Bacteria-embedded micro-nano structure substrate as well as preparation method and application thereof

Abstract

The invention relates to the technical field of micro-nano structure manufacturing, in particular to a bacterium-embedded micro-nano structure substrate and a preparation method and application thereof. According to the method, a metal film is evaporated on a quartz material, then a multi-beam laser interference photoetching technology acts on the surface of the metal film, and new energy distribution is formed at the interface of the metal film and the surface of a quartz material substrate through interaction between an interference light superposed light field redistributed light field and the metal film; the micro-nano structure is transferred to the surface of the quartz material under the action between light and the metal film, then the surface structure of the quartz material is changed, the metal film on the surface of the quartz material is removed through a chemical method, preparation of the micro-nano structure on the surface of the quartz material is achieved, and the micro-nano structure substrate embedded with bacteria is obtained. The preparation method disclosed by the invention is simple and convenient, required equipment is relatively cheap, the preparation cost of the micro-nano structure substrate embedded with bacteria is effectively reduced, the requirement on the flatness of a quartz material is not high, and the preparation of a large-area micro-nano structure is realized.

Description

Technical Field

[0001] The invention relates to the technical field of micro-nano structure manufacturing, in particular to a bacteria-embedded micro-nano structure substrate and a preparation method and application thereof. Background Art

[0002] Quartz glass has excellent optical properties. It not only has excellent visible light transmittance, but also transmits ultraviolet and infrared rays. Quartz glass is a good acid-resistant material. Except for hydrofluoric acid and hot phosphoric acid above 300°C, it can resist the corrosion of sulfuric acid, nitric acid, hydrochloric acid, aqua regia, neutral salts, carbon and sulfur at high temperatures. Its chemical stability is equivalent to 30 times that of acid-resistant ceramics and 150 times that of nickel-chromium alloys and ceramics. It also has the advantages of high temperature resistance, thermal shock resistance and a particularly small thermal expansion coefficient. In addition, quartz glass has excellent electrical properties. Its resistance at room temperature is equivalent to 10 times that of ordinary glass. The dielectric loss at all frequencies is very small, and the insulation withstand voltage strength is large. Quartz glass is mainly used in electric light sources, semiconductors, and new optical technologies. In biological detection, it is mainly used as a detection substrate.

[0003] Quartz has very stable physical and chemical properties. It is mainly composed of SiO2 and is widely used for its excellent optical properties. Since it has higher transmittance and greater hardness than K9 glass, quartz is more difficult to process, especially when high precision is required. The methods for obtaining micro-nano structures on the surface of quartz materials in the prior art include: preparing periodic structures on the glass surface using femtosecond laser coherence methods; based on ultraviolet nanoimprint technology, using soft molds and reverse imprinting processes to achieve the preparation of large-area periodic array microstructures on the surface of quartz materials; in addition, there are electron beam lithography, dry etching lithography, photolithography, nanoimprint lithography, and laser ablation dragged by a mask. Usually these preparation processes include two typical steps: one is the preparation of the mask structure, such as exposure photoresist or self-assembled colloidal crystals; the other is the subsequent etching of the target substrate, such as electron beam etching, reactive ion etching, or inductively coupled plasma etching under a protective sacrificial layer. However, these methods have their own problems in the preparation of templates, the selection of etchants and the selection of substrate materials. In particular, for hard-processed window materials such as quartz glass and sapphire, the etching process causes problems of low resist durability and material removal efficiency. In addition, these technical equipment are expensive, the processing steps are cumbersome, and there are high requirements for the flatness of the substrate, resulting in high costs.

[0004] Atomic force micro-nano manipulation technology has become a leading technology for observing and characterizing biological samples at the nanoscale in recent years due to its advantages of high resolution, fast scanning speed, quantitative detection of mechanical parameters, and detection under physiological conditions. However, due to the high activity of bacteria themselves, the preparation of bacterial samples under physiological conditions becomes complicated. The bacteria need to be fixed on a substrate first, and then the morphology and mechanical characteristics are characterized using an atomic force microscope. Currently, commonly used bioadhesives include gelatin, polylysine, Cell-TakTM and It enables observation of the physiological state of bacteria within hours.

[0005] Atomic force microscopy characterization of bacteria under physiological conditions mostly uses adsorption, cross-linking, covalent bonding and encapsulation methods. Since atomic force microscopy is a surface imaging technology, encapsulation methods are not suitable for characterization. The active groups used for covalent bonding and the reagents used for cross-linking will affect the physiological state of bacteria, so they are only suitable for fixing dead bacteria and are not conducive to the detection of live bacteria. For atomic force microscopy, if bacteria need to be firmly attached to the substrate without affecting their physiological activity, adsorption and embedding are the best choices; adsorption uses bioadhesives to enhance the electrostatic interaction between the negatively charged ions of the cell membrane and the substrate surface, thereby adsorbing the bacterial sample on the substrate, but bacterial aggregation is likely to occur after adsorption, and without bioadhesives, the bacteria cannot be observed due to their high activity.

[0006] Compared with the above methods in the prior art, the embedding method is currently considered to be the best method for observing bacteria using an atomic force microscope. The embedding method has the same working principle as the adsorption method, but the difference is that embedding requires a micro-nanostructured substrate to trap the bacteria in the surface structure. However, considering that the micro-nanostructured substrate obtained by the existing preparation method is expensive and not suitable for large-scale use, it is basically not used in atomic force microscopy characterization. Summary of the invention

[0007] In view of the deficiencies of the above-mentioned prior art, the present invention provides a bacteria-embedded micro-nano structure substrate and a preparation method and application thereof. The present invention first evaporates a metal film on a quartz material, then applies a multi-beam laser interference lithography technique to the surface of the metal film, and transfers the pattern formed by the laser interference light field distribution to the surface of the quartz material, and then removes the excess metal film by a chemical method, obtains a micro-nano structure on the surface of the quartz material, and obtains a bacteria-embedded micro-nano structure substrate. The preparation method of the present invention is simple, the preparation time is short, and the required equipment is cheaper than the equipment for preparing the bacteria-embedded micro-nano structure substrate in the prior art, which effectively reduces the preparation cost of the bacteria-embedded micro-nano structure substrate; in addition, the present invention does not have high requirements on the flatness of the quartz material, and can achieve the preparation of a large-area micro-nano structure.

[0008] To achieve the above object, the technical solution of the present invention is as follows:

[0009] The present invention provides a method for preparing a micro-nanostructure substrate for embedding bacteria, comprising the following steps:

[0010] A metal film is evaporated on a quartz material to obtain a substrate; a multi-beam laser interference lithography technique is used to act on the metal film of the substrate, and a periodic pattern formed by the laser interference light field distribution is transferred to the surface of the quartz material to obtain a pretreated substrate; the metal film on the surface of the pretreated substrate is cleaned and removed to obtain a bacteria-embedded micro-nanostructure substrate.

[0011] Preferably, the thickness of the metal film is 50-200nm; if the metal film is too thick, the laser energy may remain on the surface of the metal film and fail to leave a pattern on the quartz surface; if it is too thin, the laser energy may not be able to remain on the quartz surface due to the high transmittance of quartz crystal, that is, most of the laser energy penetrates the quartz and fails to leave a micro-nano structure.

[0012] Preferably, the metal of the metal film is selected from aluminum, copper, silver or gold.

[0013] Preferably, the conditions of the multi-beam laser interference lithography technology are: working wavelength 1064nm, pulse width 6ns, frequency 10Hz, and the energy of each single laser is 0.01-1J / cm 2 , pulse duration 7-9ns, exposure time 10-40s; if the time is too long, cracks will appear, which is caused by the excessive threshold; if the time is too short, the edge of the structure will stick together and the ideal structure height difference cannot be achieved, which will affect the embedding of bacteria and cause bacteria to agglomerate. The most important parameter in the conditions is the energy of each single laser.

[0014] Preferably, in the multi-beam laser interference lithography technology, the number of beams is ≥2.

[0015] Preferably, the surface of the pre-treated substrate is cleaned with an acid solution until no metal is left on the pre-treated substrate.

[0016] The present invention also protects the bacteria-embedded micro-nanostructure substrate obtained by the above-mentioned preparation method, the period range of the micro-nanostructure is 0.5-10μm, the depth of the micro-nanostructure is 0.05-2μm, and the period range is: the length from the same position of the smallest unit of the micro-nanostructure to the corresponding same position of the next smallest unit of the micro-nanostructure, taking the grid shape as an example, the distance from one edge of the grid to one edge of the same position of the next grid.

[0017] Preferably, the shapes of the micro-nano structures include cone, wedge, pyramid, inverted pyramid, semicircle, hexagonal column and cylinder, and the shapes of the micro-nano structures have no effect on atomic force microscopy detection.

[0018] The present invention also protects the use of the bacteria-embedded micro-nano structure substrate in preparing the bacteria-embedded micro-nano structure substrate @embedded bacteria material. The bacteria-embedded micro-nano structure substrate @embedded bacteria material is prepared according to the following steps:

[0019] The bacteria-embedded micro-nano structure substrate is loaded with a bioadhesive and then dried to obtain a modified bacteria-embedded micro-nano structure substrate; bacteria are embedded on the modified bacteria-embedded micro-nano structure substrate to obtain a bacteria-embedded micro-nano structure substrate@embedded bacteria material.

[0020] Preferably, the bioadhesive is selected from gelatin, polylysine, Cell-TakTM and

[0021] Preferably, the embedding method is: dropping the bacteria onto the modified bacteria-embedded micro-nanostructure substrate, and then dropping water onto the bacteria for liquid sealing.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] 1. The present invention first deposits a metal film on a quartz material, and then applies multi-beam laser interference lithography technology to the surface of the metal film, generates an interaction between the light field redistributed by superimposing the interference light field and the metal film, forms a new energy distribution at the interface between the metal film and the surface of the quartz material substrate, and utilizes the interaction between light and the metal film to transfer the periodic structure to the surface of the quartz material, thereby changing the structure of the surface of the quartz material, and then removes the metal film remaining on the surface of the quartz material by chemical methods, obtains a periodic structure on the substrate, realizes the preparation of a large-area periodic structure on the surface of the quartz material, and obtains a bacteria-embedded micro-nano structure substrate. The preparation method of the bacteria-embedded micro-nano structure substrate of the present invention is simple, has a fast processing speed, a large processing area, and simple structural parameter adjustment.

[0024] The present invention adopts multi-beam laser interference lithography technology to prepare a bacteria-embedded micro-nanostructure substrate. The multi-beam laser interference lithography technology realizes double-beam interference exposure, three-beam interference exposure, and four-beam interference exposure including multiple exposures. The device is simple, and the multi-beam interference of multiple exposures can easily change the period by changing the intersection angle of multiple light beams; the symmetry of the structure can be determined by regulating the rotation angle of the quartz material under continuous exposure. The operation is simple and easy, and the controllability is strong.

[0025] The method provided by the present invention realizes maskless processing, the processing time is only a few seconds to more than ten seconds, one-time molding, and can obtain structural types with different arrangements through flexible use of multiple light beams and multiple exposures.

[0026] 2. The present invention provides a novel method for preparing a bacteria-embedded micro-nanostructure substrate, thereby reducing the preparation cost of the bacteria-embedded micro-nanostructure substrate. The obtained bacteria-embedded micro-nanostructure substrate has a micro-nanostructure that can embed bacteria, making it convenient to use an atomic force microscope to characterize the morphology and physical properties of a single bacterium under a physiological state.

[0027] The present invention combines the two technologies of adsorption and embedding to slow down the agglomeration of bacteria and realize the characterization of bacteria under physiological state by atomic force microscopy; namely, laser interference lithography is used to manufacture the micro-nanostructure substrate for embedding bacteria required for embedding bacteria, and then bioadhesive is used for adsorption, so as to improve the adhesion efficiency and quality of bacteria without affecting the physiological state of bacteria. The problems of the covalently bound active groups and cross-linking reagents affecting the physiological state of bacteria, the inappropriate characterization of the encapsulation method, the cross-linking reagents affecting the physiological state of bacteria, and the easy agglomeration of adsorption are overcome, and thanks to the embedding and adsorption of the pure physical structure, the physiological state of bacteria will not be greatly affected.

[0028] 3. Compared with the prior art, the existing laser interference lithography technology can only produce structures of a single shape due to its own interference characteristics. The present invention uses multi-beam interference for modulation to achieve the preparation of micro-nano structures; at the same time, the present invention can not only increase the three-dimensional photon action contact surface within the two-dimensional unit plane area by preparing a single-shaped structure, but also adjust the interference parameters, use multi-angle multiple exposure and mobile multiple exposure methods, to achieve the preparation of large-area multi-period micro-nano structures with different shapes.

[0029] 4. The period range of the micro-nano structure prepared by the present invention is as low as 0.5 μm, which is half of the laser wavelength (1064 nm). In the prior art, the period range of 1 μm is generally the limit of micro-nano processing technology. Of course, a smaller value can be achieved through special processing such as femtosecond laser processing, but the large-area processing efficiency of 0.5 μm in the prior art is very low and the cost is relatively much higher. The reason why the present invention can obtain a period range of as low as 0.5 μm is that it is not limited by the diffraction limit of optical devices. The diffraction limit is the main technical bottleneck in the development of lithography technology, and it also leads to extremely high costs for optical lithography equipment. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is the optical path diagram of three-beam laser interference lithography.

[0031] Figure 2 This is a SEM morphology characterization image of the bacteria-embedded micro-nanostructure substrate of Example 1.

[0032] Figure 3The atomic force microscope morphology characterization images of the bacteria-embedded micro-nanostructure substrate @ embedded bacteria material of Example 1 and the modified substrate @ bacteria material of Comparative Example 1, the bacteria are Escherichia coli; wherein, a is the morphology characterization image of the modified substrate @ bacteria material of Comparative Example 1; b is the morphology characterization image of the bacteria-embedded micro-nanostructure substrate @ embedded bacteria material of Example 1; c is the 3D characterization image of the modified substrate @ bacteria material of Comparative Example 1; d is the 3D characterization image of the bacteria-embedded micro-nanostructure substrate @ embedded bacteria material of Example 1.

[0033] Figure 4 Characterization diagram of the adhesion force and Young's modulus of Escherichia coli in the modified substrate@bacterial material of Comparative Example 1 and the bacteria-embedded micro-nanostructure substrate@embedded bacterial material of Example 1.

[0034] Figure 5 These are surface morphology images of the micro-nano structure substrates embedded with bacteria of Examples 2 to 4; wherein, (a) is the surface morphology image of the micro-nano structure substrates embedded with bacteria of Example 2, (b) is the surface morphology image of the micro-nano structure substrates embedded with bacteria of Example 3, (c) and (d) are the surface morphology images of the micro-nano structure substrates embedded with bacteria of Example 4 at different magnifications.

[0035] Figure 6 These are the surface morphology images of the micro-nano structure substrates embedded with bacteria of Examples 5 to 7 and Comparative Example 1; wherein, (a) is the surface morphology image of the micro-nano structure substrates embedded with bacteria of Example 5, (b) is the surface morphology image of the micro-nano structure substrates embedded with bacteria of Example 6, (c) is the surface morphology image of the micro-nano structure substrates embedded with bacteria of Example 7, and (d) is the surface morphology image of the micro-nano structure substrates embedded with bacteria of Comparative Example 1. DETAILED DESCRIPTION

[0036] The specific embodiments of the present invention are described in detail below, but it should be understood that the protection scope of the present invention is not limited by the specific embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work belong to the protection scope of the present invention. The experimental methods described in the embodiments of the present invention are conventional methods unless otherwise specified.

[0037] The present invention uses quartz material as a substrate and adopts laser interference lithography technology to prepare a bacteria-embedded micro-nano structure substrate, wherein the bacteria-embedded micro-nano structure substrate forms a micro-nano structure on the surface of the quartz material. Laser interference lithography technology is a direct processing technology for materials using light absorption. Through the interference and diffraction characteristics of light, a specific light beam combination method is used to regulate the light intensity distribution in the interference field, and record it with a photosensitive material to produce a lithographic pattern. In particular, high-energy-density pulsed laser lithography in laser interference lithography technology has unique advantages in high precision, material selection, non-planar three-dimensional mobility, and transparent material internal processing. In the present invention, since there is no need to go through the preparation of the mask structure and the etching process of the target substrate, the present invention adopts laser interference lithography technology to overcome the shortcomings of the prior art femtosecond laser coherence method, ultraviolet nanoimprint technology, electron beam lithography, dry etching lithography, photolithography lithography, nanoimprint lithography, and laser ablation method dragged by a mask to obtain micro-nano structures on the surface of quartz materials.

[0038] In addition, since laser interference lithography does not require expensive projection optical systems, the area of ​​the exposure field is only limited by the aperture of the system, so it is particularly suitable for certain scenes that need to generate graphics over a large area. And for devices that cannot accurately control the flatness of the micro-nano structure on the surface of the substrate due to their large and thin size, the method of the present invention overcomes the technical defect that the flatness of the micro-nano structure on the surface of a large and thin substrate is difficult to accurately control, and has the potential to achieve λ / 2-λ / 4 high resolution.

[0039] The technical solution of the present invention is studied using embodiments and comparative examples below, and the specific research methods and results are as follows:

[0040] Example 1

[0041] The preparation method of the bacteria-embedded micro-nanostructure substrate comprises the following steps:

[0042] S1. Place a quartz glass with a size of 5 mm × 5 mm × 1 mm in an ultrasonic cleaner, and use acetone, anhydrous ethanol and deionized water to clean it at 100% power for 30 minutes, then dry it with nitrogen, and then evaporate a layer of aluminum film with a thickness of 50 nm to obtain a substrate.

[0043] S2, the substrate is placed on a Nd:YAG high-power frequency-doubled solid laser, and a multi-beam laser interference lithography technique is used on the aluminum film of the substrate. The working wavelength is 1064nm, the pulse width is 6ns, the frequency is 10Hz, and the three-beam laser interference lithography is used. The energy of each single laser is 0.01J / cm 2Laser interference is carried out under the conditions of 8 ns duration and 20 s exposure time, and the periodic pattern formed by the laser interference light field distribution is transferred to the quartz glass surface to obtain a pretreated substrate.

[0044] S3. Place the pretreated substrate in a dilute hydrochloric acid solution with a concentration of 4.5 mol / L, and then use ultrasonic cleaning to remove the aluminum film on the surface of the pretreated substrate. Then immerse it in acetone, alcohol and deionized water for ultrasonic oscillation to remove the deposits formed by laser ablation on the surface of the pretreated substrate to obtain a bacteria-embedded micro-nanostructure substrate.

[0045] The preparation method of the bacteria-embedded micro-nanostructure substrate@bacteria-embedded material comprises the following steps:

[0046] Step 1: Add 0.01% poly-lysine at 100 μL / cm 2 An amount of the modified bacteria-embedded micro-nano structure substrate is added dropwise onto the bacteria-embedded micro-nano structure substrate and dried overnight at room temperature to obtain a modified bacteria-embedded micro-nano structure substrate.

[0047] Here, 0.01% is a mass volume ratio, that is, 0.1 g of polylysine is dissolved in 1000 mL of distilled water, which is 0.01%.

[0048] Step 2: Pick the strain from the LB (Luria-Bertani) agar plate, culture it in 10 mL of LB culture medium at 37 ° C for 12 h, take 100 μL of the bacterial solution, centrifuge at 5000 rpm for 2 min, remove the supernatant, resuspend the cells in deionized water, repeat 3 times to remove all the LB culture medium, and obtain a bacterial solution.

[0049] The LB culture medium consisted of 1 g yeast, 2 g tryptone, 2 g NaCl and 200 mL deionized water, and then was sterilized at high temperature.

[0050] Step 3: Place the modified bacteria-embedded micro-nano structure substrate of step 1 in a small dish, then drop the bacterial solution of step 2 onto the modified bacteria-embedded micro-nano structure substrate, let stand for 30 minutes, and then take 1.5 mL of deionized water and add it to the small dish to ensure that the deionized water covers the modified bacteria-embedded micro-nano structure substrate and the bacteria are below the liquid phase to obtain the bacteria-embedded micro-nano structure substrate @ embedded bacteria material.

[0051] Example 2

[0052] The preparation method of the bacteria-embedded micro-nanostructure substrate comprises the following steps:

[0053] S1. Place a quartz glass with a size of 5 mm × 5 mm × 1 mm in an ultrasonic cleaner, and use acetone, anhydrous ethanol and deionized water to clean it at 100% power for 30 minutes, then dry it with nitrogen, and then evaporate a layer of aluminum film with a thickness of 100 nm to obtain a substrate.

[0054] S2, the substrate is placed on a Nd:YAG high-power frequency-doubled solid laser, and a multi-beam laser interference lithography technique is used to act on the aluminum film of the substrate. The working wavelength is 1064nm, the pulse width is 6ns, the frequency is 10Hz, and the three-beam laser interference lithography is used. The energy of each single laser is 280mJ / cm 2 Laser interference was carried out under the conditions of 7 ns duration and 40 s exposure time, and the periodic pattern formed by the laser interference light field distribution was transferred to the quartz glass surface to obtain a pretreated substrate.

[0055] S3. Place the pretreated substrate in a dilute hydrochloric acid solution with a concentration of 4.5 mol / L, and then use ultrasonic cleaning to remove the aluminum film on the surface of the pretreated substrate. Then immerse it in acetone, alcohol and deionized water for ultrasonic oscillation to remove the deposits formed by laser ablation on the surface of the pretreated substrate to obtain a bacteria-embedded micro-nanostructure substrate.

[0056] The preparation method of the bacteria-embedded micro-nanostructure substrate@bacteria-embedded material comprises the following steps:

[0057] Step 1: Add 0.01% poly-lysine at 100 μL / cm 2 An amount of the modified bacteria-embedded micro-nano structure substrate is added dropwise onto the bacteria-embedded micro-nano structure substrate and dried overnight at room temperature to obtain a modified bacteria-embedded micro-nano structure substrate.

[0058] Here, 0.01% is a mass volume ratio, that is, 0.1 g of polylysine is dissolved in 1000 mL of distilled water, which is 0.01%.

[0059] Step 2: Pick the strain from the LB (Luria-Bertani) agar plate, culture it in 10 mL of LB culture medium at 37 ° C for 12 h, take 100 μL of the bacterial solution, centrifuge at 5000 rpm for 2 min, remove the supernatant, resuspend the cells in deionized water, repeat 3 times to remove all the LB culture medium, and obtain a bacterial solution.

[0060] The LB culture medium consisted of 1 g yeast, 2 g tryptone, 2 g NaCl and 200 mL deionized water, and then was sterilized at high temperature.

[0061] Step 3: Place the modified bacteria-embedded micro-nano structure substrate of step 1 in a small dish, then drop the bacterial solution of step 2 onto the modified bacteria-embedded micro-nano structure substrate, let stand for 30 minutes, and then take 1.5 mL of deionized water and add it to the small dish to ensure that the deionized water covers the modified bacteria-embedded micro-nano structure substrate and the bacteria are below the liquid phase to obtain the bacteria-embedded micro-nano structure substrate @ embedded bacteria material.

[0062] Example 3

[0063] The preparation steps are the same as those in Example 2, except that the energy of each single laser is increased from 280 mJ / cm 2 Replaced with 420mJ / cm 2 .

[0064] Example 4

[0065] The preparation steps are the same as those in Example 2, except that the energy of each single laser is increased from 280 mJ / cm 2 Replaced with 560mJ / cm 2 .

[0066] Example 5

[0067] The preparation steps are the same as those in Example 2, except that the energy of each single laser is increased from 280 mJ / cm 2 Replaced with 20mJ / cm 2 .

[0068] Example 6

[0069] The preparation steps are the same as those in Example 2, except that the energy of each single laser is increased from 280 mJ / cm 2 Replaced with 30mJ / cm 2 .

[0070] Example 7

[0071] The preparation steps are the same as those in Example 2, except that the energy of each single laser is increased from 280 mJ / cm 2 Replaced with 40mJ / cm 2 .

[0072] Example 8

[0073] The preparation method of the bacteria-embedded micro-nanostructure substrate comprises the following steps:

[0074] S1. Place a quartz glass with a size of 5 mm × 5 mm × 1 mm in an ultrasonic cleaner, and use acetone, anhydrous ethanol and deionized water to clean it at 100% power for 30 minutes, then dry it with nitrogen, and then evaporate a copper film with a thickness of 150 nm to obtain a substrate.

[0075] S2, the substrate is placed on a Nd:YAG high-power frequency-doubled solid laser, and a multi-beam laser interference lithography technique is used on the copper film of the substrate. The working wavelength is 1064nm, the pulse width is 6ns, the frequency is 10Hz, and the three-beam laser interference lithography is used. The energy of each single laser is 0.7J / cm 2 Laser interference was carried out under the conditions of 7 ns duration and 40 s exposure time, and the periodic pattern formed by the laser interference light field distribution was transferred to the quartz glass surface to obtain a pretreated substrate.

[0076] S3. Place the pretreated substrate in a dilute hydrochloric acid solution with a concentration of 4.5 mol / L, and then use ultrasonic cleaning to remove the copper film on the surface of the pretreated substrate. Then immerse it in acetone, alcohol and deionized water for ultrasonic oscillation to remove the deposits formed by laser ablation on the surface of the pretreated substrate to obtain a bacteria-embedded micro-nanostructure substrate.

[0077] The preparation method of the bacteria-embedded micro-nanostructure substrate@bacteria-embedded material comprises the following steps:

[0078] Step 1: Add 0.01% poly-lysine at 100 μL / cm 2 An amount of the modified bacteria-embedded micro-nano structure substrate is added dropwise onto the bacteria-embedded micro-nano structure substrate and dried overnight at room temperature to obtain a modified bacteria-embedded micro-nano structure substrate.

[0079] Here, 0.01% is a mass volume ratio, that is, 0.1 g of polylysine is dissolved in 1000 mL of distilled water, which is 0.01%.

[0080] Step 2: Pick the strain from the LB (Luria-Bertani) agar plate, culture it in 10 mL of LB culture medium at 37 ° C for 12 h, take 100 μL of the bacterial solution, centrifuge at 5000 rpm for 2 min, remove the supernatant, resuspend the cells in deionized water, repeat 3 times to remove all the LB culture medium, and obtain a bacterial solution.

[0081] The LB culture medium consisted of 1 g yeast, 2 g tryptone, 2 g NaCl and 200 mL deionized water, and then was sterilized at high temperature.

[0082] Step 3: Place the modified bacteria-embedded micro-nano structure substrate of step 1 in a small dish, then drop the bacterial solution of step 2 onto the modified bacteria-embedded micro-nano structure substrate, let stand for 30 minutes, and then take 1.5 mL of deionized water and add it to the small dish to ensure that the deionized water covers the modified bacteria-embedded micro-nano structure substrate and the bacteria are below the liquid phase to obtain the bacteria-embedded micro-nano structure substrate @ embedded bacteria material.

[0083] Example 9

[0084] The method for preparing a bacteria-embedded micro-nanostructure substrate comprises the following steps:

[0085] S1. Place a quartz glass with a size of 5 mm × 5 mm × 1 mm in an ultrasonic cleaner, and use acetone, anhydrous ethanol and deionized water to clean it at 100% power for 30 minutes, then dry it with nitrogen, and then evaporate a silver film with a thickness of 200 nm to obtain a substrate.

[0086] S2. Place the substrate on a Nd:YAG high-power frequency-doubled solid-state laser and use multi-beam laser interference lithography technology to act on the silver film of the substrate. The working wavelength is 1064nm, the pulse width is 6ns, the frequency is 10Hz, and the three-beam laser interference lithography is performed. The energy of each single laser is 1J / cm 2 Laser interference was carried out under the conditions of 8 ns duration and 40 s exposure time, and the periodic pattern formed by the laser interference light field distribution was transferred to the quartz glass surface to obtain a pretreated substrate.

[0087] S3. Place the pretreated substrate in a dilute hydrochloric acid solution with a concentration of 4.5 mol / L, and then use ultrasonic cleaning to remove the silver film on the surface of the pretreated substrate. Then immerse it in acetone, alcohol and deionized water for ultrasonic oscillation in turn to remove the deposits formed by laser ablation on the surface of the pretreated substrate to obtain a bacteria-embedded micro-nanostructure substrate.

[0088] The preparation method of the bacteria-embedded micro-nanostructure substrate@bacteria-embedded material comprises the following steps:

[0089] Step 1: Add 0.01% poly-lysine at 100 μL / cm 2 An amount of the modified bacteria-embedded micro-nano structure substrate is added dropwise onto the bacteria-embedded micro-nano structure substrate and dried overnight at room temperature to obtain a modified bacteria-embedded micro-nano structure substrate.

[0090] Here, 0.01% is a mass volume ratio, that is, 0.1 g of polylysine is dissolved in 1000 mL of distilled water, which is 0.01%.

[0091] Step 2: Pick the strain from the LB (Luria-Bertani) agar plate, culture it in 10 mL of LB culture medium at 37 ° C for 12 h, take 100 μL of the bacterial solution, centrifuge at 5000 rpm for 2 min, remove the supernatant, resuspend the cells in deionized water, repeat 3 times to remove all the LB culture medium, and obtain a bacterial solution.

[0092] The LB culture medium consisted of 1 g yeast, 2 g tryptone, 2 g NaCl and 200 mL deionized water, and then was sterilized at high temperature.

[0093] Step 3: Place the modified bacteria-embedded micro-nano structure substrate of step 1 in a small dish, then drop the bacterial solution of step 2 onto the modified bacteria-embedded micro-nano structure substrate, let stand for 30 minutes, and then take 1.5 mL of deionized water and add it to the small dish to ensure that the deionized water covers the modified bacteria-embedded micro-nano structure substrate and the bacteria are below the liquid phase to obtain the bacteria-embedded micro-nano structure substrate @ embedded bacteria material.

[0094] Comparative Example 1

[0095] The preparation method of the modified substrate@bacteria material is the same as the preparation steps of Example 1, except that the bacteria are attached to an ordinary substrate, that is, the micro-nanostructure substrate in which the bacteria are embedded in Example 1 is replaced with an ordinary quartz material substrate, and includes the following steps:

[0096] S1. Place a quartz glass with a size of 5 mm × 5 mm × 1 mm in an ultrasonic cleaner and clean it with acetone, anhydrous ethanol and deionized water at 100% power for 30 min, then dry it with nitrogen, and then add 0.01% poly-lysine at 100 μL / cm 2 An amount of is dropped onto the quartz glass and dried at room temperature to obtain a modified substrate.

[0097] Here, 0.01% is a mass volume ratio, that is, 0.1 g of polylysine is dissolved in 1000 mL of distilled water, which is 0.01%.

[0098] S2. Pick the bacteria from the LB (Luria-Bertani) agar plate, culture it in 10 mL of LB culture medium at 37 ° C for 12 h, take 100 μL of the bacterial solution, centrifuge at 5000 rpm for 2 min, remove the supernatant, resuspend the cells in deionized water, repeat 3 times to remove all the LB culture medium, and obtain a bacterial solution.

[0099] The LB culture medium consisted of 1 g yeast, 2 g tryptone, 2 g NaCl and 200 mL water, and then was sterilized at high temperature.

[0100] S3. Place the modified substrate of step S1 in a small dish, then drop the bacterial solution of step S2 onto the modified substrate, let it stand for 30 minutes, and then take 1.5 mL of deionized water and add it to the small dish to ensure that the deionized water covers the micro-nanostructure substrate embedded with the modified bacteria and the bacteria are below the liquid phase to obtain a modified substrate@bacterial material.

[0101] Comparative Example 2

[0102] The preparation steps are the same as those in Example 2, except that the thickness of the aluminum film is changed from 200 nm to 300 nm, and the energy of each single laser is increased from 280 mJ / cm 2Replaced with 50mJ / cm 2 .

[0103] Examples 1-9 of the present invention all prepared bacteria-embedded micro-nano structure substrates that can be used to embed bacteria. The bacteria-embedded micro-nano structure substrates and bacteria-embedded micro-nano structure substrates@embedded bacteria materials of Examples 1-7 are taken as examples, and compared with Comparative Examples 1 and 2. The research methods and results are as follows:

[0104] The laser interference operation of the present invention is as follows: the present invention obtains a periodic structure substrate with multiple periods and micro-nano structures, which can be directly obtained by multi-angle superposition exposure of three-beam laser interference lithography with modulation, and can also be directly obtained by single exposure of four, five, or six-beam laser interference lithography with modulation. The three-beam laser interference lithography superposition exposure means that after the first exposure is completed, the sample is rotated by a certain angle (greater than 0° and less than 90°) or moved a certain distance by a rotating table and a displacement table for the second exposure, and after the second exposure is completed, it is moved by a certain angle or distance for the third exposure, and so on; two exposure superpositions or multiple exposure superpositions can be performed to obtain the required graphic structure, and the angle of each rotation is 0°-90°. In addition, the incident angles of the interference beams are the same or different, the spatial angles between adjacent interference beams are the same or different, and the energy of each interference beam is the same or different. In the substrate with micro-nano structure obtained in this way, its periodic structure can be any single shape structure of cone, wedge, pyramid, inverted pyramid, semicircle, hexagonal column, and cylinder, or it can be a structure spliced ​​with two or more shapes.

[0105] Figure 1 The optical path design diagram of three-beam laser interference lithography. The light is output from the laser, split into three beams by beam splitters B1 and B2, refracted by reflectors M1, M2 and M3 and converged at a point on the metal film surface of the substrate, causing interference. The polarization state and energy of the three beams can be adjusted and controlled by half-wave plates H1, H2, H3 and polarizers P1, P2, P3.

[0106] Multi-beam laser interference lithography technology is used to directly act on the surface of a quartz glass substrate coated with a metal film, and the periodic pattern formed by the laser interference light field distribution is transferred to the surface of the quartz material. The excess metal film is removed by a chemical cleaning method to obtain a periodic structure on the surface of the quartz material.

[0107] Figure 2This is a surface morphology diagram of the bacteria-embedded micro-nanostructure substrate obtained in Example 1 of the present invention. It can be seen from the figure that the surface of the bacteria-embedded micro-nanostructure substrate is composed of densely arranged grid structures, and the size of the grid structure is 6μm×3μm. The grid structure forms a unique micro-nanostructure, and the micro-nanostructure in the bacteria-embedded micro-nanostructure substrate is utilized to trap the bacteria on the surface of the bacteria-embedded micro-nanostructure substrate, thereby achieving the embedding of the bacteria.

[0108] Figure 3 In order to use atomic force microscopy to characterize the morphology of Escherichia coli on the modified substrate @ bacterial material of comparative example 1 and the bacteria-embedded micro-nanostructure substrate @ embedded bacterial material of embodiment 1, the D needle of SNL-W (elastic coefficient is 0.06N / m) was used for characterization in QI (Quality imaging) mode, and the scanning size was 128×128 pixels. Figure 3 The results show that bacteria on ordinary substrates without micro-nano structures are prone to agglomeration, making it difficult to characterize individual bacteria; while on micro-nanostructure substrates with bacteria embedded in them, the bacteria are separated by the micro-nanostructures and cannot agglomerate, making it easy to characterize individual bacteria.

[0109] Figure 4 The graphs are characterizations of the adhesion force and Young's modulus of Escherichia coli in the modified substrate@bacterial material of Comparative Example 1 and the bacteria-embedded micro-nanostructure substrate@embedded bacterial material of Example 1. Figure 4 The results showed that the bacteria had stronger adhesion to the surface of the bacteria-embedded micro-nanostructure substrate, and the adhesion showed that there was a significant difference in adhesion between the bacteria on the surface of the bacteria-embedded micro-nanostructure substrate and the bacteria on the surface of the ordinary substrate; there was no significant difference in Young's modulus between the bacteria on the surface of the bacteria-embedded micro-nanostructure substrate and the bacteria on the surface of the ordinary substrate. Young's modulus is one of the indicators to characterize the health of bacteria, indicating that physical encapsulation and physical adsorption have no effect on the physiological activity of the bacteria.

[0110] Figure 5 The surface morphology of the micro-nanostructure substrates embedded with bacteria in Example 2, Example 3, and Example 4 is shown. Figure 5 The results show that bacteria-embedded micro-nanostructure substrates can be obtained under different laser energies.

[0111] Figure 6 The surface morphology of the bacteria-embedded micro-nanostructure substrates of Example 5, Example 6, Example 7 and Comparative Example 1 show that, under the same thickness of the Al film, the shape of the micro-nanostructure formed by changing the energy of a single beam of light is also different; when the energy of the light beam becomes larger, the thickness of the film is increased to 300nm, and the structure will appear blurred without a clear outline.

[0112] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. Preparation method of bacteria-embedded micro-nanostructure substrate, It is characterized in that The steps include: Vapor-depositing a metal film on a quartz material to obtain a substrate; The multi-beam laser interference lithography technology is used to act on the metal film of the substrate, and the pattern formed by the laser interference light field distribution is transferred to the surface of the quartz material to obtain a pre-treated substrate; The metal film on the surface of the pretreated substrate is cleaned and removed to obtain a bacteria-embedded micro-nanostructure substrate.

2. The method for preparing the bacteria-embedded micro-nanostructure substrate according to claim 1, It is characterized in that The thickness of the metal film is 50-200nm.

3. The method for preparing the bacteria-embedded micro-nanostructure substrate according to claim 1, It is characterized in that The metal of the metal film is selected from aluminum, copper, silver or gold.

4. The method for preparing the bacteria-embedded micro-nanostructure substrate according to claim 1, It is characterized in that The conditions for multi-beam laser interference lithography are: working wavelength 1064nm, pulse width 6ns, frequency 10Hz, and the energy of each single laser is 0.01-1J / cm 2 , pulse duration 7-9ns, exposure time 10-40s.

5. The method for preparing the bacteria-embedded micro-nanostructure substrate according to claim 1, It is characterized in that In multi-beam laser interference lithography technology, the number of beams is ≥ 2.

6. The method for preparing the bacteria-embedded micro-nanostructure substrate according to claim 1, It is characterized in that The surface of the pre-treated substrate is cleaned with an acid solution until no metal is left on the pre-treated substrate.

7. A bacteria-embedded micro-nanostructure substrate prepared by the preparation method according to any one of claims 1 to 6, It is characterized in that The period range of the micro-nano structure is 0.5-10 μm, and the depth of the micro-nano structure is 0.05-2 μm.

8. The bacteria-embedded micro-nanostructure substrate according to claim 7, It is characterized in that The shapes of the micro-nano structures include cone, wedge, pyramid, inverted pyramid, semicircle, hexagonal column and cylinder.

9. Use of the bacteria-embedded micro-nano structure substrate according to claim 7 in preparing bacteria-embedded micro-nano structure substrate@embedded bacteria material, It is characterized in that The bacteria-embedded micro-nanostructure substrate@bacteria-embedded material is prepared according to the following steps: The bioadhesive is loaded on the bacteria-embedded micro-nano structure substrate and then dried to obtain a modified bacteria-embedded micro-nano structure substrate; Bacteria are embedded on the modified bacteria-embedded micro-nano structure substrate to obtain bacteria-embedded micro-nano structure substrate@embedded bacteria material.

10. The use according to claim 9, It is characterized in that The embedding method is: dropping the bacteria onto the modified bacteria-embedded micro-nanostructure substrate, and then using deionized water to liquid-seal the bacteria.

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