A pipette immunosensing platform with different signal amplification capabilities based on hollow polymer sleeves combined with Zr-MOF

Abstract

The present invention discloses a pipette immunosensing platform with different signal amplification capabilities based on a hollow polymer sleeve combined with Zr-MOF. Biomolecular recognition elements are coated on the inner wall of the hollow polymer sleeve through electrostatic interaction, hydrogen bond interaction or hydrophobic interaction, and the detection of various macromolecular and small molecular targets is successfully achieved. The present invention can use corresponding signal readout probes according to the different sensitivity requirements of different analytes to be measured, and combine with a picture color recognition software or a CCD camera system for quantitative analysis to meet the different sensitivity requirements of different analytes to be measured. Compared with the traditional ELISA detection method, the experiment on the hollow polymer sleeve is simple to operate, does not require additional plate washing technology, saves pipette tips, and has low cost; the sensing principle is efficient and accurate: the present invention is improved on the basis of the traditional gold standard ELISA, so it still retains the inherent advantages of accurate and stable results of ELISA; the detection sensitivity is high, rapid and on-site analysis of a large number of samples can be carried out, and the detection efficiency is extremely high.

Description

Technical Field

[0001] The present invention belongs to the field of biosensing, and relates to a pipette immunosensing platform based on hollow polymer sleeves combined with Zr-MOF with different signal amplification capabilities. Background Art

[0002] Effective and highly sensitive detection of harmful factors such as pathogenic bacteria, mycotoxins, antibiotics, biomarkers, heavy metals, etc. is of great significance in food safety, disease diagnosis, environmental monitoring, etc. Although biosensors based on signal readout methods such as optics, electrochemistry, magnetism, etc. have been developed and applied in the detection field, the enzyme-linked immunosorbent assay (ELISA) is still the most widely used immunoassay for the detection of various targets due to its good stability and strong reproducibility, and is even regarded as the standard method. Nevertheless, ELISA still has certain deficiencies in the detection process: 1. In the washing step, plate tapping operation is required, which may lead to deviations in results due to different experimental experiences of different experimenters; 2. Since different reagents are required for each step of the reaction, it is necessary to frequently change the pipette tips; 3. There are huge challenges in the sensitivity of ELISA in response to the increasing detection requirements and the interference of complex matrices. Summary of the Invention

[0003] The purpose of the present invention is to solve the problems of complex washing operation, high cost, limited sensitivity, etc. in on-site rapid detection of ELISA. By coating biorecognition molecules on the inner wall of the hollow polymer sleeve through electrostatic interaction, hydrogen bond interaction or hydrophobic interaction, the detection of various macromolecular and small molecular targets has been successfully realized. Provided is a pipette immunosensing platform based on hollow polymer sleeves combined with Zr-MOF with different signal amplification capabilities, which has low cost, good stability, strong anti-interference ability and optional sensitivity, and mainly realizes different signal amplification capabilities based on Zr-MOF and immunoassay reaction based on hollow polymer sleeves.

[0004] The technical solution of the present invention is as follows:

[0005] A pipette immunosensing platform based on Zr-MOF with different signal amplification capabilities uses a hollow polymer sleeve as the substrate, and realizes the detection of various macromolecular and small molecular targets by coating biorecognition molecules on the inner wall of the hollow polymer sleeve.

[0006] Preferably, the biorecognition molecule is a capture antibody or a detection antibody, an antibody or an antigen, a DNA capture probe or a DNA detection probe;

[0007] The target includes viruses, biomarkers, antibiotic molecules, pesticide molecules, veterinary drug molecules, biotoxins, bacteria or others; it also includes detection objects of various matrix components such as serum, milk, beverages, grain cereals or others;

[0008] The hollow polymer sleeve is made of polystyrene (PS), polycarbonate (PC), polyacrylamide (PAM), polymethyl methacrylate (PMMA), polydimethylsiloxane (PDMS), polyethylene terephthalate (PET), or others.

[0009] The operating method of the pipette immunosensing platform based on the hollow polymer sleeve combined with Zr-MOF with different signal amplification capabilities, the method comprising the following steps:

[0010] S1: The hollow polymer sleeve is incubated overnight with the biorecognition molecule, washed, and blocked with bovine serum albumin in sequence;

[0011] S2: The above-prepared biomolecule-modified hollow polymer sleeve is successively aspirated with the target to be detected for biological reaction. After the reaction, the solution is discharged and the hollow polymer sleeve is washed. An enzyme-labeled antibody or a Zr-MOF-Pt-HRP enzyme-labeled antibody or a Zr-MOF-Pt-HRP-HRP enzyme-labeled antibody-initiator strand is added for reaction. After washing the excess enzyme-labeled antibody or Zr-MOF-Pt-HRP enzyme-labeled antibody or Zr-MOF-Pt-HRP-HRP enzyme-labeled antibody-initiator strand;

[0012] S3: After aspirating the signal generation substrate (TMB chromogenic substrate) for color change, the reaction is terminated with sulfuric acid. The final color is photographed and the color intensity is analyzed with color analysis software. The color intensity is related to the concentration of the target to be detected. Or after adding the enzyme-labeled antibody for reaction, the excess enzyme-labeled antibody is washed, and then the luminescent substrate is aspirated to generate a signal. A CCD camera imaging system is combined for quantitative analysis of the target. The light intensity is related to the concentration of the target to be detected.

[0013] In step S2, adding the Zr-MOF-Pt-HRP enzyme-labeled antibody achieves dual signal amplification; if a Zr-MOF-Pt-HRP-HRP enzyme-labeled antibody-initiator strand is added, after washing, incubating the hybridization chain reaction amplification system and incubating streptavidinylated HRP achieve five-fold signal amplification.

[0014] Further preferably, the Zr-MOF-Pt-HRP enzyme-labeled antibody is prepared by the following method;

[0015] Zr-MOF is synthesized as follows: Dissolve 5 - 15 mg of H2TCPP, 15 - 45 mg of ZrOCl2·8H2O, and 350 - 350 mg of benzoic acid in 5 - 15 mL of DMF and add it to a 50 - 100 mL glass bottle; Stir at 300 rpm and 90 °C for 2 - 10 h to obtain the Zr-MOF product; Centrifuge at 14,000 rpm for 30 min, wash 3 times with DMF, and dry in a vacuum oven;

[0016] Zr-MOF-Pt was synthesized by in-situ reduction on Zr-MOF: 5-15 mg of Zr-MOF was dispersed in 10-30 mL of water, and then 100 μL of 2.5-10% H2PtCl6 was added to the suspension and stirred for 10.5-2 h (37 °C). Subsequently, 2.5 mL of 0.1-1 mg / mL NaBH4 solution was dropped into the suspension and stirred vigorously for 2-4 h (37 °C). Finally, the Zr-MOF-Pt product was centrifuged (14,000 rpm, 10 minutes), washed 3 times with water, and finally dispersed in water;

[0017] Preparation of Zr-MOF-Pt-HRP enzyme-labeled antibody: HRP enzyme-labeled antibody (100 μL, 200 μg / mL) was added to Zr-MOF-Pt and kept at 4 °C for 10-24 hours; then, the mixed solution was washed 3 times with PBS and centrifuged (12,000 rpm, 10 min, 4 °C).

[0018] More preferably, the Zr-MOF-Pt-HRP-HRP enzyme-labeled antibody-initiation strand was prepared as follows: Zr-MOF-Pt was incubated with HRP solution (0.5-2 mg / mL, 50 μL) for 1 hour (Zr-MOF-Pt-HRP); next, HRP enzyme-labeled antibody (100 μL, 100-300 μg / mL) and phosphorylated hybridization chain reaction initiation strand DNA were added and incubated at 4 °C for 12-24 hours (Zr-MOF-Pt-HRP-HRP enzyme-labeled antibody-initiation strand); then, the mixed solution was washed 3 times with PBS and centrifuged (12,000 rpm, 10 min, 4 °C); Zr-MOF-Pt-HRP-HRP enzyme-labeled antibody-initiation strand was resuspended in 1.0 mL of PBS and stored at 4 °C.

[0019] Preferably, the hybridization chain reaction amplification system is biotinylated double hairpin DNA.

[0020] The working principle of the method and device provided by the present invention is as follows:

[0021] Metal-organic framework (MOF) materials have attracted great attention from researchers due to their high porosity, large surface area, and excellent properties provided by the central atoms or ligands. In addition, MOF has been proven to be an ideal carrier for in-situ growth of noble metals. And it can interact with nucleic acids by forming coordination bonds. Among them, the large surface area of Zr-MOF can in-situ generate a large number of platinum nanoparticles (Pt NPs), and the high porosity can entrap a large amount of horseradish peroxidase (HRP). The Zr ions of Zr-MOF can form Zr-OP bonds with the terminal phosphate groups of DNA. Pt NPs can modify enzyme-labeled antibodies without the assistance of additional activating reagents. Therefore, Zr-MOF can be used as a carrier to construct probes with different signal amplification capabilities, improve the sensitivity of colorimetric detection, and perfectly solve the problem of complex coupling between MOF and biomolecules (antibodies or DNA). In this patent, we constructed two signal probes with different signal amplification capabilities based on Zr-MOF: 1. A dual-signal amplification probe based on Zr-MOF-Pt-HRP enzyme-labeled antibody, the principle of which is to in-situ generate a large number of Pt NPs on the surface of Zr-MOF, and then couple a large number of enzyme-labeled antibodies to achieve a dual-signal amplification probe; 2. A five-signal amplification probe based on Zr-MOF-Pt-HRP-HRP enzyme-labeled antibody-initiation strand, the principle of which is to in-situ generate a large number of Pt NPs on the surface of Zr-MOF, couple a large number of enzyme-labeled antibodies on the Pt NPs, entrap a large amount of HRP in the pores of Zr-MOF, covalently connect a large number of initiation strands for signal amplification of hybridization chain by Zr ions, and the initiation strand triggers a hybridization chain reaction to further enrich a large amount of HRP to achieve five-signal amplification. Secondly, when the sensitivity requirements for the detection target are not high, the enzyme-labeled antibody is directly used for detection. The different color signal intensities generated by HRP enzyme catalyzing TMB chromogenic substrate are related to the target concentration, and the quantitative analysis of the target is completed.

[0022] The beneficial effects of the present invention are as follows:

[0023] 1) Portable equipment and low cost: Experiments are carried out on a hollow polymer sleeve. The experimental operation is simple, no additional plate washing technology is required, pipette tips are saved, and the cost is low;

[0024] 2) Efficient sensing principle and good accuracy: The present invention is improved on the basis of the traditional gold standard ELISA, so it still retains the inherent advantages of accurate and stable results of ELISA;

[0025] 3) Simple coating operation: Biomolecular recognition molecules (antigens, antibodies) can be adsorbed on the hollow polymer sleeve through electrostatic adsorption, hydrophobic interaction, and hydrogen bond interaction, avoiding chemical modification;

[0026] 4) High detection sensitivity: Different signal probes based on Zr-MOF with different signal amplification capabilities achieve dual or quintuple signal amplification, greatly improving sensitivity. For different detection targets, we can adopt different signal amplification systems;

[0027] 5) High-throughput detection: Cooperating with a high-throughput pipetting gun, up to 96 channels of high-throughput detection can be performed each time, enabling rapid and on-site analysis of a large number of samples with extremely high detection efficiency. Brief Description of the Drawings

[0028] Figure 1 : Schematic diagram of the pipette immunosensing platform based on Zr-MOF with different signal amplification capabilities.

[0029] Figure 2 : Characterization diagrams of Zr-MOF and Zr-MOF-Pt (A and C are SEM and TEM diagrams of Zr-MOF; B and D are SEM and TEM diagrams of Zr-MOF-Pt;

[0030] Figure 3 : Comparison of DON detection standard curves for different signal amplification systems based on a self-written color analysis program (a: Zr-MOF-Pt-HRP-HRP enzyme-labeled antibody-initiating strand; b: Zr-MOF-Pt-HRP enzyme-labeled antibody; c: enzyme-labeled antibody);

[0031] Figure 4 : Comparison of DON detection standard curves for different signal amplification systems based on a CCD imaging system (a: Zr-MOF-Pt-HRP-HRP enzyme-labeled antibody-initiating strand; b: Zr-MOF-Pt-HRP enzyme-labeled antibody; c: enzyme-labeled antibody);

[0032] Figure 5 : PCT detection standard curve based on a self-written color analysis program;

[0033] Figure 6 : IL6 detection standard curve based on a self-written color analysis program. Detailed Embodiments

[0034] The present invention will be further described in detail below through specific embodiments. Those skilled in the art should understand that the embodiments are only for helping to understand the present invention and should not be regarded as specific limitations of the present invention.

[0035] Description of Test Materials and Related Terms

[0036] DON antibody (3.5 mg / mL), DON-BSA conjugate (5.7 mg / mL): Purchased from Shandong Landu Biotechnology Co., Ltd.

[0037] DON standard: Purchased from J&K Scientific Ltd.

[0038] 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.

[0039] The initial disposable hollow polymer cannula was designed and fabricated.

[0040] Example 1 Construction of signal probes with different signal amplification capabilities based on Zr-MOF

[0041] Synthesis of Zr-MOF: Dissolve 10 mg of H2TCPP, 30 mg of ZrOCl2·8H2O, and 280 mg of benzoic acid in 10 mL of DMF and add it to a 50 mL glass bottle. Stir at 300 rpm and 90 °C for 5 h to obtain the Zr-MOF product. Then centrifuge at 14,000 rpm for 30 min, wash 3 times with DMF, and dry in a vacuum oven, as Figure 2 shown in A and C.

[0042] Synthesis of Zr-MOF-Pt: Disperse 10 mg of Zr-MOF in 20 mL of water. Then add 100 μL of 5% H2PtCl6 to the suspension and stir for 1 h (37 °C). Subsequently, drop 2.5 mL of 0.4 mg / mL NaBH4 solution into the suspension and stir vigorously for 3 h (37 °C). Finally, centrifuge the Zr-MOF-Pt product (14,000 rpm, 10 min), wash 3 times with water, and finally disperse it in water, as Figure 2 shown in B and D.

[0043] Preparation of Zr-MOF-Pt-HRP enzyme-labeled antibody: Add HRP enzyme-labeled antibody (100 μL, 200 μg / mL) to Zr-MOF-Pt and keep it at 4 °C for 16 h. Then, wash the mixed solution 3 times with PBS and centrifuge (12,000 rpm, 10 min, 4 °C).

[0044] Synthesis of Zr-MOF-Pt-HRP-HRP-labeled antibody-initiating strand: Zr-MOF-Pt was incubated with HRP solution (1 mg / mL, 50 μL) for 1 hour (Zr-MOF-Pt-HRP). Next, HRP-labeled antibody (100 μL, 200 μg / mL) and phosphorylated hybridization chain reaction initiating strand DNA were added and incubated at 4°C for 16 hours (Zr-MOF-Pt-HRP-HRP-labeled antibody-initiating strand). Then, the mixed solution was washed 3 times with PBS and centrifuged (12,000 rpm, 10 min, 4°C). Zr-MOF-Pt-HRP-HRP-labeled antibody-initiating strand was resuspended in 1.0 mL PBS and stored at 4°C.

[0045] Table 1 Comparison of biomolecule binding performance in different media

[0046]

[0047] The results show that: our Zr-MOF-Pt material does not require additional activation reagents during the process of binding biomolecules, thus improving the coupling efficiency, simplifying the experimental steps and reducing the cost.

[0048] Example 2 Colorimetric detection of deoxynivalenol (DON)

[0049] To verify that this method has adjustable sensitivity and is suitable for the detection of different targets, taking DON in grains as an example, different signal amplification systems were used for colorimetric detection. The specific process is as follows:

[0050] 1) The hollow polymer sleeve was inhaled with deoxynivalenol antigen and incubated overnight at 4°C, and then blocked with 5% BSA at 37°C for 1 h (immunodetection hollow polymer sleeve). The immunodetection hollow polymer sleeve was inhaled with 50 μL of DON-Ab and 50 μL of different concentrations of DON, and incubated at 37°C for 30 minutes, and then washed with PBST;

[0051] 2) 100 μL of enzyme-labeled antibody was introduced into the above-mentioned hollow polymer sleeve and incubated at 37°C for 30 minutes, and then washed with PBST; then 100 μL of commercial TMB chromogenic solution was inhaled and reacted in the dark at room temperature for 15 min, and then injected into the detection well containing 50 μL of sulfuric acid to turn yellow and the result was photographed, and the B value of the yellow color was analyzed to quantitatively analyze the target content by colorimetry;

[0052] Taking the logarithm of the DON concentration as the abscissa and the absolute value of the change in the color B value ΔI B as the ordinate, a standard curve was made, as Figure 3 shown in

[0053] The principle of this DON detection method is as follows: Inside the immunodetection hollow polymer sleeve, an immunological competition reaction occurs among the DON complete antigen modified on the hollow polymer sleeve, the DON primary antibody, and DON in the sample. When there is more DON in the sample, the less the DON primary antibody binds to the DON complete antigen, and the less the HRP-labeled secondary antibody that binds to the DON primary antibody. Therefore, the concentration of DON in the sample is positively correlated with the absolute value of the change ΔI in the color B value of the chromogenic substrate B to determine DON in the sample.

[0054] Or:

[0055] 1) The immunodetection hollow polymer sleeve is inhaled with vomitoxin antigen and incubated overnight at 4°C, and then blocked with 5% BSA at 37°C for 1 h (immunodetection hollow polymer sleeve). The immunodetection hollow polymer sleeve is inhaled with 50 μL of DON-Ab and 50 μL of DON at different concentrations, incubated at 37°C for 30 minutes, and then washed with PBST;

[0056] 2) 100 μL of Zr-MOF-Pt-HRP-labeled antibody is introduced into the above-mentioned hollow polymer sleeve and incubated at 37°C for 40 minutes, and then washed with PBST; then 100 μL of commercial TMB chromogenic solution is inhaled and reacted in the dark at room temperature for 15 min, and then injected into the detection well containing 50 μL of sulfuric acid to turn yellow, and the result is photographed. The B value of the yellow color is analyzed to quantitatively analyze the target content;

[0057] Taking the logarithm of the DON concentration as the abscissa and the absolute value of the change ΔI in the color B value B as the ordinate, a standard curve is made, as Figure 3 shown in b;

[0058] The principle of this DON detection method is as follows: Inside the immunodetection hollow polymer sleeve, an immunological competition reaction occurs among the DON complete antigen modified on the hollow polymer sleeve, the DON primary antibody, and DON in the sample. When there is more DON in the sample, the less the DON primary antibody binds to the DON complete antigen, and the less the Zr-MOF-Pt-HRP-labeled antibody that binds to the DON primary antibody. Therefore, the concentration of DON in the sample is positively correlated with the absolute value of the change ΔI in the color B value of the chromogenic substrate B to determine DON in the sample.

[0059] Or:

[0060] 1) The immunodetection hollow polymer sleeve is inhaled with vomitoxin antigen and incubated overnight at 4°C, and then blocked with 5% BSA at 37°C for 1 h (immunodetection hollow polymer sleeve). The immunodetection hollow polymer sleeve is inhaled with 50 μL of DON-Ab and 50 μL of DON at different concentrations, incubated at 37°C for 30 minutes, and then washed with PBST;

[0061] 2) Introduce 100 μL of Zr-MOF-Pt-HRP-HRP enzyme-labeled antibody-initiating strand into the above hollow polymer sleeve and incubate at 37 °C for 40 minutes, then wash with PBST;

[0062] 3) Introduce 100 μL of hybridization chain reaction amplification system into the above hollow polymer sleeve and incubate at 37 °C for 90 minutes, then wash with PBST;

[0063] 4) Add SA-HRP solution to the above hollow polymer sleeve and incubate at 37 °C for 30 minutes, then wash with PBST. Aspirate 100 μL of commercial TMB chromogenic solution and react in the dark at room temperature for 10 min, then inject into the detection well containing 50 μL of sulfuric acid to turn yellow and take pictures of the results. Analyze the B value of the yellow color to quantitatively analyze the target content;

[0064] With the logarithm of DON concentration as the abscissa and the absolute value of the change in color B value △I B as the ordinate, make a standard curve, as Figure 3 shown in a;

[0065] The principle of this DON detection method is as follows: In the immunodetection hollow polymer sleeve, an immunocompetitive reaction occurs among the DON complete antigen modified on the hollow polymer sleeve, the DON primary antibody, and DON in the sample. When there is more DON in the sample, the less the DON primary antibody binds to the DON complete antigen, and the less the Zr-MOF-Pt-HRP-HRP enzyme-labeled antibody-initiating strand that binds to the DON primary antibody, and then the less the SA-HRP enzyme that binds. Therefore, the DON concentration in the sample is positively correlated with the absolute value of the change in color B value △I B of the chromogenic substrate to determine DON in the sample.

[0066] As can be seen from Figure 3 , under the same conditions, different signal amplification systems can obtain different detection linear ranges and detection sensitivities to meet different detection requirements. Therefore, it shows that this patent can select different signal amplification systems according to different analytes to achieve accurate detection.

[0067] Table 2 Comparison of ELISA and automated hollow polymer sleeve pELISA performance

[0068]

[0069] Example 3 Chemiluminescence detection of vomitoxin (DON)

[0070] To verify that this method has adjustable sensitivity and is suitable for the detection of different objects, taking DON in grains as an example, different signal amplification systems are used for chemiluminescence detection. The specific process is as follows:

[0071] 1) The hollow polymer sleeve is incubated with vomitoxin antigen at 4 °C overnight, and then blocked with 5% BSA at 37 °C for 1 h (immunodetection of the hollow polymer sleeve). The hollow polymer sleeve for immunodetection is inhaled with 50 μL of DON-Ab and 50 μL of DON at different concentrations, incubated at 37 °C for 30 minutes, and then washed with PBST;

[0072] 2) 100 μL of enzyme-labeled antibody is introduced into the above-mentioned hollow polymer sleeve and incubated at 37 °C for 30 minutes, and then washed with PBST; The catalytic luminescent substrate generates a signal, and combined with the CCD imaging system for quantitative analysis of the target. The light intensity is related to the concentration of the analyte to be measured, so as to quantitatively analyze the content of the target by chemiluminescence;

[0073] Taking the logarithm of the DON concentration as the abscissa and the absolute value of the change in light intensity ΔCL as the ordinate, a standard curve is made, as Figure 4 shown in

[0074] The principle of this DON detection method is as follows: Inside the hollow polymer sleeve for immunodetection, an immunocomplex reaction occurs among the DON complete antigen modified on the hollow polymer sleeve, DON primary antibody, and DON in the sample. When there is 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 concentration of DON in the sample is positively correlated with the absolute value of the change in the signal generated by the luminescent substrate, ΔCL, so as to determine the DON in the sample.

[0075] Or:

[0076] 1) The hollow polymer sleeve is incubated with vomitoxin antigen at 4 °C overnight, and then blocked with 5% BSA at 37 °C for 1 h (immunodetection of the hollow polymer sleeve). The hollow polymer sleeve for immunodetection is inhaled with 50 μL of DON-Ab and 50 μL of DON at different concentrations, incubated at 37 °C for 30 minutes, and then washed with PBST;

[0077] 2) 100 μL of Zr-MOF-Pt-HRP enzyme-labeled antibody is introduced into the above-mentioned hollow polymer sleeve and incubated at 37 °C for 40 minutes, and then washed with PBST; The catalytic luminescent substrate generates a signal, and combined with the CCD imaging system for quantitative analysis of the target. The light intensity is related to the concentration of the analyte to be measured, so as to quantitatively analyze the content of the target by chemiluminescence;

[0078] Taking the logarithm of the DON concentration as the abscissa and the absolute value of the change in light intensity ΔCL as the ordinate, a standard curve is made, as Figure 4 shown in

[0079] The principle of this DON detection method is as follows: In the immunodetection hollow polymer sleeve, an immunocompetitive reaction occurs among the DON complete antigen modified on the hollow polymer sleeve, the DON primary antibody, and DON in the sample. When there is more DON in the sample, the less the DON primary antibody binds to the DON complete antigen, and the less the Zr-MOF-Pt-HRP enzyme-labeled antibody that binds to the DON primary antibody. Therefore, the DON concentration in the sample is positively correlated with the absolute value of the signal change value △CL generated by the luminescent substrate, thereby determining the DON in the sample.

[0080] Or:

[0081] 1) The hollow polymer sleeve inhales the vomitoxin antigen and incubates overnight at 4°C, and then is blocked with 5% BSA at 37°C for 1 h (immunodetection hollow polymer sleeve). The immunodetection hollow polymer sleeve inhales 50 μL of DON-Ab and 50 μL of DON with different concentrations, and incubates at 37°C for 30 minutes, and then is washed with PBST;

[0082] 2) Introduce 100 μL of Zr-MOF-Pt-HRP-HRP enzyme-labeled antibody-initiation chain into the above-mentioned hollow polymer sleeve and incubate at 37°C for 40 minutes, and then wash with PBST;

[0083] 3) Introduce 100 μL of hybridization chain reaction amplification system into the above-mentioned hollow polymer sleeve and incubate at 37°C for 90 minutes, and then wash with PBST;

[0084] 4) Add SA-HRP solution to the above-mentioned hollow polymer sleeve and incubate at 37°C for 30 minutes, and then wash with PBST. Catalyze the luminescent substrate to generate a signal, and combine with the CCD camera system for quantitative analysis of the target. The light intensity is related to the concentration of the target to be measured, thereby quantitatively analyzing the content of the target by chemiluminescence;

[0085] Taking the logarithm of the DON concentration as the abscissa and the absolute value of the light intensity change value △CL as the ordinate, a standard curve is made, as Figure 4 shown in a;

[0086] The principle of this DON detection method is as follows: In the immunodetection hollow polymer sleeve, an immunocompetitive reaction occurs among the DON complete antigen modified on the hollow polymer sleeve, the DON primary antibody, and DON in the sample. When there is more DON in the sample, the less the DON primary antibody binds to the DON complete antigen, and the less the Zr-MOF-Pt-HRP-HRP enzyme-labeled antibody-initiation chain that binds to the DON primary antibody, and then the less the bound SA-HRP enzyme. Therefore, the DON concentration in the sample is positively correlated with the absolute value of the signal change value △CL generated by the luminescent substrate, thereby determining the DON in the sample.

[0087] Example 4 Colorimetric Detection of PCT

[0088] To verify that this method can meet the detection sensitivity requirements for different targets, a highly sensitive detection of procalcitonin (PCT), an inflammatory marker in human serum, was performed using a Zr-MOF-Pt-HRP enzyme-labeled antibody probe for dual signal amplification, as follows:

[0089] 1) The hollow polymer sleeve was filled with PCT capture antibody (PCT-Ab1) and incubated overnight at 4 °C, then blocked with 5% BSA at 37 °C for 1 h (immunodetection hollow polymer sleeve). The immunodetection hollow polymer sleeve was filled with 100 μL of PCT at different concentrations and incubated at 37 °C for 30 minutes, then washed with PBST;

[0090] 2) 100 μL of Zr-MOF-Pt-HRP enzyme-labeled antibody probe was introduced into the above hollow polymer sleeve and incubated at 37 °C for 30 minutes, then washed with PBST; then 100 μL of commercial TMB chromogenic solution was aspirated and reacted in the dark at room temperature for 10 min, and then injected into the detection well containing 50 μL of sulfuric acid to turn yellow and the result was photographed. The B value of the yellow color was analyzed to quantitatively analyze the target content;

[0091] With the logarithm of the PCT concentration as the abscissa and the absolute value of the change in the color B value △I B as the ordinate, a standard curve was made, as Figure 5 shown;

[0092] The standard addition method was used to detect the PCT residue in the serum sample. The process was the same as above, and the results are shown in Table 3.

[0093] The principle of this PCT detection method is as follows: Inside the immunodetection hollow polymer sleeve, the PCT-Ab1 modified on the hollow polymer sleeve undergoes an immunobinding reaction with PCT in the sample. The more PCT there is in the sample, the more PCT-Ab1 binds to PCT, and the more Zr-MOF-Pt-HRP enzyme-labeled antibody binds to the PCT-PCT-Ab1 complex. The increased sensitivity comes from the Pt NPs enriched by Zr-MOF and the HRP enzyme-labeled antibody. Therefore, the PCT concentration in the sample is positively correlated with the absolute value of the change in the color B value △I B of the chromogenic substrate to determine PCT in the sample.

[0094] Table 3. Results of detecting PCT in human serum by the standard addition method

[0095]

[0096] Example 5 Detection of interleukin 6 (IL6)

[0097] To verify that this method can meet the detection sensitivity requirements for different targets, the inflammatory marker interleukin-6 (IL6) in human serum was detected with high sensitivity using a Zr-MOF-Pt-HRP-HRP enzyme-labeled antibody-initiated chain probe as follows:

[0098] 1) The hollow polymer sleeve was filled with IL6 capture antibody (IL6-Ab1) and incubated overnight at 4 °C. Then, it was blocked with 5% BSA at 37 °C for 1 h (immunodetection hollow polymer sleeve). The immunodetection hollow polymer sleeve was filled with 100 μL of IL6 at different concentrations and incubated at 37 °C for 30 minutes, followed by washing with PBST;

[0099] 2) 100 μL of Zr-MOF-Pt-HRP-HRP enzyme-labeled antibody-initiated chain probe was introduced into the above hollow polymer sleeve and incubated at 37 °C for 40 minutes, followed by washing with PBST;

[0100] 3) 100 μL of the hybridization chain reaction amplification system was introduced into the above hollow polymer sleeve and incubated at 37 °C for 90 minutes, followed by washing with PBST;

[0101] 4) SA-HRP solution was added to the above hollow polymer sleeve and incubated at 37 °C for 30 minutes, followed by washing with PBST. 100 μL of commercial TMB chromogenic solution was added and the reaction was carried out in the dark at room temperature for 10 min. Then, it was injected into the detection well containing 50 μL of sulfuric acid to turn yellow, and the result was photographed. The B value of the yellow color was analyzed to quantitatively analyze the target content;

[0102] 5) The logarithm of the IL6 concentration was used as the abscissa, and the absolute value of the change in the color B value, ΔI B was used as the ordinate to make a standard curve, as Figure 6 shown;

[0103] 6) The standard addition method was used to detect the amount of IL6 in the serum sample. The process was the same as above, and the results are shown in Table 4.

[0104] The principle of this IL6 detection method is as follows: In the reaction device, IL6-Ab1 modified on the hollow polymer sleeve undergoes an immunobinding reaction with IL6 in the sample. The more IL6 there is in the sample, the more IL6-Ab1 binds to IL6, and the more Zr-MOF-Pt-HRP-HRP enzyme-labeled antibody-initiated chain probe binds to the IL6-IL6-Ab1 complex, and then the more SA-HRP enzyme binds. Therefore, the concentration of IL6 in the sample is positively correlated with the absolute value of the change in the color B value, ΔI B of the chromogenic substrate, and thus the IL6 in the sample can be determined.

[0105] Table 4. Results of detecting IL6 in human serum using the standard addition method

[0106]

[0107] The applicant declares that the technical solution of the present invention is explained by 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 those skilled in the art on the basis of the present invention, or equivalent replacements of the materials selected for the present invention, etc., all fall within the scope of patent protection.

Claims

1. A pipette immunosensing platform with different signal amplification capabilities based on a hollow polymer sleeve combined with Zr-MOF, characterized in that, Using a hollow polymer sleeve as a substrate and coating biorecognition molecules on the inner wall of the hollow polymer sleeve, the detection of various macromolecular and small molecular targets is realized; The operation method of the pipette immunosensing platform based on the different signal amplification capabilities of the hollow polymer sleeve combined with Zr-MOF is characterized in that: the method comprises the following steps: S1: The hollow polymer sleeve is incubated overnight with biorecognition molecules, washed, and blocked with bovine serum albumin in sequence; S2: The biorecognition molecule-modified hollow polymer sleeve obtained in step S1 sequentially aspirates the target to be detected for a biological reaction. After the reaction, the solution is discharged and the hollow polymer sleeve is washed. Zr-MOF-Pt-HRP enzyme-labeled antibody or Zr-MOF-Pt-HRP-HRP enzyme-labeled antibody-initiating strand is added for reaction. After washing the excessive Zr-MOF-Pt-HRP enzyme-labeled antibody or Zr-MOF-Pt-HRP-HRP enzyme-labeled antibody-initiating strand; S3: Aspirate the signal generation substrate to cause a color change and then terminate the reaction with sulfuric acid. Take a photo of the final color and analyze the color intensity with color analysis software. The color intensity is related to the concentration of the target to be detected. Or after adding the enzyme-labeled antibody for reaction, wash away the excessive enzyme-labeled antibody, and then aspirate the luminescent substrate to generate a signal. Combine with a CCD imaging system for quantitative analysis of the target. The light intensity is related to the concentration of the target to be detected.

2. The pipette immunosensing platform based on the different signal amplification capabilities of the hollow polymer sleeve combined with Zr-MOF according to claim 1, characterized in that: The biorecognition molecule is an antibody or antigen, DNA capture probe or DNA detection probe; The target includes viruses, biomarkers, antibiotic molecules, pesticide molecules, veterinary drug molecules, biotoxins, bacteria; The hollow polymer sleeve is made of polystyrene, polycarbonate, polyacrylamide, polymethyl methacrylate, polydimethylsiloxane or polyethylene terephthalate.

3. The pipette immunosensing platform based on a hollow polymer sleeve combined with Zr-MOF and having different signal amplification capabilities as described in claim 1, wherein: The antibody is a capture antibody or a detection antibody.

4. The pipette immunosensing platform based on a hollow polymer sleeve combined with Zr-MOF and having different signal amplification capabilities according to claim 1, wherein: The preparation method of the Zr-MOF-Pt-HRP enzyme-labeled antibody is as follows; Zr-MOF is synthesized as follows: Dissolve H2TCPP, ZrOCl2·8H2O and benzoic acid in DMF and add them to a glass bottle; stir to obtain the Zr-MOF product; and centrifuge, wash with DMF, and dry in a vacuum oven; Zr-MOF-Pt is synthesized by in-situ reduction method on Zr-MOF: Disperse Zr-MOF in water, then add H2PtCl6 to the suspension and stir. Subsequently, drop the NaBH4 solution into the suspension and stir. Finally, centrifuge the Zr-MOF-Pt product, wash with water, and finally disperse it in water to obtain Zr-MOF-Pt; Preparation of Zr-MOF-Pt-HRP enzyme-labeled antibody: Add HRP enzyme-labeled antibody to Zr-MOF-Pt for reaction; then, wash with PBS and centrifuge to obtain Zr-MOF-Pt-HRP enzyme-labeled antibody.

5. The pipette immunosensing platform based on hollow polymer sleeves combined with Zr-MOF with different signal amplification capabilities as described in claim 4, wherein: The Zr-MOF-Pt-HRP-HRP enzyme-labeled antibody-initiation strand is prepared as follows: incubate Zr-MOF-Pt with an HRP solution; next, add an HRP enzyme-labeled antibody and a phosphorylated hybridization chain reaction initiation strand DNA and incubate to obtain the Zr-MOF-Pt-HRP-HRP enzyme-labeled antibody-initiation strand.

6. The pipette immunosensing platform based on a hollow polymer sleeve combined with Zr-MOF with different signal amplification capabilities as claimed in claim 5, wherein: The hybridization chain reaction amplification system is a biotinylated double hairpin DNA.

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