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Novel signal amplification-capillary tube chemiluminescence immunosensor

A technique of chemiluminescence immunity and signal amplification, which is applied in the field of highly sensitive capillary chemiluminescence immunosensors, and can solve the problems of limited detection sensitivity of capillary immunosensors.

Inactive Publication Date: 2019-03-01
NORTHEAST NORMAL UNIVERSITY +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The purpose of the present invention is to solve the problem of limited detection sensitivity of existing capillary immunosensors, and provide a new type of signal amplification-capillary chemiluminescence immunosensor and its application

Method used

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  • Novel signal amplification-capillary tube chemiluminescence immunosensor
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  • Novel signal amplification-capillary tube chemiluminescence immunosensor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1 A Novel Signal Amplification-Capillary Chemiluminescence Immunosensor

[0032] 1. Pretreatment of capillary

[0033] Wash the capillary with 0.1M HCl and 0.1M NaOH in sequence for 20 min and 60 min respectively to activate the silicon hydroxyl groups on the inner surface; then wash with water, N 2 Blow dry; pass through 1% v / v aminopropyldiethoxymethylsilane (3-ADMS) toluene solution, make the inner surface of the capillary covered with a layer of amino groups through mutual reaction; wash with toluene, N 2 Blow dry, and then add 2.5% glutaraldehyde (GA) aqueous solution, the aldehyde group at one end interacts with the amino group on the inner wall of the capillary, and the aldehyde group at the other end is used to bind the coated antibody Ab 1 ;

[0034] 2. Antibody Ab 1 fixed

[0035] Passage 40 μg / mL procalcitonin Ab into the aldehyde-activated capillary 1 , sealed at both ends, placed in a refrigerator at 4°C overnight, washed with 0.1M pH 7.4 PBS s...

Embodiment 2

[0038] Example 2 Procalcitonin Antigen Standard Curve

[0039] 1. Optimization process and results

[0040] The standard curve was made under optimal conditions, and the condition optimization process was as follows: the incubation time of different immune reactions, Ab 1 Concentration, B-Ab 2 concentration, the ratio of streptavidin to biotin-labeled horseradish peroxidase, and the concentration of streptavidin on the signal intensity, and the conditions corresponding to the strongest signal were selected as the optimal conditions. The results are as follows image 3 shown.

[0041] 2. Standard curve

[0042] 1) Prepare a series of procalcitonin antigen standard substances with different concentrations into the sensor prepared in Example 1 with antigen diluent. Immunoreact at 37°C for 60 minutes. PBST (containing 0.05% v / v Tween-20 PBS) to wash away unbound PCT;

[0043] 2) Biotin-labeled procalcitonin antibody (B-Ab 2 ) into the sensor, immunoreact at 37°C for 60 minut...

Embodiment 3

[0048] Example 3 Interference Measurement

[0049] Use antigen diluent to prepare PCT solution with a concentration of 1000 ng / mL, and use it as a solvent to prepare a series of interfering reagents (ascorbic acid, glucose, leucine, glycine, glutamic acid, carcinoembryonic antigen, immune globulin G), the concentration ratio PCT: interfering reagent = 1:10; measure the chemiluminescence intensity according to the above method, and calculate the ratio of the chemiluminescent intensity with different interfering reagents to those without interfering reagents, as Figure 5 As shown, the inhibition or enhancement of the signal intensity detected by PCT ranged from -7.0% to +7.6%, indicating that the detection of procalcitonin was not affected in the presence of interfering reagents.

[0050] Embodiment 3 actual sample detection

[0051] 1. Sample detection method

[0052] 1) Pass the actual sample solution into the sensor prepared in Example 1, perform immunoreaction at 37°C for...

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Abstract

The invention discloses a novel signal amplification-capillary tube chemiluminescence immunosensor, which is prepared by a method with the following steps of (1) sequentially using HCl and NaOH for cleaning a capillary tube; performing cleaning by water and blow drying by N2; introducing methylbenzene solution of aminopropyl diethoxymethyl silane; performing cleaning by methylbenzene and dry blowing by N2; introducing 2 to 3 percent of glutaraldehyde water solution; (2) introducing anti-AFP into the pretreated capillary tube; sealing openings at the two ends; putting the materials at 4 DEG C over the night; washing away uncombined Ab1 by a PBS solution; (3) introducing 10 mg / mL of oxidized glutathione solution into the capillary tube; sealing the openings at the two ends; performing placement for 1.5 to 2.5 h at the room temperature; cutting the capillary tube into several sections. The invention also provides application of the novel signal amplification-capillary tube chemiluminescence immunosensor in an aspect of procalcitonin detection. The novel signal amplification-capillary tube chemiluminescence immunosensor has the advantages that the cost is low; the consumption is low; the speed is high; the instrument is simple and convenient; the sensitivity is high; the lowest detection limit is 2.5 pg / mL; the anti-interference performance is high.

Description

technical field [0001] The invention belongs to the technical field of immunodetection and analysis, and in particular relates to a high-sensitivity capillary chemiluminescence immunosensor for signal amplification. Background technique [0002] Procalcitonin (PCT), the precursor of calcitonin, is usually secreted by thyroid C cells and is mainly used to identify severe bacterial infections, viral infections, and inflammatory responses caused by autoimmune diseases. PCT is a propeptide without hormonal activity, which is composed of 116 amino acid glycoproteins. In normal human serum, the concentration is very low, generally 10-50pg / mL, but in bacterial infection, severe septic shock and other systemic inflammation After reaction syndrome, burns, trauma, and respiratory diseases, the concentration and content of PCT in plasma will increase significantly. It can be used as an effective biological indicator for the detection of diseases such as systemic inflammatory response ...

Claims

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Application Information

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IPC IPC(8): G01N33/552G01N33/543G01N21/76
CPCG01N21/76G01N33/54393G01N33/552
Inventor 杨丽聂荣彬陈翊平许雪雪冯云祥冯呈蔚
Owner NORTHEAST NORMAL UNIVERSITY
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