High-sensitivity fluorescent probe for detecting pH value as well as preparation and application of high-sensitivity fluorescent probe
By developing a high -sensitivity PH fluorescent probe, the problem of low sensitivity of the existing PH detection method is solved, and the accurate and quantitative detection of the pH value of the solution has been realized, and it has good scientific research value and application value.
Patent Information
- Application Number
- CN202411380761.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-30
- Publication Date
- 2025-01-24
Smart Images
Figure CN119350268A_ABST
Abstract
Description
Technical Field
[0001] The present application relates to the field of fluorescence detection, and in particular to a pH fluorescent probe with high sensitivity response performance and its preparation and application. Background Art
[0002] Like air, water is the basic substance for human survival, and the physical and chemical properties of water change human growth and development. Among them, pH value describes the acidity and alkalinity of aqueous solution and is the main parameter of water. According to literature research, the pH value of water is closely related to human health. From a microscopic perspective, fluctuations in intracellular pH value will significantly affect many cell behaviors, such as cell metabolism, endocytosis, apoptosis and enzyme activity. Abnormal cell pH value will lead to changes in many cell functions. For example, when the pH value changes from acidic to neutral, the activity of enzymes in lysosomes will decrease. In addition, when the intracellular pH value changes dramatically, it may cause diseases such as cancer, stroke and Alzheimer's disease. From a macroscopic perspective, the pH of water samples in rainwater, lake water, river water and other environments affects people's lives, especially the pH of drinking water directly affects human health. In short, whether in the field of environmental ecology or biomedicine, pH value is a parameter we focus on.
[0003] It is reported that many detection methods, including acid-base indicator titration and potentiometric titration, have been used to measure pH. However, these detection methods are expensive and easily interfered by metal ions and complex environments. Compared with these methods, fluorescent probes have the advantages of good selectivity, fast response, low cost, and convenient operation. A variety of pH fluorescent probes have been developed by many researchers, but most pH probes have low sensitivity, which limits their application in environmental ecology and biomedical fields. Therefore, there is an urgent need to develop highly sensitive pH fluorescent probes. Summary of the invention
[0004] The present application provides a highly sensitive fluorescent probe for detecting pH value and its preparation and application. The pH fluorescent probe of the present application has high sensitivity, a fluorescence change multiple of up to about 150 times, the signal is not interfered by ions, and can accurately and quantitatively detect the pH value of the solution.
[0005] One purpose of the present application is to provide a highly sensitive fluorescent probe for detecting pH value, wherein the structural formula of the highly sensitive fluorescent probe is as shown in formula (I):
[0006]
[0007] The second object of the present application is to provide a method for preparing the highly sensitive fluorescent probe, comprising:
[0008] Compound (II) is placed in anhydrous tetrahydrofuran, cooled to 0°C, then compound (III) and glacial acetic acid are added in sequence, and finally sodium triacetoxyborohydride is added in batches to obtain a mixed reaction liquid; the mixed reaction liquid is transferred to room temperature for reaction; after the reaction is completed, a highly sensitive fluorescent probe as shown in formula (I) is obtained by separation and purification;
[0009]
[0010] The reaction formula is as follows:
[0011]
[0012] Optionally, the molar ratio of the compound (II), the compound (III), glacial acetic acid and sodium triacetoxyborohydride is 1:1.5 to 2:10:5.
[0013] The compound (II) and compound (III) are both public compounds and purchased from a reagent company.
[0014] Optionally, the reaction time is 8-15 hours; more preferably 12 hours.
[0015] Optionally, the separation and purification includes: drying the organic solvent under reduced pressure, and separating and purifying by column chromatography, using a dichloromethane-methanol mixture in a volume ratio of 20:1 as a developing solvent to elute the target product.
[0016] The third object of the present application is to provide the application of the highly sensitive fluorescent probe in pH value detection.
[0017] Specifically, the present application provides an application of the highly sensitive fluorescent probe in the preparation of a reagent, a kit or a test strip for detecting pH value.
[0018] When applied to test strips, the test strips are prepared as follows:
[0019] The blank test paper is soaked in a dichloromethane solution containing the probe. After soaking for a certain period of time, the filter paper is taken out and placed in a ventilated dark place to finally obtain a fluorescent probe test paper for detecting pH value.
[0020] The testing process is as follows:
[0021] Use a dropper to draw a small amount of the test liquid and drop it onto the fluorescent probe test paper, then use a portable ultraviolet lamp to perform qualitative analysis of the fluorescence intensity.
[0022] Result judgment:
[0023] Under the illumination of a portable ultraviolet lamp at 365 nm, it can be seen that the fluorescence intensity of the filter paper increases with the increase of pH.
[0024] Therefore, the present application also provides a test strip for detecting pH value, wherein the test strip is made by soaking a blank test strip in a solution containing the highly sensitive fluorescent probe as described above and then drying it; the fluorescence intensity of the test strip increases with the increase of the pH of the sample to be tested under 365nm ultraviolet light.
[0025] Optionally, the solution containing the highly sensitive fluorescent probe is: a highly sensitive fluorescent probe solution prepared with dichloromethane at a concentration of 0.1 mM. Optionally, the blank test paper is immersed in the probe solution for 30 to 60 seconds.
[0026] The test paper prepared by the probe of the present application can be used for qualitative detection of pH of water samples. Therefore, the present application also provides an application of the test paper strip in qualitative detection of pH of water bodies.
[0027] The present application also provides a kit for detecting pH value, comprising the highly sensitive fluorescent probe. When used in a kit, the highly sensitive fluorescent probe can be prepared into a kit according to a mature kit preparation method in the art.
[0028] The present application also provides an application of the highly sensitive fluorescent probe in detecting the pH value of water or detecting the pH value in living cells in vitro for non-diagnostic purposes.
[0029] The present application also provides a method for quantitatively detecting pH value in water, comprising:
[0030] The highly sensitive fluorescent probe is added to the water to be tested, mixed, and the fluorescence intensity of the water to be tested is collected at an excitation wavelength of 376 nm and an emission wavelength of 460 nm, and the pH value of the water to be tested is calculated according to the standard curve.
[0031] The principle of fluorescent probe detection in this application is: under acidic conditions, the probe exists in the form of phenol structure and has almost no fluorescence; as the pH value increases, that is, OH - As the concentration increases, the probe exists in the form of a Kun structure, the conjugated system is enhanced, and the fluorescence is turned on.
[0032]
[0033] According to the test results, the fluorescence intensity of probe (I) at pH 9 is about 150 times that at pH 4, indicating that the probe has a high sensitivity to pH.
[0034] Optionally, the final concentration of the high-sensitivity fluorescent probe added to the water to be tested is 0.01 mM, which has a good linear relationship with the pH value of the water to be tested when the pH value is 6.00-8.00, and can quantitatively detect the pH value of the water to be tested within this concentration range.
[0035] Optionally, a standard curve can be prepared as follows:
[0036] 0.01 mM fluorescent probe was placed in a buffer solution with a pH value of 6.00 to 8.00, and the fluorescence intensity of the solution to be tested was collected at an excitation wavelength of 376 nm and an emission wavelength of 460 nm. The pH was used as the ordinate and Log[(I max -I) / (II min )] as the horizontal axis, and a linear standard curve can be obtained by drawing.
[0037] Compared with the prior art, the present invention has at least one of the following beneficial effects:
[0038] (1) The preparation method of the pH fluorescent probe provided in the present application is simple and has a high reaction yield.
[0039] (2) The pH fluorescent probe provided in this application has high sensitivity, with a fluorescence change multiple of up to about 150 times, the signal is not interfered by ions, and can accurately and quantitatively detect the pH value of the solution. It has good scientific research value and application value in the field of environmental and life science detection.
[0040] (3) The test strip prepared using the probe of the present application has a fluorescence intensity that increases as the pH of the sample to be tested decreases under 365nm ultraviolet light, and can be used for qualitative detection of the pH of water samples. BRIEF DESCRIPTION OF THE DRAWINGS
[0041] Figure 1 This is the H NMR spectrum of the probe (I) prepared in Example 1.
[0042] Figure 2 a is the fluorescence emission spectra of the probe (I) prepared in Example 1 added to DMSO / PBS buffer (v / v=1 / 99) of different pH values, Figure 2 Where b is pH and Log[(I max -I) / (II min )] (excitation wavelength 376nm, emission wavelength 460nm); I is the fluorescence intensity, I max is the fluorescence intensity of the probe at pH = 4.00, I min is the fluorescence intensity of the probe at pH = 9.00.
[0043] Figure 3 This is the anti-interference graph of the probe (I) prepared in Example 1 to common amino acids and ions (excitation wavelength 376nm, emission wavelength 460nm).
[0044] Figure 4 Fluorescence visualization of the probe (I) test paper prepared in Example 1 in test liquids with different pH values (under irradiation with a portable UV lamp at 365 nm). DETAILED DESCRIPTION
[0045] The following will be combined with the drawings in the embodiments of the present application to clearly and completely describe the technical solutions in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of this application.
[0046] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this application belongs. The terms used herein in the specification of this application are only for the purpose of describing specific embodiments and are not intended to limit this application.
[0047] The following is described with specific embodiments:
[0048] Example 1: Preparation of probe (I)
[0049]
[0050] In a dry 50mL round-bottom flask, compound (II) (0.15g, 0.6mmol) was dissolved in 5mL of anhydrous tetrahydrofuran and cooled to 0°C, followed by the addition of compound (III) i.e. N-(2-aminoethyl)morpholine (0.15g, 0.9mmol) and glacial acetic acid (0.35g, 6mmol), and finally sodium triacetoxyborohydride (0.62g, 3mmol) in batches according to the routine (no special requirement for the amount added in each batch). The above mixture was transferred to room temperature and reacted for 12 hours. After the reaction of the raw materials was completed, the organic solvent was dried under reduced pressure, and separated and purified by column chromatography, the eluent was dichloromethane / methanol = 20:1, v / v, to obtain 0.15g of white solid, with a yield of 69%. Its H NMR spectrum can be found at Figure 1 .
[0051] 1 H NMR (400MHz, DMSO-d6) δ8.18(d,J=2.2Hz,1H),8.13(d,J=7.5Hz,1H),8.04(d,J =8.0Hz,1H),7.54(ddd,J=8.3,7.2,1.3Hz,1H),7.44(ddd,J=8.2,7.1,1.2Hz,1 H),7.36(dd,J=8.4,2.2Hz,1H),7.03(d,J=8.4Hz,1H),3.78(s,2H),3.56(t,J= 4.6Hz, 4H), 2.68 (t, J = 6.4Hz, 2H), 2.43 (t, J = 6.4Hz, 2H), 2.33 (d, J = 4.9Hz, 4H).
[0052] Example 2: Fluorescence spectrum test of probe (I) (10 μM) in response to pH.
[0053] Accurately weigh a certain amount of probe (I) (prepared in Example 1), prepare a probe mother solution with a concentration of 1 mM using dimethyl sulfoxide, draw 5 μL with a pipette and add it to 495 μL PBS buffer to make the final mixed solution system DMSO / PBS buffer (1:99, v:v, pH 4.0, 5.01, 5.99, 6.18, 6.42, 6.6, 6.81, 7.02, 7.22, 7.38, 7.61, 7.81, 8.01, 8.48, 9), so that the final concentration of the probe is 10 μM, shake well and add it to a 96-well plate, measure the fluorescence spectrum of the probe (I) using a multifunctional microplate reader, and make a related linear curve.
[0054] Depend on Figure 2 As shown in a, with the increase of pH, the fluorescence intensity of probe (I) continues to increase, with the strongest emission fluorescence intensity at 460nm. According to the Henderson-Hasselbalch equation, the pKa of probe (I) is 7.38 ( Figure 2 b), and pH = 1.59Log [(I max -I) / (II min )] + 7.38, and has a good linear relationship with the pH value of 6.00-8.00, thus realizing the ability of the probe to quantitatively detect pH in the solution. It is more important to note that when the solution changes from pH 4 to pH 9, the fluorescence intensity of the probe (I) changes by about 150 times (at pH 4, the fluorescence intensity of the probe (I) at 460nm is 82, and at pH 9, the fluorescence intensity of the probe (I) at 460nm is 11986), which has a highly sensitive response performance.
[0055] Example 4: Study on the Selectivity of Probe (I) (10 μM) to pH
[0056] Accurately weigh a certain amount of probe (I) (prepared in Example 1), prepare a probe stock solution with a concentration of 1 mM using dimethyl sulfoxide, pipette 5 μL of the solution into 495 μL of a solution containing relevant amino acids or ions (in order: L-Leu, homoCystine, L-Cysteine, L-Arg, GSH, Ba 2+ 、Ni 2+ , K + , Mn 2+ , Fe 3+ Mg 2+ 、Co 2+ 、HCO3 - 、CH3COO - 、HSO4- 、F - ), so that the final mixed solution system is DMSO / PBS buffer (1:99, v:v, pH 5.99 or 6.81), the final concentration of the probe is 10 μM, wherein the final concentration of various ions is 0.1 mM, after shaking well, it is added to a 96-well plate, and the fluorescence intensity of the probe (I) is measured with a multifunctional microplate reader.
[0057] Fluorescence spectrum see Figure 3 The data show that in the PBS buffer environment with pH values of 5.99 and 6.81, several important related substances have little effect on the fluorescence signal of probe (I). The above experimental data show that probe (I) has good pH specificity.
[0058] Example 5: Fluorescence Visibility Test of Probe (I) (100 μM) Test Paper for Solutions of Different pH Values
[0059] Test paper preparation process: accurately weigh a certain amount of probe (I) (prepared in Example 1), prepare a probe solution with a concentration of 0.1 mM with dichloromethane, soak a blank test paper in the probe solution, soak for 45 seconds, take out the filter paper, and place it in a ventilated dark place to finally obtain a fluorescent probe test paper for detecting pH value.
[0060] Test process: Use a dropper to draw a small amount of solution (pH 4, 7, 9 respectively) and drop it onto the fluorescent probe test paper, and then use a portable ultraviolet lamp to perform qualitative analysis of the fluorescence intensity.
[0061] Fluorescence visualization see Figure 4 : Under the illumination of a portable ultraviolet lamp at 365 nm, the fluorescence intensity of the filter paper increased as the pH decreased, proving that the pH probe filter paper can qualitatively detect the pH of water samples.
[0062] The above-mentioned embodiments only express several implementation methods of the present application, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present application, and these all belong to the protection scope of the present application. Therefore, the protection scope of the patent of the present application shall be subject to the attached claims.
Claims
1. A highly sensitive fluorescent probe for detecting pH value, characterized in that: The structural formula of the highly sensitive fluorescent probe is shown in formula (I):
2. The method for preparing a highly sensitive fluorescent probe according to claim 1, characterized in that: include: Compound (II) is placed in anhydrous tetrahydrofuran, cooled to 0°C, then compound (III) and glacial acetic acid are added in sequence, and finally sodium triacetoxyborohydride is added in batches to obtain a mixed reaction liquid; the mixed reaction liquid is transferred to room temperature for reaction; after the reaction is completed, a highly sensitive fluorescent probe as shown in formula (I) is obtained by separation and purification; 3. The preparation method according to claim 2, characterized in that: The molar ratio of the compound (II), the compound (III), glacial acetic acid and sodium triacetoxyborohydride is 1:1.5 to 2:10:5; the reaction time is 8 to 15 hours; The separation and purification comprises: drying the organic solvent under reduced pressure, and separating and purifying by column chromatography, using a dichloromethane-methanol mixture in a volume ratio of 20:1 as a developing solvent, and eluting the target product.
4. Use of the highly sensitive fluorescent probe as claimed in claim 1 in preparing a reagent, a kit or a test strip for detecting pH value.
5. A kit for detecting pH value, characterized in that: Comprising the highly sensitive fluorescent probe as described in claim 1.
6. A test strip for detecting pH value, characterized in that: The test strip is made by soaking a blank test paper in a solution containing the highly sensitive fluorescent probe as claimed in claim 1 and then drying it; the fluorescence intensity of the test strip increases with the increase of the pH of the sample to be tested under 365nm ultraviolet light.
7. Use of the test strip as claimed in claim 6 in qualitative detection of pH in water.
8. Use of the highly sensitive fluorescent probe as claimed in claim 1 in detecting pH value of water or detecting pH value in living cells in vitro for non-diagnostic purposes.
9. A method for quantitatively detecting pH value in water, characterized in that: include: The highly sensitive fluorescent probe as claimed in claim 1 is added to the water body to be tested, mixed, and the fluorescence intensity of the water body to be tested is collected at an excitation wavelength of 376 nm and an emission wavelength of 460 nm, and the pH value of the water body to be tested is calculated according to the standard curve.
10. The quantitative detection method according to claim 9, characterized in that: The final concentration of the highly sensitive fluorescent probe added to the water to be tested is 0.01 mM, and the pH value of the water to be tested is 6.00-8.00.