Patent Application
20220362778
Not applicable.
Not applicable.
The present invention is directed to a system for the manipulation of particles and biological materials, and forming droplets containing these particles.
Biomedical researchers have for some time perceived the need to work with small quantities of fluid samples, and to identify compounds uniquely within these small volumes. These attributes allow large numbers of experiments to be carried out in parallel, saving time and money on equipment and reagents, and reducing the need of patients to produce large volume samples.
Indeed, the analysis of small fragments of nucleic acids and proteins suspended in small quantities of buffer fluid is an essential element of molecular biology. The ability to detect, discriminate, and utilize genetic and proteomic information allows sensitive and specific diagnostics, as well as the development of treatments. In particular, there is a need to unambiguously identify small quantities of biological material and analytes.
Most genetic and proteomic analysis requires labeling for detection of the analytes of interest. Such labelling may be referred to as “barcoding”, suggesting that the label is unique and correlated to some feature or identity. For example, in sequencing applications, nucleotides added to a template strand during sequencing-by-synthesis typically are labeled, or are intended to generate a label, upon incorporation into the growing strand. The presence of the label allows detection of the incorporated nucleotide. Effective labeling techniques are desirable in order to improve diagnostic and therapeutic results.
At the same time, precision manipulation of streams of fluids with microfluidic devices is revolutionizing many fluid-based technologies. Networks of small channels are a flexible platform for the precision manipulation of small amounts of fluids. The utility of such microfluidic devices depends critically on enabling technologies such as the microfluidic pumps and valves, electrokinetic pumping, dielectrophoretic pump or electrowetting driven flow. The assembly of such modules into complete systems provides a convenient and robust way to construct microfluidic devices.
However, virtually all microfluidic devices are based on flows of streams of fluids; this sets a limit on the smallest volume of reagent that can effectively be used because of the contaminating effects of diffusion and surface adsorption. As the dimensions of small volumes shrink, diffusion becomes the dominant mechanism for mixing leading to dispersion of reactants. This is a large and growing area of biomedical technology, as indicated by a growing number of issued patents in the field.
U.S. Pat. No. 9,440,232 describes microfluidic structures and methods for manipulating fluids and reactions. The structures and methods involve positioning fluid samples, e.g., in the form of droplets, in a carrier fluid (e.g., an oil, which may be immiscible with the fluid sample) in predetermined regions in a microfluidic network. In some embodiments, positioning of the droplets can take place in the order in which they are introduced into the microfluidic network (e.g., sequentially) without significant physical contact between the droplets. Because of the little or no contact between the droplets, there may be little or no coalescence between the droplets. Accordingly, in some such embodiments, surfactants are not required in either the fluid sample or the carrier fluid to prevent coalescence of the droplets.
U.S. Pat. No. 9,410,151 provides microfluidic devices and methods that are useful for performing high-throughput screening assays and combinatorial chemistry. This patent provides for aqueous based emulsions containing uniquely labeled cells, enzymes, nucleic acids, etc., wherein the emulsions further comprise primers, labels, probes, and other reactants. An oil based carrier-fluid envelopes the emulsion library on a microfluidic device. Such that a continuous channel provides for flow of the immiscible fluids, to accomplish pooling, coalescing, mixing, Sorting, detection, etc., of the emulsion library.
U.S. Pat. No. 9,399,797 relates to droplet based digital PCR and methods for analyzing a target nucleic acid using the same. In certain embodiments, a method for determining the nucleic acid make-up of a sample is provided.
U.S. Pat. No. 9,150,852 describes barcode libraries and methods of making and using them including obtaining a plurality of nucleic acid constructs in which each construct comprises a unique N-mer and a functional N-mer and segregating the constructs into a fluid compartments such that each compartment contains one or more copies of a unique construct
None of these references uses a small, micromechanical valving structure to control the volume of fluid surrounding the barcoded item, and to select the particle enclosed in the droplet. Accordingly, the droplets cannot be made “on demand”, and cannot be made to enclose a particle which is the object of the study.
Accordingly, it was the object of the invention to provide a microfabricated system that can separate target particles from non-target material, also separate a labelled bead, and combine the two particles in a single droplet. In addition to the target particle and the bead, the droplet may comprise a first aqueous fluid, such as a saline or buffer fluid. The droplet may be dispensed into a stream of a second fluid, immiscible with the first fluid. Thus, the droplet may maintain its integrity as a single, discrete, well defined unit because the fluids are immiscible and the droplets do not touch or coalesce.
When the target particle is a biological material such as a cell, with antigens located on its outer surface, the target particle may become attached to the bead by conjugation of these antigens with antibodies disposed on the bead. The bead may further be labelled by an identifying fluorescent signature, which may be a plurality of fluorescent tags affixed to the bead. Accordingly, each target cell, now bound to an identifiable, labelled fluorescent bead, may be essentially barcoded for its own identification. This may allow a large number of experiments to be performed on a large population of such droplets, encased in the immiscible fluid, because the particles are all identifiable and distinguishable.
In some embodiments, a genetic sequencer may be coupled to the MEMS device, which may sequence the genetic material contained in the biological particle.
Accordingly, a microfabricated droplet dispensing structure is described, which may include a MEMS micromechanical fluidic valve, configured to open and close a microfluidic channel. The opening and closing of the valve may separate a target particle and/or a bead from a fluid sample stream, and direct these two particles into a single droplet. The droplet may then be encased in a sheath of an immiscible fluid and delivered to a downstream receptacle or exit.
The system may further comprise a fluid sample stream flowing in the microfluidic channel, wherein the fluid sample stream comprises target particles and non-target material, and an interrogation region in the microfluidic channel Within the interrogation region, the target particle may be identified among non-target material, and the microfabricated MEMS fluidic valve may separate the target particle from the non-target material in response to a signal from the interrogation region, and direct the target particle into the droplet.
The system may also make use of a bead attached to a plurality of fluorescent tags, wherein the fluorescent tags specify the identity of the bead with a fluorescent signal, and wherein the microfabricated MEMS fluidic valve is configured to separate the bead and direct the bead into the droplet, wherein the bead and a target particle, are located within the same droplet. A “bead” should be understood to refer generally to a particle, and may be organic or inert. The bead may have identifying structures adhered to it, as in a “barcode” as used herein. The bead may be magnetic, but is not necessarily so.
The system may be coupled to a downstream workflow, which further processes or analyses the contents of the droplet. An interrogation region may also be included in the system downstream of the droplet formation, which may allow computer control of the process and artificial intelligence techniques to be applied.
In some embodiments, a genetic sequencer may be coupled to the MEMS device and MEMS fluidic valve, which may sequence the genetic material contained in the biological particle. The sequencer may make use of next generation sequencing techniques, including cDNA libraries, and rolling circle amplification, as described in detail below.
Various exemplary details are described with reference to the following figures, wherein:
It should be understood that the drawings are not necessarily to scale, and that like numbers may refer to like features.
The following discussion presents a plurality of exemplary embodiments of the novel microfabricated droplet dispensing system. The following reference numbers are used in the accompanying figures to refer to the following:
The system includes a microfabricated droplet dispenser that dispenses the droplets into an immiscible fluid. The system may be applied to a fluid sample stream, which may include target particles as well as non-target material. The target particles may be biological in nature, such as biological cells like T-cells, tumor cells, stem cells, for example. The non-target material might be plasma, platelets, buffer solutions, or nutrients, for example. The immiscible fluid may be a hydrophobic or non polar material. Hydrophobic molecules are molecules that do not have a charge, in other words, they are often nonpolar. Hydrophobic materials often do not dissolve in water or in any solution that contains a largely aqueous (watery) or polar environment. Examples may include alkanes, oils, fats, greasy substances, waxes, and steroids. In some embodiments, the immiscible fluid may be a fluorinated oil. Perfluoropolyether fluids (PEPE Oils) are synthetic fully fluorinated materials used as lubricants and coatings in the most extreme & critical lubrication applications. Such materials may be of use in the application described here.
The microfabricated MEMS valve may be, for example, the device shown generally in
In the figures discussed below, similar reference numbers are intended to refer to similar structures, and the structures are illustrated at various levels of detail to give a clear view of the important features of this novel device. It should be understood that these drawings do not necessarily depict the structures to scale, and that directional designations such as “top,” “bottom,” “upper,” “lower,” “left” and “right” are arbitrary, as the device may be constructed and operated in any particular orientation. In particular, it should be understood that the designations “sort” and “waste” are interchangeable, as they only refer to different populations of particles, and which population is called the “target” or “sort” population is arbitrary.
A fluid sample stream may be introduced to the microfabricated fluidic valve 110 by a sample inlet channel 120. The sample stream may contain a mixture of particles, including at least one desired, target particle and a number of other undesired, nontarget materials. The particles may be suspended in a fluid, which is generally an aqueous fluid, such as saline. For the purposes of this discussion, this aqueous fluid may be the first fluid, and this first fluid may be immiscible in a second fluid, as described below.
The target particle may be a biological material such as a stem cell, a cancer cell, a zygote, a protein, a T-cell, a bacteria, a component of blood, a DNA fragment, for example, suspended in a buffer fluid such as saline. The fluid inlet channel 120 may be formed in the same fabrication plane as the valve 110, such that the flow of the fluid is substantially in that plane. The motion of the valve 110 may also be within this fabrication plane. The decision to sort/save or dispose/waste a given particle may be based on any number of distinguishing signals.
In one embodiment, the fluid sample stream may pass through an interrogation region 170, which may be a laser interrogation region, wherein an excitation laser excites fluorescent tag affixed to a target particle. The fluorescent tag may emit fluorescent radiation as a result of the excitation, and this radiation may be detected by a nearby detector, and thus a target particle or cell may be identified. Upon identification of the target particle or cell, the microfabricated MEMS valve may be actuated, as described below, and the flow directed from the nonsort (waste) channel 145 to the sort channel 122, as illustrated in
In some embodiments, the actuation may occur by energizing an external electromagnetic coil and core in the vicinity of the valve 110. The valve 110 may include an inlaid magnetically permeable material, which is drawn into areas of changing magnetic flux density, wherein the flux is generated by the external electromagnetic coil and core. In other embodiments, other actuation mechanisms may be used, including electrostatic and piezoelectric. Additional details as to the construction and operation of such a valve may be found in the incorporated '144 patent.
In one exemplary embodiment, the decision is based on a fluorescence signal emitted by the particle, based on a fluorescent tag affixed to the particle and excited by an illuminating laser. Accordingly, these fluorescent tags may be identifiers or a barcoding system. However, other sorts of distinguishing signals may be anticipated, including scattered light or side scattered light which may be based on the morphology of a particle, or any number of mechanical, chemical, electric or magnetic effects that can identify a particle as being either a target particle, and thus sorted or saved, or an nontarget particle and thus rejected or otherwise disposed of.
This system may also be used to sort the labelled or barcoded bead. Accordingly, the “target particle” may be either a cell and/or a labelled bead. A “bead” should be understood to refer generally to a particle, and may be organic or inert. The bead may have identifying structures adhered to it, as in a “barcode” as used herein. The bead may be magnetic, but is not necessarily so.
With the valve 110 in the position shown in
The output orifice 140 may be a hole formed in the fabrication substrate, or in a covering substrate that is bonded to the fabrication substrate. Further, the valve 110 may have a curved diverting surface 112 which can redirect the flow of the input stream into a sort output stream, as described next with respect to
In this position, the movable member or valve 110 is deflected upward into the position shown in
More generally, the micromechanical particle manipulation device shown in
It should be understood that although channel 122 is referred to as the “sort channel” and orifice 140 is referred to as the “waste orifice”, these terms can be interchanged such that the sort stream is directed into the waste orifice 140 and the waste stream is directed into channel 122, without any loss of generality. Similarly, the “inlet channel” 120 and “sort channel” 122 may be reversed. The terms used to designate the three channels are arbitrary, but the inlet stream may be diverted by the valve 110 into either of two separate directions, at least one of which does not lie in the same plane as the other two. The term “substantially” when used in reference to an angular direction, i.e. substantially tangent or substantially vertical, should be understood to mean within 15 degrees of the referenced direction. For example, “substantially orthogonal” to a line should be understood to mean from about 75 degrees to about 105 degrees from the line.
When the valve is in the open or sort position shown in
Various structures may be used in this region to promote the formation of the droplet. These structures may be, for example, rounded corners or sharp edges which may influence or manipulate the strength or shape of the meniscus forces, wetting angle or surface tension of the first fluid droplet. These structures may be generally referred to as a “nozzle” indicating the region where the droplet is formed. At this nozzle point where the droplet is formed, an additional manifold may deliver an immiscible second fluid to the aqueous droplet, suspending the aqueous droplet in the fluid and preserving its general contours and boundary layers.
As mentioned, the valve 110 may be used to sort both a target cell and a bead. Laser induced fluorescence may be the distinguishing feature for either or both particles. These particles may both be delivered into a single droplet. These particles may be suspended in, and surrounded by, an aqueous first fluid, such as saline. Accordingly, the droplet may comprise primarily this first fluid, as well as the chosen particle(s), a target cell and/or a bead. The bead may be “barcoded”, that is, it may carry identifying markers. The droplet may then be surrounded by an immiscible second fluid that is provided by a source of the second fluid. These features are described further below, with respect to a number of embodiments.
Accordingly, because of the flow in the microfabricated channels, droplets may be formed at the intersection with the immiscible fluid. These droplets may be encased in an immiscible second fluid, such as a lepidic fluid or oil 200, as shown in
The pace, quality and rate of droplet formation may be controlled primarily by the dynamics of the MEMS valve 110. That is, the quantity of fluid contained in the droplet, and thus the size of the droplet, may be a function of the amount of time that the MEMS valve 110 is in the open or sort position shown in
Accordingly, the length of the sort pulse can determine the size of the generated droplet. If the pulse is too short, the oil meniscus may remain intact and no water droplet is formed. If the sort pulse is sufficiently long, a droplet may be formed at the exit and released into the stream of the second immiscible fluid.
If a target cell 5 is sorted within this time frame, the target cell 5 may be enclosed in the aqueous droplet. If the target particle is not sorted within this time frame, an empty aqueous droplet, that is, a droplet without an enclosed particle 5, may be formed. The situation is shown in
As mentioned above, the MEMS valve 110 may be made on the fabrication surface of at least one semiconductor substrate. More generally, a multi-substrate stack may be used to fabricate the MEMS valve 110. As detailed in the '144 patent, the multilayer stack may include at least one semiconductor substrate, such as a silicon substrate, and a transparent glass substrate. The transparent substrate may be required to allow the excitation laser to be applied in the laser interrogation region 170.
The droplet 300 may be formed at the edge of the semiconductor substrate, or more particularly, at the edge of the multilayer stack. The droplet 300 may be formed at the exit of the sort channel 122 from this multilayer stack. In another embodiment, the droplet is not formed at the edge of the multilayer stack, but instead may be formed at the intersection of the sort flow and oil input, within the semiconductor substrate. At this location, a structure may be formed that promotes the formation of the droplet. This structure may include sharply rounded corners so as to manipulate surface tension forces, and the formation of meniscus and wetting angles. The structure designed to promote droplet formation may be referred to herein as a nozzle 150, and the term “nozzle” may refer generally to the location at which the droplet may be formed.
In the structure shown in
Sorting Strategy Using the Valve to Form a Droplet in Oil
The method for forming a droplet in oil may be as follows. A target cell is first detected in the laser interrogation region 170. A computer or controller may monitor the signals from the laser interrogation region. Upon detecting a target particle in the region, the computer or controller may send a signal to open the MEMS valve 110 by energizing the electromagnet. Magnetic interactions then move the MEMS valve as shown in
A bead is then sorted to accompany the sorted cell as a unique barcode. A second sort pulse is long enough to cause an instability in the oil-water interface and form a water droplet in oil containing the cell and the bead.
When the valve is stationary and no sorting occurs, as depicted in
However, as oil may continue to flow, the effluent may be directed into a waste receptacle, until a target particle is detected. It may also be the case that continued leakage of the fluid sample stream through the gaps around the MEMS valve 110, may eventually cause a water droplet to form. Because no target cell has been detected, and the MEMS valve 110 has not been opened, this aqueous droplet may be empty.
Accordingly,
In another embodiment, the MEMS valve 110 may sort both a target particle 5 (here, a target cell 320) and a bead 310, as shown in
When a bead 310 is in proximity to a target cell 320, and the antibodies of the bead 310 may become conjugated with the antigens of the cell, the bead, along with its identifying fluorescent tags, may become affixed to the cell 320. Thus, the bead 310 provides an identifying marker for the cell 320, or a “barcode” which identifies the cell. A computer or controller may associate this particular barcode with the particular cell. Accordingly, a large number of such droplets may be placed in a small volume of fluid, each containing a target cell and identifying barcode and all within a field of view of a single detector. This may allow a very large number of biological assays or polymerase chain reactions, to be undertaken in parallel, and under a single detection system.
Laser Assisted Droplet Formation
Any of a variety of pulsed or continuous wave lasers may be suitable for this application. For example, a pulsed CO2 laser may be directed onto the channel as shown in
In
The embodiment shown in
Alternatively, the first droplet may contain the bead 310, and the second aqueous droplet may contain the target cell 320. In either case, the application of heat onto the channel in the laser 400 may serve to heat the fluids and allow the two droplets to merge. Accordingly, at the output of the microfabricated droplet dispenser may emerge an aqueous droplet encased in oil wherein the droplet contains both a target cell 320 and a bead 310. The bead 310 may have a fluorescent barcode affixed to it, and the bead may be conjugated to the target cell 320.
In
The reagent input channel designed into the chip may assist in the adding of reagents (enzymes) in a precise, controllable way. The reagent input channel is connected to the sort channel With each target sort (particle and cell), the valve is opening the reagent input channel—the enzymes will be added to the particles and droplet will be generated. This architecture may result in the improvement the biochemical processes within the droplet, because the amount of reagent may be carefully defined and controlled, thus controlling the amount of reagent amount per droplet, while reducing reagent costs.
The use of microfabricated flow channels may allow other technical advantages. Among these are automated verification of the success of the sorting process to isolate the correct particles in the correct number. Artificial intelligence and control parameters may be applied to this process. The techniques are discussed more fully in U.S. patent application Ser. No. 17/500,986 (the '986 application), which is incorporated by reference in its entirety. These techniques may have as input, data collected from the interrogatin of the droplet down stream of the droplet formation, such as in interrogation region 270.
After evaluation of the contents of the droplet, a number of options are available. For example, if the sort got nothing, repeat the process (try again). If the first sort got more than one thing, abort/eject the droplet. If the first sort got more than one thing, abort/eject the droplet. If the second sort failed (no second particle), repeat the process (try again). If the second sort got more than one thing, abort/eject the droplet. If possible, take a picture of the ejected droplet to verify the contents/size. The software may also record the status of each droplet.
The sorting/droplet generation process may also include an evaluation of the size of the droplet, and the control of that size. Among the controllable parameters are pressure parameters for sheath, sample and oil, valve opening time. For example, if the droplet size is too small the valve opening time can be increased. Furthermore, a defined volume of reagent may also be implemented and optimized.
All the aforementioned methods and processes may be implemented using computer control, feedback algorithms and artificial intelligence, as described in detail the incorporated '986 patent application. The size of the generated droplets may be monitored and controlled by Instrument SW. Feedback loops using control parameters and monitoring stages may be used to improve the process.
In some embodiments, the sorting/droplet generation process integrated into one disposable. In some embodiments. The sorting/droplet generation process may be integrated into one “ready to use” disposable where all reagents and buffers are filled into the cartridge (means the reagents are directly stored into the cartridge and the user may only have to add sample into the cartridge).
In some embodiments, the sorting/droplet generation may be accomplished with 2 valve openings, one to sort the target particle and the second to sort the bead. In other embodiments, the sorting/droplet generations may be accomplished in three steps, the first sorting the target particle, the second to sort a second target particle and the third to sort a bead. Of course, these step may occur in any order. Each of the sorted particles may contain different biological information.
Sorting/droplet generation process may be processed and controlled via a field programmable gate array (FPGA). The FPGA is integrated into the Instrument, interacting with the software and providing the sorting commands to the MEMS device integrated into a disposable cartridge. Further, the sorting/droplet generation process may be designed to avoid double particle or cell sorting (e.g. two or more cells or beads adhered to one another). If the adhered particles may be monitored into the sort channels the sorting process. The process may include transferring such particles into the waste. Only single particles could be transferred into the sort channel.
In some embodiments, embodiments, the sorting/droplet generation may be followed by a cell lysis and transcription module, integrated into and at the end of the droplet generation process. Details of this embodiment are described further below. In some embodiments, the cell lysis may be started after the droplets have been generated. In other embodiments, the cell lysis may be performed in the disposable cartridge (that is, without taking the sample out of the cartridge). Thermal treatments may also follow at defined points in the process.
Geometry-Induced Flow Slowdown and Downstream Interrogation Area
The embodiment shown in
Alternatively, the first droplet may contain the bead 310, and the second aqueous droplet may contain the target cell 320. In either case, the sudden widening of the channel in the merging area 500 may serve to slow the flow down within the merging area, allowing the two droplets to merge. Accordingly, at the output of the microfabricated droplet dispenser may emerge an aqueous droplet 300 encased in oil 200 wherein the droplet 300 contains a target cell 320 and a bead 310. The bead 310 may have a fluorescent barcode affixed to it, and the bead may be conjugated to the target cell 320.
The system 1000 may also include a downstream interrogation area 800, This area may allow examination or probing of the contents of the droplet, in order to assess whether the droplet contains the intended particles. The interrogation area may be a laser fluorescent interrogation, or a microscopic or video inspection. Depending on the results of the assessment, the droplet may be directed to waste disposal or to the downstream work flows for further processing. The interrogation area 800 may be coupled to a controller 900 which may execute the algorithms and implement the feedback loops.
Accordingly, described here is a microfabricated droplet dispenser, comprising a microfluidic channel formed in a substrate and a fluid flowing in the microfluidic fluid channel; a microfabricated MEMS fluidic valve, configured to open and close the microfluidic channel, a droplet comprising a first fluid dispensed at an end of the microfluidic channel, wherein a dimension of the droplet is determined by a timing of opening and closing of the microfabricated microfluidic valve, and a source of a second fluid immiscible with the first fluid wherein the droplet is dispensed from the microfluidic channel into, and immersed in, the second immiscible fluid
The droplet dispenser may further comprise a fluid sample stream flowing in the microfluidic channel, wherein the fluid sample stream comprises target particles and non-target material, an interrogation region in the microfluidic channel, wherein a target particle is identified among non-target material; and wherein the microfabricated MEMS fluidic valve is configured to separate the target particle from the non-target material in response to a signal from the interrogation region, and direct the target particle into the droplet. It may also include a bead attached to a plurality of fluorescent tags, wherein the fluorescent tags specify the identity of the bead with a fluorescent signal, and wherein the microfabricated MEMS fluidic valve is configured to separate the bead and direct the bead into the droplet, wherein the bead and a target particle, are located within the same droplet. The bead may comprise a plurality of fluorescent tags, such that the bead has an identifying fluorescent signature. The bead may also have at least one antibody, that binds to an antigen on the target particle.
The microfabricated MEMS valve may move in a single plane when opening and closing, and wherein that plane is parallel to a surface of the substrate. The droplet may be dispensed at a nozzle structure formed in the microfluidic channel in the substrate. The source of immiscible fluid is disposed symmetrically about the nozzle. Surfactant may be added to the fluid stream.
The droplet dispenser may further comprise a laser focused on the microfluidic channel upstream of the nozzle, heating the droplet to assist in severing the droplet from the fluid in the microfluidic channel, or to heat the droplet to coalesce adjacent droplets in the microfluidic channel. The microfluidic channel may have a channel widened area, wherein the cross section of the channel increases and then decreases. The microchannel may intersect the source of immiscible fluid in a butt junction. The target particles are at least one of T-cells, stem cells, cancer cells, tumor cells, proteins and DNA strands.
A method for dispensing droplets is also described. The method may include method may include forming a microfluidic channel on a substrate, providing a fluid flowing in the microfluidic fluid channel, opening and closing a microfabricated MEMS fluidic valve. The method may further comprise opening and closing a microfabricated MEMS fluidic valve, to open and close the microfluidic channel, capturing at least one of a target particle and a bead with identifiers disposed thereon, providing a source of an immiscible second fluid, immiscible with the first fluid, and dispensing a droplet of the first, wherein a dimension of the droplet is determined by a timing of opening and closing of the microfabricated microfluidic valve, and wherein the droplet encloses at least one of the bead and the target particle.
The fluid flowing in the microfluidic channel may include target particles and non-target material. The method may further include identifying a target particle among non-target material in a laser interrogation region, opening and closing the microfabricated MEMS fluidic valve to separate the identified target particle from the non-target material in response to a signal from the interrogation region, and directing the target particle into the droplet.
The method may also include providing a bead attached to a plurality of fluorescent tags, wherein the fluorescent tags specify the identity of the bead with a fluorescent signal, separating the bead using the the microfabricated MEMS fluidic valve, and directing the bead into the droplet, wherein the bead and the target particle, are located within the same droplet.
The droplet may be formed at a nozzle structure formed in the substrate. The method may further include heating the fluid with a laser focused just upstream of the nozzle.
The droplets formed by the system, MEMS device 10, described above may be coupled to a genetic sequencer, simply referred to as sequencer 600, or other cellular or genetic manipulation, and thereby obtain detailed information relating to a singular, specific biological particle or cell. The MEMS device 10 may be uniquely suited to the sequencing application because the fluid transport of the droplet containing the biological particle is enclosed throughout, and the forces used to guide the droplet and particle are gentle. This allows improved sterility and viability of the biological material. Accordingly, the MEMS device 100 may be coupled to a genetic sequencing apparatus, thus delivering a well characterized genetic sample in a droplet contained in an immiscible fluid stream.
The system 1000 is shown in
The downstream workflows may be a genetic sequencer, as described in U.S. patent application Ser. No. 17/324,570 (the '570 application), which is incorporated by reference in its entirety. Exemplary workflows 600 are illustrated in the remaining figures, which should not be understood as exhaustive or exclusive, as many other workflow options exist. For example the target particle, now encased in a droplet, may be incubated or expanded. The detailed behavior of the target particle in the presence of the reagent can also be investigated.
In
First, each droplet may further encapsulate a barcoded bead 310. Each bead encapsulated in the droplets contains many barcoded primers. The beads provides primers that contain oligo(dT) which will interact with the poly A tail of the mRNA, a unique barcode and molecular identifier (UMI) that are used to index the 3′ end of cDNA molecules during reverse transcription, thus enabling the assignment of every individual transcripts and individual cells and finally the primers provide by the beads contain a PCR handle for further amplification of the library construct.
The Sequencer 600 May Further Include a Cell Lysis & RT Stage 610.
Each functional water droplet in oil contains a single cell, a single bead with primers as described above, and RT reagents. Within each reaction vesicle, a single cell is lysed and reverse transcription of polyadenylated mRNA occurs. As a result, all cDNAs from a single cell will have the same barcode, allowing the sequencing reads to be mapped back to their original single cells of origin. After that step the droplets are pooled together and a alcohol based reagent is added to dissolve the oil water droplet solution. A washing step is introduced to get rid of unwanted left overs. The preparation of NGS libraries from these barcoded cDNAs is then carried out in a highly efficient bulk reaction. “NGS” stands for next generation sequencing and allows the determination of sequences in a massively parallel manner. The sequencer referred to herein may be an NGS sequencer, but the techniques may apply to other sequencing schemes and technologies as well.
The Sequencer 600 may further include a library preparation stage, 620: The barcoded double stranded cDNA are used to prepare an NGS library using conventional and prior art approach. The cDNA is fragmented enzymatically and post fragmentation, the ends are repaired and poly A-tailed. Adaptor oligos are then ligated to each extremity clean up with SPRI beads and amplified by PCR.
The Sequencer 600 may further include a Circularization and amplification, stage 630: The cDNA library containing adaptors is then used as a template for rolling circle amplification (RCA). The RCA reaction needs to be primed using an oligonucleotide (RCA primers) that is complementary to the common adapter portion of the circularized DNA library. This short duplex/circular template is recognized by the Polymerase performing the RCA which amplifies the DNA regardless of the target sequence into DNA rolonies containing several hundred copies or concatemers of the DNA.
The Sequencer 600 may further include a Sequencing stage, 640. The rolonies are then loaded in to a micro fluidics channel. The rolonies will randomly immobilize on a functionalized glass surface. Multiple different chemistry reagents are sequentially applied to sequence the bases on each rolony. The bases are labeled with fluorescence dyes which an optical imaging system can detect during each cycle of sequencing. A sophisticated algorithm takes all those raw images coming from the optical imaging system and does the base calling for each rolony and determines the bases.
A process or method to sequence the genetic material of a single biological particle separated from the fluid stream by the MEMS device 10 is also disclosed here, and this method is illustrated in
It should be understand that not all of these step need necessarily be performed, and they may not need to be performed in the precise order given in
In some embodiments, the cell is encapsulated with the bead in the droplet, and the bead contains barcode. The cell is lysed. In some embodiments, within the droplet, the barcode is associated with nucleic acid derived from cell. In some embodiments, all the droplets are broken and the barcoded nucleic acids are amplified in bulk using PCR. The process may then continue to library preparation and sequencing.
In another exemplary workflow, the cell is encapsulated with bead, but the bead does not contain barcode. In the droplet, the cell may be lysed. In droplet, the barcode may be associated with nucleic acid derived from cell. In some embodiments, all the droplets are broken, and a synthesis of specific barcode on the bead takes place, along with amplification of the barcoded nucleic acids by PCR (in bulk). The process may then continue to library preparation and sequencing.
Any number of alternative workflows are also envisioned. These alternatives may include heating, incubating, centrifugation, imaging, incubating, buffering, observing, PCR, sequencing.
Accordingly, a system is described for preparing and analyzing genetic material. The system may include a first microfluidic channel formed in a substrate, a first fluid, including at least one target particle and at least one bead and non-target material, flowing in the first microfluidic channel, and a microfabricated MEMS fluidic valve, configured to open and close the first microfluidic channel and formed in the same substrate wherein the MEMS valve when in the sort position, separates the target particle and redirects the target particle into a first sort channel containing the first fluid, wherein the fluidic valve forms a droplet containing the target particle. The system may further include a second fluid, immiscible with the first fluid, and in which the droplet is suspended, and a second microfluidic channel containing the second immiscible fluid. The system may further include a fluidic manifold that accepts the droplet and lyses the target particle enclosed within the particle to release genomic material; and a third microfluidic channel that contains a biologically active material, in fluid communication with the first microfluidic channel
The system may further include a reservoir for storing the biologically active material. It may also include a nozzle disposed between the first sort channel and the second microfluidic channel, wherein the nozzle forms a droplet comprising a quantity of the first fluid along with the target particle, the quantity determined by the MEMS fluidic valve opening and closing. Within the system, the droplet may contain a plurality of target cells. The droplet may also contain a barcoded bead in addition to at least one target cells. The size of the droplet may be based on motion of the microfabricated MEMS fluidic valve. In the system, the first, second and third microfluidic channels, the microfabricated MEMS fluidic valve, and the reservoirs are all contained within a disposable cartridge. The biologically active material may be at least one of a growth compound, a reagent, a drug, a hormone and an enzyme.
The system may further comprise an interrogation zone 270 downstream of the droplet formation, which characterizes the contents of the droplet. This interrogation zone may be similar to interrogation zone 170. It may also include a controller that adjusts sorting parameters based on signals from the interrogation zone, using a feedback loop. The system may also include an FPGA, which stores an algorithm that controls the sorting process and downstream interrogation and feedback.
A process for separating and analyzing a genomic sequence from a target cell is also described. The process may include forming a first fluidic channel on a substrate, providing a first fluid flowing in the first microfluidic fluid channel, opening and closing a microfabricated MEMS fluidic valve, to open and close the microfluidic channel. The process may then further include capturing at least one of a target particle and a bead with identifiers disposed thereon, providing a source of an immiscible second fluid, immiscible with the first fluid, wherein the immiscible second fluid flows in a second fluidic channel, and forming a droplet containing the target particle and suspended in the second immiscible fluid. The process may also include interrogating the droplet to acquire information characterizing its contents, and proceeding to a downstream workflow based on the droplet contents.
Within the method, the downstream workflows may comprise at least one of proteomics, genomics and transcriptomics.
In other embodiments, the downstream workflow may comprise at least one of centrifugation, heating, incubation, polymerase chain reaction, DNA sequencing and RNA sequencing. The process may further include using a feedback loop to control sorting, based on the information acquired by interrogating the droplet. The feedback loop may use artificial learning techniques to control the sorting. The laser interrogation may include at least one of laser-induced fluorescence and optical imaging.
The method may further include executing at least one of: re-sorting the target particle and re-forming the droplet, disposing of the droplet, and lysing the target cell to release its DNA. It may further include sequencing the DNA of the lysed cell. It may further include enclosing at least one target particle and at least one bead into the droplet.
While various details have been described in conjunction with the exemplary implementations outlined above, various alternatives, modifications, variations, improvements, and/or substantial equivalents, whether known or that are or may be presently unforeseen, may become apparent upon reviewing the foregoing disclosure. Accordingly, the exemplary implementations set forth above, are intended to be illustrative, not limiting.
This US nonProvisional patent application is a continuation-in-part, claiming priority to U.S. Pat. No. 17,324,570, filed May 19, 2021, which in turn is a continuation-in-part, claiming priority to Ser. No. 16/009,163, filed Jun. 14, 2018. These prior applications are incorporated by reference in their entireties.
| Number | Date | Country | |
|---|---|---|---|
| Parent | 17324570 | May 2021 | US |
| Child | 17873187 | US | |
| Parent | 16009163 | Jun 2018 | US |
| Child | 17324570 | US |
