Electrochemical biosensors are devices that detect chemical substances using biological recognition elements and electrodes as signal transducers. Amperometric electrochemical biosensors with redox enzymes have been widely used in applications such as blood glucose monitoring. Among them, the third-generation biosensor is a simple and ideal system consisting only of a direct electron transfer (DET)-type enzyme and an electrode. Since the system does not require a mediator, which is necessary in conventional technologies, it has the advantages of low cost, low risk of side reactions, and high biocompatibility. In this paper, we introduce examples of research and development of DET-type enzymes and third-generation biosensors, and describe their characteristics and future prospects.

In recent years, it has become apparent that “L-lactate”, a clinically important biomarker for various diseases, not only serves as an energy metabolite but also functions as a signaling molecule that triggers various cellular events. To date, direct observation of L-lactate at the single-cell level (and even subcellular levels) has been challenging due to the need for sample fractionation and electrode insertion at the measurement site. To investigate the emerging roles of L-lactate, it is essential to observe L-lactate dynamics with high spatiotemporal resolution. This review provides an overview of L-lactate biosensors, with a focus on genetically encoded fluorescent biosensors that offer high spatiotemporal resolution, which have been recently reported.

Small-angle neutron scattering (SANS) is a technique used for submicron-scale structural analysis of soft matter and can also be used to evaluate dynamics by taking advantage of the large difference in the scattering length between hydrogen and deuterium. The author’s group has succeeded in determining the rates of intervesicular transfer and transbilayer transfer (flip-flop) of phospholipids in vesicles using this method for the first time and has clarified the differences in these rates depending on the type of phospholipids, as well as the lipid transport activity of phospholipid transfer proteins and the induction of phospholipid flip-flop by transmembrane peptides. This technique is now being implemented by many SANS users. This comprehensive paper presents details and examples of this technique.

Herein, we propose methodologies using off-the-shelf reagents to obtain chemosensors without organic synthesis. In this system, the off-the-shelf reagents, which are selected considering their intermolecular interactions, play crucial roles as functional building blocks in preparing molecular self-assembled chemosensors. The roles of molecular self-assemblies are not only to eliminate the need for synthesis in preparing chemosensors, but also to obtain information-rich optical responses upon detecting various analytes. Such inherent cross-reactivity of the self-assembled chemosensors can contribute to obtaining fingerprint-like responses depending on chemical structures of analytes and their concentrations, and the beneficial properties of the chemosensors have been applied to pattern recognition for qualitative and quantitative discrimination. As actual approaches using off-the-shelf building blocks, this review describes three types of self-assembled chemosensors; 1) turn-on type fluorescent chemosensors for saccharides, 2) colorimetric chemosensors for oxyanions, and 3) a single fluorescent chemosensor for pattern recognition of metal ions. The high applicability of the proposed approach based on intermolecular interactions indicates the potential of off-the-shelf building blocks for real-sample analysis. The authors believe that this methodology will open a new avenue to establish chemical sensor platforms for anyone who is not familiar with chemical experiments.

Raman imaging, which detects molecular vibration, has attracted significant attention recently as an imaging technique with superior capability for multiplexed detection compared to fluorescence imaging. Raman imaging was originally proposed as a label-free method, but the recent development of vibrational tags such as small alkynes or Raman probes for multiplexed imaging enabled us to visualize a variety of biomolecules with high specificity and improved sensitivity, thus the biocompatibility of Raman imaging has been dramatically improved. We have been working on developing an activatable Raman imaging probe whose Raman signal is activated upon reaction with target enzymes. We utilized the specific phenomenon of the signal intensity of stimulated Raman scattering (SRS) increasing remarkably under electronic pre-resonance (EPR) conditions, in which a molecule is excited at a wavelength of 100–200 nm longer than its molecular absorption. Firstly, we focused on 9CN-JCP, one of the pyronin derivatives with nitrile at the 9^th position (9CN-pyronin) as a scaffold dye, and developed multicolor activatable Raman imaging probes that can detect plural enzyme activities simultaneously in living cells. More recently, we focused on rhodol derivatives with nitrile at the 9^th position (9CN-rhodol), which tend to form aggregates in aqueous solution than 9CN-pyronins, and we developed novel activatable Raman probes for enzymes that can form aggregates upon reaction with target enzymes, enabling us to perform ex vivo imaging of enzyme-expressing cells or regions in live Drosophila tissues. In this review, we first give an overview of recent trends in biological Raman imaging, and then introduce our research achievement on the development of activatable Raman probes.

We have developed a simple and compact flow-injection analysis (FIA) system using an LED-base absorbance detector and micro-ring pumps. The detector has four LEDs and can measure absorbance corresponding to each LED wavelength (465 nm, 525 nm, 625 nm, and 850 nm) sequentially. A micro-ring pump is a single roller pump whose flow contains pulsation, which imposes some noises on a flow signal. It is notable in the case that a reagent solution includes an organic solvent or colored reagent. A refractive index difference between a carrier and sample produces a pseudo peak called the Schlieren effect. Dual wavelength measurement techniques and the optimization of the reagent composition could suppress those noises. We applied this FIA system to measure nitrogen as nitrate, phosphorus as phosphate, and chromium(VI) according to the modified procedures of JIS K 0170.