CN108517284A - It is a kind of to be used to study algal grown and the micro-fluidic chip of reproduction - Google Patents

Abstract

The invention belongs to the technical field of algae growth and reproduction, in particular to a microfluidic chip for studying the growth and reproduction of algae. The microfluidic chip of the present invention is composed of a sample channel layer, a valve control layer and a substrate layer; the sample channel layer includes four parts in series: sample inlet, algae growth observation chamber, germ cell capture chamber, and sample outlet ; It adopts continuous perfusion technology to realize the functions of algae culture, germ cell release, and automatic separation, and can observe and record algae cells and subcellular structures in real time for a long time; and can quickly and accurately replace algae medium to avoid manual intervention on algae The influence of the growth state of the body; the automatically separated raw cells can be cultured separately. The chip of the invention is particularly suitable for the research on the reproduction and growth of various large algae. The manufacturing cost of the chip is low, the method is simple, and it is easy to operate and popularize.

Description

A microfluidic chip for studying algae growth and reproduction

technical field

The invention belongs to the technical field of algae growth and reproduction, and in particular relates to a microfluidic chip for studying the growth and reproduction of algae.

Background technique

In recent years, after the eutrophication of water bodies, large-scale algae outbreaks such as green tides and red tides have occurred frequently, which has become a global problem. The green tide caused by the overgrowth of large algae. When the green tide is severe, the entire sea surface near the coast is covered by algae, which consumes oxygen in the water and causes the death of zooplankton and plants in the water body. And may form swamps, produce H ₂ S gas, and cause serious damage to the ecosystem of the entire ocean. On the other hand, the green tide will also affect people's near-shore activities, damage the aquaculture industry, and affect shipping traffic. Therefore, it is very important to study the mechanism of algal blooms to solve the environmental problems caused by green tides or red tides.

Existing studies have shown that some algae that can reproduce rapidly are mainly due to its multiple reproduction methods, including sexual reproduction, asexual reproduction and vegetative reproduction. The main form of the green tide is Enteromorpha algae, which can grow on a large scale mainly by asexual reproduction of germ cells. Almost all the cells of Enteromorpha can be transformed into germ cell cysts. One sporangia produces about 8 spores, and one gamete produces about 16 gametes. According to research, 1cm ² of Enteromorpha (single side) can produce about 10 ⁶ -10 ⁷ spores and gametes, and 1g of Enteromorpha (fresh weight) can theoretically grow into 10 ⁸ -10 ⁹ new algal bodies. Two gametes of the opposite sex can combine to form a zygote to grow into a new algae, and can also grow into a new algae through asexual reproduction like spores, and the spores or gametes can also replicate themselves under suitable conditions, splitting into a large number of individuals . In short, once the right growth conditions are available, the algae will grow explosively. Moreover, spores or gametes are more sensitive to changes in environmental factors than mature algae, and are more likely to produce environmental stress. Therefore, it is very important to study the germ cells of algae to uncover the formation mechanism of green tide or red tide.

In the research on algae reproduction and growth, there are still some problems that need to be solved and optimized: in situ culture cannot be realized, and the medium control is not precise; the spatial resolution cannot meet the morphological changes of algal cells or subcellular structures (differentiation of spores or gametes) , the combination process of two gametes, etc.); the growth status of the algae cannot be observed in real time; the germ cells of the algae cannot be separated automatically. These problems are expected to be solved by microfluidics. Microfluidics can integrate the basic operation units such as sample preparation, reaction, separation, and detection in the biological, chemical, and medical analysis processes into a micron-scale chip, and automatically complete the entire analysis process.

At present, the research methods of algae are limited to macroscopic research methods, and the mechanism of germ cell production and release has not yet been clarified, so the outbreak mechanism remains to be explored. Microfluidic chips have been widely used in the cultivation of animal cells, but rarely applied in the field of plant cells. For this reason, the present invention designs a microfluidic chip to study the growth and reproduction of algae from the micron scale. The designed chip meets the needs of algae growth, germ cell release and capture, and germ cell selective cultivation alone.

Contents of the invention

The purpose of the present invention is to provide a microfluidic chip for microcosmic study of algae growth and reproduction, which can finely manipulate fluid, has a simple manufacturing method and strong versatility.

The microfluidic chip used to study the growth and reproduction of algae provided by the present invention uses optically transparent materials as the base material, adopts a microfluidic continuous perfusion system, realizes the automatic separation of algae germ cells from the microscopic level, and monitors algae cells in real time. A series of growth and reproductive changes, which solves the problems of uneven diffusion in nutrient solution and accumulation of harmful metabolites in traditional research methods. Specifically, the air pressure control valve of the microfluidic chip is used to control the algae growth unit and the germ cell capture unit, so that the free germ cell enters the germ cell capture unit, thereby realizing the capture and selective cultivation of a single germ cell. In the present invention, the chip can be placed under a fluorescent inverted microscope connected with a computer and a high-speed CCD, so as to collect and analyze changes in fluorescence intensity, and the optical signals generated by the specific combination of reagents or antibodies with germ cells can be collected and transmitted by the array of photosensitive devices Compare with the database of standard products in the microprocessor (computer), so as to realize automatic detection.

The microfluidic chip used to study the growth and reproduction of algae provided by the present invention can be provided with several parallel modules (such as 6-10, etc.) according to actual needs, and each module has three layers: sample channel layer, valve control layer, substrate layer. The substrate layer is the base of the entire microfluidic chip, used to support the valve control layer and the sample channel layer; the sample channel layer is used to accommodate algae cells and nutrient solution, and the valve control layer is used to control the flow of liquid in the sample channel layer and the movement of algae ; Wherein, the sample channel layer includes four parts: sample inlet, algae growth observation chamber, germ cell capture chamber, sample outlet, four parts are connected in sequence; algae growth observation chamber is 300-800 μ m in height, width It is a cuboid of 300-800μm, which is used to observe the growth of algae body online, and provide enough space for the growth of algal cells, and at the same time provide enough space for the formation of germ cell release port; the germ cell capture chamber is 10- 30μm, 50-150μm diameter cylindrical pipe, used for automatic separation and capture of germ cells; sample inlet is used to inject medium, injection medium is injected with a syringe, and the autosampler is connected to the syringe containing the medium; sample outlet Connected to the waste liquid tank for discharging waste liquid.

In the present invention, the valve control layer includes two control valves, which are respectively arranged at the front and back of the germ cell capture chamber; the two control valves respectively have valve control inlets; the valves are controlled by air pumps or electronic components, and then control the algae growth observation chamber Chamber and germ cell capture chamber sample channel layer valve switch. The valve is a semi-permeable valve, the reproductive cells (spores and gametes) of algae cannot pass through, and the culture liquid can pass through freely.

Let the valve behind the germ cell capture chamber be the first valve, and the valve in front of the germ cell capture chamber be the second valve; when the pneumatic pump fills the gas from the valve control inlet, the corresponding valve works, and the liquid can pass through freely at this time , but algae cells cannot pass through. When the first valve 1 is working and the second valve 2 is not working, the reproductive spores released by the algae cells can be captured by the germ cell capture chamber. When the second valve 2 is working and the first valve 1 is not working, the culture solution is injected from the sample outlet to realize the separate cultivation of the algae germ cells.

In the present invention, the height and depth of the algae growth observation chamber are relatively large, providing sufficient space for the growth of algae cells, and at the same time providing sufficient space distance for the formation of germ cell release ports. When the sample is injected, the liquid medium carries the released free germ cells into the germ cell capture unit; the germ cells are distributed in the cell capture unit in a dot-like manner, and the captured free germ cells will quickly adhere to the cell capture unit , so germ cells can be captured in the cell capture unit.

In the present invention, the germ cell capture chamber can also be used as a separate culture unit for germ cells. After the germ cells are collected, the medium is injected from the original sample outlet, and different types of medium make the algae show different states. Algal germ cells may develop into new filaments, propagules, or undergo clonal self-replication. The present invention can also perform environmental stress such as polycyclic aromatic hydrocarbons or heavy metals on the germ cells of the algae in the attached state, and then realize the in-situ automatic detection of the antioxidative enzymes of the algae by using the fluorescence analysis method.

In the present invention, the substrate of the microfluidic chip is a flexible optically transparent material, including: polydimethylsiloxane polymer (PDMS); rigid high molecular polymer: polycarbonate, polyethylene terephthalate Ethylene glycol formate, polymethyl methacrylate, polystyrene, polypropylene; inorganic materials: quartz, glass. The mold material is silicon wafer.

The present invention also provides a method for making a microfluidic chip, the specific steps are as follows:

(1) Substrate preparation: Clean the monocrystalline silicon wafer with Piranha solution to remove oxidation, blow it dry with nitrogen, and use AZ 50XT series positive resist to spin the glue through a spin coater, then dry it softly on a constant temperature heating plate to make the photoresist curing;

(2) Exposure and development: Place the mask of the sample pipeline layer and the valve control layer on the spin-coated substrate, and after exposure by a UV exposure machine, place it in a positive photoresist developer for development;

(3) Secondary exposure: After the developed silicon wafer is sprayed with a SU-8 2150 series spin coater, it is soft-baked on a constant temperature heating plate to cure the photoresist. Then carry out secondary exposure to the algae observation chamber, put it on the heating plate and then bake and solidify;

(4) Place the exposed and baked silicon wafers in a negative film developing solution for development, check the completion of development with isopropanol, and blow dry with nitrogen;

(5) Hard baking: the silicon wafer is placed on the heating plate to heat and fix;

(6) Pouring: Mix the polydimethylsiloxane monomer and curing agent at 5:1 and 20:1 respectively, pour it on the corresponding mold, and cure and peel it in the oven;

(7) Bonding: The valve layer and the sample pipe layer are calibrated and bonded to form a microchannel cavity, and then bonded to the substrate layer.

The method for using the above-mentioned microfluidic filter chip also provided by the present invention, the specific steps are as follows:

(1) Pick a small amount of algae from the female parent of the algae as an experimental sample to ensure that the algae cells are in the same growth state, and no sporangia and gametes are formed in the somatic cells;

(2) Cut out a segment of about 200 μm-500 μm from the top of the algae body, and implant the cut algae body segment into the algae observation area of the microfluidic chip;

(3) After the algae sample is successfully implanted, the sample inlet is connected to the syringe filled with the medium, and the sample outlet is connected to the waste liquid tank;

(4) Place the chip under an inverted microscope connected to a computer and a high-speed CCD, connect the syringe containing the medium to the autosampler, the injection speed is 25μl/L-60μl/L, and the capture is controlled by an air pump Pneumatic valves at both ends of the chamber are opened and closed to capture algae germ cells.

When studying germ cells alone, change the original sample outlet to an inlet, and the original inlet to a sample outlet, the injection speed is about 25μl/L-60μl/L, inject different media or perform Different environmental stresses, so as to realize the separate culture of germ cells, after the separate culture, use the fluorescence detection method to detect the antioxidant enzymes of the germ cells of algae in situ.

The present invention designs a germ cell capture chip, which has an algae growth observation chamber and a germ cell capture chamber, and forms a microfluidic technology-based algae cell automatic sensor with the chip, an automatic sampler, and a fluorescence imaging system. Training and monitoring system. The system uses a microfluidic continuous perfusion system to keep the cells in a nutrient-rich state, increase the growth rate of algae cells, and avoid manual operations during the growth cycle of algae cells to the greatest extent, realizing long-term automatic real-time monitoring of algae cells Record. The microfluidic chip provided by the invention has the advantage of finely controlling the fluid, and has a simple manufacturing method and strong versatility, and can be used in combination with various designs.

The above-mentioned microfluidic filter element can be used in the fields of on-line observation of algae, separation of germ cells of algae, separate cultivation of germ cells of algae, and the like.

Description of drawings

Figure 1 is a three-layer structure diagram of a microfluidic chip used to study algae growth and reproduction. The pipeline layer is composed of microchambers with different heights. The algae observation chamber is higher than the germ cell capture chamber, and the algae cells can only be arranged in a single layer of cells in the germ cell capture chamber. There is a pneumatic valve on both sides of the germ capture chamber, and the valve is controlled by an air pump to realize the capture and separate cultivation of germ cells.

Figure 2 is a plan view of a microfluidic chip used to study algae growth and reproduction. When the pneumatic pump is filled with gas from the valve control inlet, the corresponding valve works, and the liquid can pass through freely at this time, but the algae cells cannot pass through. When the valve 1 is working and the valve 2 is not working, the germ spores released by the algae cells can be captured by the germ cell capture chamber. When the valve 2 is working and the valve 1 is not working, the culture solution is injected from the sample outlet to realize the separate cultivation of the algae germ cells.

Detailed ways

(1) Substrate preparation: The single crystal silicon wafer was cleaned in Piranha solution (98% concentrated sulfuric acid: 30% hydrogen peroxide) for 15 minutes to deoxidize, rinsed with deionized water, dried with nitrogen, and baked at 200 ° C for 30 minutes.

(2) Spin the glue: put the prepared substrate on the spin coating position of the high-pressure adsorption spin coating machine (Spin-Coater KW-4A, ChematTechnology, Inc.), pour AZ 50XT series glue on the center of the substrate, and rotate at 2000r/min for 2min , let it stand for 10 minutes to reduce the air bubbles on the surface of the silicon wafer.

(3) Pre-baking: Place the glue-coated silicon wafer on a heating plate at 65°C, 90°C, and 65°C for 2 minutes, 7 minutes, and 2 minutes.

(4) Exposure: Place the mask of the sample pipeline layer and the valve control layer on the spin-coated substrate, and expose it with a UV exposure machine.

(5) Development: The development is carried out in a fume hood. Place the silicon wafer in the developer of the positive photoresist, first develop it in the developer with a concentration of 1/3 until the glue on the periphery of the silicon wafer shrinks inward, and then place it at a concentration of 1/5. The developer solution is complete. Rinse with deionized water and dry with nitrogen gas.

(6) Secondary exposure: After the first exposure of the sample channel layer substrate, the SU-8 2150 series is used to spin the glue through a spin coater, and then softly bake on a constant temperature heating plate to cure the photoresist. Use the mask of the algae observation unit to expose the original pipeline twice.

(7) Post-baking: After-baking the silicon wafer on a heating plate at 65°C and 95°C for 2min and 5min in turn, and cooling to room temperature.

(8) Also develop in a fume hood with a negative film developer (the main component is propylene glycol methyl ether acetate PGMEA), check that the development is complete with isopropanol, and blow dry with nitrogen.

(9) Hard baking: place the silicon wafer in the sample channel layer on a heating plate and heat and fix at 120°C.

(10) PDMS polymer pouring: mix polydimethylsiloxane monomer and curing agent at a ratio of 5:1, pour it on the silicon wafer of the sample channel layer treated with trichlorochlorosilane, and remove it with a vacuum pump After bubbling, place it in an oven at 80°C for 1 hour to cure.

(11) Valve layer and glass base layer: mix polydimethylsiloxane monomer and curing agent at a ratio of 20:1, pour them on the valve silicon wafer and glass slide respectively, and spin on a spin coater at 2000r/min Glue for 1min, then bake in an oven at 80°C for 1h to cure.

Bonding and fabrication: The sample channel layer and the valve layer chip are peeled off from the silicon wafer, the valve layer and the sample channel layer are calibrated and bonded to form a microchannel cavity, and baked in an oven at 80°C for 1 hour to make them tightly bonded together. After punching holes, it is bonded to the glass slide layer, and finally cured overnight in an oven at 80°C.

The cut algae sample is perfused into the algae observation chamber of the chip with a syringe pump, and the sample inlet of the chip is connected to the injector to perfuse different sample solutions. The released germ cells follow the fluid into the germ cell capture chamber. Germ cells are trapped in the chamber by opening and closing the air pressure controlled valve. After the germ cells are captured, the sample injection is stopped, and a specific sample solution is perfused from the original sample outlet to conduct a separate environmental stress study on the germ cells. And the change of germ cell antioxidant enzyme activity was analyzed by fluorescence analysis method.

Claims (4)

1. A microfluidic chip for studying algae growth and reproduction, characterized in that it comprises a plurality of parallel modules, and each module structure is three layers: sample channel layer, valve control layer, substrate layer; substrate layer It is the base of the entire microfluidic chip, supporting the valve control layer and the sample channel layer; the sample channel layer is used to accommodate algae cells and nutrient solution, and the valve control layer is used to control the flow of liquid in the sample channel layer and the movement of algae; where: The sample channel layer includes four parts: sample inlet, algae growth observation chamber, germ cell capture chamber, and sample outlet, and the four parts are connected in sequence; the algae growth observation chamber has a height of 300-800 μm and a width of A 300-800μm cuboid is used to observe the growth of algal bodies online, and provide enough space for the growth of algal cells, and at the same time provide enough space for the formation of germ cell release ports; the height of the germ cell capture chamber is 10-30μm , a cylindrical pipe with a diameter of 50-150 μm, which is used for automatic separation and capture of germ cells; the sample inlet is used to inject the medium, and the medium is injected using a syringe, and the autosampler is connected to the syringe containing the medium; the sample outlet is connected to the syringe containing the medium; The waste liquid tank is connected to discharge the waste liquid. The valve control layer includes two control valves, which are respectively arranged at the front and back of the germ cell capture chamber; the two control valves respectively have valve control inlets; the valves are controlled by air pumps or electronic components, and then control the algae growth observation chamber and Valve switching of the sample channel layer of the germ cell capture chamber. 2. The microfluidic chip for studying the growth and reproduction of algae according to claim 1, wherein the valve is a semi-permeable valve, through which the germ cells of the algae cannot pass, and the culture liquid can pass through freely. 3. the using method of microfluidic chip as claimed in claim 1, is characterized in that, concrete steps are as follows: (1) Pick a small amount of algae from the female parent of the algae as an experimental sample to ensure that the algae cells are in the same growth state, and no sporangia and gametes are formed in the somatic cells; (2) Cut out a segment of about 200 μm-500 μm from the top of the algae body, and implant the cut algae body segment into the algae observation area of the microfluidic chip; (3) After the algae sample is successfully implanted, the sample inlet is connected to the syringe filled with the medium, and the sample outlet is connected to the waste liquid tank; (4) Place the chip under an inverted microscope connected to a computer and a high-speed CCD, connect the syringe containing the medium to the autosampler, the injection speed is 25μl/L-60μl/L, and the capture is controlled by an air pump Pneumatic valves at both ends of the chamber are opened and closed to capture algae germ cells. 4. The method of using the microfluidic chip as claimed in claim 1, characterized in that, when the germ cells are studied separately, the original sample outlet is changed into a sample inlet, and the original sample inlet is changed into a sample outlet , the injection speed is about 25μl/L-60μl/L, inject different media or carry out different environmental stresses, so as to realize the separate culture of germ cells, and use fluorescence detection method to detect the antioxidant enzymes of algae germ cells after separate culture In situ detection.