Recent Advances in Electrochemical Detection of Cell Energy Metabolism.

Cell energy metabolism is a complex and multifaceted process by which some of the most important nutrients, particularly glucose and other sugars, are transformed into energy. This complexity is a result of dynamic interactions between multiple components, including ions, metabolic intermediates, and products that arise from biochemical reactions, such as glycolysis and mitochondrial oxidative phosphorylation (OXPHOS), the two main metabolic pathways that provide adenosine triphosphate (ATP), the main source of chemical energy driving various physiological activities. Impaired cell energy metabolism and perturbations or dysfunctions in associated metabolites are frequently implicated in numerous diseases, such as diabetes, cancer, and neurodegenerative and cardiovascular disorders. As a result, altered metabolites hold value as potential disease biomarkers. Electrochemical biosensors are attractive devices for the early diagnosis of many diseases and disorders based on biomarkers due to their advantages of efficiency, simplicity, low cost, high sensitivity, and high selectivity in the detection of anomalies in cellular energy metabolism, including key metabolites involved in glycolysis and mitochondrial processes, such as glucose, lactate, nicotinamide adenine dinucleotide (NADH), reactive oxygen species (ROS), glutamate, and ATP, both in vivo and in vitro. This paper offers a detailed examination of electrochemical biosensors for the detection of glycolytic and mitochondrial metabolites, along with their many applications in cell chips and wearable sensors.

Implementation of particle swarm optimization for complete inverse design of multilayered optical filters.

Particle swarm optimization is implemented for the complete inverse design of multilayered optical filters. To achieve this, a model is designed to optimize the thickness and material of each layer, as well as the total number of layers, simultaneously. The performance of the model is evaluated by repeating the optimization process, enabling clarification of the effects of model parameters on the final output. The designed model is also demonstrated for the optimization of various optical filters, including bandstop filters, bandpass filters, and anti-reflection coatings. The results confirm that particle swarm optimization is capable of designing arbitrary optical filters that cannot be designed using conventional design theories.

Extracellularly Detectable Electrochemical Signals of Living Cells Originate from Metabolic Reactions.

Direct detection of cellular redox signals has shown immense potential as a novel living cell analysis tool. However, the origin of such signals remains unknown, which hinders the widespread use of electrochemical methods for cellular research. In this study, the authors found that intracellular metabolic pathways that generate adenosine triphosphate (ATP) are the main contributors to extracellularly detectable electrochemical signals. This is achieved through the detection of living cells (4,706 cells/chip, linearity: 0.985) at a linear range of 7,466-48,866. Based on this discovery, the authors demonstrated that the cellular signals detected by differential pulse voltammetry (DPV) can be rapidly amplified with a developed medium containing metabolic activator cocktails (MACs). The DPV approach combined with MAC treatment shows a remarkable performance to detect the effects of the anticancer drug CPI-613 on cervical cancer both at a low drug concentration (2 µm) and an extremely short treatment time (1 hour). Furthermore, the senescence of mesenchymal stem cells could also be sensitively quantified using the DPV+MAC method even at a low passage number (P6). Collectively, their findings unveiled the origin of redox signals in living cells, which has important implications for the characterization of various cellular functions and behaviors using electrochemical approaches.

Label-free and non-destructive identification of naïve and primed embryonic stem cells based on differences in cellular metabolism.

Pluripotent stem cells (PSCs) exist in naïve or primed states based on their origin. For in vitro culture, these PSCs require different supplements and growth factors. However, owing to their similar phenotypic features, identifying both cell types without harming cellular functions is challenging. This study reports an electrochemical method that enables simple, label-free, and non-destructive detection of naïve embryonic stem cells (ESCs) derived from mouse ESCs, based on the differences in cellular metabolism. Two major metabolic pathways to generate adenosine triphosphate (ATP)-glycolysis and oxidative phosphorylation (OXPHOS)-were blocked, and it was found that mitochondrial energy generation is the origin of the strong electrochemical signals of naïve ESCs. The number of ESCs is quantified when mixed with primed ESCs or converted from naïve-primed switchable metastable ESCs. The mouse PSCs derived from doxycycline-inducible mouse embryonic fibroblasts (MEFs) are also sensitively identified among other cell types such as unconverted MEFs and primed PSCs. The developed sensing platform operates in a non-invasive and label-free manner. Thus, it can be useful in the development of stem cell-derived therapeutics.

Gold nanostructure-integrated conductive microwell arrays for uniform cancer spheroid formation and electrochemical drug screening.

Cancer spheroids, which mimic distinct cell-to-cell and cell-extracellular matrix interactions of solid tumors in vitro, have emerged as a promising tumor model for drug screening. However, owing to the unique characteristics of spheroids composed of three-dimensionally densely-packed cells, the precise characterizations of cell viability and function with conventional colorimetric assays are challenging. Herein, we report gold nanostructure-integrated conductive microwell arrays (GONIMA) that enable both highly efficient uniform cancer spheroid formation and precise electrochemical detection of cell viability. A nanostructured gold on indium tin oxide (ITO) substrate facilitated the initial cell aggregation and further 3D cell growth, while the non-cytophilic polymer microwell arrays restricted the size and shape of the spheroids. As a result, approximately 150 human glioblastoma spheroids were formed on a chip area of 1.13 cm2 with an average diameter of 224 µm and a size variation of only 5% (±11.36 µm). The high uniformity of cancer spheroids contributed to the stability of electrical signals measuring cell viability. Using the fabricated GONIMA, the effects of a representative chemotherapeutic agent, hydroxyurea, on the glioblastoma spheroids were precisely monitored under conditions of varying drug concentrations (0-0.3 mg/mL) and incubation times (24-48 h). Therefore, we conclude that the newly developed platform is highly useful for rapid and precise in vitro drug screening, as well as for the pharmacokinetic analyses of specific drugs using 3D cellular cancer models.

Recent Advances in Electrochemical Biosensors for Monitoring Animal Cell Function and Viability.

Redox reactions in live cells are generated by involving various redox biomolecules for maintaining cell viability and functions. These qualities have been exploited in the development of clinical monitoring, diagnostic approaches, and numerous types of biosensors. Particularly, electrochemical biosensor-based live-cell detection technologies, such as electric cell-substrate impedance (ECIS), field-effect transistors (FETs), and potentiometric-based biosensors, are used for the electrochemical-based sensing of extracellular changes, genetic alterations, and redox reactions. In addition to the electrochemical biosensors for live-cell detection, cancer and stem cells may be immobilized on an electrode surface and evaluated electrochemically. Various nanomaterials and cell-friendly ligands are used to enhance the sensitivity of electrochemical biosensors. Here, we discuss recent advances in the use of electrochemical sensors for determining cell viability and function, which are essential for the practical application of these sensors as tools for pharmaceutical analysis and toxicity testing. We believe that this review will motivate researchers to enhance their efforts devoted to accelerating the development of electrochemical biosensors for future applications in the pharmaceutical industry and stem cell therapeutics.

Electrically controlled mRNA delivery using a polypyrrole-graphene oxide hybrid film to promote osteogenic differentiation of human mesenchymal stem cells.

Direct messenger ribonucleic acid (mRNA) delivery to target cells or tissues has revolutionized the field of biotechnology. However, the applicability of regenerative medicine is limited by the technical difficulties of various mRNA-loaded nanocarriers. Herein, we report a new conductive hybrid film that could guide osteogenic differentiation of human adipose-derived mesenchymal stem cells (hADMSCs) via electrically controlled mRNA delivery. To find optimal electrical conductivity and mRNA-loading capacity, the polypyrrole-graphene oxide (PPy-GO) hybrid film was electropolymerized on indium tin oxide substrates. We found that the fluorescein sodium salt, a molecule partially mimicking the physical and chemical properties of mRNAs, can be effectively absorbed and released by electrical stimulation (ES). The hADMSCs cultivated on the PPy-GO hybrid film loaded with pre-osteogenic mRNAs showed the highest osteogenic differentiation under electrical stimulation. This platform can load various types of RNAs thus highly promising as a new nucleic acid delivery tool for the development of stem cell-based therapeutics. Electronic Supplementary Material: Supplementary material (electrochemical and FT-IR analysis on the film, additional SEM, AFM and C-AFM images of the film, optical and fluorescence images of cells, and the primers used for RT-qPCR analysis) is available in the online version of this article at 10.1007/s12274-022-4613-y.

Bone Regeneration Effect of Hyperbaric Oxygen Therapy Duration on Calvarial Defects in Irradiated Rats.

OBJECTIVE: In this study, we evaluated changes in bone remodeling in an irradiated rat calvarial defect model according to duration of hyperbaric oxygen therapy. MATERIALS AND METHODS: The 28 rats were divided into four groups. Radiation of 12 Gy was applied to the skull, and 5-mm critical size defects were formed on both sides of the skull. Bone grafts were applied to one side of formed defects. From the day after surgery, HBO was applied for 0, 1, and 3 weeks. At 6 weeks after bone graft, experimental sites were removed and analyzed for radiography, histology, and histomorphometry. RESULTS: Micro-CT analysis showed a significant increase in new bone volume in the HBO-3 group, with or without bone graft. When bone grafting was performed, BV, BS, and BS/TV all significantly increased. Histomorphometric analysis showed significant increases in %NBA and %BVN in the HBO-1 and HBO-3 groups, regardless of bone graft. CONCLUSION: Hyperbaric oxygen therapy was effective for bone regeneration with only 1 week of treatment.

Hybrid and Etch-Less Electrooptic Waveguide Modulator Based on Photo-Bleaching and Strain Induced Optical Waveguide Technique in Polymer.

A hybrid and etchless electrooptic (EO) polymer waveguide modulator based on both a photo-bleaching-induced optical waveguide (PBOW) and a strain-induced optical waveguide (SIOW) is described. The SIOW is defined by a metal strip line stressor deposited on top of the upper cladding that introduces the refractive index change within the core region. The PBOW technique is used to form an optical waveguide which is based on a photo-bleaching process, known as a photo-oxidation that is an irreversible decomposition of EO material, resulting in a permanent decrease in index of refraction. It is shown that this proposed fabrication idea combining two etchless techniques can be applicable to a wide range of polymer photonic integrated circuits. Preliminary results obtained from fabricated devices reveal that their half-wave voltage are ranging from 8 V to 10 V, their extinction ratio exhibits more than 15 dB, and the fiber-to-waveguide-to-lens loss is estimated to be ~9.5 dB for TM polarization at 1.55/m wavelength in the active interaction of ~1.5 cm long.

Synthesis and characterization of chitosan-PEG-Ag nanocomposites for antimicrobial application.

New chitosan nanocomposites doped with silver nanoparticles were synthesized by a simple method. The chitosan particles were prepared by desolvation followed by crosslinking with poly(ethylene glycol-di-aldehyde), this was prepared with poly(ethylene glycol) in the presence of a silver nitrate solution. The developed nanocomposites were characterized using UV-visible, FTIR, XRD, TGA, SEM and TEM to understand their physico-chemical properties. These nanocomposites were shown to have anti bacterial activity towards Escherichia coli.

Isomeric discrimination and quantification of thyroid hormones, T3 and rT3, by the single ratio kinetic method using electrospray ionization mass spectrometry.

The single ratio kinetic method is applied to the discrimination and quantification of the thyroid hormone isomers, 3,5,3'-triiodothyronine and 3,3',5'-triiodothyronine, in the gas phase, based on the kinetics of the competitive unimolecular dissociations of singly charged transition-metal ion-bound trimeric complexes [M(II)(A)(ref*)(2)-H](+) (M(II) = divalent transition-metal ion; A = T(3) or rT(3); ref* = reference ligand). The trimeric complex ions are generated using electrospray ionization mass spectrometry and the ions undergo collisional activation to realize isomeric discrimination from the branching ratio of the two fragment pathways that form the dimeric complexes [M(II)(A)(ref*)-H](+) and [M(II)(ref*)(2)-H](+). The ratio of the individual branching ratios for the two isomers R(iso) is found strongly dependent on the references and the metal ions. Various sets are tried by choosing the reference from amino acids, substituted amino acids, and dipeptides in combination with the central metal ion chosen from five transition-metal ions (Co(II), Cu(II), Mn(II), Ni(II), and Zn(II)) for the complexes in this experiment. The results are compared in terms of the isomeric discrimination for the T(3)/rT(3) pair. Calibration curves are constructed by relating the ratio of the branching ratios against the isomeric composition of their mixture to allow rapid quantitative isomer analysis of the sample pair. Furthermore, the instrument-dependence of this method is investigated by comparing the two sets of results, one obtained from a quadrupole ion trap mass spectrometer and the other from a quadrupole time-of-flight mass spectrometer.