Scanning electrochemical microscopy for determining oxygen consumption rates of cells in hydrogel fibers fabricated using an extrusion 3D bioprinter.

Three-dimensional (3D)-cultured cells have attracted the attention of researchers in tissue engineering- and drug screening-related fields. Among them, 3D cellular fibers have attracted significant attention because they can be stacked to prepare more complex tissues and organs. Cellular fibers are widely fabricated using extrusion 3D bioprinters. For these applications, it is necessary to evaluate cellular activities, such as the oxygen consumption rate (OCR), which is one of the major metabolic activities. We previously reported the use of scanning electrochemical microscopy (SECM) to evaluate the OCRs of cell spheroids. However, the SECM approach has not yet been applied to hydrogel fibers prepared using the bioprinters. To the best of our knowledge, this is the first study to evaluate the OCR of cellular fibers printed by extrusion 3D bioprinters. First, the diffusion theory was discussed to address this issue. Next, diffusion models were simulated to compare realistic models with this theory. Finally, the OCRs of MCF-7 cells in the printed hydrogel fibers were evaluated as a proof of concept. Our proposed approach could potentially be used to evaluate the OCRs of tissue-engineered fibers for organ transplantation and drug screening using in-vitro models.

Useful High-Entropy Source on Spinel Oxides for Gas Detection.

This study aimed to identify a useful high-entropy source for gas detection by spinel oxides that are composed of five cations in nearly equal molar amounts and free of impurities. The sensor responses of the spinel oxides [1# (CoCrFeMnNi)3O4, 2# (CoCrFeMnZn)3O4, 3# (CoCrFeNiZn)3O4, 4# (CoCrMnNiZn)3O4, 5# (CoFeMnNiZn)3O4, and 6# (CrFeMnNiZn)3O4] were evaluated for the test gases (7 ppm NO2, 5000 ppm H2, 3 ppm NH3, and 3 ppm H2S). In response to NO2, 1# and 2# showed p-type behavior while 3-6# showed n-type semiconductor behavior. There are three p-type and one n-type AO structural compositions in AB2O4[AO·B2O3] type spinel, and 1# showed a stable AO composition because cation migration from site B to site A is unlikely. Therefore, it was assumed that 1# exhibited p-type behavior. The p-type behavior of 2# was influenced by Cr oxide ions that were present at the B site and the stable p-type behavior of zinc oxide at the A site. The spinel oxides 3# to 6# exhibited n-type behavior with the other cationic oxides rather than the dominant p-type behavior exhibited by the Zn oxide ions that are stable at the A site. In contrast, the sensor response to the reducing gases H2, NH3, and H2S showed p-type semiconductor behavior, with a particularly selective response to H2S. The sensor responses of the five-element spinel oxides in this study tended to be higher than that of the two-element Ni ferrites and three-element Ni-Zn ferrites reported previously. Additionally, the susceptibility to sulfurization was evaluated using the thermodynamic equilibrium theory for the AO and B2O3 compositions. The oxides of Cr, Fe, and Mn ions in the B2O3 composition did not respond to H2S because they were not sulfurized. The increase in the sensor response due to sulfurization was attributed to the decrease in the depletion layer owing to electron sensitization, as the top surface of the p-type semiconductors, ZnO and NiO, transformed to n-type semiconductors, ZnS and NiS, respectively. High-entropy oxides prepared using the hydrothermal method with an equimolar combination of five cations from six elements (Cr, Mn, Fe, Co, Ni, and Zn) can be used as a guideline for the design of high-sensitivity spinel-type composite oxide gas sensors.

Magnetorheological Fluids with Surface-Modified Iron Oxide Magnetic Particles with Controlled Size and Shape.

This study is focused on surface-modified Fe3O4@SiO2 particles with precisely controlled sizes and shapes applied in magnetorheological (MR) fluids. After the preparation of the monodisperse spindle-shaped and cubic Fe3O4@SiO2 particles, surface modification with dodecyltrimethoxysilane (DTM) was carried out via a silane coupling reaction to increase the dispersion stability of the particles. Afterward, MR fluids were prepared by mixing the DTM-modified Fe3O4@SiO2 particles with silicon oil. Transmission electron microscopy observations demonstrated that spindle-shaped Fe3O4@SiO2 particles could form a more stable chain-like structure than cubic Fe3O4@SiO2 particles upon application of an external magnetic field. The rheological measurements of MR fluids also indicated that the surface modification with DTM, the introduction of anisotropic shapes, and the increase in the particle size all played positive roles in the improvement in MR properties.

Bioimaging using bipolar electrochemical microscopy with improved spatial resolution.

In this study, we developed bipolar electrochemical microscopy (BEM) using a closed bipolar electrode (cBPE) array with an electrochemiluminescence (ECL) detecting system. Because cBPEs are not directly connected to a detector, high spatio-temporal resolution imaging can be achieved by fabricating a microelectrode array in which each electrode point is arranged in a short interval. A cBPE array with individual cBPEs arranged in 41 µm intervals was successfully fabricated by depositing gold in the pores of a track-etched membrane using electroless plating. Using BEM with the cBPE array, which has a higher density of electrode points than the conventional multi-electrode array, we effectively demonstrated the imaging of [Fe(CN)6]3- diffusion and the respiratory activity of MCF-7 spheroids with high spatio-temporal resolution.

S/N Co-Doped Hollow Carbon Particles for Oxygen Reduction Electrocatalysts Prepared by Spontaneous Polymerization at Oil-Water Interfaces.

We herein report that sulfur and nitrogen co-doped hollow spherical carbon particles can be applied to oxygen reduction reaction (ORR) electrocatalysts prepared by calcination of polydopamine (PDA) hollow particles. The hollow structure of PDA was formed by auto-oxidative interfacial polymerization of dopamine at the oil and water interface of emulsion microdroplets. The PDA was used as the nitrogen source as well as a platform for sulfur-doping. The obtained sulfur and nitrogen co-doped hollow particles showed a higher catalytic activity than that of nonsulfur-doped particles and nonhollow particles. The high ORR activity of the calcined S-doped PDA hollow particles could be attributed to the combination of nitrogen and sulfur active sites and the large surface areas owing to a hollow spherical structure.

Electrochemicolor Imaging Using an LSI-Based Device for Multiplexed Cell Assays.

Multiplexed bioimaging systems have triggered the development of effective assays, contributing new biological information. Although electrochemical imaging is beneficial for quantitative analysis in real time, monitoring multiple cell functions is difficult. We have developed a novel electrochemical imaging system, herein, using a large-scale integration (LSI)-based amperometric device for detecting multiple biomolecules simultaneously. This system is designated as an electrochemicolor imaging system in which the current signals from two different types of biomolecules are depicted as a multicolor electrochemical image. The mode-selectable function of the 400-electrode device enables the imaging system and two different potentials can be independently applied to the selected electrodes. The imaging system is successfully applied for detecting multiple cell functions of the embryonic stem (ES) cell and the rat pheochromocytoma (PC12) cell aggregates. To the best of our knowledge, this is the first time that a real-time electrochemical mapping technique for multiple electroactive species, simultaneously, has been reported. The imaging system is a promising bioanalytical method for exploring complex biological phenomena.

Electrochemical Imaging of Dopamine Release from Three-Dimensional-Cultured PC12 Cells Using Large-Scale Integration-Based Amperometric Sensors.

In the present study, we used a large-scale integration (LSI)-based amperometric sensor array system, designated Bio-LSI, to image dopamine release from three-dimensional (3D)-cultured PC12 cells (PC12 spheroids). The Bio-LSI device consists of 400 sensor electrodes with a pitch of 250 µm for rapid electrochemical imaging of large areas. PC12 spheroids were stimulated with K(+) to release dopamine. Poststimulation dopamine release from the PC12 spheroids was electrochemically imaged using the Bio-LSI device. Bio-LSI clearly showed the effects of the dopaminergic drugs l-3,4-dihydroxyphenylalanine (L-DOPA) and reserpine on K(+)-stimulated dopamine release from PC12 spheroids. Our results demonstrate that dopamine release from PC12 spheroids can be monitored using the device, suggesting that the Bio-LSI is a promising tool for use in evaluating 3D-cultured dopaminergic cells and the effects of dopaminergic drugs. To the best of our knowledge, this report is the first to describe electrochemical imaging of dopamine release by PC12 spheroids using LSI-based amperometric sensors.

Material-binding peptide application--ZnO crystal structure control by means of a ZnO-binding peptide.

Recently, a zinc oxide (ZnO)-binding peptide (ZnOBP) has been identified and has been used to assist the synthesis of unique crystalline ZnO particles. We analyzed the influence of ZnOBP on the crystal growth of ZnO structures formed from zinc hydroxide. The addition of ZnOBP in the hydrothermal synthesis of ZnO suppressed [0001] crystal growth in the ZnO particles, indicating that the specificity of the material-binding peptide for specific inorganic crystal faces controlled the crystal growth. Furthermore, the dipeptides with a partial sequence of ZnO-binding "hot spot" in ZnOBP were used to synthesize ZnO particles, and we found that the presence of these dipeptides more strictly suppressed (0001) growth in ZnO crystals than did the complete ZnOBP sequence. These results demonstrate the applicability of dipeptides selected from material-binding peptides to control inorganic crystal growth.

High affinity anti-inorganic material antibody generation by integrating graft and evolution technologies: potential of antibodies as biointerface molecules.

Recent advances in molecular evolution technology enabled us to identify peptides and antibodies with affinity for inorganic materials. In the field of nanotechnology, the use of the functional peptides and antibodies should aid the construction of interface molecules designed to spontaneously link different nanomaterials; however, few material-binding antibodies, which have much higher affinity than short peptides, have been identified. Here, we generated high affinity antibodies from material-binding peptides by integrating peptide-grafting and phage-display techniques. A material-binding peptide sequence was first grafted into an appropriate loop of the complementarity determining region (CDR) of a camel-type single variable antibody fragment to create a low affinity material-binding antibody. Application of a combinatorial library approach to another CDR loop in the low affinity antibody then clearly and steadily promoted affinity for a specific material surface. Thermodynamic analysis demonstrated that the enthalpy synergistic effect from grafted and selected CDR loops drastically increased the affinity for material surface, indicating the potential of antibody scaffold for creating high affinity small interface units. We show the availability of the construction of antibodies by integrating graft and evolution technology for various inorganic materials and the potential of high affinity material-binding antibodies in biointerface applications.

Direct and selective immobilization of proteins by means of an inorganic material-binding peptide: discussion on functionalization in the elongation to material-binding peptide.

Using an artificial peptide library, we have identified a peptide with affinity for ZnO materials that could be used to selectively accumulate ZnO particles on polypropylene-gold plates. In this study, we fused recombinant green fluorescent protein (GFP) with this ZnO-binding peptide (ZnOBP) and then selectively immobilized the fused protein on ZnO particles. We determined an appropriate condition for selective immobilization of recombinant GFP, and the ZnO-binding function of ZnOBP-fused GFP was examined by elongating the ZnOBP tag from a single amino acid to the intact sequence. The fusion of ZnOBP with GFP enabled specific adsorption of GFP on ZnO substrates in an appropriate solution, and thermodynamic studies showed a predominantly enthalpy-dependent electrostatic interaction between ZnOBP and the ZnO surface. The ZnOBP's binding affinity for the ZnO surface increased first in terms of material selectivity and then in terms of high affinity as the GFP-fused peptide was elongated from a single amino acid to intact ZnOBP. We concluded that the enthalpy-dependent interaction between ZnOBP and ZnO was influenced by the presence of not only charged amino acids but also their surrounding residues in the ZnOBP sequence.

Grafting of material-binding function into antibodies Functionalization by peptide grafting.

Quite recently, a few antibodies against bulk material surface have been selected from a human repertoire antibody library, and they are attracting immense interest in the bottom-up integration of nanomaterials. Here, we constructed antibody fragments with binding affinity and specificity for nonbiological inorganic material surfaces by grafting material-binding peptides into loops of the complementarity determining region (CDR) of antibodies. Loops were replaced by peptides with affinity for zinc oxide and silver material surfaces. Selection of CDR loop for replacement was critical to the functionalization of the grafted fragments; the grafting of material-binding peptide into the CDR2 loop functionalized the antibody fragments with the same affinity and selectivity as the peptides used. Structural insight on the scaffold fragment used implies that material-binding peptide should be grafted onto the most exposed CDR loop on scaffold fragment. We show that the CDR-grafting technique leads to a build-up creation of the antibody with affinity for nonbiological materials.