CN108997255A

CN108997255A - A kind of vinyl ethers Hg2+Fluorescence probe and its preparation method and application

Info

Publication number: CN108997255A
Application number: CN201810854561.6A
Authority: CN
Inventors: 毕晶晶; 梁榕; 时蕾; 张贵生; 郝玉伟
Original assignee: Henan Normal University
Current assignee: Henan Normal University
Priority date: 2018-07-30
Filing date: 2018-07-30
Publication date: 2018-12-14

Abstract

The invention discloses a kind of vinyl ethers Hg²⁺Fluorescence probe and its preparation method and application belongs to highly selective detection Hg²⁺Fluorescent probe technique field.A kind of technical solution of the present invention main points are as follows: vinyl ethers Hg²⁺Fluorescence probe, structural formula are as follows:The invention further particularly discloses vinyl ethers Hg²⁺The preparation method of fluorescence probe and its Hg in selective enumeration method analysis water environment system or biological cell system²⁺Application.Vinyl ethers Hg produced by the present invention²⁺Fluorescence probe can highly selective identification Hg²⁺, and common coexistent metallic ion does not interfere with it；Vinyl ethers Hg obtained²⁺Fluorescence probe has good water-soluble, biocompatibility and membrane penetrating, can be used for intracellular Hg²⁺Detection and cell imaging.

Description

A kind of vinyl ethers Hg2+Fluorescence probe and its preparation method and application

Technical field

The invention belongs to chemical analyses, bioanalysis detection technique field, relate generally to highly selective detection Hg²⁺Fluorescence A kind of probe technique field, and in particular to vinyl ethers Hg²⁺Fluorescence probe and its preparation method and application.

Background technique

Mercury is silvery white liquid metal, is mainly existed in the form of mercury element, inorganic mercury, three kinds of organic mercury in nature. Mercury and mercuric compounds chemical industry, electronic apparatus industry, in terms of have extensive use, as air gauge, Thermometer, fluorescent lamp, drug etc..But mercury is widely present in as a kind of in all kinds of surrounding mediums and with serious physiological-toxicity Heavy metal has biggish harm to environmental and biological materials.Mercury simple substance can constantly be discharged with volatile mercury at room temperature Steam, after invading human body by different approaches such as respiratory tract, skin or alimentary canals, meeting Central nervous system causes seriously to damage Evil, so as to cause a series of adverse reactions, such as vomiting, diarrhea, respiratory failure are even dead；Electrodeless mercury salt is easily converted into toxicity Bigger organic mercury, organic mercury can be enriched in food chain in aquatile cylinder accumulation and constantly, thus to Man & Nature circle It causes huge harm, most typical example is exactly ' minamata disease ' occurred in Japan.Due to its persistence, property easy to migrate and height Bioconcentration, mercury become natural environment Poisoning most one of strong, the maximum heavy metal of pollution.

Therefore, the accurately and effectively content of determination of the environment and Mercury in Biological Sample ion, for bioscience, environmental protection It is crucial in terms of medicine.It so far, mainly include Atomic absorption emission spectrometry to the detection method of mercury ion (AAS/AES), electrochemical method and inductively coupled plasma mass spectrometry analysis (ICP-MS) method etc., these methods have both sensitivity The high and good advantage of selectivity, but because it detects the problems such as speed is slow, instrument price is expensive, complicated for operation, limit they Application in mercury ion detection and localization.Fluorescent molecule detection technique is to be specifically bound using fluorescence probe with targeted molecular Effect discharges the characteristic fluorescent signal that can be detected, and realizes the analysis means of particle oriented detection, the technology and traditional change Learn sensor compared to have the characteristics that high sensitivity, quick and precisely, specificity it is strong, receive the extensive concern of researcher.

Summary of the invention

Present invention solves the technical problem that be the shortcoming overcome existing fluorescent probe technique in performance and structure and Provide a kind of vinyl ethers Hg²⁺Fluorescence probe and preparation method thereof, this method are in Hg²⁺Catalytic action under, vinyl Ether compound is deprotected into alcohol compound, and then intramolecular cyclization reaction occurs with cyano and generates with hyperfluorescence property Vinyl ethers analog derivative is tested and is reacted by selectivity the difference of front and back fluorescence physical property, obtains such fluorescence probe To Hg²⁺With extremely strong selectivity and specificity, detects and be of great significance in terms of biological and chemical.

The present invention adopts the following technical scheme that solve above-mentioned technical problem, a kind of vinyl ethers Hg²⁺Fluorescence probe, It is characterized in that the structural formula of the fluorescence probe are as follows:

Vinyl ethers Hg of the present invention²⁺The preparation method of fluorescence probe, it is characterised in that specific steps are as follows:

Step S1: using acetone as solvent, 2- hydroxyl-1-naphthalene Formaldehyde 1 and carbon is added into reaction vessel under nitrogen protection Sour potassium carries out back flow reaction after adding 1,2- Bromofume, and reaction to TLC is successively washed with water after detecting raw material fully reacting Reaction system is extracted with ethyl acetate, will be concentrated in vacuo after organic phase anhydrous sodium sulfate drying, then purify to obtain through column chromatography Compound 2；

Step S2: using dimethyl sulfoxide as solvent, compound 2 is added into reaction vessel under nitrogen protection, is dripped in 0 DEG C The dimethyl sulphoxide solution for adding potassium tert-butoxide, is stirred at room temperature reaction, and reaction to TLC successively uses water after detecting raw material fully reacting Washing and ethyl acetate extract reaction system, will be concentrated in vacuo after organic phase anhydrous sodium sulfate drying, then purify through column chromatography Obtain compound 3；

Step S3: using dehydrated alcohol as solvent, compound 3 and 2- (benzo is added into reaction vessel under nitrogen protection [d] thiazol-2-yl) acetonitrile, then reacted after triethylamine is added dropwise, reaction to TLC successively uses water after detecting raw material fully reacting Washing and ethyl acetate extract reaction system, will be concentrated in vacuo after organic phase anhydrous sodium sulfate drying, then purify through column chromatography Obtain target product vinyl ethers Hg²⁺Fluorescence probe PN1；

Reaction equation in synthesis process are as follows:

Preferably, the molar ratio of 2- hydroxyl-1-naphthalene Formaldehyde 1, potassium carbonate and Bromofume described in step S1 is 1: 1:5。

Preferably, the molar ratio of compound 2 described in step S2 and potassium tert-butoxide is 1:1.2.

Preferably, the molar ratio of compound 3 described in step S3 and 2- (benzo [d] thiazol-2-yl) acetonitrile is 1: 2。

Vinyl ethers Hg of the present invention²⁺Fluorescence probe analyzes water environment system or biological cell in selective enumeration method Hg in system²⁺Application.

Preferably, the vinyl ethers Hg²⁺Fluorescence probe is for Hg in testing sample solution²⁺The detection of concentration, it is special Sign is detailed process are as follows: prepares molar concentration by solvent of dimethyl sulfoxide as the vinyl ethers Hg of 0.01mmol/L²⁺It is glimmering Light probe titer prepares the Hg of various concentration using PBS buffer solution as solvent²⁺Stock solution, by 20 μ L vinyl ethers Hg²⁺It is glimmering The light probe standard solution Hg with 2mL various concentration respectively²⁺Stock solution mixes, and measurement mixed liquor is in wavelength after standing 1h Fluorescent emission intensity at 480nm, then with Hg²⁺Concentration is abscissa, and fluorescent emission intensity is that ordinate draws standard curve simultaneously Obtain fluorescent emission intensity and Hg²⁺Concentration standard curve equation；By 20 μ L vinyl ethers Hg²⁺Fluorescence probe standard solution with 2mL testing sample solution mixes, and measurement mixed liquor is the fluorescent emission intensity at 480nm in wavelength after standing 1h, then this is glimmering Light emitting intensity substitutes into fluorescent emission intensity and Hg²⁺Concentration standard curve equation calculation obtains Hg in testing sample solution²⁺It is dense Degree, pH=6~8 of the PBS buffer solution, fluorescence spectrometry condition are exciting slit width Ex=10.0nm, transmite slit Width Em=10.0nm, excitation wavelength 426nm, response time 1s.

Preferably, the vinyl ethers Hg²⁺Fluorescence probe is in 0.1~5 μm of ol/L Hg²⁺In concentration range, show Obvious spectral response effect, being capable of Hg in the quantitative detection concentration range²⁺Content.

Preferably, the pH=7.2 of the PBS buffer solution.

Preferably, the vinyl ethers Hg²⁺Fluorescence probe is used for intracellular Hg²⁺Detection and cell imaging, feature Be detailed process are as follows: by after human cervical carcinoma cell PBS buffer solution rinse with 1 × 10^-6The vinyl ethers Hg of mol/L²⁺ Fluorescence probe solution cultivates 30min at room temperature, then by cervical cancer cell with 5 × 10^-6The HgCl of mol/L₂PBS buffer solution 30min is cultivated at room temperature, then with the observation confocal fluorescent imaging of 1000 laser scanning confocal microscopy of Olympus FV.

Compared with the prior art, the invention has the following beneficial effects:

1, vinyl ethers Hg produced by the present invention²⁺Fluorescence probe can highly selective identification Hg²⁺, and common gold coexists Belonging to ion pair, it is not interfered with；

2, vinyl ethers Hg produced by the present invention²⁺Fluorescence probe has good water-soluble, biocompatibility and film Penetrability can be used for intracellular Hg²⁺Detection and cell imaging；

3, vinyl ethers Hg produced by the present invention²⁺Fluorescence probe is in 0.1~5 μm of ol/L Hg²⁺In concentration range, performance Obvious spectral response effect out, being capable of Hg in the quantitative detection concentration range²⁺Content；

4, vinyl ethers Hg produced by the present invention²⁺Fluorescence probe is response type probe, and specificity is strong and high sensitivity, can It is applied under physiological environment, the prospect with bioluminescence imaging；

5, the present invention prepares vinyl ethers Hg²⁺The step of fluorescence probe, is simple, and yield is higher, has preferable commercialization Application value.

Detailed description of the invention

Fig. 1 is that Hg is added in fluorescence probe PN1²⁺Before (a) (b) afterwards fluorescence spectra；

Fig. 2 is Selective recognition curve of the fluorescence probe PN1 to different metal ions；

Fig. 3 is coexisting ion to fluorescence probe PN1 Selective recognition Hg²⁺Influence diagram；

Fig. 4 is fluorescence probe PN1 to various concentration Hg²⁺The fluorescence titration curve of spectrum；

Fig. 5 is fluorescence probe PN1 at λ=480nm to Hg²⁺Working curve diagram；

Fig. 6 is fluorescence probe PN1 to Hg in Hela cell²⁺Fluorescence imaging figure, fluorescence probe concentration be 1 μM, Hg²⁺It is dense Degree is 5 μM.

Specific embodiment

Above content of the invention is described in further details by the following examples, but this should not be interpreted as to this The range for inventing above-mentioned theme is only limitted to embodiment below, and all technologies realized based on above content of the present invention belong to this hair Bright range.

Embodiment 1

The synthesis of compound 2

Under nitrogen protection by compound 1 (2- hydroxyl-1-naphthalene Formaldehyde, 500mg, 2.9mmol) and potassium carbonate (400mg, It 2.9mmol) is placed in 50mL round-bottomed flask, with 10mL acetone solution, adds 1,2- Bromofume (1.3mL, 15.1mmol), Mixed solution back flow reaction 3h detects extent of reaction with TLC thin-layer chromatography, by reaction system 100mL after raw material fully reacting Water washing and ethyl acetate (3 × 40mL) extraction will be concentrated in vacuo after organic phase anhydrous sodium sulfate drying, and product is through column chromatography Purifying obtains the faint yellow compound 2 (2- (2- bromine oxethyl) -1- naphthaldehyde) of 0.5g, yield 63%.

¹H NMR(400MHz,CDCl₃) δ 10.97 (s, 1H), 9.28 (d, J=8.8Hz, 1H), 8.07 (d, J=9.2Hz, 1H), 7.79 (d, J=8.4Hz, 1H), 7.64 (t, J=8.4Hz, 1H), 7.45 (t, J=7.6Hz, 1H), 4.56 (t, J= 6.0Hz, 2H), 3.74 (t, J=6.0Hz, 2H).

The synthesis of compound 3

System temperature is down to 0 DEG C, under nitrogen protection by potassium tert-butoxide (242mg, 2.2mmol) be dissolved in 0.5mL without In water DMSO, it is then slowly dropped to the 10mL round-bottomed flask containing compound 2 (500mg, 1.8mmol are dissolved in 3mL DMSO) In, mixture is stirred at room temperature overnight, by reaction system 100mL water washing and ethyl acetate (3 after raw material fully reacting × 40mL) extraction, it will be concentrated in vacuo after organic phase anhydrous sodium sulfate drying, product purifies to obtain 164mg yellow through column chromatography Solid chemical compound 3 (2- (vinyl) -1- naphthaldehyde), yield 46%.

¹H NMR(400MHz,CDCl₃) δ 10.83 (s, 1H), 9.29 (d, J=8.4Hz, 1H), 8.07 (d, J=9.2Hz, 1H), 7.82 (d, J=8.0Hz, 1H), 7.66 (t, J=7.6Hz, 1H), 7.50 (t, J=7.6Hz, 1H), 7.28 (d, J= 8.8Hz, 1H), 6.79 (dd, J=13.6,6.4Hz, 1H), 4.83 (dd, J=13.6,2.0Hz, 1H), 4.63 (dd, J=6.0, 2.0Hz,1H)。

The synthesis of fluorescence probe PN1

Under nitrogen protection, by compound 3 (100mg, 0.5mmol) and 2- (benzo [d] thiazol-2-yl) acetonitrile (175mg, 1mmol) is placed in 100mL round-bottomed flask, is dissolved with dehydrated alcohol, then appropriate triethylamine is added dropwise, and is used after reacting 2h The detection of TLC thin-layer chromatography, extracts reaction system 50mL water washing and ethyl acetate (3 × 20mL) after raw material fully reacting, It will be concentrated in vacuo after organic phase anhydrous sodium sulfate drying, product purifies to obtain the spy of 89mg yellow solid product fluorescence through column chromatography Needle PN1, yield 50%.

¹H NMR(400MHz,CDCl₃) δ 8.68 (s, 1H), 8.14 (d, J=8.0Hz, 1H), 7.96 (t, J=9.2Hz, 2H), 7.90 (t, J=8.0Hz, 2H), 7.63-7.55 (m, 2H), 7.53-7.45 (m, 2H), 7.37 (d, J=8.8Hz, 1H), 6.78 (dd, J=2.0,14.0Hz, 1H), 4.90 (dd, J=1.6,13.6Hz, 1H), 4.62 (dd, J=2.0,6.0Hz, 1H) .¹³C NMR(100MHz,CDCl₃)δ162.10,153.81,152.71,147.78,141.93,135.26,132.96, 132.00,130.12,128.92,128.10,127.10,126.30,125.56,124.21,123.99,121.85,117.81, 117.33,115.75,113.99,97.57。

Embodiment 2

Fluorescence spectrometry condition

Exciting slit width 10.0nm, transmite slit width 10.0nm, excitation wavelength 426nm, wave-length coverage 440~ 700nm, response time 1s, sensitivity are height, and sample cell selects the bilateral quartz colorimetric utensil of 1cm light path.

Embodiment 3

The screening of fluorometric investigation condition

PH is to fluorescence probe PN1 and PN1+Hg²⁺The influence of fluorescence spectrum

The buffer solution of different systems is prepared to determine that probe PN1 detects Hg²⁺Optimum PH range, pH be 2.0~ Probe PN1 fluorescence intensity does not have significant change in the range of 12.0.And equivalent Hg is being added²⁺It afterwards, is 6.0~8.0 models in pH When enclosing, PN1+Hg²⁺Fluorescence intensity is almost unchanged, all becomes as pH changes in the range fluorescence intensity of pH<6.0 and pH>8.0 Change, therefore, fluorescence probe PN1 is to Hg when pH is 6.0~8.0 range²⁺Detection do not influenced by pH.So experiment middle probe Ion selectivity, competitiveness, concentration titrations etc. carry out in the PBS buffer solution of pH=7.2.

Influence of the time to probe steady

In the PBS buffer solution of pH=7.2, probe PN1 and mercury ion, the fluorescence intensity that same molar is added are rapid Fluorescence probe PN1 and Hg after 1h are reacted in enhancing²⁺Balance is basically reached, fluorescence intensity no longer changes.Therefore it is all test be all The detection carried out after fluorescence probe and Ar ion mixing 1h, fluorescence light of the fluorescence probe after mercury ion is not added and mercury ion is added Spectrum is as shown in Figure 1.

Embodiment 5

Fluorescence probe acts on the Selective recognition of different metal ions

With 1 μM of fluorescence probe solution of PBS buffer preparation, the Ag of 10 equivalents is then separately added into solution⁺、Al³⁺、 Ba²⁺、Cr³⁺、Ca²⁺、Cu²⁺、Fe²⁺、Fe³⁺、Li⁺、Ni²⁺、Na⁺、Zn²⁺、Pb²⁺、K⁺、Mg²⁺、Mn²⁺Plasma, and make blank solution Control.As shown in Figure 2 the result shows that, without obvious fluorescent emission at maximum absorption wavelength 480nm.In blank probe solution After the mercury ion of 5 equivalents is added, system fluorescence at 480nm enhances rapidly.Experimental data illustrates fluorescence probe PN1 to Hg²⁺Tool There is higher selectivity recognition capability.

Influence of the coexisting ion to fluorescence probe detection mercury ion

Add the Hg of 5 equivalents again in the above-mentioned solution for there are other metal ions²⁺, as shown in figure 3, in the wavelength of 480nm Similarly launch strong fluorescence in place.This illustrates that fluorescence probe PN1 has stronger anti-interference ability to other metal ions, and Identification Hg that can be highly selective²⁺。

Embodiment 6

The measurement of fluorescence titration experiment and detection limit

Fixed fluorescence probe concentration is 1 μM, and the fluorescence spectrum variation after the mercury ion of various concentration, such as Fig. 4 is added in measurement It is shown, record the fluorescence emission modal data within the scope of 440~600nm.By to fluorescence probe concentration in system (1 μM) and wave The control of long (λ=480nm), has obtained different Hg²⁺Fluorescent emission intensity and Hg under concentration²⁺The working curve of concentration is such as schemed Shown in 5, linear formula Y=4.4855 × 10 can be obtained to its linear analysis⁷X+34.6938, by formula LOD=3 σ/K, (σ is standard Variance, n=20), obtain detection limit LOD=1.37 × 10^-7mol/L(0.137μmol/L)。

Embodiment 7

Fluorescence probe and Hg²⁺Reaction mechanism is inquired into

By fluorescence probe PN1 (0.1mmol) and HgCl under room temperature₂(0.5mmol) stirs 1h in methanol solution, stops anti- It answers, obtains fluorescence probe PN1 and compound 5 after being spin-dried for solvent, high resolution mass spectrum data is as follows: (HRMS (ESI): calcd.for[M+Na]⁺C₂₀H₁₂N₂OSNa351.0563, found 351.0558).By consulting literatures (RSC Adv., 2015,5:16723) and to the careful analysis of probe structure and product, be shown below, thus it is speculated that go out fluorescence probe PN1 mercury from Hydrolytic cleavage under the catalytic action of son, and then react with cyano and generate compound 5, to generate hyperfluorescence effect.

Embodiment 8

Fluorescence probe PN1 detects Hg in living cells²⁺

Hg is detected in the cell in order to probe into the novel fluorescence probe²⁺Ability, human cervical carcinoma cell phosphate is delayed It rushes after solution (PBS) rinse with 1 × 10^-6The fluorescence probe PN1 solution of mol/L cultivates 30min at room temperature, then cervical carcinoma is thin Born of the same parents are with 5 × 10^-6The HgCl of mol/L₂PBS buffer solution cultivate 30min at room temperature, then use Olympus FV1000 laser The imaging of confocal microscopy observation confocal fluorescent, as shown in Figure 6.

Embodiment above describes basic principles and main features of the invention and advantage, the technical staff of the industry should Understand, the present invention is not limited to the above embodiments, and the above embodiments and description only describe originals of the invention Reason, under the range for not departing from the principle of the invention, various changes and improvements may be made to the invention, these changes and improvements are each fallen within In the scope of protection of the invention.

Claims

1. a kind of vinyl ethers Hg²⁺Fluorescence probe, it is characterised in that the structural formula of the fluorescence probe are as follows:

2. a kind of vinyl ethers Hg described in claim 1²⁺The preparation method of fluorescence probe, it is characterised in that specific steps Are as follows:

Step S1: using acetone as solvent, being added 2- hydroxyl-1-naphthalene Formaldehyde 1 and potassium carbonate into reaction vessel under nitrogen protection, Back flow reaction is carried out after adding 1,2- Bromofume, reaction to TLC is successively washed with water after detecting raw material fully reacting and second Acetoacetic ester extracts reaction system, will be concentrated in vacuo after organic phase anhydrous sodium sulfate drying, then purify to obtain chemical combination through column chromatography Object 2；

Step S2: using dimethyl sulfoxide as solvent, compound 2 is added into reaction vessel under nitrogen protection, in 0 DEG C of dropwise addition uncle The dimethyl sulphoxide solution of butanol potassium, is stirred at room temperature reaction, and reaction to TLC is successively washed with water after detecting raw material fully reacting Reaction system is extracted with ethyl acetate, will be concentrated in vacuo after organic phase anhydrous sodium sulfate drying, then purify to obtain through column chromatography Compound 3；

Step S3: using dehydrated alcohol as solvent, compound 3 and 2- (benzo [d] thiophene is added into reaction vessel under nitrogen protection Azoles -2- base) acetonitrile, then reacted after triethylamine is added dropwise, reaction to TLC be successively washed with water after detecting raw material fully reacting and Ethyl acetate extracts reaction system, will be concentrated in vacuo after organic phase anhydrous sodium sulfate drying, then purify to obtain mesh through column chromatography Mark product ethylene base ethers Hg²⁺Fluorescence probe PN1；

Reaction equation in synthesis process are as follows:

3. vinyl ethers Hg according to claim 2²⁺The preparation method of fluorescence probe, it is characterised in that: in step S1 The molar ratio of the 2- hydroxyl-1-naphthalene Formaldehyde 1, potassium carbonate and Bromofume is 1:1:5.

4. vinyl ethers Hg according to claim 2²⁺The preparation method of fluorescence probe, it is characterised in that: in step S2 The molar ratio of the compound 2 and potassium tert-butoxide is 1:1.2.

5. vinyl ethers Hg according to claim 2²⁺The preparation method of fluorescence probe, it is characterised in that: in step S3 The molar ratio of the compound 3 and 2- (benzo [d] thiazol-2-yl) acetonitrile is 1:2.

6. a kind of vinyl ethers Hg described in claim 1²⁺Fluorescence probe analyzes water environment system or life in selective enumeration method Hg in object cell system²⁺Application.

7. application according to claim 6, it is characterised in that: the vinyl ethers Hg²⁺Fluorescence probe is used for test sample Product Hg solution²⁺The detection of concentration, detailed process are as follows: it is 0.01mmol/L that molar concentration is prepared by solvent of dimethyl sulfoxide Vinyl ethers Hg²⁺Fluorescence probe titer prepares the Hg of various concentration using PBS buffer solution as solvent²⁺Stock solution, by 20 μ L vinyl ethers Hg²⁺The fluorescence probe standard solution Hg with 2mL various concentration respectively²⁺Stock solution mixing, measures after standing 1h Mixed liquor is the fluorescent emission intensity at 480nm in wavelength, then with Hg²⁺Concentration is abscissa, and fluorescent emission intensity is ordinate It draws standard curve and obtains fluorescent emission intensity and Hg²⁺Concentration standard curve equation；By 20 μ L vinyl ethers Hg²⁺Fluorescence Probe standard solution is mixed with 2mL testing sample solution, and measurement mixed liquor is the fluorescent emission at 480nm in wavelength after standing 1h Intensity, then the fluorescent emission intensity is substituted into fluorescent emission intensity and Hg²⁺Concentration standard curve equation calculation obtains sample to be tested Hg solution²⁺Concentration, pH=6~8 of the PBS buffer solution, fluorescence spectrometry condition are exciting slit width Ex= 10.0nm, transmite slit width Em=10.0nm, excitation wavelength 426nm, response time 1s.

8. application according to claim 7, it is characterised in that: the vinyl ethers Hg²⁺Fluorescence probe is in 0.1~5 μ mol/L Hg²⁺In concentration range, obvious spectral response effect is shown, it being capable of Hg in the quantitative detection concentration range²⁺Contain Amount.

9. application according to claim 7, it is characterised in that: the pH=7.2 of the PBS buffer solution.

10. application according to claim 6, it is characterised in that: the vinyl ethers Hg²⁺Fluorescence probe is for intracellular Hg²⁺Detection and cell imaging, detailed process are as follows: by after human cervical carcinoma cell PBS buffer solution rinse with 1 × 10^- ⁶The vinyl ethers Hg of mol/L²⁺Fluorescence probe solution cultivates 30min at room temperature, then by cervical cancer cell with 5 × 10^-6mol/ The HgCl of L₂PBS buffer solution cultivate 30min at room temperature, then with 1000 laser scanning confocal microscopy of Olympus FV see Examine confocal fluorescent imaging.