Selective Detection of Acetylcholine against Choline and In Vivo Measurement in the Mouse Brain Using the Micropipet-Supported Liquid/Liquid Interface Electrode

Abstract

Acetylcholine (ACh) is a neurotransmitter that plays critical roles in human health and diseases. To better understand ACh signaling in the brain, developing analytical capabilities for its selective and quantitative measurement in real time is essential. While electrochemical amperometry offers exceptional temporal resolution, most in vivo electrochemical ACh sensors have limited selectivity, such as against choline (the product of ACh hydrolysis) and ascorbic acid. Here, we report a micropipet-supported ITIES (interface between two immiscible electrolyte solutions), which demonstrated the selective detection of ACh against choline, ascorbic acid, and other neurotransmitters using chronoamperometry. The detection of ACh is based on its ion transfer across a water/oil interface, which was formed between an aqueous solution and a 2-nitrophenyl octyl ether (NPOE) solution containing an ACh ionophore, termed water/NPOE-ionophore ITIES. The ionophore was heptakis­(2,6-di-O-methyl)-β-cyclodextrin (DM-β-CD). The addition of DM-β-CD in the NPOE phase resulted in an easier ACh transfer at the water/NPOE-ionophore ITIES compared with that at the ITIES without the ionophore, suggesting the presence of ionophore-facilitated ion transfer in addition to the direct ion transfer at the water/NPOE-ionophore ITIES. We observed a linear detection of ACh on the water/NPOE-ionophore ITIES. When implanted in the cortex of the brain of a live mouse, the water/NPOE-ionophore ITIES tracked the dynamic concentration changes of the injected ACh in the brain. The measuring techniques are broadly applicable to quantifying real-time ACh release in the brain with negligible interference, enabling a better understanding of neurological disorders and diseases.

1. Introduction

Acetylcholine (ACh), the first neurotransmitter identified in 1914, , plays a crucial role in learning, memory, and human health; defects in its release are involved in aging, Alzheimer’s disease, and addiction. − To better understand ACh signaling in the brain, it is critical to develop a measurement platform that can measure the concentrations of ACh, as its concentration changes in diseases. After its release in the brain, the ACh hydrolysis reaction occurs, breaking ACh down into choline and acetic acid. Therefore, it is important to achieve high selectivity for ACh over choline besides other interferents, such as ascorbic acid and other neurotransmitters. The development of selective and quantitative in vivo sensors for real-time ACh is currently an area of research.

Recent advances have been made in the real-time measurement of ACh in vivo. One example is fiber photometry/genetically encoded fluorescence sensors; however, these sensors exhibit limited temporal resolution compared to amperometry. Despite their advantages, there are situations where genetically encoded fluorescent ACh sensors may not be appropriate. One caveat is that virally expressed genetically encoded ACh sensors are expressed on cell types that may represent only a subset of targets of endogenous ACh signaling. In addition, expression levels of the ACh sensor may vary between animals infused with viral constructs.

Chemical sensing with electrodes offers chemical identity, quantification, and high spatiotemporal information about biological processes in vivo. These advantages make electroanalytical chemistry one of the most widely used tools in the detection of signaling molecules as well as redox-active and redox-inactive neurotransmitters. ,−

While electrochemical amperometry offers exceptional spatial and temporal resolution, most in vivo electrochemical ACh sensors have limited selectivity. ACh is not redox-active and cannot be detected directly on an electrode. Electrodes modified with two enzymes, acetylcholinesterase (AChE) and choline oxidase (ChOx), were developed to detect ACh in the brain indirectly by measuring H₂O₂, which is the product of enzymatic reactions. AChE breaks ACh down to (acetic acid and) choline, which is subsequently converted into betaine and H₂O₂ by ChOx. Thus, H₂O₂ is generated from both ACh and choline. ACh is measured by subtracting the choline signal from the ACh/choline signal (on the AChE/ChOx-electrode). Choline is measured on a second electrode modified with ChOx. However, ChOx sensors developed sensitivity to ACh after exposure to brain tissue ostensibly due to the adsorption of endogenous AChE. , Additionally, at the oxidation potential of H₂O₂, oxidation of other redox-active species, such as ascorbic acid (AA), could happen. ,, These pose technical challenges for the in vivo selective detection of ACh against choline, AA, and other redox-active species.

ITIES (interface between two immiscible electrolyte solutions, also called liquid/liquid interface electrode) is a versatile sensor especially for the detection of non-redox-active analytes. The detection is based on analyte ion transfer across a water/oil interface, generating a Faradaic current. ,− Recently, we introduced the liquid/liquid interface electrode for the in vivo real-time detection of ACh against AA and other neurotransmitters, and it is still yet to be revealed if the liquid/liquid interface electrode distinguishes between ACh and choline. Here, we report a water/NPOE-ionophore ITIES electrode for the selective detection of ACh against choline using chronoamperometry. The ITIES is formed between an aqueous electrolyte solution and an oil electrolyte solution of 2-nitrophenyl octyl ether (NPOE) containing an ionophore (heptakis­(2,6-di-O-methyl)-β-cyclodextrin), which was used previously for ACh sensing at the solid electrodes by us and others. −

ACh and choline detections at water/NPOE-ionophore ITIES exhibit distinct potentials; choline detection requires a potential magnitude greater than that of ACh. We further demonstrated the selective detection of ACh against other interfering species, i.e., AA, glutamate, gamma-aminobutyric acid (GABA), dopamine, serotonin, norepinephrine, l-cysteine, and l-tyrosine. We observed the quantitative detection of ACh on the water/NPOE-ionophore ITIES electrode (current increased linearly with increasing concentrations). When implanted in the cortex of the brain of a live mouse, the water/NPOE-ionophore ITIES electrode tracked the dynamic changes in the injected ACh in the brain.

2. Experimental Section

2.1. Materials and Supplies

Phosphate-buffered saline (PBS) was prepared by dissolving a PBS tablet obtained from Sigma-Aldrich (St. Louis, MO). The PBS compositions are 0.01 M phosphate buffer, 0.0027 M potassium chloride, and 0.137 M sodium chloride. Serotonin hydrochloride was obtained from Fisher Scientific (Pittsburgh, PA). Acetylcholine chloride (AChCl), choline chloride (ChCl), l-glutamic acid (Glu), GABA, dopamine hydrochloride, l-ascorbic acid (AA), (−)–norepinephrine (NE), l-cysteine (l-cys), l-tyrosine (l-tyr), tetrabutylammonium (TBA) chloride hydrate, tetradodecylammonium chloride (TDDACl), N,N-dimethyltrimethylsilylamine, NPOE, and heptakis­(2,6-di-O-methyl)-β-cyclodextrin were obtained from Sigma-Aldrich (St. Louis, MO). Serotonin hydrochloride was obtained from Fisher Scientific (Pittsburgh, PA). The TFAB salt of TDDA (TDDATFAB) was prepared by metathesis. − Quartz capillaries (O.D. = 1.0 mm, I.d. = 0.7 mm, and length = 10 cm) were from Sutter Instrument (Novato, CA).

2.2. Fabrication of the MicroITIES Electrodes

We fabricated the micrometer-size pipettes using a method similar to that previously reported. First, we pulled a quartz capillary using a P-2000 laser puller (Sutter Instrument) with the following pulling parameters: heat = 680, filament (FIL) = 4, velocity (VEL) = 55, delay (DEL) = 130, and pull = 50. After pulling, the micropipettes exhibited a diameter of around 1–3 μm. To ensure a stable interface at the orifice of the micropipet, we performed a surface treatment using salinization to convert the pipet surface from hydrophilic to hydrophobic. We used a chemical vapor salinization process with N,N-dimethyltrimethylsilylamine. For the surface treatment via salinization, we placed the pulled micropipettes on a Petri dish inside a plastic desiccator. Then, we connected the desiccator to a three-way valve and applied vacuum for 45 min. Following this, we introduced N,N-dimethyltrimethylsilylamine for 1 h and 45 min by switching the three-way valve immediately after the vacuum. After the surface treatment, the micropipettes were backfilled with an organic phase filling solution containing 0.1 M TDDATFAB and 5 mM heptakis­(2,6-di-O-methyl)-β-cyclodextrin in NPOE. After that, we inserted a Pt wire (0.05 mm diameter, Goodfellow Corp, Pittsburgh, PA) into the micropipet to be around 200 μm away from the micropipet tip, followed by the fixation of the Pt wire using orthodontic wax (from HRASY). The Pt wire was etched before insertion. For making connections to the electrochemical workstation, the other end of the Pt wire was connected to a copper wire by using Ag epoxy (MG Chemicals, Burlington, Ontario, Canada).

2.3. Electrochemical Experiments

We used cyclic voltammetry (CV) and chronoamperometry to detect ACh on the new water/NPOE-ionophore microITIES electrode. All electrochemical measurements were recorded using a CHI 760 Electrochemical Workstation (CH Instruments, Austin, TX). CV was performed at a scan rate of 0.05 V/s. The sampling interval for i–t amperometry was 0.0333 s (at a frequency of 30 Hz). The water/NPOE-ionophore microITIES electrodes were immersed in an aqueous solution for the detection of ACh in PBS. PBS was chosen because it is a commonly used biological medium that mimics in vivo environments. A Ag wire coated with AgCl was placed in the PBS and served as an external reference electrode. Potentials were applied between the Pt wire inside the microITIES electrode and the external Ag/AgCl wire.

The electrochemical cell diagram shown below was used for the ACh detection measurement in PBS.

$$\mathrm{Pt}|0.1\mathrm{M}\mathrm{TDDATFAB}+5\mathrm{mM}\mathrm{heptakis}(2,6-\mathrm{di}-\mathrm{O}-\mathrm{methyl})-\beta -\mathrm{cyclodextrin}+\mathrm{NPOE}\parallel \mathrm{PBS}+x\mu \mathrm{M}\mathrm{ACh}|\mathrm{AgCl}|\mathrm{Ag}$$

The potentials of all of the electrochemical data were reported with respect to the half-wave transfer potential of TBA (E _1/2, TBA), which we used as an internal standard. We added TBACl at the end of the experiment. The E _1/2, TBA was determined from the CV. The potential conversion was performed by subtracting the E _1/2, TBA from the actual potential measured. It is critical to make sure that there is no background shift during the conversion.

2.4. Mice

Male Balb/C mice obtained from Taconic Biosciences (Germantown, NY) were used for the in vivo experiments. Mice were housed in a temperature- and humidity-controlled room on a 12-h light and 12-h dark cycle. Mice were housed in a group of a maximum of five in plastic cages with corn cob bedding. Mice had access to food and water ad libitum.

2.5. Stereotaxic Surgery for In Vivo Measurement

All of the procedures involving animals were approved by the Institutional Animal Care and Use Committee of the University of Illinois Urbana–Champaign. Stereotaxic surgery was performed to implant the water/NPOE-ionophore electrode in the cortex of a live mouse brain.

The mouse was first anesthetized in 5% inhaled isoflurane (in oxygen) in an anesthesia chamber (Kent Scientific Corporation, Torrington, CT). Once the mouse reached the stage of surgical anesthesia, it was transferred to a stereotaxic surgery frame (David Kopf Instruments, Tujunga, CA). The mouse was maintained under anesthesia throughout the surgery by the inhalation of 1–2% isoflurane. A heating pad (RWD Life Science, Sugar Land, TX) was used to maintain the body temperature of the mouse during the surgery. Incisions were made to expose the mouse brain’s skull surface. Holes were drilled in the skull for implantation of the working and reference electrodes. The anteroposterior (AP), mediolateral (ML), and dorsoventral (DV) positions referenced from bregma were used as the coordinates for the brain regions, which were obtained from the mouse brain atlas. The water/NPOE-ionophore electrode was placed at the motor cortex (AP = +1.54 mm, ML = −1.15 mm, DV = −1.00 mm). A Ag/AgCl reference electrode was implanted into the contralateral side of the brain. A Hamilton syringe was used to deliver the ACh solution in the same hole of the working electrode. A schematic diagram depicting the setup of the real time in mouse brain studies is shown in Figure .

1.

Schematic diagram of the real-time mouse brain studies.

2.6. Data Analysis

We plotted the data as mean ± standard deviation in the calibration curve and bar plots. The data points in the bar plot and calibration curve were the average values calculated from the three repeated CVs or the i–t curve over a 15 s stable region. The standard deviations were calculated and plotted as error bars. The method of least squares was used to determine the best linear fit in the calibration curve. A two-sample Student’s t test was used to determine if there was a statistically significant difference between the two groups.

The onset potentials of ACh and choline detection were determined in a similar way as we have reported previously. Briefly speaking, the baseline current (i _base) and the capacitive current (i _c) were determined from cyclic voltammograms of the PBS background. Then, we measured the onset potentials at the potentials where current i = i _base + 1.5 × i _c.

3. Results and Discussion

3.1. Cyclic Voltammograms of Choline and ACh Exhibited Distinct Potentials at the Water/NPOE-Ionophore ITIES Electrode

We used CV to study the detection of ACh and choline at the micrometer-sized water/NPOE-ionophore ITIES electrode, respectively. The ionophore we used is heptakis­(2,6-di-O-methyl)-β-cyclodextrin (DM-β-CD), which we and others have used to facilitate the detection of ACh on solid electrodes. − We first collected the cyclic voltammogram (CV) of the PBS background, with results shown in Figure A (blue curve), where we observed a nice background with a broad potential window. After that, we added ACh and choline. The CV of ACh is the red curve in Figure A. The onset potential for ACh detection occurs at −0.207 V vs E _1/2, TBA. The CV of choline detection is the black curve in Figure A, where the onset potential for choline detection occurred at −0.271 V vs E _1/2, TBA. The onset potential of choline detection was 64 mV more negative than that of ACh.

2.

Cyclic voltammograms (CVs) (A) and background-subtracted CVs (B) for 40 μM ACh and 40 μM Ch in a PBS background. ACh showed a higher current response and started to increase at a more positive potential. The scan rate was 0.05 V/s.

To better understand the shape of CV for ACh detection on the water/NPOE-ionophore ITIES, we collected CV at a potential more negative than that shown in Figure , with results shown in Figure S1. The diffusion-limited current plateau of ACh detection in the CV might occur at much more negative potentials, which lies in the background signal region. This aligns with our previous studies on the nano-NPOE/water ITIES electrode without an ionophore. To confirm this, we added an internal standard, TBA, whose transfer occurred before the background potential. The CV of TBA exhibited a very good diffusion-limited plateau (Figure S1).

For easier visualization, we overlapped the background-subtracted CVs of ACh and choline detection, with results presented in Figure B. As shown in Figure B, ACh detection is much easier than that of choline at the micrometer-sized water/NPOE-ionophore ITIES electrodes. Considering that the Gibbs free energy of their transfer is proportional to the potential of their transfer, results in Figure show that ACh has a lower Gibbs free energy of transfer at the water/NPOE-ionophore liquid/liquid junction ITIES electrodes, thus showing easier detection of ACh than choline using amperometry.

We observed that ACh and choline detection exhibit distinct potentials using CV measured on two different electrodes through two separate experiments. It is unknown how the water/NPOE-ionophore microelectrode responds to ACh in the presence of choline. To test this, we first measured the CV of choline followed by the addition of ACh. The results are shown in Figure A.

3.

Cyclic voltammograms (A) of ACh detection on a micropipet-supported NOPE-water interface probe for the selective detection of ACh against choline. The scan rate was 0.05 V/s. The bar plot at E = −0.24 V (vs E _1/2, TBA) (B) shows that when 50 μM Ch was added in the PBS background first, no difference was observed, and then when 50 μM ACh was added, in the Ch and PBS solution, a significant increase was observed.

In Figure A, we first measured the cyclic voltammogram of the PBS background, shown as a black curve. Then we added 50 μM choline to the PBS, and the measured cyclic voltammogram is the blue curve in Figure A, where we observed a small increase in current starting around −0.276 V. After that, we added 50 μM ACh, and the cyclic voltammogram is the red curve in Figure A. After adding ACh, we observed an obvious and significant current increase at a much earlier potential (less negative), i.e., at −0.232 V, indicating that ACh detection is 44 mV easier than the detection of choline.

Overall, CV results suggest that the potential region of −0.232 V to −0.276 V vs E _1/2, TBA is the potential window where the water/NPOE-ionophore ITIES electrode is sensitive to ACh but is not sensitive to choline. We measured the current at −0.24 V from the CVs in Figure A and generated a bar diagram shown in Figure B. As can be observed in Figure B, a significant increase in current happened after adding ACh to the choline and PBS solution at −0.24 V. In contrast, the current remains unchanged after adding choline to the PBS solution at −0.24 V. Results in Figure confirm that at −0.24 V vs E _1/2, TBA, selective detection of ACh against choline was achieved on the water/NPOE-ionophore ITIES electrode.

3.2. Specificity of the Water/NPOE-Ionophore ITIES Electrode toward ACh against Other Interferents

After demonstrating the selective detection of ACh against choline on the water/NPOE-ionophore ITIES electrode, we further performed a selectivity/specificity test against other interfering species using chronoamperometry at the potential where the selective detection of ACh against choline was achieved. The interferants that we tested include choline (Ch), glutamate (Glu), GABA, dopamine (DA), serotonin (5-HT), norepinephrine (NE), AA, l-cysteine (l-cys), and l-tyrosine (l-tyr). We first carried out chronoamperometry at E = −0.24 V (vs E _1/2, TBA) in the PBS background and then added these possible interfering species in sequence. Given the high concentration of AA in the brain, we tested it at a concentration of 500 μM, while the other analytes were tested at 50 μM each. The results of chronoamperometry are presented in Figure .

4.

In vitro selectivity test of the microITIES electrode toward ACh against other common interferents using chronoamperometry at E = −0.24 V (vs E _1/2, TBA) (A) with the i–t at a stable current region of each interferent and ACh (B). The sampling interval was 0.0333 s (30 Hz). The corresponding PBS background-subtracted bar plot for the stable current for each interferent (C) showed a significant increase in current after adding 50 μM ACh in the system, while no significant change was observed for other interferents.

Figure A includes the current noise associated with the addition process. For better visualization, we plotted the steady-state current from Figure A with the results shown in Figure B. We further analyzed Figure B by calculating the average and the standard deviations. We further presented this analysis in Figure C as a bar diagram, where the y-axis is the PBS background-subtracted current. In Figure , it can be observed that the current remained unchanged from the PBS background after the addition of choline, glutamate, GABA, dopamine, serotonin, norepinephrine, AA, l-cysteine, and l-tyrosine. In contrast, after adding ACh, a significant increase in current was observed. In addition, we carried out selectivity studies using CV with the results shown in Figure S2. Similarly, a significant current increase was observed after adding ACh. There is a small current increase for Ch, but at a more negative potential than ACh. For the rest of the interferents, no obvious current increase was observed after their addition. At the potential for selective ACh detection against Ch and other interferents (E = −0.24 V vs E _1/2, TBA) using chronoamperometry, only ACh exhibited a significant current increase, while others did not. These results confirm that the new water/NPOE-ionophore ITIES electrode achieved the selective detection of ACh against all of these interferants and choline.

These selectivity results highlight the advantages of the introduced water/NPOE-ionophore ITIES electrode toward the selective detection of ACh. Conventional enzyme sensors for ACh detection require two enzyme-modified electrodes, one for choline and the second one for choline/ACh, to extract the signal of ACh by subtracting the choline signal from the choline/ACh signal. Considering that it has been reported that ChOx sensors developed sensitivity to ACh after exposure to brain tissue ostensibly due to the adsorption of endogenous AChE, , concerns arise regarding the selectivity of enzyme-modified electrodes toward the selective detection of ACh against choline. The presented liquid–liquid interface electrode addressed this technical challenge.

3.3. Quantitative Detection of ACh

We investigated the quantitative detection of ACh on a micrometer-sized water/NPOE-ionophore ITIES electrode using chronoamperometry. We recorded the current at −0.24 V vs E _1/2, TBA, the potential for selective detection of ACh, as a function of time, and added ACh of different concentrations. The results are shown in Figure A, which shows that increases in the current magnitude occurred with increasing concentrations of ACh. The i–t curve at the steady-state current of each concentration of ACh is shown in Figure B. We measured the steady-state current and plotted it against the concentration to construct the calibration curve shown in Figure C. As shown in Figure C, the current of ACh detection increases linearly with its concentration. Overall, we have observed a linear quantitative detection of ACh at the potential where the selective detection of ACh against choline and other interferents was achieved. These results agree with the theory, − ,, i.e., the current measured on the microITIES is proportional to the concentration of analytes.

5.

In vitro selective detection of ACh using chronoamperometry (A) with the i–t at the stable current region of each concentration of ACh and choline (B). The sampling interval was 0.0333 s (30 Hz). We chose E = −0.24 V (vs E _1/2, TBA) for i–t amperometry based on the CV results shown in Figure . The calibration curve (C) based on the stable current for each concentration of ACh showed a good linear response of ACh. There is almost no current response toward Ch.

After the calibration curve experiment, we added choline in the ACh in a PBS solution and did not observe an obvious current increase (Figure A), confirming that water/NPOE-ionophore ITIES electrodes are selective toward ACh detection against choline.

3.4. Mechanism of ACh Detection at the Water/NPOE-Ionophore ITIES

Recently, we reported ACh detection at nanoscale water/NPOE ITIES (no ionophore), where ACh detection was achieved by direct ion transfer across the water/NPOE interface. Besides direct ion transfer, assisted ion transfer is another mechanism of analyte detection at ITIES. ,,,, For the assisted ion transfer, an ionophore can be added to the oil filling solution, which complexes with the analyte, thus enabling easier detection of the analyte. We aim to understand the mechanism of ACh detection at the presented new water/NPOE-ionophore ITIES. To achieve this, we compared the E _1/2 of ACh detection on the water/NPOE-ionophore ITIES (with DM-β-CD ionophore) with E _1/2 on the water/NPOE ITIES (without ionophore). The E _1/2 of ACh detection was calculated from the background-subtracted CVs as shown in Figures S3 and S4. For water/NPOE ITIES with ionophore, the average E _1/2, ACh was −0.318 ± 0.002 V vs E _1/2, TBA. Without an ionophore, the E _1/2, ACh was −0.338 ± 0.018 V. The E _1/2, ACh for the water/NPOE-ionophore ITIES is less negative compared to the ITIES without the ionophore. The addition of ionophore (in the filling solution) resulted in an easier ACh ion transfer at the water/NPOE-ionophore ITIES, suggesting the presence of ionophore-facilitated ion transfer in addition to the direct ion transfer at the water/NPOE-ionophore ITIES.

3.5. In Vivo Measurement of ACh on the Water/NPOE-Ionophore ITIES Electrode

We further studied the performance of the water/NPOE-ionophore ITIES electrode in measuring ACh as a function of time in mice brain. We performed stereotaxic brain surgery to implant the water/NPOE-ionophore ITIES electrode in the cortex of the brain of a live mouse. We first collected CV in the cortex of the live mouse brain during the surgery, which was later used to compare to the post-surgery CV measured in the PBS background to confirm that there was no potential shifting (Figure S5). After the CV, we performed i–t amperometry to record the current at the ACh detection potential (−0.24 V vs E _1/2, TBA) as a function of time. The result is shown in Figure A. As shown in Figure A, we observed a stable current within the first 100 s. Then, we injected 2 μL of 5 mM ACh solution into the brain at t = 100 s, as indicated by the arrows in Figure . The noise around t = 100 s likely was related to the injection process. The ACh was injected next to the working electrode to validate the capability of the introduced water/NPOE-ionophore ITIES for the real-time monitoring of ACh concentration dynamics in vivo. The experimental setup is shown in Figure . We would like to point out that the injection of ACh was not exactly at the same location as the ITIES working electrode due to technique limitations in the injection setup. The injected ACh solution took some time to transfer to the ITIES electrode surface. This might explain the gradual increase in current after ACh injection. Then the injected ACh started clearing, as shown by the decrease in the current following the increase in the current.

6.

In vivo real-time ACh (ACh) measurement on the water/NPOE-ionophore ITIES electrode implanted in the cortex of a live mouse brain. Plots of current as a function of time (A) and ACh concentration versus time (B) measured in the cortex of a live mouse brain. The ACh concentration in Figure B was calculated based on Figure A using the post-surgery calibration curve shown in Figure S6. The injection of 2 μL of a 5 mM ACh solution occurred at t = 100 s, as indicated by the arrows. The potential used for i–t amperometry in Figure A was the ACh detection potential, i.e., E = −0.24 V vs E _1/2, TBA. The sampling interval was 0.0333 s (30 Hz).

After the surgery, we took the electrode out from the brain to construct the post-surgery calibration curve using chronoamperometry. We first did the measurement in the PBS background and then added ACh of different concentrations. The post-surgery calibration results are shown in Figure S6, where a linear correlation between current and the concentration of ACh was observed with R ² = 0.995. From the post-surgery calibration curve, we converted the current to ACh concentration to generate a concentration–time curve shown in Figure B. These results demonstrate the capability of the liquid/liquid interface ITIES electrode to monitor the real-time ACh dynamics in vivo.

4. Conclusions

We introduced an advanced liquid/liquid interface amperometry measurement strategy for quantifying ACh in mice brain (against choline, AA, and other neurotransmitters). Compared to choline, ACh detection was easier and occurred at less negative potentials on the water/NPOE-ionophore ITIES electrode. The water/NPOE-ionophore ITIES electrode is insensitive to other redox-active species, such as AA, dopamine, serotonin, norepinephrine, l-cysteine, l-tyrosine, and non-redox-active species, including glutamate and GABA. Quantitative detection of ACh was achieved on the water/NPOE-ionophore ITIES electrode using amperometry. The liquid/liquid interface electrode measured the dynamic concentration changes of ACh in the cortex of the brain of a live mouse. This research highlights the technical capability of liquid–liquid interface electrodes to distinguish between small molecules of similar structures, such as ACh and choline.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsmeasuresciau.5c00117.

• Cyclic voltammograms of the PBS background, 100 μM ACh, and 100 μM TBA at the introduced water/NPOE-ionophore ITIES; selectivity of the introduced water/NPOE-ionophore ITIES toward ACh detection against other common interferents and choline studied using CV; background-subtracted CVs of 100 μM ACh at four different water/NPOE ITIES electrodes without the ionophore; background-subtracted CVs of 100 μM ACh at three different water/NPOE-ionophore ITIES electrodes; cyclic voltammograms of the in vivo background measured in the cortex of a live mouse brain and post-surgery in vitro in the PBS background; and post-surgery calibration of ACh detection after the in vivo mouse brain measurement (Figure ) to convert the measured current to the concentration of ACh (PDF)

CRediT: Peibo Xu data curation, formal analysis, investigation, methodology, validation, visualization, writing - original draft, writing - review & editing; Mei Shen conceptualization, data curation, formal analysis, investigation, funding acquisition, methodology, project administration, resources, supervision, validation, visualization, writing - original draft, writing - review & editing.

The authors declare no competing financial interest.

Published as part of ACS Measurement Science Au special issue “2025 Rising Stars in Measurement Science”.