DESC:
TECHNICAL FIELD
[0001] The present disclosure relates generally to separation of particles from a sample solution. More particularly, the present disclosure relates to an in-flow decantation apparatus that provides separation of particles from a fluid while in-flow.

BACKGROUND
[0002] Background description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.
[0003] Microfluidics, in its immense potential to offer miniaturization, cost-effectiveness, precision, automation, and use of ultra-small quantities of samples, is rapidly expanding into areas of health-care, water treatment, soil testing, biomedical research, chemical and biological sciences. In several applications involved in these areas, separation of suspended particles from sample fluid is a must as a pre-preparatory step for further investigation/research, such process also termed as microfluidic decantation.
[0004] Blood is an important body fluid that is responsible for delivery of nutrients, oxygen to the cells and take away metabolic waste from the cells. Its main constituents include plasma (˜ 55 % by volume) and blood cells (˜ 45 % by volume). About 92 % by volume of plasma is water and contains dissipated proteins, glucose, mineral ions, carbon dioxide etc. The blood cells are majorly red blood cells, white blood cells, and platelets. Many markers associated with body medical conditions reflect as either change in composition of plasma or blood cells.
[0005] Blood-based tests can be broadly categorized into plasma/serum-based tests and blood cell-based tests. Majority of plasma/serum-based tests require the separation/removal of blood cells from the whole blood as the cellular debris interferes with most of plasma/serum-based tests. Standard laboratory technique of cell separation from whole blood is based on centrifugation, which is very cumbersome, time consuming (about 30 minutes) and requires skilled personnel to operate. Microfluidics based devices/ apparatuses for plasma separation can be majorly classified into active and passive. Devices based on active techniques use some form of forces such as dielectrophoretic, electrohydrodynamic, electro-osmotic, and acoustic to achieve the desired cellular separation. The major disadvantages associated with these techniques are the inability to provide high quality plasma at higher hematocrits, complexity in fabrication and integration, and the typical dependence on the bulky external power supplies. Passive techniques take leverage on one or a combination of the parameters such as the use of filters, device geometry, inertial or dean's effects, gravity, and biophysical effects such as Fahraeus effect and Zweifach-Fung bifurcation law to achieve the desired separation. Conventional way of filtration for plasma separation was extended into microfluidics, and several variants of it were proposed. Clogging remained as one of the major drawbacks associated with these techniques due to very small pore size of filters. Plasma separation designs based on geometrical and biophysical effects were proposed. Even though these do not cause clogging problems in general, they provide high purity plasma only at lower hematocrits, hence may not be very much suitable for majority of biochemical tests. Another known art demonstrated high purity plasma separation at higher hematocrit, but at very low flow rates, thereby making the separation process very slow and limiting its use for rapid diagnosis. In addition, inertial and deans effects were utilized to separate plasma by pumping the blood at higher flow rates and were demonstrated to produce high purity plasma only at lower hematocrits. Several other passive variant techniques that demonstrated plasma separation have been extensively reviewed. However, the production of high purity plasma at larger hematocrits at optimum flow rates (without causing hemolysis) in higher yields, in a simple to fabricate device is still a challenge.
[0006] Most of the blood-cell based tests using microfluidics analyze blood cells in flow on the flow cytometric principles of imaging or scattering. One of the crucial requirements of these microfluidic devices is flow focusing of the cells into a plane or a line for interrogation widely known as flow focusing. Most of these flow focusing techniques employ external sheath fluid to focus these cells. Efforts to generate sheath-free flow focusing have led to development of both passive and active techniques. Due to the conventional drawbacks associated with active techniques such as complexity in fabrication and integration, bio-cellular incompatibility and the requirement of bulky and expensive power supplies, passive techniques remained as an attractive choice for point of care diagnostics (POCD). Most of the passive techniques operate at high flow rates by utilizing either Dean’s effects or inertial effects and are not suitable for imaging based applications. Few passive techniques that can operate at low flow rates of operation can only achieve focusing onto a plane alone but not in required alignment for imaging applications.
[0007] A prior art paper (Hydrodynamic self-focusing in a parallel microfluidic device through cross-filtration) refers to use of pillar arrays for separating particles from a sample fluid. However, apparatus therein works only for particles of larger size than the spacing between the pillars and is prone to clogging, which limits the applicability of the technique to body fluids such as blood (particle size ranges from 2 µm to 30 µm) as the pillar spacing has to be much smaller than that of 2 µm while keeping the height of the pillar (and channel) large enough to accommodate the largest particle (30 µm). This is highly difficult to achieve using regular techniques of photolithography and soft lithography normally used in manufacture of microfluidic decantation apparatuses.
[0008] Hence there is a need in the art for an apparatus for microfluidic decantation that can provide high quality plasma at higher hematocrits, is simple to fabricate, integrates easily integrate with other systems, and does not require bulky external power supplies. The apparatus should have minimal clogging of its filters and allow for in-flow decantation, while working effectively even for smaller particle sizes. Separation /decantation of high quality plasma should be achieved at good flow rates and with good plasma yields, without causing hemolysis.
[0009] All publications herein are incorporated by reference to the same extent as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. Where a definition or use of a term in an incorporated reference is inconsistent or contrary to the definition of that term provided herein, the definition of that term provided herein applies and the definition of that term in the reference does not apply.
[0010] In some embodiments, the numbers expressing quantities of ingredients, properties such as concentration, reaction conditions, and so forth, used to describe and claim certain embodiments of the invention are to be understood as being modified in some instances by the term “about.” Accordingly, in some embodiments, the numerical parameters set forth in the written description and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by a particular embodiment. In some embodiments, the numerical parameters should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as practicable. The numerical values presented in some embodiments of the invention may contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements.
[0011] As used in the description herein and throughout the claims that follow, the meaning of “a,” “an,” and “the” includes plural reference unless the context clearly dictates otherwise. Also, as used in the description herein, the meaning of “in” includes “in” and “on” unless the context clearly dictates otherwise.
[0012] The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g. “such as”) provided with respect to certain embodiments herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.
[0013] Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member can be referred to and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group can be included in, or deleted from, a group for reasons of convenience and/or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified thus fulfilling the written description of all groups used in the appended claims.

OBJECTS OF THE INVENTION
[0014] It is another object of the present disclosure to provide for a system to enable determination of time period the person user present at the location.
[0015] It is an object of the present disclosure to provide for an apparatus for microfluidic in-flow decantation that provides high quality plasma at higher hematocrits.
[0016] It is an object of the present disclosure to provide for an apparatus for microfluidic in-flow decantation that is simple to fabricate.
[0017] It is an object of the present disclosure to provide for an apparatus for microfluidic in-flow decantation that integrates easily with other systems.
[0018] It is an object of the present disclosure to provide for an apparatus for microfluidic in-flow decantation that does not require bulky external power supplies.
[0019] It is an object of the present disclosure to provide for an apparatus for microfluidic in-flow decantation that has minimum clogging of its filters and allows for in-flow decantation, while working effectively even for smaller particle sizes.
[0020] It is an object of the present disclosure to provide for an apparatus for microfluidic in-flow decantation that provides separation /decantation of high quality plasma at good flow rates and good plasma yields, without causing hemolysis.

SUMMARY
[0021] The present disclosure relates to an in-flow decantation apparatus that provides separation of particles from a fluid while in-flow. In particular, it relates to such an apparatus that provides for separation of small size particles without clogging of the apparatus, using novel configurations.
[0022] This summary is provided to introduce simplified concepts of an apparatus for microfluidic in-flow decantation, which are further described below in the Detailed Description. This summary is not intended to identify key or essential features of the claimed subject matter, nor is it intended for use in determining/limiting the scope of the claimed subject matter.
[0023] In an aspect, present disclosure elaborates upon an apparatus for in-flow decantation of a first fluid containing particles below a critical size from a sample fluid containing particles of different sizes. The apparatus can include: one or more of a second section (section II), each of the section II comprising: a channel C of depth D2; and a side channel S1 on at least one side of the channel C, the side channel S1 comprising a plurality of pillars each of length Lp, width Wp, height D3 and inter-pillar spacing Wps, the height D3 being less than the depth D2, wherein the plurality of pillars can be positioned below top edge of the channel C and aligned in a direction lateral to flow of the sample fluid through the channel C to form a stepped pillar array, and a third section ( section III) coupled to the section II, the section III comprising: a channel Ct connected to outlet of the channel C; and a side channel S1tconnected to outlet of the side channel S1, wherein any of cross-section and length of the channel Ct and cross-section and length of side channel S1t can be adapted to control flow of the sample fluid in the channel C and flow of the first fluid through any of the inter-pillar spacings (Vpg) to prevent flow of particles above the critical size through any of the inter-pillar spacings (Wps) , and so can enable in-flow decantation of the first fluid from the sample fluid..
[0024] In another aspect, cross-section of the inter-pillar spacing (Wps*D3) can be configured to be larger than the critical size.
[0025] In yet another aspect, the Vpg can be determined on the basis of the Wps, the D3, average velocity of flow of the first fluid across the inter-pillar spacings (v), and average velocity of flow of the sample fluid near the pillars along the central channel C (u).
[0026] In an aspect, the adaptation can vary hydraulic resistance of any or a combination of the channel Ct and side channel S1t to deliver required values of any of the v and the u and thereby can control ratio of rate of flow of the first fluid drawn through the side channel S1t (Qs) to rate of flow of the sample fluid drawn through the channel Ct (Qc).
[0027] In another aspect, the section II can further include a side channel S2 on other side of the channel C and the section III can further include a side channel S2t on other side of the channel Ct, wherein the side channel S2 can be identical to the side channel S1 and connected to the side channel S2t, and the side channel S2t can be identical to the side channel S1t.
[0028] In yet another aspect, the apparatus can further include a section I comprising an input channel (sample channel) of depth D1 for the sample fluid connected to the channel C wherein D1 can be less than D2 and wherein transition from the section I to the section II can bring a particles free fluid layer nearer to inner walls of the channel C.
[0029] In an aspect, the apparatus can include at least two of the sections II coupled together, and output of latter of the sections can be provided to the section III to provide for more yield of the first fluid from the sample fluid.
[0030] In another aspect, the Wp, the Wps, the Lp and number of pillars (N) in the side channel S1 can be configured according to overall size required of the apparatus.
[0031] In yet another aspect, lateral spacing Wsl between the pillars of the stepped pillar array and side wall of the side channel S1 distal from the channel C can be configured as continuously increasing from start of the pillars till end in steps of Stl per (Wp+Wps) to enable continuous extraction of small and controllable quantity of the first fluid from the spaces formed by the inter-pillar spacings Wps, D3, and length Lp.
[0032] In an aspect, the sample fluid can be blood, the first fluid can be plasma, and the apparatus can perform microfluidic in-flow decantation of the plasma from the blood.
[0033] Various objects, features, aspects and advantages of the present disclosure will become more apparent from the following detailed description of preferred embodiments, along with the accompanying drawing figures in which like numerals/alphabets represent like features.
[0034] Within the scope of this application it is expressly envisaged that the various aspects, embodiments, examples and alternatives set out in the preceding paragraphs, in the claims and/or in the following description and drawings, and in particular the individual features thereof, may be taken independently or in any combination. Features described in connection with one embodiment are applicable to all embodiments, unless such features are incompatible.

BRIEF DESCRIPTION OF THE DRAWINGS
[0035] FIG. 1A illustrates an exemplary schematic of the proposed microfluidic apparatus in accordance with an embodiment of the present disclosure, wherein (i), (ii), and (iii) represent representative schematic images at the appropriate locations.
[0036] FIGs. 1B and 1C represent exemplary schematics illustrating movement of particle due to expansion of the channel in accordance with an embodiment of the present disclosure.
[0037] FIG. 2A to 2E illustrates exemplary simulation results showing effect of parameters of section II, namely height of the pillar (D3), spacing between the pillars (Wps), number of pillars (N), slope of the side channel S (Sslp), and the sample flow rate (Q) on the side to central channel flow rate ratio (SCF), respectively in accordance with an embodiment of the present disclosure.
[0038] FIG.3 illustrates exemplary effect of side to central channel hydraulic resistance ratio (SCRR) on side to central channel flow rate ratio (SCF), wherein FIG. 3A presents simulation results demonstrating effect of SCRR on SCF, and FIG. 3B presents Experimental results showing the variation of SCF with respect to SCRR.
[0039] FIG. 4 illustrates exemplary variation of flow rate across the pillar gaps, plotted as a function of pillar number, when the slope of side channel Sslp FIG. 4a and number of pillars (N) FIG. 4b are varied in accordance with an embodiment of the present disclosure.
[0040] FIG. 5 illustrates exemplary variation of plasma purity as a function of (FIG. 5A) duration of the experiment (time), (FIG. 5B) flow rate (Q), (FIG. 5C) hematocrit (hct) and SCRR.
[0041] FIGs. 6A to 6D represent experimental images depicting concentration of RBCs in central channel C and those that escaped into the side channels S1 and S2 for different values of hematocrit.
[0042] FIGs. 6E and 6F show representative images of the beads and platelets inside the microfluidic apparatus close to the end of section II, respectively.
[0043] FIG. 7A to 7C illustrates an exemplary schematic representation of apparatus with stages 1 and 2in accordance with an embodiment of the present disclosure.
[0044] FIG. 8 illustrates an exemplary experimental set-up that was used for the validation of proposed microfluidic technique.

DETAILED DESCRIPTION OF THE INVENTION
[0045] Various objects, features, aspects and advantages of the inventive subject matter will become more apparent from the following detailed description of preferred embodiments, along with the accompanying drawing figures in which like numerals represent like components
[0046] The following is a detailed description of embodiments of the disclosure depicted in the accompanying drawings. The embodiments are in such detail as to clearly communicate the disclosure. However, the amount of detail offered is not intended to limit the anticipated variations of embodiments; on the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the present disclosure as defined by the appended claims.
[0047] Each of the appended claims defines a separate invention, which for infringement purposes is recognized as including equivalents to the various elements or limitations specified in the claims. Depending on the context, all references below to the "invention" may in some cases refer to certain specific embodiments only. In other cases it will be recognized that references to the "invention" will refer to subject matter recited in one or more, but not necessarily all, of the claims.
[0048] Various terms as used herein are shown below. To the extent a term used in a claim is not defined below, it should be given the broadest definition persons in the pertinent art have given that term as reflected in printed publications and issued patents at the time of filing.
[0049] In an aspect, present disclosure elaborates upon an apparatus for in-flow decantation of a first fluid containing particles below a critical volume from a sample fluid containing particles of different volumes. The apparatus can include: one or more of a second section (section II), each of the section II comprising: a channel C of depth D2; and a side channel S1 on at least one side of the channel C, the side channel S1 comprising a plurality of pillars each of length Lp, width Wp, height D3 and inter-pillar spacing Wps, the height D3 being less than the depth D2, wherein the plurality of pillars can be positioned below top edge of the channel C and aligned in a direction lateral to flow of the sample fluid through the channel C to form a stepped pillar array, one or more of a third section ( section III), each of the section III coupled to each of the section II, the section III comprising: a channel Ct connected to outlet of the channel C; and a side channel S1t connected to outlet of the side channel S1,wherein any of cross-section and length of the channel Ct and cross-section and length of side channel S1t can be adapted to control flow of the sample fluid in the channel C and flow of the first fluid through any of the inter-pillar spacings (Vpg) to prevent flow of particles of volumes above the critical volume through any of the inter-pillar spacings, and so enable in-flow decantation of the first fluid from the sample fluid.
[0050] In another aspect, cross-section of the inter-pillar spacing (Wps*D3) can be configured to be larger or smaller than cross-section of a particle below the critical volume.
[0051] In yet another aspect, the Vpg can be determined on the basis of the Wps, the D3, average velocity of flow of the first fluid across the inter-pillar spacings (v), and average velocity of flow of the sample fluid near the pillars along the central channel C (u).
[0052] In an aspect, the adaptation can vary hydraulic resistance of any or a combination of the channel Ct and side channel S1t to deliver required values of any of the v and the u and thereby can control ratio of rate of flow of the first fluid drawn through the side channel S1t (Qs) to rate of flow of the sample fluid drawn through the channel Ct (Qc).
[0053] In another aspect, the section II can further include a side channel S2 on other side of the channel C and the section III can further include a side channel S2t on other side of the channel Ct, wherein the side channel S2 can be identical to the side channel S1 and connected to the side channel S2t, and the side channel S2t can be identical to the side channel S1t.
[0054] In yet another aspect, the apparatus can further include a section I comprising an input channel (sample channel) for the sample fluid connected to the channel C and transition from the section I to the section II can bring a particles free fluid layer nearer to inner walls of the channel C.
[0055] In an aspect, lateral spacing Wsl between the pillars of the stepped pillar array and side wall of the side channel S1 distal from the channel C can be configured as continuously increasing from start of the pillars till end in steps of Stl per (Wp+Wps) to enable continuous extraction of small and controllable quantity of the first fluid from the spaces formed by the inter-pillar spacings Wps, D3, and length Lp.
[0056] In another aspect, any or combination of the Wp, the Wps, the Lp, the Stl and number of pillars (N) in the side channel S1 can be configured according to overall size required of the apparatus.
[0057] In yet another aspect, any or a combination of the Stl and the number of pillars (N) can be so configured so as to enable flow across all of the pillars to become similar.
[0058] In another aspect, the sample fluid can be blood, the first fluid can be plasma, and wherein the apparatus performs microfluidic in-flow decantation of the plasma from the blood.
[0059] The present disclosure relates to a separation of particles a sample fluid, which is important for several microfluidic-based sample preparation and/or sample handling techniques namely plasma separation from whole blood, sheath-free flow focusing, particle enrichment etc. This present disclosure elaborates upon an apparatus (interchangeably termed as device herein) to perform microfluidic in-flow decantation so as to separate particles from a fluid while in-flow. The design involves expansion of sample fluid channel in lateral and depth directions, thereby producing a particle-free layer towards the walls of the channel, followed by gradual extraction of this particle-free fluid through a series of tiny openings located laterally to the direction of flow of the sample fluid, as further elaborated.
[0060] In an aspect, the proposed design increases the efficiency of decantation technique and uses a novel principal termed as ‘wee-extraction’ herein. In order to demonstrate proof-of-principle, experimental characterization has been performed on beads, platelets, and blood sample at various hematocrits (2.5 % - 45 %). Experiments revealed clog-free separation of particle-free fluid for at least an hour of operation of the apparatus and demonstrated purities close to 100 % and yields as high as 15 %. The avenues to improve the yield are discussed along with several potential applications.
[0061] The present disclosure pertains to an apparatus with a design/structure/architecture that can separate particles from a fluid ( sample fluid) for a wide range of flow rates, particle sizes, and concentrations at very high purity and yield, through the principle termed as ‘wee extract’. As described further, the apparatus has a stepped pillar array configuration along with hydraulic resistance tuners that are configured to provide an independent ability to control the fluid flow through pillar gaps (interchangeably termed as inter-pillar spacings herein). Such a configuration is suitable for low flow rates as well as high flow rates and for wide variety of particle sizes without clogging the inter-pillar spacing, thereby facilitating its use for both imaging and non-imaging flow cytometric applications. The high purity (100 %) and yield (> 15 %) of the particle-free fluid separated, and the applicability, efficient working of the technique even at higher particle concentrations (> 45 %) demonstrates the potential of the proposed technique and its practical utility.
[0062] It is to be appreciated that the principle of operation of this technique has been demonstrated using beads, platelets, and blood of various particle concentrations (hematocrits). The proposed apparatus in combination with any simple to fabricate flow focusing 3D flow focusing device that uses external sheath fluid can be used to develop a sheath-free flow cytometer that accounts to the needs of blood-cell based tests.
[0063] In another aspect of the present disclosure, the proposed apparatus is simple to fabricate as it involves fabricating master mold only once and subsequent apparatus fabrication can be done in single step without the requirement of complicated alignment procedures, despite the design involves multiple heights.
[0064] In an exemplary aspect, the proposed apparatus design and its operation can be well understood by artificially dividing the whole design into three sections as shown in FIG. 1A, wherein the first section (section I) can include a sample input orifice and sample channel of depth D1 for sample inflow ( that is, for inputting a sample fluid). The second section (section II) can be further subdivided into central channel C of depth D2 and side channels S1 and S2, each of height D3. In an aspect, meeting point of Section I with Section II has a transition of depth from D1 to D2 and width W1 to W2, as shown in FIG. 1A.. The side channels S1 and S2 are identical and can be placed symmetrically on either side of central channel C, aligned to its top edge. These side channels can include a series of pillars with spacing (termed herein as inter-pillar spacing) between them illustrated as Wps. The series of pillars can each have a height D3 lesser than that of channel C (D2), and can be so positioned that their length Lp (as shown in FIG. 1A) is lateral to the direction of flow of sample fluid through channel C. Width of each of the pillar can be Wp, as shown in FIG. 1A. The pillars can be positioned below top end of the channel C and such configuration of the pillars can be termed as a stepped pillar array.
[0065] In another aspect, central channel C’s depth D2 and width W2 can be chosen larger than that of the sample channel depth D1 and width W1, so as to facilitate redistribution of sample particles present inside the sample solution along the depth and width directions.
[0066] In an aspect, for a dilute suspension of particles, as the sample passes from sample channel to central channel, average separation of particles and average distance of particles from top, bottom and side walls increases due to expansion of channel in the depth and width directions. Schematics illustrating the same are shown in FIGs. 1B and 1C. This increment in distance of particles from the walls leaves the room for large quantity of particle-free fluid to be closer to the walls. To separate this particle-free fluid, stepped pillar array design as being described along with hydraulic resistance tuners (as per section III further described) is proposed in present disclosure.
[0067] As described, stepped pillar array can include pillars of length Lp, width Wp, height D3 and inter-pillar spacing Wps as shown in FIG. 1. The width of pillars (Wp), the inter-pillar spacing between them (Wps), and the number of pillars (N) in each side channel can be configured to decide overall size of the apparatus.
[0068] In another aspect, lateral spacing Wsl ( as shown in FIG. 1A) between the pillars of the stepped pillar array and side walls of the side channels (S1 and S2) distal from the channel C can be configured as continuously increasing from start of the pillars till end in steps of Stl per (Wp+Wps) to enable continuous extraction of small and controllable quantity of the first fluid from spaces formed by the inter-pillar spacings, as further described. Dimension Wp+Wps can be considered to ‘period’ of the pillars since they are made in a repeated pattern (array). Period is the pillar width Wp plus the inter-pillar spacing Wps.
[0069] An exemplary purpose of using the pillars with constant spacing Wps between them and a variable lateral spacing Wsl can be to extract small and controllable quantity of fluid through the pillar gaps continuously. If there is zero step increment Stl, it is expected to have no further accumulation of fluid through the side channels, due to the pressure of the already existing fluid in that region, except towards the end of section II. Therefore, in order to have larger quantity of fluid to accumulate in the side channels, there is a need to have some nonzero Stl.
[0070] As can be seen in FIG. 1, motion of the particle which was originally moving at 5 µm away from the top and side walls prior to expansion of the channel is shown in FIG. 1B and FIG. 1C, as seen in side and top views, respectively. The numbers shown in the FIGs. were obtained from simulation results and indicate particles moving away from the side surfaces of channel C as they move from sample channel to channel C.
[0071] As can be understood, spacing between pillars (inter-pillar spacing Wps) and height of pillar (D3) can define a cross-section for fluid flow through the pillar gap, thereby providing a measure for fluid extraction through it. Smaller the pillar gap cross-section is, better the control will be over the quantity of fluid being drawn through the pillar gaps, for a given third section (section III) design further described. Designing this volume of extraction through each of these gaps to be smaller than the volume of a particle of a critical size to be separated from the sample fluid can ensure that fluid that flows through an inter-pillar spacing Wps carries only particles of volumes below that critical size, i.e. particles of volume equal or above the critical size can be separated/filtered out from the sample fluid and remain in channel C, while fluid containing particles each of lesser than volume of the critical size (such fluid termed as first fluid herein ) can pass through the inter-pillar spacings WPs and can be extracted from spaces ( Wps * Lp) formed by the inter-pillar spacings.
[0072] Depending upon the size of particles present in the sample fluid and upon the critical size that the proposed apparatus can be configured/designed for, it can readily be appreciated that the first fluid can achieve have negligible presence of particles ( that is, it can have very high purity). In case the sample fluid is blood, blood cells can be separated from the sample fluid and plasma of very high purity can be decanted in-flow as first fluid. In many situations the first fluid can be practically taken as particles free fluid as number of particles in a unit volume of it falls below a pre-determined limit, as defined by relevant specifications, medical standards etc.
[0073] The volume of the first fluid drawn through pillar gaps (Vpg) depends on cross-sectional area of the pillar gap (Wps* D3). If average velocity of flow across the pillar gaps is ‘v’ , and average velocity of flow of sample fluid near the pillars in the direction of flow is ‘u’, then a particle (termed critical particle, for instance) in the sample fluid will take time ‘t’ ( t= Wps/u) to travel across an inter-pillar spacing Wps. During such time, volume of fluid that can travel through the inter-pillars spacing is Wps*D3 * v* t , that is, Vpg = Wps*D3 * v* Wps/u. If velocities v and u can be so configured that the Vpg is less than the volume of the critical particle, it can be understood that the critical particle in the sample fluid will tend to move in channel C towards its outlet rather than through any of the inter-pillar spacings. So, only fluid carrying particles less than the critical particle volume will tend to move beyond inter-pillar spacings and can be extracted ( as first fluid) from spaces ( Wps*Lp) formed by such inter-pillar spacings.
[0074] Proposed invention uses principle as elaborated above. Smaller Vpg can be obtained by making the terms Wps * D3 and v / u smaller. The ratio v / u can be made dependent on cross section of an inter-pillar spacing /gap and Section III of the design of the proposed apparatus, as further described. In order to obtain a smaller Vpg, in principle Wps or D3 or v/u or any combination of them can be chosen arbitrarily small. Keeping in view of the fabrication difficulties Wps and D3 can be made small enough to conveniently fabricate (thus making apparatus proposed easy to manufacture ) and v/u can be manipulated by controlling the third section ( section III) design as further elaborated, which can ensure a smaller quantity of fluid withdrawal through the pillar gaps (inter-pillar spacings). This process of extracting smaller quantities of fluids through the pillar gaps is defined as the principle of ‘wee-extract’.
[0075] As said above, the ration v / u can be made dependent on cross section of an inter-pillar spacing /gap and third section (section III) of the design of the proposed apparatus.
[0076] In an exemplary aspect, the third section can include a central channel Ct (of hydraulic resistance Rc) and side channels S1t and S2t (each with hydraulic resistance 2*Rs), respectively. These side channels S1t and S2t can be configured as ‘hydraulic resistance tuners’ of the proposed apparatus, wherein hydraulic resistance to flow of the first fluid (particle free fluid) can be changed by changing length or cross-sectional parameters of these side channels. Rs can be representative of hydraulic resistance of the two side channels S1t and S2t combined. As these two side channels are identical, hydraulic resistance of each side channel will be 2* Rs. It is to be appreciated that an exemplary objective of the third section design is to control ratio of the fluid that gets drawn through the side channels with respect to the central channel, which in turn is controlled by the ratio of hydraulic resistances of the central and side channels Ct, S1t, and S2t. The flow rate ratio of side channels S1t and S2t (combined total flow rate = Qs) and that of central channel Ct (Qc) can be given by Qs / Qc = Rc / Rs. The side channel flow rate Qs in terms of the sample flow rate Q can be given by Qs = (Q * Rc)/ (Rc + Rs). The above analysis can be approximate and can be used to get only qualitative understanding of the separation process. In the above analysis, it can be assumed that the section II parameters do not have any significant effect on flow rates Qs and Qc, except for the distribution of flow inside the section II and was proven to be true through both simulations and experiments as presented in subsequent sections.
[0077] In an aspect, transition from the first section of the design to the second section of the design leading to a depth increment can result in bringing more quantity of particle-free fluid towards the walls of the channel (Fahraeus effect). The transition can be made in any other manner to achieve same result. The second and third sections of the design together bring out the wee-extraction of the fluid from the suspension. This later process of wee-extraction first involves extracting particle-free fluid into the side channel that was made available due to Fahraeus effect, and subsequently continues to extract the particle-free fluid present in the suspension which otherwise was not separated as particle-free layer as in Fahraeus effect. In case of lower concentration of sample particles, the former process creates larger particle-free fluid near the walls and hence the later process can extract this fluid. This helps in having greater yield and paves a way for the extracted fluid to be used for sheath-free flow focusing of particles. Whereas, as the particle concentration increases, the former process may not be able to generate larger quantities of particle-free fluid near the walls. Hence, choosing the wee-extraction volume to be smaller than the critical particle volume, the fluid separation from the particle suspension can be carried out without compromising the purity. This process ensures purity of the fluid extracted is high, while having a lower yield. However, the technique offers a scope for enhancing the yield through cascading as discussed in subsequent sections.
[0078] In an aspect, the present disclosure provides a technique that can separate particles and fluid in 100 % purity and higher yields for wide range of particle sizes, concentrations, and flow rates. The principle of operation has also been studied through theory and simulations, while the proof-of-principle was demonstrated using 5 µm beads, platelets, and blood of ranging hematocrits (2.5 % - 45 %). Lower hematocrit (2.5 %) blood could be separated in 100 % purity and high yields (15 %), whereas the high hematocrit blood (45 %) was separated in purities ranging from 98 % to 100 % depending upon the flow rate of operation (10 µlh-1 - 1500 µlh-1). The yield can be increased in k-folds without compromising on purity by cascading the proposed design k-times appropriately.
[0079] In an aspect, the proposed design/apparatus of the present invention include stepped pillar arrays and hydraulic resistance tuners, wherein the stepped pillar arrays permit fluid flow through pillar gaps in similar quantities irrespective of the flow rate of operation, thereby ensuring flow rate independent applicability of this technique, which was demonstrated for flow rates ranging from 10 µlh-1 - 1500 µlh-1. Another associated important feature of this stepped pillar arrays is that the pillar spacing cross-section can be much larger than the size of the particle to be separated from the particle-free fluid. This was demonstrated by choosing pillar gap cross-section as 12 µm X 4.5 µm and the particles to be separated as platelets (2 µm – 4 µm) with 100 % purity. The hydraulic resistance tuners majorly control the net yield and play a major role in deciding the critical particle volume to be decanted as the volume of fluid being extracted through the pillar gaps depends on the side channel to central channel hydraulic resistance ratio (SCRR), number of pillars (N), slope (Sslp), pillar spacing (Wps) and height D3.
[0080] In an aspect, the proposed design also offers several potential applications towards plasma separation from whole blood for biochemical tests, to achieve self-sheath flow focusing by utilizing the particle-free fluid to surround the central particle rich sample, and enrichment of rare cells such as CTC for cancer detection.

Simulation Results
[0081] This section pertains to assessment of effect of height of pillars (D3), spacing between pillars (Wps), number of pillars (N), slope of side channel of Section II (Sslp), flow rate (Q), and side channel to central channel resistance ratio (SCRR) on side channel to central channel flow rate ratio (SCF). It is submitted that simulations were performed on a apparatus model that was presented in simulations section with parameters D1 = 20 µm,D2 = 80 µm, N = 50, and with all other parameters as specified in FIGs.2 and 3 accordingly. Side channel slope Sslp, side channel to central channel hydraulic resistance ratio SCRR, and side channel to central channel flow rate ratio SCF are defined as Sslp = Stl/(Wp + Wps), SCRR = Rs/ Rc, and SCF = Qs / Qc, respectively. The parameters Stl is the change in distance of the side wall from the pillar array plane for a length change of pillar array period (Wp + Wps) along the pillar array direction as shown in FIG. 1. Rc is the combined hydraulic resistance of the central channels C and Ct, and Rs is the hydraulic resistance of the all the side channels S1, S2, S1t, and S2t. However, as the side channels S1 and S2 won’t contribute significantly to the net hydraulic resistance Rs due to their larger width, the effective side channel resistance was computed based on side channels S1t and S2t in this paper. Similarly, the net the hydraulic resistance of the central channel Rc is effectively due to Ct due to its small width and depth compared to C. SCF is computed based on the total flow rate (Qs) of the side channels S1t and S2t, and the flow rate (Qc) through the central channel Ct.
[0082] FIG.2 shows that there is no significant change in SCF with changes in parameters D3, Wps, N, Sslp and Q. FIG. 2A shows that SCF clearly varies with SCRR. FIG. 2B shows experimental data for apparatus with 1000 pillars while keeping all other parameters same, demonstrating a similar behavior of SCF on SCRR. The reason for this can be understood from the perspective that the fluid flow rate through a channel depends to a great deal on the hydraulic resistance offered by the channel instead of the details of the various geometrical obstructions in the path. As the hydraulic resistance of section III remained constant in all the simulations of FIG.2, it clearly showed no significant influence on SCF. As the side channel resistance of S1t and S2t was altered while keeping the central channel Ct resistance fixed, leading to a change in SCRR, the fluid flow rate in those channels got altered accordingly and manifested as shown in FIG. 2.
[0083] FIG.3 illustrates exemplary effect of side to central channel hydraulic resistance ratio (SCRR) on side to central channel flow rate ratio (SCF), wherein FIG. 3A presents simulation results demonstrating effect of SCRR on SCF, and FIG. 3B presents Experimental results showing the variation of SCF with respect to SCRR.
[0084] Simulations were further carried out to understand the effect of number of pillars (N) and side channel slope (Sslp) on uniformity of flow across pillar gaps, wherein the Simulations were performed on the model described in simulations section with D1 = 20 µm, D2 = 80 µm, D3 = 5 µm, SCRR = 50, and pillar spacing cross-section 10 µm (width) X 5 µm (depth) while varying the number of pillars (N) or the slope of the side channel (Sslp). SCRR was kept fixed at a specific value to keep the SCF constant throughout the simulations. The choice of SCRR to 50 during the simulations has no practical relevance and can be kept at any number to understand the flow behavior. Keeping the number of pillars fixed to 50, the slope of the side channel wall (Sslp) was varied from 0.015 to 0.065 by varying the Stl from 0.5 µm to 10 µm per pillar array period and the resulting flow rate variation between the pillars as a function of pillar position in the model is shown in FIG. 4A. FIG. 4 illustrates exemplary variation of flow rate across the pillar gaps, plotted as a function of pillar number, when the slope of side channel Sslp (a) and number of pillars (N) (b) are varied in accordance with an embodiment of the present disclosure. One exemplary observation is that flow rate distribution across each pillar gap was changing as a function of position of the pillar. More specifically, this variation is more pronounced towards the end pillars. The flow rate rapidly shoots up towards the end pillars. This is an undesirable feature if this section of the design is expected to extract fluid volumes smaller than the volume of the critical particle across all pillar gaps. As Sslp is increased, similar variation of flow rate continued except for one major change, the peak of this flow rate has fallen by redistribution of this increasing flow rate over larger number of pillars. This implies that larger the slope of this wall the more uniform is the distribution of flow rate across the pillars.
[0085] FIG.4B shows variation of flow rate across the pillar gaps as a function of number of pillars, for Stl as 0.5 µm per pillar array period. As the number of pillars has been increased from 50 to 200, the decrease in the peak flow rate and the redistribution of this flow across the pillars can be clearly seen. The number of pillars over which the variation is significantly present is determined by finding the pillar at which the flow rate drops to 1 % of the peak flow rate and counting pillars backwards from the peak flow rate pillar. The increase of Sslp and N seem to contribute to the increased uniformity of the flow across the pillar gaps.
[0086] In an aspect, simulations were also carried out to analyze variation of purity of plasma generated as a function of time, flow rate (Q), hematocrit, and SCRR, wherein the proposed microfluidic apparatus was fabricated with parameters D1 = 20 µm, D2 = 74 µm, D3 = 4.5 µm, Wps = 12 µm, Wp = 18 µm, Stl = 0.5 µm and with SCRR = 2.5, 10, 25, 50, and 100. Experiments were performed on the fabricated apparatus with respect to time, flow rate, hematocrit, and SCRR, and the purity of plasma collected was evaluated using image processing as discussed in section on experimental procedure. FIG.5A shows the time independence of purity of plasma generated and to a value of close to 100 % at a hematocrit of 15 % and SCRR = 25. This infers that the quality of plasma generated is very pure and is independent of time and can be used anytime during on chip analysis.
[0087] As can be observed from FIG.5B, plasma purity remains constant at around 99 % with a maximum variation of only up to 1 % with respect to variation in flow rate to about three orders of magnitude ranging from 10 µlh-1 to 1500 µlh-1 at a hematocrit of 25 % and SCRR = 50. This clearly demonstrates the flow rate independent performance of the proposed technique and can be used for a wide range of applications.
[0088] FIG. 5C shows variation of plasma purity as a function of hematocrit and SCRR. A smaller SCRR is expected to provide larger side channel flow rate as it offers low resistance to flow in side channels, however may compromise on purity at higher hematocrits. Whereas larger SCRR is expected to provide smaller side channel flow rate while offering very high purity even at higher hematocrits. FIG. 5C shows experimental results that agree with theory. Smaller SCRR of 2.5 offered 100 % purity till 2.5 % hematocrit and started dropping till 98.6 % as the hematocrit reached 5 %. However, Larger SCRR of 100 offered 100 % purity till 28 % hematocrit and the purity dropped by only 1% even at a hematocrit of 45 %. FIGs. 6A-D show the images of RBCs at various hematocrit values inside the central channel C and those that escaped into the side channels S1 and S2 at SCRR of 100. As would be appreciated from the above, FIG. 5 illustrates exemplary variation of plasma purity as a function of (a) duration of the experiment (time), (b) flow rate (Q), (c) hematocrit (hct) and SCRR.
[0089] The smaller SCRR of 2.5 has contributed to a plasma yield of about 15 % and the larger SCRR of 100 contributed to a smaller plasma yield of about 2 %. This lower yield at higher hematocrits is not an upper limit to this proposed apparatus, and it can be further increased without compromising on purity, by multiplexing this design in stages and suitably adjusting the side channel resistances as discussed in subsequent section. 5 µm beads and platelets were pumped at 100 µlh-1 into a microfluidic apparatus with SCRR = 50, the purity of the recovered particle-free fluid was close to 100 %. FIG. 6A-6D represent experimental images depicting concentration of RBCs in central channel C and those that escaped into the side channels S1 and S2 for different values of hematocrit. (a) 2.5 % hematocrit, (b) 14 % hematocrit, (c) 25 % hematocrit, and (d) 45 % hematocrit. (e) and (f) represent the experimental images depicting the localization of particles (5 µm beads and platelets) to the central region C alone. The SCRR of the apparatuses used in (a)-(d) is 100 and in (e)-(f) is 50.FIG.6E and 6F show the representative images of the beads and platelets inside the microfluidic apparatus close to the end of section II, respectively. It can be clearly seen that no platelets and beads could be observed in the side channels S1 and S2 and the whole of the particles are concentrated in the central channel.
[0090] In an exemplary aspect, in case one value of SCRR gives a specific SCF, for a sample flow rate of Q, the side channel total flow rate will be Qs = (Q * SCF) / (1 + SCF). As the apparatus has two side channels S1 and S2, the flow rate through each will be half of Qs. For N number of pillars on each side, average the flow rate through each pillar gap will be Qpg = Qs / (2 * N). For a particle of critical volume Vp not to escape through the pillar gap, the volume being drawn through the pillar gaps should be smaller than Vp. As the particle passes closely adjacent to the pillars, it will have two components of velocity u and v. u will be along the direction of main sample flow and is responsible for the particles to retain in the central channel, whereas v is perpendicular to the direction of main sample flow and into the pillar gaps, and is responsible for the particle escape into the side channels S1 and S2. Due to the motion of the particle along main sample flow, each particle spends time duration of about tpg = Wps / u. Within this duration, the volume of fluid that escapes into the pillar gaps is Wps* D3 * v * tpg = Qpg* tpg. When this volume is smaller than Vp, particles won’t escape into the side channels, thereby holding a possibility to achieve 100 % purity of side channel fluid collected. The reduction in the expected purity can arise when this criteria that has been derived for uniform flow across the pillars is not met.
[0091] During the experiments, out of the apparatuses used for experiments, a apparatus with SCRR = 100 was selected for analysis. For this SCRR, the SCF is 2 %. For Q = 400 µlh-1, Wps = 12 µm, and N = 1000, Qpg will be 0.0039 µlh-1. Simulations have been done to estimate u at a distance close enough to the pillars (2.5 µm away the center of the pillar and 2.5 µm away from the top of pillar) and was found to be 0.24 mms-1. This gave rise to time duration of 50 ms, and volume of suction as 54fL per pillar gap. In the case of plasma extraction using whole blood, the quantity of fluid being extracted is smaller than that of the RBC particle size (100 – 120 fL), hence avoiding possibility of RBC escape through the pillar gaps.
[0092] It would be appreciated that certain considerations must be made in view of the observations from simulations as shown in FIG. 4. The flow will not be uniform across the pillars as was presumed in the above analysis. The flow rate across the pillars close to the end of the array in the direction of flow will be high compared to the pillars in the beginning of the array, which may lead to that particles not meeting the desired criteria, that the fluid volume escaping through the pillar gaps to be smaller than the particle volume to be separated, towards the end pillars thereby leading to drop in purity of the fluid collected in side channels. The fabricated apparatus has 1000 pillars which is five times larger than the number of pillars used in simulation (200), hence expected to have a better uniformity of flow rate across the pillar gaps than simulated results show in FIG. 4B. At higher hematocrits (45 %) of operation of the apparatus, the purity has slightly come down to 99 %, which may be an indicator that the flow rate across the pillar gaps towards the end of the array could be still larger than the volume of RBC. Increasing the number of pillars and slope Sslp should help bring down this peak volume that is being extracted across the pillar gaps to smaller than volume of RBC, thereby providing 100% purity even at much larger hematocrits (> 45 %).
[0093] From FIG. 2E, it can be observed that SCF remains unchanged as the sample flow rate is increased, indicating that the flow rate in the side channels and central channel proportionately increase. On an average this indicates how proportionately u and v change, thereby leading to almost constant flow volume across the pillar gaps, independent of sample flow rate. This also suggests how the purity can remain almost constant even for a wide variation of flow rates. This behavior can be observed experimentally as shown in FIG.5B. The above analysis is very general and can be applied to separating particles of any size, volume and concentration at any desired flow rate. Suitably choosing the large values for SCRR, number of pillars N (> 1000), and the slope of side channels Sslp (Stl> 0.5 µm per period of the array), and smaller height D3 and spacing between the pillars Wps as per the above guidelines will help in designing a apparatus that can separate particles smaller than platelets (< 2 µm), without the need for complicated fabrication, and at dimensions that can be easily fabricated using the conventional techniques of microfabrication. The yield of this proposed apparatus can be increased by multiple folds and a way of accomplishing this is presented in the following section.
[0094] In an aspect, experiments were also carried out to evaluate the extent of enhanced yield through multiplexing, wherein one of the proposed designs that has been discussed above can be selected as stage 1, which can produce a yield of Y as shown in resistance model of the apparatus, FIG.7A. If this stage (stage 1) is added to the end of a similar such stage (stage 2), it adds additional resistance to the central channel of stage 2, thereby reducing the purity of the plasma collected. To ensure that the SCRR of the stage 2 of the newly formed combo apparatus remains at its earlier value, an additional resistance X1 needs to be added on either side of stage 2, as shown in FIG.7B. This process ensures increment in yield by a factor of 2, as the two stages contribute to the plasma generation. The inset in FIG. 7 shows a representative schematic of the apparatus with stages 1 and 2 as presented in FIG.7B. Similarly, for a k-stage apparatus the yield is expected to be k*Y, when ensured that the purity is unchanged by adding extra resistances to each stage of the apparatus (FIG.7C). As an example, the yield of the apparatus with SCRR of 2.5 at hematocrit of 2.5 % is 15 % and this can be increased to 75 % by suitably cascading five such stages, keeping in view of the extra resistances that need to be incorporated during the design. In this analysis we presumed that the concentration of the sample is such that it won’t interfere much with the separation process. FIG. 7A represents hydraulic resistance model of the proposed apparatus along with side and central channels Ct, S1t and S2t and is depicted as Stage-I, FIG. 7B represents hydraulic resistance model when Stage-II being added to Stage-I and the representative schematic is shown in the inset, and FIG. 7C represents hydraulic resistance model when k such stages are present in the apparatus.

Experimental Configuration and Results
Apparatus Fabrication
[0095] In an aspect, design of the proposed apparatus can be such that three different depths required can be obtained into a single layer of poly-di-methyl-siloxane (PDMS) and hence is very simple to fabricate. As part of the experiments, three depths on the Master were prepared by the usual process of optical lithography using three SU-8 photoresists 2005, 2015 and 2100. The resulting depths D1, D2, and height D3 were measured using the Dektak surface profiler and were found to be 20 µm, 74 µm, and 4.5 µm respectively. PDMS apparatuses were further fabricated from the Master using the standard process of soft lithography.

Sample Preparation
[0096] Fresh venous blood was collected from healthy subjects in vacutainers containing EDTA (Ethylenediaminetetraacetic acid) anticoagulant. The blood was diluted with phosphate buffer saline (PBS) (135 mMNaCl, 2.7 mM KCL, 10 mM Na2HPO4, 2 mM KH2PO4 and pH adjusted to 7.4) accordingly as per the needed hematocrit. For experiments with platelets the undiluted blood was centrifuged at 3000 rpm for 20 min and the resulting supernatant was diluted by a factor of 2 by adding PBS. 5 µm polystyrene bead solution was prepared by diluting the 10 µl of the raw suspension obtained from, for instance chemical supplier Sigma-Aldrich in 1ml of water.

Simulations
[0097] As the exact design that was used for experiments is complex and computationally intensive, simulations were performed on a simpler model that captures the essence and provides insights into the behavior of particle separation. This design consists of section I of the model discussed above, but of smaller length. The section II consists of smaller number of pillars than the actual apparatus ranging from 50 to 200, but similar other parameters. The parameters used for the simulations are D1 = 20 µm, D2 = 100 µm, D3 = 5 µm, Wps = 10 µm, Wp = 20 µm, Lp = 40 µm. The section III design has kept the hydraulic resistance ratios of the side channel (S') to central channel (Ct), abbreviated as SCRR (side channel to central channel resistance ratio), fixed while performing the simulations with respect to variations of other parameters. Fluid dynamic simulations were performed on COMSOL (computational simulation software) multi physics software version 5.2 using the inbuilt laminar flow module. The choice of fluid for flow inside the design was chosen to be water. Wide varieties of simulations were performed by changing relevant parameters and the results are presented in results and discussion section.

Experimental Procedure
[0098] Keeping in view of the practical applicability of the proposed technique for biological applications, experimental characterization was performed on the fabricated apparatuses by pumping blood at different hematocrit (hct) levels. The blood cells in flow were imaged using a Nikon microscope, 10X micro objective and Pike camera as shown in FIG.8. The number of blood cells escaping through the side channels was counted to quantify the purity of the side fluid collected. The purity of the side fluid is defined as,

[0099] where Nsampl is the total number of particles expected to enter the apparatus per second and Ns is the total number of particles entering the side channels S1 and S2 per second. Nsampl is computed based on hematocrit, flow rate (Q) and is given by Nsampl = Q * hct, whereas Ns is computed based on the images of cells entering S1 and S2. The images that were acquired have been post processed using morphological operations tool box available in MATLAB and a custom written code to obtain the number of cells entering S1 and S2.
[00100] For all the experiments the fluid from the side channels and fluid from the central channel were collected separately into vials using polyethylene tubing. The quantification of volume of fluid extracted in side channels was performed by measuring the length changes of the fluid inside the polyethylene tubing attached to side channels and multiplying with inner area of cross section of the tubing. The measurement has a precision of less than 5 % in determining the volume of fluid collected in side channels, as every measurement of length change inside the polyethylene tubing for volume estimation was larger than 2 cm and the error in measuring the length change was less than 1 mm.
[00101] The above experimental procedure was carried out by varying number of parameters to understand their effects. The parameters include side channel to central channel resistance ratio (SCRR), hematocrit value (hct), flow rate (Q) and time. Another study was carried out to characterize the purity of sheath collected by varying the particle size. Platelets and 5 µm beads were considered for these experiments in place of RBCs. Both these particle suspensions were then pumped into the microfluidic apparatus at 100 µlh-1 and imaged for estimating the purity of the sheath fluid collected.
[00102] It would be appreciated that as most of the research in microfluidics is oriented towards healthcare and the present disclosure has been explained with respect to industrial implementations and developments in separation processes being used in this industrial area. However, the subject matter of the present disclosure is not limited to healthcare in any manner, and can be applied to any other desired domain. It would further be appreciated that in healthcare majority of the diagnostic tests are performed on body fluids such as blood as the biomarkers associated with most of the body medical conditions can be found in them. Further, while some structures have been elaborated above, the principles of invention described herein may as well be implemented by other embodiments. For instance, section I may have a sample channel of a circular profile. All such embodiments and their variations are fully a part of the present disclosure. Further, apparatus described herein can be used for decantation of fluids containing deformable particles (such as red blood cells) as well as non-deformable particles (such as white blood cells).
[00103] While the foregoing describes various embodiments of the invention, other and further embodiments of the invention may be devised without departing from the basic scope thereof. The scope of the invention is determined by the claims that follow. The invention 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 invention when combined with information and knowledge available to the person having ordinary skill in the art.

ADVANTAGES OF THE INVENTION
[00104] The present disclosure elaborates upon an apparatus for microfluidic in-flow decantation that provides high quality plasma at higher hematocrits.
[00105] The present disclosure elaborates upon an apparatus for microfluidic in-flow decantation that is simple to fabricate.
[00106] The present disclosure elaborates upon an apparatus for microfluidic in-flow decantation that integrates easily with other systems.
[00107] The present disclosure elaborates upon an apparatus for microfluidic in-flow decantation that does not require bulky external power supplies.
[00108] The present disclosure elaborates upon an apparatus for microfluidic in-flow decantation that has minimum clogging of its filters and allows for in-flow decantation, while working effectively even for smaller particle sizes.
[00109] The present disclosure elaborates upon an apparatus for microfluidic in-flow decantation that provides separation /decantation of high quality plasma at good flow rates and good plasma yields, without causing hemolysis.
,CLAIMS:
1) An apparatus for in-flow decantation of a first fluid containing particles below a critical volume from a sample fluid containing particles of different volumes, said apparatus comprising:
one or more of a second section (section II), each of said section II comprising:
a channel C of depth D2; and
a side channel S1 on at least one side of said channel C, said side channel S1 comprising a plurality of pillars each of length Lp, width Wp, height D3 and inter-pillar spacing Wps, said height D3 being less than said depth D2, wherein said plurality of pillars is positioned below top edge of said channel C and aligned in a direction lateral to flow of said sample fluid through said channel C to form a stepped pillar array,
one or more of a third section ( section III), each of said section III coupled to each of said section II, said section III comprising:
a channel Ct connected to outlet of said channel C; and
a side channel S1t connected to outlet of said side channel S1,
wherein any of cross-section and length of said channel Ct and cross-section and length of side channel S1t are adapted to control flow of said sample fluid in said channel C and flow of said first fluid through any of said inter-pillar spacings (Vpg) to prevent flow of particles of volumes above said critical volume through any of said inter-pillar spacings, and so enable in-flow decantation of said first fluid from said sample fluid.
2) The apparatus of claim 1, wherein cross-section of said inter-pillar spacing (Wps*D3) is configured to be larger or smaller than cross-section of a particle below said critical volume.
3) The apparatus of claim 1, wherein said Vpg is determined on the basis of said Wps, said D3, average velocity of flow of said first fluid across said inter-pillar spacings (v) , and average velocity of flow of said sample fluid near said pillars along said central channel C (u).
4) The apparatus of claim 3, wherein said adaptation varies hydraulic resistance of any or a combination of said channel Ct and side channel S1t to deliver required values of any of said v and said u and thereby controls ratio of rate of flow of said first fluid drawn through said side channel S1t (Qs) to rate of flow of said sample fluid drawn through said channel Ct (Qc).
5) The apparatus of claim 4, wherein said section II further comprises a side channel S2 on other side of said channel C and said section III further comprises a side channel S2t on other side of said channel Ct, wherein said side channel S2 is identical to said side channel S1 and connected to said side channel S2t, and said side channel S2t is identical to said side channel S1t.
6) The apparatus of claim 5, wherein said apparatus further comprises a section I comprising an input channel (sample channel) for said sample fluid connected to said channel C and wherein transition from said section I to said section II brings a particles free fluid layer nearer to inner walls of said channel C.
7) The apparatus of claim 1, wherein lateral spacing Wsl between said pillars of said stepped pillar array and side wall of said side channel S1 distal from said channel C is configured as continuously increasing from start of the pillars till end in steps of Stl per (Wp+Wps) to enable continuous extraction of small and controllable quantity of said first fluid from said spaces formed by said inter-pillar spacings Wps, D3, and length Lp.
8) The apparatus of claim 7, wherein any or combination of said Wp, said Wps, said Lp, said Stl and number of pillars (N) in said side channel S1 are configured according to overall size required of said apparatus.
9) The apparatus of claim 8, wherein any or a combination of said Stl and said number of pillars (N) is so configured so as to enable flow across all of said pillars to become similar.
10) The apparatus of claim 1, wherein said sample fluid is blood, said first fluid is plasma, and wherein said apparatus performs microfluidic in-flow decantation of said plasma from said blood.