CN106092930B - Copper ion detection method and copper ion detection kit - Google Patents
Copper ion detection method and copper ion detection kit Download PDFInfo
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- CN106092930B CN106092930B CN201610415250.0A CN201610415250A CN106092930B CN 106092930 B CN106092930 B CN 106092930B CN 201610415250 A CN201610415250 A CN 201610415250A CN 106092930 B CN106092930 B CN 106092930B
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Abstract
The invention discloses a kind of copper ion detection methods, comprising: will likely the test sample containing copper ion and mercaptoalkyl chemical combination object the first mixed system is thoroughly mixed to form in Tris buffer solution, DNA- silver platinum cluster is added later, it is thoroughly mixed to form the second mixed system, thereafter the feature substrate of citric acid solution and peroxidase is added, it is uniformly mixed to form third mixed system, by measuring final obtained mixture in the light absorption value of visible light wave range, to realize detection to copper ion in test sample.The present invention also provides a kind of copper ion detection kits comprising feature substrate, DNA- silver platinum cluster, mercaptoalkyl chemical combination object and auxiliary reagent of peroxidase etc..Copper ion detection kit of the invention and detection method have many advantages, such as that test is easy to be quick, at low cost, and sensitivity and stability height, accuracy are good, can be applied to the detection of copper ion in environment, food etc..
Description
Technical field
The present invention relates to a kind of copper ion detection method more particularly to a kind of copper based on DNA silver platinum nanometer analogue enztme from
Sub- detection method and copper ion detection kit, belong to analytical chemistry field.
Background technique
Enzyme is a kind of biocatalyst, is a kind of protein with catalysis, can be urged in suitable environment
It chemically reacts, but the characteristic intrinsic due to enzyme, some applications are limited, for example protease is easy denaturation and hydrolysis, cost
Higher and use condition is stringent.Therefore, it is particularly important to develop a kind of analogue enztme with similar catalytic activity.Analogue enztme is a kind of
Non-protein structure but the artificial synthesized catalyst to native enzyme with similar catalytic performance.2007 Nian Yanxiyun research groups are for the first time
It was found that magnetic ferroferric oxide nano-particles (Fe3O4NPs) there is inherent Mimetic Peroxidase characteristic, nanoparticle conduct
The research of analogue enztme is paid close attention to by people.Such as CoFe2O4, magnetic nano-particles, cerium oxide nanoparicles, the metal such as FeS receive
The correlative study of the nanoparticles analogue enztme such as rice corpuscles, carbon nanomaterial works.It is the preparation of analogue enztme, pure compared with native enzyme
Change and storage is all easier, and is cheap, can be used in harsher chemical environment.Therefore, nanometer mould is developed
Quasi- enzyme has very high application prospect.
Summary of the invention
The main purpose of the present invention is to provide a kind of copper ion detection method and copper ion detection kits, existing to overcome
There is the deficiency of technology.
For realization aforementioned invention purpose, the technical solution adopted by the present invention includes:
A kind of copper ion detection method is provided in the embodiment of the present invention comprising:
(1) will likely the test sample containing copper ion be sufficiently mixed in Tris buffer solution with mercaptoalkyl chemical combination object
The first mixed system is formed, and is incubated at room temperature 30min or more；
(2) DNA- silver platinum cluster is added into step (1) finally obtained mixed system, is thoroughly mixed to form the second mixing
System, and it is incubated at room temperature 15min or more；
(3) the feature bottom of citric acid solution and peroxidase is added into step (2) finally obtained mixed system
Object is uniformly mixed to form third mixed system, and reacts 15min or more at room temperature, is existed by measuring finally obtained mixture
The light absorption value of visible light wave range, to realize the detection to copper ion in test sample.
A kind of copper ion detection kit is additionally provided in the embodiment of the present invention comprising:
The feature substrate of peroxidase,
DNA- silver platinum cluster, to peroxidase described in catalysis oxidation feature substrate and form chromogenic substrate,
Mercaptoalkyl chemical combination object,
And auxiliary reagent, include the buffering for being suitable for that Copper ioncatalyst oxidation mercaptoalkyl chemical combination object is made to form cross-linking agent
Liquid, and chromogenic substrate is formed suitable for making the feature substrate of peroxidase described in the DNA- silver platinum cluster catalysis oxidation
And the buffer that is suitable for that the DNA- silver platinum cluster and mercaptoalkyl chemical combination object is made to interact.
Compared with prior art, the invention has the advantages that
(1) a kind of copper ion detection kit and detection method provided by the invention, utilize Copper ioncatalyst oxidation sulfydryl
Alkyl compound forms cross-linking agent, inhibits mercaptoalkyl chemical combination object and DNA- silver platinum cluster (DNA-Ag/Pt NCs) and colour developing
The interaction of substrate, the substrate colour developing light absorption value for being catalyzed DNA-Ag/Pt NCs Mimetic Peroxidase restores, and is based on this
Principle develops highly sensitive copper ion (Cu2+) detection method, to Cu2+The range of linearity of detection is 10-100nM, sensitivity
It can reach 10nM.
(2) a kind of copper ion detection kit and detection method provided by the invention have easy to be quick, at low cost, stablize
The advantages that property is high, can be applied to the detection in the samples such as environment, food to copper ion.
Detailed description of the invention
In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below
There is attached drawing needed in technical description to be briefly described, it should be apparent that, the accompanying drawings in the following description is only this
The some embodiments recorded in invention, for those of ordinary skill in the art, without creative efforts,
It is also possible to obtain other drawings based on these drawings.
Fig. 1 is the TEM figure of the DNA- silver platinum cluster synthesized in the embodiment of the present invention 1；
Fig. 2 is Cu in the embodiment of the present invention 12+Concentration-light absorption value canonical plotting；
Fig. 3 is the selectivity test spectrogram in the embodiment of the present invention 1 for different heavy metal ion.
Specific embodiment
To make the object, technical solutions and advantages of the present invention clearer, with reference to the accompanying drawing to specific reality of the invention
The mode of applying is described in detail.The example of these preferred embodiments is illustrated in the accompanying drawings.Shown in attached drawing and according to
The embodiments of the present invention of attached drawing description are only exemplary, and the present invention is not limited to these embodiments.
Here, it should also be noted that, in order to avoid having obscured the present invention because of unnecessary details, in the accompanying drawings only
Show with closely related structure and/or processing step according to the solution of the present invention, and be omitted little with relationship of the present invention
Other details.
Inventor is studying for a long period of time and is largely being found surprisingly that very much in practice, and DNA- silver platinum cluster has very high
Peroxidase catalytic activity, DNA- silver platinum nanometer analogue enztme is for such as TMB-H2O2Color development system has higher
Catalytic efficiency is resistant to high temperature, high humidity, and is not influenced by enzyme inhibitor, and mercaptoalkyl chemical combination object can be with DNA- silver
Platinum cluster interaction, leads to this Mimetic Peroxidase-TMB-H2O2The color feature light absorption value of catalyzed coloration system drops
It is low；And copper ion can catalysis oxidation mercaptoalkyl chemical combination object form cross-linking agent, mercapto groups are oxidized to disulfide bond, inhibit mercapto
The interaction of base alkyl compound and DNA- silver platinum nanometer analogue enztme, so that the light absorption value of color development system be made to restore.Copper ion
Concentration in a certain range with the linear positive correlation of the light absorption value of color development system.Based on having been surprisingly found that above, this crime
Bright people is able to provide a kind of high sensitivity, and selectivity good copper ion detection kit and copper ion detection method as follows will
It is described in detail.
A kind of copper ion detection method is provided in the embodiment of the present invention comprising:
(1) will likely the test sample containing copper ion be sufficiently mixed in Tris buffer solution with mercaptoalkyl chemical combination object
The first mixed system is formed, and is incubated at room temperature 30min or more；
(2) DNA- silver platinum cluster is added into step (1) finally obtained mixed system, is thoroughly mixed to form the second mixing
System, and it is incubated at room temperature 15min or more；
(3) the feature substrate of citric acid buffer salt and peroxidase is added into step (2) finally obtained mixed system,
A series of third mixed systems are uniformly mixed to form, and react 15min or more at room temperature, by measuring finally obtained mixture
In the light absorption value of visible light wave range, to realize the detection to copper ion in test sample.
Further, the partial size of the DNA- silver platinum cluster is 2-10nm.
Further, the mercaptoalkyl chemical combination object includes any one in thioacetic acid, mercaptopropionic acid or mercaptoethylmaine
Kind or two or more combinations, but not limited to this.
Further, the feature substrate of the peroxidase includes 3,4-Dihydroxyphenylacetic acid, tetramethyl benzidine, neighbour
Any one in phenylenediamine or 2,2- connection bis- (3- ethyl benzo thiazole phenanthroline -6- sulfonic acid) diamine salts of nitrogen base or two or more groups
It closes, but not limited to this.
Further, the concentration of contained mercaptoalkyl chemical combination object is 5-20 μM in first mixed system.
Further, the concentration of contained DNA- silver platinum cluster is 0.2-2 μM in second mixed system.
Further, the concentration of the feature substrate of contained peroxidase is 1-4mM in the third mixed system.
Further, the pH value of the Tris buffer solution is 5.0-8.0.
The copper ion detection method specifically includes the following steps:
Ⅰ、
I) the standard copper ion solution, mercaptoalkyl compound solution and Tris buffer solution of a series of various concentrations are filled
Divide and be mixed to form a series of first mixed systems, is incubated at room temperature 30min or more；
II) to step I) DNA- silver platinum cluster is added in a series of mixed systems for finally being obtained, it is thoroughly mixed to form
A series of second mixed systems, and it is incubated at room temperature 15min or more；
III) to step II) citric acid solution and peroxidating are sequentially added in a series of mixed systems for finally being obtained
The feature substrate of object enzyme, and a series of third mixed systems are uniformly mixed to form, 15min or more is reacted, then measure respectively each mixed
Reactant is closed in the light absorption value of visible light wave range, establishes copper ion concentration-light absorption value standard curve accordingly；
Ⅱ、
A) test sample, mercaptoalkyl compound solution and Tris buffer solution are thoroughly mixed to form the first mixture
System, is incubated at room temperature 30min or more；
B) DNA- silver platinum cluster is added in the mixed system finally obtained to step a), is thoroughly mixed to form the second mixing
System, and it is incubated at room temperature 15min or more；
C) spy of citric acid solution and peroxidase is sequentially added in the mixed system finally obtained to step b)
Substrate is levied, and is uniformly mixed to form a series of third mixed systems, reacts 15min or more, then measure mixed reactant visible
The light absorption value of optical band, and according to aforementioned copper ion concentration-light absorption value standard curve, the copper ion measured in test sample is dense
Degree.
Further, the pH value of the citric acid-sodium citrate buffer is 4.0-5.0.
A kind of copper ion detection kit is provided in the embodiment of the present invention comprising:
The feature substrate of peroxidase,
DNA- silver platinum cluster, to peroxidase described in catalysis oxidation feature substrate and form chromogenic substrate,
Mercaptoalkyl chemical combination object,
And auxiliary reagent, include the buffering for being suitable for that Copper ioncatalyst oxidation mercaptoalkyl chemical combination object is made to form cross-linking agent
Liquid, and chromogenic substrate is formed suitable for making the feature substrate of peroxidase described in the DNA- silver platinum cluster catalysis oxidation
And the buffer that is suitable for that the DNA- silver platinum cluster and mercaptoalkyl chemical combination object is made to interact.
Further, the auxiliary reagent includes the Tris buffer solution that pH value is 5.0-8.0, and, pH value 4.0-
5.0 citric acid solution.
Additionally provided in the embodiment of the present invention above-mentioned copper ion detection kit or above-mentioned copper ion detection method in
Detect the application in copper ion.
For example, may include: by mercaptoalkyl chemical combination object and solution to be measured and Tris in a typical application scheme
Buffer solution mixing, adds DNA- silver platinum cluster solution, and reaction buffer and reaction substrate is then added, uniformly mixes shape
At hybrid reaction system, 15min or more is reacted, then measures the hybrid reaction system in visible light wave range, is preferably in wavelength
Light absorption value at 652nm, and with the standard curve control, to measure the Cu in solution to be measured2+Concentration.
Aforementioned DNA- silver platinum cluster can be using various appropriate methods preparation known to industry.For example, in a more allusion quotation
In the case study on implementation of type, the preparation method of the DNA- silver platinum cluster may include: to be by the concentration that volume ratio is 30:5:12
2.0 μM of single-chain nucleic acid solution, the silver nitrate solution that concentration is 150 μM and the Tetrachloroplatinate potassium solution that concentration is 125 μM are uniform
After mixing, it is protected from light 30min at 4 DEG C, the sodium borohydride solution that concentration is 5.0mM, the single-chain nucleic acid solution are added later
Volume ratio with sodium borohydride solution is 30:4, vibrates 3h at 37 DEG C, obtains DNA- silver platinum cluster solution.
Wherein, the single-chain nucleic acid of use can have any suitable sequence, such as in one embodiment, described single-stranded
The sequence of nucleic acid can be with are as follows: 5 '-CCCCCTAACTCCCCC-3 ' are not limited thereto certainly.
To make the object, technical solutions and advantages of the present invention clearer, with reference to the accompanying drawing and several preferred embodiments
Detailed description of the preferred embodiments.The example of these preferred embodiments is illustrated in the accompanying drawings.
The embodiments of the present invention described shown in attached drawing and with reference to the accompanying drawings are only exemplary, and the present invention is not limited to this
A little embodiments.
Here, it should also be noted that, in order to avoid having obscured the present invention because of unnecessary details, in the accompanying drawings only
Show with closely related structure and/or processing step according to the solution of the present invention, and be omitted little with relationship of the present invention
Other details.
In following embodiment, related raw material, such as DNA- silver platinum cluster solution, mercaptoalkyl chemical combination object and other
Reagent can be obtained by commercially available approach, can also be made by oneself with reference to existing document.
Embodiment 1
(1) the DNA- silver platinum cluster solution for being 4nm using oxidation-reduction method synthesis average grain diameter: by 150 μM of 50 μ L
AgNO3The K of 125 μM of solution and 120 μ L2PtCL4SsDNA solution (10mM, the phosphate of pH value 7.4 of 2 μM of solution and 300 μ L
Buffer dilution) oscillation mixing, after being protected from light 4 DEG C of reaction 30min, add the NaBH of 40 μ L 5mM4Solution (ice water dilution), it is quickly mixed
It is even, 37 DEG C are subsequently placed in, 3h is incubated on the constant temperature oscillator of 1500rpm, the DNA- silver that the average grain diameter for just obtaining 2 μM is 4nm
Platinum cluster solution.Wherein, the single-chain nucleic acid of use can have any suitable sequence, such as can be 5 '-
CCCCCTAACTCCCCC-3 ' is not limited thereto certainly.It is the DNA- silver platinum cluster synthesized in the present embodiment refering to fig. 1
TEM figure.
(2) by 10 μ L, 20 μM of mercaptopropionic acids (buffer solution containing 10mMTris), a series of 90 various concentrations of μ L copper
Solion (0-200nM) is added sequentially in ELISA Plate, is incubated for 30min after being sufficiently mixed at room temperature；
(3) DNA- silver platinum cluster solution obtained in step (1) is diluted 4 times, takes one that 10 μ L are added in step (2)
In serial mixed solution, 15min is incubated for after being sufficiently mixed at room temperature；
(4) 30 μ L citric acid solutions are sequentially added in a series of mixed solutions being incubated for into step (3)
The H of (0.01M, pH value 4.0), the TMB solution of 40 μ L 3mM, 20 μ L 1M2O2Solution, measurement reaction 15min after shaking uniformly
A series of light absorption value of mixed liquors at 652nm afterwards.Thus method obtains Cu2+Examination criteria curve as shown in fig.2, detection
Up to 10.0nM, the detection range of linearity is 10-100nM for sensitivity.
(5) Cu in tap water2+Detection: after boiling tap water is removed chlorine, filtered with 0.22 μm of micropore filtering film.Reference
Above-mentioned steps (1)-step (3) method, can be completed Cu in tap water2+Detection, Cu in sample2+Concentration referring to step
(4) standard curve established is calculated.
In addition, also being carried out to other metal ions referring to step (1)-(4) operation and essentially identical reaction condition
The detection of comparison property, final detection result see Fig. 3.Wherein Cu2+Concentration be 500nM, other metal ions be 10 μM.
Embodiment 2
(1) referring to embodiment 1, the DNA- silver platinum cluster solution that preparation average grain diameter is 4nm.
(2) by 10 μ L, 20 μM of mercaptoethylmaine solution are containing (10mM Tris buffer solution), a series of 90 various concentrations of μ L
Copper ion solution (0-200nM) is added sequentially in ELISA Plate, is incubated for 30min after being sufficiently mixed at room temperature；
(3) DNA- silver platinum cluster obtained in step (1) is diluted 4 times, takes 10 μ L to be added a series of in step (2)
In mixed solution, 15min is incubated for after being sufficiently mixed at room temperature；
(4) 30 μ L citric acid solution (0.01M, pH value are sequentially added in the mixed solution being incubated for into step (3)
For 4.0), the H of the TMB solution of 40 μ L 3.0mM, 20 μ L 1M2O2Solution, it is a series of mixed after measurement reaction 15min after shaken well
Close light absorption value of the liquid at 652nm.According to the obtained Cu of this example2+Testing result, can also obtain and mark similar in embodiment 1
Directrix curve.
(5) Cu in lake water2+Detection: 0.22 μm of micropore filtering film of lake water is filtered.Referring to above-mentioned steps (1)-step
(3) operation, can Cu in complete lake water2+Detection, Cu in sample2+The standard curve established referring to (4) of concentration calculate
It arrives.
It should be appreciated that the technical concepts and features of above-described embodiment only to illustrate the invention, its object is to allow be familiar with this
The personage of item technology cans understand the content of the present invention and implement it accordingly, and it is not intended to limit the scope of the present invention.It is all
Equivalent change or modification made by Spirit Essence according to the present invention, should be covered by the protection scope of the present invention.
Claims (9)
1. a kind of copper ion detection method, characterized by comprising:
Ⅰ、
I) the standard copper ion solution, mercaptoalkyl compound solution and Tris buffer solution of a series of various concentrations is sufficiently mixed
Conjunction forms a series of first mixed systems, is incubated at room temperature 30min or more；
II) to step I) DNA- silver platinum cluster is added in a series of mixed systems for finally being obtained, it is thoroughly mixed to form a system
The second mixed system of column, and it is incubated at room temperature 15min or more；
III) to step II) citric acid solution and peroxidase are sequentially added in a series of mixed systems for finally being obtained
Feature substrate, and be uniformly mixed to form a series of third mixed systems, react 15min or more, then to measure each mixing respectively anti-
It answers object in the light absorption value of visible light wave range, establishes copper ion concentration-light absorption value standard curve accordingly；
Ⅱ、
A) test sample containing copper ion, mercaptoalkyl compound solution and Tris buffer solution are thoroughly mixed to form first
Mixed system is incubated at room temperature 30min or more；
B) DNA- silver platinum cluster is added in the mixed system finally obtained to step a), is thoroughly mixed to form the second mixture
System, and it is incubated at room temperature 15min or more；
C) the feature bottom of citric acid solution and peroxidase is sequentially added in the mixed system finally obtained to step b)
Object, and it is uniformly mixed to form third mixed system, 15min or more is reacted, then measure mixed reactant in the suction of visible light wave range
Light value, and according to aforementioned copper ion concentration-light absorption value standard curve, measure the copper ion concentration in test sample；
Wherein, the partial size of the DNA- silver platinum cluster is 2-10nm.
2. copper ion detection method according to claim 1, it is characterised in that: the mercaptoalkyl chemical combination object includes sulfydryl
Any one in acetic acid, mercaptopropionic acid or mercaptoethylmaine or two or more combinations.
3. copper ion detection method according to claim 1, it is characterised in that: the feature substrate packet of the peroxidase
Include 3,4- dihydroxyphenyl acetic acid, tetramethyl benzidine, o-phenylenediamine or the 2,2- connection bis- (3- ethyl benzo thiazole phenanthroline -6- sulphurs of nitrogen base
Acid) any one or two or more combinations in diamine salts.
4. copper ion detection method according to claim 1, which is characterized in that the preparation side of the DNA- silver platinum cluster
Method include: by the concentration that volume ratio is 30:5:12 be 2.0 μM single-chain nucleic acid solution, the silver nitrate solution that concentration is 150 μM and
The Tetrachloroplatinate potassium solution that concentration is 125 μM after evenly mixing, is protected from light 30min at 4 DEG C, it is 5.0mM that concentration is added later
Sodium borohydride solution, the volume ratio of the single-chain nucleic acid solution and sodium borohydride solution is 30:4, vibrates 3h at 37 DEG C, obtains
DNA- silver platinum cluster solution.
5. copper ion detection method according to claim 1, it is characterised in that: contained sulfydryl in first mixed system
The concentration of alkyl compound is 5-20 μM.
6. copper ion detection method according to claim 1, it is characterised in that: contained DNA- in second mixed system
The concentration of silver-colored platinum cluster is 0.2-2 μM.
7. copper ion detection method according to claim 1, it is characterised in that: contained peroxide in the third mixed system
The concentration of the feature substrate of compound enzyme is 1-4mM.
8. copper ion detection method according to claim 1, it is characterised in that: the pH value of the Tris buffer solution is
5.0-8.0。
9. copper ion detection method according to claim 1, it is characterised in that: the pH value of the citric acid solution is
4.0-5.0。
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| CN106520972B (en) * | 2016-11-25 | 2020-11-10 | 复旦大学 | Method for detecting nucleic acid concentration by self-assembly system based on nucleic acid and platinum nano material |
| CN107462536B (en) * | 2017-05-16 | 2020-01-07 | 首都师范大学 | Method for detecting silver ions in water sample |
| CN107557431A (en) * | 2017-07-18 | 2018-01-09 | 天津大学 | A kind of kit of Visual retrieval S1 nucleases |
| CN113899731B (en) * | 2021-08-17 | 2022-06-14 | 广东省科学院测试分析研究所(中国广州分析测试中心) | One-step detection method for vibrio parahaemolyticus based on affinity difference of aptamer to target bacteria and gold nanoclusters |
| CN114807146B (en) * | 2022-06-28 | 2022-09-02 | 中国农业大学 | Preparation method and sensing application of nucleic acid nanoenzyme |
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