DESC:TECHNICAL FIELD
[0001] The present invention generally relates to the technical field of thermal cyclers. Particularly, the present disclosure provides a portable energy-efficient temperature cycler for small-volume chemical reactions. The temperature cycler of the present disclosure can find utility in myriad of applications, including usage thereof as Polymerase Chain Reaction (PCR) thermal cycler, but not limited thereto. Further aspects of the present disclosure provide a reaction cassette, a method of amplifying a sample comprising nucleic acid and a method of operating a temperature cycler.

BACKGROUND
[0002] Background description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.
[0003] Periodic heating and cooling (commonly known as “thermal cycling”), is an indispensible aspect of many chemical reactions, wherein temperature control plays a pivotal role. Thermal cycling of small volume chemical reactions (such as, chemical reactions where the volumes are 1 nanolitre to 1 millilitre (.001 to 1000 µL)) poses several fold technical challenges, and rigorous research has been done in the field. Presently, most commercial methods to perform thermal cycling make use of Peltier elements for heating and cooling. These are typically attached to a thermally conductive block and a heat sink, while the reagents/reactants are placed inside a small plastic vial (~100µL capacity). However, this is quite energy inefficient because the thermal mass of the system is much larger (>300x) than the thermal mass of the liquid (reactants/reagents), even when it is loaded to the maximum capacity. The energy required for heating is thus >300x larger than that required to heat just the liquid drop. As for cooling, even if the coefficient of performance (COP) achieves the maximum typical value of 4 [as reported in Ferrotec Thermal, “Peltier Cooler Model 9501/128/040 B - Thermoelectric”, available at https://thermal.ferrotec.com/products/peltier-thermoelectric-coolermodules/ 9501_128_040-b/, accessed on April 03, 2020], the energy required for cooling alone is still ~100x greater than the theoretical minimum required for cooling the drop alone. Such high energy requirements limit their use.
[0004] To resolve this issue, the state-of-art reported methods resort to using lower thermal mass, but they tend to use expensive materials or heating methods that must be fabricated using complex processes such as usage of micro-machined silicon [as reported in P. Neuzil, L. Novak, J. Pipper, S. Lee, L. F. P. Ng, and C. Zhang, “Rapid detection of viral RNA by a pocket-size real-time PCR system,” Lab Chip, vol. 10, no. 19, pp. 2632–2634, Oct. 2010, doi: 10.1039/c004921b], and indium-tin-oxide [as reported in K. Sun, A. Yamaguchi, Y. Ishida, S. Matsuo, and H. Misawa, “A heater-integrated transparent microchannel chip for continuous-flow PCR,” Sensors Actuators B Chem., vol. 84, no. 2–3, pp. 283–289, May 2002, doi: 10.1016/S0925-4005(02)00016-3]. Additionally, these methods and devices often use “virtual reaction chambers”, where the liquid drop is covered using mineral oil to prevent evaporation. However, this can fail for some volumes, or still allow some reagents (such as DNA amplified in a PCR) to become aerosolized [H. Terazono, H. Takei, A. Hattori, and K. Yasuda, “Development of a high-speed realtime polymerase chain reaction system using a circulating water-based rapid heat exchange,” Jpn. J. Appl. Phys., vol. 49, no. 6 PART 2, Jun. 2010, doi: 10.1143/JJAP.49.06GM05], contaminating future reactions performed in the same area.
[0005] Another method resorting to heating using air was proposed [as reported in C. T. Wittwer, G. C. Fillmore, and D. R. Hillyard, “Automated polymerase chain reaction in capillary tubes with hot air,” Nucleic Acids Res., vol. 17, no. 11, pp. 4353– 4357, Jun. 1989, doi: 10.1093/nar/17.11.4353], but this also suffers from poor energy efficiency owing to usage of high power coils (~1kW).
[0006] Still other method relies on the hot water pumped through channels in an aluminium block for heating [as reported in H. Terazono, H. Takei, A. Hattori, and K. Yasuda, “Development of a high-speed realtime polymerase chain reaction system using a circulating water-based rapid heat exchange,” Jpn. J. Appl. Phys., vol. 49, no. 6 PART 2, Jun. 2010, doi: 10.1143/JJAP.49.06GM05], but it has many moving parts. The complexity of the system proposed therein makes it difficult to make it portable and/or to implement it in point-of-care settings.
[0007] Few optical heating mechanisms have been reported in the state-of-art, to the best of the knowledge of inventors of the present application. One of the earliest methods used a high power tungsten lamp whose light was focused onto drops [C. Ke, H. Berney, A. Mathewson, and M. M. Sheehan, “Rapid amplification for the detection of Mycobacterium tuberculosis using a non-contact heating method in a silicon microreactor based thermal cycler,” Sensors Actuators, B Chem., vol. 102, no. 2, pp. 308–314, Sep. 2004, doi: 10.1016/j.snb.2004.04.083]. In some cases, infrared lasers are used to heat the droplet directly [H. Kim, S. Vishniakou, and G. W. Faris, “Petri dish PCR: Laser-heated reactions in nanoliter droplet arrays,” Lab Chip, vol. 9, no. 9, pp. 1230–1235, May 2009, doi: 10.1039/b817288a], however, this is expensive because IR lasers are expensive. Another technique reported in state-of-art uses an LED of a specific wavelength [J. H. Son et al., “Ultrafast photonic PCR,” Light Sci. Appl., vol. 4, no. 7, pp. e280– e280, Jul. 2015, doi: 10.1038/lsa.2015.53], however it uses gold nanoparticles that exhibits a phenomenon known as surface plasmon resonance (SPR) to absorb the light. As can be readily appreciated, gold is expensive, its deposition process is complex and it limits the light source that can be employed. Said PCR avoids sample evaporation by using “virtual reaction chambers” which as mentioned above are not robust for point-of-care applications. Further, temperature monitoring herein employs thermocouples in each disposable substrate which drastically increases the cost and complexity. Another device described in WO2013/100859A1 discloses the use of a laser diode that must be rotated relative to a ring of substrate and necessitates use of complex optical components like beam splitters for collimation adding to cost and complexity.
[0008] Another indispensible aspect of the temperature control, apart from the above-mentioned aspect of heating means/mechanism, is temperature detection/monitoring. In most techniques reported till date, the temperature is monitored using methods that require thermal contact, such as a thermocouple or a resistance temperature device (RTD). This removes heat from the liquid, reducing efficiency and adding significantly to the overall cost, since a thermocouple that comes into contact with the solution must usually be discarded. It is not preferred to clean a chamber used for PCR, since imperfect cleaning will lead to contamination of future reaction, and if cleaning agents are left behind, they can significantly impede the reaction.
[0009] Accordingly, there remains a long standing need in the state-of-art for a portable energy-efficient temperature cycler for small-volume chemical reactions. Need was also felt of a portable reaction cassette that can be implemented in point-of-care settings, for example in PCR based molecular diagnostics (MD) requiring thermal cycling. The present disclosure addresses the above-mentioned technical problem(s) and provides a portable energy-efficient temperature cycler and a portable reaction cassette that are particularly suited for small-volume chemical reactions that alleviates, at least in part, the shortcomings associated with the devices and methods known in the art.

OBJECTS OF THE INVENTION
[0010] An object of the present disclosure is to provide a portable, energy-efficient temperature cycler for small-volume chemical reactions.
[0011] An object of the present disclosure is to provide a portable, energy-efficient, economical and easy-to-fabricate temperature cycler.
[0012] An object of the present disclosure is to provide a portable reaction cassette that can be implemented in point-of-care settings.
[0013] An object of the present disclosure is to provide a portable reaction cassette that is easy to use and dispose and has low contamination risks.
[0014] An object of the present disclosure is to provide a temperature cycler for PCR based molecular diagnostics that is cost-effective, reliable and easy to fabricate.

SUMMARY
[0015] This summary is provided to introduce a selection of concepts in a simplified form that are further described below in Detailed Description section. This summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used as an aid in determining the scope of the claimed subject matter.
[0016] The present invention generally relates to the technical field of thermal cyclers. Particularly, the present disclosure provides a portable, energy-efficient temperature cycler for small-volume chemical reactions. The temperature cycler of the present disclosure can find utility in myriad of applications, including usage thereof as Polymerase Chain Reaction (PCR) thermal cycler, but not limited thereto. Further aspects of the present disclosure provide a reaction cassette, a method of amplifying a sample comprising nucleic acid and a method of operating a temperature cycler.
[0017] An aspect of the present disclosure provides a temperature cycler, said temperature cycler comprising: a heating unit comprising an emitter configured to emit electromagnetic radiations; a stage configured to support a reaction cassette, said reaction cassette defining at least one region made of a thermally conductive material, said at least one region being configured to host a volume of one or more reagents and defining a first surface facing the one or more reagents and a second surface opposite to the first surface, wherein the stage is configured to position the reaction cassette such that the second surface is exposed to the electromagnetic radiations emitted by the emitter; a temperature detection unit configured to detect temperature of the volume of one or more reagents, and a controller operatively connected with any or a combination of said heating unit, the stage and the temperature detection unit; wherein the at least one region at the second surface has high electromagnetic radiation absorptivity.
[0018] In an embodiment, the at least one region at the second surface has high electromagnetic radiation absorptivity. In still another embodiment, the at least one region at the first surface and the at least one region at the second surface have high electromagnetic radiation absorptivity. In an embodiment, the at least one region, at the second surface has electromagnetic radiation absorptivity higher than that of said at least one region at the first surface.
[0019] In an embodiment, the emitter may be configured to emit electromagnetic radiations in a choice of electromagnetic spectrum such as the visible spectrum (wavelength ranging from 400-900 nm). In an embodiment, the emitter may be an optothermal heater including but not limited to, Light Emitting Diode (LED) (including ultraviolet high-power LED), compact fluorescent lamp, a gas discharge tube, IR LED, laser, tungsten lamp, incandescent light source, or combinations thereof. Alternatively, the electromagnetic radiations may be generated by resistive heater, concentration of sunlight, radiation from a fire gated with a metal plate, radioactive heaters, induction heater, microwave emitter, or combinations thereof. Alternatively, the emitter may emit hot air instead of an electromagnetic radiation.
[0020] In an embodiment, the temperature detection unit comprises a non-contact based temperature detection unit including an Infrared (IR) temperature sensor, Infrared sensor array, infrared camera, fluorescence-based temperature sensor, spectroscopy based detection unit, sonic sensors, or combinations thereof; a contact based temperature detection unit including a thermocouple; or an integrated temperature detection unit including vapor-pressure based temperature unit, thermochromic liquid crystals, or resistance-based temperature detector; or combinations thereof.
[0021] In a preferred embodiment, the temperature detection unit is a non-contact based temperature detection unit, more preferably an Infrared (IR) temperature sensor. The Infrared (IR) temperature sensor may be configured to detect electromagnetic radiations having wavelength ranging from 5500 - 14000 nm. In an embodiment, the controller may be operatively connected with the heating unit and the temperature detection unit. In an embodiment, the controller is configured to control output of the emitter based on the detected temperature of the one or more reagents.
[0022] Another aspect of the present disclosure provides a reaction cassette, said cassette comprising: a platform made of a thermally insulating material, said platform defining at least one region made of a thermally conductive material, wherein said at least one region is configured to host a volume of one or more reagents, and wherein said at least one region defines a first surface facing the one or more reagents and a second surface opposite to the first surface, said at least one region, at its second surface having high electromagnetic radiation absorptivity. In an embodiment, the at least one region, at its second surface, is deposited with a material such that the region at the second surface has high electromagnetic radiation absorptivity. In still another embodiment, the at least one region at the first surface and the at least one region at the second surface have high electromagnetic radiation absorptivity.
[0023] In an embodiment, the platform defines a plurality of regions made of same or different thermally conductive materials. In an embodiment, the region defines at least one depression at its first surface hosting the volume of said one or more reagents. In an embodiment, the region defines a plurality of depressions at its first surface, one, few or all of the plurality of depressions hosting the volume of said one or more reagents.
[0024] In an embodiment, the at least one region is covered, at least in part, with a sealing material.
[0025] In embodiments, wherein a plurality of regions are defined on the platform, one, few or all of the regions can be covered with sealing material either individually (i.e. one, few or all of the regions can be covered individually with sealing material(s)) or collectively (i.e. one, few or all of the regions can be covered collectively with a sealing material). In an embodiment, the region is covered with a sealing material such that the volume of one or more reagents is not exposed to ambient environment. In an embodiment, the sealing material comprises a tape. The sealing material may be a polydimethylsiloxane (PDMS) tape, acrylic, silicone, and rubber-based tapes. In an embodiment, the sealing material is positioned such that the volume of one or more reagents may or may not be in contact with the sealing material. In an embodiment, the sealing material is of a material, which is transparent to the electromagnetic radiation. In an embodiment, the sealing material is of a material that allows transmission of electromagnetic radiations within a chosen wavelength range such as from 5500 - 14000 nm. In an embodiment, the sealing material is of a material that allows transmission of electromagnetic radiations with wavelength ranging from 280 - 400 nm.
[0026] In an embodiment, the platform comprises a slide made of a thermally insulating material and defines at least one slot having affixed thereon, one or a plurality of sheets of thermally conductive material forming the at least one region made of the thermally conductive material. In an embodiment, the at least one region, at its second surface, is deposited with a material such that the region at the second surface has electromagnetic radiation absorptivity higher than that of said region at the first surface.
[0027] In an embodiment, the platform comprises a slide made of a thermally insulating material and defines at least one slot having affixed thereon a plurality of sheets of thermally conductive material forming the region made of the thermally conductive material, wherein at least one sheet out of the plurality of sheets has high electromagnetic radiation absorptivity. In an embodiment, the at least one sheet having high electromagnetic radiation absorptivity is in vicinity of the second surface. In an embodiment, the at least one sheet having high electromagnetic radiation absorptivity is configured at the second surface.
[0028] In an embodiment, the platform comprises a plurality of overlaying slides made of a thermally insulating material, each of the plurality of overlaying slides defining at least one slot of matching profile, wherein one, few or all of the overlaying slides are affixed with one or a plurality of sheets of thermally conductive material at the at least one slot.
[0029] Other aspects of the invention will be set forth in the description which follows, and in part will be apparent from the description, or may be learnt by the practice of the invention.

BRIEF DESCRIPTION OF DRAWINGS
[0030] The following drawings form part of the present specification and are included to further illustrate aspects of the present disclosure. The disclosure may be better understood by reference to the drawings in combination with the detailed description of the specific embodiments presented herein.
[0031] FIG. 1A and 1B illustrate a schematic view of the temperature cycler realized in accordance with embodiments of the present disclosure.
[0032] FIG. 2A illustrates an exemplary view of the temperature cycler, realized in accordance with an embodiment of the present disclosure, showing positioning of a reaction cassette on the stage. FIG. 2B illustrates an exemplary view of the temperature cycler with a reaction cassette positioned on the stage, in accordance with an embodiment of the present disclosure.
[0033] FIG. 3 illustrates an exemplary schematic view of the temperature cycler in combination with the exploded view of the reaction cassette, in accordance with an embodiment of the present disclosure.
[0034] FIG. 4 illustrates an exemplary graph showing representative thermal cycle performed using the temperature cycler of the present disclosure.
[0035] FIG. 5 illustrates an exemplary graph showing a representative thermal cycle performed with the acrylic cassette, as per an embodiment of the present disclosure, using the temperature cycler of the present disclosure at 10 microlitres.
[0036] FIG. 6 illustrates an exemplary graph showing a representative thermal cycle performed with the acrylic cassette, as per an embodiment of the present disclosure, using the temperature cycler of the present disclosure at 20 microlitres.
[0037] FIG. 7 illustrates an exemplary graph for the temperature stability achieved by the temperature cycler at 90°C over 100s as per an embodiment of the present disclosure.
[0038] FIG. 8 illustrates an exemplary graph for the temperature stability achieved by the temperature cycler at 50°C over 100s as per an embodiment of the present disclosure.
[0039] FIG. 9 illustrates an exemplary reaction cassette (110) realized in accordance with an embodiment of the present disclosure. FIG. 9(a) illustrates an exemplary top view of a reaction cassette (110) realized in accordance with an embodiment of the present disclosure. FIG. 9(b) illustrates an exemplary bottom view of a reaction cassette (110) realized in accordance with an embodiment of the present disclosure. FIG. 9(c) illustrates an exemplary lateral view of the at least one region (420) realized in accordance with an embodiment of the present disclosure.
[0040] FIG. 10 illustrates briefly the process of fabrication of the exemplary reaction cassette said cassette comprising a platform made of cardboard as per an embodiment of the present disclosure.
[0041] FIG. 11 illustrates an exemplary reaction cassette (110), wherein the platform (410) is made of thermally insulating material, acrylic, in 11(a) compact form; and 11(b) exploded form, realized in accordance with an embodiment of the present disclosure. FIG. 11(c) illustrates the lateral view of the at least one region (420) realized in accordance with an embodiment of the present disclosure.
[0042] FIG. 12 provides a pictorial image of an exemplary reaction cassette (110), (a) top view and 12(b) bottom view, comprising a platform made of acrylic, realized in accordance with an embodiment of the present disclosure.
[0043] FIG. 13 illustrates an exemplary reaction cassette (110), wherein the platform (410) is made of thermally insulating material, acrylic, in (a) compact form; and (b) exploded form, realized in accordance with an embodiment of the present disclosure. FIG. 12(c) illustrates the lateral view of the at least one region (420) realized in accordance with an embodiment of the present disclosure.
[0044] FIG. 14 provides a pictorial image of an exemplary reaction cassette (110), (a) top view and 12(b) bottom view, comprising a platform made of acrylic, realized in accordance with an embodiment of the present disclosure.
[0045] FIG. 15 illustrates briefly the process of fabrication of the exemplary reaction cassette said cassette comprising a platform made of acrylic as per an embodiment of the present disclosure.
[0046] FIG. 16 illustrates exemplary reaction cassettes (110), realized in accordance with an embodiment of the present disclosure, with the aluminium foil (a) indented concavely along the first surface, and (b) indented convexly along the first surface.
[0047] FIG. 17(a) and 17(b) provides a pictorial representation of an exemplary temperature cycler in a casing as per an embodiment of the present disclosure.
[0048] FIG. 18 illustrates electrophoresis gel results for amplified DNA obtained from a conventional RT-PCR system and an optothermal temperature cycler as per an embodiment of the present disclosure.
[0049] FIG. 19 illustrates an exemplary graph of temp v/s time for a 40-cycle 2-step PCR, with no initial denaturation or final extension phase, in 10 mins for a 20 microlitre sample in a temperature cycler as per an embodiment of the present disclosure.
[0050] FIG. 20 illustrates an exemplary graph of temp v/s time for a 30-cycle 2-step PCR, with no initial denaturation or final extension phase, in 5 mins for a 10 microlitre sample in a temperature cycler as per an embodiment of the present disclosure.

DETAILED DESCRIPTION
[0051] The present invention generally relates to the technical field of thermal cyclers. Particularly, the present disclosure provides a portable energy-efficient optothermal temperature cycler for small-volume chemical reactions. The temperature cycler of the present disclosure can find utility in myriad of applications, including usage thereof as Polymerase Chain Reaction (PCR) thermal cycler, but not limited thereto. Further aspects of the present disclosure provide a portable reaction cassette, a method of amplifying a sample comprising nucleic acid and a method of operating an optothermal temperature cycler.
[0052] The following is a detailed description of embodiments of the disclosure depicted in the accompanying drawings. The embodiments are in such detail as to clearly communicate the disclosure. However, the amount of detail offered is not intended to limit the anticipated variations of embodiments; on the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the present disclosure as defined by the appended claims.
[0053] Each of the appended claims defines a separate invention, which for infringement purposes is recognized as including equivalents to the various elements or limitations specified in the claims. Depending on the context, all references below to the “invention” may in some cases refer to certain specific embodiments only. In other cases it will be recognized that references to the “invention” will refer to subject matter recited in one or more, but not necessarily all, of the claims.
[0054] As used in the description herein and throughout the claims that follow, the meaning of “a,” “an,” and “the” includes plural reference unless the context clearly dictates otherwise. Also, as used in the description herein, the meaning of “in” includes “in” and “on” unless the context clearly dictates otherwise.
[0055] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g. “such as”) provided with respect to certain embodiments herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.
[0056] An aspect of the present disclosure provides a temperature cycler, said temperature cycler comprising: a heating unit comprising an emitter configured to emit electromagnetic radiations; a stage configured to support a reaction cassette, said reaction cassette defining at least one region made of a thermally conductive material, said at least one region being configured to host a volume of one or more reagents and defining a first surface facing the one or more reagents and a second surface opposite to the first surface, wherein the stage is configured to position the reaction cassette such that the second surface is exposed to the electromagnetic radiations emitted by the emitter; a temperature detection unit configured to detect temperature of the one or a plurality of reagents, and a controller operatively connected with any or a combination of said heating unit, the stage and the temperature detection unit; wherein the at least one region at the second surface has high electromagnetic radiation absorptivity.
[0057] In still another embodiment, the at least one region at the first surface and the at least one region at the second surface have high electromagnetic radiation absorptivity. In an embodiment, the at least one region, at the second surface has electromagnetic radiation absorptivity higher than that of said at least one region at the first surface.
[0058] In an embodiment, the at least one region at the second surface may be deeply colored. In an embodiment, the at least one region at the second surface may be blackened. In an embodiment, the blackening may be performed by deposit of black ink. The deposition may be performed conveniently and cost-effectively using any method, including but not limited to, an ordinary permanent marker, spray-coating, or an inkjet printer. Alternatively, said blackening may be performed by coating of carbon.
[0059] In an embodiment, the thermally conducting material may be selected from, but is not limited to, thermally conducting metals - aluminium, tin, or copper; thermally conductive polymers - polyimide, graphene, or conductive polyethylene; thermally conductive ceramics: Aluminium Nitride, Silicon Nitride, or Beryllium oxide; thermally conductive composites: aluminium/carbon fibre, polyester/graphene, graphene/carbon-black, carbon-nanotube/graphene based composites or combinations thereof. In an embodiment, the thermally conducting material may be coated to improve its biocompatibility. Said coating may be made of biocompatible compounds, including but not limited to, biomaterials such as protein; bovine serum albumin (BSA); or polymers such as bioplastics of polypropylene, PDMS, polyethylene, PMMA, or PTFE.
[0060] In a preferred embodiment, the thermally conducting material may be Aluminium, more preferably an Aluminium foil.
[0061] In an embodiment, the emitter may be configured to emit electromagnetic radiations in a choice of electromagnetic spectrum such as the visible spectrum (wavelength ranging from 400-900 nm). In an embodiment, the emitter may be an optothermal heater including but not limited to, a Light Emitting Diode (LED) (including ultraviolet high-power LED or IR LED), compact fluorescent lamp, a gas discharge tube, laser, tungsten lamp, incandescent light source, or combinations thereof. Alternatively, the electromagnetic radiations may be generated by resistive heater (using the resistance of the thermally conductive material to heat by using electric current), concentration of sunlight, radiation from a fire gated with a plate of metal or other conductive material, radioactive heaters, induction heater, microwave emitter, or combinations thereof. In another alternative, the emitter may emit hot air instead of an electromagnetic radiation. In an embodiment, the emitter may be a combination of any of the above emitters. Preferably, the emitter is an optothermal heater, more preferably an LED. Electrical power of the LED may be in the range of about 3W to about 20W, or chosen as maybe suitable for a given application.
[0062] With regards thermal cycling, particularly, decreasing the temperature of the one or more reagents, cooling typically occurs rapidly by contact of the region holding the one or more reagents with the ambient environment. Accordingly, provision of additional heat-sink or cooling mechanism is not necessary. Nonetheless, if need be, one can configure one or more heat-sinks, vortex tube or blowers (such as, a small fan) or such other cooling mechanism for rapidly decreasing the temperature of the one or more reagents. In an embodiment, the heat-sinks or blowers may be controlled by the controller.
[0063] In an embodiment, the temperature detection unit comprises a non-contact based temperature detection unit including an Infrared (IR) temperature sensor, Infrared sensor array, infrared camera, fluorescence-based temperature sensor, spectroscopy based detection unit, sonic sensors, or combinations thereof. In fluorescence-based temperature sensor, the fluorescence of the one or more reagents may be used to infer temperature, since fluorescent dyes have a temperature-dependent fluorescence signal. Multiple fluorescent dyes can be used simultaneously to improve accuracy. In spectroscopy based detection unit: fluorescence correlation, raman spectroscopy and any other methods well known in the art may be used to infer temperature of the one or more reagents. In sonic sensors, sound waves may be used to infer temperature, such as, by measuring the change in the size of the at least one region through resonance, through photon-phonon coupling, or through change in propagation constants (such as bulk modulus). In an embodiment, the temperature detection unit comprises a contact based temperature detection unit including a thermocouple, or depositing a droplet of a liquid, (such as, ethanol) and measuring the evaporation rate (using say a camera). In an embodiment, the temperature detection unit comprises an integrated temperature detection unit including vapor-pressure based temperature unit, thermochromic liquid crystals, or resistance-based temperature detector. In vapor-pressure based temperature measurement, the vapour pressure of the liquid depends on the temperature and measurement of a parameter such as, the displacement of the sealing material, may be used to infer temperature. This may be done optically, such as by using a laser, or sonically, such as by probing resonance frequency of the at least one region. The thermochromic liquid crystals may be used with additives. Alternatively, a strip of conductive ink whose resistance depends on temperature may be used. A method of detection may be used, wherein a liquid may be added whose interface energy can change with temperature, and a camera can monitor the shape of the interface. Any other method for detection of temperature, well-known to a person of skill in the art, may be employed.
[0064] In a preferred embodiment, the non-contact based temperature detection unit comprises an Infrared (IR) temperature sensor. In an embodiment, the Infrared (IR) temperature sensor is configured to detect electromagnetic radiations having wavelength ranging from 5500 - 14000 nm.
[0065] In an embodiment, the controller may be selected from a proportional-integral-derivative (PID) controller, model-based predictive controller, adaptive controller, multipole controllers, or combinations thereof. Any conventional controller, as known to or appreciated by a person skilled in the art, may be used for controlling operations.
[0066] In an embodiment, the controller may be operatively connected with the heating unit and the temperature detection unit. In an embodiment, the controller is configured to control output of the emitter based on the detected temperature of the one or more reagents.
[0067] In an embodiment, the temperature cycler may optionally comprise an additional battery, or a recharging circuit. In an embodiment, the temperature cycler may optionally additionally comprise a barcode/QR code scanner (to automatically load programs); output devices such as a printer or a screen; network interfaces such as Bluetooth or WiFi; or external power supplies such as a mobile power bank. The temperature cycler does not necessitate the use of optical components such as focusing lenses or beam splitters or any highpower laser diodes. It is also not limited by the wavelength emitted by the emitter and shows high thermal transfer efficiency to the one or more reagents.
[0068] In an embodiment, the present disclosure provides an optothermal temperature cycler, said temperature cycler comprising: an optothermal heating unit comprising an emitter configured to emit electromagnetic radiations; a stage configured to support a reaction cassette, said reaction cassette defining at least one region made of a thermally conductive material, said at least one region being configured to host a volume of one or more reagents and defining a first surface facing the one or more reagents and a second surface opposite to the first surface, wherein the stage is configured to position the reaction cassette such that the second surface is exposed to the electromagnetic radiations emitted by the emitter; a non-contact based temperature detection unit configured to detect temperature of the volume of one or a plurality of reagents, and a controller operatively connected with any or a combination of said optothermal heating unit, the stage and the non-contact based temperature detection unit.; wherein the at least one region at the second surface has high electromagnetic radiation absorptivity.
[0069] FIG. 1A illustrates a schematic view of the temperature cycler, realized in accordance with embodiments of the present disclosure. As can be seen from FIG. 1A, the temperature cycler (100) includes a heating unit including an emitter (102) configured to emit electromagnetic radiations; a stage (104) configured to support a reaction cassette (110); a temperature detection unit (114) configured to detect temperature of the volume of one or more reagents, and a controller (116) operatively connected with any or a combination of said heating unit, the stage and the temperature detection unit. FIG. 1B illustrates a schematic view of an exemplary temperature cycler, realized in accordance with embodiments of the present disclosure, wherein the temperature cycler also comprises a fan (118).
[0070] FIG. 2A illustrates an exemplary view of the temperature cycler, realized in accordance with an embodiment of the present disclosure, showing positioning of a reaction cassette on the stage. FIG. 2B illustrates an exemplary view of the temperature cycler with a reaction cassette positioned on the stage, in accordance with an embodiment of the present disclosure. As can be seen from FIG. 2A and 2B, the temperature cycler (100) includes a heating unit including an emitter (102) configured to emit electromagnetic radiations; a stage (104) configured to support a reaction cassette (110), said reaction cassette (110) defining at least one region (420) made of a thermally conductive material, said at least one region (420) being configured to host a volume of one or more reagents and defining a first surface (shown in FIG. 9(a) and marked as 422) facing the one or more reagents and a second surface (shown in FIG. 9(b) and marked as 424) opposite to the first surface (422), wherein the stage (104) is configured to position the reaction cassette (110) such that the second surface (424) is exposed to the electromagnetic radiations emitted by the emitter (102); a temperature detection unit (114) configured to detect temperature of the one or more reagents, and a controller (shown schematically in FIG. 1 and marked as 116) operatively connected with any or a combination of said heating unit, the stage and the temperature detection unit; wherein the at least one region at the second surface(424) has high electromagnetic radiation absorptivity. FIG. 3 illustrates an exemplary schematic view of the temperature cycler in combination with an exploded view of an exemplary reaction cassette, in accordance with embodiments of the present disclosure.
[0071] As shown in FIG. 2B, once the reaction cassette (110) is positioned on the stage (104), the emitter (102) is switched-on. The electromagnetic radiations fall on region at its second surface, which absorbs, at least part of, the electromagnetic radiations and converts the absorbed light into heat. In an embodiment, the emitter (such as a LED) is configured to emit electromagnetic radiations in the visible spectrum (wavelength ranging from 400-900 nm). In a preferred embodiment, the thermally conductive material is made of Aluminium foil. The thermal mass of the volume of one or more reagents in the center is large; this means that it will be colder than the surrounding thermally conductive material. Heat thus conducts towards the one or more reagents, increasing its temperature uniformly. All objects whose temperature is above absolute zero emit light. This spectrum closely approximates the blackbody spectrum in many cases. The light (electromagnetic radiations) emitted by the heated volume of one or more reagents is of interest. The surrounding thermally conductive material has a low emissivity and is highly reflective [Roswell Flight Test Crew, “Stupid Thermal Imaging Tricks - YouTube” https://www.youtube.com/watch?v=Fx49t4sv7f0, accessed on Apr. 03, 2020]. Thus, the signal observed from it will not give the temperature of the foil itself, but of some reflected object. In order to avoid this and ensure that the signal received is from the volume of one or more reagents, in an exemplary embodiment, an IR temperature sensor (such as one available commercially as “Melexis MLX90615”) may be positioned in proximity of the one or more reagents (for example, at a distance of 0-3 mm). Accordingly, the temperature of the one or more reagents can be detected accurately without actually making any significant thermal contact with the one or more reagents.
[0072] As the temperature of the one or more reagents increases (say beyond the boiling point of the reagents), some of the reagents may be evaporated, and hence, to preclude loss of reagents, it may be beneficial to cover the reaction cassette, particularly, the region holding the volume of one or more reagents by a suitable sealing material, which is transparent to wide range of wavelengths of electromagnetic radiations. In an exemplary embodiment, the sealing material may be a polydimethylsiloxane (PDMS) tape. In such a case (where the region holding the volume of one or more reagents is sealed with a sealing material), it is possible that evaporation of the volume of one or more reagents may cause bulging, which in turn, may establish a contact between the sealing material and the IR sensor. Even in this case, the thermal loss via the contact can be effectively contained (reduced) by appropriate calibration of distance between the IR sensor and the one or more reagents held onto the cassette.
[0073] Once the temperature of the one or more reagents is detected by the IR sensor, the IR sensor transmits the detected temperature to the controller. The controller then compares the detected temperature with the pre-defined temperature (which, for example, may be selected/fed by the user or may be pre-fed into the cycler in form of temperature cycles) and controls output of the emitter based on the comparison. In an exemplary implementation, a PID controller may be used for controlling operation of the emitter based on the detected temperature. Pulse-width modulation (PWM) of ~1kHz or higher may suffice to control the LED brightness (i.e. output of the emitter), as the thermal mass of the system smoothens out the variation.
[0074] The temperature cycler is capable of running up to at least 80 cycles with a single cassette. The temperature cycler of the present disclosure is amenable to different temperature cycling needs. The temperature cycler of the present disclosure can achieve the heating speed of up to 30°C/second, can be powered even by a small battery (e.g. 10,000 mAh USB power bank), and is so energy efficient that the energy required for heating is just a few (<10) times that of the theoretical minimum. In a preferred embodiment, an RT-PCR may be performed with peak power consumption as low as 1 Watt per region. Additionally, it does not necessitate presence of any expensive or environmentally unfriendly materials for fabrication of the cassette such as silicon, thermocouples, or gold. Accordingly, the temperature cycler of the present disclosure is cost effective, disposable and portable, well suited for point-of-care/field settings. For example, the temperature cycler of the present disclosure can be used as a substitute of conventional PCR thermal cyclers.
[0075] In an embodiment, the present disclosure provides a temperature cycler for PCR thermal cycling. In an embodiment, the present disclosure provides an optothermal temperature cycler for PCR thermal cycling.
[0076] FIG. 4 illustrates an exemplary graph showing a representative thermal cycle performed using the temperature cycler of the present disclosure, wherein the scale bar is 25 seconds; three set points are 56°C, 72°C, and 95°C; black wavy line represents the measured temperature, the blue line represents the set point; and the red and green lines are 1°C above and below the set point, respectively. The temperature cycling was performed using a PI controller.
[0077] FIG. 5 illustrates an exemplary graph showing a representative thermal cycle performed with the acrylic cassette (refer FIG. 13) as per an embodiment of the present disclosure, using the temperature cycler of the present disclosure. 10µL of distilled water was loaded into the cassette, prepared as per an embodiment of the present disclosure, with a 10µL capacity. A cooling fan was used to improve cooling speed. The peak radiant flux from the LED was 2.6W. The average heating rate was 13.9°C/s and average cooling rate was 5.3°C/s, measured as the temperature difference divided by the time taken to transition between the two setpoints (denaturation (95°C) and annealing (56°C)). FIG. 6 illustrates an exemplary graph showing a representative thermal cycle performed with the acrylic cassette (refer FIG. 13) as per an embodiment of the present disclosure, using the temperature cycler of the present disclosure. The temperature cycler was setup in a similar manner except that 20 microlitres of water was loaded in a 20 microlitre cassette. The average heating rate was 11.8°C/s and average cooling rate was 5.1°C/s.
[0078] FIG. 7 illustrates an exemplary graph for the temperature stability achieved by the temperature cycler at 90°C over 100s as per an embodiment of the present disclosure. The temperature cycler’s (including an LED, current driver, IR temperature sensor, and controller) temperature stability was demonstrated by maintaining 20µL of water in a 20µL cassette at 90°C. The excellent temperature stability is visible, i.e., temperature error of not more than ±0.1°C over 100 seconds was noted.
[0079] FIG. 8 illustrates an exemplary graph for the temperature stability achieved by the temperature cycler at 50°C over 100s as per an embodiment of the present disclosure. The temperature cycler’s (including an LED, current driver, IR temperature sensor, and controller) temperature stability was demonstrated by maintaining 20µL of water in a 20µL cassette at 50°C. The excellent temperature stability is visible, i.e., temperature error of not more than ±0.15°C over 100 seconds was noted.
[0080] Another aspect of the present disclosure provides a reaction cassette, said cassette comprising: a platform made of a thermally insulating material, said platform defining at least one region made of a thermally conductive material, wherein said at least one region is configured to host a volume of one or more reagents, and wherein said at least one region defines a first surface facing the one or more reagents and a second surface opposite to the first surface, said at least one region, at its second surface having high electromagnetic radiation absorptivity.
[0081] In an embodiment, the at least one region at the first surface and the at least one region at the second surface have high electromagnetic radiation absorptivity.
[0082] In an embodiment, the at least one region, at its second surface, is deposited with a material such that the region at the second surface has electromagnetic radiation absorptivity.
[0083] In an embodiment, the at least one region at the second surface may be deeply colored. In an embodiment, the at least one region at its second surface may be blackened. In an embodiment, the blackening may be performed by deposit of black ink. The deposition may be performed conveniently and cost-effectively using any method, including but not limited to, an ordinary permanent marker, spray-coating, or an inkjet printer. Alternatively, said blackening may be performed by coating of carbon.
[0084] In an embodiment, the thermally insulating material is resistant to temperature ranging from 0-150°C (i.e. it does not exhibit any substantial change in physical properties or chemical properties thereof). In an embodiment, the thermally insulating material is resistant to rapid change in temperature (i.e. it does not exhibit any substantial change in physical properties or chemical properties thereof). In an embodiment, the thermally insulating material may be selected from cardboard, acrylic, polypropylene, high-heat polycarbonate, high-density polyethylene, polyimide, or PTFE (polytetrafluoroethylene). Preferably the thermally insulating material is cardboard or acrylic.
[0085] In an embodiment, the thermally conductive material is resistant to temperature ranging from 0-150°C (i.e. it does not exhibit any substantial change in physical properties or chemical properties thereof). In an embodiment, the thermally conductive material is resistant to rapid change in temperature (i.e. it does not exhibit any substantial change in physical properties or chemical properties thereof). In an embodiment, the thermally conductive material maybe selected from, but is not limited to, thermally conducting metals - aluminium, tin, or copper; thermally conductive polymers - polyimide, graphene, or conductive polyethylene; thermally conductive ceramics: Aluminium Nitride, Silicon Nitride, or Beryllium oxide; or combinations thereof. Preferably the thermally conductive material is an Aluminium foil. The thickness of the foil may range from about 10µm to about 20 µm.
[0086] In an embodiment, the platform defines a plurality of regions made of same or different thermally conductive materials. In an embodiment, the region defines at least one depression at its first surface hosting the volume of said one or more reagents. In an embodiment, the region defines a plurality of depressions at its first surface, one, few or all of the plurality of depressions hosting the volume of said one or more reagents.
[0087] In an embodiment, the platform may additionally comprise one or a plurality of grooves to affix the platform on the stage of the temperature cycler.
[0088] In an embodiment, the at least one region is covered, at least in part, with a sealing material. In embodiments, wherein plurality of regions are defined on the platform, one, few or all of the regions can be covered with sealing material either individually (i.e. one, few or all of the regions can be covered individually with sealing material(s)) or collectively (i.e. one, few or all of the regions can be covered collectively with a sealing material). In an embodiment, the region is covered with a sealing material such that the volume of one or more reagents is not exposed to ambient environment. The sealing material may be a polydimethylsiloxane (PDMS) tape, acrylic tape, silicone tape, or rubber-based tapes. A wide variety of chemical and mechanical sealing methods may be used. Examples include pressure sensitive tapes, heat-sealing adhesives (operated by a heat-sealer), mechanical pressure-based seals (essentially holding down a flat material tightly against the well, such as with a spring or a screw). It is also possible to load the sample into the at least one region via a microchannel, which can then be sealed using chemical or physical valves. A hollow needle may also be used to penetrate a self-sealing region and load the volume of one or more reagents. In an embodiment, the sealing material is positioned such that the volume of one or more reagents may or may not be in contact with the sealing material. In an embodiment, the sealing material is of a material, which is transparent to the electromagnetic radiation. In an embodiment, the sealing material is of a material that allows transmission of electromagnetic radiations within a chosen wavelength range such as 5500 - 14000 nm. In an embodiment, the sealing material is of a material that allows transmission of electromagnetic radiations with wavelength ranging from 280 - 400 nm.
[0089] In a preferred embodiment, the sealing material is a polydimethylsiloxane (PDMS) tape. Said polydimethylsiloxane (PDMS) tape comprises three layers – two polyester layers and one silicon adhesive layer. The silicon adhesive layer is sandwiched between the two polyester layers. Either of the polyester layers may be removed to expose the adhesive layer, optionally both the polyester layers may be removed to expose a double-sided silicon adhesive layer. In an embodiment, each of the layers maybe ~50 microns thick.
[0090] In an embodiment, the platform comprises a slide made of a thermally insulating material and defines at least one slot having affixed thereon, one or a plurality of sheets of thermally conductive material forming the at least one region made of the thermally conductive material. In an embodiment, the at least one region, at its second surface, is deposited with a material such that the region at the second surface has high electromagnetic radiation absorptivity.
[0091] In an embodiment, the platform comprises a slide made of a thermally insulating material and defines at least one slot having affixed thereon a plurality of sheets of thermally conductive material forming the region made of the thermally conductive material, wherein at least one sheet out of the plurality of sheets has high electromagnetic radiation absorptivity. In an embodiment, the at least one sheet having high electromagnetic radiation absorptivity is in vicinity of the second surface. In an embodiment, the at least one sheet having high electromagnetic radiation absorptivity is configured at the second surface.
[0092] In an embodiment, the platform comprises a plurality of overlaying slides made of a thermally insulating material, each of the plurality of overlaying slides defining at least one slot of matching profile, wherein one, few or all of the overlaying slides are affixed with one or a plurality of sheets of thermally conductive material at the at least one slot.
[0093] FIG. 9(a) illustrates an exemplary top view of a reaction cassette (110) realized in accordance with an embodiment of the present disclosure. FIG. 9(b) illustrates an exemplary bottom view of a reaction cassette (110) realized in accordance with an embodiment of the present disclosure. FIG. 9(c) illustrates an exemplary lateral view of a region (420) realized in accordance with an embodiment of the present disclosure. As can be seen from FIG. 9(a) and 9(b), the exemplary cassette includes a platform (410) made of a thermally insulating material, herein cardboard, said platform defining a region (420) made of a thermally conductive material, wherein said region (420) is configured to host a volume of one or more reagents (425), and wherein said region defines a first surface (422) facing the one or more reagents and a second surface (424) opposite to the first surface (422), said region, at the second surface having high electromagnetic radiation absorptivity. As can also be seen from FIG. 9(a) and 9(b), the region defines a depression (426) at its first surface (422) hosting the volume of said one or more reagents. As can be seen from FIG. 9(b), the region, at its second surface (424), is deposited with a material (visible as blackened region) such that the region (420) at the second surface (424) has high electromagnetic radiation absorptivity. In an exemplary embodiment, black ink is deposited onto the second surface (424) by permanent marker or black spray paint to increase its electromagnetic radiation absorptivity. When the reaction cassette is positioned on the thermal cycler, the second surface (424) having high electromagnetic radiation absorptivity faces the emitter absorbing the radiations falling thereon. The absorbed light is converted to heat, which in turn increases temperature of the volume of one or more reagents placed in the region (420). No external cooling mechanism such as a fan maybe required in the platform of low thermal mass.
[0094] In an embodiment, the region is covered, at least in part, with a sealing material (shown in FIG. 9(c) and marked as 430). In an embodiment, the region is covered with a sealing material such that the one or more reagents is not exposed to ambient environment. In an embodiment, the sealing material comprises a tape. The sealing material may be a polydimethylsiloxane (PDMS) tape. In an embodiment, the sealing material is positioned such that the volume of one or more reagents may or may not be in contact with the sealing material. The sealing material prevents any reagents from escaping and cross-contamination of reactions. In an embodiment, the sealing material may be partially or completely sealed. In a preferred embodiment, the sealing material may be partially sealed while packaging and completely sealed only after loading the volume of one or more reagents at the point-of-use.
[0095] In an embodiment, the sealing material is made of a material, which is transparent to the electromagnetic radiation. In an embodiment, the sealing material is of a material that allows transmission of electromagnetic radiations within a chosen wavelength range such as 5500-14000 nm. In an embodiment, the sealing material is of a material that allows transmission of electromagnetic radiations with wavelength ranging from 280-400 nm. In an embodiment, the sealing material is of a material that allows transmission of electromagnetic radiations with wide range of wavelengths (e.g. 180-14000 nm). This is advantageous when the cassette, post completion of thermal cycling, needs to be subjected for further analysis, for example, detection of fluorescence (potentially in the UV range) emitted by the reagents, wherein the contents of the cassette need not be removed or transferred, and the cassette can directly be inserted into the fluorescence measurement unit/device. Alternatively, the fluorescence measurement unit/device may also be integrated into the temperature cycler.
[0096] In embodiments, wherein the reaction cassette includes a region (420) covered with the sealing material (430), the reagents (e.g. dry-stored reagents or lyophilized reagents) can be pre-stored within the region (420) so as to preclude need of opening the sealing. Alternatively, one or more areas can be defined either on the platform (410) or on the region (420) itself allowing the user to add the reagent/sample therein; the one or more areas can have fluid connection with the region (420), where the reagents need to be held during temperature/thermal cycling, allowing travelling of the added reagents (such as solvent or sample or other fluid) from the one or more areas to the desired location (such as, depression(426) defined on the first surface of the region). Alternatively, few of the reagents can be pre-stored within the region (420), and rest of the reagents (e.g. a sample collected from the user that needs to be subjected to thermal cycling, other reagents, or inert fluid) can be added in the one or more areas defined either on the platform (410) or on the region (420) such that all the reagents (pre-stored as well as newly added) can be mixed and then subjected to thermal cycling.
[0097] FIG. 10 illustrates briefly the process of fabrication of the exemplary reaction cassette said cassette comprising a platform made of cardboard of FIG. 9(a) and 9(b). As shown in FIG. 10, a device (as in 10(a)) was made to reliably indent a pre-cut square of aluminium foil using a ball-bearing mounted on a shaft. The aluminium square is loaded onto a slide at the bottom of the device, which is then slid in. The shaft is then pressed downward to create the depression on the first surface of the Aluminum foil as shown in 10(b). The indented foil is then mounted onto the cardboard platform that is laser-cut 10(c), and stuck on with any ordinary adhesive tape on the sides (such as 3M pressure sensitive tape). As can be seen from FIG. 9(a), FIG. 9(b) and FIG. 10(d), the Aluminium foil is polished on the first surface(422) and deposited with black ink by permanent marker or black spray paint onto the second surface (424) to increase its electromagnetic radiation absorptivity at the second surface. PDMS tape may be used for partially or completely sealing the depression (426) on the first surface.
[0098] FIG. 11 illustrates an exemplary reaction cassette (110), wherein the platform (410) is made of thermally insulating material, acrylic, in 11(a) compact form; and 11(b) exploded form, realized in accordance with an embodiment of the present disclosure. FIG. 11(c) illustrates the lateral view of the at least one region (420). The platform (410) comprises a slide (510) made of a thermally insulating material, herein acrylic, and defines at least one slot (512) having affixed thereon, a sheet (514) of thermally conductive material, herein a flat aluminum foil, forming the at least one region (420) made of the thermally conductive material. In an embodiment, the at least one region, at its second surface (424), is deposited with a material such that the region at the second surface has high electromagnetic radiation absorptivity than that of said region at the first surface (422). As can be seen from the image, 11(b) the first surface (422) is polished and the second surface (424) is blackened. The sealing material made of PDMS tape (430) may partially or completely seal the at least one region (420). In an embodiment, the cassette may additionally comprise a PDMS tape (530) on the sheet (514) of the thermally conductive material to affix it on the slide (510). The PDMS tape (430) retains two of the three layers; one polyester layer and the silicon adhesive layer. The PDMS tape (530) retains only one of the three layers; only the silicon adhesive layer is there, with both the polyester layers removed. FIG. 12(a) top view and 12(b) bottom view provide a pictorial image of an exemplary reaction cassette (110) comprising a platform made of acrylic, realized in accordance with an embodiment of the present disclosure.
[0099] FIG. 13 illustrates an exemplary reaction cassette (110), wherein the platform (410) is made of thermally insulating material, acrylic, in (a) compact form; and (b) exploded form, realized in accordance with an embodiment of the present disclosure. FIG. 13(c) illustrates the lateral view of the at least one region (420) realized in accordance with an embodiment of the present disclosure. The platform (410) comprises a slide (510) made of a thermally insulating material, herein acrylic, and defines at least one slot (512) having affixed thereon, a sheet (514) of thermally conductive material, herein a flat aluminum foil, forming the at least one region (420) made of the thermally conductive material. In an embodiment, the at least one region, at its second surface (424), is deposited with a material such that the region at the second surface has high electromagnetic radiation absorptivity. As can be seen from the image, 13(b) the first surface (422) is polished and the second surface (424) is blackened. The sealing material made of PDMS tape (430) may partially or completely seal the at least one region (420). In an embodiment, the cassette may additionally comprise a PDMS tape (530) on the sheet (514) of the thermally conductive material to affix it on the slide (510).
[00100] In an embodiment, it may be noted that the at least one slot on the slide may be cut to reduce the thermal mass of the thermally insulating material in contact with the volume of said one or more reagents. The slot(512) in FIG. 13(b) has acrylic just enough to form the at least one region which is configured to host the volume of one or more reagents. FIG. 14(a) top view and 14(b) bottom view provide a pictorial image of an exemplary reaction cassette (110) comprising a platform made of acrylic, realized in accordance with an embodiment of the present disclosure.
[00101] FIG. 15 illustrates briefly the process of fabrication of the exemplary reaction cassette, said cassette comprising a platform made of acrylic as per an embodiment of the present disclosure. In an embodiment, the process of fabrication of an exemplary reaction cassette (110), wherein the platform (410) is made of thermally insulating material, acrylic, is provided. PDMS tape may be pre-cut to desired size as in 15(a). Aluminium foil of desired size may be pre-cut and blackened such that the first surface is polished (15(b)) and the second surface may be blackened by spray painting (15(c)). Acrylic (~ 1mm thick) may be laser cut to the desired size and shape such as in 15(d). The acrylic may be cleaned with a wipe soaked in 70% ethanol. This may be followed by pasting of a pre-cut PDMS tape by removing one of the polyester layers (15(e)). The at least one slot(512) may be laser-cut from both the acrylic frame and tape (530) to leave the minimum amount of acrylic required to reliably hold the volume of one or more reagents (as shown in 15(f)). Clean wipes soaked in alcohol (such as 70% ethanol) may be used to clean the slot to remove any burnt plastics that may interfere with the reaction. The second polyester layer may be removed to expose the silicon adhesive onto which the thermally conductive material (flat aluminum foil of thickness in the range of about 10µm to about 18 µm) that is blackened on the second surface is placed (15(g)). The excess PDMS tape and Aluminium foil that were cut larger than necessary to allow for some inaccuracy in pasting are then laser-cut to form an outline on the foil to thermally disconnect the required portion from the excess portion, so that energy is not lost on unnecessarily heating the excess portion (15(h)). Using a micropipette, Bovine Serum Albumin (BSA) solution (~10 mg/mL) is deposited in the at least one region (420) enough to fill the region. After about 10 minutes BSA is removed and replaced with the same amount of PCR-grade water. The PCR-grade water is also thereafter removed to give the final cassette (15(i)).
[00102] In an embodiment, the reaction cassette may be formed in numerous forms based on the above exemplary embodiments. They may be made using reasonable modifications, including but not limited to, modifications of the relative placements of the thermally conductive material, the sealing material, the shape or cut of the slot, at least one region or depression (including a corrugated slot or depression). This may be done to serve many purposes, including increasing contact area per unit volume, increasing volume per unit area, or improving optical readout quality by focusing incoming light. For instance, the aluminium foil may be indented in either direction (FIG. 16) or with a pattern instead of a flat sheet.
[00103] The optothermal temperature cycler and the reaction cassette of the present disclosure are particularly suited for detection of pathogens using Polymerase Chain Reaction (PCR) as explained in greater detail in New England Biosciences, “Polymerase Chain Reaction (PCR) | NEB.” available at https://international.neb.com/applications/dna-amplification-pcr-and-qpcr/polymerase-chain-reaction-pcr, contents whereof is incorporated herein, in its entirety, by way of reference. In PCR reaction, typically, genetic material (DNA or RNA) is extracted from a sample such as blood, sputum, or urine sample of a subject, and introduced into a solution containing reagents that will specifically replicate only the DNA corresponding to the pathogen. For this reaction, first, the temperature is raised to about 95°C for a few minutes, after which the temperature must be cycled between 3 different set-points, known as annealing temperature (typically between 48-72°C), extension temperatures (68-72°C), and denaturation temperatures (94-98°C). There are usually 25-50 such cycles, followed by a final extension step at the same temperature as the extension step. The timings for each step vary but are usually less than 1 minute for each step. The total time for the reaction is usually about 1 hour. However, very fast PCR reactions are also known in the art, where each cycle is performed within 10 seconds, leading to a total PCR time of less than 5 minutes.
[00104] The temperature cycler and the reaction cassette of the present disclosure can be used in any type of PCR (and other chemical reactions) that requires heating in general, and thermal cycling in particular, as part of the process/reaction. In an embodiment, the temperature cycler and the reaction cassette of the present disclosure are particularly suited for performing ultra-fast PCR reaction.
[00105] The reaction cassette may be a single-use cassette minimizing the risk of cross-contamination, while simultaneously permitting a means of safe disposal; the amplified DNA does not leave the cassette unless it is damaged. The DNA may be obtained simply by piercing the sealing material. A person skilled in the art should appreciate that the temperature cycler and the reaction cassette of the present disclosure can be used for heating and/or controlling or maintaining temperature of any reaction media, wherein the volume of one or more reagents ranges from 0.001 µl to 10000 µl, preferably from 0.01 µl to 1000 µl, and most preferably from 1 µl to 100 µl. In a preferred embodiment, the temperature cycler employed for a RT-PCR has the volume of one or more reagents in the range of about 5µl to 20 µl.
[00106] In an embodiment, the temperature cycler may be placed in a casing. The casing may be composed of a material that is reasonably strong, preferably light-weight, resistant to dimension change with weather, and may be machined fairly accurately (preferably +- 0.1 mm, or maximum +-0.5 mm). Said casing provides a compact, and easy to port, point-of-care temperature cycler. Any material may be used to prepare the casing, including but not limited to, hard plastics such as acrylic, high-density polyethylene, or polycarbonate; metals such as aluminium or stainless steel; hardwood or its substitutes such as medium density fiberboard, high density cardboard; laminates; 3D-printed PLA or ABS; clay, plaster, or combinations thereof. Pictorial representation of an exemplary temperature cycler in a casing has been provided in FIG. 17(a) and (b). Specifically, the temperature cycler is an optothermal temperature cycler. The cycler presented herein shows the temperature cycler (100), comprising an optothermal heating unit comprising an LED (102); an IR sensor (114) as the non-contact based temperature detection unit. It also shows an additional heat-sink (160) for the LED and a fan (118). The exemplary optothermal temperature cycler has been placed in a casing (162) of acrylic.
[00107] In another embodiment, the present disclosure provides a method of operating a temperature cycler, as recited herein, comprising the steps of: (a) loading the volume of one or more reagents in the at least one region made of the thermally conductive material of the reaction cassette; (b) sealing the at least one region with the sealing material; (c) positioning the reaction cassette on the stage and turning on the heating unit; (d) monitoring the temperature of the volume of one or more reagents using the temperature detection unit; and (e) adjusting the temperature using the controller. The temperature cycler has minimum scope of user error and therefore can be operated by a minimally trained user.
[00108] Any instrument may be used to load the volume of one or more reagents, including a micropipette. After loading the volume of one or more reagents the sealing material (430) may be completely sealed off. The sealing material maybe reinforced with a rolling pin or by hand pressing.
[00109] In another embodiment, the present disclosure provides a method of amplifying a sample comprising nucleic acid in a temperature cycler, as recited herein, comprising the steps of:
(a) loading the sample in the at least one region made of a thermally conductive material of the reaction cassette; (b) sealing the at least one region with the sealing material; (c) positioning the reaction cassette on the stage and turning on the heating unit; (d) monitoring the temperature of the sample using the temperature detection unit till temperature reaches a first temperature and optionally maintaining the first temperature; (e) adjusting the temperature of the sample using the controller to a second temperature; (f) optionally maintaining the second temperature; (g) adjusting the temperature of the sample using the controller to a third temperature; and (h) optionally maintaining the third temperature; wherein the steps (d) to (h) maybe performed in a loop by the controller.
[00110] The first temperature, second temperature and third temperature may correspond to the denaturation, annealing and extension temperatures of nucleic acids. The method may be employed for amplifying a sample comprising nucleic acid for diagnosis of a number of diseases including but not limited to HIV, tuberculosis, influenza, SARS-CoV-2, and/or hepatitis.
[00111] As per an embodiment of the present disclosure, the temperature cycler in a PCR can give an ultrafast thermal cycling with temperature ramp up rate of up to ~13.9°C/second and a cooling rate of up to ~5.3°C/second. In an embodiment, even higher temperature ramp rates and cooling rates may be achieved by further optimization. Therefore they are ideal for point-of-care molecular diagnostics.
[00112] It should be understood that various other modifications and combinations of the above embodiments are contemplated and will readily appear to those skilled in the art. Thus, the present invention contemplates that any and all such subject matter is included within the scope of the present invention. The invention in its broader aspects is therefore not limited to the specific details, representative apparatus and methods and illustrative examples shown and described. Accordingly, departures may be made from such details without departing from the scope or spirit of Applicants' general inventive concept.
[00113] The disclosure will now be illustrated with a working example, which is intended to illustrate the working of disclosure and not intended to take restrictively to imply any limitations on the scope of the present disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice of the disclosed methods, the exemplary methods, devices and materials are described herein. It is to be understood that this disclosure is not limited to particular methods, and experimental conditions described, as such methods and conditions may vary.
MATERIALS AND METHOD:
[00114] RT-PCR reactions were performed for DNA fragments from Mycobacterium tuberculosis. Mycobacterium tuberculosis was obtained from New England Biolabs, Ipswich, Massachusetts, United States of America (USA). Primers for the PCR were obtained from Integrated DNA Technologies, Coralville, Iowa, USA. Z-Taq was obtained from Takara Bio, Kusatsu, Shiga, Japan. Gel electrophoresis and subsequent staining (pre-staining was performed with Ethidium Bromide (EtBr) fluorescent dye) was used for identification of amplified DNA. The results are presented in FIG. 18.
[00115] Lane L depicts a DNA ladder. From the ladder, the length of the amplified DNA fragment was estimated to be 230 base pairs.
[00116] Lane RT depicts the amplified DNA from a regular 20µL 40-cycle PCR performed with recombinant Taq (rTaq, Cat #R001A) polymerase (along with the company-supplied buffer and dNTPs) obtained from Takara Bio on an Applied Biosystems Quantstudio 3 qPCR machine. The hold times for annealing (56°C), extension (72°C), and denaturation (95°C) were 15, 20, and 10 seconds respectively. The initial denaturation and final extension were both performed for 1 minute each at 95°C and 72°C respectively. The overall time taken for thermal cycling was 54 minutes and 18 seconds.
[00117] Lane RT-Z depicts the amplified DNA from a fast 20µL 40-cycle 2-step PCR performed with the same device, but with only 1 second hold time at annealing (56°C) and denaturation (95°C), with no hold for extension. No initial denaturation or final extensions were performed. The overall time taken for thermal cycling was 22 minutes and 43 seconds. However, the reagents used were different. Z-Taq, a special polymerase with 5-times normal processivity, was obtained from Takara Bio, Kusatsu, Shiga, Japan (Cat #R006A) and was used with the company-supplied buffer and dNTPs. Additionally, the concentration of Z-Taq used was 10 times the company-recommended concentration.
[00118] Lane C10m depicts the amplified DNA from an ultrafast 20µL 40-cycle PCR performed with the optothermal temperature cycler of the present invention with the same reagents as above, and with no (<1 second) hold time at either annealing or denaturation. The overall time taken for thermal cycling was 10 minutes and 10 seconds. FIG. 19 shows the temperature versus time graph for the experiment. Similar experiment was repeated but with a 10 microlitre sample, and with 30 PCR cycles (sufficient for many use cases), demonstrating 10-second thermal cycles for approximately 5 minutes (310 seconds) (refer FIG. 20). Excellent temperature accuracy and ultrafast transition rates are visible in this graph.
[00119] RESULTS: It was observed that the brightness of the bands in the fast and ultrafast PCRs, while somewhat lower, are still comparable – indicating that ultrafast PCR run on the temperature cycler of the present invention works and is feasible. The speedup (~10 minutes vs ~54 minutes) is especially significant for point-of-care nucleic acid amplification tests (NAATs), which are widely considered to be the gold standard testing method for many diseases, such as Tuberculosis, COVID-19, and HIV. The dramatically reduced time for thermal cycling allows patient wait time to be minimized. Further, the portability, cost-effectiveness, and energy-efficiency of the present invention means that even rural health care centers, with unreliable or no electricity can afford and use it (for instance, by using a small solar panel) to diagnose patients quickly. This is important since for many diseases where the results are not available on the same day, patients often do not return to collect their test results and take the appropriate treatment. This loss to follow-up leads to over 600 thousand deaths every year for tuberculosis alone. Thus, the present invention overcomes the lacunas in medical diagnostics.

ADVANTAGES
[00120] The present disclosure provides a temperature cycler that is economical, energy efficient and easy to manufacture.
[00121] The present disclosure provides a temperature cycler that does not require optical components such as focusing lenses or beam splitters.
[00122] The present disclosure provides a temperature cycler that is portable, can be used at point-of-care settings and can be powered with a light-weight, small capacity battery.
[00123] The present disclosure provides a reaction cassette that is economical and easy to fabricate.
[00124] The present disclosure provides a reaction cassette that is single use, robust and hence, minimizes the risk of cross-contamination.
,CLAIMS:1. A temperature cycler, said temperature cycler comprising: a heating unit comprising an emitter configured to emit electromagnetic radiations; a stage configured to support a reaction cassette, said reaction cassette defining at least one region made of a thermally conductive material, said at least one region being configured to host a volume of one or more reagents and defining a first surface facing the one or more reagents and a second surface opposite to the first surface, wherein the stage is configured to position the reaction cassette such that the second surface is exposed to the electromagnetic radiations emitted by the emitter;
a temperature detection unit configured to detect temperature of the volume of one or more reagents, and
a controller operatively connected with any or a combination of said heating unit, the stage and the temperature detection unit; wherein the at least one region at the second surface has high electromagnetic radiation absorptivity.
2. The temperature cycler as claimed in claim 1, wherein the at least one region at the second surface is deeply colored.
3. The temperature cycler as claimed in claim 1, wherein the at least one region at the second surface is blackened.
4. The temperature cycler as claimed in claim 1, wherein the emitter is selected from a Light Emitting Diode (LED), a compact fluorescent lamp, a gas discharge tube, a laser, a tungsten lamp, an incandescent light source, induction heater, resistive heater, microwave emitter, radioactive heaters, radiation from a fire gated with a metal plate or combinations thereof.
5. The temperature cycler as claimed in claim 1, wherein the temperature detection unit comprises a non-contact based temperature detection unit, a contact based temperature detection unit, an integrated detection unit, or combinations thereof.
6. The temperature cycler as claimed in claim 1, wherein the temperature detection unit is selected from Infrared (IR) temperature sensor, Infrared sensor array, infrared camera, fluorescence-based temperature sensor, spectroscopy based detection unit, sonic sensors, a thermocouple, vapor-pressure based temperature unit, thermochromic liquid crystals, or resistance-based temperature detector, or combinations thereof.
7. The temperature cycler as claimed in claim 1, wherein the thermally conductive material is selected from thermally conducting metals, thermally conductive composites, thermally conductive polymers, thermally conductive ceramics, or combinations thereof.
8. The temperature cycler as claimed in claim 1, wherein the thermally conductive material is selected from aluminium, tin, copper, polyimide, graphene, conductive polyethylene, Aluminium Nitride, Silicon Nitride, Beryllium oxide, aluminium/carbon fibre composite, polyester/grapheme composite, graphene/carbon-black composite, carbon-nanotube/graphene composite or combinations thereof.
9. The temperature cycler as claimed in claim 1, wherein the controller is configured to control output of the emitter based on the detected temperature of the one or more reagents.
10. The temperature cycler as claimed in claim 1, wherein the volume may range from 0.001 µl to 10000 µl.
11. A reaction cassette, said cassette comprising: a platform made of a thermally insulating material, said platform defining at least one region made of a thermally conductive material, wherein said at least one region is configured to host a volume of one or more reagents, and wherein said at least one region defines a first surface facing the one or more reagents and a second surface opposite to the first surface, said at least one region, at its second surface having high electromagnetic radiation absorptivity.
12. The cassette as claimed in claim 11, wherein the region defines at least one depression at its first surface hosting the volume of said one or more reagents.
13. The cassette as claimed in claim 11, wherein the region at the second surface is deeply colored.
14. The cassette as claimed in claim 11, wherein the region at the second surface is blackened.
15. The cassette as claimed in claim 11, wherein the thermally insulating material is selected from cardboard, acrylic, polypropylene, high-heat polycarbonate, high-density polyethylene, polyimide, or PTFE (polytetrafluoroethylene).
16. The cassette as claimed in claim 11, wherein the thermally insulating material is cardboard, or acrylic.
17. The cassette as claimed in claim 11, wherein the thermally conducting material is selected from thermally conducting metals, thermally conductive polymers, thermally conductive ceramics, or combinations thereof.
18. The cassette as claimed in claim 11, wherein the thermally conducting material is selected from aluminium, tin, copper, polyimide, graphene, conductive polyethylene, Aluminium Nitride, Silicon Nitride, Beryllium oxide, aluminium/carbon fibre composite, polyester/grapheme composite, graphene/carbon-black composite, carbon-nanotube/graphene composite or combinations thereof.
19. The cassette as claimed in claim 11, wherein the platform additionally comprises one or a plurality of grooves.
20. The cassette as claimed in claim 11, wherein the at least one region is covered, at least in part, with a sealing material.
21. The cassette as claimed in claim 20, wherein the sealing material is a polydimethylsiloxane (PDMS) tape, acrylic tape, silicone tape, or rubber-based tape.
22. The cassette as claimed in claim 11, wherein the platform comprises a slide made of a thermally insulating material and defines at least one slot having affixed thereon, one or a plurality of sheets of thermally conductive material forming the at least one region made of the thermally conductive material.
23. The cassette as claimed in claim 22, wherein the at least one region, at its second surface, is deposited with a material such that the region at the second surface has high electromagnetic radiation absorptivity.
24. The cassette as claimed in claim 22, wherein the at least one slot on the slide is cut to reduce thermal mass of the thermally insulating material in contact with the volume of said one or more reagents.
25. A method of operating an temperature cycler, as claimed in claim 1, comprising the steps of: (a) loading the volume of one or more reagents in the at least one region made of a thermally conductive material of the reaction cassette; (b) sealing the at least one region with the sealing material; (c) positioning the reaction cassette on the stage and turning on the heating unit; (d) monitoring the temperature of the volume of one or more reagents using the temperature detection unit; and (e) adjusting the temperature using the controller.
26. A method of amplifying a sample comprising nucleic acid in a temperature cycler, as claimed in claim 1, comprising the steps of:
(a) loading the sample in the at least one region made of a thermally conductive material of the reaction cassette; (b) sealing the at least one region with the sealing material; (c) positioning the reaction cassette on the stage and turning on the optothermal heating unit; (d) monitoring the temperature of the sample using the non-contact based temperature detection unit till temperature reaches a first temperature and optionally maintaining the first temperature; (e) adjusting the temperature of the sample using the controller to a second temperature; (f) optionally maintaining the second temperature; (g) adjusting the temperature of the sample using the controller to a third temperature; and (h) optionally maintaining the third temperature; wherein the steps (d) to (h) are performed in a loop by the controller.