CN107356591A - It is a kind of based on imitative enzyme nano material without one pot of glucose color developing detection method of enzyme - Google Patents
It is a kind of based on imitative enzyme nano material without one pot of glucose color developing detection method of enzyme Download PDFInfo
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Abstract
The present invention relates to nano material, catalysis and analytical chemistry field, specifically include it is a kind of based on imitative enzyme nano material without one pot of glucose color developing detection method of enzyme.This method is mixed bovine serum albumin with divalent manganesetion, normal-temperature reaction is so that manganese dioxide nano-plates generate under the stabilization of bovine serum albumin after adding sodium hydroxide using the nano structure of bovine serum albumin and its property of functional group.The material has imitative peroxidase and imitative glucose oxidase double activity, and the optimal activity condition of two kinds of Mimicry enzymes is close.Therefore, a kind of novel one-step method enzyme-free glucose color developing detection method is established, and the material has broad application prospects in analytical chemistry, environmental project and catalytic field.
Description
Technical field
The present invention relates to nano material, catalysis and analytical chemistry field, specifically include a kind of based on imitative enzyme nano material
Without one pot of glucose color developing detection method of enzyme.
Background technology
Imitative enzyme nano material is a kind of nano material with similar enzymatic activity, and it can catalytic chemistry reaction（Non-electrochemical
Catalytic reaction）.In recent years, compared with enzyme, imitate enzyme nano material because of its larger specific surface area, higher stability and activity and
It is widely used in catalytic field.Manganese dioxide（MnO2）As a kind of important inorganic nano material, receive significant attention,
A series of research shows MnO2With good peroxidase activity.Lv et al. exists《BSA-templated
MnO2nanoparticles as both peroxidase and oxidase mimics》Bovine serum albumin is utilized in one text
（BSA）Make templated synthesis MnO2Nano-particle, and its peroxidase activity is probed into by typical peroxidase substrate
（Analyst2012, 137, 4552-4558）.Han et al. exists《Phage capsid protein-directed MnO2
nanosheets with peroxidaselike activity for spectrometric biosensing and
evaluation of antioxidant behaviour》Make templated synthesis MnO using phage capsid protein in one text2Receive
Rice piece, and show peroxidase activity（Chemical Communications 2017, 53, 5216-5219）.So
And as imitating enzyme nano material with other, the MnO with imitative glucose oxidase activity is not yet found at present2And other metals
Oxide-based nanomaterial.It is noted that the nano material with electrocatalytic oxidation glucose activity reported at present is not imitative
The nano material of glucose oxidase activity, because the catalytic reaction of glucose oxidase is not required to the participation of electricity, that is, it is that electricity is urged not to be
Change reaction.
Glucose is the important Energy supply material of human body, and detection glucose is significant.In recent years, have many based on tool
There is the report of the glucose color developing detection of the nano material of imitative peroxidase activity.He et al. exists《Peroxidase-like
activity of the Co3O4 nanoparticles used for biodetection and evaluation of
antioxidant behavior》Hydrogen peroxide, Co are produced using glucose oxidase catalysis glucose3O4Nano-particle
（NPs）Pass through colorimetric determination glucose (Nanoscale 2016,8,5938-5945) as mimics of peroxidase.
Chen et al. exists《Graphene oxide–Fe3O4 magnetic nanocomposites with peroxidase-like
activity for colorimetric detection of glucose》Glucose oxidase catalysis grape is utilized in one text
Sugar produces hydrogen peroxide, utilizes graphene oxide-Fe3O4Magnetic composite nano material realizes Portugal as peroxidase substitute
The color developing detection of grape sugar（Nanoscale2012,4,3,969 3976）.Shen et al. exists《Ultrasmall Pt
nanoclusters as robust peroxidase mimics for colorimetric detection of
glucose in human serum》Hydrogen peroxide is produced using glucose oxidase catalysis glucose, then with imitating
Indirect detection glucose in the presence of the Pt nanoclusters of peroxidase activity（ACS Applied Materials &
Interfaces 2017,9,10,027 10033）.From previously reported, the imitative enzyme nano material reported for work only imitates peroxide
Compound enzymatic activity and without imitative glucose oxidase activity, therefore constructed glucose color developing detection method need to be in glucose oxidase
Carried out under the conditions of existing, detection process is complicated and whard to control, and cost is improved due to the use of biology enzyme.Therefore, make
A kind of standby nano material for having imitative peroxidase activity and imitative glucose oxidase activity concurrently is simultaneously used for the one kettle way Portugal without enzyme
Grape sugar color developing detection, there is important application and researching value.
The content of the invention
It is an object of the invention to provide it is a kind of based on imitative enzyme nano material without one pot of glucose color developing detection method of enzyme.
To achieve the above object, the technical solution adopted by the present invention is：
One kind is with bovine serum albumin templated synthesis MnO2NSs method, it is characterised in that this method comprises the following steps：
(1) bovine serum albumin is well mixed with the aqueous solution containing divalent manganesetion, be incubated 1 hour；
(2) a certain amount of sodium hydroxide is added into above-mentioned mixed solution and is mixed, oscillating reactions is for a period of time；
(3) above-mentioned reaction solution is centrifuged, removes the supernatant containing unreacted component, collect precipitation, 4 °C preserve with standby；
Also purifying manganese dioxide nano-plates can be collected by the method dialysed or filtered.
Preferably, the aqueous solution containing divalent manganesetion can be matched somebody with somebody by manganese acetate, manganese chloride, manganese sulfate, manganese nitrate etc.
System forms.
A kind of MnO with imitative peroxidase and imitative glucose oxidase double activity based on kind2NSs application, its
It is characterised by：Described MnO2NSs can be as the application of catalyst.
Preferably, described MnO2NSs can be applied to qualitative or quantitative analysis as catalyst.
It is further preferred that, described MnO2NSs is applied to glucose and qualitatively or quantitatively detected as catalyst.
More preferably, described MnO2NSs can be in faintly acid（pH 3.0−5.0）Condition, depositing without glucose oxidase
Qualitative or quantitative glucose colour developing or absorption photometry detection are carried out lower, and need not other imitative enzyme nano materials or day
The participation of right enzyme.
Organicvisualization reagent is added during the detection glucose, and the organicvisualization reagent is 2,2 '-connection nitrogen-bis-（3- ethylo benzenes
And thiazoline -6- sulfonic acid）Diamine salts（ABTS）, o-phenylenediamine（OPD）Or 3,3 ', 5,5 '-tetramethyl benzidine（TMB）Deng.
Specific glucose sensing approach：
By testing sample, MnO2NSs, organicvisualization reagent（OPD, ABTS or TMB etc.）It is mixed in pH 3.0 5.0 buffer solution
In, utilize MnO2NSs imitative peroxidase and glucose oxidase activity, grape is carried out simultaneously in the presence of inanimate object enzyme
The oxidation reaction of sugar and the oxidation reaction of organicvisualization reagent, after reacting certain time under certain temperature, sentence using color is qualitative
The concentration of glucose of disconnected determinand sample, the light absorption value of reaction system is detected using spectrophotometer or ELIASA, is carried out to be measured
The quantifying glucose detection of thing sample.
Quantitative detection is specifically to draw glucose with the light absorption value of the reaction solution of the glucose standard comprising various concentrations
Standard curve, then its concentration of glucose is calculated by the reaction solution light absorption value of testing sample.
The present invention effect be：
1. the present invention makees templated synthesis two-dimensional appearance nano material using BSA（MnO2 NSs）And with imitative peroxidase
With imitative glucose oxidase double activity.
2. the present invention utilizes MnO2 NSs dual Mimicry enzyme is realized under weakly acidic pH（pH 3.0−5.0）'s
One kettle way enzyme-free glucose development process or absorption photometry detection.
3. the MnO of the present invention2 NSs has higher temperature stability, is incubated 2 hours under 90 °C and still retains about 70%
Vigor.
4. the present invention provides the nano material with imitative peroxidase and imitative glucose oxidase double activity, can use
In food fermentation, biological medicine, chemical industry, environment, the quantitative analysis of biotechnology field and catalyst etc..
Brief description of the drawings
Fig. 1 is MnO provided in an embodiment of the present invention2NSs transmission electron microscope photo.
Fig. 2 is the MnO provided in an embodiment of the present invention using TMB as developer2NSs imitates peroxidase activity design sketch
（Figure A is reaction solution photo, and figure B corresponds to extinction spectrum figure）.
Fig. 3 is the MnO provided in an embodiment of the present invention using OPD as developer2NSs imitates peroxidase activity design sketch
（Figure A is reaction solution photo, and figure B corresponds to extinction spectrum figure）.
Fig. 4 is the MnO provided in an embodiment of the present invention using ABTS as developer2NSs imitates peroxidase activity design sketch
（Figure A is reaction solution photo, and figure B corresponds to extinction spectrum figure）.
Fig. 5 is MnO provided in an embodiment of the present invention2NSs imitates glucose oxidase activity design sketch.
Fig. 6 is MnO provided in an embodiment of the present invention2The pH effect of optimization figures of the dual Mimicry enzymes of NSs.
Fig. 7 is the quantitative measurement standard working curve of glucose provided in an embodiment of the present invention.
Fig. 8 is MnO provided in an embodiment of the present invention2NSs temperature stability design sketch.
Embodiment
In order in depth illustrate present disclosure, some embodiments will be further enumerated below, but the present invention does not limit to
In cited embodiment.Specific experiment condition or method are for example unreceipted in the following example, by the normal condition of this area
Or method is carried out.
Embodiment 1
MnO based on BSA templates2NSs preparation：
(1) by BSA（The μ g/mL of final concentration 100）With the aqueous solution of manganese acetate（The mM of final concentration 2）It is well mixed, it is incubated 1 h；
(2) aqueous solution and the mixing of sodium hydroxide, the h of oscillating reactions 24 are added into above-mentioned mixed solution；
(3) above-mentioned reaction solution is centrifuged, removes the supernatant containing unreacted component, collect precipitation, 4 °C preserve with standby；
It can be collected by the method dialysed or filtered, purifying BSA.
Embodiment 2
MnO2NSs Mimicry enzyme analysis：
MnO2NSs imitative peroxidase activity analysis：Catalystic converter system is to include H2O2（1 mmol/L）, above-mentioned implementation
The MnO that example obtains2 NSs（25 µg/ml）, organicvisualization reagent TMB（0.5 mmol/L）Acetate buffer solution（pH 3.5）.In room
Temperature（25 °C）After lower reaction 30 minutes, its light absorption value at 650 nm is detected using ELIASA.As shown in Fig. 2, using TMB as
Developer, MnO2NSs shows imitative peroxidase activity（Black curve）.Control experiment separately is done, by above-mentioned experiment
MnO2 NSs is replaced with the water of same volume, as shown in Fig. 2, is not added with MnO2Without Mimicry enzyme during NSs（Grey curves）.
Two groups of experiments separately are done, organicvisualization reagent are substituted for OPD, ABTS, in room temperature（25 °C）After lower reaction 30 minutes,
Its light absorption value at 650 nm is detected using ELIASA.As shown in Fig. 3, using OPD as developer, MnO2NSs shows to imitate
Peroxidase activity（Black curve）.Control experiment separately is done, by the MnO in above-mentioned experiment2 NSs is replaced with the water of same volume
Change, as shown in Fig. 3, be not added with MnO2Without Mimicry enzyme during NSs（Grey curves）.As shown in Fig. 4, using ABTS as developer,
MnO2NSs shows imitative peroxidase activity（Black curve）.Control experiment separately is done, by the MnO in above-mentioned experiment2 NSs with
The water of same volume is replaced, and as shown in Fig. 4, is not added with MnO2Without Mimicry enzyme during NSs（Grey curves）.
MnO2NSs imitative glucose oxidase activity analysis：Catalystic converter system is to include glucose（1 mmol/L）、
The MnO that above-described embodiment obtains2 NSs（25 µg/ml）And acetate buffer solution（pH 4.0）, after reacting 30 minutes, centrifugation, take
Clear liquid, then add organicvisualization reagent TMB（0.5 mmol/L）, peroxidase（25 µg/ml）And acetate buffer solution（pH
3.5）, in room temperature（25 °C）After lower reaction 30 minutes, its light absorption value at 650 nm is detected using ELIASA.Such as Fig. 5 institutes
Show, MnO2NSs has good imitative glucose oxidase activity.
Embodiment 3
MnO2 The pH optimizations of NSs Mimicry enzymes：
MnO2 NSs imitates the pH optimizations of peroxidase activity：Catalystic converter system is to include H2O2（1 mmol/L）、MnO2 NSs
（25 µg/ml）, organicvisualization reagent TMB（0.5 mmol/L）Different pH buffer solution（PH 1.0 2.0, glycine-HCI delays
Fliud flushing；PH 3.0 6.0, acetic acid-acetic acid acid sodium buffer solution；PH 7.0 8.0, phosphate buffer；PH 9.0, Tris- hydrochloric acid
Buffer solution）.In room temperature（25 °C）After lower reaction 10 minutes, its light absorption value at 650 nm is detected using ELIASA.Such as Fig. 6
It is shown, MnO2 NSs is under weakly acidic pH（pH 3.0−5.0）The activity of peroxidase is shown, and optimal pH is 3.5
Left and right（Black curve）.
MnO2 NSs imitates the pH optimizations of glucose oxidase activity：Catalystic converter system is to include glucose（1 mmol/L）、
MnO2 NSs（25 µg/ml）With different pH buffer solution（PH 1.0 2.0, glycine-HCI buffer solution；PH 3.0 6.0, vinegar
Acid-acetic acid acid sodium buffer solution；PH 7.0 8.0, phosphate buffer；PH 9.0, Tris- hydrochloride buffer）.In room temperature（25
°C）After lower reaction 30 minutes, centrifugation.Supernatant is added to containing organicvisualization reagent TMB（0.5 mmol/L）, peroxidase
（25 µg/ml）And acetate buffer solution（pH 3.5）, in room temperature（25 °C）It is lower reaction 15 minutes, using ELIASA detect its
Light absorption value at 650 nm.As shown in fig. 6, MnO2 NSs is under weakly acidic pH（pH 3.0−5.0）It is glycoxidative to show grape
The activity of enzyme, and optimal pH is 4. 0 or so（Grey curves）.
Embodiment 4
The quantitative detection of glucose：Catalystic converter system is the glucose comprising various concentrations（0−4 mmol/L）、MnO2 NSs
（25 µg/ml）, organicvisualization reagent TMB（0.5 mmol/L）Acetate buffer solution（pH 4.0）.Reacted 30 minutes under 37 °C
Afterwards, detect the light absorption value at its 650 nm using ELIASA and draw out Glucose standards working curve.It is as shown in fig. 7, linear
The mmol/L of scope 0 1.2.
Embodiment 5
MnO2 NSs temperature stability：
MnO2 The NSs aqueous solution is in different temperatures（4−90 °C）It is lower to be incubated 2 hours.Then, by MnO2 It is anti-that NSs is added to catalysis
Answer in system, detect its light absorption value at 650 nm, as shown in figure 8, the MnO being incubated under 40 °C2 NSs almost retains whole
Vigor, and the MnO being incubated under 90 °C2 NSs also remains about 70 % vigor.
Claims (5)
- A kind of 1. manganese dioxide nano-plates with imitative peroxidase and imitative glucose oxidase double activity（MnO2NSs）'s Using, it is characterised in that：Described MnO2NSs can use as catalyst.
- 2. the MnO as described in claim 12NSs application, it is characterised in that：Described MnO2NSs can be used as catalyst and Applied to qualitative or quantitative analysis.
- 3. as the MnO described in claim 1,22NSs application, it is characterised in that：Described MnO2NSs as catalyst and Qualitatively or quantitatively detected applied to glucose.
- 4. as the MnO described in claim 1,2,32NSs can be applied to no one pot of glucose color developing detection method of enzyme, and its feature exists In：Described MnO2NSs can be in same reaction solution（Under i.e. same pH）It is qualitative without being carried out in the presence of glucose oxidase Or quantitative glucose colour developing or absorption photometry detection, and do not need the participation of other nano enzymes or native enzyme.
- 5. the MnO as described in claim 42NSs application, it is characterised in that：Organic chromogenic is added during the detection glucose Agent；The organicvisualization reagent is 2,2 '-connection nitrogen-bis-（3- ethyl benzo thiazole phenanthroline -6- sulfonic acid）Diamine salts（ABTS）, o-phenylenediamine （OPD）Or 3,3 ', 5,5 '-tetramethyl benzidine（TMB）Deng；During the detection glucose, it is not necessary to biology enzyme（Peroxidase Or glucose oxidase）Addition.
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Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107857307A (en) * | 2017-11-20 | 2018-03-30 | 青岛农业大学 | A kind of new strategy for realizing one kettle way grape grape color developing detection |
| CN109806877A (en) * | 2019-01-23 | 2019-05-28 | 南京大学 | High-activity nano enzyme and its preparation method and application based on transition metal oxide and its derivative |
| CN109897884A (en) * | 2019-03-27 | 2019-06-18 | 河南省人民医院 | It is a kind of based on glucose oxidase/hollow manganese dioxide bifunctional enzyme compound and preparation method |
| CN110006889A (en) * | 2019-05-10 | 2019-07-12 | 中国农业大学 | A kind of glucose quick detection test paper based on monatomic nano enzyme |
| CN110170321A (en) * | 2019-06-06 | 2019-08-27 | 上海纳米技术及应用国家工程研究中心有限公司 | A method of adjusting manganese dioxide nano enzymatic activity |
| CN110231486A (en) * | 2019-06-28 | 2019-09-13 | 吉林大学 | A kind of detection method of glucose |
| CN110702675A (en) * | 2019-11-06 | 2020-01-17 | 苏州大学 | Application of two-dimensional nano material as dehydrogenase |
| CN110907249A (en) * | 2019-12-13 | 2020-03-24 | 青岛农业大学 | Glucose detection method based on composite nano enzyme system |
| CN112345474A (en) * | 2020-11-11 | 2021-02-09 | 昆明理工大学 | Method for rapidly detecting tert-butyl hydroquinone in food |
| CN112710642A (en) * | 2020-12-16 | 2021-04-27 | 青岛农业大学 | Method for detecting glucose and/or hydrogen peroxide based on mimic enzyme |
| CN112748105A (en) * | 2020-12-30 | 2021-05-04 | 临沂大学 | Preparation method of monoatomic catalyst-based colorimetric test paper for rapid detection of blood sugar/urine sugar |
| CN113466189A (en) * | 2021-05-25 | 2021-10-01 | 青岛农业大学 | Malathion colorimetric detection method based on double-enzyme activity inhibition effect |
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Cited By (19)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107857307A (en) * | 2017-11-20 | 2018-03-30 | 青岛农业大学 | A kind of new strategy for realizing one kettle way grape grape color developing detection |
| CN109806877A (en) * | 2019-01-23 | 2019-05-28 | 南京大学 | High-activity nano enzyme and its preparation method and application based on transition metal oxide and its derivative |
| CN109806877B (en) * | 2019-01-23 | 2022-04-22 | 南京大学 | High-activity nano enzyme based on transition metal oxide and derivative thereof, and obtaining method and application thereof |
| CN109897884A (en) * | 2019-03-27 | 2019-06-18 | 河南省人民医院 | It is a kind of based on glucose oxidase/hollow manganese dioxide bifunctional enzyme compound and preparation method |
| CN109897884B (en) * | 2019-03-27 | 2022-11-11 | 河南省人民医院 | Bifunctional enzyme compound based on glucose oxidase/hollow manganese dioxide and preparation method thereof |
| CN110006889A (en) * | 2019-05-10 | 2019-07-12 | 中国农业大学 | A kind of glucose quick detection test paper based on monatomic nano enzyme |
| CN110170321A (en) * | 2019-06-06 | 2019-08-27 | 上海纳米技术及应用国家工程研究中心有限公司 | A method of adjusting manganese dioxide nano enzymatic activity |
| CN110170321B (en) * | 2019-06-06 | 2022-04-05 | 上海纳米技术及应用国家工程研究中心有限公司 | Method for adjusting catalytic activity of manganese dioxide nano-enzyme |
| CN110231486A (en) * | 2019-06-28 | 2019-09-13 | 吉林大学 | A kind of detection method of glucose |
| CN110702675B (en) * | 2019-11-06 | 2022-02-15 | 苏州大学 | Application of two-dimensional nano material as dehydrogenase |
| CN110702675A (en) * | 2019-11-06 | 2020-01-17 | 苏州大学 | Application of two-dimensional nano material as dehydrogenase |
| CN110907249B (en) * | 2019-12-13 | 2022-03-04 | 青岛农业大学 | Glucose detection method based on composite nano enzyme system |
| CN110907249A (en) * | 2019-12-13 | 2020-03-24 | 青岛农业大学 | Glucose detection method based on composite nano enzyme system |
| CN112345474A (en) * | 2020-11-11 | 2021-02-09 | 昆明理工大学 | Method for rapidly detecting tert-butyl hydroquinone in food |
| CN112345474B (en) * | 2020-11-11 | 2022-06-17 | 昆明理工大学 | Method for rapidly detecting tert-butylhydroquinone in food |
| CN112710642A (en) * | 2020-12-16 | 2021-04-27 | 青岛农业大学 | Method for detecting glucose and/or hydrogen peroxide based on mimic enzyme |
| CN112748105A (en) * | 2020-12-30 | 2021-05-04 | 临沂大学 | Preparation method of monoatomic catalyst-based colorimetric test paper for rapid detection of blood sugar/urine sugar |
| CN113466189A (en) * | 2021-05-25 | 2021-10-01 | 青岛农业大学 | Malathion colorimetric detection method based on double-enzyme activity inhibition effect |
| CN113466189B (en) * | 2021-05-25 | 2024-03-08 | 青岛农业大学 | Malathion colorimetric detection method based on double enzyme activity inhibition effect |
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