CN108499619B - Membrane integrated type micro-fluidic filter chip and preparation method and application thereof - Google Patents

Abstract

The invention belongs to the technical field of microfluidic chips, in particular to a membrane-integrated microfluidic filter chip and a preparation method and application thereof. The microfluidic filter chip of the present invention is composed of a porous membrane for filtration sealed with a sealant between the upper and lower substrates of the microfluidic chip, which are engraved with microfluidic channels. The invention utilizes the filling of the sealant on the edge gap of the porous membrane and the infiltration of a small part of the membrane edge, as well as the cross-linking or mutual dissolution of the sealant and the substrate, so as to realize the complete integration of the porous membrane and the substrate, and maximize the retention in the microfluidic chip. The porous membrane has an effective filtration capacity; at the same time, the microfluidic filtration chip is constructed by using the cavity formed by the microchannel on the surface of the substrate and the membrane. The chip not only has the good hydrophilicity of the microporous filter membrane, the interception ability of ultrafine particles, and the excellent biocompatibility, but also has the advantages of fine fluid manipulation by the microfluidic chip. As a general-purpose module, it can be combined with other microfluidics It is used in combination with the control function unit, the production method is simple, the versatility is strong, and it can be used in combination with various designs.

Description

Membrane-integrated microfluidic filtration chip and preparation method and use thereof

technical field

The invention belongs to the technical field of microfluidic chips, and in particular relates to a membrane-integrated microfluidic filter chip and a preparation method and application thereof.

Background technique

Microfluidic technology has been used in the preparation and enrichment of biological samples for more than ten years. The broad market demand for the technology has stimulated the production of a large number of microfluidic-based sample preparation and enrichment research results, and a few research results have been transformed into automated and commercialized solutions. Compared with the traditional centrifugation method or membrane method, the advantage is that the use of microfluidic technology can obtain high-concentration concentrated samples in a closed system at one time, reducing sample pollution and sample loss caused by human operation, and it is convenient to achieve. Automated interface to downstream assays.

Different from the non-biological sample processing process, most biological samples need to maintain their own biological activity after separation and enrichment, and may also need to divert the samples to other tests. According to the characteristics of biological samples, it can be roughly divided into animal and plant cells, fungal spores, parasite oocysts, bacteria, viruses, proteins, nucleic acids, etc. according to the target volume. Early related researches focused on using the great flexibility in the design and processing of microfluidic chips, according to the physical and chemical properties or biological properties of the target in the sample, by constructing various morphological microstructures, directly intercepting cells with physical structures or using characteristic fluids Bacteria were isolated indirectly. However, for samples with complex components (such as blood), there are a lot of interfering substances in the samples, which will directly affect the effect of the microstructure, and even lead to the loss of chip function. For the target objects with small particle size of bacteria, if the physical structure is to be intercepted, it is necessary to achieve 0.1-1 micron precision processing, which requires extremely high process requirements, and the traditional chip manufacturing method is even more difficult to achieve. In recent years, researchers have begun to focus on integrating various new technologies with microfluidic chips to achieve more complex sample preparation and enrichment processes, such as the enrichment of nucleic acid or protein from biological samples based on adsorption fillers; Separation of circulating tumor cells in blood for immunobiology; separation of characteristic cell metabolites based on liquid-liquid extraction, solid phase extraction and separation; enrichment of cells, bacteria, fungi and yeast in samples by combining magnetic fields, electric fields, and sound fields separation etc. With the help of external devices, various related applications can be realized on microfluidic chips. However, the commercial development of these new technologies is seriously constrained by their high technical costs, resulting in their inability to meet the market’s demand for cheap and easy-to-use applications in practical applications, resulting in only a few products that stand out and can only be applied to high-end consumer products. Medical field. For low-consumption fields such as inspection and quarantine, sanitation, food, and the environment, where fungal and bacterial samples are the main samples, high-efficiency technologies are required to improve the technical level of the field, and simple operation and low cost must be considered.

In order to solve such problems, a large number of studies have introduced porous membranes into microfluidic chips. The high-density pores provided by porous membranes can physically intercept various biological samples, and their small pore sizes can be used to filter bacteria and even viruses. Go beyond the microfluidic structures that require high-precision machining, while reducing the processing cost of microfluidic chips. Integrating the hydrophilicity, bioaffinity, ultrafine filtration capability of the porous membrane and the liquid manipulation capability of microfluidics enables the microfluidic chip to complete the interception and enrichment of biological targets in samples without additional equipment. In the vast majority of studies, the simplest and most effective method of membrane fixation is to directly sandwich the porous membrane between two layers of microfluidic substrates, and use the chamber formed between the microfluidic substrate pipeline and the membrane to store samples. This method completes the isolation and enrichment of cells in the sample and performs various experiments directly on the membrane. However, the biggest problem with this method is that when the film is clamped to the substrate, a gap will be created between the edge and the substrate due to the thickness of the film. Therefore, a lot of research uses mechanical force to reduce the gap, but this does not inherently solve the problem, and it also makes the use of the chip cumbersome. Cell-targeted assays typically use this approach because the small gap between the membrane and the substrate is not sufficient for cells to pass through, and the membrane acts as a blocker. However, this method is not reliable for fungi or bacteria with smaller particle size, because there is always a small gap between the membrane edge and the substrate. During the process of intercepting the sample through filtration, some bacterial particles will escape from the gap. Leakage, especially when the target concentration is not high, leads to the fact that the filter membrane cannot fully exert its efficient interception effect, which seriously affects the enrichment efficiency. In this application, the effective integration of membranes and microfluidic chips has become a key technical problem. Filling the gap with sealant has always been a feasible method. Due to the hydrophilicity of the membrane and the influence of the micro-cavity of the microfluidic chip, when the sealant contacts the membrane or the micro-cavity, it will rapidly infiltrate the membrane and the cavity. body. With the filling of the gap at the edge of the membrane, most of the membrane and the cavity are sealed at the same time and lose their basic functions, and the production success rate is extremely low. For the above reasons, the porous membrane-integrated microfluidic filter chip cannot be applied to the treatment of bacterial samples, and there are few related studies. In order to solve this problem, it is urgent to develop a new porous membrane-integrated microfluidic filtration chip and its fabrication method to overcome the above defects.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a low-cost, high-efficiency microfluidic filter chip and its preparation method and application.

The microfluidic filtration chip provided by the present invention is based on the membrane integration technology, and the main problem to be solved is how to completely integrate the porous membrane into the microfluidic chip simply and quickly, so as to retain the high-efficiency filtration of the porous membrane. At the same time, it has the ability of microfluidic chip to control microliter fluid, solves the problem of low-cost and efficient processing of fungal spores and bacterial samples by microfluidic chip, thereby expanding the application scope of microfluidic chip. Specifically, the microfluidic pipeline is used to control the sealant, so that the gap at the edge of the porous membrane in the chip can be removed according to the production needs, so that the membrane can be completely integrated into the chip, and the problem of chip leakage can be solved.

The membrane-integrated microfluidic filter chip (also known as a microfluidic filter element) provided by the present invention is sealed by a sealant between the upper and lower substrates of the existing microfluidic chip, which are engraved with microfluidic channels. It is composed of a porous membrane for filtration to form a three-layer structure.

In the above-mentioned microfluidic filter element, the porous membrane refers to various materials with porous structures, such as textile porous membranes with irregular pore shapes, nitrocellulose membranes, polyester fibers and other polymer-based porous membranes, and Polycarbonate, polytetrafluoroethylene and other polymer porous membranes with regular pores. The sealant refers to a polymer reagent that can be cured by chemical cross-linking or physical cross-linking under certain conditions, including: temperature-sensitive reagents, such as: polydimethylsiloxane (PDMS), etc., photosensitive type reagents, such as polyacrylamide, etc., silicone glue, formula type glue, etc. The substrate refers to acrylic, glass, resin, polysiloxane, and metal as the substrate, the surface of which is processed by machining, mold injection, mold pouring and other processing processes to form microstructure pipes and cavity grooves. The encapsulant can be cross-linked or miscible with the base material.

The preparation method of the above-mentioned microfluidic filter element provided by the present invention, the specific steps are as follows:

Step 1. Make the base. The base is divided into upper and lower layers. An annular groove is formed at the corresponding positions on the surfaces of the upper and lower substrates. When the upper and lower substrates are aligned, an annular "sealed reagent control pipe" is formed; at the same time, a through hole is machined in the center of the annular groove area of the upper substrate as a sample injection hole. (that is, the liquid inlet hole of the microfluidic chip), the cavity groove (channel) of the microfluidic chip is machined at the center; 2 to 6 sealant injection holes are drilled through the annular groove (for example, 6 sealant Injection holes, numbered A1-A6) and the same number of exhaust holes (e.g., exhaust holes, numbered B1-B6), alternating between injection holes and exhaust holes along the pipe (example sequence: A1-B1-A2-B2- ...-B6-A1). One or more through holes are processed in the annular groove area of the lower substrate as sample holes;

Step 2. Cut the porous membrane to a size that conforms to the area enclosed by the annular sealing tube in the substrate, and the cut edge of the porous membrane is in the middle of the annular sealing tube. Refer to Figure 2; for example, make it satisfy: R _film =R _outer- W _density /2±W _density /4, R _film refers to the radius of the porous film, R _outer refers to the outer wall radius of the annular sealed pipe; W _dense refers to the width of the sealed pipe ;

Step 3. The cut porous membrane is sandwiched between two base layers to form a sandwich structure. The annular sealing grooves on the two base layers overlap to form an annular sealed pipe. The edge of the membrane falls in the center of the pipe. Fix and compress the base;

Step 4. Inject the sealant from the sealant injection hole at a constant speed. The sealant can infiltrate the porous membrane in the pipeline, and the air in the pipeline is discharged from the exhaust hole. When the sealant just flows to the exhaust hole, stop the injection immediately;

Step 5. Use the sealant curing method to rapidly cure the sealant, such as ultraviolet rays, heating, etc., to rapidly cure the sealant to obtain a microfluidic filter element.

In the above-mentioned manufacturing method of the microfluidic filter element, in the process of fixing and pressing the substrate by external force, an external clamp can be used to clamp the sandwich structure, and appropriate pressure can be applied to ensure that the sealant is injected into the annular pipe.

The general operation method of the above-mentioned microfluidic filter chip provided by the present invention, the specific steps are as follows:

Step 1: According to the experimental requirements, select microfluidic filter chips with different pore sizes to intercept different detection targets, which should satisfy D _target > D _{porous membrane} , D _target refers to the diameter of the detection target, and D _{porous membrane} refers to the maximum pore diameter of the porous membrane;

Step 2: The sample is injected into the microfluidic filter chip from the injection port. The injection rate of fungal solution, bacterial solution and egg solution is 15-30 μl/s, and the injection rate of cell solution is 2-10 μl/s. The injection time of sample is 10 s- 10 minutes;

Step 3: Collect the biological particles intercepted by the porous membrane for further detection and analysis.

There are many detection methods for biological particles, among which PCR detection method and fluorescence microscope method are widely used.

Method 1: PCR detection method. This method first uses a lysis buffer to lyse the biological particles captured by the porous membrane. After the cleavage is completed, use a specific primer set to identify it, the reaction can be completed in 1-2 hours, and the experimental results can be analyzed by gel electrophoresis. Recently, real-time quantitative PCR (RT-PCR) has developed rapidly. Unlike conventional PCR technology, RT-PCR does not analyze the amplification results by gel electrophoresis, but performs real-time detection of the PCR process by fluorescent signals, so it is different from PCR detection. Compared with the method, RT-PCR detection method is simpler and faster.

Method 2: Fluorescence microscopy. This method is another common method for detecting biological particles. The biological particles collected in the liquid buffer or other medium solutions are dyed with fluorescent dyes and then irradiated with incident light of suitable wavelength to emit fluorescence, so they can be counted by a fluorescence microscope. Fluorescence microscopy can enumerate all viable fungi and bacteria. In addition, if the fluorescence microscope is connected to a computer equipped with an image analysis system, high-throughput automated counting of samples can be achieved.

The invention adopts the design of the microfluidic "sealing agent control pipeline" to realize the control of the sealing agent. The complete integration of the membrane and the substrate is achieved by using the sealant to fill the gap at the edge of the membrane and to infiltrate a small part of the membrane edge, as well as the cross-linking or mutual dissolution of the sealant and the substrate. The purpose of the effective filtration capacity of the membrane. At the same time, the microfluidic filtration chip is constructed by using the cavity formed by the microchannels and the membrane on the surface of the substrate. There are many kinds of porous membrane materials, and the pore size varies widely, so it can meet the different needs of different biological particles. In addition, on the basis of ensuring high capture efficiency, the porous membrane will not cause damage to biological particles. The chip not only has the good hydrophilicity of the microporous filter membrane, the interception ability of ultrafine particles, and the excellent biocompatibility, but also has the advantages of fine fluid manipulation by the microfluidic chip. As a general-purpose module, it can be combined with other microfluidics It is used in combination with the control function unit, the production method is simple, the versatility is strong, and it can be used in combination with various designs.

The above-mentioned microfluidic filter element can be used in the fields of separation, analysis, identification or identification.

The specific effects of the present invention are as follows:

The invention utilizes the design of the liquid control pipeline to accurately control the sealant, and realizes the complete integration of the membrane and the chip substrate which is simple, efficient, and low-cost, and does not rely on external equipment. Using this new preparation method of microfluidic filter element can completely eliminate the gap between the membrane and the chip substrate, eliminate the phenomenon of liquid leakage, and at the same time ensure that more than 90% of the membrane after production is not infiltrated and sealed by the sealant, maximizing the effectiveness of retaining the membrane area. The obtained chip not only has the advantages of high hydrophilicity of porous membrane, high density pore distribution and high precision pore size, but also has the advantages of easy processing of microfluidic chip, excellent biocompatibility and fluid manipulation ability. The membrane-integrated chip has the ability to efficiently enrich bacterial samples.

Description of drawings

Figure 1. Schematic diagram of the relationship between the chip substrate and the porous membrane. Among them, the porous membrane is sandwiched between the upper and lower substrates, and an annular sealant control groove is processed at the corresponding position in the middle of the upper and lower substrates. After the upper and lower substrates are aligned, an annular sealant control pipe is formed. A sealant injection hole and several exhaust holes are processed in the annular sealant control groove; several sample outlet holes are processed in the annular sealant control groove of the lower substrate.

Figure 2. Schematic diagram of porous membrane size requirements and placement positions. Among them, R _film =R _outer- W _density /2±W _density /4, R _film refers to the multi-pore size; R _outer refers to the annular outer wall radius of the sealed pipe; W _dense refers to the width of the sealed pipe; the edge of the porous film is clamped at the sealed pipe.

Detailed ways

The present invention will be further described below in conjunction with the accompanying drawings.

Example 1: Preparation of a membrane-integrated microfluidic filter cartridge based on polydimethylsiloxane (PDMS).

Step 1. Use PDMS prepolymer and curing agent to prepare and mix in any ratio of 2:1 to 20:1 by mass ratio, and use the reverse molding method to prepare the upper and lower substrates with the sealant control pipe pattern respectively. At the same time, a through hole is machined in the center of the surrounding area of the upper substrate manipulation pipe as a sample injection hole, a cavity groove is machined at the center, and 2 to 6 sealant injection holes (numbered A1-A6) are drilled through the sealing reagent control pipe. ) and the same number of air exhaust holes (numbered B1-B6), the injection holes and exhaust holes are alternately arranged along the pipeline (order: A1-B1-A2-B2-…-B6-A1). One or more through holes are processed in the surrounding area of the manipulation pipe of the lower substrate as a liquid outlet;

Step 2. Cut the polycarbonate porous membrane with a pore size ranging from 0.1 to 12 microns to a specified size, as described with reference to Figure 2;

Step 3. Referring to Figure 2, sandwich the cut film between two layers of substrates and press tightly;

Step 4. To prepare the sealant, use PDMS prepolymer and curing agent to prepare and mix in any ratio of 2:1 to 20:1 by mass ratio. Inject the sealant from the sealant injection hole at a constant speed. The sealant can infiltrate the porous membrane in the pipeline and discharge the air in the pipeline. When the sealant just flows to the exhaust hole, stop the injection immediately;

Step 5. The chip is placed in a high temperature treatment of 50 to 140 degrees Celsius, and the sealant is rapidly cured to obtain a microfluidic filter element.

Example 2: Preparation of membrane-integrated microfluidic filter cartridges based on acrylic plastic (acrylic/PMMA).

Step 1. The upper and lower substrates with the sealant control pipe pattern are respectively prepared by engraving method or injection molding method. At the same time, a through hole is machined in the center of the surrounding area of the upper substrate manipulation pipe as a sample injection hole, a cavity groove is machined at the center, and 2 to 6 sealant injection holes (numbered A1-A6) are drilled through the sealing reagent control pipe. ) and the same number of air exhaust holes (numbered B1-B6), the injection holes and exhaust holes are alternately arranged along the pipeline (order: A1-B1-A2-B2-…-B6-A1). One or more through holes are processed in the surrounding area of the manipulation pipe of the lower substrate as a liquid outlet;

Step 2. Cut the polycarbonate porous membrane with a pore size ranging from 0.1 to 12 microns to a specified size, as described with reference to Figure 2;

Step 3. Referring to Figure 2, sandwich the cut film between two layers of substrates and press tightly;

Step 4. Using UV polymerized acrylic adhesive, inject the sealant at a constant speed from the sealant injection hole. The sealant can infiltrate the porous membrane in the pipeline and discharge the air in the pipeline. When the sealant just flows to the exhaust hole, stop the injection immediately. liquid;

Step 5. Put the chip under UV lamp radiation for 10 seconds to 5 minutes to rapidly cure the sealant to prepare a microfluidic filter element.

Claims (1)

1. a preparation method of a membrane-integrated microfluidic filter chip, is characterized in that, this microfluidic filter chip is sealed with a sealant between the upper and lower substrates of the microfluidic chip engraved with the microfluidic channel. 1. Composition of porous membrane for filtration; Specific steps are as follows: Step 1. Making the base: forming annular grooves at corresponding positions on the surfaces of the upper and lower bases respectively. When the upper and lower bases are butted together, an annular pipe is formed to control the sealant; at the same time, process 1 in the center of the annular groove area of the upper base. A through hole is used as a sample injection hole, and the cavity groove of the microfluidic chip is machined in the center; 2 to 6 sealant injection holes and the same number of exhaust holes are punched through the annular groove. Alternately arranged along the annular pipe; one or more through holes are processed in the annular groove area of the lower substrate as a sample outlet; Step 2. Cut the porous membrane to a size that matches the area surrounded by the annular conduit in the substrate, so that the edge of the porous membrane is in the middle of the annular conduit; Step 3. The cut porous membrane is sandwiched between two base layers to form a sandwich structure. The annular grooves on the two base layers overlap to form an annular pipe. The edge of the membrane falls in the center of the pipe. compress the base; Step 4. Inject the sealant from the sealant injection hole at a constant speed, so that the sealant infiltrates the porous membrane in the annular pipe, and the air in the annular pipe is discharged from the exhaust hole. When the sealant just flows to the exhaust hole, stop injecting the sealant immediately. ; Step 5. Use the sealant curing method to rapidly cure the sealant to obtain the membrane-integrated microfluidic filter chip.

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