A kind of carboxy-lesterase fluorescence probe and its preparation method and application
Technical field
The present invention relates to biochemical material technical field more particularly to a kind of carboxy-lesterase fluorescence probe and its preparation sides
Method and application.
Background technique
Mammal carboxy-lesterase (Carboxylesterase, CaE, E.C.3.1.1.1) be also ali-esterase, divides extensively
Be distributed in mammalian tissues and organ, activated centre contain serine residue, can effectively be catalyzed containing ester bond, amido bond and
The endogenous and exogenous material of thioester bond hydrolyze.The major function of mammal carboxy-lesterase may be participate in lipid transport and
Metabolism, is catalyzed some endogenous compounds, such as short chain and long acyl glycerine, long-chain acylcarnitines and long acyl CoA
Ester hydrolysis;And it participates in signal transmembrane conductor and keeps biological film integrality;It is catalyzed drug (including pro-drug) and generates phase
The free acid answered participates in a variety of drugs, environmental poisonous substance and carcinogenic removing toxic substances and metabolism.
Carboxy-lesterase mainly has hCE1 and hCE2 two types in human body, and hCE1 is primarily present in liver, and hCE2 is main
It is present in enteron aisle.HCE1 major catalytic hydrolyzes that a major class is exogenous or endogenic substrate, such as fatty acid, environmental poisonous substance and medicine
Object etc. participates in many important physiology courses, as lipid homeostasis, testosterone generate, retinol is metabolized, protein in endoplasmic reticulum
Tracking and reservation etc..Lacking hCE1 in human body will lead to a variety of diseases, as atherosclerosis, hepatic steatosis, obesity,
Hyperlipidemia and liver cancer etc..In addition recent studies have found that, carboxy-lesterase can be used as one of blood serum designated object of liver cancer.Based on carboxylic
The important physiological action of acid esters enzyme detects carboxy-lesterase and its activity to the research of many physiology courses and the therapeutic evaluation of drug
With very important scientific meaning and practical application value.
The method of traditional detection carboxy-lesterase has high performance liquid chromatography, gas chromatography mass spectrometry, chemoluminescence method and fluorescence method etc.,
Wherein, fluorescence method due to have many advantages, such as easy to operate, high sensitivity, detection limit is low, can be used for intracellular or living imaging by
The extensive concern of scientists is arrived.It is most of to exist to pH at present in the fluorescence probe of detection carboxy-lesterase reported in the literature
It is more sensitive, stability is poor, cell-penetrating ability is poor in cell culture fluid, can not be applied to living cells or tissue in be imaged etc. lack
Point.It is shorter in addition, there will be the carboxy-lesterase fluorescence probe that can be used for cell imaging required excitation wavelength in cell imaging
(< 500nm), the exciting light of short wavelength is easy to cause cell photo-damage, generate reactive oxygen species, and tissue penetration depths and at
Picture depth is shallower, limits its application in living cells and living imaging.
Summary of the invention
In view of this, the purpose of the present invention is to provide carboxy-lesterase fluorescence probe and its preparation method and application, this hair
The carboxy-lesterase fluorescence probe of bright offer can detect carboxy-lesterase under the conditions of two-photon excitation in living cells.
In order to achieve the above-mentioned object of the invention, the present invention the following technical schemes are provided:
The present invention provides a kind of carboxy-lesterase fluorescence probes, have structure shown in Formulas I:
In Formulas I, R1、R2、R3And R4It independently is hydrogen, amino, alkyl, alkoxy, alkylamino, aryl, aryloxy group or fragrant ammonia
Base.
Preferably, carbon atom number independently is 1~6 in the alkyl, alkoxy and alkylamino.
Preferably, aryl number independently is 1~2 in the aryl, aryloxy group and fragrant amino.
Preferably, the carboxy-lesterase fluorescence probe includes
The present invention provides the preparation methods of carboxy-lesterase fluorescence probe described in above-mentioned technical proposal, comprising the following steps:
(1) compound, chloroacetic chloride, alkaline reagent and organic solvent with structure shown in Formula II are mixed and carries out acetylation
Reaction, obtains the compound with structure shown in formula III;
(2) by the compound with structure shown in formula III and 1,2,3,3- tetramethyl -3H- indoles iodide, organic
Solvent mixing, condensation reaction is carried out under protective atmosphere, obtains the carboxy-lesterase fluorescence probe with structure shown in Formulas I;
In Formula II and formula III, R1、R2、R3And R4It independently is hydrogen, amino, alkyl, alkoxy, alkylamino, aryl, fragrant oxygen
Base or fragrant amino.
Preferably, the compound with structure shown in Formula II in the step (1), chloroacetic chloride, alkaline reagent and organic molten
The mixing of agent is the mixed solution that chloroacetic chloride is added to compound with structure shown in Formula II, alkaline reagent and organic solvent
In.
Preferably, the adding manner of the chloroacetic chloride is to be added dropwise;The dropwise addition carries out under conditions of -2~2 DEG C.
Preferably, the temperature of condensation reaction is 75~85 DEG C in the step (2);The time of condensation reaction is 14~18h.
The present invention provides application of the carboxy-lesterase fluorescence probe described in above-mentioned technical proposal in detection carboxy-lesterase.
Preferably, the carboxy-lesterase is the carboxy-lesterase in human cervical carcinoma cell.
The present invention provides a kind of carboxy-lesterase fluorescence probe with structure shown in Formulas I, carboxylates provided by the invention
Enzyme fluorescence probe is half cyanine compound with naphthalene core two-photon absorption unit and D- π-A structure, has two-photon absorption
And emission characteristics, it can be applied to detect intracellular carboxy-lesterase.Embodiment the experimental results showed that, carboxylic acid provided by the invention
Esterase fluorescence probe is able to extend the excitation wavelength (800nm) that carboxy-lesterase is detected in cell and tissue, reduces to cell and group
The light injury knitted increases tissue penetration depths and imaging depth;Meanwhile carboxy-lesterase fluorescence probe provided by the invention detects carboxylic
Stability is strong when acid esters enzyme (hatching 60min fluorescence intensity in the PBS solution at 37 DEG C to remain unchanged), the response time it is short (for
7min), insensitive to pH, there is specificity to the imaging of intracellular carboxy-lesterase, it can be under the conditions of two-photon excitation living thin
Carboxy-lesterase is detected in born of the same parents.
Detailed description of the invention
The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.
Fig. 1 is HCyNAc hatches fluorescence intensity after different time in PBS buffer solution variation diagram at 37 DEG C;
Fig. 2 is the variation diagram of HCyNAc fluorescence intensity in the PBS buffer solution of different pH value;
Fig. 3 is that HCyNAc is hatched not at 37 DEG C in the PBS buffer solution under the carboxy-lesterase existence condition of various concentration
With the variation diagram of fluorescence intensity after the time;
Fig. 4 is the variation of fluorescence emission spectrum after the hatching of various concentration carboxy-lesterase is added in 10 μM of HCyNAc solution
Figure;
The relationship and linearity curve of HCyNAc solution fluorescence intensity and carboxy-lesterase concentration at 567nm that Fig. 5 is 10 μM
Figure;
Fig. 6 be separately added into 10 μM of HCyNAc solution it is different competition molecules after figure of fluorescence intensity changes and 10
μM HCyNAc solution in be separately added into it is different competition molecules after add 0.05U/mL carboxy-lesterase fluorescence intensity change
Figure;
Fig. 7 is for HCyNAc to the fluorescence response of carboxy-lesterase under bis- (4- nitrobenzophenone) the phosphate existence conditions of inhibitor
Situation map;
Fig. 8 is launching light spectrogram of hydroxy compounds HCyN under the conditions of different capacity 820nm two-photon excitation;
Fig. 9 is the logarithmic relationship figure of HCyN fluorescence output spectrum integral area and input power;
Figure 10 is burnt micro- to hatch the single photon after 15min or two-photon laser copolymerization at 37 DEG C of HCyNAc and HeLa cell
Images.
Specific embodiment
The present invention provides a kind of carboxy-lesterase fluorescence probes, have structure shown in Formulas I:
In Formulas I, R1、R2、R3And R4It independently is hydrogen, amino, alkyl, alkoxy, alkylamino, aryl, aryloxy group or fragrant ammonia
Base.
In the present invention, carbon atom number is preferably independently 1~6 in the alkyl, alkoxy and alkylamino.
In the present invention, aryl number is preferably independently 1~2 in the aryl, aryloxy group and fragrant amino.
In the present invention, the carboxy-lesterase fluorescence probe preferably includes
The present invention provides the preparation methods of carboxy-lesterase fluorescence probe described in above-mentioned technical proposal, comprising the following steps:
(1) compound, chloroacetic chloride, alkaline reagent and organic solvent with structure shown in Formula II are mixed and carries out acetylation
Reaction, obtains the compound with structure shown in formula III;
(2) by the compound with structure shown in formula III and 1,2,3,3- tetramethyl -3H- indoles iodide, organic
Solvent mixing, condensation reaction is carried out under protective atmosphere, obtains the carboxy-lesterase fluorescence probe with structure shown in Formulas I;
In Formula II and formula III, R1、R2、R3And R4It independently is hydrogen, amino, alkyl, alkoxy, alkylamino, aryl, fragrant oxygen
Base or fragrant amino.
Compound, chloroacetic chloride, alkaline reagent and organic solvent with structure shown in Formula II are mixed and carry out second by the present invention
Acylation reaction obtains the compound with structure shown in formula III.In the present invention, the chemical combination with structure shown in Formula II
The molar ratio of object, chloroacetic chloride and alkaline reagent is preferably 1:(1.0~1.2): (1~1.4), more preferably 1:1:1.2.The present invention
There is no special restriction for the alkaline reagent, is suitable for carrying out acetylization reaction using well known to those skilled in the art
Alkaline reagent, it is specific such as triethylamine or pyridine.The present invention does not have the organic solvent special restriction, using ability
It is suitable for carrying out the organic solvent of acetylization reaction known to field technique personnel;Specific such as methylene chloride or tetrahydrofuran.
In the present invention, the compound with structure shown in Formula II, chloroacetic chloride, alkaline reagent and organic solvent is mixed
Close the mixed solution of the compound, alkaline reagent and organic solvent that are preferably chloroacetic chloride is added to structure shown in Formula II
In.In the present invention, the adding manner of the chloroacetic chloride is preferably added dropwise;It is described dropwise addition preferably under conditions of -2~2 DEG C into
Row, more preferably 0 DEG C.The present invention does not have the rate of the dropwise addition special restriction, using known to those skilled in the art
Dropwise addition material rate;In an embodiment of the present invention, it is specifically added dropwise with the rate of 0.6mmol/min.
In the present invention, the temperature of the acetylization reaction is preferably 20~40 DEG C, and more preferably 25~35 DEG C;In this hair
In bright embodiment, the acetylization reaction is specifically carried out at room temperature, that is, does not need additional heating or cooling.The present invention
There is no special restriction for the time of the acetylization reaction, there is the compound of structure shown in Formula II by TCL board monitoring
It completely disappears.In the present invention, the acetylization reaction preferably carries out under agitation, and the present invention is for the stirring
Rate there is no special restriction, using stirring rate well known to those skilled in the art.
After the completion of the acetylization reaction, obtained acetylation material is preferably successively washed, is dried, dense by the present invention
Contracting and recrystallization, obtain the compound with structure shown in formula III.In the present invention, washing reagent used by the washing
Preferably saturated salt solution;The number of the washing is preferably 2~5 times, and more preferably 3~4 times.The present invention is preferably washed described
Gained organic phase is dried after washing;The present invention does not have desiccant used by the drying special restriction, using this
Desiccant known to the technology of field, it is specific such as anhydrous sodium sulfate.The present invention is preferably by gained organic materials after the drying
It is concentrated;The present invention restriction not special for the concentration, using the technology of concentration well known to those skilled in the art
Scheme;In an embodiment of the present invention, gained dried material after drying is concentrated into solid particular by rotary evaporation.
The present invention preferably recrystallizes gained concentration material after the concentration;In the present invention, weight used by the recrystallization
Crystalline reagents are preferably the mixture of ethyl acetate and petroleum ether;The volume of ethyl acetate and petroleum ether in the recrystallization reagent
Than being preferably 1:(35~45), more preferably 1:40.
After obtaining the compound with structure shown in formula III, the present invention is by the compound with structure shown in formula III
With 1,2,3,3- tetramethyl -3H- indoles iodide, organic solvent mixing carry out condensation reaction under protective atmosphere, are had
The carboxy-lesterase fluorescence probe (HCyNAc) of structure shown in Formulas I.In the present invention, the chemical combination with structure shown in formula III
The molar ratio of object and 1,2,3,3- tetramethyl -3H- indoles iodide is preferably 1:(1~1.2).In the present invention, described organic
Solvent is preferably acetonitrile, toluene, acetic acid or ethyl alcohol, more preferably ethyl alcohol.
In the present invention, the temperature of the condensation reaction is preferably 75~85 DEG C, and more preferably 80 DEG C;Condensation reaction when
Between preferably 14~18h, more preferably 16h.The present invention is not special for the protective gas type for providing the protective atmosphere
Restriction, it is specific such as nitrogen using protective gas well known to those skilled in the art.
After the completion of the condensation reaction, the present invention preferably mixes obtained condensation material with ether, and red solid is precipitated,
Obtain solid-liquid mixture;The solid-liquid mixture is separated by solid-liquid separation, gained red solid material is successively washed
It washs and dries, obtain the carboxy-lesterase fluorescence probe with structure shown in Formulas I.In the present invention, the condensation material and ether
Volume ratio be preferably 1:(5~7), more preferably 1:(5.5~6.5).The present invention does not have the mode of the separation of solid and liquid
Special restriction, using solid-liquid separation method well known to those skilled in the art;In an embodiment of the present invention, it specifically adopts
The separation of solid and liquid is realized with filtering.In the present invention, washing reagent used by the washing is preferably ether;The washing
Number be preferably 2~5 times, more preferably 3~4 times.In the present invention, the drying is preferably dried in vacuo, the vacuum
Dry temperature is preferably 30~40 DEG C, and more preferably 35 DEG C;The vacuum drying time is 5~7h, more preferably 6h.
The present invention provides application of the carboxy-lesterase fluorescence probe described in above-mentioned technical proposal in detection carboxy-lesterase.?
In the present invention, the carboxy-lesterase is preferably the carboxy-lesterase in human cervical carcinoma cell.In the present invention, the carboxy-lesterase is glimmering
Light probe is half cyanine compound with naphthalene core two-photon absorption unit and D- π-A structure, has two-photon absorption and hair
Penetrate characteristic:
In the present invention, with(HCyNAc) for, the carboxy-lesterase fluorescence
The testing mechanism of probe is as follows:
There is the salt of a positive charge in HCyNAc, impart the certain water solubility of HCyNAc, therefore, HCyNAc can be used
Detection in water sample or biological sample, it is simple and practical.
In an embodiment of the present invention, by taking HCyNAc as an example, add specifically in 2mLPBS buffer solution (pH value 7.4)
Dimethyl sulfoxide (DMSO) solution (1mM) for entering 20 μ LHCyNAc, obtains 10 μM of HCyNAc solution, is then respectively adding difference
The PBS solution of the carboxy-lesterase of concentration, after hatching 7min at 37 DEG C, acquired solution after various concentration carboxy-lesterase is added in measurement
Fluorescence emission spectrum (Ex=443nm), it is abscissa by the concentration of ordinate, carboxy-lesterase of the fluorescence intensity at 567nm
The linearity curve that HCyNAc detects carboxy-lesterase is established, as shown in Figure 5.The linearity curve is specially y=2.01+1.98x,
Fluorescence intensity at 567nm for the concentration of carboxy-lesterase linear response in 0.02~0.07U/mL (R2=99.9%) it
Between.
Below in conjunction with the embodiment in the present invention, the technical solution in the present invention is clearly and completely described.It is aobvious
So, described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.Based on the reality in the present invention
Example is applied, every other embodiment obtained by those of ordinary skill in the art without making creative efforts all belongs to
In the scope of protection of the invention.
Embodiment 1
Carboxy-lesterase fluorescence probe is prepared by following reaction process:
(1) by compound 1 (172.2mg, 1mmol), triethylamine (121.4mg, 1.2mmol) and methylene chloride (anhydrous grade)
Mixing, chloroacetic chloride (94.2mg, 1.2mmol) is instilled in 2min at 0 DEG C obtained by mixed solution, progress is stirred at room temperature
Acetylization reaction, with TCL board monitoring reaction process until compound 1 completely disappears;By gained acetylation material saturated common salt
Water washing 3 times, gained organic phase is dry with anhydrous sodium sulfate, and rotary evaporation is concentrated into after solid with ethyl acetate and petroleum ether
Mixture (volume ratio of ethyl acetate and petroleum ether is 1:40) is recrystallized, and white solid is obtained.
Being computed yield is 92%;
Gained white solid is characterized, specific data are as follows:
1HNMR (400MHz, DMSO) δ 10.15 (s, 1H), 8.61 (s, 1H), 8.22 (d, J=8.9Hz, 1H), 8.06 (d,
J=8.5Hz, 1H), 7.92 (dd, J=8.5,1.1Hz, 1H), 7.81 (d, J=1.9Hz, 1H), 7.47 (dd, J=8.8,
2.2Hz,1H),2.35(s,3H)。
According to above-mentioned characterize data it is found that gained white solid is structure shown in compound 2.
(2) by compound 2 (214.2mg, 1.0mmol) and 1,2,3,3- tetramethyl -3H- indoles iodide (301.2mg,
It 1.0mmol) is mixed with ethyl alcohol, 80 DEG C of reflux carry out condensation reaction 16h under nitrogen protection;After gained condensation material cooling,
Ether (30mL) is added to be recrystallized, red solid is obtained.
Being computed yield is 94%;
Gained red solid is characterized, specific data are as follows:
1HNMR (400MHz, DMSO) δ 8.77 (s, 1H), 8.59 (d, J=16.4Hz, 1H), 8.39 (d, J=8.7Hz,
1H), 8.10 (d, J=8.8Hz, 2H), 7.94-7.87 (m, 2H), 7.85-7.78 (m, 2H), 7.68-7.61 (m, 2H), 7.46
(dd, J=8.8,2.2Hz, 1H), 4.20 (s, 3H), 2.36 (s, 3H), 1.84 (s, 6H)13C NMR(101MHz,MeOD)δ
181.93,168.94,153.18,150.51,142.91,141.20,135.66,133.09,131.24,130.51,130.14,
129.03,128.49,128.04,123.61,121.96,121.92,118.06,114.12,111.86,52.00,33.29,
24.28,19.01.HRMS(MALDI-TOF)m/z:[M]+calcd for C25H24NO2 +,370.1802,found:
370.1817.
According to above-mentioned characterize data it is found that gained white solid is HCyNAc.
Embodiment 2
The carboxy-lesterase probe prepared to embodiment 1 is tested for the property, specific as follows:
The Stability Determination of carboxy-lesterase probe: 20 μ L HCyNAc are added in 2mLPBS buffer solution (pH value 7.4)
DMSO solution (1mM), obtain 10 μM of HCyNAc solution, acquired solution hatch at 37 DEG C the different time (10,15,20,
25,30,40,50,60min), the fluorescence intensity of the solution is measured with the situation of change of brooding time.Fig. 1 is that HCyNAc exists
The variation diagram for hatching the fluorescence intensity after different time in PBS buffer solution at 37 DEG C, from fig. 1, it can be seen that fluorescence intensity is protected substantially
It holds constant, illustrates that the stability of carboxy-lesterase probe is preferable.
Carboxy-lesterase is visited to the sensitivity testing of pH: 2mL PBS buffer solution (pH value is respectively 5.0,5.5,6.0,
6.5,7.0,7.5,8.0,8.5,9.0) in the DMSO solution (1mM) of 20 μ LHCyNAc is added, obtain 10 μM of HCyNAc solution,
Then the carboxy-lesterase that 0.05U/mL is added measures the mixed solution after gained mixed solution hatches 7min at 37 DEG C
Fluorescence intensity with PBS pH value of buffer solution situation of change.Fig. 2 is HCyNAc fluorescence in the PBS buffer solution of different pH value
The variation diagram of intensity, as shown in Figure 2, carboxy-lesterase is insensitive to pH.
The response time of carboxy-lesterase probe measures: 20 μ L being added in 2mL PBS buffer solution (pH value 7.4)
The DMSO solution (1mM) of HCyNAc, obtains 10 μM of HCyNAc solution, and the carboxy-lesterase of 0.05U/mL is then added, and gained is mixed
It closes solution to hatch at 37 DEG C the different time (0.5,1,1.5,2,3,4,5,6,7,9,12,15,18,21min), described in measurement
The fluorescence intensity of mixed solution with brooding time situation of change.Fig. 3 is that there are items in the carboxy-lesterase of various concentration by HCyNAc
The variation diagram for hatching fluorescence intensity after different time in PBS buffer solution under part at 37 DEG C, from the figure 3, it may be seen that after 7min, fluorescence
Intensity basically reaches saturation, this illustrates that the carboxy-lesterase probe response time is shorter, only 7min.
The fluorescence titration of carboxy-lesterase probe is tested: 20 μ being added in 2mLPBS buffer solution (pH value 7.4)
The DMSO solution (1mM) of LHCyNAc, obtains 10 μM of HCyNAc solution, is then respectively adding the carboxy-lesterase of various concentration
PBS solution, after hatching 7min at 37 DEG C, the fluorescence emission spectrum of acquired solution after various concentration carboxy-lesterase is added in measurement
(Ex=443nm), it is that abscissa establishes HCyNAc to carboxylic using the fluorescence intensity at 567nm as the concentration of ordinate, carboxy-lesterase
The linearity curve of acid esters enzyme detection.Fig. 4 is that fluorescence after the hatching of various concentration carboxy-lesterase is added in 10 μM of HCyNAc solution
The variation diagram of emission spectrum;Fig. 5 be 10 μM of HCyNAc solution 567nm at the relationship of fluorescence intensity and carboxy-lesterase concentration and
Linear diagram, the linearity curve are specially y=2.01+1.98x, linear sound of the fluorescence intensity for the concentration of carboxy-lesterase
It should be in 0.02~0.07U/mL (R2=99.9%) between.
The selectivity test of carboxy-lesterase probe: 20 μ L are added in 2mL PBS buffer solution (pH value 7.4)
The DMSO solution (1mM) of HCyNAc, obtains 10 μM of HCyNAc solution, is separately added into 100 μM NaCl, MgCl2, hydrogen peroxide,
Tertbutanol peroxide (TBHP), bovine serum albumin(BSA) (BSA), lysozyme perhaps 50 μM of arginine, serine, Vc or
The glutathione reductase of 12.8U/mL, gained mixed solution hatch 7min at 37 DEG C respectively, then measure mixed solution
Fluorescence emission spectrum (Ex=443nm).Fig. 6 is the fluorescence being separately added into after different competition molecules in 10 μM of HCyNAc solution
Strength Changes figure (black bar);0.05U/mL is added after being separately added into different competition molecules in 10 μM of HCyNAc solution
Carboxy-lesterase figure of fluorescence intensity changes (white columns);Wherein, 1- carboxy-lesterase, 2-NaCl, 3-MgCl2, 4- hydrogen peroxide,
5-TBHP, 6- glucose, 7-Vc, 8- serine, 9- arginine, 10-BSA, 11- lysozyme, 12- glutathione reductase.By
Fig. 6 is not it is found that the fluorescence intensity that other biomolecule in addition to carboxy-lesterase react front and back with HCyNAc is remarkably reinforced together
When under the existence condition of above-mentioned disturbing molecule, add carboxy-lesterase hatching 7min later respectively measurement HCyNAc reaction front and back
Fluorescence intensity, discovery fluorescence is remarkably reinforced, and illustrates that the HCyNAc property of can choose identifies carboxy-lesterase, and not by other
The interference of the common molecule of biology.Fig. 7 is that HCyNAc is right under inhibitor bis- (4- nitrobenzophenones) phosphate (BNPP) existence condition
The fluorescence response situation map of carboxy-lesterase, wherein 1-HCyNAc, 2-HCyNAc+ carboxy-lesterase, 3-HCyNAc+ carboxy-lesterase+
0.5mM BNPP.As shown in Figure 7, glimmering after HCyNAc and carboxy-lesterase hatching 7min when being added without carboxylesterase inhibitor BNPP
Luminous intensity is remarkably reinforced;But after carboxylesterase inhibitor BNPP is added, HCyNAc and carboxy-lesterase hatching 7min mistake are added
Afterwards, fluorescence intensity no longer enhances, and illustrates that the fluorescence enhancement of HCyNAc is strictly because the specificly-response with carboxy-lesterase causes
's.
Embodiment 3
The two-phpton property of carboxy-lesterase probe prepared by embodiment 1 is measured, HCyNAc is generated after enzyme hydrolysis
HCyN, measurement HCyN whether there is two-photon absorption and emission characteristic be the carboxy-lesterase probe can be used in two-photon cell at
The basis of picture, specific as follows:
(1) it selects fluorescein as reference substance, is dissolved in being configured to 0.05 μ in sodium hydrate aqueous solution (pH=11)
The standard solution of mol/L;HCyN is configured to N,N-dimethylformamide (DMF) solution of 50 μm of ol/L;
(2) two kinds of solution of gained in 3mL step (1) are taken respectively, it is solid on SP-5W titanium-doped sapphire femtosecond pulse laser
Determining exciting power is 200mW, changes excitation wavelength from 740nm to 880nm, measures the two photon emission light of two kinds of solution respectively
Spectrum;
(3) fixed excitation wavelength is 820nm, changes exciting power from 50mW to 290mW, respectively institute in determination step (1)
Obtain the emission spectrum of two kinds of solution;
(4) two-photon cross-sections are calculated according to following formula:
δ is the two photon absorption cross section of HCyN, δ in formularefFor the two photon absorption cross section of fluorescein, Ф is HCyN's
Fluorescence quantum yield, ФrefFor the fluorescence quantum yield of fluorescein, c is the concentration of HCyN, crefFor the concentration of fluorescein, n is
The index of refraction of HCyN solution, nrefFor the index of refraction of luciferin solution, F is the emission peak integral area of HCyN, FrefFor fluorescein
Emission peak integral area.
Fig. 8 is launching light spectrogram of hydroxy compounds HCyN under the conditions of different capacity 820nm two-photon excitation, and Fig. 9 is
The logarithmic relationship figure of HCyN fluorescence output spectrum integral area and input power;By Fig. 8 and Fig. 9 it is found that HCyN has two-photon
Absorption and emissivities.
Embodiment 4
The interior carboxy-lesterase fluorescence imaging situation of human cervical carcinoma cell (HeLa) is tested, specific as follows:
HeLa cell is inoculated in 1640 culture medium of RPMI containing 10% fetal calf serum by recovery, in 37 DEG C, 5%
CO2, 100% saturated humidity incubator in cultivate, then cultivated on 18mm coverslip for 24 hours, for use.
HeLa cell after culture is immersed in the culture medium containing 5 μM of HCyNAc, in 37 DEG C, 5%CO2, 100% saturation
After cultivating 15min in the incubator of humidity, culture medium is poured out, is cleaned cell 3 times with fresh culture;Respectively in laser co-focusing
Fluorescence microscopy under the microscope, and uses 405nm as one-photon excitation light source and 800nm as two-photon excitation light source respectively, right
It take pictures under light field and dark field.Figure 10 is to hatch single photon or double light after 15min at HCyNAc and 37 DEG C of HeLa cell
Sub- Laser scanning confocal microscopy photo, wherein (A) is that HCyNAc and HeLa cell hatch the light field after 15min at 37 DEG C
Photo, (B) are that HCyNAc and HeLa cell hatchs the single photon fluorescence photo after 15min at 37 DEG C, (C) be HCyNAc and
HeLa cell hatches the two-photon fluorescence photo after 15min at 37 DEG C, and (D) is the stacking chart of (A), (B) and (C), and (E) is suppression
Preparation BNPP and HeLa cell hatchs after 30min that the photograph via bright field with HCyNAc hatching 15min, (F) are inhibition again at 37 DEG C
Agent BNPP and HeLa cell hatchs the single photon fluorescence images for hatching 15min after 30min with HCyNAc again at 37 DEG C,
(G) hatch the two photon imaging photograph for hatching 15min after 30min with HCyNAc again at 37 DEG C for inhibitor BNPP and HeLa cell
Piece, (H) are the stacking chart of (E), (F) and (G).As shown in Figure 10, strong fluorescence signal is only presented in HCyNAc in HeLa cell,
And under similarity condition, fluorescence is weaker in the HeLa cell for hatching 30min in advance with inhibitor BNPP, illustrates HCyNAc to cell
Interior carboxy-lesterase imaging has specificity.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.