CN113758886A - Multi-target object simultaneous detection method based on concentration change of latex microspheres - Google Patents

Multi-target object simultaneous detection method based on concentration change of latex microspheres Download PDF

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CN113758886A
CN113758886A CN202110898312.9A CN202110898312A CN113758886A CN 113758886 A CN113758886 A CN 113758886A CN 202110898312 A CN202110898312 A CN 202110898312A CN 113758886 A CN113758886 A CN 113758886A
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latex microspheres
microspheres
simultaneously detecting
multiple targets
latex
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CN113758886B (en
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陈翊平
周翠云
王知龙
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Huazhong Agricultural University
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Abstract

The invention belongs to the technical field of biochemical analysis. In particular to a method for simultaneously detecting multiple targets based on the concentration change of latex microspheres. Using nano magnetic particles as a magnetic separation carrier, and coupling capture antibodies of different target objects on the surface of the nano magnetic particles; taking latex microspheres with different particle sizes as signal probes, coupling detection antibodies or complete antigens of different target objects on the surfaces of the latex microspheres, wherein each particle size corresponds to one target object; carrying out immunoreaction on the magnetic separation carrier, the signal probe and a target object to be detected; collecting the unreacted signal probe solution in the separation liquid after magnetic separation; and simultaneously measuring the maximum absorbance corresponding to the ultraviolet absorption peaks of the signal probes with different particle diameters, and calculating the content of the corresponding target object according to the change of the absorbance. The invention can simultaneously detect multiple targets, greatly shortens the detection time, and has the advantages of simple and convenient operation of the ultraviolet spectrophotometer, high sensitivity, good stability and low cost.

Description

Multi-target object simultaneous detection method based on concentration change of latex microspheres
Technical Field
The invention belongs to the field of biochemical analysis, and relates to a method for simultaneously detecting multiple targets based on concentration change of latex microspheres.
Background
The rapid detection method has the advantages of simplicity, convenience, rapidness and accuracy, and plays an increasingly important role in protecting human health and protecting ecological environment. The rapid detection methods are mainly classified into optical methods, electrochemical methods, and magnetic methods according to the difference in signal readout modes. The optical-based detection method is compatible with various biochemical reactions, is a non-contact detection mode, and is widely applied to the fields of clinical diagnosis, environmental monitoring, food safety and the like.
Among the rapid detection methods based on optics, enzyme-linked immunoassay, colloidal gold immunochromatographic test strip and fluorescence spectroscopy are representative and widely used. The enzyme-linked immunoassay method and the colloidal gold immunochromatographic test strip method have low sensitivity and are not suitable for analyzing trace target substances. The fluorescent spectrum analysis can meet the requirement of trace analysis, but few substances emit fluorescence or form a fluorescence measurement system, and although part of objects to be detected can form fluorescence through chemical derivatization, the difficulty is high, and the application of the substances is greatly limited. The immunoassay method based on the fluorescent microspheres is also applied to a certain extent at present, but the price of the fluorescent microspheres is higher, and the fluorescent microspheres need to be protected from light during use, so that the further application of the fluorescent microspheres is limited. Although the method plays an important role in the rapid detection of the target, the method has limited application in the simultaneous detection of the target in a complex sample matrix and low detection efficiency.
Disclosure of Invention
In order to solve the technical problems, the invention provides a method for simultaneously detecting multiple targets based on concentration change of latex microspheres, which is based on the method for simultaneously detecting the multiple targets based on the concentration change of the latex microspheres, and realizes the simultaneous detection of the multiple targets in a complex matrix through characteristic ultraviolet spectrum of the latex microspheres. The ultraviolet spectrophotometry realizes qualitative or quantitative analysis of a target object by measuring the absorbance of a substance in a wavelength range of 190-1000 nm. In tests, the latex microspheres (100nm-5000nm) with a certain particle size have a strong ultraviolet absorption peak within the wavelength range of 190-1000 nm, and the absorption peak can generate blue shift along with the increase of the particle size, namely the latex microspheres with different particle sizes have the strongest ultraviolet absorption peaks at different wavelengths. Further experiments show that the concentration of the latex microspheres and the absorption peak intensity thereof show obvious positive correlation in a proper range, and the change of the fine concentration can cause the change of the absorption peak intensity, so that the excellent correlation is particularly suitable for constructing an immunoassay method. Meanwhile, the latex microspheres with different particle sizes have different ultraviolet absorption peaks and can be used as a plurality of signal probes, so that a plurality of target objects can be detected simultaneously.
The technical scheme adopted by the invention is as follows:
the nano magnetic particles are used as magnetic separation carriers, latex microspheres with different particle sizes are used as signal probes, capture antibodies corresponding to targets are respectively modified on the surfaces of the nano magnetic particles, and corresponding detection antibodies or complete antigens are modified on the surfaces of the latex microspheres. The existence of the target object induces or inhibits the combination of the magnetic particles and the latex microspheres, so that the concentration of the unbound free-dispersed latex microspheres in the solution is correspondingly changed, the unbound latex microspheres can be separated from the whole system through an external magnetic field, the content of the unbound latex microspheres is related to the content of the target object, meanwhile, the latex microspheres have strong ultraviolet absorption peaks, and the content of the target object can be obtained by measuring the signal intensity of the latex microspheres through an ultraviolet spectrophotometer. The method can realize high-sensitivity and rapid detection of the concentration of the target object by changing the concentration of the latex microspheres. Meanwhile, corresponding biological recognition molecules are coupled on the surfaces with different particle diameters according to the types of the objects to be detected, so that simultaneous detection of multiple objects can be realized.
A method for simultaneously detecting multiple targets based on concentration change of latex microspheres comprises the following steps:
1) the nano magnetic particles are used as magnetic separation carriers, and capture antibodies or antibodies of different target objects are coupled on the surfaces of the nano magnetic particles; taking latex microspheres with different particle sizes as signal probes, and coupling detection antibodies or complete antigens of different target objects on the surfaces of the latex microspheres;
2) carrying out sufficient immunoreaction on the magnetic separation carrier, the signal probe and a target object to be detected to obtain a mixed solution;
3) carrying out magnetic separation on the mixed solution in the step 2), quickly adsorbing a magnetic separation carrier, and taking a separation solution to obtain a signal probe solution which does not participate in the reaction;
4) and simultaneously measuring the maximum absorbance corresponding to the ultraviolet absorption peaks of the signal probes with different particle diameters in the unreacted signal probe solution obtained in the step 3), respectively establishing a quantitative relation between the concentration of the object to be measured and the maximum absorbance value, and calculating the corresponding content of the target object according to the change of the concentration.
Preferably, in the step 1), the latex microspheres are Polystyrene (PS) latex microspheres or any one of polybutadiene latex microspheres, polyisoprene latex microspheres and polyacrylic latex microspheres.
Preferably, in the step 1), the particle size of the nano-magnetic particles is 50nm-5000 nm.
Preferably, in the step 1), the particle size of the latex microspheres is 100nm-5000 nm.
Preferably, in the step 1), when the target is vomitoxin, aflatoxin and ochratoxin or staphylococcus aureus, listeria monocytogenes and salmonella are detected simultaneously, three kinds of latex microspheres are required to be used as signal probes, and the selected latex microspheres are polystyrene microspheres with particle sizes of 200nm, 1000nm and 1500 nm.
Further preferably, the wavelength corresponding to the maximum absorbance of the 200nm polystyrene latex microspheres is 228nm, the wavelength corresponding to the maximum absorbance of the 1000nm polystyrene latex microspheres is 365nm, and the wavelength corresponding to the maximum absorbance of the 1500nm polystyrene latex microspheres is 610 nm.
Preferably, in the step 2), when the sandwich immune reaction is performed, the mixed solution is a carrier-target-signaling probe complex and a signaling probe solution which does not participate in the reaction; when the competitive immune reaction is performed, the mixed solution is a carrier-signaling probe complex, a carrier-target complex, and a signaling probe solution that does not participate in the reaction.
Preferably, one kind of particle size latex microsphere is used for correspondingly detecting one kind of target object, and a plurality of kinds of target objects can be simultaneously detected by selecting latex microspheres with various particle sizes.
The method for simultaneously detecting the multiple targets is applied to simultaneously and quantitatively detecting various targets, including mycotoxin, pathogenic microorganisms, antibiotics, veterinary drugs, disease markers or the like.
The existing method for simultaneously detecting multiple targets mainly comprises counting and reading of resistive particles based on small holes and simultaneous reading of visible multi-microsphere signals based on a microscope, and the two methods count the micron-level latex microspheres and cannot read the nano-level microspheres. The invention is suitable for nanometer and micron latex microspheres, the particle counter needs to be washed again and the background removed again every time the sample is introduced, the data needs to be identified and processed by combining machine vision when the microscope is shot, the time is long, and the efficiency is low. More importantly, compared with the two methods, the method has the advantage of improving the sensitivity. As shown in fig. 1, neither particle counter nor microscopic visual readings are applicable for 200nm latex microspheres; for 1000nm latex microspheres, the particle counter can only detect the dilution of 10 at most4The microspheres with multiple times can be detected to be diluted by 5 multiplied by 10 by visual reading of a microscope4The microspheres with the multiple can be detected by an ultraviolet spectrophotometer to be diluted by 10 at most5Multiple microspheres; for 1500nm latex microspheres, the particle counter and microscopic visual reading can be analyzed to a dilution of 2X 104The multiple of microspheres can be diluted by 5 multiplied by 10 to the maximum by the ultraviolet spectrophotometry4Multiple microspheres, thus improving sensitivity to some extent. When the three methods are used for simultaneously detecting the salmonella, the effect is shown in figure 2, the particle counter and the microscope can detect the salmonella of 1000CFU/mL at the highest visual reading, and the ultraviolet spectrophotometry can reach 100CFU/mL at the highest. Fig. 1-2 show that the method has higher sensitivity than the particle counter signal reading and microscope visual reading method, compared with the counting method, the method not only solves the problem of simultaneous detection of a plurality of target objects, but also has simple method and higher sensitivity.
The invention has the beneficial effects that:
1) the operation is simple, the detection is rapid: the method has simple and convenient signal conversion, adopts one-step immunoreaction, can realize the rapid separation of the latex microspheres in a short time through magnetic separation, and greatly shortens the detection time by the ultraviolet spectrophotometer compared with a particle counter and a microscope visual reading method.
2) Simultaneously detecting multiple targets: the latex microspheres with different particle sizes have different maximum ultraviolet absorption peaks, and the simultaneous detection of multiple targets can be realized only by selecting the latex microspheres with different particle sizes (nano-scale or micron-scale) as signal probes.
3) The sensitivity is high: the absorbance is sensitive to the change of the concentration of the latex microspheres and can reach the responsivity of 10 microspheres/mL, so that high-sensitivity detection can be realized.
4) The stability is good: the latex microspheres have good stability, can be stored at room temperature for 6 months, do not need to be protected from light, and ensure the stability of the probe.
5) The cost is low: the method has simple and convenient signal conversion, does not need to label biological enzyme, luciferase and the like, and has the advantages of stable property, uniform particle size, mature preparation process and low cost.
6) The application is wide: aiming at the detection requirements of different fields, the rapid pretreatment advantage of immunomagnetic separation and the high-efficiency signal simultaneous reading of the latex microspheres are combined, and the method can be applied to different fields.
Drawings
FIG. 1 is a graph showing the sensitivity comparison of the particle count of the present invention with both particle counter and microscopic visual readings, wherein FIG. 1A is a graph showing the sensitivity comparison of three methods for detection under 1000nm latex microspheres, and FIG. 1B is a graph showing the sensitivity comparison of three methods for detection under 1500nm latex microspheres;
FIG. 2 is a comparison graph of the results of the present invention comparing with the results of the small-hole resistive particle counter and the visual readings of the microscope for detecting Salmonella, wherein FIG. 2A is a graph of the detection results of the small-hole resistive particle counter, FIG. 2B is a graph of the detection results of the microscope, and FIG. 2C is a graph of the detection results of the ultraviolet analysis of the present invention.
FIG. 3 is a linear relationship between the maximum ultraviolet absorbance and the concentration of three PS (200nm, 1000nm, 1500nm) latex microspheres with different particle sizes
FIG. 4 is a graph showing the comparison of the maximum absorbance values corresponding to three different particle sizes of PS (200nm, 1000nm, 1500nm) when latex microspheres are mixed according to different ratios, wherein FIG. 4A shows that the concentration ratio of 200nm to 1000nm to 1500nm is 1:1:1, FIG. 4B shows that 2:2:1, FIG. 4C shows that 3:1:1, and FIG. 4D shows that 3:2: 1;
FIG. 5 shows the UV absorption peaks of three PS (200nm, 1000nm, 1500nm) latex microspheres with different particle sizes when they are mixed in an optimal ratio;
FIG. 6 is a schematic diagram of the simultaneous detection of three mycotoxins in the present invention;
FIG. 7 is a relationship between the concentration of mycotoxin and the corresponding maximum absorbance when the ratio of the concentration of the magnetic separation carrier to the concentration of the signal probe is 1:1, 1:2, 1:3 and 2:1, wherein FIG. 7A is vomitoxin, FIG. 7B is aflatoxin, and FIG. 7C is ochratoxin, when mycotoxin is detected according to the invention;
FIG. 8 is a standard curve for the simultaneous detection of three mycotoxins according to the present invention;
FIG. 9 is a schematic diagram of the present invention for simultaneously detecting three pathogenic microorganisms;
FIG. 10 is a standard curve for simultaneous detection of three pathogenic microorganisms according to the present invention.
Detailed Description
The technical solution of the present invention is further explained by the following embodiments. It should be understood by those skilled in the art that the examples are only for the understanding of the present invention and should not be construed as the specific limitations of the present invention.
Description of the test materials
UV-visible spectrophotometer, available from ThermoFisher GENESYS 150.
Micro quartz cuvettes, available from Shanghai broad-spectrum optical instruments, Inc.
Carboxylated magnetic particles (MNP), 1000nm in size, 10mg/mL, were purchased from Invitrogen.
Carboxylated Polystyrene (PS) latex microspheres, particle size 200nm (10mg/mL), 1000nm (100mg/mL), 1500nm (10mg/mL), purchased from Bangs Laboratories, Inc.
1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide (EDC), N-hydroxythiosuccinimide active ester (s μ lfo-NHS): purchased from Shanghai Aladdin Biotechnology Ltd. Tween-20, Bovine Serum Albumin (BSA): purchased from Amresco Inc.
PBS buffer (10mM, pH 7.4): collecting 8.00g NaCl,0.20g KCl,0.20g KH2PO4And 2.90gNa2HPO4·12H2And (4) performing constant volume on O in a 1000mL volumetric flask, and shaking up.
MES buffer (0.1M, pH 6.0): 21.325g of MES is dissolved in deionized water to be constant volume of 1000mL to obtain solution A; dissolving 4g of NaOH in deionized water, and fixing the volume to 1000mL to obtain solution B; 1000mL of solution A and 400mL of solution B were mixed and shaken well.
PBST, MEST: 0.05% Tween-20 was added to the prepared PBS or MES buffer.
Vomitoxin, aflatoxin, ochratoxin and their corresponding complete antigens and antibodies: purchased from Shandong blue.
Staphylococcus aureus, listeria monocytogenes, salmonella and their corresponding capture and detection antibodies: purchased from Shandong blue.
To verify the effect of uv spectroscopy, the linear ranges of PS latex microspheres with particle sizes of 200nm, 1000nm and 1500nm were determined (maximum absorbance was controlled as much as possible in the range of 0.1 to 0.8, the reading was most accurate), as shown in fig. 3, the absorbance of the PS latex microspheres with particle size of 200nm was in the range of 0.14 to 0.82 in the concentration range of 5 to 30 μ g/mL, the standard curve was Y ═ 0.027X-0.001, and R was R ═ 0.027X-0.00120.9997; the light absorption value of the 1000nm PS latex microsphere is in the range of 0.13-0.79 within the concentration range of 2.5-36 mu g/mL, the standard curve is that Y is 0.044X-0.025, R is20.9956; the absorbance of 1500nm PS latex microspheres is in the range of 0.16-0.79 in the concentration range of 1-8 mu g/mL, the standard curve is that Y is 0.105X-0.003, R is2＝0.9990。
In the optimal linear range of the PS latex microspheres with different particle sizes, the ultraviolet absorption peak effects are tested by mixing according to different concentration ratios, wherein the ratio of 200nm to 1000nm to 1500nm is 1:1:1, 2:2:1, 3:1:1 and 3:2:1, as shown in figure 4, in order to obtain the optimal reading result, the maximum absorbance values of the three particle sizes are preferably close to each other, the optimal effect is finally determined when the concentration ratio of the PS latex microspheres is 200nm to 1000nm to 1500nm is 3:2:1, the ultraviolet absorption peak effect is shown in figure 5, the wavelength range of the absorption peak of the 200nm PS latex microspheres is 190-plus 270nm, the wavelength range of the absorption peak of the 1000nm PS latex microspheres is 270-plus 510nm, the wavelength range of the absorption peak of the 1500nm PS latex microspheres is 510-plus 1000nm, the three peaks are uniformly distributed, and the reading is convenient. The present invention continues to use this ratio in analyzing multiple targets.
Example 1 multiplex quantitative detection of vomitoxin, aflatoxin and ochratoxin
Simultaneously detecting vomitoxin, aflatoxin and ochratoxin by adopting a competitive immunization method, wherein a schematic diagram is shown in fig. 6, magnetic particles with the particle size of 1000nm are taken as magnetic separation carriers, and antibodies corresponding to the vomitoxin, the aflatoxin and the ochratoxin are respectively coupled to the surfaces of the magnetic separation carriers; PS latex microspheres of 200nm, 1000nm and 1500nm are taken as signal probes, complete antigens corresponding to vomitoxin, aflatoxin and ochratoxin are respectively coupled, and when a target exists, the signal probes and the target compete for antibodies on the surface of a carrier together to form a signal probe-carrier compound or a target-carrier compound. Therefore, the content of the target directly influences the number of formed signal probe-carrier complexes, after the carriers are removed by magnetic separation, only unbound signal probes and target substances are remained in the separation solution, the target substances are small molecules and trace and do not influence ultraviolet absorption peaks, the remaining amount of the signal probes is positively correlated with the concentration of the target substances, and the three signal probes can be simultaneously detected by an ultraviolet spectrophotometer (three different absorption peaks), so that the multi-element quantitative detection of the target substances can be realized.
Preparation of reagents
1) The preparation of magnetic particle coupled target antibody (magnetic separation carrier) takes vomitoxin as an example, and the specific process is as follows:
100 mu L of magnetic particles with the concentration of 10mg/mL are taken to be placed in a 1.5mL centrifuge tube, 500 mu L of LMEST is added, the magnetic particles are uniformly dispersed and then are subjected to magnetic separation to remove supernatant, the magnetic particles are repeatedly washed for 3 times in such a way to remove floating dust on the surfaces of the magnetic particles and non-specific adsorption, 80 mu L of LEDC and 40 mu L of NHS are added, MES buffer solution is used for fixing volume to 500 mu L, MEST is used for washing for 3 times after being activated for 30min at room temperature, redundant EDC and NHS are removed, 50 mu L of vomitoxin antibody is added, PBST is fixed volume to 500 mu L, coupling is carried out for 3 hours at 37 ℃, redundant antibody is removed by PBST washing for 3 times, 1000 mu L of PBST containing 1% BSA is added for sealing for 30min, redundant sealing liquid is removed by PBST washing for 3 times, the magnetic particles obtained by removing the washing liquid and are resuspended by 1000 mu L of PBST and placed at 4 ℃ for preservation.
When the magnetic particle conjugate of aflatoxin and ochratoxin is prepared, the vomitoxin antibody is only required to be changed into the corresponding antibody for conjugation.
2) Preparation of PS latex microsphere coupling target complete antigen (signal probe):
similar to the magnetic particle coupling process, only the magnetic separation process is changed into centrifugation, wherein the centrifugation condition of the 200nm PS latex microspheres is 15000rpm for 5 min; centrifuging 1000nm PS latex microspheres at 8000rpm for 5 min; the centrifugation condition of 1500nm PS latex microspheres is 6000rpm for 5min, the antibody corresponding to the target is converted into complete antigen in the coupling process, the preservation solution of 200nm PS latex microsphere-vomitoxin complete antigen conjugate, 1000nm PS latex microsphere-aflatoxin complete antigen conjugate and 1500nm PS latex microsphere-ochratoxin complete antigen conjugate is prepared, and the preservation solution is placed in a refrigerator at 4 ℃ for preservation after the coupling is completed.
Immune response and isolation
Mixing the three prepared magnetic separation carriers, the three signal probes and three target objects corresponding to a series of gradients in equal volume (100 mu L each), incubating at 37 ℃ for 30min, performing magnetic separation for 10s, and collecting separation solution.
In order to obtain the best analysis effect, the concentration ratios of the magnetic separation carrier and the signal probe are respectively optimized, fig. 7 shows the analysis effect of each target when the concentration ratios of the magnetic separation carrier and the signal probe are 1:1, 1:2, 1:3 and 2:1, obviously, the concentration ratio between the magnetic separation carrier and the signal probe of each target has a remarkable effect on the result, the result is the best when the concentration ratio of the signal probe is determined to be 200nm:1000nm:1500 nm: 3:2:1, and finally the incubation concentration ratio of the carrier of vomitoxin to the signal probe is determined to be 1:3(50 μ g/mL: 150 μ g/mL), the ratio of aflatoxin is 1:2(50 μ g/mL: 100 μ g/mL), the ratio of ochratoxin is 1:1(50 μ g/mL: 50 μ g/mL), and the linearity is the best.
Ultraviolet spectrophotometer for measuring light absorption value of separation liquid
And (3) uniformly mixing the separation solution (unbound signal probe solution), adding 200 mu L of sample into a micro cuvette, putting the micro cuvette into a cuvette rack in a sample cell, measuring the sample on the cuvette rack, and recording the maximum absorbance corresponding to the three PS latex microspheres respectively. And taking the logarithm of the concentration of the substance to be detected as an abscissa and the maximum absorbance value as an ordinate to obtain a linear relation between the logarithm of the concentration of the substance to be detected and the ordinate. As shown in fig. 8, for emetic toxin, the linear range is 1ng-1000ng/mL, the regression equation is 0.185X-0.354 for Y, R20.9819; for aflatoxins, the linear range is 0.1ng-300ng/mL, the regression equation is that Y is 0.129X-0.144, R20.9910; for ochratoxin, the linear range is 0.05ng-100ng/mL, the regression equation is that Y is 0.135X +0.069, R2＝0.9926。
The result shows that the method can be used for simultaneously detecting the mycotoxin multi-target objects, has high sensitivity and wide linear range, and can be suitable for simultaneously detecting objects to be detected with different detection requirements, the 200nm latex microspheres are used as signal probes for vomitoxin with lower detection sensitivity requirements, the 1000nm latex microspheres are used as signal probes for detecting aflatoxin, and the 1500nm latex microspheres are used as signal probes for ochratoxin with the highest detection sensitivity requirements.
Example 2 multiplex quantitative detection of Staphylococcus aureus, Listeria monocytogenes, Salmonella
Simultaneously detecting staphylococcus aureus, listeria monocytogenes and salmonella by adopting a sandwich immunoassay, wherein a schematic diagram is shown as 9, magnetic particles with the particle size of 1000nm are taken as magnetic separation carriers, and capture antibodies corresponding to the staphylococcus aureus, the listeria monocytogenes and the salmonella are respectively coupled to the surfaces of the magnetic separation carriers; 200nm, 1000nm and 1500nm PS latex microspheres are taken as signal probes and are respectively coupled with detection antibodies corresponding to staphylococcus aureus, listeria monocytogenes and salmonella, when a target exists, the target can be combined with a carrier and the signal probes to form a carrier-target-signal probe compound, so that the number of the target directly influences the number of the formed sandwich immune compound, after the carrier is removed by magnetic separation, only unbound signal probes are remained in a separation solution, the remaining amount is negatively related to the number of the target, and the three signal probes can be simultaneously detected by an ultraviolet spectrophotometer (three different absorption peaks), so that the multi-element quantitative detection of the target can be realized.
Preparation of reagents
1) The preparation of magnetic particle coupled target substance capture antibody (magnetic separation carrier) takes staphylococcus aureus as an example, and the specific process is as follows:
taking 100 mu L of magnetic particles with the concentration of 10mg/mL into a 1.5mL centrifuge tube, adding 500 mu LMEST, removing supernatant through magnetic separation after the magnetic particles are uniformly dispersed, repeatedly washing for 3 times in this way, removing floating dust on the surface of the magnetic particles and non-specific adsorption, adding 80 mu LEDC and 40 mu L NHS, fixing the volume to 500 mu L by using MES buffer solution, washing for 3 times by using MEST after activating for 30min at room temperature, removing redundant EDC and NHS, adding 50 mu L of staphylococcus aureus capture antibody, fixing the volume to 500 mu L of PBST, coupling for 3h at 37 ℃, washing for 3 times by using PBST to remove redundant antibody, adding 1000 mu L of PBST containing 1% BSA for sealing for 30min, washing for 3 times by using PBST to remove redundant sealing liquid, and resuspending the magnetic particles of the coupling antibody obtained by removing the washing liquid, using 1000 mu L of PBST and preserving at 4 ℃.
When the magnetic particle conjugate of listeria monocytogenes and salmonella is prepared, only the staphylococcus aureus capture antibody is replaced by the corresponding capture antibody for coupling.
2) Preparing a PS latex microsphere coupled target detection antibody (signal probe):
similar to the magnetic particle coupling process, only the magnetic separation process is converted into centrifugation, wherein the centrifugation condition of the 200nm PS latex microspheres is 15000rpm and 5min, the centrifugation condition of the 1000nm PS latex microspheres is 8000rpm and 5min, the centrifugation condition of the 1500nm PS latex microspheres is 6000rpm and 5min, the capture antibodies corresponding to the target in the coupling process are converted into detection antibodies, and the 200nm PS latex microspheres-staphylococcus aureus detection antibody conjugate, the 1000nm PS latex microspheres-listeria monocytogenes detection antibody conjugate and the 1500nm PS latex microspheres-salmonella detection antibody conjugate are prepared into preservation solution and placed in a refrigerator at 4 ℃.
Immune response and isolation
Mixing the three prepared carriers, the three signal probes and three targets corresponding to a series of gradients in equal volume (100 mu L each), incubating at 37 ℃ for 30min, performing magnetic separation for 10s, and collecting separation solution.
In order to obtain the best analysis effect, the concentration ratio of the carrier and the signal probe is respectively optimized, the peak effect is best when the ratio of the signal probe is determined to be 200nm:1000nm:1500nm ═ 3:2:1, and finally, the incubation concentration ratio of the carrier and the signal probe of the staphylococcus aureus is determined to be 2:3(100 mu g/mL: 150 mu g/mL), the listeria monocytogenes ratio is 1:1(100 mu g/mL: 100 mu g/mL), the salmonella ratio is 2:1(100 mu g/mL: 50 mu g/mL), and the linearity is best by referring to the mycotoxin optimization process.
Ultraviolet spectrophotometer for measuring light absorption value of separation liquid
And (3) uniformly mixing the separation solution (unreacted signal probe solution) collected in the step (a), injecting 200 mu L of sample into a micro cuvette, putting the micro cuvette on a cuvette holder in a sample cell, measuring the sample by the sample holder, and recording the maximum absorbance at the wavelength of 228nm, 365nm and 610nm respectively. And taking the logarithm of the concentration of the substance to be detected as an abscissa and the maximum absorbance value as an ordinate to obtain a linear relation between the logarithm of the concentration of the substance to be detected and the ordinate. As shown in FIG. 10, the linear range is 10 for Staphylococcus aureus3-107CFU/mL, regression equation is-0.085X +0.763, R20.9998, linear range 10 for listeria monocytogenes3-107CFU/mL, regression equation-0.092X +0.896, R20.9798, linear range 10 for salmonella2-106CFU/mL, regression equation, -0.109X +0.802, R2＝0.9995。
The results show that the method can be used for simultaneously detecting pathogenic microorganism multi-target substances, has high sensitivity and wide linear range, and can be suitable for simultaneously detecting objects to be detected with different detection requirements, the 200nm and 1000nm latex microspheres are used as signal probes for detecting staphylococcus aureus and listeria monocytogenes, and the 1500nm latex microspheres are used as signal probes for detecting salmonella with the highest detection sensitivity requirement.
The specific embodiments described herein are merely illustrative of the spirit of the invention. Various modifications or additions may be made to the described embodiments or alternatives may be employed by those skilled in the art without departing from the spirit or ambit of the invention as defined in the appended claims.

Claims (9)

1. A method for simultaneously detecting multiple targets based on concentration change of latex microspheres is characterized by comprising the following steps:
1) the nano magnetic particles are used as magnetic separation carriers, and capture antibodies or antibodies of different target objects are coupled on the surfaces of the nano magnetic particles; latex microspheres with different particle sizes are taken as signal probes, and detection antibodies or complete antigens of different target objects are coupled on the surfaces of the latex microspheres;
2) carrying out sufficient immunoreaction on the magnetic separation carrier, the signal probe and a target object to be detected to obtain a mixed solution;
3) carrying out magnetic separation on the mixed solution in the step 2), quickly adsorbing a magnetic separation carrier, and taking a separation solution to obtain a signal probe solution which does not participate in the reaction;
4) measuring the maximum absorbance corresponding to the ultraviolet absorption peak of the signal probe with different particle sizes in the signal probe solution which is not reacted in the step 3), respectively establishing a quantitative relation between the concentration of the object to be measured and the maximum absorbance value, and calculating the content of the corresponding target object according to the change of the absorbance.
2. The method for simultaneously detecting multiple targets according to claim 1, wherein in step 1), the latex microspheres are any one of polystyrene latex microspheres or polybutadiene latex microspheres, polyisoprene latex microspheres and polyacrylic latex microspheres.
3. The method for simultaneously detecting multiple targets according to claim 1, wherein in step 1), the diameter of the nanomagnetic particles is 50nm to 5000 nm.
4. The method for simultaneously detecting multiple targets according to claim 1, wherein in step 1), the particle size of the latex microspheres is 100nm to 5000 nm.
5. The method for simultaneously detecting multiple targets according to claim 1, wherein in the step 1), three kinds of latex microspheres are required to be used as signal probes when the targets are vomitoxin, aflatoxin and ochratoxin or staphylococcus aureus, listeria monocytogenes and salmonella, and the selected latex microspheres are polystyrene microspheres with the particle sizes of 200nm, 1000nm and 1500 nm.
6. The method for simultaneously detecting multiple targets according to claim 5, wherein the wavelength corresponding to the maximum absorbance of the 200nm polystyrene latex microspheres is 228nm, the wavelength corresponding to the maximum absorbance of the 1000nm polystyrene latex microspheres is 365nm, and the wavelength corresponding to the maximum absorbance of the 1500nm polystyrene latex microspheres is 610 nm.
7. The method for simultaneously detecting multiple targets according to claim 1, wherein in the step 2), when the sandwich immunoreaction is performed, the mixed solution is a "carrier-target-signaling probe" complex and a signaling probe solution that does not participate in the reaction; when the competitive immune reaction is performed, the mixed solution is a carrier-signaling probe complex, a carrier-target complex, and a signaling probe solution that does not participate in the reaction.
8. The method for simultaneously detecting multiple targets according to claim 1, wherein one latex microsphere with a single particle size is used for detecting one target, and a plurality of latex microspheres with different particle sizes are used for simultaneously detecting a plurality of targets.
9. The method for simultaneously detecting multiple targets according to claims 1-8 is applied to the simultaneous quantitative detection of various targets including mycotoxins, pathogenic microorganisms, antibiotics, veterinary drugs, disease markers, or others.
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