High sensitive and jettisonable multicomponent chemiluminescent imaging immunosensor

A chemiluminescence and immunosensing technology, which is applied in the direction of scientific instruments, instruments, analytical materials, etc., can solve the problems of insensitive chemiluminescence detection signal and high price, and achieve the effect of improving detection sensitivity, low cost and high throughput

Inactive Publication Date: 2014-02-12
JIANGSU CANCER HOSPITAL +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In recent years, the research on protein detection arrays has attracted great attention. However, protein arrays are mostly prepared by photolithography or lattice technology, and th

Method used

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  • High sensitive and jettisonable multicomponent chemiluminescent imaging immunosensor
  • High sensitive and jettisonable multicomponent chemiluminescent imaging immunosensor
  • High sensitive and jettisonable multicomponent chemiluminescent imaging immunosensor

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Embodiment 1

[0024] Embodiment 1: in combination with image 3 , Preparation of multilayer DNase-functionalized gold nanoparticle probes

[0025] Synthesize gold nanoparticles with a particle size of 13nm, and two kinds of DNA single strands modified with sulfhydryl groups at the 5' end (including: signal DNA single strands containing multiple G-quadruplex sequence repeats, and capture DNA single strands modified with biotin at the 3' end Chain) according to a certain ratio, through the gold-sulfhydryl bond loaded on the gold nanoparticles. The specific synthesis details are as follows: Add 40 μL of 1 μM biotinylated capture DNA single-strand and 24 μL of 100 μM signal DNA single-strand to 1 mL of gold nanoparticles solution with a particle size of 13 nm, the ratio of capture DNA and signal DNA is 1:60, and stir at room temperature for 16 Hour. Then, a 0.01M PBS solution containing 2M NaCl was added dropwise to the above reaction solution, so that the final concentration of NaCl in the r...

Embodiment 2

[0026] Embodiment 2: in combination with figure 2 , preparation of immunosensor, sandwich immunoreaction, probe binding reaction and chemiluminescent signal generation and acquisition process

[0027] Screen-print 4×12 silanized arrays on glass slides, drop 1.5 μL of a certain concentration of capture antibody on the surface of each array point in each row, and let it absorb overnight at 4°C and 100% humidity; The amino group is covalently bonded to the epoxy group on the silanized glass slide, rinsed after the reaction, and after drying, drip blocking solution on the surface of each sensor, rinse with rinse solution after blocking for 1 hour, and dry to obtain a capture sensor. A total of 4 arrays of target antigens.

[0028] Drop the sample on the surface of the immunosensing array for incubation to bind the antigen to be tested, then wash the sample with the washing solution and add biotinylated antibody for incubation to form a sandwich immune complex and wash away unbou...

Embodiment 3

[0029] Embodiment 3: in combination with figure 2 , taking AFP, β-HCG, CA125, and CEA as examples to illustrate the application of this highly sensitive multi-component chemiluminescent image immunosensor

[0030] (1) Drop 1.5 μL of 10 μg / mL AFP, β-HCG, CA125 and CEA capture antibodies on the silanized surface of the array, the amino groups of the antibodies are covalently bonded to the epoxy groups of the slide, rinse after the reaction, and then The blocking solution was added dropwise on the surface of each sensing point, rinsed after sealing, and dried to make a disposable multi-component immune sensing array ( figure 2 );

[0031] (2) Add 1.5 μL of different concentrations of standard antigen or serum to be tested on the surface of each sensing point of the prepared immunosensing array, incubate at room temperature for 15 minutes, and wash with washing solution; Add 1.5 μL 2 μg / mL biotinylated AFP, β-HCG, CA125 and CEA antibodies to the surface of the sensor respectiv...

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Abstract

The invention relates to a high sensitive and jettisonable multicomponent chemiluminescent imaging immunosensor. The immunosensor is prepared by: constructing a 4*12 array on a silanized glass slide by a screen printing technique, and coating the array points with different capture antibodies to construct a jettisonable multicomponent immune sensor array; immobilizing biotinylated capture DNA and multiple G-quadruplex sequence repeat signal DNA on gold nanoparticle surfaces simultaneously, combining G-quadruplex signal DNA with heme to form DNA enzyme so as to prepare a multilayer DNA enzyme functionalized gold nanoparticle probe; and based on sandwich immunoassay, forming a sandwich immune complex on the sensor array, carrying out biotin-avidin reaction, labeling different immune complexes with the multilayer DNA enzyme functionalized gold nanoparticle probe; and making use of the peroxidase characteristics of DNA enzyme to catalyze reaction between chemiluminescent substrate H2O2-luminol to obtain a sensitive chemiluminescent signal, thus realizing high sensitive image immunoassay of a variety of protein. The immunosensor has the advantages of simple design, low cost, high sensitivity, high throughput and good repeatability, etc., and has certain clinical application value.

Description

1. Technical field [0001] The invention is a high-sensitivity disposable multi-component chemiluminescent imaging immunosensor, which involves the simultaneous immunoanalysis of multiple components to be measured in multiple samples. By constructing a disposable multi-component immunosensing array, a new type of immunosensor is designed. Multilayer DNase-functionalized gold nanoparticle probes for chemiluminescence signal amplification for highly sensitive image immunoassays of various proteins. 2. Background technology [0002] As an analytical method with high selectivity and high sensitivity, immunoassay has been widely used in environmental monitoring, clinical diagnosis, food safety and other fields. According to the different immunoassay steps, it can be divided into heterogeneous immunoassay and homogeneous immunoassay, the latter is widely used because of its higher sensitivity. Chemiluminescence immunoassay combines chemiluminescence assay technology with immunoass...

Claims

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Application Information

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IPC IPC(8): G01N33/68
CPCG01N21/763G01N33/54346G01N33/6803
Inventor 严枫宗晨吴洁鞠熀先
Owner JIANGSU CANCER HOSPITAL
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