In Situ Detection of Neurotransmitters from Stem Cell-Derived Neural Interface at the Single-Cell Level via Graphene-Hybrid SERS Nanobiosensing.

In situ quantitative measurements of neurotransmitter activities can provide useful insights into the underlying mechanisms of stem cell differentiation, the formation of neuronal networks, and neurodegenerative diseases. Currently, neurotransmitter detection methods suffer from poor spatial resolution, nonspecific detection, and a lack of in situ analysis. To address this challenge, herein, we first developed a graphene oxide (GO)-hybrid nanosurface-enhanced Raman scattering (SERS) array to detect dopamine (DA) in a selective and sensitive manner. Using the GO-hybrid nano-SERS array, we successfully measured a wide range of DA concentrations (10-4 to 10-9 M) rapidly and reliably. Moreover, the measurement of DA from differentiating neural stem cells applies to the characterization of neuronal differentiation. Given the challenges of in situ detection of neurotransmitters at the single-cell level, our developed SERS-based detection method can represent a unique tool for investigating single-cell signaling pathways associated with DA, or other neurotransmitters, and their roles in neurological processes.

Silver Nanoparticle Modified Electrode Covered by Graphene Oxide for the Enhanced Electrochemical Detection of Dopamine.

Several neurological disorders such as Alzheimer's disease and Parkinson's disease have become a serious impediment to aging people nowadays. One of the efficient methods used to monitor these neurological disorders is the detection of neurotransmitters such as dopamine. Metal materials, such as gold and platinum, are widely used in this electrochemical detection method; however, low sensitivity and linearity at low dopamine concentrations limit the use of these materials. To overcome these limitations, a silver nanoparticle (SNP) modified electrode covered by graphene oxide for the detection of dopamine was newly developed in this study. For the first time, the surface of an indium tin oxide (ITO) electrode was modified using SNPs and graphene oxide sequentially through the electrochemical deposition method. The developed biosensor provided electrochemical signal enhancement at low dopamine concentrations in comparison with previous biosensors. Therefore, our newly developed SNP modified electrode covered by graphene oxide can be used to monitor neurological diseases through electrochemical signal enhancement at low dopamine concentrations.

Synthesis of copper/nickel nanoparticles using newly synthesized Schiff-base metals complexes and their cytotoxicity/catalytic activities.

Transition metal complexes compounds with Schiff bases ligand representing an important class of compounds that could be used to develop new metal-based anticancer agents and as precursors of metal NPs. Herein, 2,3-bis-[(3-ethoxy-2-hydroxybenzylidene)amino]but-2-enedinitrile Schiff base ligand and its corresponding copper/nickel complexes were synthesized. Also, we reported a facile and rapid method for synthesis nickel/copper nanoparticles based on thermal reduction of their complexes. Free ligand, its metal complexes and metals nanoparticles have been characterized based on elemental analysis, transmission electron microscopy, powder X-ray diffraction, magnetic measurements and by various spectroscopic (UV-vis, FT-IR, (1)H NMR, GC-MS) techniques. Additionally, the in vitro cytotoxic activity of free ligand and its complexes compounds were assessed against two cancer cell lines (HeLa and MCF-7 cells)and one healthy cell line (HEK293 cell). The copper complex was found to be active against these cancer cell lines at very low LD50 than the free ligand, while nickel complex did not show any anticancer activity against these cell lines. Also, the antibacterial activity of as-prepared copper nanoparticles were screened against Escherichia coli, which demonstrated minimum inhibitory concentration and minimum bactericidal concentration values lower than those values of the commercial Cu NPs as well as the previous reported values. Moreover, the synthesized nickel nanoparticles demonstrated remarkable catalytic performance toward hydrogenation of nitrobenzene that producing clean aniline with high selectivity (98%). This reactivity could be attributed to the high degree of dispersion of Ni nanoparticles.

Enzyme-free glucose sensor based on Au nanobouquet fabricated indium tin oxide electrode.

In this study, we demonstrated a simple, rapid and inexpensive fabrication method to develop a novel gold nanobouquet structure fabricated indium tin oxide (GNB/ITO) electrode based on electrochemical deposition of gold ions onto ITO substrate. The morphology of the fabricated electrode surface was characterized by scanning electron microscopy (SEM) to confirm the GNB formation. Enzyme-free detection of glucose using a GNB/ITO electrode was described with high sensitivity and selectivity based on cyclic voltammetry assay. The results demonstrate a linear relation within wide concentration range (500 nM to 10 mM) of glucose, with a correlation coefficient of 0.988. The interference effect of uric acid was effectively avoided for the detection of glucose (1 µM to 10 mM). Moreover, the developed sensor was applied to determine the concentration of glucose in the presence of human serum to indicate the ability of GNB/ITO electrodes in real samples. Hence, newly developed GNB/ITO electrode has potential application in enzyme-free glucose sensor with highly sensitivity and selectivity.

Inhibition of apoptosis and biofilm formation in Candida auris by click-synthesized triazole-bridged quinoline derivatives.

Candida auris, a recent addition to the Candida species, poses a significant threat with its association to numerous hospital outbreaks globally, particularly affecting immunocompromised individuals. Given its resistance to existing antifungal therapies, there is a pressing need for innovative treatments. In this study, novel triazole bridged quinoline derivatives were synthesized and evaluated for their antifungal activity against C. auris. The most promising compound, QT7, demonstrated exceptional efficacy with a minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) of 0.12 µg mL-1 and 0.24 µg mL-1, respectively. Additionally, QT7 effectively disrupted mature biofilms, inhibiting them by 81.98% ± 8.51 and 89.57 ± 5.47 at MFC and 2× MFC values, respectively. Furthermore, QT7 induced cellular apoptosis in a dose-dependent manner, supported by various apoptotic markers such as phosphatidylserine externalization, mitochondrial depolarization, and reduced cytochrome c and oxidase activity. Importantly, QT7 exhibited low hemolytic activity, highlighting its potential for further investigation. Additionally, the physicochemical properties of this compound suggest its potential as a lead drug candidate, warranting further exploration in drug discovery efforts against Candida auris infections.

ITO/gold nanoparticle/RGD peptide composites to enhance electrochemical signals and proliferation of human neural stem cells.

A cell chip composed of ITO, gold nanoparticles (GNP) and RGD-MAP-C peptide composites was fabricated to enhance the electrochemical signals and proliferation of undifferentiated human neural stem cells (HB1.F3). The structural characteristics of the fabricated surfaces were confirmed by both scanning electron microscopy and surface-enhanced Raman spectroscopy. HB1.F3 cells were allowed to attach to various composites electrodes in the cell chip and the material-dependent effects on electrochemical signals and cell proliferation were analyzed. The ITO/60 nm GNP/RGD-MAP-C composite electrode was found to be the best material in regards to enhancing the voltammetric signals of HB1.F3 cells when exposed to cyclic voltammetry, as well as for increasing cell proliferation. Differential pulse voltammetry was performed to evaluate the adverse effects of doxorubicin on HB1.F3 cells. In these experiments, negative correlations between cell viability and chemical concentrations were obseved, which were more sensitive than MTT viability assay especially at low concentrations (<0.1 µg/mL). FROM THE CLINICAL EDITOR: In this basic science study, a cell chip composed of ITO, gold nanoparticles and RGD-MAP-C peptide composites was fabricated to enhance electrochemical signals and proliferation of undifferentiated human neural stem cells (HB1.F3). The ITO/60 nm GNP/RGD-MAP-C composite electrode was found to best enhance the voltammetric signals of the studied cells.

Electrochemical Sensing of Dopamine Using Polypyrrole/Molybdenum Oxide Bilayer-Modified ITO Electrode.

The electrochemical sensing of biomarkers has attracted more and more attention due to the advantages of electrochemical biosensors, including their ease of use, excellent accuracy, and small analyte volumes. Thus, the electrochemical sensing of biomarkers has a potential application in early disease diagnosis diagnosis. Dopamine neurotransmitters have a vital role in the transmission of nerve impulses. Here, the fabrication of a polypyrrole/molybdenum dioxide nanoparticle (MoO3 NP)-modified ITO electrode based on a hydrothermal technique followed by electrochemical polymerization is reported. Several techniques were used to investigate the developed electrode's structure, morphology, and physical characteristics, including SEM, FTIR, EDX, N2 adsorption, and Raman spectroscopy. The results imply the formation of tiny MoO3 NPs with an average diameter of 29.01 nm. The developed electrode was used to determine low concentrations of dopamine neurotransmitters based on cyclic voltammetry and square wave voltammetry techniques. Furthermore, the developed electrode was used for monitoring dopamine in a human serum sample. The LOD for detecting dopamine by using MoO3 NPs/ITO electrodes based on the SWV technique was around 2.2 nmol L-1.

Cell chip based monitoring of toxic effects on dopaminergic cell.

Oxidative stress has been implicated in pesticide-induced neurotoxicity, base on its role in cascade of biochemical changes that lead to dopaminergic neuronal cell death. The present study examined the role of oxidative stress and the electrochemical detection by polychlorinated biphenyls (PCBs)-induced toxicant in SH-SY5Y cell. The cells were seed in the RED (Arg-Gly-Asp) nanopatterned coating gold substrate and treated with different concentration of PCBs for 24 h in culture, which induced the change of the cyclic voltammetry (CV) current peak. The CV results showed that PCB significantly decreased the current peaks in dose and time-dependent manner. After antioxidant treatment, the CV of the PCB-treated cell chip increased the current peak. Especially, gluthaione and catalase prevent PCB-induced decrease of CV current peak in the cell. The results demonstrated that the current peak decreased by the PCB and recovered by the antioxidant enzyme. In conclusion, results suggest that the electrochemical-based chip provide crucial information to improvement toward a cell chip system for drug screening application.

Nanoscale biofilm modification-method concerning a myoglobin/11-MUA bilayers for bioelectronic device.

We developed surface modification tools for the fabrication of a bioelectronic device which consists of a myoglobin monolayer self-assembled on an 11-MUA layer. To utilize a single protein as the active element, it was necessary to reduce protein aggregation on the protein layer in the nanobio electronic device, which was developed in our previous study and shown to display basic biomemory functions. Here, the reduction of myoglobin aggregation was accomplished by using 3-(3-cholamidopropyl) dimethylammonio-11-propanesulfonate (CHAPS) to fabricate a well-defined protein layer on the bioelectronic device. We investigated two different surface modification methods for making well oriented biofilm. The effects of CHAPS on the formation of a myoglobin layer self-assembled on an 11-MUA layer were examined by atomic force microscopy and Raman spectroscopy. The size of the myoglobin aggregates was reduced from 200-250 nm to 10-40 nm depending on treatment method. The sustaining redox property of the CHAPS treated myoglobin layer was examined using cyclic voltammetry. Using these techniques, we found that after surfactant CHAPS treatment, protein aggregation was dramatically reduced and the protein layer still maintained its inherent electrochemical properties.

Fabrication of gold nanodot arrays on a transparent substrate as a nanobioplatform for label-free visualization of living cells.

Two-dimensional gold (Au) nanodot arrays on a transparent substrate were fabricated for imaging of living cells. A nanoporous alumina mask with large-area coverage capability was prepared by a two-step chemical wet etching process after a second anodization. Highly ordered Au nanodot arrays were formed on indium-tin-oxide (ITO) glass using very thin nanoporous alumina of approximately 200 nm thickness as an evaporation mask. The large-area Au nanodot arrays on ITO glass were modified with RGD peptide (arginine; glycine; aspartic acid) containing a cysteine (Cys) residue and then used to immobilize human cancer HeLa cells, the morphology of which was observed by confocal microscopy. The confocal micrographs of living HeLa cells on Au nanodot arrays revealed enhanced contrast and resolution, which enabled discernment of cytoplasmic organelles more clearly. These results suggest that two-dimensional Au nanodot arrays modified with RGD peptide on ITO glass have potential as a biocompatible nanobioplatform for the label-free visualization and adhesion of living cells.

Surface enhanced Raman scattering of neurotransmitter release in neuronal cells using antibody conjugated gold nanoparticles.

This study demonstrated the potential feasibility of using antibody-conjugated gold nanoparticles as highly sensitive and homogeneous sensing probes for biological monitoring of neurotransmitters in neuronal cells. Bands at 1152 and 1322 cm(-1) were also similar to SERS of metal catecholates, and could be assigned to catechol ring vibration and carbon-oxygen stretches.

Fabrication of nanoconcave surface for cell immobilization in cell-based chip.

Nanostructured surface such as nanoconcave shape can be utilized as a bioplatform to immobilize cells. In this study, we present fabrication of Au-coated nanoconcave surface and some possibility of cell immobilization. Long-range ordered periodic patterns with concave shape were formed on aluminum substrate by electrochemical anodization process. The morphology and topography of nanoconcave surface was investigated by atomic force microscopy and scanning electron microscopy. The pore-pore distance and the pore depth of nanoconcave pattern were measured at 105 +/- 5 nm and 30 +/- 2 nm, respectively. After Au deposition, the pore depth within Au-coated concave surface was 15 +/- 2 nm. The topography of HeLa cells immobilized on the nanoconcave surface was observed by AFM combined with confocal microscopy. The result expected that the Au-coated nanoconcave surface may be used as new culture substrate for cells immobilization in cell-based chip.

Detection of the effect of environmental toxicants on the synthetic peptide modified neural cell chip.

A cell based chip was designed to differentiate and to detect the effects of environmental chemicals on the neurite outgrowth in PC12 cell. To fabricate platform of cell chip, gold surfaces were modified by RGD based synthetic oligopeptide. Nanoscale controlled self-assembled peptide layer was investigated by Atomic Force Microscopy (AFM). On the fabricated cell chip, PC12 cell was immobilized and the differentiation of neurite outgrowth in PC12 cells was done by neurite growth factor (NGF). Differentiation of PC12 cell was confirmed by immunofluorescence study. Further the differentiation and the length of neurite was confirmed by confocal microscopy study. Voltammetry behavior of the neurite induced PC12 and the electrochemical behavior of the environmental toxicants effect on the neurite outgrowth was measured by cyclic voltammetry (CV). Self-assembled layer mediated cell immobilization technique and voltammetric signal analysis system can be applied to construct the neural cell chip for the detection of large number of environmental toxins and various neurotoxicants.

Electrochemical sensor to detect neurotransmitter using gold nano-island coated ITO electrode.

Parkinson disease is a chronic neurodegenerative disorder characterized by the loss of dopamine, which is a neurotransmitter in the substantia nigra. In this study, a simple, rapid and inexpensive method to fabricate gold nano-island film (GNIF) coated ITO electrode has been developed based on electrochemical deposition of Au onto ITO substrate. The nanostructured film surface was characterized by scanning electron microscopy (SEM). Cyclic voltammetry (CV) and differential pulse voltammetry (DPV) were used to evaluate the electrochemical behavior of induvidul dopamine and uric acid solution were studied. Moreover, GNIF/ITO electrode was applied to detecte DA in the presence of Bovine Serum Albumin (50 microM) as an interference. These results demonstrate that, interfering component has no effect on the determination of DA at GNIF electrode, hence this GNIF electrode is suitable for the determination of DA with high sensitivity and selectivity. Then, GNIF coated ITO electrode was applied to monitor the electrochemical simultaneous detection of dopamine and uric acid mixtures based on CV and DPV with high sensitivity. GNIF-modified ITO electrode showed a linear range for the determination of dopamine concentration from 0.1 microM to 40 microM in the presence of 50 microM of uric acid. Based on these results, the proposed technique can be a promising method to construct a highly sensitive biosensor as well as highly efficient protein chip.

Fabrication of gold nanoparticle modified ITO substrate to detect beta-amyloid using surface-enhanced Raman scattering.

Alzheimer's disease is a progressive neurodegenerative disorder that is characterized by the deposition of beta-amyloid (Abeta) peptide and the formation of neurofibrillary tangles in neurons. The Abeta peptide is a key molecule in the pathogenesis of Alzheimer's disease and an important marker for early diagnosis. Surface-enhanced Raman scattering (SERS) has recently been attracting keen interest in various fields such as for biosensors or immunoassays. In this study, gold nanoparticles (Au NPs) were electrochemically deposited on an indium tin oxide (ITO) substrate at different heights. Abeta antibodies were immobilized on the Au-NP-coated ITO substrate, after which the interactions between the antigen and the antibody were determined via SERS spectroscopy. The SERS responses were strongest at the Au NP array height of 91 nm, with a good linear relationship that corresponded to the change in the concentration of the antigen. This Au-NP-array-mediated SERS can be applied with a highly sensitive immunodetection biosensor.

Surface-enhanced Raman scattering of dopamine on self-assembled gold nanoparticles.

Dopamine, a potent neurotransmitter in the brain, influences a variety of motivated behaviors and plays a major role in Parkinson's disease. In this study, the Raman signal of dopamine was detected on a fabricated nanoparticle-immobilized glass surface by surface-enhanced raman spectroscopy (SERS). Amine-modified glass was prepared by the self-assembly of amine-terminated silane on substrate, followed by the deposition of gold nanoparticles. The gold nanoparticles deposited on the glass surface were functionalized by anti-dopamine or dopamine. The antigen-dopamine was captured by antibody-assembled gold substrate and detected by SERS. The optical properties and morpology of the glass substrate with immobilized gold nanoparticles were analyzed by scanning electron microscopy and UV-VIS absorption spectroscopy. The Raman spectrum of dopamine displayed broad bands at 1267, 1331, 1158, 1478, 1578 and 1584 cm(-1). The strongest peaks in the spectra (at 1267 and 1478 cm(-1)) were identified as phenolic carbon-oxygen and phenyl C=C stretches, respectively. A working curve of the SERS signal constructed from cathecol ring vibration versus antigen-dopamine concentration was obtained at 1478 cm(-1), and the non-optimized detection limit for anti-dopamine surface antigen was as low as 1 ng/ml. These results suggest that SERS-based immunosensor can be a promising tool for the detection and screening of neurotransmitters.

Electrochemical Cell Chips Based on Functionalized Nanometals.

The electrochemical technique is one of the most accurate, rapid, and sensitive analytical assays, which becomes promising techniques for biological assays at a single-cell scale. Nanometals have been widely used for modification of the traditional electrodes to develop highly sensitive electrochemical cell chips. The electrochemical cell chips based on the nanostructured surface have been used as label-free, simple, and non-destructive techniques for in vitro monitoring of the effects of different anticancer drugs at the cellular level. Here, we will provide the recent progress in fabrication of nanopatterned surface and cell-based nanoarray, and discuss their applications based on electrochemical techniques such as detection of cellular states and chemicals, and non-destructive monitoring of stem cell differentiation.

Three-dimensional mesoporous gold film to enhance the sensitivity of electrochemical detection.

Cell-cell and cell-extracellular matrix (ECM) adhesion are fundamental and important in the development of a cell-based chip. In this study, a novel, simple, rapid, and one-step technique was developed for the fabrication of a uniform three-dimensional mesoporous gold thin film (MPGF) onto a gold (Au) coated glass plate based on an electrochemical deposition method. Scanning electron microscopy images demonstrated that the resulting MPGF electrode had uniformly distributed pores with diameters of about 20 nm. The cyclic voltammetric behavior of [Fe(CN)(6)](4-/3-) coupled onto MPGF and Au electrodes demonstrated that the MPGF electrode had a higher electrocatalytic sensitivity and reversibility than the bare Au electrode. The Arg-Gly-Asp (RGD) sequence containing the peptide was immobilized on the MPGF and bare Au substrates. HeLa cancer cells were then cultured on the RGD peptide layer. The successful immobilization of the peptide and cells was confirmed by atomic force microscopy. The cell proliferation and viability were evaluated by cyclic voltammetry and Trypan blue dyeing assay. These results indicated that the RGD/MPGF modified electrodes showed an electrochemical sensitivity in the detection of cancer cells which is approximately three times higher, especially at low cell density, than RGD/Au electrodes. This much improved sensitivity of the MPGF modified electrode demonstrates the potential for the fabrication of a highly sensitive and low-cost cell-based chip for rapid cancer detection.

Controlled fabrication of gold nanobipyramids/polypyrrole for shell-isolated nanoparticle-enhanced Raman spectroscopy to detect γ-aminobutyric acid.

Shell-isolated nanoparticle-enhanced Raman Spectroscopy (SHINERS) has been a non-destructive, highly sensitive, specific and powerful sensing method. Detection of γ-aminobutyric acid (GABA) and glutamate, main neurotransmitters in the human brain, is important to diagnosis the neurological disorder. The purpose of this study is preparing a simple, rapid and inexpensive fabrication of Au nanobipyramids/polymer core/shell as a SHINERS-based biosensor to detect different neurotransmitters such as GABA and glutamate with high sensitivity and specificity. Au nanobipyramids/polymer core/shell was fabricated by using two steps process. In the first Au nanobipyramids with longitude and latitude axial of about 100 nm and 10 nm, respectively, was prepared based on the chemical reduction of Au ions by using sodium borohydride as a reducing agent. Then a thin layer of polypyrrole was used for decorating the Au nanobipyramids by using direct polymerization in the presence of Au nanobipyramids. The sensor composed Au nanobipyramids with a thin layer of polypyrrole that could measure GABA within a wide range of concentrations in the presence of human serum. And this sensor was used for direct monitoring of GABA and glutamate. The proposed biosensor can be applied to monitor the level of neurotransmitters accurately for the diagnosis of various neurological disorders with optical signal enhancement.

The immobilization of animal cells using the cysteine-modified RGD oligopeptide.

RGD peptide sequence is an effective cell recognition motif and used to enhance the cell adhesion on desired solid material for cell immobilization. We have synthesized CRGD, CRGD-multiple-armed peptide (MAP), RGD-MAP-C and evaluated their comparative efficacy for cell immobilization. Each peptide was assembled on gold surface and investigated by the atomic force microscopy (AFM) technique in the contact mode. The viability of immobilized animal cells was examined by an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Our results showed that RGD-MAP-C in comparison to others was the most effective proliferation of cells on the gold surface. The goal of this present work is integration to the nano-pattern cell chip bioplatform for biomedical assays or provide valuable insights into cell biology and design of biomaterials. This RGD-MAP-C can be applicable to the nano-pattern cell chip platform.