A naked-eye visible and fluorescence “turn-on” probe for acetyl-cholinesterase assay and thiols as well as imaging of living cells

Abstract

A resorufin derivative with a DBS group (probe 1) was designed and investigated for the detection of acetylcholinesterase (AChE) and inhibitor screening. The new assay is based on cascade enzymatic and chemical reactions of ATC, AChE and probe 1, and it can be carried out in a dual-signal detection mode. Moreover, the results show that probe 1 can be used for cell fluorescence staining.

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1. Introduction

Thiol-containing molecules such as cysteine (Cys) and glutathione (GSH) play important roles in the biological systems.¹ The thiol group in Cys residues are involved in determining three dimensional structures of proteins.² GSH, a physiologically important tripeptide consisting of glutamate, cysteine and glycine, performs vital biological functions, which includes protection of the cells from oxidative stress by trapping free radicals that damage DNA and RNA.³ It is known that abnormal levels of these thiol-containing molecules can cause a number of diseases such as cancer, Alzheimer's and cardiovascular diseases.⁴ Thus, development of sensitive and selective sensors for thiols is of significant interest.⁵

Besides Cys and GSH, choline is another important thiol-containing molecule of biological interest. Choline can be generated by hydrolysis of acetylcholine (ACh) catalyzed by acetylcholinesterase (AChE). Many studies suggest that a low level of ACh is relevant to Alzheimer's disease (AD), the most common form of dementia among older people. In AD patients levels of acetylcholine (ACh) can drop by up to 90 percent. The gradual death of cholinergic brain cells results in progressive and significant loss of cognitive and behavioral function. A deficit in ACh is only one of the many factors contributing to this devastating brain disorder.^6,7 Although there is no cure for Alzheimer's, drugs currently available on the market can help to ease some of the symptoms. These drugs work to inhibit acetylcholinesterase (AChE), the enzyme which inactivates ACh at the synapse. Inhibiting this enzyme prevents the normal breakdown of ACh, which is a way of compensating for the lowered concentrations of ACh. Therefore, efficient and convenient methods for detecting AChE and corresponding inhibitor screening will be helpful to aid diagnosis and find a cure for AD.^8,9

Although many colorimetric and fluorescent AChE assay methods have been reported, there are still some drawbacks. For colorimetric methods, low sensitivity is combined with tedious signal readout.¹⁰ Compared with the other assay methods, fluorometric methods show stable positive results and good sensitivity. Many fluorescent and electrochemical assay methods have been developed to detect AChE.¹¹ We have recently developed two fluorescent assay ensembles for sensing AChE and AChE inhibitor-screening with good sensitivity based on the aggregation-induced emission feature of tetraphenylethene.¹² These two assay ensembles are convenient and continuous fluorometric methods for AChE and inhibitors screening. However, the signal readout still needs the aid of the bulk instruments. We also report the assay for AChE based on gold nanoparticles with naked-eye detection.¹³ For decades during the development of the various sensor systems convenient and simple detection methods have been pursued eagerly by scientists.

In this paper, we describe a colorimetric and fluorometric AChE assay system which is visible to the naked-eye, and this method can be used as AChE inhibitor-screening system. Probe 1 was designed based on resorufin as the chromophore and DBS (2,4-dinitrobenzenesulfonyl) group as the cleavable group through a thiolysis reaction. Resorufin is selected as the chromphore based on its excellent chromogenic and fluorogenic signal behaviors. Resorufin shows strong fluorescence at 585 nm, is visible to the naked-eye as a purple color, has good water-solubility, and can be applied in biological analysis with easy signal readout and nontoxic properties. DBS group in fluorophore probes can be deprotected by reaction with thiols leading to fluorescent changes.¹⁴ When DBS is connected with the 7-OH of resorufin, the fluorescence of resorufin is quenched.¹⁵ The design rationale for detecting AChE is illustrated in Scheme 1. The assay system contains probe 1 and ATC (acetylthiocholine iodide). ATC is a good substrate of AChE, and can be hydrolyzed to thiocholine (compound 2). Thiocholine reacts with probe 1 to cleave the DBS group, and thus releases resofurin. As a result, the color and fluorescent intensity of the mixture solution are changed accordingly. When the corresponding inhibitors for AChE are present, the hydrolyzation of ATC is restrained and the increment of fluorescent intensity is small.

Scheme 1 Illustration of the mechanism of this new assay for AChE.

Moreover, probe 1 (Scheme 1) can be thiolyzed to remove the strong electro-withdrawing group, 2,4-dinitrobenzenesulfonyl (DBS), resulting in fluorescence enhancement. Accordingly, probe 1 may be applied for the detection of thiols and imaging cells by the reaction with the thiols in living cells. The results demonstrate that the probe 1 is an efficient bright and nontoxic cell-staining reagent.

2. Experimental section

Materials and techniques

Resorufin, acetylthiocholine iodide (ATC) and acetylcholinesterase (AChE) (from Electrophorus electricus) were obtained from Sigma-Aldrich. 2,4-Dinitrobenzenesulfonyl chloride and carbofuran (CAS No. 1563-66-2) were purchased from Alfa Aesar and Dr Ehrenstorfer GmbH (Augsburg, Germany), respectively. Anhydrous THF was distilled from sodium/benzophenone, and anhydrous Et₃N was pre-dried with CaH₂, followed by distillation. Pure water was obtained with a Millipore filtration system. All other reagents, compounds, and chemicals were obtained from commercial suppliers and used without further purification. Fluorescence and absorption spectra were measured on a Hitachi F-4500 and JASCO V-570 at 37 °C, respectively.

Synthesis of probe 1

A suspension of resorufin (0.21 g, 1.2 mmol) in anhydrous THF was treated with Et₃N (0.17 mL, 1.2 mmol) at 0 °C and stirred for 30 min at the same temperature under N₂ atmosphere. To this solution, 2,4-dinitrobenzenesulfonyl chloride (0.32 g, 1.2 mmol) in 10 mL of anhydrous THF was added. The resulting mixture was stirred overnight, diluted with ethyl acetate (100 mL), washed with brine (50 mL × 3), and dried over MgSO₄. The solvent was removed by evaporation, and the residue was subjected to silica gel chromatography eluted with ethyl acetate and petroleum ether (v/v, 1 : 2) to afford probe 1 (0.12 g, 27%) as a yellow solid. ¹H NMR(400 MHz, d₆-DMSO, TMS): 9.13 (s, 1 H), 8.63 (d, 1 H, J = 8.0 Hz), 8.34 (d, 1 H, J = 8.0 Hz), 7.90 (d, 1 H, J = 8.0 Hz), 7.55 (d, 1 H, J = 8.0 Hz), 7.47 (s, 1H), 7.25 (d, 1 H, J = 8.0 Hz), 6.87 (d, 1 H, J = 20.0 Hz), 6.29 (s, 1 H); ¹³C NMR(100 MHz, d₆-DMSO): 185.7, 151.6, 149.8, 149.4, 149.1, 148.1, 144.2, 135.1, 133.6, 132.4, 131.6, 130.6, 127.7, 121.3, 119.2, 110.4, 106.3; HRMS(EI): calcd. for C₁₈H₉N₃O₉S: 443.0060; found: 443.0064

Process of cell staining

For cell culture, Hela cells in medium DMEM (Dulbecco's Modified Eagle Media, Gibco) with 10% v/v FBS (fetal bovine serum, Gibco), and penicillin/streptomycin (100 U mL⁻¹, Hyclone). The cells were maintained on 60 mm cell culture dishes (Falcon) under 5% CO₂, at 37 °C. For cell staining, cells were washed with PBS (phosphate-buffered saline), followed by the addition of probe 1 or C34551 (a commercial dye for cell stain, Invitrogen) at certain concentrations, respectively. The mixtures were incubated for 30 min.

For NEM (N-ethylmaleimide) staining, cells were first washed with PBS, NEM at certain concentrations was added. Then, the cells were incubated for 60 min in the incubator. After that, the cells were washed with PBS 3 times, and then stained with probe 1 according to above procedures.

The images were acquired with a Leica inverted microscope (DMI 6000) equipped with a digital monochrome camera (Leica DFC 350 FX) and the software provided by Leica. Phase contrast and fluorescence micrographs were obtained with a 20 × objective.

3. Results and discussion

3.1 Naked-eye visible and fluorescent assay for AChE with probe 1

Probe 1 was synthesized by the reaction of resorufin with 2,4-dinitrobenzenesulfonyl chloride in the presence of Et₃N. Resorufin itself is pink and highly emissive with emission maximum at 585 nm. Because of the electron accepting property of the DBS unit, the aqueous solution of probe 1 is colorless and weakly fluorescent. A solution of probe 1 (10 μM) and ATC (10 μM) in mixture of PBS (10 mM, pH = 8.0) and DMSO (v/v, 200/1) was prepared as the assay system for the following experiments. Fig. 1A shows the absorption spectra of the assay system in the absence and presence of AChE. For comparison, the absorption spectrum of resorufin is included in Fig. 1A. Compared to resorufin, probe 1 exhibits very weak absorption around 550 nm. However, a strong absorption around 550 nm emerged after the assay system of probe 1 and ATC was incubated with AChE (0.01 U/mL) for 30 min. Simultaneously, the color of the assay system changed from colorless to pink as shown in the inset of Fig. 1A. Such color change is easily visible to the naked-eye.

Fig. 1 (A) Absorption spectra of probe 1 [10 μM in PBS (10 mM) buffer solution, 0.5% DMSO, pH = 8.0] before reaction (green line), after reaction for 30 min (red line) in the presence of AChE (0.01 U/mL), and that of resorufin (black line); (B) Fluorescence spectra of probe 1 [10 μM in PBS (10 mM) buffer solution, 0.5% DMSO, pH = 8.0] containing ATC (10 μM) in the presence of AChE (0.01 U/mL) after incubation at 37 °C for different times (λ_ex. = 550 nm).

The fluorescent spectra of the assay system were also measured in the absence and presence of AChE. The assay system containing probe 1 and ATC showed weak fluorescence around 585 nm before the addition of AChE. However, after the addition of AChE (0.01 U/mL) to the assay system and further incubation at 37 °C for a certain time, the fluorescence intensity of the solution is increased gradually by prolonging the incubation time as demonstrated in Fig. 1B. For instance, the fluorescence intensity at 585 nm was enhanced by seven times compared to that of the assay system without AChE, and the solution emitted red fluorescence which could be easily detected by the naked-eye under UV light (365 nm) irradiation (see inset Fig. 1B). Such fluorescence enhancement for the assay system after the addition of AChE is owed to the release of the DBS unit in probe 1 and generation of free resorufin as discussed above. In fact, a mass signal corresponding to the molecular weight of resorufin (MW = 213) was detected for the assay system after incubation with AChE for 30 min (see Fig. S1†).

The fluorescence spectra of the assay system in the presence of different amounts of AChE were recorded after incubation for different times. Fig. 2 depicts the variation of the fluorescence intensity at 585 nm vs. the incubation time for the assay system containing different amounts of AChE. It is obvious that the fluorescence intensity increased notably and sharply when a high concentration of AChE was used. For instance, the fluorescence intensity of the assay system reached maximum after incubation for ca. 5.0 min if 1.0 U/mL of AChE was present in the solution. But, if 0.005 U/mL of AChE was used the fluorescence intensity increased slowly. These results are understandable since the more AChE in the assay system the more ATC available to hydrolyze to produce thiocholine that reacts with probe 1 to generate more resorufin in the solution. Nevertheless, even when the concentration of AChE in the solution was as low as 0.001 U/mL, fluorescence enhancement could still be detected after the solution was incubated for ca. 30 min (see Fig. 2). Therefore, the assay system of probe 1 and ATC can be employed for AChE analysis.

Fig. 2 A plot of fluorescence intensity at 585 nm vs. the reaction time for the probe 1 [10 μM in PBS (10 mM) buffer solution, 0.5% DMSO, pH = 8.0] and acetylthiocholine (10 μM) in the presence of different amounts of AChE (0, 0.001 U/mL, 0.005 U/mL, 0.01 U/mL, 0.05 U/mL, 1.0 U/mL) (λ_ex. = 550 nm).

The fluorescence spectra of probe 1 in the presence of ATC and AChE were also recorded in aqueous solutions of different pH values. Fig. 3 shows the variation of the fluorescence intensity at 585 nm of the ensemble (after incubation for 30 min) vs. the pH values of the solution. Obvious fluorescence enhancement was detected for probe 1 when the pH value of the solution was higher than 7.0. For comparison, the fluorescence spectrum of probe 1 was also measured in the absence of ATC and AChE similarly. As depicted in Fig. 3 the fluorescence intensity of the solution containing only probe 1 was slightly varied if the pH value of the solution was in the range of 6.0–10.0; but, it started to increase when the pH value of the solution was higher than 10.0. This may be due to the removal of DBS group from probe 1 in strongly basic solution. Therefore, the AChE assay with probe 1 can be performed in aqueous solution of pH value in the range of 7.0–10.0.

Fig. 3 The variation of fluorescence intensity at 585 nm for probe 1 [10 μM in PBS (10 mM) buffer solution, 0.5% DMSO] at different pH values after incubating with acetylthiocholine (10 μM) and AChE (0.01 U/mL) for 30 min (λ_ex. = 550 nm).

The assay system can also be employed to screen inhibitors of AChE. Carbofuran, a carbamate, can inhibit the activity of AChE, and is selected as an example to illustrate that this assay system can be used for screening inhibitors of AChE. The fluorescent spectra of the assay system containing AChE (0.01 U/mL) in the presence of different amounts of carbofuran were measured separately after each solution was incubated for different times. Fig. 4A depicts the variation of the fluorescence intensity at 585 nm vs. incubation times in the presence of different amounts of carbofuran. Clearly, the degree of fluorescence enhancement decreases after the addition of carbofuran. Moreover, the more amount of carbofuran in the assay system, the smaller degree of fluorescence enhancement as shown in Fig. 4A. This is in agreement with the fact that carbofuran can inhibit AChE activity. Additionally, the IC₅₀ value of carbofuran toward AChE was estimated to be 57.4 nM based on the plot of inhibition efficiency vs. the concentration of carbofuran as shown in Fig. 4B.

Fig. 4 (A) Variation of fluorescence intensity at 585 nm (λ_ex. = 550 nm) vs. incubation time; (B) plot of the inhibition efficiency of carbofuran toward AChE vs. Log[carbofuran]; the measurements were performed with probe 1 [10 μM in PBS (10 mM) buffer solution, pH = 8.0], AChE (0.01 U/mL), ATC (10 μM) and different amounts of carbofuran (1.0 nM, 10 nM, 100 nM and 1000 nM).

3.2 Fluorescence variation of probe 1 after incubation with Cys and GSH

As anticipated the DBS group in probe 1 can also be removed by reaction with either Cys or GSH, leading to fluorescence enhancement. Fig. S2† and Fig. S3† shows the fluorescent spectra of probe 1 after incubation with Cys and GSH, respectively. Clearly, fluorescence enhancement was detected for probe 1 after incubation with either cysteine or GSH (glutathione reduced form). But, the degree of fluorescence enhancement for probe 1 in the presence of cysteine/GSH is low, compared to that for probe 1 after incubation with ATC and AChE under the same conditions (see Fig. 5). Moreover, the interference of thiol-containing compounds such as cysteine and GSH can be eliminated by the following ways: (1) the sample under investigation was mixed with probe 1 and the solution was incubated at 37 °C for a certain time. Then, the fluorescence intensity was measured; (2) the same amount of sample was mixed with probe 1, ATC and AChE and incubated similarly. The fluorescence intensity was measured accordingly; (3) the difference in fluorescence intensity between the above two cases can be used for the AChE activity assay.

Fig. 5 Fluorescence intensity at 585 nm for the probe 1 [10 μM in PBS (10 mM) buffer solution, pH = 8.0] in the presence of Cys (10 μM), GSH (10 μM), ATC [10 μM, containing AChE (0.01 U/mL)] after incubation for 30 min (λ_ex. = 550 nm).

3.3 Fluorescence imaging of living cells with probe 1

Based on the fact that both cysteine and GSH can react with probe 1 to release free resorufin leading to fluorescence enhancement, probe 1 can be applied for fluorescence imaging of living cells through the reaction of thiol compounds including cysteine and GSH in cells with probe 1. Hela cells were selected to demonstrate the usefulness of probe 1 for cell staining. For the staining experiments, Hela cells were firstly incubated in medium on cell culture dishes under CO₂ at 37 °C. Then, probe 1(5.0 μg mL⁻¹ and 10 μg mL⁻¹) was incubated with the cells for 30 min. As shown in Fig. 6A, red fluorescence images were observed after incubation with probe 1; brighter fluorescence images were generated by increasing the concentration of probe 1 (from 5.0 μg mL⁻¹ to 10 μg mL⁻¹). For comparison, the cells were also stained with C34551 (10 μg mL⁻¹) under the same conditions; however, no fluorescence images were detected (see Fig. 6A). Additionally, if the cells were firstly incubated with N-ethylmaleimide (NEM) that is an efficient thiolblocking reagent, almost no fluorescence images were observed after further incubation with probe 1 (see Fig. 6B). This indicates that the cell staining with probe 1 is due to the reaction between probe 1 and thiol-containing compounds in cells. These results demonstrate that probe 1 can penetrate cell membranes and image the changes in thiol levels of living cells.

Fig. 6 (A) Bright field (a, b and c) and fluorescence (d, e and f) images of Hela cells stained by C34551 and probe 1 (5.0 μg mL⁻¹ and 10 μg mL⁻¹): (a and d) the images of cells with C34551 (10 μg mL⁻¹); (b and e) images of cells with probe 1 (5.0 μg mL⁻¹); (c and f) images of cells with probe 1 (10 μg mL⁻¹); (B) bright field (g and h) and fluorescence (i and j) images of Hela cells stained by probe 1 in the absence and presence of NEM (20 μg mL⁻¹): (g and i) the images of cells with probe 1 (5.0 μg mL⁻¹) only; (h and j) the images of cells with NEM (20 μg mL⁻¹), then with probe 1 in PBS buffer, respectively.

4. Conclusions

In summary, based on the cascade enzymatic and chemical reactions of ATC, AChE and probe 1, a new assay system for the detection of AChE and the inhibitor screening was successfully developed. AChE with concentration as low as 0.001 U/mL can be analyzed. Apart from the fluorescence readout signal, the assay system also shows colorimetric change which can be detected by the naked-eye. Thus, this new assay for AChE can be carried out in dual-signal detection mode. Moreover, the results also show that probe 1 can be used for cell fluorescence staining. Considering that probe 1 can be synthesized easily and ATC is commercially available, this new assay system is potentially useful for screening inhibitors of AChE and relevant drug discovery.

Acknowledgements

The present research was financially supported by NSFC, Chinese Academy of Sciences, and State Key Basic Research Program. This work was partially supported by the NSFC-DFG joint project (TRR61).

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Footnote

† Electronic supplementary information (ESI) available: Fluorescence spectra of probe 1 and cell staining. See DOI: 10.1039/c0an00456a

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