WO2018191252A1 - Système et procédé d'amplification isotherme d'acides nucléiques - Google Patents

Système et procédé d'amplification isotherme d'acides nucléiques Download PDF

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Publication number
WO2018191252A1
WO2018191252A1 PCT/US2018/026865 US2018026865W WO2018191252A1 WO 2018191252 A1 WO2018191252 A1 WO 2018191252A1 US 2018026865 W US2018026865 W US 2018026865W WO 2018191252 A1 WO2018191252 A1 WO 2018191252A1
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WIPO (PCT)
Prior art keywords
heat
phase change
change material
enclosure
measurement unit
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Application number
PCT/US2018/026865
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English (en)
Inventor
David Erickson
Ryan Snodgrass
Ethel Cesarman
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Cornell University
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Publication of WO2018191252A1 publication Critical patent/WO2018191252A1/fr

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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L7/00—Heating or cooling apparatus; Heat insulating devices
    • B01L7/52—Heating or cooling apparatus; Heat insulating devices with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/06—Auxiliary integrated devices, integrated components
    • B01L2300/0627—Sensor or part of a sensor is integrated
    • B01L2300/0654—Lenses; Optical fibres
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/18—Means for temperature control
    • B01L2300/1805—Conductive heating, heat from thermostatted solids is conducted to receptacles, e.g. heating plates, blocks
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/18—Means for temperature control
    • B01L2300/1855—Means for temperature control using phase changes in a medium
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/18—Means for temperature control
    • B01L2300/1883—Means for temperature control using thermal insulation
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L7/00—Heating or cooling apparatus; Heat insulating devices
    • B01L7/04—Heat insulating devices, e.g. jackets for flasks

Definitions

  • the present disclosure is directed generally to methods and systems for portable and energy-flexible nucleic acid analysis platforms.
  • 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,
  • Loop mediated isothermal amplification 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.
  • 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.
  • 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.
  • a nucleic acid analysis 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.
  • 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.
  • 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.
  • the device further includes a, lens configured to direct and/or amplify solar energy o the solar receiver.
  • the device further includes a power source configured to power the light source and the optical sensor during nucleic acid quantification.
  • the light source comprises a plurality of tight sources comprising at least two different wavelengths.
  • the device further includes an optical filter positioned between the light source and the optical sensor.
  • the measurement unit comprises a plurality of sample wells,.
  • the measurement unit comprises an optical sensor for each of the plurality of sample wells.
  • the phase change material comprises a Tm selected to facilitate nucleic acid analysis by the measurement unit.
  • the space between the enclosure and the second enclosure comprises a seal configured to contain the phase change material.
  • the measurement unit is configured to perform a LAMP assay using heat energy released by the phase change material
  • the enclosure and the second enclosure are cylindrical.
  • die device further includes an insulator
  • a method for nucleic acid analysis utilizing a portable device 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.
  • 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.
  • 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.
  • the device further includes a lens
  • 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.
  • FIG. 1 is a cutaway view of a portable nucleic acid analysis device, in accordance with an embodiment
  • FIG. 2 is a top view of a portion of a portable nucleic acid analysis device, in accordance with an embodiment.
  • FIG. 3 is a cutaway view of a portion of a portable nucleic acid analysis device, in accordance with an embodiment.
  • FIG. 4 is a top view of a portable nucleic acid analysis device, in accordance with an embodiment.
  • FIG. 5 is a flowchart of a method for using a portable nucleic acid analysis device, in accordance with an embodiment.
  • 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.
  • 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.
  • 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.
  • FIG. 6D is a summary of the energy required to operate the device for one hour.
  • 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.
  • 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.
  • FIG. 7B is a graph of the threshold times of samples containing the same target 0 ⁇ 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. 7 ⁇ . ⁇
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • FIG. 9 ⁇ 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.
  • 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.
  • FIG. 10B is an image of the device being heated with sunlight at the Kenya Cares Clinic in Masaka, Kenya.
  • 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.
  • 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. ⁇ ⁇ is not included as that sample was obtained after some conditions were tested.
  • FIG. 10 ⁇ 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).
  • 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,
  • 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.
  • 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.
  • 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 produced threshold times roughly between 1 ⁇ and 15 minutes.
  • Human biopsy samples blue line
  • 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.
  • 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.
  • 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.
  • 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.
  • FIG. 17 A is a schematic representation of the portable device being carried by hand, in accordance with an embodiment.
  • 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.
  • 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,
  • FIG. I7D is a schematic representation of the portable device being heated by electricity, in accordance with an embodiment.
  • FIG. ⁇ 7 ⁇ is an image of the portable device being heated by concentrated sunlight using a Fresnel lens, in accordance with an embodiment.
  • the system comprises a measurement unit and a temperature regulation unit, which can be a single device or separable components.
  • 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.
  • 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.
  • the dev ce is compared to performance of quantitative polymerase chain reaction (qPCR) machines when analyzing human skin biopsies from Kenyan patients suspected of Kaposi 's sarcoma (KS).
  • qPCR quantitative polymerase chain reaction
  • KS Kaposi's sarcoma-associated herpesvirus
  • KSHV Kaposi's sarcoma-associated herpesvirus
  • NAT herpesvirus
  • 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.
  • 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.
  • PCM phase change material
  • LAMP loop-mediated isothermal amplification
  • 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.
  • 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.
  • phase change material 150 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.
  • the phase change material may be selected to comprise a T in that facilitates nucleic acid amplification., among other types of analysis.
  • 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.
  • 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.
  • 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 .
  • 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 w3 ⁇ 4 n. the system.
  • the insulation 190 may be any material or structure configured to prevent heat loss.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • portable nucleic acid analysis device 100 can comprise many different structures.
  • 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.
  • 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.
  • 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.
  • measurement component 1 ) 0 also comprises a printed circuit board 220 on or in the top surface.
  • measurement component 1 10 is rounded i this embodiment, the component may be any shape and size.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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,
  • 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,
  • Measurement component ⁇ 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.
  • shell 260 is any heat-transmitting material, including but not limited to aluminum among many other possible materials.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the temperature-regulation unit and measurement unit were assembled together and placed into a aluminum enclosure (Protocase ® ).
  • the volume and weight of the complete system was 2.1 L and 1.1 kg, respectively.
  • two of the possible heat sources available for operating the device are electricity and sunlight.
  • 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.
  • 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.
  • 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.
  • a portable nucleic acid analysis device 100 is provided.
  • the portable nucleic acid analysis device may 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.
  • heat energy is provided to the portable nucleic acid analysts device.
  • the heat energy may be provided via any of the methods or systems described or envisioned herein.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 .
  • 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.
  • heat energy stored in the phase change material is transferred to the measurement component of the portable device.
  • 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.
  • 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.
  • the analysis can be a L AMP analysis, among many other types of nucleic acid analysis.
  • 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.
  • the portable device described or otherwise envisioned herein has numerous advantages over both commercial and research-grade systems for NAT.
  • 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.
  • the portable device is unique in that it is portable and resilient against power outages.
  • 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.
  • 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,
  • 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. ⁇ 7 ⁇ ).
  • 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).
  • FIG. 6 is a graph of heating characterization of an embodiment of the analytical device.
  • 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).
  • 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.
  • the qPCR assay 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 ⁇ i 0 " to 3.9 10 ' ' copies/reaction), but at lower concentrations threshold time no longer linearly predicted starting DNA concentration.
  • 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.
  • a field trial of tire device was conducted in partnership with two Kenyan 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 - Kenya Cares Clinic in Masaka.
  • IDi Infectious Disease Institute
  • AIDS Healthcare Foundation - Kenya 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.
  • 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 ⁇ 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
  • ROX reference dye Themo Fisher Scientific
  • 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.
  • Cylindrical (4 mm diameter) punch biopsies of skin lesions were obtained from Kenyan 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 ⁇ of Buffer AE. Total DNA concentration and purity was assessed for each sample via NanoDrop spectrophotometry.
  • qPCR assay [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 ⁇ . containing 10 ⁇ of PrimeTime Gene Expression Master Mix (IDT, eat. no. 1055770), 1.8 ⁇ of a 10 ⁇ forward and reverse primer mix (primer sequences in Table 3), 2.2 ⁇ nuclease-free water, 1 ⁇ of 5 ⁇ ORF 26 probe, and 5 ⁇ of sample.
  • IDTT PrimeTime Gene Expression Master Mix
  • 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 ⁇ containing: 5 ⁇ 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 ⁇ 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • a micro hotplate from TbermoFisher (HP23Q5BQ) was used t heat the dev ice via electricity.
  • 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.
  • 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.
  • 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.
  • 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 3 ⁇ 4> s (2.6 m£2) was selected to minimize the voltage drop across the drain and source.
  • 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 ⁇ FTP/BIP primers, 0.2 ⁇ F3/B3 primers, 0,4 uM LoopF/LoopB primers, 960 TJ/ml Glycerol-Free Bst 2.0 Warmstart ⁇ 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.

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Abstract

La présente invention concerne un dispositif d'analyse d'acide nucléique, comprenant : (i) une unité de mesure comprenant un puits d'échantillon, une source de lumière et un capteur optique, et conçue pour effectuer une analyse d'acide nucléique ; (ii) une enceinte contenant l'unité de mesure et comprenant un matériau transmettant la chaleur ; et (iii) un matériau à changement de phase conçu pour stocker de la chaleur reçue en provenance d'une source de chaleur, et conçu pour libérer de la chaleur vers l'unité de mesure par l'intermédiaire du matériau transmettant la chaleur.
PCT/US2018/026865 2017-04-10 2018-04-10 Système et procédé d'amplification isotherme d'acides nucléiques WO2018191252A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022018741A1 (fr) * 2020-07-23 2022-01-27 Indian Institute Of Technology, Kharagpur Dispositif de point d'intervention (poc) pour test à base d'acide nucléique et procédé correspondant
WO2023195945A1 (fr) * 2022-04-05 2023-10-12 Yonca Teknoloji Muhendislik Ve Elektronik Hiz. Ltd. Sti. Dispositif de pcr isotherme

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US20060145098A1 (en) * 2003-04-03 2006-07-06 Jong-Soo Baek Real-time monitoring apparatus for biochemical reaction
US20090004732A1 (en) * 2007-06-06 2009-01-01 Labarre Paul Donald Chemical Temperature Control
US20120315638A1 (en) * 2011-05-23 2012-12-13 The Trustees Of The University Of Pennsylvania Moisture-Activated Self-Heating Analysis Device
US20150027434A1 (en) * 2012-02-21 2015-01-29 Anthrogenesis Corporation Devices and methods for thawing biological material
EP1964610B1 (fr) * 2007-02-27 2017-03-29 Sony Corporation Amplificateur d'acides nucléiques

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Publication number Priority date Publication date Assignee Title
US20060145098A1 (en) * 2003-04-03 2006-07-06 Jong-Soo Baek Real-time monitoring apparatus for biochemical reaction
EP1964610B1 (fr) * 2007-02-27 2017-03-29 Sony Corporation Amplificateur d'acides nucléiques
US20090004732A1 (en) * 2007-06-06 2009-01-01 Labarre Paul Donald Chemical Temperature Control
US20120315638A1 (en) * 2011-05-23 2012-12-13 The Trustees Of The University Of Pennsylvania Moisture-Activated Self-Heating Analysis Device
US20150027434A1 (en) * 2012-02-21 2015-01-29 Anthrogenesis Corporation Devices and methods for thawing biological material

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022018741A1 (fr) * 2020-07-23 2022-01-27 Indian Institute Of Technology, Kharagpur Dispositif de point d'intervention (poc) pour test à base d'acide nucléique et procédé correspondant
WO2023195945A1 (fr) * 2022-04-05 2023-10-12 Yonca Teknoloji Muhendislik Ve Elektronik Hiz. Ltd. Sti. Dispositif de pcr isotherme

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