A biosensor based on chain displacement and dark-state silver clusters and its application

A biosensor, strand displacement technology, applied in biochemical equipment and methods, microbial determination/inspection, etc., can solve the problems of complex operation and high cost

Active Publication Date: 2021-02-02
TONGJI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The current biosensors for the detection of gene deletions mostly involve the synthesis of fluorescent labels or compounds or even the determination of the entire gene sequence, which is complicated to operate and high in cost.

Method used

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  • A biosensor based on chain displacement and dark-state silver clusters and its application
  • A biosensor based on chain displacement and dark-state silver clusters and its application
  • A biosensor based on chain displacement and dark-state silver clusters and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Embodiment 1 (preparation of various stock solutions)

[0055] 1. Preparation of PB buffer (PB: 20mM, pH=7.4): Accurately weigh Na 2 HPO 4 12H 2 O 7.16g in a 50mL beaker, dissolve it completely with triple distilled water and transfer it to a 100mL volumetric flask, wash the beaker and glass rod three times with ultrapure water, transfer the washing solution to the volumetric flask, dilute to 100mL, shake well 100mL 0.2M disodium hydrogen phosphate stock solution can be obtained for future use; accurately weigh the NaH 2 PO 4 2H 2 O 3.12g in a 50mL beaker, dissolve it completely with triple distilled water and transfer to a 100mL volumetric flask, wash the beaker and glass rod three times with ultrapure water, transfer the washing solution to a volumetric flask, dilute to 100mL, shake well 100mL 0.2M sodium dihydrogen phosphate stock solution can be obtained for later use; take the stock solution that has been prepared above: 19mL 0.2M NaH 2 PO 4 and 80 mL of 0.2...

Embodiment 2

[0068] Embodiment 2 (sensor construction-fluorescence detection based on strand displacement and dark-state silver clusters)

[0069] 1. Add C-DNA, C-DNA+P-DNA, C-DNA+P-DNA+Target to a 2.0ml centrifuge tube, the amount of each DNA (100μM) in each sample is 10μL, add PB buffer solution to make the system volume 200 μL. Incubate at 37°C for 1 hour;

[0070] 2. Add an appropriate amount of PB buffer solution to make the system volume 970 μL, add 10 μL of S-DNA (100 μM), 10 μL of AgNO 3 (1.8mM), magnetically shake for 1min, and incubate at room temperature for 30min in the dark;

[0071] 3. Add 10 μL fresh NaBH 4 (1.8mM), magnetically shaked for 1min, and cultured at 4°C in the dark for 5h;

[0072] 4. To detect fluorescence, the parameters are set to 10nm for the slit and 600V for the voltage. (excitation wavelength is 475nm, emission wavelength is 635nm), you can get image 3 .

Embodiment 3

[0073] Example 3 (Detection of AIMP2 Exon 2 Gene Deletion Based on Strand Displacement and Dark State Silver Cluster Sensors)

[0074] 1. Add 10 μL of C-DNA+P-DNA (both 100 μM) to a 2.0 ml centrifuge tube, add 10 μL of different concentrations of Target and different types of targets ((both 100 μM)), and add PB buffer solution to make the system volume 200 μL. Incubate at 37°C for 1 hour;

[0075] 2. Add an appropriate amount of PB buffer solution to make the system volume 970 μL, add 10 μL S-DNA (100 μM), 10 μL AgNO3 (1.8 mM), magnetically shake for 1 min, and incubate in the dark at room temperature for 30 min;

[0076] 3. Add 10 μL of freshly prepared NaBH4 (1.8 mM), shake magnetically for 1 min, and incubate at 4°C in the dark for 5 h;

[0077] 4. To detect fluorescence, the parameters are set to 10nm for the slit and 600V for the voltage. (excitation wavelength is 475nm, emission wavelength is 635nm), can obtain Fig. 4 and Figure 5 , to achieve a linear detection of ...

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Abstract

The present invention relates to a biosensor based on strand displacement and dark-state silver clusters and its application. The biosensor comprises three probe strands, namely C-DNA strand, P-DNA strand and S-DNA strand, and the C-DNA strand consists of two probe strands. Partial composition, the 5' end contains a G-rich sequence, the 3' end is complementary to the S-DNA part of the silver cluster template strand, and also complementary to the P-DNA part of the probe chain, and the number of bases complementary to the P-DNA is greater than that of The number of S-DNA complementary bases; the P-DNA strand contains a sequence that can be completely complementary to the target strand Target; the S-DNA strand is composed of two parts, the 5' end is the C-DNA complementary sequence, and the 3' end is dark silver Cluster synthesis template sequences. In addition, the present invention also introduces exonuclease III into the system, and releases Target to participate in the strand displacement of P-DNA / C-DNA again by cutting the P-DNA in the Target / P-DNA double strand, thereby realizing the target's Cyclic detection improves the sensitivity of detection. The invention utilizes the fluorescence change and strand displacement of the dark-state silver cluster and the amplification effect of exonuclease III, which can be used for sensitive detection of gene deletion and other related gene detection, and can realize the obvious distinction of three base deletions.

Description

technical field [0001] The present invention relates to a detection method relying on fluorescence, in particular to a biosensor based on chain displacement and dark-state silver clusters and its construction method. Background technique [0002] Gene deletion is a kind of change in DNA base sequence, which refers to the DNA sequence polymorphism caused by the deletion of a part of the DNA base sequence. Many phenotypic differences in the human body, susceptibility to drugs or diseases, etc. may be related to genes missing. For example, the amide tRNA synthetase-interacting multifunctional protein AIMP2 is a new type of tumor suppressor, and its exon 2 deletion will cause AIMP2 morphological variation, thereby losing its tumor suppressor activity, leading to lung cancer, liver cancer, skin cancer, and breast cancer. Wait for the occurrence of cancer. Therefore, at the molecular level, it is of great research significance to construct high-sensitivity biosensors and realize...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6825
CPCC12Q1/6825C12Q2537/1373C12Q2563/107C12Q2563/137C12Q2521/319
Inventor 石硕臧瑞敏
Owner TONGJI UNIV
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