Revealing the different effects induced by antipsychotic drugs using an electrochemical microsensor to measure exocytosis in living cells

Wenxue Chen^ab, Xuefeng Wang*^bc, Yuan Zhang^d, Zihui Li^be, Haoliang Li^ab, Qiongya Wan^be, Xinxin Li^bc, Dan Zheng*^a and Pengcheng Xu^bc
aSchool of Chemical and Environmental Engineering, Shanghai Institute of Technology, Shanghai 201418, China. E-mail: zhengdan@sit.edu.cn
^bState Key Laboratory of Transducer Technology, Shanghai Institute of Microsystem and Information Technology, Chinese Academy of Sciences, Shanghai 200050, China. E-mail: wangxuefeng@mail.sim.ac.cn
^cUniversity of Chinese Academy of Sciences, Beijing 100049, China
^dMaterials Genome Institute, Shanghai University, Shanghai 200444, China
^eCollege of Food Science and Technology, Laboratory of Quality & Safety Risk Assessment for Aquatic Products on Storage and Preservation (Shanghai), Ministry of Agriculture and Shanghai Engineering Research Center of Aquatic Product Processing & Preservation, Shanghai Ocean University, Shanghai 201306, China

First published on 5th August 2025

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Introduction

Researching the mechanisms of drug action is fundamental to pharmacology and drug development. By examining the biological effects of drugs, we can gain insights into how they influence physiological and pathological processes. This understanding helps ensure that drugs are specific and effective, minimize side effects, and provide a theoretical framework for clinical applications. An in-depth exploration of drug mechanisms can lead to the development of safer and more effective medicines, ultimately enhancing human health. For instance, the dopamine (DA) hypothesis of schizophrenia suggests that excessive production and transmission of DA may be a key factor in the disorder.^1–5 Antipsychotic drugs (APs) primarily treat schizophrenia by modulating the dopaminergic pathway. In treating schizophrenia, the secretion and signalling of DA can be adjusted by antipsychotic medications, which help alleviate symptoms associated with the condition. This process involves modifying DA release and inhibiting receptor activity.^6,7 Consequently, studying how APs modulate DA release is essential for thoroughly understanding their mechanisms of action.

The primary method for researching drug mechanisms is the receptor–ligand binding assay.^8–10 This assay evaluates how effectively a drug binds to its receptor through in vitro experiments and is essential for drug design and screening.¹¹ Most atypical APs have been validated using these assays, particularly their effects on DA receptors.^12–15 However, the effect of APs on cellular DA secretion has been largely overlooked, and further investigation is needed for a better understanding of their role in schizophrenia. In addition to receptor–ligand binding assays, several other methods, such as chromatography, fluorescence, and electrochemistry, have been employed to study the effects of APs on DA release.¹⁶ High-Performance Liquid Chromatography (HPLC) can measure extracellular DA levels but requires additional reagents to prevent DA oxidation, which complicates the procedure and limits real-time monitoring of living cells.¹⁷ Total Internal Reflection Fluorescence Microscopy (TIRFM) enables tracking of vesicle release sites, but it cannot provide information for quantitative analysis.^18,19 Traditional electrochemical electrodes can measure DA levels in solutions but cannot assess cell exocytosis.^20–22 Carbon fiber electrodes (CFEs) coupled with electrochemical amperometry are commonly used to study this process by oxidizing specific neurotransmitters at a fixed potential and recording the resulting transient current as an amperometric spike.^18,23–25 The CFE method, however, requires expensive equipment and a shielded environment to capture currents at picoampere levels.^26,27 It also demands precise control of the distance between the CFE and the cell membrane, which helps reduce the impact of DA diffusion. Additionally, monitoring exocytosis is only possible if the CFE is near an active site on the cell membrane, causing inefficiencies in data collection.²⁸ Similar to CFEs, various studies have reported the use of microelectrode arrays (MEA) to monitor the exocytosis of living cells, including primary cultured neurons.^29–32 However, the fabrication of MEAs involves advanced micro- and nanofabrication techniques, which pose significant challenges for their practical application. Therefore, developing new monitoring methods with easier operation and simpler fabrication is crucial for precisely measuring both DA release quantity and exocytosis frequency at single-cell resolution. Such advances would significantly enhance our understanding of APs’ mechanisms.

In previous work, we developed an electrochemical sensing chip based on a mixed-dimensional MNG-1 nanostructure, comprising vertically-aligned MOF-coated ZnO nanowires on reduced graphene oxide (rGO), for real-time monitoring of exocytotic DA release and used for assessing the neurological side effects of anticoronavirus, antiflu, and anti-inflammatory drugs.³³ In this work, a single-cell printing system was used to obtain statistically significant data more efficiently. The cells can adhere to MNG-1 sensing material naturally, allowing for the direct detection of individual exocytotic events from single cells without the need for any adjustments, simplifying the operational process. The MNG-1 sensor can capture single exocytotic events in single and multiple cells, and enables tests to be completed in minutes with a simple and low-cost experimental setup. We examined the effects of three APs, haloperidol, sulpiride, and chlorpromazine, on DA secretion from rat adrenal pheochromocytoma (PC12) cells. The effect of haloperidol concentration (ranging from 1 ng mL⁻¹ to 10 ng mL⁻¹) on cell exocytosis was also analyzed using statistically significant data. Furthermore, ELISA validation has confirmed the reliability of both the sensor platform and analytical approach, establishing a comprehensive strategy for drug evaluation. Our results revealed the distinct effects of these APs on DA release, in addition to their known receptor antagonist effects, providing new insights into their mechanisms of action.

Experimental section

Reagents

Zinc acetate dihydrate, zinc nitrate hexahydrate, hexamethylene tetramine, 2-methylimidazole, and Nafion™ perfluorinated resin solution were purchased from Sigma–Aldrich. Haloperidol, sulpiride, and chlorpromazine were purchased from Macklin Inc. Graphene Oxide dispersion was purchased from XFNANO Materials Tech. RPMI-1640 medium and fetal bovine serum (FBS) were sourced from Gibco, heat-inactivated horse serum (HS) and penicillin–streptomycin–glutamine (100×) were purchased from Sangon Biotech, and DA ELISA kit was purchased from CUSABIO.

Preparation of MNG-1

The MNG-1 material was used to efficiently detect exocytosis in living cells and was synthesized as follows: 25 μL of graphene oxide dispersion was added dropwise to a single side polished, plasma-cleaned silicon substrate, then the silicon substrate was dried at 150 °C, repeated four times. Next, 25 μL of 5 mM zinc acetate solution was added dropwise to the silicon substrate, dried at 150 °C, repeated four times, heated at 350 °C for 20 min, and cooled to room temperature. The silicon substrate was placed in 25 mM zinc nitrate and 25 mM hexamethylenetetramine solution, and the ZnO nanowire arrays were grown vertically by hydrothermal synthesis. Finally, using chemical vapor deposition (CVD), 2-methylimidazole was heated at 90 °C for 30 min to enable the growth of subclass MOF, ZIF-8, on the surface of the ZnO nanowires. After cooling to room temperature, the excess organics were removed by washing with anhydrous ethanol, and the ethanol was removed by vacuum before use.

Sensor chip preparation

To study exocytosis in living cells in real-time, we first sequentially printed the Ag-AgCl reference, carbon counter, and carbon working electrodes on a flexible polyester (PET) substrate to make screen-printed electrodes. Next, 1 mg of MNG-1 nanomaterial was ultrasonically dispersed in a mixture of ethanol (100 μL) and Nafion (25 μL) to form a uniform ink. The sensing material was then deposited uniformly and precisely onto the working electrode of the sensing chip by dropping the ink on it. After the evaporation of the solvent, the electrochemical sensing chip is ready for testing.

Cell culture and SEM sample preparation

The PC12 cell line was derived from transplantable rat pheochromocytoma and was obtained from the Chinese Academy of Sciences Cell Bank/Stem Cell Bank (ATCC source). Cell culture was performed using RPMI-1640 medium containing 5% FBS, 10% HS, and 1% penicillin–streptomycin, and PC12 cells were cultured in a constant temperature environment at 37 °C in a humidified incubator with 95% air and 5% CO₂. The SEM samples of PC12 cells were prepared by fixation with 2.5% paraformaldehyde and dehydration with ethanol. A ZEISS Sigma 300 field emission scanning electron microscope from Carl Zeiss AG to observe PC12 cells and the MNG-1 sensor interface.

Loading the accurate number of cells

PC12 cells were cultured for two days post-passaging under standard conditions to ensure complete recovery before experimental testing. To load a specific number of PC12 cells onto the MNG-1 sensor, we used the SCP4000 single-cell printing system from Shanghai Aurefluidics Technology. This system features a high-resolution optical imaging setup and advanced AI algorithms that assess cell characteristics. It can print the required number of cells on the sensor chip with single-cell precision. The SCP4000 single-cell printing system is designed to meet the MNG-1 sensor's capability of detecting from one cell to several dozen at once.

Electrochemical characterization

DA solutions with concentrations ranging from 1 pM to 100 pM were prepared through serial dilution in phosphate-buffered saline (PBS). Before electrochemical measurements, all DA solutions were purged with nitrogen for 30 min to remove dissolved oxygen. Then, 5 μL aliquots of the deoxygenated DA solution were dropped onto the working area of the MNG-1 sensor. The cyclic voltammetry (CV) measurements were performed within a potential window of −0.2 to 0.7 V at a scan rate of 50 mV s⁻¹. The amperometric current–time (i–t) curves were obtained at 0.38 V for 200 s.

Characterization and electrochemical testing instruments

We used the electrochemical workstation, CHI 660E, which was sourced from Shanghai Chenhua Instrument Co., Ltd. To enhance detection quality, we employed a CHI 200B model Faraday cage to minimize electromagnetic interference, achieving amperometric current measurements at the picoampere (pA) level.

Detection methods for exocytosis of living cells and data processing

All electrochemical data recording exocytotic events were acquired using a CHI 660E electrochemical workstation coupled with a CHI 200B Faraday cage system. To maintain accurate cell numbers during analysis, each sensor is used only once. Quantitative analysis of DA molecules released per exocytotic event was calculated via Faraday's law. With a 12 ms time interval and a continuous test duration of 200 seconds, spikes with a signal-to-noise ratio greater than 3 were counted, and the spike areas, i.e. total charge transferred (Q), were obtained by spike integration. The quantity of DA molecules released per exocytotic event was determined using Faraday's equation: Q = nNF, where N is the number of DA molecules, F is the Faraday constant (96485 C mol⁻¹), and n equals 4 in this case.³³

Validation of the proposed method using ELISA

To verify the practicality of the proposed sensor for studying the APs’ mechanism, an enzyme-linked immunosorbent assay (ELISA) was used to test the concentration of DA secreted by PC12 cells after drug treatment. Different concentrations of haloperidol were used to treat the same number of PC12 cells, followed by ELISA to test the concentration of DA in the cell supernatant, which was compared with the results of the MNG-1 sensor assay.

Statistical data analysis

Spikes were analyzed using Origin software. To avoid multiple exocytotic events, spikes with half-peak widths greater than 20 ms were excluded. Non-parametric statistical method, Mann–Whitney U Test, was used for significance analysis, with P < 0.05 as the criterion for determining significant differences, and the data variability was reported using the standard error of the mean (SEM).

Cell viability assessment

Cell viability was evaluated using Trypan Blue exclusion assay. Following treatment with either 80 mM K⁺ or varying concentrations of haloperidol, cells were incubated with 0.04% Trypan Blue (diluted by PBS) at room temperature for 5 minutes. Microscopic analysis was performed within 3 minutes post-staining to assess the cell viability.

Results and discussion

Working principle of MNG-1-based sensor chip for APs’ mechanism study

In this work, we developed an electrochemical sensor chip based on the multidimensional synergistic nanomaterial MNG-1 for real-time monitoring of exocytotic DA release from living PC12 cells, aiming to elucidate the mechanisms of three common APs (Fig. 1). The sensor's enhanced performance is attributed to the unique structural and functional integration of MNG-1: (1) rGO provides a highly conductive network for efficient electron transfer, (2) vertically aligned ZnO nanowires ensure intimate cell membrane contact, maximizing signal capture, and (3) the ZIF-8 shell coated on the nanowires selectively catalyses DA oxidation, significantly amplifying the electrochemical response. This synergistic design enables ultrasensitive DA detection with a remarkable limit of detection (LOD) of 1 pM,³³ while significantly improving the efficiency and ease of monitoring exocytotic events.

PC12 cells, which closely mimic dopaminergic neurons in DA synthesis, storage, release, and metabolism, and are commonly used as models for neurons.³⁴ By printing a specific number of PC12 cells onto a working electrode, they adhere to the MNG-1 sensing interface naturally. DA released during exocytosis is captured by the ZnO nanowires in contact with the cell membrane, generating a transient current through catalytic oxidation. The MNG-1 sensor provides high sensitivity and close contact with the cell, allowing for precise real-time monitoring of exocytosis. This method requires only a standard electrochemical workstation and a small Faraday cage, avoiding the need for advanced instrumentation or a shielded room.

Commonly used APs like haloperidol, sulpiride, and chlorpromazine primarily work by antagonizing DA receptors and blocking dopaminergic pathways.^4,35–38 However, patients with schizophrenia are hypersensitive to DA, and substances that increase DA release, such as amphetamine and L-dopa, can exacerbate positive symptoms.³⁹ An antagonist that reduces DA secretion while blocking the DA pathway may enhance therapeutic effects. In contrast, blocking DA receptors while increasing DA secretion may have adverse effects.⁴⁰ Traditional APs’ mechanism studies have focused on receptor–ligand binding and often overlook the influence of APs on DA secretion.⁴¹ Therefore, further research into how APs affect DA release is required. In this study, we examined the effects of three APs on DA secretion in PC12 cells and identified three distinct mechanisms of action.

MNG-1 nanomaterial characterization

Fig. 2a–d illustrate that the MNG-1 nanomaterial consists of rGO, ZnO nanowire arrays, and ZIF-8. Combining these three nanomaterials provides strong electrochemical catalytic activity for DA, enabling its deep oxidation to polydopamine (PDA) with four electrons transferring. Elemental analysis using energy-dispersive X-ray spectroscopy (EDS) confirmed the successful integration of rGO, ZnO, and ZIF-8.

Electrochemical characterization of the MNG-1 sensor

CV characterization (Fig. 3a) in 0.1 mM DA demonstrates that the MNG-1 material generates a stronger current response compared to bare, rGO, and ZnO on rGO-modified electrodes, highlighting the significant synergistic effect of MNG-1. A distinct oxidation peak at 0.38 V was observed, corresponding to DA oxidation, which was then applied in subsequent amperometric experiments. Fig. 3b presents the amperometric i–t curves obtained at different DA concentrations ranging from 1 to 100 pM. The oxidation current displays a linear increase with DA concentration (inset). The observed LOD of 1 pM represents the lowest concentration reliably distinguished from background noise, confirming excellent sensitivity compared to conventional DA sensors. Fig. 3c demonstrates the excellent selectivity of the MNG-1 sensor against common physiological interferents, including epinephrine, catechol, glucose, ascorbic acid, tyrosine, uric acid, and serotonin. The sensor exhibits good selectivity for 100 pM DA against 0.25 μM interfering molecules, confirming its capability for monitoring living cell exocytosis in complex cellular environments. As shown in Fig. 3d, the five randomly selected MNG-1 sensors prepared in the same batch displayed highly consistent responses to 100 pM DA (RSD = 4.58%). We also evaluated the DA detection performance of MNG-1 sensors from five different fabrication batches under identical experimental conditions, demonstrating excellent reproducibility with an RSD of 1.55% (Fig. S1). This reproducibility can provide statistically reliable data for analyzing the effects of drugs on DA exocytosis behaviour. The long-term stability and reusability of the MNG-1 sensor were evaluated through 15 consecutive measurements of 100 pM DA over a 15-day period. When stored under dry conditions at room temperature, the sensor exhibited satisfactory stability, with an RSD of only 1.82% across repeated measurements (Fig. S2).

Exocytosis analysis by MNG-1 sensor

One challenge in studying single-cell exocytosis is positioning the electrode near the cell membrane. The proposed method addresses this by using a high-precision cell printing system, which can arrange a specific number of cells on the MNG-1 sensor's working electrode with a diameter of 1 mm. After printing, the cells autonomously adhere to the MNG-1 nanomaterial, allowing their membranes to contact the tentacle-like ZnO nanowires (Fig. 4a). This method enables quick chip preparation for cell monitoring without delicate micromanipulation. The MNG-1 interface provides a larger surface area for contact, increasing the chances of capturing exocytotic signals.

The addition of 80 mM K⁺ and 2 mM Ca²⁺ solution induced the depolarization of the cell membrane, leading to the opening of ion channels. This stimulation prompted DA-containing intracellular vesicles to fuse with the cell membrane, resulting in exocytosis. As shown in the sensing trace measured at a potential of 0.38 V in Fig. 4b, the proposed method effectively tests a single exocytotic event from an individual cell without needing positional adjustments. The spike count per amperometric trace, corresponding to DA exocytotic events, showed a positive correlation with PC12 cell number during high-K⁺ stimulation (80 mM). Besides, the Ca²⁺ antagonist Cd²⁺ (100 μM CdCl₂) completely suppressed exocytosis, confirming the Ca²⁺-dependent nature of DA release. Results obtained from various cell numbers indicated that data from 10 PC12 cells provided statistically significant results, so subsequent experiments were conducted using 10 PC12 cells. By integrating the spike curves, we obtained the total charge transferred for each exocytotic event. Using Faraday's law (Q = nNF), our measurements indicate an average DA release of 157.14 zmol per exocytotic event, a value slightly lower than some previous reports,^42–44 likely reflecting inherent variability in PC12 cell subcultures.^45,46 Importantly, this quantitative difference does not impact our primary research objective, the systematic comparison of APs’ effects within our standardized experimental paradigm. The relative differences observed between drug treatments remain valid regardless of the absolute quantitation values.

Study the effect of APs on exocytotic DA secretion using the MNG-1 sensor

To investigate the effects of APs on living cell exocytosis, PC12 cells were treated with drug solutions prepared at clinically recommended concentrations according to previous research (haloperidol: 10 ng mL⁻¹, sulpiride: 300 ng mL⁻¹, chlorpromazine: 300 ng mL⁻¹).^47–49 This experimental design was implemented to elucidate the regulatory effects of APs on the exocytotic process under physiologically meaningful conditions. We conducted 13 tests (totaling 130 cells) for each AP using the MNG-1 sensor to ensure statistically significant results. The data in Fig. 5b and c indicate that haloperidol significantly reduced exocytotic events compared to untreated cells, while sulpiride had no notable effect. In contrast, chlorpromazine increased exocytotic event count. Our results suggest that sulpiride does not affect DA release, whereas chlorpromazine may increase DA secretion. Haloperidol could decrease DA secretion, potentially helping to further reduce dopaminergic function and improve positive symptoms of schizophrenia. Additionally, other research has shown that haloperidol's impact on dopaminergic activity is not solely due to its action as a receptor antagonist. This could potentially be because haloperidol inhibits vesicular transport,⁵⁰ and blocks D2 autoreceptors to eliminate the autoinhibition of DA release,⁵¹ which ultimately leads to a reduction in DA secretion. HPLC analysis of mouse striatal samples revealed that haloperidol can lower DA levels, though only 3–4 samples were examined under each condition due to challenges in sample acquisition and preparation.⁴ This study shows that certain APs unexpectedly increase DA release, revealing new mechanisms behind their clinical effects. Notably, certain APs enhance DA secretion, thereby elevating extracellular DA levels, a phenomenon that may have implications for psychiatric treatment strategies. Compared to conventional approaches, our method enables efficient analysis of a vast quantity of cells and yields statistically significant data, showing promising applicability for assessing pharmacological effects on DA secretion regulation.

We further investigated the effects of different doses of haloperidol (1 ng mL⁻¹, 5 ng mL⁻¹, and 10 ng mL⁻¹) on the exocytosis of PC12 cells, as illustrated in Fig. 6. The average count of exocytotic events decreased from 123 in the control group to 113, 92, and 32 with increasing haloperidol concentration. The count of exocytotic events obtained with 1 ng mL⁻¹ haloperidol was not significantly different from those obtained with the control group, whereas the higher concentrations of 5 ng mL⁻¹ and 10 ng mL⁻¹ haloperidol resulted in significant differences. The average quantity of DA secreted per exocytosis dropped from 157.14 zmol to 97.29, 75.30, and 31.92 zmol over 200 seconds, as shown in Fig. 6(c). To validate the reliability of the electrochemical measurements, cell viability was assessed using Trypan Blue exclusion assays both before and after 5-minute treatments with either 80 mM K⁺ or haloperidol (varying concentrations). As demonstrated in Fig. S3, all the cells remained unstained in all treatment conditions, confirming maintained membrane integrity and excellent viability throughout the experiments. This confirmation verifies that the observed reduction in DA secretion resulted specifically from the pharmacological effects of haloperidol rather than compromised cellular viability. These results demonstrate a concentration-dependent effect of haloperidol in inhibiting DA release, indicating that higher doses more effectively reduce DA signaling from neuronal cells.

ELISA is the gold standard method for measuring DA levels secreted by cells. While this method achieves excellent selectivity and specificity through antibody–antigen interactions, it nevertheless remains incapable of real-time cellular activity monitoring, exhibits relatively low sensitivity (LOD: 1.6 nM), and requires prolonged detection times exceeding 90 minutes.⁵² In this work, we analyzed DA concentrations in the supernatants of haloperidol-treated PC12 cells using both ELISA and the MNG-1 sensor (Fig. 7). ELISA requires a minimum of 10 cells for detection due to its limited sensitivity. Therefore, we used ELISA and the MNG-1 sensor with an identical number of 10 cells for comparison. Both the MNG-1 sensor and ELISA indicated that extracellular DA levels decreased as the concentration of haloperidol increased, confirming the effectiveness and accuracy of the MNG-1 sensor. Additionally, the MNG-1 sensor is more efficient and allows for real-time monitoring of cellular exocytotic events, providing statistically significant results more quickly than ELISA.

Conclusions

In conclusion, we have effectively investigated the effects of APs on DA secretion by real-time monitoring and analysis of exocytotic events in living PC12 cells using an electrochemical sensor based on the multidimensional synergistic functional nanomaterial MNG-1. Our experimental observations offer new insights into the traditionally understood mechanisms of DA receptor antagonism. The results demonstrated contrasting effects among the three APs tested: haloperidol significantly inhibited DA release from PC12 cells during exocytosis, sulpiride had no significant effect on DA release, and chlorpromazine promoted DA release. Additionally, we examined the impact of haloperidol concentration on the exocytotic secretion of DA from PC12 cells. The findings revealed that both the frequency of exocytotic events and the quantity of released DA molecules decreased as haloperidol concentration increased. This trend was consistent with results from the gold-standard ELISA, further confirming haloperidol's ability to inhibit DA secretion, in addition to its known receptor antagonist effects. The proposed MNG-1 sensing assay can monitor and quantitatively analyze exocytotic events in single or multiple cells in real-time. This provides an efficient, convenient, and cost-effective research tool for exploring the mechanisms of action of APs and holds significant potential for application in studies related to dopaminergic pathways, offering a new method for advancing drug screening and mechanistic investigations in neuropsychiatric disorders.

Author contributions

Wenxue Chen: data curation, formal analysis, validation, writing – original draft. Xuefeng Wang: formal analysis, validation, visualization, methodology, writing – original draft. Yuan Zhang: resources, writing – original draft. Zihui Li, Haoliang Li, and Qiongya Wan: data curation. Xinxin Li: resources, funding acquisition, writing – review & editing. Dan Zheng: supervision, methodology, writing – review & editing. Pengcheng Xu: resources, supervision, funding acquisition, visualization, methodology, writing – review & editing.

All authors have approved the final version of the manuscript.

Conflicts of interest

There are no conflicts to declare.

Data availability

Raw data supporting the findings of this study are available from the corresponding author upon reasonable request.

The data supporting this article have been included as part of the SI. Supplementary information (SI) available: batch-to-batch sensor reproducibility, long-term stability data, and Trypan Blue exclusion assay results for PC12 cell viability assessment. See DOI: https://doi.org/10.1039/d5an00606f.

Acknowledgements

The authors gratefully acknowledge financial support by the National Key Research and Development Program of China (2021YFB3200800), National Natural Science Foundation of China (62227815, 62271473, U21A20500).

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