WO2023129612A1

WO2023129612A1 - Systems and methods for performing plasmonic and hot or cool air chemical and/or biological assays

Info

Publication number: WO2023129612A1
Application number: PCT/US2022/054182
Authority: WO
Inventors: Nicole BLUMENFELD; Michael Anne BOLENE; Sabin ZARRANDIKOETXEA; Martin Jaspan; Mark Fasciano; Samuel K. Sia
Original assignee: Rover Diagnostics Inc.
Priority date: 2021-12-29
Filing date: 2022-12-28
Publication date: 2023-07-06

Abstract

Systems and methods for performing plasmonic and hot and/or cool air PCR are generally described. A plasmonic system is described using a first source of electromagnetic radiation as a heat source to heat a sample, a first nozzle to flow gas across the sample, a second source of electromagnetic radiation as an excitation source, and an electromagnetic radiation detector to detected excited radiation from the sample.

Description

SYSTEMS AND METHODS FOR PERFORMING PLASMONIC AND HOT OR COOL AIR CHEMICAL AND/OR BIOLOGICAL ASSAYS

GOVERNMENT SPONSORSHIP

This invention was made with Government support under Contract No. U54HL143541 awarded by the National Institute of Health. The Government has certain rights in the invention.

TECHNICAL FIELD

Systems and methods for performing plasmonic and hot air and/or cool air chemical and/or biological assays are generally described.

BACKGROUND

Chemical and/or biological assays such as RT-PCR are important processes for clinical diagnostics, particularly in the context of the current COVID- 19 pandemic. PCR requires cycling between several temperatures. This is usually done by controlling the temperature of an aluminum block which heats a PCR tube containing the sample. However, the speed of this method is limited by how long it takes to heat and cool the external aluminum block. Accordingly, improved systems and methods are desired.

SUMMARY

Systems and methods for performing plasmonic and hot air chemical and/or biological assays are generally described. The subject matter of the present disclosure involves, in some cases, interrelated products, alternative solutions to a particular problem, and/or a plurality of different uses of one or more systems, methods and/or articles.

In one aspect, a system is described, the system comprising a sample holder configured to hold a sample; one or more nozzles proximate to the sample holder each configured to flow a gas of a set temperature towards the sample holder; a first source of electromagnetic radiation proximate the sample holder configured to heat the sample; a second source of electromagnetic radiation configured to generate a signal from the sample; and a detector configured to detect the signal from the sample. In another aspect, a method for determining the presence of an analyte in a sample is described, the method comprising heating the sample with a first source of electromagnetic radiation; flowing a gas of a set temperature towards the sample to further control the temperature of (e.g., heat) the sample; and performing a chemical and/or biological reaction with the analyte in the sample.

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

BRIEF DESCRIPTION OF THE DRAWINGS

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

FIG. 1A is a schematic illustration of a system for heating a sample comprising one nozzle, according to some embodiments;

FIG. IB is a schematic illustration of a system for heating a sample comprising three nozzles, according to some embodiments;

FIGS. 2A-2F schematically depict a method for determining the presence of an analyte, according to some embodiments;

FIG. 3 is a schematic diagram of a system comprising a 3-nozzle setup, according to some embodiments;

FIG. 4 is a plot of the temperature of a sample as a function of time as different temperature control (e.g., heating) steps are applied to the sample, according to one set of embodiments;

FIG. 5 is plot illustrating the determining of an analyte after a biological reaction is performed, according to one set of embodiments; FIGS. 6A-6B is a schematic diagram of a nozzle configured to provide heated and/or cooled air, according to some embodiments;

FIG. 7 is a plot of temperature as a function of time, showing cycling of a room temperature PCT biological reaction for the detection of COVID-19, according to one set of embodiments;

FIG. 8 is a plot of a relative fluorescence response vs. cycle numbers for several analytes using a RT-PCR biological reaction and plasmonic heating, according to some embodiments;

FIG. 9 is a schematic diagram of a nozzle configuration used to provide gas flow to a sample, according to some embodiments;

FIG. 10 is a schematic diagram of a nozzle configuration used to provide ambient air to a sample holder, according to some embodiments; and

FIG. 11 is a schematic diagram of a system for providing heating and cooling to a sample, according to some embodiments.

DETAILED DESCRIPTION

Systems and methods are described herein for performing a chemical and/or biological reaction, such as a polymerase chain reaction (PCR), using a combination of internal sample temperature control (e.g., heating) and external convective heating. PCR, for example, may require several instances of thermal cycling in order to adequately prepare and analyze the sample, and the disclosed systems and methods are suitable for such cycling. Several existing techniques are known to providing heating to a sample, but no such existing techniques have yet to combine both internal sample heating and external convective heating. However, it has been discovered and appreciated by this disclosure that combining both internal sample heating and external convective heating may provide several benefits and advantages. For example, as one advantage, by using both internal heating and external heating, a desired temperature for the chemical or biological reaction may be reached much quicker relative to heating using only internal heating or only external heating, or relative to other existing sample heating techniques. As another advantage, the combination of external convective heating with internal sample heating may result in more even or homogenous heating and may minimize hot or cold spots within the sample, which can result in a more accurate determination of an analyte within the sample.

The systems and methods described herein may have one or more nozzles configured to flow gas (e.g., air) of a particular or predetermined temperature (e.g., a gas temperature) towards a sample holder (and/or a sample contained within the sample holder). By flowing gas of a particular temperature towards the sample holder, a sample contained within the sample holder can be heated (or cooled). The sample may also contain a chemical species, such as nanoparticles or gold nanoparticles, which may facilitate internal heating (e.g., temperature control) of the sample. For example, the systems and methods described herein may use an external source of electromagnetic radiation to stimulate gold nanoparticles within the sample in order to heat the sample internally. In some embodiments, the external source of electromagnetic radiation may heat the sample directly. Thus, by using both internal and external heating, a sample may be heated to a particular or desired temperature (or temperature range) and a chemical or biological reaction may be performed at this particular temperature (or temperature range). After performing the reaction, information about the analyte, such as the presence (or absence) of an analyte, within the sample may be obtained. In some instances, multiple chemical and/or biological reactions can be performed. For example, a sample may be both internally and externally heated to reach a first sample temperature, a chemical or biological reaction can be performed, and then the sample may be further heated both internally and externally to reach a second sample temperature (different from the first sample temperature), whereby another chemical or biological reaction can be performed. It may also be possible to cool the sample (e.g., after performing a chemical or biological reaction) to a third sample temperature by flowing cool or ambient temperature gas towards the sample. Details regarding the systems and methods are described in more detail below.

Turning to the figures, specific, non-limiting embodiments are described in further detail. It should be understood that the various systems, components, features, and methods described relative to these embodiments may be used either individually and/or in any desired combination as the disclosure is not limited to only the specific embodiments described herein.

In some embodiments, the system may comprise one or more nozzles proximate to the sample holder, each nozzle configured to flow a gas of a set temperature towards the sample holder. By way illustration, FIG. 1A schematically illustrates a system 100 that comprises one nozzle 110 proximate to a sample holder 120. In some embodiments, the system comprises a plurality of nozzles. For example, FIG. IB schematically illustrates a system with the inclusion of a plurality of nozzles, a first nozzle 110, in addition to a second nozzle 111 and a third nozzle 112. In some such embodiments, at least one (e.g., some, all) of the nozzles is configured to flow a gas of a set temperature towards the sample holder (or a sample contained within the sample holder). In this way, gas of a first set temperature (e.g., a first gas temperature) may flow from one nozzle, while gas of another set temperature (e.g., a second gas temperature, a third gas temperature) may flow from another nozzle. In some embodiments, additional nozzles may be included (e.g., fourth, fifth, sixth, etc. nozzles), each of which may flow a gas of a set temperature (e.g., a fourth, fifth, sixth, etc. gas temperature). Details regarding the various gas temperatures from various nozzles is described in more detail elsewhere herein.

It should be understood, however, that while some embodiments use gases of set temperatures from different nozzles, in other embodiments, just one nozzle may be used to provide gases of different set temperatures. The single nozzle may include or be operatively associated with a plurality of gas sources, each gas of the gas sources having a particular set temperature, and the one nozzle may be configured to emit gases of different set temperatures towards the sample without other nozzles being present. However, for ease of illustration, the various figures refer to configurations in which a plurality of nozzles is present.

In some embodiments, the system comprises a first source of electromagnetic radiation proximate (e.g., an infrared radiation source) to the sample holder and may be configured to heat the sample. For example, FIGS. 1A-1B schematically depict a source of electromagnetic radiation 130 within the system 100 and proximate to the sample holder 120. The first source of electromagnetic radiation may be used to internally heat a sample within the sample holder, e.g., by directly heating or cooling (e.g., controlling a temperature) a chemical species (e.g., gold nanoparticles) within the sample, and/or by heating the sample directly. Details regarding the direct heating of a chemical species within the sample are described in more detail elsewhere herein.

Various embodiments of the system include a second source of electromagnetic radiation (e.g., UV radiation, one or more lasers and/or LEDs configured to emit UV radiation) configured to generate a signal from the sample. For example, as shown schematically in FIGS. 1A-1B, a second source of electromagnetic radiation 140 is positioned proximate to the sample holder 120. The second source of electromagnetic radiation can be used to generate a signal within a sample, when a sample is present. For example, the second source of electromagnetic radiation may stimulate a species within the sample (e.g., an analyte within the sample, a species produced or consumed by an analyte in the sample after one or more chemical and/or biological reactions within the sample) to generate a signal. A detector may also be present within the system to detect this signal. As shown in FIGS. 1A-1B, for example, the system 100 includes a detector 150 configured to detect the signal from the sample.

In some embodiments, a method for determining the presence of an analyte in a sample is described. The method is described below in relation to the system 100; however, it should be understood that other systems may be used, as this disclosure is not so limited.

In some embodiments, the method comprises controlling the temperature (e.g., changing the temperature by heating) the sample with a first source of electromagnetic radiation. By way illustration, FIG. 2A schematically depicts the (first) source of electromagnetic radiation 130 emitting electromagnetic radiation (e.g., light) 132 towards the sample holder 120. The sample holder 120 holds a sample 210, which contains gold nanoparticles 220. In this example, the gold nanoparticles 220 within the sample 210 are configured to absorb the light 132 and convert its energy into heat, which can be internally dissipated within the sample 210. By this process, the temperature of the sample may change from a first sample temperature (e.g., prior to any heating) to a second sample temperature (e.g., after being heating by the source of electromagnetic radiation).

In some embodiments, the method also involves flowing a gas of a set temperature towards the sample to further heat the sample (e.g., to a third sample temperature). Illustrated schematically in FIG. 2B, the first nozzle 110 flows a gas 230 towards the sample 210. The gas may be at a particular temperature (e.g., a first gas temperature) and may further heat the sample, in addition to the heating provided by the first source of electromagnetic radiation and gold nanoparticles within the sample. Once the sample reaches a desired temperature (e.g., the third sample temperature), a chemical and/or biological reaction may be performed, and this reaction may indicate the presence (or absence) of the analyte within the sample (not pictured).

It should be understood that one or more steps of heating and/or cooling (e.g., controlling temperature steps) may be performed. By way of illustration, FIG. 2C schematically depicts the second nozzle 111 directing a gas 232 towards the sample 210. The gas 232 may be at a gas temperature (e.g., a second gas temperature) different than the gas temperature of gas 230 (e.g., a first gas temperature). In this manner, the sample can be heated to two different temperatures (e.g., a third gas temperature, a fourth gas temperature). Furthermore, the sample may be cooled (or heated) by gas of a third gas temperature. For example, as schematically depicted in FIG. 2D, the third nozzle 112 flows a gas 234 towards the sample. The gas 234 may be at a gas temperature different than the gas temperatures of gas 230 and/or gas 232. The temperature of the gas may affect the temperature of the sample. In some embodiments, a chemical and/or biological reaction is performed after the sample reaches a first sample temperature (or a second sample temperature, a third sample temperature) and after the sample reaches another sample temperature different from the previous sample temperature (e.g., a second sample temperature, a third sample temperature, a fourth sample temperature). However, in different embodiments, a chemical and/or biological reaction is performed only after the sample reaches a first sample temperature, and subsequent flowing of a gas (e.g., of a second gas temperature of a third gas temperature) towards the sample from the nozzle(s) may heat or cool the sample to a second sample temperature and/or a third sample temperature, where no further chemical and/or biological reactions are performed at the second or third sample temperatures.

It is noted that the use of ordinal terms such as “first,” “second,” “third,” etc., within this disclosure to modify an element (e.g., a first gas temperature, a second gas temperature, a first sample temperature, a second sample temperature) does not by itself connote any priority, precedence, or order of one element over another or the temporal order in which acts are performed, but are used merely as labels to distinguish one element having a certain name from another element having a same name, but for the use of the ordinal term, to distinguish the elements. For example, a sample of a first sample temperature and a second sample temperature may refer to the same sample, but at two distinct temperatures. After performing a chemical and/or biological reaction, an analyte within the sample can be determined. The analyte may be determined qualitatively and/or quantitatively. For example, in FIG. 2E, the second source of electromagnetic radiation 140 emits light 142 towards the sample 210. The sample may contain a species that interacts with the light 142 and a signal 144 (shown schematically in FIG. 2F) may be detected by the detector 150. Details regarding signal generation and detection are described further below.

As described above and elsewhere herein, the systems and methods disclosed herein may use a combination of internal sample heating (e.g., temperature control) and external sample heating (e.g., temperature control) in order to heat the sample to a desired temperature. One or more nozzles may be used to provide external heat to the sample holder (or to a sample contained within the sample holder). Without wishing to be bound by any particular theory, flowing gas from the nozzle towards the sample provides external convective flux around the sample, so that heat is more homogenously distributed around the sample, resulting in more even heating. In some embodiments, the system further comprises an insulating portion (e.g., an insulating sleeve) at least partially containing or surrounding the sample holder so that gas flowed towards the sample holder (or the sample) is not too quickly cooled by ambient conditions (e.g., the insulating portion insulates the gas, the sample holder, and/or the sample from the surrounding environment) while still allowing for adequate flow of gases within the insulating portion. The one or more nozzles (e.g., one nozzle, a plurality of nozzles) may comprise or be operatively associated with any number of conduits, channels, and/or tubing for conveying a gas within the nozzle and/or towards the sample. Those skilled in the art in view of this disclosure will be capable of selecting the appropriate number of nozzles and any associated conduits, channels, and/or tubing to convey gas towards the sample holder (or a sample contained within the sample holder) in order to heat and/or cool a sample within the system when a sample is present.

The systems and methods disclosed herein may also use a combination of sample heating and cooling (e.g., temperature control) by using internal sample heating (e.g., provided by a first source of electromagnetic radiation configured to heat the sample, for example, when the sample comprises gold nanoparticles) and/or external sample heating and/or cooling (e.g., provided by a single nozzle, provided by two or more nozzles) in order to heat the sample to a desired temperature (e.g., to perform one or more biological and/or chemical reactions) and then cool the sample down to a desired temperature. Details regarding heating and/or cooling are described elsewhere herein.

As described herein, in some embodiments, at least some of the one or more nozzles is configured to flow a gas of a set or predetermined temperature (e.g., a first gas temperature, a second gas temperature, a third gas temperature) towards the sample holder (or a sample within the sample holder). For example, the nozzle may provide heating (e.g., a source of gas higher in temperature than the sample) and/or cooling (a source of gas cooler than the temperature of the sample). When a sample is present, the gas may heat the sample to a particular temperature (e.g., a first sample temperature, a second sample temperature, a third sample temperature). In some embodiments, a first gas temperature is greater than a first sample temperature, and a gas (of the first gas temperature) may be flowed towards the sample until the first sample temperature reaches a second sample temperature. In some embodiments, the sample is heated until the second sample temperature equals the first gas temperature, or to at least a temperature greater than the first sample temperature. Similarly, in some embodiments, a second gas temperature is greater than a second sample temperature, and a gas (of the second gas temperature) may be flowed towards the sample until the second sample temperature reaches a third sample temperature. In some embodiments, the sample is heated until the third sample temperature equals the second gas temperature, or to at least a temperature greater than the second sample temperature. In some embodiments, gas of a third gas temperature, different from the first and/or second gas temperatures, is flowed towards the sample until the sample reaches a fourth sample temperature. The fourth sample temperature may be greater than or less than the third sample temperature. Those skilled in the art in view of the present disclosure will be capable of selecting appropriate gas and/or sample temperatures for heating (or cooling) a sample.

In some embodiments, at least some of the one or more nozzles is operatively associated with a heating element (e.g., a radiator, a Peltier device). The heating element can heat gas flowing to or through a nozzle to particular temperature (e.g., a first gas temperature, a second gas temperature, a third gas temperature, a fourth gas temperature). In some embodiments, the heating element comprises a thermistor, or one or more other temperature measuring devices or is associated with one or more components for determining the temperature of the sample (e.g., an optical pyrometer, IR thermal camera, or other). In some embodiments, at least one (e.g., at least some) of the nozzles comprises or is operatively associated with a cooling element. The cooling element may comprise a chiller, heat exchanger (e.g., plate heat exchangers, shell and tube heat exchanger), and/or a thermoelectric heat pump (e.g., a Peltier cooler) as non-limiting examples. Other cooling elements are possible. As mentioned elsewhere herein, in some embodiments, a sample temperature (e.g., a first sample temperature) may be greater than or equal to an ambient temperature, and at least one of the nozzles is configured to provide ambient air of a lower temperature than the sample in order to cool the sample. In some such embodiments, a single nozzle is used, and the single nozzle is configured to provide heated and/or cooled air to the system. In some embodiments, at least one (e.g., at least some) of the one or more nozzles comprises or is operatively associated with a heat source/heating element (e.g., a thermoelectric heat pump).

FIG. 6A shows a schematic illustration of a nozzle 610, according to some embodiments. FIG. 6B depicts an exploded view of the same nozzle 610 showing some components of the nozzle 610. An air flow controller 630 is used to convey gas (e.g., air) towards a nozzle diameter 615 of the nozzle 610. The air flow controller can be or can comprise a pump or a fan and can convey gas (e.g., air) towards the sample (or sample holder). As described elsewhere herein, the diameter of the nozzle (i.e., nozzle diameter) can advantageously be adjusted to control the amount of air output from the nozzle, along with the air flow controller. Also shown in FIG. 6B is temperature controller 620, which may comprise heating or cooling elements as described elsewhere herein. The air flow controller 630 can direct air through the temperature controller 620 to heat and/or cool air and then the air can exit through nozzle 610 (e.g., towards a sample in a sample holder). The nozzles may also include or be associated with housing and support enclosures. For example, in FIG. 6B, the various components of the nozzle are held together by housing and support 640 and fasteners 650. Other features may be associated with the one or more nozzles as described elsewhere herein.

In some embodiments in which multiple nozzles are used, one nozzle may comprise a cooling element for providing a gas cooler than the temperature of the sample, and another nozzle may comprise a heat source/heating element for providing a gas hotter than the temperature of the sample. Other configurations are also possible.

The one or more nozzles may each independently flow a gas at a particular temperature (e.g., a first gas temperature, a second gas temperature, a third gas temperature, a fourth gas temperature) towards the sample holder (or a sample within the sample holder). For example, in some embodiments, the temperature of a gas (e.g., a first, a second, a third, or a fourth, etc. gas temperature) may each independently be greater than or equal to 20 °C, greater than or equal to 25 °C, greater than or equal to 30 °C, greater than or equal to 40 °C, greater than or equal to 50 °C, greater than or equal to 60 °C, greater than or equal to 70 °C, greater than or equal to 80 °C, greater than or equal to 90 °C, or greater than or equal to 100 °C. In some embodiments, the temperature of a gas (e.g., a first, a second, a third, or a fourth, etc. gas temperature) may each independently be less than or equal to 100 °C, less than or equal to 90 °C, less than or equal to 80 °C, less than or equal to 70 °C, less than or equal to 60 °C, less than or equal to 50 °C, less than or equal to 40 °C, less than or equal to 30 °C, less than or equal to 25 °C, or less than or equal to 20 °C. Combinations of the above-referenced ranges are also possible (e.g., greater than or equal to 20 °C and less than or equal to 100 °C). Other ranges are possible.

A flowing gas may change a sample temperature (e.g., a first sample temperature to second temperature, a second sample temperature to a third sample temperature, a third sample temperature to a fourth sample temperature, etc.) by a particular temperature difference. In some embodiments, a flowing gas (e.g., a first gas, a second gas, a third gas, etc.) may each independently change a sample temperature (e.g., a first, second, third, fourth, etc. sample temperature) by greater than or equal to 0.1 °C, greater than or equal to 0.5 °C, greater than or equal to 1 °C, greater than or equal to 1.5 °C, greater than or equal to 2 °C, greater than or equal to 5 °C, greater than or equal to 7 °C, greater than or equal to 10 °C, greater than or equal to 15 °C, greater than or equal to 20 °C, greater than or equal to 25 °C, greater than or equal to 30 °C, greater than or equal to 35 °C, greater than or equal to 40 °C, greater than or equal to 50 °C, greater than or equal to 60 °C, greater than or equal to 70 °C, or greater than or equal to 80 °C. In some embodiments, a flowing gas (e.g., a first gas, a second gas, a third gas, etc.) changes the sample temperature by less than or equal to 80 °C, less than or equal to 70 °C, less than or equal to 60 °C, less than or equal to 50 °C, less than or equal to 40 °C, less than or equal to 35 °C, less than or equal to 30 °C, less than or equal to 25 °C, less than or equal to 20 °C, less than or equal to 15 °C, less than or equal to 10 °C, less than or equal to 7 °C, less than or equal to 5 °C, less than or equal to 2 °C, less than or equal to 1.5 °C, less than or equal to 1°C, less than or equal to 0.5 °C, or less than or equal to 0.1 °C. Combinations of the above-referenced ranges are also possible (e.g., greater than or equal to 0.1 °C and less than or equal to 80 °C). Other ranges are possible.

In some embodiments, at least some of the one or more nozzles may flow a gas that cools the sample (e.g., from one sample temperature to another, such as from a third sample temperature to the first sample temperature, from a third sample temperature to a fourth sample temperature that is less than the third sample temperature). In some such embodiments, the temperature of the flowing gas is less than or equal to the temperature of the sample prior to that gas flow. For example, in some embodiments, the step of flowing a gas of a second gas temperature cools the sample. In some embodiments, the sample is of a temperature greater than the ambient conditions of the sample, and gas of ambient temperature is flowed towards the sample in order to cool the sample. In some embodiments, the second gas temperature is less than or equal to an ambient temperature, a gas of the second gas temperature cools the sample.

The one or more nozzles may have any suitable shape. In some embodiments, the one or more nozzles are conical in shape. The one or more nozzles may also independently have a particular a maximum transverse dimension (e.g., a diameter or other maximum dimension). The maximum transverse dimension of a nozzle may advantageously control the amount of gas flowed towards the sample holder or sample. In some embodiments, the one or more nozzles has a maximum transverse dimension of greater than or equal to 0.1 mm, greater than or equal to 0.5 mm, greater than or equal to 1 mm, greater than or equal to 2 mm, greater than or equal to 3 mm, greater than or equal to 4 mm, greater than or equal to 5 mm, greater than or equal to 7 mm, or greater than or equal to 10 mm. In some embodiments, the one or more nozzles has a maximum transverse dimension of less than or equal to 10 mm, less than or equal to 7 mm, less than or equal to 5 mm, less than or equal to 4 mm, less than or equal to 3 mm, less than or equal to 2 mm, less than or equal to 1 mm, less than or equal to 0.5 mm, or less than or equal to 0.1 mm. Combinations of the above-referenced ranges are also possible (e.g., greater than or equal to 0.1 mm and less than or equal to 10 mm). Other ranges are possible.

The one or more nozzles may provide flow of a gas (e.g., air) with a particular flow rate. In some embodiments, the flow rate of gas provided by each of the one or more nozzles is, independently, greater than or equal to 0.1 L/min, greater than or equal to 1 L/min, greater than or equal to 2 L/min, greater than or equal to 3 L/min, greater than or equal to 5 L/min, greater than or equal to 7 L/min, greater than or equal to 10 L/min, greater than or equal to 12 L/min, greater than or equal to 15 L/min, or greater than or equal to 20 L/min. In some embodiments, the flow rate of gas provided by each of the one or more nozzles is, independently, less than or equal to 20 L/min, less than or equal to 15 L/min, less than or equal to 12 L/min, less than or equal to 10 L/min, less than or equal to 7 L/min, less than or equal to 5 L/min, less than or equal to 3 L/min, less than or equal to 2 L/min, less than or equal to 1 L/min, or less than or equal to 0.1 L/min. Combinations of the above-referenced ranges are also possible (e.g., greater than or equal to 0.1 L/min and less than or equal to 20 L/min). Other ranges are possible.

In some embodiments, the system may also comprise one or more solenoids connected to the one or more nozzles. The one or more solenoids can be used to position the nozzles (e.g., towards the sample holder, away from the sample holder) and may be operatively associated with the one or more nozzles to move the nozzles as desired to heat (or cool) the sample. In some embodiments, computer software and/or a controller may be used to control the solenoid and may further be operatively associated with other portions of the system, such as a heating element or a thermal sensor of the system.

In some embodiments, the system may also comprise a pump operatively associated with each of the one or more nozzles, wherein the pump is configured to provide gas flow to the one or more nozzles. The pump can convey a gas (e.g., compressed air) from a gas source (e.g., a gas cylinder) to the one or more nozzles. In some embodiments, a voltage of a heating element is held constant, and the pump voltage is modified to adjust the flow rate of a flowing gas as until the heating element reaches a particular temperature (e.g., a first gas temperature, a second gas temperature, a third gas temperature, etc.). In some embodiments, the system may also comprise a fan and/or a duct, which may also convey gas flow to and/or through a nozzle.

In some embodiments, heated gas, non-heated gas, and/or cooled gas may be combined prior to or within a nozzle in order to achieve a particular gas temperature (e.g., a first gas temperature, a second gas temperature, a third gas temperature, a fourth gas temperature). The gas may be combined by connecting or merging various gases within tubing, conduits, and/or the like. Advantageously, by combining heated gas, nonheated gas, and/or cooled gas, a single nozzle may be used to flow gas towards the sample holder (or a sample within the sample holder) of a first temperature, and the combination of heated gas, non-heated gas, and/or cooled gas can be changed from a first configuration to a second configuration, resulting in gas of a second temperature to be flowed from the single nozzle. For example, the ratio of heated gas relative to non-heated can be adjusted in order to set the temperature of gas exiting the nozzle tip. Of course, however, combinations of heated gas, non-heated gas, and/or cooled gas may be used in embodiments comprising a plurality of nozzles as well, as this disclosure is not so limited. In some embodiments, flowing the gas of a set temperature towards the sample to control the temperature of the sample maintains the temperature of the sample (e.g., a first gas temperature and a second gas temperature are the same). However, in other embodiments, flowing the gas of a set temperature towards the sample to control the temperature heats and/or cools the sample (e.g., a first gas temperature and a second gas temperature are different, such that the second temperature is greater than the first temperature or the first temperature is greater than the second temperature).

As mentioned above, a first source of electromagnetic radiation may also be used to heat the sample. In some embodiments, a sample comprises a chemical species that can be directly stimulated by the first source of electromagnetic radiation to generate or facilitate heating of the sample (i.e., internal to the sample). In some such embodiments, the chemical species comprises gold nanoparticles. Without wishing to be bound by any particular theory, it is believed that gold nanoparticles generate heat via surface plasmon resonance when stimulated by electromagnetic radiation of a particular wavelength, which in turn can generate heat within the sample when the sample contains the gold nanoparticles. Those skilled in the art in view of the present disclosure will be capable of selecting an appropriate chemical species and first source of electromagnetic radiation (i.e., a source of electromagnetic radiation configured to stimulate the chemical species to produce heat). In some embodiments, the sample comprises a fluid or a solvent containing gold nanoparticles, and wherein the step of heating the sample with the first source of electromagnetic radiation comprises heating the gold nanoparticles.

The first source of electromagnetic radiation can be configured to heat the sample by a particular amount. In some embodiments, the first source of electromagnetic radiation is configured to heat the sample by greater than or equal to 0.1 °C, greater than or equal to 0.5 °C, greater than or equal to 1 °C, greater than or equal to 1.5 °C, greater than or equal to 2 °C, greater than or equal to 5 °C, greater than or equal to 7 °C, greater than or equal to 10 °C, greater than or equal to 15 °C, greater than or equal to 20 °C, greater than or equal to 25 °C, greater than or equal to 30 °C, greater than or equal to 35 °C, greater than or equal to 40 °C, greater than or equal to 50 °C, greater than or equal to 60 °C, greater than or equal to 70 °C, or greater than or equal to 80 °C (e.g., relative to the temperature of the sample prior to application of the first source of electromagnetic radiation). In some embodiments, the first source of electromagnetic radiation is configured to heat the sample by less than or equal to 80 °C, less than or equal to 70 °C, less than or equal to 60 °C, less than or equal to 50 °C, less than or equal to 40 °C, less than or equal to 35 °C, less than or equal to 30 °C, less than or equal to 25 °C, less than or equal to 20 °C, less than or equal to 15 °C, less than or equal to 10 °C, less than or equal to 7 °C, less than or equal to 5 °C, less than or equal to 2 °C, less than or equal to 1.5 °C, less than or equal to 1°C, less than or equal to 0.5 °C, or less than or equal to 0.1 °C (e.g., relative to the temperature of the sample prior to application of the first source of electromagnetic radiation). Combinations of the above-referenced ranges are also possible (e.g., greater than or equal to 0.1 °C and less than or equal to 80 °C). Other ranges are possible. The first source of electromagnetic radiation may heat the sample from a first sample temperature to a second sample temperature, from a second sample temperature to a third sample temperature, from a third sample temperature to a fourth sample temperature, etc. The first, second, third, fourth, etc. sample temperatures may each independently have one or more of the ranges described above. The first, second, third, fourth, etc. sample temperatures may each independently be the same or different from one another.

For example, in some embodiments, the sample has a first sample temperature and the step of heating (e.g., controlling the temperature of) the sample with the first source of electromagnetic radiation comprises increasing the first sample temperature to a second sample temperature, and wherein the second sample temperature is greater than the first sample temperature by greater than or equal to 0.1 °C, greater than or equal to 0.5 °C, greater than or equal to 1 °C, greater than or equal to 1.5 °C, greater than or equal to 2 °C, greater than or equal to 5 °C, greater than or equal to 7 °C, greater than or equal to 10 °C, greater than or equal to 15 °C, greater than or equal to 20 °C, greater than or equal to 25 °C, greater than or equal to 30 °C, greater than or equal to 35 °C, greater than or equal to 40 °C, greater than or equal to 50 °C, greater than or equal to 60 °C, greater than or equal to 70 °C, or greater than or equal to 80 °C. In some embodiments, the sample has a first sample temperature and the step of heating the sample with the first source of electromagnetic radiation comprises increasing the first sample temperature to a second sample temperature, and wherein the second sample temperature is greater than the first sample temperature by less than or equal to 80 °C, less than or equal to 70 °C, less than or equal to 60 °C, less than or equal to 50 °C, less than or equal to 40 °C, less than or equal to 35 °C, less than or equal to 30 °C, less than or equal to 25 °C, less than or equal to 20 °C, less than or equal to 15 °C, less than or equal to 10 °C, less than or equal to 7 °C, less than or equal to 5 °C, less than or equal to 2 °C, less than or equal to 1.5 °C, less than or equal to 1°C, less than or equal to 0.5 °C, or less than or equal to 0.1 °C. Combinations of the above-referenced ranges are also possible (e.g., greater than or equal to 0.1 °C and less than or equal to 80 °C). Other ranges are possible. The ranges above may also be applied to a second sample temperature relative to a third sample temperature, or a third sample temperature relative to a fourth sample temperature, etc.

In some embodiments, a sample that is first heated using a first source of electromagnetic radiation may be further heated to another sample temperature by a flow of gas (e.g., by flow of a heated gas having a temperature greater than the (first) temperature of the sample). For example, in some embodiments, the sample has a second sample temperature, and the method comprises further heating the sample by flowing a gas towards the sample such that the second sample temperature reaches a third sample temperature, and wherein the third sample temperature is greater than the second sample temperature by greater than or equal to 0.1 °C, greater than or equal to 0.5 °C, greater than or equal to 1 °C, greater than or equal to 1.5 °C, greater than or equal to 2 °C, greater than or equal to 5 °C, greater than or equal to 7 °C, greater than or equal to 10 °C, greater than or equal to 15 °C, greater than or equal to 20 °C, greater than or equal to 25 °C, greater than or equal to 30 °C, greater than or equal to 35 °C, greater than or equal to 40 °C, greater than or equal to 50 °C, greater than or equal to 60 °C, greater than or equal to 70 °C, or greater than or equal to 80 °C. In some embodiments, the sample has a second sample temperature, and the method comprises further heating the sample by flowing a gas towards the sample such that the second sample temperature reaches a third sample temperature, and wherein the third sample temperature is greater than the second sample temperature by less than or equal to 80 °C, less than or equal to 70 °C, less than or equal to 60 °C, less than or equal to 50 °C, less than or equal to 40 °C, less than or equal to 35 °C, less than or equal to 30 °C, less than or equal to 25 °C, less than or equal to 20 °C, less than or equal to 15 °C, less than or equal to 10 °C, less than or equal to 7 °C, less than or equal to 5 °C, less than or equal to 2 °C, less than or equal to 1.5 °C, less than or equal to 1°C, less than or equal to 0.5 °C, or less than or equal to 0.1 °C. Combinations of the above-referenced ranges are also possible (e.g., greater than or equal to 0.1 °C and less than or equal to 80 °C). Other ranges are possible.

In some embodiments, a sample that is first heated using a first source of electromagnetic radiation may be cooled to another sample temperature by a flow of gas (e.g., by flow of a cooled gas having a temperature less than the (first) temperature of the sample, a flow of ambient air). For example, in some embodiments, the sample has a second sample temperature, and the method comprises cooling the sample by flowing a gas towards the sample such that the second sample temperature reaches a third sample temperature, and wherein the third sample temperature is less than the second sample temperature by less than or equal to 80 °C, less than or equal to 70 °C, less than or equal to 60 °C, less than or equal to 50 °C, less than or equal to 40 °C, less than or equal to 35 °C, less than or equal to 30 °C, less than or equal to 25 °C, less than or equal to 20 °C, less than or equal to 15 °C, less than or equal to 10 °C, less than or equal to 7 °C, less than or equal to 5 °C, less than or equal to 2 °C, less than or equal to 1.5 °C, less than or equal to 1°C, less than or equal to 0.5 °C, or less than or equal to 0.1 °C. In some embodiments, the sample has a second sample temperature, and the method comprises cooling the sample by flowing a gas towards the sample such that the second sample temperature reaches a third sample temperature, and wherein the third sample temperature is less than the second sample temperature by greater than or equal to 0.1 °C, greater than or equal to 0.5 °C, greater than or equal to 1 °C, greater than or equal to 1.5 °C, greater than or equal to 2 °C, greater than or equal to 5 °C, greater than or equal to 7 °C, greater than or equal to 10 °C, greater than or equal to 15 °C, greater than or equal to 20 °C, greater than or equal to 25 °C, greater than or equal to 30 °C, greater than or equal to 35 °C, greater than or equal to 40 °C, greater than or equal to 50 °C, greater than or equal to 60 °C, greater than or equal to 70 °C, or greater than or equal to 80 °C. Combinations of the above-referenced ranges are also possible (e.g., greater than or equal to 0.1 °C and less than or equal to 80 °C). Other ranges are possible.

Any suitable source of electromagnetic radiation may be used as the first source of electromagnetic radiation. In some embodiments, the first source of electromagnetic radiation and/or the second source of electromagnetic radiation comprises a lightemitting diode (LED) and/or a laser. In some embodiments, the first source of electromagnetic radiation comprises one or more LEDs and/or lasers. In an exemplary embodiment, the first source of electromagnetic radiation is configured to emit infrared (IR) radiation.

The first source of electromagnetic radiation may be configured to emit light of any suitable wavelength (i.e., a wavelength suitable heating a chemical species, such as gold nanoparticles, within the sample). In one embodiment, the first source of radiation emits IR radiation. In some embodiments, the first source of radiation emits light of a wavelength of greater than or equal to 300 nm, greater than or equal to 320 nm, greater than or equal to 350 nm, greater than or equal to 380 nm, greater than or equal to 400 nm, greater than or equal to 420 nm, greater than or equal to 450 nm, greater than or equal to 480 nm, greater than or equal to 488 nm, greater than or equal to 500 nm, greater than or equal to 520 nm, greater than or equal to 550 nm, greater than or equal to 580 nm, greater than or equal to 600 nm, greater than or equal to 650 nm, greater than or equal to 700 nm, greater than or equal to 750 nm, greater than or equal to 800 nm, greater than or equal to 850 nm, greater than or equal to 900 nm, greater than or equal to 950 nm, greater than or equal to 1000 nm, greater than or equal to 1100 nm, or greater than or equal to 1200 nm. In some embodiments, the first source of electromagnetic radiation emits light of a wavelength of less than or equal to 1200 nm, less than or equal to 1100 nm, less than or equal to 1000 nm, less than or equal to 950 nm, less than or equal to 900 nm, less than or equal to 850 nm, less than or equal to 800 nm, less than or equal to 750 nm, less than or equal to 700 nm, less than or equal to 650 nm, less than or equal to 600 nm, less than or equal to 580 nm, less than or equal to 550 nm, less than or equal to 520 nm, less than or equal to 500 nm, less than or equal to 488 nm, less than or equal to 480 nm, less than or equal to 450 nm, less than or equal to 420 nm, less than or equal to 400 nm, less than or equal to 380 nm, less than or equal to 350 nm, less than or equal to 320 nm, or less than or equal to 300 nm. Combinations of the above-referenced ranges are also possible (e.g., greater than or equal to 300 nm and less than or equal to 1200 nm). Other ranges are possible.

As described above, the system may comprise a sample holder configured to hold a sample. In some embodiments, the sample holder is a tray, a fastener, or other suitable component for holding a container containing the sample. In some embodiments, the container and/or the sample holder may comprise, a PCR tube, a capillary tube, a microfluidic chip, a microcentrifuge tube, and/or a multi-welled plate. In some embodiments, the system further comprises insulation (e.g., an insulating sleeve) around at least a portion of the sample holder.

The sample can be derived from a variety of sources, including humans, animals (e.g., a dog, a cat), and plants, fungi, bacteria, and viruses , without limitation. The sample can be derived from bodily fluids, such as saliva, blood, urine, nasal mucosa, and in some cases, the sample is dissolved or suspended in a fluid or a solvent. In some embodiments, the sample is derived from a pathogen, such as a virus, bacteria, or a fungus. In some embodiments, the sample comprises a chemical species configured to provide heat to the sample upon stimulation by the first source of electromagnetic radiation. In some embodiments, the sample contains or comprises gold nanoparticles.

In some embodiments, the sample is mixed during use of the system. The system may comprise a mixer to mix the sample, such as a magnetic stir plate, a vortexer, and/or a sonicator.

In some embodiments, the sample contains or comprises an analyte. The analyte can be a variety of chemical/biological species. In some embodiments, the analyte is a nucleic acid molecule (e.g., DNA, RNA). However, other analytes are possible. Nonlimiting examples of other analytes include antigens, antibodies, peptides, proteins, and/or carbohydrates.

In some embodiments, the analyte is present within the sample within a certain amount or concentration. In some embodiments, the concentration of an analyte within the sample is determined to be greater than or equal to 1 analyte molecule per mL of sample, greater than or equal to 10 analyte molecules per mL of sample, greater than or equal to 50 analyte molecules per mL of sample, greater than or equal to 100 molecules per mL of sample, greater than or equal to 150 analytes per mL of sample, greater than or equal to 200 analyte molecules per mL of sample, greater than or equal to 500 analyte molecules per mL of sample, greater than or equal to 1,000 analyte molecules per mL of sample, greater than or equal to 10⁴ analyte molecules per mL of sample, greater than or equal to 10⁵ analyte molecules per mL of sample, greater than or equal to 10⁶ analyte molecules per mL of sample, greater than or equal to 10⁷ analyte molecules per mL of sample, greater than or equal to 10⁸ analyte molecules per mL of sample, or greater than or equal to 10⁹ analyte molecules per mL of sample. In some embodiments, the concentration of an analyte within the sample is determined to be less than or equal to 10⁹ analyte molecules per mL of sample, less than or equal to 10⁸ analyte molecules per mL of sample less than or equal to 10⁷ analyte molecules per mL of sample less than or equal to 10⁶ analyte molecules per mL of sample, less than or equal to 10⁵ analyte molecules per mL of sample, less than or equal to 10⁴, less than or equal to 1,000 analyte molecules per mL of sample, less than or equal to 500 analyte molecules per mL of sample, less than or equal to 200 analyte molecules per mL of sample, less than or equal to 150 analyte molecules per mL of sample, less than or equal to 100 analyte molecules per mL of sample, less than or equal to 50 analyte molecules per mL of sample, less than or equal 10 analyte molecules per mL of sample, or less than or equal to 1 analyte molecules per mL of sample. Combinations of the above-referenced ranges are also possible (e.g., greater than or equal to 1 analyte molecule per mL of sample and less than or equal to 10⁹ analyte molecules per mL of sample). Other amounts and ranges are possible.

In some embodiments, a chemical and/or biological reaction is performed using a system described herein. The chemical and/or biological reaction may be performed before, during, and/or after heating the sample to a particular sample temperature. For example, in some embodiments, the step of performing the chemical and/or biological reaction with the analyte in the sample is performed at the second sample temperature, the third sample temperature, and/or a fourth sample temperature. In some embodiments, the chemical and/or biological reaction is performed after heating a sample to a first sample temperature. In some embodiments, the chemical and/or biological reaction is performed after heating the sample to a second sample temperature. In some embodiments, the chemical and/or biological reaction is performed after heating the sample to a third sample temperature.

The chemical and/or biological reaction may be any suitable reaction that may benefit from the heating and cooling processes described herein. The processes and systems described herein may be used for determining an analyte within the sample, such as the presence (or absence) of an analyte within the sample, or the amount or concentration of analyte within a sample. For example, in some embodiments, the chemical and/or biological reaction is a polymerase chain reaction (PCR) or reversetranscriptase polymerase chain reaction (RT-PCR). However, other reactions are possible. Non-limiting examples of other chemical and/or biological reactions include isothermal amplification, long-mediated isothermal amplification, other nucleic acid amplification or other natural or synthetic chemical detection methods which require controlled temperatures for suitable testing results.

Other chemical and/or biological reactions or processes are possible. Other nonlimiting examples of chemical and/or biological reactions include enzyme-facilitate reactions (e.g., restriction endonucleases, DNase, ribonucleases), sequencing reactions, isothermal amplification (e.g., DNA amplification, RNA amplification). In some embodiments, a chemical and/or biological reaction comprises inactivating a component of the sample (e.g., denaturing a protein component of the sample, degrading a biomolecule of the sample).

In some embodiments, the chemical and/or biological reaction or process (e.g., a RT-PCR reaction, a binding process) occurs within a relatively short period of time. For example, in some embodiments, a chemical and/or biological reaction or process is performed in less than or equal to 3 minutes (e.g., less than or equal to 30 seconds, less than or equal to 10 seconds). Advantageously, one or more nozzles providing heated and/or cooled gas may dramatically reduce the duration of time required for the chemical and/or biological reaction or process. In some embodiments, the duration of a chemical and/or biological reaction or process (e.g., when cool and/or heated gas is provided to the sample comprising the chemical and/or biological reaction) is less than or equal to 3 minutes, less than or equal to 2.5 minutes, less than or equal to 2 minutes, less than or equal to 1.5 minutes, less than or equal to 60 seconds, less than or equal to 45 seconds, less than or equal to 30 seconds, less than or equal to 20 seconds, less than or equal to 15 seconds, less than or equal to 10 seconds, less than or equal to 5 seconds, less than or equal to 3 seconds, less than or equal to 1 second, less than or equal to 0.1 seconds, or less than or equal to 0.01 seconds. In some embodiments, the duration of a chemical and/or biological reaction or process is greater than or equal to 0.01 seconds, greater than or equal to 0.1 seconds, greater than or equal to 1 second, greater than or equal to 3 seconds, greater than or equal to 5 seconds, greater than or equal to 10 seconds, greater than or equal to 15 seconds, greater than or equal to 20 seconds, greater than or equal to 30 seconds, greater than or equal to 45 seconds, greater than or equal to 60 seconds, greater than or equal to 1.5 minutes, greater than or equal to 2 minutes, greater than or equal to 2.5 minutes, or greater than or equal 3 minutes. Combinations of the abovereferenced ranges are also possible (e.g., greater than or equal to 0.01 seconds and less than or equal to 3 minutes). Other ranges are possible as this disclosure is not so limited. For embodiments in which heated and/or cooled gas are applied, the duration of the heated and/or cooled gas can be within any of the above-described ranges. In some embodiments, a combination heated and/or cooled air may be applied (e.g., applying hot air for greater than or equal to 0.1 second and less than or equal to 1 second and applying cool air for greater than or equal to 1 second and less than or equal 10 seconds, etc.).

In some embodiments, a series of chemical and/or biological reactions or processes (e.g., RT-PCR, binding processes) is performed on a sample. For example, in some embodiments at least 1, at least 2, at least 5, at least 10 chemical and/or biological reactions or processes is performed on a sample. Each chemical and/or biological reaction or process may occur within a certain number of times, i.e., cycles (e.g., heating and/or cooling cycles). Advantageously, chemical and/or biological reactions or processes can be conducted in relatively quick succession (e.g., less than or equal to 3 minutes), and another cycle can begin (e.g., a new heating and/or cooling). In some embodiments, greater than or equal to 2 cycles, greater than or equal to 5 cycles, greater than or equal 10 cycles, greater than equal to 15 cycles, greater than or equal to 20 cycles, greater than or equal to 30 cycles, greater than or equal to 40 cycles, greater than or equal to 50 cycles, greater than or equal to 75 cycles, or greater than or equal to 100 cycles. In some embodiments, less than or equal to 100 cycles, less than or equal to 75 cycles, less than or equal to 50 cycles, less than or equal 40 cycles, less than or equal to 30 cycles, less than or equal to 20 cycles, less than or equal to 15 cycles, less than or equal to 10 cycles, less than or equal to 5 cycles, or less than or equal to 2 cycles. Combinations of the above-referenced ranges are also possible (e.g., greater than or equal to 2 cycles and less than or equal to 100 cycles). Other ranges are possible as this disclosure is not so limited, and may, for example, depend on the nature of the chemical and/or biological reaction or processes (e.g., a reaction kinetics of the chemical and/or biological reaction). In some embodiments, the ranges in numbers of cycles above may be performed within a relatively short period of time within one or more time ranges described above.

By way of illustration and not limitation, as an example of cycling, a sample comprising a first source of electromagnetic radiation can be stimulated (e.g., by another source of electromagnetic radiation, such as light, when the sample comprises a species (e.g., gold nanoparticles) configured to absorb light) to generate electromagnetic radiation (e.g., IR radiation), and this generated electromagnetic radiation can provide heat to the sample (e.g., a solvent of the sample and/or a chemical and/or biological species of the sample), which may facilitate the occurrence of a chemical and/or biological reaction or process (e.g., a first chemical and/or biological reaction). Concurrently or subsequently, a gas flow of a first temperature (at the same or different as the desired temperature of the sample) can be provided to the sample, that can either heat, cool, or maintain a temperature of the sample. Subsequently, a gas flow of a second temperature can be provided to the sample that further heats, cools, or maintains temperature. For example, if a gas flow of a first temperature provides heat to the sample of a first sample temperature, a gas flow of a second temperature, less than the first temperature and less than the first sample temperature, can be provided to the sample in order cool the sample. Subsequently, gas flow of a third temperature (e.g., different and the second temperature) can be provided to the sample to further heat and/or cool the sample. In this manner, the sample can be relatively quickly heated and/or cool, or a temperature of the sample can be maintained. In some such embodiments, the third temperature is the same as the first temperature, and the sample can be cycled between the first temperature and second temperature over several or many cycles. Of course, other temperature and cycling options are possible as this disclosure is not so limited.

Advantageously, the system and methods herein may perform a plurality of cycles within a relatively short amount of time (e.g., before or during determining of an analyte). In some embodiments, the system is configured to perform greater than or equal to 2 and/or less than or equal to 1,000 cycles (or other ranges of cycles described herein) in less than or equal to 30 minutes. In some embodiments, the system is configured to perform greater than or equal to 2 and/or less than or equal to 1,000 cycles (or other ranges of cycles described herein) in less than or equal to 25 minutes, less than or equal to 20 minutes, less than or equal to 10 minutes, less than or equal to 5 minutes, less than or equal to 3 minutes, less than or equal to 2 minutes, less than or equal 60 seconds, less than or equal to 30 seconds, less than or equal to 15 seconds, less than or equal to 10 seconds, less than or equal to 5 seconds, or less than or equal to 1 second. In some embodiments, the system is configured to perform greater than or equal to 2 and/or less than or equal to 1,000 cycles (or other ranges of cycles described herein) in greater than or equal to 1 second, greater than or equal to 5 seconds, greater than or equal to 10 seconds, greater than or equal to 30 seconds, greater than or equal to 60 seconds, greater than or equal to 2 minutes, greater than or equal to 3 minutes, greater than or equal to 5 minutes, greater than or equal to 10 minutes, greater than or equal to 20 minutes, greater than or equal to 25 minutes, or greater than or equal to 30 minutes. Combinations of the above-referenced ranges are also possible (e.g., the system is configured to perform greater than or equal to 2 and/or less than or equal to 1,000 cycles in less than or equal to 30 minutes and greater than or equal to 1 second). Other ranges are possible.

In some embodiments, the temperature of a sample may be determined before, during, and/or after any heating step. The determined temperature can subsequently be used to determine if more heating should be provided, heating should be discontinued, and/or if cooling should be initiated, and those skilled in the art in view of the present disclosure will be capable of determining the level of heating and/or cooling based on the chemical and/or biological reaction to be performed. In some embodiments, the system comprises a thermal sensor configured to determine a temperature of the sample. In some embodiments, the thermal sensor comprises a thermocouple, or contactless devices for measuring the temperature, (e.g., an optical pyrometer).

The systems described herein may comprise a second source of electromagnetic radiation. In some embodiments, the second source of electromagnetic ration may be used to generate a signal from the sample. This signal may be used (at least in part) to determine the analyte within the sample (e.g., quantitatively or qualitatively, such as the presence (or absence) of an analyte within the sample). Any suitable source of electromagnetic radiation may be used. In some embodiments, the second source of electromagnetic radiation comprises a light-emitting diode or a laser. In some embodiments, the second source of electromagnetic radiation may generate a luminescent signal (e.g., bioluminescence, fluorescence) within the sample that can be detected by a detector. In some cases, the detector is configured to detect a reference signal and to simultaneously or subsequently detect a signal from the sample to determine the analyte (e.g., from a change in absorbance from the reference signal relative to the sample signal). In some embodiments, the method further comprises determining the presence of the analyte within the sample after the sample reaches a particular temperature (e.g., a second sample temperature, a third sample temperature), and information obtained from the signal generated by the second source of electromagnetic radiation, along with the detector, may be used to determine the analyte.

The second source of electromagnetic radiation may be configured to emit light of any suitable wavelength (i.e., light suitable for detecting the analyte in a sample, e.g., before and/or after one or more chemical and/or biological reactions has been performed within the sample). In one embodiment, the second source of radiation emits UV radiation. In some embodiments, the second source of radiation emits light of a wavelength of greater than or equal to 300 nm, greater than or equal to 320 nm, greater than or equal to 350 nm, greater than or equal to 380 nm, greater than or equal to 400 nm, greater than or equal to 420 nm, greater than or equal to 450 nm, greater than or equal to 480 nm, greater than or equal to 488 nm, greater than or equal to 500 nm, greater than or equal to 520 nm, greater than or equal to 550 nm, greater than or equal to 580 nm, greater than or equal to 600 nm, greater than or equal to 650 nm, greater than or equal to 700 nm, greater than or equal to 750 nm, or greater than or equal to 800 nm. In some embodiments, the second source of electromagnetic radiation emits light of a wavelength of less than or equal to 800 nm, less than or equal to 750 nm, less than or equal to 700 nm, less than or equal to 650 nm, less than or equal to 600 nm, less than or equal to 580 nm, less than or equal to 550 nm, less than or equal to 520 nm, less than or equal to 500 nm, less than or equal to 488 nm, less than or equal to 480 nm, less than or equal to 450 nm, less than or equal to 420 nm, less than or equal to 400 nm, less than or equal to 380 nm, less than or equal to 350 nm, less than or equal to 320 nm, or less than or equal to 300 nm. Combinations of the above-referenced ranges are also possible (e.g., greater than or equal to 300 nm and less than or equal to 800 nm). Other ranges are possible.

In some embodiments, a system further comprises a processor and/or a controller that may include one or more proportional, integral (PI), and/or derivative (PID) feedforward and/or feedback loops to adjust a parameter of the system. For example, in some embodiments, a signal from a second source of electromagnetic provides data (e.g., completion of one or more chemical and/or biological reactions) to the processor and/or controller, and the processor and/or controller provides a command to the first source of electromagnetic radiation (e.g., to provide more electromagnetic radiation to heat the sample, to provide no further electromagnetic radiation to the first source of electromagnetic radiation). In some embodiments, a temperature sensor (e.g., IR thermometer, thermocouple, IR pyrometer) is associated with the controller and/or processor, and provides data to one or more nozzles (e.g., to provide gas flow to the one or more nozzles of a higher temperature than the sample temperature, to provide gas flow to the one or more nozzles of a lower temperature than the sample temperature, to provide gas flow of the same temperature as the sample to the one or more nozzles). Advantageously, the controller and/or processor may be associated with a single nozzle, such that a single nozzle provides temperature control (e.g., heating, cooling, and/or maintaining the temperature of the sample).

The processor and/or controller may be implemented by any suitable type of analog and/or digital circuitry. In one set of embodiments, the processor and/or the controller may be implemented in a field programmable gate array (FPGA). For example, the processor and/or the controller may be implemented using hardware or a combination of hardware and software. When implemented using software, suitable software code can be executed on any suitable processor (e.g., a microprocessor, FPGA) or collection of processors. The processor and/or the controller can be implemented in numerous ways, such as with dedicated hardware, or with general purpose hardware (e.g., one or more processors) that is programmed using microcode or software to perform the functions recited above.

In this respect, it should be appreciated that one implementation of the embodiments described herein comprises at least one computer-readable storage medium (e.g., RAM, ROM, EEPROM, flash memory or other memory technology, or other tangible, non-transitory computer-readable storage medium) encoded with a computer program (e.g., a plurality of executable instructions) that, when executed on one or more processors, performs the above discussed functions of one or more embodiments. In addition, it should be appreciated that the reference to a computer program which, when executed, performs any of the above-discussed functions, is not limited to an application program running on a host computer. Rather, the terms computer program and software are used herein in a generic sense to reference any type of computer code (e.g., application software, firmware, microcode, or any other form of computer instruction) that can be employed to program one or more processors to implement aspects of the techniques discussed herein.

The systems and methods described herein may be suitable for determining the presence (or absence) of a variety of analytes. For example, the systems and methods described herein can be used to determine the presence of an analyte using PCR (e.g., RT-PCR), whereby a sample suspected of containing a nucleic acid analyte can be amplified such that the nucleic acid is within a detectable amount. For example, in some embodiments, the presence of SARS-CoV-2 can be determined by determining the presence of a nucleic acid (e.g., RNA) associated with SARS-Cov-2.

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

EXAMPLE 1

The following example describes a 3 -nozzle system for performing PCR with both internal plasmonic heating and external hot air heating.

The system combined internal plasmonic and external hot air PCR. FIG. 3 shows a schematic of the system (300). By knowing the temperature of the warm gas, the target sample (310) temperatures were reliably reached. To speed up heating in a standard PCR tube, a first source of electromagnetic radiation, IR LEDs, were turned on for a few seconds per cycle to help the sample reach approximate sample temperatures. The warm air then finished bringing the fluid of the sample to the desired temperature.

To do this, three streams of gas (air) were used that continuously flowed gas at a first gas temperature, a second gas temperature, and a third gas temperature, respectively. Each of these gasses were independently held at a constant. When a particular stream was active, a solenoid (340) connected to the nozzle pushed upwards, pointing that nozzle towards the PCR tube containing the sample. When the nozzle (330) was pointing towards the PCR tube, gas at the respective gas temperature was flowed from the pump, through a heater, through the nozzle, and vertically upwards towards the sample tube.

When a stream was inactive (i.e., was not heating or cooling the sample tube), the solenoid pulled down, pointing the nozzle away from the PCR sample tube. In this case, the gas of a particular gas temperature still flowed through the pump, heater, and nozzle but in a direction away from the PCR tube so that it would no longer heat the PCR tube.

Stream 1 blew warm air, of a gas temperature of either 50 °C (for the reverse transcription step) or 60 °C (for annealing/extension during cycling) via an air process heater. Stream 2 blew hotter air, set to a gas temperature of 95 °C (for initial denaturation and cycling denaturation) via a 2^nd air process heater. The temperatures of Stream 1 and Stream 2 were controlled by changing the voltage provided to the heater. Stream 1 and Stream 2 blew air at approximately 2 LPM. Stream 3 was not heated and blew room temperature air at 12 LPM in order to rapidly cool the fluid of the sample, as desired. For all 3 nozzles, the gas blew through a 4 mm hole. To heat the fluid rapidly, there were 3 infrared LEDs (350) equally spaced, concentrically surrounding the PCR sample tube, which could heat gold nanoparticles within the sample tube.

There was one thermistor mounted in each of the 3 nozzles to monitor the gas temperature of a particular nozzle. To determine what voltage was needed to be supplied to the heater for the gas to be warm enough so that the tube reached a steady state at a certain gas temperature, the following was employed. A thermocouple was placed in the fluid of the PCR tube. The PCR tube was placed in the system. A LabView program caused a solenoid to engage based on a readout from the thermocouple so that the desired nozzle was activated and pointed towards the PCR tube with air flowing through the nozzle. The voltage going to that nozzle’s heater was then modified until the thermocouple inside the fluid read the desired temperature. Once the desired temperature was maintained, the voltage and nozzle temperature were noted, and the voltage was kept constant. FIG. 4 shows a plot of the sample temperature as a function as time as the sample undergoes the heating and cooling described above. FIG. 5 shows a plot of before and after a biological reaction is performed, demonstrating the successful determination of an analyte once the reaction is performed.

EXAMPLE 2

The following example describes the determination of the presence of SARS- CoV-2 using a system including a single nozzle for providing cool air to a contactless, plasmonically-heated system.

The reaction mix was prepared fresh each day. All reagents were stored and prepared on a cold chamber except for the salts, which are stored at room temperature. Both Taq Polymerase and PrimeScript Reverse Transcriptase and DNTP mix were supplied by Takara Bio (USA). AuNPs were supplied by Nanopartz, Inc. (USA)), OD 2.0.

Preparation of the viral sample for a contrived positive begins with pooled Clinical Negative Saliva, which was diluted by a factor of 20. Then, from this sample, 2.5 pL of SARS-CoV-2 (WA/2020) at 1.88xl0⁵ copies/pL were used. Clinical Negative Saliva as the diluent was added to generate a IxlO⁴ copies/pL viral stock. From this stock, a variety of dilutions were made. Table 1 demonstrates the suitable amplification and detection of SARS-CoV-2 from suspected COVID- 19 patient samples. In Table 2 is demonstrated the reliable detection of low copies of SARS-CoV-2 per rection tube (25 copies/reaction). Table 1 - Clinical Sampling Testing Data Using Dilutions of COVID-19-containing Samples

*1 was indeterminate (n=60, 1.67%)

indeterminates (n=390, 0.51%)

The amplification reaction shown in FIG. 7 begins as 5 pL of the 5 copy/pL viral mix added to a PCR tube and lysed for 45-seconds at 90 °C. The PCR mixture of 15 pL was added to the sample and gently mixed. Evaporation and condensation were prevented with 15 pL of mineral oil added atop the reaction mixture. The system receives the PCR tube and thermal cycles through an optimized protocol for the developed assay. The thermal profile for this example was a 5-minute Reverse Transcription at 50 °C. The initial denature step occurs at 94 °C for 10 seconds, and 45 cycles of amplification follows with a 1-second anneal step at 61°C, 6-second extension step at 64°C, and a 0.1-second denature step at 94°C.

FIG. 7 and FIG. 8 present a data corresponding to cycling. Amplification for determining the presence of COVID-19 using the system and with an RT-PCR biological reaction, and a thermal profile consisting of 5 minutes at 50 °C, followed by 45 cycles of thermal cycling between 94 °C and 61 °C initial anneal, and 64 °C final anneal. This cycling is shown in the plot of FIG. 7. An optical pyrometer was used to drive the closed loop temperature control with IR light for reaction mixture heating, using plasmonic resonance of gold nanoparticles and stimulated by a first source of electromagnetic radiation for heat (internal to the sample), and forced air (external) to sample to directly maintain the temperature of the sample and then cool the reaction mixture. Three amplification targets were present in the reaction mix, COVID-19 N1 (diamond), COVID-19 N2 (circle), and human RNAseP (triangle), shown in FIG. 8. Amplification in the sample of the system was monitored by spectrometry by generating a signal from a second source of electromagnetic radiation and also used algorithmic signal deconvolution of the generated data.

Upon completion of the protocol, fluorescence data from the second source of electromagnetic radiation and the deconvoluted data was analyzed via a developed algorithm to give crossing threshold values for the N1 and N2 targets as well as the RP control target. The device was expandable to up to 5 detectable wavelengths.

A controlled air nozzle was attached to the bottom of the system and provided a controlled air flow directed vertically from below the sample tube. The output nozzle was 8 mm in diameter and ejected up to 12 L/min of air. The air temperature was controlled by a radiator placed between the output and the PWM controlled fan. The temperature of the air is measured by a thermistor placed at the output nozzle, which provided feedback to the temperature-controlled radiator, creating a closed-loop control system for the air temperature. Heated or cooled air flow could be provided using either a programmable heater or cooler, or simply a reversible thermoelectric Peltier device, which can provide heating or cooling to the air flow path depending on the direction of the Peltier device (i.e., whether the heating side or the cooling side of the Peltier is in contact with the air flow path).

For temperature control, an IR pyrometer pointing at the bottom of the sample tube was used as a temperature measuring device and thermal sensor. The feedback from the pyrometer is used to calculate the output power of the heating and cooling mechanisms (i.e., the Peltier device). The air flow, air temperature, and IR intensity were set based on the pyrometers measurements and the desired target temperature at any given time. Integration of a controlled air and IR LED power created relatively fast and reliable thermocycling. This device included the components shown in FIGS. 9-10. FIG. 11 shows a cross-sectional image of the same system of FIGS. 9-10.

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

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

The phrase “and/or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Other elements may optionally be present other than the elements specifically identified by the “and/or” clause, whether related or unrelated to those elements specifically identified unless clearly indicated to the contrary. Thus, as a non-limiting example, a reference to “A and/or B,” when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A without B (optionally including elements other than B); in another embodiment, to B without A (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

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

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

Some embodiments may be embodied as a method, of which various examples have been described. The acts performed as part of the methods may be ordered in any suitable way. Accordingly, embodiments may be constructed in which acts are performed in an order different than illustrated, which may include different (e.g., more or less) acts than those that are described, and/or that may involve performing some acts simultaneously, even though the acts are shown as being performed sequentially in the embodiments specifically described above.

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

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

Claims

34 CLAIMS What is claimed is:

1. A system, comprising: a sample holder configured to hold a sample; one or more nozzles proximate to the sample holder each configured to flow a gas of a set temperature towards the sample holder; a first source of electromagnetic radiation proximate the sample holder configured to heat the sample; a second source of electromagnetic radiation configured to generate a signal from the sample; and a detector configured to detect the signal from the sample.

2. The system of claim 1, wherein the first source of electromagnetic radiation is configured to heat the sample to a first temperature, and the set temperature is less than or equal to the first temperature.

3. The system of claim 1, wherein only a single nozzle for flowing a gas of a set temperature towards the sample holder is present.

4. The system of claim 1, wherein the first source of electromagnetic radiation is configured to heat the sample by greater than or equal to 0.1 °C, greater than or equal to

1 °C, greater than or equal to 5 °C, greater than or equal to 10 °C, greater than or equal to 20 °C, greater than or equal to 35 °C, greater than or equal to 50 °C, and/or greater than or equal to 80 °C; and/or less than or equal to 80 °C, less than or equal to 50 °C, less than or equal to 35 °C, less than or equal to 20 °C, less than or equal to 5 °C, less than or equal to 1 °C, and/or less than or equal to 0.1 °C.

5. The system of claim 1, further comprising a thermal sensor configured to determine a temperature of the sample. 35

6. The system of claim 1, wherein at least some of the one or more nozzles is operatively associated with a heating element.

7. The system of claim 1, wherein at least some of the one or more nozzles is operatively associated with a thermistor.

8. The system of claim 1, further comprising a solenoid connected to the one or more nozzles.

9. The system of claim 1, wherein the first source of electromagnetic radiation and/or the second source of electromagnetic radiation comprises a light-emitting diode or a laser.

10. The system of claim 1, wherein the sample holder contains a sample comprising nanoparticles, and wherein the first source of electromagnetic radiation is configured to heat the nanoparticles within the sample.

11. The system of claim 1, further comprising a pump operatively associated with each of the one or more nozzles, wherein the pump is configured to provide gas flow to the one or more nozzles.

12. The system of claim 1, further comprising a mixer configured to mix the sample.

13. The system of claim 12, wherein the mixer comprises a magnetic stir plate, a vortexer, and/or a sonic ator.

14. The system of claim 1, further comprising insulation around the sample holder.

15. A method for determining the presence of an analyte in a sample, the method comprising: heating the sample with a first source of electromagnetic radiation; flowing a gas of a set temperature towards the sample to further control a temperature of the sample; and performing a chemical and/or biological reaction with the analyte in the sample.

16. The method of claim 15, wherein the step of flowing the gas of the second gas temperature cools the sample.

17. The method of claim 15, wherein the sample has a first sample temperature and the step of heating the sample with the first source of electromagnetic radiation comprises increasing the first sample temperature to a second sample temperature, and wherein the second sample temperature is greater than the first sample temperature by greater than or equal to 0.1 °C, greater than or equal to 1 °C, greater than or equal to 5 °C, greater than or equal to 10 °C, greater than or equal to 20 °C, greater than or equal to 35 °C, greater than or equal to 50 °C, and/or greater than or equal to 80 °C and/or less than or equal to 80 °C, less than or equal to 50 °C, less than or equal to 35 °C, less than or equal to 20 °C, less than or equal to 5 °C, less than or equal to 1 °C, and/or less than or equal to 0.1 °C.

18. The method of claim 15, wherein the sample has a second sample temperature, and the method comprises further controlling the second sample temperature by flowing a gas towards the sample such that the second sample temperature reaches a third sample temperature, and wherein the third sample temperature is greater than the second sample temperature by greater than or equal to 0.1 °C, greater than or equal to 1 °C, greater than or equal to 5 °C, greater than or equal to 10 °C, greater than or equal to 20 °C, greater than or equal to 35 °C, greater than or equal to 50 °C, and/or greater than or equal to 80 °C and/or less than or equal to 80 °C, less than or equal to 50 °C, less than or equal to 35 °C, less than or equal to 20 °C, less than or equal to 5 °C, less than or equal to 1 °C, and/or less than or equal to 0.1 °C.

19. The method of claim 15, wherein the step of performing the chemical and/or biological reaction with the analyte in the sample is performed at the second sample temperature, the third sample temperature, and/or a fourth sample temperature.

20. The method of claim 15, wherein the sample comprises a fluid or a solvent containing gold nanoparticles, and wherein the step of heating the sample with the first source of electromagnetic radiation comprises heating the gold nanoparticles.

21. The method of claim 15, wherein the second gas temperature is less than the first gas temperature.

22. The method of claim 15, wherein the second gas temperature is less than or equal to an ambient temperature.

23. The method of claim 15, wherein the second gas temperature is less than or equal to 25 °C and/or less than or equal to 20 °C.

24. The method of claim 15, further comprising flowing a gas of a third gas temperature, different from the first and/or second gas temperatures, towards the sample until the sample reaches a fourth sample temperature.

25. The method of claim 15, further comprising determining a temperature of the sample prior to and/or during any one of the flowing steps.

26. The method of claim 15, further comprising mixing the sample.

27. The method of claim 15, further comprising determining the presence of the analyte within the sample after the sample reaches the second sample temperature.

28. The method of claim 15, wherein flowing the gas of the set temperature towards the sample to further control the temperature of the sample comprises cooling the sample.

29. The method of claim 15, wherein flowing the gas of the set temperature towards the sample to further control the temperature of the sample comprises heating the sample. 38

30. The method of claim 15, wherein flowing the gas of the set temperature towards the sample to further control the temperature of the sample comprises maintaining the temperature of the sample.

31. The system of claim 1, wherein at least some of the one or more nozzles comprises a thermoelectric heat pump.

32. The system of claim 1, wherein the system is configured to perform greater than or equal to 2 and less than or equal to 100 cycles in less than or equal to 30 minutes.

33. The method of claim 15, further comprising performing greater than or equal to 2 cycles and less than or equal to 100 cycles comprising the chemical and/or biological reaction.