MICROFABRICATED DROPLET DISPENSOR WITH HYDROGEL

CROSS REFERENCE TO RELATED APPLICATIONS

Not applicable.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

Not applicable.

STATEMENT REGARDING MICROFICHE APPENDIX

Not applicable.

BACKGROUND

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.

SUMMARY

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.

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.

The system may make use of a hydrogel, a material having a degree of order and viscosity that is used to suspend or encapsulate the droplet.

BRIEF DESCRIPTION OF THE DRAWINGS

Various exemplary details are described with reference to the following figures, wherein:

FIG. 1 is a schematic illustration of an embodiment of a microfabricated droplet dispenser with an immiscible fluid with the microfabricated MEMS fluidic valve in the closed position;

FIG. 2 is a schematic illustration of an embodiment of a microfabricated droplet dispenser with an immiscible fluid with the microfabricated MEMS fluidic valve in the open (sort) position;

FIG. 3 is a chart showing the functional dependence of the water droplet size on the duration that the microfabricated MEMS fluidic valve is open;

FIG. 4 is a schematic illustration of an embodiment of a microfabricated droplet dispenser with an immiscible fluid generating an empty droplet in oil;

FIG. 5 is a schematic illustration of an embodiment of a microfabricated droplet dispenser with an immiscible fluid generating a droplet, wherein the droplet contains both a particle and a bead;

FIG. 6 is a schematic illustration of an embodiment of a microfabricated droplet dispenser with an immiscible fluid in a butt junction;

FIG. 7 is a schematic illustration of an embodiment of a microfabricated droplet dispenser with a laser assisted droplet coalescence; and

FIG. 8 is a schematic illustration of an embodiment of a microfabricated droplet dispenser with a variable channel cross section;

FIG. 9 is a schematic illustration of a first embodiment of a microfabricated droplet dispenser using a hydrogel;

FIG. 10 is a schematic illustration of a second embodiment of a microfabricated droplet dispenser using a hydrogel;

FIG. 11 is a schematic illustration of a third embodiment of a microfabricated droplet dispenser using a hydrogel;

FIG. 12 is a schematic illustration of a fourth embodiment of a microfabricated droplet dispenser using a hydrogel;

FIG. 13 is a schematic illustration of a fifth embodiment of a microfabricated droplet dispenser using a hydrogel;

FIG. 14 is a schematic illustration of a sixth embodiment of a microfabricated droplet dispenser using a hydrogel;

FIG. 15 is a schematic illustration of a seventh embodiment of a microfabricated droplet dispenser using a hydrogel; and

FIG. 16 is a schematic illustration of a eighth embodiment of a microfabricated droplet dispenser using a hydrogel.

It should be understood that the drawings are not necessarily to scale, and that like numbers may refer to like features.

DETAILED DESCRIPTION

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:

- 110 microfabricated MEMS valve
- 120 fluid input channel
- 122 sort channel
- 140 waste channel
- 150 nozzle
- 170 interrogation region
- 145 non-sort flow
- 200 oil
- 220 oil input 1
- 240 oil input 2
- 260 oil flowing to outlet via
- 300 water droplet in oil
- 310 bead in water droplet
- 320 target particle in water droplet
- 400 laser heater
- 500 merging area

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 microfabricated MEMS valve may be, for example, the device shown generally in FIGS. 1 and 2. It should be understood that this design is exemplary only, and that other sorts of MEMS valves may be used in place of that depicted in FIGS. 1 and 2.

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.

FIG. 1 is an plan view illustration of the novel microfabricated fluidic MEMS droplet dispensing device 10 in the quiescent (un-actuated) position. The MEMS droplet dispensing device 10 may include a microfabricated fluidic valve or movable member 110 and a number of microfabricated fluidic channels 120, 122 and 140. The fluidic valve 110 and microfabricated fluidic channels 120, 122 and 140 may be formed in a suitable substrate, such as a silicon substrate, using MEMS lithographic fabrication techniques as described in greater detail below. The fabrication substrate may have a fabrication plane in which the device is formed and in which the movable member 110 moves. Details as to the fabrication of the valve 110 may be found in US Patent 9,372, 144 (the '144 patent) issued Jun. 21, 2016 and incorporated by reference in its entirety.

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, non-target 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 FIG. 2. The actuation means may be electromagnetic, for example. The analysis of the fluorescent signal, the decision to sort or discard a particle, and the actuation of the valve, may be under the control of a microprocessor or computer.

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.

With the valve 110 in the position shown in FIG. 1, the microfabricated MEMS fluidic valve 110 is shown in the closed position, wherein the fluid sample stream, target particles and non-target materials flow directly in to the waste channel 140. Accordingly, the input stream passes unimpeded to an output orifice and channel 140 which may be out of the plane of the inlet channel 120, and thus out of the fabrication plane of the device 10. That is, the flow is from the inlet channel 120 to the output orifice 140, from which it flows substantially vertically, and thus orthogonally to the inlet channel 120. This output orifice 140 leads to an out-of-plane channel that may be perpendicular to the plane of the paper showing FIG. 1. More generally, the output channel 140 is not parallel to the plane of the inlet channel 120 or sort channel 122, or the fabrication plane of the movable member 110.

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 FIG. 2. The contour of the orifice 140 may be such that it overlaps some, but not all, of the inlet channel 120 and sort channel 122. By having the contour 140 overlap the inlet channel, and with relieved areas described above, a route exists for the input stream to flow directly into the waste orifice 140 when the movable member or valve 110 is in the un-actuated waste position.

FIG. 2 is a schematic illustration of an embodiment of a microfabricated droplet dispenser with an immiscible fluid with the microfabricated MEMS device 10. In FIG. 2, the MEMS device 10 may include a MEMS fluidic valve 110 in the open (sort) position. In this open (sort) position, a target cell 5 as detected in the laser interrogation region 170 may be deflected into the sort channel 122, along with a quantity of the suspending (buffering) fluid.

In this position, the movable member or valve 110 is deflected upward into the position shown in FIG. 2. The diverting surface 112 is a sorting contour which redirects the flow of the inlet channel 120 into the sort output channel 122. The sort output channel 122 may lie in substantially the same plane as the inlet channel 120, such that the flow within the sort channel 122 is also in substantially the same plane as the flow within the inlet channel 120. Actuation of movable member 110 may arise from a force from force-generating apparatus (not shown). In some embodiments, force-generating apparatus may be an electromagnet, however, it should be understood that force-generating apparatus may also be electrostatic, piezoelectric, or some other means to exert a force on movable member 110, causing it to move from a first position (FIG. 1) to a second position (FIG. 2).

More generally, the micromechanical particle manipulation device shown in FIGS. 1 and 2 may be formed on a surface of a fabrication substrate, wherein the micromechanical particle manipulation device may include a microfabricated, movable member 110, wherein the movable member 110 moves from a first position to a second position in response to a force applied to the movable member, wherein the motion is substantially in a plane parallel to the surface, a fluid sample inlet channel 120 formed in the substrate and through which a fluid flows, the fluid including at least one target particle and non-target material, wherein the flow in the fluid sample inlet channel is substantially parallel to the surface, and a plurality of output channels 122, 140 into which the microfabricated member diverts the fluid, and wherein the flow in at least one of the output channels 140 is not parallel to the plane, and wherein at least one output channel 140 is located directly below at least a portion of the movable member 110 over at least a portion of its motion.

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 FIG. 2, the suspending aqueous fluid, along with at least one suspended particle, may flow into the sort channel 122, and from there to the edge of the fabrication substrate. The fluid that was flowing in the fluid sample inlet channel 120 may then form a droplet at the edge of the fabrication substrate. Alternatively, the generated droplet might flow to and accumulate in the sort chamber.

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 FIGS. 1 and 2. The oil 200 may be applied symmetrically by oil input 220 and oil input 240. The immiscible fluid may serve to maintain the separation between droplets, so that they do not coalesce, and each droplet generally contains only one target particle and only one bead. The stream of oil may exit the sort outlet via 260. The lipidic fluid may be a petroleum based lipidic fluid, or a vegetable based lipidic fluid, or an animal based lipidic fluid.

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 FIG. 2. The functional dependence of the size of the droplet on the valve open time is illustrated in FIG. 3. As can be seen in FIG. 3, the diameter of the droplet is proportional to the valve open time, over a broad range of values. Only at exceedingly large droplets and long open times (greater than about 100 usecs and 60 microns diameter) does the functional dependence vary from its linear behaviour.

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 FIG. 4.

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 FIG. 4, downstream of the microfabricated MEMS valve, and in the vicinity of the nozzle structure 150, there may be disposed a flow junction with the immiscible second fluid. In the sort channel, downstream of the valve, there may be a flow junction with oil (as a carrier for water droplets) flowing from the sides towards the sort channel 122. This flow junction may have an inlet 220 and 240 on either end of the sort channel 122, forming an oil stream 200 downstream of the nozzle 150 and sort channel 122.

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 FIG. 2. In this open (sort) position, a target cell 5 may be deflected into the sort channel, along with a quantity of the suspended fluid.

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 FIG. 1, oil continues flowing towards the sort outlet via, blocking water flow in the sort. In fact however, because of the finite gaps between the moving edges of the MEMS valve 110 shown in FIGS. 1 and 2, a small but finite amount of the fluid sample stream fluid may continue to flow down the sort channel 122. However, these leak flow rates through the valve gaps, are not sufficient to break the oil front and create a water droplet, in normal operation.

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, FIG. 4 is a schematic illustration of an embodiment of a microfabricated droplet dispenser with an immiscible fluid generating an empty first fluid droplet 300 in oil 200. This situation may occur if no target particle is present in the fluid sample stream. The MEMS valve 110 may leak slightly, causing an aqueous droplet to form but without an enclosed target particle. In this case, the droplet may be allowed to flow into a waste area of a holding receptacle.

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 FIG. 5. The bead may be a biologically inert material coated with a biologically active material, and additional compounds. The biologically active materials may be antibodies that can become conjugated to antigens appearing on a target cell surface 320. In addition to the antigens and inert materials, the bead may further be coupled to a plurality of fluorescent tags, that is, compound which fluoresces when irradiated by an excitation laser of the proper wavelength and intensity. This plurality of fluorescent tags may be different for each bead 310, and may therefore act as a signature or identifier for the bead.

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.

FIG. 5 is a schematic illustration of an embodiment of a microfabricated droplet dispenser with an immiscible fluid generating a droplet in oil, wherein the droplet contains both a particle or cell 320 and a bead 310. Accordingly, the MEMS valve 110 may first sort a particle 320, enclosing the particle 320 in an aqueous droplet as described above. The MEMS valve 110 may then also sort a barcoded bead 310, and both particle 320 and the bead 310 may be enclosed in the same aqueous droplet, as shown in FIG. 5.

FIG. 6 is a schematic illustration of another embodiment of a microfabricated droplet dispenser with an immiscible fluid in a butt junction. In this embodiment, the application of the surrounding second immiscible fluid is asymmetrical. Instead of coming both from the right and the left of the nozzle region, the oil 200, the oil junction is applied in parallel to the sort channel 122 and may exit downstream 260 of the sort channel 122 . . . . The second immiscible fluid may flow from right to left. The aqueous fluid droplet may break the oil meniscus from the side channel, as shown. As before, each droplet 300 in oil 200 may contain both a target cell 320 and an identifying bead 310.

Laser Assisted Droplet Formation

FIG. 7 is a schematic illustration of another embodiment of a microfabricated droplet dispenser with a laser assisted droplet coalescence. In this embodiment, the two particles the target cell 320 and the bead 310 are sorted separately and placed into two separate aqueous droplets in the oil stream 200. For each event, the passage of the target cell 320 and the passage of the bead 310, the sort pulse is long enough to cause an instability in the oil-water interface and form a water droplet in oil containing the cell. The two separate droplets are then merged by application of laser light 400 on to oil channel containing the aqueous droplets.

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 FIG. 7, to heat the droplets. The application of energy causes the fluids to heat, which weakens meniscus and membrane forces, allowing the droplets to merge.

In FIG. 7, as in previous embodiments, the microfabricated droplet dispenser in FIG. 7 may have a symmetric (or asymmetric) oil input configuration. In either configuration, the droplets 300 may be encased in an immiscible second fluid, such as a lepidic fluid or oil 200. The oil 200 may be applied symmetrically by oil input 220 and oil input 240. The stream of oil may exit the sort outlet via 260.

The embodiment shown in FIG. 7 may have a flow channel which is capable of sorting two aqueous droplets, and then merging them into a single larger droplet. In this embodiment, the sort pulse is long enough to cause an instability in the oil-water interface and form a water droplet in oil containing the cell. Then a bead is sorted and a separate droplet is formed. Accordingly, the first droplet may contain a target cell 320, and the second aqueous droplet may contain a bead 310 as previously described. A merging area is a portion of the sort flow channel 122 wherein the laser 400 is directed. The laser light may be focused to increase its peak intensity. The applied laser light may heat the droplet as well as the surrounding fluid, and allow the two droplets to merge. The merging may be caused by the laser-induced heating and consequent weakening of surface tension of the fluid droplet.

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.

Geometry-Induced Flow Slowdown

FIG. 8 is a schematic illustration of an embodiment of a microfabricated droplet dispenser with a variable channel cross section. Like previous embodiments, the microfabricated droplet dispenser in FIG. 8 may have a symmetric (or asymmetric) oil input configuration. In this configuration, the droplets may be encased in an immiscible second fluid, such as a lepidic fluid or oil 200. The oil 200 may be applied symmetrically by oil input 220 and oil input 240. The stream of oil may exit the sort outlet via 260.

The embodiment shown in FIG. 8 may have a flow channel which is capable of sorting two aqueous droplets, and then merging them into a single larger droplet. In this embodiment, the sort pulse is long enough to cause an instability in the oil-water interface and form a water droplet 300 in oil containing the cell. Then a bead 310 is sorted and a separate droplet is formed. Accordingly, the first droplet may contain a target cell 320, and the second aqueous droplet may contain a bead 310 as previously described. A merging area 500 is a portion of the sort channel 122 having a variable cross section 500. 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. In other words, the sudden widening may produce geometry-induced flow slowdown, which allows the droplets to merge.

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.

Another embodiment of this microfabricated droplet dispenser with immiscible fluid is shown in FIGS. 9-16. These embodiments use an immiscible fluid in the form of a hydrogel. “Hydrogel” should be understood to refer to an aqueous material having some degree of internal order which differs from a surrounding material. The hydrogel material may therefore be “micro-organized” or “gel-like”. The hydrogels may be a network or matrix of micro or macro molecules which have some degree of aqueous solubility. The hydrogel may be a cross linked polymer, for example. Or the hydrogel may be in a different phase than the surrounding material, as described further below. The hydrogel maybe continuous, or exist is discrete layers such as an outer shell encasing an inner matrix.

The matrix may be formed by a catalyst or activation mechanism. The catalyst or activation may be light induced or photoactivation, alkali/acid or pH activation, enzymatic activation or polymerization by chemical cross linking for example. The cross linking may include covalently bonded particles. The individual compounds may be aqueously soluble which is cross linked to form a semi solid or gelatin-like state, a large continuous matrix which may nonetheless be permeable by water and small molecules.

The cross linking can be initiated by photo activation by laser for example. The activation laser may be triggered by the sort timing, i.e. by the detection of fluorescence in the sample input channel. Alternatively, the activation may be timed based on the fluid speed within the channel, or viscosity, or any other convenient signal which can be used to initiate the hydrogelling.

The hydrogel may also take the form of a phase transition, wherein the hydrogel material is in a particular phase, wherein the term “phase” is used to correspond to a start of order such as exists in a liquid crystal phase.

As such, the material is pervious to water, yet can form a barrier to the contents of a droplet as described previously.

Several examples of hydrogels exist, and may include: Examples of hydrogels include polymers, proteins, gelatin, collagen, glycosaccharides, polymer-based dextran, polyethylene glycol (PEG), and further it may include any chemical modification of the aforementioned substances. These techniques may be applied to a number of target particles, and these target particles may include immune cells, CAR-T cells, Natural killer (NK) cells, and the included materials may be growth medium, a biologically active molecule or a chemical sensor.

The encapsulation may be useful for cell cloning, downstream analysis, or trapping to detect cellular secretions. The cell can be combined with a delivery particle.

Multiple cells can be encapsulated to witness the interactions between the particles. The system may thereby be used to assess the efficacy of pharmaceutical compounds, or materials such as antibiotics, or Natural Killer (NK) cells to eliminate their target cells. Accordingly, an NK cell may be encapsulated with 5 to 8 target cells, for example.

The hydrogel may be formed any of a number of ways, which may include enzymatic (protein) transglutaminase, ionic calcium cation, in situ polymerization, copolymer and endcap polymerization. Photo activation, ionic sequestration, and kinetic thermal, enzyme based oligonucleotides, and the like.

Subsequent to hydrogel formation, the hydrogel may be de-aggregated, or dissociated, or otherwise returned to the non-gel state. These release mechanisms may include enzymatic mechanisms.

Examples of de-aggregation (de-association) mechanisms are heat, enzymatic, solvent based, or enforced turbulence in the flow, for example. However, may other gels, activation or formation mechanisms and release mechanisms are envisioned, and these lists are not meant to be exhaustive.

FIG. 9 shows a further embodiment of the system, where is it instead of a invisible fluid, a hydrogel is used to encapsulate the aqueous droplet. The term hydrogel should be interpreted broadly to mean any of a group of materials that form an aqueous gel, that is a water soluble gel. Examples of hydrogels include polymers, proteins, gelatin, collagen, glycol- or oligosaccharides, polymer-based dextran, polyethylene glycol (PEG), and further it may include any chemical modification of the aforementioned substances. The hydrogel can be input into the system in a fluid form, and where in the gel forms as a result of a cross-linking, or activation process downstream of the input of the fluid. The activation mechanisms may include enzymatic activation, photo activation, ionic sequestration, kinetic, thermal, in situ polymerization, copolymers and endcap polymers, to name a few examples. Upon activation, the hydrogel may form a very viscous or even organized matrix of material that reliably may encapsulate the aqueous droplet formed as described previously. The embodiments may also use phase change to separate the fluids. The droplet can flow within the hydrogel, along with the hydrogel through the microfluidic channels as illustrated in FIG. 9.

More specifically, FIG. 9 is similar to the microfabricated structure of FIG. 8, but with the additional of a polymer input 340 upstream of the laser interrogation system 170. The sample channel contains the first fluid 120 as before, where in the sample fluid includes target particles as well as non-target material. The target particle, non-target material and polymer material may flow together through the laser interrogation region 170. The target particle may be irradiated with an excitation laser, such that a tag conjugated to the target particle may fluoresce as a result, identifying the structure of the target particle. Based on this fluorescent signal, the microfabricated sorting system may either sort the particle, or allow the fluid to flow into the waste channel, as described previously. The sorting may be accomplished by the electromagnetically activated fluid valve 110, as described previously. If the target particle is detected in the channel, the valve is activated such that the sort target particle, some non-target material, the aqueous fluid and the hydrogel proceed into the sort channel 122. The fluid components may then pass a cross linking mechanism 350 as shown in FIG. 9. This cross linking mechanism 350 may cause the hydrogel to form from the polymer components, such that the cell 310 is encased in a droplet 330, encased in the cross-linked population 300. Finally, the hydrogel flows toward the sort outlet via 360, along with the aqueous droplet and close their end.

FIG. 10 shows another exemplary embodiment of a microfabricated droplet sorter 10′ using again a polymer hydrogel to encapsulate the droplet. In this embodiment, the polymer input 340 is located downstream of the laser interrogation area 170. The polymer components than flow along with the target particle and non-target material through the sample channel 120. The fluid components once again flow to the movable valve 110, and based on the presence or absence of a fluorescent signal, the target particles either sorted, or allowed to flow into the waste channel 140. If the target particle is detected in the channel, the valve 110 is actuated, causing the particle to flow into the sort channel 122.

As before, the cross linking mechanism 350 is disposed in or adjacent to the sort channel 122. The cross-linking mechanism as a may be, for example, a photo activation mechanism, i.e. a laser for example which radiates the polymer hydrogel and causes it to cross link and become firm. The hydrogel thereafter encases the droplet, having the sorted cell 310 and sorted bead 320 inside the droplet 330. The stream with the hydrogel, droplet with cell 310 and bead 320 in the droplet 330, then flows toward the sort out that via 360.

FIG. 11 shows yet another embodiment of the microfabricated cell sorter using the hydrogel concept. In this embodiment, the polymer input 340 is disposed downstream of the movable valve 110. In other words, that polymer may be input downstream of the sorting mechanism. Accordingly, the hydrogel and bead 320 with the target particle 310, and sample stream then flow past the hydrogel activation mechanism 350. As before the activation mechanism causes the hydrogel to form from the polymer input precursors. The system with a hydrogel droplet 330 flowing toward the sort outlet via 360 then occurs as previously described.

FIG. 12 shows another embodiment of the microfabricated cell sorting device using the hydrogel concept. In this embodiment the polymer input 340 again occurs before the laser interrogation region 170. However in this embodiment the hydrogel is activated 350 just downstream of the laser interrogation region. Cross-linking in this embodiment therefore may begin earlier, but not to the point where it interferes with the sorting mechanism valve 110.

FIG. 13 shows another embodiment of the MICRA fabricate its particle sorting device 10′, using a hydrogel concept. However, in this environment, release mechanism 347 is also provided to the sample stream. This release mechanism 347 may dissolve the gel, returning it to its free fluid, or at least partially liquid or non-organized state.

FIG. 14 shows another exemplary embodiment of the micro fabricated particle sorting device 10′ and droplet dispenser, again using a hydrogel concept. In this environment, the polymer input 340 as before is inserted into the channel upstream of the laser interrogation region 170. The polymer input there by joins the sample stream come also containing a target particle and nontarget material. However, in addition, the polymer input 340, a second input channel is also provided,. This second input channel 345 may provide an enzyme input to the sample stream. this enzyme may catalyze the polymerization, or it may cause the target particle to undergo some sort of biological process, such as a denaturing or an unfolding. Subsequently, the components including the enzyme, the polymer, the target particle and the non-target material then flows through the sorting mechanism, and is sorted, or sent to waste, as before, depending on the presence of an appropriate fluorescent signal from the target particle.

As before, the assembly of components then passes by a hydrogel activation mechanism 350, which may cause the polymerization of the hydrogel. However, in this environment, yet another input channel may be provided. This input channel may provide another phase of material, for example another viscous aqueous gaseous or solid material. Illustrated in FIG. 14 may also include the release mechanism 347, described previously with respect to FIG. 13.

FIG. 15 shows another embodiment wherein an enzyme input 345 is provided.

This input port may be used to deliver the enzyme to the flowing polymer input, and may initiate the cross linking of the material.

Another exemplary embodiment of a microfabricated particle sorting device 10′ using the hydrogel concept is shown in FIG. 16. In this embodiment, the hydrogel precursors are inserted downstream of the sorting mechanism. There is a first hydrogel input 326 and a second hydrogel input 328. These components may be activated as discussed previously to form the matrix or gel which encapsulates the target particles. In this embodiment, multiple target particles may be encapsulated in the single droplet. They may be for example target particle 312, target particle 310, and target particle 314. These particles may all be enclosed in droplet 330. The three particles 310, 312 and 314 may comprised an immune cell, and a bead, and a pathogen for example.

Each of these particles 312, 310 and 314 may be chosen for a particular purpose. For example they may be oligonucleotide switch react to form a strand of DNA, they may be a labeling be, having a barcode on board. They may be pathogen, along with an agent which interferes with the functioning of the pathogen. They may be a biological cell, and nutritional growth medium, A buffer, a serum, therapeutic agent, pharmaceuticals, to name just a few. It should be understand that the this list is not exhaustive and that wide number of applications I may be available for this System and technique.

Accordingly, described here is a microfabricated droplet dispenser. The system may include a microfabricated flow channel containing a sample stream, the sample stream including at least one target particle and non-target material, a population of particles in a hydrogel, wherein the at least one target particle is encapsulated in the population of particles in the hydrogel and a microfabricated sorting device, which diverts the at least one target particle into the population of particles in the hydrogel, based on a fluorescent signal generated by the at least one target particle. The population of particles may be disposed upstream of a laser interrogation region, or upstream of the microfabricated device but downstream of the laser interrogation zone, and wherein the hydrogel encapsulates the at least one target particle in the hydrogel.

The system may also include an activation mechanism, which initiates the formation of the hydrogel, and wherein the activation mechanism is disposed either upstream of the microfabrication sorting device, or upstream of the microfabricated sorting device and downstream of the laser interrogation region. The activation mechanism may include at least one of photo-activation, enzymatic activation, chemical, mechanical or biological activation, light-induced or photoactivation, alkali/acid or pH activation, enzymatic activation or polymerization by cross linking. The system may include a release mechanism, wherein the release mechanism is disposed downstream of the microfabricated sorting device, and wherein the release mechanism includes at least one of heat, enzymatic, solvent based, or enforced turbulence in the flow, for example.

The target particle may comprise at least one of a T-cell, a car T-cell, an oligonucleotide, a serial killer cell, and a pathogen.

The microfabricated sorting mechanism may be fabricated in a plane of the substrate, and wherein the microfabricated sorting device moves in that a plane which is parallel or coplanar with this plane. The microfabricated sorting device may form a droplet, wherein the droplet dimensions are based on the timing of the microfabricated sorting device, and the droplet is suspended in the hydrogel. The droplet may enclose a plurality of particles, including at least one bead, and a quantity of fluid. The plurality of particles may include least one of a tumor cell, a t-cell, a CAR T-cell, and oligonucleotide, a bead, a fluorophore, a pathogen and an immune cell.

The hydrogel may comprise at least one of polymers, proteins, gelatin, collagen, glyco saccharides, polymer-based dextran, polyethylene glycol (PEG), and further it may include any chemical modification of the aforementioned substances.

The hydrogel may comprise a material in a phase that is different from the phase of the surrounding material.

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.

MICROFABRICATED DROPLET DISPENSOR WITH HYDROGEL

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