Microfluidic device with sensor

Abstract

A microfluidic device includes a microfluidic channel, a first impedance sensor positioned along the microfluidic channel, a second impedance sensor positioned along the microfluidic channel, and a pair of electrodes for electroporating a cell, the pair of electrodes positioned in the microfluidic channel between the first impedance sensor and the second impedance sensor.

Description

Atty. Dkt. No.: 86349808MICROFLUIDIC DEVICE WITH SENSOR BACKGROUND

[0001] 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. 3 and 4 illustrate an example microfluidic instrument.

[0005] FIGS. 5-12 illustrate example microfluidic devices.

[0006] 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

[0007] 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.Atty. Dkt. No.: 86349808[0008[ 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.

[0009] 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.

[0010] 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 repeatsAtty. Dkt. No.: 86349808(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.

[0011] 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.

[0012] In various examples, an electroporation mechanism may include electrodes within a microfluidic channel used to perform electroporation. “Microfluidic” may refer to fluid channels with widths lower than 1 millimeter, though in some cases, the channel width may be larger. As the cell, suspended in a buffer solution, 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 and number 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.

[0013] In various examples, an impedance sensor may be used to detect a cell in a microfluidic channel. An impedance sensor may also be used to determine the effectiveness or degree of the electroporation by the electroporation mechanism. As discussed above, the cell may be suspended in a buffer solution containing the transfection material as it moves through the microfluidic channel. Because the dielectric constant for cell cytoplasm (aboutAtty. Dkt. No.: 8634980860) is similar in magnitude to the dielectric constant of typical buffer solutions, such as phosphate-buffered saline, which has a dielectric constant of about 78, detecting a cell and determining the degree of operation may be difficult using a capacitive sensor. In contrast the contrast in resistivity between an insulating cell membrane and typical buffer solutions is several orders of magnitude. For example, typical values for cell membrane resistivity is in the megaohm-meters to gigaohm-meters range, in contrast to just several ohm-meters for solution resistivity. Thus, resistive detection of cells using an impedance sensor may be much more sensitive than capacitive detection. Resistance and capacitance can be calculated from impedance by assuming a particular electrical model for the cell. Approximate resistance can be determined using the impedance sensor by neglecting stray capacitance and operating the sensor below a cutoff frequency of the buffer solution. For example, forphosphate-buffered saline, the cutoff frequency is 173 MHz. Operating above this value provides capacitive detection, while operating below this value provides resistive detection. Further, at frequencies below about 10 kHz metal / solution double layer (a charge layer formed at the interface between the sensor electrodes and the buffer solution) dominates the impedance, and at frequences above about 10 MHz parasitic components (e.g., electrical components of the measurement system) dominate the impedance. Accordingly, resistive measurements may be taken in the range of about 10 kHz to about 10 MHz. Cells may have a complex electrical model such that the resistivity of the cell membrane may be more effectively determined by measuring impedance over a range of frequencies. Therefore, impedance sensors used in the embodiments disclosed herein may take multiple measurements of cell impedance at different frequencies.

[0014] In various examples, a microfluidic device is provided that includes a first impedance sensor in the microfluidic channel upstream of the electroporation mechanism and a second impedance sensor in the microfluidic channel downstream of the electroporation mechanism. The microfluidic device may also include a fluid ejector downstream of the second impedance sensor. As the fluid ejector ejects fluid from the microfluidic channel, cells and fluid upstream of the fluid ejector may move toward the fluid ejector. The first impedance sensor may detect the cell in the microfluidic channel and measure the impedance across the electrodes of the sensor as the cell moves over the sensor. After the upstream sensor detects the cell, the location of the cell can be determined and tracked based on the geometry of the microfluidic channel and the volume of fluid ejected by the fluid ejector. For example, theAtty. Dkt. No.: 86349808fluid ejector may eject a known volume of fluid with each actuation, and 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.|0015] As discussed above, the microfluidic device may include a second impedance sensor in the microfluidic channel downstream of the electroporation mechanism. The second impedance sensor may detect the cell in the microfluidic channel after the cell has been electroporated and measure the impedance across the electrodes of the sensor as the cell moves over the sensor. The pores formed in the cell membrane may decrease the resistivity of the cell. Thus, the measured impedance by the second impedance sensor may be indicative of the degree of effectiveness of the poration. In some examples, the degree of poration may be determined based on a comparison of the impedance measurement from the first impedance sensor to the impedance measurement from the second impedance sensor. In some examples, the degree of poration may be determined based on a comparison of the measurement from the second impedance sensor to a predetermined or predefined standard. For example, a standard may be established based on experimental data and may vary based on the type of cell and the geometry of the microfluidic device.

[0016] In one such experiment, the impedance of a microfluidic channel was measured at 100 mV, 200 kHz, and 1000 gain, first with only flowing buffer solution and then with flowing buffer solution with cells suspended therein. When the cells crossed over the sensor, the impedance in the microfluidic channel increased between about 1.9 percent and about 4.2 percent. An optical sensor was used to confirm that these increases in impedance coincided with the passage of a cell over the sensor. In some examples, the degree of poration may be determined by comparing the increase in impedance measured by the upstream impedance sensor to the increase in impedance measured by the downstream impedance sensor. Because the pores in the cell membrane reduce the resistivity of the cell, the decrease in impedance measured by the downstream impedance sensor may indicate the degree of poration. In some examples, it may be determined that a detected increase in impedance in the microfluidicAtty. Dkt. No.: 86349808channel above a certain threshold (e.g., 1.0 percent) but below the minimum increase in impedance caused by an un-porated cell (e.g., about 1.9 percent) indicates the detection of a porated cell. A comparison of the measured impedance of cells with conventional transfection verification testing may allow for further refinement of a range of impedance measurements indicating a sufficient degree of poration.[Q017| Referring now to FIG. 1, a top view of an example microfluidic device 100 including 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 first sensor 116 and a second sensor 117 positioned in the microfluidic channel 104, and a pair of electrodes 124 positioned in the microfluidic channel 104 between the first sensor 116 and the second sensor. The fluid ejector 106 may eject fluid (e.g., repeatedly) from the microfluidic channel 104 causing a cell 126 to move along the channel 104 in a downstream direction. The first sensor 116 may detect the cell in the microfluidic channel 104 and measure the impedance between two sensing electrodes 122 as the cell passes the first sensor 116, the pair of electrodes 124 may electroporate the cell as the cell passes between the electrodes 124 in an electroporation region 120 of the microfluidic channel 104, and the second sensor 117 may measure the impedance between two sensing electrodes 123 as the cell passes the second sensor 117. The impedance measurements from the first sensor 116 and the second sensor 117 may be transmitted to a controller, which may compare the measurements to each other and / or to a predefined standard to determine whether the cell 126 was sufficiently porated. The fluid ejector 106 may continue to eject fluid from the microfluidic channel 104 until the cell 126 approaches the fluid ejector. The fluid ejector 106 may then eject the cell 126 from the microfluidic channel 104. The measured impedance when a successfully or sufficiently porated cell crosses over the second sensor 117 may be lower than the measured impedance of a cell that has not been porated (but higher than the impedance of the microfluidic channel without a cell present), as the electric current is able to partially pass through the cell membrane of the porated cell. Thus, by, for example, comparing the impedance measurement from the first sensor 116 (before poration) to the impedance measurement from the second sensor 117 (after poration) may provide an indication of the degree of poration of the cell.Atty. Dkt. No.: 86349808[0(H8| Because the second sensor 117 is in the microfluidic channel 104, just downstream of the electroporation region 120, the impedance can be measured and the degree of poration can be determined immediately after electroporation of the cell. Because pores in the cell can begin to close immediately after electroporation, being able to immediately measure the impedance may provide the most accurate data regarding the degree of poration. Further, in some examples, this allows cells that have not been sufficiently porated to be identified and isolated rather than being mixed with sufficiently porated cells. In other examples, the immediate measurement of impedance after electroporation allows for subsequent electroporation steps if a cell has not been sufficiently porated.

[0019] 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 at a downstream end 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 (e.g., upstream) end fluidly coupled to the cell reservoir 102 to a second (e.g., downstream) end fluidly coupled to the fluid ejector 106.[0020| As discussed above, 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. For example, 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.

[0021] 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 theAtty. Dkt. No.: 86349808form 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.

[0022] 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 112. 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.

[0023] 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.

[0024] 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 bufferAtty. Dkt. No.: 86349808solution 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 TIJ 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. In some examples, the microfluidic device 100 may not have a pulsed fluid ejector. For example, a fluidic pump may be used to move the buffer solution and cells 126 along the microfluidic channel 104.

[0025] In some examples, a microfluidic device includes a microfluidic channel, a first impedance sensor positioned along the microfluidic channel, a second impedance sensor positioned along the microfluidic channel, and a pair of electrodes positioned in the microfluidic channel between the first sensor and the second sensor. In some examples, the first sensor is to measure impedance as a cell passes the first sensor in the microfluidic channel, the pair of electrodes is to electroporate the cell, and second sensor is to measure impedance as the cell passes the first sensor in the microfluidic channel.|0026] 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 first sensor 116, a second sensor 117, and an electroporation region 120 positioned between the first sensor 116 and the second sensor 117. 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 microfluidicAtty. Dkt. No.: 86349808device, 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.

[0027] The first sensor 116 is positioned along (e.g., in, proximate, under, etc.) the microfluidic channel 104 upstream of the electroporation region 120, and the second sensor 117 is positioned downstream of the electroporation region 120. In some examples, the first sensor 116 may include a pair of sensing electrodes 122 and / or may be an impedance sensor. Similarly, the second sensor 117 may include a pair of sensing electrodes 123 and / or may be an impedance sensor. In other examples, the first sensor 116 and the second sensor 117 may each be an image sensor, a light sensor, a chemical sensor, or another type of sensor. The first sensor 116 and the second sensor 117 may each be configured to detect a cell 126 in the microfluidic channel 104 and to measure the impedance as the cell crosses the respective sensor 116, 117. For example, each of the sensors 116, 117 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, 117 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. The impedance measured by the second sensor 117, after the cell 126 is electroporated by the electrodes 124 may indicate how well the cell 126 has been porated. For example, the impedance measurement from the second sensor may be compared to a predefined standard to determine whether the cell 126 has been sufficiently porated to infer that the cell has been effectively transfected. In some examples, the change in impedance that occurs after electroporation may be used to determine whether the cell has been sufficiently porated. For example, the impedance measurement from the first sensor 116 may be compared to the impedance measurement from the second sensor 117. The difference in impedance may beAtty. Dkt. No.: 86349808compared to a predefined standard to determine if the change in impedance indicated that the cell has been sufficiently porated.

[0028] As discussed above, the first sensor 116 may detect the position of a cell passing over the sensor electrodes 122, 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, in examples in which the fluid in the microfluidic channel is ejected by a pulsed fluid ejector 106, 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, the microfluidic channel 104 may be sized such that, after a cell is detected by the first, the next firing of the fluidic actuator 108 or a predetermined number of firings may cause the cell to be into the electroporation region 120. After moving the cell into the electroporation region 120, a current may be applied to the electroporation electrodes 124 to electroporate the cell for a desired amount of time. The cell may pause in the electroporation region 120 during this period (e.g., without firing the fluid ejector 106), or the fluid ejector 106 may continue to eject fluid at a rate that ensures that the cell passes through the electroporation 120 in an amount of time sufficient to electroporate the cell. Subsequent firings may pull the cell past the second sensor 117 and into the ejection chamber 112. Finally, another subsequent firing may eject the cell from the microfluidic device 100 via the nozzle 110.

[0029] The distance between the electroporation electrodes 124 may be between about 0.9 times the cell diameter and about 10 times the cell diameter, such as about 1.2 times the cell diameter. This may ensure that the channel is narrow enough to maintain the cell within the center of the electric field generated by the electroporation electrodes, providing more reliable and repeatable electroporation. If the distance between the electroporation electrodes 124 is smaller than about 0.9 times the cell diameter, the cell may clog the microfluidic channel 104. If the distance between the electroporation electrodes 124 is greater than about 10 times the cell diameter, the voltage to porate the cell may be very high, increasing the cost of the components and potentially causing electrolysis of the fluid in the microfluidic channel, generating disruptive bubbles. The length of the electroporation electrodes 124 may be between about 2 times the cell diameter and about 20 times the cell diameter, such as about 5 times the cell diameter. If the length of the electroporation electrodes 124 is smaller than about 2 times the cell diameter, it may be difficult to position a cell between of theAtty. Dkt. No.: 86349808electroporation electrodes 124 for an appropriate length of time. If the distance between the electroporation electrodes 124 is greater than about 20 times the cell diameter, the size of the microfluidic device may increase, and more fluid may be wasted to move the cell along the microfluidic channel 104.

[0030] In some examples, the distance between the sensor electrodes 122, 123 in a respective sensor 116, 117 may be between about 0.6 times the cell diameter and about 1.3 times the cell diameter. In some examples, the distance between the sensor electrodes 122, 123 in a respective sensor 116, 117 may be about equal to the cell diameter. This may increase the sensitivity of the sensor 116 because the cell will occupy most of the space between the electrodes 122, 123. If the distance between the sensor electrodes 122, 123 is lower, the vertical penetration of the electric field will cut the position of the cell and sensible volume of the cell is reduced. If the distance between the sensor electrodes 122, 123 is higher, the cell will occupy less of the sensor area, making the sensor less sensitive to the presence of the cell.10031 ] Referring now to FIG. 2, an example method 200 for transfecting a cell is shown. The method may be performed, in part, using the microfluidic device 100. In some examples, a method for transfecting a cell includes moving the cell through a microfluidic channel of a microfluidic instrument over a first impedance sensor in the microfluidic channel, through an electroporation region of the microfluidic channel comprising electroporation electrodes, and over a second impedance sensor in the microfluidic channel, measuring, using the first impedance sensor, a first impedance in the microfluidic channel as the cell moves over the first impedance sensor, applying an electric potential to the electroporation electrodes to generate an electric field in the microfluidic channel upon determining, based on the measured first impedance, that the cell is in the electroporation region, measuring, using the second impedance sensor, a second impedance in the microfluidic channel as the cell moves over the second impedance sensor, and adjusting operation of the microfluidic instrument based on the first impedance and the second impedance (e.g., based on a difference between the first impedance and the second impedance). Generating the electric field in the microfluidic channel may electroporate the cell. The method 200 may include suspending the cell in a buffer solution comprising transfection material, and some of the transfection material may the cell after the electric potential is applied to the electroporation electrodes and the cell is electroporated. The method may further include determining, based on a comparison of theAtty. Dkt. No.: 86349808first impedance to the second impedance, a degree of poration of the cell. The degree of poration may be or may correspond to a likelihood that some of the transfection material in the buffer solution has entered the cell. Adjusting operation of the microfluidic instrument may be based on or may be proportional to the determined degree of poration.

[0032] As discussed above the operation of the microfluidic instrument may be adjusted based on the measured first impedance and the measured second impedance or based on a degree of poration determined based on a comparison (e.g., a difference) between the measured first impedance and the measured second impedance. For example, the electric potential applied to the electroporation electrodes may be adjusted and / or a duration of application of the electric potential may be adjusted. A second cell may be moved through the electroporation region after adjusting the electric potential or the duration such that the second cell may be exposed to a stronger or weaker electric field than the first cell and / or may be exposed to the electric field for a different duration than the first cell. For example, if the determined degree of poration of a first cell is lower than a target degree of poration, the electric potential applied to the electroporation electrodes may be increased or the duration of application of the electric potential may be increased for subsequent cells. The amount that the electric potential and / or duration may be increased may correspond to or be proportional to how far the determined degree of poration is below the target degree of poration. For example, if the determined degree of poration is relatively far below the target degree of poration, the amount that the electric potential and / or duration may be increased may be relatively high, while if the determined degree of poration is below but relatively near target degree of poration, the amount that the electric potential and / or duration may be increased may be lower. If the determined degree of poration of a first cell is higher than a target degree of poration (e.g., if the measured second impedance suggests that the first cell has lysed), the electric potential applied to the electroporation electrodes may be decreased or the duration of application of the electric potential may be decreased for subsequent cells, such as by an amount proportional to how far the determined degree of poration is above the target degree of poration.

[0033] In some examples, adjusting the operation of the microfluidic instrument may include changing the flow of fluid in the microfluidic channel. For example, the flow may be adjusted to move the cell back into the electroporation region. After changing the flow of fluid in the microfluidic channel, an electric potential may be applied to the electroporation electrodes aAtty. Dkt. No.: 86349808second time to generate an electric field in the microfluidic channel. The second impedance sensor may then measure a third impedance in the microfluidic channel as the cell moves over the second impedance sensor. The third impedance may be compared to the first impedance to determine a degree of poration of the cell. In some examples, adjusting the flow of fluid may include reversing the flow such that the first cell is returned to the electroporation electrodes, and applying an electric field to the cell a second time by applying an electric potential to the electroporation electrodes a second time. In other examples, rather than reversing the flow of fluid, the flow may be changed to direct the cell to a recirculation loop that returns the cell to the electroporation region or to a different electroporation region.

[0034] At operation 202 of the method 200 the cell is moved through a microfluidic channel (e.g., microfluidic channel 104) of a microfluidic instrument (e.g., microfluidic instrument 300) over a first impedance sensor (e.g., sensor 116) in the microfluidic channel, through an electroporation region (e.g., electroporation region 120) of the microfluidic channel comprising electroporation electrodes, and over a second impedance sensor (e.g., sensor 117) in the microfluidic channel. As discussed above, the cell may be suspended in a buffer solution containing transfection material. Moving the cell through the microfluidic channel may include, for example, energizing a TIJ resistor or a piezoelectric actuator to eject buffer solution (e.g., repeatedly) from the microfluidic channel via a nozzle, causing buffer solution and the suspended cell to flow in toward the nozzle. For example, the fluid actuator 108 of the microfluidic device 100 may eject buffer solution via the nozzle 110 causing a cell 126 to move along the microfluidic channel 104 toward the nozzle 110. In other examples, a pump may be used to cause the buffer solution and the cell 126 to flow through the microfluidic channel 104 (e.g., in the downstream direction indicated by arrow 114).|0035] At operation 204 of the method 200, a first impedance is measured by the first impedance sensor (e.g., sensor 116) as the cell passes over the first impedance sensor. As used herein “passing over” refers to the cell crossing the impedance sensor in the longitudinal direction of the microfluidic channel and may encompass the cell passing next to or under the electroporation electrodes. The first impedance may be used, for example, to detect that the cell has passed over the first sensor, allowing the location of the cell to be determined. The location of the cell may then be inferred as the cell travels through the microfluidic channel based on the rate of fluid flow through the microfluidic channel. For example, once the cell has been detected at the first sensor 116, the cell may move a distance determined by dividingAtty. Dkt. No.: 86349808the volume of fluid ejected by the fluid ejector 106 by the cross-sectional area of the microfluidic channel. As discussed in further detail below, the first impedance may also be used as a reference point for determining the change in impedance following the electroporation of the cell.

[0036] At operation 206 of the method 200, an electric potential is applied to electroporation electrodes (e.g., electrodes 124) in an electroporation region (e.g., region 120) of the microfluidic channel. The electroporation electrodes are energized by the electric potential 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, with the cell 126 positioned between the electrodes in the electroporation region 120, the electric field causes the cell membrane to be porated in a process referred to as electroporation, as discussed above. The electroporation of the cell 126 may cause pore to form in the cell membrane. This may increase the likelihood of transfection material passing through the cell membrane and into the cell. An increased amount (e.g., duration, electric field intensity, etc.) of electroporation may allow transfection material of larger size to enter the cell. After a desired amount of time, the electrodes 124 may be de-energized. Over the course of seconds, minutes, or hours, the pores may close with the transfection material 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. Operation 206 may include determining that the cell is in the electroporation region, for example, based the volume of fluid ejected from the microfluidic channel after detecting the cell at the first impedance sensor.10037] At operation 208 of the method 200, a second impedance is measured by the second impedance sensor (e.g., sensor 117) as the cell passes over the second impedance sensor. At operation 210 of the method 200, a degree of poration of the cell may be determined based on the second impedance. The second impedance may be compared to a predetermined reference impedance and / or to the first impedance to determine whether the cell has been sufficiently porated to the target degree of poration. Sufficient poration may correspond to a likelihood of successful transfection.[0038| At operation 212 of the method 200, operation of the microfluidic instrument may be adjusted based on the determined degree of poration. For example, if it is determined that theAtty. Dkt. No.: 86349808cell has been sufficiently porated, the cell may be ejected via the nozzle (e.g., nozzle 110) of the microfluidic device into a desired location. For example, the cell may be deposited in a collection well of a multi-well plate. In some examples, one cell may be deposited in an individual well (e.g., without other cells). In other examples, sufficiently porated cells may all be deposited together in a single well. If it is determined that the cell has not been sufficiently porated, the cell may be electroporated a second time. For example, the microfluidic device may have a second electroporation region downstream of the second sensor. The electroporation electrodes of the second electroporation region may be energized to electroporate the cell a second time. A third sensor downstream of the second electroporation region may be used to measure the impedance of the cell to determine if the cell has been sufficiently porated. Additional electroporation regions and sensors may be used for subsequent electroporation steps.

[0039] In some examples, adjusting operation of the microfluidic instrument based on the determined degree of poration may include adjusting the electric potential applied to the electroporation electrodes, thereby adjusting a strength of an electric field generated by the electroporation electrode. In some examples, adjusting operation of the microfluidic instrument based on the determined degree of poration may include adjusting the duration that the electric potential is applied to the electroporation electrodes. For example, the duration may be increased if the determined degree of poration is below the target degree or poration or decreased if the determined degree of poration is above the target degree or poration. The method 200 may further include moving a second cell through the electroporation region after adjusting the electric potential or after adjusting the duration that the electric potential is applied to the electroporation electrodes. Thus, subsequent cells passing through the electroporation region may be exposed to a stronger or weaker electric field and / or may be exposed to the electric field for a different amount of time.[0040| In some examples, upon determining that the degree of poration of the cell is not sufficient the flow of fluid in the microfluidic channel may be changed (e.g., reversed, redirected, recirculated etc.) such that a cell that has not been sufficiently porated may flow back to the electroporation region and electroporated a second time. The method 200 may further include applying an electric potential to the electroporation electrodes a second time to generate an electric field in the microfluidic channel and further electroporate the cell. The method 200 may include measuring, using the second impedance sensor after electroporatingAtty. Dkt. No.: 86349808the cell a second time, a third impedance in the microfluidic channel as the cell moves over the second impedance sensor. If it is determined that a cell has not been sufficiently porated after the second electroporation operation, the flow of fluid may be changed (e.g., reversed, redirected, recirculated etc.) again, and the cell may be electroporated a third time, etc. In some examples, if it is determined that a cell has not been sufficiently porated, the cell may be deposited in a well of the multi-well plate separate from the cells that have been sufficiently porated (e.g., a waste well). As discussed above, in some examples, the flow of fluid in the microfluidic channel may be reversed to return the cell to the electroporation region. In some examples, the flow may be changed to direct the cell to a recirculation loop that returns the cell to the electroporation region. Alternatively, in some examples, the recirculation loop or another microfluidic channel may direct the cell to a different electroporation region configured to electroporate the cell a second time.

[0041] Referring now to FIG. 3 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, first and second sensors sensor positioned in the microfluidic channel, and a pair of electrodes positioned in the microfluidic channel between the sensors, e.g., with the first sensor upstream of the pair of electrodes and the second sensor downstream of the pair of electrodes. 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.[00421 In some examples, the microfluidic instrument further includes a controller to receive first sensor signals (e.g., impedance measurements, sensor data, etc.) from the first 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, cause the pair of electrodes to generate an electric field to electroporate the cell after the volume of fluid has been ejected, receive second sensor signals (e.g., impedance measurements, sensor data, etc.) from the second sensor, determine, based on the sensor signals from the second sensor,Atty. Dkt. No.: 86349808whether the cell has been sufficiently porated, and adjust operation of the microfluidic instrument based on the determined degree of poration. For example, the transfection apparatus 302 includes a controller 304 configured to control the operations of the microfluidic device 100 and the other components of the transfection apparatus 302. For example, the controller 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 instrument 300 to execute the operations of the method 200. In some examples, the degree of poration of the cell is determined based on a comparison of the first sensor data to the second sensor data. In some examples, the degree of poration of the cell is determined based on a comparison of the second sensor data to a predefined or predetermined standard. Based on the determined degree of poration, the controller 304 may adjust operation of the microfluidic instrument 300, for example, by adjusting the electric potential applied to the electroporation electrodes, thereby adjusting a strength of an electric field generated by the electroporation electrodes 124 or by adjusting the amount of time that the electric potential is applied when a cell is detected. In some examples, adjusting operation of the microfluid device 300 based on the determined degree of poration may include causing the fluid actuator 108 to eject the cell through the nozzle 110 into a different well 308 in the multi-well plate 306. For example, cells above a certain degree of poration may be ejected into a first well 308, and cells below a certain degree of poration may be ejected into a second well 308. The controller 304 may generate signals causing the actuators 312 to move the microfluidic device 100 to position the nozzle 110 above a specific well 308, and may generate signals causing the fluid actuator 108 to eject the cell.10043] 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 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 microfluidic channel 104 (e.g., in operation 202 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. The transfection apparatus 302 may supply power to the sensors 116, 117 of the microfluidic device 100 and may receive sensor signals (sensor data, impedance data, etc.) from the sensors 116, 117. For example, the controller 304 may cause alternating current to be supplied to the first sensorAtty. Dkt. No.: 86349808and the second sensor and may measure a voltage drop across electrodes of the first sensor and the second sensor. The alternating current may have a frequency between 10 kHz and 10 MHz. The controller 304 may determine, based on the sensor signals from the first sensor 116, that the first 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 first sensor 116 (e.g., an impedance sensor), that a cell has crossed over the sensing electrodes 122 of the first sensor 116 (e.g., in operation 204 of the method 200).10044] The controller 304 may continue causing 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 into the electroporation region 120. 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 206 of the method 200). As discussed above, the transfection apparatus 302 may supply power to the second sensor 117 of the microfluidic device 100, and the controller 304 may receive sensor signals (sensor data, impedance data, etc.) from the second sensor 117. The controller may determine, based on the sensor signals from the second sensor 117 or from both sensors 116, 117, whether the cell 126 has been sufficiently porated (e.g., in operation 210 of the method 200). The sensor signals from the second sensor 117 may be compared, for example, to a predetermined reference impedance or to the first impedance to determine whether the cell has been sufficiently porated. If the cell is sufficiently porated, 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.

[0045] The controller 304 may analyze the electroporation conditions (e.g., electroporation duration, electric field strength, etc.) applied to cells that are sufficiently porated and may apply similar electroporation conditions to similar cells. For example, in a batch of similar cells, the controller 304 may cause the electroporation conditions to vary for the first few cells until conditions that sufficiently porate the cells are identified. The controller 304 may then apply the same conditions to subsequent cells in the batch.Atty. Dkt. No.: 86349808[O046| 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 the isolated 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 multiwell 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 actuator 108 and fluid ejector 106 to eject the buffer solution into the waste well 310. In some examples and as discussed further below, adjusting the operation of the microfluidic instrument (e.g., as in operation 212 of the method 200) includes causing the fluid actuator to eject the cell through the nozzle into one of the plurality of wells based on the determined degree of poration.

[0047] 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. Based on the determined degree of poration (e.g., based on the determination of whether the whether the cell 126 has been sufficiently porated), the controller 304 may cause transfection apparatus 302 to eject the cell into different wells of the multi-well plate 306. For example, if the sensor data indicates that the cell has not been sufficiently porated, indicating that the transfection of the cell was likely not successful, the controller 304 may control the actuators 312 to position the nozzle of the microfluidic device 100 over the waste well 310 and may cause the fluid ej ector 106 to ej ect the cell into the waste well 310. If the sensor data indicates that the cell has been sufficiently porated, indicating that the transfection of the cell was likely successful, the controller 304 may control theAtty. Dkt. No.: 86349808actuators 312 to position the nozzle of the microfluidic device 100 over a different well, such as an individual well for a single cell. 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 electroporation region as a group or in a line in a continuous fashion and dispensed, for example, into a common reservoir. Based on the sensor data and the expected position of the cells in the microfluidic channel 104, cells that are determined not to have been sufficiently porated may be isolated from the cells that are determined not to have been sufficiently porated. This may allow for the creation of a monoculture of successfully transfected cells to be separated from those unlikely to have been successfully transfected. This may not be possible in transfection apparatuses that do not include a way to verify the degree of poration or transfection of cells before commingling them with other untested cells. In other examples (e.g., as shown in FIGS. 5-8, 11, and 12), the microfluidic device 100 may include features allowing a cell to be electroporated a second time after it is determined that the cell has not been sufficiently porated. For example, the cell may flow back to the electroporation region or may flow into a second electroporation region to be electroporated a second time. The controller 304 may receive a third impedance measurement to determine whether the cell is sufficiently porated after the second electroporation step. In either case, no further verification of transfection may be required after the measurements from the second sensor indicate that the cell has been successfully porated.[0048| As an example of the above, 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 first 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 first 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 controller 304 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 theAtty. Dkt. No.: 86349808actuators 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.[0049| Referring now to FIG. 4, an example schematic diagram of the microfluidic instrument 300 is shown. The controller 304 may control an output signal 402 (e.g., an electric potential) to one of the electrodes 122, 123 of each sensor 116, 117, generating an electric field across the electrodes 122 and an electric field across the electrodes 123. An input signal 404 from each of the respective other electrodes 122, 123 of the sensors 116, 117 may be received at a respective lock-in amplifier 406, which may extract a signal from the first sensor data from the first sensor 116 or the second sensor data from the second sensor 117. The lock-in amplifier may reduce noise and generate a cleaner signal, and may provide more reliable impedance data to the controller 304. The controller may also control a waveform generator 408 that supplies alternating current to the electroporation electrodes 124 to generate an electric field between the electrodes 124. In some examples, the microfluidic instrument 300 may not include a lock-in amplifier. 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 electroporation electrodes 124, increasing the cost of the electronic components of the microfluidic instrument 300.Atty. Dkt. No.: 86349808[0050| Using a pulsed fluid actuator 108 such as a TIJ resistor or piezoelectric actuator in combination with the first 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

[0051] FIGS. 5-12 illustrate microfluidic devices 100 according to various examples. In some examples, the pair of electroporation electrodes 124 is one of a plurality of pairs of electrodes, and the first sensor 116 and the second sensor 117 are two of a plurality of sensors. Each of the plurality of pairs of electrodes may be positioned between adjacent of the plurality of sensors (e.g., between pairs of sensors closest to each other in the longitudinal channel). Referring, for example, to FIG. 5, the microfluidic device 100 includes a second electroporation region 121 with electroporation electrodes 127 and a third sensor 119 with sensing electrodes 125. The first sensor 116 may detect a cell 126 and measure the impedance as the cell passes the first sensor 116. The cell 126 may then be electroporated by the first pair of electroporation electrodes 124 in the first electroporation region 120. The second sensor 117 may then measure the impedance as the cell passes the second sensor 117. A controller (e.g., controller 304) may then determine, based on the at least the impedance measurement from the second sensor 117, whether the cell 126 has been sufficiently porated. If the cell 126 has not been sufficiently porated, the cell 126 may be electroporated a second time in the second electroporation region 121 (e.g., by applying an electric potential to the electroporation electrodes 125). The third sensor 119 may then measure the impedance as the cell passes the third sensor 119, and the controller may determine whether the cell has been sufficiently porated. If the cell 126 was sufficiently porated in the first electroporation region 120, the cell may not be electroporated a second time in the second electroporation region 121. In other examples, the microfluidic device 100 may include three or more electroporation regions. The use of successive electroporation steps may reduce the risk of lysing the cells, as each step may slowly increase the poration until the target degree of poration is reached, whereas a single electroporation step may be more likely to lyse the cell in order to ensure sufficient poration without intermediate impedance measurements.Atty. Dkt. No.: 86349808[00521 In some examples the microfluidic device further includes a fluid ejector at each end of the microfluidic channel, with the cell reservoir fluidly coupled to the microfluidic channel between the two fluid ejectors. This may allow for reversing the flow of fluid in the microfluidic channel, for example, if a cell is not sufficiently porated after a first electroporation step. Referring, for example, to FIG. 6, the microfluidic device 100 includes a second fluid ejector 146 located in a second ejection chamber 152 and including a second fluid actuator 148 and a second nozzle 150. The second fluid ejector 146 may be substantially the same as the first fluid ejector 106. As used herein, “substantially” means that the second fluid ejector 146 may include the same components and operate in the same way as the as the first fluid ejector 106 but may differ in size or geometry. The first fluid ejector 106 and the second fluid ejector 146 may be positioned at opposite ends of the microfluidic channel 104, with the cell reservoir 102 fluidly coupled to the microfluidic channel 104 between the two fluid ejectors 106, 146. By firing the first fluid ejector 106, fluid and a cell 126 may be drawn toward the first fluid ejector 106 (e.g., to the right, as shown), and by firing the second fluid ejector 146, fluid and the cell 126 may be drawn toward the second fluid ejector 146 (e.g., to the left, as shown). By repeatedly firing the first fluid ejector 106, the cell 126 may be detected by the first sensor 116 and electroporated by the electroporation electrodes 124. The second sensor 117 may then measure the impedance as the cell 126 crosses the second sensor. If the measured impedance indicates that the cell 126 has been sufficiently porated, the cell 126 may be ejected by the fluid ejector 106 via the nozzle 110. If the measured impedance indicates that the cell 126 has been sufficiently porated, the second fluid ejector 146 may eject fluid to reverse the flow of fluid, moving the cell 126 back into the electroporation region. The electroporation electrodes 124 may then apply an electric field to the cell 126 a second time. Either the first or second fluid ejectors 106, 146 may then eject fluid to move the cell 126 in either direction, and the impedance can be measured by either of the first or second sensors 116, 117. If the cell 126 is now sufficiently porated, the cell can be ejected via either nozzle 110, 150. If the cell 126 is still not sufficiently porated, the cell 126 can be subjected to electroporation a third time, etc.[00531 The microfluidic device 100 of FIG. 6 includes a third sensor 129 that can be used to detect a cell 126 approaching the second fluid ejector 146, for example, to determine how many firings of the second fluid ejector 146 it will take for the cell 126 to move into the second ejection chamber 152. The third sensor 129 may measure the impedance as the cell crosses the third sensor 129, for example, to confirm the impedance measurement from theAtty. Dkt. No.: 86349808first sensor 116 or to determine if the pores formed in the electroporation process have begun to close.

[0054] 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. For example, FIG. 7 shows a microfluidic device 100 substantially similar to the microfluidic device of FIG. 5, except that the microfluidic device 100 of FIG. 7 includes an auxiliary microfluidic channel 804 fluidly coupled to the primary microfluidic channel 104. The auxiliary microfluidic channel 804 includes an ejection chamber 812 including an auxiliary fluid ejector 806 with an auxiliary fluid actuator 808 and a nozzle 810. The auxiliary fluid actuator 808 and nozzle 810 may be respectively smaller than the primary fluid actuator 108 and nozzle 110. Accordingly, the auxiliary fluid ejector 806 may eject less fluid per pulse than the fluid ejector 106.

[0055] The auxiliary microfluidic channel 804 and the auxiliary fluid ejector 806 may be used for fine position control of cells through the microfluidic channel 104, particularly in the electroporation regions 120, 121. The electric field generated between a pair of electroporation electrodes may be strongest at the center point between the electroporation electrodes. Thus, the auxiliary microfluidic channels 804 may be used to adjust the lateral position of cells between the electroporation electrodes 124 or between the electroporation electrodes 127. For example, ejecting fluid from the auxiliary fluid ejector 806 may cause a cell 126 in the right in the microfluidic channel 104 to move to the right a smaller distance than the cell 126 would move if fluid were ejected by the primary fluid actuator, allowing for finer control of cell position in the downstream direction.

[0056] In some examples, auxiliary microfluidic channels 804 may be positioned near the upstream end of the electroporation region 120, allowing for movement of a cell 126 in the direction opposite the fluid ejector 106. For example, FIG. 8 shows a microfluidic device 100 substantially similar to the microfluidic device of FIG. 6, except the second fluid ejector 146 is replaced with a smaller auxiliary fluid ejector 806 in an auxiliary channel 804, and the microfluidic device 100 includes another auxiliary microfluidic channel 804 near the fluid ejector 106 (e.g., similar to that shown in FIG. 7. Like the second fluid ejector 146, the auxiliary fluid ejector 806 on the left side (as shown) of the microfluidic device 100 may be fired to move a cell 126 to the left (as shown). However, because the auxiliary fluid ejectorAtty. Dkt. No.: 86349808806 is smaller than the second fluid ejector 146, the cell 126 may move a smaller distance along the microfluidic channel. The auxiliary fluid actuators on either side of the microfluidic channel may be fired to finely control the position of the cell, for example, to position the cell 126 at the center of the electroporation electrodes 124.

[0057] In some examples, a microfluidic device may include resistors (clearing resistors, debris clearing resistors, etc.) positioned along the microfluidic channel adjacent the first impedance sensor or the second impedance sensor, e.g., immediately upstream and / or downstream of the sensing electrodes, with no components therebetween. Debris may accumulate on the sensing electrodes after a period of use, reducing the sensitivity of the sensors and increasing the likelihood that cells may clog the microfluidic channel. By firing a resistor adjacent the sensing electrodes, a vapor bubble may form, similar to the vapor bubble formed by activating a TIJ resistor of a fluid ejector, and clear the debris from the sensing electrodes. For example, as shown in FIG. 9, an example microfluidic device 100 includes clearing resistors 156 on either side of each set of sensing electrodes, 122, 123. The controller 304 may supply an electric potential to any of the clearing resistors 156 causing the clearing resistors 156 to heat up and form vapor bubbles to clear debris from the sensing electrodes 122, 123. In some examples, the controller 304 may supply the electric potential to the clearing resistors 156 periodically (e.g., on a fixed schedule, after a predetermined number of firings of the fluid ejector 106) or when an anomaly is detected in the impedance measurements form the sensors 116, 117.[0058| In some examples, a microfluidic device may include a region in the microfluidic channel in the vicinity of the sensors in which an effective cross-sectional area of the microfluidic channel is gradually reduced. A minimum effective cross-sectional area of the microfluidic channel in the constriction region is positioned between the two sensing electrodes 122, 123 of the first impedance sensor 116 or the second impedance sensor 117. The sensors may provide more sensitive measurements when a cell occupies a larger percentage of the cross-sectional area of the microfluidic channel. Thus, by reducing the cross-sectional area of the channel at the sensors, the cell may occupy a larger percentage of the cross-sectional area of the microfluidic channel 104, and the sensor pay provide impedance data with higher sensitivity. For example, as shown in FIG. 10, an example microfluidic device 100 includes constriction regions 154 in the microfluidic channel at each of the sensors 116, 117 in which an effective cross-sectional area of the microfluidic channelAtty. Dkt. No.: 86349808is gradually reduced. The minimum effective cross-sectional area of the microfluidic channel 104 in the constriction region 154 is positioned between the pair of sensing electrodes 122, 123 of the respective impedance sensor 116, 117. This may ensure the highest sensitivity of impedance measurement by the sensors 116, 117. Localized constriction regions 154 may be preferable to reducing the cross-sectional area of the microfluidic channel 104, which may increase the likelihood of cells creating clogs in the microfluidic channel 104.[00591 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 cross-section. The change in effective cross-sectional area may be due to a change in the cross sectional area of the microfluidic channel 104 itself or may be due to the positioning of a protuberance in the microfluidic channel 104. 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. “Gradually reduced,” as used herein means that the cross sectional area is not immediately reduced by, for example, a protuberance with a 90-degree face extending into the microfluidic channel 104. For example, as shown in FIG.10, the upstream ends of the protuberances 158 extend a smaller distance from the wall of the microfluidic channel 104 than the middle of the protuberances 158 extend from the wall of the microfluidic channel 104.

[0060] In some examples, sensing electrodes arranged longitudinally across the bottom of the microfluidic channel may provide less sensitive impedance measurements if the cell is near the top of the microfluidic channel compared to measurements in which the cell is near the bottom of the microfluidic channel, close to the sensing electrodes. In some examples, an impedance sensor may include a pair of electrodes positioned on opposite sides of the microfluidic channel, and an electric potential applied to the pair of electrodes generates an electric field that is substantially perpendicular to a longitudinal direction of the microfluidic channel. “Substantially perpendicular,” as used herein means that, at the longitudinal centerline of the microfluidic channel, the electric field is within ten degrees of perpendicular to the longitudinal axis of the microfluidic channel. The sensing electrodes may be recessedAtty. Dkt. No.: 86349808from the microfluidic channel causing the electric field to be oriented across and perpendicular to the microfluidic channel. For example, as shown in FIG. 11, the example microfluidic device 100 includes three sensors 116, 117, 119 each including a respective pair of sensing electrodes 158, 160, 162 that are recessed from the microfluidic channel 104. When an electric potential is applied to the sensing electrodes 158, 160, 162, the electric fields 159, 161, 163 formed between the respective pairs of sensing electrodes 158, 160, 162 extend substantially perpendicular to the longitudinal axis of the microfluidic channel 104 (e.g., perpendicular to the flow direction indicated by arrow 114). This may provide more reliable impedance measurements regardless of the height of the cell 126 in the microfluidic channel 104.[00611 In some examples, the impedance sensors 116, 117 may include differential sensing electrodes. For example, the impedance sensors 116, 117 may be differential impedance sensors each with three electrodes, which may provide more sensitivity than two-electrode impedance sensors. For example, as shown in FIG. 12, the sensors 116, 117, 119 each include three electrodes 164, 166, 168. As the cell passes over, for example, sensor 116, the impedance between the left and center (as shown) electrodes 164 may be measured with the cell positioned therebetween. The impedance between the center and right (as shown) electrodes 164 may be measured simultaneously, when the cell is not between the center and right (as shown) electrodes 164, and used as a reference point that can be compared to the measurement of the impedance between the left and center (as shown) electrodes 164. This may help to reduce noise and isolate the effect of the cell 126 on the impedance measurement.[0062[ The phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting. The use of “including,” “comprising,” “having,” “containing,” “involving,” “characterized by,” “characterized in that,” and variations thereof herein, is meant to encompass the items listed thereafter, equivalents thereof, and additional items, as well as alternate implementations consisting of the items listed thereafter exclusively. In one implementation, the systems and methods described herein consist of one, each combination of more than one, or all of the described elements, acts, or components.

[0063] References to “or” may be construed as inclusive so that any terms described using “or” may indicate any of a single, more than one, and all of the described terms. References to at least one of a conjunctive list of terms may be construed as an inclusive OR to indicateAtty. Dkt. No.: 86349808any of a single, more than one, and all of the described terms. For example, a reference to “at least one of ‘A’ and ‘B’” can include only ‘A,’ only ‘B,’ as well as both ‘A’ and ‘B.’ Such references used in conjunction with "comprising" or other open terminology can include additional items. References to “is” or “are” may be construed as nonlimiting to the implementation or action referenced in connection with that term. The terms “is” or “are” or any tense or derivative thereof, are interchangeable and synonymous with "can be" as used herein, unless stated otherwise herein.

[0064] Directional indicators depicted herein are example directions to facilitate understanding of the examples discussed herein and are not limited to the directional indicators depicted herein. Any directional indicator depicted herein can be modified to the reverse direction or can be modified to include both the depicted direction and a direction reverse to the depicted direction, unless stated otherwise herein. While operations are depicted in the drawings in a particular order, such operations are not required to be performed in the particular order shown or in sequential order, and all illustrated operations are not required to be performed. Actions described herein can be performed in a different order. Where technical features in the drawings, detailed description or any claim are followed by reference signs, the reference signs have been included to increase the intelligibility of the drawings, detailed description, and claims. Accordingly, neither the reference signs nor their absence have any limiting effect on the scope of any clam elements.

[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 percent) 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 not explicitly recited or may exclude certain recited operations in some examples. Various examples of the microfluidic device 100 or the microfluidic instrument 300 may includeAtty. Dkt. No.: 86349808additional 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

Atty. Dkt. No.: 86349808WHAT IS CLAIMED IS:

1. A microfluidic device comprising:a microfluidic channel;a first impedance sensor positioned along the microfluidic channel to measure impedance as a cell passes the first impedance sensor;a second impedance sensor positioned along the microfluidic channel to measure impedance as the cell passes the second impedance sensor; anda pair of electroporation electrodes positioned in the microfluidic channel between the first impedance sensor and the second impedance sensor to electroporate the cell.

2. The microfluidic device of claim 1, further comprising a resistor positioned along the microfluidic channel adjacent the first impedance sensor or the second impedance sensor.

3. The microfluidic device of claim 1, further comprising a region in which an effective cross-sectional area of the microfluidic channel is gradually reduced, wherein a minimum effective cross-sectional area of the microfluidic channel in the region is positioned between two sensing electrodes of the first impedance sensor or the second impedance sensor.

4. The microfluidic device of claim 1, wherein the first impedance sensor comprises a pair of sensing electrodes positioned on opposite sides of the microfluidic channel, wherein an electric potential applied to the pair of sensing electrodes generates an electric field that is substantially perpendicular to a longitudinal direction of the microfluidic channel.

5. A method for transfecting a cell, the method comprising:moving the cell through a microfluidic channel of a microfluidic instrument over a first impedance sensor in the microfluidic channel, through an electroporation region of the microfluidic channel comprising electroporation electrodes, and over a second impedance sensor in the microfluidic channel;measuring, using the first impedance sensor, a first impedance in the microfluidic channel as the cell moves over the first impedance sensor;Atty. Dkt. No.: 86349808applying an electric potential to the electroporation electrodes to generate an electric field in the microfluidic channel upon determining, based on the measured first impedance, that the cell is in the electroporation region; andmeasuring, using the second impedance sensor, a second impedance in the microfluidic channel as the cell moves over the second impedance sensor; andadjusting operation of the microfluidic instrument based on the first impedance and the second impedance.

6. The method of claim 5, further comprising determining, based on a comparison of the first impedance to the second impedance, a degree of poration of the cell, wherein the operation of the microfluidic instrument is adjusted based on the determined degree of poration7. The method of claim 5, wherein adjusting the operation of the microfluidic instrument comprises:adjusting the electric potential; andmoving a second cell through the electroporation region after adjusting the electric potential.

8. The method of claim 5, wherein adjusting the operation of the microfluidic instrument comprises:adjusting a duration of application of the electric potential; andmoving a second cell through the electroporation region after adjusting the duration.

9. The method of claim 5, wherein determining the degree of poration of the cell comprises determining that a degree of poration of the cell is not sufficient, wherein adjusting the operation of the microfluidic instrument comprises:changing a flow of fluid in the microfluidic channel to move the cell into the electroporation region;applying an electric potential to the electroporation electrodes a second time to generate an electric field in the microfluidic channel; andmeasuring, using the second impedance sensor, a third impedance in theAtty. Dkt. No.: 86349808microfluidic channel as the cell moves over the second impedance sensor.

10. 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 first sensor positioned in the microfluidic channel;a pair of electrodes positioned in the microfluidic channel downstream of the first sensor; anda second sensor positioned in the microfluidic channel downstream of the pair of electrodes; anda controller to:receive first sensor data from the first sensor and second sensor data from the second sensor;determine, based on the second sensor data, a degree of poration of a cell; and adjust operation of the microfluidic instrument based on the determined degree of poration.

11. The microfluidic instrument of claim 10, wherein the degree of poration of the cell is determined based on a comparison of the first sensor data to the second sensor data.

12. The microfluidic instrument of claim 10, wherein adjusting the operation of the microfluidic instrument comprises adjusting a strength of an electric field generated by the pair of electrodes based on the determined degree of poration.

13. The microfluidic instrument of claim 10, further comprising an actuator, wherein the fluid ejector comprises a nozzle, wherein the controller is further to control the actuator to adjust a position of the nozzle relative to a multi-well plate comprising a plurality of wells, wherein adjusting the operation of the microfluidic instrument comprises causing the fluidAtty. Dkt. No.: 86349808actuator to eject the cell through the nozzle into one of the plurality of wells based on the determined degree of poration.

14. The microfluidic instrument of claim 10, further comprising a lock-in amplifier to extract a signal from the first sensor data or the second sensor data.

15. The microfluidic instrument of claim 10, wherein the controller is further to:cause alternating current with a frequency between 10 kHz and 10 MHz to be supplied to the first sensor and the second sensor; andmeasure a voltage drop across electrodes of the first sensor and the second sensor.

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