Multiplexed optofluidic device and methods thereof

Abstract

The present disclosure relates to a device or system and method for multiplexed fluorescent detection and sorting of droplets, which combines multi-parametric optical detection with dielectrophoretic sorting. More particularly, the present disclosure has developed a means in which components are encapsulated into water-in-oil droplets, which when flowed through a central flow channel at a specified rate, are excited by at least one optical source, the signal of which is collected by the signal receiving optic fiber. After receiving read-out signal, the detected target droplet is analyzed by the detection module based on pre- determined parameters and is selected to be sorted. The sorting module initiates a sorting trigger to manipulate the impulse of electrodes, to facilitate the sorting of droplets into multiple sorting channels.

Description

[0001] “MULTIPLEXED OPTOFLUIDIC DEVICE AND METHODS THEREOF”

[0002] TECHNICAL FIELD

[0003] The present disclosure relates to the field of multiplexed optofluidics. Particularly, the present disclosure relates to a device or system that is used in the field of multiplexed optofluidics. More particularly, the present disclosure relates to a multiplexed optofluidic device or system for multiplexed optical detection and sorting of microfluidic droplets, where the power of non-imaging based multiparametric on-chip optical detection and analysis of droplets using optical fibers is combined with dielectrophoretic sorting. The present disclosure also relates to a process of sorting the droplets, a method of operating the device and use of the device for sorting the droplets.

[0004] BACKGROUND AND PRIOR ART

[0005] In the field of biomedical research and diagnostic application, it is a critical requirement for the accurate detection of biomarkers and fluorophore-labelled components, whose optical interrogation helps in understanding the composition and pathophysiological state of biological components and analysis of their functionality. Droplet Microfluidics has become a key part of several high- throughput single -cell analysis techniques in biomedical research and diagnostics. Despite significant progress in the development of individual assays, multiparametric optical sensing and sorting of droplets and their encapsulated contents has been challenging. Correspondingly, this is essentially a technique used to detect and measure the physical and chemical characteristics of a population of cells, beads, particles, etc encapsulated within microfluidic droplets. The current approaches, most commonly involving microscopy-based high-speed imaging of droplets, are technically complex and require expensive instrumentation, limiting their widespread adoption. The use of microscopic imaging, and microscopic imaging-based droplet analysis, makes the setup costly, bulky, and cumbersome to operate. Due to the reliance on microscopes attached high-speed cameras, such technology is expensive and lacks the sensitivity to detect fluorescent signals from low concentration sample as well as being complex to fabricate. It is evident that there are no scalable, stand-alone, benchtop devices for the detection and sorting of the optofluidic droplets, which are based on multi-parametric on-chip optical analysis.

[0006] PCT / IB2013 / 050871 titled “Microfluidic Flow Analyzer for Pathological Detection and Method Thereof’ describes a flow cytometer that is applicable in the detection of human immunodeficiency viruses. The prior art consists of a system where the sample along with a saline buffer, flows through a central flow channel, where a plurality of lasers excites the sample. Correspondingly, it contains a plurality of first-receiving optical channels and a plurality of second- receiving optical channels. The prior art comprises a plurality of detectors placed on each of the plurality of receiving optical channels for detecting one or more of the optical signals. The control feedback in the prior art is designed solely to manipulate the syringe pump of the rate of buffer and sample solution required. However, the prior art limits itself to simple cell detection and does not include any option for selecting and sorting the target cell or droplet based on necessary parameters.

[0007] PCT / IB2020 / 056297 titled “A Microfluidic Analyser,” comprises of a droplet generator, an analyte flow channel in communication with droplet generator, at least one illumination channel, an analyte flow channel in fluid communication with droplet generator at one end and a signal detector at the distal end of the flow channel. The prior art describes a flow cytometer analyser which measures the optical signals including the absorbance, fluorescence, size scatter of analytes / cells / beads or particles which are encapsulated within water-in-oil emulsions. The multi-fluorescence signals are gathered and coupled with a detector and gives a mere read-out. The prior art focusses on a droplet generator, which comprises of a feedback control configured specifically to optimize the flow rate of droplets flowing through the central flow channel, upon receiving of at least one optical signal from an illuminated droplet. The emulsion droplets containing single or plural cells per droplet is excited by a light source, whose optical signal reading is collected, converted into digital signals and analysed for determining the droplet flow rate control. The prior art, however, lacks in using the detected optical signal of target cell or cell parameter for on-demand sorting of cells / droplets or sample components based on the parameter of choice. The existing prior art also limits itself to a feedback control configured solely for the manipulation of droplet flow rate and does not include feedback control applications for other transactions in the multiplexed optofluidic fluorescent detection and sorting.

[0008] US20220212188A1 titled “Laser induced confocal microscope and microfluidic device for dielectrophoretic fluorescence-activated droplet sorting” comprises of a system and method for sorting droplets wherein a microfluidic chip or substrate having a sorting channel includes an optical interrogation device configured to illuminate the droplets passing through the sorting channel with excitation light source and capturing emitted fluorescent light and generating an output signal correlated to the fluorescence of the droplets. The prior art includes an actuator disposed in the chip or substrate and coupled to a signal driver. However, the prior art consists of an oil-bias channel placed before the sorting junction, reducing the utility of the design and restricts the multi-way sorting to only a single sorting channel and a waste channel. Further, the prior art discloses the detection of droplet contents by the use of large external optical set-ups requiring microscopes, high speed cameras, lasers and multiple PMTs which makes the instrument extremely bulky and expensive.

[0009] US20230076378A1 titled “Cell Sorting Using a High Throughput Fluorescence Flow Cytometer” comprises of a method of sorting cells in a flow cytometry system. The prior art discloses illuminating a cell with radiation having at least two optical frequencies shifted from one another by a radiofrequency to elicit fluorescent radiation from the cell, detecting the fluorescent radiation to generate temporal fluorescence data, and processing the temporal fluorescence data to arrive at a sorting decision regarding the cell without generating an image (i.e., a pixel-by-pixel image) of the cell based on the fluorescence data. However, the prior art limits itself to detection of cells. Further, the prior art limits itself to a typical flow cytometer, wherein the sorting decision of the cell is made based on the fluorescence data obtained regarding the ratio of the size of one or more cells along 2 different dimensions, wherein 2 fluorescent markers are used- each for marking the cellular membrane and nucleus, respectively. Further, the prior art discloses the detection of cells using open-space optics and multiple Photomultiplier tubes (PMTs) which increase noise as well as make the set-up bulky. The cells in the prior art maybe damaged upon impact due to the interference of open space environments, reducing the viability. The open-space sorting architecture may also create aerosols which are not ideal for handling sensitive materials.

[0010] US10816550 titled “Systems, apparatus, and methods for sorting particles”, includes cell detection systems, fluidic devices, structures and techniques related to particle and cell sorting and detection in fluid, for example sorting specific subpopulations of cell types. It discloses a method for verification of sorting of particles includes receiving a first detection signal that is associated with optical characteristics of a particle in a first channel. A sorting channel of a plurality of second channels is determined based on the first detection signal, thereby determining the sorting of the particle into the sorting channel based on the optical characteristics of the particle. A sorting signal for sorting the particle from the first channel into the sorting channel is transmitted. A second detection signal is received that is associated with the presence of the particle in the sorting channel. The sorting of the particle from the first channel into the sorting channel is verified based on the second detection signal. However, the prior art describes the use of a piezoelectric actuator which uses mechanical sorting means to physically cause a deviation in the flow path of the cells. Further, the optical detection module utilises microscope lens combined with open space optical set-up for detection. Furthermore, the prior art fails to disclose multi-parametric sorting and limits itself to single channel sorting.

[0011] The prior art thus fails to disclose any optical fiber-based multiparametric optical analysis of components such as single cells in microfluidic droplets with on- demand droplet sorting into multiple channels using dielectrophoresis. Further, the prior art devices are bulky, expensive, laborious because of which it becomes difficult to run as many samples one would want and hence restricting its usage to very specific and limited sample set for analysis and sorting. The present disclosure overcomes one or more limitations associated with the prior art by providing a multiplexed optofluidic device or system and method thereof.

[0012] STATEMENT OF THE DISCLOSURE

[0013] The present disclosure relates to a multiplexed optofluidic device or system for a multiplexed optofluidic fluorescence detection and sorting of droplets which combines multi-parametric optical detection through optical fibers with dielectrophoretic sorting. The device comprises of at least one input channel coupled with a droplet generator wherein it consists of at least one sample inlet and one oil inlet. The device consists of an input channel at one end, a middle central flow channel, and a detection module and sorting channels at the distal end. Along the length of the central flow channel, at least one illumination source is aligned to excite the sample and at least one optic fiber is for signal acquisition. The multiplexed optofluidic device further comprises of integrated circuit and high-speed processing unit which is a programmable logic, such as Field Programmable Gate Array [FPGA] which uses input from the optic fibers. The FPGA is further connected to the Detection module and Sorting module. The Sorting module is connected to the sorting pulse generator which further manipulates the electrodes using a dielectrophoretic AC pulse.

[0014] The present disclosure also relates to a method of detecting and sorting of droplets wherein the interrogation of a sample droplet provides fluorescence signal data to the detection module. The droplets are illuminated as they flow through the central flow channel and the subsequent emissions and scattered light are detected in the form of electric signals, such as TTL Pulses but is not limited to the same, which is then transferred to the programmable logic, such as FPGA, where the processing of the hi- speed pulses is conducted. The signals are then traversed to the detection module where the real time identification and analysis of the droplet signal takes place. In an embodiment, the real time identification depends on factors such as time delay, flow rate of cells, boundary analysis of droplets, but is not limited to the same. Based on the detection, the sorting module triggers a timed activation of the High Voltage generator, while controlling the trigger pulse feature. The High Voltage Generator induces a dielectrophoretic AC Pulse to manipulate the electrodes to produce an appropriate force forcing them into appropriate sorting channels. In a preferred embodiment, the time delay calculation between the point of optical interrogation and sorting junction is determined by the speed of real-time optical data analysis, signal transmission delay, electronic delay, speed of droplets and distance between interrogation and sorting junction. In an embodiment, the time delay calculation maybe automated.

[0015] The present disclosure also relates to the use of the multiplexed optofluidic device or system for sorting of droplets.

[0016] The present disclosure also relates to a multiplexed optofluidic fluorescence detector and sorter.

[0017] In an embodiment, the droplets comprise components selected from a group comprising but not limited to cells, beads, analytes, secreted molecules, intracellular molecules, lysed cells and its contents, biomolecules and combinations thereof.

[0018] In an embodiment, a feature of the present disclosure is the ability to detect the fluorescence of the droplet components even in very small working volumes. As the droplets flow down a central flow channel, at least one illumination source excites the droplets, whose signal is acquired by the optic fiber, which is coupled into a detector for multiplexed optical detection. The digital pulse from the multiplexed optical detection is sent for signal processing which includes a Highspeed pulse counter. In another embodiment, a feature of the present disclosure is the detection and sorting module microprocessor, which may use the read-out data to either conduct downstream computation or engage a sorting trigger on a sorting pulse generator which correspondingly manipulates the dielectrophoretic sorting of droplets. In another embodiment of the present disclosure, the combination of the detection module and the sorting module in the device is a scalable, benchtop technology providing rapid and lower cost solution for diagnostics and clinical sample processing and testing.

[0019] In another embodiment, the present disclosure overcomes the drawbacks of the prior art by providing a multi-parametric on-chip optical flow cytometry-based droplet analysis with simplified optics. It would be advantageous in resourcelimited settings if the components were smaller, less expensive.

[0020] In another embodiment of the present disclosure, the device or system has the ability to detect the droplets of interest and enable multi-parametric sorting, in at- least two channels, either subsequently or simultaneously.

[0021] The present disclosure relates to a method of detecting and processing of the readout data and subsequently sorting the droplets based on multi-parametric variables into multiple sorting channel. In an embodiment, the read-out data of the receiving fibres is fed into the detecting module, wherein the droplet scatter and fluorescence intensity is processed in real-time to provide final data. The final data is mapped to the signal threshold settings to incur the sorting conditions of each droplet. The combination of the sorting conditions and fluorescence parameters cause a sorting trigger of certain intensity and pulse to at least one electrode, which would engage to draw the droplets into a negative potential, forcing the droplets to segregate into a target sorting channel. Further, the current method maybe used for droplet sorting for multiple parameters.

[0022] In an embodiment, the present disclosure is applicable in the field of, but not limited to, single-cell genomics, drug screening, point-of-care diagnostics, precision medicine, regenerative medicine, CAR-T oncotherapy and more. The present disclosure maybe applied in the field of optical analysis of single cells or components in droplets for rare cell identification in clinical samples, isolation of specific cell populations and clones of interest or both phenotype and genotype information for single cell genomics. An advantage of the present invention is the ability to obtain a fibre based multiparametric optical analysis of single microfluidic droplets with an on-demand droplet sorting into multiple channels using dielectrophoresis. Another advantage of the present invention is the realtime data visualization with single cell resolution multiplex fluorescence and multiparametric droplet sorting.

[0023] BRIEF DESCRIPTION OF ACCOMPANYING FIGURES

[0024] Figure 1: outlines the construction and functioning of an embodiment of the multiplexed optofluidic fluorescent droplet-based detector and sorter.

[0025] Figure 2(a-f): outlines different embodiments of the multiplexed optofluidic device consisting of an orientation of multiple sorting channels.

[0026] Figure 3: illustrates a 3 colour dye droplet multiplex signal capture in the detecting module.

[0027] Figure 4: illustrates the fluorescence compensation in the detection module.

[0028] Figure 5(a): illustrates the optofluidic chip components (left) including the droplet generation / reintroduction module, multiplex optical interrogation site, the central flow channel and the electrodes flanking the sorting junction. The right panel shows the inset of droplets getting sorted into a single sorting channel.

[0029] Figure 5(b) depicts the flowchart of the process followed by the detection module and sorting module to analyze the read-out data from the optical fibre to detect, analyze and sort the droplets. Figure 6(a): illustrates an exemplary example of the multiplexed optofluidic device and system detecting antibody stained MEF from a mix of MEF and THP1 cell population, in comparison with standard flow cytometers.

[0030] Figure 6(b): illustrates the sorting trigger threshold for separating MEF and THP1 from waste droplets into separate channels.

[0031] Figure 6(c): illustrates the image of the real-time sorting channel wherein the MHCI+ cells are sorted away from waste channels with empty droplets.

[0032] Figure 7: illustrates the cross-section of the sorting junction with multiple sorting channels.

[0033] DETAILED DESCRIPTION OF THE DISCLOSURE

[0034] The foregoing has broadly outlined the features and technical advantages of the present disclosure in order that the detailed description of the disclosure that follows may be better understood. Additional features and advantages of the disclosure will be described hereinafter, which form the subject of the description of the disclosure. It should also be realized by those skilled in the art that such equivalent product / methods do not depart from the scope of the disclosure.

[0035] Definitions:

[0036] Unless otherwise defined, all terms used in the disclosure, including technical and scientific terms, have meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0037] As used herein, the singular forms ‘a’, ‘an’ and ‘the’ include both singular and plural referents unless the context clearly indicates otherwise.

[0038] The term ‘comprising’, ‘comprises’ or ‘comprised of as used herein are synonymous with ‘including’, ‘includes’, ‘containing’ or ‘contains’ and are inclusive or open-ended and do not exclude additional, non-recited members, elements or method steps. Likewise, certain terms may be interchangeably used throughout the specification and thus have the same meaning even when they are referred interchangeably. For example, the terms “device” or “system” are interchangeably used throughout the specification.

[0039] The recitation of numerical ranges by endpoints includes all numbers and fractions subsumed within the respective ranges, as well as the recited endpoints.

[0040] The term ‘about’ as used herein when referring to a measurable value such as a parameter, an amount, a temporal duration, and the like, is meant to encompass variations of ±10% or less, preferably ±5% or less, more preferably ±1% or less and still more preferably ±0.1% or less of and from the specified value, insofar such variations are appropriate to perform the present disclosure. It is to be understood that the value to which the modifier ‘about’ refers is itself also specifically, and preferably disclosed.

[0041] The present disclosure relates to a multiplexed optofluidic device or system for multiplexed fluorescence detection and sorting of droplets where the power of non-imaging based multi-parametric on-chip optical detection and analysis of droplets is combined with dielectrophoretic sorting.

[0042] In an embodiment, the multiplexed optofluidic device comprise: a droplet inlet channel; an analyte flow channel wherein atleast one droplet flows through; an illumination channel wherein the droplet flowing through the analyte flow channel is interrogated; a multiplexed optical detector wherein fluorescence output of interrogated droplets is detected through optical fibres; a programmable logic comprising a signal processing unit and a Hi- speed pulse counter unit to gather the read-out data of the optical detector; a storage module which stores the read-out data of the optical detector; a detection module to process the detected signal of the logic and analyse based on optical parameters; multi-parametric sorting module which defines sorting logic and channel selection based on optical parameters, to induce a sorting trigger;

[0043] High Voltage Generator to induce a dielectric voltage to at least one electrode based on the sorting trigger; and sorting junction wherein the sorting junction separates into multiple sorting channels.

[0044] In an embodiment of the present disclosure, the detection module comprises of a fluorescence signal demultiplexing unit, real-time droplet identification unit, data storage unit and livestream visualization unit.

[0045] In an embodiment of the present disclosure, the interrogated droplets are water-in- oil droplets.

[0046] In an embodiment of the present disclosure, the droplets comprise of components selected from a group comprising but not limited to cells, beads, analytes, secreted molecules, intracellular molecules, lysed cells and its contents, biomolecules and combinations thereof.

[0047] In an embodiment of the present disclosure, the detection module comprises of a scatter analysis block and a fluorescence analysis block.

[0048] In an embodiment of the present disclosure, the sorting module comprises of a real-time fluorescence peak estimation unit, sorting condition computation unit, timed sorting trigger unit and a trigger pulse feature control unit.

[0049] In an embodiment of the present disclosure, the channel heights are all equal or have a combination of different heights. In an embodiment, the detector module encaptures the peak signals of the colour dye droplet. The dyes or fluorophores that may be used but are not limited to Fluorescein isothiocyanate (FITC), R-phycoerythrin (PE), PerCP and combinations thereof.

[0050] In an embodiment of the present disclosure, the detection module demarcates a region of interest of each droplet, by conducting a droplet boundary analysis, and identifies fluorescence peaks within.

[0051] In an embodiment of the present disclosure, the programmable logic implements a pulse counter for each electric signal and stores the received read-out data from the optical detectors, while combining the counts from each optical detector from the storage module into a single communication packet.

[0052] In an embodiment of the present disclosure, the optical detectors are but not limited to photomultiplier tubes (PMT) or Avalanche Photodiode (APD).

[0053] In an embodiment of the present disclosure, the optical detector pulse count is processed to identify leading and lagging peaks in scatter signals marking the boundary of each droplet and assigning each droplet.

[0054] In an embodiment of the present disclosure, each peak is categorized in the fluorescence of the excitation laser, based on the distance from the droplet scatter peak.

[0055] In an embodiment of the present disclosure, the sorting module initiates a General Purpose Input / Output (GPIO) Sorting trigger to the High Voltage Generator for manipulation of at least one electrode with a dielectrophoretic AC pulse.

[0056] In an embodiment of the present disclosure, fluorescence signals, time delay signals, fluid flow rates, droplet scatter characteristics are received by the detection module, for inferring the need for sorting. In an embodiment of the present disclosure, the sorting module compares the received data to pre-determined threshold values and correspondingly triggers sorting pulses to the High Voltage generator to manipulate droplets into sorting channels.

[0057] In an embodiment of the present disclosure, the number of sorting channels correspond to the number of droplet types being detected and sorted in one run.

[0058] In an embodiment of the present disclosure, the sorting module conducts electrode switching for sorting trigger initiation, based on the sorting channel of interest.

[0059] In an embodiment of the present disclosure, pulse characteristics decide the channel for sorting.

[0060] In an embodiment of the present disclosure, a lack of sorting trigger by the sorting module leads to the droplet flowing through the default waste channel.

[0061] In an embodiment of the present disclosure, the electrode(s) generate negative force to manipulate the path of the droplets.

[0062] In an embodiment of the present disclosure, the device is made of glass.

[0063] In an embodiment of the present disclosure, the device is made of polymers.

[0064] In an embodiment of the present disclosure, the sorting junction is in a 3-way Y sort with or without bend in the sorting channel.

[0065] In an embodiment of the present disclosure, the sorting junction is in a 3-way parallel sort with or without bend in the sorting channel.

[0066] In an embodiment of the present disclosure, the sorting junction is in a 5-way parallel sort with or without bend in the sorting channel. The present disclosure also relates to a method of using the multiplexed optofluidic device as mentioned in the above embodiments, wherein the method comprising interrogation of a water-in-oil droplet, wherein the output data of the interrogation droplet is received by a multiple optical detection along with signal processing by the programmable logic and transmitted to the detection module wherein the detection module performs demultiplexing of raw signals and the readout data is transferred to the sorting module, which defines the sorting logic and channel selection based on optimal parameters, upon which a sorting trigger is transmitted to a High Voltage generator which manipulates the intensity of the electrodes for sorting of droplets into multiple sorting channels.

[0067] In an embodiment of the present disclosure, the droplets comprise of components selected from a group comprising, cells, beads, analytes, secreted molecules, intracellular molecules, lysed cells and its contents, biomolecules and combinations thereof.

[0068] In an embodiment of the present disclosure, the detection module comprises a scatter analysis block wherein the baseline noise cutoff is defined for the scatter and fluorescent signals, for identification and marking of leading and lagging droplet peaks in order to define a region of interest, by conducting a droplet boundary analysis, and use the peak height and width for droplet characterization by multiplex signal allocation to each component in the droplet.

[0069] In an embodiment of the present disclosure, the detection module comprises a fluorescence analysis block, wherein regions of interest of the droplets are superimposed on fluorescence channels and fluorescent peaks are demultiplexed based on expected time delays between colour signals.

[0070] In an embodiment of the present disclosure, the fluorescence analysis block processes raw peak data for colour compensation and assigns multicolour fluorescence profile to each droplet and quantify the fluorescence. In an embodiment of the present disclosure, the programmable logic implements a pulse counter for each electric signal from and stores the read-out data from the optical detectors, while combining the counts from each optical detector from storage module while combining into a single communication packet and wherein optical detector pulse count is processed to identify leading and lagging peaks in scatter signals marking the boundary of each droplet and assigning each droplet.

[0071] In an embodiment of the present disclosure, fluorescence signals, time delay signals, fluid flow rates, droplet scatter characteristics are received by the detection module, for inferring the need for sorting and subsequently the sorting module initiates a General Purpose Input / Output (GPIO) Sorting trigger to the High Voltage Generator for the manipulation of at least one electrode with a dielectrophoretic AC pulse.

[0072] In an embodiment of the present disclosure, the sorting module defines the sorting logic and channel selection based on optical parameters and initiates a sorting trigger.

[0073] In an embodiment of the present disclosure, the sorting is multi-parametric.

[0074] The present disclosure also relate to a method of multiplex optofluidic droplet sorting comprising: atleast one droplet flowing into an analyte flow channel, wherein said droplet is interrogated through atleast one illumination channel; detecting atleast one optical signal from the interrogated droplet by at least one signal detector, plotting a time integrated pulse count over time, assessment of time-correlated independent tracks for scatter and fluorescent signals; analysing the detected optical signal in scatter analysis block wherein baseline noise cutoff is defined for the scatter and fluorescent signals, for identification and marking of leading and lagging droplet peaks, by an optical detector, in order to define a region of interest and use the peak height and width for droplet characterization; further analysing the optical signal in fluorescence analysis block, wherein regions of interest are superimposed on fluorescence channels and fluorescent peaks are demultiplexed based on expected time delays between colour signals, processing raw peak data for colour compensation and assigning multicolour fluorescence profile to each droplet and quantifying the fluorescence; defining sorting logic and channel selection based on optical parameters and generating a sorting trigger to High Voltage Generator; and manipulating intensity of electrodes for sorting of droplets into multiple sorting channels and creating a negative energy to pull the droplet to a specific sorting channel.

[0075] Thus, the present disclosure relates to a multiplexed optofluidic device or system for a multiplexed optofluidic fluorescence detection and multi-parametric on chip sorting of droplets. The device comprises of at least one input channel coupled with a droplet generator wherein it consists of at least one sample inlet and one oil inlet. The flow cytometer consists of an input channel at one end, a middle central flow channel and a sorting channel at the distal end. Along the length of the central flow channel, at least one illumination source is aligned to excite the sample through optic fiber and at least one optic fiber for signal acquisition. The multiplexed optofluidic device further comprises of a programable logic, such as Field Programmable Gate Array [FPGA] but is not limited to the same, which uses input from the optical detectors. The programmable logic is further connected to the Detection module and Sorting module. The Sorting module is connected to the sorting pulse generator which further manipulates the electrodes using dielectrophoretic AC pulse. In an embodiment, the device comprises at least one electrode. In an embodiment, the electrodes maybe selected based on a relay system triggered by the sorting module.

[0076] The present disclosure also relates to a method of detecting and sorting of droplets comprising of illumination of a sample droplet generated through the droplet generator with a combination of sample components and water-in-oil globules. The use of droplets in the platform provides significant advantages in sample handling, cell viability, detection chemistries which can be used and also the ability to measure secreted molecules, intracellular molecules, as well as contents from lysed cells. Other biomolecules such as protein, RNA and DNA can also be readily measured and sorted. The droplets are illuminated as they flow through the central flow channel and the subsequent emissions are detected in the form of electric signals, such as TTL Pulses but is not limited to the same, which is then transferred to the programmable logic, where the processing of the hi-speed pulses is done. The signals are then traversed to the detection module where the real-time identification of the droplet takes place. Based on the detection, the sorting module triggers a timed sorting of the High Voltage generator, while controlling the trigger pulse feature. The High Voltage Generator induces a dielectrophoretic AC Pulse to manipulate the electrodes to produce an appropriate force that would discriminate against droplet types, forcing them into appropriate sorting channels.

[0077] The present disclosure also relates to use of the multiplexed optofluidic device or system for sorting of droplets.

[0078] The present disclosure also relates to a multiplexed optofluidic fluorescence detector and sorter.

[0079] In an embodiment, the droplets comprise of components selected from a group comprising but not limiting to cells, beads, analytes, secreted molecules, intracellular molecules, lysed cells and its contents, biomolecules and combinations thereof..

[0080] In an embodiment, a feature of the present disclosure is the ability to detect the fluorescence of the droplet components even in very small working volumes. As the droplets flow down a central flow channel, at least one illumination source excites the droplets, whose signal is acquired by the optic fiber, which is coupled into a detector for a multiplexed optical detection. The digital pulse from the multiplexed optical detection is sent for signal processing which includes a Hi- speed pulse counter. In another embodiment, a feature of the present disclosure is the detection and sorting module microprocessor, which may use the read-out data to either conduct downstream computation or engage a sorting trigger on a sorting pulse generator which correspondingly manipulates the dielectrophoretic sorting of droplets.

[0081] In another embodiment of the present disclosure, the combination of the detection module and the sorting module in the device is a scalable, benchtop technology providing rapid and low cost solution for diagnostics and clinical sample processing and testing.

[0082] In another embodiment, the present disclosure overcomes the drawbacks of the prior art by providing a multi-parametric on-chip optical flow cytometer droplet analysis with simplified optics. It would be advantageous in resource-limited settings if the components were smaller, less expensive.

[0083] In another embodiment of the present disclosure, the device or system has the ability to detect the droplets of interest and enable a multi-parametric sorting, in at-least two channels, either subsequently or simultaneously.

[0084] In an embodiment, the present disclosure is applicable in the field of, but not limited to, single-cell genomics, drug screening, point-of-care diagnostics, precision medicine, regenerative medicine, CAR-T oncotherapy and more. The present disclosure maybe applied in the field of optical analysis of single cells or components in droplets for rare cell identification in clinical samples, isolation of specific cell populations and clones of interest or both phenotype and genotype information for single cell genomics.

[0085] An advantage of the present invention is the ability to obtain a fibre based multiparametric optical analysis of single microfluidic droplets with an on- demand droplet sorting into multiple channels using dielectrophoresis. Another advantage of the present invention is the real-time data visualization with single cell resolution multiplex fluorescence and multiparametric droplet sorting.

[0086] As shown in Figure 1, the schematic outlines the construction and functioning of the multiplexed optofluidic device or system with a water in-oil droplet-based detector and sorter. The multiplexed optofluidic chip includes at least two inlet channels, one through which the sample is pumped and another through which oil is pumped, to create a sample encapsulated in water-in-oil droplet. The droplets then flow through the central flow channel. The droplets are interrogated as they flow through the length of the flow channel. The droplets are excited based on the intensity of the optical fiber illuminating source. At least one wavelength output is received by at least one optical signal receiving fiber. The received signal is subjected to a multiplexed optical detection, upon which the TTL-level voltage pulses from the optical detectors like photomultiplier (PMT) tubes or Avalanche Photodiode (APD), but is not limited to the same, are processed to allow the current generated by the incident light to be multiplied many folds. In an embodiment, there is only one PMT or APD. The pulses are processed by implementing a pulse counter for each of the voltage pulses using a programmable logic. The Programmable logic is customized to count the pulses and store the received data in the storage module. In an embodiment, an integration time for the pulse counting may be set within the detection module, ideally being about lOOps, but is not limited to the same. The counts from each PMT may be read simultaneously from the storage module using interrupts, which may then be combined into a single communication packet, such as ethernet frame but is not limited to the same, and maybe sent out in a continuous data stream.

[0087] The received signal is analyzed by detecting the cells of interest which contain at least one pre-determined parameter. The droplets may also be selected based on a multi-parametric detection analysis. The data output from the detection module, along with its fluorescent signals, is uniquely identified in real-time. The data is then stored, visualized and relayed to the sorting module. The sorting module infers whether the droplet in question needs to be sorted into channel of interest or not. A Graphical User Interface (GUI) may stream the live data output and stores the data in the storage module. PMT outputs are overlaid on the GUI to obtain the corresponding scatter and fluorescence values at each timestamp. In an embodiment, the PMT pulse count data is processed to identify peaks using a signal extraction computer application program.

[0088] As the selected droplets, along with other droplets not of interest, flow through to the outlet end, there is a feedback control configured to trigger a Sorting Pulse Generator. The Sorting Pulse Generator is a High Voltage Generator that manipulates the dielectrophoretic AC pulse intensity of the electrodes. By manipulating the strength of electrode’s electric pulse, the droplets of interest may be sorted automatically into different channels for further sorting. The sorting may be based only on one parameter or multiple parameters. The sorting may also be based on simultaneous sorting of different cells of interest from a combination of cells in a sample mixture. The droplets maybe sorted through at least one sorted Channels. In an embodiment, sorting module initiates a GPIO Sorting trigger to the High Voltage Generator for the manipulation of at least one electrode with a dielectrophoretic AC pulse. In an embodiment, the method of multiplex optofluidic droplet sorting comprises of atleast one droplet flowing into an analyte flow channel, wherein said droplet is interrogated through atleast one illumination channel. The interrogating optical signal from the droplet is detected through atleast one optical signal from the interrogated droplet by at least one signal detector. A time integrated pulse count over time is plotted and time-correlated independent tracks for scatter and fluorescent signals is assessed and the detected optical signal in the scatter analysis block is analysed. The baseline noise cutoff is defined for the scatter and fluorescent signals, for the identification and marking of leading and lagging droplet peaks in order to define a region of interest, by conducting a droplet boundary analysis. By using peak height and width for droplet characterization, further automated multiplex fluorescence signal allocation is conducted for each component such as cell in a droplet, in the fluorescence analysis block. The regions of interest are superimposed on fluorescence channels and fluorescent peaks are demultiplexed based on expected time delays between colour signals. The raw peak data is processed for colour compensation and multicolour fluorescence [FL] profile is assigned to each droplet and fluorescence is quantified. The sorting module defines the sorting logic and channel selection based on optical parameters and generates a sorting trigger to the High Voltage Generator. The High Voltage Generator manipulates the voltage intensity to the electrodes for sorting of droplets into multiple sorting channels, creating a negative energy to pull the droplet to a specific sorting channel. The droplets are also analyzed and differentiated based on droplets containing single or multiple cells.

[0089] As illustrated in Figure 2, multiple formats of designs representing multiple channel sorting is depicted but is not limited to the same. Figure 2(a) depicts an embodiment of the design of the device, wherein the microfluidic chip comprises of a 3 way Y shaped sorting junction, without a bend in the sorting channel. Figure 2(b) depicts an embodiment of the design of the device wherein the device comprises of a simplex construction with a 3 way parallel sort with bend in the sorting channel. Figure 2(c) depicts an embodiment of the design of the device, wherein the multiplex device comprises of 3 inlet droplet generators, a single laser-multi detector based multiplex optical interrogation site with a single sorter junction. Figure 2(d) depicts an embodiment of the design of the device, wherein the microfluidic chip comprises a 5 way sorter junction without a bend in the sorting channel. In an embodiment, the sorting channel dimensions may be customized based on the droplet size. In some embodiments, the channel heights are about 10 to 130 um, but are not limited to the same. In some embodiments, the channel heights maybe a combination of different heights, such as the combination of 10 and 130um or channels are of equal heights within the range of about 10 to 130 um, but are not limited to the same. Figure 2(e) depicts an embodiment of the design of the device, wherein the microfluidic chip comprises of a single detector based multiple optical interrogation site with a single sorter without a bend in the sorting channel. Figure 2(f) depicts an embodiment of the design of the device, wherein the microfluidic chip comprises of a simplex structure with a 3 way parallel sorter without a bend in the sorting channel. In an embodiment, the design is 125um in depth, but is not limited to the same. In an embodiment, the designs may be made of polymers such as but not limiting to PDMS [Poly dimethylsiloxane], glass, 3D printed or a combination thereof, but is not limited to the same.

[0090] In an embodiment, the detection module performs signal collection, processing and demultiplexing to extract raw signals. The read-out data is processed in real time to identify droplets, scatter and fluorescence signals from the samples within. In another embodiment, there may be a detection setup, wherein the leading and lagging peaks in the scatter signals are used to mark the droplet boundaries. The droplet boundary analysis may contain individual peaks for the component scatter and fluorescence, which may be identified and assigned to respective droplets. Each peak data may be analyzed to obtain the signal intensity values which is corresponding to the peak height. As seen in Figure 3, the detector module encaptures the peak signals of the 3 colour dye droplet. The aqueous sample consists of a combination of water, Fluorescein isothiocyanate (FITC), R-phycoerythrin (PE) and PerCP dyes. Each droplet contains a mix of 3 fluorescent dyes. As a standardization means for the detection module, individual fluorescence signals are recorded as compared to blank and the combination of the 3 dyes. The height and width of the peak is calculated to detect and quantify the fluorescence peak. Figure 3(a) depicts a droplet devoid of any colour dye. Figure 3(b) depicts the droplets with FITC dye which may be detected at 66nM concentration and Figure 3(c) depicts the intensity of the PE dye, which may be used at IpM, but is not limited to the same. Figure 3(d) depicts the intensity peaks of PerCp-eFlour710 dye and Figure 3(e) depicts the cumulative intensity peaks of the combination of the 3 colour dyes. Each peak of FITC, PE or PERCP is based on the distance from the droplet scatter peak. The fluorescence maybe different FL1, FL2 and FL3 based on the excitation laser. In an exemplary example, as illustrated in Figure 3(e), a 488 nm laser output is split into 3 optical fibres. The scatter signal is collected in 1 PMT and the three fluorescence signals are coupled into the 2ndPMT, with an inherent time delay due to the placement of the optical fibres. It may further be seen that there may be spilling over of a peak of a single colour dye onto the peak of another dye. For the mitigation of the same, fluorescence compensation is conducted in the detection module. The fluorescence compensation applied, as illustrated in Figure 4, shows the representation of the data acquisition and processing from single laser-single detector based multiplex setup. The illustration depicts the droplet scatter and 3 color fluorescence data from droplets containing a mix of FITC, PE and PerCP eFluor 710 dyes. In the given figure 4, a single 488 nm laser for excitation and a single PMT is recorded for time delayed FL signals. The blue peak represents droplet scatter signals and marks the beginning of the droplet, which is the output of PMT 1. The pink line depicts the fluorescence signals corresponding to FITC, PE and PerCP dye bandwidths, which is the output of PMT 2. The detection module calls the intensity peaks and delineates the presence of specific fluorescence signals, for each droplet. The peaks are assessed in real-time and the spectral overlap is addressed through fluorescence compensation, wherein overlap is corrected by adjusting the signal from each fluorochrome, ensuring that the detected fluorescence accurately reflects the intended marker. The compensation occurs in real-time as the droplets flow across the optical interrogation point.

[0091] In an embodiment, the present disclosure may include a single- detector, wherein a multi-colour setup and an extra layer of demultiplexing the time-delay signals is performed. The embodiment would take into account the fluid flow rates, observed time delays, droplet characteristics and other conditionalities. The region of interest of the intensity-event graph is demarcated and the fluorescence peaks falling within the region of interest are identified. In an embodiment, the region of interest is the beginning of the droplet and the end of the droplet, which is the region between two scatter peaks. Further, each peak is categorized as either FITC, PE or PerCP, as per the standardization depicted in Figure 3(b), 3(c) and 3(d).

[0092] In an embodiment, peak parameters like width, height, etc are calculated above background signal noise. Compensation tracks are created for each run using single FL controls. The compensation matrix is used to process raw acquired data to give final data. Peak parameters are further used to quantify FL signals.

[0093] In an embodiment, the real-time fluorescence peak estimation unit of the sorting module takes user-inputted sorting conditions. The sorting conditions are computed by a sorting condition computation unit, based on signal threshold settings, a combination of fluorescence parameters as sorting requirements. Once the droplet qualifies for sorting, it is selected to be sorted and a trigger is sent to the high-voltage generator by the timed sorting trigger unit, where an AC Voltage pulse is produced which is precisely timed to match the droplet entering the electrode boundaries by taking into account the fluid flow rates, observed time delays, droplet characteristics and other conditionalities, either individually or in combination and monitored by a trigger pulse feature control unit. The electrode boundaries include a non-uniform electric field around the sorted channel at the distal end of the central flow channel.

[0094] In an embodiment, the droplet moves towards the direction of increasing field strength where the droplets are attracted towards at least one sorting channels. The pulse characteristics (amplitude, duration) decide which sorted channel the sample is diverted to. As elucidated in figure 5, the multiplex module includes the optical setup which illuminates the fluidics that flow through the central channel. There may be multi-fluorescent signals emanating from each droplet, which are collected by the optical signal receivers and configured with the detection unit. The detection unit selects the droplet of interest based on at least one predetermined parameter. The detection module comprises a fluorescence signal demultiplexing unit, realtime droplet identification unit, data storage unit, and livestream visualization unit. As the droplets flow to the end of the central flow channel, a feedback control in the sorting module triggers an electric impulse to such an intensity that the droplet of interest is forcefully manipulated into a predetermined tubing pathway. The sorting may occur only based on a single parameter or multi-parameter selection. In an embodiment, there may be a separation of various cells of choice, manipulated through several different tubing pathways to segregate multiple cells of interest for further processing. The sorting of the droplets is automatized and has extremely high feedback speed. The sorting frequency is optimum at about 10-20hz but is not limited to this range.

[0095] The flowchart illustrating the same process is illustrated in Figure 5(b), wherein the detection module comprises of a scatter analysis block wherein the baseline noise cutoff is defined for the scatter and fluorescent signals, for the identification and marking of leading and lagging droplet peaks in order to define a region of interest and use the peak height and width for droplet characterization. In an embodiment, the detector module may also include a fluorescence analysis block, wherein the regions of interest are superimposed on fluorescence channels and fluorescent peaks are demultiplexed based on expected time delays between colour signals. The fluorescence analysis block processes raw peak data for color compensation and assigns multicolour FL profile to each droplet and quantify the fluorescence.

[0096] An exemplary example of the multiplexed optofluidic device and system detecting and sorting MHC stained MEF sorting from a mix of MEF and THP1 cell population is illustrated in Figure 6, wherein the sample consists of THP1 cells, stained with CD14-AF488 whose fluorescence emission is in the range of 525nm and falling within the green emission spectrum, and IFNg stimulated MEF MHC 1 (PE) and MHCII (FITC) stained cells. Figure 6a depicts the scatter plot of PE-H vs FITC_H, wherein the sample comprises of blank cells depicted in grey, THP1 cells depicted in green, and MEF cells depicted in pink. The Multiplexed optofluidic device and system can detect and differentiate between atleast two different cell populations, but is not limited to the same. On the contrary, the standard flow cytometer as depicted in 6(a) may only sort one type of cell at a single time. Further, based on the read-out data processed by the detection module, the droplets are assessed based on the time delay, intensity of fluorescence, and a sorting trigger is sent by the sorting module. In an embodiment, a TTL-level voltage pulses from the PMTs were processed by implementing a pulse counter for each of them using the FPGA (Field Programmable Gate Array) chip on the Digilent Zedboard (Xilinx Zynq 7000 SoC). The integration time for pulse counting maybe set to lOOps within the hardware.

[0097] The counts from each PMT are read simultaneously from the memory using interrupts, then combined into a single communication packet and sent out in a continuous data stream. The sorting module takes user-inputted sorting conditions. Once a droplet qualifies for sorting, a trigger is sent to the High Voltage [HV] generator. The HV generator will produce an AC voltage pulse that is precisely timed to match the droplet entering the electrode boundaries. This creates a non-uniform electric field around the sorter junction. This is illustrated in Figure 6(b) wherein the trigger is communicated when the fluorescence spectrum achieves the trigger threshold previously set.

[0098] When a trigger of a particular intensity is communicated to the High Voltage generator, the droplet is forced to move towards the direction of increasing field strength and is attracted towards the sorting channel. As seen in Figure 6(c), the droplet is made to follow the path into the negative channel, based on the pulse characteristics such as but not limited to the amplitude and duration. Naturally, the droplets would flow into the waste channel 601, however on application of a specific pulse at a specific pre-determined intensity, the droplets of interest are made to follow the sorting channel of interest 602. Further, the microprocessor also sets the cooldown between triggers upon initialization. In one embodiment, as seen in Figure 7, the sorting junction cross-section illustrates unsorted droplets flow straight through the middle channel (a) and the droplets with fluorescent cells getting sorted to the bottom channel in (b). 701 depicts at least one electrode, but is not limited to the same. In a preferred embodiment, there are atleast two electrodes, but is not limited to the same and the sorting module decides which electrode is to be triggered, in order to direct the droplets into specific channels. The sorting module may also relay the switching of electrodes, based on the channel of interest. 702 illustrates the waste channel which is the default flow of droplets, wherein on triggering of an impulse, electrode 701(a) maybe engaged to divert one type of cell / droplet to flow through channel 703.

[0099] While the foregoing describes various embodiments of the disclosure, other and further embodiments of the disclosure may be devised without departing from the basic scope thereof. The disclosure is not limited to the described embodiments, versions, or examples, which are included to enable a person having ordinary skill in the art to make and use the disclosure when combined with information and knowledge available to the person having ordinary skill in the art.

Claims

We Claim:

1. A multiplexed optofluidic device comprising: a droplet inlet channel; an analyte flow channel wherein atleast one droplet flows through; an illumination channel wherein the droplet flowing through the analyte flow channel is interrogated; a multiplexed optical detector wherein fluorescence output of interrogated droplets is detected through optical fibres; a programmable logic comprising a signal processing unit and a Hi- speed pulse counter unit to gather the read-out data of the optical detector; a storage module which stores the read-out data of the optical detector; a detection module to process the detected signal of the logic and analyse based on optical parameters; multi-parametric sorting module which defines sorting logic and channel selection based on optical parameters, to induce a sorting trigger;High Voltage Generator to induce a dielectric voltage to at least one electrode based on the sorting trigger; and sorting junction wherein the said sorting junction separates into multiple sorting channels.

2. The multiplexed optofluidic device as claimed in claim 1, wherein the detection module comprises of a fluorescence signal demultiplexing unit, real-time droplet identification unit, data storage unit and livestream visualization unit.

3. The multiplexed optofluidic device as claimed in claim 1, wherein the interrogated droplets are water-in-oil droplets.

4. The multiplexed optofluidic device as claimed in claims 1, wherein the droplets comprise of components selected from a group comprising cells, beads, analytes, secreted molecules, intracellular molecules, lysed cells and its contents, biomolecules and combinations thereof.

5. The multiplexed optofluidic device as claimed in claim 1, wherein the detection module comprises of a scatter analysis block and a fluorescence analysis block.

6. The multiplexed optofluidic device as claimed in claim 1, wherein the sorting module comprises of a real-time fluorescence peak estimation unit, sorting condition computation unit, timed sorting trigger unit and a trigger pulse feature control unit.

7. The multiplexed optofluidic device as claimed in claim 1, wherein the channel heights are all equal or have a combination of different heights.

8. The multiplexed optofluidic device as claimed in claim 1, wherein the detection module demarcates a region of interest of each droplet, by conducting a droplet boundary analysis, and identifies fluorescence peaks within.

9. The multiplexed optofluidic device as claimed in claim 1, wherein the programmable logic implements a pulse counter for each electric signal and stores the received read-out data from the optical detectors, while combining the counts from each optical detector from the storage module into a single communication packet.

10. The multiplexed optofluidic device as claimed in claim 1, wherein the optical detectors are photomultiplier tubes (PMT) or Avalanche Photodiode (APD).

11. The multiplexed optofluidic device as claimed in claim 10, wherein the optical detector pulse count is processed to identify leading and lagging peaks in scatter signals marking the boundary of each droplet and assigning each droplet.

12. The multiplexed optofluidic device as claimed in claim 11, wherein each peak is, categorized in the fluorescence of the excitation laser, based on the distance from the droplet scatter peak.

13. The multiplexed optofluidic device as claimed in claim 1, wherein the sorting module initiates a General Purpose Input / Output (GPIO) Sorting trigger to the High Voltage Generator for manipulation of at least one electrode with a dielectrophoretic AC pulse.

14. The multiplexed optofluidic device as claimed in claim 1, wherein fluorescence signals, time delay signals, fluid flow rates, droplet scatter characteristics are received by the detection module, for inferring the need for sorting.

15. The multiplexed optofluidic device as claimed in claim 1, wherein the sorting module compares the received data to pre -determined threshold values and correspondingly triggers sorting pulses to the High Voltage generator to manipulate droplets into sorting channels.

16. The multiplexed optofluidic device as claimed in claim 1, wherein the number of sorting channels correspond to the number of droplet types being detected and sorted in one run.

17. The multiplexed optofluidic device, claimed in claim 1, wherein the sorting module conducts electrode switching for sorting trigger initiation, based on the sorting channel of interest.

18. The multiplexed optofluidic device as claimed in claim 16, wherein pulse characteristics decide the channel for sorting.

19. The multiplexed optofluidic device as claimed in claim 1, wherein a lack of sorting trigger by the sorting module leads to the droplet flowing through the default waste channel.

20. The multiplexed optofluidic device as claimed in claim 1, wherein the electrode(s) generate negative force to manipulate the path of the droplets.

21. The multiplexed optofluidic device as claimed in claim 1, wherein the device is made of glass.

22. The multiplexed optofluidic device as claimed in claim 1, wherein the device is made of polymers.

23. The multiplexed optofluidic device as claimed in claim 1, wherein the sorting junction is in a 3-way Y sort with or without bend in the sorting channel.

24. The multiplexed optofluidic device as claimed in claim 1, wherein the sorting junction is in a 3 -way parallel sort with or without bend in the sorting channel.

25. The multiplexed optofluidic device as claimed in claim 1, wherein the sorting junction is in a 5-way parallel sort with or without bend in the sorting channel.

26. A method of using the multiplexed optofluidic device as claimed in claim 1, wherein the method comprising interrogation of a water-in-oil droplet, wherein the output data of the interrogation droplet is received by a multiple optical detection along with signal processing by theprogrammable logic and transmitted to the detection module wherein the detection module performs demultiplexing of raw signals and the readout data is transferred to the sorting module, which defines the sorting logic and channel selection based on optimal parameters, upon which a sorting trigger is transmitted to a High Voltage generator which manipulates the intensity of the electrodes for sorting of droplets into multiple sorting channels.

27. The method as claimed in claim 26, wherein the droplets comprise of components selected from a group comprising, cells, beads, analytes, secreted molecules, intracellular molecules, lysed cells and its contents, biomolecules and combinations thereof.

28. The method as claimed in claim 26, wherein the detection module comprises a scatter analysis block wherein the baseline noise cutoff is defined for the scatter and fluorescent signals, for identification and marking of leading and lagging droplet peaks in order to define a region of interest, by conducting a droplet boundary analysis, and use the peak height and width for droplet characterization by multiplex signal allocation to each component in the droplet.

29. The method as claimed in claim 26, wherein the detection module comprises a fluorescence analysis block, wherein regions of interest of the droplets are superimposed on fluorescence channels and fluorescent peaks are demultiplexed based on expected time delays between colour signals.

30. The method as claimed in claim 29, wherein the fluorescence analysis block processes raw peak data for colour compensation and assigns multicolour fluorescence profile to each droplet and quantify the fluorescence.

31. The method as claimed in claim 26, wherein the programmable logic implements a pulse counter for each electric signal from and stores the read-out data from the optical detectors, while combining the counts from each optical detector from storage module while combining into a single communication packet and wherein optical detector pulse count is processed to identify leading and lagging peaks in scatter signals marking the boundary of each droplet and assigning each droplet.

32. The method as claimed in claim 26, wherein fluorescence signals, time delay signals, fluid flow rates, droplet scatter characteristics are received by the detection module, for inferring the need for sorting and subsequently the sorting module initiates a General Purpose Input / Output (GPIO) Sorting trigger to the High Voltage Generator for the manipulation of at least one electrode with a dielectrophoretic AC pulse.

33. The method as claimed in claim 26, wherein the sorting module defines the sorting logic and channel selection based on optical parameters and initiates a sorting trigger.

34. The method as claimed in claim 26, wherein the sorting is multipar ametric.

35. A method of multiplex optofluidic droplet sorting comprising: atleast one droplet flowing into an analyte flow channel, wherein said droplet is interrogated through atleast one illumination channel; detecting atleast one optical signal from the interrogated droplet by at least one signal detector, plotting a time integrated pulse count over time, assessment of time-correlated independent tracks for scatter and fluorescent signals;analysing the detected optical signal in scatter analysis block wherein baseline noise cutoff is defined for the scatter and fluorescent signals, for identification and marking of leading and lagging droplet peaks, by an optical detector, in order to define a region of interest and use the peak height and width for droplet characterization; further analysing the optical signal in fluorescence analysis block, wherein regions of interest are superimposed on fluorescence channels and fluorescent peaks are demultiplexed based on expected time delays between colour signals, processing raw peak data for colour compensation and assigning multicolour fluorescence profile to each droplet and quantifying the fluorescence; defining sorting logic and channel selection based on optical parameters and generating a sorting trigger to High Voltage Generator; and manipulating intensity of electrodes for sorting of droplets into multiple sorting channels and creating a negative energy to pull the droplet to a specific sorting channel.

Images