CN108676554B - Composite nano probe and preparation method and application thereof - Google Patents
Composite nano probe and preparation method and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of analytical chemistry, and particularly relates to a composite nano probe, a preparation method and an application thereof, wherein arginine (Arg) is detected based on the combination of negatively charged carbon nano particles and positively charged probe 1 through electrostatic attraction by Fluorescence Resonance Energy Transfer (FRET), the composite nano probe has high sensitivity (detection limit of 64.7 nanomole) and good selectivity and anti-interference capability, and the concentration range of arginine can be quantitatively detected by the probe from 60 to 270 micromole.
Description
Technical Field
The invention belongs to the technical field of analytical chemistry, and particularly relates to a composite nanoprobe, and a preparation method and application thereof.
Background
Amino acids have been intensively studied as the basic units of biological macromolecules and as essential components of proteins and life processes, the selective recognition and analysis of amino acids at the molecular level is of great importance in particular for the medical diagnosis of diseases, among all amino acids, arginine, a direct physiological precursor of nitric oxide, urea ornithine and agmatine, plays a key role in many biological functions such as cell division, wound healing, immune function, vasodilation and hormone release, this may cause some diseases or even life threatening if the arginine derivative is not present at the correct level, e.g. an excess of arginine may increase gastric acid levels, causing allergic reactions, but if arginine is absent, possibly resulting in blood ammonia imbalance and even coma, therefore, arginine may be used as an index for some diseases, due to the above factors, development of a feasible method for clinical arginine detection has important significance for disease diagnosis.
The major methods for detecting arginine at present are high performance liquid chromatography and gas chromatography, however, these methods usually involve professional and expensive equipment, complicated sample pretreatment and long measurement time, and therefore, many researchers try to design chemical sensors for detecting arginine, but the sensors for arginine at present are still very rare.
Disclosure of Invention
The invention aims to provide a ratiometric fluorescence detection arginine composite nano probe based on fluorescence resonance organic molecule modification, and a preparation method and application thereof.
In order to achieve the purpose, the invention adopts the technical scheme that:
a composite nano probe is prepared by combining carbon nano particles with negative charges and a probe 1 with positive charges through electrostatic attraction.
A preparation method of a composite nano probe comprises the following steps:
(1) preparation of probe 1: firstly, 8-hydroxy-2-methylquinoline reacts with acryloyl chloride at room temperature to obtain a compound 4, then the compound 4 is subjected to oxidation reaction with SeO2 at 101 ℃ to generate a compound 3, 1, 2-trimethylbenzo [ e ] indole reacts with CH3I while the compound 4 is prepared to obtain a compound 2, and finally the compound 3 and the compound 2 are subjected to Knoevenagel condensation reaction to obtain a probe 1;
(2) synthesizing carbon nanoparticles: firstly, dissolving citric acid in water, adding 1000 muL of ethylenediamine into the water, stirring the mixture to uniformly mix the mixture, transferring the solution into a hydrothermal kettle, reacting the solution in an oven at 200 ℃ for 4 hours, cooling the reaction product to room temperature to obtain a brown solution, dialyzing the solution for 48 hours by using a dialysis membrane with the molecular weight cutoff of 3000, precipitating the solution twice by using absolute ethyl alcohol, centrifuging the precipitate, and drying the obtained brown solid in a vacuum drying oven for 18 hours to obtain carbon nanoparticles;
(3) preparing a composite nano probe: placing the carbon nano-particles and the probe 1 into a volumetric flask, using ultrapure water and dimethyl sulfoxide with the volume ratio of 10:1-500:1 to perform constant volume, performing ultrasonic dissolution for 10-60 minutes, centrifuging for 3-15 minutes at 10000 r/min, and filtering to obtain the carbon nano-particles.
Further, the chemical synthesis route of the probe 1 in the step (1) is
Further, the preparation method of the compound 4 in the step (1) is as follows: adding 8-hydroxy-2-methylquinoline into a three-necked bottle, adding 15-30ml of dry dichloromethane solvent, adding 1.4-2ml of triethylamine, placing the three-necked bottle in an ice water bath, stirring for 30-60 minutes, slowly dropwise adding 4-10ml of acryloyl chloride into the reaction solution, then removing the ice water bath, stirring and reacting at room temperature for 12-36 hours, filtering after the reaction is finished, distilling the obtained filtrate under reduced pressure to obtain a crude product, and separating the crude product by column chromatography, wherein the developing agent is ethyl acetate/petroleum ether with the volume ratio of 1:4-1:10 to obtain a light yellow oily liquid compound 4, and the yield is 47.3-57.8%.
Further, the preparation method of the compound 3 in the step (1) is as follows: adding the compound 4 into a three-necked bottle, adding 13-20ml of 1, 4-dioxane, slowly heating to 65 ℃, adding selenium dioxide into the reaction liquid, continuously heating to 101 ℃, performing reflux reaction for 4-12 hours, filtering the reaction liquid while the reaction liquid is hot after the reaction is finished, adding silica gel into the filtrate, performing reduced pressure distillation to obtain a crude product, purifying the obtained crude product by a column chromatography separation method, wherein the granularity of the silica gel is 200 meshes and 300 meshes, the developing agents are ethyl acetate and petroleum ether, the volume ratio of the ethyl acetate to the petroleum ether is 1:8-1:35, performing rotary evaporation to obtain a white flocculent solid compound 3, and the yield is 71.6-80.7%.
Further, the preparation method of the compound 2 in the step (1) is as follows: dissolving 1,1, 2-trimethylbenzo [ e ] indole and methyl iodide in 15-20ml of acetonitrile, heating the mixture to 45 ℃, reacting for 13-24 hours, cooling the mixture to room temperature, pouring 40-70ml of diethyl ether into the reaction solution, and filtering to obtain the compound 2, wherein the yield is 52.6-68.2%.
Further, the preparation method of the probe 1 in the step (1) is as follows: dissolving the compound 2 and the compound 3 in ethanol, heating to 78 ℃, carrying out reflux reaction for 12-52 hours, cooling the reaction mixture to room temperature, and filtering to obtain a brown solid probe 1 with the yield of 45.1-66.5%.
The composite nano probe can be used for quantitatively detecting arginine in a near-infrared fluorescence ratio in a solution, and the concentration range of the detected arginine is 60-270 mu mol within the pH range of 5-11.
The invention has the advantages that: the invention detects arginine (Arg) based on the combination of Fluorescence Resonance Energy Transfer (FRET) with negatively charged carbon nanoparticles and positively charged probe 1 through electrostatic attraction, and has high sensitivity (detection limit of 64.7 nanomole) and good selectivity and anti-interference capability; the probe can quantitatively detect the concentration range of arginine from 60 to 270 micromolar, and in a titration curve of arginine drawn by a linear function of a composite nano probe ratio emission spectrum and the arginine concentration, the ratio of an emission peak at 440 nm to an emission peak at 607 nm can keep a good linear relation in the range of 60 to 270 mu M of arginine; the composite fluorescent probe can detect arginine changes in a wide pH range (pH from 5 to 11), which covers the pH range in organisms where various organic biomolecules Thr, Val, Ser, Phe, His, Gly, Pro, IIe, Trp, Ala, Tyr, Glu, Glc, Leu, and H are present2O2The method has the advantages that the method does not interfere detection and has good selectivity; the absorption spectrum of the gradually added arginine in the solution of the composite probe CDs-1 can be seen along withThe addition of arginine shifts the position of the absorption peak of the solution by more than 150 nm, and the color of the solution changes from yellow to colorless.
Drawings
FIG. 1 composite nanoprobe (containing 0.67. mu.g/ml carbon dots and 3X 10)−5Molar compound 1) emission spectrum of water-dimethyl sulfoxide solution (volume ratio of water to dimethyl sulfoxide is 100: 1) to titrate arginine aqueous solution (equivalent ratio to compound 1 is 0-10), excitation wavelength is 360 nm, and reaction time is 12 minutes);
FIG. 2 shows a composite probe (containing 0.67. mu.g/ml carbon dots and 3X 10−5Molal Compound 1) in water-dimethyl sulfoxide solution (volume ratio 100: 1) to detect arginine with different concentrations (0, 3 × 10 from left to right respectively−5Mol/l, 6X 10−5Mole/liter, 9X 10−5Mol/l, 12X 10−5Mol/l, 15X 10−5Mole/liter, 18X 10−5Mol/l, 21X 10−5Mol/l, 24X 10−5Mol/l, 27X 10−5Mole/liter) with an excitation wavelength of 365 nm;
FIG. 3 is a titration curve of arginine plotted as a linear function of the composite nanoprobe ratiometric emission spectrum and arginine concentration;
FIG. 4 fluorescence change of composite nanoprobes before and after arginine addition to water-dimethyl sulfoxide solution (100: 1 by volume) with different pH values (0.67. mu.g/ml carbon dots and 3X 10−5Mole/liter of compound 1);
FIG. 5 shows the selectivity and anti-interference performance of composite nanoprobes on arginine, and the organic biomolecules are Thr, Val, Ser, Phe, His, Gly, Pro, IIe, Trp, Ala, Tyr, Glu, Glc, Leu, and H2O2；
FIG. 6 is a photograph of different solutions of organic biomolecules, which are Thr, Val, Ser, Phe, His, Gly, Pro, IIe, Trp, Ala, Tyr, Glu, Glc, Leu, and H, added after arginine is recognized by the composite nanoprobe2O2；
FIG. 7 shows that with the addition of arginine at different concentrations, the fluorescence intensity ratio of the composite nanoprobe water-dimethyl sulfoxide solution (volume ratio 100: 1) at 440 nm to 607 nm changes with time, and the excitation wavelength is 360 nm;
FIG. 8 addition of arginine (concentration 3.0X 10)−4Mol/l) before and after compound 1 (concentration 3.0 × 10)−5Mol/liter, the volume ratio of water to dimethyl sulfoxide is 100: 1), the excitation wavelength is 360 nanometers, and the reaction time is 12 minutes;
FIG. 9 Water-dimethyl sulfoxide solution of Compound 1 (concentration 3.0X 10)−5Mol/l, volume ratio of water to dimethyl sulfoxide 100: 1) before and after addition of arginine 3.0X 10−4Absorption spectrum after mol/l arginine (grey line), fluorescence excitation spectrum under 360 nm light excitation and 0.67 μ g/ml carbon spot in water (rightmost grey line);
FIG. 10 is an IR spectrum of carbon dots, compound 1 and composite probe CDs-1;
FIG. 11 Zeta potentials of carbon dots and composite probe CDs-1;
FIG. 12360 nanometer light excitation arginine (3.0X 10) was added to an aqueous solution of carbon dots (0.67. mu.g/ml)−4 Mole/liter), the reaction time was 12 minutes;
FIG. 13 composite probe CDs-1 (containing 0.67. mu.g/ml carbon dots and 3X 10−5Gradually adding an absorption spectrum of arginine (equivalent ratio of the arginine to the compound 1 is 0-10) into a water-dimethyl sulfoxide solution (volume ratio of water to dimethyl sulfoxide is 100: 1) of a molar compound 1), wherein the reaction time is 12 minutes;
FIG. 14 shows that under natural light, the composite probe CDs-1 (containing 0.67. mu.g/ml carbon dots and 3X 10−5Molal Compound 1) solution was added with different concentrations of arginine (0, 3X 10 from left to right)−5Mol/l, 6X 10−5Mole/liter, 9X 10−5Mol/l, 12X 10−5Mol/l, 15X 10−5Mole/liter, 18X 10−5Mol/l, 21X 10−5Mol/l, 24X 10−5The mole ratio of the compound to the organic solvent is, 27×10−5mole/liter).
Detailed Description
Example 1
The compound 1 in the composite probe adopts the following synthetic route:
8-hydroxy-2-methylquinoline (2.41 g, 15 mmol) was added to a 100 mL three-necked flask, dried 20mL dichloromethane solvent was added, 1.5 mL triethylamine was added, the mixture was stirred in an ice-water bath for 30 minutes, 4 mL acryloyl chloride was slowly added dropwise to the reaction solution, the ice-water bath was removed, and the reaction was stirred at room temperature for 12 hours. And after the reaction is finished, filtering, and distilling the obtained filtrate under reduced pressure to obtain a crude product. And (3) separating the obtained crude product by column chromatography (the volume ratio of the developing solvent to the ethyl acetate to the petroleum ether is 1: 4) to obtain a light yellow oily liquid, namely the compound 4 (the yield is 47.3%).
And (3) characterization:1H NMR H (DMSO, 400 MHz)：8.32(d, 1H), 7.85(m, 1H), 7.57(m,2H), 7.47(d, 1H), 6.61(t, 2H), 6.20(m, 1H), 2.61(s, 3H). 13C NMR: C (100 MHz, DMSO): 164.71, 159.47, 146.77, 140.38, 136.70, 133.90, 128.25, 127.91, 126.32, 125.85, 123.31, 121.95, 25.73.
compound 4 (0.928 g, 4.4 mmol) was added to a 100 mL three-necked flask, followed by 15 mL of 1, 4-dioxane and slow warming to 65 ℃. Selenium dioxide (0.648 g, 5.8 mmol) was added to the reaction mixture, and the reaction was continued to be heated to 101 ℃ and refluxed for 4 hours. After the reaction is finished, the reaction solution is filtered while the reaction solution is hot, and silica gel is added into the filtrate to be decompressed and distilled to obtain a crude product. The obtained crude product is purified by a column chromatography separation method (silica gel is 200-mesh and 300-mesh, the volume ratio of developing solvent ethyl acetate and petroleum ether is 1:20, and the crude product is subjected to rotary evaporation to obtain white flocculent solid, namely the compound 3 (the yield is 71.6%).
And (3) characterization: 1H NMR H (DMSO, 400 MHz): 10.00(s, 1H), 8.70(d, 2H), 8.06(m, 2H), 7.84(t, 1H), 7.78(m, 1H), 6.66(t, 2H), 6.27(m, 1H). 13C NMR: C (100 MHz, DMSO): 193.74, 164.68, 152.44, 147.78, 140.64, 138.82, 134.57, 131.27, 129.85, 127.88, 126.84, 123.41, 118.31.
1,1, 2-trimethylbenzo [ e ] indole (1.045 g, 5 mmol) and methyl iodide (1 ml) were dissolved in 15 ml acetonitrile and the mixture was heated to 45 ℃. After 13 hours of reaction, the mixture was cooled to room temperature and 50 ml of diethyl ether was poured into the reaction solution. Filtration gave product 2 (0.9234 g, yield: 52.6%).
And (3) characterization: 1H NMR H (DMSO, 400 MHz): 8.38 (d, 1H), 8.31 (d, 1H), 8.22 (d, 1H), 8.12 (d, 1H), 7.78 (t, 1H), 7.76 (t, 1H), 4.10 (s, 3H), 2.88 (s, 3H), 1.76 (s, 6H). 13C NMR: C (DMSO, 100 MHz): 196.37, 133.49, 130.98, 130.21, 128.84, 127.59, 127.58, 123.88, 113.62, 55.72, 35.59, 21.75, and 14.48.
Compound 2 (0.3513 g, 1 mmol) and 3 (0.2771 g, 1 mmol) were dissolved in 10ml ethanol, heated to 78 ℃, refluxed for 12 hours, and then the reaction mixture was cooled to room temperature. Final product Probe 1 was obtained as a brown solid after filtration (0.2528 g, 45.1%).
And (3) characterization: HRMS (EI) m/z calcd for C29H25N2O2 [M-I], 433.1911; found, 433.1839. 1H NMR H (DMSO, 400 MHz): 8.75 (d, 1H), 8.66 (d, 1H), 8.35 (t, 3H), 8.28 (d, 1H), 8.22 (d, 1H), 8.14 (d, 1H), 8.05, (m, 1H), 7.86 (t, 1H), 7.79 (m, 3H), 6.78 (d, 2H), 6.36 (t, 1H)4.25 (s, 3H), and 2.05 (s, 6H). 13C NMR: C (DMSO, 100 MHz): 182.49, 164.86, 152.03, 148.53, 147.75, 139.95, 138.48, 134.68, 133.98, 131.61, 130.53, 130.20, 129.09, 128.69, 128.12, 127.11, 126.63, 125.29, 123.98, 123.06, 117.15, 114.02, 54.71, 36.01, 25.08.
Example 2
8-hydroxy-2-methylquinoline (2.006 g, 12.5 mmol) was added to a 100 mL three-necked flask, dried 30mL dichloromethane solvent was added, 2.0 mL triethylamine was added, the mixture was stirred in an ice-water bath for 45 minutes, 10mL acryloyl chloride was slowly added dropwise to the reaction mixture, the ice-water bath was removed, and the reaction was stirred at room temperature for 36 hours. And after the reaction is finished, filtering, and distilling the obtained filtrate under reduced pressure to obtain a crude product. And (3) separating the obtained crude product by column chromatography (the volume ratio of the developing solvent to the ethyl acetate to the petroleum ether is 1: 10) to obtain a light yellow oily liquid, namely the compound 4 (the yield is 57.8%). Compound 4 (0.742 g, 3.5 mmol) was added to a 100 mL three-necked flask, followed by 13 mL of 1, 4-dioxane and slow warming to 65 ℃. Selenium dioxide (0.518 g, 4.6 mmol) was added to the reaction mixture, and the temperature was further raised to 101 ℃ to conduct a reflux reaction for 12 hours. After the reaction is finished, the reaction solution is filtered while the reaction solution is hot, and silica gel is added into the filtrate to be decompressed and distilled to obtain a crude product. The resulting crude product was purified by column chromatography separation (silica gel 200-300 mesh, developing solvent ethyl acetate and petroleum ether in a volume ratio of 1:8, spin-evaporated to obtain a white flocculent solid, i.e. compound 3 (yield 77.2%). 1,1, 2-trimethylbenzo [ e ] indole (1.254 g, 6 mmol) and iodomethane (1 ml) were dissolved in 20ml of acetonitrile, the mixture was heated to 45 ℃ for 24 hours, the mixture was cooled to room temperature and 70ml of diethyl ether was poured into the reaction solution, filtration was carried out to obtain product 2 (1.174 g, yield 55.7%). compound 2 (0.3513 g, 1 mmol) and 3 (0.2771 g, 1 mmol) were dissolved in 15 ml of ethanol, heated to 78 ℃ and refluxed for 36 hours, the reaction mixture was cooled to room temperature, the final product probe 1 was filtered to obtain (0.3212 g, 57.3%).
Example 3
8-hydroxy-2-methylquinoline (1.605 g, 10 mmol) was added to a 100 mL three-necked flask, dried 15 mL dichloromethane solvent was added, 1.4 mL triethylamine was added, the mixture was stirred in an ice-water bath for 60 minutes, 5 mL acryloyl chloride was slowly added dropwise to the reaction mixture, the ice-water bath was removed, and the reaction was stirred at room temperature for 34 hours. And after the reaction is finished, filtering, and distilling the obtained filtrate under reduced pressure to obtain a crude product. And (3) separating the obtained crude product by column chromatography (the volume ratio of the developing solvent to the ethyl acetate to the petroleum ether is 1: 7) to obtain a light yellow oily liquid, namely the compound 4 (the yield is 55.0%). Compound 4 (1.265 g, 6.0 mmol) was added to a 100 mL three-necked flask, 20mL of 1, 4-dioxane was added, and the temperature was slowly raised to 65 ℃. Selenium dioxide (0.905 g, 8.1 mmol) was added to the reaction mixture, and the temperature was further raised to 101 ℃ to conduct a reflux reaction for 10 hours. After the reaction is finished, the reaction solution is filtered while the reaction solution is hot, and silica gel is added into the filtrate to be decompressed and distilled to obtain a crude product. The obtained crude product was purified by column chromatography separation (silica gel 200-300 mesh, developing solvent ethyl acetate and petroleum ether in a volume ratio of 1:35, rotary evaporation to obtain a white flocculent solid, i.e. compound 3 (yield 80.7%). 1,1, 2-trimethylbenzo [ e ] indole (0.627 g, 3 mmol) and iodomethane (0.5 ml) were dissolved in 20ml of acetonitrile, the mixture was heated to 45 ℃ for 24 hours, the mixture was cooled to room temperature and 40 ml of diethyl ether was poured into the reaction solution, filtration was performed to obtain product 2 (0.599 g, yield 68.2%). Compound 2 (0.3513 g, 1 mmol) and 3 (0.2771 g, 1 mmol) were dissolved in 15 ml of ethanol, heated to 78 ℃ and refluxed for 52 hours, the reaction mixture was cooled to room temperature, the final product 1 was filtered to obtain (0.3728 g, 66.5%).
The method for preparing the composite nano probe for detecting arginine by fluorescence in mitochondrial localization ratio by using organic molecules comprises the following steps:
1) synthetic carbon nanoparticles
Firstly weighing 1 g of citric acid, dissolving the citric acid in 10mL of water, adding 100-1000 muL of ethylenediamine, stirring to mix the solution uniformly, transferring the solution into a hydrothermal kettle, reacting for 4 hours at 200 ℃ in an oven, cooling to room temperature to obtain a brown solution, and dialyzing the brown solution for 48 hours by using a dialysis membrane with the molecular weight cutoff of 3000. And precipitating twice by using absolute ethyl alcohol, centrifuging, and drying the obtained brown solid in a vacuum drying oven for 18 hours to obtain the carbon nano-particles.
2) Preparation of composite nanoprobes
Weighing 10 mg of carbon nano-particles and 5 mg of probe molecules 1 in a 10mL volumetric flask, performing constant volume by using ultrapure water and dimethyl sulfoxide (the volume ratio is 10:1-500: 1), performing ultrasonic dissolution for 10-60 minutes, centrifuging for 3-15 minutes by using a centrifuge with 10000 revolutions, and filtering.
The nano composite material can be used for detecting arginine (Arg) in a near-infrared fluorescence ratio manner, has high sensitivity (detection limit of 64.7 nanomole) and good selectivity and anti-interference capability, and is a probeThe concentration of arginine can be quantitatively detected to range from 60 to 270 micromoles, and the luminescent color of the solution with different arginine concentrations changes from red to blue (as shown in figure 2); in the titration curve of arginine drawn by the linear function of the ratio emission spectrum of the composite nano probe and the concentration of arginine, the ratio of the emission peak at 440 nm to the emission peak at 607 nm can keep a good linear relation in the range of 60 to 270 mu M of arginine, which indicates that arginine can be quantitatively obtained from the ratio fluorescence intensity in the range (as shown in figure 3); the composite fluorescent probe can detect arginine changes in a wide pH range (pH from 5 to 11), which covers the pH range in vivo (FIG. 4), and various organic biomolecules Thr, Val, Ser, Phe, His, Gly, Pro, IIe, Trp, Ala, Tyr, Glu, Glc, Leu, and H in vivo2O2No interference to detection and good selectivity (fig. 5); the absorption spectrum of gradually adding arginine into the solution of the composite probe CDs-1 can see that the position of the solution absorption peak moves by more than 150 nanometers along with the addition of arginine (as shown in FIG. 13), FIG. 7 shows that the fluorescence intensity ratio of 440 nm and 607 nm of the composite nano probe solution changes with time when arginine with different concentrations is added, the fluorescence intensity ratio is finally close to saturation with time, and the saturation time is about 30 minutes; FIG. 8 is an emission spectrum of a water-dimethylsulfoxide solution of Compound 1 before and after arginine addition, with a shift of approximately 170 nanometers in luminescence color of the solution over 12 minutes; water-dimethyl sulfoxide solution of Compound 1 in FIG. 9 before and after addition of arginine 3.0X 10−4The absorption spectrum (black line) after mol/l arginine (grey) matches the fluorescence excitation spectrum (rightmost grey line) of an aqueous solution of carbon dots that emit light that is not absorbed by the compound as it is initially absorbed; the infrared spectrum of the carbon dot, the compound 1 and the composite probe CDs-1 in FIG. 10 shows that the composite probe is loaded with the organic compound; the electronegativity of the composite probe was calculated from-1.35 mV to 0.57 mV based on the carbon dots in FIG. 11 and the Zeta potential of the CDs-1 composite probe. FIG. 12 shows the addition of arginine (3.0X 10) to an aqueous solution of carbon dots (0.67. mu.g/ml) under 360 nm light excitation−4Mole/liter) before and afterEmission spectrum, before and after adding arginine, the emission spectrum of the carbon dot solution does not change greatly; FIG. 14 is a photograph of different concentrations of arginine added to a composite probe CDs-1 solution under natural light, and the color of the solution changed from yellow to colorless as the concentration of arginine increased.
In summary, a composite nanoprobe detects arginine (Arg) based on the combination of a negatively charged carbon nanoparticle and a positively charged probe 1 through electrostatic attraction by Fluorescence Resonance Energy Transfer (FRET), and has high sensitivity (detection limit of 64.7 nanomolar) and good selectivity and anti-interference capability. The probe can quantitatively detect arginine at concentrations ranging from 60 to 270 micromolar.
Claims (7)
1. A method for preparing a composite nano probe is characterized in that the composite nano probe is prepared by combining carbon nano particles with negative charges and a probe 1 with positive charges through electrostatic attraction, and comprises the following steps:
(1) preparation of probe 1: firstly, 8-hydroxy-2-methylquinoline reacts with acryloyl chloride at room temperature to obtain a compound 4
Then adding compound 4
With SeO at 101 deg.C2Oxidation reaction takes place to produce compound 3
In the preparation of compound 4
While simultaneously reacting 1,1, 2-trimethylbenzo [ e ]]Indole and CH3I reaction to give Compound 2
Finally, compound 3 is added
And compound 2
Performing Knoevenagel condensation reaction to obtain a probe 1
(2) Synthesizing carbon nanoparticles: firstly, dissolving citric acid in water, adding 1000 mu L of ethylenediamine with 100-;
(3) preparing a composite nano probe: mixing carbon nanoparticles with the probe 1
Putting into a volumetric flask, performing constant volume by using ultrapure water and dimethyl sulfoxide with the volume ratio of 10:1-500:1, performing ultrasonic dissolution for 10-60 minutes, centrifuging for 3-15 minutes at 10000 r/min, and filtering to obtain the product.
2. The method of preparing a composite nanoprobe according to claim 1, wherein: the chemical synthesis route of the probe 1 in the step (1) is
3. The method of preparing a composite nanoprobe according to claim 1, wherein: compound 4 in said step (1)
The preparation method comprises the following steps: adding 8-hydroxy-2-methylquinoline into a three-necked bottle, adding 15-30mL of dry dichloromethane solvent, adding 1.4-2mL of triethylamine, placing the three-necked bottle in an ice water bath, stirring for 30-60 minutes, slowly dropwise adding 4-10mL of acryloyl chloride into the reaction solution, then removing the ice water bath, stirring and reacting for 12-36 hours at room temperature, filtering after the reaction is finished, carrying out reduced pressure distillation on the obtained filtrate to obtain a crude product, and separating the crude product by column chromatography, wherein the developing agent is ethyl acetate/petroleum ether with the volume ratio of 1:4-1:10 to obtain a light yellow oily liquid compound 4
The yield thereof was 47.3-57.8%.
4. The method of preparing a composite nanoprobe according to claim 1, wherein: compound 3 in said step (1)
The preparation method comprises the following steps: adding the compound 4 into a three-necked bottle, adding 13-20mL of 1, 4-dioxane, slowly heating to 65 ℃, adding selenium dioxide into the reaction liquid, continuously heating to 101 ℃, performing reflux reaction for 4-12 hours, filtering the reaction liquid while the reaction is hot after the reaction is finished, adding silica gel into the filtrate, performing reduced pressure distillation to obtain a crude product, purifying the obtained crude product by a column chromatography separation method, wherein the particle size of the silica gel is 200 meshes and 300 meshes, the developing agents are ethyl acetate and petroleum ether, the volume ratio of the ethyl acetate to the petroleum ether is 1:8-1:35, and performing rotary evaporation to obtain a white flocculent solid compound 3
The yield thereof was 71.6 to 80.7%.
5. The method of preparing a composite nanoprobe according to claim 1, wherein: the compound in the step (1)2 
The preparation method comprises the following steps: 1,1, 2-trimethylbenzo [ e ]]Dissolving indole and methyl iodide in 15-20mL acetonitrile, heating the mixture to 45 deg.C, reacting for 13-24 hr, cooling the mixture to room temperature, pouring 40-70mL diethyl ether into the reaction solution, and filtering to obtain compound 2The yield thereof is 52.6 to 68.2 percent.
6. The method of preparing a composite nanoprobe according to claim 1, wherein: the probe 1 in the step (1)
The preparation method comprises the following steps: reacting the compound 2
And compound 3
Dissolving in ethanol, heating to 78 deg.C, refluxing for 12-52 hr, cooling the reaction mixture to room temperature, and filtering to obtain brown solid probe 1
The yield thereof is 45.1 to 66.5 percent.
7. Use of a nanoprobe prepared by the method of preparation of a composite nanoprobe according to any of claims 1 to 6, characterized in that: the composite nano probe is used for quantitatively detecting arginine in a near-infrared fluorescence ratio in a solution, and the concentration of the detected arginine ranges from 60 to 270 mu mol within the pH range of 5 to 11.
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