CN118033119A

CN118033119A - Washing-free biosensing method for carrying out multiple detection on site

Info

Publication number: CN118033119A
Application number: CN202410164960.5A
Authority: CN
Inventors: 陈翊平; 鲁鹏; 詹晨
Original assignee: Huazhong Agricultural University
Current assignee: Huazhong Agricultural University
Priority date: 2024-02-05
Filing date: 2024-02-05
Publication date: 2024-05-14

Abstract

The invention relates to a washing-free biosensing method for carrying out multiple detection on site, wherein an object reacts with a separation carrier A and a signal probe B in an immune manner to form a separation carrier A-object-signal probe B complex, namely an A-object-B complex, an unreacted signal probe B is suspended in a supernatant A, and the unreacted signal probe B in the supernatant A is removed; adding an agent capable of breaking the binding chemical bond of the biological recognition molecule to the complex in the A-target object-B, and then releasing the signaling probe B, wherein the signaling probe B is released into the supernatant B; detecting the signaling probes B in the supernatant B by using a particle counter, and counting the number and particle size of the signaling probes B; calculating the concentration of the target; and (5) finishing detection of the target object. The invention discloses a detection method for realizing separation of a signal probe and a reaction carrier based on gravity and buoyancy differences of microspheres with different sizes, and realizes washing-free multi-target rapid screening.

Description

Washing-free biosensing method for carrying out multiple detection on site

Technical Field

The invention relates to the fields of clinical diagnosis, food safety and biosensing, in particular to a washing-free biosensing method for carrying out multiple detection on site.

Background

Severe acute respiratory viruses (e.g., novel coronaviruses, influenza viruses, parainfluenza viruses) are highly infectious and diverse, highlighting the need for accurate identification and screening of respiratory virus infected patients for precise management and treatment. Since these respiratory viruses cause similar symptoms, such as cold, fever, asthma, recurrent respiratory infections, etc., and even fatal pneumonia, it is challenging to determine appropriate treatment methods for different patients. In addition, simultaneous infection with multiple respiratory viruses increases viral infectivity, thereby exacerbating the patient's condition. The effects of mycotoxins on the patient's immune system have also attracted considerable attention. Toxicity of mycotoxins (such as aflatoxin B1, vomitoxin, and ochratoxin a) to the immune system is manifested by damage to immune organs, programmed cell death of immune cells, and altered secretion of immune factors, thereby increasing the risk of infection with respiratory viruses. Thus, the simultaneous detection of respiratory viruses and mycotoxins at an early stage of disease is critical to guiding clinical treatment of patients.

Currently, the real-time quantitative polymerase chain reaction (Q-PCR) is considered as a gold standard for the detection of respiratory viruses. However, it requires specialized equipment and certified laboratories, which are not available in many remote areas. The Lateral Flow Immunoassay (LFIA) is used as a point of care testing technology (POCT), has the advantages of low cost, quick response, simple operation and rapid and convenient diagnosis. LFIA is of great interest because it can provide real-time diagnostic results to users. However, LFIA has the disadvantages of low sensitivity, narrow dynamic range, matrix interference, non-specific binding, and difficulty in multiplex detection. In addition, the method for accurately detecting the harmful factors such as mycotoxins in food is mainly an instrument analysis method, an immunoassay method and a biosensing method, the instrument analysis method mainly detects by large-scale precise instruments such as a gas chromatograph, a high performance liquid chromatograph-mass spectrometer and the like, the detection sensitivity is high, the linear range is wide, but the instrument is used for analyzing the sample, a large amount of pretreatment is needed, the instrument is expensive, the detection cost is high, and the method is not suitable for on-site rapid detection. The immunoassay method mainly comprises an enzyme-linked immunosorbent assay (ELISA) and the like, and the ELISA has the advantages of relatively simple operation, high throughput and the like, but the sensitivity is generally in ng/mL level, the linear range is narrow, and the ELISA is not suitable for trace detection. Therefore, developing a diagnostic platform that can rapidly and sensitively detect multiple targets, for early large-scale screening, is critical to preventing the transmission of hazard factors.

In the previous work, the inventor proposes a biosensing detection method (publication No. CN112595759 a) based on the change of the state of the insulating microsphere, which causes the resistance of the microchannel to change, in which a solution enters the microchannel under the action of electroosmotic flow and occupies a certain space in the channel, the insulating microsphere in the solution causes the concentration of conductive ions in the microchannel to change, so that the resistance of the microchannel changes correspondingly with the different aggregation states of the microsphere, and the concentration of the insulating microsphere can be measured by measuring the current change at two ends of the microchannel. The concentration of the microsphere and the concentration of the target substance, which are changed based on the immune response, have a certain linear relationship. The invention has lower resolution, and needs larger concentration difference between samples to detect obvious signal change, so that the accuracy is not high when the method is applied to the detection of actual samples, and more importantly, the method can not detect multiple targets simultaneously. In addition, the inventor also provides a multi-target simultaneous detection method (publication number CN 202111511613.8) based on the integral of the ultraviolet absorption spectrum peak area of latex microspheres, the method uses latex microspheres with different sizes and coupled with biological recognition molecules as signal probes, immune magnetic particles with the surfaces coupled with the biological recognition molecules as magnetic separation carriers, and the immune magnetic particles and the target to be detected are subjected to full immune reaction to obtain mixed solution; and after magnetic separation, scanning the whole wavelength of the signal probe solution which does not participate in the reaction, and selecting a wavelength range corresponding to the latex microsphere to integrate the peak area, thereby realizing accurate analysis and quantification of each signal probe. The method is based on the ultraviolet absorption peak difference of latex microspheres with different sizes, well avoids the background interference of a solution matrix, and can realize simultaneous quantification of multiple targets. However, the ultraviolet analyzer used in this method is bulky and requires multiple magnetic separation steps, which is suitable for laboratory detection, and it is difficult to perform rapid detection on site.

Disclosure of Invention

The invention provides a washing-free biosensing method for carrying out multiple detection on site, which is simple and convenient to operate and can be used for rapidly screening a plurality of pathogenic factors. This strategy relies solely on the gravity and buoyancy differences of the PS microspheres for separation without the need for complex reaction and washing steps. The sedimentation of microspheres with different sizes in the solution accelerates the isobaric line change of the solution, so that the microspheres collide more severely, and the combination of recognition molecules and target objects is facilitated. The target may interact specifically with recognition factors on the microspheres to form precipitated complexes of micron-sized (PS _μm) and millimeter-sized (PS _1000μm) microspheres. The subsequent introduction of NaOH disrupts this interaction, (the protein structure of the bound biorecognition molecules on the microspheres and the chemical bonds formed are denatured and cleaved in alkaline conditions, and the resulting complex after NaOH treatment eventually remains millimeter-sized and micrometer-sized microspheres), releasing the PS _μm microspheres from the complex. PS _1000μm microspheres sink rapidly within 3 seconds, and PS _μm microspheres can remain suspended for several hours, which provides a simple and easy-to-use tool for separating microspheres of different sizes. The released PS _μm microspheres are in a relationship (proportional or inversely) to the target concentration, and the size and number can be quantified by a portable particle counter, allowing simultaneous detection of different analytes. In addition, by using the method for accurately detecting the hazard factors in clinical diagnosis and food, different target probes can be designed according to different types of target objects to be detected, and further the requirement of simultaneously detecting various target objects is met to the greatest extent.

The technical scheme of the invention is as follows:

a wash-free biosensing method for performing multiplex assays in situ, the method comprising the steps of:

1) The target substance, the separation carrier A and the signal probe B are subjected to immune reaction to form a separation carrier A-target substance-signal probe B complex, namely an A-target substance-B complex, wherein the A-target substance-B complex is settled at the bottom of the solution, the unreacted signal probe B is suspended in the supernatant A, and the unreacted signal probe B in the supernatant A is removed;

2) Adding alkaline solution or organic solution or other reagent capable of breaking biological recognition molecule binding chemical bond to the complex in the A-target object-B, then releasing the signal probe B bound on the complex in the A-target object-B, and releasing the signal probe B into the supernatant B;

3) Detecting the signaling probe B in the supernatant B by using a portable particle counter, and identifying and counting the particle size and the number of the signaling probe B;

4) Calculating the concentration of the target object by taking the logarithmic value of the standard stock solution of the target object as an abscissa and the number of signal probes B with different particle diameters as an ordinate;

and (5) finishing detection of the target object.

Preferably, in the step 1), the separation carrier a is a microsphere coupled with a biological recognition molecule, the signal probe B is a microsphere coupled with a biological recognition molecule, and the microsphere is any one of a polystyrene latex microsphere or a polybutadiene latex microsphere, a polyisoprene latex microsphere, and a polyacrylic acid latex microsphere.

Preferably, the sedimentation rate of the separation carrier A is greater than the sedimentation rate of the signaling probe B in the same solution system.

Preferably, in the method, the time for which the signaling probe B is suspended in the supernatant a is greater than the reaction time+detection time, the reaction time being the total process time of step 1) and step 2); the detection time is the operation time of the step 3).

Preferably, in the method, the time for which the signaling probe B is suspended in the supernatant B is longer than the suspension time of the substance in the supernatant B after the release of the signaling probe B by the A-target-B complex.

Preferably, in the step 2), the density of the complex in the a-target-B is greater than the density of the solution.

Preferably, the alkaline solution in the step 2) is sodium hydroxide or potassium hydroxide, and the organic solvent is absolute ethanol solution or methanol, acetone solution or others.

The wash-free biosensing method for performing multiplex detection on site is applied to simultaneous quantitative detection of various targets, including mycotoxins, pathogenic microorganisms, antibiotics, agricultural and veterinary drugs, disease markers or others.

Preferably, the biological recognition molecules include, but are not limited to, an analyte-specific antibody and an analyte-complete antigen, a detection antibody and a capture antibody, an antibody and an antigen, an antigen and an antibody, a DNA capture probe and a DNA detection probe, a DNA detection probe and a DNA capture probe, a phage and an antibody, an antibody and a phage, a phage and a polypeptide, and the biological recognition molecules on the separation carrier a and the signal probe B can only specifically bind to the target but not bind to other targets.

The beneficial effects of the invention are as follows:

1. The detection method is a detection method for realizing separation of the signal probe and the reaction carrier based on the gravity and buoyancy differences of microspheres with different sizes, and the microspheres with different sizes are used as the signal probe, so that the washing-free multi-target rapid screening is realized.

2. The size difference of the microspheres is obvious (millimeter level and micron level), the sedimentation of the microspheres causes the liquid isobaric line to change drastically, the collision frequency between the microspheres is higher, and the reaction efficiency is higher.

Drawings

FIG. 1 is a schematic diagram of a wash-free biosensing strategy for multiplex detection in situ.

Figure 2 simultaneously measures the size distribution of three polystyrene microspheres using a portable particle counter.

FIG. 3 relationship between the location of target complex in solution and solution density.

FIG. 4 is a graph showing a simulation of the sedimentation velocity of a millimeter-sized microsphere in a solution, the change of the isobaric line of the solution, and the diffusion effect of a micrometer-sized microsphere in the solution.

FIG. 5 density of the complex in solution (with target, without target and after NaOH treatment).

Figure 6 is a conditional optimization of the simultaneous detection of three respiratory viruses.

Figure 7 shows the standard curve and linear range for simultaneous detection of three respiratory viruses.

FIG. 8 condition optimization for simultaneous detection of three mycotoxins.

FIG. 9 shows the standard curve and linear range for simultaneous detection of three mycotoxins.

Detailed Description

The present invention will be described in further detail with reference to the accompanying drawings and examples, but embodiments of the present invention are not limited thereto. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art. The test methods for specific experimental conditions are not noted in the examples below, and are generally performed under conventional experimental conditions or under experimental conditions recommended by the manufacturer. The reagents and starting materials used in the invention may be prepared by commercial or conventional methods unless otherwise specified.

Test materials and related term description

Carboxyl modified polystyrene microsphere (diameter 4, 6, 10 [ mu ] m;10 mg/mL): available from Thermo FISHER SCIENTIFIC company.

Carboxyl modified polystyrene microsphere (diameter 1000 μm): purchased from su zhou known beneficial microsphere technologies.

Mouse anti-SARS-CoV-2 coronavirus nucleocapsid protein (N protein) monoclonal antibodies (catalog numbers: CSB-DA701BmN-7 and CSB-DA701 BmN-8) and SARS-CoV-2 coronavirus nucleocapsid protein (N protein) were purchased from Wohan Meinai Biotechnology company.

Anti-influenza a H1N1 nucleocapsid protein antibody (ab 104870), anti-influenza a nucleoprotein antibody [ C43] (ab 128193), recombinant influenza a hemagglutinin protein (His tag) strain California/07/2009/H1N1 (ab 217662), anti-parainfluenza iii antibody (ab 28584), anti-parainfluenza iii hemagglutinin antibody [ B289M ] (ab 252769) and native parainfluenza iii protein (ab 274662) were purchased from Shanghai Able ANTI TRADING Co. (CHINA SHANGHAI).

Vomitoxin, aflatoxin, ochratoxin and corresponding complete antigens and antibodies: purchased from shandong blue all.

NaOH (analytically pure), naCl (analytically pure): purchased from national pharmaceutical group chemical company, inc.

Acetonitrile, methanol (all analytically pure): purchased from Shanghai Aba Ding Shenghua technologies Co.

PBS buffer (10 mM, ph=7.4): 8.00g NaCl, 0.20 g KCl, 0.20 g KH ₂PO₄ and 2.90 g Na ₂HPO₄·12H₂ O were taken and shaken well in 1000 mL volumetric flasks.

MES buffer (0.1M, ph=6.0): taking 21.325 g MES, dissolving in deionized water to constant volume to 1000 mL, and obtaining solution A; dissolving 4 g NaOH in deionized water to constant volume to 1000 mL to obtain solution B; mixing 1000 mL A liquid and 400 mL B liquid, and shaking.

PBST, MEST: to the formulated PBS or MES buffer was added 0.05% Tween-20.

Example 1 principle description and verification of a Wash-free biosensing method for multiplex detection in situ

In the invention, a washing-free biosensing strategy for carrying out multiple detection on site is developed, and the method is simple to operate, has multiplexing capability and can be used for rapidly screening various pathogenic factors. The method relies solely on the gravity and buoyancy differences of the PS microspheres for separation without the need for complex reaction and washing steps, as shown in fig. 1. The target analyte can interact specifically with recognition molecules on the PS microspheres to form a precipitated complex of micron-sized (PS _μm) -millimeter-sized (PS _1000μm) microspheres. The introduced NaOH solution disrupts this interaction, releasing the bound PS _μm microspheres from the complex. Because the gravity and the buoyancy of the microspheres with different sizes are obviously different, the PS _1000μm microspheres can quickly sink within 3 seconds, and the PS _μm microspheres can keep a suspension state within a plurality of hours, so that a simple and easy-to-use tool is provided for separating the microspheres with different sizes. The size and number of the separated PS _μm microspheres can be quantified by a portable particle counter, allowing simultaneous multiplexed detection of different targets.

According to the principle of resistance counting, the resistance between two electrodes is equivalent to the resistance of the electrolyte in the vicinity of the microwells. As the particles pass through the micropores, the electrolyte circulation area across the micropores decreases, resulting in an increase in resistance and a consequent change in voltage across the electrodes. When the particle diameter is between 2% and 40% of the micropores, the voltage pulse amplitude is proportional to the particle volume. Thus, the size and distribution of particles can be deduced from the amplitude of the voltage pulses. PS _μm microspheres with particle size distribution of 4 meters, 6, 10 μm and PS _μm microsphere mixtures of gradient concentration were validated using a portable particle counter. The verification result is shown in fig. 2, and the resistance counting principle can accurately identify PS _μm microspheres with various sizes at the same time and has high sensitivity.

Further, to investigate whether the presence of targets, antibodies and PS _μm microspheres would affect the sedimentation effect of PS _1000μm microspheres, the present invention used a model protein to mimic the actual antigen-antibody interactions. The specific process is as follows:

The sandwich complex was formed by adding goat anti-rabbit antibody (20. Mu.g/mL) conjugated PS _1000μm microspheres, human IgG (20. Mu.g/mL) conjugated PS _4μm microspheres, and rabbit anti-human antibody (0.5. Mu.g/mL) for an immune reaction. The compound was found to deposit at the bottom of the container. In addition, 2. Mu.L of NaCl solution (2M) was added successively until the microspheres began to float, the density of the solution after each addition of NaCl solution was recorded, and the density of the final solution was compared with the density of the solution used for the actual sample analysis (1.040-1.0600 g/cm ³). The verification result (as shown in fig. 3) shows that: a) The analyte, the antibody and the PS _4μm microsphere do not influence the sinking state of the PS _1000μm microsphere in the solution; b) The PS _1000μm microspheres remain submerged even in the high concentration of the actual sample solution. Further, by using COMSOL simulation software to simulate the movement of the microspheres in the solution, the results show that PS _1000μm microspheres can quickly sink to the bottom of the solution within 3 seconds (fig. 4 a), and that PS _1000μm microspheres sink to cause a change in the isobars, making the movement of PS _4μm microspheres in the solution more vigorous, facilitating the binding of PS _1000μm microspheres to PS _4μm microspheres (fig. 4b, c).

Finally, the densities of the compound samples with the target (negative sample), without the target (positive sample) and treated with the NaOH solution were verified. The results showed (as shown in fig. 5) that the complex density of the surface bound PS _4μm microspheres was higher, while the complex density of the NaOH solution treated was reduced, which demonstrated the breaking of covalent bonds and release of PS _4μm microspheres from the complex. The above verification and calculation provides powerful theoretical support for the successful use of the method for rapid detection of pathogenic agents.

EXAMPLE 2 modification of millimeter-sized and micron-sized microspheres with biological recognition molecules

1. Activation of microspheres

(1) 2Mg PS microspheres (average diameter 4, 6, 10 μm) were placed in a centrifuge tube, washed 2 times with 500. Mu.L MEST (10 mM MES,0.05% Tween 20, pH 6.0), centrifuged (10000 rpm,6 min) and the supernatant removed;

(2) 5 mg/mL EDC solution and 5 mg/mL NHS solution were prepared with 10 mM MES (pH 6.0);

(3) 100. Mu.L EDC (5 mg/mL) and 50. Mu.L NHS (5 mg/mL) are respectively added into a centrifuge tube filled with PS microspheres, the PS microspheres are fully suspended by using a vortex machine, diluted to 500. Mu.L by MES, placed on a rotary mixer and activated for 30min at 37 ℃;

(4) Centrifuging (10000 rpm,6 min), removing supernatant, and washing with 500 μl MEST 3 times;

through the above steps, carboxyl groups on the surface of the PS microsphere have been activated.

2. Coupling of PS microspheres with biological recognition molecules

By taking a 4-micrometer microsphere coupled SARS-CoV-2 recombinant protein antibody as an example, the coupling process is described, and other biological recognition molecules including influenza A virus, parainfluenza virus, mycotoxin antibody and the like can also adopt similar methods.

Preparation of PS microsphere-recombinant protein antibody conjugate (PS _4μm -Ab)

(1) Adding 20 mug of recombinant protein antibody into the centrifuge tube filled with PS microspheres, regulating the total volume to 500 mug by PBST, and mixing the PS microspheres and the recombinant protein antibody by light shaking;

2) Placing the mixture on a rotary mixer for reaction at 37 ℃ for 3 h;

3) Centrifuging (10000 rpm,6 min), removing supernatant, adding 500 μl of PBST (pH 7.4) containing 1% BSA, suspending PS microsphere again, placing on rotary mixer, and sealing at 37deg.C for 30 min;

4) Centrifuging (10000 rpm,6 min), removing supernatant, washing with 500 μl PBST 3 times;

5) Centrifugation (10000 rpm,6 min), removal of supernatant, resuspension of the resultant recombinant protein antibody modified PS microspheres with 1 mL PBST (pH 7.4, containing 0.02% NaN ₃, 0.5% BSA) and storage at 4 ℃. Activation of millimeter-sized microspheres (PS _1000μm) and coupling of biorecognition molecules were the same as described above. 1-2 μg of the biological recognition molecule is coupled to each millimeter-sized microsphere.

Example 3 Using the present accurate immunosensor method to detect SARS-CoV-2, influenza Virus, parainfluenza Virus as an example, the reaction conditions were optimized

The immune reaction time, the PS _μm microsphere coupled antibody concentration and the PS _1000μm microsphere coupled antibody concentration were optimized, the reaction time was set to be 10 min, 20 min and 30min, the PS _1000μm microsphere coupled antibody concentration was set to be 0.1. Mu.g, 0.5. Mu.g, 1. Mu.g, 2. Mu.g and 5. Mu.g, the PS _μm microsphere coupled antibody concentration was set to be 1. Mu.g, 5. Mu.g, 10. Mu.g, 20. Mu.g and 50. Mu.g, respectively, and the other conditions were unchanged.

The respiratory viruses were detected as follows:

1) Respectively diluting PS _μm microspheres modified by three virus recombinant protein antibodies to 100 mug/mL by PBS;

2) Preparing a standard stock solution of the virus recombinant protein with the concentration of 1 mg/mL by using a CBS buffer solution, and preparing standard working solutions with the concentration of 0, 0.01, 0.1, 1, 10, 100 and 1000 ng/mL by using PBS (phosphate buffer solution);

3) 100. Mu.L of PS _μm-Ab₁ dilutions (100. Mu.g/mL, PS _μm microsphere-conjugated antibody concentrations of 1. Mu.g, 5. Mu.g, 10. Mu.g, 20. Mu.g, 50. Mu.g, respectively), 100. Mu.L of virus recombinant protein standard solutions (0, 0.01, 0.1, 1, 10, 100, 1000 ng/mL), and PS _1000μm-Ab₂ (one PS _1000μm microsphere was used for each target, PS _1000μm microsphere-conjugated antibody concentrations of 0.1. Mu.g, 0.5. Mu.g, 1. Mu.g, 2. Mu.g, 5. Mu.g, respectively) were added to the vessel, and the reaction was shaken at 37℃ (reaction times of 10min, 20 min, 30min, respectively);

4) After the reaction, sucking the supernatant, and adding 100 mu L of NaOH (1M) solution to react for 10 min;

5) Sucking the supernatant by using a portable particle counter for detection;

6) The logarithmic value of the standard stock solution of the virus recombinant protein is taken as an abscissa, and the number of the microspheres with different particle diameters is taken as an ordinate, as shown in figure 6; the optimized results show that the results of detecting three respiratory viruses under different conditions are different, the final selection condition is that the reaction time is 20 min, the concentration of PS _1000μm microsphere coupled antibody is 2 mug, and the concentration of PS _4μm microsphere coupled antibody is 20 mug as the optimal reaction condition for the subsequent experiment.

Example 4 for the simultaneous detection of SARS-CoV-2, influenza Virus, parainfluenza Virus, standard Curve and Linear Range Using the present accurate immunosensor method

The three respiratory viruses were tested simultaneously as follows:

The experiment of this example was performed using the optimal conditions obtained by the optimization in example 3, i.e., the reaction time was 20 min, the concentration of PS _1000μm. Mu.g of the microsphere-conjugated antibody was 2. Mu.g, the concentration of PS _4μm microsphere-conjugated antibody was 20. Mu.g, and the other conditions were unchanged.

1) Respectively diluting PS _4μm microspheres modified by three virus recombinant protein antibodies to 100 mug/mL by PBS;

3) 100 mu L of PS _μm-Ab₁ diluent, 100 mu L of virus recombinant protein standard solution and PS _1000μm-Ab₂ are respectively added into a container, and the mixture is subjected to shaking reaction at 37 ℃ for 20 min;

5) Sucking the supernatant by using a portable particle counter for detection;

6) Taking the logarithmic value of the virus recombinant protein standard stock solution as an abscissa and the number of the microspheres with different particle diameters as an ordinate, and making a standard curve for three optimization results, as shown in figure 7; along with the increasing concentration of the respiratory viruses, the quantity of PS _μm-Ab₁ is gradually increased, the SARS-CoV-2 has a good linear relation with the quantity of PS _4μm-Ab₁ in 0.01-50 ng/mL, parainfluenza virus has a good linear relation with the quantity of PS _6μm-Ab₁ in 0.01-100 ng/mL, influenza A virus has a good linear relation with the quantity of PS _10μm-Ab₁ in 0.1-100 ng/mL, and therefore, the rapid detection of various respiratory viruses can be realized by the method.

Example 5 optimization of reaction conditions for Simultaneous detection of aflatoxin B1, vomit toxin, ochratoxin Using the present accurate immunosensor method

The immune reaction time, the total antigen concentration of PS _μm microsphere coupled mycotoxin and the total antigen concentration of PS _1000μm microsphere coupled mycotoxin were optimized, the reaction time was set to 10min, 20min and 30min, the total antigen concentration of PS _1000μm microsphere coupled mycotoxin was set to 0.1. Mu.g, 0.5. Mu.g, 1. Mu.g, 2. Mu.g and 5. Mu.g, the total antigen concentration of PS _μm microsphere coupled mycotoxin was set to 1. Mu.g, 5. Mu.g, 10. Mu.g, 20. Mu.g and 50. Mu.g, and the remaining conditions were unchanged.

The respiratory viruses were detected as follows:

1) Respectively diluting the PS _μm microspheres modified by the three mycotoxin antigens to 100 mug/mL by PBS;

2) Preparing a mycotoxin standard stock solution with the concentration of 1 mg/mL by using a PBS buffer solution, and preparing standard working solutions with the concentration of 0, 0.01, 0.1, 1, 10, 100 and 1000 ng/mL by using PBS;

3) 100. Mu.L of PS _μm -BSA dilutions (1. Mu.g, 5. Mu.g, 10. Mu.g, 20. Mu.g, 50. Mu.g, respectively) were added to the vessel, 100. Mu.L of mycotoxin standard solutions (0, 0.01, 0.1, 1, 10, 100, 1000 ng/mL) and PS _1000μm -Ab (one PS _1000μm microsphere was used for each target, PS _1000μm microsphere conjugated antibody concentrations were set to 0.1. Mu.g, 0.5. Mu.g, 1. Mu.g, 2. Mu.g, 5. Mu.g, respectively) were reacted by shaking at 37℃with shaking (reaction times were set to 10 min, 20 min, 30 min, respectively);

4) After the reaction, sucking the supernatant, and adding 100 mu L of NaOH (1M) solution for reaction for 10 min;

5) Sucking the supernatant by using a portable particle counter for detection;

6) Calculating the difference between the number of microspheres without target and the number of microspheres with target (Δcount=count (no target) -Count (target)) on the abscissa of the logarithmic scale of the mycotoxin standard stock solution, as shown in fig. 8; the optimized results show that the results of detecting three types of mycotoxins under different conditions are different, the final selection condition is that the reaction time is 20 min, the concentration of PS _1000μm microsphere coupled antibody is 2 mug, and the concentration of PS _μm microsphere coupled antigen is 20 mug as the optimal reaction condition for subsequent experiments.

Example 6 for the simultaneous detection of aflatoxin B1, vomit toxin, ochratoxin standard curve and linear range using the present accurate immunosensor method

The simultaneous detection steps for three mycotoxins were as follows:

The experiment of this example was performed using the optimal conditions obtained by the optimization in example 5, i.e., the reaction time was 20 min, the concentration of PS _1000μm microsphere-coupled antibody was 2. Mu.g, the concentration of PS _μm microsphere-coupled antigen was 20. Mu.g, and the other conditions were unchanged.

1) The mycotoxin antigen modified PS _4μm microsphere is diluted to 100 mug/mL by PBS;

3) 100. Mu.L of PS _μm -BSA diluent, 100. Mu.L of mycotoxin standard solution and PS _1000μm -Ab are added into a container respectively, and the mixture is subjected to shaking reaction at 37 ℃ for 20 min;

5) Sucking the supernatant by using a portable particle counter for detection;

6) Calculating the difference between the number of microspheres without target and the number of microspheres with target (Δcount=count (no target) -Count (target)) with the logarithmic value of the mycotoxin standard stock solution as the abscissa and making a standard curve for the three optimization results as shown in fig. 9; as the concentration of mycotoxins increases, the quantity change (delta Count) of PS _μm-Ab₁ gradually increases, AFB1 has a good linear relationship between the quantity of PS _4μm-Ab₁ and the quantity of 0.1-500 ng/mL, DON has a good linear relationship between the quantity of PS _6μm-Ab₁ and the quantity of 10-500 ng/mL, and OTA has a good linear relationship between the quantity of PS _10μm-Ab₁ and the quantity of 0.1-500 ng/mL, so that the method can realize rapid detection of various mycotoxins.

The technical solution of the present invention is explained by the above embodiments, but the present invention is not limited to the above embodiments, i.e. it does not mean that the present invention must be implemented depending on the above specific embodiments. Any modifications, or equivalent substitutions of materials for the invention, which are made by those skilled in the art based on the present invention, fall within the scope of protection of the patent.

Claims

1. A wash-free biosensing method for multiplex detection in situ, characterized by: the method comprises the following steps:

3) Detecting the signaling probe B in the supernatant B by using a particle counter, and identifying and counting the particle size and the number of the signaling probe B;

4) Calculating the concentration of the target;

and (5) finishing detection of the target object.

2. The method according to claim 1, wherein in step 1), the separation carrier a is a microsphere coupled with a biological recognition molecule, the signal probe B is a microsphere coupled with a biological recognition molecule, and the microsphere is any one of a polystyrene latex microsphere, a polybutadiene latex microsphere, a polyisoprene latex microsphere, and a polyacrylic acid latex microsphere.

3. The method of claim 2, wherein the sedimentation rate of the separation carrier a is greater than the sedimentation rate of the signaling probe B in the same solution system.

4. The method of claim 1, wherein the time for suspending signaling probe B in supernatant a is greater than the reaction time + detection time, the reaction time being the total process time of step 1) and step 2); the detection time is the operation time of the step 3).

5. The wash-free biosensing method for performing multiplexed assays on-site according to claim 1, wherein signal probe B is suspended in supernatant B for a longer period of time than the suspension of material in supernatant B after release of signal probe B by a-target-B complex.

6. The method of claim 1, wherein in step 2), the density of the complex in the a-target-B is greater than the density of the solution.

7. The method according to claim 1, wherein the alkaline solution in step 2) is sodium hydroxide or potassium hydroxide, and the organic solvent is absolute ethanol solution or methanol, acetone solution or others.

8. The use of a wash-free biosensing method according to any one of claims 1-8 for multiplex detection in situ for simultaneous quantitative detection of various targets, including mycotoxins, pathogenic microorganisms, antibiotics, agro-veterinary drugs, disease markers or others.

9. The wash-free biosensing method for performing multiplexed assays in situ according to claim 1, wherein the biological recognition molecules include, but are not limited to, analyte-specific antibodies and analyte-complete antigens, detection antibodies and capture antibodies, antibodies and antigens, antigens and antibodies, DNA capture probes and DNA detection probes, DNA detection probes and DNA capture probes, phage and antibodies, antibodies and phage, phage and polypeptides, and the biological recognition molecules on separation carrier a and signal probe B can only specifically bind to the target and cannot bind to other targets.