WO2023141244A1

WO2023141244A1 - Devices and methods for droplet electroporation

Info

Publication number: WO2023141244A1
Application number: PCT/US2023/011196
Authority: WO
Inventors: Harold G. Craighead; Thomas N. Corso; Jacob Vanderburgh
Original assignee: CyteQuest, Inc.
Priority date: 2022-01-21
Filing date: 2023-01-20
Publication date: 2023-07-27

Abstract

A microfluidic device for droplet electroporation includes a channel having an inlet, and outlet, a channel thickness, a channel width, and a first end and a second end defining a channel length, wherein the channel is configured to receive a flow medium having one or more droplets disposed therein. The microfluidic device for droplet electroporation includes at least one pair of electrodes disposed along the channel length and configured to provide an electric potential difference across at least a portion of the channel, wherein each electrode of the pair of electrodes is disposed opposite one another.

Description

DEVICESAND METHODS FOR DROPLET ELECTROPORATION

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority to U.S. Provisional Patent Application Ser. No. 63/301,893, filed January 21, 2022, which is hereby incorporated by reference.

BACKGROUND

Electroporation is a technique in which an electrical field is applied to one or more cells in order to increase the permeability of the cell membrane, allowing a target material (e.g., one or more chemicals, drugs, or other molecules) to be introduced into the cell (also called electrotransfer). Electroporation may be used to modify cells via the insertion of one or more biomolecules, such as DNA or RNA. These cell modifications are important for biological and biomedical research, and form the basis of a new class of cell-based therapies that require careful and reliable treatment of cells.

A specific group of electroporation techniques includes electroporating liquid drops. Various such methods have been developed in the prior art. Some of the prior approaches include electroporating liquid drops on surfaces or aqueous drops in oil, using electrical control of surface tension (electrowetting), using electrodes to mix a droplet containing cells with a droplet containing material to be delivered (mixed droplet), and generating droplets containing cells with cargo for delivery through dispersion within non-conductive oil and multiple tubular electrodes separated by a gap of variable distance.

Droplet electroporation provides advantages for electroporation through the controlled delivery of cargo to single cells or small groups of cells. However, the devices performing one of the known techniques described above have many disadvantages. For example, some devices require complex mechanical, fluidic, and electrical subsystems (e.g., with multiple coaxial, tubular electrodes), which imposes significant manufacturing challenges. Other disadvantages include an increase in un-wanted electrochemistry at the electrode surface, depletion of charged cargo such as DNA due to electrophoretic drive, and the inability to precisely control the electrical (e.g., voltage) waveform delivered to the droplet.

Accordingly, there is a need for novel electroporation devices and methods that can overcome the limitations of existing devices and utilize the advantages of droplet-based electroporation. SUMMARY

To overcome the described limitations of droplet electroporation, the present disclosure provides systems, devices, and methods for droplet-triggered electroporation using an electroporation chip. In various embodiments, methods are provided for applying a voltage waveform (e.g., an arbitrary time-varying voltage) continuously or by triggering based on the arrival of a droplet through a channel.

In various embodiments, a microfluidic device for droplet electroporation includes a channel having an inlet, and outlet, a channel thickness, a channel width, and a first end and a second end defining a channel length. The channel is configured to receive a flow medium having one or more droplets disposed therein. The microfluidic device further includes at least one pair of electrodes disposed along the channel length and configured to provide an electric potential difference across at least a portion of the channel. Each electrode of the pair of electrodes may be disposed opposite one another. Alternatively, each electrode of the pair of electrodes may be disposed on the same side on the channel (see, e.g., Fig. 1C and Fig. ID).

In various embodiments, a device includes a droplet-forming region configured to form one or more droplets, an electroporation region comprising one or more pairs of electrodes, and a collection device configured to collect or extract the droplets. At least one of the one or more droplets have at least one cell and at least one biological material contained therein.

In various embodiments, a device capable of inserting a biologically active molecule into a living cell includes a fluid channel having a fluid input and a fluid output configured to allow plug fluid flow. The plug fluid flow has at least a first plug comprising a first fluid and a second plug comprising a second fluid. The first plug and the second plug alternate along a length of the channel. The first fluid has a different fluid property from the second fluid such that the first and second plugs remain substantially separate during plug fluid flow. At least one of the first plug and the second plug includes one or more living cells and one or more biologically active molecules. The device further includes a first electrode and a second electrode disposed on opposite sides of the fluid channel to which a voltage can be applied to generate an electric field directed across the fluid channel when the first and second plugs pass between the first and second electrodes. The first and second electrodes are separated by a distance that enables the first and second plugs to pass therethrough. The strength of the electric field to which the one or more living cells is exposed is sufficient to form pores within a cell membrane of the one or more living cells through which the one or more biologically active molecule can traverse the cell membrane, but not lyse the one or more living cell.

BRIEF DESCRIPTION OF FIGURES

Fig. 1A illustrates a top view of a microfluidic device for droplet electroporation with a single pair of electrodes and multiple droplet-forming structures in accordance with an embodiment of the present disclosure.

Fig. IB illustrates a side view of a microfluidic device for droplet electroporation with a single pair of electrodes and multiple droplet-forming structures in accordance with an embodiment of the present disclosure.

Figs. 1C and ID illustrate a side view of a microfluidic device for droplet electroporation with a single pair of electrodes and multiple droplet-forming structures in accordance with an embodiment of the present disclosure.

Fig. 2A illustrates a side view of a microfluidic device for droplet electroporation with a single pair of electrodes in accordance with an embodiment of the present disclosure

Fig. 2B illustrates a top view of a microfluidic device for droplet electroporation with a single pair of electrodes in accordance with an embodiment of the present disclosure.

Fig- 3 illustrates a microfluidic device for droplet electroporation with two pairs of electrodes in accordance with an embodiment of the present disclosure.

Fig- 4 illustrates a microfluidic device for droplet electroporation with two pairs of electrodes and an output receptacle in accordance with an embodiment of the present disclosure.

Fig- 5 illustrates a microfluidic device for triggered electroporation operating with plug flow in accordance with an embodiment of the present disclosure.

Fig. 6 illustrates an exemplary time-varying voltage waveform used for electroporation in accordance with an embodiment of the present disclosure.

Fig. 7 illustrates a top view of a microfluidic device for droplet electroporation with multiple parallel channels where electrodes for each channel are controlled by a multiplexer in accordance with an embodiment of the present disclosure.

DETAILED DESCRIPTION

General principles and workings of droplet electroporation have previously been described elsewhere; thus, they are not repeated here. For example, US 6,911,132 (incorporated by reference), US 2016/0108432 (incorporated by reference), US 2016/0333302 (incorporated by reference), US 2020/0122138 (incorporated by reference), Electroporation of cells in microfluidic droplets (Zhan et al., 2009), Droplet electroporation in microfluidics for efficient cell transformation with or without cell wall removal (Qu et al., 2012), and Transfection of Jurkat T cells by droplet electroporation (Im & Jeong, 2017) describe various approaches to droplet electroporation.

Various aspects of these systems and methods are illustrated in the provided examples. The microfluidic devices described here can have many configurations. The simplest contains a single uniform flow channel and a single pair of electrodes. Also encompassed are devices with multiple channels and a single pair of electrodes connecting to each channel. Multiple electrodes can readily be incorporated in the channels with independently addressable connections. The channels can also be made in different configurations with additional functions such as varying the width or thickness of the flow channel(s) to apply hydrodynamic forces to cells in addition to or instead of electric fields. Integrating with other on-chip microfluidic device functions such as cell sorting or filtering is also possible.

Definitions

The articles “a” and “an” are used herein to refer to one or to more than one (i.e. to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.

Unless specifically stated or obvious from context, as used herein, the term "about" is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. About can be understood as within 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from context, all numerical values provided herein are modified by the term “about.”

All numerical ranges provided herein are understood to be shorthand for all of the decimal and fractional values within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50, as well as all intervening decimal values between the aforementioned integers such as, for example, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, and 1.9 and all intervening fractional values between the aforementioned integers such as, for example, 1/2, 1/3, 1/4, 1/5, 1/6, 1/8, and 1/9, and all multiples of the aforementioned values. With respect to sub-ranges, "nested sub-ranges" that extend from either end point of the range are specifically contemplated. For example, a nested sub-range of an exemplary range of 1 to 50 may comprise 1 to 10, 1 to 20, 1 to 30, and 1 to 40 in one direction, or 50 to 40, 50 to 30, 50 to 20, and 50 to 10 in the other direction.

As used herein, the term “chip” is used interchangeably with the term “device” or “microfluidic device.”

As used herein, the term “voltage waveform” (or related terms, e.g., “time-dependent voltage waveform,” “time-varying voltage waveform,” “time-varying voltage,” “temporal voltage wave form,” etc.) refers to the voltage that varies in time as supplied to the electrodes by the voltage controller or other source of voltage. This may be described by a periodically repeated time varying function, but it can also vary arbitrarily in time and not be repeated.

Microfluidic device for electroporation

In some embodiments, an electroporation device comprises at least one planar flow channel flanked by at least one pair of electrodes on opposite sides of the channel to which electrical potentials can be applied to create an electric field across the channel between the electrode pair. In some embodiments, the dimension of the height of the fluid channel is approximately equal to the droplet diameter. In some embodiments, one or more dimensions of the fluid channel is approximately equal to the droplet diameter, for example, the diameter of the fluid channel. The channel, in some embodiments, has no restriction on distance in the other two dimensions of channel length and opposing channel walls not flanked by the electrodes. The droplets flow through the channel at a set flux, and these features enable a user to apply precise electric fields to the droplets containing the cell. The strength of the electric field is strong enough to form pores within the membrane of the living cell through which heterologous objects (e.g., biological molecules) can traverse the cell membrane, but weak enough to not lyse the cell.

The device includes one or more ports (e.g., fluid inputs and fluid outputs). When the device includes a single fluid input, a single fluid stream is created. The single fluid stream contains droplets. Some of the droplets contain living cells in combination with a heterologous object (e.g., biologically active molecules) for introduction of the heterologous object (e.g., biologically active molecule) into the living cell by electroporation. Suitable spacing between the electrodes includes about 0.5 to 5 times larger than the diameter of the droplet, or smaller than approximately five times the typical droplet diameter, forcing the droplets to pass through the space between the electrodes in a single layer. Suitable distance between the electrodes of an electrode pair includes a range of from about 50 micrometers to about 100 micrometers, or less than about 100 micrometers (e.g., 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 micrometers).

In various embodiments, a suitable distance between the electrodes of an electrode pair includes from about 50 micrometers to about 500 micrometers. Another advantage of an embodiment of the disclosure is that the user can manipulate the chemical and electrical properties of the environment at different positions along the length of the channel. Furthermore, some embodiments of the disclosure allow the user to monitor various properties of the droplets and/or the solution to modify and optimize the flow and voltage parameters in real time.

In some embodiments, a microfluidic device for electroporation comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 planar flow channels. In some embodiments, a microfluidic device comprises 1 planar flow channel.

In some embodiments, a microfluidic device for electroporation comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 pairs of electrodes. In some embodiments, electrical current is applied to only one pair of electrodes. In some embodiments, at least one pair of electrodes is configured to detection of droplets. In some embodiments, one pair of electrodes is configured to detect the presence of a droplet and apply a voltage waveform in response to the detection.

In some embodiments, a microfluidic device comprises at least one pair of electrodes that extend to at least one edge of the microfluidic device such that electrical control module can connect to the electrodes at one end of the planar device. In some embodiments, the at least one pair of electrodes extend to the edge of the microfluidic device that is distal to the at least one second port. In some such embodiments, the at least one second port may be the outlet port that dispenses electroporated cells into a multi-well module. Fig. 1A illustrates a top view of a microfluidic device 100 for droplet electroporation with a single pair of electrodes and multiple droplet-forming structures 120a-120d. Fig. IB illustrates a side view of the microfluidic device 100 for droplet electroporation with a single pair of electrodes and multiple droplet-forming structures 120a-120d. As shown in Figs. 1A and IB, the microfluidic device includes a substantially planar channel having a width, w, and a thickness, t. In various embodiments, the droplet-forming structures 120a-120d are individual channels configured to form droplets 105a- 105k of an aqueous liquid in the channel 104. In various embodiments, the droplet-forming structures 120a-120d are configured to form droplets 105a-105k of an aqueous liquid in a non-polar flow medium (e.g., oil). The droplet forming structures may be one or more tubular structures wherein a fluid passes through and forms a droplet upon contact with and through said droplet forming structure. The droplet forming structures may be one or more features configured to introduce turbulence to the flow of the droplet fluid to create the one or more droplets. In various embodiments, the flow medium flows from the inlet of the microfluidic device 100, through one or more pairs of electrodes 110a- 110b, and out of the outlet 103 of the microfluidic device 100. In various embodiments, there is at least one outlet 103 consistent with the entirety of the disclosure. In various embodiments, there is a plurality of outlets 103 configured to provide an exit path, passively or actively to the fluid within channel 104. In various embodiments, the width, w, of the channel 104 is wide enough to accommodate two or more (e.g., four) droplet widths flowing across the channel. In various embodiments, the channel 104 is configured to only accommodate one droplet flowing across the channel (e.g., a single file line) as shown in Figs. IB-4. In various embodiments, droplets are released from the droplet-forming structures 120a-120d in a staggered manner so that the droplets do not contact one another while flowing through the channel 104. In various embodiments, the microfluidic device 100 is configured to perform electroporation. In various embodiments, the microfluidic device 100 includes one pair of electrodes 110a-l 10b disposed on opposite sides of the channel 104. In various embodiments, one electrode 110a is disposed along one side (e.g., top side) of the channel 104 and the other electrode 110b is disposed along an opposite side (e.g., bottom side) of the channel 104. In various embodiments, the electrodes 110a, 110b may be disposed opposite each other across the channel 104 in a differing arrangement, such as horizontally (e.g., left to right). In various embodiments, the microfluidic device 100 includes an outlet allowing passage into a subsequent analytical device for collection after electroporation. In various embodiments, one or more pairs of electrodes may be disposed on the same side of the channel, as shown in Figs. 1C-1D. In various embodiments, as shown in Fig. 1C, a distance between the two electrodes may be smaller than a length of individual droplets (e.g., from end to end along a length of the droplet measured along the channel length). In various embodiments, a distance between the two electrodes may be equal to a length of individual droplets. In various embodiments, as shown in Fig. ID, a distance between the two electrodes may be greater than a length of individual droplets. In various embodiments, a larger distance between the electrodes allows for a more uniform electric field through the channel and between the two electrodes.

In various embodiments, any (e.g., all) of the droplets 105a- 105k may include one or more cells. In various embodiments, one or more droplets 105a- 105k may not include any cells. In various embodiments, because droplet formation follows Poisson statistics, some droplets contain zero cells and about the same number of droplets contain one cell. In various embodiments, a portion of the formed droplets will contain no cells while another portion of droplets contain one or more cells. In various embodiments, one or more droplets 105a- 105k may include one or more biological material (e.g., DNA, RNA) to be electroporated into the one or more cells.

In various embodiments, a system, method, and device is provided for the introduction of a biologically active molecule into a living cell by electroporation on a microfluidic platform, where the cell has been incorporated into a droplet. In various embodiment, an electroporation device contains a fluid channel having a pair of electrodes on opposite sides of the channel to which an electrical potential difference (e.g., a predetermined voltage waveform) can be applied to create an electric field across the channel between the electrode pair, as well as a structure for encapsulating one or more cells in droplets. In various embodiments, the dimensions of the fluid channel provide sufficient control to maintain the droplets within the fluid flow at similar positions with respect to proximity to the electrode pair they are passing through. In various embodiments, the droplet flow is one layer thick in the channel dimension between the opposing electrode pairs so that the droplets are independently exposed to the same electrical current formed when passing between the electrode pairs. The fluid channel, in some embodiments, has no restriction on distance in the other two dimensions of channel length and opposing channel walls not flanked by the electrodes. In various embodiments, the strength (e.g., voltage or current) of the electric field is suitable to form pores within the membrane of the living cells inside the droplets through which biologically active molecules can traverse the cell membrane, but weak enough to not lyse the cell. In various embodiments, an arbitrary voltage waveform (e.g., as shown in Fig. 6) may be constantly applied across the pair of electrodes. In various embodiments, the arbitrary voltage waveform is any suitable voltage. In various embodiments, the arbitrary waveform is formed by an arbitrary waveform generator or other electronic device suitable to generate an arbitrary waveform as is known in the art. In various embodiments, the arbitrary waveform generator generates any arbitrarily-defined waveform as output. In various embodiments, the waveforms can be either repetitive or single-shot. In various embodiments, the voltage waveform is defined as a series of "waypoints" (specific voltage targets occurring at specific times along the waveform). In various embodiments, the arbitrary voltage waveform generator can jump to those specific waypoint levels or use any suitable method(s) to interpolate between those levels. In various embodiments, the arbitrary voltage waveform generator operates as a conventional function generator. In various embodiments, the arbitrary voltage waveform generator generates one or more standard waveforms, such as sine, square, ramp, triangle, noise and/or pulse. In various embodiments, the arbitrary waveform may be randomly-generated.

In various embodiments, a pair of electrodes continuously applies a predetermined voltage waveform (rather than a continuously-applied arbitrary voltage waveform). In various embodiments, the predetermined voltage waveform is a voltage waveform selected before performing electroporation and suitable to perform electroporation of one or more cell(s). In various embodiments, the continuously-applied predetermined voltage waveform has a specified, limited duration. In various embodiments, the continuously-applied voltage waveform is a predetermined voltage waveform repeated in a loop. In various embodiments, between each repetition of the predetermined voltage waveform, the continuously-applied voltage waveform includes a buffer (e.g., a short duration of zero voltage). In various embodiments, the continuously-applied voltage waveform is suitable for electroporation of living cells to allow a biological material (e.g., DNA, RNA) to enter the cell membrane.

In various embodiments, as described in more detail below, a pair of electrodes may be used to trigger the application of a predetermined voltage waveform in place of the arbitrary voltage waveform. In various embodiments, no voltage waveform may be applied until the electrodes are triggered to apply the predetermined voltage waveform.

The separation between electrodes located across the thickness of the fluidic device is small, therefore requiring an applied voltage of only a few volts to perform the electroporation. This contrasts with the need for voltages up to several thousand volts that are normally required for standard electroporation. For example, it is known in the literature that a transmembrane electric field of less than 1 kV / cm is required to porate the cell membrane (Weaver and Chizmadzhev, 1996). However, for a distance between the electrode pairs of 100 micrometers, this requires approximately a 5 V potential difference to porate an average mammalian cell in accord with the present device. Suitable voltage differences across a living mammalian cell include the following range: 0.1 V to 10 V. For example, for a distance between the electrodes of 100 micrometers this range corresponds to an electric field of 10 V/cm to 1000 V/cm.

The flow channel can have one or several electrically independent electrode pairs. For example, it can have four sets of electrode pairs 101. Connections to the electrodes are made by using clips, conductive pins or conduction adhesive to connect these to a variable-voltage power supply, function generator, computer via a data acquisition card or amplifier, or batteries with a voltage divider. An ammeter can be used to monitor the current flowing between any pair of electrodes for monitoring and controlling the process.

The electrodes can be configured to apply either a constant, pulsating, or continuously time varying voltage, thereby creating an electric field perpendicular to the direction of flow or along the direction of flow. If a pulsating voltage is desired, a pulse duration from about 0.01 millisecond to about 100 milliseconds is suitable. The plurality of electrode pairs can be patterned to create spatially and temporally varying electric fields. The electrodes may be patterned using a photomask in the photolithographic process or by a shadow mask in the sputtering or deposition process. Patterning allows for the fabrication of electrodes with varying geometric shape. The variation of the shape combined with the fluid flow characteristics provides for controlling the time that cells are subject to the electric field.

The invention provides for the ability to pattern electrodes at different locations on the surface of the flow channel that can be individually connected to various electrical sources, where the electrical sources can have different voltage and current characteristics. The disclosed planar fluid systems consisting of electrically insulating material(s) enable the patterning of various electrode structures.

In various embodiments, any one or more of the following can be done: (1) one electrode or group of electrodes can be activated with time-dependent voltage characteristics to open pores in the cells; (2) another electrode or group of electrodes can be activated to drive charged molecules into cells; (3) another electrode or group of electrodes can be used to measure the electrical properties of the cell-containing fluid; (4) another electrode or group of electrodes can be used to concentrate nucleic acids or other molecules at the interface between fluid layers of varying conductivity; (5) another electrode or group of electrodes can be activated to move the droplets, and thereby cells actively, or passively by a creating flow in the fluid, to a prescribed location in the flow channel for droplet and/or cell sorting or other purposes; and (6) another electrode or group of electrodes can be activated to rotate the cells to increase the surface area exposed for electroporation.

Importantly, some of the disclosed embodiments permit the application of an arbitrary time-varying voltage to different electrodes. The voltage signals can be formed by computer generation of the desired time varying voltage waveform, which is converted to an applied voltage by digital to analog conversion and amplification to the desired voltage range.

A simple voltage waveform could be as shown in Fig. 6. An amplitude portion of the voltage waveform needs to be sufficient to permeabilize the cells. This, in some embodiments, requires a voltage drop of approximately 1 V over the typical 10 micrometers size of a mammalian cell within the fluidic device. This implies that the amplitude of the voltage waveform could be about 5V, with a range extending from 0.1 V to 100 V depending on the depth of the fluidic device (e.g., chip) and the ionic composition of the fluid layers. The frequency of the voltage waveform depends on the impedance characteristics of the circuit, specifically on the capacitive aspects of the so-called double layer that is known to form at the surface of the electrode due to the presence of free moving ions in the aqueous solution as well as the resistance of the fluid, or fluid layers of varying conductivity. The impedance of the capacitive double layer depends inversely on the frequency. Consequently, the frequency should preferably be around 10 kHz so that the impedance of the fluid layers dominates, leading to most of the voltage change occurring within the fluid layer and not at the electrode-electrolyte interface. The frequency might range from 100 Hz to 1 MHz depending on the fluidic device dimensions and the ionic composition of the fluid layers. The impedance of the circuit may depend on a complicated manner on the ionic conductivity of the fluid layers. The resistance of the fluid scales inversely with the ionic concentration, while the double layer capacitance is proportional to the ionic concentration raised to some power. The circuit at the electrolyte-electrode interface is often approximated as a capacitor due to the double layer in parallel with a frequency dependent impedance that is in series with a resistance due to charge transferred across the electrode (referred to as the Randles equivalent circuit model). The ability to control the time variation of the voltage waveform means that the current charging the double layer and the current due to charge transferred across the electrodes may be modulated according to the optimum configuration for electroporating the cells. The voltage waveform could also be composed of the sum of a sinusoidal wave in addition to a constant DC voltage offset, resulting in a net flow of current.

Another periodic voltage waveform, according to some embodiments, has a short duration voltage to open pores followed by a lower voltage of longer duration to move charged molecules into proximity to the cells. The movement of the charged molecule can be due to an electrophoretic force, or due to electrophoresis from a net fluid motion induced by the electrodes, or due to a dielectrophoretic force on the charged molecule or cell.

The continuous repeating nature of the voltage waveform is useful for the continuous flow systems. The applied voltage can vary from positive to negative or remain at zero or another constant voltage for portions of the voltage waveform.

A voltage waveform of arbitrary shape can be created by adding together any number of sinusoidal voltage waveforms each with their own frequency and amplitude, in addition to a constant voltage offset. The net time-average voltage can be chosen to be positive, negative, or zero providing the ability to control the net direction of charge flow. This would be of utility for controlling surface electrochemistry on electrodes and for directing charged molecules in a chosen direction.

The voltage waveform may also be chosen to open pores in the cells or cell nucleus and allow time for diffusion of neutral molecules into the cells before another pore-opening voltage application.

In some embodiments, the spatial arrangement of sets of counterpart electrodes across the surfaces of the fluid channel allows creating an electric field within the fluid channel that varies as a function of time and position without a need for a user to create discrete electrical pulses (e.g., via multiple voltage suppliers providing a voltage waveform to each set of counterpart electrodes, which can be a sinusoidal voltage waveform for any set, and which can be different between the different sets).

Electrodes can be patterned by a variety of methods, including inkjet printing, silk screening, lithographic patterning, vapor deposition through a shadow mask and other methods for patterning electrical conducting material on a variety of substrates including plastics.

In various embodiments, a distance between the electrodes of an electrode pair is from about 5 micrometers to about 250 micrometers. In various embodiments, a distance between the electrodes of an electrode pair is from about 50 micrometers to about 100 micrometers. In various embodiments, a distance between the electrodes of an electrode pair is less than about 250 micrometers. In various embodiments, a distance between the electrodes of an electrode pair is less than about 100 micrometers. In various embodiments, a distance between the electrodes of an electrode pair is about 5 micrometers. In various embodiments, a distance between the electrodes of an electrode pair is about 10 micrometers. In various embodiments, a distance between the electrodes of an electrode pair is about 15 micrometers. In various embodiments, a distance between the electrodes of an electrode pair is about 20 micrometers. In various embodiments, a distance between the electrodes of an electrode pair is about 25 micrometers. In various embodiments, a distance between the electrodes of an electrode pair is about 30 micrometers. In various embodiments, a distance between the electrodes of an electrode pair is about 35 micrometers. In various embodiments, a distance between the electrodes of an electrode pair is about 40 micrometers. In various embodiments, a distance between the electrodes of an electrode pair is about 45 micrometers. In various embodiments, a distance between the electrodes of an electrode pair is about 50 micrometers. In various embodiments, a distance between the electrodes of an electrode pair is about 55 micrometers. In various embodiments, a distance between the electrodes of an electrode pair is about 60 micrometers. In various embodiments, a distance between the electrodes of an electrode pair is about 65 micrometers. In various embodiments, a distance between the electrodes of an electrode pair is about 70 micrometers. In various embodiments, a distance between the electrodes of an electrode pair is about 75 micrometers. In various embodiments, a distance between the electrodes of an electrode pair is about 80 micrometers. In various embodiments, a distance between the electrodes of an electrode pair is about 85 micrometers. In various embodiments, a distance between the electrodes of an electrode pair is about 90 micrometers. In various embodiments, a distance between the electrodes of an electrode pair is about 95 micrometers. In various embodiments, a distance between the electrodes of an electrode pair is about 100 micrometers. In various embodiments, a distance between the electrodes of an electrode pair is about 150 micrometers. In various embodiments, a distance between the electrodes of an electrode pair is about 200 micrometers. In various embodiments, a distance between the electrodes of an electrode pair is about 250 micrometers.

In various embodiments, the dimension of the channel along the fluid flow is from about 0.5 mm to 250 mm. In various embodiments, the dimension of the channel along the fluid flow is from about 0.5 mm to 100 mm. In various embodiments, the dimension of the channel along the fluid flow is about 0.5 mm. In various embodiments, the dimension of the channel along the fluid flow is about 1 mm. In various embodiments, the dimension of the channel along the fluid flow is about 5 mm. In various embodiments, the dimension of the channel along the fluid flow is about 10 mm. In various embodiments, the dimension of the channel along the fluid flow is about 20 mm. In various embodiments, the dimension of the channel along the fluid flow is about 30 mm. In various embodiments, the dimension of the channel along the fluid flow is about 40 mm. In various embodiments, the dimension of the channel along the fluid flow is about 50 mm. In various embodiments, the dimension of the channel along the fluid flow is about 60 mm. In various embodiments, the dimension of the channel along the fluid flow is about 70 mm. In various embodiments, the dimension of the channel along the fluid flow is about 80 mm. In various embodiments, the dimension of the channel along the fluid flow is about 90 mm. In various embodiments, the dimension of the channel along the fluid flow is about 100 mm.

The dimension of the width of the microfluidic channel, specified as the dimension perpendicular to the fluid flow and perpendicular to the dimension between the electrodes, can be from about 1 mm to about 200 mm, depending on the volumetric throughput required. Figs. 2A and 2B illustrate a microfluidic device 200 for droplet electroporation with a single pair of electrodes 210a, 210b. As shown in Figs. 2A and 2B, the microfluidic device has an inlet 202 for receiving a flow medium having one or more droplets 205a-205f, an outlet 203, top electrode 210a, and bottom electrode 210b (collectively, a pair of electrodes 210a, 210b). As shown in Fig- 2B, the width of the fluid channel is w and its length is L. Fluid streams interface to the device via tubing, fittings, interconnects, a manifold, or discreet fluid path connections. One or more of these parts can be part of the fluid interface. The fluid interface serves to reformat the tubing or conduits into the receiving slit-port of the device. The fluid interface may have changes in surface area as well as varying geometries for delivering fluid to the device. The fluid interface may have features to enhance mixing or maintain laminar flow characteristics. This includes geometric changes that may aid in turbulent flow, diffusion rate changes, or residence time in the flow path. The fluid path may have geometries tailored to avoid the trapping gas (bubbles) or seeding to avoid gas bubble formation due to gas coming out of solution.

The fluid path components may be machined, molded (e.g., injection molding), casted, extruded, or the like. The fluid interface may be fabricated as part of the channel device (one piece) or bonded (integrated) to the device via a permanent or non-permanent bond.

Alternatively, the fluidic interface could be manufactured as part of the device as one integrated component, for example via injection molding where the device and fluid interface are both formed during the molding process. Sealing between the fluid interface and the device may be hermetic, compression-based, O-ring-based, gasket-based, adhesion-based, fused, luer locked (quick connect), flat bottom compression-based, tapered ferrule-based, frusto-conical compression-based, friction fit, barbed connection, or the like. The fluid interface component may be composed of one or more fluid paths and is not limited to the location or number of inlet or outlet features. Fluid transfer lines may be soft, semi-hard, or hard where the leak tight seal between components are made with connections known to those in the art.

Tubing and fluid conduits may be manufactured via extrusion or molding.

For some manifold designs, portions of the system may not contain tubing and fluid will be routed via the manifold structure.

In some embodiments, the fluid interface to the device may be via a leak-tight seal to the planar device with a compressive material such as an O-ring or gasket. The device can be interfaced to a fluid delivery system. A fluid delivery apparatus or pump is configured to displace fluid from a vessel to establish a fluid flow within the fluid path. The fluid vessel may contain a pure fluid or a solution. The fluid may contain cells, small molecules, or large molecules including chemical entities for the transfection process. The fluid displacement apparatus can provide positive and/or negative displacement of the fluid. This allows fluid to be pushed or pulled through the device and the fluid path components.

The delivery pump may include mechanisms comprising peristaltic, syringe, gear pump, diagram, gas pressure (positive or negative), centrifugal, piston, check-valve, or mechanical displacement, hydrostatic or gravity driven flow.

Preferably, the fluid is indirectly displaced by the pump without the liquid directly contacting any of the moving parts of the apparatus, such as, for example, a peristaltic pump acting upon a fluid filled tube. Alternatively, a positive pressure displacement mechanism may be used where a head pressure displaces liquid from a pressurized vessel, or a negative displacement where a vacuum is used to pull liquid into the electroporation device; vacuum via pressure regulator or a peristaltic pump. The use of negative displacement allows for limited system components to be implemented on the inlet side of the device.

Conversely, fluid may be directly displaced by an apparatus, when the fluid is displaced by directly contacting any of the moving parts of the apparatus, such as, for example, the plunger of a syringe pump. Alternatively, the syringe pump could pull liquid through the device with the target fluids not traveling to the point of reaching the syringe barrel. The syringe may be re-usable or disposable. The syringe may be integrated in the fluid path or connected at the time of use.

Fluid control may be open-loop or may have closed-loop feedback control.

Pumping systems established to date have several weaknesses for controlling flow rate accuracy and precision, and may have performance limitations around controlling stable non-pulsing flows. Controlling of fluid pulsing for the electroporation device is most preferably controlled on the time frame of less than 30 seconds, more preferably less than 10 seconds, and most preferable faster than 1 second. Pulsing control is better than that of 20% for the given time period mentioned in the latter.

For the electroporation device, some preferred embodiments may comprise the peristaltic pump mechanism and or a gas pressure pump-based mechanism. Both types may operate to pull or push liquid. Traditional peristaltic pumps suffer from high pulsing delivery because the fix rate of mechanical contact on the pump tube via rollers (or linear compression mechanisms) which continuously alter the cross-section area by compressing the tube resulting in tube ID change. Pulsing results from the cross-sectional change of the tube ID. Additionally, peristaltic pumps suffer from accuracy issues that result from tubing compliance changes and tube wear characteristic changes over time and use. This wear is difficult to be compensated or adjusted for without direct measurement of the fluid flow rate or measuring the output with a balance or volumetric measurement. Measuring liquid flow rate with a balance is not preferred, as then an additional instrument must be added that requires an adequate environment (e.g., temperature, humidity, vibration, and space). Also, the fluid path then becomes dependent on access to the relatively large footprint/ space requirement of a balance.

Pressure pumps deliver relatively non-pulsatile flow but can suffer from accuracy issues because of fluid path dimensional tolerances, viscosity and temperature changes (fluid and ambient temperature), and liquid height changes as vessels are emptied and filled. Measuring liquid flow rate with a balance is not preferred as then an additional instrument must be added that requires an adequate environment (e.g., temperature, humidity, vibration, and space). Also, the fluid path then becomes dependent on access to the relatively large footprint/ space requirement of a balance.

To counter these limitations a flow-sensor may be used to provide closed-loop feedback to the liquid displacement mechanism. Here is proposed the addition of a fluid flow rate sensor (in line with system components) to measure the flow rate in near-real-time with the ability to provide feedback to the fluid displacement mechanism. For example, a flow rate sensor with a peristaltic pump or a gas pressure control system acting on a fluid vessel. The flow rate sensor may control the fluid displacement continuously or intermittently. The sensor may also be used to measure the flow rate as a check in the case of open-loop operation.

Most preferably, in some embodiments, the sensor does not contact the fluid and is not in communication with the device, tubing, or conduit.

The sensor may be reusable where it is used in conjunction with a disposable fluid component s). Or the sensor maybe disposable.

Most preferably, the two types of sensor that may be used include, but are not limited to: (1) ultrasonic-based sensor that is in communication with the fluid path (non-contact), which sensor is in communication with a component the liquid is traveling through; and (2) thermal flow sensor that is in communication with the fluid path (non-contact), which sensor is in communication with a component the liquid is traveling through.

The sensors may be re-used where they temporarily interfaced with a fluid path component that is to be changed, or the sensor may be part of the path and be disposable in nature. In some embodiments, the disposable sensor is integrated in the fluid path.

Interfacing of the liquid entering the device may occur via one or more components, such as a tube or conduit, and/or a fluid interface. The fluid component may comprise one or more features that allow for distributing or altering the flow profile and path of the fluid. This component may a wetted path where the cross-section area and shape may be varying from that of the fluid component exiting cross sectional area or shape. The fluid path change may be part of an assembly or may be molded as part of the electroporation device.

This may include geometric shape(s) that redistribute or format the liquid flow from the tube conduit to a format that is compatible with the device inlet. This architecture of the fluid path depends on the incoming fluid source tubing, fitting, or fluid interface as well as the device fluid inlet shape.

The fluid interface component may be composed of one or more fluid paths and is not limited to the location or number of inlet or outlet features.

A fluidic interface may serve to allow for various formats and types fluid components to make a fluid seal to the microfluidic device inlet. The device inlet may be a circular shape or may have a non-cylindrical geometry or shape. A fluidic interface component may for example allow for one or more incoming fluid lines or conduits to connect to the fluidic interface inlet where the fluid may then traverse a changed cross-section or geometric shape, followed by the fluid exiting the fluidic interface in a cross-section or shape that matches the device fluid inlet geometry. The fluid device inlet geometry would correspond to the fluid interface component output geometry. For example, the fluidic interface may serve to allow for a traditional tube to then supply fluid to a split on the device. Interfacing the fluid can be accomplished in many ways (e.g., different geometric shapes for different types of conduits/tubes), which are available to a person of ordinary skill in the art.

The microfluidic device 100 includes an inlet channel 209a that is substantially vertical, and an outlet channel 209b that is substantially vertical. In various embodiments the intlet channel 209a and the outlet channel 209b are disposed at an angle to the channel 204 and fluidly connected thereto. In various embodiments, the input channel 209a and the outlet channel 209b are generally planar and disposed parallel or coextensive with the channel 104. In various embodiments, the channel 204 is coupled to the inlet channel 209a and the outlet channel 209b and is substantially planar. In various embodiments, the width of the channel 204 is larger than the droplet size and the thickness, /, is sufficiently small to allow only one droplet at a time in the thickness dimension. In various embodiments, the channel 204 has a length, L. The fluid can flow through the channel at a rate of 0.1 cm/s, with a relevant range of flow rate between 0.001 cm/s and 10 cm/s. The volume of fluid flowing through the channel relates to the cross-sectional area of the flow channel. For example, for a channel 2 cm wide and 100 micrometers high, the volumetric flow rates would be in the range of from about 0.2 microliters/s to 2 milliliters/s.

The use of multiple inputs of fluid can prevent various types of fouling or contamination. For example, the molecules or nucleic acids to be inserted into the cells can exist in a separate solution from the droplets. This can be useful because certain molecules, like RNA, may not be stable in the vicinity of living cells due to enzymes on the cell surface or cell culture media. Also, it is known that degradation of the electrodes can result in the release of contaminants that are toxic to cells. The separate fluid layers, such as the flow media and the droplets, may ensure that the cells remain free from contaminants from the electrodes. Further, the cells themselves are kept out of contact with both the surface of the support block and the electrodes by containment within the droplets, thereby preventing possible contamination.

In some embodiments, using separate fluid streams allows maintaining different components in different media optimal for them for a longer time. For example, one fluid stream can be utilized to form the droplets that contain the cells to be electroporated. The fluid of the droplet can contain the cells in a medium that is optimal for them (e.g., for their survival) before electroporation. The droplets may be allowed to mix with an electroporation medium during the actual electroporation time window, such as the region proximate the one or more electrodes or electrode pairs. The embodiments disclosed herein thus allow dynamically controlling the chemical environment of the cells and the reagents to be electroporated into the cells separately, for example as a function of time and/or position within the fluid channel.

In various embodiments, the microfluidic device may be formed of a transparent polymer (e.g., poly dimethylsiloxane). In various embodiments, the device also includes one or more droplet forming structures or structures in the same microfluidic device or a mechanism for coupling to droplet forming structures on a separate device.

In various embodiments, the flow after electroporation is directed to an output for collection or coupled to one or more additional devices for analysis or processing. The processing could include, for example, addition of additional chemicals, application of heat or application of light. As each droplet 205a-205f passes through the pair of electrodes 210a, 210b, the droplet completes the circuit between the top electrode 210a and the bottom electrode 210b allowing an electric potential difference to be generated therebetween. Thus, electrical energy is provided only to the droplet(s) passing through the pair of electrodes 210a, 210b. In various embodiments, for a given flow rate, the length of the electrode may be selected such that a flow-through time of the droplet corresponds to at least the time necessary for electroporation to occur in the one or more cells disposed therein.

In various embodiments, as shown in Figs. 2A and 2B, microfluidic devices 200 are provided for droplet electroporation and for forming droplets. In various embodiments, microfluidic devices 200 are provided for performing electroporation. In various embodiments, the microfluidic devices 200 include a mechanism to flow the droplets into a device for subsequent processing or analysis. In various embodiments, the droplet forming device produces droplets in a surrounding fluid-flow medium wherein said droplet comprises a fluid different than the surrounding fluid-flow medium and at least one biological material and zero, one or multiple cells. In various embodiments, the device that is capable of introducing a flow from the droplet formation device into the channel where electroporation takes place containing at least one pair of electrodes disposed across the channel. In various embodiments, the microfluidic device includes an outlet allowing passage into a subsequent analytical device for collection after electroporation.

In various embodiments, a microfluidic device 200 is provided for droplet electroporation that includes a channel having a channel length and a channel thickness and a channel width, wherein said thickness allows only one droplet to fit in the thickness dimension of the channel. In various embodiments, the microfluidic device is capable of creating droplets wherein said droplet comprises a fluid different to the surrounding fluid, at least one biological material, and zero, one or multiple cells in a surrounding medium. In various embodiments, the device is capable of introducing a flow from the droplet formation device into the channel where electroporation takes place containing at least one pair of electrodes disposed across the channel. In various embodiments, the microfluidic device includes an outlet for allowing passage into a subsequent analytical device for collection after electroporation.

After electroporation, a mixture of all the fluid streams can leave the device via one or more outlet. The solution may be transferred to sterile polymer bags, T-flasks, conical tubes, media bottles, well plates, or the like and allowed to recover at 37 °C. The cells may then be re-suspended in standard tissue culture medium and plated for immediate use in cellular assays, cryopreserved for future use, or used as desired. Post Electroporation Cell Manipulation: After electroporation, cells may be moved to an additional region in the device for secondary processing or transferred. The cells may be transferred from the device fluid outlet (or fluid interface component) to a sterile, multi-well dish or vessel and exposed to a secondary set of conditions. For example, to be exposed to for 30-40 minutes at 37°C. The cells are suspended in cell medium and either cultured for immediate use or cryopreserved.

In various embodiments, the microfluidic device provides improved and more efficient electroporation by directing electrical current primarily through the more electrically conducting droplet that is surrounded by a fluid medium of lower electrical conductivity. For example, the droplet may consist of an aqueous salt solution surrounded by less-conducting (e.g., non-conductive) oil. In this example, most of the electrical current passes through the droplet rather than the surrounding less-conducting medium (e.g., non-conductive oil), thus reducing the power dissipation that creates undesirable heat. The reduced electrical current also reduces undesirable electrochemical processes such as hydrolysis at the electrode surfaces.

In various embodiments, microfluidic devices for droplet electroporation include a channel having a channel length and a channel thickness and a channel width, wherein said thickness and width are sufficiently small to allow flowing droplets through the channel in a single file; an inlet for introducing a flow of a flow medium and a droplet into the channel, wherein some droplets droplet comprises at least one cell and at least one biological material; an outlet for allowing collection of the cell after it is electroporated to contain the biological material; and at least one pair of electrodes disposed across the channel. In various embodiments, said pair of electrodes comprise an electrode length that is greater than the average spacing between drops, permitting more than one drop at a time to reside between the electrodes. In various embodiments, the pair of electrodes comprise an electrode length that is smaller than the average spacing between drops, permitting on average only one drop at a time to reside between the electrodes.

Further advantages occur when the width and thickness of the channel and length of the electrode are sufficiently small to permit only one droplet at a time to fit between the electrodes. This condition allows electroporation to be addressed to individual droplets and to trigger a time varying voltage for electroporation based on the detected position of the droplet. In various embodiments, methods are provided for triggering the application of the applied voltage waveform based on the arrival of a droplet at an electrode in a flowing system. In various embodiments, using an electrode configuration and electrical operation in the disclosed electroporation chip, the applied electroporation voltage applied to each droplet can be essentially identical, permitting a more uniform electroporation condition for the cells. In various embodiments, the microfluidic devices advantageously trigger the applied voltage in multiple parallel channels upon the passing of a droplet through the respective electrode.

In various embodiments, the channel thickness and width are uniform across the channel length. In various embodiments, the channel thickness or channel width may be non- uniform along the channel length. In various embodiments, the channel may narrow in any dimension along the flow direction of the fluid and drops. In various embodiments the channel may widen along the flow direction of the fluid and drops. In various embodiments the channel may be configured to direct drops within the channel, placing them in an optimal position between the at least one pair of electrodes. In various embodiments, the channel may be configured to direct droplets in single file order, should they not be staggered or in single file already, for example, a slight taper, shoulder, protrusion or other feature may push or direct drops to a desired location within the channel, such as the center line of the flow path. In various embodiments, the channel thickness is at least 10 micrometers and at most 900 micrometers. In various embodiments, the channel width is at least 10 micrometers and at most 900 micrometers. In various embodiments, the thickness or width may be smaller than the droplet diameter at one or more locations along the channel length and the electrodes may be placed at any position along the channel length.

In various embodiments, the pair of electrodes also senses the presence of a droplet between the electrodes, thereby triggering the delivery of the electrical voltage waveform. In various embodiments, the pair of electrodes senses the presence of the droplet based on the droplet having a higher conductivity than the surrounding flow medium. In various embodiments, the pair of electrodes senses the presence of the droplet based on the droplet having a different conductivity than the flow medium. In various embodiments, the pair of electrodes senses the presence of the droplet based on the droplet having a lower conductivity than the flow medium. In various embodiments, the pair of electrodes senses the presence of the droplet based on the droplet having a higher resistance than the flow medium. In various embodiments, the pair of electrodes senses the presence of the droplet based on the droplet having a lower resistance than the flow medium. In various embodiments, the pair of electrodes senses the presence of the droplet based on the droplet having a different resistance than the flow medium. In various embodiments, the pair of electrodes senses the presence of the droplet based on the droplet having a different electrical permittivity than the flow medium. In various embodiments, the pair of electrodes senses the presence of the droplet based on the droplet having a lower permittivity than the flow medium. In various embodiments, the flow medium is a non-conductive oil. In various embodiments, the non- conductive oil is selected from hexadecane, silicone oil, FC-70, or equivalent. In some embodiments, the flow medium is a non-polar fluid.

In various embodiments, the microfluidic device further comprises a sensing module for sensing the presence of a droplet upstream of said pair of electrodes, wherein said sensing module triggers the pair of electrodes to deliver the electrical voltage waveform. In various embodiments, the sensing module is another pair of electrodes. In various embodiments, another pair of electrodes senses the presence of the droplet based on the droplet having a higher conductivity than the flow medium. In various embodiments, another pair of electrodes senses the presence of the droplet based on the droplet having a lower conductivity than the flow medium. In various embodiments, the pair of electrodes senses the presence of the droplet based on the droplet having a lower conductivity than the flow medium. In various embodiments, the pair of electrodes senses the presence of the droplet based on the droplet having a higher resistance than the flow medium. In various embodiments, the pair of electrodes senses the presence of the droplet based on the droplet having a lower resistance than the flow medium. In various embodiments, the pair of electrodes senses the presence of the droplet based on the droplet having a different resistance than the flow medium. In various embodiments, another pair of electrodes senses the presence of the droplet based on the droplet having a higher permittivity than the flow medium. In various embodiments, another pair of electrodes senses the presence of the droplet based on the droplet having a lower permittivity than the flow medium. In various embodiments, the flow medium is a non- conductive oil. In various embodiments, the non-conductive oil is selected from hexadecane, silicone oil, FC-70, or equivalent. In some embodiments, the flow medium is a non-polar fluid.

In various embodiments, the sensing module is a diode laser and imaging device that detects light scattering from the droplet. In various embodiments, the laser may be aimed across the channel such that the drop is detected upon crossing the laser during flow past the location along the length of the channel where the diode is disposed. In various embodiments, the laser is directed at an angle relative to the channel. In various embodiments, more than one laser are disposed in the same orientation relative to the channel. In various embodiments, the one or more laser are disposed in differing orientations from each other relative to the channel. In some embodiments, said sensing module is a fluorescence-based detector that detects a fluorescent label within the droplet. In various embodiments, the sensing module is an optical detector that detects the droplet via image-processing. In various embodiments, the sensing module is a video camera. In various embodiments the sensing module is an infrared camera. In various embodiments the sensing module is an optical sensor configured to capture images in the visible spectrum. In various embodiments, the sensing module is an optical sensor configured to capture images in the non-visible spectrum. In various embodiments the sensing module is a sensor configured to detect a change in pressure in the flow medium associated with one or more drops. In various embodiments the sensing module is configured to detect a change or threshold flow rate associated with the presence of one or more droplets. In various embodiments, the sensing module detects a change due to a change in refractive index, thereby detecting the presence of one or more droplets in the flow medium. This change may occur from the chemical makeup within the droplet or the droplet relative to the bulk carrying stream.

In various embodiments, the wavelength range of light for the source and detection is between about 175 nm to about 3300 nm.

In various embodiments, the microfluidic device further comprises a collectiontriggering sensing module for sensing the presence of a droplet downstream of said pair of electrodes, wherein said collection-triggering sensing module triggers a collection system to receive the droplet from the outlet. In various embodiments, the collection system comprises a multi-well plate. In various embodiments, similarly to how the sensing module is for the initial droplet detection, the sensing module can be another pair of electrodes, a diode laser, a fluorescence-based detector, or another optical detector.

In various embodiments, methods of electroporating a cell comprise running a flow medium and at least one droplet comprising one or more cells and one or more biological molecules through any of the microfluidic devices disclosed herein; and collecting the one or more cell after it is electroporated to contain the biological molecule. In various embodiments, droplets may not contain any cells and/or biological material.

In various embodiments, the methods further comprise adjusting or selecting a flow rate for running the flow medium. In various embodiments, the methods further comprise selecting a droplet size for the droplet. In various embodiments, the methods of selecting an electroporation condition for droplet electroporation comprise varying the flow rate in the method of electroporating a cell. In various embodiments, the methods further comprise collecting cells using any of the described collection-triggering sensing modules.

In various embodiments, the microfluidic device may contain a plurality of parallel fluid channels that each contain their own pair of electrodes for either sensing or triggering on droplets within that channel. In various embodiments each a plurality of parallel fluid channels includes an input, an output, one or more pairs of electrodes, one or more sensing modules and any other component as described in reference to a single channel. In various embodiments each of the plurality of fluid channels includes the identical configuration. In various embodiments, each of the plurality of fluid channels includes at least one variation configuration, wherein at least one component is different than the plurality of configurations. In various embodiments, each of the parallel fluid channel includes a different configuration, that is to say each may include a different sensing module type, for example. In various embodiments, the electrodes are connected to a common input so that any may be selected by use of a multiplexer for applying the electroporating voltage waveform in the appropriate fluidic channel. In various embodiments, if any unwanted electric field is generated between the fluid channel that is triggered and in an adjacent fluid channel kept at ground, this effect can be minimized by having the distance between the parallel electrodes be greater than the distance between the electrode pairs that are on opposite sides of a given fluid channel (i.e., across the thickness of that channel). In various embodiments, for sensing, optical systems could be used to focus the diode laser on individual spots within each parallel fluid channel or shape the laser into a beam that is focused across all the fluid channels. In various embodiments, a separate photodetector could be used to sense scattered or fluorescent light from each fluid channel or an array of photodetectors could be used. In various embodiments, the sensing module in one or more channel is a diode laser and imaging device that detects light scattering from the droplet. In some embodiments, the sensing module in one or more channel is a fluorescence-based detector that detects a fluorescent label within the droplet. In various embodiments, the sensing module in one or more channel is an optical detector that detects the droplet via image-processing. In various embodiments, the sensing module in one or more channel is a video camera. In various embodiments the sensing module in one or more channel is an infrared camera. In various embodiments the sensing module in one or more channel is an optical sensor configured to capture images in the visible spectrum. In various embodiments, the sensing module in one or more channel is an optical sensor configured to capture images in the non-visible spectrum. In various embodiments the sensing module in one or more channel is a sensor configured to detect a change in pressure in the flow medium associated with one or more drops. In various embodiments the sensing module in one or more channel is configured to detect a change or threshold flow rate associated with the presence of one or more droplets. In various embodiments, the sensing module in one or more channel detects a change due to a change in refractive index, thereby detecting the presence of one or more droplets in the flow medium. This change may occur from the chemical makeup within the droplet or the droplet relative to the bulk carrying stream.

In various embodiments, the droplets are created in the same microfluidic device containing the electrodes for triggering and sensing the droplets. In various embodiments, the droplets may be formed outside the device and introduced into the channel after formation. In various embodiments, at least one droplet is formed outside the device and introduced into the channel including a plurality of droplets formed within the device.

In various embodiments, an additional marker (such as a fluorescent dye) may be added to the droplet in addition to the electroporation cargo. In various embodiments, another optical sensor could be used to detect changes in the droplet fluorescent intensity after the electroporation voltage waveform has been applied to determine whether the marker was also successfully electroporated into the cell. In various embodiments, these cells may be collected into an output fluid reservoir.

Fig- 3 illustrates a microfluidic device 300 for droplet electroporation with two pairs of electrodes (first pair of electrodes 310a, 300b and second pair of electrodes 312a, 312b) where the length and width of the channel 304 are sufficiently narrow to cause the droplets to move in single file. In various embodiments, the first pair of electrodes 310a, 310b include an arbitrary voltage waveform (e.g., a constant voltage) applied between the electrodes. In various embodiments, the device includes a pair of electrodes 312a, 312b that can sense changes in conductivity due to a drop arriving at the electrodes 312a, 312b and trigger the delivery of a predetermined voltage waveform applied on the next electrode pair 310a, 310b for electroporation. Thus, the droplet receives a precise predetermined voltage waveform of correct phase. In various embodiments, each pair of electrodes has an electrode length (measured along the length of the channel) of about 0.1 mm to about 20 mm. In various embodiments, the pairs of electrodes extend the entire width of the channel 104.

In various embodiments, as shown in Fig. 3, droplet-triggered electroporation involves using an electrode pair to both 1) trigger and 2) deliver a predetermined electrical voltage waveform. In various embodiments, the predetermined voltage waveform can be triggered without a trigger electrode (applying a trigger voltage). In various embodiments, the channel of the microfluidic device is narrowed such that droplets are forced to flow through the electrodes single file. In various embodiments, triggering the predetermined voltage waveform is performed in channels having a width configured to allow droplets to pass through in a single file. In various embodiments, the cells and cargo to be delivered are encapsulated into aqueous drops within non-conductive oil. In various embodiments, the resulting emulsion flows in between the electrode pair. In various embodiments, as a conductive droplet contacts the electrode pair, it changes the conductance of the circuit and acts as a trigger for an electrical voltage to the electrode pair. In various embodiments, the electrodes may not contact the liquid (e.g., the electrodes may be positioned at a predetermined distance from the liquid). In various embodiments, the electrodes may not directly contact the droplet or plurality of droplets. In various embodiments, the electrodes may contact at least one of a plurality of droplets. In various embodiments, the electrodes may contact droplets of a certain or variable size threshold. In various embodiments, the electrodes are disposed in the channel or proximate the channel at a variable distance. In various embodiments, the electrodes apply a substantially direct current (DC) e.g., the voltage waveform is substantially constant, which includes an AC to DC converted voltage). In various embodiments, the electrodes apply an alternating current (AC). In various embodiments, minimal current is required to sense the presence of a droplet at the electrode pair and enables precise delivery of a predetermined voltage waveform triggered by the electrical signal generated as the droplet passes through the second pair of electrodes 312a, 312b. In various embodiments, the electrodes are configured to deliver a predetermined voltage waveform. In various embodiments, the electrodes are configured to deliver a variable voltage waveform. In various embodiments, the electrodes are configured to deliver a voltage waveform based on a measured parameter of the droplet. In various embodiments, the electrodes are configured to deliver a voltage waveform based on more than one parameter of the droplet. In various embodiments, the electrodes are configured to deliver a predetermined voltage waveform a predetermined number of cycles. In various embodiments, the electrodes may be configured to deliver a predetermined number of voltage waveforms to each droplet.

In various embodiments, droplet size and flow speed determine the temporal resolution for triggering, but electrical triggering can occur on the order of nanoseconds which exceeds the requirements for effective triggering. In various embodiments, the flow speed also determines the transit time of the droplet underneath, between, over, or otherwise proximate the electrode. In various embodiments, the rate that droplets pass through the channel can be modulated to match the desired testing capacity. Triggered electroporation of droplets enables multiple advantages over the prior art including 1) precise delivery of an arbitrary voltage waveform of correct phase to single cells or small groups of cells and 2) triggering an electrical voltage limits the amount of time the electrode pair is energized for electroporation. The correct voltage waveform may be modulated or configured to precisely electroporate the droplet that triggered or is located proximate the electrode or electrode pair, in embodiments.

In various embodiments, as shown in Fig. 3, droplet-triggered electroporation is performed using two electrode pairs such that one electrode pair acts as a trigger for the second electrode pair to deliver an electrical voltage. In various embodiments, the first electrode pair can be narrow and positioned immediately upstream of the second electrode pair. As a conductive droplet contacts the first electrode pair, the droplet would again change the conductance of the circuit and act as a trigger for an electrical voltage to the second electrode pair. In various embodiments, as the droplet enters the second electrode pair, the droplet will receive a precise electrical voltage, triggered by the first electrode pair. In various embodiments, dedicating an electrode pair to sensing the presence of a droplet enables the second electrode pair to only be energized during voltage waveform delivery and may further reduce unwanted electrochemistry or prolong electrode life.

In various embodiments, the triggering produced by the droplet completing the circuit within the electrode pair is replaced by a diode laser and imaging device that detects scattering by the droplet. In various embodiments, the trigger is provided by optical information such as image-processing recognition that a cell has entered the field of view. In various embodiments, the triggering is through fluorescent imaging and recognition of fluorescent markers purposefully encapsulated with the cells/cargo.

Fig- 4 illustrates a microfluidic device 400 for droplet electroporation with two pairs of electrodes (first pair of electrodes 410a, 410b and additional pair of electrodes 414a, 414b) and an output receptacle 408. The microfluidic device 400 is similar to those devices shown in Figs. 2A, 2B, and 3. The microfluidic device 400 includes an inlet 302, an outlet 403, channel 404, droplets 405a-405f, and a first pair of electrodes 410a, 410b for delivering an electric potential difference as the droplet (e.g., droplet 405d) passes through. In various embodiments, the additional pair of electrodes 414a, 414b is used for quality control.

As shown in Fig. 4, another electrode pair 414a, 414b is included after the electroporation electrode 410a, 410b to detect the presence of the droplet and trigger an automated collection system 408 to move over a new receptacle to receive the electroporated droplet 407. In various embodiments, the outlet 403 includes an output nozzle 406 configured to interface with the collection system 408. In various embodiments, the automated collection system 408 deposits individual processed droplets 407 within wells of a culture plate (e.g., 96-well culture plate). As the droplet enters the last electrode pair, the change in electrical conductivity is detected and the cell(s) is/are counted. After a predetermined number of droplets have been processed the output nozzle is moved to a different area of the output receptacle e.g., another well of a multi -well plate). Rather than moving the nozzle relative to the collection receptacle an electrostatic field can direct the flow to another receptacle in a manner analogous to a cell sorter. In various embodiments, the post-processing electrodes can also be used to detect the presence of a cell in the droplet or to sense electrical modifications to a cell or cells in the droplet. One of skill in the art will understand that the number of wells in the plate is not limited and includes known commercially-available formats such as 24, 48, 96, 384 and 1536-well plates. In various embodiments, integration of triggered electroporation with an automated collection cycle triggered by another electrode pair enables a closed-loop system that could rapidly screen cell response to a large number of arbitrary voltage waveforms.

Fig- 5 illustrates a microfluidic device for triggered electroporation operating with plug flow. In various embodiments, a microfluidic device capable of inserting a biologically active molecule into a living cell includes a fluid channel having a fluid input and a fluid output configured to allow plug fluid flow as described herein. One of skill in the art would appreciate that the description herein allows for a non-limiting amount and arrangement of fluid inputs and outputs to facilitate a flow of a flow medium and droplets therein. The plug fluid flow has at least a first plug comprising a first fluid and a second plug comprising a second fluid. In various embodiments, the first plug may be delivered to the microfluidic device by a first fluid input and the second plug may be delivered to the microfluidic device by a second fluid input. The arrangement of the fluid inputs may allow for alternative delivery of the first and second plug fluid flows, thereby providing alternating plugs of fluid within a channel of the microfluidic device. In various embodiments, the alternating fluids may be provided simultaneously, serially, or a combination of both to at least two channels as described herein. The first plug and the second plug alternate along a length of the channel. The first fluid has a different fluid property from the second fluid such that the first and second plugs remain substantially separate during plug fluid flow. In various embodiments, the first and second plug fluids have a measureable conductivity difference, thereby providing an identifiable characteristic to one or more sensors, such as a pair of electrodes. In various embodiments the first and second plug fluids may be miscible fluids. At least one of the first plug and the second plug includes one or more living cells and one or more biologically active molecules.

As shown in Fig. 5, plug flow having two or more alternating plugs (e.g., a low- conductivity electroporation buffer, a high conductivity media) of fluid. In various embodiments, plug flow is an alternative to forming droplets (e.g., aqueous droplets in a non- conductive oil flow medium). At least one of the plugs includes one or more cells and one or more biological material to be electroporated into the one or more cells. In various embodiments, segments of plug flow may be utilized in addition to droplets suspended in an oil flow medium. In various embodiments, the cells and/or biological material are disposed within one type of fluid of the two or more plugs. For example, in a plug microfluidic device having two plugs of fluid alternating with one another, one plug has the cell(s) and molecules to be electroporated and an adjacent plug includes a different fluid, such as a non-conducting oil or an aqueous medium with no cells and electrical conductivity different than the plug with the cells and molecules. That is to say that a first plug including the cells and the biological material would be sandwiched between a second plug with an identifiable parameter or characteristic. A plug, as used herein, is a series of different (e.g., alternating) fluid compositions (e.g., chemical and/or fluid property difference). Each fluid composition has a respective length along the channel and substantially fills the entire thickness and the width of the channel defining a respective plug volume for each different fluid composition in the plug flow. In various embodiments, as the plug (containing, for example, cells and cargo diluted in low conductivity electroporation buffer) enters the electrode pair, the current decreases below a threshold value and triggers an electrical voltage to the electrode pair. In various embodiments, the aqueous solution and non-conductive oil are replaced by two miscible aqueous solutions of significantly different conductivities. In various embodiments, the cells/cargo in low conductivity electroporation buffer are injected as plugs into a fluid flow of high conductivity media lacking cells or cargo. In various embodiments, the electrode pair triggers when the current decreases below a threshold value. In various embodiments, the triggering is based on optical, fluorescent, or scattering methods. In various embodiments, the first or second plug flow is intermittently injected in a continuous flow of the other of the first and second plug flow into the microfluidic device channel. In various embodiments, diffusion between the solutions would limit the temporal resolution of the triggering apparatus. In various embodiments, exclusive use of aqueous solutions reduces known cytotoxic effects of oil contact with cells. Fig- 6 illustrates an exemplary voltage waveform used for electroporation. As used herein, “voltage” and “electric potential difference” are interchangeable. In various embodiments, applied voltage waveforms have a maximum amplitude of about 1 V to about 80 V. In various embodiments, the applied voltage waveforms have a maximum amplitude of about 1 V. In various embodiments, the applied voltage waveforms have a maximum amplitude of about 2 V. In various embodiments, the applied voltage waveforms have a maximum amplitude of about 3 V. In various embodiments, the applied voltage waveforms have a maximum amplitude of about 4 V. In various embodiments, the applied voltage waveforms have a maximum amplitude of about 5 V. In various embodiments, the applied voltage waveforms have a maximum amplitude of about 6 V. In various embodiments, the applied voltage waveforms have a maximum amplitude of about 7 V. In various embodiments, the applied voltage waveforms have a maximum amplitude of about 8 V. In various embodiments, the applied voltage waveforms have a maximum amplitude of about 9 V. In various embodiments, the applied voltage waveforms have a maximum amplitude of about 10 V. In various embodiments, the applied voltage waveforms have a maximum amplitude of about 15 V. In various embodiments, the applied voltage waveforms have a maximum amplitude of about 20 V. In various embodiments, the applied voltage waveforms have a maximum amplitude of about 25 V. In various embodiments, the applied voltage waveforms have a maximum amplitude of about 30 V. In various embodiments, the applied voltage waveforms have a maximum amplitude of about 35 V. In various embodiments, the applied voltage waveforms have a maximum amplitude of about 40 V. In various embodiments, the applied voltage waveforms have a maximum amplitude of about 45 V. In various embodiments, the applied voltage waveforms have a maximum amplitude of about 50 V. In various embodiments, the applied voltage waveforms have a maximum amplitude of about 55 V. In various embodiments, the applied voltage waveforms have a maximum amplitude of about 60 V. In various embodiments, the applied voltage waveforms have a maximum amplitude of about 65 V. In various embodiments, the applied voltage waveforms have a maximum amplitude of about 70 V. In various embodiments, the applied voltage waveforms have a maximum amplitude of about 75 V. In various embodiments, the applied voltage waveforms have a maximum amplitude of about 80 V. In various embodiments, the duration of the voltage waveform ranges from about 10 ps to about 5000 ps. In various embodiments, the duration of the voltage waveform may correspond to the amount of time a droplet takes to traverse the electrode length. In various embodiments, voltages for the voltage waveform ranges from about 0.1 V to about 5 V. In various embodiments, the duration ranges from about 1000 ps to about 10,000 ps. More complex voltage waveforms than the one pictured here are contemplated. Fig. 6 illustrates that the voltage waveform may have variable voltage levels during one period of the voltage waveform.

Fig- 7 illustrates a top view of a microfluidic device for droplet electroporation with multiple parallel channels where electrodes for each channel are controlled by a multiplexer. In particular, Fig. 7 illustrates a droplet-triggered electroporation device with pairs of electrodes in multiple parallel fluidic channels. The electrodes are connected to a common electrical input where the active electrode pair is selected by use of a multiplexer. In various embodiments, the electrodes are on opposite sides of the fluidic channels (located across the thickness in one dimension of the fluid channel).

In various embodiments, as shown in Fig. 7, the microfluidic chip contains parallel flow channels, each containing at least one electrode pair located on opposite sides of the given flow channel. In various embodiments, each flow channel has dimensions such that the droplets travel along the flow direction in single file. In various embodiments, the concentration of the droplets is maintained such that the average spacing between the droplets is larger than the width of the electrode dimension along the direction of the flow, ensuring that on average only a single droplet is in the volume between an electrode pair in a given flow channel. In various embodiments, each electrode pair is capable of detecting the presence of a droplet, in one of the manners previously described due to a change in the conductivity, refractive index, permittivity, or other, of the fluid volume between an electrode pair. In various embodiments, each pair is capable of delivering a voltage waveform sufficient to electroporate the cell or cells within the droplet. In various embodiments, another sensing module previously described is used to determine the presence of a droplet in the volume between an electrode pair. In an embodiment, all the electrodes on one side of the fluid channel are connected to a multiplexer that is connected to a single voltage controller and a logic device. In various embodiments, the logic device may be used to select the given electrode pair to which the voltage waveform is applied at a time suitable based on the passage of a droplet through that electrode pair’s volume. In various embodiments, multiple pairs of electrodes are included in each parallel fluid channel. In various embodiments, one pair of electrodes is used to determine the presence of a droplet and a second pair of electrodes is used to deliver a voltage waveform. Manufacturing the device

A person of ordinary skill in the art would understand that there are many ways of fabricating the device or parts thereof using various materials known in the art. Exemplary methods of fabricating the device are presented herein.

Some embodiments of the device are constructed from a three-layer stack of polymer substrates or plastics. All three layers may be laser cut with a small beam spot, high resolution CO2 laser. The layers on which the electrodes are fixed may be cut from 1 mm thick acrylic slabs, creating opposite surfaces of the channel. A middle layer defines the distance between the electrode pairs. In some embodiments, the three dimensions of the layers are the same. In preferred embodiments, the central layer that defines the channel height is much thinner than the outer two layers on which the electrodes are deposited and which provide the mechanical stability of the device. Although it is most practical for the layers to be the same in dimensions in the plane that the stream flows, these dimensions can be different from one another. One way to manufacture these layers is to use a laser to cut acrylic pieces similar in dimension to a microscope slide 25 x 75 mm, add fluid inlet slits or ports and add alignment holes to facilitate assembly. A thin film electrode (50 nm) of a gold (Au) is deposited by physical vapor deposition through a shadow mask on the inside surface of each acrylic piece. The middle layer polymer film with medical adhesive on each side is cut to shape and receives the corresponding alignment holes via the laser cutting process. After laser cutting, the three pieces are placed on a jig containing alignment pins corresponding to the alignment holes in each layer. The sandwich assembly is then compression-bonded in a press. This two-step process of laser cutting and compression assembly is amenable to mass production and allows for a cost-effective consumable to be created. The process can be used to manufacture hundreds of thousands of devices per year. This contrasts with many other types of standard non-electroporation microfluidic devices that typically require expensive capital equipment and a large number of chemical processing steps.

Alignment of the device layers may be conducted by optical positioning or a physical means such as alignment pins or structures. The device layers may have receiving features for use with a jig alignment piece or system. Alternatively, the alignment features may reside in the device layers as so no jig or peripheral alignment system is necessary. These may include pin-like structures or features that snap together. The flow cell could also be produced by an injection molding process to form one or more of the three layers, where the volume can scale to millions of single-use devices per year, using one injection molding press with a multi-cavity mold.

This disclosure allows for architectures for manufacturing the device that are readily amenable to injection molding. In this device, all the layers may be formed via injection molding. The fluidic channel may be formed in one layer at full depth or, alternatively, the channel may span two or more layers, where the full depth is achieved upon assembly. Injection ports may be created via core pins. Alternatively, the fluid inlets may be added post molding as a secondary operation or structure. The layers may be molded from the planar surface or from the edges. Appropriate and efficient part release from the mold cavity is known in the art.

The molded layers may be assembled together through mechanical connection, adhesion, bonding, welding (including ultrasonic and laser), fusing, melting, or the like. Additionally, there may be another material between the layers for connection and sealing such as, but not limited to, a gasket, O-ring, washer, or the like. Alternatively, sealing can be achieved through press tight or bonding features.

Circular entrance ports can be connected with various fittings to conventional tubing such as that from an automated cell manufacturing platform. Low cost manufacturing methods are desirable because the flow cell and material that comes in contact with cellcontaining media should typically be discarded after one use to prevent cross-contamination. There are many ways to injection mold including using one mold or more than one over molding technique. Multiple layers may also be bonded post molding using, but not limited to, such techniques as ultrasonic, laser, thermal heat compression, adhesion, or alike.

In some embodiments, the fluid channel may reside in one layer and the opposing sealing structure is a non-injected molded part such as a film, tape, or planar material containing necessary fluid inlets.

In some embodiments, the device may be created by three-dimensional printing or additive manufacturing processes. Other fabrication techniques include compression molding, casting, and embossing.

In some embodiments, devices are made from glass via lithography and wet or dry etching. Alternatively, the devices may be physically machined via computer numeric control (CNC) or ultrasonic machining. In other embodiments, the devices can be made from various materials, such as, for example, where at least one layer is glass, where at least one layer is plastic, where one of the layers is optically transparent, or where the channel material is electrically insulating. Manufacturing the electrodes

The formation of patterned electrodes on the flow channel surface can be accomplished with a variety of readily available techniques and materials known in the art. Exemplary methods are presented herein.

One method is to use the process of sputtering for deposition of a metallic conducting layer such as gold, platinum, aluminum, palladium, other metals, or alloys of multiple metals. Gold-palladium is an example of a metallic alloy that can be used to compose the electrodes. The electrodes can be made of an optically transparent material to allow observation of the motion of the living cells in the fluid channel of the device. To generate transparent conducting layers, films of indium-tin oxide (ITO) are frequently used. After metal deposition, these conducting layers can be patterned by masking and etching to remove material where it is not wanted to form the desired patterned electrode shapes. Appropriate masks may be formed from photoresist using common photolithographic exposure processes.

One exemplary method for forming electrodes is to deposit electrically conducing films made of metals or other conducting layers such as ITO. By depositing them through a prepositioned mask, sometimes called a shadow mask, the masks are positioned in proximity to the surface to be coated so that the conducting layer reaches the surface only where previously opened regions have been formed in the mask. In addition, a related technique called “lift-off’ can be used, in which a patterned photoresist layer can be used to shape the pattern of deposited conducing material. Another exemplary method for patterning deposited electrodes is ablation by laser or ion etching to remove metal to form the electrode pattern.

The deposition of layers of conducting ink can be performed by brushing or spraying, followed by heating to form patterned conducting films.

These thin film patterning processes are well known to those skilled in the art. In this case, the thickness of the films is desired to be in the range of from 5 nm to 5 micrometers, with a preferred range of from 10 nm to 100 nm.

In some embodiments of the device, electrodes can be formed by inlaying wires or metal bars in grooves formed in the support block instead of affixing the electrodes to the support blocks. In this embodiment, grooves are machined into the support block, for example, a plastic support block, and the electrodes are metal. The wires or bars can be formed of metals such as aluminum, nickel, copper, stainless steel, and may be gold plated. The wires or bars may be glued into the groove or held by a tight compression fitting.

Some embodiments of a system include an electroporation device, fluid delivery system including a pump, temperature control and optical and electrical monitor of the cells to obtain real-time feedback on the cell modification process. Feedback can be obtained by monitoring the electrical current passing between the two electrodes to provide information about living cell modifications, imaging of the living cells to provide information about living cell modifications or monitoring fluorescence of the living cells to provide information about living cell modifications.

Some embodiments include a system for inserting a biologically active molecule into a living cell, which system includes an electroporation device capable of performing a cell modification process including inserting a biologically active molecule into a living cell contained in a fluid flow by flowing fluid including living cells and biologically active molecules through a channel between two electrodes, each electrode disposed on opposite sides of the channel; passing the cells through a space between the two electrodes in a single layer so a living cell in the fluid flow is maintained in a similar position as other living cells in the fluid flow as they pass between the two electrodes; and applying an electric voltage across the two electrodes while the living cell is passing between the two electrodes in a manner that prevents one living cell from shielding another living cell from the applied electric field, in which the strength of the electric field to which the living cell is exposed is sufficient to form pores within the membrane of the living cell through which the biologically active molecule can traverse the cell membrane, but not lyse the living cell; a fluid delivery system including a fluid source and a fluid pump in fluid communication with the electroporation device; an electrical current source in electrical communication with the pair of electrodes; a temperature control in thermal communication with in the fluid flow; and an optical and electrical monitor of the living cell capable of obtaining real-time feedback on the cell modification process.

An advantage to the electroporation device is the ability to optically and electrically monitor the cells to obtain real-time feedback on the cell modification process. In some embodiments of the device,: a microfluidic electroporation system comprises an observation microscope. Accordingly, the fluid flow controller or voltage controller can be adjusted as required to optimize the process efficiency and cell viability. In some such embodiments, the microscope may be positioned so that it views a reservoir that contains biologically active material. For example, this could be nucleic acids. The fluid from input cell reservoir flows through the channel of the microfluidic electroporation device and across the field of view of the microscope, and into a cell collection reservoir, thus enabling the user make adjustments as necessary to improve the efficiency of transformation. As used herein, the cell collection reservoir refers to any vessels, bags, plates, dishes, or containers that are capable of collecting cells flowing out of the microfluidic device.

Temperature control of the solutions or materials in contact with the fluids may be implemented at any instance(s) in the system, including heating and cooling. This may include static control or temperature cycling.

The device can be interfaced to a fluid delivery system. A fluid delivery system (e.g., a pump) operating with a flow controller is configured to displace, preferably, indirectly displace, the fluid from the input cell reservoir to establish a fluid flow within the fluid path. The fluid displacement apparatus can provide positive and/or negative displacement of the fluid. The delivery pump includes mechanisms based on peristalsis, pneumatics (pressure displacement), hydraulics, pistons, vacuum, centrifugal force, manual or mechanic pressure from a syringe, and the like. Preferably, the fluid is indirectly displaced by the pump without the fluid directly contacting any of the moving parts of the apparatus, such as, for example, a peristaltic pump acting upon a fluid filled tube. Alternatively, a pneumatic displacement mechanism may be used where a head pressure displaces liquid from a pressurized vessel. Conversely, fluid may be directly displaced by an apparatus, when the fluid is displaced by directly contacting any of the moving parts of the apparatus, such as, for example, the plunger of a syringe pump.

In addition to common mechanical pumping mechanisms, such as syringe and peristaltic pumps, the fluid delivery means may include gravity driven or hydrostatic pressure driven liquid flow. Here the fluid containing vessel is positioned at a given height (relatively to the device fluid outlet) to provide the desired flow rate. The fluid height is chosen based on the overall system fluid restriction circuit (cross-sectional area, internal diameters, and lengths of the fluid path). In addition to the device’s internal flow path dimensions, external components such as tubing internal diameters may be chosen to obtain a desired restriction for controlling the flowrate.

The liquid containing vessel may accept application of controlled gas head-pressure to aid in the displacement of the liquid from the vessel to the device.

The fluid delivery system (e.g., pump) may include a flow sensor for monitoring the flow rate or the flow sensor may provide closed loop feedback to the pump control system. The closed loop feedback can ensure accuracy and reduce pulsing. The pump displaces fluid contained in flexible tubing to create a fluid stream. The system may operate with an inline flow sensor configured to directly measure directly the fluid flow rate as the fluid passes the sensor. The system, in some embodiments, includes a feedback control in communication with the fluid displacement apparatus and the inline flow sensor. The inline flow sensor measures the flow and communicates with a feedback control mechanism. Suitable types of flow sensor mechanisms include thermal pulse, ultrasonic wave, acoustic wave, mechanical, and the like. The inline sensor may be mechanical-based, electrical-based, motion-based, or microelectromechanical systems (MEMS)-based. The sensor mechanism may be thermal, ultrasonic or acoustic, electromagnetic, or differential pressure. One example of a sensor suitable for use in accord with the present disclosure is a thermal-type flow sensor where the sensor typically has a substrate that includes a heating element and a proximate heatreceiving element or two. When two sensing elements are used, they are preferably positioned at upstream and downstream sides of the heating element relative to the direction of the fluid (liquid or gas) flow to be measured. When fluid flows along the substrate, it is heated by the heating element at the upstream side, and the heat is then transferred non- symmetrically to the heat-receiving elements on either side of the heating element. Because the level of non-symmetry depends on the rate of fluid flow and that non-symmetry can be sensed electronically, such a flow sensor can be used to determine the rate and the cumulative amount of the fluid flow. This mechanism allows the flow to be measured in either direction. In preferred embodiments, the temperature sensors and the heating element are in thermal contact with the exterior of the fluid transporting tube and as the fluid stream only contacts the internal surfaces of the tube, the fluid media avoids direct contact with the sensor and heating elements. This format type allows highly accurate and highly sensitive flow measurements to be performed.

Heterologous Objects

In certain aspects, the methods and devices described herein are used to introduce a heterologous object into a cell. The heterologous object can be any object that is small enough to be encompassed by a cell (e.g., small enough to pass through the temporary pore created by electroporation). Such an object can be a nucleic acid (e.g., DNA, RNA), a protein, a peptide, a peptidomimetic, a bead, a dye, a chemical compound, and/or any object that is known in the art to have been introduced into a cell.

In some embodiments, the heterologous object is a nucleic acid. In some embodiments, the nucleic acid is DNA. In other embodiments, the nucleic acid is RNA. In some embodiments, RNA may comprise e.g., mRNA, RNP, small RNA (e.g., siRNA, miRNA, piRNA, etc.), RNAi agent, CRISPR/Cas agent (e.g., gRNA).

In some embodiments, the heterologous object modulates gene expression or modulates/alters the genome of a cell (e.g., creates a double-strand break, introduces into the genome a deletion, a substitution, an addition, a mutation (or corrects a mutation present in the genome), or a combination thereof). Systems for altering the genome (e.g., genomic sequence) is well known in the art. Non-limiting examples are provided below.

CRISPR/CAS

It is art-recognized that CRISPR/Cas system is effective in altering the genome. CRISPR/Cas systems are found in 40% of bacteria and 90% of archaea and differ in the complexities of their systems. See, e.g., U.S. Patent No. 8,697,359 (incorporated by reference). The CRISPR loci (clustered regularly interspaced short palindromic repeat) are regions within the organism's genome where short segments of foreign DNA are integrated between short repeat palindromic sequences. These loci are transcribed and the RNA transcripts ("pre-crRNA") are processed into short CRISPR RNAs (crRNAs). There are three types of CRISPR/Cas systems which all incorporate these RNAs and proteins known as "Cas" proteins (CRISPR associated). Types I and III both have Cas endonucleases that process the pre-crRNAs, that, when fully processed into crRNAs, assemble a multi-Cas protein complex that is capable of cleaving nucleic acids that are complementary to the crRNA.

In type II systems, crRNAs are produced using a different mechanism where a transactivating RNA (tracrRNA) complementary to repeat sequences in the pre-crRNA, triggers processing by a double strand-specific RNase III in the presence of the Cas9 protein or a variant thereof. Cas9 is then able to cleave a target DNA that is complementary to the mature crRNA however cleavage by Cas9 is dependent both upon base-pairing between the crRNA and the target DNA, and on the presence of a short motif in the crRNA referred to as the PAM sequence (protospacer adjacent motif) (see Qi et al (2013) Cell 152: 1173). In addition, the tracrRNA must also be present as it base pairs with the crRNA at its 3' end, and this association triggers Cas9 activity.

The Cas9 protein has at least two nuclease domains: one nuclease domain is similar to a HNH endonuclease, while the other resembles a Ruv endonuclease domain. The HNH-type domain appears to be responsible for cleaving the DNA strand that is complementary to the crRNA while the Ruv domain cleaves the non-complementary strand. The variants of Cas9 are art-recognized, e.g., Cas9 nickase mutant that reduces off-target activity (see e.g., Ran et al. (2014) Cell 154(6): 1380-1389), nCas, Cas9-D10A.

The requirement of the crRNA-tracrRNA complex can be avoided by use of an engineered "single-guide RNA" (sgRNA) that comprises the hairpin normally formed by the annealing of the crRNA and the tracrRNA (see Jinek et al (2012) Science 337:816 and Cong et al (2013) Sciencexpress/10.1126/science.1231143). Thus, exogenously introduced CRISPR endonuclease (e.g., Cas9 or a variant thereof) and a guide RNA (e.g., sgRNA or gRNA) can induce a DNA break at a specific locus within the genome of a target cell. Nonlimiting examples of single-guide RNA or guide RNA (sgRNA or gRNA) sequences suitable for targeting are shown in Table 1 in US Application 2015/0056705, which is incorporated herein in its entirety by reference.

In some embodiments, the gene editing nucleic acid sequence encodes a gene editing nucleic acid molecule selected from the group consisting of: a sequence specific nuclease, one or more guide RNA (gRNA), CRISPR/Cas, a ribonucleoprotein (RNP) or any combination thereof. In some embodiments, the sequence -specific nuclease comprises: a TAL-nuclease, a zinc-finger nuclease (ZFN), a meganuclease, a megaTAL, or an RNA guide endonuclease of a CRISPR/Cas system (e.g., Cas proteins e.g. CAS 1-9, Csy, Cse, Cpfl, Cmr, Csx, Csf, cpfl, nCAS, or others). These gene editing systems are known to those of skill in the art, See for example, TALENS described in International Patent Application No. PCT/US2013/038536, and U.S. Patent Publication No. 2017/-0191078-A9 which are incorporated by reference in their entirety. CRISPR cas9 systems are known in the art and described in U.S. Patent Application No. 13/842,859 filed on March 2013, and U.S. Patent Nos. 8,697,359, 8771,945, 8795,965, 8,865,406, 8,871,445 (all of which are incorporated by reference). The devices and methods described herein are also useful for introducing into a cell the deactivated nuclease systems, such as CRISPRi or CRISPRa dCas systems, nCas, or Casl3 systems.

GUIDE RNAS (gRNAS)

In general, a guide sequence is any polynucleotide sequence having sufficient complementarity with a target polynucleotide sequence to hybridize with the target sequence and direct sequence-specific targeting of an RNA-guided endonuclease complex to the selected genomic target sequence. In some embodiments, a guide RNA binds to a target sequence and e.g., a CRISPR associated protein that can form a ribonucleoprotein (RNP), for example, a CRISPR/Cas complex. In some embodiments, the guide RNA (gRNA) sequence comprises a targeting sequence that directs the gRNA sequence to a desired site in the genome, is fused to a crRNA and/or tracrRNA sequence that permit association of the guide sequence with the RNA- guided endonuclease. In some embodiments, the degree of complementarity between a guide sequence and its corresponding target sequence, when optimally aligned using a suitable alignment algorithm, is at least 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or more. Optimal alignment can be determined with the use of any suitable algorithm for aligning sequences, such as the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on the Burrows-Wheeler Transform (e.g., the Burrows Wheeler Aligner), ClustalW, Clustal X, BLAT, Novoalign (Novocraft Technologies, ELAND (Illumina, San Diego, Calif.), SOAP, and Maq.

A guide sequence can be selected to target any target sequence. In some embodiments, the guide RNA can be complementary to either strand of the targeted DNA sequence. It is appreciated by one of skill in the art that for the purposes of targeted cleavage by an RNA-guided endonuclease, target sequences that are unique in the genome are preferred over target sequences that occur more than once in the genome. Bioinformatics software can be used to predict and minimize off-target effects of a guide RNA (see e.g., Naito et al. “CRISPRdirect: software for designing CRISPR/Cas guide RNA with reduced off-target sites” Bioinformatics (2014), epub; Heigwer et al. “E-CRISP: fast CRISPR target site identification” Nat. Methods 11 : 122-123 (2014); Bae et al. “Cas-OFFinder: a fast and versatile algorithm that searches for potential off-target sites of Cas9 RNA-guided endonucleases” Bioinformatics 30(10): 1473-1475 (2014); Aach et al. “CasFinder: Flexible algorithm for identifying specific Cas9 targets in genomes” BioRxiv (2014)).

In general, a “crRNA/tracrRNA fusion sequence,” as that term is used herein refers to a nucleic acid sequence that is fused to a unique targeting sequence and that functions to permit formation of a complex comprising the guide RNA and the RNA-guided endonuclease. Such sequences can be modeled after CRISPR RNA (crRNA) sequences in prokaryotes, which comprise (i) a variable sequence termed a “protospacer” that corresponds to the target sequence as described herein, and (ii) a CRISPR repeat. Similarly, the tracrRNA (“transactivating CRISPR RNA”) portion of the fusion can be designed to comprise a secondary structure similar to the tracrRNA sequences in prokaryotes (e.g., a hairpin), to permit formation of the endonuclease complex. In some embodiments, the single transcript further includes a transcription termination sequence, such as a polyT sequence, for example six T nucleotides. In some embodiments, a guide RNA can comprise two RNA molecules and is referred to herein as a “dual guide RNA” or “dgRNA.” In some embodiments, the dgRNA may comprise a first RNA molecule comprising a crRNA, and a second RNA molecule comprising a tracrRNA. The first and second RNA molecules may form a RNA duplex via the base pairing between the flagpole on the crRNA and the tracrRNA. When using a dgRNA, the flagpole need not have an upper limit with respect to length.

In other embodiments, a guide RNA can comprise a single RNA molecule and is referred to herein as a “single guide RNA” or “sgRNA.” In some embodiments, the sgRNA can comprise a crRNA covalently linked to a tracrRNA. In some embodiments, the crRNA and tracrRNA can be covalently linked via a linker. In some embodiments, the sgRNA can comprise a stem-loop structure via the base-pairing between the flagpole on the crRNA and the tracrRNA. In some embodiments, a single-guide RNA is at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120 or more nucleotides in length (e.g., 75-120, 75-110, 75-100, 75-90, 75-80, 80-120, 80-110, 80-100, 80-90, 85-120, 85-110, 85-100, 85-90, 90-120, 90-110, 90-100, 100-120, 100-120 nucleotides in length). In some embodiments, a nucleic acid or a composition thereof comprises a nucleic acid that encodes at least 1 gRNA. For example, the second polynucleotide sequence may encode between 1 gRNA and 50 gRNAs, or any integer from 1-50. Each of the polynucleotide sequences encoding the different gRNAs can be operably linked to a promoter. In some embodiments, the promoters that are operably linked to the different gRNAs may be the same promoter. The promoters that are operably linked to the different gRNAs may be different promoters. The promoter may be a constitutive promoter, an inducible promoter, a repressible promoter, or a regulatable promoter.

In some embodiments, a nucleic acid for integration into an endogenous locus is introduced in conjunction with another nucleic acid that encodes a Cas nickase (nCas; e.g., Cas9 nickase or Cas9-D10A). It is contemplated herein that such an nCas enzyme is used in conjunction with a guide RNA that comprises homology to an endogenous locus and can be used, for example, to release physically constrained sequences or to provide torsional release. Releasing physically constrained sequences can, for example, “unwind” the nucleic acid such that a homology directed repair (HDR) template homology arm(s) are exposed for interaction with the genomic sequence.

In some embodiments, zinc finger nuclease is used to induce a DNA break that facilitates integration of the desired nucleic acid. “Zinc finger nuclease” or “ZFN” as used interchangeably herein refers to a chimeric protein molecule comprising at least one zinc finger DNA binding domain effectively linked to at least one nuclease or part of a nuclease capable of cleaving DNA when fully assembled. “Zinc finger” as used herein refers to a protein structure that recognizes and binds to DNA sequences. The zinc finger domain is the most common DNA-binding motif in the human proteome. A single zinc finger contains approximately 30 amino acids and the domain typically functions by binding 3 consecutive base pairs of DNA via interactions of a single amino acid side chain per base pair.

In some embodiments, a nucleic acid for integration described herein is integrated into a target genome in a nuclease-free homology-dependent repair systems, e.g., as described in Porro et al., Promoterless gene targeting without nucleases rescues lethality of a Crigler- Najjar syndrome mouse model, EMBO Molecular Medicine, (2017). In some embodiments, the in vivo gene targeting approaches are suitable for the insertion of a donor sequence, without the use of nucleases. In some embodiments, the donor sequence may be promoterless.

In some embodiments, the nuclease located between the restriction sites can be a RNA-guided endonuclease. As used herein, the term “RNA-guided endonuclease” refers to an endonuclease that forms a complex with an RNA molecule that comprises a region complementary to a selected target DNA sequence, such that the RNA molecule binds to the selected sequence to direct endonuclease activity to a selected target DNA sequence. CRISPR/CAS9 and Variants

As art-recognized and described above, a CRISPR-CAS9 system includes a combination of protein and ribonucleic acid (“RNA”) that can alter the genetic sequence of an organism (see, e.g., US publication 2014/0170753; incorporated by reference). CRISPR- Cas9 provides a set of tools for Cas9- mediated genome editing via nonhomologous end joining (NHEJ) or homologous recombination in mammalian cells. One of ordinary skill in the art may select between a number of known CRISPR systems such as Type I, Type II, and Type III. In some embodiments, a nucleic acid can be designed to include the sequences encoding one or more components of these systems such as the guide RNA, tracrRNA, or Cas (e.g., Cas9 or a variant thereof). In certain embodiments, a single promoter drives expression of a guide sequence and tracrRNA, and a separate promoter drives Cas (e.g., Cas9 or a variant thereof) expression. One of skill in the art will appreciate that certain Cas nucleases require the presence of a protospacer adjacent motif (PAM) adjacent to a target nucleic acid sequence.

RNA-guided nucleases including Cas (e.g., Cas9 or a variant thereof) are suitable for initiating and/or facilitating the integration of a nucleic acid delivered using the devices and methods described herein. The guide RNAs can be directed to the same strand of DNA or the complementary strand.

In some embodiments, the methods and compositions described herein can comprise and/or be used to deliver CRISPRi (CRISPR interference) and/or CRISPRa (CRISPR activation) systems to a host cell. CRISPRi and CRISPRa systems comprise a deactivated RNA-guided endonuclease (e.g., Cas9 or a variant thereof) that cannot generate a double strand break (DSB). This permits the endonuclease, in combination with the guide RNAs, to bind specifically to a target sequence in the genome and provide RNA-directed reversible transcriptional control.

Accordingly, in some embodiments, the nucleic acid compositions can comprise a deactivated endonuclease, e.g., RNA-guided endonuclease and/or Cas9 or a variant thereof, wherein the deactivated endonuclease lacks endonuclease activity, but retains the ability to bind DNA in a site-specific manner, e.g., in combination with one or more guide RNAs and/or sgRNAs. In some embodiments, the nucleic acid can further comprise one or more tracrRNAs, guide RNAs, or sgRNAs. In some embodiments, the de-activated endonuclease can further comprise a transcriptional activation domain.

In some embodiments, the nucleic acid compositions for integration of a nucleic acid of interest into an endogenous locus can comprise a hybrid recombinase. For example, Hybrid recombinases based on activated catalytic domains derived from the resolvase/invertase family of serine recombinases fused to Cys2-His2 zinc-finger or TAL effector DNA-binding domains are a class of reagents capable improved targeting specificity in mammalian cells and achieve excellent rates of site-specific integration. Suitable hybrid recombinases include those described in Gaj et al. Enhancing the Specificity of Recombinase -Mediated Genome Engineering through Dimer Interface Redesign, Journal of the American Chemical Society, (2014).

The nucleases described herein can be altered, e.g., engineered to design sequence specific nuclease (see, e.g., US Patent 8,021,867; incorporated by reference). Nucleases can be designed using the methods described in e.g., Certo et al. Nature Methods (2012) 9-.Q13- 975; U.S. Patent Nos. 8,304,222; 8,021,867; 8,119,381; 8,124,369; 8,129,134; 8,133,697; 8,143,015; 8,143,016; 8,148,098; and 8,163,514, the contents of each are incorporated herein by reference in their entirety. Alternatively, nuclease with site specific cutting characteristics can be obtained using commercially available technologies e.g., Precision BioSciences’ Directed Nuclease Editor™ genome editing technology.

MEGATALS In some embodiments, the nuclease described herein can be a megaTAL. MegaTALs are engineered fusion proteins which comprise a transcription activator-like (TAL) effector domain and a meganuclease domain. MegaTALs retain the ease of target specificity engineering of TALs while reducing off-target effects and overall enzyme size and increasing activity. MegaTAL construction and use is described in more detail in, e.g., Boissel et al. 2014 Nucleic Acids Research 42(4):259L601 and Boissel 2015 Methods Mol Biol 1239: 171- 196. Protocols for megaTAL-mediated gene knockout and gene editing are known in the art, see, e.g., Sather et al. Science Translational Medicine 2015 7(307):ral56 and Boissel et al. 2014 Nucleic Acids Research 42(4):259L601. MegaTALs can be used as an alternative endonuclease in any of the methods and compositions described herein. CAR Molecules and CAR Therapy

The devices and methods of the present disclosure provide a particular utility for introducing a heterologous object into a cell for a CAR therapy. Chimeric antigen receptors (CARs) are transmembrane proteins that have been engineered to give the cells (e.g., T cells, macrophages, NK cells) the new ability to target/bind a specific protein. The receptors are chimeric because they combine both antigen-binding and certain cellular functions (e.g., T cell activating function) into a single receptor. For example, the receptor can comprise an extracellular antigen-binding domain (e.g., scFv) that binds to a specific antigen (e.g., those highly and specifically expressed on the surface of cancer cells) fused to a transmembrane domain and an intracellular costimulatory domain/activation domain. CAR T THERAPY

Chimeric antigen receptor T cells (CAR T cells) are T cells that are engineered to express the CAR proteins for cancer therapy. CARs enable T cells to recognize tumor- associated antigens (TAAs) in a major histocompatibility complex (MHC)-independent manner. CAR T therapy can use T cells that are autologous or allogeneic to the patient. After CAR T cells are infused into a patient, they act as a “living drug” against cancer cells. When they come in contact with their targeted antigen on a cell, CAR T cells bind to it and become activated, then proceed to proliferate and become cytotoxic. CAR T cells destroy cells through several mechanisms, including extensive stimulated cell proliferation, increasing the degree to which they are toxic to other living cells (cytotoxicity) and by causing the increased secretion of factors that can affect other cells such as cytokines, interleukins and growth factors. The first CAR T cell therapies were FDA-approved in 2017, and there are now 6 approved CAR T therapies. There are several variations/generations of CAR designs. The first reports of tumortargeting CARs demonstrated that an scFv recognizing antigens such as human epidermal growth factor receptor 2 (HER2) fused to the CD3(^ signaling domain can elicit tumorspecific cytotoxicity, but T cells expressing these “first-generation” CARs that included only the CD3(^ chain for T-cell signaling generally failed to elicit potent antitumor effects. In the following years, second- and third-generation CARs emerged that included one or two costimulatory domains, respectively, drawing from the biological understanding that the endogenous TCR requires association with other costimulatory or accessory molecules for robust signaling. Most commonly derived from CD28 or 4- IBB, these costimulatory domains conferred more potent antitumor cytotoxicity, increased cytokine production, and improved proliferation and persistence of CAR-T cells. The choice of costimulatory domain has an impact on a wide range of properties, including metabolic pathways, T-cell memory development, and antigen-independent tonic signaling, prompting further research into other costimulatory domains. For example, a third-generation CAR with 0X40 and CD28 costimulatory domains repressed CD28-induced secretion of interleukin (IL)- 10, an antiinflammatory cytokine that compromises T-cell activity. In addition, the inducible T cell costimulator (ICOS) costimulatory domain in combination with either CD28 or 4-1BB costimulation increased in vivo persistence and MyD88/CD40 costimulation improved in vivo proliferation of CAR-T cells. More recently, fourth-generation CARs that incorporate additional stimulatory domains, commonly referred to as “armored” CARs, have been reported. In one example, the engineered armored CAR-T cells termed “T cells redirected for universal cytokine-mediated killing’ ’ (TRUCK) have been engineered to secrete the proinflammatory cytokine IL-12 to stimulate innate immune cells against the tumor and resist inhibitory elements of the TME, including regulatory T (Treg) cells and myeloid-derived suppressor cells (MDSCs). The secretion of other soluble factors has been studied, including IL- 15 or IL- 18 to enhance T cell proliferation, as well as the combination of CCL19 and IL-7 to recruit endogenous immune cells and establish a memory response against tumors.

The devices and methods of the present disclosure can be used in introducing a nucleic acid to a T cell for generations of the CAR T cells for use as e.g., a cancer therapy. DUAL CAR THERAPY

CAR T cells with ability to target two antigens on a cancer cell surface have been proven to be effective clinically. For example, CAR T cells with dual targeting of CD19 and CD22 in adult patients with recurrent or refractory B cell maglignancies showed improved efficacy (Spiegel et al. (2021) Nature Medicine, 27: 1419-1431). In addition, dual CAR T demonstrated effectiveness in targeting tumor cells with heterogeneous antigen expression. For example, CAR-T cells targeting simultaneously two tumor-associated antigens with trans-acting CD28 and 4- IBB co-stimulation caused rapid antitumor effects in in vivo stress conditions, protection from tumor re-challenge and prevention of tumor escape due to low antigen density. Molecular and signaling studies indicated that T cells engineered with the dual CAR design demonstrated sustained phosphorylation of T-cell-receptor-associated signaling molecules and a molecular signature supporting CAR-T-cell proliferation and long-term survival. Furthermore, metabolic profiling of CAR-T cells displayed induction of glycolysis that sustains rapid effector T-cell function, but also preservation of oxidative functions, which are critical for T-cell long-term persistence (Hirabayashi et al. (2021) Nature Cancer, 2:904-918).

CAR-M THERAPY

Programming CARs into cell types other than T cells can further expand the versatility of the therapy by realizing new functions unachievable by CAR T cells. It was recently demonstrated that primary macrophages can be engineered with CARS via adenoviral transduction (Klinchinsky et al. (2020) Nat Biotechnol). The resulting CAR M cells exhibited tumor-specific phagocytosis, inflammatory cytokine production, polarization of bystander macrophages to the immunostimulatory Ml phenotype, and cross-presentation of the tumor associated antigen (TAA) to bystander T cells. CAR-NK THERAPY

CD19-targeting CAR-NK cells have achieved robust clinical efficacy without inducing cytokine release syndrome (CRS), neurotoxicity, or graft-versus-host syndrome (GvHD) in patients with B-cell lymphoid tumors. CAR NK cells have been shown to exert potent and specific cytotoxicity toward a variety of tumor models, including leukemia, multiple myeloma, ovarian cancer, and glioblastoma, as well as toward immunosuppressive cell types such as myeloid-derived suppressor cells (MDSCs) and follicular helper T cells (TFH). Lastly, natural killer T (NKT) cells possess antitumor and tumor-homing capabilities, and GD2-targeting CAR NKT cells that harness these inherent advantages exhibited effective localization to and lysis of neuroblastoma cells without significant toxicity (Xu et al., (2019) Clin Cancer Res 25 : 7126-7138).

Systems

In various embodiments, microfluidic devices for droplet electroporation are provided. In various embodiments, the devices have a channel with a thickness that allows flowing droplets through such that only one drop will fit within the thickness dimension of the channel.

In various embodiments, the width and thickness of the channel may be sufficiently small that the droplets must pass through the channel in single file. In addition, the devices have at least one pair of electrodes disposed across the channel or on one of the surfaces of the channel. This pair of electrodes can deliver a pre-selected voltage waveform to a droplet when the droplet is passing between the pair of electrodes.

In various embodiments, the voltage waveform is delivered after there is a triggering event, such as the detection of the presence of a droplet. In various embodiments, the detection, is performed by the same pair of electrodes that delivers the voltage, whereas in others it is performed by another pair of (upstream) electrodes or by another sensing module. In various embodiments, when the detection is by an electrode pair (either the same one or a different one), the presence of the droplet is sensed based on the droplet having a higher or lower conductivity than the flow medium. As an example, the flow medium can be a non- conductive oil to allow such detection of aqueous droplets. In various embodiments, when the detection is by another sensing module, it can be by a diode laser that detects scattering from the droplet, by a fluorescence-based detector that detects a fluorescent label within the droplet, or by an optical detector that detects the droplet via image-processing.

In various embodiments, the microfluidic device (or system) includes a collectiontriggering sensing module for sensing the presence of a droplet downstream of said pair of electrodes, wherein said collection-triggering sensing module triggers a collection system to receive the droplet from the outlet. In various embodiments, the collection system includes a multi-well plate. In various embodiments, the downstream sensing module can be similar to the sensing module upstream of the electrode pair that delivers the voltage. For example, it can be another pair of electrodes, a diode laser that detects scattering from the droplet, a fluorescence-based detector that detects a fluorescent label within the droplet, or an optical detector that detects the droplet via image-processing.

Methods

In various embodiments, methods of electroporating a cell are provided. In various embodiments, a flow medium and a droplet comprising zero, one, or more than one cell and at least one biological molecule are flowed through a microfluidic device, and any cell(s) after electroporation to contain one or more biological molecule are collected.

In various embodiments, the biological material is a nucleic acid (e.g., DNA, mRNA). In various embodiments, the methods also include adjusting or selecting a flow rate for running the flow medium. In some embodiments, the methods also include selecting a droplet size for the droplet.

These methods of electroporating a cell can also be used for screening, for example for selecting an electroporation condition for droplet electroporation. In such methods, as an example, one may vary the flow rate. As another example, in screening methods, one may use any of the described collection-triggering sensing modules.

Exemplary Embodiments

1. A microfluidic device for droplet electroporation, the microfluidic device, comprising: a channel having an inlet, and outlet, a channel thickness, a channel width, and a first end and a second end defining a channel length, wherein the channel is configured to receive a flow medium having one or more droplets disposed therein; and at least one pair of electrodes disposed along the channel length and configured to provide an electric potential difference across at least a portion of the channel, wherein each electrode of the pair of electrodes is disposed opposite one another.

2. The microfluidic device of 1, wherein the channel thickness is uniform along the channel length.

3. The microfluidic device of 1, wherein the channel thickness is non-uniform along the channel length.

4. The microfluidic device of any one of 1 to 3, wherein the channel thickness allows flow of the one or more droplets through the channel in a single file (e.g., 1B-D).

5. The microfluidic device of any one of 1 to 4, wherein the channel thickness is about 10 micrometers to about 900 micrometers.

6. The microfluidic device of any one of 1 to 5, wherein the channel width allows flow of the one or more droplets through the channel in a single file.

7. The microfluidic device of any one of 1 to 5, wherein the channel width allows flow of two or more droplets through the channel (e.g., Fig. 1 A). 8. The microfluidic device of any one of 1 to 7, wherein the channel width is about 10 micrometers to about 900 micrometers.

9. The microfluidic device of any one of 1 to 8, wherein the pair of electrodes are external to the channel.

10. The microfluidic device of any one of 1 to 9, wherein the at least one pair of electrodes is a single pair of electrodes.

11. The microfluidic device of 10, wherein each electrode of the single pair of electrodes has an electrode length of about 1 mm to about 20 mm.

12. The microfluidic device of 10 or 11, wherein the single pair of electrodes is configured to continuously deliver a voltage comprising a pre-selected waveform (e.g., Fig. 6).

13. The microfluidic device of 10 or 11, wherein the single pair of electrodes is configured to continuously deliver a voltage comprising an arbitrary waveform.

14. The microfluidic device of 10 or 11, wherein the single pair of electrodes is configured to, upon being triggered, deliver a voltage comprising a pre-selected waveform.

15. The microfluidic device of 14, wherein the at least one pair of electrodes is configured to determine the presence of the one or more droplets between the electrodes, thereby triggering the delivery of the voltage.

16. The microfluidic device of 15, wherein the at least one pair of electrodes is configured to determine the presence of the one or more droplets based on conductivity.

17. The microfluidic device of 16, wherein the at least one pair of electrodes is configured to determine the presence of the one or more droplets based on the one or more droplets having a higher conductivity than the flow medium.

18. The microfluidic device of 16, wherein the at least one pair of electrodes is configured to determine the presence of the one or more droplets based on the one or more droplets having a lower conductivity than the flow medium. 19. The microfluidic device of any one of 1 to 9, wherein the at least one pair of electrodes is two or more pairs of electrodes, wherein a first pair of the two or more pairs of electrodes is configured to determine the presence of the one or more droplets between the electrodes, wherein a second pair of the two or more pairs of electrodes is configured to deliver a voltage.

20. The microfluidic device of 19, wherein the first pair has an electrode length of about 1 mm to about 20 mm.

21. The microfluidic device of 19 or 20, wherein the second pair has an electrode length of about 0.5 mm to about 1 mm.

22. The microfluidic device of any one of 19 to 21, wherein the first pair of electrodes is spaced at a distance away from the second pair of electrodes, wherein the distance is less than about 100 micrometers.

23. The microfluidic device of any one of 19 to 22, wherein the second pair of electrodes is downstream from the first pair of electrodes.

24. The microfluidic device of any one of 1 to 23, further comprising a sensing module configured to determine the presence of the one or more droplets, wherein said sensing module triggers the at least one pair of electrodes to deliver the voltage.

25. The microfluidic device of 24, wherein the sensing module comprises a diode laser that detects scattering from the one or more droplets.

26. The microfluidic device of 24, wherein the sensing module comprises a fluorescencebased detector configured to detect a fluorescent label within the one or more droplets.

27. The microfluidic device of 24, wherein said sensing module comprises an optical detector configured to detect the one or more droplets via image-processing.

28. The microfluidic device of any one of 1 to 27, further comprising a collection system and a collection-triggering sensing module configured to sense the presence of the one or more droplets downstream of the at least one pair of electrodes, wherein said collectiontriggering sensing module triggers the collection system to receive the one or more droplets from the outlet (e.g., Fig. 4). 29. The microfluidic device of 28, wherein said collection system comprises a multi-well plate (e.g., Fig. 4).

30. The microfluidic device of 28 or 29, wherein the sensing module comprises an additional pair of electrodes, wherein the additional pair of electrodes are configured to cause the energizing of the at least one pair of electrodes upon detection of the one or more droplets.

31. The microfluidic device of 28 or 29, wherein the sensing module comprises a diode laser that detects scattering from the one or more droplets.

32. The microfluidic device of 28 or 29, wherein the sensing module comprises a fluorescence-based detector that detects a fluorescent label within the one or more droplets.

33. The microfluidic device of 28 or 29, wherein the sensing module comprises an optical detector configured to detect the one or more droplets via image-processing.

34. The microfluidic device of any one of 1 to 33, wherein the flow medium is a non- conductive oil.

35. The microfluidic device of 34, wherein the non-conductive oil is selected from the group consisting of: hexadecane, silicone oil, FC-70.

36. The microfluidic device of any one of 1 to 35, wherein the channel has a length of about 0.5 mm to about 100 mm.

37. The microfluidic device of any one of 1 to 36, wherein the at least one pair of electrodes is configured to deliver a voltage of about 1 V to about 50 V.

38. The microfluidic device of any one of 1 to 37, wherein at least one of the one or more droplets comprises at least one cell and at least one biological material.

39. A method of electroporating one or more cells, comprising: providing a microfluidic device of any one of 1-38; providing the flow medium at the inlet, the flow medium having the one or more droplets disposed therein; providing an electric potential difference between the at least one pair of electrodes to thereby electroporate the at least one cell to contain the at least one biological molecule as the droplet in which the at least one cell is contained passes between the at least one pair of electrodes; and collecting the at least one cell from the outlet (e.g., Fig. 4).

40. The method of 39, wherein the flow medium is provided at a predetermined flow rate.

41. The method of 39, wherein the flow medium is provided at a varying flow rate.

42. The method of 40, wherein the flow rate is selected to provide a droplet rate of about

1,000 to about 10,000,000 droplets per minute.

43. The method of any one of 39 to 42, wherein the one or more droplets have a mean diameter of about 1 micrometer to about 200 micrometers.

44. A device capable of inserting a biologically active molecule into a living cell, the device comprising: a fluid channel comprising a fluid input and a fluid output configured to allow a fluid flow comprising one or more living cells and one or more biologically active molecules enclosed in at least one droplet through the channel; and a first electrode and a second electrode disposed on opposite sides of the fluid channel to which a voltage can be applied to generate an electric field directed across the fluid channel when the droplet passes between the first and second electrodes, wherein the first and second electrodes are separated by a distance between the first and second electrodes enables the droplets to pass through the space between the electrodes in a single layer, wherein the strength of the electric field to which the living cell is exposed is sufficient to form pores within the membrane of the living cell through which the biologically active molecule can traverse the cell membrane, but not lyse the living cell.

45. The device of 44, wherein the distance between the first and second electrodes is less than about 100 micrometers.

46. The device of 44, wherein the distance between the first and second electrodes is from about 100 micrometers to about 250 micrometers.

47. The device of 44, wherein the first and second electrodes are formed by coating the channel on opposite sides with electrically conducting layers. 48. The device of 44, wherein the channel comprises an optically transparent material.

49. The device of 44, wherein the electrodes are arranged to generate spatially varying electric fields

50. The device of 44, wherein the electrodes are arranged to generate a constant electric field.

51. The device of 44, wherein the electrodes are arranged to generate a varying electric field.

52. The device of 44, wherein the droplet comprises an aqueous solution contained in an oil-based flow medium.

53. A device comprising: a droplet-forming region configured to form one or more droplets, wherein at least one of the one or more droplets have at least one cell and at least one biological material contained therein; an electroporation region comprising one or more pairs of electrodes; and a collection device configured to collect or extract the droplets.

54. A device capable of inserting a biologically active molecule into a living cell, the device comprising: a fluid channel comprising a fluid input and a fluid output configured to allow plug fluid flow, the plug fluid flow having at least a first plug comprising a first fluid and a second plug comprising a second fluid, the first plug and the second plug alternating along a length of the channel, wherein the first fluid has a different fluid property from the second fluid such that the first and second plugs remain substantially separate during plug fluid flow, wherein at least one of the first plug and the second plug comprises one or more living cells and one or more biologically active molecules; and a first electrode and a second electrode disposed on opposite sides of the fluid channel to which a voltage can be applied to generate an electric field directed across the fluid channel when the first and second plugs passes between the first and second electrodes, wherein the first and second electrodes are separated by a distance that enables the first and second plugs to pass therethrough, wherein the strength of the electric field to which the one or more living cells is exposed is sufficient to form pores within a cell membrane of the one or more living cells through which the one or more biologically active molecules can traverse the cell membrane, but not lyse the one or more living cells.

EXAMPLES

The disclosure will be further illustrated with reference to the following specific examples. These examples are given by way of illustration and are not meant to limit the disclosure or the claims that follow.

Triggered electroporation of mammalian cells with a DNA plasmid while being encapsulated in non-conductive oil

This example describes an embodiment where the aqueous solution containing cells with plasmid DNA is emulsified within a non-conductive oil. The emulsified cells are flowed through a device with one electrode pair that 1) detects droplets for triggering and 2) conducts electroporation.

Jurkat cells (Sigma) are electroporated with a plasmid expressing green fluorescent protein (GFP). Cells are cultured in an incubator at 37 °C and 5% CO2 and cultured in RPMI 1640 (Fisher) supplemented with 10% fetal bovine serum (Fisher). The day before an electroporation experiment, the cells are seeded at a suspension density of 4 x 10⁵ cells/mL in culture media. On the day of electroporation, 16 million cells are withdrawn from the suspension and centrifuged at 500 g for 5 minutes. The cells are then washed two times in a proprietary, low conductivity electroporation buffer. After the second wash, cells are resuspended with 2 mL of electroporation buffer bringing the cell suspension density to 8 x 10⁶ cells/mL. Plasmid DNA is subsequently added to bring the concentration to 50 pg/mL.

The aqueous solution containing cells and plasmid DNA is loaded into a syringe which is then loaded into a syringe pump. A second syringe containing non-conductive oil is prepared and loaded into a second syringe pump. The non-conductive oil is hexadecane with 5% (weight) of surfactant (Span80, Sigma). The syringe pump outputs connect to a T- junction where the aqueous and oil solutions mix to produce droplets. Flow rates are adjusted to create droplets containing 1-2 cells per droplet on average.

The resulting emulsion flows through the microfluidic device as an oil stream carrying aqueous droplets. As an aqueous droplet passes through the electrode pair, the droplet completes the circuit and triggers an electrical voltage to the same electrode pair. The droplet is subjected to an arbitrary voltage waveform, with an example of such as waveform shown in Fig. 6. In various embodiments, voltages for the highest voltage range from 7 V to 20 V while the duration ranges from 10 gs to 1000 gs. In various embodiments, voltages for the lower voltage ranges from 0. IV to 5 V while the duration ranges from 10 gs to 10,000 gs. The transit time of the droplet under the electrode pair will depend on the flow rate of the emulsion and determines the minimum electrode length required for the droplet to experience the desired duration of the applied voltage waveform.

Electroporated droplets exit the output stream of the device into a 24-well plate containing 500 gL of cell media per well. Droplets are collected for 30 seconds per voltage waveform. Cells are permitted to settle within the well plate for 30 minutes after electroporation. The media containing cells, oil and other fluid components are then centrifuged at 500 g for 5 minutes. The supernatant is subsequently removed carefully via a pipette. 1 mL of media is added to the centrifuge tube, and the cells are centrifuged again at 500 g for 5 minutes. The cells are washed two more times with fresh media, then resuspended with 0.5 mL media and added to a 24-well plate.

Cell viability is measured 24 hours after electroporation by incubation with the 7- AAD viability dye and measurement of positive staining cells via flow cytometry. Transfection efficiency is measured by GFP positive cells via flow cytometry. Control cells are subjected to similar conditions but receive no electrical voltage waveform, according to embodiments.

Incorporation by Reference

All U.S. patents, and U.S. and PCT patent application publications mentioned herein are hereby incorporated by reference in their entirety as if each individual patent or patent application publication was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, will control.

Equivalents

Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation many equivalents to the specific embodiments of the present invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

CLAIMS What is claimed is:

1. A microfluidic device for droplet electroporation, comprising: a channel having an inlet, and outlet, a channel thickness, a channel width, and a first end and a second end defining a channel length, wherein the channel is configured to receive a flow medium having one or more droplets disposed therein; and at least one pair of electrodes disposed along the channel length and configured to provide an electric potential difference across at least a portion of the channel, wherein each electrode of the pair of electrodes is disposed opposite one another.

2. The microfluidic device of claim 1, wherein the channel thickness is uniform along the channel length.

3. The microfluidic device of claim 1, wherein the channel thickness is non-uniform along the channel length.

4. The microfluidic device of any one of claims 1 to 3, wherein the channel thickness allows flow of the one or more droplets through the channel in a single file.

5. The microfluidic device of any one of claims 1 to 4, wherein the channel thickness is about 10 micrometers to about 900 micrometers.

6. The microfluidic device of any one of claims 1 to 5, wherein the channel width allows flow of the one or more droplets through the channel in a single file.

7. The microfluidic device of any one of claims 1 to 5, wherein the channel width allows flow of two or more droplets through the channel.

8. The microfluidic device of any one of claims 1 to 7, wherein the channel width is about 10 micrometers to about 900 micrometers.

9. The microfluidic device of any one of claims 1 to 8, wherein the pair of electrodes are external to the channel. The microfluidic device of any one of claims 1 to 9, wherein the at least one pair of electrodes is a single pair of electrodes. The microfluidic device of claim 10, wherein each electrode of the single pair of electrodes has an electrode length of about 1 mm to about 20 mm. The microfluidic device of claim 10 or 11, wherein the single pair of electrodes is configured to continuously deliver a voltage comprising a pre-selected waveform. The microfluidic device of claim 10 or 11, wherein the single pair of electrodes is configured to continuously deliver a voltage comprising an arbitrary waveform. The microfluidic device of claim 10 or 11, wherein the single pair of electrodes is configured to, upon being triggered, deliver a voltage comprising a pre-selected waveform. The microfluidic device of claim 14, wherein the at least one pair of electrodes is configured to determine the presence of the one or more droplets between the electrodes, thereby triggering the delivery of the voltage. The microfluidic device of claim 15, wherein the at least one pair of electrodes is configured to determine the presence of the one or more droplets based on conductivity. The microfluidic device of claim 16, wherein the at least one pair of electrodes is configured to determine the presence of the one or more droplets based on the one or more droplets having a higher conductivity than the flow medium. The microfluidic device of claim 16, wherein the at least one pair of electrodes is configured to determine the presence of the one or more droplets based on the one or more droplets having a lower conductivity than the flow medium. The microfluidic device of any one of claims 1 to 9, wherein the at least one pair of electrodes is two or more pairs of electrodes, wherein a first pair of the two or more pairs of electrodes is configured to determine the presence of the one or more droplets between the electrodes, wherein a second pair of the two or more pairs of electrodes is configured to deliver a voltage. The microfluidic device of claim 19, wherein the first pair has an electrode length of about 1 mm to about 20 mm. The microfluidic device of claim 19 or 20, wherein the second pair has an electrode length of about 0.5 mm to about 1 mm. The microfluidic device of any one of claims 19 to 21, wherein the first pair of electrodes is spaced at a distance away from the second pair of electrodes, wherein the distance is less than about 100 micrometers. The microfluidic device of any one of claims 19 to 22, wherein the second pair of electrodes is downstream from the first pair of electrodes. The microfluidic device of any one of claims 1 to 23, further comprising a sensing module configured to determine the presence of the one or more droplets, wherein said sensing module triggers the at least one pair of electrodes to deliver the voltage. The microfluidic device of claim 24, wherein the sensing module comprises a diode laser that detects scattering from the one or more droplets. The microfluidic device of claim 24, wherein the sensing module comprises a fluorescence-based detector configured to detect a fluorescent label within the one or more droplets. The microfluidic device of claim 24, wherein said sensing module comprises an optical detector configured to detect the one or more droplets via image-processing. The microfluidic device of any one of claims 1 to 27, further comprising a collection system and a collection-triggering sensing module configured to sense the presence of the one or more droplets downstream of the at least one pair of electrodes, wherein said collection-triggering sensing module triggers the collection system to receive the one or more droplets from the outlet. The microfluidic device of claim 28, wherein said collection system comprises a multi-well plate. The microfluidic device of claim 28 or 29, wherein the sensing module comprises an additional pair of electrodes, wherein the additional pair of electrodes are configured to cause the energizing of the at least one pair of electrodes upon detection of the one or more droplets. The microfluidic device of claim 28 or 29, wherein the sensing module comprises a diode laser that detects scattering from the one or more droplets. The microfluidic device of claim 28 or 29, wherein the sensing module comprises a fluorescence-based detector that detects a fluorescent label within the one or more droplets. The microfluidic device of claim 28 or 29, wherein the sensing module comprises an optical detector configured to detect the one or more droplets via image-processing. The microfluidic device of any one of claims 1 to 33, wherein the flow medium is a non-conductive oil. The microfluidic device of claim 34, wherein the non-conductive oil is selected from the group consisting of: hexadecane, silicone oil, FC-70. The microfluidic device of any one of claims 1 to 35, wherein the channel has a length of about 0.5 mm to about 100 mm. The microfluidic device of any one of claims 1 to 36, wherein the at least one pair of electrodes is configured to deliver a voltage of about 1 V to about 50 V. The microfluidic device of any one of claims 1 to 37, wherein at least one of the one or more droplets comprises at least one cell and at least one biological material. A method of electroporating one or more cells, comprising: providing a microfluidic device of any one of claims 1-38; providing the flow medium at the inlet, the flow medium having the one or more droplets disposed therein; providing an electric potential difference between the at least one pair of electrodes to thereby electroporate the at least one cell to contain the at least one biological molecule as the droplet in which the at least one cell is contained passes between the at least one pair of electrodes; and collecting the at least one cell from the outlet. The method of claim 39, wherein the flow medium is provided at a predetermined flow rate. The method of claim 39, wherein the flow medium is provided at a varying flow rate. The method of claim 40, wherein the flow rate is selected to provide a droplet rate of about 1,000 to about 10,000,000 droplets per minute. The method of any one of claims 39 to 42, wherein the one or more droplets have a mean diameter of about 1 micrometer to about 200 micrometers. A device capable of inserting a biologically active molecule into a living cell, the device comprising: a fluid channel comprising a fluid input and a fluid output configured to allow a fluid flow comprising one or more living cells and one or more biologically active molecules enclosed in at least one droplet through the channel; and a first electrode and a second electrode disposed on opposite sides of the fluid channel to which a voltage can be applied to generate an electric field directed across the fluid channel when the droplet passes between the first and second electrodes, wherein the first and second electrodes are separated by a distance between the first and second electrodes enables the droplets to pass through the space between the electrodes in a single layer, wherein the strength of the electric field to which the living cell is exposed is sufficient to form pores within the membrane of the living cell through which the biologically active molecule can traverse the cell membrane, but not lyse the living cell. The device of claim 44, wherein the distance between the first and second electrodes is less than about 100 micrometers. The device of claim 44, wherein the distance between the first and second electrodes is from about 100 micrometers to about 250 micrometers. The device of claim 44, wherein the first and second electrodes are formed by coating the channel on opposite sides with electrically conducting layers. The device of claim 44, wherein the channel comprises an optically transparent material. The device of claim 44, wherein the electrodes are arranged to generate spatially varying electric fields The device of claim 44, wherein the electrodes are arranged to generate a constant electric field. The device of claim 44, wherein the electrodes are arranged to generate a varying electric field. The device of claim 44, wherein the droplet comprises an aqueous solution contained in an oil-based flow medium. A device comprising: a droplet-forming region configured to form one or more droplets, wherein at least one of the one or more droplets have at least one cell and at least one biological material contained therein; an electroporation region comprising one or more pairs of electrodes; and a collection device configured to collect or extract the droplets. A device capable of inserting a biologically active molecule into a living cell, the device comprising: a fluid channel comprising a fluid input and a fluid output configured to allow plug fluid flow, the plug fluid flow having at least a first plug comprising a first fluid and a second plug comprising a second fluid, the first plug and the second plug alternating along a length of the channel, wherein the first fluid has a different fluid property from the second fluid such that the first and second plugs remain substantially separate during plug fluid flow, wherein at least one of the first plug and the second plug comprises one or more living cells and one or more biologically active molecules; and a first electrode and a second electrode disposed on opposite sides of the fluid channel to which a voltage can be applied to generate an electric field directed across the fluid channel when the first and second plugs passes between the first and second electrodes, wherein the first and second electrodes are separated by a distance that enables the first and second plugs to pass therethrough, wherein the strength of the electric field to which the one or more living cells is exposed is sufficient to form pores within a cell membrane of the one or more living cells through which the one or more biologically active molecules can traverse the cell membrane, but not lyse the one or more living cells.