Water-soluble pH fluorescent probe as well as preparation and application thereof

Abstract

The invention discloses a water-soluble pH fluorescent probe as well as preparation and application thereof, the structural formula of the water-soluble pH fluorescent probe is as shown in formula (I): # imgabs0, the probe (I) takes naphthalimide as a fluorophore and takes hydroxyl as a pH recognition group, and the water solubility of the probe is improved by utilizing an ethylene glycol long chain. The fluorescence change multiple of the probe for identifying pH is relatively high (47 times), the Stokes shift is large (170nm), the ion interference resistance is strong, and the probe has broad-spectrum pH (2.5-12) test capability and can be used for qualitative detection of food spoilage. In a word, the probe is excellent in fluorescence response performance and has good practical application value in the fields of environment, life science, food and the like.

Description

Technical Field

[0001] The present application relates to the technical field of fluorescent probes, and particularly relates to a water-soluble pH fluorescent probe and its preparation and application. Background Art

[0002] As a main indicator parameter, the pH value plays a crucial role in fields such as life science, food science, and environmental science.

[0003] In the field of life science, the fluctuation of the pH value affects the normal physiological functions of living organisms. The pH value of human blood usually maintains within the range of 7.35 - 7.45, and deviation from this value will have a significant impact on health. Intracellular pH affects many cellular behaviors such as cell metabolism, endocytosis, apoptosis, and enzyme activity. A sharp change in intracellular pH may cause diseases such as Alzheimer's disease, cancer, and stroke.

[0004] The pH value is also an important indicator for environmental monitoring, reflecting the health status of the ecosystem. Changes in the pH values of water bodies, soils, and the atmosphere are directly related to ecological safety and human well-being. Monitoring the pH value can effectively reduce environmental risks and promote the sustainable development of society.

[0005] During the storage of food, the pH value will change with the progress of microbial activities and chemical reactions. The pH value is an important reference index for quickly judging whether food is fresh or has deteriorated. By monitoring the pH value of food, the degree of food spoilage can be quickly judged.

[0006] In summary, achieving effective detection of the pH value has important research value and application value in fields such as life, environment, and food.

[0007] It is reported that fluorescent probes have the advantages of good selectivity, fast response, low cost, and simple operation. Many researchers have designed and developed various types of pH fluorescent probes and used them for pH detection in various fields. However, most of the reported pH probes still have some defects, such as poor water solubility of the probe, small Stokes shift, limited pH response range, etc., which limit their further application. Therefore, developing a new type of pH fluorescent probe with excellent performance has important practical significance. Summary of the Invention

[0008] The present application provides a water-soluble pH fluorescent probe and its preparation and application. The pH fluorescent probe of the present application has good water solubility, a high fluorescence change multiple (at pH = 2.5, the relative fluorescence intensity is 16650; at pH = 12, the relative fluorescence intensity is 354, and the fluorescence change multiple is as high as 47 times), a large Stokes shift (170 nm), strong specificity for pH, good fluorescence signal stability, has the ability to perform broad-spectrum pH testing, and can be used for qualitative detection of food spoilage.

[0009] An object of the present application is to provide a water-soluble pH fluorescent probe, and the structural formula of the water-soluble pH fluorescent probe is shown in formula (I):

[0010]

[0011] For the water-soluble pH fluorescent probe of the present application: the fluorophore is naphthalimide; the pH-responsive group is the hydroxyl group at the 4-position of naphthalimide, which can be protonated or deprotonated under different pH conditions, thereby changing the fluorescence signal; the water-soluble group is a long-chain glycol, which is used to improve the water solubility of the probe.

[0012] The present application also provides a preparation method of the water-soluble pH fluorescent probe, including:

[0013] Using compound (II), compound (III), glacial acetic acid and sodium triacetoxyborohydride as raw materials, reacting in an organic solvent to prepare the water-soluble pH fluorescent probe shown in formula (I);

[0014]

[0015] The reaction formula is as follows:

[0016]

[0017] The compound (II) is a publicly known compound, and its preparation method can refer to the literature (Tang Xinxue, Sun Hao, Nie Jing, et al. An o-hydroxyl aldehyde structure based naphthalimide derivative: Reversible photochromic properties and its application in ClO - detection in living cells[J]. Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, 2019, 219, 154-163.)

[0018] The compound (III) is a publicly known compound 1-amino-3,6,9-trioxaundecan-11-ol (CAS No.: 86770-74-3), and is purchased from a reagent company.

[0019] Optionally, the molar ratio of the compound (II), compound (III), glacial acetic acid and sodium triacetoxyborohydride is 1:1.5-2:10:5.

[0020] Optionally, the organic solvent is anhydrous tetrahydrofuran.

[0021] Optionally, the reaction time is 8 - 15 hours; more preferably 12 hours.

[0022] Optionally, first place compound (II) and the organic solvent in a reaction vessel, cool to 0 °C, then sequentially add compound (III) and glacial acetic acid, and finally add sodium triacetoxyborohydride portionwise, and transfer the reaction solution to room temperature for reaction; after the reaction is completed, separate and purify the obtained reaction solution to finally obtain the water-soluble pH fluorescent probe shown in formula (I).

[0023] Optionally, the separation and purification includes: rotary evaporation of the organic solvent under reduced pressure, and separation and purification by column chromatography, using a dichloromethane - methanol mixture with a volume ratio of 25:1 as the eluent to elute the target product, thus obtaining it.

[0024] The third object of the present application is to provide the application of the described water-soluble pH fluorescent probe in pH value detection.

[0025] Specifically, the present application provides an application of the described water-soluble pH fluorescent probe in the preparation of a reagent, kit or test strip for detecting pH value.

[0026] The present application also provides a pH detection kit, including the described water-soluble pH fluorescent probe. When applied to a kit, the water-soluble pH fluorescent probe can be prepared into a kit according to the mature kit preparation method in the art.

[0027] The present application also provides an application of the water-soluble pH fluorescent probe in detecting the pH value of water bodies, detecting the intracellular pH value of living cells in vitro for non-diagnostic purposes or food detection.

[0028] The present application also provides a method for quantitatively detecting the pH value in water bodies, including:

[0029] Adding the described water-soluble pH fluorescent probe to the water body to be measured, mixing evenly, collecting the fluorescence intensity of the water body to be measured at an excitation wavelength of 375 nm and an emission wavelength of 545 nm, and calculating the pH value of the water body to be measured according to the standard curve.

[0030] According to the test results, it can be seen that probe (I) has the ability of broad-spectrum pH testing, that is, at pH 2.5 to 5.5, pH 5.5 to 10, and pH 10 to 12, probe (I) has corresponding response situations respectively.

[0031] Optionally, the final concentration of the water-soluble pH fluorescent probe added to the water body to be measured is 0.01 mM, and it has a good linear relationship with the pH value of the water body to be measured in the range of 3.00 to 5.00, enabling quantitative detection of the pH value of the sample to be measured within this concentration range.

[0032] Optionally, the standard curve is prepared as follows:

[0033] Place 0.01 mM fluorescent probe in buffer solutions with pH values ranging from 3.00 to 5.00, collect the fluorescence intensity of the solution to be measured at an excitation wavelength of 375 nm and an emission wavelength of 545 nm, and plot a linear standard curve with the fluorescence intensity as the ordinate and pH as the abscissa.

[0034] This application also provides a method for qualitative detection of pH value during food spoilage, including:

[0035] Add the above-mentioned water-soluble pH fluorescent probe to coconut water (coconut water is placed at 4 and 30 °C for 0, 3, and 5 days respectively), and measure the fluorescence intensity of coconut water under the conditions of an excitation wavelength of 375 nm and an emission wavelength of 545 nm.

[0036] Compared with the prior art, this application has at least one of the following beneficial effects:

[0037] (1) The Stokes shift of the pH fluorescent probe provided by this application is large, up to 170 nm, reducing background interference.

[0038] (2) The fluorescence change multiple of the pH fluorescent probe provided by this application for recognizing pH is high (47 times).

[0039] (3) The pH fluorescent probe provided by this application has good water solubility and can perform pH detection under the condition that DMSO is 0.1%, providing the possibility for pH detection of actual samples and live cell imaging.

[0040] (4) Compared with most of the reported fluorescent probes, the pH fluorescent probe provided by this application has a wider measurement range, can achieve qualitative detection of pH values from 2 to 12, and can be used for qualitative detection of food spoilage.

[0041] (5) The signal of the pH fluorescent probe provided by this application is not interfered by ions, and can accurately quantitatively detect the pH value of the solution. The reaction conditions for probe preparation are mild, suitable for large-scale production, and have good scientific research value and application value. Description of the Drawings

[0042] Figure 1 1H NMR spectrum of the probe (I) prepared in Example 1.

[0043] Figure 213C NMR spectrum of the probe (I) prepared in Example 1.

[0044] Figure 3 In a, it is the fluorescence emission spectra of the probe (I) prepared in Example 1 added at different pH values;

[0045] Figure 3 In b, it is the graph of the relationship between fluorescence intensity and pH value;

[0046] Figure 3 In c, it is the linear relationship between fluorescence intensity and pH value.

[0047] Figure 4 It is the anti-interference ability spectrum of the probe (I) prepared in Example 1 against common ions.

[0048] Figure 5 It is the reversible response spectrum of the probe (I) prepared in Example 1.

[0049] Figure 6 It is the application spectrum of the probe (I) prepared in Example 1 for fluorescence detection during food spoilage. Detailed implementation manners

[0050] Next, the technical solutions in the embodiments of the present application will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present application.

[0051] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the technical field to which this application belongs. The terms used in the specification of this application herein are only for the purpose of describing specific embodiments, and are not intended to limit this application.

[0052] The following is illustrated with specific examples:

[0053] Example 1: Preparation of the probe (I)

[0054]

[0055] Weigh compound (II) (0.3 g, 1 mmol) and anhydrous tetrahydrofuran (8 mL) and place them in a dry round-bottom flask (50 mL). Cool the mixture to 0 °C at low temperature. Then, add compound (III), namely 1-amino-3,6,9-trioxaundecan-11-ol (0.29 g, 1.5 mmol), and glacial acetic acid (0.6 g, 10 mmol) in sequence. Finally, add the reducing agent sodium triacetoxyborohydride (1 g, 5 mmol) batch by batch in the conventional way (there is no special requirement for the addition amount of each batch). Transfer the above reaction solution to room temperature and react for 12 hours. After the reaction is completed, concentrate the reaction solution under reduced pressure and separate and purify it by silica gel column chromatography. The eluent is dichloromethane / methanol = 25:1, v / v, to obtain 0.25 g of white solid with a yield of 53%. The 1H NMR spectrum is shown in Figure 1 、 Figure 2 。

[0056] 1 H NMR (400 MHz, DMSO-d6) δ 8.45 (dd, J = 8.0, 1.4 Hz, 1H), 8.28 (dd, J = 7.4, 1.4 Hz, 1H), 8.14 (s, 1H), 7.39 (t, J = 7.6 Hz, 1H), 4.18 (s, 2H), 4.06 - 3.97 (m, 2H), 3.70 (t, J = 5.2 Hz, 2H), 3.58 (s, 4H), 3.52 (m, 4H), 3.47 (d, J = 5.1 Hz, 2H), 3.41 (d, J = 5.2 Hz, 2H), 3.10 (t, J = 5.2 Hz, 2H), 1.57 (p, J = 7.5 Hz, 2H), 1.33 (q, J = 7.4 Hz, 2H), 0.92 (t, J = 7.3 Hz, 3H). 13 C NMR (101 MHz, DMSO-d6) δ 176.45, 164.66, 163.12, 136.49, 132.37, 131.14, 130.77, 127.35, 122.03, 114.19, 99.41, 72.77, 70.27, 70.20, 70.19, 70.12, 66.20, 60.66, 49.54, 45.74, 39.04, 30.50, 20.35, 14.30.

[0057] Example 2: Fluorescence spectrum test of probe (I) (10 μM) in response to pH.

[0058] Accurately weigh a certain amount of probe (I) (prepared in Example 1), prepare a probe stock solution with a concentration of 10 mM using dimethyl sulfoxide, and then dilute it with ultrapure water to obtain a 1 mM probe solution. Pipette 5 μL and add it to 495 μL of PBS buffer to make the final mixture system DMSO / PBS buffer (the volume of DMSO accounts for 0.1%, and the pH values are 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 10, 11, 11.5, 12 in sequence), so that the final concentration of the probe is 10 μM. After shaking well, add it to a 96-well plate. Using 375 nm as the excitation wavelength, measure the fluorescence spectrum of probe (I) with a multifunctional microplate reader and make relevant linear curves.

[0059] The probe can be used under the condition that the volume of DMSO accounts for 0.1%, indicating that the probe has good water solubility. As Figure 3 shown in a, with the increase of pH, the fluorescence intensity of probe (I) continuously decreases, and it has the strongest emission fluorescence intensity at 545 nm, that is, the Stokes shift of this probe is 170 nm.

[0060] According to Figure 3 the results in b, within the range of pH from 2.5 to 5.5, the fluorescence intensity of probe (I) decreases with the increase of pH; within the range of pH from 5.5 to 10, the fluorescence intensity of probe (I) remains basically unchanged; within the range of pH from 10 to 12, the fluorescence intensity of probe (I) decreases with the increase of pH; in short, probe (I) has the ability to test a wide range of pH values.

[0061] Furthermore, from Figure 3 c, it is obtained that the fluorescence intensity of probe (I) has a good linear relationship with the pH value (3 - 5) (y = -6355.44x + 35250, R 2 = 0.999, y represents the fluorescence intensity, and x represents the pH value), thus realizing the ability of the probe to quantitatively detect the pH in the solution.

[0062] It should be noted that during the process of the solution from pH 2.5 to pH 12, the fluorescence change of the probe is as high as 47 times (pH = 2.5, the relative fluorescence intensity is 16650; pH = 12, the relative fluorescence intensity is 354).

[0063] Example 3: Selectivity study of probe (I) (10 μM) for pH

[0064] Accurately weigh a certain amount of probe (I) (prepared in Example 1), prepare a probe stock solution with a concentration of 10 mM using dimethyl sulfoxide, and then dilute it with ultrapure water to obtain a 1 mM probe solution. Pipette 5 μL and add it to 495 μL of a solution containing relevant metal ions (in sequence Mg 2+ , K + , Al3+ , Cu 2+ , Cu + , Sr 2+ , Ce 3+ , Co 2+ , Zn 2+ , Cs + , Ag + , Fe 3+ , Pb 2+ , Mn 2+ , Cd 2+ , Ni 2+ , Ba 2 + , (blank), so that the final mixed solution system is DMSO / PBS buffer (DMSO volume accounts for 0.1%, pH is 3 or 11 respectively), the final concentration of the probe is 10 μM, and the final concentration of each ion is 0.1 mM. After shaking well, add it to a 96-well plate. Using 375 nm as the excitation wavelength, measure the fluorescence intensity of the probe (I) with a multifunctional microplate reader.

[0065] The fluorescence spectrum is shown in Figure 4 : The data shows that in the PBS buffer environment with pH 3 and 11, the fluorescence enhancement of the probe (I) varies little in the presence of different metal ions, indicating that the probe (I) has good pH specificity.

[0066] Example 4: Reversibility of the pH response of probe (I) (10 μM)

[0067] Accurately weigh a certain amount of probe (I) (prepared in Example 1), and prepare a probe mother liquor with a concentration of 10 mM using dimethyl sulfoxide. Subsequently, dilute it with ultrapure water to obtain a 1 mM probe solution. Pipette 20 μL and add it to 1980 μL of PBS buffer (DMSO volume accounts for 0.1%, pH is 2.5), the final concentration of the probe is 10 μM. Then alternately adjust the pH of the solution, from acidic to alkaline and then back to acidic, and adjust the acid-base cycle 4 times. Using 375 nm as the excitation wavelength and 545 nm as the emission wavelength, record the fluorescence signal change with a multifunctional microplate reader.

[0068] The test results are shown in Figure 5 , with the pH cycle change of pH from acidic to alkaline, the fluorescence enhancement of the probe (I) changes accordingly. On the one hand, it shows that the recognition mechanism of the probe for pH is based on the protonation and deprotonation of hydroxyl groups. On the other hand, it also shows that the probe has good reversibility to the pH response.

[0069] Example 5: Test on the fluorescence detection ability of probe (I) (10 μM) during food spoilage

[0070] Fresh coconut juice was obtained from coconuts and filtered to obtain a filtrate. The coconut juice filtrate was placed in environments of 4°C and 30°C respectively, and the probe solution was added on days 0, 3, and 5. With an excitation wavelength of 375 nm and an emission wavelength of 545 nm, a multifunctional microplate reader was used to record the fluorescence values.

[0071] Test detection process: A certain amount of probe (I) (prepared in Example 1) was accurately weighed and prepared into a probe stock solution with a concentration of 10 mM using dimethyl sulfoxide. Subsequently, it was diluted with ultrapure water to obtain a 1 mM probe solution. 5 μL was pipetted with a pipette gun and added to 495 μL of coconut juice filtrate (the volume of DMSO accounted for 0.1%). With an excitation wavelength of 375 nm and an emission wavelength of 545 nm, a multifunctional microplate reader was used to record the fluorescence values.

[0072] As Figure 6 shown, in the 30°C environment, as the placement time of the coconut juice became longer, the fluorescence value became larger, indicating that the coconut juice became sour during the placement process; in the 4°C environment, the fluorescence value changed little after the coconut juice was placed for 5 days, indicating that the acidity and alkalinity of the coconut juice basically did not change during the placement process; the above results show that the coconut juice is more likely to deteriorate at 30°C, and it is consistent with the fact that the coconut juice becomes sour during the deterioration process, proving that this pH probe can be used for qualitative detection during the food spoilage process.

[0073] The above-described embodiments merely represent several implementation manners of the present application. Their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present application, several deformations and improvements can still be made, 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 appended claims.

Claims

1. A water-soluble pH fluorescent probe, characterized in that, The structural formula of the water-soluble pH fluorescent probe is shown in Formula (I):

2. Use of the water-soluble pH fluorescent probe according to Claim 1 in the preparation of a reagent, test kit or test strip for detecting pH value.

3. A pH detection kit, characterized in that, Comprising the water-soluble pH fluorescent probe according to Claim 1.

4. Use of the water-soluble pH fluorescent probe according to Claim 1 in detecting the pH value of water, the pH value inside living cells in vitro for non-diagnostic purposes or food detection.

5. A quantitative detection method for pH value in water body, characterized in that, Comprising: Adding the water-soluble pH fluorescent probe according to Claim 1 into the water to be tested, mixing evenly, collecting the fluorescence intensity of the water to be tested at an excitation wavelength of 375 nm and an emission wavelength of 545 nm, and calculating the pH value of the water to be tested according to the standard curve.

6. The quantitative detection method according to claim 5, wherein The final concentration of the water-soluble pH fluorescent probe added to the water to be tested is 0.01 mM, and the pH value of the water to be tested is 3-5.

7. The preparation method of the water-soluble pH fluorescent probe according to claim 1, wherein, Comprising: Using compound (II), compound (III), glacial acetic acid and sodium triacetoxyborohydride as raw materials, reacting in an organic solvent to prepare the water-soluble pH fluorescent probe shown in Formula (I); 8. The preparation method according to claim 7, characterized in that, The molar ratio of compound (II), compound (III), glacial acetic acid and sodium triacetoxyborohydride is 1:1.5-2:10:5; the organic solvent is anhydrous tetrahydrofuran; the reaction time is 8-15 hours.

9. The preparation method according to claim 7, wherein First, place compound (II) and the organic solvent in a reaction vessel, cool to 0 °C, then sequentially add compound (III) and glacial acetic acid, finally add sodium triacetoxyborohydride in batches, and transfer the reaction solution to room temperature for reaction; after the reaction is completed, separate and purify the obtained reaction solution to finally obtain the water-soluble pH fluorescent probe shown in Formula (I).

10. The preparation method according to claim 9, characterized in that, The separation and purification includes: rotary evaporating the organic solvent under reduced pressure and separating and purifying by column chromatography, using a dichloromethane-methanol mixed solution with a volume ratio of 25:1 as the eluent to elute the target product to obtain it.

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