Yusuke Mizutani

The number distribution of complex shear modulus of single cells measured by atomic force microscopy

The viscoelastic properties of a large number of mouse fibroblast NIH3T3 cells (n approximately 130) were investigated by combining atomic force microscopy (AFM) with a microarray technique. In the experiments, the cells were arranged and cultured in the wells of a microarray substrate, and a force modulation mode experiment was used to measure the complex shear modulus, G*, of individual cells in a frequency range 0.5-200Hz. The frequency dependence of G* of the cells exhibited a power-law behavior and similar frequency dependencies have been observed in several cell types cultured on flat substrates. This indicated that the NIH3T3 cells cultured in the wells of a microarray have analogous structural organization to those cells cultured on flat substrates. The number distribution of both the storage and loss moduli of G* fitted well to a log-normal distribution function, whereas the power-law exponent estimated by a power-law structural damping model showed a normal distribution function. These results showed that combining AFM with a microarray technique was a suitable approach for investigating the statistics of rheological properties of living cells without the requirement of cell surface modification.

Elasticity of Living Cells on a Microarray during the Early Stages of Adhesion Measured by Atomic Force Microscopy

The number distribution of the elastic modulus of fibroblast cells was successfully measured during the early stages of adhesion using an atomic force microscope (AFM) combined with a microarray as a substrate, which allowed us to arrange and culture cells so that a large number of cells could be measured in a short time period. We confirmed that the cells deposited in the wells of the microarray could be cultured for at least 12 h without any significant migration. Histograms of the Youngs modulus, E, of the cells during the early stages of adhesion produced from force curve measurements of cells (n\cong300) cultured for 3–9 h were well fitted to a log-normal distribution function. With increasing incubation time, the average value of E increased significantly, while the standard deviation of the distribution remained almost constant. The results are discussed in terms of the cytoskeleton inside cells.

Power-Law Stress and Creep Relaxations of Single Cells Measured by Colloidal Probe Atomic Force Microscopy

We measured stress and creep relaxations of mouse fibroblast cells arranged and cultured on a microarray, by colloidal probe atomic force microscopy (AFM). A hydrophobic monolayer coating of perfluorodecyltrichlorosilane (FDTS) on the surface of colloidal silica beads significantly reduced the adhesion force of live cells, compared with untreated beads. The rheological behaviors of cells were estimated by averaging several relaxation curves of cells measured by the AFM. Longer-time tailing of both stress and creep relaxation curves followed single power-law behavior over a time scale of 60 s, with exponents in the range 0.1–0.4, varying with cells. The results were in good agreement with previous measurements of the frequency-domain rheology of cells using the force modulation mode.

Oxidative Debromination and Degradation of Tetrabromo-bisphenol A by a Functionalized Silica-Supported Iron(III)-tetrakis(p-sulfonatophenyl)porphyrin Catalyst

Tetrabromobisphenol A (TBBPA), a commonly used brominated flame retardant, also functions as an endocrine disruptor. Thus, the degradation of TBBPA has attracted considerable interest among the scientific community. Iron(III)-porphyrin complexes are generally regarded as “green” catalysts and have been reported to catalyze the efficient degradation and dehalogenation of halogenated phenols in environmental wastewaters. However, they are quickly deactivated due to self-degradation in the presence of an oxygen donor, such as KHSO5. In the present study, an iron(III)-tetrakis(p-sulfonatophenyl)-porphyrin (FeTPPS) was immobilized on imidazole-modified silica (FeTPPS/IPS) via coordination of the Fe(III) with the nitrogen atom in imidazole to suppress self-degradation and thus enhance the catalyst reusability. The oxidative degradation and debromination of TBBPA and the influence of humic acid (HA), a major component in leachates, on the oxidation of TBBPA was investigated. More than 95% of the TBBPA was degraded in the pH range from 3 to 8 in the absence of HA, while the optimal pH for the reaction was at pH 8 in the presence of HA. Although the rate of degradation was decreased in the presence of HA, over 95% of the TBBPA was degraded within 12 h in the presence of 28 mg-C L−1 of HA. At pH 8, the FeTPPS/IPS catalyst could be reused up to 10 times without any detectable loss of activity for TBBPA for degradation and debromination, even in the presence of HA.

Influence of Halogen Substituents on the Catalytic Oxidation of 2,4,6-Halogenated Phenols by Fe(III)-Tetrakis(p-hydroxyphenyl) porphyrins and Potassium Monopersulfate

The influence of halogen substituents on the catalytic oxidation of 2,4,6-trihalogenated phenols (TrXPs) by iron(III)-porphyrin/KHSO5 catalytic systems was investigated. Iron(III)-5,10,15,20-tetrakis(p-hydroxyphenyl)porphyrin (FeTHP) and its supported variants were employed, where the supported catalysts were synthesized by introducing FeTHP into hydroquinone-derived humic acids via formaldehyde poly-condensation. F (TrFP), Cl (TrCP), Br (TrBP) and I (TrIP) were examined as halogen substituents for TrXPs. Although the supported catalysts significantly enhanced the degradation and dehalogenation of TrFP and TrCP, the oxidation of TrBP and TrIP was not enhanced, compared to the FeTHP catalytic system. These results indicate that the degree of oxidation of TrXPs is strongly dependent on the types of halogen substituent. The order of dehalogenation levels for halogen substituents in TrXPs was F > Cl > Br > I, consistent with their order of electronegativity. The electronegativity of a halogen substituent affects the nucleophilicity of the carbon to which it is attached. The levels of oxidation products in the reaction mixtures were analyzed by GC/MS after extraction with n-hexane. The most abundant dimer product from TrFP via 2,6-difluoroquinone is consistent with a scenario where TrXP, with a more electronegative halogen substituent, is readily oxidized, while less electronegative halogen substituents are oxidized less readily by iron(III)-porphyrin/KHSO5 catalytic systems.

Potassium monopersulfate oxidation of 2,4,6-tribromophenol catalyzed by a SiO2-supported iron(III)-5,10,15,20-tetrakis(4-carboxyphenyl)porphyrin

Iron(III)-porphyrin complexes are generally regarded as green catalysts, since they mimic the catalytic center of cytochrome-P450 and widely used as green catalysts for degrading halogenated phenols in wastewater, such as landfill leachates. However, iron(III)-porphyrins are deactivated by self-oxidation in the presence of an oxygen donor, such as KHSO5. In the present study, to enhance the reusability of an iron(III)-porphyrin catalyst, iron(III)-5,10,15,20-tetrakis(4-carboxyphenyl) porphyrin (FeTCPP) was immobilized on a functionalized silica gel. The oxidative degradation of 2,4,6-tribromophenol (TrBP), a widely used brominated flame retardant that is found in landfill leachates, was examined using the prepared catalyst. In addition, the influence of humic substances (HSs), major components of leachates, on the TrBP oxidation was investigated. Concerning the effect of pH, more than 90% of the TrBP was degraded in the pH range of 3–8 in the absence of HS, while the optimal pH for the reaction was in the range of pH 5-7 in the presence of HS. Although the oxidation of TrBP was inhibited in the presence of HSs, more than 90% of the TrBP was degraded in the presence of 50 mg L−1 of HS. Thus, the prepared catalyst, SiO2-FeTCPP, showed a high catalytic activity and could be reused up to 10 times even in the presence of HS.

Nanoscale fluctuations on epithelial cell surfaces investigated by scanning ion conductance microscopy

Nanoscale fluctuations on the apical surfaces of epithelial cells connected to neighboring cells were investigated by scanning ion conductance microscopy. Mapping the ion current as a function of the tip–surface distance revealed that in untreated cells, the apparent fluctuation amplitude increased towards the cell center. We found that the spatial dependence was less correlated with the heterogeneities of cell stiffness but was significantly reduced when actin filaments were disrupted. The results indicate that apical surface fluctuations are highly constrained at the cell–cell interface, in the vertical direction to the surface and by the underlying actin filaments.

The oxidation of tetrabromobisphenol A by potassium monopersulfate with an iron(III)-phthalocyanine-tetrasulfonic acid catalyst in the presence of humic acid

Tetrabromobisphenol A (TBBPA), a type of brominated flame retardant that shows endocrine disruption effects, has been identified in leachates from landfills. Iron(III)-porphyrins that mimic the active site of peroxidases have been shown to be effective in oxidizing halogenated phenols, such as TBBPA. In the present study, TBBPA was subjected to oxidation with potassium monopersulfate (KHSO5) using an iron(III)-phthalocyanine-tetrasulfonic acid (FePcTS), structural analogue of iron(III)-porphyrin, in the presence of humic acid (HA), a major component in landfill leachates. When TBBPA was oxidized using the above system, the levels of degradation and debromination increased with increasing pH in the presence of HA. Because of landfill leachates are weakly alkaline (around pH 8), oxidation products derived from TBBPA were investigated at pH 8. Approximately 48% of the bromine in the degraded TBBPA was incorporated into HA, and hydroxy-tribromobisphenol A was determined to be the major brominated intermediate in the HA fraction. In the iron(III)-porphyrin catalytic systems, the brominated intermediate incorporated into HA is mainly TBBPA, and no hydroxy-substituted bromophenols are found. Thus, the catalytic power of FePcTS is higher than that of iron(III)-porphyrin catalysts.

Comparison of the oxidation products produced by tetrahalobisphenol A flame retardants as a result of potassium monopersulfate oxidation with an iron(III)-tetrakis(p-sulfonatophenyl)porphyrin in the presence of humic acid

Tetrabromobisphenol A (TBBPA) and tetrachlorobisphenol A (TCBPA), commercially used halogenated flame retardants, can be found in leachates from landfills, because hydrophobic interactions with humic acids (HAs), major organic components in landfills, result in an increase in their solubility. The oxidation characteristics of TBBPA and TCBPA in the presence of HA were compared using a catalytic system comprised of a combination of iron(III)-tetrakis(p-sulfophenyl)porphyrin (FeTPPS) and KHSO5 that can mimic the enzymatic reactions that occur in landfills. The levels of degradation and dehalogenation of TBBPA and TCBPA at pH 4 were significantly lower than at pH 8, which is a typical pH value for landfill leachates. In the presence of HA at pH 8, 2-hydroxyisopropyl-2,6 -dihalophenols (2HIP-26DXPs) were detected as major by-products. These compounds are likely produced via the β-carbon scission of the substrates, and their levels decreased with increasing reaction time. The levels of coupling compounds between 2,6-dihalopnenols and TBBPA or TCBPA increased with reaction time. The 27% of Br in the degraded TBBPA and 50% of Cl in the degraded TCBPA were incorporated into the HA as a result of catalytic oxidation via the FeTPPS/KHSO5 system. These results suggest that TCBPA is incorporated into HA more readily than TBBPA. The coupling compounds between HA and halogenated intermediates from TBBPA or TCBPA were assigned by pyrolysis-gas chromatography/mass spectrometry.

Rheological Properties of Growth-Arrested Fibroblast Cells under Serum Starvation Measured by Atomic Force Microscopy

The rheological properties of growth-arrested and quiescent (G0 phase) mouse fibroblast cells under serum starvation were investigated by atomic force microscopy (AFM) with a microarray technique. The number distribution of complex shear modulus, G*, of quiescent cells at the serum concentration, CS=0.1%, followed a log-normal distribution, and the frequency dependence of G* exhibited a power law behavior, which were similar to those under a control condition at CS=10%. On the other hand, we found that the Newtonian viscosity coefficient of the quiescent cells significantly increased, and the distribution broadened, as compared with the control cells, whereas the power-law exponent was unchanged. The result indicated that the rheological properties of quiescent fibroblast cells were not identical to those in the G1 phase during cell cycle. This finding suggests that the Newtonian viscosity of cells is one of the useful indicators for evaluating growth-arrested cells under serum starvation.