Online detection device and detection method for bacterial biofilm in liquid and application

Abstract

The invention belongs to the field of on-line detection, and particularly relates to an on-line detection device and method for bacterial biofilms in liquid and application of the on-line detection device and method. The on-line detection device comprises a support (1), a rotating wheel (2) and a detection piece (3); the center of the rotating wheel (2) is connected to the end part of the bracket (1) through a bolt; the detection sheet (3) is embedded into an insertion opening formed in the edge of the rotating wheel (2); and the detection sheet (3) is a substrate for fluorescence detection. Compared with the prior art, the method has the advantages that the problem that a main detection method of the bacterial biofilm in the prior art is not suitable for online detection is solved, and online detection of the bacterial biofilm in the liquid is realized.

Description

Technical Field

[0001] The invention belongs to the field of online detection, and in particular relates to an online detection device, a detection method and an application of bacterial biofilm in liquid. 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. Compared with the free state, bacteria in biofilms have unique physiological synergy and spatial organization capabilities, which improve metabolic capacity and better resist changes in the external environment and the host's immune response. This also makes it difficult to remove biofilms by conventional means. The impact of biofilms in the food industry is particularly serious. On the one hand, biofilm attachment causes corrosion of metal surfaces, resulting in decreased performance of equipment and increased maintenance costs; on the other hand, specific bacteria such as Pseudomonas and Bacillus proliferate in large quantities through biofilms, and the proteolytic enzymes and lipolytic enzymes secreted by them will cause the product to deteriorate and produce a rancid odor. Escherichia coli, Salmonella, etc. may further cause large-scale food poisoning incidents. If biofilms are not prevented and controlled before they are formed in large quantities, problems may occur in the entire batch of products. Biofilms have caused great economic losses in production and life, and have become a major threat to the health of the people. Therefore, it is urgent to propose methods and techniques to assess the degree of sample contamination by detecting bacterial biofilms.

[0003] The existing methods for biofilm detection mainly include crystal violet staining, inverted fluorescence microscopy technology, scanning electron microscope (SEM) or confocal laser scanning microscope (CLSM) observation. Among them, the crystal violet staining method is to culture the sample containing bacterial biofilm, and then use crystal violet staining to perform semi-quantitative detection of the biofilm, and then obtain the absorbance value of the experimental group under a certain wavelength of light. For example, after the well plate forming Staphylococcus aureus was treated with depolymerase B, statistically significant results were obtained under 570nm light. 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, which requires specialist operation. The inverted fluorescence microscope observation method requires staining the bacterial biofilm first, and then using a microscope for observation. For example, after staining the biofilm with DAPI or propidium iodide and observing it under a microscope, it was found that the number of colonies in the experimental group and the control group was significantly different. The electron scanning microscope and laser confocal microscope observation methods are to treat the samples in a certain way and then observe them under SEM and CLSM respectively. For example, SEM and CLSM are used to observe biofilms and study the effects of alginate oligosaccharides on biofilms. However, this microscope observation method has high requirements for sample processing. For example, SEM requires strict vacuum and dehydration preparation, which results in only being able to observe the general shape of the target object, and can only be judged qualitatively by visual means, without a good quantitative standard. Moreover, the strict requirements for the preparation environment will inevitably lead to an increase in detection costs and an increase in the difficulty of promotion.

[0004] Therefore, the main detection methods of bacterial biofilms are generally applicable to laboratory detection, but inevitably difficult to be applied in real production and life, and have certain limitations. Therefore, the advent of more convenient, easy-to-operate, and low-cost bacterial biofilm detection devices is an urgent need of society and the market. Summary of the invention

[0005] The purpose of the present invention is to solve at least one of the above problems and to provide an online detection device, detection method and application of bacterial biofilm in liquid, so as to solve the problem that the main detection method of bacterial biofilm in the prior art is not suitable for online detection. This scheme realizes the online detection of bacterial biofilm in liquid.

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

[0007] The first aspect of the present invention discloses an online detection device for bacterial biofilm in liquid, comprising a bracket, a rotating wheel and a detection sheet;

[0008] The center of the rotating wheel is connected to the end of the bracket through a latch;

[0009] The detection piece is embedded in the socket opened on the edge of the rotating wheel;

[0010] The detection sheet is a substrate used for fluorescence detection.

[0011] Preferably, the rotating wheel comprises three equally divided sector-shaped areas, and the edge of each sector-shaped area is provided with a socket for embedding the detection sheet.

[0012] Preferably, the socket is opened in the middle of the edge of the fan-shaped area.

[0013] Preferably, the bracket includes a bottom plate and an ear plate;

[0014] A pair of ear plates are provided, and the ear plates are connected to the same side of the bottom plate in parallel with each other;

[0015] The rotating wheel is connected between the ear plates through a latch.

[0016] Preferably, the detection sheet is an Au fluorescence enhancement substrate having a three-dimensional periodic structure of a butterfly wing.

[0017] Preferably, the detection sheet is prepared by the following steps:

[0018] The butterfly wing template is metallized by chemical plating, and then phosphoric acid is added to remove the biological template to obtain the metallized butterfly wing; the metallized butterfly wing is immersed in a human serum protein solution and a fluorescein isothiocyanate solution in turn, and then washed and dried to obtain a detection sheet.

[0019] The second aspect of the present invention discloses an online detection method of bacterial biofilm in liquid, using any of the above-mentioned liquid food bacterial biofilm regular detection devices;

[0020] The detection method is:

[0021] An opening is cut in the side wall of the container, a portion of the rotating wheel embedded with the detection sheet is inserted into the opening, and a bracket is fixed; the liquid to be tested is poured into the container for long-term storage, and regular testing is performed during the long-term storage;

[0022] The periodic detection is:

[0023] Before sampling, the surface of the rotating wheel is disinfected, and then the rotating wheel is rotated to rotate the test piece immersed in the liquid to be tested, and the blank test piece is transferred into the container and immersed in the liquid to be tested. The test piece that is rotated out is removed and fluorescein isothiocyanate solution is added to it, and the fluorescence spectrum of the test piece is detected.

[0024] Preferably, the interval of the regular detection is 3 to 14 days; and the liquid to be tested is liquid food or liquid industrial raw material.

[0025] Preferably, the surface disinfection includes washing with chemical detergents and disinfection with ultraviolet rays.

[0026] The third aspect of the present invention discloses the use of any of the above-mentioned online detection devices for bacterial biofilm in liquid in online testing of bacterial biofilm.

[0027] The working principle of the present invention is:

[0028] The present invention first embeds a detection sheet made based on butterfly wings on a rotating wheel so that it can rotate with the rotating wheel. This allows an unused and uncontaminated detection sheet to enter the liquid to be tested through the rotating wheel, and also allows a detection sheet that has adsorbed a sufficient amount of bacterial cell membrane to the surface of the container through the rotating wheel, and then can be taken out for the next step of testing.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] The invention is easy to use and simple in method, and can reduce the cost of training professional testing personnel compared with the prior art;

[0031] The use of the present invention does not require stopping the rotation or stirring of the food storage tank itself, and can detect the number of liquid bacteria in the tank in real time and evaluate the degree of contamination. Compared with the prior art, the probability of the stored liquid food being contaminated during the detection process can be greatly reduced;

[0032] The detection sheet of the present invention can be reloaded on the rotating wheel after disinfection treatment and recycled for detection again, which is more environmentally friendly and sustainable compared with the prior art;

[0033] The detection sheet of the present invention fully utilizes the butterfly wing hierarchical structure, has higher precision than traditional detection methods, and has more accurate detection performance.

[0034] The present invention solves the defects of the existing sampling detection method, such as high loss, high risk of contamination, difficult operation and complicated process; the detection sample loss of the present invention is small, the risk of contamination is low, and the operation method is simple and safe. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 It is a schematic diagram of the structure of an online detection device for bacterial biofilm in liquid;

[0036] Figure 2 Schematic diagram of the preparation process of the test piece;

[0037] In the figure: 1- bracket; 2- rotating wheel; 3- detection piece. DETAILED DESCRIPTION

[0038] The present invention is described in detail below with reference to the accompanying drawings and specific embodiments.

[0039] Example 1

[0040] An on-line detection device for bacterial biofilm in liquid, such as Figure 1 As shown, it includes a bracket 1, a rotating wheel 2 and a detection sheet 3;

[0041] The center of the rotating wheel 2 is connected to the end of the bracket 1 through a latch;

[0042] The detection piece 3 is embedded in the socket opened on the edge of the rotating wheel 2;

[0043] The detection sheet 3 is a substrate for fluorescence detection.

[0044] More specifically, in this embodiment:

[0045] The rotating wheel 2 can be divided into three equally divided sector areas, and the rotating wheel 2 is a cylinder with a height of 30 mm and a diameter of 60 mm. In the middle of the edge of each sector area, a socket for inserting the detection sheet 3 is provided, and the distance between adjacent sockets is about 1 / 3 of the circumference. The socket is an opening with a convex structure in cross section. After the detection sheet 3 is inserted, it can have a large space for the liquid to be tested to enter the socket and contact with the detection sheet 3 for detection, and the opening is sufficient to provide space for liquid exchange, and try to avoid forming a dead zone inside the socket.

[0046] The bracket 1 is composed of a base plate and a pair of ear plates, and the pair of ear plates are connected to the same side of the base plate in parallel to form a Π structure; the rotating wheel 2 is rotatably connected between the ear plates through a latch.

[0047] The detection piece 3 uses an Au fluorescence enhancement substrate with a butterfly wing three-dimensional periodic structure, such as Figure 2 As shown, the butterfly wing template is first metallized by chemical plating, and then phosphoric acid is added to remove the biological template to obtain the metallized butterfly wing; the metallized butterfly wing is immersed in a human serum protein solution and a fluorescein isothiocyanate solution in turn, and then washed and dried to obtain a detection sheet 3.

[0048] Specifically, test piece 3:

[0049] (1) Apply metal coating on the original butterfly wing template using chemical plating process

[0050] The original butterfly wings (Morpho Sulkowskyi (M. sulkowskyi) butterfly wing scales) were 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.

[0051] 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.

[0052] In this embodiment, the metal subjected to chemical plating is gold. The specific chemical plating solution composition is 10 g / L hydrogen tetrachloroaurate, 6 g / L sodium chloride, 4 g / L tartaric acid, 50 g / L sodium hydroxide, and 70 mL / L ethanol. The reaction temperature is controlled at 20-30° C., and the reaction time is controlled within the range of 10-20 min.

[0053] 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.

[0054] (2) Use phosphoric acid solution to remove the original template of butterfly wings

[0055] 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.

[0056] 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.

[0057] (3) Preparation of test piece 3

[0058] The butterfly wings were immersed in 20 μM human serum albumin (HSA) solution and 30 μM fluorescein isothiocyanate (FITC) solution successively, and then dried to obtain the detection sheet 3.

[0059] The butterfly wings were placed in a human serum albumin solution for 30 hours, and then washed with deionized water to remove the human serum albumin molecules that were not adsorbed on the surface of the metal butterfly wings; the butterfly wings were placed in a fluorescein isothiocyanate solution for 12 hours, and then washed with deionized water to remove the FITC molecules that were not adsorbed on the sample. The drying method was vacuum drying.

[0060] The present invention is inspired by the detection of bacteria by butterfly wing hierarchical structure guided by the concept of heritage materials. The butterfly wing hierarchical structure or butterfly wing-like micro-nanostructure metal functional materials use butterfly wings as biological templates, transform them into Au fluorescence enhancement substrates with three-dimensional periodic structures of butterfly wings, and study the influence and mechanism of the three-dimensional micro-nanostructure of Au butterfly wings on the fluorescence enhancement of fluorescent molecules-human serum albumin markers. In addition, through structural regulation, fluorescence enhancement effects with different enhancement multiples can be produced on different structures. Further study the coupling effect between the three-dimensional periodic structure of butterfly wings and fluorescent molecules. On the one hand, by analyzing the variation of surface plasmon resonance spectra with structural size, and using fluorescence lifetime research methods,

[0061] On the one hand, the mechanism of structure-induced fluorescence enhancement is revealed. On the other hand, fluorescence radiation is used as a means to explore the application of Au fluorescence enhancement substrates with butterfly wing three-dimensional periodic structures in cell imaging, providing a theoretical basis and practical approach for the application of metal butterfly wing structure-enhanced fluorescence effects in the field of biological detection.

[0062] Example 2

[0063] An online detection method for bacterial biofilm in liquid, using the liquid food bacterial biofilm regular detection device as described in Example 1;

[0064] The detection method is:

[0065] An opening is cut in the side wall of the container, a portion of the rotating wheel 2 embedded with the detection sheet 3 is inserted into the opening, and the bracket 1 is fixed; the liquid to be tested is poured into the container for long-term storage, and regular testing is performed during the long-term storage;

[0066] The periodic detection is:

[0067] Before sampling, the surface of the rotating wheel 2 is disinfected, and then the rotating wheel 2 is rotated to rotate the test piece 3 immersed in the liquid to be tested, and the blank test piece 3 is transferred into the container and immersed in the liquid to be tested, the rotated test piece 3 is taken out and fluorescein isothiocyanate solution is added thereto, and the fluorescence spectrum of the test piece 3 is detected.

[0068] More specifically, in this embodiment:

[0069] The detection method comprises the following steps:

[0070] (1) cutting an opening in the side wall of the container as a socket for inserting the detection piece 3;

[0071] (2) Place the rotating wheel 2 with the detection sheet 3 embedded in the device into the opening so that the detection sheet 3 extends into the container;

[0072] (3) Fix the bracket 1 of the device around the opening;

[0073] (4) Pour the liquid to be tested that needs long-term regular testing into the container;

[0074] (5) After a period of storage, the surface of the rotating wheel 2 exposed to the outside of the container is disinfected;

[0075] (6) The rotating wheel 2 is turned to rotate the portion of the rotating wheel 2 originally immersed in the liquid in the container out, and the next portion of the rotating wheel 2 loaded with the detection sheet 3 is put into the liquid;

[0076] (7) Take out the transferred detection sheet 3, add FITC fluorescent substance and perform subsequent detection operations.

[0077] More specifically,

[0078] The detection method comprises the following steps:

[0079] (1) Cut an opening in the side wall of the container

[0080] When the container is not loaded with liquid, an opening is cut out of the middle side wall of the container by a cutting machine.

[0081] Among them, the uses of the container include but are not limited to liquid food storage, liquid industrial raw material storage, etc.; the material of the container includes but is not limited to aluminum cans, iron cans, steel cans, etc.; the size of the opening is 50mm×30mm (matching the height of the rotating wheel 2), and the edge of the opening is polished to make it as smooth as possible to prepare for the installation of the liquid food bacterial cell membrane periodic detection device.

[0082] (2) Place the device wheel 2 into the opening

[0083] When the container is not loaded with liquid, the device wheel 2 is placed into the opening with part facing downward, so that the detection piece 3 extends into the interior of the container.

[0084] The dimensions of the runner 2 of the device are 60 mm in cross-sectional diameter and 30 mm in height.

[0085] (3) Fix the T-shaped bracket 1 of the device around the opening

[0086] When the container is not loaded with liquid and the device's partial wheel 2 is inserted into the opening, the upper end (bottom plate) of the device's bracket 1 and the container wall just above / below it are connected with long screws so that the device can be stably installed on the container wall.

[0087] (4) Pour the liquid that needs to be tested regularly into the container

[0088] After the device is installed, the liquid to be tested that requires long-term, regular online real-time testing is poured into the container.

[0089] Among them, the long-term time is up to one year; the regular time varies from 3 to 14 days depending on different needs; liquids include but are not limited to milk, juice and other liquid foods and industrial raw materials.

[0090] (5) After a period of storage, the surface of the rotating wheel 2 is disinfected

[0091] After the liquid is poured into the container, after a period of time, the surface of the rotating wheel 2 is disinfected.

[0092] The storage time varies from 3 to 14 days depending on the demand; the disinfection methods for the surface of the rotating wheel 2 include but are not limited to washing with chemical detergents that have little impact on the stored liquid, disinfection with ultraviolet light, etc.

[0093] (6) Turn the wheel 2 to rotate the part originally immersed in the liquid in the container out.

[0094] After storing for a certain period of time and disinfecting the surface of the rotating wheel 2, the rotating wheel 2 is turned to rotate the portion originally immersed in the liquid in the container out, and the next portion of the rotating wheel 2 loaded with the detection sheet 3 enters the liquid.

[0095] (7) Take out the test piece 3 and perform subsequent testing operations

[0096] FITC fluorescein is added to the surface of the detection sheet 3, and a fluorescence spectrum test is performed to read the number of bacteria on the surface of the detection sheet 3 and evaluate the overall bacterial content in the stored liquid.

[0097] Taking milk as an example, the regular testing in steps (4) and (5) is to take samples once every 7 days.

[0098] 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. An on-line detection device for bacterial biofilm in liquid, characterized in that: It comprises a support (1), a rotating wheel (2) and a detection sheet (3); The center of the rotating wheel (2) is connected to the end of the bracket (1) through a latch; The detection piece (3) is embedded in the socket opened on the edge of the rotating wheel (2); The detection sheet (3) is a substrate used for fluorescence detection.

2. The on-line detection device for bacterial biofilm in liquid according to claim 1, characterized in that: The rotating wheel (2) comprises three equally divided sector-shaped areas, and the edge of each sector-shaped area is provided with an insertion opening for embedding the detection sheet (3).

3. The on-line detection device for bacterial biofilm in liquid according to claim 2, characterized in that: The socket is opened in the middle of the edge of the fan-shaped area.

4. The on-line detection device for bacterial biofilm in liquid according to claim 1, characterized in that: The bracket (1) comprises a bottom plate and an ear plate; A pair of ear plates are provided, and the ear plates are connected to the same side of the bottom plate in parallel with each other; The rotating wheel (2) is connected between the ear plates via a latch.

5. The on-line detection device for bacterial biofilm in liquid according to claim 1, characterized in that: The detection sheet (3) is an Au fluorescence enhancement substrate with a three-dimensional periodic structure of butterfly wings.

6. The on-line detection device for bacterial biofilm in liquid according to claim 5, characterized in that: The detection sheet (3) is prepared by the following steps: The butterfly wing template is metallized by chemical plating, and then phosphoric acid is added to remove the biological template to obtain a metallized butterfly wing; the metallized butterfly wing is immersed in a human serum protein solution and a fluorescein isothiocyanate solution in turn, and then washed and dried to obtain a detection sheet (3).

7. An on-line detection method for bacterial biofilm in liquid, characterized in that: A device for periodic detection of bacterial biofilm in liquid food as claimed in any one of claims 1 to 6; The detection method is: An opening is cut in the side wall of the container, a portion of the rotating wheel (2) embedded with the detection sheet (3) is inserted into the opening, and a bracket (1) is fixed; a liquid to be tested is poured into the container for long-term storage, and regular testing is performed during the long-term storage period; The periodic detection is: Before sampling, the surface of the rotating wheel (2) is disinfected, and then the rotating wheel (2) is rotated to rotate the test piece (3) immersed in the liquid to be tested, and the blank test piece (3) is transferred into the container and immersed in the liquid to be tested, and the transferred test piece (3) is removed and a fluorescein isothiocyanate solution is added thereto, and the fluorescence spectrum of the test piece (3) is detected.

8. The method for online detection of bacterial biofilm in liquid according to claim 7, characterized in that: The interval time of the regular detection is 3 to 14 days; the liquid to be detected is liquid food or liquid industrial raw material.

9. The method for online detection of bacterial biofilm in liquid according to claim 7, characterized in that: The surface disinfection includes washing with chemical detergents and disinfection with ultraviolet rays.

10. Use of the on-line detection device for bacterial biofilm in liquid as claimed in any one of claims 1 to 6 in on-line testing of bacterial biofilm in liquid.

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