CN103575715B - A kind of method detecting mitoxantrone based on luminescent gold nano-cluster - Google Patents
A kind of method detecting mitoxantrone based on luminescent gold nano-cluster Download PDFInfo
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- CN103575715B CN103575715B CN201310544623.0A CN201310544623A CN103575715B CN 103575715 B CN103575715 B CN 103575715B CN 201310544623 A CN201310544623 A CN 201310544623A CN 103575715 B CN103575715 B CN 103575715B
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Abstract
The invention discloses a kind of method detecting mitoxantrone based on luminescent gold nano-cluster, comprise the following steps: prepare gold nanoclusters, under the exciting of 469nm wavelength light, measure gold nanoclusters in the fluorescence intensity at 619nm wavelength place, be designated as F
0; Add different quality mitoxantrone in step one after, the gold nanoclusters of mensuration containing variable concentrations mitoxantrone is under 469nm wavelength light excites, in the fluorescence intensity at 619nm wavelength place, be designated as F<i>, </i> is according to after adding different quality mitoxantrone, the changing value of fluorescence intensity, calculates and adds the changing value of gold nanoclusters fluorescence intensity and the linear relationship of mitoxantrone concentration before and after mitoxantrone; Testing sample is added in gold nanoclusters, test it and add front and back under the exciting of 469nm wavelength light, at the changing value of the fluorescence intensity at 619nm wavelength place, according to the linear relationship obtained, calculate the detection of quality or the concentration foundation adding mitoxantrone.The method has high selectivity, high sensitivity, the feature such as easy, easy.
Description
Technical field
The invention belongs to mitoxantrone drug tests, be specifically related to the bio-sensing fluoroscopic examination cancer therapy drug mitoxantrone new method based on golden bunch of water-soluble Illuminant nanometer.
Background technology
Mitoxantrone is by widely used a kind of new type anticancer medicine clinically, belong to Anthraquinones cancer therapy drug, it all has significant clinical efficacy [see Thackery to most of malignant tumour, Ellen.TheGaleEncyclopediaofCancer:L-Z.Detroit:ThomsonGal e.2002, pp:708-710.], clinical diagnose is contributed to the quantitative measurement of this medicine.Current, the assay method of Anthraquinones cancer therapy drug mainly contains high performance liquid chromatography, capillary electrophoresis, HPLC-MS coupling method, and spectral analysis research seldom, and only fluorescent spectrometry and spectrophotometric method mostly low, the poor selectivity of defectiveness such as sensitivity, therefore, find new highly sensitive, high selectivity, easy, fast spectral detection new method there is important Research Significance.Volume in " analytical chemistry " Dec 8 in 2012 12 interim " electrochemical research of mitoxantrone on gold electrode and application thereof " reports a kind of method utilizing gold electrode to detect mitoxantrone, and its experimental technique is step one: the process of gold electrode, by gold electrode Al
2o
3powder polishing, difference supersound washing 5min in absolute ethyl alcohol, acetone and pure water, and repeat 3 times, then carry out electrochemical activation process (namely at 0.5mol/LH
2sO
4in in-0.3 ~ 1.5V potential range, carry out cyclic voltammetric (CV) scanning), until obtain the response of stable cyclic voltammetric; Gold electrode is first carried out repeatedly cyclic voltammetry scan (sweep limit is-0.5 ~ 1.6V) until obtain stable cyclic voltammetry curve by step 2 cyclic voltammetric (CV) experiment in BR buffer solution, then appropriate mitoxantrone standard solution is added, stir 1min, electrode is put into after letting nitrogen in and deoxidizing 2min, wait for 4min, in-0.5 ~ 1.6V potential range, carry out cyclic voltammetry scan with the sweep speed of 80mV/s.After each mensuration is complete, electrode redistilled water is rinsed, and in liquid of the blank end cyclic voltammetry scan to current stabilization, the material of electrode surface is adsorbed on removing, reach the object of activated electrode, experiment shows, the oxidation peak current of mitoxantrone and its concentration are 1.0 × 10
-9~ 1.0 × 10
-8mol/L, 1.0 × 10
-8~ 1.0 × 10
-7mol/L, 1.0 × 10
-7~ 1.0 × 10
-6all in good linear relationship within the scope of mol/L, linear equation and related coefficient are respectively y=1.2421x+0.5639, r=0.9906; Y=1.6952x+10.3, r=0.9998; Y=3.7636x+23.575, r=0.9994.Detection limit can reach 5.6 × 10
-10mol/L; To 1.0 × 10
-7the relative standard deviation of the mitoxantrone replicate determination 10 times of mol/L is 3.8%.
Summary of the invention
In recent years, noble-metal nanoclusters is progressively becoming the novel fluorescence probe of alternative conventional fluorescent group and is receiving much concern, this is mainly because it has super-small, high quantum yield, light stability, the characteristic such as nontoxic [see ZhengJ., NicovichP.R., DicksonR.M.Annu.Rev.Phys.Chem., 2007,58,409-431.].Especially using biomacromolecule material as protein as templated synthesis go out there is water-soluble, good biocompatibility, luminous gold nanoclusters attracted to pay close attention to quite widely [see WeiH., WangZ., YangL., TianS., HouC., LuY.Analyst, 2010,135,1406-1410.].Up to now, there is not been reported in the work of the fluorescence nano gold bunch mensuration mitoxantrone utilizing BSA to be templated synthesis.
The object of this invention is to provide a kind of bio-sensing fluoroscopic examination cancer therapy drug mitoxantrone new method based on golden bunch of water-soluble Illuminant nanometer, to solve the deficiency in existing detection mitoxantrone technology.Technical scheme of the present invention is as follows: a kind of method detecting mitoxantrone based on luminescent gold nano-cluster, comprises the following steps: step one, prepare gold nanoclusters, under the exciting of 469nm wavelength light, measures gold nanoclusters in the fluorescence intensity at 619nm wavelength place, is designated as F
0; Step 2, add different quality mitoxantrone in step one after, the gold nanoclusters of mensuration containing variable concentrations mitoxantrone, under 469nm wavelength light excites, in the fluorescence intensity at 619nm wavelength place, is designated as F
,after adding different quality mitoxantrone, the changing value of fluorescence intensity, calculates and adds the changing value of gold nanoclusters fluorescence intensity and the linear relationship of mitoxantrone concentration before and after mitoxantrone; Step 3, adds testing sample in gold nanoclusters, tests it and adds front and back under the exciting of 469nm wavelength light, at the changing value of the fluorescence intensity at 619nm wavelength place, according to the linear relationship that step 2 obtains, calculate the quality or concentration that add mitoxantrone.
During described fluorescence intensity test, the slit width of instrument is set as 5nm.
In described step one, the preparation process of gold nanoclusters is: the tetra chlorauric acid solution of preparation 10mg/mL, and the solution getting just preparation joins in bovine serum albumin solution, and period carries out vigorous stirring, make it fully to be mixed, after 2 minutes, then add NaOH solution, continue stirring 12 hours, maintain the temperature at 37
?c.
Advantageous Effects of the present invention:
1, detection method of the present invention has high selectivity, high sensitivity, the characteristic such as easy, easy.
2, this detection method can be generalized to the mensuration of Anthraquinones cancer therapy drug, and in the detection of other medicines.
3, the foundation of the method can provide new approaches for highly sensitive, the quick detection of medicine.
Accompanying drawing explanation
Fig. 1 Illuminant nanometer gold bunch fluorescent quenching detects mitoxantrone schematic diagram
Fig. 2 adds the fluorescence intensity level of the gold nanoclusters after the mitoxantrone medicine of variable concentrations in embodiment 1.
embodiment:
embodiment 1
Take BSA as the nm of gold bunch that templated synthesis has fluorescent characteristic: the synthesis of fluorescence nano gold bunch is that method according to bibliographical information is [see XieJ., ZhengY., YingJ.Y.
j.Am.Chem.Soc., 2009,131,888-889.], have made some improvements, process is as follows: first, and 1g tetra chlorauric acid is dissolved in 100mL distilled water, forms the storing solution of 10mg/mL.Then, getting solution that 4.1mL just prepared, to join 10mL concentration be in 50mg/mL bovine serum albumin solution, temperature of reaction 37
?c, period carries out vigorous stirring, makes it fully to be mixed.After 2 minutes, then to add 1mL concentration be 1.0mol/LNaOH solution, continues stirring 12 hours, maintain the temperature at 37
?c.After improvement, generated time shortened to 12 hours by three days.The gold nanoclusters probe prepared, has stable fluorescent characteristic, and at 619nm wavelength, there is maximum emission peak at place, and excitation wavelength is 469nm.
Utilize the fluorogold nano-cluster probe in detecting cancer therapy drug mitoxantrone concrete steps prepared: get nm of gold bunch sample prepared by 300 μ L, join in centrifuge tube, then add the mitoxantrone medicine working solution of a series of concentration, detect variable concentrations mitoxantrone respectively to the impact (final concentration is 0,2,4,6,8,10,12,14,16,18,20,22,24,26,28,30,32,34,36,38,40,42,44,46,48,50,52,54 μ g/mL) of the fluorescence signal of gold nanoclusters probe.Being diluted to final volume with distilled water is 3mL, mixes, measures.Under the exciting of 469nm wavelength light, the fluorescence spectrum of test gold nanoclusters, the slit width of instrument is set as 5nm, detects the emission peak fluorescence intensity at 619nm wavelength place.Test adds the fluorescence signal of sample after medicine, is recorded in the fluorescence intensity of this emission maximum peak position, is expressed as: Δ
f=
f 0 –
f, wherein,
f 0 with
fbe respectively nm of gold bunch self and the fluorescence intensity after adding cancer therapy drug.Adding medicine, there is Fluorescence-quenching in the fluorescence signal of probe, and along with the increase of drug concentration, fluorescence probe signal intensity weakens thereupon, in 2 ~ 20 μ g/mL concentration ranges, the fluorescence intensity of mitoxantrone concentration and probe presents good linear relationship, and it detects and is limited to 1 μ g/mL.
Human serum sample tests: get Freshman blood serum sample, add the protein in appropriate phenol-chloroform-isoamylol (v/v/v=25:24:1) precipitation sample, get supernatant and dilute 100 times as solution to be measured after high speed centrifugation.In PE pipe, add 300 μ L blood samples successively, nm of gold bunch, a certain amount of mitoxantrone that 300 μ L synthesize, be diluted to 3mL with distilled water, mix, then carry out analysiss mensuration, the results are shown in Table 1.
The test result of table 1 human serum sample
embodiment 2
Utilize the fluorogold nano-cluster probe in detecting Daunomycin concrete steps prepared: get nm of gold bunch sample prepared by 300 μ L, join in centrifuge tube, then add the daunorubicin medicine working solution of a series of concentration, detect variable concentrations daunorubicin respectively to the impact (final concentration is 0,2,4,10,20,50,70,100,110,120,130,140 μ g/mL) of the fluorescence signal of gold nanoclusters probe.Being diluted to final volume with distilled water is 3mL, mixes, measures.Under the exciting of 469nm wavelength light, the fluorescence spectrum of test gold nanoclusters, the slit width of instrument is set as 5nm, detects the emission peak fluorescence intensity at 619nm wavelength place.Test adds the fluorescence signal of sample after medicine, is recorded in the fluorescence intensity of this emission maximum peak position, is expressed as: Δ
f=
f 0 –
f, wherein,
f 0 with
fbe respectively nm of gold bunch self and the fluorescence intensity after adding cancer therapy drug.Adding medicine, there is Fluorescence-quenching in the fluorescence signal of probe, and along with the increase of drug concentration, fluorescence probe signal intensity weakens thereupon, in 1 ~ 18 μ g/mL concentration range, the fluorescence intensity of daunorubicin concentration and probe presents good linear relationship, and it detects and is limited to 0.5 μ g/mL.
Human serum sample tests: get Freshman blood serum sample, add the protein in appropriate phenol-chloroform-isoamylol (v/v/v=25:24:1) precipitation sample, get supernatant and dilute 100 times as solution to be measured after high speed centrifugation.In PE pipe, add 300 μ L blood samples successively, nm of gold bunch, a certain amount of daunorubicin that 300 μ L synthesize, be diluted to 3mL with distilled water, mix, then carry out analysiss mensuration, the results are shown in Table 2.
The test result of table 2 human serum sample
More than show and describe ultimate principle of the present invention and principal character and advantage of the present invention.The technician of the industry should be appreciated that; the present invention is not restricted to the described embodiments; what describe in above-described embodiment and instructions just illustrates principle of the present invention; without departing from the spirit and scope of the present invention; the present invention also has various changes and modifications; these changes and improvements all fall in the claimed scope of the invention, and application claims protection domain is defined by its equivalent of appending claims.
Claims (3)
1. detect a method for mitoxantrone based on luminescent gold nano-cluster, comprise the following steps: step one, prepare gold nanoclusters, under the exciting of 469nm wavelength light, measure gold nanoclusters in the fluorescence intensity at 619nm wavelength place, be designated as F
0; Step 2, add different quality mitoxantrone in step one after, the gold nanoclusters of mensuration containing variable concentrations mitoxantrone, under 469nm wavelength light excites, in the fluorescence intensity at 619nm wavelength place, is designated as F
,after adding different quality mitoxantrone, the changing value of fluorescence intensity, calculates and adds the changing value of gold nanoclusters fluorescence intensity and the linear relationship of mitoxantrone concentration before and after mitoxantrone; Step 3, adds testing sample in gold nanoclusters, tests it and adds front and back under the exciting of 469nm wavelength light, at the changing value of the fluorescence intensity at 619nm wavelength place, according to the linear relationship that step 2 obtains, calculate the quality or concentration that add mitoxantrone.
2. detect the method for mitoxantrone as claimed in claim 1 based on luminescent gold nano-cluster, when it is characterized in that fluorescence intensity is tested, the slit width of instrument is set as 5nm.
3. the method for mitoxantrone is detected as claimed in claim 1 or 2 based on luminescent gold nano-cluster, it is characterized in that the preparation process of gold nanoclusters in step one is: the tetra chlorauric acid solution of preparation 10mg/mL, the solution getting just preparation joins in bovine serum albumin solution, period carries out vigorous stirring, make it fully to be mixed, after 2 minutes, then add NaOH solution, continue stirring 12 hours, maintain the temperature at 37
?c.
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| CN106198478B (en) * | 2016-08-03 | 2018-12-07 | 陕西师范大学 | The method of molecularly imprinted polymer detection mitoxantrone based on quantum dot ratio fluorescent |
| CN110174459A (en) * | 2019-06-14 | 2019-08-27 | 广西师范大学 | Application of the golden cluster in Matrix-assisted laser desorption ionization detection |
| CN113292987B (en) * | 2021-05-26 | 2022-06-28 | 四川中科微纳科技有限公司 | Double-emitting gold cluster ratiometric fluorescent probe, preparation method thereof and doxycycline detection method |
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| CN103006565A (en) * | 2011-09-28 | 2013-04-03 | 国家纳米科学中心 | Nano granular medicinal composition and preparation method thereof |
| CN103196883A (en) * | 2013-04-19 | 2013-07-10 | 中国科学院化学研究所 | Method for detecting activity of chymotrypsin by adopting gold nanoclusters |
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| CN103196883A (en) * | 2013-04-19 | 2013-07-10 | 中国科学院化学研究所 | Method for detecting activity of chymotrypsin by adopting gold nanoclusters |
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