CN101387606A - Method for detecting hydrogen peroxide or glucose based on enzyme simulation by ferroferric oxide magnetic nanometer particle - Google Patents
Method for detecting hydrogen peroxide or glucose based on enzyme simulation by ferroferric oxide magnetic nanometer particle Download PDFInfo
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- CN101387606A CN101387606A CNA2008100510403A CN200810051040A CN101387606A CN 101387606 A CN101387606 A CN 101387606A CN A2008100510403 A CNA2008100510403 A CN A2008100510403A CN 200810051040 A CN200810051040 A CN 200810051040A CN 101387606 A CN101387606 A CN 101387606A
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Abstract
The invention relates to a method for detecting hydrogen peroxide or glucose, based on ferroferric oxide nanometer particle mimetic enzyme, wherein the ferroferric oxide nanometer particle mimetic enzyme is obtained by co-precipitation method, the ferroferric oxide nanometer particle mimetic enzyme has the catalysis property similar to peroxidase, and the ferroferric oxide nanometer particle mimetic enzyme can be used to detect hydrogen peroxide for real-time presenting the colorimetric detection of hydrogen peroxide. The detected limit of hydrogen peroxide is 3x10<-6>mol/L, the linear range is 5x10<-6> to 1x10<-4>mol/L. The ferroferric oxide nanometer particle mimetic enzyme can be combined with glucose oxidase to realize the colorimetric detection of glucose. The detection limit of glucose is 3x10<-5>mol/L and the linear range is 5x10<-5> to 1x10<-3>mol/L. The colorimetric detection of glucose has high selectivity and sensitivity, without complex instruments.
Description
Technical field
The present invention relates to detect the method for hydrogen peroxide or glucose, belong to technical field of analytical chemistry based on enzyme simulation by ferroferric oxide magnetic nanometer particle.
Background technology
Native enzyme has high substrate specificity and high catalytic efficiency under temperate condition.But the native enzyme changeableness, wasted time and energy by proteasome degradation and preparation and purifying easily.Therefore, people are seeking all kinds of substitutes of native enzyme (being analogue enztme) hardy.The analogue enztme of finding has at present: artificial ribozyme and DNAzyme, Cytochrome P450 analogue enztme, serineprotein kinase analogue enztme, two oxygenation analogue enztme, peroxophosphoric acid diester oxygen analogue enztme, link enzyme simulation enzyme, nuclease analogue enztme and superoxide analogue enztme, or the like.Wherein, the superoxide analogue enztme comprises: ferriheme, hematin, haemoglobin, cyclodextrin and porphyrin, or the like.This class superoxide analogue enztme has been used to the detection of hydrogen peroxide and ascorbic acid.
Recent studies show that the efficient catalytic ability of nano material mainly comes from its big specific surface area.People have carried out research in depth to the magnetic Nano material as the tri-iron tetroxide, and it is widely used in fields such as magnetic resonance imaging, drug delivery, biological sample separation and living things catalysis.It has been generally acknowledged that magnetic Nano material is chemistry and biologically inert, thereby need wait the function that provides extra at the further modified metal catalyzer in magnetic Nano material surface, enzyme or antibody in the practical application.But it is the viewpoint of chemistry and biologically inert that the nearest result of study that Yan tin accumulates research group has changed magnetic material.They find that ferroferric oxide magnetic nano-particles has the catalytic activity of similar peroxidase, and have proposed notion (Gao, the L.Z. of enzyme simulation by ferroferric oxide magnetic nanometer particle; Zhuang, J.; Nie, L.; Zhang, J.B.; Zhang, Y.; Gu, N.; Wang, T.H.; Feng, J.; Yang, D.L.; Perrett, S.; Yan, X., Intrinsic peroxidase-like activity of ferromagnetic nanoparticles.Nat.Nanotechnology 2007,2, (9), 577-583).
The present invention is based on similar thought, the ferriferrous oxide nano-particle analogue enztme that utilizes us to prepare provides the method for a kind of colorimetric detection hydrogen peroxide or glucose.
Summary of the invention
Based on the notion of enzyme simulation by ferroferric oxide magnetic nanometer particle, one of the object of the invention provides the method based on enzyme simulation by ferroferric oxide magnetic nanometer particle colorimetric detection hydrogen peroxide.This method can be used for the colorimetric detection of hydrogen peroxide, from 5 * 10
-6To 1 * 10
-4Detection presents good linear response to hydrogen peroxide in the mol/L concentration range.
Two of purpose of the present invention provides a kind of with the ferriferrous oxide nano-particle analogue enztme and the method for coming colorimetric detection glucose in conjunction with glucose oxidase.This method can be finished the sensitivity of glucose, special detection.This method is 5 * 10 for the linear response range of glucose colorimetric detection
-5To 1 * 10
-3Mol/L.
The process of chemical reaction involved in the present invention, suc as formula (1) and (2):
The preparation process of ferriferrous oxide nano-particle analogue enztme is as follows:
(1) be that 5: 1 concentration is that 1M ferric chloride in aqueous solution and concentration are that the iron protochloride hydrochloric acid solution of 2M mixes logical nitrogen deoxygenation 10 minutes with volume ratio;
(2) when stirring, above-mentioned mixed solution is added drop-wise to the ammonia spirit that concentration is 0.7M, this ammonia spirit deoxygenation and nitrogen protection, 1M ferric chloride in aqueous solution in the described mixed solution and concentration are that the volume ratio of the ammonia spirit of 0.7M is 1: 10, room temperature reaction 30 minutes, the black ferroferric oxide that obtains precipitation;
(3) the black ferroferric oxide precipitation that reaction is obtained is centrifugal, washes with water, is scattered in again in the water, obtains ferriferrous oxide nano-particle analogue enztme stock solution.Room temperature storage is standby.
Utilize the step of ferriferrous oxide nano-particle analogue enztme colorimetric estimation hydrogen peroxide as follows:
(1) with concentration is 2,2 of 60mM '-Lian nitrogen-two (3-ethyl benzo thiazole phenanthroline-6-sulfonic acid) di-ammonium salts, ferriferrous oxide nano-particle analogue enztme stock solution and H to be determined
2O
2The concentration that sample joins is that described concentration is 2,2 of 60mM '-Lian nitrogen-two (3-ethyl benzo thiazole phenanthroline-6-sulfonic acid) di-ammonium salts: ferriferrous oxide nano-particle analogue enztme stock solution: H in the hac buffer of pH=4.0 of 0.2M
2O
2Sample: concentration is that the volume ratio of hac buffer of the pH=4.0 of 0.2M is 24: 10: 24: 185;
(2) gained mixed liquor in (1) was reacted 10 minutes in 45 ℃ of water-baths;
(3) remove ferriferrous oxide nano-particle in the reaction solution with externally-applied magnetic field;
(4) reactant liquor that will remove ferriferrous oxide nano-particle is added to the water, and mixes, and measures absorption spectrum; The reactant liquor of described removal ferriferrous oxide nano-particle: the volume ratio of water is 1: 9.
Utilize sign that colorimetric method of the present invention detects hydrogen peroxide as depicted in figs. 1 and 2.
From Fig. 1 data as can be seen, our method has well than colour response for hydrogen peroxide.
From Fig. 2 data as can be seen, this method is limited to 3 * 10 for detecting of hydrogen peroxide
-6Mol/L, the range of linearity is 5 * 10
-6To 1 * 10
-4Mol/L.
Based on ferriferrous oxide nano-particle analogue enztme and as follows in conjunction with the step of glucose oxidase colorimetric estimation glucose:
(1) 20mg/mL glucose oxidase and glucose phosphate buffer solution to be measured are mixed,, obtain glucose response liquid in 37 ℃ of reactions 30 minutes; Described 20mg/mL glucose oxidase: the volume ratio of glucose phosphate buffer solution to be measured is 1: 10; Described glucose phosphate buffer concentration scope to be measured is: 1 * 10
-5Mol/L to 1 * 10
-2Mol/L, this solution are formulated in the phosphate buffer solution of 10mM pH=7.0;
(2) be 60mM2 with concentration, 2 '-Lian nitrogen-two (3-ethyl benzo thiazole phenanthroline-6-sulfonic acid) di-ammonium salts, ferriferrous oxide nano-particle analogue enztme stock solution and concentration are that the hac buffer of the pH=4.0 of 0.2M joins in the glucose response liquid of above-mentioned steps (1);
Described concentration is 60mM2,2 '-Lian nitrogen-two (3-ethyl benzo thiazole phenanthroline-6-sulfonic acid) di-ammonium salts: ferriferrous oxide nano-particle analogue enztme stock solution: concentration is the hac buffer of the pH=4.0 of 0.2M: the volume ratio of glucose response liquid 12: 5: 400: 110;
(3) with the mixed liquor in (2) in 45 ℃ water-bath 10 minutes, remove ferriferrous oxide nano-particle in the reaction solution with externally-applied magnetic field, obtain end reaction liquid;
(4) get end reaction liquid in the step (3), carry out absorption spectromtry.
Utilize colorimetric method of the present invention as shown in Figure 3 and Figure 4 for the sign of glucose detection.
From Fig. 3 data as can be seen, this method can be finished the sensitivity of glucose, special colorimetric detection.
From Fig. 4 data as can be seen, this method is limited to 3 * 10 for detecting of glucose
-5Mol/L, the range of linearity is 5 * 10
-5To 1 * 10
-3Mol/L.
Beneficial effect of the present invention: the ferriferrous oxide nano-particle that is synthesized among the present invention, function with peroxidase, be a kind of analogue enztme of new peroxidase, not only can be used for colorimetric analysis, and in can being applied to galvanochemistry etc. in principle other analyzing; The present invention prepared based on the ferriferrous oxide nano-particle analogue enztme, can be used for the colorimetric detection of hydrogen peroxide, hydrogen peroxide is detected good sensitivity; The prepared ferriferrous oxide nano-particle analogue enztme of the present invention can be used for the colorimetric detection of glucose in conjunction with glucose oxidase, and glucose detection is had good selectivity and sensitivity.
Description of drawings
Fig. 1 adds 24 μ L 60mM2 in 185 μ L 0.2M pH, 4.0 hac buffers, 2 '-Lian nitrogen-two (3-ethyl benzo thiazole phenanthroline-6-sulfonic acid) di-ammonium salts, investigate the catalytic activity figure that the tri-iron tetroxide analogue enztme reacts hydrogen peroxide oxidation ABTS in the time of 45 ℃.Colorimetric photo among the figure is taken after finishing for reaction and is got.From left to right be followed successively by: do not contain H
2O
2But contain Fe
3O
4, contain 10mM H
2O
2But do not contain Fe
3O
4, contain 10mM H
2O
2And contain Fe
3O
4
Fig. 2 is the hydrogen peroxide concentration-417 nanometers absorption value response curve (A) that obtains when detecting hydrogen peroxide with the ferriferrous oxide nano-particle analogue enztme; The linearity correction curve (B) that hydrogen peroxide is measured.As can be seen, this sensor is limited to 3 * 10 for detecting of hydrogen peroxide among the figure
-6Mol/L, the range of linearity is 5 * 10
-6To 1 * 10
-4Mol/L.
Fig. 3 is the colorimetric photo figure that takes when detecting glucose with glucose oxidase and ferriferrous oxide nano-particle analogue enztme.From left to right be followed successively by: 500 μ M glucose, buffer solution, 5mM fructose, 5mM lactose and 5mM maltose.As can be seen, this sensor can be finished the sensitivity of glucose, special colorimetric detection among the figure.
Fig. 4 is the concentration of glucose-417 nanometers absorption value response curve (A) that obtains when detecting glucose with glucose oxidase and ferriferrous oxide nano-particle analogue enztme; The linearity correction curve map (B) of glucose measurement.As can be seen, this sensor is limited to 3 * 10 for detecting of glucose among the figure
-5Mol/L, the range of linearity is 5 * 10
-5To 1 * 10
-3Mol/L.
Embodiment
Embodiment 1
The preparation process of ferriferrous oxide nano-particle analogue enztme is as follows:
(1) be that 1M ferric chloride in aqueous solution and 10mL concentration are iron protochloride hydrochloric acid solution (2M) mixing of 2M with 50mL concentration, logical nitrogen deoxygenation 10 minutes;
(2) concentration that above-mentioned mixed solution is added drop-wise to 500mL in vigorous stirring is the ammonia spirit of 0.7M, this ammonia spirit deoxygenation and nitrogen protection, room temperature reaction 30 minutes, the black ferroferric oxide that obtains precipitation;
(3) the black ferroferric oxide precipitation that reaction is obtained is centrifugal, washes with water three times; Again be scattered in the water, obtain the ferriferrous oxide nano-particle analogue enztme, room temperature storage is standby.
Embodiment 2
Utilize the ferriferrous oxide nano-particle analogue enztme to come the step of colorimetric detection hydrogen peroxide as follows:
(1) be that the concentration of 2,2 of 60mM '-Lian nitrogen-two (3-ethyl benzo thiazole phenanthroline-6-sulfonic acid) di-ammonium salts, 10 μ L ferriferrous oxide nano-particle analogue enztme stock solutions and 24 μ L is the H of 100mM with the concentration of 24 μ L
2O
2The concentration that joins 185 μ L is in the hac buffer of pH=4.0 of 0.2M;
(2) gained mixed liquor in (1) was reacted 10 minutes in 45 ℃ of water-baths;
(3) remove ferriferrous oxide nano-particle analogue enztme in the reaction solution with externally-applied magnetic field, obtain removing the reactant liquor of ferriferrous oxide nano-particle analogue enztme;
(4) with gained reactant liquor among the 100 μ L (3), join in the 900 μ L water, mix, measure absorption spectrum.
Embodiment 3
Utilize the ferriferrous oxide nano-particle analogue enztme, and come the step of colorimetric detection glucose as follows in conjunction with glucose oxidase:
(1) glucose solution with 20 μ L 20mg/mL glucose oxidases and 200 μ L variable concentrations mixed, in 37 ℃ of reactions 30 minutes; The concentration of described glucose solution is 5 * 10
-5, 1 * 10
-4, 5 * 10
-4, 1 * 10
-3, 5 * 10
-3With 1 * 10
-2Mol/L;
(2) be that 60mM 2,2 '-Lian nitrogen-two (3-ethyl benzo thiazole phenanthroline-6-sulfonic acid) di-ammonium salts, 10 μ L ferriferrous oxide nano-particle analogue enztme stock solutions and 800 μ L concentration are in the glucose response liquid of the pH=4.0 hac buffer of the 0.2M 220 μ L that join above-mentioned steps (1) with 24 μ L concentration;
(3) with the mixed liquor in the above-mentioned steps (2) in 45 ℃ of water-baths 10 minutes, remove ferriferrous oxide nano-particle in the reaction solution with externally-applied magnetic field;
(4) get end reaction liquid among the 900 μ L (3), carry out absorption spectromtry.
Claims (2)
1, detect the method for hydrogen peroxide based on the ferriferrous oxide nano-particle analogue enztme, it is characterized in that step is as follows:
The preparation process of ferriferrous oxide nano-particle analogue enztme is as follows:
(1) be that the concentration of 5:1 is that 1M ferric chloride in aqueous solution and concentration are that the iron protochloride hydrochloric acid solution of 2M mixes logical nitrogen deoxygenation 10 minutes with volume ratio;
(2) when stirring, above-mentioned mixed solution is added drop-wise to the ammonia spirit that concentration is 0.7M, this ammonia spirit deoxygenation and nitrogen protection, 1M ferric chloride in aqueous solution in the described mixed solution and concentration are that the volume ratio of the ammonia spirit of 0.7M is 1:10, room temperature reaction 30 minutes, the black ferroferric oxide that obtains precipitation;
(3) the black ferroferric oxide precipitation that reaction is obtained is centrifugal, washes with water, is scattered in again in the water, obtains ferriferrous oxide nano-particle analogue enztme stock solution;
Utilize the step of ferriferrous oxide nano-particle analogue enztme colorimetric estimation hydrogen peroxide as follows:
(1) with concentration is 2,2 of 60mM '-Lian nitrogen-two (3-ethyl benzo thiazole phenanthroline-6-sulfonic acid) di-ammonium salts, ferriferrous oxide nano-particle analogue enztme stock solution and H to be determined
2O
2The concentration that sample joins is that described concentration is 2,2 of 60mM '-Lian nitrogen-two (3-ethyl benzo thiazole phenanthroline-6-sulfonic acid) di-ammonium salts: ferriferrous oxide nano-particle analogue enztme stock solution: H in the hac buffer of pH=4.0 of 0.2M
2O
2Sample: concentration is that the volume ratio of hac buffer of the pH=4.0 of 0.2M is 24:10:24:185;
(2) gained mixed liquor in (1) was reacted 10 minutes in 45 ℃ of water-baths;
(3) remove ferriferrous oxide nano-particle in the reaction solution with externally-applied magnetic field;
(4) reactant liquor that will remove ferriferrous oxide nano-particle is added to the water, and mixes, and measures absorption spectrum; The reactant liquor of described removal ferriferrous oxide nano-particle: the volume ratio of water is 1:9.
2, based on the ferriferrous oxide nano-particle analogue enztme and the method for coming colorimetric detection glucose, it is characterized in that step is as follows in conjunction with glucose oxidase:
(1) preparation of ferriferrous oxide nano-particle analogue enztme
1. be that the ferric chloride in aqueous solution of 1M and iron protochloride hydrochloric acid solution (2M) that concentration is 2M mix with concentration, logical nitrogen deoxygenation 10 minutes; Described concentration is the ferric chloride in aqueous solution of 1M: the volume ratio of iron protochloride hydrochloric acid solution (2M) is 5:1;
2. in vigorous stirring, above-mentioned mixed solution is added drop-wise to the ammonia spirit that concentration is 0.7M, this ammonia spirit deoxygenation and nitrogen protection, room temperature reaction 30 minutes, the black ferroferric oxide that obtains precipitation; Described concentration is the ferric chloride in aqueous solution of 1M: iron protochloride hydrochloric acid solution (2M): the volume ratio of the ammonia spirit of 0.7M is 5:1:50;
3. the black ferroferric oxide that reaction obtained precipitation is centrifugal, wash with water and be scattered in again in the water, obtains ferriferrous oxide nano-particle analogue enztme stock solution;
(2) come the step of colorimetric detection glucose as follows based on the ferriferrous oxide nano-particle analogue enztme and in conjunction with glucose oxidase:
(1) 20mg/mL glucose oxidase and glucose phosphate buffer solution to be measured are mixed,, obtain glucose response liquid in 37 ℃ of reactions 30 minutes; Described 20mg/mL glucose oxidase: the volume ratio of glucose phosphate buffer solution to be measured is 1:10; Described glucose phosphate buffer concentration scope to be measured is: 1 * 10
-5Mol/L to 1 * 10
-2Mol/L, this solution are formulated in the phosphate buffer solution of 10mM pH=7.0;
(2) be that 60mM 2,2 '-Lian nitrogen-two (3-ethyl benzo thiazole phenanthroline-6-sulfonic acid) di-ammonium salts, ferriferrous oxide nano-particle analogue enztme stock solution and concentration are that the hac buffer of the pH=4.0 of 0.2M joins in the glucose response liquid of above-mentioned steps (1) with concentration;
Described concentration is 60mM 2,2 '-Lian nitrogen-two (3-ethyl benzo thiazole phenanthroline-6-sulfonic acid) di-ammonium salts: ferriferrous oxide nano-particle analogue enztme stock solution: concentration is the hac buffer of the pH=4.0 of 0.2M: the volume ratio 12:5:400:110 of glucose response liquid;
(3) with the mixed liquor in (2) in 45 ℃ water-bath 10 minutes, remove ferriferrous oxide nano-particle in the reaction solution with externally-applied magnetic field, obtain end reaction liquid;
(4) get end reaction liquid in the step (3), carry out absorption spectromtry.
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