A detection substrate based on polymer passivation self-polymerization thin film coating modification and supporting detection method and equipment

Abstract

The present invention discloses a detection substrate, detection method and equipment based on the modification of polymer passivation self-polymerizing thin film coating. A layer of self-polymerizing polymer thin film coating is modified inside the gun head, and the reaction activity is controlled by a passivating agent to reduce the background signal of the actual detection, thereby preparing a gun head modified with a specific antigen / antibody. By using an automated device matched therewith, the following experimental operations can be realized in sequence: immune reaction based on antibody-antigen recognition, washing, incubation of enzyme-labeled antibodies, catalytic signal generation substrate and color signal self-identification reading; in addition, different signal reading probes can be accurately selected, thereby realizing accurate detection according to the content and standard of the object to be detected. The present invention realizes that in the entire detection process, only the relevant corresponding reagent consumables need to be prepared and placed in the corresponding position, and the automatic detection of different objects can be satisfied by setting the corresponding program, and the target content result can be directly obtained. The equipment is portable and can be operated automatically.

Description

Technical Field

[0001] The present invention belongs to the field of biosensing, and relates to an immunosensing method and supporting device based on polymer passivation self-polymerizing thin film coating modification and nano-microsphere signal amplification, especially a detection substrate based on polymer self-polymerizing thin film coating modification and supporting detection equipment and method. In addition, according to the different requirements of different detection objects for the sensitivity of the method, the present invention also relates to a signal amplification strategy using self-polymerizing nano-microspheres as carriers. During the entire detection process, it is only necessary to prepare the corresponding reagent consumables and put them in the automatic feeding tray position, and the automatic detection of different detection objects can be controlled by program. Background Art

[0002] Effective and highly sensitive detection of harmful factors such as pathogens, mycotoxins, antibiotics, biomarkers, heavy metals, etc. is of great significance in food safety, disease diagnosis, environmental monitoring, etc. Although biosensors based on optical, electrochemical, magnetic and other signal readout methods have been developed and applied in the detection field, enzyme-linked immunosorbent assay (ELISA) is still the most widely used immunoassay method for various target detections due to its good stability and strong reproducibility, and is even regarded as the standard method. Despite this, ELISA still has certain shortcomings in the detection process: (1) The plate washing operation is required in the washing step. If it is done manually, it may lead to errors in the results due to different experimental experiences of different experimenters. If it is automated, the equipment is expensive, which limits its application in on-site and rapid detection; (2) Since different reagents are required for each step of the reaction, the pipette tip needs to be replaced frequently, which increases the cost and is also prone to environmental pollution; (3) Proteins and ELISA plates are bound by physical adsorption between the hydrophobic groups on the protein molecular structure and the hydrophobic groups on the surface of the solid phase carrier. This physical adsorption is affected by molecular weight, isoelectric point, concentration, etc., and requires a higher coating concentration to achieve the expected experimental results. Biorecognition molecules are expensive, resulting in high costs; (4) The sensitivity of ELISA faces huge challenges in responding to the increasing detection requirements and the interference of complex matrices.

[0003] If the immune response carrier can be placed directly on the surface of the automated pipette, such as inside the pipette tip, it is important to effectively and efficiently modify the antibody inside the pipette tip. The self-polymerized thin film coating modified tip can bind to the protein through chemical covalent bonds, which can improve the coupling efficiency, greatly reduce the concentration of the coated protein, save the tip and thus reduce the cost. However, the self-polymerized thin film coating is too active and can easily lead to high background signals in the actual detection process. Therefore, we add a certain passivating agent to the self-polymerized molecule to effectively regulate its activity, thereby reducing the nonspecific adsorption and background signals of the actual detection; the high precision of the pipette (tip) can be combined with an automated device to achieve automated detection, avoid experimental differences and be more suitable for on-site detection; the self-polymerized nanoparticles synthesized based on the mild hydrothermal method can realize the signal amplification strategy and obtain different sensitivities according to the needs. After searching, no reports have been found so far on the realization of efficient antibody coating based on the passivated self-polymerized thin film coating, the realization of multiple signal amplification strategies based on the self-polymerized nanoparticles and the automated portable detection equipment suitable for on-site rapid detection. Summary of the invention

[0004] The purpose of the present invention is to solve the problems of complex washing operation, high cost and limited sensitivity in ELISA rapid on-site detection, and to provide an analysis mode with a passivated self-polymerizing film coating gun tip as a carrier with low cost, good stability, high sensitivity and high automation, and a mobile automated detection method and portable detection equipment that realizes a signal amplification strategy with self-polymerizing nano-microspheres.

[0005] The method and device provided by the present invention have the following working principles:

[0006] Dopamine (DA) is a biological molecule with catechol and amino groups. It can self-polymerize under alkaline conditions to form polydopamine (PDA). PDA has good stability and biocompatibility, and is easy to attach to the surface of organic and inorganic materials as a surface modifier. At the same time, the wide adhesion and secondary reactivity of PDA can modify biological molecules again to fix biorecognition molecules. It has a good adhesion effect on protein molecules such as antibodies. The reason is that the surface of PDA has many groups, and efficient chemical coupling is achieved through catechol groups, avoiding the physical adsorption of the traditional ELISA plate surface and antibodies. It also avoids the shortcomings of traditional chemical coupling methods such as chemical reagents and multi-step operations. Polydopamine and antibody coupling only need to be mixed at room temperature, thereby maximizing the activity of the antibody. Polydopamine nanospheres (PDA NS) can be synthesized at low cost under mild conditions from a mixed solution of ammonia, ethanol and deionized water, and have excellent biocompatibility and adsorption properties. PDANS can enrich HRP enzyme-labeled antibodies to achieve the first signal amplification. Biotin-tyramine is rapidly covalently bound to the stationary phase protein by hydrogen peroxide oxidation products under the catalysis of horseradish peroxidase (HRP), and can bind a large amount of streptavidin-HRP enzyme to achieve the second signal amplification. The intensity of different color signals produced by the double enriched HRP enzyme-catalyzed TMB colorimetric substrate is related to the target concentration, completing the quantitative analysis of the target. For analytes with high content in the sample, only signal amplification of polydopamine nanospheres or separate HRP-detection antibodies are needed. Therefore, through this patent, accurate detection can be achieved according to the requirements of different analytes.

[0007] A detection substrate modified based on a polymer self-polymerizing thin film coating, wherein a layer of self-polymerizing polymer thin film coating is modified on a substrate, and the coating fixes biological recognition molecules on the substrate to prepare a detection substrate modified with specific antigens / antibodies.

[0008] Preferably, the raw material of the self-polymerizing polymer film coating is dopamine.

[0009] Preferably, the raw material of the self-polymerizing polymer film coating may also be chitosan.

[0010] Preferably, in the process of modifying a layer of self-polymerizing polymer thin film coating on the substrate, a passivating agent is also added, and the passivating agent is amino polyethylene glycol or polyacrylamide or polyvinylamine.

[0011] Preferably, the substrate is glass or fiber or quartz or plastic.

[0012] Further preferably, the plastic is shaped like a gun tip model.

[0013] Preferably, the biorecognition molecule is an antibody and its antigen that can compete with the target to be detected for immune reaction; or the biorecognition molecule is a coated antibody and its labeled antibody that can undergo double antibody sandwich immune reaction with the target to be detected.

[0014] The preparation method of the detection substrate based on the modification of the polymer self-polymerized thin film coating comprises the following steps:

[0015] 1) The substrate is placed in a mixed solution of dopamine and amino polyethylene glycol (PEG-NH2) for 3 hours and then washed with deionized water;

[0016] 2) the substrate treated in step 1) is placed in the biorecognition molecule and incubated at 37° C. for 2 hours, and then washed with PBST solution;

[0017] 3) The substrate treated in step 1) is placed in bovine serum albumin for blocking operation to obtain a detection substrate modified with a polymer self-polymerizing thin film coating.

[0018] Preferably, in the step 1), in the mixed solution of dopamine and amino polyethylene glycol (PEG-NH2), the concentration of dopamine is 5 μg / mL, the concentration of PEG-NH2 is 100 μg / mL, and the concentration of H2O2 is 0.2 mol / L.

[0019] Preferably, in the step 2), in the PBST solution, the molar concentration of PBS is 0.01M, and the mass fraction of Tween is 0.05%; in the step 3), the mass fraction of bovine serum albumin is 8%.

[0020] A detection device based on a detection substrate modified by a polymer passivation self-polymerizing thin film coating,

[0021] The equipment consists of five parts: box, reaction liquid carrying platform, pipette moving platform, control system and detection system;

[0022] The box body is a constant temperature insulation box with heat-insulating material; the inner wall of the insulation box is provided with a temperature control device, which can provide the required constant temperature for the entire reaction process.

[0023] The reaction liquid carrying platform is provided with a detection substrate modified by a polymer self-polymerizing thin film coating, a detection substrate box, a detection system and an automatic feeding platform;

[0024] The pipette moving platform is provided with a pipette, on which a detection substrate modified by a polymer self-polymerized thin film coating is provided;

[0025] The control system consists of two parts: the control knob and the touch screen.

[0026] Preferably, the box body is a constant temperature insulation box with heat-insulating material; the inner wall of the insulation box is provided with a temperature control device, which can provide the required constant temperature for the entire reaction process;

[0027] The detection substrate is placed on the detection substrate box; the detection system is an optical camera that can perform colorimetric detection on the final reaction product; the automatic feeding platform includes a rotating platform that can perform continuous feeding for up to several times, and there are several ELISA plates containing reaction liquids on the rotating platform. The rotating platform rotates clockwise to achieve automatic sampling of the reaction liquid; a waste liquid pool is provided at the rear of the rotating platform; there are leveling knobs at the upper and lower parts of the reaction liquid carrying platform, and the reaction liquid carrying platform can be manually leveled; a first stepper motor is installed at the bottom of the reaction liquid carrying platform, and the first stepper motor drives the carrying platform through a belt drive to achieve forward and backward movement of the Y-axis.

[0028] Preferably, a second stepper motor and a third stepper motor are respectively provided on both sides of the pipette moving platform and the side of the pipette. The second stepper motor on the side of the pipette realizes the left and right movement of the pipette on the X-axis through a conveyor belt transmission, and the third stepper motors on both sides of the pipette moving platform realize the up and down movement of the pipette on the Z-axis through a worm gear transmission.

[0029] Preferably, the control system receives control instructions issued by the operator through the touch screen, controls the contacts of the corresponding relays to open and close, and thus controls the first stepper motor, the second stepper motor, and the third stepper motor to rotate forward and reverse, thereby realizing the movement of the reaction liquid carrying platform on the Y axis and the movement of the pipette on the X axis and the Z axis. The control system can adjust the reaction temperature in real time through the temperature control device on the box to achieve temperature control of the entire reaction process. The timer in the control system can control the time interval and rotation angle of each rotation of the linear motor to achieve automatic sampling of the reaction liquid. The control knob can be rotated and pressed to achieve manual selection and control of the control system.

[0030] The detection substrate modified by polymer self-polymerizing thin film coating or the detection substrate or detection device prepared by the preparation method is used in a method for detecting a target object. During the detection process, an HRP enzyme-labeled antibody or an HRP enzyme-labeled antibody enriched in self-polymerizing nano-microspheres or an HRP enzyme-labeled secondary antibody probe enriched in self-polymerizing nano-microspheres can be used in combination with a tyramide amplification system.

[0031] Preferably, the material of the self-polymerizing nano-microspheres is self-polymerizing nano-microspheres dopamine hydrochloride microspheres or polystyrene microspheres or magnetic nano-ferroferric oxide microspheres.

[0032] Preferably, the detection method comprises the following steps:

[0033] S1: The detection substrate modified by the polymer self-polymerized thin film coating absorbs the target to be detected to undergo a biological reaction, and after the reaction, the solution is discharged and the detection substrate is washed;

[0034] S2: According to the different requirements of different detection objects for sensitivity, different signal amplification probes a, b, and c are used for reaction, wherein a is HRP enzyme-labeled antibody, b is HRP enzyme-labeled antibody enriched by self-polymerizing nano-microspheres, and c is HRP enzyme-labeled secondary antibody probe enriched by self-polymerizing nano-microspheres combined with tyramide amplification system; after the biological reaction, the non-specifically adsorbed signal amplification probe is washed to remove, and then the signal generating substrate (TMB color developing substrate) is aspirated to change the color and then sulfuric acid is used to terminate the reaction, the final color is automatically photographed and the color intensity is analyzed by color, the yellow intensity is related to the concentration of the target to be detected, and the detection of the target is completed.

[0035] Preferably, the biological reaction time of the target object to be detected in step S1 is 30 minutes.

[0036] Preferably, in step S2, when using HRP enzyme-labeled antibody or HRP enzyme-labeled antibody enriched by self-polymerizing nanospheres, the reaction time is 30 minutes.

[0037] Preferably, step S2 is when the HRP enzyme-labeled secondary antibody probe enriched by the self-polymerizing nanospheres is combined with the tyramide amplification system: after adding the HRP enzyme-labeled antibody enriched by the self-polymerizing nanospheres for washing, a biotin tyramide diluent is added for reaction, after washing, a streptavidin-labeled HRP enzyme is added for incubation and washing to achieve double enrichment of the HRP enzyme; then a signal generating substrate (TMB color developing substrate) is aspirated for color change and sulfuric acid is used to terminate the reaction, the final color is automatically photographed and the color intensity is analyzed by color, and the yellow intensity is related to the concentration of the target to be measured.

[0038] More preferably, the reaction time of the biotin tyramide diluent is 15 min; the incubation time of the streptavidin-labeled HRP enzyme is 30 min; and the color development time of the TMB color developing solution is 10 min.

[0039] More preferably, the amount of biotin tyramine is 20 μg / mL, 100 μL.

[0040] In order to achieve the purpose of automated detection, the present invention provides a signal amplification strategy based on a disposable pipette tip modified by a passivation type molecular self-polymerizing thin film coating and self-polymerizing nano-microspheres as signal amplification carriers. In addition, a mobile automated portable detection device is designed based on the disposable pipette tip modified by a passivation type molecular self-polymerizing thin film coating. During the entire detection process, it is only necessary to prepare the corresponding reagents and consumables for pre-packaging and place them on the corresponding detection device carrier platform. By setting the corresponding program, the automated detection of different objects can be met.

[0041] Compared with the traditional ELISA detection method, the passivated molecular self-polymerized thin film coating of the present invention can reduce the antibody coating concentration and reduce the amount of gun tips used in the washing process, thereby achieving ultra-low-cost detection; secondly, the gun tip as a reaction carrier can avoid the clapping step in the washing process, making the operation simple and highly automated; finally, the self-polymerized nano-microspheres can achieve a dual signal amplification effect and greatly improve the sensitivity to meet the challenges of high sensitivity requirements of different detection objects. This method has good analytical performance and has good application prospects in the fields of in vitro diagnosis, food safety, and environmental monitoring. The beneficial effects of the present invention are as follows:

[0042] 1) The equipment is portable and low-cost: the experiment is carried out on the gun tip, which is easy to operate. The gun tip is modified with a self-polymerizing film coating, and the protein is bound by chemical covalent bonds, which can greatly reduce the concentration of the coating protein, save the amount of antibodies and gun tips, and the cost is low;

[0043] 2) Simple operation and good accuracy: The present invention is improved on the basis of the traditional gold standard ELISA, thus still retaining the inherent advantages of ELISA in terms of accuracy and stability, while avoiding complex operations such as plate washing;

[0044] 3) High detection sensitivity: The polydopamine-modified carrier has a large specific surface area and a strong ability to bind antibodies, so the sensitivity of the entire method is one order of magnitude higher than that of the traditional ELISA method; at the same time, the self-polymerizing nanoparticles synthesized by the mild hydrothermal method achieve dual signal amplification, greatly improving the sensitivity. For different detection objects, we can use different signal amplification systems to achieve accurate detection;

[0045] 4) High-throughput detection: The detection equipment can perform high-throughput detection of up to 96 channels at a time. Combined with 96-well plates, it can realize rapid and on-site analysis of large quantities of samples, with extremely high detection efficiency;

[0046] 5) The detection equipment has a high degree of automation: temperature control, sample injection, reaction time, washing, color signal analysis and result reading can all be controlled by pre-set programs to achieve unmanned operation. No additional plate washing technology is required. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 : Blank tip (A) and tip modified with self-polymerizing film coating (B).

[0048] Figure 2: Characterization diagram of self-polymerizing nanospheres (PDANS) and their enriched HRP enzyme-labeled antibody probes (A and B are SEM images of PDANS; C is a comparison of the ultraviolet absorption of dopamine and PDA NS; D is an elemental analysis diagram of PDA NS-coupled HRP enzyme antibody (PDA NS-HRP-Ab2); E is a Zeta potential analysis diagram before and after PDANS-HRP-Ab2; F is a comparison of the catalytic effects of PDA NS-HRP-Ab2 and PDANS-coupled HRP enzyme antibody-biotin tyramide amplification system (PDA NS-HRP-Ab2-TSA)).

[0049] Figure 3 : The effect of different enrichment carriers on the enrichment of HRP-Ab2 based on a self-written color analysis program (a: PDA NS is dopamine self-polymerized nanospheres; b: Fe3O4 is nanoparticles; c: PS MS is polystyrene microspheres).

[0050] Figure 4 : Detailed schematic diagram of the surface mobile automated portable detection device.

[0051] Figure 5 : Overall schematic diagram of mobile automated portable testing equipment.

[0052] Figure 6 :Schematic diagram of the control original diagram of mobile automation portable detection equipment.

[0053] Figure 7 : Comparison of detection of DON antigen coated with different concentrations in gun tip ELISA and traditional ELISA (pELISA is DON-ELISA under a specific gun tip, and cELISA is DON-ELISA under traditional enzyme-labeled well coating).

[0054] Figure 8 : Graph of the results of the one-time specific gun tip experiment.

[0055] Fig. 9 :Comparison of DON detection standard curves for different signal amplification systems based on self-written color analysis program (a: PDANS-HRP enzyme antibody-biotin tyramide amplification system (PDA NS-HRP-Ab2-TSA); b: PDA NS-HRP enzyme antibody (PDA NS-HRP-Ab2); c: HRP-Ab2)

[0056] Fig.10 : Standard curve for PCT detection based on self-written color analysis program.

[0057] Fig.11 : Standard curve for IL6 detection based on self-written color analysis program.

[0058] Description: box 1, reaction liquid carrying platform 2, pipette moving platform 3, control system 4, detection system 5, temperature control device 101; detection substrate 201 modified by polymer self-polymerizing thin film coating, detection substrate box 202, detection system 203, automatic feeding platform 204, rotating platform 205, ELISA plate 206, waste liquid pool 207, leveling knob 208, first stepper motor 209, pipette moving platform 3, pipette 301, second stepper motor 302, third stepper motor 303, conveyor belt 304, worm gear 305, control system 4, control knob 401, touch screen 402. DETAILED DESCRIPTION

[0059] The present invention is further described in detail below by specific examples. It should be understood by those skilled in the art that the examples are only to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0060] Test materials and related terms

[0061] Deoxynivalenol (DON) antibody (3.5 mg / mL) and DON-BSA conjugate (5.7 mg / mL) were purchased from Shandong Landu Biotechnology Co., Ltd.

[0062] DON standard: purchased from J&K Technology Co., Ltd.

[0063] Interleukin 6 (IL6), interleukin 6 capture antibody (IL6-Ab1), interleukin 6 enzyme-labeled antibody, PCT, PCT capture antibody (PCT-Ab1), PCT enzyme-labeled antibody: purchased from abcam.

[0064] The initial disposable gun tips were purchased from AXYGEN.

[0065] We design and build the automated testing equipment ourselves.

[0066] Example 1 Construction of self-polymerized nanospheres

[0067] Ammonia solution (3mL, NH4OH, 28-30%), ethanol (40mL) and deionized water (90mL) were stirred at 30°C for 30min. Dopamine hydrochloride (0.5g) was then dissolved in deionized water (10mL) and injected into the mixture solution for 24 hours. Self-polymerized nanospheres were obtained by centrifugation and washed 3 times with deionized water. The average diameter of the prepared self-polymerized nanospheres was 280nm.

[0068] Example 2: Construction of modified gun tips for specific detection, HRP-labeled secondary antibodies, and self-polymerizing nanoparticles

[0069] Taking the antibody against vomitoxin as an example, the modification of the gun tip to construct a gun tip for specifically detecting vomitoxin is described. A similar method can also be used for the other gun tips for detecting interleukin 6 and procalcitonin (PCT).

[0070] 1) Aspirate 120 μL of a mixed solution of dopamine (5 mg / mL), PEG-NH2 (100 μg / mL) and H2O2 (0.2 M) into the tip of the pipette, keep it at room temperature for 3 h, and then wash it with deionized water;

[0071] 2) 100 μL of bovine serum albumin coupled to vomitoxin DON-BSA (500 ng / mL) was aspirated into the above-mentioned pipette tip and incubated at 37°C for 2 hours and then washed with PBST;

[0072] 3) 200 μL of BSA solution (8%, w / v) was aspirated into the above-mentioned tip to block non-specific sites at 37° C. for 1 hour, and then washed with PBST and stored at 4° C. in the dark.

[0073] We performed transmission characterization on the morphology of the gun tip modified with the self-polymerizing film coating and compared it with a blank gun tip. The results showed that the gun tip modified with the self-polymerizing film coating ( Figure 1 B) Rougher than the blank tip ( Figure 1 A), a thin film of uniform and dense nanoparticles can be seen.

[0074] At the same time, we also conducted a series of characterizations on the self-aggregating nanospheres and the HRP-labeled antibodies enriched in the self-aggregating nanospheres ( Figure 2 ), demonstrating the successful synthesis of self-polymerizing nanospheres and their enriched HRP enzyme-labeled antibodies.

[0075] Taking the antibody against vomitoxin as an example, the coupling process is explained here. A similar method can also be used for the HRP-labeled secondary antibodies of other biorecognition molecules, including interleukin-6-HRP-labeled antibody, PCT-HRP-labeled antibody, etc.

[0076] 1) The HRP enzyme-labeled secondary antibody and the self-polymerized nanospheres at a mass ratio of 1:20 were reacted on a rotary mixer at 37°C for 3 h;

[0077] 2) 100 μL BSA (8%) was added to the above mixture and blocked at 37°C for 1 h;

[0078] 3) Wash and separate by centrifugation, remove the supernatant, resuspend with 1 mL PBST (pH 7.4, 0.5% BSA), and store at 4°C.

[0079] We selected a reported HRP enzyme-labeled antibody coupling process using polystyrene microspheres and ferroferric oxide nanoparticles as enrichment carriers for comparison:

[0080] 5 mg of carboxyl polystyrene microspheres with a particle diameter of 1000 nm were taken into a 1.5 mL centrifuge tube, and 150 μL of the above-mentioned phosphate-sodium hydroxide buffer solution was added for re-dissolution, and 10 μL of N-hydroxysulfosuccinimidesodium salt (NHS) with a mass concentration of 50 mg / ml and 10 μL of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride [1-Ethyl-3-(3-dimethylaminopropyl)carbodiimidehydrochloride, EDC] with a mass concentration of 50 mg / ml were added, and shaken for 20 minutes; centrifuged at 5000 rpm for 10 minutes, then the waste liquid was poured out, and washed with 200 μL of PBS with a molar concentration of 0.01 M and pH = 7.4 each time, washed 2-3 times, and then dissolved in 200 μL of the above-mentioned phosphate buffer solution to obtain an activated polystyrene microsphere suspension. Take 100 μL of the above activated microsphere suspension in a centrifuge tube, add 0.3 mg of enzyme-labeled antibody (protein content: 10 mg / mL), shake for 1 hour, centrifuge at 8000 rpm for 6 minutes, pour out the waste liquid, and then dissolve in 600 μL of the above blocking solution to obtain microspheres fixed with antibodies, i.e., composite enrichment reagents, which are stored in a 4°C refrigerator.

[0081] The coupling process of the 150 nm carboxylated ferroferric oxide nanoparticle enrichment carrier and the HRP enzyme-labeled antibody is similar to the above coupling process, wherein the washing process is completed by magnetic separation operation on a magnetic separation rack.

[0082] The results show that no additional activation reagent is required in the enrichment coupling process of the present invention, the coupling efficiency is high, the experimental steps are simplified, and the cost is reduced.

[0083] Table 1 Comparison of performance of different enrichment media for enzyme-labeled antibodies

[0084]

[0085] At the same time, we used vomitoxin as an analysis model and enriched HRP-Ab2 with the above three carriers for comparison of enrichment effects. Figure 3 The results showed that dopamine self-polymerized nanospheres had better linear quantitative range and detection sensitivity.

[0086] Example 3: Construction of portable automated testing equipment

[0087] refer to Figure 4-6 , the device is composed of five parts: a box 1, a reaction liquid carrying platform 2, a pipette moving platform 3, a control system 4, and a detection system 203;

[0088] The box body 1 is a constant temperature insulation box with heat-insulating material; the inner wall of the insulation box is provided with a temperature control device 101, which can provide the required constant temperature for the entire reaction process.

[0089] The reaction liquid carrying platform 2 has a detection substrate 201 modified by a polymer self-polymerizing thin film coating, a detection substrate box 202, a detection system 203 and an automatic feeding platform 204;

[0090] The pipette moving platform 3 is provided with a pipette 301, which is a 96-channel pipette with adjustable range. The pipette 301 is provided with a detection substrate 201 modified by a polymer self-polymerized thin film coating;

[0091] The control system 4 consists of two parts: a control knob 401 and a touch screen 402 .

[0092] Preferably, the box body 1 is a constant temperature insulation box with a heat-insulating material; the inner wall of the insulation box is provided with a temperature control device 101, which can provide the required constant temperature for the entire reaction process;

[0093] The detection substrate 201 is placed on the detection substrate box 202; the detection system 203 is an optical camera that can perform colorimetric detection on the final reaction product; the automatic feeding platform 204 includes a rotating platform 205 that can perform continuous feeding for up to several times, and there are several ELISA plates 206 for containing reaction liquid on the rotating platform 205. The rotating platform 205 rotates clockwise to realize automatic sampling of the reaction liquid; a waste liquid tank 207 is provided at the rear of the rotating platform 205; a leveling knob 208 is provided at the lower part of the reaction liquid carrying platform 2, and the reaction liquid carrying platform 2 can be manually leveled; a first stepper motor 209 is installed at the bottom of the reaction liquid carrying platform 2, and the first stepper motor 209 drives the carrying platform through a belt drive to realize the forward and backward movement of the Y axis.

[0094] Preferably, a second stepper motor 302 and a third stepper motor 303 are respectively provided on both sides of the pipette moving platform 3 and the side of the pipette 301. The second stepper motor 302 on the side of the pipette 301 is driven by a conveyor belt 304 to realize the left and right movement of the pipette 301 on the X-axis, and the third stepper motor 303 on both sides of the pipette moving platform 3 is driven by a worm gear 305 to realize the up and down movement of the pipette on the Z-axis.

[0095] Preferably, the control system receives control instructions issued by the operator through the touch screen 402, controls the contacts of the corresponding relays to open and close, thereby controlling the first stepper motor 209, the second stepper motor 302, and the third stepper motor 303 to rotate forward and reverse, thereby realizing the movement of the reaction liquid carrying platform 2 on the Y axis and the movement of the pipette 301 on the X axis and the Z axis. The control system 4 can adjust the reaction temperature in real time through the temperature control device 101 on the box 1 to achieve temperature control of the entire reaction process. The timer in the control system 4 can control the time interval and rotation angle of each rotation of the linear motor to achieve automatic sampling of the reaction liquid. The control knob can be rotated and pressed to achieve manual selection and control of the control system.

[0096] In addition, the detection platform also has power supply, switch, guide slide bar, support frame and other parts. Power input parameters: 100-240VAC, 1.2Amax, 50-60HZ; the switch controls the power on and off of the entire detection platform; the guide slide bar provides a moving track for the reaction liquid carrying platform and the pipette; the support frame plays a fixing role for the entire detection platform.

[0097] Example 4 Optimization of PEG-NH2 concentration

[0098] In order to achieve low background and high efficiency antibody coupling, the optimal PEG-NH2 concentration is of great significance. The specific process is as follows:

[0099] We used PCT as the analysis object and conducted optimization experiments with DA and PEG-NH2 concentration ratios of 500:1, 100:1, 50:1, and 10:1, respectively. The signal label was an enzyme-labeled antibody and the PCT concentration was 1 μg / mL.

[0100] Table 2 Comparison of signal-to-noise ratios at different DA and PEG-NH2 concentration ratios

[0101] <![CDATA[C DA :C PEG -NH2]]> Signal-to-Noise Ratio (S / N) 500：1 2.4 100:1 4.3 50:1 8.1 10：1 4.7

[0102] Therefore, we chose a DA to PEG-NH2 concentration ratio of 50:1 as the condition for the entire experiment.

[0103] At the same time, we used vomitoxin as the analysis object and compared the detection effects of different DON antigen coating concentrations in the tip ELISA (pELISA) and the traditional ELISA (cELISA), in which the signal label was an enzyme-labeled antibody. The results showed that ( Figure 7 ), our pELISA can achieve equivalent results to cELISA with 1000 ng / mL of coated antigen and competitive detection antibody when both the coated antigen and competitive detection antibody are at 500 ng / mL, which verifies that pELISA can reduce the amount of antigen and antibody by half while achieving equivalent detection results.

[0104] in, Figure 8 This was one of the preliminary experiments to complete the DON competition experiment using the pELISA strategy, which showed that our strategy is indeed feasible.

[0105] Example 5 Colorimetric Detection of DON

[0106] In order to verify that this method can be applied to the detection sensitivity requirements of different objects, taking DON in grain as an example, different signal amplification systems are used for detection. The specific process is as follows:

[0107] 1) After the automated detection equipment captured the specific detection of DON through system control, 50 μL of DON-Ab and 50 μL of different concentrations of DON were aspirated, and incubated at 37°C for 30 minutes, followed by PBST washing;

[0108] 2) Introduce 100 μL of HRP enzyme-labeled secondary antibody into the reaction device and incubate at 37°C for 30 minutes, then wash with PBST; then aspirate 100 μL of TMB commercial colorimetric solution and react at room temperature in the dark for 10 minutes, then inject into the detection well containing 50 μL of sulfuric acid to turn yellow and take a photo of the result, analyze the B value of the yellow color to quantitatively analyze the target content;

[0109] The logarithm of DON concentration is used as the horizontal axis, and the absolute value of the color B value change is △I B As the vertical axis, draw a standard curve, such as Fig. 9 As shown in c;

[0110] The principle of the DON detection method is as follows: In the reaction device, the DON complete antigen modified on the gun tip, the DON primary antibody and the DON in the sample undergo an immune competitive reaction. The more DON in the sample, the less the DON primary antibody binds to the DON complete antigen, and the less the HRP enzyme-labeled secondary antibody binds to the DON primary antibody. Therefore, the absolute value of the change in the color B value of the DON concentration in the sample and the color substrate is △I B There is a positive correlation between the DON in the sample.

[0111] or:

[0112] 1) After the automated detection equipment captured the specific detection of DON through system control, 50 μL of DON-Ab and 50 μL of different concentrations of DON were aspirated, and incubated at 37°C for 30 minutes, followed by PBST washing;

[0113] 2) Introduce 100 μL of HRP-labeled secondary antibody enriched with self-polymerizing nanospheres into the reaction device and incubate at 37°C for 40 minutes, then wash with PBST; then aspirate 100 μL of TMB commercial colorimetric solution and react at room temperature in the dark for 10 minutes, then inject into the detection well containing 50 μL of sulfuric acid to turn yellow and take a photo of the result, analyze the B value of the yellow color to quantitatively analyze the target content;

[0114] The logarithm of DON concentration is used as the horizontal axis, and the absolute value of the color B value change is △I B As the vertical axis, draw a standard curve, such as Fig. 9 As shown in b;

[0115] The principle of the DON detection method is as follows: In the reaction device, the DON complete antigen modified on the gun tip, the DON primary antibody and the DON in the sample undergo an immune competitive reaction. The more DON in the sample, the less the DON primary antibody binds to the DON complete antigen, and the less HRP enzyme-labeled secondary antibody enriched in the self-polymerized nanospheres bound to the DON primary antibody. Therefore, the absolute value of the change in the color B value of the DON concentration in the sample and the color substrate is △I B There is a positive correlation between the DON in the sample.

[0116] or:

[0117] 1) After the automated detection equipment captured the specific detection of DON through system control, 50 μL of DON-Ab and 50 μL of different concentrations of DON were aspirated, and incubated at 37°C for 30 minutes, followed by PBST washing;

[0118] 2) Introduce 100 μL of HRP-labeled secondary antibody enriched with self-polymerizing nanospheres into the reaction device and incubate at 37°C for 40 minutes, followed by PBST washing;

[0119] 3) Introduce 100 μL of tyramide and H2O2 mixed solution into the reaction device and incubate with HRP enzyme-labeled secondary antibody and self-polymerized nanoparticles at 37°C for 20 minutes, and then wash with PBST;

[0120] 4) Add SA-HRP solution to the reaction device and incubate at 37°C for 30 minutes, then wash with PBST, aspirate 100 μL TMB commercial colorimetric solution and react for 10 minutes at room temperature in the dark, then inject into the detection well containing 50 μL sulfuric acid to turn yellow and take a photo of the result, analyze the B value of the yellow color to quantitatively analyze the target content;

[0121] The logarithm of DON concentration is used as the horizontal axis, and the absolute value of the color B value change is △I B As the vertical axis, draw a standard curve, such as Fig. 9 As shown in a;

[0122] The principle of the DON detection method is as follows: In the reaction device, the DON complete antigen modified on the gun tip, the DON primary antibody and the DON in the sample undergo an immune competitive reaction. The more DON in the sample, the less the DON primary antibody binds to the DON complete antigen, and the less HRP enzyme-labeled secondary antibody enriched in the self-polymerizing nano-microspheres bound to the DON primary antibody. The less tyramide is bound to the self-polymerizing nano-microspheres labeled with the above-mentioned HRP enzyme-labeled secondary antibody, and then the less SA-HRP enzyme is bound. Therefore, the absolute value of the change in the color B value of the DON concentration in the sample and the color substrate is △I B There is a positive correlation between the DON in the sample.

[0123] from Fig. 9 It can be seen that under the same conditions, different signal amplification systems can be used to obtain different detection linear ranges and detection sensitivities to meet different detection requirements. Therefore, this patent can select different signal amplification systems according to different analytes to achieve accurate detection.

[0124] Table 3 Performance comparison between ELISA and automated pipette tip pELISA

[0125]

[0126] Example 6 Detection of PCT

[0127] In order to verify that this method can be applied to the detection sensitivity requirements of different subjects, the inflammatory marker procalcitonin (PCT) in human serum was detected with high sensitivity by initial signal amplification of HRP enzyme-labeled secondary antibody probe enriched with self-polymerizing nanospheres, as follows:

[0128] 1) After the automated detection equipment captured the specific detection PCT tip through system control, 100 μL of PCT of different concentrations was aspirated and incubated at 37°C for 30 minutes, followed by PBST washing;

[0129] 2) Introduce 100 μL of HRP-labeled secondary antibody enriched with self-polymerizing nanospheres into the reaction device and incubate at 37°C for 30 minutes, then wash with PBST; then aspirate 100 μL of TMB commercial colorimetric solution and react at room temperature in the dark for 10 minutes, then inject into the detection well containing 50 μL of sulfuric acid to turn yellow and take a photo of the result, analyze the B value of the yellow color to quantitatively analyze the target content;

[0130] The logarithm of PCT concentration is used as the horizontal axis, and the absolute value of the color B value change is △I B As the vertical axis, draw a standard curve, such as Fig.10 As shown;

[0131] The PCT residues in serum samples were detected by the standard addition method, the process was the same as above, and the results are shown in Table 4.

[0132] The principle of the PCT detection method is as follows: In the reaction device, the PCT-Ab1 modified on the gun tip reacts with the PCT in the sample by immune binding reaction. The more PCT in the sample, the more PCT-Ab1 binds to PCT, and the more HRP enzyme-labeled secondary antibodies enriched in the self-polymerized nano-microspheres that bind to the PCT-PCT-Ab1 complex. The improved sensitivity comes from the HRP enzyme-labeled secondary antibodies enriched in the self-polymerized nano-microspheres. Therefore, the absolute value of the change in the color B value of the PCT concentration in the sample and the color substrate is △I B There is a positive correlation between the PCT in the sample.

[0133] Table 4. Results of PCT detection in human serum using the standard addition method

[0134]

[0135] Example 7 Detection of interleukin 6 (IL6)

[0136] In order to verify that this method can be applied to the detection sensitivity requirements of different subjects, the inflammatory marker interleukin 6 (IL6) in human serum was detected with high sensitivity using a self-polymerizing nanosphere-enriched HRP enzyme-labeled secondary antibody probe combined with a tyramide amplification system, as follows:

[0137] 1) After the automated detection equipment captured the specific IL6 detection tip through system control, 100 μL of IL6 of different concentrations was aspirated and incubated at 37°C for 30 minutes, followed by PBST washing;

[0138] 2) Introduce 100 μL of HRP-labeled secondary antibody enriched with self-polymerizing nanospheres into the reaction device and incubate at 37°C for 40 minutes, followed by PBST washing;

[0139] 3) Introduce 100 μL of tyramide and H2O2 mixed solution into the reaction device and incubate with HRP enzyme-labeled secondary antibody and self-polymerized nanoparticles at 37°C for 20 minutes, and then wash with PBST;

[0140] 4) Add SA-HRP solution to the reaction device and incubate at 37°C for 30 minutes, then wash with PBST, aspirate 100 μL TMB commercial colorimetric solution and react for 10 minutes at room temperature in the dark, then inject into the detection well containing 50 μL sulfuric acid to turn yellow and take a photo of the result, analyze the B value of the yellow color to quantitatively analyze the target content;

[0141] 5) Taking the logarithm of IL6 concentration as the horizontal axis, the absolute value of the color B value change △I B As the vertical axis, draw a standard curve, such as Fig.11 As shown;

[0142] 6) The IL6 residue in the serum samples was detected by the standard addition method, the process was the same as above, and the results are shown in Table 5.

[0143] The principle of the IL6 detection method is as follows: In the reaction device, the IL6-Ab1 modified on the gun tip reacts with the IL6 in the sample by immune binding reaction. When the sample contains more IL6, the more IL6-Ab1 binds to IL6, the more self-polymerized nano-microspheres labeled with HRP enzyme-labeled secondary antibody bind to the IL6-IL6-Ab1 complex, the more tyramide binds to the self-polymerized nano-microspheres labeled with the HRP enzyme-labeled secondary antibody, and then the more SA-HRP enzyme binds. Therefore, the absolute value of the change in the color B value of the IL6 concentration in the sample and the color substrate is △I B There is a positive correlation between the IL6 in the sample.

[0144] Table 5. Results of the detection of IL6 in human serum using the standard addition method

[0145]

[0146] The applicant declares that the technical solution of the present invention is explained through the above embodiments, but the present invention is not limited to the above embodiments, that is, it does not mean that the present invention must rely on the above specific embodiments to be implemented. Any improvements made by technicians in the relevant field based on the present invention, or equivalent replacement of the materials selected by the present invention, etc., fall within the scope of protection of the patent.

Claims

1. A detection substrate modified by a polymer self-polymerized thin film coating, characterized in that: The detection substrate is: a layer of self-polymerizing polymer thin film coating is modified on the substrate, and the coating fixes the biological recognition molecules on the substrate to prepare a detection substrate modified with specific antigens / antibodies; the substrate is glass or fiber or quartz or plastic; the substrate is shaped like a gun tip model; in the process of modifying the layer of self-polymerizing polymer thin film coating on the substrate, a passivator is also added, and the passivator is amino polyethylene glycol or polyacrylamide or polyethylene amine.

2. The detection substrate modified by polymer self-polymerizing thin film coating according to claim 1, characterized in that: The raw material of the self-polymerizing polymer film coating is dopamine or chitosan; the biorecognition molecule is an antibody and its antigen that can compete with the target to be detected for immune reaction; or the biorecognition molecule is a coated antibody and its labeled antibody that can have a double antibody sandwich immune reaction with the target to be detected.

3. The method for preparing a detection substrate based on modification of a polymer self-polymerizing thin film coating according to any one of claims 1 to 2, characterized in that: 1) The substrate was placed in a mixed solution of dopamine and amino polyethylene glycol (PEG-NH2) for 3 hours and then washed with deionized water; 2) The substrate treated in step 1) was placed in the biorecognition molecule and incubated at 37°C for 2 hours, and then washed with PBST solution; 3) The substrate treated in step 1) is placed in bovine serum albumin for blocking operation to obtain a detection substrate modified with a polymer self-polymerized thin film coating.

4. The detection device based on the detection substrate modified by the polymer self-polymerizing thin film coating according to any one of claims 1-2, characterized in that: The device is composed of five parts: a box (1), a reaction liquid carrying platform (2), a pipette moving platform (3), a control system (4), and a detection system (203); A detection substrate (201) modified by a polymer self-polymerizing thin film coating, a detection substrate box (202), a detection system (203) and an automatic feeding platform (204) are provided on the reaction liquid carrying platform (2); A pipette (301) is mounted on the pipette moving platform (3), and a detection substrate (201) modified by a polymer self-polymerizing thin film coating is mounted on the pipette (301); The control system (4) consists of two parts: a control knob (401) and a touch screen (402).

5. The detection device based on the detection substrate modified by the polymer passivation self-polymerizing thin film coating according to claim 4 is characterized in that: The box body (1) is a constant temperature insulation box made of heat-insulating material; the inner wall of the insulation box is provided with a temperature control device (101) which can provide the required constant temperature for the entire reaction process; The detection substrate (201) is placed on the detection substrate box (202); the detection system (203) is an optical camera capable of performing color development detection on the final reaction product; the automatic feeding platform (204) comprises a rotating platform (205) capable of performing a maximum of several times of continuous feeding, a plurality of enzyme-labeled plates (206) for holding reaction liquids are arranged on the rotating platform (205), and the rotating platform (205) rotates clockwise to achieve automatic injection of the reaction liquid; a waste liquid pool (207) is arranged behind the rotating platform (205); a leveling knob (208) is arranged at the bottom of the reaction liquid carrying platform (2), and the reaction liquid carrying platform (2) can be manually leveled; a first stepper motor (209) is arranged at the bottom of the reaction liquid carrying platform (2), and the first stepper motor (209) drives the carrying platform through a belt drive to achieve forward and backward movement of the Y axis; A second stepping motor (302) and a third stepping motor (303) are respectively arranged on both sides of the pipette moving platform (3) and on the side of the pipette (301); the second stepping motor (302) on the side of the pipette (301) is driven by a conveyor belt (304) to achieve left-right movement of the pipette (301) on the X-axis; and the third stepping motor (303) on both sides of the pipette moving platform (3) is driven by a worm gear (305) to achieve up-and-down movement of the pipette on the Z-axis.

6. A method for detecting a target object using the detection substrate modified by a polymer self-polymerizing thin film coating as described in any one of claims 1 to 2 or the detection substrate modified by a polymer self-polymerizing thin film coating prepared by the preparation method as described in claim 3 or the detection device as described in any one of claims 4 to 5, characterized in that: During the detection process, according to the different sensitivity required by the target, three different signal amplification systems are used: HRP enzyme-labeled antibody, HRP enzyme-labeled antibody enriched by self-polymerizing nanospheres, or HRP enzyme-labeled secondary antibody probe enriched by self-polymerizing nanospheres combined with tyramide amplification system.

7. The method for detecting a target object according to claim 6, characterized in that: The material of the self-polymerizing nano-microspheres is self-polymerizing nano-microspheres dopamine hydrochloride microspheres or polystyrene microspheres or magnetic nano-ferroferric oxide microspheres.

8. The method for detecting a target object according to claim 7, characterized in that: The detection method comprises the following steps: S1: The detection substrate modified by the polymer self-polymerized thin film coating absorbs the target to be detected to undergo a biological reaction, and after the reaction, the solution is discharged and the detection substrate is washed; S2: According to the different requirements of different detection objects for sensitivity, different signal amplification probes a, b, and c are used for reaction, wherein a is HRP enzyme-labeled antibody, b is HRP enzyme-labeled antibody enriched by self-polymerizing nano-microspheres, and c is HRP enzyme-labeled secondary antibody probe enriched by self-polymerizing nano-microspheres combined with tyramide amplification system; after the biological reaction, the non-specifically bound signal probe is washed away, and then the signal-generating substrate is inhaled for color change and sulfuric acid is used to terminate the reaction, and the final color is automatically photographed and the color intensity is analyzed by color, and the yellow intensity is related to the concentration of the target to be detected, thereby completing the detection of the target; The step S2 is when the HRP enzyme-labeled secondary antibody probe enriched by the self-polymerizing nanospheres is combined with the tyramide amplification system: after adding the HRP enzyme-labeled antibody enriched by the self-polymerizing nanospheres for washing, a biotin tyramide diluent is added for reaction, after washing, a streptavidin-labeled HRP enzyme is added for incubation and washing to achieve double enrichment of the HRP enzyme; then, a signal-generating substrate is aspirated for color change and sulfuric acid is used to terminate the reaction, the final color is automatically photographed and the color intensity is analyzed by color, and the yellow intensity is related to the concentration of the target to be measured.

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