Miniaturized Implantable Fluorescence Probes Integrated with Metal-Organic Frameworks for Deep Brain Dopamine Sensing.

Continuously monitoring neurotransmitter dynamics can offer profound insights into neural mechanisms and the etiology of neurological diseases. Here, we present a miniaturized implantable fluorescence probe integrated with metal-organic frameworks (MOFs) for deep brain dopamine sensing. The probe is assembled from physically thinned light-emitting diodes (LEDs) and phototransistors, along with functional surface coatings, resulting in a total thickness of 120 µm. A fluorescent MOF that specifically binds dopamine is introduced, enabling a highly sensitive dopamine measurement with a detection limit of 79.9 nM. A compact wireless circuit weighing only 0.85 g is also developed and interfaced with the probe, which was later applied to continuously monitor real-time dopamine levels during deep brain stimulation in rats, providing critical information on neurotransmitter dynamics. Cytotoxicity tests and immunofluorescence analysis further suggest a favorable biocompatibility of the probe for implantable applications. This work presents fundamental principles and techniques for integrating fluorescent MOFs and flexible electronics for brain-computer interfaces and may provide more customized platforms for applications in neuroscience, disease tracing, and smart diagnostics.

Heterometallic MIL-125(Ti-Al) frameworks for electrochemical determination of ascorbic acid, dopamine and uric acid.

The detection of ascorbic acid (AA), dopamine (DA), and uric acid (UA) is not only of great significance in the areas of biomedicine and neurochemistry but also helpful in disease diagnosis and pathology research. Due to their diverse structures, designability, and large specific surface areas, metal-organic frameworks (MOFs) have recently caught considerable attention in the electrochemical field. Herein, a family of heterometallic MOFs with amino modification, MIL-125(Ti-Al)-xNH2 (x = 0%, 25%, 50%, 75%, and 100%), were synthesized and employed as electrochemical sensors for the detection of AA, DA, and UA. Among them, MIL-125(Ti-Al)-75%NH2 exhibited the most promising electrochemical behavior with 40% doping of carbon black in 0.1 M PBS (pH = 7.10), which displayed individual detection performance with wide linear detection ranges (1.0-6.5 mM for AA, 5-100 µM for DA and 5-120 µM for UA) and low limits of detection (0.215 mM for AA, 0.086 µM for DA, and 0.876 µM for UA, S/N = 3). Furthermore, the as-prepared MIL-125(Ti-Al)-75%NH2/GCE provided a promising platform for future application in real sample analysis, owing to its excellent anti-interference performance and good stability.

A wearable flexible electrochemical biosensor with CuNi-MOF@rGO modification for simultaneous detection of uric acid and dopamine in sweat.

BACKGROUND: In health assessment and personalized medical services, accurate detection of biological markers such as dopamine (DA) and uric acid (UA) in sweat is crucial for providing valuable physiological information. However, there are challenges in detecting sweat biomarkers due to their low concentrations, variations in sweat yield among individuals, and the need for efficient sweat collection. RESULTS: We synthesized CuNi-MOF@rGO as a high-activity electrocatalyst and investigated its feasibility and electrochemical mechanism for simultaneously detecting low-concentration biomarkers UA and DA. Interaction between the non-coordinating carboxylate group and the sample produces effective separation signals for DA and UA. The wearable biomimetic biosensor has a wide linear range of 1-500 µM, with a detection limit of 9.41 µM and sensitivity of 0.019 µA µM-1 cm-2 for DA, and 10-1000 µM, with a detection limit of 9.09 µM and sensitivity of 0.026 µA µM-1 cm-2 for UA. Thus, our sensor performs excellently in detecting low-concentration biomarkers. To improve sweat collection, we designed a microfluidic-controlled device with hydrophilic modification in the microchannel. Experimental results show optimal ink flow at 2% concentration. Overall, we developed an innovative and highly active electrocatalyst, successfully enabling simultaneous detection of low-concentration biomarkers UA and DA. SIGNIFICANCE: This study provides a strategy for sweat analysis and health monitoring. Moreover, the sensor also showed good performance in detecting real sweat samples. This study has shown great potential in future advances in sweat analysis and health monitoring.

Reusable graphite-based electrochemical sensors for L-dopa and dopamine detection.

A fully reusable electrochemical device is proposed for the first time made from laser cutting and a homemade conductive ink composed of carbon and nail polish. As a sensor substrate, we applied polymethyl methacrylate, which allows the surface to be renewed by simply removing and reapplying a new layer of ink. In addition to the ease of renewing the sensor's conductive surface, the design of the device has allowed for the integration of different forms of analysis. The determination of L-Dopa was performed using DPV, which presented a linear response range between 5.0 and 1000.0 µmol L-1, and a LOD of 0.11 µmol L-1. For dopamine, a flow injection analysis system was employed, and using the amperometric technique measurements were performed with a linear ranging from 2.0 to 100.0 µmol L-1 and a LOD of 0.26 µmol L-1. To demonstrate its applicability, the device was used in the quantification of analytes in pharmaceutical drug and synthetic urine samples.

Waste Coffee Ground-Derived Porous Carbon for Neurochemical Detection.

We present an optimized synthetic method for repurposing coffee waste to create controllable, uniform porous carbon frameworks for biosensor applications to enhance neurotransmitter detection with fast-scan cyclic voltammetry. Harnessing porous carbon structures from biowastes is a common practice for low-cost energy storage applications; however, repurposing biowastes for biosensing applications has not been explored. Waste coffee ground-derived porous carbon was synthesized by chemical activation to form multivoid, hierarchical porous carbon, and this synthesis was specifically optimized for porous uniformity and electrochemical detection. These materials, when modified on carbon-fiber microelectrodes, exhibited high surface roughness and pore distribution, which contributed to significant improvements in electrochemical reversibility and oxidative current for dopamine (3.5 ± 0.4-fold) and other neurochemicals. Capacitive current increases were small, showing evidence of small increases in electroactive surface area. Local trapping of dopamine within the pores led to improved electrochemical reversibility and frequency-independent behavior. Overall, we demonstrate an optimized biowaste-derived porous carbon synthesis for neurotransmitter detection for the first time and show material utility for viable neurotransmitter detection within a tissue matrix. This work supports the notion that controlled surface nanogeometries play a key role in electrochemical detection.

Quantification of Plasma Dopamine in Depressed Patients Using Silver-Enriched Silicon Nanowires as SERS-Active Substrates.

A decrease in the levels of dopamine (DA)─a key catecholamine biomarker for major depressive disorder─highlights the need for quantitative analysis of biological fluids to aid in the early diagnosis of diverse neuropsychiatric disorders. This study developed silicon nanowires enriched with silver nanoparticles to serve as a surface-enhanced Raman scattering (SERS) substrate to enable precise and sensitive quantification of blood plasma DA levels in humans. The silver-enriched silicon nanowires (SiNWs@Ag) yielded flower-like assemblies with densely populated SERS "hot spots," allowing sensitive DA detection. By correlating DA concentration with Raman intensity at 1156 cm-1, the plasma DA levels in treatment-naïve patients with major depression (n = 18) were 2 orders of magnitude lower than those in healthy controls (n = 18) (6.56 × 10-10 M vs 1.43 × 10-8 M). The plasma DA concentrations differed significantly between the two groups (two-tailed p = 5.77×10-7), highlighting a distinct demarcation between depression patients and healthy controls. Furthermore, the SiNWs@Ag substrate effectively differentiated between DA and norepinephrine (NE) in mixtures at nanomolar levels, demonstrating its selective detection capability. This study represents the first report on the quantitative detection of DA levels in human blood samples from individuals with major depression using an SERS technique, emphasizing its potential clinical utility in the evaluation and diagnosis of neuropsychiatric disorders.

Sensitive and selective electrochemical aptasensing method for the voltammetric determination of dopamine based on AuNPs/PEDOT-ERGO nanocomposites.

In this study, the effects of phosphate buffered saline (PBS) and graphene oxide (GO) as supporting electrolytes and dopants on the electropolymerization process of 3,4-ethylenedioxythiophene (EDOT) on glassy carbon electrode (GCE) were investigated. It was found that the PEDOT-ERGO nanocomposites obtained by a simple one-step electrochemical redox polymerization method using GO as the only supporting electrolyte and dopant possess excellent electrochemical properties. Then, the PEDOT-ERGO nanocomposites were used as electrode substrate to further modify with AuNPs, and an electrochemical aptasensor based on AuNPs/PEDOT-ERGO nanocomposites was successfully constructed for the sensitive and selective determination of dopamine (DA). Comparison of the cyclic voltammetric response of different neurotransmitters before and after aptamer assembly showed that the aptamer significantly improved the selectivity of the sensor for DA. The low detection limit of 1.0 µM (S/N = 3) indicated the good electrochemical performance of the PEDOT-ERGO nanocomposite film. Moreover, the aptasensor showed good recoveries in 50-fold diluted fetal bovine serum with RSD values all less than 5 % (n = 5), indicating that the PEDOT-ERGO nanocomposites and the electrochemical aptasensor have promising applications in other neurochemicals assay and biomedical analysis.

New insight into interference-free and highly sensitive dopamine electroanalysis.

Early diagnosis of Parkinson's disease and hyperprolactinemia based on electrochemical dopamine (DA) sensing appears as an efficient and promising practical diagnostic method. However, the coexistence of DA in real samples with ascorbic acid (AA) and uric acid (UA), which oxidize at potentials close to its own, prevents the accurate electrochemical DA sensing and therefore, hinders the effective diagnosis of these diseases. In this work, we successfully combined the electrostatic proprieties of GO, the electron transfer properties of an AuNPs@MWCNTs nanocomposite and the ability of thiol group of the amino acid l-cysteine to react chemically with carbonyl groups of UA, to develop a novel approach that enabled complete suppression of interference from AA and UA and hence, accurate DA electroanalysis in the conditions close to those of human blood serum. The chemical reaction between l-cysteine and UA was evidenced by monitoring the DPV responses of UA under different conditions. XRD, Raman spectroscopy, XPS and FE-SEM revealed the successful synthesis of GO and AuNPs@MWCNTs. The study of the electrode material (GO-AuNPs@MWCNTs) morphology via FE-SEM and HR-TEM showed that AuNPs@MWCNTs are distributed throughout the exfoliated GO layers. The fabricated sensor was calibrated in the concentration range of 0.5-5 µM, in the presence of the highest blood concentrations of AA and UA for healthy individuals. A linear relationship was observed and the LOD was found to be 1.31 nM (S/N = 3). Furthermore, the sensor showed good electron transfer kinetics, good repeatability and reproducibility, satisfactory long-term stability, and recoveries in human blood serum.

An ultra-sensitive platinized nanocavity electrode for analysis of cytosolic catecholamines in one living cell.

The catecholamines, mainly dopamine (DA), are present in the cellular cytosol with low abundance, while, play key roles in various neurodegenerative disorders. Here, platinized nanocavity carbon electrodes are employed to analyze cytosolic catecholamines in a single living PC12 cell, which is not easily quantified using the classic electrodes. The confined structure and excellent conductivity in the platinized nanocavity accelerate the electron transfer of the DA, resulting in a low detection limit down to 50 nM. The sensitivity of DA detection is improved to be 10.73 pA mM-1 nm-1 in the response range of 50 nM-100 µM, which guarantees quantitative analysis of cytosolic catecholamines with low abundance. Eventually, the platinized nanocavity electrode is employed to detect cytosolic catecholamines in a single PC12 cell without an obvious interruption of cellular catecholamine level. The cytosolic catecholamines in a single PC12 cell is measured in situ to be 0.1 µM, which is achieved for the first time at the single cell level using the electrochemical method. The results demonstrate that the nanocavity electrode with a high sensitivity could offer a promising means to dynamically track catecholamines in a single cell.

Ultrastructural analysis of nigrostriatal dopaminergic terminals in a knockin mouse model of DYT1 dystonia.

DYT1 dystonia is associated with decreased striatal dopamine release. In this study, we examined the possibility that ultrastructural changes of nigrostriatal dopamine terminals could contribute to this neurochemical imbalance using a serial block face/scanning electron microscope (SBF/SEM) and three-dimensional reconstruction to analyse striatal tyrosine hydroxylase-immunoreactive (TH-IR) terminals and their synapses in a DYT1(ΔE) knockin (DYT1-KI) mouse model of DYT1 dystonia. Furthermore, to study possible changes in vesicle packaging capacity of dopamine, we used transmission electron microscopy to assess the synaptic vesicle size in striatal dopamine terminals. Quantitative comparative analysis of 80 fully reconstructed TH-IR terminals in the WT and DYT1-KI mice indicate (1) no significant difference in the volume of TH-IR terminals; (2) no major change in the proportion of axo-spinous versus axo-dendritic synapses; (3) no significant change in the post-synaptic density (PSD) area of axo-dendritic synapses, while the PSDs of axo-spinous synapses were significantly smaller in DYT1-KI mice; (4) no significant change in the contact area between TH-IR terminals and dendritic shafts or spines, while the ratio of PSD area/contact area decreased significantly for both axo-dendritic and axo-spinous synapses in DYT1-KI mice; (5) no significant difference in the mitochondria volume; and (6) no significant difference in the synaptic vesicle area between the two groups. Altogether, these findings suggest that abnormal morphometric changes of nigrostriatal dopamine terminals and their post-synaptic targets are unlikely to be a major source of reduced striatal dopamine release in DYT1 dystonia.

Alkaline liquid-derived NaxTi11.5MoVOx/C-40 material with controlled electron transfer rate for sensitive electrochemical detection of dopamine.

The neurotransmitter dopamine (DA) is associated with many physiological and pathological processes, so the importance of low detection limits and high sensitivity analysis cannot be overstated, especially for early disease detection. Here, 2 M NaOH aqueous solution is used to precipitate metal ions in an ethanol solution containing carbon black (CB), and then nanocomposite catalysts (NaxTi11.5MoVOx/C-40 (40 denoted as 40 mg CB)) were obtained by calcining the precipitation. When used for DA detection, NaxVOx acts as the main active site for electrochemical oxidation of DA and NaxTi11.5MoOx plays a role in facilitating the binding of DA to the active site and stabilizing the active site. The NaxTi11.5MoVOx/C-40 electrochemical biosensor has a limit of detection (LOD) of 0.003 µM with a linear range of 0.005-51.665 µM for DA. This sensor can be used to sensitively identify the concentration of DA in human blood and urine. Catalysts containing varying amounts of CB exhibit diverse electron transfer rates, and surprisingly, we found that the appropriate electron transfer rate is optimal for the detection of low concentrations of DA. Because the performance of the electrochemical biosensors is affected by both the activity of the catalysts and the accuracy of the electrochemical testing instrumentation. To better explain this phenomenon, we propose the concept of resolution (Rn) and present the formula to derive it, offering a new approach to evaluating the performance of electrochemical biosensors.

COF-Coated Microelectrode for Space-Confined Electrochemical Sensing of Dopamine in Parkinson's Disease Model Mouse Brain.

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.

Early life exposure to F-53B induces neurobehavioral changes in developing children and disturbs dopamine-dependent synaptic signaling in weaning mice.

BACKGROUND: Previous studies have shown that F-53B exposure may be neurotoxic to animals, but there is a lack of epidemiological evidence, and its mechanism needs further investigation. METHODS: Serum F-53B concentrations and Wisconsin Card Sorting Test (WCST) were evaluated in 314 growing children from Guangzhou, China, and the association between them were analyzed. To study the developmental neurotoxicity of F-53B, experiments on sucking mice exposed via placental transfer and breast milk was performed. Maternal mice were orally exposed to 4, 40, and 400 µg/L of F-53B from postnatal day 0 (GD0) to postnatal day 21 (PND 21). Several genes and proteins related to neurodevelopment, dopamine anabolism, and synaptic plasticity were examined by qPCR and western blot, respectively, while dopamine contents were detected by ELISA kit in weaning mice. RESULTS: The result showed that F-53B was positively associated with poor WCST performance. For example, with an interquartile range increase in F-53B, the change with 95 % confidence interval (CI) of correct response (CR), and non-perseverative errors (NPE) was -2.47 (95 % CI: -3.89, -1.05, P = 0.001), 2.78 (95 % CI: 0.79, 4.76, P = 0.007), respectively. Compared with the control group, the highest exposure group of weaning mice had a longer escape latency (35.24 s vs. 51.18 s, P = 0.034) and a lesser distance movement (34.81 % vs. 21.02 %, P < 0.001) in the target quadrant, as observed from morris water maze (MWM) test. The protein expression of brain-derived neurotrophic factor (BDNF) and growth associated protein-43 (GAP-43) levels were decreased, as compared to control (0.367-fold, P < 0.001; 0.366-fold, P < 0.001; respectively). We also observed the upregulation of dopamine transporter (DAT) (2.940-fold, P < 0.001) consistent with the trend of dopamine content (1.313-fold, P < 0.001) in the hippocampus. CONCLUSION: Early life exposure to F-53B is associated with adverse neurobehavioral changes in developing children and weaning mice which may be modulated by dopamine-dependent synaptic plasticity.

Nitrocellulose membranes in situ grown with Prussian blue nanoparticles as stable nanozyme pads for colorimetric detection of dopamine.

Prussian blue (PB) is a typical peroxidase mimic with simple preparation, low cost and high eco-friendliness, but it still has drawbacks of poor stability (e.g., decomposition in aqueous dispersions) and intrinsic optical interference (e.g., high extinction coefficient over a wide wavelength range) in colorimetric assays. Herein, we used nitrocellulose (NC) membranes as synthesis hosts of PB nanoparticles (NPs) to develop a new type of three-dimensional (3D) porous nanozyme pad. By means of an in situ synthesis route, PB NPs were uniformly grown on the surfaces of the fiber scaffolds with desirable stability, which also avoided signal interference from PB NPs owing to the easy handling of the pads in a quantitative solid state. The pads showed significant peroxidase-mimicking activity toward the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) with the output of colorimetric signals. Based on the reduction of oxidized TMB (oxTMB) by dopamine (DA), the pads were exploited for simple and quantitative colorimetric detection of DA with a limit of detection (LOD) of 0.59 µM and a satisfactory accuracy for analysis of real human urine samples. This is the first attempt at exploiting NC membranes as the synthesis hosts to develop nanozyme pads, which solves the above drawbacks of traditional PB-based peroxidase mimics and has promise for various colorimetric bioanalyses, given the structural benefits of NC membranes and their broad applications in biosensors.

Synthesis of 2D VO2 Nanosheets for the Dual Optical Sensor Method by Colorimetric and Fluorometric Sensing of Catecholamines.

For the first time, we report a dual optical sensor method (DOSM) using novel 2D VO2 nanosheets to act as fluorometric and colorimetric sensors to perform quantitative analysis of epinephrine (EP) and dopamine (DA). The wide color spectrum of the 2D vanadium oxidation series and specifically metastable blue 2D VO2 nanosheets were used to develop a DOSM biosensor. DA and EP are the major catecholamines in the human body that play vital roles as neurotransmitters and stress-responsive hormones of the endocrine system, respectively. Accurate and selective detection of these biomolecules can assist in the diagnosis of many neuroendocrine system-related diseases. The newly synthesized 2D VO2 nanosheet sensor showed bluish-green fluorescence as the first-ever fluorescence from 2D VO2 nanosheets. This sensor showed dual-function sensing toward EP by a dominant color change and fluorescence quenching. It is capable of individually detecting and quantifying both EP and DA with high selectivity and sensitivity by using both colorimetry and fluorometry simultaneously, with the detection limits of 1.07 and 5.54 µM for colorimetric analysis, respectively, and 48.07 and 3.98 µM for fluorescence analysis, respectively. The DOSM sensor was directly applied to real urine samples and gained satisfactory recovery above 90% by means of spiked concentrations. This study has opened a new platform using the DOSM and the vanadium oxidation spectrum in a much more effective way for biosensing. The fluorescence capabilities of this metal oxide can be further applied to many sensor applications based on both fluorescence and colorimetric detection.

Porous carbon nanosheets derived from two-dimensional Fe-MOF for simultaneous voltammetric sensing of dopamine and uric acid.

It is of great significance for electrochemical sensors to simultaneously detect dopamine (DA) and uric acid (UA) related to biological metabolism. In this work, two-dimensional (2D) porous carbon nanosheets (CNS) was prepared as electrocatalysts to improve the sensitivity, the selectivity, and the detection limit of the simultaneous detection. First, 2D amorphous iron-metal organic frameworks (Fe-MOF) was synthesized with Fe3+and terephthalic acid via a facile wet chemistry method at room temperature. And then, CNS was prepared by pyrolysis and pickling of Fe-MOF. CNS had large specific surface area, good electrical conductivity and lots of carbon defects. The response currents of the CNS modified electrode was larger than those of the control electrodes in the simultaneous determination. The simultaneous determination was measured via differential pulse voltammetry to reduce the effect of capacitive currents on quantitative analysis. The CNS modified electrodes showed high sensitivity and low detection limit for the simultaneous detection of DA and UA. The modified electrodes have been successfully used to detect DA and UA in normal human serum.

Efficient mercury(II) removal by corn bract/dopamine@ZnS composites.

In this study, we have utilized corn bract, a green agricultural by-product, as a carrier. It is subsequently modified with zinc sulfide to synthesize an efficient composite material termed as corn bract/polydopamine@zinc sulfide (CB/PDA@ZnS). This novel composite demonstrates significant potential for biomass removal of mercury ions (Hg(II)). The composition, structure, and morphology of CB/PDA@ZnS composites are characterized by Fourier transform infrared (FT-IR) spectrum, thermogravimetric analysis (TGA), X-ray powder diffraction (XRD), and scanning electron microscope (SEM). The effect of pH value, adsorbent dosage, initial Hg(II) concentration, adsorption time and temperature, and coexistence ions on the adsorption behavior is investigated. The results show that CB/PDA@ZnS can efficiently remove Hg(II) from water with uptake capacities of 333.03 mg/g and removal efficiency of 99.91% under an optimal conditions (pH of 3, the adsorbent dosage of 0.015 g, contact time of 90 min, and initial concentration of 100 mg/L) at room temperature. The fitting analysis of the experimental data reveals that the adsorption process of Hg(II) follows the quasi-secondary adsorption kinetic model as well as the Langmuir isothermal adsorption model, which is a spontaneous heat absorption process. In addition, the composite adsorbent obtained exhibit excellent selectivity for Hg(II) ions and anti-coexisting ion interference performance. After five cycles of adsorption-desorption experiments, the corresponding adsorption capacity is 331.11 mg/g, accounting for 93.33% of the first adsorption capacity, indicating that the adsorbent has excellent regeneration performance. The stability of the adsorbent and the adsorption mechanism of Hg(II) ion are systematically discussed using FT-IR, XRD, and X-ray photoelectron spectroscopy (XPS). Finally, this adsorbent is tested for the removal of industrial wastewater containing Hg(II), and the adsorption and removal efficiency are 331.67 mg/g and 99.50%, respectively. This study provides a very valuable information for future Hg(II) removal from aqueous solutions.

Response to comment on "Food Wanting is Mediated by Transient Activation of Dopaminergic Signaling in the Honeybee Brain".

In a technical comment, Barron et al. (1) criticized the work of Huang et al. (2) putting the accent on the quantification of dopamine levels via high-performance liquid chromatography (HPLC), yet also including data interpretation through alternative hypotheses aimed at invalidating the original ones proposed by Huang et al. We thank the authors of this technical comment, which allows us to clarify technical aspects of our work that may have been unclear, and for promoting discussion around the conclusions of our work. Below we provide answers to the points raised in their comment.

Crumpled MXene nanosheets for sensing of ascorbic acid in food, biological fluids, and erythrocytes in-vitro microenvironment.

In this work, a simple and facile method was developed to achieve controlled oxidation and enhance the surface area of MXene nanosheets and their utilization in the efficient sensing of ascorbic acid (AA or vitamin C). After etching of MAX phase to MXene via the MILD technique, controlled flash oxidation was carried out in the open air environment for 1.5 h, followed by flocculation of oxidized MXene nanosheets by using H2SO4, consequently achieving crumpled MXene possessing anatase phase, porosity, and improved surface area as revealed and confirmed by SEM, TEM, Raman, and BET analysis results. The as-prepared crumpled MXene was coated over a glassy carbon electrode (GCE) and used to determine AA successfully via cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), and differential pulse voltammetry (DPV) with a linear concentration range of 300 µM to 0.005 µM with a detection limit (LOD) of 2 nM (2.8 % RSD and S/N = 3). The developed electrochemical sensor was used to determine the AA in various actual samples such as juice, urine, serum, and erythrocytes spiked with AA with excellent recoveries in the 94-103 % range. The sensor also demonstrated excellent reproducibility (~1 % RSD for five repetitive assays) and a shelf life of nearly one month with a negligible decrease in response. Furthermore, it lost only 10 % of its response for the next ten days. It also showed satisfactory selectivity toward AA in the presence of other similar compounds, including uric acid (UA), dopamine (DA), and glucose.

A Reliable Method Based on Liquid Chromatography-Tandem Mass Spectrometry for the Simultaneous Quantification of Neurotransmitters in Caenorhabditis elegans.

Neurotransmitters like dopamine (DA), serotonin (SRT), γ-aminobutyric acid (GABA) and acetylcholine (ACh) are messenger molecules that play a pivotal role in transmitting excitation between neurons across chemical synapses, thus enabling complex processes in the central nervous system (CNS). Balance in neurotransmitter homeostasis is essential, and altered neurotransmitter levels are associated with various neurological disorders, e.g., loss of dopaminergic neurons (Parkinson's disease) or altered ACh synthesis (Alzheimer's disease). Therefore, it is crucial to possess adequate tools to assess precise neurotransmitter levels, and to apply targeted therapies. An established in vivo model to study neurotoxicity is the model organism Caenorhabditis elegans (C. elegans), as its neurons have been well characterized and functionally are analogous to mammals. We have developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method including a sample preparation assuring neurotransmitter stability, which allows a simultaneous neurotransmitter quantification of DA, SRT, GABA and ACh in C. elegans, but can easily be applied to other matrices. LC-MS/MS combined with isotope-labeled standards is the tool of choice, due to its otherwise unattainable sensitivity and specificity. Using C. elegans together with our analytically validated and verified method provides a powerful tool to evaluate mechanisms of neurotoxicity, and furthermore to identify possible therapeutic approaches.