Colorimetric/fluorescent probe for detecting hypochlorite ions and preparation method and application thereof
阅读说明：本技术 一种检测次氯酸根离子的比色/荧光探针及其制备方法与应用 (Colorimetric/fluorescent probe for detecting hypochlorite ions and preparation method and application thereof ) 是由 张然 苗保喜 赵鑫榆 倪中海 于 2021-08-11 设计创作，主要内容包括：一种检测次氯酸根离子的比色/荧光探针及其制备方法与应用,荧光探针命名为TPE-acylamide-Rh,其化学结构式如式I所示；由1-(4-甲酸苯基)-1,2,2-三苯乙烯溶于氯化亚砜中,加热反应后,在惰性气氛和催化作用下与罗丹明B酰肼反应合成、纯化得到。该探针在溶液状态和聚集状态下对ClO～(-)识别性能表现出优良的选择性、抗离子干扰能力以及灵敏度。另外,该探针在聚集状态形成纳米颗粒,容易细胞染色,有利于细胞成像应用。Hela细胞成像研究表明：TPE-acylamide-Rh表现出对细胞中ClO～(-)优异的识别特性。该探针制备工艺简单、合成路线短。将探针应用于检测次氯酸根离子中,拓宽了探针的应用范围。(A colorimetric/fluorescent probe for detecting hypochlorite ions, a preparation method and application thereof are disclosed, wherein the fluorescent probe is named as TPE-acylamide-Rh, and the chemical structural formula of the fluorescent probe is shown as formula I; dissolving 1- (4-phenyl formate) -1,2, 2-triphenylethylene in thionyl chloride, heating for reaction, reacting with rhodamine B hydrazide under the action of an inert atmosphere and catalysis, and purifying to obtain the rhodamine B hydrazide. The probe is used for ClO in a solution state and an aggregation state ‑ The identification performance shows excellent selectivity, anti-ion interference capability and sensitivity. In addition, the probe forms nanoparticles in an aggregation state, is easy to dye cells and is beneficial to cell imaging application. Hela cell imaging studies showed that: TPE-acylamide-Rh showed to be directed to ClO in cells ‑ Excellent identification characteristics. The probe has simple preparation process and synthesisThe route is short. The probe is applied to detecting hypochlorite ions, so that the application range of the probe is widened.)
1. The colorimetric/fluorescent probe for detecting hypochlorite ions is characterized in that the fluorescent probe is named as TPE-acrylamide-Rh, and the corresponding chemical structural formula is shown as formula I:
2. the method for preparing the colorimetric/fluorescent probe for detecting hypochlorite ions according to claim 1, which comprises the following steps:
dissolving 1- (4-phenyl formate) -1,2, 2-triphenylethylene in thionyl chloride, heating and stirring for 4-8h at the temperature of 60-80 ℃, distilling under reduced pressure to remove the thionyl chloride, cooling to room temperature, sequentially adding acetonitrile and triethylamine under the protection of inert atmosphere, finally adding rhodamine B hydrazide, heating and stirring for 10-18h at the temperature of 40-85 ℃; the molar ratio of the 1- (4-phenyl formate) -1,2, 2-triphenylethylene to the rhodamine B hydrazide is 1: 1.03; after the reaction is finished, extracting the reaction liquid, combining organic phases, drying to obtain a crude product, and purifying the crude product by silica gel column chromatography to obtain TPE-acylamide-Rh; the structural formula of the rhodamine B hydrazide is
3. The method for preparing a colorimetric/fluorometric probe for detecting hypochlorite ion according to claim 2, wherein after the reaction, the reaction solution is poured into deionized water and extracted with dichloromethane three times.
4. The method for preparing a colorimetric/fluorometric probe for detecting hypochlorite ions according to claim 2 or 3, wherein the eluent used for column chromatography purification is a dichloromethane/methanol solution with a volume ratio of 30: 1.
5. Use of the fluorescent probe of claim 1 to detect hypochlorite ion content.
Technical Field
The invention belongs to the field of organic fluorescent molecular probes, and particularly relates to a colorimetric/fluorescent probe for detecting hypochlorite ions, and a preparation method and application thereof.
Background
Hypochlorous acid, as a strong oxidant, is widely applied to tap water disinfection, clothes bleaching and the like in daily life; is also an important active oxygen small molecule in organisms and is involved in the processes of immune regulation, antigen response, signal transduction, cell withering and the like of the organisms. Breaking in vivo ClO-The balance can cause various cardiovascular diseases, tissue inflammation and even cancer. Therefore, to prevent from ClO-To research rapid, efficient, accurate detection of ClO in the environment and in vivo-There is a great need for methods of (1).
With conventional ClO-Compared with detection methods (iodometry, coulometry, polarography, electrochemistry, potentiometry and the like), the fluorescent probe has the advantages and potentials of rapidness, sensitivity, good selectivity, in-situ real-time detection and the like, is widely applied, and has great advantages in hypochlorous acid development imaging technology in cells. ClO-Has no coordination ability of metal ions and no CN-Strong nucleophilic property of or F-Is strongly electronegative, and thus, ClO-The design and synthesis of fluorescent probes present certain challenges.
In recent years, researchers have designed and synthesized many clos based on different detection mechanisms-Fluorescent probes, such as fluorescence quenching type, OFF-ON type, ratiometric type, etc. Compared with a fluorescence quenching type probe, although the OFF-ON type decarburized light probe has a certain improvement ON detection sensitivity and is easy to realize visual detection, the detection is carried out under a single wavelength, and the interference of the light intensity, photobleaching and background light of detection equipment is easy to occur; in the detection process, the concentration of the probe sample and the change of the micro-environment (such as the uniformity, viscosity and overflow degree of a test sample) borne by the probe sample can cause deviation to the experimental result; the ratio type fluorescent probe has two related emission signals, can avoid detection errors caused by instruments and environmental factors, and can show better sensitivity, dynamic response range and the like. In addition, some of the current designs and reports are for ClO-Fluorescent imaging probe, few probes can realize ClO in biological system-Real-time quantitative fluorescence imaging. Therefore, the design and synthesis of the ratio-type ClO with high sensitivity and capability of real-time quantitative fluorescence imaging-Fluorescent probe and application thereof to ClO in living body-The real-time quantitative fluorescence imaging is still ClO-The center of gravity of the probe study work.
Disclosure of Invention
One of the objectives of the present invention is to provide a colorimetric/fluorescent probe for detecting hypochlorite ions, which has high sensitivity and can realize real-time quantitative fluorescence imaging.
The invention also aims to provide the preparation method of the colorimetric/fluorescent probe for detecting hypochlorite ions, which has the advantages of simple preparation process and short synthetic route.
The invention also aims to provide the application of the colorimetric/fluorescent probe for detecting hypochlorite ions, and the application range of the probe is widened.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows: a colorimetric/fluorescent probe for detecting hypochlorite ions is named as TPE-acrylamide-Rh, and the corresponding chemical structural formula is shown as formula I:
the preparation method of the colorimetric/fluorescent probe for detecting hypochlorite ions comprises the following steps:
dissolving 1- (4-phenyl formate) -1,2, 2-triphenylethylene in thionyl chloride, heating and stirring for 4-8h at the temperature of 60-80 ℃, distilling under reduced pressure to remove the thionyl chloride, cooling to room temperature, sequentially adding acetonitrile and triethylamine under the protection of inert atmosphere, finally adding rhodamine B hydrazide, heating and stirring for 10-18h at the temperature of 40-85 ℃; the molar ratio of the 1- (4-phenyl formate) -1,2, 2-triphenylethylene to the rhodamine B hydrazide is 1: 1.03; after the reaction is finished, extracting the reaction liquid, combining organic phases, drying to obtain a crude product, and then obtaining the crude productPurifying by silica gel column chromatography to obtain TPE-acylamide-Rh; the structural formula of the rhodamine B hydrazide is
Further, after the reaction, the reaction solution was poured into deionized water and extracted with dichloromethane three times.
Preferably, the eluent used for column chromatography purification is a 30:1 volume ratio dichloromethane/methanol solution.
The specific synthetic route is as follows:
the invention also provides application of the fluorescent probe in detecting the content of hypochlorite ions.
The tetraphenyl ethylene is a molecule with aggregation-induced emission mechanism (AIE) property, the red light characteristic of rhodamine enables the rhodamine to be an excellent fluorescent group, and under the mutual coupling effect of the tetraphenyl ethylene and the rhodamine, the tetraphenyl ethylene not only can show colorimetric/fluorescent double-channel response in a solution state, but also can obtain a ratio type fluorescent probe in an aggregation state. The fluorescent probe can be calibrated by a colorimetric channel and a fluorescent channel in a solution state; interference can also be eliminated in the aggregated state by dual wavelength response. The invention takes tetraphenylethylene as a fluorescent group to modify and regulate different positions of rhodamine and design and synthesize reactive ClO-And fluorescent probe TPE-acrylamide-Rh. Due to aggregation induction effect of tetraphenylethylene, the fluorescent probe shows the property of a ratio type fluorescent probe in an aggregated state, and recognition sites of the fluorescent probe are all amide structures of rhodamine, wherein the amide structures are in ClO-Under the action of strong oxidizing property, decomposition reaction occurs, so that rhodamine is subjected to ring opening reaction, and colorimetric and fluorescent response of the probe solution is caused.
Compared with the prior art, the invention has the following advantages:
the invention designs and synthesizes rhodamine amide group as a recognition reaction siteRatio type ClO of tetraphenylethylene coupled rhodamine reaction-The probe shows AIE properties, and shows colorimetric/fluorescent double-channel response in a solution state (in a 50% ethanol-water system) and ClO-Under the action of the solution, the color of the solution generates a response (changes from colorless to red) visible to the naked eye; under the excitation of a 365nm ultraviolet lamp, orange yellow fluorescence is emitted; and shows better sensitivity (the lowest detection limit of ultraviolet absorption spectrum is 1.3 mu M; the lowest detection limit of fluorescence spectrum is 2.5 mu M); in an aggregated state (in a 60% ethanol-water system), exhibits a ratiometric fluorescence characteristic, recognizing ClO-Also has good sensitivity (the lowest detection limit of fluorescence spectrum is 2.8 mu M). Therefore, the probe is directed to ClO in a solution state and an aggregation state-The identification performance shows excellent selectivity, anti-ion interference capability and sensitivity, and real-time quantitative fluorescence imaging is realized. In addition, the probe forms nanoparticles in an aggregation state, is easy to dye cells and is beneficial to cell imaging application. Hela cell imaging research shows that TPE-acylamide-Rh shows that ClO in cells-Excellent recognition characteristics, therefore, the probe is successfully applied to ClO in living cells-Fluorescence imaging techniques. The fluorescent probe has simple preparation process and short synthetic route. The probe is applied to detecting hypochlorite ions, so that the application range of the probe is widened.
Drawings
FIG. 1 is a nuclear magnetic hydrogen spectrum of TPE-acylamide-Rh compound;
FIG. 2 is a mass spectrum of TPE-acylamide-Rh compound;
FIG. 3 is a graph showing the trend of the peak fluorescence value of TPE-acrylamide-Rh (2 μ M) in ethanol-water solutions with different water contents as a function of the water content;
FIG. 4 is the fluorescence emission spectrum of TPE-acrylamide-Rh (2 μ M) in ethanol-water solution with different water content;
FIG. 5 shows the reaction of TPE-acrylamide-Rh (2. mu.M) with ClO in ethanol-water solutions of different water contents-A fluorescence spectrum;
FIG. 6 is a graph of the UV absorption spectra of TPE-acrylamide-Rh (2 μ M) in a 50% ethanol-water system for different analytes;
FIG. 7 shows TPE-acrylamide-Rh (2 μ M) in 50% ethanol-water system for different analytes and ClO addition-The subsequent ultraviolet absorption spectrogram;
FIG. 8 is a fluorescence spectrum of TPE-acylamide-Rh (2 μ M) in a 50% ethanol-water system for different analytes;
FIG. 9 shows TPE-acrylamide-Rh (2. mu.M) in 50% ethanol-water system for different analytes and ClO addition-The subsequent fluorescence spectrogram;
FIG. 10 is a fluorescence spectrum of TPE-acrylamide-Rh (2 μ M) in 60% ethanol-water system for different analytes;
FIG. 11 shows TPE-acrylamide-Rh (2. mu.M) in 60% ethanol-water system for different analytes and ClO addition-The subsequent fluorescence spectrogram;
FIG. 12 is a graph of the fluorescence titration spectrum of TPE-acylamide-Rh (2. mu.M) in a 50% ethanol-water system;
FIG. 13 shows the fluorescence intensity of TPE-acylamide-Rh (2. mu.M) in 50% ethanol-water system with ClO-A linear plot of concentration;
FIG. 14 is a graph of the fluorescence titration spectrum of TPE-acylamide-Rh (2. mu.M) in a 60% ethanol-water system;
FIG. 15 shows the ratio of fluorescence intensity of TPE-acylamide-Rh (2. mu.M) in 60% ethanol-water system to ClO-A linear plot of concentration;
FIG. 16 is a graph of the UV absorption titration spectrum of TPE-acylamide-Rh (2. mu.M) in a 50% ethanol-water system;
FIG. 17 shows the UV absorption intensity of TPE-acrylamide-Rh (2 μ M) in 50% ethanol-water system and ClO-A linear plot of concentration;
FIG. 18 is a statistical chart of the results of the HeLa cytotoxicity test of TPE-acylamide-Rh;
FIG. 19 is a photograph of the fluorescent image of the TPE-acrylamide-Rh in HeLa cells: (a) imaging HeLa cells in a TPE-acrylamide-Rh bright field; (b) imaging HeLa cells in TPE-acrylamide-Rh green channel; (c) HeLa cells were cultured in TPE-acrylamide-Rh and 20. mu.M ClO-Imaging of cells in red channel; (d) the graph b and the graph c are superposed.
Detailed Description
The invention is further described with reference to the following figures and specific examples.
The starting materials and reagents used in the following examples are commercially available products unless otherwise specified, and the purity thereof was analytical purity and above.
Example 1: synthesis of probe compound TPE-acylamide-Rh
Weighing 0.50g (1.33mmol) of 1- (4-phenyl formate) -1,2, 2-triphenylethylene, adding the weighed solution into 5mL of thionyl chloride solution, and heating and refluxing the solution for 4 hours; after the reaction is finished, distilling under reduced pressure to evaporate the thionyl chloride solution. When the temperature is reduced to room temperature, adding 30mL of acetonitrile solution, and then adding 1mL of triethylamine; finally, 0.62g (1.37mmol) of rhodamine B hydrazide is weighed and added into the reaction solution, and the mixture is heated and refluxed under the protection of nitrogen and reacted overnight. After the reaction is finished, pouring the reaction liquid into water, extracting the reaction liquid for three times by using dichloromethane, combining organic phases, drying the mixture by using anhydrous magnesium sulfate, and performing spin drying to obtain a crude product, wherein the weight ratio of dichloromethane: methanol volume ratio of 30:1 as mobile phase, passing through silica gel column to obtain 0.35g pure product, yield: 31.4%, melting point: 173 ℃ and 175 ℃. The hydrogen spectrum is shown in FIG. 1, the mass spectrum is shown in FIG. 2,1H NMR(600MHz,DMSO-d6)δ10.10(s,1H),7.84(d,J＝7.3Hz,1H),7.63–7.51 (m,2H),7.40(d,J＝8.1Hz,2H),7.19–7.05(m,10H),7.01–6.91(m,8H),6.55(d,J＝8.8Hz, 2H),6.35–6.25(m,4H),3.33–3.19(m,8H),1.06(t,J＝7.0Hz,12H).13C NMR(151MHz, Chloroform-d)δ156.87.,153.15,151.88,148.75,143.83,140.48,136.04,133.80,133.05, 131.24,131.22,131.18,128.91,128.10,128.02,127.63,127.60,127.52,126.34,126.12, 126.04,123.75,123.47,123.26,110.08,107.89,106.26,97.95,65.84,55.37,44.32,12.67. MALDI-MS:m/z calcd for C55H50N4O3:815.0105,found:814.9627[M]+.
example 2: probe TPE-acrylamide-Rh ratio type fluorescence characteristic and AIE property
The probe TPE-acylamide-Rh is ClO with AIE characteristic and is connected with rhodamine B through amido bond by tetraphenyl ethylene-A fluorescent probe. The recognition site of the probe TPE-acrylamide-Rh is a rhodamine amide structure which is in ClO-Under the action of strong oxidizing property, amido bond is broken, ring-opening reaction of rhodamine spiro ring occurs, and color comparison/fluorescence of the probe changes. The probe TPE-acrylamide-Rh shows the fluorescence of tetraphenyl ethylene in an aggregation state and reacts with ClO-After the reaction, the aggregation-induced luminescence of tetraphenylethylene of the probe disappears, and the fluorescence of a rhodamine structure is displayed, so that the probe becomes a typical ratiometric fluorescent probe.
The probe TPE-acrylamide-Rh shows typical aggregation-induced emission properties due to the tetraphenylethylene group with a "propeller" structure (as shown in FIGS. 3 and 4). The probe TPE-acrylamide-Rh has good solubility in an ethanol-water system, and is in a dissolved state and free of fluorescence emission when the water content of the solution is 0-50%; when the water content of the solution reaches 60%, the TPE-acrylamide-Rh is subjected to nano aggregation (the average particle size is 396nm), and an obvious fluorescence emission peak is generated at the position of 487nm, wherein the fluorescence emission peak is a typical tetraphenyl ethylene fluorescence emission peak; when the water content of the solution is 70%, the fluorescence intensity reaches the maximum value; then, as the water content of the solution increases, the fluorescence intensity gradually decreases.
Example 3: solvent ratio selection of probe TPE-acrylamide-Rh
TPE-acylamide-Rh is a reactive ClO-The sensitivity of the fluorescent probe is influenced by the selection of the proportion of the fluorescent probe and the solvent. Testing the addition of ClO to ethanol-water systems of different water contents-The optimal solvent ratio was investigated according to the fluorescence intensity of (2), and the results are shown in FIG. 5. The water content of the TPE-acrylamide-Rh solution is between 0% and 50%, the TPE-acrylamide-Rh solution is in a dissolved state, has no fluorescence emission, and is in a ClO state-Under the action, a fluorescence emission peak appears at 583nm, and the intensity of the emission peak is maximum when the water content is 50%, which indicates that: TPE-acrylamide-Rh in solution state to ClO-Has good sensitivity to ClO in an ethanol-water system with 50 percent of water content-The response sensitivity is highest. Therefore, TPE-acrylamide-Rh was in solution to ClO-The identified solvent ratios were selected for the 50% ethanol-water system. When the water content of the solution reaches 60%, the probe is in a nano-aggregation state, and emission peaks appear at 583nm and 478nm at the same time, which shows typical characteristicsThe ratio-type fluorescence characteristic is that when the water content is more than 70%, the fluorescence emission peak intensity at 583nm is very weak, and almost no response is caused to ClO-ions, so that the optimal solvent ratio condition of the probe TPE-acylamide-Rh in the aggregation state is as follows: 60% ethanol-water system.
Example 4: probe TPE-acrylamide-Rh couple ClO-Selectivity and anti-ion interference ability of identification
Preparation of TPE-acrylamide-Rh solution: 2.0X 10 of TPE-acrylamide-Rh-3mol L-1The solution was prepared by using chromatographically pure ethanol as a solvent, weighing the calculated amount of the probe prepared in example 1, adding the solution to a 3mL volumetric flask, fixing the volume with ethanol, performing ultrasonic treatment until the sample is completely dissolved, and storing the solution in a sealed manner for later use.
ClO-And preparing an interference ion solution: with NO2 -、NO3 -、HCO3 -、ACO-、Br-、ClO4 -、SO4 2-、 S2-、H2O2、NO、ONOO-DTBP and ClO-Dissolving in deionized water to prepare an ionic solution. The concentration of the ionic solution is 1.0X 10- 2mol L-1Weighing the ionic salts with calculated amount, putting the ionic salts into a 5mL volumetric flask, fixing the volume to the graduation line of a volumetric product by using deionized water, shaking and carrying out ultrasonic treatment to completely dissolve each ionic salt, and sealing and storing the ionic salts for later use.
As shown in FIG. 6, the probe TPE-acrylamide-Rh was in solution in a 50% ethanol-water system except in ClO-The solution changed from colorless to red in the presence of the ion, and no other interfering ions changed significantly. Meanwhile, in the presence of other ions, adding ClO continuously-As shown in FIG. 7, the probe TPE-acrylamide-Rh showed good anti-ion interference ability. Similarly, the probe TPE-acrylamide-Rh also shows excellent selectivity and anti-ion interference capability in the fluorescence spectrum (as shown in FIGS. 8-9).
The probe TPE-acrylamide-Rh shows the property of a ratio type fluorescent probe in an ethanol-water system with the water content of 60 percent. Probe TPE-acrylamide-Rh (2. mu.M) in 60% ethanol-waterIn (1), 10 times of equivalent of test ions are added respectively, and the selectivity of the probe is studied by the fluorescence spectrum intensity. As shown in FIG. 10, in the 60% ethanol-water system, since the probe is in an aggregated state, the fluorescence spectrum of TPE-acrylamide-Rh has a fluorescence emission peak at 487nm before the test ion is added, and only ClO is added after the test ion is added-The fluorescence in the fluorescent probe solution emits orange-red fluorescence, and the emission wavelength of the fluorescence is 583 nm; the emitted fluorescence intensity of other ion solutions at 487nm has only a small change, which indicates that the probe TPE-acylamide-Rh has ClO-Has a higher selectivity. At the same time, continuously adding ClO in the presence of other ions-As shown in FIG. 11, the probe TPE-acrylamide-Rh shows better ion interference resistance.
Example 5: probe TPE-acrylamide-Rh couple ClO-Titration of fluorescence spectra
The probe TPE-acrylamide-Rh has fluorescence intensity and ClO under the solution state (50% ethanol-water)-The trend of the concentration change is shown in FIG. 12. The TPE-acrylamide-Rh blank solution has weak fluorescence; adding ClO-Then, the fluorescence spectrum shows a new fluorescence emission peak at 583nm, along with ClO-The fluorescence intensity is gradually enhanced when the concentration is increased, and when the concentration is increased, the ClO is added-Concentration up to 30 μ M, slow increase in fluorescence intensity, ClO-The concentration reached a saturated state. As can be seen from FIG. 13, the fluorescence intensity of the probe TPE-acylamide-Rh is in ClO-A good linear relationship was exhibited between 0.5. mu.M and 30. mu.M. Obtaining fluorescence intensity and ClO through linear fitting-The concentration relation is as follows: 14680.71x +106666.31, R20.993. The lowest detection limit of the probe TPE-acrylamide-Rh in a 50% ethanol-water system can be calculated by the lowest detection limit formula LOD ═ 3 sigma/k: 1.3. mu.M.
The probe TPE-acrylamide-Rh has fluorescence intensity and ClO under an aggregation state (60% ethanol-water)-The trend of the concentration change is shown in FIG. 14. When the TPE-acrylamide-Rh blank solution is excited at 365nm, the solution generates a strong tetraphenylethylene fluorescence emission peak (maximum emission wavelength: 476nm), and ClO is added-Then, the fluorescence spectrum shows a new fluorescence emission peak at 583nm, along with ClO-The concentration is increased, the fluorescence intensity at 476nm is gradually reduced, and the fluorescence intensity at 583nm is gradually enhanced; when ClO is present-The fluorescence intensity hardly changed at the two positions at a concentration of 30. mu.M. As can be seen from FIG. 15, the ratio of fluorescence intensity of TPE-acrylamide-Rh probe at 583nm to that at 476nm is in ClO-A good linear relationship was exhibited between 0.5. mu.M and 30. mu.M. Linear fitting to obtain I579nm/I485nmAnd ClO-The concentration relation is as follows: 0.08394x +0.16389, R20.998. The lowest detection limit of the probe TPE-acrylamide-Rh in a 60% ethanol-water system is calculated according to the lowest detection limit formula LOD which is 3 sigma/k: 2.8. mu.M.
Example 6: probe TPE-acrylamide-Rh couple ClO-Titration by ultraviolet absorption spectroscopy
The probe TPE-acrylamide-Rh is designed and synthesized based on a rhodamine structure, so that the probe TPE-acrylamide-Rh can identify ClO-Then, not only will there be a fluorescent response, but also will cause a colorimetric response. As shown in FIG. 16, the TPE-acrylamide-Rh blank solution has no ultraviolet absorption peak, the solution is colorless, and ClO is added-Later, a new absorption peak in the ultraviolet absorption spectrum appears at 556nm, along with ClO-The concentration is increased, the absorption peak intensity is gradually enhanced when the ClO is added-After the concentration reached 30. mu.M, the UV absorption peak intensity stopped increasing. As shown in FIG. 17, the UV absorption intensity of the probe TPE-acrylamide-Rh is in ClO-The concentration between 2 and 7. mu.M shows a good linear relationship. Obtaining ultraviolet absorption spectrum intensity and ClO through linear fitting-The concentration relation is as follows: 0.02688x-0.17201, R20.978. The lowest detection limit of the probe TPE-acrylamide-Rh in a 50% ethanol-water system can be calculated by the formula LOD of the lowest detection limit being 3 sigma/k: 2.5. mu.M.
Example 7: cytotoxicity research of probe TPE-acrylamide-Rh
Cell culture: cervical cancer cells for cell imaging (Hela cells) were purchased from bio, Hela cell culture process: the purchased Hela cells were transferred to a culture dish containing 2mL of DMEM culture solution containing 10% fetal bovine serum, and the culture dish was put into a sterile incubator maintained at a constant temperature of 37 ℃ with a carbon dioxide content for 24 hours for cell imaging.
Cytotoxicity test: hela cells are transferred to a 96-well culture dish containing 100 mu L of culture solution, incubated for 24h in an environment with 5% of carbon dioxide content and constant temperature of 37 ℃, then 100 mu L of probe solutions with the concentrations of 0 mu M, 5 mu M, 10 mu M, 20 mu M and 30 mu M are prepared by using the culture solution, and are respectively placed into a 96-well culture dish with a mark, 5 groups of parallel experiments are carried out on each concentration of probe for the accuracy of the experiments, and the culture is continued for 24 h. Then 20. mu.L of MTT (mg/mL) was added to each well and incubated for 4 h. And finally, adding 100 mu L of dimethyl sulfoxide solution, uniformly mixing, adding an enzyme-labeling instrument, and calculating the cell survival rate by measuring the ultraviolet absorption intensity of the mixture at 570nm so as to reflect the toxicity of the fluorescent probe.
The results of the probe TPE-acrylamide-Rh in Hela cytotoxicity test (MTT) are shown in FIG. 18. Hela cells were incubated in different concentrations (0. mu.M, 5. mu.M, 10. mu.M, 20. mu.M and 30. mu.M) of the probe TPE-acrylamide-Rh for the same time, and the cell viability was observed. The cell survival rate is reduced slightly along with the increase of the concentration of TPE-acrylamide-Rh, and when the concentration of the probe reaches 30 mu M, the cell survival rate is over 95 percent. The result shows that the low-concentration TPE-acrylamide-Rh probe has low cytotoxicity and is suitable for cell imaging technology.
Example 8: cellular imaging of Probe TPE-acylamide-Rh
Cell imaging is mainly used for detecting exogenous ClO-Cell imaging experiments. Two groups were used for cell imaging. In the first group, Hela cells are put into a culture dish containing a culture solution to be cultured for 24 hours, washed three times by using a PBS buffer solution with the pH value of 7.4, then 20 mu L of a fluorescent probe standard solution is added into the cell culture solution to be cultured for 1 hour, finally washed three times by using a PBS buffer solution with the pH value of 7.4, cell metabolites and dead cells are washed away, and the rest cells are used for cell imaging; the second group cultured Hela cells in a culture dish containing a culture solution for 24 hours, washed three times with a PBS buffer solution having a pH of 7.4, added 20. mu.L of a fluorescent probe standard solution to the cell culture solution, cultured for 1 hour, and added 10. mu.L of ClO-Standard of meritThe solution was incubated for 1h, washed three times with PBS buffer pH 7.4, and cell metabolites and dead cells were washed away, leaving the cells for cell imaging.
As shown in FIG. 19, after the TPE-Acylamide-Rh solution was added to Hela cell culture medium for culturing, the cells were transferred to a confocal microscope, and excited by ultraviolet light of 405nm, and the TPE-Acylamide-Rh probe was obtained by observing a blue channel (425nm-475nm), which easily entered the cells and emitted a weak yellow-green fluorescence (as shown in FIG. 19-b). When 20. mu.M ClO was added-After the culture was continued, the cells were again placed under a confocal microscope for photographing and observation, and the cells were found to emit red fluorescence (as shown in FIG. 19-c). The cell imaging experiment of the probe TPE-acrylamide-Rh shows that the probe is used for treating the ClO in the cell-The recognition effect (red fluorescence in cell imaging) was more pronounced than in the 60% ethanol-water system (orange-yellow fluorescence in solution). This is because the confocal microscope has two channels (blue light channel and red light channel), wherein the excitation wavelength of the blue light channel is 405nm, the excitation wavelength of the red light channel (570-620nm) is 561nm, and in the cell imaging experiment, the picture obtained after the probe reacts with the ions is collected through the red light channel under the excitation of 561nm, so the fluorescence is stronger than the fluorescence displayed by the fluorescence titration solution. The result shows that the probe TPE-acrylamide-Rh can sensitively detect ClO in cells-. Therefore, the probe TPE-acrylamide-Rh can realize the effect of ClO in Hela living cells-Detecting and can realize ratio type cell fluorescence imaging.