Acetylcholine Sensor Research Articles

Medial prefrontal cortex acetylcholine signaling mediates the ability to learn an active avoidance response following learned helplessness training.

Increased brain levels of acetylcholine (ACh) have been observed in patients with depression, and increasing ACh levels pharmacologically can precipitate stress-related behaviors in humans and animals. Conversely, optimal ACh levels are required for cognition and memory. We hypothesize that excessive ACh signaling results in strengthening of negative encoding in which memory formation is aberrantly strengthened for stressful events. The medial prefrontal cortex (mPFC) is critical for both top-down control of stress-related circuits, and for encoding of sensory experiences. We therefore evaluated the role of ACh signaling in the mPFC in a learned helplessness task in which mice were exposed to repeated inescapable stressors followed by an active avoidance task. Using fiber photometry with a genetically-encoded ACh sensor, we found that ACh levels in the mPFC during exposure to inescapable stressors were positively correlated with later escape deficits in an active avoidance test in males, but not females. Consistent with these measurements, we found that both pharmacologically- and chemogenetically-induced increases in mPFC ACh levels resulted in escape deficits in both male and female mice, whereas chemogenetic inhibition of ACh neurons projecting to the mPFC improved escape performance in males, but impaired escape performance in females. These results highlight the adaptive role of ACh release in stress response, but also support the idea that sustained elevation of ACh contributes to maladaptive behaviors. Furthermore, mPFC ACh signaling may contribute to stress-based learning differentially in males and females.

Analyzing Fusion Pore Dynamics and Counting the Number of Acetylcholine Molecules Released by Exocytosis.

Acetylcholine (ACh) is a critical neurotransmitter influencing various neurophysiological functions. Despite its significance, quantitative methods with adequate spatiotemporal resolution for recording a single exocytotic ACh efflux are lacking. In this study, we introduce an ultrafast amperometric ACh biosensor that enables 50 kHz electrochemical recording of spontaneous single exocytosis events at axon terminals of differentiated cholinergic human SH-SY5Y neuroblastoma cells with sub-millisecond temporal resolution. Characterization of the recorded amperometric traces revealed seven distinct current spike types, each displaying variations in shape, time scale, and ACh quantities released. This finding suggests that exocytotic release is governed by complex fusion pore dynamics in these cells. The absolute number of ACh molecules released during exocytosis was quantified by calibrating the sensor through the electroanalysis of liposomes preloaded with varying ACh concentrations. Notably, the largest quantal release involving approximately 8000 ACh molecules likely represents full exocytosis, while a smaller release of 5000 ACh molecules may indicate partial exocytosis. Following a local administration of bafilomycin A1, a V-ATPase inhibitor, the cholinergic cells exhibited both a larger quantity of ACh released and a higher frequency of exocytosis events. Therefore, this ACh sensor provides a means to monitor minute amounts of ACh and investigate regulatory release mechanisms at the single-cell level, which is vital for understanding healthy brain function and pathologies and optimizing drug treatment for disorders.

Flexible Acetylcholine Neural Probe with a Hydrophobic Laser-Induced Graphene Electrode and a Fluorous-Phase Sensing Membrane.

This work develops the first laser-induced graphene (LIG)-based electrochemical sensor with a superhydrophobic fluorous membrane for a flexible acetylcholine (ACh) sensor. ACh regulates several physiological functions, including synaptic transmission and glandular secretion. The ACh sensing membrane is doped with a fluorophilic cation-exchanger that can selectively measure ACh based on the inherent selectivity of the fluorous phase for hydrophobic ions, such as ACh. The fluorous-phase sensor improves the selectivity for ACh over Na+ and K+ by 2 orders of magnitude (compared to traditional lipophilic membranes), thus lowering the detection limit in artificial cerebrospinal fluid (aCSF) from 331 to 0.38 μ M, thereby allowing measurement in physiologically relevant ranges of ACh. Engraving LIG under argon creates a hydrophobic surface with a 133.7° contact angle, which minimizes the formation of a water layer. The flexible solid-contact LIG fluorous sensor exhibited a slope of 59.3 mV/decade in aCSF and retained function after 20 bending cycles, thereby paving the way for studying ACh's role in memory and neurodegenerative diseases.

Widefield in vivo imaging system with two fluorescence and two reflectance channels, a single sCMOS detector, and shielded illumination.

Widefield microscopy of the entire dorsal part of mouse cerebral cortex enables large-scale ("mesoscopic") imaging of different aspects of neuronal activity with spectrally compatible fluorescent indicators as well as hemodynamics via oxy- and deoxyhemoglobin absorption. Versatile and cost-effective imaging systems are needed for large-scale, color-multiplexed imaging of multiple fluorescent and intrinsic contrasts. We aim to develop a system for mesoscopic imaging of two fluorescent and two reflectance channels. Excitation of red and green fluorescence is achieved through epi-illumination. Hemoglobin absorption imaging is achieved using 525- and 625-nm light-emitting diodes positioned around the objective lens. An aluminum hemisphere placed between objective and cranial window provides diffuse illumination of the brain. Signals are recorded sequentially by a single sCMOS detector. We demonstrate the performance of our imaging system by recording large-scale spontaneous and stimulus-evoked neuronal, cholinergic, and hemodynamic activity in awake, head-fixed mice with a curved "crystal skull" window expressing the red calcium indicator jRGECO1a and the green acetylcholine sensor . Shielding of illumination light through the aluminum hemisphere enables concurrent recording of pupil diameter changes. Our widefield microscope design with a single camera can be used to acquire multiple aspects of brain physiology and is compatible with behavioral readouts of pupil diameter.

Non-enzymatic sensor based on doped polyindole/multi-walled carbon nanotube for detecting neurotransmitter acetylcholine

A facile non-enzymatic acetylcholine sensor composed of doped-polyindole/multi-walled carbon nanotube (dPIn-MWCNT) composite was designed, fabricated, and presented here. The negative current response toward acetylcholine concentration was first found and reported here. The confirmation of acetylcholine detection mechanism was elucidated by XPS, where the acetylcholine interaction resulted in a decrease in the % (dPIn doping level and an increase of the dPIn structural defect. The response optimizations with dPIn concentrations, applied potentials, and incubation times were systematically investigated. The 45dPIn-15MWCNT operated at + 0.6 V vs. Ag/AgCl with 5 min of incubation time was the optimum condition to obtain the highest acetylcholine current response. Chronoamperometric acetylcholine detection revealed two distinct current regimes from the acetylcholine surface interaction and the de-doping of dPIn. Regime I was related to the diffusion-controlled process as observed in the low concentration range of 10−8–10−5 M. Regime II corresponded to the mixed diffusion-controlled and adsorption-controlled process as observed in the concentration range of 10−5–10−2 M. For regime I, the calculated LOD was as low as 1.27 nM with a very high acetylcholine response sensitivity. Regime I is suggested for the point-of-care utility, where the detectable concentration range covered the acetylcholine level normally found in the healthy human blood. Regime II can be applicable and useful in the treatment monitoring of neurological diseases.

Cholinergic Basal Forebrain Connectivity to the Basolateral Amygdala Modulates Food Intake.

Obesity results from excessive caloric input associated with overeating and presents a major public health challenge. The hypothalamus has received significant attention for its role in governing feeding behavior and body weight homeostasis. However, extrahypothalamic brain circuits also regulate appetite and consumption by altering sensory perception, motivation, and reward. We recently discovered a population of basal forebrain cholinergic (BFc) neurons that regulate appetite suppression. Through viral tracing methods in the mouse model, we found that BFc neurons densely innervate the basolateral amygdala (BLA), a limbic structure involved in motivated behaviors. Using channelrhodopsin-assisted circuit mapping, we identified cholinergic responses in BLA neurons following BFc circuit manipulations. Furthermore, in vivo acetylcholine sensor and genetically encoded calcium indicator imaging within the BLA (using GACh3 and GCaMP, respectively) revealed selective response patterns of activity during feeding. Finally, through optogenetic manipulations in vivo, we found that increased cholinergic signaling from the BFc to the BLA suppresses appetite and food intake. Together, these data support a model in which cholinergic signaling from the BFc to the BLA directly influences appetite and feeding behavior.

Functionally refined encoding of threat memory by distinct populations of basal forebrain cholinergic projection neurons.

Neurons of the basal forebrain nucleus basalis and posterior substantia innominata (NBM/SIp) comprise the major source of cholinergic input to the basolateral amygdala (BLA). Using a genetically encoded acetylcholine (ACh) sensor in mice, we demonstrate that BLA-projecting cholinergic neurons can 'learn' the association between a naive tone and a foot shock (training) and release ACh in the BLA in response to the conditioned tone 24 hr later (recall). In the NBM/SIp cholinergic neurons express the immediate early gene, Fos following both training and memory recall. Cholinergic neurons that express Fos following memory recall display increased intrinsic excitability. Chemogenetic silencing of these learning-activated cholinergic neurons prevents expression of the defensive behavior to the tone. In contrast, we show that NBM/SIp cholinergic neurons are not activated by an innately threatening stimulus (predator odor). Instead, VP/SIa cholinergic neurons are activated and contribute to defensive behaviors in response to predator odor, an innately threatening stimulus. Taken together, we find that distinct populations of cholinergic neurons are recruited to signal distinct aversive stimuli, demonstrating functionally refined organization of specific types of memory within the cholinergic basal forebrain of mice.

How can I measure brain acetylcholine levels in vivo? Advantages and caveats of commonly used approaches.

The neurotransmitter acetylcholine (ACh) plays a central role in the regulation of multiple cognitive and behavioral processes, including attention, learning, memory, motivation, anxiety, mood, appetite, and reward. As a result, understanding ACh dynamics in the brain is essential for elucidating the neural mechanisms underlying these processes. In vivo measurements of ACh in the brain have been challenging because of the low concentrations and rapid turnover of this neurotransmitter. Here, we review a number of techniques that have been developed to measure ACh levels in the brain in vivo. We follow this with a deeper focus on use of genetically encoded fluorescent sensors coupled with fiber photometry, an accessible technique that can be used to monitor neurotransmitter release with high temporal resolution and specificity. We conclude with a discussion of methods for analyzing fiber photometry data and their respective advantages and disadvantages. The development of genetically encoded fluorescent ACh sensors is revolutionizing the field of cholinergic signaling, allowing temporally precise measurement of ACh release in awake, behaving animals. Use of these sensors has already begun to contribute to a mechanistic understanding of cholinergic modulation of complex behaviors.

Acetylcholine (ACh) enhances Cd tolerance through transporting ACh in vesicles and modifying Cd absorption in duckweed (Lemna turionifera 5511)

Acetylcholine (ACh), an important neurotransmitter, plays a role in resistance to abiotic stress. However, the role of ACh during cadmium (Cd) resistance in duckweed (Lemna turionifera 5511) remains uncharacterized. In this study, the changes of endogenous ACh in duckweed under Cd stress has been investigated. Also, how exogenous ACh affects duckweed's ability to withstand Cd stress was studied. The ACh sensor transgenic duckweed (ACh 3.0) showed the ACh signal response under Cd stress. And ACh was wrapped and released in vesicles. Cd stress promoted ACh content in duckweed. The gene expression analysis showed an improved fatty acid metabolism and choline transport. Moreover, exogenous ACh addition enhanced Cd tolerance and decreased Cd accumulation in duckweed. ACh supplement reduced the root abscission rate, alleviated leaf etiolation, and improved chlorophyll fluorescence parameters under Cd stress. A modified calcium (Ca2+) flux and improved Cd2+ absorption were present in conjunction with it. Thus, we speculate that ACh could improve Cd resistance by promoting the uptake and accumulation of Cd, as well as the response of the Ca2+ signaling pathway. Also, plant-derived extracellular vesicles (PDEVs) were extracted during Cd stress. Therefore, these results provide new insights into the response of ACh during Cd stress.

Detection of Acetylcholine in an Enzyme‐Free System Based on a GCE/V2O5 NRs/BPM Modified Sensor

AbstractIn this approach, vanadium pentoxide nanorods (V2O5NRs) were prepared by utilizing a facile wet‐chemical approach (WCA) in an alkaline phase at low temperatures then applied to detect acetylcholine. Various optical techniques, such as Ultra‐Violet spectroscopy (UV‐Visible), Fourier Transform Infra‐red spectroscopy (FTIR), Powder X‐ray Diffraction (XRD), Field Emission Scanning Emission Microscopy (FESEM), X‐ray Electron Dispersive Spectroscopy (XEDS) and X‐ray Photo‐electron Spectroscopy (XPS) were applied to analyse the V2O5NRs for structural, optical, functional, morphological and elemental analyses. Adjustment of the light weight fabrication of NRs onto Glassy carbon electrode (GCE) exhibited sensitive and selective acetylcholine sensor probe. Based on the stable current‐voltage relationship, analytical sensing parameters like sensitivity, Linear dynamic range (LDR), Limit of detection (LOD), Limit of quantification (LOQ), durability and interference measurement of the fabricated sensor (GCE/V2O5NRs/BPM) for acetylcholine detection were evaluated and reported in this paper. Here, Nafion was used as a binding polymer matrix (BPM) for the fabrication of flat GCE with V2O5NRs. Acetylcholine calibration curve was found to be linear throughout in a broad range of concentration of target acetylcholine analyte. Calibration curve was employed to calculate the sensing parameters such as sensitivity (101.27 nAμM−1cm−2), LOD down to 11.58 pM, LOQ (38.60 pM) and LDR (100 pM∼100 μM) of the developed GCE/V2O5NRs/BPM sensor probe. The use of the facile WCA method for the synthesis of low‐dimensional V2O5NRs is a good strategy for developing an un‐doped nano‐material based sensor for enzyme‐free bio‐molecule recognition and detection via this electrochemical approach. The developed (GCE/V2O5NRs/BPM) sensor probe could be used to selectively detect acetylcholine in real biological samples by electrochemical method with satisfactory outcomes.

Hippocampal acetylcholine modulates stress-related behaviors independent of specific cholinergic inputs.

Acetylcholine (ACh) levels are elevated in actively depressed subjects. Conversely, antagonism of either nicotinic or muscarinic ACh receptors can have antidepressant effects in humans and decrease stress-relevant behaviors in rodents. Consistent with a role for ACh in mediating maladaptive responses to stress, brain ACh levels increase in response to stressful challenges, whereas systemically blocking acetylcholinesterase (AChE, the primary ACh degradative enzyme) elicits depression-like symptoms in human subjects, and selectively blocking AChE in the hippocampus increases relevant behaviors in rodents. We used an ACh sensor to characterize stress-evoked ACh release, then used chemogenetic, optogenetic and pharmacological approaches to determine whether cholinergic inputs from the medial septum/diagonal bands of Broca (MSDBB) or ChAT-positive neurons intrinsic to the hippocampus mediate stress-relevant behaviors in mice. Chemogenetic inhibition or activation of MSDBB cholinergic neurons did not result in significant behavioral effects, while inhibition attenuated the behavioral effects of physostigmine. In contrast, optogenetic stimulation of septohippocampal terminals or selective chemogenetic activation of ChAT-positive inputs to hippocampus increased stress-related behaviors. Finally, stimulation of sparse ChAT-positive hippocampal neurons increased stress-related behaviors in one ChAT-Cre line, which were attenuated by local infusion of cholinergic antagonists. These studies suggest that ACh signaling results in maladaptive behavioral responses to stress if the balance of signaling is shifted toward increased hippocampal engagement.

Enzymeless copper microspheres@carbon sensor design for sensitive and selective acetylcholine screening in human serum

Follow up of neuronal disorders, such as Alzheimer’s and Parkinson’s diseases using simple, sensitive, and selective assays is urgently needed in clinical and research investigation. Here, we designed a sensitive and selective enzymeless electrochemical acetylcholine sensor that can be used in human fluid samples. The designed electrode consisted of a micro spherical construction of Cu-metal decorated by a thin layer of carbon (CuMS@C). A simple and one-pot synthesis approach was used for Cu-metal controller formation with a spherical like structures. The spherical like structure was formed with rough outer surface texture, circular build up, homogeneous formation, micrometric spheres size (0.5 −1 µm), and internal hollow structure. The formation of a thin layer of carbon materials on the surface of CuMS sustained the catalytic activity of Cu atoms and enriched negatively charge of the surface. CuMS@C acted as a highly active mediator surface that consisted of Cu metal as a highly active catalyst and carbons as protecting, charge transport, and attractive surface. Therefore, the CuMS@C surface morphology and composition played a key role in various aspects such as facilitated ACh diffusion/loading, increased the interface surface area, and enhanced the catalytic activity. The CuMS@C acted as an electroactive catalyst for ACh electrooxidation and current production, due to the losing of two electrons. The fabricated CuMS@C could be a highly sensitive and selective enzymeless sensor for detecting ACh with a detection limit of 0.1 µM and a wide linear range of 0.01 – 0.8 mM. The designed ACh sensor assay based on CuMS@C exhibited fast sensing property as well as sensitivity, selectivity, stability, and reusability for detecting ACh in human serum samples. This work presents the design of a highly active electrode surface for direct detection of ACh and further clinical investigation of ACh levels.

Cholinergic suppression of hippocampal sharp-wave ripples impairs working memory

Learning and memory are assumed to be supported by mechanisms that involve cholinergic transmission and hippocampal theta. Using G protein-coupled receptor-activation-based acetylcholine sensor (GRABACh3.0) with a fiber-photometric fluorescence readout in mice, we found that cholinergic signaling in the hippocampus increased in parallel with theta/gamma power during walking and REM sleep, while ACh3.0 signal reached a minimum during hippocampal sharp-wave ripples (SPW-R). Unexpectedly, memory performance was impaired in a hippocampus-dependent spontaneous alternation task by selective optogenetic stimulation of medial septal cholinergic neurons when the stimulation was applied in the delay area but not in the central (choice) arm of the maze. Parallel with the decreased performance, optogenetic stimulation decreased the incidence of SPW-Rs. These findings suggest that septo-hippocampal interactions play a task-phase-dependent dual role in the maintenance of memory performance, including not only theta mechanisms but also SPW-Rs.

Research on Competitive Enzymatic Hydrolysis-Assisted Liquid Crystal-based Acetylcholine Sensor

1. 报道了一种基于液晶的简单、低成本、无标记的检测乙酰胆碱的方法。 2. 氯化十四酰胆碱与乙酰胆碱发生竞争性的酶解反应，引起液晶分子在界面上的排列方式及液晶的光学形貌发生了改变。 3. 乙酰胆碱的检测限低至14.5 nmol/L。 4. 基于液晶实现了对乙酰胆碱高灵敏度、高特异性的检测。 A simple, convenient and label-free approach was developed for detection of acetylcholine (ACh) based on competitive enzymatic hydrolysis by use of liquid crystals (LCs) as a sensing medium. Self-assembled monolayers of myristoylcholine (Myr) were obtained at the aqueous/LC interface when the surfactants were in contact with LCs, resulting in a perpendicular orientation of LCs at the interface. Thus, LCs displayed a dark optical image. However, enzymatic hydrolysis of Myr by acetylcholinesterase (AChE) disturbed the Myr monolayers, which led to a horizontal orientation of LCs at the interface, thereby inducing a dark-to-bright shift of the LCs optical appearance. Interestingly, as ACh was also a substrate of AChE, the bright appearance of LCs was prevented when incubating the mixture of AChE and ACh with Myr decorated aqueous/LC interface due to the competitive enzymatic hydrolysis between Myr and ACh by AChE. By using this strategy, ACh could be detected selectively and sensitively, with a broad linear range from 0.01 μmol/L to 1 mmol/L and a low detection limit of 14.5 nmol/L. The results showed that the competitive enzymatic hydrolysis-assisted LC-based sensor had potential applications in ACh detection. A simple, low-cost and label-free assay was reported for detection of acetylcholine (ACh) based on liquid crystals (LCs). Variations in the arrangement and optical appearance of LCs are due to the competitive enzymatic hydrolysis between myristoylcholine (Myr) and ACh by acetylcholinesterase (AChE). By using this strategy, highly sensitive and highly specific detection of ACh is achieved.

An optimized acetylcholine sensor for monitoring in vivo cholinergic activity.

The ability to directly measure acetylcholine (ACh) release is an essential step towards understanding its physiological function. Here, we optimized the GRABACh (GPCR-Activation‒Based-ACh) sensor to achieve substantially improved sensitivity in ACh detection, as well as reduced downstream coupling to intracellular pathways. The improved version of the ACh sensor retains the sub-second response kinetics, physiological-relevant affinity and precise molecular specificity to ACh of its predecessor. Using this sensor, we revealed compartmental ACh signals in the olfactory center of transgenic flies in response to external stimuli including odor and body shock. Using fiber photometry recording and two-photon imaging, our ACh sensor also enabled sensitive detection of single-trial ACh dynamics in multiple brain regions in mice in a variety of behaviors.

Acetylcholine is released in the basolateral amygdala in response to predictors of reward and enhances the learning of cue-reward contingency.

The basolateral amygdala (BLA) is critical for associating initially neutral cues with appetitive and aversive stimuli and receives dense neuromodulatory acetylcholine (ACh) projections. We measured BLA ACh signaling and activity of neurons expressing CaMKIIα (a marker for glutamatergic principal cells) in mice during cue-reward learning using a fluorescent ACh sensor and calcium indicators. We found that ACh levels and nucleus basalis of Meynert (NBM) cholinergic terminal activity in the BLA (NBM-BLA) increased sharply in response to reward-related events and shifted as mice learned the cue-reward contingency. BLA CaMKIIα neuron activity followed reward retrieval and moved to the reward-predictive cue after task acquisition. Optical stimulation of cholinergic NBM-BLA terminal fibers led to a quicker acquisition of the cue-reward contingency. These results indicate BLA ACh signaling carries important information about salient events in cue-reward learning and provides a framework for understanding how ACh signaling contributes to shaping BLA responses to emotional stimuli.

SPARC_A DNA‐based optical nanosensor for in vivo imaging of acetylcholine in the peripheral nervous system

The study of neurotransmitter signaling helps to understand fundamental neuronal circuits and develop strategies against neurodegenerative diseases. Acetylcholine (ACh) plays a pivotal role in modulating neuron functions. Monitoring its physiological level in real‐time and probing its biological distribution is of crucial importance. However, the current techniques are subject to temporal and spatial limitations which impede their further in vivo applications. Herein we report the development of a ACh nanosensor by using DNA as a scaffold, acetylcholinesterase as a recognition component, pH‐sensitive fluorophores as signal generator, and α‐bungarotoxin as a targeting ligand. The nanosensor was delivered to the submandibular ganglion in living mice by microinjection for in vivo imaging of endogenous ACh release. The ACh nanosensor selectively binds to acetylcholine receptors on the post‐synaptic neurons, and display a sensitive response to physiological ACh range from 0.228 μM to 358 μM in ex vivo calibration. The ACh nanosensors also respond to endogenous ACh release triggered by electrical stimulation in a reversible and dose‐dependent manner. We also found that the treatment of cholinergic inhibitor, Vesamicol, significant suppresses the response of the ACh nanosensor. This work provides a tool for in vivo imaging of ACh dynamics with unprecedented capabilities to enhance the spatial and temporal resolution of detection. We envision such a sensor platform could be applied to imaging other neurotransmitters in mammalian system as well as extended to other organ systems in the peripheral nervous systemSupport or Funding InformationNIH common fund_SPARC_Project number: 1OT2OD024909‐01DNA‐based acetylcholine (ACh) nanosensors detect the ACh in the submandibular ganglion (SMG) of living mice. (A) ACh sensors were microinjected into the SMG of a living mouse. With the targeting ability of bungarotoxin, the ACh nanosensor can immobilize in the synaptic cleft of the mouse. (B) The mechanism of ACh nanosensors. (D) ΔF/F from pHAb channel in response to electrical stimulation (indicated by red arrow).Figure 1

Acetylcholine biosensor based on the electrochemical functionalization of graphene field-effect transistors

We present a new strategy of Acetylcholinesterease (AchE) immobilization on graphene field-effect transistors (gFETs) for building up Acetylcholine sensors. This method is based on the electrosynthesis of an amino moiety-bearing polymer layer on the graphene channel. The film of the copolymer poly(3-amino-benzylamine-co-aniline) (PABA) does not only provide the suitable electrostatic charge and non-denaturing environment for enzyme immobilization, but it also improves the pH sensitivity of the gFETs (from 40.8 to 56.3 μA/pH unit), probably due to its wider effective pKa distribution. The local pH changes caused by the enzyme-catalyzed hydrolysis produce a shift in the Dirac point of the gFETs to more negative values, which are evidenced as differences in the gFET conductivity and thereby constituted the signal transduction mechanism of the modified transistors. In this way, the constructed biosensors showed a LOD of 2.3 μM and were able to monitor Ach in the range from 5 to 1000 μM in a flow configuration. Moreover, they showed a sensitivity of −26.6 ± 0.7 μA/Ach decade and also exhibited a very low RSD of 2.6%, revealing good device-to-device reproducibility. The biosensors revealed an excellent selectivity to interferences known to be present in the extracellular milieu, and the response to Ach was recovered by 97.5% after the whole set of interferences injected. Finally, the biosensors showed a fast response time, with an average value of 130 s and a good long-term response.

Communication—Microfluidic Electrochemical Acetylcholine Detection in the Presence of Chlorpyrifos

An amperometric acetylcholine sensor was developed for use in a microfluidic system and characterized with chlorpyrifos and its metabolite, chlorpyrifos oxon. This enzymatic sensor was highly selective for acetylcholine, with a detection limit of 0.2 μM and sensitivity of 1.7 nA μM−1 from 1–150 μM. Though chlorpyrifos had no effect on sensor function, chlorpyrifos oxon significantly inhibited response across a range of concentrations (0.5–50 μM). Inhibition was reversed by 2-pyridine aldoxime methyl chloride. This platform can be used to both quantify acetylcholine in the presence of chlorpyrifos and as a biorecognition method for chlorpyrifos oxon.

Low energy nitrogen ion beam implanted tungsten trioxide thin films modified indium tin oxide electrode based acetylcholine sensor

Abstract This paper reports the fabrication of non-enzymatic acetylcholine (ACh) electrochemical sensor based on low energy nitrogen ion (100 keV) implanted tungsten trioxide (WO3) thin films modified indium tin oxide (ITO) electrode. The WO3 thin films deposited on ITO by spin coating technique was subjected to nitrogen ion implantation with the optimum fluence of 1 × 1015 ions/cm2. The implanted electrode was characterized by X-ray diffraction, field emission scanning electron microscopy (FESEM), photoluminescence spectra, Hall Effect measurements, cyclic voltammetry and amperometry. The implanted WO3 modified ITO electrode induces desirable changes in the crystal structure, surface morphology and electron mobility. Compared with pristine WO3/ITO, nitrogen ion implanted WO3/ITO displayed improved electrical conductivity and electrocatalytic activity towards ACh oxidation in 0.1 M potassium hydroxide solution (KOH). Amperometric measurements showed that the fabricated electrode had a wide linear response range of 0.1–8000 µM, higher sensitivity of 140.57 ± 0.62 mA/M/cm2 and the lowest detection limit of 28 nM, which are superior among the non-enzymatic ACh electrochemical sensors. The proposed sensor exhibits an excellent anti-interferential ability, good stability and reproducibility and successfully applied for the determination of ACh levels in human serum samples.