Simulation Study of the Effect of Nanoporous Surfaces on the Adhesion Properties of Cross-Linked Polymer Networks.

We have investigated the effect of surface nanopores on the adhesion behavior between cross-linked polymer networks and metal substrates by molecular dynamics simulations. By increasing the cross-linking ratio of the polymer network, the fracture behavior in tensile mode changed from cohesive failure to interfacial failure. In the case of polymers without cross-links, the breaking strengths were almost the same for systems with flat and porous metal substrates. Conversely, in the case of cross-linked polymer networks, the tensile behavior for the porous metal substrates depended on the cross-linking ratio and structure of the polymer chains. For polymer networks consisting of long polymer chains, the force curves in extension mode before the yield points were almost the same for the systems regardless of the surface roughness caused by nanopores. Meanwhile, for highly cross-linked resin networks consisting of short rigid molecules, the yielding strength of the porous metal surfaces showed slightly higher values than that of the flat metal surfaces. The simulation results revealed that the adhesion behavior between cross-linked polymer networks and rough metal surfaces is related not only to the interfacial area but also to the detailed networking topology of the polymers.

Disentangling Origins of Adhesive Bonding at Interfaces between Epoxy/Amine Adhesive and Aluminum.

Joining metals by adhesive bonding is essential in widespread fields such as mobility, dentistry, and electronics. Although adhesive technology has grown since the 1920s, the roles of interfacial phenomena in adhesive bonding are still elusive, which hampers the on-demand selection of surface treatment and adhesive types. In the present study, we clarified how chemical interactions and mechanical interlocking governed adhesive bonding by evaluating adhesion properties at the interfaces between epoxy/amine adhesive and two kinds of Al adherends: a flat aluminum hydroxide (AlxOyHz) and technical Al plate with roughness. Spectroscopic and microscopical data demonstrate that the protonation of the amino groups in an amine hardener converts Al(OH)3 on the AlxOyHz surface to AlO(OH). The interfacial protonation results in an interfacial dipole layer with positive charges on the adhesive side, whose electrostatic interaction increases the interfacial fracture energy. The double cantilever beam tests for the flat AlxOyHz and technical Al substrates clarify that the mechanical interlocking originating from the surface roughness further increases the fracture energy. This study disentangles the roles of the chemical interactions and mechanical interlocking occurring at the epoxy adhesive/Al interface in the adhesion mechanism.

Simulation Study of the Effects of Interfacial Bonds on Adhesion and Fracture Behavior of Epoxy Resin Layers.

The adhesion and fracture behavior of tetraglycidyl-4,4'-diaminodiphenylmethane (TGDDM)/4,4'-diaminodiphenyl sulfone (44DDS)-bisphenol A diglycidyl ether (DGEBA)/44DDS layer interfaces were investigated by molecular dynamics (MD) simulation, mainly focusing on the role of covalent and noncovalent interactions. To accurately investigate the bond dissociation processes, the force field parameters of several bond potentials of the epoxy resin polymers were optimized by density functional theory calculations. In the MD simulations under a tensile load, small voids gradually developed without covalent bond dissociation in the plateau region. In the final large strain region, the stress rapidly increased with bond breaking, leading to failure. When the chemical bonds across the interface between the two layers were removed, the stress-strain curve in the initial elastic region was almost the same as that with interfacial bonds. This showed that the nonbonded interactions governed adhesion strength in the initial elastic region. In contrast, the bonded interactions at interfaces played important roles in the hardening regions because the bonded interactions made the major contribution to the fracture energies. We also investigated the effect of the etherification reaction in cross-linking. It was found that the etherification reaction mainly contributed to the behavior in the late region with large strain. These simulation results revealed that the nonbonded interactions, especially, van der Waals interactions, are important factors for adhesion of the different polymer layers in the small strain region up to the yield point.

Urinary adiponectin as a new diagnostic index for chronic kidney disease due to diabetic nephropathy.

Objective: The chronic kidney disease (CKD) is widely diagnosed on the basis of albuminuria and the glomerular filtration rate. A more precise diagnosis of CKD, however, requires the assessment of other factors. Urinary adiponectin recently attracted attention for CKD assessment, but evaluation is difficult due to the very low concentration of urinary adiponectin in normal subjects. Research design and methods: We developed an ultrasensitive ELISA coupled with thionicotinamide-adenine dinucleotide cycling to detect trace amounts of proteins, which allows us to measure urinary adiponectin at the subattomole level. We measured urinary adiponectin levels in 59 patients with diabetes mellitus (DM) and 24 subjects without DM (normal) to test our hypothesis that urinary adiponectin levels increase with progression of CKD due to DM. Results: The urinary adiponectin levels were 14.88±3.16 (ng/mg creatinine, mean±SEM) for patients with DM, and 3.06±0.33 (ng/mg creatinine) for normal subjects. The threshold between them was 4.0 ng/mg creatinine. The urinary adiponectin levels increased with an increase in the CKD risk. Furthermore, urinary adiponectin mainly formed a medium-molecular weight multimer (a hexamer) in patients with DM, whereas it formed only a low-molecular weight multimer (a trimer) in normal subjects. That is, the increase in urinary adiponectin in patients with DM led to the emergence of a medium-molecular weight form in urine. Conclusions: Our new assay showed that urinary adiponectin could be a new diagnostic index for CKD. This assay is a non-invasive test using only urine, thus reducing the patient burden.

Simulation Study of the Effects of Nanoporous Structures on Mechanical Properties at Polymer-Metal Interfaces.

We investigated the effect of nanopores on the adhesion behavior at polymer-metal interfaces by molecular dynamics simulation. The effects of shear and extension behavior were examined. In the shear mode, samples with porous substrates showed larger shear forces than those with flat substrates. Meanwhile, the breaking strengths in the extension mode were almost the same for systems with flat and porous substrates. The similar behavior in the extension mode was ascribed to the formation of voids in the polymer layer, which was related to the increase of total system volume and not affected by the presence of pores. We also investigated the relationship between the mechanical properties of polymer-metal interfaces in the shear mode and pore size in detail. Even a very shallow pore with a depth of 0.5 nm produced a large shear force comparable to that of a pore with a depth of 2.0 nm. The shear force increased gradually as the pore diameter became wider. These simulation results revealed that the adhesion forces between polymers and rough metal surfaces are not simply related to the interface area but depend on the pulling mode, pore size, and polymer chain length in a complicated manner.

Simulation Study of the Effect of the Side-Chain Structure on the Initial Nucleation Process of Polythiophene Derivatives.

We have investigated the initial nucleation process of polythiophene derivatives by molecular dynamics simulations to elucidate the role of flexible side chains in the crystallization process. We considered poly(3-hexylthiophene) (P3HT), poly(3,3‴-dihexylquarterthiophene) (PQT6), poly(3-dodecylthiophene) (P3DDT), and poly(3,3‴-didodecylquaterthiophene) (PQT12), in which the lengths and densities of the alkyl side chains are different. In the case of the short side-chain molecules (P3HT and PQT6), the initial nucleation process is based on the following steps. At the beginning, the thiophene rings align and ordering of the main chains commences shortly afterward. Ordering of the flexible side chains is induced after formation of a stacked structure of rigid main chains. In the case of a long and dense side-chain molecule (P3DDT), the initial nucleation process shows different features. The ordering process of the thiophene rings or main chains becomes very slow, while that of side chains becomes fast. In addition, the local order of the main chains becomes lower than those of P3HT and PQT6. These simulation results reveal that long and dense side chains suppress the nucleation process, whereas long but low-density side chains lead to formation of stacked nuclei in a different manner from those observed for short side-chain molecules.

Ultrasensitive detection of proteins and sugars at single-cell level.

Each cell produces its own responses even if it appears identical to other cells. To analyze these individual cell characteristics, we need to measure trace amounts of molecules in a single cell. Nucleic acids in a single cell can be easily amplified by polymerase chain reaction, but single-cell measurement of proteins and sugars will require de novo techniques. In the present study, we outline the techniques we have developed toward this end. For proteins, our ultrasensitive enzyme-linked immunosorbent assay (ELISA) coupled with thionicotinamide-adenine dinucleotide cycling can detect proteins at subattomoles per assay. For sugars, fluorescence correlation spectroscopy coupled with glucose oxidase-catalyzed reaction allows us to measure glucose at tens of nM. Our methods thus offer versatile techniques for single-cell-level analyses, and they are hoped to strongly promote single-cell biology as well as to develop noninvasive tests in clinical medicine.

Immunoreactive insulin in diabetes mellitus patient sera detected by ultrasensitive ELISA with thio-NAD cycling.

To minimize patient suffering, the smallest possible volume of blood should be collected for diagnosis and disease monitoring. When estimating insulin secretion capacity and resistance to insulin in diabetes mellitus (DM), increasing insulin assay immunosensitivity would reduce the blood sample volume required for testing. Here we present an ultrasensitive ELISA coupled with thio-NAD cycling to measure immunoreactive insulin in blood serum. Only 5 µL of serum was required for testing, with a limit of detection (LOD) for the assay of 10(-16) moles/assay. Additional recovery tests confirmed this method can detect insulin in sera. Comparisons between a commercially available immunoreactive insulin kit and our ultrasensitive ELISA using the same commercially available reference demonstrated good data correlation, providing further evidence of assay accuracy. Together, these results demonstrate our ultrasensitive ELISA could be a powerful tool in the diagnosis and treatment of not only DM but also many other diseases in the future.

Formation of Hydroxyapatite Skeletal Materials from Hydrogel Matrices via Artificial Biomineralization.

Several kinds of hydrogels were prepared as mimics for the collagen/acidic protein hydrogel employed as the polymer matrix for mineralization in natural bone formation. The hydrogels prepared as mineralization matrices were employed for synthesizing artificial bones. The artificial bone made from a network of poly(vinyl alcohol) (PVA) and poly(acrylic acid) (PAA) prepared by heating (PVA/PAA-h-network) exhibited mechanical properties comparable with those of fish scales. To elucidate the formation mechanism of the artificial bone, we synthesized four further kinds of matrix. Artificial bones were obtained from both a PVA/PAA network prepared by repeated freezing and thawing (PVA/PAA-ft-network) and a chitosan/PAA network, in which hydrogen bonding exists between the two constituent polymers, similar to that observed in a natural collagen/acidic protein network. The artificial bone made from the chitosan/PAA network was confirmed to be formed by the phase transformation of a cartilaginous precursor by a process similar to the transformation of cartilaginous tissue to natural bone. In addition, skeletal phase material, i.e., a homogeneous solid phase of hydroxyapatite/polymers, was formed in the cartilaginous phase, i.e., the hypercomplex gel. The skeletal phase grew thicker at the expense of the cartilaginous phase until it formed the entirety of the composite. Artificial bones were also obtained from a gelatin/PAA network and a poly[N-(2-hydroxyethyl)acrylamide]-co-(acrylic acid) network. These experimental results suggested that the coexistence of proton donor and proton acceptor functions in the hydrogel is a key factor for bone formation. The hydroxyapatite content of our artificial bones was almost conterminous with those of natural bones.

Detection of HIV-1 p24 at Attomole Level by Ultrasensitive ELISA with Thio-NAD Cycling.

To reduce the window period between HIV-1 infection and the ability to diagnose it, a fourth-generation immunoassay including the detection of HIV-1 p24 antigen has been developed. However, because the commercially available systems for this assay use special, high-cost instruments to measure, for example, chemiluminescence, it is performed only by diagnostics companies and hub hospitals. To overcome this limitation, we applied an ultrasensitive ELISA coupled with a thio-NAD cycling, which is based on a usual enzyme immunoassay without special instruments, to detect HIV-1 p24. The p24 detection limit by our ultrasensitive ELISA was 0.0065 IU/assay (i.e., ca. 10(-18) moles/assay). Because HIV-1 p24 antigen is thought to be present in the virion in much greater numbers than viral RNA copies, the value of 10(-18) moles of the p24/assay corresponds to ca. 10(3) copies of the HIV-1 RNA/assay. That is, our ultrasensitive ELISA is chasing the detection limit (10(2) copies/assay) obtained by PCR-based nucleic acid testing (NAT) with a margin of only one different order. Further, the detection limit by our ultrasensitive ELISA is less than that mandated for a CE-marked HIV antigen/antibody assay. An additional recovery test using blood supported the reliability of our ultrasensitive ELISA.

Effects of surface affinity on the ordering dynamics of self-assembled monolayers of chain molecules: Transition from a parallel to a perpendicular structure.

The effects of surface interactions on the ordering dynamics of self-assembled monolayers (SAM) of chain molecules were studied using molecular dynamics simulations. When the strength of surface-chain interactions was equal to or less than that of chain-chain interactions, domains of chain molecules adsorbed perpendicular to the surface ("upright" chains) formed on the surface. Although chain molecules adsorbed parallel to the surface ("lying" chains) were initially observed on the surface, they did not develop into two-dimensionally aligned structures. In contrast, when the strength of surface-chain interactions was at least twice that of chain-chain interactions, the proportion of upright chain molecules was initially small, and the reorientation of lying chains was observed shortly afterwards. In this case, the reorientation from lying to upright configuration developed slowly from the domain boundaries of two-dimensionally aligned structures late in the calculation period. Although the orientation processes of chain molecules on surfaces were strongly influenced by the strength of surface-chain interactions, the total adsorption rate on the surface was not. We also analyzed the maximum area of domains formed by lying chains. The development of two-dimensionally aligned domains required strong surface-chain interactions to prevent the spontaneous formation of nuclei of upright domains.

Diffusion Influenced Adsorption Kinetics.

When the kinetics of adsorption is influenced by the diffusive flow of solutes, the solute concentration at the surface is influenced by the surface coverage of solutes, which is given by the Langmuir-Hinshelwood adsorption equation. The diffusion equation with the boundary condition given by the Langmuir-Hinshelwood adsorption equation leads to the nonlinear integro-differential equation for the surface coverage. In this paper, we solved the nonlinear integro-differential equation using the Grünwald-Letnikov formula developed to solve fractional kinetics. Guided by the numerical results, analytical expressions for the upper and lower bounds of the exact numerical results were obtained. The upper and lower bounds were close to the exact numerical results in the diffusion- and reaction-controlled limits, respectively. We examined the validity of the two simple analytical expressions obtained in the diffusion-controlled limit. The results were generalized to include the effect of dispersive diffusion. We also investigated the effect of molecular rearrangement of anisotropic molecules on surface coverage.

The peroxidase activity of ADM-Fe(3+) cooperates with lipid peroxidation: The participation of hydroperoxide and hydroxyl radicals in the damage to proteins and DNA.

To investigate the mechanisms of cardiotoxicity induced by adriamycin (ADM), the enzymatic activities of ADM-Fe(3+), including the peroxidase and lipoxygenase (LOX) activity, and participation of active oxygen species in the damage to biological components were examined. ADM-Fe(3+), but not ADM, steadily oxidized tetramethyl-p-phenylenediamine in the presence of peroxides, indicating that ADM-Fe(3+) acts as a peroxidase. However, the activity of ADM-Fe(3+) as peroxidase was very low compared with that of heme peroxidase, but was similar to that of LOX, which has a known peroxidase activity. Conversely, the activity of ADM-Fe(3+) as a LOX was also very low compared with that of LOX itself. However, the lipid hydroperoxides (LOOH) produced by ADM-Fe(3+) were the substrate for ADM-Fe(3+) as a peroxidase. These findings indicate that lipid peroxidation cooperates with the peroxidase activity of ADM-Fe(3+). Hydroxyl radicals (HO) were generated when ADM-Fe(3+) was incubated with H2O2, but not with LOOH. Alcohol dehydrogenase was inactivated by LOOH. Conversely, DNA was mainly damaged by ADM-Fe(3+) with H2O2. A small amount of DNA remained at the starting point on agarose gels during incubation with ADM-Fe(3+) with LOOH and ADM-Fe(3+) with H2O2. It seems that HO and compound I-like species participate in the strand breaks and the aggregation of DNA, respectively.

Subattomole detection of adiponectin in urine by ultrasensitive ELISA coupled with thio-NAD cycling.

Adiponectin is a hormone secreted from adipocytes, and it demonstrates antidiabetic, anti-atherosclerotic, antiobesity and anti-inflammatory effects. However, the patterns of change in urinary adiponectin levels in various diseases remain unknown, because only trace amounts of the hormone are present in urine. In the present study, we applied an ultrasensitive ELISA coupled with thio-NAD cycling to measure urinary adiponectin levels. Spikeand-recovery tests using urine confirmed the reliability of our ultrasensitive ELISA. The limit of detection for adiponectin in urine was 2.3×10(-19) moles/assay (1.4 pg/mL). The urinary adiponectin concentration ranged between 0.04 and 5.82 ng/mL in healthy subjects. The pilot study showed that the urinary adiponectin levels, which were corrected by the creatinine concentration, were 0.73±0.50 (ng/mg creatinine, N=6) for healthy subjects, versus 12.02±3.85 (ng/mg creatinine, N=3) for patients with diabetes mellitus (DM). That is, the urinary adiponectin levels were higher (P<0.05) in DM patients than in healthy subjects. Further, these urinary adiponectin levels tended to increase with the progression of DM accompanied with nephropathy. Our method is thus expected to provide a simple, rapid and reasonably priced test for noninvasive monitoring of the progression of DM without the requirement of special tools.

The peroxidase activity of bleomycin-Fe3+ is associated with damage to biological components.

In this study, bleomycin-Fe(3+) steadily oxidized tetramethylbenzidine (TMB) in the presence of peroxides. However, the ability of bleomycin-Fe(3+) to function as a peroxidase was extremely low compared with that of other peroxidases. A characteristic property of bleomycin-Fe(3+) different from that observed for other peroxidases is its ability to oxidize TMB at the similar rate at both a pH 5 and 8 in the presence of lipid hydroperoxide (LOOH). In the present experiments, hydroxyl radicals (HO•) were generated only when bleomycin-Fe(3+) was incubated with H2O2 at a pH of 5. No generation of HO• was observed during the incubation of bleomycin-Fe(3+) with LOOH. Meanwhile, bleomycin-Fe(3+) induced the formation of LOOH from linoleic acid and alcohol dehydrogenase was inactivated by bleomycin-Fe(3+) with peroxides. Thiobarbituric acid reactive substances were formed from DNA by bleomycin-Fe(3+) with H2O2, and strand breaks were caused by bleomycin-Fe(3+) with LOOH. The oxidative substrates for bleomycin-Fe(3+) blocked the damage to biological components induced by bleomycin-Fe(3+). These results suggest that compound I-like species contribute to the process of damage to biological components induced by bleomycin-Fe(3+).

Ultrasensitive enzyme-linked immunosorbent assay (ELISA) of proteins by combination with the thio-NAD cycling method.

An ultrasensitive method for the determination of proteins is described that combines an enzyme-linked immunosorbent assay (ELISA) and a thionicotinamide-adenine dinucleotide (thio-NAD) cycling method. A sandwich method using a primary and a secondary antibody for antigens is employed in an ELISA. An androsterone derivative, 3α-hydroxysteroid, is produced by the hydrolysis of 3α-hydroxysteroid 3-phosphate with alkaline phosphatase linked to the secondary antibody. This 3α-hydroxysteroid is oxidized to a 3-ketosteroid by 3α- hydroxysteroid dehydrogenase (3α-HSD) with a cofactor thio-NAD. By the opposite reaction, the 3-ketosteroid is reduced to a 3α-hydroxysteroid by 3α-HSD with a cofactor NADH. During this cycling reaction, thio-NADH accumulates in a quadratic function-like fashion. Accumulated thio-NADH can be measured directly at an absorbance of 400 nm without any interference from other cofactors. These features enable us to detect a target protein with ultrasensitivity (10(-19) mol/assay) by measuring the cumulative quantity of thio-NADH. Our ultrasensitive determination of proteins thus allows for the detection of small amounts of proteins only by the application of thio-NAD cycling reagents to the usual ELISA system.

Detection of H2O2 by fluorescence correlation spectroscopy.

Fluorescence correlation spectroscopy (FCS) is a technique in which measurement of fluorescence intensity fluctuations is used to clarify dynamic molecular interactions within a very small space in a solution containing a small number of fluorescent molecules. The FCS-based analysis gives the average number and average diffusion time of the fluorescent molecules during their passage through a very small space. One advantage of FCS is that physical separation between free and bound fluorescent probes is not required because the properties of fluorescence fluctuations are accounted for. Therefore, when fluorescent probes are bound with proteins by peroxidase and hydrogen peroxide (H2O2), FCS enables us to detect H2O2 with high sensitivity. In addition, because H2O2 is generated by oxidase-catalyzed reactions, a highly sensitive method for detecting H2O2 is applicable to the measurement of low levels of various oxidases and their substrates, such as glucose. We here describe the protocol of a de novo, highly sensitive method for the measurement of H2O2 and glucose using FCS.

Simulation study of the initial crystallization processes of poly(3-hexylthiophene) in solution: ordering dynamics of main chains and side chains.

We study the initial nucleation dynamics of poly(3-hexylthiophene) (P3HT) in solution, focusing on the relationship between the ordering process of main chains and that of side chains. We carried out Langevin dynamics simulation and found that the initial nucleation processes consist of three steps: the ordering of ring orientation, the ordering of main-chain vectors, and the ordering of side chains. At the start, the normal vectors of thiophene rings aligned in a very short time, followed by alignment of main-chain end-to-end vectors. The flexible side-chain ordering took almost 5 times longer than the rigid-main-chain ordering. The simulation results indicated that the ordering of side chains was induced after the formation of the regular stack structure of main chains. This slow ordering dynamics of flexible side chains is one of the factors that cause anisotropic nuclei growth, which would be closely related to the formation of nanofiber structures without external flow field. Our simulation results revealed how the combined structure of the planar and rigid-main-chain backbones and the sparse flexible side chains lead to specific ordering behaviors that are not observed in ordinary linear polymer crystallization processes.

Spectrophotometric method for the assay of steroid 5α-reductase activity of rat liver and prostate microsomes.

A simple spectrophotometric method for the assay of steroid 5α-reductase (5α-SR) was developed in which 5α-dihydrotestosterone (5α-DHT) and 5α-androstane-3α,17ß-diol (5α-diol), metabolites formed in the NADPH-dependent reduction of testosterone with enzyme sources of 5α-SR, were measured by enzymatic cycling using 3α-hydroxysteroid dehydrogenase in the presence of excess thionicotinamide-adenine dinucleotide (thio-NAD) and NADH. It was found that 5α-SR activity was proportional to the accumulated thio-NADH having an absorption maximum at 400 nm. Because of the high cycling rate (> 600 cycle per min) and no interference from testosterone, enzymatic cycling can determine the sum of 5α-DHT and 5α-diol at the picomole level without separation from excess testosterone. The present method was readily applicable to the assay of 5α-SR activity of rat liver and prostate microsomes as well as to the assay of inhibitory activity of finasteride, a synthetic inhibitor of 5α-SR.