second channel 150 is positioned between second electrode 120B and third electrode 120C. Accordingly, as the first channel is in fluidic communication with the second channel, when a liquid is introduced to the first channel via the inlet, at least a portion of the liquid may flow along the first channel, between the first electrode and the second electrode, before entering the second channel and flowing between the second electrode and the third electrode. As an example, turning again to FIG. 1C, when a liquid is introduced to first channel 115 via inlet 105, at least a portion of the liquid may flow along first channel 115, between first electrode 120 A and second electrode 120B, before entering second channel 150 and flowing between second electrode 120B and third electrode 120C. In some embodiments, the first vertical flow region and the second vertical flow region are in physical contact. The first vertical flow region in physical contact with the second vertical flow region may allow for at least a portion of the liquid, when present, to be transported from the first channel to the second channel.

[0119] In some embodiments, one or more channels (e.g., the first channel and / or the second channel) of the fluidic device have any of a variety of suitable thicknesses or heights. In some embodiments, the one or more channels of the fluidic device (e.g., the first channel and / or the second channel) have a thickness or height of greater than or equal to 50 microns, greater than or

[0120] #14670283vl equal to 75 microns, greater than or equal to 100 microns, greater than or equal to 125 microns, greater than or equal to 150 microns, greater than or equal to 200 microns, greater than or equal to 250 microns, greater than or equal to 300 microns, greater than or equal to 400 microns, greater than or equal to 500 microns, or greater than or equal to 750 microns. In some embodiments, the one or more channels of the fluidic device (e.g., the first channel and / or the second channel) have a thickness or height of less than or equal to 1 millimeter, less than or equal to 750 microns, less than or equal to 500 microns, less than or equal to 400 microns, less than or equal to 300 microns, less than or equal to 250 microns, less than or equal to 200 microns, less than or equal to 150 microns, less than or equal to 125 microns, or less than or equal to 100 microns. Combinations of the above-referenced ranges are also possible (e.g., greater than or equal to 50 microns and less than or equal to 1 millimeter, greater than or equal to 50 microns and less than or equal to 500 microns, or greater than or equal to 50 microns and less than or equal to 100 microns). Other ranges are also possible.

[0121] In some embodiments, one or more channels (e.g., the first channel and / or the second channel) of the fluidic device have any of a variety of suitable widths. In some embodiments, the one or more channels (e.g., the first channel and / or the second channel) of the fluidic device have a width of greater than or equal to 0.2 cm, greater than or equal to 0.5 cm, greater than or equal to 1 cm, greater than or equal to 1.5 cm, greater than or equal to 2 cm, or greater. In some embodiments, the one or more channels (e.g., the first channel and / or the second channel) of the fluidic device have a width of less than or equal to 10 cm, less than or equal to 7.5 cm, less than or equal to 5 cm, less than or equal to 3 cm, less than or equal to 2 cm, or less than or equal to 1.5 cm, or less. Combinations of the above-referenced ranges are also possible (e.g., greater than or equal to 0.2 cm and less than or equal to 10 cm, greater than or equal to 0.2 cm and less than or equal to 5 cm, greater than or equal to 0.5 cm and less than or equal to 10 cm, or greater than or equal to 1.5 cm and less than or equal to 3 cm). Other ranges are also possible.

[0122] In some embodiments, one or more channels (e.g., the first channel and / or the second channel) of the fluidic device have any of a variety of suitable lengths. In some embodiments, the one or more channels of the fluidic device have a length of greater than or equal to 4 millimeters, greater than or equal to 5 millimeters, greater than or equal to 7.5 millimeters, greater than or equal to 10 millimeters, greater than or equal to 15 millimeters, greater than or equal to 20 millimeters, greater than or equal to 25 millimeters, greater than or equal to 30 millimeters, or greater than or equal to 35 millimeters. In some embodiments, the one or more channels of the fluidic device have a length of less than or equal to 40 millimeters, less than or equal to 35 millimeters, less than or equal to 30 millimeters, less than or equal to 25 millimeters,

[0123] #14670283vl less than or equal to 20 millimeters, less than or equal to 15 millimeters, less than or equal to 10 millimeters, less than or equal to 7.5 millimeters, or less than or equal to 5 millimeters. Combinations of these ranges are also possible (e.g., greater than or equal to 4 millimeters and less than or equal to 40 millimeters). Other ranges are also possible.

[0124] In some embodiments, one or more channels (e.g., the first channel and / or the second channel) of the fluidic device have any of a variety of suitable aspect ratios (i.e., ratios of the channel length to the channel width). In some embodiments, the one or more channels (e.g., the first channel and / or the second channel) of the fluidic device have an aspect ratio of greater than or equal to 0.5, greater than or equal to 1, greater than or equal to 1.2, greater than or equal to 1.5, greater than or equal to 2, greater than or equal to 3, or greater. In some embodiments, the one or more channels (e.g., the first channel and / or the second channel) of the fluidic device have an aspect ratio of less than or equal to 5, less than or equal to 3, less than or equal to 2, less than or equal to 1.5, less than or equal to 1.2, or less. Combinations of the above-referenced ranges are also possible (e.g., greater than or equal to 0.5 and less than or equal to 5). Other ranges are also possible.

[0125] In some embodiments, one or more channels (e.g., the first channel and / or the second channel) of the fluidic device have any of a variety of suitable volumes. In some embodiments, one or more channels (e.g., the first channel and / or the second channel) of the fluidic device have a volume greater than or equal to 1 microliter, greater than or equal to 2 microliters, greater than or equal to 5 microliters, greater than or equal to 10 microliters, greater than or equal to 15 microliters, greater than or equal to 20 microliters, greater than or equal to 30 microliters, greater than or equal to 40 micro liters, greater than or equal to 50 microliters, greater than or equal to 75 microliters, greater than or equal to 100 microliters, greater than or equal to 150 microliters, greater than or equal to 200 microliters, greater than or equal to 300 microliters, greater than or equal to 400 microliters, greater than or equal to 500 microliters, or greater than or equal to 750 microliters. In some embodiments, one or more channels (e.g., the first channel and / or the second channel) of the fluidic device have a volume less than or equal to 1 mL, less than or equal to 750 microliters, less than or equal to 500 microliters, less than or equal to 400 microliters, less than or equal to 300 microliters, less than or equal to 200 microliters, less than or equal to 150 microliters, less than or equal to 100 microliters, less than or equal to 75 microliters, less than or equal to 50 microliters, less than or equal to 40 microliters, less than or equal to 30 microliters, less than or equal to 20 microliters, less than or equal to 15 microliters, less than or equal to 10 microliters, less than or equal to 5 microliters, or less than or equal to 2 microliters. Combinations of the above -referenced ranges are also possible (e.g., greater than or

[0126] #14670283vl equal to 1 microliter and less than or equal to 1 mL, greater than or equal to 1 microliter and less than or equal to 50 microliters, or greater than or equal to 100 microliters and less than or equal to 300 microliters). Other ranges are also possible.

[0127] In some embodiments, one or more channels of the fluidic device comprise vertical flow regions. For example, as shown in FIG. 1C, first channel 115 comprises first vertical flow region 135 and second channel 150 comprises second vertical flow region 140. In some embodiments, the first vertical flow region is in fluidic communication with the first channel. In some embodiments, the second vertical flow region is in fluidic communication with the second channel. Accordingly, the first channel may be in fluidic communication with the second channel via the first vertical flow region and the second vertical flow region.

[0128] In some embodiments, one or more layers of a fluidic device described herein comprise a porous material. The porous material may be absorbent, or may not be absorbent. A porous, absorbent material may, upon exposure to a fluid, wick the fluid into the layer and / or wick the fluid through the layer. When layers comprising channels comprise a porous, absorbent material, the porous, absorbent material may wick the fluid into the channels therein and / or through the channels therein. In some embodiments, a fluid may flow into and / or through a porous, absorbent material due to capillarity (capillary action) or by wicking. In some embodiments, a porous, absorbent material will, upon exposure to a fluid (e.g., a fluid sample of interest, a fluid sample for which it is absorbent), transport the fluid into the interior of the porous, absorbent material (i.e., the fluid sample may penetrate into the interior of the material in which the pores are positioned, such as into the interior of fibers making up a porous, absorbent material that comprises fibers). In some embodiments, a porous, absorbent material will, upon exposure to a fluid, experience an increase in mass due to the fluid absorbed therein. It should be understood that some layers comprising porous absorbent materials may have one or more of the properties described above with respect to porous, absorbent materials.

[0129] In some embodiments, a porous, absorbent material is a cellulose-based material. The cellulose-based material may comprise cellulose derived from wood (e.g., it may be a woodbased material), cellulose derived from cotton (e.g., it may be a cotton-based material), and / or nitrocellulose.

[0130] In some embodiments, a porous, absorbent material comprises a synthetic material and / or a glass. Non-limiting examples of suitable synthetic materials include polyester, poly(ethersulfone), nylon, and / or nitrocellulose.

[0131] Porous materials described herein (e.g., porous, absorbent materials described herein) may have a variety of designs. In some embodiments, a fluidic device comprises a porous

[0132] #14670283vl material that is a fibrous material (e.g., a fibrous material comprising fibers formed from a cellulose-based material). The fibrous material may be a non-woven material, or may be a woven material. The fibers may have a variety of suitable diameters and distributions of diameters, and, if woven, may be woven in a variety of suitable weaves. In some embodiments, the non-woven material is a paper, such as a cellulose-based paper. A wide variety of commercially available cellulose-based papers may be employed, such as those manufactured by Whatman, those manufactured by Ahlstrom, and / or those manufactured by Munktell.

[0133] Fibrous materials may comprise fibers having any suitable average fiber diameter. The average fiber diameter of the fibers may be greater than or equal to 0.1 micron, greater than or equal to 0.2 microns, greater than or equal to 0.5 microns, greater than or equal to 1 micron, greater than or equal to 2 microns, greater than or equal to 5 microns, greater than or equal to 10 microns, greater than or equal to 15 microns, greater than or equal to 20 microns, greater than or equal to 25 microns, greater than or equal to 30 microns, greater than or equal to 40 microns, greater than or equal to 50 microns, greater than or equal to 60 microns, or greater than or equal to 70 microns. The average fiber diameter of the fibers may be less than or equal to 75 microns, less than or equal to 70 microns, less than or equal to 60 microns, less than or equal to 50 microns, less than or equal to 40 microns, less than or equal to 30 microns, less than or equal to 25 microns, less than or equal to 20 microns, less than or equal to 15 microns, less than or equal to 10 microns, less than or equal to 5 microns, less than or equal to 2 microns, less than or equal to 1 micron, less than or equal to 0.5 microns, or less than or equal to 0.2 microns. Combinations of the above-referenced ranges are also possible (e.g., greater than or equal to 0.1 micron and less than or equal to 75 microns). Other ranges are also possible. The average fiber diameter may be determined using electron microscopy.

[0134] Porous materials (e.g., porous, absorbent materials) and layers comprising porous materials (e.g., layers comprising porous, absorbent materials) described herein may have a variety of suitable porosities. The porosity of a porous material and / or a layer comprising a porous material may be greater than or equal to 1 vol%, greater than or equal to 2 vol%, greater than or equal to 5 vol%, greater than or equal to 10 vol%, greater than or equal to 20 vol%, greater than or equal to 50 vol%, greater than or equal to 55 vol%, greater than or equal to 60 vol%, greater than or equal to 65 vol%, greater than or equal to 70 vol%, or greater than or equal to 75 vol%, or greater than or equal to 80 vol%. The porosity of a porous material and / or a layer comprising a porous material may be less than or equal to 85 vol%, less than or equal to 80 vol%, less than or equal to 75 vol%, less than or equal to 70 vol%, less than or equal to 65 vol%, less than or equal to 60 vol%, less than or equal to 55 vol%, less than or equal to 50 vol%, less

[0135] #14670283vl than or equal to 20 vol%, less than or equal to 10 vol%, less than or equal to 5 vol%, or less than or equal to 2 vol%. Combinations of the above-referenced ranges are also possible (e.g., greater than or equal to 1 vol% and less than or equal to 85 vol%, greater than or equal to 1 vol% and less than or equal to 80 vol%, or greater than or equal to 50 vol% and less than or equal to 80 vol%). Other ranges are also possible. The porosity of a material or a layer may be determined by mercury intrusion porosimetry.

[0136] Porous materials (e.g., porous, absorbent materials) and layers comprising porous materials (e.g., layers comprising porous, absorbent materials) described herein may comprise pores with a variety of suitable sizes. The average pore size of a porous material and / or a layer comprising a porous material may be greater than or equal to 0.1 micron, greater than or equal to 0.2 microns, greater than or equal to 0.5 microns, greater than or equal to 1 micron, greater than or equal to 2 microns, greater than or equal to 5 microns, greater than or equal to 10 microns, greater than or equal to 15 microns, greater than or equal to 20 microns, greater than or equal to 25 microns, greater than or equal to 30 microns, greater than or equal to 35 microns, greater than or equal to 40 microns, greater than or equal to 50 microns, greater than or equal to 75 microns, greater than or equal to 100 microns, or greater than or equal to 125 microns. The average pore size of a porous material and / or a layer comprising a porous material may be less than or equal to 150 microns, less than or equal to 125 microns, less than or equal to 100 microns, less than or equal to 75 microns, less than or equal to 50 microns, less than or equal to 40 microns, less than or equal to 35 microns, less than or equal to 30 microns, less than or equal to 25 microns, less than or equal to 20 microns, less than or equal to 15 microns, less than or equal to 10 microns, less than or equal to 5 microns, less than or equal to 2 microns, less than or equal to 1 micron, less than or equal to 0.5 microns, or less than or equal to 0.2 microns. Combinations of the above-referenced ranges are also possible (e.g., greater than or equal to 0.1 micron and less than or equal to 150 microns, or greater than or equal to 2 microns and less than or equal to 40 microns). Other ranges are also possible. The average pore size of a porous material or a layer comprising a porous material may be determined by mercury intrusion porosimetry.

[0137] In some embodiments, one or more layers of the fluidic device comprises a polymeric material.

[0138] In some embodiments, one or more layers of the fluidic device comprises a functionalized material. That is, one or more layers of the fluidic device may be functionalized such that one or more properties of the functionalized layer(s) may be altered by the functionalization (e.g., the functionalized layer may have greater hydrophilicity after functionalization than the hydrophilicity prior to functionalization). In some embodiments, one

[0139] #14670283vl or more layers of the fluidic device comprises a physically functionalized material. In some embodiments, the one or more layers of the fluidic device comprises nanoparticles.

[0140] Fluidic devices described herein may comprise an inlet. In some embodiments, the fluidic device comprises an inlet in fluidic communication with the first and / or the second channel. In some embodiments, the inlet is configured to receive at least a portion of the liquid such that at least a portion of the liquid may flow through the inlet such that at least some of the liquid enters the first channel. In some embodiments, a filter is positioned over the inlet such that at least some of the liquid, prior to entering the channel, permeates through the filter such that portions of the liquid not capable of permeating through the filter are substantially prevented from entering the first channel.

[0141] Any of a variety of suitable liquids may be flowed through fluidic devices described herein. In some embodiments, the liquid comprises a liquid from biological origin (e.g., a bodily fluid). In some embodiments, the liquid comprises a bodily fluid. In some embodiments, the bodily fluid comprises blood, tears, saliva, wound exudate, urine, cerebrospinal fluid, and / or sweat. In some embodiments, the liquid comprises a fluid derived from the bodily fluid (e.g., plasma derived from blood). In some embodiments, the liquid comprises an aqueous solution.

[0142] In some embodiments, the liquid comprises a target analyte. In some embodiments, the presence of the target analyte may alter the current measured between any two electrodes of the fluidic device when an electrical potential is applied across the electrodes of the fluidic device. In some embodiments, the target analyte comprises glucose and / or Pb(II).

[0143] In some embodiments, liquids described herein may flow through fluidic devices. In some embodiments, the liquid may be absorbed by one or more layers (e.g., the first layer and / or the second layer) of the fluidic device such that the liquid flows along the channels of the fluidic device. The liquid may flow through the fluidic device via any of a variety of mechanisms. In some embodiments, the liquid may flow via capillarity. That is, the liquid may flow along one or more channels of the fluidic device due to capillary flow of the liquid. In some embodiments, the liquid may flow via an applied pressure (e.g., applied via a pump and / or a syringe). Pressure may be applied to facilitate flow of the liquid through the fluidic device and may allow for liquids having relatively high viscosities to flow through the fluidic device. It should be understood that the liquid may flow through the fluidic device via other mechanisms as well as.

[0144] In some embodiments, after a liquid has flowed through a fluidic device, the fluidic device may facilitate the detection of a target analyte. For example, as shown in FIG. 1C, a liquid, when present, can flow through fluidic device 100 along first channel 115 between first electrode 120A and second electrode 120B. When an electrical potential is applied across any of

[0145] #14670283vl electrodes 120 A, 120B, and / or 120C, the target analyte in the liquid may alter the current between any of electrode 120A, 120B, and / or 120C. In some embodiments, detecting the target analyte comprises applying a voltage across two or more electrodes of the fluidic device (e.g., using a voltage source).

[0146] In some embodiments, fluidic devices described herein may be configured to facilitate any of a variety of electrochemical techniques such that a target analyte may be detected. In some embodiments, the target analyte may be detected via chrono amperometry and / or squarewave anodic stripping voltammetry (SWASV). Other electrochemical techniques may also be used.

[0147] The following examples are intended to illustrate certain embodiments of the present invention, but do not exemplify the full scope of the invention.

[0148] EXAMPLE 1

[0149] In this example, 3D electrochemical paper-based analytical devices (ePAD) were developed. This design of these devices allows for controllable interelectrode distances that are defined by the thickness of the paper used to make the device and the adhesive layers used (ca. 10-500 pm) to adhere the paper layers together. Since each electrode is deposited on a separate layer, the design supports interchangeable electrode configurations. Electrical leads connected to each individual electrode can be simply swapped to change configurations. The design also allows for relatively scalable fabrication process since each electrode is deposited on a separate layer. The 3D ePADs described in this example have relatively low limits of detection (LOD) and wide detection ranges for a variety of compounds.

[0150] Three-dimensional device architecture

[0151] The 3D ePADs comprise three electrodes (a working electrode, a counter electrode, and a reference electrode) that were stencil printed on separate layers of paper (FIG. 4A). The separated electrodes allowed for greater control over electrode positioning such the uncompensated resistance between electrodes was reduced. Additionally, the formal order of each electrode (working / counter / reference vs. counter / working / reference) could be altered. Each electrode comprised stencil printed Ag / AgCl pads such that each cell electrode was capable of connecting to their corresponding electrical lead from the electrochemical workstation (FIG. 5). Each electrode was positioned in the center of a channel that also begins and ends with circular zones. Accordingly, the layers of the device are both duplicates but are also uniquely

[0152] #14670283vl addressable. This generally simplified fabrication while also permitting flexibility in device design. Layers were affixed to each other by sheets of patterned, double- sided adhesives (FIG. 4B). As a result, two electrodes were separated from one another by a distance equal to the combined thickness of the paper and adhesive used in the device. The overall fluidic pathway in the device was a serpentine-shape. That is, the sample, when present, may wet the device inlet, travel down the top layer to the outlet, then travel vertically to the layer below where the sample changes directions. Patterned holes in the adhesive film separating the channels on subsequent layers allow for two electrodes to be wetted by the sample simultaneously (FIG. 4C). This process repeated until the sample reaches the bottom layer of the device. The adhesive films in between layers of paper are included to reduce the likelihood of leaks and / or contamination.

[0153] Stencil printed carbon or Ag / AgCl inks generally do not reside on the surface of the paper layer but rather penetrate a depth into the paper. Such penetration can be difficult to control in a reproducible manner and can result in an obstruction of sample flow. Accordingly, the device was fabricated using two different grades of paper: TFN and Ahlstrom 601. TFN was selected as an example for relatively thick papers (ca. 460 pm), while Ahlstrom 601 (A601; ca. 160 pm) was selected as an example for relatively thin papers.

[0154] For 3D ePADs made from TFN, the flow of an applied sample was not hindered by the printed electrodes, and accordingly, these devices were prepared using only three layers. 3D- ePADs made from A601, however, suffered from impeded sample flow due to the permeation of electrode inks into the paper channels. For devices prepared from thinner papers like A601, a duplicate layer was included: the first was sacrificial and included the electrode while the second functioned as a wicking conduit (e.g., a channel) to direct flow. As a result, the devices made from A601 were prepared using five layers of paper.

[0155] Interchangeability of electrode configurations

[0156] The design of the 3D ePAD allows for the configuration of printed electrodes to be interchangeable such that individual, electrode-patterned layers may adopt different roles in a measurement by repositioning the electrical leads, connected to the potentiostat, to each electrode. That is, when modifications to the working electrode (WE) are not required, 3D ePADs can adopt an arrangement of electrodes that are arranged in two distinct configurations: (i) working / counter / reference (WCR) or (ii) counter / working / reference (CWR). The fabrication, assembly, and / or treatment of the device does not need to be altered to adopt either configuration. The final electrode is the reference electrode (RE) for two reasons. First, it may be desirable for the RE order to not affect the distance between the working electrode and the

[0157] #14670283vl reference (WE-RE distance) electrode when comparing the CWR and WCR configurations. The WE-RE distance is relatively important because it is proportional to the uncompensated resistance of the electrochemical system. As an example, if the RE is positioned in the middle between the working electrode and the counter electrode (CE), the distance between the WE and the RE will be approximately the same as in the CWR configuration. If the RE is positioned above the WE and CE, the order of the electrodes in the WCR and CWR configurations are effectively inverted, depending on WE and CE orders in their respective configurations and assuming that there is no change in the distance between the WE and CE. Second, the RE was not reconfigured as a WE nor CE because the RE has a different composition than the WE and / or the CE. The RE was Ag / AgCl-based while the WE and CE were carbon-based. It is important to note that the limited difference in surface area between the WE and the CE may also facilitate interchangeability of electrode configurations while mitigating any loss of current transfer through the circuit.

[0158] A modification in the electrode configuration of the device may result in a considerable change in the magnitude of the measured electrochemical signal. This variation in signal output may be attributed to the change in distance between device electrodes, which contributes to the overall resistance of the device. These differences may impact the performance or even applicability of a measurement based on the electrode configuration. Accordingly, the device was evaluated using a variety of paper thicknesses and electrode configurations operating using various electrochemical techniques. In this example, the following electrochemical techniques were demonstrated using 3D ePADs: (i) electrochemical activity using cyclic voltammetry against potassium ferricyanide (Ks[Fe(CN)6]), (ii) chronoamperometry to detect glucose, and (iii) square wave anodic stripping voltammetry (SWASV) to detect Pb(II).

[0159] Results and Discussion

[0160] Electrochemical activity using cyclic voltammetry against potassium ferricyanide (Ks[Fe(CN)6]) using the 3D ePAD

[0161] K3[Fe(CN)e] was used as a model for a redox-active compound to evaluate and compare the electrochemical activity of 3D ePADs fabricated using the grades of paper and the electrode configurations described above.

[0162] The anticipated linear relationship between the square root of CV scan rate applied and the resulting current values for all 3D-ePADs was obtained (FIGS. 6A-6B). Surprisingly, the

[0163] #14670283vl measured currents differed substantially with respect to configuration and paper grade as shown in FIG. 7. In the WCR configuration, absolute values of currents were in the microampere range (approximately 20-200 pA). Conversely, absolute values of currents in the CWR configuration were in the milliampere range (approximately 1-15 mA) representing almost a 100-fold difference in magnitude. The difference in magnitude, without wishing to be bound by any particular theory, may be attributed to the electrode arrangement of CWR format in which WE is in the middle vertical position between CE (top) and RE (bottom). This configuration allows for a relatively low distance between the WE and the CE and a relatively low distance between the WE and the RE. However, the WCR format allows for a relatively low distance between the WE and the CE while that the distance between the WE and the RE is doubled as the CE separates the WE from the RE. As the distance between WE and RE increases, uncompensated resistance increases, which can lead to lower current.

[0164] For all scan rates between 100 - 500 mV, the ratio between the anodic and cathodic current was close to unity (ipa / ipc=l). The produced peak shapes revealed a Nernstian electrochemical reaction whose rate is governed by electroactive species diffusion to the WE surface. Also, in the configurations tested in this example, peak-to-peak separation potential (AEp) increased with increasing the CV scan rate. With increasing scan rates, the electrochemical system has a correspondingly amount of time to reach equilibrium at each potential point. This decreased time results in larger separation between the potential values at which oxidation and reduction of the electroactive species happen, or polarization overpotential. CV curves for screen-printed electrodes (SPE) were used as a reference that can be compared to the output obtained by different 3D ePAD formats (FIGS. 8A-8B). 3D ePADs made using both paper types having WCR configurations produced current values that are relatively similar in magnitude to the SPE devices with the respect to the square root of the applied scan rate. Peak- to-peak separation resulted from SPE cyclic voltammograms at different scan rates are also close to that of the WCR configurations. The similarity could indicate, without wishing to be bound by any particular theory, that the SPE devices and the 3D ePAD having the WCR configuration possibly exhibit similar charge transfer kinetics at the surface of their working electrodes. With that, the CWR configuration devices showed larger peak-to-peak separation which could indicate a quasi-reversible electrochemical system influenced by the change in electrode position exhibited by this configuration. However, the current magnitude generated by cyclic voltammograms in SPE devices is approximately two orders of magnitude lower than that of CWR configuration of both paper grades. This current magnitude difference between the two

[0165] #14670283vl types of devices may be attributed to the relatively larger interelectrode distance with which these SPEs were designed.

[0166] Chronoamperometric detection of glucose

[0167] Glucose detection is important for clinical medicine and the food industry, especially for diagnostic applications for those diagnosed with diabetes. Enzymatic paper-based electrochemical sensors are a promising tool for glucose detection at the point-of-care (POC) in which sensitivity, specificity, low cost, and fast response are relevant application factors. To test the ability of the devices to perform enzymatic electrochemical detection, glucose was measured using chronoamperometry technique. Glucose oxidase was deposited around the WE surface. Detection was carried out using both electrode configurations and paper grades described above. For both electrode configurations, the high charging current decays to a steady state Faradaic current within 80 s after applying step potential of 500 mV (FIG. 9). Accordingly, the steady state current was determined as the average current value measured between 80 - 100 s after applying the step potential.

[0168] For both the 3D ePADs tested in this example, the detected analyte concentration was proportional to the Faradaic current detected, as per Cottrell equation. FIGS. 10A-10B shows the calibration plots, EODs, and sensitivities produced by both configurations and paper grades. All proposed 3D ePAD iterations in this example showed linear relationships between glucose concentrations and their corresponding Faradaic current. For E-A601 and TFN paper grades, the CWR electrode configuration generated current values that were two orders of magnitude greater than those produced by the WCR configuration. Without wishing to be bound by any particular theory, this difference in magnitude can be attributed to the minimized uncompensated resistance between WE and RE in the CWR configuration. The distance between the CE (top layer) and the WE (middle layer) is equal to the distance between the WE and the RE (lower layer). According to this difference in signal magnitude, CWR configurations produced an LOD of 0.7 mM for both L-A601 and TFN devices, while WCR configurations produced LODs of 2.5 mM for L-A601 and 3.8 mM for TFN devices. The linear ranges offered by both configurations and paper grades encompass the relevant ranges for normal glucose concentrations in blood. Advantageously, the lower LOD exhibited by CWR configurations may allow for detection of glucose levels in other biological matrices such as tears and / or saliva.

[0169] Anodic stripping voltammetric analysis of Pb( II)

[0170] #14670283vl Lead (Pb(II)) is one of the most potent water pollutant heavy metal ions, posing a serious threat to public health and global ecosystem. Using paper-based electrochemical sensors for Pb(II) detection can be a powerful tool to utilize, especially in resource-limited regions where a fast selective sensor is needed while minimal access to sophisticated lab equipment is available.

[0171] To test the ability of the 3D ePAD to perform a trace-level detection of ionic species, a wide range of Pb(II) concentrations were chosen to be measured using a square- wave anodic stripping voltammetry (SWASV) technique. SWASV was chosen for Pb(II) detection because it generally produces a high signal-to-noise ratio, reducing the background noise resulting from the charging current during the potential scan. Without any modification of the WE surface, Pb(II) detection was carried out using both electrode configurations and paper grades described above.

[0172] The devices exposed to Pb(II) in concentrations ranging from 10 ppb to 1000 ppm. The wide concentration range allowed the correlation between the generated current signal and Pb(II) concentration to fit to a nonlinear model (FIG. 11). For the WCR configuration, both A601 and TFN-based devices showed current signals to Pb(II) concentrations from 10 ppm to 1000 ppm, with an limit of detection (LOD) of 2.0 ppm for A601 and 3.3 ppm for TFN. This configuration showed limited sensitivity against lower Pb(II) concentrations which could be attributed to the relatively large uncompensated resistance exhibited by WCR configuration (FIG. 12).

[0173] For the CWR configuration, devices having paper of both thicknesses showed higher sensitivities towards Pb(II) concentrations. For the same Pb(II) concentration, the CWR TFN ePADs generated approximately one order of magnitude higher current than generated by their corresponding WCR devices. This enhanced sensitivity, in this example, allowed for signals to be generated over a wider Pb(II) concentration range than those of WCR configuration. The CWR TFN ePADs produced a signal for a Pb(II) concentration range from 50 ppb to 1000 ppm, with an LOD of 35.5 ppb. An even higher current magnitude was generated by CWR A601 devices, in this example, as the CWR A601 devices showed around a 50 times increase in the current produced by the corresponding WCR devices. Owing to their higher sensitivity, the signals generated by CWR A601 devices detected Pb(II) when the Pb(II) concentration was in a range from 10 ppb to 1000 ppm, and the respective LOD was 29.0. The detection range exhibited by CWR devices, in this example, covers the permissible limit of Pb(II) concentration in fresh water. This diversity in detection range depending on electrode configuration is indicative of the advantages associated with the interchangeability of configurations offered by this device design.

[0174] Similar SWASV measurements using SPEs as a reference were conducted. Similar to the CWR TFN devices, SPEs showed a Pb(II) detection range from 50 ppb to 1000 ppm, with an

[0175] #14670283vl LOD of 233.0 ppb (FIGS. 13A-13B). The similarity in detection range between commercially available SPEs and the CWR TFN devices suggests the applicability of 3D ePAD devices for practical use. Even though SPEs showed similar current magnitudes compared to those produced by CWR A601 for the same Pb(II) concentrations, the CWR A601 devices showed a lower LOD than that of the SPE devices. Without wishing to be bound by any particular theory, the lower LOD may be attributed to the lower background signal exhibited by CWR A601 potentially resulting from less contamination of the WE as it is positioned as the middle layer in the device.

[0176] Since the CWR L-A601 devices seemed to have the most sensitive device tested in this example, the impact of the interelectrode distances is evident. By reducing the separation between electrodes in this device design, the overall device resistance may decrease leading to a sensitivity that is even higher than that of commercial SPEs.

[0177] EXAMPLE 2

[0178] This example describes design and testing of non-limiting device architectures comprising three electrodes (working, counter, and reference) that were stencil printed on separate layers of paper. FIG. 14A presents an exploded perspective illustration of the device, FIG. 14B presents a cross-sectional schematic diagram of the device, and FIG. 14C presents a top-view photograph of the device, according to some embodiments. In the figures, separate layers are enumerated as layers 1, 2, 3, 4, and 5. The unique device geometry allowed versatile definition of electrode configuration and separation distance between electrode surfaces equal to only paper + adhesive layers thickness. Based on the desired electrode configuration, the 3 electrode pads of the device were connected to their corresponding potentiostat metal clamps. Numbers to the left represent the order of paper-based device layers. Layers 1, 3, and 5 host working electrode (WE), counter electrode (CE), and reference electrode (RE), respectively. Layers 2 and 4 served as flow layers between each two consecutive electrode-hosting layers. Light blue dashed lines indicate fluid flow through and between layers. Upon sample introduction, fluid flowed in a serpentine fashion to create an electrolytic connection among the three-electrode paper-based electrochemical cell. Discontinuation in adhesive layers refers to laser cut holes created to direct sample flow within the device and allow electrolytic connection between electrodes.

[0179] The device was designed to separate the cell electrodes to allow control over electrode positioning to minimize the effect of uncompensated resistance between electrodes and also modify the formal order of each electrode (working / counter / reference vs. counter / working / reference).

[0180] #14670283vl Each electrode was stencil-printed over a hydrophilic rectangle that was patterned by wax printing on each electrode-hosting layer. The middle and lower electrodes, layers 3 and 5, were printed to be wider than their hosting hydrophilic rectangle, as shown in FIG. 15, which presents a schematic of the face and back of each electrode-hosting layer in the 3D ePAD design. The top and middle electrodes were double- sided to help ensure carbon ink penetration across the paper layer thickness. The Ag / AgCl RE, however, was top-sided printed since electrolyte does not pass beneath its hosting layer. The electrode areas were wider in the middle and lowest layers to allow for a separate connection space with the Ag / AgCl electrode pads. The extra electrode width served as a contact area with the electrode pad, which helped to separate the Ag / AgCl pads from the electrolytic pathway in the flow layers.

[0181] Contact between the Ag / AgCl pad and the electrolyte could lead to Ag / AgCl oxidation, which takes the form of a tarnish color to the electrode pad. This uncontrolled electrode pad oxidation could compromise the electrical conductivity of the pad, resulting in less consistent signal output. While the excess width of the middle electrode goes to one direction, right in this device design, it goes the opposite direction for the lower electrode to allow for pad connection to minimize chances of contact between clamps and leave room for more pads when multiplexing. The need for an excess electrode area does not exist for the top electrode since the electrode pad was not in contact with the electrolytic flow. Unlike middle and lower flow pads, no color tarnishing to the silver color of this electrode pad was observed.

[0182] To each printed electrode, stencil printed Ag / AgCl pads were added to connect the cell electrodes to their corresponding metal clamps from the electrochemical workstation. Each electrode was positioned to match the center of the hydrophilic flow channel(s) above and / or underneath. Each electrode-hosting layer includes a circular hydrophilic zone to the right or the left of the electrode to direct the sample flow. In this way, every electrode hosting layer of the device was a mirror image to the subsequent one, yet also uniquely addressable, thus simplifying manufacturing while also permitting flexibility in device design. Upon wicking, the circular hydrophilic zone to the right of the RE indicates the device filling such that all device layers hydrophilic channels / zones have been wetted by the sample added. Device layers were affixed to each other by sheets of patterned, double-sided adhesives. The adhesive patterns were designed to allow electrolytic connection between each two subsequent electrode surfaces. As a result, two electrodes were separated from one another by a distance equal to the combined thickness of the paper and adhesive used.

[0183] The overall fluidic pathway in the device was serpentine: the sample wetted the device inlet, wicked vertically to the first flow layer, layer 2, traveled down the flow layer laterally to

[0184] #14670283vl the outlet, then vertically through the two layers below, layers 3 and 4, where it changed directions. Patterned holes in the adhesive film separating channels on subsequent layers permitted two electrodes to be wetted by the sample simultaneously, allowing electrolytic connection between each two facing electrode surfaces. This process repeated until the sample reached the circular zone of the bottom layer of the device. Adhesive films in between layers of paper ensured that leaks or contamination do not occur.

[0185] Stencil printed carbon or Ag / AgCl inks do not ordinarily reside solely on the surface of the paper layer — they often penetrate a depth into the paper that was challenging to control reproducibly and can result in an obstruction of sample flow. Carbon ink was stencil-printed on both sides of the layers hosting the top and middle electrodes, layers 1 and 3. The double-sided printing allowed for a more direct electrolytic contact with electrodes. Thus, the lower surface of the top electrode and both the upper and lower surfaces of the middle electrodes were in contact with the electrolyte passing through the flow layers.

[0186] FIGS. 16A-16B presents a schematic cross-sectional view of the 3D ePAD layers, constructed using (FIG. 16A) double-sided printed and (FIG. 16B) top-sided printed electrodes. To the left of electrodes, brackets represent the distance between facing surfaces of each two consecutive electrodes. Red brackets refer to the combined distance created by flow layer plus adhesive sheet thicknesses. Yellow brackets, in B, represent the additional thickness added by the lower midsection of the paper when electrode ink was only top-sided printed. To the right of electrodes, brackets represent the distance between the bottom surface and the top surface of the upper and lower electrodes, respectively. For FIG. 16A, this distance components, in blue, include hydrophilic flow layers, adhesive sheets, and conductive carbon ink. For FIG. 16B, this distance was combined with the lower midsection of the paper containing the top-sided printed electrodes, in yellow. Discontinuation in adhesive layers refers to laser cut holes created to allow electrolytic connection between electrodes.

[0187] Carbon electrodes were only printed on one side because printing carbon ink only on the upper surface of the electrode-hosting layer caused interrupted electrolytic contact, since the printed electrode rendered the remaining midsection of the paper more hydrophobic. Thus, if only the upper midsection was stencil printed, the lower midsection would not exhibit full contact with the electrolyte in the flow layer underneath. In addition, the top- sided printing of the electrode increases the interelectrode distance by an amount equal to the lower midsection of the paper layer hosting the single-sided electrode FIG. 18B. This additional lower midsection would have increased the distance between each two consecutive electrodes and, of course, the upper and lower electrodes. When electrodes were double-sided printed, the overall distance

[0188] #14670283vl between the upper and lower electrodes would comprise (i) hydrophilic section (flow layer), (ii) electrolyte-filled adhesive thickness, and (iii) conductive electrode paste-printed paper (electrode-hosting layer). When electrodes were only top-sided printed, however, the overall distance would differ in two ways: (1) by an increased distance between upper and lower electrodes, and (2) by a reduction in overall conductive thickness. Both differences were due to partial conductive ink penetration. An unexplored alternative was printing the lower midsection of the top electrode-hosting layer with ink, but that option could have limited access to the electrode pad. Hence, both sides of the top-layer electrode were printed — although the upper midsection of the top-layer electrode was not in direct contact with the electrolyte path — to allow for easier access between the electrode pad and the corresponding metal clamp connected to the electrochemical workstation. The device was fabricated using two different grades of paper: TFN and Ahlstrom 601. TFN was selected as an example for thick papers (ca. 460 pm), Ahlstrom 601 (A601; ca. 270 pm) was selected as an example for medium thickness papers, while Kimtech Kimwipes (KW; ca. 70 pm) were selected as an example for thin papers.

[0189] The design of the 3D-ePAD allowed the configuration of printed electrodes to be interchangeable by repositioning the metal clamps connecting electrode pads to the potentiostat. Thus, when no WE modification was required, without needing to alter how a device was fabricated, assembled, or treated, 3D-ePADs could adopt an arrangement of electrodes that were ordered (i) working / counter / reference (WCR) or (ii) counter / working / reference (CWR). The final electrode was always the reference because of two reasons. First, changing RE order will not affect WE-RE distance when compared to CWR and WCR configurations. WE-RE distance was important because it was proportional to the uncompensated resistance of the electrochemical system. If the RE were moved to the middle position, layer 3, the WE-RE distance will be the same as in the CWR configuration. If the RE were moved to the top position, layer 1, the order of the electrodes in the WCR and CWR configurations would merely be inverted, depending on WE and CE orders in their respective configurations, without changing the inter-electrode distance. Second, the RE was not reconfigured as a WE nor CE because the RE has different chemical compositions - RE was Ag / AgCl-based while WE and CE were carbon-based.

[0190] This configuration interchangeability was not supported by most of the ePAD designs reported in literature so far where all electrodes were coplanar. Many of these designs mainly focus on minimizing their WE-RE distance more than their WE-CE distance. This was because minimizing WE-RE was associated with lowering the uncompensated resistance between these two electrodes, while the potentiostat automatically compensates for the resistance arising from

[0191] #14670283vl WE-CE distancing. Although these device designs supports sensitive performance, their coplanar electrode configuration limits their versatility. In fact, interchanging electrode configuration between counter electrode and working electrode in conventional coplanar ePAD designs might be impractical because of their relatively large WE-CE distance. While the WE- CE resistance was mitigated by the potentiostat to some extent, aka iR compensation, the associated effects of increased resistance might not be eliminated. For instance, higher resistance in the electrochemical system could still introduce additional noise to the measurement, compromising the sensitivity and precision of the system. In addition, while WE and RE have minimal separation distance in many coplanar ePAD designs, they cannot be interchanged since they have different chemical composition. Commonly, the WE was made of carbon-based ink while RE was made of Ag / AgCl-based ink.

[0192] Additionally, the surface area of CE in conventional ePADs was much higher than that of WE. The reason for this enlarged surface area was to avoid limiting current transfer in the paperbased electrochemical circuit by the CE surface area. However, the considerable variation in electrode surface area poses a challenge to changing the electrode configuration. This was because if the CE were to be used as the WE, or vice versa, the CE surface area would be much smaller than that of WE, which would limit the current transfer in the circuit.

[0193] Controlling flow layer thicknesses and / or modifying electrode configuration in 3D ePADs could result in considerable change in the magnitude of a measured electrochemical current. This variation in signal output was attributed to the change in distancing between device electrodes, which contributes to the overall resistance of the device. These differences may ultimately impact the performance or even applicability of a measurement based on electrode configuration. These variables (i.e., device construction dictated by paper thickness and overall electrode configuration) were evaluated using different electrochemical techniques: (i) electrochemical activity using cyclic voltammetry against potassium ferricyanide (Ks[Fe(CN)6]), (ii) chronoamperometry to detect glucose, and (iii)square wave anodic stripping voltammetry (SWASV) to detect Pb(II).

[0194] The influence of ink penetration on both sample flow and signal output were tested using different devices. FIG. 17 shows cross sectional schematic and images of the ink penetration associated with printing (A) top and (B) both sides of different papers with carbon ink. While images were not to scale, the heights of black rectangles in the schematic illustrations reflect the ink penetration percentage in their corresponding paper types. KW, A601, and TFN represent thin, medium, and thick papers. Although pristine carbon ink was used to stencil print electrodes on KW and TFN papers, 80% ink concentration was used to deposit electrodes on A601 papers

[0195] #14670283vl to ensure deeper ink penetration. Ratios to the right represent the number of times the sample successfully flowed under the electrode in its hosting layer. These numbers were not applicable for double- sided printed electrode since sample flow was completely interrupted and a separate flow layer was introduced.

[0196] The influence of ink penetration was tested using 3D ePADs made of different paper layer thicknesses; namely KW, A601, and TFN. FIGS. 18A-18C illustrate sample flow under electrodes deposited on papers of different thicknesses. FIG. 18A shows images of sample flow after being introduced to paper layers from the circular zone to the right of each electrode, wicking towards the left-hand side. While flow was interrupted when relatively thin papers were used, typically KW and A601, sample flow proceeded underneath TFN thick papers. The initial device design used to evaluate sample flow consisted of 3 electrode hosting layers without flow layers. FIG. 18B schematizes the 3 electrode-hosting-layer device used to evaluate the top-sided printed TFN-based devices. In this design, each electrode was positioned in the center of a hydrophilic channel that also begins and ends with circular zones. These zones were in contact through patterned holes in the adhesive sheets binding layers together to create the final serpentine sample flow. These TFN-based devices were tested using WCR electrode configuration and linear sweep voltammetry (LSV) technique against samples of 1 mM K3[Fe(CN)e] in 0.1 M KC1. Dashed blue lines indicate the lateral and vertical fluid flow across device layers. FIG. 18C presents a table showing the variability in peak current and potential produced by running linear sweep voltammetry (LSV) using the top- sided printed TFN-based devices. For LSV, WCR electrode configuration and 100 pL samples of 10 mM Ks[Fe(CN)6] in 1 M KC1. (N = 4) were used.

[0197] For 3D-ePADs made from TFN, the flow of an applied sample was not hindered by topside printed electrodes. Printed ink penetrated approximately 61.4% [95% CI: 58.3 - 64.5] of the TFN paper thickness, which allowed for sample flow in the lower midsection underneath the printed ink. FIG. 19 shows cross sectional schematic and actual images of the ink penetration associated with printing both faces of A601 papers with carbon ink. Different ink concentrations, diluted with EtOH, of carbon ink produced penetration of variable depths. The ink penetration % represents the combined penetrations from both faces of the paper layer, related to the large variability in ink penetration across the electrode width. Despite the successful sample flow and the relatively simpler design, the current measured from the linear sweep voltammograms showed high variability (CoV = 65% for peak current). The compromised reproducibility of this design could be attributed to the inconsistent interfacial contact between the sample (electrolyte) and the electrode surface. This inconsistency might be

[0198] #14670283vl 3D-ePADs made from A601 and KW, however, suffered from impeded or no sample flow as electrode inks blocked up to 100% of paper thickness. Ink penetration consumes 90.0% [95% CI: 83.2 - 96.7] and 71.3% [95% CI: 60.5 - 82.2] of KW and A601 thicknesses, respectively. LSV measurements using devices made of either of these paper types due were not performed to the interrupted sample flow - no electrolytic connection among electrodes. Alternatively, carbon electrode ink was printed on both sides for devices made of all paper types (TFN, A601, and KW). The double-sided printing aimed to eliminate the ink penetration variability across the paper thickness, providing more consistent electrode access to sample flow. While double-sided printing was utilized only for carbon ink forming top and middle electrodes, stencil printing was continued for Ag / AgCl ink forming RE, lower electrode, on one side of the paper, top side. This was because only one side of the RE surface was needed to ensure consistent contact with electrolyte, since the sample only wicks the flow layer above the RE.

[0199] As double-sided printing was expected to obstruct the flow in the electrode-hosting layer, due to deeper ink penetration, this printing style was tested with the 5-layered device described earlier. In the 5-layered devices, all 3 electrode-hosting and 2 flow layers were made of the same paper type.

[0200] While double sided printing on TFN electrode-hosting layers resulted in 93.2% [95% CI: 86.3 - 100.1] ink penetration, A601 showed less collective ink penetration across the paper thickness using pristine carbon ink, 63.4% [95% CI: 54.5 - 72.3]. As a result, several solvent dilutions were tested and it was and found that 80% carbon ink concentration (wt / wt) provided the deepest ink penetration, 89.4% [95% CI: 81.3 - 97.6]. This ink dilution was used for carbon ink printing over all A601 paper sheets. The Ag / AgCl ink was not diluted for any device, including A601 -based, since it was only printed on one side, top side, of the lowest layer, layer 5. As previously mentioned, top-sided printing of KW resulted in 100% ink penetration. However, to use KW-based devices for the 5 layered device, the electrode was double-sided printed.

[0201] The KW-based devices made of double-sided printed electrodes showed less LSV peak current variance than top- sided printed devices. FIGS. 20A-20B illustrate the results of printing top or both sides of KW paper grade. FIG. 20A shows front and back sides of KW paper layer when hosting only top-sided printed electrodes. Dashed red rectangle highlights the irregularity of the ink filled area, since printed wax does not stay in shape after melting over KW paper layers. FIG. 20B presents a table showing the variability in LSV peak current and potential produced using top-sided and double-sided printed KW-based devices. For LSV, the WCR

[0202] #14670283vl electrode configuration was used and 100 |aL samples of 1 mM K3[Fe(CN)6] in 100 mM KC1. (N = 4).

[0203] The mean LSV peak current varied across devices with respect to the paper thickness forming the layers of each device. For instance, the mean LSV peak current increased with decreasing the paper thickness when KW was used as a flow layer. Although the TFN-based devices showed the lowest current (1.4 (± 92 %) pA) among other paper types, this current output was more than three times higher than that obtained from singlesided printed TFN-based devices prepared from 3-layered devices. This increase in current suggested that double-sided printing could provide closer contact between the electrode surface and the electrode.

[0204] Additionally, it was hypothesized that the current output variability, both the mean values and their associated CoV%, among double-sided ink devices could be related to the thickness of both (i) the electrode-hosting layer, which probably influences the amount of ink deposited in the paper, and (ii) the flow layer, which defines the interelectrode thickness. To test this hypothesis, the LSV measurements were extended by employing devices constructed from a matrix of various paper thickness combinations.

[0205] Due to the versatility that the 3D ePAD design provides, the impact of changing the paper thickness of the electrode-hosting and flow layers on the signal output was studied. In this study, it was examined whether changing the flow layer thickness could be correlated with the interelectrode distance, which was associated with the overall device resistance. The variability in peak current and potential was evaluated by employing a combinatorial matrix of paper thicknesses. In this matrix, each paper type was utilized as the electrode-hosting layer and tested against flow layers of varying thicknesses, and vice versa. For all combinations, WCR and CWR electrode configurations were tested using linear sweep voltammetry against samples of 1 mM K3[Fe(CN)6] in 0.1 M KC1.

[0206] The measured currents differed substantially with respect to configuration and paper grade for all paper thickness combinations. FIGS. 21A-21B present the measured relationship between the thicknesses of electrode-hosting and the flow layers based on the resulting LSV current. For all devices, both WCR (FIG. 21 A) and CWR (FIG. 2 IB) electrode configurations were tested. Devices were fabricated using combinations of electrode-hosting and flow paper layers, using KW (70 um), A601 (270 um), and TFN (460 um) paper grades to represent different thicknesses. Different electrode-hosting layers were represented in red, blue, and green based on their thicknesses. For every combination, the same paper type was used for all electrode-hosting layers, and another consistent type was used for all flow layers. Devices were

[0207] #14670283vl tested with LSV using 100 pL samples of 1 mM Ks[Fe(CN)6] in 100 mM KC1. Error bars represent the standard error of the mean. (N = 4).

[0208] In the WCR configuration, absolute values of currents ranged approximately 0.3-14.0 pA. Conversely, absolute values of currents in the CWR configuration were ranged approximately 2.5-68.2 pA in which many combinations demonstrated higher than one order of magnitude difference in electrochemical current. This result was believed to be attributable to the electrode arrangement of CWR format in which WE was in the middle vertical position between CE (top) and RE (bottom). This electrode arrangement allowed minimal WE-CE and WE-RE distance, unlike WCR format that allowed for only minimal WE-CE distance while that distance was doubled between the WE (top layer) and RE (lowest layer). As distance between WE and RE increased, uncompensated resistance of the overall cell increased, which led to lower current.

[0209] For all paper thickness combinations, all WCR configurations showed a correlation between the current magnitude and the flow layer thickness. For devices with the same electrode-hosting layer, thicker flow layers resulted in lower current response, which was in agreement with the fact that larger interelectrode distances increase the overall device resistance. Furthermore, the thinner electrode-hosting layers exhibited higher current responses. This trend was consistently observed across all KW flow layers and in both WCR and CWR configurations. The increased current density could possibly be attributed to faster redox species diffusion at the electrode surface demonstrated by thinner electrode-hosting paper layer. This trend shown by KW flow layers fails to persist with thicker papers, namely, A601 and TFN, for both WCR and CWR electrode configurations. This deviation from the trend might indicate that the increased resistance resulting from the greater interelectrode distance substantially contributes to the overall system resistance. It could also be related to porosity influence, which was not consistent across different paper types, since change in thickness was not necessarily the only factor affecting the current. Changing porosity, for instance, could impact the amount of electrolyte the hydrophilic area could accommodate, which changes the density of charge carriers, i.e. ions, in the space between electrodes. Collectively, these effects could probably outweigh the enhanced conductivity due to the improved diffusion of redox species at the electrode surface.

[0210] Using KW as electrode-hosting layers resulted in relatively higher CoV % than corresponding A601 -based devices, up to three-fold more variability. This observation might be related to the lower mechanical stability of KW as compared to thicker paper types

[0211] While TFN layers provided the highest mechanical stability for their devices, most devices that incorporated TFN layers demonstrated the lowest reproducibility. This case was

[0212] #14670283vl applicable whether TFN was purposed as an electrode-hosting or a flow layer. Except for all TFN-based CWR devices, all TFN electrode-hosting devices showed CoV % that ranged between 35 % to 61 %. While the increased interelectrode distance may partially explain the observed TFN behavior, further investigation was required to fully understand the factors contributing to this electrochemical response in TFN-containing matrices. Inversely, electrodehosting A601 layers showed the lowest CoV % that ranged from 8 % to 14 % except when TFN paper was used as flow layers.

[0213] As a result, the electrochemical characterization and utility of devices design using CWR configuration were tested for of all A601 -based 3D ePADs.

[0214] To serve as a model for the device design, the CWR electrode configuration of the device was selected such that all electrode-hosting and flow layers were made of A601. Although all KW devices showed higher current response, all A601 devices were for manufacturability and repeatability reasons.

[0215] Ks[Fe(CN)6] was used as a model for a redox-active compound to evaluate the electrochemical activity of the 3D-ePAD. By adding 75 pF samples of 1 mM Ks[Fe(CN)6] in 0.1 M KC1, cyclic voltammetry measurements were performed using scan rates of 100 - 500 mV / s. Over the range of scan rates, the anticipated linear relationship between the square root of CV scan rate applied and the resulting current values for the CWR all-A601 3D-ePADs were obtained. FIGS. 22A-22B show non-limiting cyclic voltammetry using all A601-based 3D ePAD with CWR electrode configuration. FIG. 22A shows cyclic voltammograms produced by introducing samples of 100 pL of 1 mM Ks[Fe(CN)6] in 100 mM KC1 and performed CV experiments using scan rates of 100 - 500 mV / s. FIG. 22B shows linear relationships between square root of scan rates and their measured anodic (blue squares) and cathodic (red circles) peak currents. Error bars represent the standard error of the mean. (N = 4).

[0216] This correlation confirms the consistent electrochemical behavior of the 3D ePAD design. As described by the Randles- Sevcik equation, the obtained linearity suggests that the device operates within a diffusion-controlled regime, which was important for its intended future analytical applications at the POC. This diffusion-controlled regime represents the mass transfer of potassium ions in the electrode plating phase of the electrochemical reaction. This predictable electrochemical response of the 3D ePAD also suggested its suitability for quantitative analysis.

[0217] For all scan rates between 100 - 500 mV, the ratio between the anodic and cathodic current was close to unity (ipa / ipc~ 1) (Table 1). The produced peak shapes revealed a Nernstian electrochemical reaction whose rate was governed by electroactive species diffusion to the WE surface.

[0218] #14670283vl Table 1. Peak current analysis of CV data generated by all A601 -based 3D ePAD using a CWR electrode configuration

[0219] Scan rate (mV / s) 100 200 300 400 500 ipa / ipc1.03 0.90 0.92 0.95 0.99

[0220] Expectedly, the peak-to-peak separation potential (AEp) increased with increasing the CV scan rate (Table 2). As scan rates increased, the electrochemical system was allowed less time to reach equilibrium at each potential point. This decreased time resulted in larger separation between the potential values at which oxidation and reduction of the electroactive species happen, aka polarization overpotential.

[0221] Table 2. Peak-to-peak separation analysis of CV data generated by all A601 -based 3D ePAD using a CWR electrode configuration

[0222] Scan rate (mV / s) 100 200 300 400 500

[0223] AEp (V) 0.91 0.95 0.99 1.04 1.07

[0224] Theoretically, at 298 K, this polarization overpotential was 59 mV per n, where n was the number of electrons involved in the charge transfer process. So, for the redox-active couple Fe(CN)63“ / Fe(CN)64“, where the number of transferred electrons n = 1, the expected overpotential value was 59 mV. In the 3D ePAD, the increased polarization overpotential could be attributed to the paper substrate in which adsorption profile and capillary action could lead to uneven distribution of redox active species at the electrode surface, suggesting a quasi-reversible electrochemical system.

[0225] As a frame of reference, CV for SPE was also recorded, allowing comparison of the peak potential values obtained by the 3D ePAD CWR electrode configuration. FIGS. 23A-23B show non-limiting cyclic voltammetry using SPEs. FIG. 23A Cyclic voltammograms produced by SPE devices. The sample was 10 pL of 10 mM Ks[Fe(CN)6] in IM KC1 and performed CV experiments using scan rates of 100 - 500 mV / s. FIG. 23B Linear relationships between square root of scan rates and their measured anodic (blue squares) and cathodic (red circles) peak currents. Error bars represent the standard error of the mean. (N = 4).

[0226] #14670283vl The SPE peak-to-peak separation potentials resulted from cyclic voltammograms at different scan rates were closer than that of the proposed 3D ePAD, indicating faster charge transfer kinetics at the surface of SPE working electrode.

[0227] Enzymatic paper-based electrochemical sensors were a promising tool for glucose detection at the POC in which sensitivity, specificity, low cost, and fast response were important application factors. To test the ability of the device design to perform enzymatic electrochemical detection, glucose was measured by using chronoamperometry technique such that glucose oxidase was deposited in multiple locations in the flow layers. Detection was performed using the CWR electrode configuration in which all device layers were made of A601 paper grade. For all measurements, it was found that the high charging current decays to a steady state Faradaic current within 40 s after applying step potential of 500 mV. FIGS. 24A-24B show chronoamperometric detection of glucose using all A601 -based 3D ePAD with CWR electrode configuration. FIG. 24A shows chronoamperometry plots for detecting 0 - 20 mM of glucose concentration ranges over a period of 50 s. FIG. 24B shows the resulting calibration curve, which showed a linear relationship between the glucose concentration (X) and the average resulting current in the duration between 40 s and 50 s (Y). This relationship was described by the equation Y (uA) = 0.6369*X (mM) + 2.570. Sample was 100 pF of varying glucose concentrations in DI water solutions. The applied a step potential was 500 mV. Error bars represent the standard error of the mean. (N = 3).

[0228] Accordingly, it was determined the steady state current as the average current value measured between 40 - 50 s after applying the step potential.

[0229] For the tested electrode configuration and paper grade, it was found that the glucose concentration was proportional to the Faradaic current detected, as per Cottrell equation. As shown in FIG. 24B, the linear calibration plot was described by the equation y = 0.6369*x + 2.570 where x was the glucose concentration and y was the corresponding Faradaic current response. All proposed 3D ePAD iterations showed linear relationships between glucose concentrations and their corresponding Faradaic current. While the normal level of glucose in whole blood was 3.5 - 5.3 mM and 2.5 - 5.3 in plasma, the LOD for glucose detection using the proposed 3D ePAD was 1.75 mM (N = 10). The LOD exhibited by this device pave the way for utilizing the device design to detect glucose levels in other biological matrices such as urine, tears, and saliva.

[0230] Lead (Pb(II)) was one of the most potent water pollutant heavy metal ions, posing a serious threat to public health and global ecosystem. Using paper-based electrochemical sensors for Pb(II) detection could be a powerful tool to utilize, especially in resource-limited regions

[0231] #14670283vl where a fast selective sensor was needed while minimal access to sophisticated lab equipment was available. To test the ability of the device design to perform a trace level detection of ionic species, a wide range of Pb(II) concentrations were measured using square- wave anodic stripping voltammetry (SWASV) technique because it produces high signal-to-noise ratio, reducing the background noise resulting from the charging current during the potential scan. Without any modification of the WE surface, Pb(II) detection was performed using the CWR electrode configuration in which all device layers were made of A601 paper grade.

[0232] CWR electrode configurations were tested against Pb(II) concentration range of 10 ppb - 1000 ppb. Because of that wide concentration range, the correlation between the generated current signal and Pb(II) concentration fitted a nonlinear three-parameter logistic model. FIGS. 25A-25B show SWASV for detection of Pb(II) using 3D ePAD. FIG. 25A shows SWASV plots for Pb(II) detection using CWR configurations of A601-based paper layers. FIG. 25B shows the calibration curve of SWASV detection of Pb(II). Data were fit to a 3-parameter logistic (3PE) model with the following parameters: bottom was 0.1375, top was 16.09, and EC o was 426.9. For all devices, 100 pF of Pb(II) concentrations in the range of 0 - 1000 ppb were dissolved in 0.1 M acetate buffer solutions (pH = 5). Step potential was 500 mV and deposition time was 600 s. Error bars represent the standard error of the mean. (N = 4).

[0233] Owing to their higher sensitivity, the signals generated by CWR A601 devices the tested covered a Pb(II) concentration range with an EOD of 4.4 ppb. The detection range exhibited by CWR configuration covers the permissible limit of Pb(II) concentration in fresh water. The sensitivity of this device, being only a result of electrode configuration, signifies the usability of the configuration interchangeability offered by the device design. Since the devices were tested using only commercial unmodified carbon ink, the analytical performance of the proposed design was expected to increase if the working electrode surface was chemically modified to capture different analytes.

[0234] These experiments demonstrated that interelectrode distance, dictated and controlled by the thickness of the mediating layers, extensively impacts the device resistance and, hence, current output. These results emphasized the importance of material selection and layer construction for achieving reproducible and sensitive electrochemical signals. Moreover, the design versatility in adopting different electrode configurations could be employed to minimize the cell uncompensated resistance, enhancing device performance across multiple electrochemical techniques.

[0235] Reagents and Device Materials

[0236] #14670283vl Kimtech 34133 Kimwipes, 1-Ply, were purchased from Amazon. Alhstrom chromatography paper grade 601 was purchased from Thomas Scientific. Munktell TFN paper was purchased from Laboratory Sales and Services (Somerville, NJ). Flexmount Select DF051521 (permanent adhesive double-faced liner) was purchased from FLEXcon. 1 / 4” clear acrylic sheets were purchased from McMaster-Carr. Avery laminating sheets were purchased from Amazon. Elikliv 7" digital microscope with IPS screen - 1300X was purchased from Amazon. E3178 carbon ink and E2414 AG / AGCL ink was purchased from Ercon Inc.

[0237] Glucose Oxidase was purchased from Aspergillus niger from Sigma. Lead(II) nitrate, 99+% was purchased from Thermo Scientific Chemicals. Samsill Economy Transparent Printer Sheets were purchased from Amazon. Potassium ferricyanide was purchased from Acros Organics. Carbon screen printed electrodes (WED-3mm-carbon / pseudo RE-Ag / AgCl / CE- carbon) were purchased from Zensor R&D (Taiwan), potassium chloride [4 M] saturated with silver chloride was purchased from Ricca Chemical Company. We purchased glacial acetic acid and sodium acetate anhydrous from Fisher Scientific. 99.5% anhydrous ethanol was purchased from Thermo Fisher. ASTM Type I water was purchased from Ricca Chemical Company. A CH Instrument 650E workstation was used to run all electrochemical measurements.

[0238] For cyclic voltammetry and linear sweep voltammetry measurements, solutions of 10 and 1 mM potassium ferricyanide in 1 and 0.1 M potassium chloride solution, respectively, were prepared. Potassium chloride solution was diluted from the 4 M solution from Ricca Chemical Company. 0.1 M acetate buffer solution (pH 5) was prepared by mixing amounts of acetic acid and sodium acetate solutions and adjusting the pH to 5 using drops of 1 M hydrochloric acid and sodium hydroxide solutions. Fresh samples of different concentrations of lead (II) nitrate solutions (1 ppb - 10 ppm) in acetate buffer (pH 5) were prepared daily by serial dilution.

[0239] For glucose assay, 3.25 pL of 250 U / mL glucose oxidase in 600 mM potassium ferricyanide in 1 M potassium chloride solution was deposited on each flow layer of the A601based device. The treated paper layers were dried for 20 min at 4 °C in dark over a silica gel bed. The sample was 150 pL of different glucose concentrations. The increased sample volume mitigated the sample evaporation effect.

[0240] Electrodes were stencil printed using both carbon and Ag / AgCl inks. For KW and TFN paper types, ink was deposited as received. For carbon ink deposited on A601, 80 % (wt / wt) ink

[0241] #14670283vl was used after dilution with 99.5% anhydrous ethanol. Dilution was done by mechanical mixing until a homogeneous paste was formed. Ag / AgCl ink was stencil printed to deposit all electrode pads. To dry deposited ink, hosting device layers were incubated in the oven for 20 minutes at 65 °C.

[0242] Adobe Illustrator was used to design the device layers and then printed the hydrophobic wax barriers using a Xerox ColorQube 8570 wax printer. All adhesive, stencil, and paper layers were cut using a Boss laser cutter (BOSS-LS1630). VWR® Forced Air Microbiological Incubator was used for drying conductive ink on paper sheets. An Elikliv 7" digital microscope to image paper cross sections for ink penetration studies. A Promo Heat CS-15 T-shirt press (45 seconds at 138 °C) was used to melt the wax onto the chromatography paper to form hydrophobic barriers. Using a custom acrylic alignment jig, the device layers were belted bottom up, starting with the RE-hosting layer. Using the jig, the device layers were laid and compressed them as layers compiled up. Next, the assembled device layers were compressed in the unheated T-shirt press to remove any air bubbles trapped between the layers.

[0243] EXAMPLE 3

[0244] This non-limiting example describes design and testing of a non-limiting multiplexed 3D ePAD design. According to some embodiments, the design was evaluated its capability for simultaneous glucose detection using two adjacent working electrodes. The objective of this experiment was to determine whether the current output from both electrodes would be consistent. Consistent current from both working electrodes would indicate uniform electrochemical behavior, regardless of the physical position of the working electrode in its hosting layer. For this test, A601 paper layers were used and positioned both working electrodes on the center of the layer. The working electrode-hosting layer was positioned in the middle of the other two electrode-hosting layers, CE and RE, forming a CWR configuration.

[0245] FIG. 26 shows a schematic of the first iteration of the non-limiting multiplexed 3D ePAD design. The layers were displayed in assembly order from bottom to top, with blue labels indicating the electrode-hosting layers (CE, 2 WE’s, and RE). The design features two distinct working electrodes (WE 1 on the right, WE 2 on the left) deposited on the middle electrodehosting layer. Working electrodes were positioned to create a CWR configuration. While CE and RE were wider to cover both working electrodes, their pads orientation remains consistent with the singleplexed device design. The laser-cut adhesive holes, red outlines, allow for fluid

[0246] #14670283vl passage between layers while maintaining device integrity. Light blue dashed lines indicate fluid flow through and between layers.

[0247] The electrode pads of the working electrodes were aligned in the same direction, while the counter electrode (CE) and reference electrode (RE) pad orientations remained unchanged from the singleplexed design of Example 2.

[0248] The upper flow layer, Layer 2, was treated with 16 pL 250 U / mL glucose oxidase in 200 mM potassium ferricyanide in 1 M potassium chloride. Evaluating the obtained current from both electrodes serves as a metric to check if there was an influence from the electrode position, right or left in the electrode hosting layer, on the electrochemical connection among cell electrodes. For all tested multiplexed iterations of the 3D ePAD, both CE and RE were wider in dimensions to be in contact with the full width of both working electrodes. Also, the wider CE aims to minimize the chances of limiting the current transfer in the circuit. This modification could enhance circuit stability and reduce the risk of performance variability between the electrodes.

[0249] To address issues of signal interference and reproducibility associated with close proximity of the working electrodes, a compartmentalized version of the multiplexed 3D ePAD was developed. This design strategically separates the two working electrodes using a hydrophobic wax-printed barrier, creating isolated compartments for each working electrode. FIG. 27 presents a detailed schematic of the dual-input compartmentalized iteration of the multiplexed 3D ePAD. The device layers were displayed in assembly order from bottom to top, with blue labels indicating the electrode-hosting layers (CE, two WEs, and RE). This design features distinct sample introduction points for each working electrode (right: WE 1, left: WE 2), enabling independent filling of each compartment. The flow paths ensure that each sample remains isolated within its respective compartment, preventing cross contamination. Laser-cut adhesive holes, outlined in red, facilitate fluid passage between layers while maintaining electrolytic contact and overall layer integrity. The wider CE and RE pads ensure full contact with both working electrodes, enhancing signal stability. The mirror- image configuration of the working electrode pads minimizes asymmetry, contributing to more uniform signal output between the two electrodes. This setup allows for simultaneous yet separate detection of different analytes introduced through distinct sample inputs. Light blue dashed lines indicate the fluid flow pathways through and between the layers.

[0250] For a CWR configuration, the compartmentalized design still maintains consistent interelectrode distances to both the upper and lower electrodes - counter and reference electrodes, respectively. By segregating the working electrodes into distinct compartments, this

[0251] #14670283vl design minimizes the risk of cross-talk and signal interference, even when both assays rely on similar detection chemistries, such as the redox cycling of a mediator like potassium ferricyanide.

[0252] This compartmentalization enhanced the versatility of the device, allowing two parallel assays to be conducted simultaneously. The concurrent reagent separation and rehydration aims to perform both assays without compromising their selectivity or the accuracy of current signal measured from each working electrode. In all compartmentalized multiplexed devices, carbon ink was used not only to print counter and both working electrodes, but also to print pseudo reference electrodes. Carbon-based pseudo-reference electrodes provide improved chemical stability, especially when exposed to varying sample matrices or redox environments, reducing the risk of leaching or contamination.

[0253] The fluidic architecture of the compartmentalized device design employed a comb-like sample flow structure. FIGS. 28A-28B show cross-sectional illustration of the dual-input compartmentalized iteration of the multiplexed 3D ePAD. FIG. 28A shows a detailed and FIG. 28B shows a compact illustrations show the layer configuration and fluidic architecture. Light blue dashed lines in FIG. 28B indicates fluid flow through and between layers. After their introduction to layer 1, dual samples wick their respective compartments within lower layers. Upon sample introduction, fluid flows in both sides, following a comb-like pattern to create an electrolytic connection among the three-electrode paper-based electrochemical cell while preventing interference across assays conducted in parallel on each working electrode. Discontinuation in adhesive layers refers to laser cut holes created to direct sample flow within the device and allow electrolytic connection between electrodes.

[0254] Upon sample introduction, the fluid wicks across each layer from one side of the device until it encounters the hydrophobic barrier at the center. This barrier diverts the flow towards the lower layers of the same compartment, preventing cross-contamination between the two sides. This comb-like flow pattern ensures efficient sample distribution within each compartment while maintaining chemical isolation between the assays conducted on either side of the device.

[0255] In this iteration, the working electrode pads were designed to be stencil-printed in opposite directions. This mirror-image configuration divides the device symmetrically into two superimposable halves, reducing the likelihood of signal variability due to asymmetry. This design feature mitigates potential discrepancies in sample volume and analyte distribution between the two compartments, which could otherwise result in differences in reagent concentration and, consequently, signal output.

[0256] #14670283vl -M -

[0257] This compartmentalized multiplexed device was tested using a dual-input sample introduction layer (Layer 1), where samples were added separately to each side of the device. This dual-sample capability allows for independent parallel analysis of two distinct analytes, enhancing the functionality of the device for multi-analyte detection. Additionally, the sample flow layer (Layer 2) could be treated with different reagents specific to the target analytes, enabling tailored detection chemistries for simultaneous analysis.

[0258] To further enhance the versatility of the compartmentalized 3D ePAD and address sample volume limitations, a single-input iteration of this multiplexed device was used. FIG. 29 provides a detailed schematic of the single-input compartmentalized iteration of the multiplexed 3D ePAD. The device layers were displayed in assembly order from bottom to top, with blue labels indicating the electrode-hosting layers (CE, two WEs, and RE). This design features distinct sample introduction points for each working electrode (right: WE 1, left: WE 2), enabling independent filling of each compartment. The flow paths ensure that each sample remains isolated within its respective compartment, preventing crosscontamination. Laser-cut adhesive holes, outlined in red, facilitate fluid passage between layers while maintaining electrolytic contact and overall layer integrity. The wider CE and RE pads ensure full contact with both working electrodes, enhancing signal stability. The mirror- image configuration of the working electrode pads minimizes asymmetry, contributing to more uniform signal output between the two electrodes. This setup allows for simultaneous yet separate detection of different analytes introduced through distinct sample inputs. Light blue dashed lines indicate the fluid flow pathways through and between the layers.

[0259] This version introduces a separate sample introduction / distribution layer, Layer 1. This layer aims to facilitate simultaneous filling of both compartments using a single sample containing the analytes of interest. The compartmentalized structure ensures even sample distribution and flow across both sides of the device, while allowing for rehydration of the dried recognition elements stored within each compartment. FIGS. 30A-30B provide non-limiting cross-sectional illustration of the single-input compartmentalized iteration of the multiplexed 3D ePAD. FIG. 30A shows a detailed and FIG. 30B shows a compact illustrations show the layer configuration and fluidic architecture. Light blue dashed lines in FIG. 30B indicate fluid flow through and between layers. After its introduction to layer 1, the sample was distributed to respective compartments within lower layers. Upon sample introduction, fluid flows in a comblike pattern to create an electrolytic connection among the three-electrode paperbased electrochemical cell while preventing interference across assays conducted in parallel on each

[0260] #14670283vl working electrode. Discontinuation in adhesive layers refers to laser cut holes created to direct sample flow within the device and allow electrolytic connection between electrodes.

[0261] This single-input design of this multiplexed 3D ePAD offers several advantages. First, it reduces the required sample volume, which was particularly beneficial in applications where obtaining sufficient biological sample volume was challenging, such as in tear or sweat analysis. Second, it minimizes the risk of device irreproducibility by reducing the number of user interactions with the device — requiring only a single sample introduction instead of two. Third, by utilizing a single sample that contains both analytes, the device demonstrates improved analyte specificity. Each analyte interacts selectively with its respective recognition element. For instance, glucose reacts only with the glucose oxidase enzyme on one side of the device, while lactate reacts with lactate oxidase on the other side. This selective interaction minimizes crossreactivity and interference, reinforcing the capability of the device to perform accurate multiplexed detection. This iteration further underscores the robustness of the device architecture, offering a streamlined approach to multiplexed detection while maintaining the chemical isolation between parallel assays within the same sample matrix.

[0262] Since a single-channel potentiostat was used for running multiplexed assays, sequential measurements were performed by manually switching the working electrode clamp from WE 1 (glucose side) to WE 2 (lactate side). This approach introduces a time delay, as WE 2 inherently receives 100 seconds more than WE 1 due to the order of measurement. To determine if this additional time affects signal intensity, an experiment was used to evaluate the correlation between signal output and elapsed time across multiple measurement cycles.

[0263] In this experiment, the single-input compartmentalized 3D ePAD was used for multiplexed detection of glucose and lactate. Upon complete device filling, the chronoamperometric measurement was conducted by first connecting the working electrode clamp to WE 1 (glucose side) for 100 seconds. Immediately after this, the clamp was switched to WE 2 (lactate side) to perform the second chronoamperometric measurement under identical conditions. Together, these two sequential runs constituted one measurement cycle, consuming a total of 200 seconds. This process was repeated over 5 consecutive cycles, with a total duration of 1000 seconds, and monitored the current output for each working electrode.

[0264] The signals obtained from both working electrodes were analyzed and compared. In the initial trials, the correlation between current intensity and glucose concentration was weak for both electrodes. The R2values were 0.652 for WE 1 and 0.402 for WE 2, indicating poor linearity. A similar trend was observed when the devices were tested using lactate assays on both

[0265] #14670283vl TFN-based and A601-based iterations. The lack of strong correlation between current signal and analyte concentration could be due to two main factors.

[0266] First, interference in signals generated by each electrode which could result from having two working electrodes in close proximity. This interference could be related to the competition over the H2O2 molecules produced as byproducts in the detection reaction of both glucose and lactate using glucose oxidase and lactate oxidase, respectively. While H2O2 molecules were oxidized at the working electrode surface to generate the signal, the close proximity of the two working electrodes could lead to having both electrodes competing for the same H2O2 molecules. This competition could result in uneven current among the working electrodes.

[0267] Second, mass transport limitations that could arise from the close inter-working-electrode distance, ~ 1 mm. This distance could lead to diffusion layer overlap, where the analyte transport to each electrode becomes inefficient. Theoretically, under steady-state reaction conditions, the typical thickness of a diffusion layer was on the order of 100 to 500 pm. While the two working electrodes were, by design, 1 mm apart, this distance could shift due to manufacturing limitations that were related to printing, handling, and dimensional change after ink drying. This distance shifting could result in diffusion layer overlap which could culminate in mass transport limitations. Eventually, this overlap could probably result in competition for the available H2O2, as both working electrodes were reacting with the same limited pool of reaction products. In addition, the current output obtained from either working electrodes was a result of the same glucose oxidation reaction in the presence of the redox mediator, potassium ferricyanide. This means that if 2 assays were used that work with the same chemistry, the current device design will not has the ability to accurately tell which signal was a result from which assay. This compromises the device reliability as it leads to a higher chance of signal interference.

[0268] We utilized the dual-input iteration of the compartmentalized 3D ePAD for multiplexed detection of glucose and lactate. For glucose detection, the sample flow layer (Layer 2) was treated with 250 U / mL glucose oxidase in 600 mM potassium ferricyanide dissolved in 1 M KC1. The glucose assay was performed on the right side of the device, where the working electrode (WE 1) was dedicated for this detection. Consequently, treatment of the flow layers with glucose oxidase was only limited to the right-side of Layer 2. Also, glucose samples of varying concentrations were introduced on the right side of the device.

[0269] The lactate detection assay was conducted on the left side of the device, utilizing the second working electrode (WE 2). For this assay, the left side of the sample flow layer, Layer 2, was treated with 50 U / mL lactate oxidase in 200 mM potassium ferricyanide in 1 M KC1. Instantly after introducing glucose samples to the right side, lactate samples of varying

[0270] #14670283vl concentrations were introduced to the left side. Following complete device filling with sample, the potentiostat's working electrode clamp was connected to WE 1 to run the chronoamperometric measurement for glucose detection, over a duration of 100 seconds. Immediately after the glucose measurement, the working electrode clamp was connected to WE 2 while counter and reference electrode clamps stayed untouched. This setup allowed to perform the lactate detection assay under identical experimental conditions such as interelectrode distance and electrode connections.

[0271] Both the glucose and lactate assays demonstrated a linear relationship between the analyte concentration and the resulting chronoamperometric current. FIGS. 31A-31B present chronoamperometric detection of both glucose (FIG. 31 A) and lactate (FIG. 3 IB) using the dual input iteration of the multiplexed 3D ePAD with CWR electrode configuration. All paper layers were made of A601 grade. The calibration curves show a linear relationship between both analyte concentrations (X) and the average resulting current (Y) over the 0 - 20 mM range in the duration between 80 s and 100 s. Chronoamperometric measurements for glucose, WE 1, were conducted first, followed immediately by lactate measurements, WE 2. Dual samples of 75 pL each were used, containing varying concentrations of glucose and lactate in DI water. The applied step potential was 500 mV. Error bars represent the standard error of the mean (N = 3).

[0272] The linear calibration plots obtained for each assay indicate a consistent and proportional response from the respective working electrodes, validating the effectiveness of the compartmentalized design in supporting simultaneous multi-analyte detection without signal interference. This result underlines the versatility and robustness of the device, as the compartmentalization mitigated crosstalk, allowing each assay to be performed independently even with the same redox mediator chemistry employed on both sides.

[0273] The single-input iteration of the compartmentalized 3D ePAD was used for multiplexed detection of glucose and lactate using a sample that contained both analytes. Similar to the dualinput iteration, the sample flow layers were treated with enzyme solutions specific to each analyte: glucose oxidase on the side corresponding to the glucose assay (WE 1, right) and lactate oxidase on the side for the lactate assay (WE 2, left). This compartmentalized approach maintained the distinct chemical environment for each assay despite the single-sample input, ensuring selective interactions with the respective recognition elements.

[0274] Except for sample introduction, multiplexed detection experiments in this device iteration were conducted exactly like those for the dual-input iteration. Upon introducing the glucose-lactate mixture solution, the sample filled both compartments simultaneously. While filling the device, the sample rehydrated the enzyme reagents pre-stored and dried on each side.

[0275] #14670283vl Following complete device filling, the potentiostat’s working electrode clamp was connected to WE 1 to perform the chronoamperometric measurement for glucose detection over a period of 100 seconds. Immediately after completing the glucose assay, the working electrode clamp was switched to WE 2, while keeping the counter and reference electrode connections unchanged, to run the chronoamperometric experiment for lactate detection under identical conditions. The device exhibited a linear relationship between the concentrations of glucose and lactate and the corresponding chronoamperometric currents generated by each working electrode.

[0276] FIGS. 32A-32B present chronoamperometric detection of both glucose and lactate using the single input iteration of the multiplexed 3D ePAD with a CWR electrode configuration. All paper layers were made from A601 grade. The chronoamperometric response plots for FIG. 32A glucose and FIG. 32B lactate show current profiles across the 0-20 mM concentration range. The calibration curves for FIG. 32C glucose and FIG. 32D lactate indicate a linear relationship between analyte concentration (X) and the average resulting current (Y) in the duration between 80 and 100 seconds. Chronoamperometric measurements for glucose, at WE 1, were conducted first, followed immediately by lactate measurements, at WE 2. Samples were 150 pL of mixtures containing varying concentrations of glucose and lactate in DI water. In each sample mixture, both glucose and lactate were present at equimolar concentrations. The applied step potential was 500 mV. Error bars represent the standard error of the mean (N = 3).

[0277] The LOD of the glucose assay was 3.3 mM while that of the lactate assay was 2.9 mM. This outcome demonstrates that the single-input design successfully mitigates cross-reactivity, even when the sample contains both analytes, while keeping a sensitive performance. By isolating the interactions of each analyte with its recognition enzyme in separate compartments, the device provided specific and reproducible signals for both assays. While highlighting the robustness of the 3D ePAD design, this iteration also offers a practical advantage for scenarios requiring simultaneous analysis of multiple biomarkers from a single, limited sample volume.

[0278] The results indicated that both device sides maintained consistent current intensities over the first 3 cycles - 600 seconds. FIG. 33 presents a stability assessment of the chronoamperometric current response for both glucose (red bars) and lactate (blue bars). Signals were measured using the single-input iteration of the multiplexed 3D ePAD, CWR configuration with A601 papers. Measurements were conducted over five consecutive cycles, with each cycle consisting of a 100-second glucose detection (WE 1) followed by a 100-second lactate detection (WE 2), totaling 200 seconds per cycle. The current output remains stable across the first three cycles for both analytes. Sample was 150 pL of 15 mM glucose and lactate in DI water. In each

[0279] #14670283vl sample mixture, both glucose and lactate were present at equimolar concentrations. The applied step potential was 500 mV. Error bars represent the standard error of the mean (N = 3).

[0280] During the subsequent two cycles, however, a noticeable current decile was observed for both glucose and lactate assays. This signal reduction could potentially be attributed to several factors: (i) enzyme deactivation or denaturation; (ii) depletion of active sites on the working electrodes, possibly caused by product buildup over time; (iii) sample evaporation, especially given the prolonged measurement duration, which might concentrate the sample and alter reagent rehydration dynamics; and / or (iv) electrode fouling, where reaction byproducts or intermediates may adsorb onto the electrode surface, inhibiting effective electron transfer and reducing the overall current response. This experiment highlights the importance of considering time-dependent effects when using a single-channel potentiostat for multiplexed assays. Future improvements may involve employing a multi-channel potentiostat or a multiplexer unit, optimizing the reagent formulation, and / or implementing strategies to mitigate evaporation and fouling, thereby enhancing signal stability over extended measurement periods.

[0281] To further investigate the stability of the signal output over time, the experiment was repeated for the first 3 measurement cycles using a range of analyte concentrations for both glucose and lactate assays. This extended analysis aimed to determine if the observed consistency in current held true across different concentrations. The tested concentrations ranged from 5 mM to 20 mM for both analytes. FIGS. 34A-34B present extended analysis of chronoamperometric current stability across three measurement cycles for glucose (FIG. 34A) and lactate (FIG. 34B). Signals were measured using the single-input iteration of the multiplexed 3D ePAD, CWR configuration with A601 papers. Samples were 150 pF of glucose and lactate mixture solutions with 0 - 20 mM concentration range. In each sample mixture, both glucose and lactate were present at equimolar concentrations. Data points represent the mean current measurements for each cycle (Cycle 1: dark, Cycle 2: medium, Cycle 3: light). Error bars represent the standard error of the mean (N = 3).

[0282] The results confirmed that the maintained signal intensity over the initial 3 cycles was consistent across nearly all tested concentrations. However, the highest concentration of 20 mM did not maintain the same signal consistency. At this elevated concentration, a gradual decline in current intensity was observed from one cycle to the next. Despite this diminishing trend, the signal remained proportional to the expected analyte response. Thus, although the current output for the 20 mM sample decreased over the cycles, it maintained a higher intensity than the 15 mM sample, indicating that the relative signal intensity was preserved.

[0283] #14670283vl These findings suggest that high analyte concentration might lead to additional measurement challenges. These challenges could include enzyme saturation, increased product buildup, or higher fouling rate at the electrode surface, contributing to the observed signal decline. Further optimization of the assay conditions, such as adjusting enzyme loading or altering the mediator concentration, may be necessary to mitigate these effects and ensure stable performance even at higher analyte levels. Future studies should also consider exploring strategies to enhance the longevity of the signal response, particularly when detecting high concentrations of target analytes.

[0284] In another experiment, an advanced standalone version of the 3D Epad was designed. This iteration replaces the wax-printed barriers with hydrophilic channels supported by laser-cut mylar layers, eliminating the reliance on wax printing, which many manufacturers no longer support. This modification not only improves fabrication reproducibility but also mitigates potential leakage issues, preserving the integrity of sample flow and reagent distribution. Additionally, the standalone design allows for selective replacement of the hydrophilic channels while retaining the mylar-based framework and copper electrode pads — another modification introduced in this device iteration — thereby enabling device reusability. This modular approach promotes resource efficiency and contributes to waste reduction, aligning with the growing emphasis on sustainable practices in analytical device development.

[0285] In this iteration of the device, a standalone version of the 3D ePAD was developed wherein the paper-based electrodes and fluid flow channels were integrated and supported by Mylar layers. This Mylar-supported 3D ePAD retains the core architecture of the fully paperbased version, featuring three electrodes — working, counter, and reference — stencil printed on separate paper layers. In the original fully paper-based 3D ePAD, silver / silver chloride (Ag / AgCl) pads were used to connect each electrode to the corresponding metal clamps from the electrochemical workstation. However, the Mylar- supported 3D ePAD replaced these connections with 3 mm- wide copper tape, which serves as the link between the stencil-printed electrodes and their respective potentiostat metal clamps. For each electrode, the copper tape was partially adhered to the surface of the electrode, while the remaining portion was affixed to the Mylar layer that supports the electrode-hosting paper layer. This dual function of the copper tape not only ensures a reliable connection between the electrode and the potentiostat clamp but also secures the electrode-hosting paper layer to the patterned Mylar layer, enhancing the structural integrity of the device.

[0286] This design modification also streamlines the manufacturing process by reducing reliance on stencil printing. In the fully paper-based version, the fabrication process involved

[0287] #14670283vl two stages of stencil printing. The first stage involved printing carbon ink for the working and counter electrodes and Ag / AgCl ink for the reference electrode, followed by an ink drying step. The second stage involved stencil printing Ag / AgCl pads for all electrodes, with subsequent drying under the same conditions of time and temperature. Conversely, the use of copper tape in the Mylar- supported 3D ePAD eliminates the need for the second stencil printing stage. As a result, fabricating this iteration saves drying time and minimizes the potential for manufactural defects related to misalignment, ink costs, energy consumption, and material waste. Specifically, avoiding the second stage of stencil printing significantly reduces the risk of defects that could arise from misalignment of the stencil-printed pads with their corresponding electrodes. In cases where misalignment occurs in the fully paper-based device, the entire sheet was rendered unusable, necessitating a complete reprint of both stages. The use of copper tape, on the other hand, allows for more precise manual placement, effectively mitigating this risk and reducing material waste during device fabrication associated with stencil printing. This adaptation not only enhanced the efficiency and reliability of the device production process, but also underscores the potential for scaling up the manufacturing of 3D ePADs for broader applications.

[0288] Since this device iteration only modifies the hydrophilic layer support and not the interelectrode distancing, each two electrodes were still separated from one another by a distance equal to the combined thickness of the paper and adhesive used - similar to fully paper-based 3D ePAD. The overall fluidic pathway in the device maintains the serpentine fashion: the sample wets the device inlet, travels down the top layer to the outlet, then vertically to the layer below where it changes directions. Patterned holes in the adhesive film separating channels on subsequent layers permit two electrodes to be wetted by the sample simultaneously. This process repeated until the sample reaches the bottom layer of the device. Adhesive films in between layers of paper ensure that leaks or contamination do not occur. In addition, the standalone 3D ePAD iteration still supports electrode interchangeability, since each electrode was still defined on a unique layer. Hence, this device version allows for further studying the influence of electrode configuration on different electrochemical measurements.

[0289] To accommodate the varying ink penetration extents, the Mylar-supported 3D ePAD was tested using both TFN and A601 paper grades. For TFN devices, each electrode was positioned in the center of a hydrophilic channel that also begins and ends with circular zones. In this way, every layer of the device was both a duplicate yet still uniquely addressable, maintaining the simple manufacturing and flexible device design offered in the fully paper-based 3D ePAD. While layers were affixed to each other by sheets of patterned, double-sided adhesives, the

[0290] #14670283vl Mylar-supported 3D ePAD also includes two laminate sheets on top of and underneath the compacted layers.

[0291] FIG. 35 provides a non-limiting exploded perspective schematic of the non-limiting TFN-based standalone version of the 3D ePAD. The device was assembled in sequential order from bottom to top, illustrating the individual paper and adhesive layers. The TFN paper serves as the substrate for electrode stencil printing. Due to their thickness, the same TFN serve as both electrode-hosting and flow layer, allowing sample to flow underneath the deposited electrode. While RE remains the lowest electrode, the top and middle electrodes could be reconfigured as working or counter electrodes, based on potentiostat clamp attached. Copper stickers were used as electrode pads and were affixed to Mylar layers, which form the structural skeleton of the device. Top and bottom laminates aim to ensure structural integrity of device layers. Laser-cut adhesive holes guide the sample flow path to facilitate controlled wicking of all layers and electrolytic connection among electrodes.

[0292] The laminate sheets serve to further affix the upper and lower paper conduits, including their electrodes, in position, since only one face of both layers was affixed with the double-sided adhesive sheet. The top laminate layer was patterned to provide access to the sample introduction port in the device. The lower laminate layer, however, was not patterned, since it serves as a preventive layer to avoid dripping of the sample and washing the reagents away. This could be particularly helpful in case the assay run by the device needs any layer pretreatment, such as the chronoamperometric glucose assay described in Example 2.

[0293] In the A601 version of the Mylar- supported device, the hydrophilic area was confined to the two flow layers, Layers 7 and 9. The electrodes, however, were printed as rectangles on a separate sheet then cut and manually placed to be in the middle position over these hydrophilic flow layers, Layers 2, 6, and 10.

[0294] The placement of electrodes was a critical aspect, with each electrode being precisely positioned at the center over these flow layers. This precise alignment was facilitated by placeholders on the Mylar frame, Layers 3, 6, and 9, ensuring accurate and reproducible electrode positioning. These placeholders were etched into the Mylar frame using laser cutting at a specific power level that marks the surface without penetrating the layer entirely. This controlled engraving process was essential to prevent sample leakage, as penetration through the Mylar could compromise the device's integrity and result in signal variability.

[0295] The minimized hydrophilic area within these layers was engineered to maintain an efficient electrolytic connection between the centrally aligned electrodes while reducing the required sample volume. The reduced sample volume necessary for device filling, 50 pL as

[0296] #14670283vl compared to 100 pL in fully paper-based device, was a substantial feature of this standalone iteration, potentially enabling the device to be used for analyte measurements where sample volume was limited. FIG. 36 presents a non-limiting exploded-perspective schematic of the A601 -based standalone version of the 3D ePAD. The device was assembled in sequential order from bottom to top, illustrating the individual paper and adhesive layers. The A601 paper serves as the (i) substrate for electrode stencil printing and (ii) sample flow layers. Due to the deep ink penetration in A601 papers, the device features dedicated layers for sample flow. While RE remains the lowest electrode, the top and middle electrodes could be reconfigured as working or counter electrodes, based on potentiostat clamp attached. Copper stickers were used as electrode pads and were affixed to Mylar layers, which form the structural skeleton of the device. Top and bottom laminates aim to ensure structural integrity of device layers. Laser-cut adhesive holes guide the sample flow path to facilitate controlled wicking of all layers and electrolytic connection among electrodes.

[0297] As described in Example 2, the fully paper-based 3D ePAd iteration utilizes Ag / AgCl electrode pads which take three different directions. The different directions of electrode pads aimed to facilitate easier manual connection to external measurement systems and reduce the risk of accidental connections when attached to metal clamps. However, the Mylar-supported iteration employs copper tape-based electrode pads which were aligned next to each other such that they resemble the electrode pads in commercial SPEs. From this perspective, the aligned copper tape-pads represent a natural design evolution from a prototype-focused design to one optimized for standardized instrumentation. This Mylar-supported version was more suitable for use with commercial potentiostats and glucometers, where metal clamps were not always supported.

[0298] The electrochemical activity of the standalone version of the 3D ePAD was tested by running cyclic voltammetry experiments using a range of scan rates, 50 - 500 mV / S. As a demonstration for this device design, the electrode-hosting layers in these devices were TFN- based, in which the electrode ink was only deposited on the upper side of each paper channel. Ks[Fe(CN)6] was used as a model for a redox-active compound to evaluate and compare the electrochemical activity of this Mylar-based 3D-ePADs iteration using both electrode configurations (i.e., CWR vs. WCR). For WCR configuration, a potential range from 0 V to 0.7 V was used, while for CWR configuration, a -0.5 V to 0.4 V potential range was used. For both configurations, cyclic voltammetry experiments over 50 - 500 mV / s scan rate range was run.

[0299] Electrochemical impedance spectroscopy (EIS) was used to evaluate the difference in resistance elements of both WCR and CWR configurations using the standalone 3D ePAD

[0300] #14670283vl iteration. EIS was a powerful technique commonly used to investigate the electrical properties and interfacial processes at electrode surfaces. EIS measures the response of the system towards a small, alternating potential signal over a range of frequencies. These measurements provide a comprehensive analysis of both the resistive and capacitive elements in electrochemical systems. By providing insight into the resistance to charge transfer, double-layer capacitance, and diffusion processes, EIS was instrumental in characterizing various aspects of electrode performance. EIS measurements were relevant for analyzing the 3D ePADs as they allow us to study the complex interfacial processes that could affect device performance and reliability.

[0301] In the EIS analysis, several key electrical elements were studied, including the solution resistance (Rs), charge transfer resistance (Ret), Warburg impedance (W), and doublelayer capacitance (Cai), each representing distinct aspects of the electrochemical system. While the solution resistance represents the resistive component arising from the electrolyte solution, the charge transfer resistance reflects the energy barrier associated with electron transfer between the electrode and the analyte. Warburg impedance, an important element in systems involving diffusive transport, accounts for the impedance caused by mass transfer limitations, especially at lower frequencies. The double-layer capacitance represents the capacity of the electrode to store charge at the electrolyte interface. A Warburg circuit fitting was used to accurately model and interpret the EIS data, as this fitting method captures the contributions of diffusive processes to the overall impedance. In addition to showing high fitting accuracy (> 85%), using the Warburg model to study the system helps quantify diffusion-related behavior and identify potential issues related to mass transport limitations in both electrode configurations. This approach not only provides a comprehensive understanding of interfacial phenomena, but also allows for comparative analysis between the WCR and CWR configurations. This output could highlight any design-specific influences on electrochemical performance of either configuration. For all EIS measurements, an initial potential of 0 V, high frequency of 1 x 10 Hz, low frequency of 0.1 Hz, and amplitude of 5 mV were employed.

[0302] The cyclic voltammograms of this Mylar-based device iteration showed an output similar to that obtained from the fully paper-based 3D ePADs in multiple ways. FIGS. 37A-37B present the results of cyclic voltammetry produced by standalone TFN-based 3D ePAD using both WCR and CWR electrode configurations. FIG. 37A presents voltammograms recorded over a scan rate range of 50 - 500 mV / s as samples of 100 pL of 10 mM K3[Fe(CN)e] in 1 M KC1 were introduced. FIG. 37B presents the linear relationships between square root of scan rates and their measured anodic (black triangles) and cathodic (red circles) peak currents. Error bars represent the standard error of the mean. (N = 4).

[0303] #14670283vl For instance, the measured current magnitude still maintained the considerable difference with respect to the electrode configuration. In the WCR configuration, absolute values of currents were in the microampere range (approximately 20-200 pA). In opposition, absolute values of currents in the CWR configuration were in the milliampere range (approximately 0.5 - 3 mA) representing a difference of more than one order of magnitude. This result suggests that the standalone version of the device did not negatively impact the influence of interelectrode distance with respect to electrode interchangeability. As discussed in Example 2, the CWR configuration allows minimal WECE and WE-RE distance. On the other hand, WCR format allows for only minimal WECE distance, while that distance was doubled between WE (top layer) and RE (lowest layer). As distance between WE and RE increases, uncompensated resistance increases, which leads to lower current.

[0304] For both WCR and CWR electrode configurations, the standalone 3D ePAD iteration exhibited the anticipated linear relationship between the square root of the applied scan rate and the resulting peak current magnitude (FIG. 37B). For WCR configuration, the linear relationship of the cathodic current was described as Y = -9.298*X + 6.372 while the anodic current equation was Y = 9.015*X - 4.526. On the other hand, the linear relationship of the CWR cathodic current was described as Y = -0.1120*X + 0.3280 while the anodic current equation was Y = 0.1280*X - 0.6273, where X was the square root of the applied scan rate and Y was the corresponding peak current. The obtained linearity was characteristic of a process where the rate of electron transfer was limited by the diffusion of the analyte to the electrode surface. This relationship was described by the Randles Sevcik equation: where i was the peak current, n was the number of electrons transferred in the redox process, A was the electrode area, D was the diffusion coefficient of the analyte, C was the concentration of the analyte, and v was the scan rate.

[0305] For both electrode configurations, the peak current intensity was obtained in both scan directions of cyclic voltammetry, i.e. anodic and cathodic (Table 3). For WCR configuration, all scan rates between 100 - 500 mV demonstrated ratios between the anodic and cathodic current that were close to unity (ipa / ipc=l). The produced peak shapes revealed a Nernstian electrochemical reaction whose rate was governed by electroactive species diffusion to the WE surface. Also, the WCR configuration showed an increasing peak-to-peak separation potential (AEp) with increasing the CV scan rate (Table 4). As previously discussed in Example 2, the higher scan rates allow the electrochemical system less time to reach equilibrium at each

[0306] #14670283vl potential point. This decreased time results in larger separation between the potential values at which oxidation and reduction of the electroactive species happen, aka polarization overpotential. The CWR configuration, however, showed less regular cyclic voltammograms than those of WCR counterparts. For instance, CWR devices demonstrated an increase in the anodic current for both 100 and 200 mV / s scan rates (ipa / ipcvalues were 1.47 and 1.28, respectively) (Table 3). The increased anodic current might indicate a partial accumulation of the electroactive species near the working electrode. In CWR, there may be an uneven distribution of species in the diffusion layer, leading to a concentration polarization. This polarization could cause the anodic peak current to rise more than expected, as the reduced species has less chance to fully diffuse away from the electrode surface before the reverse scan.

[0307] In general, both electrode configurations of the Mylar- supported version showed closer peak-to-peak separations than that exhibited by fully paper-based 3D ePADs (Table 4). While this closer potential separation was only slightly shown in WCR electrode configuration, the CWR configuration exhibited a substantial difference in peak-to-peak separation, as compared to CWR results obtained from fully paper-based cyclic voltammograms. The lower peak-to-peak separation offered by standalone CWR configuration suggests that it demonstrated higher reversibility. This influence might be related to the fact that this device iteration does not depend on wax printing to define electrolytic path, avoiding the probability of increasing cell resistance due to wax diffusion.

[0308] Table 3. Peak current analysis of cyclic voltammetry data generated by standalone TFN-based 3D ePAD using both WCR and CWR electrode configurations.

[0309] Scan rate (mV / s) 50 100 200 300 400 500 0.99 0.99 0.98 0.98 0.98 0.98 0.95 1.47 1.28 1.08 0.89 0.95

[0310] Additionally, CWR devices showed an increasing AEp with most increasing CV scan rates (Table 4). However, this AEp showed a drop at 300 mV / s scan rate and did not show a difference between 400 and 500 mV / s rates. In general, the irregularity of CV shapes could be related to an electrolyte pathway disturbance, in which Mylar frames could has introduced some inconsistencies in the electrolytic path. Since the hydrophilic channels were manually aligned in their Mylar frames, accidental slight misalignment could compromise the electrolyte flow path. This might affect the uniformity of electroactive species reaching the electrode surface.

[0311] #14670283vl Table 4. Peak-to-peak separation analysis of cyclic voltammetry data generated by standalone TFN-based 3D ePAD using both WCR and CWR electrode configurations.

[0312] Scan rate (mV / s) 50 100 200 300 400 500

[0313] ,4WCR 0.15 0.20 0.27 0.31 0.35 0.40

[0314] AE (V) CWR 0.39 0.45 0.51 0.37 0.60 0.59

[0315] The Nyquist plots of the EIS measurements demonstrated major differences between the WCR and CWR electrode configurations of the 3D ePAD system. FIGS. 38A-38B. Nyquist plots representing the electrochemical impedance spectroscopy responses for both the WCR (FIG. 38A) and CWR (FIG. 38B) electrode configurations of the standalone 3D ePAD. Inset in B shows the equivalent circuit used for fitting both configurations according to the Warburg model. Circuit elements include solution resistance (Rs), double-layer capacitance (C), charge transfer resistance (Ret), and Warburg impedance (W). The semicircle observed in the high- frequency region reflects the charge transfer resistance, while the linear tail at low frequencies indicates diffusion-controlled processes characterized by the Warburg impedance.

[0316] For the WCR configuration, the data revealed a pronounced semicircle at high frequencies. The diameter of this WCR semicircle was significantly larger than in the CWR, covering two higher orders of magnitude. This indicates higher charge transfer resistance, as compared to that of the CWR configuration. The higher impedance to electron transfer exhibited by the WCR format could possibly be due to increased interelectrode distances. This difference in charge transfer resistance could impact the sensitivity and response time of the WCR configuration - findings that align with the minimized current response obtained from WCR cyclic voltammograms in the previous section. These data could potentially favor the CWR configuration for applications that require rapid and efficient electron transfer.

[0317] The comparison of solution resistance and double-layer capacitance values between WCR and CWR configurations reinforces the distinction in their electrochemical characteristics. FIGS. 39A-39C present quantitative comparison of ElS-derived parameters for WCR and CWR configurations of the standalone 3D ePAD version. Plots display solution resistance, Rs (FIG. 39A), charge transfer resistance, Ret (FIG. 39B), and double-layer capacitance, C (FIG. 39C). WCR configuration exhibits higher Rs and Ret values, suggesting increased electrolyte and

[0318] #14670283vl charge transfer resistances. CWR, however, shows a higher C value, consistent with an inverse relationship between Ret and C. These differences highlight the distinct interfacial properties of each configuration. Whiskers represent the minimum and maximum values. (N = 3).

[0319] In the WCR configuration, Rsvalues were notably higher, indicating greater resistance through the electrolyte, which could result from a less direct electrochemical pathway across electrodes. While WCR devices showed higher Rsand Rct values, they showed lower Cai than CWR configuration. This observation aligns with the inverse proportionality between Rct and Cai. This relationship was described by the equation:

[0320] 1 Cdl~ 2nfRctwhere C was the double-layer capacitance, R was the charge transfer resistance, and f was the frequency of the applied AC signal at which the relationship holds. This equation highlights that as R increases, C decreases, reflecting a reduced capacity to store charge at the interface in configurations with higher impedance to charge transfer.

[0321] Accordingly, the lower capacitance in the CWR setup might lead to more stable signals. These findings suggest that there would be fewer fluctuations associated with changes in the double-layer structure during electrochemical processes performed by the CWR configuration. However, this suggestion does not perfectly align with the irregularities demonstrated by CWR cyclic voltammograms.

[0322] Additionally, the capacitance values also vary across configurations, with the CWR configuration showing lower capacitance. This difference in capacitance could relate to differences in electrode surface area accessibility, where the CWR configuration might allow a more compact double-layer formation at the electrode interface. Lower capacitance in the CWR format might lead to more stable signals, as there would be fewer fluctuations associated with changes in the double-layer structure during electrochemical processes.

[0323] The Warburg element, evident in the low-frequency tail of both Nyquist plots, provides additional insight into the diffusion-dominated processes in each configuration. The linear segment of the Nyquist plot for CWR extends further along the real axis than in the WCR configuration. This extension indicates that mass transport limitations could be more prominent in the CWR format. This suggests that while the CWR configuration may benefit from lower Rct and Rsvalues, it may also encounter diffusion-related constraints, potentially impacting the device's performance under high-demand scenarios where rapid analyte diffusion to the electrode surface was required.

[0324] #14670283vl TFN-based standalone 3D ePAD for chronoamperometric detection of glucose was attempted. In these trials, 8 pL of glucose oxidase solution was deposited in the hydrophobic midsection underneath the electrode printed on the top TFN layer. Compared to blank samples, glucose samples showed an increased chronoamperometric current. However, sample flow experienced frequent interruptions which increased flow time and manifested the sample evaporation effects. This issue likely stemmed from the limited hydrophilic area available beneath the stencil-printed electrode. Alternatively, the A601 -based standalone 3D ePAD described earlier was tested to run the same experiment. In this device iteration, smooth sample flow was demonstrated since the recognition enzyme was deposited on the sample flow layer while the electrode was printed on a separate layer. Despite the improved flow, no correlation between glucose concentration and the measured current was observed. Potential improvements for this experiment include using ink dilution and double-sided printing to deposit electrodes on A601 layers — techniques that were not implemented in this initial trial.

[0325] These experiments demonstrated that the multiplexed version of the 3D ePAD was developed to enable simultaneous detection of multiple analytes within a single device architecture. The dual-input and single-input compartmentalized iterations showcased the versatility of this design, allowing independent assays to run in parallel without crossinterference. The standalone version was primarily optimized for singleplexed detection, with a focus on maximizing structural stability. By leveraging TFN and A601 paper substrates, various strategies to improve structural integrity while maintain controlled sample flow dynamics to ensure reliable electrochemical responses were identified. Utilizing both WCR and CWR configurations, the standalone version demonstrated the electrode interchangeability primarily offered by fully paper-based devices while eliminating the need for wax printing.

[0326] Reagents and Device Materials

[0327] Alhstrom chromatography paper grade 601 were purchased from Thomas Scientific. Munktell TFN paper was purchased from Laboratory Sales and Services (Somerville, NJ). Flexmount Select DF051521 (permanent adhesive double-faced liner) was purchased from FLEXcon. Uxcell Double-Sided Conductive Tape Copper Foil Tape Adhesive was purchased from Amazon. Banltre 7.5 mil Mylar Sheets was purchased from Amazon. 1 / 4” clear acrylic sheets were purchased from McMaster-Carr. Avery laminating sheets were purchased from Amazon. A Elikliv 7" digital microscope with IPS screen - 1300X was purchased from Amazon. E3178 carbon ink and E2414 AG / AGCL ink were purchased from Ercon Inc. Glucose Oxidase from Aspergillus niger and Lactate Oxidase from Aerococcus viridans were purchased from

[0328] #14670283vl Sigma. Lead(II) nitrate, 99+% from Thermo Scientific Chemicals. Samsill Economy Transparent Printer Sheets was purchased from Amazon. Potassium ferricyanide from Acros Organics. Carbon screen printed electrodes (WE-D-3mm-carbon / pseudo RE-Ag / AgCl / CE-carbon) were purchased from Zensor R&D (Taiwan). Potassium chloride [4 M] saturated with silver chloride was purchased from Ricca Chemical Company. Glacial acetic acid and sodium acetate anhydrous were purchased from Fisher Scientific. 99.5% anhydrous ethanol were purchased from Thermo Fisher. ASTM Type I water were purchased from Ricca Chemical Company. A CH Instrunment 650E workstation to was used run all electrochemical measurements.

[0329] Solutions were prepared following the same conditions described in Example 2. For multiplexed assays, 4 pL of either 250 U / mL glucose oxidase in 600 mM potassium ferricyanide in 1 M potassium chloride solution or 50 U / mL lactate oxidase in 200 mM potassium ferricyanide in 1 M potassium chloride solution were deposited on the opposing compartments of WE / CE flow layer. The treated paper layers were dried for 20 min at 4 °C in dark over a silica gel bed. The sample was 150 pL of different concentrations of glucose and lactate mixture. The increased sample volume was used to mitigate sample evaporation.

[0330] Electrodes were stencil printed using both carbon and Ag / AgCl inks. For multiplexed assays, all electrodes were deposited on a single (top) side of their respective paper layers. All ink was used as received without dilutions for both A601 and TFN-based devices. Ag / AgCl ink was stencil-printed to deposit all electrode pads. To dry deposited ink, the hosting device layers were incubated in the oven for 20 minutes at 65 °C.

[0331] Multiplexed paper-based devices were fabricated following the same protocol described in Example 2.

[0332] Fabrication of standalone devices

[0333] Adobe Illustrator was used to design the Mylar skeleton. Maylar, adhesive, stencil, and paper layers were cut using a Boss laser cutter (BOSS-LS1630). Using a custom acrylic alignment jig, the device layers were belted bottom up, starting with the lower Fellowes laminating sheets. Next the RE-hosting Mylar frame were introduced. The laser cut hydrohphilic

[0334] #14670283vl papers were then manually placed in their designated spaces in the Mylar frame. The same sequence was repeated with all Mylar-paper-based layers, whether electrodehosting or flow layers, with adhesive layers inserted between each two layers to ensure device integrity. Finally, a strip of a Fellowes laminating sheet was cut and adhered as a top layer with a hole to access the sample introduction port. The uppoer and lower laminating sheet serves to further support device layer integrity and prevents evaporation or dripping of sample or product formed in the cell. After assembling the device layers, the device was sent through a laminator (Trulam) to remove any air bubbles trapped between the layers.

[0335] While several embodiments of the present invention have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and / or structures for performing the functions and / or obtaining the results and / or one or more of the advantages described herein, and each of such variations and / or modifications is deemed to be within the scope of the present invention. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and / or configurations will depend upon the specific application or applications for which the teachings of the present invention is / are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, the invention may be practiced otherwise than as specifically described and claimed. The present invention is directed to each individual feature, system, article, material, and / or method described herein. In addition, any combination of two or more such features, systems, articles, materials, and / or methods, if such features, systems, articles, materials, and / or methods are not mutually inconsistent, is included within the scope of the present invention.

[0336] The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.”

[0337] The phrase “and / or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Other elements may optionally be present other than the elements specifically identified by the “and / or” clause, whether related or unrelated to those elements specifically identified unless clearly indicated to

[0338] #14670283vl the contrary. Thus, as a non-limiting example, a reference to “A and / or B,” when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A without B (optionally including elements other than B); in another embodiment, to B without A (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

[0339] As used herein in the specification and in the claims, “or” should be understood to have the same meaning as “and / or” as defined above. For example, when separating items in a list, “or” or “and / or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of’ or “exactly one of,” or, when used in the claims, “consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e. “one or the other but not both”) when preceded by terms of exclusivity, such as “either,” “one of,” “only one of,” or “exactly one of.” “Consisting essentially of,” when used in the claims, shall have its ordinary meaning as used in the field of patent law.

[0340] As used herein in the specification and in the claims, the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, “at least one of A and B” (or, equivalently, “at least one of A or B,” or, equivalently “at least one of A and / or B”) can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

[0341] Some embodiments may be embodied as a method, of which various examples have been described. The acts performed as part of the methods may be ordered in any suitable way. Accordingly, embodiments may be constructed in which acts are performed in an order different

[0342] #14670283vl than illustrated, which may include different (e.g., more or less) acts than those that are described, and / or that may involve performing some acts simultaneously, even though the acts are shown as being performed sequentially in the embodiments specifically described above.

[0343] Use of ordinal terms such as “first,” “second,” “third,” etc., in the claims to modify a claim element does not by itself connote any priority, precedence, or order of one claim element over another or the temporal order in which acts of a method are performed, but are used merely as labels to distinguish one claim element having a certain name from another element having a same name (but for use of the ordinal term) to distinguish the claim elements.

[0344] In the claims, as well as in the specification above, all transitional phrases such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,” and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases “consisting of’ and “consisting essentially of’ shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03.

[0345] #14670283vl

Claims

CLAIMSWhat is claimed is:

1. A fluidic device, comprising: a first layer comprising a first channel; a second layer comprising a second channel; a first electrode; a second electrode; and a third electrode, wherein: the first layer is disposed on the second layer; the first layer and the second layer each comprise a porous, absorbent material; the first channel is positioned between the first electrode and the second electrode; the second channel is positioned between the second electrode and the third electrode; and the first channel comprises a first vertical flow region, the second channel comprises a second vertical flow region, and the first and second vertical flow regions are in fluidic communication.

2. A method, comprising: flowing a liquid through a first layer comprising a first channel, and a second layer comprising a second channel, wherein: the first layer is disposed on the second layer; the first layer and the second layer comprise a porous, absorbent material; the first channel is positioned between a first electrode and a second electrode; the second channel is positioned between the second electrode and a third electrode; and the first channel comprises a first vertical flow region, the second channel comprises a second vertical flow region, and the first and second vertical flow regions are in fluidic communication.#14670283vl3. The fluidic device or method of any one of the preceding claims, wherein the distance between the first electrode and the second electrode is less than or equal to 1 mm.

4. The fluidic device or method of any one of the preceding claims, wherein the distance between the second electrode and the third electrode is less than or equal to 1 mm.

5. The fluidic device or method of any one of the preceding claims, wherein the first electrode extends through at least 5% of the total thickness of the first layer.

6. The fluidic device or method of any one of the preceding claims, wherein the second electrode extends through at least 5% of the total thickness of the second layer.

7. The fluidic device or method of any one of the preceding claims, wherein the first layer has a thickness less than or equal to 1 mm.

8. The fluidic device or method of any one of the preceding claims, wherein the second layer has a thickness less than or equal to 1 mm.

9. The fluidic device or method of any one of the preceding claims, wherein the third layer has a thickness less than or equal to 1 mm.

10. The fluidic device or method of any one of the preceding claims, wherein the liquid comprises a target analyte.

11. The method of any one of the preceding claims, further comprising detecting a target analyte.

12. The method of claim 11, wherein the target analyte comprises glucose and / or Pb(II).#14670283vl13. The method of any one of the preceding claims, further comprising detecting an amount of glucose in the liquid.

14. The method of any one of the preceding claims, further comprising detecting an amount of Pb(II) in the liquid.

15. The fluidic device or method of any one of the preceding claims, wherein the second electrode is positioned at least partially between the first and the third electrodes.

16. The fluidic device or method of any one of the preceding claims, where the first layer, the second layer, and / or the third layer comprises a polymeric material.

17. The fluidic device or method of any one of the preceding claims, wherein the liquid comprises a bodily fluid.

18. The fluidic device or method of claim 16, wherein the bodily fluid comprises blood, tears, saliva, and / or sweat.

19. The fluidic device or method of any one of the preceding claims, wherein the liquid comprises a fluid derived from the bodily fluid.

20. The fluidic device or method of any one of the preceding claims, wherein the porous, absorbent material comprises a cellulose-based material.

21. The fluidic device or method of claim 20, wherein the cellulose-based material is a type of paper.

22. The fluidic device or method of any one of the preceding claims, wherein the porous, absorbent material is fibrous.

23. The fluidic device or method of any one of the preceding claims, wherein the porous, absorbent material is a synthetic material.#14670283vl24. The fluidic device or method of any one of the preceding claims, wherein the porous, absorbent material is a non-woven material.

25. The fluidic device or method of any one of claims 23-24, wherein the synthetic material comprises polyester, poly(ethersulfone), nylon, and / or nitrocellulose.

26. The fluidic device or method of any one of the preceding claims, wherein the porous, absorbent material is functionalized.

27. The fluidic device or method of claim 26, wherein the functionalization comprises physical functionalization.

28. The fluidic device or method of claim 27, wherein the physical functionalization comprises nanoparticles.

29. The method of any one of the preceding claims, wherein the fluid is flowed by capillarity.

30. The fluidic device or method of any one of the preceding claims, wherein the porous, absorbent material has an average pore size of less than or equal to 15 microns.

31. The fluidic device or method of any one of the preceding claims, wherein the first channel and / or the second channel has a thickness less than or equal to 1 mm.

32. The fluidic device or method of any one of the preceding claims, wherein the first channel and / or the second channel has a width of greater than or equal to 0.5 millimeters.

33. The fluidic device or method of any one of the preceding claims, wherein the first channel and / or the second channel has a length greater than or equal to 4 millimeters.#14670283vl34. The fluidic device of any one of the preceding claims, further comprising an inlet configured to receive the liquid.

35. The fluidic device of any one of the preceding claims, further comprising a filter over the inlet.

36. The fluidic device or method of any one of the preceding claims, wherein the first electrode comprises a first lead portion, the second electrode comprises a second lead portion, and the third electrode comprises a third lead portion.

37. The fluidic device or method of claim 36, wherein the first, second, and third lead portions are electrically isolated from each other.

38. The fluidic device of any one of claims 36-37, wherein the fluidic device further comprises a third layer, and wherein the second layer is disposed on the third layer.

39. The fluidic device of claim 38, wherein a portion of a side of the third layer adjacent the second layer is not covered by the second layer, and wherein a portion of a side of the second layer adjacent the first layer is not covered by the first layer.

40. The fluidic device of any one of claims 38-39, wherein the first lead portion is disposed on the first layer, the second lead portion is disposed on the portion of the side of the second layer adjacent the first layer is not covered by the first layer, and wherein the third lead portion is disposed on the portion of the side of the third layer adjacent the second layer is not covered by the second layer.

41. The fluidic device or method of any one of the preceding claims, wherein the first vertical flow region and the second vertical flow region are in physical contact.#14670283vl

Images