Homogeneous visualization and bifluorescence signal analysis method based on multiple selective recognition reactions and application

A signal analysis method and fluorescence signal technology, which are applied in the field of homogeneous visualization and dual fluorescence signal analysis, and can solve problems such as difficult visualization and reading.

Active Publication Date: 2021-03-12
WEST CHINA HOSPITAL SICHUAN UNIV +1
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] One of the objectives of the present invention is to provide a homogeneous visualization and dual-fluorescence signal analysis method based on multiple selective recognition reactions to solve the problem of relying on a single signal molecule to quantify the target in the prior art, and it is difficult to read visually

Method used

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  • Homogeneous visualization and bifluorescence signal analysis method based on multiple selective recognition reactions and application
  • Homogeneous visualization and bifluorescence signal analysis method based on multiple selective recognition reactions and application
  • Homogeneous visualization and bifluorescence signal analysis method based on multiple selective recognition reactions and application

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Embodiment 1

[0039] This example provides the synthesis method of CdTe QDs.

[0040] First, 0.5mmol CdCl 2 and 0.20 g of trisodium citrate were dissolved in 50 ml of water, and 52 μL of mercaptopropionic acid (MPA) was added to the above solution. Using NaOH solution, adjust the pH of the above mixture solution to 10.5;

[0041] Then, add 0.1mmol Na 2 TeO 3 and 50mg KBH 4 Add to the above solution, reflux for 1 hour, until the solution is red, showing strong red fluorescence under the irradiation of ultraviolet light;

[0042] Finally, the CdTe QDs solution was purified by precipitation (using n-propanol) and centrifugation (11000 rpm, 30 min).

[0043] The MPA-CdTe QDs synthesized above were stored at 4 °C before use.

Embodiment 2

[0045] The present embodiment provides the analysis steps of PPase, specifically as follows:

[0046] (1) Add 77 μL Tris-HCl buffer (pH=7.4, 10 mM, 500 mM NaCl, 100 mM MgCl 2 ), 10 μL pyrophosphate (PPi, 17.5 mM) and 50 μL pyrophosphatase (PPase) of different concentrations, incubated at 37°C for 0.5h to complete the hydrolysis reaction;

[0047] (2) Subsequently, 10 μL of CuCl was added to the centrifuge tube 2 Solution (100μM), continue to incubate at 37°C for 0.5h to complex copper ions and PPi;

[0048] (3) Next, add 2 μL Ce(NO 3 ) 3 Solution (0.5mM) and 1μL CdTe QDs stock solution, reacted at room temperature for 12min to complete the cation exchange reaction and coordination complexation reaction;

[0049] (4) Transfer the above solution to a cuvette, use a fluorometer to test and record the data (excitation wavelength: 295nm, emission wavelength range: 310nm-800nm).

Embodiment 3

[0051] This embodiment provides PPase analysis-excitation wavelength selectivity and selectivity identification phenomenon verification, as follows:

[0052] Considering that the optimal excitation wavelength required for PPi-Ce CPNs is 295nm and the optimal excitation wavelength for CdTe QDs is 365nm, the excitation wavelength of the dual fluorescence signal analysis system was selected first. Such as figure 2 As shown in A, when excited at 295nm, both PPi-Ce CPNs and QDs have corresponding emission peak shapes, but when excited at 365nm, PPi-Ce CPNs do not emit a peak at 348nm, but show a peak shape at the excitation wavelength of 365nm.

[0053] In summary, the final selectivity of 295nm is the excitation wavelength of the system.

[0054] At the same time, it was further verified that different concentrations of PPi-Cu 2+ -The effect of PPi complex on the fluorescence signal of QDs, and the effect of different PPi on Ce 3+ Influence of fluorescent signal. Such as fi...

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Abstract

The invention provides a homogeneous visualization and bifluorescence signal analysis method based on multiple selective recognition reactions, which relates to the technical field of diagnosis analysis. The method comprises the steps of selectively recognizing Cu<2+> and a compound formed by Cu<2+> and PPi based on QDs, and selectively recognizing PPi and other phosphates through Ce<3+>, so as toobtain a fluorescence signal of QDs and a fluorescence signal of PPi-Ce CPNs, and quantifying a single target object based on the fluorescence signals of the QDs and the fluorescence signals of the PPi-Ce CPNs. According to the method, PPi-Ce CPNs and QDs are used as double fluorescence signal molecules to quantitatively analyze a target object, so that the accuracy of the method is improved, andmeanwhile, a luminescent nano material Ce<3+> is introduced into biochemical and medical diagnosis to realize visual POCT analysis. The analysis method provided by the invention can be used for analyzing pyrophosphatase or alkaline phosphatase.

Description

technical field [0001] The invention relates to the technical field of biomedical diagnostic analysis methods, in particular to a homogeneous visualization and dual fluorescence signal analysis method and application based on multiple selective recognition reactions. Background technique [0002] In the existing biochemical and medical diagnostic systems, the quantitative analysis of a single target is mainly based on the signal intensity of a single signal molecule. For example, the enzyme-linked immunosorbent assay (ELISA) based on the immune recognition reaction uses a UV-visible spectrophotometer to monitor the ultraviolet signal, an electrochemiluminescence strategy to monitor the electrochemical signal of ruthenium tripyridin, etc., and a fluorescence strategy to monitor the fluorescence signal of the fluorescent dye (such as FITC, FAM, etc.) etc. [0003] Although, a small number of researchers use dual signal molecules to achieve target analysis, which mainly uses o...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/64
CPCG01N21/6428G01N21/6486G01N2021/6432
Inventor 陈飘飘应斌武瞿润连何雅秦
Owner WEST CHINA HOSPITAL SICHUAN UNIV
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