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Method for detecting activity of acid phosphatase based on light-controlled enzyme cascade reaction

An acid phosphatase, activity detection technology, applied in the field of nano-biological analysis and detection, can solve the problem of no literature report and so on

Active Publication Date: 2018-04-13
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

To the best of our knowledge, there is no literature report on the use of photoactive nanomaterials formed in situ for enzyme-catalyzed reactions to construct enzyme cascade catalytic systems.

Method used

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  • Method for detecting activity of acid phosphatase based on light-controlled enzyme cascade reaction
  • Method for detecting activity of acid phosphatase based on light-controlled enzyme cascade reaction
  • Method for detecting activity of acid phosphatase based on light-controlled enzyme cascade reaction

Examples

Experimental program
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Effect test

Embodiment 1

[0015] a. Preparation of titanium dioxide nanomaterials: Slowly add 1.0mL of titanium tetrachloride to 10mL of deionized water at 0°C, stir to obtain a white solution, and then heat to obtain a milky white titanium dioxide suspension; the obtained suspension is filtered Washing, drying the obtained product to obtain the solid powder of titanium dioxide nanomaterials;

[0016] b. Determination of acid phosphatase activity: Add 10 μL of acid phosphatase of different concentrations and 10 μL of 5 mmol / L catechol phosphate solution in a microwell plate, mix and shake well in an acetate buffer solution with pH=6.0, and store at 37°C Incubate for 1 h with shaking; then add 10 μL of 0.15 mg / mL TiO 2 Nanoparticle solution, 40 μL 0.4g / L horseradish peroxidase, 100 μL 40 mmol / L acetate buffer solution with pH 4.0 and 20 μL 5 mmol / L 3,3',5,5'-tetramethylbenzidine solution, After mixing evenly, place the microplate under a xenon lamp (λ≥400nm) to irradiate for 15min, and measure the abso...

Embodiment 2

[0018] a. Preparation of titanium dioxide nanomaterials: Slowly add 1.5mL of titanium tetrachloride into 10mL of deionized water at 0°C, stir to obtain a white solution, and then heat to obtain a milky white titanium dioxide suspension; the obtained suspension is filtered Washing, drying the obtained product to obtain the solid powder of titanium dioxide nanomaterials;

[0019] b. Determination of acid phosphatase activity: Add 10 μL of different concentrations of acid phosphatase and 10 μL of 5 mmol / L salicylic acid phosphate solution in a microwell plate, mix and shake well in an acetate buffer solution with pH=6.0, and then store at 37°C Shake and incubate for 1 h; then add 10 μL of 0.15 mg / mL TiO 2 Nanoparticle solution, 40μL 0.4g / L horseradish peroxidase, 100μL 40mmol / L acetate buffer solution with pH 4.0 and 20μL 0.01mol / L 2,2'-azinobis(3-ethylbenzo Thiazoline-6-sulfonic acid) diammonium salt solution, after mixing evenly, place the microplate under a xenon lamp (λ≥400n...

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Abstract

The invention relates to a method for detecting activity of acid phosphatase based on light-controlled enzyme cascade reaction. The method is characterized in that a novel enzyme cascade catalyzing reaction system is established by a photoactivity nanometer material which is produced by natural enzyme through catalyzing reaction in situ, namely the catalyzing product of the acid phosphatase can becombined to the surface of titanium dioxide nanoparticle in situ to form the photoactivity nanometer material; the photoactivity nanometer material is used for activating horse radish peroxidase to catalyze and oxidize the reaction of feature substrate under the radiation of visible light, so as to form the enzyme cascade catalyzing system which can be controlled by light to realize accurate regulating and controlling effects. The novel light-controlled enzyme cascade catalyzing system has the advantages that the high-efficiency triple signal amplifying function is realized, and the acid phosphatase can be simply and conveniently determined at high efficiency and high selectivity; the detection limit of ALP activity can reach 1.0mU / L.

Description

Technical field: [0001] The invention relates to the field of nano-biological analysis and detection, in particular to a novel nano-material-mediated enzyme cascade catalytic reaction system and its application in the detection of acid phosphatase activity. Background technique: [0002] Enzyme-catalyzed cascade reactions have many advantages, such as high reaction efficiency, mild reaction conditions, and high selectivity. Therefore, its role in biocatalysis [You C.; Myung S.; Zhang Y.-H.P. Angew. Chem., Int. Ed. 2012, 51, 8787-8790; Simon R C.; Nina Richter N.; Busto E. ; Wolfgang Kroutil W.ACS Catal.2014,4,129-143], Biosensing [Freeman R.; Sharon E.; Tel-Vered R.; Itamar Willner I.J.Am.Chem.Soc.2009,131,5028– 5029; Eguílaz M.; Villalonga R.; P.; J.M.Anal.Chem.2011, 83, 7807-7814; Hu Y.W.; Wang F.; Lu C.H.; Girsh J.; Synthetic biology [Dueber J.E.; Wu G.C.; Malmirchegini G.R.; Moon T.S.; Petzold C.J.; Ullal A.V.; Prather K.L.J.; Keasling J.D.Nat.Biotechnol. Especial...

Claims

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Application Information

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IPC IPC(8): G01N33/573G01N33/535G01N21/78
Inventor 王光丽曹根霞李苹董玉明
Owner JIANGNAN UNIV
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