CN100346154C - Method for producing internal reference nano PH sensor and using for in-cell PH non-trauma monitoring - Google Patents
Method for producing internal reference nano PH sensor and using for in-cell PH non-trauma monitoring Download PDFInfo
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- CN100346154C CN100346154C CNB2004100467941A CN200410046794A CN100346154C CN 100346154 C CN100346154 C CN 100346154C CN B2004100467941 A CNB2004100467941 A CN B2004100467941A CN 200410046794 A CN200410046794 A CN 200410046794A CN 100346154 C CN100346154 C CN 100346154C
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Abstract
The present invention discloses a method for preparing a fluorescence nanometer pH sensor with a silicon shell and internal reference, and non-traumatic monitoring used for the pH of living cells, which aims at synthesizing a nanometer pH sensor with stable performance for simultaneously embedding reference dye and sensitive dye so as to realize the non-traumatic monitoring of the dynamic variation of the pH of the cells. The method for preparing the sensor comprises: oil phase cyclohexane, Triton X-100 as a surfactant and hexanol as a cosurfactant are uniformly mixed according to the volume ratio of 4.2 to 1 to 1 so as to serve as a dispersed phase by adding water and obtain water-in-oil microemulsion, and a mixed solution of the sensitive dye and the reference dye is added for agitation; tetraethyl orthosilicate (TEOS) as a silylating reagent and ammonia water as a catalyst in the same volume are added, and acetone is used for breaking the microemulsion after a reaction is completed; the microemulsion is washed by absolute alcohol and ultrapure water in a centrifugal way for obtaining the fluorescence nanometer sensor with a silicon shell and internal reference. The sensor is used for the non-traumatic monitoring of the pH of the living cells, and the variation of the pH of the cells is visually reflected by the variation of the fluorescence ratio of the sensitive dye to the reference dye in the nanometer sensor for realizing the dynamic monitoring of the variation of the pH of the cells.
Description
Technical field:
The present invention relates to field of nanometer material technology, be specifically related to a kind of preparation method of the nanometer pH sensor with interior reference and be applied to the real-time monitoring that internal pH changes.
Background technology:
Many important vital movements such as cell growth are all closely relevant with intracellular pH with differentiation, calcium adjusting, Apoptosis etc., because the size of cell is between several microns are to tens microns, measure the sensor that intracellular pH changes needs development small-sized, early the nanometer of development and optical fiber, the optoelectronic pole of micron level need penetration cell could detect intracellular pH, but also can damage by pair cell simultaneously, cause that cell physiological changes and the measurement of interference pH.The development non-intrusion type, highly sensitive, to respond real-time detection method rapidly be new demand to the sensor development, the PEBBLES sensor preparation process complexity of arising at the historic moment, the sensitive dye of embedding also is confined to the lipophilicity dyestuff, and sensor enters cell by the method for micro-injection, particle gun transmission and needs expensive instrument and need professional's operation.Though and the liposome sensor of new development enters cell easily, after but propping material and cell membrane merge, the sensitive dye of parcel is discharged into the murder by poisoning that can cause pair cell in the kytoplasm, and the sensitive dye interference that is vulnerable to the background fluorescence signal causes the error of measurement.Because the fluctuation of excitation source, photobleaching, the transmission variation of optical fiber, the light scattering of sample etc. all cause the variation of measuring-signal easily, therefore the sensor of the single sensitive dye of embedding can not reflect the variation of pH exactly by the variation of fluorescence signal, so the sensor variation that is difficult to satisfy pH in the pair cell realizes non-intrusion type, highly sensitive, accurate, real-time dynamic monitoring.
Summary of the invention:
Defective at the sensor, overcome the measurement inaccuracy that cell puncture or dyestuff leak or the light signal fluctuation brings, technical matters to be solved by this invention is synthetic a kind of nanometer pH sensor that is embedded with the stable performance of reference dyestuff and sensitive dye simultaneously, realizes the no wound monitoring of pH dynamic change in the living cells.
For this reason, the technical scheme that the present invention solves the problems of the technologies described above employing is: a kind of preparation method with silicon shell fluorescence nano pH sensor of interior reference, it is characterized in that preparation technology's flow process is: oil phase cyclohexane, surfactant triton x-100 (TritonX-100) and cosurfactant n-hexyl alcohol were mixed in 4.2: 1: 1 by volume, add suitable quantity of water as disperse phase, be stirred to solution and be the bright water-in-oil microemulsion of clarification; Add sensitive dye and reference dyestuff mixed liquor, stir; Add isopyknic silylating reagent ethyl orthosilicate (TEOS) and catalyst ammonia water, reaction continues 24h.Back acetone breakdown of emulsion is finished in reaction, and absolute ethyl alcohol and ultrapure water centrifuge washing obtain the silicon shell fluorescence nano sensor of while embedding sensitive dye and reference dyestuff, promptly have the silicon shell fluorescence nano sensor of interior reference.
Described sensitive dye prepares by the following method: the preferred fluorescein isothiocynate FITC of pH sensitive dye, fluorescein isothiocynate FITC solution is mixed with amino silane reagent γ-An Jibingjisanyiyangjiguiwan moles such as (APTES), react under the room temperature lucifuge condition and spend the night, obtain the sensitive dye of product γ-An Jibingjisanyiyangjiguiwan-fluorescein isothiocynate APTES-FITC as preparation fluorescence nano sensor
Described reference dyestuff is for to change insensitive ruthenium pyridine Rubpy to pH.
Utilize the hydrolysis of the amino silane reagent A PTES that is connected with sensitive dye fluorescein isothiocynate FITC to be covalently bonded in the network structure of teos hydrolysis formation, utilize simultaneously reference dyestuff connection ruthenium pyridine Rubpy with positive charge and electrostatic attraction is fitted in this network structure, realize that embedding sensitive dye and reference dyestuff are in silicon shell fluorescence nano sensor simultaneously.
Described silicon shell fluorescence nano sensor with interior reference is used for the no wound monitoring of pH in the living cells, its concrete steps are: the cell and the nanometer pH sensor of in vitro culture are cultivated altogether, nano-sensor is easy to do not entered cell by cytophagy with having wound, the H in the born of the same parents
+Passing network structure and its interior dyestuff of embedding of silicon dioxide shell has an effect, the fluorescence ratio of sensitive dye and reference dyestuff changes, fluorescence ratio by sensitive dye in the nano-sensor and reference dyestuff changes the variation that intuitively reflects pH in the born of the same parents, realizes the dynamic monitoring that internal pH changes.
Silicon shell fluorescence nano sensor with interior reference is used for the no wound monitoring of pH in the living cells, it is characterized in that: described nanometer pH sensor is spherical in shape, be of a size of 40 ± 5nm, in aqueous solution, have good dispersiveness, be easy to not had wound and enter cell by cytophagy.
The present invention has following advantage:
First silicon shell of the present invention fluorescence nano pH size sensor is about 40nm, the size homogeneous, good dispersion in aqueous solution, be easy to during with cytosis do not entered cell with can having wound, exempted the other types sensor or penetrate or enter cell and the cellular damage that causes changes with injection systems such as particle guns by cytophagy.
It two is that silicon shell fluorescence nano pH sensor construction of the present invention is to be the nm-class core-and-shell particles of dyestuff embedding in it of shell with silicon dioxide, the shell physicochemical property of silicon dioxide is stable, has biocompatibility, and can avoid the leakage of dyestuff and the measurement that brings is inaccurate and the toxic action of pair cell, its interior anti-photobleaching performance of dyestuff of embedding simultaneously increases greatly, has improved measuring reliability.
It three is that nano particle with simultaneously embedding reference dyestuff and sensitive dye is as the pH sensor, the variation of pH in the ratio reflection born of the same parents by sensitive dye fluorescence signal and reference dye fluorescence signal, the measuring error that can avoid the fluctuation, photobleaching, the transmission variation of optical fiber, the light scattering of sample etc. of excitation source to cause.
Nanometer pH sensor of the present invention can realize not having the quantitative test of pH in the wound born of the same parents, for the vital movement of studying under cell normal function and the pathological state provides a kind of accurately sensitive method and instrument, for the relation of explaining between cellular pH variation and the many important vital movements of cell such as cell growth and differentiation, calcium adjusting, the Apoptosis provides a kind of research means.
Description of drawings:
Now in conjunction with the accompanying drawings the present invention is explained.
Fig. 1 is the atomic force scanning shape appearance figure of silicon shell fluorescence nano sensor of the present invention.
The nano particle diameter of preparation is typical sphere about 40nm, have good dispersiveness in water, is easy to by cytophagy.
Fig. 2 is the response curve of silicon shell fluorescence nano sensor of the present invention in the pH damping fluid.
The response curve of 1 representative fluorescence nano sensor in the damping fluid of pH9.18 among the figure
The response curve of 2 representatives fluorescence nano sensor in the damping fluid of pH7.2
The response curve of 3 representatives fluorescence nano sensor in the damping fluid of pH4.49
The fluorescence peak at 520nm place is the characteristic peak of sensitive dye fluorescein isothiocynate FITC, and the fluorescence peak at 595nm place is the characteristic peak of reference dyestuff connection ruthenium pyridine Rubpy.Two kinds of dyestuffs can be embedded in the nano particle simultaneously, and sensitive dye fluorescein isothiocynate FITC has good pH response, fluorescent value significantly descends along with the pH reduction of damping fluid, the reference dyestuff is then insensitive to pH, fluorescence intensity remains unchanged substantially, therefore with the ratio of the fluorescence intensity of fluorescein isothiocynate FITC and the fluorescence intensity that joins ruthenium pyridine Rubpy as measuring-signal science and reflect the variation of pH exactly more.
Fig. 3 silicon shell of the present invention fluorescence nano sensor is to the typical curve of pH response.
Fig. 4 is used for the monitoring of pH in the macrophage for silicon shell fluorescence nano sensor of the present invention.
Wherein A is the fluorescence imaging figure of the fluorescein isothiocynate FITC of nano particle in the cell,
B is the connection ruthenium pyridine Rubpy fluorescence imaging figure of nano particle in the cell,
C is a cell transmitted light image,
D is the stacking diagram of A and B.
Fig. 4-1 is contrast, the distribution of the interior fluorescence of mouse macrophage, I at this moment when promptly not adding the processing of medicine chloroquine
FITC/ I
Rubpy=0.4823, pH4.75 in the lysosome is learnt in calculating according to typical curve)
The distribution of the interior fluorescence of mouse macrophage, I at this moment when 4-2 handles 5min for adding the medicine chloroquine
FITC/ I
Rubpy=0.7553, calculating learns that pH rises to 6.50 in the lysosome according to typical curve
The distribution of the interior fluorescence of mouse macrophage, I at this moment when 4-3 handles 15min for adding the medicine chloroquine
FITC/ I
Rubpy=0.7865, calculating learns that pH rises to 6.70 in the lysosome according to typical curve
The distribution of the interior fluorescence of mouse macrophage, I at this moment when 4-4 handles 20min for adding the medicine chloroquine
FITC/ I
Rubpy=0.7943, calculating learns that pH rises to 6.75 in the lysosome according to typical curve
The distribution of the interior fluorescence of mouse macrophage, I at this moment when 4-5 handles 25min for adding the medicine chloroquine
FITC/ I
Rubpy=0.8145, calculating learns that pH rises to 6.88 in the lysosome according to typical curve
The distribution of the interior fluorescence of mouse macrophage, I at this moment when 4-6 handles 40min for adding the medicine chloroquine
FITC/ I
Rubpy=0.8083, calculating learns that pH fluctuates 6.84 in the lysosome according to typical curve
Fig. 5 is used for the dynamic monitoring of pH in the hepatoma carcinoma cell for silicon shell fluorescence nano sensor of the present invention.
4 situations of change among the figure for pH in the hepatoma carcinoma cell behind the adding 0.1mmol/L succinic acid
5 situations of change for pH in the hepatoma carcinoma cell behind the adding 1mmol/L succinic acid
6 situations of change for pH in the hepatoma carcinoma cell behind the adding 3mmol/L succinic acid
7 situations of change for pH in the hepatoma carcinoma cell behind the adding 4mmol/L succinic acid
8 situations of change for pH in the hepatoma carcinoma cell behind the adding 5mmol/L succinic acid
9 situations of change for pH in the hepatoma carcinoma cell behind the adding 10mmol/L succinic acid
Embodiment:
Embodiment one, and preparation has the silicon shell fluorescence nano pH sensor of interior reference:
The first step, at first dispose the solution of sensitive dye and reference dyestuff: the saturated fluorescein isothiocynate FITC solution of 0.1mol/L is mixed with amino silane reagent γ-An Jibingjisanyiyangjiguiwan moles such as (APTES), react under the room temperature lucifuge condition and spend the night, the product FITC-APTES (80 μ L) that obtains and the connection ruthenium pyridine solution (10 μ L) of 0.1mol/L are mixed, stand-by.
Second step, with cyclohexane (7.5mL), surfactant triton x-100 (TritonX-100,1.8mL) and cosurfactant n-hexyl alcohol (1.8mL) mix, add 310 μ L water as disperse phase, be stirred to solution and be the bright water-in-oil microemulsion of clarification, add the sensitive dye and the reference dyestuff mixed liquor of first step preparation, stir, add silylating reagent ethyl orthosilicate (TEOS) 200 μ L and catalyst ammonia water 200 μ L, reaction continues 24h.FITC-APTES hydrolysis in the microemulsion is covalently bonded in the network structure that forms after the TEOS hydrolysis, connection ruthenium pyridine Rubpy then utilize its with positive charge and be entrenched in the inside of network structure by electrostatic attraction, reach like this two kinds of dyestuffs be embedded into purpose in the nano particle simultaneously.Acetone breakdown of emulsion after reaction is finished, distinguish centrifuge washing 4 times with absolute ethyl alcohol and ultrapure water, obtain the silicon shell fluorescence nano sensor (silicon shell fluorescence nano sensor) of while embedding sensitive dye and reference dyestuff with interior reference, the nano-sensor nanoparticle form material as shown in Figure 1 that generates, spherical in shape from the visible nano-sensor of Fig. 1 photo, size evenly, size about 40nm, good dispersion.Sensitive dye fluorescein isothiocynate in the nano-sensor has the response performance good to pH simultaneously, and reference dyestuff connection ruthenium pyridine is then insensitive to the variation of pH, as shown in Figure 2.
Embodiment two, and the silicon shell fluorescence nano pH sensor with interior reference is used for the no wound monitoring of pH in the macrophage
The first step, draw the typical curve of nano-sensor to the pH response, detailed process is: 5 μ L nano particles are joined in the 200 μ L damping fluids of different pH, behind the reaction 5min, solution is dripped on the microslide that is fixed on the laser confocal microscope objective table, sensitive dye fluorescein isothiocynate FITC and reference dyestuff join ruthenium pyridine Rubpy relative intensity of fluorescence separately in the record nano particle, calculate both fluorescence ratio (I
FITC/ I
Rubpy), the experiment triplicate.With pH is horizontal ordinate, I
FITC/ I
RubpyFor ordinate is drawn the response curve of nano particle to pH, as Fig. 3, nano-sensor is 4-7.5 to the range of linearity of pH response, because just in the scope of pH5-7, therefore nano-sensor of the present invention is fit to the measurement of PH in the cell to the variation of the pH under the normal physiological conditions in tenuigenin and the organelle very much.
Second step, the mouse of axenic cultivation is put to death rapidly, under the prerequisite of not destroying its internal organs and blood vessel, collect ascites cells carry out in vitro culture (37 ℃, 5%CO
2) behind the 4h, remove not adherent neutrophil leucocyte and leucocyte, renew bright nutrient culture media, continue overnight incubation.
The 3rd step, to ultrasonic uniform 8 μ L nano-sensor suspending liquid in advance, disperse with the dilution of 1mL anteserum-less substrate, the macrophage that cultivates with the first step is cultivated 30min altogether under 37 ℃ then, abandon nutrient culture media, (PBS, pH7.4) the rinse cell is three times, removes not by nano-sensor unnecessary in the cytophagic nutrient culture media and the non-specific adsorption nano-sensor on the macrophage surface with cold phosphate buffer before observing.Because the size of nano-sensor is about 40nm, macrophage be easy to by lysosomal pathway with the sensor endocytosis in cell, with the AT transfered cell of nanometer pH sensor.
In the 4th step, the interior pH of drug treating cell induction born of the same parents changes and dynamically monitors.Go on foot the cell fixation of cultivation to the laser confocal microscope objective table with the 3rd, a selected cell or a representational visual field, identical when being transferred to the instrument parameters with the drawing standard curve write down the fluorescence ratio I of fluorescein isothiocynate FITC and connection ruthenium pyridine Rubpy then
FITC/ I
Rubpy(control group).The strong and weak variation of fluorescence that adds chloroquine (final concentration 200 μ mol/L) back nano particle in the different time sections observation of cell, record fluorescence ratio I
FITC/ I
RubpyIt in the lysosome sour environment, the pH value is between 4.5-5, fluorescence nano sensor with the macrophage cultivation mainly passes through the lysosomal pathway internalization in advance, therefore contain more nano-sensor in the lysosome, sensitive dye fluorescein isothiocynate FITC fluorescence is extinguished in the nano-sensor under the lysosomal acid condition, and the connection of the reference dyestuff in sensor ruthenium pyridine Rubpy is insensitive to pH, so the I in the control group
FITC/ I
RubpyRatio is low, as Fig. 4-1.Diffuse into cytosis after chloroquine adds and cause that in lysosome pH raises, the fluorescence of fluorescein isothiocynate recovers in the nano-sensor, so I
FITC/ I
RubpyRatio raises after adding chloroquine, as shown in Figure 4, and I after adding medicine 20min
FITC/ I
RubpyRatio reaches capacity, and no longer raises.Fluorescence ratio by sensitive dye and reference dyestuff changes the variation that can accurately reflect internal pH adding medicine front and back, realizes the not damaged dynamic monitoring of pH in the born of the same parents.
Embodiment three, and the silicon shell fluorescence nano pH sensor with interior reference is used for the no wound monitoring of pH in the hepatoma carcinoma cell
The first step, 24h cultivates hepatoma carcinoma cell to 70% coverage rate in advance before the interior pH of observation of cell, abandons nutrient culture media.To ultrasonic uniform 8 μ L nano-sensor suspending liquid in advance, disperse with the dilution of 1mL anteserum-less substrate, joining then to cultivate has in the double dish of hepatoma carcinoma cell, under 37 ℃, cultivate 4h altogether, because the size of nano-sensor is about 40nm, hepatoma carcinoma cell can realize the AT importing hepatoma carcinoma cell of nanometer pH sensor with the sensor endocytosis in cell.(PBS, pH7.4) the rinse cell is three times, removes not by nano-sensor unnecessary in the cytophagic nutrient culture media and the non-specific adsorption nano-sensor on the hepatoma carcinoma cell surface, adds the PBS of 1mL pH6.5 then with cold phosphate buffer before observing.
Second step, the weak organic acid such as the succinic acid that in the hepatoma carcinoma cell of second step preparation, add variable concentrations, weak organic acid can be diffused into rapidly in the cell and the tumour cell of acidifying in vitro culture with non-ionic form, the fluorescence of the nano-sensor in the born of the same parents correspondingly changes, thereby realize the pH dynamic monitoring in the born of the same parents, as Fig. 5, the influence of the succinic acid pair cell pH of low concentration is little, when concentration is increased to 4mmol/L, pH descends rapidly in the born of the same parents, cause the nonvolatil acidifying of cell inside irreversible, the monitoring by pH can provide a kind of new anticancer strategy for treatment of cancer.
Claims (3)
1. preparation method with silicon shell fluorescence nano pH sensor of interior reference, it is characterized in that preparation technology's flow process is: oil phase cyclohexane, surfactant Qu Latong TritonX-100 and cosurfactant n-hexyl alcohol were mixed in 4.2: 1: 1 by volume, add suitable quantity of water as disperse phase, be stirred to solution and be the bright water-in-oil microemulsion of clarification; Add sensitive dye and reference dyestuff mixed liquor, stir; Add isopyknic silylating reagent ethyl orthosilicate TEOS and catalyst ammonia water, reaction continues 24h; Acetone breakdown of emulsion after reaction is finished, absolute ethyl alcohol and ultrapure water centrifuge washing, obtain the silicon shell fluorescence nano sensor of while embedding sensitive dye and reference dyestuff, the silicon shell fluorescence nano sensor that promptly has interior reference, described sensitive dye prepares by the following method, fluorescein isothiocynate FITC solution is mixed with moles such as amino silane reagent γ-An Jibingjisanyiyangjiguiwan APTES, react under the room temperature lucifuge condition and spend the night, obtain the sensitive dye of product γ-An Jibingjisanyiyangjiguiwan-fluorescein isothiocynate APTES-FITC as preparation fluorescence nano sensor.
2. the preparation method with silicon shell fluorescence nano pH sensor of interior reference as claimed in claim 1 is characterized in that: described reference dyestuff is to the insensitive ruthenium pyridine of pH Rubpy.
3. the preparation method with silicon shell fluorescence nano pH sensor of interior reference as claimed in claim 2, it is characterized in that: utilize the hydrolysis of the amino silane reagent A PTES that is connected with sensitive dye fluorescein isothiocynate FITC to be covalently bonded in the network structure of teos hydrolysis formation, utilize simultaneously reference dyestuff connection ruthenium pyridine Rubpy with positive charge and electrostatic attraction is fitted in this network structure, realize that embedding sensitive dye and reference dyestuff are in silicon shell fluorescence nano sensor simultaneously.
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| CN100427594C (en) * | 2006-06-13 | 2008-10-22 | 山西大学 | Silica gel granules of imbedment enzyme or cells of microbe |
| CN102841080B (en) * | 2012-08-06 | 2014-10-15 | 上海交通大学 | Two-parameter optical fiber sensor used for measurement of pH value and dissolved oxygen |
| CN102830104A (en) * | 2012-08-29 | 2012-12-19 | 浙江大学 | Method of preparing microcapsule pH (Potential Of Hydrogen) sensor |
| CN104388079A (en) * | 2014-10-28 | 2015-03-04 | 江苏大学 | Preparation method of composite fluorescent microspheres |
| US10736550B2 (en) * | 2017-06-01 | 2020-08-11 | Cnoga Medical Ltd. | Apparatus and method of generating pH of subject from at least three wavelengths |
| CN108169199B (en) * | 2018-02-09 | 2020-07-24 | 大连理工大学 | Method for quickly quantifying exosome by using fluorescence ratio |
| PL240562B1 (en) * | 2018-04-16 | 2022-05-02 | Inst Chemii Fizycznej Polskiej Akademii Nauk | Method for producing chemically inert silica nanoobjects containing a fluorescent core |
| CN108896522A (en) * | 2018-07-27 | 2018-11-27 | 香港科技大学深圳研究院 | One kind is based on nano fluorescent dyestuff to aquatile intestinal pH method for measuring |
| CN113533267A (en) * | 2021-06-03 | 2021-10-22 | 浙江大学医学院附属邵逸夫医院 | FITC-based cell microenvironment pH determination method and application thereof in osteoclast blocking zone |
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