CN109876157B - Composite material of ion-specific filter membrane/mesoporous silicon, nano sensor, product of nano sensor and application of nano sensor - Google Patents
Composite material of ion-specific filter membrane/mesoporous silicon, nano sensor, product of nano sensor and application of nano sensor Download PDFInfo
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- CN109876157B CN109876157B CN201910143844.4A CN201910143844A CN109876157B CN 109876157 B CN109876157 B CN 109876157B CN 201910143844 A CN201910143844 A CN 201910143844A CN 109876157 B CN109876157 B CN 109876157B
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Abstract
The invention relates to an ion-specific filter membrane/mesoporous silicon composite material, a nano sensor, a product and application thereof. The composite material of the ion-specific filter membrane/mesoporous silicon comprises mesoporous silica nanoparticles and an ion-specific filter membrane deposited on the surfaces of the mesoporous silica nanoparticles. The nano sensor comprises an ion-specific filter membrane/mesoporous silicon composite material and a corresponding ion indicator adsorbed in mesoporous silica nanoparticles. The nano sensor can perform dynamic imaging on the change of the ion concentration outside the freely moving living cells with high selectivity and high sensitivity, thereby reflecting the brain nerve activity condition.
Description
Technical Field
The invention relates to the field of preparation of a nano sensor, in particular to an ion-specific filter membrane/mesoporous silicon composite material, a nano sensor, a product and application thereof.
Background
There has long been a need to understand the principles of brain operation using techniques for recording neuronal activity. To this end, scientists have actively tried to construct a scalable electrode array for mapping tissue electrophysiology and design various sensors capable of non-invasively measuring calcium or voltage changes. These advances open new avenues for brain research and bring many new biological insights.
K+Extracellular K, one of the key factors determining the membrane potential+Concentration ([ K ]+]o) Can directly affect membrane potential and broadly affect neuronal activity such as activation and inactivation of voltage-gated channels, synaptic transmission, and the bioelectrical transport of neurotransmitters. Thus, K+Specific permeation through cell membranes fundamentally affects the electrical signal and forms the basis for information storage and transfer. In addition, abnormal [ K ]+]oSignals are associated with many brain disorders, such as epilepsy, cortical spreading depression and ischemic stroke. In view of K+Plays an important role in various physiological and pathological functions of the brain, and precisely measures [ K ] in complex behaviors+]oThe dynamics have important scientific significance. However, due to the available K+The limitation of sensitive probes, which remains challenging.
Available to date, K+Optical sensors (e.g., Xiangxing, K.; Fengyu, S.; Liqiang, Z.; Jordan, Y.; Fred, L.; Zhengwei, S.; Yanqing, T.; Meldrum, D.R., A high selectivity Mitochondria-Targeting Fluorecent K (+) sensor. Angewandte Chemie 2015,127 (41)), 12221-;
V.R.; gooding, j.j.; bakker, E.S., Light-Addressable Ion Sensing for Real-Time Monitoring of Extracellular conference chemical International Edition 2018,57(51),16801-+]oConventional measurements of spatio-temporal dynamics were performed, but measurements in freely moving animals were not possible. Freely moving animal brain [ K ] due to animal behavior affecting brain state+]oDynamic imaging of changes is crucial for the direct comparative analysis of their behavior and neural activity.
However, such dynamic imaging has not been reported so far, and there are mainly the following reasons. First, during nerve discharge, [ K ]+]oThe maximum change was only ten-fold (from 3nM to 30nM, however extracellular Ca2+Can vary up to 104Multiple); k+The sensitivity of the optical sensor is not high enough to detect such narrow K+]oThe fluctuation range. Second, although K+The selectivity of the sensor is significantly improved, but Na is still not well distinguished+And K+. During nerve discharge, K+Outflow is often accompanied by corresponding amounts of Na+Internal flow, if K+The selectivity of the sensor is not high enough, in combination with K+While at the same time can combine Na+Then extracellular Na+The decrease in concentration will offset the moiety [ K ]+]oThe induced signal change amplitude is changed.
Thus, novel Ks having high sensitivity and high selectivity were constructed+Sensor pair [ K+]oIs of great importance.
Disclosure of Invention
The invention aims to provide an ion-specific filter membrane/mesoporous silicon composite material, a nano sensor, a product and an application thereof, aiming at the defects of the prior art.
The technical scheme provided by the invention is as follows:
an ion-specific filter membrane/mesoporous silicon composite material comprises mesoporous silica nanoparticles and an ion-specific filter membrane deposited on the surfaces of the mesoporous silica nanoparticles;
the ion specificity filter membrane is assembled by molecules of a structural formula (1).
According to the invention, the ion specificity filter membrane is assembled by the molecules of the structural formula (1), and the pore canal diameter of the filter membrane is suitable and is rich in carbon base, so that the composite material of the ion specificity filter membrane/mesoporous silicon can selectively capture potassium ions. The ion specificity filter membrane captures potassium ions, then the ions are diffused to the mesoporous silica nano particles, the local ion concentration is increased to be beneficial to signal amplification, meanwhile, the mesoporous silica nano particles can carry ion indicators, and dynamic imaging can be carried out on the change of the concentration of the potassium ions outside the freely moving living cells with high selectivity and high sensitivity, so that the brain nerve activity condition is reflected.
The thickness of the ion-specific filter membrane is 0.5-100 nm. Preferably, the thickness of the ion-specific filter membrane is 0.5-30 nm. More preferably 1.5 to 20 nm.
The particle size of the mesoporous silica nano particles is 5-500 nm. Preferably, the mesoporous silica nanoparticles have a particle size of 5 to 100 nm. More preferably 30 to 50 nm.
The carbonyl groups on the ion-specific filter membrane of the present invention are partially oxidized to carboxyl groups. Because carbonyl on the filter membrane is partially oxidized into carboxyl, the affinity of the pore channel to ions is changed, and the diameter of the pore channel is reduced, so that the ion-specific filter membrane/mesoporous silicon composite material can selectively capture sodium ions.
The surface of the ion-specific filter membrane/mesoporous silicon composite material is modified with PEG. After PEG modification, the dispersibility and stability of the PEG modified polypeptide in vivo are improved, and the high-selectivity and high-sensitivity monitoring of the dynamic change of the ion concentration outside the freely moving living brain cells can be realized, so that the brain nerve activity condition is reflected.
The invention provides a nano sensor which comprises the ion-specific filter membrane/mesoporous silicon composite material and a corresponding ion indicator adsorbed in mesoporous silica nanoparticles. The mesoporous silica nano particles have extremely large pore volume, can carry ion indicators and are used for monitoring the dynamic change condition of the extracellular ion concentration of the brain; and secondly, the surface-coated ion-specific filter membrane has higher affinity for certain ions, selectively captures certain ions and diffuses the certain ions to the mesoporous silicon, so that the local ion concentration is improved, and high-selectivity and high-sensitivity monitoring can be realized.
Preferably, when the ion-specific filter membrane is used for specifically capturing potassium ions, the ion indicator is K+Indicators APG, K+Indicator PBFI, and the like.
Preferably, when the ion-specific filter membrane is used for specifically capturing sodium ions, the ion indicator is Na+Indicator SBFI, and the like.
The invention provides an application of the nano sensor in monitoring dynamic change of the concentration of ions outside cells of the brain of a living body. Preferably, there is provided the use of a nanosensor for monitoring the dynamic change in the concentration of free-moving, living brain extracellular ions. The nano sensor is used for reflecting the brain nerve activity condition by dynamically imaging the change of the free moving potassium ion concentration outside the living cells, and can be used for monitoring the brain nerve activity.
The invention provides a preparation method of an ion-specific filter membrane/mesoporous silicon composite material, which comprises the following steps:
1) adding N-benzyl salicylamide and anhydrous potassium carbonate into N, N-dimethylformamide, heating to 85-95 ℃, and continuously adding 1,1,1-tri (p-toluenesulfonyloxy-methyl) ethane and 2-aminoterephthalic acid to react to obtain a filter membrane precursor;
2) and depositing the filter membrane precursor on the surface of the mesoporous silica nanoparticle to obtain the potassium ion specific filter membrane/mesoporous silicon composite material.
The filter membrane precursor in step 1) is a molecule of formula (1): 1,1,1-tris- { [ (2' -benzylaminoformyl) phenoxy ] methyl } ethane.
The specific reaction formula is as follows:
preferably, the charge ratio of the N-benzyl salicylamide, anhydrous potassium carbonate, 1,1,1-tri (p-toluenesulfonyloxy-methyl) ethane and 2-amino terephthalic acid in the step 1) is as follows: 3-4 g: 2-3 g: 2.5-3.5 g: 0.2-0.4 ml.
Preferably, in the step 1), N-benzyl salicylamide and anhydrous potassium carbonate are added into N, N-dimethylformamide, the mixture is heated to 85-95 ℃,1,1, 1-tris (p-toluenesulfonyloxy-methyl) ethane and 2-aminoterephthalic acid are continuously added to react for 10-15 hours, the obtained product is subjected to silica gel column chromatography, and a petroleum ether-ethyl acetate mixed solution is used as an eluent to obtain a white filter membrane precursor. The volume ratio of the petroleum ether to the ethyl acetate in the eluent is 1.5-2.5: 1.
Preferably, the preparation of the mesoporous silica nanoparticles comprises the following steps: dissolving hexadecyl trimethyl ammonium chloride and triethanolamine in deionized water, reacting for 0.5-5 h at 90-100 ℃, and continuously adding tetraethoxysilane for reacting for 0.5-5 h; and extracting the product by using a sodium chloride methanol solution to obtain the mesoporous silica nano particle. The feed ratio of the hexadecyl trimethyl ammonium chloride to the triethanolamine to the tetraethoxysilane is as follows: 1.5-2.5 g: 0.05-0.09 g: 1.4-1.6 ml. Preferably, 0.5-1.5 wt% sodium chloride methanol solution is adopted to extract the mesoporous silica nanoparticles for 2-6 h, and hexadecyl trimethyl ammonium chloride is removed.
Preferably, in the step 2), the mesoporous silica nanoparticles are dispersed in an acetonitrile solution under an ultrasonic condition; under the condition of violent stirring, adding a filter membrane precursor acetonitrile solution into the solution for reaction, and depositing the filter membrane precursor on the surfaces of the mesoporous silica nanoparticles by an in-situ loading method; annealing for 10-15 h at room temperature to obtain the potassium ion specific filter membrane/mesoporous silicon composite material.
Preferably, the mass ratio of the mesoporous silica nanoparticles to the filter membrane precursor is 45-55: 1.
The preparation method of the ion-specific filter membrane/mesoporous silicon composite material comprises the step of modifying PEG on the surface of the potassium ion-specific filter membrane/mesoporous silicon composite material. After PEG modification, the dispersibility and stability of the PEG modified polypeptide in vivo are improved, and the high-selectivity and high-sensitivity monitoring of the dynamic change of the ion concentration outside the freely moving living brain cells can be realized, so that the brain nerve activity condition is reflected.
Preferably, the surface-modified PEG comprises: activating the composite material of the potassium ion specific filter membrane/mesoporous silicon by using N-hydroxysuccinimide and 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride under the condition of pH 5-6, and reacting for 0.5-1 h; dissolving monofunctional group amino-terminated PEG in N-hydroxysuccinimide and 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride solution with the pH value of 8-9; and mixing the two solutions, mechanically stirring overnight to obtain the PEG modified potassium ion specific filter membrane/mesoporous silicon composite material.
Preferably, the feeding ratio of the potassium ion specific filter membrane/mesoporous silicon composite material, N-hydroxysuccinimide and 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride is as follows: 1 mg: 8-12 mM: 8 to 12 mM. The charge ratio of the monofunctional amino-terminated PEG, N-hydroxysuccinimide and 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride is 5 mg: 8-12 mM: 8 to 12 mM.
The preparation method of the ion-specific filter membrane/mesoporous silicon composite material comprises the step of oxidizing the potassium ion-specific filter membrane/mesoporous silicon composite material by adopting potassium permanganate to obtain the sodium ion-specific filter membrane/mesoporous silicon composite material. The carbonyl groups on the ion-specific filter are partially oxidized to carboxyl groups. Because carbonyl on the filter membrane is partially oxidized into carboxyl, the affinity of the pore channel to ions is changed, and the diameter of the pore channel is reduced, so that the ion-specific filter membrane/mesoporous silicon composite material can selectively capture sodium ions.
Preferably, the potassium ion specific filter membrane/mesoporous silicon composite material is dispersed in deionized water, a potassium permanganate solution is added, the reaction is carried out for 8-15 hours at the temperature of 20-30 ℃, and the sodium ion specific filter membrane/mesoporous silicon composite material is obtained through precipitation and washing.
Preferably, the feeding ratio of the potassium ion specific filter membrane/mesoporous silicon composite material to potassium permanganate is 500 mug: 0.4 to 0.6 mM.
The preparation method of the ion-specific filter membrane/mesoporous silicon composite material comprises the step of modifying PEG on the surface of the sodium ion-specific filter membrane/mesoporous silicon composite material. After PEG modification, the dispersibility and stability of the PEG modified polypeptide in vivo are improved, and the high-selectivity and high-sensitivity monitoring of the dynamic change of the ion concentration outside the freely moving living brain cells can be realized, so that the brain nerve activity condition is reflected.
Preferably, the surface-modified PEG comprises: activating the sodium ion specific filter membrane/mesoporous silicon composite material by using N-hydroxysuccinimide and 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride under the condition of pH 5-6, and reacting for 0.5-1 h; dissolving monofunctional group amino-terminated PEG in N-hydroxysuccinimide and 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride solution with the pH value of 8-9; and mixing the two solutions, mechanically stirring overnight to obtain the PEG modified sodium ion specific filter membrane/mesoporous silicon composite material.
Preferably, the feeding ratio of the sodium ion specific filter membrane/mesoporous silicon composite material, N-hydroxysuccinimide and 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride is as follows: 1 mg: 8-12 mM: 8 to 12 mM. The charge ratio of the monofunctional amino-terminated PEG, N-hydroxysuccinimide and 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride is 5 mg: 8-12 mM: 8 to 12 mM.
Compared with the prior art, the invention has the beneficial effects that:
(1) the nano sensor can selectively capture certain specific ions, then the ions are diffused to the mesoporous silicon, and the local ion concentration is increased to facilitate signal amplification; meanwhile, the ion indicator can be carried, and the dynamic change condition of certain ion concentration outside the brain cells of the freely moving living body can be monitored with high selectivity and high sensitivity.
(2) The preparation method has the advantages of mild reaction system and controllable conditions, and the prepared materials have good biocompatibility and good clinical transformation possibility.
Drawings
FIG. 1 is a TEM photograph of mesoporous silica nanoparticles of example 1;
FIG. 2 shows K in example 1+A TEM photograph (a) and an EDS element line scanning result chart (b) of the specific filter membrane/mesoporous silicon composite material;
FIG. 3 shows polyethylene glycol-modified K in example 1+TEM photograph of the specific filter membrane/mesoporous silicon composite material;
FIG. 4 shows the results of application example 1 containing K at different concentrations+A specific filter membrane/APG/mesoporous silicon nano sensor is used for carrying out quantitative analysis on the activity of the MTT cells with the biocompatibility of HEK 293 cells (a) and a quantitative analysis result graph on the activity of the MTT cells at different incubation times (b);
FIG. 5 shows K in application example 1+Specific filter membrane/APG/mesoporous silicon nano sensor, APG/mesoporous silicon nano sensor and APG to epileptic seizure nerve [ K ]+]oA dynamic imaging result graph (a) of the change and a corresponding ICP-AES analysis result graph (b);
FIG. 6 shows K in application example 1+Specific filter membrane/APG/mesoporous silicon nano sensor, APG/mesoporous silicon nano sensor and APG pair electric ignition mouse hippocampus [ K ]+]oVariation and fluorescence signal variation amplitude (a) and duration (b) result plots;
FIG. 7 shows Na in application example 2+Specific filter membrane/SBFI/mesoporous silicon nano-sensor, SBFI in 150mM Na+/K+The results of the change in fluorescence intensity in the solution are shown.
Detailed Description
The invention is further described with reference to the following specific embodiments and the accompanying drawings.
Example 1
(1) Synthesizing mesoporous silica nanoparticles: 2g of cetyltrimethylammonium chloride and 0.07g of triethanolamine are dissolved in 20ml of deionized water in sequence, and the temperature is raised to 95 ℃ under the condition of vigorous stirring for reaction for 1 hour. Thereafter, 1.5ml of ethyl orthosilicate was added dropwise, and the reaction was continued with stirring for 1 hour. Washing with methanol for several times to remove impurities, and centrifuging to collect the product. And extracting the collected product by using 1 wt% sodium chloride methanol solution for 3h, and washing away the template agent to obtain the mesoporous silica nano particles.
And (3) carrying out appearance characterization on the prepared mesoporous silica nanoparticles by using a transmission electron microscope, wherein the diameter of the mesoporous silica nanoparticles is about 30-50 nm as shown in figure 1.
(2) Synthesis of potassium ion specific filter membrane/mesoporous silicon composite material: 3.4g N-benzylsalicylamide and 2.5g anhydrous potassium carbonate were added sequentially to 25mL anhydrous dimethylformamide to raise the temperature to 90 ℃ followed by 2.9g 1,1,1-tris (p-toluenesulfonyloxy-methyl) ethane and 0.3mL 2-aminoterephthalic acid and stirred for 12 h. After cooling to room temperature, the reaction mixture was added to 200ml of deionized water. The obtained solid product was subjected to silica gel column chromatography using petroleum ether-ethyl acetate (2: 1) as an eluent to obtain a white solid product of a filter precursor.
Adding 10ml of filter membrane precursor acetonitrile solution with the concentration of 0.1mg/ml into 50ml of acetonitrile solution containing mesoporous silica nano particles with the concentration of 1mg/ml under vigorous stirring, heating to 50 ℃ for reaction for 20min, annealing at room temperature for 12h, washing once with methanol and washing twice with water to obtain K+The thickness of the filter membrane layer of the composite material of the specific filter membrane/mesoporous silicon is about 3.5 nm.
For the prepared K+The morphology characterization (a) and the EDS element line scanning characterization (b) of the specific filter membrane/mesoporous silicon composite material are carried out by a transmission electron microscope, and are shown in an attached figure 2.
(3) Synthesis of polyethylene glycol modified composite material: 1mg of K+The specific filter membrane/mesoporous silicon composite material is dispersed in 1ml of N-hydroxysuccinyl with the concentration of 10mM and the pH value of 5.6Reacting imine with 10mM 1-ethyl-3- (3-dimethyl amino propyl) carbodiimide hydrochloride aqueous solution for 30 min; subsequently, 5mg of monofunctional amino-terminated polyethylene glycol (MW 5000) was dissolved in an aqueous solution containing N-hydroxysuccinimide at a concentration of 10mM and 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride at a concentration of 10mM at pH 8.6, and added to the above solution to mechanically stir overnight, whereby polyethylene glycol-modified K was obtained+A composite material of a specific filter membrane/mesoporous silicon.
To the prepared polyethylene glycol modified K+The morphology of the specific filter membrane/mesoporous silicon composite material is characterized by a transmission electron microscope, as shown in figure 3.
Example 2
(1) Synthesizing mesoporous silica nanoparticles: 2g of cetyltrimethylammonium chloride and 0.07g of triethanolamine are dissolved in 20ml of deionized water in sequence, and the temperature is raised to 95 ℃ under the condition of vigorous stirring for reaction for 1 hour. Thereafter, 1.5ml of ethyl orthosilicate was added dropwise, and the reaction was continued with stirring for 1 hour. Washing with methanol for several times to remove impurities, and centrifuging to collect the product. And extracting the collected product by using 1 wt% sodium chloride methanol solution for 3h, and washing away the template agent to obtain the mesoporous silica nano particles.
(2) Synthesis of potassium ion specific filter membrane/mesoporous silicon composite material: 3.4g N-benzylsalicylamide and 2.5g anhydrous potassium carbonate were added sequentially to 25mL anhydrous dimethylformamide to raise the temperature to 90 ℃ followed by 2.9g 1,1,1-tris (p-toluenesulfonyloxy-methyl) ethane and 0.3mL 2-aminoterephthalic acid and stirred for 12 h. After cooling to room temperature, the reaction mixture was added to 200ml of deionized water. The obtained solid product was subjected to silica gel column chromatography using petroleum ether-ethyl acetate (2: 1) as an eluent to obtain a white solid product of a filter precursor.
Adding 10ml of filter membrane precursor acetonitrile solution with the concentration of 0.1mg/ml into 50ml of acetonitrile solution containing mesoporous silica nano particles with the concentration of 1mg/ml under violent stirring, heating to 50 ℃ for reaction for 10min, annealing at room temperature for 12h, washing once by using methanol and washing twice by using water to obtain K+Specific filter membrane/mesoporous silicon composite material and filterThe thickness of the film layer is about 1.5 nm.
(3) Synthesis of polyethylene glycol modified composite material: 1mg of K+The composite material of the specific filter membrane/mesoporous silicon is dispersed in 1ml of an aqueous solution with the pH of 5.6, the aqueous solution contains N-hydroxysuccinimide with the concentration of 10mM and 1-ethyl-3- (3-dimethyl amino propyl) carbodiimide hydrochloride with the concentration of 10mM and reacts for 30 min. Subsequently, 5mg of monofunctional amino-terminated polyethylene glycol (MW 5000) was dissolved in an aqueous solution containing N-hydroxysuccinimide at a concentration of 10mM and 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride at a concentration of 10mM at pH 8.6, and added to the above solution to mechanically stir overnight, whereby polyethylene glycol-modified K was obtained+A composite material of a specific filter membrane/mesoporous silicon.
Example 3
(1) Synthesizing mesoporous silica nanoparticles: 2g of cetyltrimethylammonium chloride and 0.07g of triethanolamine are dissolved in 20ml of deionized water in sequence, and the temperature is raised to 95 ℃ under the condition of vigorous stirring for reaction for 1 hour. Thereafter, 1.5ml of ethyl orthosilicate was added dropwise, and the reaction was continued with stirring for 1 hour. Washing with methanol for several times to remove impurities, and centrifuging to collect the product. And extracting the collected product by using 1 wt% sodium chloride methanol solution for 3h, and washing away the template agent to obtain the mesoporous silica nano particles.
(2) Synthesis of potassium ion specific filter membrane/mesoporous silicon composite material: 3.4g N-benzylsalicylamide and 2.5g anhydrous potassium carbonate were added sequentially to 25mL anhydrous dimethylformamide to raise the temperature to 90 ℃ followed by 2.9g 1,1,1-tris (p-toluenesulfonyloxy-methyl) ethane and 0.3mL 2-aminoterephthalic acid and stirred for 12 h. After cooling to room temperature, the reaction mixture was added to 200ml of deionized water. The obtained solid product was subjected to silica gel column chromatography using petroleum ether-ethyl acetate (2: 1) as an eluent to obtain a white solid product of a filter precursor.
Adding 10ml of filter membrane precursor acetonitrile solution with the concentration of 0.1mg/ml into 50ml of acetonitrile solution containing mesoporous silica nano particles with the concentration of 1mg/ml under vigorous stirring, heating to 50 ℃ for reaction for 40min, annealing at room temperature for 12h, washing once by using methanol and washing twice by using water to obtain K+Specific forThe thickness of the filter membrane layer of the sexual filter membrane/mesoporous silicon composite material is about 10 nm.
(3) Synthesis of polyethylene glycol modified composite material: 1mg of K+The composite material of the specific filter membrane/mesoporous silicon is dispersed in 1ml of an aqueous solution with the pH of 5.6, the aqueous solution contains N-hydroxysuccinimide with the concentration of 10mM and 1-ethyl-3- (3-dimethyl amino propyl) carbodiimide hydrochloride with the concentration of 10mM and reacts for 30 min. Subsequently, 5mg of monofunctional amino-terminated polyethylene glycol (MW 5000) was dissolved in an aqueous solution containing N-hydroxysuccinimide at a concentration of 10mM and 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride at a concentration of 10mM at pH 8.6, and added to the above solution to mechanically stir overnight, whereby polyethylene glycol-modified K was obtained+A composite material of a specific filter membrane/mesoporous silicon.
Example 4
(1) Synthesizing mesoporous silica nanoparticles: 2g of cetyltrimethylammonium chloride and 0.07g of triethanolamine are dissolved in 20ml of deionized water in sequence, and the temperature is raised to 95 ℃ under the condition of vigorous stirring for reaction for 1 hour. Thereafter, 1.5ml of ethyl orthosilicate was added dropwise, and the reaction was continued with stirring for 1 hour. Washing with methanol for several times to remove impurities, and centrifuging to collect the product. And extracting the collected product by using 1 wt% sodium chloride methanol solution for 3h, and washing away the template agent to obtain the mesoporous silica nano particles.
(2) Synthesis of potassium ion specific filter membrane/mesoporous silicon composite material: 3.4g N-benzylsalicylamide and 2.5g anhydrous potassium carbonate were added sequentially to 25mL anhydrous dimethylformamide to raise the temperature to 90 ℃ followed by 2.9g 1,1,1-tris (p-toluenesulfonyloxy-methyl) ethane and 0.3mL 2-aminoterephthalic acid and stirred for 12 h. After cooling to room temperature, the reaction mixture was added to 200ml of deionized water. The obtained solid product was subjected to silica gel column chromatography using petroleum ether-ethyl acetate (2: 1) as an eluent to obtain a white solid product of a filter precursor.
Adding 10ml of filter membrane precursor acetonitrile solution with the concentration of 0.1mg/ml into 50ml of acetonitrile solution containing mesoporous silica nano particles with the concentration of 1mg/ml under vigorous stirring, heating to 50 ℃, reacting for 60min, annealing at room temperature for 12h, washing once by using methanol and washing by using waterTwice, K can be obtained+The thickness of the specific filter membrane/mesoporous silicon nano sensor is about 17 nm.
(3) Synthesis of polyethylene glycol modified composite material: 1mg of K+The composite material of the specific filter membrane/mesoporous silicon is dispersed in 1ml of an aqueous solution with the pH of 5.6, the aqueous solution contains N-hydroxysuccinimide with the concentration of 10mM and 1-ethyl-3- (3-dimethyl amino propyl) carbodiimide hydrochloride with the concentration of 10mM and reacts for 30 min. Subsequently, 5mg of monofunctional amino-terminated polyethylene glycol (MW 5000) was dissolved in an aqueous solution containing N-hydroxysuccinimide at a concentration of 10mM and 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride at a concentration of 10mM at pH 8.6, and added to the above solution to mechanically stir overnight, whereby polyethylene glycol-modified K was obtained+A composite material of a specific filter membrane/mesoporous silicon.
Example 5
(1) Synthesizing mesoporous silica nanoparticles: 2g of cetyltrimethylammonium chloride and 0.07g of triethanolamine are dissolved in 20ml of deionized water in sequence, and the temperature is raised to 95 ℃ under the condition of vigorous stirring for reaction for 1 hour. Thereafter, 1.5ml of ethyl orthosilicate was added dropwise, and the reaction was continued with stirring for 1 hour. Washing with methanol for several times to remove impurities, and centrifuging to collect the product. And extracting the collected product by using 1 wt% sodium chloride methanol solution for 3h, and washing away the template agent to obtain the mesoporous silica nano particles.
(2) Synthesis of the sodium ion specific filter membrane/mesoporous silicon composite material: 3.4g N-benzylsalicylamide and 2.5g anhydrous potassium carbonate were added sequentially to 25mL anhydrous dimethylformamide to raise the temperature to 90 ℃ followed by 2.9g 1,1,1-tris (p-toluenesulfonyloxy-methyl) ethane and 0.3mL 2-aminoterephthalic acid and stirred for 12 h. After cooling to room temperature, the reaction mixture was added to 200ml of deionized water. The obtained solid product was subjected to silica gel column chromatography using petroleum ether-ethyl acetate (2: 1) as an eluent to obtain a white solid product of a filter precursor.
Adding 10ml of filter membrane precursor acetonitrile solution with the concentration of 0.1mg/ml into 50ml of acetonitrile solution containing mesoporous silica nano particles with the concentration of 1mg/ml under vigorous stirring, heating to 50 ℃, reacting for 20min, annealing at room temperature for 12h, washing the filtrate once with methanol and twice with water to obtain K with the thickness of the filter membrane layer of about 3.5nm+A composite material of a specific filter membrane/mesoporous silicon.
500 mu g K+Dispersing the composite material of the specific filter membrane/mesoporous silicon in 5ml of deionized water, adding 5ml of potassium permanganate solution with the concentration of 1M, stirring and reacting for 12 hours at 25 ℃, washing with the deionized water for three times to obtain Na+The thickness of the filter membrane layer of the composite material of the specific filter membrane/mesoporous silicon is about 3.5 nm.
(3) Synthesis of polyethylene glycol modified composite material: adding 1mg of Na+The composite material of the specific filter membrane/mesoporous silicon is dispersed in 1ml of an aqueous solution with the pH of 5.6, the aqueous solution contains N-hydroxysuccinimide with the concentration of 10mM and 1-ethyl-3- (3-dimethyl amino propyl) carbodiimide hydrochloride with the concentration of 10mM and reacts for 30 min. Subsequently, 5mg of monofunctional amino-terminated polyethylene glycol (MW 5000) was dissolved in an aqueous solution containing N-hydroxysuccinimide at a concentration of 10mM and 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride at a concentration of 10mM at pH 8.6, and added to the above solution to mechanically stir overnight, thereby obtaining polyethylene glycol-modified Na+A composite material of a specific filter membrane/mesoporous silicon.
Application example 1: k+Application of specific filter membrane/APG/mesoporous silicon nano sensor in freely moving living brain [ K ]+]oDynamic imaging
(1) Evaluation of in vitro biocompatibility
Preparation of sensor solutions of different concentrations: 50mg of the product prepared in example 1 (polyethylene glycol-modified K) was used+Specific filter membrane/mesoporous silicon composite) is dispersed in 10ml containing 5mg of APG (K)+Indicator) at room temperature for 24 hours, centrifuging and washing to obtain K+The specific filter membrane/APG/mesoporous silicon nano sensor has APG drug loading of 4.0 wt%, and the obtained product is re-dispersed with sterilized PBS solution of different volumes to obtain sensor solutions of different concentrations (5 mug/ml, 10 mug/ml, 20 mug/ml, 50 mug/ml, 100 mug/ml).
Selection of human Kidney epithelial cell line (HEK 293) for varying concentrations of K+Specific filter membraneThe in vitro biocompatibility of the/APG/mesoporous silicon nano sensor.
The results of quantitative analysis of MTT cell activity are shown in FIG. 4(a), and the control group (0. mu.g/ml) shows that the cell survival rate was 90% or more in the groups with different drug concentrations when only the cell culture solution was used for incubation, indicating that K was present+The specific filter membrane/APG/mesoporous silicon nano sensor has good in-vitro biocompatibility.
MTT cell activity quantitative analysis at different incubation times, as shown in FIG. 4(b), a sensor solution with a concentration of 20 μ g/ml was selected, and the cell survival rates were all above 90% at different incubation times of 0h,6h,12h,24h,36h, and 48 h.
(2) Epileptic seizure nerve [ K ]+]oDynamic monitoring of changes
Preparation of specific sensor solution: the product prepared in example 1 (polyethylene glycol-modified K) was selected+Specific filter membrane/mesoporous silicon composite) 50mg is dispersed in 10ml of aqueous solution containing 5mg of APG, the mixture is stirred for 24 hours at room temperature and then centrifuged and washed to obtain the composite material, wherein the drug loading of the APG is 4.0 wt%. The resulting product was redispersed with a volume of artificial cerebrospinal fluid to give a sensor solution with a concentration of 20.0. mu.g/ml.
Preparation of non-specific sensor solution: 50mg of the mesoporous silica nanoparticles prepared in the embodiment 1 are dispersed in 10ml of aqueous solution containing 5mg of APG, stirred at room temperature for 24 hours, centrifuged and washed to obtain the APG/mesoporous silicon nano sensor. The resulting product was redispersed with a volume of artificial cerebrospinal fluid to give a sensor solution with a concentration of 14.8. mu.g/ml.
Establishing a cell model: hippocampus somatic cells were obtained from newborn Sprague-Dawley rat pups.
An imaging group: 1ml of K containing a concentration of 20.0. mu.g/ml was administered+Specific filter membrane/APG/mesoporous silicon nano-sensor and 10 mu M coriaria sinica lactone artificial cerebrospinal fluid.
Control group 1: 1ml of artificial cerebrospinal fluid containing APG/mesoporous silicon nanosensor at a concentration of 14.8. mu.g/ml and 10. mu.M coriaria lactone was administered.
Control group 2: 1ml of artificial cerebrospinal fluid containing APG at a concentration of 0.8. mu.g/ml and 10. mu.M coriamyrtin was administered.
After adding the artificial cerebrospinal fluid, recording the change of fluorescence intensity for 0-30 min, and treating the K outside the nerve cells by ICP-AES+The concentration change was quantitatively measured, and the results are shown in FIG. 5. K+The specific filter membrane/APG/mesoporous silicon nano sensor can effectively distinguish K+And Na+Can be used for treating nerve cell exterior [ K ]+]oDynamic imaging is performed on the change of (2); k cannot be effectively distinguished between APG/mesoporous silicon nano-sensor and APG+And Na+Effective imaging cannot be performed.
(3) Epilepsy-induced changes in mouse neural activity cause [ K+]oEvaluation of varying dynamic imaging capabilities
Preparation of specific sensor solution: the product prepared in example 1 (polyethylene glycol-modified K) was selected+Specific filter membrane/mesoporous silicon composite) 50mg is dispersed in 10ml of aqueous solution containing 5mg of APG, the mixture is stirred for 24 hours at room temperature and then centrifuged and washed to obtain the composite material, wherein the drug loading of the APG is 4.0 wt%. The resulting product was redispersed with a volume of sterile PBS solution to give a sensor solution with a concentration of 20.0. mu.g/ml.
Preparation of non-specific sensor solution: 50mg of the mesoporous silica nanoparticles prepared in the embodiment 1 are dispersed in 10ml of aqueous solution containing 5mg of APG, stirred at room temperature for 24 hours, centrifuged and washed to obtain the APG/mesoporous silicon nano sensor. The resulting product was redispersed with a volume of artificial cerebrospinal fluid to give a sensor solution with a concentration of 14.8. mu.g/ml.
Establishing an animal model: the right ventral hippocampus of the C57/BL6 mouse was continuously subjected to the electrical ignition stimulation.
An imaging group: mice were given 1. mu.l K by intracranial injection at a concentration of 20.0. mu.g/ml+Artificial cerebrospinal fluid of the specific filter membrane/APG/mesoporous silicon nano sensor.
Control group 1: mice were given 1 μ l of artificial cerebrospinal fluid containing APG/mesoporous silicon nanosensors at a concentration of 14.8 μ g/ml by intracranial injection.
Control group 2: mice were given 1 μ l of artificial cerebrospinal fluid containing APG at a concentration of 0.8 μ g/ml by intracranial injection.
Multiple electric ignition stimulation is carried out on the right ventral hippocampus of the C57/BL6 mouse, and each time of the electric ignition stimulation is carried out on the right ventral hippocampus+]oThe results of the fluorescence signal change amplitude and duration are shown in FIG. 6. K+The [ K ] can be obviously observed by a specific filter membrane/APG/mesoporous silicon nano sensor+]oThe amplitude and the duration of the change of the fluorescence signal are increased, but the APG/mesoporous silicon nano-sensor and the APG cannot detect the change.
Application example 2: na (Na)+Application of specific filter membrane/SBFI/mesoporous silicon nano sensor to freely moving living brain [ K +]oDynamic imaging
(1) Evaluation of Selectivity and sensitivity
Preparation of a specific sensor: the product obtained in example 5 (Na) was used+Specific filter membrane/mesoporous silicon composite) 50mg is dispersed in 10ml containing 5mg SBFI (Na)+Indicator), stirring for 24 hours at room temperature, centrifuging and washing to obtain Na+A specific filter membrane/SBFI/mesoporous silicon nano sensor.
Preparation of non-specific sensor solution: and dispersing 50mg of the mesoporous silica nanoparticles prepared in the embodiment 5 in 10ml of aqueous solution containing 5mg of SBFI, stirring at room temperature for 24 hours, centrifuging, and washing to obtain the SBFI/mesoporous silicon nano sensor.
SBFI, SBFI/mesoporous silicon nano-sensor and Na+The specific filter membrane/SBFI/mesoporous silicon nano-sensor is respectively dispersed in Na+Detecting the intensity of signal change in a solution with the concentration of 150 mM; SBFI, SBFI/mesoporous silicon nano-sensor and Na+The specific filter membrane/SBFI/mesoporous silicon nano-sensor is respectively dispersed in K+The intensity of the change in the detection signal in the 150mM solution is shown in FIG. 7. Na (Na)+The specific filter membrane/SBFI/mesoporous silicon nano sensor has higher sensitivity in Na+At the same concentration, Na+The fluorescence intensity of the specific filter membrane/SBFI/mesoporous silicon nano sensor is changed maximally; and Na+The specific filter membrane/SBFI/mesoporous silicon nano sensor has higher selectivity and K+The case of the same concentrationThen, Na+The fluorescence intensity of the specific filter membrane/SBFI/mesoporous silicon nano sensor is basically unchanged.
The above embodiments are described in detail to explain the technical solutions and advantages of the present invention, and it should be understood that the above embodiments are only specific examples of the present invention and are not intended to limit the present invention, and any modifications, additions, equivalents, etc. made within the scope of the principles of the present invention should be included in the scope of the present invention.
Claims (7)
1. The composite material of the ion-specific filter membrane/mesoporous silicon is characterized by comprising mesoporous silica nanoparticles and an ion-specific filter membrane deposited on the surfaces of the mesoporous silica nanoparticles;
the ion specificity filter membrane is assembled by molecules of a structural formula (1);
the carbonyl groups on the ion-specific filter membrane are partially oxidized to carboxyl groups.
2. The ion-specific filter membrane/mesoporous silicon composite material of claim 1, wherein the thickness of the ion-specific filter membrane is 0.5-100 nm; the particle size of the mesoporous silica nanoparticles is 5-500 nm.
3. The ion-specific filter membrane/mesoporous silicon composite material according to claim 1 or 2, wherein the surface of the ion-specific filter membrane/mesoporous silicon composite material is modified with PEG.
4. A nanosensor comprising the ion-specific filter/mesoporous silicon composite of any of claims 1-3, and a corresponding ion indicator adsorbed within mesoporous silica nanoparticles.
5. Use of the nanosensor of claim 4, in the manufacture of a device for monitoring the dynamic change of ion concentration outside a cell of a living brain.
6. A preparation method of an ion-specific filter membrane/mesoporous silicon composite material is characterized by comprising the following steps:
1) adding N-benzyl salicylamide and anhydrous potassium carbonate into N, N-dimethylformamide, heating to 85-95 ℃, and continuously adding 1,1,1-tri (p-toluenesulfonyloxy-methyl) ethane and 2-aminoterephthalic acid to react to obtain a filter membrane precursor;
2) depositing the filter membrane precursor on the surface of the mesoporous silica nanoparticle to obtain a potassium ion specific filter membrane/mesoporous silicon composite material;
oxidizing the potassium ion specific filter membrane/mesoporous silicon composite material by potassium permanganate to obtain the sodium ion specific filter membrane/mesoporous silicon composite material.
7. The method as claimed in claim 6, which comprises modifying the surface of the composite of Na ion-specific membrane/mesoporous silicon with PEG.
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