CN106583747A - Preparation of protamine gold nanoclusters and application in analogue enzyme color comparison and fluorescence detection - Google Patents

Preparation of protamine gold nanoclusters and application in analogue enzyme color comparison and fluorescence detection Download PDF

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CN106583747A
CN106583747A CN201611109927.4A CN201611109927A CN106583747A CN 106583747 A CN106583747 A CN 106583747A CN 201611109927 A CN201611109927 A CN 201611109927A CN 106583747 A CN106583747 A CN 106583747A
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gold nanoclusters
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fluorescence
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CN106583747B (en
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王永生
黄艳琴
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University of South China
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Abstract

The invention relates to preparation of protamine gold nanoclusters and application in analogue enzyme color comparison and fluorescence detection. The protamine gold nanoclusters not only have activity of horseradish peroxidase and oxidase, but also have fluorescent characteristic. Hereby, a protamine gold nanocluster analogue enzyme visual color comparator and a fluorescence sensor are established respectively and are used for detecting heavy metal ions. The protamine gold nanoclusters are prepared simply and conveniently, are low in cost, and have excellent fluorescence stability and chemical stability, and the shortcomings that fluorescence of traditional dyestuff is short in service life, photobleaching occurs easily and the like can be overcome; a fluorescence method is high in sensitivity and good in stability, and expensive instruments are not required; and analogue enzyme catalytic color comparison is sensitive and environment-friendly, and can be used for field real-time monitoring. The protamine gold nanoclusters are expected to be expanded and applied to detection and analysis of related substances in the fields of environment and biomedicines and disease markers, and have wide application prospect.

Description

The preparation of protamine gold nanoclusters and in analogue enztme colorimetric and fluoroscopic examination Using
Technical field
The present invention relates to material science and analytical chemistry field, specially a kind of preparation of protamine gold nanoclusters And the application in analogue enztme colorimetric and fluoroscopic examination.
Background technology
Metal nano clustered materials cause in recent years extensive research interest.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 big, quantum is produced Rate height and good biocompatibility；On the other hand, metal nanometre cluster analogue enztme compares, people also by the pro-gaze of people with native enzyme Work analogue enztme has the advantages such as easy modification, preparation, cheap and high specificity, sensitivity height, can overcome natural enzyme source Inactivation limited, easily affected by environment and it is expensive the shortcomings of.Although metal nanometre cluster investigation of materials achieves impressive progress, The nanocluster material of report still can not meet in terms of species, quantity and sensitivity, specificity chemical (biology) sense, environment and The demand of biomedical sector, especially both can serve as fluorescent dye, and the rarely seen report of the nanocluster material with analogue enztme activity Road.
The invention provides a kind of preparation method of protamine gold nanoclusters, the nano-cluster is both with horseradish peroxidase Enzyme and oxidase active, can be used for colorimetric detection heavy metal ion；But also as fluorometric reagent, with a fluorimetry detection huge sum of money Category ion.As far as we know, this is that the protamine gold nanoclusters for finding first had both had horseradish peroxidase and oxidase Activity, may replace existing organic fluorescent dye again, for pollutant such as fluoroscopic examination heavy metals, it is expected to for other biological mark The detection of will thing such as disease marker, has broad application prospects.
The content of the invention
It is an object of the invention to provide a kind of preparation method of protamine gold nanoclusters；Meanwhile, using the milt for preparing The analogue enztme activity and fluorescent characteristic of albumen gold nanoclusters, be respectively applied to protamine 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 described protamine gold nanoclusters, comprises the following steps：
(1) 10～50mL 10mM chlorauric acid solutions are pipetted, 10～50mL, 0.625～2.5mg/mL protamine sulfates are placed in In (protamine sulfate, PS) solution, 25～50 DEG C of water-baths, 2 minutes are stirred vigorously；
(2) 1M NaOH solutions adjustment pH to 12 is added in the solution that step (1) is obtained, in 25～50 DEG C 8～24 is persistently stirred Hour；
(3) the protamine gold nanoclusters (PS-AuCNs) for preparing keep in dark place in 4 DEG C.
The method of protamine gold nanoclusters analogue enztme colorimetric determination mercury ion of the present invention, including following step Suddenly:
(1) 25～50 μ L protamine gold nanoclusters (PS-AuCNs), appropriate TMB (TMB) are pipetted And H2O2, in being placed in NaAc-HAc buffer (pH5.5), it is subsequently adding the Hg of variable concentrations2+Standard solution, sterilizing ultra-pure water is determined Hold to 500 μ L, placing response 30min is fully mixed under room temperature；
(2) pipette the solution obtained in step (1) appropriate, carry out absorption spectrum with UV-2550 ultraviolet-uisible spectrophotometers and sweep Retouch, with the absorbance (A of λ=450nm450) carry out quantitatively.
The range of linearity of protamine gold nanoclusters analogue enztme colorimetric determination mercury ion of the present invention is：4.0× 10-9～1.0 × 10-6Mol/L, detection is limited to 1.16 × 10-9mol/L。
The method of protamine gold nanoclusters fluoremetry heavy metal ion of the present invention, can be arranged by following technology Apply to realize：
(1) 30～70 μ L protamine gold nanoclusters (PS-AuCNs), appropriate lead or uranyl ion standard solution, ultra-pure water are pipetted The μ L of cumulative volume 500 are settled to, 25～35 DEG C are incubated 20～35 minutes；
(2) solution obtained in step (1) is pipetted appropriate, on F-4500 spectrofluorophotometers, in 550～650nm wavelength In the range of scan (λex=300nm, exciting slit 5cm, transmite slit 10cm, photomultiplier tube negative voltage is 700V), in λem= 599nm determines the fluorescence change of system, draws standard curve；
(3) by regulation experiment condition (mainly pH value and temperature), may be used to determine heavy metal ion such as lead and uranyl ion Concentration；
(4) in above-mentioned steps (3), for determining lead ion (Pb2+) experiment condition be：Trisodium citrate-HCl buffer solution (pH=6.5)；PS-AuCNs:30～70 μ L；Experimental temperature：35℃；Response time：20min；
(5) in above-mentioned steps (3), for determining uranyl ion (UO2 2+) experiment condition be：NaAc-HAc buffer solution (pH5.5)；PS-AuCNs:30～70 μ L；Experimental temperature：25℃；Response time：35min.
The method of protamine gold nanoclusters Fluorometric assay heavy metal ion of the present invention, can be used for determine lead from Son concentration range be：8.0×10-8～2.0 × 10-6mol/L；Detection is limited to 2.4 × 10–8mol/L.Can be used to determine 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 has the following advantages and effect relative to prior art：
(1) find prepared by protamine gold nanoclusters material simple, it is with low cost；Because it is closely sized to Fermi's ripple of electronics Long, these metal nanometre clusters have the electron transition of size adjustable, so as to show stronger luminescent properties, with superior Fluorescence and chemical stability, can overcome conventional organic dyes fluorescence lifetime short, the shortcomings of easy photobleaching；But also as Radix Cochleariae officinalises peroxide Compound analogue enztme and oxidase, for being catalyzed colorimetric analysiss；
(2) based on protamine gold nanoclusters (PS-AuCNs) catalytic colorimetric determination mercury ion, catalysis activity is high, and catalytic condition is more Plus environment-friendly and green, Visual retrieval can be carried out by bore hole, detection limit reaches 1.16 × 10–9Mol/L, can be used for live real When monitor；
(3) it is easy based on protamine gold nanoclusters (PS-AuCNs) Fluorometric assay heavy metal ion method, good stability, no Expensive instrument, detection limit is needed to respectively reach 6.1 × 10-9mol·L-1(UO2 2+) and 2.4 × 10–8mol·L-1(Pb2+)；
(4) above-mentioned (2) and (3) methods described have good selectivity；
(5) present invention presents good potential, it is expected to which expansion is applied to environment and biomedical sector related substanceses and disease The detection and analysis of mark.
Description of the drawings
Fig. 1 is the protamine gold nanoclusters morphology characterization transmission electron microscope photo of synthesis,
Fig. 2 is the fluorescence spectra of the protamine gold nanoclusters of synthesis,
Fig. 3 is H2O2-TMB-PS-AuCNs-Hg2+Kinetics-the time graph of system,
Fig. 4 is the PS-AuCNs for measuring its fluorescent value attenuation in month, is with first day fluorescence relative activity 100%, it is seen that PS-AuCNs its photoluminescent property in month is relatively stable,
Fig. 5 is the ultraviolet-visible absorption spectroscopy figure based on protamine gold nanoclusters analogue enztme colorimetric determination mercury ion, in figure a: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 protamine gold nanoclusters analogue enztme colorimetric determination mercury ion, cTMB=0.4mM；cH2O2= 12mM；NaAc-HAc buffer (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 protamine gold nanoclusters Spectrophotometric Determination of Mercury ion,
Fig. 8 be based on protamine gold nanoclusters detect uranyl ion fluorophotometric figure, NaAc-HAc buffer (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 protamine gold nanoclusters.Sodium citrate-HCl buffer solution (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 embodiment
With reference to specific embodiment, the present invention is described further, but embodiments of the present invention not limited to this. Following experimental techniques if no special instructions, are conventional method, and the experiment material for being used if no special instructions, can be easily Obtain from commercial company.
Embodiment 1：Synthesis Multifunctional fish protamine gold nanoclusters, specifically include following steps：
(1) glass drying oven used uses in advance HCl in testing:HNO3The chloroazotic acid soaked overnight of preparation is then thorough with ultra-pure water Cleaning；
(2) HAuCl of 0.02g/mL is prepared4The protamine sulfate solution (PS) of stock solution and 0.625～2.5mg/mL； HAuCl4The solution used time is diluted to 10mmol/L；
(3) 10～50mL 10mmol/L HAuCl are pipetted4Solution in the PS solution of 10～50mL, 0.625～2.5mg/mL, After being stirred vigorously 2 minutes under 37 DEG C of water bath conditions, the NaOH solution for adding 1mol/L adjusts pH to 12, and mixed solution is 25～50 DEG C persistently stir 8～24 hours；
(4) PS-AuCNs for preparing keeps in dark place at 4 DEG C.
The sign and Performance of the Multifunctional fish protamine gold nanoclusters that the present invention is obtained.Sample is chosen embodiment 1 and is made Standby Multifunctional fish protamine gold nanoclusters (PS-AuCNs),
(1) characterized by transmission electron microscope, the Multifunctional fish protamine gold nanoclusters particle diameter prepared in the present embodiment It is homogeneous, about 2nm or so, as shown in Figure 1；
(2) PS-AuCNs of synthesis sends green-emitting fluorescent under the ultra violet lamp of 365nm；By scanning fluorescence spectrum, excite Wavelength is 300nm, and launch wavelength has maximum fluorescence value for 599nm, as shown in Figure 2.Prove the protamine gold nano of the present invention Cluster has fluorescent characteristic.
(3) Fig. 3 is H2O2-TMB-PS-AuCNs-Hg2+Kinetics-the time graph of system.As seen from the figure, only H2O2With 3, 3 ', 5, (curve c), absorbance of the solution at 652nm wavelength is changed in the presence of 5 '-tetramethyl benzidine (TMB) Very little；Adding the absorbance of PS-AuCNs, solution in the solution increases linearly over time (curve b), it was demonstrated that PS-AuCNs has There is Mimetic Peroxidase activity, H can be catalyzed2O2The reaction of oxidation TMB；In PS-AuCNs-H2O2Hg is added in-TMB solution2 +, the absorbance of solution is increased dramatically (curve a), it was demonstrated that Hg with the time2+PS-AuCNs analogue enztme activities can be strengthened；
(4) fluorescent value of the PS-AuCNs for preparing is as shown in Figure 4 with time attenuation.As seen from the figure, the PS- of preparation AuCNs its photoluminescent property in month is relatively stable.
Embodiment 2：Mercury ion is determined based on protamine gold nanoclusters (PS-AuCNs) catalytic colorimetry,
Mercury ion measurement result and determination of recovery rates in the water sample of table 1
(8) sample analysis, take respectively laboratory tap water (sample 1), the water (sample 2) of University Of Nanhua's pond sugar, Xiang River water (sample 3a,3b, 3c).First filtered respectively with quantitative filter paper and be placed in twice in beaker, be placed on heated and boiled on electric furnace, keep being taken after boiling 10min Under, placement makes its natural cooling, precipitation, to be measured with 0.22 μm of membrane filtration.Sample determination result is as shown in table 1.
(7) under optimum experimental condition, according in environmental water sample can compatible material, test various interfering materials to determining As a result impact, when relative error control is ± 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+Equal not interference measurements；
(6) present invention can visualize measure mercury ion, as shown in Figure 6；
(5) concentration range that protamine gold nanoclusters catalytic colorimetry of the present invention determines mercury ion is：4.0×10-9 ～1.0 × 10-6Mol/L, detection limit 1.16 × 10-9mol/L.Taking ion concentration of mercury respectively is：8.0×10-7Mol/L, 8.0 × 10-8Mol/L, 8.0 × 10-9Mol/L, carries out 11 parallel assays, tries to achieve relative standard deviation and is respectively：3.82%th, 3.87%th, 4.57%；
(4) pipette the solution obtained in step (3) appropriate, carry out absorption spectrum with UV-2550 ultraviolet-uisible spectrophotometers and sweep Retouch, determine the absorbance (A of λ=450nm450), standard curve standard measure.Such as Fig. 5, with the increase of ion concentration of mercury, system Absorbance increases successively (Fig. 5, d-f)；,
(3) Hg of variable concentrations is added in the solution that (2) obtain2+Standard solution, sterilizing ultra-pure water is settled to 500 μ L, room temperature It is lower fully to mix placing response 30min；It is subsequently adding appropriate 0.2mol/L H2SO4Solution terminating reaction；
(2) appropriate TMB (TMB), H are sequentially added in (1)2O2With NaAc-HAc buffer (pH5.5)；
(1) the μ L of protamine gold nanoclusters (PS-AuCNs) solution 25～50 for pipetting preparation clean the EP of sterilization treatment in 2mL Guan Zhong；
Embodiment 3：Based on protamine gold nanoclusters (PS-AuCNs) Fluorometric assay uranyl ion (UO2 2+),
(1) 30～70 μ L protamine gold nanoclusters (PS-AuCNs) are pipetted in EP pipes, appropriate NaAc-HAc buffer is added (pH5.5) and variable concentrations UO2 2+Standard solution, with aquesterilisa 500 μ L are settled to, and are fully mixed, and react 35min；
(2) solution obtained in step (1) is pipetted appropriate, on F-4500 spectrofluorophotometers, setting is excited and launches narrow Seam is respectively 5cm and 10cm, and photomultiplier tube negative voltage is 700V, and (λ is scanned in 550～650nm wave-length coveragesex= 300nm), in λem=599nm determines the fluorescence change of system, draws standard curve；
(3) present invention can be used to determine 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 conditionses, respectively configured in parallel 11 manage, 3 kinds of concentration working solutions： 4.0×10-8Mol/L, 4.0 × 10-7Mol/L, 4.0 × 10-6Mol/L carries out Precision Experiment, tries to achieve relative standard deviation difference For 3.86%, 1.41%, 1.71%；
(4) under most suitable experiment condition, impact of various interfering materials to this measuring is detected.When relative error control ± When 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 disturbed to determine；
(5) sample analysis and determination of recovery rates.Respectively gather laboratory tap water, University Of Nanhua's Flos Nelumbinises water, Xiang River upper water, Swimming, Lower Xiangjiang water, are first filtered respectively with quantitative filter paper and are placed in twice in beaker in Xiang River, are placed on heating on electric furnace and are boiled Boiling, keeps being removed after boiling 30min, and placement makes its natural cooling, precipitation.With 0.22 μm of filter syringe Aspirate supernatant, then It is secondary to be filtered, filter is removed, the water sample after processing in syringe is placed in 2mL EP pipes, surveyed by the method set up It is fixed, as a result such as table 2.
Uranyl ion measurement result and determination of recovery rates in the water sample of table 2
Embodiment 4：Based on protamine gold nanoclusters (PS-AuCNs) Fluorometric assay lead ion (Pb2+),
(1) 30～70 μ L protamine gold nanoclusters (PS-AuCNs) are pipetted in appropriate trisodium citrate-HCl buffer solution (pH =6.5) in, add the lead standard solution of variable concentrations, ultra-pure water to be settled to the μ L of cumulative volume 500,35 DEG C are incubated 20 minutes；
(2) solution obtained in step (1) is pipetted appropriate, on F-4500 spectrofluorophotometers, in 550～650nm wavelength In the range of scan (λex=300nm, exciting slit 5cm, transmite slit 10cm, photomultiplier tube negative voltage is 700V), in λem= 599nm determines the fluorescence change of system, draws standard curve；
(3) concentration range of protamine 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.Under the experiment condition of optimization, it is parallel prepare it is final concentration of 1.0 × 10–7 mol/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 interfering material that may be present in water sample is carried out Interference measurement.When relative error is not more than ± 5%, 100 times of Zn2+、Mg2+、K+、Cu2+、Mn2+、Na+、Cd2+、NH4 +、80 Ag again+, 50 times of UO2 2+、Hg2+、Ca2+, 20 times of Al3+, 10 times of Fe3+Measure is not interfered with；
(5) sample analysis.With cleaning water bottle collection laboratory tap water, University Of Nanhua's Flos Nelumbinises pond water, on Xiang River sewage draining exit in Downstream water, is recorded as 1,2 and 3a、3b、3c.First filtered respectively with quantitative filter paper and be placed in twice in beaker, be placed on electric furnace and heat Boil, keep being removed after boiling 10min, placement makes its natural cooling, precipitation.Filtered with 0.22 μm of filter, by the inventive method Determine, as a result as shown in table 3.
The measurement result of Pb in the water sample of table 3
Better embodiment only of the invention, but embodiments of the present invention above is not limited by above-described embodiment System, above-mentioned design of the invention, other it is any without departing from the change made under the principle of the invention and spirit, modification, Substitute, combine, simplify, be regarded as equivalent substitute mode, be included within protection scope of the present invention.

Claims (6)

1. a kind of preparation of protamine gold nanoclusters, it is characterised in that：Comprise the following steps：
(1) 10～50mL 10mM chlorauric acid solutions are pipetted, 10～50mL, 0.625～2.5mg/mL protamine sulfates are placed in In solution, 25～50 DEG C of water-baths, 2 minutes are stirred vigorously；
(2) 1M NaOH solutions adjustment pH to 12 is added in the solution that step (1) is obtained, in 25～50 DEG C 8～24 is persistently stirred Hour；
(3) the protamine gold nanoclusters for preparing keep in dark place in 4 DEG C.
2., according to the method for claim 1 gained protamine gold nanoclusters analogue enztme colorimetric determination mercury ion, its feature exists In comprising the following steps：
(2.1) 25～50 μ L protamine gold nanoclusters are pipetted, appropriate 3,3 ', 5,5 '-tetramethyl benzidine and H2O2, put In NaAc-HAc buffer,
(2.2) it is subsequently adding Hg2+Standard solution, sterilize ultra-pure water constant volume, fully mixes under room temperature, reacts 30min；Can be used for Visualization colorimetric detection mercury ion.
3. the method for protamine gold nanoclusters analogue enztme colorimetric determination mercury ion according to claim 2, its feature It is that the range of linearity for detecting mercury ion is：4.0 ×10-9~1.0×10-6Mol/L, detection is limited to 1.16 × 10-9mol/L。
4. according to the method for claim 1 gained protamine gold nanoclusters fluoremetry heavy metal ion, it is characterised in that logical Cross following steps to realize：
(4.1) 30～70 μ L protamine gold nanoclusters, appropriate lead or uranyl ion standard solution are pipetted, ultra-pure water is settled to The μ L of cumulative volume 500,25 ~ 35 DEG C are incubated 20 ~ 35 minutes；
(4.2) solution obtained in step (4.1) is pipetted appropriate, on F-4500 spectrofluorophotometers, in 550 ~ 650nm ripples Scanning in long scope,λ ex=300nm, exciting slit 5cm, transmite slit 10cm, photomultiplier tube negative voltage is 700V, inλ em= 599nm determines the fluorescence change of system, draws standard curve.
5. the method for protamine gold nanoclusters fluoremetry heavy metal ion according to claim 4, it is characterised in that Experiment condition for determining lead ion is：Trisodium citrate-HCl buffer solution, pH=6.5；PS-AuCNs:30～70 μ L； Experimental temperature：35℃；Response time：20min.
6. the method for protamine gold nanoclusters fluoremetry heavy metal ion according to claim 4, it is characterised in that Experiment condition for determining uranyl ion is：NaAc-HAc buffer solution, pH=5.5；PS-AuCNs:30～70 μ L；Experiment Temperature：25℃；Response time：35min.
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