CN110903960A

CN110903960A - Preparation method of chip for measuring soil microbial chemotaxis

Info

Publication number: CN110903960A
Application number: CN201911258118.3A
Authority: CN
Inventors: 吴金水; 葛体达; 王东东; 祝贞科; 魏晓梦; 袁红朝
Original assignee: Institute of Subtropical Agriculture of CAS
Current assignee: Institute of Subtropical Agriculture of CAS
Priority date: 2019-12-10
Filing date: 2019-12-10
Publication date: 2020-03-24
Anticipated expiration: 2039-12-10
Also published as: CN110903960B

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 micro-chamber 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

Preparation method of chip for measuring soil microbial chemotaxis

Technical Field

The invention belongs to the technical field of biochemistry and environment, and particularly relates to a preparation method of a chip for measuring soil microbial chemotaxis.

Background

The soil is the largest biological resource bank on the earth, and a special pore structure bears a large number of microbial communities with extremely rich diversity, and plays an important role in the biogeochemical cycles of soil organic matter decomposition, nutrient release, energy transfer and the like. The soil has certain ion concentration and nutrient content suitable for the growth and propagation of microorganisms, and the microorganisms can sense chemical substances in the surrounding environment and directionally move along the concentration gradient of the chemical substances, and the property is called chemotaxis. The chemotaxis of the microorganism is a basic attribute of the survival of the microorganism adapting to the environmental change, can help the microorganism to better obtain the conditions which are beneficial to the microorganism in the external environment, and more quickly obtain the carbon source or the energy required by the microorganism, or when the microorganism feels the stimulation of harmful substances in the environment, the microorganism is far away from the harmful substances and avoids the adverse effect of the environment, and the chemotaxis of the microorganism is an important ecological factor for the colonization of the microorganism in the specific environment. The chemotaxis of microorganisms in soil is ubiquitous due to the uneven distribution of nutrients, heterologous pollutants, water conditions and the like, and chemotactic microorganisms participate in the regulation of the diversity of soil microorganisms and the circulation and distribution of soil nutrients and the like by utilizing a carbon source and a nitrogen source in the environment for metabolism, and have significance in the research of soil pollution prevention, soil microorganism remediation and the like.

The traditional methods for studying chemotaxis of microorganisms are many and have advantages and disadvantages. Capillary detection, group plate detection and other methods can perform chemotaxis qualitative analysis on microorganisms, but can not realize quantitative detection; the motile cell automatic tracking detection method and the thrombocyte detection method can track and observe the movement of a single cell, and provide a method for researching a bacterial chemotaxis mechanism and a theoretical model, but the motile cell automatic tracking detection method can only track one cell at a time and needs highly complex data recording equipment, and the thrombocyte detection method cannot react to a concentration gradient. This makes quantitative analysis of bacterial chemotaxis and accurate characterization of bacterial movement difficult to achieve using traditional detection methods.

Microfluidic chips are a relatively new detection technology emerging today, and have great advantages in detecting chemotaxis of microorganisms as a means of micromachining and micromanipulation. First, microfluidic chips, combined with high-level automated manipulation techniques, can control not only the configuration of the microfluidic chip channels, but also the flow state of the internal fluid, providing a more precisely controllable method of concentration gradients of the chemotactic agent. And secondly, the micro-channel of the micro-fluidic chip has small size and high transparency, so that the response of the microorganism to the concentration gradient can be observed conveniently through a microscope. Cell counting is carried out by a microscopic camera system and an image analysis technology, so that the microbial quantity can be accurately counted, and the chemotactic response of bacteria individuals can be directly observed. In a word, the chemical gradient environment based on accurate micro-fluidic control brings a brand new idea for researching the influence of chemical signals on the dynamics and functions of microorganisms, has the advantages of being more microscopic, controllable and visualized, and is incomparable to the traditional means. However, the current research mainly focuses on the common single strains which are easy to culture, such as escherichia coli and pseudomonas aeruginosa, and more focuses on the concentration gradient regulation of a chemotactic agent, and other factors of the microenvironment where the microorganism is located are lack of regulation, so that the behaviors of the microorganism in the real complex environment cannot be reflected. Therefore, the construction of the in-situ controllable visualization-based soil microorganism chemotaxis quantitative determination platform is of great significance to understanding the spatial distribution of soil microorganisms, the formation of plant rhizosphere microorganism hot areas and the utilization of microorganism treatment environment.

Disclosure of Invention

In view of the above, the present invention provides a method for preparing a chip for measuring soil microbial chemotaxis, which can measure soil microbial chemotaxis in situ, quantitatively and visually.

In order to achieve the above purpose, the invention provides the following technical scheme:

the invention provides a preparation method of a chip for measuring soil microbial chemotaxis, which comprises 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, and then drying and hardening to obtain a male die;

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.

Preferably, every two microchambers of step 1) are connected by one channel to obtain a connecting microchamber, and every two connecting microchambers are connected by one channel to one mixing region.

Preferably, 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.

Preferably, the photoresist of step 2) comprises a photoresist SU-8.

Preferably, 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.

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

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

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

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

Preferably, 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.

The invention provides a preparation method of a chip for measuring soil microbial chemotaxis, which comprises 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, and then drying and hardening to obtain a male die; 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.

The soil microorganism chemotaxis chip prepared by the preparation method is embedded in soil to detect the chemotaxis of the microorganisms in situ, and then the pump pushing method is combined with the fluorescent dye dyeing method to detect the microbial biomass and activity of different chemotactic agent chambers, thereby quantitatively and visually representing the selective chemotaxis of the soil in situ microorganisms to different chemotactic agents.

Drawings

FIG. 1 is a schematic view of an entire photolithographic mask;

FIG. 2 is a schematic diagram of a photolithographic mask for a single PDMS stamp;

FIG. 3 is a visual gray scale of dead bacteria and live bacteria of rhizosphere and non-rhizosphere chips in different days of culture;

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

Detailed Description

2) placing photoresist in the center of a silicon wafer, sequentially carrying out glue homogenizing and drying, aligning the photoetching mask obtained in the step 1) with the 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 (positive electrode active electron assembly), and drying and hardening to obtain a male mold;

The invention prints the chip containing the micro-chamber, the channel and the mixing area to obtain the photoetching mask. The invention preferably uses Auto CAD to draw a chip containing a micro-cavity, a channel and a mixing area to obtain a pattern, the pattern is sent to a film company, and the film company prints the pattern on a film to prepare a film with a specific pattern, namely a photoetching mask.

In the present invention, every two micro-chambers are preferably connected by one channel, resulting in a connecting micro-chamber, and every two connecting micro-chambers are preferably connected by one channel to one mixing zone. In the present invention, the diameter of the micro chamber is preferably 2mm to 3mm, the number of the micro chambers is preferably 4, a chemotactic agent can be placed in the micro chamber, the placement amount of the chemotactic agent in each micro chamber is preferably 2 to 4 μ L, the chemotactic agent is preferably a solution containing carbon element, the concentration of the carbon element in the solution is preferably 0 to 12g/L, and in the present invention, the chemotactic agent functions as: through the diffusion effect of the chemotactic agent in the micro-chamber and the channel, bacteria near the channel port feel the concentration gradient of the chemotactic agent to make corresponding chemotactic reaction, swim into the micro-chamber, and perform staining counting through the bacteria in the micro-chamber to quantitatively analyze the chemotactic reaction of the bacteria. In the present invention, the diameter of the channel is preferably 0.3mm to 0.8mm, and the number of the channels is preferably 2. In the present invention, the length of the mixing zone is preferably 6mm to 7mm, the width of the mixing zone is preferably 2mm to 4mm, the number of mixing zones is preferably 1, and the mixing zones function as: the microbes entering the soil microbe chemotaxis chip are mixed uniformly, and different micro-chambers are selected according to the chemotaxis. In the present invention, the gray area of the mask is a transparent area, and the rest is opaque. In the present invention, the photolithographic mask is a circular film with a diameter of 100 mm.

The method comprises the steps of placing photoresist in the center of a silicon wafer, sequentially carrying out photoresist homogenizing and first drying to obtain a silicon wafer with homogenized photoresist, aligning an obtained photoresist mask with an outer frame of the silicon wafer with homogenized photoresist, 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, and then drying and hardening to obtain a male mold.

In the present invention, the silicon wafer preferably has a diameter of 100mm and a thickness of 0.4 mm. In the invention, the photoresist preferably comprises the photoresist SU-8, the source of the photoresist SU-8 is not particularly limited, and the photoresist SU-8 can be obtained by adopting a conventional commercially available product, and in the invention, the usage amount of the photoresist SU-8 is preferably 1-2 ml per silicon wafer. In the present invention, the spin coating preferably comprises: firstly processing at 800 rpm-1000 rpm for 5 s-15 s, then processing at 1200 rpm-1800 rpm for 20 s-40 s, more preferably processing at 900rpm for 10s, and then processing at 1500rpm for 30 s. In the present invention, the first drying preferably includes: firstly treating at 60-70 ℃ for 10-20 min, then treating at 90-100 ℃ for 1-2 h, more preferably treating at 65 ℃ for 15min, and then treating at 95 ℃ for 2 h. In the present invention, the second drying preferably includes: firstly treating at 60-70 ℃ for 4-6 min, and then treating at 90-100 ℃ for 40-50 min. In the present invention, the time for the ultraviolet irradiation is preferably 6 to 8 seconds. After ultraviolet irradiation, the light-transmitting area, the ultraviolet ray and the photoresist are reacted and cured, and the light-tight area is not cured. In the present invention, the conditions for the hardening preferably include: firstly treating at 65 ℃ for 5min and then at 135 ℃ for 2 h.

According to the invention, a PDMS prepolymer and a curing agent are mixed and then placed on an obtained male mold, and the mixture is solidified after standing to obtain the PDMS stamp.

In the present invention, the positive mold is preferably a silicon wafer after development hardening.

In the invention, the mass ratio of the PDMS prepolymer to the curing agent is preferably 10-15: 1, more preferably 13:1, the sources of the PDMS prepolymer and the curing agent are not particularly limited, and the PDMS prepolymer and the curing agent are preferably purchased from Shanghai Korea company of China, Shanghai, and the product type is

184, the PDMS prepolymer and the curing agent are

184, the product set. In the invention, the PDMS prepolymer and the curing agent are used for preparing PDMS. According to the invention, the PDMS prepolymer and the curing agent are preferably mixed under stirring, bubbles in PDMS are removed by a vacuum pump, and then the mixture is placed on a male mold, wherein the thickness is preferably 4 mm-6 mm. In the present invention, the time for the standing is preferably 15min to 25min, and more preferably 20 min; the curing temperature is preferably 60 ℃ to 70 ℃, more preferably 65 ℃, and the curing time is preferably 2h to 4h, more preferably 3 h. In the invention, the cured male mold is preferably cut according to the edge on the graph in fig. 2 to obtain the PDMS stamp. In the present invention, the length of the PDMS stamp is preferably 10mm to 13mm, the width is preferably 6.5mm to 9.5mm, and the thickness is preferably 2mm to 4 mm.

The method comprises the following steps of carrying out surface hydrophilic modification treatment on an obtained 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 a chip for measuring soil microbial chemotaxis; the surface of the glass slide is subjected to hydrophilization modification treatment.

The method for carrying out surface hydrophilization modification treatment on the PDMS stamp and the glass slide is not particularly limited, and a conventional method can be adopted. In the present invention, the amount of the chemotactic agent placed in each micro-chamber is preferably 2 to 4 μ L, the chemotactic agent is preferably a solution containing carbon element, the carbon element is preferably in the form of glucose, the concentration of the glucose in the solution is preferably 0 to 12g/L, and in the present invention, the chemotactic agent functions as: through the diffusion effect of the chemotactic agent in the micro-chamber and the channel, bacteria near the channel port feel the concentration gradient of the chemotactic agent to make corresponding chemotactic reaction, swim into the micro-chamber, and perform staining counting through the bacteria in the micro-chamber to quantitatively analyze the chemotactic reaction of the bacteria. In the present invention, the PDMS stamp and the glass slide subjected to the hydrophilization modification treatment are bonded together by the formation of the silicon-oxygen-silicon bond. In the present invention, the size of the slide is preferably the same as the PDMS stamp.

The prepared chip for measuring soil microbial chemotaxis is preferably put into soil, after one week of culture, microorganisms in different micro chambers are dyed by a fluorescent dye through a peristaltic pump, and the microorganisms are observed under a microscope.

The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.

Example 1

The construction method of the soil microorganism chemotaxis chip comprises the steps of structural design, SU-8 template manufacture, PDMS stamp manufacture and chip construction. The method comprises the following steps:

A. structural design:

the structure was designed using an AutoCAD aided drawing tool to fabricate a chip containing microchambers (circle segment, d 2.6mm in fig. 1) and channels (connecting channel between two microchambers, d 0.5mm in fig. 1). As shown in FIG. 1, the two channels were provided with an inlet for the microorganisms from the mixing zone (length. times. width: 6.5 mm. times.3 mm) into the microchamber containing the chemotactic agent (3. mu.L of 0g/L and 10g/L glucose solution per microchamber). And sending the designed structure to a company in a PDF format for printing to obtain the photoetching mask. The gray areas in the mask are light-transmitting areas, while the rest are non-light-transmitting areas.

B. Making an SU-8 template:

dropping a proper amount of photoresist SU-8 on the center of a cleaned and dried silicon wafer, homogenizing the photoresist at 900rmin 10s and 1500rmin 30s, and placing the silicon wafer on a hot plate for pre-drying (drying at 65 ℃ for 15min, and then drying at 95 ℃ for 2 h). And aligning the mask with the outer frame of the silicon wafer subjected to glue homogenizing, and carrying out ultraviolet irradiation for 6-8 seconds. The light-transmitting area, the ultraviolet ray and the photoresist are cured by reaction, and the light-proof area is not cured. Placing the exposed silicon wafer on a flat heater for post-baking (baking at 65 ℃ for 5min, and then baking at 95 ℃ for 45 min); the photoresist which is not exposed by ultraviolet is dissolved by adopting an organic reagent PEGMEA, and then the solution is placed on a hot flat plate for hardening (firstly, the solution is baked for 5min at 65 ℃ and then baked for 2h at 135 ℃) to form an SU-8 male mold, and the SU-8 male mold is stored for later use.

C. Preparing a PDMS stamp:

PDMS prepolymer and curing agent: (

184) Mixing at a mass ratio of 13:1, fully stirring, removing bubbles in PDMS by using a vacuum pump, pouring the PDMS on an SU-8 male mold with a thickness of between 12 and 24, and standing for 20min at normal temperature. And then curing the substrate at 65 ℃ for 3h, taking out the substrate, and demolding the PDMS to cut the PDMS according to the edge of the pattern, namely the PDMS stamp for later use.

D. Chip construction:

and carrying out surface hydrophilic modification treatment on the clean chip carrying piece and the PDMS stamp by adopting oxygen plasma, adding a chemotactic agent into the corresponding micro-chamber, and then attaching the glass slide and the PDMS, wherein the PDMS and the glass slide are adhered together due to the formation of silica-silicon bonds, and the chip is manufactured.

Example 2

The soil microorganism chemotaxis chip prepared in example 1 was used to perform selective chemotaxis of non-rhizosphere soil microorganisms in rice soil rhizosphere to carbon sources.

Selecting a typical paddy field in a subtropical region, embedding the prepared chip into the soil of the rhizosphere and the non-rhizosphere of the rice, detecting the number of living microorganisms by using a pump-push method and a dead cell staining method, and discussing the selective tropism of the in-situ microorganisms of the soil under the non-rhizosphere condition of the rhizosphere. And (3) taking glucose as a carbon source, respectively adding sterilized water and 10g/L glucose solution into the micro-chamber, respectively sampling at 5d, 10d and 20d, and detecting the change of microorganisms in different micro-chambers of the chip.

As shown in the figure, FIG. 3 is a visual gray scale of the viable bacteria of the rhizosphere and non-rhizosphere chip dead bacteria in different days of culture, and FIG. 4 is a trend of the gray scale of the viable bacteria of the rhizosphere and non-rhizosphere chip dead bacteria in different days of culture. The following CK means: the number of microorganisms in the micro chamber to which the sterile aqueous solution is added. As can be seen from the results obtained in FIG. 4, the maximum gray values of viable and dead bacteria were CK-rhizosphere treatment at the time of 5d culture, and the minimum gray values were CK-non-rhizosphere treatment; when the culture is carried out for 10d, the maximum gray values of viable bacteria and dead bacteria are respectively C-rhizosphere and CK-non-rhizosphere, and the minimum gray values are respectively CK-non-rhizosphere and C-rhizosphere; and when the culture is carried out for 20d, the maximum gray values of live bacteria and dead bacteria are CK-rhizosphere and C-rhizosphere respectively, and the minimum gray values are C-rhizosphere. Probably because the carbon concentration of the exogenous glucose added in the chip is different from the carbon concentration of the rhizosphere, the inhibition effect on the growth of microorganisms exists, and the gray value of the C treatment viable bacteria is lower than that of CK treatment at 5 d; along with the increase of the culture time, the microorganisms are gradually adapted to a new living environment through self adjustment, the addition of exogenous carbon provides nutrients needed by the microorganisms, the reproduction quantity is greater than that of CK treatment, and C-rhizosphere treatment carbon provides more sufficient carbon, so that the gray value of viable bacteria treated by C is greater than that of CK treatment, and the gray value of dead bacteria treated by C is smaller than that of C-non-rhizosphere at 10 d; at the 20d, due to the limited addition of the exogenous carbon, the improvement of carbon nutrient elements is less than the requirement of microorganism growth, and the nutrition deficiency of the C-non-rhizosphere treatment is more obvious, so that the gray value of live bacteria of the C-treatment is less than that of CK treatment, and the gray value of dead bacteria of the C-non-rhizosphere treatment is greater than that of C-rhizosphere.

Claims

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

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, and drying and hardening to obtain a male die;

2. The preparation method according to claim 1, wherein step 1) every two micro-chambers are connected by one channel to obtain a connecting micro-chamber, and every two connecting micro-chambers are connected by one channel to one mixing zone.

3. The production method according to claim 1 or 2, wherein the diameter of the micro chamber is 2mm to 3 mm;

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

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

5. 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.

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

7. The preparation method according to claim 1, wherein the ultraviolet irradiation time in the step 2) is 6-8 s.

8. 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.

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

10. 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.