Claims:We Claim:
1. A bi-phasic method for sorting of a micro droplets in a micro fluidic channel, the method comprising;
a preliminary phase, wherein a line scan image of a section of a micro fluidic channel is obtained to identify at least two objects wherein each object is contained within a single micro droplet;
estimation of a plurality of parameters associated with the object; and
a secondary phase, wherein the identified object is sorted downstream of the micro fluidic channel depending on a threshold based on the estimated parameters.
2. The method as claimed in claim 1, wherein the line scan image of the micro fluidic channel is obtained as a 1D line scan or a low 2D line scan, further wherein the 1D line scan or low 2D line scan is obtained either in scattering mode or fluorescence mode.
3. The method as claimed in claim 1, wherein the plurality of parameters includes width of the object, length of the object, location of the object in the micro fluidic channel, fluorescent intensity of the object, scattering intensity of the object and relative location of two successive close lying objects.
4. The method as claimed in claim 1, wherein the threshold is a preset value associated with the parameters.
5. The method as claimed in claim 1, wherein the sorting of the objects is achieved through a dielectrophoretic electrodes, a microswitch, a MEM switch or a piezoelectric switch.
6. The method as claimed in claim 1, wherein the object comprises of a biological cell, a microparticle, a hydrogel beads or a combination thereof.
7. A bi-phasic method for sorting of a micro droplets in a micro fluidic channel, the method comprising;
a preliminary phase, wherein a line scan image of a section of a micro fluidic channel is obtained to identify at least two objects;
estimation of a plurality of parameters associated with the object;
allowing a micro droplet to be formed downstream of the micro fluidic channel;
a secondary phase, wherein the objects are identified within a single micro droplet based on the estimated parameters; and
sorting the objects depending on a threshold based on the estimated parameters.
8. The method as claimed in claim 7, wherein the line scan image of the micro fluidic channel is obtained as a 1D line scan or a low 2D line scan, further wherein the 1D line scan or low 2D line scan is obtained either in scattering mode or fluorescence mode.
9. The method as claimed in claim 7, wherein the plurality of parameters includes width of the object, length of the object, location of the object in the micro fluidic channel, fluorescent intensity of the object, scattering intensity of the object and relative location of two successive close lying objects.
10. The method as claimed in claim 7, wherein the threshold is a preset value associated with the parameters.
11. The method as claimed in claim 7, wherein the sorting of the objects is achieved through a dielectrophoretic electrodes, a microswitch, a MEM switch or a piezoelectric switch.
12. The method as claimed in claim 7, wherein the object comprises of a biological cell, microparticle, hydrogel beads or a combination thereof.

Bangalore ANJU RAWAT
08 April 2022 (IN/PA/3151)
AGENT FOR APPLICANT
(INTELLOCOPIA IP SERVICES)
, Description:A BI-PHASIC METHOD FOR SORTING MICRODROPLETS
FIELD OF INVENTION
The invention generally relates to the field of microfluidics and particularly to a bi-phasic method for sorting of microdroplets in a microfluidic channel.
BACKGROUND
Droplet-based microfluidics allows for the generation and
control of droplets in ideal micro-environments for a variety
of chemical applications, biological applications and processes. After their generation, droplets move in microchannels in a continuous fashion. To fulfil specific functions and objectives, actuation technologies are needed for the manipulation of these droplets according to their contents. Basic manipulation tasks include droplet coalescence, mixing, sorting, and phase changing. Since cells and other biological particles are encapsulated in a micro droplet, sorting of micro droplets based on its content is of immense importance for subsequent analysis. Sorting of droplets require the distinction of their properties, therefore, the first step in droplet sorting is droplet detection, which provides a feedback to the actuator to sort droplets.
Various techniques are available and used for sorting of microdroplets in a microfluidic channel. One of the existing approaches eg, flow cytometery, is the use of optical detection for screening of cells or particles. Light from a source is made incident on a cell or a particle at an interrogation point in the flow path. A signal in the form of scattered light or emitted fluorescent light from the object is captured and directed onto a photodetector. The cell or particle are then encapsulated in droplets and the captured signal triggers selection of desirable droplets. One of the significant disadvantage of the technique is risk of damage to cells, by the pressure exerted by the flow cytometer. Other disadvantage of the method is that only one cell is screened at a time and do not allow co-encapsulation of particles/objects in droplets.
Another existing approach as disclosed in WO 2009/011808 Al includes formation of droplets encapsulating one or more cells along with a florescent signalling entity and interrogation of the droplets using a light source. The emitted fluorescent light from within the droplet is captured and directed onto a photodetector. One of the significant disadvantage of the technique is that it requires multiple fluorescence measurement setup to detect different cells co-encapsulated within the droplets. This setup is complex and restricts interrogation to fluorescently labelled cells only.
Yet another existing technique as disclosed in US8936762B2 includes screening of cells of a plurality of different samples simultaneously by using line-scan images and analyze the line scan image via a cell-typing algorithm and emitted fluorescence. One of the significant disadvantage of the technique is that it is limited to analyze the samples flowing in parallel microchannels and not to co-localization of multiple objects flowing in same microchannel.
Yet another existing technique as disclosed in US11015165B2 includes transporting a cell through a flow channel, capturing an image of the cell from plurality of different angles and analyzing the image using a deep learning algorithm to sort the cells. One significant disadvantage of the technique is that the real time operation for screening large number of cells require expensive and complex setup for imaging and running deep learning algorithms.
Thus, there is need for a technique for sorting of microdroplet encapsulating multiple objects which is rapid, accurate and do not require complex setup for imaging and running deep learning algorithms.
BRIEF DESCRIPTION OF DRAWINGS
So that the manner in which the recited features of the invention can be understood in detail, some of the embodiments are illustrated in the appended drawings. It is to be noted, however, that the appended drawings illustrate only typical embodiments of this invention and are therefore not to be considered limiting of its scope, for the invention may admit to other equally effective embodiments.
Fig.1 shows a schematic of a bi-phasic method for sorting micro droplets in a microfluidic channel, according to an embodiment of the invention.
Fig. 2 shows a schematic of a bi-phasic method for sorting micro droplets in a microfluidic channel, according to an alternate embodiment of the invention.

SUMMARY OF THE INVENTION
One aspect of the invention provide a bi-phasic method for sorting of micro droplets in a micro fluidic channel. The method includes, a preliminary phase of obtaining a line scan image of a section of a micro fluidic channel to identify at least two objects wherein each object is contained within a single micro droplet, estimation of a plurality of parameters associated with the object and a secondary phase of sorting of the identified object down stream of the micro fluidic channel depending on a threshold based on the estimated parameters.
Another aspect of the invention provides a biphasic method for sorting micro droplets in a micro fluidic channel. The method includes a preliminary phase of obtaining a line scan image of a section of a micro fluidic channel to identify at least two objects, estimation of a plurality of parameters associated with the object, allowing a micro droplet to be formed downstream of the micro fluidic channel, a secondary phase, wherein the objects are identified within a single micro droplet based on the estimated parameters, and sorting the objects depending on a threshold based on the estimated parameters.
DETAILED DESCRIPTION OF THE INVENTION
Various embodiments of the invention provide a bi-phasic method for sorting of micro droplets in a micro fluidic channel. In one embodiment of the invention, the bi-phasic method for sorting of micro droplets includes, a preliminary phase of obtaining a line scan image of a section of a micro fluidic channel to identify at least two objects wherein each object is contained within a single micro droplet, estimation of a plurality of parameters associated with the object and a secondary phase of sorting of the identified object down stream of the micro fluidic channel depending on a threshold based on the estimated parameters. In another embodiment of the invention, the bi-phasic method of sorting of microdroplets include a preliminary phase of obtaining a line scan image of a section of a micro fluidic channel to identify at least two objects, estimation of a plurality of parameters associated with the object, allowing a micro droplet to be formed downstream of the micro fluidic channel. A secondary phase, wherein the objects are identified within a single micro droplet based on the estimated parameters and sorting the objects depending on a threshold based on the estimated parameters.
Fig.1 shows a schematic of a bi-phasic method for sorting micro droplets in a microfluidic channel, according to an embodiment of the invention. The bi-phasic method of sorting of micro droplets includes a preliminary phase of obtaining a line scan image of a section of a micro fluidic channel to identify at least two objects. Objects 101 include but are not limited to a biological cell, a microparticle, a hydrogel bead or a combination thereof.
A plurality of objects 101 are allowed to flow in a micro fluidic channel through a plurality of inlets 103. Objects 101 flowing in a media in the micro channel combine with oil to generate water in oil emulsion micro droplets 105. The micro droplet generation is under observation by means of an optical arrangement 107 to image objects encapsulated within a micro droplet 105. In one example, the line scan image of a section of micro fluidic channel is obtained by a line scan camera 109. The line scan image of the micro fluidic channel is obtained as a 1D line scan or a low 2D line scan in either scattering mode or fluorescence mode. The line-scan camera 109 outputs one line (1xN array) of pixels at a time. Consecutive line scans of the section of the micro fluidic channel are obtained as the micro droplets encapsulating the objects pass through the section of the micro fluidic channel. The 1xN captured array of pixels is transferred to a data processor for estimation of a plurality of parameters associated with the object 101. The plurality of parameters associated with the object 101 includes but are not limited to width of the object, length of the object, location of the object in the micro fluidic channel, fluorescent intensity of the object, scaterring intensity of the object and relative location of two successive close lying objects. Based on the parameter an algorithm calculates a threshold value for selecting the micro droplets containing atleast two objects. After generation of microdroplet encapsulating objects, the microdroplets 105 flow dowmstream of the micro channel, the travel time taken by the microdroplet 105 depends on factors including but not limited to the flow rate of media, flow rate of oil and dimensions of the microfluidic channel. In secondary phase, the micro droplet 105 containing atleast two objects is sorted downstream of the micro fluidic channel. Sorting of the micro droplets 105 downstream of the micro channel is enabled by a micro processor, the micro processor activates sorting of the desired micro droplet selected on the basis of parameters. The sorting of the desired microdroplet encapsulating identified objects is achieved through a dielectrophoretic electrodes, a microswitch, a MEM switch or a piezoelectric switch. The micro processor enables the sorting of the desired micro droplet 105 encapsulating identified objects by taking in consideration, the travel time required by the micro droplet 105 to reach downstream of the microfluidic channel. In one example of the invention, the micro processor activates a pair of dielectrophoretic electrodes 111 by applying voltage to sort the desired microdroplet 105 encapsulating identified objects.
Fig.2 shows a schematic of a bi-phasic method for sorting micro droplets in a microfluidic channel, according to an alternate embodiment of the invention. The bi-phasic method for sorting of micro droplets includes a preliminary phase of obtaining a line scan image of a section of a micro fluidic channel to identify at least two objects. Objects 201 include but are not limited to a biological cell, a microparticle, a hydrogel bead or a combination thereof.
A plurality of objects 201 are allowed to flow in a micro fluidic channel through a plurality of inlets 203. To image objects flowing in a micro channel, a portion of the micro fluidic channel is under observation by means of an optical arrangement 205. In one example, the line scan image of a section of micro fluidic channel is obtained by a line scan camera 207. The line scan image of the micro fluidic channel is obtained as a 1D line scan or a low 2D line scan in either scattering mode or fluorescence mode. The line-scan camera 207 outputs one line (1xN array) of pixels at a time. Consecutive line scans of the section of the micro fluidic channel are obtained to identify atleast two objects passing though the section of the micro fluidic channel. The 1xN captured array of pixels is transferred to a data processor for estimation of a plurality of parameters associated with the object 201. The plurality of parameters includes but are not limited to width of the object, length of the object, location of the object in the micro fluidic channel, fluorescent intensity of the object, scattering intensity of the object and relative location of two successive close lying objects. The objects flowing in media are allowed to combine with oil to form a water in oil emulsion micro droplet 209 downstream of the microchannel. Based on the parameter an algorithm calculates a threshold value for selecting the micro droplets 209 containing atleast two objects. After generation of micro droplet 209 encapsulating objects, the micro droplets 209 flow dowmstream of the micro channel. The travel time taken by the micro droplet depends on factors including but not limited to the flow rate of media, flow rate of oil and dimensions of the microfluidic channel. The micro droplet 209 containing atleast two objects is sorted further downstream of the micro fluidic channel. Sorting of the micro droplets 209 downstream of the micro channel is enabled by a micro processor, the micro processor activates sorting of the desired micro droplet selected on the basis of parameters. The sorting of the desired micro droplet encapsulating identified objects is achieved through a dielectrophoretic electrodes, a microswitch, a MEM switch or a piezoelectric switch. The micro processor enables the sorting of the desired micro droplet 209 encapsulating identified objects by taking in consideration, the travel time required by the micro droplet to reach downstream of the microfluidic channel. In one example of the invention, the micro processor activates a pair of dielectrophoretic electrodes 211 by applying voltage to sort the desired microdroplet 209 encapsulating identified objects.
The bi-phasic method for sorting of micro droplets in a micro fluidic channel as provided by the invention, allows rapid and efficient sorting of micro droplets encapsulating atleast two objects. Imaging and data acqusition as line scans reduces the need for complex optic arrangement and computation algorithm. Further the invention provides identification and sorting of micro droplets encapsulating atleast two objects.
The foregoing description of the invention has been set merely to illustrate the invention and is not intended to be limiting. Since modifications of the disclosed embodiments incorporating the spirit and substance of the invention may occur to person skilled in the art, the invention should be construed to include everything within the scope of the appended claims and equivalents thereof.