CN114441490B - Method for detecting hypochlorite ions - Google Patents
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Abstract
A method for detecting hypochlorite ions, comprising the steps of: step 1: determining a curcumin standard curve; step 2: synthesizing curcumin liposome; step 3: assembling double-emission ratio fluorescent probe [ Ru (bpy) 3 ] 2+ @ CCM-NPs; step 4: construction of hypochlorite ionFluorescence detection regression equation; step 5: pretreatment of a sample containing hypochlorite ions; step 6: sample detection and hypochlorite ion concentration calculation. The invention designs and assembles the ratio type fluorescent probe, and the ratio type fluorescent probe can effectively eliminate or reduce the irrelevant factor interference of the analyte by self-calibrating the two emission wavebands, thereby improving the detection sensitivity and accuracy.
Description
Technical Field
The invention relates to a method for detecting hypochlorite ions, and belongs to the technical field of detection.
Background
Hypochlorous acid is a highly active oxygen widely present in living bodies, and can react with biomolecules such as proteins, nucleic acids, fatty acids, and the like, and plays an important role in the human immune system. However, excessive hypochlorous acid in the human body causes oxidative stress and cell damage to the body, and eventually causes a series of diseases such as lung injury, atherosclerosis, arthritis, neurodegenerative diseases and cancer. Hypochlorite solutions are also widely used as antibacterial, bleaching and disinfectant agents in everyday life. The excessive use of hypochlorite solution can hurt respiratory system of human body, can lead semicarbazide in food to exceed standard, can react with organic matters in water to form cancerogenic substances such as carbon tetrachloride, and further causes harm to human body and environment. Therefore, development of an analytical method capable of rapidly and sensitively detecting hypochlorite ions is of great importance to food safety and environmental protection.
The traditional method for detecting hypochlorite ions mainly comprises a potential method, a chemiluminescence method and a chromatography method, but the method is complex in operation and low in sensitivity. In contrast, fluorescence spectroscopy has been widely used for detection of hypochlorite ions in foods and environments due to its advantages of simple operation, high sensitivity, low detection limit, non-invasiveness, and the like.
The existing fluorescence spectrometry for detecting hypochlorite ions lacks effective internal reference, often shows single-wavelength emission and is easily interfered by instrument parameters, photobleaching, micro-environmental changes around the probe and uneven probe distribution. The ratiometric fluorescence method is an analytical method for determining a target by measuring the ratio of fluorescence intensities at two different wavelengths as a signal parameter. Because the measured fluorescence ratio signal is not affected by the intensity of the light source and the sensitivity of the instrument, the ratio fluorescent probe has higher sensitivity, selectivity and linear range. In view of this, development of a ratio-type fluorescent probe for realizing rapid, efficient and stable detection of hypochlorite ions is urgently required.
Disclosure of Invention
The invention aims to provide a method for detecting hypochlorite ions.
To achieve the above and other related objects, the present invention provides the following technical solutions: a method for detecting hypochlorite ions, comprising the steps of:
step 1: determining a curcumin standard curve;
step 2: synthesizing curcumin liposome;
step 3: assembling double-emission ratio fluorescent probe [ Ru (bpy) 3 ] 2+ @CCM-NPs；
Step 4: constructing a hypochlorite ion fluorescence detection regression equation;
step 5: pretreatment of a sample containing hypochlorite ions;
the sample to be measured is tap water, liquid food or environmental water; directly detecting tap water and liquid food; filtering the environmental water body by using filter paper or filter membrane with the aperture of 0.22 mu m before detecting, and collecting filtrate for detecting;
step 6: sample detection and hypochlorite ion concentration calculation;
first, the dual-emission ratio fluorescent probe [ Ru (bpy) ] is removed 3 ] 2+ The @ CCM-NPs solution is put in a PE tube, then a sample to be detected is added to a volume of 200.0 mu L, the sample is transferred to a cuvette after reaction, the fluorescence emission spectrum of the sample under 365nm excitation is measured by a fluorescence spectrophotometer, and the fluorescence emission spectrum intensity F at 503nm and 603nm is recorded 503nm 、F 603nm The method comprises the steps of carrying out a first treatment on the surface of the F to be measured 503nm 、F 603nm Substituting the value into the hypochlorite ion fluorescence detection regression equation to obtain the secondary in the sample to be detectedThe value of the chlorate ions is the residual value of hypochlorite ions.
The preferable technical scheme is as follows: the determining curcumin standard curve comprises:
step one: weighing curcumin powder, and then adding dimethyl sulfoxide solution to prepare curcumin solution with concentration of 1 mmol/L;
step two: 1.0. Mu.L, 1.6. Mu.L, 2.2. Mu.L, 2.8. Mu.L, 3.4. Mu.L and 4.0. Mu.L curcumin solutions with concentration of 1mmol/L are removed, 199.0. Mu.L, 198.4. Mu.L, 197.8. Mu.L, 197.2. Mu.L, 196.6. Mu.L and 196.0. Mu.L mixed solution of dimethyl sulfoxide-deionized water are respectively added, and after mixing, ultraviolet absorption spectra are respectively measured;
step three: according to the corresponding ultraviolet absorption intensities measured by curcumin solutions with different concentrations, a corresponding curcumin concentration standard curve is prepared, and a regression equation is as follows: a=0.0376C Curcumin -0.0206, correlation coefficient of 0.9994, A being the ultraviolet absorption intensity of the sample to be measured, C Curcumin Is the concentration of curcumin.
The preferable technical scheme is as follows: the volume ratio of dimethyl sulfoxide to deionized water in the dimethyl sulfoxide-deionized water mixed solution is 1/4.
The preferable technical scheme is as follows: the synthetic curcumin liposome comprises: adding methoxy phospholipid polyethylene glycol and curcumin into a brown reagent bottle, uniformly mixing, performing ultrasonic treatment at room temperature, and stirring the mixed solution at a dark place overnight; after the completion of stirring, the mixed solution was evaporated at 37.0 ℃ to remove the organic solvent, and then transferred into an ultrafiltration tube of 10.0KD, centrifuged at 4000rpm, and washed 3 times with deionized water to obtain purified curcumin liposome nanoparticle having green fluorescence emission.
The preferable technical scheme is as follows: the ultrasonic power of the strong ultrasonic treatment is 150W, the ultrasonic frequency is 37kHz, and the ultrasonic treatment is continuously carried out for 5min.
The preferable technical scheme is as follows: the assembled dual emission ratio fluorescent probe [ Ru (bpy) 3 ] 2+ The @ CCM-NPs include: curcumin liposome and [ Ru (bpy) 3 ] 2+ Adding into PBS buffer solution, stirring and mixing uniformly to obtain dual-emission ratio fluorescent probe[Ru(bpy) 3 ] 2+ @CCM-NPs。
The preferable technical scheme is as follows: the construction of hypochlorite ion fluorescence detection regression equation comprises: double emission ratio fluorescent probe [ Ru (bpy) 3 ] 2+ Adding the @ CCM-NPs solution and hypochlorite ion solutions with different concentrations into PBS buffer solution, transferring to a cuvette after reaction, measuring fluorescence emission spectra of all reaction samples under 365nm excitation by using a fluorescence spectrophotometer, and recording fluorescence emission spectrum intensities F at 503nm and 603nm 503nm 、F 603nm The method comprises the steps of carrying out a first treatment on the surface of the As hypochlorite ion concentration increases, a dual emission ratio fluorescent probe [ Ru (bpy) ] is caused 3 ] 2+ Fluorescence intensity of @ CCM-NPs gradually decreased, using F 603nm /F 503nm And the hypochlorite ion concentration to make a regression equation: f (F) 603nm /F 503nm ＝0.18166C ClO - +0.70179, the correlation coefficient is 0.9952; the linear range is 0.0601-24 mu mol/L; the detection limit is 18.03nmol/L.
Due to the application of the technical scheme, compared with the prior art, the invention has the advantages that:
1. the invention adopts DSPE-PEG 2000 OMe encapsulates curcumin. First: DSPE-PEG 2000 OMe encapsulates curcumin, which will make curcumin more stable and more specific in response to hypochlorite ions. Second,: curcumin itself is a fat-soluble substance, DSPE-PEG 2000 The curcumin liposome formed by coating curcumin by OMe has good water solubility and wider application range; third,: curcumin liposome has negative charges on the surface, and can lead the positively charged tris (2, 2' -bipyridine) ruthenium (II) chloride hexahydrate to be electrostatically bound on the surface, thereby assembling the ratio type fluorescent probe ([ Ru (bpy)) for detecting hypochlorite ions 3 ] 2+ @CCM-NPs)。
2. The invention designs and assembles a new ratio type fluorescent probe ([ Ru (bpy)) 3 ] 2+ @ CCM-NPs). Common single wavelength emitting fluorescent probes for detecting hypochlorite ions are easily interfered by instrument parameters, photobleaching, micro-environmental changes around the probes, uneven distribution of the probes and the like, and cause root causes of the problemsIs a lack of effective internal reference in fluorescence sensing processes. In contrast, the ratio-type fluorescent probe can effectively eliminate or reduce the interference of irrelevant factors of analytes by self-calibrating two emission wavebands, and improves the detection sensitivity and accuracy.
Drawings
FIG. 1 (a) shows the ultraviolet absorption spectra of curcumin at different concentrations; (b) is a curcumin standard curve.
FIG. 2 (a) is [ Ru (bpy) 3 ] 2+ @CCM-NPs with 0.0 to 32.0. Mu. Mol/L ClO - Fluorescence spectrum of the reaction; (b) Is [ Ru (bpy) 3 ] 2+ Ratio fluorescence intensity F of @ CCM-NPs 603nm /F 503nm With 0.0 to 32.0. Mu. Mol/L ClO - Is a fit of the curve.
FIG. 3 is [ Ru (bpy) 3 ] 2+ TEM image of @ CCM-NPs.
Fig. 4 is an ultraviolet-visible absorption spectrum: (1) CCM-NPs; (2) [ Ru (bpy) 3 ] 2+ ；(3)[Ru(bpy) 3 ] 2+ @CCM-NPs；(4)[Ru(bpy) 3 ] 2+ @CCM-NPs and [ Ru (bpy) 3 ] 2+ Is a difference in absorbance spectra of (a).
Fig. 5 is a fluorescence spectrum: individual [ Ru (bpy) 3 ] 2+ The @ CCM-NPs spectrum; [ Ru (bpy) 3 ] 2+ @CCM-NPs and ClO - Spectrum after reaction; individual [ Ru (bpy) 3 ] 2+ And (3) spectrum.
FIG. 6 is [ Ru (bpy) 3 ] 2+ Ratio fluorescence intensity F of 154.0 seconds of @ CCM-NPs reacted in PBS buffer (1X) at different pH (4.0-10.0) 603nm /F 503nm 。
Detailed Description
Further advantages and effects of the present invention will be readily apparent to those skilled in the art from the following disclosure of the present invention by reference to the specific embodiments.
Please refer to fig. 1-6. It should be understood that the structures, proportions, sizes, etc. shown in the drawings are shown only in the drawings and should not be taken as limiting the invention to those having ordinary skill in the art, since modifications, changes in proportions, or adjustments of sizes, etc. could be made without departing from the spirit or essential characteristics of the invention. The following examples are provided for a better understanding of the present invention, but are not intended to limit the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the examples described below were purchased from conventional biochemical reagent stores unless otherwise specified.
Example 1: method for detecting hypochlorite ions
The hypochlorite ion detecting process includes the following steps.
(1) Determination of curcumin standard curve
Curcumin powder was used, purchased from ala Ding Shiji (Shanghai) limited, with a purity of more than 98%.
Step one: weighing curcumin powder, placing in a 1.5mL PE tube, adding dimethyl sulfoxide solution, and preparing into curcumin solution with concentration of 1mmol/L for later use.
Step two: 6 PE tubes (1.5 mL) were taken and numbered 1-6 in sequence. 1.0. Mu.L, 1.6. Mu.L, 2.2. Mu.L, 2.8. Mu.L, 3.4. Mu.L, 4.0. Mu.L of curcumin solution at a concentration of 1mmol/L were sequentially put into a 1-6-well, and 199.0. Mu.L, 198.4. Mu.L, 197.8. Mu.L, 197.2. Mu.L, 196.6. Mu.L, 196.0. Mu.L of dimethyl sulfoxide-deionized water mixed solution (V Dimethyl sulfoxide /V Water and its preparation method =1/4), and thoroughly mixed. Then, the solution was pipetted into a cuvette to measure the ultraviolet absorption A1, A2, A3, A4, A5, A6, and three parallel experiments were performed for each concentration of curcumin solution.
As can be seen from FIG. 1 (a), the characteristic absorption peak of curcumin was detected at 425nm by ultraviolet absorption spectroscopy.
Step three: according to the corresponding ultraviolet absorption intensities measured by curcumin solutions with different concentrations, a corresponding curcumin concentration standard curve is made, and a regression equation is as follows: a=0.0376C Curcumin -0.0206, correlation coefficient of 0.9994 (see fig. 1 (b)). Through a regression equation, the corresponding curcumin concentration can be calculated according to the ultraviolet absorption intensity A of the sample to be detected.
(2) Synthetic curcumin liposomes (CCM-NPs)
2.0mg DSPE-PEG 2000 OMe (dissolved in 1.5mL dichloromethane solvent) and 50.0 μg CCM (dissolved in 1.5mL deionized water) were added to a brown reagent bottle and thoroughly mixed, followed by intense sonication at room temperature for 5.0 min. Subsequently, the mixed solution was fixed on a magnetic stirrer and stirred well overnight in the absence of light. After the stirring was completed, the mixed solution was spin-distilled at 37.0 ℃ to remove the organic solvent, and then rapidly transferred into a 10.0KD ultrafiltration tube, centrifuged at 4000rpm, and washed 3 times with deionized water, to finally obtain purified curcumin liposome nanoparticle having green fluorescence emission, the concentration of which was measured with CCM.
The DSPE-PEG 2000 OMe is methoxy phospholipid polyethylene glycol with purity of more than 95%.
The CCM is curcumin, and the purity is more than 98%.
The ultrasonic power of the specific method is 150W, the ultrasonic frequency is 37kHz, and the ultrasonic is continuously carried out for 5min.
The rotary evaporation is operated by a rotary evaporator, the rotary evaporation temperature is 37 ℃, the vacuum degree is-0.095 to-0.1 mPa, and the refrigeration temperature is-20 ℃; after the organic reagent in the evaporating container is evaporated, collecting the rest materials for standby.
The 10.0KD ultrafiltration tube is subjected to centrifugal treatment, namely, the synthesized material is put into the ultrafiltration tube, and if the material is successfully coated by phospholipid, the molecular weight of the material is more than 10.0KD; by centrifugation, the non-encapsulated curcumin (molecular weight less than 10.0 KD) can be filtered.
(3) Assembling double-emission ratio fluorescent probe [ Ru (bpy) 3 ] 2+ @CCM-NPs
Based on the negatively charged nature of the phospholipid layer of CCM-NPs, a dual emission ratio fluorescent probe [ Ru (bpy) ] is assembled by electrostatic interaction 3 ] 2+ @ CCM-NPs. Specifically, 50.0 μg of CCM-NPs and different masses of [ Ru (bpy) 3 ] 2+ (30.4, 38.0, 60.9, 91.3 and 152.2 μg) were mixed well in PBS buffer (1 x, ph=7.4), respectively. By further evaluating their analytical parameters for hypochlorite ions, the optimal duplex for hypochlorite ions is determinedEmission proportion fluorescent probe [ Ru (bpy) 3 ] 2+ The @ CCM-NPs consist of 50.0 μg CCM-NPs and 38.0 μg [ Ru (bpy) 3 ] 2+ Composition, the concentration of which was also determined by CCM.
Said "[ Ru (bpy) 3 ] 2+ ", refer to Ru (bpy) 3 Cl 2 ·6H 2 O, chinese name is tris (2, 2' -bipyridine) ruthenium (II) chloride hexahydrate, purity>98％。
The "PBS buffer (1 x, ph=7.4)", was obtained by diluting 20-fold with 20x PBS buffer purchased from the company of the division of bioengineering (Shanghai).
(4) Construction of hypochlorite ion fluorescence detection regression equation
4.0 mu L [ Ru (bpy) 3 ] 2+ @ CCM-NPs solution and 10.0. Mu.L of ClO at various concentrations - The solution was added to 186.0 μlpbs buffer (1 x, ph=7.4) and reacted together. After 154.0 seconds of reaction at 25 ℃, the reaction mixture was transferred to a cuvette, and the fluorescence emission spectra of all the reaction samples under 365nm excitation were measured by a fluorescence spectrophotometer, and the fluorescence emission spectrum intensities F at 503nm and 603nm were recorded 503nm 、F 603nm . Along with ClO - An increase in concentration results in a ratiometric fluorescent probe [ Ru (bpy) 3 ] 2+ Fluorescence intensity of @ CCM-NPs gradually decreased, using F 603nm /F 503nm Ratio of (C) and ClO - Regression equation is made for concentration: f (F) 603nm /F 503nm ＝0.18166C ClO - +0.70179, the correlation coefficient is 0.9952; the linear range is 0.0601-24 mu mol/L; the detection limit is 18.03nmol/L.
The said ClO - ", refers to hypochlorite ions.
PBS buffer used (1 x, ph=7.4): PBS buffer (20 x, ph=7.4) was purchased directly from bio-engineering (Shanghai) stock, directly diluted 20-fold for use.
Used "[ Ru (bpy) 3 ] 2+ @ CCM-NPs ", concentration checking method: depending on the concentration of curcumin. Remove 4.0. Mu.L of the synthesized ratiometric fluorescent probe material in a 1.5mL PE tube, add 196.0. Mu.L PBS buffer (1X, pH=7.4), mix thoroughlyThe ultraviolet absorption intensity at 425nm, a= 0.2426, was measured, bringing into the curcumin standard curve a=0.0376C Curcumin -0.0206, C can be calculated Curcumin Ratio fluorescent probe [ Ru (bpy) =7.0. Mu. Mol/L 3 ] 2+ Curcumin concentration contained in @ CCM-NPs was 7.0. Mu. Mol/L.
The resulting regression equation, fit, was obtained using origin 8.5 software fit.
(5) Pretreatment of hypochlorite ion-containing samples
The hypochlorite ion residue is detected, and tap water, liquid food or environmental water is detected; tap water and liquid food can be directly used without further treatment. The environmental water body is filtered by filter paper or filter membrane with the pore diameter of 0.22 mu m before detection, and the filtrate is collected.
(6) Sample detection and hypochlorite ion concentration calculation
First, 4.0. Mu.L of [ Ru (bpy) was removed 3 ] 2+ The @ CCM-NPs solution was placed in a 1.5mL PE tube and 196.0. Mu.L of the sample to be tested was then added to a volume of 200.0. Mu.L. After 154.0 seconds of reaction at 25 ℃, the sample was transferred to a cuvette, and the fluorescence emission spectrum of the sample under 365nm excitation was measured with a fluorescence spectrophotometer, and the fluorescence emission spectrum intensities F at 503nm and 603nm were recorded 503nm 、F 603nm . F to be measured 503nm 、F 603nm Substituting the values into the regression equation F 603nm /F 503nm ＝0.18166C ClO - +0.70179 to obtain C in the sample ClO - The value of (2) is the residual value of hypochlorite ions.
Double-emission ratio fluorescent liposome [ Ru (bpy) 3 ] 2+ Determination of @ CCM-NPs Properties:
10 mu mol/L [ Ru (bpy) 3 ] 2+ Dripping @ CCM-NPs on a 230-mesh common carbon support membrane copper net, and naturally air-drying for half an hour at 25 ℃; and then dropwise adding a drop of phosphotungstic acid solution with the concentration of 3% for negative dyeing for 2 minutes, sucking redundant solution by taking filter paper, naturally air-drying at 25 ℃, and storing in a copper net box. TEM images were taken with a transmission electron microscope model JEM-1200EX (120 KV) to give FIG. 3.
The 230 mesh common carbon support film copper mesh is purchased from Beijing middle mirror department instrument technology Co.
TEM image of FIG. 3 shows [ Ru (bpy) 3 ] 2+ The @ CCM-NPs have good dispersibility and a size of about 21.6nm, indicating that they have nanometer size.
FIG. 4 is for further explanation [ Ru (bpy) 3 ] 2+ Successful Assembly of @ CCM-NPs, CCM-NPs and [ Ru (bpy) were studied 3 ] 2+ UV visible absorbance spectra before and after incubation in PBS buffer. By using [ Ru (bpy) 3 ] 2+ @CCM-NPs (curve 3) minus free [ Ru (bpy) 3 ] 2+ The absorption spectrum of (Curve 2) gives (Curve 4) a spectrum of [ Ru (bpy) 3 ] 2+ CCM-NP as reference. Curve 4 shows a red shift of the characteristic peak from-425 nm to-429 nm with a pronounced fading effect, compared with the UV-visible absorption spectrum of CCM-NPs with PBS buffer as reference (Curve 1), indicating an increase in the polarity of the liposomal nanoparticles and their surface, [ Ru (bpy) 3 ] 2+ Successfully assembled on the surface of CCM-NPs.
The detection method comprises the following steps: 1. 7.0 mu mol/L [ Ru (bpy) was removed 3 ] 2+ The @ CCM-NPs solution was placed in a 1.5mL PE tube, PBS buffer (1X, pH=7.4) was added to a constant volume of 200mL, and mixed well to test fluorescence. 2. 7.0 mu mol/L [ Ru (bpy) was removed first 3 ] 2+ The @ CCM-NPs solution was added to 32.0. Mu. Mol/LClO in a 1.5mL PE tube - The solution was finally added with PBS buffer (1 x, ph=7.4) to a volume of 200mL, mixed well, reacted at 25 ℃ for 154.0 seconds and then measured for fluorescence. 3. 3.5 mu mol/L [ Ru (bpy) was removed 3 ] 2+ The solution was placed in a 1.5mL PE tube, and PBS buffer (1 x, ph=7.4) was added to a constant volume of 200mL, mixed well and fluorescent to be measured. Fluorescence spectra were separately detected, yielding FIG. 5.
FIG. 5 further demonstrates the ratiometric fluorescent probe [ Ru (bpy) 3 ] 2+ ClO detection by @ CCM-NPs - Feasibility of (2); also can explain [ Ru (bpy) 3 ] 2+ The reason for the slight decrease in @ CCM-NPs at 603 nm. As can be seen from FIG. 5, ru alone (bpy) 3 ] 2+ At 365nm excitation wavelength, the @ CCM-NPs initially have two fluorescence emission peaks at 503nm and 603 nm. At the same time with ClO - After the reaction, [ Ru (bpy) 3 ] 2+ The fluorescence emission of @ CCM-NPs at 503nm was significantly reduced while the fluorescence emission peak at 603nm was slightly reduced, thus enabling the construction of a typical dual emission ratio fluorescent probe.
[Ru(bpy) 3 ] 2+ The decrease in fluorescence at 503nm of @ CCM-NPs is mainly due to ClO - Oxidation of o-methoxyphenol of curcumin in the liposome nanoparticle to benzoquinone leads to a sharp decrease in ultraviolet absorption of curcumin in the range of 300-510nm, and finally leads to disappearance of fluorescence emission peak of curcumin. The slight decrease in fluorescence at 603nm is due to the sharp decrease in ultraviolet absorption of curcumin in the 300-510nm range, blocking [ Ru (bpy) 3 ] 2+ Internal curcumin orientation [ Ru (bpy) @ CCM-NPs 3 ] 2+ Initial energy transferred.
FIG. 6 shows [ Ru (bpy) 3 ] 2+ Detection of ClO in PBS buffer (1X) at pH @ CCM-NPs - The effect of (2) is optimal.
An example of the method for detecting the concentration of hypochlorite ions in tap water is given
The tap water was taken from the energy building 1110 laboratory of the university of joint fertilizer industry at 2022, 1 and 10 days, and the water temperature measured in the laboratory was 25 ℃.
First, 7.0. Mu. Mol/L [ Ru (bpy) was removed 3 ] 2+ @ CCM-NPs in a 1.5mL PE tube, tap water was then added to fix volume to 200mL. After 154.0 seconds of reaction at 25 ℃, the sample was transferred to a cuvette, and the fluorescence emission spectrum of the sample under 365nm excitation was measured with a fluorescence spectrophotometer, and the fluorescence emission spectrum intensities F at 503nm and 603nm were recorded 503nm 、F 603nm . Measurement of F 603nm /F 503nm = 0.92043, substituting it into the regression equation F 603nm /F 503nm ＝0.18166C ClO - +0.70179, giving a hypochlorite concentration of 1.2036. Mu. Mol/L.
Example 2: method for detecting hypochlorite ions
The difference from example 1 is that: the lake water is taken from the Hefei emerald lake in 2022 1/10 days, and the water temperature is measured to be 25 ℃ in the experiment.
Taking a certain volume of lake water, and filtering with a 0.22 mu m pore-size filter membrane to remove impurities for later use. 7.0 mu mol/L [ Ru (bpy) was removed 3 ] 2+ The @ CCM-NPs were placed in a 1.5mL PE tube and then lake water was added to a constant volume of 200mL. After 154.0 seconds of reaction at 25 ℃, the sample was transferred to a cuvette, and the fluorescence emission spectrum of the sample under 365nm excitation was measured with a fluorescence spectrophotometer, and the fluorescence emission spectrum intensities F at 503nm and 603nm were recorded 503nm 、F 603nm . Measurement of F 603nm /F 503nm = 0.74087, substituting it into the regression equation F 603nm /F 503nm ＝0.18166C ClO - +0.70179, giving a hypochlorite concentration of 0.2151. Mu. Mol/L.
Example 3: method for detecting hypochlorite ions
A method for detecting hypochlorite ions, comprising the steps of:
step 1: determining a curcumin standard curve;
step 2: synthesizing curcumin liposome;
step 3: assembling double-emission ratio fluorescent probe [ Ru (bpy) 3 ] 2+ @CCM-NPs；
Step 4: constructing a hypochlorite ion fluorescence detection regression equation;
step 5: pretreatment of a sample containing hypochlorite ions;
the sample to be measured is tap water, liquid food or environmental water; directly detecting tap water and liquid food; filtering the environmental water body by using filter paper or filter membrane with the aperture of 0.22 mu m before detecting, and collecting filtrate for detecting;
step 6: sample detection and hypochlorite ion concentration calculation;
first, the dual-emission ratio fluorescent probe [ Ru (bpy) ] is removed 3 ] 2+ The @ CCM-NPs solution is put in a PE tube, then a sample to be detected is added to a volume of 200.0 mu L, the sample is transferred to a cuvette after reaction, the fluorescence emission spectrum of the sample under 365nm excitation is measured by a fluorescence spectrophotometer, and the fluorescence emission spectrum intensity F at 503nm and 603nm is recorded 503nm 、F 603nm The method comprises the steps of carrying out a first treatment on the surface of the F to be measured 503nm 、F 603nm Value substitution into the saidAnd (3) obtaining a hypochlorite ion fluorescence detection regression equation, wherein the value of the hypochlorite ion in the sample to be detected is the hypochlorite ion residual value.
The preferred embodiments are: the determining curcumin standard curve comprises:
step one: weighing curcumin powder, and then adding dimethyl sulfoxide solution to prepare curcumin solution with concentration of 1 mmol/L;
step two: 1.0. Mu.L, 1.6. Mu.L, 2.2. Mu.L, 2.8. Mu.L, 3.4. Mu.L and 4.0. Mu.L curcumin solutions with concentration of 1mmol/L are removed, 199.0. Mu.L, 198.4. Mu.L, 197.8. Mu.L, 197.2. Mu.L, 196.6. Mu.L and 196.0. Mu.L mixed solution of dimethyl sulfoxide-deionized water are respectively added, and after mixing, ultraviolet absorption spectra are respectively measured;
step three: according to the corresponding ultraviolet absorption intensities measured by curcumin solutions with different concentrations, a corresponding curcumin concentration standard curve is prepared, and a regression equation is as follows: a=0.0376C Curcumin -0.0206, correlation coefficient of 0.9994, A being the ultraviolet absorption intensity of the sample to be measured, C Curcumin Is the concentration of curcumin.
The preferred embodiments are: the volume ratio of dimethyl sulfoxide to deionized water in the dimethyl sulfoxide-deionized water mixed solution is 1/4.
The preferred embodiments are: the synthetic curcumin liposome comprises: adding methoxy phospholipid polyethylene glycol and curcumin into a brown reagent bottle, uniformly mixing, performing ultrasonic treatment at room temperature, and stirring the mixed solution at a dark place overnight; after the completion of stirring, the mixed solution was evaporated at 37.0 ℃ to remove the organic solvent, and then transferred into an ultrafiltration tube of 10.0KD, centrifuged at 4000rpm, and washed 3 times with deionized water to obtain purified curcumin liposome nanoparticle having green fluorescence emission.
The preferred embodiments are: the ultrasonic power of the strong ultrasonic treatment is 150W, the ultrasonic frequency is 37kHz, and the ultrasonic treatment is continuously carried out for 5min.
The preferred embodiments are: the assembled dual emission ratio fluorescent probe [ Ru (bpy) 3 ] 2+ The @ CCM-NPs include: curcumin liposome and [ Ru (bpy) 3 ] 2+ Adding inIn PBS buffer solution, stirring and mixing uniformly to obtain a dual-emission ratio fluorescent probe [ Ru (bpy) 3 ] 2+ @CCM-NPs。
The preferred embodiments are: the construction of hypochlorite ion fluorescence detection regression equation comprises: double emission ratio fluorescent probe [ Ru (bpy) 3 ] 2+ Adding the @ CCM-NPs solution and hypochlorite ion solutions with different concentrations into PBS buffer solution, transferring to a cuvette after reaction, measuring fluorescence emission spectra of all reaction samples under 365nm excitation by using a fluorescence spectrophotometer, and recording fluorescence emission spectrum intensities F at 503nm and 603nm 503nm 、F 603nm The method comprises the steps of carrying out a first treatment on the surface of the As hypochlorite ion concentration increases, a dual emission ratio fluorescent probe [ Ru (bpy) ] is caused 3 ] 2+ Fluorescence intensity of @ CCM-NPs gradually decreased, using F 603nm /F 503nm And the hypochlorite ion concentration to make a regression equation: f (F) 603nm /F 503nm ＝0.18166C ClO - +0.70179, the correlation coefficient is 0.9952; the linear range is 0.0601-24 mu mol/L; the detection limit is 18.03nmol/L.
The foregoing description of the preferred embodiment of the invention is not intended to be limiting in any way, but rather, it is intended to cover all modifications or variations of the invention which fall within the spirit and scope of the invention.
Claims (7)
1. A method for detecting hypochlorite ions is characterized in that: comprises the following steps:
step 1: determining a curcumin standard curve;
step 2: synthesizing curcumin liposome;
step 3: assembling double-emission ratio fluorescent probe [ Ru (bpy) 3 ] 2+ @CCM-NPs；
Step 4: constructing a hypochlorite ion fluorescence detection regression equation;
step 5: pretreatment of a sample containing hypochlorite ions;
the sample to be measured is tap water, liquid food or environmental water; directly detecting tap water and liquid food; filtering the environmental water body by using filter paper or filter membrane with the aperture of 0.22 mu m before detecting, and collecting filtrate for detecting;
step 6: sample detection and hypochlorite ion concentration calculation;
first, the dual-emission ratio fluorescent probe [ Ru (bpy) ] is removed 3 ] 2+ The @ CCM-NPs solution is put in a PE tube, then a sample to be detected is added to a volume of 200.0 mu L, the sample is transferred to a cuvette after reaction, the fluorescence emission spectrum of the sample under 365nm excitation is measured by a fluorescence spectrophotometer, and the fluorescence emission spectrum intensity F at 503nm and 603nm is recorded 503nm 、F 603nm The method comprises the steps of carrying out a first treatment on the surface of the F to be measured 503nm 、F 603nm Substituting the value into the hypochlorite ion fluorescence detection regression equation to obtain the numerical value of the hypochlorite ion in the sample to be detected, namely the residual numerical value of the hypochlorite ion.
2. The method for detecting hypochlorite ions according to claim 1, wherein: the determining curcumin standard curve comprises:
step one: weighing curcumin powder, and then adding dimethyl sulfoxide solution to prepare curcumin solution with concentration of 1 mmol/L;
step two: 1.0. Mu.L, 1.6. Mu.L, 2.2. Mu.L, 2.8. Mu.L, 3.4. Mu.L and 4.0. Mu.L curcumin solutions with concentration of 1mmol/L are removed, 199.0. Mu.L, 198.4. Mu.L, 197.8. Mu.L, 197.2. Mu.L, 196.6. Mu.L and 196.0. Mu.L mixed solution of dimethyl sulfoxide-deionized water are respectively added, and after mixing, ultraviolet absorption spectra are respectively measured;
step three: according to the corresponding ultraviolet absorption intensities measured by curcumin solutions with different concentrations, a corresponding curcumin concentration standard curve is prepared, and a regression equation is as follows: a=0.0376C Curcumin -0.0206, correlation coefficient of 0.9994, A being the ultraviolet absorption intensity of the sample to be measured, C Curcumin Is the concentration of curcumin.
3. The method for detecting hypochlorite ions according to claim 2, wherein: the volume ratio of dimethyl sulfoxide to deionized water in the dimethyl sulfoxide-deionized water mixed solution is 1/4.
4. The method for detecting hypochlorite ions according to claim 1, wherein: the synthetic curcumin liposome comprises: adding methoxy phospholipid polyethylene glycol and curcumin into a brown reagent bottle, uniformly mixing, performing ultrasonic treatment at room temperature, and stirring the mixed solution at a dark place overnight; after the completion of stirring, the mixed solution was evaporated at 37.0 ℃ to remove the organic solvent, and then transferred into an ultrafiltration tube of 10.0KD, centrifuged at 4000rpm, and washed 3 times with deionized water to obtain purified curcumin liposome nanoparticle having green fluorescence emission.
5. The method for detecting hypochlorite ions according to claim 4, wherein: the ultrasonic power of the ultrasonic treatment is 150W, the ultrasonic frequency is 37kHz, and the ultrasonic treatment is continuously carried out for 5min.
6. The method for detecting hypochlorite ions according to claim 4, wherein: the assembled dual emission ratio fluorescent probe [ Ru (bpy) 3 ] 2+ The @ CCM-NPs include: curcumin liposome and [ Ru (bpy) 3 ] 2+ Adding into PBS buffer solution, stirring and mixing uniformly to obtain dual-emission ratio fluorescent probe [ Ru (bpy) 3 ] 2+ @CCM-NPs。
7. The method for detecting hypochlorite ions according to claim 1, wherein: the construction of hypochlorite ion fluorescence detection regression equation comprises: double emission ratio fluorescent probe [ Ru (bpy) 3 ] 2+ Adding the @ CCM-NPs solution and hypochlorite ion solutions with different concentrations into PBS buffer solution, transferring to a cuvette after reaction, measuring fluorescence emission spectra of all reaction samples under 365nm excitation by using a fluorescence spectrophotometer, and recording fluorescence emission spectrum intensities F at 503nm and 603nm 503nm 、F 603nm The method comprises the steps of carrying out a first treatment on the surface of the As hypochlorite ion concentration increases, a dual emission ratio fluorescent probe [ Ru (bpy) ] is caused 3 ] 2+ Fluorescence intensity of @ CCM-NPs gradually decreased, using F 603nm /F 503nm And the hypochlorite ion concentration to make a regression equation: f (F) 603nm /F 503nm ＝0.18166C ClO - +0.70179, the correlation coefficient is 0.9952; the linear range is 0.0601-24 mu mol/L; the detection limit is 18.03nmol/L.
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