CN114716360A - Preparation and application of colorimetric/spectral dual-mode fluorescence sensor for detecting acidic pH change - Google Patents
Preparation and application of colorimetric/spectral dual-mode fluorescence sensor for detecting acidic pH change Download PDFInfo
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Abstract
Based on an intramolecular charge transfer mechanism, the invention discloses a high-sensitivity colorimetric/spectral dual-mode fluorescent probe (H4) for detecting acidic pH change and a preparation method thereof. The H4 fluorescent probe has the characteristics of excellent sensitivity, short response time (less than 10s), low hemolytic effect (less than 0.3%), good light stability (more than 120min) and the like, and has a good linear relation (R) within the pH range of 2.1-7.42＝0.9901，Y＝22.0409X‑58.3397)，pKaWas 3.2. In addition, the probe can also be used as a fluorescence test piece and further used as a portable sensor, so that the screening speed is improved, and H is realized+The method has great application potential in food freshness evaluation.
Description
Technical Field
The invention relates to the technical field of preparation and application of fluorescent probes, in particular to a high-performance fluorescent probe which is high in sensitivity, quick in response, good in stability and capable of detecting acidic pH change.
Background
pH plays an irreplaceable role in cells, organisms, the environment and food. Especially, abnormal changes in acidic pH value affect the normal metabolic function of the biological system, thereby causing various symptoms. Such as gastric cancer and alzheimer's disease. In environmental aspects, acid abnormalities can lead to acid rain, water and soil loss, crop loss, and the like. In addition, in the aspect of food safety, an abnormal pH value may also cause food to be rotten. For example, protein-based foods, such as milk and tofu, deteriorate upon contact with acid. Therefore, it is essential to accurately track the acidic pH changes of the environment and biological systems (such as plants and food). To date, many analytical and testing methods for monitoring pH changes have been widely used, including plasma mass spectrometry, atomic absorption spectroscopy, atomic fluorescence spectroscopy, and the like. However, some techniques are complex in operation, complex in sample pretreatment and long in time consumption. Fluorescence analysis techniques have recently been favored by researchers due to their characteristics of accuracy, quantitative and qualitative detection, real-time monitoring, colorimetric visualization, and the like. In addition, the fluorescent probe is combined with a fluorescent imaging technology, so that the quantitative and qualitative detection of small molecules (such as ions, pH and ROS) in the environment and a living system can be realized. In recent years, various fluorescent probes have been designed and reported, but some of them are complicated in synthesis and post-treatment and have low sensitivity. Therefore, it is necessary to develop a fluorescent probe for real-time detection of substances, which has the advantages of simple operation, good colorimetric effect, high sensitivity and strong selectivity.
Based on the design, the invention designs a micromolecule fluorescent probe material, and a D-Pi-A structure is formed by taking a vinyl bridge formed by connecting 4-diphenylamine benzaldehyde and benzene dicarboxylic acyl as a probe. The probe can be prepared by a one-step method, and the synthesis and post-treatment methods are simple. In order to realize real-time and accurate detection of acid abnormality in environment and food, the invention also prepares a portable and simple fluorescent sensor combined test paper, and can carry out instant response on proton signals by a colorimetric method. In addition, the fluorescent probe also shows great advantages of fluorescent and visual bimodulus, high selectivity, photostability, compatibility to aqueous solutions and food samples (zebrafish and onion tissues) and sensitive monitoring of acidic pH changes, which undoubtedly arouses greater research interest in the food and bio-related fields.
Disclosure of Invention
The invention mainly solves the problems of complex synthesis and post-treatment, complex purification method and the like of the existing fluorescent probe, and simultaneously solves the problems of complex detection process, easy interference from other external environments during detection, insensitive detection and the like of the existing H + fluorescent probe, and provides the H + fluorescent probe which is simple to operate, good in colorimetric effect, high in sensitivity and strong in selectivity, and the spectral performance and the application thereof. The structural formula of the high-sensitivity colorimetric/spectral dual-mode fluorescent probe for detecting the change of acidic pH is as follows:
the preparation method of the high-sensitivity colorimetric/spectral dual-mode fluorescent probe (H4) for acidic pH change comprises the following steps:
firstly, sequentially adding 2, 3, 3-trimethyl-3H-benzo [ e ] indole, 4-diphenylamine benzaldehyde, hydrochloric acid and absolute ethyl alcohol into a reaction container with a reflux device; heating to 75-85 deg.C, maintaining reflux for reaction, tracking reaction process by thin layer chromatography (developer petroleum ether/ethyl acetate volume ratio is 1: 1), and stopping reaction when new product point appears on silica gel plate and two raw materials disappear.
Secondly, concentrating the reactant in the reaction container to 30mL, cooling to room temperature, collecting precipitate, then recrystallizing with absolute ethyl alcohol, and drying for 6H to obtain the fluorescent probe which has high sensitivity colorimetric/spectral dual-mode recognition on acidic pH change and is a red solid, and is abbreviated as H4.
The synthesis preparation process of the invention is as follows:
the fluorescent probe with high-sensitivity colorimetric/spectral dual-mode identification on the change of acidic pH can be prepared by one-step method, the solvent adopted in the synthetic process is non-toxic, the purification operation is simple, and the fluorescent probe shows the great advantages of fluorescence and vision dual modes. In addition, the probe has high sensitivity, selectivity, correctability and reliability within the pH range of 7.4-2.1, and can quantitatively detect the pH change in a solution. The fluorescent probe has extremely high application value in detecting the pH change of living organisms (zebra fish and onion tissues).
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the description of the embodiments will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without inventive exercise. Wherein:
FIG. 1 is a schematic representation of a colorimetric/spectroscopic dual mode fluorescent probe for detecting acidic pH changes prepared in example 1 in a buffer solution (H)2And (3) ultraviolet absorption spectra under different pH values (pH value of 7.4-2.1) in O/EtOH (99: 1, v/v). The abscissa is wavelength and the ordinate is absorbance.
FIG. 2 is a schematic representation of the colorimetric/spectroscopic dual mode fluorescent probe for detecting acidic pH changes prepared in example 1 in buffer solution (H)2Fluorescence emission spectra at different pH values (pH 7.4-2.1) in O/EtOH 99: 1, v/v), wherein lambdaex436nm and a slit width of 10/12 nm. The abscissa is wavelength and the ordinate is fluorescence intensity.
FIG. 3 is a change in scattergram between the fluorescence intensity and pH at 545nm of the colorimetric/spectroscopic dual-mode fluorescent probe for detecting acidic pH change prepared in example 1. The abscissa is the pH value and the ordinate is the fluorescence intensity.
FIG. 4 is a fluorescent image of the colorimetric/spectroscopic dual-mode fluorescent probe for detecting acidic pH change prepared in example 1 incubated with zebrafish in a mixed solution of different pH values for various periods of time.
Detailed Description
The first embodiment is as follows: a high-sensitivity colorimetric/spectroscopic dual-mode fluorescent probe for detecting acidic pH changes has the structural formula as follows:
the second embodiment is as follows: the preparation method of the high-sensitivity colorimetric/spectroscopic dual-mode fluorescent probe (H4) for detecting acidic pH change comprises the following steps:
firstly, sequentially adding 2, 3, 3-trimethyl-3H-benzo [ e ] indole, 4-diphenylamine benzaldehyde, hydrochloric acid and absolute ethyl alcohol into a reaction container with a reflux device; heating to 75-85 deg.C, maintaining reflux for reaction, tracking reaction process by thin layer chromatography (developer petroleum ether/ethyl acetate volume ratio is 1: 1), and stopping reaction when new product point appears on silica gel plate and two raw materials disappear.
Secondly, concentrating the reactant in the reaction container to 30mL, cooling to room temperature, collecting precipitate, then recrystallizing with absolute ethyl alcohol, and drying for 6H to obtain the fluorescent probe which has high sensitivity colorimetric/spectral dual-mode recognition on acidic pH change and is a red solid, and is abbreviated as H4.
The third concrete implementation mode: the present embodiment is different from the second embodiment in that the molar ratio of 2, 3, 3-trimethyl-3H-benzo [ e ] indole to 4-diphenylaminobenzaldehyde in the first step is 1: (1.5 to 3.5), and the other steps are the same as the second embodiment.
The fourth concrete implementation mode: the second or third embodiment is different from the second or third embodiment in that the ratio of the amount of 2, 3, 3-trimethyl-3H-benzo [ e ] indole to the volume of absolute ethanol in the first step is 1 mmoL: (20.0-25.0) mL, and the other embodiments are the same as the second or third embodiment.
The following examples are used to demonstrate the beneficial effects of the present invention:
example 1: the preparation method of the high-sensitivity colorimetric/spectral dual-mode fluorescent probe for detecting the change of the acidic pH value comprises the following steps of:
firstly, sequentially adding 2, 3, 3-trimethyl-3H-benzo [ e ] indole, 4-diphenylamine benzaldehyde, hydrochloric acid and absolute ethyl alcohol into a reaction container with a reflux device; heating to 75-85 deg.C, maintaining reflux for reaction, tracking reaction process by thin layer chromatography (volume ratio of developing agent petroleum ether/ethyl acetate is 1: 1), and stopping reaction when new product point appears on silica gel plate and two raw materials disappear.
Concentrating the reactant in the reaction container to 30mL, cooling to room temperature, collecting precipitate, then recrystallizing with absolute ethyl alcohol, and drying for 6H to obtain the fluorescent probe with high sensitivity colorimetric/spectral dual-mode recognition on acidic pH change, wherein the probe is a red solid and is abbreviated as H4.
The structure of the high-sensitivity colorimetric/spectroscopic dual-mode fluorescent probe for detecting acidic pH change prepared in this example was characterized by a nuclear magnetic resonance spectrum and a mass spectrum, and the obtained data were as follows:
1H-NMR(600MHz，DMSO-d6)：δ2.50[s，6H，C(CH3)2]，6.88(d，J＝8.1Hz，2H，ArH)，7.01(d，J＝8.2Hz，2H，ArH)，7.12(d，J＝14.8Hz，1H，CH＝CH)，7.21(m，J＝8.3Hz，1H，ArH)，7.26(d，J＝8.1Hz，2H，ArH)，7.44(t，J＝8.3Hz，1H，ArH)，7.59(m，2H，ArH)，7.68(d，J＝7.6Hz，1H，ArH)，7.73(m，J＝7.9Hz，1H，ArH)，7.84(d，J＝8.8Hz，2H，ArH).HRMS(ESI-TOF)m/z calcd for[C34N28N2+H]+464.23；found：465.2334.Z-Avg：2.021(nm).
from the above characterization results, the structural formula of the high-sensitivity colorimetric/spectroscopic dual-mode fluorescent probe (H4) for detecting acidic pH change is as follows:
10mM H4 prepared in example 1 was added to a 100mL volumetric flask and the volume was adjusted with ethanol to obtain a probe stock solution. 0.5mL of the probe stock solution was transferred to a 5mL volumetric flask using VH2O∶VEtOHThe volume of the mixed solution is 1: 1, wherein the pH value of the buffer solution is 2.1-7.4, and the concentration of the high-sensitivity colorimetric/spectral dual-mode fluorescent probe for detecting the change of acidic pH is 10-20 mu mol/L; the following tests were performed using the probe solution.
A small amount of the H4 probe prepared above was taken in buffer solutions (H) at different pH values2O/EtOH 99: 1, v/v), the UV-visible absorption at 350-650nm was determinedThe spectrum changes as shown in fig. 1. From fig. 1 it can be seen that H4 shows a clear photophysical effect, which is well matched by acidic pH changes. As the pH of the probe solution is reduced from 7.4 to 2.1, the absorbance at 418nm is obviously reduced, and the absorbance at 545nm is obviously increased. It can be seen that the H4 fluorescent probe has a sensitive response to pH changes, which can be attributed to the protonation process of the probe under acidic conditions.
A small amount of the H4 probe prepared above was taken in buffer solutions (H) at different pH values2O/EtOH 99: 1, v/v), the change in fluorescence emission spectrum at 450-700nm was determined, as shown in fig. 2. As can be seen from FIG. 2, the maximum fluorescence intensity of the H4 probe solution under neutral conditions was 549nm (. lamda.) (λ)ex436 nm). The fluorescence intensity of probe H4 was gradually quenched as the acidity of the system increased, indicating that H4 responded significantly to pH changes. As shown in FIG. 3, Sigmoidal fitting yields pKaThe value is 3.2, is very close to the absorption measurement value, and the pH-dependent fluorescence intensity has a good linear relation (R) in the pH range of 2.1-7.42＝0.9901，Y＝22.0409X-58.3397)。
From the above results, it was found that probe H4 has excellent recognition and detection effects on changes in acidic pH in a solution. Subsequently, fluorescence imaging experiments were further performed on zebrafish in H4 solutions at different pH, as shown in fig. 4. Under acidic conditions (pH 3.4), the probe showed a weak fluorescence effect in zebrafish. However, as the acidity of the culture system is reduced, the green fluorescence in the zebrafish is gradually increased, which is consistent with the spectrum result, and the probe has the potential of detecting the pH change of the organism. Under neutral conditions, the green fluorescence is gradually enhanced along with the increase of the culture time (10s-60s), and no obvious change occurs until 90s, which indicates that H4 has good light stability. The fluorescence imaging results further prove that H4 can be used as a fluorescence probe for in vivo correlation detection, and has good feasibility and high sensitivity. On the other hand, CIE1993 analysis software and RGB value analysis show that the probe can be made into a simple and portable colorimetric sensor for qualitatively monitoring the change of acidic pH value in the environment, and has high practical application value.
It should be noted that the above-mentioned embodiments are only used for illustrating the technical solutions of the present invention and not for limiting, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that several modifications and amendments can be made without departing from the spirit and scope of the technical solutions of the present invention, which should be covered by the claims of the present invention.
Claims (4)
1. The structural formula of the high-sensitivity colorimetric/spectroscopic dual-mode fluorescent probe for detecting the change of acidic pH is as follows:
2. the preparation of a highly sensitive colorimetric/spectroscopic dual-mode fluorescent probe for detecting acidic pH changes according to claim 1, characterized in that the method is carried out by the following steps:
firstly, sequentially adding 2, 3, 3-trimethyl-3H-benzo [ e ] indole, 4-diphenylamine benzaldehyde, hydrochloric acid and absolute ethyl alcohol into a reaction container with a reflux device; heating to 75-85 deg.C, maintaining reflux for reaction, tracking reaction process by thin layer chromatography (volume ratio of developing agent petroleum ether/ethyl acetate is 1: 1), and stopping reaction when new product point appears on silica gel plate and two raw materials disappear.
Secondly, concentrating the reactant in the reaction container to 30mL, cooling to room temperature, collecting the precipitate, then recrystallizing with absolute ethyl alcohol, and drying for 6h to obtain the fluorescent probe with high-sensitivity colorimetric/spectral dual-mode identification on acidic pH change.
3. The method for preparing a high-sensitivity colorimetric/spectroscopic dual-mode fluorescence probe for detecting acidic pH change according to claim 2, wherein the molar ratio of 2, 3, 3-trimethyl-3H-benzo [ e ] indole to 4-diphenylaminobenzaldehyde in the step one is 1: 1.5-3.5.
4. The method for preparing a high-sensitivity colorimetric/spectroscopic dual-mode fluorescent probe for detecting acidic pH change according to claim 2 or 3, wherein the ratio of the amount of 2, 3, 3-trimethyl-3H-benzo [ e ] indole to the volume of absolute ethyl alcohol in the step one is 1mmoL to (20.0-25.0) mL.
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