CN116554860A - Ratio fluorescent probe for detecting listeria monocytogenes and preparation method thereof - Google Patents
Ratio fluorescent probe for detecting listeria monocytogenes and preparation method thereof Download PDFInfo
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- 241000186779 Listeria monocytogenes Species 0.000 title claims abstract description 44
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Abstract
The invention discloses a ratio fluorescent probe for detecting listeria monocytogenes and a preparation method thereof, belonging to the technical field of food safety. Is prepared by uniformly mixing a first fluorescent probe serving as an energy donor and a second fluorescent probe serving as an energy acceptor; the first fluorescent probe is a vancomycin modified carbon quantum dot; the second fluorescent probe is an aptamer modified silicon nanoparticle; the aptamer is used for specifically recognizing listeria monocytogenes. The linear fitting coefficient of the ratio fluorescent probe for detecting listeria monocytogenes is 0.99332, and the detection limit is 2.06CFU/mL. Compared with the traditional method, the method has lower detection time and better specificity; in addition, the established linear regression equation still shows excellent applicability under the interference of the milk sample.
Description
Technical Field
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
Food-borne diseases are common human diseases causing infection or poisoning by intake of pathogenic factors into the body through food intake, and cause serious public health burden on the global scale, and have adverse effects on economic and social development. According to the data of the world health organization 2020-2022, about 5.5-6 hundred million people are ill each year worldwide due to the consumption of contaminated food, wherein about 23 ten thousand people die and food-borne pathogenic bacteria are a major cause of food-borne diseases, and listeria monocytogenes is a common pathogenic bacteria which is widely present in environmental, food, human and animal hosts, can survive under extreme conditions and cause food contamination, and the eating of food contaminated by the bacteria causes listeriosis with a mortality rate as high as 20% -30%, and has a great hazard to public health safety. For detection of common pathogenic bacteria, the traditional culture method is still a recognized and reliable method, but the traditional culture method is complicated by a plurality of steps such as bacteria separation culture, morphological observation, biochemical identification and the like; conventional polymerase chain reaction (polymerase chain reaction, PCR) methods are limited by the long detection times. ELISA kit based on immunological technology is high in price and difficult to popularize and use in industrialization on a large scale. Thus, there is an urgent need to develop simple, rapid, highly sensitive, accurate strategies and analytical methods for real-time detection of listeria monocytogenes in foods and environments.
The detection method of the emerging fluorescent biological probe is widely applied to the determination of target substances due to the advantages of low cost, simple and convenient operation, quick response, high stability and the like. Among them, fluorescence resonance energy transfer (FRET, ffos) is widely used in fluorescent probe detection technology as a non-radiative energy transfer that transfers energy of a donor excited state to an acceptor excited state through intermolecular electric dipole interaction. At present, the main fluorescent markers used as detection probes comprise organic fluorescent dyes and quantum dots. Among them, the conventional organic fluorescent dyes have problems of low brightness and serious photobleaching, which prevent their practical application. In addition, quantum dots have limited applications due to the disadvantages of complicated synthesis steps, poor stability, incompatibility with various commercial media, and the like. Relatively speaking, carbon Dots (CDs) are also called carbon nanodots or carbon quantum dots, and are zero-dimensional carbon nanomaterials with excellent fluorescence properties, and can be used as potential nontoxic substitutes for semiconductor quantum dots. Typically the carbon point is generally defined by sp 2 /sp 3 The hybrid carbon, the nitrogen/oxygen-containing group and the post-modification functional group are formed, the morphology is spherical, the particle size is about 2nm-10nm, and the hybrid carbon can be divided into two types of amorphous and crystalline. In addition, silicon nanoparticles (silicon-containing nanoparticles, SNPs) have been relatively rarely studied, and are often prepared by catalytic reduction of a silicon-containing coupling agent. Methods based on fluorescence resonance energy transfer are mostly achieved by shortening the distance between donor and acceptor, for example, by using proximity of a ratio-type fluorescent probe coupled to the surface of microbial cells. The ratio-type fluorescent probe has the advantages of high sensitivity, simple operation, short response time to a target object and the like, and is attracting attention in detection of food microorganisms. The method simultaneously pays attention to the signal change of two different fluorophores, normalizes the two signals or takes the ratio of the two signals as an output signal, which is equivalent to introducing an internal standard, can be implemented to a certain degreeAnd the noise of the instrument and the interference of the environment of the sample to be detected are eliminated, and the accuracy of the method is improved. Currently, 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 constitute an effective FRET-type ratio fluorescent probe, especially a non-quenching-type ratio fluorescent probe, but since the microbial cell structure is special, FRET effect is not easily formed, and the FRET is easily detached due to rapid metabolic activity or immune reaction of the microbial cell surface.
Disclosure of Invention
Aiming at the problems of high detection difficulty, long time consumption and low sensitivity of the existing detection technology of the listeria monocytogenes in food, the invention provides a ratio fluorescent probe for detecting the listeria monocytogenes and a preparation method thereof.
A ratio fluorescent probe for detection of listeria monocytogenes is composed of a first fluorescent probe as an energy donor and a second fluorescent probe as an energy acceptor in a volume ratio of 1:1, uniformly mixing to prepare the mixture;
the first fluorescent probe is vancomycin modified carbon quantum dots;
the second fluorescent probe is an aptamer modified silicon nanoparticle;
the vancomycin is vancomycin hydrochloride, and the CAS number is 1404-93-9;
the DNA sequence of the aptamer is shown as SEQ ID No. 1, and the 5' end of the aptamer is modified by amino;
the linear fitting coefficient of the ratio fluorescent probe for detecting listeria monocytogenes is 0.99332, and the detection limit is 2.06CFU/mL.
The preparation procedure for the ratiometric fluorescent probe for detection of listeria monocytogenes is as follows:
(1) Preparation of activated Mixed solution
According to the volume ratio of 1:1, mixing 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride with the concentration of 10mM and N-hydroxysuccinimide with the concentration of 5mM to obtain an activated mixed solution;
(2) Preparation of first fluorescent Probe
According to the mass volume ratio of 12mg: mixing and dissolving guanidine hydrochloride and ultrapure water by 2mL to obtain a mixed solution;
transferring the mixed solution into a reaction kettle, reacting at 200 ℃ for 8 hours, and cooling to room temperature to obtain a reaction solution;
centrifuging the reaction solution for 20min at the rotation speed of 8000rpm and the temperature of 4 ℃ and collecting supernatant;
according to the volume ratio of 2:1 mixing the supernatant with the activation mixed solution in the step (1), and incubating for 25min at room temperature; adding a vancomycin solution with the concentration of 50uM, wherein the addition amount of the vancomycin solution is 0.006 times of the volume of the supernatant, and incubating for 2.5 hours in a shaking table under the conditions of the rotating speed of 180rpm and the room temperature to prepare a first fluorescent probe;
(3) Preparation of the second fluorescent Probe
According to the volume ratio of 1:1, mixing sodium ascorbate solution with the molar concentration of 100mM with 3-aminopropyl trimethoxysilane, adding eight times of volume of ultrapure water, stirring and mixing uniformly, and stirring and mixing uniformly for 80min;
dialyzing with 500Da dialysis bag for 18h to obtain a retentate;
according to the volume ratio of 2:1, mixing the retention solution with the activation mixed solution in the step (1), and incubating for 30min at room temperature; adding an aptamer solution with the concentration of 100uM, wherein the addition amount of the aptamer solution is 0.03 times of the volume of the retention solution; incubating the mixture for 2.5 hours in a shaking table at the rotating speed of 180rpm and at the room temperature to prepare a second fluorescent probe;
(4) Preparation of ratio fluorescent probes
According to the volume ratio of 1:1 mixing the first fluorescent probe with the second fluorescent probe to prepare a ratio fluorescent probe;
when the fluorescence emission wavelength of the carbon quantum dots is shifted from 452nm to 458nm, the vancomycin in the ratio fluorescent probe is successfully modified on the carbon quantum dots, and when the fluorescence emission wavelength of the silicon nanoparticles is shifted from 500nm to 488nm, the aptamer in the ratio fluorescent probe is successfully modified on the silicon nanoparticles.
The beneficial technical effects of the invention are as follows:
1. proved by verification, the ratio fluorescent probe prepared by the invention has higher linear fitting degree (the correlation coefficient is up to 0.99332) and lower detection limit (as low as 2.06 CFU/mL), and the linear fitting degree of the current prior art is generally in the range of 0.90-0.99; meanwhile, compared with the traditional technology (a strand displacement reaction method, a PCR amplification method and an ELISA kit method), the detection time is as long as 12h-3d, the specificity of the probe is different for part of common mode strains, the specific fluorescent probe has lower detection time (only 25 min), better specificity (for common mode strains such as staphylococcus aureus and escherichia coli) and the like, for example, the traditional strand displacement reaction utilizes the free energy difference of hybridization of DNA molecules, and uses a single-stranded DNA to displace a target single strand in a double-helix structure of the hybridized DNA, and the efficiency of the strand displacement reaction is relatively low because the phosphate groups with negative charges on the single-stranded DNA have an intramolecular electrostatic repulsion effect on the double-stranded DNA.
2. Through tests, the linear regression equation established under the standard environment can be directly applied to the detection of the milk/LB broth sample, bacteria are not required to be re-cultured to establish a standard curve, the established linear regression equation is only required to be directly applied, time is saved, and the method is suitable for rapidly estimating the primary concentration of Listeria monocytogenes in the food sample under the emergency environment. For example, in a milk sample, the concentration of listeria monocytogenes measured by a ratiometric fluorescent probe in the context of artificial inoculation of listeria monocytogenes in milk is 3.86 x 10 6 CFU/mL, almost the same as its true concentration (3.9 x 10 6 CFU/mL) is close, it can be seen that even if the corresponding standard curve and linear regression equation under the milk sample environment are not established, the standard curve and linear regression equation established under the pure PBS sample environment can still have better universality. For another example, in an environment in which Listeria monocytogenes is artificially inoculated in LB broth, the measured concentration of Listeria monocytogenes is 3.83×10 6 CFU/mL, almost the same as its true concentration (3.9 x 10 6 CFU/mL), it can be seen that even if the corresponding standard curve and linear regression equation under the LB broth sample environment are not established, the standard curve and linear regression equation established under the existing tap water environment still have better universality, so that the method has better universality and can be rapidly applied to different sample detection。
3. In summary, the application selects the vancomycin-modified carbon quantum dot and aptamer-modified silicon nanoparticle composition ratio type fluorescent probe, which is the first combination of two different types of probes, and the selection of proper pairing fluorescent probes from tens of thousands of similar quantum dots or nanoparticles requires great creative labor, meanwhile, the prior art does not disclose that the carbon quantum dot/silicon nanoparticle is applied to the detection of food-borne listeria monocytogenes, because the original carbon quantum dot is used for the detection of small molecular alpha-glucosidase and the carbon nanoparticle is also used for the detection of small molecular alpha-glucosidase, whether the carbon quantum dot/silicon nanoparticle can be applied to the detection of bacteria can not be known, whether the complexation reaction occurs between the carbon quantum dot/silicon nanoparticle or the chelation occurs with the outer surface of the listeria monocytogenes or not only needs to be considered, and the combination of the carbon quantum dot/silicon nanoparticle ratio fluorescent probe also needs to consider whether the effective FRET effect can occur or not, and the degree of overlapping of fluorescence emission-ultraviolet absorption between the carbon quantum dot and the silicon nanoparticle needs to be considered; in addition, the covalent coupling between the carbon quantum dots and vancomycin, the covalent coupling between the silicon nano-particles and the aptamer, and the use concentration and volume ratio between the two probes are considered as a plurality of complex factors, which are not disclosed in the prior art and are not suggested by the corresponding common general knowledge in the field.
Drawings
FIG. 1 is a schematic diagram of the application of a ratiometric fluorescent probe for detection of Listeria monocytogenes in the present invention.
FIG. 2 is a graph showing fluorescence spectra before and after covalent coupling of a first fluorescent probe with vancomycin according to the present invention.
FIG. 3 is a graph showing fluorescence spectra before and after covalent coupling of a second fluorescent probe with a nucleic acid aptamer according to the present invention.
FIG. 4 is a graph showing fluorescence spectra of different concentrations of Listeria monocytogenes in the present invention.
FIG. 5 is a standard graph of Listeria monocytogenes of the present invention.
FIG. 6 is a diagram showing the detection of Listeria monocytogenes specificity of the present invention.
Detailed Description
The invention is further described below in connection with specific embodiments.
Unless defined otherwise, 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.
The raw materials used in the following examples are all conventional biochemical reagents unless otherwise specified; the experimental methods are all conventional methods unless specified; the quantitative tests in the following examples were carried out by setting three repeated experiments, and taking the average value of the results, unless otherwise specified; in the following examples, the percentages are by mass unless otherwise indicated.
In the following examples, the aptamers used were purchased from the biological engineering (Shanghai) stock Co.Ltd; 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride, N-hydroxysuccinimide, guanidine hydrochloride, vancomycin, sodium ascorbate and 3-aminopropyl trimethoxysilane used were purchased from Shanghai Ala-dine Biochemical technologies Co., ltd; the 500Da dialysis bag used was purchased from Shanghai Seiyaka Biotechnology Co., ltd; unless otherwise indicated, all other materials used were purchased from national pharmaceutical group chemical company, inc.
The PBS buffer used in this application was a sterile PBS buffer with a value of 7.4 of 0.1M, pH.
All species used in this application were purchased from the American Type Culture Collection (ATCC),
wherein listeria monocytogenes is numbered ATCC 43251, wherein staphylococcus aureus is numbered ATCC 29213, wherein salmonella is numbered ATCC 14028, wherein escherichia coli is numbered ATCC 25922, wherein cronobacter sakazakii is numbered ATCC29544, and wherein pseudomonas aeruginosa is numbered ATCC 15442. In addition, the above strain was purchased by foreign agency through Hefei Xueyue biotechnology Co., ltd.
The instrumentation, starting reagents, or method steps used in the present application ensure that the process is performed under sterile conditions.
The instrumentation, starting reagents or method steps not mentioned in the present application, which are conventional or known technical methods for the person skilled in the art, are not described in detail in the present application, and reference may be made to the following paper documents (yuwei Ren, lu 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 detectionof Vibrio parahaemolyticus, food Control, volume 145,2023,109412,ISSN 0956-7135, https:// doi.org/10.1016/j.foodcon.2022.109412).
Example 1
The preparation procedure for the ratiometric fluorescent probe for detection of listeria monocytogenes is as follows:
(1) Preparation of activated Mixed solution
According to the volume ratio of 1:1, mixing 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride with the concentration of 10mM and N-hydroxysuccinimide with the concentration of 5mM, performing vortex mixing treatment in a vortex machine to prepare an activated mixed solution, and storing the activated mixed solution in a refrigerator with the temperature of 4 ℃ for later use;
(2) Preparation of first fluorescent Probe
Referring to a in fig. 1, guanidine hydrochloride and ultrapure water were mixed in a mass volume ratio of 12mg:2mL is mixed and dissolved, and a vortex machine is adopted for full oscillation to obtain a mixed solution; transferring the mixed solution into a hydrothermal synthesis reaction kettle with a polytetrafluoroethylene lining for reaction, heating to 200 ℃ by using an oven for reaction, controlling the reaction time to 8 hours to obtain a reaction solution, and cooling the reaction solution to room temperature after the reaction is finished; centrifuging the reaction solution at 8000rpm and 4 ℃ for 20min, and collecting supernatant; according to the volume ratio of 2:1 respectively taking 500uL of supernatant and 250uL of activated mixed solution, mixing, and incubating for 25min at room temperature at 180rpm by using a shaking table; adding 3.2uL of vancomycin solution with the concentration of 50uM, wherein the volume of the vancomycin solution is 0.006 times that of the supernatant, and incubating for 2.5 hours by a shaker at 180rpm and room temperature to prepare a first fluorescent probe;
(3) Preparation of the second fluorescent Probe
See B in fig. 1, in volume ratio 1:1, mixing sodium ascorbate solution with the molar concentration of 100mM with 3-aminopropyl trimethoxysilane, adding eight times of volume of ultrapure water, stirring and mixing uniformly, and stirring and mixing uniformly for 80min;
dialyzing with 500Da dialysis bag for 18 hr to obtain retentate, and diluting with fifteen times;
according to the volume ratio of 2:1 mixing 500uL of the retention solution and 250uL of the activation mixed solution, and incubating for 30min at room temperature; adding 16uL of an aptamer solution with the concentration of 100uM, wherein the addition amount of the aptamer solution is 0.03 times of the volume of the retention solution; incubating the mixture for 2.5 hours in a shaking table at the rotating speed of 180rpm and at the room temperature to prepare a second fluorescent probe;
(4) Preparation of ratio fluorescent probes
According to the volume ratio of 1:1, mixing 50uL of a first fluorescent probe and 50uL of a second fluorescent probe to prepare a ratio fluorescent probe;
vancomycin in the ratio fluorescent probe is successfully modified on the carbon quantum dot, and the aptamer is successfully modified on the silicon nanoparticle.
Referring to fig. 2, a curve in fig. 2 represents a fluorescence spectrum curve before covalent coupling, and a curve B in fig. 2 represents a fluorescence spectrum curve after covalent coupling; specifically, the curve a in fig. 2 is a graph of fluorescence emission wavelength of carbon quantum dots not modified by vancomycin, and the curve B in fig. 2 is a graph of fluorescence emission wavelength of carbon quantum dots modified by vancomycin, when the fluorescence emission wavelength of the carbon quantum dots shifts from 452nm to 458nm, the vancomycin in the ratiometric fluorescent probe is successfully modified on the carbon quantum dots.
Referring to fig. 3, a curve in fig. 3 represents a fluorescence spectrum curve before covalent coupling, and a curve B in fig. 3 represents a fluorescence spectrum curve after covalent coupling; specifically, the curve a in fig. 3 is a graph of the fluorescent emission wavelength of the aptamer-modified silicon nanoparticle, and the curve B in fig. 3 is a graph of the fluorescent emission wavelength of the silicon nanoparticle without the aptamer modification, when the fluorescent emission wavelength of the silicon nanoparticle is shifted from 500nm to 488nm, indicating that the aptamer in the ratiometric fluorescent probe is successfully modified onto the silicon nanoparticle.
Example 2
Referring to FIG. 1C, the ratio fluorescent probe prepared in example 1 was used for detection of Listeria monocytogenes as follows:
(1) Linear regression formulaAnd (3) program establishment: firstly, preparing a to-be-detected solution of a to-be-detected sample without bacteria, setting the to-be-detected solution as a blank control to-be-detected solution, carrying out artificial pollution on a plurality of blank control to-be-detected solutions to obtain listeria monocytogenes, setting a plurality of bacteria-containing to-be-detected solutions which are diluted in a linear gradient, and then, mixing the prepared ratio fluorescent probe with the bacteria-containing to-be-detected solution or the blank control to-be-detected solution according to a volume ratio of 1:1, incubating for 25min to obtain an incubation liquid, then respectively measuring fluorescence intensities of the incubation liquid at two emission wavelengths of 458nm and 488nm at an excitation wavelength of 285nm, and finally, obtaining a fluorescence intensity ratio F 458 /F 488 As an ordinate, drawing a standard curve by taking the logarithmic values of the bacterial concentrations of a plurality of bacteria-containing solutions to be detected as an abscissa, and obtaining a linear regression equation to obtain a correlation coefficient and a detection limit;
(2) Detection of listeria monocytogenes in a sample to be tested: firstly, preparing a to-be-detected solution of a to-be-detected sample, and mixing the prepared ratio fluorescent probe with the to-be-detected solution according to a volume ratio of 1:1, incubating for 25min to obtain an incubation liquid, then respectively measuring fluorescence intensities of the incubation liquid at two emission wavelengths of 458nm and 488nm at an excitation wavelength of 285nm, and finally obtaining a fluorescence intensity ratio F according to the obtained fluorescence intensity ratio 458 /F 488 Substituting the linear regression equation obtained in the step (1) to obtain the concentration of the listeria monocytogenes in the sample to be detected.
During experiments, the specific operation is that PBS buffer solution is selected as a blank control solution to be tested, meanwhile, bacterial liquid of Listeria monocytogenes is centrifuged and then washed three times by the PBS buffer solution, and then the PBS buffer solution is used for resuspension and gradient dilution, so that the preparation concentrations are respectively 3.9 x 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 CFU/mL of bacteria-containing solution to be tested. Firstly taking 100uL of the ratio fluorescent probe prepared in the embodiment 1, respectively incubating with 100uL of a blank control to-be-detected solution and the seven gradient diluted 100uL of a bacteria-containing to-be-detected solution for 25min to obtain an incubation liquid, then respectively measuring the fluorescent intensity of the incubation liquid at 458nm and 488nm at an excitation wavelength of 285nm, and finally, using a fluorescent intensity ratio F 458 /F 488 And drawing a standard curve by taking the logarithmic values of the bacterial concentrations of a plurality of bacteria-containing solutions to be detected as the ordinate and taking the logarithmic values as the abscissa to obtain a linear regression equation, thereby obtaining a correlation coefficient and a detection limit.
The ratio fluorescent probe prepared by the invention has a distinct fluorescent emission peak at 458nm, and the second fluorescent probe has a distinct fluorescent emission peak at 488nm, and meanwhile, the ratio between the fluorescent intensity value at 458nm and the fluorescent intensity value at 488nm is in linear relation with the logarithmic value of the bacterial concentration of listeria monocytogenes.
Meanwhile, referring to fig. 4, from top to bottom, respectively: FIG. 4 shows a fluorescence spectrum curve (blank test solution) with a bacterial concentration of 0CFU/mL, and FIG. 4 shows a fluorescence spectrum curve with a bacterial concentration of 3.9X10 1 Fluorescence spectrum curve of CFU/mL, curve No. 3 in FIG. 4, shows that the concentration of bacteria is 3.9X10 2 Fluorescence spectrum curve of CFU/mL, curve 4 in FIG. 4 shows that the concentration of bacteria is 3.9×10 3 Fluorescence spectrum curve of CFU/mL, curve No. 5 in FIG. 4, shows that the concentration of bacteria is 3.9×10 4 Fluorescence spectrum curve of CFU/mL, curve 6 in FIG. 4, shows that the concentration of bacteria is 3.9×10 5 Fluorescence spectrum curve of CFU/mL, curve 7 in FIG. 4, shows that the concentration of bacteria is 3.9X10 6 Fluorescence spectrum curve of CFU/mL, curve 8 in FIG. 4, shows that the concentration of bacteria is 3.9×10 7 Fluorescence spectrum curve of CFU/mL; meanwhile, as seen from fig. 5, taking the quotient of the fluorescent intensity value of 458nm and the fluorescent intensity value of 488nm on each of the fluorescent spectrum curves in fig. 4 as the ordinate, taking the logarithmic value of the bacterial concentration corresponding to each of the fluorescent spectrum curves as the abscissa, the linear regression equation is y= -0.03708x+0.64827, the correlation coefficient (r 2 ) 0.99332, the detection limit is 2.06CFU/mL.
Example 3
Detection of authentic food samples by ratiometric fluorescent probes prepared in example 1, milk samples were selected for testing:
in order to verify whether the standard curve according to example 2 can be effectively used for measurement in a disturbing environment (milk), the difference between the number of bacteria detected by the probe in the artificially quantified bacteria-stained and milk samples, respectively, was performed to know that the ratio fluorescent probe established in the present application can be effectively detected under the influence of high protein and high fat even in a real food sample.
The milk sample according to example 3 was purchased from a school supermarket, and the milk was stored in a refrigerator at 4℃during purchase, which was "Mongolian Niu Chun milk", which was a whole sterilized milk, which was prepared as cow milk with a protein content of 3.2g and a fat content of 4.0g per 100g of milk.
At the time of the experiment, 1mL of milk was artificially inoculated with listeria monocytogenes, wherein the final concentration of listeria monocytogenes after inoculation was 3.9x10 6 Centrifuging CFU/mL at 5000 Xg for 5min, discarding supernatant, re-suspending precipitate with 1mL PBS buffer solution to obtain solution to be tested, mixing 500uL solution to be tested with ratio fluorescent probe prepared 500uL, incubating for 25min to obtain incubation liquid, measuring fluorescent intensity of the incubation liquid at excitation wavelength 285nm at 458nm and 488nm respectively, and finally determining fluorescent intensity ratio F according to obtained fluorescent intensity ratio F 458 /F 488 Substituting the linear regression equation obtained in the step (1) in the example 2 to obtain the concentration of the listeria monocytogenes in the sample to be detected.
The test result shows that the concentration of the listeria monocytogenes measured by the ratio fluorescent probe is 3.86 x 10 under the environment of artificially inoculating the listeria monocytogenes in milk 6 CFU/mL, almost the same as its true concentration (3.9 x 10 6 CFU/mL) is close, it can be seen that even if the corresponding standard curve and linear regression equation under the milk sample environment are not established, the standard curve and linear regression equation established under the pure PBS sample environment still have better universality, so that the ratio fluorescent probe prepared by the invention has better universality, and can be rapidly applied to different sample detection.
Example 4
Substantially the same as in example 2, except that:
the listeria monocytogenes is respectively replaced by staphylococcus aureus, salmonella, escherichia coli, cronobacter sakazakii and pseudomonas aeruginosa;and respectively preparing the components with the concentration of 3.9 x 10 6 CFU/mL of bacteria-containing solution to be tested.
Referring to FIG. 6, a solution to be tested containing bacteria after adding Staphylococcus aureus, salmonella, escherichia coli, cronobacter sakazakii, pseudomonas aeruginosa, has a fluorescence intensity ratio F 458 /F 488 The ratio of the two is 0.70866, 0.71062, 0.70962, 0.70676 and 0.71123 respectively, and meanwhile, the fluorescence intensity ratio F of the bacteria-containing solution to be detected after the listeria monocytogenes is added under the same condition 458 /F 488 The ratio of the fluorescent light to the fluorescent light is 0.40643, and in addition, the fluorescent light is F 458 /F 488 The ratio of the fluorescent probe to the listeria monocytogenes is 0.70760, so that the fluorescent probe has good specificity for listeria monocytogenes.
It will be readily appreciated by those skilled in the art that the above embodiments are merely preferred embodiments of the invention and are not intended to limit the invention, but any modifications, equivalent substitutions and improvements made within the spirit and principles of the invention should be included in the scope of the invention.
Claims (2)
1. A ratiometric fluorescent probe for detecting listeria monocytogenes, comprising: the volume ratio of the first fluorescent probe serving as an energy donor to the second fluorescent probe serving as an energy acceptor is 1:1, uniformly mixing to prepare the mixture;
the first fluorescent probe is vancomycin modified carbon quantum dots;
the second fluorescent probe is an aptamer modified silicon nanoparticle;
the vancomycin is vancomycin hydrochloride, and the CAS number is 1404-93-9;
the DNA sequence of the aptamer is shown as SEQ ID No. 1, and the 5' end of the aptamer is modified by amino;
the linear fitting coefficient of the ratio fluorescent probe for detecting listeria monocytogenes is 0.99332, and the detection limit is 2.06CFU/mL.
2. The method for preparing a ratiometric fluorescent probe for detection of listeria monocytogenes according to claim 1, characterized by the following operative steps:
(1) Preparation of activated Mixed solution
According to the volume ratio of 1:1, mixing 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride with the concentration of 10mM and N-hydroxysuccinimide with the concentration of 5mM to obtain an activated mixed solution;
(2) Preparation of first fluorescent Probe
According to the mass volume ratio of 12mg: mixing and dissolving guanidine hydrochloride and ultrapure water by 2mL to obtain a mixed solution;
transferring the mixed solution into a reaction kettle, reacting at 200 ℃ for 8 hours, and cooling to room temperature to obtain a reaction solution;
centrifuging the reaction solution for 20min at the rotation speed of 8000rpm and the temperature of 4 ℃ and collecting supernatant;
according to the volume ratio of 2:1 mixing the supernatant with the activation mixed solution in the step (1), and incubating for 25min at room temperature; adding a vancomycin solution with the concentration of 50uM, wherein the addition amount of the vancomycin solution is 0.006 times of the volume of the supernatant, and incubating for 2.5 hours in a shaking table under the conditions of the rotating speed of 180rpm and the room temperature to prepare a first fluorescent probe;
(3) Preparation of the second fluorescent Probe
According to the volume ratio of 1:1, mixing sodium ascorbate solution with the molar concentration of 100mM with 3-aminopropyl trimethoxysilane, adding eight times of volume of ultrapure water, stirring and mixing uniformly, and stirring and mixing uniformly for 80min; dialyzing with 500Da dialysis bag for 18h to obtain a retentate;
according to the volume ratio of 2:1, mixing the retention solution with the activation mixed solution in the step (1), and incubating for 30min at room temperature; adding an aptamer solution with the concentration of 100uM, wherein the addition amount of the aptamer solution is 0.03 times of the volume of the retention solution; incubating the mixture for 2.5 hours in a shaking table at the rotating speed of 180rpm and at the room temperature to prepare a second fluorescent probe;
(4) Preparation of ratio fluorescent probes
According to the volume ratio of 1:1 mixing the first fluorescent probe with the second fluorescent probe to prepare a ratio fluorescent probe;
when the fluorescence emission wavelength of the carbon quantum dots is shifted from 452nm to 458nm, the vancomycin in the ratio fluorescent probe is successfully modified on the carbon quantum dots, and when the fluorescence emission wavelength of the silicon nanoparticles is shifted from 500nm to 488nm, the aptamer in the ratio fluorescent probe is successfully modified on the silicon nanoparticles.
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