HEK293T cell lines were purchased from Cell resource center, IBMS, and cultured at 37 °C, 5% CO₂, and 95% air in DMEM (Dulbecco’s Modified Eagle Medium containing 4.5 g glucose/mL; 11965092, Gibco) supplemented with 1% Penicillin/Streptomycin (CC004, MACGENE) and 10% Fetal Bovine Serum (FBS; 10099141C, Gibco). This culture medium was used for all experiments.

The following reagents were purchased from commercial sources: puromycin (R23002, YUANYE Inc), G418 disulfate salt (A1720, Sigma), Lipofectamine™ 3000 transfection kit (L3000015, Invitrogen), Opti-MEM™ (31985070, Gibco), calcium phosphate transfection kit (CTK001, MACGENE), PrimeSTAR® HS DNA Polymerase (R010A, Takara), NEBuilder HiFi DNA Assembly Master Mix (E2621L, NEB), Acetylcholine chloride (249495, J&K), Neostigmine Bromide (N838484, Macklin), Physostigmine (P922786, Macklin), dimethyl sulfoxide (DMSO, D2650, Sigma), aspirin (A2093, Sigma), serotonin (S31021, YUANYE), dimethoate (D109819, Aladdin), chlorpyrifos (C109843, Aladdin), Donepezil Hydrochloride (D849374, Macklin).

Antibodies were as follows: Goat anti-Rabbit IgG (H + L) Cross-Adsorbed Secondary Antibody (A11012, Invitrogen), Rabbit anti-Flag Antibody (14793T, Sigma), polyclonal rabbit anti-Flag (F1804, Sigma), Anti-rabbit IgG, HRP-linked Antibody (7074, CST), Cy3-conjugated goat anti-mouse (A0521, Beyotime).

The expression cassette of ACh3.0-IRES-mcherry-CAAX was amplified from the pDisplay-Ach3.0 plasmids from Li Lab (Jing et al., 2020) and inserted into the pCDH-CMV-MCS-EF1-puro lentiviral vector between the XbaI and EcoRI restriction sites, to generate the pCDH-ACh-puro plasmid. The AChE gene (Genbank: NM_000665) with 3×Flag Tag in the 3′ terminal was amplified from a cloning vector purchased from Youbio (G152420) and inserted into GV350 lentiviral vector (GeneChem) to generate the Ubc-ACHE-3Flag-SV40-Neomycin plasmid. All the constructs were confirmed by DNA sequencing. Lentivirus was obtained following the standard three-plasmid packaging protocol.

Total RNA was isolated from cultured cells using the RNeasy Kit (QIAGEN). cDNA was synthesized by reverse transcription conducted with the PrimeScript™ RT Kit (Takara Bio) under programmed thermal cycling: 37 °C (15 min), 85 °C (5 s), and 4 °C. Gene expression was quantified on a Bio-Rad CFX-96 thermocycler using SYBR Green PCR-Mix (Takara Bio). The ddCt method was used to calculate the relative change of gene expressions. Quantitation of target gene was normalized to GAPDH.

The primer sequences for qRT-PCR analysis are as followed:

CAAX-qF: GGT​ACT​GCT​GCT​CTG​GGT​TC.

CAAX-qR: GCT​GCT​CGA​GAA​ACA​TTG​TAG​C.

GAPDH-qF: CGT​CAT​GGG​TGT​GAA​CCA​TG.

GAPDH-qR: GGA​CTG​TGG​TCA​TGA​GTC​CT.

For protein extraction, cultured HEK293T cells infected with indicated lentivirus were collected and lysed in RIPA buffer containing 20 mM Tris–HCI pH 7.5, 150 mM NaCl, 10 mM EDTA, 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride (PMSF), and complete protease inhibitor. Cell lysates were sonicated and centrifuged at 12000 rpm. The protein concentration of each sample was determined with a BCA assay kit, following the manufacturer’s instructions. Equal amounts of protein were separated by SDS-PAGE on 8%–12% polyacrylamide gels and subsequently transferred onto PVDF membranes. The membranes were blocked with 5% non-fat milk in Tris-buffered saline containing 0.1% Tween-20 (TBST) for 1 h at room temperature, followed by incubation with specific primary antibodies overnight at 4 °C. After washing three times with TBST, the membranes were incubated with appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 h at room temperature. Protein bands were visualized using an enhanced chemiluminescence (ECL) detection system and imaged with a chemiluminescence imaging system (Gel DocTM XR + imaging system, Bio-Rad).

The HEK293T cell line that stably expresses GACh3.0 and AChE (human), were plated in a 24-well tissue culture plate at a density of 5 × 10⁴ cells/well and incubated overnight. Cells were fixed in 4% paraformaldehyde for 20 min at room temperature, washed with PBS and then blocked with blocking buffer for 30 min at room temperature, followed by incubation with primary antibody anti-Flag antibody (1:500, polyclonal rabbit anti-Flag, Sigma, F1804) overnight at 4 °C. Next day, wash the primary antibody with PBS, cells were incubation with secondary antibody labeled with Cy3-conjugated goat anti-mouse (1:500, Beyotime, A0521) for 1 h at room temperature in the dark. After thorough washing with PBS, 2 drops per well nuclear dye (Hoechst 33342, 1:2,000, Invitrogen) for 1 h at room temperature in the dark, adding liquid antifade and images were obtained with the confocal microscope (Nikon AX), the acquisition software was NIS-Elements V5.24.01.

The detection of the fluorescence changes of GACh3.0 was performed as previously described (Jing et al., 2020). Briefly, cultured HEK293T cells expressing the GACh3.0 sensor were first imaged using the Opera Phenix High-Content Screening System (PerkinElmer) equipped with a ×20/1.15-NA water-immersion objective, a 488-nm laser and a 561-nm laser; the GACh3.0 sensor’s signal was obtained using a 525/50-nm emission filter, and the mCherry–CAAX signal was obtained using a 600/30-nm emission filter. The ratio between green (G) and red (R) fluorescence was calculated before and after application of 100 μM ACh, and the change in the G/R ratio was used as the fluorescence response.

HEK293T cells stably expressing GACh3.0 and AChE (human) were plated in a 96-well tissue culture plate at a density of 1 × 10⁴ cells/well and incubated overnight. Test compounds were added to the cell culture medium at a 1:100 ratio to achieve a final concentration of 10 μM, followed by incubation at 37 °C for 5 min. Subsequently, 100 μM acetylcholine was added to each well, and incubation continued at 37 °C for 2 min. The 96-well plate was then placed into the Opera Phenix High-Content Screening System (PerkinElmer) to detect fluorescence intensity in different samples, following the method described in Section 2.7. If a compound effectively inhibits AChE activity, a significant increase in fluorescence intensity will be detected in that sample group.

Statistical analyses were performed with a two-tailed unpaired t-test or as indicated in the legends. P values are indicated by asterisks in the figures as followed: *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001. Differences with a P value of 0.05 or less were considered significant. Data visualization was conducted using GraphPad Prism 10.

This study aimed to design a novel, low-cost, high-throughput method for screening AChE activity by leveraging the specific recognition of acetylcholine by a genetically encoded biosensor. First, a stable cell line co-expressing the fluorescent probe and AChE was constructed. During drug screening, the cells are first co-incubated with the test compound, followed by the addition of a specific concentration of ACh. If the compound inhibits AChE activity, the ACh present in the system will induce a significant upregulation of the fluorescent probe signal. Conversely, if the test compound lacks inhibitory activity, the ACh will be rapidly hydrolyzed by the cell-expressed AChE, resulting in no significant change in the detected fluorescence signal. A schematic diagram of this principle is shown in Figure 1.

The GRAB probe is a genetically encoded fluorescent sensor that can be efficiently expressed on the cell membrane. In this study, the acetylcholine probe GACh3.0, developed by Li Lab (Jing et al., 2020), was cloned into a lentiviral vector to produce GACh3.0 lentivirus. GACh3.0 primarily consists of two modules: the muscarinic receptor M3R (a natural receptor for ACh sensing) with an inserted cpGFP, and the red fluorescent protein mCherry carrying a CAAX membrane localization sequence. mCherry serves as an internal reference for fluorescence intensity to accurately measure changes in green fluorescence. These modules were integrated into a lentiviral transfer plasmid (Figure 2). The transfer plasmid and helper plasmids were transfected into HEK293T cells using the calcium phosphate method, and virus was collected after 48 h. The titer of the concentrated virus reached 2 × 10⁸ TU/mL.

Subsequently, HEK293T cells were infected with the virus at an MOI of 5, and stable HEK293T cell lines expressing GACh3.0 (GACh-Lenti) were obtained via puromycin selection. Expression of the target gene post-lentiviral infection was assessed by quantitative PCR (qPCR) and confocal microscopy. qPCR results showed that the relative quantification (RQ) value of the target gene in lentivirus-infected 293T cells reached 7420.25 compared to wild-type 293T cells, indicating high-efficiency probe expression (Figure 3A). Confocal microscopy results (Figure 3B) revealed significant expression of both green and red fluorescent proteins 48 h post-infection. Both GACh3.0 and mCherry-CAAX exhibited proper membrane localization in 293T cells (Figure 3C). These results demonstrate that the constructed stable cell line achieves efficient and active expression of GACh3.0.

To achieve stable AChE expression in the aforementioned cells, we then constructed another lentiviral plasmid for AChE (Figure 4A), and produced lentiviral particles with a concentrated viral titer 1.4 × 10⁹ TU/mL. The GACh-Lenti cells stably expressing GACh3.0 were infected with the prepared lentivirus to achieve co-expression of AChE and GACh3.0. Polyclonal stable cell lines were selected using puromycin and G418, yielding AARC cells (Acetylcholinesterase Activity Reporter Cells). The expression of AChE in AARC cells was verified by Western blotting (Figure 4B) and Immunofluorescence (Figure 4C).

Furthermore, to verify whether the AChE expressed in the cell line possessed normal enzymatic activity, AARC cells were pre-treated with 10 μM physostigmine (acetylcholinesterase inhibitor) or DMSO solvent for 5 min. Then 100 μM acetylcholine was added to the culture medium. It was observed that acetylcholine in the cells without physostigmine was rapidly hydrolyzed, resulting in a fluorescence intensity significantly lower than that of the DMSO control group (Figure 4D). This result indicates that the AChE expressed in the cells exhibits robust hydrolytic activity, demonstrating that the functionality of the co-expressing cell line aligns with our design expectations.

After achieving stable activity expression of GACh3.0 and AChE in AARC cells, we designed an acetylcholinesterase inhibitor screening platform based on AARC cells, as shown in Figure 5A. First, the test compounds were co-incubated with the cells for 5 min to ensure sufficient binding between the compounds and the targets. Then acetylcholine was added to detect the activation of GACh3.0. Due to the rapid hydrolysis of acetylcholine catalyzed by acetylcholinesterase, a significant change in fluorescence signal intensity could be observed shortly after the addition of acetylcholine.

We next tested and optimized both the concentration of acetylcholine added and the duration of the signal. Considering the high hydrolytic activity of AChE towards ACh, the ACh concentration needed to be set at an appropriate level. If too low, ACh would be easily hydrolyzed, making signal detection difficult; if too high, it could lead to false positives. Given that the EC₅₀ of the GACh3.0 probe’s response to ACh is at the micromolar level (Pundir et al., 2019), (Jing et al., 2020), we tested six concentration gradients (10 μM, 50 μM, 100 μM, 200 μM, 400 μM, and 800 μM), using serotonin (inactive on GACh3.0) as a control, to assess the cellular fluorescence response. To simulate chemical toxin inhibition of AChE, 100 μM neostigmine (an AChE inhibitor) was pre-added to the cell culture medium. The results (Figure 5B) showed that 50 μM ACh was sufficient to achieve a highly significant difference (P < 0.001). Considering a wider Screening window is needed to meet the requirements for screening compounds with varying levels of AChE inhibitory activity, 100 μM ACh was selected for standard protocol. Regarding signal duration, we monitored the fluorescence signal intensity continuously for 7 min after acetylcholine addition and observed no significant signal decay (Figure 5C). This timeframe is sufficient to complete the scanning of an entire 96-well plate.

The final optimized system requires less than 10 minutes to complete a batch detection (using 96-well or 384-well plates), with an average detection cost of 0.86 RMB per sample (see the cost breakdown in Supplementary Table S1). Compared to an existing commercial AChE activity assay kits (e.g., Sigma, MAK119), this represents a cost reduction of approximately two orders of magnitude.

To further validate the sensitivity of the screening system, we tested two AChE inhibitors, neostigmine bromide (100 μM) and physostigmine (10 μM). Aspirin was set as the negative control which has no inhibitory activity on AChE. Compared to the inactive aspirin and solvent control groups, both inhibitor compounds showed significant differences on the fluorescence signal (Figure 5E). In terms of sensitivity, the results indicated an EC₅₀ value for physostigmine of approximately 8.8 μM, which generally meets the requirements for compound high-throughput screening (Figure 5D).

Next the inhibitory activities of two pesticides, dimethoate and chlorpyrifos, and an approved drug for Althamer’s, donepezil hydrochloride, were evaluated by the screening method. AARC cells were pretreated these three chemicals (20 μM) for 5 min, followed by the addition of ACh with centration of 100 μM. Since these compounds are water-soluble, we employed Phosphate buffer saline (PBS) as the solvent and vehicle control. The results showed that all these chemicals induced significant fluorescence signal increase (Figure 5F), indicating that our screening methods is suitable for the test of different kinds of compounds.