A fluorescent probe and preparation method, and a portable method for detecting acetylcholinesterase

Abstract

The present invention proposes a fluorescent probe and a preparation method, a portable method for detecting acetylcholinesterase, which belongs to the technical field of acetylcholinesterase detection, and is used to solve the technical problems of complex on-site detection process of acetylcholinesterase, low sensitivity and selectivity. The structural formula of the probe material of the present invention is: #imgabs0# The probe LSS-AChE recognizes acetylcholinesterase (AChE) based on the catalytic hydrolysis of the ester group, releasing significant green fluorescence. The LSS-AChE has high selectivity and sensitivity to AChE (LOD=0.0097mU / mL), good water solubility and a large Stokes shift (105nm). A portable detection platform is constructed in combination with a smart phone using chitosan hydrogel as a carrier, which realizes on-site instant detection of AChE.

Description

Technical Field

[0001] The invention belongs to the technical field of acetylcholinesterase detection, and in particular relates to a fluorescent probe and a preparation method, and a portable method for detecting acetylcholinesterase. Background Art

[0002] Acetylcholinesterase (AChE) possesses both carboxypeptidase and aminopeptidase activities and plays a crucial role in animal neurotransmission. Its primary physiological function is to hydrolyze acetylcholine (ATCh) to produce acetic acid and choline (TCh). Maintaining AChE levels within the nervous system within a reasonable range hinders nerve impulse transmission and promotes neuronal development and regeneration. Studies have shown that AChE can accelerate the abnormal aggregation of amyloid-β peptides, forming Alzheimer's amyloid fibrils in the central nervous system, thereby inducing Alzheimer's disease. Furthermore, abnormal mood swings in depression may also be influenced by AChE activity. Therefore, understanding changes in AChE activity is crucial for in-depth analysis of the molecular mechanisms underlying these neurological disorders. However, the dynamic regulation of AChE activity in these diseases remains largely unknown. To further understand the role of AChE in these diseases, the development of real-time tools for monitoring AChE in vitro is crucial.

[0003] Traditional AChE detection methods typically employ conventional chromatographic techniques such as high-performance liquid chromatography, gas chromatography, and mass spectrometry. However, these methods have drawbacks such as cumbersome pre-treatment processes, long analysis times, and high instrument and drug costs. Consequently, establishing simple, rapid, and highly sensitive AChE detection methods has become a research priority. Existing AChE detection methods often utilize large instruments as fluorescence detectors, which are bulky and difficult to carry, and are not simple or rapid enough for on-site AChE detection. Therefore, providing a highly sensitive and selective method for on-site AChE detection is a pressing issue for those skilled in the art. Summary of the Invention

[0004] To address the technical issues of complex on-site detection of acetylcholinesterase, the present invention proposes a fluorescent probe and preparation method, as well as a portable method for detecting acetylcholinesterase. This solution can use mobile phones and portable devices instead of large instruments as fluorescence detectors. The detection method is simpler, more portable, and more sensitive, facilitating on-site detection of AChE.

[0005] In order to achieve the above object, the technical solution of the present invention is achieved as follows:

[0006] A fluorescent probe, wherein the structure of the fluorescent probe is:

[0007]

[0008] The preparation method of the fluorescent probe is characterized in that the synthesis route is as follows:

[0009]

[0010] The method comprises the following steps: mixing LSS-OH, propionyl chloride, an alkaline catalyst and a solvent to prepare a reaction solution, and reacting to obtain a fluorescent probe LSS-AChE.

[0011] The molar ratio of the LSS-OH, propionyl chloride and base is 1:(1-5):(1-3).

[0012] The concentration of LSS-OH in the reaction solution is 0.05-0.1 mmol / mL.

[0013] The alkaline catalyst is any one of triethylamine, pyridine or N,N-diisopropylethylamine.

[0014] The solvent is any one of dichloromethane, chloroform, acetone, ethyl acetate, ethyl formate, methanol, ethanol, isopropanol, butanol, isopropylamine, dimethylformamide, dimethyl sulfoxide, ether and petroleum ether.

[0015] The reaction conditions are: time of 8 to 24 hours and temperature of 20°C to 30°C.

[0016] The preparation method further comprises purifying the reaction solution after the reaction, and the purification is selected from: column chromatography, recrystallization, beating method and thin layer chromatography.

[0017] A portable method for detecting acetylcholinesterase, using the fluorescent probe according to claim 1 to detect acetylcholinesterase, comprising the following steps:

[0018] Step 1: Mix agarose, fluorescent probe and Tris-HCl buffer to prepare target-responsive hydrogel;

[0019] Step 2: preparing n portions of acetylcholinesterase standard solutions of different concentrations, wherein n≥2;

[0020] Step 3: n portions of acetylcholinesterase standard solutions of different concentrations are reacted with the target response hydrogel respectively, and a color image of each standard solution to be detected is obtained under ultraviolet irradiation conditions. The color image is analyzed to obtain the RGB value, and a linear relationship between the intensity ratio G / R+B and the acetylcholinesterase concentration is established.

[0021] In the step 1, agarose and a fluorescent probe are dissolved in water to prepare a fluorescent probe solution and an agarose solution, which are then mixed with agarose and a Tris-HCl buffer. The volume ratio of the agarose solution, the fluorescent probe solution, and the Tris-HCl buffer is (1-2): (1-4): (1-2).

[0022] The concentration of the agarose solution is 4-5 mM; the concentration of the fluorescent probe solution is 5-10 μM; the concentration of the Tris-HCl buffer solution is 50 mM, and the pH is 7.5.

[0023] In the step 2, 5≤n≤10, the concentrations of n portions of acetylcholinesterase standard solutions are increased, the initial concentration is 0, and the concentration difference between two adjacent portions of acetylcholinesterase standard solutions is 1-10 mU / mL.

[0024] Beneficial effects of the present invention: A new indene-chalcone-based fluorescent probe, LSS-AChE, was designed and synthesized in the present invention. LSS-AChE has no fluorescence emission at 505 nm. Upon encountering AChE, the ester bond within its molecule is hydrolyzed and cleaved, resulting in strong fluorescence emission at 505 nm. LSS-AChE has high selectivity and sensitivity to AChE (LOD = 0.0097 mU / mL), good water solubility, and a large Stokes shift (105 nm). Furthermore, using hydrogel as a carrier, a portable intelligent detection platform was constructed with a smartphone, enabling on-site instant detection of AChE.

[0025] This solution can use mobile phones and portable devices instead of large instruments as fluorescence detectors. The detection method is simpler, more portable, and more sensitive, making it easier to detect AChE on site. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0027] Figure 1 This is the nuclear magnetic resonance image of the fluorescent probe LSS-AChE of the present invention.

[0028] Figure 2 This is the mass spectrum of the fluorescent probe LSS-AChE of the present invention.

[0029] Figure 3 This is a graph showing the fluorescence changes of AChE detected by the fluorescent probe LSS-AChE of the present invention.

[0030] Figure 4 This is a color change diagram of the portable hydrogel of the present invention.

[0031] Figure 5 is a linear relationship diagram of the hydrogel RGB and AChE of the present invention, wherein R 2 =0.9938. DETAILED DESCRIPTION

[0032] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without creative work are within the scope of protection of the present invention.

[0033] Example 1

[0034] A fluorescent probe, the synthesis route is as follows:

[0035]

[0036] The preparation method is:

[0037] Dissolve LSS-OH (1.4 mmol, 0.458 g) in dichloromethane (20 mL). Add triethylamine (4.2 mmol, 580 mL) and stir in an ice bath for 10 min. Slowly add a solution of propionyl chloride (2.8 mmol, 0.291 g) in dichloromethane (5 mL). After the addition is complete, stir at 25°C for 12 h. Extract with ethyl acetate, concentrate, and purify by column chromatography to yield LSS-AChE (0.527 g, 93%).

[0038] Figure 1 and 2 The H NMR spectrum and mass spectrum of LSS-AChE prepared in Example 1 are shown below. 1 HNMR(400MHz, DMSO-d6)δ(ppm): δ7.78(m,J=6.6Hz,3H),7.49(m,J=7.4Hz,5H),7.26(dd,J=8.4,2.1H z,1H),4.16(d,J=2.6Hz,2H),2.67(q,J=7.5Hz,2H),1.16(t,J=7.5Hz,3H).ESI(+)-HRMS(m / z):[M+H] + calcd.forC 19 H 17 O3:293.1172; found293.1198.

[0039] Figure 3The fluorescence change diagram of the fluorescent probe LSS-AChE of the present invention for detecting AChE is shown in FIG. LSS-AChE has no fluorescence emission at 505 nm. When AChE is added, the system has strong fluorescence emission at 505 nm (λ ex =400nm,λ em =505nm), Stokes shift 105nm.

[0040] Example 2

[0041] A fluorescent probe was prepared by dissolving LSS-OH (2.8 mmol, 0.916 g) in dichloromethane (30 mL), adding triethylamine (8.4 mmol, 1160 μL), and stirring in an ice bath for 10 minutes. A solution of propionyl chloride (2.8 mmol, 0.291 g) in dichloromethane (5 mL) was then slowly added dropwise. After the addition was complete, the mixture was stirred at 25°C for 10 hours. Extraction and concentration with ethyl acetate were performed, and purification by column chromatography afforded LSS-AChE (0.2635 g, 46%).

[0042] Example 3

[0043] A fluorescent probe was prepared by dissolving LSS-OH (1.4 mmol, 0.458 g) in dichloromethane (20 mL), adding triethylamine (4.2 mmol, 580 μL), and stirring in an ice bath for 10 min. A solution of propionyl chloride (7 mmol, 0.728 g) in dichloromethane (5 mL) was then slowly added dropwise. After the addition was complete, the mixture was stirred at room temperature for 12 h. Extraction and concentration with ethyl acetate were performed, and purification by column chromatography afforded LSS-AChE (0.510 g, 90%).

[0044] Example 4

[0045] A fluorescent probe was prepared by dissolving LSS-OH (1.4 mmol, 0.458 g) in dichloromethane (20 mL), adding triethylamine (1.4 mmol, 193 μL), and stirring in an ice bath for 10 minutes. A solution of propionyl chloride (2.8 mmol, 0.291 g) in dichloromethane (5 mL) was then slowly added dropwise. After the addition was complete, the mixture was stirred at 20°C for 24 hours. Extraction and concentration with ethyl acetate were performed, and purification by column chromatography afforded LSS-AChE (0.2535 g, 43%).

[0046] Example 5

[0047] A fluorescent probe was prepared by dissolving LSS-OH (1.4 mmol, 0.458 g) in dichloromethane (20 mL), adding triethylamine (2.8 mmol, 580 μL), and stirring in an ice bath for 10 minutes. A solution of propionyl chloride (2.8 mmol, 0.291 g) in dichloromethane (5 mL) was then slowly added dropwise. After the addition was complete, the mixture was stirred at 30°C for 8 hours. Extraction and concentration with ethyl acetate were performed, and purification by column chromatography afforded LSS-AChE (0.340 g, 60%).

[0048] Example 6

[0049] A method for detecting acetylcholinesterase using a portable detection platform, using the fluorescent probe LSS-AChE prepared in Example 1 to detect acetylcholinesterase. The specific steps are:

[0050] (1) 1 mL (4.2 mM) agarose solution, 1 mL (8 μM) LSS-AChE, and a mixture thereof were dissolved in 2 mL (50 mM) Tris-HCl buffer (pH 7.5) to obtain the target responsive hydrogel. Then, 200 μL of the target responsive hydrogel was transferred to the cap of a centrifuge tube for later use.

[0051] (2) Prepare AChE solutions with the following concentrations (0, 2, 4, 6, 8, 10, 12, 13 mU / mL) and allow the hydrogels in the centrifuge tubes to react for ten minutes.

[0052] (3) In a UV light box, under UV irradiation, a color digital image of the centrifuge tube lid is obtained using a smartphone camera, e.g. Figure 4 As shown in Figure 2, the color of the hydrogel gradually turned green with the increase of AChE. The color digital image was analyzed by ImageJ software to obtain the RGB value, and a linear fit was performed by fitting G / R+B with the AChE concentration, where R 2 =0.9938( Figure 5 ), LSS-AChE has high selectivity and sensitivity to AChE (LOD = 0.0097 mU / mL).

[0053] Example 7

[0054] A method for detecting acetylcholinesterase using a portable detection platform, using the fluorescent probe LSS-AChE prepared in Example 1 to detect acetylcholinesterase. The specific steps are:

[0055] (1) 2 mL (5 mM) agarose solution, 4 mL (10 μM) LSS-AChE, and a mixture thereof were dissolved in 1 mL (50 mM) Tris-HCl buffer (pH 7.5) to obtain the target responsive hydrogel. Then, 200 μL of the target responsive hydrogel was transferred to the cap of a centrifuge tube for later use.

[0056] (2) Prepare AChE solutions with the following concentrations (0, 1, 3, 5, 10, 15, 20, 30, 40, 50 mU / mL) and allow the hydrogels in the centrifuge tubes to react for ten minutes.

[0057] (3) In a UV light box, under UV irradiation, a color digital image of the centrifuge tube lid was obtained using a smartphone camera. The color digital image was analyzed using ImageJ software to obtain RGB values, and a linear fit was performed by fitting the G / R+B ratio with the AChE concentration.

[0058] Example 8

[0059] A method for detecting acetylcholinesterase using a portable detection platform, using the fluorescent probe LSS-AChE prepared in Example 1 to detect acetylcholinesterase. The specific steps are:

[0060] (1) 1.5 mL (4 mM) agarose solution, 4 mL of 5 μM LSS-AChE, and the mixture were dissolved in 1.5 mL of 50 mM Tris-HCl buffer at pH 7.5 to obtain the target responsive hydrogel. Then, 200 μL of the target responsive hydrogel was transferred to the cap of a centrifuge tube for later use.

[0061] (2) Prepare AChE solutions with the following concentrations (0, 5, 10, 15, 20 mU / mL) and allow the hydrogels in the centrifuge tubes to react for ten minutes.

[0062] (3) In a UV light box, under UV irradiation, a color digital image of the centrifuge tube lid was obtained using a smartphone camera. The color digital image was analyzed using ImageJ software to obtain RGB values, and a linear fit was performed by fitting the G / R+B ratio with the AChE concentration.

[0063] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A fluorescent probe, characterized in that The structure of the fluorescent probe is:

2. The method for preparing the fluorescent probe according to claim 1, characterized in that: The synthetic route is as follows: The method comprises the following steps: mixing LSS-OH, propionyl chloride, an alkaline catalyst and a solvent to prepare a reaction solution, and reacting to obtain a fluorescent probe LSS-AChE.

3. The method for preparing a fluorescent probe according to claim 2, wherein: The molar ratio of the LSS-OH, propionyl chloride and base is 1:(1-5):(1-3).

4. The method for preparing a fluorescent probe according to claim 2, wherein: The alkaline catalyst is any one of triethylamine, pyridine or N,N-diisopropylethylamine.

5. The method for preparing a fluorescent probe according to claim 2, wherein: The solvent is any one of dichloromethane, chloroform, acetone, ethyl acetate, ethyl formate, methanol, ethanol, isopropanol, butanol, isopropylamine, dimethylformamide, dimethyl sulfoxide, ether and petroleum ether.

6. The method for preparing a fluorescent probe according to claim 2, wherein: The reaction conditions are: time of 8 to 24 hours and temperature of 20°C to 30°C.

7. A portable method for detecting acetylcholinesterase, characterized in that: The method of detecting acetylcholinesterase using the fluorescent probe according to claim 1 comprises the following steps: Step 1: Mix agarose, fluorescent probe and Tris-HCl buffer to prepare target-responsive hydrogel; Step 2: preparing n portions of acetylcholinesterase standard solutions of different concentrations, wherein n≥2; Step 3: n portions of acetylcholinesterase standard solutions of different concentrations are reacted with the target response hydrogel respectively, and a color image of each standard solution to be detected is obtained under ultraviolet irradiation conditions. The color image is analyzed to obtain the RGB value, and a linear relationship between the intensity ratio G / R+B and the acetylcholinesterase concentration is established.

8. The portable method for detecting acetylcholinesterase according to claim 7, characterized in that: In the step 1, agarose and a fluorescent probe are dissolved in water to prepare a fluorescent probe solution and an agarose solution, which are then mixed with agarose and a Tris-HCl buffer. The volume ratio of the agarose solution, the fluorescent probe solution, and the Tris-HCl buffer is (1-2): (1-4): (1-2).

9. The portable method for detecting acetylcholinesterase according to claim 8, characterized in that: The concentration of the agarose solution is 4-5 mM; the concentration of the fluorescent probe solution is 5-10 μM; the concentration of the Tris-HCl buffer solution is 50 mM, and the pH is 7.

5.

10. The portable method for detecting acetylcholinesterase according to claim 7, characterized in that: In the step 2, 5≤n≤10, the concentrations of n portions of acetylcholinesterase standard solutions are increased, the initial concentration is 0, and the concentration difference between two adjacent portions of acetylcholinesterase standard solutions is 1-10 mU / mL.

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