A sensitive and selective fluorescent probe for acetylcholinesterase: Synthesis, performance, mechanism and application

2.1 Reagents and instruments

Dimethylcarbamic acid 2-formyl-5-(diethylamino) phenyl ester (BDFAM) was provided by Chemieliva Pharmaceutical Co., Ltd. 2-Benzothiazoleacetonitrile and other chemicals were commercially available from Innochem and used without further purification. Acetylcholinesterase (AChE, 200 U/g, extracted from electric eel), trypsin (TPS, 2500 U/mg), lysozyme (LZM, 2000 U/mg), bovine serum albumin (BSA, biotechnology grade), β-glucosidase (CB, 20 U/mg) were purchased from Macklin. Butyrylcholinesterase (BChE, 10 U/mg, extracted from horse serum) was purchased from Sigma-Aldrich. Ultrapure water (18.25 MΩ.cm, 25 °C) was used in all experiments.

NMR spectra were recorded on a Bruker Avance III spectrometer at 500 MHz for ¹H NMR and 125 MHz for ¹³C NMR. High-resolution mass spectrometry (HRMS) was measured on an Agilent 6520 Accurate-Mass Q-TOF LC/MS spectrometer. Fluorescence spectroscopic studies were performed on a Hitachi F-7000 spectrometer. Fluorescence decay dynamics were observed using an Edinburgh-Fluorescence Spectrometer FLS980 (Edinburgh, U.K). UV–visible spectra were measured on a TU-1901 spectrometer.

2.2 Synthesis and characterization

Synthesis of BDFA: BDFAM (0.264 g, 1 mmol), 2-benzothiazoleacetonitrile (0.174 g, 1 mmol) and pyridine (2 drops) were dissolved in CH₃OH (30 mL) and stirred at room temperature for 8 h. Then, the solvent was removed under reduced pressure to produce a crude product, which was further purified by silica gel column chromatography (CH₃OH/CH₂Cl₂ = 1:20, v/v) to obtain BDFA as a yellow powder (21.0 mg, 0.5 mmoL): yield 50%. ¹H NMR (500 MHz, Chloroform-d) δ 8.48 (d, J = 9.2 Hz, 1H), 8.25 (s, 1H), 8.00 (d, J = 8.2 Hz, 1H), 7.84 (d, J = 8.0 Hz, 1H), 7.47 (t, J = 7.7 Hz, 1H), 7.36 (t, J = 7.6 Hz, 1H), 6.60 (d, J = 9.2 Hz, 1H), 6.49 (s, 1H), 3.43 (q, J = 7.1 Hz, 4H), 3.28 (s, 3H), 3.08 (s, 3H), 1.23 (t, J = 7.1 Hz, 6H) (Fig. S4). ¹³C NMR (125 MHz, Chloroform-d) δ 165.07, 154.06, 153.91, 153.67, 151.92, 140.05, 134.47, 129.95, 126.50, 125.16, 123.06, 121.39, 118.01, 112.39, 109.25, 104.99, 98.17, 77.31, 77.06, 76.80, 44.88, 37.05, 36.75, 12.67 (Fig. S5). ESI-MS m/z calculated for [C₂₃H₂₄N₄O₂S + Na]⁺ 443.1512, measured 443.1517 (Fig. S6) Scheme 1.

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Scheme 1

Synthetic route of BDFA.

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Synthesis of the BDFA-AChE reaction product (BDFAP): BDFA (20 mg, 0.48 mmoL) was fully dissolved in PBS solution (10 mM, pH = 7, containing 5% DMSO) and a sufficient amount of AChE was gradually added, and then the mixture was incubated at 37 °C for 40 min. The above solution was extracted with CH₂Cl₂ and the organic phase was concentrated under reduced pressure to obtain a crude product, which was further purified by silica gel column chromatography (CH₃OH/CH₂Cl₂ = 1:20, v/v) to obtain BDFAP as a yellow powder. ¹H NMR (500 MHz, Methanol‑d₄) δ 8.16 (s, 1H), 8.00 (dd, J = 15.4, 8.0 Hz, 2H), 7.53 (t, J = 7.7 Hz, 1H), 7.44 (dt, J = 7.7, 3.2 Hz, 2H), 6.69 (dd, J = 9.1, 2.5 Hz, 1H), 6.53 (d, J = 2.4 Hz, 1H), 3.53 (q, J = 7.1 Hz, 4H), 1.26 (t, J = 7.1 Hz, 6H) (Fig. S7). ESI-MS m/z calculated for [C₂₀H₁₉N₃OS + H]⁺ 350.1322, measured 350.1320 (Fig. S8).

2.3 General procedures for fluorescence measurement

5 µl of BDFA stock solution (200 μM, DMSO) were added to the PBS solution (pH = 7.0, 10 mM), and then the stock solutions of various analytes were subsequently introduced. These analytes include: acetylcholinesterase (AChE, 10 U/mL), butyrylcholinesterase (BChE, 10 U/mL), glutamic acid (Glu, 10 mM), lysine (Lys, 10 mM), arginine (Arg, 10 mM), Tyrosine (Tyr, 10 mM), cysteine (Cys, 10 mM), tryptophan (Trp, 10 mM), glutathione (GSH, 10 mM), glucose (Glc, 10 mM), sucrose (Suc, 10 mM), dextran sulfate (DS, 10 mg/mL), trypsin (TPS, 400 U/mL), lysozyme (LZM, 100 U/mL), β-glucosidase (CB, 20 U/mL) and bovine serum albumin (BSA, 1 mg/mL). Subsequently, the above mixture was incubated at 37 °C for 40 min, and then its total volume was adjusted to 500 μL with a PBS solution (pH = 7.0, 10 mM). Finally, the solution was subjected to fluorescence measurement under excitation at 470 nm, with 900 V photomultiplier voltage and 5 nm excitation and emission bandwidth.

2.4 Molecular docking calculations

The molecular docking simulation was carried out on the software AutoDock 1.5.6. The crystal structure of AChE was found in the protein data bank (PDB ID: 4bc1). The BDFA structure was optimized at the level of B3LYP/6-31G* on the solvation model of water by using Gaussian 09. The BDFA-AChE docking conformation was visualized using Pymol software, and its schematic diagram was also drawn using the same software.

2.5 Determination of AChE activity in blood

Blood samples collected from three healthy students in our laboratory were diluted 20 times with PBS solution (pH 7.0, 10 mM) to prevent blood clotting. The samples were divided into three parts. Two of which were added with Dichlorvos (final concentration: 100 μg/mL) and Carbaway (final concentration: 100 μg/mL) and then incubated at room temperature for 30 min. Subsequently, the diluted blood (95 μL), pesticide-pretreated blood (95 μL) and PBS solution (95 μL) were added to BDFA (final concentration: 2 μM) in PBS solution (105 μL). The above mixture was incubated at 37 °C for 40 min, and finally fluorescence measurement was performed.

3.1 Spectral response of BDFA to AChE

The optical properties of BDFA are closely related to its molecular configuration, which was first studied at the level of B3LYP/6-31G* on the solvation model of water using Gaussian 09. The results in Fig. 2a show that in the energy minimization configuration, the benzene ring and benzothiazole ring of BDFA are not distributed in a plane due to intramolecular steric hindrance, resulting in a distorted π-conjugated skeleton. Such non-planar structure is detrimental to fluorescence emission, and will reduce the background fluorescence from BDFA. In PBS solution, BDFA presents a main absorption band at 370–550 nm with a peak at 460 nm, which undergoes a noticeable enhancement and a small blue shift upon the addition of AChE (Fig. 2b). Under parallel conditions, BDFA emits weak fluorescence, which is consistent with the molecular configuration we simulated above. As an increasing concentration of AChE was introduced into the BDFA solution, a gradually enhanced emission centered at 525 nm appears, as shown in Fig. 2c. The fluorescence intensity at 525 nm rises sharply, and reaches a stable level after adding 2.0 U/mL AChE (Fig. 2d). The relationship between the fluorescence intensity at 525 nm and the AChE concentration in the range of 0.0045–1.0 U/mL conforms to the linear equation F = 416.7 * [AChE] + 39.1 (R² = 0.9952), and the detection limit (DL) was calculated as 4.5 mU/mL (DL = 3σ/k, where σ is the standard deviation (0.63) of the BDFA fluorescence intensity F₅₂₅ in the absence of AChE under 11 parallel measurements, and k is the slope (416.7) of the linear equation plotting fluorescence intensity F₅₂₅ vs AChE concentration). In general, BDFA exhibits a highly sensitive fluorescence response to AChE, which is expected to be applied to quantify AChE activity.

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Fig. 2

(a) Energy-minimized conformation of BDFA. The white, gray, blue and yellow balls represent hydrogen, carbon, nitrogen and sulfur atoms, respectively. (b) Absorption spectra of BDFA (5 μM) in the absence and presence of AChE (3.0 U/mL). (c) Fluorescence spectra of BDFA (2 μM) and (d) its emission intensity at 525 nm varies with the AChE concentration in the range of 0–3.0 U/mL. All spectra were measured in PBS solution (pH 7.0, 10 mM) with excitation at 470 nm.

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3.2 The selectivity of BDFA to AChE

To evaluate the selectivity in response to AChE, the fluorescence spectra of BDFA in the presence of various compounds were subsequently examined, and the results are shown in Fig. 3. It can be seen that the fluorescence shows an evident increase upon the addition of AChE or BChE, while other compounds, including common cations, anions, thiols, carbohydrates and enzymes only cause limited fluorescence changes. We also noticed that after adding the same concentration of AChE and BChE, the fluorescence intensity at 525 nm increases by 16.9 times and 5.2 times, respectively, which proves that the specificity of BDFA for AChE was much higher than that of BChE. Such results are consistent with the results of previous studies (Wang et al., 2019; Ma et al., 2020a, 2020b; He et al., 2021), which is speculated to be due to the better spatial match between the N, N-dimethyl carbamate group and the active cavity of AChE, thus causing the carbamate group to be easily hydrolyzed by AChE. Even in the coexistence of various compounds, BDFA still maintains a significant and relatively stable fluorescence response to AChE. The presence of BChE only increases the fluorescence response signal by 21%, so its interference is limited.

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Fig. 3

(a) Fluorescence spectra of BDFA (2 μM) and (b) its emission intensity at 525 nm in the coexistence of various compounds, including Na⁺ (1 mM), K⁺ (1 mM), Ca²⁺ (1 mM), Mg²⁺ (1 mM), Zn²⁺ (1 mM), Fe³⁺ (1 mM), Cu²⁺ (1 mM), NO₃⁻ (1 mM), CO₃²⁻ (1 mM), PO₄³⁻ (1 mM), Cl⁻ (1 mM), EDTA (1 mM), Glu (1 mM), Lys (1 mM), Arg (1 mM), Tyr (1 mM), Cys (1 mM), GSH (1 mM), Trp (1 mM), GLC (1 mM), Suc (10 mM), DS (1 mg/mL), TPS (10 U/mL), LZM (10 U/mL), CB (10 U/mL), BSA (0.1 mg/mL), BChE (2.0 U/mL) and AChE (2.0 U/mL).

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3.3 Response rate of BDFA to AChE

The BDFA-AChE reaction rate was studied by monitoring the dynamics of the fluorescence intensity over the reaction time. As displayed in Fig. 4a, BDFA emits extremely weak fluorescence, while the fluorescence gradually rises to a stable level within 20 min after adding AChE. Therefore, BDFA shows a fast response to AChE, which is suitable for rapidly detecting AChE activity.

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Fig. 4

(a) Time-dependent fluorescence intensity of BDFA (5 μM) after adding different concentrations (0, 0.4, 1.0, 1.6, 2.0 U/mL) of AChE. (b) Fluorescence intensity of BDFA (2 μM) in the absence and presence of AChE (2 U/mL) in PBS solutions of pH 3–11. (c) Changes in the fluorescence intensity of BDFA and BDFA-AChE reaction product with storage time. The fluorescence intensity was measured at 525 nm, with excitation at 470 nm.

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3.4 pH effect

The effect of pH on the response of BDFA to AChE was subsequently evaluated. As shown in Fig. 4b and Fig. S1, the fluorescence of BDFA maintains a relatively stable level in the range of pH 5–10, and shows a significant and consistent response to AChE under neutral and weakly alkaline conditions (pH 7–9). These results indicate that BDFA is suitable for detecting AChE in near-neutral biological matrices such as blood and cytoplasm.

3.5 Impact of test time window on results

As shown in Fig. 4c, within 3 h, BDFA emits weak and stable fluorescence, while the BDFA-AChE reaction product only presents a small decrease in emission intensity. These results reveal that these two compounds have good time-dependent stability, providing a long-time window for reading the test results.

3.6 Response mechanism of BDFA to AChE

To explore the internal reaction mechanism, the BDFA-AChE molecular docking was simulated on Autodock software. The results in Fig. 5a reveal that there are multiple hydrophobic interactions and hydrogen bonds in the BDFA-AChE docking model, and the binding energy was calculated to be −8.53 Kcal/mol, suggesting that BDFA and AChE are spontaneously combined. As shown in Fig. 5b, the fluorescence decay kinetics of BDFA slows down slightly after reacting with AChE, and its average fluorescence lifetime increases from 2.71 ns to 2.90 ns, indicating that a strong fluorescent compound may be produced.

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Fig. 5

(a) Molecular docking mode of BDFA and AChE. (b) Fluorescence decay kinetics of BDFA (5 μM) in the absence and presence of AChE (2.0 U/mL).

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To further clarify the reaction mechanism, we prepared and purified the BDFA-AChE reaction product (see section 2.2), and characterized its chemical structure by ¹H NMR (Fig. S6) and HRMS (Fig. S7). Moreover, we also performed a detailed analysis of the ¹H NMR spectra of BDFA and its reaction product with AChE (referred to as BDFAF) in Fig. 6a. It can be seen that the hydrogen atom signal (3.08 ppm, 3H; 3.28 ppm, 3H) attributed to the N, N-dimethyl carbamate group disappears in the NMR spectrum of BDFAP, confirming that the carbamate group is separated from the BDFA molecular under the catalysis of AChE. Fig. 6a also reveals an apparent redistribution of aromatic hydrogen atoms at 7.83–8.49 ppm, indicating that BDFAF has a different π-conjugated skeleton from BDFA. Concerning our previous and other studies (Cui et al., 2019; Zhang et al., 2021), we proposed the BDFA-AChE reaction mechanism, as described in Fig. 6b. First, the intramolecular ester bond of BDFA is hydrolyzed by AChE, which leads to the removal of the carbamate protecting group and produces an intermediate product (termed BDFAI). Subsequently, the phenolic hydroxyl group of BDFAI undergoes a nucleophilic reaction with its cyano group, causing an intramolecular cyclization reaction to generate the final product BDFAP with strong fluorescence emission. Our proposed mechanism for the BDFA-AChE reaction was also confirmed by the HPLC method (Fig. S3).

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Fig. 6

(a) Comparison of the ¹H NMR spectra of BDFA and BDFAP. (b) Proposed reaction pathways for BDFA and AChE.

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3.7 Determination of AChE activity in blood

Medical studies indicate that the AChE activity in serum can be used as a biomarker for detecting pesticide poisoning, assessing the severity of acute pancreatitis and predicting death (Xu et al., 2019; Badiou et al., 2008). Hence, we checked the feasibility of BDFA in detecting AChE activity in the serum matrix. As shown in Fig. 7, the fluorescence displays a significant increase after introducing the blood samples collected from three healthy individuals, demonstrating that the AChE in these samples maintains normal activity. As we all know, organophosphorus and carbamate pesticides severely inhibit the AChE activity due to the strong binding of pesticide molecules to the active sites of AChE (Bajgar, 2004). Therefore, we further monitored the changes in AChE activity of the blood samples in the presence of Dichlorvos and Carbaway (Guo et al., 2021). After adding the blood samples pre-incubated with the above two pesticides, the increase in fluorescence intensity is significantly reduced, indicating that the AChE activity in the blood samples is partially inhibited. These results confirm that BDFA is suitable for detecting AChE activity in blood matrix. In addition, we measured AChE activity in the blood samples using BDFA-based fluorometry and Ellman assay, a widely-used standard method for determining ChE activity. The detection results of the two methods are in good agreement (Table S2). The detected activities of AChE in the blood are also consistent with that in the literature (Xu et al., 2019).

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Fig. 7

(a) Fluorescence spectra and (b) emission intensity of BDFA (2 μM) upon the addition of PBS solution (pH 7.0, 10 mM), blood samples collected from three healthy individuals and these blood samples pre-treated with Dichlorvos (100 μg/mL) and Carbaway (100 μg/mL).

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