CN104014370A - Peroxide mimic enzyme and preparation and applications thereof - Google Patents
Peroxide mimic enzyme and preparation and applications thereof Download PDFInfo
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- CN104014370A CN104014370A CN201410241361.5A CN201410241361A CN104014370A CN 104014370 A CN104014370 A CN 104014370A CN 201410241361 A CN201410241361 A CN 201410241361A CN 104014370 A CN104014370 A CN 104014370A
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Abstract
The invention discloses a peroxide mimic enzyme and preparation and applications thereof, and relates to a horse radish peroxidase bio-mimetic technology, namely a technology of orderly fixing natural enzyme prothetic groups that are porphyrin molecules on the surface of a mass-produced carbon nitride nanosheet in an axial coordination manner so as to produce a mimic enzyme with a structure approaching a natural enzyme protein structure. The various catalytic kinetics indexes of the mimic enzyme are equivalent with that of a natural enzyme. On this basis, a general peroxide mimic enzyme CoII-Hemin@g-C3N4 nanometer biological probe is designed, and a high-sensitivity high-specificity biosensor used for detection of the H7N9 avian influenza virus is constructed, wherein the biosensor has characteristics of stable electrogenerated chemiluminescence output signals and a low detection lower limit. The mimic enzyme can be used in the aspects of biomedicine, clinical diagnosis, environment monitoring, judicial expertise, and the like.
Description
Technical field
The present invention relates to biological electroanalytical chemistry field, be specifically related to a kind of Mimetic Peroxidase, preparation and application thereof.
Background technology
Peroxidase is the class oxidoreducing enzyme being produced by microorganism or plant, can catalysis much react.It is with H
2o
2enzyme for the oxidation of electron acceptor catalytic substrate.Mainly be present in the peroxisome of cell, take ferriporphyrin as prothetic group, can catalytic oxidation phenols and the reacting of aminated compounds and hydrogen peroxide, there is the double action of eliminating hydrogen peroxide, phenols, amine toxicity simultaneously.Structure, characteristic, action principle and the enzyme that Mimetic Peroxidase is simulated peroxidase by artificial synthetic organic/inorganic composite matter chemical reaction process in vivo.Research analogue enztme is mainly as a class, to have the protein of catalytic activity in order to solve enzyme, is easily subject to the impact of multiple physics, chemical factor and inactivation, can not extensively replace the shortcoming of industrial catalyst.
Existing Mimetic Peroxidase is general direct to be prepared by ferriporphyrin and derivative thereof, and simulates its biologically active.As the ferroheme aptamer sequence of Willner seminar discovery, document 1:(Angew.Chem.Int.Ed.2011,50,11710-11714), its albuminoid " DNA enzyme " that can associate into G tetrad configuration with ferroheme monomer specificity (DNAzyme).The nano composite structure of the Graphene support ferroheme of Huang research team design, document 2:(Angew.Chem.Int.Ed.2012,124,3888-3891), its unit catalytic activity is suitable with horseradish peroxidase.There is following defect in said method:
(1) nucleic acid is expensive, and the inadequate storage-stable condition of structure is harsh, is not suitable for large-scale industry synthetic, and catalytic activity is still difficult to protease shoulder to shoulder, as document 1.
(2) Graphene cost is high, and biological functional difficulty and Graphene easily pollute and restricted its batch production and application while producing, as document 2.
(3) more seriously, in the preparation of these analogue enztmes, custom is with the fixing porphyrin prothetic group of simple chemical suction type, another key factor of constructing corresponding analogue enztme is always out in the cold, is deeply embedded in heme group between native enzyme subunit and is with the histidine terminal residue axial coordination of contiguous peptide chain! If the conformation of cobalamin is exactly the corrin (corrin) of AMP grafting histidine end axial coordination, synthetic biology and biophysics experiment have confirmed that axial coordination has basic impact to the catalytic activity of enzyme in view of " structure-activity relationship " rule!
Above-mentioned defect causes up to now, applies existing process and is difficult to obtain stable in properties, and cheap, structure approaches native enzyme, and environmental protection and the Mimetic Peroxidase suitable with native enzyme catalytic activity limit it in the further application in bioanalysis field.
Summary of the invention
The object of the invention is to prepare a kind of simple and easy, cheapness, structure and catalytic activity approach native enzyme, the Mimetic Peroxidase of stable in properties, and apply this Mimetic Peroxidase catalysis pyrogallic acid oxidation reaction and detect viral gene.
The technical solution that realizes the object of the invention is:
A Mimetic Peroxidase, take azotized carbon nano lamella as carrier, and the axial coordination by the pyridine nitrogen in azotized carbon nano sheet repetitive structure unit and porphyrin center cobalt ions, is combined into Mimetic Peroxidase.
Described Mimetic Peroxidase has following structure:
Above-mentioned oxide analogue enztme is prepare by the following method and obtain:
(1) select melamine to heat up at Muffle furnace Program as reactant, generate b-C
3n
4, then thermal oxide under oxygen atmosphere, ultrasonic generation g-C
3n
4;
(2) get g-C
3n
4the methanol solution mixing constant temperature oscillation of the aqueous solution and cobalt porphyrin, centrifugal abandoning supernatant, sedimented particle vacuum drying obtains Mimetic Peroxidase.
Wherein, the temperature programming described in step (1) refers to that 3～5 ℃ of intensifications per minute are warming up to 500 ℃; Oxidate temperature is 500～600 ℃;
The thermal oxide time described in step (1) is 2～4h, ultrasonic time 30-60min.
Cobalt porphyrin described in step (2) is selected cobalt protoporphyrin, 1mg/mlg-C
3n
4the methanol solution mixed volume of the aqueous solution and 1mM cobalt porphyrin is than being 3:1.
Constant temperature oscillation temperature described in step (2) is 20～25 ℃, and the constant temperature oscillation time is 12～16h.
Above-mentioned Mimetic Peroxidase can be used for catalysis pyrogallic acid oxidation reaction.
Above-mentioned Mimetic Peroxidase can be used as biological response element testing viral gene.The process that detects viral gene is as follows:
(1) polishing is carried out in glass-carbon electrode surface;
(2) with schiff base reaction, the biotinylated capture probe DNA of end is fixed on to CdTe quantum dot electrode surface, seals non-Characteristic Adsorption site, obtain biological nucleic acid sensor;
(3) by certain density target DNA and the hybridization of step 2 gained nucleic acid sensor incubation, the Mimetic Peroxidase of Streptavidin functionalization drips at electrode surface, after incubation drip washing, adopt electrogenerated chemiluminescence method, the electrogenerated chemiluminescence response of detecting electrode to target DNA.
Compared with prior art, its remarkable advantage is in the present invention:
(1) this Mimetic Peroxidase adopts raw material of industry melamine synthetic, cheap for manufacturing cost by simply adding thermal response, operates easyly, and building-up process can not cause environmental pollution.
(2) this Mimetic Peroxidase mesoporphyrin molecule and nano silicon nitride carbon, by the mode combination of axial coordination, approach natural horseradish peroxidase, have realized the human simulation to natural biologic enzyme.
(3) the effective catalysis pyrogallic acid oxidation reaction of this Mimetic Peroxidase, catalytic performance surpasses other several existing analogue enztmes, and quite active with native enzyme.
(4) this Mimetic Peroxidase can detect viral gene as biological response combination of elements biological nucleic acid sensor after biological functional, its good biocompatibility, and labeling effciency is high.
(5) this Mimetic Peroxidase detects that its response of viral gene is sensitive, stable in properties, specificity are high, detectability reaches Asia and flies mol level.
Accompanying drawing explanation
Fig. 1 is the three-dimensional structure schematic diagram of Mimetic Peroxidase.
Fig. 2 is the HRTEM figure of Mimetic Peroxidase in embodiment 1.
Fig. 3 is g-C in embodiment 1
3n
4and the in-situ AFM figure of Mimetic Peroxidase (B) and corresponding cross-sectional height distribution map (A).
Fig. 4 is Co in embodiment 2
iI-hemin (a) and Mimetic Peroxidase (b) are modified the CV figure of GCE in the saturated detection liquid of nitrogen; Interpolation: (on) linear graph of sweep speed (υ) and reduction peak spike potential (E), (under) linear graph of sweep speed (υ) and reduction peak peak current (i).
Fig. 5 is GCE in embodiment 2 (a), g-C
3n
4(b), Co
iI-hemin (c) and Mimetic Peroxidase (d) electro-catalysis H
2o
2cV figure.
Fig. 6 is Co in embodiment 2
iIthe XPSN1s spectrogram of-hemin (A) and Mimetic Peroxidase (B) and matching split swarming; Co
iIthe XPSCo2p spectrogram of-hemin (C) and Mimetic Peroxidase (D) and matching split swarming.
Fig. 7 is the molecule modeling of Mimetic Peroxidase in embodiment 2 and the Co that local dense functional calculates gained
iI-hemin is at g-C
3n
4the design sketch of adsorption.
Fig. 8 is the ultraviolet kinetic effect figure of Mimetic Peroxidase in embodiment 3, the double reciprocal plot that illustration is Mimetic Peroxidase.
Fig. 9 is the structure schematic diagram of genetic analysis biology sensor figure in embodiment 4 and embodiment 5.
Figure 10 (A) is the corresponding electrogenerated chemiluminescence signal strength signal intensity of variable concentrations HA1RTarget and corresponding canonical plotting in embodiment 5; (B) the electrogenerated chemiluminescence signal strength signal intensity of answering for mispairing group gene pairs.
The specific embodiment
A Mimetic Peroxidase, take azotized carbon nano lamella as carrier, and the axial coordination by the pyridine nitrogen in azotized carbon nano sheet repetitive structure unit and porphyrin center cobalt ions, is combined into Mimetic Peroxidase.
Described Mimetic Peroxidase has following structure:
Above-mentioned oxide analogue enztme is prepare by the following method and obtain:
(1) select melamine to heat up at Muffle furnace Program as reactant, generate b-C
3n
4, then thermal oxide under oxygen atmosphere, ultrasonic generation g-C
3n
4;
(2) get g-C
3n
4the methanol solution mixing constant temperature oscillation of the aqueous solution and cobalt porphyrin, centrifugal abandoning supernatant, sedimented particle vacuum drying obtains Mimetic Peroxidase.
Above-mentioned Mimetic Peroxidase can be used for catalysis pyrogallic acid oxidation reaction.
Above-mentioned Mimetic Peroxidase can be used as biological response element testing viral gene.The process that detects viral gene is as follows:
(1) polishing is carried out in glass-carbon electrode surface;
(2) with schiff base reaction, the biotinylated capture probe DNA of end is fixed on to CdTe quantum dot electrode surface, seals non-Characteristic Adsorption site, obtain biological nucleic acid sensor;
(3) by certain density target DNA and the hybridization of step 2 gained nucleic acid sensor incubation, the Mimetic Peroxidase of Streptavidin functionalization drips at electrode surface, after incubation drip washing, adopt electrogenerated chemiluminescence method, the electrogenerated chemiluminescence response of detecting electrode to target DNA.
Below in conjunction with drawings and Examples, further describe the present invention.
Embodiment 1
Mimetic Peroxidase structure be take azotized carbon nano lamella as shown in Figure 1 as carrier, by the axial coordination key of pyridine nitrogen atom abundant in its skeleton and cobalt porphyrin center cobalt ions, prepares Mimetic Peroxidase, and concrete preparation process is as follows:
(1) select cheap raw material of industry melamine in heat up 3～5 ℃ of the intensifications per minute of Muffle furnace Program, to be warming up to 500～600 ℃ as reactant, reaction 4h, generates yellow particle shape b-C
3n
4.By easy thermal oxide-liquid phase, strip off tandem plan again and prepare g-C
3n
4, 500～600 ℃, the thermal oxide time is 2～4h, ultrasonic time 30-60min; Finally with intermediate water, disperseing to be diluted to mass concentration is 1.0mgmL
-1the aqueous solution.
(2) get 1.0mgmL
-1g-C
3n
4the aqueous solution and 1mMCo
iIthe methanol solution of-hemin, with the volume ratio mix and blend of 3:1, after 20～25 ℃ of constant temperature oscillation 12～16h, obtains bronzing precipitum after centrifugal abandoning supernatant, obtain analogue enztme solid particle after vacuum drying.This pressed powder is disperseed with methyl alcohol homogeneous phase, take AFM and HRTEM microphoto.
The g-C preparing as shown in Figures 2 and 3
3n
4dispersed fine, and structure homogeneous, Co confirmed
iI-hemin is at g-C
3n
4the monolayer adsorption on surface, the height distribution peak averaging of horizontal cross-section has increased about 0.3nm, approaches unimolecule thickness, and volatile methanol molecules can not cause the variation of height.In addition, also can be observed g-C
3n
4distribution of sizes homogeneous comparatively, because the fluctuating of basal plane fold causes its average thickness to be about 1.2nm.
Embodiment 2
Checking Co
iI-hemin and g-C
3n
4cross combination and verify catalytic property and architectural feature:
First verify the electrochemical properties of peroxidase, detecting step is:
(1) GCE is used respectively to 0.3 and 0.05 μ m γ-Al
2o
3polishing, ultrasonic cleaning in absolute ethyl alcohol and intermediate water, finally dries up with high pure nitrogen respectively, and room temperature is deposited standby.
(2) get respectively the Co of equivalent
iI-hemin solution and Mimetic Peroxidase solution drip on two bare electrode surfaces, and naturally dry.By electrochemical techniques, measure the catalytic current of above-mentioned modified electrode in nitrogen saturation detection liquid, and add a certain amount of H in nitrogen saturation detection liquid
2o
2catalytic current.
(3) employing standard three electrodes configurations: saturated calomel electrode is reference electrode, platinum electrode as to electrode, and GCE is working electrode.With cyclic voltammetry measurement, sweep limits :-0.6～0V, sweep speed: 50mVs
-1.Letting nitrogen in and deoxidizing 20min.
As shown in Figure 4, Co
iI-hemin and g-C
3n
4load rear oxidation reduction peak current is greatly improved, himself electrochemical signals result show the electrochemical reaction that this reaction is controlled for surface; While is Co as shown in Figure 5
iI-hemin and g-C
3n
4after load, it is to H
2o
2catalytic current also has obvious enhancing.As shown in Figure 6 this sample has been carried out to xps energy spectrum analysis, can find out by the Co of the mode combination of axial coordination
iI-PyridinicN minimum energy, valence state is the most stable.And in conjunction with having carried out molecule modeling and Quantum mechanical calculation as Fig. 7, jointly confirm Co with electrochemical behavior
iI-hemin in the one-tenth key mode of axial coordination at g-C
3n
4surface conjunction, therefore, the Mimetic Peroxidase that name the present invention prepares is Co
iI-Hemin@g-C
3n
4.
Embodiment 3
Mimetic Peroxidase (the Co that selects respectively the present invention to prepare
iI-Hemin@g-C
3n
4) and other several Mimetic Peroxidases as catalyst, be applied to following catalysis pyrogallic acid oxidation reaction, carry out kinetic determination, its assay method is as follows:
After the Mimetic Peroxidase making is disperseed with PBS, the pyrogallol oxidation reaction of usining is measured it as the enzyme kinetics parameter of catalyst as benchmark.Concrete assay method is: add in batches as shown in Figure 8 the standard water solution of variable concentrations metagallic acid, and 2.0,1.0,0.5,0.25,0.125mM, according to classical Lineweaver-Burk equation:
1/υ＝(K
M/V
max)·1/[S]+1/V
max
Wherein υ is enzymatic instantaneous reaction rate, K
mfor being that Michaelis (Michaelis-Menten) constant, [S] are substrate for enzymatic activity concentration, V
maxfor the maximum reaction rate of enzyme when completely saturated by substrate, use double-reciprocal plot method, by slope and the intercept of fitting a straight line, obtain respectively K
mand V
maxvalue.Basis again
V
max＝k
cat·[E
T]
[E wherein
t] total concentration, the k of corresponding enzyme
catfor the molecular number of enzyme unit per second enzyme molecule conversion substrate when saturated by substrate, definition k
cat/ K
mfor apparent secondary rate constant.
Its measurement result is as shown in table 1:
The kinetic constant comparison of each Mimetic Peroxidase catalytic oxidation of table 1 pyrogallol.
The comparing result of table 1 can be seen Mimetic Peroxidase (Co of the present invention
iI-Hemin@g-C
3n
4) Michaelis constant K
mvalue is 0.61, lower than the Michaelis constant of native enzyme HRP.Second order reaction speed constant (k
cat/ K
m) over the analogue enztme of graphene oxide and ferriporphyrin load, also reached the suitable order of magnitude of native enzyme, show the advantage of the surface area load porphyrin prothetic group that nanostructured is huge, can be used as the substitute of native enzyme.
Embodiment 4
As shown in Figure 9, Mimetic Peroxidase (Co of the present invention
iI-Hemin@g-C
3n
4) can be used as biological response element testing viral gene, wherein first build biological nucleic acid sensor, concrete steps are:
(1) GCE is used respectively to 0.3 and 0.05 μ m γ-Al
2o
3polishing, ultrasonic cleaning in absolute ethyl alcohol and intermediate water, finally dries up with high pure nitrogen respectively, and room temperature is deposited standby.
(2) drip the water-soluble CdTe quantum dots solution 20 μ L of 10 μ M, after room temperature is dried naturally, to the chitosan-acetic acid solution of the coated 10 μ L0.025wt.% of this electrode surface; After dry, 2.0% glutaraldehyde solution to its surface-coated 10 μ L, surperficial with 10mMpH7.4PBS drip washing GCE after 37 ℃ of standing 1h, then drip the 1.0 μ M capture probe DNA that are coated with 10 μ L, as the HA1FStem-LoopCaptureProbe in table 2 or HA1RStem-Loop Capture Probe.In 37 ℃ of incubation casees, hatch after 1h standing over night in 4 ℃ of refrigerators.The PBS drip washing electrode surface that is 7.4 with 10mMpH after taking out, and seal non-specific adsorption site with the 1.0wt.% bovine serum albumin solution of 10 μ L, the PBS that is 7.4 with 10mMpH after incubation 1h rinses surface, obtains nucleic acid sensor.
Embodiment 5
Utilize the Mimetic Peroxidase that the prepared biological nucleic acid sensor of embodiment 4 is prepared in conjunction with the present invention to detect viral gene, concrete grammar is as follows:
(1) with 0.1 μ M～1.0fM of 10 μ L, obtain nucleic acid sensor hybridization as the HA1F Target in table 2 or HA1R Target and step (3), at 37 ℃ of incubation case reaction 30～50min, after brewing with the PBS that 10mMpH is 7.4, to electrode surface, drip the Mimetic Peroxidase biological detection probe solution of 10 μ L Streptavidin functionalization, at 37 ℃ of incubation 1h and after cleaning by the same method, can detect.
(2) in electrogenerated chemiluminescence instrument, adopt three-electrode system: saturated calomel electrode is reference electrode, platinum electrode as to electrode, using the electrode in step 1 as working electrode; All electrodes are placed in 0.1MpH9.0PBS, include 0.1MKNO
3as supporting electrolyte and 1.0mMH
2o
2as exogenous coreagent, detect in solution, regulate electric potential scanning scope: electronegative potential-1.25V, high potential 0.2V; Sweep speed is 100mVs
-1; PMT:-800V, detects electrogenerated chemiluminescence signal strength signal intensity.
This ECL biology sensor can be realized the highly sensitive detection of viral gene as shown in Figure 10 A, and it detects the range of linearity is 0.1nM～1.0fM.It is linear good, detects lower limit lower.
Embodiment 6
Specificity and stability that detection method described in check embodiment 5 detects viral gene, concrete grammar is as follows:
(1) 0.1pM that gets successively 10 μ L is as the mispairing group (HA1ROne-BaseMismatch in table 2, HA1R Two-BaseMismatch, HA1R Three-Base Mismatch) or mispairing group (HA1F One-BaseMismatch, HA1FTwo-BaseMismatch, HA1FThree-BaseMismatch) obtain nucleic acid sensor hybridization with step (3) respectively, at 37 ℃ of incubation case reaction 30～50min, after brewing with the PBS that 10mMpH is 7.4, to electrode surface, drip the Mimetic Peroxidase biological detection probe solution of 10 μ L Streptavidin functionalization, at 37 ℃ of incubation 1h and after cleaning by the same method, can detect.
(2) in electrogenerated chemiluminescence instrument, adopt three-electrode system: saturated calomel electrode is reference electrode, platinum electrode as to electrode, using the electrode in step 1 as working electrode; All electrodes are placed in 0.1MpH9.0PBS, include 0.1MKNO
3as supporting electrolyte and 1.0mMH
2o
2as exogenous coreagent, detect in solution, regulate electric potential scanning scope: electronegative potential-1.25V, high potential 0.2V; Sweep speed is 100mVs
-1; PMT:-800V, detects electrogenerated chemiluminescence signal strength signal intensity.
B shown in Figure 10 B, c, d, be respectively single base, double alkali yl, the electrogenerated chemiluminescence signal strength signal intensity electrogenerated chemiluminescence signal strength signal intensity contrast corresponding with a target gene that three base mispairings are corresponding, can find out the base mismatch group quantum dot electrogenerated chemiluminescence signal strength signal intensity that can not decay, representing this sensor has well selective and stability.
Table 2 hrp gene assay kit includes each primer table
Claims (10)
1. a Mimetic Peroxidase, is characterized in that: take azotized carbon nano lamella as carrier, the axial coordination by the pyridine nitrogen in azotized carbon nano sheet repetitive structure unit and porphyrin center cobalt ions, is combined into Mimetic Peroxidase.
2. Mimetic Peroxidase according to claim 1, is characterized in that: described Mimetic Peroxidase has following structure:
3. a preparation method for Mimetic Peroxidase, is characterized in that described method comprises:
(1) select melamine to heat up at Muffle furnace Program as reactant, generate b-C
3n
4, then thermal oxide under oxygen atmosphere, ultrasonic generation g-C
3n
4;
(2) get g-C
3n
4the methanol solution mixing constant temperature oscillation of the aqueous solution and cobalt porphyrin, centrifugal abandoning supernatant, sedimented particle vacuum drying obtains Mimetic Peroxidase.
4. the preparation method of Mimetic Peroxidase according to claim 3, is characterized in that: the temperature programming described in step (1) refers to that 3～5 ℃ of intensifications per minute are warming up to 500 ℃; Oxidate temperature is 500～600 ℃.
5. the preparation method of Mimetic Peroxidase according to claim 3, is characterized in that: the thermal oxide time described in step (1) is 2～4h, ultrasonic time 30-60min.
6. the preparation method of Mimetic Peroxidase according to claim 3, is characterized in that: the cobalt porphyrin described in step (2) is selected cobalt protoporphyrin, 1mg/mlg-C
3n
4the methanol solution mixed volume of the aqueous solution and 1mM cobalt porphyrin is than being 3:1.
7. the preparation method of Mimetic Peroxidase according to claim 3, is characterized in that: the constant temperature oscillation temperature described in step (2) is 20～25 ℃, and the constant temperature oscillation time is 12～16h.
8. an application for Mimetic Peroxidase as claimed in claim 1, is characterized in that: described Mimetic Peroxidase is for catalysis pyrogallic acid oxidation reaction.
9. an application for Mimetic Peroxidase as claimed in claim 1, is characterized in that: described Mimetic Peroxidase can be used as biological response element testing viral gene.
10. the application of Mimetic Peroxidase according to claim 9, is characterized in that: the process that detects viral gene is as follows:
(1) polishing is carried out in glass-carbon electrode surface;
(2) with schiff base reaction, the biotinylated capture probe DNA of end is fixed on to CdTe quantum dot electrode surface, seals non-Characteristic Adsorption site, obtain biological nucleic acid sensor;
(3) by certain density target DNA and the hybridization of step 2 gained nucleic acid sensor incubation, the Mimetic Peroxidase of Streptavidin functionalization drips at electrode surface, after incubation drip washing, adopt electrogenerated chemiluminescence method, the electrogenerated chemiluminescence response of detecting electrode to target DNA.
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| CN104596956A (en) * | 2015-01-05 | 2015-05-06 | 上海纳米技术及应用国家工程研究中心有限公司 | Application of nano-nickel oxide as mimetic peroxidase for detecting hydrogen peroxide |
| CN105413750A (en) * | 2015-12-11 | 2016-03-23 | 安徽师范大学 | Preparation method of mimic enzyme |
| CN107217050A (en) * | 2017-06-29 | 2017-09-29 | 中国农业科学院油料作物研究所 | A kind of preparation method of the surface immobilized enzyme of graphite phase carbon nitride nanometer sheet |
| CN108273554A (en) * | 2018-01-09 | 2018-07-13 | 西安交通大学 | A kind of g-C3N4The preparation and application of@Hemin compound analogue enztmes |
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| CN104596956A (en) * | 2015-01-05 | 2015-05-06 | 上海纳米技术及应用国家工程研究中心有限公司 | Application of nano-nickel oxide as mimetic peroxidase for detecting hydrogen peroxide |
| CN105413750A (en) * | 2015-12-11 | 2016-03-23 | 安徽师范大学 | Preparation method of mimic enzyme |
| CN107217050A (en) * | 2017-06-29 | 2017-09-29 | 中国农业科学院油料作物研究所 | A kind of preparation method of the surface immobilized enzyme of graphite phase carbon nitride nanometer sheet |
| CN108273554A (en) * | 2018-01-09 | 2018-07-13 | 西安交通大学 | A kind of g-C3N4The preparation and application of@Hemin compound analogue enztmes |
| CN108816286A (en) * | 2018-04-11 | 2018-11-16 | 湖北大学 | A kind of Cu-Ag/g-C3N4The preparation method of/ZIF tri compound analogue enztme |
| CN108704662A (en) * | 2018-06-22 | 2018-10-26 | 南京白云环境科技集团股份有限公司 | A kind of metalloporphyrin/graphite phase carbon nitride composite photo-catalyst |
| CN111239219A (en) * | 2020-01-17 | 2020-06-05 | 青岛科技大学 | Novel composite material for detecting β -amyloid protein and preparation method and application thereof |
| CN111239111A (en) * | 2020-02-14 | 2020-06-05 | 西北师范大学 | g-C3N4VB (vitamin B) detection by nanosheet12In (1) |
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