Patent Application
20230356231
Microfluidic devices and methods have revolutionized high-throughput and high-fidelity bioassays. Precise control of temperature within a microfluidic device has been a challenge in the field. Accordingly, there is a need for improved devices and methods that provide precise temperature control of microfluidic devices.
The invention provides methods for rapidly setting, adjusting, and controlling the temperature of a microfluidic device (e.g., to improve assay quality and reproducibility).
In one aspect, the invention provides a method for controlling temperature of a microfluidic device by providing an instrument for performing an assay at a first temperature, the instrument having a housing and a tray for inserting the microfluidic device into the housing and removing the microfluidic device from the housing, the tray having a thermal energy source, and the instrument having a first temperature sensor to determine ambient temperature and a second temperature sensor to determine a temperature of the tray and/or microfluidic device and placing the microfluidic device in the tray and inserting the microfluidic device into the housing. The instrument determines a difference between the ambient temperature and the first temperature and energizes the thermal energy source to heat or cool the microfluidic device by overshooting the first temperature and then reducing the overshoot until the microfluidic device reaches the first temperature or by preheating or precooling the tray relative to ambient temperature until the microfluidic device reaches the first temperature, e.g., as determined from the second temperature sensor.
In some embodiments, the thermal energy source includes a thermoelectric cooler (TEC), e.g., having a heatsink.
In some embodiments, the first and second temperature sensors include independently located thermistors. In some embodiments, the instrument includes a third temperature sensor (e.g., an infrared sensor) for the microfluidic device, e.g., when the second temperature sensor is in the tray.
In some embodiments the thermal energy source heats or cools the microfluidic device by overshooting the first temperature and then reducing the overshoot until the microfluidic device reaches the first temperature. In some embodiments, the overshoot initiates with insertion of the tray into the instrument. In some embodiments, the thermal energy source preheats or precools the tray prior to insertion into the housing. In some embodiments, the microfluidic device is placed in a thermally conductive holder, which is placed in the tray.
In some embodiments, the ambient temperature is between 15° C. and 30° C. In some embodiments, the first temperature is between 20° C. and 25° C. In some embodiments, the temperature of the microfluidic device reaches a temperature within 1° C. of the first temperature in less than 60 seconds, e.g., less than 30 seconds or less than 15 seconds. In some embodiments, the temperature of the microfluidic device reaches a temperature within 0.5° C. of the first temperature in less than 60 seconds, e.g., less than 30 seconds or less than 15 seconds.
In another aspect, the invention provides a device having a housing and a tray for inserting a microfluidic device into the housing and removing the microfluidic device from the housing and a thermal energy source, a first temperature sensor to determine ambient temperature and a second temperature sensor to determine a temperature of the tray and/or microfluidic device. The device is programmed to determine a difference between the ambient temperature and a first temperature and energizes the thermal energy source to heat or cool the microfluidic device by overshooting the first temperature and then reducing the overshoot until the microfluidic device reaches the first temperature, e.g., as determined from the second temperature sensor, or by preheating or precooling the tray relative to ambient until the microfluidic device reaches the first temperature, e.g., as determined from the second temperature sensor.
In some embodiments, the thermal energy source includes a thermoelectric cooler (TEC), e.g., having a heatsink. In some embodiments, the first and second temperature sensors include independently located thermistors. In some embodiments, the device includes a third temperature sensor (e.g., an infrared sensor) for the microfluidic device, e.g., when the second temperature sensor is in the tray. In some embodiments, the third temperature sensor includes an infrared sensor. In some embodiments, the thermal energy source heats or cools the microfluidic device by overshooting the first temperature and then reducing the overshoot until the microfluidic device reaches the first temperature. In some embodiments, the overshoot initiates with insertion of the tray into the instrument. In some embodiments, the thermal energy source preheats or precools the tray prior to insertion into the housing.
In some embodiments, the microfluidic device is placed in a thermally conductive holder, which is placed in the tray. In some embodiments, ambient temperature is between 15° C. and 30° C. In some embodiments, the first temperature is between 20° C. and 25° C. In some embodiments, the temperature of the microfluidic device reaches a temperature within 1° C. of the first temperature in less than 60 seconds, e.g., less than 30 seconds or less than 15 seconds. In some embodiments, the temperature of the microfluidic device reaches a temperature within 0.5° C. of the first temperature in less than 60 seconds, e.g., less than 30 seconds or less than 15 seconds.
In one aspect, the invention provides a method for controlling temperature of a microfluidic device by providing an instrument for performing an assay at a first temperature, the instrument having a housing and a tray for inserting the microfluidic device into the housing and removing the microfluidic device from the housing, the tray having a thermal energy source, and the instrument having a first temperature sensor to determine ambient temperature and a coupling for a second temperature sensor in the microfluidic device and placing the microfluidic device in the tray and inserting the microfluidic device into the housing. The instrument determines a difference between the ambient temperature and the first temperature and energizes the thermal energy source to heat or cool the microfluidic device by overshooting the first temperature and then reducing the overshoot until the microfluidic device reaches the first temperature, e.g., as determined from the second temperature sensor, or by preheating or precooling the tray relative to ambient temperature until the microfluidic device reaches the first temperature, e.g., as determined from the second temperature sensor.
In some embodiments, the thermal energy source includes a thermoelectric cooler (TEC), e.g., having a heatsink.
In some embodiments, the first and second temperature sensors include thermistors.
In some embodiments the thermal energy source heats or cools the microfluidic device by overshooting the first temperature and then reducing the overshoot until the microfluidic device reaches the first temperature. In some embodiments, the overshoot initiates with insertion of the tray into the instrument. In some embodiments, the thermal energy source preheats or precools the tray prior to insertion into the housing. In some embodiments, the microfluidic device is placed in a thermally conductive holder, which is placed in the tray.
In some embodiments, the ambient temperature is between 15° C. and 30° C. In some embodiments, the first temperature is between 20° C. and 25° C. In some embodiments, the temperature of the microfluidic device reaches a temperature within 1° C. of the first temperature in less than 60 seconds, e.g., less than 30 seconds or less than 15 seconds. In some embodiments, the temperature of the microfluidic device reaches a temperature within 0.5° C. of the first temperature in less than 60 seconds, e.g., less than 30 seconds or less than 15 seconds.
In another aspect, the invention provides a device having a housing and a tray for inserting a microfluidic device into the housing and removing the microfluidic device from the housing and a thermal energy source, a first temperature sensor to determine ambient temperature and a coupling for a second temperature sensor in the microfluidic device. The device is programmed to determine a difference between the ambient temperature and a first temperature and energizes the thermal energy source to heat or cool the microfluidic device by overshooting the first temperature and then reducing the overshoot until the microfluidic device reaches the first temperature, e.g., as determined from the second temperature sensor, or by preheating or precooling the tray relative to ambient until the microfluidic device reaches the first temperature, e.g., as determined from the second temperature sensor.
In some embodiments, the thermal energy source includes a thermoelectric cooler (TEC), e.g., having a heatsink. In some embodiments, the first and second temperature sensors include thermistors. In some embodiments, the third temperature sensor includes an infrared sensor. In some embodiments, the thermal energy source heats or cools the microfluidic device by overshooting the first temperature and then reducing the overshoot until the microfluidic device reaches the first temperature. In some embodiments, the overshoot initiates with insertion of the tray into the instrument. In some embodiments, the thermal energy source preheats or precools the tray prior to insertion into the housing.
In some embodiments, the microfluidic device is placed in a thermally conductive holder, which is placed in the tray. In some embodiments, ambient temperature is between 15° C. and 30° C. In some embodiments, the first temperature is between 20° C. and 25° C. In some embodiments, the temperature of the microfluidic device reaches a temperature within 1° C. of the first temperature in less than 60 seconds, e.g., less than 30 seconds or less than 15 seconds. In some embodiments, the temperature of the microfluidic device reaches a temperature within 0.5° C. of the first temperature in less than 60 seconds, e.g., less than 30 seconds or less than 15 seconds.
Where values are described as ranges, it will be understood that such disclosure includes the disclosure of all possible sub-ranges within such ranges, as well as specific numerical values that fall within such ranges irrespective of whether a specific numerical value or specific sub-range is expressly stated.
As used herein, the term “about” refers to a value that is within 10% above or below the value being described.
As used herein, the term “overshooting” refers to setting the set point of a thermal energy device to reach an equilibrium temperature than is higher (when heating) or lower (when cooling) than the desired assay temperature.
As used herein, the term “reaches a temperature” refers to the state of at least a portion of an object measured as being at that temperature±at most 1° C., e.g., at most 0.5° C. or at most 0.1° C., including normal fluctuations. The term does not require that the entirety of an object be at the same temperature. The temperature of the object may also be measured directly, e.g., with an embedded sensor, a sensor in contact with the device, or a remote sensor, e.g., an IR sensor. The temperature of the object may also be inferred from the temperature of another component in thermal contact with the device, e.g., a secondary holder for a microfluidic device.
In general, the invention features methods and devices for control of temperature of a microfluidic device. The invention allows for reaching an assay temperature more rapidly, thereby shortening total assay time. Temperature adjustment from ambient temperature to a first temperature may be automated by appropriate software and/or hardware.
Instrument and Components
Assay Instrument
The assay instrument of the invention can be any instrument for performing biological, biochemical, chemical, physical, or biophysical assays (e.g., sequencing of genetic information, e.g., DNA sequencing, RNA sequencing, protein sequencing, peptide sequencing, amplifying genetic information (e.g., DNA amplification, RNA amplification, e.g., polymerase chain reaction), biomarker detection, droplet generation, and single-cell assays (e.g., RNA-seq, encapsulation of single cells in droplets). The instrument includes a housing and a tray and brings at least one microfluidic device (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 microfluidic devices) to a desired first temperature prior to performing an assay (
The housing generally provides an environment for at least one microfluidic device to be maintained at a desired first temperature. The housing need not be airtight. The housing also may enclose various components for carrying out one or more assays including controllers, reagent reservoirs, pumps, gas or liquid manifolds, detectors, etc. The housing may be of any suitable shape and constructed of any suitable material, e.g., a metal, a plastic, a glass, or a ceramic material. The instrument also includes at least one tray (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 trays). The tray transports a microfluidic device into and out of the housing. Transport may occur manually, e.g., by a spring and catch mechanism), or in an automated fashion, e.g., controlled by a controller in the housing. In some embodiments, the microfluidic device may be placed in a secondary holder which is placed in the tray. In some embodiments, the tray is made of a thermally conductive material (e.g., a metal or metalized material). Multiple trays may work in coordination or independently and transport at least one microfluidic device each into and out from the instrument. The housing will also typically enclose other components necessary to carry out an assay, e.g., pneumatic manifold or other pumps, computer or other controller for directing the operation of the instrument, fans, and any other necessary component for a particular assay.
The instrument of the invention includes a thermal energy source that can heat or cool the microfluidic device. In some embodiments, the thermal energy source is a thermoelectric cooler (TEC). Other thermal energy sources are known in the art, e.g., resistive heaters, circulating fluid baths, etc. The thermal energy source can also include a heatsink on at least one of its sides. In some embodiments, the thermal energy source heats or cools the microfluidic device, e.g., disposed in a holder, to an assay temperature (within ±1° C., e.g., within ±0.5° C. or within ±0.1° C.) in less than 90 seconds, e.g., less than 60, less than 30 seconds or less than 15 seconds, such as between 5 and 90 seconds, e.g., between 10 and 60 seconds, or about 60, about 30, or about 15 seconds. In some embodiments, the thermal energy source has a temperature resolution of 0.1° C.
The thermal energy source may be configured to respond to a command from a controller to either heat or cool the tray. In some embodiments the thermal energy source can transition from heating mode to cooling mode repeatedly and in response to a series of commands from the controller. In some embodiments, the thermal energy source can cool or heat at varying rates whereby the trajectory of heating or cooling can change over time. In some embodiments, the thermal energy source responds to a computer program to control the rate of heating or cooling over time, also called the temperature profile.
The instrument of the invention may include more than one thermal energy source (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 thermal energy sources). Multiple thermal energy sources may be employed with a single microfluidic device, or a single thermal energy source may be used for two or more microfluidic devices. Thermal energy sources may be positioned to heat or cool the microfluidic device directly, e.g., in contact with the microfluidic device, or indirectly, e.g., via a conductive medium, such as air or metal, e.g., in a holder. Conductive greases, liquids, or pastes may also be employed. Thermal energy sources may be present in the tray, the housing, or both.
The instrument of the invention includes a plurality of temperature sensors, (e.g., thermistors or infrared sensors). In some embodiments the temperatures sensors can measure and report the temperature measurements at a frequency between about 1 Hz and 100 Hz (e.g., between about 1 and about 10 Hz, between about 10 and about 100 Hz, e.g., about 10 Hz, about 11 Hz, about 12 Hz, about 13 Hz, about 14 Hz, about 15 Hz, about 16 Hz, about 17 Hz, about 18 Hz, about 19 Hz, about 20 Hz, about 21 Hz, about 22 Hz, about 23 Hz, about 24 Hz, about 25 Hz, about 26 Hz, about 27 Hz, about 28 Hz, about 29 Hz, about 30 Hz, about 31 Hz, about 32 Hz, about 33 Hz, about 34 Hz, about 35 Hz, about 36 Hz, about 37 Hz, about 38 Hz, about 39 Hz, about 40 Hz, about 41 Hz, about 42 Hz, about 43 Hz, about 44 Hz, about 45 Hz, about 46 Hz, about 47 Hz, about 48 Hz, about 49 Hz, about 50 Hz, about 51 Hz, about 52 Hz, about 53 Hz, about 54 Hz, about 55 Hz, about 56 Hz, about 57 Hz, about 58 Hz, about 59 Hz, about 60 Hz, about 61 Hz, about 62 Hz, about 63 Hz, about 64 Hz, about 65 Hz, about 66 Hz, about 67 Hz, about 68 Hz, about 69 Hz, about 70 Hz, about 71 Hz, about 72 Hz, about 73 Hz, about 74 Hz, about 75 Hz, about 76 Hz, about 77 Hz, about 78 Hz, about 79 Hz, about 80 Hz, about 81 Hz, about 82 Hz, about 83 Hz, about 84 Hz, about 85 Hz, about 86 Hz, about 87 Hz, about 88 Hz, about 89 Hz, about 90 Hz, about 91 Hz, about 92 Hz, about 93 Hz, about 94 Hz, about 95 Hz, about 96 Hz, about 97 Hz, about 98 Hz, about 99 Hz, or about 100 Hz). In some embodiments, the instrument includes at least one temperature sensor that measures an ambient temperature. In some embodiments, the instrument includes at least one temperature sensor that measures the temperature of the microfluidic device or the tray. The instrument may include temperature sensors for both the tray and the microfluidic device (e.g.,
The instrument may also operatively couple, e.g., by wired or wireless connection, with temperature sensors in or on the microfluidic device, e.g., adjacent the channels or on the outside of the device (e.g.,
The instrument and methods of the invention are configured to control the temperature of at least one microfluidic device. The microfluidic device may be configured and used for various applications, such as, for example, to generate droplets, to evaluate and/or quantify the presence of a biological particle or organism (e.g., microbiome analysis, environmental testing, food safety testing, epidemiological analysis), to process a single analyte (e.g., bioanalytes, e.g., RNA, DNA, or protein) or multiple analytes (e.g., bioanalytes, e.g., DNA and RNA, DNA and protein, RNA and protein, or RNA, DNA and protein) from a single cell or from multiple cells, to process, for example, proteomic, transcriptomic, and/or genomic analysis of a cell or population of cells (e.g., simultaneous proteomic, transcriptomic, and/or genomic analysis of a cell or population of cells), or to modify analytes. Exemplary microfluidic devices for producing droplets are described in WO 2019/040637, WO 2020/123657, WO 2020/176882, and WO 2015/157567, the microfluidic devices of which are incorporated by reference. Holders for such devices are known in the art, e.g., as described in U.S. Pat. No. 9,975,122, the holders of which are incorporated by reference herein.
The invention provides control of the temperature of a microfluidic device. In general, the instrument determines an ambient temperature, e.g., via a first temperature sensor, and a tray (
In some embodiments, the change in temperature employs overshooting. In these methods, the instrument sets the set point of the thermal energy source to an equilibrium point higher or lower than the assay temperature, e.g., at least about 5° C., such as about 5-20° C., e.g., about 8-15° C., e.g., about 8° C. or about 10° C. As the microfluidic device approaches the assay temperature, the instrument changes the set point of the thermal energy source to allow the microfluidic device to reach the first temperature. The microfluidic device may pass the assay temperature during the overshoot prior to reaching the equilibrium assay temperature. The overshooting may begin before the tray is opened, as the tray is opened, once a microfluidic device is inserted, or once the tray is being closed or closed. After the overshooting, the temperature of the tray may be held at the assay temperature or at a temperature (e.g., ±about 1-6° C.) above or below to offset heat loss or gain. The overshoot may last for a specified period of time, e.g., between 5 and 90 seconds, e.g., between 15 and 60 seconds, between 20 and 45 second, or about 15, about 30, about 45, or about 60 seconds.
In some embodiments, overshooting can bring at least one microfluidic device to a first temperature in less than about 90 seconds (e.g., less than 60 seconds, less than 30 seconds, or less than 15 seconds, or between 10 and 90 seconds, 15 and 60 seconds, 10 and 45 seconds, or 45 and 90 seconds, e.g., about 15 seconds, about 30 seconds, about 45 seconds, or about 60 seconds).
In some embodiments, the methods of the invention employ preheating or precooling of the tray or microfluidic device. Preheating or precooling may be performed prior to insertion of the microfluidic device into the housing. In some embodiments, the instrument preheats or precools and maintains the tray at a baseline temperature. In some embodiments, the instrument heats or cools the tray while the tray is open to bring the microfluidic device to temperature quicker. The preheating or precooling may begin before the tray is opened, as the tray is opened, once a microfluidic device is inserted, or once the tray is being closed. The temperature of the tray may be held at the assay temperature or at a temperature (e.g., ±about 1-6° C.) above or below to offset heat loss or gain.
In some embodiments, preheating or precooling can bring at least one microfluidic device to a first temperature in less than about 90 seconds (e.g., less than 60 seconds, less than 30 seconds, or less than 15 seconds, or between 10 and 90 seconds, 15 and 60 seconds, 10 and 45 seconds, or 45 and 90 seconds, e.g., about 15 seconds, about 30 seconds, about 45 seconds, or about 60 seconds).
In some embodiments, the instrument is capable of running various assays at different temperatures, e.g., where the assay temperature is determined by reading instructions or indicia on the microfluidic device. In these embodiments, the tray may be heated or cooled to a starting temperature from which the instrument will heat or cool to reach the final assay temperature that depends on the assay being performed. In such embodiments, overshooting may be employed once the assay is determined.
Combinations of preheating/precooling and overshooting are also contemplated, where the preheating or precooling is to a starting temperature and the overshooting allows the device to reach the assay temperature faster.
In some embodiments, the temperature of a tray or microfluidic device is controlled based on a computer program that determines a heating or cooling profile based on inputs from one or more temperature sensors, e.g., a sensor for ambient or a sensor for the tray. The profile may be a linear ramp up (e.g., heating) or ramp down (e.g., cooling) until the device temperature reaches the assay temperature. Other profiles, e.g., exponential or stepped, may be employed. The profile may be calculated based on inputs, or the profile may be stored in a look up table, where the ramp and power requirements are determined based on the difference between the ambient temperature or starting tray temperature and the assay temperature. Once the assay temperature is reached, the thermal energy source may be held at a temperature (e.g., ±about 1-6° C.) to offset heat loss or heat gain to maintain the microfluidic device at the assay temperature.
The following examples are put forth so as to provide those of ordinary skill in the art with a description of how the compositions and methods described herein may be used, made, and evaluated, and are intended to be purely exemplary of the invention and are not intended to limit the scope of what the inventors regard as their invention.
In this example, the microfluidic device, the thermal energy source, and ambient temperature were below the desired setpoint temperature (27° C.).
In this example, the microfluidic device, the thermal energy source, and ambient temperature were above the desired setpoint temperature (14° C.).
In this example, the microfluidic device and ambient temperature were below the desired setpoint temperature (26° C.). The thermal energy source was preheated and maintained at an offset temperature (27° C.).
In this example, the microfluidic device and ambient temperature are above the desired setpoint temperature (16° C.). The thermal energy source is precooled and maintained at an offset temperature (14° C.).
Thermistor 1 was adjacent the microfluidic channels, and thermistor 2 was on the side of the microfluidic device. Thermistor 2 illustrated the temperature gradient across the microfluidic device, which can be used to determine an offset for the temperature of the tray versus the microfluidic device. The output of a thermistor placed on the thermal energy source was also monitored. The steep downward slope of their initial response to the tray temperature shows the effect of the overshoot temperature.
While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the invention that come within known or customary practice within the art to which the invention pertains and may be applied to the essential features hereinbefore set forth, and follows in the scope of the claims. Other embodiments are within the claims.
| Number | Date | Country | |
|---|---|---|---|
| 63139000 | Jan 2021 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/US2022/012918 | Jan 2022 | US |
| Child | 18223903 | US |
