A fluorescence quenching biosensor for detecting Listeria monocytogenes and preparation method thereof

Abstract

The present invention discloses a fluorescence quenching biosensor for detecting Listeria monocytogenes and a preparation method thereof, and belongs to the technical field of food safety. It is prepared by uniformly mixing a fluorescent probe as an energy donor and a quenching probe as an energy acceptor in a volume ratio of 1:2; the fluorescent probe is a silicon nanoparticle modified by a first aptamer, and the quenching probe is a gold nanoparticle modified by a second aptamer, and both the first aptamer and the second aptamer specifically recognize Listeria monocytogenes. It has been verified that it has a higher degree of linear fitting and a lower detection limit. Compared with traditional methods, it also has a lower detection time and better specificity; in addition, under the interference of LB broth samples, the established linear regression equation still shows excellent applicability.

Description

Technical Field

[0001] The invention belongs to the technical field of food detection, and particularly relates to a fluorescence quenching biosensor for detecting Listeria monocytogenes and a preparation method thereof. Background Art

[0002] Listeria monocytogenes is a foodborne pathogen that can cause invasive listeriosis. Contaminated ready-to-eat foods are a high-risk source of human listeriosis. Since the 1980s, Listeria monocytogenes has had a huge economic impact on the food industry, and the association between human listeriosis outbreaks and contaminated food has always been a concern of health departments. Currently, there are several methods for detecting Listeria monocytogenes, such as immunomagnetic beads, digital PCR, and biosensors. Most of them have not been widely promoted due to their high costs. The national standard method and real-time fluorescence PCR are the two most commonly used methods. The national standard method (GB 4789.30-2016) requires the isolation and purification of strains for the detection of Listeria monocytogenes, and the results are accurate based on biochemical tests, but it takes at least 4 days to get the results. This method has limitations in real-time monitoring of food production. Real-time fluorescence PCR (SN / T1870-2016) does not require strain isolation for the detection of Listeria monocytogenes. It uses molecular biological technology and has a fast detection speed, but its minimum detection limit and amplification effects in different types of food are rarely compared and reported. Emerging fluorescent biological probe detection methods are widely used in the determination of target substances due to their advantages such as low cost, simple operation, fast response, and high stability. Among them, fluorescence resonance energy transfer (FRET) is a non-radiative energy transition that transfers donor excited state energy to acceptor excited state through intermolecular electric dipole interactions. It is widely used in fluorescent probe detection technology. There are few corresponding studies on silicon nanoparticles (SNPs), which are often prepared by catalytic reduction of silicon-containing coupling agents. Most methods based on fluorescence resonance energy transfer are achieved by shortening the distance between the donor and the acceptor. For example, energy transfer is carried out by using ratiometric fluorescent probes coupled to the surface of microorganisms to move closer to each other. Ratio-type fluorescent probes have the advantages of high sensitivity, simple operation, and short response time to the target. At present, it is necessary to select a suitable first fluorescent probe that can be used as an energy donor and a suitable second fluorescent probe that can be used as an energy acceptor to form an effective FRET-type fluorescence quenching biosensor. However, due to the high failure rate of the double aptamer sandwich method, new nucleic acid aptamers need to be used in combination. It is easy for the bacterial surface to be completely occupied by one of the fluorescent probes, making the other fluorescent probe unusable. Summary of the invention

[0003] In view of the problems of great detection difficulty, long time consumption and low sensitivity in the existing detection technology of Listeria monocytogenes in food, the present invention provides a fluorescence quenching biosensor for detecting Listeria monocytogenes, and also provides a method for preparing the fluorescence quenching biosensor for detecting Listeria monocytogenes.

[0004] A fluorescence quenching biosensor for detecting Listeria monocytogenes is prepared by uniformly mixing a fluorescent probe as an energy donor and a quenching probe as an energy acceptor in a volume ratio of 1:2;

[0005] The fluorescent probe is a silicon nanoparticle modified with a first aptamer;

[0006] The quenching probe is a gold nanoparticle modified with a second aptamer;

[0007] The DNA sequence of the first aptamer is shown in SEQ ID No: 1, and the 5' end of the first aptamer is modified with a carboxyl group;

[0008] The DNA sequence of the second aptamer is shown in SEQ ID NO: 2, and the 5' end of the second aptamer is modified with a thiol group;

[0009] The linear fitting coefficient of the fluorescence quenching biosensor for detecting Listeria monocytogenes is 0.98154, and the detection limit is 19.27 CFU / mL.

[0010] The preparation steps of a fluorescence quenching biosensor for detecting Listeria monocytogenes are as follows:

[0011] (1) Preparation of the first activation mixture

[0012] Mix 10 mM 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 5 mM N-hydroxysuccinimide in a volume ratio of 1:1 to obtain a first activation mixture;

[0013] (2) Preparation of the second activation mixture

[0014] Adding pure water to 3-(2-formylethyl)phosphine hydrochloride to obtain a 3-(2-formylethyl)phosphine hydrochloride solution with a final concentration of 2.867 g / L, i.e., the second activated mixed solution;

[0015] (3) Preparation of fluorescent probes

[0016] 6 mL of deionized water was added to 1 mL of N-(β-aminoethyl-γ-aminopropyl)methyldimethoxysilane; 8.0 mg of amikacin was added and stirred continuously for 30 min; the mixture was heated at 200°C for 6 h and cooled to room temperature; pure water was added to dilute the mixture 20 times to obtain a reaction solution; 500 uL of the reaction solution and 250 uL of the first activation mixture were vortexed and mixed, and incubated on a shaker at 180 rpm and room temperature for 30 min; 160 uL of the first aptamer solution with a volume concentration of 10 umol / L was added and vortexed, and incubated on a shaker at 180 rpm and room temperature for 2.5 h to obtain a fluorescent probe, which was then wrapped in tin foil and stored at 4°C in a dark place;

[0017] The DNA sequence of the first aptamer in the first aptamer solution is as shown in SEQ ID No: 1, and the 5' end of the first aptamer is modified with a carboxyl group;

[0018] (4) Preparation of quenching probe

[0019] (4.1) Add 1 mL of 1% tetrachloroauric acid solution to 100 mL of distilled water and boil. Quickly add 2.5 mL of 1% trisodium citrate solution and continue boiling for 15 min. Cool naturally to room temperature to obtain a gold nanoparticle solution.

[0020] (4.2) Place 100 μL of the second aptamer solution with a volume concentration of 10 μM in a water bath at 95°C for 5 min, and cool at 4°C for 5 min to obtain an annealing solution; add 10 μL of 3-(2-formylethyl)phosphine hydrochloride solution with a mass volume concentration of 2.867 g / L to the annealing solution, and allow to react at room temperature for 30 min to obtain a reaction solution; add 1 mL of the gold nanoparticle solution to the reaction solution, incubate at 37°C for 12 h, centrifuge at 12000 rpm for 20 min, remove the supernatant, and obtain a precipitate;

[0021] The DNA sequence of the second aptamer in the second aptamer solution is as shown in SEQ ID NO: 2, and the 5' end of the second aptamer is modified with a thiol group;

[0022] (4.3) Add 400 μL of PBS solution with a mass concentration of 0.1 M and a pH value of 7.4 to the precipitate, and store it at 4°C wrapped in tin foil and protected from light to prepare a quenching probe;

[0023] (5) Preparation of biosensors

[0024] The fluorescent probe and the quenching probe are mixed at a volume ratio of 1:1.5 to prepare a fluorescence quenching biosensor;

[0025] When the fluorescence emission wavelength of silicon nanoparticles shifts from 520nm to 525nm, it indicates that the first aptamer in the fluorescence quenching biosensor is successfully modified onto the silicon nanoparticles. When the ultraviolet absorption wavelength of gold nanoparticles shifts from 522nm to 527nm, it indicates that the second aptamer in the fluorescence quenching biosensor is successfully modified onto the gold nanoparticles.

[0026] The beneficial technical effects of the present invention are embodied in the following aspects:

[0027] 1. It has been verified that the fluorescence quenching biosensor prepared by the present invention has a high degree of linear fitting (correlation coefficient as high as 0.98154) and a low detection limit (as low as 19.27 CFU / mL). The linear fitting degree of the current existing technology generally ranges from 0.90 to 0.99; at the same time, compared with traditional technologies (strand displacement reaction method, PCR amplification method, ELISA kit method), their detection time varies from 12h to 3d, and the specificity for some common model strains is cross-specific, while the fluorescence quenching biosensor has a lower detection time (only 35min) and better specificity (for common model strains such as Staphylococcus aureus and Escherichia coli). For example, the traditional chain displacement reaction utilizes the free energy difference of DNA molecular hybridization to replace the target single strand in the hybrid DNA double helix structure with a single-stranded DNA. Since the negatively charged phosphate group on the single-stranded DNA has an intramolecular electrostatic repulsion effect on the double-stranded DNA, the efficiency of the chain displacement reaction is relatively low. In addition, for dual-aptamer sandwich biosensors, the prior art usually uses the same two aptamers for sandwich treatment, which causes one of the probes to quickly occupy the bacterial surface, making it impossible for the other probe to be adsorbed to the bacterial surface; while in the fluorescence quenching biosensor of the present invention, one probe uses an existing well-known nucleic acid aptamer, and the other uses a new nucleic acid aptamer screened by the present invention. Since the binding properties of nucleic acid aptamers have not been clearly studied so far, it is very difficult to effectively select and combine existing public nucleic acid aptamers.

[0028] 2. After testing, the linear regression equation established under the standard environment can be directly applied to the detection of LB broth samples. There is no need to re-cultivate bacteria to establish a standard curve. You only need to directly apply the established linear regression equation, which saves time and is suitable for quickly estimating the initial concentration of Listeria monocytogenes in common samples under emergency conditions. For example, in LB broth samples, under the environment of artificial inoculation of Listeria monocytogenes in LB broth, the concentration of Listeria monocytogenes measured by the fluorescence quenching biosensor is 3.82*10 6 CFU / mL, almost the same as its actual concentration (3.9*10 6It can be seen that even if the corresponding standard curve and linear regression equation in the LB broth sample environment are not established, the standard curve and linear regression equation established in the pure PBS sample environment can still have good versatility.

[0029] 3. In summary, the present application selects silicon nanoparticles modified by the first aptamer and gold nanoparticles modified by the second aptamer to form a fluorescence quenching sensor. This is the first time that two different types of probes are used in combination. It takes a lot of creative work to select a suitable pair of fluorescent probes from tens of thousands of similar quantum dots or nanoparticles. At the same time, the prior art does not disclose the application of silicon nanoparticles / gold nanoparticles to the detection of foodborne Listeria monocytogenes. Since the original silicon nanoparticles are used for the detection of small molecule rutin, it is unknown whether they can be applied to the detection of bacteria. It is necessary to consider whether there is a complexation reaction between silicon nanoparticles / gold nanoparticles or whether they react with monocytogenes. Chelation occurs on the outer surface of Listeria monocytogenes, and the combination of the above-mentioned silicon nanoparticles / gold nanoparticles into a fluorescence quenching biosensor also needs to consider whether an effective FRET effect can occur, and the degree of overlap of fluorescence emission-ultraviolet absorption between silicon nanoparticles and gold nanoparticles needs to be considered; in addition, whether one of the aptamers will completely occupy the surface of the bacteria, the covalent coupling between the silicon nanoparticles and the first aptamer, the covalent coupling between the gold nanoparticles and the second aptamer, and the use concentration and volume ratio of the two probes, etc., all of which are not disclosed in the prior art and cannot be technically inspired by the corresponding common sense in the field. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is a schematic diagram of the application principle of the fluorescence quenching biosensor used for detecting Listeria monocytogenes in the present invention.

[0031] Figure 2 This is a fluorescence spectrum diagram of the fluorescent probe before and after covalent coupling with the first aptamer in the present invention.

[0032] Figure 3 This is the UV absorption diagram before and after the covalent coupling of the quenching probe and the second aptamer in the present invention.

[0033] Figure 4 It is the fluorescence spectra of Listeria monocytogenes at different concentrations in the present invention.

[0034] Figure 5 This is a standard curve diagram of Listeria monocytogenes in the present invention.

[0035] Figure 6 This is a specific detection diagram of Listeria monocytogenes in the present invention.

[0036] Figure 7 It is the standard curve diagram of the tap water sample in the present invention.

[0037] Figure 8 It is the standard curve diagram of the egg sample in the present invention.

[0038] Fig. 9 It is the standard curve diagram of cheese samples in the present invention. DETAILED DESCRIPTION

[0039] The present invention is further described below in conjunction with specific embodiments.

[0040] Unless otherwise defined, technical and scientific terms used in the following examples have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0041] The raw materials used in the following examples are all conventional biochemical reagents unless otherwise specified; the experimental methods are all conventional methods unless otherwise specified; the quantitative tests in the following examples are all repeated three times, and the results are averaged; the % in the following examples are all mass percentages unless otherwise specified.

[0042] In the following examples, the aptamers used were purchased from Shanghai Bioengineering Co., Ltd.; 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride, N-hydroxysuccinimide, guanidine hydrochloride, vancomycin, sodium ascorbate and 3-aminopropyltrimethoxysilane used were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; the 500Da dialysis bag used was purchased from Shanghai Yuanye Biotechnology Co., Ltd.; unless otherwise specified, other raw materials used were purchased from Sinopharm Chemical Reagent Co., Ltd.

[0043] It should be noted that the PBS phosphate buffer used in the present invention is a sterile PBS buffer with a pH value of 7.4 and a 0.1 M concentration.

[0044] It should be noted that all the strains used in the present invention were purchased from the American Type Culture Collection (ATCC).

[0045] The number of Listeria monocytogenes is ATCC 43251, the number of Staphylococcus aureus is ATCC29213, the number of Salmonella is ATCC 14028, the number of Escherichia coli is ATCC 25922, the number of Cronobacter sakazakii is ATCC29544, and the number of Pseudomonas aeruginosa is ATCC 15442. In addition, the above strains are purchased through overseas agents of Hefei Xiyue Biotechnology Co., Ltd.

[0046] It should be noted that the instruments, equipment, raw materials, reagents or method steps used in the present invention are ensured to be processed under sterile conditions.

[0047] It should be noted that the instruments, equipment, raw materials, reagents or method steps not mentioned in the present invention are conventional or well-known technical methods for those skilled in the art and will not be described in detail in this application. Please refer to the following paper materials (yuwei Ren, Lulu Cao, Xiyan Zhang, Rui Jiao, Dexin Ou, Yang Wang, Danfeng Zhang, Yizhong Shen, Na Ling, Yingwang Ye, A novel fluorescence resonance energy transfer (FRET)-based paper sensor with smartphone for quantitative detection of Vibrio parahaemolyticus, Food Control, Volume 145, 2023, 109412, ISSN 0956-7135, https: / / doi.org / 10.1016 / j.foodcont.2022.109412.).

[0048] Example 1

[0049] The preparation steps of a fluorescence quenching biosensor for detecting Listeria monocytogenes are as follows:

[0050] (1) Preparation of the first activation mixture

[0051] 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride at a concentration of 10 mM and N-hydroxysuccinimide at a concentration of 5 mM were mixed at a volume ratio of 1:1 to obtain a first activation mixture.

[0052] (2) Preparation of the second activation mixture

[0053] Pure water was added to 3-(2-formylethyl)phosphine hydrochloride to obtain a 3-(2-formylethyl)phosphine hydrochloride solution with a final concentration of 2.867 g / L, namely, the second activated mixed solution.

[0054] (3) Preparation of fluorescent probes

[0055] See also Figure 1A in the mixture was prepared by adding 6 mL of deionized water to 1 mL of N-(β-aminoethyl-γ-aminopropyl)methyldimethoxysilane; then adding 8.0 mg of amikacin and stirring continuously for 30 min; heating at 200°C for 6 h, cooling to room temperature; adding pure water to dilute 20 times to obtain a reaction solution; 500 uL of the reaction solution and 250 uL of the first activated mixed solution were vortexed and mixed, and incubated on a shaker at 180 rpm and room temperature for 30 min; then 160 uL of the first aptamer solution with a volume concentration of 10 umol / L was added and vortexed, and incubated on a shaker at 180 rpm and room temperature for 2.5 h to obtain a fluorescent probe, which was then wrapped in tin foil and stored at 4°C in the dark.

[0056] The DNA sequence of the first aptamer in the first aptamer solution is shown as SEQ ID No: 1, and the 5' end of the first aptamer is modified with a carboxyl group.

[0057] (4) Preparation of quenching probe

[0058] See also Figure 1 In B, the specific operations are as follows:

[0059] (4.1) Add 1 mL of 1% tetrachloroauric acid solution to 100 mL of distilled water and boil. Quickly add 2.5 mL of 1% trisodium citrate solution and continue boiling for 15 min. Stop boiling and cool naturally to room temperature to obtain a gold nanoparticle solution.

[0060] (4.2) Place 100 μL of the second aptamer solution with a volume concentration of 10 μM in a water bath at 95°C for 5 min, and cool at 4°C for 5 min to obtain an annealing solution; add 10 μL of 3-(2-formylethyl)phosphine hydrochloride solution with a mass volume concentration of 2.867 g / L to the annealing solution, and let it react at room temperature for 30 min to obtain a reaction solution; add 1 mL of gold nanoparticle solution to the reaction solution, incubate at 37°C for 12 h, centrifuge at 12000 rpm for 20 min, remove the supernatant, and obtain a precipitate.

[0061] The DNA sequence of the second aptamer in the second aptamer solution is shown in SEQ ID NO: 2, and the 5' end of the second aptamer is modified with a thiol group.

[0062] (4.3) Add 400 μL of PBS solution with a mass concentration of 0.1 M and a pH value of 7.4 to the precipitate, and store it at 4°C wrapped in tin foil and protected from light to prepare a quenching probe;

[0063] (5) Preparation of biosensors

[0064] The fluorescent probe and the quenching probe are mixed at a volume ratio of 1:1.5 to prepare a fluorescence quenching biosensor;

[0065] See also Figure 2 , Figure 2 Curve A in FIG. 1 is a graph showing the fluorescence emission wavelength of silicon nanoparticles that have not been modified with the first aptamer. Figure 2 Curve B in FIG. 1 is a graph of the fluorescence emission wavelength of silicon nanoparticles modified with the second aptamer. When the fluorescence emission wavelength of the silicon nanoparticles shifts from 520 nm to 525 nm, it indicates that the first aptamer in the fluorescence quenching biosensor is successfully modified onto the silicon nanoparticles.

[0066] See also Figure 3 , Figure 3 Curve A in FIG. 1 is the UV absorption graph of gold nanoparticles without modification by the second aptamer. Figure 3 Curve B in FIG. 1 is the UV absorption graph of gold nanoparticles decorated with the second aptamer. When the UV absorption wavelength of the gold nanoparticles shifts from 522 nm to 526 nm, it indicates that the second aptamer in the fluorescence quenching biosensor is successfully modified onto the gold nanoparticles.

[0067] Example 2

[0068] Detection of Listeria monocytogenes using the fluorescence quenching biosensor prepared in Example 1

[0069] See also Figure 1 The specific detection steps are as follows:

[0070] (1) Establishment of linear regression equation: Prepare a test solution of the test sample that does not contain bacteria, set it as a blank control test solution, and artificially contaminate the blank control test solution with Listeria monocytogenes, and set a test solution containing bacteria in a linear gradient dilution.

[0071] The fluorescence quenching biosensor was mixed with the blank control test solution and the bacteria-containing test solution at a volume ratio of 1:1, and incubated for 35 minutes to obtain an incubation solution.

[0072] Under an excitation wavelength of 435 nm, the fluorescence intensity of the incubation solution at an emission wavelength of 525 nm was measured, wherein the fluorescence intensity of the blank control test solution was recorded as F0, and the fluorescence intensity of the bacteria-containing test solution was recorded as F; a standard curve was plotted with the fluorescence intensity ratio F / F0 as the ordinate and the logarithm of the bacterial concentration of the bacteria-containing test solution as the abscissa to obtain a linear regression equation, and the correlation coefficient and detection limit were obtained.

[0073] (2) Detection of Listeria monocytogenes in the test sample: prepare a test solution of the test sample, mix the fluorescence quenching biosensor and the test solution in a volume ratio of 1:1, incubate for 35 minutes, and obtain an incubation solution; measure the fluorescence intensity of the incubation solution at an emission wavelength of 525 nm under an excitation wavelength of 430 nm.

[0074] Substitute the obtained fluorescence intensity ratio F1 / F0 into the linear regression equation obtained in step (2) to obtain the concentration of Listeria monocytogenes in the sample to be tested.

[0075] During the experiment, PBS buffer was selected as the blank control test solution. At the same time, the bacterial solution of Listeria monocytogenes was centrifuged and washed three times with PBS buffer, and then resuspended and graded diluted with PBS buffer to make concentrations of 3.9*10 2 CFU / mL, 3.9*10 3 CFU / mL, 3.9*10 4 CFU / mL, 3.9*10 5 CFU / mL, 3.9*10 6 CFU / mL, 3.9*10 7 CFU / mL bacteria-containing test solution. First, take 100uL of the prepared fluorescence quenching biosensor, and incubate with 100uL of blank control test solution and 100uL of gradient diluted bacteria-containing test solution at 37°C for 35min to obtain an incubation solution. Then, under an excitation wavelength of 435nm, measure the fluorescence intensity of the incubation solution at an emission wavelength of 525nm, where the fluorescence intensity of the blank control test solution is recorded as F0, and the fluorescence intensity of the bacteria-containing test solution is recorded as F; use the fluorescence intensity ratio F / F0 as the ordinate and the logarithm of the bacterial solution concentration of the bacteria-containing test solution as the abscissa to draw a standard curve, obtain a linear regression equation, and obtain the correlation coefficient and detection limit.

[0076] The fluorescence quenching biosensor prepared by the present invention has a fluorescent probe with an obvious fluorescence emission peak at 525nm, and the ratio of the fluorescence intensity value F at 525nm to the fluorescence intensity value F0 at 525nm is linearly related to the logarithmic value of the bacterial solution concentration of Listeria monocytogenes.

[0077] Also, see Figure 4 , the fluorescence spectrum curves from top to bottom are: Figure 4 Curve 0 in the figure represents the fluorescence spectrum curve with a bacterial concentration of 0 CFU / mL (blank control test solution). Figure 4 Curve 3 in the figure indicates that the bacterial concentration is 3.9*10 3 Fluorescence spectrum curve of CFU / mL, Figure 4 Curve 4 in the figure shows that the bacterial concentration is 3.9*10 4 Fluorescence spectrum curve of CFU / mL, Figure 4 Curve 5 in the figure shows that the bacterial concentration is 3.9*10 5 Fluorescence spectrum curve of CFU / mL, Figure 4 Curve 6 in the figure shows that the bacterial concentration is 3.9*10 6Fluorescence spectrum curve of CFU / mL, Figure 4 Curve 7 in the figure shows that the bacterial concentration is 3.9*10 7 CFU / mL fluorescence spectrum curve; at the same time, combined with Figure 5 Let's take the above Figure 4 The quotient ratio between the fluorescence intensity value F at 525 nm and the fluorescence intensity value F0 at 525 nm on the fluorescence spectrum curve is taken as the ordinate, and the logarithm of the bacterial concentration corresponding to each fluorescence spectrum curve is taken as the abscissa. Figure 5 From the above, the linear regression equation is y=-0.10577x+1.25589, and its correlation coefficient (r 2 ) was 0.98154, and its detection limit was 19.27 CFU / mL.

[0078] In order to increase the detection of real food samples, LB broth samples were selected for testing:

[0079] It should be noted that in order to verify whether the above-mentioned standard curve can be effectively applied to the determination under the interference environment (LB broth), the difference between the number of bacteria detected by the probe in the artificial quantitative infection and LB broth samples was respectively carried out to find out that even in the real samples, the fluorescence quenching biosensor established by the present invention can effectively perform detection under the influence of nutrients such as high protein.

[0080] It should be noted that the LB broth involved in this Example 2 was purchased from Qingdao High-Tech Industrial Park Haibo Biotechnology Co., Ltd., and its ingredients (g / L) are as follows: 10.0 g / L tryptone, 5.0 g / L yeast extract powder, 10.0 g / L sodium chloride, pH 7.0±0.1.

[0081] In the experiment, Listeria monocytogenes was artificially inoculated in 1 mL of LB broth, and the final concentration of Listeria monocytogenes after inoculation was 3.9*10 6 CFU / mL, centrifuge at 5000×g for 5 min, discard the supernatant, resuspend the precipitate with 1 mL of PBS buffer as the bacteria-containing test solution, then take 500uL of the test solution and mix it with 500uL of the prepared fluorescence quenching biosensor, incubate for 35 min to obtain an incubation solution, then measure the fluorescence intensity of the incubation solution at an emission wavelength of 525nm under an excitation wavelength of 425nm, wherein the fluorescence intensity of the blank control test solution is recorded as F0, and the fluorescence intensity of the bacteria-containing test solution is recorded as F, finally, according to the obtained fluorescence intensity ratio F / F0, it is substituted into the linear regression equation obtained above to obtain the concentration of Listeria monocytogenes in the test sample.

[0082] The experimental results show that, according to calculation, the concentration of Listeria monocytogenes measured by the fluorescence quenching biosensor in the LB broth sample artificially inoculated with Listeria monocytogenes is 3.82*10 6 CFU / mL, almost the same as its actual concentration (3.9*10 6 CFU / mL), it can be seen that even if the corresponding standard curve and linear regression equation in the LB broth sample environment are not established, the standard curve and linear regression equation established in the pure PBS sample environment can still have good versatility. It can be seen that the fluorescence quenching biosensor prepared by the present invention has good versatility and can be quickly applied to different sample detections.

[0083] In addition, specific detection tests are performed:

[0084] Listeria monocytogenes was replaced with Staphylococcus aureus, Salmonella, Escherichia coli, Cronobacter sakazakii, and Pseudomonas aeruginosa, and the concentrations were 3.9*10 6 CFU / mL of the test solution containing bacteria.

[0085] Combination Figure 6 It can be seen that the fluorescence intensity ratios F / F0 of the bacteria-containing test solutions after adding Staphylococcus aureus, Salmonella, Escherichia coli, Cronobacter sakazakii and Pseudomonas aeruginosa are 0.98, 0.99, 0.99, 0.99 and 0.98, respectively. At the same time, the fluorescence intensity ratio F / F0 of the bacteria-containing test solution after adding Listeria monocytogenes under the same conditions is 0.57. In addition, the fluorescence intensity ratio F0 / F0 of the blank control test solution under the same conditions is 1. This shows that the fluorescence quenching biosensor prepared by the present invention has good specificity for Listeria monocytogenes.

[0086] Example 3

[0087] The fluorescence quenching biosensor prepared in Example 1 was used to detect Listeria monocytogenes in real food samples.

[0088] (1) Testing of tap water samples

[0089] Tap water: taken from the tap water pipes in the school toilets. The tap water temperature is 15℃.

[0090] The operation steps of the fluorescence quenching biosensor for visual detection of Listeria monocytogenes in tap water are as follows:

[0091] Preparation of tap water solution: 1 mL of tap water was artificially inoculated with Listeria monocytogenes to obtain a final concentration of 3.9*10 3 CFU / mL, 3.9*10 4CFU / mL, 3.9*10 5 CFU / mL, 3.9*10 6 CFU / mL, 3.9*10 7 CFU / mL; centrifuge the five solutions at 5000×g for 5 min respectively, and discard the supernatant; resuspend the precipitate of the five solutions with 1 mL of phosphate buffered saline respectively to obtain five tap water solutions.

[0092] Preparation of test solution: Mix the ratio fluorescent probe with five parts of tap water solution at a volume ratio of 1:1, incubate for 35 minutes, and obtain five parts of test solution;

[0093] Testing the test liquid:

[0094] The five samples of the test solution are respectively measured by a red, green and blue three-color imager under an excitation wavelength of 435 nm to generate images protected from light, and the red, green and blue values ​​of the images are read;

[0095] The ratio of the five red values ​​to the blue values ​​among the read red, green and blue values ​​was selected as the ordinate, and the logarithm of the bacterial concentration of the test liquid was selected as the abscissa to draw a standard curve, and the correlation coefficient and detection limit were obtained according to the linear regression equation.

[0096] Under the same conditions, uninoculated tap water was used as the blank control test solution, in which the concentration of Listeria monocytogenes was 0 CFU / mL.

[0097] The five test liquids were excited at 435 nm using a micro-fluorescence spectrometer (UV ultraviolet light equipment produced by Taiwan Guanghong Co., Ltd., model number Taiyi), and the RGB red, green and blue three-color imager (online test colorimeter produced by Admesy, the Netherlands, model number Hyperion) was used to analyze the RGB color values ​​of the five test liquids after the excitation treatment. Finally, a standard curve was drawn with the ratio of the red value to the blue value as the ordinate and the logarithmic value of the bacterial concentration of the five test liquids as the abscissa, and the correlation coefficient and the detection limit were obtained based on the linear regression equation.

[0098] Tap water solution:

[0099] See also Figure 7 In A, from left to right ("0", "3", "4", "5", "6", "7") are: detection images with a bacterial concentration of 0 CFU / mL (blank control test solution), bacterial concentration of 3.9*10 3 CFU / mL detection image, bacterial concentration is 3.9*10 4 CFU / mL detection image, bacterial concentration is 3.9*10 5CFU / mL detection image, bacterial concentration is 3.9*10 6 CFU / mL detection image, bacterial concentration is 3.9*10 7 The detection images of CFU / mL, and the ratios between the red value and the blue value corresponding to the above six detection images are 1.37, 1.26, 1.18, 1.11, 1.07, and 1.03 respectively. Figure 7 B in the image is used to analyze the RGB red, green and blue color values ​​of the detection image. Figure 1 The ratio of the red value to the blue value of each detection image in A is taken as the ordinate, and the logarithm of the bacterial concentration corresponding to each detection image is taken as the abscissa. The linear regression equation is y=-0.05591x+1.44309, and the correlation coefficient (r 2 ) was 0.97501, and its detection limit was 88.12 CFU / mL.

[0100] For the tap water solution, when the ratio between the red value and the blue value in the read red, green and blue values ​​is greater than 1.26, Listeria monocytogenes is not detected in the tap water; when the ratio is less than 1.26, Listeria monocytogenes is contained in the tap water. The concentration of Listeria monocytogenes in the tap water solution can be obtained by substituting the ratio into the linear regression equation.

[0101] The test results of tap water taken from the water pipes in the school toilets showed that the concentration of Listeria monocytogenes was 0 CFU / mL.

[0102] (2) Testing egg samples

[0103] Eggs: Purchased from the supermarket on campus, the eggs were stored in a refrigerator at 4°C. The manufacturer is Wen's Food Group Co., Ltd., its brand is "Wen's Tianlu Fresh Eggs", its product code is 65280985486, and its place of origin is Yancheng, Jiangsu.

[0104] The operation steps of the fluorescence quenching biosensor for visual detection of Listeria monocytogenes in eggs are as follows:

[0105] Preparation of egg solution: 1 mL of egg white was inoculated with Listeria monocytogenes to obtain a final concentration of 3.9*10 3 CFU / mL, 3.9*10 4 CFU / mL, 3.9*10 5 CFU / mL, 3.9*10 6 CFU / mL, 3.9*10 7 CFU / mL; centrifuge the five solutions at 5000×g for 5 min respectively and discard the supernatant; resuspend the precipitate of the five solutions with 1 mL of phosphate buffered saline respectively to obtain five egg solutions.

[0106] Preparation of test solution: Mix the ratio fluorescent probe with five egg solutions at a volume ratio of 1:1, incubate for 35 minutes, and obtain five test solutions;

[0107] Testing the test liquid:

[0108] The five samples of the test solution are respectively measured by a red, green and blue three-color imager under an excitation wavelength of 435 nm to generate images protected from light, and the red, green and blue values ​​of the images are read;

[0109] The ratio of the five red values ​​to the blue values ​​among the read red, green and blue values ​​was selected as the ordinate, and the logarithm of the bacterial concentration of the test liquid was selected as the abscissa to draw a standard curve, and the correlation coefficient and detection limit were obtained according to the linear regression equation.

[0110] Under the same conditions, uninoculated eggs were used as blank control test solution, in which the concentration of Listeria monocytogenes was 0 CFU / mL.

[0111] The five test liquids were excited at 435 nm using a micro-fluorescence spectrometer (UV ultraviolet light equipment produced by Taiwan Guanghong Co., Ltd., model number Taiyi), and the RGB red, green and blue three-color imager (online test colorimeter produced by Admesy, the Netherlands, model number Hyperion) was used to analyze the RGB color values ​​of the five test liquids after the excitation treatment. Finally, a standard curve was drawn with the ratio of the red value to the blue value as the ordinate and the logarithmic value of the bacterial concentration of the five test liquids as the abscissa, and the correlation coefficient and the detection limit were obtained based on the linear regression equation.

[0112] Egg solution:

[0113] See also Figure 8 In A, from left to right ("0", "3", "4", "5", "6", "7") are: detection images with a bacterial concentration of 0 CFU / mL (blank control test solution), bacterial concentration of 3.9*10 3 CFU / mL detection image, bacterial concentration is 3.9*10 4 CFU / mL detection image, bacterial concentration is 3.9*10 5 CFU / mL detection image, bacterial concentration is 3.9*10 6 CFU / mL detection image, bacterial concentration is 3.9*10 7 The detection images of CFU / mL, and the ratios between the red value and the blue value corresponding to the above six detection images are 1.41, 1.20, 1.12, 1.06, 1.04, and 0.99 respectively. Figure 8B in the image is used to analyze the RGB red, green and blue color values ​​of the detection image. Figure 1 The ratio of the red value to the blue value of each detection image in A is taken as the ordinate, and the logarithm of the bacterial concentration corresponding to each detection image is taken as the abscissa. The linear regression equation is y=-0.05079x+1.36724, and the correlation coefficient (r 2 ) was 0.95902, and its detection limit was 194.12 CFU / mL.

[0114] For the egg solution, when the ratio between the red value and the blue value in the read red, green and blue values ​​is greater than 1.20, Listeria monocytogenes is not detected in the eggs; when the ratio is less than 1.20, Listeria monocytogenes is contained in the eggs. The concentration of Listeria monocytogenes in the egg solution can be obtained by substituting the ratio into the linear regression equation.

[0115] The test results from eggs showed that the concentration of Listeria monocytogenes was 0 CFU / mL.

[0116] (3) Testing cheese samples

[0117] Cheese: Purchased from the supermarket on campus. The cheese was stored in a refrigerator at 4°C. The manufacturer is Three Squirrels Co., Ltd., and its brand is "Little Deer Blue Freeze-Dried Cheese Blocks Original Flavor". Its nutritional components are as follows: 2279kJ of energy, 14.3g of protein, 32.1g of fat, 49.9g of carbohydrates, 430mg of sodium, and 437mg of calcium per 100g.

[0118] The operation steps of the fluorescence quenching biosensor for visual detection of Listeria monocytogenes in cheese are as follows:

[0119] Preparation of cheese solution: Inoculate cheese with Listeria monocytogenes at an inoculum of 3.9*10 8 CFU / g, and let it stand for 2 hours at room temperature to obtain inoculated cheese; cut the inoculated cheese into pieces; in a sterilization bag, mix 25g of the inoculated cheese cut into pieces with 25mL of phosphate buffered saline solution, homogenize for 2 minutes to obtain a phosphate buffered saline solution mixture, let the mixture stand for 5 minutes, and take the supernatant; by adding phosphate buffered saline solution, the final concentration of Listeria monocytogenes in the supernatant was adjusted to 3.9*10 3 CFU / mL, 3.9*10 4 CFU / mL, 3.9*10 5 CFU / mL, 3.9*10 6 CFU / mL, 3.9*10 7 CFU / mL is five parts of solution, that is, five parts of cheese solution.

[0120] Preparation of test solution: Mix the ratio fluorescent probe with five parts of cheese solution in a volume ratio of 1:1, incubate for 35 minutes, and obtain five parts of test solution;

[0121] Testing the test liquid:

[0122] The five samples of the test solution are respectively measured by a red, green and blue three-color imager under an excitation wavelength of 435 nm to generate images protected from light, and the red, green and blue values ​​of the images are read;

[0123] The ratio between the five red values ​​and the blue value among the read red, green and blue values ​​was selected as the ordinate, and the logarithm of the bacterial concentration of the test liquid was selected as the abscissa to draw a standard curve, and the correlation coefficient and detection limit were obtained according to the linear regression equation.

[0124] Under the same conditions, uninoculated cheese was used as a blank control test solution, in which the concentration of Listeria monocytogenes was 0 CFU / mL.

[0125] The five test liquids were excited at 435 nm using a micro-fluorescence spectrometer (UV ultraviolet light equipment produced by Taiwan Guanghong Co., Ltd., model number Taiyi), and the RGB red, green and blue three-color imager (online test colorimeter produced by Admesy, the Netherlands, model number Hyperion) was used to analyze the RGB color values ​​of the five test liquids after the excitation treatment. Finally, a standard curve was drawn with the ratio of the red value to the blue value as the ordinate and the logarithmic value of the bacterial concentration of the five test liquids as the abscissa, and the correlation coefficient and the detection limit were obtained based on the linear regression equation.

[0126] Cheese solution:

[0127] See also Fig. 9 In A, from left to right ("0", "3", "4", "5", "6", "7") are: detection images with a bacterial concentration of 0 CFU / mL (blank control test solution), bacterial concentration of 3.9*10 3 CFU / mL detection image, bacterial concentration is 3.9*10 4 CFU / mL detection image, bacterial concentration is 3.9*10 5 CFU / mL detection image, bacterial concentration is 3.9*10 6 CFU / mL detection image, bacterial concentration is 3.9*10 7 The detection images of CFU / mL, and the ratios between the red value and the blue value corresponding to the above six detection images are 1.40, 1.20, 1.13, 1.05, 1.02, and 1.00 respectively. Fig. 9 B in the image is used to analyze the RGB red, green and blue color values ​​of the detection image. Figure 1 The ratio between the red value and the blue value of each detection image in A is taken as the ordinate, and the logarithm of the bacterial concentration corresponding to each detection image is taken as the abscissa. The linear regression equation is y=-0.04514x+1.33332, and the correlation coefficient (r 2 ) was 0.92661, and its detection limit was 323.73 CFU / mL.

[0128] For the cheese solution, when the ratio between the red value and the blue value in the read red, green and blue values ​​is greater than 1.20, Listeria monocytogenes is not detected in the cheese; when the ratio is less than 1.20, Listeria monocytogenes is contained in the cheese, and the concentration of Listeria monocytogenes in the cheese solution can be obtained by substituting the ratio into the linear regression equation.

[0129] The test results from the cheese showed that the concentration of Listeria monocytogenes was 0 CFU / mL.

[0130] It is easy for those skilled in the art to understand that the above embodiment 1 is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A fluorescence quenching biosensor for detecting Listeria monocytogenes, characterized in that: The fluorescent probe as an energy donor and the quenching probe as an energy acceptor are uniformly mixed in a volume ratio of 1:2; The fluorescent probe is a silicon nanoparticle modified with a first aptamer; The quenching probe is a gold nanoparticle modified with a second aptamer; The DNA sequence of the first aptamer is shown in SEQ ID No: 1, and the 5' end of the first aptamer is modified with a carboxyl group; The DNA sequence of the second aptamer is shown in SEQ ID NO: 2, and the 5' end of the second aptamer is modified with a thiol group; The linear fitting coefficient of the fluorescence quenching biosensor for detecting Listeria monocytogenes is 0.98154, and the detection limit is 19.27 CFU / mL.

2. The method for preparing a fluorescence quenching biosensor for detecting Listeria monocytogenes according to claim 1, characterized in that: The steps are as follows: (1) Preparation of the first activation mixture Mix 10 mM 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 5 mM N-hydroxysuccinimide in a volume ratio of 1:1 to obtain a first activation mixture; (2) Preparation of the second activation mixture Adding pure water to 3-(2-formylethyl)phosphine hydrochloride to obtain a 3-(2-formylethyl)phosphine hydrochloride solution with a final concentration of 2.867 g / L, i.e., the second activated mixed solution; (3) Preparation of fluorescent probes Add 6 mL of deionized water to 1 mL of N-(β-aminoethyl-γ-aminopropyl)methyldimethoxysilane; then add 8.0 mg of amikacin and stir continuously for 30 min; heat at 200°C for 6 h, cool to room temperature; add pure water to dilute 20 times to obtain a reaction solution; vortex mix 500 uL of the reaction solution and 250 uL of the first activation mixture, and incubate on a shaker at 180 rpm and room temperature for 30 min; then add 160 uL of the first aptamer solution with a volume concentration of 10 umol / L and vortex mix, and incubate on a shaker at 180 rpm and room temperature for 2.5 h to obtain a fluorescent probe, which is wrapped in tin foil and stored at 4°C in a dark place; The DNA sequence of the first aptamer in the first aptamer solution is as shown in SEQ ID No: 1, and the 5' end of the first aptamer is modified with a carboxyl group; (4) Preparation of quenching probe (4.1) Add 1 mL of 1% tetrachloroauric acid solution to 100 mL of distilled water and boil. Quickly add 2.5 mL of 1% trisodium citrate solution and continue boiling for 15 min. Cool naturally to room temperature to obtain a gold nanoparticle solution. (4.2) Place 100 μL of the second aptamer solution with a volume concentration of 10 μM in a water bath at 95°C for 5 min, and cool at 4°C for 5 min to obtain an annealing solution; add 10 μL of 3-(2-formylethyl)phosphine hydrochloride solution with a mass volume concentration of 2.867 g / L to the annealing solution, and allow to react at room temperature for 30 min to obtain a reaction solution; add 1 mL of the gold nanoparticle solution to the reaction solution, incubate at 37°C for 12 h, centrifuge at 12000 rpm for 20 min, remove the supernatant, and obtain a precipitate; The DNA sequence of the second aptamer in the second aptamer solution is as shown in SEQ ID NO: 2, and the 5' end of the second aptamer is modified with a thiol group; (4.3) Add 400 μL of PBS solution with a mass concentration of 0.1 M and a pH value of 7.4 to the precipitate, and store it at 4°C wrapped in tin foil and protected from light to prepare a quenching probe; (5) Preparation of biosensors The fluorescent probe and the quenching probe are mixed at a volume ratio of 1:1.5 to prepare a fluorescence quenching biosensor; When the fluorescence emission wavelength of silicon nanoparticles shifts from 520nm to 525nm, it indicates that the first aptamer in the fluorescence quenching biosensor is successfully modified onto the silicon nanoparticles. When the ultraviolet absorption wavelength of gold nanoparticles shifts from 522nm to 527nm, it indicates that the second aptamer in the fluorescence quenching biosensor is successfully modified onto the gold nanoparticles.

Images