The present invention relates to a cell sorting systems used in medical diagnoses and biological studies by employing the advancements in the field of microfluidic technology. Most specifically relates to rapid extraction of the target cells from droplets without any damage to the cells.
Fluorescence Activated Cell Sorter (FACS) is an instrument, which interrogates a small volume of fluid to detect and sort biological cells present in a sample fluid [J. S. Kim, et al., PAN Stanford Publishing, Singapore, 2010]. Presently, due to its capability for detailed analysis, FACS is the state of the art for biological sample analysis [R. B. L. Gwatkin., et al., Practical flow cytometry, 1994; Mol. Reprod. Dev., 1995]. FACS finds numerous applications including biomedical research for immunology, single cell analysis and molecular biology. However, conventional FACS systems are very expensive and thus are available only in centralized research facilities and major health care centres [R. B. L. Gwatkin., et al., Practical flow cytometry, 1994; Mol. Reprod. Dev., 1995]. Similarly, due to its complexity, regular maintenance and skilled expertise are required to operate the machine, analyse data and make reports. In addition, skilled technicians are required for fixing any functional failure and troubleshooting. These factors add a considerable cost to the maintenance of the machine and increase the cost per test in diagnosis using conventional FACS. In the last few years, research work has been carried out to design cost-effective, portable MicroFACS by employing the advancements in the field of microfluidic technology. However, one of the main hindrances in the development of a MicroFACS is the complicated techniques required for three dimensional focusing of biological cells flowing inside the microchannel and controlling interdistance between them in the optical window [P. K. Shivhare, et al., Microfluid. Nanofluidics, 2016]. Another challenge in the development of MicroFACS is the isolation of target cells downstream after detection. In literature, various techniques have been reported to achieve the isolation of target cells such as hydrodynamic [A. Wolff et al., Lab Chip, 2003], dielectrophoresis [D. Holmes et al., Micro Total Anal. Syst, 2004], optical [M. M. Wang et al., Nat. Biotechnol 2005] and piezoelectric [A. Wolff et al., Lab Chip, 2003]. However, such techniques require high voltage or high shear thus affecting cell viability and cell property, offer low throughput, employ complicated instrumentation and thus are not amenable to the development of a microfluidic sorter [S. H. Cho et al., Biomicrofluidics, 2010]. Also, none of these techniques are suitable for the extraction and isolation of target cells in single-cell format.
Many publications showed that an electric field has been employed for coalescence of droplets for microparticle extraction and droplet sorting [K. Ahn C et al., Appl. Phys. Lett., 2006; L. M. Fidalgo et al., Angew. Chemie, 2008; L. Mazutis et al., Lab Chip, 2012; T. Szymborski et al., Appl. Phys. Lett, 2011; A. R. Thiam et al., Phys. Rev. Lett, 2009]. Coalescence of droplets in an emulsion along the direction of the flow has been explored [Keunho Ahn et al., Appl. Phys. Lett, 2006]. Coalescence of aqueous droplets with a parallel stream of aqueous phase in a direction normal to the flow direction has also been investigated [V. Chokkalingam et al., Lab Chip, 2014]. However, the later device requires very high voltage (thousands of volts) and electric field (107 V/m) thus not suitable for biological applications due to cell viability issue.
Thus the present invention relates to a technique in which cells are focused into a single-file stream and subsequently encapsulated inside droplets at a channel junction. The cell encapsulating droplets self-align toward the centre of the channel due to non-inertial lift force and move into the detection window as single-file thus solving the challenges stated above. Once the droplet-encapsulating target cells are detected, electro-coalescence is used to extract these cells either in single-cell format inside droplets or into an aqueous phase for downstream analysis
The present invention relates to a cell sorting systems by employing the advancements in the field of microfluidic technology. Most specifically relates to rapid extraction of the target cells from droplets without any damage to the cells.
The detected droplet-encapsulating target cells are electro-coalesced to extract these cells either in single-cell format inside droplets or into an aqueous phase for downstream analysis. Wherein the aqueous droplets containing the cells are in continuous contact with the interface between the continuous phase and a co-flowing aqueous phase before entering the electric field region thus require significantly lower voltage and electric field. This approach enables rapid extraction of the target cells microparticles from droplets into a co-flowing stream of aqueous phase or in single-cell format without any damage to the cells.
In one embodiment, the present invention develops a MicroFACS for the isolation of target cells in which MicroFACS has three different modules which can be used independently for various applications and together for analysis and sorting of biological cells and microparticles. The three different modules are (i) focusing and encapsulation module, (ii) optical detection module and (iii) electro-coalescence module.
In other embodiment, the present invention provides a technique in which cells are focused into a single-file stream and subsequently encapsulated inside droplets at a channel junction. The encapsulated droplets are self-aligned toward the centre of the channel due to non-inertial lift force and move into the detection window as single-file stream.
In yet other embodiment, the present invention show that the encapsulated droplets are moved towards the detection modules where the target cells are detected using fluorescence signals and scattering signals received from labeled and non labeled cells respectively. The detected droplets are move towards electro-coalescence module. The electro-coalescence is used to sort target cells. This module consists of a microchannel with two inlets, one to introduce the immiscible continuous phase (oil) containing the droplets (containing cells or microparticles) and the other to introduce the co-flowing aqueous stream, and one or more pairs of electrodes connected to an alternating current (AC) power source. The electrical pressure is required to coalesce the droplet into fluid stream. Wherein, the droplets flowing in the immiscible continuous phase (oil) come in contact with the interface due to the positioning of the aqueous stream. The required voltage is 25 V or the corresponding electric field (105 V/m) is at least two orders of magnitude smaller as compared to the existing methods.
In another embodiment, the present invention provides a method for continuous or on-demand coalescence of aqueous droplets containing target cells or microparticles with an aqueous phase for extraction of cells and microparticles from the discrete droplets and further processing of such cells or microparticles downstream. Continuous coalescence of droplets containing cells or microparticles or droplets (without any cells or microparticles) can be achieved using a continuous electric field. However, on-demand electro-coalescence requires activation of the electrodes only when a target cell, microparticle or droplet is detected in the optical detection module.
In yet another embodiment, the present invention provides a MicroFACS method by integration of the optical detection and electro-coalescence modules. The target cells or microparticles are detected optically, sorting of these target cells or microparticles into the co-flowing aqueous phase stream is achieved by triggering the electrodes in the electro-coalescence module. This method used for on-demand coalescence of droplets containing the target fluid or droplets of particular size.
Referring to the drawings, the embodiments of the present invention are further described. The figures are not necessarily drawn to scale, and in some instances the drawings have been exaggerated or simplified for illustrative purposes only. One of ordinary skill in the art may appreciate the many possible applications and variations of the present invention based on the following examples of possible embodiments of the present invention.
In the following detailed description, a reference is made to the accompanying drawings that form a part hereof, and in which the specific embodiments that may be practiced is shown by way of illustration. The embodiments are described in sufficient detail to enable those skilled in the art to practice the embodiments and it is to be understood that the logical, mechanical and other changes may be made without departing from the scope of the embodiments. The following detailed description is therefore not to be taken in a limiting sense.
The proposed invention relates to a cell sorting systems by employing the advancements in the field of microfluidic technology. Most specifically relates to rapid extraction of the target cells from droplets without any damage to the cells. The present invention develops a MicroFACS for the isolation of target cells in which MicroFACS has three different modules which can be used independently for various applications and together for analysis and sorting of biological cells and microparticles. The three different modules are (i) focusing and encapsulation module, (ii) optical detection module and (iii) electro-coalescence module.
The hydrodynamic focusing and encapsulation module (
The optical detection module consists of a fluidic channel, a number of optical grooves placed at a predetermined angle with the fluid channel, laser source, fibres, filter and high-speed detectors (
The detection module can be used for the detection of target droplets (without any cell or microparticle) that containing a fluid of interest based on the fluorescence signature of the fluid contained inside the droplet.
The electro-coalescence module consists of a microchannel with two inlets: one to introduce the immiscible continuous phase (oil) containing the droplets (containing cells or microparticles) and the other to introduce the co-flowing aqueous stream, and one or more pairs of electrodes connected to an alternating current (AC) power source (
The ratio of the flow rate of the co-flowing aqueous stream is adjusted so that the droplets flowing in the immiscible continuous phase (oil) come in contact with the interface. If there is a variation in the size of the droplets, the interface location is adjusted such that even the smallest droplet comes in contact and automatically the larger droplets are in contact with the interface. In this case, an aqueous droplet and a stream of aqueous phase are separated by a very thin film of surfactant for droplet stabilization (
When droplet and planar-interface are stabilized by the surfactant are in contact with each other as shown in
To achieve coalescence the electric field has to deform the droplet and planar-interface and make the contact between interfaces. Once the contact is established the electric field has to overcome the repulsive disjoining pressure created by surfactant molecules. The electric field strength required to deform the droplets are very high compared to the electric field strength require to overcome the disjoining pressure. So the electric field required to coalesce the droplet not in contact with the other interface (˜107 V/m) is one to two orders of magnitude greater compare to droplet in contact with the other interface (˜105 V/m) [Liu, Z, et al., Lab on a Chip, 2014] [V. Chokkalingam Y, et al., Lab Chip, 2014]. If the droplet is in contact with the other droplet or planar interface it can be coalesced easily by applying less electric field (˜105 V/m). The cell damage problems are averted completely at electric field strength less than 5×105 V/m [Gascoyne P. R. C, et al., Cancers, 2014].
The method proposed here can be used for continuous or on-demand coalescence of aqueous droplets containing target cells or microparticles with an aqueous phase for extraction of cells and microparticles from the discrete droplets and further processing of such cells or microparticles downstream. The method can be used for continuous or on-demand coalescence of droplets (without any cells or particles) present in the immiscible continuous oil phase with an aqueous phase for demulsification or sorting of droplets which has importance in various applications. Continuous coalescence of droplets containing cells or microparticles or droplets (without any cells or microparticles) can be achieved using a continuous electric field. However, on-demand electro-coalescence requires activation of the electrodes only when a target cell, microparticle or droplet is detected in the optical detection module.
The optical detection and electro-coalescence modules are integrated to provide a MicroFACS (
Similarly, for applications that require single-cell analysis, the target cells encapsulated inside droplets in single-cell format can be obtained at the device outlet (
It may be appreciated by those skilled in the art that the drawings, examples and detailed description herein are to be regarded in an illustrative rather than a restrictive manner.
Number | Date | Country | Kind |
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201741012180 | Apr 2017 | IN | national |
Filing Document | Filing Date | Country | Kind |
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PCT/IN2018/050194 | 4/5/2018 | WO | 00 |