Methylene blue in electrochemical (Bio)sensing: Historical evolution, mechanistic insights, and emerging...

Methylene blue (MB), a redox-active phenothiazine dye, has transitioned from a historical textile colorant to a central component in modern electrochemical biosensing. Its reversible redox behavior, distinct color change, and compatibility with biological redox systems make it an ideal mediator and indicator. However, challenges such as mediator leaching, film degradation, and interference in complex matrices hinder broader application. This review addresses these limitations by analyzing MB's electrochemical properties, immobilization strategies, and sensor integration techniques.

MB exhibits a proton-coupled, two-step electron transfer process with radical intermediates, revealed by voltammetry and spectroelectrochemistry. Four immobilization strategies-adsorption, covalent binding, polymer entrapment, and electropolymerization-impact sensor performance in terms of stability and signal reproducibility. Applications span enzyme-based glucose and H₂O₂ biosensors (nanomolar detection), nucleic acid sensors using hybridization and DNA-mediated charge transport, and electrocatalytic chemical sensing (e.g., Cr(VI), NADH). Integration with nanomaterials enhances surface area and sensitivity. Anti-fouling coatings and ratiometric detection improve selectivity and operational longevity, demonstrating MB's compatibility with advanced materials and fabrication platforms.

The integration of MB with emerging nanomaterials and immobilization techniques reinforces its role in next-generation electrochemical biosensors. Its dual optical-electrochemical function and mediator versatility enable the design of sensitive, stable, and miniaturized point-of-care diagnostics.