Utilizing a Disposable Sensor with Polyaniline-Doped Multi-Walled Carbon Nanotubes to Enable Dopamine Detection in Ex Vivo Mouse Brain Tissue Homogenates.

Disposable sensors are inexpensive, user-friendly sensing tools designed for rapid single-point measurements of a target. Disposable sensors have become more and more essential as diagnostic tools due to the growing demand for quick, easy-to-access, and reliable information related to the target. Dopamine (DA), a prevalent catecholamine neurotransmitter in the human brain, is associated with central nervous system activities and directly promotes neuronal communication. For the sensitive and selective estimation of DA, an enzyme-free amperometric sensor based on polyaniline-doped multi-walled carbon nanotubes (PANI-MWCNTs) drop-coated disposable screen-printed carbon electrodes (SPCEs) was fabricated. This PANI-MWCNTs-2/SPCE sensor boasts exceptional accuracy and sensitivity when working directly with ex vivo mouse brain homogenates. The sensor exhibited a detection limit of 0.05 µM (S/N = 3), and a wide linear range from 1.0 to 200 µM. The sensor's high selectivity to DA amidst other endogenous interferents was recognized. Since the constructed sensor is enzyme-free yet biocompatible, it exhibited high stability in DA detection using ex vivo mouse brain homogenates extracted from both Parkinson's disease and control mice models. This research thus presents new insights into understanding DA release dynamics at the tissue level in both of these models.

Amelioration of Astrocyte-Mediated Neuroinflammation by EI-16004 Confers Neuroprotection in an MPTP-induced Parkinson's Disease Model.

Parkinson's disease (PD) is a neurodegenerative disorder that results in motor impairment due to dopaminergic neuronal loss. The pathology of PD is closely associated with neuroinflammation, which can be characterized by astrocyte activation. Thus, targeting the inflammatory response in astrocytes might provide a novel therapeutic approach. We conducted a luciferase assay on an in-house chemical library to identify compounds with anti-inflammatory effects capable of reducing MPP+-induced NF-κB activity in astrocytes. Among the compounds identified, EI-16004, a novel 3-benzyl-N-phenyl-1H-pyrazole-5-carboxamides, exhibited a significant anti-inflammatory effect by significantly reducing MPP+-induced astrocyte activation. Biochemical analysis and docking simulation indicated that EI-16004 inhibited the MPP+-induced phosphorylation of p65 by attenuating ERK phosphorylation, and EI-16004 reduced pro-inflammatory cytokine and chemokine levels in astrocytes. In vivo studies on the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD model in male C57BL/6 mice showed that EI-16004 ameliorated motor impairment and protected against dopaminergic neuronal loss, and EI-16004 effectively mitigated the MPTP-induced astrocyte activation in striatum (STR) and substantia nigra (SN). These results indicate EI-16004 is a potential neuroprotective agent for the prevention and treatment of astrocyte-mediated neuroinflammatory conditions in PD.

Glutamate oxidase sheets-Prussian blue grafted amperometric biosensor for the real time monitoring of glutamate release from primary cortical neurons.

Glutamate (GLU) is a primary excitatory neurotransmitter, and its dysregulation is associated with several neurodegenerative disorders. A major challenge in GLU estimation is the existence of other biomolecules in the brain that could directly get oxidized at the electrode. Hence, highly selective electroenzymatic biosensors that enable rapid estimation of GLU are needed. Initially, a copolymer, poly(2-dimethylaminoethyl methacrylate- styrene) was synthesized through reversible addition-fragmentation chain transfer polymerization to noncovalently functionalize reduced graphene oxide (rGO), named DS-rGO. Glutamate oxidase macromolecule immobilized DS-rGO formed enzyme nanosheets, which was drop-coated over Prussian blue electrodeposited disposable electrodes to fabricate the GLU biosensor. The interconnectivity between the enzyme nanosheets and the Prussian blue endows the biosensor with enhanced conductivity and electrochemical activity. The biosensor exhibited a linearity: 3.25-250 µM; sensitivity: 3.96 µA mM-1 cm-2, and a limit of detection: 0.96 µM for GLU in the Neurobasal Medium. The biosensor was applied to an in vitro primary rat cortical model to discriminate GLU levels in Neurobasal Medium, before and after KCl mediated depolarization, which provides new insights for elucidating neuronal functioning in the brain.

Ruthenium-Anchored Carbon Sphere-Customized Sensor for the Selective Amperometric Detection of Melatonin.

Melatonin (MT), a pineal gland hormone, regulates the sleep/wake cycle and is a potential biomarker for neurodegenerative disorders, depression, hypertension, and several cancers, including prostate cancer and hepatocarcinoma. The amperometric detection of MT was achieved using a sensor customized with ruthenium-incorporated carbon spheres (Ru-CS), possessing C- and O-rich catalytically active Ru surfaces. The non-covalent interactions and ion-molecule adducts between Ru and CS favor the formation of heterojunctions at the sensor-analyte interface, thus accelerating the reactions towards MT. The Ru-CS/Screen-printed carbon electrode (SPCE) sensor demonstrated the outstanding electrocatalytic oxidation of MT owing to its high surface area and heterogeneous rate constants and afforded a lower detection limit (0.27 µM), high sensitivity (0.85 µA µM -1 cm-2), and excellent selectivity for MT with the co-existence of crucial neurotransmitters, including norepinephrine, epinephrine, dopamine, and serotonin. High concentrations of active biomolecules, such as ascorbic acid and tyrosine, did not interfere with MT detection. The practical feasibility of the sensor for MT detection in pharmaceutical samples was demonstrated, comparable to the data provided on the product labels. The developed amperometric sensor is highly suitable for the quality control of medicines because of its low cost, simplicity, small sample size, speed of analysis, and potential for automation.

Enzyme Nanosheet-Based Electrochemical Aspartate Biosensor for Fish Point-of-Care Applications.

Bacterial infections in marine fishes are linked to mass mortality issues; hence, rapid detection of an infection can contribute to achieving a faster diagnosis using point-of-care testing. There has been substantial interest in identifying diagnostic biomarkers that can be detected in major organs to predict bacterial infections. Aspartate was identified as an important biomarker for bacterial infection diagnosis in olive flounder (Paralichthys olivaceus) fish. To determine aspartate levels, an amperometric biosensor was designed based on bi-enzymes, namely, glutamate oxidase (GluOx) and aspartate transaminase (AST), which were physisorbed on copolymer reduced graphene oxide (P-rGO), referred to as enzyme nanosheets (GluOx-ASTENs). The GluOx-ASTENs were drop casted onto a Prussian blue electrodeposited screen-printed carbon electrode (PB/SPCE). The proposed biosensor was optimized by operating variables including the enzyme loading amount, coreactant (α-ketoglutarate) concentration, and pH. Under optimal conditions, the biosensor displayed the maximum current responses within 10 s at the low applied potential of -0.10 V vs. the internal Ag/AgCl reference. The biosensor exhibited a linear response from 1.0 to 2.0 mM of aspartate concentrations with a sensitivity of 0.8 µA mM-1 cm-2 and a lower detection limit of approximately 500 µM. Moreover, the biosensor possessed high reproducibility, good selectivity, and efficient storage stability.

Screen-printed carbon electrode modified with de-bundled single-walled carbon nanotubes for voltammetric determination of norepinephrine in ex vivo rat tissue.

A voltammetric sensor for norepinephrine (NE) detection was developed by modifying a disposable screen-printed carbon electrode (SPCE) with de-bundled single-walled carbon nanotubes (D-SWCNTs). The de-bundling was carried out using a newly synthesized polymeric dispersant, a co-polymer of polystyrene sulfonate and methacrylate of lipoic acid. The D-SWCNTs/SPCE showed better sensitivity towards NE compared to the bare SPCE and that modified with bundled SWCNTs. The sensor was optimized for detecting NE by differential pulse voltammetry (DPV) in terms of the D-SWCNTs concentration, DPV parameters, and solution pH. Under the optimum conditions, the sensor exhibited a dynamic linear range of 100 nM-2.0 µM NE, and the detection limit was 62.0 nM (S/N = 3). Additionally, the effects of possible interferents were investigated. The relative standard deviation for five successive measurements of 2.0 µM NE was 7.6%, and approximately 75.8% of the sensor activity was retained after four weeks of storage. The practical potential of this sensor was demonstrated by quantifying NE in ex vivo rat tissue samples.

Robust Nanozyme-Enzyme Nanosheets-Based Lactate Biosensor for Diagnosing Bacterial Infection in Olive Flounder (Paralichthys olivaceus).

Bacterial infections in fish farms increase mass mortality and rapid detection of infection can help prevent its widespread. Lactate is an important biomarker for early diagnosis of bacterial infections in farmed olive flounder (Paralichthys olivaceus). To determine the lactate levels, we designed a disposable amperometric biosensor based on Prussian blue nanozyme and lactate oxidase (LOX) entrapped in copolymer-reduced graphene oxide (P-rGO) on screen-printed carbon electrodes. Because LOX is inherently unstable, P-rGO nanosheets were utilized as a base matrix to immobilize it. After optimization in terms of enzyme loading, operating potential, and pH, the biosensor displayed maximum current responses within 5 s at the applied potential of -0.1 V vs. internal Ag/AgCl. The biosensor had Langmuir-type response in the lactate concentration range from 10 µM to 1.6 mM, a dynamic linear response range of 10-100 µM, a sensitivity of 15.9 µA mM-1 cm-2, and a lower detection limit of 3.1 µM (S/N = 3). Additionally, the biosensor featured high reproducibility, good selectivity, and stability till four weeks. Its practical applicability was tested in olive flounder infected by Streptococcus parauberis against the uninfected control. The results were satisfactory compared to those of a standard colorimetric assay kit, validating our method.

Polymer-dispersed reduced graphene oxide nanosheets and Prussian blue modified biosensor for amperometric detection of sarcosine.

A new disposable amperometric biosensor for sarcosine (Sar, a biomarker for prostate cancer) was designed based on screen-printed carbon electrodes, Prussian blue, polymer dispersed reduced graphene oxide (P-rGO) nanosheets, and sarcosine oxidase (SOx). Poly(sodium 4-styrenesulfonate-r-LAHEMA) denoted as PSSL was newly synthesized as dispersant for rGO. The P-rGO was utilized for SOx immobilization, the sulfonate and disulfide functionalities in PSSL enable physical adsorption of SOx and its bioactivity and stability properties were improved. The biosensor was optimized by various enzyme concentration, applied potential, and operating pH. Under the optimized conditions, the biosensor exhibited maximum current responses within 5 s at an applied potential of -0.1 V vs. integrated Ag/AgCl reference electrode. The biosensor had a dynamic linear range of 10-400 µM, with a sensitivity of 9.04 µA mM-1 cm-2 and a low detection limit of 0.66 µM (S/N = 3). Additionally, the biosensor possesses strong anti-interference capability, high reproducibility, and storage stability over 3 weeks. Furthermore, its clinical applicability was tested in urine samples from both prostate cancer patients and healthy control, and the analytical recoveries were satisfactory. Therefore, this biosensor has significant potential in the rapid and non-invasive point-of-care testing for prostate cancer diagnosis.

Cost-Effective Electrochemical Activation of Graphitic Carbon Nitride on the Glassy Carbon Electrode Surface for Selective Determination of Serotonin.

A simple one-step electrochemical deposition/activation of graphitic carbon nitride (g-C3N4) is highly desired for sensor configurations and remains a great challenge. Herein, we attempt an electrochemical route to exfoliate the g-C3N4 nanosheets in an aqueous solution of pH 7.0 for constructing a sensor, which is highly sensitive for the detection of serotonin (5-HT). The significance of our design is to exfoliate the g-C3N4 nanosheets, a strong electrocatalyst for 5-HT detection. Investigations regarding the effect of neutral pH (pH 7.0) on the bulk g-C3N4 and g-C3N4 nanosheets, physical characterization, and electrochemical studies were extensively carried out. We demonstrate that the g-C3N4 nanosheets have a significant electrocatalytic effect for the 5-HT detection in a dynamic linear range from 500 pM to 1000 nM (R2 = 0.999). The limit of detection and sensitivity of the designed 5-HT sensor was calculated to be 150 pM and 1.03 µA µM-1 cm-2, respectively. The proposed sensor has great advantages such as high sensitivity, good selectivity, reproducibility, and stability. The constructed g-C3N4 nanosheets-based sensor platform opens new feasibilities for the determination of 5-HT even at the picomolar/nanomolar concentration range.