A ratiometric fluorescent probe for detecting Listeria monocytogenes and preparation method thereof

Abstract

The present invention discloses a ratio fluorescent probe for detecting Listeria monocytogenes and a preparation method thereof, and belongs to the field of food safety technology. It is prepared by uniformly mixing a first fluorescent probe as an energy donor and a second fluorescent probe as an energy acceptor; the first fluorescent probe is carbon quantum dots modified with vancomycin; the second fluorescent probe is silicon nanoparticles modified with an aptamer; and the aptamer is used to specifically identify Listeria monocytogenes. It has been verified that the linear fitting coefficient of the ratio fluorescent probe of the present invention for detecting Listeria monocytogenes is 0.99332, and the detection limit is 2.06 CFU / mL. Compared with traditional methods, it also has a lower detection time and better specificity; in addition, under the interference of milk 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 ratio fluorescent probe for detecting Listeria monocytogenes and a preparation method thereof. Background Art

[0002] Foodborne diseases are common human diseases that cause infection or poisoning by ingesting food and allowing pathogenic factors to enter the body. They cause serious public health burdens worldwide and have adverse effects on economic and social development. According to data from the World Health Organization from 2020 to 2022, about 550 million to 600 million people worldwide become ill each year from eating contaminated food, of which about 230,000 die. Foodborne pathogens are a major cause of foodborne diseases, among which Listeria monocytogenes is a common pathogen. It is widely present in the environment, food, humans and animal hosts. It can survive under extreme conditions and cause food contamination. Eating food contaminated by this bacterium can cause listeriosis, with a mortality rate of up to 20%-30%, which poses a great threat to public health safety. For the detection of common pathogens, traditional culture methods are still recognized and reliable methods, but they require multiple steps such as bacterial isolation and culture, morphological observation, and biochemical identification, which are relatively cumbersome; conventional polymerase chain reaction (PCR) methods are limited by the long detection time. However, ELISA kits based on immunological technology are expensive and difficult to promote on a large scale. Therefore, there is an urgent need to develop simple, rapid, highly sensitive and accurate strategies and analytical methods for real-time detection of Listeria monocytogenes in food and the environment.

[0003] The detection methods of emerging fluorescent biological probes 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, Förster resonance energy transfer) is a non-radiative energy transition that transfers the energy of the donor excited state to the acceptor excited state through the electric dipole interaction between molecules, and is widely used in fluorescent probe detection technology. At present, the main fluorescent markers used as detection probes include organic fluorescent dyes and quantum dots. Among them, traditional organic fluorescent dyes have low brightness and serious photobleaching problems, which hinder their practical application. In addition, quantum dots are limited in application due to their cumbersome synthesis steps, poor stability, and incompatibility with various commercial media. Relatively speaking, carbon dots (CDs), also known as carbon nanodots or carbon quantum dots, are a type of zero-dimensional carbon nanomaterials with excellent fluorescence properties, which can be used as a potential non-toxic alternative to semiconductor quantum dots. Usually carbon dots are generally composed of sp 2 / sp 3It is composed of three parts: hybrid carbon, nitrogen / oxygen-containing groups and post-modified functional groups. It is spherical in shape and has a particle size of about 2nm-10nm. It can be divided into two types: amorphous and crystalline. In addition, 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 the proximity of ratiometric fluorescent probes coupled to the surface of microbial cells. Ratiometric fluorescent probes have attracted much attention in the detection of food microorganisms because of their advantages such as high sensitivity, simple operation and short response time to the target. This method focuses on the signal changes of two different fluorophores at the same time, normalizes the two signals or uses their ratio as the output signal, which is equivalent to introducing an internal standard, which can eliminate the interference of instrument noise and sample environment to a certain extent and improve the accuracy of the method. Currently, it is necessary to select a suitable first fluorescent probe that can serve as an energy donor and a suitable second fluorescent probe that can serve as an energy acceptor to form an effective FRET-type ratio fluorescent probe, especially a non-quenching ratio fluorescent probe. However, due to the special structure of microbial cells, it is not easy to form a FRET effect and it is easily affected by the rapid metabolic activity or immune response on the surface of the cells and fall off. Summary of the invention

[0004] In view of the problems that the existing detection technology of Listeria monocytogenes in food has great detection difficulty, long time consumption and low sensitivity, the present invention provides a ratio fluorescent probe for detecting Listeria monocytogenes and a preparation method thereof.

[0005] A ratio fluorescent probe for detecting Listeria monocytogenes is prepared by uniformly mixing a first fluorescent probe as an energy donor and a second fluorescent probe as an energy acceptor in a volume ratio of 1:1;

[0006] The first fluorescent probe is vancomycin-modified carbon quantum dots;

[0007] The second fluorescent probe is aptamer-modified silicon nanoparticles;

[0008] The vancomycin is vancomycin hydrochloride, with a CAS number of 1404-93-9;

[0009] The DNA sequence of the aptamer is shown in SEQ ID No: 1, and the 5' end of the aptamer is modified with an amino group;

[0010] The linear fitting coefficient of the ratio fluorescent probe used to detect Listeria monocytogenes is 0.99332, and the detection limit is 2.06 CFU / mL.

[0011] The preparation steps of the ratiometric fluorescent probe for detecting Listeria monocytogenes are as follows:

[0012] (1) Preparation of activation mixture

[0013] Mix 10 mM 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 5 mM N-hydroxysuccinimide in a volume ratio of 1:1 to obtain an activated mixed solution;

[0014] (2) Preparation of the first fluorescent probe

[0015] Guanidine hydrochloride and ultrapure water were mixed and dissolved in a mass volume ratio of 12 mg: 2 mL to obtain a mixed solution;

[0016] The mixed solution was transferred to a reaction kettle, the reaction temperature was 200°C, the reaction time was 8h, and it was cooled to room temperature to obtain a reaction solution;

[0017] The reaction solution was centrifuged at 8000 rpm and 4°C for 20 min, and the supernatant was collected;

[0018] The supernatant was mixed with the activation mixture in step (1) at a volume ratio of 2:1, and incubated at room temperature for 25 minutes; a 50 uM vancomycin solution was added, and the amount of the vancomycin solution added was 0.006 times the volume of the supernatant, and the mixture was incubated on a shaker at a speed of 180 rpm and room temperature for 2.5 hours to obtain a first fluorescent probe;

[0019] (3) Preparation of the second fluorescent probe

[0020] A 100 mM sodium ascorbate solution and 3-aminopropyltrimethoxysilane were mixed in a volume ratio of 1:1, and eight times the volume of ultrapure water were added and stirred for 80 minutes;

[0021] Dialyze for 18 h using a 500 Da dialysis bag to obtain the retentate;

[0022] The retention solution was mixed with the activation mixture in step (1) at a volume ratio of 2:1, and incubated at room temperature for 30 minutes; an aptamer solution with a concentration of 100 uM was added, and the amount of the aptamer solution added was 0.03 times the volume of the retention solution; and the mixture was incubated on a shaker at a speed of 180 rpm and room temperature for 2.5 hours to obtain a second fluorescent probe;

[0023] (4) Preparation of ratiometric fluorescent probes

[0024] The first fluorescent probe and the second fluorescent probe are mixed at a volume ratio of 1:1 to prepare a ratio fluorescent probe;

[0025] When the fluorescence emission wavelength of the carbon quantum dots shifts from 452nm to 458nm, it indicates that vancomycin in the ratio fluorescence probe is successfully modified onto the carbon quantum dots. When the fluorescence emission wavelength of the silicon nanoparticles shifts from 500nm to 488nm, it indicates that the aptamer in the ratio fluorescence probe is successfully modified onto the silicon nanoparticles.

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

[0027] 1. It has been verified that the ratio fluorescent probe prepared by the present invention has a high degree of linear fitting (correlation coefficient as high as 0.99332) and a low limit of detection (as low as 2.06 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 ratio fluorescent probe of the present invention has a lower detection time (only 25min) 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 hybridized 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.

[0028] 2. After testing, the linear regression equation established in the standard environment of the present invention can be directly applied to the detection of milk / LB broth samples. There is no need to re-cultivate bacteria to establish a standard curve. It is only necessary to directly apply the established linear regression equation, which saves time and is suitable for quickly estimating the initial concentration of Listeria monocytogenes in food samples under emergency conditions. For example, in a milk sample, under the environment of artificial inoculation of Listeria monocytogenes in milk, the concentration of Listeria monocytogenes measured by the ratio fluorescence probe is 3.86*10 6 CFU / mL, almost the same as its actual concentration (3.9*10 6 CFU / mL) is close. It can be seen that even if the corresponding standard curve and linear regression equation are not established in the milk sample environment, the standard curve and linear regression equation established in the pure PBS sample environment can still have good versatility. For example, in the environment of artificial inoculation of Listeria monocytogenes in LB broth, the measured concentration of Listeria monocytogenes is 3.83*10 6 CFU / mL, almost the same as its actual concentration (3.9*10 6CFU / 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 existing tap water environment can still have good versatility. It can be seen that the method of the present application has good versatility and can be quickly applied to different sample detection.

[0029] 3. In summary, the present application selects vancomycin-modified carbon quantum dots and aptamer-modified silicon nanoparticles to form a ratio-type fluorescent probe. 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 carbon quantum dots / silicon nanoparticles to the detection of foodborne Listeria monocytogenes. Since the original carbon quantum dots are used for the detection of small molecule α-glucosidase, and carbon nanoparticles are also used for the detection of small molecule α-glucosidase, it is unknown whether they can be applied to the detection of bacteria. It is necessary to consider not only Whether a complexation reaction occurs between the carbon quantum dots / silicon nanoparticles or chelation occurs with the outer surface of Listeria monocytogenes, and whether an effective FRET effect can occur when the above-mentioned carbon quantum dots / silicon nanoparticles are combined into a ratiometric fluorescent probe, the overlap degree of fluorescence emission-UV absorption between the carbon quantum dots and the silicon nanoparticles needs to be considered; in addition, the covalent coupling between the carbon quantum dots and vancomycin, the covalent coupling between the silicon nanoparticles and the aptamer, and the use concentration and volume ratio of the two probes and other complex factors need to be considered, 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 ratiometric fluorescent probe used to detect Listeria monocytogenes in the present invention.

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

[0032] Figure 3 This is a fluorescence spectrum diagram of the second fluorescent probe before and after covalent coupling with the nucleic acid 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. DETAILED DESCRIPTION

[0036] The present invention is further described below in conjunction with specific embodiments.

[0037] 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.

[0038] 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.

[0039] 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.

[0040] The PBS buffer used in this application is a sterile PBS buffer with a pH value of 7.4 and a 0.1 M content.

[0041] All bacterial strains used in this application were purchased from the American Type Culture Collection (ATCC).

[0042] 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.

[0043] The instruments, equipment, raw materials, reagents or method steps used in this application are ensured to be processed under sterile conditions.

[0044] The instruments, equipment, raw materials, reagents or method steps not mentioned in this application are conventional or well-known technical methods for those skilled in the art and are not 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 Vibrioparahaemolyticus, Food Control, Volume 145, 2023, 109412, ISSN 0956-7135, https: / / doi.org / 10.1016 / j.foodcont.2022.109412.).

[0045] Example 1

[0046] The preparation steps of the ratiometric fluorescent probe for detecting Listeria monocytogenes are as follows:

[0047] (1) Preparation of activation mixture

[0048] Mix 10 mM 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 5 mM N-hydroxysuccinimide in a volume ratio of 1:1, vortex mix in a vortex machine to obtain an activated mixed solution, and store it in a refrigerator at 4°C for later use;

[0049] (2) Preparation of the first fluorescent probe

[0050] See also Figure 1A in the mixture, guanidine hydrochloride and ultrapure water are mixed and dissolved at a mass volume ratio of 12 mg: 2 mL, and a vortex machine is used for sufficient shaking to obtain a mixed solution; the mixed solution is transferred to a hydrothermal synthesis reactor with a polytetrafluoroethylene liner for reaction, and the temperature is raised to 200°C in an oven for reaction, and the reaction time is controlled to be 8 hours to obtain a reaction solution, and the reaction solution is cooled to room temperature after the reaction is completed; the reaction solution is centrifuged at 8000 rpm and 4°C for 20 minutes, and the supernatant is collected; 500 uL of the supernatant and 250 uL of the activated mixed solution are respectively taken at a volume ratio of 2:1, and incubated at 180 rpm at room temperature for 25 minutes using a shaker; 3.2 uL of a 50 uM vancomycin solution is added, wherein the volume of the vancomycin solution is 0.006 times the volume of the supernatant, and the mixture is incubated at 180 rpm at room temperature for 2.5 hours on a shaker to obtain a first fluorescent probe;

[0051] (3) Preparation of the second fluorescent probe

[0052] See also Figure 1 In B, a 100 mM sodium ascorbate solution and 3-aminopropyltrimethoxysilane were mixed in a volume ratio of 1:1, and eight times the volume of ultrapure water was added and stirred for 80 minutes;

[0053] The solution was dialyzed for 18 h using a 500 Da dialysis bag to obtain the retentate and diluted fifteen times;

[0054] 500uL of the retention solution was mixed with 250uL of the activation mixture at a volume ratio of 2:1, and incubated at room temperature for 30min; 16uL of 100uM aptamer solution was added, and the amount of the aptamer solution added was 0.03 times the volume of the retention solution; the mixture was incubated on a shaker at a speed of 180rpm and room temperature for 2.5h to obtain a second fluorescent probe;

[0055] (4) Preparation of ratiometric fluorescent probes

[0056] 50uL of the first fluorescent probe and 50uL of the second fluorescent probe were mixed at a volume ratio of 1:1 to prepare a ratio fluorescent probe;

[0057] In the ratiometric fluorescent probe, vancomycin was successfully modified onto carbon quantum dots, and the aptamer was successfully modified onto silicon nanoparticles.

[0058] See also Figure 2 , Figure 2 Curve A in the figure represents the fluorescence spectrum curve before covalent coupling. Figure 2 Curve B in the figure represents the fluorescence spectrum curve after covalent coupling; specifically, Figure 2 Curve A in the figure is the fluorescence emission wavelength diagram of carbon quantum dots without vancomycin modification. Figure 2Curve B in the figure is the fluorescence emission wavelength diagram of vancomycin-modified carbon quantum dots. When the fluorescence emission wavelength of the carbon quantum dots shifts from 452nm to 458nm, it indicates that vancomycin in the ratiometric fluorescent probe is successfully modified onto the carbon quantum dots.

[0059] See also Figure 3 , Figure 3 Curve A in the figure represents the fluorescence spectrum curve before covalent coupling. Figure 3 Curve B in the figure represents the fluorescence spectrum curve after covalent coupling; specifically, Figure 3 Curve A in the figure is the fluorescence emission wavelength diagram of aptamer-modified silicon nanoparticles. Figure 3 Curve B in the figure is the fluorescence emission wavelength diagram of silicon nanoparticles without aptamer modification. When the fluorescence emission wavelength of silicon nanoparticles shifts from 500nm to 488nm, it means that the aptamer in the ratiometric fluorescence probe is successfully modified onto the silicon nanoparticles.

[0060] Example 2

[0061] See also Figure 1 C, the ratiometric fluorescent probe prepared in Example 1 is used for the detection of Listeria monocytogenes in the following steps:

[0062] (1) Establishment of linear regression equation: First, prepare a test solution of a non-bacterial test sample, set it as a blank control test solution, and artificially contaminate multiple blank control test solutions with Listeria monocytogenes. Set up multiple bacterial test solutions with linear gradient dilution. Then, mix the prepared ratio fluorescent probe with the bacterial test solution or the blank control test solution at a volume ratio of 1:1 and incubate for 25 minutes to obtain an incubation solution. Then, measure the fluorescence intensity of the incubation solution at two emission wavelengths of 458 nm and 488 nm under an excitation wavelength of 285 nm. Finally, use the fluorescence intensity ratio F 458 / F 488 As the ordinate, and the logarithmic value of the bacterial concentration of multiple bacteria-containing test solutions as the abscissa, a standard curve is drawn to obtain a linear regression equation, and the correlation coefficient and detection limit are obtained;

[0063] (2) Detection of Listeria monocytogenes in the sample to be tested: First, prepare the test solution of the sample to be tested, mix the prepared ratio fluorescence probe with the test solution at a volume ratio of 1:1, incubate for 25 minutes, and obtain an incubation solution. Then, measure the fluorescence intensity of the incubation solution at two emission wavelengths of 458 nm and 488 nm under an excitation wavelength of 285 nm. Finally, according to the obtained fluorescence intensity ratio F 458 / F 488 , substitute into the linear regression equation obtained in step (1) to obtain the concentration of Listeria monocytogenes in the sample to be tested.

[0064] During the experiment, the specific operation was as follows: 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 gradiently diluted with PBS buffer to make concentrations of 3.9*10 1 CFU / mL, 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 First, 100uL of the ratio fluorescence probe prepared in Example 1 was taken and incubated with 100uL of the blank control test solution and 100uL of the seven gradient dilutions of the test solution containing bacteria for 25min to obtain an incubation solution. Then, the fluorescence intensity of the incubation solution at the two emission wavelengths of 458nm and 488nm was measured under an excitation wavelength of 285nm. Finally, the fluorescence intensity ratio F was used. 458 / F 488 As the ordinate, and the logarithmic value of the bacterial concentration of multiple bacteria-containing test solutions as the abscissa, a standard curve is drawn to obtain a linear regression equation, and the correlation coefficient and detection limit are obtained.

[0065] The ratio fluorescent probe prepared by the present invention has a first fluorescent probe with an obvious fluorescence emission peak at 458 nm, and a second fluorescent probe with an obvious fluorescence emission peak at 488 nm. At the same time, the ratio between the fluorescence intensity value at 458 nm and the fluorescence intensity value at 488 nm is linearly related to the logarithmic value of the bacterial concentration of Listeria monocytogenes.

[0066] Also, see Figure 4 , from top to bottom are: Figure 4 Curve 1 in the middle represents the fluorescence spectrum curve with a bacterial concentration of 0 CFU / mL (blank control test solution), Figure 4 Curve No. 2 indicates that the bacterial concentration is 3.9*10 1 Fluorescence spectrum curve of CFU / mL, Figure 4 Curve No. 3 in the middle indicates that the bacterial concentration is 3.9*10 2 Fluorescence spectrum curve of CFU / mL, Figure 4 Curve No. 4 in the middle indicates that the bacterial concentration is 3.9*10 3 Fluorescence spectrum curve of CFU / mL, Figure 4 Curve No. 5 indicates that the bacterial concentration is 3.9*10 4 Fluorescence spectrum curve of CFU / mL, Figure 4 Curve No. 6 indicates that the bacterial concentration is 3.9*105 Fluorescence spectrum curve of CFU / mL, Figure 4 Curve No. 7 indicates that the bacterial concentration is 3.9*10 6 Fluorescence spectrum curve of CFU / mL, Figure 4 Curve No. 8 in the middle indicates 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 at 458 nm and the fluorescence intensity value at 488 nm on each 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. The linear regression equation is y=-0.03708x+0.64827, and the correlation coefficient (r 2 ) was 0.99332, and its detection limit was 2.06CFU / mL.

[0067] Example 3

[0068] The ratiometric fluorescent probe prepared in Example 1 was used to detect real food samples, and milk samples were selected for testing:

[0069] In order to verify whether the standard curve according to Example 2 can be effectively applied to the determination in an interference environment (milk), this Example 3 conducts artificial quantitative bacterial contamination and the difference between the number of bacteria detected by the probe in the milk sample to find out that even in real food samples, the ratio fluorescence probe established in this application can effectively detect under the influence of high protein and high fat.

[0070] The milk samples involved in this Example 3 were purchased from a supermarket on campus. When purchased, the milk was stored in a refrigerator at 4°C. It was "Mengniu Pure Milk", its type was full-fat sterilized milk, its ingredients were raw cow's milk, and the protein content in every 100g of milk was 3.2g, and the fat content was 4.0g.

[0071] In the experiment, Listeria monocytogenes was artificially inoculated in 1 mL of milk, 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 test solution, then take 500uL of the test solution and mix it with 500uL of the prepared ratio fluorescence probe, incubate for 25 min to obtain the incubation solution, then measure the fluorescence intensity of the incubation solution at the two emission wavelengths of 458nm and 488nm at the excitation wavelength of 285nm, and finally, according to the obtained fluorescence intensity ratio F 458 / F 488 , substitute into the linear regression equation obtained in step (1) of Example 2 to obtain the concentration of Listeria monocytogenes in the sample to be tested.

[0072] The experimental results show that, according to calculations, the concentration of Listeria monocytogenes measured by the ratio fluorescence probe is 3.86*10 6 CFU / mL, almost the same as its actual concentration (3.9*10 6 CFU / mL) is close. It can be seen that even if the corresponding standard curve and linear regression equation are not established in the milk sample environment, 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 ratio fluorescent probe prepared by the present invention has good versatility and can be quickly applied to different sample detections.

[0073] Example 4

[0074] Basically the same as Example 2, except that:

[0075] Listeria monocytogenes was replaced with Staphylococcus aureus, Salmonella, Escherichia coli, Cronobacter sakazakii, and Pseudomonas aeruginosa; and the concentrations of the bacteria were 3.9*10 6 CFU / mL of the test solution containing bacteria.

[0076] See also Figure 6 , the fluorescence intensity ratio F of the solution containing bacteria after adding Staphylococcus aureus, Salmonella, Escherichia coli, Cronobacter sakazakii, and Pseudomonas aeruginosa 458 / F 488 The ratios between them were 0.70866, 0.71062, 0.70962, 0.70676, and 0.71123, respectively. At the same time, the fluorescence intensity ratio F 458 / F 488 The ratio is 0.40643. In addition, under the same conditions, the fluorescence intensity ratio of the blank control solution to be tested is F 458 / F 488 The ratio is 0.70760, which indicates that the ratio fluorescent probe prepared by the present invention has good specificity for Listeria monocytogenes.

[0077] It will be easily understood by those skilled in the art that the above embodiments are merely preferred embodiments of the present invention and are 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 ratiometric fluorescent probe for detecting Listeria monocytogenes, characterized in that: The first fluorescent probe as an energy donor and the second fluorescent probe as an energy acceptor are uniformly mixed in a volume ratio of 1:1; The first fluorescent probe is vancomycin-modified carbon quantum dots; The second fluorescent probe is aptamer-modified silicon nanoparticles; The vancomycin is vancomycin hydrochloride, with a CAS number of 1404-93-9; The DNA sequence of the aptamer is shown in SEQ ID No: 1, and the 5' end of the aptamer is modified with an amino group; The linear fitting coefficient of the ratiometric fluorescent probe for detecting Listeria monocytogenes was 0.99332, and the detection limit was 2.06 CFU / mL; The preparation steps of the ratiometric fluorescent probe for detecting Listeria monocytogenes are as follows: (1) Preparation of 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 an activated mixed solution; Preparation of the first fluorescent probe Guanidine hydrochloride and ultrapure water were mixed and dissolved in a mass volume ratio of 12 mg: 2 mL to obtain a mixed solution; The mixed solution was transferred to a reaction kettle, the reaction temperature was 200°C, the reaction time was 8h, and it was cooled to room temperature to obtain a reaction solution; The reaction solution was centrifuged at 8000 rpm and 4°C for 20 min, and the supernatant was collected; The supernatant was mixed with the activation mixture in step (1) at a volume ratio of 2:1, and incubated at room temperature for 25 minutes; a 50 uM vancomycin solution was added, and the amount of the vancomycin solution added was 0.006 times the volume of the supernatant, and the mixture was incubated on a shaker at a speed of 180 rpm and room temperature for 2.5 hours to obtain a first fluorescent probe; Preparation of the second fluorescent probe A 100 mM sodium ascorbate solution and 3-aminopropyltrimethoxysilane were mixed in a volume ratio of 1:1, and eight times the volume of ultrapure water was added and stirred for 80 minutes; dialyzed for 18 hours with a 500Da dialysis bag to obtain a retentate; The retention solution was mixed with the activation mixture in step (1) at a volume ratio of 2:1, and incubated at room temperature for 30 minutes; an aptamer solution with a concentration of 100 uM was added, and the amount of the aptamer solution added was 0.03 times the volume of the retention solution; and the mixture was incubated on a shaker at a speed of 180 rpm and room temperature for 2.5 hours to obtain a second fluorescent probe; Preparation of ratiometric fluorescent probes The first fluorescent probe and the second fluorescent probe are mixed at a volume ratio of 1:1 to prepare a ratio fluorescent probe; When the fluorescence emission wavelength of the carbon quantum dots shifts from 452nm to 458nm, it indicates that vancomycin in the ratio fluorescence probe is successfully modified onto the carbon quantum dots. When the fluorescence emission wavelength of the silicon nanoparticles shifts from 500nm to 488nm, it indicates that the aptamer in the ratio fluorescence probe is successfully modified onto the silicon nanoparticles.

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