CN113046062A - Multi-fluorescence micro sensor, preparation method and application thereof - Google Patents
Multi-fluorescence micro sensor, preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to the technical field of biological detection, in particular to a multi-fluorescence micro-sensor, a preparation method and application thereof. The sensor takes amino-polystyrene microspheres as a carrier, a rhodamine B fluorescent probe is filled in the microspheres for detecting temperature, and a BCECF fluorescent probe is modified on the surfaces of the microspheres for measuring pH. The invention also discloses a method for measuring the temperature and pH of the microenvironment inside and outside the oocyte. The prepared multi-fluorescence microsensor is injected into the perivitelline space of the oocyte by utilizing a piezoelectric ultrasonic microinjection technology, so that the measurement of the temperature and the pH value in the oocyte is realized. The multi-fluorescence micro sensors are arranged in the array type micro-pits at the bottom of the culture dish by utilizing a micro-operation technology to form a sensor array, so that the temperature and the pH of the micro environment outside the oocyte can be measured simultaneously.
Description
Technical Field
The invention relates to the technical field of biological detection, in particular to a multi-fluorescence micro-sensor, a preparation method and application thereof.
Background
With the development and popularization of micromanipulation techniques, biomedical research objects have been miniaturized from individuals, organs to single cells, organelles. The need for acquisition of environmental parameters at micron size has become increasingly strong during research on cells. Temperature and pH are very important physiological parameters for cells, affecting many physiological activities of cells, including regulating cell cycle, affecting cell metabolism, cell proliferation and apoptosis, substance transport and degradation, etc. There are often large differences in temperature and pH between different cells. Even in the same cell, the temperature and pH of different organelles inside the cell are different. For example: the pH of lysosome is 4.0-4.5, the pH of cell nucleus is 7.2-7.4, and the pH of mitochondria is about 8.0; as an energy supply station for cells, the temperature of mitochondria is often 6 to 9 ℃ higher than that of other parts. Within cells, abnormal fluctuations and changes in temperature and pH will significantly affect various metabolic activities of cells, such as protein synthesis, gene expression, cell division, etc., cause functional disorders of cells and induce various disorders, such as inflammation, neurodegenerative diseases, cancer, etc.
Under external stimuli, such as drugs or other signals, cells may rapidly change their metabolic activity, resulting in drastic changes in the temperature and pH inside the cells from normal states. However, such changes in intracellular temperature are typically small-scale and transient due to the thermal influence of the extracellular environment, which makes them difficult to measure using conventional temperature detection methods. Throughout the somatic cloning process, oocytes need to undergo enucleation, injection, reconstitution, activation, cleavage, etc., which is necessarily accompanied by changes in temperature and pH. Studying the temperature and pH changes in oocytes during this period helps to understand the intrinsic mechanisms of somatic cloning and to improve the success rate of cloning. The sensors currently available for measuring the temperature and pH of oocytes have the following disadvantages: 1. due to the movement of the oocyte and the surrounding culture medium, the interference to the measurement process is large, the reliability of the measurement result is poor, the measurement precision is not high 2, and the measurement of the temperature and the pH value can not be completed simultaneously.
Disclosure of Invention
In view of this, the present invention provides a multi-fluorescence micro-sensor and a method for manufacturing the same. The fluorescence microsensor can simultaneously realize the measurement of microenvironment temperature and pH in the oocyte.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a multi-fluorescence microsensor, which comprises amino-modified amino-polystyrene microspheres, rhodamine B filled in the amino-polystyrene microspheres, and BCECF fluorescent probes modified on the surfaces of the amino-polystyrene microspheres.
BCECF contains carboxyl groups on the surface, but is very weak in acidity. The surface of the polystyrene microsphere is modified with amino. BCECF can be combined on the surface of the amino-polystyrene microsphere through dehydration condensation reaction (concentrated sulfuric acid is used as a dehydrating agent).
In the multi-fluorescence microsensor provided by the invention, the BCECF fluorescent probe is used for measuring pH; the rhodamine B fluorescent probe is used for measuring temperature.
The invention also provides a preparation method of the multi-fluorescence microsensor, which comprises the following steps:
step 1: preparing amino-polystyrene microspheres;
step 2: adding amino-polystyrene microspheres into a saturated ethanol solution of the rhodamine fluorescent probe, ultrasonically stirring, reacting, centrifuging, and washing with deionized water to obtain amino-polystyrene microspheres filled with the rhodamine fluorescent probe;
and step 3: and (3) carrying out dehydration condensation reaction on the amino-polystyrene microspheres obtained in the step (2) and the carboxyl fluorescein derivative fluorescent probe to obtain the multi-fluorescence microsensor.
In some embodiments, in step 1, the amino-polystyrene microspheres are prepared from polystyrene microspheres with a diameter of 4.5-5.5 um by nitration reaction.
In some embodiments, in step 2, the time of the sonication is 30min, and the speed of the centrifugation is 6000 rpm; the washing is with deionized water.
The invention also provides application of the multi-fluorescence microsensor in detecting the pH and the temperature of a microenvironment in a cell.
Wherein the cell is an oocyte.
The invention also provides a method for measuring the temperature and pH of the microenvironment inside and outside the oocyte, and the multifluorescent microsensor is injected into the perivitelline space of the oocyte.
The multi-fluorescence microsensor comprises amino-modified amino-polystyrene microspheres, rhodamine fluorescent probes filled in the amino-polystyrene microspheres, and carboxyl fluorescein derivative fluorescent probes modified on the surfaces of the amino-polystyrene microspheres. The invention has the following advantages:
(1) the multi-fluorescence microsensor has good stability: the invention uses the amino-polystyrene microsphere as the carrier of the sensor to fix the fluorescent probe, solves the problem that the fluorescent probe is easy to run off, and improves the stability of the sensor. And secondly, the multi-fluorescence microsensor is injected into the perivitelline space of the oocyte, so that the influence of the flow of an extracellular culture medium and the movement of the cell on the measurement is avoided, and the stability is further improved.
(2) The multi-fluorescence micro-sensor has no toxic and side effects on cells: most fluorescent probes are cytotoxic. The amino-polystyrene microsphere is used as a carrier of the fluorescent probe, so that the cells are prevented from being dyed by the fluorescent probe, and the toxic and side effects of the cells are reduced. The micro-sensor is injected into perivitelline space instead of cell membrane, and the cell membrane protection can further reduce the toxic and side effect of cells.
(3) The invention realizes double sensing of temperature and pH: the selected fluorescent probe rhodamine B is filled in the amino-polystyrene microsphere, and the fluorescent probe rhodamine B is not in direct contact with the outside, so that the fluorescence intensity of the fluorescent probe rhodamine B is only sensitive to temperature. The selected fluorescent probe BCECF is modified on the surface of the amino-polystyrene microsphere, the fluorescence intensity of the fluorescent probe BCECF is only sensitive to pH, and the pKa of the fluorescent probe BCECF is 6.98, so that the fluorescent probe BCECF is suitable for measuring a neutral oocyte environment. The excitation wavelength of rhodamine B is 561nm, and the corresponding maximum emission wavelength is 591 nm. The excitation wavelength of BCECF is 488nm, and the corresponding maximum emission wavelength is 535 nm. The working wavelength ranges of the two are far away from each other, and the two can work simultaneously without mutual influence.
Drawings
FIG. 1 shows a schematic diagram of a sensor configuration;
FIG. 2 is a schematic diagram showing the working wavelengths of fluorescent probes rhodamine B and BCECF;
FIG. 3 shows a sensing curve of a multi-fluorescent microsensor versus temperature;
FIG. 4 shows a sensing curve of a multi-fluorescent microsensor versus pH;
FIG. 5 shows a schematic diagram of the measurement of temperature and pH inside oocytes using a multi-fluorescence microsensor;
FIG. 6 shows a schematic of the measurement of the extracellular temperature and pH using a multi-fluorescence microsensor.
Detailed Description
The invention provides a multi-fluorescence microsensor, a preparation method and application thereof. Those skilled in the art can modify the process parameters appropriately to achieve the desired results with reference to the disclosure herein. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.
As shown in figure 1, the multi-fluorescence microsensor disclosed by the invention comprises amino-modified amino-polystyrene microspheres, rhodamine fluorescent probes filled in the amino-polystyrene microspheres and carboxyl fluorescein derivative fluorescent probes modified on the surfaces of the amino-polystyrene microspheres.
The preparation method of the multi-fluorescence microsensor comprises the following steps:
(1) preparing polystyrene microspheres with the diameter of about 5um by using a dispersion polymerization method;
(2) filling a layer of nitro on the surface of the polystyrene microsphere through nitration reaction, and reducing the nitro into amino through reduction reaction to prepare the amino-polystyrene microsphere;
(3) filling a fluorescent probe rhodamine B in the amino-polystyrene microsphere by an expansion method;
(4) modifying a layer of fluorescent probe BCECF on the surface of the amino-polystyrene microsphere through dehydration condensation reaction;
(5) and measuring the luminescence property of the multi-fluorescence microsensor, and characterizing the temperature and pH sensing characteristics.
Specifically, the method comprises the following steps:
the step (1) specifically comprises the following steps: taking the ratio of ethanol to water as 9: 1 as a dispersion medium, adding 0.5g each of polyvinylpyrrolidone and calcium hydroxy phosphate as dispersants, heating to 40 ℃ in a water bath, and stirring until the dispersants are fully dissolved. The resulting solution was charged into a 250ml three-necked flask equipped with a spherical condenser, a thermometer and a nitrogen gas inlet tube, and a magnetic rotor was placed inside. 1.77g of azodiisobutyronitrile serving as an initiator is added into 28ml of styrene monomer, heated to 40 ℃ in a water bath and stirred until the initiator is fully dissolved. The mixture of initiator and styrene was added to a three-necked flask, which was heated to 70 ℃ by a water bath and purged with nitrogen and stirred at 100 rpm. After the reaction is carried out for 12 hours, the heating and the stirring are stopped, after the reaction is cooled to room temperature, the product is centrifugally separated at the speed of 6000rpm and washed by deionized water, and the polystyrene microspheres with the product diameter of about 5um are obtained. The polystyrene microspheres were stored in pure water at a concentration of 10mg/ml for later use.
The step (2) specifically comprises the following steps: and (2) adding 20ml of mixed acid with the ratio of sulfuric acid to nitric acid being 3: 2 into 10ml of the polystyrene microsphere solution prepared in the step (1), heating in a water bath at 40 ℃, stirring at the speed of 500rpm, and reacting for 2-3 h. Then centrifugally separating at 6000rpm and washing by deionized water to obtain the product of the nitro-polystyrene microspheres. Adding reducing agent Na into the prepared nitro-polystyrene microspheres2S2O4And 20ml of mixed solution of NaOH, heating in a water bath at 70 ℃, stirring at the speed of 500rpm, reacting for 4 hours, centrifuging at the speed of 6000rpm, and washing with deionized water to obtain the product, namely the amino-polystyrene microspheres. The amino-polystyrene microspheres were stored in pure water at a concentration of 10mg/ml for later use.
The step (3) specifically comprises the following steps: and (3) adding 10ml of the amino-polystyrene microsphere prepared in the step (2) into 10ml of a saturated ethanol solution of rhodamine B, ultrasonically stirring, reacting for 30min, then performing centrifugal separation at the speed of 6000rpm, and washing with deionized water. The amino-polystyrene microspheres can expand in an ethanol solution, so that rhodamine B enters the microspheres. The microspheres shrink when washed with deionized water, immobilizing rhodamine B within the microspheres. Repeating the steps for 3 times to ensure that enough rhodamine B is filled in the amino-polystyrene microspheres.
The step (4) specifically comprises the following steps: and (3) adding 1g of the amino-polystyrene microsphere with the rhodamine B dissolved inside, which is obtained in the step (3), into a BCECF aqueous solution, wherein amino groups on the surface of the polystyrene microsphere and carboxyl groups of the BCECF undergo a dehydration condensation reaction, so that the BCECF is fixed on the surface of the polystyrene microsphere. After reacting for 1h, centrifuging at 6000rpm to separate the product and washing with deionized water to obtain the multi-fluorescence microsensor.
The test materials adopted by the invention are all common commercial products and can be purchased in the market.
The invention is further illustrated by the following examples:
EXAMPLE 1 preparation of multiple fluorescent microsensors
(1) Preparing polystyrene microspheres with the diameter of about 5um by using a dispersion polymerization method; the specific method comprises the following steps: taking the ratio of ethanol to water as 9: 1 as a dispersion medium, adding 0.5g each of polyvinylpyrrolidone and calcium hydroxy phosphate as dispersants, heating to 40 ℃ in a water bath, and stirring until the dispersants are fully dissolved. The resulting solution was charged into a 250ml three-necked flask equipped with a spherical condenser, a thermometer and a nitrogen gas inlet tube, and a magnetic rotor was placed inside. 1.77g of azodiisobutyronitrile serving as an initiator is added into 28ml of styrene monomer, heated to 40 ℃ in a water bath and stirred until the initiator is fully dissolved. The mixture of initiator and styrene was added to a three-necked flask, which was heated to 70 ℃ by a water bath and purged with nitrogen and stirred at 100 rpm. After the reaction is carried out for 12 hours, the heating and the stirring are stopped, after the reaction is cooled to room temperature, the product is centrifugally separated at the speed of 6000rpm and washed by deionized water, and the polystyrene microspheres with the product diameter of about 5um are obtained. The polystyrene microspheres were stored in pure water at a concentration of 10mg/ml for later use.
(2) Filling a layer of nitro on the surface of the polystyrene microsphere through nitration reaction, and reducing the nitro into amino through reduction reaction to prepare the amino-polystyrene microsphere; the specific method comprises the following steps: and (2) adding 20ml of mixed acid with the ratio of sulfuric acid to nitric acid being 3: 2 into 10ml of the polystyrene microsphere solution prepared in the step (1), heating in a water bath at 40 ℃, stirring at the speed of 500rpm, and reacting for 2-3 h. Followed by centrifugation at 6000rpm and washing with deionized water to give the product nitro-polystyrene microspheres. Adding reducing agent Na into the prepared nitro-polystyrene microspheres2S2O4And 20ml of mixed solution of NaOH, heating in a water bath at 70 ℃, stirring at the speed of 500rpm, reacting for 4 hours, centrifuging at the speed of 6000rpm, and washing with deionized water to obtain the product, namely the amino-polystyrene microspheres. The amino-polystyrene microspheres were stored in pure water at a concentration of 10mg/ml for later use.
(3) Filling a fluorescent probe rhodamine B in the amino-polystyrene microsphere by an expansion method; the method specifically comprises the following steps: and (3) adding 10ml of the amino-polystyrene microsphere prepared in the step (2) into 10ml of a saturated ethanol solution of rhodamine B, ultrasonically stirring, reacting for 30min, then performing centrifugal separation at the speed of 6000rpm, and washing with deionized water. The amino-polystyrene microspheres can expand in an ethanol solution, so that rhodamine B enters the microspheres. The microspheres shrink when washed with deionized water, immobilizing rhodamine B within the microspheres. Repeating the steps for 3 times to ensure that enough rhodamine B is filled in the amino-polystyrene microspheres.
(4) Modifying a layer of fluorescent probe BCECF on the surface of the amino-polystyrene microsphere through dehydration condensation reaction; the method specifically comprises the following steps: and (3) adding 1g of the amino-polystyrene microsphere with the rhodamine B dissolved inside, which is obtained in the step (3), into a BCECF aqueous solution, wherein amino groups on the surface of the polystyrene microsphere and carboxyl groups of the BCECF undergo a dehydration condensation reaction, so that the BCECF is fixed on the surface of the polystyrene microsphere. After reacting for 1h, centrifuging at 6000rpm to separate the product and washing with deionized water to obtain the multi-fluorescence microsensor.
Example 2 measurement of the luminescence Performance of a multiple fluorescence microsensor, characterization of temperature and pH sensing characteristics
And measuring the luminous performance of the multi-fluorescence microsphere by using a laser confocal fluorescence microscope. Under the condition of constant pH, the temperature is controlled to rise from 30 ℃ to 40 ℃ at intervals of 0.1 ℃, 561nm of excited fluorescence is used for irradiating the multi-fluorescence microsensor, and the emission fluorescence intensity (591 nm) of the fluorescence probe rhodamine B is detected. Under the condition of constant temperature, the microspheres are placed in solutions with different pH values (pH value: 6-8 and interval of 0.1pH value), 488nm excited fluorescence is used for irradiating the multi-fluorescence microsensor, and the emission fluorescence intensity (535nm position) of the fluorescence probe BCECF is detected. Since the amount of fluorescent materials (rhodamine B and BCECF) filled or surface-modified in the microspheres cannot be controlled, it is not generally meaningful to calibrate the temperature and pH with the absolute fluorescence intensity of the multi-fluorescence microsensor. The invention adopts relative fluorescence intensity to calibrate the temperature and the pH.
Calibrating the temperature, taking the absolute fluorescence intensity of the multi-fluorescence microsensor at 40 ℃ as a reference, and then calculating according to the following formula:
the resulting temperature sensing curve is shown in fig. 3.
And (3) calibrating the pH sensing curve, taking the absolute fluorescence intensity when the pH value is 5 as a reference, and then calculating according to the formula:
the resulting pH sensing curve is shown in FIG. 4.
Example 3 method for measuring the temperature and pH of the microenvironment inside and outside the oocyte
The multi-fluorescence microsensor is injected into the perivitelline space of the oocyte by utilizing a piezoelectric ultrasonic microinjection technology, so that the detection of the temperature and the pH value in the oocyte is realized. The solution containing the multi-fluorescence microsensor is diluted to 0.01mg/ml, a micro-injection needle (the needle point inner diameter is 6um) connected with a high-precision air pump is inserted into the solution, and negative pressure is started to suck the multi-fluorescence microsensor. Inserting a micro-suction needle (the inner diameter of the needle point is 15um, the outer diameter is 100um) connected with a high-precision air pump into a culture medium containing the oocyte, and starting negative pressure to suck one oocyte. And moving the micro-injection needle to slightly extrude the oocyte, starting the piezoelectric driver to output piezoelectric ultrasonic membrane breaking pulse, driving the micro-injection needle to puncture the zona pellucida of the oocyte to be tested, and immediately closing the piezoelectric driver after the puncture is finished. The positive pressure is started by the injection needle air pump, the multi-fluorescence microsensor is pushed into the perivitelline space, and the multi-fluorescence microsensor can spread in the perivitelline space along with the movement of the oocyte; the temperature and pH value in the oocyte can be obtained by detecting the fluorescence intensity of the multi-fluorescence microsensor.
SU-8 photoresist with good biocompatibility was spin-coated on the bottom of a glass petri dish to a thickness of 7 um. The mask plate was covered on SU-8 photoresist and placed on an ultraviolet lithography machine for exposure. And placing the exposed SU-8 photoresist on a hot plate for baking heat treatment to reduce the internal stress of the SU-8 photoresist microstructure. And then carrying out ultrasonic development on the photoresist pattern to obtain a photoresist pattern. And finally, heating and curing the SU-8 photoresist microstructure at 150-200 ℃. Thus, array type micro-pits (the diameter of the micro-pit is 7um, and the distance between centers of circles in the direction of X, Y is 14um) are processed at the bottom of the culture dish. The solution containing the multi-fluorescence microsensor is diluted to 0.01mg/ml, a micro-injection needle (the needle point inner diameter is 6um) connected with a high-precision air pump is inserted into the solution, and negative pressure is started to suck the multi-fluorescence microsensor. The needle point of the injection needle is moved to the upper part of the micro pit through the mechanical arm, the air pump is started to realize positive pressure to push the microspheres into the micro pit, and therefore the array type sensor is formed at the bottom of the culture dish. The temperature and pH of the external microenvironment of the oocyte can be obtained by detecting the fluorescence intensity of the multi-fluorescence microsensor.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that it is obvious to those skilled in the art that various modifications and improvements can be made without departing from the principle of the present invention, and these modifications and improvements should also be considered as the protection scope of the present invention.
Claims (8)
1. The multi-fluorescence microsensor is characterized by comprising amino-modified amino-polystyrene microspheres, rhodamine B filled in the amino-polystyrene microspheres and a BCECF fluorescent probe modified on the surfaces of the amino-polystyrene microspheres.
2. The multi-fluorescent microsensor of claim 1, wherein the BCECF fluorescent probe is used to measure pH; the rhodamine B fluorescent probe is used for measuring temperature.
3. The method of manufacturing a multi-fluorescent microsensor according to claim 1 or 2, comprising:
step 1: preparing amino-polystyrene microspheres;
step 2: adding amino-polystyrene microspheres into a saturated ethanol solution of the rhodamine fluorescent probe, ultrasonically stirring, reacting, centrifuging, and washing with deionized water to obtain amino-polystyrene microspheres filled with the rhodamine fluorescent probe;
and step 3: and (3) carrying out dehydration condensation reaction on the amino-polystyrene microspheres obtained in the step (2) and the carboxyl fluorescein derivative fluorescent probe to obtain the multi-fluorescence microsensor.
4. The preparation method according to claim 3, wherein in the step 1, the amino-polystyrene microspheres are prepared from polystyrene microspheres with a diameter of 4.5-5.5 um by nitration reaction.
5. The preparation method according to claim 4, wherein in the step 2, the ultrasonic time is 30min, and the speed of the centrifugation is 6000 rpm; the washing is with deionized water.
6. Use of the multi-fluorescent microsensor according to any one of claims 1 to 3 or the multi-fluorescent microsensor prepared by the preparation method according to any one of claims 3 to 5 for detecting the pH and temperature of the microenvironment in a cell.
7. The use according to claim 6, wherein the cell is an oocyte.
8. A method for measuring the temperature and pH of the microenvironment inside and outside an oocyte, which is characterized in that the multi-fluorescence microsensor of claim 1 or 2 or the multi-fluorescence microsensor prepared by the preparation method of any one of claims 3 to 5 is injected into the perivitelline space of the oocyte.
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| CN104017129A (en) * | 2014-05-30 | 2014-09-03 | 吉林大学 | Fluorescence functional polymer nanometer microsphere with dual responsiveness to temperature and pH, preparing method and applications |
| CN108535169A (en) * | 2018-04-17 | 2018-09-14 | 中国农业科学院特产研究所 | A kind of internal pH value detection method |
| CN111117606A (en) * | 2019-12-31 | 2020-05-08 | 北京市计量检测科学研究院 | Carbon quantum dot fluorescent microsphere standard substance and preparation method thereof |
| CN111426659A (en) * | 2020-03-24 | 2020-07-17 | 深圳唯公生物科技有限公司 | Magnetic fluorescent coding microsphere and preparation method thereof |
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| CN104017129A (en) * | 2014-05-30 | 2014-09-03 | 吉林大学 | Fluorescence functional polymer nanometer microsphere with dual responsiveness to temperature and pH, preparing method and applications |
| CN108535169A (en) * | 2018-04-17 | 2018-09-14 | 中国农业科学院特产研究所 | A kind of internal pH value detection method |
| CN111117606A (en) * | 2019-12-31 | 2020-05-08 | 北京市计量检测科学研究院 | Carbon quantum dot fluorescent microsphere standard substance and preparation method thereof |
| CN111426659A (en) * | 2020-03-24 | 2020-07-17 | 深圳唯公生物科技有限公司 | Magnetic fluorescent coding microsphere and preparation method thereof |
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