CN111024943A - Switch-on/off type composite fluorescent nano probe for rapid detection of salmonella and preparation method thereof - Google Patents
Switch-on/off type composite fluorescent nano probe for rapid detection of salmonella and preparation method thereof Download PDFInfo
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Abstract
A switch-on/switch-off type composite fluorescent nano probe for rapid detection of salmonella and a preparation method thereof belong to the technical field of food safety. The invention is based on the fluorescence resonance energy transfer effect between quantum dots and gold nanoparticles, assembles the chemically modified functional magnetic beads, antibodies and quantum dots, and constructs the magnetic bead-antibody-quantum dot switch type composite fluorescent nano probe which integrates the functions of magnetic enrichment separation and rapid presentation of detection results. After the target bacteria are added, the target bacteria are combined with the fluorescent probe through antigen-antibody reaction, the distance between the fluorescent probe and the gold nanoparticles is increased, the fluorescence resonance energy transfer effect is weakened, and the fluorescence signal quenched by the fluorescence resonance energy transfer is recovered. The fluorescent nano probe provided by the invention is low in interference of a background matrix, has the advantages of high selectivity, rapidness and sensitivity, and provides an accurate and effective method for rapid detection of salmonella.
Description
Technical Field
The invention belongs to the technical field of food safety, and particularly relates to a switch-type composite fluorescent nano probe for rapid detection of salmonella and a preparation method thereof.
Background
Salmonella (Salmonella) is a gram-negative brevibacterium of enterobacteriaceae, and has been reported to share more than 2600 serotypes, is one of important zoonosis pathogenic bacteria causing outbreak of food-borne diseases, is generally transmitted by polluted food, becomes a main public health threat worldwide, can cause various diseases such as gastroenteritis, typhoid fever, septicemia and the like, and can cause shock and disturbance of consciousness in severe cases. In China, food poisoning incidents caused by Salmonella annually account for 10% to 20% of all food poisoning incidents, and Salmonella is one of the major pathogenic bacteria causing outbreaks of food-borne diseases in recent years.
In recent years, the main rapid detection methods for salmonella include Polymerase Chain Reaction (PCR), nucleic acid probe technology, enzyme-linked immunosorbent assay (ELISA), Surface Plasmon Resonance (SPR), and the like. Although the methods are obviously improved in the aspects of detection speed, sensitivity, specificity and the like compared with the traditional methods, most detection methods are limited to be applied to the aspect of rapid detection on site due to the defects of precise instruments, complex sample pretreatment and the like. Therefore, it is necessary to develop a rapid detection method which is sensitive, simple, easy to operate and highly selective.
With the development of modern nanometer technology, nanometer materials have gained wide attention due to their excellent properties, and researchers at home and abroad will use a method of combining nanometer materials with fluorescence technology for rapid detection of microorganisms. The quantum dots are also called semiconductor Quantum Dots (QDs) or semiconductor nanocrystals, are novel semiconductor nanomaterials, have excellent spectral characteristics and photochemical characteristics, have the characteristics of good stability, wide and continuous excitation spectrum, narrow and symmetrical emission spectrum, high fluorescence intensity and the like compared with the traditional fluorescent dye as a novel marking material, are often used as fluorescent probes, have excellent detection performance in the aspects of molecular recognition probes and biosensor technologies, and are widely applied to the field of food and drug detection.
Disclosure of Invention
The invention provides a switch-type composite fluorescent nano probe with high selectivity and sensitivity and a preparation method thereof by utilizing the advantages of the specific recognition and fluorescence resonance energy transfer effect of a salmonella antibody.
The invention assembles aminated magnetic beads, antibodies and quantum dots based on Fluorescence Resonance Energy Transfer (FRET) between QDs and gold nanoparticles (AuNPs), and constructs the magnetic bead-antibody-quantum dot switch type composite fluorescent nano probe which integrates the functions of magnetic enrichment separation and rapid presentation of detection results. The quantum dot modified by mercaptopropionic acid in the fluorescent probe contains carboxyl (-COOH) and has negative charges; gold nanoparticle solution (CS-AuNPs) modified by mercaptoethylamine contains amino (-NH)2) Is positively charged; the distance between the two nano materials is shortened due to the electrostatic effect, the absorption spectrum of AuNPs and the emission spectrum of QDs are well overlapped, effective fluorescence resonance energy transfer can occur, and the fluorescence of the fluorescent probe is remarkably quenched. After the target bacteria are added, the target bacteria are combined with the fluorescent probe through antigen-antibody reaction, the distance between the fluorescent probe and the CS-AuNPs is increased, the FRET effect is weakened, and the fluorescent signal quenched by FRET is recovered. The invention constructs a fluorescent 'off-on' system based on FRET effect between QDs and AuNPs, and establishes an off-on type composite fluorescent nano probe for rapid detection of salmonella and a preparation method and a use method thereof. The method is used for backingThe interference of the scenic substrate is low, and the method has the advantages of high selectivity, rapidness and sensitivity, and provides an accurate and effective method for rapid detection of salmonella.
The invention relates to a preparation method of a switch-type composite fluorescent nano probe for rapid detection of salmonella, which comprises the following steps:
(1) preparation of immunomagnetic beads
Weighing 4-5 g of FeCl3·6H2O and 2-3 g of Fe3O4·7H2Dissolving O in 150-300 mL of ultrapure water, introducing N2Stirring until dissolved; adding 3-3.5 g of NaOH to enable the solution to immediately turn black, and continuously stirring for 0.5-2.0 h under the condition of water bath at the temperature of 80-90 ℃ to obtain Fe3O4Nano magnetic beads; multiple washing of Fe with ethanol and ultrapure water by magnetic decantation3O4The nano magnetic beads are neutral, and then are dried in vacuum at 50-70 ℃ to obtain naked magnetic beads; adding 0.3-0.8 g of naked magnetic beads and 4-8 mL of Tetraethoxysilane (TEOS) into 200-300 mL of ethanol and water according to the volume ratio of 1:1, performing ultrasonic dispersion for 15-30 min, continuously stirring at 50-70 ℃ for reaction for 2-4 h, separating by using an external magnetic field, and fully cleaning by using ultrapure water to obtain silanized magnetic beads (Fe)3O4@SiO2) Drying at 55-65 ℃ under vacuum; taking 0.1-0.5 g of Fe3O4@SiO2Adding the mixture into 15-25 mL of anhydrous ethanol, performing ultrasonic treatment to uniformly disperse the mixture, adding 1-5 mL of 3-Aminopropyltriethoxysilane (APTES), adjusting the pH to 11 with ammonia water, stirring the mixture in a water bath at 55-65 ℃ for overnight reaction, washing the mixture for multiple times with ultrapure water to remove the uncoupled APTES, and performing vacuum drying at 55-65 ℃ to obtain chemically modified functionalized magnetic beads, namely Fe3O4@SiO2@NH2。
30-60 mg of Fe3O4@SiO2@NH2Dispersing by using 3-5 mL of PBS buffer solution with the pH value of 7.0, and carrying out ultrasonic treatment for 20-40 min; adding 2-3 mL of glutaraldehyde solution with the mass fraction of 25%, oscillating at room temperature for 2-4 h, storing in 0.2M PBS buffer solution with the pH of 5.0, adding 0.8-1.3 mL of the magnetic bead solution into 5-8 mL of salmonella specific antibody (prepared by adding salmonella intoSpecific antibodies were mixed with PBS buffer at 1: diluting according to the volume ratio of 500), and performing oscillation reaction at room temperature for 1-3 h to obtain an immunomagnetic bead solution;
(2) preparation of quantum dots
Taking 30-60 mL of 0.024mol/L Na230-60 mL of S solution and 0.032mol/L ZnCl2Dropwise and alternately dropwise adding the solution into 30-60 mL of purchased MPA-CdTe QDs solution of which the concentration is 0.05 mu mol/L under the conditions of rapid stirring and nitrogen introduction, carrying out closed stirring reaction for 0.8-1.5 h at the temperature of 55-65 ℃ to obtain MPA-CdTe @ ZnS QDs solution, standing, and mixing the MPA-CdTe @ ZnS QDs solution with acetone according to the ratio of 1:1, oscillating for 3-8 min, centrifuging for 3-8 min under the condition of 8000-15000 r/min, then centrifugally washing and precipitating by using ultrapure water, suspending by using ultrapure water to obtain a purified MPA-CdTe @ ZnS QDs solution, and storing at 4 ℃ for later use;
(3) performing coupling reaction on the immunomagnetic beads and the quantum dots together to obtain the composite fluorescent nano probe
EDC/NHS is selected to activate the carboxyl groups on the surface of the quantum dots in the step (2). Adding 20-30 mu L of 33.4 mmol/L1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride aqueous solution (EDC) and 20-30 mu L of 70.9mmol/L N-hydroxysuccinimide solution (NHS) into 80-120 mu L of the quantum dot solution obtained in the step (2), fully mixing uniformly, and carrying out oscillation reaction on a constant temperature oscillator at 20-30 ℃ for 10-20 min to obtain an activated quantum dot solution; adding 40-50 mu L of the immunomagnetic bead solution prepared in the step (1) into the activated quantum dot solution, carrying out magnetic separation after slight oscillation at a constant temperature of 20-30 ℃ in the dark for 1.5-3.0 h, and repeatedly washing with PBS solution for 2-4 times to obtain the composite fluorescent nano probe;
(4) preparation of gold nano-meter, construction of switch-off type composite fluorescent nano-probe
Mixing 400-700 mu L of cysteamine hydrochloride of 0.213mol/L and 40-60 mL of tetrachloroauric acid of 1.4mmol/L, stirring in the dark at room temperature for 15-30 min, quickly adding 10-15 mu L of sodium borohydride solution of 10mmol/L, continuously and violently stirring for 20-40 min to prepare wine red solution, filtering by a 0.45 mu m microporous filter membrane to prepare mercaptoethylamine modified gold nano solution, namely CS-AuNPs; the amount of the solution is 5-20 mu L,0.2mol/L of K2CO3And (3) adjusting the pH value of the CS-AuNPs to 8, putting 0.8-1.2 mL of CS-AuNPs diluted by 15 times and having a pH value of 8 into a centrifugal tube, adding 80-120 mu L of the composite fluorescent nano probe prepared in the step (3), uniformly mixing, and standing for reacting for 10-20 min to obtain the quenched off-switch composite fluorescent nano probe.
The method for detecting salmonella by using the switch type composite fluorescent nano probe prepared by the invention comprises the following steps:
(1) adding the salmonella liquid to be detected into the quenched switch-type composite fluorescent nano probe, and combining the salmonella liquid with the quenched switch-type composite fluorescent nano probe for reaction;
(2) detecting the fluorescence value of the probe before and after adding the salmonella liquid by using a fluorescence spectrometer under the excitation wavelength of 365nm, and respectively recording the detection results as F, F0By F-F0Showing the fluorescence recovery degree of the probe after adding the salmonella liquid, and comparing and analyzing the fluorescence recovery degrees of the probe after adding the salmonella with different concentrations to find the logarithm (x) of the concentration of the salmonella liquid and the fluorescence recovery degree (y represents F-F)0) Has good linear relation, and the linear equation is that y is 103.5x +121.4 (R)20.9956). Therefore, the concentration of the salmonella can be quantitatively calculated by substituting the measured fluorescence difference value before and after adding the bacterial liquid into the linear equation. Compared with the prior art, the invention has the following advantages:
(1) the invention adopts gold nano modified by mercaptoethylamine as a quenching agent for the first time, and the gold nano solution modified by mercaptoethylamine contains amino (-NH)2) The quantum dots in the composite fluorescent nano probe are positively charged, contain carboxyl (-COOH) after being modified by MPA and are negatively charged, and due to the electrostatic self-assembly effect, compared with unmodified gold nanoparticles, the quantum dots are more tightly combined, so that the firmness of connection among nano materials in the probe is enhanced, and the sensitivity of the probe is improved.
(2) The invention utilizes the advantages of the specific recognition and fluorescence resonance energy transfer effect of the salmonella antibody, and orderly assembles the functionalized magnetic beads, the antibody and the Quantum Dots (QDs), thereby constructing the magnetic bead-antibody-quantum dot composite fluorescent nano probe which integrates the functions of magnetic enrichment separation and rapid presentation of the detection result, and having higher detection efficiency;
(3) the detection method is simple and convenient to operate, the corresponding salmonella concentration can be obtained by only mixing the sample with the fluorescent probe and then measuring the fluorescence intensity of the sample, and the method has the characteristics of strong specificity and high sensitivity, and the detection limit can reach 102CFU/mL；
Drawings
FIG. 1: fe3O4(a) And Fe3O4@SiO2@NH2(b) (ii) a near infrared spectrum;
FIG. 2: a fluorescence spectrum chart before quenching (a) and after quenching (b) by the composite fluorescent nano probe;
FIG. 3: the ultraviolet absorption spectrum of CS-AuNPs;
FIG. 4: zeta potential value of CS-AuNPs;
FIG. 5: the Zeta potential value of MPA-CdTe @ ZnS QDs;
FIG. 6: a schematic diagram of detection results of the off-type composite fluorescent nano-probe for salmonella with different concentrations;
FIG. 7: a relation curve graph of the fluorescence recovery degree of the closed composite fluorescent nano probe along with the change of the salmonella bacterial liquid concentration;
FIG. 8: a schematic diagram of the specific detection result; target bacteria: salmonella; non-target bacteria: shigella, staphylococcus aureus, and escherichia coli;
FIG. 9: a fluorescence microscopic imaging picture after the salmonella and the non-target mixed bacteria are combined with the fluorescent probe;
Detailed Description
The invention will be further illustrated with reference to the following specific examples. It should be understood that the specific embodiments described herein are merely illustrative of the invention and do not limit the invention.
The invention provides a composite fluorescent nano probe. The fluorescent composite nano probe for rapid detection of salmonella is composed of a salmonella antibody, functionalized magnetic beads and quantum dots. The antibody reacts with the functionalized magnetic beads and the quantum dots to form a coupling system to construct a fluorescent composite nano probe; the probe and the gold nano-particles generate fluorescence resonance energy transfer to obtain a quenched switch-type composite fluorescence nano-probe; detecting salmonella based on the quenched composite fluorescent nanoprobe; the detection performance of the detection probe was evaluated.
Example 1: the preparation method of the composite fluorescent nano probe for rapid detection of salmonella comprises the following steps:
(1) preparation of immunomagnetic beads
Weighing 4.78g FeCl3·6H2O and 2.78g Fe3O4·7H2Dissolving O in 200mL of ultrapure water, introducing N2Stirring until dissolved. Adding 3.2g NaOH, and continuously stirring for 1h at the temperature of 85 ℃ to obtain Fe3O4And (4) nano magnetic beads. Washing the magnetic beads for multiple times until the magnetic beads are neutral, drying the magnetic beads in vacuum at 60 ℃ to obtain naked magnetic beads, taking 0.5g of the naked magnetic beads, adding the naked magnetic beads and 6mL of Tetraethoxysilane (TEOS) into 250mL of ethanol: ultrasonically dispersing for 20min in a dispersion system with water being 1:1, continuously stirring at 60 ℃ for reacting for 3h, then carrying out magnetic separation, and fully cleaning with ultrapure water to obtain silanized magnetic beads Fe3O4@SiO2And vacuum drying at 60 deg.C for use. Adding 0.3g of the magnetic beads into 20mL of absolute ethanol, adding 3mL of 3-Aminopropyltriethoxysilane (APTES) after uniform ultrasonic dispersion, adjusting the pH to 11 with ammonia water, stirring in a water bath at 60 ℃ for reaction overnight, washing with ultrapure water for multiple times to remove the uncoupled APTES, and drying in vacuum at 60 ℃ to obtain the functionalized magnetic beads for later use.
50mg of the functionalized magnetic beads are taken, dispersed by 4mL of PBS buffer solution with pH7.0, ultrasonically treated for 30min, added with 2.5mL of 25% glutaraldehyde, reacted for 3h at room temperature with shaking, and then stored in the PBS buffer solution with pH 5.00.2M. 1mL (about 10mg) of the magnetic beads are taken, 6mL of salmonella specific antibody (diluted by 1: 500) is added, and the mixture is shaken at room temperature for reaction for 2h to obtain the immunomagnetic beads. Mixing bare magnetic beads (Fe)3O4) And amino group-modified silanized immunomagnetic beads (Fe)3O4@SiO2@NH2) And (4) after vacuum drying, characterizing the functional groups with characteristic peaks in a near infrared spectrometer. As shown in FIG. 1, at 574cm-1A strong absorption peak appears for Fe-O stretching in oxygen tetrahedral position and oxygen octahedral positionAnd (4) contracting the vibration peak. Usually Fe3O4Has an absorption peak at 580cm-1The small difference may be due to the size effect of the nanoparticles, the frequency of the radicals is shifted, thus proving that the phase of the sample is Fe3O4. In FIG. 2b, at 3402cm-1The strong absorption peak is attributed to the stretching vibration of the O-H bond in the carboxyl, which indicates that-OH is introduced on the surface of the modified magnetic bead. 1080cm-1The strong absorption peak is the stretching vibration peak of Si-O-Si bond, 800cm-1The stretching vibration peak of Si-O shows that SiO2Successfully wrapped in Fe3O4And (4) the surface of the magnetic beads. At 3402cm-1Is in the form of-NH2Characteristic absorption peak of 1633cm-1The bending vibration peak of the N-H bond appears, which indicates that the amino group is successfully modified in Fe3O4And (4) the surface of the magnetic beads. The results of the near infrared spectroscopy show that the immunized magnetic bead is successfully made of SiO2Coated and successfully modified with amino through chemical modification.
(2) Preparing quantum dots, connecting with immunomagnetic beads to construct composite fluorescent nano probe
50mL of 0.024mol/L Na is taken2S solution and 50mL of 0.032mol/L ZnCl2The solution is dripped into mercaptopropionic acid (MPA) modified CdTe QDs solution drop by drop under the conditions of rapid stirring and nitrogen introduction, and is stirred and reacted for 1h in a closed manner at the temperature of 60 ℃ to obtain MPA-CdTe @ ZnS QDs solution. The solution was transferred to a clean and dry glass bottle, sealed and allowed to stand at room temperature. Mixing the prepared QDs solution with acetone of the same volume, oscillating for 5min, centrifuging at 10000r/min for 5min, washing precipitate with ultrapure water, and suspending with ultrapure water for use. To 100. mu.L of the quantum dot solution were added 28.75. mu.L of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) (33.4mmol/L, as prepared immediately before use) and 27. mu.L of N-hydroxysuccinimide (NHS) (70.9 mmol/L). Mix well and shake the reaction on a 25 ℃ constant temperature shaker for 15 min. Adding 45 mu L of the immunomagnetic beads synthesized in the step (1) into the activated quantum dot solution. And (3) oscillating for 2h at constant temperature of 20-30 ℃ in a dark place, carrying out magnetic separation, and repeatedly washing for 3 times by using a PBS (phosphate buffer solution) solution to obtain the composite fluorescent nano probe.
(3) Preparing gold nano-particles, and performing fluorescence quenching on the composite fluorescent nano-probe by using the gold nano-particles as a quenching agent to obtain the switch-on/off type composite fluorescent nano-probe
Mixing 500 mu L of 0.213mol/L cysteamine hydrochloride and 50mL of 1.4mmol/L tetrachloroauric acid, stirring at room temperature in the dark for 20min, quickly adding 12.5 mu L of 10mmol/L newly prepared sodium borohydride solution, continuously stirring vigorously for 30min, filtering the prepared wine red solution with a 0.45 mu m microporous filter membrane, and storing at 4 ℃.
And (3) putting 1mL of CS-AuNPs diluted by 15 times in proportion and having the pH value of 8 into a centrifuge tube, adding 100 mu L of the composite fluorescent nano probe, uniformly mixing, and standing at room temperature for 15min to obtain the quenched composite fluorescent nano probe. As can be seen from FIG. 2, the fluorescence emission peak of the probe is located at about 516nm, and after CS-AuNPs are added, the CS-AuNPs effectively quench the composite fluorescent nano-particles, and the fluorescence intensity of the quenched probe is obviously reduced. As can be seen from the UV spectrum of CS-AuNPs in FIG. 3, the maximum absorption peak of CS-AuNPs is at 524nm, and the spectrum shows an asymmetric distribution. This substantially coincides with the wavelength of the fluorescence peak of the probe. When the wavelength of the ultraviolet absorption peak of the quencher is basically coincident with the wavelength of the fluorescence peak of the fluorescent substance, the quencher can quench the fluorescent substance well, so that the colloidal gold serving as the fluorescence quencher can meet the requirement of quantum dot fluorescence quenching. From the Zeta potential value of the CS-AuNPs in figure 4 and the Zeta potential value of the MPA-CdTe @ ZnS QDs in figure 5, the Zeta potentials of the CS-AuNPs and the MPA-CdTe @ ZnS QDs in the system are respectively-35.7 mV and 15.4mV, and further proves that the gold nano modified with mercaptoethylamine and the quantum dots modified with MPA are tightly combined to generate effective fluorescence resonance energy transfer due to electrostatic attraction.
Example 2: the use method of the composite fluorescent nano probe for the rapid detection of the salmonella comprises the following steps:
(1) dispersing the switch-on/off type composite fluorescent nano-probe quenched by the gold nano-particles in the step (3) of the embodiment 1 in a PBS buffer solution for later use;
(2) the concentration of the bacterial liquid is 1 × 101、1×102、1×103、1×104、1×105、1×106、1×107、1×108CFU/Respectively adding mL of salmonella bacterial liquid into the reaction system, placing the salmonella bacterial liquid in a shaking table for oscillation reaction at 37 ℃ for 1h, and measuring the change condition of the fluorescence signal value before and after adding the salmonella bacterial liquid into the reaction system by using a fluorescence spectrometer, wherein the fluorescence of the quenched reaction system is obviously recovered after adding the salmonella as can be seen from figure 6;
(3) difference in fluorescence F-F0The log of the salmonella bacteria liquid concentration is expressed by x, which is recorded as the fluorescence recovery degree y, and the linear equation can be made as 103.5x +121.4 (R) from FIG. 620.9956), i.e., as shown in fig. 7. When the closed-type composite fluorescent nano probe is used for detecting the salmonella with unknown concentration, the fluorescence recovery degree y obtained by the measured fluorescence difference value before and after the salmonella is added is substituted into the linear equation to quantitatively calculate the concentration logarithm x of the salmonella, so that the concentration of the salmonella is obtained.
Example 3: the detection performance evaluation of the composite fluorescent nano probe for the rapid detection of the salmonella comprises the following steps:
(1) the detection effect on different concentrations of salmonella is shown in figure 7, and the data in the figure show that the concentration of salmonella is less than 102The detection result is negative when the concentration is higher than 10 when the concentration is CFU/mL2The detection result is positive when CFU/mL is obtained, so that the detection limit of the salmonella is determined to be 102CFU/mL；
(2) In order to detect the specificity of the system to salmonella, different food-borne pathogenic bacteria are analyzed. The response of non-target bacteria (shigella, staphylococcus aureus and escherichia coli) to chemiluminescence was studied, and as shown in fig. 8, the fluorescence intensity of the composite fluorescent nanoprobe solution gradually increased with the increase of salmonella concentration, while the addition of the non-target bacteria did not cause the fluorescence intensity of the system to change significantly. FIG. 9 is a fluorescent microscope photograph showing the target bacteria and the non-target mixed bacteria added (a, b are bright field and dark field images of the target bacteria under the fluorescent microscope, respectively, and c, d are bright field and dark field images of the non-target mixed bacteria under the fluorescent microscope, respectively). As can be seen from the comparison between the bright field and the dark field, the target bacteria in FIG. 9-b emit orange fluorescence, indicating that the quenched fluorescence is recovered; little fluorescence was observed in FIG. 9-d, indicating that the non-target bacteria did not restore the quenched fluorescence. The results show that the method has good specificity for detecting the salmonella.
(3) In order to further illustrate the detection performance of the rapid detection method for the salmonella based on the fluorescence resonance energy transfer effect, the salmonella existing in the artificially contaminated milk sample is detected. As shown in Table 1, salmonella suspensions with different concentrations were randomly added to fresh milk samples, and cultured in a 37 ℃ incubator for 18 hours to obtain artificially contaminated milk samples. Compared with the detection result of the national standard (GB/T4789.4-2016 food microbiology detection salmonella test), the analysis data shows that the numerical values obtained by the two methods are basically consistent, and the square of the correlation coefficient R reaches 0.9998, which indicates that the detection method not only can detect and analyze the salmonella under the laboratory condition, but also can analyze the salmonella in the milk.
Table 1: detection result of artificially simulating polluted milk sample
The above embodiments are merely preferred embodiments of the present invention, which should not be construed as limiting the present invention, and any modifications, equivalents or improvements made within the spirit and principle of the present invention should be included in the scope of the present invention.
Claims (2)
1. A preparation method of a switch-type composite fluorescent nano probe for rapid detection of salmonella comprises the following steps:
(1) preparation of immunomagnetic beads
Weighing 4-5 g of FeCl3·6H2O and 2-3 g of Fe3O4·7H2Dissolving O in 150-300 mL of ultrapure water, introducing N2Stirring until dissolved; adding 3-3.5 g of NaOH, and continuously stirring for 0.5-2.0 h under the condition of water bath at the temperature of 80-90 ℃ to obtain Fe3O4Nano magnetic beads; using ethanol and ultra-high by magnetic pouring methodMultiple washing of Fe with pure water3O4The nano magnetic beads are neutral, and then are dried in vacuum at 50-70 ℃ to obtain naked magnetic beads; adding 0.3-0.8 g of naked magnetic beads and 4-8 mL of tetraethoxysilane into 200-300 mL of ethanol and water according to the volume ratio of 1:1, performing ultrasonic dispersion for 15-30 min, continuously stirring at 50-70 ℃ for reaction for 2-4 h, separating by using an external magnetic field, and fully cleaning by using ultrapure water to obtain silanized magnetic beads, namely Fe3O4@SiO2Drying at 55-65 ℃ under vacuum; taking 0.1-0.5 g of Fe3O4@SiO2Adding the mixture into 15-25 mL of anhydrous ethanol, performing ultrasonic treatment to uniformly disperse the mixture, adding 1-5 mL of 3-aminopropyltriethoxysilane, adjusting the pH to 11 with ammonia water, stirring the mixture in a water bath at 55-65 ℃ for reaction overnight, washing the mixture for multiple times with ultrapure water to remove the uncoupled 3-aminopropyltriethoxysilane, and drying the mixture in vacuum at 55-65 ℃ to obtain chemically modified functionalized magnetic beads, namely Fe3O4@SiO2@NH2；
30-60 mg of Fe3O4@SiO2@NH2Dispersing by using 3-5 mL of PBS buffer solution with the pH value of 7.0, and carrying out ultrasonic treatment for 20-40 min; adding 2-3 mL of glutaraldehyde solution with the mass fraction of 25%, oscillating at room temperature for 2-4 h, storing in 0.2M PBS (phosphate buffer solution) with the pH of 5.0, adding 0.8-1.3 mL of the magnetic bead solution into 5-8 mL of PBS buffer solution, and mixing the solution with the solution in the ratio of 1: carrying out shake reaction on the salmonella specific antibody solution diluted by 500 volume ratios at room temperature for 1-3 h to obtain an immunomagnetic bead solution;
(2) preparation of quantum dots
Taking 30-60 mL of 0.024mol/L Na230-60 mL of S solution and 0.032mol/L ZnCl2Dropwise and alternately adding the solution into 30-60 mL of 0.05 mu mol/L MPA-CdTe QDs solution under the conditions of rapid stirring and nitrogen introduction, carrying out closed stirring reaction for 0.8-1.5 h at the temperature of 55-65 ℃ to obtain an MPA-CdTe @ ZnS QDs solution, standing, and then mixing the MPA-CdTe @ ZnSQDs solution with acetone according to the ratio of 1:1, uniformly mixing, oscillating for 3-8 min, centrifuging for 3-8 min under the condition of 8000-15000 r/min, then centrifugally washing and precipitating by using ultrapure water, suspending by using ultrapure water to obtain a purified MPA-CdTe @ ZnS QDs solution, and storing at 4 ℃;
(3) performing coupling reaction on the immunomagnetic beads and the quantum dots together to obtain the composite fluorescent nano probe
Adding 20-30 mu L of 33.4 mmol/L1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride aqueous solution and 20-30 mu L of 70.9mmol/L N-hydroxysuccinimide solution into 80-120 mu L of the quantum dot solution obtained in the step (2), fully mixing uniformly, and carrying out oscillation reaction on a constant temperature oscillator at 20-30 ℃ for 10-20 min to obtain an activated quantum dot solution; adding 40-50 mu L of the immunomagnetic bead solution prepared in the step (1) into the activated quantum dot solution, carrying out magnetic separation after 1.5-3.0 h of constant temperature light-proof slight oscillation, and repeatedly washing with PBS solution for 2-4 times to obtain a composite fluorescent nano probe;
(4) preparation of gold nano-meter, construction of switch-off type composite fluorescent nano-probe
Mixing 400-700 mu L of cysteamine hydrochloride of 0.213mol/L and 40-60 mL of tetrachloroauric acid of 1.4mmol/L, stirring in the dark at room temperature for 15-30 min, quickly adding 10-15 mu L of sodium borohydride solution of 10mmol/L, continuously and violently stirring for 20-40 min to prepare wine red solution, filtering by a 0.45 mu m microporous filter membrane to prepare mercaptoethylamine modified gold nano solution, namely CS-AuNPs; using 5-20 μ L of 0.2mol/L K2CO3And (3) adjusting the pH value of the CS-AuNPs to 8, putting 0.8-1.2 mL of CS-AuNPs diluted by 15 times and having a pH value of 8 into a centrifugal tube, adding 80-120 mu L of the composite fluorescent nano probe prepared in the step (3), uniformly mixing, and standing for reacting for 10-20 min to obtain the quenched off-switch composite fluorescent nano probe.
2. A switch-on/switch-off type composite fluorescent nano probe for rapid detection of salmonella is characterized in that: is prepared by the method of claim 1.
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