CN103969445B - The preparation of heme-manganese dioxide composites and the method for detecting human IgG thereof - Google Patents

Abstract

The invention discloses the preparation of heme-manganese dioxide complex and its method for detecting human IgG. In the present invention, the heme is dissolved in the phosphate buffer solution under the action of ammonia water, and the MnAc ₂ solution and the NaOH solution are added in turn to react to obtain the heme-manganese dioxide complex (Hemin-MnO ₂ ); Hemin- MnO ₂ has peroxidase activity, which can catalyze hydrogen peroxide to oxidize TMB to turn it from colorless to blue. Through the traditional "sandwich" sandwich structure method, Hemin-MnO ₂ is used as a rabbit anti-human IgG marker to conduct human For the detection of immunoglobulin IgG antigen, the colorimetric immunoassay using Hemin- _MnO2 has the advantages of good stability, high sensitivity, good selectivity, excellent reproducibility, and fast analysis speed; the immunosensing method is simple and inexpensive It is cheap, its color change can be observed with naked eyes, and has wide application prospects.

Description

Preparation of heme-manganese dioxide complex and its method for detecting human IgG

technical field

The invention relates to the preparation of a heme-manganese dioxide complex and a method for detecting human IgG, belonging to the field of biosensing.

Background technique

In recent years, the construction and application of new biosensing technologies have developed rapidly, and have been widely used in early clinical diagnosis, environmental monitoring, food testing and other fields. However, many sensing methods and their applications cannot be popularized in many developing countries due to the limitations of instruments and related technical professionals. Therefore, building a simple, economical sensing method that does not require the use of sophisticated instruments and equipment has become a hot research field. The biological immunosensing method based on color change is favored by many scientific researchers because of its rapid response, convenience, economy, and simple operation.

Existing methods for quantitative detection of human IgG include electrochemical detection, fluorescence detection, chemiluminescence and the like. However, these methods rely on certain instruments and equipment, and cannot detect multiple samples at the same time, which takes a long time.

Contents of the invention

The object of the present invention is to provide a method for preparing a heme-manganese dioxide complex with stable structure and peroxidase activity, which is simple and low in cost.

Another object of the present invention is to provide a method for detecting human IgG based on the heme-manganese dioxide complex, which utilizes the heme-manganese dioxide complex to catalyze the oxidation of 3,3' ,5,5'-Tetramethylbenzidine (TMB) produces a color change and realizes the detection of human IgG. Compared with the traditional enzyme-linked immunosorbent method, this method does not need to use enzymes as antibody markers, and has good stability and simplicity. , high sensitivity, good selectivity, low price and other advantages, realize the high-throughput detection of IgG in human body.

The invention provides a preparation method of a heme-manganese dioxide complex. The preparation method is to mix the heme with a phosphate buffer solution, add ammonia water to promote the dissolution of the heme, and then mix the heme in the mixed solution. Add excess MnAc ₂ solution and excess NaOH solution in turn, stir evenly, and after centrifugation, remove unreacted MnAc ₂ and NaOH, that is.

The preparation method of the heme-manganese dioxide complex of the present invention includes the following preferred schemes:

The centrifugation treatment is centrifugation at a speed of 8000-10000 rpm for 15-25 minutes.

The pH of the phosphate buffer solution is 7.4.

The prepared heme-manganese dioxide complex PBS buffer solution is washed 2 to 3 times, and stored in an environment not higher than 4°C for future use.

The present invention also provides a method for detecting human IgG based on the heme-manganese dioxide complex. The method is to first fix goat anti-human IgG on the bottom surface of each well of a polystyrene 96-well plate, and use bovine Serum albumin seals the bottom surface of each well; and then assembles different molar amounts of human IgG on the bottom surface of each well sealed by bovine serum albumin, and then further assembles the heme-manganese dioxide composite After the assembly is completed, add an equal amount of citrate buffer containing H ₂ O ₂ and 3,3',5,5'-tetramethylbenzidine to each well for reaction, and the reaction is complete Afterwards, the absorbance value at 652 nm of the oxidation product of 3,3',5,5'-tetramethylbenzidine in each well was measured with a microplate reader for colorimetric analysis.

The method for detecting human IgG based on the heme-manganese dioxide complex of the present invention also includes the following preferred schemes:

The preferred method is to add goat anti-human IgG to each well of a polystyrene 96-well plate and let it stand for 8 to 12 hours to remove excess goat anti-human IgG, then add bovine serum albumin to each well and let stand 2~3h, remove excess bovine serum albumin; then add equal volumes of human IgG solutions of different concentrations to each well, incubate at 37°C for 0.8~1.2h, remove the remaining human IgG solution in each well, and then Add heme-manganese dioxide complex-modified rabbit anti-human IgG to each well, incubate at 37°C for 1.2-1.7h, and remove the remaining heme-manganese dioxide complex-modified rabbit in each well. Anti-human IgG; finally, add an equal amount of citrate buffer containing H ₂ O ₂ and 3,3',5,5'-tetramethylbenzidine to each well for 10-20 minutes of reaction, and use a microplate reader to The absorbance value at 652 nm of the oxidation product of 3,3',5,5'-tetramethylbenzidine in each well was measured for colorimetric analysis.

The rabbit anti-human IgG modified by the heme-manganese dioxide complex is prepared by the following method, placing the heme-manganese dioxide complex in chitosan solution and glutaraldehyde solution in sequence React with rabbit anti-human IgG and centrifuge to obtain it.

The heme-manganese dioxide complex is sequentially placed in 0.3-0.8wt% chitosan solution to react for 1.8-2.2h, centrifuged for 3-7min, and placed in 0.2-0.3wt% glutaraldehyde solution React for 0.4~0.6h, centrifuge for 3~7min, put in 40~60μg mL ^-1 rabbit anti-human IgG for 0.8~1.2h, after centrifugation, disperse the centrifuge in phosphate buffer solution to obtain heme— Manganese dioxide complex modified rabbit anti-human IgG solution. The pH value of the phosphate buffer solution is preferably pH=7.4. The concentration of the rabbit anti-human IgG solution modified by the heme-manganese dioxide complex is 1 mg mL ^-1 .

Beneficial effects of the present invention: the present invention synthesized the heme-manganese dioxide complex (Hemin-MnO ₂ ) through a simple method for the first time, and found that it has peroxidase activity and can catalyze the oxidation of 3,3', 5,5'-tetramethylbenzidine (TMB) makes it change from colorless to blue; the application of the present invention further applies the heme-manganese dioxide complex to the detection of human IgG as an antibody marker, which has With high sensitivity, the color change can be discerned with the naked eye. The present invention adopts the traditional "sandwich" sandwich structure method, immobilizes goat anti-human IgG on a polystyrene 96-well microtiter plate, and uses the synthesized Hemin- _MnO2 complex as a rabbit anti-human IgG marker to carry out human immunization In the detection of globulin IgG antigen, it was found that as the concentration of the antigen increased, the color of the final oxidized product also changed regularly (colorless-light blue-dark blue), and the quantification of human IgG can be realized by means of the absorbance value measured by a microplate reader analyze. Compared with the traditional enzyme-linked immunoassay in the prior art, the colorimetric immunoassay using the Hemin-MnO ₂ complex does not need to use enzymes as antibody markers, and has good stability, high sensitivity, good selectivity, and excellent reproducibility , analysis speed and so on. The immunosensing method is simple, inexpensive, and its color change can be observed with naked eyes, and has potential application prospects in biotechnology, bioassay and biomedicine.

Description of drawings

[Fig. 1] is a transmission electron microscope image of the Hemin-MnO ₂ composite prepared in Example 1 of the present invention.

[Fig. 2] is the energy dispersive X-ray spectrogram of the Hemin-MnO ₂ composite prepared in Example 1 of the present invention.

[Fig. 3] is the Hemin-MnO ₂ composite prepared in Example 1 of the present invention and Hemin, MnO ₂ and TMB catalyzed hydrogen peroxide oxidation TMB color change chart and the corresponding absorbance-wavelength curve; 1 is TMB; 2 is MnO ₂ , 3 is Hemin, and 4 is Hemin-MnO ₂ complex; the color change chart corresponds to curves 4, 3, 2 and 1 from top to bottom.

[Fig. 4] Hemin- _MnO complex catalyzed oxidation equivalent TMB color change diagram and corresponding absorbance-wavelength curve diagram for different concentrations prepared in Example 1 of the present invention; Curves 9, 8, 7, 6 and 5 Corresponding to the Hemin-MnO ₂ complex concentration from high to low, the color change diagram corresponds to 9, 8, 7, 6 and 5 from top to bottom.

[ FIG. 5 ] is a standard curve diagram of the Hemin-MnO ₂ complex prepared in Example 1 of the present invention using the immunosensor modified by the double-antibody sandwich method to measure different concentrations of human IgG antigen.

Detailed ways

The following examples are intended to further illustrate the content of the present invention, but not to limit the protection scope of the present invention.

Example 1

Synthesis of a novel heme-manganese dioxide complex (Hemin-MnO ₂ ): mix 15 mg of Hemin with 10 mL of PBS buffer solution with pH=7.4, add 50 μL of ammonia water, stir to dissolve, and then add 1.6 mL of 100mM MnAc ₂ solution, stirred at room temperature for 5min, then added 100μL of 1M NaOH solution, continued to stir for 6-8h, and finally obtained the Hemin-MnO ₂ complex, and then centrifuged at 9000rpm for 20min to remove excess MnAc ₂ and NaOH, and then Wash with PBS buffer solution for 2-3 times, and finally store the product obtained by centrifugation in a refrigerator at 4°C for future use.

Hemin-manganese dioxide complex modified rabbit anti-human IgG synthesis method of the present invention: the synthesized Hemin- _MnO complex is dispersed in 0.5wt% chitosan solution, reacted for 2h, and centrifuged for 5min; 0.25wt% glutaraldehyde was dispersed for 0.5h, centrifuged for 5min; then reacted with 50μg mL ^-1 rabbit anti-human IgG for 1h, centrifuged; finally dispersed in PBS with pH=7.4 to prepare 1mg mL ^-1 The solution was stored in a refrigerator at 4 °C for later use.

The heme-manganese dioxide complex of the present invention is used in the immunoassay to detect immunoglobulin IgG. The detection method: add 150 μL goat anti-human IgG (diluted to 10 μg mL ^-1 with buffer) into polystyrene 96 wells Incubate overnight at 4°C in a microtiter plate to immobilize the antibody into the wells. The unfixed goat anti-human IgG solution was washed three times with 300 μL washing buffer to wash away. Then at 37°C, 250 μL of blocking buffer was added and incubated for 2.5 hours to reduce the non-specific adsorption of human IgG, and excess BSA was washed three times with 300 μL of washing buffer. Prepare different concentrations of IgG solutions (0, 1, 2, 5, 10, 50, 100, 500, 1000 pg mL ^-1 ) with PBS solution at pH = 7.4, and then add 150 μL of human IgG solutions with different concentrations to wells, incubated for 1 h, and washed three times. Then 150 μL of Hemin-MnO ₂ modified rabbit anti-human IgG was added and incubated for 1.5 h. Wash four times with 300 μL of wash buffer. Finally, add 150 μL of 2.5 mM TMB prepared in citrate buffer containing 4.3 mM hydrogen peroxide to each well, react for 15 min, and then use a microplate reader to measure the absorbance of TMB oxidation products. The prepared standard curve is shown in Figure 5 .

Example 2

Using the heme-manganese dioxide complex for clinical immunoassay detection to measure the recovery rate of human IgG in human serum: the human serum sample is diluted to ^10-10 times with PBS buffer solution of pH=7.4, and then Human IgG solutions at different concentrations of 0pg mL ^-1 , 10pg mL ^-1 , 50pg mL ^-1 , 100pg mL ^-1 and 500pg mL ^-1 were added. Taking the pure serum solution added with 0 pg mL ^-1 human IgG as blank reference solution, the absorbance value of the TMB oxidation product was measured according to the specific operation of Example 1, and the recovery rate obtained is shown in Table 1. From the data in Table 1, it can be seen that human IgG The recovery rate is high, which can prove the feasibility and accuracy of the detection method in Example 1.

Table 1. Determination of the recovery rate of human IgG in human serum (pg mL ^-1 )

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Claims (6)

1. detect the method for human IgG based on heme-manganese dioxide composites, it is characterized in that, first fix goat anti-human igg at the basal surface in each hole of polystyrene 96 orifice plate, and close with the basal surface of bovine serum albumin(BSA) by each hole described; After basal surface again in each hole that described bovine serum albumin(BSA) was closed assembles the human IgG of different molar weight respectively, assemble heme-manganese dioxide composites further and modify rabbit anti-human igg; After having assembled, what in each hole, add equivalent contains H ₂o ₂react with the citrate buffer solution of TMB, after having reacted, measure the absorbance of oxidation product at 652nm of TMB in each hole by microplate reader, carry out colorimetric analysis;

Described heme-manganese dioxide composites is obtained by following preparation method: after being mixed with phosphate buffered solution by heme, adds ammoniacal liquor and impels heme to dissolve, then add excessive MnAc successively in mixed solution ₂solution and excessive NaOH solution, stir, then after centrifugal treating, the MnAc that removing unreacted is complete ₂and NaOH, to obtain final product.

2. the method for claim 1, is characterized in that, described centrifugal treating is centrifugal 15 ~ 25min under the speed of 8000 ~ 10000rpm.

3. the method for claim 1, is characterized in that, described phosphate buffered solution pH is 7.4.

4. the method for claim 1, it is characterized in that, in each hole of polystyrene 96 orifice plate, first add goat anti-human igg leave standstill 8 ~ 12h, remove unnecessary goat anti-human igg, in each described hole, add bovine serum albumin(BSA) again leave standstill 2 ~ 3h, remove unnecessary bovine serum albumin(BSA); The human IgG solution of equal-volume variable concentrations is added again respectively in each hole, incubation 0.8 ~ 1.2h at 37 DEG C, remove the residue human IgG solution in each hole, in each hole, add heme-manganese dioxide composites again modify rabbit anti-human igg, incubation 1.2 ~ 1.7h at 37 DEG C, the residue heme-manganese dioxide composites removed in each hole modifies rabbit anti-human igg; What in each hole, finally add equivalent contains H ₂o ₂after carrying out reaction 10 ~ 20min with the citrate buffer solution of TMB, measure the absorbance of oxidation product at 652nm of TMB in each hole by microplate reader, carry out colorimetric analysis.

5. the method for claim 1, it is characterized in that, described heme-manganese dioxide composites is modified rabbit anti-human igg and is prepared by the following method, described heme-manganese dioxide composites is placed in successively chitosan solution, glutaraldehyde solution and rabbit anti-human igg reaction, centrifugal treating, to obtain final product.

6. the method for claim 1, it is characterized in that, described heme-manganese dioxide composites is placed in the chitosan solution reaction 1.8 ~ 2.2h of 0.3 ~ 0.8wt% successively, centrifugal 3 ~ 7min, 0.4 ~ 0.6h is reacted in the glutaraldehyde solution of 0.2 ~ 0.3wt%, centrifugal 3 ~ 7min, in 40 ~ 60 μ g mL ^-1rabbit anti-human igg in 0.8 ~ 1.2h, after centrifugal treating, centrifugal thing is distributed in phosphate buffered solution, obtains heme-manganese dioxide composites and modify rabbit anti-human igg's solution.