CN101692023B - Chemical preparing method of molecular recognition of ultratrace explosive TNT based on fluorescence resonance energy transfer - Google Patents
Chemical preparing method of molecular recognition of ultratrace explosive TNT based on fluorescence resonance energy transfer Download PDFInfo
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- CN101692023B CN101692023B CN 200910144974 CN200910144974A CN101692023B CN 101692023 B CN101692023 B CN 101692023B CN 200910144974 CN200910144974 CN 200910144974 CN 200910144974 A CN200910144974 A CN 200910144974A CN 101692023 B CN101692023 B CN 101692023B
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Abstract
The invention discloses a chemical preparing method of molecular recognition of ultratrace explosive TNT molecule based on fluorescence resonance energy transfer, comprising: taking silica ball-shaped nanoparticles as a supporting body; decorating the surface with aminopropyl and 6-FAM hydroxysuccinimide ester; and causing the surface to have special functional group. The invention has the following two steps in the preparing process: firstly, forming a coupled body by covalent coupling of a reacted monomer APTS and fluorescein C1609 at certain reaction condition; then, taking ammonia water as catalyst to hydrolyze and condensate TEOS to obtain silica particles; and finally, mixing the coupling containing excessive APTS with the silica nanoparticles and reacting so as to prepare silica nanoparticles which have expertise identification function to TNT. The silica nanoparticle prepared with the method has large specific surface area and can form a nano-structure probe with high density identification locus, favourable selectivity, favourable sensitiveness and molecular recognition performance.
Description
Technical field the present invention relates to material science, particularly has based on the chemical preparation process of FRET (fluorescence resonance energy transfer) to ultratrace explosive TNT molecular recognition.
Background technology is from the angle of social safety, nitroaromatic has the high explosivity and the harmfulness of environment, therefore, in recent years, caused that in exploration society research mechanism pays close attention to and fruitful exploration widely to the detection of ultratrace nitroaromatic and relevant sensor array.The laboratory of specific nitryl aromatic family's explosive and vapor signal thereof is surveyed and is carried out widely by the method for gas chromatograph-mass spectrometer, ion mobility spectrometry and neutron activity analysis etc.These traditional analysis technology can satisfy the basic demand in the analysis, as selectivity, reliability, accuracy and repeatability, but these detection methods are expensive, consuming time and loaded down with trivial details heaviness, because sample must be to break away from the detection scene to be sent to the laboratory and to go to analyze in detecting, can not accomplish the detection of real-time on-site.In sum, be necessary to seek a kind of can be fast and detect the method for TNT easily.In application facet, fluorescence molecule is a desirable sensitive material of constructing nano-scale structures.And the high surface area of nano silicon, the surface is easy to functionalization, is desirable nano fluorescent sensor carrier material.In order to solve the explosive detection problem, need urgently chemical sensor can provide the target analytes in liquid phase and the vapour phase environment a kind of high selectivity, high response, high responsive, fast, low cost and in-situ investigation.
In various signal transducers, the optically addressable sensor of " opening " mechanism based on fluorescence " pass " or fluorescence has been proved to be researcher's method that expectation detects various micromolecule target analytes in the environment of many challenges, because the high signal output and the reliable result of this detection method.It is extremely beneficial that the chemical sensor of fluorescence " pass " mechanism detects with fluorescent method nitroaromatic.The nitryl aromatic explosive that has the aromatic rings of short of electricity is the acceptor of an electronics, and the fluorescent material surface that so just demonstrates electron rich has very high affinity.This photoluminescence is at electron accepter with to passing through the direct cancellation of electron transfer π compound mechanism between the body, and this cancellation mainly depends on the electronic capability of accepting of nitro aromatic thing.Recently, U.S. Swager scientific research group surveys the nitro-aromatic compound steam of ultratrace with the semiconductive organic polymer that has very high fluorescence quantum yield and has manifested a series of examples of many successful.Be coated with on the surface of optical fiber and spread conducting polymer, aromatic nitro explosive detection signal has been amplified 30 times.Meanwhile, other photoluminescence polymer material is as poly-tetraphenyl silane, and the porous silica microballoon of poly-tetraphenyl germane and physisorption dyestuff all has been proved to be the effective sensor to the nitryl aromatic explosive steam detection of substandard.Recently, U.S. swager research group report fluorescence " is opened " chemical sensor by being that the reduction reaction of media is carried out the direct detection method to non-aromatic nitro compound hexogen (RDX) and pentaerythritol tetranitrate (PETN) with the organized enzyme.
Be exactly that semiconductor-quantum-point replaces fluorescein as one of alternative method in these methods at present, because it has very high-fluorescence quantum yield, fluorescence semiconductor quantum dots mixes Nano particles of silicon dioxide and has shown for optics for basic chemical/biological sensors detects great potential is arranged recently.Development will provide a kind of novel strategy that has more the chemical/biological sensors of dirigibility in " laboratory on the nano particle " technology, the strategy of this dirigibility allows optics can be in harmonious proportion the assembling of specific complex and acceptor, it is better contact target analyte that high-specific surface area can be provided, and has the assembling ability to various sensors simultaneously.On principle, can be by two components of covalency coupling connection based on the sensor of nano particle: a kind of be identification acceptor combining target molecule and another kind of sensor (chromophoric group), the i.e. acceptor of signal.Most typical is exactly that people such as U.S. Goldman has reported recently and is used between quantum dot part antibody and the organic dyestuff based on the FRET (fluorescence resonance energy transfer) chemical/biological sensors.This sensor that maltose and enzymatic activity are had a specific measuring ability is successfully prepared.
In recent years, be carrier with the Nano particles of silicon dioxide, modify fluorophor has attracted large quantities of researchers as probe interest.The patent of invention (US2006183246) of Wiesner scientific research group in 2006 discloses " Fluorescent silica-based nanoparticles " preparation method.It has adopted following step: (1) dyestuff TRITC is the core of core-shell type nanoparticles; (2) being the presoma of reaction with the ethyl orthosilicate, is that the particle diameter that Preparation of Catalyst goes out core-shell-type is a 30nm fluorescent silicon dioxide nano particle with ammoniacal liquor.The patent of invention (WO2007074722) of Aizawa Hideki scientific research group in 2007 discloses " Fluorescentsilica nano-particle; fluorescent nano-material; biochip using the material, andassay method " preparation method.It has adopted following step: (1) is the presoma of reaction with the ethyl orthosilicate, is 30nm or less than Cy3 and Cy5 fluorescent dye on the fluorescent silicon dioxide nano-sized surface modification of 30nm, this fluorescent silicon dioxide particle can be used as biochip and come the detection of biological molecule with ammoniacal liquor for the Preparation of Catalyst particle diameter.
But the Nano particles of silicon dioxide of these core-shell-types with finishing the fluorescent silicon dioxide nano particle selectivity of fluorescent dye TRITC, Cy3 and Cy5 relative relatively poor, susceptibility is poor, because the group of special knowledge is not carried on its surface.Therefore, the fluorescent silicon dioxide nano-probe preparation method of synthetic high selectivity and hypersensitivity realizes that ultratrace TNT molecular recognition is had its necessity.
In the present invention, we have reported in the detection to ultratrace TNT in solution and gas phase of a kind of fluorescence resonance energy resonance transfer strategy on Nano particles of silicon dioxide surface.Nano particles of silicon dioxide is especially to be suitable as the fluorescence chemical sensor supporter because Nano particles of silicon dioxide optical clear and photon physics inertia with and surface energy enough be easy to subunit by modifying new assembling function with the silylating reagent condensation reaction.The aminopropyl on Nano particles of silicon dioxide surface can pass through to form charge-transfer complex with the TNT target molecule, owing to there is electron delocalization, this charge-transfer complex has shown a very strong visible absorbance peak in 500-600nm wavelength coverage class.This result's discovery excites us to explore based on the detection of FRET (fluorescence resonance energy transfer) chemical sensor to the special knowledge of TNT target molecule by covalent cross-linking 3-aminopropyl triethoxysilane (APTS) and 6-Fluoresceincarboxylic acid succinimide ester (C1609) on the Nano particles of silicon dioxide surface.When the absorption spectrum of the ultraviolet-visible of selecting its fluorescence emission spectrum that has of suitable luminescent dye molecule and APTS-TNT compound coincides, its fluorescence resonance will be absorbed by the APTS-TNT compound, therefore, the photoluminescence of Nano particles of silicon dioxide will be by effective cancellation.Since have on the Nano particles of silicon dioxide surface many to TNT in conjunction with the very strong aminopropyl subunit of susceptibility, will cause so optionally TNT target molecule in the environment being formed response fast.And do not see bibliographical information about aminopropyl in the Nano particles of silicon dioxide finishing and 6-Fluoresceincarboxylic acid succinimide ester at present.This finishing the Nano particles of silicon dioxide of APTS and C1609 detect nanomolar concentration level TNT molecule and several ppb levels in gas phase by FRET (fluorescence resonance energy transfer) selectivity in solution.This have the fluorescent silicon dioxide nano particle of special knowledge effect to the TNT molecule, is that recognition site shows the detection to TNT high selectivity, high sensitivity and ultratrace with fluorescence as probe, aminopropyl.
Summary of the invention goal of the invention: at the weak point of present prior art existence, the present invention utilizes nano silicon to synthesize as supporter to have a kind of based on the chemical preparation process of FRET (fluorescence resonance energy transfer) to ultratrace explosive TNT molecular recognition of aminopropyl and fluorophor first, and first nano-silica surface is carried out functional modification and be used for ultratrace explosive TNT molecular recognition.Described method is a chemical synthesis, and at first Zhi Bei single dispersion gel Nano particles of silicon dioxide carries out the aminopropyl and the modification of 6-Fluoresceincarboxylic acid succinimide ester on surface, makes its functionalization.
Technical scheme of the present invention is: a kind of based on the chemical preparation process of FRET (fluorescence resonance energy transfer) to ultratrace explosive TNT molecular recognition, comprise with the spherical nano particle of silicon dioxide and make supporter, aminopropyl and 6-Fluoresceincarboxylic acid succinimide ester is characterized in that in its finishing; The electron rich aminopropyl is with shifting formation peony compound by electric charge between the sub-TNT target molecule of short of electricity in the spherical nano particle of the silicon dioxide of described modification, this compound has maximum visible absorbance peak at the 525nm place, and the 6-Fluoresceincarboxylic acid succinimide ester steady-state fluorescence emission spectrum on surface just in time overlaps, according to the FRET (fluorescence resonance energy transfer) principle, just in time can be absorbed by the compound of the TNT of this wavelength, decline by fluorescence intensity, thereby realize that preparation process of the present invention comprises following two steps to the TNT molecular recognition:
1.1 with 3-aminopropyl triethoxysilane (3-aminopropyltriethoxysilane, APTS) aminopropyl in and 6-Fluoresceincarboxylic acid succinimide ester (6-Carboxyfluorescein N-succinimideester, C1609) succinimide ester in is coupled on the silylating reagent APTS with the covalent bond form by nucleophilic substitution.Typical synthetic method is in the ethanol solution that is distributed to 10mL of the dyestuff C1609 of APTS that contains excessive 0.5mL and 2.4mg, with 250rpm rotating speed lucifuge successive reaction 12h at room temperature relatively slowly, obtain the reaction product of bright orange green with the constant temperature rocking bed;
1.2 the reaction of second step is the single neck ground flask that the absolute ethyl alcohol of 89.1mL is placed 500mL, 3.4mL TEOS be added in the ethanolic solution rapidly, under 500rpm, stir 3min, then the ammoniacal liquor of 10.9mL is joined in the above-mentioned reaction solution, and rapidly strong stir three minutes with 750rpm after, speed of agitator is reduced to 500rpm, at room temperature reacts 18h, and obtaining particle diameter approximately is the silicon oxide particle solution of 200nm.The reaction product that the resultant fluorescent dye C1609 of reaction is coupled to the bright orange green on the APTS joins the synthetic back of reaction particle diameter together together with 50 μ L TEOS and is approximately in the above-mentioned reaction solution of 200nm and goes, and at room temperature stirs lucifuge reaction 24h with the 500rpm rotating speed.
It is inferior that centrifugal, the ultrasonic dispersion of the product of above-mentioned gained is given a baby a bath on the third day after its birth with absolute ethyl alcohol, is distributed in the ethanol again again.Finally obtain the Nano particles of silicon dioxide of C1609, APTS end-blocking, had the fluorescent silicon dioxide nano particle probe that TNT high selectivity, hypersensitivity and ultratrace are surveyed.
As a further improvement of existing technologies, said supporter is a nano-silicon dioxide particle, and its particle diameter is controlled can be controlled by the ratio between the amount of conditioned reaction thing; Aminopropyl and fluorescein base group are rich in surperficial modified its surface that makes of the spherical nano particle supporter of said silicon dioxide; Said silylating reagent is: the 3-aminopropyl triethoxysilane.Said fluorescein is a 6-Fluoresceincarboxylic acid succinimide ester.The said molecule that is identified is TNT.The aminopropyl on said Nano particles of silicon dioxide surface specially the property known in conjunction with the TNT target molecule.
Beneficial effect with respect to prior art:
One: with respect to prior art, the patent of invention (US2006183246) of Wiesner scientific research group in 2006 discloses " Fluorescent silica-based nanoparticles " preparation method.It has adopted following step: (1) dyestuff TRITC is the core of core-shell type nanoparticles; (2) being the presoma of reaction with the ethyl orthosilicate, is that the particle diameter that Preparation of Catalyst goes out core-shell-type is a 30nm fluorescent silicon dioxide nano particle with ammoniacal liquor.The patent of invention (WO2007074722) of Aizawa Hideki scientific research group in 2007 discloses " Fluorescent silica nano-particle; fluorescentnano-material; biochip using the material, and assay method " preparation method.It has adopted following step: (1) is the presoma of reaction with the ethyl orthosilicate, is 30nm or less than Cy3 and Cy5 fluorescent dye on the fluorescent silicon dioxide nano-sized surface modification of 30nm, this fluorescent silicon dioxide particle can be used as biochip and come the detection of biological molecule with ammoniacal liquor for the Preparation of Catalyst particle diameter.This fluorescent nano particles does not have the specially recognition site of the property known, and poor selectivity does not relate to FRET (fluorescence resonance energy transfer) mechanism, and the fluorophor of modification is not by on the covalent bonding, the easy wash-out of fluorescence molecule, instability.
It is that the spherical nano particle of silicon dioxide of 200nm is a supporter as template that the present invention has adopted mean grain size, modify aminopropyl and fluorescein C1609 by bonding on its surface, the spherical nano particle of silicon dioxide after the modification has the electron rich functional group-aminopropyl of special knowledge, preparation process of the present invention comprises two steps: at first, the 3-aminopropyl triethoxysilane of 0.5mL and the 6-Fluoresceincarboxylic acid succinimide ester of 2.4mg are dispersed in the absolute ethyl alcohol of 10mL, and aminopropyl and the succinimide ester among the C1609 among the APTS are coupled on the silylating reagent APTS with the covalent bond form by nucleophilic substitution.
Then, it is presoma that above-mentioned reaction solution is added the TEOS that contains 3.4mL together together with the TEOS of 50 μ L, 10.9mL ammoniacal liquor be that the absolute ethyl alcohol of catalyzer and 89.1mL is that synthetic particle diameter is approximately in the mixed liquor of 200nm Nano particles of silicon dioxide after the reaction of solvent, at room temperature stir lucifuge reaction 24h with the 500rpm rotating speed.
It is inferior that centrifugal, the ultrasonic dispersion of the product of above-mentioned gained is given a baby a bath on the third day after its birth with absolute ethyl alcohol, is distributed in the ethanol again again.Obtain the surperficial Nano particles of silicon dioxide that is rich in aminopropyl and fluorescein, prepared the fluorescent silicon dioxide nano particle probe that the TNT target molecule is had high selectivity, high sensitivity and ultratrace detection.
In sum, the nano molecular fluorescence probe especially has the fluorescence probe of molecular recognition group at nano-silica surface, both increased the specific surface area of namo fluorescence probe, and having has increased the molecular recognition site, improves selectivity, identity and susceptibility.
Its two: finishing the Nano particles of silicon dioxide of aminopropyl and fluorescein C1609 molecule, can be to the TNT molecular recognition.For example, add a certain amount of TNT target molecule and can form bolarious compound with the aminopropyl of silica surface, this compound absorbs the emission light of silica surface fluorescein C1609, descends by fluorescence intensity, realizes the TNT molecular recognition.Synthetic surface is rich in the Nano particles of silicon dioxide ball of aminopropyl and fluorescein, also can selectivity be used for detecting other nitro explosive molecules simultaneously, as 2, and 4-dinitrotoluene (DNT) (DNT), nitrobenzene (NB) and hexogen (RDX).And the fluorescein among the present invention is a 6-Fluoresceincarboxylic acid succinimide ester.As seen, method provided by the present invention is general, and usage range is more extensive.
Its three: compare with traditional fluorescent molecular probe, finishing Nano particles of silicon dioxide molecular probe has bigger specific surface area, more recognition site, high selectivity, hypersensitivity improves the molecular recognition performance.Fig. 9 be among the present invention nano particle chip cancellation percentage along with comparison (B) at variation function relation figure (A) with the nano particle of the having modified different fluorescent dyes cancellation percentage function of time in the TNT saturated vapour of target analytes saturated vapour time.Wherein Fig. 9 (A) has modified the Nano particles of silicon dioxide of C1609 to different target analytes saturated vapour testing results, and wherein cancellation percentage is maximum under the identical condition of TNT concentration.Fig. 9 (B) has modified the silicon dioxide granule of C1609 and has modified the comparison of the silicon dioxide of 6-Carboxy-X-rhodamine N-succinimidyl ester (ROX) to TNT cancellation percentage, as can be seen finishing the silicon dioxide granule of C1609 to be better than the susceptibility of ROX from far away to the susceptibility of TNT.
Its four: in the method provided by the present invention, the controlled Nano particles of silicon dioxide of particle diameter can be controlled by the ratio between the conditioned reaction thing amount.Promptly can control, therefore, control the particle diameter of Nano particles of silicon dioxide by the amount of regulating ammoniacal liquor and TEOS with the particle diameter of the silica fluorescent nanoparticle of function recognition group.
Its five: select the colloidal sol nano SiO 2 particle as the spherical template purpose because it has the following advantages: (1) synthetic easily colloidal sol monox nanometer particle, its particle size range can be from 5nm-2000nm, bigger surface area, relatively low cost; (2) stability that has chemistry and heat in course of reaction is not reacted with organic solvent; (3) the easy grafting organo-functional group in surface; (4) environmental sound.
Description of drawings
The synthetic synoptic diagram of the Nano particles of silicon dioxide of Fig. 1 has been finishing of the present invention APTS and C1609.
Fig. 2 is that APTS of the present invention and TNT target molecule shift red complex (A) and the normalization ultraviolet-visible light spectrogram of APTS-TNT red complex and the steady-state fluorescence emission spectrum figure (B) of C1609 that interacts and form by electric charge in solution.
Fig. 3 is that the Nano particles of silicon dioxide of aminopropyl and C1609 is rich in to TNT target molecule detection principle schematic in surface of the present invention.
Fig. 4 is sem photograph (A) and the perspective Electronic Speculum figure (B) that the Nano particles of silicon dioxide of aminopropyl and C1609 is rich on surface of the present invention.
The ultraviolet-visible light spectrogram (a) and the steady-state fluorescence spectrogram (b) thereof of the Nano particles of silicon dioxide of Fig. 5 aminopropyl that has been the resulting finishing of the present invention and C1609.
Fig. 6 is the Nano particles of silicon dioxide and the comparison diagram of its pure dye to the fluorescent quenching constant of different nitro compounds of the different fluorescent dye of the resulting modification of the present invention.
Fig. 7 is the brightness of TNT solution phosphor dot to the chip of C1609-APTS-silica nano particle assembling of the resulting dropping 10 μ L variable concentrations of the present invention and the fluorescence co-focusing microphotograph of change in size.
Fig. 8 be among the present invention fluorescent nano particles with the variation (A) and its variation (B) of the increase fluorescence intensity of TNT concentration to the cancellation percentage of different analyte concentrations.
Fig. 9 be among the present invention nano particle chip cancellation percentage along with comparison (B) at variation function relation figure (A) with the nano particle of the having modified different fluorescent dyes cancellation percentage function of time in the TNT saturated vapour of target analytes saturated vapour time.
Further explain embodiment with reference to the accompanying drawings
The synthetic synoptic diagram of the Nano particles of silicon dioxide of Fig. 1 has been finishing of the present invention APTS and C1609.At first APTS and C1609 carry out covalency coupling connection in Fig. 1, and aminopropyl and fluorescein C1609 on silica dioxide gel is nanoparticle surface modified have obtained the fluorescent silicon dioxide nano particle then.
The Nano particles of silicon dioxide surface must be modified, and makes the surface be with the particular functional group, so that the Nano particles of silicon dioxide surface has recognition site.What the present invention adopted is at pure silicon dioxide nanoparticle surface covalency coupling hydrazine propyl group and fluorescein C1609, make its surface coverage aminopropyl and fluorescein C1609, the target analytes that helps short of electricity like this forms compound with the electron rich aminopropyl on the Nano particles of silicon dioxide surface;
At first be that silylating reagent APTS and fluorescein C1609 are joined by the covalency coupling under the certain reaction condition, it is conjuncted to form coupling.Then, be that catalyzer hydrolysis and condensation TEOS obtain silicon dioxide granule with ammoniacal liquor, at last that coupling is conjuncted with the mixed liquid reaction of the Nano particles of silicon dioxide that makes, thus preparation has the fluorescent silicon dioxide nano particle of special knowledge recognition reaction to TNT.
Fig. 2 is that APTS of the present invention and TNT target molecule shift red complex (A) and the normalization ultraviolet-visible light spectrogram of APTS-TNT red complex and the steady-state fluorescence emission spectrum figure (B) of fluorescein C1609 that interacts and form by electric charge in solution.The APTS-TNT red complex has maximum visible absorbance peak at the 525nm place, the absorption of its compound is just in time with the steady-state fluorescence emission spectrum figure overlaid of fluorescein C1609, according to FRET (fluorescence resonance energy transfer), light positive is good is absorbed by the APTS-TNT red complex for the emission of the steady-state fluorescence of fluorescein C1609, make and realize the decline of C1609 fluorescence intensity the TNT molecular recognition.
Fig. 3 is that the Nano particles of silicon dioxide that aminopropyl and C1609 are rich in surface of the present invention detects principle schematic to target molecule.Under the condition that the TNT target molecule exists, form red compound by the electric charge transferance between the sub-TNT target molecule of the aminopropyl of fluorescent silicon dioxide particle surface electron rich and short of electricity, according to FRET (fluorescence resonance energy transfer), light positive is good is absorbed by the APTS-TNT red complex for the emission of the steady-state fluorescence of fluorescein C1609, make the change of C1609 fluorescence intensity, thereby make fluorescent quenching, realize the TNT molecular recognition.
Fig. 4 is sem photograph (A) and the transmission electron microscope picture (B) that the Nano particles of silicon dioxide of aminopropyl and fluorescein is rich on surface of the present invention.Owing to adopt two-step reaction, at first, by covalency coupling hydrazine propyl group and fluorescein, then, preparation pure silicon dioxide nano particle obtains both hybrid reactions scanning electron microscope and the transmission electron microscope photo that the Nano particles of silicon dioxide of aminopropyl and fluorescein is rich on the surface at last again.
The ultraviolet-visible light spectrogram (a) and the steady-state fluorescence spectrogram (b) thereof of the Nano particles of silicon dioxide of Fig. 5 aminopropyl that has been the resulting finishing of the present invention and C1609.Absorb spectrogram from ultraviolet-visible and can be clearly seen that the absorption peak of the Nano particles of silicon dioxide of the fluorescein in the modification, also clearly see its maximum emission intensity from the steady-state fluorescence emission spectrum simultaneously at 518nm at 502nm.
Fig. 6 is the Nano particles of silicon dioxide and the comparison diagram of pure dye to the fluorescent quenching constant of different nitro compounds that the resulting different fluorescent dyes of the present invention are modified.As can be seen from the figure modified the cancellation constant maximum of the Nano particles of silicon dioxide of aminopropyl and C1609 dyestuff to TNT, selectivity and susceptibility are remarkable.
Fig. 7 is the fluorescence co-focusing microphotograph of the variation of the brightness of TNT solution phosphor dot to the chip of C1609-APTS-silica nano particle assembling of the resulting dropping 10 μ L variable concentrations of the present invention and size.From figure, can clearly find out the molecular chip array of increase fluorescent silicon dioxide nanoparticle intensity of fluorescence reduction gradually in the drawings along with TNT concentration.
Fig. 8 be among the present invention fluorescent nano particles with the variation (A) of the increase fluorescence intensity of TNT concentration and to the variation (B) of the cancellation percentage of different analyte concentrations.This as can be seen Nano particles of silicon dioxide ball of having modified fluorescein can be low to moderate 10 to the detection of TNT from Fig. 8 A
-9Mol L
-1From Fig. 8 B the surface this nano particle be limited to minimum to the detection of TNT.
Fig. 9 be among the present invention nano particle chip cancellation percentage along with comparison (B) at variation function relation figure (A) with the nano particle of the having modified different fluorescent dyes cancellation percentage function of time in the TNT saturated vapour of target analytes saturated vapour time.Wherein Fig. 9 A has modified the Nano particles of silicon dioxide of C1609 to different target analytes saturated vapour testing results, and wherein cancellation percentage is maximum under the identical condition of TNT concentration.Fig. 9 B has modified the silicon dioxide granule of fluorescein C1609 and has modified the comparison of the silicon dioxide of fluorescein ROX to TNT cancellation percentage, as can be seen finishing the silicon dioxide granule of C1609 to be better than the susceptibility of ROX from far away to the susceptibility of TNT.
Embodiment: a kind of based on the chemical preparation process of FRET (fluorescence resonance energy transfer) to ultratrace explosive TNT molecular recognition, comprise with the spherical nano particle of silicon dioxide as supporter, aminopropyl and 6-Fluoresceincarboxylic acid succinimide ester in its finishing, it is characterized in that: the spherical nanoparticle surface of described silicon dioxide has aminopropyl and 6-Fluoresceincarboxylic acid succinimide ester, its surperficial aminopropyl can be further and the effect of TNT target molecule, the aminopropyl that makes its surperficial electron rich is as forming wine-colored compound by the electric charge transferance between the TNT acceptor of electron donor with short of electricity, this peony compound, can absorb the fluorescence of the spherical nanoparticle surface 6-of silicon dioxide Fluoresceincarboxylic acid succinimide ester by FRET (fluorescence resonance energy transfer), utilize the change of fluorescence intensity, realization is to ultratrace explosive TNT molecular recognition, and preparation process of the present invention comprises following two steps:
The first step, be that aminopropyl in the 3-aminopropyl triethoxysilane and 6-Fluoresceincarboxylic acid succinimide ester are coupled on the silylating reagent 3-aminopropyl triethoxysilane with the covalent bond form by addition reaction, typical synthetic method is in the ethanol solution that is distributed to 10mL～20mL of the 6-Fluoresceincarboxylic acid succinimide ester of the 3-aminopropyl triethoxysilane that contains excessive 0.5mL～1mL and 2.0mg～10mg, with the constant temperature rocking bed with 100rpm～300rpm rotating speed lucifuge successive reaction 12h～24h at room temperature relatively slowly;
Second step, reaction is the single neck ground flask that the absolute ethyl alcohol of 50mL～90mL is placed 500mL, the ethyl orthosilicate of 1mL～3.6mL (Tetraethyl orthosilicate, TEOS) be added in the ethanolic solution rapidly, under 500rpm, stir 3min～10min, then the ammoniacal liquor of 1mL～15mL is joined in the above-mentioned reaction solution, and rapidly strong stir three minutes with 500rpm～750rpm after, speed of agitator is reduced to 400rpm～600rpm, at room temperature react 15h～24h, in the 10mL～20mL ethanol solution of the 6-Fluoresceincarboxylic acid succinimide ester of the 3-aminopropyl triethoxysilane that contains excessive 0.5mL～1mL and 2.0mg～10mg, react reaction product that resultant 6-Fluoresceincarboxylic acid succinimide ester is coupled to the bright orange green on the 3-aminopropyl triethoxysilane and join reaction together together with 10 μ L～50 μ L TEOS and obtain after synthetic going in the above-mentioned reaction solution of Nano particles of silicon dioxide, at room temperature stir lucifuge reaction 15h～24h with 400rpm～600rpm rotating speed.
The Nano particles of silicon dioxide that has aminopropyl and fluorescein of above-mentioned gained is centrifugal, giving a baby a bath on the third day after its birth with the ultrasonic dispersion of absolute ethyl alcohol time is distributed in the ethanol more again, finally obtain the Nano particles of silicon dioxide of aminopropyl and fluorescein C1609 end-blocking, had the fluorescent silicon dioxide nano particle probe that TNT high selectivity, extremely sensitive and ultratrace are surveyed.
Add target molecule in the Nano particles of silicon dioxide of finishing, can change its recognition performance.As adding the TNT molecule, the amino on Nano particles of silicon dioxide surface and TNT form compound, absorb the fluorescein institute emitted fluorescence of silica particle surface, thereby obtain the molecular recognition to TNT.
Embodiment: according to the covalency coupled reaction, utilize nano silicon, adopt two-step reaction can obtain the Nano particles of silicon dioxide that aminopropyl and fluorescein are rich in the surface as supporter.
1, the coupled reaction of APTS and fluorescein C1609: in the ethanol solution that is distributed to 10mL of the dyestuff C1609 of APTS that contains excessive 0.5mL and 2.4mg, with the constant temperature rocking bed with 250rpm rotating speed lucifuge successive reaction 12h at room temperature relatively slowly;
2, modified the silica dioxide gel nano particle preparation of aminopropyl and fluorescein C1609: the single neck ground flask that the absolute ethyl alcohol of 89.1 mL is placed 500mL, 3.4mL TEOS be added in the ethanolic solution rapidly, under 500rpm, stir 3min, then the ammoniacal liquor of 10.9mL is joined in the above-mentioned reaction solution, and rapidly strong stir three minutes with 750rpm after, speed of agitator is reduced to 500rpm, at room temperature reacts 18h.In the 10mL ethanol solution that contains excessive 0.5mL APTS and 2.4mg C1609, react reaction product that resultant fluorescent dye C 1609 is coupled to the bright orange green on the APTS and join the synthetic back of reaction particle diameter together together with 50 μ LTEOS and be approximately in the above-mentioned reaction solution of 200nm and go, at room temperature stir lucifuge reaction 24h with the 500rpm rotating speed.The modification of comparative dye ROX is similar to dyestuff C1609.
It is inferior that centrifugal, the ultrasonic dispersion of the product of above-mentioned gained is given a baby a bath on the third day after its birth with absolute ethyl alcohol, is distributed in the ethanol again again.Finally obtain the Nano particles of silicon dioxide of C1609, APTS end-blocking, had the fluorescent silicon dioxide nano particle probe that TNT high selectivity, hypersensitivity and ultratrace are surveyed.
Claims (3)
1. one kind based on the chemical preparation process of FRET (fluorescence resonance energy transfer) to ultratrace explosive TNT molecular recognition, comprise with the spherical nano particle of silicon dioxide as supporter, aminopropyl and 6-Fluoresceincarboxylic acid succinimide ester in its finishing, it is characterized in that: the spherical nanoparticle surface of described silicon dioxide has aminopropyl and 6-Fluoresceincarboxylic acid succinimide ester, its surperficial aminopropyl can be further and the effect of TNT target molecule, the aminopropyl that makes its surperficial electron rich is as forming wine-colored compound by the electric charge transferance between the TNT acceptor of electron donor with short of electricity, this peony compound can absorb the fluorescence of the spherical nanoparticle surface 6-of silicon dioxide Fluoresceincarboxylic acid succinimide ester by FRET (fluorescence resonance energy transfer), utilize the change of fluorescence intensity, realization is to ultratrace explosive TNT molecular recognition, and this preparation process comprises following two steps:
1.1 first step reaction is that the succinimide ester in aminopropyl in the 3-aminopropyl triethoxysilane and the 6-Fluoresceincarboxylic acid succinimide ester is passed through nucleophilic substitution, with the covalent bond form 6-Fluoresceincarboxylic acid succinimide ester is coupled on the silylating reagent 3-aminopropyl triethoxysilane, its synthetic method is in the ethanol solution that is distributed to 10mL～20mL of the 6-Fluoresceincarboxylic acid succinimide ester of the 3-aminopropyl triethoxysilane that contains excessive 0.5mL～1mL and 2.0mg～10mg, with 100rpm～300rpm rotating speed lucifuge successive reaction 12h～24h at room temperature relatively slowly, obtain the reaction product that 6-Fluoresceincarboxylic acid succinimide ester is coupled to the bright orange green on the 3-aminopropyl triethoxysilane with the constant temperature rocking bed;
1.2 the reaction of second step is the single neck ground flask that the absolute ethyl alcohol of 50mL～90mL is placed 500mL, the ethyl orthosilicate of 1mL～3.6mL (Tetraethyl orthosilicate, TEOS) be added in the ethanolic solution rapidly, under 500rpm, stir 3min～10min, then the ammoniacal liquor of 1mL～15mL is joined in the above-mentioned reaction solution, and rapidly strong stir three minutes with 500rpm～750rpm after, speed of agitator is reduced to 400rpm～600rpm, at room temperature react 15h～24h, obtain the spherical nano particle of silicon dioxide, the first step is reacted the reaction product that resultant 6-Fluoresceincarboxylic acid succinimide ester is coupled to the bright orange green on the 3-aminopropyl triethoxysilane, join the second step reaction together together with 10 μ L～50 μ L ethyl orthosilicates and obtain after synthetic going in the reaction solution of the spherical nano particle of silicon dioxide, at room temperature stir lucifuge reaction 15h～24h with 400rpm～600rpm rotating speed;
The spherical nano particle of the silicon dioxide that has aminopropyl and fluorescein of gained is centrifugal, giving a baby a bath on the third day after its birth with the ultrasonic dispersion of absolute ethyl alcohol inferiorly is distributed in the ethanol more again, finally obtained the spherical nano particle of silicon dioxide of fluorescein and aminopropyl end-blocking, the spherical nano particle of this silicon dioxide has TNT high selectivity, extremely sensitive and ultratrace detection.
2. according to claim 1 a kind of based on the chemical preparation process of FRET (fluorescence resonance energy transfer) to ultratrace explosive TNT molecular recognition, it is characterized in that: said supporter is the spherical nano particle of silicon dioxide, and its particle diameter is controlled can be controlled by the ratio between the amount of conditioned reaction thing.
3. according to claim 1 a kind of based on the chemical preparation process of FRET (fluorescence resonance energy transfer) to ultratrace explosive TNT molecular recognition, it is characterized in that: the aminopropyl of the spherical nanoparticle surface of said silicon dioxide specially the property known in conjunction with the TNT molecule.
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