Enzyme-assisted and Label-Free Imaging from Cells to Organs: Advances and Perspectives in Scanning Electrochemical Microscopy

Accurate spatial mapping of biomolecules in complex biological systems is essential for advancing diagnostics and understanding disease mechanisms. Traditional imaging methods, while powerful, often lack the capability to provide quantitative electrochemical information with high spatial resolution. Scanning electrochemical microscopy (SECM) addresses these gaps by enabling detailed, label-free, non-optical quantitative mapping from single cells to whole organs. This review focuses on SECM as a transformative tool in biological imaging, particularly through enzyme-assisted, label-free techniques for detecting proteins, metabolites, cytokines, and nanomaterials. We detail key operational modes, including feedback, generation-collection, redox competition, potentiometric, and dual-electrode approaches, and highlight core applications, including enzymatic amplification with alkaline phosphatase and horseradish peroxidase, aptamer-based detection, and oxidase-driven metabolite mapping. Recent advances, including single-cell receptor imaging, label-free metabolic profiling, integration with microfluidics, and soft-probe technologies that enable high-resolution imaging from single cells to tissues and whole organs, are discussed. Current challenges, including probe miniaturization, signal specificity, and data interpretation in heterogeneous systems, are critically discussed, along with future directions toward multiplexed detection and clinical translation. Overall, SECM represents a promising approach for next-generation biosensing and electrochemical imaging, bridging the gap between molecular recognition and functional biological analysis.