Numerical Modeling for Sensitive and Rapid Molecular Detection by Membrane-Based Immunosensors.

Rapid, simple, and sensitive point-of-care testing (POCT) has attracted attention in recent years due to its excellent potential for early disease diagnosis and health monitoring. The flow-through biosensor design is a candidate for POCT that utilizes the small-sized pores of a porous membrane as a recognition space where it emits a signal comparable to that of a conventional enzyme-linked immunosorbent assay within 35 min of detection time. In this paper, we present a numerical model for this immunosensing technology to systematically design an improved recognition system. The model considers mass transfer into the pore (convection and diffusion), the kinetics between the immobilized receptor and the target molecule, and the flow conditions, successfully leading to a bottleneck step (capture of secondary antibody) in sandwich-type detection. Our simulation results also show that this problem can be solved by adopting both appropriate receptors and analytical conditions. Eventually, the requirements to achieve the sensitivity required for POCT were fulfilled, which will allow for further development of immunosensing devices for disease detection.

Suitable acid groups and density in electrolytes to facilitate proton conduction.

Proton conducting materials suffer from low proton conductivity under low-relative humidity (RH) conditions. Previously, it was reported that acid-acid interactions, where acids interact with each other at close distances, can facilitate proton conduction without water movement and are promising for overcoming this drawback [T. Ogawa, H. Ohashi, T. Tamaki and T. Yamaguchi, Chem. Phys. Lett., 2019, 731, 136627]. However, acid groups have not been compared to find a suitable acid group and density for the interaction, which is important to experimentally synthesize the material. Here, we performed ab initio calculations to identify acid groups and acid densities as a polymer design that effectively causes acid-acid interactions. The evaluation method employed parameters based on several different optimized coordination interactions of acids and water molecules. The results show that the order of the abilities of polymer electrolytes to readily induce acid-acid interactions is hydrocarbon-based phosphonated polymers > phosphonated aromatic hydrocarbon polymers > perfluorosulfonic acid polymers ≈ perfluorophosphonic acid polymers > sulfonated aromatic hydrocarbon polymers. The acid-acid interaction becomes stronger as the distance between acids decreases. The preferable distance between phosphonate moieties is within 13 Å.

Differentiating Grotthuss proton conduction mechanisms by nuclear magnetic resonance spectroscopic analysis of frozen samples.

Available methods to analyze proton conduction mechanisms cannot distinguish between two proton-conduction processes derived from the Grotthuss mechanism. The two mechanistic variations involve structural diffusion, for which water movement is indispensable, and the recently proposed "packed-acid mechanism," which involves the conduction of protons without the movement of water and is typically observed in materials consisting of highly concentrated (packed) acids. The latter mechanism could improve proton conductivity under low humidity conditions, which is desirable for polymer electrolyte fuel cells. We proposed a method with which to confirm quantitatively the packed-acid mechanism by combining (2)H and (17)O solid-state magic-angle-spinning nuclear magnetic resonance (MAS-NMR) measurement and (1)H pulsed-field gradient (PFG)-NMR analysis. In particular, the measurements were performed below the water-freezing temperature to prevent water movement, as confirmed by the (17)O-MAS-NMR spectra. Even without water movement, the high mobility of protons through short- and long-range proton conduction was observed by using (2)H-MAS-NMR and (1)H-PFG-NMR techniques, respectively, in the composite of zirconium sulfophenylphosphonate and sulfonated poly(arylene ether sulfone) (ZrSPP-SPES), which is a material composed of highly concentrated acids. Such behavior contrasts with that of a material conducting protons through structural diffusion or vehicle mechanisms (SPES), in which the peaks in both (2)H and (17)O NMR spectra diminished below water-freezing temperature. The activation energies of short-range proton movement are calculated to be 2.1 and 5.1 kJ/mol for ZrSPP-SPES and SPES, respectively, which indicate that proton conduction in ZrSPP-SPES is facilitated by the packed-acid mechanism. Furthermore, on the basis of the mean-square displacement using the diffusivity coefficient below water-freezing temperature, it was demonstrated that long-range proton movement, of the order of 1.3 µm, can take place in the packed-acid mechanism in ZrSPP-SPES.

Non-humidified proton conduction between a Lewis acid-base pair.

Proton conduction in zirconium sulphate (ZrSO4) composed of a Lewis acid-base pair was studied. ZrSO4 exhibits non-humidified proton conductivity, comparable to other proton conductors under similar conditions. Ab initio calculation shows that a proton transfers in ZrSO4 from a Lewis acid to a Lewis base without a proton carrier.

Biomolecule-recognition gating membrane using biomolecular cross-linking and polymer phase transition.

We present for the first time a biomolecule-recognition gating system that responds to small signals of biomolecules by the cooperation of biorecognition cross-linking and polymer phase transition in nanosized pores. The biomolecule-recognition gating membrane immobilizes the stimuli-responsive polymer, including the biomolecule-recognition receptor, onto the pore surface of a porous membrane. The pore state (open/closed) of this gating membrane depends on the formation of specific biorecognition cross-linking in the pores: a specific biomolecule having multibinding sites can be recognized by several receptors and acts as the cross-linker of the grafted polymer, whereas a nonspecific molecule cannot. The pore state can be distinguished by a volume phase transition of the grafted polymer. In the present study, the principle of the proposed system is demonstrated using poly(N-isopropylacrylamide) as the stimuli-responsive polymer and avidin-biotin as a multibindable biomolecule-specific receptor. As a result of the selective response to the specific biomolecule, a clear permeability change of an order of magnitude was achieved. The principle is versatile and can be applied to many combinations of multibindable analyte-specific receptors, including antibody-antigen and lectin-sugar analogues. The new gating system can find wide application in the bioanalytical field and aid the design of novel biodevices.

Connected iridium nanoparticle catalysts coated onto silica with high density for oxygen evolution in polymer electrolyte water electrolysis.

We propose connected Ir nanoparticle catalysts (Ir/SiO2) by coating 1.8 nm Ir particles with high density onto silica for the oxygen evolution reaction. Nanoparticles form electron-conducting networks, which can eliminate the need for an electron-conducting support. Ir/SiO2 showed a high electrochemical surface area, mass activity, and water electrolysis performance.

Immobilization of hydroquinone through a spacer to polymer grafted on carbon black for a high-surface-area biofuel cell electrode.

We immobilized hydroquinone through a spacer to polymer grafted on carbon black and achieved a high-surface-area biofuel cell electrode. Quinone compounds are well-known to transfer electrons in the respiratory chain and have been considered prospective mediators in biofuel cells because of their relatively negative redox potentials. Evaluation of three different spacer arms tethering hydroquinone to linear polymers revealed that only the hydrophilic and flexible di(ethylene oxide) spacer made it possible for immobilized hydroquinone to transfer electrons from glucose oxidase (GOD) to an electrode; direct immobilization and an alkyl spacer did not. The electrode comprising hydroquinone immobilized through di(ethylene oxide) spacer to polymer grafted on carbon black transferred electrons from GOD to the electrode. The potential at which an anodic current began to increase was more negative by about 0.2 V than that for a vinylferrocene-mediated electrode, while the increase in the anodic current density was of the same order.

Beneficial Role of Copper in the Enhancement of Durability of Ordered Intermetallic PtFeCu Catalyst for Electrocatalytic Oxygen Reduction.

Design of Pt alloy catalysts with enhanced activity and durability is a key challenge for polymer electrolyte membrane fuel cells. In the present work, we compare the durability of the ordered intermetallic face-centered tetragonal (fct) PtFeCu catalyst for the oxygen reduction reaction (ORR) relative to its counterpart bimetallic catalysts, i.e., the ordered intermetallic fct-PtFe catalyst and the commercial catalyst from Tanaka Kikinzoku Kogyo, TKK-PtC. Although both fct catalysts initially exhibited an ordered structure and mass activity approximately 2.5 times higher than that of TKK-Pt/C, the presence of Cu at the ordered intermetallic fct-PtFeCu catalyst led to a significant enhancement in durability compared to that of the ordered intermetallic fct-PtFe catalyst. The ordered intermetallic fct-PtFeCu catalyst retained more than 70% of its mass activity and electrochemically active surface area (ECSA) over 10 000 durability cycles carried out at 60 °C. In contrast, the ordered intermetallic fct-PtFe catalyst maintained only about 40% of its activity. The temperature of the durability experiment is also shown to be important: the catalyst was more severely degraded at 60 °C than at room temperature. To obtain insight into the observed enhancement in durability of fct-PtFeCu catalyst, a postmortem analysis of the ordered intermetallic fct-PtFeCu catalyst was carried out using scanning transmission electron microscopy-energy dispersive X-ray spectroscopy (STEM-EDX) line scan. The STEM-EDX line scans of the ordered intermetallic fct-PtFeCu catalyst over 10 000 durability cycles showed a smaller degree of Fe and Cu dissolution from the catalyst. Conversely, large dissolution of Fe was identified in the ordered intermetallic fct-PtFe catalyst, indicating a lesser retention of Fe that causes the destruction of ordered structure and gives rise to poor durability. The enhancement in the durability of the ordered intermetallic fct-PtFeCu catalyst is ascribed to the synergistic effects of Cu presence and the ordered structure of catalyst.

Effect of length of molecular recognition moiety on enzymatic activity switching.

We site-specifically conjugated biotin-PEG derivatives with spacer arms of different lengths to mutant P450cam (3mD) and evaluated the activity of and structural changes in the conjugates as a first step toward clarifying the mechanism whereby the activity of the 3mD conjugate is inhibited. 3mD was prepared by site-specific mutation to inhibit its enzymatic activity artificially, after which the derivative compounds were conjugated to the enzyme. 3mD has one cysteine on its surface with a reactive thiol group that can react with compounds near the active site, where a conformational change will be induced after conjugation. The activity of 3mD was retained in the biotin-PEG2-3mD conjugate, but was dramatically reduced in the biotin-PEG11-3mD conjugate. To investigate the effect of poly(ethylene glycol) (PEG) length on the enzymatic activity after conjugation, PEGs of different lengths, exceeding that in biotin-PEG11, and whose termini were not biotin, were conjugated to 3mD. The activity of 3mD decreased in all these conjugates. This indicates that the activity of 3mD in these conjugates decreased after its conjugation with PEG molecules that exceeded a certain length. The biotin-PEG2-3mD, which retains enzymatic activity after conjugation, showed avidin responsiveness; the enzymatic activity decreased after avidin binding.

Molecular recognition moiety and its target biomolecule interact in switching enzyme activity.

We conjugated a molecular recognition moiety, biotin, with an enzyme site-specifically near to its active site and succeeded in inactivating the enzyme by binding the specific target biomolecule avidin to biotin. Bacterial P450 was used as a model enzyme, which has attracted much attention in several fields. Site-directed mutagenesis was conducted to produce a mutant P450 that could attach biotin site-specifically. The activity of the conjugate decreased markedly to one tenth of that of biotinylated P450 after binding to avidin. Ultraviolet-visible spectroscopy of the carbon monoxide-bound P450, circular dichroism data, and the ratio of the active form to the sum of the active form and the inactive form indicated that this decrease in activity was because of a conformational change in the tertiary structure surrounding the active center after avidin binding, while the secondary structure of P450 remained unchanged.

Physical re-examination of parameters on a molecular collisions-based diffusion model for diffusivity prediction in polymers.

Molecular collisions, which are the microscopic origin of molecular diffusive motion, are affected by both the molecular surface area and the distance between molecules. Their product can be regarded as the free space around a penetrant molecule defined as the "shell-like free volume" and can be taken as a characteristic of molecular collisions. On the basis of this notion, a new diffusion theory has been developed. The model can predict molecular diffusivity in polymeric systems using only well-defined single-component parameters of molecular volume, molecular surface area, free volume, and pre-exponential factors. By consideration of the physical description of the model, the actual body moved and which neighbor molecules are collided with are the volume and the surface area of the penetrant molecular core. In the present study, a semiempirical quantum chemical calculation was used to calculate both of these parameters. The model and the newly developed parameters offer fairly good predictive ability.