CN111638212B - Method for detecting content of glucose-6-phosphate based on nano enzyme - Google Patents
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Abstract
The invention discloses a method for detecting the content of glucose-6-phosphate based on nanoenzyme, belonging to the field of nano-bioanalysis detection. The method generates the photoactive nano material mimic enzyme in situ by coupling glucose-6-phosphate dehydrogenase (G6 PD)/P-hydroxybenzoic acid hydroxylase (PHBH), has multiple signal amplification effects, and realizes the sensitive detection of glucose-6-phosphate (G-6-P); in particular to 3, 4-dihydroxybenzoic acid (PCA) and strontium titanate (SrTiO) generated by G6PD/PHBH enzyme cascade reaction3) The surface complex is combined to form a surface complex, has super strong mimic oxidase activity, and can oxidize and discolor a typical chromogenic substrate, namely 3,3',5,5' -Tetramethylbenzidine (TMB)/2,2' -biazabis-3-ethylbenzthiazoline-6-sulfonic Acid (ABTS). The method can detect G-6-P within the range of 0.05-100 mu M, has the detection limit as low as 0.022 mu M, and has very excellent application prospect.
Description
Technical Field
The invention relates to the field of nano biological analysis and detection, in particular to a method for detecting the content of glucose-6-phosphate based on nano enzyme.
Background
Phosphorylated carbohydrates are key intermediates in biochemical pathways, and different phosphorylation of glucose is associated with different biological processes. Glucose-6-phosphate (G-6-P) is the central point of glycolysis and glycogenesis, and is also the initial metabolite in the pentose phosphate pathway [ Nguyen a.h.; deutsch j.m.; xiao l.; schultz Z.D.anal.chem.2018,90,11062-11069 ]. Monitoring the G-6-P concentration in blood or human tissues is particularly important because it can directly reflect the relative activities of enzymes associated with many catabolic pathways, such as phosphoglucosidase, hexokinase, and phosphoglucose isomerase, etc. [ Banerjee s; sarkar p.; turner A.anal.biochem.2013,439, 194-200 ]. Furthermore, monitoring of G-6-P levels has been used to gain insight into human red blood cells [ Kirkman h.n.; gaetani g.f.j.biol.chem.1986,261, 4033-4038 ] and regulation of glucose-6-phosphate dehydrogenase (G6PDH) in rat hepatocytes [ Sapag-Hagar m.; lagunas r.; sols A. biochem. Biophys. Res. Commun.1973,50, 179-185 ]. Several methods for determining G-6-P concentration based on radiology, chromatography and spectroscopic techniques have been reported [ Kirkman h.n.; gaetani g.f.j.biol.chem.1986,261, 4033-4038; Sapag-Hagar M.; lagunas r.; sols A. biochem. Biophys. Res. Commun.1973,50, 179-185 ], but these methods are time consuming, labor intensive and require expensive instrumentation reagents.
The analysis method based on the nano material draws wide attention due to the advantages of rapidness, sensitivity, easy miniaturization and the like. The nano material mimic enzyme has high-efficiency mimic enzyme activity, and also has the characteristics of good stability, easy regulation of enzyme activity and the like [ Wei H ]; wang E.chem.Soc.Rev.2013,42, 6060-ion 6093; lin y.h.; ren J.S.; qu X.G., Acc.chem.Res.2014,47, 1097-1105 ]. Although we have been studying and reporting the application of nanoenzymes based on titanium dioxide composites in bioassays [ Jin l.y.; dong y.m.; wu x.m.; cao g.x.; wang G.L.anal.chem.2015,87,10429-10436], but its mimic enzyme activity is not ideal enough, and it is still necessary to find new nano-enzyme. The perovskite type metal oxide strontium titanate has excellent electronic and optical properties, good photochemical stability, higher catalytic efficiency and low cost, [ Kang h.w.; lim s.n.; park S.B.Int.J.hydrogen Energy 2012,37, 5540-. However, most of the current research on strontium titanate is limited to photocatalytic hydrogen production, photodegradation/reduction of organic pollutants [ Lee j.t.; chen y.j.; su e.c.; wey M.Y.Int.J.hydrog.energy 2019,44, 21413-21423; wu Z.; zhang y.; wang X.; zuuz.new j.chem.2017,41, 5678-; wan c.; park n.h.; tsuruta k; seo w.s.; koumoto k.acs appl.mater.interfaces 2013,5, 10933-; ede S.R.; nithiyananthambc u.; kundu S.New J.chem.2017,41,3473-3486, etc.; at present, no research on analysis and detection of strontium titanate applied to nano-enzyme is reported.
Disclosure of Invention
The invention provides a sensitive detection method of glucose-6-phosphate (G-6-P) with multiple signal amplification effects, which is used for generating an optically active nano material mimic enzyme in situ by coupling glucose-6-phosphate dehydrogenase (G6 PD)/P-hydroxybenzoic acid hydroxylase (PHBH). The invention utilizes the characteristic that strontium titanate is excellent and is easy to combine with alkylene glycol to form a surface complex, and the strontium titanate is used as nano enzyme to be applied to colorimetric biological detection for the first time, thus having better innovation. Due to the specific and high-efficiency affinity of the enediol ligand to the strontium titanate, the 3, 4-dihydroxy benzoic acid (PCA) and the strontium titanate (SrTiO) generated by the G6PD/PHBH enzyme cascade reaction3) The surface complex is combined to form a surface complex, has super strong mimic oxidase activity, and can oxidize and discolor a typical chromogenic substrate, namely 3,3',5,5' -Tetramethylbenzidine (TMB)/2,2' -biazabis-3-ethylbenzthiazoline-6-sulfonic Acid (ABTS).
The invention aims to provide a glucose-6-phosphate detection method based on a photoactive nano material mimic enzyme, wherein the photoactive nano material mimic enzyme can be generated in situ through a G6PD/PHBH enzyme cascade reaction; the high-efficiency enzyme-like catalytic activity of the photoactive nano material mimic enzyme formed by the G6PD/PHBH enzyme cascade reaction is utilized to realize signal amplification, and glucose-6-phosphate can be conveniently, sensitively and rapidly detected.
A method for determining the content of glucose-6-phosphate based on nanoenzyme, which comprises the following steps:
(1) mixing glucose-6-phosphate with different concentrations with glucose-6-phosphate dehydrogenase and oxidized coenzyme respectively, and incubating; then adding p-hydroxybenzoic acid, p-hydroxybenzoic acid hydroxylase and buffer solution respectively, and continuing to incubate to obtain a mixed solution;
(2) after incubation is finished, adding strontium titanate, a characteristic chromogenic substrate and a buffer solution into the mixed solution, and performing spectral scanning by using an enzyme-linked immunosorbent assay under the irradiation of visible light to obtain absorbance increment under different concentrations; the increase in absorbance (A-A)0) For different glucose-6-phosphatesThe increase in absorbance at a concentration of 0 relative to the concentration of glucose-6-phosphate;
(3) and (3) constructing a linear relation by utilizing the concentration of the glucose-6-phosphate and the absorbance increment to obtain a linear measurement model.
In one embodiment of the invention, the method further comprises: according to the step (1) and the step (2), carrying out incubation treatment on a sample to be detected, and measuring a corresponding absorbance increment; and (4) calculating to obtain the concentration content of glucose-6-phosphate in the sample to be detected through the linear determination model in the step (3).
In one embodiment of the invention, the characteristic chromogenic substrate is 3,3',5,5' -Tetramethylbenzidine (TMB) and/or 2,2' -diazylbis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS).
In one embodiment of the invention, the oxidized coenzyme is selected from oxidized coenzyme I (NAD)+) And/or oxidized coenzyme II (NADP)+)。
In one embodiment of the present invention, in step (1), 0.05mM oxidized coenzyme, 14U/mL glucose-6-phosphate dehydrogenase, and glucose-6-phosphate of different concentrations are mixed and incubated at 30-40 ℃ for 10-15 min.
In one embodiment of the present invention, step (1) is to add 1.0mM parahydroxybenzoic acid, 11U/mL parahydroxybenzoic acid hydroxylase and 50mM buffer solution (pH 7.8), and incubate at 30-40 ℃ for 10-15 min.
In one embodiment of the present invention, the strontium titanate in step (2) can be prepared by the following method:
dissolving a titanium-containing compound in ethylene glycol to obtain a clear solution, then dripping a strontium salt solution into the clear solution, finally adding a NaOH solution, and uniformly mixing to form a mixed system; transferring the mixed system to a high-pressure kettle for reaction; and after the reaction is finished, washing and drying to obtain a white powder strontium titanate product.
In one embodiment of the invention, the molar ratio of titanium containing compound to strontium salt is 1: (1-5).
In one embodiment of the present invention, the titanium-containing compound is selected from tetrabutyl titanate and titanium tetrachloride.
In one embodiment of the present invention, the strontium salt is selected from strontium chloride and strontium nitrate.
In one embodiment of the invention, the concentration of the strontium salt is 0.1-0.5 mol/L.
In one embodiment of the present invention, the temperature of the reaction is 150-; the reaction time is 20-24 h.
In one embodiment of the present invention, the preparation method of strontium titanate specifically includes the following steps:
synthesizing the strontium titanate nano material: dissolving 5.0mmol of titanium-containing compound in 10mL of ethylene glycol to form a clear solution, then dripping 10mL of strontium salt solution with certain concentration into the clear solution, and finally adding 5.0mL of 5.0M NaOH solution; stirring for 30 minutes, transferring the mixture into a stainless steel autoclave with a polytetrafluoroethylene lining, and then heating for a certain time at a certain temperature; the resulting product was washed with water and ethanol until pH 7.0 was reached, then dried overnight at 70 ℃ to finally give a white powder product.
In one embodiment of the present invention, the method for measuring glucose-6-phosphate is specifically as follows:
(1)0.05mM of oxidized coenzyme, 14U/mL of glucose-6-phosphate dehydrogenase and glucose-6-phosphate with different concentrations are mixed and added to a 96-well plate and incubated at 30 ℃ for 10min, after the incubation is finished, 1.0mM of p-hydroxybenzoic acid, 11U/mL of p-hydroxybenzoic acid hydroxylase and 50mM of Tris-HCl buffer solution (pH 7.8) are added thereto, and the incubation is continued at 37 ℃ for 10 min;
(2) after incubation, sequentially adding 0.1mg/mL strontium titanate, 0.5mM nano material mimic enzyme characteristic chromogenic substrate and 0.2M NaAc/HAc buffer solution (pH is 4.0) into the mixed solution, irradiating for 10min under visible light, and performing spectrum scanning by using an enzyme labeling instrument to obtain absorbance increment corresponding to different concentrations;
(3) and (3) constructing a linear relation by utilizing the concentration of the glucose-6-phosphate and the corresponding absorbance increment to obtain a linear determination model.
The invention has the beneficial effects that:
the invention provides a method for colorimetric detection of G-6-P by enzyme cascade multiple signal amplification, which can detect G-6-P within the range of 0.05-100 mu M, and the detection limit is as low as 0.022 mu M. The method is a novel, simple and economic G-6-P detection method, the strontium titanate nano material is applied to optical colorimetric biological detection for the first time, and 3, 4-dihydroxy benzoic acid (PCA) and strontium titanate (SrTiO)3) The combination of (A) and (B) enables the enzyme to have super-strong simulated oxidase activity, and can obtain a very large detection signal in a short time, thereby avoiding the interference of background and false signals.
Drawings
FIG. 1 is a transmission electron micrograph of the strontium titanate nanomaterial prepared in example 1.
FIG. 2 is a graph of the absorption spectra of different substances under light conditions: (a)3, 3',5,5' -tetramethylbenzidine; (b) a mixture of 3,3',5,5' -tetramethylbenzidine and 3, 4-dihydroxybenzoic acid; (c) a mixture of 3,3',5,5' -tetramethylbenzidine and strontium titanate nanomaterial; (d)3, 4-dihydroxy benzoic acid, 3,3',5,5' -tetramethyl benzidine and strontium titanate nano material. The concentration of 3,3',5,5' -tetramethylbenzidine is 5X 10-4mol/L。
FIG. 3 is an absorption spectrum of the system in the presence of different concentrations of G-6-P (0,0.05,0.5,1.0,5.0,10,20,50, 100. mu.M) in example 1.
FIG. 4 is a graph showing the linear relationship between the increase in absorbance and the logarithmic value of G-6-P concentration in example 1.
FIG. 5 is a graph showing a G-6-P detection pattern in example 2 using ABTS as a chromogenic substrate; wherein (A) the absorption spectrum of the system in the presence of G-6-P (0,0.05, 0.1, 0.5,1.0,5.0,10, 50,100 mu M) with different concentrations; (B) a linear plot of the increase in absorbance versus the log of G-6-P concentration.
FIG. 6 is a detection spectrum of G-6-P performed with the titanium dioxide nanomaterial in comparative example 1; wherein (A) the absorption spectrum of the system in the presence of G-6-P (0,1,3,5,10,30,50,100, 130 mu M) with different concentrations; (B) a linear plot of the increase in absorbance versus the log of G-6-P concentration.
Detailed Description
Example 1:
a. synthesizing the strontium titanate nano material: dissolving 5.0mmol of titanium tetrachloride in 10mL of ethylene glycol to form a clear solution, then dropping 10mL of 0.5M strontium chloride solution thereto, and finally adding 5.0mL of 5.0M NaOH solution; after stirring for 30 minutes, the mixture was transferred to a stainless steel autoclave lined with polytetrafluoroethylene and then heated at 180 ℃ for 24 hours; washing the obtained product with water and ethanol until the pH reaches 7.0, and then drying at 70 ℃ overnight to finally obtain a white powder product;
b. determination of glucose-6-phosphate: 0.05mM oxidized coenzyme I, 14U/mL glucose-6-phosphate dehydrogenase, glucose-6-phosphate dehydrogenase at various concentrations (0,0.05,0.5,5,1,5,10,20,50, 100. mu.M) were mixed and added to a 96-well plate and incubated at 30 ℃ for 10min, after the incubation was completed, 1.0mM p-hydroxybenzoic acid, 11U/mL p-hydroxybenzoic acid hydroxylase and 50mM Tris-HCl buffer (pH 7.8) were added thereto, and the incubation was continued at 37 ℃ for 10 min; after the incubation, 0.1mg/ml of strontium titanate, 0.5mM of 3,3',5,5' -Tetramethylbenzidine (TMB), and 0.2M of NaAc/HAc buffer solution (pH 4.0) were added to the above mixed solution in this order, and after irradiation with visible light for 10min, the absorbance values at characteristic absorption (maximum wavelength 652nm) of samples of different concentrations were obtained by performing spectrum scanning using a microplate reader (see fig. 3), and the absorbance increase values (a-a) at different concentrations were calculated0) Wherein A is the absorbance value at that concentration; a. the0The absorbance value is 0;
c. constructing a linear measurement model: using the concentration and absorbance increment A-A of glucose-6-phosphate0And constructing a linear relation to obtain a measurement model. The results are shown in FIG. 4, where the linear model is: 0.1238LogCG-6-P+ 0.1617; coefficient of correlation R2Comprises the following steps: 0.9759, respectively; the linear range is: 0.05-100 μ M; the detection limit is as low as 0.016. mu.M.
Example 2:
a. synthesizing the strontium titanate nano material: dissolving 5.0mmol of tetrabutyl titanate in 10mL of ethylene glycol to form a clear solution, then dripping 10mL of 0.5M strontium nitrate solution into the clear solution, and finally adding 5.0mL of 5.0M NaOH solution; after stirring for 30 minutes, the mixture was transferred to a stainless steel autoclave lined with polytetrafluoroethylene and then heated at 180 ℃ for 24 hours; washing the obtained product with water and ethanol until the pH reaches 7.0, and then drying at 70 ℃ overnight to finally obtain a white powder product;
b. determination of glucose-6-phosphate: 0.05mM oxidized coenzyme II, 14U/mL glucose-6-phosphate dehydrogenase, glucose-6-phosphate dehydrogenase at various concentrations (0,0.05,0.5,5,1,5,10,20,50, 100. mu.M) were mixed and added to a 96-well plate and incubated at 30 ℃ for 10min, after the incubation was completed, 1.0mM p-hydroxybenzoic acid, 11U/mL p-hydroxybenzoic acid hydroxylase and 50mM Tris-HCl buffer (pH 7.8) were added thereto, and the incubation was continued at 37 ℃ for 10 min; after the incubation, 0.1mg/ml of strontium titanate, 0.5mM of 2,2' -diazylbis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) and 0.2M of NaAc/HAc buffer solution (pH 4.0) were added to the above mixed solution in this order, and after 10min of irradiation with visible light, a microplate reader was used to perform spectral scanning to obtain absorbance values at characteristic absorption (maximum wavelength 471nm) of samples of different concentrations, and absorbance increase values (a-a) at different concentrations were calculated0) Wherein A is the absorbance value at that concentration; a. the0The absorbance value is 0;
c. constructing a linear measurement model: using the concentration and absorbance increment A-A of glucose-6-phosphate0And constructing a linear relation to obtain a measurement model.
The result of detection of G-6-P using ABTS as a chromogenic substrate is shown in FIG. 5. The linear model constructed by the method is as follows: 0.1288LogCG-6-P+ 0.1992; the correlation coefficient is: 0.9895, respectively; the linear range is: 0.05-100 μ M; the detection limit is as low as 0.022 mu M.
Comparative example 1:
referring to step b of example 1, the photoactive material was replaced with titanium dioxide from strontium titanate, the other conditions were not changed, after irradiation for 10min under visible light, a microplate reader was used to perform spectral scanning (as shown in fig. 6), absorbance values at characteristic absorption (maximum wavelength 652nm) of samples with different concentrations were obtained, and absorbance increase values (a-a) at different concentrations were calculated0)；
Among them, titanium dioxide can be prepared by the following preparation processes (refer to anal. chem.2015,87, 10429-10436): firstly, 4.5mL of tetrabutyl titanate is added into 75mL of deionized water, stirred at room temperature for 2h, then the mixture is transferred into a stainless steel autoclave with a Teflon lining and heated at 433K for 24h, and then the obtained product is centrifugally washed and dried at 333K for 12h, thus obtaining TiO2And (4) nano powder.
Then the concentration and absorbance increment A-A of glucose-6-phosphate are utilized0Constructing a linear relation, and finding out that: under the same conditions, using TiO2The detection of glucose-6-phosphate is carried out, the obtained result is shown in figure 6, and the linear model constructed by the method is as follows: 0.1138LogCG-6-P+0.0712, correlation coefficient 0.9786, linear range 1.0-100 μ M, detection limit 0.36 μ M.
Claims (8)
1. A method for determining the glucose-6-phosphate content, comprising the steps of:
(1) mixing glucose-6-phosphate with different concentrations with glucose-6-phosphate dehydrogenase and oxidized coenzyme respectively, and incubating; then adding p-hydroxybenzoic acid, p-hydroxybenzoic acid hydroxylase and buffer solution respectively, and continuing to incubate to obtain corresponding mixed solution;
(2) after incubation is finished, respectively adding strontium titanate, a characteristic chromogenic substrate and a buffer solution into the mixed solution, and performing spectral scanning by using an enzyme-labeling instrument under the irradiation of visible light to obtain the absorbance increment under different concentrations; the absorbance increment is the absorbance increment when the concentration of different glucose-6-phosphate is 0 relative to the concentration of glucose-6-phosphate;
(3) constructing a linear relation by utilizing the increment of the concentration of the glucose-6-phosphate and the absorbance to obtain a linear measurement model;
mixing 0.05mM oxidized coenzyme, 14U/mL glucose-6-phosphate dehydrogenase and glucose-6-phosphate with different concentrations, and incubating for 10-15min at 30-40 ℃;
the preparation method of strontium titanate in the step (2) comprises the following steps:
dissolving a titanium-containing compound in ethylene glycol to obtain a clear solution, then dripping a strontium salt solution into the clear solution, finally adding a NaOH solution, and uniformly mixing to form a mixed system; transferring the mixed system to a high-pressure kettle for reaction; and after the reaction is finished, washing and drying to obtain a white powder strontium titanate product.
2. The method according to claim 1, wherein the characteristic chromogenic substrate is 3,3',5,5' -tetramethylbenzidine and/or 2,2' -diazylbis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt.
3. The method of claim 1, wherein the oxidized coenzyme is selected from oxidized coenzyme I and/or oxidized coenzyme II.
4. The method according to any one of claims 1-3, further comprising: according to the step (1) and the step (2), carrying out incubation treatment on the sample to be detected, and measuring a corresponding absorbance difference; and (4) calculating to obtain the concentration of glucose-6-phosphate in the sample to be detected through the linear determination model in the step (3).
5. The method according to claim 1, wherein the molar ratio of titanium-containing compound to strontium salt is 1: (1-5).
6. The method of claim 1 wherein the titanium-containing compound is selected from the group consisting of tetrabutyl titanate, titanium tetrachloride; the strontium salt is selected from strontium chloride and strontium nitrate.
7. The method according to claim 1, wherein the concentration of said strontium salt solution is 0.1-0.5 mol/L.
8. The method as claimed in claim 1, wherein the temperature of the reaction is 150-200 ℃; the reaction time is 20-24 h.
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