CN106583747B - The preparation of nucleoprotamine gold nanoclusters and the application in analogue enztme colorimetric and fluoroscopic examination - Google Patents
The preparation of nucleoprotamine gold nanoclusters and the application in analogue enztme colorimetric and fluoroscopic examination Download PDFInfo
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Abstract
The present invention relates to a kind of preparation of nucleoprotamine gold nanoclusters and the applications in analogue enztme colorimetric and fluoroscopic examination.The nucleoprotamine gold nanoclusters of invention not only have horseradish peroxidase and oxidase active, but also have fluorescent characteristic.Accordingly, nucleoprotamine gold nanoclusters analogue enztme visualization colorimetric and fluorescent optical sensor are constructed respectively, are applied to detection of heavy metal ion.The shortcomings of nucleoprotamine gold nanoclusters prepare simplicity, of low cost, have superior fluorescence and chemical stability, conventional dyes fluorescence lifetime can be overcome short, easy photobleaching；Fluorescence method high sensitivity, stability is good, is not required to expensive instrument；Mimetic enzyme catalysis colorimetric, it is sensitive, environmentally friendly, it can be used for real time monitoring.The present invention is expected to expand the detection and analysis for being applied to environment and biomedical sector related substances and disease marker, has broad application prospects.
Description
Technical field
The present invention relates to materials science field and analytical chemistry field, specially a kind of preparation of nucleoprotamine gold nanoclusters
And the application in analogue enztme colorimetric and fluoroscopic examination.
Background technology
Metal nano clustered materials cause extensive research interest in recent years.This is because：One side fluorescence metal nanometer
Cluster has the advantages that conventional organic dyes are incomparable, and such as toxicity is low, good light stability, Stokes shift are big, quantum production
Rate height and good biocompatibility；On the other hand, metal nanometre cluster analogue enztme is also by the pro-gaze of people, compared with native enzyme, people
Work analogue enztme, which has, is easy the advantages such as modification, preparation, cheap and high specificity, sensitivity height, can overcome natural enzyme source
The shortcomings of inactivating and is expensive limited, easily affected by environment.Although metal nanometre cluster investigation of materials achieves impressive progress,
The nanocluster material of report cannot still meet in terms of type, quantity and sensitivity, specificity chemical (biology) sense, environment and
The demand of biomedical sector especially not only can serve as fluorescent dye, but also the rarely seen report of nanocluster material with analogue enztme activity
Road.
The present invention provides a kind of preparation method of nucleoprotamine gold nanoclusters, which both has horseradish peroxidase
Enzyme and oxidase active can be used for colorimetric detection heavy metal ion；But also as fluorescent reagent, a huge sum of money is detected with fluorimetry
Belong to ion.As far as we know, this, which is the nucleoprotamine gold nanoclusters found for the first time, both has horseradish peroxidase and oxidizing ferment
Activity, and existing organic fluorescent dye is may replace, for pollutants such as fluoroscopic examination heavy metals, it is expected to be used for other biological mark
The detection of will object such as disease marker, has broad application prospects.
Invention content
The object of the present invention is to provide a kind of preparation methods of nucleoprotamine gold nanoclusters；Meanwhile utilizing the milt of preparation
The analogue enztme activity and fluorescent characteristic of albumen gold nanoclusters, be respectively applied to nucleoprotamine gold nanoclusters analogue enztme colorimetric sensing and
Fluoremetry heavy metal ion.
The purpose of the present invention can be achieved by the following technical measures：
The preparation method of the nucleoprotamine gold nanoclusters, includes the following steps：
(1) 10~50mL 10mM chlorauric acid solutions are pipetted, 10~50mL, 0.625~2.5mg/mL sulfuric acid milt eggs are placed in
In vain in (protamine sulfate, PS) solution, 25~50 DEG C of water-baths are vigorously stirred 2 minutes；
(2) 1M NaOH solutions are added in the solution that step (1) obtains and adjust pH to 12,8 are persistently stirred in 25~50 DEG C
~24 hours；
(3) the nucleoprotamine gold nanoclusters (PS-AuCNs) being prepared are kept in dark place in 4 DEG C.
The method of nucleoprotamine gold nanoclusters analogue enztme colorimetric determination mercury ion of the present invention, including following step
Suddenly:
(1) 25~50 μ L nucleoprotamine gold nanoclusters (PS-AuCNs), appropriate 3,3',5,5'-tetramethylbenzidine are pipetted
(TMB) and H2O2, it is placed in NaAc-HAc buffer solutions (pH5.5), the Hg of various concentration is then added2+Standard solution, sterilizing are super
Pure water is settled to 500 μ L, mixes well placing response 30min at room temperature；
(2) it is appropriate to pipette the solution obtained in step (1), absorption light is carried out with UV-2550 ultraviolet-uisible spectrophotometers
Spectrum scanning, with the absorbance (A of λ=450nm450) quantified.
The range of linearity of nucleoprotamine gold nanoclusters analogue enztme colorimetric determination mercury ion of the present invention is：4.0×
10-9~1.0 × 10-6Mol/L, detection are limited to 1.16 × 10-9mol/L。
The method of nucleoprotamine gold nanoclusters fluoremetry heavy metal ion of the present invention, can be arranged by following technology
It applies to realize：
(1) 30~70 μ L nucleoprotamine gold nanoclusters (PS-AuCNs), appropriate lead or uranyl ion standard solution are pipetted, is surpassed
Pure water is settled to 500 μ L of total volume, and 25~35 DEG C are incubated 20~35 minutes；
(2) it is appropriate to pipette the solution obtained in step (1), on F-4500 sepectrophotofluorometers, in 550~650nm
(λ is scanned in wave-length coverageex=300nm, exciting slit 5cm, transmite slit 10cm, photomultiplier negative voltage are 700V), in
λem=599nm measures the fluorescence change of system, draws standard curve；
(3) it by adjusting experiment condition (mainly pH value and temperature), can be used to measure heavy metal ion such as lead and uranyl
The concentration of ion；
(4) in above-mentioned steps (3), for measuring lead ion (Pb2+) experiment condition be：Trisodium citrate-HCl bufferings are molten
Liquid (pH=6.5)；PS-AuCNs:30~70 μ L；Experimental temperature：35℃；Reaction time：20min；
(5) in above-mentioned steps (3), for measuring uranyl ion (UO2 2+) experiment condition be：NaAc-HAc buffer solutions
(pH5.5)；PS-AuCNs:30~70 μ L；Experimental temperature：25℃；Reaction time：35min.
The method of nucleoprotamine gold nanoclusters Fluorometric assay heavy metal ion of the present invention, can be used for measure lead from
Son concentration range be：8.0×10-8~2.0 × 10-6mol/L；Detection is limited to 2.4 × 10–8mol/L.It can be used for measuring uranyl
Ion (UO2 2+) concentration range be 2.0 × 10-8Mol/L~1.0 × 10-5mol/L；Detection is limited to 6.1 × 10-9mol/L。
The present invention compared with the existing technology, has the following advantages and effect：
(1) found nucleoprotamine gold nanoclusters material preparation is simple, of low cost；Since it is closely sized to the expense of electronics
Metric wave is long, these metal nanometre clusters have the electron transition of size adjustable, to show stronger luminescent properties, has superior
Fluorescence and chemical stability, conventional organic dyes fluorescence lifetime can be overcome short, the shortcomings of easy photobleaching；But also as horseradish mistake
Oxide analogue enztme and oxidizing ferment, for being catalyzed colorimetric analysis；
(2) it is based on nucleoprotamine gold nanoclusters (PS-AuCNs) catalytic colorimetric determination mercury ion, catalytic activity is high, is catalyzed item
The more environmentally-friendly green of part can carry out Visual retrieval by bore hole, and detection limit reaches 1.16 × 10–9Mol/L can be used for existing
Field monitoring in real time；
(3) it is easy to be based on nucleoprotamine gold nanoclusters (PS-AuCNs) Fluorometric assay heavy metal ion method, stability
It is good, expensive instrument is not needed, detection limit can respectively reach 6.1 × 10-9mol·L-1(UO2 2+) and 2.4 × 10–8mol·L-1
(Pb2+)；
(4) above-mentioned (2) and (3) the method have good selectivity；
(5) present invention presents good potential, it is expected to expansion be applied to environment and biomedical sector related substances and
The detection and analysis of disease marker.
Description of the drawings
Fig. 1 is the nucleoprotamine gold nanoclusters morphology characterization transmission electron microscope photo of synthesis,
Fig. 2 is the fluorescence spectra of the nucleoprotamine gold nanoclusters of synthesis,
Fig. 3 is H2O2-TMB-PS-AuCNs-Hg2+Dynamics-time graph of system,
Fig. 4 is the PS-AuCNs that measures its fluorescent value attenuation in one month, is with first day fluorescence relative activity
100%, it is seen that PS-AuCNs its photoluminescent property in one month is stablized relatively,
Fig. 5 is the ultraviolet-visible absorption spectroscopy figure based on nucleoprotamine gold nanoclusters analogue enztme colorimetric determination mercury ion,
A in figure:TMB；b:TMB+H2O2；c:TMB+H2O2+PS-AuCNs；D~f:TMB+H2O2+PS-AuCNs+Hg2+,cHg 2+(d~f)/
Nmol/L=8.0；80.0；800,
Fig. 6 is the photo based on nucleoprotamine gold nanoclusters analogue enztme colorimetric estimation mercury ion, cTMB=0.4mM；cH2O2=
12mM；NaAc-HAc buffer solutions (pH 5.5)；cHg 2+(1~9):0.0,10.0,20.0,50.0,100.0,200.0,400.0,
800.0,1000.0nmol/L；
Fig. 7 is the standard curve of nucleoprotamine gold nanoclusters Spectrophotometric Determination of Mercury ion,
Fig. 8 is the fluorophotometric figure that uranyl ion is detected based on nucleoprotamine gold nanoclusters, NaAc-HAc buffer solutions
(pH5.5),cUO2 2+(μm ol/L)/(a~g):0.00,0.05,1.00,2.00,4.00,6.00,10.00,
Fig. 9 is the fluorophotometric figure that lead ion is detected based on nucleoprotamine gold nanoclusters.Sodium citrate-HCl buffer solutions
(pH6.5), cPb 2+(μm ol/L)/(a~h):0.00,0.10,1.00,2.00,4.00,8.00,10.00,12.00.
Specific implementation mode
With reference to specific embodiment, the present invention is described further, and embodiments of the present invention are not limited thereto.
Following experimental methods are conventional method unless otherwise instructed, and used experiment material unless otherwise instructed, can be easily
It is obtained from commercial company.
Embodiment 1：Multifunctional fish protamine gold nanoclusters are synthesized, following steps are specifically included：
(1) glassware used uses HCl in advance in testing:HNO3The chloroazotic acid soaked overnight of preparation, then uses ultra-pure water
Thoroughly cleaning；
(2) HAuCl of 0.02g/mL is prepared4The protamine sulfate solution of stock solution and 0.625~2.5mg/mL
(PS)；HAuCl4The solution used time is diluted to 10mmol/L；
(3) 10~50mL 10mmol/L HAuCl are pipetted4Solution is in the PS solution of 10~50mL, 0.625~2.5mg/mL
In, after 2 minutes are vigorously stirred under 37 DEG C of water bath conditions, the NaOH solution tune pH to 12 of 1mol/L is added, mixed solution is 25
~50 DEG C are persistently stirred 8~24 hours；
(4) PS-AuCNs being prepared is kept in dark place at 4 DEG C.
The characterization and Performance for the Multifunctional fish protamine gold nanoclusters that the present invention obtains.Sample is chosen embodiment 1 and is made
Standby Multifunctional fish protamine gold nanoclusters (PS-AuCNs),
(1) it is characterized by transmission electron microscope, the Multifunctional fish protamine gold nanoclusters being prepared in the present embodiment
Uniform particle diameter, about 2nm or so, as shown in Figure 1；
(2) PS-AuCNs synthesized sends out green-emitting fluorescent under the ultra violet lamp of 365nm；By scanning fluorescence spectrum,
Excitation wavelength is 300nm, and launch wavelength, which is 599nm, maximum fluorescence value, as shown in Figure 2.Prove the nucleoprotamine gold of the present invention
Nano-cluster has fluorescent characteristic.
(3) Fig. 3 is H2O2-TMB-PS-AuCNs-Hg2+Dynamics-time graph of system.As seen from the figure, only H2O2With
(curve c), absorbance value of the solution at 652nm wavelength become at any time in the presence of 3,3',5,5'-tetramethylbenzidine (TMB)
Change very little；PS-AuCNs is added in the solution, the absorbance value of solution increases (curve b), it was demonstrated that PS-AuCNs linearly over time
With Mimetic Peroxidase activity, H can be catalyzed2O2Aoxidize the reaction of TMB；In PS-AuCNs-H2O2It is added in-TMB solution
Hg2+, the absorbance of solution increased dramatically (curve a), it was demonstrated that Hg at any time2+PS-AuCNs analogue enztme activities can be enhanced；
(4) attenuation is as shown in Figure 4 at any time for the fluorescent value for the PS-AuCNs being prepared.As seen from the figure, preparation
PS-AuCNs its photoluminescent property in one month is stablized relatively.
Embodiment 2：Mercury ion is measured based on nucleoprotamine gold nanoclusters (PS-AuCNs) catalytic colorimetry,
(1) 25~50 μ L of nucleoprotamine gold nanoclusters (PS-AuCNs) solution for pipetting preparation clean sterilization treatment in 2mL
EP pipes in；
(2) suitable 3,3',5,5'-tetramethylbenzidine (TMB), H are sequentially added in (1)2O2It is buffered with NaAc-HAc
Liquid (pH5.5)；
(3) Hg of various concentration is added in the solution that (2) obtain2+Standard solution, sterilizing ultra-pure water are settled to 500 μ L,
Placing response 30min is mixed well at room temperature；Then appropriate 0.2mol/L H are added2SO4Solution terminates reaction；
(4) it is appropriate to pipette the solution obtained in step (3), absorption light is carried out with UV-2550 ultraviolet-uisible spectrophotometers
Spectrum scanning, measures the absorbance (A of λ=450nm450), standard curve standard measure.As Fig. 5 is with the increase of ion concentration of mercury
The absorbance value of system successively increases (Fig. 5, d-f)；,
(5) concentration range that nucleoprotamine gold nanoclusters catalytic colorimetry of the present invention measures mercury ion is：4.0×
10-9~1.0 × 10-6Mol/L, detection limit 1.16 × 10-9mol/L.The ion concentration of mercury is taken to be respectively：8.0×10-7Mol/L, 8.0
×10-8Mol/L, 8.0 × 10-9Mol/L carries out 11 parallel determinations, and acquiring relative standard deviation is respectively：3.82%,
3.87%, 4.57%；
(6) present invention can visualize measurement mercury ion, as shown in Figure 6；
(7) under optimum experimental condition, according in environmental water sample can compatible substance, test a variety of interfering substances pair
The influence of measurement result, when relative error is controlled ± 5%, 1000 times of Ca2+, Mg2+；100 times of K+, Fe3+, Zn2+, Na+, UO2 2+, NH4 +, Cd2+, Al3+, Mn2+, Cu2+；90 times of Ag+；5 times of Pb2+Not interference measurement；
(8) sample analysis takes laboratory tap water (sample 1), the water (sample 2) of University Of Nanhua's pond sugar, Xiang River water (sample respectively
3a,3b,3c).It is first filtered with quantitative filter paper and is placed in beaker twice respectively, be placed on heating on electric furnace and boil, keep boiling 10min
After remove, placement make its natural cooling, precipitation, it is to be measured with 0.22 μm of membrane filtration.Sample measurement result is as shown in table 1.
Mercury ion measurement result and determination of recovery rates in 1 water sample of table
Embodiment 3：Based on nucleoprotamine gold nanoclusters (PS-AuCNs) Fluorometric assay uranyl ion (UO2 2+),
(1) 30~70 μ L nucleoprotamine gold nanoclusters (PS-AuCNs) are pipetted in EP pipes, it is slow that appropriate NaAc-HAc is added
The UO of fliud flushing (pH5.5) and various concentration2 2+Standard solution is settled to 500 μ L with aqua sterilisa, mixes well, and reacts 35min；
(2) it is appropriate to pipette the solution obtained in step (1), on F-4500 sepectrophotofluorometers, setting excitation and hair
It is respectively 5cm and 10cm to penetrate slit, and photomultiplier negative voltage is 700V, in scanning (λ in 550~650nm wave-length coveragesex=
300nm), in λem=599nm measures the fluorescence change of system, draws standard curve；
(3) present invention can be used for measuring uranyl ion (UO2 2+) concentration range be 2.0 × 10-8Mol/L~1.0 × 10- 5mol/L；Detection is limited to 6.1 × 10-9mol·L-1.Under optimum reaction conditions, the pipe of configured in parallel 11,3 kinds of concentration work respectively
Liquid：4.0×10-8Mol/L, 4.0 × 10-7Mol/L, 4.0 × 10-6Mol/L carries out Precision Experiment, acquires relative standard deviation
Respectively 3.86%, 1.41%, 1.71%；
(4) under most suitable experiment condition, influence of a variety of interfering substances to this measuring is detected.When relative error controls
At ± 5%, 500 times of Mg2+、Ca2+, 100 times of Na+、NH4 +、Mg2+、Ag+、Mn2+、Ca2+, 90 times of K+、Zn2+、Cd2+、Cu2 +、Al3+, 50 times of Hg2+, 30 times of Fe3+、Pb2+Uranyl ion is not interfered to measure；
(5) sample analysis and determination of recovery rates.Laboratory tap water, University Of Nanhua's lotus water are acquired respectively, on Xiang River
Swimming, Lower Xiangjiang water, are first filtered with quantitative filter paper and are placed in beaker twice respectively in swimming, Xiang River, are placed on electric furnace and are added
Heat is boiled, and is removed after keeping boiling 30min, and placement makes its natural cooling, precipitation.Supernatant is drawn with 0.22 μm of filter syringe
Liquid is filtered again, removes filter, and by treated in syringe, water sample is placed in 2mL EP pipes, by establish method into
Row measures, as a result such as table 2.
Uranyl ion measurement result and determination of recovery rates in 2 water sample of table
Embodiment 4：Based on nucleoprotamine gold nanoclusters (PS-AuCNs) Fluorometric assay lead ion (Pb2+),
(1) 30~70 μ L nucleoprotamine gold nanoclusters (PS-AuCNs) are pipetted in appropriate trisodium citrate-HCl buffer solutions
(pH=6.5) in, the lead standard solution of various concentration is added, ultra-pure water is settled to 500 μ L of total volume, and 35 DEG C are incubated 20 minutes；
(2) it is appropriate to pipette the solution obtained in step (1), on F-4500 sepectrophotofluorometers, in 550~650nm
(λ is scanned in wave-length coverageex=300nm, exciting slit 5cm, transmite slit 10cm, photomultiplier negative voltage are 700V), in
λem=599nm measures the fluorescence change of system, draws standard curve；
(3) concentration range of nucleoprotamine gold nanoclusters fluorescence spectrometry lead ion of the present invention is：8.0×10-8
~2.0 × 10-6mol/L；Detection is limited to 2.4 × 10–8mol/L.It is parallel to prepare final concentration of 1.0 under the experiment condition of optimization
×10–7mol/L、4.0×10–6mol/L、1.5×10–5The Pb of mol/L2+Titer carries out Precision Experiment (n=11), relatively
Standard deviation is respectively 1.62%, 2.53% and 1.86%；
(4) present invention is respectively with 1.0 × 10–6mol/L Pb2+Standard solution to interfering substance that may be present in water sample
Interference measurement is carried out.When relative error is not more than ± 5%, 100 times of Zn2+、Mg2+、K+、Cu2+、Mn2+、Na+、Cd2+、NH4 +, 80 times of Ag+, 50 times of UO2 2+、Hg2+、Ca2+, 20 times of Al3+, 10 times of Fe3+Measurement is not interfered with；
(5) sample analysis.Laboratory tap water, University Of Nanhua's lotus pond water, Xiang River sewage draining exit are acquired with cleaning water bottle
Upper, middle and lower is swum, and is recorded as 1,2 and 3a、3b、3c.It is first filtered with quantitative filter paper and is placed in beaker twice respectively, is placed on electric furnace
Heating is boiled, and is removed after keeping boiling 10min, and placement makes its natural cooling, precipitation.It is filtered with 0.22 μm of filter, by the present invention
Method measures, and the results are shown in Table 3.
The measurement result of Pb in 3 water sample of table
Above is only the better embodiment of the present invention, but embodiments of the present invention are not limited by above-described embodiment
System, according to the above-mentioned design, other it is any without departing from change made by under the principle of the invention and Spirit Essence, modification,
It substitutes, combination, simplify, be regarded as equivalent substitute mode, be included within the scope of the present invention.
Claims (5)
1. the method for nucleoprotamine gold nanoclusters analogue enztme colorimetric determination mercury ion prepares nucleoprotamine gold in the following ways
Nano-cluster,
(1) 10~50mL 10mM chlorauric acid solutions are pipetted, it is molten to be placed in 10~50mL, 0.625~2.5mg/mL protamine sulfates
In liquid, 25~50 DEG C of water-baths are vigorously stirred 2 minutes；
(2) 1M NaOH solutions are added in the solution that step (1) obtains and adjust pH to 12,8~24 are persistently stirred in 25~50 DEG C
Hour；
(3) the nucleoprotamine gold nanoclusters being prepared are kept in dark place in 4 DEG C；
It is characterized in that, after preparing nucleoprotamine gold nanoclusters, nucleoprotamine gold nanoclusters analogue enztme colorimetric determination mercury ion
Specifically include following steps：
(2.1) 25~50 μ L nucleoprotamine gold nanoclusters, appropriate 3,3',5,5'-tetramethylbenzidine and H are pipetted2O2, it is placed in
In NaAc-HAc buffer solutions,
(2.2) then be added Hg2+Standard solution, sterilize ultra-pure water constant volume, mixes well at room temperature, reacts 30min；It can be used to
Visualize colorimetric detection mercury ion.
2. the method for nucleoprotamine gold nanoclusters analogue enztme colorimetric determination mercury ion according to claim 1, feature
It is, the range of linearity for detecting mercury ion is：4.0×10-9~1.0 × 10-6Mol/L, detection are limited to 1.16 × 10-9mol/L。
3. the method for nucleoprotamine gold nanoclusters fluoremetry heavy metal ion, prepares nucleoprotamine gold nano in the following ways
Cluster,
(1) 10~50mL 10mM chlorauric acid solutions are pipetted, it is molten to be placed in 10~50mL, 0.625~2.5mg/mL protamine sulfates
In liquid, 25~50 DEG C of water-baths are vigorously stirred 2 minutes；
(2) 1M NaOH solutions are added in the solution that step (1) obtains and adjust pH to 12,8~24 are persistently stirred in 25~50 DEG C
Hour；
(3) the nucleoprotamine gold nanoclusters being prepared are kept in dark place in 4 DEG C；
It is characterized in that, after preparing nucleoprotamine gold nanoclusters, nucleoprotamine gold nanoclusters fluoremetry heavy metal ion passes through
Following steps are realized：
(4.1) 30~70 μ L nucleoprotamine gold nanoclusters, appropriate lead ion or uranyl ion standard solution, ultra-pure water constant volume are pipetted
To 500 μ L of total volume, 25~35 DEG C are incubated 20~35 minutes；
(4.2) it is appropriate to pipette the solution obtained in step (4.1), on F-4500 sepectrophotofluorometers, in 550~650nm
Scanning, λ in wave-length coverageex=300nm, exciting slit 5cm, transmite slit 10cm, photomultiplier negative voltage are 700V, in
λem=599nm measures the fluorescence change of system, draws standard curve.
4. the method for nucleoprotamine gold nanoclusters fluoremetry heavy metal ion according to claim 3, which is characterized in that
Experiment condition for measuring lead ion is：Trisodium citrate-HCl buffer solutions, pH=6.5；PS-AuNCs:30~70 μ L；
Experimental temperature：35℃；Reaction time：20min.
5. the method for nucleoprotamine gold nanoclusters fluoremetry heavy metal ion according to claim 3, which is characterized in that
Experiment condition for measuring uranyl ion is：NaAc-HAc buffer solutions, pH=5.5；PS-AuNCs :30~70 μ L；Experiment
Temperature：25℃；Reaction time：35min.
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