Patent ID: 12220652

DETAILED DESCRIPTION

The disclosed technology relates to a microfluidic cooling device for liquid-liquid phase separation. A sample including a first liquid, a second liquid, and a plurality of soluble particles, can be inserted into a microfluidic cooling device. The sample can be inserted into the microfluidic cooling device at a first temperature such that the first liquid and second liquid are substantially miscible and the soluble particles are homogenously distributed throughout. At least a portion of the microfluidic pathway can be in thermal communication with a thermoelectric cooling element of the microfluidic cooling device. Upon initiation of thermoelectric cooling, the thermoelectric cooling element can transition the sample from the first temperature to a second temperature. At the second temperature, the sample can separate into a first phase including a majority of the first liquid and a portion of the soluble particles that is more soluble in the first liquid than the second liquid, and a second phase including a majority of the second liquid and a portion of the soluble particles that is more soluble in the second liquid than the first liquid.

The disclosed technology will be described more fully hereinafter with reference to the accompanying drawings. This disclosed technology can, however, be embodied in many different forms and should not be construed as limited to the examples set forth herein. The components described hereinafter as making up various elements of the disclosed technology are intended to be illustrative and not restrictive. Many suitable components that would perform the same or similar functions as components described herein are intended to be embraced within the scope of the disclosed electronic devices and methods. Such other components not described herein may include, but are not limited to, for example, components developed after development of the disclosed technology.

In the following description, numerous specific details are set forth. But it is to be understood that examples of the disclosed technology can be practiced without these specific details. In other instances, well-known methods, structures, and techniques have not been shown in detail in order not to obscure an understanding of this description. References to “one embodiment,” “an embodiment,” “example embodiment,” “some embodiments,” “certain embodiments,” “various embodiments,” etc., indicate that the embodiment(s) of the disclosed technology so described may include a particular feature, structure, or characteristic, but not every embodiment necessarily includes the particular feature, structure, or characteristic. Further, repeated use of the phrase “in one embodiment” does not necessarily refer to the same embodiment, although it may.

Throughout the specification and the claims, the following terms take at least the meanings explicitly associated herein, unless the context clearly dictates otherwise. The term “or” is intended to mean an inclusive “or.” Further, the terms “a,” “an,” and “the” are intended to mean one or more unless specified otherwise or clear from the context to be directed to a singular form.

Unless otherwise specified, the use of the ordinal adjectives “first,” “second,” “third,” etc., to describe a common object, merely indicate that different instances of like objects are being referred to, and are not intended to imply that the objects so described should be in a given sequence, either temporally, spatially, in ranking, or in any other manner.

Unless otherwise specified, the use of the term “hydrophilic” refers to any particle, plurality of particles, analyte, plurality of analytes, or any chemical constituent that is entirely hydrophilic, substantially hydrophilic, or has a polarity tendency that is more hydrophilic than hydrophobic.

Unless otherwise specified, the use of the term “hydrophobic” refers to any particle, plurality of particles, analyte, plurality of analytes, or any chemical constituent that is entirely hydrophobic, substantially hydrophobic, or has a polarity tendency that is more hydrophobic than hydrophilic.

FIG.1illustrates a front view of an example microfluidic cooling device100. The microfluidic cooling device100can include a thermoelectric cooling element104disposed proximate a heat sink108. The thermoelectric cooling element104can be many different cooling elements (or coolers) known in the art, including, but not limited to, a Peltier cooler. The microfluidic cooling device100can include any number of thermoelectric cooling elements104. As illustrated inFIG.1, the microfluidic cooling device100can include two thermoelectric cooling elements104. Thermoelectric cooling can be initiated when electrical power is supplied the thermoelectric cooling element104via any standard power supply. During thermoelectric cooling via the thermoelectric cooling element104, one face of the thermoelectric cooling element104can become cooled, while an opposite face can become heated. The heated face of the thermoelectric cooling element104can be positioned proximate the heat sink108such that the heat sink108can absorb heat being transferred from the cooled face to the heated face. A fan102can be positioned proximate the heat sink104to facilitate thermoelectric cooling.

The microfluidic cooling device100can further include a microfluidic pathway102configured to receive a sample200. As the sample200flows through the microfluidic pathway102, the sample200can undergo thermoelectric cooling. During thermoelectric cooling, the sample200can transition from an input temperature to a separation temperature. A microfluidic pathway102can be positioned proximate the thermoelectric cooling element104. The microfluidic pathway102can be positioned proximate the cooled face of the thermoelectric cooling element104such that the sample200flowing through the microfluidic pathway102can be cooled. In some embodiments, only a portion of the microfluidic pathway102can be in thermal communication with the thermoelectric cooling element104. Alternatively, in some embodiments, an entire length of the microfluidic pathway102can be in thermal communication with the thermoelectric cooling element104. In some embodiments, the microfluidic pathway102is positioned directly on the thermoelectric cooling element104. By way of example, the microfluidic pathway102can be positioned directly on the thermoelectric cooling element104using any attachment means, including an adhesive, or without an attachment means.

In some embodiments, a cover106can be placed proximate the microfluidic pathway102. The cover106can secure the microfluidic pathway102to the thermoelectric cooling element104. The cover106can be affixed to the microfluidic cooling device100via any attachment means, including but not limited to, a screw, bolt, adhesives, welding, or the like. In some embodiments, the cover106can be transparent to provide a visual indicator of the microfluidic pathway102and/or the sample200within the microfluidic pathway102during operation of the microfluidic cooling device100.

The microfluidic cooling device100can have an exterior housing made of insulating material. The exterior housing can optimize thermoelectric cooling of the sample200. In some embodiments, the microfluidic cooling device100can be portable, thereby facilitating use and transport to various locations.

FIG.2illustrates a schematic diagram of the sample200prior to thermoelectric cooling and after thermoelectric cooling. The sample200inserted into the microfluidic pathway102can include a first liquid202and a second liquid204. The first liquid202can be water, including deionized water. The second liquid204can be acetonitrile. The sample200can include a plurality of soluble particles, including hydrophobic particles206and hydrophilic particles208. The hydrophobic particles206and the hydrophilic particles208can be substantially dissolved within the sample200when the sample is inserted into the microfluidic pathway102at the input temperature.

The sample200can be inserted into the microfluidic cooling device100at the input temperature. The input temperature can be above a phase transition temperature of the sample200. In some embodiments, the input temperature can be based at least in part on the composition of the sample200. In some embodiments, the input temperature can be between approximately 20° C. and approximately 30° C. At the input temperature, the first liquid202and the second liquid204can be substantially miscible, and the hydrophobic particles206and the hydrophilic particles208can be homogenously distributed throughout the sample200.

In some embodiments, the sample200can include a plurality of analytes210. The plurality of analytes210can be metabolites, chemical constituents, or any component of interest in an analytical procedure. The plurality of analytes210can be of any molecular weight or chemical composition. By way of example, the plurality of analytes210can be amino acids, peptides, lipids, metabolites, proteins, or the like. The plurality of analytes210can be entirely or substantially hydrophilic. In some embodiments, the plurality of analytes210can be more hydrophilic than hydrophobic. Alternatively, in some embodiments, the plurality of analytes210can be entirely or substantially hydrophobic. In some embodiments, the plurality of analytes210can be more hydrophobic than hydrophilic. In some embodiments, the plurality of analytes210can include hydrophilic analytes and hydrophobic analytes. The plurality of analytes210can be dissolved within the sample200and homogenously distributed throughout the sample200when the sample200is inserted into the microfluidic cooling device100at the input temperature.

When the microfluidic cooling device100is operating, as the sample200flows through the microfluidic pathway102, the sample200can undergo thermoelectric cooling via the thermoelectric cooling element104and thereby transition from the input temperature to the separation temperature. Upon the thermoelectric cooling, the first liquid202and the second liquid204can become substantially immiscible, thereby separating from one another into a first phase212and a second phase214. The temperature at which the sample200can separate into the first phase211and the second phase214can be below the phase transition temperature of the sample200. The separation temperature can depend on the composition of the sample200. By way of example, when the sample200includes water as the first liquid202and acetonitrile as the second liquid204, and no additional solutes or analytes, separation into the first phase212and the second phase214can occur at a separation temperature of approximately −1.3° C. Alternatively, the sample200can separate into the first phase212and the second phase214at a separation temperature of less than −1.3° C. In some embodiments, the sample200can separate into the first phase212and the second phase212at a temperature of between approximately −10° C. and approximately −20° C. In some embodiments, the thermoelectric cooling element104can transition the sample200from the input temperature to the separation temperature within 60 seconds. In some embodiments, the thermoelectric cooling element104can transition the sample200from the input temperature to the separation temperature within between approximately 61 seconds and approximately 5 minutes. In some embodiments, the thermoelectric cooling element104can transition the sample200from the input temperature to the separation temperature within between approximately 5 minutes and approximately 30 minutes. The ability to transition the sample from the input temperature to the separation temperature rapidly, and thereby separating the sample200into the first phase212and the second phase214rapidly can provide increased throughput of sample analysis as compared to other liquid-liquid phase separation methods. Additionally, the rapid nature of the thermoelectric cooling via the thermoelectric cooling device100can allow for dynamic, sensitive, and/or reactive analytes210to be analyzed.

When the first liquid202is water and the second liquid204is acetonitrile, the first phase212can include a majority of the first liquid202and a majority of the hydrophilic particles208. In some embodiments, the first phase212can include all of the hydrophilic particles208. The second phase214can include a majority of the second liquid204and a majority of the hydrophobic particles206. In some embodiments, the second phase214can include all of the hydrophobic particles206. The ratio of the first liquid202to second liquid204in the first phase212and the second phase214can depend at least in part on the initial compositions and volumes of the first liquid202and the second liquid204. The ratio of the first liquid202and the second liquid204in the first phase212and the second phase214can also depend on the separation temperature. In some embodiments, when the sample200separates into the first phase212and the second phase214, the first phase212can include approximately all of the first liquid202and the second phase214can include approximately all of the second liquid204. Alternatively, and by way of a non-limiting example, the first phase can include approximately 75% of the first liquid202and 25% of the second liquid204, while the second phase can include approximately 75% of the second liquid204and approximately 25% of the first liquid202.

The plurality of analytes210can separate into either the first phase212or the second phase214depending on the polarity of the plurality of analytes210. By way of example, when the plurality of analytes210is more soluble in the first liquid202than the second liquid204, and thereby more hydrophilic or more hydrophilic than hydrophobic, the first phase212can include the plurality of analytes210, as illustrated inFIG.2. Alternatively, when the plurality of analytes210is more soluble in the second liquid204than the first liquid202, and thereby more hydrophobic than hydrophilic, the second phase214can include the plurality of analytes210. When the plurality of analytes210includes both hydrophilic analytes and hydrophobic analytes, the first phase212can include the portion of the plurality of analytes210that is more soluble in the first liquid202than the second liquid204, and thereby more hydrophilic than hydrophobic. The second phase214can include the portion of the plurality of analytes210that is more soluble in the second liquid204than the first liquid202, and thereby more hydrophobic than hydrophilic. Upon separation, the first phase212and the second phase214can be outputted from the microfluidic pathway102of the microfluidic cooling device100. The first phase212and the second phase214can be outputted from the microfluidic cooling device100at the separation temperature to ensure the first phase212and the second phase214remain separated.

FIGS.3A-3Fillustrate schematic diagrams of a top view of the microfluidic pathway102. The microfluidic pathway102can have one or more inlets302configured to receive the sample200, and one or more outlets304configured to output the first phase212and the second phase214upon phase separation. The sample200can be inserted into the inlet302of the microfluidic pathway102using any standard fluidic introduction method or device, including a syringe, a pipette, or the like. Upon separation, the first phase212and the second phase214can be outputted from the outlet304via any standard fluidic output method or device, including suction, a pipette, a syringe, or the like.

The microfluidic pathway102can include one or more flow channels configured to direct the sample200through the microfluidic cooling device100from the inlet302to the outlet304. In some embodiments, the flow channel or flow channels can be a tube or tubes extending from the inlet302to the outlet304. The tube can have an inner diameter of between approximately 10 microns and approximately 250 microns. In some embodiments, adhesives can be used to secure the tube to the thermoelectric cooling element104such that the tube and the thermoelectric cooling element104are in thermal communication. Alternatively, the cover106can be disposed proximate the tube such that the tube and the thermoelectric cooling element104can remain in thermal communication. In some embodiments, the microfluidic pathway102can include a self-contained microfabricated separation chamber, and the flow channels can be fabricated into the separation chamber using photolithography and standard etching procedures. By way of example, the flow channels can be etched from a silicon substrate or glass substrate. Similarly, in some embodiments, the flow channels can be fabricated from a patterned polydimethylsiloxane (PDMS) substrate bonded to glass. Fully integrated fluidics can provide seamless flow of the sample200between the microfluidic pathway102and the separation chamber. In some embodiments, the flow channels can be elongated recesses. The recesses can be cut from any material, including a polymeric material, a thermoplastic material, or the like. By way of example, the flow channels can be cut from a 1/16-inch rubber gasket material. The material including the etched flow channels can be affixed directly to the thermoelectric cooling element104such that the microfluidic pathway102can be in thermal communication with the thermoelectric cooling element104. The cover106can then be positioned to secure the material to the thermoelectric cooling element104. In some embodiments, the flow channels can be microfabricated into the cover106. The cover106including the flow channels can be affixed directly to the thermoelectric cooling element104such that the microfluidic pathway102is in thermal communication with the thermoelectric cooling element104.

The microfluidic pathway102can have a variety of configurations. As illustrated inFIG.3A, the microfluidic pathway102can include a single flow channel (e.g., a single tube or single elongated recess) configured to direct the sample from the inlet302to the outlet304. The single flow channel can be substantially linear. As illustrated inFIG.3B, the microfluidic pathway102can include a single flow channel that can diverge into two distinct flow channels. In this configuration, the sample200can be inserted into the microfluidic pathway102via the inlet302and can be outputted from the microfluidic pathway102via a first outlet304aand a second outlet304b. In some embodiments, the first phase212can exit the microfluidic pathway102via the first outlet304aand the second phase214can exit the microfluidic pathway102via the second outlet304b. As illustrated inFIG.3C, the microfluidic pathway102can include two flow channels that can converge into a single flow channel. In this configuration, the microfluidic pathway102can include a first inlet302aand a second inlet302band a single outlet304. As illustrated inFIG.3D, the microfluidic pathway102can have a winding, twisting, and/or serpentine configuration. The microfluidic pathway102can include any number of flow channels. As illustrated inFIG.3E, the microfluidic pathway102can include two separate and distinct linear flow channels with each flow channel including an inlet302a,302band an outlet304a,304b. As illustrated inFIG.3F, the microfluidic pathway102can include two separate and distinct winding, twisting, and/or serpentine flow channels with each flow channel including an inlet302a,302band an outlet304a,304b. When the microfluidic pathway102includes multiple flow channels, the sample200can be inserted via the inlet302into each flow channel, thereby providing a high throughput of separation of the sample200into the first phase212and the second phase214. In some embodiments, the configuration and/or dimensions of the microfluidic pathway102can be based at least in part on optimizing thermoelectric cooling and phase separation of the sample200.

AlthoughFIGS.3A through3Fillustrate various example microfluidic pathways102, it is contemplated the microfluidic pathway102can include any number of flow channels having any dimensions, any number of inlets302and outlets304, and any flow channel configuration.

In some embodiments, the inlet302and/or outlet304of the microfluidic flow path102can be configured to be integrated with an existing fluidic flow path. By way of example, the outlet304can be configured to direct the first phase212and/or the second phase214to mass spectrometry for further analysis. By integrating the microfluidic flow path102with an existing fluidic flow path, unintentional loss and/or dilution of the sample200can be minimized and analyte concentration for maximum detectability in downstream analysis can be preserved.

In some embodiments, the microfluidic cooling device100can be configured to receive any number of microfluidic pathways102. By way of example, a first microfluidic pathway102having a first configuration and a first set of dimensions can be positioned proximate the thermoelectric cooling element104. Upon thermoelectric cooling and phase separation of the sample200, the sample200can be outputted from the first microfluidic pathway102. The first microfluidic pathway102can then be removed from the microfluidic cooling device100. Subsequently, a second microfluidic pathway having a second configuration and a second set of dimensions can be positioned proximate the thermoelectric cooling element104such that the sample200can undergo thermoelectric cooling within the second microfluidic pathway. Accordingly, the first microfluidic pathway102can be easily interchanged with a second microfluidic pathway having a different configuration and/or different dimensions such that thermoelectric cooling can be tailored depending on the sample200composition, desired composition and volume of the first phase212and the second phase214, and/or separation temperature.

FIG.4is a diagram of a microfluidic cooling system400. The microfluidic cooling system400can include the microfluidic cooling device100, a controller402, and one or more temperature sensors406. The controller402can be in electrical communication with the microfluidic cooling device100and the one or more temperature sensors406. The controller402can be configured to send one or more signals to various components of the microfluidic cooling device100and receive one or more signals from various components of the microfluidic cooling device100in order to optimize thermoelectric cooling of the sample200.

The controller402can include a graphical user interface404for receiving user input for operative parameters of the thermoelectric cooling, thereby allowing a user to tailor the thermoelectric cooling via the microfluidic cooling device100based on known characteristics of the sample200prior to thermoelectric cooling and desired parameters of the first phase212and the second phase214upon separation. A user can input data pertaining to characteristics of the sample200in order to achieve optimal thermoelectric cooling and the resulting phase separation into the first phase212and the second phase214. By way of example, the user can input data indicative of the sample200, including but not limited to, chemical composition of the sample200, volume of the sample200, ratio of the first liquid202to the second liquid204, characteristics of the hydrophilic particles206and hydrophobic particles208, and characteristics of the plurality of analytes210. The user can input data indicative of a desired flow rate of the sample200through the microfluidic pathway102. The user can input data indicative of desired characteristics of the first phase212and the second phase214including compositions of the first phase212and/or the second phase214and volume fractions of the first phase212and/or the second phase214. Based at least in part on the data inputted and phase equilibrium models, the controller402can determine operative parameters of the thermoelectric cooling. By way of example, the controller402can determine the separation temperature required to achieve separation of the sample200into the first phase212and the second phase214. In this sense, unlike a traditional freezer cooling method to induce phase separation that can only provide a single cooling temperature, the microfluidic cooling device100can be configured to provide thermoelectric cooling at a first cooling temperature (e.g., output temperature) for a first sample and upon separation and removal of the first sample, can provide a second cooling temperature for a second sample. The controller402can determine an approximate length of time the sample200will need to be in thermal communication with the thermoelectric cooling element104in order to have phase separation into the first phase212and the second phase214. The controller402can further determine volume of a buffer solution, including acetonitrile buffer and/or aqueous buffer, that can be inserted into the microfluidic pathway102in order for the desired resulting compositions and volumes to be achieved. After determining operative parameters of thermoelectric cooling of the sample200, the controller402can output one or more signals to various components of the microfluidic cooling device100to implement such operative parameters. Unlike traditional liquid-liquid phase separation techniques that were often limited to a single cooling temperature and were unable to specifically tailor phase separation based on characteristics of the sample200or desired resulting phases212,214, the microfluidic cooling system400can provide rapid thermoelectric cooling that can be precisely controlled using phase equilibrium data and characteristics of the sample200.

The microfluidic cooling device100can include one or more temperature sensors406in electrical communication with the controller402. A temperature sensor406can be disposed on, near, or proximate the thermoelectric cooling element104to determine a temperature of the thermoelectric cooling element. A temperature sensor406can be disposed proximate the inlet302of the microfluidic pathway102to determine a temperature of the sample200prior to thermoelectric cooling and/or phase separation. A temperature sensor406can be disposed proximate the outlet304of the microfluidic pathway102to determine a temperature of the sample200after thermoelectric cooling and/or phase separation. The temperature sensors406can provide real-time thermal management and monitoring of the sample200during thermoelectric cooling. In some embodiments, the output temperature (e.g., cooling temperature) can be dynamically controlled during thermoelectric cooling by continuous electrical communication between the temperature sensor406and the controller402, such that a user can set the output temperature before thermoelectric cooling and/or vary the output temperature during thermoelectric cooling.

FIG.5Ais a schematic diagram of the microfluidic cooling device100configured to operate in a batch mode. In the batch mode operation, the sample200can be inserted into the microfluidic pathway102of the microfluidic cooling device100. The sample200can be maintained within the microfluidic pathway102for a predetermined amount of time during which thermoelectric cooling via the thermoelectric cooling element104can be initiated and the sample200can accordingly separate into the first phase212and the second phase214. The resulting first phase212and the second phase214can be outputted from the microfluidic pathway102.

When the microfluidic cooling device100is operating in a batch mode, the microfluidic pathway102can first be filled with a buffer solution, including an aqueous buffer and/or an acetonitrile buffer, via a first valve502prior to inserting the sample200into the microfluidic pathway102. The sample200can then be inserted into the microfluidic pathway102via the first valve502. The first valve502can be configured to insert a predetermined amount of the sample200into the microfluidic pathway102at a predetermined rate. In some embodiments, a predetermined amount of the first liquid202can be inserted into the microfluidic pathway102prior to a predetermined amount of the second liquid204. Alternatively, a predetermined amount of the second liquid204can be inserted into the microfluidic pathway102prior to a predetermined amount of the first liquid202. In some instances, the first liquid202and the second liquid204can be inserted into the microfluidic pathway simultaneously. As the sample200is inserted into the microfluidic pathway102, the buffer solution can be displaced and can exit the microfluidic pathway102via a second valve504. In some embodiments, the buffer solution that exits the microfluidic pathway102can be rinsed and re-inserted into the microfluidic pathway102until the desired composition and volume of the sample200within the microfluidic pathway102is achieved. The first valve502and the second valve504can then be closed, thereby isolating the sample200within the microfluidic pathway102. The sample200can remain within the microfluidic pathway102for a predetermined amount of time while thermoelectric cooling via the thermoelectric cooling element104is initiated. The thermoelectric cooling via the thermoelectric cooling element104can transition the sample200from the input temperature to the separation temperature. The temperature sensors406can monitor the temperature of the thermoelectric cooling element, and thereby the approximate temperature of the sample200, to provide feedback to the controller402. At the separation temperature, the sample200can separate into the first phase212and the second phase214. In some embodiments, the first phase212, including the first liquid (e.g., water)202and the plurality of hydrophilic particles208, can settle to the bottom of the microfluidic pathway, while the second phase214, including the second liquid (e.g., acetonitrile)204and the plurality of hydrophobic particles208, can be a distinct layer above the first phase212. Upon separation, the second valve504can be configured to remove the first phase212and the second phase214from the microfluidic pathway102. In some embodiments, the first phase212and the second phase214can be removed by applying suction at the outlet304of the microfluidic pathway102.

In some embodiments, after removal, the first phase212can be directed to a first fluid reservoir510via a third valve506, and/or the second phase214can be directed to a second fluid reservoir512via a fourth valve508. In some embodiments, the first phase212within the first fluid reservoir510and the second phase214within the second fluid reservoir512can be subjected to secondary thermoelectric cooling in order to freeze the first phase212and the second phase214before transport. In some embodiments, after removal, the first phase212and/or the second phase214can be directly infused into a downstream process flows via the third valve506and the fourth valve508, respectively. After the first phase212and the second phase214are removed from the microfluidic pathway102, the microfluidic pathway102can be rinsed with a buffer solution in order to prepare the microfluidic pathway102for receiving an additional sample or an additional volumetric amount of the same sample200. The buffer solution can be inserted into the microfluidic pathway102via the first valve502, and the second valve504, the third valve506, and the fourth valve508can be configured to receive the buffer solution such that the entire system can be rinsed.

FIG.5Bis a cross-section view of the microfluidic cooling device100ofFIG.5A. The microfluidic pathway102can be disposed proximate the thermoelectric cooling element104. In some embodiments, the microfluidic pathway102can be disposed directly on the thermoelectric cooling element104. The cover106can be positioned to secure the microfluidic pathway102to the thermoelectric cooling element104such that the microfluidic pathway102can be in thermal communication with the thermoelectric cooling element104. Although inFIG.5B, the microfluidic pathway102is in thermal communication with the thermoelectric cooling element104over the entire length of the microfluidic pathway102, it is contemplated that in some embodiments only a portion of the microfluidic pathway102can be in thermal communication with the thermoelectric cooling element104. When the thermoelectric cooling is initiated by the controller402and a voltage is applied to the thermoelectric cooling element104, a temperature gradient can be established. In particular, thermoelectric cooling via the thermoelectric cooling element104can create a cooled face514proximate the microfluidic pathway102and a heated surface516proximate the heat sink108. The fan110positioned proximate the heat sink108can facilitate establishing the temperature gradient. The sample200within the microfluidic pathway102can be in thermal communication with the cooled surface514of the thermoelectric cooling element104, thereby causing the sample200to transition from the input temperature to the separation temperature, and the sample200to subsequently separate into the first phase212and the second phase214.

FIG.6Aillustrates a schematic diagram of the microfluidic cooling device100configured to operate in a continuous mode. The microfluidic pathway102can be prefilled with a buffer solution. The sample200can be continuously inserted into the microfluidic pathway102via a first valve602and a second valve604disposed upstream of the microfluidic pathway102. The first valve602and the second valve604can be in electrical communication with the controller402such that a predetermined amount of the sample200at a predetermined flow rate can be inserted into the microfluidic pathway102. In some embodiments, the first valve602and the second valve604can be configured to automatically insert the second liquid204upon the first liquid202being inserted into the microfluidic pathway102. In some embodiments, the first valve602and the second valve604can be configured to automatically insert the first liquid202upon the second liquid204being inserted into the microfluidic pathway102. Alternatively, in some embodiments, the first valve602and the second valve604can be configured to simultaneously insert the first liquid202and the second liquid204. As the sample200flows through the microfluidic pathway102, the initial volume of buffer solution can be displaced and can exit the microfluidic cooling device100via a third valve606and a fourth valve608. The third valve606and the fourth valve608can be configured to direct the buffer solution to refuse lines.

In some embodiments, upon insertion into the microfluidic pathway102, the sample200can flow through a mixing portion610of the microfluidic pathway102. The mixing portion610can be a winding, twisting, and/or serpentine portion of the microfluidic pathway102. The mixing portion610can ensure the plurality of analytes210is homogenously distributed throughout the sample200. The mixing portion610can be disposed on or proximate an insulating base620such that the mixing portion610is not in thermal communication with the thermoelectric cooling element104. This configuration can ensure that the sample200does not prematurely transition to the separation temperature, and thereby separate into the first phase212and the second phase214. Upon flowing through the mixing portion610, the sample200can flow through a separation portion612of the microfluidic pathway102. The separation portion612can be in thermal communication with the thermoelectric cooling element104such that as the sample200flows through the separation portion612, the temperature of the sample200can gradually decrease from the input temperature to the separation temperature. When the sample200is approximately the separation temperature, the sample200can separate into the first phase212and the second phase214. As illustrated inFIG.6A, in the continuous operation mode, upon separation, the first phase212and the second phase214can be alternating liquid slugs separated by a consistent length. Accordingly, the first phase212and the second phase214can continuously exit the microfluidic pathway102in an alternating pattern via the third valve606and the fourth valve608, respectively. Once a predetermined volume of the sample200has been inserted into the microfluidic pathway102and subsequently separated and removed from the microfluidic pathway102, thermoelectric cooling can be stopped and each valve can be rinsed with buffer solution. The buffer solution can be inserted into the microfluidic pathway102via the second valve604. The third valve606and the fourth valve608can be configured to direct the buffer solution to refuse lines during rinsing.

Operating in continuous mode can provide various advantages, including the ability to provide inline introduction of the sample200and automated, precise flow control via the plurality of valves, enabling user free operation with maximum repeatability.

FIG.6Bis a cross-section view of the microfluidic cooling device100ofFIG.6A. The microfluidic pathway102can be disposed proximate the thermoelectric cooling element104and an insulating base620. The cover106can be positioned proximate the microfluidic pathway102to secure the microfluidic pathway102to the insulating base620and the thermoelectric cooling element104. The mixing portion610of the microfluidic pathway102can be disposed proximate the insulating base620, while the separation portion612of the microfluidic pathway102can be disposed proximate the thermoelectric cooling element104. In this configuration, the sample200can ensure homogenous distribution of analytes210prior to thermoelectric cooling. As discussed herein, thermoelectric cooling via the thermoelectric cooling element104can create the cooled face514proximate the separation portion610and a heated surface516proximate the heat sink108. The fan110positioned proximate the heat sink108can facilitate establishing the temperature gradient. The sample200within the separation portion610of the microfluidic pathway102can be in thermal communication with the cooled surface514of the thermoelectric cooling element104, thereby causing the sample200to transition from the input temperature to the separation temperature, and the sample200to subsequently separate into the first phase212and the second phase214.

AlthoughFIGS.6A and6Billustrate one example configuration of a microfluidic cooling device100configured to operate in a continuous mode, it is contemplated that any configuration that provides for continuous flow of the sample200through the microfluidic pathway102and thus continuous output of the first phase212and the second phase214can be applied.

FIG.7is a flow diagram outlining a method700of liquid-liquid phase separation via the microfluidic cooling device100. The method can include inserting702a sample200into a microfluidic cooling device100at a first temperature (i.e., the input temperature) via one or more inlets302. The sample can include the first liquid202, the second liquid, and the plurality of soluble particles. The first liquid and the second liquid can be substantially miscible at the input temperature.

In some embodiments, the first liquid202can be inserted into the microfluidic cooling device100before inserting the second liquid204. Alternatively, the second liquid204can be inserted into the microfluidic cooling device100before inserting the first liquid202. In some embodiments, the first liquid202and the second liquid204can be inserted into the microfluidic cooling device simultaneously. In some embodiments, the first liquid202and the second liquid204are mixed prior to inserting, thus, the pre-mixed solution of the first liquid202and the second liquid204can then be inserted into the microfluidic cooling device100.

The method700can further include cooling704the sample200to a second temperature (e.g., the separation temperature). The controller402can be in electrical communication with the one or more temperature sensors406in order to control the temperature of the thermoelectric cooling element104during thermoelectric cooling and provide precise and dynamic temperature regulation.

The method700can further include separating706, as a result of the cooling, the sample200into a first phase212and a second phase214. The first phase212can include a majority of the first liquid202and a portion of the soluble particles that are more soluble in the first liquid202than the second liquid204. The second phase214can include a majority of the second liquid204and at a portion of the soluble particles that are more soluble in the second liquid204. By way of example, the first phase212can include water as the first liquid202and the portion of the soluble particles that have a hydrophilic polarity, while the second phase214can include acetonitrile as the second liquid204and the portion of the soluble particles that have a hydrophobic polarity.

In some embodiments, the sample200can separate into the first phase212and the second phase214within approximately 5 seconds and approximately 60 seconds. In some embodiments, the sample200can separate into the first phase212and the second phase214within approximately 60 seconds an approximately 5 minutes. In some embodiments, the sample200can separate into the first phase212and the second phase214within between approximately 5 minutes and approximately 30 minutes.

The method700can further include analyzing the first phase212. Alternatively or in addition to, the method700can further include analyzing the second phase214. The first phase212and/or the second phase214can be analyzed by any known analysis technique, including but not limited to, mass spectrometry, chromatography, and the like.

The microfluidic cooling device100and the method700of liquid-liquid phase separation as discussed herein can be used in a variety of applications. By way of example, the microfluidic cooling device100can be used for desalination of aqueous samples for mass spectrometry analysis of hydrophobic and/or hydrophilic compounds. Additionally, the microfluidic cooling device100can be used for inline removal of acetonitrile as an intermediate step when performing high performance liquid chromatography and mass spectrometry for protein analysis. In this application, the first phase (e.g., the aqueous phase including proteins)212can be further analyzed, while the second phase (e.g. acetonitrile rich phase)214can be discarded. The microfluidic cooling device100can also be used for sample preparation with integrated cell lysis for intracellular metabolomics. In this application, a cell laden sample can be inserted into the microfluidic pathway102. The microfluidic cooling device100can be configured to operate in the batch mode. After rinsing the sample with aqueous buffer, the cells can be lysed. Acetonitrile can be inserted into the microfluidic pathway102until the desired final compositions of the first phase212and second phase214have been achieved. Thermoelectric cooling can be initiated, and the sample can separate into a first phase212and the second phase214. The second phase214, including a majority of the acetonitrile and hydrophobic compounds can be further analyzed.

Additionally, the microfluidic cooling device100can be used to facilitate control of chemical reactions. By way of example, the sample200can include one or more reactive species. The reactive species can react within the sample200for a predetermined time such that the reactive species reacts to a predetermined degree. Upon the reactive species reacting to the predetermined degree, thermoelectric cooling via the microfluidic cooling device100can be initiated, thereby separating the initial reactive agents and/or intermediate reactive products based on solubility. The thermoelectric cooling and subsequently separation can prevent further reaction of the reactive species. This application can be particularly advantageous as it can provide an initially high mass transfer because the sample200can be a single homogenous phase, and subsequently significantly reduce mass transfer through thermoelectric cooling and phase separation.

In another application, the microfluidic cooling device100can be used for preparation of the sample200for Nuclear Magnetic Resonance (NMR) and Raman spectroscopy. Lipids can disrupt identification of the plurality of analytes210, including metabolites and proteins, within the sample200during NMR spectroscopy. Accordingly, thermoelectric cooling via the microfluidic cooling device100can be used to isolate lipids within the second phase214(e.g., the acetonitrile rich phase) while a majority of the plurality of analytes210can remain in the first phase212(e.g., aqueous rich phase). The first phase212and the second phase214can subsequently be analyzed by NMR spectroscopy. Moreover, the presence of water can produce background fluorescence, thereby reducing and/or hiding signals from analytes during Raman spectroscopy. Accordingly, the second phase214can be analyzed by Raman spectroscopy in order to obtain better identification of the analytes210.

In an additional application, the portability of the microfluidic cooling device100can facilitate in situ preparation of the sample200. Accordingly, the microfluidic cooling system400can provide a rapid method for collection of the sample200, preparation of the sample200using thermoelectric cooling via the microfluidic cooling device100, and analysis of the resulting phases212,214at a single location. Similarly, in some embodiments, the sample200can be collected and prepared using the microfluidic cooling device100at a first location. The resulting phases212,214can then be preserved by freezing and transporting to a second location (e.g., a lab) for further analysis.

It is to be understood that the embodiments and claims disclosed herein are not limited in their application to the details of construction and arrangement of the components set forth in the description and illustrated in the drawings. Rather, the description and the drawings provide examples of the embodiments envisioned. The embodiments and claims disclosed herein are further capable of other embodiments and of being practiced and carried out in various ways. Also, it is to be understood that the phraseology and terminology employed herein are for the purposes of description and should not be regarded as limiting the claims.

Accordingly, those skilled in the art will appreciate that the conception upon which the application and claims are based may be readily utilized as a basis for the design of other structures, methods, and systems for carrying out the several purposes of the embodiments and claims presented in this application. It is important, therefore, that the claims be regarded as including such equivalent constructions.

Furthermore, the purpose of the foregoing Abstract is to enable the United States Patent and Trademark Office and the public generally, and especially including the practitioners in the art who are not familiar with patent and legal terms or phraseology, to determine quickly from a cursory inspection the nature and essence of the technical disclosure of the application. The Abstract is neither intended to define the claims of the application, nor is it intended to be limiting to the scope of the claims in any way. Instead, it is intended that the invention is defined by the claims appended hereto.