Bioorthogonal Chemistry in Biomolecule Quantification: A Review of Reactions and Strategies.

Bioorthogonal chemistry has emerged as a transformative strategy for detecting and quantifying biomolecules in complex biological systems. This review highlights recent advances in catalyst-free bioorthogonal reactions specifically applied to semi-quantitative and quantitative biomolecular analysis. We exclude reactions that require toxic or complex catalysts and focus on four reactions: Staudinger ligation, strain-promoted azide-alkyne cycloaddition, inverse electron-demand Diels-Alder reaction, and 2-cyanobenzothiazole-cysteine condensation. For each, we discuss reaction kinetics and strategies for representative applications in biomolecular quantification. The scope of target biomolecules varies by reaction, including proteins, nucleic acids, glycans, and small molecules. Quantification techniques such as fluorescence spectroscopy, luminescence spectroscopy, and mass spectrometry are examined, with reported limits of detection typically ranging from nanomolar to micromolar, and a few advanced techniques reaching femtomolar or attomolar sensitivity. Each reaction is discussed in terms of kinetics, molecular compatibility, and analytical sensitivity. Finally, we outline key challenges and future opportunities, emphasizing the need for faster reaction kinetics, improved probe design, enhanced integration with advanced analytical platforms, and standardized methods to improve reproducibility and cross-study comparability in biomolecular quantification.