CN109142703B - Detection method of aflatoxin M1 based on water-soluble perovskite nanocrystal - Google Patents
Detection method of aflatoxin M1 based on water-soluble perovskite nanocrystal Download PDFInfo
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Abstract
The invention discloses a method for detecting aflatoxin M1 based on water-soluble perovskite nanocrystals. The method comprises the steps of CsX and PbX2The raw materials of (1) are added with a small amount of surfactant oleylamine, water-soluble perovskite nanocrystalline which can be dispersed in water is obtained through ball milling, then the surface of the water-soluble perovskite nanocrystalline is negatively charged through further modifying an anionic surfactant on the surface of the water-soluble perovskite nanocrystalline, the water-soluble perovskite nanocrystalline is combined with an antibody of the aflatoxin M1, and quantitative detection is carried out on the aflatoxin M1 by adopting a fluorescence immunoassay method. The perovskite nanocrystalline prepared by the invention has the advantages of simple process, low cost and high fluorescence efficiency, can be used for quantitatively detecting aflatoxin M1, has the detection sensitivity of 0.01ng/mL, expands the application range of perovskite materials, and has good application prospect.
Description
Technical Field
The invention relates to a detection method of aflatoxin M1 based on water-soluble perovskite nanocrystals, belonging to the field of immunoassay.
Background
The halide perovskite material has excellent photovoltaic performance and higher energy conversion rate, and has great potential in the field of backlight fluorescent powder. With organic-inorganic hybrid perovskites (CH)3NH3PbX3) In contrast, inorganic perovskites (CsPbX)3) The compound has excellent stability and has great potential application value in optoelectronics.
The perovskite has excellent luminescence property and has application potential in biological imaging, fluorescence immunoassay probes and the like, however, the water stability of the perovskite is still a technical bottleneck, and the water solubility is difficult to realize, so that the application of the perovskite in the aspect of biology is greatly limited. In order to realize water solubility, research is carried out on the water solubility by preparing oil-soluble perovskite nanocrystals and then wrapping the perovskite nanocrystals with solid lipid nanoparticles, but the preparation method is complex, and the usage amount of organic solvents is large, which is not beneficial to mass production and environmental protection requirements (1. Gomez L, de Weerd C, Hueso J L, et al. color-stable water-dispersed complex mineral nanoparticles J. Nanoscale,2017,9(2): 631).
In immunoassay, the fluorescence immunoassay is rapid and easy to operate, and is an excellent immunoassay method. Aflatoxin M1 is a toxic substance that is easily exposed to the human body, and is often present in dairy products such as milk, and its exposure to the target includes infants, so its detection is essential. At present, the detection method of aflatoxin M1 at home and abroad includes high performance liquid Chromatography, fluorescence probe method and the like ([2] Wang Y, et al. HPLC determination of aflatoxin M1in liquid mill and mill powder using solid phase extraction on 0ASIS HLB. food Control, 2012; 28 (1): 13 l-134. [3] Cavalire C, Foglia P, et al. Anotoxin M1determination in chemical by liquid Chromatography. J Chromatography A, 2006; 1135 (2): 135. 141.[4] Tabib M, et al. determination of total aflatoxin-using cadmium polysaccharides. CdS. J. fluorescence. reaction, 2016. 9. fluorescence detection method and the like (2. Wang Y, et al. HPLC determination of aflatoxin M1. 3. calcium C, Fogliquid P, et al. detection of total aflatoxin-protein amplification in chemical by Chromatography. J. Chromatography A. 294, 2016. fluorescence detection method of polypeptide and protein by PCR. J. Chromatography. 10. fluorescence. mu. fluid Chromatography. J. 2016. 9. fluorescence. detection method). The fluorescence probe method is rapid and convenient, does not need expensive instruments, and is a very active research field. At present, the application of perovskite in the detection of aflatoxin M1 is not reported yet.
Disclosure of Invention
The invention aims to provide a detection method of aflatoxin M1 based on water-soluble perovskite nanocrystals.
The technical scheme for realizing the purpose is as follows:
the aflatoxin M1 detection method based on the water-soluble perovskite nanocrystal comprises the following specific steps:
step 3, adding the aflatoxin M1 antibody solution into the water-soluble perovskite solution, uniformly mixing, standing at room temperature for adsorption to obtain a mixed solution of the antibody and the water-soluble perovskite;
step 4, mixing the aflatoxin M1 solution to be detected with a coating solution, incubating at 37 ℃ for coating, removing the liquid after coating, cleaning, spin-drying, adding 4% milk diluent, incubating at 37 ℃, cleaning, and spin-drying;
step 5, dripping the mixed solution of the antibody and the water-soluble perovskite into the aflatoxin M1 to be detected coated in the step 4, detecting the absorbance, and obtaining the initial absorbance F0Then incubating at 37 ℃, after the incubation is finished, cleaning, spin-drying, detecting the absorbance to obtain the final absorbance F, and according to the concentration of the aflatoxin M1 and the F/F0Calculating to obtain the concentration of the aflatoxin M1 to be detected.
In the step 2, the anionic surfactant is Sodium Dodecyl Sulfate (SDS), and the mass ratio of the perovskite nanocrystal to the water-soluble anionic surfactant is 5: 1-10: 1.
And 3, standing for 15-30 min for adsorption.
And 4, coating and incubating for 1-2 h.
And 5, incubating for 20-30 min.
Compared with the prior art, the invention has the following remarkable advantages:
1) the water-soluble perovskite nanocrystalline is prepared by adopting a one-step ball milling method, the method is simple, can realize batch amplification production, has low cost, and the prepared perovskite is stable in water;
2) the water-soluble perovskite nanocrystal is used for detecting the aflatoxin M1, the detection sensitivity can reach 0.01ng/mL, the requirement of practical application is met, and the fluorescence detection range of the aflatoxin M1 is widened.
According to the invention, the perovskite is applied to the detection of aflatoxin M1 for the first time, so that the application range of the perovskite is widened.
Drawings
Fig. 1 is an XRD pattern of perovskite nanocrystals prepared in examples 1 and 3.
Fig. 2 is a TEM image of the perovskite nanocrystal prepared in example 1.
Fig. 3 is a PL plot of the perovskite nanocrystals prepared in example 1.
FIG. 4 is a graph of the UV absorption spectrum of the perovskite nanocrystals prepared in example 1 as a function of water concentration.
FIG. 5 is a TEM image of perovskite nanocrystals prepared in example 1 coupled with an antibody.
FIG. 6 is a graph showing the change of fluorescence detection at different pH values when perovskite nanocrystals prepared in example 1 are coupled with an antibody.
FIG. 7 is a standard curve for detecting aflatoxin M1in the perovskite nanocrystals of example 2.
FIG. 8 is a photograph of an aqueous perovskite nanocrystal solution prepared in example 4.
Fig. 9 is a PL plot of the perovskite nanocrystals prepared in example 5.
Fig. 10 is a graph of the perovskite nanocrystals prepared in example 1 and comparative example 1 after being bound to an antibody and then left to stand.
FIG. 11 is a graph showing the dispersion of the perovskite nanocrystals obtained in comparative example 2 in water.
Detailed Description
The present invention will be described in more detail with reference to the following examples and the accompanying drawings.
The invention is in CsX, PbX2The raw materials of the fluorescent nano-crystal are added with a small amount of surfactant, the luminescent nano-crystal which can be dispersed in water is obtained by a ball milling method, the surface is further modified with anionic surfactant to make the surface have negative charges, the surface is combined with an antibody of aflatoxin M1, and quantitative detection can be carried out on aflatoxin M1 by a fluorescence immunoassay method.
Example 1
The aflatoxin M1 detection method based on the water-soluble perovskite nanocrystal specifically comprises the following steps:
1) mu.L oleylamine was added to 2mmol of CsBr and PbBr2Powder ofFilling argon for protection, sealing a ball milling tank, and ball milling at the rotating speed of 400rpm for 2 hours, wherein the mass ratio of ball materials is 30: 1;
2) adding 10mg of SDS powder into 80mg of ball milled powder, adding 2mL of Tris buffer solution, stirring for dissolving, and adjusting the pH value to 7.4 to obtain a water-soluble perovskite solution;
3) taking 8 parts of 75 mu L of the solution obtained in the step 2), respectively adding 425 mu L of pure water for dilution, and using 0.1M K2CO3Adjusting the pH value of the solution by using the solution, respectively dropwise adding 1-8 mu L of the solution, and uniformly mixing the solution by using a uniformly mixing device;
4) respectively adding 10 mu L of aflatoxin M1 antibody solution into the mixed solution in the step 3), uniformly mixing, and standing for half an hour at room temperature for adsorption;
5) the concentration of the prepared aflatoxin M1 antigen solution is 0.25ug/mL, and the coating solution (Na)2CO3、NaHCO3) Dripping 100uL into each hole, dripping 100uL of antigen solution, shaking a plate in an enzyme-labeling instrument for multiple times, uniformly mixing, incubating at 37 ℃ for 2h for coating, pouring off the solution in the holes, automatically washing the plate, and drying; adding milk diluent (4% concentration) into the wells, culturing at 37 ℃ for 1h, and then automatically washing the plate for three times for later use;
6) respectively adding 100 mu L of the solutions in the step 4) with different pH gradients into a pore plate, shaking uniformly, and measuring an initial value by using an enzyme-labeling instrument;
7) and (3) culturing the solution in the step 6) at 37 ℃ for half an hour, automatically washing the plate for three times, drying the plate, and measuring the final fluorescence value by using a microplate reader.
CsPbBr prepared in this example3The crystallinity of the perovskite nanocrystal is characterized by XRD (1) shown in figure 1, and the peak position and the strength of the crystal phase are matched with those of a standard card of the perovskite, which indicates good crystallinity of the perovskite nanocrystal. The TEM image is shown in FIG. 2, and the grain size distribution is 50-100 nm. The PL luminescence spectrum of the aqueous solution is shown in FIG. 3, the central wavelength of the luminescence peak is 522nm, and the half-peak width is only 18 nm. The ultraviolet absorption spectrum of the compound changes with the water soluble concentration as shown in figure 4, and the solubility of the compound in water can reach 3.4 mg/mL. TEM image of antibody conjugation is shown in FIG. 5, with CsPbBr in order from a to c3Perovskite nanocrystal, antibody and coupled fluorescent probe. The graph of the change of fluorescence detection at different pH is shown in FIG. 6And when the pH value is higher, the detection effect is good.
Example 2
1) mu.L oleylamine was added to 2mmol of CsBr and PbBr2Filling argon into the powder for protection, sealing a ball milling tank, and carrying out ball milling for 2 hours at the rotating speed of 400rpm, wherein the mass ratio of ball materials is 30: 1;
2) adding 10mg of SDS powder into 80mg of ball milled powder, adding 2mL of Tris buffer solution, stirring for dissolving, and adjusting the pH value to 7.4 to obtain a water-soluble perovskite solution;
3) taking 75 μ L of the solution obtained in the step 2), adding 425 μ L of pure water for dilution, and using 0.1M K2CO3The solution adjusts the pH value of the solution, 8 mu L of the solution is dripped and mixed by a mixer;
4) respectively adding 10 mu L of aflatoxin M1 antibody solution into the mixed solution in the step 3), uniformly mixing, and standing for half an hour at room temperature for adsorption;
5) preparing aflatoxin M1 antigen solutions with different concentrations, and coating solution (Na)2CO3、NaHCO3) Dripping 100uL into each hole, dripping 100uL of antigen solution, shaking a plate in an enzyme-labeling instrument for multiple times, uniformly mixing, incubating at 37 ℃ for 2h for coating, pouring off the solution in the holes, automatically washing the plate, and drying; adding milk diluent (4% concentration) into the wells, culturing at 37 ℃ for 1h, and then automatically washing the plate for three times for later use;
6) adding 100 mu L of the solution obtained in the step 4) into a pore plate, shaking uniformly, and measuring an initial value by using an enzyme-labeling instrument;
7) and (3) culturing the solution in the step 6) at 37 ℃ for half an hour, automatically washing the plate for three times, drying the plate, and measuring the final fluorescence value by using a microplate reader.
The standard curve for detecting aflatoxin M1 for the perovskite nanocrystals is shown in fig. 7. The aflatoxin M1 shows linear change in the concentration range of 0.02to 10.1ng/mL, the detection sensitivity can reach 0.01ng/mL, and the requirements of practical application can be met.
Example 3
1) 50 μ L oleylamine was added to 2mmol CsBr and PbBr2Filling argon into the powder for protection, sealing a ball milling tank, and carrying out ball milling for 2 hours at the rotating speed of 400rpm, wherein the mass ratio of ball materials is 30: 1;
2) adding 10mg of SDS powder into 50mg of ball milled powder, adding 2mL of Tris buffer solution, stirring for dissolving, and adjusting the pH value to 7.4 to obtain a water-soluble perovskite solution;
3) taking 8 parts of the solution in the step 2), diluting with 425 μ L of pure water, and using 0.1M K2CO3Adjusting the pH value of the solution by using the solution, and uniformly mixing by using a uniformly mixing device;
4) respectively adding 10 mu L of aflatoxin M1 antibody solution into the mixed solution in the step 3), uniformly mixing, and standing for half an hour at room temperature for adsorption;
5) the concentration of the prepared aflatoxin M1 antigen solution is 0.25ug/mL, and the coating solution (Na)2CO3、NaHCO3) Dripping 100uL into each hole, dripping 100uL of antigen solution, shaking a plate in an enzyme-labeling instrument for multiple times, uniformly mixing, incubating at 37 ℃ for 2h for coating, pouring off the solution in the holes, automatically washing the plate, and drying; adding milk diluent (4% concentration) into the wells, culturing at 37 ℃ for 1h, and then automatically washing the plate for three times for later use;
6) adding the solution in the step 4) into a pore plate in an amount of 100 mu L respectively, shaking uniformly, and measuring an initial value by using an enzyme-labeling instrument;
7) and (3) culturing the solution in the step 6) at 37 ℃ for half an hour, automatically washing the plate for three times, drying the plate, and measuring the final fluorescence value by using a microplate reader.
CsPbBr prepared in this example3The perovskite nanocrystal has XRD shown in 2 of figure 1, and the crystallinity is still good.
Example 4
This example is essentially the same as example 3, except that the volume of the oleic amine in step 1) of example 1 was changed to 200. mu.L, and the other conditions were kept the same. The photograph of the perovskite nanocrystal aqueous solution prepared in this example is shown in fig. 8, and the water solubility is good.
Example 5
This example is essentially the same as example 3, except that CsBr and PbBr were added in step 1) of example 12The powder is changed into CsI and PbI2Other conditions were kept consistent. The luminescent spectrum of the perovskite nanocrystal prepared by the embodiment is shown in figure 9, and the luminescent performance is still good。
Comparative example 1
This comparative example is essentially the same as example 3, except that Tris in step 2) of example 1 was changed to PBS, and the other conditions were kept the same. CsPbBr prepared in this comparative example3The perovskite nanocrystalline is used for fluorescence immunoassay of aflatoxin M1. The detection effect is slightly poor, and the antibody is combined with the nanocrystal and then placed with a precipitate, as shown in figure 10.
Comparative example 2
This comparative example is substantially the same as example 3, except that the solubility of the perovskite nanocrystals obtained in this comparative example in water was deteriorated by changing the amount of oleylamine in step 1) of example 1 to 250. mu.L, and the other conditions were kept the same, as shown in FIG. 11, and most of the perovskite nanocrystals precipitated and could not be dispersed in water.
Claims (5)
1. The aflatoxin M1 detection method based on the water-soluble perovskite nanocrystal is characterized by comprising the following specific steps:
step 1, add oleylamine to CsX and PbX2Ball-milling the powder for 1-7 h at the rotating speed of 300-500 rpm, wherein the mass ratio of ball materials is 15: 1-30: 1, X is Cl, Br, I, CsPbX3The volume ratio of the molar weight of the (D) to the oleylamine is 10-40: 1, and mol: L;
step 2, adding a water-soluble anionic surfactant into the ball-milled powder, adding a Tris buffer solution, stirring and dissolving, and adjusting the pH value to 7-8 to obtain a water-soluble perovskite solution;
step 3, adding the aflatoxin M1 antibody solution into the water-soluble perovskite solution, uniformly mixing, standing at room temperature for adsorption to obtain a mixed solution of the antibody and the water-soluble perovskite;
step 4, mixing the aflatoxin M1 solution to be detected with a coating solution, incubating at 37 ℃ for coating, removing the liquid after coating, cleaning, spin-drying, adding 4% milk diluent, incubating at 37 ℃, cleaning, and spin-drying;
step 5, dripping the mixed solution of the antibody and the water-soluble perovskite into the aflatoxin M1 to be detected coated in the step 4, detecting the absorbance, and obtaining the initial absorbanceF0Then incubating at 37 ℃, after the incubation is finished, cleaning, spin-drying, detecting the absorbance to obtain the final absorbance F, and according to the concentration of the aflatoxin M1 and the F/F0Calculating to obtain the concentration of the aflatoxin M1 to be detected.
2. The detection method according to claim 1, wherein in the step 2, the anionic surfactant is sodium dodecyl sulfate, and the mass ratio of the perovskite nanocrystal to the water-soluble anionic surfactant is 5: 1-10: 1.
3. The detection method according to claim 1, wherein in the step 3, the standing adsorption time is 15-30 min.
4. The detection method according to claim 1, wherein in step 4, the coating incubation time is 1-2 h.
5. The detection method according to claim 1, wherein in the step 5, the incubation time is 20-30 min.
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| CN102495210A (en) * | 2011-12-06 | 2012-06-13 | 扬州大学 | Method for non-competitive homogeneous immunoassay of quantum dot labeled antibody of organic substance with low molecular weight |
| WO2017020137A1 (en) * | 2015-07-31 | 2017-02-09 | Nanograde Ag | Luminescent crystals and manufacturing thereof |
| CN105759045A (en) * | 2016-03-18 | 2016-07-13 | 南昌大学 | Method for detecting aflatoxin B1 |
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