DESC:FIELD OF THE INVENTION
[001] The present invention relates to separation of microparticles from a multicomponent colloidal solution.

BACKGROUND OF THE INVENTION
[002] In conventional microfluidic devices, specifically microfluidic devices using continuous flow-based separation, inertial or hydrodynamic forces of microparticles for separation from the suspension fluid are utilised. Alternatively, separation can also be achieved by application of external forces such as dielectrophoretic force, magnetic force, optical force, acoustic force, centrifugal force etc., or a combination thereof. Irrespective of the forces used, the separation efficiency and yield of these devices are constrained to a narrow range of inlet flow rate, a constrained range of inlet microparticle concentration and limited to a single step separation process.
[003] On the basis of separation, multicomponent colloidal suspensions are divided into three distinct components namely, the suspension fluid, which constitutes the fluid medium in which microparticles are dispersed; the characteristic microparticles, which participate in the separation process and whose concentration decrease in the suspension fluid outlet line and increase in the microparticle enriched outlet line; and other microparticles which do not participate in the separation process and generally pass through the suspension fluid outlet line.
[004] The net effect of separation on the characteristics microparticles is captured as their effective concentration in the outlet suspension fluid line. When concentration of the microparticles in the inlet fluid is beyond a workable value, separation using existing microfluidic flow-based separation devices is not feasible.
[005] While subjected to very high or low inlet flow rates, the efficiency of these microfluidic separation devices reduce drastically, making them ineffectual for use in applications with continuously flowing inlet streams, as in the case of real-time sampling and testing of blood parameters during procedures such as open heart surgeries, blood exchange transfusion, extracorporeal membrane oxygenation, kidney dialysis etc.
[006] For example, during open heart surgeries, the functioning of the patient’s heart and lungs are temporarily taken over by a cardiopulmonary bypass pump or heart-lung machine. While connected to the heart-lung machine, vital blood or biological parameters are monitored in order to maintain effective functioning of the patient’s body. Blood sample is withdrawn from the patient every 20 to 30 minutes and the plasma is separated for carrying out laboratory testing procedures for multiple parameters such as blood coagulation, arterial blood gas analysis, mixed venous oxygen saturation, blood base deficit, lactate concentration etc. This process is elaborate, time consuming and the reported results have a time lag, hence the values don’t correspond to the condition of the patient undergoing the procedure at the given instant. This lag can prove fatal especially when an undetected blood clot accidently blocks the heart-lung machine or there is inadequate end-organ perfusion which can result in organ failure.
[007] Thus, there is a need in the art for which addresses at least the aforementioned problems.

SUMMARY OF THE INVENTION
[008] In one aspect, the present invention is directed at a device for separation of characteristic microparticles from a multicomponent colloidal solution. The device has a separation module having at least one microfluidic separation channel. Herein, the microfluidic separation channel is configured to separate the multicomponent colloidal solution into a suspension fluid and a microparticles enriched fluid. The microfluidic separation channel has an inlet for receiving a sample of the multicomponent colloidal solution, a first outlet for exiting the suspension fluid, and a second outlet for exiting the microparticles enriched fluid. The device further has a replenishing module that is configured to receive the microparticles enriched fluid from the second outlet and a replenishing fluid to form a replenished colloidal solution suitable for being supplied/supplied back to the inlet for separation into the suspension fluid and the microparticles enriched fluid, thereby achieving an enhanced yield of the microparticles enriched fluid or the suspension fluid as per need..
[009] In an embodiment of the invention, the separation module has the microfluidic separation channels connected in series connection. In an alternate embodiment, the separation module has the microfluidic separation channels connected in parallel connection.
[010] In an embodiment of the invention, the replenishing module has a port and valve arrangement for holding and supplying the replenishing fluid.
[011] In another embodiment of the invention, the device has an inlet module configured for supplying desired volume of the sample of the multicomponent colloidal solution to the separation module. The inlet module is connected to the inlet of the separation module and the replenishing module. In an embodiment, the inlet module has one or more port and valve arrangements for supplying desired volume of the sample of the multicomponent colloidal solution to the separation module.
[012] In another embodiment of the invention, the device has an outlet module configured for receiving the suspension fluid exiting the first outlet and the microparticles enriched fluid from the second outlet, and transmitting the microparticles enriched fluid exiting the second outlet to the replenishing module. In an embodiment, the outlet module has two or more port and valve arrangements for exiting the suspension fluid and microparticles enriched fluid from the separation module.
[013] In a further embodiment of the invention, the device has a pumping module connecting the inlet module to the separation module, for achieving a desired flow rate in the separation module.
[014] In another embodiment of the invention, the device has a venting module with a port and valve arrangement for removing any pre-filled air, priming fluid or rinsing fluid from the device.
[015] In a further embodiment of the invention, the multicomponent colloidal solution is one of pigmented ink, paint, blood, urine, cerebrospinal fluid (CSF), biochemical samples, water, food or fuel with impurities.
[016] In a further embodiment of the invention, the multicomponent colloidal solution is blood, and the replenishing fluid received by the replenishing module, is blood.
[017] In a further embodiment of the invention, the multicomponent colloidal solution is blood and the replenishing fluid received by the replenishing module, is a buffer solution. In an embodiment, the buffer solution is selected from a group comprising distilled water, 0.8% sodium chloride solution or saline, phosphate buffered saline (PBS), or RBC diluting fluids.
[018] In a further embodiment of the invention, the inlet module, the replenishing module and the venting module are pooled together to form a single pool module.
[019] In a further embodiment of the invention, the minimum sample volume of blood required for the device is 20µl. In an embodiment, the device has an upstream processing module for adding and mixing reagents to the blood entering the separation module, and a downstream processing module for processing separated plasma from the separation module. In an embodiment, the device has an inlet control module for checking quality of the blood entering the device and an outlet control module for checking quality of the suspension fluid or plasma existing the device.
[020] In another embodiment of the invention, the inlet control module has a flow pulsation damper for reducing pulsations in flow or pressure of blood in the device, and a clot capturing channel to capture clots and impurities in the blood from entering the device. Further, the outlet control module has a microparticle concentrator to increase the separation efficiency by removing any stray microparticles from the suspension fluid.
[021] In another embodiment of the invention, the inlet module has a miniature evacuated or non-evacuated chamber covered with an accessible top rubber sealing. In an alternative embodiment, the inlet module is configured to receive barrel of a syringe directly on the device such that the barrel itself acts as a sample chamber. In another alternative embodiment, the inlet module has a needle valve arrangement configured to receive samples from vacutainer tubes or sealed chambers accessible through a rubber sealing.
[022] In a further embodiment of the invention, the minimum sample volume of blood required for the device ranges from 100µl to 100ml. Herein, the device has a working fluid storage module to store finite quantity of blood. In an embodiment, the device is adapted in an Automated Plasma Culture Testing Device. In another embodiment, the device is adapted for a fully-automated plasma separation device, which is integrated with downstream automated immunoassay or diagnostic testing. In an embodiment, the device is adapted for a downstream automated HIV TRI-DOT testing.
[023] In a further embodiment of the invention, the sample of the blood for the device is a continuous flow source. In an embodiment, the device has a sample collection module connected to the working fluid storage module to store finite quantity of the blood. In another embodiment, the device has a disposal collection module or collecting any overflow such as rinsing fluid, unused plasma, or suspension fluid, from the venting module and the output module. In an embodiment, the device is adapted for a downstream continuous blood quality monitoring system.

BRIEF DESCRIPTION OF THE DRAWINGS
[024] Reference will be made to embodiments of the invention, examples of which may be illustrated in accompanying figures. These figures are intended to be illustrative, not limiting. Although the invention is generally described in context of these embodiments, it should be understood that it is not intended to limit the scope of the invention to these particular embodiments.
Figure 1 is a schematic representation of a device for separation of characteristic microparticles from a multicomponent colloidal solution, in accordance with an embodiment of the invention.
Figures 2A and 2B illustrate the device with blood as replenishing fluid for separation of blood as multicomponent colloidal solution, and priming and working configuration, in accordance with an embodiment of the invention.
Figure 3 illustrates a graphical comparison of yield and purity of plasma separated using the device with increasing number of multi-passes for blood as replenishing fluid, in accordance with an embodiment of the invention.
Figure 4A and 4B illustrate the device with buffer solution as replenishing fluid for separation of blood as multicomponent colloidal solution, and priming and working configuration, in accordance with an embodiment of the invention.
Figure 5 illustrates a graphical comparison of yield and purity of plasma separated using the device with increasing number of multi-passes for buffer solution as replenishing fluid, in accordance with an embodiment of the invention.
Figure 6 illustrates a single pool module configuration of the device, in accordance with an embodiment of the invention.
Figure 7 illustrates configuration of the device for handling various input volumes of the multicomponent colloidal solution, in accordance with an embodiment of the invention.
Figure 8 illustrates a flow pulsation damper of an inlet control module, in accordance with an embodiment of the invention.
Figure 9 illustrates the flow pulsation damper of the inlet control module, in accordance with an embodiment of the invention.
Figure 10 illustrates a clot capturing channel of the inlet control module, in accordance with an embodiment of the invention.
Figure 11 illustrates a microparticle concentrator of an outlet control module, in accordance with an embodiment of the invention.
Figure 12 illustrates the implementation of the device for a droplet plasma separation device, in accordance with an embodiment of the invention.
Figure 13 illustrates the implementation of the device for an Automated Plasma Culture Testing Device, in accordance with an embodiment of the invention.
Figure 14 illustrates the implementation of the device for a fully-automated plasma separation device, in accordance with an embodiment of the invention.
Figure 15 illustrates the implementation of the device for a downstream continuous blood quality monitoring system, in accordance with an embodiment of the invention.
Figure 16 illustrates an exemplary parallel connection of the microfluidic separation channels and a routing layer, in accordance with an embodiment of the invention.
Figures 17A-17F illustrates various configurations of parallel combination of the microfluidic separation channels, in accordance with corresponding embodiments of the invention.
Figure 18A-18C illustrates various configurations of the routing layer, in accordance with corresponding embodiments of the invention.
Figure 19 illustrates the device with microfluidic separation channels of the separation module connected in series with each other, in accordance with an embodiment of the invention.
Figure 20 illustrates the device with microfluidic separation channels of the separation module connected in series with each other, in accordance with an alternate embodiment of the invention.

DETAILED DESCRIPTION OF THE INVENTION
[025] The present invention relates to separation of microparticles from a colloidal solution. More particularly, the present invention relates to a device for separation of microparticles from a multicomponent colloidal solution.
[026] Figure 1 illustrates a device 100 for separation of characteristic microparticles from a multicomponent colloidal solution, in accordance with an embodiment of the invention. As illustrated in Figure 1, the device 100 has a separation module 110. The separation module 110 has one or more microfluidic separation channels 112 (shown in Figure 2) that are configured to separate the multicomponent colloidal solution into a suspension fluid and a microparticles enriched fluid. Herein, the microfluidic separation channel 112 has an inlet (i) (shown in Figure 2) for receiving a sample of the multicomponent colloidal solution, a first outlet (d) (shown in Figure 2) for exiting the suspension fluid, and a second outlet (o) (shown in Figure 2) for exiting the microparticles enriched fluid. In an embodiment of the invention, the microfluidic separation channels 112 are configured for continuous flow separation with a known maximum yield ?max for an optimum flow rate ƒopt and separation efficiency ?max. Separation efficiency ? is defined as the percentage of characteristic microparticles present in the microparticles enriched fluid being separated from the multicomponent colloidal suspension. For instance, if the multicomponent colloidal solution is blood, separation efficiency ? is defined as the ratio of percentage of RBCs remaining in the separated plasma to that of the blood.
[027] Herein, the multicomponent colloidal solution can be one of pigmented ink, paint, blood, urine, cerebrospinal fluid (CSF), biochemical samples, water, food or fuel with impurities.
[028] The device 100 further has a replenishing module 150. Herein, the replenishing module 150 is configured to receive the microparticle enriched fluid from the second outlet (o) and a replenishing fluid to form a replenished colloidal solution. In that, the replenished colloidal solution is suitable for being supplied back to the inlet (i) for separation into the suspension fluid and the microparticles enriched fluid. This enables multi-passing of multicomponent colloidal solution through the separation module 110. In an embodiment, the replenishing module 150 has a port and valve arrangement for holding and supplying the replenishing fluid. The multi-passing of the multicomponent colloidal solution thereby achieves an enhanced yield of the microparticles enriched fluid or the microparticle free suspension fluid as per need. Yield for the purpose of this invention, is understood as the ratio of volume of microparticles separated from the multicomponent colloidal suspension to volume of the inlet volume of the multicomponent colloidal solution. For instance, if the multicomponent colloidal solution is blood, and the microparticles that are to be separated from blood is plasma, yield is defined as the percentage ratio of volume of plasma separated to the inlet volume of blood.
[029] The enhanced yield achievable by the device 100 for the multicomponent colloidal suspension with an initial microparticle concentration of R1 following ‘n’ number of multi-passes through the separation module 110, is given by the equation:

where, is the yield of the microfluidic separation channel 112 as a function of particle concentration and flow rate for the ith multi-pass; Vi is the inlet volume of the multicomponent colloidal solution; is the separation efficiency of the microfluidic separation channel 112 as a function of microparticle concentration and flow rate for the ith multi-pass; Dr is the dilution ratio which represents the dilution of the suspension fluid with the replenishing fluid, automatically controlled by the device 100 for any effective concentration of characteristic microparticles in the inlet (i). The cumulative dilution fraction following ‘n’ number of multi-passes through the microfluidic separation channel 112 is given by the equation:

where, is the buffer volume fraction in the microfluidic separation channel 112 before ith pass; is the flow rate in the microfluidic separation channel 112 before ith pass and R1 is the initial microparticle concentration for the multicomponent colloidal solution and is the microparticle concentration in microfluidic separation channel 112 before the ith pass. Therefore, the device 100 can achieve desired concentration of microparticles in the separated microparticles enriched fluid. This allows the user to obtain the microparticles at a user specified concentration.
[030] As further illustrated in the embodiment depicted in Figure 1, the separation module 110 is connected to an inlet module 120 which has one or more inlet port and valve arrangements, for inputting desired volume of blood sample for separation. The inlet module 120 is connected to the inlet (i) of the microfluidic separation channel 112 in the separation module 110 and the replenishing module 150, to receive the replenished colloidal solution from the replenishing module 150 and supply the same to the separation module 110. To further facilitate multi-passing of the multicomponent colloidal solution, the device 100 comprises a pumping module 130 connecting the inlet module 120 to the separation module 110, for achieving a desired flow rate in the separation module 110. The pumping module 130 comprises of one or more pumping systems selected from a group comprising peristaltic pumps, ring pump, gear pump, piston or syringe pump, membrane-based pumps like piezoelectric pumps, solenoid pump, manual pumps etc. The pumping module 130 also holds any required pump accessories such as controls for the pump, ports and valves, and reservoirs for generating the pumping action.
[031] In an embodiment, the device 100 further comprises of an outlet module 140 configured for receiving the suspension fluid exiting the first outlet (d) and the microparticles enriched fluid from the second outlet (o), and transmitting the microparticle enriched fluid exiting the second outlet (o) to the replenishing module 150. Herein, the outlet module 140 has two or more port and valve arrangements for exiting the suspension fluid and microparticles enriched fluid from the separation module 110.
[032] As further illustrated in the embodiment depicted in Figure 1, the device 100 has a venting module 160 with a port and valve arrangement for removing any pre-filled air, priming fluid or rinsing fluid from the device 100.
[033] Figures 2A and 2B illustrate an embodiment of the device 100 wherein the multicomponent colloidal solution is blood and the replenishing fluid received by the replenishing module 150, is blood. As illustrated in Figures 2A and 2B, a single valve 114 configuration switches the device 100 between a priming circuit as illustrated in Figure 2A and a working circuit as illustrated in Figure 2B. When the device 100 is in the priming circuit mode, blood is filled in the device 100 for removal of any prefilled air or other impurities from the device 100 before initiating the separation. In this embodiment, the inlet module 120 also acts as the replenishing module 150 since the multicomponent colloidal solution and the replenishing fluid are the same, that is blood. Initially, the valve 114 is in position as shown in Figure 2A, when blood is added into the inlet module 120. In this valve 114 position, the pumping module 130 is started for priming the device 100, wherein the blood is drawn into the device 100 and pushed through the separation module 110. Following priming of the device 100, the blood reaches the venting module 160 and the pumping module 130 is stopped momentarily.
[034] Thereafter, the device 100 is changed into a working circuit by positioning the valve 114 as shown in Figure 2B. In that, the inlet module 120 starts acting as the replenishing module 150 for replenishing the microparticle enriched fluid from the separation module 110 for multi-passing. The pumping module 130 facilitates initiation of separation, and plasma is collected from the first outlet (d). Figure 3 illustrates a graphical comparison of yield and purity of plasma separated using the device 100 from 1 ml of blood having varied hematocrit concentration (Hct) namely (a) 37% Hct blood (b) 45% Hct blood and (c) 60% Hct blood respectively. Herein, hematocrit concentration is defined as the ratio of volume of red blood cells to the total volume of blood.
[035] Figures 4A and 4B illustrate an embodiment of the device 100 wherein the multicomponent colloidal solution is blood and the replenishing fluid received by the replenishing module 150, is a buffer solution. The buffer solution can be selected from a group comprising distilled water, 0.8% sodium chloride solution or saline, phosphate buffered saline (PBS), RBC diluting fluids etc. Initially, the valve 114 is in position as shown in Figure 4A when blood is added into the inlet module 120. In this valve 114 position, the pumping module 130 is started for priming the device 100, wherein blood is drawn into the device 100 and pushed through the separation module 110. Following priming of the device 100, the blood reaches the replenishing module 150 and the pumping module 130 is stopped momentarily.
[036] Thereafter, the device 100 is changed to working configuration by changing the position of the valve 114 as shown in Figure 4B and the inlet module 120 is disconnected from the separation module 110. The replenishing module 150 is filled with the buffer solution as replenishing fluid and the pumping module 130 is started to initiate separation. The running time of the device 100 depends on the desired plasma volume. Plasma is collected from the first outlet (d) of the microfluidic separation channel 112 of the separation module 110. Figure 5 illustrates a graphical comparison of yield and purity of plasma separated using the device 100 with increasing number of multi-passes and buffer as replenishing fluid, from 1 ml of (a) 37% Hct blood, (b) 45% Hct blood and (c) 60% Hct blood respectively.
[037] Figure 6 illustrates the device 100 being configured in a single pool module 162 configuration, in accordance with an embodiment of the invention. As illustrated in Figure 6, in this configuration, the inlet module 120, the replenishing module 150 and the venting module 160 are pooled together to form the single pool module 162, thereby eliminating need for a valve for switching the device 100 between initial priming and working configurations. The single pool module 162 also acts as the second outlet (o) for retrieving the microparticles enriched fluid.
[038] Herein, for instance, if the multicomponent colloidal solution and the replenishment fluid is blood, blood is added into the single pool module 162 having two connections, one for the pumping module 130 and other connects to the separation module 110. The pumping module 130 is started to draw the blood from the single pool module 162 and push it into the separation module 110, from where it is pumped back into the single pool module 162 following separation. This configuration of the device 100 helps in easy pre-processing of the blood, such as addition of anticoagulants to blood sample, before initiating separation. The entire volume of blood sample is recirculated through the separation module 110 and plasma is collected from the first outlet (d).
[039] Figure 7 illustrates the configuration of the device 100 so as to receive different volumes of the multicomponent colloidal solution for separation. In an embodiment, the device 100 is configured to receive a droplet input of the multicomponent colloidal solution, which is especially relevant for blood. Herein, for instance, if the multicomponent colloidal solution is blood, the device 100 as illustrated in Figure 7 is integrated with an upstream processing module 170, a downstream processing module 180, along with an inlet control module 124 and an outlet control module 144. The upstream processing module 170 is responsible for adding and mixing of reagents to the blood entering the separation module 110, and the downstream processing module 180 is responsible for processing separated plasma from the separation module 110. In an embodiment, the reagents are added using pipettes, or are prepared into prefilled tablets or bags which can be squeezed into a special mixing chamber. Alternatively, microfluidic mixing channels with reagents coated inner walls are used for adding and mixing of the reagents by passing blood or any inlet fluid through the mixing channel.
[040] Further, the downstream processing module 180 is configured for either processing of separated plasma into dried spots or pooled into a reservoir for storage, directly filled into vials, tubes and containers, or analyzed using sensors for disease screening and therapeutic monitoring applications. In an embodiment, a sensor integrated in the downstream processing module 180 is capable of directly inputting the separated plasma of desired volume into the sensors which can detect either a physical, electrical, mechanical, optical and/or chemical change and provide corresponding output.
[041] Further, the inlet control module 124 acts as a quality check for the separation fluid before it is pumped into the separation module 110 in order to achieve maximum separation.
[042] As illustrated in the embodiment depicted in Figure 8, the inlet control 124 module can comprise a flow pulsation damper 126 for reducing pulsations in flow or pressure of blood in the device 100. Herein the flow pulsation damper 126 is formed of a single or an array of orifices to reduce the pulsations in the fluid flow or pressure in the device 100. The orifice dimensions are kept either equal or larger than the smallest channel in the device 100 to avoid any blockage.
[043] In an alternate embodiment as illustrated in Figure 9, the flow pulsation damper 126 is formed by one or more closed pressure chambers filled with air or other compressible gases. The closed pressure chambers are made such that their opening is connected to the microfluidic separation channel 112. As the pressure in the microfluidic separation channel 112 fluctuates, the air inside the chamber compresses and relaxes faster than the blood thereby reducing the net flow fluctuation in the microfluidic separation channel 112 downstream. The chambers are filled at atmospheric pressure by sealing them at the time of manufacturing by trapping air or other compressible gas inside. Alternatively, high pressure gas is filled in the chambers through a pneumatic port after manufacturing the separation module 110 or the device 100. These pneumatic ports in the chambers are sealed after a desired air pressure is reached. It is also possible to keep the pneumatic source connected with the chambers, allowing continuous pressure monitoring and control for improved flow damping using a pneumatic control system.
[044] As illustrated in the embodiment depicted in Figure 10, the inlet control module 124 further comprises of a clot capturing channel 128 or filter membranes to capture clots and impurities in the blood from entering the device 100. The clot capturing channel 128 has a capturing channel with a bifurcation, connected to a narrow c-shaped channel 128a and one or more orifices or flow restrictors for capturing any coagulated mass or impurities. In an embodiment, multiple orifices are designed for different size-based capturing. Once the capturing channel 128 gets clogged, the flow continues through the narrow c-shaped channel 128a.
[045] As illustrated in the embodiment depicted in Figure 11, the outlet control module 144 has a microparticle concentrator 146 to increase the separation efficiency by removing any stray microparticles from the suspension fluid. Herein, the first outlet (d) of the separation module 110 for the suspension fluid is configured to include small pit like structures 146a at the base of the microfluidic separation channel 112. This allows stray microparticles such as RBCs in the case of plasma separation, to settle in the lower pit 146a while the suspension fluid will flow uninterrupted. The pits 146a are designed to increase particle settlement in the pit by reducing their velocity component in the direction of the flow. Further, small sized closed compartments are made along the first outlet (d) and in, or around the pit. Small vortices get formed in these compartments due to flow, creating suction which traps stray particles and helps improve particle capture percentage. In an embodiment, before introducing a new sample of blood, the microfluidic separation channels 112 are flushed out using rinsing fluid from a rinsing module 194. The rinsing fluid can be selected from a group comprising deionized water, saline or buffer solutions, RBC diluting fluids and hypochlorite solution.
[046] Figure 12 illustrates the implementation of the device 100 as a droplet plasma separation device 300, in accordance with an embodiment of the invention. Herein, sample of blood is either pipetted from a previously collected blood sample, or directly dropped from a finger prick. or from blood sample collected from conventional syringes. A small volume is injected into the inlet module 120 on the droplet plasma separation device 300. In an embodiment, the inlet module 120 is a miniature evacuated or non-evacuated chamber covered with an accessible top rubber sealing which allows for an easy perforation with the needle for sample insertion. In an alternate embodiment, the inlet module 120 receives a barrel of a syringe directly on the droplet plasma separation device 300 such that the barrel itself becomes a sample chamber. In another embodiment, the inlet module 120 comprises a needle valve arrangement which receives samples from vacutainer tubes or sealed chambers accessible through a rubber sealing.
[047] The droplet plasma separation device 300 further includes the outlet module 140 for collection of plasma. The separated plasma is collected in a storage pool in the droplet plasma separation device 300 or in a detachable vial for easy transport. In an embodiment, the outlet module 140 has a plasma collection needle which is connected either directly for collecting the plasma in vacutainer tubes, sealed chambers etc., or via a tubing, to fill and store plasma in tubes, bottles or containers for storage, transport and processing.
[048] In an alternate embodiment, the outlet module 140 is integrated with a downstream sensor cartridge port for blood analysis or processing with plasma. Drops of separated plasma are allowed to fall or inputted on a sensor cartridge placed in a port for carrying out desired blood analysis, disease screening or therapeutic monitoring applications using the droplet plasma separation device 300. Since the blood volume used is very small, the plasma collected varies between 1 to 40 percent of total blood volume supplied and further depends on the hematocrit of the supplied blood.
[049] Reference is made to Figure 7, wherein in an embodiment of the invention, the device 100 is configured to receive a finite volume batch input of the multicomponent colloidal solution. Herein, if the multicomponent colloidal solution is blood, the minimum sample volume of blood required for the device 100 ranges from 100µl to 100ml. In this embodiment, the device 100 as shown in Figure 7 additionally comprises a working fluid storage module 190, which incorporates a container to store finite quantity of multicomponent colloidal solution, namely blood in the case of plasma separation. The additional components for this embodiment such as the working fluid storage module 190 are depicted by dashed lines in Figure 7. In operation, if the multicomponent colloidal solution is blood, the blood sample is collected and placed in the container in the working fluid storage module 190 and then pumped into the inlet module 120 of the device 100. Thereafter, the sample is passed through the separation module 110 and after multi-passing, the microparticle enriched fluid comes back from the outlet module 140 into the container in the working fluid storage module 190. This microparticle enriched fluid can be used for further applications.
[050] In an embodiment, the working fluid storage module 190 has a magnetic stirrer or mechanical rocker for continuously stirring the container for preventing settling of microparticles in the multicomponent colloidal solution. In another embodiment, the working fluid storage module 190 has two containers, an inlet container containing blood for plasma separation and an output container for collecting the microparticle enriched fluid from outlet module 140.
[051] Figure 13 illustrates the adaptation of the device 100 in an Automated Plasma Culture Testing Device 400 which receives a finite volume batch input. Herein, the separated plasma is directly collected downstream in culture bottles 402. The working fluid storage module 190 has the container 404 and the separation module 110 as illustrated. The features of this plasma culture testing device 400 include automation of plasma collection from the outlet module 140 in the separation module 110 for a user defined volume directly injected into the culture bottles 402. The input from the user is obtained using a user-interface provided with the plasma culture testing device 400. The separation module 110 is capable of stopping the multi-passing automatically after achieving the required plasma volume and load the culture bottles 402.
[052] Figure 14 illustrates the adaptation of the device 100 for a fully-automated plasma separation device 500, which is integrated with downstream automated immunoassay or diagnostic testing. In an embodiment, the device 100 is adapted for a downstream automated HIV TRI-DOT testing. In that, the plasma separation device 500 inputs blood sample from a container 502 which acts as the working fluid storage module 190, and directly pumps the blood into the separation module 110 for separating plasma. The separation of plasma is carried out as per the user input from a user-interface. The plasma separation device 500 further has provisions for accepting different reagents or analytes needed for the HIV TRI-DOT+ Ag test in different forms of reagent containers 504.
[053] The plasma separation device 500 has provisions for accepting a testing chip in a testing chip port 506, which can be manually or automatically positioned to carry out the HIV TRI-DOT+ Ag testing in meticulous order by inputting plasma and the required analytes from the reagent containers 504, into the testing chip. In operation, the plasma separation device 500 observes the following steps, (i) loading an unused microfluidic separation channel 112 in the separation module 110, (ii) placing blood sample in a compatible container 502 designed to fit in the working fluid storage module 190, (iii) connect reagent containers 504 filled with required analytes, (iv) insert testing chip into the testing chip port 506, (v) specify volume of plasma required for the testing chip in the user-interface. The plasma separation device 500 runs the separation module 110 for predefined time period calculated based on inputs from the user. The plasma separation device 500 automatically adds the required quantity of separated plasma on to the testing chip and the necessary analytes, as specified in the procedure for HIV TRI-DOT+Ag testing. The results of the testing chip are read automatically and displayed in the user-interface.
[054] Reference is made to Figure 7, wherein in an embodiment of the invention, the device 100 is configured to receive the sample of the blood from a continuous flow source 210. In this embodiment, the device 100 additionally comprises a sample collection module 196 which in turn is connected to a working fluid storage module 190. The additional components for this embodiment are depicted by dotted lines in Figure 7. Herein, the blood or any multicomponent colloidal solution as the case may be, is passed through the separation module 110 continuously. In this embodiment, the device 100 additionally comprises a disposal collection module 192 for collecting any overflow such as rinsing fluid, unused plasma, separation fluid etc., from the venting module 160 and output module 140. A system incorporating the device 100 with continuous flow source and downstream diagnostics or analysis modules will be useful for continuous or intermittent monitoring in various applications such as plasma extraction and testing from continuous blood flow during procedures such as open heart surgeries, extracorporeal membrane oxygenation, kidney dialysis, blood exchange transfusion etc.
[055] Figure 15 illustrates the adaptation of the device 100 for a downstream continuous blood quality monitoring system 600. In this embodiment, the system 600 has one or more inlet ports 602 for the inlet module 120 and one or more outlet ports 604 for the outlet module 140, connected to a heart-lung machine. The system is connected to a flow line 210 having continuous blood flowing in through the inlet 602 and exiting out through the system outlet 604. The blood is pumped into the separation module 110 and the separated plasma is utilized downstream for blood quality sensing cartridge 606. The results of the blood quality sensing cartridge 606 can be read manually or automatically. The system 600 is made reusable without contaminating different inlet sources by replacing components like the microfluidic separation channels 112 in the separation module 110, sensing cartridge 606, rinsing the inlet 602 and outlet 606 channels before introducing a new inlet source etc.
[056] In another embodiment of the invention, the device 100 is adapted for a liquid biopsy application. Herein, the device 100 achieves plasma separation from blood by making use of passive hydrodynamic techniques, as opposed to external forces. This not only prevents lysis of White Blood Cells, but also preserves a ctDNA (cell-free tumor DNA) to cfDNA (circulating Free DNA) ratio of the blood sample in the extracted plasma. Further, the device allows for maintenance of the quality of ctDNA, and simplifying and optimizing the preanalytical sample workflow, so as to maximize the accuracy and efficacy of the existing ctDNA isolation and detection techniques. This further allows for standardization and optimization of liquid biopsy protocols. In a further embodiment, the device 100 is implemented in the analysis of other delicate cells and molecules in the blood, such as fetal cell markers, which are too delicate to be separated and captured using centrifugation based plasma separation methods.
[057] It is a feature of the device 100 that it allows for the user to customise either the yield of the microparticles enriched fluid or the suspension fluid in a given time frame or to customise the specific concentration of the microparticles enriched fluid in the given time frame. In an embodiment, where the user desires high yield within a given time frame, more than one microfluidic separation channels 112 are stacked or connected in parallel to achieve the same. The high yield is achieved while minimizing the negative impact on the performance of individual microfluidic separation channels 112. This is achieved by incorporation of routing layers 116 which combine inlets (i) or outlets (o,d) of all parallel microfluidic separation channels 112, as per requirement. The paths in the routing layers 116 are designed with specific dimensions such that the effective resistance ratios of the microfluidic separation channels 112 are maintained. The paths may also utilize gravity to its advantage for minimizing pressure heads on individual microfluidic separation channels 112 thus ensure their optimal separation performance. Herein, an exemplary separation module 110 with the microfluidic separation channels 112 connected in parallel, along with the routing layer 116 is illustrated in Figure 16.
[058] Figures 17A-17F illustrate various embodiments of the microfluidic separation channels 112 being connected in parallel connection, in accordance with various embodiment of the invention. Figure 17A illustrates a parallel connection of 6 microfluidic separation channels 112 having a single combined inlet (i), and individual first outlets (d) and second outlets (o). This parallel connection is combined with the routing layer 116 as illustrated in Figure 18B, resultantly providing a single inlet (i), a single first outlet (d) and a single second outlet (o), in accordance with an embodiment. In an alternate embodiment, the parallel connection as illustrated in Figure 17A is combined with the routing layer 116 as illustrated in Figure 18C, resultantly providing the single inlet (i), the single first outlet (d) and the single second outlet (o).
[059] Figure 17B illustrates a parallel combination of 4 microfluidic separation channels 112 having the single combined inlet (i), and individual first outlets (d) and second outlets (o). Figure 17C illustrates a parallel combination of 10 microfluidic separation channels 112 having the single combined inlet (i), and individual first outlets (d) and second outlets (o). Figure 17D illustrates a parallel combination of 2 microfluidic separation channels 112 having combined inlet (i), first outlet (d) and second outlet (o). Figure 17E illustrates a parallel combination of 8 microfluidic separation channels 112 having the single combined inlet (i), and combined second outlets (o), which have been combined to form 4 distinct second outlets (o) and individual first outlets (d). In an embodiment, the parallel connection as illustrated in Figure 17E is combined with the routing layer 116 as illustrated in Figure 18A, resultantly providing the single inlet (i), the single first outlet (d) and the single second outlet (o). Figure 17F illustrates a parallel combination of 8 microfluidic separation channels 112 having multiple inlets (i), which have been combined to form 5 distinct inlets (i), and combined second outlets (o), which have been combined to form 5 distinct second outlets (o) and a single combined first outlets (d).
[060] In another embodiment, where the user desires to achieve specific concentrations of characteristic microparticles, more than one microfluidic separation channels 112 are connected in series in the separation module 110 to achieve the desired separation, or alternatively more than one separation modules 110 are connected in series with each other. When more than one separation modules 110 are connected in series, the pressure drop across each of the connected separation modules 110 will influence the efficiency of the corresponding separation module 110. Therefore, each separation module 110 can output different concentrations of the suspension fluid and the microparticles enriched fluid. By configuring the pressure drop and efficiency for each separation module 110 connected in series, the user can obtain different desired concentrations of characteristic microparticles from the outlet module 140 for each separation module 110. The outlet (o,d) lines can be combined all together or selected outlets (o,d) together to achieve specific concentrations of characteristic microparticles.
[061] Figure 19 illustrates the more than one microfluidic separation channels 112 of the separation modules 110 connected in series, in accordance with an embodiment of the invention. Herein, the separation modules 110 of desired characteristics are connected in series i.e microparticles enriched fluid form the second outlet (o1) of one separation module 1101 is the inlet of the immediate next separation module 1102, and the separation modules 1101, 1102, 110N combine to form the single effective separation module 110. Herein, separation modules 1101, 1102, 110N are configured so as to achieve the desired yield and separation efficiency. Different concentration of the suspension fluid is achieved at the first outlets (d1, d2, dN) of each subsequent separation module 1101, 1102, 110N which could separately be utilized for downstream processing. In operation, blood is drawn from the working fluid storage module 190 and is pushed to the separation module 1101 using the pumping module 130, and thereafter sent to subsequent separation modules 1102, 110N. Upstream and downstream processes are performed as needed for a given application by the concerned upstream processing module 170 and downstream processing module 180. Further, the venting module 160 connects the outlets (d1, d2, dN) and allows the user to push out the stagnant suspension fluid in the outlets (d1, d2, dN), thereby increasing the overall yield of the microparticles enriched fluid.
[062] Figure 20 illustrates the more than one microfluidic separation channels 112 of the separation modules connected 110 in series, in accordance with an alternate embodiment of the invention. In this configuration, the suspension from one separation module 1101 acts as input for each subsequent separation modules 1102, 110N. Separation modules 1101, 1102, 110N at each stage are configured to achieve desired yield and efficiency. Herein, blood drawn from the working fluid storage module 190 is pushed to the separation module 1101 using the pumping module 130. In this configuration, very high purity of suspension fluid could be obtained. In general, in series configuration, by suitably configuring the separation module 100, the user achieves greater control over the desired output. For instance, the embodiment depicted in Figure 19 allows the user to obtain various different concentrations of the suspension fluid at the same time while the embodiment depicted in Figure 20 allows for multistage separation to optimize the throughput and purity of the suspension fluid by configuration of separation modules 110 at each stage.
[063] Advantageously, the present invention provides a device for separation of characteristic microparticles from a multicomponent colloidal solution, which allows for multi-passing of the multicomponent colloidal solution thought the separation module. The multi-passing resultantly provides a wide band of achievable separation efficiency and enhanced yield of the microparticle free suspension fluid or microparticle enriched fluid as per need.
[064] Further, the device of the present invention is operable over a wide range of inlet flow rate and a wide range of inlet microparticle concentration. This allows for effective use of the device in applications with continuously flowing inlet streams, as open heart surgeries, blood exchange transfusion, extracorporeal membrane oxygenation, kidney dialysis etc.
[065] Further, the device in the present invention provides separation of characteristic microparticles from the multicomponent colloidal solution without any significant time lag, allowing for the downstream processing modules to process the information from the microparticles enriched fluid. This ensures that the values correspond to the condition of the patient undergoing the procedure at the given instant.
[066] While the present invention has been described with respect to certain embodiments, it will be apparent to those skilled in the art that various changes and modification may be made without departing from the scope of the invention as defined in the following claims. ,CLAIMS:WE CLAIM:
1. A device (100) for separation of characteristic microparticles from a multicomponent colloidal solution, comprising:
a separation module (110) having at least one microfluidic separation channel (112) configured to separate the multicomponent colloidal solution into a suspension fluid and a microparticles enriched fluid, the microfluidic separation channel (112) having an inlet (i) for receiving a sample of the multicomponent colloidal solution, a first outlet (d) for exiting the suspension fluid, and a second outlet (o) for exiting the microparticles enriched fluid; and
a replenishing module (150) configured to receive the microparticle enriched fluid from the second outlet (o) and a replenishing fluid to form a replenished colloidal solution suitable for being supplied to the inlet (i) for separation into the suspension fluid and the microparticles enriched fluid, thereby achieving an enhanced yield of the microparticles enriched fluid or the suspension fluid as per need.

2. The device (100) as claimed in claim 1, wherein the separation module (110) comprises the microfluidic separation channels (112) connected in series connection.

3. The device (100) as claimed in claim 1, wherein the separation module (110) comprises the microfluidic separation channels (112) connected in parallel connection.

4. The device (100) as claimed in claim 1, wherein the replenishing module (150) comprises a port and valve arrangement for holding and supplying the replenishing fluid.

5. The device (100) as claimed in claim 1, comprising an inlet module (120) configured for supplying desired volume of the sample of the multicomponent colloidal solution to the separation module (110), the inlet module (120) being connected to the inlet (i) of the separation module (110) and the replenishing module (150).

6. The device (100) as claimed in claim 5, wherein the inlet module (120) comprises one or more port and valve arrangements for supplying desired volume of the sample of the multicomponent colloidal solution to the separation module (110).

7. The device (100) as claimed in claim 1, comprising an outlet module (140) configured for receiving the suspension fluid exiting the first outlet (d) and the microparticles enriched fluid from the second outlet (o), and transmitting the microparticle enriched fluid exiting the second outlet (o) to the replenishing module (150).

8. The device (100) as claimed in claim 1, wherein the outlet module (140) comprises two or more port and valve arrangements for exiting the suspension fluid and microparticles enriched fluid from the separation module (110).

9. The device (100) as claimed in claims 1 and 5, comprising a pumping module (130) connecting the inlet module (120) to the separation module (110) for achieving a desired flow rate in the separation module (110).

10. The device (100) as claimed in claim 1, comprising a venting module (160) with a port and valve arrangement for removing any pre-filled air, priming fluid or rinsing fluid from the device (100).

11. The device (100) as claimed in claim 1, wherein the multicomponent colloidal solution is one of pigmented ink, paint, blood, urine, cerebrospinal fluid (CSF), biochemical samples, water, food or fuel with impurities.

12. The device (100) as claimed in claim 1, wherein the multicomponent colloidal solution is blood, and the replenishing fluid received by the replenishing module (150), is blood.

13. The device (100) as claimed in claim 1, wherein the multicomponent colloidal solution is blood and the replenishing fluid received by the replenishing module (150), is a buffer solution.

14. The device (100) as claimed in claim 13, wherein the buffer solution is selected from a group comprising distilled water, 0.8% sodium chloride solution or saline, phosphate buffered saline (PBS), or RBC diluting fluids.

15. The device (100) as claimed in claims 1, 5 and 10, wherein the inlet module (120), the replenishing module (150) and the venting module (160) are pooled together to form a single pool module (162).

16. The device (100) as claimed in claim 12 or 13, wherein the minimum sample volume of blood required for the device (100) is 20µl.

17. The device (100) as claimed in claim 16, comprising an upstream processing module (170) for adding and mixing reagents to the blood entering the separation module (110), and a downstream processing module (180) for processing separated plasma from the separation module (110).

18. The device (100) as claimed in claim 16, comprising an inlet control module (124) for checking quality of the blood entering the device (100) and an outlet control module (144) for checking quality of the suspension fluid or plasma existing the device (100).

19. The device (100) as claimed in claim 18, wherein the inlet control module (124) comprises a flow pulsation damper (126) for reducing pulsations in flow or pressure of blood in the device (100), and a clot capturing channel (128) to capture clots and impurities in the blood from entering the device (100).

20. The device (100) as claimed in claim 18, wherein the outlet control module (144) comprises a microparticle concentrator (146) to increase the separation efficiency by removing any stray microparticles from the suspension fluid.

21. The device (100) as claimed in claim 16, wherein the inlet module (120) comprises a miniature evacuated or non-evacuated chamber covered with an accessible top rubber sealing.

22. The device (100) as claimed in claim 16, wherein the inlet module (120) is configured to receive barrel of a syringe directly on the device (100) such that the barrel itself acts as a sample chamber.

23. The device (100) as claimed in claim 16, wherein the inlet module (120) comprises a needle valve arrangement configured to receive samples from vacutainer tubes or sealed chambers accessible through a rubber sealing.

24. The device (100) as claimed in claim 12 or 13, wherein the minimum sample volume of blood required for the device ranges from 100µl to 100ml.

25. The device (100) as claimed in claim 24, comprising a working fluid storage module (190) to store finite quantity of blood.

26. The device (100) as claimed in claim 24, wherein the device (100) is adapted in an Automated Plasma Culture Testing Device (400).

27. The device (100) as claimed in claim 24, wherein the device (100) is adapted for a fully-automated plasma separation device (500), which is integrated with downstream automated immunoassay or diagnostic testing.

28. The device (100) as claimed in claim 27, wherein the device (100) is adapted for a downstream automated HIV TRI-DOT testing or other similar plasma based tests.

29. The device (100) as claimed in claim 12 or 13, wherein the sample of the blood for the device is a continuous flow source (210).

30. The device (100) as claimed in claim 29, comprising a sample collection module (196) connected to the working fluid storage module (190) to store finite quantity of the blood.

31. The device (100) as claimed in claim 29, comprising a disposal collection module (192) or collecting any overflow such as rinsing fluid, unused plasma, or suspension fluid, from the venting module (160) and the output module (140).

32. The device (100) as claimed in claim 29, wherein the device (100) is adapted for a downstream continuous blood quality monitoring system (600).
Dated this 21 Day of April 2021

EMBRYYO TECHNOLOGIES PRIVATE LIMITED and
Indian Institute of Technology Bombay
By their Agent & Attorney

(Jayesh Varavadekar)
of Khaitan & Co
Reg No IN-PA-2544