A kind of thrombin detection method and kit thereof

Abstract

The application discloses a thrombin detection method and a kit thereof, the method comprising the following steps: 1) synthesizing a complementary PNA chain according to the DNA sequence of the thrombin aptamer; 2) combining the DNA sequence of the thrombin aptamer with The PNA chain synthesized in the step 1) is mixed to prepare a substrate solution; 3) adding a chromogen to the substrate solution to observe the color of the solution; 4) adding the sample to be tested, observing the color of the solution, and judging the The concentration of thrombin in the sample to be tested. The detection method of the present application is fast and convenient, has extremely low requirements on instruments and equipment, and has high sensitivity and good specificity.

Description

Technical field

[0001] The present application relates to a prothrombin detection method and a kit thereof, belonging to the technical field of chemistry and biosensor. Background technique

[0002] The thrombin is used as a hemostatic agent, which can catalyze fibrin element into fibrin to promote blood solidification, commonly used for local hemostasis of capillary hemorrhage and healing after surgery. The deletion or mechanism of thrombin will result in a variety of hemorrhagic diseases, such as cerebral hemorrhage, digestive tract bleeding, etc., severe cancer, so thrombin is often used as a biological marker for diagnosing related diseases. Currently detecting thrombin method mainly includes electrochemical methods, nano-gold color development method, surface enhanced Raman Law (SERS), fluorescent signal detection, etc., but there is a certain disadvantage, such as the applicable range, and needs to be used Large-scale detection instruments or synthesis of probes that requir...

Examples

Embodiment 1

[0038] (1) Design and synthesis of nucleic acids for detection: Unmodified thrombin suitable ligand DNA sequence: 5'-AGT CCGTGG TAG GGC AGG TTG GGG TGA CT-3 ', can be used without further purification; Synthesis method manually synthesized 10 base lengths of complementary PNA chain: AAC CTG CCT C, cleaves PNA crude in a polypeptide synthesis, and washed three times with anhydrous ether after hydrocephalium. Ultra pure water dissolves. The sample was purified on HPLC, and the target peak was subjected to mass spectrometry, and the target peak product was collected multiple times and then lyophilized and dissolved with ultrapure water, and the concentration was quantified with an ultraviolet visible absorption spectrometer.

[0039] (2) Exploration of thrombin detection and its detection: The same concentration of PNA and DNA (2 μm) were added to the Tris-HCl buffer as a substrate solution, which was heated at 95 ° C for 10 minutes and placed at room temperature to cool. The 180 μm ...

Embodiment 2

[0043](1) Design and synthesis of nucleic acids for detection: Unmodified thrombin suitable ligand DNA sequence: 5'-AGT CCGTGG TAG GGC AGG TTG GGG TGA CT-3 ', can be used without further purification; Synthesis method manually synthesized 10 base lengths of complementary PNA chain: AAC CTG CCT C, cleaves PNA crude in a polypeptide synthesis, and washed three times with anhydrous ether after hydrocephalium, and the sample was blown out with nitrogen. Ultra pure water dissolves. The sample was purified on HPLC, and the target peak was subjected to mass spectrometry, and the target peak product was collected multiple times and then lyophilized and dissolved with ultrapure water, and the concentration was quantified with an ultraviolet visible absorption spectrometer.

[0044] (2) Exploration of thrombin detection and its detection limit: The same concentration of PNA and DNA (1.5 μm) were added to the TRIS-HCl buffer as a substrate solution, and heated at 80 ° C for 15 minutes and pl...

Embodiment 3

[0046] (1) Design and synthesis of nucleic acids for detection: Unmodified thrombin suitable ligand DNA sequence: 5'-AGT CCGTGG TAG GGC AGG TTG GGG TGA CT-3 ', can be used without further purification; Synthesis method manually synthesized 10 base lengths of complementary PNA chain: AAC CTG CCT C, cleaves PNA crude in a polypeptide synthesis, and washed three times with anhydrous ether after hydrocephalium, and the sample was blown out with nitrogen. Ultra pure water dissolves. The sample was purified on HPLC, and the target peak was subjected to mass spectrometry, and the target peak product was collected multiple times and then lyophilized and dissolved with ultrapure water, and the concentration was quantified with an ultraviolet visible absorption spectrometer.

[0047] (2) Exploration of thrombin detection and its detection limit: The same concentration of PNA and DNA (2.5 μm) were added to the Tris-HCl buffer as a substrate solution, and heated at 100 ° C for 5 minutes and p...

Technical field

[0001] The present application relates to a prothrombin detection method and a kit thereof, belonging to the technical field of chemistry and biosensor. Background technique

[0002] The thrombin is used as a hemostatic agent, which can catalyze fibrin element into fibrin to promote blood solidification, commonly used for local hemostasis of capillary hemorrhage and healing after surgery. The deletion or mechanism of thrombin will result in a variety of hemorrhagic diseases, such as cerebral hemorrhage, digestive tract bleeding, etc., severe cancer, so thrombin is often used as a biological marker for diagnosing related diseases. Currently detecting thrombin method mainly includes electrochemical methods, nano-gold color development method, surface enhanced Raman Law (SERS), fluorescent signal detection, etc., but there is a certain disadvantage, such as the applicable range, and needs to be used Large-scale detection instruments or synthesis of probes that requir...

Examples

Embodiment 1

[0038] (1) Design and synthesis of nucleic acids for detection: Unmodified thrombin suitable ligand DNA sequence: 5'-AGT CCGTGG TAG GGC AGG TTG GGG TGA CT-3 ', can be used without further purification; Synthesis method manually synthesized 10 base lengths of complementary PNA chain: AAC CTG CCT C, cleaves PNA crude in a polypeptide synthesis, and washed three times with anhydrous ether after hydrocephalium. Ultra pure water dissolves. The sample was purified on HPLC, and the target peak was subjected to mass spectrometry, and the target peak product was collected multiple times and then lyophilized and dissolved with ultrapure water, and the concentration was quantified with an ultraviolet visible absorption spectrometer.

[0039] (2) Exploration of thrombin detection and its detection: The same concentration of PNA and DNA (2 μm) were added to the Tris-HCl buffer as a substrate solution, which was heated at 95 ° C for 10 minutes and placed at room temperature to cool. The 180 μm ...

Embodiment 2

[0043](1) Design and synthesis of nucleic acids for detection: Unmodified thrombin suitable ligand DNA sequence: 5'-AGT CCGTGG TAG GGC AGG TTG GGG TGA CT-3 ', can be used without further purification; Synthesis method manually synthesized 10 base lengths of complementary PNA chain: AAC CTG CCT C, cleaves PNA crude in a polypeptide synthesis, and washed three times with anhydrous ether after hydrocephalium, and the sample was blown out with nitrogen. Ultra pure water dissolves. The sample was purified on HPLC, and the target peak was subjected to mass spectrometry, and the target peak product was collected multiple times and then lyophilized and dissolved with ultrapure water, and the concentration was quantified with an ultraviolet visible absorption spectrometer.

[0044] (2) Exploration of thrombin detection and its detection limit: The same concentration of PNA and DNA (1.5 μm) were added to the TRIS-HCl buffer as a substrate solution, and heated at 80 ° C for 15 minutes and pl...

Embodiment 3

[0046] (1) Design and synthesis of nucleic acids for detection: Unmodified thrombin suitable ligand DNA sequence: 5'-AGT CCGTGG TAG GGC AGG TTG GGG TGA CT-3 ', can be used without further purification; Synthesis method manually synthesized 10 base lengths of complementary PNA chain: AAC CTG CCT C, cleaves PNA crude in a polypeptide synthesis, and washed three times with anhydrous ether after hydrocephalium, and the sample was blown out with nitrogen. Ultra pure water dissolves. The sample was purified on HPLC, and the target peak was subjected to mass spectrometry, and the target peak product was collected multiple times and then lyophilized and dissolved with ultrapure water, and the concentration was quantified with an ultraviolet visible absorption spectrometer.

[0047] (2) Exploration of thrombin detection and its detection limit: The same concentration of PNA and DNA (2.5 μm) were added to the Tris-HCl buffer as a substrate solution, and heated at 100 ° C for 5 minutes and p...

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