WO2021250060A1

WO2021250060A1 - Plural microfabricated valve sorter with immiscible fluid

Info

Publication number: WO2021250060A1
Application number: PCT/EP2021/065393
Authority: WO
Inventors: Alexander ROCKENBACH; Stefan Miltenyi
Original assignee: Miltenyi Biotec B.V. & Co. KG
Priority date: 2020-06-12
Filing date: 2021-06-09
Publication date: 2021-12-16

Abstract

A microfabricated droplet dispensing structure is described, which may include a plurality of MEMS microfluidic fluidic valves, configured to open and close a microfluidic channel. The opening and closing of the valve may separate a target particle and a bead from a sample stream, and direct these two particle into a single droplet formed at the edge of the substrate. At least one additional microfabricated valve may sort a barcoded bead, wherein the sorted bead is conjugated with the sorted cell to make a bead/cell complex. The complex may be enclosed in an aqueous droplet and the droplet dispensed into a stream of a second immiscible fluid.

Description

PLURAL MICROFABRICATED VALVE SORTER WITH IMMISCIBLE FLUID

BACKGROUND

[0001] The present invention is directed to a system for the manipulation of particles and biological materials, and forming droplets containing these particles.

[0002] 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.

[0003] 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.

[0004] 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.

[0005] 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 micro fluidic devices.

[0006] 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.

[0007] USP 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 micro fluidic 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.

[0008] USP 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.

[0009] USP 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.

[0010] USP 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

[0011] 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

[0012] 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.

[0013] 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.

[0014] 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.

[0015] 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.

[0016] 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. At least one additional microfabricated valve may be used to separate a particular barcoded bead, for attachment to the sorted target cell, to for a bead cell complex. This complex may be encased in an aqueous droplet and dispensed into a stream of an immiscible fluid. The formation of the droplet may be accomplished by yet another microfabricated valve. [0017] Accordingly, a microfabricated droplet dispensing structure is described, which may include a plurality of MEMS microfluidic fluidic valves, configured to open and close a microfluidic channel. The opening and closing of the valve may separate a target particle and a bead from a sample stream, and direct these two particle into a single droplet formed at the edge of the substrate. At least one additional microfabricated valve may sort a barcoded bead, wherein the sorted bead is conjugated with the sorted cell to make a bead/cell complex. The complex may be enclosed in an aqueous droplet and the droplet dispensed into a stream of a second immiscible fluid.

BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Various exemplary details are described with reference to the following figures, wherein:

[0019] 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;

[0020] 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;

[0021] 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;

[0022] Fig. 4 is a schematic illustration of an embodiment of a microfabricated droplet dispenser with an immiscible fluid generating an empty droplet in oil;

[0023] 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;

[0024] Fig. 6 is a schematic illustration of an embodiment of a microfabricated droplet dispenser with an immiscible fluid in a butt junction;

[0025] Fig. 7 is a schematic illustration of an embodiment of a microfabricated droplet dispenser with a laser assisted droplet coalescence; and

[0026] Fig. 8 is a schematic illustration of an embodiment of a microfabricated droplet dispenser with a variable channel cross section.

[0027] Fig. 9 illustrates an embodiment of the microfabricated sorting valve, wherein a second sorting valve is used to sort barcoded beads in addition to the target cells; [0028] Fig. 10 illustrates an embodiment of the microfabricated sorting valve, wherein a second sorting valve is used to sort barcoded beads in addition to the target cells;.

[0029] Fig. 11 illustrates an embodiment of the microfabricated sorting valve, wherein a second sorting valve is used to sort barcoded beads in addition to the target cells;

[0030] It should be understood that the drawings are not necessarily to scale, and that like numbers may refer to like features.

DETAILED DESCRIPTION

[0031] 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

[0032] 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. [0033] 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.

[0034] 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.

[0035] 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 June 21, 2016 and incorporated by reference in its entirety.

[0036] 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.

[0037] 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.

[0038] 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.

[0039] 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.

[0040] 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.

[0041] 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.

[0042] 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.

[0043] 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.

[0044] 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.

[0045] 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).

[0046] 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.

[0047] 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.

[0048] 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.

[0049] Various structures may be used in this region to promote the formation of the droplet. These structures may be, for example, rounded comers 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.

[0050] 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.

[0051] 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.

[0052] 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 psecs and 60 microns diameter) does the functional dependence vary from its linear behaviour.

[0053] 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.

[0054] 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.

[0055] 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.

[0056] 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.

[0057] 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

[0058] 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.

[0059] 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.

[0060] 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.

[0061] 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.

[0062] 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.

[0063] 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.

[0064] 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. [0065] 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.

[0066] 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

[0067] 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.

[0068] 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.

[0069] 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.

[0070] 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.

[0071] 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

[0072] 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.

[0073] 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.

[0074] 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 micro fabricated 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.

[0075] 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

[0076] 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.

[0077] 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.

[0078] 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 micro fluidic 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.

[0079] 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.

[0080] The fluid flowing in the micro fluidic 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.

[0081] 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 microfabricated MEMS fluidic valve, and directing the bead into the droplet, wherein the bead and the target particle, are located within the same droplet.

[0082] 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.

[0083] Because of the very small size of the microfabricated structures shown in these embodiments, and because of the batch fabrication processing techniques used to make the structures, it may be possible to build a plurality of similar microfabricated fluidic valves on a single wafer. This can be done cost-effectively, because of the economy of batch- and wafer- photolithographic processing used to make the movable valves 110.

[0084] In some applications, it may be particularly advantageous to build multiple microfabricated valves on a substrate surface. In the embodiment described below, a first microfabricated valve may be used to sort a target cell from the remainder of a fluid stream, as described above, and to enclose this target particle in an aqueous droplet. [0085] However, a second microfabricated fluid valve may also be used to sort a “bar- coded” target magnetic bead from a population of similar beads. Here, the term “barcode” or “bar-code” or “bar code” is a term used to refer to an identifying marker, which can be attached to a particle as an identifying tag. The tag may be conjugated to a particular type of particle, a cell or bead for example, using antibody/antigen conjugation. DNA/RNA barcoding may also be used color codes, that is, fluorescent tags, may also be an option in addition or alone. Bar codes may use a fluorescent signature, for example, to uniquely identify the attached particle, when the marker binds with the particle. Examples of bar codes are set forth more fully below.

[0086] The target bar coded bead may then be combined with the target particle, to form a bead/cell complex, and this complex may then be encased in an aqueous droplet (water being the “first fluid”) and dispensed into a stream of immiscible fluid (oil, for example, being the “second immiscible fluid). The cell/bead complex encased in the droplet and flowing in the stream of immiscible fluid may then be used for downstream purposes, such as further analysis, expansion or experimentation. The second immiscible fluid may flow, with the aqueous droplet, within a microfluidic channel, all on the same fabrication substrate. Embodiments of this idea are described next.

[0087] Fig. 9 shows an embodiment of this structure with a plurality of microfabricated valves included. In Fig. 9, there is a first and a second microfabricated sorting valve. The first valve 30 may be used to sort cells, and the second valve 40 may be used to sort barcoded particles. The term “particle” should be understood to refer to any biological object, whether it is an actual biological material such as a cell, or a cell fragment such as DNA or RNA, or an inorganic material such as a magnetic bead, but used for a biological purpose such as magnetic separation. Downstream, the sorted cells are associated with the sorted beads to form a barcoded complex.

[0088] Accordingly, the first microfabricated structure in Fig. 9 is a microfabricated sample valve 30. This microfabricated sample valve 30 may have a design and operation similar to microfabricated sample valves 110 described above. Adjacent to microfabricated sample valve 30 is a similar microfabricated bead valve 40. In this embodiment, the first microfabricated valve 30 is used to sort a target particle, for example a particular target cell 10, from the rest of a sample stream. The other microfabricated structure, bead valve 40, is used to separate a particular target bead 20 from a population of beads stored in a bead reservoir 22.

[0089] In Fig. 9, a population of cells including target cells 10 may be stored in a sample reservoir or chamber 12. This target cell 10 may be suspended in a sample fluid along with other particles and non-target materials, all of which is contained in a sample chamber 12. The target fluid along with target cells 10, may proceed under hydrostatic pressure through microfabricated channels 120, and to a laser interrogation zone, here a cell detection zone 170, depicted in Fig. 9. In the laser detection or laser interrogation zone 170, a laser may irradiate the target cells 10 as described above with respect to other earlier laser interrogation zones 170, in order to identify the particles passing through the laser interrogation zone 170. Zone 170 is therefore an area wherein an excitation laser excites fluorescent tag affixed to a target particle or bead. 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 140 to the sort channel 122, as was 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.

[0090] Ordinarily, the sample valve 30 as shown in Fig. 9, is in the quiescent position, such that the whole sample fluid flows from the sample chamber 12 into the waste chamber 140 (not shown in Fig. 9). However, when a target particle 10 is detected in the laser interrogation zone 170 by the laser induced florescence, a computer directs the sample valve 30 to move, in order to separate the target cell 10 from the remainder of the sample fluid. The sort position is as was illustrated in Fig. 2. This motion may result from the generation of magnetic flux by the flux generating apparatus, which may interact with the magnetically permeable inlaid material as described previously. This action diverts the target cell 10 from the waste channel 140 to the sort channel 122, as illustrated by Figs. 1 and 2.

[0091] On the right hand side of the page, a second microfabricated valve 40 may separate the target bead from a population of similar beads. The beads may be provided with a fluorescent tag, similar to the target particles 10. Accordingly, a population of beads may be stored in bead chamber 22. These beads 20 may be suspended in a fluid, which flows down the microfabricated channel 220 to another laser interrogation zone 170’. These beads may also be tagged with fluorescent tag which identifies them. This tag is detected at the interrogation zone 170’ after the radiation with a laser, in a manner similar to that described above with respect to the first laser interrogation zone 170. Accordingly, as mentioned, the structures on the right hand side of this figure may be analogous to the structures on the left- hand side of Fig. 9, except that the right hand side may pertain to beads, whereas the left-hand side may pertain to cells.

[0092] In the second laser interrogation (bead detection) zone (170’) the same decision is made for a bead coming from an bead input reservoir (22) and the corresponding bead valve (40) is open also according to user criteria defined in software executed by a controller or computer. These steps must be performed after each other within a short period of time. The sample consisting of different beads may be led from a bead reservoir (20) into the second detection zone or laser interrogation zone (170’). Here, the fluorescent label on the cell, which identifies the bead is read. Therefore, one or more lasers may illuminate the bead (20) and the bead valve (40) may be triggered depending on the criteria implemented in the software.

[0093] Not sorted cells and not sorted beads may be recovered and led back from the waste reservoir through two separate circulation channels (126, 226) to their specific reservoirs (12, 22). This allows a reduction in the number of wasted cells and wasted beads to almost zero. This may also reduce the requirements for buffer and reagent. Only the fluorescent staining might be reduced, by irradiation in the laser interrogation zone. This might lead to a reduction of the maximum number of sort cycles before cells must be stained again. To allow a re-circulation like this, another microfluidic pump or another pressure drop may be integrated in a way to realize this circulation. As the provision of a pumping mechanism should be within the level of ordinary skill, the pump is not shown in Figs. 9, 10 or 11.

[0094]

Behind both valves 30 and 40, the sorted particles may travel down their respective sort paths 122 and 222. Sorted cells may travel down the sort output path 122 and sorted beads may travel down the sorted bead output path 222. The sorted cell 10 and the sorted bead 20 may be received in a collection area 160. In this zone 160, cells and beads are waiting here on each other and in close proximity. When a bead 20 is in proximity to a target cell 10, and the antibodies of the bead 20 may become conjugated with the antigens of the cell, the bead, along with its identifying fluorescent tags, may become affixed to the target cell 10. Thus, the bead 20 provides an identifying marker for the cell 10, or a “barcode” which identifies the cell. A computer or controller may associate this particular barcode with the particular cell. It should be understood that it is not necessary to combine the barcode in the sorter. It is also possible to sort beads to cells and only detect the barcode in the sequencing on DNA/RNA level. But in any case, 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.

[0095] Barcode labelling may be in the form of oligonucleotides that are bound to the surface of the target particle. In some applications, oligonucleotides C (color specific barcode), B (bead specific barcode) and U (unique molecular identifier) are referred to as “barcode” since they allow identifying a single target by their unique sequence. Also, fluorescent tags which emit a photon of fluorescence having a specific, identifying color upon laser stimulation or irradiation can be added to the bead as barcode in addition or solely.

[0096] In some applications, oligonucleotides C (color specific barcode), B (bead specific barcode) and U (unique molecular identifier) are referred to as “barcode” since they allow identifying a single target by their unique sequence.

[0097] Due to the now increased pressure, sorted cell 10 and sorted bead 20 may form a cell/bead complex within the collection zone 160. The pressure increase may be a method to transport cells and beads downstream. A pressure drop may occur from sort channel to collection zone. Only if an additional pump or pressure channel is provided, there may be an increased pressure as long as the drop is not formed. As soon as the drop is formed, this pressure is relaxed again and a new drop/bead combination may be sorted. Accordingly, the pressure may be increased again to form the drop. So, both means are possible: building the drop by switching of the valves, or by applying an additional pressure pulse. This cell/bead complex may then be encased in an aqueous droplet 300 and dispensed into the immiscible fluid simply under hydrostatic pressure that drives the fluid in the microchannel 322. Accordingly, this water droplet 300 in oil may be formed at the intersection between the collection zone 160 and oil input (120) and output channel (322).

[0098] Accordingly, the embodiment shown in Fig. 9 may simply use the aqueous flow into the immiscible fluid to passively form droplets 300 into the output channel 322. Cell and bead are thereby encapsulated in an hydrophobic liquid. The sorted portion of buffer with cell and bead are led as droplet 300 into the output microchannel 322 containing the immiscible fluid. This immiscible fluid may be an hydrophobic fluid such as oil as shown in Fig. 9. A different embodiment will be described next, wherein the droplet is formed by the active use of a third microfabricated valve. This next embodiment may therefore be somewhat more complex, but may offer better control over the droplet formation.

[0099] The embodiment described above may make use of multiple magnetically driven valves. The plurality of valves on one chip may lead to interference of the magnetic pulses between both valves. This can be reduced by using shielding (150) of the valves by magnetic material. The design of the magnetic shielding is discussed further below with respect to Fig. 11.

[00100] Accordingly, a microfabricated droplet dispenser is disclosed. The dispenser may include at least one microfluidic channel formed in a substrate; a first source of target particles and non-target material; a second source of identifying markers for the target particles, wherein the target particles and the identifying markers are suspended in a first fluid flowing in the at least one microchannel. The dispenser may also include a first microfabricated MEMS fluidic valve, configured to open and close the at least one microfluidic channel, to separate the target particles, and at least one additional microfabricated MEMS fluidic valve, configured to open and close the at least one microfluidic channel, to separate the identifying markers. The dispenser may produce a droplet comprising a quantity of the first fluid, and containing the separated target particle and the separated identifying marker, and may therefore include a source of a second fluid immiscible with the first fluid wherein the droplet is dispensed into, and immersed in, the second immiscible fluid.

[00101] In Fig. 9, the droplet is formed passively simply by allowing the sample stream with the beads and cells suspended therein to flow at some rate. By allowing this flow, aqueous droplets of a characteristic size may be released into the immiscible fluid contained in the microchannel 120. Another method of droplet formation is illustrated in Fig. 10.

[00102] The embodiment illustrated in Fig. 10 differs from that illustrated in Fig. 9 by its method of droplet formation. In the embodiment shown in Fig. 10, the droplet is not formed passively simply by the flow of fluid water-based fluid into the immiscible oil fluid. Instead, in this environment, a third microfabricated valve 70 forms the droplet actively. In other words, as the fluid flows to the encapsulation valve 70, quantity is allowed to pass into the immiscible fluid stream in the form of a droplet, 300 as shown in Fig. 10. The size of a droplet can be controlled by the duration for which the valve is open.

[00103] Accordingly, Fig. 10 illustrates an embodiment of the plural microfabricated valves used in the sorting system. As before, a first microfabricated valve 30 sorts target cells 10, a second microfabricate valve sorts barcoded beads, and a third microfabricated valve 70 forms the droplet encasing the cell/bead complex in an aqueous droplet.

[00104] The embodiment illustrated in Fig. 10 may also have laser interrogation zones 170 and 170’, which may operate as described previously with respect to Fig. 9.

[00105] As in the embodiment shown in Fig. 9, behind both valves 30 and 40, sorted cell 10 and bead 20 are combined in a collection zone (160). Cells and beads are waiting here on each other, and after a period in this zone 160, may be combined correctly. The encapsulation valve (70) is then triggered. Cell and bead are encapsulated in an aqueous droplet 300 and dispensed into the microchannel 322. Accordingly, the channel leading from the collection zone 160 then is the input channel to the droplet- forming microfabricated valve 70. In this embodiment, the bead/cell complex is placed in a droplet 300 and dispensed into the immiscible second fluid stream by encapsulation valve 70.

[00106] Droplet formation may begin when the valve 70 is activated in a manner illustrated in Fig. 2. During the period in which the valve 70 is in the actuated position, the droplet may increase in size by accepting the fluid flowing from input channels 120 and 220 into collection zone 160, and then into the actuated encapsulation valve 70. The droplet is released into the output channel 322 when the valve is closed again, returning to the position shown in Fig. 1.

[00107] Within each dispensed droplet 300, the target cells are associated with an identifying barcoded bead to form a barcoded complex, encased within a droplet 300 by encapsulation valve 70. As mentioned previously, the bead 20 may provide an identifying marker for the cell 10, 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.

[00108] As described, in the embodiment illustrated in Fig. 10, the droplet formation may be actively controlled by encapsulation valve 70. As a result, droplets may be produced of demand, and of a well-controlled size and content.

[00109] Fig. 11 illustrates yet another embodiment of the plural microfabricated valves used in the sorting system. As before, a first microfabricated valve 30 sorts target cells 10, a second microfabricate valve sorts barcoded beads, and a third microfabricated valve 70 forms the droplet encasing the cell/bead complex in an aqueous droplet.

[00110] The embodiment illustrated in Fig. 11 may also have laser interrogation zones 170 and 170’, which may operate as described previously with respect to Fig. 9.

[00111] As in the embodiment shown in Figs. 9 and 10, behind both valves 30 and 40, sorted cell 10 and bead 20 are combined in a collection zone (160). Cells and beads are waiting here on each other, and after a period in this zone 160, may be combined correctly. The encapsulation valve (70) is then triggered. Cell and bead are encapsulated in an aqueous droplet 300 and dispensed into the microchannel 322. Accordingly, the channel leading from the collection zone 160 then is the input channel to the droplet- forming microfabricated valve 70.

[00112] The target cells are thereby associated with an identifying barcoded bead to form a barcoded complex, encased within a droplet 300 by encapsulation valve 70. In this embodiment, the bead/cell complex is placed in a droplet 300 and dispensed into the immiscible second fluid stream 322.

[00113] Because microfabricated valves 30, 40, and 70, are all magnetic in nature, it may be important to provide magnetic shielding between these magnetic structures to reduce cross talk or interference. The shields 150 are depicted in Fig. 11, and are shown surrounding each of the microfabricated valves 30, 40, and 70.

[00114] Multiple magnetically driven valves on one chip may led to interference of the magnetic pulses between both valves. This can be reduced by using shielding 150 of the valves by magnetic material. The shielding may use a permeable magnetic material. Ni/Fe “permalloy” consists of 80% nickel and 20% iron. The deposition by plating or sputtering of 80/20 permalloy is well known in the art. Accordingly, permeable structures 150 may be deposited around the structures to shield them from stray magnetic fields- The shielding may only be applied on 3 sides. The magnetic flux may be led from the flux-generating solenoid tip into the actuator and into the shielding favouring the shielding as path back to close the flux line. The flux density may thereby be reduced outside the shielding and therefore also the interference with other magnetic fields which may be driving the other actuators. The permalloy shield thickness may be at least about 10 microns. More generally, the permalloy film thickness may be from a fraction of a micron to hundreds of microns, for example. Generally, the lower limit on thickness is determined by fracture resistance, but may be as low as 0.1 to 0.5 microns. 5-10 microns may be a typical thickness.

[00115] Accordingly, a microfabricated droplet dispenser is disclosed. The dispenser may include at least one microfluidic channel formed in a substrate, a first source of target particles and non-target material, and a second source of identifying markers for the target particles, wherein the target particles and the identifying markers are suspended in a first fluid flowing in the at least one microchannel. The dispenser may further include a first microfabricated MEMS fluidic valve, configured to open and close the at least one microfluidic channel, to separate the target particles into a sort channel and non-target material into a waste channel, and at least one additional microfabricated MEMS fluidic valve, configured to open and close the at least one microfluidic channel, to separate the identifying markers. Dispensed may be a droplet comprising a quantity of the first fluid, and containing the separated target particle and the separated identifying marker, and also provided may be a source of a second fluid, the second fluid immiscible with the first fluid, wherein the droplet is dispensed into, and immersed in, the second immiscible fluid.

[00116] The microfabricated droplet dispenser may further comprise a fluid collection zone which accepts the separated target particle from the first microfabricated MEMS fluidic valve and the separated identifying marker from the second microfabricated MEMS fluidic valve and concentrates the target particle and the identifying marker.

[00117] The microfabricated droplet dispenser may further include a droplet forming structure that forms a volume of the first fluid wherein this volume contains both the target particle and the identifying marker, and releases the volume into the second immiscible fluid as the droplet.

[00118] The microfabricated droplet dispenser may further include at least one recirculation path which may provide fluid communication between the waste channel and at least one of the first source and the second source.

[00119] The microfabricated droplet dispenser may further include a permeable magnetic material disposed adjacent to at least one of the first and at least one additional microfabricated valve, wherein the permeable material may be configured to reduce interference from magnetic flux directed to the microfabricated valves.

[00120] The microfabricated droplet dispenser may further include a bead disposed in the first fluid, wherein the bead is attached to at least one fluorescent tag, wherein the fluorescent tag identifies 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.

[00121] Within the microfabricated droplet dispenser, the microfabricated MEMS fluidic valve may move in a single plane when opening and closing, and wherein that plane is parallel to a surface of the substrate. Within the microfabricated droplet dispenser, the microfabricated MEMS fluidic valve may move in a single plane when opening and closing, and moves as a result of electromagnetic forces acting on the microfabricated MEMS fluidic valve. Within the microfabricated droplet dispenser, the droplet may include at least one of a target cell and a fluorescently-labelled bead. Within the microfabricated droplet dispenser, the source of immiscible fluid may be disposed symmetrically about a nozzle structure formed in the substrate. Within the microfabricated droplet dispenser a surfactant may be added to the first fluid. Within the microfabricated droplet dispenser, the bead may include a plurality of fluorescent tags, such that the bead has an identifying fluorescent signature. Within the microfabricated droplet dispenser, the bead may also comprise at least one antibody, that binds to an antigen on the at least one target particle. Within the microfabricated droplet dispenser, the bead may also comprise at least one of DNA and RNA. Within the microfabricated droplet dispenser, the source of immiscible fluid may be disposed asymmetrically about the nozzle.

[00122] The microfabricated droplet dispenser may further include a laser focused on the microfluidic channel and directed onto the droplet, wherein the laser is configured to heat the droplet to coalesce adjacent droplets in the microfluidic channel. Within the microfabricated droplet dispenser, the microfluidic channel may have a channel widened area, wherein the cross section of the channel increases and then decreases. Within the microfabricated droplet dispenser, the microchannel may intersect the source of immiscible fluid in a butt junction. Within the microfabricated droplet dispenser, the target particles may comprise at least one of T-cells, stem cells, cancer cells, tumor cells, proteins and DNA strands.

[00123] Further disclosed is a method for forming a droplet in an immiscible fluid. The method 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, 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 fluid into the immiscible second fluid, 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.

[00124] Within the method, the first fluid flowing in the microfluidic channel may comprise target particles and non-target material, and the target particles comprise at least one of T-cells, stem cells, cancer cells, tumor cells, proteins and DNA strands. 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 further 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 microfabricated MEMS fluidic valve, and directing the bead into the droplet, wherein the bead and the target particle, are located within the same droplet. Within the method, the droplet may be formed at a nozzle structure formed in the substrate.

[00125] The method may further include heating the droplet of fluid with a laser directed to the droplet. The method may further include generating a first sort pulse to capture a labelled bead, and then subsequently generating a second sort pulse to capture a target cell, wherein the sort pulses are generated such that the bead and a target particle are located within the same droplet dispensed into the second immiscible fluid.

[00126] The method may further include heating the droplet of fluid with a laser directed to the droplet. The method may further include generating a first sort pulse to capture a labelled bead, and then subsequently generating a second sort pulse to capture a target cell, wherein the sort pulses are generated such that the bead and a target particle are located within the same droplet dispensed into the second immiscible fluid. The method may further include generating a first sort pulse to capture a target cell; and then subsequently generating a second sort pulse to capture a labelled bead, wherein the sort pulses are generated such that the bead and a target particle are located within the same droplet dispensed into the second immiscible fluid.

[00127] 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.

Claims

WHAT IS CLAIMED IS:

1. A micro fabricated droplet dispenser, comprising: at least one microfluidic channel formed in a substrate; a first source of target particles and non-target material; a second source of identifying markers for the target particles, wherein the target particles and the identifying markers are suspended in a first fluid flowing in the at least one microchannel; a first microfabricated MEMS fluidic valve, configured to open and close the at least one micro fluidic channel, to separate the target particles into a sort channel and non-target material into a waste channel; at least one additional microfabricated MEMS fluidic valve, configured to open and close the at least one microfluidic channel, to separate the identifying markers; a droplet comprising a quantity of the first fluid, and containing the separated target particle and the separated identifying marker; and a source of a second fluid immiscible with the first fluid wherein the droplet is dispensed into, and immersed in, the second immiscible fluid.

2. The microfabricated droplet dispenser of claim 1, further comprising: a fluid collection zone which accepts the separated target particle fromthe first microfabricated MEMS fluidic valve and the separated identifying marker from the second microfabricated MEMS fluidic valve and concentrates the target particle and the identifying marker.

3. The microfabricated droplet dispenser of claim 1, further comprising: a droplet forming structure that forms a volume of the first fluid wherein this volume contains both the target particle and the identifying marker, and releases the volume into the second immiscible fluid as the droplet.

4. The microfabricated droplet dispenser of claim 1, further comprising: at least one recirculation path which provides fluid communication between the waste channel and at least one of the first source and the second source.

5. The microfabricated droplet dispenser of claim 1, further comprising: permeable magnetic material disposed adjacent to at least one of the first and at least one additional microfabricated valve, wherein the permeable material is configured to reduce interference from magnetic flux directed to the microfabricated valves.

6. The microfabricated droplet dispenser of claim 1, further comprising: a bead disposed in the first fluid, wherein the bead is attached to at least one fluorescent tag, wherein the fluorescent tag identifies 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.

7. The microfabricated droplet dispenser of claim 1, wherein the microfabricated MEMS fluidic valve, moves in a single plane when opening and closing, and wherein that plane is parallel to a surface of the substrate.

8. The microfabricated droplet dispenser of claim 1, wherein the microfabricated MEMS fluidic valve, moves in a single plane when opening and closing, and moves as a result of electromagnetic forces acting on the microfabricated MEMS fluidic valve.

9. The microfabricated droplet dispenser of claim 1, wherein the droplet includes at least one of a target cell and a fluorescently-labelled bead.

10. The microfabricated droplet dispenser of claim 6, wherein the source of immiscible fluid is disposed symmetrically about a nozzle structure formed in the substrate.

11. The microfabricated droplet dispenser of claim 1, wherein a surfactant is added to the first fluid.

12. The microfabricated droplet dispenser of claim 3, wherein the bead comprises a plurality of fluorescent tags, such that the bead has an identifying fluorescent signature.

13. The microfabricated droplet dispenser of claim 9, wherein the bead also comprises at least one antibody, that binds to an antigen on the at least one target particle.

14. The microfabricated droplet dispenser of claim 9, wherein the bead also comprises at least one of DNA and RNA

15. The microfabricated droplet dispenser of claim 7, wherein the source of immiscible fluid is disposed asymmetrically about the nozzle.

16. The microfabricated droplet dispenser of claim 3, further comprising a laser focused on the microfluidic channel and directed onto the droplet, wherein the laser is configured to heat the droplet to coalesce adjacent droplets in the microfluidic channel.

17. The microfabricated droplet dispenser of claim 3, wherein the micro fluidic channel has a channel widened area, wherein the cross section of the channel increases and then decreases.

18. The microfabricated droplet dispenser of claim 3, wherein the microchannel intersects the source of immiscible fluid in a butt junction.

19. The microfabricated droplet dispenser of claim 3, wherein the target particles comprise at least one of T-cells, stem cells, cancer cells, tumor cells, proteins and DNA strands.

20. A method for forming a droplet in an immiscible fluid, comprising: 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; 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 fluid into the immiscible second fluid, 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.

21. The method of claim 16, wherein the first fluid flowing in the micro fluidic channel comprises target particles and non-target material, and the target particles comprise at least one of T-cells, stem cells, cancer cells, tumor cells, proteins and DNA strands.

22. The method of claim 16, further comprising: 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.

23. The method of claim 16, further comprising: 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 microfabricated MEMS fluidic valve; and directing the bead into the droplet, wherein the bead and the target particle, are located within the same droplet.

24. The method of claim 16, wherein the droplet is formed at a nozzle structure formed in the substrate.

25. The method of claim 16, further comprising: heating the droplet of fluid with a laser directed to the droplet.

26. The method of claim 16, further comprising: generating a first sort pulse to capture a labelled bead; and then subsequently generating a second sort pulse to capture a target cell, wherein the sort pulses are generated such that the bead and a target particle are located within the same droplet dispensed into the second immiscible fluid.

27. The method of claim 16, further comprising: generating a first sort pulse to capture a target cell; and then subsequently generating a second sort pulse to capture a labelled bead, wherein the sort pulses are generated such that the bead and a target particle are located within the same droplet dispensed into the second immiscible fluid.