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

Recording of microvascular dimensions with an image-splitter television microscope.

<Introduction> It has recently been revealed that oxytocin (OT), a peptide hormone known for its role in lactation and parturition, works as a neurotransmitter as well. Due to a series of findings of positive effects on social behaviors such as trust in human, oxytocin has been of keen interest to neuroscientists. What is required now for further understanding of oxytocin science is a real-time measurement of oxytocin in vivo. On the other hand, in the region where oxytocin is secreted in the hypothalamus, another peptide, vasopressin (VP), is also secreted. Vasopressin is also a nonapeptide with a quite similar structure to oxytocin. Therefore, selective measurement of oxytocin and vasopressin is inevitable. Electrochemical detection allows a real-time measurement in millisecond order with high sensitivity. Compared to conventional electrodes, boron-doped diamond (BDD) electrode has a variety of outstanding properties such as wide potential window and low background current, which have led to a number of reports about sensitive measurement of substances which cannot be detected by other electrodes. Using BDD microelectrodes, in vivomeasurement of biomolecules have been achieved and applied to medical and physiological studies. In addition, surface termination of BDD can be changed from original state (hydrogen termination) to oxygen termination by some oxidation treatment, which enables selective measurement. In the present study, electrochemical detection of oxytocin was investigated using BDD electrodes. Two types of BDD, as-deposited (AD-BDD) and anodically-oxidized (AO-BDD), were used in order to achieve selective measurement. <Experiments> BDD electrodes were prepared by growing polycrystalline BDD thin films on silicon substrates or needle tungsten wires using microwave plasma-assisted chemical vapor deposition (MPCVD) system. Electrochemical measurements were conducted in a three-electrode system with BDD, platinum, and Ag/AgCl (KCl saturated) electrode as a working electrode, counter electrode, and reference electrode, respectively. Phosphate buffer saline (PBS) and Tris buffer were used as buffer solution after adjusted to pH 7.4. Surface transformation of BDD electrodes to oxygen termination was conducted on AD-BDD by means of anodic oxidation of 3.0 V application for 20 minutes in PBS. <Results and discussion>The electrochemical behavior of oxytocin was studied using AD-BDD. Cyclic voltammetry (CV) was performed for 0.1 mM oxytocin in 0.1 M PBS with a scan rate of 100 mV/s. Oxidation peak was observed at 0.7 V (vs. Ag/AgCl). Among the 9 amino acids constructing oxytocin, only tyrosine (Tyr) has been reported to be electrochemically oxidized. Tyrosine also gave oxidation signal at 0.7 V and the shape of voltammogram was quite similar to that of oxytocin. Consequently, it was deduced that oxidation of oxytocin is occurred at tyrosine moiety. When compared to other electrodes, BDD showed 4 times higher signal to background ratio than glassy carbon electrode. No signal was observed in the case of platinum electrode. These results showed that sensitive measurement of oxytocin is possible by using BDD electrode. Since vasopressin also contains tyrosine in its structure, it should show the oxidation signal. CV of vasopressin was compared to that of oxytocin. Exactly the same voltammograms were observed. On the other hand, AO-BDD, which has oxygen-terminated surface, showed apparent difference in voltammograms (Figure). Although the peak potential of vasopressin was maintained at the same position as AD-BDD, oxytocin showed a broad signal shifted to more positive potential region. On-set potential of tyrosine was still higher than that of oxytocin. One possible explanation to the results is the electrostatic interaction between the electrode surface and the molecules. The surface state of AO-BDD rich in C-O functional groups can be expected to have electrostatically negative state. On the other hand, tyrosine is negatively charged, oxytocin is almost neutral, and vasopressin is positively charged in the condition of pH 7.4. These facts and the results of CV well explain the mechanism of electrostatic interaction. Aiming at in vivo and selective measurement, chronoamperometry combined with flow injection analysis (FIA) by BDD microelectrodes was conducted. AD-BDD microelectrode applied at 1.0 V gave high linearity (R2 = 0.994) of the current peaks over the oxytocin concentration range from 0.1 to 10.0 μM with a detection limit of 50 nM (S/N =3). On the other hand, using AO-BDD microelectrode, selective measurement of oxytocin and vasopressin was achieved by the use of applied potential of 0.54 V, which gave the signals of only vasopressin. Consequently, the concentration of oxytocin can be obtained by subtracting the concentration of vasopressin measured on AO-BDD from that of oxytocin + vasopressin measured on AD-BDD. Figure 1

Parkinson's disease (PD) is a progressive neurodegenerative disorder causing the loss of dopaminergic neurons in the substantia nigra and the drastic depletion of dopamine (DA) in the striatum; thus, DA can act as a marker for PD diagnosis and therapeutic evaluation. However, detecting DA in the brain is not easy because of its low concentration and difficulty in sampling. In this work, we report the fabrication of a covalent organic framework (COF)-modified carbon fiber microelectrode (cCFE) that enables the real-time detection of DA in the mouse brain thanks to the outstanding antibiofouling and antichemical fouling ability, excellent analytical selectivity, and sensitivity offered by the COF modification. In particular, the COF can inhibit the polymerization of DA on the electrode (namely, chemical fouling) by spatially confining the molecular conformation and electrochemical oxidation of DA. The cCFE can stably and continuously work in the mouse brain to detect DA and monitor the variation of its concentration. Furthermore, it was combined with levodopa administration to devise a closed-loop feedback mode for PD diagnosis and therapy, in which the cCFE real-time monitors the concentration of DA in the PD model mouse brain to instruct the dose and injection time of levodopa, allowing a customized medication to improve therapeutic efficacy and meanwhile avoid adverse side effects. This work demonstrates the fascinating properties of a COF in fabricating electrochemical sensors for in vivo bioanalysis. We believe that the COF with structural tunability and diversity will offer enormous promise for selective detection of neurotransmitters in the brain.

Monoamine oxidases A and B (MAO‐A and MAO‐B) are key enzymes involved in neurotransmitter metabolism and are critical biomarkers for neurodegenerative and psychiatric disorders. Here, we present a highly sensitive nanopore‐based method for the selective detection and quantification of MAO‐A and MAO‐B using specifically designed peptide probes. These probes undergo enzyme‐specific oxidation, generating distinct nanopore translocation signatures that enable precise identification. Our method achieves picomolar‐level detection, outperforming conventional assays such as ELISA. Importantly, we demonstrate its applicability in complex biological samples, including cell lysates (SH‐SY5Y and HepG2) and mouse brain and liver tissues, with results strongly correlating with ELISA. The ability to selectively detect both enzymes within a single sample highlights its advantage in studying enzyme interplay in biological systems. This label‐free, real‐time approach offers a powerful tool for biomedical research, disease diagnostics, and drug screening, with potential for expanding to other clinically relevant enzymes.

We have investigated the capability of nanoparticle-assisted laser desorption ionization mass spectrometry (NP-LDI MS), matrix-assisted laser desorption ionization (MALDI) MS, and gas cluster ion beam secondary ion mass spectrometry (GCIB SIMS) to provide maximum information available in lipid analysis and imaging of mouse brain tissue. The use of Au nanoparticles deposited as a matrix for NP-LDI MS is compared to MALDI and SIMS analysis of mouse brain tissue and allows selective detection and imaging of groups of lipid molecular ion species localizing in the white matter differently from those observed using conventional MALDI with improved imaging potential. We demonstrate that high-energy (40 keV) GCIB SIMS can act as a semi-soft ionization method to extend the useful mass range of SIMS imaging to analyze and image intact lipids in biological samples, closing the gap between conventional SIMS and MALDI techniques. The GCIB SIMS allowed the detection of more intact lipid compounds in the mouse brain compared to MALDI with regular organic matrices. The 40 keV GCIB SIMS also produced peaks observed in the NP-LDI analysis, and these peaks were strongly enhanced in intensity by exposure of the sample to trifluororacetic acid (TFA) vapor prior to analysis. These MS techniques for imaging of different types of lipids create a potential overlap and cross point that can enhance the information for imaging lipids in biological tissue sections.Graphical abstractSchematic of mass spectral imaging of a mouse brain tissue using GCIB-SIMS and MALDI techniquesElectronic supplementary materialThe online version of this article (doi:10.1007/s00216-016-9812-5) contains supplementary material, which is available to authorized users.

Recognition unit-free and self-cleaning photoelectrochemical sensing platform on TiO2 nanotube photonic crystals for sensitive and selective detection of dopamine release from mouse brain

Monoamine oxidases A and B (MAO-A and MAO-B) are key enzymes involved in neurotransmitter metabolism and are critical biomarkers for neurodegenerative and psychiatric disorders. Here, we present a highly sensitive nanopore-based method for the selective detection and quantification of MAO-A and MAO-B using specifically designed peptide probes. These probes undergo enzyme-specific oxidation, generating distinct nanopore translocation signatures that enable precise identification. Our method achieves picomolar-level detection, outperforming conventional assays such as ELISA. Importantly, we demonstrate its applicability in complex biological samples, including cell lysates (SH-SY5Y and HepG2) and mouse brain and liver tissues, with results strongly correlating with ELISA. The ability to selectively detect both enzymes within a single sample highlights its advantage in studying enzyme interplay in biological systems. This label-free, real-time approach offers a powerful tool for biomedical research, disease diagnostics, and drug screening, with potential for expanding to other clinically relevant enzymes.

Sensitive and selective monitoring of sialic acid (SA) in cerebral nervous system is of great importance for studying the role that SA plays in the pathological process of Alzheimer's disease (AD). In this work, we first reported an electrochemical biosensor based on a novel stimuli-responsive copolymer for selective and sensitive detection of SA in mouse brain. Notably, through synergetic hydrogen-bonding interactions, the copolymer could translate the recognition of SA into their conformational transition and wettability switch, which facilitated the access and enrichment of redox labels and targets to the electrode surface, thus significantly improving the detection sensitivity with the detection limit down to 0.4 pM. Besides amplified sensing signals, the proposed method exhibited good selectivity toward SA in comparison to potential interference molecules coexisting in the complex brain system due to the combination of high affinity between phenylboronic acid (PBA) and SA and the directional hydrogen-bonding interactions in the copolymer. The electrochemical biosensor with remarkable analytical performance was successfully applied to evaluate the dynamic change of SA level in live mouse brain with AD combined with in vivo midrodialysis. The accurate concentration of SA in different brain regions of live mouse with AD has been reported for the first time, which is beneficial for progressing our understanding of the role that SA plays in physiological and pathological events in the brain.

The development and validation of an assay for the determination of paclitaxel in human plasma, human brain tumor tissue, mouse plasma and mouse brain tumor tissue is described. Paclitaxel was extracted from the matrices using liquid-liquid extraction with tert-butyl methyl ether, followed by chromatographic analysis using an alkaline eluent. Positive ionization electrospray tandem mass spectrometry was performed for selective and sensitive detection. The method was validated according to the FDA guidelines on bioanalytical method validation. Validation results indicate that calibration standards in human plasma can be used to quantify paclitaxel in all tested matrices. In human samples, the validated range for paclitaxel was from 0.25-1000 ng ml(-1) using 200 microl plasma aliquots and from 5 to 5000 ng g(-1) using 50 microl tumor homogenate aliquots (0.2 g tissue ml(-1) control human plasma). In mice, the ranges were 1-1000 ng ml(-1) and 5-5000 ng g(-1) using 50 microl of mouse plasma and 50 microl of tumor homogenate aliquots (0.2 g tissue ml(-1) control human plasma), respectively. The method can be applied to studies generating only small sample volumes (e.g. mouse plasma and tumor tissue), but also to studies in human plasma requiring a lower limit of quantitation. The assay was applied successfully to several studies with both human and mouse samples.

In vitro and in vivo dose-dependent inhibition of methylmercury on glutamine synthetase in the brain of different species

Noninvasive imaging of tau and amyloid-β pathologies would facilitate diagnosis of Alzheimer's disease (AD). Recently, we have developed [(18)F]THK-5105 for selective detection of tau pathology by positron emission tomography (PET). The purpose of this study was to clarify biological properties of optically pure [(18)F]THK-5105 enantiomers. Binding for tau aggregates in AD brain section was evaluated by autoradiography (ARG). In vitro binding assays were performed to evaluate the binding properties of enantiomers for AD brain homogenates. The pharmacokinetics in the normal mouse brains was assessed by ex vivo biodistribution assay The ARG of enantiomers showed the high accumulation of radioactivity corresponding to the distribution of tau deposits. In vitro binding assays revealed that (S)-[(18)F]THK-5105 has slower dissociation from tau than (R)-[(18)F]THK-5105. Biodistribution assays indicated that (S)-[(18)F]THK-5105 eliminated faster from the mouse brains and blood compared with (R)-[(18)F]THK-5105. (S)-[(18)F]THK-5105 could be more suitable than (R)-enantiomer for a tau imaging agent.

Soluble amyloid β (Aβ) aggregates have attracted attention as therapeutic targets and biomarkers of Alzheimer's disease. We previously reported a fluorescent probe, BAOP-16, which targets soluble Aβ aggregates. However, its limited aqueous solubility has hindered its application in biological assays. In this study, we developed aqueous-soluble fluorescent probes that target soluble Aβ aggregates. Among the synthesized candidates, sBAOP-2-3, which is substituted with two CH2C-Tf2 groups and a diphenyl amino group, exhibited markedly improved aqueous solubility compared with BAOP-16. It also showed a marked increase in fluorescence intensity in the presence of Aβ oligomers, and the highest selectivity for Aβ oligomers over Aβ fibrils. During the aggregation process of Aβ, sBAOP-2-3 detected soluble Aβ aggregates in an early phase. Furthermore, staining experiments using mouse brain sections suggested that sBAOP-2-3 selectively stained soluble Aβ aggregates in mouse brains. These results suggest that sBAOP-2-3 shows promise as a probe for the selective detection of soluble Aβ aggregates in biological systems.

A covalent organic frameworks based sensor for adsorptive stripping voltammetric detection of nanomolar dopamine in living mouse brain

Selective detection of reactive oxygen species (ROS) is vital for studying their role in brain diseases. Fluorescence probes can distinguish ONOO- species from other ROS; however, their selectivity toward ONOO- species depends on the ONOO- recognition group. Aryl-boronic acids and esters, which are common ONOO- recognition groups, are not selective for ONOO- over H2O2. In this study, we developed a diaminonaphthalene (DAN)-protected boronic acid as a new ONOO- recognition group that selectively reacts with ONOO- over H2O2 and other ROS. Three DAN-protected boronic acid (DANBA)-based fluorophores that emit fluorescence over visible to near-infrared (NIR) regions, Cou-BN, BVP-BN, and HDM-BN, and their aryl-boronic acid-based counterparts (Cou-BO, BVP-BO, and HDM-BO), were developed. The DANBA-based probes exhibited enhanced selectivity toward ONOO- over that of their control group, as well as universality in solution assays and in vitro experiments with PC12 cells. The NIR-emissive HDM-BN was optimized to delineate in vivo ONOO- levels in mouse brains with Parkinson's disease. This DAN-protected boronic acid belongs to a new generation of recognition groups for developing ONOO- probes, and this strategy could be extended to other common hydroxyl-containing dyes to detect ONOO- levels in complex biological systems and processes.

Hydrogen peroxide (H2O2), a pivotal reactive oxygen species (ROS), is closely linked to oxidative stress in the pathogenesis of Alzheimer's disease (AD). Herein, we report a dual-mode probe (Re-PS) integrating turn-on fluorescence and ratiometric electrochemistry for the selective detection of H2O2 in brain microdialysates of AD model mice. The probe is constructed using resorufin (Re) as a dual-signal reporter and a pentafluorobenzenesulfonyl (PS) group as the H2O2-responsive unit. Upon reaction with H2O2, the PS group undergoes nucleophilic substitution, leading to the release of Re; this process triggers a fluorescence "turn-on" response and generates a ratiometric electrochemical signal. Compared with ester-based probes, Re-PS shows superior stability due to the strong electron-withdrawing effect of fluorine atoms in the PS group. The fluorescence mode achieves a detection limit (LOD) of 50 nM, while the electrochemical detection mode (using a carbon fiber microelectrode modified with carbon nanotubes (CFME/CNT)) has a detection range of 1.0-50 μM. Both modes exhibit excellent selectivity against other ROS and biomolecules. In vivo microdialysis analysis reveals significantly elevated H2O2 levels in the brains of AD mice (28.6 ± 3.2 μM) compared with wild-type mice (10.3 ± 1.8 μM). This dual-mode strategy enables cross-validation, providing a reliable tool for monitoring oxidative stress in neurodegenerative diseases.