Microfluidic device with sensor

Abstract

A microfluidic device includes a fluid ejector including a fluid actuator, a microfluidic channel fluidly coupled to the fluid ejector, a sensor positioned along the microfluidic channel, a constriction region positioned between the sensor and the fluid ejector in which an effective cross-sectional area of the microfluidic channel is gradually reduced, and a pair of electrodes positioned in the microfluidic channel between the constriction region and the fluid ejector.

Description

MICROFLUIDIC DEVICE WITH SENSORBACKGROUND[00011 Cell transfection is a process of introducing transfection material, such as nucleic acids, proteins, or other molecules or particles, inside a cell. Cell transfection may be used for a variety of different applications, including but not limited to gene therapy, gene editing, and pharmaceutical applications. Cell transfection may be performed by suspending a cell in a buffer solution with transfection material and forming pores in the cell membrane, allowing the transfection material to enter the cell through the pores.BRIEF DESCRIPTION OF THE DRAWINGS

[0002] FIG. 1 illustrates an example microfluidic device.

[0003] FIG. 2 illustrates an example method for transfecting a cell.

[0004] FIGS. 3A-3E illustrates the operations of the method of FIG. 2 performed using the microfluidic device of FIG. 1.

[0005] FIG. 4 illustrates an example microfluidic instrument.

[0006] FIG. 5 illustrates a portion of the microfluidic device of FIG. 1.

[0007] FIG. 6 illustrates an example cross-section of a microfluidic channel of the microfluidic device of FIG. 1.

[0008] FIGS. 7A-9B illustrate portions of example microfluidic devices.

[0009] It will be recognized that the figures are schematic representations of examples for purposes of illustration. The figures are provided for the purpose of illustrating example implementations with the explicit understanding that the figures will not be used to limit the scope of the meaning of the claims. Thus, the description is not limited to the examples and / or implementations provided in the drawings.DETAILED DESCRIPTION

[0010] In the following detailed description, reference is made to the accompanying drawings which form a part hereof, and in which are shown by way of illustration of specific examples in which the disclosure may be practiced. It is to be understood that other examples may be utilized, and structural or logical changes may be made without departing from the scope of the present disclosure. The following detailed description, therefore, is not to be taken in a limiting sense, and the scope of the present disclosure is defined by the appended claims. It is to be understood that features of the various examples described herein may be combined, in part or whole, with each other, unless specifically noted otherwise.

[0011] Biological cells are the basic building blocks of skin, tissues, and other biological materials. For example, Eukaryotic cells have a nuclease and other membrane-bound organelles enclosed within a cell membrane. The membrane-bound organelles enclosed within the cell membrane, such as the nucleus, mitochondria, and endoplasmic reticulum, may be associated with cell properties and functionalities that are of interest for testing, research, and other purposes. A gelatinous liquid fills the inside of the cell, referred to as the cytosol contained in the cytoplasm, which includes water, salts, and organic molecules. Some of the membrane-bound organelles may be enclosed by another membrane, such as a nuclear membrane that encloses the nucleus, to separate such organelles from the cytoplasm. In a biological sample, a cell of interest or a target cell may be intermixed with other cells and components. The other cells and components may interfere with manipulation and / or analysis of the target cell. Isolating the target cell from other cells and components of the biological sample may allow for subsequent analysis or processing of the target cell without further interference. Although the above describes Eukaryotic cells, examples are not so limited and may include Prokaryotic cells. Prokaryotic cells include a nucleoid region that contains genetic material (e.g., nucleic acid), ribosomes that make proteins, and cytosol that contains a cytoskeleton that organizes the cellular materials that are enclosed by the cell membrane. Prokaryotic cells lack a nucleus and other organelles, as well as internal membranes.[0012| As noted above, cell transfection refers to the process of introducing transfection material, such as nucleic acids, proteins, or other molecules or particles, inside a cell (e.g., a Eukaryotic cell or a Prokaryotic cell). Transfection of cells can be used for research or production of certain biological products. Transfection allows the behavior of the cell to be changed. For example, by diffusing a reagent, such as a particular DNA along with proteins that incorporate the DNA, into a genome of a cell, the genome may be altered to create a genetically modified organism. Cellular processes, organelles, and more can be studied by transfecting specific DNA molecules and proteins.

[0013] Transfecting cells may be performed using viral transfection, lipofection, electrotransfection, and mechanical transfection techniques. Viral transfection is laborious and results in the introduction of viral components into the cell, which may be unwanted for various applications. Viral transfection additionally is limited in the size of transfection material that may be moved into the intracellular space, which inhibits transfecting transfection material such as Cluster regularly interspaced short palindromic repeats (CRISPR) associated protein 9 (Cas9) protein for CRISPR, quantum dots, deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) above a threshold size, peptides, proteins, antibodies and nanoparticles. Lipofection is also laborious and introduces surfactants into the cell or cell membrane.

[0014] Electrotransfection and mechanical transfection both involve forming pores in the cell membrane, allowing the transfection material to pass or diffuse through an aperture to the inside of the cell. This is generally referred to herein as “cell poration” or “cell porating.” Cell poration may be performed by a cell-poration mechanism, such as those implementing electroporation and / or mechanical poration techniques. Electroporation, as used herein, refers to or includes forming apertures or pores in a cell membrane using electric fields. Mechanical poration refers to or includes forming apertures or pores in a cell membrane using mechanical forces.

[0015] In various examples, an electroporation mechanism may include electrodes within a microfluidic channel used to perform electroporation. “Microfluidic” may refer to fluidchannels with widths lower than 1 millimeter, though in some cases, the channel width may be larger. As the cell passes through the cell-poration region, the electrodes are used to apply an electric field and / or shear force to or across the cell to cause formation of apertures in the cell membrane, which may be referred to as “pores.” As used herein, an electric field refers to or includes a physical field or region surrounding a charged particle, e.g., the cell, which exerts forces on the charged particle by attraction or repulsion. The strength of the electric field may affect the size of the apertures. The strength of the electric field applied may be set to cause apertures of sufficient size for the selected cell transfection material to pass through. Depending, for example, on the type of cell being transfected or the type of transfection material, the electric field may be a static electric field created by a static voltage applied to the electroporation electrodes or an alternating electric field, which oscillates in time, created by an alternating current or alternating voltage applied to the electroporation electrodes.

[0016] In various examples, a mechanical portion mechanism may include a constriction region or constriction portion within a microfluidic channel. The constriction region may include a protuberance positioned in the microfluidic channel that reduces the diameter and / or cross-sectional area of a fluid flow region of the channel or may be a narrowing and reduction of the diameter and / or cross-sectional area of the channel itself. The diameter of the constriction region may be smaller than the diameter of a cell, or the cross-sectional area of the constriction region may be smaller than the cross-sectional area of a cell. The cell may thus be subjected to shear force as it “squeezes” through the constriction region. Shear force refers to or includes a force caused by parallel forces acting on the cell in opposite directions and at different parts of the cell. The shear force may cause the formation of apertures in the cell membrane, and the amount of shear force may affect the size of the apertures. By adjusting the diameter and / or cross-sectional area of the constriction region, the amount of shear force applied may be set to cause apertures of sufficient size for the selected cell transfection material to pass through.

[0017] In various examples, a microfluidic device is provided that includes both a mechanical portion mechanism and an electroporation mechanism in a microfluidic channel, as well as a sensor to detect a cell in the microfluidic channel and a fluid ejector to eject fluidand cells from the microfluidic channel. The mechanical portion mechanism may include a constriction region as described above. The electroporation mechanism may include electrodes to which a current or voltage can be applied to generate an electric field in the microfluidic channel. The electroporation mechanism may be positioned downstream of the mechanical poration mechanism and may further electroporate a cell after an initial mechanical poration step. In some examples, the sensor may be a separate sensor, such as an impedance sensor, which may be positioned upstream of the mechanical portion mechanism and an electroporation mechanism or in other locations along the microfluidic channel. In other examples, the electroporation electrodes may also function as the sensor by detecting changes in the electric field that occur when a cell is in the vicinity of the electroporation electrodes. In some examples, the sensor is an impedance sensor. In some examples, the impedance sensor may function to detect a cell in the microfluidic channel.

[0018] The fluid ejector may eject a known volume of fluid with each actuation. After the sensor detects the cell, the location of the cell can be determined based on the geometry of the microfluidic channel and the volume of fluid ejected by the fluid ejector. For example, each actuation of the fluid ejector may cause the cell to move a known distance along the microfluidic channel based on the volume of fluid ejected and the cross-sectional area of the microfluidic channel. Because the location of the cell is known, it can be determined when (e.g., upon which actuation of the fluid ejector) the cell will be ejected from the microfluidic channel. This may allow cells to be individually ejected into specific locations, for example, into individual wells of a multi-well plate. The microfluidic device may thus allow cells to be individually transfected and isolated for further investigation and study.

[0019] Referring now to FIG. 1, a top view of an example microfluidic device 100 including both a mechanical portion mechanism and an electroporation mechanism is shown. The microfluidic device, which may be, for example, a microfluidic chip or a microfluidic cartridge, includes a fluid ejector 106 including a fluid actuator 108, a microfluidic channel 104 fluidly coupled to the fluid ejector 106, a sensor 116 positioned in the microfluidic channel 104, a constriction region 118 positioned between the sensor 116 and the fluid ejector 106 in which an effective cross-sectional area of the microfluidic channel 118 isgradually reduced, and a pair of electrodes 124 positioned in the microfluidic channel 104 between the constriction region 118 and the fluid ejector 106. The sensor 116 may detect a cell in the microfluidic channel 104, the constriction region 118 may mechanically porate the cell as the cell passes through the constriction region 118, the pair of electrodes 124 may electroporate the cell, and the fluid ejector 106 may eject fluid and the cell from the microfluidic channel 104. In some examples, a separate sensor 116 may not be included, and the pair of electrodes 124 may also function as a sensor to detect a cell in the microfluidic channel. For example, changes in the electric field caused by the presence of the cell 216 may be detected by the electroporation electrodes 124, and signals may be sent to a controller. The controller may determine, based on the signals, that the cell is in the vicinity of the electroporation electrodes 124 (e.g., in an electroporation region). Accordingly, in some examples, the pair of electrodes further function as the sensor to detect a cell in the microfluidic channel. Thus, in examples of the microfluidic device 100 that include a pair of electrodes and a sensor, both of these components may refer to the electroporation electrodes 124.

[0020] In some examples, the microfluidic device 100 may include a cell reservoir 102 fluidly coupled to an upstream end of the microfluidic channel 104. The fluid ejector 106 may be positioned in an ejection chamber 112 of the microfluidic channel 104. The fluid ejector 106 may include a nozzle 110. The fluid actuator 108 may be, for example, a resistor, such as a thermal inkjet (TIJ) resistor or a piezoelectric actuator, and is configured to eject fluid from the microfluidic channel out of the nozzle 110. Other example fluid actuators 108 include electrodes, a fluidic pump, a magnetostrictive element, an ultrasound source, mechanical / impact driven membrane actuators, and magneto-restrictive drive actuators, among others. The microfluidic channel 104 extends in a longitudinal direction from a first end fluidly coupled to the cell reservoir 102 to a second end fluidly coupled to the fluid ejector 106.[0021 J In some examples, the fluidic actuator 108 is or includes a TIJ resistor. Activation of the TIJ resistor may create a flow of fluid by rapidly heating up and creating a vapor bubble, firing drops or bursts of fluid from the microfluidic channel 104 out of the nozzle 110. Forexample, a pulse of current may be passed through the fluidic actuator 108 (e.g., the TIJ resistor). The TIJ resistor acts as a heater, and heat from the TIJ resistor causes vaporization of fluid in the ejection chamber 112 to form the vapor bubble, which causes a pressure increase that propels the fluid droplet of fluid from the nozzle 110.10022] In some examples, different types of resistors or fluidic actuators may be used instead of a TIJ resistor. In some examples, the fluidic actuator 108 is or includes a piezoelectricbased actuator (element, pump, etc.). The piezoelectric-based actuator may generate pressure pulses that force fluid droplets of the reaction fluid out of the nozzle 110. In such piezoelectric-based actuator, a voltage may be applied to the fluidic actuator 108 that is in the form of a piezoelectric element (e.g., piezoelectric material) located in the ejection chamber 112. When the voltage is applied, the piezoelectric element changes shape, which generates a pressure pulse that forces a fluid droplet of the reaction fluid from the fluid ejector 106.

[0023] As fluid in the ejection chamber 112 is ejected, fluid from the microfluidic channel 104 and the cell reservoir 102 upstream of the ejection chamber 112 is pulled towards the fluid ejector 106, for example, filling the volume of the ejected droplet. The arrow 114 indicates a downstream direction (e.g., opposite an upstream direction) of the microfluidic channel 104 along a longitudinal direction of the microfluidic channel 104 from its first end at the cell reservoir 102 to its second end at the fluid ejector 106. As fluid is ejected (or after fluid is ejected) from the ejection chamber 112 by the fluid ejector 106, fluid in the cell reservoir 102 and the microfluidic channel travels in the downstream direction toward the ejection chamber. It should be understood that, when the fluid actuator 108 is a resistor, the vapor bubble created during a firing may cause some of the fluid in the microfluidic channel to be pushed briefly in the upstream direction before flowing in the downstream direction as the resistor cools and the ejection chamber 112 refills.

[0024] A similar effect may occur with a piezoelectric actuator. The piezoelectric actuator may deform towards the nozzle 110, ejecting fluid while also causing a small amount of back-flow of fluid in the upstream direction. When the piezoelectric actuator is released or deactivated, the fluid may be pulled back into the ejection chamber 112. In some examples,the piezoelectric actuator may be “pre-charged,” or deformed in the opposite direction to draw fluid into the ejection chamber, and fluid may be ejected by releasing the actuator. In this case, a cell in the microfluidic channel may be pulled downstream along with the fluid when the piezoelectric actuator is pre-charged. In some examples, the piezoelectric actuator may be pre-charged to draw fluid in and then actively actuated toward the nozzle 110. In this case, a cell in the microfluidic channel may be pulled downstream along with the fluid both when the piezoelectric actuator is pre-charged and when the piezoelectric actuator is deactivated after deforming toward the nozzle 110. In any case, it should be generally understood that the ejection of fluid from the ejection chamber 112 causes the fluid and cells 126 to travel downstream toward the ejection chamber 112.

[0025] In some examples, the microfluidic device 100 is a cell transfection device. Cells 126 (e.g., eukaryotic cells, prokaryotic cells, etc.) may be deposited in the cell reservoir 102 in a buffer solution (or other fluid medium) that fills the microfluidic channel 104. The buffer solution may be an isotonic solution with a similar pH as the cells 126 and may contain transfection material. Upon ejecting fluid from the nozzle 110, a cell (e.g., a single cell at a time) or cells may be pulled into the microfluidic channel 104 along with the buffer solution. Subsequent firings may move the cell in the downstream direction along the microfluidic channel toward the ejection chamber 112. In examples in which the fluid actuator 108 generates pressure pulses to eject fluid, such as with a TH resistor or piezoelectric actuator, the fluid actuator 108 may be configured to eject a specific volume of fluid from the nozzle 110 with each pulse or firing. Based on the volume of fluid ejected and the cross-sectional area of the microfluidic channel 104, the distance traveled by fluid and cell in the microfluidic channel 104 can be determined and / or controlled. For example, in a microfluidic device 100 that has a microfluidic channel 104 with a cross-sectional area of 100 square micrometers and a fluid ejector 106 configured to eject 1000 cubic micrometers of fluid per pulse, a cell in the microfluidic channel 104 may be expected to travel 10 micrometers in the downstream direction with each pulse.

[0026] In some examples, a microfluidic device includes a fluid ejector including a fluid actuator, a microfluidic channel fluidly coupled to the fluid ejector, a sensor positioned alongthe microfluidic channel, a constriction region positioned between the sensor and the fluid ejector in which an effective cross-sectional area of the microfluidic channel is gradually reduced, and a pair of electrodes positioned in the microfluidic channel between the constriction region and the fluid ejector. In some examples, the sensor is to detect a cell in the microfluidic channel, the constriction region is to mechanically porate the cell as the cell passes through the constriction region, the pair of electrodes is to electroporate the cell, and the fluid ejector is to eject fluid and the cell from the microfluidic channel. For example, as discussed above, the microfluidic device 100 may be a cell transfection device. In the example shown in FIG. 1, the microfluidic device 100 includes a sensor 116, a constriction region 118, and an electroporation region 120. The sensor 116 is positioned along (e.g., in, proximate, under, etc.) the microfluidic channel 104 upstream of the constriction region 118 and the electroporation region 120, and the constriction region 118 is positioned upstream of the electroporation region 120. In some examples, the sensor 116 may include a pair of sensing electrodes 122 and / or may be an impedance sensor. In some examples, the sensor 116 may be an image sensor, a light sensor, a chemical sensor, or another type of sensor. The sensor 116 may be configured to detect a cell in the microfluidic channel 104 in the vicinity of the sensor 116. For example, the sensor 116 may be an impedance sensor with a pair of electrodes 122 (e.g., sensing electrodes). The buffer solution in the microfluidic channel 104 may be nonconductive, while the cells may be conductive. When a cell is in the vicinity of (e.g., crossing over or past) the impedance sensor and a current or voltage is applied to one of the electrodes 122, the impedance signal detected by the sensor 116 may change. Thus, the change in the impedance signal from the sensor 116 may indicate that a cell is positioned above, crossing over, or otherwise in the vicinity of the sensor 116. Because the sensor 116 is upstream of the constriction region 118, the electroporation region 120, and the fluid ejector 106, the cell 126 may be detected before it is mechanically porated in the constriction region 118, electroporated in the electroporation region 120, and ejected by the fluid ejector 106. In some examples, the sensor 116 may be an optical sensor (e.g., a photodiode) that is integrated into the substrate below the microfluidic channel 104. The optical sensor may include an external light source that illuminates the microfluidic channel 104. When a cell126 passes over the photodiode, the photodiode signal may change, indicating the position of the cell 126.

[0027] The constriction region 118 may be configured to mechanically porate the cells that pass therethrough. In some examples, the constriction region 118 may be a narrowing (e.g., a reduction in the cross-sectional area) of the microfluidic channel 104 itself. In some examples, the constriction region 118 may include a protuberance or protuberances 119 (or obstructions, projections, etc.) that reduce the effective cross-sectional area of the microfluidic channel 104. The “effective cross-sectional area” refers to the cross-sectional area of the fluid in the microfluidic channel 104 on a plane perpendicular to the longitudinal direction of the microfluidic channel 104 (e.g., perpendicular to the fluid flow direction). Similarly, the “effective width” and “effective height” of the microfluidic channel 104 refer respectively to the width and height of the fluid in the microfluidic channel along the crosssection. The effective cross-sectional area may be gradually reduced in the downstream direction from the cross-sectional area of the upstream portions of the microfluidic channel 104, reaching a minimum effective cross-sectional area before gradually expanding back to a larger cross-sectional area. The portions of the microfluidic channel 104 outside the constriction region may be referred to as the “other portions of the microfluidic channel 104.” The minimum effective cross-sectional area of the constriction region 118 may be smaller than the cross-sectional area of the cell 126 when the cell 126 is in the buffer solution and undistorted in shape. Thus, the cell 126 must be deformed in order to fit or “squeeze” through the constriction region 118. This “squeeze” may cause the mechanical poration of the cell due to shear forces on the cell membrane as the cell 126 passes through the constriction region 118. In some examples, the microfluidic device 100 may include multiple constriction regions 118 to increase the probability of successful mechanical poration. For example, in some examples, two or more constriction regions 118 may be arranged in series between the sensor 116 and the electroporation electrodes 124. In some examples, for example, a second constriction region 118 may be positioned downstream of the electroporation electrodes 124.[00281 In examples in which one of the effective width or the effective height remains constant throughout the constriction region 118, the effective height or effective width may be gradually reduced in the downstream direction from the effective height or effective width of the other portions of the microfluidic channel 104, reaching a minimum effective height or effective width before gradually expanding back to the effective height or effective width of the other portions of the microfluidic channel 104. The minimum effective height or effective width of the constriction region 118 may be smaller than the diameter of the cell 126 when the cell 126 is in the buffer solution and undistorted in shape. Thus, the cell 126 must be deformed in order to squeeze through the constriction region 118.

[0029] The electroporation region 120 includes a pair of electroporation electrodes 124. By applying an electrical current or voltage to the electrodes 124, an electrical field is generated between the electrodes in the microfluidic channel 104. A cell exposed to the electric field may be electroporated. The strength of the electric field, the position of the cell relative to the electrodes 124, and the duration the electric field is applied may determine the extent of the poration. In some examples of a microfluidic device, the pair of electrodes extend parallel to a longitudinal direction of the microfluidic channel. For example, the electroporation electrodes 124 may be arranged to extend parallel to the longitudinal direction of the microfluidic channel 104 (e.g., parallel to the downstream direction indicated by the arrow 114). For example, the electroporation electrodes 124 may be elongate in shape and may extend along the microfluidic channel 104, rather than across the microfluidic channel 104. The electroporation electrodes 124 may be positioned on opposite sides of the microfluidic channel 104 such that the electric field extends across the entire microfluidic channel 104. For example, the electroporation electrodes 124 may be positioned on opposite sides of a lower portion of the microfluidic channel 104.

[0030] As discussed above, the sensor 116 may detect the position of a cell, and the distance traveled by the cell as a result of each firing of the fluidic actuator 108 may be determined based on the cross-sectional area of the microfluidic channel and the volume of fluid ejected by the fluid ejector 106. Thus, once a cell is detected by the sensor, the position of the cell may be “tracked” as it travels along the microfluidic channel 104. For example, themicrofluidic channel 104 may be sized such that, after a cell is detected by the sensor, the next firing of the fluidic actuator 108 may cause the cell to be pulled through the constriction region. A subsequent firing may pull the cell into the electroporation region 120, and another subsequent firing may pull the cell into the ejection chamber 112. Finally, another subsequent firing may eject the cell from the microfluidic device 100 via the nozzle 110.

[0031] Referring now to FIGS. 2 and 3 A-3E, an example method 200 for transfecting a cell and illustrations of an example microfluidic device 100 during the operations of the method 200 are shown. In some examples, a method for transfecting a cell includes activating a fluid actuator at least once to eject fluid from the microfluidic channel to move the cell through a constriction region of the microfluidic channel and into an electroporation region of the microfluidic channel including electroporation electrodes, detecting the location of the cell in the microfluidic channel, applying an electric potential to the electroporation electrodes to generate an electric field in the microfluidic channel upon determining, based on the detected location of the cell, that the cell is in the electroporation region, and activating the fluid actuator to eject the cell from the microfluidic channel. In some examples, wherein determining that the cell is in the electroporation region comprises detecting the cell upstream of the electroporation region and determining the number of times the fluid actuator is activated after detecting the cell. For example, determining that the cell is in the electroporation region may include detecting the cell upstream of the electroporation region 120 using the sensor 116 and activating the fluid actuator 108 one or more times to move the cell into the electroporation region 120. Based on the geometry of the microfluidic channel 104 and the volume of fluid ejected per actuation of the fluid actuator 108 it can be determined how many activations of the fluid actuator 108 are needed after detecting the cell for the cell to move into the electroporation region. In some examples, determining that the cell is in the electroporation region may include detecting the cell using the electroporation electrodes 124 themselves, for example, where the electroporation electrodes 124 also function as a sensor.

[0032] A cell may be suspended in a buffer solution in a reservoir fluidly coupled to a microfluidic channel. The buffer solution may contain transfection material for transfectingthe cell using the method 200. At operation 202 of the method 200, the cell is detected in the microfluidic channel. For example, as shown in FIG. 3A, the cell 126 is positioned above the sensing electrodes 122 of the sensor 116. The cell 126 is suspended in a buffer solution or other fluid medium containing transfection material 128. “Transfection material” refers to material (e.g., nucleic acids, proteins, protein fragments, particles, etc.) suspended in the buffer solution or fluid medium to be transfected into the cell 126. The sensor 116 may detect the cell based on, for example, a change in impedance across the electrodes 122.

[0033] In some examples, the method further includes repeatedly activating the fluid actuator to eject fluid from the microfluidic channel until the cell is detected in the microfluidic channel, wherein the fluid actuator is activated to move the cell through a constriction region of the microfluidic channel upon detecting the cell in the microfluidic channel. For example, before detecting the cell 126, the method 200 may include actuating or activating the fluid actuator 108 to eject fluid (e.g., buffer solution) from the nozzle until the cell 126 travels out of the cell reservoir 102, into the microfluidic channel 104, and downstream to the sensor 116. For example, if the fluid actuator 108 is a TIJ resistor, the TH resistor may be repeatedly fired until the cell 126 is detected by the sensor 116 at operation 202 of the method 200.

[0034] At operation 204 of the method 200, a fluid actuator (e.g., the fluid actuator 108) is activated to eject fluid (e.g., buffer solution) from the microfluidic channel 104 to move the cell 126 through a constriction region (e.g., the constriction region 118) of the microfluidic channel. In some examples, moving the cell through a constriction region imparts shear forces to mechanically porate the cell. As shown in FIG. 3B, upon detecting the cell 126 passing over the sensor 116, the fluid actuator 108 is fired to eject fluid from the nozzle. This causes the fluid and the cell in the microfluidic channel 104 to be pulled downstream toward the ejection chamber 112. As discussed above, the smallest effective cross-sectional area of the microfluidic channel 104 in the constriction region 118 is smaller than the cross-sectional area of the undistorted cell 126. The cell deforms as it is pulled and “squeezed” through the constriction region 118, temporarily reducing the cross-sectional area of the cell 126. As the cell is “squeezed,” the constriction region 118 imparts shear forces on cell membrane of thecell 126. This may cause pores 130 to form in the cell membrane in a process referred to as mechanical poration, as discussed above. Transfection material 128 may enter the cell through the pores 130 formed in the mechanical poration process.

[0035] Operation 204 may be repeated as necessary to pull the cell 126 through the entire constriction region 118. For example, a first firing of the fluid actuator 108 after the cell 126 is detected in operation 202 may pull the cell 126 halfway through the constriction region 118, and a second firing may pull the cell 126 through the remainder of the constriction region 118. The number of firings used to move the cell 126 through the constriction region 118 may have little effect on the effectiveness of the poration. The velocity of the cell 126 through the constriction region may affect the shear force experienced by the cell 126 and affect the size and quantity of the pores. Larger pores may allow for larger transfection material 128 to be transfected into the cell 126, while more numerous pores increase the opportunities for transfection material 128 to enter the cell and thus the likelihood of successful transfection.

[0036] At operation 206 of the method 200, the fluid actuator is activated to move the cell into a region of the microfluidic channel including electroporation electrodes (e.g., an electroporation region). In some examples, generating the electric field in the microfluidic channel electroporates the cell. For example, as shown in FIG. 3C, activating the fluid actuator 108 causes the cell to move downstream from the constriction region 118 (or an area between the constriction region 118 and the electroporation region 120) into the electroporation region 120. In some examples, operations 204 and 206 may be combined into a single operation. For example, a single firing of the fluid actuator 108 may cause a cell 126 to be positioned above the sensor 116 to be pulled through the constriction region 118 into the electroporation region 120. Thus, it is explicitly contemplated that activating the fluid actuator to move the cell through the constriction region and activating the fluid actuator to move the cell into the region of the microfluidic channel with electroporation electrodes may refer to a single activation (e.g., firing) of the fluid actuator. As discussed above, the distance traveled by the cell 126 in a single firing of the fluid actuator 108 can be determined or controlled based on the volume of fluid ejected by the fluid ejector 106 and the cross-sectional area of the microfluidic channel 104. It should be understood that passing through the constriction region 118 may cause the cell 126 to travel a greater distance along the microfluidic channel 104 in a single firing due to the decreased effective cross-sectional area in the constriction region.10037] At operation 208 of the method 200, the electroporation electrodes are energized to create an electric field in the microfluidic channel to electroporate the cell. At least some of the transfection material in the buffer solution may enter the pores formed in the mechanically porated and electroporated cell. For example, as shown in FIG. 3D, an electrical current or voltage may be supplied to the electrodes 124 causing the electrodes 124 to generate an electric field therebetween. With the cell 126 positioned between the electrodes in the electroporation region 120, the electric field causes the cell membrane to be further porated in a process referred to as electroporation, as discussed above. The electroporation of the cell 126 may cause new pores 130 to form in the cell membrane and may cause pores 130 formed in the mechanical poration process to increase in size. This may increase the likelihood of transfection material 128 passing through the cell membrane and into the cell and may allow transfection material 128 of larger size to enter the cell. As shown in FIG. 3D, two pieces of transfection material 128 have passed through the pores in the cell membrane and into the cell 126. After a desired amount of time, the electrodes 124 may be de-energized, allowing the pores 130 to close with the transfection material 128 trapped within the cell 126. In some examples, the electrodes 124 may remain energized, and the pores may close or begin to close when the cell 126 exits the electroporation region 120.

[0038] At operation 210 of the method 200, the fluid actuator is activated to eject the cell from the microfluidic channel. The fluid actuator may be actuated (e.g., repeatedly) to move the cell towards the fluid actuator and an ejection nozzle and again to eject the cell through the nozzle. For example, as shown in FIG. 3E, a first activation of the fluid actuator 108 may move the cell into the ejection chamber 112. The pores 130 formed in operations 204 and 208 may close or begin to close after the electrodes A second activation of the fluid actuator 108 may eject the cell 126 from the microfluidic channel 104 via the nozzle 110. In some examples, the method further includes suspending the cell in a buffer solution includingtransfection material, the fluid ejected from the microfluidic channel including a portion of the buffer solution, wherein at least some of the transfection material enters the cell after the cell is mechanically porated and electroporated.

[0039] The microfluidic device 100 and the method 200 allow for the controlled transfection of a single cell 126. By detecting the cell 126 in the microfluidic channel 104, the location of the cell 126 in the microfluidic channel 104 can be tracked throughout the method 200 to ensure that the cell 126 is positioned between the electroporation electrodes 124 when the electrodes are energized and to determine with which firing the cell 126 is ejected from the microfluidic device 100. This may allow the cell 126 to be transfected and the transfected cell 126 to be isolated.

[0040] Referring now to FIG. 4 an example microfluidic instrument 300 is shown. The microfluidic instrument may be referred to as a cell transfection system or cell transfection instrument. In some examples, the microfluidic instrument includes a cell reservoir, a fluid ejector including a fluid actuator, a microfluidic channel including a first end fluidly coupled to the cell reservoir and a second end fluidly coupled to the fluid ejector, the microfluidic channel defining a downstream direction from the first end to the second end, a sensor positioned in the microfluidic channel, a constriction region positioned downstream of the sensor in which the cross-sectional area of the microfluidic channel is gradually reduced, and a pair of electrodes positioned in the microfluidic channel downstream of the constriction region. For example, as discussed above, the microfluidic device 100 may be a microfluidic chip or cartridge for transfecting cells. The microfluidic device 100 may be inserted into a transfection apparatus 302 including control circuitry, actuators, and other components for operating the microfluidic device 100 and depositing transfected cells. In some examples, the microfluidic instrument further includes a controller to receive sensor signals from the sensor, determine, based on the sensor signals, the presence of a cell proximate the sensor, cause the fluid actuator to eject a volume of fluid from the microfluidic channel via a nozzle, and cause the pair of electrodes to generate an electric field to electroporate the cell after the volume of fluid has been ejected. For example, the transfection apparatus 302 includes a controller 304 configured to control the operations of the microfluidic device 100. For example, thecontroller 304 includes at least one processor 303 and at least one memory 305 storing machine-readable instructions that, when executed by the at least one processor 308, may cause the microfluidic device 100 to execute the operations of the method 200. The transfection apparatus 302 may be connected to an electric power source and may supply power to the microfluidic device 100 based on commands from the controller 304. For example, the transfection apparatus 302 may supply power to the sensor 116 of the microfluidic device 100 and may receive sensor signals (sensor data, impedance data, etc.) from the sensor 116. The controller 304 may determine, based on the sensor signals, that the sensor 116 has detected a cell in the microfluidic channel 104 proximate the sensor 116. For example, the controller 304 may determine, based on a sudden change in impedance measured by the sensor (e.g., an impedance sensor), that a cell has crossed over the electrodes of the sensor 116 (e.g., in operation 202 of the method 200).

[0041] The controller 304 may cause the transfection apparatus 302 to deliver an electrical current to the fluid ejector 106 to eject a volume of fluid from the nozzle 110 (e.g., by energizing a TIJ resistor) and move the cell 126 through the constriction region 118 and into the electroporation region 120 (e.g., in operations 204 and 206 of the method 200). The controller 304 may cause the fluid ejector 106 to eject fluid multiple times until the desired volume of fluid is ejected, causing the cell 126 to move a desired distance along the microfluidic channel 104. After the desired volume of fluid has been ejected and the cell 126 has moved into a desired position (e.g., between the electroporation electrodes 124), the controller 304 may cause the transfection apparatus 302 to deliver an electrical current or voltage to the electroporation electrodes 124 to generate an electric field in the microfluidic channel 104 to electroporate the cell 126 (e.g., in operation 208 of the method 200). The controller 304 may cause the transfection apparatus 302 to deliver an electrical current or voltage to the fluid ejector 106 to eject fluid from the nozzle 110 to eject the cell from the microfluidic channel 104 via the nozzle 110 (e.g., in operation 210 of the method 200).

[0042] As discussed above, the microfluidic device 100 may allow for the isolation and transfection of a single cell at a time. The transfection apparatus 302 may be configured to position the microfluidic device 100 (and more specifically the nozzle 110) to eject theisolated transfected cell in a desired location. For example, as shown in FIG. 4, the transfection apparatus 302 may be configured to eject fluid and cells into wells 308 of a multi-well plate 306. The multi-well plate 306 includes a waste well 310 for ejections of fluid without a cell 126. For example, before a cell 126 is detected by the sensor 116, the transfection apparatus 302 may cause the microfluidic device 100 to eject buffer solution into the waste well 310. In some examples, the microfluidic instrument further includes an actuator, and the controller is further to control the actuator to adjust the position of the nozzle relative to a multi-well plate including a plurality of wells and cause the fluid actuator to eject the cell into a different well than a well into which the fluid ejected before the cell is electroporated is ejected. For example, transfection apparatus 302 may include actuators 312 configured to adjust the position of the microfluidic device 100 based on commands from the controller 304. The controller 304 may control the actuators 312 to position the nozzle of the microfluidic device 100 over the waste well and may cause the fluid ejector 106 to eject the buffer solution into the waste well 310. After detecting a cell 126 based on signals from the sensor 116, the controller 304 may determine or be pre-programmed with the number of firings of the fluid actuator 108 before the cell 126 is ejected from the nozzle 110. The controller 304 may then cause the fluid actuator 108 to fire to eject fluid into the waste well 310 one fewer time than the number of firings needed to eject the cell 126.

[0043] In some examples, the microfluidic device 100 may allow for “bulk transfection,” in which cells 126 are not isolated individually. Instead, the cells are pulled through the constriction region 118 and between the electroporation electrodes 124 as a group or in a line in a continuous fashion and dispensed, for example, into a common reservoir. In such an embodiment, the cell sensor 116 may or may not be included.

[0044] For example, based on the volume of fluid ejected and the cross-sectional area of the microfluidic channel 104, the controller 304 may determine that the cell 126 will be ejected on the fifth firing following detecting the cell 126 at the sensor 116. The controller 304 may cause the microfluidic device 100 to eject fluid into the waste well four times after detecting the cell 126 at the sensor 116. After the fourth firing, the cell 126 may be positioned in the ejection chamber 112 and be positioned to be ejected on the subsequent firing. The controller304 may cause the actuators 312 to move the microfluidic device 100 to position the nozzle 110 over a different desired well 308 and may then cause the microfluidic device 100 to eject the cell 126 into the desired well 308 (e.g., different than the waste well 310). The process may be repeated, with fluid being ejected into the waste well 310 until the next cell 126 is positioned in the ejection chamber 112. This process may allow for a single cell 126 to be positioned into a desired well 308 for further inspection and / or processing. In some examples, the microfluidic device 100 may remain stationary, while the actuators 312 of the transfection apparatus 302 are configured to move the multi-well plate 306 to move the desired well 308 under the nozzle 110.

[0045] As discussed above, the geometry of the microfluidic device 100 may affect the efficacy of the poration processes. In some examples, the mean cell diameter of the cells 126 transfected using the microfluidic device 100 may be between 5 micrometers and 50 micrometers, though in some cases the mean cell diameter may fall outside of this range. In some examples, the mean cell diameter of the cells 126 transfected using the microfluidic device 100 may be 14 micrometers. FIG. 5, shows an example microfluidic device 100 with dimension markings. The cell reservoir 102 is not shown in FIG. 5. The distance DI across the narrowest point of the constriction region is between 0.3 and 0.9 times the mean cell diameter of the cells 126 being transfected by the microfluidic device 100. In some examples, the constriction region 118 reduces the effective width of the microfluidic channel 104 without reducing the effective height of the microfluidic channel 104. In other examples, the constriction region 118 reduces the effective height of the microfluidic channel 104 without reducing the effective width of the microfluidic channel 104. In other examples, both the height and width (e.g., the effective height and width) of the microfluidic channel 104 are reduced. For example, the protuberances 119 may extend from the sides, top, and bottom of the microfluidic channel to form a circular or rectangular opening. The distance DI may refer to the distance across the width of the opening or the distance across the height of the opening. If the distance DI is less than 0.3 times the mean cell diameter, the cell 126 may be lysed or may block and clog the constriction region 118. If the distance DI is greater than 0.9 times the mean cell diameter, the cell 126 may not be sufficiently deformed to porate the cell membrane, and the cell may pass through the constriction region 118 without being porated.[0046| The distance D2 indicates the length of the constriction region 118 where the distance across the constriction region 118 (e.g., in a width direction or a height direction) is less than the mean cell diameter of the cells 126 being transfected. Thus, for example, the portions of the constriction region 118 in which the cells 126 are smaller than the distance between the protuberances 119 is not included in the distance D2. The distance D2 may be between 0.1 and 10 times the mean cell diameter. If D2 is below 0.1 times the mean cell diameter, the microfluidic device 100 may be difficult to fabricate and may break during operation. Increasing the distance D2 may marginally improve the poration of the cells 126. However, if the distance D2 is greater than 10 times the mean cell diameter, the cells 126 may become stuck in the constriction region 118 and clock the microfluidic channel 104. Further, if the distance D2 is greater than 10 times the mean cell diameter, the fluidic resistance will increase, requiring a higher-pressure pump.

[0047] The distance D3 indicates the distance between the sensor 116 and the location of the minimum width of the constriction region 118 along the downstream direction and is between 1 and 10 times the mean cell diameter of the cells 126 being transfected. If the distance D3 is less than the mean cell diameter of the cells 126 being transfected, the motion of the cell 126 through the constriction may influence the signal from the sensor, complicating the signal processing and making it more difficult to detect the cell 126. If the distance D3 is greater than 10 times the mean cell diameter of the cells 126 being transfected, a second cell 126 from the cell reservoir 102 may be detected by the sensor 116 before a first cell reaches the constriction region 118. The first cell may become temporarily stuck upstream of the constriction region 118 before being pulled through, which may cause the second cell 126 to be too close to the first cell 126 to maintain separation when the cells 126 are ejected into the multi-well plate 306. The distance D3 being no more than 10 times the mean cell diameter of the cells 126 being transfected, may ensure that the first cell detected is pulled through the constriction region 118 before a second cell 126 is detected, thus maintaining separation between the cells 126. However, as discussed above, if the microfluidic device 100 is being used for bulk transfection, the distance D3 may not be limited to this range, and, in some examples, the sensor 116 may not be included.

[0048] The height or thickness of the microfluidic channel 104 (e.g., into the page, as shown in FIG. 5) may be between 0.5 and 5 times the mean cell diameter of the cells 126 being transfected. If the height or thickness of the microfluidic channel 104 is below 0.5 times the mean cell diameter of the cells 126 being transfected, the cells 126 may be lysed or may clog the microfluidic channel 104. If the height or thickness of the microfluidic channel 104 is above 5 times the mean cell diameter of the cells 126 being transfected, the strength of the electric field generated by the electroporation electrodes 124 may be too weak to sufficiently electroporate the cells 126. For example, the electrodes may be positioned on or adjacent a bottom portion or “floor” of the microfluidic channel 104. The electric field generated by the electroporation electrodes 124 weakens with increasing distance from the electroporation electrodes 124. Thus, if a cell is positioned near a top portion or “ceiling” of the microfluidic channel 104, the electroporation electrodes 124 on the “floor” may not generate a strong enough electric field to electroporate the cells if the “ceiling” is more than 5 times the mean cell diameter of the cells 126 being transfected above the “floor” and the mean cell diameter of the cells 126 being transfected.

[0049] The speed of the cells 126 through the constriction region 118 may be between 1000 and 100,000 times the mean cell diameter of the cells 126 being transfected per second. The maximum amount of cell transfection may occur at the moment of fastest velocity of the cell 126 through the constriction region 118. The speed of cell 126 through the constriction region 118 is related to the rate at which the fluid is ejected, or, more specifically, the refill rate (or refill velocity) of the ejection chamber 112. So, for example, in a microfluidic device 100 that has a microfluidic channel 104 with a cross-sectional area of 100 square micrometers and a fluid ejector 106 configured to eject 1000 cubic micrometers of fluid per pulse with the ejection chamber refilling in .001 seconds, a cell 126 may travel 10 micrometers in .001 seconds, or 10,000 micrometers per second, along the microfluid channel 104. If the speed of the cells 126 through the constriction region 118 is below 1000 times the mean cell diameter of the cells 126 being transfected per second, the cells 126 may not be sufficiently porated. If the speed of the cells 126 through the constriction region 118 is above 100,000 times the mean cell diameter of the cells 126 being transfected per second, the cells 126 may be lysed when passing through the constriction region 118. The refill rate of the ejection chamber112, and therefore the speed of the cell 126 through the constriction region 118, can be controlled by several factors including the width and height of constriction region 118, the width and height of other portions of the microfluidic channel 104, the overall length of the microfluidic channel 104, and the viscosity and surface tension of the fluid (e.g., the buffer solution).

[0050] The distance D4 between the electroporation electrodes 124 may be between 0.9 and 10 times the mean cell diameter of the cells 126 being transfected. The electroporation electrodes 124 may be positioned just outside the microfluidic channel 124, with the walls of the microfluidic channel 104 substantially aligned with the inner edges of the electroporation electrodes 124. The width of the microfluidic channel 104 may be slightly larger than the distance D4. Thus, a distance D4 that is less than 0.9 times the mean cell diameter of the cells 126 being transfected, may indicate that the width of the microfluidic channel 104 is less than the mean cell diameter. If the width of the microfluidic channel 104 is less than 0.9 times the mean cell diameter, the cells 126 may clog the microfluidic channel 104. As discussed above, the strength of the electric field generated by the electroporation electrodes 124 decreases with increasing distance from the electroporation electrodes 124. If the distance D4 is greater than 10 times the mean cell diameter of the cells 126 being transfected, the electric field near the middle of the microfluidic channel 104 may not be strong enough to electroporate the cells 126. The voltage across the electroporation electrodes 124 can be increased to account for larger distances between the electrodes 124, but increasing the voltage above an upper limit can cause electrolysis in the buffer solution, creating bubbles and disrupting the transfection process.(0051 j The distance D5 indicates the length of the electroporation electrodes 124. The distance D5 may be between 2 and 10 times the mean cell diameter of the cells 126 being transfected. In some examples, the distance D5 may be 5 times the mean cell diameter of the cells 126 being transfected. If the distance D5 is below 2 times the mean cell diameter of the cells 126 being transfected, it may be difficult to ensure that a cell 126 dwells in the electroporation region 120 between pulses of the fluid actuator 108 and is exposed to a strong enough electric field to electroporate the cell 126. If the electroporation electrodes 124 aretoo short, the cell may “skip over” the electroporation region 120 during a pulse. The electric field generated by the electroporation electrodes 124 is strongest between the electrodes and becomes weaker upstream and downstream of the electroporation electrodes 124. If the distance D5 is above 10 times the mean cell diameter of the cells 126 being transfected, the size of the microfluidic device 10 increases with minimal or no benefit. This may reduce the number of microfluidic devices 100 that can be manufactured on a die. Extending the length of the microfluidic channel 104 to account for a distance D5 longer than 10 times the mean cell diameter may also reduce the refill velocity of the ejection chamber 112, reducing the efficacy of the mechanical poration. The reduction in refill velocity also limits the firing rate of the fluid actuator 108, reducing the throughput of the microfluidic device 100. Further, longer electroporation electrodes 124 may increase the difficulty of controlling the exposure of the cell to the electric field because the cell 126 may remain between the electroporation electrodes 124 for multiple periods between pulses.

[0052] In some examples, the cells 126 may be exposed to the electric field in the electroporation region 120 for between .001 seconds and 3.0 seconds. In some examples, the cells 126 may be exposed to the electric field for approximately .700 seconds. The amount of time that the cell is exposed to the electric field may depend on the type and size of the cells and may be determined experimentally. In some examples, the voltage applied across the electroporation electrodes 124 may be between 1 V rms and 40 V rms. In some examples, the voltage applied across the electroporation electrodes 124 may be approximately 7 V rms. Lower voltages may fail to electroporate the cells 126, while higher voltages may lyse the cells 126. In some examples, the frequency of the electric field generated by the electroporation electrodes 124 may be between 1 kHz and 1 MHz or between 100 kHz and 200 kHz. Lower frequencies may cause electrolysis of the buffer solution, while higher frequencies may not effectively porate the cells 126. In some examples, the electrical conductivity of the buffer solution may be between 0.02 S / m to 1.6 S / m. In some examples, the electrical conductivity of the buffer solution may be 0.3 S / m. Higher conductivity may cause excessive heating, outgassing (bubbles), and / or lysis of the cells 126. Low conductivity may cause excessive voltage drop across the buffer solution and low voltage across the cell. This may require high voltage to be applied across the electroporationelectrodes 124, increasing the cost of the electronic components of the microfluidic instrument 300.

[0053] The geometric dimensions (e.g., the distances DI -D6) of the microfluidic device 100 may be tailored to the size of the cells to be transfected. For example, a first microfluidic device 100 may be used for cells with a mean cell diameter of 10 micrometers, and a second microfluidic device 100 may be used for cells with a mean cell diameter of 20 micrometers. The microfluidic devices 100 may be removably coupled to the transfection apparatus 302, such that one transfection apparatus 302 can be used to transfect cells of varying size.

[0054] Using a pulsed fluid actuator 108 such as a TIJ resistor or piezoelectric actuator in combination with the sensor 116 allows for accurate position control of cells to ensure that the cells are properly positioned between the electroporation electrodes 124 exposed to an electric field strong enough to electroporate the cells to a desired poration. Further, this allows for control of the amount of time that the cell is exposed to the electric field, which allows for more precise control of pore size and pore distribution, for example, to control the size of transfection material allowed to enter the cell. Control of the amount of time the cell rests after being electroporated also contributes to the success of transfection by ensuring that the pores are allowed to close before subsequent firings that may cause shear forces on the cell. The distance D6 between the constriction region 118 and the electroporation electrodes 124 also ensures that the electroporation of the cells by the electroporation electrodes 124 builds upon and improves the poration of the cells after passing through the constriction region and being mechanically porated.

[0055] The distance D6 between the location of the minimum width of the constriction region 118 and the electrodes 124 along the downstream direction may be between 2 and 20 times the mean cell diameter of the cells 126 being transfected. If D6 is less than 2 times the mean cell diameter of the cells 126 being transfected, the cell 126 may be exposed to the electric field while being mechanically porated in the constriction region 118. This may affect the total amount of time the cell 126 is exposed to the electric field, making the electroporation less reproducible. If D6 is greater than 20 times the mean cell diameter of thecells 126 being transfected, the pores generated in the cell 126 during mechanical poration may close up before the cell is electroporated, potentially eliminating the benefits of sequentially mechanically porating and electroporating the cell 126. Extending the length of the microfluidic channel 104 to account for a distance D6 longer than 20 times the mean cell diameter may also reduce the refill velocity of the ejection chamber 112, reducing the efficacy of the mechanical poration as well as limiting the firing rate of the fluid actuator 108 and reducing the throughput of the microfluidic device 100.

[0056] In some examples, the distance D4 may be approximately 1.2 times the mean cell diameter of the cells 126 being transfected, which may reduce or eliminate clogging while minimizing the voltage across the electroporation electrodes 124 and accounting for normal variation in cell diameter. The other distances DI, D2, D3, D5, and D6 may be defined relative to the distance D4. For example, because the distance D4 may be 1.2 times the mean cell diameter of the cells 126 being transfected, the width or height of a narrowest portion of the constriction region 118 (e.g., distance DI, which may be between 0.3 and 0.9 times the mean cell diameter) may be between 0.25 (0.3 divided by 1.2) and 0.75 (0.9 divided by 1.2) times the distance D4. The distance between the narrowest portion of the constriction region 118 and the pair of electrodes 124 along a longitudinal direction of the microfluidic channel 104 (e.g., distance D6, which may be between 2 and 20 times the mean cell diameter) may be between 1.67 (2 divided by 1.2) and 16.67 (20 divided by 1.2) times the distance between the pair of electrodes. The lengths of the pair of electrodes 124 in the longitudinal direction (e.g., distance D5, which may be between 2 and 10 times the mean cell diameter) may be between 1.67 (2 divided by 1.2) and 8.33 (10 divided by 1.2)times the distance between the pair of electrodes. Thus, in some examples, a width or height of a narrowest portion of the constriction region is between 0.25 and 0.75 times the distance between the pair of electrodes, wherein a distance between the narrowest portion of the constriction region and the pair of electrodes along a longitudinal direction of the microfluidic channel is between 1.67 and 16.67 times the distance between the pair of electrodes, and wherein the lengths of the pair of electrodes in the longitudinal direction is between 1.67 and 8.33 times the distance between the pair of electrodes.

[0057] FIG. 6 shows a cross-section of a portion of the electroporation region 120 of the microfluidic device 100 according to some examples. Specifically, FIG. 6 shows side walls 401 of the microfluidic channel 104 and a portion of the floor 410 of the microfluidic channel 104. The sides wall 401 of the microfluidic channel 104 each include a first wall portion 402 and a second wall portion 404. The first wall portion 402 and the second wall portion 404 may be made from a photoresist material such as SU-8. The floor 410 may include first, second, and third floor portions 406, 412, 407 in areas away from the electroporation electrodes 124. For example, the second floor portion 412 may extend across the bottom of the microfluidic channel 104 to the second electroporation electrode 124. Each side wall 401 may be substantially the same as (e.g., a mirror image of) the opposite side wall 401, with the third floor portion 407 positioned underneath the opposite side wall 401, which is substantially the same as (e.g., a mirror image of) the first floor portion 406. The floor portions 406, 412, 407 may be made of dielectric material. Thus, a lower portion of the microfluidic channel may include a dielectric floor portion (e.g., floor portions 406, 412, 407) defining a lower surface of the microfluidic channel 104 with openings 414 fluidly coupling the microfluidic channel 104 to the pair of electrodes 124.

[0058] The electroporation electrodes 124 may be positioned below openings 414 in the dielectric material of the floor portions 406, 412 that expose the electroporation electrodes 124 to the buffer solution in the microfluidic channel 104. It should be understood that the floor portions 406, 412 may be attached in the areas outside of the electroporation region 120 and may thus be referred to as a single “floor portion” defining a lower surface of the microfluidic channel 104. For example, the floor portions 406, 412 may be contiguous across the floor 410 of the microfluidic channel 104 with the openings 414 forming “windows” that expose portions of the electroporation electrodes 124, fluidly coupling the microfluidic channel to the pair or electroporation electrodes 124. The electroporation electrodes 124 may be made of gold or other highly conductive metals. The electroporation electrodes 124 may be positioned on a substrate layer 408, which may be made of tantalum.

[0059] Referring now to FIGS. 7A and 7B, top views of portions of example microfluidic devices 100 are shown with dimensions indicated. Specifically, only the portions of themicrofluidic devices 100 near the electroporation region 120 and the ejection chamber 112 are shown. The cell reservoir 102 and constriction region 118 are not shown but would be positioned to the left of the portions shown in FIGS. 7 A and 7B. In the microfluidic devices 100 of both FIGS. 7A and 7B, the openings 414 exposing portions of the electroporation electrodes 124 are between about 1 micrometer and about 10 micrometers wide and between about 15 micrometers and about 25 micrometers long. For example, the openings 414 may be 3 micrometers by 25 micrometers. The electroporation electrodes 124 are between about 5 micrometers and about 15 micrometers wide, extending about 1 micrometer to about 7 micrometers on either side of the openings 414. For example, the electroporation electrodes 124 may be may be 9 micrometers wide, extending 3 micrometers on either side of the openings 414. The substrate layers 408 are between about 7 and about 25 micrometers wide, extending about 1 micrometer to about 10 micrometers on either side of the electroporation electrodes 124. The substrate layers 408 may be 15 micrometers wide, extending 3 micrometers on either side of the electroporation electrodes 124. The nozzles 110 of the microfluidic devices 100 of FIGS. 7A and 7B are between about 10 and about 50 micrometers in diameter. For example, the nozzle 110 of the microfluidic device 100 of FIG. 7 A may be 30 micrometers in diameter, and the nozzle 110 of the microfluidic device 100 of FIG. 7 A may be 40 micrometers in diameter. The microfluidic device 100 of FIG. 7 A includes a rectangular fluid actuator 108 (e.g., TH resistors) with a length and a width between about 20 micrometers and about 40 micrometers by 40 micrometers. For example, the fluid actuator 108 of FIG. 7 A may be 30 micrometers by 30 micrometers and the fluid actuator 108 of FIG. 7B may be 37 micrometers by 37 micrometers.

[0060] As shown in FIGS. 7A and 7B, the microfluidic channel 104 may vary in width along the length of the microfluidic channel 104. The variation in width can allow for more precise control of the movement of cells 126 along the microfluidic channel 104. For example, cells 126 may travel larger distances per pulse of the fluid ejectors 108 in the narrower portions of the microfluidic channel 104 than in the wider portions. Additionally, the diameter of the nozzle 110 may affect the volume of fluid ejected per pulse of the fluid ejectors 108. The microfluidic channel 104 may widen as it approaches the ejection chamber 112 to accommodate larger nozzles 110.

[0061] In some examples, the microfluidic device further includes an auxiliary microfluidic channel fluidly coupled to a side of the microfluidic channel, and an auxiliary fluid ejector including an auxiliary fluid actuator fluidly coupled to the auxiliary microfluidic channel. Referring now to FIGS. 8A-9B, top views of portions of example microfluidic devices 100 are shown with auxiliary microfluidic channels 804 fluidly coupled to the respective primary microfluidic channel 104. Setting aside the auxiliary microfluidic channels 804, the dimensions of the microfluidic devices 100 of FIGS. 8 A and 9 A may be the same as the dimensions of the microfluidic device 100 of FIG. 7 A, and the dimensions of the microfluidic devices 100 of FIGS. 8B and 9B may be the same as the dimensions of the microfluidic device 100 of FIG. 7B. The auxiliary microfluidic channels 804 include ejection chambers 812 including an auxiliary fluid ejector 806 with four auxiliary fluid actuators 808 and a nozzle 810. The auxiliary fluid actuators 808 may each be 3.5 micrometers by 15 micrometers. The nozzle 810 of each auxiliary fluid ejector 806 may have a diameter of 13 micrometers. Accordingly, the auxiliary fluid ejector 106 may eject less fluid per pulse than the fluid ejectors 106.

[0062] The auxiliary microfluidic channels 804 and their respective auxiliary fluid ejectors 806 may be used for fine position control of cells 126 in the electroporation region 120. The electric field generated between the electroporation electrodes 124 may be strongest at the center point between the electroporation electrodes 124. Thus, the auxiliary microfluidic channels 804 may be used to adjust the lateral position of cells between the electroporation electrodes 124. For example, ejecting fluid from an auxiliary fluid ejector 806 on the left side of the microfluidic channel 104 may cause a cell 126 in the electroporation region 120 to move to the left in the microfluidic channel 104, and ejecting fluid from an auxiliary fluid ejector 806 on the right side of the microfluidic channel 104 may cause a cell 126 in the electroporation region 120 to move to the right in the microfluidic channel 104.

[0063] Because the auxiliary microfluidic channels 804 are positioned near the downstream end of the electroporation region 120, ejecting fluid from the auxiliary fluid ejectors 806 may also move a cell 126 in the electroporation region 120 in the downstream direction. Because less fluid is ejected via the auxiliary fluid ejectors 806 than the fluid ejector 106, the cell 126may move a shorter distance in the downstream direction than when the fluid ejector 106 is activated, allowing for finer control of cell position in the downstream direction. In some examples, auxiliary microfluidic channels 804 may be positioned near the upstream end of the electroporation region 120, allowing for upstream movement of a cell 126. While the auxiliary microfluidic channels 804 allow for more precise control of the position of a cell 126 in the electroporation region 120, the auxiliary microfluidic channels 804 also increase the footprint of the microfluidic device 100, which may reduce the number of microfluidic devices 100 that can fit onto a die.

[0064] In some examples, the electroporation electrodes 124 may also function as a sensor to determine the position of a cell in the electroporation region 120. For example, as discussed above, changes in the electric field caused by the presence of the cell may be detected by the electroporation electrodes 124, and signals may be sent to the controller 304. The controller 304 may determine, based on the signals the approximate location of the cell in the electroporation region and may send commands to the auxiliary fluid ejectors 806 in the auxiliary microfluidic channels 804 to activate the auxiliary fluid actuators 808 to eject fluid and adjust the position of the cell 126. This process may be repeated until the cell 126 is approximately in the center of the electroporation electrodes 124.

[0065] The various ranges provided herein include the stated range and any value or subrange within the stated range. Furthermore, when “about” is utilized to describe a value or percentage this includes, refers to, and / or encompasses variations (up to + / - ten %) from the stated value or percentage. In describing and claiming the examples disclosed herein, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise.

[0066] Although specific examples have been illustrated and described herein, a variety of alternate and / or equivalent implementations may be substituted for the specific examples shown and described without departing from the scope of the present disclosure. This application is intended to cover any adaptations or variations of the specific examples discussed herein. For example, the method 200 may include additional operations notexplicitly recited or may exclude certain recited operations in some examples. Various examples of the microfluidic device 100 or the microfluidic instrument 300 may include additional components not explicitly recited, may exclude certain recited components, or may include the recited components in different relative positions than shown in the examples described above. Therefore, it is intended that the scope of this disclosure be limited only by the claims and the equivalents thereof.

Claims

WHAT IS CLAIMED IS:

1. A microfluidic device comprising: a fluid ejector comprising a fluid actuator; a microfluidic channel fluidly coupled to the fluid ejector; a sensor positioned along the microfluidic channel; a constriction region positioned between the sensor and the fluid ejector in which an effective cross-sectional area of the microfluidic channel is gradually reduced; and a pair of electrodes positioned in the microfluidic channel between the constriction region and the fluid ejector.

2. The microfluidic device of claim 1, wherein the pair of electrodes extend parallel to a longitudinal direction of the microfluidic channel.

3. The microfluidic device of claim 1, wherein the sensor is an impedance sensor.

4. The microfluidic device of claim 1, wherein the sensor is to detect a cell in the microfluidic channel, the constriction region is to mechanically porate the cell as the cell passes through the constriction region, the pair of electrodes is to electroporate the cell, and the fluid ejector is to eject fluid and the cell from the microfluidic channel.

5. The microfluidic device of claim 4, wherein the pair of electrodes further function as the sensor to detect a cell in the microfluidic channel.

6. The microfluidic device of claim 1, wherein a width or height of a narrowest portion of the constriction region is between 0.25 and 0.75 times the distance between the pair of electrodes, wherein a distance between the narrowest portion of the constriction region and the pair of electrodes along a longitudinal direction of the microfluidic channel is between 1.67 and 16.67 times the distance between the pair of electrodes, and wherein the lengths of the pair of electrodes in the longitudinal direction is between 1.67 and 8.33 times the distance between the pair of electrodes.

7. The microfluidic device claim 1, further comprising: an auxiliary microfluidic channel fluidly coupled to a side of the microfluidic channel; and an auxiliary fluid ejector comprising an auxiliary fluid actuator fluidly coupled to the auxiliary microfluidic channel.

8. A method for transfecting a cell, the method comprising: activating a fluid actuator at least once to eject fluid from a microfluidic channel to move the cell through a constriction region of the microfluidic channel and into an electroporation region of the microfluidic channel comprising electroporation electrodes; detecting the location of the cell in the microfluidic channel; applying an electric potential to the electroporation electrodes to generate an electric field in the microfluidic channel upon determining, based on the detected location of the cell, that the cell is in the electroporation region; and activating the fluid actuator to eject the cell from the microfluidic channel.

9. The method of claim 8, wherein moving the cell through a constriction region imparts shear forces to mechanically porate the cell.

10. The method of claim 8, wherein generating the electric field in the microfluidic channel electroporates the cell.

11. The method of claim 8, further comprising suspending the cell in a buffer solution comprising transfection material, the fluid ejected from the microfluidic channel comprising a portion of the buffer solution, wherein at least some of the transfection material enters the cell after the cell is mechanically porated and electroporated.

12. The method of claim 8, wherein determining that the cell is in the electroporation region comprises detecting the cell upstream of the electroporation region and determining the number of times the fluid actuator is activated after detecting the cell.

13. A microfluidic instrument comprising: a cell reservoir; a fluid ejector comprising a fluid actuator; a microfluidic channel comprising a first end fluidly coupled to the cell reservoir and a second end fluidly coupled to the fluid ejector, the microfluidic channel defining a downstream direction from the first end to the second end; a sensor positioned in the microfluidic channel; a constriction region positioned downstream of the sensor in which the cross-sectional area of the microfluidic channel is gradually reduced; and a pair of electrodes positioned in the microfluidic channel downstream of the constriction region.

14. The microfluidic instrument of claim 13, further comprising a controller to: receive sensor signals from the sensor; determine, based on the sensor signals, the presence of a cell proximate the sensor; cause the fluid actuator to eject a volume of fluid from the microfluidic channel via a nozzle; and cause the pair of electrodes to generate an electric field to electroporate the cell after the volume of fluid has been ejected.

15. The microfluidic instrument of claim 14, further comprising an actuator, wherein the controller is further to: control the actuator to adjust the position of the nozzle relative to a multi-well plate comprising a plurality of wells; and cause the fluid actuator to eject the cell into a different well than a well into which the fluid ejected before the cell is electroporated is ejected.

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