Preparation method of a wood-based cell culture chip with low fluorescence background

Abstract

The present invention discloses a preparation method of a wood-based cell culture chip with low fluorescence background. The wood is subjected to water immersion treatment with low-concentration alkali solution, laser cutting is performed on the water-soaked wood, the wood is crosslinked with an epoxy-based crosslinking agent, and then lignin is removed. The wood pores are filled with polysaccharide nanofibers to obtain a wood-based cell culture chip with low fluorescence background. Based on the original pores of the wood as cell culture cavities, the present invention uses laser cutting to obtain a flat surface, and then uses crosslinking means to fix the wood framework to keep it stable during the subsequent lignin removal process. Finally, the filling of polysaccharide nanofibers is used to control the depth of the nanopores, thereby controlling the number of cells that can be stored in a single pore and the chemical environment of the cells after entering the wood pores, and completing the preparation of the wood-based cell culture chip with low fluorescence background.

Description

Technical Field

[0001] The present invention belongs to the field of biomass-based functional materials, and particularly relates to a preparation method of a wood-based cell culture chip with a low fluorescence background. Background Art

[0002] During the process of cell proliferation, differentiation, and metabolism, due to the combined action of internal and external factors, even seemingly identical homologous cells will have significant differences in their morphology, function, composition, and genetic properties. The analysis results of population cells can only give an "average value" and cannot characterize the heterogeneity between cells. Therefore, single-cell analysis plays an important role in revealing the heterogeneity between cells and explaining the mechanism of intracellular life processes. Single-cell analysis can overcome the difficulties caused by cell heterogeneity in diagnosis, thereby enabling the establishment and research of target disease models. Through single-cell culture and proliferation research, it mainly focuses on the observation of cell proliferation morphology and the research of active components secreted by cells (proteins, DNA, RNA, exosomes, metabolites). Single-cell analysis is of great significance in improving biological principles, diagnosing and treating diseases, and revealing the mysteries of life.

[0003] The current mainstream single-cell analysis and culture tool is a polydimethylsiloxane (PDMS) biochip produced by "microwell arrary" in Sweden. The microstructure is as Figure 1 shown. Micron-sized pores are obtained by processing the PDMS matrix film, and its thickness and depth can be adjusted, which has been widely used in single-cell culture. Even so, this biochip still has the following three problems: 1. PDMS is a hydrophobic matrix, and there are certain problems with the immersion of cell culture medium into the pores; 2. If long-term culture is carried out, there are certain problems in replacing the culture medium in the microchannels without changing the original growth state of the cells; 3. The value of the PDMS biochip is quite high, and the price of a 4000-hole chip is about $250, which is too high. Summary of the Invention

[0004] The present invention provides a preparation method of a wood-based cell culture chip with a low fluorescence background. By virtue of the porous structure of wood itself, first, the wood skeleton is fixed using a cross-linking technique, and then lignin with a fluorescence background in the wood template is removed using a chemical oxidation method. Polysaccharide nanofibers are used to control the depth of the pores for customized design of specific cell culture.

[0005] The technical solution of the present invention is as follows:

[0006] A preparation method of a wood-based cell culture chip with low fluorescence background, which uses low-concentration alkali solution to carry out water immersion cooking treatment on wood, uses laser cutting to slice the wood after water treatment, cross-links the wood with an epoxy-based cross-linking agent, then removes lignin, repeatedly rinses the lignin-removed wood with distilled water, fills the wood pores with polysaccharide nanofibers, thereby controlling the number of cells that can be stored in a single pore and the chemical environment where the cells are located after entering the wood pores. The filled wood chips are soaked in an anhydrous ethanol solution for 24 h, then soaked in a tert-butanol solution 3 times, 12 h each time. After soaking, the wood chips are placed in a freeze dryer for freeze-drying treatment for 12 h, and finally the preparation of the wood-based cell culture chip with low fluorescence background is completed.

[0007] When the low-concentration alkali solution is used to carry out water immersion cooking treatment on wood, the low-concentration alkali solution used is sodium hydroxide solution, lithium hydroxide solution, potassium hydroxide solution, and the concentration of the low-concentration alkali solution is 1 wt% - 4 wt%, and the cooking treatment time is 6 - 12 hours.

[0008] The laser cutting is to slice the water-soaked wood. The laser cutting power is 80 - 150 watts, and the wood is cut into wood chips with a thickness of 1.5 - 2.0 cm.

[0009] The cross-linking is to place 1 - 2 g of wood, 5 - 10 mL of cross-linking agent, and 15 - 30 mL of deionized water in a small beaker, seal it with plastic wrap, mix, and place it on a shaker to shake well for 30 - 40 min. Then add 2 - 4 mL of sodium hydroxide solution with a concentration of 6.5 M, and place it in a water bath at 75 - 85 °C for 2 h.

[0010] The cross-linking agent for cross-linking the wood with an epoxy-based cross-linking agent includes epichlorohydrin, polyethylene glycol diglycidyl ether, BDDE, etc.

[0011] The method for removing lignin: Prepare 1 L of acetic acid solution with a concentration of 0.1 mol / L, add 10 g of sodium chlorite, add NaOH solution to adjust the pH value to 4.6 to obtain a delignification solution, place the cross-linked wood in the prepared solution (1 g of wood: 100 mL of delignification solution), and treat it under the condition of a water bath at 60 - 80 °C to remove lignin; among them, spruce wood and beech wood are treated for 12 h, and balsa wood is treated for 10 h.

[0012] The preparation method of the polysaccharide nanofiber dispersion: Take polysaccharide nanocellulose and add deionized water, stir evenly, and then pour the mixed solution into a blender for treatment for 1 min to obtain a polysaccharide nanofiber dispersion with a mass fraction of 0.1 - 1%.

[0013] The method for filling the pores of wood with polysaccharide nanofibers is as follows: Place the wood on the suction filter membrane of a suction filtration device, and slowly add a 0.1-1 wt% aqueous dispersion of polysaccharide nanofibers to the suction filtration device. Then, turn on the water pump switch for suction filtration. The suction filter membrane is purchased from Shanghai Xingya Purification Materials Factory, and the mixed cellulose ester microporous membrane (aqueous system) has a specification of 110 mm * 0.22 μm.

[0014] For the filling of wood pores with polysaccharide nanofibers, the polysaccharide nanofibers include cellulose nanofibers, cellulose nanocrystals, chitin nanofibers, chitin nanocrystals, etc., which can be prepared or purchased on the market.

[0015] Advantages of the present invention:

[0016] 1. The present invention utilizes the existing micron-sized pores in wood, and the pore-forming cost is low.

[0017] The present invention makes full use of the existing micron-sized pores in wood, successfully avoiding the construction work of microchannels, and greatly reducing the preparation cost, which has a great promoting effect on the analysis and culture work of single cells.

[0018] 2. The wood template of the present invention features "no fluorescence or less fluorescence".

[0019] One of the major features of the wood-based biochip prepared by the present invention is "no fluorescence or less fluorescence". After cross-linking + bleaching treatment, the wood can remove a large amount of lignin while retaining the mechanical strength of the wood chips. At the same time, it ensures that the subsequent culture and observation of single cells are not affected by the fluorescence of lignin.

[0020] 3. The wood-based chip of the present invention has the great advantage of being reusable.

[0021] There are still two major problems with polydimethylsiloxane (PDMS) biochips: 1. PDMS is a hydrophobic matrix, and there are certain problems with the immersion of cell culture medium into the pores; 2. For long-term culture, there are certain problems with replacing the culture medium in the microchannels without changing the original growth state of the cells; the biochip designed by the present invention solves the above two problems well. The wood-based biochip is filled with nanocellulose, avoiding the problem of unsmooth entry of cell culture medium into the pores. Experiments have proved that under the action of a circulating water vacuum pump and a sintered filter, the culture medium can enter the pores of the wood-based biochip very smoothly, and the chip of the present invention achieves reusable. Description of the drawings

[0022] Figure 1 It is the microscopic structure of a polydimethylsiloxane (PDMS) biochip produced by "microwell arrary" in Sweden.

[0023] Figure 2 Scanning diagram of the porous structure of wood;

[0024] Figure 3 Comparison of laser confocal microscopy images of the original wood, non-crosslinked delignified wood, and crosslinked + delignified wood slices in Example 1;

[0025] Figure 4 Scanning electron microscope image of the low-fluorescence background wood-based cell culture chip prepared in Example 1;

[0026] Figure 5 Optical image of the low-fluorescence background wood-based cell culture chip prepared in Example 1. Detailed implementation mode

[0027] Abbreviations in the text: BDDE: 1,4-butanediol diglycidyl ether; TEMPO: 2,2,6,6-tetramethylpiperidine-N-oxide.

[0028] The present invention will be further described in detail below with reference to specific examples, but it should be noted that the protection scope of the present invention is not limited by these examples. The wood raw materials used in the examples of the present invention are balsa wood, spruce wood, and beech wood.

[0029] Example 1

[0030] A preparation method of a low-fluorescence background wood-based cell culture chip, the specific steps are as follows:

[0031] (1) Take balsa wood, Figure 2 Scanning diagram of the porous structure of wood. Immerse the balsa wood in a 1wt% sodium hydroxide solution, cook for 6h, and then take it out and keep it moist for later use;

[0032] (2) Put the balsa wood cooked in step (1) into a laser cutter and cut it into 1.8 cm thick slices at a power of 80W;

[0033] (3) After the wood slices in step (2) are naturally air-dried, put 5 mL of BDDE, 15 mL of deionized water, and 1 g of dry wood chip raw material into a small beaker, seal it with plastic wrap, mix, and shake it evenly on a shaker for 30 min. Then add 2 mL of 6.5 M sodium hydroxide solution to it, and then place it in a 75°C water bath for 2 h to complete the crosslinking of the wood for later use;

[0034] (4) First, weigh 6 g of acetic acid (content > 99%) in a small beaker, then completely transfer the acetic acid to a 1000 mL volumetric flask to prepare a 1000 mL 0.1 mol / L acetic acid solution. Then pour the prepared acetic acid solution into a beaker, add 10 g of sodium chlorite solid, and stir evenly. Then adjust the pH value to 4.6 using a high-concentration NaOH solution. Finally, obtain the delignification solution for standby;

[0035] (5) Take out the wood crosslinked in step (3) and soak it in the delignification solution prepared in step (4) (1 g of wood: 100 mL of delignification solution). Perform the delignification treatment at 60 °C in a water bath for 10 h. After the treatment, repeatedly rinse the wood chips with distilled water until the delignification solution on the wood is washed clean;

[0036] (6) Preparation of cellulose nanofibers: Based on softwood fibers, prepare a nanofibrillated cellulose dispersion by TEMPO-mediated oxidation. The specific steps are as follows: Suspend 35 g of softwood fibers in 3500 mL of deionized water containing 0.84 g of TEMPO, 5.25 g of sodium bromide, and 328.05 g of sodium hypochlorite. Add 0.1 mol / L HCl and 0.1 mol / L NaOH to adjust the pH value to 10. TEMPO-mediated oxidation starts, stir at 500 rpm at room temperature, and continuously add sodium hydroxide solution to adjust the pH value during the stirring process until the pH value is 10 and constant. Then pour the mixture into a Buchner funnel and filter and wash it with deionized water until the washing liquid is neutral. Place the washed cellulose nanofibers in a beaker, seal it with plastic wrap, and store it in a refrigerator at 4 °C;

[0037] (7) Take the cellulose nanofibers prepared in step (6), add deionized water, stir evenly, and pour it into a blender for 1 min to obtain 500 g of a 0.1 wt% cellulose nanofiber dispersion;

[0038] (8) Place the lignin-removed wood block (1 cm * 1 cm * 1.8 mm) on the filter membrane of the suction filtration device, and slowly add 500 g of the 0.1 wt% cellulose nanofiber dispersion prepared in step (7) to the suction filtration device. Then turn on the water pump switch for suction filtration. The filter membrane is purchased from Shanghai Xingya Purification Materials Factory, and the specifications of the mixed cellulose ester microporous filter membrane (aqueous system) are 110 mm * 0.22 μm;

[0039] (9) Immerse the wood chips filled in step (8) in absolute ethanol for 24 h, then soak it in tert-butanol 3 times, each time for 12 h. After the immersion, place the wood chips in a freeze dryer for freeze-drying for 12 h. Finally, complete the preparation of the low-fluorescence background wood-based cell culture chip.

[0040] The untreated wood chips and the wood chips obtained by experimental treatment were respectively placed under a confocal laser microscope for observation, and Figure 3 it can be seen from the comparison that the wood chips obtained by experimental treatment showed the expected low fluorescence background effect.

[0041] The prepared wood chips were placed under a scanning electron microscope for observation, and Figure 4 it can be seen from this that nanocellulose was successfully filled into the micropores of the wood, proving the feasibility of using nanofibers for filling; the optical picture of the wood-based cell culture chip with low fluorescence background is as Figure 5 shown.

[0042] Example 2

[0043] A preparation method of a wood-based cell culture chip with low fluorescence background, the specific steps are as follows:

[0044] (1) Immerse beech wood in a 2wt% sodium hydroxide solution, cook for 8 h, and then take it out and keep it moist for later use;

[0045] (2) Put the balsa wood after cooking in step (1) into a laser cutting machine and cut it into 1.5 cm thick slices at a power of 90 W;

[0046] (3) After the wood slices in step (2) are naturally air-dried, first place 7.5 mL of BDDE, 22.5 mL of deionized water and 1.5 g of dry wood chip raw materials in a small beaker, seal it with plastic wrap, mix it, and place it on a shaker and shake it evenly for 35 min. Then add 3 mL of 6.5 M sodium hydroxide solution to it, and then place it in a water bath at 80 °C for 2 h to complete the cross-linking of the wood for later use; (4) First weigh 6 g of acetic acid (content > 99%) in a small beaker, then completely transfer the acetic acid to a 1000 mL volumetric flask, prepare 1000 mL of 0.1 mol / L acetic acid solution, then pour the prepared acetic acid solution into a beaker, and then add 10 g of sodium chlorite solid to it and stir evenly. Then adjust the pH value to 4.6 with a high-concentration NaOH solution to finally obtain a delignification solution for later use;

[0047] (5) Take out the cross-linked wood in step (3), soak it in the delignification solution prepared in step (4) (1 g of wood: 100 mL of delignification solution), and carry out the delignification treatment at 70 °C in a water bath for 12 h. After the treatment is completed, repeatedly rinse the wood chips with distilled water until the delignification solution on the wood is washed away;

[0048] (6) Preparation of chitin nanofibers: Place 30 g of shrimp shells in a 5000 mL beaker. First, treat them with 3000 mL of 4 wt% hydrochloric acid at room temperature for 12 hours or heat-treat them at 60 °C for 4 hours to remove most of the mineral salts. Then, rinse with tap water until neutral and then treat with 3000 mL of 4 wt% NaOH at room temperature for 12 hours or heat-treat at 60 °C for 4 hours to remove most of the proteins, and then rinse with tap water. Treat alternately continuously until the raw material turns white, and pay attention to removing impurities during this process. Add the purified chitin powder to a 33 wt% NaOH solution containing a small amount of NaBH4 (mass fraction 0.3%) (the ratio of chitin to the alkali solution is 1:20, that is, 20 mL of NaOH solution is used for every 1 g of absolute dry chitin), and then heat at 90 °C for 2 h, filter to remove the alkali, and then wash with 50 wt% ethanol-water to obtain chitin with amino groups. Treat the amino chitin with a cell disruptor for 10 min to prepare amino chitin nanofibers;

[0049] (7) Take the amino chitin nanofibers prepared in step (6), add deionized water, and prepare 500 g of a 0.17 wt% amino chitin nanofiber dispersion;

[0050] (8) Place the lignin-removed wood blocks (1 cm * 1 cm * 1.5 mm) from step (5) on the filter membrane of the suction filtration device, and slowly add 500 g of the 0.17 wt% amino chitin nanofiber dispersion prepared in step (7) into the suction filtration device, and then turn on the water pump switch for suction filtration. The filter membrane is purchased from Shanghai Xingya Purification Materials Factory, and the specifications of the mixed cellulose ester microporous filter membrane (aqueous system) are 110 mm * 0.22 μm;

[0051] (9) Immerse the wood chips filled in step (8) in absolute ethanol for 24 h, then soak in tert-butanol 3 times, 12 h each time. After the soaking is completed, place the wood chips in a freeze dryer for freeze-drying for 12 h, and finally complete the preparation of the low-fluorescence background wood-based cell culture chip.

[0052] Example 3

[0053] A preparation method of a low-fluorescence background wood-based cell culture chip, the specific steps are as follows:

[0054] (1) Immerse spruce wood in a 4 wt% sodium hydroxide solution, cook for 12 h, and then take it out and keep it moist for later use;

[0055] (2) Put the balsa wood after cooking in step (1) into a laser cutting machine and cut it into 2.0 cm thick slices at a power of 150 W;

[0056] (3) After the wood flakes in step (2) are naturally air-dried, first place 10 mL of BDDE, 30 mL of deionized water, and 2 g of dry wood chip raw materials in a small beaker, seal it with plastic wrap, mix, and place it on a shaker and shake well for 40 min. Then add 4 mL of 6.5 M sodium hydroxide solution to it, and then place it in a water bath at 85 °C for 2 h to complete the cross-linking of the wood for standby;

[0057] (4) First, weigh 6 g of acetic acid (content > 99%) in a small beaker, then completely transfer the acetic acid to a 1000 mL volumetric flask, prepare 1000 mL of 0.1 mol / L acetic acid solution, then pour the prepared acetic acid solution into a beaker, add 10 g of sodium chlorite solid to it and stir evenly, and then adjust the pH value to 4.6 with a high-concentration NaOH solution. Finally, obtain the delignification solution for standby;

[0058] (5) Take out the cross-linked wood in step (3) and soak it in the delignification solution prepared in step (4) (1 g of wood: 100 mL of delignification solution), and carry out the delignification treatment at 80 °C in a water bath for 12 h. After the treatment is completed, wash the wood chips with distilled water; (6) Weigh commercially available chitin nanocrystals and add water to prepare 500 g of a 0.5 wt% chitin nanocrystal dispersion;

[0059] (7) Place the delignified wood block (1 cm * 1 cm * 2.0 mm) in step (5) on the filter membrane of the suction filtration device, and slowly add 500 g of the 0.5 wt% chitin nanocrystal dispersion prepared in step (8) to the suction filtration device, and then turn on the water pump switch for suction filtration. The filter membrane is purchased from Shanghai Xingya Purification Materials Factory, and the specifications of the mixed cellulose ester microporous filter membrane (water system) are 110 mm * 0.22 μm;

[0060] (8) Immerse the wood chips filled in step (7) in absolute ethanol for 24 h, then soak them in tert-butanol 3 times, each time for 12 h. After the soaking is completed, place the wood chips in a freeze dryer for freeze-drying treatment for 12 h, and finally complete the preparation of the low-fluorescence background wood-based cell culture chip.

Claims

1. A preparation method of a low-fluorescence-background wood-based cell culture chip, characterized in that, The wood is subjected to water immersion cooking treatment with low-concentration alkali solution, sliced by laser cutting after the water immersion treatment, crosslinked with an epoxy-based crosslinking agent, then lignin is removed, the lignin-removed wood is repeatedly rinsed with distilled water, the pores of the wood are filled with a polysaccharide nanofiber dispersion, the filled wood chips are soaked in absolute ethanol for 24 h, then soaked in tert-butanol three times, 12 h each time, and after the soaking is completed, the wood chips are freeze-dried for 12 h to complete the preparation of the low-fluorescence background wood-based cell culture chip; The method for filling the pores of the wood with the polysaccharide nanofiber dispersion: Place the wood on a suction filter membrane, add a polysaccharide nanofiber dispersion with a mass fraction of 0.1-1% to the suction filtration device for suction filtration, and the specification of the suction filter membrane is 110 mm * 0.22 μm; The polysaccharide nanofibers in the polysaccharide nanofiber dispersion include cellulose nanofibers, cellulose nanocrystals, chitin nanofibers, and chitin nanocrystals.

2. The preparation method of the low-fluorescence background wood-based cell culture chip according to claim 1, wherein When the low-concentration alkali solution is used for water immersion cooking treatment of the wood, the low-concentration alkali solution is sodium hydroxide solution, lithium hydroxide solution, or potassium hydroxide solution, the mass fraction of the low-concentration alkali solution is 1%-4%, and the cooking treatment time is 6-12 hours.

3. The preparation method of the low-fluorescence background wood-based cell culture chip according to claim 1, characterized in that, The laser cutting power is 80-150 watts, and the wood is cut into wood chips with a thickness of 1.5-2.0 cm.

4. The preparation method of the low-fluorescence background wood-based cell culture chip according to claim 1, characterized in that, The crosslinking is to place 1-2 g of wood, 5-10 mL of crosslinking agent, and 15-30 mL of deionized water in a small beaker, seal it, shake it evenly on a shaker for 30-40 min, then add 2-4 mL of sodium hydroxide solution with a concentration of 6.5 mol / L, and perform a water bath treatment at 75-85 °C for 2 h.

5. The preparation method of the low-fluorescence-background wood-based cell culture chip according to claim 4, characterized in that, The crosslinking agent is epichlorohydrin, polyethylene glycol diglycidyl ether, or 1,4-butanediol diglycidyl ether.

6. The preparation method of the low-fluorescence-background wood-based cell culture chip according to claim 1, characterized in that, The method for removing lignin: Prepare 1 L of acetic acid solution with a concentration of 0.1 mol / L, add 10 g of sodium chlorite, adjust the pH value to 4.6 to obtain a delignification solution, place the crosslinked wood in the prepared delignification solution, where 1 g of wood is added to 100 mL of delignification solution, and perform a treatment under a water bath condition of 60-80 °C to remove lignin; among them, spruce wood and beech wood are treated for 12 h, and balsa wood is treated for 10 h.

7. The preparation method of the low-fluorescence background wood-based cell culture chip according to claim 1, characterized in that The preparation method of the polysaccharide nanofiber dispersion: Take polysaccharide nanofibers and add deionized water, stir evenly, and then pour the mixed solution into a wall breaker and process it for 1 min to obtain a polysaccharide nanofiber dispersion with a mass fraction of 0.1-1%.

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