CN104897846A - Alkaline phosphatase activity assay method based on in-situ formation of optical active nanometer material mimic enzyme - Google Patents
Alkaline phosphatase activity assay method based on in-situ formation of optical active nanometer material mimic enzyme Download PDFInfo
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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:
The present invention relates to nanosecond science and technology field and bioanalysis detection field, particularly relate to novel non-photoactive nanoparticles material simulation enzyme and the application in alkaline phosphatase activities detects thereof.
Background technology:
Alkaline phosphatase (ALP) is a kind of enzyme be extensively present in various biological tissue.Closely related [the Colombatto P. of generation that there is the various diseases such as concentration and skeletal diseases, hepatitis and prostate cancer of ALP; 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.; Brunetto M.R.J.Viral Hepatitis 1996,3,301-306].In addition, it is high that ALP has catalytic activity, good stability, and cost is low, and the advantage such as substrate specificity, is a kind of immunoassay label be widely used, is used to [Lei J. in enzyme-linked immunoassay widely; Ju H.Chem.Soc.Rev.2012,41,2122-2134; Akanda M.R.; Tamilavan V.; Park S.; Jo K.; Hyun M.H.; Yang H.Anal.Chem.2013,85,1631-1636; Xian Y.L.; Wang Z.; Jiang X.Y.ACS Nano 2014,8,12741 – 12747; Geraud E.; Prevot V.; Forano C.; Mousty C.Chem.Commun.2008,13,1554 – 1556].In enzyme-linked immunoassay, between antigen and antibody, specific combination can be produced, produce measuring-signal by the catalytic reaction being marked at the ALP on antibody.Because single enzyme molecule can produce a large amount of products by catalysis many substrate molecules, thus detection signal is made to amplify [Zhang B.; Tang D.; Goryacheva I.; Niessner R.; Knopp D.Chem.-Eur.J.2013,19,2496-2503].At present, be that the Enzyme Linked Immunoadsorbent Assay of label has grown into one of important technology [Qu W. in environment measuring, food security and clinical diagnosis etc. with native enzyme; LiuY.; Liu D.; Wang Z.; Jiang X.Angew.Chem.Int.Ed.2011,50,3442-3445].Therefore, the detection of ALP activity is significant in clinical disease diagnosis and immunoassay research.
At present, although major part is comparatively simple for the detection method of ALP, electrochemical signals or the light signal of the product relying on merely ALP catalysis to produce due to these class methods detect, lack signal and amplify strategy, the sensitivity of method is not very high [Gao Z.; Xu M.; Hou L.; Chen G.; Tang D.Anal.Chem.2013,85,6945 – 6952; Zhang L.; Zhao J.; Duan M.; Zhang H.; Jiang J.; Yu R.Anal.Chem.2013,85,3797-3801; Wei H.; Chen C.; Han B.; Wang E.Anal.Chem.2008,80,7051 – 7055; Qian Z.; Chai L.; Tang C.; Huang Y.; Chen J.; Feng H.Anal.Chem.2015,87,2966-2973].So, explore efficient signal and amplify strategy remains needs solution problem with the detection method setting up high-sensitive ALP activity.
Along with the development of nanosecond science and technology, because of it, there is quick, sensitive feature based on the analytical approach of nano material and be more prone to the advantage such as microminiaturized and cause everybody and pay close attention to widely.It is active that nano material analogue enztme has efficient analogue enztme, and have good stability, and enzymatic activity such as easily to regulate at feature [the Wei H.; Wang E.Chem.Soc.Rev.2013,42,6060 – 6093; Lin Y.H.; Ren J.S.; Qu X.G., Acc.Chem.Res.2014,47,1097 – 1105].Although the nano material with peroxidase activity has good activity, lack means easily and control and trigger its class enzymatic activity.In addition, this fermentoid operationally rely on there is harmfulness hydrogen peroxide as electron accepter.These problems seriously limit the activity of superoxide nano material analogue enztme, biocompatibility and [the Tao Y. of the application in bioanalysis thereof; Lin Y.H.; Huang Z.Z.; Ren J.S.; Qu X.G.Adv.Mater.2013,25,2594 – 2599].Current nano material analogue enztme is nearly all adopt certain chemical reagent reaction to prepare rear use.Different from current research, the present invention utilizes the catalytic reaction original position of native enzyme enzyme to define a kind of non-photoactive nanoparticles material simulation enzyme with higher catalytic activity.Can there is specific complex reaction with the titanium on titanium dioxide nano material surface (IV) in the compound containing two hydroxyl that alkaline phosphatase (ALP) catalytic phosphatase ester class substrate produces as the hydrolysis reaction of catechol phosphate, salicylic acid phosphate, form charge transfer complex at nano-material surface.Independent titanium dioxide nano material does not show photolytic activity analogue enztme character, and the titanium dioxide nano material combining dihydroxyl compound can produce higher simulation oxidase active under visible ray illumination condition.This oxidasic activity can be regulated and controled easily by illumination; Under the existence not relying on hydrogen peroxide, show very high quasi-enzyme catalytic active, show that it has better stability and biocompatibility.Compared with most of nano material analogue enztme, the feature of this method is that this nanometer photolytic activity analogue enztme is produced by the situ catalytic reaction of ALP; (rely on merely the galvanochemistry/optical signalling of the product of catalytic reaction) compared with the ALP activity test method of routine, the nanometer photolytic activity analogue enztme that the catalysate original position of ALP is formed creates good signal amplification to the detection of ALP activity further.Achieve the highly sensitive detection of ALP activity, detectability is low to moderate 0.01U/L, far below content 40-190U/L [the Hausamen T.U. of ALP in HAS; Helger R.; Rick W.; Gross W.Clin.Chim.Acta 1967,15,241-245].Meet the detection demand of ALP in actual sample.As far as we know, the non-photoactive nanoparticles material simulation enzyme utilizing enzymic catalytic reaction original position to be formed carries out Enzyme assay, there is no bibliographical information.The method the detection of ALP and with ALP be label immunoassay in have broad application prospects.
Summary of the invention:
The object of this invention is to provide a kind of alkaline phosphatase activities detection method based on non-photoactive nanoparticles material simulation enzyme, this non-photoactive nanoparticles material simulation enzyme can be produced by the catalytic reaction original position of alkaline phosphatase; The efficient quasi-enzyme catalytic activity of the non-photoactive nanoparticles material simulation enzyme utilizing alkaline phosphate ester enzymic catalytic reaction to be formed realizes signal and amplifies, can facilitate, sensitive, detect alkaline phosphatase activities rapidly.
Object of the present invention realizes by following technical measures:
The preparation of a, titanium dioxide nano material: 5mL titanium-containing compound is dropwise joined in 75mL ultrapure water, mixed solution at room temperature Keep agitation.Then above-mentioned solution to be transferred in reactor and to be heated to 160 DEG C of lasting 24h.Centrifugal acquisition product and with milli-Q water repeatedly, finally dry and obtain white titania nano particle;
The mensuration of b, alkaline phosphatase activities: join in 96 microwell plates after the substrate of the alkaline phosphatase of 5 μm of ol/L mixes with the alkaline phosphatase of 10 μ L variable concentrations, react a period of time under room temperature; After adding the titanium dioxide nano material reaction 15min of 10 μ L 1.5mg/mL, add the feature substrate of the nano material analogue enztme of hac buffer that 100 μ L pH are the 0.2mol/L of 4.0 and 20 μ L 5mmol/L, after irradiating 10min with visible ray (λ >=400nm) under being placed on xenon lamp, and in microplate reader, measure absorption spectrum or the fluorescence spectrum of system.
Object of the present invention also realizes by following technical measures:
Described titanium-containing compound, is selected from butyl titanate, titanium tetrachloride; Described phosphate substrate, is selected from catechol phosphate, salicylic acid phosphate; The feature substrate of described non-photoactive nanoparticles material simulation enzyme has TMB, 2, two (3-ethyl benzo thiazole phenanthroline-6-sulfonic acid) di-ammonium salts of 2 '-Lian nitrogen base, 10-acetyl group-3,7-dihydroxyphenazine.
Accompanying drawing illustrates:
Fig. 1 is (A) scanning electron microscope and (B) transmission electron microscope picture of the titanium dioxide nano material of invention preparation.
Fig. 2 is the abosrption spectrogram of different material under illumination condition: (a) TMB; The potpourri of (b) TMB and catechol; The potpourri of (c) TMB and titanium dioxide nano material; (d) catechol phosphate, alkaline phosphatase, the potpourri of TMB and titanium dioxide nano material.The concentration of TMB is 5 × 10
-4mol/L.
Fig. 3 is that different reactive intermediate scavenger is on the impact (substrate is TMB) of the photolytic activity analogue enztme performance of the titania nanoparticles in conjunction with catechol.
Fig. 4 is with catechol phosphate for alkaline phosphate ester zymolyte, detects linear relationship chart (a) and the selectivity (b) of alkaline phosphatase when using TMB to make nano material analogue enztme substrate.
Embodiment 1:
A, 5mL butyl titanate is dropwise joined in 75mL ultrapure water, mixed solution at room temperature Keep agitation.Then above-mentioned solution to be transferred in reactor and to be heated to 160 DEG C of lasting 24h.Centrifugal acquisition product and with milli-Q water repeatedly, finally, dry and obtain white titania nano particle;
Join in 96 orifice plates after the catechol phosphate of b, 5 μm of ol/L mixes with the alkaline phosphatase of 10 μ L variable concentrations, under room temperature, react 1h; After adding the titanium dioxide nano material reaction 15min of 10 μ L 1.5mg/mL, add the feature substrate 3 of hac buffer that 100 μ L pH are the 0.2mol/L of 4.0 and 20 μ L 5mmol/L, 3 ', 5,5 '-tetramethyl benzidine, after irradiating 10min with visible ray (λ>=400nm) under being placed on xenon lamp, 3,3 ', 5, the characteristic absorption (λ of the oxidation product of 5 '-tetramethyl benzidine
max=652nm) place's mensuration absorption spectrum.
Embodiment 2:
A, 5mL titanium tetrachloride is dropwise joined in 75mL ultrapure water, mixed solution at room temperature Keep agitation.Then above-mentioned solution to be transferred in reactor and to be heated to 160 DEG C of lasting 24h.Centrifugal acquisition product and with milli-Q water repeatedly, finally obtain white titania nano particle in oven dry;
Join in 96 orifice plates after the salicylic acid phosphate of b, 5 μm of ol/L mixes with the alkaline phosphatase of 10 μ L variable concentrations, under room temperature, react 1h; After adding the titanium dioxide nano material reaction 15min of 10 μ L 1.5mg/mL, add the feature substrate 2 of hac buffer that 100 μ L pH are the 0.2mol/L of 4.0 and 20 μ L 5mmol/L, two (3-ethyl benzo thiazole phenanthroline-6-sulfonic acid) di-ammonium salts of 2 '-Lian nitrogen base, after irradiating 10min with visible ray (λ>=400nm) under being placed on xenon lamp, at the characteristic absorption (λ of the oxidation product of two (3-ethyl benzo thiazole phenanthroline-6-sulfonic acid) di-ammonium salts of 2,2 '-Lian nitrogen bases
max=417nm) place's mensuration absorption spectrum.
Embodiment 3:
A, 5mL butyl titanate is dropwise joined in 75mL ultrapure water, mixed solution at room temperature Keep agitation.Then above-mentioned solution to be transferred in reactor and to be heated to 160 DEG C of lasting 24h.Centrifugal acquisition product and with milli-Q water repeatedly, finally obtain white titania nano particle in oven dry;
Join in 96 orifice plates after the catechol phosphate of b, 5 μm of ol/L mixes with the alkaline phosphatase of 10 μ L variable concentrations, under room temperature, react 1h; After adding the titanium dioxide nano material reaction 15min of 10 μ L 1.5mg/mL, add the feature substrate 2 of hac buffer that 100 μ L pH are the 0.2mol/L of 4.0 and 20 μ L 5mmol/L, two (3-ethyl benzo thiazole phenanthroline-6-sulfonic acid) di-ammonium salts of 2 '-Lian nitrogen base, after irradiating 10min with visible ray (λ>=400nm) under being placed on xenon lamp, at the characteristic absorption (λ of the oxidation product of two (3-ethyl benzo thiazole phenanthroline-6-sulfonic acid) di-ammonium salts of 2,2 '-Lian nitrogen bases
max=417nm) place's mensuration absorption spectrum.
Claims (4)
1. form an alkaline phosphatase activities detection method for non-photoactive nanoparticles material simulation enzyme based on original position, it is characterized in that:
The preparation of a, titanium dioxide nano material: 5mL titanium-containing compound is dropwise joined in 75mL ultrapure water, mixed solution at room temperature Keep agitation.Then above-mentioned solution to be transferred in reactor and to be heated to 160 DEG C of lasting 24h.Centrifugal acquisition product and with milli-Q water repeatedly, finally dry and obtain white titania nano particle;
The mensuration of b, alkaline phosphatase activities: join in 96 orifice plates after the substrate of the alkaline phosphatase of 5 μm of ol/L mixes with the alkaline phosphatase of 10 μ L variable concentrations, react a period of time under room temperature; After adding the titanium dioxide nano material reaction 15min of 10 μ L 1.5mg/mL, add the feature substrate of the nano material analogue enztme of hac buffer that 100 μ L pH are the 0.2mol/L of 4.0 and 20 μ L 5mmol/L, after irradiating 10min with visible ray (λ >=400nm) under being placed on xenon lamp, microplate reader measures absorption spectrum or fluorescence spectrum.
2. a kind of alkaline phosphatase activities detection method forming non-photoactive nanoparticles material simulation enzyme based on original position according to claim 1, the raw material selected when preparing titanium dioxide nano material shown in it is characterized in that, is selected from butyl titanate, titanium tetrachloride.
3. a kind of alkaline phosphatase activities detection method forming non-photoactive nanoparticles material simulation enzyme based on original position according to claim 1, is characterized in that the substrate of described alkaline phosphatase, is selected from catechol phosphate, salicylic acid phosphate.
4. a kind of alkaline phosphatase activities detection method forming non-photoactive nanoparticles material simulation enzyme based on original position according to claim 1; it is characterized in that the feature substrate of described nano material analogue enztme has 3; 3 '; 5; 5 '-tetramethyl benzidine, 2; 2 '-Lian nitrogen base two (3-ethyl benzo thiazole phenanthroline-6-sulfonic acid) di-ammonium salts, 10-acetyl group-3,7-dihydroxyphenazine.
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| CN107907683A (en) * | 2017-12-17 | 2018-04-13 | 江南大学 | A kind of activity of acid phosphatase detection method based on light-operated enzyme cascade |
| CN108896506A (en) * | 2018-07-16 | 2018-11-27 | 济南大学 | The method of detection of alkaline phosphatase activity and Inhibitors of Alkaline Phosphatase concentration |
| CN110501317A (en) * | 2019-08-27 | 2019-11-26 | 中国科学院长春应用化学研究所 | A kind of fluorescence detection method of alkaline phosphatase activities |
| CN111220609A (en) * | 2020-02-05 | 2020-06-02 | 江苏大学 | Based on CeVO4Colorimetric detection method of alkaline phosphatase Activity |
| CN111229193A (en) * | 2020-01-15 | 2020-06-05 | 重庆师范大学 | Application of zirconium dioxide nano particles as alkaline phosphatase nano mimics |
| CN111638212A (en) * | 2020-06-15 | 2020-09-08 | 江南大学 | Method for detecting content of glucose-6-phosphate based on nano enzyme |
| CN115057472A (en) * | 2022-06-21 | 2022-09-16 | 中国医学科学院基础医学研究所 | Novel fluorescence sensing system and application thereof in PTP-1B detection |
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| CN107907683A (en) * | 2017-12-17 | 2018-04-13 | 江南大学 | A kind of activity of acid phosphatase detection method based on light-operated enzyme cascade |
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| CN111229193B (en) * | 2020-01-15 | 2022-10-18 | 重庆师范大学 | Application of zirconium dioxide nano particles as alkaline phosphatase nano mimics |
| CN111220609B (en) * | 2020-02-05 | 2022-04-26 | 江苏大学 | Based on CeVO4Colorimetric detection method of alkaline phosphatase Activity |
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