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

A 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 non-invasive pattern-recognition-based cell-sensing approach could potentially be employed as a versatile tool for characterizing cells. </p

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

Dense and Acidic Organelle-Targeted Visualization in Living Cells: Application of Viscosity-Responsive Fluorescence Utilizing Restricted Access to Minimum Energy Conical Intersection

Cell-imaging methods with functional fluorescent probes are an indispensable technique to evaluate physical parameters in cellular mi-croenvironments. In particular, molecular rotors, which take advantage of the twisted intramolecular charge transfer (TICT) process, have helped evaluate microviscosity. However, the involvement of charge-separated species in the fluorescence process potentially limits the quantitative evaluation of viscosity. Herein we developed viscosity-responsive fluorescent probes for cell imaging that are not depend-ent on the TICT process. We synthesized AnP2-H and AnP2-OEG, both of which contain 9,10-di(piperazinyl)anthracene, based on 9,10-bis(N,N-dialkylamino)anthracene that adopt a non-flat geometry at minimum energy conical intersection. AnP2-H and AnP2-OEG exhibited enhanced fluorescence as the viscosity increased, with sensitivities comparable to those of conventional molecular rotors. In living cell systems, AnP2-OEG showed low cytotoxicity and, reflecting its viscosity-responsive property, allowed specific visualization of dense and acidic organelles such as lysosomes, secretory granules and melanosomes under washout-free conditions. These results provide a new direction for developing functional fluorescent probes targeting dense organelles