CN103323440B - Method for detecting sulfide ions - Google Patents

Abstract

The invention provides a fluorescence detection method for sulfide ions, which is a method for quantitatively detecting the sulfide ions based on perylene-3, 4, 9, 10-tetracarboxylic acid dianhydride. Specifically, the method comprises the following steps of: adding sulfide ions into HEPES (4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid) containing the perylene-3, 4, 9, 10-tetracarboxylic acid dianhydride with the pH of 7.0 (without fluorescence emission), producing strong fluorescence emission and realizing the detection of the sulfide ions. According to the detection method, high sensitivity and selectivity are showed against the sulfide ions, and the method has the advantages of low price of a detection reagent, simplicity and convenience in detection process, sensitivity, quickness and accurate detection result.

Description

A kind of method detecting sulphion

Technical field

The present invention relates to sulphion detection technique, specifically belong to perylene-3,4,9,10-tetracarboxylic dianhydride is as fluorescent reagent, and quick, quantitative fluorescence detects the method for sulphion.

Background technology

Sulfuretted hydrogen (H ₂s), there is unpleasant rotten-egg odour, be considered to a kind of toxic gas traditionally.But nearest research shows, sulfuretted hydrogen is a kind of endogenous gas signal compound (gas conveyer), its importance and other two known endogenous gas signaling molecules: nitrogen monoxide (NO) and carbon monoxide (CO) are mentioned in the same breath.But in recent years, research evidence shows, in mammal, this Small molecular can as a cell signalling agent.Endogenous and external source H ₂the generation of S has been proved to be has protective effect in much pathology.Such as, H ₂s relaxes vascular smooth muscle, causes myocardium vessel to expand, reduces blood pressure.H ₂s participates in the enhancing of blood pressure and its abnormal concentrations and central nervous system disease, as relevant with other mental disease in Down syndrome, Alzheimer's.Therefore, the detection of sulphion has important biological significance.Sulphion measures the several method existed, and comprises based on the stoichiometer of organic chromophores or fluorophore, electrochemical process, phosphorimetry and atomic emissions etc.Above method major part relates to the synthesis detecting reagent, or testing process is loaded down with trivial details.

Therefore, development business can obtain, cheap reagent, and high, the highly sensitive sulphion quantitative detecting method of easy to operate, selectivity is particularly important.

Summary of the invention:

The object of the invention is, in order to overcome Problems existing in the detection of prior art sulphion, to provide the method for the fluoroscopic examination sulphion that a kind of system is cheap, easy to operate, selectivity is high, highly sensitive.

Shang industry Ke get perylene-3,4,9,10-tetracarboxylic dianhydride applies in detection sulphion as fluorescent reagent by the present invention.Suo Shu perylene-3,4,9,10-tetracarboxylic dianhydride, its structural formula is:

A kind of method detecting sulphion provided by the invention: step is:

(1), preparation pH=7.0, concentration is the HEPES buffer solution of 10mM, the DMSO solution of preparation 2mM perylene-3,4,9,10-tetracarboxylic dianhydride;

(2), 200:1 by volume, be added in clean fluorescence cuvette by the DMSO solution of HEPES buffer solution and perylene-3,4,9,10-tetracarboxylic dianhydride, Fluorescence spectrophotometer detects, and along with treating adding of test sample, the fluorescence intensity of 499nm strengthens gradually;

(3), the HEPES buffer solution of 2mL, 10 μ L perylene-3,4,9,10-tetracarboxylic dianhydride DMSO solution are added in another fluorescence cuvette, are adding S respectively ^{2 –}when the volume of solution is 10,20,40,60,80,100,120,140,160,180,200 μ L, it is 76,119,205,281,370,448,517,599,677,742,828 that Fluorescence spectrophotometer measures fluorescence intensity F corresponding to 499nm, with S ^2-concentration is horizontal ordinate, with relative intensity of fluorescence F-F ₀for ordinate draws figure, F ₀﹦ 35, obtains S ^{2 –}the working curve of concentration; Equation of linear regression is: F-F ₀the unit of=-2.302+4.336c, c is 10 ^-6mol/L;

(4), working sample solution time, the fluorescence intensity that records is substituted into equation of linear regression, can S be tried to achieve ^{2 –}concentration c.

Compared with prior art, tool of the present invention has the following advantages and effect: 1, detection system is with low cost, and reagent business can obtain and cheap; 2, detection method of the present invention, to S ^{2 –}show high sensitivity and selectivity; 3, testing process is carried out in aqueous phase; 4, detection means is simple, only needs can realize by Fluorescence spectrophotometer.

Accompanying drawing illustrates:

Fig. 1 embodiment 1 perylene-3,4,9,10-tetracarboxylic dianhydride and S ^{2 –}the fluorescent emission figure of effect

The fluorescence histogram of Fig. 2 embodiment 2 perylene-3,4,9,10-tetracarboxylic dianhydride and various analysis thing

Fig. 3 embodiment 3 working curve

The fluorescent emission figure of Fig. 4 embodiment 4 working sample

Fig. 5 embodiment 5 cell imaging figure

Embodiment:

Embodiment 1

Preparation pH=7.0, concentration are the HEPES buffer solution of 10mM, and prepare 2mM perylene-3,4,9,10-tetracarboxylic dianhydride's solution with DMSO; The HEPES buffer solution of 2mL and 10 μ L perylene-3,4,9,10-tetracarboxylic dianhydride DMSO solution are added in clean fluorescence cuvette, get S ^{2 –}solution, be added in this cuvette with microsyringe gradually, application of sample limit, limit is detected, along with S on Fluorescence spectrophotometer ^{2 –}add, 499nm place fluorescence intensity strengthens gradually.Fluorescent emission figure is shown in Fig. 1.

Embodiment 2

Preparation pH=7.0, concentration are the HEPES buffer solution of 10mM, and prepare 2mM perylene-3,4,9,10-tetracarboxylic dianhydride's solution with DMSO; In 15 fluorescence cuvette, respectively add HEPES buffer solution and 10 μ L perylene-3,4,9, the 10-tetracarboxylic dianhydride DMSO solution of 2mL, then add the S of 20 molar equivalents respectively ^{2 –}, and other various analysis things of 100 molar equivalents: Cys, Hcy, GSH, CN ^–, SCN ^–, HSO ₃ ^–, HCO ₃ ^–, SO ₄ ^{2 –}, N ₃ ^–, P ₂o ₇ ^{4 –}, citrate, HPO ₄ ^{2 –}, F ^–, Fluorescence spectrophotometer detects, draws the different histogram analyzing 499nm fluorescence intensity corresponding to thing, obtain fluorescent emission figure (see figure 2).S ^{2 –}the fluorescence intensity of Shi get perylene-3,4,9,10-tetracarboxylic dianhydride changes to about 830 by 35, the basic change not having Yin Qi perylene-3,4,9,10-tetracarboxylic dianhydride's fluorescence intensity of other analysis thing.

The experiment proved that, other analyze thing not interference system to S ^{2 –}mensuration.

Embodiment 3

Preparation pH=7.0, concentration are the HEPES buffer solution of 10mM, and prepare 2mM perylene-3,4,9,10-tetracarboxylic dianhydride's solution with DMSO, with the S of distilled water preparation 2mM ^{2 –}solution; The HEPES buffer solution of 2mL and 10 μ L perylene-3,4,9,10-tetracarboxylic dianhydride DMSO solution are added in fluorescence cuvette, are adding S respectively ^{2 –}when the volume of solution is 10,20,40,60,80,100,120,140,160,180,200 μ L, it is 76,119,205,281,370,448,517,599,677,742,828 that Fluorescence spectrophotometer measures fluorescence intensity F corresponding to 499nm, with S ^2-concentration is horizontal ordinate, with relative intensity of fluorescence F-F ₀for ordinate draws figure, F ₀﹦ 35, obtains S ^{2 –}the working curve (see figure 3) of concentration; Equation of linear regression is: F-F ₀the unit of=-2.302+4.336c, c is 10 ^-6mol/L.

Embodiment 4

Preparation pH=7.0 HEPES(10mM) buffer solution, preparation 2mM S ^2-aqueous solution, and prepare 2mM perylene-3,4,9,10-tetracarboxylic dianhydride's solution with DMSO; The HEPES buffer solution of 2mL and 10 μ L perylene-3,4,9,10-tetracarboxylic dianhydride DMSO solution are added in clean fluorescence cuvette, get S ^2-solution 145 μ L, be added in this cuvette with microsyringe, the fluorescence intensity F simultaneously measuring the correspondence of 499nm in fluorescence spectrophotometer is 414, relative intensity of fluorescence F ﹣ F ₀﹦ 382, by the equation of linear regression of embodiment 4, tries to achieve c=87.9 × 10 ^-6mol/L, deviation is 2.25%, sees Fig. 4.

Embodiment 5

2mM perylene-3,4,9,10-tetracarboxylic dianhydride DMSO solution is prepared with DMSO; Jiang perylene-3,4,9,10-tetracarboxylic dianhydride DMSO solution adds in hepatoma carcinoma cell nutrient solution, makes its concentration be 10 μMs, adds the S of external source simultaneously ^{2 –}, make its concentration be 100 μMs, under 37 ° of C, reaction 30min, system nothing under fluorescence co-focusing imager shows strong green fluorescence.Fig. 5 is that hepatoma carcinoma cell a(unstressed configuration original under fluorescence co-focusing imager is launched) and Yu perylene-3,4,9,10-tetracarboxylic dianhydride and S ^{2 –}the cell b(green fluorescence of effect) image.

Claims (2)

1. perylene-3,4,9,10-tetracarboxylic dianhydride is detecting the application in sulphion as fluorescent reagent.

2. one kind is detected the method for sulphion: it is characterized in that, step is:

(1), preparation pH=7.0, concentration is the HEPES buffer solution of 10mM, the DMSO solution of preparation 2mM perylene-3,4,9,10-tetracarboxylic dianhydride;

(2), 200:1 by volume, by HEPES buffer solution with perylene-3,4, the DMSO solution of 9,10-tetracarboxylic dianhydride is added in clean fluorescence cuvette, and Fluorescence spectrophotometer detects, along with treating adding of test sample, the fluorescence intensity of 499nm strengthens gradually;

(3), with the S of distilled water preparation 2mM ^{2 –}solution, is added to the HEPES buffer solution of 2mL, 10 μ L perylene-3,4,9,10-tetracarboxylic dianhydride DMSO solution in another fluorescence cuvette, is adding S respectively ^{2 –}when the volume of solution is 10,20,40,60,80,100,120,140,160,180,200 μ L, it is 76,119,205,281,370,448,517,599,677,742,828 that Fluorescence spectrophotometer measures fluorescence intensity F corresponding to 499nm, with S ^2-concentration is horizontal ordinate, with relative intensity of fluorescence F-F ₀for ordinate draws figure, F ₀﹦ 35, obtains S ^{2 –}the working curve of concentration; Equation of linear regression is: F-F ₀the unit of=-2.302+4.336c, c is 10 ^-6mol/L;

(4), working sample solution time, the fluorescence intensity that records is substituted into equation of linear regression, can S be tried to achieve ^{2 –}concentration c.