CN105092551B - Nitric oxide production method is detected based on the fluorescence noble-metal nanoclusters that bovine serum albumin is modified - Google Patents
Nitric oxide production method is detected based on the fluorescence noble-metal nanoclusters that bovine serum albumin is modified Download PDFInfo
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- CN105092551B CN105092551B CN201510500384.8A CN201510500384A CN105092551B CN 105092551 B CN105092551 B CN 105092551B CN 201510500384 A CN201510500384 A CN 201510500384A CN 105092551 B CN105092551 B CN 105092551B
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Abstract
Nitric oxide production method is detected the present invention relates to the fluorescence noble-metal nanoclusters modified based on bovine serum albumin, including：(1) the bovine serum albumin aqueous solution is configured, precious metal salt solution is added, it is 11~12 to be adjusted with sodium hydroxide to pH, is reacted 8~12 hours under the conditions of lucifuge, to prepare the fluorescence noble-metal nanoclusters of bovine serum albumin modification；(2) NO saturated solutions are configured, the NO standard liquids of various concentrations are diluted to, the concentration of each nitric oxide solution is surveyed with Griess reagent；(3) the fluorescence noble-metal nanoclusters and the nitric oxide solution reaction of various concentrations modified certain density bovine serum albumin, determine its fluorescence intensity under different NO concentration, the linear relationship between NO concentration and fluorescence intensity is built, the fluorescence noble-metal nanoclusters NO fluorescence probes modified based on bovine serum albumin are obtained.Compared with prior art, the present invention is convenient and simple, economical, and the fluorescence probe selectivity of preparation is high, and sensitivity is good, has huge application value to NO efficient detection.
Description
Technical field
The invention belongs to biomaterial, fluorescence probe and nanometer pharmaceutical technology field, and in particular to one kind is based on cow's serum
The preparation method of the nitric oxide fluorescent probe of protein modified fluorescence metal nano-cluster.
Background technology
Nitric oxide (nitric oxide, NO) has immunological regulation, neurotransmission, blood as messenger molecule in human body
Press the particularly important physiological actions such as physiological regulating control.Nitric oxide is a kind of gas radicals, with short life, and activity is high,
The characteristics of being easy to react with other materials.Therefore, in vivo delicately, selectively detection nitric oxide just into
It is also one of study hotspot in recent years for a kind of challenging work.Existing nitric oxide production detection method is main
There are the methods such as chemoluminescence method, AAS, electrochemical method, electron paramagnetic resonance spectrum (EPR) method, but these methods have
Time-consuming, and the range of linearity is narrow, the shortcomings of accuracy is not high, relative to these methods, and fluorescence probe method has sensitivity height, selection
The good, simple operation and other advantages of property.
Existing nitric oxide fluorescent probe mainly includes the fluorescence probe containing metal ion and o-phenylenediamine class is organic
Fluorescence probe, although these molecules have good selectivity and sensitivity, has certain cytotoxicity, therefore, research
A kind of nitric oxide fluorescent probe with high sensitivity, selectivity, high-biocompatibility and low cytotoxicity turns into
One important topic.
The features such as fluorescence noble metal nanocluster has good biocompatibility and low cytotoxicity, in the side such as biomedicine
There is very big purposes in face.Bovine serum albumin has fabulous biocompatibility and no cytotoxicity, so bovine serum albumin is modified
Fluorescence noble-metal nanoclusters have very big application value in terms of biomarker and imaging and medical science.Ox can be made based on NO
The structure of haemocyanin changes, so that the mechanism of the fluorescence noble-metal nanoclusters fluorescent quenching of bovine serum albumin modification,
The high-efficiency fluorescence probe of a kind of high sensitivity and selectivity can be just designed, has huge application valency to the detection of NO in organism
Value.
So far, the stable fluorescence noble-metal nanoclusters of bovine serum albumin are had no to detect nitric oxide production related China
The report of patent.Therefore, the nitric oxide fluorescent probe for the noble-metal nanoclusters modified based on bovine serum albumin, develops organism
Interior NO fluorescence detection, these researchs are a significant job predictive of this special NO probes, are also currently associated
Key technical problem urgently to be resolved hurrily in research.
The content of the invention
It is an object of the present invention to overcome the above-mentioned drawbacks of the prior art and provide a kind of simple, economical, high
Biocompatibility, hypotoxicity, the fluorescence based on bovine serum albumin modification for possessing applications well prospect in biomedicine field are expensive
Metal nanometre cluster detects nitric oxide production method.
The purpose of the present invention can be achieved through the following technical solutions：A kind of fluorescence based on bovine serum albumin modification is expensive
Metal nanometre cluster detects nitric oxide production method, it is characterised in that this method specifically includes following steps：
(1) the bovine serum albumin aqueous solution is configured, precious metal salt solution is added, it is 11~12 to be adjusted with sodium hydroxide to pH,
Reacted 8~12 hours under the conditions of lucifuge, to prepare the fluorescence noble-metal nanoclusters of bovine serum albumin modification；
(2) NO saturated solutions are configured, the NO standard liquids of various concentrations are diluted to, each nitric oxide is surveyed with Griess reagent
The concentration of solution；
(3) the fluorescence noble-metal nanoclusters and the nitric oxide of various concentrations modified certain density bovine serum albumin are molten
Liquid reacts, and determines its fluorescence intensity under different NO concentration, builds the linear relationship between NO concentration and fluorescence intensity, and then
Obtain the fluorescence noble-metal nanoclusters NO fluorescence probes modified based on bovine serum albumin.
Precious metal salt solution described in step (1) is HAuCl4.4H2O or AgNO3, its concentration is 5~10Mm；Cow's serum
The concentration of protein solution is 10~50mg/mL.
Step (1) regulation pH value is to add sodium borohydride as reducing agent after 11~12, the concentration of boron sodium oxide molybdena for 0.5~
10mM。
The concentration of NO standard liquids described in step (2) is 0.01~2.1mM.
The reaction solution of the fluorescence noble-metal nanoclusters and nitric oxide solution of bovine serum albumin modification described in step (3)
The concentration of middle noble-metal nanoclusters is 0.5~2mg/mL.
It is glimmering that the present invention uses fabulous biocompatibility and do not have that cytotoxic bovine serum albumin modifies for raw material
Light noble-metal nanoclusters, there is very big application value in terms of biomarker and imaging and medical science.Ox blood can be made based on NO
Albuminised structure changes, so that the mechanism of the fluorescence noble-metal nanoclusters fluorescent quenching of bovine serum albumin modification, if
The high-efficiency fluorescence probe of a kind of high sensitivity and selectivity has been counted, NO in organism has been detected.
Compared with prior art, the present invention is with simply, and economic method prepares the expensive gold of fluorescence of bovine serum albumin modification
Belong to nano-cluster, the relation between the fluorescence intensity of the system and NO concentration is determined, so as to obtain the nitric oxide based on the system
Fluorescence probe.Compared with the existing nitric oxide production technology of detection, the inventive method is simple, economical, and the nitric oxide of preparation is glimmering
Light probe good biocompatibility, hypotoxicity, fluorescence intensity is high, has huge application value for NO efficient detection.
Embodiment
Embodiments of the invention are elaborated below, the present embodiment is carried out lower premised on technical solution of the present invention
Implement, give detailed embodiment and specific operating process, but protection scope of the present invention is not limited to following implementations
Example.
Embodiment 1
Nitric oxide production method, detailed step process are detected based on the fluorescence noble-metal nanoclusters that bovine serum albumin is modified
It is as follows：50mg/mL bovine serum albumen solution is configured, 10mM HAuCl is added4.4H2O solution, hydroxide is added after two minutes
Sodium solution regulation pH is about 12, is reacted 12 hours under the conditions of lucifuge, to prepare the fluorescence gold nanoclusters of bovine serum albumin modification.Match somebody with somebody
The nitric oxide saturated solution that concentration is 2.1mM is put, concentration is diluted to respectively for 0.08mM, 0.18mM, 0.26mM, 0.32mM,
0.45mM, 0.85mM nitric oxide solution.With concentration all for 1mg/mL bovine serum albumin modify gold nanoclusters solution with
The nitric oxide solution reaction of above-mentioned various concentrations, then surveys its fluorescence intensity under different NO concentration, obtain NO concentration with
Linear relationship between fluorescence intensity, so that the fluorescence gold nanoclusters for obtaining modifying based on bovine serum albumin detect nitric oxide production
Method.
Embodiment 2
25mg/mL bovine serum albumen solution is configured, 5mM HAuCl is added4.4H2O solution, hydrogen-oxygen is added after two minutes
It is about 12 to change sodium solution regulation PH, is reacted 12 hours under the conditions of lucifuge, to prepare the fluorescence gold nanoclusters of bovine serum albumin modification.
Configuration concentration is 2.1mM nitric oxide saturated solution, and concentration 0.09mM, 0.21mM, 0.25mM, 0.31mM are diluted to respectively,
0.43mM, 0.81mM nitric oxide solution.The gold nanoclusters solution all modified with concentration for 0.5mg/mL bovine serum albumin
With the nitric oxide solution reaction of above-mentioned various concentrations, then survey it and fluorescence intensity is obtained under different NO concentration, obtain NO concentration
Linear relationship between fluorescence intensity, so that the fluorescence gold nanoclusters for obtaining modifying based on bovine serum albumin detect nitric oxide
Method.
Embodiment 3
50mg/mL bovine serum albumen solution is configured, 10mM AgNO is added3Solution, adds sodium hydroxide solution regulation
PH is about 12, and the sodium borohydride solution for adding 10mM does and reacted 12 hours under the conditions of reducing agent, lucifuge, to prepare cow's serum egg
The fluorescence silver nanoclusters modified in vain.Configuration concentration is 2.1mM nitric oxide saturated solution, and concentration is diluted to respectively is
0.13mM, 0.38mM, 0.56mM, 0.86mM, 0.94mM, 1.20mM nitric oxide solution.All it is 1mg/mL ox with concentration
The nitric oxide solution reaction of the silver nanoclusters and above-mentioned various concentrations of haemocyanin modification, then surveys it under different NO concentration
Fluorescence intensity, obtain the linear relationship between NO concentration and fluorescence intensity, thus obtain based on bovine serum albumin modify silver
Nano-cluster detects nitric oxide production method.
Embodiment 4
25mg/mL bovine serum albumen solution is configured, 5mM AgNO is added3Solution, adds sodium hydroxide solution regulation PH
About 12, the sodium borohydride solution for adding 5mM does and reacted 12 hours under the conditions of reducing agent, lucifuge, to prepare bovine serum albumin
The fluorescence silver nanoclusters of modification.Configuration concentration is 2.1mM nitric oxide saturated solution, and concentration is diluted to respectively for 0.11mM,
0.41mM, 0.55mM, 0.83mM, 0.91mM, 1.08mM nitric oxide solution.All it is 1mg/mL bovine serum albumin with concentration
The fluorescence silver nanoclusters of modification and the nitric oxide solution reaction of above-mentioned various concentrations, then survey it glimmering under different NO concentration
Luminous intensity, obtains the linear relationship between NO concentration and fluorescence intensity, so as to obtain the silver nanoparticle modified based on bovine serum albumin
Cluster detects nitric oxide production method.
Embodiment 5
10mg/mL bovine serum albumen solution is configured, 8mM AgNO is added3Solution, adds sodium hydroxide solution regulation pH
For 11~12, add 8mM sodium borohydride solution do reducing agent, lucifuge under the conditions of react 8 hours, to prepare bovine serum albumin
The fluorescence silver nanoclusters of modification.Configuration concentration is 2.1mM nitric oxide saturated solution, and concentration is diluted to respectively for 0.01mM,
0.41mM, 0.55mM, 0.83mM, 0.91mM, 1.08mM nitric oxide solution.All it is 2mg/mL bovine serum albumin with concentration
The fluorescence silver nanoclusters of modification and the nitric oxide solution reaction of above-mentioned various concentrations, then survey it glimmering under different NO concentration
Luminous intensity, obtains the linear relationship between NO concentration and fluorescence intensity, so as to obtain the silver nanoparticle modified based on bovine serum albumin
Cluster detects nitric oxide production method.
Claims (5)
1. a kind of detect nitric oxide production method based on the fluorescence noble-metal nanoclusters that bovine serum albumin is modified, it is characterised in that
This method specifically includes following steps：
(1) the bovine serum albumin aqueous solution is configured, precious metal salt solution is added, it is 11~12 to be adjusted with sodium hydroxide to pH, is being kept away
Reacted 8~12 hours under optical condition, to prepare the fluorescence noble-metal nanoclusters of bovine serum albumin modification；
(2) NO saturated solutions are configured, the NO standard liquids of various concentrations are diluted to, each nitric oxide solution is surveyed with Griess reagent
Concentration；
(3) the fluorescence noble-metal nanoclusters and the nitric oxide solution of various concentrations modified certain density bovine serum albumin are anti-
Should, its fluorescence intensity under different NO concentration is determined, the linear relationship between NO concentration and fluorescence intensity is built, and then obtain
The fluorescence noble-metal nanoclusters NO fluorescence probes modified based on bovine serum albumin.
2. according to claim 1 detect nitric oxide production side based on the fluorescence noble-metal nanoclusters that bovine serum albumin is modified
Method, it is characterised in that the precious metal salt solution described in step (1) is HAuCl4﹒ 4H2O or AgNO3, its concentration is 5~10mM；
The concentration of the bovine serum albumin aqueous solution is 10~50mg/mL.
3. according to claim 1 detect nitric oxide production side based on the fluorescence noble-metal nanoclusters that bovine serum albumin is modified
Method, it is characterised in that step (1) regulation pH value be 11~12 after add reducing agent, reducing agent is sodium borohydride, boron sodium oxide molybdena
Concentration is 0.5~10mM.
4. according to claim 1 detect nitric oxide production side based on the fluorescence noble-metal nanoclusters that bovine serum albumin is modified
Method, it is characterised in that the concentration of the NO standard liquids described in step (2) is 0.01~2.1mM.
5. according to claim 1 detect nitric oxide production side based on the fluorescence noble-metal nanoclusters that bovine serum albumin is modified
Method, it is characterised in that the fluorescence noble-metal nanoclusters and nitric oxide solution of the bovine serum albumin modification described in step (3)
The concentration of noble-metal nanoclusters is 0.5~2mg/mL in reaction solution.
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| CN105798322B (en) * | 2016-03-17 | 2018-04-24 | 湖北大学 | The preparation method and applications of silver nanoclusters |
| CN108896750B (en) * | 2018-05-11 | 2021-05-25 | 江苏大学 | Preparation method and application of BSA-Au/Ag NCs/OPD/HRP proportional type fluorescence sensor |
| CN110161002A (en) * | 2019-04-28 | 2019-08-23 | 浙江工业大学 | A kind of ratio fluorescence imaging system and its construction method, application |
| CN111849467B (en) * | 2020-08-11 | 2021-08-24 | 苏州大学 | Infrared II-region fluorescence gold nanocluster and preparation and application thereof |
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| US6636652B1 (en) * | 1999-08-02 | 2003-10-21 | The Regents Of The University Of Michigan | Optical sensors for the detection of nitric oxide |
| CN102585417A (en) * | 2012-01-04 | 2012-07-18 | 武汉理工大学 | Quantum dot/polymer composite nitric oxide fluorescent probe and preparation method thereof |
| CN104614355A (en) * | 2015-02-06 | 2015-05-13 | 盐城工学院 | Method for detecting concentration of Cu<2+> based on bovine serum albumin functionalized gold nanocluster light scattering probe |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| US6636652B1 (en) * | 1999-08-02 | 2003-10-21 | The Regents Of The University Of Michigan | Optical sensors for the detection of nitric oxide |
| CN102585417A (en) * | 2012-01-04 | 2012-07-18 | 武汉理工大学 | Quantum dot/polymer composite nitric oxide fluorescent probe and preparation method thereof |
| CN104614355A (en) * | 2015-02-06 | 2015-05-13 | 盐城工学院 | Method for detecting concentration of Cu<2+> based on bovine serum albumin functionalized gold nanocluster light scattering probe |
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