Kit for detecting trypsin and inhibitor thereof based on platinum nanoclusters

A detection kit, trypsin technology, applied in the field of nano biosensing, can solve the problems of time-consuming, complex modification steps, etc., and achieve the effects of high sensitivity, good stability and high detection specificity

Inactive Publication Date: 2019-05-21
FUJIAN MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

These methods are relatively time-consuming, or some require complex mo

Method used

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  • Kit for detecting trypsin and inhibitor thereof based on platinum nanoclusters
  • Kit for detecting trypsin and inhibitor thereof based on platinum nanoclusters
  • Kit for detecting trypsin and inhibitor thereof based on platinum nanoclusters

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Add 1ml of 16 mmol / L chloroplatinic acid solution and 1 mL of 40 mmol / L sodium citrate into 38ml of ultrapure water, stir and mix at room temperature for two minutes, then add 200 μl of 50 mmol / L sodium borohydride dropwise Used to reduce chloroplatinic acid; the mixture was stirred continuously at room temperature for 1 hour, and after the reaction, the solution was put into an ultrafiltration tube with a molecular weight cut-off of 3k, centrifuged and ultrafiltered at 6000 r / min for 24 hours, and washed 3 times with water to obtain a stable citric acid- Platinum nanocluster aqueous solution, all glassware used in the preparation process were soaked in aqua regia, washed thoroughly with double distilled water and dried. The Zeta potential of the as-prepared platinum nanocluster material is -26.7±1.9 mV (see figure 1 ); using X-ray photoelectron spectroscopy to carry out elemental analysis on citric acid stabilized-platinum nanocluster materials, it can be seen from the...

Embodiment 2

[0037] The platinum nanocluster material obtained in Example 1 was characterized by a transmission electron microscope, and it was found that the platinum nanoclusters were regularly and uniformly dispersed, with a size of 2.95 ± 0.53 nm (see image 3 ); protamine is added to the prepared platinum nanocluster material, because protamine is rich in arginine and has a strong positive charge, it can electrostatically interact with negatively charged platinum nanoclusters to make platinum nanoclusters The surface state of the material changes and then destroys the stability of platinum nanoclusters, resulting in the aggregation of platinum nanoclusters. The surface state of platinum nanoclusters is characterized by transmission electron microscopy, and the results are as follows Figure 4 As shown, protamine causes the change of the surface state of platinum nanoclusters to cause aggregation, which leads to the reduction of its catalytic activity.

Embodiment 3

[0039] Add 10 µL of 0.2 mg / mL protamine to 10 µL of 10 ng / mL trypsin and 30 ng / mL trypsin solution, respectively, add 480 µL of Tris-HCl buffer solution with a concentration of 10 mmol / L and pH 8, in Incubate at 37°C for 2 hours; after that, add 50 μL of platinum nanoclusters, N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3- Methylaniline sodium salt and 3-methyl-2-benzothiazolinone hydrazone hydrochloride, after incubating at 40°C for 25 minutes to obtain color-developed products, visually observe the color change or measure the UV-visible absorption at 590nm degree, the result is as Figure 5 Shown: curve (a) blank, (b) 10ng / mL trypsin, (c) 30ng / mL trypsin. When 20ng / mL inhibitor was added, the absorbance decreased (see Figure 5 Curve d), indicating that the inhibitor acts to inhibit the hydrolysis of protamine by trypsin.

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Abstract

The invention discloses a kit for detecting trypsin and the inhibitor thereof based on platinum nanoclusters. The kit is characterized in that synthesized platinum nanocluster simulated oxidase is utilized to catalyze the color development of the coupling product of 3-Methyl-2-benzothiazolinone hydrazone hydrochloride monohydrate and N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline sodium salt;protamine makes platinum nanoclusters aggregate by means of an electrostatic effect, so that the catalytic activity of the nanoclusters is decreased; the protamine is hydrolyzed by trypsin, and so that the trypsin can effectively inhibit the effect of the protamine on the aggregation of the platinum nanoclusters. Therefore, on the basis of solution color change and ultraviolet absorption spectrumfeature change, the kit can be directly used for the determination of the trypsin and the content of the inhibitor of the trypsin. The rapid detection method provided by the invention does not need complicated chemical modification or signal labeling, has strong anti-interference capacity, can quickly and accurately realize the rapid detection of the trypsin, and is suitable for clinical application, for example the diagnosis of diseases such as pancreatitis.

Description

technical field [0001] The invention relates to a novel trypsin content detection method and a detection kit, in particular to a trypsin rapid detection method based on platinum nano-cluster simulated oxidase and a detection kit thereof, which belong to the technical field of nanobiological sensing. Background technique [0002] Trypsin (TRY) is a kind of serine protease produced by the pancreas. Its main function is to hydrolyze the protein discrete cells between cells. It widely exists in the digestive system of vertebrates and also plays a key role in regulating the exocrine function of the pancreas. Studies have shown that the level of trypsin in the blood of patients with acute pancreatitis or pancreas transplantation (1.4±0.6 μg / mL) is higher than that of normal healthy people (0.25±0.1 μg / mL). The levels in body fluids can be used as biomarkers of pancreatic function and pathological changes. To sum up, we can diagnose whether the human pancreas is functioning normal...

Claims

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Application Information

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IPC IPC(8): G01N21/78G01N21/33G01N21/31
Inventor 林丽清林新华林晓昀赵成飞吴丽娜
Owner FUJIAN MEDICAL UNIV
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