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Acid phosphatase detection method based on cysteamine-N-acetyl-L-cysteine-gold nanocluster fluorescent material

A gold nanocluster, acid phosphatase technology, applied in analytical chemistry and nano fields, can solve the problems of using toxic metal ions, complicated operation steps, interference, etc., and achieve the effects of strong specificity, high detection sensitivity and low cost

Active Publication Date: 2019-12-20
FUJIAN MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the currently developed acid phosphatase fluorescence analysis method has deficiencies in varying degrees, such as cumbersome operation steps, the use of toxic metal ions, and the interference of other enzymes, etc.

Method used

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  • Acid phosphatase detection method based on cysteamine-N-acetyl-L-cysteine-gold nanocluster fluorescent material
  • Acid phosphatase detection method based on cysteamine-N-acetyl-L-cysteine-gold nanocluster fluorescent material
  • Acid phosphatase detection method based on cysteamine-N-acetyl-L-cysteine-gold nanocluster fluorescent material

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Preparation of gold nanoclusters: Mix 0.75 mL cysteamine (30 mmol / L) and 0.25 mL N-acetyl-L-cysteine ​​(30 mmol / L) in advance, add 8 mL ultrapure water and mix well, then Add 1 mL of chloroauric acid (20 mmol / L), incubate in a 90°C water bath for 1.5 h, take out and centrifuge to remove large particles of nanoparticles, use a dialysis bag with a molecular weight cut-off of 3500, and dialyze in double distilled water for 24 h to obtain Co-protected gold nanocluster solution with cysteamine and N-acetyl-L-cysteine. All glassware used in the preparation was soaked in aqua regia, washed thoroughly with double-distilled water, and air-dried.

Embodiment 2

[0033] Add 0.2 mL of pyridoxal phosphate solution with a concentration of 25 μmol / L and 0.2 mL of acid phosphatase solution with a final concentration of 5 U / L into 1.45 mL of acetate buffer (pH 5.0, 50 mmol / L), After mixing, place in a 37°C water bath for 30 min. After the end, 0.15 mL of the gold nanocluster solution prepared in Example 1 was mixed with the above reaction solution, and the emission spectrum was measured immediately after mixing (excitation wavelength was 425 nm). Set up a control group. It can be seen from the figure that the gold nanocluster solution itself has obvious emission at 590nm ( figure 1 A in A); when pyridoxal phosphate was added, the fluorescence of the gold nanocluster solution was significantly inhibited ( figure 1 B in B); and when the system contains acid phosphatase, the fluorescence quenching of gold nanocluster solution caused by pyridoxal phosphate can be restored ( figure 1 in C).

Embodiment 3

[0035] Add 0.2 mL of pyridoxal phosphate solution with a concentration of 25 μmol / L and 0.2 mL of acid phosphatase solution with a final concentration of 5 U / L to 1.45 mL of acetate buffer (50 mmol / L), mixed well and placed in a 37°C water bath for 30 min. After the end, 0.15 mL of the gold nanocluster solution prepared in Example 1 was mixed with the above reaction solution, and then the fluorescence emission intensity value of the solution at 590 nm (F 590 ) (excitation wavelength is 425 nm), each group was measured three times in parallel. A control group (without acid phosphatase) was set under each pH condition. According to the fluorescence emission intensity F between the experimental group and the control group 590 The difference (△F) to judge the optimal reaction conditions of the reaction system. Depend on figure 2 It can be seen that the optimal reaction pH of the acid phosphatase assay system is 5.0.

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Abstract

The invention discloses an acid phosphatase detection method based on a cysteamine-N-acetyl-L-cysteine-gold nanocluster fluorescent material. After specificities of a cysteamine-N-acetyl-L-cysteine-gold nanocluster and pyridoxal phosphate are interacted, the fluorescence of the gold nanocluster is inhibited; acid phosphatise hydrolyzes pyridoxal phosphate to release pyridoxal; the inhibiting effect of pyridoxal phosphate on the fluorescence of the cysteamine-N-acetyl-L-cysteine-gold nanocluster can be relieved; therefore, change of light emitting spectral intensity characteristic is shown; therefore, the material is used for content detection of acid phosphatise; the linear range for acid phosphatise detection is 0.1-5 U / L; and the detection limit is 0.05 U / L. The detection method has theadvantages of being simple and convenient to operate, short in time consumption, high in sensitivity, high in specificity, green, environment-friendly and the like, and is easy for popularization anduse.

Description

technical field [0001] The invention provides a novel acid phosphatase activity detection method based on cysteamine-N-acetyl-L-cysteine-gold nano-cluster fluorescent material, and belongs to the field of analytical chemistry and nanotechnology. Background technique [0002] As we all know, acid phosphatase is an enzyme that can catalyze the hydrolysis of monophosphate under mildly acidic conditions. The change of acid phosphatase content is an important indicator for the diagnosis of prostate cancer, thrombophlebitis, Gaucher disease, Paget's disease, hepatitis, hyperparathyroidism and multiple myeloma. Therefore, it is of great clinical significance to develop a quick and easy detection method for acid phosphatase. [0003] Fluorescence analysis is widely used in the determination of acid phosphatase because of its advantages of high sensitivity, good selectivity, low cost, fast signal response time, and simple operation. However, the currently developed acid phosphatase...

Claims

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Application Information

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IPC IPC(8): G01N21/64
CPCG01N21/6428G01N21/6486G01N2021/6432
Inventor 邓豪华黄开源陈伟查代君孙伟明
Owner FUJIAN MEDICAL UNIV
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