Method for carrying out cyclic polymerization label-free fluorescence detection on PD-1 (Programmed Cell Death-1) based on nucleic acid aptamer

A nucleic acid aptamer, PD-1 technology, applied in the field of analytical chemistry and biosensing detection, to achieve the effect of high actual sample detection capability, high sensitivity and selectivity, and high sensitivity detection

Inactive Publication Date: 2018-11-16
OCEAN UNIV OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the use of nucleic acid aptamer sensing technology for the de...

Method used

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  • Method for carrying out cyclic polymerization label-free fluorescence detection on PD-1 (Programmed Cell Death-1) based on nucleic acid aptamer
  • Method for carrying out cyclic polymerization label-free fluorescence detection on PD-1 (Programmed Cell Death-1) based on nucleic acid aptamer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1: Establishment of a method for label-free detection of PD-1 based on nucleic acid aptamer-based cyclic polymerization

[0026] 1. Reaction time optimization for cyclic polymerization

[0027] The fluorescence signal intensity corresponding to different reaction times after adding hairpin DNA4 was systematically investigated. The results show that within 2 to 5 hours, the fluorescent signal becomes stronger with the increase of the cyclic polymerization reaction time. When the reaction time is 6 hours, the signal is almost the same as that of 5 hours. Therefore, we choose the cyclic polymerization reaction time in the experiment for 5 hours.

[0028] 2. Fluorescence sensor sensitivity

[0029] Different concentrations of PD-1 were added to the reaction system, and the results showed that the fluorescence intensity was the strongest at the excitation wavelength of 416 nm and the emission wavelength of 590. The fluorescence intensity of the reaction system inc...

Embodiment 2

[0032] Example 2: Detection of PD-1 in serum

[0033] Take 1mL of rat serum, and then detect according to the optimized conditions, PD-1 was not detected. Another rat serum was taken, and PD-1 standard substances of different concentrations were added to the serum, and then the detection was carried out according to the optimized conditions. The results showed that PD-1 could be detected, and the recovery rate was between 89.6% and 104.3%. It shows that the method has good specificity and specificity, and can be used for the detection of biological samples such as serum containing PD-1.

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Abstract

The invention discloses a method for carrying out cyclic polymerization label-free fluorescence detection on PD-1 (Programmed Cell Death-1) based on a nucleic acid aptamer, and relates to an establishing method and application of a fluorescence spectrum sensor in the field of a signal amplification technology. The method comprises the following steps: complementarily hybridizing a self-designed DNA (Deoxyribonucleic Acid) single chain with the PD-1 nucleic acid aptamer, adding a to-be-detected matter PD-1, and unwinding DNA double chains because a binding force between the to-be-detected matter PD-1 and the PD-1 nucleic acid aptamer is greater than the coordination ability of DNA double helices; carrying out cyclic amplification on released DNA single chains under the action of various tool enzyme, and carrying out cascade signal amplification; changing a trigger fluorescence signal of an amplification product through conformation, thus realizing high-sensitivity and high-selectivity nucleic acid optical sensory detection of the PD-1. By virtue of the method disclosed by the invention, the detection limit of the PD-1 can be up to 0.3ng/mL, and detection of the PD-1 in biological samples such as serum can be met.

Description

technical field [0001] The invention belongs to the field of analytical chemistry and biosensing detection, and relates to the construction and application of a signal amplification technology fluorescence spectrum sensor. In addition, the present invention also relates to a method for detecting programmed death receptor (PD-1) by using the fluorescent spectrum sensor. Background technique [0002] Programmed cell death-1 (PD-1) is a co-receptor mainly expressed on T cells, binding to its ligand (PD-L1 / PD-L2) can inhibit the activation of T cells, making The body is protected from its own immune system (Tumeh P. C., Harview C. L., Yearley J. H., et al. Nature, 2014, 515:568-571). In addition to regulating and maintaining autoimmune tolerance, the expression of PD-L1 is upregulated in tumor cells, and the expression of PD-1 is also upregulated in virus-infected T cells. The PD-1 / PD-L1 signaling pathway is involved in tumor The immune escape of cells and infectious pathogens...

Claims

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Application Information

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IPC IPC(8): C12Q1/6804
CPCC12Q1/6804C12Q2525/205C12Q2563/107
Inventor 姜廷福吕爽
Owner OCEAN UNIV OF CHINA
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