CN109387500B - Method for detecting escherichia coli based on magnetic graphene oxide composite star @ gold-silver alloy nanoparticles - Google Patents
Method for detecting escherichia coli based on magnetic graphene oxide composite star @ gold-silver alloy nanoparticles Download PDFInfo
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Abstract
The invention discloses a method for detecting escherichia coli based on magnetic graphene oxide composite star @ gold-silver alloy nanoparticles, and belongs to the technical field of food safety detection. Firstly, respectively preparing a magnetic graphene oxide nano material and a 4-ATP marked aventurine @ gold-silver alloy Raman substrate material, respectively modifying nucleic acid aptamers of target bacteria on the surfaces of the magnetic graphene oxide nano material and the 4-ATP marked aventurine @ gold-silver alloy Raman substrate material, and fixing aventurine @ gold-silver alloy nano particles on the magnetic graphene oxide nano material through the combination of the surface nucleic acid aptamers and the target bacteria when the target bacteria exist. The unfixed Venus @ gold-silver alloy nanoparticles were removed by magnetic separation. And (3) establishing a linear relation between the concentration of pathogenic bacteria and the 4-ATP Raman signal intensity by using the Raman spectrometer for scanning, and realizing the quantitative detection of the escherichia coli. The invention realizes the rapid and high-sensitivity detection of pathogenic microorganisms. The invention provides an effective method for rapidly detecting harmful microorganisms in food and environment.
Description
Technical Field
The invention relates to a method for detecting escherichia coli based on magnetic graphene oxide composite star @ gold-silver alloy nanoparticles, and belongs to the technical field of food safety detection.
Background
The food-borne pathogenic bacteria such as escherichia coli and the like can pollute food in the food processing and production process, cause food deterioration and secrete toxic substances, induce diseases such as hematoenteritis, hemolytic uremic syndrome, septicemia and the like, and even cause death for children with weak resistance. The infection has the characteristics of short incubation period, fierce coming, simultaneous attack of a plurality of people in a short time and the like, and is very easy to cause the outbreak of large-scale food-borne diseases. The establishment and research of the fast detection method of food-borne pathogenic bacteria have great significance for guaranteeing food safety and human health.
In the detection of food-borne pathogenic bacteria, time and sensitivity are the two most important indexes, because microorganisms grow vigorously and multiply quickly, even one pathogenic bacterium in food can possibly cause infection, and accurate trace detection of the pathogenic bacterium is required within the shortest time. The traditional pathogenic bacteria detection method mostly adopts a bacteria isolation culture method, and although the method has better stability, the method is far from meeting the requirement of the current food safety rapid detection due to the complexity, time consumption (generally 3-5 days), large workload, need of abundant experience foundation and the like. In recent years, many novel rapid detection methods such as enzyme-linked immunoassay, PCR, fluorescence labeling and the like have been developed for food-borne pathogenic bacteria, and although these methods have made some progress in high-sensitivity or high-throughput detection, they still have some disadvantages, which restrict their further application and popularization. For example, enzyme-linked immunoassay generally requires enrichment culture of a sample, and a detection process of 1-2 hours, which seriously increases the time and cost and is not favorable for rapid detection. Although the PCR method does not need enrichment culture, the method has serious problems of high requirement on professional technology and easy pollution caused by the operation process to cause false positive. By adopting a fluorescent labeling method, the detection result is easily influenced by a complex food matrix, and the stability and the repeatability are poor. Therefore, the establishment of a rapid, stable and high-sensitivity food-borne pathogenic bacteria detection method is a problem which needs to be solved urgently at present.
Surface-enhanced Raman scattering (SERS) spectrum is a phenomenon that Raman signals absorbed on metal Surface molecules are enhanced by utilizing Surface plasmon resonance of nano materials, and enhancement factors can reach 10 at most8-1014And the method has the advantage of single molecule detection. With redCompared with external absorption spectrum, the peak width of SERS is narrower, more detailed and understandable chemical information can be provided in a limited wavelength range, and the method is more suitable for measurement of biological samples. The SERS has the advantages that the rapid, repeatable and more importantly nondestructive quantitative analysis can be carried out on the sample, namely, the specific trace components in the complex mixed system can be directly detected without carrying out any pretreatment or extraction separation on the sample, and the SERS is particularly suitable for detecting toxic and harmful substances in complex food substrates. The development of a novel Raman sensor for rapid and high-sensitivity detection of pathogenic bacteria is a development direction of technical innovation.
Disclosure of Invention
The invention aims to provide a method for detecting escherichia coli based on magnetic graphene oxide composite Venus @ gold-silver alloy nanoparticles, which can be used for quickly and highly sensitively detecting a small amount of escherichia coli in a solution and provides an effective method for quickly screening harmful pathogenic bacteria in food.
The purpose of the invention is realized by the following technical scheme:
a method for detecting escherichia coli based on magnetic graphene oxide composite Venus @ gold-silver alloy nanoparticles is characterized by comprising the following steps:
1) preparing a magnetic graphene oxide nano material: respectively weighing ammonium ferric sulfate and ammonium ferrous sulfate in N2Under the protection condition, adding ultrapure water at the temperature of 50-90 ℃, adjusting the pH value to 3-5 by using ammonium hydroxide, then adding a graphene oxide solution, adjusting the pH value to 9-11, stirring and cooling to room temperature, respectively alternately cleaning by using absolute ethyl alcohol and ultrapure water, and freeze-drying to obtain the magnetic graphene oxide nanomaterial;
2) preparation of 4-ATP-labeled Venus @ gold-silver alloy nanoparticles: adding chloroauric acid solution, phosphate buffer salt solution, polyvinylpyrrolidone solution and hydroxylamine hydrochloride solution into the prepared 4-ATP-labeled aventurine @ silver nanoparticle solution, and uniformly mixing to obtain 4-ATP-labeled aventurine @ gold-silver alloy nanoparticle solution;
3) the surface modification nucleic acid aptamer of the nano material: taking the magnetic oxidized graphene nano material and the Venus @ gold-silver alloy nano particles prepared in the steps 1) and 2), respectively adding an escherichia coli aptamer solution into the two nano materials, reacting overnight, and then carrying out magnetic separation or centrifugation to remove supernatant so as to respectively obtain the magnetic oxidized graphene modified by the aptamer and the Venus @ gold-silver alloy nano particles;
4) establishing a standard curve of an escherichia coli Raman detection system: mixing the aptamer modified magnetic graphene oxide prepared in the step 3) with the Venus @ gold-silver alloy nanoparticles, adding target bacteria solutions with different concentrations into the mixture, fixing the Venus @ gold-silver alloy nanoparticles marked by 4-ATP on the surface of the magnetic graphene oxide by combining the target bacteria when the target bacteria exist, enriching the magnetic graphene oxide nano material to one side of a magnetic field through an external magnetic field, retaining the unfixed Venus @ gold-silver alloy nanoparticles in a supernatant, removing the supernatant, and adding a small amount of sterile water to resuspend the precipitate; scanning the heavy suspension by adopting a Raman spectrum, establishing a linear relation between the bacterial concentration and a 4-ATP Raman signal, and calculating a minimum detection limit;
5) detecting escherichia coli in a food sample: purchasing qualified raw milk from a supermarket, adding escherichia coli liquid with different concentrations into a sample, uniformly mixing, adding a mixed solution of a magnetic graphene oxide nano material and Venus @ gold-silver alloy nano particles, enriching the magnetic graphene oxide nano material to one side of a magnetic field through an external magnetic field, retaining the unfixed Venus @ gold-silver alloy nano particles in a supernatant, removing the supernatant, and adding a small amount of sterile water to carry out heavy suspension and precipitation; and performing Raman spectrum measurement on the heavy suspension, substituting the intensity of a scanning peak into a standard curve, and quantitatively calculating the number of the target bacteria in the food sample.
The technical scheme of the invention is as follows: the molar ratio of the ammonium ferric sulfate to the ammonium ferrous sulfate in the step 1) is 1-3: 1; the concentration of the graphene oxide solution is 0.5-1.5mg/mL, and the concentration of ferric ammonium sulfate: the mass ratio of the ultrapure water to the graphene oxide solution is (0.5-1.5) g, (180-220) g: (30-60) mg.
The technical scheme of the invention is as follows: the preparation method of the 4-ATP marked aventurine @ silver nanoparticle solution in the step 2) comprises the following steps:
the first step is as follows: surface modification of the venus nanoparticles with raman beacon molecules: adding a 4-aminothiophenol solution into the Venus citriodora nanoparticle solution, uniformly stirring, centrifuging to remove a supernatant, adding ultrapure water for resuspension, and obtaining the Venus citriodora nanoparticle solution with the molar concentration of 1-30 nmol/L and labeled by Raman beacon molecules;
the second step is that: venus @ silver nanoparticles: uniformly mixing the aventurine nanoparticle solution labeled by the Raman beacon molecules prepared in the step (1) with a polyvinylpyrrolidone solution, an ascorbic acid solution and a phosphate buffer solution, then adding a silver nitrate solution, adjusting the solution to be alkaline by using sodium hydroxide, stirring for 10-60 min, centrifuging to remove a supernatant after the reaction is finished, and adding ultrapure water to prepare an aventurine @ silver nanoparticle solution labeled by the Raman beacon molecules with the concentration of 1-20 nmol/L;
in some preferred embodiments: the preparation parameters of the 4-ATP labeled aventurine @ silver nanoparticle solution are as follows:
in the first step: the concentration of the venomous nano particle solution is 1-15 nmol/L, and the concentration of the 4-aminothiophenol solution is 0.1-5 mmol/L;
preferably: the concentration of the venomous nano particle solution is 3-10 nmol/L, and the concentration of the 4-aminothiophenol solution is 0.1-3 mmol/L;
further preferably: the volume ratio of the venomous nano particle solution to the 4-aminothiophenol solution is 100: 1-10;
most preferably: the volume ratio of the venomous nano particle solution to the 4-aminothiophenol solution is 100: 3-8;
in the second step: the concentration of the phosphate buffer solution is 5-20 mmol/L; the mass concentration of the polyvinylpyrrolidone solution is 0.1-5%; the molar concentration of the ascorbic acid solution is 50-150 mmol/L; the molar concentration of the silver nitrate solution is 1-15 mmol/L;
preferably: in the second step: the concentration of the phosphate buffer solution is 8-15 mmol/L; the mass concentration of the polyvinylpyrrolidone solution is 0.1-3%; the molar concentration of the ascorbic acid solution is 80-120 mmol/L; the molar concentration of the silver nitrate solution is 3-8 mmol/L;
further preferably: in the second step: raman beacon molecule labeled Venus nanoparticle solution: phosphate buffered saline solution: polyvinylpyrrolidone solution: ascorbic acid solution: the volume ratio of the silver nitrate solution is 1-10: 10-30: 5-15: 1-10: 1-5;
most preferably: in the second step: raman beacon molecule labeled Venus nanoparticle solution: phosphate buffered saline solution: polyvinylpyrrolidone solution: ascorbic acid solution: the volume ratio of the silver nitrate solution is 3-8: 15-25: 8-12: 3-8: 1 to 5.
The technical scheme of the invention is as follows: in the step 2), the mass concentration of the polyvinylpyrrolidone solution is 0.1-5%, the molar concentration of the hydroxylamine hydrochloride solution is 1-20 mmol/L, the molar concentration of the chloroauric acid solution is 1-20 mmol/L, and the molar concentration of the phosphate buffer solution is 1-20 mmol/L;
preferably: in the step 2), the mass concentration of the polyvinylpyrrolidone solution is 0.1-3%, the molar concentration of the hydroxylamine hydrochloride solution is 8-15 mmol/L, the molar concentration of the chloroauric acid solution is 5-15 mmol/L, and the molar concentration of the phosphate buffer solution is 5-15 mmol/L;
further preferably: the method comprises the following steps of (1) preparing a Raman beacon molecule labeled Venus @ silver nanoparticle solution: polyvinylpyrrolidone solution: hydroxylamine hydrochloride solution: chloroauric acid solution: the volume ratio of the phosphate buffer salt solution is 1-10: 1-10: 0.1-5: 0.1-5: 5-15;
most preferably: the method comprises the following steps of (1) preparing a Raman beacon molecule labeled Venus @ silver nanoparticle solution: polyvinylpyrrolidone solution: hydroxylamine hydrochloride solution: chloroauric acid solution: the volume ratio of the phosphate buffer salt solution is 3-8: 2-8: 0.1-3: 0.1-3: 5 to 15.
The technical scheme of the invention is as follows: the preparation method of the magnetic graphene oxide surface modified aptamer in the step 3) comprises the following steps:
a) dissolving the magnetic graphene oxide nano material prepared in the step 1) in a phosphate buffer solution to obtain a mixed solution 1;
b) adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxy thiosuccinimide into the mixed solution 1, removing the supernatant after the reaction is finished, adding a phosphate buffer solution, and uniformly mixing to obtain a mixed solution 2;
c) adding a DNA2 solution into the mixed solution 2, carrying out oscillation reaction, removing supernatant, adding a phosphate buffer solution again, and uniformly mixing to obtain a magnetic graphene oxide surface modified aptamer;
preferably: the mass-to-volume ratio of the magnetic graphene oxide nano material to the phosphate buffer solution in the step a) is (0.1-3) mg: 1mL, wherein the concentration of the phosphate buffer solution is 5-15 mmol/L;
preferably: in the step b), the concentration of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride is 1-5 mg/mL, and the concentration of N-hydroxy thiosuccinimide is 1-5 mg/mL; mixed solution 1: 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride salt: n-hydroxy thiosuccinimide: the volume ratio of the phosphate buffer solution is 3-8: 0.1-0.5: 0.1-0.5: 3-8; the concentration of the phosphate buffer solution is 5-15 mmol/L;
preferably: mixed solution 2 in step c): aptamer DNA21 solution: the volume ratio of the phosphate buffer solution is (1-30) mL: (40-60) μ L: (3-8) mL, wherein the concentration of the aptamer DNA2 solution is 50-150 mu mol/L;
wherein: the sequence of DNA2 is 5' NH2-ATC CGT CAC ACC TGC TCT ACT GGC CGG CTC AGC ATG ACT AAG AAG GAA GTT ATG TGG TGT TGG CTC CCG TAT TTT TTT TTT-3’。
The technical scheme of the invention is as follows: the preparation method of the gold star @ gold-silver alloy nanoparticle surface modification aptamer comprises the steps of adding a 4-ATP-labeled gold star @ gold-silver alloy nanoparticle solution prepared in the step 2) into an aptamer DNA1 solution, carrying out oscillation reaction at room temperature, centrifuging, removing a supernatant, adding 5mL of phosphate buffer solution, and carrying out heavy suspension to obtain the gold star @ gold-silver alloy nanoparticle surface modification aptamer;
preferably: the concentration of the 4-ATP-labeled aventurine @ gold-silver alloy nanoparticle solution is 5-15 nmol/L, and the concentration of the aptamer DNA1 solution is 50-150 mu mol/L; 4-ATP labeled aventurine @ gold-silver alloy nanoparticle solution: aptamer DNA1 solution: the volume ratio of the phosphate buffer solution is (1-10) mL: (1-20) μ L: (1-10) mL;
the sequence of the DNA1 is 5 'SH-ATC CGT CAC ACC TGC TCT ACT GGC CGG CTC AGC ATG ACT AAG AAG GAA GTT ATG TGG TGT TGG CTC CCG TAT TTT TTT TTT-3';
the technical scheme of the invention is as follows: the standard curve of the Escherichia coli detection system is established according to 1082cm-1And (3) processing the intensity value of the peak, wherein the scanning conditions of the Raman spectrum are as follows: excitation at 532nm and scan time 30 s.
The technical scheme of the invention is as follows: and in the step 4) and the step 5), the oxidized graphene surface modified aptamer and the Venus @ gold-silver alloy nanoparticle surface modified aptamer are mixed in equal volumes, and the concentration of the magnetic oxidized graphene nano material after mixing is 0.5-1.5 mg/mL.
The technical scheme of the invention is as follows: the linear range in the step 4) is 5-105cfu/mL, the minimum detection limit in step 5) was 3 cfu/mL.
The invention has the beneficial effects that:
1. the aventurine @ gold-silver alloy nanoparticles with strong Raman signals are fixed on the surface of the magnetic graphene oxide through the specificity recognition effect of the nucleic acid aptamer and the target bacteria, and the unfixed aventurine @ gold-silver alloy nanoparticles are removed through magnetic separation, so that the specificity of the detection method is effectively improved, and the influence of other substances in a detection system on a detection result is avoided.
2. The Venus @ gold-silver alloy nanoparticle is used as a Raman substrate material, a strong hot spot region is formed in a narrow gap of the Venus @ gold-silver alloy nanoparticle, a Raman signal of 4-ATP in the interval can be greatly enhanced, and compared with a Raman substrate material (high hollow and reproducible surface-enhanced Raman scattering from DNA-target nanoparticles with 1-nm inter gap. nat. Nanotechnol.2011,6 (7)) which is modified on the outer layer by other beacon molecules, the Venus @ gold-silver alloy nanoparticle has a stable, repeatable and extremely strong Raman signal, and is a key for realizing high-sensitivity detection of pathogenic bacteria.
3. The Venus @ gold-silver alloy nanoparticles are used as a Raman substrate material, and a novel Raman sensing detection method of escherichia coli is established through enrichment and separation of magnetic graphene oxide, wherein the minimum detection limit is 3cfu/mL, and the sensitivity is 5cfu/mL-105cfu/mL, much higher than normalThe method of nano-sensing detection (Simultaneous biosensor for multiple pathological bacteria detection based on multiple upper conversion nanoparticles. anal. chem.2014,86(6), 3100-7).
4. The invention aims to provide a method for detecting escherichia coli based on magnetic graphene oxide composite Venus @ gold-silver alloy nanoparticles, which can be used for detecting other harmful substances and can realize multiple detection.
Drawings
FIG. 1: a transmission electron micrograph of the magnetic graphene oxide nanomaterial described in example 1;
FIG. 2: enrichment of magnetically oxidized graphene described in example 1 with applied magnetic field;
FIG. 3: transmission electron microscopy of Venus @ gold-silver alloy nanoparticles as described in example 1;
FIG. 4: establishing a standard curve of the escherichia coli Raman detection system described in example 1; the concentration of the Escherichia coli is 0cfu/mL, 5cfu/mL, 10cfu/mL and 10cfu/mL in sequence2cfu/mL，103cfu/mL，104cfu/mL， 105cfu/mL；
FIG. 5: e.coli assay standard plots for the food samples described in example 1;
FIG. 6: e.coli assay standard curve of the food sample described in example 2;
FIG. 7: e.coli assay standard plots for the food samples described in example 3;
the specific implementation mode is as follows:
the present invention is further illustrated by the following specific embodiments.
The first step is as follows: preparing a magnetic graphene oxide nano material:
preparation of magnetic graphene oxide nanomaterial of example 1:
1) preparing a magnetic graphene oxide nano material: 1.736g of NH were taken4Fe(SO4)2·12H2O, 0.784g of (NH)4)2Fe(SO4)2·6H2O, in N2Under the protection condition of (3), 180mL of 70 DEG CThe ultrapure water is stirred evenly. Adding 1M NH4Adjusting the pH value to 3 by OH, adding 40mL of 0.9mg/mL graphene oxide solution, and stirring for 30 min. The pH value is adjusted to 9, and stirring is continued for 1 h. Cooling to room temperature, alternately cleaning for 2 times by using absolute ethyl alcohol and ultrapure water, and freeze-drying to obtain the magnetic graphene oxide nano material;
a TEM representation of the magnetic graphene oxide is shown in fig. 1, and it can be observed from the TEM image that a large number of magnetic nanoparticles are attached to the surface of the sheet layer of the magnetic graphene oxide, and the distribution is relatively uniform, wherein the diameter of the magnetic nanoparticles is 10-20 nm. Fig. 2 is an enrichment diagram of magnetic graphene oxide under the action of an external magnetic field, and it can be seen from the diagram that the prepared magnetic graphene oxide has better magnetism.
Preparation of magnetic graphene oxide nanomaterial of example 2:
1) preparing a magnetic graphene oxide nano material: 1.736g of NH were taken4Fe(SO4)2·12H2O, 0.784g of (NH)4)2Fe(SO4)2·6H2O, in N2Under the protection condition of (3), the mixture is added into 200mL of 70 ℃ ultrapure water and stirred uniformly. Adding 1M NH4And regulating the pH value to 4 by OH, adding 40mL of 1.150mg/mL graphene oxide solution, and stirring for 30 min. The pH value is adjusted to 10, and stirring is continued for 1 h. Cooling to room temperature, alternately cleaning for 2 times by using absolute ethyl alcohol and ultrapure water, and freeze-drying to obtain the magnetic graphene oxide nano material;
preparation of magnetic graphene oxide nanomaterial of example 3:
1) preparing a magnetic graphene oxide nano material: 1.736g of NH were taken4Fe(SO4)2·12H2O, 0.784g of (NH)4)2Fe(SO4)2·6H2O, in N2Under the protection condition of (3), the mixture is added into 220mL of 70 ℃ ultra-pure water and stirred uniformly. Adding 1M NH4And regulating the pH value to 5 by OH, adding 40mL of 1.40mg/mL graphene oxide solution, and stirring for 30 min. The pH was adjusted to 11 and stirring was continued for 1 h. Cooling to room temperature, alternately cleaning with anhydrous ethanol and ultrapure water for 2 times, and freeze-drying to obtain magnetic graphene oxideA nanomaterial;
the second step is that: preparation of the Venus @ gold-silver alloy nanoparticle solution:
the preparation method comprises the following steps:
preparation of a venus nanoparticle solution:
1) synthesis of gold species
Adding 50mL of 1mM chloroauric acid solution into a clean conical flask, and heating and stirring until the solution is boiled; adding 7.5mL of 1% trisodium citrate solution rapidly, continuing to stir and heat until the color becomes wine red and remains unchanged, stopping heating and stirring, and cooling to room temperature.
2) Preparation of Venus nano-particles
Another clean conical flask is taken, 200mL of 0.25mM chloroauric acid solution and 200 μ L of gold seeds are sequentially added at room temperature, 20 μ L of hydrochloric acid is used for regulating the pH value to 3, and after uniform mixing, 200 μ L of 2mM silver nitrate solution and 100 μ L of 0.1M ascorbic acid are simultaneously added, so that the color is changed into dark blue. Centrifuging for 10 minutes at 3000 r/min, removing supernatant, adding ultrapure water for resuspension, and preparing the aventum passeris nanoparticle solution with the molar concentration of 1-15 nmol/L.
Preparation of 4-ATP-labeled Venus @ gold-silver alloy nanoparticles:
(1) surface modification Raman beacon molecule of Venus nano-particles
And (3) taking the prepared gold star nanoparticle solution, quickly adding a 4-ATP solution into the gold star nanoparticle solution, stirring at 25 ℃ for reaction overnight, centrifuging for 10 minutes, removing a supernatant, and adding ultrapure water for heavy suspension to prepare the gold star nanoparticle solution labeled by the Raman beacon molecules.
(2) Preparation of Venus @ silver nanoparticles
And (2) taking the gold star nanoparticle solution labeled by the Raman beacon molecule prepared in the step (1), uniformly mixing the polyvinylpyrrolidone solution, the phosphate buffer solution and the ascorbic acid solution which are sequentially shown in the following table, then adjusting the pH to about 9 with an alkali liquor, finally quickly adding silver nitrate, stirring and reacting at room temperature in a dark place for 30 minutes, then performing 8000 rpm centrifugation for 10 minutes, removing a supernatant, adding ultrapure water for heavy suspension, and preparing the gold star @ silver nanoparticle solution labeled by the Raman beacon molecule.
(3) Preparation of Venus @ gold-silver alloy nanoparticle solution
And (3) taking 100 mu L of the Venus @ silver nanoparticle solution prepared in the step (2), sequentially adding a phosphate buffer salt solution, a polyvinylpyrrolidone solution and a hydroxylamine hydrochloride solution, uniformly mixing, then quickly adding a chloroauric acid solution, stirring at room temperature in a dark place for reaction for 2 hours, then 5000 rpm, centrifuging for 10 minutes, removing the supernatant, and adding ultrapure water for heavy suspension to prepare the 4-ATP-labeled Venus @ gold-silver alloy nanoparticle solution.
Example 1-34 Experimental parameters for the preparation of ATP-labeled Venus @ gold-silver alloy nanoparticles
Note: the volume ratio of the solution of each component in the step (2) is the gold star nanoparticle solution marked by the Raman beacon molecules: phosphate buffered saline solution: polyvinylpyrrolidone solution: ascorbic acid solution: the volume ratio of the silver nitrate solution;
the volume ratio of the solution of each component in the step (3) is the golden star @ silver nanoparticle solution marked by the Raman beacon molecules: polyvinylpyrrolidone solution: hydroxylamine hydrochloride solution: chloroauric acid solution: volume ratio of phosphate buffered saline solution.
A TEM representation of the 4-ATP-labeled star @ gold-silver alloy nanoparticle prepared in example 1 is shown in fig. 3, and it can be known from the TEM that the star @ gold-silver alloy nanoparticle has a core-shell structure, a narrow gap is formed between the core and the shell, and the diameter of the nanoparticle is 50-80 nm.
The third step: nano material surface modification nucleic acid aptamer
Wherein: the sequence of DNA2 is 5' NH2-ATC CGT CAC ACC TGC TCT ACT GGC CGG CTC AGC ATG ACT AAG AAG GAA GTT ATG TGG TGT TGG CTC CCG TAT TTT TTT TTT-3’。
Preparation of magnetic graphene oxide surface-modified aptamer of example 1:
dissolving 3mg of magnetic graphene oxide in 3mL of 5mmol/L phosphate buffer solution, adding 0.2mL of 1mg/mL1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and 0.1mL of 1mg/mL N-hydroxy thiosuccinimide into the solution respectively, carrying out shake reaction at 37 ℃ for 2h, carrying out magnetic separation to remove the supernatant, adding 3mL of 5mmol/L phosphate buffer solution, mixing uniformly, adding 50 mu L of 50 mu mol/L aptamer DNA2 solution, and carrying out shake reaction at 37 ℃ for 2 h. The supernatant was removed by magnetic separation and 3mL of phosphate buffer was added.
Preparation of magnetic graphene oxide surface-modified aptamer of example 2:
5mg of magnetic graphene oxide is dissolved in 5mL of 10mmol/L phosphate buffer solution, then 0.4mL of 2mg/mL1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and 0.2mL of 2mg/mL N-hydroxy thiosuccinimide are respectively added into the solution, the mixture is shaken at 37 ℃ for 2 hours, the supernatant is removed by magnetic separation, 5mL of 10mmol/L phosphate buffer solution is added, after uniform mixing, 50 muL of 100 mumol/L aptamer DNA2 solution is added, and the mixture is shaken at 37 ℃ for 2 hours. The supernatant was removed by magnetic separation and 5mL of phosphate buffer was added.
Preparation of magnetic graphene oxide surface-modified aptamer of example 3:
8mg of magnetic graphene oxide is dissolved in 8mL of 15mmol/L phosphate buffer solution, then 0.6mL of 5mg/mL1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and 0.4mL of 5mg/mL N-hydroxy thiosuccinimide are respectively added into the solution, the mixture is shaken at 37 ℃ for 2 hours, the supernatant is removed by magnetic separation, 8mL of 15mmol/L phosphate buffer solution is added, after uniform mixing, 50 μ L of 150 μmol/L aptamer DNA2 solution is added, and the mixture is shaken at 37 ℃ for 2 hours. The supernatant was removed by magnetic separation and 8mL of phosphate buffer was added.
The sequence of the DNA1 is 5 'SH-ATC CGT CAC ACC TGC TCT ACT GGC CGG CTC AGC ATG ACT AAG AAG GAA GTT ATG TGG TGT TGG CTC CCG TAT TTT TTT TTT-3';
preparation of 4-ATP labeled gold star @ gold-silver alloy nanoparticle surface-modified aptamer of example 1:
5mL of 5nmol/L aventurine @ gold-silver alloy nanoparticle solution is taken, 5 mu L of 50 mu mol/L aptamer DNA1 solution is added into the solution, the mixture is subjected to shaking reaction at room temperature for 4h and 5000r/min, after centrifugation for 10min, the supernatant is removed, and 5mL of phosphate buffer solution is added for resuspension.
Preparation of 4-ATP labeled gold star @ gold-silver alloy nanoparticle surface-modified aptamer of example 2:
10mL of 10nmol/L aventurine @ gold-silver alloy nanoparticle solution is taken, 10 muL of 100 mumol/L aptamer DNA1 solution is added into the solution, shaking reaction is carried out for 4h at room temperature, 5000r/min is carried out, after centrifugation is carried out for 10min, supernatant is removed, and 10mL of phosphate buffer solution is added for resuspension.
Preparation of 4-ATP labeled gold star @ gold-silver alloy nanoparticle surface-modified aptamer of example 3:
taking 10mL of 15nmol/L aventurine @ gold-silver alloy nanoparticle solution, adding 20 muL of 150 mumol/L aptamer DNA1 solution, carrying out shake reaction at room temperature for 4h, carrying out 5000r/min, centrifuging for 10min, removing supernatant, and adding 10mL of phosphate buffer solution for resuspension.
The fourth step: establishment of Raman sensing detection system for escherichia coli
Establishment of E.coli Raman sensing detection System described in example 1
The construction of the Escherichia coli Raman detection method based on the magnetic graphene oxide composite Venus @ gold-silver alloy nanoparticles comprises the following specific steps: taking 7 centrifuge tubes, adding 1mL of different concentrations (0cfu/mL, 5cfu/mL, 10 cfu/mL) into each tube2cfu/mL，103cfu/mL，104cfu/mL，105cfu/mL) of escherichia coli, and then 1mL of a mixed solution of aptamer-modified gold star @ gold-silver alloy nanoparticles and magnetic graphene oxide nanoparticles (example 1) was added thereto. After magnetic enrichment separation, washing with ultrapure water for 3 times, then adding 0.5mL of ultrapure water for resuspension, and irradiating the resuspension solution with 532nm laser for 30s to obtain Raman spectra with different intensities (as shown in FIG. 4). According to 1082cm-1And establishing a standard curve of the Raman signal intensity and the target bacteria concentration. As shown in FIG. 5, the detection limit of Escherichia coli obtained by the method is 5cfu/mL, and the linear range is 5cfu/mL-105cfu/mL。
Establishment of E.coli Raman sensing detection System described in example 2
Based on magnetic oxidation of graphiteThe construction method of the escherichia coli Raman detection method of the alkene composite Venus @ gold-silver alloy nanoparticles comprises the following specific steps: taking 7 centrifuge tubes, adding 1mL of different concentrations (0cfu/mL, 5cfu/mL, 10 cfu/mL) into each tube2cfu/mL，103cfu/mL，104cfu/mL，105cfu/mL) of escherichia coli, and then 1mL of a mixed solution of aptamer-modified gold star @ gold-silver alloy nanoparticles and magnetic graphene oxide nanoparticles was added thereto (example 2). After magnetic enrichment separation, washing with ultrapure water for 3 times, adding 0.5mL of ultrapure water for resuspension, and irradiating the resuspension solution with 532nm laser for 30s to obtain Raman spectrograms with different intensities. According to 1082cm-1And establishing a standard curve of the Raman signal intensity and the target bacteria concentration. As shown in FIG. 6, the detection limit of Escherichia coli obtained by the method is 3cfu/mL, and the linear range is 5cfu/mL-105 cfu/mL。
Establishment of E.coli Raman sensing detection System as described in example 3
The construction of the Escherichia coli Raman detection method based on the magnetic graphene oxide composite Venus @ gold-silver alloy nanoparticles comprises the following specific steps: taking 7 centrifuge tubes, adding 1mL of different concentrations (0cfu/mL, 5cfu/mL, 10 cfu/mL) into each tube2cfu/mL，103cfu/mL，104cfu/mL，105cfu/mL) of escherichia coli, and then 1mL of a mixed solution of aptamer-modified gold star @ gold-silver alloy nanoparticles and magnetic graphene oxide nanoparticles was added thereto (example 3). After magnetic enrichment separation, washing with ultrapure water for 3 times, adding 0.5mL of ultrapure water for resuspension, and irradiating the resuspension solution with 532nm laser for 30s to obtain Raman spectrograms with different intensities. According to 1082cm-1And establishing a standard curve of the Raman signal intensity and the target bacteria concentration. As shown in FIG. 7, the detection limit of Escherichia coli obtained by the method is 4cfu/mL, and the linear range is 5cfu/mL-105 cfu/mL。
The fifth step: detecting escherichia coli in a food sample:
purchasing qualified Weigang fresh milk from supermarket, taking 4 centrifuge tubes, adding 5mL fresh milk into each tube, and adding 0.5mL fresh milk into each tube with different concentrations (4.0 × 10, 6.0 × 10)2，2.0×103，1.0×104) After the escherichia coli liquid is uniformly mixed, adding 1mL of mixed liquid of the magnetic graphene oxide nano material and the Venus @ gold-silver alloy nano particles, enriching the magnetic graphene oxide nano material to one side of a magnetic field through an external magnetic field, removing supernatant, and adding a small amount of sterile water to resuspend and precipitate; and (4) carrying out Raman spectrum measurement on the heavy suspension, bringing the intensity of a scanning peak into a standard curve, and quantitatively calculating the number of escherichia coli in the fresh milk sample.
TABLE 1 detection of E.coli in food samples as described in example 1
Table 2 food sample e.coli assay as described in example 2
| Sample (I) | Additive concentration (cfu/mL) | Measured concentration (cfu/mL) | Recovery (%) |
| 1 | 4.0×10 | 4.1×10 | 102.5％±3.0 |
| 2 | 6.0×102 | 5.9×102 | 98.3％±0.6 |
| 3 | 2.0×103 | 2.2×103 | 110％±4.2 |
| 4 | 1.0×104 | 0.97×104 | 97％±1.6 |
TABLE 3 detection of E.coli in food samples as described in example 3
| Sample (I) | Additive concentration (cfu/mL) | Measured concentration (cfu/mL) | Recovery (%) |
| 1 | 4.0×10 | 4.2×10 | 105％±2.4 |
| 2 | 6.0×102 | 6.1×102 | 101％±1.3 |
| 3 | 2.0×103 | 1.98×103 | 99％±3.2 |
| 4 | 1.0×104 | 1.15×104 | 115％±1.4 |
As can be seen from the measurement results of tables 1 to 3, the sample recovery rate measured by the method is between 95% and 115%, which shows that the method has better accuracy.
It will be understood that the above-described embodiments are merely illustrative of the principles of the invention, which is not limited thereto, and that various modifications and changes can be made by those skilled in the art without departing from the spirit of the invention, which also falls within the scope of the invention.
Claims (22)
1. A method for detecting escherichia coli based on magnetic graphene oxide composite Venus @ gold-silver alloy nanoparticles is characterized by comprising the following steps: the method comprises the following steps:
1) preparing a magnetic graphene oxide nano material: respectively weighing ammonium ferric sulfate and ammonium ferrous sulfate in N2Under the protection condition, adding ultrapure water at the temperature of 50-90 ℃, adjusting the pH value to 3-5 by using ammonium hydroxide, then adding a graphene oxide solution, adjusting the pH value to 9-11, stirring and cooling to room temperature, respectively and alternately cleaning by using absolute ethyl alcohol and ultrapure water, and freeze-drying to obtain the magnetic graphene oxideA nanomaterial;
2) preparation of 4-ATP-labeled Venus @ gold-silver alloy nanoparticles: adding chloroauric acid solution, phosphate buffer salt solution, polyvinylpyrrolidone solution and hydroxylamine hydrochloride solution into the prepared 4-ATP-labeled aventurine @ silver nanoparticle solution, and uniformly mixing to obtain 4-ATP-labeled aventurine @ gold-silver alloy nanoparticle solution;
the preparation method of the 4-ATP-labeled aventurine @ silver nanoparticle solution in the step 2) comprises the following steps:
the first step is as follows: surface modification of the venus nanoparticles with raman beacon molecules: adding a 4-aminothiophenol solution into the Venus citriodora nanoparticle solution, uniformly stirring, centrifuging to remove a supernatant, adding ultrapure water for resuspension, and obtaining the Venus citriodora nanoparticle solution with the molar concentration of 1-30 nmol/L and labeled by Raman beacon molecules;
the second step is that: venus @ silver nanoparticles: uniformly mixing the gold star nanoparticle solution labeled by the Raman beacon molecules prepared in the first step with a polyvinylpyrrolidone solution, an ascorbic acid solution and a phosphate buffer solution, then adding a silver nitrate solution, adjusting the solution to be alkaline by using sodium hydroxide, stirring for 10-60 min, centrifuging to remove a supernatant after the reaction is finished, and adding ultrapure water to prepare a gold star @ silver nanoparticle solution labeled by the Raman beacon molecules with the concentration of 1-20 nmol/L;
3) the surface modification nucleic acid aptamer of the nano material: taking the magnetic oxidized graphene nano material and the Venus @ gold-silver alloy nano particles prepared in the steps 1) and 2), respectively adding an escherichia coli aptamer solution into the two nano materials, reacting overnight, and then carrying out magnetic separation or centrifugation to remove supernatant so as to respectively obtain the magnetic oxidized graphene modified by the aptamer and the Venus @ gold-silver alloy nano particles;
4) establishing a standard curve of an escherichia coli Raman detection system: mixing the aptamer modified magnetic graphene oxide prepared in the step 3) with the Venus @ gold-silver alloy nanoparticles, adding target bacteria solutions with different concentrations into the mixture, fixing the Venus @ gold-silver alloy nanoparticles marked by 4-ATP on the surface of the magnetic graphene oxide by combining the target bacteria when the target bacteria exist, enriching the magnetic graphene oxide nano material to one side of a magnetic field through an external magnetic field, retaining the unfixed Venus @ gold-silver alloy nanoparticles in a supernatant, removing the supernatant, and adding a small amount of sterile water to resuspend the precipitate; scanning the heavy suspension by adopting a Raman spectrum, establishing a linear relation between the bacterial concentration and a 4-ATP Raman signal, and calculating a minimum detection limit;
5) detecting escherichia coli in a food sample: purchasing qualified raw milk from a supermarket, adding escherichia coli liquid with different concentrations into a sample, uniformly mixing, adding a mixed solution of a magnetic graphene oxide nano material and Venus @ gold-silver alloy nano particles, enriching the magnetic graphene oxide nano material to one side of a magnetic field through an external magnetic field, retaining the unfixed Venus @ gold-silver alloy nano particles in a supernatant, removing the supernatant, and adding a small amount of sterile water to carry out heavy suspension and precipitation; and performing Raman spectrum measurement on the heavy suspension, substituting the intensity of a scanning peak into a standard curve, and quantitatively calculating the number of the target bacteria in the food sample.
2. The detection method according to claim 1, characterized in that: the molar ratio of the ammonium ferric sulfate to the ammonium ferrous sulfate is 1-3: 1; the concentration of the graphene oxide solution is 0.5-1.5mg/mL, and the concentration of ferric ammonium sulfate: the mass ratio of the ultrapure water to the graphene oxide solution is (0.5-1.5) g, (180-220) g: (30-60) mg.
3. The detection method according to claim 1, characterized in that: the preparation method of the 4-ATP-labeled aventurine @ gold-silver alloy nanoparticle comprises the following steps: the concentration of the venomous nano particle solution is 1-15 nmol/L, and the concentration of the 4-aminothiophenol solution is 0.1-5 mmol/L;
in the second step: the concentration of the phosphate buffer solution is 5-20 mmol/L; the mass concentration of the polyvinylpyrrolidone solution is 0.1-5%; the molar concentration of the ascorbic acid solution is 50-150 mmol/L; the molar concentration of the silver nitrate solution is 1-15 mmol/L.
4. The detection method according to claim 3, characterized in that: the concentration of the venomous nano-particle solution is 3-10 nmol/L, and the concentration of the 4-aminothiophenol solution is 0.1-3 mmol/L.
5. The detection method according to claim 3, characterized in that: the volume ratio of the venomous nano particle solution to the 4-aminothiophenol solution is 100: 1 to 10.
6. The detection method according to claim 5, characterized in that: the volume ratio of the venomous nano particle solution to the 4-aminothiophenol solution is 100: 3 to 8.
7. The detection method according to claim 1, characterized in that: the second step of the preparation method of the 4-ATP marked aventurine @ gold-silver alloy nano-particles comprises the following steps: the concentration of the phosphate buffer solution is 8-15 mmol/L; the mass concentration of the polyvinylpyrrolidone solution is 0.1-3%; the molar concentration of the ascorbic acid solution is 80-120 mmol/L; the molar concentration of the silver nitrate solution is 3-8 mmol/L.
8. The detection method according to claim 1, characterized in that: the second step of the preparation method of the 4-ATP marked aventurine @ gold-silver alloy nano-particles comprises the following steps: raman beacon molecule labeled Venus nanoparticle solution: phosphate buffered saline solution: polyvinylpyrrolidone solution: ascorbic acid solution: the volume ratio of the silver nitrate solution is 1-10: 10-30: 5-15: 1-10: 1 to 5.
9. The detection method according to claim 8, characterized in that: in the second step: raman beacon molecule labeled Venus nanoparticle solution: phosphate buffered saline solution: polyvinylpyrrolidone solution: ascorbic acid solution: the volume ratio of the silver nitrate solution is 3-8: 15-25: 8-12: 3-8: 1 to 5.
10. The detection method according to claim 1, characterized in that: in the step 2), the mass concentration of the polyvinylpyrrolidone solution is 0.1-5%, the molar concentration of the hydroxylamine hydrochloride solution is 1-20 mmol/L, the molar concentration of the chloroauric acid solution is 1-20 mmol/L, and the molar concentration of the phosphate buffer solution is 1-20 mmol/L.
11. The detection method according to claim 10, characterized in that: in the step 2), the mass concentration of the polyvinylpyrrolidone solution is 0.1-3%, the molar concentration of the hydroxylamine hydrochloride solution is 8-15 mmol/L, the molar concentration of the chloroauric acid solution is 5-15 mmol/L, and the molar concentration of the phosphate buffer solution is 5-15 mmol/L.
12. The detection method according to claim 1, characterized in that: the method comprises the following steps of (1) preparing a Raman beacon molecule labeled Venus @ silver nanoparticle solution: polyvinylpyrrolidone solution: hydroxylamine hydrochloride solution: chloroauric acid solution: the volume ratio of the phosphate buffer salt solution is 1-10: 1-10: 0.1-5: 0.1-5: 5 to 15.
13. The detection method according to claim 12, characterized in that: the method comprises the following steps of (1) preparing a Raman beacon molecule labeled Venus @ silver nanoparticle solution: polyvinylpyrrolidone solution: hydroxylamine hydrochloride solution: chloroauric acid solution: the volume ratio of the phosphate buffer salt solution is 3-8: 2-8: 0.1-3: 0.1-3: 5 to 15.
14. The detection method according to claim 1, characterized in that: the preparation method of the magnetic graphene oxide surface modified aptamer in the step 3) comprises the following steps:
a) dissolving the magnetic graphene oxide nano material prepared in the step 1) in a phosphate buffer solution to obtain a mixed solution 1;
b) adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxy thiosuccinimide into the mixed solution 1, removing the supernatant after the reaction is finished, adding a phosphate buffer solution, and uniformly mixing to obtain a mixed solution 2;
c) adding a DNA2 solution into the mixed solution 2, carrying out shaking reaction, removing supernatant, adding phosphate buffer solution again, and uniformly mixing to obtain the magnetic graphene oxide surface modified aptamer.
15. The detection method according to claim 14, characterized in that: the mass-to-volume ratio of the magnetic graphene oxide nano material to the phosphate buffer solution in the step a) is (0.1-3) mg: 1mL, and the concentration of the phosphate buffer solution is 5-15 mmol/L.
16. The detection method according to claim 14, characterized in that: in the step b), the concentration of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride is 1-5 mg/mL, and the concentration of N-hydroxy thiosuccinimide is 1-5 mg/mL; mixed solution 1: 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride salt: n-hydroxy thiosuccinimide: the volume ratio of the phosphate buffer solution is 3-8: 0.1-0.5: 0.1-0.5: 3-8; the concentration of the phosphate buffer solution is 5-15 mmol/L.
17. The detection method according to claim 14, characterized in that: mixed solution 2 in step c): aptamer DNA2 solution: the volume ratio of the phosphate buffer solution is (1-30) mL: (40-60) μ L: (3-8) mL, wherein the concentration of the aptamer DNA2 solution is 50-150 mu mol/L;
the sequence of DNA2 is 5' NH2-ATC CGT CAC ACC TGC TCT ACT GGC CGG CTC AGC ATG ACT AAG AAG GAA GTT ATG TGG TGT TGG CTC CCG TAT TTT TTT TTT-3’。
18. The detection method according to claim 1, characterized in that: the preparation method of the gold star @ gold-silver alloy nanoparticle surface modification aptamer comprises the steps of adding the aptamer DNA1 solution into the 4-ATP-labeled gold star @ gold-silver alloy nanoparticle solution prepared in the step 2), carrying out oscillation reaction at room temperature, centrifuging, removing the supernatant, adding 5mL of phosphate buffer solution, and carrying out heavy suspension to obtain the gold star @ gold-silver alloy nanoparticle surface modification aptamer.
19. The detection method according to claim 18, characterized in that: the concentration of the 4-ATP-labeled aventurine @ gold-silver alloy nanoparticle solution is 5-15 nmol/L, and the concentration of the aptamer DNA1 solution is 50-150 mu mol/L; 4-ATP labeled aventurine @ gold-silver alloy nanoparticle solution: aptamer DNA1 solution: the volume ratio of the phosphate buffer solution is (1-10) mL: (1-20) μ L: (1-10) mL;
the sequence of DNA1 is 5 'SH-ATC CGT CAC ACC TGC TCT ACT GGC CGG CTC AGC ATG ACT AAG AAG GAA GTT ATG TGG TGT TGG CTC CCG TAT TTT TTT TTT-3'.
20. The detection method according to claim 1, characterized in that: the standard curve of the Escherichia coli detection system is established according to 1082cm-1And (3) processing the intensity value of the peak, wherein the scanning conditions of the Raman spectrum are as follows: excitation at 532nm and scan time 30 s.
21. The detection method according to claim 1, characterized in that: and in the step 4) and the step 5), the oxidized graphene surface modified aptamer and the Venus @ gold-silver alloy nanoparticle surface modified aptamer are mixed in equal volumes, and the concentration of the magnetic oxidized graphene nano material after mixing is 0.5-1.5 mg/mL.
22. The detection method according to claim 1, characterized in that: the linear range in the step 4) is 5-105cfu/mL, the minimum detection limit in step 5) was 3 cfu/mL.
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