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High-sensitivity chemiluminescence immune assay method based on analogue enzyme labeled silver nanoparticles

A technology of silver nanoparticles and immunoassay, which is applied in the field of high-sensitivity chemiluminescence immunoassay based on imitating enzyme-labeled silver nanoparticles, can solve the problems of harsh conditions for synthesis or biomodification, difficulties for non-professionals, complicated steps, etc., and achieve The effect of simple equipment and operation, good storage stability and low cost

Inactive Publication Date: 2017-07-21
CHINA PHARM UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, common protein array chips are mostly prepared by photolithography, dot matrix or printing technology, the steps are very complicated, and need to be equipped with corresponding machines, it is difficult for non-professionals to complete
[0006] In addition, in the field of practical application, the demand for highly sensitive methods in immunoassays is also increasing day by day. In order to achieve accurate detection of low-abundance components, most immunoassay methods use natural enzymes such as horseradish peroxidase and alkaline phosphate Enzymes are loaded onto nanoparticles to achieve signal amplification, but natural enzymes often affect their catalytic activity due to slight conformational changes, resulting in deterioration. Nanoparticles used to load enzymes, such as gold nanoparticles, carbon nanotubes, graphene, etc., Regardless of synthesis or biomodification, the conditions are harsh and expensive

Method used

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  • High-sensitivity chemiluminescence immune assay method based on analogue enzyme labeled silver nanoparticles
  • High-sensitivity chemiluminescence immune assay method based on analogue enzyme labeled silver nanoparticles
  • High-sensitivity chemiluminescence immune assay method based on analogue enzyme labeled silver nanoparticles

Examples

Experimental program
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Effect test

Embodiment 1

[0030] Embodiment 1: in combination with image 3 , Preparation of Mimetic Enzyme-labeled Silver Nanoparticle Probes

[0031] Silver nanoparticles with a particle size of 6nm were synthesized, and the signal antibody Ab2 and G-quadruplex DNA were loaded on the silver nanoparticles through electrostatic adsorption and silver-sulfhydryl bonds in a certain proportion, respectively. The specific synthesis details are as follows: First, 3.5 μL of 2 mg / mL signal antibody Ab2 was added to 1.0 mL of silver nanoparticle solution (adjusted to pH 9.0 with 0.4 M NaOH in advance), stirred gently at room temperature, and incubated for 30 minutes. Then 150 μL of 100 μM G-quadruplex DNA was slowly added, stirred constantly, and incubated for 16 hours. Another 122 μL of phosphate buffer was added to continue the reaction for 6 hours. Then 21 [mu]L of 2M NaCl solution was slowly added, and this step was repeated after 3 hours. After 12 hours, 26 μL of 2M NaCl solution was added, and similarl...

Embodiment 2

[0032] Embodiment 2: in combination with figure 2 , preparation of immunosensing chip and process of sandwich immunoreaction, probe binding reaction and chemiluminescent signal generation

[0033] A layer of non-photosensitive film designed with 48 grooves was manually pasted on the aldehyde-based substrate to obtain an array of 4 rows×12 columns, and the surface of each sensing cell was coated with the capture antibody Ab1, so that it was stored at 4°C and 100% humidity Adsorbed overnight under the condition, the amino group of the antibody is covalently bonded to the aromatic aldehyde group of the substrate, rinsed after the reaction, dried, and dripped the blocking solution on the surface of each sensor, after blocking for 2 hours, rinsed with the rinse solution, after drying, An array is produced that captures the antigen of interest.

[0034] Drop the standard solution or sample on the surface of the sensing cell for incubation. After the standard solution or sample rea...

Embodiment 3

[0035] Embodiment 3: in combination with figure 2 , taking cTnT as an example to illustrate the application of this highly sensitive chemiluminescence imaging immunoassay

[0036] (1) Drop 8 μL of 10 μg / mL cTnT capture antibody Ab1 on the surface of the aldehyde group array, the amino group of the antibody is covalently bonded to the aromatic aldehyde group of the substrate, rinse after the reaction is completed, and then drop the blocking agent on the surface of each sensing point solution, rinsed after sealing, and dried to make a disposable immune sensor chip ( figure 2 );

[0037] (2) Add 8 μL of standard solutions or samples to be tested at different concentrations on the surface of each sensing pool of the prepared immune sensor chip, incubate at room temperature for 30 minutes, wash with the washing solution, and then add 6 μL of the solution in Example 1 dropwise. The prepared imitative enzyme-labeled silver nanoparticle probe was incubated at room temperature for ...

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Abstract

The invention relates to a high-sensitivity chemiluminescence immune assay method based on analogue enzyme labeled silver nanoparticles. The high-sensitivity chemiluminescence immune assay method based on analogue enzyme labeled silver nanoparticles comprises the following steps: constructing a 4*12 array on an aldehyde substrate by using a manual film adhering technology; preparing an analogue enzyme labeled silver nanoparticle probe by modifying a signal antibody Ab2 and high-proportion G-quadruplex DNA / heme compound on the surfaces of the silver nanoparticles; capturing the analogue enzyme labeled silver nanoparticle probe into a sensing pool by the analogue enzyme labeled silver nanoparticle probe through sandwich immunoreaction; loading a large number of analogue enzymes with characteristics of peroxidase on the silver nanoparticles; and catalyzing hydrogen peroxide oxidized luminol to obtain a high-strength chemiluminescence signal so as to realize high-sensitivity imaging immunoassay of protein. The immunosense method has the advantages of high sensitivity, high flux, good stability, easiness in operation, low cost and the like, and has certain clinical application value.

Description

[0001] 1. Technical field [0002] The invention is a highly sensitive chemiluminescence immunoassay method based on imitating enzyme-labeled silver nanoparticles. By constructing a disposable immunosensing chip and designing a novel DNA-imitating enzyme-labeled silver nanoparticle probe, the chemiluminescent signal is amplified. Sensitive and high-throughput imaging immunoassays for proteins. [0003] 2. Background technology [0004] The detection of protein markers plays an important role in the clinical diagnosis and monitoring of cardiovascular diseases. Immunoassay has been widely used in the detection of protein markers because of its high specific recognition ability for biomolecules. As the most popular immunoassay method, enzyme-linked immunoassay requires professionals to operate, complex instruments, cumbersome steps, time-consuming and expensive, these shortcomings greatly limit its further application. [0005] The development of simple, easy-to-operate, low-cos...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N33/58G01N33/543G01N21/76
CPCG01N33/6893G01N21/76G01N33/54346G01N33/581G01N2800/32
Inventor 李萍宗晨张多多
Owner CHINA PHARM UNIV
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