Devices and methods for dissociating and manipulating samples in microfluidic systems

Abstract

Microfluidic devices and methods for dissociating and manipulating cells or particles within a microfluidic channel are disclosed. The microfluidic device includes an inlet, an outlet, and a microfluidic channel arranged on a substrate between the inlet and the outlet. The microfluidic device includes a first set of piezoelectric actuators arranged adjacent to the inlet channel and configured to dissociate particles of a fluid sample within the microfluidic channel. The microfluidic device includes a second set of piezoelectric actuators arranged between the inlet and outlet channels and configured to manipulate particles of a fluid sample as particles move through the microfluidic channel. The microfluidic device includes a third set of piezoelectric actuators arranged above the outlet channel and adjacent to the outlet and configured to discharge a portion of the fluid sample from the microfluidic channel through the outlet.

Description

Technical Field

[0001] [Priority and Related Applications] This application is a continuation application of U.S. Provisional Patent Application No. 63 / 467,903, titled "Microfluidic chip for 3D scanning of single cells using piezoelectric actuators in a sensing zone", filed on May 19, 2023, and U.S. Patent Application No. 18 / 605,476, titled "Devices and Methods for Sample Dissociation and Manipulation in a Microfluidic System", filed on Mar. 14, 2024, and claims the priority of U.S. Provisional Patent Application No. 63 / 457,507, titled "Integrated microfluidics chip with Plurality of individually controllable thin film piezoelectric vibrating devices for sample preparation, sorting, levitation, rotation, regulating flow and droplet formation", filed on Apr. 6, 2023.

[0002] [Technical Field] This application generally relates to the flow control of cells or particles, and more specifically, to the dissociation and manipulation of particles in a microfluidic flow channel.

Background Art

[0003] Conventional sample preparation for single-cell sample analysis requires chemical (enzymatic) processes, mechanical processes, and careful handling. Specifically, in the case of microfluidic-based single-cell analysis techniques, it is crucial that the introduced sample does not aggregate, because aggregation renders the microfluidic chip useless. Therefore, it is important to have a mechanism that can dissociate the cell sample before it is inserted into the microfluidic chip. Centrifugation is the most common method for mechanically breaking up aggregates, and at the microscopic scale, surface acoustic waves (SAW) and bulk acoustic waves (BAW) have also been demonstrated to break up aggregates. These methods have significant limitations because centrifugation is a macroscopic-scale process, an additional step requiring dilution and subsequent verification, and can sometimes damage fragile cells. SAW and BAW require high power, are very slow, have limited operating regimes (they cannot effectively induce flow and are easily overwhelmed by lateral flow), and have been shown not to be readily incorporated into microfluidic devices for manufacturing. [Overview of the Initiative]

[0004] As described above, dissociation of cell samples is a challenging problem in microfluidic devices. In some systems, centrifugation, SAW, and BAW have been demonstrated to break up aggregates. However, these methods present challenges to microfluidic chip design and, in particular, are not readily incorporated into microfluidic devices for manufacturing. Devices and methods for dissociating and manipulating cell samples in microfluidic devices and systems are described herein. Such devices and methods may address the challenges associated with conventional devices and methods for dissociating and manipulating cell samples in microfluidic devices or systems.

[0005] For example, a particle sensing system (for example, for cells, bacteria, and / or viruses) having an integrated set of piezoelectric actuators (for example, to form laminar flow within a microfluidic device) is described. The set of piezoelectric actuators dissociates and manipulates particles in a fluid sample. Additional sets of piezoelectric actuators can discharge particles and / or at least a portion of the fluid sample from the microfluidic device.

[0006] According to several embodiments, the microfluidic device includes (i) an inlet channel, (ii) an outlet channel, (iii) a microfluidic channel, (iv) a first set of piezoelectric actuators, (v) a second set of piezoelectric actuators, and (vi) a third set of piezoelectric actuators. The microfluidic channel is arranged on a substrate between the inlet channel and the outlet channel such that the outlet of the microfluidic channel is located on at least a portion of the outlet channel. The first set of piezoelectric actuators is arranged adjacent to the inlet channel and configured to dissociate particles of a fluid sample within the microfluidic channel. The second set of piezoelectric actuators is arranged between the inlet channel and the outlet channel. The second set of piezoelectric actuators is configured to manipulate particles of a fluid sample as particles move through the microfluidic channel. The third set of piezoelectric actuators is arranged above and adjacent to the outlet channel. The third set of piezoelectric actuators is configured to discharge a portion of the fluid sample from the microfluidic channel through the outlet.

[0007] According to several embodiments, the method includes (i) supplying a fluid sample containing a plurality of particles through an inlet to a microfluidic channel of a microfluidic device having an outlet; (ii) selectively dissociating two or more particles of the fluid sample using a first set of piezoelectric actuators positioned adjacent to the inlet; (iii) selectively manipulating one or more particles of the fluid sample flowing through the microfluidic channel using a second set of piezoelectric actuators; and (iv) selectively discharging a portion of the fluid sample from the microfluidic channel using a third set of piezoelectric actuators positioned adjacent to the outlet.

[0008] Accordingly, the disclosed devices and methods relate to techniques for dissociating and manipulating cell samples, which are implemented in or as part of a microfluidic device using a set of piezoelectric actuators. Such techniques provide reliable preparation, localization, and analysis of single-cell samples. The disclosed devices and methods may replace or complement conventional devices and methods.

[0009] The features and advantages described herein are not necessarily exhaustive, and in particular, several additional features and advantages will be apparent to those skilled in the art in consideration of the drawings, specification and claims provided herein. Furthermore, it should be noted that the language used herein has been chosen primarily for readability and explanatory purposes and is not necessarily chosen to limit or restrict the subject matter described herein. [Brief explanation of the drawing]

[0010] To enable a more detailed understanding of this disclosure, a more specific description can be provided by referring to the features of various embodiments, some of which are shown in the accompanying drawings. However, the accompanying drawings only illustrate the relevant features of this disclosure and should not be considered limiting, for the description may allow for other valid features, as a reader skilled in the art will understand. [Figure 1A]A plan view of an exemplary microfluidic device according to several embodiments is shown. [Figure 1B] Figure 1A shows a cross-sectional view of a microfluidic device according to several embodiments. [Figure 1C] Figure 1A shows a plan view and a corresponding cross-sectional view of the input area of ​​the microfluidic device according to several embodiments. [Figure 1D] Figure 1A shows a plan view and a corresponding cross-sectional view of the control region of the microfluidic device according to several embodiments. [Figure 1E] Figure 1A shows a plan view and a corresponding cross-sectional view of the output region of the microfluidic device according to several embodiments. [Figure 2A] Alternative embodiments of the pattern of a first set of piezoelectric actuators in the input region of a microfluidic device are shown according to several embodiments. [Figure 2B] Alternative embodiments of the pattern of a first set of piezoelectric actuators in the input region of a microfluidic device are shown according to several embodiments. [Figure 2C] Alternative embodiments of the pattern of a first set of piezoelectric actuators in the input region of a microfluidic device are shown according to several embodiments. [Figure 2D] Alternative embodiments of the pattern of a first set of piezoelectric actuators in the input region of a microfluidic device are shown according to several embodiments. [Figure 2E] Alternative embodiments of the pattern of a first set of piezoelectric actuators in the input region of a microfluidic device are shown according to several embodiments. [Figure 2F] Alternative embodiments of the pattern of a first set of piezoelectric actuators in the input region of a microfluidic device are shown according to several embodiments. [Figure 2G] Alternative embodiments of the pattern of a first set of piezoelectric actuators in the input region of a microfluidic device are shown according to several embodiments. [Figure 3]This is a block diagram illustrating exemplary electrical components for a microfluidic device according to several embodiments. [Figure 4] This flowchart illustrates an exemplary method for dissociating and manipulating cells or particles within a microfluidic channel, according to several embodiments.

[0011] In accordance with common practice, the various features shown in the drawings are not necessarily drawn to a consistent scale, and similar reference numbers may be used throughout this specification and the drawings to represent similar features. [Modes for carrying out the invention]

[0012] Embodiments are referenced, and examples thereof are shown in the accompanying drawings. The following description includes numerous specific details to provide a full understanding of the various embodiments described. However, it will be apparent to those skilled in the art that the various embodiments described can be carried out without these specific details. In other examples, methods, procedures, components, circuits, and networks well known to those skilled in the art are not described in detail so as not to unnecessarily obscure the aspects of the embodiments.

[0013] The microfluidic devices described herein enable the electrical and / or optical sensing of one or more cells (or other particles such as bacteria, viruses, elements, compounds, etc.). The microfluidic aspects of the devices enable the precise dissociation and manipulation of particles within the microfluidic channels (e.g., using a set of piezoelectric actuators), as well as the accurate / precise measurement of particle properties. In addition, microfluidic devices having an outlet port (e.g., a nozzle) with a corresponding piezoelectric actuator enable the direct discharge (e.g., ejection) of cells (e.g., after they have been processed) and the regulation of the flow rate through the microfluidic device.

[0014] The microfluidic devices described herein offer improved design flexibility and can address a wider design space by utilizing the placement of piezoelectric materials. For example, piezoelectric structures ranging from small (e.g., less than 20 microns (μm)) to large (e.g., greater than 200 μm) can be implemented and used, thereby providing a wider range of performance attributes. In addition, the piezoelectric films described herein can improve processing (e.g., ease of processing and repeatability). For example, the piezoelectric film can be placed between a silicon-on-insulator (SOI) handle layer and a glass top layer, and encapsulated within an inert material (e.g., a dielectric material), so that the film is not subjected to mechanical stress. For example, this film can be formed by selectively etching the SOI. This can also enable easier wafer handling. The devices described herein can also improve packaging flexibility and / or robustness (improve yield) by mechanically protecting the piezoelectric actuator and piezoelectric film.

[0015] Figure 1A shows a plan view of a microfluidic device 100 according to several embodiments. The microfluidic device 100 includes a fluid channel 102 (e.g., a microfluidic channel) formed on a substrate. In some embodiments, the fluid channel 102 is formed by connecting a first substrate having a recess, recess, or notch to a second substrate so that the fluid channel 102 is formed between the first substrate and the second substrate. The fluid channel 102 has an inlet 103 and a plurality of outlets 107a-107c (e.g., three outlets as shown in Figure 1A). The positions of the inlet 103 and the plurality of outlets 107a-107c shown with respect to the fluid channel 102 in Figure 1A are just examples. The inlet 103 and the plurality of outlets 107a-107c may be configured at other positions along the longitudinal direction of the fluid channel 102 or the microfluidic device 100.

[0016] In some embodiments, the microfluidic device 100 includes more or fewer outlets (e.g., one, two, four, five, or six outlets) than those shown in FIG. 1A. In some embodiments, the length of the fluid channel 102 (e.g., measured from the inlet 103 to the plurality of outlets 107a - 107c) is in the range of 1 millimeter (mm) to 50 mm (e.g., 15 mm). In some embodiments, the width of the microfluidic device 100 is in the range of 0.2 mm to 5 mm (e.g., 0.7 mm). In some embodiments, the width of the fluid channel 102 is configured based on the size of the particles to be analyzed. For example, for cell measurements, the width of the fluid channel 102 can be configured according to the size of the cells such that only a single cell is detected at a time (e.g., the width of the fluid channel 102 in the sensing region 110). In some embodiments, the fluid channel 102 includes one or more portions having different respective widths. For example, the fluid channel 102 can include a portion having a shape that protrudes (where the width of that portion is greater than the width of the fluid channel 102 in the sensing region 110) (e.g., the width of the fluid channel 102 in the control region 120). Similarly, the fluid channel 102 can include one or more portions having a width narrower than the width of the fluid channel 102 in the sensing region 110. In some embodiments, a wider fluid channel 102 results in a slower velocity of the particles flowing within the corresponding portion of the fluid channel 102 (e.g., when the fluid channel 102 has a uniform height). Thus, for example, a wider portion can be used to reduce the velocity of the particles (e.g., immobilize the particles), thereby providing more time for analyzing the particles.

[0017] The device 100 also includes an input region 104 (sometimes called the inlet region) for receiving a sample fluid containing particles (e.g., cells) as input to the microfluidic device 100, and for supplying the sample fluid from the inlet port to the fluid channel 102 via the inlet 103. According to some embodiments, the microfluidic device 100 includes a plurality of pillars 101 adjacent to (or within) the input region 104. The pillars 101 are shaped and arranged to interfere with (e.g., separate) particles in the sample fluid. The shape and size of the input region 104 in Figure 1A are merely examples. The microfluidic device 100 includes a first set of piezoelectric actuators 105 around the input region 104 (e.g., around the inlet 103). In some embodiments, the first set of piezoelectric actuators 105 is a single piezoelectric actuator (e.g., as shown in Figure 1A). In some embodiments, a first set of piezoelectric actuators 105 is positioned adjacent to the inlet 103 and configured to induce laminar flow from the inlet 103 toward a plurality of outlets 107a to 107c. In some embodiments, the first set of piezoelectric actuators 105 is configured to generate inertial and acoustic turbulence within the asymmetric channel to induce chaotic advection and drive local nonlinear behavior within the laminar flow field. In some embodiments, in response to the application of an electrical signal from an actuation circuit (e.g., actuation circuit 330 as described with respect to Figure 3), the first set of piezoelectric actuators 105 generates displacements causing mixing and dissociation of the sample fluid, as well as vibrations that generate acoustic waves, thereby controlling the local inertial motion of particles to induce laminar flow within the fluid channel 102. In some embodiments, the first set of piezoelectric actuators 105 is configured to regulate particle mixing, particle dissociation, particle aggregation, particle solidification, and particle clogging in the fluid sample (e.g., to dissociate any clogged particles and / or to prevent clogging in the input region 104 and / or sensing region 110). In some embodiments, a first set of piezoelectric actuators 105 is configured to adjust the particles and / or fluid viscosity and / or density of a fluid sample.In some embodiments, the first set of piezoelectric actuators 105 is configured to control and / or prevent the formation of bubbles in the fluid sample.

[0018] The microfluidic device 100 further includes an output region 106 for collecting at least a portion of the sample fluid from the fluid channel 102 and for discharging or delivering a portion of the sample fluid via a plurality of outlets (e.g., nozzles) 107a-107c for disposal or further processing or analysis. In some embodiments, the output region 106 includes a plurality of output sub-regions, each sub-region including one of the plurality of outlets 107a-107c. The shape and size of the output region 106 and the output sub-regions in FIG. 1A are merely examples. The output region 106 includes a third set of piezoelectric actuators 109a-109c disposed adjacent to each of the plurality of outlets 107a-107c for discharging a portion of the fluid within the fluid channel 102. In some embodiments, each output sub-region includes a subset of the third set of piezoelectric actuators 109a-109c, each subset being adjacent to a respective one of the plurality of outlets 107a-107c. Thus, different portions of the sample fluid may be collected and discharged from corresponding output sub-regions. In some embodiments, the microfluidic device 100 is configured such that each output sub-region and each of the plurality of outlets 107a-107c are associated with the type and / or quality of particles in the fluid sample. For example, the control region 120 is configured such that different types of particles in the fluid sample flow to different ones of the plurality of outlets 107a-107c.

[0019] The microfluidic device 100 further includes a control region 120 for regulating the flow of the sample fluid and manipulating particles in the sample fluid (e.g., levitating, rotating, localizing, and / or deflecting cells). The shape and size of the control region 120 in Figure 1A are merely examples. The control region 120 includes a second set of piezoelectric actuators 121a and 121b. In some embodiments, the second set of piezoelectric actuators 121a and 121b are located between the inlet 103 and a plurality of outlets 107a-107c (for example, the inlet 103 may be located in the upstream region of the fluid channel 102, the plurality of outlets 107a-107c may be located in the downstream region of the fluid channel 102, and the second set of piezoelectric actuators may be located in the midstream region of the fluid channel 102). In some embodiments, the second set of piezoelectric actuators 121a and 121b are located between the sensing region 110 and the output region 106. In some embodiments, the second set of piezoelectric actuators 121a and 121b includes actuators arranged on both sides of the fluid channel 102 (e.g., the two actuators shown in Figure 1A). In some embodiments, the second set of piezoelectric actuators 121a and 121b are configured to deflect particles in the fluid sample to designated output sub-regions of the output region 106 (e.g., each output sub-region corresponding to a particular cell or cell type) based on one or more properties of particles in the fluid sample. Manipulation of particles in the sample fluid can be achieved, for example, by vibration and displacement caused by activation of the second set of piezoelectric actuators 121a and 121b. For example, the vibration and displacement provided by the second set of piezoelectric actuators 121a and 121b can control the movement, position, rotation, and / or acceleration of particles in the fluid channel 102. In some embodiments, the second set of piezoelectric actuators 121a and 121b are configured to vibrate in a direction perpendicular to the flow of the sample fluid in the fluid channel 102.For example, two actuators positioned on either side of the fluid channel 102 work together to either deflect particles downstream to one of the output sub-regions and / or adjust the position of the particles (e.g., sorting may be performed based on phenotypic analysis without cell labeling to isolate and / or enrich various cell population types). Particle manipulation may be based on commands from the sensing region 110 and / or the actuation circuit 330. In some embodiments, the second set of piezoelectric actuators includes more than two piezoelectric actuators (e.g., two pairs, three pairs, or four pairs of piezoelectric actuators). In some embodiments, the piezoelectric actuators in the second set of piezoelectric actuators are offset from each other (e.g., piezoelectric actuator 121a is positioned upstream of piezoelectric actuator 121b, or vice versa). In some embodiments, the piezoelectric actuators in the second set of piezoelectric actuators have different sizes and / or shapes.

[0020] In some embodiments, each of the three sets of piezoelectric actuators (a first set of piezoelectric actuators 105, a second set of piezoelectric actuators 121a-121c, and a third set of piezoelectric actuators 109a-109c) includes one or more piezoelectric actuators (e.g., piezoelectric micro-electro-mechanical system (MEMS) actuators). In some embodiments, each of the three sets of piezoelectric actuators includes two or more piezoelectric actuators. In some embodiments, the first set of piezoelectric actuators 105 includes one or more actuators having a first size, the second set of piezoelectric actuators 121a and 121b includes one or more actuators having a second size different from the first size, and the third set of piezoelectric actuators 109a-109c includes one or more actuators having a third size different from the first and second sizes (e.g., the first size is three times the second size and twice the third size). In some embodiments, each of the three sets of piezoelectric actuators has a thickness in the range of 0.1 μm to 5 μm. In some embodiments, each of the three sets of piezoelectric actuators is arranged on a film (for example, the film is made up of a substrate having a thickness in the range of 1 μm to 5 μm).

[0021] In some embodiments, the microfluidic device 100 includes an actuation circuit (e.g., actuation circuit 330 described with respect to Figure 3) electrically connected to each of three sets of piezoelectric actuators. In some embodiments, each of the three sets of piezoelectric actuators is configured to vibrate at a configurable frequency (e.g., in the range of 1 kilohertz (kHz) to 100 gigahertz (GHz)). In some embodiments, operating within a frequency range includes vibrating within a frequency range. In some embodiments, at least a subset of the three sets of piezoelectric actuators described herein is configured to use an operating frequency within a frequency range based on the type of particles in the sample fluid and / or the intended behavior (e.g., dissociation, levitation, rotation, and / or mixing). For example, a subrange of megahertz (MHz) may be used to levitate particles (e.g., cells), a subrange of kHz and / or MHz may be used to rotate particles, and a range of kHz may be used to sort particles. As another example, the first set of piezoelectric actuators can operate in the kHz to MHz range to perform sonication of a sample, and the selected frequency may depend on the desired function (e.g., breaking up agglomerates, breaking up blockages, and / or pushing / stimulating fluid flow) and the type of particles in the sample.

[0022] In some situations, the piezoelectric actuators disclosed herein operate at low power, and their operating modes are tunable based on the desired operating function. In some embodiments, each of the three sets of piezoelectric actuators is configured to have an operating voltage in the range of 0.1 volts (V) to 100 V (e.g., 0.1 V to 30 V), based on the actuator type and / or the sample type. For example, a PZT actuator may have an operating voltage up to 30 V, while a polymer actuator may have an operating voltage in the range of 50 V. In some embodiments, each of the three sets of piezoelectric actuators is configured to have a deflection in the range of 5 nanometers (nm) to 50 μm (e.g., 100 nm to 10 μm), based on the actuator type and / or the sample type. In some embodiments, when an electrical signal is applied from the actuation circuit, each of the three sets of piezoelectric actuators generates displacement and vibrations that produce acoustic waves (either individually or synchronously), thereby controlling the local inertial motion of particles in the fluid channel 102 in the three-dimensional x, y, and z planes with sub-μm level control. In some embodiments, each of the three sets of piezoelectric actuators induces laminar flow from the inlet 103 toward a plurality of outlets 107a to 107c. In some configurations, the sample fluid flows through the fluid channel 102 at a rate of 1 μL / min to 1 mL / min.

[0023] In some embodiments, the microfluidic device 100 further includes a sensing region 110 for sensing one or more properties of particles in a fluid sample. In some embodiments, the sensing region 110 overlaps with (e.g., is the same as) the control region 120. The sensing region 110 comprises a set of electrodes 125 and / or other sensing elements for sensing one or more properties. In some embodiments, the echo responses of a first set of piezoelectric actuators 105 in the input region 104 and / or a second set of piezoelectric actuators 121a and 121b in the control region 120 are sensed to determine the properties of the sample and / or the microfluidic channel (e.g., the echo responses of the actuators will change if foreign matter is present on the actuators). The location of the sensing region 110 in Figure 1A is merely an example. In some embodiments, the sensing region includes at least one filter (e.g., a filter pillar array). The location of the set of electrodes 125 and the sensing region 110 may vary along the length of the fluid channel 102. For example, to obtain accurate measurements, it may be beneficial to make the distance between each electrode in the electrode set equal. In some embodiments, the electrode set detects electrical signals from particles (e.g., cells) flowing through a fluid channel 102 adjacent to the electrode set. In some embodiments, the microfluidic device 100 includes a readout circuit (e.g., a drive / readout circuit 340 described with respect to Figure 3) electrically connected to the electrode set. In some embodiments, the readout circuit receives electrical signals from the electrode set and relays the electrical signals (with or without processing such as filtering) to one or more processors in the microfluidic device 100, or to one or more processors operably connected to the microfluidic device 100.

[0024] In some embodiments, the set of electrodes charges particles flowing through the fluid channel 102 by applying an electric field to the sample fluid to charge the particles so that the particles can be manipulated by another electric field. In some embodiments, the distance between the pair of electrodes is configured so that only a single cell is charged and / or manipulated at a time. In some embodiments, the microfluidic device 100 includes a drive circuit (e.g., a drive circuit 340 described with respect to Figure 3) electrically connected to the set of electrodes. In some embodiments, the drive circuit is configured to generate electrical signals in the MHz and GHz frequency ranges. In some embodiments, the frequency of the electrical signals supplied to the electrodes depends on one or more types of particles being analyzed using the microfluidic device 100. In some embodiments, the drive circuit is configured to generate electrical signals having voltages in the range of 1V to 100V. In some embodiments, the drive circuit is configured to generate electrical signals having pulses in the range of 1μs to 20μs. In some embodiments, the pulses are sawtooth pulses, square pulses, or sinusoidal pulses.

[0025] In some embodiments, each particle may pass near one or more pairs of electrodes in a period of 0.1 milliseconds (ms) to 100 ms. In some embodiments, each particle may pass near one or more pairs of second electrodes in a period of 0.1 ms to 100 ms. In some embodiments, the separation distance between the pairs of electrodes, and the distance between the first and second electrodes, are configured based on one or more types of particles to be analyzed using device 100. In some embodiments, the particle processing rate in the microfluidic device 100 is 100 particles / min to 1 million particles / min.

[0026] In some embodiments, sets of electrodes are arranged on the same substrate as each other and / or the piezoelectric components. In some embodiments, pairs of electrodes are arranged on different substrates (e.g., one electrode of a pair is on a lower substrate and the other electrode of a pair is on an upper substrate). In some embodiments, the electrodes have a thickness in the range of 0.01 μm to 2 μm. In some embodiments, the electrodes are made of gold (Au) and / or platinum (Pt). In some embodiments, an additional set of electrodes is shielded from each of the three sets of piezoelectric actuators. For example, the electrodes are on a first substrate / chip, and each of the sets of piezoelectric actuators is on a separate substrate / chip. In some embodiments, the operation of the electrodes is based on the operation of each of the sets of piezoelectric actuators (e.g., the electrode operation is timed to reduce / minimize interference from the operation of each of the sets of piezoelectric actuators).

[0027] Figure 1B shows a cross-sectional view of a microfluidic device 100 according to several embodiments. As shown in Figure 1B, the microfluidic device 100 includes an optical layer 152 (e.g., made of a transparent or translucent material such as glass) having an inlet channel 164 (e.g., inlet 103 shown in Figure 1A). The optical layer 152 is connected to a substrate 159 via a polymer layer 154. The polymer layer 154 constitutes a fluid channel 170 (e.g., fluid channel 102 shown in Figure 1A). In some embodiments, the fluid channel 170 is a microfluidic channel. According to some embodiments, an outlet channel 166 is configured within the substrate 159 (e.g., etched within a handle layer 162). In some embodiments, three sets of piezoelectric actuators, as described with reference to Figure 1A, are arranged on a portion of the substrate from which the silicon base layer has been removed (e.g., to form a film beneath the actuators) (e.g., as shown in Figures 1B to 1E). In the example shown in Figure 1B, the substrate 159 includes a handle layer 162 (e.g., composed of silicon), a buried oxide (BOX) layer 160 (e.g., composed of SiO2), and a device layer 158. According to some embodiments, an outlet 168 (e.g., one of the multiple outlets 107a-107c shown in Figure 1A) is located within the substrate 159 (e.g., through the BOX layer 160 and the device layer 158). An inactivating layer 156 (e.g., a sealing layer) (e.g., composed of nitrides, silicon nitride, silicon carbide, SiO2, aluminum nitride, aluminum oxide, and / or a photosensitive polymer) is connected to the substrate 159. According to some embodiments, electrodes are arranged within a fluid channel 170. In some embodiments, the device layer 158 has a thickness in the range of 1 μm to 5 μm. In some embodiments, the handle layer 162 has a thickness in the range of 200 μm to 600 μm. In some embodiments, the inactivation layer 156 has a thickness in the range of 0.01 μm to 1 μm.

[0028] In some embodiments, the fluid channel 170 is composed of an optical layer 152, a polymer layer 154, and a device layer 158. In some embodiments, the channel 170 has a height of 10 μm to 1 mm (for example, 10 μm, 20 μm, 30 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100 μm, 200 μm, 300 μm, 400 μm, 500 μm, 600 μm, 700 μm, 800 μm, 900 μm, or 1 mm, or within the range of any two of the aforementioned values). In some embodiments, the optical layer 152 has a thickness of 5 μm to 2 mm (for example, 5 μm, 6 μm, 7 μm, 8 μm, 9 μm, 10 μm, 20 μm, 30 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100 μm, 200 μm, 300 μm, 400 μm, 500 μm, 600 μm, 700 μm, 800 μm, 900 μm, 1 mm, 1.1 mm, 1.2 mm, 1.3 mm, 1.4 mm, 1.5 mm, 1.6 mm, 1.7 mm, 1.8 mm, 1.9 mm, or 2 mm, or within the range of any two of the aforementioned values). In some embodiments, the substrate 159 has a thickness of 500 μm. In some embodiments, the inlet channel 164 is configured within the substrate 159 (for example, in addition to, or alternative to, being configured within the optical layer 152).

[0029] In some embodiments, the polymer layer 154 is positioned between the optical layer 152 and the substrate 159 (for example, between the optical layer 152 and the device layer 158). In some embodiments, the polymer layer 154 is composed of polyvinylidene fluoride (PVDF) and its copolymers, polyamides, as well as paralyn-C, polyimide and polyvinylidene chloride (PVDC), and diphenylalanine peptide nanotubes (PNTs). In some embodiments, the polymer layer 154 is fitted and / or positioned to bond the optical layer 152 (e.g., a first substrate) and the substrate 159 (e.g., a second substrate) to each other. For example, if the polymer layer 154 is not included, the optical layer 152 may not bond to the substrate 159. In some embodiments, the polymer layer 154 is composed of a photosensitive material. For example, by drawing the polymer layer 154, definitions such as the width, height, and curvature of the fluid channel 170 can be provided (e.g., the signal-to-noise ratio (SNR) for single-cell sensing can be improved). In some embodiments, the polymer layer 154 is fitted and / or positioned to provide stress relief to the microfluidic device 100 (e.g., to prevent stress cracks when the chip is assembled into a package). In some embodiments, the polymer layer 154 is cured / solidified (e.g., subjected to multiple stages of curing / solidification). In some embodiments, the polymer layer 154 is exposed to a temperature above the transition temperature of the polymer layer 154 (e.g., 150 degrees Celsius), thereby curing the polymer layer 154 and bonding the optical layer 152 (e.g., glass) to the substrate 159 (e.g., silicon). In some embodiments, the polymer layer 154 is composed of a liquid or a dry film. The polymer layer 154 may be a negative or positive photoresist. In some embodiments, the polymer layer 154 is composed of epoxy (e.g., bisphenol A) and / or polyimide, along with a photoinitiator (e.g., added to drive crosslinking based on the wavelength of light).

[0030] Figures 1C, 1D, and 1E show cross-sectional views of the microfluidic device 100 in each of a sub-region of the input region 104, control region 120, and output region 106, respectively, according to several embodiments. Figure 1C shows the input region 104 having an input channel 164, which in some embodiments is configured within an optical layer 152. The input region 104 further includes a first set of piezoelectric actuators 105 located below the input channel 164. Figure 1D shows the control region 120 having one of a second set of piezoelectric actuators 121a and 121b. Figure 1E shows a sub-region of the output region 106 having an output channel 166 and an output 168 (e.g., one of a plurality of outputs 107a to 107c), which in some embodiments is configured within a substrate 159. The sub-region further includes a subset of a third set of piezoelectric actuators 109a to 109c located around the output 168.

[0031] Each of the first set of piezoelectric actuators 105, the second set of piezoelectric actuators 121a and 121b, and the third set of piezoelectric actuators 109a to 109c includes a lower electrode 174, an upper electrode 180, and a piezoelectric layer 178. Each piezoelectric actuator further includes a lower contact 176 connected to the lower electrode 174 and an upper contact 188 connected to the upper electrode 180. In some embodiments, the lower electrode 174 and / or the upper electrode 180 are each composed of a conductive material (e.g., copper, aluminum, gold, or platinum), strontium oxide (SRO), and / or titanium. In some embodiments, the lower electrode 174 and / or the upper electrode 180 have a thickness in the range of 10 nm to 50 nm.

[0032] In some embodiments, a first conductive layer is connected to an upper contact 188, and a second conductive layer is connected to a lower contact 176. For example, the first and second conductive layers include conductive wiring configured to supply electrical signals (e.g., actuation signals) to the piezoelectric actuator. In some embodiments, the first and second conductive layers connect their respective piezoelectric actuators to a control circuit (e.g., actuation circuit 330). In some embodiments, each conductive layer is made of a conductive material (e.g., copper, aluminum, gold, or platinum). In some embodiments, each bond pad is connected to each of the conductive layers (e.g., each bond pad provides an off-chip electrical connection to the piezoelectric actuator via the conductive layer). A deactivating layer 156 separates the piezoelectric layer 178 and electrodes 174 and 180 from the fluid channel 170. In some embodiments, an intermediate adhesive layer is arranged between the piezoelectric layer 178 and the deactivating layer 156. In some embodiments, the intermediate adhesive layer is configured to match the mechanical impedance between the piezoelectric layer 178 and the deactivating layer 156. In some embodiments, the intermediate adhesive layer is made of a metal (e.g., titanium (Ti)). In some embodiments, the intermediate adhesive layer is configured to reduce cracking of the inactivating layer 156. In some embodiments, the intermediate adhesive layer has a thickness in the range of 10 nm to 300 nm.

[0033] In some embodiments, the piezoelectric layer 178 has a thickness of 0.1 μm to 100 μm (for example, 0.1 μm, 0.5 μm, 1 μm, 2 μm, 5 μm, 10 μm, 20 μm, 30 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, or 100 μm, or within the range of any two of the aforementioned values). In some embodiments, the piezoelectric layer 178 is located on a silicon-on-insulator (SOI) layer. In some embodiments, the silicon-on-insulator (SOI) layer is connected to one or more of the contacts (for example, contact 176). In some embodiments, the piezoelectric layer 178 is made of PZT and has a thickness in the range of 0.1 μm to 10 μm (for example, 2 μm).

[0034] Figure 1C further shows a plan view of the first piezoelectric film 131 according to several embodiments. The first piezoelectric film 131 is located in the insertion region 104. The first piezoelectric film 131 may include a substrate 159 (e.g., an embedded oxide layer and / or device layer), an inactivation layer 156, a piezoelectric material 178 (e.g., PZT), and associated conductive layers (e.g., electrodes, vias, contacts, and / or wiring). For example, the cross-sectional view in Figure 1C shows the first piezoelectric film 131 along the diameter A-A'.

[0035] Figure 1D further shows a plan view of a second piezoelectric film 132 according to several embodiments. The second piezoelectric film 132 is located in a control region 120. The second piezoelectric film 132 may include a substrate 159, an inactivating layer 156, a piezoelectric material 178, and associated conductive layers (e.g., electrodes, vias, contacts, and / or wiring). In some embodiments, the second piezoelectric film 132 includes first contacts 135 (e.g., upper contacts 188) electrically connected to each electrode (e.g., upper electrode 180). The first electrodes may be connected to one or more piezoelectric layers to actuate the piezoelectric material, regulate the flow of the sample fluid, and manipulate particles in the sample fluid. For example, the cross-sectional view in Figure 1D shows the second piezoelectric film 132 along the diameter B-B'.

[0036] Figure 1E further shows a plan view of a third piezoelectric film 133 according to several embodiments. The third piezoelectric film 133 is located in the output region 106. The third piezoelectric film 133 may include a substrate 159, an inactivating layer 156, a piezoelectric material 178, and associated conductive layers (e.g., electrodes, vias, contacts, and / or wiring). In some embodiments, the third piezoelectric film 133 includes a first contact 135 (e.g., a lower contact 176) electrically connected to each electrode (e.g., a lower electrode 174), a second contact 136 (e.g., an upper contact 188) electrically connected to each second electrode (e.g., an upper electrode 180), and an outlet 168 (e.g., one of a plurality of outlets 107a-107c) (e.g., a nozzle). The first and second electrodes may be connected to one or more piezoelectric layers to actuate the piezoelectric material and discharge the sample fluid through the outlet 137. In some embodiments, the outlet 168 has a diameter of less than 200 μm (e.g., 30 μm, 60 μm, or 120 μm). For example, the cross-sectional view in Figure 1E shows a third piezoelectric film 133 along the diameter C-C'.

[0037] In some embodiments, the first piezoelectric material is any of the families of ceramics having polyvinylidene fluoride, gallium phosphate, sodium bismuth titanate, lead zirconate titanate, quartz, berinite (AlPO4), sucrose (table sugar), Rochelle salt, topaz, tourmaline group minerals, lead titanate (PbTiO3), langasite (La3Ga5SiO14), gallium orthophosphate (GaPO4), lithium niobate (LiNbO3), lithium tantalate (LiTaO3), or perovskite. These include tungsten bronze, potassium niobate (KNbO3), sodium tungstate (Na2WO3), Ba2NaNb5O5, Pb2KNb5O15, potassium sodium niobate ((K,Na)NbO3) (e.g., NKN or KNN), bismuth ferrite (BiFeO3), sodium niobate (NaNbO3), barium titanate (BaTiO3), bismuth titanate (Bi4Ti3O12), sodium bismuth titanate (NaBi(TiO3)2), zincblende crystal, GaN, InN, AlN, and ZnO. In some embodiments, the piezoelectric material is one of the BKT-BMT-BFO set. For example, KNN such as (K0.5Na0.5)NbO3, BKT such as (Bi0.5K0.5)TiO3, BMT such as Bi(Mg0.5Ti0.5)O3, BFO such as BiFeO3, BNT such as (Bi0.5Na0.5)TiO3, and / or BT such as BaTiO3.

[0038] Figures 2A to 2G show alternative embodiments of the pattern of the first set of piezoelectric actuators 105 in the input region 104 according to several embodiments. Figure 2A shows a first configuration according to several embodiments, in which the first set of piezoelectric actuators 105 are aligned with the inlet 103 and have a diameter larger than the diameter of the inlet 103 (for example, as shown in Figure 1A). Figure 2B shows a second configuration according to several embodiments, in which the first set of piezoelectric actuators 105 are aligned with the inlet 103 and have the same diameter as the diameter of the inlet 103. Figure 2C shows a third configuration in which the first set of piezoelectric actuators 105 are aligned with the inlet 103 and have a diameter smaller than the diameter of the inlet 103. Figure 2D shows a fourth configuration in which the first set of piezoelectric actuators 105 are offset from the inlet 103 (for example, as shown in Figure 2D, in which the centers of the first set of piezoelectric actuators 105 are closer to the control region 120 relative to the inlet 103) and have the same diameter as the diameter of the inlet 103. Figure 2E shows a fifth configuration in which the first set of piezoelectric actuators 105 are offset from the inlet 103 (for example, as shown in Figure 2E, the center of the first set of piezoelectric actuators 105 is closer to the control region 120 relative to the inlet 103) and have a diameter smaller than the diameter of the inlet 103. Figure 2F shows a sixth configuration in which the first set of piezoelectric actuators 105 are offset from the inlet 103 (for example, as shown in Figure 2F, the center of the first set of piezoelectric actuators 105 is closer to the control region 120 relative to the inlet 103) and have a diameter larger than the diameter of the inlet 103. Figure 2G shows a seventh configuration in which the first set of piezoelectric actuators 105 are offset from the inlet 103 (for example, as shown in Figure 2G, the center of the first set of piezoelectric actuators 105 is closer to the control region 120 relative to the inlet 103) and are oval-shaped.

[0039] According to some embodiments, the pattern of the inlet 103 and / or the pattern of the first set of piezoelectric actuators 105 are of different shapes (e.g., circular, oval, rectangular, etc.). According to some embodiments, the pattern of the inlet 103 and / or the pattern of the first set of piezoelectric actuators 105 are selected based on the desired function of the input area 104, the quality of the sample fluid, and / or the quality of the particles. For example, the pattern of the inlet 103 and / or the pattern of the first set of piezoelectric actuators and / or their relative positions may be selected as desired to maximize the dissociation of particles in the fluid sample, achieve a desired layer flow rate in the fluid channel 102, and / or prevent the formation of bubbles in the fluid sample.

[0040] Figure 3 is a block diagram illustrating exemplary electrical components for a microfluidic device according to several embodiments. In some embodiments, the device (e.g., microfluidic device 100) includes one or more processors 302 (e.g., one or more microcontrollers (MCUs), one or more CPUs, and / or other types of control circuits) and a memory 304. In some embodiments, the memory 304 contains instructions for execution by one or more processors 302. In some embodiments, the stored instructions include instructions for providing operating signals to each of three sets of piezoelectric actuators (e.g., a first set of piezoelectric actuators 105, a second set of piezoelectric actuators 121a and 121b, and a third set of piezoelectric actuators 109a-109c). In some embodiments, the operating signals for different piezoelectric actuators are configured such that each piezoelectric actuator generates vibrations at a different frequency. For example, one or more of the piezoelectric actuators may operate at a frequency in the range of 1 kHz to 100 kHz, for example, based on a desired flow rate. In some embodiments, the stored instructions include instructions for providing activation signals to electrodes 305 to charge particles flowing through the fluid channel 102, thereby allowing the particles to be manipulated using an electric field. In some embodiments, the device also includes an electrical interface 306 connected to one or more processors 302 and memory 304. In some embodiments, the device further includes an actuation circuit 330 connected to one or more piezoelectric actuators 301, such as a first set of piezoelectric actuators 105, a second set of piezoelectric actuators 121, and / or a third set of piezoelectric actuators 209a-209c. For example, the actuation circuit 330 transmits an electrical signal to the piezoelectric actuator to initiate operation of the piezoelectric actuator.

[0041] In some embodiments, the device further includes a drive circuit 340 connected to the electrodes 305. For example, the drive circuit 340 transmits an electrical signal to one or more electrodes to generate an electric field using one or more electrodes to charge particles flowing through a fluid channel. In some embodiments, the device further includes a readout circuit (e.g., a drive / readout circuit 340) connected to one or more electrodes 305 (e.g., electrode 125). In some embodiments, the drive / readout circuit is further connected to a piezoelectric actuator 301 (e.g., a second set of piezoelectric actuators 121). The readout circuit receives an electrical signal from one or more electrodes 305 and provides the electrical signal (with or without processing) to one or more processors 302 via an electrical interface 306.

[0042] In some embodiments, the device further includes a measurement / analysis circuit 350 connected to one or more electrodes 352 (e.g., electrode 125) and / or one or more piezoelectric actuators 354 (e.g., any of the piezoelectric actuators described herein). In some embodiments, the measurement / analysis circuit 350 is configured to detect the electrical impedance of particles in a microfluidic channel (e.g., fluid channel 102). In some embodiments, the measurement / analysis circuit 350 is connected to an actuation circuit 330 to inform the actuation circuit 330 how to actuate one or more piezoelectric actuators 301 (e.g., based on impedance measurements). In some embodiments, the actuation circuit 330 is configured to adjust the operation of one or more piezoelectric actuators (e.g., adjust frequency and / or magnitude) based on particle analysis results from the measurement / analysis circuit 350. For example, if the measurement / analysis circuit 350 indicates the presence of particle agglomerations, one or more piezoelectric actuators 301 are configured to break up the agglomerations. In some embodiments, one or more electrodes are shared between electrode 305 and electrode(s) 352. In some embodiments, one or more piezoelectric actuators are shared between one or more piezoelectric actuators 301 and one or more piezoelectric actuators 354.

[0043] Figure 4 is a flow diagram illustrating a method 400 for controlling the flow of cells or particles in microfluidic channels (e.g., fluid channel 102 and / or fluid channel 170) according to some embodiments. In some embodiments, method 400 is performed in a microfluidic device (e.g., microfluidic device 100).

[0044] Method 400 includes supplying a fluid sample containing a plurality of particles (e.g., cells, molecules, and / or other types of particles) through an inlet (e.g., inlet 103) to a microfluidic channel (e.g., fluid channel 102) of a microfluidic device (e.g., microfluidic device 100) having an outlet (e.g., one of a plurality of outlets 107a to 107c) (402). For example, a sample fluid containing particles is supplied into the fluid channel 102 having an inlet 103 and a plurality of outlets 107a to 107c.

[0045] In some embodiments, method 400 further includes determining the state of a microfluidic device (404). In some embodiments, determining the state of a microfluidic device includes operating one or more actuators from a first set of piezoelectric actuators (e.g., first set of piezoelectric actuators 105) and / or a second set of piezoelectric actuators (second set of piezoelectric actuators 121a and 121b) in an activated state to generate vibration signals (406). In some embodiments, determining the state of a microfluidic device further includes switching the operation of one or more actuators to a sensing state to sense the echo response corresponding to the vibration signals (408). For example, if large aggregates of cells are present on one or more actuators, the response will change. In some embodiments, the actuators of the first subset operate in an activated state to generate one or more vibration signals, and the actuators of the second subset operate in a sensing state to sense the echo response of one or more vibration signals. As an example, the acoustic signal received by the second subset of piezoelectric actuators after rebound may indicate the viscosity and / or density of the sample and can be used to evaluate aggregation / coagulation / clogging of the sample.

[0046] Method 400 includes selectively dissociating two or more particles of a fluid sample using a first set of piezoelectric actuators positioned adjacent to the inlet (410).

[0047] In some embodiments, Method 400 includes using a set of electrodes (e.g., electrode 352) and / or a second set of piezoelectric actuators (e.g., piezoelectric actuator 354) to sense one or more properties of one or more particles of a fluid sample flowing through a microfluidic channel (412). In some embodiments, Method 400 further includes selectively providing actuation signals to a first set, a second set, and a third set of piezoelectric actuators (e.g., via actuation circuit 330) based on acquired sensing data (e.g., via measurement / analysis circuit 350). In some embodiments, actuation signals are generated to produce vibration frequencies in the actuators based on the fluid composition in the microfluidic channel (e.g., particle size and / or concentration). For example, the third set of actuators may be configured to vibrate so that only one cell is ejected at a time. For example, the sensing signals may include information about the state of the fluid.

[0048] Method 400 includes using a second set of piezoelectric actuators to selectively manipulate one or more particles of a fluid sample flowing through a microfluidic channel (414). In some embodiments, selectively manipulating one or more particles of a fluid sample flowing through a microfluidic channel using a second set of piezoelectric actuators includes selectively levitating, rotating, and / or sorting one or more particles. In some embodiments, selectively manipulating one or more particles of a fluid sample flowing through a microfluidic channel using a second set of piezoelectric actuators includes adjusting the operating frequency of the second set of piezoelectric actuators to perform different types of operations.

[0049] Method 400 includes selectively discharging a portion of the fluid sample from the microfluidic channel using a third set of piezoelectric actuators (e.g., a third set of piezoelectric actuators 109a-109c) positioned adjacent to the outlet (416). In some embodiments, the third set of piezoelectric actuators and the outlet are sized to discharge particles having a diameter in the range of 2 μm to 50 μm.

[0050] In light of the above disclosure, specific embodiments are described below.

[0051] (A1) In one embodiment, several embodiments include a microfluidic device (e.g., microfluidic device 100), which includes (i) an inlet channel (e.g., inlet channel 164), (ii) an outlet channel (e.g., outlet channel 166), (iii) microfluidic channels (e.g., fluid channel 102 and / or fluid channel 170), (iv) a first set of piezoelectric actuators (e.g., a first set of piezoelectric actuators 105), (v) a second set of piezoelectric actuators (e.g., a second set of piezoelectric actuators 121a and 121b), and (vi) a third set of piezoelectric actuators (e.g., a third set of piezoelectric actuators 109a to 109c). The microfluidic channels are arranged on a substrate (e.g., substrate 159) between the inlet channel and the outlet channel such that the outlets of the microfluidic channels (e.g., one of a plurality of outlets 107a to 107c and / or outlet 168) are located on at least a portion of the outlet channel. A first set of piezoelectric actuators is arranged adjacent to the inlet channel and configured to dissociate particles (e.g., cells) of the fluid sample within the microfluidic channel. In some embodiments, the first set of piezoelectric actuators is configured to pull, separate, and / or dissociate particles of the fluid sample from one another. A second set of piezoelectric actuators is arranged between the inlet channel and the outlet channel. The second set of piezoelectric actuators is configured to manipulate particles of the fluid sample as they move through the microfluidic channel. A third set of piezoelectric actuators is arranged above the outlet channel and adjacent to the outlet. The third set of piezoelectric actuators is configured to discharge a portion of the fluid sample from the microfluidic channel through the outlet. In some embodiments, at least some of the piezoelectric actuators described herein are configured to vibrate at a configurable frequency.

[0052] In some embodiments, the first set of piezoelectric actuators consists of a single actuator positioned below the inlet channel. In some embodiments, the first set of piezoelectric actuators is configured to generate inertial and acoustic turbulence within an asymmetric channel to induce chaotic advection and drive local nonlinear behavior in a laminar flow field. In some embodiments, the first set of piezoelectric actuators is configured to ultrasonically treat a fluid sample (e.g., to dissociate any clogged particles and / or to prevent clogging in the inlet region and / or sensing region). In some embodiments, the first set of piezoelectric actuators is positioned between the inlet channel and the filter region (e.g., a filter pillar array). In some embodiments, the first set of piezoelectric actuators consists of a single actuator positioned below the inlet channel. In some embodiments, the first set of piezoelectric actuators is configured to generate inertial and acoustic turbulence within an asymmetric channel to induce chaotic advection and drive local nonlinear behavior in a laminar flow field. In some embodiments, the first set of piezoelectric actuators is positioned asymmetrically with respect to the inlet channel and closer to the filter region to localize and direct energy toward the pillars. In some embodiments, a first set of piezoelectric actuators can be configured to selectively dissociate, clear blockages, adjust flow rates, and / or address bubble formation when required by a controller.

[0053] In some embodiments, a second set of piezoelectric actuators includes actuators arranged on both sides of the microfluidic channel, and the actuators are configured to vibrate perpendicular to the flow of the fluid sample in the microfluidic channel. In some embodiments, the second set of piezoelectric actuators are configured to levitate, rotate, and / or localize, and / or deflect particles to a specified downstream position (e.g., based on commands from the sensing region 110). For example, actuators positioned on both sides of the flow channel work together to either deflect particles downstream or adjust the position of particles. In this way, sorting can be performed based on phenotypic analysis without cell labeling, and various cell population types can be isolated and enriched.

[0054] In some embodiments, a third set of piezoelectric actuators includes actuators arranged adjacent to an outlet channel (or a plurality of outlet channels). In some embodiments, the third set of piezoelectric actuators is configured to discharge and / or deliver a portion of the sample fluid through the outlet channel (or a plurality of outlet channels).

[0055] (A2) In some embodiments of A1, at least one of the first set of piezoelectric actuators, the second set of piezoelectric actuators, and the third set of piezoelectric actuators includes one or more microelectromechanical system (MEMS) actuators. For example, a microfluidic device may include a set of MEMS-fabricated vibratory piezoelectric devices, which operate in the kHz to GHz range, are positioned at various locations in a laminar flow field, and are configured to generate inertia and acoustic perturbations, either individually or synchronously with other piezoelectric devices, for mixing cells and / or particles, clearing blockages, levitating, and / or rotating. In some embodiments, the piezoelectric actuators disclosed herein are configured to have an operating voltage in the range of 0.1V to 100V (e.g., 0.1V to 30V), based on the type of actuator and / or the type of sample. In some embodiments, the piezoelectric actuators disclosed herein are configured to have a deflection in the range of 5nm to 50μm (e.g., 100nm to 10μm), based on the type of actuator and / or the type of sample.

[0056] (A3) In some embodiments of A1 or A2, one or more actuators from the first set of piezoelectric actuators and / or the second set of piezoelectric actuators have a rounded shape. In some embodiments, at least some of the piezoelectric actuators described herein have a circular or elliptical shape. In some embodiments, at least some of the piezoelectric actuators described herein have a non-rounded shape (e.g., square or rectangular).

[0057] (A4) In some embodiments of A1 to A3, one or more actuators of the third set of piezoelectric actuators have an annular shape, and the annular shape is configured to be discharged through the center of the annular shape. In some embodiments, the third set of piezoelectric actuators are arranged on a membrane above an outlet channel. In some embodiments, the third set of piezoelectric actuators have a rounded shape. In some embodiments, the third set of piezoelectric actuators are MEMS actuators. In some embodiments, the third set of piezoelectric actuators are configured to discharge droplets containing particles into another system for further analysis.

[0058] (A5) In some embodiments of A1 to A4, at least one of the first set of piezoelectric actuators, the second set of piezoelectric actuators, and the third set of piezoelectric actuators is configured to operate in a frequency range of 1 kHz to 100 GHz. In some embodiments, operating within a frequency range includes vibrating within a frequency range. In some embodiments, at least some of the piezoelectric actuators described herein are configured to adjust the operating frequency within a frequency range based on the type of particles in a fluid sample and / or the intended operation (e.g., dissociation, buoyancy, rotation, and / or mixing). For example, a subrange of MHz may be used to buoy particles (e.g., cells), a subrange of kHz and / or MHz may be used to rotate particles, and a range of kHz may be used to sort particles. As another example, the first set of piezoelectric actuators may operate in a range of kHz to MHz to perform sonication of a sample, and the selected frequency within the range may depend on the desired function (e.g., breaking up agglomerations, breaking up blockages, and / or pushing / stimulating fluid flow) and the type of particles in the sample. In some embodiments, a frequency at the lower end of the range (e.g., 20 kHz) is used for a first sonication operation, and if the first sonication operation is unsuccessful (e.g., aggregation is still present in the sample), the frequency is increased (e.g., up to 200 kHz) and a second sonication operation is performed. In some embodiments, the sonication operation is repeated at increasing frequencies until the operation is determined to be successful.

[0059] (A6) In some embodiments of A1 to A5, at least one of the first set of piezoelectric actuators, the second set of piezoelectric actuators, and the third set of piezoelectric actuators is arranged on a film. For example, the film may consist of a thin layer of substrate (e.g., with a thickness in the range of 1 μm to 5 μm).

[0060] (A7) In some embodiments of A1 to A6, the first set of piezoelectric actuators includes one or more actuators having a first size, and the second set of piezoelectric actuators includes one or more actuators having a second size different from the first size. For example, an actuator in the first set of actuators may have a size twice or three times that of an actuator in the second set of actuators.

[0061] (A8) In some embodiments of A1 to A7, the microfluidic device further includes an optical layer (e.g., optical layer 152), and the inlet channel is formed by the optical layer. In some embodiments, the optical layer is made of a transparent or translucent material such as glass. For example, the optical layer may be made of glass. In some embodiments, the optical layer includes a top layer. In some embodiments, the optical layer is made of a transparent and / or translucent material.

[0062] (A9) In some embodiments of A1 to A8, the microfluidic device further includes an inactivating layer (e.g., inactivating layer 156) that separates the first set, second set, and third set of piezoelectric actuators from the fluid sample in the microfluidic channel. In some embodiments, the inactivating layer is composed of nitrides, silicon nitride, silicon carbide, SiO2, aluminum nitride, aluminum oxide, and / or photosensitive polymers. For example, the inactivating layer may be composed of a nitride. In some embodiments, the inactivating layer has a thickness in the range of 0.01 μm to 1 μm. Exemplary inactivating materials include silicon nitride, silicon carbide, SiO2, aluminum nitride, aluminum oxide, and photosensitive polymers.

[0063] (A10) In some embodiments of A1 to A9, the microfluidic device further includes an inactivating layer (e.g., inactivating layer 156) arranged between the set of piezoelectric actuators and the microfluidic channel. For example, the inactivating layer may be composed of a nitride. In some embodiments, the inactivating layer has a thickness in the range of 0.01 μm to 1 μm. Exemplary inactivating materials include silicon nitride, silicon carbide, SiO2, aluminum nitride, aluminum oxide, and photosensitive polymers.

[0064] (A11) In some embodiments of A1 to A10, the microfluidic device further includes an intermediate adhesive layer arranged between a set of piezoelectric actuators and the deactivating layer. In some embodiments, the intermediate adhesive layer is configured to match the mechanical impedance between the piezoelectric layer 178 and the deactivating layer 156. In some embodiments, the intermediate adhesive layer is configured to reduce cracking of the deactivating layer 156. In some embodiments, the intermediate adhesive layer is configured to match the impedance between the set of piezoelectric actuators and the deactivating layer. In some embodiments, the intermediate adhesive layer is made of a metal such as titanium (Ti). In some embodiments, the intermediate adhesive layer is configured to reduce cracking of the deactivating layer.

[0065] (A12) In some embodiments of A1 to A11, the intermediate adhesive layer is made of titanium. For example, the titanium has a thickness in the range of 10 nm to 300 nm.

[0066] (A13) In some embodiments of A1 to A12, the microfluidic device further includes a polymer layer (e.g., polymer layer 154) that constitutes at least a portion of the microfluidic channel. In some embodiments, the polymer layer is configured to bond the substrate to the optical layer.

[0067] (A14) In some embodiments of A1 to A13, the microfluidic device further includes one or more electrodes, which are arranged adjacent to a microfluidic channel between an inlet and an outlet and are configured to apply an electric field to a fluid sample in the microfluidic channel and / or to sense one or more properties of the fluid sample. In some embodiments, the first and second sets of piezoelectric actuators and one or more electrodes are arranged on the same layer. In some embodiments, one or more electrodes have a thickness in the range of 0.01 μm to 1 μm.

[0068] (A15) In some embodiments of A1 to A14, the microfluidic device further includes a control circuit (e.g., the control circuit described with reference to Figure 3), which is electrically connected to first and second sets of piezoelectric actuators and configured to provide operating signals to the first, second, and third sets of piezoelectric actuators (e.g., piezoelectric actuator 301). In some embodiments, the control circuit is further configured to provide activation signals to one or more electrodes (e.g., electrode 305) to selectively charge particles of a fluid sample flowing through a microfluidic channel and / or selectively sense at least one characteristic of the fluid sample. In some embodiments, the control circuit comprises one or more microcontrollers (e.g., one or more processors 302). In some embodiments, the control circuit monitors signals from one or more sensing elements (e.g., one or more piezoelectric actuators 354 and / or one or more electrodes 352 in sensing mode) and adjusts the operation of the actuators accordingly. For example, if a sensing signal indicates the presence of agglomerates, the actuator is configured to break up the agglomerates.

[0069] (A16) In some embodiments of A1 to A15, the microfluidic device further comprises a set of electrodes (e.g., electrode 352) configured to provide an actuation signal from the control circuit to at least one of a first set of piezoelectric actuators and a second set of piezoelectric actuators. In some embodiments, the operation of the set of electrodes is based on the operation of the actuators (e.g., the electrode operation is timed to reduce / minimize interference from the operation of the actuators).

[0070] (B1) In other embodiments, some embodiments include a method (e.g., Method 400) that includes: (i) supplying a fluid sample containing a plurality of particles (e.g., cells) through an inlet (e.g., inlet 103) to a microfluidic channel (e.g., fluid channel 102) of a microfluidic device (e.g., microfluidic device 100) having an outlet (e.g., one of a plurality of outlets 107a to 107c); (ii) selectively dissociating two or more particles of the fluid sample using a first set of piezoelectric actuators (e.g., first set of piezoelectric actuators 105) positioned adjacent to the inlet; (iii) selectively manipulating one or more particles of the fluid sample flowing through the microfluidic channel using a second set of piezoelectric actuators (e.g., second set of piezoelectric actuators 121a and 121b); and (iv) selectively discharging a portion of the fluid sample from the microfluidic channel using a third set of piezoelectric actuators (e.g., third set of piezoelectric actuators 109a to 109c) positioned adjacent to the outlet. In some embodiments, a third set of piezoelectric actuators and outlets are sized to discharge particles having a diameter in the range of 2 μm to 50 μm. In some embodiments, the method further includes using a first and / or second set of piezoelectric actuators to induce laminar flow from the inlet to the outlet of a microfluidic channel. In some embodiments, at least one of the first and second sets of piezoelectric actuators is manufactured using SOI PZT and a combination of dry reactive ion etching (DRIE), polymer layered microfluidics, and top glass bonding to a polymer.

[0071] (B2) In some embodiments of B1, the method further includes selectively providing actuation signals to a first set, a second set, and a third set of piezoelectric actuators (e.g., via an actuation circuit 330) based on acquired sensing data (e.g., via a measurement / analysis circuit 350). In some embodiments, the actuation signals are generated to produce vibration frequencies in the actuators based on the fluid composition (e.g., particle size and / or concentration) in the microfluidic channel. For example, the third set of actuators may be configured to vibrate so that only one cell is ejected at a time. For example, the sensing signals may include information about the state of the fluid.

[0072] (B3) In some embodiments of B1-B2, the method further includes determining the state of a microfluidic device, which includes (i) operating one or more actuators from a first set of piezoelectric actuators and / or a second set of piezoelectric actuators in an operating state to generate an vibration signal, and (ii) switching the operation of one or more actuators to a sensing state to sense an echo response corresponding to the vibration signal. For example, if large aggregates of cells are present on one or more actuators, the response will change. In some embodiments, a first subset of actuators operates in an operating state to generate one or more vibration signals, and a second subset of actuators operates in a sensing state to sense an echo response to one or more vibration signals. As an example, the acoustic signal received by the piezoelectric actuator after rebound indicates the viscosity and / or density of the sample and can be used to evaluate aggregation / coagulation / clogging of the sample.

[0073] (B4) In some embodiments of B1 to B3, selective manipulation of one or more particles of a fluid sample flowing through a microfluidic channel using a second set of piezoelectric actuators includes selectively levitating, rotating, and / or sorting one or more particles.

[0074] (B5) In some embodiments of B1 to B4, selectively manipulating one or more particles of a fluid sample flowing through a microfluidic channel using a second set of piezoelectric actuators includes adjusting the operating frequencies of the second set of piezoelectric actuators to perform different types of operations.

[0075] (B6) In some embodiments of B1 to B5, the method further includes (i) sensing the flow rate of a fluid sample flowing through a microfluidic channel and one or more characteristics of one or more particles of the fluid sample via a sensing component; (ii) determining the operating parameters of a first set of piezoelectric actuators and a third set of piezoelectric actuators according to at least the sensed flow rate; and (iii) determining the operating parameters of a second set of piezoelectric actuators according to at least one or more sensed characteristics of one or more particles. In some embodiments, the sensing component includes a set of electrodes (e.g., electrode 352) and / or a second set of piezoelectric actuators (e.g., piezoelectric actuator 354). In some embodiments, the operating parameters include an operating frequency (e.g., vibration frequency). In some embodiments, one or more characteristics of one or more particles include the measured electrical impedance of each particle and / or the size of each particle.

[0076] Terms such as "first," "second," etc., may be used herein to describe various elements, but it will be understood that these elements should not be limited by these terms. These terms are used solely to distinguish one element from another. For example, without departing from the scope of the various described embodiments, a first array can be called a second array, and similarly, a second array can be called a first array. Both the first array and the second array are arrays, but they are not the same array.

[0077] The terms used in the description of embodiments herein are for the sole purpose of describing specific embodiments and are not intended to limit the scope of the claims. In the description and the appended claims, the singular forms “a,” “an,” and “the” are intended to include the plural form unless otherwise specifically indicated in the context. In this specification, the terms “and / or” will also be understood to refer to and encompass one or more any and all possible combinations of the listed related items. In this specification, the terms “comprises” and / or “comprising” specify that the described features, integers, steps, actions, elements, and / or components exist, but will not be understood to exclude the existence or addition of one or more other features, integers, steps, actions, elements, components, and / or groups thereof.

[0078] The above description is provided with reference to specific embodiments for illustrative purposes. However, the above exemplary description is not intended to be exhaustive or to limit the claims to the exact form disclosed. Many modifications and variations are possible in light of the above teachings. The embodiments have been selected and described to best illustrate the principles and practical applications of the various embodiments described, thereby enabling those skilled in the art to best utilize the principles and the various embodiments described, along with various modifications suitable for a particular intended use.

Claims

1. Inlet channel and Exit channel and A microfluidic channel arranged on a substrate between the inlet channel and the outlet channel, wherein the outlet of the microfluidic channel is positioned on at least a portion of the outlet channel, A first set of piezoelectric actuators, arranged adjacent to the inlet channel and configured to dissociate particles of the fluid sample in the microfluidic channel, A second set of piezoelectric actuators, arranged between the inlet channel and the outlet channel and configured to manipulate the particles in the fluid sample as the particles move through the microfluidic channel, A third set of piezoelectric actuators, arranged adjacent to the outlet on the outlet channel and configured to discharge a portion of the fluid sample from the microfluidic channel through the outlet, A microfluidic device equipped with the following features.

2. The microfluidic device according to claim 1, wherein at least one of the first set of piezoelectric actuators, the second set of piezoelectric actuators, and the third set of piezoelectric actuators comprises one or more micro-electromechanical system (MEMS) actuators.

3. The microfluidic device according to claim 1 or 2, wherein one or more actuators from the first set of piezoelectric actuators and / or the second set of piezoelectric actuators have a rounded shape.

4. The microfluidic device according to any one of claims 1 to 3, wherein one or more actuators among the third set of piezoelectric actuators have an annular shape, and the annular shape is configured to be discharged through the center of the annular shape.

5. The microfluidic device according to any one of claims 1 to 4, wherein at least one of the first set of piezoelectric actuators, the second set of piezoelectric actuators, and the third set of piezoelectric actuators is configured to operate in a frequency range of 1 kilohertz to 100 gigahertz.

6. The microfluidic device according to any one of claims 1 to 5, wherein at least one of the first set of piezoelectric actuators, the second set of piezoelectric actuators, and the third set of piezoelectric actuators is arranged on a film.

7. The microfluidic device according to any one of claims 1 to 6, wherein the first set of piezoelectric actuators includes one or more actuators having a first size, and the second set of piezoelectric actuators includes one or more actuators having a second size different from the first size.

8. The microfluidic device according to any one of claims 1 to 7, further comprising an optical layer, wherein the inlet channel is formed by the optical layer.

9. The microfluidic device according to any one of claims 1 to 8, further comprising an inactivation layer that separates the piezoelectric actuators of the first set, the second set, and the third set from the fluid sample in the microfluidic channel.

10. The microfluidic device according to any one of claims 1 to 9, further comprising an inactivation layer arranged between the piezoelectric actuator and the microfluidic channel.

11. The microfluidic device according to claim 10, further comprising an intermediate adhesive layer arranged between the set of piezoelectric actuators and the deactivating layer.

12. The microfluidic device according to claim 11, wherein the intermediate adhesive layer is a metal layer.

13. The microfluidic device according to any one of claims 1 to 12, further comprising a polymer layer constituting at least a portion of the microfluidic channel.

14. The microfluidic device according to any one of claims 1 to 13, further comprising one or more electrodes arranged adjacent to the microfluidic channel between the inlet channel and the outlet channel, and configured to apply an electric field to the fluid sample in the microfluidic channel and / or to sense one or more characteristics of the fluid sample.

15. The microfluidic device according to any one of claims 1 to 14, further comprising a control circuit electrically connected to the first set and the second set of piezoelectric actuators and configured to provide operating signals to the first set, the second set and the third set of piezoelectric actuators.

16. A method performed in a microfluidic device, A fluid sample containing multiple particles is supplied through an inlet to a microfluidic channel having an outlet of the microfluidic device, A first set of piezoelectric actuators positioned adjacent to the inlet is used to selectively dissociate two or more particles of the fluid sample, A second set of piezoelectric actuators is used to selectively manipulate one or more particles of the fluid sample flowing through the microfluidic channel, A third set of piezoelectric actuators, positioned adjacent to the outlet, is used to selectively discharge a portion of the fluid sample from the microfluidic channel. Methods that include...

17. The further includes determining the state of the microfluidic device, and determining the state of the microfluidic device is To generate a vibration signal, one or more actuators from the first set of piezoelectric actuators and / or the second set of piezoelectric actuators are operated in an operating state, In order to sense the echo response corresponding to the vibration signal, the operation of one or more actuators is switched to a sensing state, The method according to claim 16, including the method described in claim 16.

18. The method according to claim 16 or 17, wherein the selective manipulation of one or more particles of the fluid sample flowing through the microfluidic channel using the second set of piezoelectric actuators includes adjusting the operating frequencies of the second set of piezoelectric actuators to perform different types of operations.

19. The method according to any one of claims 16 to 18, further comprising selectively providing operating signals to the first set, the second set, and the third set of piezoelectric actuators via a control circuit based on acquired sensing data.

20. The sensing component allows for sensing the flow rate of the fluid sample flowing through the microfluidic channel, and one or more characteristics of one or more particles in the fluid sample. Determining the operating parameters of the first set of piezoelectric actuators and the third set of piezoelectric actuators to operate according to at least the sensed flow rate, Determining a second set of operating parameters for operating the second set of piezoelectric actuators according to at least one of the sensed characteristics of the one or more particles, It further includes, The operating signals are selectively provided according to the determined operating parameters of the first set and the second set. The method according to claim 19.

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