Cross-Reference to Related Application

_. [0001] Thi application claims priority to co-pending U.S. Provisional Patent Application Ser. No. 62/483,563, filed April 10. 2017, and entitled "TTNY; Tiny Isothermal Nucleic Acid Amplification sYstem," the entire disclosure of which is incorporated herein by reference.

Statement Regarding Federally Sponsored Research

[0002] This invention was made with government support under Grant Numbers CA2Q2723 & EB016803 awarded by the National institutes of Health. The government has certain rights in the invention,

Field of the Disclosure

[0003] The present disclosure is directed generally to methods and systems for portable and energy-flexible nucleic acid analysis platforms.

Background

[0004] Infectious diseases such as HIV, malaria, and respiratory infections are among the leading causes of death in developing countries. While treatment for many infectious diseases is available worldwide, effective and widespread diagnosis remains a challenge, for example, a nucleic acid test (NAT) is required for early infancy diagnosis of HIV, but in 2014 there were an estimated 1.2 million infants exposed to HIV, of which only half received a diagnostic test. Furthermore,, quantitative NATS- are in demand for applications such as HIV viral load monitoring, but such tests are still largely unavailable in settings where infectious diseases are most common,

[0005] Traditional diagnostics in low- and middle-income countries may be burdened by lengthy procedures for transporting human samples from rural healthcare clinics to central laboratories. Modern tools have aimed to disrupt this dependency on centralized laboratories to improve the time to treatment of infectious diseases. For example, tuberculosis time to treatment in Cape Town, South Africa was decreased from 71 days (centralized) to 8 days (decentralized) following implementation of the GeneXpert system. The GeneXpert (Cepheid) is a fully automated platform for NAT and has reported good clinical performance; however, the GetieXpert TV is not portable, has an instalment cost of about 17,000 USD, and requires a dedicated electricity supply. Electricity dependence is a critical issue for using such tools in developing. For example, in 1 1 countries in the south of Africa, about one-fourth of healthcare facilities have no access to electricity and about three- fourths of healthcare facilities lack access to reliable electricity.

[0006] Because they negate the need for thermal cycling, many forms of isothermal nucleic acid amplification have been used in point-of-care diagnostic tools. Loop mediated isothermal amplification (LAMP) is one such isothermal method, and is capable of nucleic acid quantification. Simple systems for performing isothermal amplification in resource limited settings exist, although many are only qualitative, and those that are quantitative use mierofluidie chips as consumables, making them impractical to use i the field. For heat input, these systems either use exothennie chemical reaction packets or stable electricity. None have the flexibility to use electricity whe it is available while also being configured to use alternative heat sources when electricity is not available.

Summary of the Disclosure

[Θ007] There is thus a continued need for portable nucleic acid analysis platforms that are capable of utilizing variety of energy sources, including electricity and alternative energy sources.

[0008] The present disclosure is directed to inventive methods and systems for nucleic acid analysis using a portable and highly adaptable analytical platform. Various embodiments and implementations herein are directed to a system with a measurement unit. configured to perform nucleic acid analysis and including a sample well,, a light source, and an optical sensor. The system includes a first enclosure made of a heat-transmitting material and comprising the measurement unit, and optionally a second enclosure made of a heat-transnnttiiig material and surrounding the first enclosure. Disposed ithin the device is a phase change material configured to store heat energy. The system includes one or more mechanisms to transfer energy to the system, including: (1) a solar receiver configured to receive solar energy and transfer the received solar energy to the phase change material; (2) a electric heater configured to transfer heat to the phase change material when the device has access to electricity; (3) and/or a heat- receiving element configured to receive heat from an external heat source and transfer the received heat to the phase change material. The phase change material is configured to absorb heat from one Or more of the electric heater, the solar receiver, and/or the heat- eceiving element _* and to release heat during nucleic acid analysis by the measurement unit.

[0009] According to an embodiment is a nucleic acid analysis device. The device includes: (i) a measurement unit comprising a sample well, a ligh source, and an optical sensor, the measurement unit configured to perform nucleic acid analysis; (ii) an enclosure containing the measurement unit arid comprising a heat-transmitting material; arid (in) a phase change material configured to store heat received from a heat source, and configured to release heat to the measurement unit via the heat-transmitting material.

JOG 10] According to an embodiment, the heat source comprises a solar receiver configured to receive solar energ and transfer the received solar energy to the phase change material, an electric heating eiemeni configured to transfer heat to the phase change material when the device has access to electricity, a heat-receiving element configured to receive heat from an external heat source and transfer the received heat to the phase change material, or any combination of a solar receiver, ail electric heating element, and/or a heat-receiving element.

[0011] According to an embodiment, the device further includes a second enclosure surrounding the enclosure and comprising a heat-transmitting material, where the phase change material is disposed within a space between the enclosure and the second enclosure.

[0012J According to an embodiment, the device further includes a, lens configured to direct and/or amplify solar energy o the solar receiver.

[0013] According to a embodiment, the device further includes a power source configured to power the light source and the optical sensor during nucleic acid quantification.

[0014] According to an embodiment, the light source comprises a plurality of tight sources comprising at least two different wavelengths.

10015] According to an embodiment, the device further includes an optical filter positioned between the light source and the optical sensor. [001 ] According to an embodiment, the measurement unit comprises a plurality of sample wells,.

[0017] According to an embodiment, the measurement unit comprises an optical sensor for each of the plurality of sample wells.

[0018] According to an embodiment, the phase change material comprises a Tm selected to facilitate nucleic acid analysis by the measurement unit.

[0019] According to an embodiment, the space between the enclosure and the second enclosure comprises a seal configured to contain the phase change material.

[0020] According to an embodiment, the measurement unit is configured to perform a LAMP assay using heat energy released by the phase change material

[0021] According to an embodiment, the enclosure and the second enclosure are cylindrical.

[0022] According to an embodiment, die device further includes an insulator

[0023] According to an embodiment is a method for nucleic acid analysis utilizing a portable device. The method includes: providing a portable nucleic acid device, comprising: (i) a measurement unit comprising a sample well, a light source, and an optical sensor; (ii); an enclosure containing the measurement unit and comprisin a heat-transmitting material; and (Hi) a phase change material configured to store heat received from a heat source, and configured to release heat to the measurement unit via the heat-transmitting material; providing heat energy to the phase change material; storing the heat, energy in the phase change materiai; transferring the stored heat energy from the phase change materia! to the measurement unit; and performing, usin the transferred heat energy, a nucleic acid analysis by the measurement unit.

[0Θ24] Accordin to an embodiment, the device further includes: a solar receiver configured ^• to receive solar energy and transfer the received solar energy to the phase change material, an electric heating element configured to transfer heat to the phase change material when the device has access to electricity, a heat-receiving element configured to receive heat from an external heat source and transfer the received heat to the phase change material, or any combination of a solar receiver, an electric heating element and/or a heat-receiving element. [0025] According to an embodiment the device further includes a second enclosure surrounding the enclosure and comprising a heat-transmitting material, where the phase change material is disposed within a space between the enclosure and the second enclosure.

[0026] According to an embodiment, the device further includes a lens, and the step of providing heat energy to the phase change material comprises directing and/or amplifying solar energy on the solar receiver via the lens.

[0027] Additional features and advantages of the invention will be set forth in the detailed description which follows, and in part will be readily apparent to those skilled in the art from that description or recognized by practicing the invention as described herein, including the detailed description which follows, the claims, as well as the appended drawings.

[0028] Tt is to be understood that both the foregoing general description and the following detailed description are merely examples of the invention, and are intended to provide an overview or framework for understanding the nature and character o the invention as it is claimed. The accompanying drawings are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification. The drawings ill ustrate various embodiments of the in vention and together w ith the description serve to explain the princi ples and operati on of the in v enti on.

Brief Description of the Drawings

[0029] The present invention will be more fully understood and appreciated by reading the following Detailed Description in conjunction with the accompanying drawings, in which;

[0030] FIG. 1 is a cutaway view of a portable nucleic acid analysis device, in accordance with an embodiment,

[0031] FIG. 2 is a top view of a portion of a portable nucleic acid analysis device, in accordance with an embodiment.

[0032] FIG. 3 is a cutaway view of a portion of a portable nucleic acid analysis device, in accordance with an embodiment.

[0033] FIG. 4 is a top view of a portable nucleic acid analysis device, in accordance with an embodiment. [0034] FIG. 5 is a flowchart of a method for using a portable nucleic acid analysis device, in accordance with an embodiment.

[0035] FIG . 6 A i s a graph of temperature profi les of the device at the location where samples are placed when heated by a hotplate for a variety of times. Heating started at 0 minutes, and at the respective heating time the device was taken off the hotplate and allowed to cool. Dashed horizontal lines sho the isothermal temperature range (68 ± PC). Thick colored lines show the isothermal dwell.

[0036] FIG. 6B is a comparison of th cooldown temperature profile inside the device w'hen different materials were placed between the two concentric aluminum cylinders. Isothermal time for each material is shown.

[0037] FIG. 6C is is a graph of the temperature profile of the device when heated by a cartridge heater. A microcontroller is used to automatically turn on and off the heating.

[0038] FIG. 6D is a summary of the energy required to operate the device for one hour.

[0039] FIG. 6E are temperature profiles of the device when heated via sunlight (i, ii), hotplate (Hi), or Bunsen burner (iv). Heating conditions are displayed in each subfigure, along with the duration of ^' the following isothermal dwell (marked with the right-facing arrow). FIG. 6F shows the device heated using sunlight on a partly cloudy day.

[0040] FIG. 7 'A is a graph of the temperature of the samples inside the device during three separate LAMP reactions, with each experiment using a different heating method. Average temperatures are reported in the top-right corner. Samples were inserted into the device at 0 minutes.

[0041] FIG. 7B is a graph of the threshold times of samples containing the same target 0ΜΑ concentration ( 12,000 copies/reaction) but heated using different sources. The average time of four samples is displayed above each method. The data in FTG. 7B are from the same experiments shown in FIG. 7Α.·

[0042] FIG. 7C is a graph of the fluorescent signal measured in the device during nucleic acid amplification. The threshold time (large data point) is taken as the time the fluorescence passes a predefined threshold (green horizontal line). Samples were inserted into the device at 0 minutes. [0043] FIG. 8A is a graph of amplification results for BC-3 cell line standards, as tested by qPCR and LAMP (LAMP results include trials from both the device and the ViiA 7). At the bottom is shown the number of Samples which amplified using the LAMP assay at a particular concentration, di vided by the number of total trials performed. Each sample was run in duplicate using the qPCR assay _. , and error bars represent standard deviation.

[0044] FIG. 8B is a graph of standard curves as measured by the device and the ViiA 7 commercial machine, both performing the LAMP assay using BC-3 ceil line standards. Error bars represent standard deviation.

[0045] FIG. 9A is a graph of true KSHV DNA concentration of 42 human skin samples (as determined by qPCR), grouped by LAMP result from the device. Each sample was amplified in the device twice. Samples with detectable levels of KSHV were those that amplified for both trials with threshold times < 24 minutes (later threshold times were ver rare). One sample had mixed results for the two trials, and was classified as uncertain.

[0046] FIG. 9B is a graph of KSHV DNA quantification by qPCR and LAMP (in the device) for the 33 detectable samples from (A). Dashed line shows where the two assays match.

[0047] FIG. 9C is a graph of the order -of-magnitude difference in KSFIV quantification between duplicates for each assay/system. Media difference of the 33 detectable samples is listed for each assay/system. Technical replicates were samples amplified twice on the same qPCR plate, while experimental replicates were samples amplified in different qPCR experiments.

[0048] FIG. 9Ό is a graph of a comparison of absorbance and fluorescence threshold times for the 33 detectable samples. LAMP quantification reported in FIG. 9B was calculated using fluorescence threshold time.

[0049] FIG. 10A is a detailed timeline from biopsy to the device result, for 3 patients. Gray boxes for patients A and B show when electricity outages were experienced. Light blue boxes detail how the device was heated before LAMP.

[0050] FIG. 10B is an image of the device being heated with sunlight at the Uganda Cares Clinic in Masaka, Uganda. [0051] FIG. I OC is a graph of threshold times of 8 human samples (target: KSHV DNA) when analyzed at a variety of locations and via different heating conditions or operating procedures.

[0052] FIG. 10D is a graph of the threshold times of the same S human samples, grouped by patient instead of location or heating condition. Patient Z from Fig. Ι θΑ is not included as that sample was obtained after some conditions were tested.

[0053] FIG. 10Έ is a graph of the temperature profiles inside the device during LAMP from FIGS, OC and D, with color indicating the heating method. Dashed lines show the target temperature range (6.8 ± 1°C).

[0054] FIG. 1 1 A is a graph of raw data collected from photodiodes during three different LED excitation states (blue, yellow, red). Blue light excites Evagreen dye, yellow light excites ROX dye, and red light provides an absorbance measurement. Photodiodes convert irradiatio to frequency, which were measured using the Teensy microcontroller,

[0055] FIG. I IB is a graph of raw data collected from photodiodes, where Evagreen values were normalized by the ROX values and then applied a fit (dashed line) to the linear portion of the response. The fluorescence data reported in FIG. 7C is the difference between the normalized data (solid line) and the fit line seen here. This data was collected from a device prototype with wells for four samples instead of six samples.

[0056] FIG. 12 is a graph of calculation of threshold time via difference data. The difference between successive points of the Evagree data normalized by the ROX data. All the device threshold times reported in this article were found via this method, and were calculated as the time that the data passed a pre-defined threshold (green horizontal line). The large circles mark the calculated threshold times. It would found that this method of threshold time calculation was the most reliable, as it did not depend on line fitting. This data was collected from a device prototype with wells lor four samples instead of six samples.

[0057] FIG. 13 is a graph of LAMP amplification. Three different sample types were tested via the LAMP assay in the device. Two types were samples for standard curve preparation (piasmid DNA and BC-3 cell line DNA}, and the third sample type was the extracted DNA from human skin biopsy samples. The true concentration of all three sample types was determined via qPCR. When amplified via LAMP, a difference in efficiency was observed for the three sample types. That is, although all three sample types had similar KSHV concentrations (as determined by qPCR), threshold time in the LAMP assay was not consistent between sample types. Plasmid DNA standards (green data) amplified the most efficiently, producing threshold times roughly between 10 and 13 minutes. BC-3 cell line standards (orange data) produced threshold times roughly between 1 ί and 15 minutes. Human biopsy samples (blue line) amplified the least efficiently, with threshold times roughly between 13,5 and 17 minutes. The dashed blue line is a best fit of the 33 human samples with detectable amounts of KSHV (as determined by L AMP in the device). The discrepancy in amplification efficiency may be explained by sample composition and/or extraction, procedure used. DNA from the BC-3 cell line samples was extracted using the same extraction procedure as the human skin samples (DNeasy, Qiagen). The lower amplificatio efficiency when amplifying ^' human samples explains wh the quantification of those samples via LAMP is lower than when quantified via qPCR.

[ΘΘ58] FIG. 14 is a graph of the difference between qPC and LAMP quantification. The difference in quantification between the two assays is reported in orders of magnitude of copies/reaction. Only die 33 human samples with detectable amounts of KSHV (as determined by LAMP i the device) are considered.

ΙΘ059] FIG. 15 is a graph of ViiA 7 quantification of human skin samples. Quantification of the Ugandan skin samples for KSHV DNA by LAMP performed in the ViiA 7 commercial machine (triangles). For comparison, quantification by LAMP performed in the device (circies) is included. Only the samples with detectable amounts of KSHV DNA b the LAMP assay are considered (31 samples for the ViiA 7, 33 samples for the device). Dashed line represents where quantification from LAMP and qPCR agree perfectly.

[0060] FIG. 16 is an image of gel electrophoresis of LAMP products. Plasmid samples of differing target concentrations were amplified in the device for 50 minutes.

[0061] FIG. 17 A is a schematic representation of the portable device being carried by hand, in accordance with an embodiment.

[0062] FIG. 17B is a schematic representation of the portable device in comparison to a GeneXperi IV by Cepheid and a ViiA 7 Real-Time PCR System by Thermo Fisher Scientific, in accordance with an embodiment. [0063] FIG. 7C is an image of the portable device being heated by a Runsen burner through an opening in the bottom of the system, in accordance with an embodiment,

[0064] FIG. I7D is a schematic representation of the portable device being heated by electricity, in accordance with an embodiment.

[0065] FIG. Γ7Ε is an image of the portable device being heated by concentrated sunlight using a Fresnel lens, in accordance with an embodiment.

Detailed Description of Embodiments

[0066] The present disclosure describes methods and systems for nucleic acid quantification using a portable and highly adaptable analytical platform. As described or otherwise envisioned herein, the system comprises a measurement unit and a temperature regulation unit, which can be a single device or separable components. To ensure reliable access to energy even whe electricity is not available, the system includes one or more mechanisms to capture energy in a phase change material for the steady release of energy during analysis. The mechanisms include: (1) a solar cover configured to receive solar energy and transfer the received solar energy to the phase change material; (2) an electric heater configured to transfer heat to the phase change material whe the device has access to electricity; and/or (3) a heat-receivin element configured to receive heat from a external heat source and transfer the received heat to the phase change material. The phase change material is configured to absorb heat from the electric heater, the cover, and/or the heat-receiving element, and to release heat during nucleic acid analysis by the measurement unit.

[0067] A decentralized approach to diagnostics has shown to decrease the time to treatment of infectious diseases in resource limited settings, but modem diagnostic tools still rely o stable electricity, are not portable, and are too expensive. The heat required to operate the portable nucleic acid analysis device described herein may be supplied via electricity, but may also be supplied via sunlight or flame for operation in locations without electricity. In one set of experiments, described below, the dev ce is compared to performance of quantitative polymerase chain reaction (qPCR) machines when analyzing human skin biopsies from Ugandan patients suspected of Kaposi 's sarcoma (KS). KS is caused by the Kaposi's sarcoma-associated herpesvirus (KSHV, also formally known as human he es ίrus 8), and is most common in HIV- infected individuals. Diagnosis of S via NAT for KSHV DHA in skin lesions ma be a alternative to current diagnostics because the accuracy of those methods in developing regions is often tow. Furthermore, when deployed at multiple Ugandan healt clinics, th device demonstrated equivalent performance whether using stable electricity, interrupted electricity, or sunlight as a heat source, thus demonstrating a reliable, ^' energy-flexible system for decentralizing nucleic acid diagnostics.

f0068] The weight and volume of the device are orders of magnitude smaller when compared to commercial qPCR machines. Additionally, the devic can use a variety of heat sources because it store heat isothermally through use of a phase change material (PCM), and thermal cycling i not required as the device performs loop-mediated isothermal amplification (LAMP) of DNA. The latent heat of the melted phase change material inside the device keeps the system isothermal in-case of power outages when heated b electricity, or in-case of variable cloud coverage whe heated via sunlight, it also enables the collection of heat from external sources such as fire or similar sources,

{0069J Referring to FIG. 1 _¾ in one embodiment, is a portable nucleic acid analysis device 100. The portable nucleic acid analysis comprises a measurement component 1 10 configured to track the nucleic acid analysis, and a temperature regulation component 120 configured for heat collection and isothermal stabilization.

[0070J According to an embodiment, temperature regulatio component 220 comprises an inner or first, enclosure 130 with a pocket or otherwise configured to a least partiall enclose or contai the measurement component 1 10. First enclosure 130 comprises an upper opening via which the measurement component 110 can be accessed. The first enclosure comprises a heat- transmitting material such as a metal, including but not limited to aluminum. Many other heat- transmitting materials are possible. Temperature regulation component 120 comprises an outer or second enclosure 140 configured to at least partially enclose or contain the first enclosure 130. Second enclosure 140 comprises an upper opening via which the measurement component 110 can be accessed.. The second enclosure comprises a heat-transmitting material such as a metal, including but not limited to aluminum. Many other heat-transmitting materials are possible. Referring to FIG, 1, for example, first enclosure 130 and second enclosure 140 are cylinders, although many other shapes and sizes are possible.

[0071] Disposed within a space 142 betwee first enclosure 130 and second enclosure 140 is a phase change material 150. The phase change material may be any material configured to release heat at a temperature suitable for the nucleic acid analysis by measurement component 1 10. The phase change material may be selected to comprise a T _w selected to facilitate nucleic acid analysis by the measurement unit. For example, the phase change material may be selected to comprise a T _in that facilitates nucleic acid amplification., among other types of analysis.

[GG72] For example, one suitable phase change material is PureTemp* 63 (nominal T _m : 63 ^C C), although many other phase change materials are possible. Space 142 between first enclosure 130 and second enclosure 140 may also comprise a seal 144 or other containment mechanism configured to keep the phase change material 150 within the space, particularly in a liquid or semi-!iquid form. According to an embodiment, the phase change material is configured to absorb and store energy from an electric heater, a solar receiver, and/or a heat-receiving element, and to release that stored energy during nucleic acid analysis by the measurement, unit.

[0073] Accordin to an embodiment, PureTemp 68 can be used as a phas change material because its melting temperature (68°C) is suitable for a ^' LA P reaction. The phas chang material can serve two functions. First, it can act as a thermal buffer to make sure that the temperature of the samples does not get too high: heat input may be attenuated before temperature increase begins after the melting stage. Second, it can serve as a large heat reservoir for operation with unreliable heat sources. For example, solar energ may be collected in excess when available and stored in the form of latent heat, allowing for isothermal operatio even if clouds block the sun during the LAMP assay .

[0074] ^' Temperature regulation component 120 further comprises an insulator 190 surrounding or enclosin at least a portion of second enclosure 140. This keeps the heat gathered and stored in the phase change material w¾ n. the system. The insulation 190 may be any material or structure configured to prevent heat loss.

[0075] ^' Temperature regulation component 120 further comprises a solar receiver ISO configured to removably cover at least a portion of the upper opening of the first enclosure 130 and/or the second enclosure 140. The solar receiver is configured to receive solar energy, and to transfer that received solar energy to the phase change material 150 via either the heat- transmitting material of the first enclosure 130 and/or the second enclosure 140. Although not shown in FIG. i, the system 100 may comprise a lens, such as a Fresne lens, configured to concentrate solar energy on the solar receiver to facilitate energy eoliection and heating of the phase change material.

[0076 Temperature regulation component 120 further comprises an electric heating element 160 which is electrically controlled. Electric heating element 160 is configured to transfer heat to the phase change material when the device has access to electricity. The electric heating element 1 0 is configured to be heated when the system has access to electricity, and thus may comprise or be configured to receive electricity' from an outlet, solar charger, generator, or othe source of electricity. Electric heating element 160 may be placed anywhere within system 100, including but not limited to within first enclosure 130, second enclosure 140, space 142, and insulation 190, among other locations.

[0077] Temperature regulation component 120 further comprises a heat-receiving element 170 disposed on or in an external portion of the device. The heat-receiving element 170 is configured to receive heat from an external heat source 172, and to transfer the received heat to th phase change material 150. According to an embodiment, the heat-receiving element 170 transfers the heat received from the external heat source 172 to the phase change material 150 via the second enclosure 140, although the heat-receiving element 170 ma be configured to transfer the received heat directly to the phase change material 150, The heat-receiving element 170 may be a panel, exposure, or other heat-receiving and heat-transmitting structure, and may be composed of a metal such as aluminum, among many other materials. According to one embodiment, the heat-receiving element 170 is a metal element on the bottom of th device 100,. and can receive heat energy from a heat source such as fire to heat the phase change material. The fire can be any fire such as a wood fire, a Bun sen burner, a torch, or any other heat source.

[0078] Although a specific embodiment is depicted in FIG. 1 , it will be recognized that portable nucleic acid analysis device 100 can comprise many different structures. For example, device 100 may comprise a measurement unit 120 with a sample well, light source, and optical sensor. Device 100 may also comprise a single enclosure containing the measurement unit and composed of a heat-transmitting material. The device may also comprise a phase change material configured to store heat received from a heat source and configured to release heat to the measurement unit via the heat-transmitting material. Many other configurations and structures are possible.

[0079] Referring to FTG. 2, in one embodiment, is a top view of the measurement component 1 10 of the portable nucleic acid analysis device 100. According to a embodiment, measurement component 1 10 comprises one or more sample wells 210 into whic samples or tubes or other containers comprising samples may be inserted for analysis. Device 00 in FIG. 2 comprises six sample wells 210 _? althoug it should be understood that more or fewer sample wells are possible. According to an embodiment measurement component 1 ) 0 also comprises a printed circuit board 220 on or in the top surface. Although measurement component 1 10 is rounded i this embodiment, the component may be any shape and size.

[ΘΘ80] Referring to FIG. 3, in one embodiment, is a side, cutaway view of the measurement component 1 10 of the portabl nucleic acid analysis device 500. The measurement component comprises one or more sample wells 210 into which samples or tubes, such as PG tubes, or other containers comprising samples ma be inserted for analysis. The measurement component comprises an upper circuit board 220 on or i the top surface which may facilitate one or more functions of the system, and a lower circuit board 230 on or in a bottom surface or location of the component which ma facilitate one or more function of the system. For example, upper circuit board 220 may comprise or control a light source 230, which can be any light source. The light source may comprise one or more light sources, such as LEDs of one or more colors. For example, blue, yellow, and red LEDs affixed to the upper circuit board ma excite commonly used fJuorophores in the sample.

[Θ.Θ81] Lower circuit board 230 may comprise or control an optical sensor 240, which ca be any optical sensor configured to detect wavelengths necessary for the nucleic acid analysis. The optical sensor may be one optical sensor of multiple optical sensors. For example, the system may comprise one optical sensor for each sample well, among other possible embodiments. Measurement component 1 10 may also comprise an optical filter 250 to enhance or control the functionality of the system. For example, optical fil ter 250 may be a dual bandpass optical filter configured to allow the device to measure bot fluorescence and absorbance by cycling the active LED,

[0082] Accordingly, measurement component 1 10 comprises an optical path from the light source 230, through one or more sample wells 210, optionally through an optical filter 250, to optical sensor 240. There may be a single optical path for each sample well,

[0083] Measurement component ί 10 may comprise or be surrounded by a shell 260 which may insulate the component, and'or facil itate the transfer of stored energy from the phase change materia? to the sample wells via the shell and first enclosure 130. According to an embodiment, shell 260 is any heat-transmitting material, including but not limited to aluminum among many other possible materials.

[0084] Referring to FIG. 4 is a top vie of an embodiment of portable nucleic acid analysis device 100. The device comprises a measurement component 1 10 positioned within a first enclosure 130, a second enclosure 140 positioned around the first enclosure and separated by a space 142 comprising the phase change material (not shown). The second enclosure is surrounded by insulation 1 0 to keep captured energy within the system.

[0085] According to an embodiment, portable nucleic acid analysis device 00 may comprise a controller 410 or other electronics r circuitry configured to facilitate one or more functions of the system. For example, controller 410 may be in wired and/or wireless communication with the measurement component 1 10, including the ligh source, optical sensor, and circuit boards.

[0086] All or a portio of the portable nucleic acid analysis device may be placed or situated or constructed within a housing 420, which may be a component of the device. Although not shown, housing 420 may comprise an opening, panel, or other access for heat-receiving element 170 to receive heat energy from an external heat source 172 such as fire to heat the phase change material. According to one embodiment, the temperature-regulation unit and measurement unit were assembled together and placed into a aluminum enclosure (Protocase ^® ). According to one constructed embodiment of the device, the volume and weight of the complete system was 2.1 L and 1.1 kg, respectively. [0087] According to an embodiment, during operation, two of the possible heat sources available for operating the device are electricity and sunlight. To be resistant to electricity outages and cloud coverage, the device stores a large amount, of heat (14 kj) in the latent heat of a phase change material. The heat storage enables an extended isothermal dwell under a variety of heating conditions. According to an embodiment, even in the case of heat-source disruption, the device stayed isothermal for about 65 minutes, sufficient time for about two LAMP reactions. The temperature stability provided by the phase change material is illustrated well when compared with water. The phase change material was replaced with water, and it was found that the system stayed isothermal for only 11% as long. An electric heating element 160 can be used to automatically heat the device when eleetiieity is available, and the controller can control the temperature and keep the system isothermal indefinitely.

[0088] While the heating of the device need not be provided by electricity, as described or otherwise envisioned herein, eleetiieit is required to power the device's light sources, optical sensors, and controller. According to one embodiment, only a small amount of the device's total energy requirement is electrical. Thus, one most sensible use of the device in the field would be to use a battery or photovoltaic cell to power the electronics, but to provide energy for heating by either sunlight or flame. This method would allow for extended system operation away from dedicated electricity. For example, an iPhone 6S battery (capacity: 6.9 Wh) could power the device's electronics for over 24 hours, while more than one (130%) of the .same battery would be required to heat the device and power the electronics for a single hour. Referring to TABLE 1 , in one embodiment, is a summary of the energy requirements for an embodiment of the device.

[0089] TABLE 1. Portable nucleic acid analysis device energy requirements.

[0090] Referring to FIG. 5, in one embodiment, is a flowchart of a method 500 for nucleic acid analysis. At step 10 of the method, a portable nucleic acid analysis device 100 is provided. The portable nucleic acid analysis device ma be any of the devices or systems disclosed or otherwise described herein. Motably, due to its portability and energy flexibility, portable nucleic acid analysis device 100 may be field-deployed, and may be powered by a wide variety of energy sources.

{0091] At step 520 of the method, heat energy is provided to the portable nucleic acid analysts device. The heat energy ma be provided via any of the methods or systems described or envisioned herein. Fo example, the heat energy may be provided by solar energy. The solar energy may be provided via solar receiver 1 SO configured to removably cover at least a portio of the upper opening of the first enclosure 130 and/or the second enclosure 1 0. The solar receiver is configured to receive solar energy, and to transfer that received solar energy to the phase change materia! 150 via either the heat-transmitting material of the first enclosure 130 and/or the second enclosure 140. [0092] According to an embodiment, the heat energy may be provided by an electric heating element 160 configured to transfer heat to the phase change material when the device has access to electricity _* The electric heating element 160 is configured to be heated when the system has access to electricity, and thus may comprise or be configured to receive electricity from an outlet, solar charger, generator, or other source of electricity. Electric heating element 160 may be placed anywhere within system 100, including but not limited to within first enclosure 130, second enclosure 140, space 142, and insulation 1 0, among other locations.

[0093] According to an embodiment, the heat energy may be provided by an external energ source. The device may comprise a heat-receiving element 170 disposed on or in an external portio of the device and configured to receive heat from an external heat source 172, and to transfer the received heat to the phase change material 150. The heat-receiving element 170 may be a panel, exposure, or other heat-receiving and -transmitting structiue, and may be composed of a metal such as aluminum, among many other materials. According to one embodiment, the heat-receiving element. 170 is a metal element on the bottom of the device 100, and ca receive heat energy frOni a heat sourc such as fire to heat the phase change material. Tile fire can be any fire such as a wood fire, a Bunsen burner, a torch, or any other heat source.

[0094] At step 530 of the method, the heat energy is stored in the phase change material 150 of the portable device. Step 530 of the method can be perfonned simultaneously with step 520 of the method, so that the phase change material is effectively a heat sink that receives and stores the provided heat energy . According to an embodiment, the phase change material is selected or designed such that it can accept heat energy which will be provided at the temperature or temperatures generated by the one or more heat sources. The heat energy ca be stored in the phase change material until the heat energy is no longer available, and/or until the device is ready or needed for nucleic acid analysis.

[0095] At step 540 of the method, heat energy stored in the phase change material is transferred to the measurement component of the portable device. For example, the stored energy may be transferred to the measurement component from the phase change material at any time, which may be a passive or active transfer. [0096] At step 550 of the method, the measurement component 1 10 of the portable device utilizes the transferred heat energy to perform a nucleic acid analysis. The nucleic acid analysis can be any analysis that can utilize the heat transferred from the phase change material. For example, the analysis can be a L AMP analysis, among many other types of nucleic acid analysis.

[0097] At step 560 of the method, which can occur at any stage of the method, the system again captures heat energy provided via any of the methods or systems described or envisioned herein. The heat energy stored in the phase change material may be depleted, by the nucleic acid analysis and/or by general heat dissipation, and thus will operate again as a heat sink for new heat energy introduced to the system.

[0098] According to an embodiment, the portable device described or otherwise envisioned herein has numerous advantages over both commercial and research-grade systems for NAT. Compared to devices in the literature, the portable device is the only system that can use electricity when it is available, but also use other heat sources when electricity is unavailable, making it practical in both the laboratory and the field. Compared to commercial machines, the portable device is unique in that it is portable and resilient against power outages. Moreover, the throughput of the portable device (6 samples/test) is higher than that, of popular commercial systems (4 sampies test), and higher tha other systems in the literature using ubiquitous consumables. Even though the cost of the optical and electrical components inside the portable device is only about 250 USD, it was shown that, quantification by the portable device is on-par with commercial systems performing the same LAMP assay. As described in detail herein, the portable device was tested using human skin samples from Uganda for KSHV DNA. Device- qPCR agreement was 41 of 42 patients (98%).

[0099] Field trials, described in detail herein, confirm that the portable device is particularly useful for operation i resource limited settings. The small size of the portable device makes it convenient to transport to multiple Ugandan clinics, and the device performed equivalently under a, variety of locations, device-operators, and heating conditions including sunlight. The device successfully completed multiple LAMP reactions even though electricity outages were experienced mid-assay. During the outages that the device successfully operated through, commercial machines running diagnostics in the same laboratory had their assays ruined, even though a generator and backup batteries were installed for such situations, and the generator failed to start. Several KS-suspect patients arrived at the Ugandan clinics during the time there, and it was possible to go from biopsy to the device result in just 2,5 hours, with DMA extraction being the time-iimiting step. This time could be even shorter if the disease of interest is one that can be tested using fluid samples. For S, the lysis of a skin sample is necessary, a step that can take multiple hours,

[001001 Although the device was used to perform the LAMP assay, replacement of the phase change material with one that melts at other temperatures would allow the system to perform other isothermal assays, making it broadly useful. It is foreseen that the device being particularly suited for multiple applications in developing countries. First, it could be carried by a healthcare worker traveling between communities to provide diagnostics to those patients unable to travel to urban healthcare institutions. Second, it could be used as a stationary tool in district-level clinics and hospitals, where the device's unique ability to operate using unreliable ^' electricity would be of value. Both applications represent opportunities for nucleic acid diagnostics to reach more of the population in developing countries, with the potential to reduce die time to treatment, of infectious diseases,

[001O1J According to an embodiment depicted i FIG. 17, the device is portable and easily carried in one hand (FIG. I7A), in contrast to other nucleic acid quantification systems such as the GeneXpert IV by Cepheid o the ViiA 7 Real-Time PGR System b Thermo Fisher Scientific (FIG. Γ7Β). The device can be heated by a, Bunsen burner through an opening in the bottom of the system (FIG. 17C), by electricity (FIG. 17D), and/or by sunlight (FIG. 17E).

[001O2J EXAMPLES

[001031 Referring to FIG. 6, in one embodiment, is a graph of heating characterization of an embodiment of the analytical device. As shown in FIG. 6A, heat storage enables an extended isodieniial dwell under a variety of heating conditions, Even in the case of heat-source disruption, the device stays isothermal for about 65 minutes— sufficient time for abou two LAMP reactions. The temperature stability provided by the phase change material is illustrated well whe compared with water: when the phase change material was replaced with water, the system stayed isothermal for only 11% as long (FIG. 6B). A cartridge heater can be used to automatically heat the device when electricity is available, and a microcontroller can control the temperature and keep the system isothermal indefinitely (FIG, 6C). While the heating of the device need not be provided by electricity, electricit is required to power the device's sensors. Only a small amount (3%) of the device's total energy requirement is electrical (FIG, 6D).

[00104J According to an embodiment, the LAMP assa in the device is independent of the heat source. The device was heated usin a variety of heat sources, with the hypothesis that all heat sources would be able to reach the isothermal condition desired for the LAMP reaction, FIG, 6E depicts temperature profiles of the device during heat-up using a Bunsen burner, a small hotplate, and sunlight. It was found that heating the device for about half an hour i sunlight was sufficient to melt all the phase change material and to sustain the long isothermal dwell, although this is dependent upon ambient conditions. Once while collecting sunlight, the device experienced complete cloud coverage for about six minutes, but the effect of the cloud was to only delay heatmg of the device (FIG. 6F). In contrast, previously developed microfluidic devices that performed PGR via solar thermal heating are only capable of operation during clear-sky operation.

[00105} It was hypothesized that the device would perform a LAMP assay equivalently using any of the heating methods. According to an embodiment, LAMP reactions were performed when the device was heated by a hotplate, a Bunsen burner, and by sunlight. The average sample temperature for each of these experiments was just above 68 ^Q C, and only deviated by 0.3 ^Q C ^' between the heating metliods, as shown in FIG. 7 A. Similar threshold times were observed when the same sample was amplified in the device, no matter the heating method, as shown in FIG. 7B. Threshold times Were calculated by tracking fluorescence data in real-time (FIGS. 7( ^' . 11, and 12).

[00106J The device's capabilit to perform quantitative NAT using skin biopsy samples from patients suspected of having Kaposi ^' s sarcoma Was evaluated. To quantify KSHV load in unknowii-coneenftation skin biopsy samples, standard curves with known copy numbers of the KS target gene, QRF 26, were generated fro recombinant plasmid DNA, and DMA extracted from a KSHV* cell line, BC-3. Amplification data from the plasmid or cell line standards was compared against that obtained from the unknown biopsy samples to approximate KSHV content. The following observations are drawn from the KSHV+ cell line (BC-3) standards, as the D A in these samples was extracted using the same procedure as for the human biopsy samples (D easy, Qiagen). The qPCR assay (the gold-standard) proved quantitative for all concentrati ons of standards (FIG . 8 A ). The LAMP assay produced repeaiable threshold ti mes for the four highest standards tested (3.2 χ i 0 ^" to 3.9 10 ^' ' copies/reaction), but at lower concentrations threshold time no longer linearly predicted starting DNA concentration. At the lowest concentration tested (5 KSHV copies/reaction), the LAMP assay amplified in 7 of 8 trials, and at the second lowest concentration ( 135 copies/reaction), the L AMP assay amplified in 8 of 8 trials. A 2007 study using a very similar assay found a limit of detection of approximately 100 copies/reaction. It was also observed that the amplification efficiency of the LAMP assay was dependent upon the type of sample being amplified, as shown in FIG. 13.

[0O1G7J Standard samples were amplified using LAMP i both the device and a commercial qPCR machine (ViiA 7 from Thermo Fisher Scientific, set to a constant 68°C). Similar threshold times were observed for both machines, as shown in FIG. 8B, confirming that the device can perform quantitative, isothermal assays with results that are equivalent to those from commercial systems.

100108] Forty-two human biopsy samples were collected from Ugandan patients suspected of having Kaposi's sarcoma, and these samples were tested via LAJVIP in the device, via LAMP in the V jA 7 _> and via traditional qPCR in an Applied Biosystems 7500 Fast. Samples were collected at the Infectious Disease institute of Makerere University (Kampala, Uganda), and then transferred to the U.S. for analysis. Analysis of the human samples was first considered on a binary, deteetable/not-detectable basis. Device-qPCR agreement was 41/42 (98%) on a binary, detectable/not-detectable basis, with both systems finding the same 8 patients negative, as shown in FIG. 9A. For the sample with the lowest KSHV concentration, the device gave a mixed positive/negative result (all samples were tested twice). It is noted that the diagnostic value of this analysis cannot be assessed without histological confirmation and a larger sample size.

[00109] Next, the 33 samples with device-detectable KSHV levels were analyzed quantitatively. Quantification by qPCR was compared with quantification by LAM (performed in the device), finding a coefficient of determination; r ² - 0.38 (Fig. 6B). A similar coefficient of determination (r = 0.48) was found n a previous study that compared LAMP and qPCR quantification, in all cases except for one, quantification obtained from the LAMP assay was tower than the quantification obtained from the qPCR assay, as shown in FIG. M. This observation has been previously reported in study comparing digital LAMP and digital PCR. Quantificatio of the human samples via LAMP was similar whether performed in the device or the ViiA 7 commercial machine, as shown in FTG. 15.

[0011 1 It was also observed that successive trials of qPCR gave more repeatable quantification than successive trials of LAMP, as shown in FTG. 9C, Replicate trials of qPCR quantified the same sample with high ^' reproducibility* while replicate trials of LAMP could often disagree in quantification by art order of magnitude or more. As the difference in quantification was similar for both the device and Vii A 7, it was hypothesized that this observation is a result of the assay itself, and is not largely dependent on the machine used for LAMP. Quantifying samples using either fluorescence or absorbance data from the device was considered, and it was found that the two methods were equally capable, as shown in FIG. 9D,

[00111 j According to an embodiment, a field trial of tire device was conducted in partnership with two Ugandan health clinics that regularly diagnose KS-suspect patients using visual inspection and/or histology. The field trial took place at the Infectious Disease Institute (IDi) in Kampala, and the AIDS Healthcare Foundation - Uganda Cares Clinic in Masaka. One of the goals of this effort was to characterize the sample-to-answer timeline and to demonstrate that results from the device could be obtained on a clinically relevant timescale. Three KS-suspect patients presented at the clinics during the field trial. Biopsies Were taken from the patients and a portion of each biopsy was immediately sent for DNA extraction and subsequent analysis by the device. Results from the device were obtained about 2,5 hours following the start of the biopsy procedure, as shown in FIG. 10A. DNA extraction was the longest part of the process, about 85 minutes on average.

[00112J it Was hypothesized that results from the device would not be dependent on the location of the test (U.S. vs. Uganda), the heating method used (electricity vs. sunlight), or the device operator (the device developers vs. locally trained staff). DNA was extracted from eight KS-suspect biopsies at the IDI in Uganda and was amplified under different experimental conditions. It was found that the same five samples were positive for KSHV D A regardless of the location, heating method, or device operator for the device (Table 2), including samples amplified using sunlight, as shown in FIG. 10B. The threshold times for these eight samples were similar across a large variety of conditions, as shown in FIG. IOC. Furthermore, when threshold times were grouped by patient the resulting clustering shows that quantification by the device is possible across all locations and heating methods, as shown in FTG. 10D. The device gave comparable results using both lyophilized reagents and liquid reagents, demonstrating that the cold chain may not be necessary- for the assay.

[00113] TABLE 2. Results for eight human samples tested in Uganda,

[00114J Three electricity outages (durations: 62 minutes, 1 minute, and 1 minute) were experienced during the amplification of two of the eight biopsy samples. During the longest electricity outage, the device was heated at a neighboring building, as shown in FIG. 1 OA. Upon bringing the device back to the laboratory after heating, the device stayed within the target temperature and finished the assay without electricity. The temperature inside the device for all experiments performed in Uganda was within the 68 ± 1°C target temperature, regardless of heating method or electricity outages, as shown hi FIG. 1QE.

[OOllSJ The high throughput of the device made it possible to analyze many samples by a variety of device operators. Nineteen 6-saniple experiments ( 114 samples) were performed in 5 days. After training the local staff how to operate the device, they were proficient at. operating the system autonomously, and they obtained the same results as did Cornell staff, as shown in Table 2.

[001161 Materials and Methods

[00117 J LAMP assay composition.

[00118 J LAMP uses a strand displacement polymerase and a set of four to six D A primers to create aoipiicons that resemble cauliflower-like, stem-loop D3MA structures in less than an hour. The LAMP assay eontamed 320 U niL of Bst 2.0 WaniiStart DNA Polymerase, IX Isothermal Amplification Buffer, 6 niM MgSG ₄ , L4 m!vi dNTP mix (all from New England BioLabs Inc.), along with primers; 1.6 pM FIP/BtP, 0.2 μΜ F3/B3, and 0.4 pM LodpF LoopB. Isothermal primers were designed previously with Q F 26 as the target, as shown in TABLE 3. Evagreen fluorescent dye (Biotium) was also added to final concentration IX, and ROX reference dye (Themo Fisher Scientific) to final concentration 2X. LAMP ampiicons were confirmed via gel electrophoresis, as shown i FIG. 16.

[00119]! TABLE 3. isothermal primers for the LAMP assay.

[00120} Sample preparation for amplification in the device and ViiA 7,

[00121| Four mL of master mix was made prior to performing quantification experiments. This mix was aiiquoted into tubes for individual experiments to be performed in the device, and then frozen. The _. large -master mix was made to minimize variation in assay composition that might arise from pipetting errors during the preparation of multiple master mixes, so that threshold times could be compared between experiments. The master mix contained all reagents except for Bsl 2.0 WarmStart polymerase, nuclease-free water, and ^' DMA sample. To prepare a sample for amplification in the device, Bsl 2.0 Warmstart DNA Polymerase and water were added to ^' the master mix, and then 35 μΐ, of this mixture was aliquOted into a. PCR tube. ^" Next, 5 pL of DNA sample was added to the PCR tubs and mixed by repeated pipetting. Filially, 50 μ,Τ of paraffin oil was placed on top of the LAMP assay to prevent evaporation. For amplifications performed in the ViiA 7 qPCR machine, the same assay was used except 2,5 pL of DNA sample and 17.5 pL of the mixture containing all other reagents were combined in individual wells in a 96 well qPCR plate. No oil was used for Vii A 7 amplifications.

[901.22] _. Isothermal amplification in the device.

[00123] ^' All nucleic acid amplification experiments in the device started with heating the system to at or above 67°C. If too much heat was put into the system, the inner system temperature (sample temperature) was cooled to at least 70°C before beginning LAMP. When the temperature was suitable for amplification, the l id of the device was removed, the PCR tubes were inserted into sample holes, and the lid was replaced. A microcontroller (Teensy 3.2) ranning an Arduino program was used to track the temperature, fluorescence, and absorbance of the samples throughout the course of the LAMP reaction (at least 50 minutes). Sampling rate was 0.2 Hz. Data from all sensors was analyzed by a MATLAB script.

[00124] Isothermal amplification in the ViiA 7 Real-Time PCR System.

[00125] The normal thermal cycling profile in the ViiA 7 was replaced with a single ramp from room temperature to 68°C, followed by a repeated dwell at 68°C so that fluorescence was recorded ever 30 seconds and total amplification time was 60 min. Threshold times were calculated by the QiiantSiiidio™ Real-Time PG Software using default settings. Thirty seconds were added to the threshold time of all samples ran in the ViiA 7 to account for an. initial 30 second hold that is not considered the first cycle.

[00126] Plasmid DNA standards preparation.

[00127] Circular pBSK-ORF26 plasmid DNA was transformed into competent TOP 10 E. coli (Invitrogen, cat. no. C404OO3) via heat shock. Transformed E. coli were incubated on LB agar plates with ampicillin overnight. Presence of ORF 26 was confirmed via PCR and a single colony was expanded in LB broth with ampiciHin. Resulting DNA was extracted (Zymo Research, cat, no, D4036) and measured via NanoDrop. The circular pBSK-GRF26 plasmid DNA was linearized with EcoRT for 1 hour at .37 ^° C. followed fey heat inaetivation for 20 minutes. Resulting linearized DNA was measured via Qubit 2.0 HS DNA assay and diluted in water until a minimally detectable concentration was reached (-0.1 ug/ L). Further dilutions were performed in 1 ng μΤ salmon sperm DNA (Life Technologies, cat. no. 1563201 1 ) until an estimated target concentration of 0.216 pg/uL was reached, corresponding to 300,000 copies of ORF 26 per iL. 1:5 serial dilutions wer performed such t at a set of standards wa created containing 300000, .60000, 12000, 2400, 480, 96, 19.2, and 0 copies of ORF 26 per 5 £· reaction. f00128]i Cell culture DNA standards preparation.

[00129] DNA was extracted from KSHV+ BC-3 cells cultured in RPMI 1640 + 20% FBS using the DNeasy Blood & Tissue kit (Qiagen, cat. no. 69504). Total starting DNA concentratio was measured via Qubit 2.0 HS DNA assay and the sample was diluted in water to a minimally detectable concentration. 1:5 serial dilutions were performed in salmon sperm DNA and eac sample was run in duplicate against the plasmid standard curve to estimate copy number. Resulting BC-3 standards used in LAMP amplified linearly via qPCR and were estimated to contain cop numbers on the same order of magnitude as the plasmid standard curve.

[00130] DNA extraction from human samples.

[00131] Cylindrical (4 mm diameter) punch biopsies of skin lesions were obtained from Ugandan adults who had at least some level of clinical suspicion for Kaposi ^' s sarcoma and who were referred to the Infectious Diseases Institute in Kampala, for a diagnostic biopsy. Biopsies were stored in RNAIater (Qiagen, cat. no. 76104) and later bisected. Half of the biopsy was processed using the Puiification of Total DNA fiom Animal Tissues protocol of the DNeasy Blood & Tissue kit (Qiagen, cat. no. 69504) and resulting DNA was eluted in 75 Τ of Buffer AE. Total DNA concentration and purity was assessed for each sample via NanoDrop spectrophotometry.

[00132] qPCR assay. [00133] Taqf an assays were used for real-time amplification and detection of viral ORF 26 and control gene GAPDH i qPCR. Eac reaction of the custom ORF 26 assay was performed at a total volume of 20 μΐ. containing 10 μΕ of PrimeTime Gene Expression Master Mix (IDT, eat. no. 1055770), 1.8 ΐ of a 10 μΜ forward and reverse primer mix (primer sequences in Table 3), 2.2 Ε nuclease-free water, 1 μΤ of 5 μΜ ORF 26 probe, and 5 μΤ of sample. The ORF 26 assay was thermal-cycled with holding at 95°C for 20 seconds before cycling 40 times between 95°C for 3 seconds and 60°C for 30 seconds. Each reaction of the GAPDH assay was performed at a total reactio volume of 10 μΕ containing: 5 μΕ of TaqMan Genotyping Master Mix (Thermo Scientific, cat. no. 4371355), 0.5 pL of a 20X GAPDH TaqMan Copy Number Assay (Thermo Scientific, cat. no. 44002 2-Hs0048311 I_cn), and 4.5 Τ of sample. The GAPDH assay was thermal-cycled with holding at 50°C for 2 minutes, 95°C fo 10 minutes, then cycling 40 times between .95 °C for 1 seconds and 6Q°C for 1 minute. All samples were run in duplicate against a standard plasmid curve. Late Ct values amplifying outside the range of the standard curve were considered inconclusive/negative. Raw tissue biopsy DNA extracts were run directly as the assay input and verified with, standard 10 ng dilutions in both assays. All samples showed high copy number of GAPDH.

[00134 J Quantification experiment replication.

[00135]; According to an embodiment, a series of experiments was completed to detemime the reproducibility of quantification by the qPCR. and LAMP assays. All experiment were conducted on human biopsy samples. For technical replicates, each human sample was amplified twice on the same qPCR plate, and quantified the samples by comparison to standard samples amplified on the same plate. For experimental replicates, a plate containing all human samples and all standard samples was amplified in two separate experiments, using the same qPCR master mix. Each plate was then quantified using an average standard curve. An average standard curve was used to mimic the quantification method used in the device, as the device's throughput tn this particula embodiment (4 samples/test) did not enable standard samples to be amplified in the same experiment as human samples. Master mix was frozen and thawed between experimental replicates for the qPCR assay. For the LAMP assay, technical replicates were performed in the ViiA 7 (two of each human sample was amplified in the same experiment), and experimental replicates were performed in the device (human samples were amplified in different experiments, but using the same master mix, which was frozen and thawed between replicates).

00136| Photodiodes used in the device.

[00137J The photodiodes (TSL237SM, ASM sensors) were capable of transducing small light signals to a square wave signal with frequency proportional to irradiation. Light-to-frequency converters were used over Mgbt-to-vohage converters so that the resolution of the measurement would not be limited by the analog to digital converter of the microcontroller (Teensy 3.2), A Teeiisy was used because it is capable of simultaneously measuring frequencies from multiple inputs, with a standardized frequency measurement library. Many other photodiodes and optical sensors could be utilized.

[00138} Threshold time calculation from phoiodiode data.

[00139] Accordin to an embodiment, each frequency was first smoothed using a 10-point moving average method. Then, the Evagree fluorescence (blue LED) and absorhance (red LED) smoothed frequencies were normalized by the ROX smoothed frequencies (yellow LED). Two different algorithms were used to calculate threshold time from this normalized frequency data. Tn the first method, a line was fit to the pre-exponential-amplifi cation normalized frequencies, and the difference between the normalized frequenc and the fit was calculated (FIG. 1 1 ), Once exponential amplification began, this difference would raise above a threshold and the algorithm would calculate this time as the threshold time; The second algorithm calculated the. difference i normalized frequency between successive data points (FIG. 12). Since the second algorithm did not depend o a fit line, it was found to be more reliable. All threshold times reported from the device were calculated using this second method.

[001 0] Phase change material selection.

[00141] During initial prototyping of the device, isothermal reactions were tested with PureTemp 63 (nominal T _m : 63°C) and it was suitable for LAMP. However, more instances were observed of late-stage amplification of negative control samples when using PureTemp 63 , arid therefore chose to work with PureTem 68 (nominal T _m : 68°C) for all experiments presented i this paper. Phase change materials were donated by Entropy Solutions LLC. Although PureTemp materials were used, it should be recognized that many other phase change materials are possible. Additionally, the system may comprise a plurality of phase change materials, which may have the same or different nominal T,„s.

[001421 Solar absorber pla te construction.

[00143} According to an embodiment, the plate used for absorbing sunlight was an aluminum disk painted _, with flat, black paint A Teflon o-ring and acrylic- disk were fixed by high- temperature epo y onto the top of th black aluminum disk. The acrylic disk functions to slow epnvective heat loss to the ambient. The Teflon o-ring has high temperature tolerance and serves to separate the acrylic disk from the hot absorber plate.

[00144f Solar heating of the device.

[00145] According to an embodiment, to heat the device using sunlight, a relativ ely flat working surface that had no obstructions of the sun was identified. A support structure was attached to the device that mounted a 28 x 28 cm square Fresnet lens (Edmund Optics part #32- 597). Both the dev ice and the support, structure for the lens are capable of rotation for alignment with the sun. After alignment, the lens was used to concentrate sunlight onto the absorber plate until enough heat was collected and the isothermal temperature was reached.

[00146] Hoi plate heating of the de vice.

[001 7] According to an embodiment a micro hotplate from TbermoFisher (HP23Q5BQ) was used t heat the dev ice via electricity. To heat, a cutout in the bottom of the device enclosure was removed, and the bottom aluminum surface of the outer cylinder in the device was set onto the hotplate. For the data displayed in FTG. 7A, both the dev ice and the hotplate began at room temperature. The hotplate was then set to level 5 and the device was placed on the hotplate for the reported heating time. After heating, the bottom aluminum cutout was reattached to slow heat loss to the ambient.

[00148] Bunsen burner healing of the device.

[00149] According to an embodiment, a portable, butane-fueled Bunsen burner (Fisher Scientific, item S6514S) was used to heat the device v ia flame. Three support legs were mounted to the: bottom of the device to raise: the system an appropriate height above the Bunsen burner. Then, the cutout was removed from the bottom of the device enclosure to expose the bottom aluminum surface of the outer cylinder. The Bunsen burner was placed beneath this aluminum surface and turned on a low setting to heat the device. After heating, the bottom aUimiitum cutout was reattached to slow heat loss to the environment.

[OOISOJ Automated heating of the device via cartridge heater.

[00151] According to an embodiment, a 12-yolt DC cartridge heater rated at 54 W (Comstat Inc., part MCH1 -240W-004) was used for automated heating of the device. An AC-to-DC adapter ( 12-volt) was used to power a central PCB with a switch, fuse, and MOSFET in series with the cartridge heater. The gate of the MOSFET was controlled by a digital signal from the Teertsy microcontroller, which used a simple code to cycle the heater o or off based on the temperature of the outer cylinder and the temperature of the bottom PCB. A surface-mount power MOSFET (IRLR7843PbF, international Rectifier) with low ¾> _s (2.6 m£2) was selected to minimize the voltage drop across the drain and source.

[00152] Lyopkiiized reagents used in Uganda.

[001531 According to an embodiment, a two-part reaction was setup, and consisted of a lyophiiization mixture and a rehydration mixture. The lyophiiization mixture contained a final concentratio of 1.4 mM dWTPs, 1.6 μΜ FTP/BIP primers, 0.2 μΜ F3/B3 primers, 0,4 uM LoopF/LoopB primers, 960 TJ/ml Glycerol-Free Bst 2.0 Warmstart ΌΝΑ Polymerase (Ne England Biolabs, cat, no. M04O2Z), IX EvaGreen, and I RQX. Tin ^' s mixture was added in equal parts to 2X Lyophiiization Reagent (OPS Diagnostics, cat. no. LR2X 500-02) before being frozen at -80°C and then iran.sfe.tTed. for overnight lyophiiization in. a Labconco Freeze Dryer. After lyophiiization, samples were vacuum packed and stored at room temperature. The rehydratio mixture consisted of IX Isothermal amplificatio buffer and 6 mM MgSO-j. Samples were stored at room temperature for 8 days before experiments in Uganda, and were rehydrated immediately before performing the LAMP assay.

[00154J LAMP experiments performed in Uganda.

[00155] The same operatin procedures were used i Uganda as those outlined in the previous methods sections. For solar experiments, the device was heated outside in the sun to a temperature slightly above 68°C. Following heating, the device was brought back inside the clinic and then LAMP was performed, with no further heat input. All LAMP reactions performed in Uganda were conducted for at least 50 minutes; however, samples which amplified past 24 minutes were to be considered negative (although late amplification was never observed). Since any amplification that started beyond 24 minutes was to be considered negative, 24 minutes wa used as the time for the device amplification during the timeline analysis for biopsy-to-result f00156| ^' While embodiments of the present invention have been particularly shown and described with reference to certain exemplary embodiments, it will be understood by one skilled in the art that various changes in detail may be effected therein without departing from the spirit and scope of the invention as defined by claims that can be supported by the written description and drawings. Further, where exemplary embodiments are described with reference to a certain number of elements it will be understood that the exemplar embodiments can be practiced utilizing either less tha or more than the certain number of elements.