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Nano gold biological composite probe, detection method and application thereof

A composite probe and nano-gold technology, which is applied in biochemical equipment and methods, microbial determination/inspection, and measurement devices, can solve the problems of low sensitivity of enzyme labeling, limited detection range, radioactive contamination, etc., and achieve high sensitivity High, easy to operate, short time-consuming effect

Inactive Publication Date: 2009-09-30
SHANGHAI INST OF MICROSYSTEM & INFORMATION TECH CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Different labeling methods have different advantages and disadvantages. Due to the limitation of tracer properties, the sensitivity of enzyme labeling is not high, and the detection range is limited. Although radioactive labeling is relatively sensitive, there is a certain problem of radioactive contamination. Fluorescent labeling is currently a A dominant detection method

Method used

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  • Nano gold biological composite probe, detection method and application thereof
  • Nano gold biological composite probe, detection method and application thereof
  • Nano gold biological composite probe, detection method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Embodiment 1: detect human CEA positive serum (see the schematic diagram figure 1 )

[0037] 1 CEA monoclonal capture antibody labeled magnetic beads

[0038] 1.1 Activation of magnetic beads: Wash the magnetic beads twice with MES (pH6), add ice-bathed EDC solution and NHS solution, incubate at room temperature for 30 min; then wash twice with MES (pH6).

[0039] 1.2 Labeling: Add CEA antibody at a concentration of 50 μg / mg magnetic beads, and place at room temperature for 30 minutes; then wash 4 times with PBS buffer and resuspend the magnetic beads, store at 2-8°C for later use.

[0040] 2 Preparation of nano-gold biocomposite probes

[0041] 2.1 Determine the optimal amount of antibody using K 2 CO 3Solution Adjust the pH value of the nano-gold solution to 8.5-9, take different amounts of antibodies (respectively 0 μg, 0.5 μg, 1 μg, 2 μg, 3 μg, 5 μg, 6 μg, 7 μg, 8 μg) into 1 ml of nano-gold solution, Let stand for 5 minutes, then add NaCl solution to determine ...

Embodiment 2

[0049] Embodiment 2: detection people contain CEA, P 53 serum (see schematic diagram figure 2 )

[0050] 1 Capture antibody-labeled magnetic beads

[0051] 1.1 Activation of magnetic beads: Wash the magnetic beads twice with MES (pH6), add ice-bathed EDC solution and NHS solution, incubate at room temperature for 30 min; then wash twice with MES (pH6).

[0052] 1.2 Labeling: CEA antibody, P 53 The antibodies were mixed according to a certain ratio, added to the antibody mixture, the antibody concentration was 50 μg / mg magnetic beads, and left at room temperature for 30 minutes; then washed 4 times with PBS buffer and resuspended magnetic beads, stored at 2-8°C for later use.

[0053] 2 CEA detection antibody and DNA probe labeled gold nanoparticles

[0054] 2.1 Determine the optimal amount of antibody using K 2 CO 3 Solution Adjust the pH value of the nano-gold solution to 8.5-9, take different amounts of antibodies (respectively 0 μg, 0.5 μg, 1 μg, 2 μg, 3 μg, 5 μg, 6 ...

Embodiment 3

[0066] After adding the enhancement solution, shake gently at room temperature for 5-10 minutes, and measure the fluorescence intensity at 615nm and 545nm. Measure according to concentration gradient (CEA protein concentration is respectively 125pg / ml, 62.5pg / ml, 31.25pg / ml, 15.63pg / ml, 7.81pg / ml; P 53 Concentrations are respectively 528pg / ml, 264pg / ml, 132pg / ml, 66pg / ml, 33pg / ml) diluted positive serum and negative control serum, and the measurement results are as follows: Figure 5 , 6 Shown is a histogram of the correlation between fluorescence intensity and protein concentration. Embodiment 3: detect people's serum containing CEA, P53, NSE

[0067] 1 Capture antibody-labeled magnetic beads

[0068] 1.1 Activation of magnetic beads: Wash the magnetic beads twice with MES (pH6), add ice-bathed EDC solution and NHS solution, incubate at room temperature for 30 min; then wash twice with MES (pH6).

[0069] 1.2 Labeling: CEA antibody, P53 antibody, and NSE antibody were mix...

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Abstract

The invention relates to a nano gold biological composite probe, a detection method and application thereof. The detection method is characterized in that: the method comprises the following steps: firstly marking a bead through a monoclonal antibody of a protein to be tested; marking the polyclonal antibody of the protein to be tested on nano gold while and simultaneously marking a DNA probe with a biotin label; carrying out a biotin-streptavidin reaction on the DNA probe on the nano gold to make a lanthanide bonded with colloidal gold to construct the nano gold biological composite probe; mixing the bead of marked monoclonal antibody of the protein to be tested, the nano gold biological composite probe and a protein sample to be tested, carrying out the incubation on the mixture for a certain time at 37 DEG C; cleaning away the nano gold probe which does not react; adding a reinforcing liquid; and determining the fluorescence intensity so as to realize the aim of carrying out the quantitative determination on the protein to be tested. The method obviously improves the detection sensitivity of biomolecules, and is used for detecting a plurality of kinds of biomolecules synchronously. The method is widely applied in the fields of clinical diagnosis, antigen, antibody and nucleic acid detection, health quarantine, environmental tests, and the like.

Description

technical field [0001] The invention relates to a nano-gold biocomposite probe, a detection method and its application, in particular to a nano-gold biocomposite probe based on nano-gold and lanthanide elements, a highly sensitive detection method, and a method for simultaneously detecting multiple applications in biomolecules. It belongs to the technical field of biological detection. Background technique [0002] It is always the direction of people's efforts to detect biomolecules simply, quickly, with high selectivity and high sensitivity. It is of great significance in the fields of early diagnosis of diseases, rapid diagnosis, health detection, and environmental monitoring. According to different labeling means, people have developed a series of effective detection methods, such as radioactive labeling, fluorescent labeling, enzyme labeling and so on. Different labeling methods have different advantages and disadvantages. Due to the limitation of tracer properties, t...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11G01N33/533G01N33/577G01N33/543G01N21/64
Inventor 刘美英贾春平金庆辉赵建龙
Owner SHANGHAI INST OF MICROSYSTEM & INFORMATION TECH CHINESE ACAD OF SCI
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