CN108410449B - Preparation and application of water-soluble fluorescent silicon nanoparticles for detecting alkaline phosphatase - Google Patents
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Abstract
The invention discloses a preparation method and application of silicon nanoparticles for detecting alkaline phosphatase, and the preparation method comprises the steps of adding N- [3- (trimethoxysilyl) propyl ] ethylenediamine (DAMO) into water under stirring, then respectively adding hydroquinone, resorcinol and catechol, and stirring at room temperature and normal pressure to obtain yellow fluorescent silicon nanoparticles (yellow-Si NPs), blue fluorescent silicon nanoparticles (blue-Si NPs) and orange fluorescent silicon nanoparticles (orange-Si NPs). The prepared water-soluble orange fluorescent silicon nanoparticles can be used for high-selectivity sensitive detection of alkaline phosphatase (ALP).
Description
Technical Field
The invention belongs to the field of chemistry and chemical engineering, and particularly relates to preparation and application of water-soluble fluorescent silicon nanoparticles for detecting alkaline phosphatase.
Background
Alkaline phosphatase (ALP) is an enzyme capable of dephosphorylating a corresponding substrate and is widely distributed in various organs of the human body, most of which are liver, and secondly, tissues such as kidney, bone, intestine, and placenta. ALP detection is mainly used for detecting obstructive jaundice, primary liver cancer, secondary liver cancer, cholestatic hepatitis and the like. In these diseases, the hepatic cells overproduce ALP, enter the blood via the lymphatic channel and hepatic sinus, and simultaneously, the serum ALP is significantly increased due to bile excretion disorder in the hepatic and biliary tracts and reflux into the blood. Therefore, establishing a method for accurately detecting ALP in serum plays a crucial role in human health. To date, methods for detecting ALP based on CdS/CdTe/CdSe QDs, Au/Ag NCs, C dots, polymer probes, and small molecule probes have been developed. The detection material has the problems of low selectivity, complex preparation, high cost, high toxicity and the like, and compared with the silicon nanoparticles, the silicon nanoparticles have the advantages of simple preparation, low toxicity, strong stability, good biocompatibility, biodegradability, abundant and cheap raw materials, no need of complex modification and the like. Therefore, it is especially necessary to establish a new method for detecting ALP with high selectivity and high sensitivity by using silicon nanoparticles as probes. To date, the preparation of silicon nanoparticles has typically required high temperatures and pressures or complicated instrumentation. Most of the currently prepared silicon nanoparticles are blue fluorescence or green fluorescence, and no report is found about the preparation of silicon nanoparticles with long-wavelength fluorescence emission or fluorescence adjustability. Therefore, it is necessary to rapidly prepare the silicon nanoparticles with fluorescence adjustability by a simple and mild method at room temperature and normal pressure to realize the adjustment from blue fluorescence to yellow or red fluorescence.
Disclosure of Invention
In view of the disadvantages of the prior art and the problems described above, an object of the present invention is to provide a novel method for preparing water-soluble silicon nanoparticles having multi-color fluorescence tunability. On the basis, a novel method for simply, highly selectively and sensitively detecting alkaline phosphatase (ALP) is established by taking the silicon nanoparticles as fluorescent probes.
The invention provides a water-soluble fluorescent silicon nanoparticle for detecting alkaline phosphatase, which is synthesized from N- [3- (trimethoxysilyl) propyl ] ethylenediamine (DAMO) and benzenediol.
Further, the hydroquinone is hydroquinone, resorcinol or catechol.
Further, when the hydroquinone is hydroquinone, the preparation method of the water-soluble fluorescent silicon nanoparticle comprises the following steps:
(1) adding N- [3- (trimethoxysilyl) propyl ] ethylenediamine (DAMO) into water;
(2) adding hydroquinone and stirring to obtain yellow fluorescent water-soluble fluorescent silicon nanoparticles (yellow-Si NPs);
(3) and dialyzing the prepared water-soluble fluorescent silicon nanoparticles by using a dialysis bag of 500Da to obtain the purified water-soluble fluorescent silicon nanoparticles. Wherein the mol ratio of N- [3- (trimethoxysilyl) propyl ] ethylenediamine (DAMO) to hydroquinone is 1: 10.
further, when the hydroquinone is resorcinol, the preparation method of the water-soluble fluorescent silicon nano-particles comprises the following steps:
(1) adding N- [3- (trimethoxysilyl) propyl ] ethylenediamine (DAMO) into water;
(2) adding resorcinol and stirring to obtain blue fluorescent water-soluble fluorescent silicon nano particles (blue-Si NPs);
(3) and dialyzing the prepared water-soluble fluorescent silicon nanoparticles by using a dialysis bag of 500Da to obtain the purified water-soluble fluorescent silicon nanoparticles. Wherein the molar ratio of N- [3- (trimethoxysilyl) propyl ] ethylenediamine (DAMO) to resorcinol is 1: 140.
further, when the hydroquinone is catechol, the preparation method of the water-soluble fluorescent silicon nanoparticle comprises the following steps:
(1) adding N- [3- (trimethoxysilyl) propyl ] ethylenediamine (DAMO) into water;
(2) adding catechol and stirring to obtain orange fluorescent water-soluble fluorescent silicon nanoparticles (orange-Si NPs);
(3) and dialyzing the prepared water-soluble fluorescent silicon nanoparticles by using a dialysis bag of 500Da to obtain the purified water-soluble fluorescent silicon nanoparticles. Wherein the molar ratio of N- [3- (trimethoxysilyl) propyl ] ethylenediamine (DAMO) to catechol is 1: 6.
a method for detecting alkaline phosphatase comprising water-soluble fluorescent silica nanoparticles, comprising the steps of:
(1) adding 0.7mol/L of ascorbic acid 2-phosphate (AAP) and alkaline phosphatase (ALP) at different concentrations to Tris-HCl buffer solution with pH of 7.4, incubating in water bath at 37 deg.C for 25 min;
(2) sequentially adding the prepared orange fluorescent water-soluble fluorescent silicon nanoparticle (orange-Si NPs) solution and KMnO with the concentration of 10mM into the solution4A solution;
(3) setting the excitation wavelength to 481nm, measuring the fluorescence intensity at the emission wavelength of 558nm, making a standard working curve, and calculating the ALP content according to the fluorescence intensity and the standard curve.
Further, the above-described method for detecting alkaline phosphatase can be applied to the detection of alkaline phosphatase in serum.
The invention has the beneficial effects that:
(1) the method for preparing the water-soluble fluorescent silicon nano-particles is simple and low in cost.
(2) The water-soluble fluorescent silicon nano-particles prepared by the method have the advantages of low toxicity, water solubility, good stability, stronger salt resistance, photobleaching resistance, pH stability and good biocompatibility.
(3) The invention establishes a new method for detecting alkaline phosphatase in serum, and the method has good selectivity and high sensitivity.
Drawings
FIG. 1: TEM images and size distribution maps of the prepared nanoparticles:
wherein A is yellow fluorescent water-soluble fluorescent silicon nanoparticles (Y-Si NPs), B is orange fluorescent water-soluble fluorescent silicon nanoparticles (O-Si NPs), and C is blue fluorescent water-soluble fluorescent silicon nanoparticles (B-Si NPs);
FIG. 2: fourier transform infrared spectroscopy (FT-IR) of the nanoparticles prepared:
wherein line a represents orange fluorescent water-soluble fluorescent silicon nanoparticles (O-Si NPs), line B represents yellow fluorescent water-soluble fluorescent silicon nanoparticles (Y-Si NPs), and line c represents blue fluorescent water-soluble fluorescent silicon nanoparticles (B-Si NPs);
FIG. 3: excitation spectrum, emission spectrum and ultraviolet-visible absorption spectrogram of the prepared nano particles are as follows:
wherein A is an excitation spectrum (line a), an emission spectrum (line b) and an ultraviolet visible absorption spectrum (line c) of orange fluorescent water-soluble fluorescent silicon nanoparticles (O-Si NPs); b is an excitation spectrum (line a), an emission spectrum (line B) and an ultraviolet visible absorption spectrum (line c) of yellow fluorescent water-soluble fluorescent silicon nanoparticles (Y-Si NPs); c is an excitation spectrum (line a) and an emission spectrum (line B) of blue fluorescent water-soluble fluorescent silicon nanoparticles (B-Si NPs);
FIG. 4: the prepared water-soluble fluorescent silicon nanoparticles are used for detecting the fluorescence spectrogram and the standard curve of alkaline phosphatase:
wherein A is a fluorescence spectrogram obtained by adding alkaline phosphatase with different concentrations into orange fluorescence water-soluble fluorescent silicon nanoparticles (orange-Si NPs); b is a standard working curve;
Detailed Description
The technical solutions in the embodiments of the present invention will be described clearly and completely with reference to the drawings in the specification, and it is obvious that the described embodiments are only a part of the present invention, and not all of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1 preparation of Water-soluble fluorescent silicon nanoparticles and structural characterization thereof
(1) Preparation of yellow fluorescent water-soluble fluorescent silicon nanoparticles
1mL of N- [3- (trimethoxysilyl) propyl ] ethylenediamine (DAMO) was added to 5mL of water with stirring. Then 5.0mg of hydroquinone was added and stirred at room temperature and normal pressure for 25 minutes to obtain 1.13g of yellow fluorescent water-soluble fluorescent silicon nanoparticles (yellow-Si NPs). And dialyzing the prepared silicon nanoparticles for 10 hours by using a 500Da dialysis bag to obtain the purified silicon nanoparticles.
(2) Preparation of blue fluorescent water-soluble fluorescent silicon nanoparticles
1mL of N- [3- (trimethoxysilyl) propyl ] ethylenediamine (DAMO) was added to 5mL of water with stirring. Then, 70.0mg of resorcinol was added and stirred at room temperature and normal pressure for 25 minutes, whereby 0.97g of blue fluorescent silicon nanoparticles (blue-Si NPs) was obtained. And dialyzing the prepared silicon nanoparticles for 10 hours by using a 500Da dialysis bag to obtain the purified silicon nanoparticles.
(3) Preparation of orange fluorescent water-soluble fluorescent silicon nanoparticles
1mL of N- [3- (trimethoxysilyl) propyl ] ethylenediamine (DAMO) was added to 5mL of water with stirring. Then 3.0mg of catechol was added and stirred at room temperature under normal pressure for 2 hours to obtain 1.17g of orange fluorescent silicon nanoparticles (orange-Si NPs). And dialyzing the prepared silicon nanoparticles for 10 hours by using a 500Da dialysis bag to obtain the purified silicon nanoparticles.
A TEM image and a size distribution graph of the prepared nanoparticles as shown in fig. 1, wherein a is yellow fluorescent water-soluble fluorescent silicon nanoparticles (yellow-Si NPs), B is orange fluorescent water-soluble fluorescent silicon nanoparticles (orange-Si NPs), and C is blue fluorescent water-soluble fluorescent silicon nanoparticles (blue-Si NPs);
a fourier transform infrared (FT-IR) spectrum of the nanoparticles prepared as shown in fig. 2, wherein line a represents orange fluorescent water-soluble fluorescent silicon nanoparticles (orange-Si NPs), line b represents yellow fluorescent water-soluble fluorescent silicon nanoparticles (yellow-Si NPs), and line c represents blue fluorescent water-soluble fluorescent silicon nanoparticles (blue-Si NPs);
the excitation spectrum, emission spectrum and ultraviolet-visible absorption spectrum of the prepared nanoparticles are shown in fig. 3, wherein a is the excitation spectrum (line a), emission spectrum (line b) and ultraviolet-visible absorption spectrum (line c) of orange fluorescent water-soluble fluorescent silicon nanoparticles (orange-Si NPs); b is an excitation spectrum (line a), an emission spectrum (line B) and an ultraviolet visible absorption spectrum (line c) of yellow fluorescent water-soluble fluorescent silicon nanoparticles (yellow-Si NPs); c is an excitation spectrum (line a) and an emission spectrum (line b) of blue fluorescent water-soluble fluorescent silicon nanoparticles (blue-Si NPs).
The water-soluble fluorescent silica nanoparticles prepared as shown in FIG. 4 detected fluorescence spectra (shown in A) and standard work curves (shown in B) of alkaline phosphatase at different concentrations.
Example 2 a method for detecting alkaline phosphatase comprising water-soluble fluorescent silica nanoparticles
A method for detecting alkaline phosphatase comprising water-soluble fluorescent silica nanoparticles comprising:
(1) to 2.83mL of a 10mM Tris-HCl buffer solution at pH 7.4, 30. mu.L of 0.7mol/L Ascorbic Acid Phosphate (AAP) and alkaline phosphatase (ALP) at concentrations of 0U/L, 0.01U/L, 0.1U/L, 1U/L, 5U/L, 50U/L and 500U/L, respectively, were added, and incubated in a water bath at 37 ℃ for 25 minutes.
(2) To the above solution were added 20. mu.L of the orange fluorescent water-soluble fluorescent silicon nanoparticle (orange-Si NPs) solution prepared in example 1 and 90. mu.L of KMnO with a concentration of 10mM in this order4And (3) solution.
(3) The fluorescence intensity was measured at an excitation wavelength of 481nm and an emission wavelength of 558nm
(4) Making a standard working curve and calculating the content of ALP: with orange-Si NPs + KMnO4Fluorescence intensity of the solution F1；orange-Si NPs+KMnO4Fluorescence intensity of + AAP + ALP solution is F2(ii) a With F2-F1Is ordinate, logcALPIs a horizontal coordinate; the width of the slit of the excitation wavelength and the emission wavelength are both 5nm, and the fluorescence intensity and the standard curve F are obtained2-F1＝116.17logcALP100.52 calculating the ALP content.
EXAMPLE 3 detection of alkaline phosphatase in serum by a method for detecting alkaline phosphatase comprising Water-soluble fluorescent silica nanoparticles
(1) Purchased human serum samples were diluted 100-fold with Tris-HCl buffer solution (pH 7.4; concentration 10mM), 30. mu.L of ascorbic acid 2-phosphate (AAP) at a concentration of 0.7mol/L and ALP at a concentration of 50U/L, 100U/L and 150U/L, respectively, were added to the diluted solution to give a final total volume of 2.89mL, and incubated in a water bath at 37 ℃ for 25 minutes.
(2) To the above solution were added 20. mu.L of orange-Si NPs solution prepared in example 1, 90. mu.L of KMnO with a concentration of 10mM in sequence4And (3) solution.
(3) The excitation wavelength was set to 481nm, and the fluorescence intensity was measured at an emission wavelength of 558 nm.
(4) Making a standard working curve and calculating the content of ALP: with orange-Si NPs + KMnO4Fluorescence intensity of the solution F1；orange-Si NPs+KMnO4Fluorescence intensity of + AAP + ALP solution is F2(ii) a With F2-F1Is ordinate, logcALPIs a horizontal coordinate; the width of the slit of the excitation wavelength and the emission wavelength are both 5nm, and the fluorescence intensity F is measured1、F2And standard curve F2-F1＝116.17logcALP100.52 calculating the ALP content.
(5) The experimental results are as follows: table 1 the results of the measurement of alkaline phosphatase in serum using the water-soluble fluorescent silica nanoparticles prepared in example 1 are shown:
TABLE 1 determination of ALP in human serum samples
The experimental results prove that the method can sensitively detect the content of the alkaline phosphatase in the serum, meet the requirement of detecting the serum alkaline phosphatase in clinic and have certain guiding significance for clinical monitoring and analysis; the result of the invention for detecting alkaline phosphatase is more accurate, and the result obtained by the experiment is consistent with the result of the PNPP colorimetric method, thus proving that the method is reliable and feasible.
Claims (10)
1. The water-soluble fluorescent silicon nanoparticles are used for detecting alkaline phosphatase and are characterized in that the water-soluble fluorescent silicon nanoparticles are synthesized by N- [3- (trimethoxysilyl) propyl ] ethylenediamine and benzenediol.
2. The method of claim 1, wherein the hydroquinone is hydroquinone, resorcinol, or catechol.
3. The method of claim 1, wherein the hydroquinone is hydroquinone, and the method of preparing the water-soluble fluorescent silica nanoparticles comprises:
(1) adding N- [3- (trimethoxysilyl) propyl ] ethylenediamine into water;
(2) adding hydroquinone and stirring to obtain yellow fluorescent water-soluble fluorescent silicon nanoparticle yellow-Si NPs;
(3) and dialyzing the prepared water-soluble fluorescent silicon nanoparticles by using a dialysis bag of 500Da to obtain the purified water-soluble fluorescent silicon nanoparticles.
4. The method of claim 3, wherein the molar ratio of N- [3- (trimethoxysilyl) propyl ] ethylenediamine to hydroquinone is 1: 10.
5. the method of claim 1, wherein the hydroquinone is resorcinol, and the method of preparing the water-soluble fluorescent silica nanoparticles comprises:
(1) adding N- [3- (trimethoxysilyl) propyl ] ethylenediamine into water;
(2) adding resorcinol and stirring to obtain blue fluorescent water-soluble fluorescent silicon nano-particles blue-Si NPs;
(3) and dialyzing the prepared water-soluble fluorescent silicon nanoparticles by using a dialysis bag of 500Da to obtain the purified water-soluble fluorescent silicon nanoparticles.
6. The method of claim 5, wherein the molar ratio of N- [3- (trimethoxysilyl) propyl ] ethylenediamine (DAMO) to resorcinol is 1: 140.
7. the method of claim 1, wherein the catechol is catechol, and the method of preparing the water-soluble fluorescent silica nanoparticles comprises:
(1) adding N- [3- (trimethoxysilyl) propyl ] ethylenediamine into water;
(2) adding catechol and stirring to obtain orange fluorescent water-soluble fluorescent silicon nano-particles orange-Si NPs;
(3) and dialyzing the prepared water-soluble fluorescent silicon nanoparticles by using a dialysis bag of 500Da to obtain the purified water-soluble fluorescent silicon nanoparticles.
8. The method as claimed in claim 2, wherein the molar ratio of N- [3- (trimethoxysilyl) propyl ] ethylenediamine to catechol is 1: 6.
9. a method for detecting alkaline phosphatase using water-soluble fluorescent silica nanoparticles, comprising the steps of:
(1) adding 30 μ L of 0.7mol/L vitamin C phosphate AAP and alkaline phosphatase ALP with different concentrations into 2.83mL of 10mM Tris-HCl buffer solution with pH 7.4, placing in a water bath at 37 ℃ and incubating for 25 min;
(2) to the above solution were added 20. mu.L of orange-Si NPs solution prepared according to claim 7, 90. mu.L of 10mM KMnO in sequence4A solution;
(3) setting the slit width of excitation and emission to be 5nm and the excitation wavelength to be 481nm, and measuring the fluorescence intensity orange-Si NPs + KMnO at the emission wavelength of 558nm4The fluorescence intensity of the solution isF 1，orange-Si NPs + KMnO4The fluorescence intensity of the + AAP + ALP solution wasF 2To do so byF 2-F 1Is ordinate, logc ALPFor the abscissa, a standard working curve was made.
10. The method for detecting alkaline phosphatase comprising the water-soluble fluorescent silica nanoparticles according to claim 9, wherein the probe for detecting alkaline phosphatase is applicable to the detection of alkaline phosphatase in serum, comprising the steps of:
(1) the purchased human serum samples were diluted 100-fold with 10mM Tris-HCl buffer solution, pH 7.4, and 30. mu.L of 0.7mol/L AAP and 30. mu.L of ALP at various concentrations were added to the diluted human serum samples to a final total volume of 2.89mL, and incubated in a 37 ℃ water bath for 25 minutes;
(2) to the above solution were added 20. mu.L of the prepared orange-Si NPs solution, 90. mu.L of 10mM KMnO in this order4A solution;
(3) setting the width of a slit for excitation and emission to be 5nm and the excitation wavelength to be 481nm, and measuring the fluorescence intensity at the emission wavelength to be 558 nm;
(4) the ALP content was calculated from the fluorescence intensity and the standard curve.
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| One-Pot Synthesis of Fluorescent Silicon Nanoparticles for Sensitive and Selective Determination of 2,4,6-Trinitrophenol in Aqueous Solution;Yangxia Han，等;《Anal. Chem.》;20170210;第89卷;3001-3008 * |
| Tuning surface states to achieve the modulated fluorescence of carbon dots for probing the activity of alkaline phosphatase and immunoassay of α-fetoprotein;Xin Fang，等;《Sensors and Actuators B: Chemical》;20171103;第257卷;620-628 * |
| Yangxia Han，等.One-Pot Synthesis of Fluorescent Silicon Nanoparticles for Sensitive and Selective Determination of 2,4,6-Trinitrophenol in Aqueous Solution.《Anal. Chem.》.2017,第89卷3001-3008. * |
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