CN116590008B - Background elimination-based ratio fluorescent probe for escherichia coli O157: H7 detection and preparation method thereof - Google Patents
Background elimination-based ratio fluorescent probe for escherichia coli O157: H7 detection and preparation method thereof Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
- C09K11/08—Luminescent, e.g. electroluminescent, chemiluminescent materials containing inorganic luminescent materials
- C09K11/65—Luminescent, e.g. electroluminescent, chemiluminescent materials containing inorganic luminescent materials containing carbon
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
- C09K11/02—Use of particular materials as binders, particle coatings or suspension media therefor
- C09K11/025—Use of particular materials as binders, particle coatings or suspension media therefor non-luminescent particle coatings or suspension media
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- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
- C09K11/08—Luminescent, e.g. electroluminescent, chemiluminescent materials containing inorganic luminescent materials
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention discloses a background elimination-based ratio fluorescent probe for detecting escherichia coli O157:H27 and a preparation method thereof, and belongs to the technical field of food safety. The ratiometric fluorescent probe based on background elimination comprises a quantum dot compound serving as a fluorescence donor, a carbon dot probe serving as a fluorescence acceptor and a magnetic carbon nano tube compound carrier for eliminating background interference. When the escherichia coli O157:H27 exists, a fluorescence donor and a fluorescence acceptor simultaneously capture target bacteria, the space distance between the fluorescence donor and the fluorescence acceptor is shortened, FRET is turned on, energy is transferred from the donor fluorophore to the acceptor fluorophore, and fluorescence double signals are changed reversibly, so that the escherichia coli O157:H27 is detected; in addition, the superfluous fluorescent receptor which is not combined with the target bacteria in the system is adsorbed on the surface of the magnetic carbon nano tube composite carrier due to pi-pi stacking effect, and is efficiently separated through magnetic separation, so that the background interference is eliminated. The invention has simple operation and low detection cost, and improves the detection sensitivity to 2.6cfu/mL by matching with a magnetic separation technology.
Description
Technical Field
The invention belongs to the technical field of food safety, and particularly relates to a background elimination-based ratio fluorescent probe for detecting escherichia coli O157H 7 and a preparation method thereof.
Background
Bacterial infections have become a major problem in global public health. Coli O157H 7 was first identified in 1982 as a pathogenic bacterium, a gram-negative, facultative anaerobic bacterium, a major causative factor in human hemorrhagic diarrhea and enteritis. It is counted that about 280 ten thousand people worldwide are infected with E.coli O157:H2 7 each year. Symptoms and diseases caused after infection include abdominal cramps, watery diarrhea, ulcerative colitis, hemorrhagic colitis, hemolytic uremic syndrome, thrombotic thrombocytopenic purpura, etc., and even death can result when severe. In addition, even low concentrations of infection pose a high risk to public health. Therefore, the establishment of a sensitive and rapid escherichia coli O157:H27 detection method has important significance for promoting food quality safety.
Currently, most detection mechanisms still predominate in the detection of pathogenic bacteria by traditional bacterial culture and colony counting. However, the traditional detection technology is complex in operation and long in detection period, is easy to cause delay of treatment and diagnosis, and cannot meet the requirement of rapid screening of escherichia coli O157:H27. Therefore, various rapid, highly sensitive and highly specific detection methods have been proposed by researchers. Such as molecular biological detection methods, immunological methods, and the like. The molecular biological detection method mainly comprises Polymerase Chain Reaction (PCR), nucleic acid isothermal amplification technology, nuclease auxiliary signal amplification and the like. The identification time is greatly shortened compared to the conventional microorganism culture method, but these methods require complicated nucleic acid sequence design to ensure high sensitivity and accuracy. In practical operation, the experiment is very easy to generate false positive results due to cross contamination and nonspecific amplification, so that the probes are required to be redesigned and reagents are required to be replaced, and the application of the probes is seriously hindered. The immunological detection has the advantages of simplicity, rapidness, high sensitivity, high specificity and the like. Among them, enzyme-linked immunosorbent assay, immunomagnetic separation technique and lateral flow immunochromatography technique are three common forms of immunological detection. However, the antibody serving as a specific recognition element is expensive and is greatly influenced by the external environment, so that the further popularization of the antibody is limited. Therefore, there is an urgent need to develop new technologies that meet the all-round performance requirements of rapidity, quantification, sensitivity, specificity, and economy.
In order to meet the current detection needs and address the above limitations, various new identification strategies have been developed, including fluorescence, colorimetric, electrochemical methods, and the like. The fluorescence method has the advantages of high sensitivity, short detection time, strong specificity and the like, and is widely applied to the detection of food-borne pathogenic bacteria. Traditional fluorophores comprise 6-carboxyfluorescein (FAM), rhodamine B and Cy5, and the application range of the traditional fluorophores is limited due to the defects of photobleaching, sensitivity to pH, low quantum yield, narrow excitation spectrum, complex modification process and the like. Most of high-performance quantum dots have excellent characteristics of easy synthesis, stable fluorescence characteristic, wide spectrum excitation response and the like compared with the traditional fluorescent dye, but contain metal elements (such as lead, cadmium and arsenic) with biotoxicity. Therefore, development of a fluorescent marker which is efficient, stable, excellent in biocompatibility and inexpensive is necessary. In addition, single peak based fluorescence detection is susceptible to interference from non-target substances in the food matrix, reducing the detection accuracy and dynamic response range of food contaminants. In order to reduce the influence of external factors, an effective pollution early warning mechanism is established, false positive output in food media is avoided, and a response ratio fluorescence analysis platform shows great application prospect. However, the ratio fluorescence has a good internal reference correction effect in detection, but it usually uses bacteria as a carrier, and signal conversion is achieved by pulling the distance between a fluorescence donor and an acceptor. This results in a smaller reaction system that can still trigger energy transfer of both groups when no target bacteria are present in the sample matrix, resulting in an adverse effect of detection background signal being too high. Thus, there is a need to develop a simple fluorescence background reduction strategy to further improve the sensitivity and linear response range of a ratiometric fluorescence platform for detecting food-borne pathogenic bacteria. In addition, most of the current ratio fluorescence detection strategies take vancomycin as a specific identification element, so that the detection of gram-positive bacteria is realized, and a ratio detection method utilizing a negative bacteria identification element is still rarely reported.
Disclosure of Invention
In order to realize simple, rapid, high-precision and high-sensitivity detection of the escherichia coli O157:H27, the invention provides a ratio fluorescent probe based on background elimination for the escherichia coli O157:H27 detection.
A ratiometric fluorescent probe based on background elimination comprises a quantum dot compound serving as a fluorescence donor, a carbon dot probe serving as a fluorescence acceptor and a magnetic carbon nano tube compound carrier for eliminating background interference;
the quantum dot compound is polymyxin B modified nitrogen-sulfur co-doped graphene quantum dots;
the polymyxin B is polymyxin B sulfate, and the CAS number is 1405-20-5;
the carbon point probe is an aptamer modified yellow carbon point;
the DNA sequence of the aptamer is shown as SEQ ID No. 1, and the 5' end of the aptamer is modified by carboxyl;
the magnetic carbon nanotube composite carrier is a multiwall carbon nanotube modified by carbonyl iron powder;
the CAS number of the carbonyl iron powder is 7439-89-6;
the multiwall carbon nanotube is carboxylated multiwall carbon nanotube with CAS number of 1333-86-4;
when the escherichia coli O157:H27 does not exist in the detected object, the carbon point probe in the detection system is adsorbed by the magnetic carbon nano tube composite carrier, and fluorescence of the quantum dot composite is not quenched;
when the Escherichia coli O157: H7 exists in the object to be detected, the quantum dot complex and the carbon dot probe in the detection system capture target bacteria simultaneously, and the space distance between the fluorescence donor and the fluorescence acceptor is shortened, so that fluorescence resonance energy transfer occurs, fluorescence quenching of the quantum dot complex is achieved, and fluorescence of the carbon dot probe is enhanced.
The preparation operation steps of the ratiometric fluorescent probe based on background elimination are as follows:
(1) Preparation of Quantum dot complexes
(1.1) preparation of Nitrogen-sulfur co-doped graphene quantum dot solid
(1.1.1) uniformly mixing 1.0g of citric acid and 0.3. 0.3g L-cysteine, heating an oil bath to 200 ℃, stopping heating when the color of a reactant changes from light yellow to orange, and naturally cooling to room temperature to obtain an orange product;
(1.1.2) dissolving the orange product into 10mL of ultrapure water to obtain a nitrogen-sulfur co-doped graphene quantum dot stock solution;
(1.1.3) freeze-drying the nitrogen-sulfur co-doped graphene quantum dot stock solution for 12 hours to prepare a nitrogen-sulfur co-doped graphene quantum dot solid;
(1.2) preparation of Quantum dot Complex
(1.2.1) dissolving 1mg of nitrogen-sulfur co-doped graphene quantum dot solid in 10mL of deionized water, and performing ultrasonic treatment for 10min to obtain a dispersion solution;
(1.2.2) to the dispersion solution were added 6mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and 5mg of N-hydroxysuccinimide (NHS), and stirred at room temperature for 2 hours to obtain an activated quantum dot solution;
(1.2.3) adding 3mg of polymyxin B into the activated quantum dot solution, and stirring at 200rpm for 7h at room temperature to obtain a quantum dot compound;
the maximum emission wavelength of the nitrogen-sulfur co-doped graphene quantum dot is 418nm, and the Zeta potential is 6.77mV; when the maximum emission wavelength of the quantum dot compound is shifted to 422nm and the Zeta potential is changed to 17.37mV, the polymyxin B in the quantum dot compound is successfully modified to the nitrogen-sulfur co-doped graphene quantum dot;
(2) Preparation of carbon dot probes
(2.1) preparation of yellow carbon dot solid
(2.1.1) adding 216mg of o-phenylenediamine into 30mL of deionized water, and vibrating and uniformly mixing to obtain a mixed solution;
(2.1.2) heating the mixed solution at 160 ℃ for 4 hours, naturally cooling to room temperature, and centrifuging for 12 minutes under the centrifugal force of 9289×g to remove impurities, thereby obtaining a reaction solution;
(2.1.3) adding the reaction solution into a dialysis bag with molecular permeation of 3000Da, and dialyzing with deionized water for 24 hours to obtain a dialysate;
(2.1.4) freeze-drying the dialysate for 12 hours to obtain yellow carbon dot solid;
(2.2) preparation of carbon Point Probe
(2.2.1) to 300. Mu.L of 1. Mu.M aptamer solution, 50. Mu.L of 4mg/mL 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) solution and 50. Mu.L of 4mg/mL N-hydroxysuccinimide (NHS) solution were added, and incubated at 180rpm for 30min at room temperature to obtain an activated aptamer solution;
(2.2.2) dissolving 5mg of yellow carbon dot solid in 5mL of deionized water, adding the activated aptamer solution, and incubating for 2.5h at a rotation speed of 180rpm under the condition of room temperature to obtain a carbon dot probe; the maximum emission wavelength of the yellow carbon point is 562nm, and the Zeta potential is-4.01 mV; when the maximum emission wavelength of the carbon point probe is shifted to 566nm and the zeta potential is changed to-7.84 mV, the aptamer is modified on the surface of the yellow carbon point, and the carbon point probe is successfully constructed;
(3) Preparation of magnetic carbon nanotube composite carrier
(3.1) weighing 1.5mg of multi-wall carbon nano tube in a glass bottle, adding 1.5mL of morpholine ethanesulfonic acid monohydrate acid solution with the concentration of 0.1M, pH and the value of 5.5, and fully oscillating to obtain multi-wall carbon nano tube solution;
(3.2) adding 200. Mu.L of a solution of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) having a concentration of 6.2mg/mL and 200. Mu.L of a solution of N-hydroxysuccinimide (NHS) having a concentration of 4.6mg/mL to the multi-wall carbon nanotube solution, and incubating at room temperature for 2 hours to obtain an activated multi-wall carbon nanotube solution;
(3.3) adding 3mg of carbonyl iron powder into the activated multi-wall carbon nano tube solution, carrying out ultrasonic treatment for 1h, and drying in an oven at 85 ℃ for 20min to obtain a magnetic carbon nano tube composite carrier;
the multi-wall carbon nano tube is nonmagnetic, the Zeta potential is-23.01 mV, the carbonyl iron powder is magnetic, and the Zeta potential is 4.25mV; when the magnetic carbon nano tube composite carrier is placed on the magnetic frame, the magnetic carbon nano tube composite carrier can be adsorbed to the wall of the centrifuge tube under the action of magnetic force, so that the magnetic carbon nano tube composite carrier has magnetism; the Zeta potential of the magnetic carbon nano tube composite carrier is-2.38 mV and is between-23.01 mV and 4.25mV, which proves that the magnetic carbon nano tube composite carrier is a combination of multi-wall carbon nano tubes and carbonyl iron powder; in summary, the successful preparation of the magnetic carbon nanotube composite carrier is demonstrated.
The application of the ratio fluorescent probe based on background elimination in the detection of escherichia coli O157:H27 comprises the following specific operation steps:
taking 400 mu L of liquid to be detected in a 1.5mL sterilized centrifuge tube, adding 60 mu L of carbon dot probe and 100 mu g of magnetic carbon nanotube composite carrier into the liquid to be detected, and incubating the liquid at 37 ℃ for 70min to obtain a composite solution; placing the composite solution in a magnetic rack, standing for 1min, and taking supernatant to obtain a magnetic separation solution; adding 40 mu L of quantum dot compound into the magnetic separation solution, and incubating for 20min at 37 ℃ to obtain a solution to be detected; 200 mu L of the liquid to be measured is taken in a quartz cuvette, the excitation wavelength of a fluorescence spectrophotometer is set to 355nm, and the fluorescence intensity values of the liquid to be measured at 422nm and 566nm are measured.
The detection and analysis principle of the method of the invention is as follows:
taking the quantum dot compound as an energy donor, taking a carbon dot probe as an energy acceptor, and when the quantum dot compound and the carbon dot probe simultaneously capture escherichia coli O157:H7, the spatial distance between donor and acceptor molecules is shortened, then FRET is opened, energy is transferred from a donor fluorophore to an acceptor fluorophore, and fluorescence double signals are reversibly changed; in addition, the aptamer in the redundant carbon point probe which is not combined with the target bacteria in the system is adsorbed on the magnetic carbon nano tube composite carrier due to pi-pi stacking effect and is separated efficiently through magnetic separation, so that the interference of background noise is eliminated, and the simple, sensitive and rapid detection of the escherichia coli O157:H27 is realized.
The beneficial technical effects of the invention are as follows:
(1) Compared with a single-emission fluorescence method, the ratio fluorescence strategy adopted by the invention has a built-in self-calibration function, avoids the interference of various target independent factors, reduces the output of false positive results, and thus realizes higher accurate detection sensitivity and wider dynamic linear range. Fluorescence Resonance Energy Transfer (FRET) mechanisms are an effective strategy for ratiometric fluorescence, which typically consists of two fluorophores forming an energy donor-acceptor pair at a distance within 10nm, allowing energy transfer from the donor fluorophore to the acceptor fluorophore and resulting in a shift in the dual emission intensity, thereby achieving a ratiometric response to food contaminants by enabling or disabling the FRET process. However, when the target bacteria are not present in the sample matrix, the energy transfer between the two groups triggered by the smaller reaction system may also cause adverse effects such as too high detection background signal. In order to solve the defects in the prior art, the invention utilizes the magnetic carbon nano tube composite carrier to build the magnetic separation platform. Wherein, the multiwall carbon nanotube can be used as a nano carrier due to the large specific surface area and surface pi bond, and can adsorb a large amount of biomolecules (such as carbon point probes) based on non-covalent effect. After the carbonyl iron powder is doped to endow the fluorescent probe with magnetism, the high-efficiency separation of the redundant fluorescent receptor probe (carbon point probe) can be further realized, and the fluorescent receptor probe (carbon point probe) fixed on the escherichia coli O157: H7 is remained in the system, so that the high-precision sensitive detection purpose is achieved.
(2) The magnetic carbon nano tube composite carrier synthesized by the invention can replace immunomagnetic beads and becomes an effective means for resisting disturbance of food matrixes. In addition, compared with the traditional magnetic beads, highly delocalized pi bonds are formed among carbon atoms distributed in a hexagonal grid shape in the multiwall carbon nanotubes. The microcosmic uniqueness enables the multiwall carbon nanotube to adsorb a large amount of single-stranded DNA nucleic acid aptamer according to pi-pi stacking effect, and complicated and expensive chemical coupling is not needed to be carried out on the aptamer and the multiwall carbon nanotube in advance, so that flexible adsorption-desorption switching is realized, and the aptamer is used as an adjustable signal switch to improve the signal-to-noise ratio of a detection sensor.
(3) Compared with the traditional fluorescent material, the nitrogen-sulfur co-doped graphene quantum dot and the yellow carbon dot used in the method have the advantages of good biocompatibility, adjustable photoluminescence, high chemical stability, easiness in surface functionalization and the like. In addition, the incorporation of nitrogen and sulfur in the graphene structure of the nitrogen-sulfur co-doped graphene quantum dot enables the graphene quantum dot to have good photoluminescence and higher fluorescence quantum yield, and the cross-linking between the L-cysteine molecule and the graphene core effectively prevents aggregation of the graphene quantum dot, so that the graphene quantum dot becomes an excellent fluorescence analysis tool. The amino groups rich in the surface of the yellow carbon dots can not only improve fluorescence, but also provide covalent binding sites for carboxylated aptamer probes.
(4) The invention takes polymyxin B and an aptamer as molecular recognition elements, and respectively shows broad-spectrum and high-specificity combination on the surface of escherichia coli. Wherein polymyxin B is a cationic cyclic polypeptide isolated from Bacillus polymyxa, and is used as an antibiotic for treating gram-negative bacteria, and can target lipopolysaccharide of outer membrane of gram-negative bacteria at low concentration. The aptamer is a single-stranded DNA or RNA oligonucleotide, can identify and bind a specific target, and has the advantages of low cost, easy synthesis and modification, good repeatability and the like.
(5) The invention constructs a novel ratio type fluorescence biosensor based on FRET effect. Because the fluorescence emission spectrum of the nitrogen-sulfur co-doped graphene quantum dot overlaps with the excitation spectrum of the yellow carbon dot, the sensor takes the nitrogen-sulfur co-doped graphene quantum dot modified by polymyxin B as an energy donor, takes the yellow carbon dot modified by an aptamer as an energy acceptor, ingeniously takes bacteria as a carrier to pull the distance between the two, and realizes accurate and reliable detection of escherichia coli O157:H7.
(6) The invention uses polymyxin B and the aptamer to replace the antibody as the recognition element, and the detection cost is reduced by about 75%. Meanwhile, the raw materials for synthesizing the nitrogen-sulfur co-doped graphene quantum dots, the yellow carbon dots and the magnetic carbon nanotube composite carrier are low in price, so that the detection cost is further reduced, and the market popularization is facilitated.
Drawings
FIG. 1 is a schematic diagram of a preparation method and detection analysis of a ratio fluorescent probe based on background elimination in the invention;
FIG. 2 is a fluorescence spectrum diagram before and after covalent coupling of nitrogen-sulfur co-doped graphene quantum dots and polymyxin B;
FIG. 3 shows the Zeta potential of the nitrogen-sulfur co-doped graphene quantum dot and quantum dot composite in the invention;
FIG. 4 is a graph showing fluorescence spectra before and after covalent coupling of yellow carbon dots with an aptamer in accordance with the present invention;
FIG. 5 shows Zeta potentials of yellow carbon dot and carbon dot probes according to the present invention;
FIG. 6 is a photograph showing an aqueous solution of a magnetic carbon nanotube composite carrier according to the present invention placed before and after a magnetic shelf;
FIG. 7 shows the Zeta potential of the multi-walled carbon nanotube, carbonyl iron powder and magnetic carbon nanotube composite carrier according to the present invention;
FIG. 8 is a fluorescence spectrum of E.coli O157:H27 detected by the ratio fluorescent biosensor in example 2 of the present invention;
FIG. 9 is a graph showing the linear relationship between the detection of E.coli O157 to H7 by the ratio fluorescent biosensor in example 2 of the present invention;
FIG. 10 is a diagram showing the specificity of the detection method in example 3 of the present invention.
Detailed Description
The invention is further illustrated, but is not limited, by the following examples.
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 test reagent consumables 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 all set up in triplicate and the results averaged.
In the following examples, polymyxin B, citric acid, L-cysteine were purchased from Shanghai Biotechnology Co., ltd; multiwall carbon nanotubes are purchased from Nanjing Xianfeng nanomaterial technologies, inc.; carbonyl iron powder is purchased from Shanghai microphone Biochemical technology Co., ltd; o-phenylenediamine, morpholinoethanesulfonic acid monohydrate, commercially available from ala Ding Shiji inc; n-hydroxysuccinimide and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride were purchased from Sigma-Aldrich, USA; PBS solution with pH value of 7.4 and concentration of 0.1M is purchased from Shanghai Biotechnology Co., ltd; sequence reference "An aptamer-exonuclease III (Exo III) -assisted amplification-based lateralflow assay for sensitive detection of for aptamerEscherichia coliO157H 7 in mill ", purchased from Shanghai Biotechnology Co., ltd.
The preparation method of the ratio fluorescent probe based on background elimination and the detection principle thereof are shown in figure 1.
A ratiometric fluorescent probe based on background elimination comprises a quantum dot compound serving as a fluorescence donor, a carbon dot probe serving as a fluorescence acceptor and a magnetic carbon nano tube compound carrier for eliminating background interference;
the quantum dot compound is polymyxin B modified nitrogen-sulfur co-doped graphene quantum dots;
the polymyxin B is polymyxin B sulfate, and the CAS number is 1405-20-5;
the carbon point probe is an aptamer modified yellow carbon point;
the DNA sequence of the aptamer is shown as SEQ ID No. 1, and the 5' end of the aptamer is modified by carboxyl;
the magnetic carbon nanotube composite carrier is a multiwall carbon nanotube modified by carbonyl iron powder;
the CAS number of the carbonyl iron powder is 7439-89-6;
the multiwall carbon nanotube is carboxylated multiwall carbon nanotube with CAS number of 1333-86-4;
when the escherichia coli O157:H27 does not exist in the detected object, the carbon point probe in the detection system is adsorbed by the magnetic carbon nano tube composite carrier, and fluorescence of the quantum dot composite is not quenched;
when the Escherichia coli O157: H7 exists in the object to be detected, the quantum dot complex and the carbon dot probe in the detection system capture target bacteria simultaneously, and the space distance between the fluorescence donor and the fluorescence acceptor is shortened, so that fluorescence resonance energy transfer occurs, fluorescence quenching of the quantum dot complex is achieved, and fluorescence of the carbon dot probe is enhanced.
Example 1
The preparation operation steps of the ratio fluorescent probe based on background elimination for detecting the escherichia coli O157:H27 are as follows:
(1) Preparation of Quantum dot complexes
(1.1) preparation of Nitrogen-sulfur co-doped graphene quantum dot solid
(1.1.1) uniformly mixing 1.0g of citric acid and 0.3. 0.3g L-cysteine, heating an oil bath to 200 ℃, stopping heating when the color of a reactant changes from light yellow to orange, and naturally cooling to room temperature to obtain an orange product;
(1.1.2) dissolving the orange product into 10mL of ultrapure water to obtain a nitrogen-sulfur co-doped graphene quantum dot stock solution;
(1.1.3) freeze-drying the nitrogen-sulfur co-doped graphene quantum dot stock solution for 12 hours to prepare a nitrogen-sulfur co-doped graphene quantum dot solid;
(1.2) preparation of Quantum dot Complex
(1.2.1) dissolving 1mg of nitrogen-sulfur co-doped graphene quantum dot solid in 10mL of deionized water, and performing ultrasonic treatment for 10min to obtain a dispersion solution;
(1.2.2) to the dispersion solution were added 6mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and 5mg of N-hydroxysuccinimide (NHS), and stirred at room temperature for 2 hours to obtain an activated quantum dot solution;
(1.2.3) to the activated quantum dot solution was added 3mg of polymyxin B and stirred at 200rpm for 7 hours at room temperature to obtain a quantum dot complex.
Referring to fig. 2, the maximum emission wavelength of the nitrogen-sulfur co-doped graphene quantum dot is 418nm, the maximum emission wavelength of the quantum dot compound is 422nm, and the red shift of the emission wavelength proves successful coupling of the polymyxin B and the nitrogen-sulfur co-doped graphene quantum dot; in addition, referring to fig. 3, the Zeta potential of the nitrogen-sulfur co-doped graphene quantum dot is 6.77mV, the Zeta potential of the quantum dot compound is 17.37mV, and the Zeta potential test result shows that the polymyxin B is successfully modified on the surface of the nitrogen-sulfur co-doped graphene quantum dot; the above results indicate that quantum dot complexes were successfully constructed.
(2) Preparation of carbon dot probes
(2.1) preparation of yellow carbon dot solid
(2.1.1) adding 216mg of o-phenylenediamine into 30mL of deionized water, and vibrating and uniformly mixing to obtain a mixed solution;
(2.1.2) heating the mixed solution at 160 ℃ for 4 hours, naturally cooling to room temperature, and centrifuging for 12 minutes under the centrifugal force of 9289×g to remove impurities, thereby obtaining a reaction solution;
(2.1.3) adding the reaction solution into a dialysis bag with molecular permeation of 3000Da, and dialyzing with deionized water for 24 hours to obtain a dialysate;
(2.1.4) the dialysate was freeze-dried for 12h to give a yellow carbon dot solid.
(2.2) preparation of carbon Point Probe
(2.2.1) to 300. Mu.L of 1. Mu.M aptamer solution, 50. Mu.L of 4mg/mL 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) solution and 50. Mu.L of 4mg/mL N-hydroxysuccinimide (NHS) solution were added, and incubated at 180rpm for 30min at room temperature to obtain an activated aptamer solution;
(2.2.2) 5mg of yellow carbon dot solid was dissolved in 5mL of deionized water, and the activated aptamer solution was added, and incubated at 180rpm for 2.5 hours at room temperature to obtain a carbon dot probe.
Referring to fig. 4, the maximum emission wavelength of the yellow carbon dot is 562nm, the maximum emission wavelength of the carbon dot probe is 566nm, and the red shift of the emission wavelength demonstrates successful coupling of the aptamer and the yellow carbon dot; in addition, referring to FIG. 5, the Zeta potential of the yellow carbon dot is-4.01 mV, the Zeta potential of the carbon dot probe is-7.84 mV, and the Zeta potential test result shows that the aptamer is successfully modified on the surface of the yellow carbon dot; the above results indicate that the carbon dot probe was successfully constructed.
(3) Preparation of magnetic carbon nanotube composite carrier
(3.1) weighing 1.5mg of multi-wall carbon nano tube in a glass bottle, adding 1.5mL of morpholine ethanesulfonic acid monohydrate acid solution with the concentration of 0.1M, pH and the value of 5.5, and fully oscillating to obtain multi-wall carbon nano tube solution;
(3.2) adding 200. Mu.L of a solution of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) having a concentration of 6.2mg/mL and 200. Mu.L of a solution of N-hydroxysuccinimide (NHS) having a concentration of 4.6mg/mL to the multi-wall carbon nanotube solution, and incubating at room temperature for 2 hours to obtain an activated multi-wall carbon nanotube solution;
(3.3) adding 3mg of carbonyl iron powder into the activated multi-wall carbon nano tube solution, carrying out ultrasonic treatment for 1h, and drying in an oven at 85 ℃ for 20min to obtain the magnetic carbon nano tube composite carrier.
Referring to fig. 6, the magnetic carbon nanotube composite carrier is a uniform and stable dispersion system in the centrifuge tube, and is placed on a magnetic rack, and the magnetic carbon nanotube composite carrier is adsorbed to the wall of the centrifuge tube under the action of magnetic force, so that successful doping of carbonyl iron powder is demonstrated; in addition, referring to fig. 7, the Zeta potential of the multi-wall carbon nanotube is-23.01 mV, the Zeta potential of the carbonyl iron powder is 4.25mV, the Zeta potential of the magnetic carbon nanotube composite carrier is-2.38 mV, and the Zeta potential test result shows that the magnetic carbon nanotube composite carrier is a combination of the multi-wall carbon nanotube and the carbonyl iron powder; in summary, the characterization proves that the magnetic carbon nano tube composite carrier is successfully prepared.
Example 2
The detection equation is established, and the specific operation steps are as follows:
(1) Culturing Escherichia coli O157:H27 in LB broth for 12H till the late logarithmic growth phase, taking 1mL, transferring into a sterilized centrifuge tube, centrifuging at 5000 Xg for 5min, discarding supernatant, and re-suspending in sterile PBS buffer with an equal volume concentration of 0.1 and M, pH value of 7.4 to obtain bacterial suspension; gradient dilution of the bacterial suspension to obtain E.coli O157H 7 final concentration of 10 respectively 1 、10 2 、10 3 、10 4 、10 5 、10 6 、10 7 cfu/mL of bacteria-containing liquid to be detected;
(2) Respectively taking 400 mu L of the bacteria-containing liquid to be detected in a 1.5mL sterilized centrifuge tube, adding 60 mu L of a carbon point probe and 100 mu g of a magnetic carbon nano tube composite carrier into the liquid to be detected, and incubating the liquid for 70min at 37 ℃ to obtain seven composite solutions;
the carbon point probe was prepared from example 1;
the magnetic carbon nanotube composite carrier was prepared by example 1;
(3) Placing the seven composite solutions on a magnetic rack respectively, standing for 1min, and taking supernatant to obtain seven magnetic separation solutions;
(4) Adding 40 mu L of quantum dot compound into the seven magnetic separation solutions respectively, and incubating for 20min at 37 ℃ to obtain seven corresponding solutions to be detected;
the quantum dot composite was prepared from example 1;
(5) Respectively taking 200 mu L of seven to-be-measured liquids in a quartz cuvette, setting the excitation wavelength of a fluorescence spectrophotometer to 355nm, and measuring the fluorescence intensity values of the to-be-measured liquids at 422nm and 566nm, wherein the result is shown in figure 8; by F 566 /F 422 In the ordinate Y, in largeThe logarithmic value of the concentration (cfu/mL) of the enterobacteria O157:H7 bacterial liquid is represented by an abscissa X, a standard curve is drawn, the result is shown in FIG. 9, and a detection equation is obtained: y= 0.1531X-0.0603, correlation coefficient 0.9979, detection limit 2.6cfu/mL;
the F is 566 F is the fluorescence intensity value of the liquid to be measured at 566nm 422 The fluorescence intensity value of the liquid to be measured at 422 nm.
Example 3
Specific investigation of ratiometric fluorescent biosensors
(1) Sample pretreatment
Culturing Escherichia coli O157:H27 and 8 common food-borne pathogenic bacteria (strain information is shown in Table 1) in LB broth for 12H to logarithmic growth later stage, transferring 1mL into sterilized centrifuge tube, centrifuging at 5000 Xg for 5min, discarding supernatant, and resuspending in sterile PBS buffer with equal volume concentration of 0.1 and M, pH value of 7.4 to obtain bacterial suspension, and gradient diluting the bacterial suspension to test strain concentration of about 10 4 cfu/mL; in addition, a sterile PBS buffer solution with the concentration of 0.1M, pH value of 7.4 is taken as a blank control solution, and the concentration of Escherichia coli O157:H2 7 in the blank control solution is 0cfu/mL;
(2) Detection of
(2.1) taking 400 mu L of to-be-detected liquid in a 1.5mL sterilized centrifuge tube, adding 60 mu L of carbon point probe and 100 mu g of magnetic carbon nano tube composite carrier into the centrifuge tube, and incubating the mixture at 37 ℃ for 70min to obtain a composite solution;
the carbon point probe was prepared from example 1;
the magnetic carbon nanotube composite carrier was prepared by example 1;
(2.2) placing the composite solution in a magnetic rack, standing for 1min, and taking supernatant to obtain a magnetic separation solution;
(2.3) adding 40 mu L of quantum dot compound into the magnetic separation solution, and incubating for 20min at 37 ℃ to obtain a liquid to be detected;
the quantum dot composite was prepared from example 1;
(2.4) taking 200 mu L of the liquid to be measured in a quartz cuvette, setting the excitation wavelength of a fluorescence spectrophotometer to 355nm, and measuring the fluorescence intensity values of the liquid to be measured at 422nm and 566 nm;
(3) Analysis of detection results
As shown in FIG. 10, F of the solution to be tested when E.coli O157: H7 is present in the system due to the specific recognition of E.coli O157: H7 by the recognition element 566 /F 422 Values were much higher than the blank control solution; when the system does not contain escherichia coli O157: H7, the shigella flexneri, cronobacter sakazakii, salmonella typhimurium, listeria monocytogenes, vibrio parahaemolyticus, pseudomonas putida, enterococcus faecalis and Pseudomonas aeruginosa are tested to obtain F 566 /F 422 The value is far lower than that of Escherichia coli O157H 7, and has no obvious change compared with a blank control solution; therefore, the ratio-type fluorescence biosensor has good specificity;
example 4
In order to verify the feasibility of the detection equation established in the embodiment 2 for detecting the escherichia coli O157:H2 7 in the complex matrix sample, a milk sample is selected as a test object, and the test object is artificially and quantitatively infected with bacteria to enable the milk sample to contain the escherichia coli O157:H2 7 with known concentration, and the test object is compared with the quantitative detection result of the ratio fluorescent biosensor constructed by the detection system so as to acquire the detection effect of the ratio fluorescent probe established by the invention in an actual sample.
The milk sample of this example 4 was purchased from a local market with a synthetic fertilizer and was formulated as raw milk, whole sterilized milk of the type GB 25190, and 3.6g protein, 4.4g fat, 5.0g carbohydrate, 58mg sodium and 120mg calcium per 100mL milk.
The invention relates to application of escherichia coli O157:H27 in detection of a labeled polluted milk sample, which comprises the following specific detection operation steps:
(1) Preparation of artificial pollution milk to-be-detected liquid
A concentration of 2.72X10 was added to 1mL of milk sample 2 cfu/mL of E.coli O157:H27 standard, inCentrifuging for 5min at 5000 Xg, discarding supernatant, repeatedly washing with sterile PBS buffer solution with concentration of 0.1 and M, pH value of 7.4 for three times, and re-suspending and precipitating in 1mL PBS buffer solution to obtain solution to be detected;
(2) Detection of
(2.1) taking 400 mu L of to-be-detected liquid in a 1.5mL sterilized centrifuge tube, adding 60 mu L of carbon point probe and 100 mu g of magnetic carbon nano tube composite carrier into the centrifuge tube, and incubating the mixture at 37 ℃ for 70min to obtain a composite solution;
the carbon point probe was prepared from example 1;
the magnetic carbon nanotube composite carrier was prepared by example 1;
(2.2) placing the composite solution in a magnetic rack, standing for 1min, and taking supernatant to obtain a magnetic separation solution;
(2.3) adding 40 mu L of quantum dot compound into the magnetic separation solution, and incubating for 20min at 37 ℃ to obtain a liquid to be detected; the quantum dot composite was prepared from example 1;
(2.4) taking 200 mu L of the liquid to be measured in a quartz cuvette, setting the excitation wavelength of a fluorescence spectrophotometer to 355nm, and measuring the fluorescence intensity values of the liquid to be measured at 422nm and 566 nm;
(3) Analysis of detection results
(3.1) calculating a detection result according to a detection equation, wherein the detection equation is as follows: y= 0.1531X-0.0603, wherein X represents the logarithmic value of the concentration of E.coli O157 to H7 bacterial liquid and Y is F 566 /F 422 The method comprises the steps of carrying out a first treatment on the surface of the Wherein F is 422 F is the fluorescence intensity value of the liquid to be measured at 422nm 566 The fluorescence intensity value of the liquid to be measured at 566 nm; the detection equation is obtained in example 2;
(3.2) calculating to obtain F 566 /F 422 The value is 0.3095, and the Escherichia coli O157/H7 concentration in the artificially contaminated milk is 2.60 multiplied by 10 2 cfu/mL; the method established by the research has good universality and can meet the detection requirement of actual samples.
Example 5
The milk sample of this example 5 was purchased from a local market with a synthetic fertilizer and was formulated as raw milk, whole sterilized milk of the type GB 25190, and 3.6g protein, 4.4g fat, 5.0g carbohydrate, 58mg sodium and 120mg calcium per 100mL milk.
The invention relates to application of escherichia coli O157:H27 in detection of untagged polluted milk samples, which comprises the following specific detection operation steps:
(1) Preparation of liquid to be tested
Taking 1mL of commercial milk sample, centrifuging for 5min at 5000 Xg, discarding supernatant, repeatedly washing with 1mL of sterile PBS buffer solution with the concentration of 0.1M, pH value of 7.4 for three times, and re-suspending and precipitating in the PBS buffer solution with equal volume to obtain a liquid to be detected;
(2) Detection of
(2.1) taking 400 mu L of to-be-detected liquid in a 1.5mL sterilized centrifuge tube, adding 60 mu L of carbon point probe and 100 mu g of magnetic carbon nano tube composite carrier into the centrifuge tube, and incubating the mixture at 37 ℃ for 70min to obtain a composite solution;
the carbon point probe was prepared from example 1; the magnetic carbon nanotube composite carrier was prepared by example 1;
(2.2) placing the composite solution in a magnetic rack, standing for 1min, and taking supernatant to obtain a magnetic separation solution;
(2.3) adding 40 mu L of quantum dot compound into the magnetic separation solution, and incubating for 20min at 37 ℃ to obtain a liquid to be detected; the quantum dot composite was prepared from example 1;
(2.4) taking 200 mu L of the liquid to be measured in a quartz cuvette, setting the excitation wavelength of a fluorescence spectrophotometer to 355nm, and measuring the fluorescence intensity values of the liquid to be measured at 422nm and 566 nm;
(3) Analysis of detection results
F of the liquid to be tested 566 /F 422 The value is almost unchanged compared with the blank control solution, which indicates that the coliform O157 and H7 are not detected in the commercial milk sample; wherein F is 422 F is the fluorescence intensity value of the liquid to be measured at 422nm 566 The fluorescence intensity value of the liquid to be measured at 566 nm; f of the blank solution 566 /F 422 The values were determined from example 3.
It should be understood by those skilled in the art that the foregoing is merely illustrative of the preferred embodiments of the present invention and that various modifications, equivalents, and improvements may be made within the spirit and principles of the present invention.
Claims (2)
1. A background elimination-based ratiometric fluorescent probe for the detection of e.coli O157: H7, characterized in that: the ratio fluorescent probe based on background elimination comprises a quantum dot compound serving as a fluorescent donor, a carbon dot probe serving as a fluorescent acceptor and a magnetic carbon nano tube compound carrier for eliminating background interference;
the quantum dot compound is polymyxin B modified nitrogen-sulfur co-doped graphene quantum dots;
the polymyxin B is polymyxin B sulfate, and the CAS number is 1405-20-5;
the carbon point probe is an aptamer modified yellow carbon point;
the DNA sequence of the aptamer is shown as SEQ ID No. 1, and the 5' end of the aptamer is modified by carboxyl;
the magnetic carbon nanotube composite carrier is a multiwall carbon nanotube modified by carbonyl iron powder;
the CAS number of the carbonyl iron powder is 7439-89-6;
the multiwall carbon nanotube is carboxylated multiwall carbon nanotube with CAS number of 1333-86-4;
when the escherichia coli O157:H27 does not exist in the detected object, the carbon point probe in the detection system is adsorbed by the magnetic carbon nano tube composite carrier, and fluorescence of the quantum dot composite is not quenched;
when the Escherichia coli O157: H7 exists in the object to be detected, the quantum dot complex and the carbon dot probe in the detection system capture target bacteria simultaneously, and the space distance between the fluorescence donor and the fluorescence acceptor is shortened, so that fluorescence resonance energy transfer occurs, fluorescence quenching of the quantum dot complex is achieved, and fluorescence of the carbon dot probe is enhanced.
2. The method for preparing the background elimination-based ratiometric fluorescent probe according to claim 1, wherein the method comprises the following operation steps:
(1) Preparation of Quantum dot complexes
(1.1) preparation of Nitrogen-sulfur co-doped graphene quantum dot solid
(1.1.1) uniformly mixing 1.0g of citric acid and 0.3. 0.3g L-cysteine, heating an oil bath to 200 ℃, stopping heating when the color of a reactant changes from light yellow to orange, and naturally cooling to room temperature to obtain an orange product;
(1.1.2) dissolving the orange product into 10mL of ultrapure water to obtain a nitrogen-sulfur co-doped graphene quantum dot stock solution;
(1.1.3) freeze-drying the nitrogen-sulfur co-doped graphene quantum dot stock solution for 12 hours to prepare a nitrogen-sulfur co-doped graphene quantum dot solid;
(1.2) preparation of Quantum dot Complex
(1.2.1) dissolving 1mg of nitrogen-sulfur co-doped graphene quantum dot solid in 10mL of deionized water, and performing ultrasonic treatment for 10min to obtain a dispersion solution;
(1.2.2) to the dispersion solution, 6mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and 5mg of N-hydroxysuccinimide were added, and stirred at room temperature for 2 hours to obtain an activated quantum dot solution;
(1.2.3) adding 3mg of polymyxin B into the activated quantum dot solution, and stirring at 200rpm for 7h at room temperature to obtain a quantum dot compound;
the maximum emission wavelength of the nitrogen-sulfur co-doped graphene quantum dot is 418nm, and the Zeta potential is 6.77mV; when the maximum emission wavelength of the quantum dot compound is shifted to 422nm and the Zeta potential is changed to 17.37mV, the polymyxin B in the quantum dot compound is successfully modified to the nitrogen-sulfur co-doped graphene quantum dot;
(2) Preparation of carbon dot probes
(2.1) preparation of yellow carbon dot solid
(2.1.1) adding 216mg of o-phenylenediamine into 30mL of deionized water, and vibrating and uniformly mixing to obtain a mixed solution;
(2.1.2) heating the mixed solution at 160 ℃ for 4 hours, naturally cooling to room temperature, and centrifuging for 12 minutes under the centrifugal force of 9289×g to remove impurities, thereby obtaining a reaction solution;
(2.1.3) adding the reaction solution into a dialysis bag with molecular permeation of 3000Da, and dialyzing with deionized water for 24 hours to obtain a dialysate;
(2.1.4) freeze-drying the dialysate for 12 hours to obtain yellow carbon dot solid;
(2.2) preparation of carbon Point Probe
(2.2.1) to 300. Mu.L of 1. Mu.M aptamer solution, 50. Mu.L of 4mg/mL 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride solution and 50. Mu.L of 4mg/mL N-hydroxysuccinimide solution were added, and incubated at 180rpm for 30 minutes at room temperature to obtain an activated aptamer solution;
(2.2.2) dissolving 5mg of yellow carbon dot solid in 5mL of deionized water, adding the activated aptamer solution, and incubating for 2.5h at a rotation speed of 180rpm under the condition of room temperature to obtain a carbon dot probe;
the maximum emission wavelength of the yellow carbon point is 562nm, and the Zeta potential is-4.01 mV; when the maximum emission wavelength of the carbon point probe is shifted to 566nm and the zeta potential is changed to-7.84 mV, the aptamer is modified on the surface of the yellow carbon point, and the carbon point probe is successfully constructed;
(3) Preparation of magnetic carbon nanotube composite carrier
(3.1) weighing 1.5mg of multi-wall carbon nano tube in a glass bottle, adding 1.5mL of morpholine ethanesulfonic acid monohydrate acid solution with the concentration of 0.1M, pH and the value of 5.5, and fully oscillating to obtain multi-wall carbon nano tube solution;
(3.2) adding 200. Mu.L of a solution of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride with a concentration of 6.2mg/mL and 200. Mu.L of a solution of N-hydroxysuccinimide with a concentration of 4.6mg/mL to the multi-wall carbon nanotube solution, and incubating at room temperature for 2 hours to obtain an activated multi-wall carbon nanotube solution;
(3.3) adding 3mg of carbonyl iron powder into the activated multi-wall carbon nano tube solution, carrying out ultrasonic treatment for 1h, and drying in an oven at 85 ℃ for 20min to obtain a magnetic carbon nano tube composite carrier;
the multi-wall carbon nano tube is nonmagnetic, the Zeta potential is-23.01 mV, the carbonyl iron powder is magnetic, and the Zeta potential is 4.25mV; the magnetic carbon nano tube composite carrier has magnetism, and the Zeta potential is-2.38 mV and is between-23.01 mV and 4.25 mV.
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