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 the microfluidic chip 100 shown in fig. 1-5B and the microfluidic 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 a microfluidic chip 100. The microfluidic 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.

The microfluidic 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 the chip 100 at an appropriate length so long as the objectives of the present invention are achieved. The desired flow rate through the microfluidic chip 100 may be controlled by a predetermined introduction flow rate introduced into the chip 100 by a pumping mechanism, by maintaining proper microchannel specifications within the chip 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 the microfluidic chip 100, which provide access to the microchannels/channels. In one embodiment, as shown in fig. 1-2C, the sample input port 106 is used to introduce sample components 160 in a sample fluid mixture 120 (see fig. 4-5B) from a reservoir source (see fig. 9) into a sample input channel 164A of the microfluidic chip 100. The microfluidic 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 the microfluidic chip 100, which include a sheath/buffer input 107 and a sheath/buffer input 108, both of which are disposed proximate to the sample input 106, and both of which introduce sheath or buffer fluid into the microfluidic 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 the chip 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 into ejection 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 the separation component 160 and/or sheath or buffer fluids) that have flowed through the microfluidic chip 100. In one embodiment as shown in fig. 1-2C, there are three output channels 140-142, including a left output channel 140, a center output channel 141, and a right output channel 142. The left output channel 140 ends at the first output port 111, the center output channel 141 ends at the second output port 112, and the right output channel 142 ends at the third output port 113. However, the number of output ports may be less or greater depending on the number of components 160 to be separated from the fluid mixture 120.

In one embodiment, instead of straight edges, a plurality of notches or grooves 146 are provided at the bottom edge of the microfluidic 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, the second output port 112, and the third output port 113 via output channels 140 and 142 originating from the interrogation chamber 129 (see fig. 2A-4).

In one embodiment, the microfluidic 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 a microfluidic 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 the microfluidic 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, a first gasket 105 is disposed at one end of the microfluidic chip 100 and is connected to the layer 104 or adhered to the layer 104 with an epoxy. A plurality of apertures 144 are disposed in the first gasket 105 and are configured to align with the sample 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, a second gasket 143 is disposed at the other end of the microfluidic chip 100 opposite the first gasket 105 and is connected to the top structural layer 104 or bonded to the top structural layer 104 with an epoxy. The second gasket 143 is configured to assist in sealing and stabilizing or balancing the microfluidic chip 100 in the microfluidic chip holder 200 (see fig. 6).

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

In one embodiment, a sample fluid mixture 120 including a component 160 is introduced into the sample input port 106, and the fluid mixture 120 flows through the primary 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 through channel 114, channel 115 and channel 116, channel 117, respectively, and into main channel 164 and toward interrogation chamber 129 before exiting through output channel 140 and 142.

In one embodiment, if the chambers 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 the ejection chambers 130, 131 through the air vents 121, 122 after the microfluidic chip 110 is manufactured to fill the chambers 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, the fluid mixture 120 from the primary channel 164 is combined with the sheath or buffer fluid 163 from the channels 114, 115 at an intersection 161 in the same plane of the microfluidic chip 100. In one embodiment, downstream of the second junction 162, the buffer fluid 163 from the channels 116, 117 combines the combined fluid mixture 120 and the sheath or buffer fluid 163 from the first junction 161. In one embodiment, channels 114, 115 are substantially the same gauge as channels 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 in microfluidic 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, the channels 114, 123, 124, 140, 142, 125a, 125b, 126a, 126b, 127, 128 of the microfluidic chip 100 may not only vary in size, but may also have a tapered shape at the entry point of other channels in the chip 100 in order to control the flow of fluid through these channels. For example, the primary channel 164 may be tapered at the point of entry of the intersection 161 (see fig. 5B) to control and expedite the flow of the sample 120 into the intersection 161, and to allow the sheath or buffer fluid 163 from the channels 114, 115 to compress the sample 120 in a first direction (i.e., horizontally) on at least two sides, provided not on all sides (depending on the location of the tapered channel 164 in conjunction with the channel 164A). Thus, the sample 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 the channel 164A. However, one of ordinary skill in the art will appreciate that the primary channels 164 entering the intersection 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 the channels 116, 117 is disposed in a different structural layer of the microfluidic chip 100 than the layer in which the channels 164 are disposed. For example, channel 116 may be disposed in layer 103 and channel 117 may be disposed in layer 101 (see fig. 1) such that when sheath or buffer fluid 163 joins fluid mixture 120 at intersection 162, channel 116, channel 117 are in a different plane than other channels 164 and 114, channel 115 (in layer 102). In one embodiment, the primary channels 164 are disposed between the layers 102, 103 (see fig. 3); however, one of ordinary skill in the art will appreciate that channels 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, although channels 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 on chip 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 in channels 116, 117 combines the fluid mixture via holes cut in layers 101 and 103 at locations substantially perpendicular above and below intersection 162. The sheath or buffer fluid from the channels 116, 117 compresses the flow of the fluid mixture 120 in a perpendicular manner relative to the channel 164B such that the components 160 in the fluid mixture 120 are compressed or flattened and oriented in a selected or desired direction (see below) while still maintaining laminar flow in the channel 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 by sample input 106. Thus, in one embodiment, channels 114, 115 and 116, 117 are disposed in a substantially parallel arrangement, with channels 114, 115 and 116, 117 being equidistant from primary 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 join intersection points 161 in the same plane at an angle of 45 degrees or less, while channels 116, 117 of parallel sample input channels 164A join intersection 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 described microfluidic chip 100 may be different so long as they achieve the desired features of the present invention.

In one embodiment, downstream of the intersection 162, the component 160 in the fluid mixture 120 flows through the channel 164B into the interrogation chamber 129 where the component 160 is interrogated.

In one embodiment, ejection chambers 130, 131 are covered by flexible 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 of channel 164B and interrogation chamber 129 (see fig. 2A and 2B) to mechanically displace diaphragms 170, 171 to eject or push sheath or buffer fluid 163 from one of ejection chamber 130, ejection chamber 131 on that side of channel 164B to push ingredient 160 from channel 164C into one of output channels 140, 142 on the other side of channel 164B. In other words, the actuator ejects the sheath or buffer fluid 163 from the ejection chamber 130 into the channel 164C and pushes the target component 160 of the channel 164C into the output channel 142 to separate the target component from the fluid mixture 120. This embodiment is useful when only one type of target component 160 is separated, which may, for example, only require two output channels 141, 142 instead of three output 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 the channel 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 the channel 164B and connected to the channel 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 the chip 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 activate diaphragm 170, diaphragm 171 and eject sheath or buffer fluid 163 from chamber 130, chamber 131 into channel 164B, two external, stacked piezoelectric actuator assemblies 209, 210 (see fig. 6 and 7) are provided, the two piezoelectric actuator assemblies 209, 210 aligning with diaphragm 170, diaphragm 171 and actuating diaphragm 170, diaphragm 171. External, stacked piezoelectric actuator assemblies 209, 210 are disposed in the microfluidic chip holder 200. Stacked piezoelectric actuator assemblies 209, 210 each include a piezoelectric 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 with diaphragm 170, 171 to squeeze sheath or buffer fluid 163 from chamber 130, 131 into channel 164C.

The microfluidic chip holder 200 may be of any type known to those of ordinary skill in the art and is configured to precisely position the piezoelectric actuators 219, 220 such that the piezoelectric actuators 219, 220 may maintain continuous contact with the diaphragms 170, 171 of the microfluidic 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 on lockable adjustment screw 201 and thumbscrew 202, respectively, lockable adjustment screw 201 causing piezoelectric actuator 219, piezoelectric actuator 220 to move into position against diaphragm 170, diaphragm 171, respectively; the threaded body of the thumb screw 202 is used to move the screw 202 against the diaphragm 170, 171 for stability. The spacers 203 attached to the piezoelectric actuators 219, 220 allow a feasible contact between the spacers 203 and the membranes 170, 171 of the microfluidic chip 100. Adjustment screws 201 allow a user to adjust the position of piezoelectric actuators 209, 210 relative to microfluidic chip 100 for coarse and fine adjustments. The thumb screws 202 may be tightened to secure the piezoelectric assemblies 209, 210 to the main die body 100, or the thumb screws 202 may be loosened to detach the piezoelectric actuator assemblies 209, 210 from the main 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 the microfluidic chip 100. The adjustment screw 201 is mounted on the holder 200 and can be extended and retracted by turning the screw 201. The tip of the adjusting screw 201 abuts against the plate. When the screw 201 is extended, the plate and the piezoelectric actuators 209, 210 are pushed in a translational movement towards the diaphragm 170, 171, so as to make possible contact between the piezoelectric actuators 209, 210 and the diaphragm 170, 171. With this approach, the positioning of piezoelectric actuators 209, 210 is adjusted solely by the translation of piezoelectric actuators 209, 210, whereas in previous embodiments where piezoelectric actuators 209, 210 were mounted directly to adjustment screw 201, the positioning of piezoelectric actuators 209, 210 was a combination of the translation and rotation of piezoelectric actuators 209, 210, during which damage to fragile piezoelectric actuators 209, 210 may be caused.

In another embodiment, the electronic circuitry is coupled to the stacked piezoelectric actuator assemblies 209, 210 prior to driving the stacked piezoelectric actuator assemblies 209, 210. When each of the stacked piezoelectric actuators 219, 220 is in contact with the respective diaphragm 170, 171, the resistance from the diaphragms 170, 171 induces a strain on the stacked piezoelectric 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 stacked piezoelectric actuators 219, 220 are in contact with the diaphragms 170, 171, indicating that contact is made between the stacked piezoelectric actuators 219, 220 and the diaphragms 170, 171. This contact sensing allows sufficient force for actuators 219, 220 to compress chambers 130, 131, thereby injecting fluid 163 into channel 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 one diaphragm 170, 171 to form at least one piezoelectric actuator assembly 109, 110 (see fig. 2A and 4) to displace (flex) the respective diaphragm 170, 171 and drive fluid in the respective ejection chamber 130, 131 into channel 164C, respectively. The piezoelectric material is permanently bonded to the flexible diaphragm 170, 171, previously described, by an adhesive mechanism. Thus, in this embodiment, when a voltage is applied across the electrodes of the piezoelectric actuator assembly 109, 110, the entire diaphragm 170, 171 flexes into the chamber 130, 131 and squeezes the fluid 163 in the chamber 130, 131 into the channel 164C to deflect the target or selected component 160 into the side output channel 140, 142.

As described above with respect to externally stacked piezoelectric 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 from ejection chamber 130 into channel 164C and push target component 160 in channel 164C into output channel 142 to separate the target component from fluid mixture 120, as shown in fig. 2B.

In one embodiment, the piezoelectric actuator assemblies 109, 110 are used to seal the ejection 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 the microfluidic chip 100 is not affected by fluid leakage, for example, after the chambers 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 on diaphragms 170, 171 in view of the relatively small bending displacements of diaphragms 170, 171, as compared to the large displacements and strong forces exerted by externally stacked piezoelectric 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 the actuator assemblies 109, 110, 209, 210 used in the microfluidic 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 the diaphragms 170, 171 operates as a strain sensor to determine how much strain or displacement the outer stacked piezoelectric actuator assemblies 209, 210 generate when the outer stacked piezoelectric actuator assemblies 209, 210 are triggered by an electrical signal to displace the respective diaphragms 170, 171. The diameter and thickness of the piezoelectric film depend on the cross section of the externally stacked piezoelectric actuator 219, piezoelectric actuator 220 and the forces generated on the diaphragms 170, 171. The piezoelectric film and diaphragms 170, 171 may be different from the corresponding components discussed above in alternative embodiments.

The filling of ejection chambers 130, 131 will now be described. In one embodiment, air vents 121, 122 are provided to remove air from ejection chambers 130, 131 respectively after fabrication of chambers 130, 131 filling with sheath or buffer fluid 163 (forcing air out through air vents 121, 122) and before sealing chambers 130, 131 with sheath or buffer fluid 163 in chambers 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 the chambers 130, 131, provided this is not done during the manufacturing process. The sheath or buffer fluid or other fluid 163 disposed in ejection chamber 130, 131 may be the same or different than the sheath or buffer fluid 163 input through channel 114, channel 115, channel 116 or channel 117.

In one embodiment, if sheath or buffer fluid 163 is used to fill ejection chambers 130, 131, they may be input through input port 121, input port 122, and flow through channels 123, 124, respectively, to enter ejection chamber 130 via channels 125a and 125b, and to enter ejection chamber 131 via channels 126a and 126 b.

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

In one embodiment, to form a strong, transient jet, the jet channels 127, 128 may be tapered as they connect to the primary channel 164C. However, one of ordinary skill in the art will appreciate that the injection 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 flex diaphragm 170, diaphragm 171, respectively, and inject or squeeze sheath or buffer fluid 163 into channel 164C. However, when diaphragms 170, 171 return to the neutral (unbent) position, ejection channels 127, 128 emanating from ejection chambers 130, 131 operate as dispersers to ensure that a net fluid volume is maintained from ejection chambers 130, 131 to channel 164C and to ensure that chambers 130, 131 are easily refilled with sheath or buffer fluid 163.

In one embodiment, output channels 140-142 are from channel 164C within interrogation chamber 129 to output port 111-113. As described above, in one embodiment, more than one on-chip piezoelectric actuator assembly 109, piezoelectric actuator assembly 110, or externally stacked piezoelectric actuator assembly 209, piezoelectric actuator assembly 210 (in any size or in any location) may be used to connect to each of ejection channel 127, 128 to provide additional motive force to eject sheath or buffer fluid 163 from ejection chamber 130, 131 into channel 164C. In one embodiment, the distance from each injection channel 127, 128 of the inlet channel 164C to each output channel 140-142 should be shorter than the distance between the components 160 to avoid mixing of the target 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 where channels 116, 117 enter channel 164B. In one embodiment, the channel 164B tapers into the interrogation chamber 129, which accelerates the flow of the fluid mixture through the interrogation chamber 129. However, one of ordinary skill in the art will appreciate that the channel 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 the constituent 160 of the fluid mixture in the channel 164B that passes through the interrogation chamber 129. Note that the channel 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, the interrogation chamber 129 includes an opening or window 149 (see fig. 3) cut into the microfluidic chip 100 in at least the uppermost layer (i.e., layer 104 or other layer), and another opening or window 152 is cut into the chip 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, the top window 149 is configured to receive the first cover 133 and the bottom window 152 is configured to receive the second cover 132. However, the windows 149, 152 may be located in any suitable layer, not necessarily in the uppermost/lowermost layer. The covers 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 of window 149, window 152, and opening 150 are shown in fig. 3, these may vary depending upon manufacturing considerations.

In one embodiment, the first cover 133 and the second cover 132 mentioned above are configured to enclose the challenge chamber 129. The window 149, window 152 and covers 133, 132 (see FIG. 3) allow viewing of the composition 160 (see FIG. 5A) flowing through the challenge chamber 129 in the fluid mixture 120 in the channel 164B through the opening 150, and the light source 147 is configured to emit a high intensity light beam 148 having any wavelength matching the stimulable composition in the fluid mixture 120 by acting on the composition 160 through a suitable light source 147. Although a laser 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 a suitable 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 a laser beam 148 that passes through a window 149 in layer 104, through cover 133 at the uppermost portion of chip 100, through opening 150, and through cover 132 and window 152 in layer 101 of chip 100 to irradiate composition 160 flowing through channel 164B in interrogation region 129 of chip 100.

In one embodiment, the light beam 148 may be delivered to the component 160 through an optical fiber embedded in the microfluidic chip 100 at the opening 150.

The high intensity light beam 148 interacts with the component 160 (see detailed discussion below) and passes through the first cover 133 and the second cover 132 to exit from the bottom window 152 such that the emitted light 151 induced by the light beam 148 is received by the objective lens 153. The objective lens 153 may be disposed at any suitable location with respect to the microfluidic chip 100. Because the challenge chamber 129 is sealed by the first cover 133 and the second cover 132, the high intensity light beam 148 does not impinge on the microfluidic chip 100 and damage the layer 101 and 104. Thus, the first cover 133 and the second cover 132 help prevent the high intensity light beam 148 and the photonic noise caused by the microfluidic chip material (i.e., plastic) from damaging the microfluidic chip 100.

In one embodiment, the emitted light 151 received by the objective lens 153 is converted into an electrical signal by an optical 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) based controller 156. The DSP-based controller 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 the interrogation 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 bonded piezoelectric actuator assemblies 109, 110, the thickness of the diaphragms 170, 171 may be different and depend on the voltage applied through the wires by the actuator assemblies 109, 110 on the chip 100. When an electrical signal is sent directly to the actuator assembly (i.e., 109, 110) through the electronic circuitry, the diaphragms 170, 171 flex and change (increase) the pressure in the chambers 130, 131.

After interrogation, at least one of the piezoelectric actuator assemblies (109, 110 or 209, 210) is used to act on the desired component 160 in the fluid mixture in the channel 164C as the component 160 exits the opening 150 for interrogation of the region 129. Although the actuation driver 157b and the piezoelectric actuator assembly 110 are not shown in fig. 4, the operation and configuration of the actuation driver 157b and the piezoelectric actuator assembly 110 are the same as those of the actuation driver 157a and the piezoelectric actuator assembly 109. Accordingly, the piezoelectric actuator 157b acts to deflect the component 160 in the flow stream in channel 164C to the right output channel 142 and to the third output port 113. The same operation applies to piezoelectric actuator assembly 110, which ejects sheath or buffer fluid 163 from ejection chamber 131 via ejection channel 128 and deflects the target or selected component 160 to left output channel 140 and third output port 113.

In an alternative embodiment, a piezoelectric actuator assembly 106A (i.e., a piezoelectric disk similar to piezoelectric 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 pump sample fluid 120 in channel 164 toward junction 161. The sample piezoelectric actuator assembly 106A would be disposed at the sample input port 106. By pumping the sample fluid mixture 120 into the primary channel 164, control measures can be implemented in separating the components 160 therein such that a more controlled relationship can be made between the components 160 as the components 160 enter the primary channel 164.

If no piezoelectric actuator assembly 109, 110 is employed, the (target) component 160 proceeds from the primary channel 164 to the central output channel 141 and to the secondary output port 112, and the sheath or buffer fluid 163 proceeds through the output channels 140, 142 to the output ports 110, 112, respectively.

In one embodiment, the size of output channels 140-142 increases from channel 164C to exit interrogation chamber 129 such that the output ratio for improved separation of components 160 increases through one or more associated channels.

Chip operation

In one embodiment, the microfluidic 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 the microfluidic chip 100 may be cleaned by draining the microfluidic chip 100 or by flowing a sheath or buffer fluid 153 or other solution through the microfluidic chip 100, according to known methods. Once the microfluidic chip 100 is ready and the ejection 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 the chambers 130, 131 during operation.

In one embodiment, as described above, the components 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. The composition 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 of components 160 can be measured simultaneously, where each component 160 is examined for a different or similar number of flow patterns (e.g., multiplexed measurements), or components 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 the component 160 in the channel 164B for interrogation in the interrogation chamber 129.

In one embodiment, the first focusing step of the present invention is achieved by inputting a fluid sample 120 containing components 160 (e.g., sperm cells, etc.) through the sample input port 106 and inputting a sheath or buffer fluid 163 through the sheath or buffer input port 107, the sheath or buffer input port 108. In one embodiment, the composition 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 in ejection chambers 130, 131, and input ports 121, 122 are sealed.

In one embodiment, as shown in fig. 5A, the components 160 in the sample fluid mixture 120 flow through the primary channel 164 and have random orientations and locations (see inset a). At the intersection 161, when the sheath or buffer fluid 163 encounters the sample mixture 120, the sample mixture 120 flowing in the primary channel 164 is compressed by the sheath or buffer fluid 163 from the channel 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 the primary channel 164 enters the intersection 161). Thus, the composition 160 is focused around the channel 164, and the composition 160 may be compressed into a thin ribbon over the depth of the channel 164A. The intersection 161 leading into the channel 164A is the focal region. Thus, at intersection 161, the component 160 (i.e., sperm cells) moves toward the center of the width of the channel 164 as the sample 120 is compressed toward the center of the channel 164A by the sheath or buffer fluid 163 from the channels 114, 115.

In one embodiment, the present invention includes a second focusing step, wherein at the intersection 162 the sample mixture 120 containing the component 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 the channel 116, channel 117. The intersection 162 leading into channel 164B is the second focal region. Note that although the entrances to the intersections 162 from the channels 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 the channels 116, 117 (which may be disposed in a different layer of the microfluidic chip 100 than the channels 164A-164B) enters the channels 164A-164B at different planes to align the component 160 with the center of the channel 164B in width and depth (i.e., horizontal and vertical) as the component 160 flows along the channel 164B.

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

In one embodiment, the components 160 are sperm cells 160, and due to their flattened or flat teardrop-shaped heads, the sperm 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, the sperm cells 160 not only form a single row at the center of the channel 164B, but they also achieve a consistent orientation with their flat surfaces orthogonal to the direction of compression.

Thus, all of the components 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 the components 160 to move through the channel 164B in a single row, in a more consistent orientation (depending on the type of component 160), which allows for easier interrogation of the components 160.

In one embodiment, further downstream in channel 164B, the component 160 is detected with light source 147 through cover 132, cover 133, and in the interrogation chamber 129 at opening 150. The light source 147 emits a light beam 148, which may be emitted via an optical fiber, the light beam 148 being focused at the center of the channel 164C at the opening 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 the component 160 faces the light beam 148. In addition, all of the components 160 are moved by focusing into a single row as all of the components 160 pass under the beam 148. As component 160 passes under light source 147 and light beam 148 acts on component 160, component 160 fluoresces indicating the desired component 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, the composition 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 the objective lens 153 and subsequently converted into an electrical signal by the optical sensor 154. The electrical signal is then digitized by an analog-to-digital converter (ADC) 155 and sent to an electronic 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-based controller 156 monitors the electrical signal and may then trigger at least one actuation driver (i.e., 157a or 157b) upon detection of the desired constituent 160 to drive one of the two piezoelectric actuator assemblies (109, 110 or 219, 220, i.e., a portion of the respective piezoelectric 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 desired constituent 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 respective piezoelectric actuator assembly 109, 110, 209, 210).

Thus, in one embodiment, the selected or desired component 160 in channel 164C is separated in interrogation chamber 129 by a jet of sheath or buffer fluid 163 from one of jet channel 127, jet channel 128, depending on which output channel 140, 142 is desired for the selected component 160. In one exemplary embodiment, at the time the target or selected component 160 reaches the intersection of jet channel 127, jet channel 128, and main channel 164C, an electrical signal activates a driver to trigger the external stacked piezoelectric actuator 219 (or activates driver 157a to trigger actuator 109). This causes the outer stacked piezoelectric actuator assembly 209 (or 109) to contact diaphragm 170 and push diaphragm 170, compress ejection chamber 130, and squeeze a strong jet of buffer or sheath fluid 163 from ejection chamber 130 into main channel 164C via ejection channel 127, which pushes selected or desired component 160 into output channel 142. Note that similar to the performance of stacked external piezoelectric actuator assembly 209, the firing of piezoelectric actuator assembly 210 (or 110) will push the desired constituent 160 from ejection channel 128 into output channel 140 on the opposite side.

Thus, sheath or buffer fluid 163 ejected from one of ejection channel 127, 128 diverts target components or selected components 160 from their ordinary path in channel 164C to one of the selected or desired respective output channels 140, 142, separates those target components 160, and improves flow in those output channels 140, 142, and depletes the flow, if any, in sample fluid 120 that continues straight through output channel 141, accompanied by unselected components. Thus, the absence of triggering of the piezoelectric actuator assembly 109, the piezoelectric actuator assembly 110 means that the unselected component 160 in the fluid mixture 120 continues to pass straight through the output channel 141.

In one embodiment, the separated components 160 are collected from the first output 111 or the third output 113, for storage, for further separation, or for processing (e.g., cryopreservation), for example, using methods known in the art. Of course, the components 160 that are not separated into the output ports 111, 113 may also be collected from the second output port 112. Portions of the first output port 111, the second output port 112, and the third 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 the component 160 is accomplished by other means. Thus, portions of the microfluidic chip 100 or outputs from the microfluidic 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 interrogated region 129 can be used in conjunction with additives, such as chemicals that bind to or affect the components of the sample 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 other parameters characterizing component 160.

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

In one embodiment, the component 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 the component 160 or the fluorescence of a substance associated with the component 160 as an identification or background marker or to meet a selected size, specification, or surface characteristic, or the like.

In one embodiment, which components 160 are discarded and which components are collected may be selected via the computer 182 (which computer 182 monitors the electrical signals and triggers the piezoelectric 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 the computer system 182 includes a computer screen that displays the components 160 in the field of view captured by the CCD camera 183 on the microfluidic chip 100.

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

Chip box and holder

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

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

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

The details of these cartridge 212 and holder 200 and the mechanism for attaching the chip 100 to the cartridge 212 and holder 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 a microfluidic 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 a pressurized gas 235 that provides pressure for pumping the sample fluid mixture 120 from the reservoir 233 (i.e., the sample tube) to the sample input port 106 of the chip 100.

A collapsible container 237 having a sheath or buffer fluid 163 therein is disposed in the pressurized container 236, and the pressurized gas 235 pushes the fluid 163 to a manifold 238 having a plurality of different output ports, such that the fluid 163 is delivered via conduits 231a, 231b to the sheath or buffer input ports 107, 108, respectively, of the chip 100.

A pressure regulator 234 regulates the pressure of the gas 235 within the reservoir 233 and a pressure regulator 239 regulates the pressure of the gas 235 within the container 236. Mass flow regulators 232a, 232b control the fluid 163 pumped via conduits 231a, 231b into the sheath or buffer input port 107, sheath or buffer input port 108, respectively. Thus, the tubing 230, 231a, 231b is used to initially load the fluid 120 into the chip 100, and the tubing 230, 231a, 231b may be used throughout the chip 100 to load the sample fluid 120 into the sample input port 106 or the sheath or buffer input port 107, sheath or buffer input port 108. Additionally, for example, in one embodiment, tubing (not shown) may provide fluid 163 from manifold 238 into air vents 121, 122 to fill chambers 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.

Images