CROSS REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims priority to United States Provisional Patent Application No. 63/342,880 filed May 17, 2022, the entire contents of which is hereby incorporated by reference in its entirety.

BACKGROUND

[0002] Continuous bioprocessing methods are widely understood as having the potential to streamline processing, reduce costs and safeguard quality. To achieve these goals, an enhanced fluid manipulating capability is essential. For example, cell sorter, cell filter, and reagents mixer are necessary parts of lab-on-a-chip (LOC) devices and micro-total analysis systems (p-TAS).

SUMMARY

[0003] In some aspects, the present disclosure provides microfluidic systems for continuous bioprocessing. Bioprocessing, as described herein, includes sorting, filtering, mixing, etc. of two of more agents (e.g. biological agents). In continuous bioprocessing, for example, filtration can partially or completely remove suspended cells, thereby reducing the liquid conditioning time and consequently increasing the accuracy of metabolite analysis. Furthermore, the mixing capability of micromixers may affect the overall performance of the entire systems, i.e., fast mixing of cells, reagents, and organic solutions are essential in bioprocessing.

[0004] The present disclosure provides microfluidic microfluidic devices for providing enhanced fluid manipulating capabilities. In some embodiments, the present disclosure provides a microfluidic device that comprises a spiral tangential flow filtration (STFF) and a mixing apparatus (e.g. a mixer). The present disclosure provides a STFF developed with a cross-flow centrifugal microfluidic platform for the separation of permeate solution from a retentate solution. The present disclosure uses centrifugal microfluidics to effectively perform densitybased separations. Centrifugal microfluidics allows for any suspended particles in a fluid to be trapped and focused near an outer wall of a microfluidic device where strong vortices exist (Dean vortices). Smaller particles in a fluid are trapped inside Dean vortices and remain near the outer wall, while bigger particles are focused near an inner wall of a microfluidic device, thus allowing for particle separation at outlets.

[0005] In some embodiments, a STFF comprises at least one inlet. In some embodiments, a STFF comprises one or more outlets (e.g. two outlets). In some embodiments, a mixing apparatus comprises one or more inlets (e.g. two inlets). In some embodiments, a mixing apparatus comprises at least one outlet. In some embodiments, a STFF comprises a first microchannel having a first end that is connected to a first port (e.g. an inlet). In some embodiments, a first portion of a first microchannel comprises a spiral path around a first port. Furthermore, in some embodiments, a STFF comprises a second microchannel having a spiral portion adjacent to a first portion of a first microchannel. In some embodiments, a second microchannel is separated from a first microchannel by a membrane. In some embodiments, a second end of a first microchannel is connected to a second port (e.g. an outlet).

[0006] In some embodiments, a microfluidic device as described herein comprises a mixing apparatus that comprises a third microchannel. In some embodiments, a first end of a third microchannel may be connected to a third port (e g. an inlet). In some embodiments, a STFF is connected to a mixing apparatus. In some embodiments, a microfluidic device comprises a second microchannel of a STFF connected to a mixing apparatus. For example, in some embodiments, a mixing apparatus comprises two microchannels (e.g. a second microchannel of a STFF and a third microchannel) that converge into the mixing apparatus. In some embodiments, two microchannels of a mixing apparatus join together at an angle ranging between zero degrees and 180 degrees. In some embodiments, a mixing apparatus comprises a splitter. In some embodiments a mixing apparatus comprises a mixer channel. Furthermore, in some embodiments, a splitter may split flow from a microchannel (e.g. a second microchannel of a STFF) into a first flow and a second flow. In some embodiments, a first flow from a splitter and flow from a third microchannel may converge into a first end of a mixer channel. In some embodiments, a second end of a mixer channel may be connected to a fourth port (e.g. an outlet). [0007] In some embodiments, a stream of fluid is capable of flowing through each microchannel of a microfluidic device. In some embodiments, a stream flows and separates in to multiple (e.g. two or more) streams. For example, in some embodiments, an inlet stream (e.g. comprising particles suspended in solution) may flow through a STFF, as described herein, and separate in to a permeate solution and a retentate solution. In some embodiments, flow of one or more streams is laminar.

[0008] In some embodiments, streams join and flow into the mixing apparatus. In some embodiments, at least one stream comprises a solvent solution. In some embodiments, at least one stream comprises a reference solution (e.g. NMR solution (e.g. NMR reference solution)). In some embodiments, at least one stream comprises a permeate solution. In some embodiments, flow of one or more streams is laminar.

[0009] In some embodiments, microchannels have a circular cross-section. In some embodiments, microchannels have a rectangular cross-section. In some embodiments, microchannels are of uniform shape. In some embodiments, microchannels are not of uniform shape. In some embodiments, a width or a height of each micro channel ranges from approximately 1 pm to approximately 1000 pm. In some embodiments, a length of each microchannel ranges from approximately 100 pm to approximately 10 cm.

[0010] Among other things, the present disclosure provides a microfluidic chip. In some embodiments, a microfluidic chip comprises a first port. In some embodiments, a microfluidic chip comprises a first microchannel. In some embodiments, a first microchannel has a first end connected to a first port. In some embodiments, a first microchannel has a first portion with a spiral path around a first port. In some embodiments, a microfluidic chip comprises a membrane. In some embodiments, a microfluidic chip comprises a second microchannel. In some embodiments, a second microchannel has a spiral portion adjacent to a first portion of a first microchannel. In some embodiments, a second microchannel is separated from a first microchannel by a membrane.

[0011] In some embodiments, a spiral path of a first microchannel circles a first port three or more times. In some embodiments, a microfluidic chip further comprises a second port. In some embodiments, a second end of a first microchannel is connected to a second port. In some embodiments, a microfluidic chip further comprises a third port. In some embodiments, a microfluidic chip further comprises a third microchannel. In some embodiments, a third microchannel is connected to a third port.

[0012] In some embodiments, a microfluidic chip further comprises a mixer. In some embodiments, a mixer comprises a second microchannel. In some embodiments, a mixer comprises a third microchannel. In some embodiments, a second microchannel and a third microchannel are connected thereto.

[0013] In some embodiments, a mixer comprises (a) a splitter, and (b) a mixer channel. In some embodiments, a splitter splits flow from a second microchannel in to a first flow and a second flow. In some embodiments, a mixer channel mixes a first flow from a second microchannel and a flow from a third microchannel.

[0014] In some embodiments, a first portion of a first microchannel is longer in length than a spiral portion of a second microchannel. In some embodiments, a first portion of a first microchannel is shorter in length than a spiral portion of a second microchannel. In some embodiments, a first portion of a first microchannel is the same in length as a spiral portion of a second microchannel.

[0015] In some embodiments, a height of a first microchannel, a second microchannel, a third microchannel, and a mixer channel are the same. In some embodiments, a height of a first microchannel, a second microchannel, a third microchannel, and a mixer channel are not the same. In some embodiments, a height of a first microchannel, a second microchannel, a third microchannel, and a mixer channel ranges from about 1 pm to about 1000 pm, about 50 pm to about 500 pm, or about 100 pm to about 250 pm. In some embodiments, a height of a first microchannel, a second microchannel, a third microchannel, and a mixer channel is about 150 pm.

[0016] In some embodiments, a width of a first microchannel, a second microchannel, a third microchannel, and a mixer channel are the same. In some embodiments, a width of a first microchannel, a second microchannel, a third microchannel, and a mixer channel are the not the same. In some embodiments, a width of a first microchannel, a second microchannel, a third microchannel, and a mixer channel ranges from about 1 pm to about 5000 pm, about 10 pm to about 400 pm, about 50 pm to about 3000 pm, about 400 m to about 2000 pm or about 500 pm to about 1500 pm. In some embodiments, a width of a first microchannel is about 400 pm. In some embodiments, a width of a second microchannel is about 800 pm. In some embodiments, a width of a third microchannel is about 600 pm. In some embodiments, a width of a mixer channel is about 3000 pm or about 5000 pm. In some embodiments, a width of a mixer channel is about 2000 pm.

[0017J In some embodiments, a microfluidic chip comprises one or more stages. In some embodiments, a microfluidic chip comprises at least one stage, at least two stages, or at least three stages. In some embodiments, a microfluidic chip comprises more than four stages. In some embodiments, a microfluidic chip comprises between 1 to 100 stages, 5 to 90 stages, 10 to 75 stages, 15 to 60 stages, 20 to 50 stages, or 25 to 30 stages. In some embodiments, a microfluidic chip comprises at most 100 stages. In some embodiments, each stage comprises a further microchannel having a spiral portion adjacent to a portion of a microchannel of a previous stage. In some embodiments, a microchannel of each stage is separated from a microchannel of a previous stage by a membrane.

[0018J In some embodiments, a microfluidic chip comprises three stages. In some embodiments, a first stage comprises a total microchannel width of about 400 pm. In some embodiments, a second stage comprises a total microchannel width of about 1200 pm. In some embodiments, a third stage comprises a total microchannel width of about 2000 pm.

[0019] In some embodiments, a microfluidic chip further comprises a fourth microchannel. In some embodiments, a fourth microchannel has a spiral portion adjacent to a portion of a spiral portion of a second microchannel. In some embodiments, a fourth microchannel is separated from a second microchannel by a second membrane.

[0020] In some embodiments, a width of a fourth microchannel is about 800 pm. In some embodiments, a height of a fourth microchannel is about 150 pm.

[0021] Among other things, the present disclosure provides bioprocessing methods. In some embodiments a method comprises the steps of inputting a fluid sample into a first inlet of a microfluidic chip; flowing a fluid sample in a spiral path through a first microchannel; filtering a permeate portion of a fluid sample from a first microchannel into a second microchannel; and outputting a permeate portion from a second microchannel. In some embodiments, a first microchannel is connected to a first inlet and a spiral path spiraling around a first inlet. In some embodiments, a second microchannel has a spiral portion adjacent to a first microchannel. . In some embodiments, a second microchannel is separated from a first microchannel by a membrane. In some embodiments, a fluid sample flow is predominantly tangential to a membrane.

[0022] In some embodiments, a method further comprises retaining a retentate portion of a fluid sample from a first microchannel. In some embodiments, a method further comprises outputting a retentate portion from a first microchannel. In some embodiments, a first microchannel is connected to a first outlet.

[0023] In some embodiments, a method further comprises inputting a permeate portion into a mixer. In some embodiments, inputting a permeate portion comprises a step of splitting a permeate portion from a second microchannel in to a first flow and a second flow. In some embodiments, a step of splitting is performed using a splitter. In some embodiments, inputting a permeate portion further comprises inputting a first flow into a mixer channel.

[0024] In some embodiments, a retentate portion comprises one or more particles. In some embodiments, one or more particles comprise one or more of a microparticle, a nanoparticle, a lipid nanoparticles (LNP), a liposome, a micelle, a cell, a cellular particle, or a combination thereof.

[0025] In some embodiments, a method further comprises: inputting a mixing solution into a second inlet of a microfluidic chip; flowing a mixing solution in a path through a third microchannel; mixing a first flow from a second microchannel and a fluid solution; and outputting a metabolite from the mixer channel. In some embodiments, a third microchannel is connected to a second inlet. In some embodiments, a mixer channel is connected to a second outlet.

[0026] In some embodiments, a fluid sample is introduced into a microfluidic chip at a specified flow rate. In some embodiments, a flow rate is regulated. In some embodiments, a flow rate is not regulated. In some embodiments, a flow rate of a fluid sample is in the range of about 0.001 pl/min to about 20.0 ml/min, about 1 pl/min to about 20.0 ml/min, or about 1 ml/min to about 20.0 ml/min. In some embodiments, a flow rate of a fluid sample is 1.6 ml/min, 3 ml/min, 4 ml/min, 5 ml/min, or 6 ml/min.

[0027] In some embodiments, a step of inputting a mixing solution comprises inputting a mixing solution using a pressure-driven system. In some embodiments, a pressure of a pressure driven system is between about 0.1 psi to about 50 psi, about 1 psi to about 25 psi, or about 5 psi to about 10 psi. In some embodiments, a pressure of a pressure driven system is about 3 psi.

[0028] These, and other aspects encompassed by the present disclosure, are described in more detail below and in the claims.

BRIEF DESCRIPTION OF THE DRAWING

[0029] FIG. 1A shows an integrated spiral tangential flow fdtration (STFF) with mixer microfluidic chip (i.e. a mixer-tailed microfluidic chip or a mixer-tailed microfluidic device, as described herein) for providing an enhanced fluid manipulating capability. FIG. IB shows a cross section of a portion of a microchannels and illustrates how inertial focusing provides for separation of cells within a fluid sample. FIG. 1C shows an example of operation of a chip, according to an illustrative embodiment.

[0030] FIG. 2 shows a photograph of a mixer-tailed microfluidic chip, according to some embodiments.

[0031] FIG. 3A shows a comparison of focusing dynamics of 15pm and 20pm particles in a portion of chip #100 with specific channel thickness (t=l 50pm) at the optimal flow rates. The scale bars are 100pm. FIG. 3B shows the recovery efficiency for 15pm and 20pm particles for an illustrative embodiment of a mixer-tailed microfluidic chip. FIG. 3C shows dimensions of a chip, as described herein, in millimeters.

[0032] FIG. 4 shows a computer system operably connected to various actuators (e.g., pumps), sensors, and other instrumentation modules to control use of a microfluidic chip, according to an illustrative embodiment.

[0033] FIG. 5 shows a poster titled “Mixer-Tailed Filtration Fluidic Chip for Continuous Bioprocessing”. [0034] FIG. 6 shows a design and fabrication of microfluidic devices (e.g. a mixer-tailed filtration fluidic chip) as described herein, according to an embodiment.

[0035] FIG. 7 shows viewgraphs of a fluidic setup for a microfluidic device, as described herein.

[0036] FIG. 8 shows a spiral tangential flow filtration (STFF) system with varying channel widths for filtering polymer microspheres, according to an illustrative embodiment.

[0037] FIG. 9 shows a photograph of a mixer-tailed microfluidic chip (i.e. an STFF with mixer) and its dimensions, according to some embodiments.

DEFINITIONS

[0038] About, Approximately: The term “about” or “approximately”, when used herein in reference to a value, refers to a value that is similar, in context to the referenced value. In general, those skilled in the art, familiar with the context, will appreciate the relevant degree of variance encompassed by “about” in that context. For example, in some embodiments, the term “about” may encompass a range of values that within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less of the referred value.

[0039] Agent : In general, the term “agent”, as used herein, may be used to refer to a compound or entity of any chemical class including, for example, a polypeptide, nucleic acid, saccharide, lipid, small molecule, metal, or combination or complex thereof. In appropriate circumstances, as will be clear from context to those skilled in the art, the term may be utilized to refer to an entity that is or comprises a cell or organism, or a fraction, extract, or component thereof. Alternatively or additionally, as context will make clear, the term may be used to refer to a natural product in that it is found in and/or is obtained from nature. In some instances, again as will be clear from context, the term may be used to refer to one or more entities that is manmade in that it is designed, engineered, and/or produced through action of the hand of man and/or is not found in nature. In some embodiments, an agent may be utilized in isolated or pure form; in some embodiments, an agent may be utilized in crude form. In some embodiments, potential agents may be provided as collections or libraries, for example that may be screened to identify or characterize active agents within them. In some cases, the term “agent” may refer to a compound or entity that is or comprises a polymer; in some cases, the term may refer to a compound or entity that comprises one or more polymeric moieties. In some embodiments, the term “agent” may refer to a compound or entity that is not a polymer and/or is substantially free of any polymer and/or of one or more particular polymeric moieties. In some embodiments, the term may refer to a compound or entity that lacks or is substantially free of any polymeric moiety.

[0040] Comprising: A composition or method described herein as "comprising" one or more named elements or steps is open-ended, meaning that the named elements or steps are essential, but other elements or steps may be added within the scope of the composition or method. To avoid prolixity, it is also understood that any composition or method described as "comprising" (or which "comprises") one or more named elements or steps also describes the corresponding, more limited composition or method "consisting essentially of' (or which "consists essentially of) the same named elements or steps, meaning that the composition or method includes the named essential elements or steps and may also include additional elements or steps that do not materially affect the basic and novel characteristic(s) of the composition or method. It is also understood that any composition or method described herein as "comprising" or "consisting essentially of' one or more named elements or steps also describes the corresponding, more limited, and closed-ended composition or method "consisting of (or "consists of') the named elements or steps to the exclusion of any other unnamed element or step. In any composition or method disclosed herein, known or disclosed equivalents of any named essential element or step may be substituted for that element or step.

[0041] Particle, Microparticle, Microspherc. As used herein, a “particle”, “microparticle”, or “microsphere” refers to any entity having a diameter of less than 100 microns (pm). Typically, particles have a greatest dimension (e.g., diameter) of 1000 pm or less. In some embodiments, particles have a diameter of 300 pm or less. In some embodiments, particles have a diameter of 200 pm or less. In some embodiments, particles have a diameter of 100 pm or less. In some embodiments, nanoparticles have a diameter of 1000 nm or less. In some embodiments, nanoparticles have a diameter of 500 nm or less. In some embodiments, nanoparticles have a diameter of 300 nm or less. In some embodiments, nanoparticles have a diameter of lOOOnm or less. In some embodiments, a population of particles may be relatively uniform in terms of size, shape, charge, and/or composition. In some embodiments, particles as described herein can be solid or hollow and can comprise one or more layers. In some embodiments, particles are spheres, spheroids, flat, plate-shaped, cubes, cuboids, ovals, ellipses, cylinders, cones, or pyramids. In some embodiments, particles can comprise a matrix of one or more polymers. In some embodiments, the matrix is cross-linked. In some embodiments, formation of the matrix involves a cross-linking step. In some embodiments, the matrix is not substantially cross-linked. In some embodiments, formation of the matrix does not involve a cross-linking step. In some embodiments, particles may be microparticles, nanoparticles, lipid nanoparticles (LNPs), liposomes, micelles, and/or other cellular particles. As used herein, the term “nanoparticle” refers to any particle having a diameter of less than 1000 nm. In some embodiments, particles are organic particles such as particles made from organic polymer, lipids, sugars, or other organic materials. Such organic particles may optionally contain some inorganic material. In some embodiments, for example, the amount of inorganic material is less than 50%, less than 25%, less than 10%, less than 5%, or less than 1%. In some embodiments, particles are polymeric particles with a substantial portion of a matrix of the particle being polymeric.

[0042] Program, Software: The terms “program” or “software” are used herein to refer to any type of computer code or plurality of computer-executable instructions that may be employed to program one or more processors to implement various aspects of the present disclosure as discussed herein.

[0043] Reference: As used herein describes a standard or control relative to which a comparison is performed. For example, in some embodiments, an agent, animal, individual, population, sample, sequence or value of interest is compared with a reference or control agent, animal, individual, population, sample, sequence or value. In some embodiments, a reference or control is tested and/or determined substantially simultaneously with the testing or determination of interest. In some embodiments, a reference or control is a historical reference or control, optionally embodied in a tangible medium. Typically, as would be understood by those skilled in the art, a reference or control is determined or characterized under comparable conditions or circumstances to those under assessment. Those skilled in the art will appreciate when sufficient similarities are present to justify reliance on and/or comparison to a particular possible reference or control.

[0044] Substantially: As used herein, the term “substantially” refers to the qualitative condition of exhibiting total or near-total extent or degree of a characteristic or property of interest. One of ordinary skill in the scientific arts will understand that, for example, biological and chemical phenomena rarely, if ever, go to completion and/or proceed to completeness or achieve or avoid an absolute result. The term “substantially” is therefore used herein to capture the potential lack of completeness inherent in many scientific phenomena.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

Microfluidics

[0045] Microfluidics is the science and technology of manipulating flows in microscale channels, typically ranging from 10 pm - 100 pm in size (Whitesides, 2006, Nature, 442:368). Microfluidics as a field of research emerged in the 1990s, pioneered by the idea of Manz et al. (1992, J. Chromatography, 593:253) that several time consuming, laborious steps in chemical and biological analysis could be efficiently carried out in miniaturized channels and chambers with faster throughput and time-to-result, smaller sample consumption, and lower cost. Since then, microfluidics has emerged as an active research area, with applications ranging from biological analysis to chemical synthesis, and electronic cooling to optics.

Microfluidic Devices

[0046] The present disclosure provides microfluidic devices (i.e. microfluidic chips) for providing enhanced fluid manipulating capabilities. In some embodiments, the present disclosure provides a microfluidic device that comprises a spiral tangential flow filtration (STFF) and a mixing apparatus (e.g. a mixer). That is, in some embodiments, a STFF and a mixing apparatus are components of a microfluidic device as described herein. [0047] The present disclosure provides a STFF developed with a cross-flow centrifugal microfluidic platform for the separation of permeate solution from a retentate solution. The present disclosure uses centrifugal microfluidics to effectively perform density-based separations. Centrifugal microfluidics allows for any suspended particles in a fluid to be trapped and focused near an outer wall of a microfluidic device where strong vortices exist (Dean vortices). Smaller particles in a fluid are trapped inside Dean vortices and remain near the outer wall, while bigger particles are focused near an inner wall of a microfluidic device, thus allowing for particle separation at outlets.

[0048] The present disclosure provides a mixing apparatus that works by utilizing the Coanda effect to split an approaching stream and then redirect one of the diverged flows so that it will meet a second tributary (i.e. a second microchannel) of a mixing apparatus.

[0049] In some embodiments, flow of streams through microchannels is laminar. In some embodiments, flow of the streams is characterized by a Reynolds number of approximately 10' ⁵ , approximately 10' ⁴ , approximately 0.001, approximately 0.01, approximately 0.1, approximately 1.0, approximately 10, approximately 100, approximately 1000, or approximately 2000.

[0050] In some embodiments, microchannels have or comprise a circular cross-section. In some embodiments, microchannels have or comprise an oval or ovaloid cross-section. In some embodiments, microchannels have or comprise an elliptical or an ellipsoid cross-section. In some embodiments, microchannels have or comprise a cross-section of irregular shape. In some embodiments, microchannels that converge are of uniform shape. In some embodiments, microchannels that converge are not of uniform shape.

[0051] In some embodiments, a width or a height of a microchannel ranges from approximately 1 pm to approximately 5000 pm. In some embodiments, a width or a height of a microchannel ranges from approximately 10 pm to approximately 4000 pm. In some embodiments, a width or a height of a microchannel ranges from approximately 50 pm to approximately 3000 pm. In some embodiments, a width or a height of a microchannel ranges from approximately 400 pm to approximately 2000 pm. In some embodiments, a width or a height of a microchannel ranges from approximately 500 pm to approximately 1500 pm. In some embodiments, a width or a height of a microchannel ranges from approximately 400 pm to approximately 1200 pm. In some embodiments, a width or a height of a microchannel ranges from approximately 600 pm to approximately 1200 pm. In some embodiments, a width or a height of a microchannel ranges from approximately 10 pm to approximately 10000 pm. In some embodiments, a width or a height of a microchannel ranges from approximately 25 pm to approximately 100 pm. In some embodiments, a width or a height of a microchannel ranges from approximately 50 pm to approximately 100 pm. In some embodiments, a width or a height of a microchannel ranges from approximately 75 pm to approximately 100 pm. In some embodiments, a width or a height of a microchannel ranges from approximately 10 pm to approximately 75 pm. In some embodiments, a width or a height of a microchannel ranges from approximately 10 pm to approximately 50 pm. In some embodiments, a width or a height of a microchannel ranges from approximately 10 pm to approximately 25 pm.

[0052] In some embodiments, a width or a height of a microchannel is approximately 1 pm, approximately 5 pm, approximately 10 pm, approximately 20 pm, approximately 30 pm, approximately 40 pm, approximately 50 pm, approximately 60 pm, approximately 70 pm, approximately 80 pm, approximately 90 pm, approximately 100 pm, approximately 150 pm, approximately 200 pm, approximately 250 pm, approximately 300 pm, approximately 350 pm, approximately 400 pm, approximately 450 pm, approximately 500 pm, approximately 550 pm, approximately 600 pm, approximately 650 pm, approximately 700 pm, approximately 750 pm, approximately 800 pm, approximately 850 pm, approximately 900 pm, approximately 950 pm, approximately 1000 pm, approximately 1100 pm, approximately 1200 pm, approximately 1300 pm, approximately 1400 pm, approximately 1500 pm, approximately 1600 pm, approximately 1700 pm, approximately 1800 pm, approximately 1900 pm, approximately 2000 pm, approximately 3000 pm, approximately 4000 pm, or approximately 5000 pm.

[0053] In some embodiments, a width of a microchannel ranges from approximately 5 pm to approximately 5000 pm. In some embodiments, a width of a microchannel is approximately 5 pm, approximately 10 pm, approximately 15 pm, approximately 20 pm, approximately 25 pm, approximately 30 pm, approximately 35 pm, approximately 40 pm, approximately 45 pm, approximately 50 pm, approximately 60 pm, approximately 70 pm, approximately 80 pm, approximately 90 pm, approximately 100 pm, approximately 150 pm, approximately 200 pm, approximately 250 pm, approximately 300 pm, approximately 350 pm, approximately 400 pm, approximately 450 pm, approximately 500 pm, approximately 550 pm, approximately 600 pm, approximately 650 pm, approximately 700 pm, approximately 750 pm, approximately 800 pm, approximately 850 pm, approximately 900 pm, approximately 950 pm, approximately 1000 pm, approximately 1100 pm, approximately 1200 pm, approximately 1300 pm, approximately 1400 pm, approximately 1500 pm, approximately 1600 pm, approximately 1700 pm, approximately 1800 pm, approximately 1900 pm, approximately 2000 pm, approximately 3000 pm, approximately 4000 pm, or approximately 5000 pm.

[0054] In some embodiments, a height of a microchannel ranges from approximately 10 pm to approximately 10000 pm. In some embodiments, a height of a microchannel is approximately 10 pm, approximately 100 pm, approximately 150 pm, approximately 250 pm, approximately 400 pm, approximately 500 pm, approximately 600 pm, approximately 750 pm, approximately 1000 pm, approximately 2000 pm, approximately 3000 pm, approximately 4000 pm, approximately 5000 pm, approximately 6000 pm, approximately 7000 pm, approximately 8000 pm, approximately 9000 pm, or approximately 10000 pm. In some embodiments, a height of a microchannel(s) is approximately 150 pm.

[0055] In some embodiments, a length of a microchannel ranges from approximately 100 pm to approximately 60 cm. In some embodiments, length of a ranges is approximately 100 pm, approximately 1 .0 mm, approximately 10 mm, approximately 100 mm, approximately 100 mm, approximately 200 mm, approximately 300 mm, approximately 400 mm, approximately 500 mm, approximately 600 mm, approximately 700 mm, approximately 800 mm, approximately 900 mm, approximately 1.0 cm, approximately 1.1 cm, approximately 1.2 cm, approximately 1.3 cm, approximately 1.4 cm, approximately 1.5 cm, approximately 5 cm, approximately 10 cm, approximately 20 cm, approximately 25 cm, approximately 30 cm, approximately 35 cm, approximately 40 cm, approximately 45 cm, approximately 50 cm, approximately 55 cm, or approximately 60 cm.

[0056] In some embodiments, a length of a microfluidic device ranges from approximately 10 cm to 100 cm. In some embodiments, a length of a microfluidic device ranges from approximately 10 cm to 75 cm. In some embodiments, a length of a microfluidic device ranges from approximately 10 cm to 60 cm. In some embodiments, a length of a microfluidic device is approximately 57.5 cm.

[0057] In some embodiments, microchannels are of uniform width, height, and/or length. In some embodiments, microchannels are not of uniform width, height, and/or length.

[0058] In some embodiments, microchannels are arranged in one plane relative to one another. In some embodiments, microchannels, STFF(s), and/or mixing apparatus are all arranged in one plane relative to one another. In some embodiments, microchannels are arranged in different planes relative to one another. In some embodiments, microchannels, STFF(s), and/or mixing apparatus are all arranged in different planes relative to one another. In some embodiments, microchannels are arranged in one plane relative to one another, and a mixing apparatus is arranged in a different plane relative to microchannels. In some embodiments, microchannels are arranged in one plane relative to one another, and a STFF is arranged in a different plane relative to microchannels. In some embodiments, a mixing apparatus is arranged in one plane and a STFF is arranged in a different plane relative to a mixing apparatus.

[0059] In general, microchannels converge at an angle greater than zero degrees. In some embodiments, microchannels converge at an angle of approximately 90 degrees. In some embodiments, microchannels converge at an angle of approximately 10 degrees, approximately 20 degrees, approximately 30 degrees, approximately 40 degrees, approximately 50 degrees, approximately 60 degrees, approximately 70 degrees, approximately 80 degrees, approximately 90 degrees, approximately 100 degrees, approximately 110 degrees, approximately 120 degrees, approximately 130 degrees, approximately 140 degrees, approximately 150 degrees, approximately 160 degrees, approximately 170 degrees, or approximately 180 degrees.

[0060] A microfluidic device may be composed of any material suitable for the flow of fluid through microchannels comprised in a device’s STFF(s), and/or mixing apparatus. Typically, the material is one that is resistant to solvents and non-solvents that are used in bioprocessing methods. In general, the material is not one that will dissolve or react with a solvent or non-solvent. In some embodiments, a microfluidic device and/or its components are composed of one or more of glass, silicon, metal, metal alloys, polymer(s), plastic(s), epoxy, ceramic(s), or combination thereof. In some embodiments, a microfluidic device and/or its components are composed of a metal and/or metal alloys (e.g. iron, titanium, aluminum, gold, platinum, chromium, molybdenum, zirconium, silver, niobium, alloys thereof, etc.). In some embodiments, a microfluidic device and/or its components are composed of a polymer, as described herein. In some embodiments, a microfluidic device and/or its components are composed of plastic, including, but not limited to, polycarbonate, polyethylene terephthalate (PET) polyethylene terephthalic ester (PETE), polytetrafluoroethylene (PTFE), polydimethylsiloxane (PDMS), polyurethane, bakelite, polyester, etc. In some embodiments, a microfluidic device and/or its components are composed of photocurable epoxy. In some embodiments, a microfluidic device and/or its components are composed of polydimethylsiloxane. In some embodiments, a microfluidic device and/or its components are composed of ceramics (e.g. silicon nitride, silicon carbide, titania, alumina, silica, etc.).

[0061] Microfluidic devices and its components (e.g. STFF, microchannels, mixing apparatus) described herein may be constructed from one or more materials. For example, in some embodiments, a material a microfluidic device is constructed from may include, but is not limited to, for example, polydimethylsiloxane (PDMS). In some embodiments, other suitable materials may be used to construct a microfluidic device. In some embodiments, a PDMS microfluidic channel may be constructed on a glass substrate.

[0062] In some embodiments, a microfluidic device may optionally comprise an apparatus for controlling temperature. Tn some embodiments, bioprocessing occurs in a microfluidic device at approximately room temperature. In some embodiments, bioprocessing occurs in a microfluidic device at a temperature ranging from approximately 0 °C to approximately 10 °C. In some embodiments, bioprocessing occurs in a microfluidic device at a temperature ranging from approximately 10 °C to approximately 20 °C. In some embodiments, bioprocessing occurs in a microfluidic device at a temperature ranging from approximately 20 °C to approximately 30 °C. In some embodiments, bioprocessing occurs in a microfluidic device at a temperature ranging from approximately 30 °C to approximately 40 °C.

[0063] FIG. 1A show an integrated microfluidic chip #100, according to an illustrative embodiment, for providing an enhanced fluid manipulating capability. In FIG. 1A, a fluid sample entering at inlet #150 is pumped through the spiral microchannels #110. Upon reaching the end of the spiral microchannels, a portion of the fluid sample will have passed through the structural membrane (the permeate) and a second portion of the fluid sample will not have passed through the structural membrane (the retentate). The retentate is directed along path #120 and exits chip #100 at outlet #121. The permeate is directed along path #130.

[0064] Path #130 may connect to mixing apparatus #160 on chip #100. In some embodiments, an NMR reference solution is pumped into chip #100 at inlet #140, down path #141, and mixed with the permeate in mixer #160. The structure shown in the exemplary mixer #160 utilizes the Coanda effect to split an approaching stream and then redirect one of the diverged flows so that it will meet the second tributary. For example, the stream from path #130 may be split and one of the diverged flows is redirected so that it meets the flow from path #140. The example, mixer #160, repeats this mixing stage multiple times before the resultant metabolite exits the chip #100 at outlet #161.

[0065] In some embodiments, a microfluidic device and/or its components is formed by lithography, etching, embossing, or molding of a polymeric surface. In general, the fabrication process may involve one or more of any of the processes described below (or similar processes), and different parts of a device may be fabricated using different methods and assembled or bonded together.

[0066] Lithography involves use of light or other form of energy such as electron beam to change a material. Typically, a polymeric material or precursor (e.g. photoresist, a light- resistant material) is coated on a substrate and is selectively exposed to light or other form of energy. Depending on the photoresist, exposed regions of the photoresist either remain or are dissolved in subsequent processing steps known generally as “developing.” This process results in a pattern of the photoresist on the substrate. In some embodiments, a photoresist is used as a master in a molding process. In some embodiments, a polymeric precursor is poured on the substrate with photoresist, polymerized (i.e. cured) and peeled off. The resulting polymer is bonded or glued to another flat substrate after drilling holes for inlets and outlets.

[0067] In some embodiments, a photoresist is used as a mask for an etching process. For example, after patterning photoresist on a silicon substrate, microchannels can be etched into the substrate using a deep reactive ion etch (DRIE) process or other chemical etching process known in the art (e.g. plasma etch, KOH etch, HF etch, etc.). The photoresist is removed, and the substrate is bonded to another substrate using one of any bonding procedures known in the art (e.g. anodic bonding, adhesive bonding, direct bonding, eutectic bonding, etc.). Multiple lithographic and etching steps and machining steps such as drilling may be included as required.

[0068] In some embodiments, a polymeric substrate may be heated and pressed against a master mold for an embossing process. The master mold may be formed by a variety of processes, including lithography and machining. The polymeric substrate is then bonded with another substrate to form microchannels that form a STFF and/or a mixing apparatus.

Machining processes may be included if necessary.

[0069] In some embodiments, a molten polymer or metal or alloy is injected into a suitable mold and allowed to cool and solidify for an injection molding process. The mold typically consists of two parts that allow the molded component to be removed. Parts thus manufactured may be bonded to result in the device.

[0070] In some embodiments, sacrificial etch may be used to form microchannels that form a STFF and/or a mixing apparatus. Lithographic techniques may be used to pattern a material on a substrate. This material is covered by another material of different chemical nature. This material may undergo lithography and etch processes, or other machining process. The substrate is then exposed to a chemical agent that selectively removes the first material. Microchannels are formed in the second material, leaving voids where the first material was present before the etch process.

[0071] In some embodiments, microchannels are directly machined into a substrate by laser machining or CNC machining. Several layers thus machined may be bonded together to obtain the final device. In specific embodiments, laser machining may be performed on polymer sheets commonly used for lamination (e.g., of certificates, identity cards, etc.). Lamination of machined sheets is used to form the device.

[0072] Typically, a source of fluid is attached to each channel, and the application of pressure to the source causes the flow of the fluid in the channel. In some embodiments, pressure is applied by a syringe. Tn some embodiments, pressure is applied by a pump. Tn some embodiments, pressure is applied by gravity. In some embodiments, applied pressure is regulatable (i.e. applied pressure may be increased, decreased, or held constant). In some embodiments, flow rate is regulatable by adjusting applied pressure. In some embodiments, flow rate is regulatable by adjusting size (e.g. length, width, and/or height) of a microchannel.

[0073] In some embodiments, flow rate may range from approximately 0.001 pl/min to approximately 20.0 ml/min. In some embodiments, flow rate is approximately 0.01 pl/min, approximately 0.1 pl/min, approximately 0.5 pl/min, approximately 1.0 pl/min, approximately 5 pl/min, approximately 10 pl/min, approximately 50 pl/min, approximately 100 pl/min, approximately 1.0 ml/min, approximately 1.5 ml/min, approximately 1.6 ml/min, approximately 2.0 ml/min, approximately 3.0 ml/min, approximately 4.0 ml/min, approximately 5.0 ml/min, approximately 6.0 ml/min, approximately 7.0 ml/min, approximately 8.0 ml/min, approximately 9.0 ml/min, approximately 10.0 ml/min, approximately 11.0 ml/min, approximately 12.0 ml/min, approximately 13.0 ml/min, approximately 14.0 ml/min, approximately 15.0 ml/min, approximately 16.0 ml/min, approximately 17.0 ml/min, approximately 18.0 ml/min, approximately 19.0 ml/min, or approximately 20.0 ml/min. In some embodiments, flow rate is ranges between approximately 3 ml/min to 6 ml/min.

[0074] In some embodiments, a same amount of pressure is applied to all of microchannels. In some embodiments, different amounts of pressure are applied to different microchannels. Thus, in some embodiments, flow rate may be the same through all microchannels, or the flow rate may be different in different microchannels.

[0075] The present invention provides microfluidic systems in which a plurality of inlet streams converge and mix, and the resulting mixture is the polymeric stream that flows into a mixing apparatus (for example, see Figure 4). In some embodiments, a microfluidic system may comprise at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or more inlet streams. The flow of each inlet stream is regulated by a source of fluid, wherein the application of pressure to the source causes the flow of fluid in the inlet stream. In some embodiments, the pressure is applied by a syringe. In some embodiments, the pressure is applied by a pump. In some embodiments, the pressure is applied by gravity. In some embodiments, the applied pressure is regulatable (i.e. the applied pressure may be increased, decreased, or held constant). In some embodiments, the flow rate is regulatable by adjusting the applied pressure. [0076] In some embodiments, the same amount of pressure is applied to all of the inlet streams. In some embodiments, different amounts of pressure are applied to different inlet streams. Thus, in some embodiments, the flow rate may be the same through all inlet streams, or the flow rate may be different in different inlet streams.

[0077] In some embodiments, microfluidic systems or devices as described herein fdter particles at a rate ranging from approximately 0.1 mg/min to approximately 1 g/min. In some embodiments, microfluidic systems or devices filter particles at a rate ranging from approximately 1.0 mg/min to approximately 500 mg/min. In some embodiments, microfluidic systems or devices filter particles at a rate ranging from approximately 10 mg/min to approximately 100 g/min.

[0078] In specific embodiments, microfluidic systems or devices as described herein which involve the use of mixing apparatuses (e.g. mixers), such as those described herein, filter particles at a rate ranging from 0.01 mg/min to 100 mg/min. In some embodiments, microfluidic systems or devices which involve the use of mixing apparatuses, such as those described herein, filter particles at a rate of approximately 0.01 mg/min to 1.0 mg/min. In some embodiments, microfluidic systems or devices as described herein which involve the use of mixing apparatuses filter particles at a rate of approximately 0.01 mg/min, approximately 0.1 mg/min, approximately 1.0 mg/min, approximately 10 mg/min, approximately 100 mg/min, or greater than 100 mg/min.

[0079] In some embodiments, microfluidic systems or devices as described herein filter particles at an average rate of approximately 0.1 mg/min. In some embodiments, microfluidic systems or devices filter particles at an average rate of approximately 1.0 mg/min. In some embodiments, microfluidic systems or devices filter particles at an average rate of approximately 10 mg/min. In some embodiments, microfluidic systems or devices filter particles at an average rate of approximately 100 mg/min. In some embodiments, microfluidic systems or devices filter particles at an average rate of approximately 500 mg/min. In some embodiments, microfluidic systems or devices filter particles at an average rate of approximately 1.0 g/min.

[0080] Purification steps may be optionally performed after filtration of particles. In some embodiments, further analyses of filtered particles may be performed. For example, in some embodiments, Nuclear magnetic resonance (NMR) may be performed on a permeate solution. In some embodiments, NMR may be performed on a permeate solution that is mixed with a reference solution (e.g. an NMR reference solution). In some embodiments, a permeate solution and a reference solution are mixed in a mixing apparatus of a microfluidic device as described herein.

[0081] FIG. 2 and FIG. 9 show photographs of a mixer-tailed microfluidic chip, according to an illustrative embodiment of the present disclosure. In the example of FIG. 9, a STFF is connected to a mixing apparatus. The STFF of this example comprises three stages. As shown in FIG. 9, a first stage comprises a microchannel of width of approximately 400 pm, a second stage comprises a microchannel of width of approximately 1200 pm, and a third stage comprises a microchannel of width of approximately 2000 pm. Additionally, a microfluidic device of FIG. 9 comprises a mixer input channel comprises a microchannel of width of approximately 600 pm and a mixer channel of width of approximately 2000 pm. Each of these stages comprise microchannels with a height of approximately 150 pm. The photograph of FIG.

9 shows an exemplary embodiment in which a sample solution (e.g. comprising microspheres) is inputted into an STFF inlet port using a pump system at a follow rate of 1.6 ml/min. The microspheres are collected from an outlet port of the STFF. The permeate solution is then provided as input to a mixing apparatus and an output is collected from an outlet of the mixing apparatus.

[0082] FIG. 3 A shows a comparison of focusing dynamics of 15pm and 20pm particles in a portion of a microfluidic device with specific channel thickness (t=l 50pm) at the optimal flow rates. The scale bars are 100pm. FIG. 3C shows dimensions for this example in millimeters. In this example, the first stage microchannel has a width of 0.4mm, in the second stage, the microchannel has a width of 1.2mm, and in the third stage the microchannel width is 2.0mm. Note also that in the example, the mixing apparatus (i.e. mixer unit) dimensions are 5mm x 3mm (W ^X H). It should be appreciated that any suitable dimensions may be used for the microchannels, mixer, and other features of a microfluidic device as described herein.

[0083] FIG. 3B shows the recovery efficiency for 15pm and 20pm particles for an illustrative embodiment of a mixer-tailed microfluidic chip. It should be appreciated that these results are provided merely to illustrate performance of an example embodiment and that other embodiments may have different recovery performance. [0084] Spiral Tangential Flow Filtration (STFF):

[0085] In some aspects, the present disclosure provides a STFF developed with a crossflow centrifugal microfluidic platform for the separation of permeate solution from a retentate solution.

[0086] In some embodiments, a microfluidic device as described herein is or comprises a STFF. In some embodiments, a microfluidic device as described herein is or comprises at least one STFF. In some embodiments, a microfluidic device as described herein is or comprises two STFFs. In some embodiments, a microfluidic device as described herein is or comprises two or more STFFs. For example, FIG. 6 shows design and fabrication of microfluidic devices comprising one or more STFFs.

[0087] The present disclosure provides a STFF developed with a cross-flow centrifugal microfluidic platform for the separation of permeate solution from a retentate solution. The present disclosure uses centrifugal microfluidics to effectively perform density-based separations. Centrifugal microfluidics allows for any suspended particles in a fluid to be trapped and focused near an outer wall of a microfluidic device where strong vortices exist (Dean vortices). Smaller particles in a fluid are trapped inside Dean vortices and remain near the outer wall, while bigger particles are focused near an inner wall of a microfluidic device, thus allowing for particle separation at outlets.

[0088] In some embodiments, a STFF comprises at least one inlet. In some embodiments, a STFF may comprise two or more inlets. In some embodiments, a STFF comprises one or more outlets. In some embodiments, a STFF comprises two outlets. For example, a STFF may comprise a first outlet for a permeate solution and a second outlet for a retentate solution.

[0089] In some embodiments, a STFF may be connected to a mixing apparatus as described herein. In some embodiments, two STFFs may be connected to generate a high yield STFF (for example, see FIG. 6). In such embodiments, a first outlet of a first STFF may be connected to a first outlet of a second STFF. Additionally, in such embodiments, a second outlet of a first STFF may be connected to a second outlet of a second STFF. In some embodiments, a first outlet of a first STFF may be the same as a first outlet of a second STFF. In some embodiments, a second outlet of a first STFF may be the same as a second outlet of a second STFF

[0090] In some embodiments, a STFF comprises a first microchannel having a first end that is connected to a first port (e g. an inlet). In some embodiments, a first portion of a first microchannel comprises a spiral path around a first port. Furthermore, in some embodiments, a STFF comprises a second microchannel having a spiral portion adjacent to a first portion of a first microchannel. In some embodiments, a second microchannel is separated from a first microchannel by a membrane. In some embodiments, a second end of a first microchannel is connected to a second port (e.g. an outlet).

[0091] In some embodiments, a first microchannel circles a first port once. In some embodiments, a first microchannel circles a first port at least once. In some embodiments, a first microchannel circles a first port two or more times. In some embodiments, a first microchannel circles a first port at least three times. In some embodiments, a first microchannel circles a first port three or more times (e.g. 4, 5, 6, 7, 8, 9, or 10 times).

[0092] The present disclosure contemplates a STFF comprising a structural membrane in microchannels with multiple stages in order to enhance filtering capabilities. In some embodiments, microchannels may comprise one stage. In some embodiments, microchannels may comprise at least one stage. In some embodiments, microchannels may comprise two or more stages. In some embodiments, microchannels may comprise three stages. In some embodiments, microchannels may comprise three or more stages. In some embodiments, microchannels may comprise at most 100 stages.

[0093] FIG. 1A show an integrated microfluidic chip #100 for providing an enhanced fluid manipulating capability. In some embodiments, a microfluidic chip (e.g. chip #100) is used to enhance fluid manipulating capability for continuous bioprocessing. For example, fluid manipulating capabilities include, but are not limited to, cell separation, filtering, and chemical mixing. A microfluidic chip, as shown in FIG. 1A, i.e. chip #100 may utilize spiral tangential flow filtration through a combination of inertial focusing of cells and cross-flow between multistage membranes in microchannels #110. Furthermore, chip #100 has curved rectangular (i.e. curved to form spirals with rectangular cross-section) microchannels #110 with geometric structural membranes that are connected to a mixing channel. The microchannels work by utilizing hydrodynamic flow effect to split an approaching stream. One of the diverged flows may be redirected so that it meets a second tributary (e.g., path #130 meets path #141).

[0094] As discussed above, a structural membrane in microchannels (e g. microchannels #110 of FIG. 1A) may have multiple stages to enhance filtering capability. In the example shown in FIG. 1 A, microchannels #110 have three stages, though, any suitable number of stages may be used. In this example, the first stage generates inertial focusing in a fluid sample. The inertial focusing sorts the initially randomly distributed cells into well-defined positions. In a second stage, the membrane transfers cells into the inside wall of the membrane through downstream cross-flow channels. In some embodiments, after being transferred into the downstream channel, the cells are refocused into the channel and then cross-flow filtration is performed again at the third stage. By repeating inertial focusing and cross-flow filtration, cell-free fluid is separated from a focused cell, allowing for a significant reduction in filtration time and for high throughput.

[0095] FIG. 8 shows a micrograph of a STFF with varying channel widths for filtering polymer microspheres, according to an illustrative embodiment. In this example, a first stage comprises a microchannel of width of approximately 400 pm, a second stage comprises a microchannel of width of approximately 1200 pm, and a third stage comprises a microchannel of width of approximately 2000 pm. Each of these stages comprise microchannels with a height of approximately 150 pm. The micrograph shows an exemplary embodiment in which a solution of polymer microspheres (e.g. polystylene microspheres) with an average diameter of approximately 15-20 pm suspended in PBS is separated in to microspheres and media when passed through the three stages of a STFF.

[0096] Structural membranes may be comprise pores. In some embodiments, a structural membrane is constructed of a perforated channel divider. In some embodiments, a structural membrane comprises micron size pores. In some embodiments, pore size may be, for example, in a range from 100pm to 500pm.

[0097] Microfluidic devices and its components (e.g. STFF, microchannels, mixing apparatus) described herein may be constructed from one or more materials. For example, in some embodiments, a material a microfluidic device is constructed from may include, but is not limited to, for example, polydimethylsiloxane (PDMS). In some embodiments, other suitable materials may be used to construct a microfluidic device. In some embodiments, a PDMS microfluidic channel may be constructed on a glass substrate.

[0098] The structural membrane in microchannels (e.g. #110 of FIG. 1 A) may be used to implement cross-flow (i.e., tangential) filtration whereby the majority of the feed flow travels across the surface of the membrane, rather than directly into a filter. Thus, during the process of cross-flow filtration, the liquid sample passes tangentially across a membrane, which is achieved via the centrifugal force acting on the platform. Components smaller than the membrane’s pores are driven through the filter as pressure increases, while larger components pass over the membrane surface. Any filter cake is substantially washed away during the filtration process, increasing the length of time that the chip can be operational. Thus, in some embodiments of, a microfluidic device (e g. chip #100) may be used in a continuous or stop-flow process and need not be limited to a batch-wise processes as generally required by dead-end filtration.

[0099] FIG. IB shows a cross section of a portion of a microchannels #110 and illustrates how inertial focusing provides for separation of cells within a fluid sample. The inventors have recognized and appreciated that centrifugal microfluidics is inherently effective for density-based separations. Suspended particles inside the fluid are trapped and focused near the outer wall where strong vortices exist (Dean vortices). The smaller particles are trapped inside the Dean vortices and remain near the outer wall while bigger particles are focused near the inner wall, thus allowing particle separation at the outlets.

[0100] An example of operation of chip #100 (e.g. of a STFF of a microfluidic chip as described herein) is described with reference to FIGs. 1A & 1C. The fluid sample (e.g., broth media) may be pumped into chip #100 at inlet #150. Initially, cells in the fluid sample flowing through the spiral-shaped microchannel #110 are randomly distributed as illustrated at box #151. As the fluid sample continues to flow along microchannel #110, inertial focusing causes the initially randomly distributed cells to separate into well-defined positions within microfluidic channels as illustrated at box #152. For example, where the cells are size-dependent differently sized cells focus at slightly different positions by the hydrodynamic forces in microchannel #110. At microchannel outlet #154 the sorted cells flow into respective paths #155 as further illustrated at box #153.

[0101] It should be appreciated that while the number of outlet paths in the embodiment of a microfluidic device shown in FIG. 1A (i.e. chip #100) is two, and three of the five different outlet paths are used in the embodiment of FIG. 1C, different embodiments of a microfluidic device described herein may have any suitable number of outlet paths. Further, the downstream processing of the separated portions of the fluid sample is exemplary, and other embodiments and applications may utilize other processing steps which may be integrated into a microfluidic chip #100, performed off-chip, or a combination thereof.

[0102] In some embodiments, two or more microfluidic devices (i.e. microfluidic chips) may be connected in parallel to expand the processing capacity. In some embodiments, two or more microfluidic devices (i.e. microfluidic chips) may be connected in series.

[0103] Mixing Apparatus:

[0104] In some embodiments, a microfluidic device as described herein comprises a mixing apparatus. In some embodiments, a mixing apparatus comprises a third microchannel. In some embodiments, a first end of a third microchannel may be connected to a third port (e.g. an inlet). In some embodiments, a STFF is connected to a mixing apparatus. In some embodiments, a microfluidic device comprises a second microchannel of a STFF connected to a mixing apparatus. For example, in some embodiments, a mixing apparatus comprises two microchannels (e.g. a second microchannel of a STFF and a third microchannel) that converge into the mixing apparatus. In some embodiments, two microchannels of a mixing apparatus j oin together at an angle ranging between zero degrees and 180 degrees.

[0105] In some embodiments, a microfluidic device comprises two microchannels of a mixing apparatus that converge. In some embodiments, microchannels join together at an angle ranging between zero degrees and 180 degrees. In some embodiments, microchannels join together at an angle of approximately 10 degrees, approximately 20 degrees, approximately 30 degrees, approximately 40 degrees, approximately 50 degrees, approximately 60 degrees, approximately 70 degrees, approximately 80 degrees, approximately 90 degrees, approximately 100 degrees, approximately 110 degrees, approximately 120 degrees, approximately 130 degrees, approximately 140 degrees, approximately 150 degrees, approximately 160 degrees, approximately 170 degrees, or approximately 180 degrees. A stream of fluid is capable of flowing through each channel, and streams join and flow into a mixing apparatus. Typically, in some embodiments, microfluidic devices comprise an outlet from a mixing apparatus.

[0106] In some embodiments, a mixing apparatus comprises one or more inlets (e g. two inlets). In some embodiments, a mixing apparatus comprises at least one outlet.

[0107] FIG. 5 shows a poster titled “Mixer-Tailed Filtration Fluidic Chip for Continuous Bioprocessing” which provides further description of some embodiments described herein.

[0108] Having thus described several aspects of at least one embodiment, it is to be appreciated that various alterations, modifications, and improvements will readily occur to those skilled in the art. Such alterations, modifications, and improvements are intended to be within the spirit and scope of the present disclosure. Accordingly, the foregoing descriptions and drawings are by way of example only.

[0109] Automation:

[0110] In some embodiments, a computer system may be operably connected to various actuators (e.g., pumps), sensors, and other instrumentation modules may be used to control use of a microfluidic device as described herein. An example configuration such a microfluidic system is shown in FIG. 4.

[OHl] The viewgraphs of FIG. 7 provide further description of a microfluidic system, according to an illustrative embodiments of the present disclosure. In some embodiments, a microfluidic system comprises a diaphragm metering pump. In some embodiments, a microfluidic system comprises a microfluidic chip. In some embodiments, a microfluidic system comprises a monitoring screen. In some embodiments, a microfluidic system comprises a pump that allows to control flow rate and volume through a microfluidic chip.

[0112] The above-described embodiments of the present disclosure can be implemented in any of numerous ways. For example, the embodiments may be implemented using hardware, software, or a combination thereof. When implemented in software, the software code may be executed on any suitable processor or collection of processors, whether provided in a single computer or distributed among multiple computers.

[0113] Also, the various methods or processes outlined herein may be coded as software that is executable on one or more processors running any one of a variety of operating systems or platforms. Such software may be written using any of a number of suitable programming languages and/or programming tools, including scripting languages and/or scripting tools. In some instances, such software may be compiled as executable machine language code or intermediate code that is executed on a framework or virtual machine. Additionally, or alternatively, such software may be interpreted.

[0114] The techniques disclosed herein may be embodied as a non-transitory computer- readable medium (or multiple non-transitory computer-readable media) (e.g., a computer memory, one or more floppy discs, compact discs, optical discs, magnetic tapes, flash memories, circuit configurations in Field Programmable Gate Arrays or other semiconductor devices, or other non-transitory, tangible computer storage medium) encoded with one or more programs that, when executed on one or more processors, perform methods that implement the various embodiments of the present disclosure discussed above. The computer-readable medium or media may be portable, such that the program or programs stored thereon may be loaded onto one or more different computers or other processors to implement various aspects of the present disclosure as discussed above.

[0115] The terms “program” or “software” are used herein to refer to any type of computer code or plurality of computer-executable instructions that may be employed to program one or more processors to implement various aspects of the present disclosure as discussed above. Moreover, it should be appreciated that according to one aspect of this embodiment, one or more computer programs that, when executed, perform methods of the present disclosure need not reside on a single computer or processor, but may be distributed in a modular fashion amongst a number of different computers or processors to implement various aspects of the present disclosure. [0116] Computer-executable instructions may be in many forms, such as program modules, executed by one or more computers or other devices. Program modules may include routines, programs, objects, components, data structures, etc. that perform particular tasks or implement particular abstract data types. Functionalities of the program modules may be combined or distributed as desired in various embodiments.

[0117] Also, data structures may be stored in computer-readable media in any suitable form. For simplicity of illustration, data structures may be shown to have fields that are related through location in the data structure. Such relationships may likewise be achieved by assigning storage for the fields to locations in a computer-readable medium that convey how the fields are related. However, any suitable mechanism may be used to relate information in fields of a data structure, including through the use of pointers, tags, or other mechanisms that establish how the data elements are related.

[0118] Various features and aspects of the present disclosure may be used alone, in any combination of two or more, or in a variety of arrangements not specifically discussed in the embodiments described in the foregoing, and are therefore not limited to the details and arrangement of components set forth in the foregoing description or illustrated in the drawings. For example, aspects described in one embodiment may be combined in any manner with aspects described in other embodiments.

[0119] Also, the techniques disclosed herein may be embodied as methods, of which examples have been provided. The acts performed as part of a method may be ordered in any suitable way. Accordingly, embodiments may be constructed in which acts are performed in an order different from illustrated, which may include performing some acts simultaneously, even though shown as sequential acts in illustrative embodiments.

[0120] Use of ordinal terms such as “first,” “second,” “third,” etc. in the claims to modify a claim element does not by itself connote any priority, precedence, or order of one claim element over another or the temporal order in which acts of a method are performed, but are used merely as labels to distinguish one claim element having a certain name from another element having a same name (but for use of the ordinal term) to distinguish the claim elements. [0121] Also, the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting. The use of “including,” “comprising,” “having,” “containing,” “involving,” “based on,” “according to,” “encoding,” and variations thereof herein, is meant to encompass the items listed thereafter and equivalents thereof as well as additional items.

EQUIVALENTS

[0122] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. The scope of the present invention is not intended to be limited to the above Description, but rather is as set forth in the following claims: