Abstract:
The present invention pertains to methods and systems for on demand droplet generation and impedance based detection. The microfluidic system can utilize an electrical sensor for detecting an electrical property of a fluid and for generating an electrical signal indicating a change in the electrical property of the fluid; a droplet generator coupled to the electrical sensor configured to generate the droplet from the fluid in response to the electrical signal from the electrical sensor; and a microfluidic channel coupled to the droplet generator for receiving the droplet. The method for generating droplets involves measuring an electrical property of the fluid, transmitting an electrical signal indicative of the electrical property, and forming the droplets from the fluid based on the electrical signal.

Description:
RELATED U.S. APPLICATION 
       [0001]    This application claims priority to the co-pending U.S. provisional patent application Ser. No. 61/390,948, Attorney Docket Number 2011-162-1, entitled “METHODS AND SYSTEMS FOR ON DEMAND DROPLET GENERATION AND IMPEDANCE BASED DETECTION,” with filing date Oct. 7, 2010, which is hereby incorporated by reference in its entirety. 
     
    
     BACKGROUND 
       [0002]    Cell encapsulation has developed into an integral part of droplet microfluidic research, with various groups demonstrating technologies to improve the encapsulation process. However, many challenges still need to be overcome for microfluidic technology to become a truly viable platform for the biomedical field. 
         [0003]      FIGS. 1 ,  2 ,  3 , and  4  are diagrams showing a variety of cell encapsulation processes that have been developed on microfluidic systems.  FIG. 5  is a diagram showing a process for controlled encapsulation of single-cells into monodisperse picolitre drops.  FIG. 6  is a diagram showing a process for overcoming Poisson encapsulation challenges. Drawbacks of conventional systems include the concentration dependency of encapsulation efficiency, temporariness of overcoming Poisson challenges and an assumption of a concentration of 1×10 6  cells per ml. 
         [0004]    In conventional continuous and on-demand droplet generation schemes the entire volume of the dispersed phase is made into droplets in a sequential manner. This results in high percentages of empty droplets as compared to cell-encapsulating droplets over a large volume. 
       SUMMARY 
       [0005]    In one embodiment, a microfluidic system for generating a droplet is provided. The microfluidic system can utilize an electrical sensor for detecting an electrical property of a fluid and for generating an electrical signal indicating a change in the electrical property of the fluid; a droplet generator coupled to the electrical sensor configured to generate the droplet from the fluid in response to the electrical signal from the electrical sensor; and a microfluidic channel coupled to the droplet generator for receiving the droplet. The electrical sensor can be an impedance sensor and can utilize a plurality of electrodes for determining size of an object in the fluid. The electrical sensor can also utilize three electrodes for determining size and speed of an object in the fluid. The droplet generator can utilize a deformable chamber. The droplet generator can also utilize an activation delay circuit coupled to the electrical sensor. The delay can be less than 30 ms. The droplet generator can utilize an actuation timing circuit for controlling size of the droplet and can be variable. The actuation time can be less than 500 ms. 
         [0006]    In another embodiment, a method for generating droplets from a fluid is provided. An electrical property of the fluid is measured; an electrical signal indicative of the electrical property is transmitted; and the droplets from the fluid is formed based on the electrical signal. The electrical property can be impedance. The size of an object in the fluid can be determined. The speed of an object in the fluid can be calculated. The electrical signal indicative of the electrical property can be delayed. The delay can be between 10 ms and 30 ms. The electrical property can be sampled at a sampling rate of at least 48 kHz. A chamber containing the fluid can be actuated. The actuation time can be between 10 ms and 500 ms. The chamber containing the fluid can be deformed at a rate of at least 10 Hz. 
         [0007]    These and other objects and advantages of the various embodiments of the present invention will be recognized by those of ordinary skill in the art after reading the following detailed description of the embodiments that are illustrated in the various drawing figures. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0008]    The accompanying drawings, which are incorporated in and form a part of this specification, illustrate embodiments of the invention and, together with the description, serve to explain the principles of the invention: 
           [0009]      FIGS. 1 ,  2 ,  3 , and  4  are diagrams showing a variety of cell encapsulation processes that have been developed on microfluidic systems. 
           [0010]      FIG. 5  is a diagram showing a process for controlled encapsulation of single-cells into monodisperse picolitre drops. 
           [0011]      FIG. 6  is a diagram showing a process for overcoming Poisson encapsulation challenges. 
           [0012]      FIG. 7  is a diagram showing droplet generation region of a droplet generation device of one embodiment of the present invention. 
           [0013]      FIG. 8  is a diagram showing a labeled schematic of a device of one embodiment of the present invention. 
           [0014]      FIG. 9  is a diagram showing components of a device of one embodiment of the present invention. 
           [0015]      FIG. 10  is a graph illustrating the alignment of impedance signal of cells with the chamber activation to create droplets encapsulating the corresponding cells. 
           [0016]      FIG. 11  is a diagram showing a sequence of a single cell being encapsulated by the droplet generation process of one embodiment of the present invention. 
           [0017]      FIG. 12  is a diagram showing a view of a device of one embodiment of the present invention. 
           [0018]      FIG. 13  is a diagram showing a portion of a device during droplet generation of one embodiment of the present invention. 
           [0019]      FIG. 14  is a diagram showing the droplet generation mechanism of a device during different stages of droplet generation of one embodiment of the present invention. 
           [0020]      FIG. 15  is a diagram illustrating the impedance measurement of cells according to one embodiment of the present invention. 
           [0021]      FIG. 16  is a graph illustrating cell impedance data that are associated with impedance measurement operations of one embodiment of the present invention. 
           [0022]      FIG. 17  is a diagram illustrating device components that are utilized as a part of impedance measurement operations of one embodiment of the present invention. 
           [0023]      FIG. 18  is a graph of cell impedance data that is used in impedance signal processing operations of one embodiment of the present invention. 
           [0024]      FIG. 19  is a diagram illustrating a LabVIEW interface for system integration of one embodiment of the present invention. 
           [0025]      FIG. 20  is a graph that illustrates features of the encapsulation control scheme of one embodiment of the present invention. 
           [0026]      FIGS. 21 ,  22  and  23  are diagrams showing problems of conventional systems that can be addressed by embodiments of the present invention. 
           [0027]      FIG. 24  is a graph of droplet size versus actuation time of one embodiment of the present invention. 
           [0028]      FIG. 25  is a graph of droplet size versus actuation pressure of one embodiment of the present invention. 
           [0029]      FIG. 26  is a flowchart of a process for actuating a droplet generator of one embodiment of the present invention. 
           [0030]      FIG. 27  is a diagram illustrating a screenshot of an interface of one embodiment of the present invention. 
       
    
    
       [0031]    The drawings referred to in this description should not be understood as being drawn to scale except if specifically noted. 
       DETAILED DESCRIPTION 
       [0032]    Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings. While the invention will be described in conjunction with these embodiments, it will be understood that they are not intended to limit the invention to these embodiments. On the contrary, the invention is intended to cover alternatives, modifications and equivalents, which may be included within the spirit and scope of the invention as defined by the appended claims. Furthermore, in the following detailed description of embodiments of the present invention, numerous specific details are set forth in order to provide a thorough understanding of the present invention. However, it will be recognized by one of ordinary skill in the art that the present invention may be practiced without these specific details. In other instances, well-known methods, procedures, components, and circuits have not been described in detail as not to unnecessarily obscure aspects of the embodiments of the present invention. 
         [0033]    In one embodiment, a microfluidic system capable of encapsulating cells at very high efficiency is provided. The device is intended to address the problem of empty droplets generated using conventional encapsulation methods. The device integrates impedance measurement with an on-demand droplet generation process to enable the selective generation of droplets when the presence of a cell is detected. This ensures that a high percentage of droplets that are generated actually contain droplets, regardless of initial cell concentration. In one embodiment, the system includes but is not limited to the utilization of droplets, cell encapsulation, and on-demand impedance detection. In one embodiment, a specifically selected volume within the dispersed fluid stream is converted into droplets, enabling the selective encapsulation of cells. 
         [0034]      FIG. 7  is a diagram showing droplet generation region of a droplet generation device of one embodiment of the present invention.  FIG. 8  is a diagram showing a labeled schematic of a device of one embodiment of the present invention. Contacts for the electrodes are shown at the edge of the device. The rectangle near the droplet generation region is a deformable chamber that is used to generate cell-encapsulating droplets. In one embodiment the device consists of two main components: (1) an impedance sensor; and (2) an on-demand droplet generator. In one embodiment, the impedance sensor is comprised of three electrodes patterned on glass and positioned below a cell inlet channel as shown in  FIG. 7 . In one embodiment, the device is made of poly dimethylsiloxane (PDMS) on a glass substrate. The cell suspension fluid provides a baseline impedance measurement as it flows past the electrodes. Due primarily to the presence of the cell membrane, a cell may present a different impedance measurement when compared to the surrounding fluid. In one embodiment, this difference (e.g., change in impedance) is detected by the sensing electrodes and used to indicate the presence of cells inside the channel. In one embodiment, the on-demand generation component may consist of a modified T junction connected to an added ground line. 
         [0035]    In one embodiment, the droplet generation system (e.g., device shown in  FIG. 8 ) functions by directing all dispersed phase fluid into a ground line as the default state and dispensing or directing fluid into the main channel only when activated.  FIG. 9  is a diagram showing components of a device of one embodiment of the present invention. More specifically,  FIG. 9  shows an encapsulation region of the device, where a top arrow shows a default path of dispersed phase towards the ground line and a bottom arrow shows the path of dispersed phase when the chamber is activated to create a droplet in the main fluidic channel. Referring to  FIG. 9 , in one embodiment, the control line is used to deform the chamber near the droplet generation region (see inlet line). As a result of this deformation, an influx of fluid is forced out of the chamber and into a droplet shearing junction, forming a single droplet (that is directed into the main channel). 
         [0036]    In one embodiment, when the pressure is released, the flow returns to the previous pattern where the dispersed phase fluid flows into the ground line. By coupling the impedance detection of cells with droplet generation, droplets are produced that may always contain cells. More specifically, in one embodiment, empty droplets are not produced. A number of parameters govern the behavior of the devices, and critical of which is the timing of the initiation of the droplet generation process and the length of the chamber actuation time as shown in  FIG. 10 .  FIG. 10  is a graph illustrating the alignment of impedance signal of cells with the chamber activation to create droplets encapsulating the corresponding cells. Sampling frequency in this embodiment can be 48 kHz. As shown in  FIG. 10 , chamber activation may be delayed by Δt 1  to account for the time it takes for the cell to travel from the detection region to the encapsulation region. Moreover, Δt 2  is the amount of time the chamber is activated. The length of Δt 2  can be varied to control the size of the droplet generated. 
         [0037]    Referring to  FIG. 10 , Δt 1  is the delay between a detection of the presence of the cell and the activation of the droplet generation process and is a function of the velocity of the fluid and the distance between the electrodes and the droplet generation region. In one embodiment, additional delay due to signal processing time and chamber activation, which may be between 10 ms to 30 ms, may also be taken into account to produce the final value. 
         [0038]    In one embodiment, in normal operational range, to generate droplets of 50 nm in diameter, the value is 50 ms. In one embodiment, activation pressure can be increased to lower Δt 2  to improve the throughput of the system. In one embodiment, chamber actuation time may be varied from 10 ms to 500 ms and the frequency of generation may be around 10 Hz. Exemplary embodiments enable the scaling up of throughput through a parallelization of the platform. 
         [0039]      FIG. 11  is a diagram showing a sequence of a single cell being encapsulated by the droplet generation process of one embodiment of the present invention. More specifically,  FIG. 11  shows sequential images of a single cell being encapsulated in a droplet. Circles highlight the cell inside the fluidic channel and inside the droplet. The arrow indicates the time point at which the membrane is deformed. The sequence shown: a) the cell in the channel just before the chamber—dispersed phase is directed towards the ground line; b) the cell right after the chamber was deformed to initiate droplet generation; c) membrane pressure is released and droplet was formed containing the cell; d) membrane returned to normal state the dispersed phase is directed to the ground line again. In one embodiment, this system not only enables the potential generation of 100% cell containing droplets, but serves as the basis of microfluidic platforms that are capable of performing small volume sampling out of a larger, continuously processed volume; thus providing an important stepping stone for droplet microfluidics to be integrated into large scale fluidic processes. 
         [0040]      FIG. 12  is a diagram showing a view of a device of one embodiment of the present invention. A unique feature is the K junction. As illustrated in  FIG. 12 , the suspended cells inlet normally flows to the ground outlet unless the membrane deflects and generates a droplet that exits into the continuous phase channel. This configuration enables high efficiency encapsulation or selective encapsulation of cells or other particulates in the dispersed phase.  FIG. 13  is a diagram showing a portion of a device during droplet generation of one embodiment of the present invention.  FIG. 14  is a diagram showing the droplet generation mechanism of a device during different stages of droplet generation of one embodiment of the present invention.  FIG. 15  is a diagram illustrating the impedance measurement of cells according to one embodiment of the present invention. In one embodiment, differential measures between 3 electrodes may be used, and information such as size and speed may be extracted.  FIG. 16  is a graph illustrating cell impedance data that are associated with impedance measurement operations of one embodiment of the present invention.  FIG. 17  is a diagram illustrating device components that are utilized as a part of impedance measurement operations of one embodiment of the present invention. 
         [0041]      FIG. 18  is a graph of cell impedance data that is used in impedance signal processing operations of one embodiment of the present invention.  FIG. 19  is a diagram illustrating a LabVIEW interface for system integration of one embodiment of the present invention.  FIG. 20  is a graph that illustrates features of the encapsulation control scheme of one embodiment of the present invention.  FIGS. 21 ,  22  and  23  are diagrams showing problems of conventional systems that can be addressed by embodiments of the present invention. In one embodiment, the droplet generation rate may be about 10 droplets per second with 50 ms membrane actuation and 50 ms spacing. In one embodiment, LabVIEW based processing may provide 10 k samples per second. In one embodiment, a real time system embodiment may have much higher sampling rates. In one embodiment, based on the speed of the cells in the channel, densities of around 1×10 6  cells per mL can be accommodated. 
         [0042]      FIG. 24  is a graph of droplet size versus actuation time of one embodiment of the present invention.  FIG. 25  is a graph of droplet size versus actuation pressure of one embodiment of the present invention.  FIG. 26  is a flowchart of a process for actuating a droplet generator of one embodiment of the present invention.  FIG. 27  is a diagram illustrating a screenshot of an interface of one embodiment of the present invention. 
         [0043]    Exemplary embodiments provide a microfluidic cell encapsulation device integrated with an impedance-based detector that enables high efficiency production of cell-containing droplets. In one embodiment, this platform also can support a sampling process that allows the selective extraction of precise, digitized volumes out of a larger, continuous volume of fluid. Under another embodiment, a sub population of cells that have a unique impedance signature is selected. For example, the technique may selectively encapsulate white blood cells from whole blood or even circulating tumor cells from blood. 
         [0044]    The foregoing descriptions of specific embodiments of the present invention have been presented for purposes of illustration and description. They are not intended to be exhaustive or to limit the invention to the precise forms disclosed, and many modifications and variations are possible in light of the above teaching. The embodiments were chosen and described in order to best explain the principles of the invention and its practical application, to thereby enable others skilled in the art to best utilize the invention and various embodiments with various modifications as are suited to the particular use contemplated. It is intended that the scope of the invention be defined by the claims appended hereto and their equivalents.