Methods and compositions for sample filtration

Abstract

The present application relates to methods and compositions for sample filtration, and in particular discloses methods and compositions for producing a filter structure de novo for filtering a sample prior to further processing, e.g., chemical, biological, or other sample. In certain embodiments, provided herein are methods for providing a first filter element, comprising (i) providing a first suspension of a plurality of first microparticles in a first liquid, wherein the first microparticles have a first hydrodynamic diameter of between 1 um and 1000 um; (ii) applying the first suspension to at least one non-circular opening having a short dimension and a long dimension, wherein the short dimension is less than the hydrodynamic diameter of the first microparticles, and the long dimension is at least 2 times the hydrodynamic diameter of the first particles, such that the first microparticles accumulate at the opening and form a first filter element comprising the first microparticles.

Description

[0001] This application is a divisional application of the Chinese national phase patent application with application number 202080068861.7, which corresponds to the PCT application with international application number PCT / US2020 / 046033, international application date of August 12, 2020, entitled "Method and Composition for Sample Filtration", which entered the Chinese national phase on March 30, 2022.

[0002] Cross-referencing

[0003] This application claims the benefit of U.S. Provisional Application No. 62 / 886,299, filed August 13, 2019, which is incorporated herein by reference. Technical Field

[0004] This application relates to the field of analytical liquid handling, and more particularly to methods and compositions for sample filtration. Background Technology

[0005] The analytical liquids handling field has a growing need for microfluidic devices capable of handling small to medium-volume samples rapidly, with high recovery rates, resistance to fouling and clogging, and with minimal potential intersample contamination. These attributes include low cost, ease of use, and suitability for microfluidic analytical liquids handling workflows—meaning these solutions should not hinder the user's workflow. Summary of the Invention

[0006] In some embodiments, this document provides a method for providing a first filter element, comprising (i) providing a first suspension of a plurality of first microparticles in a first liquid, wherein the first microparticles have a first hydrodynamic diameter between 1 μm and 1000 μm; and (ii) applying the first suspension to at least one non-circular opening having a short dimension and a long dimension, wherein the short dimension is smaller than the hydrodynamic diameter of the first microparticles and the long dimension is at least twice the hydrodynamic diameter of the first particles, such that the first microparticles accumulate at the opening and form a first filter element comprising the first microparticles. In some embodiments, the first suspension is applied to at least one constriction by allowing the first liquid to flow into and through the opening. In some embodiments, the opening includes an opening leading to a conduit leading to a treatment system. In some embodiments, the method further comprises dispersing the first filter element formed in step (ii) by allowing a second liquid to flow out and through the opening to disperse the microparticles at the opening. In some embodiments, the method further includes forming a second filter element by applying a second suspension of a plurality of second microparticles in a third liquid to the at least one opening to form a second filter element containing the second microparticles at the opening, wherein the second microparticles have a second hydrodynamic diameter between 1 μm and 1000 μm, wherein the short dimension of the opening is smaller than the hydrodynamic diameter of the second microparticles, and the long dimension of the opening is at least twice the hydrodynamic diameter of the second microparticles. In some embodiments, the method further includes dispersing the second filter element by allowing a fourth liquid to flow out and through the opening to disperse the microparticles at the constriction. In some embodiments, the first microparticles include a surface that adsorbs at least one component of the first liquid. In some embodiments, the first microparticles include a surface that does not bind nucleic acids. In some embodiments, the surface includes a fluorinated surface. In some embodiments, the conduit includes an internal fluorinated surface. In some embodiments, the first liquid contains nucleic acids, and the processing system includes a polymerase chain reaction system. In some embodiments, the first microparticles are spherical or substantially spherical, and the opening has a rectangular or substantially rectangular shape. In some embodiments, the method further includes applying a first suspension of the first microparticle to a plurality of openings, wherein the plurality of openings are non-circular, having a short dimension and a long dimension, wherein the short dimension is smaller than the hydrodynamic diameter of the first microparticle, and the long dimension is at least twice the hydrodynamic diameter of the first particle.

[0007] In some embodiments, this document provides an apparatus comprising (i) a plurality of sample containers, each containing microparticles and a liquid therein suspending the microparticles, wherein the microparticles have a hydrodynamic diameter between 1 μm and 1000 μm; (ii) a conduit having at least one non-circular opening having a short dimension and a long dimension, wherein the short dimension is smaller than the hydrodynamic diameter of the microparticles and the long dimension is at least twice the hydrodynamic diameter of the microparticles; (iii) a system configured to sequentially immerse the one or more openings of the conduit into the plurality of samples; (iv) a system configured to allow the liquid to flow through the one or more openings into the conduit, such that the microparticles accumulate at the openings and form a filter element containing the microparticles; and (v) a system configured to allow a second liquid to flow out of the conduit through the one or more openings to disperse the microparticles at the openings. In some embodiments, the plurality of first microparticles have a diameter in the range of 10 μm to 100 μm. In some embodiments, the first liquid is an aqueous liquid. In some embodiments, the microparticles are spherical or substantially spherical.

[0008] In some embodiments, this document provides a method for loading polymerase chain reaction (PCR) samples into a digital PCR analyzer, comprising (i) providing a plurality of samples containing nucleic acids in an aqueous solution, wherein each sample further comprises a plurality of microparticles, and wherein said microparticles have a hydrodynamic diameter; (ii) placing a sample catheter into a first sample of the plurality of samples, wherein said catheter has an opening, and wherein said opening has a critical size smaller than the hydrodynamic diameter of said microparticles in the first sample, and wherein said catheter is configured to be fluidly connected to said PCR analyzer; (iii) allowing said nucleic acids in the aqueous solution of the first sample to flow into said opening in said catheter and into said catheter, wherein said microparticles in the first sample cannot flow into said PCR analyzer. (iv) After step (iii), liquid is allowed to flow through the conduit and out of the opening of the conduit to remove the one or more microparticles from the first sample from the opening of the conduit, and the first filter element is removed; (v) The sample conduit is placed into a second sample of the plurality of samples, wherein the second sample is different from the first sample, and nucleic acids in an aqueous solution of the second sample are allowed to flow into the opening of the conduit and into the conduit, wherein the microparticles in the second sample cannot flow into the conduit, but a second filter element different from the first filter element is formed at the opening of the conduit, containing one or more microparticles. In some embodiments, the method further includes (vi) after step (v), allowing liquid to flow through the conduit and out of the opening of the conduit to remove the one or more microparticles from the second sample from the opening of the conduit, and removing the second filter element. In some embodiments, at least 10 different samples are allowed to flow into the conduit and into the PCR analyzer, forming and removing at least 10 different filter elements. In some embodiments, at least 100 different samples are fed into the catheter and into the PCR analyzer, forming and removing at least 100 different filter elements. In some embodiments, the microparticles have a hydrodynamic diameter of 1 μm to 1000 μm. In some embodiments, the method further includes generating multiple partitions from the aqueous solution of the one or more samples.

[0009] Incorporate by reference

[0010] All publications, patents and patent applications mentioned in this specification are incorporated herein by reference to the extent that each individual publication, patent or patent application is expressly and individually indicated to be incorporated herein by reference. Attached Figure Description

[0011] Novel characteristic compositions and methods are particularly set forth in the appended claims. A better understanding of the features and advantages of the compositions and methods of the invention will be obtained by referring to the following detailed description and accompanying drawings illustrating illustrative embodiments, which utilize the principles of the compositions and methods, as shown in the drawings:

[0012] Figure 1 This shows the various stages in the formation of the de novo filter.

[0013] Figure 2 Micrographs showing the various stages of de novo filter formation and filter dispersion.

[0014] Figure 3 One embodiment is shown, wherein the opening in the sample conduit is a slit, and the particles used for filter formation are spheres.

[0015] Figure 4 One embodiment is shown, wherein the opening in the sample conduit is a slit, the particles used for filter formation are spheres, and the length dimension of the slit is several times the average diameter of the sphere. Detailed Implementation

[0016] The methods and compositions disclosed herein relate to methods for filtering liquid solutions, and more specifically to the on-demand generation of microporous filter elements, comprising the steps of: providing a liquid suspension of microparticles; applying the liquid suspension to a nozzle having orifices smaller than the particle size in the suspension; drawing the liquid into the nozzle, causing the formation of a microporous filter element at the orifices of the nozzle, until the flow is reversed, disrupting the filter element. Typically, sample filtration for microfluidic applications can be performed before or during the use of a microfluidic device. The microparticles for the liquid solution can be exogenous, such as microparticles added to a sample (e.g., an analytical sample) or microparticles formed within a sample (e.g., an analytical sample).

[0017] Typically, in applications where filtration precedes the use of a microfluidic device, the liquid solution is filtered using a suitable filter unit with an pore size smaller than the minimum critical size of the microfluidic device to be used. This ensures that the liquid entering the device has been adequately removed of debris and / or particulate matter that could impede the device's function, for example, by clogging passages within the device. However, transferring the filtered liquid from its storage container to a reservoir on the device, and other steps in this process, can introduce substances from a number of sources, including but not limited to the surface of a new container, air, or the user during handling.

[0018] To overcome this challenge, filtration units are often integrated into the fluid flow of the device to allow for continuous filtration during use. These filters can exist as tubular filters, such as discs, meshes, or frits, or as structural features incorporated into the microfluidic device itself, such as weir filters, column filters, cross-flow filters, or membrane filters. A typical approach is to use a microfluidic device equipped with microfabricated columns, each with a defined spacing in an array. The microfluidic columns allow liquid components with a hydrodynamic diameter smaller than the column spacing to pass through, while trapping larger particles that could compromise the device's function. However, as with all static filtration methods, such devices are prone to rapid fouling as trapped particles accumulate, clogging portions of the filter and reducing the number of openings. This clogging effectively alters the hydrodynamic resistance across the fouled filter until the device becomes unusable. Furthermore, and even more problematic when using the same microfluidic device to process multiple subsequent samples, filter features and / or debris from clogging can effectively transfer sample components from previous samples to subsequent samples, thereby compromising sample integrity.

[0019] Therefore, most devices with integrated microfluidic channels often require pre-filtration of samples using disposable, single-use filtration units, or the microfluidic itself is a single-use item that needs to be exchanged between samples.

[0020] Therefore, there is a need for a filtration strategy that allows for continuous use of microfluidic instruments without the need for disposable microfluidic devices or sample pre-filtration units.

[0021] The methods and compositions disclosed herein allow the introduction of multiple consecutive samples into a processing system, wherein the samples have been adequately filtered to avoid disrupting the function of the processing system without interrupting sampling, such as to replace or clean filters, and with little or no residue from one sample to the next due to filtration. In some embodiments, the methods and compositions allow the introduction of at least 5, 10, 20, 30, 50, 100, 200, 500, 1000, 2000, or 5000 consecutive samples into a processing system, wherein the samples have been adequately filtered to avoid disrupting the function of the processing system without interrupting sampling, such as to replace or clean filters, and with little or no residue from one sample to the next due to filtration. In some embodiments, this is achieved by adding or forming exogenous microparticles in each sample, wherein the microparticles have a critical dimension larger than the maximum size of a constriction (e.g., an opening; generally, the terms “constriction” and “opening” are used synonymously herein unless otherwise stated), such as the diameter of a larger spherical particle. In some embodiments, the microparticles are spherical or substantially spherical, and the constriction (e.g., opening) is non-circular, such as rectangular or substantially rectangular, wherein the diameter of the microparticle is larger than the shorter dimension of the non-circular constriction (opening), for example, a rectangle. In some embodiments, the rectangular constriction has a longer dimension that is at least 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, or 10 times the shorter dimension. However, any suitable geometry of the constriction can be used, as long as it is constructed in a way that does not allow microparticles to pass through. Microparticles accumulate in the constriction, thereby forming a de novo filter that prevents particles larger than the space between the microparticles in the sample from moving into the sampling conduit. Typically, between samples, the flow in the conduit is reversed to remove all or substantially all microparticles from one sample from the constriction of the conduit before it is placed in the next sample. This process is then repeated in the next sample. Therefore, it is not necessary to replace the filter element, as the filter element is formed de novo in each sample and then removed before the next sample. There is also virtually no cross-contamination between samples from de novo filter elements, because no or virtually no de novo filter elements from one sample are transferred to the next. For example, on average, less than 5%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.01%, 0.005%, 0.001%, 0.0005%, or 0.0001% of de novo filter elements (formed from, for example, exogenously added or formed microparticles) from one sample are transferred to the next sample.

[0022] Therefore, we disclose herein a dynamically formed filter unit constructed at a predetermined location inside or outside a microfluidic tube, channel, or conduit by the accumulation of microparticles in a liquid solution at a constriction or orifice with a critical size smaller than the microparticles. The accumulation of microparticles at the constriction creates a micrometer-sized filter unit that prevents particulate matter from passing through the filter unit without impeding the entry of the liquid solution into the microfluidic device. After the desired amount of liquid solution is extracted, backflush of any liquid solution of interest disrupts the microparticle filter unit, returning the device to its initial state.

[0023] In its broadest aspect, the present invention relates to a method for producing a filter element by generating a microporous element, comprising the steps of: providing a liquid solution of microparticles, applying a liquid suspension to at least one constriction portion smaller than the hydrodynamic diameter of the microparticles in the suspension, and causing the microparticles to accumulate in the constriction portion.

[0024] While the microparticles may contain any suitable material, in some embodiments the material is selected from resins, fluoropolymers, ceramics, metals, silicates and their derivatives, agarose, acrylamide, etc.

[0025] In some cases, the surface material of the microparticles is a substance that does not interact with or substantially does not interact with one or more components of interest in the material passing through the microparticles; for example, one or more components of a sample (such as an aqueous sample) filtered through a de novo filter produced by the microparticles. In samples where nucleic acids are of interest, this substance does not interact with or substantially does not interact with nucleic acids. In samples where proteins or peptides are of interest, this substance does not interact with or substantially does not interact with proteins or peptides. In some cases, the substance includes a fluorinated surface to prevent sample adsorption. Suitable resins include poly(tetrafluoroethylene) (PTFE) and optionally fluoropolymer resins or derivatized silica microparticles coated with a fluorinated surface.

[0026] In some cases, microparticles include, for example, derivatized surfaces to adsorb certain components from the liquid solution as the liquid solution passes through the microparticle aggregates, while remaining inert or substantially inert to those molecules being analyzed in the device. For example, in applications related to polymerase chain reaction (PCR), microparticles can selectively bind to common blood-based PCR inhibitors such as proteases, nucleases, and heme, or soil-based inhibitors such as humic acid, fulvic acid, polysaccharides, and polyphenols.

[0027] While microfluidic tubes, channels, or conduits may contain any suitable material, preferred materials include silica, polyetheretherketone, polydimethylsiloxane, or fluoropolymer plastics.

[0028] In some cases, tubes, channels, or conduits include fluorinated surfaces to prevent sample adsorption. Suitable materials include tubes, channels, or conduits constructed from fluoropolymers such as PTFE, PFA, etc., or from derivatized silica fragments coated with fluorinated surfaces.

[0029] In some cases, the microparticle size can range from 100 nm to 2 mm, or 500 nm to 1 mm, or 1 μm to 1000 μm, or 1 μm to 500 μm, or 2 μm to 500 μm, or 2 μm to 200 μm, or 5 μm to 500 μm, or 5 μm to 200 μm in diameter. In some embodiments, the range is a hydrodynamic diameter of 10 μm to 100 μm. It will be understood that in embodiments where the microparticles are spherical or substantially spherical, the hydrodynamic diameter and the actual diameter are equal.

[0030] The contraction or its relevant dimensions have a size smaller than (e.g., slightly smaller than) the hydrodynamic diameter of the microparticle, ensuring that the microparticle does not pass through the contraction or become embedded within it. The relevant or critical dimension of the contraction (opening) is the size that the microparticle must fit to pass through the contraction (opening). For example, in a circular contraction, the relevant or critical dimension is the diameter; for a rectangle, it is the length of the shorter side. Similar analyses can be performed for other shapes. The contraction (opening) may have a critical dimension with a diameter of 50 nm to 1 mm, or 100 nm to 1 mm, or 500 nm to 1 mm, or 1 μm to 1000 μm, or 1 μm to 500 μm, or 2 μm to 500 μm, or 2 μm to 200 μm, or 5 μm to 500 μm, or 5 μm to 200 μm. In some cases, this range can be from 5 μm to 95 μm.

[0031] In some cases, microparticles may have the same shape as the constriction in a microfluidic tube, channel, or conduit. In other cases, microparticles may have a different shape than the constriction. For example, if the microparticles are spherical and the constriction is non-spherical or vice versa, the likelihood of constriction blockage due to complementary shape matching of one of the microparticles in the suspension is lower.

[0032] In some cases, the constriction zone in a microfluidic tube, channel, or conduit can be smaller than the constriction zone in only one (critical) dimension. For example, the constriction zone can take the form of an elongated slit, with the shorter dimension smaller than the diameter of the microparticles in the solution, while the larger slit dimension can be one or more times the size of the microparticles. For example, the larger dimension can be at least 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, or 10 times the shorter dimension. This example will limit the possibility of constriction zone clogging.

[0033] In some cases, channels, conduits, or pipes may have multiple boreholes (such as openings or constrictions as used herein) that allow fluid to enter the microfluidic device. When a suspension of microparticles is drawn into the microfluidic device, fluid enters each fluid inlet, resulting in the formation of multiple microparticle aggregates. In the event of blockage of one or more fluid inlets, the remaining unblocked fluid inlets allow the device to continue functioning. Furthermore, increasing the number of fluid inlets compared to a single fluid inlet allows for an increase in the overall flow rate. Therefore, in some embodiments, multiple constrictions (openings) are employed, all having a critical size smaller than the microparticles, for example, at least 2, 3, 4, 5, 6, 7, 8, 10, 12, 15, 20, 30, 40, 50, 75, or 100 constrictions (openings) and / or no more than 3, 4, 5, 6, 7, 8, 10, 12, 15, 20, 30, 40, 50, 75, 100, or 150 constrictions (openings).

[0034] In some cases, microparticles can be attracted to the fluid inlet by forces different from those of fluid flow. Non-limiting examples of these may include magnetic and magnetic particles, electric fields and charged particles, dielectric fields and corresponding particles, and particles that settle to the bottom of the container by gravity as a tube is inserted into the particle bed.

[0035] In the case of liquid handling for analysis of liquid samples, microparticles may be part of the initial sample, added as an external reagent, or pre-deposited into the container and resuspended when the sample is added to the container.

[0036] A non-limiting example of pre-depositing microparticles into a container could be preparing a solution or suspension of microparticles in a support phase such as sorbitol, adding a predetermined volume of the sample to the container, and then lyophilizing the solvent until dry. The sugar matrix of the microparticles effectively remains bound to the container until the solvated sample dissolves. Sorbitol is merely exemplary, and it will be understood that any suitable support phase can be used, as long as it can be lyophilized and does not interact with, or substantially does not interact with, the possible samples to be used in a way that alters their use in the processing system in which the sample is introduced.

[0037] In some embodiments, the microparticle filtration strategy can be used in analytical liquid handling systems. These methods and compositions can be applied to any analytical liquid handling system capable of effectively filtering the substance of interest by size.

[0038] A non-limiting example of an analytical liquid handling system is a digital PCR instrument. In this example, PCR samples are prepared by a liquid handling robot or the user, injected into the analytical liquid handling system, converted into an emulsion using a droplet generator and a continuous phase carrier such as a fluorinated oil carrier, thermally cycled to carry out the PCR reaction, and then passed through a detector to analyze the extent of the PCR reaction in any suitable manner. In this example, each sample passes through the same analytical liquid handling system, requiring a high level of sample filtration and a low level of inter-sample contamination. The number of samples can range from one to thousands.

[0039] The introduction of the microparticle filtration strategy represents a significant advancement in the ease of use and robustness of the digital PCR instrument. For example, a 2x concentrated PCR master mixture containing the desired concentration of microparticles can be prepared. After a one-to-one dilution with the required amounts of sample, primers, and reagents, the sample can be added to a 96-well plate and inserted into the digital PCR instrument. A sample injection tube with perforations smaller than the diameter of the microparticles in the PCR solution is immersed in the sample, drawing the sample into the instrument. This forms a microparticle filter on the surface of the tube, allowing the sample to be drawn into the instrument for digital PCR. After sample extraction, the sample injection tube is moved to a cleaning station where a cleaning solution is dispensed via the tip, effectively removing the microparticle filter, washing away PCR material from the tube, and effectively returning the sample injection tube to its initial state before contacting the sample.

[0040] Therefore, in some embodiments, a method is provided for loading polymerase chain reaction (PCR) samples into a PCR analysis instrument such as a digital PCR instrument, comprising (i) providing a plurality of samples containing nucleic acids in an aqueous solution, wherein each sample further comprises a plurality of microparticles, and wherein said microparticles have a hydrodynamic diameter; (ii) placing a sample catheter into a first sample of the plurality of samples, wherein said catheter has an opening (constriction), and wherein the critical dimension of said opening is smaller than the hydrodynamic diameter of said microparticles in the first sample, and wherein said catheter is configured to be fluidly connected to said PCR analysis instrument; (iii) allowing said nucleic acids in the aqueous solution of the first sample to flow into said opening in said catheter and into said catheter, wherein said microparticles in the first sample do not flow into said catheter, but instead form a first filter element containing one or more microparticles at said opening of said catheter. The opening (constriction) can be of any suitable shape. In some embodiments, the opening is circular or substantially circular, and the critical dimension is the diameter. In some embodiments, the opening is rectangular or substantially rectangular, and the critical dimension is the length of the shorter side of the rectangle; in such cases, the length of the longer side of the rectangle can be any suitable length, such as at least 1.5, 2, 2.5, 3, 3.5, 4, 5, 6, 7, 8, 9, 10, 12, 15, or 20 times the length of the shorter side. In some embodiments, the method includes (iv) after step (iii), allowing liquid to flow through the conduit and out of the opening of the conduit to remove the one or more microparticles from the first sample from the opening of the conduit and to remove the first filter element; for example, by introducing fluid away from the opening into the conduit and allowing the fluid to flow through the conduit and out of the opening, thereby displacing the microparticles. In some cases, the conduit remains in the sample container from which the sample is removed, and the flowing fluid enters the container; in some cases, the conduit is moved to another container, such as a waste container, and the flowing fluid enters the other container. The method may further include (v) placing the sample catheter into a second sample of the plurality of samples, wherein the second sample is different from the first sample, and allowing nucleic acids in an aqueous solution of the second sample to flow into the opening of the catheter and into the catheter, wherein the microparticles in the second sample cannot flow into the catheter, but instead form a second filter element different from the first filter element at the opening of the catheter, containing one or more microparticles.This process can be repeated any suitable number of times, wherein a de novo filter of microparticles is formed at each sample and then dispersed after the sample or a portion of the sample has been moved into the PCR instrument, without any additional filtration steps, such as the need to add or replace filters (in the conventional sense) during the process; for example, continuous sampling at least 2, 3, 4, 5, 7, 10, 12, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 150, 200, 300, 400, 500, 600, 700, 80 0, 900, or 1000 samples are taken to add new or clean filter elements (beyond the needs of forming and dispersing the de novo filter) and / or no more than 3, 4, 5, 7, 10, 12, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 3000, 4000, or 5000 samples are taken continuously to add new or clean filter elements (beyond the needs of forming and dispersing the de novo filter). Instead, for each sample, a new filter element is formed and then removed. The microparticles have any suitable size, as long as they cannot pass through one or more openings, such as the sizes described elsewhere in this document, for example, hydrodynamic diameters of 1-1000 μm. The microparticles can be of any suitable shape, such as spherical or substantially spherical. The catheter can contain any suitable number of openings, as described herein, such as 1-100 openings, 1-10 openings, 1-5 openings, 2-100 openings, 2-10 openings, 2-5 openings, 3-100 openings, 3-10 openings, 3-5 openings, 5-100 openings, 5-50 openings, 5-10 openings, 10-100 openings, or 10-50 openings.

[0041] Figure 1 A substrate 101 is displayed containing channels, conduits, or orifices 102 with a specific critical diameter (size). This substrate 101 may be a microfluidic chip or conduit containing microfluidic channels. A solution containing microparticles 103 with a hydrodynamic diameter larger than the specific critical diameter of the channels or orifices 102 in the substrate 101 is drawn through the channels or orifices 102. During solution drawing, the microparticles begin to accumulate at the entry point. This accumulation of microparticles forms a matrix 104 with pores 105, the size of which is specified by the packing properties of the microparticles at the entry point. The size of these pores 105 is significantly smaller than the entry point on the microfluidic substrate 101. The accumulated microparticle aggregates prevent the entry of particulate matter from the solution, which could potentially hinder the function of the microfluidic device.

[0042] Figure 2 Tests illustrating one embodiment of the methods and compositions disclosed herein were performed. A custom-made PFA conduit 201 was prepared by inserting a 50 μm metal wire 203 into a 1 / 16” OD conduit 202 with an inner diameter of 0.01” and heating it. The PFA conduit 201 softened and deformed around the metal wire 203 upon heating. After removing the metal wire 203, the PFA conduit 201 effectively had an orifice 206 with a diameter of 50 μm. The custom-made conduit 201 was connected to a syringe pump and then immersed in a preparation solution 204 of suspended 53-63 μm fluorescent polystyrene microspheres 205. The preparation solution 204 was prepared at a concentration of 0.1 g of microspheres 205 dispersed in 10 mL of deionized water, according to the manufacturer's instructions. The tube and solution were placed under a fluorescence microscope at 40x magnification and imaged while being withdrawn at a rate of 100 μL / min. After a specified amount of time, the liquid flow was reversed. Frames selected from the video are placed in chronological order. Arrows indicate the speed of liquid flow. Liquid extraction began at time t = 1. Liquid reversal begins at time t=5. Over time, it becomes apparent that microparticles 207 accumulate at the pipe borehole and disperse immediately upon liquid reversal.

[0043] Figure 3 Another embodiment of the methods and compositions disclosed herein is shown. In some cases, the microparticles may have the same shape as the contraction portion. In other cases, the microparticles may have a different shape than the contraction portion. For example, if the microparticles are spherical and the contraction portion is non-spherical or vice versa, the likelihood of the contraction portion becoming blocked is lower.

[0044] In one embodiment, a liquid sample of spherical microparticles 301 is drawn into a non-circular microchannel 302 with a critical diameter smaller than that of the microparticles in the solution. As the microparticle solution is drawn into the non-circular microchannel 302 in the substrate 303, the microparticles 301 aggregate at the fluid inlet. This shape mismatch between the microparticles and the channel ensures that the microparticles do not clog the fluid inlet.

[0045] Figure 4 Further embodiments of the methods and compositions disclosed herein are shown. In some cases, the contraction portion can be smaller than the microparticles in only one critical dimension. For example, the contraction portion can be formed by creating a slit with a short diameter smaller than that of the microparticles, while a larger slit size can be one or more times the size of the microparticles. This example limits the possibility of the contraction portion becoming clogged and preventing liquid flow through it.

[0046] In one embodiment, a liquid sample of spherical microparticles 403 is drawn into an elongated microchannel 401 whose critical diameter (size) is smaller than that of the microparticles 403 in the solution. The elongated microchannel 401 has a geometry that prevents the microparticles from entering, but is large enough for multiple microparticles to span the entire inlet. As the microparticle solution is drawn into the elongated microchannel 401 in the substrate 402, the microparticles 403 aggregate at the fluid inlet. The shape mismatch between the microparticles 403 and the elongated microchannel 401 ensures that the microparticles 403 can block the fluid inlet.

[0047] In some embodiments, this document provides a method for providing a first filter element, comprising (i) providing a first suspension of a plurality of first microparticles in a first liquid, wherein the first microparticles have a first hydrodynamic diameter within any suitable range as described herein, such as between 1 μm and 1000 μm or between 10 μm and 100 μm; and (ii) applying the first suspension to at least one non-circular opening having a short dimension and a long dimension, such as an elliptical or substantially elliptical shape or a rectangular or substantially rectangular shape, wherein the short dimension is smaller than the hydrodynamic diameter of the first microparticles, and the long dimension is a suitable multiple of the short dimension, as described herein, for example, at least 1.5, 2, 3, 4, or 5 times, for example, at least 2 times, the hydrodynamic diameter of the first particles, such that the first microparticles accumulate at the opening and form a first filter element comprising the first microparticles. The opening may be an opening leading to a conduit leading to an instrument, or configured to be located leading to an instrument for treating liquid or components of liquid passing through the opening. Liquid can be passed through the opening by any suitable method, for example, by allowing liquid to flow. In some embodiments, the filter element formed in step (ii) can be dispersed by allowing the second liquid to flow back through an opening (e.g., reversing the direction of fluid flow through the opening). The method may further include forming a second filter element by applying a second suspension of a plurality of second microparticles in a third liquid to the at least one opening to form a second filter element containing the second microparticles at the opening, wherein the second microparticles have a second hydrodynamic diameter within any suitable range as described herein, such as between 1 μm and 1000 μm or between 10 μm and 100 μm, wherein the short dimension of the opening is smaller than the hydrodynamic diameter of the second microparticles, and the long dimension of the opening is a suitable multiple of the short dimension, such as, as described herein, for example, at least 1.5, 2, 3, 4, or 5 times, for example, at least 2 times, the hydrodynamic diameter of the second microparticles. The second filter can then be dispersed similarly to the first filter in some cases. The microparticles in each fluid can be of any suitable shape; in some cases, the microparticles are spherical or substantially spherical. The surfaces of some or all of the microparticles may enable them to interact with certain components of one or more liquids, and / or not interact with or substantially not interact with other components. For example, in the case of a liquid containing nucleic acids to be analyzed, the microparticles may have surfaces that do not interact with or substantially do not interact with the nucleic acids; said surfaces may further be capable of interacting with other components, as described elsewhere herein. In some cases, multiple openings are used, all with a critical dimension smaller than the hydrodynamic diameter of the particle.

[0048] In some embodiments, this document provides an apparatus comprising (i) a plurality of sample containers, each containing microparticles and a liquid therein suspending the microparticles, wherein the microparticles have any suitable hydrodynamic diameter, for example, between 1 μm and 1000 μm or between 10 μm and 100 μm; and (ii) a conduit having at least one non-circular opening having a short dimension and a long dimension, wherein the short dimension is smaller than the hydrodynamic diameter of the microparticles, and the long dimension is any suitable multiple of the hydrodynamic diameter of the microparticles, such as the hydrodynamic diameter of the microparticles. (iii) a system configured to sequentially immerse the one or more openings of the conduit into the plurality of samples, such as a sampling system for a digital PCR instrument; (iv) a system configured to allow the liquid to flow through the one or more openings into the conduit, such as a pump, causing the microparticles to accumulate at the openings and form a filter element containing the microparticles; and (v) a system configured to allow a second liquid to flow out of the conduit through the one or more openings to disperse the microparticles at the openings. Therefore, the device is configured to sequentially sample material from sample containers, form a filter during each sampling, disperse the filter after sampling is complete, and move it between containers. Each filter may be dispersed back into the container from which the sample was taken, or dispersed into different containers, such as one or more waste containers.

[0049] While preferred embodiments of the compositions and methods of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Many variations, modifications, and substitutions will be contemplated by those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the methods and compositions described herein can be used to practice the methods and compositions. The following claims are intended to define the scope of the compositions and methods, and thereby cover methods and structures within the scope of these claims and their equivalents.

Claims

1. A method for filtering a liquid solution, comprising: The liquid flows into and through a non-circular opening in the sampling conduit leading to the processing system. The opening comprises a short dimension and a long dimension, which are configured to limit the likelihood of blockage and prevent microparticles present in the liquid from entering the sampling catheter. The longer dimension is a multiple of at least one of 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9 or 10 times the shorter dimension.

2. The method of claim 1, wherein the short dimension is the critical dimension of the opening.

3. The method of claim 1 or 2, wherein the short dimension is the length of the short side of the opening.

4. The method of claim 2, wherein the critical dimension includes a length between 1 µm and 500 µm.

5. The method of claim 2, wherein the critical dimension includes a length between 5 µm and 95 µm.

6. The method of claim 1 or 2, wherein the opening is rectangular or elliptical.

7. The method of claim 1 or 2, wherein the opening is a slit.

8. The method of claim 1 or 2, wherein the long dimension is at least 6 times the short dimension.

9. The method of claim 1 or 2, wherein the sampling catheter comprises a material selected from the group consisting of: silica, polyetheretherketone, polydimethylsiloxane, fluoropolymers, and any combination thereof.

10. The method of claim 1 or 2, wherein the sampling catheter comprises a fluorinated surface.

11. The method of claim 1 or 2, wherein the processing system comprises a polymerase chain reaction system.

12. The method of claim 11, wherein the processing system comprises a digital polymerase chain reaction system.

13. An analytical liquid handling system, comprising: A sampling conduit leading to a processing system, wherein the sampling conduit includes an opening through which a liquid sample enters the sampling conduit. The opening includes a short dimension and a long dimension, which are configured to prevent clogging of the opening and to prevent particles present in the liquid sample from entering the sampling catheter. The longer dimension is a multiple of at least one of 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9 or 10 times the shorter dimension.

14. The system of claim 13, wherein the analytical liquid handling system is a digital polymerase chain reaction instrument.

15. The system of claim 13 or 14, wherein the analytical liquid processing system includes a droplet generator configured to convert the liquid sample into an emulsion in a continuous phase carrier.

16. A method for providing a first filter element, comprising: (i) A first suspension of a plurality of first microparticles in a first liquid is provided, wherein the first microparticles have a first hydrodynamic diameter between 1 µm and 1000 µm; (ii) The first suspension is applied to a non-circular opening in at least one sampling conduit having a short dimension and a long dimension, wherein the short dimension is smaller than the hydrodynamic diameter of the first microparticle and the long dimension is at least twice the hydrodynamic diameter of the first microparticle, such that the first microparticle accumulates at the opening and forms a first filter element containing the first microparticle, wherein the first suspension is applied to at least one constriction by allowing the first liquid to flow in and through the opening.

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