WO2017014828A2 - Device and method for electrochemical detection - Google Patents

Abstract

A microfluidic, electrochemical device for detecting a genetic material is described, including: one or more cellulosic layers comprising at least one of a hydrophilic test zone and a hydrophilic sample deposition zone in fluid communication with each other; one or more amplification agents selected for amplifying a genetic material and embedded in the hydrophilic test zone or the sample deposition zone; one or more binding agents embedded in the test zone or the sample deposition zone and selected for binding the amplified genetic material to result in a change of the concentration of a signaling chemical, wherein the signaling chemical is either embedded in the test zone or the sample deposition zone prior to the binding or is a newly generated product of the binding; and an electrode assembly comprising one or more electrodes in fluidic contact with the test zone and configured to interact with the signaling chemical to result in a current change readable by an electrochemical reader. Methods of using the device and kit containing the device are also described.

Description

Device and Method for Electrochemical Detection

Incorporation by Reference

[0001] All patents, patent applications and publications cited herein are hereby incorporated by reference in their entirety in order to more fully describe the state of the art as known to those skilled therein as of the date of the invention described herein.

Related Applications

[0002] This application claims priority to U. S. Provisional Patent Application

No. 62/164,079, filed on May 20, 2015, the content of which is hereby incorporated by reference in its entirety.

Government Funding Clause

[0003] This invention was made with support from the United States government under Grant No. HDTRA1-14-C-0037 awarded by the Defense Threat Reduction Agency. The United States government has certain rights to this invention.

Background

[0004] Detection and analysis of nucleic acids are among the current approaches for a range of important applications including pre-symptomatic detection of infectious diseases, examination of perinatal genetics and inherent genetic disorders, and speciation of pathogens in food and water samples. Because these analyses involve typically sophisticated procedures that require expensive equipment operated by trained personnel, they are confined to centralized laboratories. If made inexpensive and portable, however, analyses of nucleic acids could be particularly useful in resource-limited settings, where it could be used at the point-of-care or promote public health. Existing instruments, however, are too expensive and cumbersome for widespread use.

[0005] The primary challenges to the creation of a useful point-of-care analyzer of nucleic acids and the extension of the benefits of nucleic acid analysis to resource-limited settings are to simplify the instrumentation and procedures need for (i) amplification and (ii) measurement. Analysis of nucleic acids requires amplification because the DNA in a biological sample (e.g., in pre-symptomatic patients) is present only at such low concentrations that it cannot be detected directly. Methods of nucleic acid amplification methods enable deoxy-ribonucleic acid (DNA) detection even at ultra-low, picomolar concentrations (e.g., as in pre-symptomatic samples), that are otherwise precluded with direct measurements. For amplification, modern systems often use polymerase chain reaction (PCR) for amplification; but i) it requires sophisticated equipment to control the sequence of thermal cycling it uses, and ii) it requires a high degree of temperature stability. These characteristics have so far made PCR unsuitable for use in low-cost, portable detectors.

Recent isothermal alternatives to PCR eliminate thermocycling, but they require costly proprietary reagents and sophisticated procedures.

[0006] Amplification of the initially low concentration of target DNA produces a high concentration of amplicon (i.e., replicated DNA) that can be quantified with conventional analytical techniques at the end of the reaction (end-point measurement). To enable accurate quantification during the amplification (real-time measurement), one method of detection uses fluorimetry, and relies on the indirect measurement of fluorescent molecules that interact with DNA. The detection of fluorescence requires the use of advanced optical systems. Fluorescence-based methods can be either non-specific or specific to the DNA target sequence. Non-specific intercalating molecules bind to double-stranded (ds) DNA, as soon as the product is synthesized during amplification, in a sequence-independent fashion. SYBR Green I is one example of a DNA intercalator, which binds to the minor groove of DNA. The binding yields a change in optical signal. The kinetics of this reaction depends on the initial amount of target DNA. Although accurate, fluorometry is challenging to implement in a rugged, low-cost, portable form factor because optical components can be bulky, costly, and fragile. Specific real-time fluorescence detection uses fluorescent oligonucleotide probes, which are complementary to the DNA target sequence. A wide variety of probes are now available, and the state-of-art has been recently reviewed. TaqMan probes were the first to be developed for use with PCR amplification.

[0007] Although less developed for these purposes, electrochemical detection offers an attractive alternative to optical methods because (i) the required instrumentation can be implemented easily with low-cost components and ii) the measured electrochemical signal is not dependent on lighting conditions. The electrochemical approach may directly measure the native electrochemistry of DNA, or (in a process conceptually similar to the optical alternative) use molecules that interact with dsDNA. These molecules, however, are chosen to be electrochemically (rather than optically) active so that they can be detected by electroanalytical techniques (e.g., cyclic voltammetry (CV) or square-wave voltammetry (SWV)).

[0008] Although the first integrated, miniaturized analytical DNA system was published in the 1990s, low-cost DNA analyzers are yet to be commercially available. The challenge for developing an integrated system is the complex steps required for the processing of a clinical sample. These steps include cell purification, concentration and lysis, DNA

purification and extraction, DNA amplification and detection.

[0009] The existing experiments are performed in bulk solutions, with a benchtop thermocycler for thermal control, and a benchtop electrochemical analyzer for detection.

There is only one example of an integrated, commercial, bench-top fluorescence-based system, which performs all the steps from sample to result analysis. The GeneExpert

(Cepheid Inc.) is endorsed by the WHO for TB detection and performs excellently. However, it still needs to be within a laboratory environment and is far too expensive for the developing world and point-of-care applications, even at its discounted price of -$15,000.

[0010] Other DNA amplification methods have been implemented with non-specific electrochemical detection by intercalators. Defever et al. employed osmium complexes, such as Os[(bpy)₂phen]²⁺, to detect the real-time PCR amplification of cDNA. See, Defever, T., et al.; Real-Time Electrochemical PCR with a DNA Intercalating Redox Probe. Analytical Chemistry 2011, 83 (5), 1815-1821. Methylene blue was also used as an electroactive mediator for real-time PCR of Chlamydia trachomatis. Ahmed et al. used hexaamine ruthenium (III) for real-time detection of bench-top loop mediated isothermal amplification (LAMP) of genomic DNA from Staphylococcus aureus in solution. See, Ahmed, M. U. et al.; Real-time electrochemical detection of pathogen DNA using electrostatic interaction of a redox probe. Analyst 2013, 138 (3), 907-915. LAMP is also isothermal, similarly to recombinase polymerase amplification (RPA). A single publication has been shown thus far for electrochemical sequence-specific detection of PCR amplification of human cDNA. All afore-mentioned examples were performed in solution using a bench-top thermocycler and commercial potentiostat for electrochemical detection. Atlas Genetics Ltd. (Trowbridge, UK) has developed the only integrated bench-top platform for electrochemical DNA amplification. It uses ultra-fast PCR and electroactive sequence-specific probes for C. trachomatis and Neisseria gonorrhoeae . Electrochemical DNA amplification has never been shown on cellulosic layers, e.g., paper, before.

Summary

[0011] Described herein are microfluidic, electrochemical devices for detecting a genetic material. The device includes one or more cellulosic layers comprising at least one of a sample deposition zone and a test zone with one or more amplification agents and binding agents embedded therein. The amplification agent amplifies a targeted genetic material which then binds to the binding agent to provide in a change of the concentration of a signaling chemical and, in turn, a change in a current detectable by an electrode assembly comprising one or more electrodes in fluidic contact with the sample deposition zone and/or test zone and connectable to an electrochemical reader.

[0012] In one aspect, a microfluidic, electrochemical device for detecting a genetic material is described, including: one or more cellulosic layers comprising at least one of a hydrophilic test zone and a hydrophilic sample deposition zone in fluid communication with each other; one or more amplification agents selected for amplifying a genetic material and embedded in the hydrophilic test zone or the sample deposition zone; one or more binding agents embedded in the test zone or the sample deposition zone and selected for binding the amplified genetic material to provide a change of concentration of a signaling chemical, wherein the signaling chemical is either embedded in the test zone or the sample deposition zone prior to the binding or is a newly generated product of the binding; and an electrode assembly comprising one or more electrodes in fluidic contact with the test zone and configured to interact with the signaling chemical to result in a current change readable by an electrochemical reader.

[0013] In any one of embodiments described herein, at least a portion of the electrode assembly is sized and arranged to be insertable into the electrochemical reader. [0014] In any one of embodiments described herein, the device further includes an isothermal subsystem in contact with the test zone configured to maintain a constant temperature of the test zone.

[0015] In any one of embodiments described herein, the test zone and the sample deposition zone are the same zone or two different zones.

[0016] In any one of embodiments described herein, the genetic material is selected from the group consisting of DNA and RNA.

[0017] In any one of embodiments described herein, the genetic material is a specific nucleic acid sequence of a DNA or RNA or a portion thereof.

[0018] In any one of embodiments described herein, the genetic material is selected from the group consisting of the specific nucleic acid sequences of a bacterium, virus, and eukaryotic organism.

[0019] In any one of embodiments described herein, the genetic material is a tuberculosis virus.

[0020] In any one of embodiments described herein, the genetic material is a human gene.

[0021] In any one of embodiments described herein, the amplification agent comprises one or more recombinase polymerase amplification agent.

[0022] In any one of embodiments described herein, the amplification agent comprises one or more primers specific for the genetic material.

[0023] In any one of embodiments described herein, the binding agent is selected to generally bind to the genetic material.

[0024] In any one of embodiments described herein, the binding agent is selected to specifically bind to the genetic material.

[0025] In any one of embodiments described herein, the signaling chemical is the one of the binding agents. [0026] In any one of embodiments described herein, the binding agent is hexaamine ruthenium (III), osmium complexes, or a ferrocene-containing probe specific to the genetic material.

[0027] In any one of embodiments described herein, the signaling chemical is hexaamine ruthenium (III) or ferrocene.

[0028] In any one of embodiments described herein, the change of the concentration of the signaling chemical results in an increase or decrease current change readable by the electrochemical reader.

[0029] In any one of embodiments described herein, the one or more cellulosic layers comprising a first cellulosic layer comprising the sample deposition zone and a second cellulosic layer comprising the test zone.

[0030] In any one of embodiments described herein, the device further includes a spacer layer disposed between the first and second cellulosic layers, said spacer layer comprising an opening in alignment with at least a portion of the test zone and the sample deposition zone.

[0031] In any one of embodiments described herein, the electrochemical reader is a universal mobile electrochemical detector.

[0032] In any one of embodiments described herein, the electrochemical reader further includes one or more lysis agent embedded in the hydrophilic test zone or the sample deposition zone.

[0033] In any one of embodiments described herein, the electrochemical reader further includes a partial or complete circuitry in electrical communication with the electrode assembly.

[0034] In any one of embodiments described herein, the cellulosic layer comprises paper.

[0035] In any one of embodiments described herein, the test zone and the sample deposition zone are in fluidic communication through a connection channel.

[0036] In any one of embodiments described herein, the test zone and the sample deposition zone are defined by a fluid impermeable material in the cellulosic layer. [0037] In any one of embodiments described herein, the fluid-impermeable material includes polymerized photoresist.

[0038] In any one of embodiments described herein, the fluid-impermeable material further includes one or more lysis agent embedded in the hydrophilic test zone or the sample deposition zone.

[0039] In any one of embodiments described herein, the fluid-impermeable material further includes a partial or complete circuitry in electrical communication with the electrode assembly.

[0040] In any one of embodiments described herein, the device further includes an isothermal subsystem in contact with the test zone and configured to maintain a constant temperature of the test zone.

[0041] In any one of embodiments described herein, the temperature is from about 37° to about 45°.

[0042] In another aspect, a method of detecting a genetic material is described, including: providing the device of any one of the embodiments described herein; depositing a sample in the sample deposition zone; contacting the device with an electrochemical reader; and obtaining a readout by the electrochemical reader indicative of the presence or absence of the genetic material.

[0043] In any one of embodiments described herein, the method further includes lysing a cell in the sample. In any one of embodiments described herein, the method further includes maintaining a constant temperature of the test zone. In any one of embodiments described herein, the temperature is from about 37° to about 45°.

[0044] In any one of embodiments described herein, contacting the device with an electrochemical reader results in electrical contact between the electrode and the circuity of the electrochemical reader.

[0045] In yet another aspect, a kit is described, including: a device of any one of the embodiments described herein; and a first set of instructions for obtaining a readout by an electrochemical reader for the detection of the genetic material.

[0046] In any one of embodiments described herein, the first set of instructions includes instructions to deposit a sample containing a genetic material in the sample deposition zone, instructions to contact the device with an electrochemical reader, and/or instructions to maintain a constant temperature of the test zone. In any one of embodiments described herein, the temperature is from about 37° to about 45°.

[0047] In yet another aspect, a kit is described, including: a microfluidic, electrochemical device for detecting a genetic material, comprising: one or more cellulosic layers comprising at least one of a hydrophilic test zone and a hydrophilic sample deposition zone in fluid communication with each other; one or more cellulosic layers comprising a sample deposition zone and a test zone; and an electrode assembly comprising one or more electrodes in fluidic contact with the test zone; a first set of instructions for embedding, in the test zone or the sample deposition zone, one or more amplification agents selected for the amplification of a genetic material and one or more binding agents selected for binding the amplified genetic material to result in a change of the concentration of a signaling chemical; wherein the signaling chemical is selected to interact with the electrode to result in a current change readable by an

electrochemical reader; and at least a portion of the device is sized and arranged to be insertable into the electrochemical reader; and a second set of instructions for obtaining a readout by an electrochemical reader for the detection of the genetic material.

[0048] In any one of embodiments described herein, the second set of instructions includes instructions to deposit a sample containing a genetic material in the sample deposition zone, instructions to contact the device with an electrochemical reader, and/or instructions to maintain a constant temperature of the test zone. In any one of embodiments described herein, the temperature is from about 37° to about 45°. [0049] It is contemplated that any embodiment disclosed herein may be properly combined with any other embodiment disclosed herein. The combination of any two or more embodiments disclosed herein is expressly contemplated.

[0050] Unless otherwise defined, used or characterized herein, terms that are used herein (including technical and scientific terms) are to be interpreted as having a meaning that is consistent with their accepted meaning in the context of the relevant art and are not to be interpreted in an idealized or overly formal sense unless expressly so defined herein.

[0051] It will be understood that the spatially relative terms, as well as the illustrated configurations, are intended to encompass different orientations of the apparatus in use or operation in addition to the orientations described herein and depicted in the figures.

Spatially relative terms, such as "above," "below," "left," "right," "in front," "behind," and the like, may be used herein for ease of description to describe the relationship of one element to another element, as illustrated in the figures. For example, if the apparatus in the figures is turned over, elements described as "below" or "beneath" other elements or features would then be oriented "above" the other elements or features. Thus, the exemplary term, "above," may encompass both an orientation of above and below. The apparatus may be otherwise oriented (e.g., rotated 90 degrees or at other orientations) and the spatially relative descriptors used herein interpreted accordingly. Further still, in this disclosure, when an element is referred to as being "linked to," "on," "connected to," "coupled to," "in contact with," etc., another element, it may be directly linked to, on, connected to, coupled to, or in contact with the other element or intervening elements may be present unless otherwise specified.

[0052] The terminology used herein is for the purpose of describing particular embodiments and is not intended to be limiting of exemplary embodiments. As used herein, singular forms, such as "a" and "an," are intended to include the plural forms as well, unless the context indicates otherwise. Additionally, the terms, "includes," "including,"

"comprises" and "comprising," specify the presence of the stated elements or steps but do not preclude the presence or addition of one or more other elements or steps. Description of the Drawings

[0053] The invention is described with reference to the following figures, which are presented for the purpose of illustration only and are not intended to be limiting. In the Drawings:

[0054] Figs. 1 A-1C show three-dimensional schematics of the portable detector device described herein, in which Fig. 1A shows the device fitted into an electrochemical reader, e.g., a universal mobile electrochemical detector (uMED) portable device, according to one or more embodiments described herein; Fig. IB is an exploded view showing detailed diagrams of the device 100, according to one or more embodiments described herein; and. Fig. 1C shows the microfluidic subsystem 103 including disposable cellulosic layers 109 and 111, according to one or more embodiments described herein.

[0055] Fig. 2A is a schematic summarizing the steps of the recombinase polymerase amplification (RPA) reaction with real-time exonuclease probes, according to one or more embodiments described herein; Fig. 2B is a schematic showing the non-specific

electrochemical RPA approach, according to one or more embodiments described herein; and Fig. 2C is a schematic illustrating the sequence-specific electrochemical RPA approach, according to one or more embodiments described herein.

[0056] Fig. 3 A shows gel electrophoresis results (Agilent TapeStation™) of the RPA reaction, according to one or more embodiments described herein; and Fig. 3B shows realtime fluorescence results for RPA assays at variable amounts of genomic target DNA (500 pg, 50 pg, 5 pg and 500 fg, n=3), according to one or more embodiments described herein.

[0057] Figs. 4A-4D illustrate electroanalysis results comparing the performance of the universal mobile electrochemical detector (uMED) and a bench-top potentiostat at variable concentration of hexaamine ruthenium (III) in 40 mM Tris- Acetate buffer (pH7.4), according to one or more embodiments described herein, in which Figs. 4A and 4B show cyclic voltammograms collected on the bench-top potentiostat and the UMED, respectively

(100 mV scan rate, 5 mV steps), according to one or more embodiments described herein; and Figs. 4C and 4D show square wave voltammograms collected on the bench-top potentiostat and the UMED, respectively, (n=4, 50 μΐ., 14.7 Hz frequency, 50 mV amplitude, and 50 mV scan rate), according to one or more embodiments described herein. [0058] Figs. 5 A-5D show square-wave voltammograms collected on the uMED portable device using paper-based test strips at t=0 and t=20 minutes of the RPA reaction (25 μΐ., 14.7 Hz frequency, 50 mV amplitude, and 50 mV/s scan rate) having initial amounts of genomic DNA from M.smegmatis of 0, 1, 35 and 140 ng, respectively, according to one or more embodiments described herein.

[0059] Fig. 6 shows the redox couple for [Ru( H₃)₆]³⁺.

[0060] Fig. 7 shows the Electroanalytical results on the uMED portable device, according to one or more embodiments described herein.

[0061] Fig. 8 shows cyclic voltammograms collected on the uMED portable device (100 mV/s scan rate), according to one or more embodiments described herein.

[0062] Figs. 9A-9D show square-wave voltammograms collected on the uMED portable device using paper-based test strips at t=0 and t=20 minutes of the RPA reaction (25 μΐ., 14.7 Hz frequency, 50 mV amplitude, and 50 mV/s scan rate), at 10, 100, 250 and 500 μΜ of [Ru( H₃)₆]³⁺, respectively, according to one or more embodiments described herein.

[0063] Fig. 10A shows square-wave voltammograms collected on the uMED portable device using paper-based test strips at t=0 and t=20 minutes of the RPA reaction (25 μΐ., 14.7 Hz frequency, 50 mV amplitude, and 50 mV/s scan rate), according to one or more embodiments described herein; and Fig. 10B shows gel electrophoresis results (Agilent TapeStation™) for the same RPA reaction on the uMED portable device for 140 ng of smegmatis DNA, according to one or more embodiments described herein.

[0064] Fig. 11 shows electroanalytical results on the benchtop electrochemical analyzer.

Detailed Description

[0065] In one aspect, a microfluidic, electrochemical device for detecting a genetic material is described, including: one or more cellulosic layers comprising at least one of a hydrophilic test zone and a hydrophilic sample deposition zone in fluid communication with each other; one or more amplification agents selected for amplifying a genetic material and embedded in the hydrophilic test zone and/or the sample deposition zone; one or more binding agents embedded in the test zone and/or the sample deposition zone and selected for binding the amplified genetic material to result in a change of the concentration of a signaling chemical, wherein the signaling chemical is either embedded in the test zone or the sample deposition zone prior to the binding of the binding agent to the genetic material or is a newly generated product of the binding; and an electrode assembly comprising one or more electrodes in fluidic contact with the test zone and configured to interact with the signaling chemical to result in a current change readable by an electrochemical reader. Methods of using the device and kit containing the device are also described. In some embodiments, the sample deposition zone and the test zone may be the same zone or two different zones in fluidic communication with one another. In some embodiments, at least a portion of the electrode assembly of the electrochemical device is sized and arranged to be insertable into an electrochemical reader so that the electrode with the electrode assembly is in electrical contact with the electrochemical reader's circuitry. In some embodiments, the

electrochemical device also includes a partial or complete circuitry. The circuitry may be built-in, e.g., printed, in or on the one or more cellulosic layers and in electrical contact with the electrode assembly of the device.

[0066] During use, a user may deposit a fluid sample possibly containing a genetic material into the sample deposition zone. The source of the genetic material may be from any of the kingdoms (i.e., Plants, Animals, Protists, Fungi, Archaebacteria, Eubacteria), and be thus derived from a natural source, or be synthesized by means of biotechnology. The type of the genetic material may be: (1) naturally-derived double stranded DNA; (2) naturally- derived single stranded DNA, either sense or antisense strand; (3) synthetic double stranded DNA; (4) synthetic single stranded DNA, either sense or antisense stand; (5) naturally- derived single stranded RNA, either sense or antisense strand; (6) synthetic single stranded RNA, either sense or antisense strand. The genetic material may be a DNA or RNA, or a specific nucleic acid sequence (e.g., a specific tuberculosis genetic material) or a portion thereof. In certain embodiments, the genetic material is selected from the group consisting of the specific nucleic acid sequences of a bacterium, virus, and eukaryotic organism. Non- limiting examples of the genetic material include a tuberculosis virus and a human gene. Through microfluidic forces, the fluid sample is wicked into the test zone (e.g., through a hydrophilic channel connecting the test zone with the sample deposition zone), where one or more amplification agents and binding agents are embedded. In other embodiments, the test zone and the sample deposition zone may also be the same zone, so that the fluid sample is applied to the zone containing the amplification agents and binding agents. The test zone and the sample deposition zone may be in the same cellulosic layer or different cellulosic layers. In some embodiments, the test zone and the sample deposition zone are in direct contact (e.g., they are located on different cellulosic layers and are aligned vertically which renders them in direct contact). In certain embodiments, the one or more cellulosic layers comprise a first cellulosic layer comprising the sample deposition zone and a second cellulosic layer comprising the test zone. In other embodiments, the test zone and the sample deposition zone are in fluidic communication through a connection channel. In certain embodiments, the test zone, the sample deposition zone, and/or the connection channel are defined by a fluid impermeable material in the cellulosic layer, as explained in further detail below. Additional examples of suitable cellulosic layers are described in PCT application WO2008/049083, the contents of which are incorporated by reference.

[0067] In certain embodiments, the device further includes an isothermal subsystem in contact with the test zone and configured to maintain a constant temperature of the test zone. In certain embodiments, the constant temperature is about 30, 35, 38, 39, 40, 41, 42, 43, 44, 45, 46, 50°C, or in a range bounded by any of the two values disclosed herein. For example, the isothermal subsystem can be selected to operate at temperatures of about 30-50, 39-42, 39-45, or 37-42°C.

[0068] An exemplary microfluidic, electrochemical device disclosed herein is described with reference to Figs. l(a)-(c). A microfluidic, electrochemical device 100 upper lid 102 and a lower lid 115 for detection of genetic material as described in certain embodiments is shown in Fig. 1(a). As described in detail below, device 100 includes a microfluidic system 103 including amplification and detection reagents (e.g., signaling chemicals and/or biding agents), an isothermal subsystem, e.g., heater 105, for isothermal heating and circuitry for electrochemical detection. Device 100 is configured to be insertable into an electrochemical reader 101 for electronic readings. In certain embodiments, the electrochemical reader is a universal mobile electrochemical detector (μΜΕϋ). See, Nemiroski, A. et al.; Universal mobile electrochemical detector designed for use in resource-limited applications.

Proceedings of the National Academy of Sciences 2014, 111 (33), 11984-11989; the content of which is expressly incorporated by reference. In other embodiments, a commercially available glucose reader can be used. For instance, the current commercial CVS glucometer True trackTM, are fully automated and can be used in conjunction with the device disclosed herein. The use of other commercial electronic readers is also contemplated. The detection of signals such as electrical current, voltage, and other electric signals known in the art is contemplated.

[0069] Fig. 1(b) is an exploded view of the microfluidic, electrochemical device 100. The device 100 optionally includes a protective case that has an upper lid 102 and a lower lid 115 (Fig. 1(b)). Device 100 includes a microfluidic, electrochemical subsystem or device 103 that includes one or more cellulosic layers, which are described in greater detail below.

[0070] As shown in Fig. 1(b), subsystem 103 may be disposed in a holding layer 104 with an opening space 116 designed to hold 103. The device 103 may include an isothermal subsystem, e.g., a heating element 105 and thermocouple 124, in contact with the subsystem 103 and/or the test zone and configured to maintain a constant temperature of the subsystem 103 and/or the test zone. Heating element 105 has two contact pads 122 (one positive, one negative) that are wired to a port on the electrical port 123. Thermocouple 124 has two leads (one positive, one negative), which are wired (see dotted lines in Fig. 1(b)) directly to port

123. A connector 121 (for example, a 3-pin, modular contact) mates with the contacts in 103 and connects them to port 123. Port 123 connects 100 to the uMED to enable thermal regulation of heating element 105 based on temperature input received from thermocouple

124, and simultaneous electrochemical interrogation of electrochemical subsystem 103. The temperature may be maintained by a proportional-integral-derivative (PID) feedback loop that uses the temperature measured by the thermocouple to adjust the power delivered to the heating mat. The heating element may be, for example, a resistive heater or a thermoelectric heater.

[0071] As shown in Fig. 1(c), electrochemical subsystem 103 includes a first cellulosic layer 109 which includes one or more amplification agents in a sample deposition zone 112. Using cellulosic layer (e.g., paper)-based test strips reduces the sample volume and therefore cost of reagents. In some embodiments, the cellulosic layer is a patterned layer, e.g., a patterned paper layer. For example, the sample deposition zone 112 is a hydrophilic porous area in the cellulosic layer defined by a fluid-impermeable material 110 which permeates through the thickness of layer 109 and surrounds the sample deposition zone 112.

[0072] The one or more amplification agents are selected to specifically amplify a target genetic material. The target genetic material may be a specific DNA or RNA, or a specific nucleic acid sequence (e.g., a specific tuberculosis genetic material) or a portion thereof. In certain embodiments, the amplification agent is configured for recombinase polymerase amplification (RPA) of the target genetic material. RPA is an isothermal alternative to PCR that operates at a temperature, e.g., 39-45°C. RPA is insensitive to temperature variations (±1 °C) and rapid (10-15 minutes). The detail of the amplification and detection is described further below with reference to Figs. 2(a)-2(c).

[0073] In certain embodiments, the amplification agent is configured for PCR

amplification of the target genetic material. Other non-limiting examples of methods for the amplification of the target material include recombinase polymerase amplification (RPA), nucleic acid sequence-based amplification (NASBA), simple amplification- based assay (SAMBA) and loop-mediated isothermal amplification (LAMP).

[0074] Continuing with reference to Fig. 1(c), the device 100's subsystem 103 further includes a second cellulosic layer 111, with an electrode assembly 114 printed or attached onto layer 111. Electrode assembly 114 may contain two electrodes (cathode and anode). In other embodiments, electrode assembly 114 contains 3, 4, or more electrodes, e.g., positive, negative and reference electrodes. The electrodes in assembly 114, e.g., screen-printed electrodes (SPE) 119, connect electrically to a set of contact pads 125 from the test zone 118.

[0075] Optionally, a spacer layer 106 is disposed between layers 109 and 111. In certain embodiments, the spacer layer 106 is non-porous, e.g., plastic or glass. In certain

embodiments, the spacer can be made from double-sided tape to joint layers 109 and 111. This spacer includes an opening 113 to allow fluidic contact between portions of layers 109 and 111, e.g., the sample deposition zone 112 and the test zone 118. After a fluidic sample 107 is deposited in the sample deposition zone 112, an optional cover layer 108 may be placed on top of layer 109 to prevent fluid evaporation. The sample deposition zone 112 is in fluidic communication with a test zone 118 on layer 111. In certain embodiments, one or more amplification agents are embedded in the test zone 118 and/or the sample deposition zone 112. Amplification agents interact with the genetic material to provide copies of the genetic material using one of many methods disclosed herein. In certain embodiments, one or more binding agents are embedded in the test zone 118 and/or the sample deposition zone 112 and selected for binding the amplified genetic material to result in a change of the concentration of a signaling chemical. The test zone 118 is in fluidic communication with the electrode assembly 114. In some embodiments, a hydrophilic channel connecting the test zone to the electrode assembly fludically. In other embodiments, at least part of the electrode assembly is located in, printed in, or overlaps with the test zone. When the signaling chemical's concentration is changed as a result of the binding between the binding agents and the amplified genetic material, such a change may be detected by the electrochemical reader 101 to generate an electronic readout. Other variations in the arrangement of the cellulosic layers, sample deposition zone and detection zone are contemplated and will be apparent to one of skill in the art.

[0076] In some embodiments, the amplifying and binding reagents are selected for conducting a Recombinase Polymerase Amplification (RPA). RPA is an isothermal process, e.g., there is no need for temperature cycling in the amplification process. Because it is isothermal, RPA reactions are simpler to run than PCR, which needs a thermal cycler. This makes RPA a good candidate for low-cost, rapid, point-of-care molecular tests. Fig. 2a shows a schematic of the RPA reaction summarizing the steps of the RPA reaction with realtime exonuclease probes. Briefly, the RPA process employs three core enzymes - a recombinase, a single-stranded DNA-binding protein (SSB) and strand-displacing

polymerase. Recombinases are capable of pairing oligonucleotide primers with homologous sequence in duplex DNA. SSB binds to displaced strands of DNA and prevent the primers from being displaced. Finally, the strand displacing polymerase begins DNA synthesis where the primer has bound to the target DNA. By using two opposing primers, much like PCR, if the target sequence is indeed present, an exponential DNA amplification reaction is initiated. There is no other sample manipulation such as thermal or chemical melting is required to initiate amplification. At optimal temperatures (about 37-42°C), the reaction progresses rapidly and results in specific DNA amplification from just a few target copies to detectable levels, typically within 5 - 10 minutes. As shown in Fig. 2(a), in some embodiments, the one or more amplification agents include a recombinase-primer complex (with two primers A and B) and a Bsu polymerase. Double-stranded (ds) template DNA hybridizes with recombinase- primer complexes. Primers are extended by Bsu polymerase in D-loop structure from both 5' and 3' directions. This results in two copies of the original ds DNA.

[0077] In other embodiments, other amplification methods that produces ds DNA are incorporated into the device using a non-specific binding agent. Non-limiting examples of such methods include polymerase chain reaction (PCR), nucleic acid sequence-based amplification (NASBA), simple amplification- based assay (SAMBA), loop-mediated isothermal amplification (LAMP), rolling circle amplification (RCA), Strand Invasion Based Amplification (SIBA), helicase-dependent amplification (HDA), and strand displacement amplification (SDA). For the specific binding agent, the amplification method may include an enzyme with exonuclease III activity or endonuclease IV (e.g., RPA, nicking endonuclease signal amplification (NESA)/nicking endonuclease assisted nanoparticle activation

(NEANA)). Alternatively, an exonuclease III or endonuclease IV is added to the reaction mixture.

[0078] In some embodiments, the presence of a target genetic material is detected in the test zone, where it interacts with a binding agent. The binding interaction can be specific or non-specific. In certain embodiments, the binding of the amplified targeted genetic material with the one or more binding agents is not specific to the targeted genetic material. Thus, the non-specific binding agent binds to any DNA or RNA genetic material, without

differentiating between different DNA or RNA sequences or specific binding to any specific DNA or RNA sequences. In some specific embodiments, the non-specific binding agent itself is the signaling chemical, which optionally can interact with or bind to the electrode to increase or decrease the current. As a result of the binding, the concentration of the free binding agent (signaling chemical) will decrease and a change in the electric current can be detected. Fig. 2(b) is a schematic showing the non-specific electrochemical RPA approach. In certain embodiments, non-specific binding agent, e.g., hexaamine ruthenium (III), is used to bind to the groove of ds DNA. At the start of the RPA reaction, the amount of DNA present is negligible, allowing the signaling chemical, e.g., hexaamine ruthenium (III) ([Ru(NH3)₆]³⁺), to diffuse to the negatively charged surface of the electrode and to produce a high signal in current. As the reaction progresses, more ds DNA is synthesized and the concentration of the signaling chemical, e.g., hexaamine ruthenium (III), decreases, causing a decrease in current, which can be detected using an electrochemical reader. Other non- limiting examples of non-specific binding agents include methylene blue and osmium complexes (e.g., Os[(bpy)₂phen]²⁺). See, T. Defever, M. Druet, D. Evrard, D. Marchal, B. Limoges, Anal. Chem. 2011, 83, 1815-1821; the contents of which are incorpoeated herein by reference.

[0079] Thus, in certain embodiments, non-specific detection involves DNA intercalator hexaamine ruthenium (III), which binds to the groove of ds DNA via hydrogen-bonding and electrostatic interactions (intercalative stacking). The RPA reaction may be implemented with hexaamine ruthenium (III) and thus observe electrochemically its reversible one-electron reduction hexaamine ruthenium (III). At the start of the RPA reaction, the amount of DNA present is negligible, allowing the electroactive positively molecules to diffuse to the negatively charged surface of the electrode and to produce a high signal in current. As the reaction progresses, more ds DNA is synthesized and the concentration of free electroactive molecules decreases, causing a decrease in current.

[0080] In certain embodiments, the binding of the amplified targeted genetic material with the one or more binding agents is specific to the targeted genetic material. Thus, the specific binding agent binds only to a specific DNA or RNA sequence, without binding to any other DNA or RNA sequences. In some specific embodiments, the specific binding between the binding agents and the target specific genetic material is a chemical reaction which results in the formation of a signaling chemical which can interact with the electrode, e.g., bind to the electrode, to increase or decrease the current. As the binding reaction progresses, the concentration of the newly formed signaling chemical increases and a change in the electric current can be detected. In certain embodiments, the binding agent is a fluorescent oligonucleotide probe, which is complementary to the target genetic material sequence. Fig. 2(c) is a schematic illustrating the sequence-specific electrochemical RPA approach. In certain specific embodiments, the binding agent is a ferrocene-containing oligonucleotide probe, where the oligonucleotide is complimentary to the target genetic material sequence. Upon binding, the signaling chemical, e.g., ferrocene, is released. At the start of the reaction, electrostatic repulsion prevents the diffusion of the electroactive oligonucleotide probe to the electrode surface, producing a low signal in current. In certain embodiments, a cleaving agent is used to cleave the probe-genetic material complex to release the signaling chemical, e.g., the fluorescent ferrocene. Non-limiting examples of the cleaving agent include Exonuclease III, which cleaves the amplified DNA in the 3' to

5 'direction to yield nucleoside 5 'phosphates and ferrocene. During the course of the reaction, the concentration of free ferrocene molecules increases, causing an increase in the current detectable by the electrochemical reader. Other non-limiting examples of specific binding agents include biotinylated primers conjugated with an electroactive molecule like ferrocene binding to a streptavidin-coated electrode.

[0081] Thus, as used herein, "signaling chemical" refers to a chemical whose

concentration changes as a result of the binding between the binding agent and the amplified genetic material. In certain embodiments, the signaling chemical is a new chemical formed as the result of the binding, e.g., ferrocene. In certain embodiments, the signaling chemical is an existing chemical, e.g., the binding agent, whose concentration changes as a result of the binding, e.g., hexaamine ruthenium (III). Other non-limiting examples of the signaling chemicals include other ion complexes (e.g., osmium complexes such as Os[(bpy)₂phen]²⁺, [(bpy)₂DPPZ²⁺(Os)] DPPZ: dipyrido[3,2-a:2',3'-c] phenazine, Os-[(bpy)₂ DPPZ]²⁺, Os[(4,4'- dimethyl-bpy)₂ DPPZ]²⁺, and Os[(4,4'-diamino-bpy)₂ DPPZ]²⁺ (with bpy = 2,2' -bipyridine, phen =phenanthroline, and DPPZ = dipyrido[3,2-a:2',3'-c]phenazine)), ruthenium complexes such as [Ru( H3)₆]³⁺, and iron complexes such as [Fe(CN)₆])⁴⁺ and Fe(phen)₃ ²⁺.

[0082] In some embodiments, the RPA-based amplification approach as described herein can be controlled with components lower in cost than those required by PCR. In some embodiments, the cellulosic layer (e.g., paper)-based 3D microfluidic device for multiplexed assay has several significant features: (i) it is fairly inexpensive, in terms of cost for materials of devices (paper is the main substrate) and the electrochemical reader; it may be used with well-developed, commercially available, and inexpensive readers such as the glucose meters, without spending a large amount of expenditure or sophisticated engineering work to develop new electrochemical readers; (ii) it provides higher density of biomarker information than other POC devices commercially available; (iii) it is portable; (iv) cavity valves (e.g., opening 113 in Fig. 1(c)) can be included in such a device to control fluids, without the use of any complicated pneumatic setup and external electric source; and (v) it can provide multiple replicas of data from a single run assay.

[0083] In another aspect, a method of detecting a genetic material is described, including: providing the device of any one of the embodiments described herein; depositing a sample in the sample deposition zone; contacting the device with an electrochemical reader; and obtaining a readout by the electrochemical reader indicative of the presence or absence of the genetic material.

[0084] In some embodiments, the method further includes maintaining a constant temperature of the test zone. In some embodiments, the temperature is from about 37° to about 45°. In some embodiments, the method further includes lysing a cell in the sample. [0085] In some embodiments, contacting the device with an electrochemical reader results in electrical contact between the electrode and the circuity of the electrochemical reader.

[0086] In yet another aspect, a kit is described, including: a device of any one of the embodiments described herein; and a first set of instructions for obtaining readout by an electrochemical reader for the detection of the genetic material.

[0087] In some embodiments, the first set of instructions includes instructions to deposit a sample containing a genetic material in the sample deposition zone, instructions to contact the device with an electrochemical reader, and/or instructions to maintain a constant temperature of the test zone. In some embodiments, the temperature is from about 37° to about 45°.

[0088] In yet another aspect, a kit is described, including: a microfluidic, electrochemical device for detecting a genetic material, comprising: one or more cellulosic layers comprising at least one of a hydrophilic test zone and a hydrophilic sample deposition zone in fluid communication with each other; one or more cellulosic layers comprising a sample deposition zone and a test zone; and an electrode assembly comprising one or more electrodes in fluidic contact with the test zone; a first set of instructions for embedding, in the test zone or the sample deposition zone, one or more amplification agents selected for the amplification of a genetic material and one or more binding agents selected for binding the amplified genetic material to provide in a change of the concentration of a signaling chemical; wherein the signaling chemical is selected to interact with the electrode to result in a current change readable by an

electrochemical reader; and at least a portion of the device is sized and arranged to be insertable into the electrochemical reader; and a second set of instructions for obtaining a readout by an electrochemical reader for the detection of the genetic material. [0089] In some embodiments, the second set of instructions includes instructions to deposit a sample containing a genetic material in the sample deposition zone, instructions to contact the device with an electrochemical reader, and/or instructions to maintain a constant temperature of the test zone. In some embodiments, the temperature is from about 37° to about 45°.

[0090] Porous, hydrophilic or cellulosic layers include any hydrophilic substrate that wicks fluids by capillary action. In one or more embodiments, the cellulosic layer is paper. Non-limiting examples of cellulosic layers include chromatographic paper, filter paper, nitrocellulose and cellulose acetate, cellulosic paper, filter paper, paper towels, toilet paper, tissue paper, notebook paper, Kim Wipes, VWR Light-Duty Tissue Wipers, Technicloth Wipers, newspaper, any other paper that does not include binders, cloth, and porous polymer film. In general, any paper that is compatible with the selected patterning method may be used. In certain embodiments, porous, hydrophilic layers include Whatman chromatography paper No. 1.

[0091] Electrodes can be attached or taped on the paper layer to enable contact with the test zone. In other embodiments, electrodes can be screen-printed electrodes using conductive carbon ink, and circuitry, e.g., wires, can be screen-printed using silver ink because of its good conductivity. Carbon ink can also be used for wire material as well. The electrodes made from conductive ink have several advantages: (i) they are less expensive, compared to Au or Pt electrodes; (ii) the fabrication process is simple, and has less requirements on cleanroom facilities; (iii) those materials are well developed, and easy to obtain, because they are widely used in both industrial and academic research; and (iv) screen printing is capable of mass production at low cost.

[0092] In some embodiments, the electrode and the hydrophilic regions can be treated with chemicals to increase the hydrophilicity. Non-limiting examples of such chemical agents include 3-aminopropyldimethylethoxysilane (APDES).

[0093] In some embodiments, the cellulosic layer is patterned by a fluid impermeable (e.g., water-impermeable) material. In some embodiments, paper is used as the substrate for electrochemical detection and can be patterned using wax printing. In certain embodiments, the fluid-impermeable material permeates through the thickness of the cellulosic layer and defines the test zone, the sample deposition zone, and/or a connection channel through which the test zone and the sample deposition zone are in fluidic communication.

[0094] Non-limiting examples of fluid-impermeable material comprise wax and polymerized photoresist. The photoresist used for patterning porous, hydrophilic material include SU-8 photoresist, SC photoresist (Fuji Film), poly(methylmethacrylate), nearly all acrylates, polystyrene, polyethylene, polyvinylchloride, and any photopolymerizable monomer that forms a hydrophobic polymer.

[0095] In some embodiments, patterned paper-based microfluidic channels, test zone, and/or sample deposition zone can be fabricated by wax-printing on chromatography paper. The fabrication process is simpler and less expensive, compared to the photolithography technique used in cleanroom. The testing strips are sized and shaped to be insertable into a commercial glucose meter.

[0096] Further examples of the electrode assembly and cellulosic layers patterned by fluid-impermeable materials that define one or more hydrophilic channels or regions on the patterned cellulosic layer can be found in PCT Application No. US 12/53930, the content of which is incorporated in its entirety by reference. The method of preparing patterned hydrophilic layers is described in detail in PCT Publication No. 2008/049083, the content of which is incorporated in its entirety by reference. The method of preparing a microfluidic device including one or more electrode assemblies is described in details in PCT Application Nos. PCT/US2010/026499 and PCT/US2010/26547, the contents of which are incorporated in their entirety by reference.

[0097] In certain specific embodiments, a low-cost, portable device for amplification and detection of nucleic acids is described, using isothermal amplification of DNA coupled with electrochemical readout. The device may comprise: (i) consumable paper strips with preserved reagents for isothermal amplification and (ii) a handheld electronic controller for thermoregulation and electrochemical detection.

[0098] In certain specific embodiments, for amplification, recombinase polymerase amplification (RPA) is used, which is an isothermal alternative to PCR that operates at 39- 45°C, is insensitive to temperature variations (±1 °C), and is rapid (10-15 minutes). The reaction is chemically initiated with the addition of magnesium acetate to the mixture. See, Figs. 2(a)-(c). These characteristics make RPA a candidate for applications that target resource-limited environments.

[0099] This approach to amplification can be controlled with components lower in cost than those required by PCR. Using paper-based test strips enables us to reduce the sample volume and therefore cost of reagents. In some embodiments, electrochemical detection is used for measurement, because of its sensitivity and compatibility with inexpensive analyzers for point-of-care use, such as the recently developed universal mobile electrochemical detector (uMED). See, Nemiroski, A. et al; Universal mobile electrochemical detector designed for use in resource-limited applications. Proceedings of the National Academy of Sciences 2014, 111 (33), 11984-11989; the content of which is expressly incorporated by reference.

[0100] In some embodiments, by adding closed-loop circuitry and software for thermoregulation and developing custom-made, disposable, paper-based test strips that mate with an advanced version of the uMED to perform the test at the point-of-care, the

functionality of the universal mobile electrochemical detector (uMED) is enhanced. The use of paper-based test strips enabled us to reduce the sample volume and therefore the cost of the reagents. With appropriate components and algorithms to control the amplification of DNA and perform electrochemical detection automatically, the test is able to set and stabilize the temperature of the RPA reaction to within 0.1 °C, in addition to providing full capabilities for electrochemical readout using a variety of pulse sequences (e.g., cyclic and square-wave voltammetry), on screen readout, and capabilities for interfacing the acquired data to "the cloud" using any phone. In certain embodiments, the uMED measures the current as a function of time during an electrochemical reaction. In the case of square-wave voltammetry, it extracts the position and height of the oxidation or reduction peak and correlates that to a concentration of the analyte, using saved calibration data. The uMED then displays this information to the user on the screen. The uMED provides full capabilities for

electrochemical detection using a variety of pulse sequences (e.g., CV and SWV), on-screen readout, and versatile capabilities for interfacing the acquired data to "the cloud" by communicating through audio, USB, or Bluetooth. In certain embodiments, to communicate the data to the cloud, the uMED may encode any acquired or computed data into a series of frequency tones and transmit them through an audio cable connected to audio port of a cellular phone. The uMED may also communicate with a computer by universal serial bus (USB). In other embodiments, an added radio (e.g., Bluetooth, WiFi, Zigbee) enables the uMED to communicate wirelessly through radio frequency communication with a computer or local area network.

[0101] In some specific embodiments, it is shown that coupling isothermal amplification of deoxyribonucleic acid (DNA) with electrochemical readout enables accurate and rapid detection of DNA. Additionally, an affordable, handheld device is disclosed herein in certain embodiments that couples to custom-made, disposable, paper-based test strips and screen- printed electrodes (SPE) to perform these analyses at the point-of-care.

Electrochemical Detection of Tuberculosis

[0102] The low-cost portable device for DNA detection could easily be modified for similar applications by simply altering the reagents on the disposable paper strips. In some embodiments, the device disclosed herein combines recombinase polymerase amplification — an isothermal alternative to the polymerase chain reaction- with an electroactive mediator to enable simple and accurate detection of DNA in the field using a custom-made, low-cost, portable electrochemical analyzer.

[0103] In some embodiments, the performance of this system is demonstrated in the electrochemical detection of tuberculosis. The device disclosed herein has been

demonstrated for the detection of Mycobacterium smegmatis as a simulant for tuberculosis (TB). TB is the second most lethal infectious disease globally and is caused by a single agent, M.tuberculosis. Early diagnoses of active TB and characterization of latent and silent TB infections can help to control the disease. Present assays available for TB point-of-care diagnosis commonly use nucleic acid or immunological analytical methods.

[0104] In some specific embodiment, a bench-top RPA assay that targets all members of genus Mycobacterium was designed and optimized. Mycobacterium smegmatis is used as a surrogate strain for simulant forM tuberculosis. We performed RPA on the custom-made disposable strips and investigated end-point detection by using two approaches: (i) a fluorescence exonuclease III RPA oligonucleotide probe and (ii) an electroactive RPA oligonucleotide probe.

[0105] In some embodiments, the device disclosed herein using benchtop RPA assay is used to detect 19 members of genus Mycobacterium. Our results on the portable system confirmed that we can electrochemically detect DNA from M. tuberculosis using the same assay.

[0106] In certain specific embodiments, this device as disclosed herein is a handheld device having one or more of the following attributes: (1) disposable, paper-based strips that incorporate screen-printed carbon electrodes, (2) thermoregulation at ±0.1°C temperature accuracy, and (3) electrochemical detection in various pulse sequences, such as square-wave and cyclic voltammetry. In certain specific embodiments, detection of about or lower than about 40 pg/μί of genomic DNA from Mycobacterium smegmatis (a surrogate forM tuberculosis— the main cause of tuberculosis), and M tuberculosis is achieved.

[0107] In certain specific embodiments, a fully integrated, point-of-care device for isothermal DNA amplification combined with electrochemical detection is described. We used the RPA reaction for DNA amplification (e.g., Fig. 2a); we chose RPA because the cost of the components for thermoregulation is lower than those required by PCR. In certain specific embodiments, the reaction with [Ru(NH₃)6]³⁺ is used as an electroactive mediator for the electrochemical detection of DNA (e.g., Fig. 2b), and the electrochemical detection coupled with RPA is demonstrated on the same device. Fig. 6 shows the redox couple for [Ru(NH₃)₆]³⁺. When the ruthenium complex binds with dsDNA, the concentration of free [Ru(NH₃)₆]³⁺ drops in proportion to the concentration of DNA present in the initial sample; this drop produces a decrease in the cathodic current measured by, for example, SWV. We chose electrochemical detection because of its speed, sensitivity and compatibility with inexpensive analyzers for point-of-care use, such as the universal mobile electrochemical detector (uMED) previously developed in our laboratory, because it is insensitive to color or particulates in the sample, and because it allows easy transfer of information electronically.

[0108] Thus, in some embodiments, the devices disclosed herein utilize isothermal amplification and electrochemical detection of DNA provide low-cost, portable detection suitable for resource-limited settings and not relying on the use of expensive benchtop equipment. The procedures and instrumentation necessary to detect nucleic acids is greatly simplified. In certain specific embodiments, the device combines isothermal amplification (RPA) with electrochemical readout of redox-active ([Ru(NH₃)₆]³⁺) to enable the entire process to be performed automatically by a low-cost ($30) device. In certain embodiments, the microfluidic subsystem (e.g., 103 in Fig. 1(b); also referred to as test strip) is disposable. The microfluidic subsystem enables pre-storage of all reagents needed for RPA as well as the redox probe, and yields sensitive and accurate measurements of low-concentration DNA. Using low-cost electrodes also enables a simplification of the process for disposal of potentially harmful or pathogenic samples, and, through reduction of the volume of reagents consumed, decreases the cost of consumable components. I n certain embodiments, as low as 5 pg of DNA per sample can be detected with the device disclosed herein.

[0109] In some embodiments, electroactive sequence-specific probes is used with the RPA assay to increase the sensitivity. In some embodiments, lysis agent (e.g., guanidinium thiocyanate) is used to enable the use of the device or microfluidic subsystem disclosed herein for samples of whole blood.

[0110] The foregoing and other features and advantages of various aspects of the invention(s) will be apparent from the following, more-particular description of various concepts and specific, non-limiting embodiments within the broader bounds of the

invention(s). Various aspects of the subject matter introduced above and discussed in greater detail below may be implemented in any of numerous ways, as the subject matter is not limited to any particular manner of implementation. Examples of specific implementations and applications are provided primarily for illustrative purposes.

Experimental

Materials

[0111] All chemicals, unless otherwise stated, were purchased from Sigma Aldrich (Montana, USA) and were of molecular biology grade. All solutions were prepared in sterile glassware or certified nuclease-free plasticware. All pipette tips were sterile aerosol filter tips.

Cell Cultures

[0112] M. smegmatis strain ATCC 700084 was purchased from the American Type Culture Collection (ATCC, Virginia, USA) as a lyophilized pellet. The pellet was rehydrated in sterile 7H9 broth base. The broth base consisted of 5 g/L Middlebrook 7H9 solid broth base, 0.2% (v/v) glycerol, 10% (v/v) albumin dextrose catalase Middlebrook growth supplement (ADC), 1 mM CaCl₂, 50 μg/mL carbenecillin and 10 μg/mL cycloheximide. An aliquot of the cell suspension was spotted onto a 7H10 agar plate and grown in a static incubator (C24, New Brunswick Scientific, Connecticut, USA) at 37°C for four days until colonies formed. Each 7H10 agar plate consisted of 10 mL of a sterile solidified solution of 20 g/L 7H10 agar powder, 0.5% (v/v) glycerol, 10% (v/v) oleic acid albumin dextrose catalase Middlebrook growth supplement (OADC), 1 mM CaCl₂, 50 μg/mL carbenecillin and 10 μg/mL cycloheximide. One colony from the agar plate was inoculated into 50 mL of 7H9 broth base enriched with 0.05% (v/v) Tween 80. The culture was grown in an incubator shaker (C24, New Brunswick Scientific, Connecticut, USA) at 37°C for three days. Finally, an aliquot (1 mL) of this culture was used to inoculate 50 mL of the 7H9 broth base and the resulting solution was grown in an incubator shaker at 37°C overnight. The following day, the culture was used for DNA extraction and purification.

Cell Lysis and DNA Extraction

[0113] An aliquot (2 mL) of theM smegmatis was lysed using a custom-made lysis buffer for Gram-positive bacteria (20 mM Tris-HCl, 2 mM sodium EDTA, 1.2% Triton X- 100, 20 mg/mL lysozyme). Using the DNeasy kit (Qiagen, California, USA), we next extracted the DNA of the lysate following the instructions of the manufacturer. We finally eluted the purified DNA in 100 iL of nuclease-free water (Ambion® RT-PCR Grade Water, Life Technologies, New York, USA). The yield and purity of DNA was assessed by UV-VIS spectrophotometry (Nanodrop 2000c, Thermo Scientific, New York, USA). Specifically, we used the ratio between the absorbance peaks at 260 nm and 280 nm to assess DNA purity. The intensity of the peak at 260 nm was used to estimate the yield of DNA. BEI Resources (NIAID, NIH) provided genomic DNA from Mycobacterium tuberculosis, strain H37Rv, NR-48669, free of charge.

Design and Bench-top Validation of the RPA Assay

[0114] The target sequence was a 213-nucleotide region within the 16S rRNA sequence of genus Mycobacterium. Using the Primerslist open source software, we designed the primer sequences and checked in silico for self- and cross-dimers. Table 1 lists the sequences that we used. We purchased the primers from Integrated DNA Technologies (IDT, Iowa, USA) and ran the DNA amplification reaction (RPA Basic Kit, TwistDX, Cambridge, UK) in 50-μΕ reaction volumes according the instructions of the manufacturer.

Table 1: The sequences of the primers and target DNA used in the RPA assay.

Name DNA Sequence (GenBank X52922.1 ) Forward primer 5'-TGA GTA AC A CGT GGG TGA TCT GCC CTG CAC TTT GG-3'

Reverse primer 5'-AGT CCC AGT GTG GCC GGT CAC CCT CTC AGG CCG GC-3'

Target sequence 5' -TGA GTA AC A CGT GGG TGA TCT GCC CTG CAC TTT GGG ATA AGC CTG GGA AAC TGG GTC TAA TAC CGA ATA CAC CCT GCT GGT CGC ATG GCC TGG TAG GGG AAA GCT TTT GCG GTG TGG GAT GGG CCC GCG GCC TAT CAG CTT GTT GGT GGG GTG ATG GCC TAC CAA GGC GAC GAC GGG TAG CCG GCC TGA GAG GGT GAC CGG CCA CAC TGG GAC T-3'

* The target DNA sequence is a 213-nucleotide region within the 16S rRNA sequence of genus Mycobacterium of 16S rRNA M smegmatis gene (GenBank X52922.1).

[0115] To validate the RPA assay, we performed fluorescence measurements in real time on a commercial benchtop system for PCR-based detection (CFX96, Bio-Rad, Massachusetts, USA). We used SYBR Safe (lx, Life Technologies, New York, USA) as a fluorescent realtime intercalator. To validate the electrochemical version of the RPA assay, we performed electrochemical measurements using a commercial, benchtop electrochemical analyzer (AutoLab PGSTAT12, Metrohm Florida, USA). For the electrochemical RPA experiments, the standard benchtop RPA reaction was implemented with 250 μΜ [Ru(NH₃)₆]Cl₃.

Paper-based RPA for M. smegmatis DNA on Paper Devices

[0116] We adapted the 50-μΤ bench-top RPA reaction to enable use with smaller volumes of 25 [iL on paper. The paper-based strips provided a disposable matrix to pre-store all reagents as dehydrated powders. In addition to the previously mentioned reagents, we added 8% v/v of preservative (DNA Stable™ Plus, Biomatrica, Sigma Aldrich, Montana, USA). The buffer was dehydrated in vacuo at room temperature for 3 hours. Prepared devices were stored in air-sealed pouches at 4°C until use. After the addition of the DNA sample on the test zone, we sealed the test zone with PCR polyethylene transparent tape (PCR sealing tape, Corning, Sigma Aldrich, Montana, USA).

Design and Fabrication of Portable Device For Thermoregulation and Electrochemical Detection

[0117] Figs. l(a)-(c) describe the main components of the portable device. We fabricated an advanced version of the uMED with additional temperature control through a

proportional-integral-derivative (PID) loop implemented in the Arduino programming language and executed by the same microcontroller (Atmega328, ATMEL, California, USA) that runs the uMED. The heating subsystem consists of a flexible, resistive heating mat (0.75 mm thick, Kapton T-36067-00, Cole Parmer, Illinois, USA) powered by a high-voltage boost converter (TPS61170, Texas Instruments) to convert the 3.3 V battery supply to the voltage needed to power the heater with output current controlled by an NPN transistor

(MMBT2222A-TP, Micro Commercial Components, California, USA). To tune the flow of current through the heater (and therefore, the temperature), we connected the gate of the transistor to a digital output from the microcontroller, and used pulse-width modulation to control the duty-cycle of the applied voltage. The temperature sensing subsystem consists of a temperature-to-voltage converter (MAX31855K, Maxim, California, USA) that

communicates with the microcontroller by the serial-peripheral-interface protocol and is connected to the thermocouple inside the heating module through a seven-pin connector. The microcontroller uses the measured temperature as input to a software-based PID loop, and uses pulse-width modulation to adjust the temperature of the heater. Using this device, we routinely achieved ±0.1°C temperature stability at the set-point temperature (39.0°C) within two minutes of initial activation of the PID control.

Design and Fabrication of the Heating Module to Interface with the Paper-based Test Strip

[0118] The heating module was designed in SolidWorks (Dassault Systemes,

Massachusetts, USA) and fabricated the components from acrylonitrile butadiene styrene (ABS) plastic by 3D printing (Fortus 250mc, Stratsys, Minnesota, USA). The base of the heating module included a slot for the test strip, as well as space for a flexible, resistive heating mat (0.75-mm thick, Kapton T-36067-00, Cole Parmer, Illinois, USA), and a seven- pin connector to mate with the uMED. The lid contained a slot for the thermocouple (61161- 372, VWR, Chicago, USA), and a three-pin modular contact (70AAJ-3-M0G, Bourns Inc., California, USA) to connect to the SPE on the test strip, when the lid was closed. The module also included space to run wires that interconnect the thermocouple, heating mat, and modular contact with the seven-pin connector.

Design and Fabrication of Paper -based Test Strips

[0119] The test strips consisted of a stack of the following four components: i) a commercial, ceramic substrate with SPE (DRP-110, manufactured by Dropsens (Llanera, Spain) and supplied by Metrohm, (Florida, USA)); ii) a spacer layer formed from double- sided tape (Flexmount® Select™ DF052521, Flexon, New York, USA) with a 1.8-mm diameter hole cut by a laser cutter (Epilog Mini 18, Epilog Laser Systems, Colorado, USA) aligned with the electrode test area; and iii) a disposable, paper-based test strip composed of Whatman cellulose chromatography paper (1 Chr, 200 x 200 mm, Sigma Aldrich, Montana, USA) with wax barriers, printed by a Xerox ColorCube 8870W printer, defining the reagent storage area. The spacer layer formed from double-sided tape enabled the cellulose layer to be fastened adhesively to the ceramic electrode. We conditioned each new SPE by first performing a CV on a sample of Tris Acetate buffer (40 mM, pH7.4). We rinsed the electrode with ultrapure deionized water and dried with N₂. The paper and tape layers were then attached on the SPE and the paper-based test strip was fabricated. Fig. 1(c) shows the different layers of the test strip.

Design and Validation of RPA assay

[0120] After identifying a 213-bp region common to bothM tuberculosis and M smegmatis, we designed appropriate primers for the RPA assay to amplify this sequence. To determine the efficacy of the RPA assay, we performed the assay in bulk solution with a benchtop thermocycler and confirmed the efficiency of the amplification process with gel electrophoresis (Agilent TapeStation™). Fig. 3a summarizes the electrophoresis results. The 213-bp band in the RPA reactions confirms that efficient amplification of the reaction product occurred and that the forward primer (positions 69-103 of 16S rRNAM smegmatis gene, GenBank X52922.1) and the reverse primer (positions 248-282) successfully amplified the target DNA sequence. We also confirmed that our assay amplified the same target sequence in Mycobacterium tuberculosis (Strain H37Rv, GenBank AL123456). We next tested the real-time efficiency using a fluorescent DNA intercalating dye. Fig. 3b shows realtime RPA results for four different concentrations of genomic DNA on a benchtop fluorescence thermocycler. The threshold of detection (TOD) was at 22±2.1 AU of fluorescence. This value was calculated as 3σ above the average of three negative control experiments. The minimum concentration of genomic DNA detected above the TOD was 5 Pg-

Validation of the Electrochemical Probe

[0121] To characterize the electrochemical properties of [Ru( H₃)₆]³⁺, we first performed a titration curve at different concentrations in Tris-Acetate buffer (40 mM, pH7.4). For each measurement, we added a 50-μΙ. droplet of the sample, which contained the mycobacterial DNA, onto a bare SPE. Using Figure 4a as an example, at 1 mM of [Ru(NH₃)₆]³⁺, we observed two processes: (1) a cathodic peak at -0.279 V, and (2) an anodic peak at -0.173 V. The difference between peak potentials (E_pa-Ep_C = 70 mV) and the ratio between peak currents (z_pa/z_Pc =-0.7) indicate the presence of an almost reversible process. Figs. 4(a)-(b) shows CVs and SWVs comparing measurements performed by the uMED and a benchtop analyzer for different concentrations of [Ru(NH₃)₆]³⁺ and establish the equivalence between measurements performed with both. The intensity of the peak current (either anodic or cathodic in CV) was proportional to the concentration of [Ru(NH₃)₆]³⁺. Calibration curves were linear in the range of concentrations tested (R² >0.99). Figure 11 shows these calibration curves for z_pa and z_pc. Although CV could be used as the basis for analysis, SWV is more sensitive, and we have thus chosen this technique for recording the current due to the electron transfer processes involving [Ru(NH₃)₆]³⁺.

Design and Validation of Paper-based Test Strip

[0122] We designed and fabricated a paper-based test strip that included the SPE and layers of paper and tape. We first evaluated the effect of the paper-layer on the

electrochemical performance of the redox probe. Fig. 7 shows SWV for the same

concentration of the redox probe on the disposable paper-based test strip vs. a liquid droplet on the bare SPE measured by the uMED, specifically, square-wave voltammograms (n=2, 50 μΐ,, 14.7 Hz frequency, 50 mV amplitude, and 50 mV/s scan rate) of 250 μΜ [Ru(NH₃)₆]³⁺ in Tris-Acetate buffer (pH 7.4) measured in a liquid droplet and on a disposable paper strip. We observed that using the paper matrix reduces the anodic peak by approximately 8.9% and shifts it by approximately 33.5 mV, compared to the case of the free droplet. Maximum current in SWV is proportional to the square root of D₀. Most likely, paper has this effect because of the [Ru(NH₃)₆]³⁺ having a lower diffusion coefficient (D₀) in a porous matrix than in the homogeneous liquid droplet. DNA Detection by RPA with Electrochemical Readout on a Portable Device

[0123] To detect DNA with our approach, we first prepared a paper-based test strip that included the necessary reagents {i.e., RPA reagents, and [Ru(NH₃)₆]³⁺). We then performed the analysis by: i) adding a 25-μΙ. sample that contained Mycobacterium DNA to the reagent zone, ii) affixing a cover layer to prevent evaporation, iii) inserting the test strip into the heating module and closing the lid, and iv) connecting the module to the uMED to perform the analysis. The uMED then executed the following sequence automatically: i) it performed SWV to measure the background concentration of the redox probe at t = 0 min, ii) it controlled the RPA reaction by incubating the test strip at 39°C for 20 min, and iii) it performed SWV again to measure the reduction in the unbound concentration of the redox probe.

[0124] We programmed the uMED using a desktop computer to send all raw data to a custom application written in MATLAB (Mathworks) and also to perform all relevant signal analysis (e.g., baseline corrections) automatically. The uMED displays the measured signal of maximum measured current to the user. For an initial peak current I₀ at t = t₀ and a final peak current ly at time t, we define the signal s(t) = [1 - I(t) 11₀] x 100% as the % decrease of peak current vs. time. This type of quantification has been previously used for detecting DNA, for example, by Won et al (Won, B. Y.; Shin, S.; Baek, S.; Jung, Y. L.; Li, T.; Shin, S. C; Cho, D.-Y.; Lee, S. B.; Park, H. G. Analyst 2011, J 36 (8), 1573-1579) in real-time electrochemical PCR and by Ahmed et al. (Ahmed, M. U.; Nahar, S.; Safavieh, M.; Zourob, M. Analyst 2013, 138 (3), 907-915) in real-time LAMP. We used t₀ = 5 min because the reaction mastermix took this time to reach 39°C.

Implementation of the Electrochemical Probe to the RPA Reaction on Paper

[0125] We implemented the electrochemical probe, [Ru(NH₃)₆]³⁺, to the RPA reaction, and studied its electrochemical behavior. Fig. 8 shows CVs of the reaction mastermix in the presence and absence of 1 mM of [Ru(NH₃)₆]³⁺. We observed significant double layer capacitance in the CV for the mastermix. The CV for [Ru(NH₃)₆]³⁺ showed (1) a cathodic peak at -0.038 V, and (2) an anodic peak at 0.023 V), in addition to the same double layer charging. We then performed the same experiment at different concentrations of the

[Ru(NH₃)₆]³⁺ to identify the optimal concentration of the probe for the RPA assay.

Figs. 9(a)-9(d) show SWVs at 10, 100, 250 and 500 μΜ of [Ru(NH₃)₆]³⁺, respectively, with

35 ng DNA as the initial amount before initiation of the RPA reaction. We found that a concentration of 250 μΜ [Ru(NH₃)₆] yielded the largest % decrease in current for SWV measurement, and therefore, provided the optimal sensitivity for detection of DNA.

[0126] We observed two electrochemical processes that occurred during the RPA reaction when implemented with [Ru(NH₃)₆]³⁺. We hypothesize that the process at 0.15 V is diffusion-controlled, while the process at -0.15 V is surface-confined. The diffusion controlled process is caused by electron transfer of free [Ru(NH₃)₆]³⁺ that diffuses to the electrode. The surface confined process is due to [Ru(NH₃)₆]³⁺ adsorbing to dsDNA and proteins. At the start of the RPA reaction, the amount of DNA present was negligible, allowing for the electroactive molecules to diffuse into the negatively charged surface of the electrode and to yield a high reduction signal. As the reaction progressed, more dsDNA was synthesized and the concentration of free electroactive molecules decreased, causing a decrease in current. We used the signal decrease to the 0.15 V peak current to quantify the initial amount of DNA present. This decrease was expected because of binding of the redox probe to dsDNA by hydrogen-bonding and electrostatic interactions (intercalative stacking). These interactions decrease the number of probe molecules available for reduction at the surface of the electrode.

Detection ofM. smegmatis andM. tuberculosis on Paper on a Portable Device

[0127] We performed the reaction on the paper-based test strip with different initial concentrations of genomic target DNA present at the start of the reaction. Figs. 5(a)-(d) show SWVs at 0, 1, 35 and 140 ng of M smegmatis DNA with 250 μΜ of [Ru(NH₃)₆]³⁺. We observed the two peaks at 0.15 V and -0.15 V, and the former was relative to the initial amount of DNA present; the more the DNA, the higher the peak current at -0.15 V. We used the peak at -0.15 V to calculate the % decrease of peak current. Fig. 10a shows the SWV for an RPA reaction with 140 ng of genomic DNA from M.smegmatis performed on the uMED on a paper-based test strip. We calculated an s(t) of 100%. We also collected an aliquot of the same RPA reaction, and confirmed efficient amplification by gel electrophoresis

(Fig. 10b).

[0128] Upon review of the description and embodiments described above, those skilled in the art will understand that modifications and equivalent substitutions may be performed in carrying out the invention without departing from the essence of the invention. Thus, the invention is not meant to be limiting by the embodiments described explicitly above.

Claims

Claims We claim:

1. A microfluidic, electrochemical device for detecting a genetic material, comprising: one or more cellulosic layers comprising at least one of a hydrophilic test zone and a hydrophilic sample deposition zone in fluid communication with each other; one or more amplification agents selected for amplifying a genetic material and embedded in the hydrophilic test zone or the sample deposition zone; one or more binding agents embedded in the test zone or the sample

deposition zone and selected for binding the amplified genetic material to provide a change of concentration of a signaling chemical, wherein the signaling chemical is either embedded in the test zone or the sample deposition zone prior to the binding or is a newly generated product of the binding; and an electrode assembly comprising one or more electrodes in fluidic contact with the test zone and configured to interact with the signaling chemical to result in a current change readable by an electrochemical reader.

2. The device of claim 1, wherein at least a portion of the electrode assembly is sized and arranged to be insertable into the electrochemical reader.

3. The device of claim 1 or 2, further comprising an isothermal subsystem in contact with the test zone configured to maintain a constant temperature at the test zone.

4. The device of any one of the preceding claims, wherein the test zone and the sample deposition zone are the same zone or two different zones.

5. The device of any one of the preceding claims, wherein the genetic material is selected from the group consisting of DNA and RNA.

6. The device of any one of the preceding claims, wherein the genetic material is a specific nucleic acid sequence of a DNA or RNA or a portion thereof.

7. The device of any one of the preceding claims, wherein the genetic material is selected from the group consisting of the specific nucleic acid sequences of a bacterium, virus, and eukaryotic organism.

8. The device of any one of the preceding claims, wherein the genetic material is a tuberculosis virus.

9. The device of any one of the preceding claims, wherein the genetic material is a human gene.

10. The device of any one of the preceding claims, wherein the amplification agent comprises one or more recombinase polymerase amplification agent.

11. The device of any one of the preceding claims, wherein the amplification agent comprises one or more primers specific for the genetic material.

12. The device of any one of the preceding claims, wherein the binding agent is selected to generally bind to the genetic material.

13. The device of any one of the preceding claims, wherein the binding agent is selected to specifically bind to the genetic material.

14. The device of any one of the preceding claims, wherein the signaling chemical is the one of the binding agents.

15. The device of any one of the preceding claims, wherein the binding agent is hexaamine ruthenium (III), osmium complexes, or a ferrocene-containing probe specific to the genetic material.

16. The device of any one of the preceding claims, wherein the signaling chemical is hexaamine ruthenium (III) or ferrocene.

17. The device of any one of the preceding claims, wherein the change of the concentration of the signaling chemical results in an increase or decrease current change readable by the electrochemical reader.

18. The device of any one of the preceding claims, wherein the one or more cellulosic layers comprises a first cellulosic layer comprising the sample deposition zone and a second cellulosic layer comprising the test zone.

19. The device of claim 18, further comprising a spacer layer disposed between the first and second cellulosic layers, said spacer layer comprising an opening in alignment with at least a portion of the test zone and the sample deposition zone.

20. The device of any one of the preceding claims, wherein the electrochemical reader is a universal mobile electrochemical detector.

21. The device of any one of the preceding claims, wherein the cellulosic layer comprises paper.

22. The device of any one of the preceding claims, wherein the test zone and the sample deposition zone are in fluidic communication through a connection channel.

23. The device of any one of the preceding claims, wherein the test zone and the sample deposition zone are defined by a fluid impermeable material in the cellulosic layer.

24. The device of claim 23, wherein the fluid-impermeable material comprises polymerized photoresist.

25. The device of any one of the preceding claims, further comprising one or more lysis agent embedded in the hydrophilic test zone or the sample deposition zone.

26. The device of any one of the preceding claims, further comprising a partial or complete circuitry in electrical communication and the electrode assembly.

27. The device of any one of the preceding claims, further comprising an isothermal subsystem in contact with the test zone and configured to maintain a constant temperature of the test zone.

28. The device of claim 27, wherein the temperature is from about 37° to about

45°.

29. A method of detecting a genetic material, comprising: providing the device of any one of the preceding claims; depositing a sample in the sample deposition zone; contacting the device with an electrochemical reader; and obtaining a readout by the electrochemical reader indicative of the presence or absence of the genetic material.

30. The method of claim 29, further comprising lysing a cell in the sample.

31. The method of claim 29, further comprising maintaining a constant temperature of the test zone.

32. The method of claim 31, wherein the temperature is from about 37° to about

45°.

33. The method of claim 29, wherein contacting the device with an electrochemical reader results in electrical contact between the electrode with the circuity of the electrochemical reader.

34. A kit comprising: a device of any one of claims 1-28; and a first set of instructions for obtaining a readout by an electrochemical reader for the detection of the genetic material.

35. The kit of claim 34, wherein the first set of instructions comprises instructions to deposit a sample containing a genetic material in the sample deposition zone, instructions to contact the device with an electrochemical reader, and/or instructions to maintain a constant temperature of the test zone.

36. The kit of claim 35, wherein the temperature is from about 37° to about 45°.

37. A kit compri sing : a microfluidic, electrochemical device for detecting a genetic material, comprising: one or more cellulosic layers comprising a sample deposition zone and a test zone which are in the same or different cellulosic layers and in fluidic communication with each other; and an electrode assembly comprising one or more electrodes in fluidic contact with the test zone; a first set of instructions for embedding, in the test zone or the sample deposition zone, one or more amplification agents selected for the amplification of a genetic material and one or more binding agents selected for binding the amplified genetic material to result in a change of the concentration of a signaling chemical; wherein the signaling chemical is selected to interact with the electrode to result in a current change readable by an electrochemical reader; and at least a portion of the device is sized and arranged to be insertable into the electrochemical reader; and a second set of instructions for obtaining a readout by an electrochemical reader for the detection of the genetic material.

38. The kit of claim 37, wherein the second set of instructions comprises instructions to deposit a sample containing a genetic material in the sample deposition zone, instructions to contact the device with an electrochemical reader, and/or instructions to maintain a constant temperature of the test zone.

39. The kit of claim 38, wherein the temperature is from about 37° to about 45°.