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Alkaline phosphatase activity assay method based on in-situ formation of optical active nanometer material mimic enzyme

A nanomaterial, phosphatase technology, applied in the field of nanotechnology and biological analysis and detection

Active Publication Date: 2015-09-09
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 enzyme activity detection using photoactive nanomaterials mimicking enzymes formed in situ for enzyme-catalyzed reactions.

Method used

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  • Alkaline phosphatase activity assay method based on in-situ formation of optical active nanometer material mimic enzyme
  • Alkaline phosphatase activity assay method based on in-situ formation of optical active nanometer material mimic enzyme
  • Alkaline phosphatase activity assay method based on in-situ formation of optical active nanometer material mimic enzyme

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Experimental program
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Effect test

Embodiment 1

[0016] a. Add 5 mL of tetrabutyl titanate dropwise into 75 mL of ultrapure water, and keep stirring the mixed solution at room temperature. The above solution was then transferred into a reaction kettle and heated to 160 °C for 24 h. The product was obtained by centrifugation and washed several times with ultrapure water, and finally, dried to obtain white titanium dioxide nanoparticles;

[0017] b. 5 μmol / L catechol phosphate was mixed with 10 μL alkaline phosphatase of different concentrations and added to a 96-well plate, and reacted at room temperature for 1 h; after adding 10 μL 1.5 mg / mL titanium dioxide nanomaterials and reacted for 15 min, Add 100 μL of 0.2 mol / L acetate buffer solution with a pH of 4.0 and 20 μL of 5 mmol / L characteristic substrate 3,3',5,5'-tetramethylbenzidine, place it under a xenon lamp with visible light (λ≥400nm) After irradiating for 10min, the characteristic absorption (λ max =652nm) to measure the absorption spectrum.

Embodiment 2

[0019] a. Add 5 mL of titanium tetrachloride dropwise to 75 mL of ultrapure water, and keep stirring the mixed solution at room temperature. The above solution was then transferred into a reaction kettle and heated to 160 °C for 24 h. The product was obtained by centrifugation and washed several times with ultrapure water, and finally dried to obtain white titanium dioxide nanoparticles;

[0020]b. 5 μmol / L salicylic acid phosphate was mixed with 10 μL alkaline phosphatase of different concentrations and added to a 96-well plate, and reacted at room temperature for 1 hour; 100 μL of 0.2 mol / L acetate buffer solution with a pH of 4.0 and 20 μL of 5 mmol / L characteristic substrate 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt, After placed under a xenon lamp and irradiated with visible light (λ≥400nm) for 10 minutes, the characteristic absorption of the oxidation product of 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt ( lambda ...

Embodiment 3

[0022] a. Add 5 mL of tetrabutyl titanate dropwise into 75 mL of ultrapure water, and keep stirring the mixed solution at room temperature. The above solution was then transferred into a reaction kettle and heated to 160 °C for 24 h. The product was obtained by centrifugation and washed several times with ultrapure water, and finally dried to obtain white titanium dioxide nanoparticles;

[0023] b. 5 μmol / L catechol phosphate was mixed with 10 μL alkaline phosphatase of different concentrations and added to a 96-well plate, and reacted at room temperature for 1 h; after adding 10 μL 1.5 mg / mL titanium dioxide nanomaterials and reacted for 15 min, Add 100 μL of 0.2 mol / L acetate buffer solution with pH 4.0 and 20 μL of 5 mmol / L characteristic substrate 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt , placed under a xenon lamp and irradiated with visible light (λ≥400nm) for 10 minutes, the characteristic absorption of the oxidation product of 2,2'-azinobi...

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Abstract

The invention provides an alkaline phosphatase activity assay method with high-efficiency signal amplifying function based on in-situ formation of optical active nanometer material mimic enzyme. Hydrolysate products of ALP like catechol or salicylic acid can be combined to the surface of TiO2 nanometer material specially to form charge transfer complex, so that the TiO2 nanometer material can express efficient mimic enzymatic activity under irradiation of visible light. Compared with conventional nanometer mimic enzyme, the optical active mimic enzyme is generated in situ by catalytic reaction of natural enzyme and has excellent biocompatibility without high-concentration H2O2 during use. The optical active nanometer mimic enzyme generated in situ by catalytic reaction of alkaline phosphatase can generate high-efficiency signal amplifying function for activity assay of alkaline phosphatase, so that detection limit of ALP activity is up to 0.01 U / L. The alkaline phosphatase activity assay method has the advantages of simplicity, flexibility and fastness.

Description

Technical field: [0001] The invention relates to the field of nanotechnology and the field of biological analysis and detection, in particular to a novel photoactive nanometer material imitating enzyme and its application in the detection of alkaline phosphatase activity. Background technique: [0002] Alkaline phosphatase (ALP) is an enzyme widely present in various biological tissues. The concentration of ALP is closely related to the occurrence of various diseases such as bone disease, hepatitis and prostate cancer [Colombatto P.; Randone A.; Civitico G.; Gorin J.M.; Dolci L.; Medaina N.; Oliveri F.; Verme G .; Marchiaro G.; Pagni R.; Karayiannis P.; Thomas H.C.; Hess G.; Bonino F.; In addition, ALP has the advantages of high catalytic activity, good stability, low cost, and broad substrate specificity. It is a widely used immunoassay marker and is used in enzyme-linked immunoassays [Lei J.; Ju H.Chem.Soc.Rev.2012,41,2122-2134; Akanda M.R.; Tamilavan V.; Park S.; Jo K.;...

Claims

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Application Information

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IPC IPC(8): G01N31/10G01N21/33G01N21/64
Inventor 王光丽金璐怡吴秀明曹根霞
Owner JIANGNAN UNIV
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