CN110903960B - Preparation method of chip for measuring soil microbial chemotaxis - Google Patents

Abstract

The invention provides a preparation method of a chip for measuring soil microbial chemotaxis, which belongs to the technical field of biochemistry and environment and comprises the following steps: printing a chip containing a micro-cavity, a channel and a mixing area to obtain a photoetching mask; placing photoresist in the center of a silicon wafer, sequentially carrying out glue homogenizing and drying, aligning a photoetching mask with an outer frame of the silicon wafer with the glue homogenized, carrying out ultraviolet exposure to obtain an exposed silicon wafer, drying the exposed silicon wafer, developing by using an organic reagent PEGEMEA, drying and hardening to obtain a male die; mixing the PDMS prepolymer and a curing agent, placing the mixture on a male mold, standing and curing to obtain a PDMS stamp; and (3) carrying out surface hydrophilic modification treatment on the PDMS stamp to obtain a treatment stamp, adding a chemotactic agent into a microcavity of the treatment stamp, and attaching the treatment stamp to a glass slide to obtain the chip for measuring soil microbial chemotaxis. The chip prepared by the invention can be used for in-situ, quantitative and visual determination of soil microorganism chemotaxis.

Description

A preparation method for measuring soil microbial chemotaxis chip

technical field

The invention belongs to the technical field of biochemistry and environment, and in particular relates to a preparation method of a chip for determining the chemotaxis of soil microorganisms.

Background technique

Soil is the largest biological resource pool on the earth, and its unique pore structure carries a large number and extremely rich diversity of microbial communities, which play an important role in biogeochemical cycles such as soil organic matter decomposition, nutrient release, and energy transfer. The places suitable for the growth and reproduction of microorganisms in the soil have a certain ion concentration and nutrient content. Microorganisms can sense the chemical substances in the surrounding environment and move directionally along the concentration gradient of the chemical substances. This property is called chemotaxis. Microbial chemotaxis is a basic attribute for microorganisms to adapt to environmental changes and survive. It can help microorganisms to better obtain favorable conditions in the external environment, obtain the carbon sources or energy they need faster, or When microorganisms feel the stimulation of harmful substances in the environment, staying away from harmful substances and avoiding the adverse effects of the environment is an important ecological factor for microorganisms to settle in a specific environment. The uneven distribution of nutrients, heterogeneous pollutants, and water conditions lead to the widespread occurrence of microbial chemotaxis in soil. Chemotaxis microorganisms use carbon and nitrogen sources in the environment for metabolism, not only participating in the regulation of soil microbial diversity and soil The cycle and distribution of nutrients are of great significance in the research of soil pollution prevention and soil microbial remediation.

There are many traditional methods for studying microbial chemotaxis, each with its own advantages and disadvantages. Methods such as capillary detection and group plate detection can perform qualitative analysis on the chemotaxis of microorganisms, but cannot achieve quantitative detection; automatic tracking detection of swimming cells and tethered cell detection can track and observe the movement of individual cells, which provide a good basis for bacteria. The study of chemotaxis mechanism and theoretical model provides a method, but the automatic tracking detection method of swimming cells can only track one cell at a time, and requires highly complex data recording equipment, while the plug cell detection method cannot respond to concentration gradients. This makes quantitative analysis of bacterial chemotaxis and accurate characterization of bacterial motility difficult to achieve using traditional detection methods.

Microfluidic chip is a relatively new detection technology emerging today. As a means of micro-processing and micro-operation, it has great advantages in detecting the chemotaxis of microorganisms. First of all, the combination of microfluidic chip and high-level automatic operation technology can not only control the configuration of the microfluidic chip channel, but also precisely control the flow state of the internal fluid, providing more precise and controllable chemotaxis. method of concentration gradient. Secondly, the microchannel size of the microfluidic chip is small and the transparency is high, which is convenient for observing the response of microorganisms to the concentration gradient through a microscope. Cell counting by microscopic camera system and image analysis technology can accurately count the number of microorganisms, and can also directly observe the chemotaxis response of individual bacteria. In short, these precise and controllable chemical gradient environments based on microfluidics bring a new concept to the study of the influence of chemical signals on microbial dynamics and functions. They have more microscopic, controllable and visualized advantages, which are unmatched by traditional methods. However, the current research mainly focuses on common and easy-to-cultivate single strains such as Escherichia coli and Pseudomonas aeruginosa, and more focuses on the regulation of chemoattractant concentration gradients, lack of regulation on other elements of the microenvironment where microorganisms live, and cannot reflect Behavior of microorganisms in real complex environments. Therefore, the construction of an in-situ, controllable visualization-based quantitative assay platform for soil microbial chemotaxis is of great significance for understanding the spatial distribution of soil microorganisms, the formation of microbial hotspots in the rhizosphere of plants, and the use of microorganisms to control the environment.

Contents of the invention

In view of this, the object of the present invention is to provide a method for preparing a chip for determining the chemotaxis of soil microorganisms, which can measure the chemotaxis of soil microorganisms in situ, quantitatively and visually.

In order to realize the above-mentioned purpose of the invention, the present invention provides the following technical solutions:

The invention provides a method for preparing a chip for measuring the chemotaxis of soil microorganisms, comprising the following steps:

1) Printing a chip containing microchambers, channels and mixing regions to obtain a photolithography mask;

2) Place the photoresist in the center of the silicon wafer and then carry out uniform glue and first drying successively to obtain a silicon wafer with uniform glue. After aligning the outer frame, carry out ultraviolet exposure to obtain an exposed silicon wafer, after the second drying of the exposed silicon wafer, develop with an organic reagent PEGEMEA, and then dry the hard mold to obtain a positive mold;

3) After mixing the PDMS prepolymer and the curing agent, place it on the positive mold obtained in step 2), and solidify after standing to obtain a PDMS stamp;

4) The PDMS stamp obtained in step 3) is subjected to surface hydrophilic modification treatment to obtain a treated stamp, and after adding a chemotactic agent to the microchamber of the treated stamp, it is attached to a glass slide to obtain a soil microbial assay. Chemotaxis chip;

The surface of the slide glass is treated with hydrophilic modification.

Preferably, in the step 1) every two microchambers are connected through a channel to obtain connected microchambers, and every two connected microchambers are connected to a mixing zone through a channel.

Preferably, the microchamber has a diameter of 2 mm to 3 mm;

The diameter of the channel is 0.3 mm to 0.8 mm;

The length of the mixing zone is 6mm-7mm, and the width of the mixing zone is 2mm-4mm.

Preferably, the step 2) photoresist includes photoresist SU-8.

Preferably, the step 2) homogenizing includes: firstly treating at 800rpm-1000rpm for 5s-15s, and then treating at 1200rpm-1800rpm for 20s-40s.

Preferably, the step 2) first drying includes: firstly treating at 60°C-70°C for 10min-20min, and then treating at 90°C-100°C for 1h-2h.

Preferably, the time of the step 2) ultraviolet irradiation is 6-8s.

Preferably, the step 2) second drying includes: firstly treating at 60°C to 70°C for 4min to 6min, and then treating at 90°C to 100°C for 40min to 50min.

Preferably, the mass ratio of the step 3) PDMS prepolymer to curing agent is 10-15:1.

Preferably, the standing time of step 3) is 15 minutes to 25 minutes, the curing temperature is 60° C. to 70° C., and the curing time is 2 hours to 4 hours.

The invention provides a method for preparing a chip for measuring the chemotaxis of soil microorganisms, which comprises the following steps: 1) printing a chip containing a microchamber, a channel and a mixing area to obtain a photolithographic mask; 2) applying the photoresist After being placed in the center of the silicon wafer, perform uniform glue and first drying in sequence to obtain a uniform glue silicon wafer, align the photolithographic mask obtained in step 1) with the outer frame of the uniform glue silicon wafer, and then perform UV exposure to obtain the exposed silicon wafer, after the second drying of the exposed silicon wafer, develop with the organic reagent PEGEMEA, and then dry the hard mold to obtain the male mold; 3) After mixing the PDMS prepolymer and the curing agent, place On the positive mold obtained in step 2), solidify after standing to obtain a PDMS stamp; 4) carry out surface hydrophilic modification treatment on the PDMS stamp obtained in step 3) to obtain a treated stamp, add a chemoattractant After arriving at the microchamber where the seal is processed, it is attached to a glass slide to obtain a chip for measuring the chemotaxis of soil microorganisms; the surface of the slide glass is treated with hydrophilic modification.

The soil microbial chemotaxis chip prepared by the preparation method of the present invention is embedded in the soil to detect microbial chemotaxis in situ, and then uses the pumping method combined with the fluorescent dye staining method to detect the microbial biomass and activity of different chemotaxis chambers, and quantitative visualization Characterizing the selective chemotaxis of soil in situ microorganisms to different chemoattractants.

Description of drawings

FIG. 1 is a schematic diagram of the entire photolithography mask;

Figure 2 is a schematic diagram of a photolithographic mask of a single PDMS stamp;

Fig. 3 is the visual situation of the gray scale of the dead bacteria and live bacteria of the rhizosphere and non-rhizosphere chips at different culture days;

Figure 4 shows the gray scale change trend of dead and live bacteria in the rhizosphere and non-rhizosphere chips at different culture days.

Detailed ways

The invention provides a method for preparing a chip for measuring the chemotaxis of soil microorganisms, comprising the following steps:

1) Printing a chip containing microchambers, channels and mixing regions to obtain a photolithographic mask;

2) After placing the photoresist in the center of the silicon wafer, carry out homogenization and drying successively, align the photolithography mask obtained in the step 1) with the outer frame of the silicon wafer with the homogenization, and then carry out ultraviolet exposure to obtain Expose the silicon wafer, dry the exposed silicon wafer and develop it with the organic reagent PEGEMEA, then dry the hard mold to obtain the positive mold;

3) After mixing the PDMS prepolymer and the curing agent, place it on the positive mold obtained in step 2), and solidify after standing to obtain a PDMS stamp;

4) The PDMS stamp obtained in step 3) is subjected to surface hydrophilic modification treatment to obtain a treated stamp, and after adding a chemotactic agent to the microchamber of the treated stamp, it is attached to a glass slide to obtain a soil microbial assay. Chemotaxis chip;

The surface of the slide glass is treated with hydrophilic modification.

The invention prints the chip containing the microchamber, the channel and the mixing area to obtain a photolithography mask. The present invention preferably uses Auto CAD to draw the chip that contains microchamber, passage and mixing area, obtains pattern, and described pattern is sent to film company, pattern is printed on film by film company, makes the film of specific pattern, namely for photolithography masks.

In the present invention, every two microchambers are preferably connected through a channel to obtain connecting microchambers, and every two connecting microchambers are preferably connected to a mixing zone through a channel. In the present invention, the diameter of the microchamber is preferably 2 mm to 3 mm, and the number of the microchamber is preferably 4. Chemotactic agents can be placed in the microchamber, and chemotactic agents can be placed in each microchamber. The placement amount of the chemoattractant is preferably 2 to 4 μl. The chemoattractant preferably contains a solution of carbon, and the concentration of the carbon in the solution is preferably 0 to 12 g/L. In the present invention, the effect of the chemoattractant is Yes: through the diffusion of chemoattractants in the microchamber and channel, the bacteria near the channel mouth feel the concentration gradient of the chemoattractant, make a corresponding chemotactic response, swim into the microchamber, and pass through the microchamber. The bacteria were stained and counted to quantitatively analyze the chemotaxis response of the bacteria. In the present invention, the diameter of the channel is preferably 0.3mm-0.8mm, and the number of the channels is preferably 2. In the present invention, the length of the mixing zone is preferably 6 mm to 7 mm, the width of the mixing zone is preferably 2 mm to 4 mm, the number of the mixing zone is preferably 1, and the function of the mixing zone is: Microorganisms in the soil microbial chemotaxis chip are mixed, and different microchambers are selected according to their chemotaxis. In the present invention, the gray area of the photolithography mask is the light-transmitting area, and the rest are opaque. In the present invention, the photolithographic mask is a circular film film with a diameter of 100mm.

In the present invention, after the photoresist is placed in the center of the silicon wafer, the glue is uniformed and the first drying is carried out successively to obtain a silicon wafer with a uniform glue, and the obtained photolithography mask is aligned with the outer frame of the silicon wafer with a uniform glue UV exposure is carried out to obtain an exposed silicon wafer, and the exposed silicon wafer is subjected to second drying and then developed with an organic reagent PEGEMEA, and then dried to harden the mold to obtain a positive mold.

In the present invention, the diameter of the silicon wafer is preferably 100 mm, and the thickness is preferably 0.4 mm. In the present invention, the photoresist preferably includes photoresist SU-8. The source of the photoresist SU-8 is not particularly limited in the present invention, and conventional commercially available products can be used. In the present invention, the The usage amount of the photoresist SU-8 is preferably 1-2 ml per silicon wafer. In the present invention, the homogenization preferably includes: firstly treating at 800rpm-1000rpm for 5s-15s, then at 1200rpm-1800rpm for 20s-40s, more preferably first at 900rpm for 10s, then at 1500rpm for 30s. In the present invention, the first drying preferably includes: firstly treating at 60°C-70°C for 10min-20min, then at 90°C-100°C for 1h-2h, more preferably first at 65°C for 15min, then at 95°C for 2h. In the present invention, the second drying preferably includes: firstly treating at 60° C. to 70° C. for 4 minutes to 6 minutes, and then treating at 90° C. to 100° C. for 40 minutes to 50 minutes. In the present invention, the ultraviolet irradiation time is preferably 6s-8s. After ultraviolet irradiation, the light-transmitting area is cured by the reaction of the ultraviolet light and the photoresist, and the opaque area is not cured. In the present invention, the conditions for hardening the mold preferably include: firstly treating at 65° C. for 5 minutes and then treating at 135° C. for 2 hours.

In the present invention, after mixing the PDMS prepolymer and the curing agent, they are placed on the obtained positive mold, and then solidified after standing to obtain the PDMS stamp.

In the present invention, the positive mold is preferably a developed silicon wafer.

184即可，PDMS预聚体和固化剂为

184中的配套产品。在本发明中，所述PDMS预聚体和固化剂的作用是制备PDMS。本发明优选将PDMS预聚体和固化剂在搅拌下混合，采用真空泵将PDMS中的气泡排除后，置于阳模上，厚度优选为4mm～6mm。在本发明中，所述静置的时间优选为15min～25min，更优选为20min；所述固化的温度优选为60℃～70℃，更优选为65℃，所述固化的时间优选为2h～4h，更优选为3h。本发明优选将固化后的阳模按照图2中图形上的边进行切割，得到PDMS印章。在本发明中，所述PDMS印章的长度优选为10mm～13mm，宽度优选为6.5mm～9.5mm，厚度优选为2mm～4mm。In the present invention, the mass ratio of the PDMS prepolymer to the curing agent is preferably 10 to 15:1, more preferably 13:1, and the source of the PDMS prepolymer and the curing agent is not particularly limited in the present invention, preferably purchased Made in Yilu Trading (Shanghai) Co., Ltd., Shanghai, China, the product model is

184, the PDMS prepolymer and curing agent are

Complementary products in 184. In the present invention, the function of the PDMS prepolymer and curing agent is to prepare PDMS. In the present invention, the PDMS prepolymer and the curing agent are preferably mixed under stirring, and the air bubbles in the PDMS are removed by using a vacuum pump, and then placed on the positive mold, and the thickness is preferably 4mm-6mm. In the present invention, the standing time is preferably 15min to 25min, more preferably 20min; the curing temperature is preferably 60°C to 70°C, more preferably 65°C, and the curing time is preferably 2h to 4h, more preferably 3h. In the present invention, the cured male mold is preferably cut according to the edges on the graph in Figure 2 to obtain the PDMS stamp. In the present invention, the length of the PDMS stamp is preferably 10mm-13mm, the width is preferably 6.5mm-9.5mm, and the thickness is preferably 2mm-4mm.

In the present invention, the obtained PDMS stamp is subjected to surface hydrophilization modification treatment to obtain the treated stamp, after the chemotactic agent is added to the microchamber of the treated stamp, it is attached to a glass slide to obtain a chip for determining the chemotaxis of soil microorganisms; The surface of the slide glass is treated with hydrophilic modification.

In the present invention, the method for surface hydrophilic modification of the PDMS stamp and slide glass is not particularly limited, and conventional methods can be used. In the present invention, the placement amount of the chemoattractant in each microchamber is preferably 2-4 μl, and the chemoattractant preferably contains a solution of carbon element, and the carbon element preferably exists in the form of glucose. The concentration of glucose is preferably 0～12g/L, and in the present invention, the effect of described chemotactic agent is: by the diffusion action of chemotactic agent in microchamber and channel, the bacterium near channel mouth feels chemotactic agent The concentration gradient of the bacteria is used to make corresponding chemotactic reactions, swim into the microchamber, and count the bacteria in the microchamber to quantitatively analyze the chemotactic reactions of the bacteria. In the present invention, the hydrophilized PDMS stamp and the glass slide are bonded together through the formation of silicon-oxygen-silicon bonds. In the present invention, the size of the glass slide is preferably the same as the PDMS stamp.

In the present invention, the prepared chip for measuring the chemotaxis of soil microorganisms is preferably put into the soil, and after culturing for one week, the microorganisms in different microchambers are stained with fluorescent dyes by means of a peristaltic pump, and observed under a microscope.

The technical solutions provided by the present invention will be described in detail below in conjunction with the examples, but they should not be interpreted as limiting the protection scope of the present invention.

Example 1

The construction method of the soil microbial chemotaxis chip includes structure design, SU-8 template making, PDMS stamp making and chip construction. The steps are:

A. Structural design:

Adopt AutoCAD auxiliary drafting tool to design the structure, make the chip that contains microcavity (circle part in Fig. 1, d=2.6mm) and channel (connection pipeline between two microchambers in Fig. 1, d=0.5mm) . As shown in Figure 1, two channels are provided with one inlet, and microorganisms enter the mixture containing chemoattractant (3 μL of 0 g/L and 10 g/L glucose respectively) from the mixing zone (length × width: 6.5 mm × 3 mm). solution) microchambers. Send the designed structure to the company in PDF format for printing to obtain a photolithography mask. The gray area in the mask is the transparent area, while the rest is the opaque area.

B. Production of SU-8 template:

Drop an appropriate amount of photoresist SU-8 on the center of the cleaned and dried silicon wafer, after 900rmin 10s and 1500rmin 30s, place the silicon wafer on a hot plate and bake it (65°C for 15min, then 95°C 2h). Align the mask with the outer frame of the silicon wafer that has been evenly glued, and irradiate with ultraviolet light for 6-8 seconds. The light-transmitting area is cured by the reaction of ultraviolet rays and photoresist, while the opaque area is not cured. Place the exposed silicon wafer on a flat heater and bake (65°C for 5 minutes, and then 95°C for 45 minutes); use the organic reagent PEGMEA to dissolve the photoresist that has not been exposed to ultraviolet light, and then place it on a hot plate hard mold (First bake at 65°C for 5 minutes, then bake at 135°C for 2 hours). It is the male mold of SU-8, and it is stored for future use.

C. PDMS stamp production:

184)13:1质量比混合，充分搅拌后，采用真空泵将PDMS中气泡排除后，倒在SU-8阳模上，厚度在.12～.24之间，常温静置20min。然后在65℃下固化3h后取出，将PDMS脱模按照图形上的边切割，即PDMS印章，待用。PDMS prepolymer and curing agent (

184) Mix at a mass ratio of 13:1. After fully stirring, use a vacuum pump to remove the air bubbles in PDMS, pour it on the SU-8 male mold with a thickness between .12 and .24, and let it stand at room temperature for 20 minutes. Then take it out after curing at 65°C for 3 hours, remove the PDMS from the mold and cut it according to the edge of the graph, that is, the PDMS stamp, ready for use.

D. Chip construction:

Use oxygen plasma to modify the surface of the clean slide and PDMS seal to hydrophilize, add the chemoattractant to the corresponding microchamber, and attach the glass slide to the PDMS. At this time, the PDMS and the glass slide will Oxygen-silicon bond formation and adhesion together, the chip is completed.

Example 2

Using the soil microbial chemotaxis chip prepared in Example 1, the selective tropism of paddy soil rhizosphere and non-rhizosphere soil microorganisms to carbon sources was carried out.

Select a typical paddy field in the subtropical region, bury the prepared chip in the rice rhizosphere and non-rhizosphere soil, use the pump-push method combined with the dead and alive cell staining method to detect the number of living microorganisms, and explore the soil in situ under the rhizosphere and non-rhizosphere conditions. Selective tropism of microorganisms. Using glucose as a carbon source, sterilized water and 10g/L glucose solution were added to the microchamber, and samples were taken at 5d, 10d, and 20d to detect the changes of microorganisms in different microchambers of the chip.

As shown in the figure, Figure 3 shows the visual gray scale of dead bacteria and live bacteria in the rhizosphere and non-rhizosphere chips at different days of culture, and Figure 4 shows the gray scale of dead and live bacteria in the rhizosphere and non-rhizosphere chips at different days of culture changing trend situation. The following CK refers to: the number of microorganisms in the microchamber with sterile aqueous solution. From the results obtained in Figure 4, it can be seen that on the 5th day of culture, the maximum gray value of live bacteria and dead bacteria is CK-rhizosphere treatment, and the lowest is CK-non-rhizosphere; on the 10th day of culture, the maximum gray value of live bacteria and dead bacteria is The gray values of C-rhizosphere and CK-non-rhizosphere were respectively, and the lowest values were CK-non-rhizosphere and C-rhizosphere respectively; on the 20th day of culture, the maximum gray values of live and dead bacteria were respectively CK-rhizosphere and C-rhizosphere, the lowest is C-rhizosphere. It may be due to the difference between the carbon concentration of exogenous glucose added in the chip and the carbon concentration of the rhizosphere, which inhibited the growth of microorganisms. At 5 days, the gray value of viable bacteria in C treatment was lower than that in CK treatment; with the growth of culture time, the microbial Gradually adapt to the new living environment through self-adjustment, the addition of exogenous carbon provides the nutrients needed by the microorganisms, the reproduction number is greater than that of CK treatment, and the C-rhizosphere treatment provides more sufficient carbon. The gray value of the dead bacteria in the C-rhizosphere treatment was greater than that of the CK treatment, and the gray value of the dead bacteria in the C-rhizosphere treatment was smaller than that of the C-non-rhizosphere treatment; on the 20th day, due to the limited addition of exogenous carbon, the increase of carbon nutrients was less than the demand for microbial growth, and C -Non-rhizosphere treatment has more significant nutrient deficiency, therefore, the gray value of live bacteria in C treatment is smaller than that in CK treatment, and the gray value of dead bacteria in C-non-rhizosphere treatment is greater than that in C-rhizosphere.

Claims (9)

1. A preparation method of a chip for measuring soil microbial chemotaxis is characterized by comprising the following steps:

1) Printing a chip containing a micro-cavity, a channel and a mixing area to obtain a photoetching mask;

2) Placing photoresist in the center of a silicon wafer, sequentially carrying out glue homogenizing and first drying to obtain a silicon wafer with homogenized glue, aligning the photoetching mask obtained in the step 1) with the outer frame of the silicon wafer with homogenized glue, carrying out ultraviolet exposure to obtain an exposed silicon wafer, carrying out second drying on the exposed silicon wafer, developing by using an organic reagent PEGEMEA (positive electrode assembly film), and drying and hardening the mold to obtain a male mold;

3) Mixing the PDMS prepolymer and a curing agent, placing the mixture on the male mold obtained in the step 2), standing and curing to obtain a PDMS stamp;

4) Carrying out surface hydrophilic modification treatment on the PDMS stamp obtained in the step 3) to obtain a treatment stamp, adding a chemotactic agent into a microcavity of the treatment stamp, and attaching the treatment stamp to a glass slide to obtain a chip for measuring soil microbial chemotaxis;

the surface of the glass slide is subjected to hydrophilization modification treatment;

in the step 1), every two micro chambers are connected through one channel to obtain a connecting micro chamber, and every two connecting micro chambers are connected with one mixing region through one channel;

the diameter of the micro chamber is 2 mm-3 mm;

the diameter of the channel is 0.3 mm-0.8 mm;

the length of the mixing zone is 6 mm-7 mm, and the width of the mixing zone is 2 mm-4 mm.

2. The method according to claim 1, wherein the step 2) photoresist comprises a photoresist SU-8.

3. The method of claim 1, wherein the step 2) of homogenizing comprises: firstly processing at 800 rpm-1000 rpm for 5 s-15 s, and then processing at 1200 rpm-1800 rpm for 20 s-40 s.

4. The manufacturing method according to claim 1, wherein the step 2) first drying includes: firstly processing at 60-70 ℃ for 10-20 min, and then processing at 90-100 ℃ for 1-2 h.

5. The method according to claim 1, wherein the time of the ultraviolet irradiation in the step 2) is 6 to 8 seconds.

6. The manufacturing method according to claim 1, wherein the step 2) second drying includes: firstly treating at 60-70 ℃ for 4-6 min, and then treating at 90-100 ℃ for 40-50 min.

7. The preparation method according to claim 1, wherein the mass ratio of the PDMS prepolymer and the curing agent in the step 3) is 10-15.

8. The preparation method of claim 1, wherein the standing time in the step 3) is 15-25 min, the curing temperature is 60-70 ℃, and the curing time is 2-4 h.

9. Use of the chip obtained by the preparation method according to claim 1 for measuring chemotaxis of soil microorganisms, wherein the chemotaxis of soil microorganisms can be measured in situ, quantitatively and visually.

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