Biological membrane detection substrate based on butterfly wing biomorphic material as well as preparation method, detection method and application of biological membrane detection substrate

Abstract

The invention belongs to the field of biology, and particularly relates to a biological membrane detection substrate based on a butterfly wing biomorphic material as well as a preparation method, a detection method and application thereof, the detection substrate comprises a biomorphic material substrate and a fluorescence detection substance loaded on the biomorphic material substrate; the biomorphic material substrate is a metalized butterfly wing with an original butterfly wing structure; and the fluorescence detection substance is an FITC-HSA associated compound fluorescence molecule. Compared with the prior art, the problems that in the prior art, biofilm detection is high in cost and complex in treatment are solved, the fluorescence intensity is enhanced through the butterfly wing structure, and the detection sensitivity is improved; compared with other detection means, the operation is more convenient, the biological membrane detection substrate is renewable, and the cost of the detection method is reduced.

Description

Technical Field

[0001] The invention belongs to the biological field, and specifically relates to a biofilm detection substrate based on butterfly wing relic material, and a preparation method, a detection method and application thereof. Background Art

[0002] Bacteria generally exist in the form of a complex microbial community in nature. They often attach to a surface and are interconnected by an extracellular polymer (EPS) matrix. This form is called a biofilm. When the cell density of the biofilm reaches a threshold value, or when the nutrients in the environment are sufficient for the planktonic bacteria to multiply in large numbers, the biofilm begins to disintegrate, releasing a large number of bacteria and causing infection. In production and life, on the one hand, biofilm attachment causes corrosion of metal surfaces, causing equipment performance degradation and increased maintenance costs; on the other hand, after specific bacteria such as Pseudomonas and Bacillus proliferate in large numbers through biofilms, the proteolytic enzymes and lipolytic enzymes secreted will cause the product to deteriorate and produce a rancid odor. Escherichia coli, Salmonella, etc. may further cause large-scale food poisoning incidents, causing great economic losses and endangering the health of the people. There is a need for a method to detect bacterial biofilms to assess the degree of contamination of samples.

[0003] The existing methods for biofilm detection mainly include crystal violet staining, inverted fluorescence microscopy technology, and electron scanning microscope or laser confocal microscope observation. Among them, the crystal violet staining method is to culture the sample containing the bacterial biofilm, and then use crystal violet staining to perform semi-quantitative detection of the biofilm. However, crystal violet has certain toxicity. When testing during food production and storage, it is necessary to take samples before testing, and the technical threshold is high, requiring specialist operation. The inverted fluorescence microscope observation method requires the bacterial biofilm to be stained first, and then observed under a microscope, and the electron scanning microscope and laser confocal microscope observation methods are to treat the sample in a certain way, and then observe it under an electron scanning microscope and a laser confocal microscope respectively. However, this microscope observation method has high requirements for sample processing, high detection costs, and is difficult to promote and apply in production and life.

[0004] Therefore, it is necessary to design a low-cost and easy-to-operate biofilm detection method. Summary of the invention

[0005] The purpose of the present invention is to solve at least one of the above-mentioned problems and to provide a biofilm detection substrate based on butterfly wing relic materials and its preparation method, detection method and application, so as to solve the problems of high cost and complex processing of biofilm detection in the prior art. The present scheme utilizes the butterfly wing structure to enhance the fluorescence intensity and improve the sensitivity of detection. Compared with other detection methods, the operation of the present invention is more convenient, the biofilm detection substrate is renewable, and the cost of the detection method is reduced.

[0006] The purpose of the present invention is achieved through the following technical solutions:

[0007] The first aspect of the present invention discloses a biofilm detection substrate based on butterfly wing relic material, wherein the detection substrate comprises a relic material substrate and a fluorescent detection substance loaded on the relic material substrate;

[0008] The heritage material substrate is a metalized butterfly wing with an original butterfly wing structure;

[0009] The fluorescent detection substance is a FITC-HSA complex fluorescent molecule.

[0010] The second aspect of the present invention discloses a method for preparing a biofilm detection substrate based on butterfly wing remains material as described above, comprising the following steps:

[0011] S1: applying a metal coating on the butterfly wing template by chemical plating process, and then removing the biological template to obtain a metallized butterfly wing;

[0012] S2: Soaking the metallized butterfly wings obtained in step S1 in human serum protein solution and fluorescein isothiocyanate solution in turn to obtain the biofilm detection substrate.

[0013] Preferably, in step S1, the butterfly wing template is a pretreated butterfly wing, and the pretreatment includes demineralization treatment, amination treatment, activation treatment and reduction treatment implemented in sequence.

[0014] Preferably, the pretreatment comprises the following steps:

[0015] 1) Demineralization: Soak the butterfly wings in a dilute nitric acid solution for demineralization, let it stand for 1 to 2 hours, then take it out, wash it with distilled water and dry it in the air;

[0016] 2) Amination treatment: soak the demineralized butterfly wings obtained in step 1) in an ethylenediamine solution, let it stand for 4 to 6 hours, then take it out, wash it with distilled water and dry it in the air;

[0017] 3) Activation treatment: soak the aminated butterfly wings obtained in step 2) in an aqueous solution of chloroauric acid, let stand for 2 to 4 hours, then take them out and wash them with distilled water;

[0018] 4) Reduction treatment: The activated butterfly wings obtained in step 3) are immersed in a sodium borohydride aqueous solution to reduce the AuCl4 adsorbed on the butterfly wing template. - ions, then rinsed with deionized water and dried.

[0019] Preferably, in step S1, the chemical plating comprises: immersing the butterfly wing template in a chemical plating solution for plating, and then washing with deionized water; the metals used in the chemical plating include silver, gold, cobalt, copper, nickel, palladium and platinum.

[0020] Preferably, in step S1, the biological template is removed by dropping phosphoric acid on the butterfly wing template with a metal coating to remove the biological skeleton of the original butterfly wing, followed by washing with deionized water and drying.

[0021] Preferably, in step S2, the concentration of the human serum albumin solution is 10-30 μM, and the immersion time is 20-40 h; the concentration of the fluorescein isothiocyanate solution is 20-40 μM, and the immersion time is 8-16 h.

[0022] More preferably, in step S2, the concentration of the human serum albumin solution is 20 μM, and the immersion time is 30 h; the concentration of the fluorescein isothiocyanate solution is 30 μM, and the immersion time is 12 h.

[0023] The third aspect of the present invention discloses a detection method for a biofilm detection substrate based on butterfly wing remains material as described above, comprising the following steps:

[0024] S3: Mixing and culturing the biofilm detection substrate and the sample to be tested, removing the culture solution after the culturing and taking out the biofilm detection substrate after the culturing;

[0025] S4: the biofilm detection substrate after culture in step S3 is washed with a buffer solution, then paraformaldehyde is added and allowed to stand, then washed with a buffer solution again, then fluorescein isothiocyanate solution is added and allowed to stand, then washed with a buffer solution again;

[0026] S5: Characterize the biofilm content in the sample to be tested by detecting the biofilm detection substrate treated in step S4 through FITC molecular spectroscopy.

[0027] Preferably, the detection method comprises the following steps: mixing the biofilm detection substrate with the sample to be tested, adding the culture solution of the sample to be tested, placing it in an incubator for several hours, and then absorbing the culture solution; washing the biofilm detection substrate twice with phosphate buffer solution (PBS); soaking it in paraformaldehyde, and then washing it twice with PBS. Soaking it in fluorescein isothiocyanate (FITC) solution, and then washing it twice with PBS; detecting the FITC molecular spectrum on the biofilm detection substrate.

[0028] Preferably, the mass fraction of the paraformaldehyde is 1% to 3%, and the paraformaldehyde immersion is 10 to 20 minutes; the concentration of FITC is 0.1 to 0.3 μg / mL, and the FITC immersion time is 20 to 30 minutes; when detecting the FITC molecular spectrum, for each group of fluorescence spectra, a method of measuring multiple times at different positions and averaging is adopted to ensure the reliability of the experiment.

[0029] More preferably, the mass fraction of the paraformaldehyde is 2%, and the paraformaldehyde immersion is 20 minutes; the concentration of FITC is 0.1 μg / mL, and the FITC immersion time is 30 minutes.

[0030] Preferably, the method further comprises a step of regenerating the biofilm detection substrate:

[0031] S6: sterilizing, cleaning and drying the biofilm detection substrate characterized in step S5 to achieve regeneration of the metallized butterfly wings;

[0032] S7: Soaking the metallized butterfly wings regenerated in step S6 in human serum protein solution and fluorescein isothiocyanate solution in turn to complete the regeneration of the biofilm detection substrate.

[0033] Preferably, the sterilization adopts laser sterilization and / or ultrasonic sterilization.

[0034] The fourth aspect of the present invention discloses an application of the above-mentioned biofilm detection substrate based on butterfly wing relic material in biofilm detection, specifically, the detection of biofilm in food processing, production pipelines and equipment, clinical and other aspects.

[0035] The working principle of the present invention is:

[0036] The surface plasmon formed on the metal surface has unique surface localization and near-field enhancement properties to enhance the fluorescence signal intensity of the substance to be tested. When the incident light acts on the metal micro-nano structure to resonate, a strong local field will be generated around it, and this strong local electric field improves the excitation efficiency of the fluorescent molecules, thereby enhancing the fluorescence emission intensity, that is, local electric field enhancement. The other part is that the fluorescence emitted by the fluorescent molecules under the action of the excitation light acts on the metal micro-nano structure, and is absorbed by the metal micro-nano structure to form surface plasmon resonance. The coupling effect between the fluorescent molecules and the metal micro-nano structure shortens the life of the fluorescent molecules, increases the fluorescence quantum yield, and thus enhances the fluorescence radiation intensity, that is, radiation resonance enhancement.

[0037] The butterfly wing scales have a complex three-dimensional structure, which expands the local enhancement area of ​​the electromagnetic field along the third dimension, which is conducive to exciting more fluorescent molecules, greatly improving the excitation efficiency and radiation efficiency, and greatly enhancing the emission intensity of fluorescent molecules. The present invention uses butterfly wings as a template to make a detection substrate, improves the fluorescence radiation intensity, and uses the fluorescence spectrum of the substrate as an indicator for evaluating the content of the biofilm, which has a high sensitivity.

[0038] After the biofilm detection substrate is tested, it is sterilized by laser and ultrasonic means and cleaned with deionized water to remove the bacterial biofilm, fluorescein FITC and human blood protein molecules grown on the biofilm detection substrate without destroying the microscopic three-dimensional structure of the metal butterfly wings, thereby achieving the regeneration of the metal butterfly wings. The biofilm detection substrate can be re-prepared by subsequently soaking it in human serum albumin (HSA) solution and fluorescein isothiocyanate (FITC) solution.

[0039] Compared with the prior art, the present invention has the following beneficial effects:

[0040] The present invention utilizes the three-dimensional structure of butterfly wings to enhance the fluorescence radiation intensity of the substrate. When a single scale is used for fluorescence detection, the Au butterfly wing scale can enhance the fluorescence emission intensity of the important fluorescent dye FITC by 31 times, which is twice the enhancement effect of other similar substrates currently available. When the entire Au metal butterfly wing is used, the fluorescence emission intensity of the important fluorescent dye FITC can be enhanced by about 25.4 times before contacting the sample cells to be tested. When the sample cells to be tested have been inoculated, the fluorescence emission intensity of the important fluorescent dye FITC can be enhanced by about 24 times, which has a higher sensitivity.

[0041] The relative standard deviation of the fluorescence spectra of metal butterfly wings in different areas is small and highly repeatable. The fluorescence spectrum can be used as an indicator to evaluate the biofilm content for quantitative analysis.

[0042] After the detection, the metal butterfly wings are sterilized without destroying the three-dimensional structure of the metal butterfly wings, so that the metal butterfly wings can be regenerated and the cost of detection is reduced.

[0043] After the metal butterfly wings are made, they can be placed in the original sampling environment for culture, and then fluorescence spectroscopy detection can be performed as an indicator for evaluating the biofilm content. The operation is portable. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 Fluorescence spectra of 30 μM FITC on different substrates (λ max =545nm): (I) Au butterfly wing scale prepared in Example 1; (III) blank cover glass.

[0045] Figure 2The fluorescence spectra of FITC detected on the whole Au butterfly wing and the blank glass sheet in Example 2 correspond to curve I and curve I0 respectively. The illustration in the upper left corner is the fluorescence spectrum of FITC detected on the biofilm detection substrate that has not yet contacted the sample to be tested.

[0046] Figure 3 The bioimaging of FITC-labeled HeLa cells on Au butterfly wings and glass slides in Example 2. (a)-(c) correspond to the results on Au butterfly wing scales: (a) overlapping micrographs of bright field and dark field, green bright spots are HeLa cells, (b) dark field photo, (c) HeLa cells, fluorescence spectrum of the red marked area in (b); (e)-(g) correspond to the results on glass slides; scale length: (a), (b), (e), (f): 100 μm, (c), (g): 15 μm.

[0047] Figure 4 for Figure 3 (c) and Figure 3 (e) The average fluorescence spectrum after scanning a line area selected in the fluorescence imaging area, corresponding to curve d and curve h respectively.

[0048] Figure 5 The fluorescence spectra of 30 μM FITC in different areas on 30 groups of different substrates in Example 1 are shown.

[0049] Figure 6 The fluorescence imaging photos of the Au butterfly wing biofilm detection substrate, ordinary gold foil, and commercial gold substrate with an inverted pyramid structure on the surface under an upright fluorescence microscope in Example 9. (a) Au butterfly wing biofilm detection substrate, (b) ordinary gold foil, (c) commercial gold substrate with an inverted pyramid structure on the surface; scale length: (a), (b), (c): 100 μm. DETAILED DESCRIPTION

[0050] The present invention is described in detail below with reference to the accompanying drawings and specific embodiments.

[0051] In the following description, unless otherwise specified, the reagents used are conventional commercially available products, and the methods used are well known in the art.

[0052] A method for preparing and detecting a biofilm based on butterfly wing remains material comprises the following steps:

[0053] (1) A metal coating is applied to the original butterfly wing template using a chemical plating process; (2) A phosphoric acid solution is used to remove the biological template; (3) After the metal butterfly wing is dried, it is immersed in 20 μM human serum albumin and 30 μM fluorescein isothiocyanate, and then dried to obtain a biofilm detection substrate; (4) After the sample to be tested is co-cultured with the biofilm detection substrate, fluorescence detection is performed to detect the biofilm; (5) Laser sterilization and ultrasonic sterilization are used to achieve the regeneration of the biofilm detection substrate.

[0054] Specific:

[0055] (1) Apply metal coating on the original butterfly wing template using chemical plating process

[0056] The original butterfly wings are subjected to demineralization treatment, amination treatment, activation treatment and reduction treatment in sequence, and then immersed in a corresponding chemical plating solution for plating, and then washed with deionized water.

[0057] Among them, butterfly wings are the flight organs of butterflies. Their structure is a translucent film, usually composed of three layers. The middle layer of film is used to fix the scales on the back and abdomen. It has a multi-level and multi-dimensional hierarchical structure composed of microstructures such as ridges, ribs, and short ribs; its main component is composed of polysaccharide organic matter - chitin.

[0058] The main operation of demineralization treatment in this step is: immersing the original butterfly wing template with a fine hierarchical structure in a dilute nitric acid solution for 1 to 2 hours at room temperature, then washing it with distilled water and drying it in the air; the main operation of amination treatment is: immersing the butterfly wings in an ethylenediamine (EDA) solution, taking them out after 4 to 6 hours, washing and drying them; the main operation of activation is: immersing the butterfly wings in a chloroauric acid (HAuCl4) aqueous solution, taking them out after 2 to 4 hours, washing them; the main operation of reduction treatment is: immersing the butterfly wing template in a sodium borohydride (NaBH4) solution, then washing it with deionized water and drying it.

[0059] The metals that can be plated by chemical plating include silver, gold, cobalt, copper, nickel, palladium and platinum. Different metals should use different chemical plating solutions and corresponding reaction conditions, as shown in the following table:

[0060]

[0061]

[0062] In the embodiment of this patent, gold is used for chemical plating, and the chemical plating solution is 10g / L hydrogen tetrachloroaurate, 6g / L sodium chloride, 4g / L tartaric acid, 50g / L sodium hydroxide, 70mL / L ethanol, the reaction temperature is 20-30°C, and the reaction time is 10-20min.

[0063] In this step, the original butterfly wing template was aminated with ethylenediamine to increase the amino functional groups on the butterfly wing surface, which is beneficial for the butterfly wing to adsorb and complex Au. Ⅲ ions; surface activation can provide uniformly distributed catalytic cores for the subsequent metal chemical plating process, which plays a key role in the subsequent formation of various highly consistent continuous metal coatings.

[0064] (2) Use phosphoric acid solution to remove the original template of butterfly wings

[0065] The metallized butterfly wings after chemical plating are moved to a pre-prepared silicon wafer substrate, phosphoric acid is dripped in to remove the biological skeleton of the original butterfly wing template, and then washed with deionized water and dried in a vacuum box to obtain a functional material of a metal micro-nano structure that retains the original butterfly wing structure.

[0066] In this step, the chitosan organic template can be directly removed using a phosphoric acid solution at room temperature, which not only prevents the nano-metal particles from coarsening, melting and agglomerating and failing to maintain the fine structure of the butterfly wing template, but also avoids the template shrinkage problem caused by the original template during high-temperature sintering, which is conducive to obtaining high-quality metal micro-nano functional materials.

[0067] (3) Preparation of biofilm detection substrate

[0068] The butterfly wings were immersed in human serum albumin (HSA) solution and fluorescein isothiocyanate (FITC) solution successively, and then dried to obtain a biofilm detection substrate.

[0069] The butterfly wings were placed in a human serum albumin solution for 25 to 35 hours, and human serum albumin molecules that were not adsorbed on the surface of the metal butterfly wings were washed with deionized water after being taken out; the butterfly wings were placed in a fluorescein isothiocyanate solution for 8 to 16 hours, and FITC molecules that were not adsorbed on the sample were washed with deionized water after being taken out. The drying method was vacuum drying.

[0070] More preferably, the butterfly wings are placed in a 20 μM human serum albumin solution for 30 hours, and the human serum albumin molecules that are not adsorbed on the surface of the metal butterfly wings are washed with deionized water after being taken out; the FITC molecules that are not adsorbed on the sample are washed with deionized water after being placed in a 30 μM fluorescein isothiocyanate solution for 12 hours. After being taken out, the biofilm detection substrate is obtained after vacuum drying. In this step, human serum albumin is selected to form FITC-HSA complex fluorescent molecules with FITC, and then adsorbed to the substrate surface, mainly to avoid the direct adsorption of FITC on the metal substrate that may cause severe fluorescence quenching.

[0071] (4) Co-culturing the sample to be tested with the biofilm detection substrate and then performing fluorescence detection

[0072] Mix the biofilm detection substrate with the sample to be tested, add the culture solution of the sample to be tested, put it in an incubator and culture it for several hours, then drain the culture solution. Wash the biofilm detection substrate twice with phosphate buffer solution (PBS). Soak it in paraformaldehyde, then wash it twice with PBS. Soak it in fluorescein isothiocyanate (FITC) solution, then wash it twice with PBS. Detect the FITC molecular spectrum on the biofilm detection substrate.

[0073] Among them, the culture conditions are determined according to different samples to be tested; the mass fraction of paraformaldehyde is 1wt% to 3wt%, and the standing time is 10 to 20 minutes; the density of FITC solution is 0.1 to 0.3μg / ml, and the standing time is 20 to 30 minutes. When testing the fluorescence spectrum, for each group of fluorescence spectra, the method of measuring multiple times at different positions to obtain the average value is adopted to ensure the reliability of the experiment.

[0074] More preferably, the culture conditions are: the mass fraction of paraformaldehyde is 2 wt %, and the standing time is 20 min; the density of the FITC solution is 0.1 μg / ml, and the standing time is 30 min.

[0075] In this step, the sample cells to be tested grow evenly on the surface of the metal butterfly wings, with clear morphology and distinct surface texture, which is conducive to the observation of fluorescence imaging.

[0076] (5) Use sterilization methods to achieve regeneration of biofilm detection substrates

[0077] The used biofilm detection substrate was sterilized, washed twice with deionized water and placed in a vacuum oven for drying to achieve the regeneration of the metallized butterfly wings. The regenerated metallized butterfly wings were immersed in 20 μM HSA solution and 30 μM FITC solution in sequence again, and the biofilm detection substrate was regenerated after drying.

[0078] Among them, sterilization methods include laser sterilization and ultrasonic sterilization.

[0079] Example 1

[0080] A method for preparing and detecting a biofilm based on butterfly wing remains material comprises the following steps:

[0081] Step (1) uses a chemical plating process to apply a metal coating on the original butterfly wing template. The specific steps are as follows: select the Morpho Sulkowskyi (M. sulkowskyi) butterfly wing scale as a template, immerse it in a dilute nitric acid solution for demineralization at room temperature for 2 hours, then wash it with distilled water and place it in the air to dry for 4 hours. After drying, immerse it in an ethylenediamine solution, take it out after 4 hours, wash it, and dry it for 4 hours. Then immerse the amino butterfly wing template in a chloroauric acid aqueous solution, take it out after 2 hours, and wash it. After washing, immerse it in a sodium borohydride solution, wash it with deionized water after 2 hours, and dry it for 4 hours. Use a chemical plating solution with the ingredients of 10g / L tetrachloroauric acid hydrogen, 6g / L sodium chloride, 4g / L tartaric acid, 50g / L sodium hydroxide, and 70mL / L ethanol, immerse the activated butterfly wing template in the plating solution for plating, adjust the temperature to 25°C, let it stand for 20 minutes, and then wash it with deionized water.

[0082] Step (2) uses phosphoric acid solution to remove the original template of the butterfly wing. The specific steps are as follows: place the treated gold-plated butterfly wing under an ordinary stereo microscope, use a fine needle to move the selected single scale from the butterfly wing to a pre-prepared silicon wafer substrate, and then drip phosphoric acid on the gold-plated butterfly wing scale to remove the skeleton of the original butterfly wing scale template, then wash it with deionized water, and place it in a vacuum box to dry for 2 hours to obtain a gold micro-nanostructure functional material that retains the original butterfly wing scale structure.

[0083] Step (3) Preparation of biofilm detection substrate The specific steps are as follows: Soak the Au butterfly wing scale in 20μM human serum albumin solution, take it out after 30 hours, and wash it with deionized water. Then soak the Au butterfly wing scale in 30μM fluorescein isothiocyanate, take it out after 12 hours, and wash it with deionized water. Put it in a vacuum box and dry it for 2 hours.

[0084] Step (4) is to co-culture the sample to be tested with the biofilm detection substrate and then perform fluorescence detection. The specific steps are as follows: select human cervical cancer (HeLa) cells as the sample to be tested, mix the biofilm detection substrate with the sample to be tested, add 10% calf serum, 1×10 5 U / L penicillin and streptomycin in DMEM culture medium, put it in an incubator with 5% CO2 and a temperature of 37°C for 24 hours, and then dry the culture medium. Wash the biofilm detection substrate twice with phosphate buffer solution (PBS). Add 2wt% paraformaldehyde and soak for 20 minutes, then wash twice with PBS. Add 0.1μg / mL fluorescein isothiocyanate (FITC) solution and soak for 30 minutes, then wash twice with PBS.

[0085] Step (5) uses sterilization means to achieve the regeneration of the biofilm detection substrate. The specific steps are as follows: place the used biofilm detection substrate in an ultrasonic sterilization device, add 3M70508BMEC all-purpose high-efficiency multi-enzyme cleaning solution at a dilution ratio of 1:400, adjust the ultrasonic frequency to 24kHz, and the time is 30min. After the ultrasonication, wash it twice with deionized water and put it in a vacuum box to dry.

[0086] The FITC molecular spectrum on the biofilm detection substrate that has not yet been exposed to the sample to be tested was detected using the Renishaw invia confocal micro-Raman spectrometer of the British Renishaw Company. A blank cover glass was used as the control group. First, a certain part of the substrate was selected under the field of view of the ×10 objective lens, and then tested under the ×50 short-focus objective lens (numerical aperture: NA=0.75). The scanning range was 490-590nm, the laser wavelength was 488nm, the incident power on the substrate surface was about 1.0mW, the integration time was 10s, and a single measurement was performed. For each group of fluorescence spectra, multiple measurements at different positions were used to obtain the average value. The fluorescence spectrum characterization results are shown in Figure 2. Figure 1 As shown. Among them, substrate I is an Au scale with butterfly wing structure, and substrate III is a blank cover glass. The fluorescence signal on substrate III is very weak, while the fluorescence intensity on substrate I is very strong, which shows that the structure of butterfly wing scale has good fluorescence enhancement performance, and the enhancement factor is 31.04 times.

[0087] A laser confocal fluorescence microscope (TCS SP5-II, Leica) was used to measure the fluorescence microscopy imaging on the biofilm detection substrate that had not yet contacted the sample to be tested. During the test, the substrate surface was first found under a 10x optical microscope, and then under a 20x optical microscope, the excitation wavelength was selected as 488nm, and the excitation module with the same absorption wavelength as the FITC dye was selected. The acquisition image and spectral band were automatically set, and the bright field and fluorescence imaging images were displayed at the same time. The fluorescence imaging results also showed that the fluorescence signal of the fluorescent molecules on the Au butterfly wing scales was enhanced.

[0088] Ten Au butterfly wing scale substrates were selected, and 30 different areas were randomly selected to detect the fluorescence spectrum of FITC molecules. Figure 5 For the 30 groups of FITC fluorescence spectra, the repeatability analysis of the FITC fluorescence spectra showed that the relative standard deviation (RSD) was 6.5%, indicating that the Au butterfly wing scales had good signal repeatability.

[0089] Example 2

[0090] This embodiment is basically the same as Embodiment 1, except that in the preparation process in this embodiment, no single scale is selected from the butterfly wing, and the entire Au butterfly wing is directly moved to a pre-prepared silicon wafer substrate, phosphoric acid is dripped in, and the skeleton of the original butterfly wing scale template is removed, and the subsequent operations are the same.

[0091] A laser confocal fluorescence microscope (TCS SP5-II, Leica) was used to image the biofilm detection substrate and HeLa cells on the surface of Au butterfly wings that had not yet contacted the sample to be tested. During the test, the substrate surface was first found under a 10x optical microscope, and then under a 20x optical microscope, the excitation wavelength was selected as 488nm, and the excitation module with the same absorption wavelength as the FITC dye was selected. The acquisition image and spectral band were automatically set, and the bright field and fluorescence imaging images were displayed simultaneously.

[0092] All the pictures were taken under the same experimental conditions. Figure 2 As shown, curve I is the fluorescence detection result of the whole Au butterfly wing, curve I0 is the fluorescence detection result of the blank coverslip, and the illustration in the upper left corner is the fluorescence spectrum of FITC detected by the biofilm detection substrate that has not yet contacted the sample to be tested. It can be seen from the figure that the whole Au butterfly wing still has a strong fluorescence spectrum, and the enhancement factor is 25.4 times. Figure 3 The fluorescence imaging results of HeLa cells on Au butterfly wings and blank coverslips are shown in Figure 2. The fluorescence signal of HeLa cells on the surface of the blank coverslip is very weak, and its morphology cannot be clearly seen ( Figure 3 (e)-(g)). In contrast, the morphology of HeLa cells can be clearly observed on the Au butterfly wing substrate, and even the cytoplasm, pseudopodia, nucleus and other organelles can be distinguished ( Figure 3 (c)). Figure 4 The curves d and h in Figure 3 (c) and Figure 3 (e) The average fluorescence spectrum after scanning a line area in the fluorescence imaging area shows that the average signal intensity of FITC fluorescent molecules on the Au butterfly wing is about 120 cps, while the average signal intensity on the blank coverslip is only 5 cps, with an enhancement factor of 24 times.

[0093] Example 3

[0094] This embodiment is basically the same as the embodiment 1, except that the chemical plating solution in this embodiment is selected to have 10 g / L silver nitrate, 20 mL / L ammonium hydroxide, and 50 g / L potassium sodium tartrate, and the standing temperature is 25°C for 20 minutes. Finally, Ag butterfly wing scales that can be used for biofilm detection are obtained.

[0095] Example 4

[0096] This embodiment is basically the same as the embodiment 1, except that the chemical plating solution in this embodiment is selected to contain 30 g / L copper sulfate, 40 g / L sodium hydroxide, 120 g / L potassium sodium tartrate, and 100 ml / L formaldehyde, and the standing temperature is 25°C for 10 minutes. Finally, Cu butterfly wing scales that can be used for biofilm detection are obtained.

[0097] Example 5

[0098] This example is basically the same as Example 1, except that the chemical plating solution in this example is selected to contain 25 g / L cobalt sulfate, 25 g / L sodium succinate, and 4 g / L dimethylamine borane, and the standing temperature is 25°C for 10 minutes. Finally, Co butterfly wing scales that can be used for biofilm detection are obtained.

[0099] Example 6

[0100] This embodiment is basically the same as the embodiment 1, except that the chemical plating solution in this embodiment is selected to have 40 g / L nickel sulfate, 20 g / L sodium citrate, 10 g / L lactic acid, 2 g / L dimethylamine borane, 30 ml / L ammonium hydroxide, pH 6.8 chemical plating solution, and the standing temperature is 25°C for 20 minutes. Finally, Ni butterfly wing scales that can be used for biofilm detection are obtained.

[0101] Example 7

[0102] This embodiment is basically the same as the embodiment 1, except that the chemical plating solution in this embodiment is selected to have 10 g / L palladium chloride, 41 g / L disodium ethylenediaminetetraacetic acid, 198 ml / L ammonium hydroxide, 5.6 ml / L hydrazine hydrate, pH 10.2, and the standing temperature is 25°C for 20 minutes. Finally, Pd butterfly wing scales that can be used for biofilm detection are obtained.

[0103] Example 8

[0104] This embodiment is basically the same as embodiment 1, except that the chemical plating solution in this embodiment is selected as 4g / L chloroplatinic acid, 4g / L hydroxylamine hydrochloride, 400ml / L ammonium hydroxide, 30ml / L hydrazine hydrate, pH 11 chemical plating solution, the standing temperature is 50°C, and the time is 300 minutes. Finally, Pt butterfly wing scales that can be used for biofilm detection are obtained.

[0105] Example 9

[0106] This embodiment is basically the same as embodiment 1, except that in this embodiment, fluorescent Pseudomonas NCIMB10586 is selected as the sample to be tested, LB culture solution containing 8g / L NaCl, 10g / L peptone, and 5g / L yeast extract is added to the test tube, and the culture is placed in an incubator at a temperature of 30°C for 12 hours, and then the cultured stable period bacterial solution is taken, and the bacterial solution is diluted to a solution absorbance of 0.05 at a wavelength of 600nm, and then the bacterial solution is transferred to a 96-well plate containing an Au butterfly wing biofilm detection substrate, and then placed in an incubator at a temperature of 30°C for 96 hours. After the culture is completed, the culture solution is sucked dry, and the biofilm detection substrate is washed twice with a phosphate buffer solution (PBS). Add 2wt% paraformaldehyde and soak for 20min, then wash twice with PBS. Add 0.1μg / mL of fluorescein isothiocyanate (FITC) solution and soak for 30min, then wash twice with PBS.

[0107] An upright fluorescence microscope (Olympus BX 53) was used to image the Au butterfly wing biofilm detection substrate, with ordinary gold foil (model: Au9999) purchased from Hefei Wenghe Metal Materials Co., Ltd. and a commercial gold substrate with an inverted pyramid structure on the surface purchased from Foriin Technology (Shanghai) CO., LTD as the control group. During the test, under a 20x optical microscope, the excitation wavelength was selected as 488nm, and the excitation module with the same absorption wavelength as the FITC dye was selected, and the acquisition image and spectral band were automatically set, and the bright field and fluorescence imaging images were displayed at the same time. Figure 6 a shows the fluorescence imaging photo of the Au butterfly wing biofilm detection substrate under an upright fluorescence microscope. Figure 6 b shows the fluorescence imaging photo of ordinary gold foil under an upright fluorescence microscope. Figure 6 c shows the fluorescence imaging photo of the commercial gold substrate with an inverted pyramid structure on the surface under an upright fluorescence microscope. The fluorescence intensity of multiple images was analyzed using ImageJ image analysis software to obtain multiple unit area grayscale values, and the average grayscale value of the fluorescence intensity of the Au butterfly wing biofilm detection substrate was 117.93, the average grayscale value of the ordinary gold foil was 59.46, and the average grayscale value of the commercial gold substrate with an inverted pyramid structure on the surface was 60.13. It can be seen that the fluorescence intensity on the Au butterfly wing biofilm detection substrate is about twice that of the ordinary gold foil and the commercial gold substrate with an inverted pyramid structure on the surface.

[0108] In summary, the present invention is a method for preparing and detecting a biofilm based on butterfly wing relic materials, comprising the following steps: applying a metal coating on the original butterfly wing template using a chemical plating process; removing the biological template using a phosphoric acid solution; drying the metal butterfly wing, successively soaking it in 20μM human serum albumin and 30μM fluorescein isothiocyanate, and obtaining a biofilm detection substrate after drying; after the sample to be tested is co-cultured with the biofilm detection substrate, fluorescence detection is performed to detect the biofilm; laser and ultrasonic sterilization methods are used to achieve the regeneration of the biofilm detection substrate. The present invention utilizes the butterfly wing structure to enhance the fluorescence intensity and improve the sensitivity of the detection; compared with other detection methods, the operation of the present invention is more convenient; the biofilm detection substrate can be regenerated, which reduces the cost of the detection method.

[0109] The above description of the embodiments is to facilitate the understanding and use of the invention by those skilled in the art. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative work. Therefore, the present invention is not limited to the above embodiments, and improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the present invention should be within the scope of protection of the present invention.

Claims

1. A biofilm detection substrate based on butterfly wing remains material, characterized in that: The detection substrate comprises a legacy material substrate and a fluorescent detection substance loaded on the legacy material substrate; The heritage material substrate is a metalized butterfly wing with an original butterfly wing structure; The fluorescent detection substance is a FITC-HSA complex fluorescent molecule.

2. A method for preparing a biofilm detection substrate based on butterfly wing remains material as claimed in claim 1, characterized in that: The steps include: S1: applying a metal coating on the butterfly wing template by chemical plating process, and then removing the biological template to obtain a metallized butterfly wing; S2: Soaking the metallized butterfly wings obtained in step S1 in human serum protein solution and fluorescein isothiocyanate solution in turn to obtain the biofilm detection substrate.

3. The method for preparing a biofilm detection substrate based on butterfly wing remains material according to claim 2, characterized in that: In step S1, the butterfly wing template is a pre-treated butterfly wing, and the pre-treatment includes demineralization treatment, amination treatment, activation treatment and reduction treatment implemented in sequence.

4. The method for preparing a biofilm detection substrate based on butterfly wing remains material according to claim 3, characterized in that: The pretreatment comprises the following steps: 1) Demineralization: Soak the butterfly wings in a dilute nitric acid solution for demineralization, let it stand for 1 to 2 hours, then take it out, wash it with distilled water and dry it in the air; 2) Amination treatment: soak the demineralized butterfly wings obtained in step 1) in an ethylenediamine solution, let it stand for 4 to 6 hours, then take it out, wash it with distilled water and dry it in the air; 3) Activation treatment: soak the aminated butterfly wings obtained in step 2) in an aqueous solution of chloroauric acid, let stand for 2 to 4 hours, then take them out and wash them with distilled water; 4) Reduction treatment: The activated butterfly wings obtained in step 3) are immersed in a sodium borohydride aqueous solution to reduce the AuCl4 adsorbed on the butterfly wing template. - ions, then rinsed with deionized water and dried.

5. The method for preparing a biofilm detection substrate based on butterfly wing remains material according to claim 2, characterized in that: In step S1, the chemical plating comprises: immersing the butterfly wing template in a chemical plating solution for plating, and then washing with deionized water; the metals used in the chemical plating include silver, gold, cobalt, copper, nickel, palladium and platinum.

6. The method for preparing a biofilm detection substrate based on butterfly wing remains material according to claim 2, characterized in that: In step S1, the biological template is removed by dropping phosphoric acid on the butterfly wing template with a metal coating to remove the biological skeleton of the original butterfly wing, followed by washing with deionized water and drying.

7. The method for preparing a biofilm detection substrate based on butterfly wing remains material according to claim 2, characterized in that: In step S2, the concentration of the human serum protein solution is 10-30 μM, and the immersion time is 20-40 h; the concentration of the fluorescein isothiocyanate solution is 20-40 μM, and the immersion time is 8-16 h.

8. A method for detecting a biofilm detection substrate based on butterfly wing remains material as claimed in claim 1, characterized in that: The steps include: S3: Mixing and culturing the biofilm detection substrate and the sample to be tested, removing the culture solution after the culturing and taking out the biofilm detection substrate after the culturing; S4: the biofilm detection substrate after culture in step S3 is washed with a buffer solution, then paraformaldehyde is added and allowed to stand, then washed with a buffer solution again, then fluorescein isothiocyanate solution is added and allowed to stand, then washed with a buffer solution again; S5: Characterize the biofilm content in the sample to be tested by detecting the biofilm detection substrate treated in step S4 through FITC molecular spectroscopy.

9. The method for detecting a biofilm detection substrate based on butterfly wing remains material according to claim 8, characterized in that: Also included are steps for regeneration of the biofilm detection substrate: S6: sterilizing, cleaning and drying the biofilm detection substrate characterized in step S5 to achieve regeneration of the metallized butterfly wings; S7: Soaking the metallized butterfly wings regenerated in step S6 in human serum protein solution and fluorescein isothiocyanate solution in turn to complete the regeneration of the biofilm detection substrate.

10. Use of the biofilm detection substrate based on butterfly wing remains material as claimed in claim 1 in biofilm detection.

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