Biology subjects

Minagawa, Y.

Publications and source records attributed to Minagawa, Y..

9 recordsLinked to original sources

bioRxiv2026

Open Digital Bioassays Enabled by Aqueous Two-Phase Microreactor Arrays

Digital bioassays enable precise single-molecule quantification but are difficult to adapt to point-of-care testing (POCT) because conventional protocols include off-chip complex processes for sample treatment and sealing, requiring hardware and workflow complexity. We present OASSIS (Open Aqueous two-phase Separation System for Integrated Single- molecule digital bioassay platform), an oil-free and open ATPS platform that localizes both targets and signals in femtoliter-scale dextran (DEX) droplets beneath a polyethylene glycol (PEG) phase. We integrated a CRISPR-Cas13a assay system with a novel, branched fluorescent reporter conjugated to a dextran-binding domain (DBD), which ensures signal retention within the DEX droplets after cleavage. Fluorescence recovery after photobleaching experiments confirmed this robust signal confinement. OASSIS not only performs amplification-free digital RNA detection but also enables serial sample introductions through its open-format architecture that progressively improve sensitivity: the limit of detection (LOD) improved from 1.08 fM (first introduction) to 0.34 fM (third introduction). Furthermore, OASSIS demonstrated specific detection and [~]10-fold enrichment from a complex, denaturant-treated nasopharyngeal swab matrix. Together, these results demonstrate that the open-format architecture of OASSIS provides a practical route toward sensitive, low-complexity POCT and clinical diagnostic applications.

biophysics↗

bioRxiv2025

Assessing analysis methods of brain synchrony in social interaction: Simulation-based comparison

Inter-brain synchrony is an essential measure for investigating social interactive behaviour via hyperscanning. While functional near-infrared spectroscopy is a unique modality for measuring this index in dynamic, real-world interactions, methods to adequately assess inter-brain relationships have not been firmly established, and the overall picture remains unclear. Consequently, in this article, we first briefly summarize analysis methods for examining social interaction by dividing them into static and dynamic measures. Among these, we focus on measures of synchrony and their assessment in correlating behaviours, conducting a simulation-based comparison and analysis. Specifically, we directly compared static and dynamic variants of wavelet transform coherence (WTC), Pearsons correlation coefficient (CC), and phase mutual information (pMI) using a real fNIRS dataset. Results showed a significant divergence between WTC and CC, while WTC and pMI exhibited similar patterns as static measures. Overall, WTC was suggested to better identify synchrony due to its non-linear, instantaneous, and robust nature. For the latter part, based on other simulation analyses, we propose a new method using generalized linear model (GLM) regression. Simulations with synthetic fNIRS data supported the effectiveness of our proposed method, which can capture the dynamic relationships between inter-brain synchrony and behaviour, even during free interaction.

neuroscience↗

bioRxiv2025

Low-Background Cancer Imaging With a Bioorthogonal Fluorescence Probe and Engineered Reporter Enzyme Bearing a Targeting Moiety

Combinatorial use of an antibody-reporter enzyme conjugate and a fluorescence probe activated by the enzyme is a powerful strategy for fluorescence-guided cancer surgery. However, conventional probes for typical reporter enzymes are insufficiently bioorthogonal, resulting in high background signals in non-target tissues. We screened a library of HMRef (rhodol derivative)-based fluorescence probes bearing various sugar moieties, and discovered that HMRef-{beta}-D-fucose is bioorthogonal in mammalian systems, but is activated by a metagenomic glycosidase, Td2F2. Directed evolution generated a mutant with a kcat/Km value for HMRef-{beta}-D-fucose of 3.3 x 105/M/sec, 7.3 times higher than that of wild-type Td2F2 and comparable to that of {beta}-galactosidase (LacZ) with a corresponding probe. Theoretical calculation suggested that E296G mutation in Td2F2 causes structural changes that facilitate the probes access to the enzymes active site. In a proof-of-concept study, cancer cells were visualized with a minimal background in the mesentery of a mouse model of peritoneally disseminated human-ovarian-cancer-derived SKOV-3 cells, which endogenously express HER2, by using HMRef-{beta}-D-fucose together with engineered Td2F2 conjugated/fused to a HER2-binding antibody/nanobody.

bioengineering↗

bioRxiv2024

Metastable phase-separated droplet generation and long-time DNA enrichment by laser-induced Soret effect

Spatiotemporally controlled laser-induced phase separation (LIPS) offers unique research avenues and has a potential for biological and biomedical applications. However, LIPS conditions often have drawbacks for practical use, which limit their applications. For instance, LIPS droplets are tentative and diminish after the laser is terminated. Here, we developed a novel LIPS method using laser-induced Soret effect with a simple setup to solve these problems. We generate liquid-liquid phase-separated (LLPS) droplets using LIPS in an aqueous two-phase system (ATPS) of dextran (DEX) and polyethylene glycol (PEG). When DEX-rich droplets were generated in the DEX/PEG mix on the phase boundary, the droplets showed unprecedently high longevity; the DEX droplets were retained over 48 h. This counterintuitive behavior suggests that the droplet is in an unknown metastable state. By exploiting the capability of DEX-rich droplets to enrich nucleic acid polymers, we achieved stable DNA enrichment in LIPS DEX droplets with a high enrichment factor of 1400 {+/-} 400. Further, we patterned DNA-carrying DEX-rich droplets into a designed structure to demonstrate the stability and spatiotemporal controllability of DEX-rich droplet formation. This is the first report for LIPS droplet generation in a DEX/PEG system with flexible conditions for the usage of LIPS droplets, opening new avenues for biological and medical applications of LIPS.

biophysics↗

bioRxiv2024

Self-growing protocell models in aqueous two-phase system induced by internal DNA replication reaction

The bottom-up reconstitution of self-growing artificial cells is a critical milestone toward realizing autonomy and evolvability. However, building artificial cells that exhibit self-growth coupled with internal replication of gene-encoding DNA has not been achieved yet. Here, we report self-growing artificial cell models based on dextran-rich droplets in an aqueous two-phase system of poly(ethylene glycol) (PEG) and dextran (DEX). Motivated by the finding that DNA induces the generation of DEX-rich droplets, we integrated DNA amplification system with DEX-rich droplets, which exhibited active self-growth. We implemented the protocells with cell-free transcription-translation (TXTL) systems coupled with DNA amplification/replication, which also showed active self-growth. We also observed self-growth activity of protocells carrying a single copy of DNA. Considering the simplicities in terms of the chemical composition and the mechanism, these results underscore the potential of DEX droplets as a foundational platform for engineering protocells, giving implications for the emergence of protocells under prebiotic conditions.

synthetic biology↗

bioRxiv2024

Artificial cells with all-aqueous droplet-in-droplet structures for spatially separated transcription and translation

The design of functional artificial cells involves compartmentalizing biochemical processes to mimic cellular organization. To emulate the complex chemical systems in biological cells, it is necessary to incorporate an increasing number of cellular functions into single compartments. Artificial organelles that spatially segregate reactions inside artificial cells will be beneficial in this context by rectifying biochemical pathways. In our study, we developed artificial cells featuring all-aqueous droplet-in-droplet structures that separate transcription and translation processes, mimicking the nucleus and cytosol in eukaryotic cells. This droplet-in-droplet architecture utilizes intrinsically disordered protein (IDP) to form coacervate droplets for the inner compartments, and aqueous two-phase systems (ATPS) for the outer compartments, with the outer interfaces stabilized by colloidal emulsifiers. The inner droplet was designed to enrich DNA and RNA polymerase to conduct transcription, which was coupled to translation at the outer droplet, realizing the cascade reaction mediated by mRNA. We also demonstrate that these processes proceed independently within each artificial cell compartment, maintaining the correspondence between genotype and phenotype. The modular configuration of these artificial organelles could be extended to other enzymatic reactions. Coupled with the ease of manufacturing these artificial cells, which only requires simple agitation in an all-aqueous mixture, this approach provides a practical and accessible tool for exploring complex systems of artificial organelles within large ensembles of artificial cells.

synthetic biology↗

bioRxiv2024

Functional reorganization of brain regions supporting non-adjacent dependency learning across the first half year of life

Pre-babbling infants can track nonadjacent dependencies (NADs) in the auditory domain. While this forms a crucial prerequisite for language acquisition, the neurodevelopmental origins of this ability remain unknown. We applied functional near- infrared spectroscopy in neonates and 6-7-month-old infants to investigate the neural substrate supporting NAD learning using tone sequences in an artificial grammar learning paradigm. Detection of NADs was indicated by left prefrontal activation in neonates while by left supramarginal gyrus (SMG), superior temporal gyrus (STG), and inferior frontal gyrus activation in 6-7-month-olds. Functional connectivity analyses further indicated that the neonate activation pattern during the test phase benefited from a brain network consisting of prefrontal regions, left SMG and STG during the rest and learning phases. These findings suggest a left-hemispheric learning-related functional brain network may emerge at birth and be strengthened by complex auditory input across the first half year of life, providing a neural basis for language acquisition.

neuroscience↗

bioRxiv2020

Multi-dimensional digital bioassay platform based on an air-sealed femtoliter reactor array device

Single-molecule experiments have been helping us to get deeper inside biological phenomena by illuminating how individual molecules actually work. Digital bioassay, in which analyte molecules are individually confined in small compartments to be analyzed, is an emerging technology in single-molecule biology and applies to various biological entities (e.g., cells and virus particles). However, digital bioassay is not compatible with multi-conditional or multi-parametric assays, hindering understanding of analytes. This is because current digital bioassay lacks a repeatable solution-exchange system that keeps analytes inside compartments. To address this challenge, we developed a new digital bioassay platform with easy solution exchanges, called multi-dimensional (MD) digital bioassay, and tested its quantitativity and utility. We immobilized single analytes in arrayed femtoliter (10-15 L) reactors and sealed them with airflow. The solution in each reactor was stable and showed no cross-talk via solution leakage for more than 2 h, and over 30 rounds of perfect solution exchanges were successfully performed. To show the utility of our system, we investigated neuraminidase inhibitor (NAI) sensitivity on single influenza A virus (IAV) particles in a multi-conditional assay. We proved that IAV particles show a heterogeneous response to the NAI. Further, to demonstrate multi-parametric assays, we examined the sensitivity of individual IAV particles or model enzyme molecules to two different inhibitors. Our results support that MD digital bioassay is a versatile platform to unveil heterogeneities of biological entities in unprecedented resolution.

biophysics↗

bioRxiv2020

Elucidation and control of low and high active populations of alkaline phosphatase molecules for quantitative digital bioassay

Alkaline phosphatase (ALP), a homo-dimeric enzyme has been widely used in various bioassays as disease markers and enzyme probes. Recent advancements of digital bioassay revolutionized ALP-based diagnostic assays as seen in rapid growth of digital ELISA and the emerging multiplex profiling of single-molecule ALP isomers. However, the intrinsic heterogeneity found among ALP molecules hampers the ALP-based quantitative digital bioassays. This study aims quantitative analysis of single-molecule activities of ALP from Escherichia coli and reveals the static heterogeneity in catalytic activity of ALP with two distinct populations: half-active and fully active portions. Digital assays with serial buffer exchange uncovered single-molecule Michaelis-Menten kinetics of ALP; half-active molecules have halved values of the catalytic turnover rate, kcat, and the rate constant of productive binding, kon, of the fully active molecules. These findings suggest that half-active ALP molecules are heterogenic dimers composed of inactive and active monomer units, while fully active ALP molecules comprise two active units. Static heterogeneity was also observed for ALP with other origins: calf intestine or shrimp, showing how the findings can be generalized across species. Cell-free expression of ALP with disulfide bond enhancer and spiked zinc ion resulted in homogenous population of ALP of full activity, revealing that inactive monomer units of ALP are deficient in disulfide bond formation and zinc ion coordination, and also offering the way to prepare homogenous and active populations of ALP for quantitative digital bioassays of ALP.

biophysics↗