Fluorescent probe as well as preparation method and application thereof

Abstract

The invention relates to the technical field of fluorescent probes, in particular to a fluorescent probe as well as a preparation method and application thereof, the fluorescent probe is silicon quantum dots (SiQDs), the fluorescent probe is blue under ultraviolet radiation and has an obvious absorption peak and a symmetrical fluorescence peak at 357nm and 461nm respectively, and the position of the fluorescence peak is irrelevant to excitation wavelength. The fluorescent probe provided by the invention is a blue fluorescent water-soluble silicon quantum dot with a large amount of amino groups and hydroxyl groups on the surface, and has excellent light stability and pH stability. H2O2 can effectively quench fluorescence of the fluorescent probe, a good linear relation exists between the fluorescence quenching rate and the logarithm of the H2O2 concentration, H2O2 can be quantitatively detected, the detection system has the advantage of being high in anti-interference capacity, and the established fluorescence detection method is successfully used for analyzing H2O2 in human serum and has good application prospects. The method is expected to be expanded to imaging analysis of active oxygen H2O2 in cells subsequently.

Description

Technical Field

[0001] The present invention relates to the technical field of fluorescent probes, and in particular to a fluorescent probe, a preparation method and application thereof. Background Art

[0002] Hydrogen peroxide (H 2 O 2 ) is considered to be one of the most important reactive oxygen species. It plays a very important regulatory role in living organisms. Excessive levels can cause damage to DNA, RNA and proteins, and even lead to cell death, skin damage, cancer, Alzheimer's disease, Parkinson's disease, etc. Therefore, the establishment and development of quantitative detection of H 2 O 2 The method of detecting H 2 O 2 The methods include chemiluminescence, colorimetry, chromatography and fluorescence spectrophotometry. Among them, fluorescence spectrophotometry has the advantages of high sensitivity, simple operation and rapidity, and is increasingly used in the field of biochemical detection.

[0003] In recent years, researchers have designed and constructed various new types of H 2 O 2 Fluorescent probes. For example, researchers prepared coumarin derivative probes containing boron ester groups, which react with H 2 O 2 After the reaction, blue fluorescence is generated, and the intensity of the fluorescence signal is related to the concentration range of H 2 O 2 Quantum dots (QDs) have the advantages of wide excitation wavelength range, narrow emission wavelength range, and high quantum yield, and are widely used in biological imaging and fluorescence detection. For example, Zhou et al. used H 2 O 2 Ag generated by silver oxide nanoclusters + It can quench the fluorescence of CdSe@ZnS QDs and establish a detection method for H 2 O 2 A new method for fluorescence detection (Anal. Chim. Acta, 2020, 1106: 176–182). However, the QDs used in this work contain toxic heavy metal Cd, which will pollute the environment. With the advancement of nanotechnology, the development of low-toxic nanomaterials has become one of the important research hotspots in the field of fluorescence detection.

[0004] SiQDs are zero-dimensional nanomaterials with ultra-small particle sizes. They have lower toxicity than group II–VI, III–V, etc. QDs and possess advantages such as excellent optical properties, good biocompatibility, and controllable surface functionalization. These characteristics make SiQDs very likely to replace traditional Cd-containing QDs and become a highly promising "green" luminescent material. In the past decade, researchers have synthesized water-soluble SiQDs suitable for the biochemical field using various methods. For example, the research group of He Zhike at Wuhan University synthesized blue-fluorescent water-soluble SiQDs (2.5 nm) with amino groups on the surface by the hydrothermal method and further used them for the detection and imaging analysis of targets in cells (Anal. Chem., 2018, 90: 9796–9804). The research group of He Yao at Soochow University developed an ultraviolet and microwave-assisted method to prepare blue-to-red fluorescent SiQDs (1.8–3.8 nm) in an aqueous phase and applied them to cell and in vivo imaging studies (Nano Today, 2019, 26: 149–163). However, current biological applications of SiQDs mainly focus on fluorescence imaging, and there are few applications for detecting molecules and ions. Summary of the Invention

[0005] One object of the present invention is to provide a fluorescent probe that is easily soluble in water and has excellent photostability and pH stability.

[0006] Another object of the present invention is to provide a preparation method for the fluorescent probe, which is simple and easy to adjust.

[0007] A third object of the present invention is to provide an application of the fluorescent probe. Based on fluorescence quenching, a new method for detecting H 2 O 2 has been established.

[0008] A fourth object of the present invention is to provide another application of the fluorescent probe.

[0009] The solution adopted by the present invention to achieve the first object is: a fluorescent probe, the fluorescent probe is silicon quantum dots SiQDs, the fluorescent probe appears blue under ultraviolet irradiation, has an obvious absorption peak and a symmetric fluorescence peak at 357 nm and 461 nm respectively, and the position of the fluorescence peak is independent of the excitation wavelength.

[0010] Preferably, when the excitation light is 330 nm, the fluorescent probe has the strongest fluorescence intensity.

[0011] Preferably, the fluorescent probe is water-soluble silicon quantum dots, and its surface is rich in amino groups and hydroxyl groups.

[0012] The second solution adopted to achieve the object of the present invention is: A method for preparing the fluorescence probe, comprising the following steps: At room temperature, dissolve a certain amount of sodium ascorbate in ultrapure water, then add a certain volume of diethylenetriaminepropyltrimethoxysilane, mix evenly, react completely at a certain temperature, after the reaction is completed, cool to room temperature, purify the obtained solution, centrifuge to collect the precipitate, and dry to obtain the fluorescence probe.

[0013] Preferably, the mass-volume ratio of sodium ascorbate to diethylenetriaminepropyltrimethoxysilane is 100-130 mg: 1 mL.

[0014] Preferably, the mass-volume ratio of sodium ascorbate to diethylenetriaminepropyltrimethoxysilane is 100 mg: 1 mL

[0015] Preferably, the reaction temperature is 180-220 °C.

[0016] Preferably, the reaction temperature is 200 °C.

[0017] Preferably, the obtained solution is purified with acetonitrile.

[0018] The third solution adopted to achieve the object of the present invention is: An application of the fluorescence probe, using the fluorescence probe to detect H in a liquid environment 2 O 2 .

[0019] The fourth solution adopted to achieve the object of the present invention is: An application of the fluorescence probe, using the fluorescence probe to detect H in a serum environment 2 O 2 .

[0020] Preferably, the fluorescence probe is used for quantitative detection of H 2 O 2 , and the detection limit is 1.7 μM.

[0021] The present invention has the following advantages and beneficial effects:

[0022] The fluorescence probe of the present invention is a blue fluorescent water-soluble SiQDs with a large number of amino and hydroxyl groups on the surface, having excellent photostability and pH stability.

[0023] H 2 O 2 can effectively quench the fluorescence of the fluorescence probe SiQDs of the present invention, and there is a good linear relationship between the fluorescence quenching rate and the logarithm of the H 2 O 2 concentration (10–1000 μM), and it can quantitatively detect H 2 O 2, and this detection system has the advantage of strong anti-interference ability. The established fluorescence detection method has been successfully applied to the analysis of H in human serum. 2 O 2 The spiked recovery rate is 92% - 102%, and it is expected to be extended to the imaging analysis of reactive oxygen species H 2 O 2 in cells in the future.

[0024] The preparation method of the present invention has a simple process and is easy to adjust. Water-soluble SiQDs have been successfully prepared by the hydrothermal method. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 is a schematic diagram of the synthesis of SiQDs (silicon quantum dots);

[0026] Figure 2 is the optical detection diagram of SiQDs; among them, (A) UV-visible absorption spectrum and fluorescence emission spectrum of SiQDs solution (the inset shows the luminescence of SiQDs solution under natural light (left) and ultraviolet light (right)); (B) fluorescence emission spectrum of SiQDs solution at different excitation wavelengths; (C) photostability of SiQDs solution; (D) TEM image of SiQDs (the inset is the HRTEM image of SiQDs); (E) particle size distribution diagram of SiQDs; (F) XRD spectrum of SiQDs;

[0027] Figure 3 is the detection diagram of the composition and surface groups of SiQDs, among which (A) FTIR spectrum of SiQDs; (B) full-scan XPS spectrum of SiQDs; (C) XPS spectrum of Si2p; (D) XPS spectrum of C1s; (E) XPS spectrum of N1s; (F) XPS diagram of O1s;

[0028] Figure 4 is the analysis diagram of the influence of pH on SiQDs, among which (A) fluorescence spectra of SiQDs in different pH buffers; (B) influence of pH on the fluorescence intensity of SiQDs at 461 nm;

[0029] Figure 5 is the analysis diagram of the influence of ionic strength on SiQDs;

[0030] Figure 6 is the schematic diagram of SiQDs for detecting H 2 O 2 ;

[0031] Figure 7 is the schematic diagram of the result of the interaction between SiQDs and H 2 O 2 , among which (A) the interaction between SiQDs and H 2 O2 Fluorescence spectra at different times of action; (B) Influence of the action time on the fluorescence intensity of SiQDs;

[0032] Figure 8 Schematic diagram of the results of the fluorescence intensity of SiQDs (black column) and its co-action with H 2 O 2 for 2 h (red column) in the presence of interfering substances, where 1: Blank, 2: 150 mM KCl, 3: 50 μM FeCl 3 ·6H 2 O, 4: 50 μM Zn(NO 3 ) 2 ·6H 2 O, 5: 50 μM Cu(NO 3 ) 2 ·3H 2 O, 6: 50 μM NiCl 2 ·6H 2 O, 7: 25 μM (CH 3 COO) 2 Co·4H 2 O, 8: 10 μM AlCl 3 , 9: 10 mM CaCl 2 , 10: 20 mM MgCl 2 ·6H 2 O, 11: 20 mM Glucose, 12: 80 μM histidine, 13: 80 μM cysteine, 14: 100 mM NaCl (10 mM PBS buffer, pH 7.4);

[0033] Figure 9 Schematic diagram of the performance analysis results for the detection of H 2 O 2 by SiQDs, where (A) Variation of the fluorescence intensity of SiQDs with different concentrations (0–2000 μM) of H 2 O 2 ; (B) Variation of the fluorescence quenching rate of SiQDs with lg[C H2O2 . Specific embodiments

[0034] To better understand the present invention, the following examples are further descriptions of the present invention, but the content of the present invention is not limited to the following examples.

[0035] Table 1 shows the main experimental instruments in the examples of the present invention.

[0036] Table 1 Main experimental instruments

[0037]

[0038]

[0039] Table 2 shows the main experimental reagents used in the embodiments of the present invention.

[0040] Table 2 Main Experimental Reagents

[0041]

[0042] Example 1

[0043] As Figure 1 shown, a preparation method of a fluorescent probe includes the following steps. At room temperature, 100 mg of sodium ascorbate is dissolved in 4 mL of ultrapure water, then 1 mL of AEEA (diethylenetriaminepropyltrimethoxysilane) is added, and the mixture is stirred for 10 min. The obtained light yellow solution is transferred to a reaction kettle and heated at 200 °C for 5.5 h. After cooling to room temperature, the SiQDs solution is purified with a suitable organic reagent, acetonitrile, and the volume ratio of acetonitrile to the SiQDs solution is 4:1. The precipitate is collected by centrifugation (8000 rpm, 3 min), and the obtained product is vacuum dried at 60 °C for 24 h. Subsequently, the SiQDs are stored in an environment at 4 °C.

[0044] Optical Properties and Morphology Characterization of SiQDs

[0045] The optical properties of SiQDs were studied by ultraviolet lamp irradiation, fluorescence spectroscopy, and ultraviolet–visible absorption spectroscopy. Under daylight, the SiQDs solution is light yellow and transparent, while under 365 nm ultraviolet irradiation, it is bright blue ( Figure 2 A, inset), indicating that SiQDs have good water solubility and fluorescence properties. In addition, as Figure 2 shown in Figure 2 A, the SiQDs solution has an obvious absorption peak at 357 nm and a symmetric fluorescence peak at 461 nm, and the position of the fluorescence peak is independent of the excitation wavelength. When the excitation wavelength is 330 nm, the fluorescence intensity of SiQDs is the strongest ( Figure 2 B). At the optimal excitation wavelength, using quinine sulfate (0.1 M sulfuric acid, QY = 58%) as a standard, the quantum yield of SiQDs in aqueous solution was calculated to be 19.5%. We further studied the photostability of SiQDs. After continuous irradiation with xenon light for 442 min, the fluorescence intensity of the SiQDs solution still remained at 95% of its initial intensity (

[0046] C), indicating that the prepared SiQDs have good anti-photobleaching ability. Figure 2D is the TEM image of SiQDs. The SiQDs are spherical particles with an average diameter of 3.2 ± 0.6 nm( Figure 2 E). The high-resolution transmission electron microscopy (HRTEM) image shows that some SiQDs have a lattice structure with a lattice spacing of 0.19 nm. The XRD spectrum shows that most SiQDs are amorphous structures( Figure 2 F).

[0047] Composition and surface groups of SiQDs

[0048] To further explore the composition elements and surface groups of SiQDs, FTIR and XPS characterizations were performed on them. Figure 3 A is the FTIR image of SiQDs. The main absorption peaks are analyzed as follows: The absorption bands at 3405–3298 cm -1 and 1125–1041 cm -1 are the stretching vibrations of N–H / O–H and Si–O–Si, respectively; The absorption peaks at 2935, 1631, 1473, 1315, 929, 763 and 692 cm -1 are the C–H stretching vibration, N–H bending vibration, C–H bending vibration, C–N stretching vibration, Si–OH stretching vibration, N-H rocking vibration and vibration of secondary amine in SiQDs, respectively. In the full-scan XPS spectrum of SiQDs( Figure 3 B), four elements can be observed, including Si (8.7%, 101.6 eV, 153.1 eV), C (59.4%, 284.5 eV), N (12.5%, 398.5 eV) and O (19.4%, 530.9 eV). In addition, the XPS spectra of Si 2p, C 1s, N 1s and O 1s were measured and peak shape fitting was performed. In the XPS spectrum of Si 2p( Figure 3 C), three fitting peaks are shown, corresponding to Si-C (101.1 eV), Si-N (101.8 eV) and Si-O (103.0 eV), respectively. In the XPS spectrum of C 1s( Figure 3 D), it includes C-Si (283.8 eV), C-C / C=C (284.5 eV), C-N / C-O (285.5 eV) and C=O (287.4 eV) groups. In the XPS spectrum of N 1s( Figure 3 E), the three peaks at 398.0, 398.6 and 400.2 eV are N-Si, C-N-C and N-H, respectively. In the XPS spectrum of O 1s( Figure 3F), the two peaks at 530.1 eV and 531.2 eV represent Si-O, while the peaks at 532.0 eV and 534.7 eV represent C-O and O-H, respectively. The characterization results of XPS are consistent with those of FTIR. It can be concluded therefrom that the surface of SiQDs is rich in amino and hydroxyl groups, and these hydrophilic groups endow it with good water solubility.

[0049] pH and ionic strength

[0050] The effects of pH and ionic strength on the fluorescence of SiQDs were further investigated. As Figure 4 shown, the fluorescence signal of SiQDs does not change with pH (pH 5.24 - 8.75), indicating that the prepared SiQDs have good pH stability. The reason for its insensitivity to pH may be that the positively charged amino groups and negatively charged hydroxyl groups on the surface of SiQDs balance each other, thus being less affected by pH. In addition, the fluorescence signal of SiQDs remains unchanged in PBS (5 - 400 mM) buffer solutions with different ionic strengths ( Figure 5 ). These results suggest that SiQDs may be applicable for detecting target substances in physiological environments.

[0051] Example 2

[0052] H 2 O 2 detection

[0053] As Figure 6 shown, 50 mg of SiQDs was ultrasonically dispersed in 1 mL of ultrapure water. Then, 10 μL of the SiQDs solution was mixed with different concentrations of H 2 O 2 and diluted to 1 mL with PB solution (10 mM, pH 7.4). Then it was mixed well, and after standing at room temperature for 2 h, the fluorescence spectrum of the resulting solution was measured with a fluorescence spectrophotometer, and the experiment was repeated three times in parallel.

[0054] It was found experimentally that H 2 O 2 can effectively quench the fluorescence of SiQDs ( Figure 7 ). After interacting for 10 min, the quenching rate can reach 60%. As the interaction time increases, the fluorescence intensity of SiQDs gradually decreases and stabilizes after 2 h, and the quenching rate reaches more than 90%, with the most obvious quenching effect. Therefore, 2 h was selected for subsequent experiments.

[0055] Selectivity

[0056] It was further investigated whether the SiQDs probe can selectively respond to H 2 O 2. Under physiological pH (10 mM PBS buffer, pH 7.4), some common substances have basically no effect on the fluorescence signal of SiQDs, and the response of SiQDs to H 2 O 2 is not affected by interfering substances ( Figure 8 ). This indicates that the SiQDs probe has good anti-interference ability and selectivity.

[0057] Analytical performance

[0058] Under the optimal experimental conditions, the analytical performance of SiQDs for detecting H 2 O 2 was investigated. As Figure 9 shown, as the concentration of H 2 O 2 increases, the fluorescence intensity of SiQDs decreases (Figure 9A); when the concentration of H 2 O 2 is 10–1000 μM, there is a good linear relationship between the fluorescence quenching rate of SiQDs and the logarithm of the concentration of H 2 O 2 ( Figure 9 Figure 9B). In addition, the detection limit of this method is 1.7 μM, and the relative standard deviation is 4.5% (C = 20 μM, n = 7). Comparing this method with other methods for fluorescence detection of H 2 O 2 based on nanomaterials, the linear range and detection limit of this method are comparable to some extent (Table 3).

[0059] Table 3 Comparison of this method with other fluorescence sensing methods

[0060]

[0061] Example 3

[0062] To investigate the applicability of the SiQDs probe, the probe was used to detect H 2 O 2 in 10% human serum samples, and the results are shown in Table 4. The spiked recovery rate of the target H 2 O 2 in the samples is in the range of 92%–102%, indicating that the SiQDs probe can be used for the detection of H 2 O 2 in relatively complex matrices.

[0063] Table 4 Analytical detection of H 2 O 2 in human serum

[0064]

[0065]

[0066] The present invention prepares water-soluble SiQDs by a hydrothermal method and conducts systematic characterization on them, including morphology, composition, surface groups, optical properties, etc. The prepared SiQDs have excellent photo-stability and pH-stability. Further, based on fluorescence quenching, a new method for detecting H 2 O 2 is established. This method has the advantages of high sensitivity, good selectivity, and strong anti-interference ability, and realizes the quantitative analysis of H 2 O 2 in human serum samples. Subsequently, it is expected to be extended to the imaging analysis of reactive oxygen species H 2 O 2 in cells.

[0067] The above is the preferred embodiment of the present invention. Of course, the scope of the rights of the present invention cannot be limited thereby. It should be pointed out that for those of ordinary skill in the art of this technology, without departing from the principle of the present invention, several improvements and changes can still be made, and these improvements and changes are also regarded as the protection scope of the present invention.

Claims

1. A fluorescent probe, characterized in that: The fluorescent probe is silicon quantum dots SiQDs, which are blue under ultraviolet irradiation, and have an obvious absorption peak and a symmetrical fluorescence peak at 357nm and 461nm respectively, and the position of the fluorescence peak is independent of the excitation wavelength.

2. The fluorescent probe according to claim 1, characterized in that: When the excitation light is 330 nm, the fluorescence intensity of the fluorescent probe is the strongest.

3. The fluorescent probe according to claim 1, characterized in that: The fluorescent probe is a water-soluble silicon quantum dot, the surface of which is rich in amino groups and hydroxyl groups.

4. A method for preparing a fluorescent probe as claimed in any one of claims 1 to 3, characterized in that: The method comprises the following steps: dissolving a certain amount of sodium ascorbate in ultrapure water at room temperature, then adding a certain volume of diethylenetriaminopropyltrimethoxysilane, mixing evenly, reacting completely at a certain temperature, cooling to room temperature after the reaction is completed, purifying the obtained solution, collecting the precipitate by centrifugation, and drying to obtain the fluorescent probe.

5. The method for preparing a fluorescent probe according to claim 4, characterized in that: The mass volume ratio of the sodium ascorbate to diethylenetriaminopropyltrimethoxysilane is 100-130 mg:1 mL.

6. The method for preparing a fluorescent probe according to claim 4, characterized in that: The reaction temperature is 180-220°C.

7. The method for preparing a fluorescent probe according to claim 4, characterized in that: The resulting solution was purified using acetonitrile.

8. Use of the fluorescent probe according to any one of claims 1 to 3 or the fluorescent probe prepared by the preparation method according to any one of claims 4 to 7, characterized in that: The fluorescent probe is used to detect H2O2 in a liquid environment.

9. Use of the fluorescent probe according to any one of claims 1 to 3 or the fluorescent probe prepared by the preparation method according to any one of claims 4 to 7, characterized in that: The fluorescent probe was used to detect H2O2 in a serum environment.

10. The use according to claim 8 or 9, characterized in that: The fluorescent probe was used to quantitatively detect H2O2 with a detection limit of 1.7 μM.

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