CN102435592B - Method for determining Nevirapine by utilizing ZnS nano fluorescence probe - Google Patents
Method for determining Nevirapine by utilizing ZnS nano fluorescence probe Download PDFInfo
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- CN102435592B CN102435592B CN 201110344793 CN201110344793A CN102435592B CN 102435592 B CN102435592 B CN 102435592B CN 201110344793 CN201110344793 CN 201110344793 CN 201110344793 A CN201110344793 A CN 201110344793A CN 102435592 B CN102435592 B CN 102435592B
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Abstract
The invention relates to a method for rapidly determining Nevirapine by utilizing mercaptoacetic-acid-modified ZnS nano particles as a nano fluorescence probe, and belongs to the technical field of optical analysis detection. In the method provided by the invention, a principle that the fluorescence intensity changes after the ZnS nano fluorescence probe interacts with Nevirapine to be determined is mainly utilized, thus sensitive and quantitative analysis and determination are carried out on the Nevirapine through spectrofluorimetry. In the method provided by the invention, by optimizing experiment conditions such as temperature, addition sequence, pH value and reaction time, the fluorescence probe is applied to quantitative detection of the Nevirapine in optimal conditions. The determination process provided by the invention has the characteristics of simpleness, rapidness, sensitivity, accuracy and the like.
Description
Technical field
The present invention relates to a kind of semiconductor nano material zinc sulphide that utilizes as the method for fluorescence probe mensuration NVP, belong to the light technical field of analysis and detection.
Background technology
Nano material is an important directions in the modern scientific research as the important research object of nanometer technology.Quantum confined effect makes nano material compare with the macroscopic body phase material to have many unusual performances, as surface effect, quantum size effect and macroscopical tunnel effect etc.Be subjected to the influence of these effects, nano particle has shown performances such as the special light that is different from bulk matter, electricity, magnetic, heat, mechanics, machinery, utilizes its special luminescent properties, and people can be applied in the every field and go it as namo fluorescence probe.Compare with traditional organic fluorescent dye, namo fluorescence probe has more superior performance, for example wide and continuous excitation spectrum, narrow and symmetrical emission spectrum, can be accurately tuning emission wavelength, long fluorescence lifetime and insignificant photobleaching etc., these features make namo fluorescence probe more and more replace organic fluorescent dye to be applied to fluorescence labeling, quantitative various aspects such as detection.Wherein, the application of namo fluorescence probe in life science received the concern of height, and become the focus of a research rapidly, the major part of having reported concentrates on identification and the imaging aspect of fluorescence probe, as the interaction between research and biomacromolecule such as protein, nucleic acid, DNA, the application of aspects such as the fluorescence labeling of cell and imaging and living imaging.Meanwhile, also very fast based on the fluorescence probe quantitative measurement technology development of fluorescent quenching or enhanced sensitivity.Nowadays Bei Jiance material mainly contains heavy metal ion, inorganic ions, protein, vitamin etc., and utilizes the application of semi-conductor nano particles mensuration medicine aspect also actually rare.
NVP is a kind of novel non-nucleoside reverse transcriptase inhibitor, and it can suppress the reverse transcriptase of HIV-1, suppresses virus replication, reduces that it is pathogenic, has that oral absorption is good, low toxin.Clinically with antiretroviral drugs (as Zidovudine, Da Nuoxin) share and carry out triple therapy, be used for the treatment of AIDS viral infection, separately medication can be used for preventing HIV to infect and mother-to-baby transmission.The patient must strictly observe the dosage requirement when taking NVP, and in initial 8 weeks for the treatment of phase, should carry out tight monitoring to patient's situation, in order in time find potential serious and life-threatening dermoreaction or serious hepatitis, hepatic failure.Have report to use among the patient of NVP treatment and once produced serious life-threatening dermoreaction, comprise Stevens-Johnson syndrome (SJS), toxicity epidermis necrolysis (TEN), impaired with fash, constitutional symptom and internal organ be the allergic reaction of characteristics.Therefore, set up a kind of simple, fast, sensitive, accurately detect NVP analysis on Content method and having great significance aspect clinical medicine and the pharmaceutical research.Assay method for NVP has at present: vapor-phase chromatography, the high liquid phase look of imitating are composed – UV method, using high performance liquid chromatography tandem mass spectrum method, micellar electrokinetic chromatography method, capillary zone electrophoresis method etc., but the method that is to use namo fluorescence probe to measure this medicine yet there are no report.
Summary of the invention
The purpose of this invention is to provide a kind of method of utilizing semiconductor nano material zinc sulphide to measure the NVP medicine as namo fluorescence probe.
A kind of semiconductor nano material zinc sulphide that utilizes is characterized in that having following process and step as the method for namo fluorescence probe mensuration NVP:
A. the preparation of ZnS namo fluorescence probe: get the synthetic white nanometer ZnS nano particle of 0.0100 g in beaker, add a certain amount of redistilled water, place on the self-poking arrangement and constantly stir; Slowly splash into the NaOH solution of 0.10 M for preparing in the beaker, about the pH value to 12 of regulator solution; Get the mercaptoacetic acid of about 2.0 ml in another beaker, with redistilled water it being diluted to the pH value is about 2; Then with the dilution after mercaptoacetic acid solution dropwise add in the nanometer ZnS solution, until the pH value of solution value about 9; Stir about 3 h of above-mentioned solution with keeping.Nanometer ZnS solution upper strata after the modification is colloidal sol, the visible a small amount of leucosol precipitation of beaker bottom, and drawing upper solution with dropper is the ZnS namo fluorescence probe.
B. the selection of fluorometric investigation condition: it is 268 nm that excitation wavelength is set, and this wavelength place NVP does not have absorption peak to exist, and the fluorescence emission peak peak shape is narrower, intensity is stronger; Setting excites and launches slit width and is 5 nm, and sweep limit is set to 278 nm to 350 nm;
C. the ZnS namo fluorescence probe is to the mensuration of NVP: add the NVP standard solution of a series of concentration known and 1 ml synthetic good mercaptoacetic acid modified ZnS solution in a series of brown, dry 25 ml volumetric flasks.Be settled to scale after then it being diluted with phosphate buffer solution.The solution for preparing after placing the regular hour, the dark place is done fluorometric investigation.Find that by fluorescent spectroscopy the fluorescence intensity of ZnS namo fluorescence probe reduces with the increase of NVP concentration.In 4.6 μ M ~ 200 μ M scopes, the relative intensity of fluorescence of ZnS namo fluorescence probe therewith in the concentration range concentration of NVP linear.
Advantage of the present invention and characteristics are as described below:
The present invention utilizes the optical characteristics of semi-conductor nano particles uniqueness, and big specific surface area and the character that some are relevant with size are gone detection of drugs to semi-conductor nano particles as namo fluorescence probe.By optimizing the reaction time of temperature, addition sequence, pH value and ZnS fluorescence probe and NVP, drawn the top condition of measuring.Because the optical characteristics of quantum dot depends on its surface nature, the quantum dot surface can cause the dynamic change of its optical property with the interaction of amalyzing substances, so can utilize it as fluorescence probe amalyzing substances to be detected under top condition.
It is a kind of novel fluorescent reagent that mercaptoacetic acid among the present invention is modified the ZnS namo fluorescence probe, by optimizing test condition, can be directly used in the quick mensuration of NVP, has characteristics such as simple, quick, sensitive, accurate.
Description of drawings
Fig. 1 is for measuring the fluorescence spectrum figure of variable concentrations NVP with mercaptoacetic acid modified ZnS namo fluorescence probe among the present invention.C (10
– 7Mol/L) be :) (A): 0, (B): 46, (C): 160, (D): 280, (E): 400, (F): 640, (G): 880, (H): 1200, (S): 1600, (J): 2000.As can be seen from the figure, along with the increase of NVP concentration, the fluorescence intensity of ZnS namo fluorescence probe reduces gradually.
Fig. 2 is the relative intensity of fluorescence under ZnS namo fluorescence probe among the present invention and the existence of variable concentrations NVP and the linear relationship chart of NVP concentration.
Embodiment
After now specific embodiments of the invention being described in.
The step of utilizing mercaptoacetic acid modified ZnS namo fluorescence probe mensuration NVP in the present embodiment is as follows:
A. the preparation of ZnS nano particle: accurately take by weighing 0.7161 g zinc powder and 0.1172 g sulphur powder (mol ratio 3:1), putting into specification together is the liner of the teflon autoclave of 30 ml, adds the NaOH solution of 3 M that 25 ml prepare then; Sealing is placed on reacts 24 h under 180 ℃ of temperature in the baking oven.Reaction finishes and closes baking oven, treats that the teflon high pressure vessel naturally cools to room temperature; Draw the upper strata stillness of night, pour the liner bottom product into beaker, add ultrasonic, centrifugal, the washing of deionized water, then with absolute ethyl alcohol replace that deionized water is ultrasonic, centrifugal, washing is for several times till ultrasonic back beaker bottom noresidue solid particle; Product after centrifugal is disperseed slightly with an amount of absolute ethyl alcohol, be poured on the surface plate, place in the baking oven, at 80 ℃ of following heating, drying products, obtain 0.12794 g ZnS nano particle.
B. the preparation of ZnS namo fluorescence probe: get the synthetic white nanometer ZnS nano particle of 0.0100 g in beaker, add a certain amount of redistilled water, place on the self-poking arrangement and constantly stir; Think slowly to splash in the beaker NaOH solution of 0.10 M for preparing, about the pH value to 12 of regulator solution; Get the mercaptoacetic acid of about 2.0 ml in another beaker, with redistilled water it being diluted to the pH value is about 2; Then with the dilution after mercaptoacetic acid solution dropwise add in the nanometer ZnS solution, until the pH value of solution value about 9; Stir about 3 h of above-mentioned solution with keeping.Nanometer ZnS solution upper strata after the modification is colloidal sol, the visible a small amount of leucosol precipitation of beaker bottom, and drawing upper solution with dropper is the ZnS namo fluorescence probe.
C. the using method of this namo fluorescence probe and measure as follows: in a series of brown, dry 25 ml volumetric flasks, add the NVP standard solution of a series of concentration known and 1 ml synthetic good mercaptoacetic acid modified ZnS solution.By optimizing experiment condition, being settled to scale after the phosphate buffer solution dilution of the solution that adds with pH=7, the solution for preparing after placing 1 h, the dark place is done fluorometric investigation.It is 268 nm that excitation wavelength is set, and this wavelength place NVP does not have absorption peak to exist.Setting excites and launches slit width and is 5 nm, and sweep limit is set to 278 nm to 350 nm.
Fluorescence analysis method detects NVP
Under best test condition, as shown in Figure 1, the fluorescence intensity of ZnS namo fluorescence probe reduces with the increase of NVP concentration.In 4.6 μ M ~ 200 μ M scopes, the relative intensity of fluorescence of ZnS namo fluorescence probe therewith in the concentration range concentration of NVP linear, as shown in Figure 2.Linear equation is log (F
0/ F)=0.00729+6.00846*10
3C (C, 10
– 7M), linearly dependent coefficient is 0.9996.
The method that the present invention utilizes mercaptoacetic acid modified ZnS namo fluorescence probe to measure NVP has simply, quick, sensitive, characteristic of accurate.When measuring NVP with the ZnS namo fluorescence probe, some common metal cations, negative ion, little molecular biosciences molecule are to mensuration and noiseless.By recovery measuring, the recovery is between 95.9% to 101.3%.Its minimum detectability is 0.73 μ M.
Claims (1)
1. utilize the zinc sulfide nano fluorescence probe to measure the method for NVP, it is characterized in that this method has following step:
A. the preparation of ZnS namo fluorescence probe: get the synthetic white nanometer ZnS nano particle of 0.0100 g in beaker, add a certain amount of redistilled water, place on the self-poking arrangement and constantly stir; Slowly splash into the NaOH solution of 0.10 M for preparing in the beaker, regulate the pH value to 12 of suspending liquid; Get the mercaptoacetic acid of 2.0 ml in another beaker, with redistilled water it being diluted to the pH value is 2; Then with the dilution after mercaptoacetic acid solution dropwise add in the nanometer ZnS suspending liquid, until the pH value 9; Stir above-mentioned suspending liquid 3 h with keeping; Nanometer ZnS solution upper strata after the modification is colloidal sol, the visible a small amount of leucosol precipitation of beaker bottom, and drawing upper solution with dropper is the ZnS namo fluorescence probe;
B. the selection of fluorometric investigation condition: it is 268 nm that excitation wavelength is set, and arranges to excite and launch slit width to be 5 nm, and sweep limit is set to 278 nm to 350 nm;
C. the ZnS namo fluorescence probe is to the mensuration of NVP: add the NVP standard solution of a series of concentration known and 1 ml synthetic good mercaptoacetic acid modified ZnS solution in a series of brown, dry 25 ml volumetric flasks; Be settled to scale after then it being diluted with the pH=7 phosphate buffer solution; The solution for preparing after placing 1 h, the dark place is done fluorometric investigation; In 4.6 μ M ~ 200 μ M scopes, utilize the relative intensity of fluorescence of ZnS namo fluorescence probe and the linear relationship of NVP concentration to measure NVP.
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| CN101281131A (en) * | 2008-05-26 | 2008-10-08 | 南开大学 | Method for detecting enoxacin in biological body fluid with Mn doping ZnS quantum point room temperature phosphorescent |
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Non-Patent Citations (8)
| Title |
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| Fluorometric Measurement of Reverse Transcriptase Activity with 4",6-Diamidino-2-phenylindole;Surendra J.Chavan et al.;《Analytical Biochemistry》;19951231;第225卷;第54-59页 * |
| Rapid determination of nevirapine in human plasma by ion-pair reversed-phase high-performance liquid chromatography with ultraviolet detection;Rolf P.G. van Heeswijk et al.;《Journal of Chromatography B》;19981231;第713卷;第395-399页 * |
| Rolf P.G. van Heeswijk et al..Rapid determination of nevirapine in human plasma by ion-pair reversed-phase high-performance liquid chromatography with ultraviolet detection.《Journal of Chromatography B》.1998,第713卷第395-399页. |
| SurendraJ.Chavanetal..FluorometricMeasurementofReverseTranscriptaseActivitywith4" 6-Diamidino-2-phenylindole.《Analytical Biochemistry》.1995 |
| 两种新型镍-邻菲咯啉类核酸荧光探针的合成及性质研究;庄惠生;《化学试剂》;20031231;第25卷(第6期);第325-328页 * |
| 吴翠敏等.荧光淬灭法分析奈韦拉平含量.《福建医科大学学报》.2006,第40卷(第5期),第523-525页. |
| 庄惠生.两种新型镍-邻菲咯啉类核酸荧光探针的合成及性质研究.《化学试剂》.2003,第25卷(第6期),第325-328页. |
| 荧光淬灭法分析奈韦拉平含量;吴翠敏等;《福建医科大学学报》;20060930;第40卷(第5期);第523-525页 * |
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