CN104792758B - The preparation method of hydrogen sulfide fluorescence chemical sensor based on silicon nanowires or silicon nanowire array - Google Patents
The preparation method of hydrogen sulfide fluorescence chemical sensor based on silicon nanowires or silicon nanowire array Download PDFInfo
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- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
Abstract
The present invention relates to the preparation method and application based on silicon nanowires or the hydrogen sulfide fluorescence chemical sensor of silicon nanowire array.The present invention is by 3 aminopropyltriethoxywerene werenes and 4 amino 1,8 naphthalic anhydrides successively covalent modification to the surface of silicon nanowires or silicon nanowire array, obtained surface modification has 4 amino 1, the silicon nanowires or silicon nanowire array of 8 aphthalimide fluorogens, amino is further subjected to Azide with natrium nitrosum, reaction of sodium azide, obtains the hydrogen sulfide fluorescence chemical sensor based on silicon nanowires or silicon nanowire array.The hydrogen sulfide fluorescence chemical sensor based on silicon nanowires or silicon nanowire array of the present invention can be used for the detection of hydrogen sulfide in solution, and can be using the substrate based on the sensor of silicon nanowire array as cell growth, the change of real-time, in-situ monitoring cell periphery hydrogen sulfide.
Description
Technical field
The invention belongs to the fluorescence chemical sensor of one-dimensional nano structure, more particularly to based on silicon nanowires or silicon nanowires
The preparation method of the hydrogen sulfide fluorescence chemical sensor of array.
Background technology
Hydrogen sulfide (H2S a kind of endogenic gaseous signal molecule) is used as, take part in numerous physiology courses in organism,
Protected including blood vessel dilatation, angiogenesis, oxygen sensing, Apoptosis, tissue inflammation, nerve modulation process and wound and ischemic
Deng.Hydrogen sulfide levels height in human body, imply the presence of some diseases in human body, including Alzheimer disease, Tang Shi synthesis
Disease, diabetes and hepatic sclerosis.At present, detecting the method for hydrogen sulfide includes fluorescence method, colorimetric method, electrochemistry, chromatography etc., and
Fluorescent technique is due to its high sensitivity, easy to operate and have in sulfurated hydrogen detection the features such as be easy to monitor in real time unique excellent
Gesture.It has been reported that the fluorescence probe of a variety of hydrogen sulfide, such as M.K.Thorson, Angew.Chem.Int.Ed.2013 in document,
52,1；L.W.He,Chem.Commun.2015,51,1510；P.Sathyadevi,Biosensors and
Still these probes are all by being dyed to cell, carrying out sulphur in the solution to Bioelectronics 68 (2015) 681.
Change the detection of hydrogen, this brings some inconvenients to the use of sensor.And the mechanism of action of hydrogen sulfide in vivo is still
Without final conclusion, the physiology and pathology of the real-time of hydrogen sulfide in living cells environment, in situ detection to research hydrogen sulfide in cell are realized
Process is significant.Therefore, a kind of probe pair that can be used in hydrogen sulfide in real-time, the unicellular environment of in-situ monitoring is developed
It is very important in biology and clinical research.
Silicon nanowires is good and beneficial to integrated due to having the advantages that non-toxic, bio-compatibility, is widely answered
For constructing for fluorescent optical sensor, and good detection performance is shown.Silicon nanometer especially in silicon nanowire array
The tip of line and the micro-structural of cell surface can interact, and enhancing cell is in the adhesiveness on silicon nanowire array surface, rush
Enter cell in its superficial growth, therefore silicon nanowire array has been successfully applied to capture and the in situ detection of specific cells
Middle Nano Lett.2014,14,3124.Patent 201410429525.7 is based on silicon nanowires battle array using copper ion competition law structure
The sulfide ion sensor of row, although there is response to hydrogen sulfide, copper ion competition law when for biosystem sulfurated hydrogen detection,
Can be by the severe jamming of sulfur-containing amino acid, glutathione etc. in cell, it is impossible to be used in the detection of hydrogen sulfide in biosystem.
So, structure can have specific recognition ability and the hydrogen sulfide sensor based on silicon nanowire array to hydrogen sulfide, and
The detection of hydrogen sulfide in cellular environment is used it for, avoids the interference of other sulfur-containing amino acid and glutathione in biosystem,
Real-time in-situ detection provider's rule for hydrogen sulfide in cellular environment becomes with realistic meaning.
The content of the invention
An object of the present invention is to provide to be sensed based on the hydrogen sulfide fluorescence chemical of silicon nanowires or silicon nanowire array
Device, the hydrogen sulfide fluorescence chemical sensor based on silicon nanowires or silicon nanowire array is somebody's turn to do as real-time, in situ detection cell periphery
Hydrogen sulfide provide a method that.
The second object of the present invention is to provide to be sensed based on the hydrogen sulfide fluorescence chemical of silicon nanowires or silicon nanowire array
The preparation method of device.
The third object of the present invention is to provide to be sensed based on the hydrogen sulfide fluorescence chemical of silicon nanowires or silicon nanowire array
Application of the device in real-time, in-situ monitoring cell periphery hydrogen sulfide.
The present invention is that covalent modification arrives successively by APTES and 4- amino -1,8- naphthalic anhydrides
The surface of silicon nanowires or silicon nanowire array, obtained surface modification have the silicon of 4- amino -1,8- aphthalimide fluorogens
Further with natrium nitrosum, sodium azide azido reaction occurs for nano wire or silicon nanowire array, obtains being based on silicon nanowires
Or the hydrogen sulfide fluorescence chemical sensor of silicon nanowire array.
The preparation method bag of the hydrogen sulfide fluorescence chemical sensor based on silicon nanowires or silicon nanowire array of the present invention
Include following steps：
1) at room temperature, by silicon nanowires or silicon nanowire array, 10~35mL of the 30~60mg by hydroxylating processing
Dry toluene and 0.1~0.45mL APTES are added in reactor, under inert gas shielding
After being heated to 50~90 DEG C, isothermal reaction 12~48 hours, room temperature is subsequently cooled to, it is not anti-to be cleaned by ultrasonic removing with organic solvent
The APTES answered, collection, which obtains surface modification, the silicon nanowires of APTES
Or silicon nanowire array；
2) surface modification that at room temperature, step 1) is obtained has the silicon nanowires or silicon of APTES
Nano-wire array is immersed in the 0.5~2.0mol/L ethanol solution of 4- amino -1,8- naphthalic anhydrides, in indifferent gas
40~90 DEG C are heated under body protection, isothermal reaction 5~15 hours, is cooled to room temperature, then ultrasound is clear repeatedly with absolute ethyl alcohol
Unreacted 4- amino -1,8- naphthalic anhydride molecule is removed in washout, and obtaining surface modification has 4- amino -1,8- aphthalimides glimmering
The silicon nanowires or silicon nano-array of light blob；
3) surface modification for obtaining step 2) has the silicon nanowires of 4- amino -1,8- aphthalimide fluorogens or silicon to receive
Nanowire arrays are scattered or are immersed in 20~40mL acetonitriles/dichloromethane (v/v 2:1) in the mixed solvent, ice bath are cooled to 0 DEG C,
20~40 μ L trifluoroacetic acids are added, 30~50 μ L isoamyl nitrites, are reacted 2~3 hours under condition of ice bath, it is added dropwise 15~
The aqueous solution of 30mg sodium azide, stir 1.5~2 hours, add 40~60mL saturated sodium bicarbonate solutions, filter, washing is cold
Freeze and preserve, obtain the hydrogen sulfide fluorescence chemical sensor based on silicon nanowires or silicon nanowire array.
The preparation of the hydrogen sulfide fluorescence chemical sensor based on silicon nanowires of the present invention, from the ethoxy of 3- aminopropyls three
Base silane is as connector, by hydrolysis by connector covalent modification to silicon nanowire array surface, by 4- amino -1,8-
Naphthalic anhydride and amino terminal react, and the fluorogen in surface of silicon nanowires covalent modification, further passes through diazotising, nitrine
Change reaction, the azido group to the selective response of hydrogen sulfide is introduced on fluorogen, is obtained to the selective response of hydrogen sulfide
The fluorescence chemical sensor based on silicon nanowires.
Described silicon nanowires is the silicon nanowires that chemical vapour deposition technique is prepared, a diameter of 10~15nm.
Described silicon nanowire array is the silicon nanowire array that chemical etching method is prepared, in silicon nanowire array
A diameter of 200~400nm of silicon nanowires, length are 15~20 μm.
Described hydroxylating handles adoptable method：Silicon nanowires or silicon nanowire array are immersed in into volume ratio is
1:1~8:1 concentrated sulfuric acid (mass fraction is 50%~98%) and H2O2In the mixed liquor of (mass fraction is 5%~30%),
Temperature is after carrying out processing 30~90 minutes at 70~95 DEG C, to be washed with deionized to neutrality, soaking at room temperature is in volume ratio
For 3:1:1~9:1:1 H2O:H2O2(mass fraction is 5%~30%)：NH41~2.5 hour in OH mixed liquor, it is washed to
Neutrality, vacuum drying.
Described organic solvent is methanol, ethanol, dichloromethane or acetone.
The vulcanization based on silicon nanowires or silicon nanowire array to hydrogen sulfide with selective fluorescence response of the present invention
Hydrogen fluorescence chemical sensor can be used in solution and real-time, the in situ detection of the outer hydrogen sulfide of living cells.
The hydrogen sulfide fluorescence chemical sensor based on silicon nanowires of the present invention is placed in the solution system containing hydrogen sulfide,
Detected on Fluorescence spectrophotometer, with the addition of hydrogen sulfide, the fluorescence intensity of solution system to be detected gradually increases
By force；By the concentration of hydrogen sulfide known to drafting and the calibration curve of fluorescent characteristics peak relative intensity, by described based on silicon nanometer
The Fluorescence Increasing at the fluorescent characteristics peak that the fluorescence chemical sensor of line detects determines the concentration of hydrogen sulfide in system.It is used to excite
Light source is xenon lamp (excitation wavelength is 400~500nm).
Hydrogen sulfide fluorescence chemical sensor based on silicon nanowire array is examined as the active substrate and fluorescence of cell growth
The substrate of survey, be combined laser confocal microscope, it is above-mentioned be loaded with living cells the hydrogen sulfide based on silicon nanowire array it is Fluoresceinated
Learn sensor and Fluorescence Increasing occurs in the system containing hydrogen sulfide.Pass through the concentration of hydrogen sulfide known to drafting and Fluorescence Increasing
Calibration curve, the Fluorescence Increasing detected by the fluorescence chemical sensor based on silicon nanowire array determine cell periphery
The concentration of hydrogen sulfide.Excitation source used is the laser that excitation wavelength is 488nm.
What the preparation method of the present invention obtained is sensed based on the hydrogen sulfide fluorescence chemical of silicon nanowires or silicon nanowire array
The light that device is launched in detection architecture is green glow.
The advantage of the invention is that：
1. preparation method is simple.
2. be prepared the hydrogen sulfide fluorescence chemical sensor based on silicon nanowire array in real time, in situ detection it is extracellular
The hydrogen sulfide enclosed provides a method that.
With reference to specific embodiment and accompanying drawing, the present invention is further illustrated.
Brief description of the drawings
Fig. 1 shows the hydrogen sulfide fluorescence chemical based on silicon nanowires or silicon nanowire array in 1-6 of the embodiment of the present invention
Sensing schematic diagram of the sensor to hydrogen sulfide；
Fig. 2 shows the hydrogen sulfide fluorescence chemical sensor and 300 micromole's sulphur based on silicon nanowires of the embodiment of the present invention 1
Change the fluorescence after hydrogen effect to change with time；
Fig. 3 shows that the relative fluorescence of the hydrogen sulfide fluorescence chemical sensor based on silicon nanowires of the embodiment of the present invention 1 is strong
The linearity curve of degree and concentration of hydrogen sulfide；
Fig. 4 shows choosing of the hydrogen sulfide fluorescence chemical sensor to hydrogen sulfide based on silicon nanowires of the embodiment of the present invention 1
Selecting property fluorescence response；
Fig. 5 a and 5b show the hydrogen sulfide fluorescence chemical sensor conduct based on silicon nanowire array of the embodiment of the present invention 2
Substrate is detected, real-time, in-situ monitoring the fluorescence picture applied to the concentration of hydrogen sulfide change of living cells periphery.
Embodiment
Embodiment 1
1) a diameter of 10~15nm prepared by chemical vapour deposition technique silicon nanowires is immersed in volume ratio as 1:1
The concentrated sulfuric acid (mass fraction 98%) and H2O2In the mixed liquor of (mass fraction 5%), processing 30 is carried out at being 95 DEG C in temperature
After minute, it is washed with deionized to neutrality, soak at room temperature is 3 in volume ratio:1:1 H2O:H2O2(mass fraction is
30%)：NH42.5 hours in OH mixed liquor, neutrality is washed to, is dried in vacuo, obtains the silicon nanowires of hydroxylating processing；
2) silicon nanowires handled by hydroxylating that at room temperature, dry 30mg steps 1) are obtained, 10mL's is anhydrous
Toluene and 0.1mL APTES are added in reactor, permanent after being heated to 50 DEG C under nitrogen protection
Temperature reaction 48 hours, is subsequently cooled to room temperature, is cleaned with EtOH Sonicate and removes unreacted APTES,
Collection, which obtains surface modification, the silicon nanowires of APTES；
3) silicon nanowires that the surface modification that at room temperature, step 2) is obtained has APTES immerses
Into the ethanol solution of 0.5mol/L 4- amino -1,8- naphthalic anhydrides, 40 DEG C are heated under nitrogen protection, constant temperature
Reaction 15 hours, after being cooled to room temperature, it is cleaned by ultrasonic repeatedly with absolute ethyl alcohol and removes unreacted 4- amino -1,8- naphthalenedicarboxylic acid
Acid anhydride molecule, obtaining surface modification has the silicon nanowires of 4- amino -1,8- aphthalimide fluorogens (see Fig. 1)；
4) silicon nanowires that the surface modification for obtaining step 3) has 4- amino -1,8- aphthalimide fluorogens is dispersed in
20mL acetonitriles/dichloromethane (v/v 2:1) in the mixed solvent, ice bath are cooled to 0 DEG C, add 20 μ L trifluoroacetic acids, and 30 μ L are sub-
Isoamyl nitrate, react 3 hours under condition of ice bath, the aqueous solution of 15mg sodium azide is added dropwise, stir 2 hours, add
40mL saturated sodium bicarbonate solutions, filter, washing, freezen protective, obtain the hydrogen sulfide fluorescence chemical sensing based on silicon nanowires
Device.
The HEPES bufferings that hydrogen sulfide fluorescence chemical sensor obtained above based on silicon nanowires is dispersed in 20mM are molten
Suspension is formed in liquid system, is added in fluorescence cuvette, is detected on Fluorescence spectrophotometer.300 are added micro- to rub
After your hydrogen sulfide, excited with 400nm light, the fluorescence chemical sensing based on silicon nanowires of solution system to be detected
Device can produce Fluorescence Increasing, and find as the increase of action time, the fluorescence intensity of solution system to be detected gradually strengthen.
Fig. 2 gives change of the shown fluorescence chemical sensor based on silicon nanowires with hydrogen sulfide effect with the fluorescence spectrum of time
Change.
In the HEPES buffer solution systems of the 2mL 20mM containing the fluorescence chemical sensor based on silicon nanowires
In, the hydrogen sulfide of various concentrations known to addition, excited with 400nm light, the fluorescence chemical based on silicon nanowires passes
Sensor carries out fluoroscopic examination after being reacted 30 minutes with known hydrogen sulfide, finds with the increase of known concentration of hydrogen sulfide, molten
The intensity at the fluorescent characteristics peak (maximum emission wavelength 535nm) of liquid system gradually strengthens.Pass through hydrogen sulfide known to drafting
The calibration curve (Fig. 3) of concentration and fluorescent characteristics peak relative intensity, and in the solution system with being considered as to be detected by described
The Fluorescence Increasing at fluorescent characteristics peak that detects of silicon nanowires fluorescence chemical sensor be compared, it is determined that be considered as to be checked
The concentration of hydrogen sulfide in the solution system of survey, it is achieved thereby that to be considered as to be detected solution system in hydrogen sulfide inspection
Survey
The sensor has selectivity well.Fig. 4 gives the fluorescence to other sulphur-containing substances or bioactive species
Response condition, it can be seen that the addition of only hydrogen sulfide causes the Fluorescence Increasing of the solution system containing the sensor.
Embodiment 2
1) silicon nanowire array (in silicon nanowire array prepared by chemical etching method for being 1cm × 1cm by silicon chip size
Silicon nanowires a diameter of 200~400nm, length is 15~20 μm) to be immersed in volume ratio be 1:1 concentrated sulfuric acid (quality point
98%) and H number is2O2In the mixed liquor of (mass fraction 5%), temperature be 95 DEG C at carry out processing 30 minutes after, spend from
After sub- water washing to neutrality, soak at room temperature is 3 in volume ratio:1:1 H2O:H2O2(mass fraction 30%)：NH4OH mixing
2.5 hours in liquid, neutrality is washed to, is dried in vacuo, obtains the silicon nanowire array of hydroxylating processing；
2) silicon nanowire array handled by hydroxylating that at room temperature, dry step 1) is obtained, 10mL's is anhydrous
Toluene and 0.1mL APTES are added in reactor, permanent after being heated to 50 DEG C under nitrogen protection
Temperature reaction 48 hours, is subsequently cooled to room temperature, is cleaned with EtOH Sonicate and removes unreacted APTES,
Collection, which obtains surface modification, the silicon nanowire array of APTES；
3) surface modification that at room temperature, step 2) is obtained has the silicon nanowire array of APTES
It is immersed in the 0.5mol/L ethanol solution of 4- amino -1,8- naphthalic anhydrides, is heated to 40 DEG C under nitrogen protection,
Isothermal reaction 15 hours, after being cooled to room temperature, it is cleaned by ultrasonic repeatedly with absolute ethyl alcohol and removes unreacted 4- amino -1,8- naphthalene two
Formic anhydride molecule, obtaining surface modification has the silicon nanowire array of 4- amino -1,8- aphthalimide fluorogens；
4) surface modification for obtaining step 3) has the silicon nanowire array point of 4- amino -1,8- aphthalimide fluorogens
It is dispersed in 20mL acetonitriles/dichloromethane (v/v 2:1) in the mixed solvent, ice bath are cooled to 0 DEG C, add 20 μ L trifluoroacetic acids, 30 μ
L isoamyl nitrites, react 3 hours under condition of ice bath, the aqueous solution of 15mg sodium azide is added dropwise, stir 2 hours, add
40mL saturated sodium bicarbonate solutions, filter, washing, freezen protective, obtain the hydrogen sulfide fluorescence chemical based on silicon nanowire array
Sensor.
Hydrogen sulfide fluorescence chemical sensor obtained above based on silicon nanowire array is cut into 0.5cm × 0.5cm, will
It is placed in a hole of 24 porocyte culture plates, adds the cell suspending liquid that 1mL cell concentrations are 106 cells/mls, will
Tissue Culture Plate is placed in cell culture incubator (CO2：5% temperature：37 DEG C) cell culture is carried out, after 12 hours, taking out growth has
The sensor array of cell, washed five times with degerming PBS (pH 7.4).For the ease of the observation of cell, will grow
The sensor array for having cell is transferred in another hole, adds 1mL cell culture fluids, then add 2mL2mg/mL into the hole
The DAPI aqueous solution, (the CO in cell culture incubator2：5%；Temperature：37 DEG C) in be incubated 20 minutes after, buffered with degerming PBS
Liquid (pH 7.4), which washs, to be obtained growing the sensor array for having the cell by DAPI dyeing three times.
The sensor array that growth obtained above has cell is inverted in and fills the degerming PBS (pH of 1mL
7.4) in copolymerization Jiao's culture dish, laser confocal microscope is combined, real-time, original is carried out to the hydrogen sulfide of living cell growth environment
The detection of position.When the concentration of hydrogen sulfide that growth has the sensor array system of cell increases to 250 μ Μ from 0, sensor array
Fluorescence Increasing (see Fig. 5 a and Fig. 5 b, the concentration of scheming hydrogen sulfide in a is that the concentration of hydrogen sulfide in 0, b is 250 μM.).By painting
The concentration of hydrogen sulfide known to system and the calibration curve of Fluorescence Increasing, and with the fluorescence chemical sensor based on silicon nanowire array
The Fluorescence Increasing detected is compared, and determines the concentration of the hydrogen sulfide in system, it is achieved thereby that cell periphery hydrogen sulfide
Detection.
Embodiment 3
1) a diameter of 10~15nm prepared by chemical vapour deposition technique silicon nanowires is immersed in volume ratio as 8:1
The concentrated sulfuric acid (mass fraction 50%) and H2O2In the mixed liquor of (mass fraction 30%), handled at being 70 DEG C in temperature
After 90 minutes, it is washed with deionized to neutrality, soak at room temperature is 9 in volume ratio:1:1 H2O:H2O2(mass fraction is
5%)：NH41 hour in OH mixed liquor, neutrality is washed to, is dried in vacuo, obtains the silicon nanowires of hydroxylating processing；
2) at room temperature, by 60mg steps 1) obtain by hydroxylating handle silicon nanowires, 35mL dry toluene and
0.45mL APTES is added in reactor, and after being heated to 90 DEG C under nitrogen protection, constant temperature is anti-
Answer 12 hours, be subsequently cooled to room temperature, be cleaned by ultrasonic with acetone and remove unreacted APTES, filtering
Collection, which obtains surface modification, the silicon nanowires of APTES；
3) silicon nanowires that the surface modification that at room temperature, step 2) is obtained has APTES immerses
Into the ethanol solution of 2.0mol/L 4- amino -1,8- naphthalic anhydrides, 90 DEG C are heated under nitrogen protection, constant temperature
Reaction 5 hours, after being cooled to room temperature, it is cleaned by ultrasonic repeatedly with absolute ethyl alcohol and removes unreacted 4- amino -1,8- naphthalenedicarboxylic acid
Acid anhydride molecule, obtaining surface modification has the silicon nanowires of 4- amino -1,8- aphthalimide fluorogens；
4) silicon nanowires that the surface modification for obtaining step 3) has 4- amino -1,8- aphthalimide fluorogens is dispersed in
40mL acetonitriles/dichloromethane (v/v 2:1) in the mixed solvent, ice bath are cooled to 0 DEG C, add 40 μ L trifluoroacetic acids, and 50 μ L are sub-
Isoamyl nitrate, react 2 hours under condition of ice bath, the aqueous solution of 30mg sodium azide is added dropwise, stir 1.5 hours, add
60mL saturated sodium bicarbonate solutions, filter, washing, freezen protective, obtain the hydrogen sulfide fluorescence chemical sensing based on silicon nanowires
Device.
The HEPES bufferings that hydrogen sulfide fluorescence chemical sensor obtained above based on silicon nanowires is dispersed in 20mM are molten
Suspension is formed in liquid system, is added in fluorescence cuvette, is detected on Fluorescence spectrophotometer.After adding NaSH,
Excited with 500nm light, the fluorescence chemical sensor based on silicon nanowires of solution system to be detected can produce glimmering
Light strengthens, and finds as the increase of action time, the fluorescence intensity of solution system to be detected gradually strengthen.
In the HEPES buffer solution systems of the 2mL 20mM containing the fluorescence chemical sensor based on silicon nanowires
In, the hydrogen sulfide of various concentrations known to addition, excited with 500nm light, described silicon nanowires fluorescence chemical sensor with
Known hydrogen sulfide carries out fluoroscopic examination after reacting 30 minutes, finds with the increase of known concentration of hydrogen sulfide, solution system
The intensity at fluorescent characteristics peak (maximum emission wavelength 535nm) gradually strengthen.By the concentration of hydrogen sulfide known to drafting and
The calibration curve of fluorescent characteristics peak relative intensity, and it is glimmering by described silicon nanowires in the solution system with being considered as to be detected
The Fluorescence Increasing at the fluorescent characteristics peak that Optochemical sensor detects is compared, it is determined that is considered as solution system to be detected
In hydrogen sulfide concentration, it is achieved thereby that to be considered as to be detected solution system in hydrogen sulfide detection
Embodiment 4
1) silicon nanowire array (in silicon nanowire array prepared by chemical etching method for being 1cm × 1cm by silicon chip size
Silicon nanowires a diameter of 200~400nm, length is 15~20 μm) to be immersed in volume ratio be 8:1 concentrated sulfuric acid (quality point
50%) and H number is2O2In the mixed liquor of (mass fraction 30%), after carrying out processing 90 minutes at being 70 DEG C in temperature, spend
After ion water washing to neutrality, soak at room temperature is 9 in volume ratio:1:1 H2O:H2O2(mass fraction 5%)：NH4OH's is mixed
Close in liquid 1 hour, be washed to neutrality, be dried in vacuo, obtain the silicon nanowire array of hydroxylating processing；
2) silicon nanowire array handled by hydroxylating that at room temperature, dry step 1) is obtained, 35mL's is anhydrous
Toluene and 0.45mL APTES are added in reactor, after being heated to 90 DEG C under nitrogen protection,
Isothermal reaction 12 hours, is subsequently cooled to room temperature, is cleaned by ultrasonic with methanol and removes unreacted 3- aminopropyls triethoxysilicane
Alkane, collection, which obtains surface modification, the silicon nanowire array of APTES；
3) surface modification that at room temperature, step 2) is obtained has the silicon nanowire array of APTES
It is immersed in the 2.0mol/L ethanol solution of 4- amino -1,8- naphthalic anhydrides, is heated to 90 DEG C under nitrogen protection,
Isothermal reaction 5 hours, after being cooled to room temperature, it is cleaned by ultrasonic repeatedly with absolute ethyl alcohol and removes unreacted 4- amino -1,8- naphthalene two
Formic anhydride molecule, obtaining surface modification has the silicon nanowire array of 4- amino -1,8- aphthalimide fluorogens；
4) surface modification for obtaining step 3) has the silicon nanowire array point of 4- amino -1,8- aphthalimide fluorogens
It is dispersed in 40mL acetonitriles/dichloromethane (v/v 2:1) in the mixed solvent, ice bath are cooled to 0 DEG C, add 40 μ L trifluoroacetic acids, 50 μ
L isoamyl nitrites, react 2 hours under condition of ice bath, the aqueous solution of 30mg sodium azide is added dropwise, stir 1.5 hours, add
Enter 60mL saturated sodium bicarbonate solutions, filter, washing, freezen protective, it is Fluoresceinated to obtain the hydrogen sulfide based on silicon nanowire array
Learn sensor.
Sensor array obtained above is cut into 0.5cm × 0.5cm, is placed on a hole of 24 porocyte culture plates
In, the cell suspending liquid that 1mL cell concentrations are 106 cells/mls is added, Tissue Culture Plate is placed in cell culture incubator
(CO2：5%；Temperature：37 DEG C) cell culture is carried out, after 12 hours, taking out growth has the sensor array of cell, with degerming
PBS (pH 7.4) washs five times.For the ease of the observation of cell, the sensor array that growth has cell is transferred to separately
In an outer clean hole, 1mL cell culture fluids are added, then the μ g/mL of the 2 μ L 2 DAPI aqueous solution is added into the hole, in thin
(CO in born of the same parents' incubator2：5%；Temperature：37 DEG C) in be incubated 20 minutes after, wash three with degerming PBS (pH 7.4)
Secondary, obtaining growth has the sensor array of the cell by DAPI dyeing.
The sensor array that growth obtained above has cell is inverted in and fills the degerming PBS (pH of 1mL
7.4) in copolymerization Jiao's culture dish, laser confocal microscope is combined, real-time, original is carried out to the hydrogen sulfide of living cell growth environment
The detection of position.When growth has the concentration of hydrogen sulfide change of the sensor array system of cell, the fluorescence of sensor array base
Change.
The fluorescence of concentration by hydrogen sulfide known to drafting and the fluorescence chemical sensor based on silicon nanowire array increases
Strong calibration curve, and compared with the Fluorescence Increasing that described silicon nanowires fluorescence chemical sensor detects, determine body
The concentration of hydrogen sulfide in system, it is achieved thereby that the detection of cell periphery hydrogen sulfide.
Embodiment 5
1) a diameter of 10~15nm prepared by chemical vapour deposition technique silicon nanowires is immersed in volume ratio as 4:1
The concentrated sulfuric acid (mass fraction 70%) and H2O2In the mixed liquor of (mass fraction 15%), handled at being 80 DEG C in temperature
After 60 minutes, it is washed with deionized to neutrality, soak at room temperature is 6 in volume ratio:1:1 H2O:H2O2(mass fraction is
5%)：NH41.5 hours in OH mixed liquor, neutrality is washed to, is dried in vacuo, obtains the silicon nanowires of hydroxylating processing；
2) at room temperature, by 45mg steps 1) obtain by hydroxylating handle silicon nanowires, 20mL dry toluene and
0.3mL APTES is added in reactor, after being heated to 75 DEG C under nitrogen protection, isothermal reaction
30 hours, room temperature is subsequently cooled to, is cleaned by ultrasonic with acetone and removes unreacted APTES, filtering is received
Collection, which obtains surface modification, the silicon nanowires of APTES；
3) silicon nanowires that the surface modification that at room temperature, step 2) is obtained has APTES immerses
Into the ethanol solution of 1.5mol/L 4- amino -1,8- naphthalic anhydrides, 70 DEG C are heated under nitrogen protection, constant temperature
Reaction 10 hours, after being cooled to room temperature, it is cleaned by ultrasonic repeatedly with absolute ethyl alcohol and removes unreacted 4- amino -1,8- naphthalenedicarboxylic acid
Acid anhydride molecule, obtaining surface modification has the silicon nanowires of 4- amino -1,8- aphthalimide fluorogens；
4) silicon nanowires that the surface modification for obtaining step 3) has 4- amino -1,8- aphthalimide fluorogens is dispersed in
30mL acetonitriles/dichloromethane (v/v 2:1) in the mixed solvent, ice bath are cooled to 0 DEG C, add 30 μ L trifluoroacetic acids, and 40 μ L are sub-
Isoamyl nitrate, react 2.5 hours under condition of ice bath, the aqueous solution of 25mg sodium azide is added dropwise, stir 1.8 hours, add
Enter 50mL saturated sodium bicarbonate solutions, filter, washing, freezen protective, obtain the hydrogen sulfide fluorescence chemical based on silicon nanowires and pass
Sensor.
The HEPES bufferings that hydrogen sulfide fluorescence chemical sensor obtained above based on silicon nanowires is dispersed in 20mM are molten
Suspension is formed in liquid system, is added in fluorescence cuvette, is detected on Fluorescence spectrophotometer.Add hydrogen sulfide
Afterwards, excited with 435nm light, the fluorescence chemical sensor based on silicon nanowires of solution system to be detected can produce
Fluorescence Increasing, and find as the increase of action time, the fluorescence intensity of solution system to be detected gradually strengthen.
In the HEPES buffer solution systems of the 2mL 20mM containing described silicon nanowires fluorescence chemical sensor, add
Enter the hydrogen sulfide of known various concentrations, excited with 435nm light, described silicon nanowires fluorescence chemical sensor with it is known
Hydrogen sulfide carries out fluoroscopic examination after reacting 30 minutes, finds with the increase of known concentration of hydrogen sulfide, the fluorescence of solution system
The intensity of characteristic peak (maximum emission wavelength 535nm) gradually strengthens.It is special by the concentration and fluorescence of hydrogen sulfide known to drafting
The calibration curve (Fig. 3) of peak relative intensity is levied, and it is glimmering by described silicon nanowires in the solution system with being considered as to be detected
The Fluorescence Increasing at the fluorescent characteristics peak that Optochemical sensor detects is compared, it is determined that is considered as solution system to be detected
In hydrogen sulfide concentration, it is achieved thereby that to be considered as to be detected solution system in hydrogen sulfide detection
Embodiment 6
1) silicon nanowire array (in silicon nanowire array prepared by chemical etching method for being 1cm × 1cm by silicon chip size
Silicon nanowires a diameter of 200~400nm, length is 15~20 μm) to be immersed in volume ratio be 4:1 concentrated sulfuric acid (quality point
70%) and H number is2O2In the mixed liquor of (mass fraction 15%), after carrying out processing 60 minutes at being 80 DEG C in temperature, spend
After ion water washing to neutrality, soak at room temperature is 6 in volume ratio:1:1 H2O:H2O2(mass fraction 5%)：NH4OH's is mixed
Close in liquid 1.5 hours, be washed to neutrality, be dried in vacuo, obtain the silicon nanowire array of hydroxylating processing；
2) silicon nanowire array handled by hydroxylating that at room temperature, dry step 1) is obtained, 20mL's is anhydrous
Toluene and 0.3mL APTES are added in reactor, permanent after being heated to 75 DEG C under nitrogen protection
Temperature reaction 30 hours, is subsequently cooled to room temperature, is cleaned by ultrasonic with methanol and removes unreacted APTES,
Collection, which obtains surface modification, the silicon nanowire array of APTES；
3) surface modification that at room temperature, step 2) is obtained has the silicon nanowire array of APTES
It is immersed in the 1.5mol/L ethanol solution of 4- amino -1,8- naphthalic anhydrides, is heated to 70 DEG C under nitrogen protection,
Isothermal reaction 10 hours, after being cooled to room temperature, it is cleaned by ultrasonic repeatedly with absolute ethyl alcohol and removes unreacted 4- amino -1,8- naphthalene two
Formic anhydride molecule, obtaining surface modification has the silicon nanowire array of 4- amino -1,8- aphthalimide fluorogens；
4) surface modification for obtaining step 3) has the silicon nanowire array point of 4- amino -1,8- aphthalimide fluorogens
It is dispersed in 30mL acetonitriles/dichloromethane (v/v 2:1) in the mixed solvent, ice bath are cooled to 0 DEG C, add 30 μ L trifluoroacetic acids, 40 μ
L isoamyl nitrites, react 2.5 hours under condition of ice bath, the aqueous solution of 25mg sodium azide be added dropwise, stir 1.8 hours,
50mL saturated sodium bicarbonate solutions are added, are filtered, washing, freezen protective, obtain the hydrogen sulfide fluorescence based on silicon nanowire array
Chemical sensor.
Sensor array obtained above is cut into 0.5cm × 0.5cm, is placed on a hole of 24 porocyte culture plates
In, the cell suspending liquid that 1mL cell concentrations are 106 cells/mls is added, Tissue Culture Plate is placed in cell culture incubator
(CO2：5%；Temperature：37 DEG C) cell culture is carried out, after 12 hours, taking out growth has the sensor array of cell, with degerming
PBS (pH 7.4) washs five times.For the ease of the observation of cell, the sensor array that growth has cell is transferred to separately
In an outer clean hole, 1mL cell culture fluids are added, then the μ g/mL of the 2 μ L 2 DAPI aqueous solution is added into the hole, in thin
(CO in born of the same parents' incubator2：5%；Temperature：37 DEG C) in be incubated 20 minutes after, wash three with degerming PBS (pH 7.4)
Secondary, obtaining growth has the sensor array of the cell by DAPI dyeing.
The sensor array that growth obtained above has cell is inverted in and fills the degerming PBS (pH of 1mL
7.4) in copolymerization Jiao's culture dish, laser confocal microscope is combined, real-time, original is carried out to the hydrogen sulfide of living cell growth environment
The detection of position.When growth has the concentration of hydrogen sulfide change of the sensor array system of cell, the fluorescence of sensor array occurs
Change.
The fluorescence of concentration by hydrogen sulfide known to drafting and the fluorescence chemical sensor based on silicon nanowire array increases
Strong calibration curve, and compared with the Fluorescence Increasing that the fluorescence chemical sensor based on silicon nanowire array detects, really
The concentration of the hydrogen sulfide in system is determined, it is achieved thereby that the detection of cell periphery hydrogen sulfide.
Obviously, the above embodiment of the present invention is only intended to clearly illustrate example of the present invention, and is not pair
The restriction of embodiments of the present invention, for those of ordinary skill in the field, may be used also on the basis of the above description
To make other changes in different forms, all embodiments can not be exhaustive here, it is every to belong to this hair
Row of the obvious changes or variations that bright technical scheme is extended out still in protection scope of the present invention.
Claims (8)
1. a kind of preparation method of the hydrogen sulfide fluorescence chemical sensor based on silicon nanowires or silicon nanowire array, its feature exist
In described preparation method comprises the following steps：
1) at room temperature, by 30 ~ 60mg by hydroxylating processing silicon nanowires or silicon nanowire array, 10 ~ 35mL without water beetle
Benzene and 0.1 ~ 0.45mL APTES are added in reactor, and 50 are heated under inert gas shielding
After ~ 90 DEG C, isothermal reaction 12 ~ 48 hours, room temperature is subsequently cooled to, is cleaned by ultrasonic with organic solvent and removes unreacted 3- amino
Propyl-triethoxysilicane, collection, which obtains surface modification, the silicon nanowires or silicon nanowires of APTES
Array；
2) surface modification that at room temperature, step 1) is obtained has the silicon nanowires or silicon nanometer of APTES
Linear array is immersed in 0.5 ~ 2.0 mol/L ethanol solution of 4- amino -1,8- naphthalic anhydrides, is protected in inert gas
40 ~ 90 DEG C are heated under shield, isothermal reaction 5 ~ 15 hours, room temperature is cooled to, is then cleaned by ultrasonic removing repeatedly with absolute ethyl alcohol
Unreacted 4- amino -1,8- naphthalic anhydride molecule, obtaining surface modification has 4- amino -1,8- aphthalimide fluorogens
Silicon nanowires or silicon nano-array；
3) surface modification for obtaining step 2) has the silicon nanowires or silicon nanowires of 4- amino -1,8- aphthalimide fluorogens
Array is scattered or is immersed in 20 ~ 40 mL acetonitriles/dichloromethane v/v 2:1 in the mixed solvent, ice bath are cooled to 0 DEG C, add
20 ~ 40 μ L trifluoroacetic acids, 30 ~ 50 μ L isoamyl nitrites, react 2 ~ 3 hours under condition of ice bath, 15 ~ 30 mg are added dropwise
The aqueous solution of sodium azide, stir 1.5 ~ 2 hours, add 40 ~ 60 mL saturated sodium bicarbonate solutions, filter, washing, freezing is protected
Deposit, obtain the hydrogen sulfide fluorescence chemical sensor based on silicon nanowires or silicon nanowire array.
2. preparation method according to claim 1, it is characterised in that method is used by described hydroxylating processing：
Silicon nanowires or silicon nanowire array are immersed in volume ratio as 1:1~8:1 concentrated sulfuric acid and H2O2Mixed liquor in, be in temperature
After processing being carried out at 70 ~ 95 DEG C 30 ~ 90 minutes, it is washed with deionized to neutrality, soaking at room temperature is 3 in volume ratio:1:1~
9:1:1 H2O: H2O2：NH41 ~ 2.5 hour in OH mixed liquor, neutrality is washed to, is dried in vacuo.
3. preparation method according to claim 2, it is characterised in that the mass fraction of the described concentrated sulfuric acid is 50% ~ 98%,
H2O2Mass fraction be 5% ~ 30%.
4. preparation method according to claim 1 or 2, it is characterised in that：Described silicon nanowires is chemical vapor deposition
The silicon nanowires that method is prepared, a diameter of 10 ~ 15nm；
Described silicon nanowire array is the silicon nanowire array that chemical etching method is prepared, and the silicon in silicon nanowire array is received
A diameter of 200 ~ 400nm of rice noodles, length are 15 ~ 20 μm.
5. preparation method according to claim 1, it is characterised in that：Organic solvent described in step 1) be methanol, ethanol,
Dichloromethane or acetone.
6. a kind of hydrogen sulfide fluorescence chemical sensor based on silicon nanowires or silicon nanowire array, it is by claim 1 to 5
Preparation method described in any one is prepared.
7. a kind of hydrogen sulfide fluorescence chemical sensor based on silicon nanowires or silicon nanowire array described in claim 6 should
With, it is characterized in that：The described hydrogen sulfide fluorescence chemical sensor based on silicon nanowires or silicon nanowire array is used in solution
Or the detection of cell periphery hydrogen sulfide.
8. application according to claim 7, it is characterized in that：The detection of described hydrogen sulfide, it is to be received with described based on silicon
The hydrogen sulfide fluorescence chemical sensor of rice noodles or silicon nanowire array is combined XRF or laser co-focusing shows as substrate
Micro mirror, in having system existing for hydrogen sulfide, the sensor based on silicon nanowires or silicon nanowire array can produce glimmering
Light strengthens, by the concentration of hydrogen sulfide known to drafting and the calibration curve of fluorescent characteristics peak relative intensity, by described based on silicon
The Fluorescence Increasing at the fluorescent characteristics peak that the fluorescence chemical sensor of nano wire or silicon nanowire array detects determines sulphur in system
Change the concentration of hydrogen.
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