Microfiltration device and preparation method thereof, and method and kit for separating biological material

Abstract

Disclosed herein is a microfiltration device comprising a microfilter coated with a positively charged degradable layer, and a method of making the microfiltration device. In particular, the positively charged degradable layer comprises a polymer matrix and a cation, and wherein the polymer matrix is an alginate gel and the cation is calcium (II). A method of separating biological material from a sample using the microfiltration device and a kit including the microfiltration device are also disclosed. In particular, the biological material is negatively charged bacteria, fungi or viruses. The microfiltration devices and methods of separating biological materials described herein exhibit good compatibility with various downstream analysis techniques.

Description

[0001] Citation of Related Applications

[0002] This application claims priority to U.S. Application No. 63 / 377,775, filed Sep. 30, 2022, with the United States Patent and Trademark Office, the content of which is incorporated herein by reference. Technical Field

[0003] The present invention generally relates to a microfiltration device for separating biological materials and a method for preparing a microfiltration device as described herein. The present invention also relates to a method for separating biological materials using a microfiltration device as described herein, and a kit for separating biological materials comprising a microfiltration device as described herein. Background Art

[0004] The detection and analysis of biological materials, especially microorganisms (bacteria, fungi, viruses) from various samples including biological fluids, wastewater, foodborne samples, and biomanufactured products, are important for both clinical diagnosis and monitoring of microorganisms. However, even the most sensitive techniques, such as digital polymerase chain reaction (dPCR), are severely limited in their detection performance by the low microbial abundance relative to the large background in bulk samples.

[0005] Commonly used techniques for microbial separation include size-based separation techniques such as centrifugation and hollow fiber filtration. However, these size-based separation techniques have serious drawbacks. Centrifugation is labor-intensive and has the defect of significant loss of low-abundance microorganisms, while hollow fiber filtration is severely affected by clogging of complex contents in crude samples and significant loss of low-abundance microorganisms during the recovery step, which is caused by non-specific adhesion of microorganisms.

[0006] In addition to the above-mentioned size-based separation techniques, there are also charge-based methods for separating and recovering microorganisms from liquid / water samples, such as negative electrofiltration and positive electrofiltration. To avoid the need for preconditioning steps associated with negative electrofiltration, positive electrofiltration is usually used as an alternative. However, this technique is mainly limited by traditional filter membranes, such as biofouling and clogging. In addition, these traditional electropositive filters are still plagued by concentration bottlenecks because the elution solution volume is large (about 10 mL), so a second concentration is required to enrich the analyte of interest (microorganisms).

[0007] In addition to the above-mentioned drawbacks, it is very difficult and inefficient to release and recover the captured microorganisms from the above filters because it is difficult to break or overcome the strong electrostatic attraction between the microorganisms and the filters. In addition, the sponge-like structure of these filters limits the efficient release of the captured microorganisms, thus forming a bottleneck between the separation and analysis times.

[0008] Accordingly, there is a need to provide a microfiltration device that can enrich and recover microorganisms even when the microbial abundance is very low to improve downstream detection. There is also a need to provide a separation method that can interface with downstream detection methods to minimize the time spent between separation and analysis. SUMMARY OF THE INVENTION

[0009] In one aspect, the present disclosure relates to a microfiltration device for separating biological materials from a sample, the microfiltration device including a microfilter coated with a positively charged degradable layer.

[0010] Advantageously, the microfiltration devices described herein can be used for the separation and recovery of various biological materials. In particular, it has been found that the microfiltration devices described herein are capable of effectively separating various microorganisms, including bacteria, fungi, and viruses. The effective recovery of various microorganisms can be attributed to the positively charged degradable layer coated on the microfilter, which allows separation to occur through electrostatic interactions in addition to size exclusion. Thus, the microfiltration devices and methods described herein provide a general sample preparation method for capturing and concentrating biological materials for downstream processing.

[0011] In another aspect, the present disclosure relates to a method for preparing a microfiltration device for separating biological materials from a sample, the method including the step of coating a positively charged degradable layer onto the surface of a microfilter.

[0012] In another aspect, the present disclosure relates to a method for separating biological materials using a microfiltration device as described herein, the method including the step of passing a sample containing biological materials through a microfiltration device as described herein, whereby the biological materials are captured on the positively charged degradable layer on the microfilter, thereby separating the biological materials from the sample.

[0013] Advantageously, the method for separating biological materials using the microfiltration device described herein achieves a high capture efficiency, even for samples containing low concentrations of biological materials. In one embodiment, a capture efficiency of approximately 85% can be achieved even for a sample containing 1 colony forming unit (CFU) / mL of bacteria. The high capture efficiency achieved using the microfiltration device can be attributed to the positively charged degradable material coated on the microfilter. Specifically, negatively charged biological materials can form strong electrostatic interactions with the positively charged degradable layer coated on the microfilter, thereby allowing the negatively charged biological materials to be effectively separated from the sample.

[0014] Advantageously, due to the small volume of the solution used for releasing / recovering the captured biomaterial from the positively charged degradable layer (about 60 μL is used herein), the biomaterial separation method as described herein provides high capture efficiency (>90%) and high enrichment factor (higher than 150-fold and can be increased to 8333-fold). It can be understood that by doing so, the biomaterial will be concentrated. Therefore, when the biomaterial is recovered using a volume smaller than the input volume, the method of separating the biomaterial can also be interpreted as a method of concentrating the biomaterial by electrostatic microfiltration (i.e., EM concentration).

[0015] More advantageously, when using digital loop-mediated isothermal amplification (dLAMP), the biomaterial separation method using the microfiltration device as described herein provides a lower limit of detection (about 100-fold lower). Samples of 1 - 1000 CFU / mL (>10 mL) are undetectable based on current centrifugation-based protocols, but are detectable by dPCR based on the concentration supported by the high-throughput (≥1 mL per minute) electrostatic microfiltration of the present invention.

[0016] More advantageously, the biomaterial separation method as described herein allows it to be compatible with other downstream methods due to the high enrichment factor, such as polymerase chain reaction (PCR)-based methods, sequencing, and MALDI-TOF mass spectrometry, etc. The high enrichment factor enables good compatibility at the interface with downstream analysis after separation and reduces the processing time as no further enrichment steps are required.

[0017] In another aspect, the present disclosure relates to a kit that includes a microfiltration device as described herein, a sampling tube, a discharge tube, and a housing container.

[0018] The present disclosure also relates to a microfluidic capture device including a positively charged degradable layer.

[0019] The present disclosure also relates to a microfilter coated with a positively charged degradable layer.

[0020] The present disclosure also relates to a kit component that includes a microfilter having a positively charged degradable layer thereon, a sampling area, and a collection area. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The present invention can be better understood when considered in conjunction with non-limiting examples and the drawings, in which:[[]]END]]

[0022] Figure 1 A schematic diagram showing the working principle of a microfiltration device including a microfilter coated with a positively charged degradable layer and its application in a method for separating biomaterials from a sample is shown. Figure 1Row a) shows the preparation of the positively charged degradable layer 117 from the polymer matrix solution 101 and the crosslinking agent 103, and the effect of contacting the positively charged degradable layer 117 with the degrading agent 109. Figure 1 Row b) shows coating the microfilter 115 with the positively charged degradable layer 117, and removing the positively charged degradable layer 117 using the degrading agent 109. Figure 1 Row c) shows the method of separating biological materials using the microfiltration device 106, and the subsequent step of removing the concentrated or separated biological material 125 from the microfilter 115.

[0023] Figure 2a Shows a scanning electron microscope (SEM) image of the micropores of the microfilter before coating with calcium alginate gel, Figure 2b Shows an SEM image of the micropores with a calcium alginate gel coating layer, Figure 2c Shows an SEM image of the micropores after incubating with EDTA (or alginate lyase) to degrade the coated calcium alginate gel. All scales are 5 μm.

[0024] Figure 3a Shows a schematic diagram of the method of separating biological materials using the microfiltration device as described herein, which method has additional processing steps for detecting biological materials such as captured bacteria. The method may include the following steps: a) introducing a sample containing biological materials into the microfiltration device; b) passing the sample through the microfiltration device under the action of gravity, optionally accelerating by an applied force (such as centrifugal force, pressure or vacuum); c) removing the microfilter from the microfiltration device and contacting the microfilter containing the captured bacteria with a lysis buffer; d) inducing lysis of the captured bacteria by a cycle of vortexing and heating; e) separating the supernatant from the waste precipitate; and f) detecting the presence of bacteria using the dLAMP program. Figure 3b Is a photograph of the microfiltration device as described herein. The microfiltration device may include a sampling container 317, a fixing device 319 for attaching the support 321 to the sampling container 317, and a discharge pipe 323. Figure 3c Is a top view of the microfilter 325 on the microfilter support 321, which microfilter support 321 may include an opening 327 on which the microfilter 325 may be placed. Figure 3d Is a side view of the microfiltration device as described herein. Figure 3e Is a photograph of the method of separating biological materials as described herein, wherein a sample may be introduced into the sampling container 317 and passed through the microfilter 325 fixed on the support 321. Using the microfiltration device as described herein, gravity-driven filtration of a 10 mL sample can be achieved in just 1.5 minutes.

[0025] Figure 4aShows the fluorescence distribution image of the dLAMP detection signal of nucleic acids obtained from electrostatic microfiltered bacteria according to Example 2, Figure 4b shows the fluorescence distribution image of the dLAMP detection signal of nucleic acids obtained from a conventional centrifugation technique (10,000×g for 10 minutes).

[0026] Figure 5a shows the partition scatter plot in the dLAMP detection of nucleic acids obtained from electrostatic microfiltered bacteria according to Example 2, Figure 5b shows the partition scatter plot in the dLAMP detection of nucleic acids obtained from a conventional centrifugation technique (10,000×g for 10 minutes). The x-axis is the partition number, and the y-axis is the fluorescence intensity expressed in relative fluorescence units.

[0027] Figure 6 shows the SEM image (scale bar is 5 μm) of Staphylococcus aureus (S. aureus) (Sa.6538, ATCC) electrostatically captured on the alginate gel layer of the microfilter according to Example 2.

[0028] Figure 7a shows the SEM image of bacteria (Staphylococcus aureus, Klebsiella pneumoniae (K. pneumoniae), Pseudomonas aeruginosa) captured on the alginate layer according to Example 4, with a scale bar of 10 μm. Figure 7b shows Staphylococcus aureus detected by dLAMP in a sample containing a mixture of different concentrations of Staphylococcus aureus, Klebsiella pneumoniae, and Pseudomonas aeruginosa according to Example 4 Klebsiella pneumoniae and Pseudomonas aeruginosa in a bar graph of bacterial DNA. Figure 7c shows the SEM image of fungi (Candida albicans) captured on the alginate layer, with a scale bar of 10 μm; while Figure 7d shows the SEM image of Herpes simplex virus (HSV) particles captured on the alginate layer according to Example 7, with a scale bar of 1 μm. Embedded in Figure 7d is the SEM image of the captured HSV at 10 times higher magnification (scale bar is 100 nm).

[0029] Figure 8a shows the SYTO9 (live and dead) signal of bacteria (Klebsiella pneumoniae) in an unfiltered or unreleased control sample, while Figure 8b shows the PI (dead) signal of bacteria from the same control sample. Figure 8cShows the SYTO9 (live and dead) signal of bacteria (Klebsiella pneumoniae) released from alginate gel after filtering a sample through the microfiltration device described herein, while Figure 8d shows the PI (dead) signal of bacteria captured and released from alginate gel after filtering the same sample according to Example 3;

[0030] Figure 9a is a photograph of an LB agar plate with colonies of cultured Klebsiella pneumoniae recovered from the microfiltration device. Figure 9b is a photograph of an LB agar plate with colonies of cultured Pseudomonas aeruginosa recovered from the alginate gel of the microfiltration device described herein. Figure 9c is a photograph of an LB agar plate with colonies of cultured Staphylococcus aureus according to Example 3;

[0031] Figure 10a is a bar graph showing the recovery efficiency of recovering Staphylococcus aureus from a sample with low abundance of Staphylococcus aureus using the method for separating biomaterials described herein; Figure 10b is a bar graph showing the recovery efficiency of recovering Klebsiella pneumoniae from a sample containing low-abundance bacteria using the method for separating biomaterials described herein; while Figure 10c is a bar graph showing the recovery efficiency of recovering from a Luria Borth (LB) sample containing low-abundance Pseudomonas aeruginosa using the electrostatic microfiltration method described herein according to Example 2. The recovery efficiency of the electrostatic microfiltration method described herein represented by the solid-colored columns (■) is compared with the columns represented by the conventional centrifugation method. The x-axis in the figure is the concentration of microorganisms in the sample, and the y-axis in the figure is the recovery efficiency of microorganisms in the sample. Figure 10d is a scatter plot of the dLAMP signal of bacterial DNA of Staphylococcus aureus recovered from a sample containing low-abundance bacteria using the separation method described herein; Figure 10e is a box plot of the median and range within 1.5 interquartile ranges (IQR) of the dLAMP signal of bacterial DNA of Staphylococcus aureus recovered from a sample containing low-abundance bacteria using the separation method described herein compared with the conventional centrifugation method and the original sampling; Figure 10f is a scatter plot of the dLAMP signal of bacterial DNA of Klebsiella pneumoniae recovered from a sample containing low-abundance bacteria using the method for separating biomaterials described herein compared with the conventional centrifugation method and the original sampling; Figure 10g is a box plot of the median and range within 1.5 IQR of the dLAMP signal of bacterial DNA of Klebsiella pneumoniae recovered from a sample containing low-abundance bacteria using the method for separating biomaterials described herein compared with the conventional centrifugation method and the original sampling; Figure 10hScatter plot of dLAMP signals of bacterial DNA of Pseudomonas aeruginosa recovered from samples containing low-abundance bacteria using the method for separating biological materials described herein as compared to traditional centrifugation methods and crude sampling; Figure 10i Box plot of the median and range within 1.5 IQR of dLAMP signals of bacterial DNA of Pseudomonas aeruginosa recovered from samples containing low-abundance bacteria using the method for separating biological materials described herein as compared to traditional centrifugation methods and crude sampling. This was obtained according to Example 2. The x-axis is the abundance of bacteria in the sample, and the y-axis is the dLAMP signal reading. Samples containing 0 CFU / mL or NTC bacteria indicate that there are no microorganisms in the sample. In Figure 10d , Figure 10f and Figure 10h In each of the figures, the dLAMP readings of bacterial DNA detected in each replicate using the method for separating biological materials described herein are represented by open square markers (□); the dLAMP signals of bacterial DNA detected in each replicate using the conventional centrifugation method are represented by open triangle markers (△), and the dLAMP readings of bacterial DNA detected in each unfiltered crude sample replicate are represented by open star markers (☆). In Figure 10e , Figure 10g and Figure 10i In each of the figures, the average dLAMP signal of bacterial DNA detected in bacterial samples recovered using the method for separating biological materials described herein is represented by solid square markers (■); the average dLAMP signal of bacterial DNA detected in bacterial samples recovered from the sample using the conventional centrifugation method is represented by solid triangle markers (▲), and the average dLAMP signal of bacterial DNA in the crude sample containing bacteria without any filtration or separation method is represented by solid star markers (★). In Figure 10e , Figure 10g and Figure 10i In each box plot in, the box drawn with a solid line but without pattern filling (□) represents the dPCR or dLAMP data of bacterial samples recovered using the method for separating biological materials described herein; the box drawn with a solid line and filled with a horizontal line represents the dPCR or dLAMP data of bacterial samples recovered using the conventional centrifugation method; and the box drawn with a solid line and filled with diagonal bricks represents the dPCR or dLAMP data of the crude bacterial sample. The height of each marked box represents the 1.5 IQR range of the dLAMP signal obtained from the replicates, and the line within the box indicates the median of the dLAMP signal. The bars extending from the box represent the range of the remaining signals. The data sets marked with parentheses and * indicate that the significance test P value is less than 0.05, and the limit of detection can be determined based on the significance test of the signal relative to the NTC group (P<0.05);

[0032] Figure 11a Scatter plot of dLAMP signals of bacterial DNA of recovered Staphylococcus aureus detected using the method for separating biological materials described herein Figure 11b is a scatter plot of dLAMP signals of bacterial DNA of recovered Klebsiella pneumoniae detected using the method for separating biological materials described herein, while Figure 11c is a scatter plot of bacterial DNA of recovered Pseudomonas aeruginosa detected using the method for separating biological materials described herein. The x-axis is the abundance of bacteria in the sample, while the y-axis is the dLAMP signal reading. The dLAMP signals of bacterial DNA of bacteria recovered from bacterial samples in pure LB are represented by open square markers (□), while the dLAMP signals of bacterial DNA of bacteria recovered from samples containing bacteria in serum-containing LB are represented by open circular markers are shown. This was obtained according to Example 5

[0033] Figure 12a is a scatter plot of bacterial DNA of Staphylococcus aureus detected by dLAMP after recovering bacteria from 100 mL and 500 mL samples using the method for separating biological materials described herein Figure 12b is a scatter plot of bacterial DNA of Klebsiella pneumoniae detected by dLAMP after recovering bacteria from 100 mL and 500 mL water samples using the method for separating biological materials described herein, while Figure 12c is a scatter plot of bacterial DNA of Pseudomonas aeruginosa recovered from 100 mL and 500 mL water samples using the method for separating biological materials described herein. The x-axis is the abundance of bacteria in the water sample, while the y-axis is the dLAMP signal reading. For each replicate, the dLAMP signal of the bacterial DNA detected after recovering bacteria from 100 mL of the water sample is represented by an open circular marker are shown. The average dLAMP signal of the bacterial DNA detected after recovering bacteria from 500 mL of the water sample is represented by an open square marker (□). This was obtained according to Example 6

[0034] Figure 13a is a scatter plot of fungal DNA detected by dLAMP after recovering Candida albicans from 10 mL of brain heart infusion (BHI) broth samples using the method for separating biological materials described herein Figure 13b is a scatter plot of dPCR signals of viral DNA when recovering HSV from cell spent medium (DMEM) samples containing herpes simplex virus (HSV) using the method for separating biological materials described herein. The open square marker (□) is the dLAMP or dPCR signal for each replicate. This was obtained according to Example 7

[0035] Definition

[0036] As used herein, the term "biomaterial" refers to, but is not limited to, bacteria, microorganisms (or their cells, which may include bacteria, fungi, and viruses), pathogens, nucleic acids (such as cell-free nucleic acids), proteins, and exosomes.

[0037] The term "microorganism" refers to microscopic-sized organisms that may or may not be capable of causing disease, including but not limited to bacteria, fungi, viruses, protozoa, algae, and parasites.

[0038] As used herein, the term "pathogen" refers to a microorganism, such as a bacteria, fungus, virus, or any other microorganism capable of causing disease.

[0039] The term "parylene" refers to the common name for a polymer whose main chain consists of p-phenylene rings -C6H4- connected by 1,2-ethanediyl bridges -CH2-CH2-. It can be obtained by the polymerization of p-xylene H2C=C6H4=CH.

[0040] The term "enrichment factor" refers to the input / starting sample volume divided by the volume used to recover or resuspend the biomaterial (such as microorganisms) retained on or by the microfilter after the sample has passed through the microfiltration device.

[0041] As used herein, the term "porosity" refers to the ratio of the area of the pore openings to the total area of the microfilter.

[0042] Unless otherwise specified, the term "comprising" and its grammatical variations are intended to denote an "open" or "inclusive" language, such that they include the recited elements, but also permit the inclusion of additional, unrecited elements.

[0043] As used herein, the term "about" in the context of the concentration of formulation components generally refers to ±5% of the stated value, more generally to ±4% of the stated value, more generally to ±3% of the stated value, more generally to ±2% of the stated value, more generally to ±1% of the stated value, and further more generally to ±0.5% of the stated value.

[0044] In the present disclosure, certain embodiments may be disclosed in a range format. It should be understood that the description in range format is only for convenience and brevity and should not be construed as imposing a rigid limitation on the disclosed range. Thus, the description of a range should be considered as specifically disclosing all possible sub-ranges as well as individual values within that range. For example, the description of a range from 1 to 6 should be considered as specifically disclosing sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., and individual numbers within that range, such as 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range. Detailed Embodiments

[0045] Exemplary and non-limiting embodiments of a microfiltration device for separating biological materials from a sample will now be disclosed, wherein the microfiltration device includes a microfilter coated with a positively charged degradable layer.

[0046] The microfiltration device may include a microfilter. The microfilter may include pores suitable for size exclusion filtration, preferably including a microporous array.

[0047] The microfilter may be made of a biocompatible material that does not react adversely with biological materials, such as biomolecules and microorganisms, including pathogens. The biocompatible material may be a biocompatible material approved by the FDA (U.S. Food and Drug Administration). The biological material does not degrade or inactivate upon contact with the material. When the biological material is a microorganism, such as a pathogen, the microorganism or pathogen remains viable upon contact with the material and can then be used for downstream studies where a viable microorganism or pathogen is required.

[0048] Surprisingly, it has been found that the microfiltration device described herein is capable of effectively recovering various microorganisms, including bacteria, fungi, and viruses. This may be attributed to the working principle of the microfilter, which relies on the electrostatic interaction between the negative charge on the cell surface of the microorganism and the positively charged degradable layer used for microorganism isolation.

[0049] The microfilter may be made of an inert, biocompatible material, such as an inert, biocompatible polymer. The polymer may be an unsubstituted hydrocarbon polymer. The microfilter may be made of a biocompatible polymer selected from parylene C, parylene N, parylene D, parylene AF-4, and combinations thereof. The biocompatible material may be parylene C.

[0050] The material of the microfilter may also be chemically inert and thus compatible with various downstream chemical treatments or chemical extractions.

[0051] The microfilter can have a thickness in the range of from about 4 micrometers (μm) to about 10 μm, from about 6 μm to about 10 μm, from about 8 μm to about 10 μm, from about 4 μm to about 8 μm, or from about 4 μm to about 6 μm. The thickness of the microfilter can be about 4 μm, 4.2 μm, 4.4 μm, 4.6 μm, 4.8 μm, 4.9 μm, or 5 μm.

[0052] The microfilter can include pores or an array of pores adapted to separate biological materials from a suspension. The pores can have an average diameter in the range of from about 1 μm to about 10 μm, from about 2 μm to about 10 μm, from about 4 μm to about 10 μm, from about 6 μm to about 10 μm, from about 8 μm to about 10 μm, from about 1 μm to about 8 μm, from about 1 μm to about 6 μm, from about 1 μm to about 4 μm, or from about 1 μm to about 2 μm. For example, the average diameter of the pores can be about 1 μm, 1.2 μm, 1.4 μm, 1.5 μm, 1.6 μm, 1.8 μm, or 2 μm.

[0053] When the microfilter is coated with a positively charged degradable layer, the average pore size of the coated microfilter may decrease. The pores of the coated microfilter can have an average diameter of from about 1 μm to about 10 μm, from about 2 μm to about 10 μm, from about 4 μm to about 10 μm, from about 6 μm to about 10 μm, from about 8 μm to about 10 μm, from about 1 μm to about 8 μm, from about 1 μm to about 6 μm, from about 1 μm to about 4 μm, or from about 1 μm to about 2 μm. For example, the average diameter of the pores of the microfilter coated with a positively charged degradable layer can be about 1.2 μm.

[0054] The average diameter of the pores of the microfilter can be about 1.5 μm. The pore pitch of the microfilter can be from about 1 μm to about 10 μm, 1 μm to about 9 μm, 1 μm to about 8 μm, 1 μm to about 7 μm, 1 μm to about 6 μm, 1 μm to about 5 μm, 1 μm to about 4 μm, 1 μm to about 3 μm, 2 μm to about 3 μm. For example, the pore pitch can be about 1.5 μm, 1.8 μm, 2 μm, 2.5 μm, or 3 μm. The pore pitch of the microfilter can be 2.5 μm.

[0055] The microfilter can have a pitch in the range of from about 2 μm to about 6 μm, from about 4 μm to about 6 μm, or from about 2 μm to about 4 μm. The pitch of the microfilter is preferably from about 3 μm to about 5 μm, for example 3 μm, 3.2 μm, 3.5 μm, 3.8 μm, 4 μm, 4.2 μm, 4.5 μm, 4.8 μm, or 5 μm. The pitch of the microfilter can be about 4 μm. The microfilter can be referred to as a microporous array.

[0056] The spacing (pore diameter, pore pitch) of the microfilter can be optimized to achieve higher flux microfiltration. As used herein, the term "spacing" refers to the distance between repeating elements in a structure with translational symmetry. For the microfilter described above that can be characterized as a microporous array, the spacing can refer to the distance between the centers of adjacent pores or the distance from one edge of a pore to the equivalent edge of the next pore. Thus, this can be considered mathematically equivalent to the sum of the average pore diameter and pore pitch of the microfilter. The pore pitch of the microfilter can be optimized to adjust the porosity of the microfilter to filter a larger volume of sample. In particular, the smaller the distance between the pores of the microfilter, the higher the porosity of the microporous array, and thus the higher the flux of microfiltration. This enables a larger sample volume to be processed in a given time.

[0057] The porosity of the microfilter can be greater than 10%, greater than about 14%, greater than 20%, greater than 25%, greater than about 30%, greater than about 40%, greater than about 50%, greater than about 60%, greater than about 70%, greater than about 80%. The porosity of the microfilter can be in the range of about 10% to about 90.9%, for example about 12%, 14%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90%. The porosity of the microfilter can be about 14%.

[0058] After coating with a positively charged degradable layer, the porosity of the microfilter may change. The porosity of the microfilter coated with a positively charged degradable layer can be lower than that of the microfilter before coating. For example, the porosity of the microfilter coated with a positively charged degradable layer can be greater than about 5%, greater than about 10%, greater than about 20%, greater than about 30%, greater than about 40% or greater than about 50%. The porosity of the coated microfilter can be in the range of about 5% to about 90.9%, for example about 6%, 8%, 10%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90%. The porosity of the microfilter coated with a positively charged degradable layer can be 9% or higher, or about 9%.

[0059] The porous microfilter can allow the sample to pass through the microfilter quickly with minimal clogging. For example, it takes less than 30 minutes to process a sample with a volume exceeding 100 mL using a microfilter coated with a positively charged degradable layer having a pore diameter of about 1.2 μm and a pore-to-pore distance of about 2.8 μm (i.e., 4 μm spacing), and a porosity of about 9%.

[0060] Advantageously, the microfilter of the present disclosure has a high filtration rate due to its thickness (5 μm) and porosity (≥9%). In particular, the porosity of the microfiltration device described herein allows for the filtration of a large volume of sample. For example, the microfiltration device can be used to filter a sample of approximately 500 mL. The ability to filter a large volume is particularly advantageous for the recovery of biological materials, such as microorganisms provided at low concentrations. In particular, the microfilter allows for the efficient recovery of microorganisms from a large volume of sample that may contain low concentrations of such microorganisms. In one embodiment, the microfiltration device described herein is capable of achieving a limit of detection (LOD) as low as 10 CFU when filtering a 500 mL sample.

[0061] More advantageously, the parylene C material of the microfilter ensures that the microfilter remains chemically inert, thus making it compatible with various downstream chemical treatments, biological studies, or nucleic acid extractions. In addition, parylene C is an FDA-approved biocompatible material, thus ensuring the viability of the separated biological materials.

[0062] The microfilter is not limited to a specific size or shape and can be modified or adapted based on the size of the microfiltration device, sampling area, collection area, or housing. The microfilter can be circular or any quadrilateral, such as square or rectangular, where the microfiltration device is housed in a centrifuge tube or microcentrifuge tube, and the size of the microfilter can be adjusted based on the inner diameter of the housing. For example, in the case where the microfiltration device is housed in a Falcon tube, the size of the microfilter can be adjusted to approximately 17 mm × 17 mm. The microfiltration device can be in the form of a gravity-driven filtration device, where the microfilter can be disposed within a core that can be contained within a housing.

[0063] The positively charged degradable layer can include a polymer matrix. The polymer matrix can be a gel, including a biocompatible hydrogel. Such gels can include gelatin, agar, IOTA carrageenan, alginate gels, and natural gums, such as gellan gum, xanthan gum, guar gum, Gum arabic, and Acacia gum. The polymer matrix can be an alginate gel or IOTA carrageenan. In one embodiment, the polymer matrix can be an alginate gel. Thus, the positively charged degradable layer can be referred to as a positively charged alginate gel layer.

[0064] The polymer matrix can be modified with cations, such as monovalent or divalent cations, to form a positively charged layer. The cations can form crosslinks within the polymer matrix to impart a net positive charge, thereby creating the positively charged degradable layer.

[0065] The positively charged degradable layer can contain a divalent cation. The divalent cation can be selected from the group consisting of calcium (II) (Ca2+ ) Strontium (II) (Sr 2+ ) Barium (II) (Ba 2+ ) Iron (II) (Fe 2+ ) Copper (II) (Cu 2+ ) Zinc (II) (Zn 2+ ) and mixtures thereof. The divalent cation can be calcium (II) (Ca 2+ ). Alternatively or additionally, the positively charged degradable layer can comprise a monovalent cation, which can include potassium (K + ), sodium (Na + ), lithium (Li + ), cuprous (I) (Cu + ) or mixtures thereof. The positively charged degradable layer can comprise a mixture of divalent cations and monovalent cations, each of the divalent cations and monovalent cations being independently selected.

[0066] Advantageously, the presence of the divalent cation imparts a strong net positive charge on the positively charged degradable layer. This may contribute to a stronger attraction of the biomaterial to the positively charged degradable layer, thereby improving the separation of the biomaterial from the sample, even for samples with low concentrations of biomaterial.

[0067] In the case where the polymer matrix is alginate, introducing calcium ions into the alginate solution is beneficial for the formation of calcium alginate gel because of crosslinking with calcium ions. The presence of calcium cations in the gel also imparts a net positive charge on the alginate.

[0068] The positively charged degradable layer can be regarded as coated on the microfilter and can be referred to as a positively charged degradable coating. The positively charged degradable layer can be in the form of a gel or a gel layer. The positively charged degradable layer can be a controllably degradable hydrogel (to be further explained in detail below).

[0069] The positively charged degradable layer coated on the microfilter can have a thickness in the range of about 0.2 microns to about 2 microns, about 0.5 microns to about 2 microns or about 1 micron to about 2 microns. For example, the positively charged degradable layer can have a thickness of about 0.5 microns, 0.75 microns, 1 micron, 1.2 microns, 1.4 microns, 1.5 microns, 1.6 microns, 1.8 microns or 2 microns. The thickness of the positively charged degradable layer can be 1.25 microns.

[0070] The positive charge on the degradable layer can be removed by reacting the positively charged degradable layer with a suitable degrading agent that reacts with the cations on the degradable layer to form a complex and simultaneously dissolves the degradable layer to form a polymer matrix or a solution of the polymer matrix. Thus, the term "degradable" refers to the removal of cations from the degradable layer due to reaction with the degrading agent and the dissolution of the degradable layer into a solution of the polymer matrix. The degradation can be controlled as it depends on when the degrading agent is added. The degrading agent can be alginate lyase, ethylenediaminetetraacetic acid (EDTA), ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), sodium citrate, nitrilotriacetic acid, or N-(2-hydroxyethyl)ethylenediaminetriacetic acid (hEDTA). The degrading agent can additionally or alternatively be an organic acid such as oxalic acid, malic acid, rubeanic acid, or citric acid. The degrading agent can be EDTA or alginate lyase. When using alginate, the positively charged alginate gel layer can be referred to as a degradable positively charged alginate gel layer.

[0071] Due to the positive charge on the positively charged degradable layer, negatively charged or charge-carrying biomaterials (which will be referred to herein for simplicity as "negatively charged biomaterials") will be attracted to the positively charged degradable layer by electrostatic attraction or interaction and thus be captured on the microfilter. Biomaterials that are not negatively charged (such as positively charged or uncharged biomaterials) can be captured on the microfilter by size exclusion or simply pass through the microfilter. The separation by electrostatic interaction described herein has been found to be surprisingly effective for capturing various microorganisms, including bacteria, fungi, and viruses.

[0072] Advantageously, the separation of biomaterials by electrostatic interaction allows for the recovery of intact microorganisms even in the presence of other biomaterials. In particular, it has been found that the microfiltration device described herein is capable of effectively recovering microorganisms in samples containing serum, proteins, and other nutrients commonly found in biological samples. In one embodiment, it has been found that the recovery of microorganisms from a sample containing a high concentration of protein is comparable to the recovery of microorganisms from cell culture broth. This is thought to be due to the electrostatic interaction between such microorganisms (which have a negatively charged cell surface) and the positively charged degradable layer, which facilitates separation by electrostatic interaction.

[0073] The microfiltration device can be a gravity-driven microfiltration device. The microfiltration device can include a microfilter having a positively charged degradable layer thereon, a sampling area where a sample can be introduced and passed through the microfilter, and a collection area located on the opposite side of the microfilter for collecting the filtrate or the filtered sample, whereby the amount or quantity of the biological material contained in the filtered sample is reduced compared to the (initial) sample. For example, the sampling area can be an area above the surface of the positively charged degradable layer, while the collection area can be located below the microfilter. The sampling area can be located within a sampling container such as a sampling bowl, and the collection area can be contained within a collection container such as a collection tube. The microfiltration device can further include a support having an opening, to which the microfilter can be placed or attached. The support can include a discharge tube that can direct the filtrate flow towards the collection container. The support can be integrally formed with the sampling container or can be provided as a separate component that can be detached from the sampling container. Fixing means can also be provided on the support to attach the support to the sampling container. The fixing means can also be used to fix the microfilter above the opening of the support. This can prevent the microfilter from moving during the execution of the method for separating biological materials.

[0074] The microfiltration device can be adjusted based on the sample to be separated. Thus, the dimensions of the microfiltration device, including the microfilter, the sampling container, the collection container, the support, the opening on the support, the discharge tube, and the fixing means, can be of any shape or size. For example, the width of the opening provided on the support can be adapted to facilitate high throughput and the inflow of the filtrate into the collection tube. The width of the opening can be smaller than the width of the microfilter. For example, in the case of providing a microfilter with a width of 17 mm, the opening can have a width of 13 mm, and the diameter of the discharge tube can be 13 mm.

[0075] After passing the sample through the microfiltration device, the microfilter having the positively charged degradable layer can be removed from the microfiltration device and then treated with a degrading agent to release the negatively charged biological material from the microfilter. Alternatively, the degrading agent can be added to the sampling area of the microfiltration device, and the negatively charged biological material released by the degradation through the positive charge layer can be collected from the collection area of the microfiltration device. Then, the negatively charged biological material can be processed downstream as needed.

[0076] Exemplary, non-limiting embodiments of a method for preparing a microfiltration device for separating biological materials from a sample will now be disclosed.

[0077] The method can include the step of coating a positively charged degradable layer on the surface of the microfilter. The positively charged degradable layer can be a positively charged alginate gel layer.

[0078] The method may further include the step of preparing a positively charged degradable layer by mixing a crosslinking agent with a polymer matrix solution such as an alginate solution. The step of preparing a mixture comprising the crosslinking agent and the polymer matrix solution may be carried out before the coating step.

[0079] The crosslinking agent may be a metal salt, wherein the metal may be selected from the group consisting of calcium (Ca), strontium (Sr), barium (Ba), iron (Fe), copper (Cu), zinc (Zn), potassium (K), sodium (Na), and lithium (Li + )]. The metal salt may be a water-soluble metal salt. For example, the metal salt may be a metal halide salt, such as a metal chloride salt, a metal bromide salt, or a metal iodide salt. The metal salt may also be a metal nitrate, a metal sulfate, a metal phosphate, a metal bicarbonate, or a water-soluble metal carbonate. The crosslinking agent may be CaCl2.

[0080] The alginate solution may comprise an alginate precursor. The alginate precursor may be selected from the group consisting of alginic acid, sodium alginate, potassium alginate, ammonium alginate, and propylene glycol alginate. The alginate solution may be a sodium alginate solution.

[0081] The coating step may be carried out by spin-coating a mixture comprising the crosslinking agent and the polymer matrix solution onto the surface of the microfilter, thereby coating the surface of the microfilter with a positively charged degradable layer. The positively charged degradable layer may be coated onto the microfilter by spin-coating at a rotational speed (rpm) of about 500 rpm to about 2000 rpm, about 1000 rpm to about 2000 rpm, about 1500 rpm to about 2000 rpm, about 500 rpm to about 1500 rpm, or about 500 rpm to about 1000 rpm, and for a duration of about 10 seconds to about 60 seconds, about 20 seconds to about 60 seconds, about 30 seconds to about 60 seconds, about 40 seconds to about 60 seconds, about 50 seconds to about 60 seconds, about 10 seconds to about 50 seconds, about 10 seconds to about 40 seconds, about 10 seconds to about 30 seconds, or about 10 seconds to about 20 seconds.

[0082] The rotation rate may be controlled to obtain a positively charged degradable layer of a desired thickness. The rotation rate may preferably be 2000 rpm.

[0083] The positively charged degradable layer may include at least two sub-layers, each sub-layer being the same as or different from each other. For example, in the case of having three Ca 2+ -alginate sub-layers, the positively charged degradable layer may be considered to be composed of Ca 2+ -alginate / Ca 2 + -alginate / Ca 2+- A layer composed of an alginate sublayer, wherein the total thickness of the entire layer can range from 0.2 microns to 2 microns, from 0.5 microns to about 2 microns, or from about 1 micron to about 2 microns. Each sublayer can be spin-coated and allowed to stand, and then the next sublayer can be coated thereon.

[0084] In the case where the positively charged degradable layer is a positively charged alginate gel layer, the method can further include the step of curing the alginate gel on the microfilter by allowing the alginate gel to stand. The coated microfilter can be allowed to stand for a time ranging from about 10 minutes to about 30 minutes, from about 20 minutes to about 30 minutes, or from about 10 minutes to 20 minutes to cure the alginate gel layer.

[0085] Exemplary, non-limiting embodiments of a method for separating biomaterials from a sample using the microfiltration device described herein will now be disclosed.

[0086] The biomaterial to be separated can be negatively charged. The biomaterial can be a negatively charged biomolecule or a whole microorganism. The biomaterial that can be captured on the positively charged degradable layer can be a whole microorganism, especially a pathogenic microorganism. The microorganism can be a bacterium, a fungus, or a virus. For example, the biomaterial can be a pathogenic bacterium and a fungus having a net negative charge on its cell wall, or a virus containing a capsid having a net negative charge. For example, the microfiltration device described herein can capture Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa, Candida albicans, Herpes simplex virus (HSV).

[0087] The method for separating biomaterials from a sample can include the step of passing a sample containing the biomaterial through the microfiltration device described herein, wherein the biomaterial is captured on the positively charged degradable layer of the microfilter. The biomaterial can be a negatively charged biomaterial. The step can include capturing or intercepting the negatively charged biomaterial on the microfiltration device, especially on the positively charged degradable layer coated on the microfilter. For example, when the sample passes through the microfiltration device, the negatively charged biomaterial can be captured by the positively charged degradable layer, whereby the negatively charged biomaterial can be attracted to the positively charged degradable layer of the microfilter by electrostatic interaction, thereby separating the negatively charged biomaterial from the sample.

[0088] The sample can include a liquid. The sample can have a volume ranging from about 100 milliliters to about 1 liter, from about 200 milliliters to about 1 liter, from about 400 milliliters to about 1 liter, from about 800 milliliters to about 1 liter, from about 100 milliliters to about 800 milliliters, from about 100 milliliters to about 400 milliliters, or from about 100 milliliters to about 200 milliliters.

[0089] The sample can be passed through the microfiltration device by gravity-driven filtration, ultrafiltration, vacuum filtration, centrifugation, or any combination thereof.

[0090] Before passing the sample through the microfiltration device, the sample can undergo a pre-separation or pre-filtration step to remove larger-sized contents from the sample. The pre-separation step can include, but is not limited to, techniques based on multi-layer microfiltration, low-speed centrifugation, and any combination thereof.

[0091] The method can also include the step of isolating the microfilter containing the negatively charged biomaterial adsorbed thereon from the microfiltration device. The negatively charged biomaterial can be separated from the sample mainly by size exclusion and, where applicable, also by charge attraction of a positively charged layer. Preferably, the negatively charged biomaterial can be separated from the sample by charge attraction of a positively charged alginate gel layer.

[0092] In the case where the biomaterial is a microorganism, there can be another method of culturing the microorganism (e.g., by inoculation / plating), which includes the following steps:

[0093] a) Removing the separated microorganism from the microfilter with a release solution; and

[0094] b) Streaking an inoculation loop of the release solution with the microorganism in step a) onto a culture plate for functional testing.

[0095] The "release solution" described herein can refer to a solution containing a degrading agent and the captured or separated microorganism.

[0096] The culture plate in step b) can contain an agar selected from the group consisting of Luria agar (LB agar) and Mueller-Hinton agar (MH agar), or any other agar suitable for culturing the captured / released microorganism. The culture plate can contain LB agar.

[0097] Functional tests can be performed on the microorganism, such as antimicrobial susceptibility testing and mechanism studies.

[0098] The method can include the step of separating the negatively charged biomaterial from the microfilter by contacting or incubating the microfilter coated with a positively charged degradable layer with a solution containing a degrading agent. The degrading agent can cause the degradation of the positively charged alginate gel layer, resulting in the separation of the negatively charged biomaterial from the microfilter. In particular, the degrading agent can be adapted to remove the positive charge of the positively charged degradable layer. When contacted with the degrading agent, the positive charge of the positively charged degradable layer can be removed, resulting in the dissolution of the positively charged degradable layer and the release of the negatively charged biomaterial captured on the positively charged degradable layer. The solution containing the degrading agent can be an aqueous solution.

[0099] The degrading agent can be alginate lyase, ethylenediaminetetraacetic acid (EDTA), ethylene glycol bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), sodium citrate, nitrilotriacetic acid or N-(2-hydroxyethyl)ethylenediaminetriacetic acid (hEDTA) or a combination thereof. The degrading agent can additionally or alternatively be an organic acid, such as oxalic acid, malic acid, rhodanine, citric acid or a combination thereof. The degrading agent can be EDTA or alginate lyase.

[0100] Advantageously, the method of separating a biomaterial by removing positive charges from a positively charged degradable layer allows for the recovery of viable biomaterials for further functional studies. In particular, it has been found that degrading agents that remove positive charges from the positively charged degradable layer described herein do not have an adverse effect on the structure and function of the recovered biomaterials. For example, it has been found that microorganisms such as bacteria, fungi and viruses are viable and culturable after being recovered / released using the method described herein. Before culturing, the microorganisms can be released from the positively charged degradable layer or not. In the case of releasing the microorganisms from the positively charged degradable layer, a solution containing the degrading agent and the released microorganisms can be brought into contact with an agar plate for subsequent culturing of the microorganisms. Alternatively, a microfilter containing the positively charged degradable layer on which the microorganisms are captured can be transferred onto an agar plate for direct culturing of the microorganisms without first releasing or separating the microorganisms from the microfilter coated with the positively charged degradable layer. Advantageously, the use of a microfilter made of a biocompatible material enables direct culturing of microorganisms from the microfilter coated with a positively charged degradable layer and on which the microorganisms are captured. Thus, the method described herein advantageously allows for the collection and recovery of viable biomaterials, which can then be used for downstream studies.

[0101] Alternatively, the method can include the step of removing the microfilter from the microfiltration device and incubating the removed microfilter with the degrading agent, whereby the microfilter comprises a positively charged alginate gel layer on which a negatively charged biomaterial is adsorbed. Here, the degrading agent degrades or lyses the biomaterial (and can degrade or not degrade the microfilter).

[0102] Incubation can be carried out at a temperature and for a duration sufficient to degrade the positively charged degradable layer and / or cleave the biomaterial. There is no restriction on the temperature, which can be in the range of about 25°C to about 45°C, about 30°C to about 45°C, about 35°C to about 45°C, about 40°C to about 45°C, about 25°C to about 40°C, about 25°C to about 35°C, or about 25°C to about 30°C, and the duration can be about 10 minutes to about 30 minutes, about 20 minutes to about 30 minutes, or about 10 minutes to about 20 minutes. It should be understood that depending on the type of biomaterial and the downstream process required, this determines the temperature and duration required for incubation. The temperature and duration can be selected to ensure the viability of the biomaterial, or the temperature and duration outside the viability limit of the biomaterial can be selected to assist in the degradation of the biomaterial.

[0103] The isolated negatively charged biomaterial can be collected in a collection tube containing an appropriate amount of solution. The volume of the collection solution is smaller compared to the initial sample to concentrate the recovered negatively charged biomaterial.

[0104] In the case where the negatively charged biomaterial is a microorganism, there can be further analysis methods, such as culturing the microorganism (as described above) or lysing the microorganism. In the case of preferably lysing the microorganism, the method can include the following steps:

[0105] a) Transfer the microorganism into a lysis solution;

[0106] b) Lyse the microorganism in the solution of step (a) to extract nucleic acid or protein from the microorganism;

[0107] c) Centrifuge the solution in step (b) for a period of time to separate the waste precipitate from the supernatant containing the nucleic acid or protein of the microorganism; and

[0108] d) Mix the supernatant containing the nucleic acid or protein with a solution suitable for subsequent analysis.

[0109] In step (a), the microorganism may have been recovered from the microfilter or may still be adsorbed to the positively charged degradable layer coated on the microfilter.

[0110] The lysis solution can be suitable for extracting nucleic acid or protein from the microorganism or pathogen. The lysis solution (depending on whether nucleic acid or protein is to be extracted) can contain a buffer solution with an ionic salt and optionally a detergent. For example, the lysis solution can be a buffer selected from the plant lysis buffer from Lucigen, QuickExtract TM DNA from Lucigen, the one-step lysis buffer from Chai Bo, or the platinum direct lysis buffer from Invitrogen.

[0111] The lysis step b) may further include the steps of i) homogenization and ii) heating, wherein the homogenization step i) and the heating step ii) are optionally repeated one or more times in an alternating manner.

[0112] The homogenization step may be carried out using vortexing for a duration of about 5 seconds to about 60 seconds, about 10 seconds to about 60 seconds, about 20 seconds to about 60 seconds, about 40 seconds to about 60 seconds, about 5 seconds to about 40 seconds, about 5 seconds to about 20 seconds, about 5 seconds to about 10 seconds, or about 10 seconds to about 20 seconds. The homogenization step may be carried out for a duration of about 15 seconds.

[0113] The heating step may be carried out in a temperature range of about 40 °C to about 100 °C, about 60 °C to about 100 °C, about 80 °C to about 100 °C, about 40 °C to about 80 °C, or about 40 °C to about 60 °C, for a duration of about 2 minutes to about 10 minutes, about 4 minutes to about 10 minutes, about 6 minutes to about 10 minutes, about 8 minutes to about 10 minutes, about 2 minutes to about 8 minutes, about 2 minutes to about 6 minutes, or about 2 minutes to about 4 minutes. It should be understood that depending on the type of lysis buffer used, this determines the temperature and duration required for the heating step.

[0114] The first homogenization step may be carried out by vortexing for a duration of 15 seconds, and the first heating step may be carried out at a temperature of 65 °C for about 6 minutes. The second homogenization step may be carried out by vortexing for a duration of 15 seconds, and the second heating step may be carried out at a temperature of 98 °C for a duration of 2 minutes. The third homogenization step may be carried out by vortexing for a duration of about 15 seconds.

[0115] The heating step may be carried out in a water bath or a dry bath / block heater.

[0116] The centrifugation step may be carried out at a relative centrifugal force of about 4000 × g to about 10000 × g, about 6000 × g to about 10000 × g, about 8000 × g to about 10000 × g, about 4000 × g to about 8000 × g, or about 4000 × g to about 10000 × g for a duration of about 10 seconds to about 60 seconds, about 20 seconds to about 60 seconds, about 40 seconds to about 60 seconds, about 10 seconds to about 40 seconds, or about 10 seconds to about 20 seconds. The centrifugation step is preferably carried out at a relative centrifugal force of about 5000 × g for a duration of 30 seconds.

[0117] The waste precipitate may contain a microfilter and unwanted biological materials from the microorganism (i.e., biological materials other than nucleic acids) or only contain unwanted biological materials.

[0118] Using a micropipette, the supernatant containing nucleic acid or protein can be mixed with the solution at a concentration of about 0.01× to about 1.0×, about 0.05× to about 1.0×, about 0.1× to about 1.0×, about 0.2× to about 1.0×, about 0.4× to about 1.0×, about 0.6× to about 1.0×, about 0.8× to about 1.0×, about 0.01× to about 0.8×, about 0.01× to about 0.6×, about 0.01× to about 0.4×, about 0.01× to about 0.2×, about 0.01× to about 0.1×, or about 0.01× to about 0.05×.

[0119] The nucleic acid can be deoxyribonucleic acid, ribonucleic acid, or a mixture thereof.

[0120] In step (d), depending on whether nucleic acid or protein is extracted, the downstream analysis of nucleic acid or protein is determined. In the case of extracting nucleic acid, subsequent analyses can be PCR (sequencing can be performed later if needed), electrophoresis, Southern blotting, etc. In the case of using PCR, the solution is a PCR master mix solution containing an isothermal amplification buffer. For example, the isothermal amplification buffer can be an isothermal buffer package containing Tris-HCl, (NH4)2SO4, KCl, MgSO4, 0.1% 20 of the isothermal buffer package, the pH of which is adjusted to 8.8 for 25 °C.

[0121] The master mix solution can contain nucleotides. The nucleotides can be deoxynucleotides (dNTPs), ribonucleotides, or a combination thereof. The nucleotides can be deoxynucleotides (dNTPs).

[0122] The master mix solution can contain buffer salts. The buffer salts can be selected from the group consisting of magnesium sulfate, ammonium sulfate, sodium sulfate, potassium chloride, and sodium chloride. The buffer salt can be magnesium sulfate (MgSO4).

[0123] The master mix solution can contain amino acids. The amino acids are not limited and depend on the type of PCR used. For example, the amino acid can be 2-aminoethanesulfonic acid (taurine).

[0124] The master mix solution can contain dyes. The dyes can have an excitation wavelength of about 600 nanometers to about 800 nanometers. The dye can be a 680 dye (i.e., a dye having an excitation wavelength of about 680 nanometers).

[0125] The master mix solution may contain a polymerase. The polymerase may be selected from the group consisting of DNA polymerases and RNA polymerases. The polymerase may be a DNA polymerase, such as Bst 2.0.

[0126] The master mix solution may contain a primer set. The primer set may further contain a forward inner primer, a reverse inner primer, a forward outer primer, a reverse outer primer, a forward loop primer, and a reverse loop primer.

[0127] Additionally, and / or, the master mix solution may contain an isothermal amplification buffer, taurine, dNTPs, magnesium sulfate (MgSO4), Bst 2.0, 680 dye, and a primer set.

[0128] A solution containing nucleic acid and the master mix may be loaded onto a polymerase chain reaction (PCR) chip for amplification. The amplification technique may be PCR or digital loop-mediated isothermal amplification (dLAMP). The amplification technique may be dLAMP. The amplification technique may be heat-initiated CRISPR / Cas-based dLAMP.

[0129] The loaded PCR chip may undergo a dLAMP program.

[0130] In the case of protein extraction, protein analysis techniques such as immunoblotting, protein gel analysis, Edman degradation, or mass spectrometry may be applied to the protein.

[0131] When separating a biological material from a sample, the biological material may be considered recovered from the sample, and, depending on the volume of the solution used to collect the recovered biological material, the biological material may be considered concentrated. Thus, the microfiltration device (such as the microfiltration device described herein or a microfiltration device prepared by the method described herein) may also be used to recover (and additionally concentrate) biological materials from a sample. The method for separating a biological material from a sample may be a method for recovering (and additionally concentrating) biological materials from a sample.

[0132] Exemplary, non-limiting embodiments of a kit including a microfiltration device will now be disclosed.

[0133] The microfiltration device is as described above and may include a microfilter having a positively charged degradable layer thereon, a sampling zone that receives a sample and directs the sample to the positively charged degradable layer coated on the microfilter, and a collection zone located on the opposite side of the microfilter for collecting the filtered sample or filtrate. For example, the sampling zone may be the area above the positively charged degradable layer, and the collection zone may be the area below the microfilter.

[0134] The kit may further include a component for receiving a sample. For example, the component may be a sampling tube. The sampling component may further include a rubber O-ring to seal the sampling tube in the sampling area of the microfiltration device.

[0135] The kit may further include a component for sample discharge. For example, the component may be a discharge tube. The discharge tube may be connected to the collection area. The discharge component may further include a component for locking the sampling component and the discharge component, and the microfiltration device is sealed between the sampling component and the discharge component.

[0136] The kit may further include a housing container for accommodating the locked sampling component and discharge component. The housing container may be a tube, such as a Falcon tube.

[0137] Exemplary, non-limiting embodiments of a microfluidic capture device including a positively charged degradable layer will now be disclosed.

[0138] The microfluidic capture device may include a positively charged degradable layer. The positively charged degradable layer may be a positively charged alginate gel layer prepared according to the method described herein. The alginate gel layer may be coated onto a filter, a filtration membrane, or a non-filter structure, but is not limited thereto.

[0139] The coated filter, filtration membrane, and non-filter structure may be incorporated into a device for microfluidic capture of analytes from a sample.

[0140] The microfluidic capture device may separate biological materials from a large-volume sample with a complex background and concentrate the separated biological materials to be compatible with downstream analysis techniques.

[0141] Exemplary, non-limiting embodiments of a microfilter will now be disclosed.

[0142] The microfilter includes a positively charged degradable layer coated thereon. The positively charged degradable layer may be a positively charged alginate gel layer. The microfilter and the positively charged degradable layer are as described herein.

[0143] Exemplary, non-limiting embodiments of a kit will now be disclosed.

[0144] The kit includes a microfilter having a positive charge layer thereon, a sampling area, and a collection area. The positively charged degradable layer may be a positively charged alginate gel layer. In use, the microfilter may be connected or coupled to a sampling area on one side of the microfilter and a collection area on the opposite side of the microfilter to form a microfiltration device. The microfiltration device, the sampling area, and the collection area are as described herein.

[0145] The kit may further include a sampling tube or sampling container and / or a discharge tube.

[0146] Advantageously, the microfiltration device described herein provides a simpler method for separating biological materials from a sample in a rapid manner (taking less than 30 minutes for a crude sample of >100 mL) without the need for bulky facilities or instruments (such as centrifuges). When separating biological materials from a sample, the microfiltration device described herein allows for a higher enrichment factor, which improves compatibility with various downstream methods.

[0147] Even more advantageously, the microfiltration device described herein is both portable and single-use convenient, thus making it convenient for use in point-of-care testing scenarios, even in remote or resource-limited areas.

[0148] Detailed Description of the Drawings

[0149] Please refer to Figure 1 , and now the working principle of the microfiltration device including a microfilter coated with a positively charged degradable layer and its use in a method for separating biological materials from a sample will be explained. In Figure 1 row a) of , the positively charged degradable layer 117 can be prepared from a polymer matrix solution 101 and a crosslinking agent 103. The crosslinking agent 103 can contain a cation 105, which promotes the formation of crosslinks 107 in the polymer matrix 101 and imparts a net positive charge when forming the positively charged degradable layer 117. A mixture 102 of the crosslinking agent 103 and the polymer matrix 101 can be coated onto the microfilter 115 to provide a positively charged degradable layer 117 on the surface of the microfilter 115 constituting the microfiltration device 116, as shown in Figure 1 row (b) of . A degrading agent 109 that can disrupt the crosslinks 107 in the positively charged degradable layer 117 can be used to dissolve or degrade the positively charged degradable layer 117. For example, the degrading agent 109 can form a complex 113 with the cation 105 to dissolve or degrade the positively charged degradable layer 117, resulting in the formation of the complex 113 and the polymer matrix solution 101. The dissolution or degradation of the positively charged degradable layer 117 also results in the loss of the surface charge of the microfilter 119. Please refer to Figure 1 row c) of . When used for separating biological materials, a sample 121 containing biological materials can be passed through the microfiltration device 106 to capture the biological materials on the positively charged degradable layer 117. The captured biological materials 123 can be recovered by contacting the positively charged degradable layer 117 with the degrading agent 109 to dissolve or degrade the positively charged degradable layer 117 and release the captured biological materials. This can allow for the recovery of concentrated biological materials 125 for downstream processing or detection. Then, the resulting microfilter 119 (as the microfilter 115) can be used again and the positively charged degradable layer 117 can be coated again.

[0150] Examples

[0151] Example 1: Method for Preparing a Microfiltration Device

[0152] Here, a parylene C microfilter with a thickness of approximately 5 μm and a high porosity greater than 14% was first purchased from Hangzhou Branemagic Medical Technology Co., Ltd. (Hangzhou, China). The SEM image of the surface of the parylene C microfilter is as Figure 2a shown.

[0153] Subsequently, an alginate gel layer was prepared from sodium alginate and calcium chloride (CaCl2) (product numbers A1112 and C5670, purchased from Sigma Aldrich). A 0.4% sodium alginate and 10 mM CaCl2 solution was prepared, and a volume of 0.2 mL to 0.8 mL of the solution was added to the microfilter in a spin coater (obtained from POLOS). The spin coater was continuously operated at 500 revolutions per minute for 10 seconds and then continuously operated at 2000 revolutions per minute for 60 seconds to produce a first calcium alginate gel layer. Then, a second calcium alginate gel layer was added on the first calcium alginate gel layer according to the method just described herein to obtain a total layer thickness of 0.2 to 2 microns. Refer to Figure 1 row a) of Figures 3b to 3d and Figure 1 row b) of Figure 2b shown. The surface of the microfilter coated with the calcium alginate gel coating was studied by SEM.

[0154] Example 2: Results of Bacteria Capture and dLAMP Detection

[0155] The method of Figure 3 was followed here. Refer to Figure 3a step a) of Figure 3eAs shown, a sample containing biological materials with a volume of approximately 1 - 10 mL is introduced into a microfiltration device (with a pore size of approximately 1.2 μm and a porosity of 9%) manufactured according to Example 1. The biological materials in the sample include Staphylococcus aureus (Sa.6538), Klebsiella pneumoniae, or Pseudomonas aeruginosa (Pa.9027) bacteria (purchased from the American Type Culture Collection). In step b), the sample containing biological materials is passed through the microfiltration device using ultrafiltration by centrifuging the microfiltration device at a speed of 500×g for 1 minute to recover the biological materials on the positively charged alginate gel layer. As shown in step c), the microfilter 301 containing the separated bacteria on the alginate gel layer is removed from the microfiltration device and transferred to a 1.5 mL microcentrifuge tube containing 0.06 mL of lysis solution 303. The lysis solution is a plant lysis buffer obtained from Lucigen. After sample filtration, the SEM image of the microfilter coated with the alginate gel layer shows that Staphylococcus aureus is captured on the alginate gel layer of the microfilter ( Figure 6 ). In step d), the tube containing the microfilter and the lysis solution is subjected to homogenization and heating cycles to induce bacterial lysis. Initially, the microcentrifuge tube is vortexed at a speed of 1000 rpm for 15 seconds (using a vortex machine purchased from ThermoFischer Scientific), then heated at 65 °C for the first time for 6 minutes, followed by vortexing at a speed of 1000 rpm for the second time for 15 seconds, and then heated at 98 °C for the second time for 2 minutes. After that, in step e), the solution is centrifuged at 5000×g for 30 seconds (using a centrifuge purchased from ThermoFisher Scientific) to separate the lysate into a waste precipitate 305 and a supernatant 311 containing nucleic acids.

[0156] In step f) of Figure 3, 8 μL of the supernatant 311 containing nucleic acids is pipetted into 32 μL of the master mix solution 307 to obtain a concentration of 0.2× (with a total volume of 40 μL). It should be understood that the total volume may vary based on the dPCR machine used. In addition, the volume ratio of the supernatant to the master mix solution may vary according to the dPCR protocol employed. Then, 40 μL of the resulting solution is transferred to the wells of a PCR chip 309 (obtained from Qiagen) (in triplicate for each sample), and then analyzed by the digital loop-mediated isothermal amplification (dLAMP) protocol. The master mix solution is prepared according to Table 1 below.

[0157] Table 1. Reaction system for dLAMP

[0158]

[0159]

[0160] For comparison, a traditional centrifugation technique was used to concentrate a sample containing Staphylococcus aureus (Sa.6538) bacteria. The obtained pellet was subjected to the lysis steps of steps c) to f) in the method of Figure 3 for dLAMP analysis. It can be seen that when using the electrostatic microfiltration (EM) bacteria capture method as described herein, compared with the centrifuged (C) sample ( Figure 4b ), the EM sample ( Figure 4a ) has a higher fluorescence distribution intensity. In the dLAMP detection analysis, each fluorescent spot represents a positive signal of a droplet / partition. Therefore, Figure 4a the higher proportion / percentage of fluorescent spots in Figure 5b indicates that the use of the microfiltration device can better recover and concentrate microorganisms. In addition, compared with 38 copies / μL of the C sample ( Figure 5a ), the partition scatter plot of the EM sample (

[0161] ) shows 1635 copies / μL, thus indicating that the microorganism separation method using the microfiltration device described herein provides a much higher enrichment factor.

[0162] The results of bacteria capture using the method described herein compared with the traditional centrifugation method are shown in Table 2 below.

[0163] Table 2. Results of bacteria capture

[0164]

[0165] The capture efficiency was calculated as follows:

[0166]

[0167] As shown in Table 2, for an abundance of approximately 10 4 CFU / mL (taking Staphylococcus aureus 6538 as an example), the average capture efficiency of electrostatic microfiltration was 87.8 ± 21.0%, which is much higher than the average capture efficiency of centrifugation (10000×g @ 10 min) of 17.9 ± 2.8%. In addition, compared with the centrifugation-based process, the high-fold (>20×) enrichment (from 1 mL to 50 μL) of the sample preparation based on electrostatic microfiltration increased the dLAMP detection readings by approximately 37 ± 9 times (from 27.78 ± 19 to 957 ± 528 copies / μL, normalized for 10 4 CFU, n = 3).

[0168] To further study the capture efficiency of the microfiltration device, the above steps were repeated for 10 mL samples containing Staphylococcus aureus, Klebsiella pneumoniae, or Pseudomonas aeruginosa at concentrations of 1 CFU / mL, 10 CFU / mL, 100 CFU / mL, or 1000 CFU / mL. For comparison, bacteria were also captured or recovered by centrifuging samples containing Staphylococcus aureus, Klebsiella pneumoniae, or Pseudomonas aeruginosa at the above concentrations at a speed of 10,000×g for 10 minutes. In Figure 10a the capture / recovery efficiency of the microfiltration device for capturing Staphylococcus aureus is shown, while in Figure 10b the capture / recovery efficiency of the microfiltration device for Klebsiella pneumoniae is shown. Figure 10c shows the capture / recovery efficiency of the microfiltration device for capturing Pseudomonas aeruginosa.

[0169] Figure 10d and Figure 10e show a graph of the dLAMP signal of the bacterial DNA of Staphylococcus aureus recovered after degradation of the positively charged degradable layer. Figure 10f and Figure 10g show the dLAMP signal obtained from the bacterial DNA of Klebsiella pneumoniae recovered from the microfilter, while Figure 10h and Figure 10i show the dLAMP signal of the bacterial DNA of Pseudomonas aeruginosa recovered from the microfilter. In Figures 10d to 10i the dLAMP signals of the bacterial DNA of Staphylococcus aureus, Klebsiella pneumoniae, or Pseudomonas aeruginosa in the sample before filtration are also shown (indicated by star markers ★ and ☆). For comparison, in Figures 10d to 10i the dLAMP signals of the bacterial DNA of Staphylococcus aureus, Klebsiella pneumoniae, or Pseudomonas aeruginosa in the precipitate recovered by the conventional centrifugation method are also shown (indicated by ▲ and Δ).

[0170] The developed microfiltration device exhibited excellent capture efficiency for various bacteria (three of the six ESKAPE pathogens, including Staphylococcus aureus, Klebsiella pneumoniae, and Pseudomonas aeruginosa, were tested as typical examples), even at low concentrations of microorganisms (as low as 1 CFU / mL). For the cases of 1000 CFU / mL and 1 CFU / mL, compared with centrifugation, the method for separating biomaterials described herein was able to achieve capture efficiencies higher than 5-fold and higher than 40-fold, respectively ( Figures 10a to 10i ).

[0171] Compared with the traditional centrifugation method, electrostatic microfiltration-based sample preparation combined with digital PCR (dPCR) was able to push the limit of detection (LOD) to approximately 100-fold lower (for Staphylococcus aureus and Pseudomonas aeruginosa, Figure 10d ,Figure 10e , Figure 10h and Figure 10i ) and 10-fold lower (for Klebsiella pneumoniae, Figure 10f and Figure 10g ). Compared to the original sampling, for the capture of Staphylococcus aureus and Pseudomonas aeruginosa, the LOD is approximately 1000-fold lower, and for the capture of Klebsiella pneumoniae, the LOD is approximately 100-fold lower.

[0172] Example 3: Capture and Release of Viable Biomaterials

[0173] Separation of biological materials was performed using the microfiltration device of Example 1. A sample containing Klebsiella pneumoniae at a concentration of 1×10 6 CFU / mL was prepared using the microfiltration device for separation. Refer to Figure 1 c) row, and a sample containing biological material 121 (a sample containing 1×10 6 CFU / mL of Staphylococcus aureus, a sample containing 1×10 6 CFU / mL of Klebsiella pneumoniae, or a sample containing 1×10 6 CFU / mL of Pseudomonas aeruginosa) was passed through the microfiltration device 106, where the microfilter 115 coated with a positively charged degradable layer (in the form of calcium alginate gel) had an aperture of approximately 1.2 μm and a porosity of 9%.

[0174] The microfilter containing the biological material 123 (such as Klebsiella pneumoniae) captured by calcium alginate gel was incubated with a solution of the degrading agent 109 (EDTA at a concentration of 10 mM or alginate lyase at a concentration of 0.1 U / mL). The degradation products were removed together with the captured biological material 123, leaving the microfilter 119 without surface charge. Figure 2c The SEM image of the microfilter after removing the calcium alginate gel is shown. Aliquots of the solution of the degrading agent, the degraded positively charged degradable layer, and the captured biological material (Klebsiella pneumoniae) were removed and applied to an agar plate for subsequent culturing. Flow cytometry studies were performed using the remaining solution containing the captured biological material to determine the viability of the captured biological material. A photograph of well-grown Klebsiella pneumoniae colonies recovered from the microfiltration device is shown in Figure 9a . Flow cytometry studies showed that the microorganisms recovered after capture on the positively charged degradable layer were highly viable ( Figure 8c and Figure 8d ), and comparable to the control samples ( Figure 8a and Figure 8b ).

[0175] The above steps were repeated to obtain from a concentration of 1×10 6Staphylococcus aureus samples at CFU / mL and concentrations of 1×10 6 Recover microorganisms from Pseudomonas aeruginosa samples at CFU / mL. Use a degrading agent (EDTA at a concentration of 10 mM or alginate lyase at a concentration of 0.1 U / mL) to release the captured microorganisms, and apply the release solution containing the degrading agent and the captured microorganisms to an agar plate for further culture. Figure 9b Shows the cultured colonies of the recovered Pseudomonas aeruginosa, Figure 9c Shows the cultured colonies of the recovered Staphylococcus aureus.

[0176] Example 4: Detection of Microorganisms in a Sample of a Mixture Containing Microorganisms

[0177] Filter a sample of a mixture containing biological materials using the microfiltration device of Example 1 (pore size approximately 1.2 μm, porosity ≥ 9%). A sample of a mixture containing 1×10 6 CFU / mL of Staphylococcus aureus, 1×10 6 CFU / mL of Klebsiella pneumoniae, and 1×10 6 CFU / mL of Pseudomonas aeruginosa is filtered through the microfilter of Example 1. The SEM image of the microfilter coated with a positively charged degradable layer shows the bacterial capture on the microfilter coated with the positively charged degradable layer ( Figure 7a ). The captured Staphylococcus aureus is indicated by an arrow , the captured Pseudomonas aeruginosa is indicated by an arrow , and the captured Klebsiella pneumoniae is indicated by .

[0178] Please refer to Figure 1 Line c), and then incubate the microfilter containing the biological material 123 captured by calcium alginate gel with a solution of the degrading agent 109 (EDTA at a concentration of 10 mM or alginate lyase at a concentration of 0.1 U / mL). Remove the degradation products together with the captured biological material 123, leaving the microfilter 119 without surface charge. Figure 2c Shows the SEM image of the microfilter after removing the calcium alginate gel. Then incubate the solution of the captured biological material and the degrading agent with the main mixture containing the reagents listed in Table 3. Detection of bacterial DNA is performed by dLAMP.

[0179] Table 3. Reaction system for the detection of multiple bacteria

[0180]

[0181]

[0182] At Figure 7bdLAMP readings for the detection of bacterial DNA of Staphylococcus aureus, Klebsiella pneumoniae, and Pseudomonas aeruginosa are provided.

[0183] Repeat the above steps using samples containing: i) a mixture of 10 CFU / mL Staphylococcus aureus and 100 CFU / mL Klebsiella pneumoniae, ii) a mixture of 100 CFU / mL Staphylococcus aureus and 100 CFU / mL Klebsiella pneumoniae, iii) a mixture of 10 CFU / mL Staphylococcus aureus and 100 CFU / mL Pseudomonas aeruginosa, iii) a mixture of 100 CFU / mL Staphylococcus aureus and 100 CFU / mL Pseudomonas aeruginosa, iv) a mixture of 10 CFU / mL Staphylococcus aureus, 100 CFU / mL Klebsiella pneumoniae, and 100 CFU / mL Pseudomonas aeruginosa, v) a mixture of 100 CFU / mL Staphylococcus aureus, 10 CFU / mL Klebsiella pneumoniae, and 10 CFU / mL Pseudomonas aeruginosa, and v) a mixture of 100 CFU / mL Staphylococcus aureus, 100 CFU / mL Klebsiella pneumoniae, and 100 CFU / mL Pseudomonas aeruginosa. In Figure 7b the dLAMP signals of the bacterial DNA are shown. This demonstrates the versatility of the microfiltration method described herein for the capture and downstream detection of microorganisms, even when provided in mixture form.

[0184] Example 5: Capture of Bacteria from a Sample with High Background

[0185] A high-concentration protein background is typically present in liquid samples of biological materials. Thus, bacteria are captured from samples spiked with fetal bovine serum using the microfiltration device conjugated with dPCR described herein.

[0186] Add 10 mL of fetal bovine serum (FBS) to 10 mL of samples containing Staphylococcus aureus at concentrations of 10 CFU / mL and 100 CFU / mL in Luria Borth (LB). For comparison, prepare 10 mL of samples containing Staphylococcus aureus in pure LB (i.e., without serum). Filter the samples using the microfilter prepared in Example 1, and perform subsequent detection of the captured bacteria according to the dLAMP procedure described in Example 2 (steps a to f of Figure 3). Repeat the above steps for samples containing Klebsiella pneumoniae and samples containing Pseudomonas aeruginosa. Figure 11a The dLAMP signals of the bacterial DNA of the recovered Staphylococcus aureus are shown, Figure 11b the dLAMP signals of the bacterial DNA of the recovered Klebsiella pneumoniae are shown, while Figure 11c the dLAMP signals of the bacterial DNA of Pseudomonas aeruginosa are shown. The results show that the LOD of the bacteria recovered from the serum-spiked samples (circular markers ) is comparable to the LOD (square marker □) in pure Luria Borth (LB), as Figures 11a to 11c shown. This is consistent for all 3 tested bacteria ( Figures 11a to 11c ).

[0187] Example 6: Capture of Bacteria from a Large-Volume Sample

[0188] To study the flux of the microfiltration device, large volume samples containing bacteria were filtered through the microfilter prepared in Example 1. Water samples of 100 mL and 500 mL containing Staphylococcus aureus at concentrations of 10 CFU / mL and 100 CFU / mL were passed through the microfiltration device of Example 1. Biological materials were separated from the samples according to the method of Example 2 (steps a to f in Figure 3), and the captured biological materials were subsequently detected. The above procedure was repeated for samples of 100 mL and 500 mL containing Klebsiella pneumoniae or Pseudomonas aeruginosa at concentrations of 10 CFU / mL and 100 CFU / mL. Figure 12a shows the dLAMP signal of the recovered Staphylococcus aureus, while Figure 12b and Figure 12c show the digital signals of Klebsiella pneumoniae and Pseudomonas aeruginosa recovered from 100 mL samples (round marker ) and 500 mL samples (square marker □).

[0189] Due to the high porosity of the microfilter coated with a positively charged degradation layer, the method of separating biological materials using the microfiltration device described herein advantageously allows for a high volume flux, which ensures the feasibility of processing ultra-large volume samples. The results show that for the recovery of bacteria, for 500 mL samples, the LOD can be as low as 10 CFU (i.e., 1 CFU / 50 mL). Thus, the concentration of bacterial samples from 500 mL to 60 μL provides an enrichment factor of approximately 8333.

[0190] Example 7: Capture of Viruses or Fungi

[0191] To demonstrate the general applicability of the microfiltration device for microbial separation, samples containing Candida albicans at a concentration of 1×10 6 CFU / mL and samples containing Herpes simplex virus at a concentration of 1×10 6 PFU / mL were prepared and filtered through the microfiltration device of Example 1. Figure 7c shows the SEM image of Candida albicans captured on the microfilter coated with a positively charged degradable layer, while Figure 7d shows the SEM image of HSV captured on the microfilter coated with a positively charged degradable layer.

[0192] In addition, 10 mL of spent culture medium of a blended brain heart infusion (BHI) broth containing Candida albicans at concentrations of 1 CFU / mL and 10 CFU / mL and 10 mL of a mock cell therapy product containing herpes simplex virus (HSV) at concentrations of 100 PFU / mL and 10,000 PFU / mL were tested. The samples were filtered using the microfilter prepared in Example 1. Detection of fungi was performed using the dLAMP system in Table 2 based on the procedure in Example 2, and detection of HSV was performed using the dPCR reaction system in Table 4 below based on the procedure in Example 2. The dLAMP signal of fungal DNA of Candida albicans recovered from the microfilter is shown in Figure 13a and the dPCR signal of HSV recovered from the microfilter is shown in Figure 13b .

[0193] Table 4. Reaction system for HSV detection

[0194] Reagents Concentration 4×QIAcuity Probe PCR Master Mix (Qiagen) 1× Forward Primer 800 nM Reverse Primer 800 nM HSV Fluorescent Probe 400 nM HSV (Virus) Lysate 0.2×

[0195] The results showed that the LOD of Candida albicans in the 10 mL sample could be reduced to 1 CFU / mL ( Figure 13a ), and the LOD of HSV in the 10 mL sample could be reduced to at least 100 PFU / mL ( Figure 13b ). The dPCR signal of the 100 PFU / mL sample was higher than 10 copies / μL (a one-order-of-magnitude difference from the NTC sample), and the LOD was expected to decrease by at least 10 PFU / mL.

[0196] Industrial applicability

[0197] The microfiltration device and the method for separating biomaterials can also be used as a sample preparation method, which can be carried out before the analysis or detection of biomaterials. For example, the microfiltration device and the method for separating biomaterials described herein can be used to detect and monitor the level of biomaterials in a collected sample, which can be a sample collected from the environment. The microfiltration device and the method for separating biomaterials described herein can be combined with various downstream analysis methods and technologies, and even allow the recovery of viable biomaterials for further research. The microfiltration device and the method for separating biomaterials can also achieve the recovery of biomaterials from a sample, and the concentration of the recovered biomaterials will generally increase.

[0198] It is obvious that various other modifications and transformations of the present invention made by those skilled in the art after reading the foregoing disclosure are obvious without departing from the spirit and scope of the present invention, and all such modifications and transformations are within the scope of the appended claims.

Claims

1. A microfiltration device comprising a microfilter coated with a positively charged degradable layer.

2. The microfiltration device according to claim 1, wherein the microfilter is made of a biocompatible material.

3. The microfiltration device according to claim 2, wherein the biocompatible material is a biocompatible polymer selected from the group consisting of parylene C, parylene N, parylene D, parylene AF-4, and combinations thereof.

4. The microfiltration device according to any one of the preceding claims, wherein the positively charged degradable layer comprises a polymer matrix and cations.

5. The microfiltration device according to claim 4, wherein the polymer matrix is gelatin, agar, IOTA carrageenan, alginate gel, gellan gum, xanthan gum, guar gum, gum arabic, or acacia gum.

6. The microfiltration device according to claim 4 or 5, wherein the cation is selected from the group consisting of calcium (II) (Ca 2+ ), strontium (II) (Sr 2+ ), barium (II) (Ba 2+ ), iron (II) (Fe 2+ ), copper (II) (Cu 2+ ), zinc (II) (Zn 2+ ), potassium (K + ), sodium (Na + ), lithium (Li + ), cuprous (I) (Cu + ) and mixtures thereof.

7. The microfiltration device according to any one of claims 4 to 6, wherein the polymer matrix is an alginate gel and the cation is calcium (II) (Ca 2+ ).

8. The microfiltration device according to any one of the preceding claims, wherein the microfilter coated with the positively charged degradable layer has a porosity of 9% or higher.

9. The microfiltration device according to any one of the preceding claims, wherein the positively charged degradable layer has a thickness of about 0.2 μm to 2 μm, and / or wherein the microfilter coated with the positively charged degradable layer comprises pores having an average diameter of about 1 μm to about 10 μm.

10. A method for separating biological materials from a sample, comprising the following steps: Pass a sample containing a biological material through the microfiltration device according to any one of the preceding claims, whereby the biological material is captured on the positively charged degradable layer on the microfilter.

11. The method according to claim 10, further comprising the step of contacting the microfilter coated with the positively charged degradable layer with a solution containing a degrading agent, wherein the degrading agent releases the captured biological material.

12. The method according to claim 11, wherein the degrading agent is alginate lyase, ethylenediaminetetraacetic acid (EDTA), ethylene glycol bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), sodium citrate, nitrilotriacetic acid, N-(2-hydroxyethyl)ethylenediaminetriacetic acid (hEDTA), oxalic acid, malic acid, rubeanic acid, citric acid, or combinations thereof.

13. The method according to any one of claims 10 to 12, wherein the biological material is a negatively charged biological material.

14. The method according to claim 13, wherein the negatively charged biological material is bacteria, fungi, or viruses.

15. A method for preparing a microfiltration device, comprising the step of coating a positively charged degradable layer onto the surface of a microfilter.

16. The method according to claim 15, further comprising, before the coating step, the step of preparing a mixture comprising a crosslinking agent and a polymer matrix solution.

17. The method according to claim 16, wherein the crosslinking agent is a metal salt.

18. The method according to claim 17, wherein the metal of the metal salt is selected from the group consisting of calcium (Ca), strontium (Sr), barium (Ba), iron (Fe), copper (Cu), zinc (Zn), potassium (K), sodium (Na), and lithium (Li).

19. The method according to any one of claims 10 to 18, wherein the coating step comprises the step of spin-coating the mixture onto the surface of the microfilter.

20. A kit comprising the microfiltration device according to any one of claims 1 to 9, a sampling tube, a discharge tube, and a housing container.

Images