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A no-cleaning universal ELISA fluorescent immunoprobe and its preparation method and application

A fluorescent immune, general-purpose technology, applied in the field of analysis and inspection, can solve problems such as complex chemical reaction mechanisms, and achieve the effects of simple production method, fast reaction, and simple operation

Inactive Publication Date: 2021-07-09
NORTHEASTERN UNIV LIAONING
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these newly developed methods usually require complex chemical reaction mechanisms, so further improvement and refinement of these methods are required for widespread application

Method used

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  • A no-cleaning universal ELISA fluorescent immunoprobe and its preparation method and application
  • A no-cleaning universal ELISA fluorescent immunoprobe and its preparation method and application
  • A no-cleaning universal ELISA fluorescent immunoprobe and its preparation method and application

Examples

Experimental program
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Effect test

Embodiment 1

[0043] A method for preparing a no-cleaning universal ELISA fluorescent immunoprobe, comprising the following steps:

[0044] (1) g-C 3 N 4 Synthesis of powder: Weigh 3g of dicyandiamide into a 50mL alumina crucible, cover the lid and wrap it with aluminum foil, put it in a muffle furnace, and heat it at 3°C ​​min -1 The speed is raised to 550 ° C and maintained for 2 hours; the reactant is cooled to room temperature and taken out, and the obtained light yellow solid is g-C 3 N 4 , using an agate mortar and mortar to place the g-C 3 N 4 Grind into uniform solid powder and set aside;

[0045] (2) Boronic acid modification: Take 166mg of 4-carboxyphenylboronic acid and 400mg of HBTU in 30mL of anhydrous DMF, stir to dissolve, and continue to stir at 50°C for 30min to obtain a mixture; then add 100mg of the g-C 3 N 4 and 0.5mL DIEA, and continue to stir at 50°C for 12h to complete the reaction; the reacted product was collected by centrifugation, washed with absolute etha...

Embodiment 2

[0050] A kind of no-cleaning universal ELISA fluorescent immunoprobe prepared by the method of Example 1 is applied to the detection of targets with specific antibodies. The specific experiment is for three cardiac markers in human serum, troponin I (cTnI), Myoglobin (Mb) and creatine kinase isoenzyme MB (CK-MB) were detected.

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

[0052] Preparation: Using the method of Example 1, a kind of no-cleaning universal ELISA fluorescent immunoprobe Ab was prepared using cTnI antibody cTnI -BCNNS.

[0053] Detection method and result: to Ab cTnI -Add different concentrations of cTnI directly to BCNNS, mix well and incubate in a water bath at 37°C for 20 minutes, then measure the fluorescence spectrum of the mixture under excitation at 330nm to obtain figure 2 , as can be seen from the figure, with the increase of cTnI concentration (0-100ng mL -1 ), the fluorescence intensity of the system gradually decreased. Record the fluorescence inte...

Embodiment 3

[0067] A no-wash universal ELISA fluorescent immunoprobe prepared by the method in Example 1 was applied to detect cTnI, Mb and CK-MB in human serum.

[0068] Under optimal conditions, to Ab cTnI - Add different human serum samples (can be divided into three groups according to negative, low positive, high positive, four samples in each group, 12 samples in total) in BCNNS, measure the fluorescence spectrum of the mixture under the same conditions, and record at 440nm Fluorescence intensity, and calculate the content of cTnI in each human serum sample. At the same time, the same batch of serum samples was tested with the commercialized cTnI ELISA kit. The test value of the serum sample with the cTnI ELISA kit was on the abscissa, and the test value of the serum sample by the fluorescence immunoassay method in this experiment was The ordinate, get the linear correlation diagram of the two methods ( Figure 12 ), it can be seen from the figure that the correlation between the ...

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Abstract

The invention provides a no-cleaning universal ELISA fluorescent immunoprobe and its preparation method and application, which is characterized in that carboxyphenylboronic acid is modified to g-C by HBTU / DIEA coupling reaction 3 N 4 After ultrasonic stripping, weakly fluorescent boric acid-modified graphitic carbon nitride nanosheets (BCNNS) can be obtained, and then the antibody (Ab) is labeled on the BCNNS, and the strong fluorescent Ab‑BCNNS composite (ON) can be obtained , with the addition of the target antigen, the fluorescence of the Ab-BCNNS composite material is quenched (OFF), based on this ON-OFF phenomenon, the specific detection of the target antigen can be achieved; and by changing the Ab-BCNNS Labeled antibodies can achieve selective detection of different target antigens.

Description

technical field [0001] The invention belongs to the technical field of analysis and testing, and relates to an immunodetection method, in particular to a no-cleaning universal ELISA fluorescent immunoprobe and its preparation method and application. Background technique [0002] Biomarkers generally refer to biochemical indicators of certain characteristics in the physiological, pathological, and therapeutic processes that can be objectively measured and evaluated. Through the determination of biomarkers, the progress of the current biological process of the body can be known. The examination of biomarkers can play a certain role in the identification of diseases, early diagnosis and prevention, and monitoring of the treatment process. [0003] Therefore, finding and discovering valuable biomarkers has become a research hotspot in related fields, and in the process of biomarker research, people find that the key problem is: how to improve the accuracy of biomarker detection...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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 LIAONING
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