Cleaning-free universal ELISA fluorescence immunoassay probe and preparation method and application thereof

A general-purpose technique of fluorescence immunity, which is applied in the field of analysis and testing, can solve problems such as complex chemical reaction mechanisms, and achieve the effects of simple production methods, rapid reactions, and simple and fast detection methods

Inactive Publication Date: 2020-06-09
NORTHEASTERN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, these newly developed methods usually require complex chemical reaction mechanism...

Method used

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  • Cleaning-free universal ELISA fluorescence immunoassay probe and preparation method and application thereof
  • Cleaning-free universal ELISA fluorescence immunoassay probe and preparation method and application thereof
  • Cleaning-free universal ELISA fluorescence immunoassay probe and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] A preparation method of a no-clean universal ELISA fluorescent immune probe includes the following steps:

[0044] (1)g-C 3 N 4 Synthesis of powder: Weigh 3g of dicyandiamide into a 50mL alumina crucible, cover it and wrap it with aluminum foil, and put it in a muffle furnace at 3℃min -1 Raise the temperature to 550℃ and keep it for 2h; when the reactant is cooled to room temperature, take it out, the obtained light yellow solid is g-C 3 N 4 , Use an agate mortar to combine the g-C 3 N 4 Grind into uniform solid powder for use;

[0045] (2) Boric acid modification: Take 166 mg of 4-carboxyphenylboronic acid and 400 mg of HBTU in 30 mL of anhydrous DMF, stir to dissolve, and continue to stir at 50°C for 30 minutes to obtain a mixture; then add 100 mg of the mixture to the mixture gC 3 N 4 And 0.5mL DIEA, continue to stir at 50℃ for 12h to complete the reaction; the product after the reaction is collected by centrifugation, and washed 2-3 times with absolute ethanol and seconda...

Embodiment 2

[0050] The method of Example 1 will be used to prepare a no-clean general-purpose ELISA fluorescent immunological probe for the detection of targets with specific antibodies. The specific experiment is to test three myocardial markers in human serum, troponin I (cTnI), Myoglobin (Mb) and creatine kinase isoenzyme MB (CK-MB) were tested.

[0051] 1. Detection of troponin I (cTnI)

[0052] Preparation: The method of Example 1 was used to prepare a no-clean universal ELISA fluorescent immunoprobe Ab using cTnI antibody cTnI -BCNNS.

[0053] Detection methods and results: to Ab cTnI -Directly add cTnI of different concentrations to BCNNS, mix well and incubate in a 37℃ water bath for 20 minutes, and then measure the fluorescence spectrum of the mixture under excitation at 330nm to obtain figure 2 , As can be seen from the figure, as the concentration of cTnI increases (0-100ng mL -1 ), the fluorescence intensity of the system gradually decreases. Record the fluorescence intensity of ...

Embodiment 3

[0067] The method of Example 1 was used to prepare a no-clean universal ELISA fluorescent immunological probe and applied to the detection of cTnI, Mb and CK-MB in human serum.

[0068] Under optimal conditions, to Ab cTnI -BCNNS adds different human serum samples (can be divided into three groups according to negative, low positive, high positive, four samples in each group, a total of 12 samples), measure the fluorescence spectrum of the mixed solution under the same conditions, and record at 440nm And calculate the cTnI content in each human serum sample. At the same time, use the commercial cTnI enzyme-linked immunoassay kit to test the same batch of serum samples. The test value of the cTnI enzyme-linked immunoassay kit for the serum sample is the abscissa, and the test value of the serum sample by the fluorescence immunoassay in this experiment is On the ordinate, the linear correlation diagram of the two methods is obtained ( Picture 12 ), it can be seen from the figure ...

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Abstract

The invention provides a cleaning-free universal ELISA fluorescence immunoassay probe as well as a preparation method and application thereof. The probe is characterized in that through an HBTU/DIEA coupling reaction, carboxyphenylboronic acid is modified onto g-C3N4, after ultrasonic exfoliation, a weak-fluorescence boric acid modified graphite phase carbon nitride nanosheet (BCNNS) can be obtained; an antibody (Ab) is labeled on the BCNNS to obtain a strong-fluorescence Ab-BCNNS composite material (ON), the fluorescence of the Ab-BCNNS composite material is quenched (OFF) along with additionof a target antigen, and the specific detection of the target antigen can be realized based on the ON-OFF phenomenon. And moreover, by changing the labeled antibody in the Ab-BCNNS, the selective detection of different target antigens can be realized.

Description

Technical field [0001] The invention belongs to the technical field of analysis and inspection, and relates to an immunological detection method, in particular to a non-cleaning universal ELISA fluorescent immunological probe, and a preparation method and application thereof. Background technique [0002] Biomarkers generally refer to biochemical indicators of a certain characteristic in the course of physiology, pathology, and treatment that can be objectively measured and evaluated. Through the measurement of biomarkers, the progress of the biological process of the body can be known. The examination of biomarkers can play a certain role in disease identification, early diagnosis and prevention, and monitoring of the treatment process. [0003] Therefore, searching for and discovering valuable biomarkers has become a research hotspot in related fields. In the process of biomarker research, people have discovered the key question: how to improve the accuracy of biomarker detectio...

Claims

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Application Information

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IPC IPC(8): G01N33/533G01N33/543G01N33/58G01N33/68G01N33/573G01N33/574G01N33/53
CPCG01N33/5308G01N33/533G01N33/54353G01N33/573G01N33/57473G01N33/582G01N33/6887G01N2333/4712G01N2333/805G01N2333/9123
Inventor 杨婷王怡婷王建华
Owner NORTHEASTERN UNIV
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