CN110975947A - Device for identifying components in a fluid mixture and device for producing a fluid mixture - Google Patents

Abstract

The present invention relates to an apparatus for identifying components in a fluid mixture and an apparatus for producing a fluid mixture. An apparatus for identifying a constituent in a fluid mixture comprising: (i) a microfluidic chip comprising: (a) a sample input channel disposed in the first structural layer, including a first intersection and a second intersection; (b) a first sheath-like fluid channel disposed in the first structural layer that intersects the sample input channel at a first intersection point; (c) a second sheath fluid channel disposed in the second structural layer that intersects the sample input channel at a second intersection point; and (d) a plurality of output channels fluidly connected to and branching from the sample input channel, each output channel disposed between a pair of recessed portions of the ends of the first structural layer and the second structural layer; (ii) an interrogation device disposed downstream of the second intersection that distinguishes the plurality of components into selected components and unselected components; and (iii) a focusing energy device acting on the selected component.

Description

Device for identifying components in a fluid mixture and device for producing a fluid mixture

The present application is a divisional application of a parent application having an application date of 2013, 16/7, application No. 201380079634.4 and an invention name of "microfluidic chip".

Technical Field

The present invention relates to microfluidic chip designs that utilize laminar flow to separate particles or cellular material into various components and fractions.

Background

1. Field of the invention

The present invention relates to microfluidic chip designs that utilize laminar flow to separate particles or cellular material into various components and fractions.

2. Description of the related Art

In connection with the separation of various particles or cellular material (e.g., separation of sperm into viable and motile sperm and non-viable and non-motile sperm, or by sex), under strict volume constraints, this process is often a time-consuming task. Thus, for example, existing separation techniques do not produce the desired benefits, or process volumes of cellular material in a timely manner.

Accordingly, there is a need for separation techniques and separation devices that are continuous, have high productivity, provide time savings, and cause negligible or minimal damage to the various separated components. In addition, such devices and methods should also have application in the biological and medical fields, not only to sperm sorting, but also to the separation of blood and other cellular material, including viruses, organelles, globular tissues, colloidal suspensions, and other biological materials.

Disclosure of Invention

The present invention relates to a microfluidic chip system comprising a microfluidic chip loaded on a microfluidic chip cartridge mounted on a microfluidic chip holder.

In one embodiment, the microfluidic chip comprises a plurality of layers in which a plurality of channels are disposed, the plurality of channels comprising: a sample input channel into which a sample fluid mixture having components to be separated is input; a plurality of first sheath-like fluid channels into which sheath-like fluid is input, the plurality of first sheath-like fluid channels intersecting the sample input channel at a first intersection point such that the sheath-like fluid compresses the sample fluid mixture on at least two sides such that the sample fluid mixture becomes a relatively small, narrower stream bounded by the sheath-like fluid while maintaining laminar flow in the sample input channel; a plurality of second sheath fluid channels having substantially the same specifications as the plurality of first sheath fluid channels into which sheath fluid is input, the plurality of second sheath fluid channels intersecting the sample input channel at a second intersection point downstream of the first intersection point in a second direction substantially 90 degrees above and below the sample input channel such that the sheath fluid from the plurality of second sheath fluid channels compresses the sample fluid mixture such that the components in the sample fluid mixture are compressed and oriented in a predetermined direction while still maintaining laminar flow in the sample input channel; and a plurality of output channels originating from the sample input channel, the plurality of output channels moving the components and the sheath fluid out of the microfluidic chip.

In one embodiment, the microfluidic chip comprises an interrogation device that interrogates and identifies the component in the sample fluid mixture in the sample input channel in an interrogation chamber disposed downstream of the second intersection.

In one embodiment, the microfluidic chip includes a separation mechanism that separates the selected component of the sample fluid mixture downstream of the interrogation chamber by moving a trajectory of the flow of the sample fluid mixture in the sample input channel and pushing the selected component of the moved flow of the sample fluid mixture into one of the plurality of output channels leading from the interrogation chamber.

In one embodiment, the microfluidic chip further comprises at least one ejection chamber containing a sheath fluid introduced into the ejection chamber through at least one air vent; and at least one ejection channel connected to the at least one ejection chamber, the at least one ejection channel entering the sample input channel at the interrogation chamber.

In one embodiment, the separation mechanism comprises at least one piezoelectric actuator assembly disposed on at least one side of the sample input channel.

In one embodiment, the piezoelectric actuator assembly is an externally stacked piezoelectric actuator assembly.

In one embodiment, the microfluidic chip further comprises a membrane covering each of the ejection chambers; and wherein the outer stacked piezoelectric actuator assembly is aligned with and moves the diaphragm to drive the sheath fluid in the ejection chamber into the sample input channel to move the trajectory of the stream of the sample fluid mixture in the sample input channel into one of the plurality of output channels.

In one embodiment, the external stacked piezoelectric actuator assembly is disposed in a microfluidic chip holder.

In one embodiment, the microfluidic chip further comprises electronic circuitry connected to the piezoelectric actuator assembly, the electronic circuitry amplifying electrical signals generated by resistance from the piezoelectric actuator in contact with the membrane.

In one embodiment, the electrical signal from the piezoelectric film indicates how much strain is generated by the outer stacked piezoelectric actuator assembly.

In one embodiment, an indicator of contact is automatically activated when contact is made between the piezoelectric actuator and the diaphragm.

In one embodiment, when the sensing of contact is made, the electrical signal exceeds a set threshold and the piezoelectric actuator assembly compresses the ejection chamber to eject sheath fluid from the ejection chamber into the sample fluid channel.

In one embodiment, the indicator of contact comprises light, sound, tactile, or any combination thereof.

In one embodiment, the piezoelectric actuator assembly includes a flexible diaphragm covering the ejection chamber; and a piezoelectric material bonded on the top surface of the diaphragm by a bonding mechanism.

In one embodiment, when a voltage is applied across the electrodes of the piezoelectric actuator assembly, the flexible membrane flexes into the ejection chamber and squeezes the sheath fluid from the ejection chamber into the sample input channel to deflect the selected constituent into one of the plurality of output channels.

In one embodiment, the injection channel is tapered when it is connected to the sample input channel.

In one embodiment, the microfluidic chip further comprises a plurality of output ports disposed at ends of the plurality of output channels.

In one embodiment, the plurality of output channels increase in size from the sample input channel.

In one embodiment, the microfluidic chip further comprises a plurality of indentations disposed at a bottom edge of the microfluidic chip for spacing the plurality of output ports.

In one embodiment, the sample input channel and the plurality of sheath channels are disposed in one or more planes of the microfluidic chip.

In one embodiment, the sample input channel and the plurality of sheath channels are disposed in one or more structural layers of the microfluidic chip or between structural layers of the microfluidic chip.

In one embodiment, at least one of the plurality of sheath channels is disposed in a different plane than the plane in which the sample input channel is disposed.

In one embodiment, at least one of the plurality of sheath channels is disposed in a different structural layer than the structural layer in which the sample input channel is disposed.

In one embodiment, the sample input channel tapers at an entrance point into the first intersection with the plurality of sheath channels.

In one embodiment, the sample input channel tapers into the interrogation chamber.

In one embodiment, at least one of the first intersection or the second intersection, the plurality of sheath-like fluid channels taper at an entry point into the sample input channel.

In one embodiment, the interrogation chamber includes an opening cut through the structural layer in the microfluidic chip; and a first cover configured to be received in an opening in at least one of the structural layers; and a second cover configured to be received in an opening in at least one of the structural layers.

In one embodiment, the interrogation chamber includes an opening cut through the plane in the microfluidic chip; and a first cover configured to be received in an opening in at least one of the planes of the microfluidic chip; and a second cover configured to be received in an opening in at least one of the planes of the microfluidic chip.

In one embodiment, the interrogation device includes a light source configured to emit a light beam through a first cover to illuminate and stimulate the constituent in the sample fluid mixture; and wherein emitted light induced by the light beam passes through the second cover and is received by the objective lens.

In one embodiment, the interrogation device includes a light source configured to emit a light beam through a structural layer of the microfluidic chip to illuminate and stimulate the component in the sample fluid mixture; and wherein emitted light induced by the light beam is received by the objective lens.

In one embodiment, the interrogation device includes a light source configured to emit a light beam through the plane of the microfluidic chip to illuminate and stimulate the component in the sample fluid mixture; and wherein emitted light induced by the light beam is received by the objective lens.

In one embodiment, the emitted light received by the objective lens is converted into an electrical signal that triggers the piezoelectric actuator assembly.

In one embodiment, one of the sample fluid mixture or the sheath fluid is pumped into the microfluidic chip by a pumping device.

In one embodiment, an external conduit delivers fluid to the microfluidic chip.

In one embodiment, the component is a cell.

In one embodiment, wherein the cells to be isolated comprise at least one of: viable and motile sperm separated from non-viable and non-motile sperm; sperm separated by gender and other gender classification variations; stem cells isolated from a population of cells; one or more labeled cells separated from unlabeled cells including sperm cells; cells including sperm cells differentiated by a desired or undesired characteristic; genes isolated in nuclear DNA according to defined characteristics; cells isolated based on surface markers; cells isolated based on membrane integrity or viability; cells isolated based on a potential or predicted reproductive state; cells isolated based on their ability to survive freezing; cells separated from contaminants or debris; healthy cells separated from damaged cells; red blood cells separated from white blood cells and platelets in a plasma mixture; or any cell separated into corresponding parts with any other cellular components.

In one embodiment, the separated components are moved into one of the plurality of output channels and the unselected components flow out through another of the plurality of output channels.

In one embodiment, the microfluidic chip further comprises a computer that controls pumping of one of the sample fluid mixture or the sheath fluid into the microfluidic chip.

In one embodiment, the microfluidic chip further comprises a computer that displays the components in a field of view captured by a CCD camera disposed over the opening in the microfluidic chip.

In one embodiment, a microfluidic chip system comprises: a microfluidic chip loaded on a microfluidic chip cartridge mounted on a microfluidic chip holder, the microfluidic chip having a sample input for introducing a sample fluid into the microfluidic chip and a sheath input for introducing a sheath fluid into the microfluidic chip; and a pumping mechanism that pumps the sample fluid from a reservoir into the sample input port of the microfluidic chip and pumps the sheath fluid into the sheath input port of the microfluidic chip.

In one embodiment, a method of orienting and separating components in a fluid mixture, the method comprising: inputting a sample fluid mixture comprising components into a sample input channel of a microfluidic chip; inputting a sheath fluid into a plurality of first sheath fluid channels of the microfluidic chip, the sheath fluid from the first sheath fluid channels combining the sample fluid mixture in the sample input channel at a first intersection of the plurality of first sheath fluid channels with the sample input channel; wherein the sheath fluid from the first sheath fluid channel compresses the sample fluid mixture in the sample input channel in a direction to focus the components in the sample fluid mixture around a center of the sample input channel; and inputting a sheath fluid into a plurality of second sheath fluid channels of the microfluidic chip, the sheath fluid from the plurality of second sheath fluid channels combining the sample fluid mixture in the sample input channel at a second intersection of the plurality of second sheath fluid channels and the sample input channel downstream of the first intersection; wherein at the second intersection point, the sheath fluids from the plurality of second sheath fluid channels also compress the sample fluid mixture in a second direction such that the components are focused and aligned with a center of the sample input channel in width and depth as the components flow through the sample input channel; and wherein the sheath fluid acts on the component to compress and orient the component in a selected direction as it flows through the sample fluid channel.

There has thus been outlined, some of the features according to the invention in order that the detailed description thereof that follows may be better understood, and in order that the present contribution to the art may be better appreciated. There are, of course, additional features of the invention that will be described hereinafter and which will form the subject matter of the claims appended hereto.

In this respect, before explaining at least one embodiment in accordance with the invention in detail, it is to be understood that the invention is not limited in its application to the details of construction and to the arrangements of the components set forth in the following description or illustrated in the drawings. The methods and apparatus according to the present invention are capable of other embodiments and of being practiced and carried out in various ways. Also, it is to be understood that the phraseology and terminology employed herein, as well as the abstract included below, are for the purpose of description and should not be regarded as limiting.

As such, those skilled in the art will appreciate that the conception upon which this disclosure is based may readily be utilized as a basis for the designing of other structures, methods and systems for carrying out the several purposes of the present invention. It is important, therefore, that the claims be regarded as including such equivalent constructions insofar as they do not depart from the spirit and scope of the methods and apparatuses according to the present invention.

Drawings

The objects, features and advantages of the present invention will be more readily appreciated by reference to the following disclosure when considered in connection with the accompanying drawings wherein:

FIG. 1 shows an exploded perspective view of a schematic embodiment of a microfluidic chip according to an embodiment of the present invention;

fig. 2A-2C show top views of the assembled microfluidic chip of fig. 1, according to a variant embodiment of the present invention;

FIG. 3 illustrates a cross-sectional view of an interrogation chamber of the microfluidic chip of FIGS. 1-2, according to one embodiment of the present invention;

FIG. 4 shows a cross-sectional internal view of a graphical interrogation by a light source of a component flowing in a fluid mixture through the microfluidic chip of FIGS. 1-2, and a graphical action of one of two (mirrored) piezoelectric actuator assemblies, according to one embodiment of the present invention;

FIG. 5A shows an oblique view of a perspective interior of a schematic operation of the composition and two-step focusing flowing through the microfluidic chip of FIGS. 1-2, according to one embodiment of the present invention;

FIG. 5B shows a perspective oblique view of the channels and interrogation chambers provided in the microfluidic chip of FIGS. 1-2C, according to one embodiment of the present invention;

FIG. 6 shows a schematic illustration of a front view of a body of a microfluidic chip holder according to one embodiment of the present invention;

FIG. 7 shows a schematic illustration of a side view of a piezoelectric actuator assembly of the microfluidic chip holder of FIG. 6, according to one embodiment of the present invention;

FIG. 8 shows a schematic illustration of a front view of a microfluidic chip holder according to an embodiment of the present invention;

figure 9 illustrates a pumping mechanism that pumps a sample fluid and a sheath or buffer fluid into a microfluidic chip, according to one embodiment of the present invention.

Detailed Description

Before turning to the figures, which illustrate the embodiments in detail, it is to be understood that the invention is not limited to the details or methodology set forth in the description or illustrated in the figures. It is also to be understood that the terminology is for the purpose of description only and is not to be interpreted as limiting. Work has been done to refer to the same or similar parts throughout the drawings using the same or similar reference numbers.

The present invention relates to a microfluidic chip design that utilizes laminar flow to separate particle or cellular material (e.g., sperm and other particles or cells) into various components and fractions.

Various embodiments of the invention provide for the separation of components in a mixture, such as: separating viable and motile sperm from non-viable and non-motile sperm; separating the sperm according to gender and other gender classification variations; isolating stem cells from the cell population; separating one or more labeled cells differentiated by the desired/undesired characteristic from unlabeled cells; isolating genes in the nuclear DNA according to a defined characteristic; isolating cells based on the surface markers; isolating cells based on membrane integrity (viability), potential or predicted reproductive status (fertility), ability to survive after freezing, and the like; separating the cells from the contaminants or debris; separating healthy cells from damaged cells (i.e., cancer cells) (e.g., in bone marrow extract); separating the red blood cells from the white blood cells and platelets in the plasma mixture; and separating any cell from any other cellular components into corresponding fractions.

In addition, the subject matter of the present invention is also suitable for other medical applications. For example, the various laminar flows discussed below may be used as part of a renal dialysis procedure in which whole blood is cleared of waste and returned to the patient. In addition, the various embodiments of the present invention may be further applied in other biological or medical fields, for example for the separation of cells, viruses, bacteria, organelles or cell subsets, globular tissues, colloidal suspensions, lipids and fat particles, gels, immiscible particles, blastomeres, aggregated cells, microorganisms and other biological substances. For example, component separation according to the present invention may include cell "washing", in which contaminants (e.g., bacteria) are removed from a cell suspension, which is particularly useful in medical and food industry applications. Notably, the prior art flow-based techniques have not recognized any application to the separation of inactive cellular components, as recognized in the present invention.

The subject matter of the present invention can also be utilized to move species from one solution to another in cases where separation by filtration or centrifugation is impractical or unsatisfactory. In addition to the applications discussed above, additional applications include, for example, separating colloids of a given size from colloids of other sizes (for research or commercial applications), and washing particles such as cells, ova, etc. (effectively replacing the medium containing them and removing contaminants) or washing particles such as nanotubes from solutions of salts and surfactants with different salt concentrations or salt solutions without surfactants.

The action of separating species may depend on several physical properties of the object or component, including automotility, self-diffusion, free fall velocity, or action under an external force (e.g. an actuator, an electric field, or a holographic optical trap). For example, properties that may be classified include cell motility, cell activity, object size, object mass, object density, tendency of objects to attract or repel each other or other objects in a flow, object charge, object surface chemistry, and tendency of certain other objects (i.e., molecules) to adhere to objects.

Various embodiments of microfluidic chips as discussed below utilize one or more flow channels, with multiple independently present laminar flows, allowing one or more components to be interrogated for identification and separated into streams exiting into one or more outlets. In addition, the various components of the mixture can be separated on the chip, for example, by removal using additional separation mechanisms (e.g., flow mechanisms) or optical tweezers or holographic optical traps, or by magnetism (i.e., using magnetic beads). Various embodiments of the present invention thus provide for separation of components on a continuous bottom (e.g., within a continuous, closed system) without potential damage and contamination of existing processes, particularly as provided in sperm separation. The continuous process of the present invention also provides significant time savings in separating the components.

While the following discussion focuses on separating sperm into viable and motile sperm and non-viable and non-motile sperm, or separating sperm by gender and other gender classification variations, or separating one or more labeled cells from unlabeled cells that differentiate between desired/undesired characteristics, etc., the devices, methods, and systems of the present invention may be extended to other types of particles, biological substances, or cellular substances that can be interrogated within a fluid flow by fluorescence techniques, or that can be manipulated between different fluids flowing into different outlets.

Although the present subject matter is discussed in detail with reference to themicrofluidic chip 100 shown in fig. 1-5B and themicrofluidic chip holder 200 shown in fig. 6-9, it should be understood that the discussion applies equally to the various other embodiments discussed herein or any variations thereof.

Microfluidic chip assembly

Fig. 1 is an illustrative embodiment of amicrofluidic chip 100. Themicrofluidic chip 100 is made of a suitable thermoplastic (e.g., a low autofluorescent polymer, etc.) by a molding process or an injection molding process (as known to those of ordinary skill in the art) and has suitable dimensions.

Themicrofluidic chip 100 includes a plurality of structural layers in which microchannels, sheath-like or buffer fluid channels, output channels, and the like, serving as sample input channels are disposed. The microchannels are of suitable size to accommodate laminar flow of particles and may be provided in any layer of thechip 100 at an appropriate length so long as the objectives of the present invention are achieved. The desired flow rate through themicrofluidic chip 100 may be controlled by a predetermined introduction flow rate introduced into thechip 100 by a pumping mechanism, by maintaining proper microchannel specifications within thechip 100, by providing narrowed microchannels at various locations, and/or by providing obstructions or partitions within the microchannels.

A plurality of input ports are provided into themicrofluidic chip 100, which provide access to the microchannels/channels. In one embodiment, as shown in fig. 1-2C, thesample input port 106 is used to introducesample components 160 in a sample fluid mixture 120 (see fig. 4-5B) from a reservoir source (see fig. 9) into asample input channel 164A of themicrofluidic chip 100. Themicrofluidic chip 100 also includes at least one sheath/buffer input (in one embodiment, sheath/buffer input 107, sheath/buffer input 108) for introducing a sheath fluid or buffer fluid. In one embodiment, there are two sheath/buffer inputs in themicrofluidic chip 100, which include a sheath/buffer input 107 and a sheath/buffer input 108, both of which are disposed proximate to thesample input 106, and both of which introduce sheath or buffer fluid into themicrofluidic chip 100. Sheath or buffer fluids are known in the microfluidic art and may, in one embodiment, contain nutrients known in the art to maintain the activity of the components 160 (i.e., sperm cells) in the fluid mixture. The positions of the sheath/buffer input 107, sheath/buffer input 108 may vary and they may enter microchannels in thechip 100 in the same or different structural layers.

In one embodiment, fill holes or air vents 121, 122 (assuming no seals) may be used to introduce sheath or buffer fluids intoejection chambers 130, 131 (described below).

In one embodiment, a plurality of output channels originating from the main channel 164 (see fig. 2A) are provided to remove fluids (including theseparation component 160 and/or sheath or buffer fluids) that have flowed through themicrofluidic chip 100. In one embodiment as shown in fig. 1-2C, there are three output channels 140-142, including aleft output channel 140, acenter output channel 141, and aright output channel 142. Theleft output channel 140 ends at the first output port 111, thecenter output channel 141 ends at thesecond output port 112, and theright output channel 142 ends at thethird output port 113. However, the number of output ports may be less or greater depending on the number ofcomponents 160 to be separated from thefluid mixture 120.

In one embodiment, instead of straight edges, a plurality of notches orgrooves 146 are provided at the bottom edge of themicrofluidic chip 100, if necessary, to separate the output ports (i.e., output ports 111 and 113), for attachment of external tubing, and the like. To the first output port 111, thesecond output port 112, and thethird output port 113 viaoutput channels 140 and 142 originating from the interrogation chamber 129 (see fig. 2A-4).

In one embodiment, themicrofluidic chip 100 has multiple structural layers with microchannels disposed in the multiple structural layers. The channels may be provided in one or more layers or between layers. In one embodiment, as shown in FIG. 1, four layers of structural plastic 101-104 are shown, by way of example, to form amicrofluidic chip 100. However, one of ordinary skill in the art will appreciate that fewer or additional layers may be used and that the channels may be provided in any layer so long as the objectives of the present invention are achieved.

Gaskets or O-rings of any desired shape may be provided to maintain a tight seal between themicrofluidic chip 100 and the microfluidic chip holder 200 (see fig. 6). In the case of a gasket, it may be a single sheet or multiple components in any configuration, or the desired material (i.e., rubber, silicone, etc.). In one embodiment, as shown in fig. 1, afirst gasket 105 is disposed at one end of themicrofluidic chip 100 and is connected to thelayer 104 or adhered to thelayer 104 with an epoxy. A plurality ofapertures 144 are disposed in thefirst gasket 105 and are configured to align with thesample input port 106, the sheath/buffer input port 107, the sheath/buffer input port 108, and the air vents 121, 122 to provide access thereto.

In one embodiment, asecond gasket 143 is disposed at the other end of themicrofluidic chip 100 opposite thefirst gasket 105 and is connected to the topstructural layer 104 or bonded to the topstructural layer 104 with an epoxy. Thesecond gasket 143 is configured to assist in sealing and stabilizing or balancing themicrofluidic chip 100 in the microfluidic chip holder 200 (see fig. 6).

In one embodiment, wells andalignment posts 145 are provided at various convenient locations inmicrofluidic chip 100 to secure and align the various layers (i.e., layers 101-104) during chip fabrication.

In one embodiment, asample fluid mixture 120 including acomponent 160 is introduced into thesample input port 106, and thefluid mixture 120 flows through theprimary channel 164 and toward the interrogation chamber 129 (see fig. 2A, 4, 5A, and 5B). Sheath or buffer fluid 163 is introduced into sheath/buffer input 107, sheath/buffer input 108, and flows throughchannel 114,channel 115 andchannel 116,channel 117, respectively, and intomain channel 164 and towardinterrogation chamber 129 before exiting throughoutput channel 140 and 142.

In one embodiment, if thechambers 130, 131 are not filled with the sheath or buffer fluid 163 during the manufacturing process, the sheath or buffer fluid 163 may be introduced into theejection chambers 130, 131 through the air vents 121, 122 after themicrofluidic chip 110 is manufactured to fill thechambers 130, 131. As mentioned above, the sheath or buffer fluid 163 used is well known to those of ordinary skill in the art of microfluidics.

In one embodiment, thefluid mixture 120 from theprimary channel 164 is combined with the sheath or buffer fluid 163 from thechannels 114, 115 at anintersection 161 in the same plane of themicrofluidic chip 100. In one embodiment, downstream of thesecond junction 162, the buffer fluid 163 from thechannels 116, 117 combines the combinedfluid mixture 120 and the sheath or buffer fluid 163 from thefirst junction 161. In one embodiment,channels 114, 115 are substantially the same gauge aschannels 116, 117, so long as the desired flow rate is achieved to achieve the objectives of the present invention.

In one embodiment,channels 114 and 117,channels 123, 124, 140 and 142, 125a, 125b, 126a, 126b, 127 and 128 may have substantially the same dimensions, however, one of ordinary skill in the art will recognize that the dimensions of any or all of the channels inmicrofluidic chip 100 may vary in dimension (i.e., between 50 and 500 microns) as long as the desired flow rate is achieved for the purposes of the present invention.

In one embodiment, thechannels 114, 123, 124, 140, 142, 125a, 125b, 126a, 126b, 127, 128 of themicrofluidic chip 100 may not only vary in size, but may also have a tapered shape at the entry point of other channels in thechip 100 in order to control the flow of fluid through these channels. For example, theprimary channel 164 may be tapered at the point of entry of the intersection 161 (see fig. 5B) to control and expedite the flow of thesample 120 into theintersection 161, and to allow the sheath or buffer fluid 163 from thechannels 114, 115 to compress thesample 120 in a first direction (i.e., horizontally) on at least two sides, provided not on all sides (depending on the location of the taperedchannel 164 in conjunction with thechannel 164A). Thus, thesample fluid mixture 120 becomes a relatively small, narrow fluid stream bounded or surrounded by the sheath or buffer fluid 163 while maintaining laminar flow in thechannel 164A. However, one of ordinary skill in the art will appreciate that theprimary channels 164 entering theintersection 161 may have any physical arrangement, such as rectangular or circular shaped channels, as long as the objectives of the present invention are achieved.

In one exemplary embodiment, at least one of thechannels 116, 117 is disposed in a different structural layer of themicrofluidic chip 100 than the layer in which thechannels 164 are disposed. For example,channel 116 may be disposed inlayer 103 andchannel 117 may be disposed in layer 101 (see fig. 1) such that when sheath or buffer fluid 163 joinsfluid mixture 120 atintersection 162,channel 116,channel 117 are in a different plane thanother channels 164 and 114, channel 115 (in layer 102). In one embodiment, theprimary channels 164 are disposed between thelayers 102, 103 (see fig. 3); however, one of ordinary skill in the art will appreciate thatchannels 114 and 117,channels 164,channels 123,channels 124,channels 140 and 142, channels 125a,channels 125b,channels 126a,channels 126b,channels 127,channels 128, and the like may be disposed in any layer or between any two layers. In addition, althoughchannels 114 and 117,channels 164,channels 123,channels 124,channels 140 and 142,channels 125a, 125b,channels 126a, 126b,channels 127,channels 128, etc. are described in the exemplary embodiment as shown in the figures, one of ordinary skill in the art will appreciate that the particular arrangement or layout of the channels onchip 100 may be any desired arrangement so long as they achieve the features described in the present invention.

In one embodiment, the sheath or buffer fluid inchannels 116, 117 combines the fluid mixture via holes cut inlayers 101 and 103 at locations substantially perpendicular above and belowintersection 162. The sheath or buffer fluid from thechannels 116, 117 compresses the flow of thefluid mixture 120 in a perpendicular manner relative to thechannel 164B such that thecomponents 160 in thefluid mixture 120 are compressed or flattened and oriented in a selected or desired direction (see below) while still maintaining laminar flow in thechannel 164B.

In one embodiment, as shown in fig. 1-2C,channels 114, 115 and 116, 117 are depicted as being partially coaxial with each other relative to a center point defined bysample input 106. Thus, in one embodiment,channels 114, 115 and 116, 117 are disposed in a substantially parallel arrangement, withchannels 114, 115 and 116, 117 being equidistant fromprimary channel 164. However, one of ordinary skill in the art will recognize that the configurations described may be different so long as the desired features of the invention are achieved.

Additionally, in one embodiment,channels 114, 115 preferably joinintersection points 161 in the same plane at an angle of 45 degrees or less, whilechannels 116, 117 of parallelsample input channels 164A joinintersection points 162 from different layers at an angle of substantially 90 degrees. However, one of ordinary skill in the art will appreciate that the configuration, angle, and structural arrangement of the layers or channels of the describedmicrofluidic chip 100 may be different so long as they achieve the desired features of the present invention.

In one embodiment, downstream of theintersection 162, thecomponent 160 in thefluid mixture 120 flows through thechannel 164B into theinterrogation chamber 129 where thecomponent 160 is interrogated.

In one embodiment,ejection chambers 130, 131 are covered byflexible diaphragm 170, 171 (see fig. 1) made of a suitable material, such as one of stainless steel, brass, titanium, nickel alloy, polymer, or other suitable material having a desired elastic response. In one embodiment, an actuator is disposed on at least one side ofchannel 164B and interrogation chamber 129 (see fig. 2A and 2B) to mechanically displacediaphragms 170, 171 to eject or push sheath or buffer fluid 163 from one ofejection chamber 130,ejection chamber 131 on that side ofchannel 164B to pushingredient 160 fromchannel 164C into one ofoutput channels 140, 142 on the other side ofchannel 164B. In other words, the actuator ejects the sheath or buffer fluid 163 from theejection chamber 130 into thechannel 164C and pushes thetarget component 160 of thechannel 164C into theoutput channel 142 to separate the target component from thefluid mixture 120. This embodiment is useful when only one type oftarget component 160 is separated, which may, for example, only require twooutput channels 141, 142 instead of threeoutput channels 140, 142 (see fig. 2B).

The actuator may be of a piezoelectric type, a magnetic type, an electrostatic type, a hydraulic type, or a pneumatic type. Although a disc-shaped actuator assembly (i.e., 109, 110) is shown in fig. 1-2C, one of ordinary skill in the art will appreciate that any type or shape of actuator may be used that performs the desired function.

In other embodiments, the actuators are disposed on either side of thechannel 164B (as shown in fig. 2A), but in other embodiments more than one (relatively smaller sized) actuator may be disposed on one or more sides of thechannel 164B and connected to thechannel 164B via a jetting channel (see fig. 2C).

The function of the actuator will be described below with reference to fig. 2A, although any type of actuator disposed at a location on thechip 100 is known to those of ordinary skill in the art to be acceptable as long as it implements the features of the present invention.

In one embodiment, to activatediaphragm 170,diaphragm 171 and eject sheath or buffer fluid 163 fromchamber 130,chamber 131 intochannel 164B, two external, stackedpiezoelectric actuator assemblies 209, 210 (see fig. 6 and 7) are provided, the twopiezoelectric actuator assemblies 209, 210 aligning withdiaphragm 170,diaphragm 171 andactuating diaphragm 170,diaphragm 171. External, stackedpiezoelectric actuator assemblies 209, 210 are disposed in themicrofluidic chip holder 200. Stackedpiezoelectric actuator assemblies 209, 210 each include apiezoelectric actuator 219, 220, respectively,piezoelectric actuator assembly 209, 210 having a high resonant frequency and each disposed at a location centered on and in contact withdiaphragm 170, 171 to squeeze sheath or buffer fluid 163 fromchamber 130, 131 intochannel 164C.

Themicrofluidic chip holder 200 may be of any type known to those of ordinary skill in the art and is configured to precisely position thepiezoelectric actuators 219, 220 such that thepiezoelectric actuators 219, 220 may maintain continuous contact with thediaphragms 170, 171 of themicrofluidic chip 100. For example, in one embodiment, this is accomplished by mounting (or bonding with a suitable epoxy)piezoelectric actuator assembly 209,piezoelectric actuator assembly 210 onlockable adjustment screw 201 andthumbscrew 202, respectively,lockable adjustment screw 201 causingpiezoelectric actuator 219,piezoelectric actuator 220 to move into position againstdiaphragm 170,diaphragm 171, respectively; the threaded body of thethumb screw 202 is used to move thescrew 202 against thediaphragm 170, 171 for stability. Thespacers 203 attached to thepiezoelectric actuators 219, 220 allow a feasible contact between thespacers 203 and themembranes 170, 171 of themicrofluidic chip 100. Adjustment screws 201 allow a user to adjust the position ofpiezoelectric actuators 209, 210 relative tomicrofluidic chip 100 for coarse and fine adjustments. The thumb screws 202 may be tightened to secure thepiezoelectric assemblies 209, 210 to themain die body 100, or the thumb screws 202 may be loosened to detach thepiezoelectric actuator assemblies 209, 210 from themain die body 100.

In one embodiment, at least one piezoelectric actuator (209 or 210) is mounted on a plate (not shown) that can translate in a direction orthogonal to the diaphragm (170 or 171) of themicrofluidic chip 100. Theadjustment screw 201 is mounted on theholder 200 and can be extended and retracted by turning thescrew 201. The tip of the adjustingscrew 201 abuts against the plate. When thescrew 201 is extended, the plate and thepiezoelectric actuators 209, 210 are pushed in a translational movement towards thediaphragm 170, 171, so as to make possible contact between thepiezoelectric actuators 209, 210 and thediaphragm 170, 171. With this approach, the positioning ofpiezoelectric actuators 209, 210 is adjusted solely by the translation ofpiezoelectric actuators 209, 210, whereas in previous embodiments wherepiezoelectric actuators 209, 210 were mounted directly toadjustment screw 201, the positioning ofpiezoelectric actuators 209, 210 was a combination of the translation and rotation ofpiezoelectric actuators 209, 210, during which damage to fragilepiezoelectric actuators 209, 210 may be caused.

In another embodiment, the electronic circuitry is coupled to the stackedpiezoelectric actuator assemblies 209, 210 prior to driving the stackedpiezoelectric actuator assemblies 209, 210. When each of the stackedpiezoelectric actuators 219, 220 is in contact with therespective diaphragm 170, 171, the resistance from thediaphragms 170, 171 induces a strain on the stackedpiezoelectric actuators 219, 220, which generates an electrical signal. Thus, the electronic circuitry can amplify the electrical signal to a predetermined value to trigger a Light Emitting Diode (LED). The LED automatically turns on when the stackedpiezoelectric actuators 219, 220 are in contact with thediaphragms 170, 171, indicating that contact is made between the stackedpiezoelectric actuators 219, 220 and thediaphragms 170, 171. This contact sensing allows sufficient force foractuators 219, 220 to compresschambers 130, 131, thereby injecting fluid 163 intochannel 164B.

It will be clear to one of ordinary skill in the art that an LED is one example of a contact indicator. For example, once a contact is made and the electrical signal exceeds a set threshold, feedback is generated to the user, which may be in any of the following forms: light (i.e., LED), sound (i.e., buzzer), tactile (i.e., vibrator), or any combination thereof. Thus, the user may stop adjusting the contact and maintain the contact. Of course, in one embodiment, the process described above may be automated.

In an alternative embodiment, instead of at least one external, stacked piezoelectric actuator assembly, a thin film of piezoelectric material (as is well known to those of ordinary skill in the art) is disposed directly on the top surface of at least onediaphragm 170, 171 to form at least onepiezoelectric actuator assembly 109, 110 (see fig. 2A and 4) to displace (flex) therespective diaphragm 170, 171 and drive fluid in therespective ejection chamber 130, 131 intochannel 164C, respectively. The piezoelectric material is permanently bonded to theflexible diaphragm 170, 171, previously described, by an adhesive mechanism. Thus, in this embodiment, when a voltage is applied across the electrodes of thepiezoelectric actuator assembly 109, 110, theentire diaphragm 170, 171 flexes into thechamber 130, 131 and squeezes the fluid 163 in thechamber 130, 131 into thechannel 164C to deflect the target or selectedcomponent 160 into theside output channel 140, 142.

As described above with respect to externally stackedpiezoelectric actuator assemblies 209, 210 or 109, 110, in one embodiment, only one piezoelectric actuator assembly may be required to eject sheath or buffer fluid 163 fromejection chamber 130 intochannel 164C and pushtarget component 160 inchannel 164C intooutput channel 142 to separate the target component fromfluid mixture 120, as shown in fig. 2B.

In one embodiment, thepiezoelectric actuator assemblies 109, 110 are used to seal theejection chambers 130, 131 at the layer 103 (but one of ordinary skill in the art would know can be in any structural layer) so that themicrofluidic chip 100 is not affected by fluid leakage, for example, after thechambers 130, 131 are filled with sheath or buffer fluid 163, respectively.

Thus,piezoelectric actuator assemblies 109, 110 meet the low flow rate requirements and the low force requirements ondiaphragms 170, 171 in view of the relatively small bending displacements ofdiaphragms 170, 171, as compared to the large displacements and strong forces exerted by externally stackedpiezoelectric actuator assemblies 209, 210, which are capable of operating at very high flow rates. However, one of ordinary skill in the art will appreciate that theactuator assemblies 109, 110, 209, 210 used in themicrofluidic chip 100 may be independently selected based on different operating speeds and flow rate requirements.

In one embodiment, the piezoelectric film disposed on top of thediaphragms 170, 171 operates as a strain sensor to determine how much strain or displacement the outer stackedpiezoelectric actuator assemblies 209, 210 generate when the outer stackedpiezoelectric actuator assemblies 209, 210 are triggered by an electrical signal to displace therespective diaphragms 170, 171. The diameter and thickness of the piezoelectric film depend on the cross section of the externally stackedpiezoelectric actuator 219,piezoelectric actuator 220 and the forces generated on thediaphragms 170, 171. The piezoelectric film anddiaphragms 170, 171 may be different from the corresponding components discussed above in alternative embodiments.

The filling ofejection chambers 130, 131 will now be described. In one embodiment, air vents 121, 122 are provided to remove air fromejection chambers 130, 131 respectively after fabrication ofchambers 130, 131 filling with sheath or buffer fluid 163 (forcing air out through air vents 121, 122) and before sealingchambers 130, 131 with sheath or buffer fluid 163 inchambers 130, 131 (see fig. 2A). Alternatively, in another embodiment, if the air vents 121, 122 remain open, the sheath or buffer fluid 163 may be directed through the vents 121, 122 into thechambers 130, 131, provided this is not done during the manufacturing process. The sheath or buffer fluid or other fluid 163 disposed inejection chamber 130, 131 may be the same or different than the sheath or buffer fluid 163 input throughchannel 114,channel 115,channel 116 orchannel 117.

In one embodiment, if sheath or buffer fluid 163 is used to fillejection chambers 130, 131, they may be input through input port 121, input port 122, and flow throughchannels 123, 124, respectively, to enterejection chamber 130 viachannels 125a and 125b, and to enterejection chamber 131 viachannels 126a and 126 b.

In one embodiment,ejection channel 127exits ejection chamber 130 andejection channel 128exits ejection chamber 131, and bothejection channel 127 andejection channel 128 enter interrogation chamber 129 (see FIG. 2A). Theejection chambers 127,ejection channels 128 may be disposed in any layer of thechip 100 and enter thechannels 164C in the same plane at any angle.

In one embodiment, to form a strong, transient jet, thejet channels 127, 128 may be tapered as they connect to theprimary channel 164C. However, one of ordinary skill in the art will appreciate that theinjection channels 127, 128 may have a particular angle or have different configurations as long as they achieve the features described in the present invention.

In one embodiment,injection channels 127, 128 operate to displace or flexdiaphragm 170,diaphragm 171, respectively, and inject or squeeze sheath or buffer fluid 163 intochannel 164C. However, whendiaphragms 170, 171 return to the neutral (unbent) position,ejection channels 127, 128 emanating fromejection chambers 130, 131 operate as dispersers to ensure that a net fluid volume is maintained fromejection chambers 130, 131 to channel 164C and to ensure thatchambers 130, 131 are easily refilled with sheath or buffer fluid 163.

In one embodiment, output channels 140-142 are fromchannel 164C withininterrogation chamber 129 to output port 111-113. As described above, in one embodiment, more than one on-chippiezoelectric actuator assembly 109,piezoelectric actuator assembly 110, or externally stackedpiezoelectric actuator assembly 209, piezoelectric actuator assembly 210 (in any size or in any location) may be used to connect to each ofejection channel 127, 128 to provide additional motive force to eject sheath or buffer fluid 163 fromejection chamber 130, 131 intochannel 164C. In one embodiment, the distance from eachinjection channel 127, 128 of theinlet channel 164C to each output channel 140-142 should be shorter than the distance between thecomponents 160 to avoid mixing of thetarget component 160 with undesired components 160 (described further below). In one embodiment, the cross-section and length of the output channels 140-142 should be maintained at a predetermined volume ratio (i.e., 2:1:2 or 1:2:1, etc.) to achieve the desired hydraulic resistance of the output channels 140-142.

In one embodiment, interrogation devices are positioned downstream of the location wherechannels 116, 117enter channel 164B. In one embodiment, thechannel 164B tapers into theinterrogation chamber 129, which accelerates the flow of the fluid mixture through theinterrogation chamber 129. However, one of ordinary skill in the art will appreciate that thechannel 164B need not be tapered and can have any size and dimensions so long as the invention performs as desired.

The interrogation device is used to interrogate and identify theconstituent 160 of the fluid mixture in thechannel 164B that passes through theinterrogation chamber 129. Note that thechannel 164B may be disposed in a single layer (i.e., layer 102), or may be disposed between layers (i.e.,layer 102, layer 103). In one embodiment, theinterrogation chamber 129 includes an opening or window 149 (see fig. 3) cut into themicrofluidic chip 100 in at least the uppermost layer (i.e.,layer 104 or other layer), and another opening orwindow 152 is cut into thechip 110 in at least the lowermost layer (i.e.,layer 101 or other layer).

In one embodiment, opening 150 is cut through layer 101-104 into the microfluidic chip. In one embodiment, thetop window 149 is configured to receive thefirst cover 133 and thebottom window 152 is configured to receive thesecond cover 132. However, thewindows 149, 152 may be located in any suitable layer, not necessarily in the uppermost/lowermost layer. Thecovers 133, 132 may be made of any material, such as plastic, glass, or even a lens, depending on the desired transmission requirements. Note that although the relative diameters ofwindow 149,window 152, andopening 150 are shown in fig. 3, these may vary depending upon manufacturing considerations.

In one embodiment, thefirst cover 133 and thesecond cover 132 mentioned above are configured to enclose thechallenge chamber 129. Thewindow 149,window 152 and covers 133, 132 (see FIG. 3) allow viewing of the composition 160 (see FIG. 5A) flowing through thechallenge chamber 129 in thefluid mixture 120 in thechannel 164B through theopening 150, and thelight source 147 is configured to emit a highintensity light beam 148 having any wavelength matching the stimulable composition in thefluid mixture 120 by acting on thecomposition 160 through a suitablelight source 147. Although alaser 147 is shown, any other suitable light source (e.g., a light emitting diode, an arc lamp, etc.) may be used to emit a beam that stimulates the component.

In one embodiment, a high intensity laser beam 148 (e.g., a 355nm Continuous Wave (CW) (or quasi-CW) laser 147) having a preselected wavelength from asuitable laser 147 is required to stimulate the components 160 (i.e., sperm cells) in the fluid mixture. In one embodiment, a laser 147 (see FIG. 3) emits alaser beam 148 that passes through awindow 149 inlayer 104, throughcover 133 at the uppermost portion ofchip 100, throughopening 150, and throughcover 132 andwindow 152 inlayer 101 ofchip 100 to irradiatecomposition 160 flowing throughchannel 164B ininterrogation region 129 ofchip 100.

In one embodiment, thelight beam 148 may be delivered to thecomponent 160 through an optical fiber embedded in themicrofluidic chip 100 at theopening 150.

The highintensity light beam 148 interacts with the component 160 (see detailed discussion below) and passes through thefirst cover 133 and thesecond cover 132 to exit from thebottom window 152 such that the emitted light 151 induced by thelight beam 148 is received by theobjective lens 153. Theobjective lens 153 may be disposed at any suitable location with respect to themicrofluidic chip 100. Because thechallenge chamber 129 is sealed by thefirst cover 133 and thesecond cover 132, the highintensity light beam 148 does not impinge on themicrofluidic chip 100 and damage thelayer 101 and 104. Thus, thefirst cover 133 and thesecond cover 132 help prevent the highintensity light beam 148 and the photonic noise caused by the microfluidic chip material (i.e., plastic) from damaging themicrofluidic chip 100.

In one embodiment, the emitted light 151 received by theobjective lens 153 is converted into an electrical signal by anoptical sensor 154, for example, a photomultiplier tube (PMT) or photodiode, among others. The electrical signal may be digitized by an analog-to-digital converter (ADC) 155 and sent to a Digital Signal Processor (DSP) basedcontroller 156. The DSP-basedcontroller 156 monitors the electrical signal and may then trigger one of the two actuation drivers (i.e., 157a, 157b) at a predetermined value to drive an associated one of the two piezoelectric actuator assemblies (109, 110 or 209, 210). In one embodiment (as shown in FIG. 2A), the piezo drivers and piezo actuators (158a, 158b or 219, 220) are portions of two piezo actuator assemblies (109, 110 or 209, 210) disposed on either side of theinterrogation chamber 129, respectively. The trigger signal sent to the piezoelectric actuator (109, 110 or 219, 220) is determined by the raw sensor signal to activate the particular piezoelectric actuator assembly (109, 110, 209, 210) when the selected component is detected.

In embodiments with bondedpiezoelectric actuator assemblies 109, 110, the thickness of thediaphragms 170, 171 may be different and depend on the voltage applied through the wires by theactuator assemblies 109, 110 on thechip 100. When an electrical signal is sent directly to the actuator assembly (i.e., 109, 110) through the electronic circuitry, thediaphragms 170, 171 flex and change (increase) the pressure in thechambers 130, 131.

After interrogation, at least one of the piezoelectric actuator assemblies (109, 110 or 209, 210) is used to act on the desiredcomponent 160 in the fluid mixture in thechannel 164C as thecomponent 160 exits theopening 150 for interrogation of theregion 129. Although the actuation driver 157b and thepiezoelectric actuator assembly 110 are not shown in fig. 4, the operation and configuration of the actuation driver 157b and thepiezoelectric actuator assembly 110 are the same as those of theactuation driver 157a and thepiezoelectric actuator assembly 109. Accordingly, the piezoelectric actuator 157b acts to deflect thecomponent 160 in the flow stream inchannel 164C to theright output channel 142 and to thethird output port 113. The same operation applies topiezoelectric actuator assembly 110, which ejects sheath or buffer fluid 163 fromejection chamber 131 viaejection channel 128 and deflects the target or selectedcomponent 160 to leftoutput channel 140 andthird output port 113.

In an alternative embodiment, a piezoelectric actuator assembly 106A (i.e., a piezoelectric disk similar topiezoelectric actuator assembly 109,piezoelectric actuator assembly 110 and of suitable dimensions, see fig. 2C) or a suitable pumping system (see fig. 9, e.g., discussed below) is used to pumpsample fluid 120 inchannel 164 towardjunction 161. The sample piezoelectric actuator assembly 106A would be disposed at thesample input port 106. By pumping thesample fluid mixture 120 into theprimary channel 164, control measures can be implemented in separating thecomponents 160 therein such that a more controlled relationship can be made between thecomponents 160 as thecomponents 160 enter theprimary channel 164.

If nopiezoelectric actuator assembly 109, 110 is employed, the (target)component 160 proceeds from theprimary channel 164 to thecentral output channel 141 and to thesecondary output port 112, and the sheath or buffer fluid 163 proceeds through theoutput channels 140, 142 to theoutput ports 110, 112, respectively.

In one embodiment, the size of output channels 140-142 increases fromchannel 164C to exitinterrogation chamber 129 such that the output ratio for improved separation ofcomponents 160 increases through one or more associated channels.

Chip operation

In one embodiment, themicrofluidic chip 100 is placed in a sterile state and one or more solutions (i.e., sheath or buffer fluid 163) may be prepared, or any fluid or substance of themicrofluidic chip 100 may be cleaned by draining themicrofluidic chip 100 or by flowing a sheath orbuffer fluid 153 or other solution through themicrofluidic chip 100, according to known methods. Once themicrofluidic chip 100 is ready and theejection chambers 130, 131 are filled with sheath or buffer fluid 163 (either during or after fabrication (as described above)), the air vents 121, 122 are sealed. As described above, in another embodiment, the air vents 121, 122 may remain open for adding additional sheath or buffer fluid 163 to thechambers 130, 131 during operation.

In one embodiment, as described above, thecomponents 160 to be separated include, for example: viable and motile sperm separated from non-viable and non-motile sperm; sperm separated by gender and other gender classification variations; stem cells isolated from a population of cells; one or more labeled cells separated from unlabeled cells that are differentiated by a desired/undesired characteristic; sperm cells having different desired characteristics; genes isolated in nuclear DNA according to defined characteristics; cells isolated based on surface markers; cells isolated based on membrane integrity (viability), potential or predicted reproductive status (fertility), ability to survive after freezing, and the like; cells separated from contaminants or debris; healthy cells isolated from damaged cells (i.e., cancer cells) (e.g., in bone marrow extracts); red blood cells separated from white blood cells and platelets in a plasma mixture; and any cell separated into corresponding portions with any other cellular components; damaged cells, or contaminants or debris, or any other biological substance desired to be separated.Component 160 may be a cell or bead treated or coated with a linker molecule or a label molecule embedded with fluorescence or luminescence. Thecomposition 160 may have a variety of physical or chemical properties, such as size, shape, material, texture, and the like.

In one embodiment, a heterogeneous population ofcomponents 160 can be measured simultaneously, where eachcomponent 160 is examined for a different or similar number of flow patterns (e.g., multiplexed measurements), orcomponents 160 can be examined and distinguished based on label (e.g., fluorescence), image (due to size, shape, different absorption, scattering, fluorescence, luminescent characteristics, fluorescent or luminescent emission profiles, decay lifetime of fluorescence or luminescence), and/or particle location, among other things.

In one embodiment, as shown in FIG. 5A, a two-step focusing method of a component sorting system according to the present invention may be used to position thecomponent 160 in thechannel 164B for interrogation in theinterrogation chamber 129.

In one embodiment, the first focusing step of the present invention is achieved by inputting afluid sample 120 containing components 160 (e.g., sperm cells, etc.) through thesample input port 106 and inputting a sheath or buffer fluid 163 through the sheath orbuffer input port 107, the sheath orbuffer input port 108. In one embodiment, thecomposition 160 is pre-stained with a stain (e.g., the stain of Hoechst) to allow fluorescence and for imaging to be detected. In one embodiment, sheath or buffer fluid 163 is disposed inejection chambers 130, 131, and input ports 121, 122 are sealed.

In one embodiment, as shown in fig. 5A, thecomponents 160 in thesample fluid mixture 120 flow through theprimary channel 164 and have random orientations and locations (see inset a). At theintersection 161, when the sheath or buffer fluid 163 encounters thesample mixture 120, thesample mixture 120 flowing in theprimary channel 164 is compressed by the sheath or buffer fluid 163 from thechannel 114, 115 in a first direction (i.e., at least horizontally, on at least two sides of the flow, assuming not all sides, depending on where theprimary channel 164 enters the intersection 161). Thus, thecomposition 160 is focused around thechannel 164, and thecomposition 160 may be compressed into a thin ribbon over the depth of thechannel 164A. Theintersection 161 leading into thechannel 164A is the focal region. Thus, atintersection 161, the component 160 (i.e., sperm cells) moves toward the center of the width of thechannel 164 as thesample 120 is compressed toward the center of thechannel 164A by the sheath or buffer fluid 163 from thechannels 114, 115.

In one embodiment, the present invention includes a second focusing step, wherein at theintersection 162 thesample mixture 120 containing thecomponent 160 is further compressed by a sheath or buffer fluid 163 in a second direction (i.e., a vertical direction from the top and bottom) entering from thechannel 116,channel 117. Theintersection 162 leading intochannel 164B is the second focal region. Note that although the entrances to theintersections 162 from thechannels 116, 117 are shown as rectangles, those of ordinary skill in the art will appreciate that any other suitable configuration (i.e., tapered, circular) may be used. The sheath or buffer fluid 163 in thechannels 116, 117 (which may be disposed in a different layer of themicrofluidic chip 100 than thechannels 164A-164B) enters thechannels 164A-164B at different planes to align thecomponent 160 with the center of thechannel 164B in width and depth (i.e., horizontal and vertical) as thecomponent 160 flows along thechannel 164B.

Thus, in one embodiment, with the second focusing step of the present invention, thesample mixture 120 is again compressed by the vertical sheath or buffer fluid 163 entering at thechannel 116,channel 117, and as shown in fig. 5A, thesample 120 stream is focused at the center of the depth of thechannel 164B, and thecomponents 160 flow along the center of thechannel 164B in an approximately single file.

In one embodiment, thecomponents 160 aresperm cells 160, and due to their flattened or flat teardrop-shaped heads, thesperm cells 160 will reorient themselves in a predetermined direction as they undergo the second focusing step, i.e., with their flat surfaces perpendicular to the direction of the light beam 148 (see fig. 5A). Thus,sperm cells 160 exhibit a preference in their bulk orientation when subjected to the two-step focusing process. In particular,sperm cells 160 tend to be more stable with their flat body perpendicular to the direction of compression. Thus, with the control of the sheath or buffer fluid 163,sperm cells 160 that start in a random orientation now achieve a consistent orientation. Thus, in the second focusing step, thesperm cells 160 not only form a single row at the center of thechannel 164B, but they also achieve a consistent orientation with their flat surfaces orthogonal to the direction of compression.

Thus, all of thecomponents 160 introduced into the sample input port 106 (which may be other types of cells or other substances, etc. as described above) undergo a two-step focusing step that allows thecomponents 160 to move through thechannel 164B in a single row, in a more consistent orientation (depending on the type of component 160), which allows for easier interrogation of thecomponents 160.

In one embodiment, further downstream inchannel 164B, thecomponent 160 is detected withlight source 147 throughcover 132,cover 133, and in theinterrogation chamber 129 atopening 150. Thelight source 147 emits alight beam 148, which may be emitted via an optical fiber, thelight beam 148 being focused at the center of thechannel 164C at theopening 150. In one embodiment, the component 160 (e.g., sperm cell 160) is oriented by focusing the flow (i.e., the flow of sheath or buffer fluid 163 acting on the sample flow 120) such that the flat surface of thecomponent 160 faces thelight beam 148. In addition, all of thecomponents 160 are moved by focusing into a single row as all of thecomponents 160 pass under thebeam 148. Ascomponent 160 passes underlight source 147 andlight beam 148 acts oncomponent 160,component 160 fluoresces indicating the desiredcomponent 160. For example, with regard to sperm cells, X chromosome cells fluoresce at different intensities than Y chromosome cells; alternatively, cells carrying one property may fluoresce at a different intensity or wavelength than cells carrying a different set of properties. In addition, thecomposition 160 may be viewed with respect to shape, size, or any other distinguishing indicia.

In the embodiment of beam-induced fluorescence, the emission beam 151 (in fig. 3) is then collected by theobjective lens 153 and subsequently converted into an electrical signal by theoptical sensor 154. The electrical signal is then digitized by an analog-to-digital converter (ADC) 155 and sent to anelectronic controller 156 for signal processing. The electronic controller may be any electronic processor with sufficient Processing power, such as a DSP, a MicroController Unit (MCU), a Field Programmable Gate Array (FPGA), or even a Central Processing Unit (CPU). In one embodiment, the DSP-basedcontroller 156 monitors the electrical signal and may then trigger at least one actuation driver (i.e., 157a or 157b) upon detection of the desiredconstituent 160 to drive one of the two piezoelectric actuator assemblies (109, 110 or 219, 220, i.e., a portion of the respectivepiezoelectric actuator assembly 109, 110, 209, 210). In another embodiment, the FPGA-based controller monitors the electrical signal and then communicates or acts independently with the DSP controller upon detection of the desiredconstituent 160 to trigger at least one actuation driver (157a or 157b) to drive one of the two piezoelectric actuator assemblies (109, 110 or 219, 220, i.e., a portion of the respectivepiezoelectric actuator assembly 109, 110, 209, 210).

Thus, in one embodiment, the selected or desiredcomponent 160 inchannel 164C is separated ininterrogation chamber 129 by a jet of sheath or buffer fluid 163 from one ofjet channel 127,jet channel 128, depending on whichoutput channel 140, 142 is desired for the selectedcomponent 160. In one exemplary embodiment, at the time the target or selectedcomponent 160 reaches the intersection ofjet channel 127,jet channel 128, andmain channel 164C, an electrical signal activates a driver to trigger the external stacked piezoelectric actuator 219 (or activatesdriver 157a to trigger actuator 109). This causes the outer stacked piezoelectric actuator assembly 209 (or 109) to contactdiaphragm 170 and pushdiaphragm 170, compressejection chamber 130, and squeeze a strong jet of buffer or sheath fluid 163 fromejection chamber 130 intomain channel 164C viaejection channel 127, which pushes selected or desiredcomponent 160 intooutput channel 142. Note that similar to the performance of stacked externalpiezoelectric actuator assembly 209, the firing of piezoelectric actuator assembly 210 (or 110) will push the desired constituent 160 fromejection channel 128 intooutput channel 140 on the opposite side.

Thus, sheath or buffer fluid 163 ejected from one ofejection channel 127, 128 diverts target components or selectedcomponents 160 from their ordinary path inchannel 164C to one of the selected or desiredrespective output channels 140, 142, separates thosetarget components 160, and improves flow in thoseoutput channels 140, 142, and depletes the flow, if any, insample fluid 120 that continues straight throughoutput channel 141, accompanied by unselected components. Thus, the absence of triggering of thepiezoelectric actuator assembly 109, thepiezoelectric actuator assembly 110 means that theunselected component 160 in thefluid mixture 120 continues to pass straight through theoutput channel 141.

In one embodiment, the separatedcomponents 160 are collected from the first output 111 or thethird output 113, for storage, for further separation, or for processing (e.g., cryopreservation), for example, using methods known in the art. Of course, thecomponents 160 that are not separated into theoutput ports 111, 113 may also be collected from thesecond output port 112. Portions of the first output port 111, thesecond output port 112, and thethird output port 113 are electronically characterized to detect concentrations of components, PH measurements, cell counts, electrolyte concentrations, and the like.

In one embodiment, interrogation of the sample 120 (i.e., biological material) containing thecomponent 160 is accomplished by other means. Thus, portions of themicrofluidic chip 100 or outputs from themicrofluidic chip 100 may be optically or visually inspected. In general, methods for interrogation may include direct visual imaging (e.g., visual imaging with a camera) and may utilize direct brightness imaging or fluorescence imaging; alternatively, more sophisticated techniques may be used, such as spectroscopic techniques, transmission spectroscopic techniques, spectroscopic imaging techniques or scattering (e.g. dynamic light scattering or dispersive wave spectroscopy) techniques.

In some cases, the optically interrogatedregion 129 can be used in conjunction with additives, such as chemicals that bind to or affect the components of thesample mixture 120, or beads that are functionalized to bind and/or fluoresce in the presence of certain substances or diseases. These techniques can be used to measure cell concentration, to detect disease, or to measure otherparameters characterizing component 160.

However, in another embodiment, if fluorescence is not used, a polarized light backscattering method may also be used. Thegeological query component 160 is described above using a spectroscopic method. The spectra of thosecomponents 160 that have a positive result and fluoresce (i.e., thosecomponents 160 that react with the label) are identified for separation by activatingpiezoelectric assembly 109,piezoelectric assembly 110,piezoelectric assembly 209,piezoelectric assembly 210.

In one embodiment, thecomponent 160 may be identified and selected for separation based on reacting or binding the component with the additive or sheath or buffer fluid 163, or by using the natural fluorescence of thecomponent 160 or the fluorescence of a substance associated with thecomponent 160 as an identification or background marker or to meet a selected size, specification, or surface characteristic, or the like.

In one embodiment, whichcomponents 160 are discarded and which components are collected may be selected via the computer 182 (whichcomputer 182 monitors the electrical signals and triggers thepiezoelectric assembly 109,piezoelectric assembly 110,piezoelectric assembly 209, piezoelectric assembly 210) and/or the operator based on the completed analysis.

In one embodiment, the user interface of thecomputer system 182 includes a computer screen that displays thecomponents 160 in the field of view captured by theCCD camera 183 on themicrofluidic chip 100.

In one embodiment, thecomputer 182 controls any external device (if used), such as a pump (i.e., the pumping mechanism of fig. 9), to pump anysample fluid 120, sheath or buffer fluid 163 into themicrofluidic chip 100, and also controls any heating device that sets the temperature of the fluid 120, 163 input into themicrofluidic chip 100.

Chip box and holder

Themicrofluidic chip 100 is loaded on achip cartridge 212, and thechip cartridge 212 is mounted on thechip holder 200. Thechip holder 200 is mounted to a translation stage (not shown) to allow fine positioning of theholder 200. Themicrofluidic chip holder 200 is configured to hold themicrofluidic chip 100 in place such that thelight beam 148 can intercept thecomponent 160 at theopening 150 in the manner described above. The gasket layer 105 (see fig. 1) forms a substantially leak-free seal between thebody 211 and themicrofluidic chip 100 when themicrofluidic chip 100 is in the closed position.

As shown in fig. 6, in one embodiment, themicrofluidic chip holder 200 is made of a suitable material (e.g., an aluminum alloy or other suitable metal/polymeric material) and includes abody 211 and at least one stacked externalpiezoelectric actuator 209, 210.

Thebody 211 of theholder 200 may be any suitable shape, but its configuration depends on the layout of thechip 100. For example, stacked externalpiezoelectric actuators 209, 210 must be placed on thediaphragms 170, 171 such that contact is made between the tips of thepiezoelectric actuators 219, 220 and thediaphragms 170, 171 of themicrofluidic chip 100. Thebody 211 of theholder 200 is configured to receive and engage external tubing (see fig. 9) for delivering fluid/sample to themicrofluidic chip 100.

The details of thesecartridge 212 andholder 200 and the mechanism for attaching thechip 100 to thecartridge 212 andholder 200 are not described in detail, as one of ordinary skill in the art will appreciate, these devices are well known and may have any configuration that accommodates amicrofluidic chip 100 so long as the objectives of the present invention are met.

As shown in fig. 9, in one embodiment, the pumping mechanism includes a system with apressurized gas 235 that provides pressure for pumping thesample fluid mixture 120 from the reservoir 233 (i.e., the sample tube) to thesample input port 106 of thechip 100.

Acollapsible container 237 having a sheath or buffer fluid 163 therein is disposed in thepressurized container 236, and thepressurized gas 235 pushes the fluid 163 to a manifold 238 having a plurality of different output ports, such that the fluid 163 is delivered viaconduits 231a, 231b to the sheath orbuffer input ports 107, 108, respectively, of thechip 100.

Apressure regulator 234 regulates the pressure of thegas 235 within thereservoir 233 and apressure regulator 239 regulates the pressure of thegas 235 within thecontainer 236.Mass flow regulators 232a, 232b control the fluid 163 pumped viaconduits 231a, 231b into the sheath orbuffer input port 107, sheath orbuffer input port 108, respectively. Thus, thetubing 230, 231a, 231b is used to initially load the fluid 120 into thechip 100, and thetubing 230, 231a, 231b may be used throughout thechip 100 to load thesample fluid 120 into thesample input port 106 or the sheath orbuffer input port 107, sheath orbuffer input port 108. Additionally, for example, in one embodiment, tubing (not shown) may provide fluid 163 frommanifold 238 into air vents 121, 122 to fillchambers 130, 131.

According to the illustrated embodiments, any of the operations, steps, control options, etc. may be implemented by instructions stored on a computer readable medium, such as a computer memory, a database, etc. When executed, instructions stored on a computer-readable medium may cause a computer device to perform any of the operations, steps, control options, and the like described herein.

The operations described in this specification may be implemented as operations performed by data processing apparatus or processing circuitry on data stored on one or more computer-readable storage devices or received from other sources. A computer program (also known as a program, software application, script, or code) can be written in any form of programming language, including compiled or interpreted languages, declarative or procedural languages, and it can be deployed in any form, including as a stand-alone program or as a module, component, subroutine, object, or other unit suitable for use in a computing environment. A computer program may, but need not, correspond to a file in a file system. A program can be stored in a portion of a file that holds other programs or data (e.g., one or more scripts stored in a markup language document), in a single file dedicated to the program in question, or in multiple coordinated files (e.g., files that store one or more modules, sub programs, or portions of code). A computer program can be deployed to be executed on one computer or on multiple computers at one site or distributed across multiple sites and interconnected by a communication network. Processing circuitry adapted to execute computer program modules comprises: such as general and special purpose microprocessors, and any one or more processors of any kind of digital computer.

It should be noted that the orientation of the various elements may be changed in accordance with other illustrated embodiments, and such variations are intended to be encompassed by the present invention.

As shown in the various illustrated embodiments, the construction and arrangement of the microfluidic chip is illustrative only. Although only a few embodiments have been described in detail in this disclosure, many modifications are possible (e.g., variations in sizes, dimensions, structures, shapes and proportions of the various elements, values of parameters, mounting arrangements, use of materials, colors, orientations, etc.) without materially departing from the novel teachings and advantages of the subject matter described herein. Some elements shown as integrally formed may be constructed of multiple parts or elements, the position of elements may be reversed or otherwise varied, and the nature or number of discrete elements or positions may be altered or varied. The order or sequence of any process, logical computer, or method steps may be varied or re-sequenced according to alternative embodiments. Other substitutions, modifications, changes and omissions may be made in the design, operating conditions and arrangement of the various illustrated embodiments without departing from the scope of the present invention.

Claims (19)

1. An apparatus for identifying a constituent in a fluid mixture, comprising:

(i) a microfluidic chip, the microfluidic chip comprising:

(a) a sample input channel disposed in the first structural layer, the sample input channel for receiving a fluid mixture comprising a plurality of components, wherein the sample input channel comprises:

a first intersection point; and

a second intersection point;

(b) a plurality of first sheath fluid channels disposed in the first structural layer, wherein the plurality of first sheath fluid channels intersect the sample input channel at the first intersection point;

(c) a plurality of second sheath-like fluid channels disposed in a second structural layer, wherein the plurality of second sheath-like fluid channels intersect the sample input channel at the second intersection; and

(d) a plurality of output channels fluidly connected to and branching from the sample input channel, wherein each of the plurality of output channels is disposed between a pair of recessed portions of the ends of the first structural layer and the second structural layer;

(ii) a challenge device disposed downstream of the second intersection, the challenge device distinguishing the plurality of components into selected components and unselected components; and

(iii) a focused energy device acting on said selected component.

2. The apparatus of claim 1, wherein the sample input channel is tapered at an entry point into the first intersection such that sheath fluid from the plurality of first sheath fluid channels compresses the plurality of components in the fluid mixture on at least two sides.

3. The apparatus of claim 2, wherein sheath fluid from the plurality of first sheath fluid channels compresses the plurality of components in the fluid mixture on all sides.

4. The apparatus of claim 1, wherein the sample input channel tapers into the interrogation device to increase a velocity of the fluid mixture passing through the interrogation device.

5. The apparatus of claim 1, wherein the focused energy device emits a focused energy beam to destroy selected components.

6. The apparatus of claim 1, wherein the interrogation device includes a light source that emits a light beam to illuminate and excite the plurality of components in the fluid mixture.

7. The apparatus of claim 1, further comprising:

a pumping mechanism that pumps the fluid mixture into the sample input channel and sheath fluid into the plurality of first sheath fluid channels and the plurality of second sheath fluid channels.

8. The apparatus of claim 1, wherein the plurality of components is a plurality of cells.

9. The apparatus of claim 8, wherein the focused energy device emits a focused energy beam to kill selected cells.

10. The device of claim 9, wherein the interrogation means interrogates the plurality of cells to differentiate selected cells based on viability, motility, gender, tags, desired characteristics, DNA content, surface markers, membrane integrity, predicted reproductive status, health or survival characteristics.

11. The apparatus of claim 10, wherein the plurality of cells are sperm.

12. An apparatus for producing a fluid mixture of living and killed cells, comprising:

(i) a microfluidic chip, the microfluidic chip comprising:

(a) a sample input channel disposed in the first structural layer, the sample input channel for receiving a fluid mixture comprising a plurality of cells, wherein the sample input channel comprises a first intersection;

(b) a plurality of first sheath fluid channels disposed in the first structural layer, wherein the plurality of first sheath fluid channels intersect the sample input channel at the first intersection point;

wherein the sample input channel tapers at an entry point into the first intersection point such that the plurality of cells in the fluid mixture are compressed vertically and horizontally by sheath fluid from the plurality of first sheath fluid channels;

(c) at least one output channel fluidly connected to the sample input channel;

(ii) an interrogation device that interrogates the plurality of cells to select cells to be killed; and

(iii) a focused energy device to kill the selected cells, wherein the at least one output channel is disposed downstream of the focused energy device to receive a fluid mixture of the living and killed cells.

13. The apparatus of claim 12, wherein the microfluidic chip further comprises:

(d) a plurality of second sheath-like fluid channels disposed in a second structural layer, the plurality of second sheath-like fluid channels intersecting the sample input channel at a second intersection between the first intersection and the interrogation device.

14. The apparatus of claim 13, wherein sheath fluid from the plurality of second sheath fluid channels further vertically compresses the plurality of cells in the fluid mixture.

15. The apparatus according to claim 12, wherein the interrogation device includes a light source that emits a light beam to illuminate and excite the plurality of cells in the fluid mixture.

16. The apparatus of claim 13, further comprising:

a pumping mechanism that pumps the fluid mixture into the sample input channel and sheath fluid into the plurality of first sheath fluid channels and the plurality of second sheath fluid channels.

17. The device of claim 12, wherein the interrogation means interrogates the plurality of cells to select cells based on viability, motility, gender, tags, desired characteristics, DNA content, surface markers, membrane integrity, predicted reproductive status, health or survival characteristics.

18. The apparatus of claim 12, wherein the plurality of cells are sperm.

19. The device of claim 18, wherein the interrogation device interrogates the sperm to select cells based on gender.

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