CN105907601B - A kind of microorganism three-dimensional COMMUNITY STRUCTURE chip and application based on Solid Double gel - Google Patents

Abstract

The present invention provides a kind of micro-fluidic chips for microorganism three-dimensional COMMUNITY STRUCTURE and inter-species communication research.The chip is double-layer structure, is prepared using mold method, and lower layer is mainly constituted by liquid injection hole, for the microchannel of first time package and the wide pipeline for wrapping up for the second time；Micro-valve door is contained on upper layer, control can be opened and closed to the chip pipeline positioned at lower layer.The present invention carries out double wrapped by the different microorganism of two kinds of three kinds of solid gels pair, forms solid-state microorganism three-D space structure.By changing chip into flow velocity, mode can be wrapped up to the accumulation of solid gel microballoon and be changed, is adjusted so as to the three dimensions distance easily between not same species of microorganism.

Description

Microbial three-dimensional community structure analysis chip based on solid-state double gel and its application

technical field

The invention belongs to the field of environmental biological analysis, and in particular relates to a microfluidic chip that double-wraps microorganisms through two kinds of porous solid gels to establish a three-dimensional community structure of solid microorganisms.

Background technique

Microorganisms exist in the form of communities in nature, and different species of microorganisms in the community transmit information through interspecific interactions and perform biological functions through the metabolic process of different substrates. The population structure, diversity, and community function of environmental microbial communities are always changing dynamically, and play a vital role in the balance of environmental ecosystems.

In recent years, the three-dimensional cultivation of microbial populations using biocompatible materials has become one of the research hotspots in the field of environmental microbiology. This method can "assemble" a variety of microorganisms with different metabolic pathways and functions for cultivation, thereby reflecting Its synergistic metabolism simulates the life and metabolism of real microorganisms in the environment. For example, several microorganisms that secrete different enzymes and substrates are three-dimensionally cultured to form a simulated biological community, and their secretions produce products with certain environmental functions through enzymatic reactions. This three-dimensional simulated biological community analysis method has also become a method for many drugs or A new research platform for environmental toxicology analysis. However, there are many problems in the existing methods for establishing the three-dimensional structure of microorganisms. For example, the construction cost of the commonly used hydrogel space support structure is high, the steps are cumbersome, and the small pore size of the gel is not conducive to the transmission of information between microorganisms. The physical distance between communities has changed, and the lack of automatic operation has resulted in large errors and poor repeatability. These defects urgently need to be solved by new technical methods.

Microfluidic chip technology is developing rapidly in the field of environmental biology. Using mature chip processing and manufacturing methods and micro-droplet (Droplet) technology, microbial culture chambers can be built in the chip, and a single microorganism can be wrapped in a diameter of 20- 200μm microspheres, so as to achieve precise manipulation and growth and metabolism analysis of individual microorganisms. Microbes can still contact and absorb external environmental stimuli, cytokines and nutrients inside the microspheres with a porous structure. In addition, the solid gel is a loose porous structure, which can provide the space required for the growth of microorganisms, so this method can ensure the normal growth and metabolism of microorganisms. The microfluidic chip research platform based on this technical principle can break through many technical bottlenecks. In addition, the cultivation and manipulation of microorganisms in the microfluidic chip can effectively avoid external contamination, and the automatic control of the chip also makes it easier to control tiny microorganisms. accurate.

At present, the research on using solid gel to enclose environmental microorganisms in a second solid state to construct a three-dimensional microbial community in a chip is still blank. The microfluidic chip of the present invention can automatically control the encapsulation and cultivation of microorganisms and the formation and analysis of three-dimensional communities in the chip, and can simulate the real three-dimensional community state of microorganisms in a natural environment, which is of great significance to environmental biology research.

Contents of the invention

The invention provides a microfluidic chip capable of wrapping three microorganisms in solid gel twice to form a solid three-dimensional spatial structure, which can be used for the analysis of the three-dimensional community structure of microorganisms and the research of inter-species communication of microorganisms.

Technical scheme of the present invention is:

A microbial three-dimensional community structure analysis chip based on solid double gel, the microbial three-dimensional community structure analysis chip has a double-layer structure, including a pipeline flow layer 12 and a micro-valve layer 11; it is functionally divided into a gel microsphere wrapping unit, A gel microsphere packing unit and a solid gel secondary wrapping unit.

The micro-valve layer 11 includes a micro-valve 5 and a valve pipe for air pressure control, and the micro-valve 5 controls the opening or closing of the pipe in the flow-through layer of the chip pipe.

The pipeline flow layer 12 includes injection holes 1, 2, and 3 for the mixture of three kinds of microorganisms and low melting point liquid agarose gel, injection hole 4 for silicon and surfactant, injection hole 6 for liquid trehalose gel, Injection hole 7 for calcium chloride solution, injection hole 8 for silicone oil and surfactant, collection hole 9 for solid microbial three-dimensional community gel, microchannel for liquid circulation and gel microsphere wrapping, and for gel microsphere Wide channel for ball packing and solid gel secondary wrapping. The micropipe in the chip has a depth of 40-60.0 μm and a width of 40-50 μm, and is a smooth and straight groove; a wide pipeline has a depth of 40-60.0 μm and a width of 600-800 μm.

The material of the microbe three-dimensional community structure analysis chip is polydimethylsiloxane polymer (PDMS) with good optical permeability and elasticity.

The liquid circulation layer 12 and the microvalve layer 11 of the microbiological three-dimensional community structure analysis chip are respectively irradiated with 365nm ultraviolet light for 10-20 minutes, plasma treated for 2-5 minutes, and then irreversibly sealed and bonded by docking.

Using the above-mentioned microbial three-dimensional community structure analysis chip to construct and collect the microbial solid-state three-dimensional gel structure, specifically includes the following steps:

(1) After mixing three different microorganisms with liquid low-melting point agarose gel and liquid culture medium heated to 41°C, inject the micro-channels through the mixed liquid injection holes 1, 2, and 3 respectively, and at the same time mix with the The mixed liquid of silicon and surfactant is injected into the micropipe through the injection hole 4 at the same liquid inlet speed as the liquid, and agarose gel micro-droplets containing three kinds of microorganisms are formed under the action of the mixed liquid of silicon and surfactant. Closing the micro-valve 5 makes the wide pipeline in a closed state, and the agarose gel micro-droplets located in the wide pipeline are blocked and trapped by the micro-valve; the accumulated agarose gel micro-droplets solidify into agarose gel after standing at room temperature Microspheres realize one-time solid-state encapsulation of three microorganisms. The low-melting point agarose gel is liquid at high temperature, and solidifies into a porous gel state when the temperature drops to room temperature.

(2) Open the microvalve 5, inject the trehalose gel solution through the injection hole 6, and make it fill the interstitial space between the agarose gel microspheres. Calcium chloride solution is injected through the injection hole 7, and the trehalose gel is solidified under the action of the calcium chloride solution to form a porous colloidal solid structure, thereby fixing the agarose gel microspheres containing microorganisms in their three-dimensional space position , forming solid microbial gel microspheres 10 wrapped in trehalose gel to form a three-dimensional stacking structure, realizing secondary solid-state encapsulation of three microorganisms.

(3) Collect the solid microbial gel microspheres through the collection hole 9 and place them in the culture solution. The culture solution can penetrate into the interior of the three-dimensional gel through the porous structure of the two solid gels, and perform three-dimensional culture on the microorganisms wrapped therein. By adding fluorescent dyes, it can be used to study the three-dimensional community structure of microorganisms and the communication between species in the community.

The invention can adjust the three-dimensional spatial distance between different kinds of microorganisms by changing the flow rate of the liquid entering the chip and changing the stacking and wrapping mode of the gel microspheres.

Beneficial effects of the present invention: the microfluidic chip provided by the present invention can significantly reduce the consumption of microorganisms, reagents and culture fluid; the microvalve in the chip can be precisely controlled by a computer control system. In the chip, three kinds of microorganisms are double-wrapped by two different porous solid gels, which can simulate the real state of the growth of the three-dimensional structure of microorganisms in nature, and can be used for the study of the three-dimensional community structure of microorganisms and interspecies communication.

Description of drawings

Fig. 1 is a design diagram of the microfluidic chip of the present invention.

Fig. 2 is a schematic diagram of a three-dimensional microbial community formed by solid gel encapsulation in a chip.

Fig. 3 is a schematic cross-sectional view of the chip pipeline.

In the figure: 1-3 mixed solution injection hole; 4 injection hole; 5 micro valve; 6 injection hole; 7 injection hole; 8 injection hole; 9 collection hole; 10 solid microbial gel microsphere; 11 valve layer; 12 pipeline circulation Floor.

Detailed ways

Figure 1 shows the structure diagram of the microfluidic chip used for the analysis of the three-dimensional community structure and interspecies communication of microorganisms. Inject the low-melting point agarose gel mixture containing three different microorganisms into the microchannels of the microfluidic chip into microwells No. 1, No. 2, and No. 3, and inject silicone oil through microwell No. 4 at the same velocity. And surfactants, can form liquid agarose gel micro-droplets that wrap three kinds of microorganisms in micro-channels. Then, increase the air pressure (no more than 15psi) in the No. 5 micropore, so that the microvalve in the valve layer of the chip is in a closed state, and the agarose gel microdroplet located in the wide pipeline is blocked and retained by the microvalve. After standing still at room temperature, the accumulated agarose gel micro-droplets solidify into loose porous solid agarose gel microspheres, encapsulating microorganisms in the microspheres, and realizing one-time solid-state encapsulation of three microorganisms. Re-open the microvalve, inject the liquid trehalose gel solution into the chip through the No. 6 hole, and make it fill the interstitial space between the agarose gel microspheres. Then inject calcium chloride solution through No. 7 hole to mix with the liquid trehalose gel solution in the wide pipeline, and at the same time inject silicon and surfactant mixture through No. 8 hole to form a liquid trehalose gel solution in the wide pipeline of the chip Section, under the action of calcium chloride, the trehalose gel encapsulating the agarose gel microspheres will solidify into a loose and porous solid structure, forming a solid dimensional structure, and realizing the secondary solid-state encapsulation of the three microorganisms.

The solid gel containing three kinds of microorganisms is collected through the No. 9 hole and placed in the culture solution. The culture solution can penetrate into the interior of the three-dimensional gel through the porous structure of the two solid gels, and the microorganisms wrapped in it are three-dimensionally cultivated. The addition of fluorescent dyes can be used to analyze the three-dimensional community structure of microorganisms and study the communication between species in the community.

Both the chip pipeline and the microvalve are made of PDMS, and the preparation method is as follows:

(1) Preparation of silicon wafers: use Prianha solution to boil and clean single crystal silicon wafers for 30-60 minutes. ~60 minutes to cure;

(2) Micro-valve mold preparation: pour the positive photoresist on the surface of the silicon wafer, spin-coat it on a spin coater at a speed of 2000 rpm, bake at 115°C for 10-20 minutes and solidify, then slowly cool to room temperature;

(3) Ultraviolet exposure: Place a mask plate with a liquid flow channel and a micro-valve structure on the surface of the spin-coated silicon wafer, and use an ultraviolet exposure machine to perform 365nm ultraviolet irradiation treatment for 3 to 5 minutes;

(4) Silicon wafer development: use developer to clean and develop the exposed monocrystalline silicon wafer for 10 to 30 minutes, clean the surface of the silicon wafer with isopropanol solution, and dry it with nitrogen;

(5) Chip pouring: Mix the polydimethylsiloxane monomer and curing agent at a volume ratio of 5:1 and 20:1, pour them on the silicon wafer mold of the pipeline flow layer and the microvalve layer, and bake at 80°C 40-60 minutes to make it solidify;

(6) Chip sealing: After peeling off the cured PDMS and the silicon wafer mold and punching holes, use oxygen ion to treat the surface of the PDMS chip in the pipeline flow layer and the microvalve layer for 2 to 5 minutes, and then bond the two layers of chips. Combined, baked at 80°C for 8-12 hours to complete chip construction.

Claims (2)

1. a kind of microorganism three-dimensional COMMUNITY STRUCTURE chip based on Solid Double gel, it is characterised in that, the microorganism is three-dimensional COMMUNITY STRUCTURE chip is double-layer structure, including micro-valve gate layer (11) and pipeline flow layer (12)；Functionally microorganism is three-dimensional COMMUNITY STRUCTURE chip is divided into gel micro-ball wrapping unit, gel micro-ball stacked units and solid gel secondary capsulation unit；

The micro-valve gate layer (11) includes micro-valve door (5) and valve pipe containing pressure control, and micro-valve door (5) is to chip pipe Pipeline in road circulation layer carries out closure or openness control；

The pipeline flow layer (12) include three kinds of microorganisms with the mixed liquor injection hole of low melting point liquid Ago-Gel (1, 2,3), the first injection hole (4) of silicone oil and surfactant, the injection hole (6) of liquid seaweed sugar gel, the note of calcium chloride solution Enter second injection hole (8) in hole (7), silicone oil and surfactant, the collection hole (9) of solid-state microorganism three-dimensional group gel is used In the microchannel that liquid communication and gel micro-ball are wrapped up, and for the wide pipe of gel micro-ball accumulation and solid gel secondary capsulation Road；Microchannel in the chip is smooth straight groove, and depth is 40~60.0 μm, and width is 40~50 μm；Width pipe The depth in road is 40~60.0 μm, and width is 600~800 μm；

The material of the microorganism three-dimensional COMMUNITY STRUCTURE chip is polydimethylsiloxanepolymer polymer PDMS；The liquid Body circulation layer (12) and micro-valve gate layer (11) pass through 365nm ultraviolet lights 10~20 minutes respectively, corona treatment 2~5 After minute, realize that irreversible sealing-in is bonded by docking.

It is coagulated 2. being built using microorganism three-dimensional COMMUNITY STRUCTURE chip described in claim 1 and collecting microbe in solid state three-dimensional The method of plastic structure, which is characterized in that include the following steps：

The first step trains three kinds of different microorganisms with the low melting-point agarose gel of the liquid that is heated to 41 DEG C and liquid respectively After supporting base mixing, microchannel is injected by mixed liquor injection hole (1,2,3) respectively, while with feed speed identical with mixed liquor The mixed liquor of silicone oil and surfactant is injected into microchannel by the first injection hole (4), is mixed in silicone oil and surfactant The Ago-Gel microlayer model for including three kinds of microorganisms is formed under the action of liquid；Being closed micro-valve door (5) makes wide pipeline be in closing State is located at the Ago-Gel microlayer model in wide pipeline by micro-valve gate resistance gear, retention；The Ago-Gel microlayer model of accumulation passes through It is solidified as agarose gel microsphere after being stored at room temperature, realizes and a solid-state for three kinds of microorganisms is wrapped up；

Second step opens micro-valve door (5), injects trehalose gel solution by injection hole (6), trehalose gel solution is full of fine jade Clearance space between sepharose microballoon；Calcium chloride solution is injected by injection hole (7), while passing through silicone oil and surface-active The second injection hole (8) the injection silicone oil and surfactant mixed liquor of agent, the agarose gel microsphere containing microorganism is fixed Three-dimensional space position in place forms the solid-state microorganism gel micro-ball that trehalose gel is wrapped to form three-dimensional packed structures (10), it realizes and the secondary solid-state of three kinds of microorganisms is wrapped up；

Third walks, and collects solid-state microorganism gel micro-ball by collecting hole (9), is placed in culture solution, culture solution passes through two kinds The porous structure of solid gel penetrates into inside three dimensional gel, dimensional culture is carried out to wrapping up microorganism therein, by adding Add fluorescent dye microorganisms three-dimensional structure of community and group's inter-species communication.