CN108499619B - Membrane integrated type micro-fluidic filter chip and preparation method and application thereof - Google Patents
Membrane integrated type micro-fluidic filter chip and preparation method and application thereof Download PDFInfo
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- B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
- B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
- B01L3/502753—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by bulk separation arrangements on lab-on-a-chip devices, e.g. for filtration or centrifugation
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- B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
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- B01L3/502707—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by the manufacture of the container or its components
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Abstract
The invention belongs to the technical field of microfluidic chips, and particularly relates to a membrane integrated microfluidic filter chip and a preparation method and application thereof. The micro-fluidic filter chip is formed by sealing a porous membrane for filtering between an upper substrate and a lower substrate of the micro-fluidic chip, wherein the upper substrate and the lower substrate are provided with micro-fluidic channels in an engraved mode. The invention utilizes the filling of the sealant to the edge gap of the porous membrane, the infiltration to the edge of a small part of the membrane, and the crosslinking or mutual dissolution of the sealant and the substrate to realize the complete integration of the porous membrane and the substrate and maximally retain the effective filtering capacity of the porous membrane in the microfluidic chip; meanwhile, the micro-fluidic filter chip is constructed by utilizing a cavity formed by the micro-pipeline on the surface of the substrate and the membrane. The chip has the advantages of good hydrophilicity of the microporous filter membrane, ultrafine particle interception capability and excellent biocompatibility, has the advantage of fine control of the microfluidic chip on fluid, can be used as a universal module to be used with other microfluidic functional units, is simple in manufacturing method and high in universality, and can be used in combination with various designs.
Description
Technical Field
The invention belongs to the technical field of microfluidic chips, and particularly relates to a membrane integrated microfluidic filter chip and a preparation method and application thereof.
Background
The micro-fluidic technology has been used for the preparation and enrichment research of biological samples for more than ten years, and the wide market demand of the fields of life science, clinical medicine, new drug screening, quarantine, health, food, environment and the like on the analysis technology of the biological samples is benefited, so that the generation of a large amount of sample preparation and enrichment research results based on micro-fluidic is stimulated, and a few of the research results are converted into an automatic and commercialized solution. Compared with the traditional centrifugal mode or filter membrane mode, the method has the advantages that the high-concentration concentrated sample can be obtained in a closed system at one time by adopting the microfluidic technology, the sample pollution and the sample loss caused by manual operation are reduced, and the automatic connection with the downstream detection and analysis is conveniently realized.
Unlike non-biological sample processing techniques, most biological samples need to maintain their own biological activity after separation and enrichment, and may need to be drained to other assays. According to the characteristics of biological samples, the biological samples can be roughly classified into animal and plant cells, fungal spores, parasite oocysts, bacteria, viruses, proteins, nucleic acids and the like according to target volumes. Early related studies focused on utilizing the great flexibility in the design and processing of microfluidic chips, and directly intercepting cells by physical structures or indirectly separating bacteria by using characteristic fluids by constructing various morphological microstructures according to the physicochemical or biological characteristics of targets in samples. However, in the case of a sample with complex composition (such as blood), a large amount of interfering substances exist in the sample, which directly affect the effect of the microstructure and even cause the loss of the chip function. For the target object with tiny particle size of bacteria, the processing with 0.1-1 micron precision needs to be realized if the interception of the physical structure is carried out, the process requirement is extremely high, and the traditional chip manufacturing method is difficult to realize. In recent years, researchers have focused on integrating various new technologies with microfluidic chips to achieve more complex sample preparation and enrichment processes, such as enrichment of biological sample nucleic acids or proteins based on adsorption packing; separation of circulating tumor cells in blood based on hydrodynamics or immunobiology; separation of characteristic cellular metabolites based on liquid-liquid extraction, solid phase extraction and separation; and the enrichment and separation of cells, bacteria and fungi in the sample are realized by combining a magnetic field, an electric field, an acoustic field and the like. Various related applications can be realized on the microfluidic chip by means of external equipment. However, the commercial development of these new technologies is severely limited by their high technical cost, so that they cannot meet the needs of cheap and easy-to-use market in practical application, and only a few products are made out of the question and can be applied to the medical field of high-end consumption. For low consumption fields such as inspection and quarantine, sanitation, food, environment and the like mainly comprising fungi and bacteria samples, high-efficiency technology is needed to improve the technical level of the field, and simple operation and low cost are considered.
In order to solve the problems, a large number of researches are carried out to introduce a porous membrane into a microfluidic chip, various biological samples can be physically intercepted by virtue of high-density pores provided by the porous membrane, the fine pore diameter of the porous membrane can be used for filtering bacteria and even viruses, the performance of the porous membrane is completely superior to that of a microfluidic structure which can be realized only by high-precision processing, and the processing cost of the microfluidic chip is reduced. The hydrophilicity, the biological affinity, the superfine filtering capacity and the microfluidic liquid control capacity of the porous membrane are integrated, so that the microfluidic chip can finish the interception and enrichment of biological targets in a sample without additional equipment. In most of researches, the simplest and most effective membrane fixing method is to directly sandwich a porous membrane between two microfluidic substrates, and store a sample by using a chamber formed between a microfluidic substrate pipeline and the membrane. However, in this method, the biggest problem is that when the film is sandwiched between the substrates, a gap is generated between the edge and the substrate due to the thickness of the film. Therefore, a lot of research has been done to reduce the gap by mechanical force, but this does not essentially solve the problem and also makes the use of the chip cumbersome. This is often the case for cell-targeted assays because the small gap between the membrane and the substrate is insufficient for the cell to pass through and the membrane acts as a barrier. However, this method is not reliable for fungi or bacteria with smaller particle size, because there is always a tiny gap between the edge of the membrane and the substrate, and some bacterial particles will leak out from the gap during the process of intercepting the sample by filtration, especially in the case of low target concentration, which results in that the membrane cannot fully exert its efficient intercepting effect, thus seriously affecting the enrichment efficiency. In this application, the effective integration of the membrane with the microfluidic chip becomes a key technical problem. The gap is filled with the sealant which seems to be a feasible method all the time, and due to the influence of the hydrophilicity of the membrane and the micro-cavity of the micro-fluidic chip, when the sealant contacts the membrane or the micro-cavity, the membrane and the cavity can be quickly infiltrated. With the filling of the film edge gap, most of the film and the cavity are simultaneously sealed and lose the basic functions, and the manufacturing power is extremely low. Due to the reasons, the porous membrane integrated microfluidic filter chip cannot be applied to the treatment of bacterial samples, and related researches are few. In order to solve the problem, it is necessary to develop a new integrated microfluidic filter chip with a porous membrane and a method for manufacturing the same, so that the above drawbacks can be overcome.
Disclosure of Invention
The invention aims to provide a microfluidic filter chip with low cost and high efficiency, and a preparation method and application thereof.
The microfluidic filter chip provided by the invention is based on a membrane integration technology, and the main problem to be solved is how to simply and quickly integrate a porous membrane in the microfluidic chip completely, so that the porous membrane not only retains the efficient filtering effect of the porous membrane, but also has the control capability of the microfluidic chip on micro-liter fluid, and the problems of low cost and high efficiency treatment of the microfluidic chip on fungal spore and bacterial samples are solved, thereby expanding the application range of the microfluidic chip. Specifically, the sealant is controlled by the microfluidic pipeline, so that the gap at the edge of the porous membrane in the chip can be removed according to the manufacturing requirement, the membrane is completely integrated into the chip, and the problem of chip leakage is solved.
The invention provides a membrane integrated micro-fluidic filter chip (also called micro-fluidic filter element), which is formed by sealing a porous membrane for filtering by a sealant between an upper substrate and a lower substrate of the existing micro-fluidic chip, wherein the upper substrate and the lower substrate are provided with micro-fluidic channels, so that a three-layer structure is formed.
In the above microfluidic filter element, the porous membrane refers to various materials having a porous structure, such as a textile porous membrane having an irregular pore shape, a nitrocellulose membrane, a polyester fiber, and other polymer-based porous membranes, and a polycarbonate having a regular pore shape, a polytetrafluoroethylene, and other polymer porous membranes. The sealant refers to a high molecular agent which can be cured through chemical crosslinking or physical crosslinking under certain conditions, and comprises the following components: temperature sensitive reagents, such as: polydimethylsiloxane (PDMS), etc., a photosensitive type agent such as polyacrylamide, etc., a silicone adhesive, a formula adhesive, etc. The substrate is characterized in that acrylic, glass, resin, polysiloxane and metal are used as a substrate, and a microstructure pipeline and a cavity groove are formed on the surface of the substrate through machining, mold injection, mold pouring and other processing technologies. The sealant may be cross-linked or miscible with the substrate material.
The preparation method of the microfluidic filter element provided by the invention comprises the following specific steps:
step 1, manufacturing a substrate, wherein the substrate is divided into an upper layer and a lower layer. Annular grooves are respectively processed at corresponding positions on the surfaces of the upper substrate and the lower substrate, and an annular sealing reagent control pipeline is formed after the upper substrate and the lower substrate are combined; meanwhile, 1 through hole is processed at the center of the annular groove area of the upper substrate to be used as a sample inlet hole (namely a liquid inlet hole of the microfluidic chip), and a cavity groove (channel) of the microfluidic chip is processed at the center; 2 to 6 sealant injection holes (for example, 6 sealant injection holes, No. A1-A6) and the same number of exhaust holes (for example, exhaust holes, No. B1-B6) are punched on the annular groove, and the injection holes and the exhaust holes are alternately arranged along the pipeline (for example, the sequence is A1-B1-A2-B2- … … -B6-A1). Processing 1 or more through holes in an annular groove area of the lower substrate to serve as sample outlet holes;
and 2, cutting the porous membrane into a size which is consistent with the size of the area surrounded by the annular sealed pipeline in the substrate, wherein the cut edge of the porous membrane is positioned in the middle of the annular sealed pipeline. As shown in fig. 2; for example, it is made to satisfy: rFilm=ROuter cover-WSecret key/2±WSecret key/4，RFilmMeans radius of porous film, ROuter coverThe radius of the outer wall of the annular sealing pipeline is pointed; wSecret keyThe width of the sealed pipeline;
step 3, clamping the cut porous membrane between two substrate layers to form a sandwich structure, overlapping annular sealing grooves on the two substrate layers to form an annular sealing pipeline, enabling the edge of the membrane to fall in the center of the pipeline, and fixing and pressing the substrate by means of the self viscosity or external force of the material;
step 4, injecting the sealant from the sealant injection hole at a constant speed, wherein the sealant can infiltrate the porous membrane in the pipeline, air in the pipeline is exhausted from the exhaust hole, and the liquid injection is stopped immediately when the sealant just flows to the exhaust hole;
and 5, rapidly curing the sealant by adopting a sealant curing method, such as ultraviolet curing, heating and the like, so as to rapidly cure the sealant, and obtain the microfluidic filter element.
In the manufacturing method of the microfluidic filter element, in the process of fixing and pressing the substrate by the external force, the sandwich structure can be clamped by an external clamp, and proper pressure is applied to ensure that the sealant is injected into the annular pipeline.
The invention provides a general operation method of the microfluidic filter chip, which comprises the following specific steps:
step 1: according to the experimental requirements, the microfluidic filter chips with different apertures are selected to intercept different detection targets, and D is satisfiedTarget>DPorous membrane，DTargetFinger diameter of target, DPorous membraneRefers to the maximum pore diameter of the porous membrane;
step 2: injecting the sample into the micro-fluidic filter chip from the sample inlet, wherein the injection speed of the fungus solution, the bacteria solution and the worm egg solution is 15-30 mul/s, the injection speed of the cell solution is 2-10 mul/s, and the injection time of the sample is 10 s-10 min;
and step 3: and collecting the biological particles intercepted by the porous membrane for further detection and analysis.
The detection method of the biological particles is various, and the PCR detection method and the fluorescence microscopy are widely applied.
The method comprises the following steps: and (3) a PCR detection method. The method firstly uses a lysis solution to lyse biological particles intercepted by a porous membrane. After the cracking is finished, identifying the DNA chip by using a specific primer group, finishing the reaction within 1-2 h, and analyzing the experimental result by gel electrophoresis. Recently, real-time quantitative fluorescent PCR (RT-PCR) is rapidly developed, and unlike conventional PCR techniques, RT-PCR does not perform amplification result analysis by gel electrophoresis, but performs real-time detection of PCR process by fluorescent signals, so that RT-PCR detection methods are simpler, more convenient and faster than PCR detection methods.
The method 2 comprises the following steps: fluorescence microscopy. This method is another commonly used method for detecting biological particles. Biological particles collected in a liquid buffer or other medium solution can emit fluorescence after being dyed by a fluorescent dye and irradiated by incident light with a proper wavelength, so that the counting can be completed through a fluorescence microscope. Fluorescence microscopy allows the enumeration of all active 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 a microfluidic sealant control pipeline to realize sealant control. The complete integration of the membrane and the substrate is completed by utilizing the filling of the sealant to the gap at the edge of the membrane, the infiltration of the edges of a small part of the membrane and the cross-linking or mutual dissolution of the sealant and the substrate, and the purpose of maximally retaining the effective filtering capacity of the porous membrane in the microfluidic chip is realized for the first time. Meanwhile, the micro-fluidic filter chip is constructed by utilizing a cavity formed by the micro-pipeline on the surface of the substrate and the membrane. The porous membrane has various types of materials and large pore diameter variation range, thereby meeting different requirements of different biological particles. In addition, on the basis of ensuring high capture efficiency, the porous membrane can not cause damage to biological particles. The chip has the advantages of good hydrophilicity of the microporous filter membrane, ultrafine particle interception capability and excellent biocompatibility, has the advantage of fine control of the microfluidic chip on fluid, can be used as a universal module to be used with other microfluidic functional units, is simple in manufacturing method and high in universality, and can be used in combination with various designs.
The microfluidic filter element can be used in the fields of separation, analysis, identification or the like.
The invention has the following specific effects:
the invention utilizes the design of the liquid control pipeline to accurately control the sealant, and realizes the complete integration of the film and the chip substrate which are simple, efficient, low in cost and independent of external equipment. By utilizing the novel preparation method of the microfluidic filter element, the gap between the membrane and the chip substrate can be completely eliminated, the liquid leakage phenomenon is eliminated, meanwhile, more than 90% of the membrane after the preparation is ensured not to be soaked and sealed by the sealant, and the effective area of the membrane is reserved to the maximum extent. The chip has the advantages of porous membrane high hydrophilicity, high density pore distribution and high precision pore diameter, and has the advantages of easy processing, excellent biocompatibility and fluid control capability. The membrane integrated chip has the capability of efficiently enriching bacterial samples.
Drawings
FIG. 1 is a schematic diagram showing the combination of a chip substrate and a porous membrane. The porous membrane is clamped between an upper layer substrate and a lower layer substrate, an annular sealant control groove is processed at the corresponding position of the middle part of the upper layer substrate and the lower layer substrate, an annular sealant control pipeline is formed after the upper layer substrate and the lower layer substrate are combined, a sample inlet hole is processed at the central part of the upper layer substrate, and a sealant injection hole and a plurality of exhaust holes are processed at the annular sealant control groove; a plurality of sample outlet holes are processed in the annular sealant control groove of the lower substrate.
FIG. 2 is a schematic diagram of the porous membrane size requirements and placement. Wherein R isFilm=ROuter cover-WSecret key/2±WSecret key/4 ，RFilmRefers to a plurality of apertures; rOuter coverThe radius of the annular outer wall of the sealed pipeline is defined; wSecret keyThe width of the sealed pipeline; the edge of the porous membrane is clamped at the position of the sealed pipeline.
Detailed Description
The invention will be further explained with reference to the drawings.
Example 1: preparation of Polydimethylsiloxane (PDMS) based membrane-integrated microfluidic filter elements.
Step 1, mixing PDMS prepolymer and curing agent according to any mass ratio of 2:1 to 20:1, and preparing an upper substrate and a lower substrate with sealant control pipeline patterns by a reverse mold method. Meanwhile, 1 through hole is processed in the center of the surrounding area of the upper-layer substrate operation pipeline to be used as a sample inlet hole, a cavity groove is processed in the center position, 2 to 6 sealant injection holes (numbered A1-A6) and the same number of air exhaust holes (numbered B1-B6) are processed in the sealing agent operation pipeline in a penetrating way, and the injection holes and the exhaust holes are alternately arranged along the pipeline (the sequence is: A1-B1-A2-B2- … … -B6-A1). Processing 1 or more through holes as liquid outlet holes in the surrounding area of the control pipeline of the lower substrate;
step 2. cutting a polycarbonate porous membrane with a pore size in the range of 0.1 to 12 μm to a specified size, as described with reference to FIG. 2;
step 3, clamping the cut film between two layers of substrates and pressing the films with reference to the figure 2;
and 4, preparing a sealant, namely preparing a PDMS prepolymer and a curing agent, and mixing the PDMS prepolymer and the curing agent in any ratio of 2:1 to 20:1 by mass. Injecting the sealant from the sealant injection hole at a constant speed, wherein the sealant can infiltrate the porous membrane in the pipeline and discharge air in the pipeline, and stopping injecting the sealant immediately after the sealant just flows to the exhaust hole;
and 5, placing the chip at a high temperature of between 50 and 140 ℃ for treatment, and quickly curing the sealant to obtain the microfluidic filter element.
Example 2: preparation of a membrane-integrated microfluidic filter element based on acrylic plastic (acrylic/PMMA).
Step 1, preparing an upper layer substrate and a lower layer substrate with sealant control pipeline patterns respectively by adopting a carving method or an injection molding method. Meanwhile, 1 through hole is processed in the center of the surrounding area of the upper-layer substrate operation pipeline to be used as a sample inlet hole, a cavity groove is processed in the center position, 2 to 6 sealant injection holes (numbered A1-A6) and the same number of air exhaust holes (numbered B1-B6) are processed in the sealing agent operation pipeline in a penetrating way, and the injection holes and the exhaust holes are alternately arranged along the pipeline (the sequence is: A1-B1-A2-B2- … … -B6-A1). Processing 1 or more through holes as liquid outlet holes in the surrounding area of the control pipeline of the lower substrate;
step 2. cutting a polycarbonate porous membrane with a pore size in the range of 0.1 to 12 μm to a specified size, as described with reference to FIG. 2;
step 3, clamping the cut film between two layers of substrates and pressing the films with reference to the figure 2;
step 4, injecting the sealant from the sealant injection hole at a constant speed by adopting an ultraviolet polymerization type acrylic adhesive, wherein the sealant can infiltrate the porous membrane in the pipeline and exhaust air in the pipeline, and stopping injecting liquid immediately after the sealant just flows to the exhaust hole;
and 5, placing the chip under ultraviolet lamp radiation for 10 seconds to 5 minutes, and quickly curing the sealant to obtain the microfluidic filter element.
Claims (1)
1. The preparation method of a membrane integrated microfluidic filter chip is characterized in that the microfluidic filter chip is formed by sealing a porous membrane for filtering between an upper substrate and a lower substrate of the microfluidic chip, wherein the upper substrate and the lower substrate are provided with microfluidic channels;
the method comprises the following specific steps:
step 1, manufacturing a substrate: processing the corresponding positions on the surfaces of the upper substrate and the lower substrate to form annular grooves respectively, and forming an annular pipeline for controlling the sealant after the upper substrate and the lower substrate are jointed; meanwhile, 1 through hole is processed at the center of the annular groove area of the upper substrate to be used as a sample inlet, and a cavity groove of the microfluidic chip is processed at the center; 2 to 6 sealant injection holes and exhaust holes with the same number are punched on the annular groove, and the injection holes and the exhaust holes are alternately arranged along the annular pipeline; processing 1 or more through holes in an annular groove area of a lower substrate to serve as sample outlet holes;
step 2, cutting the porous membrane into a size which is consistent with the size of the area surrounded by the annular pipeline in the substrate, and enabling the edge of the porous membrane to be positioned in the middle of the annular pipeline;
step 3, clamping the cut porous membrane between two substrate layers to form a sandwich structure, overlapping annular grooves on the two substrate layers to form an annular pipeline, enabling the edge of the membrane to fall in the center of the pipeline, and fixing and pressing the substrate by means of the self viscosity or external force of the material;
step 4, injecting the sealant from the sealant injection hole at a constant speed to enable the sealant to infiltrate the porous membrane in the annular pipeline, discharging air in the annular pipeline from the exhaust hole, and immediately stopping injecting the sealant when the sealant just flows to the exhaust hole;
and 5, rapidly curing the sealant by adopting a sealant curing method to obtain the membrane integrated microfluidic filter chip.
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| CN112791755B (en) * | 2021-03-31 | 2021-07-02 | 苏州大学 | Multilayer microfluidic chip packaging device, multilayer microfluidic chip and application thereof |
| CN113941379B (en) * | 2021-10-15 | 2023-02-17 | 重庆大学 | Micro-fluidic chip and method for sorting, enriching and detecting bacteria and fungi |
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