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Fluorescent biological probe for detecting mucin 1, sensor, application and detection method

A fluorescent bioprobe and biosensor technology, applied in the field of fluorescent bioprobes, can solve the problems of long operation time (more than 15 hours, complicated steps in the sensor preparation process, difficult to control the particle size, etc., to avoid covalent labeling, The effect of lowering the detection limit and improving the detection sensitivity

Active Publication Date: 2020-06-30
JIANGSU INST OF NUCLEAR MEDICINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, there are the following defects in the above scheme: 1. The gold electrode used in the electrochemiluminescence sensor needs to be sequentially modified with polydiallyldimethylammonium chloride (PDDA), gold nanoparticles (AuNPs), SH-DNA and mercaptohexanol (MCH) is closed, the preparation process of the whole sensor is complicated, and the operation takes a long time (more than 15 hours); Second, gold nanoparticles (AuNPs) are made of gold chloride (HAuCl 4 ) reduction method, the preparation is cumbersome, the particle size is difficult to control, and certain experience in organic synthesis is required

Method used

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  • Fluorescent biological probe for detecting mucin 1, sensor, application and detection method
  • Fluorescent biological probe for detecting mucin 1, sensor, application and detection method
  • Fluorescent biological probe for detecting mucin 1, sensor, application and detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0078] The design of embodiment 1 fluorescent biological probe

[0079] The fluorescent biological probes used for mucin 1 detection provided in this example include aptamer probes and hairpin probes;

[0080] The aptamer probe is self-assembled from sequences P1, P2 and P3;

[0081] The sequence P1 sequentially includes sequence a, sequence b and at least two sequences c from the 5' end to the 3' end;

[0082] The sequence P2 sequentially includes sequence a, sequence b, and sequence c* complementary to sequence c bases from the 5' end to the 3' end;

[0083] The sequence P3 includes sequence b* complementary to the base of sequence b and sequence a* complementary to the base of sequence a from the 5' end to the 3' end; the sequence P3 is the aptamer sequence of the target, which can specifically sexual binding target;

[0084] The hairpin probes include hairpin probes H1 and hairpin probes H2;

[0085] The hairpin probe H1 comprises sequentially from the 5' end to the 3'...

Embodiment 2

[0092] Example 2 Preparation of Fluorescent Bioprobes for Mucin 1 Detection

[0093] 1. Preparation of aptamer probes

[0094] (1) The dry powders of the oligonucleotide sequences P1, P2 and P3 in Table 1 were centrifuged at 12000rpm for 5 minutes, and then dissolved in 20mM Tris-HNO 3 Buffer (containing 20mM NaNO 3 ,10mM NH 4 NO 3 ,2mM Mg(NO 3 ) 2 , pH7.4), to obtain 100 μM stock solutions respectively;

[0095] (2) Pipette 10 μL of the stock solution of sequence P1, pipette 20 μL of the stock solution of sequence P2, pipette 30 μL of the stock solution of sequence P3, and add the above-mentioned stock solution to 40 μL of reaction buffer (containing 20 mM Tris-HNO 3 ,50mM KNO 3 ,10mM Mg(NO 3 ) 2 , 1 mM DTT, pH 7.9), incubated at 37°C for 20 minutes to obtain M-shaped aptamer probes P1-P2-P3 at a concentration of 10 μM, and stored at 4°C.

[0096] 2. Preparation of fluorescent hairpin probes

[0097] (1) Centrifuge the dry powder of oligonucleotide sequence H1 at 1...

Embodiment 3

[0101] Example 3 Fluorescence biosensor for mucin 1 detection

[0102] This example provides a fluorescent biosensor for mucin 1 detection, including the aptamer probe prepared in Example 2 and fluorescent hairpin probes AgNCs-H1 and AgNCs-H2.

[0103] Further, the first reaction reagents are included, based on a total volume of 49 μL, including:

[0104] Aptamer probe, 1 μM, 5 μL;

[0105] Reaction buffer, 32 μL;

[0106] Exo I solution, 10U / μL, 2μL;

[0107] 1× Exo I buffer, 10 μL.

[0108] Further, the second reaction reagent is included, based on a total volume of 50 μL, including:

[0109] Fluorescent hairpin probe AgNCs-H1, 1 μM, 15 μL;

[0110] Fluorescent hairpin probe AgNCs-H2, 1 μM, 15 μL;

[0111] GO solution, 1 mg / mL, 20 μL.

[0112] Further, the Exo I solution contains 10U / μL of Exo I;

[0113] The 1×Exo I buffer contains 67mM glycine-KNO 3 ,6.7mM Mg(NO 3 ) 2 , and 1 mM DTT, pH9.4;

[0114] The reaction buffer contains 20mM Tris-HNO 3 ,50mM KNO 3 ,10...

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Abstract

The invention relates to the technical field of mucin 1 detection, in particular to a fluorescent biological probe for mucin 1 detection, a sensor, application and a detection method. In the present invention, the aptamer probe is used for an Exo I assisted target circulation (EATR) reaction. The hairpin probe comprises a hairpin probe H1 and a hairpin probe H2 which are used for GO-assisted hybridization chain reaction (HCR); the aptamer probe and the hairpin probe are combined to be used for mucin 1 detection, so organic combination of Exo I-assisted target circulation reaction and GO-assisted hybridization chain reaction can be realized; therefore, cascade amplification of fluorescence signals is realized, the detection sensitivity of mucoprotein 1 is remarkably improved, the detectionlimit is reduced, quantitative detection of low-concentration mucin 1 can be realized, and theprobe has high specificity and can be used for specifically identifying and detecting mucin 1.

Description

technical field [0001] The invention relates to the technical field of detection of mucin 1, in particular to a fluorescent biological probe, sensor, application and detection method for detection of mucin 1. Background technique [0002] Mucin 1 (MUC1) is a high molecular weight, highly glycosylated protein that forms a complete transmembrane domain through the gel matrix, and its polypeptide backbone consists of three parts: extracellular segment, transmembrane segment and intracellular segment. MUC1 plays an important role in the signal transduction process. MUC1 can down-regulate the expression of E-cadherin, mediate the combination between cells, and play an inhibitory role in tumor metastasis. MUC1 is mainly expressed in the epithelial cells near the lumen or gland cavity surface of various tissues and organs. MUC1 is slightly expressed in normal epithelial cells, but highly expressed in many malignant tumor cells, such as breast cancer, gastric cancer, lung cancer, p...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N21/64C12N15/115C12N15/10
CPCG01N33/68G01N21/6486C12N15/115G01N2333/4725C12N2310/16Y02A50/30
Inventor 吴昊邹霈刘娅灵王洪勇吴军韩国庆
Owner JIANGSU INST OF NUCLEAR MEDICINE
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