Colorimetric method for simple detection of thrombin

A thrombin, to-be-detected technology, applied in the field of analysis and detection, can solve problems such as the difficulty of biological macromolecules, and achieve the effect of high selectivity, specificity and high specificity

Inactive Publication Date: 2019-03-29
TONGJI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, at present, nucleotide aptamers are mainly used to detect small biological molecules, and there are still many technical difficulties to be overcome in order to detect biological macromolecules.

Method used

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  • Colorimetric method for simple detection of thrombin
  • Colorimetric method for simple detection of thrombin
  • Colorimetric method for simple detection of thrombin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0072] (1) Synthesis of Fe-MIL-88A

[0073] First, 5 mM ferric chloride hexahydrate (FeCl 3 ·6H 2 O) and 5mM fumaric acid were dissolved in 25mL ultrapure water, and then the entire homogeneous solution was transferred to a 60mL reactor and heated to 65°C for 12 hours. After cooling to room temperature, the reactant was repeatedly washed with water and ethanol, centrifuged and dried overnight in a vacuum oven (lower than 80° C.) to obtain the product Fe-MIL-88A powder. Weigh 4.0 mg of the powder and dissolve it in 10.0 mL of ultrapure water to form a 0.4 mg / mL Fe-MIL-88A solution after ultrasonic mixing.

[0074] from figure 1 It can be seen that Fe-MIL-88A is a spindle-shaped crystal. from figure 2 It can be seen that Fe-MIL-88A has a specific crystal structure.

[0075] (2) Preparation of thrombin MOF-nucleic acid aptamer mixture

[0076] 100 μL thrombin aptamer 5'-TCT CTC AGT CCG TGG TAG GGC AGG TTG GGG TGA CT-3' (1 μM concentration) was reacted with 50 μL thrombin ...

Embodiment 2

[0085] Embodiment 2: Anti-interference test

[0086] In order to explore the anti-interference ability of the method of the present invention to other potential interfering proteins and amino acids with different isoelectric points, we tested bovine serum albumin (BSA), immunoglobulin G (IgG), lysozyme (lysozyme), lysine (lysine ), arginine (arginine), glycine (glycine), sodium glutamate (L-sodium glutamate, LSG) carried out parallel interference test experiments. from Figure 5 It can be seen that only in the presence of thrombin, the absorbance reduction rate increases significantly, and the solution becomes almost colorless. In the presence of other macromolecular proteins or amino acids with various isoelectric points, the catalytic activity of Fe-MIL-88A mimic enzymes has not been significantly inhibited, and the solution is still blue. Experimental results show that the detection method of the present invention has good anti-interference performance and thrombin recogn...

Embodiment 3

[0087] Example 3: Addition experiment in serum

[0088] Standard addition method was used to add standard thrombin to bovine serum to prepare serum samples with different concentrations. Serum is the plasma left after the blood is coagulated and the fibrin is removed. Serum is similar in chemical composition to plasma but does not contain clotting proteins. Three sets of parallel experiments were repeated for each set of spiked experiments, and the final data in Table 1 is the average of the three sets of parallel experiments. The results show that the recovery rate of thrombin is between 99.3% and 100.4%, and the relative standard deviation is between 0.3% and 1%. The invention has good accuracy and repeatability and can meet the quantitative requirements.

[0089] Table 1 Recovery rate of thrombin sample in serum spiked experiment

[0090]

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Abstract

The invention provides a colorimetric method for simple detection of thrombin. By utilization of a fact that a metal organic skeleton compound Fe-MIL-88A has the characteristics of a peroxide mimeticenzyme under acidic conditions, a thrombin tetramer is formed by reaction of a thrombin nucleic acid aptamer and thrombin and then is subjected to catalytic oxidation reaction with Fe-MIL-88A and a 3,3',5,5'-tetramethylbiphenylamine (TMB)-H2O2 system; the thrombin concentration is determined by ultraviolet spectrophotometry and colorimetry. The method can detect thrombin sensitively, accurately, specifically and visually by the catalytic performance of the Fe-MIL-88A mimetic enzyme and the specific binding effect of the nucleic acid aptamer to a substrate.

Description

technical field [0001] The invention relates to the technical field of analysis and detection, in particular to a simple colorimetric method for detecting thrombin, in particular to a colorimetric method using metal organic framework compound material Fe-MIL-88A for detecting thrombin. Background technique [0002] Thrombin (thrombin) is a very important serine protease in the body's blood coagulation system. It consists of 308 amino acid residues and has a molecular weight of 37kD. Thrombin has both procoagulant and anticoagulant functions in the coagulation system, plays an important role in inflammation regulation, angiogenesis, tissue repair and tumor biology, and also has pathological and physiological roles in the central nervous system. Thrombin is considered an important measure of the coagulation mechanism and a biomarker for some disease diagnoses such as lung metastasis and cartilage synovial inflammation. Therefore, thrombin detection is of great significance to...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/37G01N21/33
CPCC12Q1/37G01N21/33
Inventor 王颖李风亭张冰如
Owner TONGJI UNIV
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