Nano-enzyme catalysis assisted ratio type colorimetric heparin detection method

A ratio-type, nano-enzyme technology, applied in the field of analytical chemistry, can solve the problems of complex detection operation steps, susceptibility to interference from external factors, complicated detection process, etc., and achieve ultra-sensitive and specific quantitative detection with simple experimental steps , the effect of reliable results

Active Publication Date: 2021-11-12
JIANGSU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] (1) The chromatographic detection method requires more expensive instruments and equipment, and the detection operation steps are relatively complicated;
[0010] (2) Most colorimetric and fluorescence methods are based on target-induced single-signal changes, which are easily disturbed by external factors in practical applications, resulting in poor repeatability and robustness of detection methods;
[0011] (3) Most fluorescence and colorimetric methods involve the preparation of chromogenic substrates, which increases the detection cost and operational difficulty;
[0012] (4) Some ratiometric detection methods involve the use of multiple chromogenic substrates, which complicates the detection process

Method used

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  • Nano-enzyme catalysis assisted ratio type colorimetric heparin detection method
  • Nano-enzyme catalysis assisted ratio type colorimetric heparin detection method
  • Nano-enzyme catalysis assisted ratio type colorimetric heparin detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] FeMoO 4 Nanorods exhibit oxidase-like activity and catalyze the oxidation of colorless TMB to generate blue TMBox

[0038] (1) Add 900 μL of 0.1M phosphate buffer (pH 7.0) to a 1.5mL centrifuge tube and shake well;

[0039] (2) Add 50 μL of 1 mg / mL FeMoO to the above buffer solution 4 Nanorod aqueous solution and 50 μL 5mMTMB solution (prepared with ethanol), ensure that the total volume of the solution is 1.0mL, and react for 1-10min;

[0040] (3) Measure the light absorption signal of the above solution with an ultraviolet-visible spectrometer, and obtain the absorbance value at 652 nm.

[0041] The result is as figure 1 as shown, figure 1 Medium TMB substrates cannot be directly oxidized by dissolved oxygen, and low concentrations of FeMoO 4 The aqueous solution (50 μg / mL) also has no background color. However, adding FeMoO to a TMB solution containing dissolved oxygen 4 After the nanorods, an obvious color reaction occurs quickly, and an obvious visible absor...

Embodiment 2

[0043] Heparin induces the aggregation of TMBox and affects the color development of the solution

[0044] (1) Add 900 μL of 0.1M phosphate buffer (pH 7.0) to a 1.5mL centrifuge tube and shake well;

[0045] (2) Add 50 μL of 1 mg / mL FeMoO to the above buffer solution 4 Nanorod aqueous solution and 25 μL 5mMTMB solution (prepared with ethanol), react for 5 minutes;

[0046] (3) Add 3 μg / mL heparin solution to the above solution and react for 2 minutes;

[0047] (4) Measure the absorbance signal of the above-mentioned solution with a UV-visible spectrometer, and obtain the absorbance values ​​at 652nm and 565nm.

[0048] The result is as figure 2 shown, since the FeMoO 4 Under the catalysis of nanorods, TMB is oxidized into TMBox, making FeMoO 4 +TMB solution turns blue. When heparin was added (3.0μg / mL, equivalent to 0.51U / mL or 0.486μM), the solution turned purple, and a new obvious signal appeared around 565nm, while the absorbance at 652nm decreased. Furthermore, onl...

Embodiment 3

[0050] The effect of the adding order of heparin on the detection system

[0051] In this assay, the analyte heparin was added in two ways ( image 3 ): One is to add after TMB is completely catalyzed and oxidized into blue TMBox species, forming a two-step detection method; the other is to add TMB at the same time, forming a one-pot detection method. In order to check whether the two methods of operation will cause differences in the detection results, the detection results of the same heparin concentration (1.0 μg / mL) by different addition methods were compared. The results are as follows: image 3 shown. It was found that the above two addition methods had no significant difference in the detection of heparin. In order to save detection time, a one-pot method was optimally used for the detection of heparin.

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Abstract

The invention belongs to the technical field of analytical chemistry, and relates to a nano-enzyme catalysis assisted ratio type colorimetric heparin detection method, which comprises the steps of adding 900 [mu]L of a 0.1 M phosphate buffer solution into a plurality of 1.5 mL centrifuge tubes, uniformly shaking, and standing; respectively adding 50 [mu]L of a 1mg / mL FeMoO4 nanorod aqueous solution, 25 [mu]L of a 5mM TMB solution and heparin solutions with different concentrations, and ensuring that the total volume of the solutions is 1mL; acquiring absorbance values at 652nm and 565nm by using an ultraviolet-visible spectrograph, and drawing a standard working curve by taking the heparin concentration as an abscissa and the absorbance ratio (A652 / A565) at the 652nm and 565nm as an ordinate; and repeatedly operating a sample to be detected, and comparing with the standard working curve to obtain the concentration of the heparin in the sample to be detected. Based on specific electrostatic interaction between a to-be-detected object and positively charged species generated by nano-enzyme catalysis, the method disclosed by the invention is simple and rapid in experimental steps, can realize ultrasensitive and specific quantitative detection of heparin, and provides a reliable result for determination of heparin in a clinical blood sample.

Description

technical field [0001] The invention belongs to the technical field of analytical chemistry, and relates to a method for detecting heparin, in particular to a method for detecting heparin by ratiometric colorimetry assisted by nanozyme catalysis. Background technique [0002] As an anticoagulant widely used clinically, heparin can prevent the formation of blood clots in cardiopulmonary surgery. In addition, it can accelerate the inactivation rate of various coagulation factors, and is often used to treat acute venous thrombosis. However, excessive use of heparin can cause a series of serious complications, including heparin-induced thrombocytopenia and bleeding. The guiding therapeutic dose of heparin is 0.2-1.2 U / mL in postoperative care and 2-8 U / mL in cardiovascular surgery. In addition, heparin is also involved in the regulation of some physiological and pathological processes, such as anti-inflammatory, anti-allergic and so on. Therefore, it is very important to moni...

Claims

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Application Information

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IPC IPC(8): G01N21/31G01N21/33
CPCG01N21/31G01N21/33
Inventor 牛湘衡汪梦珠朱恒佳刘朋刘邦祥
Owner JIANGSU UNIV
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