CN109490261B

CN109490261B - Efficient porous superparamagnetic fluorine ion dual-fluorescence detection probe and preparation method thereof

Info

Publication number: CN109490261B
Application number: CN201811125562.3A
Authority: CN
Inventors: 庄琳; 李晓磊; 许跃; 刘伍明; 王玮; 郑昂
Original assignee: Sun Yat Sen University
Current assignee: Sun Yat Sen University
Priority date: 2018-09-26
Filing date: 2018-09-26
Publication date: 2021-12-24
Anticipated expiration: 2038-09-26
Also published as: CN109490261A

Abstract

The invention discloses a high-efficiency porous superparamagnetic fluorine ion dual-fluorescence detection probe and a preparation method thereof. The probe comprises an autofluorescent marker and a quenched fluorescent probe; the autofluorescence marker is mesoporous silicon dioxide containing organosilane functionalized carbon points, and the quenching fluorescence probe is a chelate of pure carbon points and nickel ions. The probe has double fluorophores, can be repeatedly used, can well monitor the real-time concentration of fluorine ions in a water environment, has the lowest detection limit of F ions as low as 65nM, has the linear response range of 1-25 MuM, and has good sensitivity and selectivity; meanwhile, the fluorine ions can be efficiently adsorbed and removed, the effective rate of removing the fluorine ions in tap water is up to 96%, and the fluorine ions can be repeatedly recycled; and the method has excellent biocompatibility, good cell permeability and low cytotoxicity, shows great potential in the direction of detecting the fluorine ions in the intracellular and the external environments, is green and environment-friendly, and has wide application prospect.

Description

Efficient porous superparamagnetic fluorine ion dual-fluorescence detection probe and preparation method thereof

Technical Field

The invention belongs to the technical field of functional nano materials. More particularly, relates to a high-efficiency porous superparamagnetic fluorine ion dual-fluorescence detection probe and a preparation method thereof.

Background

Fluoride ion (F)^-) Widely distributed in environmental system and biological process, the proper amount of fluorine ion intake is beneficial to the health of teeth and bones, but too much or too little fluorine ion intake has adverse effect on human body, and systemic diseases such as fluorosis and osteoporosis can be causedAnd the like. Therefore, accurate detection and control of fluoride ion concentration in water resources is of great importance to humans.

Various efforts have been attempted by the scientific community to develop new methods and strategies for detecting fluoride ions, including photophysical mechanisms and fluoride-induced chemical reaction mechanisms. Most of the photophysical mechanism methods can sensitively respond to the deprotonation fluorescence transduction of fluorine ions, and the sensing probes are applied by matching with organic macromolecules, such as anthracene and pyrene substances with potential toxicity and organic solvent dependence. Meanwhile, interference of acid ions is inevitable, and the selectivity of the probe will be seriously affected. The chemical reaction mechanism method induced by high-selectivity fluoride is only researched on the basis of a small organic molecule probe, and the final aim is to enhance the luminescence of a luminophor. However, most fluoride-induced chemical probes are applied in organic media and cannot be used for detection of fluoride ions in tap water. Thus, the water-insolubility, non-regenerability and biotoxicity of the above-mentioned chemical reaction mechanism probes limit their practical application in industrial manufacturing and environmental systems.

Carbon quantum dots (CDs) are an attractive alternative to conventional organic molecules in the imaging field due to their small size, good biocompatibility, good water solubility, and rich functionality. At present, a large number of experiments are focused on exploring the use of CDs as detection probes. However, the currently reported probes have the disadvantages of potential biological toxicity, low specific surface area, unsatisfactory dispersibility and difficult application to practical use by naked eyes, and most of the probes are single fluorophores, the probes have weak fluorescence and low selectivity and sensitivity to metal ions, and the application of the probes in the ecological environment is questionable and influences further application in real life.

In order to achieve effective sensitivity and practical application in biological media, a fluorine ion detection probe which is easy to identify, has good luminescence characteristics in the visible spectrum, and has high sensitivity and selectivity, excellent dispersibility and low toxicity is urgently needed.

Disclosure of Invention

The technical problem to be solved by the present invention is to overcome the defects and shortcomings of the prior art, and to provide a highly efficient, reusable and double fluorophore compounded superparamagnetic fluorine ion double fluorescence detection probe, which has excellent dispersibility, excellent stability and excellent biocompatibility, good selectivity and high sensitivity, and can be used for detecting and removing fluorine ions in tap water.

The invention aims to provide a high-efficiency porous superparamagnetic fluorine ion dual-fluorescence detection probe.

The invention also aims to provide a preparation method of the fluorine ion dual-fluorescence detection probe.

The above purpose of the invention is realized by the following technical scheme:

a high-efficiency porous superparamagnetic fluorine ion dual-fluorescence detection probe comprises an autofluorescence marker and a quenching fluorescence probe; the autofluorescence marker is mesoporous silicon dioxide containing organosilane functionalized carbon points, and the quenching fluorescence probe is a chelate of pure carbon points and nickel ions.

The invention creatively takes a mesoporous silicon dioxide layer fixed with carbon quantum dots (SiCDs) as an autofluorescent marker, and simultaneously takes fluorophore pure carbon dots (NCDs) and F ion acceptor nickel ions (Ni)²⁺) The chelate is a quenching fluorescent probe to form a double-fluorophore composite superparamagnetic fluorescent probe to quickly, efficiently and specifically detect the F ions. Since F ions compete with complexed NCDs in aqueous solution for binding to Ni²⁺The mechanism, NCDs separate from the quenching fluorescent probe and recover fluorescence with a maximum emission wavelength of 430nm as the concentration of F ions increases. At the same time, the excitation wavelength (lambda) is the same_ex565nm) under the condition of autofluorescence_em565nm) and NCDs (. lamda.)_em430nm), the fluorescence intensity is gradually restored with the increase of F ions, and thus the color of the emitted light of the whole aqueous solution is changed from green to blue, so that fluorine ions can be identified and quantitatively detected according to the change of the light emission spectrum of the fluorescent probe. In addition, F ions adsorbed on the fluorescent probe of the present invention can be removed by calcium salt deposition, and the fluorescent probe has excellent magnetic properties and can be recovered and reused without being clarifiedThe activity of the compound is obviously lost, and F ions are convenient to quickly evaluate and separate, so that the monitoring and collection of the F ions are realized.

Preferably, the fluorine ion double-fluorescence detection probe comprises an inner core and an outer shell; the inner core is Fe₃O₄Nanoparticles, the shell being modified in the Fe₃O₄Mesoporous silica containing organosilane functionalized carbon dots on the outer surface of the nanoparticles. The mesoporous silicon dioxide layer has high surface area, excellent biocompatibility and a large number of surface modification sites, and ensures high load of the F ion receptor.

Preferably, the particle size of the fluorine ion dual-fluorescence detection probe is 200-300 nm.

More preferably, the particle size of the fluorine ion dual-fluorescence detection probe is 250 nm.

In addition, the invention also provides a preparation method of the fluorine ion dual-fluorescence detection probe, which comprises the following steps:

s1, synthesizing an autofluorescent marker:

s11, mixing hydrophilic Fe₃O₄After the core, deionized water and ethanol are subjected to ultrasonic dispersion and mixing, adding an ammonia water solution and a tetraethyl orthosilicate-ethanol solution, and stirring to obtain a mixed solution A;

s12, mixing cetyl trimethyl ammonium bromide, deionized water and ethanol, and stirring until a clear solution B is obtained;

s13, transferring the mixed solution A into a clear solution B, adding an organosilane functionalized carbon dots (SiCDs) -ethanol solution under the stirring condition, carrying out magnetic separation and washing a product, and refluxing with an ammonium nitrate-ethanol solution to remove hexadecyl trimethyl ammonium bromide to obtain an autofluorescence marker;

s2, synthesizing magnetic nano particles coated by fluorescent silicon dioxide;

s3, shaking and mixing the magnetic nanoparticles coated by the fluorescent silicon dioxide, the deionized water and the pure carbon dot solution for 0.5-1.5 h, filtering, washing and drying to obtain the fluorine ion double-fluorescence detection probe.

Preferably, in step S11, the hydrophilic Fe₃O₄A core,The ratio of the addition amount of deionized water to ethanol is 0.1g:1 to 5mL:5 to 15mL, more preferably 0.1g:2.5mL:10 mL.

Preferably, in step S11, the ultrasonic dispersion mixing conditions are: after ultrasonic dispersion and mixing for 5min, stirring was carried out at 1000 rpm.

Preferably, in step S11, the volume ratio of the ammonia water solution to the tetraethyl orthosilicate-ethanol solution is 0.1-0.7: 2.25, and more preferably 0.4: 2.25.

More preferably, in step S11, the tetraethyl orthosilicate-ethanol solution is a 10% v/v tetraethyl orthosilicate-ethanol solution.

Preferably, in step S11, the stirring conditions are: mechanically stirred at 40 ℃ for 10 h.

Preferably, the hydrophilic Fe of step S11₃O₄The preparation method of the core comprises the following steps:

s111, adding anhydrous FeCl₃Dissolving trisodium citrate and ethylene glycol in a mass ratio of 0.1-1.5: 1, and stirring to form an orange yellow solution;

s112, adding sodium acetate under the stirring condition (preferably magnetic stirring) until a uniform yellowish-brown solution is obtained, keeping the solution at the temperature of 150-250 ℃ for 5-15 h, cooling to room temperature, carrying out magnetic separation on a black product, washing and drying to obtain the hydrophilic Fe₃O₄And (4) a core.

More preferably, in step S111, the anhydrous FeCl₃And trisodium citrate in a mass ratio of 0.8125: 1.

More preferably, in step S112, the amount of sodium acetate added is 5 to 10 times, most preferably 7.5 times, the mass of trisodium citrate.

More preferably, in step S112, after obtaining a uniform yellowish-brown solution, the solution is maintained at 200 ℃ for 10 hours.

More preferably, in step S112, the washing and drying conditions are: washing with ethanol for 4-6 times, then washing with deionized water for 1-2 times, and finally vacuum drying at 40 ℃.

Preferably, in step S12, the ratio of the addition amounts of the cetyltrimethylammonium bromide, the deionized water and the ethanol is 0.1g: 1-11 mL: 10-20 mL, and more preferably 0.1g:6mL:15 mL.

Preferably, in step S12, the stirring is mechanical stirring, and the rotation speed is 600 rpm.

Preferably, in step S13, the stirring conditions are: vigorous mechanical stirring was carried out at 40 ℃ and 1000 rpm.

Preferably, in step S13, the washing conditions are: washing with ethanol and/or deionized water for 4-6 times.

Preferably, the hydrophilic Fe of step S11₃O₄The mass-to-volume ratio of the core to the organosilane-functionalized carbon dot-ethanol solution of step S13 is 0.1 g/0.1-0.5 mL, more preferably 0.1 g/0.5 mL.

Preferably, in step S13, the method for removing cetyl trimethyl ammonium bromide by refluxing with ammonium nitrate-ethanol solution is as follows: refluxing with ammonium nitrate-ethanol solution (0.6 wt% ethanol solution) at 60-90 deg.C for 8-16 h; more preferably: reflux with ammonium nitrate-ethanol solution (0.6 wt% ethanol solution) at 75 deg.C for 12 h.

The invention also provides a preparation method of the synthetic fluorescent silica-coated magnetic nanoparticles in the step S2, which comprises the following steps:

s21, adding the autofluorescent marker into an anhydrous toluene solution of (3-aminopropyl) triethoxysilane, and reacting in N₂Refluxing under the atmosphere;

s22, magnetically separating the product obtained in the step S21, washing and drying, adding the product into an acetic acid-ethanol solution, adding diethylenetriamine pentaacetic dianhydride, and further refluxing;

s23, filtering, recovering and washing the product obtained in the step S22, dispersing the product in deionized water, adding excessive nickel salt, stirring and adjusting the pH value to obtain the fluorescent silicon dioxide coated magnetic nanoparticles.

Preferably, the refluxing time in the step S21 is 20-28 h; in the step S22, the refluxing time is 12-20 hours, and the refluxing temperature is 60-100 ℃.

More preferably, the refluxing time in step S21 is 24 h; the refluxing time in step S22 was 16h, and the refluxing temperature was 80 ℃.

Preferably, in the anhydrous toluene solution of (3-aminopropyl) triethoxysilane in step S21, the volume ratio of the anhydrous toluene to the (3-aminopropyl) triethoxysilane is 30: 0.1-1.3, and more preferably 30: 0.7.

Preferably, the washing in step S22 is washing with acetone.

Preferably, the acetic acid-ethanol solution in the step S22 is an acetic acid-ethanol solution of 40% -60% v/v, more preferably an acetic acid-ethanol solution of 50% v/v.

Preferably, the method for recovering and washing the product of the step S22 by filtration in the step S23 is as follows: after recovering the product of step S22 by vacuum filtration, washing was performed with excess acetone and deionized water.

Preferably, the nickel salt is Ni (NO) in step S23₃)₂、NiCl₂。

Preferably, the pH value in step S23 is 5-7, and more preferably 6.

Preferably, in step S3, the mass-to-volume ratio of the fluorescent silica-coated magnetic nanoparticles to the pure carbon dot solution is 1-20: 1mg/mL, and more preferably 10:1 mg/mL.

The organosilane functionalized carbon dot (SiCDs) -ethanol solution, the pure carbon dot solution and the diethylene triamine pentaacetic dianhydride can be simply prepared after being sold in the market, and can also be prepared according to the following method.

The invention also provides a preparation method of the organosilane functionalized carbon dots (SiCDs) -ethanol diluted solution, which comprises the following steps:

s131. under the high-temperature condition, under N₂SiCDs were synthesized in an atmosphere: placing N-beta- (aminoethyl) -gamma-aminopropyltrimethoxysilane (AEAPMS) in an oil bath in N₂Refluxing for 3-10 min at 200-240 ℃ in the atmosphere;

s132, under the condition of vigorous stirring, quickly adding anhydrous citric acid into the solution to react for 1-5 min to obtain an orange transparent solution;

and S133, extracting the final product for 2-3 times by using petroleum, and dissolving the final product in ethanol to obtain a SiCDs-ethanol diluted solution.

Preferably, in step S131, in N₂Reflux at 220 ℃ for 5min in an atmosphere.

The invention also provides a preparation method of the pure Carbon Dot Solutions (NCDs), which comprises the following steps:

s31, mixing deionized water and trisodium citrate, and carrying out hydrothermal reaction for 3-7 h at a constant temperature of 180-220 ℃;

s32, after cooling, adding an ammonia solution, reacting at 180-220 ℃ for 8-12 h, dialyzing, soaking, and stirring overnight;

s33, adjusting the pH value to 5.5-6.5 (preferably 6) to obtain the NCDs solution.

Preferably, in step S31, the hydrothermal reaction is carried out for 5h at a constant temperature of 200 ℃.

Preferably, in step S32, the reaction is carried out at 200 ℃ for 10 h.

The invention also provides a preparation method of the diethylene triamine pentaacetic dianhydride, which comprises the following steps:

s221, adding diethylenetriamine pentaacetic acid (DTPA) into pyridine, and then adding acetic anhydride; general formula (N)₂Removing dissolved oxygen, and stirring the solution at 50-90 ℃ for reaction for 12-36 h to obtain DTPA anhydride;

s222, washing DTPA anhydride in excessive acetic anhydride for 1-3 times, washing in excessive ethyl ether for 2-4 times, and drying in vacuum to obtain the diethylene triamine pentaacetic dianhydride.

Preferably, in step S221, the solution is reacted for 24 hours with stirring at 70 ℃.

The invention uses carbon dots and Ni for the first time²⁺The chelate compounds synthesize a fluorescent probe to detect fluorine ions, and the synthesis conditions are green, hydrophilic and cheap. The fluorescent probe can well monitor the real-time concentration of fluorine ions in a water environment, the lowest detection limit of F ions is as low as 65nM, the linear response range is 1-25 mu M, and the fluorescent probe has good sensitivity and selectivity; meanwhile, the method can efficiently adsorb and remove the fluoride ions, has the effective rate of removing the fluoride ions in the tap water as high as 96 percent, and has the advantages of rapid and efficient adsorption effect and excellent regenerationThe performance is favorable for recycling treatment, and secondary pollution can not be caused; and has excellent biocompatibility, good cell permeability and low cytotoxicity, shows huge potential in the direction of detecting F ions in the intracellular and the external environments, and can be successfully applied to the induction and imaging of the F ions in living cells.

Accordingly, the application of the fluorine ion dual-fluorescence detection probe in detecting and/or removing fluorine ions is also within the protection scope of the invention.

Preferably, said use refers to the use in the detection and/or removal of fluoride ions in an aqueous medium.

The invention provides a method for detecting the concentration of fluorine ions, which is to detect the fluorine ions by using the fluorine ion double-fluorescence detection probe and determine the concentration of the fluorine ions according to the fluorescence intensity.

The invention also provides a method for removing fluorine ions, which is used for removing the fluorine ions in the water phase by using the fluorine ion dual-fluorescence detection probe.

Compared with the prior art, the invention has the following beneficial effects:

(1) the fluorine ion dual-fluorescence detection probe provided by the invention is completely suitable for rapid detection and separation of F ions in a water environment.

(2) The fluorine ion dual-fluorescence detection probe (FSMN) provided by the invention has stable and good luminescence characteristics under a visible spectrum. When the fluorine ions are added, the color of the solution is changed from green to blue, the color change before and after the reaction is obvious, and the luminescence of FSMN and NCDs can be distinguished by naked eyes under the excitation of lambda ex-360 nm, so that the concentration of the fluorine ions can be quickly evaluated.

(3) The fluorine ion dual-fluorescence detection probe has the linear response range of 1-25 mu M, the detection limit of 65nM, wide linear detection range, low detection lower limit, high sensitivity and good selectivity, is not interfered by other anions, and has practical application value in the fields of biochemistry, environmental science and the like.

(4) The porous superparamagnetic fluorine ion dual-fluorescence detection probe has excellent dispersity, excellent biocompatibility, excellent cell permeability and low cytotoxicity, shows huge potential in the direction of detecting F ions in an intracellular environment and an extracellular environment, and can be successfully applied to the induction and imaging of the F ions in living cells.

(5) The F ions adsorbed on the fluorine ion dual-fluorescence detection probe can be removed through calcium salt deposition, and the fluorine ion dual-fluorescence detection probe has excellent stability and excellent superparamagnetic property, can be recycled without obviously losing the activity, has excellent reusability, is green and environment-friendly in preparation conditions and easy to control, and is suitable for batch production.

Drawings

FIG. 1 is a scheme showing the preparation of diethylenetriaminepentaacetic acid (DTPA) anhydride.

FIG. 2 is a flow chart of the preparation of pure carbon dots (NCDs).

FIG. 3 is a multi-step synthetic route of fluoride ion dual fluorescence detection probes (FSMN-NCDs nanoparticles).

FIG. 4 is a microscopic morphology of FSMN-NCDs nanoparticles.

FIG. 5 (a) pure cetyltrimethylammonium bromide (CTAB) and Fe₃O₄@mSiO₂-infrared spectrogram of SiCDs nanoparticles; (b) is a magnetic core (Fe)₃O₄Core); (c to d) are Fe₃O₄@mSiO₂HRTEM image of SiCDs nanoparticles.

FIG. 6 is a TEM and HRTEM image of organosilane functionalized carbon dots (SiCDs) and pure carbon dots (NCDs); wherein (a) SiCDs (200kV, TEM), (b) SiCDs (200kV, HRTEM), (c) NCDs (200kV, TEM), and (d) NCDs (200kV, HRTEM).

FIG. 7 is a 1H-NMR spectrum of a standard DTPA anhydride and a synthetic DTPA anhydride of the present invention; Δ H (400MHz, DMSO-d6):3.70(8H, s),3.29(2H, s), 2.83-2.54 (8H, m).

FIG. 8 is an XPS spectrum of FSMN-NCDs nanoparticles; (a) broad spectrum scanning of FSMN particles, (b) C1 s of FSMN particles, (C) O1 s of FSMN particles, (d) N1 s of FSMN particles, (e) Ni 2p of FSMN particles, (F) F1 s of FSMN-F-nanoparticles.

FIG. 9 (a) is an FTIR chart of NCDs; (b) FTIR plots of SiCDs; (c) XRD patterns of NCDs; (d) XRD patterns of SiCDs are shown.

FIG. 1 shows a schematic view of a0 wherein (a) is (i) Fe₃O₄Nucleus, (ii) Fe₃O₄@mSiO₂-FTIR spectra of SiCDs nanoparticles, (iii) FSMN and (iv) FSMN-NCDs nanoparticles; (b) additional zeta potentials during the synthesis are monitored; (c) is a low-angle XRD pattern of the FSMN-NCDs nano particles; (d) and (e) nitrogen adsorption/desorption isotherms of FSMN and FSMN-NCDs nanoparticles; (f) is Fe₃O₄Core, Fe₃O₄@mSiO₂Hysteresis curves of SiCDs nanoparticles, FSMN-NCDs nanoparticles.

FIG. 11 shows fluorescence emission and excitation spectra of aqueous FSMN solution (150ppm) and aqueous FSMN-NCDs nanoparticle solution (150ppm) in deionized water.

Fig. 12 shows the electron transfer mechanism of FSMN.

FIG. 13 is a graph showing fluorescence lifetime curves of NCDs at different concentrations of nickel ions.

FIG. 14 is a graph of the effect of different influencing factors on the efficiency of detection of a probe (a) in the presence of different cations; (b) in the presence of different anions; (c) different pH value to Fe₃O₄@mSiO₂-the effect of SiCDs nanoparticles; (d) the effect of the number of cycles on FSMN-NCDs nanoparticles.

FIG. 15 is (a) the reaction of FSMN-NCDs nanoparticles on F ion concentration in different concentrations of F ion in deionized water; (b) peak scatter plots of FSMN-NCDs nanoparticles against F ion concentration in F ion-deionized water solutions of different concentrations; (c) the detection limit of the reaction of FSMN-NCDs nanoparticles to the concentration of F ions in F ion-deionized water solutions with different concentrations; (d) peak scatter plots of FSMN-NCDs nanoparticles versus F ion concentration in different concentrations of F ion-tap water; (e) in different concentrations of F ion-tap water solution; (f) detection limits of the reaction of FSMN-NCDs nanoparticles to F ion concentration in different concentrations of F ion-tap water solution.

FIG. 16 is a graph showing the macroscopic color change of FSMN-NCDs nanoparticles at different F ion concentrations.

FIG. 17 is a schematic representation of the reusability of FSMN-NCDs nanoparticles.

FIG. 18 is (a) the detection capability of FSMN-NCDs nanoparticles for F ions under different types and concentrations of interfering anions; (b) the FSMN-NCDs nanoparticle F ion detection has complete response time.

FIG. 19 is (a) CCK-8 cell viability assay for FSMN; (b) CCK-8 cell activity detection of FSMN-NCDs nano-particles; (c) co-culture photomorphogram of SCC-15 cells and FSMN-NCDs nanoparticles; (d) co-culture photomicroscopy of MC3T3 cells and FSMN-NCDs nanoparticles; (e) confocal laser mapping of SCC-15 cells, MC3T3 cells, co-cultured with FSMN-NCDs nanoparticles.

FIG. 20 is Fe₃O₄、Fe₃O₄@mSiO₂-SiCDs nanoparticles and FSMN-NCDs nanoparticles low magnetic field strength hysteresis curves.

FIG. 21 is (a) fluorescence excitation-generation spectra of NCDs; (b) fluorescence excitation-generation spectra of SiCDs.

FIG. 22 is an XPS spectrum of NCDs. (a) C1 s of NCDs; (b) NCDs/Ni²⁺C1 s of (1).

Detailed Description

The invention is further described with reference to the drawings and the following detailed description, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.

Unless otherwise indicated, reagents and materials used in the following examples are commercially available.

Example 1 hydrophilic Fe₃O₄Synthesis of core, organosilane functionalized carbon dots (SiCDs) dilute solutions and autofluorescent labels (Fe)₃O₄@mSiO₂Preparation of-SiCDs)

1. Hydrophilic Fe₃O₄Synthesis of the core, comprising the steps of:

(1) 0.325g of anhydrous FeCl₃And 0.4g trisodium citrate dissolved in 40mL ethylene glycol and magnetically stirred until an orange yellow solution is formed;

(2) after adding 3g of sodium acetate with magnetic stirring until a homogeneous yellowish-brown solution is obtained, the solution is transferred to a 50mL polytetrafluoroethylene (Teflon) -lined autoclave and maintained at 200 ℃ for 10 h; coldCooling to room temperature, magnetically separating black product, washing with ethanol for 5 times, washing with deionized water for 1 time, and vacuum drying at 40 deg.C to obtain hydrophilic Fe₃O₄And (4) a core.

2. Synthesis of dilute solutions of organosilane functionalized carbon dots (SiCDs) comprising the steps of:

(1) under high temperature conditions in N₂SiCDs were synthesized in an atmosphere: 10mL of AEAPMS were placed in a 100mL three-necked flask in an oil bath under N₂Refluxing in atmosphere at 220 deg.C for 5 min;

(2) under the condition of vigorous stirring (1000rpm), 0.5g of anhydrous citric acid is quickly added into the solution to react for 1min, and an orange transparent solution is obtained;

(3) the final product was purified 3 times by extraction with 50mL petroleum and then dissolved in 100mL ethanol to obtain a SiCDs-ethanol solution.

3. Auto-fluorescent marker (Fe)₃O₄@mSiO₂-SiCDs) comprising the steps of:

(1) mixing the above 0.1g hydrophilic Fe₃O₄After core, 2.5mL deionized water and 10mL ethanol were dispersed and mixed under ultrasonic conditions for 5min, they were transferred under vigorous mechanical stirring (1000rpm) (water bath) to a 100-mL three-necked round bottom flask; then, 0.4mL of an ammonia solution and 2.25mL of a tetraethyl orthosilicate (TEOS) -ethanol solution (10% v/v) were added by a micropipette, and mechanically stirred at 40 ℃ for 10 hours to obtain a mixed solution A;

(2) while in a 100-mL three-necked flask with moderate speed mechanical stirring (600rpm), 0.1g cetyltrimethylammonium bromide (CTAB), 6mL deionized water and 15mL ethanol were mixed uniformly until a clear solution B was obtained;

(3) transferring the mixed solution A into a clear solution B, adding 0.5mL of organosilane functionalized carbon dots (SiCDs) -ethanol solution into the mixture under further intense mechanical stirring (1000rpm) at 40 ℃, stirring for 2h, collecting the product with a magnet, washing with ethanol and deionized water for 4-6 times, refluxing the washed product with ammonium nitrate-ethanol solution (0.6 wt% ethanol solution) at 75 ℃ for 12h to fully remove CTAB, and obtaining Fe₃O₄@mSiO₂-SiCDs nanoparticles, i.e. autofluorescent markers.

The Fe₃O₄@mSiO₂the-SiCDs nanoparticles can be stable in ethanol for several months and have good dispersibility. Continuously freeze-drying at-70 deg.C under vacuum degree of 133ubar or below for 24h to obtain Fe₃O₄@mSiO₂SiCDs powders, which can be stored at room temperature.

Example 2 DTPA anhydride and fluorescent silica-coated magnetic nanoparticles (Fe)₃O₄@mSiO₂-SiCDs@DTPA-Ni²⁺FSMN) preparation

1. The preparation process of Diethylene Triamine Pentaacetic Acid (DTPA) anhydride is shown in figure 1, and specifically comprises the following steps:

(1) 33.7g of diethylenetriaminepentaacetic acid (DTPA) was added to a 250mL round bottom flask containing 40mL of pyridine followed by 33mL of acetic anhydride;

(2) general formula (N)₂Removing dissolved oxygen, and reacting the solution at 70 ℃ under vigorous stirring (1000rpm) for 24h to obtain DTPA anhydride;

(3) washing DTPA anhydride in excessive acetic anhydride for 2 times, washing in excessive ethyl ether for 3 times, and drying in vacuum to obtain diethylenetriamine pentaacetic anhydride.

2. Fluorescent silica-coated magnetic nanoparticles (Fe)₃O₄@mSiO₂-SiCDs@DTPA-Ni²⁺FSMN), comprising the steps of:

(1) 0.3g of Fe as in example 1 above₃O₄@mSiO₂The SiCDs nanoparticles were added to 30mL of dry toluene (29.3 mL volume of dry toluene) containing 0.7mL (3mmol) of (3-aminopropyl) triethoxysilane (APTES) in N₂Refluxing for 24h under the atmosphere to obtain APTES modified nanoparticles;

(2) magnetic separation of APTES modified nanoparticles, after washing with acetone and drying thoroughly, 0.1g of APTES modified nanoparticles was added to 36mL of ethanolic acid solution (50% v/v), the previously prepared diethylenetriaminepentaacetic acid (DTPA) anhydride was added and further refluxed at 80 ℃ for 16 h;

(3) finally, the Fe was recovered by vacuum filtration₃O₄@mSiO₂-SiCDs @ DTPA nanoparticles, washed with excess acetone for 2 times, then with deionized water for 2 times, the volume used for each wash being 20 mL; then dispersed in 50mL deionized water, and a slight excess of Ni (NO) was added₃)₃The salt was stirred for 1h and the pH was adjusted to 6.0 and further stirred for 24h to give FSMN (Fe)₃O₄@mSiO₂-SiCDs@DTPA-Ni²⁺) Namely, the magnetic nano particles coated by the fluorescent silicon dioxide can be collected by freeze drying.

EXAMPLE 3 Synthesis of pure carbon dots (NCDs) and fluoride ion Dual fluorescence detection probes (Fe)₃O₄@mSiO₂-SiCDs@DTPA-Ni²⁺Preparation of-NCDs, FSMN-NCDs)

1. The preparation process of pure carbon dots (NCDs) is shown in fig. 2, and specifically comprises the following steps:

(1) 20mL of deionized water was mixed with 0.84g of trisodium citrate, transferred to a 50mL polytetrafluoroethylene-lined hydrothermal reactor, and reacted at a constant temperature of 200 ℃ for 5 hours;

(2) after cooling, adding an ammonia solution, reacting for 10 hours at 200 ℃, dialyzing the obtained yellowish-brown solution in a dialysis bag, soaking the dialyzed yellowish-brown solution in 1000mL of deionized water, and stirring overnight by mild magnetic force (200-600 rpm);

(3) after adjusting the pH to 6, NCDs solution was obtained.

2. Fluorine ion dual-fluorescence detection probe (Fe)₃O₄@mSiO₂-SiCDs@DTPA-Ni²⁺-NCDs, FSMN-NCDs) comprising the steps of:

(1) 10mg of the fluorescent silica-coated magnetic nanoparticles of example 2 above (FSMN powder) were added to the 1mL of pure carbon dots (NCDs) solution above (mass to volume ratio of FSMN to NCDs solution 10:1mg/mL) along with 4mL of deionized water with gentle shaking, mixed for 1h with shaking,

(2) filtering, washing with deionized water for 3 times, and freeze drying to obtain FSMN-NCDs powder as the dual-fluorescence detection probe for fluorine ions.

Example 4 fluoride ion Dual fluorescence detection Probe (Fe)₃O₄@mSiO₂-SiCDs@DTPA-Ni²⁺Preparation of-NCDs, FSMN-NCDs)

The other conditions were the same as in example 3, with the only difference that: and adding 1mg of fluorescent silica-coated magnetic nanoparticles (FSMN powder) and 4mL of deionized water into 1mL of pure carbon dots (NCDs) solution together, wherein the mass-to-volume ratio of the FSMN to the NCDs solution is 1:1mg/mL, so as to obtain the high-efficiency porous superparamagnetic fluorine ion dual-fluorescence detection probe.

Example 5 fluoride ion Dual fluorescence detection Probe (Fe)₃O₄@mSiO₂-SiCDs@DTPA-Ni²⁺Preparation of-NCDs, FSMN-NCDs)

The other conditions were the same as in example 3, with the only difference that: and adding 20mg of fluorescent silica-coated magnetic nanoparticles (FSMN powder) and 4mL of deionized water into 1mL of pure carbon dots (NCDs) solution together, wherein the mass-volume ratio of the FSMN to the NCDs solution is 20:1mg/mL, so as to obtain the high-efficiency porous superparamagnetic fluorine ion dual-fluorescence detection probe.

Example 6 microstructure of a Fluoronium BiFluorogenic detection Probe and demonstration thereof

(1) The fluorine ion dual-fluorescence detection probe uses Fe₃O₄The nanoparticles serve as an inner core and mesoporous silica serves as an outer shell, so that a large specific surface area is provided. The multistep synthetic route for magnetic fluoride fluorescent probes (FSMN-NCDs nanoparticles) is shown in FIG. 3. In the preparation process of the FSMN-NCDs nano-particles, TEOS is used as a silicon source, SiCDs are used as a labeling fluorophore, and CTAB is used as a structure directing agent.

(2) FIG. 4, panel a, shows the microscopic morphology of FSMN-NCDs nanoparticles, confirming that the FSMN-NCDs nanoparticles are almost spherical and have good dispersibility, and the diameter of the FSMN-NCDs nanoparticles is about 250 nm. In the b-picture High Resolution TEM (HRTEM) image of FIG. 4, the FSMN-NCDs nanoparticles can easily distinguish the mesoporous silica layer (about 75nm) from the irregular mesoporous silica layer. SEDS in the d, e and f plots of fig. 4 show the gray scale distribution of Fe, Si and Ni in the c plot of fig. 4. In a silicon dioxide layerIn and uniformly distributed Ni²⁺Ion indicates, Ni²⁺Successfully chelated with DTPA.

(3) Fourier transform infrared spectroscopy (FTIR) analysis of the invention verified successful removal of CTAB. Fe₃O₄@mSiO₂-SiCDs nanoparticles at 1470cm^-1There was no cetyl trimethyl split absorption peak, which also laterally confirmed the removal of CTAB (panel a in fig. 5).

(4) The FSMN-NCDs nanoparticles have magnetic cores as shown in b of FIG. 5, and are easily separated by an external magnetic field. Diffraction peaks of FSMN-NCDs nanoparticles and Fe₃O₄The normal characteristic diffraction patterns of inverse spinel structures (PCPDFWIN v.2.02, PDF No.89-0691) are closely matched. The lattice parameter was calculated to be 8.38A. Except for Fe₃O₄Besides the characteristic peak, the 20-27-degree broadband can belong to an amorphous silicon shell (PCPDFWIN v.2.02, PDF No.29-0085) and an irregular graphite structure. The HRTEM analysis in panels c and d of fig. 5 further illustrates the successful immobilization of SiCDs in the silicon dioxide layer. The clear fringe distance at 0.207nm matches the (102) lattice distance of the SiCDs in FIG. 6.

(5) The invention uses APTES to lead Fe₃O₄@mSiO₂-SiCDs nanoparticles-NH₂Functionalized, APTES was further reacted with the-CO-O-OC-group of DTPA anhydride in an acetic anhydride/ethanol environment (FIG. 7).

(6) To elucidate the attachment of DTPA to FSMN and study its surface functional groups, the present inventors performed XPS investigations on FSMN (panel a of FIG. 8 to panel e of FIG. 8) and FSMN-F- (panel F of FIG. 8) nanoparticles to eliminate the disruption from NCDs. The deconvolution XPS spectrum of panel C1 s in fig. 8 b consists of four peaks corresponding to C ═ C (sp2 carbon), CC/CH (sp3 carbon), CN/CO (sp3 carbon) centered at 284.1, 284.7, 285.7 and 288.6 eV) and-COOH/-CONH- (oxidized carbon). Due to carboxyl and Ni²⁺Coordination between ions-COOH peak was slightly higher than reported results 31. The high strength of CN/CO (sp3 carbon) is believed to be the CN bond of the DTPA molecule and the presence of SiCDs prepared by aeapms.fitted peaks of 24 and C ═ C (sp2 carbon) again confirm that the immobilized SiCDs are in graphitic structures. The broad peaks of the high resolution O1 s light emission at 530.5 and 532.8eV in plot c in FIG. 8 correspond toThe strong peak at 532.0eV refers to the-OH of the mesoporous silica shell. In the d plot in FIG. 8, a high resolution scan of N1 can be fitted to the three peaks of the N-H, C-N and-CONH-bonds. The peak at 399.5eV is presumed to represent pyrrole N (NH) in the SiCD and unreacted-NH 2 groups on the silica shell the peak at 26 at 400.1eV represents graphite N (CN) of the SiCD in the silica shell, which is the same FTIR spectrum as the SiCD in panel a of FIG. 9. The peak appearing at 401.0eV is attributed to the nitrogen atom in the CO-NH bond of the peptide bond between DTPA and the silica layer. The presence of high resolution Ni3d 3/2 and Ni3d5/2 photoelectron energies 837.1 and 853.8eV and multiple spilts in plot e in FIG. 8 was confirmed with Fe₃O₄@mSiO₂-SiCDs @ DTPA nanoparticles form nickel chelates. The difference between the main peak and the multiplet was 4.6eV, indicating that Ni²⁺and-COOH was oxidized to the NiO state. The high resolution spectrum of F1 s at 687.0eV corresponds to Ni²⁺The ion-bound half-ion-bonded fluoride (graph f in fig. 8).

(7)(i)Fe₃O₄A core, (ii) Fe₃O₄@mSiO₂FTIR spectra of the-SiCDs nanoparticles, (iii) FSMN and (iv) FSMN-NCDs nanoparticles are shown in graph a in FIG. 10. Fe₃O₄The Fe-O content in the magnetic core is 559cm^-1Strong absorption in the vicinity. 2880cm^-1(ii)，2890cm^-1(iii) And 2885cm^-1(iv) The methylene peak of (a) confirms the presence of SiCD, compared to the FTIR spectrum of SiCD in panel a of fig. 9. NH bond at 1460cm^-1The feature widths indicated successful bonding of the DTPA and silicon dioxide layers, while at 3366 and 3296cm^-1Asymmetric and symmetric stretching vibrations of NH bond were not clearly observed, probably because they were covered through 3200 to 3400cm^-1The characteristic stretching vibration peak of-OH at 32 for FSMN (iii) and FSMN-NCDs nanoparticle (iv), 1610cm^-1The nearby strong peak corresponds to the coordinate carboxyl group of vas (COO-), but is 1650cm away from the silica layer^-1Hydration of H₂O OH vibratory superposition [32-33 ]]. 1695cm in FSMN (3) caused by CO-NH bond^-1The spike in (A) indicates that DTPA is in this state indicating oxygen incompatibility in CO-NH. At 1697cm^-1Tips on FSMN-NCDs nanoparticles (iv)The sharp peak shows a relative increase in the strength of the CO-NH bond for the combination of pCD. The above results are also consistent with the XPS survey results, which revealed that SiCD has been successfully immobilized in the mesoporous silica layer, DTPA and Fe₃O₄@mSiO₂-SiCDs nanoparticles bound, with many hydrophilic functional groups present on the FSMN-1 surface, pCD nanoparticles.

(8) Additional zeta potentials during synthesis are monitored in panel b in fig. 10, revealing surface modification and potential changes during the establishment of FSMN-pCD nanoparticles. Fe due to surface modification of trisodium citrate dehydrate₃O₄Core (i) showed a negative potential of-25.4 mV, due to the-OH group. Fe₃O₄@mSiO₂-SiCDs nanoparticles (ii) showed positive potential +28.7 mV. This is mainly due to SiCDs on the surface of the mesoporous silica shell. However, after APTES modification, the potential was shifted to +41.5mV (iii), indicating-NH at the surface of the silica layer₂The results obtained by XPS were combined and confirmed. Fe₃O₄@mSiO₂The potential of the-SiCDs @ DTPA nanoparticles (iv) is-32.7 mV due to the binding of DTPA, while the potential of FSMN (v) is-17.3 mV, indicating Ni²⁺Chelation of ions.34 and the potential of FSMN-NCDs nanoparticle (vi) to become more negative (-45.1eV) indicate attachment of NCDs.

(9) As a particular feature in the following applications after SiCDs immobilization, the mesoporous structure provides a larger surface and binding site for chemical reactions. The low angle XRD pattern (see inset of c in fig. 10) shows strong diffraction peaks from 2 ° to 4.5 ° which represent the typical two-dimensional mesoporous structure of the outer silica layer in FSMN-NCDs nanoparticles. The broad weak peak from 4.5 ° to 8.0 ° may be due to the disordered immobilization process of SiCDs resulting in non-uniform distribution and irregular structure of mesopores, similar to previous reports on uniform and non-uniform mesoporous silica structures. The nitrogen adsorption/desorption isotherms of 35-36FSMN and FSMN-NCDs nanoparticles exhibit a hysteresis type IV isotherm curve (plot d in fig. 10 and plot e in fig. 10), which is characteristic of mesoporous materials. The surface area before bonding of NCDs and the total pore volume were calculated to be 282.34m respectively²G and 0.630cm³A/g is much higher than Fe₃O₄@SiO₂Nanoparticles 37-38. The pore size distribution determined from the desorption portion of the isotherm confirmed the formation of mesopores with an average diameter of 3.28 nm. After attachment of the NCDs, the surface area and total pore volume dropped to 231.21m²G and 0.413cm³G, and the pore size becomes smaller (2.939 nm). Interestingly, due to the chelation of NCDs, the pore diameter showed a sharp decrease from 2.5 to 4.5nm, which well satisfies the diameter of NCDs.

(10) FSMN-NCDs nanoparticles are easily separated by external magnets due to their magnetic properties. The hysteresis curves in fig. 10 f and fig. 11 show that the intermediate and final products show ideal superparamagnetism at 300K, which is a satisfactory property for magnetic separation 39, fluorescent probes 40, targeted drug delivery 41 and other applications 42. For Fe₃O₄Core, Fe₃O₄@mSiO₂-SiCDs nanoparticles and FSMN-NCDs nanoparticles, with values of saturation magnetization of 59.3, 41.1 and 19.0emu/g, respectively. The FSMN-NCDs nano-particle has higher saturation magnetization compared with the reported silicon dioxide composite material, which shows that the FSMN-NCDs nano-particle has better application potential 43 because of superparamagnetism and high saturation magnetization, the FSMN-NCDs nano-particle shows strong magnetic sensitivity in lower external magnetic field in practical application.

Example 7 fluoride ion detection mechanism and application

(1) FIG. 11 shows fluorescence emission and excitation spectra of aqueous FSMN solution (150ppm) and aqueous FSMN-pCD nanoparticle solution (150ppm) in deionized water. Aqueous FSMN solution at lambda_ex-FSMNShows the strongest fluorescence emission peak lambda under the irradiation of 360nm ultraviolet light_em-FSMN565nm (green light) at λ_em-FSMN470nm with a broad and weak emission peak. As shown in FIG. 12, the strongest fluorescence emissions of NCDs and SiCDs are located at λ under 338nm and 370nm excitation, respectively_em-NCDs430nm (blue) and λ _em-SiCDs470 nm. Thus, the present invention can be used at λ_exLuminescence of FSMN and NCDs was distinguished by the naked eye under excitation at 360 nm. Obviously, there is a difference in fluorescence properties between aqueous solutions of FSMN-NCDs of the same concentration as compared to the fluorescence spectra of aqueous solutions of FSMN. Emission of aqueous solutions of FSMN-NCDsThe fluorescence intensity in the short wavelength region is higher than the spectrum of the FSMN aqueous solution. In the inset of FIG. 11, due to Ni²⁺The fluorescence quenching effect of the ions, the difference in value between the aqueous FSMN solution and the aqueous FSMN-NCDs solution is consistent with the spectral pattern of the NCDs, and the difference in intensity is much lower than that of the NCDs at the usual concentration.

(2) Under normal conditions, nickel coordinates to water molecules in a chelated state, [ Ni ]_(II)2(DTPA)₂(H₂O)]^4-A binuclear structure is formed by sharing a pair of carboxyl oxygen atoms in a DTPA ligand, and only one coordination water molecular site Ni_(II)One side of the cation, thus this Ni_(II)Is decaoordinated, while another Ni (II) that does not coordinate water molecules is uncoordinated. When the dimeric chelate is dissolved in an aqueous solution, the central Ni_(II)With Gd_(III)The ions are identical in character, consisting of five carboxyl oxygen atoms, one coordinated water molecule and three nitrogen atoms from the DTPA ligand. FIG. 10, a (iii) 1695cm of FSMN in FTIR spectrum^-1The spike in (b) was due to the presence of a-CONH-bond, indicating that DTPA is attached to the surface of the mixed nanoparticle and that oxygen in the-CONH-bond is bound to Ni²⁺The connection is not matched. Thus, at the surface of FSMN, one carboxyl group of DTPA (attached to a tertiary amine group) binds to the ATPES-modified amine group, resulting in a dehydrated synthesis, and to Ni²⁺The unsaturated coordination of the ions produces brightness. The original position of-OH is represented by H₂O/OH-occupation and finally becomes the non-coordinating chelate Ni coordinated with two water molecules or two hydroxyl groups in FIG. 12_(II)(DTPA)(H₂O)₂. Thus, it is presumed that hydroxyl oxygen and C-N-C bond in DTPA are bonded to Ni²⁺Ion coordination constitutes the F-ion acceptor. And Ni²⁺The carboxyl oxygen and hydroxyl oxygen of ion-chelated NCDs are F^-The electron transfer and fluorescence quenching mechanisms necessary for ion detection applications are shown in fig. 12.

(3) To further elucidate the mechanism of the fluorescence quenching phenomenon, the present invention performed time-resolved fluorescence emission studies on fluorescence quenching probes and performed stringent control experiments. In FIG. 13, a is a graph showing the difference between λ and_exexcitation of samples at 360nm and varying concentrations of Ni²⁺Ion(s)Lower monitor lambda_emEmission wavelength at 430nm, in the absence and presence of Ni²⁺Time resolved emission decay traces of NCDs in the case of ions. Interestingly, with Ni²⁺The emission decay curves of NCDs become faster with increasing ions. This observation indicates that Ni²⁺Increase in ion concentration from NCDs to Ni²⁺Electron transfer of the ions is facilitated. Thus, addition of Ni is observed²⁺Faster emission decay curve of ions. Similar studies were also performed by exciting FSMN at 360nm and monitoring emission at 430 nm. The time-resolved emission decay traces for NCDs in the presence and absence of FSMN are graphically shown in fig. 13 b. The present invention finds similar features and faster traces of emission decay when adding FSMN as compared to adding FSMN. It is therefore presumed that the fluorescence quenching is caused by the transfer of non-radiative electrons from an excited state to a metal ion via the carboxyl group on NCDs and Ni²⁺Soft-soft interactions between ion centers. To confirm this, the present invention uses Ni in FIG. 13²⁺Ion pairs NCDs were further investigated by XPS. XPS analysis showed Ni²⁺The ions bind to the-COOH groups on the NCDs. This indicates that Ni²⁺Ions bind directly, resulting in higher luminescence quenching and faster decay traces. Thus, NCDs and Ni²⁺The ion complexes are highly chelated, unlike traditional F-ion detection mechanisms. According to the XPS study and Ni_(II)(DTPA)(H₂O)₂In Ni²⁺Coordination of ions to carboxyl groups on the surface of NCDs to form Ni_(II)(DTPA) (NCDs-COO) increases the electron density of NCDs, which reduces electron defects and causes the fluorescence quenching phenomenon in FIG. 12. This ultimately results in the transfer of electrons from NCDs to Ni of FSMN²⁺Ions. Whereas the recovery of fluorescence is attributable to the replacement of bound NCDs in solution by F-ions. Adding F^-The fluorescence intensity then recovers because it has Ni in comparison with FSMN²⁺High capacity for ion binding. Adding F^-After ionization, it occupies the binding site, thereby displacing NCDs from the silica surface in aqueous solution. The binding of fluoride ions to FSMN in the high resolution F1 xps spectra in the F plot in fig. 8 clearly indicates the semiionic fluoride.

(4) As for the autofluorescent labeling (SiCDs), graphs c in FIG. 13 and d in FIG. 13 show different concentrations of Ni²⁺Ionic SiCDs and Fe₃O₄@mSiO₂Time-resolved emission decay curves of SiCDs nanoparticles. It is clear that with Ni²⁺The emission attenuation of the ions is almost unchanged due to the increase of the ions. This indicates that the organosilane functionalization process of SiCDs and the covalent linkage between SiCDs and the silica shell blocks a large amount of-COOH and contributes to good stability of autofluorescent labeling under complicated aqueous solution conditions. Also, unlike NCDs and reported carbon dot complexes, SiCDs and Fe₃O₄@mSiO₂SiCDs nanoparticles are stable in many types of saline solutions and have different fluorescence quenching properties (graph a in fig. 14 and graph b in fig. 14). Explore Fe₃O₄@mSiO₂Stability of SiCDs nanoparticles in acidic and basic solutions (c diagram in fig. 14). The results show that Fe₃O₄@mSiO₂the-SiCDs nano particles are stable at a pH value of 3-10.

(5) The addition of FSMN powder to NCDs solutions results in a "turn-off" phenomenon of fluorescence quenching effects in the formation of FSMN-NCDs nanoparticles. In diagram a in FIG. 15, at F^-After ions are gradually increased (1-1000 mu M), the NCDs recover fluorescence by an opening effect, the emission wavelength of the aqueous solution is changed from 470nm to 430nm, and the peak value of the fluorescence intensity is increased along with the gradual increase of the concentration of the F-ions, which indicates that the addition of the F-ions can effectively recover the fluorescence of the NCDs. Meanwhile, the emission light of the mixture gradually moved from green to blue, and the change in fluorescence was easily recognized by the naked eye (fig. 16). To evaluate the sensitivity of the probes, different concentrations of F were used^-Ion experiments were performed and the addition of 700. mu. M F in panel b of FIG. 15 was observed^-The fluorescence intensity after ionization was recovered by 90%. The limit of detection (LOD) is estimated from statistical processing of the calibration curve. At the same time, according to the formula 1,

relative strength of solution to fluoride concentration (F-F)₀/F₀) Shows good linearity in the range of 1-25 mu M, R²0.9997 (panel c in fig. 15). LOD was calculated as 65 nM. Magnetic carrier and F under the application of external magnetic field^-The ions together can be easily removed from the solution as shown in fig. 17.

(6) As previously mentioned, NCDs/Ni²⁺The ion system can be used to detect F^-Ions; however, in any environmental sample, many other competing ions are also present. FSMN-NCDs nanoparticles have important selectivity to F-ions, and fluorescence response in the presence of different anions is measured. Graph a in fig. 18 shows the fluorescence response of different anions in aqueous solution. Adding Cl respectively^-、Br^-、SO₄ ^2-、PO₄ ^2-、CO₃ ^2-、HPO₄ ^-And NO₃ ^-Negligible recovery was observed after ionization. Interestingly, even if other anions were present, upon addition of F^-The fluorescence is almost completely recovered after the ion reaction, which indicates that the fluorescent detection probe pair synthesized by the invention is F^-The ions are highly specific and selective. F^-The ion being capable of recognizing a positively charged center (Ni)²⁺Ions) and competes with-COOH on NCDs, resulting in the strongest and smallest electronegative ions that release pCD from FSMN and result in enhanced fluorescence. Para Cl in contrast to other acid radicals^-Ions and Br^-The higher fluorescence response of the ions indicates that the ion diameter may also participate in the detection probe's recognition mechanism.

(7) In order to study the response time of the acceptor to fluoride, the present invention measures the change in fluorescence intensity with time after addition of fluoride ion. The b-plot in FIG. 18 clearly shows that the fluorescence intensity increases rapidly from 0-40 min, slowly from 40-60 min, and finally remains constant after equilibrium is established.

(8) The effectiveness and reproducibility of fluoride ion detection in tap water samples were evaluated.

The detection probes of the invention were used to detect F-ions in tap water filtered through a 200nm nitrocellulose membrane (to remove bacteria and suspended particles).

Experiments show that the fluorine ion detection probe has good detection effect in tap water, and when F is detected^-At an ion concentration of 600. mu.M, the recovery rate was 90% (d in FIG. 15 and e in FIG. 15). The fluorescence intensity was observed as a function of F^-The concentration increases. In the concentration range of more than 0-25 mu M, R²0.9929 hr pair F^-The ions have a linear response (plot f in fig. 15). However, the rate of change of fluorescence in tap water of different concentrations is higher than in deionized water, probably due to the presence of organic impurities, minerals, Cl in the tap^-Ions and H₂PO^4-Ions, etc.

The present invention further calculates the probe reusability by using an external magnet to separate FSMN in combination with FS. With Ca (NO)₃)₂After complete reaction and recombination with NCDs solution, the FSMN separated from the detection probe of the invention can be successfully used for another four cycles. Furthermore, it was found that the detection probe of the present invention had very little loss of sensing ability during repeated cycling (panel d in FIG. 14). Determination of F in each cycle using inductively coupled plasma Mass Spectrometry (ICP-AES)^-The quantitative separation of ions shows that 21.4mg of F can be magnetically adsorbed and separated by using 1000mg of the fluorine ion dual-fluorescence detection probe designed by the invention^-Ions (table 1).

TABLE 1 repeated adsorption and separation Performance of the fluoride ion Bifluoric detection probes of the present invention for fluoride ions

Example 8 in vitro cytotoxicity and fluorescence imaging Studies of fluoride ion Dual fluorescence detection probes

1. In vitro cytotoxicity detection method

(1) Human squamous cell carcinoma cell line SCC-15 and human osteoblast cell line MC3T3 (2X 10)³Individual cells) were seeded into 96-well plates, respectively, and the cells were plated at 100 μ L of a solution containing 10 μ M DMEM: f12 (1: 1) in FBS (i.e. DMEM: F12 in a total volume of 100. mu.L with FBS) for 48h (37 ℃, 5% CO)₂)；

(2) The above cells were incubated with FSMN or FSMN-NCDs nanoparticles at various concentrations (0, 10, 25, 50, 75, 100, 150, 200, 300 and 500 μ g/mL) in fresh DMEM: f12 (1: 1), and then culturing for 24h in a serum-free culture medium;

(3) after co-cultivation, viable cell concentrations were examined by CCK8 assay. All experiments were repeated three times.

2. Method for imaging living cells

(1) SCC-15 and MC3T3 cells were incubated with FSMN nanoparticles, FSMN-pCD nanoparticles (150. mu.g/mL), respectively, in humidified 5% CO at 37 ℃₂Culturing for 24h in an incubator;

(2) washing with fresh PBS for 3 times, and imaging the cells under 360nm ultraviolet excitation by a confocal microscope;

(3) PBS solution containing 500. mu. M F ions was added and co-incubated for 2h, and after 3 washes with PBS, the cells were imaged again with UV light (360nm) of the same excitation wavelength.

3. Results

The experiment shows that the activity of SCC-15 and MC3T3 cells is not obviously reduced. Therefore, the probe is nontoxic, biocompatible and non-cytotoxic, and can be used for F in living cells^-Bioimaging of ions (panels a in fig. 19 and b in fig. 19). In the absence of F-ions, little green fluorescence was observed at 360nm excitation. However, when cells were co-cultured with F-ions, a clear green signal was observed in lysosomes surrounding the nuclear region, indicating the ability of the probes of the invention to exhibit "on" behavior in the presence of F-ions (panel c in FIG. 19 to panel e in FIG. 19).

As can be seen from FIG. 20, Fe₃O₄Nanoparticles, Fe₃O₄@mSiO₂both-SiCDs nanoparticles and FSMN-NCDs nanoparticles exhibit superparamagnetism.

As can be seen from FIG. 21, the fluorescence emission of NCDs is concentrated in the blue wavelength band, and the fluorescence emission of SiCDs is shifted to the red wavelength band as compared with NCDs.

As is clear from FIG. 22, Ni ions are mainly bonded to the C-O bonds of NCDs.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Claims

1. A high-efficiency porous superparamagnetic fluorine ion dual-fluorescence detection probe is characterized by comprising an autofluorescence marker and a quenching fluorescence probe; the autofluorescence marker is mesoporous silicon dioxide containing organosilane functionalized carbon points, and the quenching fluorescence probe is a chelate of pure carbon points and nickel ions;

the double-fluorescence detection probe comprises an inner core and an outer shell, and the particle size is 200-300 nm; wherein the core is Fe₃O₄Nanoparticles, the shell of which is modified in the Fe₃O₄Mesoporous silica containing organosilane functionalized carbon dots on the outer surface of the nanoparticles;

the preparation method of the double-fluorescence detection probe comprises the following steps:

s1, synthesizing an autofluorescent marker:

s13, transferring the mixed solution A into a clear solution B, adding an organosilane functionalized carbon dot-ethanol solution under the stirring condition, carrying out magnetic separation, washing a product, and refluxing with an ammonium nitrate-ethanol solution to remove hexadecyl trimethyl ammonium bromide to obtain an autofluorescence marker;

s2, synthesizing magnetic nano particles coated by fluorescent silicon dioxide;

2. The dual-fluorescence detection probe for fluorine ions according to claim 1, wherein in step S3, the mass-to-volume ratio of the fluorescent silica-coated magnetic nanoparticles to the pure carbon dot solution is 1-20: 1 mg/mL.

3. The dual fluorescent fluoride ion detection probe of claim 1, wherein the hydrophilic Fe is at step S11₃O₄The preparation method of the core comprises the following steps:

s112, adding sodium acetate under the stirring condition until a uniform yellowish-brown solution is obtained, keeping the solution at the temperature of 150-250 ℃ for 5-15 hours, cooling the solution to room temperature, magnetically separating a black product, washing and drying the product to obtain the hydrophilic Fe₃O₄And (4) a core.

4. The dual fluorescent fluoride ion detection probe of claim 1, wherein the synthetic fluorescent silica-coated magnetic nanoparticles of step S2 comprise the steps of:

5. The dual-fluorescence detection probe for fluorine ions according to claim 4, wherein the time of the reflux in step S21 is 20-28 h; in the step S22, the refluxing time is 12-20 hours, and the refluxing temperature is 60-100 ℃.

6. The dual fluorescence detection probe of claim 4, wherein the acetic acid-ethanol solution in step S22 is 40% to 60% v/v acetic acid-ethanol solution.

7. The dual fluorescent detection probe of claim 4, wherein the nickel salt is Ni (NO) in step S23₃)₂、NiCl₂。

8. The dual fluorescence detection probe of claim 4, wherein the pH value in step S23 is 5-7.