Displacement Assay in a Polythiophene Sensor System Based on Supramacromolecuar Disassembly-Caused Emission Quenching.

Exploring new methodologies for simple and on-demand methods of manipulating the emission and sensing ability of fluorescence sensor devices with solid-state emission molecular systems is important for realizing on-site sensing platforms. In this regard, although conjugated polymers (CPs) are some of the best candidates for preparing molecular sensor devices owing to their luminescent and molecular recognition properties, the development of CP-based sensor devices is still in its early stages. In this study, we herein propose a novel strategy for preparing a chemical stimuli-responsive solid-state emission system based on supramacromolecular assembly-induced emission enhancement (SmAIEE). The system was spontaneously developed by mixing only the component polymers (i.e., polythiophene and a transient cross-linking polymer). The proposed strategy can be applied to the facile preparation of molecular sensor devices. The analyte-induced fluorescent response of polythiophene originated from the dynamic displacement of the transient cross-linker in the polythiophene ensemble and the generation of the polythiophene-analyte complex. Our successful demonstration of the spontaneous preparation of the fluorescence sensor system by mixing two component polymers could lead to the development of on-site molecular analyzers including the determination of multiple analytes.

Pseudo-Luciferase Activity of the SARS-CoV-2 Spike Protein for Cypridina Luciferin.

Enzymatic reactions that involve a luminescent substrate (luciferin) and enzyme (luciferase) from luminous organisms enable a luminescence detection of target proteins and cells with high specificity, albeit that conventional assay design requires a prelabeling of target molecules with luciferase. Here, we report a luciferase-independent luminescence assay in which the target protein directly catalyzes the oxidative luminescence reaction of luciferin. The SARS-CoV-2 antigen (spike) protein catalyzes the light emission of Cypridina luciferin, whereas no such catalytic function was observed for salivary proteins. This selective luminescence reaction is due to the enzymatic recognition of the 3-(1-guanidino)propyl group in luciferin at the interfaces between the units of the spike protein, allowing a specific detection of the spike protein in human saliva without sample pretreatment. This method offers a novel platform to detect virus antigens simply and rapidly without genetic manipulation or antibodies.

Arrayed labeling-free cultivation and growth evaluation from a single microorganism.

The development of high-throughput screening methods for microorganisms is desired because microorganisms are useful and sustainable resources with which valuable substances utilized in various industries can be produced. Micro-space-based methods are the best candidates for the efficient screening of microorganisms owing to their low reagent consumption and compact integration. In this study, we developed a picoliter-sized incubator array for quantitative and labeling-free evaluation of the growth process of Escherichia coli (E. coli) by autofluorescence. Because the array with 8464 incubators is able to compartmentalize single E. coli individually utilizing the Poisson distribution, the array can evaluate 100 single E. coli simultaneously. Our incubator array not only realized the high-throughput screening of microorganisms, but also provided an analytical tool for assessing individual differences in E. coli.

Phase-Separation Propensity of Non-ionic Amino Acids in Peptide-Based Complex Coacervation Systems.

Uncovering the sequence-encoded molecular grammar that governs the liquid-liquid phase separation (LLPS) of proteins is a crucial issue to understand dynamic compartmentalization in living cells and the emergence of protocells. Here, we present a model LLPS system that is induced by electrostatic interactions between anionic nucleic acids and cationic oligolysine peptides modified with 12 different non-ionic amino acids, with the aim of creating an index of "phase-separation propensity" that represents the contribution of non-ionic amino acids to LLPS. Based on turbidimetric titrations and microscopic observations, the lower critical peptide concentrations where LLPS occurs (Ccrit) were determined for each peptide. A correlation analysis between these values and known amino acid indices unexpectedly showed that eight non-ionic amino acids inhibit the generation of LLPS, whereby the extent of inhibition increases with increasing hydrophobicity of the amino acids. However, three aromatic amino acids deviate from this trend and rather markedly promote LLPS despite their high hydrophobicity. A comparison with double-stranded DNA and polyacrylic acid revealed that this is primarily due to interactions with DNA nucleobases. Our approach to quantify the contribution of non-ionic amino acids can be expected to help to provide a more accurate description and prediction of the LLPS propensity of peptides/proteins.

Quantum yield enhancement of firefly bioluminescence with biomolecular condensates.

The enzymatic luminescence reactions of fireflies are accelerated in the presence of biomolecular condensates comprising a positively charged peptide and ATP. We revealed that this acceleration is caused by the enrichment of reaction elements, local pH changes, and promotion of inhibitory intermediate dissociation, improving the bioluminescence quantum yield by approximately 10%.

Damage-free evaluation of cultured cells based on multivariate analysis with a single-polymer probe.

We present a pattern-recognition-based sensor that targets cell-derived components in culture media and evaluates cultured cells without damaging them. An array sensor made of a single-polymer probe was employed to obtain fluorescence response patterns of the analyte media, allowing successful identification of the type and state of the cells via multivariate analysis.

Polymer-based chemical-nose systems for optical-pattern recognition of gut microbiota.

Gut-microbiota analysis has been recognized as crucial in health management and disease treatment. Metagenomics, a current standard examination method for the gut microbiome, is effective but requires both expertise and significant amounts of general resources. Here, we show highly accessible sensing systems based on the so-called chemical-nose strategy to transduce the characteristics of microbiota into fluorescence patterns. The fluorescence patterns, generated by twelve block copolymers with aggregation-induced emission (AIE) units, were analyzed using pattern-recognition algorithms, which identified 16 intestinal bacterial strains in a way that correlates with their genome-based taxonomic classification. Importantly, the chemical noses classified artificial models of obesity-associated gut microbiota, and further succeeded in detecting sleep disorder in mice through comparative analysis of normal and abnormal mouse gut microbiota. Our techniques thus allow analyzing complex bacterial samples far more quickly, simply, and inexpensively than common metagenome-based methods, which offers a powerful and complementary tool for the practical analysis of the gut microbiome.

Direct Capture and Amplification of Small Fragmented DNAs Using Nitrogen-Mustard-Coated Microbeads.

Circulating cell-free DNA (cfDNA) has been implicated as an important biomarker and has been intensively studied for "liquid biopsy" applications in cancer diagnostics. Owing to its small fragment size and its low concentration in circulation, cfDNA extraction and purification from serum samples are complicated, and the extraction yield affects the precision of subsequent molecular diagnostic tests. Here, we report a novel approach using nitrogen-mustard-coated DNA capture beads (NMD beads) that covalently capture DNA and allow direct subsequent polymerase chain reaction (PCR) amplification from the NMD bead without elusion. The complex DNA extraction and purification processes are not required. To illustrate the diagnostic use of the NMD beads, we detected short DNA fragments (142 bp) that were spiked into fetal bovine serum (as a model serum sample). The spiked DNAs were captured directly from serum samples and detected using real-time PCR at concentrations as low as 10 fg/mL. We anticipate that this DNA capture bead technique has the potential to simplify the preanalytical processes required for cfDNA detection, which could significantly expand the diagnostic applications of liquid biopsy.

A polymer-based chemical tongue for the non-invasive monitoring of osteogenic stem-cell differentiation by pattern recognition of serum-supplemented spent media.

The development of non-invasive techniques to characterize cultured cells is invaluable not only to ensure the reproducibility of cell research, but also for quality assurance of industrial cell products for purposes such as regenerative medicine. Here, we present a polymer-based 'chemical tongue', i.e., a biosensing technique that mimics the human taste system, that is capable of non-invasively generating fluorescence response patterns that reflect the proteins secreted, and also partially consumed, by cultured cells, even from serum-supplemented media containing abundant interferants. Analysis of the spent media collected during cell culture using our chemical tongue, which consists of cationic polymers of different scaffolds appended with environmentally responsive dansyl fluorophores, led to the successful (i) identification of human-derived cell lines, (ii) monitoring of the differentiation process of stem cells, even at the stage when conventional staining was negative, and (iii) detection of cancer-cell contamination in stem cells. Since the characterization of cultured cells is usually performed via invasive methods that result in cell death, our chemical-tongue approach, which is of high practical utility, will offer a new means of addressing the growing demand for highly controlled cell production in the medical and environmental fields.

Mix-and-read bioluminescent copper detection platform using a caged coelenterazine analogue.

Serum copper levels are biomarkers for copper-related diseases. Quantification of levels of free copper (not bound to proteins) in serum is important for diagnosing Wilson's disease, in which the free copper concentration is elevated. Bioluminescence is commonly used in point-of-care diagnostics, but these assays require genetically engineered luciferase. Here, we developed a luciferase-independent copper detection platform. A luminogenic caged coelenterazine analogue (TPA-H1) was designed and synthesized to detect copper ions in human serum. TPA-H1 was developed by introducing a tris[(2-pyridyl)-methyl]amine (TPA) ligand, which is a Cu+ cleavable caging group, to the carbonyl group at the C-3 position of the imidazopyrazinone scaffold. The luciferin, named HuLumino1, is the product of the cleavage reaction of TPA-H1 with a copper ion and displays "turn-on" bioluminescence signals specifically with human serum albumin, which can be used to quantitatively analyse copper ions. TPA-H1 exhibited a fast cleavage of the protective group, high specificity, and high sensitivity for copper over other metal ions. This novel caged coelenterazine derivative, TPA-H1, can detect free copper ions in serum in a simple "mix-and-read" manner.

Uncharged Components of Single-Stranded DNA Modulate Liquid-Liquid Phase Separation With Cationic Linker Histone H1.

Liquid-liquid phase separation (LLPS) of proteins and DNAs has been recognized as a fundamental mechanism for the formation of intracellular biomolecular condensates. Here, we show the role of the constituent DNA components, i.e., the phosphate groups, deoxyribose sugars, and nucleobases, in LLPS with a polycationic peptide, linker histone H1, a known key regulator of chromatin condensation. A comparison of the phase behavior of mixtures of H1 and single-stranded DNA-based oligomers in which one or more of the constituent moieties of DNA were removed demonstrated that not only the electrostatic interactions between the anionic phosphate groups of the oligomers and the cationic residues of H1, but also the interactions involving nucleobases and deoxyriboses (i) promoted the generation of spherical liquid droplets via LLPS as well as (ii) increased the density of DNA and decreased its fluidity within the droplets under low-salt conditions. Furthermore, we found the formation of non-spherical assemblies with both mobile and immobile fractions at relatively higher concentrations of H1 for all the oligomers. The roles of the DNA components that promote phase separation and modulate droplet characteristics revealed in this study will facilitate our understanding of the formation processes of the various biomolecular condensates containing nucleic acids, such as chromatin organization.

Quadruplex Folding Promotes the Condensation of Linker Histones and DNAs via Liquid-Liquid Phase Separation.

Liquid-liquid phase separation (LLPS) of proteins and DNA has recently emerged as a possible mechanism underlying the dynamic organization of chromatin. We herein report the role of DNA quadruplex folding in liquid droplet formation via LLPS induced by interactions between DNA and linker histone H1 (H1), a key regulator of chromatin organization. Fluidity measurements inside the droplets, binding assays using G-quadruplex-selective probes, and structural analyses based on circular dichroism demonstrated that quadruplex DNA structures, such as the G-quadruplex and i-motif, promote droplet formation with H1 and decrease molecular motility within droplets. The dissolution of the droplets in the presence of additives and the LLPS of the DNA structural units indicated that, in addition to electrostatic interactions between the DNA and the intrinsically disordered region of H1, π-π stacking between quadruplex DNAs could potentially drive droplet formation, unlike in the electrostatically driven LLPS of duplex DNA and H1. According to phase diagrams of anionic molecules with various conformations, the high LLPS ability associated with quadruplex folding arises from the formation of interfaces consisting of organized planes of guanine bases and the side surfaces with a high charge density. Given that DNA quadruplex structures are well-documented in heterochromatin regions, it is imperative to understand the role of DNA quadruplex folding in the context of intranuclear LLPS.

Withanolide Derivative 2,3-Dihydro-3ß-methoxy Withaferin-A Modulates the Circadian Clock via Interaction with RAR-Related Orphan Receptor α (RORa).

Withanolide derivatives have anticancer, anti-inflammatory, and other functions and are components of Indian traditional Ayurvedic medicine. Here, we found that 2,3-dihydro-3ß-methoxy withaferin-A (3ßmWi-A), a derivative of withaferin-A (Wi-A) belonging to a class of withanolides that are abundant in Ashwagandha (Withania somnifera), lengthened the period of the circadian clock. This compound dose-dependently elongated circadian rhythms in Sarcoma 180 cancer cells and in normal fibroblasts including NIH3T3 and spontaneously immortalized mouse embryonic fibroblasts (MEF). Furthermore, 3ßmWi-A dose-dependently upregulated the mRNA expression and promoter activities of Bmal1 after dexamethasone stimulation and of the nuclear orphan receptors, Rora and Nr1d1, that comprise the stabilization loop for Bmal1 oscillatory expression. We showed that 3ßmWi-A functions as an inverse agonist for RORa with an IC50 of 11.3 µM and that 3ßmWi-A directly, but weakly, interacts with RORa (estimated dissociation constant [Kd], 5.9 µM). We propose that 3ßmWi-A is a novel modulator of circadian rhythms.

Affinity Diversification of a Polymer Probe for Pattern-recognition-based Biosensing Using Chemical Additives.

Pattern-recognition-based sensing has attracted attention as a promising alternative to conventional sensing methods that rely on selective recognition. Here, we report on novel strategy using chemical additives with the ability to modulate probe/analyte interactions to more easily construct pattern-recognition-based sensing systems for proteins and cells. The fluorescence of dansyl-modified cationic poly-L-lysine (PLL-Dnc) is enhanced upon binding to proteins in aqueous solution, while the addition of salts, inert polymers, or alcohols modulates the protein/PLL-Dnc interactions via a variety of mechanisms. Subsequent readout of the fluorescence changes produces response patterns that reflect the characteristics of the analytes. Multivariate analysis of the response patterns allowed for accurate identification of not only eight structurally similar albumin homologues, but also four mammalian cells. This strategy, which uses inexpensive and common additives, significantly improves the accessibility of pattern-recognition-based sensing, which will offer new opportunities for the detection of various bioanalytes.

Coelenterazine Analogue with Human Serum Albumin-Specific Bioluminescence.

A synthetic luciferin comprising an imidazopyrazinone core, named HuLumino1, was designed to generate specific bioluminescence with human serum albumin (HSA) in real serum samples. HuLumino1 was developed by attaching a methoxy-terminated alkyl chain to C-6 of coelenterazine and by eliminating a benzyl group at C-8. HSA levels were quantified within 5% error margins of an enzyme-linked immunosorbent assay without the need for any sample pretreatments because of the high specificity of HuLumino1.

Microfluidic Sensing System with a Multichannel Surface Plasmon Resonance Chip: Damage-Free Characterization of Cells by Pattern Recognition.

The development of a versatile sensing strategy for the damage-free characterization of cultured cells is of great importance for both fundamental biological research and industrial applications. Here, we present a pattern-recognition-based cell-sensing approach using a multichannel surface plasmon resonance (SPR) chip. The chip, in which five cysteine derivatives with different structures are immobilized on Au films, is capable of generating five unique SPR sensorgrams for the cell-secreted molecules that are contained in cell culture media. An automatic statistical program was built to acquire kinetic parameters from the SPR sensorgrams and to select optimal parameters as "pattern information" for subsequent multivariate analysis. Our system rapidly (∼10 min) provides the complex information by merely depositing a small amount of cell culture media (∼25 µL) onto the chip, and the amount of information obtained is comparable to that furnished by a combination of conventional laborious biochemical assays. This noninvasive pattern-recognition-based cell-sensing approach could potentially be employed as a versatile tool for characterizing cells.

A Multichannel Pattern-Recognition-Based Protein Sensor with a Fluorophore-Conjugated Single-Stranded DNA Set.

Recently, pattern-recognition-based protein sensing has received considerable attention because it offers unique opportunities that complement more conventional antibody-based detection methods. Here, we report a multichannel pattern-recognition-based sensor using a set of fluorophore-conjugated single-stranded DNAs (ssDNAs), which can detect various proteins. Three different fluorophore-conjugated ssDNAs were placed into a single microplate well together with a target protein, and the generated optical response pattern that corresponds to each environment-sensitive fluorophore was read via multiple detection channels. Multivariate analysis of the resulting optical response patterns allowed an accurate detection of eight different proteases, indicating that fluorescence signal acquisition from a single compartment containing a mixture of ssDNAs is an effective strategy for the characterization of the target proteins. Additionally, the sensor could identify proteins, which are potential targets for disease diagnosis, in a protease and inhibitor mixture of different composition ratios. As our sensor benefits from simple construction and measurement procedures, and uses accessible materials, it offers a rapid and simple platform for the detection of proteins.

Quantitative analysis of global 5-methyl- and 5-hydroxymethylcytosine in TET1 expressed HEK293T cells.

DNA methylation at the 5-position of cytosine bases (5-methylcytosine, 5mC) in genomic DNA is representative epigenetic modification and is involved in many cellular processes, including gene expression and embryonic development. The hydroxylation of 5mC provide 5-hydroxymethylcytosine (5hmC), the so-called sixth base rediscovered recently in mammalian cells, is also considered to act as an epigenetic regulator. We report herein the immunochemical assessment of 5hmC achieved by an enzyme-linked immunosorbent assay (ELISA) using our linker technology. The keys to this assay are 1) the immobilization of genomic DNA with the bifunctional linker molecule, and 2) quantitative analysis by using guaranteed standard samples containing defined amounts of 5hmC. We succeeded in the sensitive and quantitative detection of 5hmC as well as 5mC in HEK293T cells transfected with TET1, and also monitored the effect of ascorbate on the TET1 catalyzed conversion of 5mC to 5hmC. Our linker technology enables the rapid and stable immobilization of genomic samples and thus contributes to the realization of a reproducible 5hmC evaluation method.

The Power of Assemblies at Interfaces: Nanosensor Platforms Based on Synthetic Receptor Membranes.

Synthetic sensing materials (artificial receptors) are some of the most attractive components of chemical/biosensors because of their long-term stability and low cost of production. However, the strategy for the practical design of these materials toward specific molecular recognition in water is not established yet. For the construction of artificial material-based chemical/biosensors, the bottom-up assembly of these materials is one of the effective methods. This is because the driving forces of molecular recognition on the receptors could be enhanced by the integration of such kinds of materials at the 'interfaces', such as the boundary portion between the liquid and solid phases. Additionally, the molecular assembly of such self-assembled monolayers (SAMs) can easily be installed in transducer devices. Thus, we believe that nanosensor platforms that consist of synthetic receptor membranes on the transducer surfaces can be applied to powerful tools for high-throughput analyses of the required targets. In this review, we briefly summarize a comprehensive overview that includes the preparation techniques for molecular assemblies, the characterization methods of the interfaces, and a few examples of receptor assembly-based chemical/biosensing platforms on each transduction mechanism.