Thiophenyl quinoline-based squaraine amide fluorescent probe for recognizing adenosine triphosphate and preparation method thereof

Abstract

The application belongs to the field of chemical analysis test, and particularly relates to a thiophene alkyl quinoline-based squaramide fluorescent probe for identifying adenosine triphosphate and a preparation method. 8-aminoquinaldine and 2-thiophene formaldehyde are dissolved in ethanol, acetic acid is used as an acidic reaction condition, and an intermediate 1 is obtained. 2,3,3-trimethyl-3H-indole and iodoethane are dissolved in acetonitrile and refluxed, and an intermediate 2 is obtained. The intermediate 2 and squaric acid diethyl ester are dissolved in ethanol, and triethylamine is used as an alkaline reaction condition to obtain an intermediate 3. The intermediate 3 is dissolved in acetonitrile, acetic acid and hydrochloric acid are added, and reaction is carried out to obtain an intermediate 4. The intermediate 1 and the intermediate 4 are dissolved in n-butanol and refluxed, the obtained crude product is separated by column chromatography, and a final pink product SQN-1 is obtained. The squaramide fluorescent probe obtained in the application can quickly identify adenosine triphosphate in a cetyltrimethylammonium bromide surfactant aqueous solution.

Description

Technical Field

[0001] This invention belongs to the field of chemical analysis and testing, specifically relating to a thienene-quinoline-based squaric acid amide fluorescent probe for recognizing adenosine triphosphate and its preparation method. Background Technology

[0002] Adenosine triphosphate (ATP) is a nucleotide that serves as the energy currency within living cells, participating in energy storage and transfer. It also acts as a signaling mediator in various biological processes, such as the tricarboxylic acid cycle, ion channels, nerve transmission, cell division, and DNA synthesis. Furthermore, changes in ATP concentration are directly related to several pathological processes, such as ischemic injury, platelet aggregation, and Parkinson's syndrome.

[0003] Currently, the main methods for detecting nucleotides are high-performance liquid chromatography (HPLC), gel electrophoresis, polymerase chain reaction (PCR), and nuclear magnetic resonance (NMR). These methods can usually only measure the content of nucleoside triphosphates, and the operation procedures are complex and the detection cycle is long. Fluorescence analysis is a simple, sensitive and rapid detection method.

[0004] In recent years, numerous ATP sensing methods have been developed, including sensors that form metal ion complexes, small organic molecule sensors based on multiple interactions, and sensors containing polythiophene. However, many existing fluorophores suffer from poor photostability, low fluorescence quantum yield, or short wavelength ranges that are easily affected by biological background. Therefore, developing simple and rapid detection techniques for adenosine triphosphate (ATP) is of great significance for human health. Summary of the Invention

[0005] This invention provides a squaric acid amide-based fluorescent probe SQN-1 based on thieneneylquinoline, the structural formula of which is:

[0006]

[0007] This invention also provides a method for preparing a squaric acid amide-based fluorescent probe based on thienylquinoline, wherein the chemical reaction formula for preparing the fluorescent probe is as follows:

[0008]

[0009] The specific preparation method steps are as follows:

[0010] (1) Dissolve 8-aminoquinalidine and 2-thiophenecaraldehyde in ethanol solution at a molar equivalent ratio of 1:1 to 1.2, then add 1 molar equivalent of glacial acetic acid and heat at 60°C for 12 hours. After the reaction is completed, remove the solvent, extract the crude product with dichloromethane and water, remove the organic solvent under reduced pressure, and finally purify by thin-layer chromatography using a mixed solvent of petroleum ether and ethyl acetate at a volume ratio of 5:1 as the developing solvent to obtain yellow solid intermediate 1.

[0011] (2) 2,3,3-trimethyl-3H-indole and iodoethane were dissolved in acetonitrile solution at a molar ratio of 1:4, and the mixture was heated under reflux at 80°C for 24 hours. After the reaction was completed, the solvent was removed, and the mixture was washed three times with diethyl ether to obtain purple solid intermediate 2.

[0012] (3) Intermediate 2 and diethyl squaric acid were dissolved in an ethanol solution at a molar ratio of 1:1. Triethylamine was then added as an alkaline condition (the volume ratio of ethanol to triethylamine was 8:1), and the mixture was heated at 60°C for 8 hours. After the reaction was completed, the solvent was removed, and the crude product was extracted with dichloromethane and water. The organic solvent was removed under reduced pressure, and finally purified by thin-layer chromatography using a mixed solvent of petroleum ether and ethyl acetate at a volume ratio of 6:1 as the developing solvent to obtain orange solid intermediate 3.

[0013] (4) Intermediate 3 was dissolved in a mixed solution of acetonitrile, glacial acetic acid, and hydrochloric acid in a volume ratio of 15:3:1, and heated at 60°C for 6 hours. After the reaction was completed, the solvent was removed, and the crude product was extracted with dichloromethane and water. The organic solvent was removed under reduced pressure, and finally purified by thin-layer chromatography using a mixed solvent of dichloromethane and methanol in a volume ratio of 6:1 as the developing solvent to obtain a yellow solid intermediate 4. Each 50 mg of intermediate 3 was dissolved in 1.5 ml of acetonitrile.

[0014] (5) Intermediate 1 and Intermediate 4 were dissolved in n-butanol solution at a 1:1 molar equivalent ratio and heated under reflux at 90°C for 3-6 hours. After the reaction was completed, the solvent was removed, and the crude product was extracted with dichloromethane and water. The organic solvent was removed under reduced pressure, and finally purified by thin-layer chromatography using a mixed solvent of petroleum ether and ethyl acetate at a volume ratio of 5:1 as the developing solvent to obtain a pink solid pure product.

[0015] This invention also provides applications of the above-mentioned fluorescent probes: the prepared thienylquinoline-based squaric acid amide fluorescent probes can rapidly recognize adenosine triphosphate (ATP) in aqueous solution of CTAB surfactant.

[0016] The concentration of the CTAB surfactant aqueous solution is 0.5mM-1.0mM.

[0017] The specific application method is as follows: Add 2 μL of a 10 mM nucleotide and analogue solution (the nucleotides in each well are adenosine triphosphate (ATP), cyclic adenosine monophosphate (cAMP), guanosine monophosphate (GMP), guanine (G), O-6-benzylguanine (O6-BG), deoxyguanosine (dG), adenine (A), cytosine (C), thymidine acetate (TAA), guanosine, β-nicotinamide mononucleotide (NMN), oxidized coenzyme I (NAD), sodium alendronate (AST), and blank) to a 96-well plate, 196 μL of cetyltrimethylammonium bromide (CTAB surfactant) solution (0.5 mM), and 2 μL of a thienene-quinoline-based squaric acid amide fluorescent probe (concentration 1 × 10⁻⁶). -3 The fluorescence intensity of each well was measured using a microplate reader (μmol / L) and compared with a solution containing a squaric acid amide fluorescent probe without nucleotides (blank). The solutions in each well were thoroughly mixed, and the fluorescence intensity of each solution was measured using a microplate reader. The results showed that after the addition of adenosine triphosphate (ATP), the fluorescence intensity of the probe at 570 nm was 60 times that of the nucleotide-free solution; furthermore, the fluorescent probe did not exhibit significant changes in fluorescence intensity for other nucleotides and analogs, demonstrating the selective recognition of ATP by this fluorescent probe in a cetyltrimethylammonium bromide (CTAB) surfactant solution.

[0018] The squaric acid amide used in this fluorescent probe is a squaric acid cyanine derivative, which possesses excellent optical properties and stability, and can produce strong fluorescence when excited at a specific wavelength. The final squaric acid amide fluorescent probe is obtained by linking thienenylquinoline and hemisquaric acid cyanine through nucleophilic substitution. When it recognizes ATP, the fluorescence intensity is significantly enhanced, while it does not have the same effect on other nucleotide analogs, thus achieving the purpose of ATP detection.

[0019] Beneficial effects

[0020] The raw materials used in this invention are readily available, the synthesis method is simple, the reaction conditions are easy to control, and the pure product can be obtained through simple post-processing. The probe SQN-1 reported in this invention solves the problem of rapid ATP recognition in the CTAB solvent system. From a fluorescence perspective, the fluorescence intensity of probe SQN-1 at 570 nm significantly increased after the addition of ATP, thus demonstrating the ATP recognition function of probe SQN-1 in this system. Attached image description:

[0021] Figure 1 The probe prepared for Example 1 was prepared at a ratio of 1 × 10⁻⁶. -3 Fluorescence spectra of CTAB at concentrations of mol / L after interaction with different nucleotides and analogs.

[0022] Figure 2 The probe prepared for Example 1 was prepared at a ratio of 1 × 10⁻⁶. -3 Fluorescence spectra of ATP at different concentrations in CTAB at a concentration of mol / L.

[0023] Figure 3 The fluorescent probe prepared for Example 1 was used at 1×10 -3 Fluorescence intensity at 570 nm of CTAB surfactant aqueous solution at a concentration of mol / L reacting with ATP after the addition of five times the amount of interfering nucleotides and analogues.

[0024] Figure 4 The proton spectrum of the probe prepared in Example 1.

[0025] Figure 5 The diagram shows the recognition ability of the probe prepared in Example 1 for ATP in aqueous solutions of CTAB surfactant at different concentrations.

[0026] Figure 6 The diagram shows the ability of the probe prepared in Example 1 to recognize ATP in different buffer solutions. Detailed Implementation

[0027] The present invention will be further described in detail below with reference to specific embodiments:

[0028] Example 1

[0029] (1) 8-Aminoquinaldine (160 mg, 1.00 mmol) and 2-thiophenecaraldehyde (110 mg, 1.00 mmol) were dissolved in a round-bottom flask with 4 mL of ethanol as solvent, followed by the addition of glacial acetic acid (60 μL, 1.00 mmol), and the mixture was heated under reflux at 80 °C for 12 hours. After the reaction was completed, the solvent was removed, and the crude product was extracted with dichloromethane and water. The organic solvent was removed under reduced pressure, and finally purified by thin-layer chromatography using a mixture of petroleum ether and ethyl acetate in a volume ratio of 5:1 as the developing solvent to obtain 185 mg of yellow solid intermediate 1.

[0030] (2) 2,3,3-trimethyl-3H-indole (320 μL, 2.00 mmol) and iodoethane (630 μL, 8.00 mmol) were dissolved in 5 mL of acetonitrile solution and heated under reflux at 80 °C for 24 hours. After the reaction was completed, the solvent was removed and the mixture was washed three times with diethyl ether to obtain 1.5 g of purple solid intermediate 2.

[0031] (3) The intermediate 2 (315 mg, 1.00 mmol) obtained in step (2) and diethyl squaric acid (170 mg, 1.00 mmol) were dissolved in a mixed solution of 6 mL ethanol and 750 μL triethylamine and heated under reflux at 60 °C for 8 hours. After the reaction was completed, the solvent was removed, and the crude product was extracted with dichloromethane and water. The organic solvent was removed under reduced pressure, and finally purified by thin-layer chromatography using a mixed solvent of petroleum ether and ethyl acetate in a volume ratio of 6:1 as the developing solvent to obtain 220 mg of orange solid intermediate 3.

[0032] (4) The intermediate 3 (200 mg, 0.64 mmol) obtained in step (3) was dissolved in 4 mL of acetonitrile solution, followed by the addition of 0.8 mL of acetic acid and 0.28 mL of hydrochloric acid. The mixture was heated under reflux at 60 °C for 6 hours. After the reaction was completed, the solvent was removed, and the crude product was extracted with dichloromethane and water. The organic solvent was removed under reduced pressure, and finally purified by thin-layer chromatography using a 6:1 (v / v) mixture of dichloromethane and methanol as the developing solvent to obtain 120 mg of yellow-green solid intermediate 4.

[0033] (5) Intermediate 1 (50 mg, 0.25 mmol) obtained in step (1) and intermediate 4 (70 mg, 0.25 mmol) obtained in step (4) were dissolved in 3 mL of n-butanol solution and heated under reflux at 90 °C for 3 hours. After the reaction was completed, the solvent was removed, and the crude product was extracted with dichloromethane and water. The organic solvent was removed under reduced pressure, and finally purified by thin-layer chromatography using a mixed solvent of petroleum ether and ethyl acetate in a volume ratio of 5:1 as the developing solvent to obtain 55 mg of pink solid final product, with a yield of 53%.

[0034] Figure 1 The fluorescence spectra of the thienene-quinoline-based squaric acid amide fluorescent probe prepared in Example 1 after reaction with different nucleotides and analogs (adenosine triphosphate (ATP), cyclic adenosine monophosphate (cAMP), guanosine monophosphate (GMP), guanine (G), O-6-benzylguanine (O6-BG), deoxyguanosine (dG), adenine (A), cytosine (C), thymidine acetate (TAA), guanosine, β-nicotinamide mononucleotide (NMN), oxidized coenzyme I (NAD), alendronate sodium (AST), and blank) in 0.5 mM CTAB surfactant solution are shown. The figures illustrate the changes in fluorescence intensity of the probe after the addition of different nucleotide and analog solutions. When 10 mM ATP solution was added, the fluorescence intensity of the fluorescent probe solution at 570 nm increased significantly (curve indicated by arrow); while in CTAB surfactant solution, the probe did not show any particularly obvious fluorescence change for other nucleotides or analogs, thus demonstrating the selective recognition of ATP by the probe in this system.

[0035] Figure 2 The fluorescent probe prepared for Example 1 was used at 1×10 -3 Fluorescence spectra of SQN-1 at a concentration of mol / L reacting with different concentrations of ATP in CTAB aqueous solution. The figures show that the fluorescence intensity of the probe solution gradually increases with increasing ATP concentration. At a concentration of 50 μM, the fluorescence intensity of SQN-1 tends to plateau. Even at its lowest concentration of 10 μM, the fluorescence peak at 570 nm is still distinguishable from the background fluorescence curve without nucleotides, indicating that this probe has a low detection limit and high sensitivity for ATP.

[0036] Figure 3 The fluorescent probe prepared for Example 1 was used at 1×10 -3 Fluorescence intensity at 570 nm of CTAB at a concentration of mol / L in aqueous solution reacting with ATP after the addition of five times the amount of interfering nucleotides and analogues. The figure shows that when only ATP solution was added, the fluorescence intensity of the probe solution at 570 nm was significantly higher than that of the blank. However, the probe did not show any significant fluorescence changes for other nucleotides (ATP, cAMP, GMP, guanine (G), O-6-benzylguanine (O6-BG), deoxyguanosine (dG), adenine (A), cytosine (C), thymidine acetate (TAA), guanosine, β-nicotinamide mononucleotide (NMN), oxidized coenzyme I (NAD), alendronate sodium (AST), and blank). Subsequently, the fluorescence intensity of the probe at 570 nm was changed after adding 5 times the amount of other nucleotides and analogues to the detection system. This shows that the addition of other proteins and redox substances did not lead to a decrease in the ATP detection results of the probe, indicating that the fluorescent probe has strong anti-interference ability during the detection process.

[0037] Figure 4 The proton spectrum of the thiophene-enylquinoline-based squaric acid amide fluorescent probe prepared in Example 1. 1H NMR(400MHz,Chloroform)δ10.38(s,1H),8.91(dd,J=7.5,1.4Hz,1H),8.03(d,J=8.6Hz,1H ),7.83(d,J=16.0Hz,1H),7.66(dd,J=5.7,3.3Hz,2H),7.55(d,J=8.6Hz,1H),7.47(dd,J=5 .7,3.2Hz,2H),7.32(d,J=7.4Hz,1H),7.24(d,J=3.6Hz,1H),7.19(s,1H),7.11(d,J=6.4Hz ,1H),6.97(d,J=7.8Hz,1H),5.92(s,1H),4.02(s,2H),1.35(t,J=7.4Hz,3H),1.18(s,6H).

[0038] Example 2:

[0039] (1) 8-Aminoquinaldine (480 mg, 3.00 mmol) and 2-thiophenecaraldehyde (330 mg, 3.00 mmol) were dissolved in a round-bottom flask with 8 mL of ethanol as solvent, followed by the addition of glacial acetic acid (180 μL, 3.00 mmol), and the mixture was heated under reflux at 80 °C for 12 hours. After the reaction was completed, the solvent was removed, and the crude product was extracted with dichloromethane and water. The organic solvent was removed under reduced pressure, and finally purified by column chromatography using a mixture of petroleum ether and ethyl acetate in a volume ratio of 5:1 as the developing solvent to obtain 500 mg of yellow solid intermediate 1.

[0040] (2) 2,3,3-trimethyl-3H-indole (150 μL, 1.00 mmol) and iodoethane (300 μL, 4.00 mmol) were dissolved in 2 mL of acetonitrile solution and heated under reflux at 80 °C for 24 hours. After the reaction was completed, the solvent was removed and the mixture was washed three times with diethyl ether to obtain 720 mg of purple solid intermediate 2.

[0041] (3) The intermediate 2 (150 mg, 0.50 mmol) obtained in step (2) and diethyl squaric acid (80 mg, 0.50 mmol) were dissolved in a mixed solution of 3 mL ethanol and 325 μL triethylamine and heated at 60 °C for 8 hours. After the reaction was completed, the solvent was removed, the crude product was extracted with dichloromethane and water, the organic solvent was removed under reduced pressure, and finally purified by column chromatography using a mixed solvent of petroleum ether and ethyl acetate in a volume ratio of 6:1 as the developing solvent to obtain 100 mg of orange solid intermediate 3.

[0042] (4) The intermediate 3 (100 mg, 0.32 mmol) obtained in step (3) was dissolved in 2 mL of acetonitrile solution, followed by the addition of 400 μL of acetic acid and 140 μL of hydrochloric acid, and heated at 60 °C for 6 hours. After the reaction was completed, the solvent was removed, and the crude product was extracted with dichloromethane and water. The organic solvent was removed under reduced pressure, and finally purified by thin-layer chromatography using a 6:1 (v / v) mixture of dichloromethane and methanol as the developing solvent to obtain 55 mg of yellow-green solid intermediate 4.

[0043] (5) Intermediate 1 (25 mg, 0.125 mmol) obtained in step (1) and intermediate 4 (35 mg, 0.125 mmol) obtained in step (4) were dissolved in 2 mL of n-butanol solution and heated under reflux at 90 °C for 3 hours. After the reaction was completed, the solvent was removed, and the crude product was extracted with dichloromethane and water. The organic solvent was removed under reduced pressure, and finally purified by thin-layer chromatography using a mixed solvent of petroleum ether and ethyl acetate in a volume ratio of 5:1 as the developing solvent to obtain 30 mg of pink solid final product, with a yield of 62.5%.

[0044] Example 3:

[0045] Add 2 μL of a 1×10⁻⁶ solution to each well of a 96-well plate. -2 Adenosine triphosphate solution of mol / L was used in 13 parallel groups. Thirteen different concentrations of hexadecyltrimethylammonium bromide solution (0 mM, 0.1 mM, 0.2 mM, 0.3 mM, 0.4 mM, 0.5 mM, 0.6 mM, 0.7 mM, 0.8 mM, 0.9 mM, 1.0 mM, 2.0 mM, 3.0 mM) and 2 μL of squaric acid amide fluorescent probe (concentration 1.0 × 10⁻⁶) were added to each group. -3 The solutions in each well were mixed thoroughly using a microplate reader (0.5-1.0 mM CTAB aqueous solution), and the fluorescence intensity of each solution was detected. The results showed that the fluorescent probe could effectively recognize ATP in the 0.5-1.0 mM CTAB aqueous solution system. When the CTAB concentration was greater than 1.0, other nucleotide analogs also enhanced the fluorescence intensity of the probe.

[0046] The specific recognition ability of the probe for dTTP was studied in aqueous solutions of CTAB surfactant at different concentrations. Specific data are as follows: Figure 5 As shown, the horizontal axis represents the concentration of the CTAB surfactant aqueous solution, and the vertical axis represents the difference in fluorescence intensity of the probe before and after the addition of dTTP, denoted by I-I0. The probe exhibits significant recognition ability for ATP at CTAB concentrations of 0.5 mM-1.0 mM.

[0047] Example 4:

[0048] 2 μL of 1.0 mM squaric acid amide fluorescent probe was added to each well of a 96-well plate. Then, 198 μL of each of seven different buffer solutions were added: HEPES (10 mM), SDS (4 mM), Tris-HCl (10 mM), PBS (10 mM), CTAB (0.5 mM), EtOH, and pure water. The control group consisted of 2 μL of 1.0 mM squaric acid amide fluorescent probe and 2 μL of 1×10⁻⁶ HCl. -2 Adenosine triphosphate (ATP) at a concentration of mol / L was added to 196 μL of each of seven different buffer solvents. The solutions in each well were mixed thoroughly, and the fluorescence intensity of the solution in each well was detected using a microplate reader. The results showed that the fluorescent probe's recognition ability in the CTAB surfactant aqueous solution was significantly greater than that in other buffer solvents.

Claims

1. A squaric acid amide-based fluorescent probe based on thieneneylquinoline, characterized in that: The structural formula of the probe is: 。 2. A method for preparing a squaric acid amide-based fluorescent probe based on thienene-quinoline as described in claim 1, characterized in that: The preparation method steps are as follows: (1) Using ethanol as a solvent, 8-aminoquinaldine and 2-thiophenecarboxaldehyde were dissolved in a round-bottom flask, and then glacial acetic acid was added. The mixture was heated at 60°C for 12 hours. After the reaction was completed, the solvent was removed, and the mixture was extracted with dichloromethane and water. The organic solvent was removed under reduced pressure, and finally purified by thin-layer chromatography using petroleum ether and ethyl acetate as the developing solvent to obtain a yellow solid intermediate 1. Its structural formula is as follows: ; (2) Using acetonitrile as solvent, 2,3,3-trimethyl-3H-indole and iodoethane were dissolved in a round-bottom flask and heated under reflux at 80°C for 24 hours. After the reaction was completed, the solvent was removed, and the mixture was washed three times with diethyl ether to obtain a purple solid intermediate 2. Its structural formula is: ; (3) The intermediate 2 obtained in step (2) was dissolved in an ethanol solution with diethyl squaric acid, and then triethylamine was added as an alkaline condition. The mixture was heated at 60°C for 8 hours. After the reaction was completed, the solvent was removed, and the crude product was extracted with dichloromethane and water. The organic solvent was removed under reduced pressure, and finally purified by thin-layer chromatography using petroleum ether and ethyl acetate as the developing solvent to obtain an orange solid intermediate 3. Its structural formula is as follows: ; (4) Dissolve intermediate 3 obtained in step (3) in acetonitrile solution, then add glacial acetic acid and hydrochloric acid, and heat at 60°C for 6 hours; after the reaction is complete, remove the solvent, extract the crude product with dichloromethane and water, remove the organic solvent under reduced pressure, and finally purify it by thin-layer chromatography using dichloromethane and methanol as the developing solvent to obtain yellow solid intermediate 4; its structural formula is ; (5) Dissolve intermediate 1 obtained in step (1) and intermediate 4 obtained in step (4) in n-butanol solution and reflux at 90°C for 3-6 hours. After the reaction is completed, remove the solvent, extract the crude product with dichloromethane and water, remove the organic solvent under reduced pressure, and finally purify it by thin-layer chromatography with petroleum ether and ethyl acetate as the developing solvent to obtain a pink thiophene-based quinoline-based squaric acid amide fluorescent probe.

3. The method for preparing the squaric acid amide fluorescent probe based on thienene-quinoline as described in claim 2, characterized in that: In step (1), the molar equivalent ratio of 8-aminoquinaldine, 2-thiophenecarboxaldehyde and glacial acetic acid is 1:1 to 1.2:1; the developing solvent is a mixture of petroleum ether and ethyl acetate with a volume ratio of 5:

1.

4. The method for preparing the squaric acid amide fluorescent probe based on thienene-quinoline as described in claim 2, characterized in that: In step (2), the molar equivalent ratio of 2,3,3-trimethyl-3H-indole to iodoethane is 1:

4.

5. The method for preparing the squaric acid amide fluorescent probe based on thienene-quinoline as described in claim 2, characterized in that: In step (3), the volume ratio of ethanol to triethylamine is 8:1, the molar equivalent ratio of intermediate 2 to diethyl squaric acid is 1:1, and the developing solvent is a mixture of petroleum ether and ethyl acetate with a volume ratio of 6:

1.

6. The method for preparing the squaric acid amide fluorescent probe based on thienene-quinoline as described in claim 2, characterized in that: In step (4), each 50 mg of intermediate 3 is dissolved in 1.5 ml of acetonitrile. The volume ratio of acetonitrile, glacial acetic acid and hydrochloric acid is 15:3:

1. The developing solvent is a mixture of dichloromethane and methanol with a volume ratio of 6:

1.

7. The method for preparing the squaric acid amide fluorescent probe based on thienene-quinoline as described in claim 2, characterized in that: In step (5), the molar equivalent ratio of intermediate 1 and intermediate 4 is 1:1; the developing solvent is a mixture of petroleum ether and ethyl acetate with a volume ratio of 5:

1.

8. The application of the thienylquinoline-based squaric acid amide fluorescent probe as described in claim 1 in the preparation of a probe for detecting adenosine triphosphate (ATP) in an aqueous solution of a cetyltrimethylammonium bromide surfactant by fluorescence enhancement.

9. The application of the squaric acid amide fluorescent probe based on thienylquinoline as described in claim 8, characterized in that: The concentration of the aqueous solution of hexadecyltrimethylammonium bromide surfactant is 0.5-1.0 mM.

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