Layer-by-layer fluorescent film detector for bacteria detection and its preparation method and application

Abstract

The present invention belongs to the field of sensor technology, and discloses a layer-by-layer fluorescent film detector for bacteria detection, a preparation method thereof, and an application in bacteria detection. The preparation method is to use a biocompatible unit, a fluorescent group unit, and an anion / cation unit as co-block polymer monomers, and adopt a reversible addition-fragmentation chain transfer polymerization reaction to prepare fluorescent polymers with negative / positive charges respectively; then prepare positively charged and negatively charged fluorescent polymer solutions respectively, and alternately deposit fluorescent polyanions / cations on a negatively charged glass carrier by a layer-by-layer self-assembly method to obtain. The present invention obtains fluorescent polyanions / cations by polymerizing monomers with different functions, and then adopts a layer-by-layer self-assembly technology to prepare a fluorescent polymer film, and by regulating different chain lengths and fluorescent groups, the enrichment and fluorescent staining of Escherichia coli are improved, and the visual rapid detection of Escherichia coli is realized, and the detection method is easy to operate and can be recycled.

Description

Technical Field

[0001] The invention relates to the technical field of sensors, and in particular to a layered fluorescent film detector for bacteria detection, a preparation method thereof, and an application of the detector in fluorescence detection of bacteria. Background Art

[0002] The content of bacteria in food and drinking water is an important biochemical indicator for evaluating their safety. The traditional method is to detect bacteria by agar culture and then colony counting, but it is time-consuming (37-48 hours) and has limited strains that can be detected. The bacterial detection method based on electrochemical impedance spectroscopy and surface plasmon resonance technology has the advantages of high sensitivity and fast detection speed, but it still has problems such as complex equipment and high requirements for operators.

[0003] Therefore, the development of simple and rapid fluorescence detection methods has important practical significance. Compared with the fluorescence liquid phase detection method, the fluorescence film detection method is more convenient to operate, has a shorter action time and can be reused. Summary of the invention

[0004] The purpose of the present invention is to provide a layer-by-layer fluorescent film detector for bacteria detection and a preparation method thereof and application in bacteria detection in view of the defects of the existing bacteria detection methods.

[0005] To achieve the above object, the present invention adopts the following technical solutions:

[0006] The first aspect of the present invention is to provide a method for preparing a layer-by-layer fluorescent film detector for bacteria detection, using a biocompatible unit, a fluorescent group unit, and an anion / cation unit as co-block polymer monomers, and using a reversible addition-fragmentation chain transfer polymerization reaction (RAFT) to prepare negatively / positively charged fluorescent polymers respectively;

[0007] The negatively charged / positively charged fluorescent polymers are then prepared into positively charged / negatively charged fluorescent polymer solutions, respectively. Based on electrostatic adsorption, fluorescent polycations and fluorescent anions are alternately deposited on a negatively charged glass carrier through layer-by-layer assembly to obtain a nanoscale thin film detector with fluorescent properties.

[0008] Specifically, the method for preparing the layer-by-layer fluorescent film detector for bacteria detection comprises the following steps:

[0009] (1) The biocompatible unit, the fluorescent group unit, the anion / cation unit and the chain transfer agent TTCA are added in a round-bottom flask containing a dioxane solvent in proportion, and then the initiator ACPA is added under vigorous stirring. After the mixture is deoxygenated in an ice bath for 25 to 40 minutes, a polymerization reaction is carried out at 78 to 82° C.;

[0010] (2) The progress of the polymerization reaction was monitored by nuclear magnetic resonance spectroscopy. After the polymerization was completed, the round-bottom flask was placed in ice water to quench the polymerization reaction. The obtained product was further P polyacrylamide gel chromatography column was used for purification to obtain negatively / positively charged fluorescent polymers;

[0011] (3) preparing the products obtained in step (2) into positively / negatively charged fluorescent polymer aqueous solutions of different concentrations, adding different doses of NaCl, adjusting the pH value of the solution, and obtaining fluorescent polyanion / cation deposition solutions of different concentrations;

[0012] (4) The glass slide is processed into a glass carrier with a negatively charged surface, and then alternately immersed in fluorescent polyanion / cation deposition solutions, and the surface is blown dry with a nitrogen dust gun to obtain a nanoscale thin film detector with fluorescent characteristics.

[0013] Preferably, in step (1), the anion / cation unit is one or more of acrylic acid (AA), 2-(dimethylamino)ethyl acrylate (DMEA), [2-(acryloyloxy)ethyl]trimethylammonium chloride (TMEA), and 3-sulfopropyl methacrylate (SPMP). It should be noted that, when preparing polycations, only cations are added; when preparing polyanions, anions are added. Anions and cations cannot be added to a polymer at the same time.

[0014] Preferably, in step (1), the fluorescent group unit is one or more of BODIPY-based, rhodamine-based, fluorescein, and fluorescent quantum dots.

[0015] Preferably, in step (1), the molar ratio of the biocompatible unit, the fluorescent group unit, and the anionic / cationic unit is 3:1:3, and the degree of polymerization n of the negatively charged or positively charged fluorescent polymer is 2 and 14, respectively.

[0016] Preferably, in step (1), the molar ratio of the biocompatible unit, the fluorescent group unit, and the anion / cation unit is 3:0.8-2:2-4, and the degree of polymerization n of the negatively charged or positively charged fluorescent polymer is 2 and 14, respectively.

[0017] Preferably, in step (1), the deoxygenation process is to introduce nitrogen or argon into the round-bottom flask to remove oxygen in the round-bottom flask.

[0018] Preferably, in step (3), the concentration of the positively charged and negatively charged fluorescent polymer solutions is 1.3×10 -7 ~1.3×10 -4 mol / L.

[0019] Preferably, in step (3), the concentration of the positively charged and negatively charged fluorescent polymer solutions is 1.3×10 -4 mol / L,1.3×10 -6 mol / L,5.0×10 -7 mol / L,1.3×10 -7 mol / L.

[0020] Preferably, in step (3), the dosage of NaCl is 0.01-0.1 mol / L; and the pH value is 3.2-5.5.

[0021] Preferably, in step (3), the dosage of NaCl is 0.1 mol / L, 0.05 mol / L, 0.01 mol / L; the pH values ​​of the positively / negatively charged fluorescent polymer aqueous solution are 5.5 and 3.2 respectively.

[0022] Preferably, in step (4), the glass slide is a common commercial glass slide or an ITO glass slide, which is treated by a Piranha solution treatment method or a plasma treatment method to become a negatively charged glass carrier.

[0023] Preferably, in step (4), the layer-by-layer self-assembly method includes but is not limited to electrostatic adsorption, hydrogen bonding, coordination bonding, and chemical bonding forces.

[0024] Preferably, in step (4), the dust blowing gun includes but is not limited to nitrogen, compressed air, and argon.

[0025] The second aspect of the present invention is to provide a layer-by-layer fluorescent polymer film detector prepared by the method described above.

[0026] The third aspect of the present invention is to provide an application of the layer-by-layer fluorescent polymer film detector in fluorescence detection of bacteria.

[0027] Preferably, the bacteria include Escherichia coli, Staphylococcus aureus, Bacillus subtilis in food or drinking water.

[0028] Preferably, the method for fluorescence detection of the bacteria is: using a layer-by-layer fluorescent film detector, dropping a bacterial suspension on the film surface, and observing the fluorescence imaging after culturing for 10 minutes.

[0029] Preferably, the bacteria is Escherichia coli, and 2 μL of an Escherichia coli suspension with an OD@600nm value of 0.2 is dripped onto the obtained fluorescent film with 12 layers of fluorescent polymer deposited thereon, and after culturing for 10 minutes, it is washed twice with M9 culture medium, and then 2 μL of M9 culture medium is dripped on it, and then a coverslip is applied and observed under a fluorescence microscope.

[0030] The present invention adopts the above technical solution, which has the following technical effects compared with the prior art:

[0031] (1) The present invention creatively obtains fluorescent polyanions and fluorescent polycations by polymerizing monomers with different functions, and further utilizes electrostatic adsorption to prepare fluorescent polymer film detectors using layer-by-layer self-assembly technology, which exhibits excellent staining rate in bacterial fluorescence imaging;

[0032] (2) The present invention prepares layer-by-layer fluorescent film detectors with different characteristics by regulating different chain lengths and fluorescent groups, which can greatly improve the enrichment and fluorescent staining of Escherichia coli, realize the visual rapid detection of Escherichia coli, and the imaging effect is conveniently adjustable with the characteristics of the fluorescent film;

[0033] (3) Compared with the existing fluorescent liquid phase detection method, the fluorescent thin film detection method based on the layer-by-layer fluorescent thin film detector of the present invention has the advantages of convenient operation, short action time and recyclable use for bacteria detection due to its good fluorescence characteristics. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 (a) is a structural diagram of the fluorescent polycation prepared in Example 1, and (b) is a structural diagram of the fluorescent polyanion prepared in Example 1;

[0035] Figure 2 (a) is the UV-visible absorption and fluorescence emission spectra of the sample prepared in Example 1, and (b) is the fluorescence lifetime decay diagram of the prepared sample;

[0036] Figure 3 (a) is a UV-visible absorption graph of the sample obtained in Example 2, and (b) is a graph showing the relationship between the UV-visible absorption value of the sample obtained in Example 2 and the number of film layers;

[0037] Figure 4 The UV-visible absorption and fluorescence emission diagrams of the samples obtained in Example 2;

[0038] Figure 5 (a) is a graph showing the effect of the fluorescent polymer obtained in Example 3 on the growth curve of Escherichia coli in M9 culture medium, and (b) is a graph showing the effect of the fluorescent film obtained in Example 3 on the growth curve of Escherichia coli;

[0039] Figure 6These are fluorescence imaging images of Escherichia coli produced by the layer-by-layer fluorescent films prepared at different concentrations in Example 4. DETAILED DESCRIPTION

[0040] The present invention is described in detail and specifically by specific examples below to provide a better understanding of the present invention, but the following examples do not limit the scope of the present invention.

[0041] It should be noted that the experimental methods used in the following examples are conventional methods unless otherwise specified, and the materials, reagents and instruments used therein can be purchased through commercial channels unless otherwise specified. All reagents used are analytically pure. Milli-Q high-purity water (18.2 MΩ) was used during the experiments.

[0042] Example 1: Synthesis of fluorescent polyanions and polycations

[0043] Preparation of fluorescent polyanion: 0.179 g BODIPY acrylate (BDPMA), 0.506 g polyethylene glycol (APEG) and 0.083 g acrylic acid (AA) were added to 5 mL dioxane in a molar ratio of 1:3:3, TTCA (0.1 mmol, 36.4 mg) as chain transfer agent and DMF (0.54 mmol, 39.7 mg) as 1 The internal standard reference material for H NMR detection was added with initiator ACPA (0.029 mL of 0.3 mol / L solution). The mixture was deoxygenated by nitrogen in an ice bath for 30 min, and then the polymerization reaction was started in an 80°C oil bath. The reaction process was monitored by NMR. 1 H NMR was used to analyze the residual amount of ethylene protons in the reaction mixture and calculate the polymer conversion rate. The green fluorescent polyanion (denoted as GFPC - ).

[0044] Preparation of fluorescent polycation: Different from the above-mentioned preparation method of fluorescent polyanion, AA was replaced by 2-(dimethylamino)ethyl acrylate (DMEA), and the other steps were the same. A green fluorescent polycation (denoted as GFPC) was prepared. + ).

[0045] Figure 1 (a) is the green fluorescent polycation (GFPC + ) and (b) fluorescent polyanion (GFPC -) Chemical structure composition diagram. It can be seen from the figure that the polymer is composed of three functional units: biocompatible unit, fluorescent unit and ionic unit. The molecular weight of the polymer is about 5kg / mol (n=2); when the ionic unit is DMEA (2-(dimethylamino)ethyl acrylate), the polycationic GFPC + When AA (acrylic acid) is used, polyanion GFPC is obtained. - .

[0046] Figure 2 The green fluorescent polycation (GFPC + ) and fluorescent polyanions (GFPC - ) dispersed in water (a) Normalized absorption and fluorescence emission spectra (λ ex =495nm) diagram; (b) fluorescence decay lifetime diagram (λ ex =495nm,λ em =540nm), where [GFPC + ]=[GFPC - ]=4.5×10 -7 M). It can be seen from the figure that the prepared GFPC polymer has good water solubility and maintains the fluorescence characteristics of BODIPY. + and GFPC - The fluorescence lifetimes are 5.6 and 5.9 ns, respectively.

[0047] Example 2: Preparation of layer-by-layer fluorescent film detector

[0048] Commercial glass slides were immersed in Piranha solution (30% H2O2 + 70% H2SO4) for 30 min, washed three times with deionized water, and dried with nitrogen. + (12mL, 1.3×10 - 4 mol / L, [NaCl] = 0.1 mol / L, pH = 5.5) and GFPC - (12mL, 1.3×10 -4 mol / L, [NaCl]=0.1mol / L, pH=5.5) solution for 10 min each. After 6 cycles of alternate immersion (12 times in total), the film was dried with nitrogen to obtain a fluorescent film (referred to as GFPC film).

[0049] Figure 3 (a) is the green fluorescent polycation (GFPC + ) and fluorescent polyanions (GFPC -) UV-Vis absorption spectrum of LbL self-assembled GFPC layer on activated glass; (b) is the correlation curve between the maximum absorbance value of the film at 529nm and the number of film layers. LbL self-assembly is at [GFPCs] = 0.95 × 10 -5 It is carried out at M. From the figure, it can be seen that the maximum absorbance increases linearly with the number of film layers.

[0050] Figure 4 The absorption and fluorescence emission spectra of the green fluorescent film (GFPC LbL film) prepared above, where [GFPCs] = 5.0 × 10 -7 M. It can be seen from the figure that the prepared fluorescent film maintains the spectral characteristics of the fluorescent polymer.

[0051] Example 3: Toxicity evaluation of fluorescent polymer GFPCs and layer-by-layer fluorescent films on Escherichia coli

[0052] The obtained GFPC + GFPC - The fluorescent polymer was added to a 96-well flat-bottomed cell culture plate with a lid containing an E. coli solution. After gradual dilution, the final concentration of the fluorescent polymer in the E. coli solution was 4.0 × 10 -6 M, 70 μL of mineral oil was added to each sample to prevent evaporation, and the sample was incubated in a microplate reader at 37°C in the dark for 18 hours. In the example, only bacteria and only M9 culture medium were used as comparative experiments with the same other steps.

[0053] Figure 5 (a) is the growth curve of E. coli by different fluorescent polymers ([FPCs] = 4.0 × 10 -6 M), it can be seen from the figure that after 18 hours of cultivation, the growth curve of bacteria containing GFPC- is very similar to that of bacteria in M9 culture medium, while the growth curve of bacteria containing GFPC- is very similar to that of bacteria in M9 culture medium. + The bacterial growth curve of + It has a certain inhibitory effect on the growth of Escherichia coli; (b) is the growth curve of Escherichia coli in M9 culture medium containing GFPC film. It can be seen from the figure that the fluorescent film has no toxic effect on the growth of Escherichia coli.

[0054] Example 4: Detection performance of layer-by-layer fluorescent films on bacteria

[0055] The detection performance test method is as follows: Escherichia coli (K-12, BW25113) with an OD value of 0.2 and M9 mixed solution (1.1-5.5×10 7cell / mL) as the detection object, and the obtained layered fluorescent film is used as a slide. 2 μL of bacterial suspension is added to the surface of the film, and after the bacterial suspension is incubated for 10 minutes, it is washed twice with M9 culture solution, and then covered with a cover glass and observed under a fluorescence microscope. In the embodiment, there is no bacteria and other steps are the same as a comparative experiment. The collected fluorescent images and their fluorescence intensity are analyzed by Image J software.

[0056] In this embodiment:

[0057] The E. coli (K-12, BW25113) M9 mixture with an OD value of 0.2 was used as the test object. 2 μL of bacterial suspension (1.1-5.5×10 7 cell / mL), after the bacterial suspension was treated for 10 min, it was washed 3 times with M9 culture medium, and then covered with a cover glass and observed under a fluorescence microscope. The collected fluorescence images and their fluorescence intensity were analyzed by Image J software. In the embodiment, a glass carrier without a fluorescent polymer film and other steps were the same as those in the comparative experiment.

[0058] Figure 6 Fluorescence imaging of Escherichia coli (E. coli) by the layered fluorescent films prepared from the above fluorescent polymer deposition solutions with different concentrations: (a, b) [GFPCs] = 1.3 × 10 -4 M; (c, d) [GFPCs] = 5.0 × 10 -7 M. It can be seen from the figure that the staining rate of the fluorescent film prepared in the deposition solution with a higher concentration can reach 94% for Escherichia coli, while when the concentration of the deposition solution is lower, no Escherichia coli is stained on the fluorescent film.

[0059] The specific embodiments of the present invention are described in detail above, but they are only examples, and the present invention is not limited to the specific embodiments described above. For those skilled in the art, any equivalent modifications and substitutions made to the present invention are also within the scope of the present invention. Therefore, the equalization changes and modifications made without departing from the spirit and scope of the present invention should be included in the scope of the present invention.

Claims

1. The application method of the layer-by-layer fluorescent polymer film detector in the fluorescence detection of bacteria is characterized in that: The bacterial detection method is to use a layer-by-layer fluorescent film detector, drip a bacterial suspension on the film surface, and observe the fluorescent imaging after culturing for 10 minutes; the preparation method of the layer-by-layer fluorescent film detector includes the following steps: (1) The biocompatible unit, the fluorescent group unit, the anion / cation unit and the chain transfer agent TTCA are added in a round-bottom flask containing a dioxane solvent in proportion, and then the initiator ACPA is added under vigorous stirring. After the mixture is deoxygenated in an ice bath for 25 to 40 minutes, a polymerization reaction is carried out at 78 to 82° C.; (2) The progress of the polymerization reaction was monitored by nuclear magnetic resonance spectroscopy. After the polymerization was completed, the round-bottom flask was placed in ice water to quench the polymerization reaction. The obtained product was further P polyacrylamide gel chromatography column was used for purification to obtain negatively / positively charged fluorescent polymers; (3) preparing the products obtained in step (2) into positively / negatively charged fluorescent polymer aqueous solutions of different concentrations, adding different doses of NaCl, adjusting the pH value of the solution, and obtaining fluorescent polyanion / cation deposition solutions of different concentrations; (4) treating a glass slide to be a glass carrier with a negative charge on the surface, then alternately immersing the slide in fluorescent polyanion / cation deposition solutions, and blowing the surface dry with a nitrogen dust gun to obtain a nanoscale thin film detector with fluorescent properties; Wherein: in step (1), the molar ratio of the biocompatible unit, the fluorescent group unit, and the anion / cation unit is 3:1:3, and the polymerization degree n of the negatively charged / positively charged fluorescent polymer is 2 and 14 respectively; In step (3), the concentration of the positively charged / negatively charged fluorescent polymer aqueous solution is 1.3×10 -7 ~1.3×10 -4 mol / L.

2. The application method according to claim 1, characterized in that: In step (1), the anion / cation unit is one or more of acrylic acid (AA), 2-(dimethylamino)ethyl acrylate (DMEA), [2-(acryloyloxy)ethyl]trimethylammonium chloride (TMEA), and 3-sulfonate propyl methacrylate (SPMP).

3. The application method according to claim 1, characterized in that: In step (1), the fluorescent group unit is one or more of BODIPY-based, rhodamine-based, fluorescein, and fluorescent quantum dots.

4. The application method according to claim 1, characterized in that: In step (3), the dosage of NaCl is 0.01-0.1 mol / L, and the pH value is 3.2-5.

5.

5. The application method according to claim 1, characterized in that: In step (4), the glass slide is a common commercial glass slide or an ITO glass slide, which is treated by a Piranha solution treatment method or a plasma treatment method to become a negatively charged glass carrier.

6. The application method according to claim 1, characterized in that: The bacteria include Escherichia coli, Staphylococcus aureus, and Bacillus subtilis.

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