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Substrate concentration control type time-resolved homogeneous chemiluminescence biological detection method

A homogeneous chemiluminescence and biological detection technology, applied in the field of biochemical analysis, can solve the problems that the detection results cannot truly reflect the content of the analyte, no enzymatic signal amplification, and complex detection signal interference, etc., so as to improve the detection accuracy and Linear range, reduce the interference of non-specific signals, and facilitate the effect of on-site detection

Pending Publication Date: 2021-06-04
HUAZHONG AGRI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

This method can detect chlorpyrifos under the homogeneous condition that does not wash and separate, but also has following shortcoming: (1) detection accuracy is low
The markers in the solution that did not participate in the immunocompetitive reaction were added to the H 2 o 2 Finally, a non-specific detection signal will be generated, which will interfere with the detection signal of the complex, resulting in the detection result not being able to truly reflect the content of the analyte.
(2) The chemiluminescent reaction is carried out under alkaline conditions, the reaction rate is poorly controllable, and the stability is not good; (3) There is no signal amplification by the enzyme, resulting in low sensitivity and other problems

Method used

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  • Substrate concentration control type time-resolved homogeneous chemiluminescence biological detection method

Examples

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Effect test

Embodiment 1

[0052] Example 1 Verification of substrate concentration-controlled time-resolved homogeneous chemiluminescence biochemical analysis method

[0053] With HRP-H 2 o 2 - acridinium ester system as an example to verify the substrate concentration-controlled time-resolved homogeneous chemiluminescent biosensing theory. First, a sufficient amount of chemiluminescence substrate acridinium ester is mixed with a small amount of HRP (ratio of the amount of substances is 50000:1), by adding a sufficient amount of H 2 o 2 Trigger the chemiluminescence reaction, and record the chemiluminescence kinetic curve. Such as figure 1 Shown, adding H 2 o 2 Afterwards, the chemiluminescent reaction proceeds immediately, and its rate gradually reaches and maintains the maximum, reflecting that the chemiluminescent signal intensity gradually increases and finally reaches a plateau. At this time, the reaction rate only depends on the concentration of HRP, and in the reaction, HRP is the catalyst...

Embodiment 2

[0055] Example 2 Detection of Pesticide Residual Chlorpyrifos

[0056] According to the substrate concentration-controlled time-resolved homogeneous chemiluminescence theory, if the chemiluminescent reaction rate is too fast, it will lead to the enhancement and early generation of non-specific signals, thereby interfering with the specific signal; if the reaction rate is too slow, it will lead to specificity. The signal tails and thus overlaps with non-specific signals, again detrimental to time resolution. Therefore, it is very important to select a chemiluminescent system with a moderate reaction rate. Since different chemiluminescent systems have different chemical reaction kinetics, the common HRP-H 2 o 2 -Acridinium ester, HRP-H 2 o 2 -luminol, ALP-AMPPD and other chemiluminescent systems, the time resolution effects of specific and non-specific signals were investigated. Such as Figure 4 shown, for HRP-H 2 o 2 -luminol system, because its reaction rate is too fa...

Embodiment 3

[0075] Example 3 Detection of inflammatory marker procalcitonin

[0076] Procalcitonin is a polypeptide and is analyzed by the sandwich method. The principle is as follows: firstly, the capture antibody of procalcitonin is modified by the chemiluminescent substrate acridinium ester, then mixed with the HRP-labeled detection antibody, added to the sample to be tested for biorecognition reaction, the capture antibody, calcitonin The original and detection antibodies will form a sandwich complex, and the concentration of acridinium ester in the sandwich complex and the HRP in the surrounding microenvironment is higher than that of the solution body. Introduce sufficient trigger H without any washing steps 2 o 2 , HRP in the sandwich complex will catalyze H 2 o 2 A large number of oxidative products are produced, which will react rapidly with the acridinium ester in the surrounding microenvironment, and the luminescent substrate acridinium ester is completely consumed, produci...

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Abstract

The invention discloses a substrate concentration control type time-resolved homogeneous chemiluminescence biochemical analysis method. The method comprises the following steps: (1) respectively marking a catalyst and a luminescent substrate on a biological recognition molecule; (2) mixing the biological recognition molecule solution marked by the catalyst and the biological recognition molecule solution marked by the luminous substrate, then adding the solution of the substance to be detected, and carrying out biological recognition reaction to generate a compound; and (3) adding a triggering agent into the reaction system, and carrying out quantitative analysis on the to-be-detected object by reading the specific flash signal intensity. According to the invention, separation of specific and non-specific light-emitting signals in a homogeneous system is realized by controlling the concentration of the light-emitting substrate, so that interference of the non-specific signals is reduced, a washing step is not needed, and the detection accuracy is improved.

Description

technical field [0001] The invention belongs to the field of biochemical analysis, and relates to an analysis detection method, in particular to a homogeneous chemiluminescent biological detection method. Background technique [0002] Biochemical analysis technology is playing an increasingly important role in various sectors of the national economy, such as food safety, disease diagnosis, and environmental monitoring. In recent years, a research hotspot in the field of biochemical analysis is how to simplify the measurement process while achieving high-sensitivity detection, which is crucial to improving detection efficiency. [0003] Chemiluminescence has high sensitivity and wide linear range, and is widely used in the field of analysis and testing. In particular, chemiluminescent biosensors have both the high sensitivity of chemiluminescence and the high specificity of biorecognition reactions, and are suitable for the quantitative analysis of trace targets in complex s...

Claims

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Application Information

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IPC IPC(8): G01N33/569G01N33/74G01N33/535G01N33/532G01N33/53G01N21/76C12Q1/6818C12Q1/10C12Q1/06C12R1/42
CPCG01N33/56916G01N33/74G01N33/532G01N33/535G01N33/5308G01N21/76C12Q1/6818G01N2333/255G01N2333/585G01N2430/10C12Q2563/103
Inventor 陈翊平何慧禹聂荣彬
Owner HUAZHONG AGRI UNIV