Point-of-care testing (POCT) of biomarkers, such as proteins and nucleic acids, is a hot topic in modern medical engineering toward the early diagnosis of various diseases including cancer. Although microfluidic chips show great promise as a new platform for POCT, external pumps and valves for driving those chips have hindered the realization of POCT on the chips. To eliminate the need for pumps and valves, a power-free microfluidic pumping method utilizing degassed poly(dimethylsiloxane) (PDMS) was invented in 2004. In this article, the working principle of the degas-driven power-free microfluidic chip is first described, and then applications of those chips to biomarker analysis are reviewed. The biomarker analysis on the chip was typically achieved with a small sample volume of ∼1 μL and a short analysis time of ∼20 min. For protein analysis, the sandwich immunoassay format was adopted. The limit of detection (LOD) was improved by three orders of magnitude by using laminar flow-assisted dendritic amplification (LFDA), which was a newly devised amplification method specialized for microfluidic chips. For analysis of nucleic acids such as DNA and microRNA, the sandwich hybridization format was adopted, and the LFDA was also effective to reduce the LOD. With the LFDA, typical LOD values for proteins and nucleic acids were both around 1 pM.

A nonsense suppressor tRNA (sup-tRNA) allows a natural or non-natural amino acid to be assigned to a nonsense codon in mRNA. Sup-tRNAs were utilized initially for studying tRNA functions but lately are used more for protein engineering and gene regulation. In the latter application, a sup-tRNA that is aminoacylated with a natural amino acid by the corresponding aminoacyl-tRNA synthetase is used to express a full-length natural protein from its mutated gene with a nonsense codon in the middle. This type of sup-tRNA has recently been artificially evolved to develop biosensors. In these biosensors, an analyte induces the processing of an engineered premature sup-tRNA into a mature sup-tRNA, which suppresses the corresponding nonsense codon incorporated into a gene, encoding an easily detectable reporter protein. This review introduces sup-tRNA-based biosensors that the author’s group has developed by utilizing bacterial and eukaryotic cell-free translation systems.

Anisotropic gold nanoparticles have attracted great interest due to their unique physicochemical properties derived from the shape anisotropy. Manipulation of their interfacial interactions, and thereby the assembling behaviors are often requisite in their applications ranging from optical sensing and diagnosis to self-assembly. Recently, the control of interfacial force based on base pair stacking of DNA terminals have offered a new avenue to surface engineering of nanostructures. In this review, we focus on the DNA base stacking-induced assembly of anisotropic gold nanoparticles, such as nanorods and nanotriangles. The fundamental aspects of anisotropic gold nanoparticles are provided, including the mechanism of the anisotropic growth, the properties arising from the anisotropic shape, and the construction of DNA-grafted anisotropic gold nanoparticles. Then, the advanced applications of their functional assemblies in biosensing and ordered assembly are summarized, followed by a comparison with gold nanospheres. Finally, conclusions and the direction of outlooks are given including future challenges and opportunities in this field.

Enzymes play a central role in the detection of target molecules in biotechnological fields. Most probes used in detection are bifunctional proteins comprising enzymes and binding proteins conjugated by chemical reactions. To create a highly sensitive detection probe, it is essential to increase the enzyme-to-binding protein ratio in the probe. However, if the chemical reactions required to prepare the probe are insufficiently site-specific, the detection probe may lose functionality. Genetic modifications and enzyme-mediated post-translational modifications (PTMs) can ensure the site-specific conjugation of proteins. They are therefore promising strategies for the production of detection probes with high enzyme contents, i.e., polymeric bifunctional proteins. Herein, we review recent advances in the preparation of bifunctional protein conjugates and polymeric bifunctional protein conjugates for detection. We have summarized research on genetically fused proteins and enzymatically prepared polymeric bifunctional proteins, and will discuss the potential use of protein polymers in various detection applications.

Graphene-based FRET aptasensors can be realized only by unique combinations of aptamer that can be freely functionalized by chemical modification, and graphene/graphene oxide that works as an excellent fluorescence acceptor at the same time as aptamer adsorbates. This review describes the principles of the sensor, several applications to microchannel devices, improvement of the sensing performance by molecular design of the aptamer and remarks on future prospects based on an introduction of recent works and achievements, including the author’s paper. The sensor employs DNA modified with graphene/graphene oxide at the terminal as the molecular probe. This system is supported by the excellent property of DNA that does not lose the molecular recognition ability even due to a chemical modification at the terminal. I hope that this review will be useful for developing research on higher performance of graphene aptasensors in the future.

To establish a guideline for the design of cell culture substrates to control human mesenchymal stem cell (MSC) differentiation, we quantitatively characterized the heterogeneity in the responsiveness of MSCs to the elastic modulus of culture substrates. We analyzed the elastic modulus-dependent dynamics of a mechanotransducer, YAP, and an osteogenic differentiation factor, RUNX2, in three different MSC lots using a styrenated gelatin gel with controllable elastic modulus. The percentage of cells with YAP in the nucleus increased linearly with increases in the elastic modulus, reaching a plateau at 10 kPa for all the lots analyzed. The increase in the percentage with the substrate elastic modulus was described by the same linear function. The percentage of cells with RUNX2 nuclear localization also increased linearly with increases in the substrate elastic modulus, plateauing at 5 kPa, although the regression lines to the linearly increasing regions varied between lots. These similarities and differences in YAP and RUNX2 dynamics among cell populations are basis to design the substrate elastic modulus to manipulate YAP and RUNX2 localizations.

Immunochromatography assay is an easy and rapid on-site detection method. However, conventional sandwich immunochromatographies using two antibodies can only detect target molecules above a threshold size. Small molecules below 1000 in molecular weight are usually detected using competitive immunoassay. However, competitive immunoassay is not suitable for visual detection of low concentration samples. Based on the principles of open sandwich immunoassay, which detects small molecules via interchain interaction of separated variable region fragments (V_H and V_L) from a single antibody, we developed non-competitive open sandwich immunochromatography. Bone Gla protein (BGP)-C7, a peptide containing the seven C-terminal amino acids of human osteocalcin, was selected as the target. By using V_L fragments fixed on a nitrocellulose membrane, and colored cellulose bead-labeled V_H fragments, we specifically detected 10 ng/mL of BGP-C7. This is the first report of open sandwich immunochromatography, which is an easy and rapid method for on-site, signal-on detection of small molecules.

We prepared microspheres densely covered with oligo-DNA strands by immobilizing amino-terminated oligo-DNA strands on the surface of carboxylate polystyrene latex (PS) particles via the amide bond formation. The obtained microspheres (ssDNA-PS) stably dispersed in neutral pH buffer containing high concentrations of NaCl. For the ssDNA-PS ≥1 μm diameter, only 3 – 5% of surface-immobilized oligo-DNA could form a duplex with the complementary strands. Nevertheless, the resulting ssDNA-PS showed a distinct duplex terminal dependency in their dispersion behavior under neutral pH and high NaCl conditions; the microspheres with fully-matched duplexes on the surface spontaneously aggregated in a non-crosslinking manner. By contrast, the microspheres with terminal-mismatched duplexes remained dispersed under the identical conditions. These results suggest that the micrometer-scale particles covered with oligo-DNA strands also have high susceptibility to a duplex terminal sequence in their dispersion property, similar to previously reported DNA-functionalized nanoparticles. This property could potentially be used in various applications including analytical purposes.

In this paper, we report on stimuli-responsive behaviors for room temperature fluorescent liquid materials based on N-heteroacene frameworks in response to HCl vapor. These liquid materials as well as their mixtures prepared by varying the combination can provide various emission colors and stimuli-responsive properties in liquid states. Also, we achieved an improvement in total synthetic yield (>40%) by redesigning the molecular structures of liquid materials as compared to previous liquid materials (<10%).

Demand for the detection of estradiol, which is a naturally occurring hormone, has been increasing. Gold nanoparticle-based colorimetric aptasensors have been developed for estradiol detection; however, the long sequence of aptamers due to the formation of the secondary structure likely affects the sensitivity of the aptasensors. Herein, a sensitive colorimetric biosensor is developed for label-free detection of estradiol by using an estradiol-specific split aptamer. The results demonstrate that a superior response is observed when a split aptamer with a high free energy of the secondary structure (ΔG > –3 kcal/mol) is used, in comparison to that observed using a split aptamer with a low free energy of the secondary structure (ΔG < –3 kcal/mol) at 27°C. After selecting the appropriate split aptamer, the standard calibration curve obtained for estradiol has a detection limit of 6.7 nM, with a linear range of 6.7 nM – 66.7 μM in the logarithmic scale. Furthermore, this assay is sensitive, easy-to-operate, inexpensive, and non-time-consuming (provides results within 50 min), thereby showing potential for clinical applications (detection of other small molecular targets).

Fluorescence imaging using the over-thousand-nanometer (OTN) near-infrared (NIR) light is an emerging method for an in vivo imaging analysis of deep tissues without physical sectioning. Polymer micelle nanoparticles (PNPs) composed of organic polymers encapsulating an OTN-NIR fluorescent dye, IR-1061, in their hydrophobic core are expected to be biocompatible probes. Because IR-1061 quickly quenches due to the vibration of polar hydroxyl bonding in its surroundings, the influence of hydroxyl ions should be minimized. Herein, we investigated the effect of the hydrogen ion concentration during the preparation process using IR-1061 and an organic polymer, poly(ethylene glycol)-block-polystyrene (PEG-b-PSt), on the emission properties of the obtained OTN-PNPs. The OTN-PNP has a hydrodynamic diameter of 20 – 30 nm and emits 1110-nm fluorescence that is applicable to angiography. The loading efficiency of IR-1061 in the OTN-PNPs increased when prepared in an aqueous solution with a low hydroxyl ion concentration. In this solution (pH 3.0), highly emissive OTN-PNPs was obtained with IR-1061 at lower nominal concentrations. Decreasing the hydroxyl ion concentration during the preparation process yields highly emissive OTN-PNPs, which may improve the in vivo imaging analysis of biological phenomena in deep tissues.

Various cells and tissues are highly organized in vivo by basement membranes (BMs) and thus promising artificial BMs (A-BMs) constructed by electrospinning and layer-by-layer (LbL) assembly have recently attracted much attention in the tissue engineering field. However, control of cell adhesion, morphology, and migration of the attached cells on the A-BMs has not been reported yet. In this study, we investigated both thickness and roughness-dependent effects of A-BMs on the functions of endothelial cells (ECs), which resulted from different assembly concentrations. The results indicated that the roughness of A-BMs increased gradually with the increase of nanofilm thickness. EC adhesion, spreading and proliferation were inhibited on thicker A-BM surfaces with larger roughness, while interendothelial junctions and the barrier effect of confluent EC monolayers on thicker A-BM surfaces were compensated by increasing seeding cell number and expanding culture time. Our study highlights the influence of LbL assembly conditions on endothelial functions, which offers a new criterion for the design of A-BMs in well-organized 3D tissues.

The quantitatively controlled organellar transfer between living single cells provides a unique experimental platform to analyze the contribution of organellar heterogeneity on cellular phenotypes. We previously developed a microfluidic device which can perform quantitatively controlled mitochondrial transfer between live single cells by promoting strictured cytoplasmic connections between live single cells, but its application to other organelles is unclear. In this study, we investigated the quantitative properties of peroxisome transfer in our microfluidic device. When cells were fused through a 10 or 4 μm long microtunnel by a Sendai virus envelope-based method, a strictured cytoplasmic connection was achieved with a length corresponding to that of the microtunnel, and a subsequent recovery culture disconnected the fused cells. The peroxisome number being transferred through a 10 μm length of the microtunnel was smaller than that of 4 μm. These data suggest that our microfuidic device can perform a quantitative control of peroxisome transfer.

Gold nanoparticles (AuNPs) are commonly used in biosensing applications. In this study, AuNPs were synthesized by using reduced bovine serum albumin (rBSA) as the reducing agent. The rBSA conjugated with AuNPs via Au-Sulfur interactions to form rBSA-functionalized AuNPs (rBSA-AuNPs). The interaction of the rBSA moieties on the rBSA-AuNP surface with an anti-BSA antibody (anti-BSA) led to AuNP aggregation, which enabled the successful detection of anti-BSA at a concentration as low as 20 nM through darkfield microscopy (DFM). This study demonstrates the potential applications of protein-functionalized AuNPs in the bioanalysis of substances through DFM.

Single-molecule DNA/RNA sequencing based on single-molecule measurement is a prominent method for higher throughput sequencing. In a previous report, the single-molecule DNA/RNA sequencing method has applied to detect each base-conductance difference in the tunneling current time profiles, and determined the sequence. However, discrimination of identical base lengths has not yet been achieved. The number of the identical contiguous bases has importance in biology because some homopolymers of nucleic acid control gene expression. In this study, we aimed to develop a method for discriminating the length of homopolymer of nucleic acids of adenosine monophosphate (AMP) using a single-molecule sequencing technique. We carried out single-molecule conductance measurements of adenine pentamer, hexamer and heptamer. The single-molecule signals of the oligomers are not distinguishable from current and duration time histograms. The three oligomers were discriminated by our developed machine learning-based analysis with accuracy of 0.54 for a single signal, and 99% for 40 signals. This method will be applied to the single signals and identify the contiguous bases in the sequence and provide new biological insights.

The bioinert interfaces that prevent adhesion of proteins and cells are important for biomaterial applications. In order to design a bioinert interface, the immobilization of an appropriate functional group and the control of molecular density is required. Dendrimer was modified with triethylene glycol (TEG) to display a dense brush structure. TEG with different density and terminal groups were immobilized with a dendrimer template and thiol terminated molecules. The inhibitory effect on protein and bacteria binding was investigated. The physical property of the interface was measured by QCM-admittance to clarify the factor of the bioinert property.

We investigated a host-guest complex between cucurbit[7]uril and malachite green, and its effect on the toxicity to human liver cells. The host-guest complexation was evaluated by a UV/vis titration and electrospray-ionization mass spectrometry. Interestingly, the host-guest complex resulted in remarkable suppression of the toxicity of malachite green in its practical concentration range (ca. ∼6 μM). This study is one step forward to the active control of the biological effects of potent toxicants utilizing host-guest chemistry.

We previously developed a hydrolase-based fluorescence amplification method for antigen-specific cell labelling, in which fluorescent substrates stained cells by a non-covalent hydrophobic interaction. To improve the substrates retention in cells, we examined the effect of a chloroacetyl group modification on the substrate retention. We found that the chloroacetyl group suppressed the dissociation of the substrate after forming a covalent bond with intracellular proteins. However, the slow reaction speed of the chloroacetyl group allowed dissociation for cells in the early stage of the staining reaction.

The electrochemical signal from ferrocene on a DNA probe was successfully modulated in a homogeneous solution by the template-directed formation and dissociation of an inclusion complex with β-cyclodextrin on another probe. The electrochemical response was amplified by combining with a DNA circuit, in which the target DNA served as a catalyst. This system did not require any modification of a complementary DNA with the ferrocene-modified probe on the electrode surface to separate the bound/free probe for the detection of 200 nM target DNA.

Soft nanofibrous oligopeptide hydrogels consisting of self-assembled l- and d-form Fmoc-Phe-Phe-Cys networks photo-cross-linked by poly(ethylene glycol)-dimethacrylate, which are referred to as l- and d-gel, respectively, were developed for investigation of chirality effects on the undifferentiated state of mesenchymal stem cells. Encapsulated mesenchymal stem cells in d-gel showed slower growth and less spreading, resulting in higher maintenance of the undifferentiated state, compared to in l-gel. This indicates that d-form peptide materials might be useful as scaffold materials for regenerative medicine using stem cells.