Aggregation Induced Emission Fluorescent Probes for the Determination and Discrimination of Collagen I and II

Rapid and reliable discrimination of collagen subtypes is critical for biomaterials evaluation, tissue engineering, and disease-related extracellular matrix studies. However, this task remains difficult when conventional fluorescence readouts are used. In this study, two tetraphenylethene (TPE)-based aggregation-induced emission (AIE) fluorescent probes, bis[12-(N,N-dimethyl-N′,N′,N′-trimethylethane-1,2-diammonium)dodecyloxy]-substituted tetraphenylethene (TPE-NN) and bis[12-(N-methylimidazolium)dodecyloxy]-substituted tetraphenylethene (TPE-MZ), were developed as environment-sensitive reporters for viscosity and collagen determination. Both probes displayed viscosity-enhanced emission, consistent with restricted intramolecular motion. TPE-NN exhibited a fluorescence turn-on response at 488 nm with a strong linear relationship between emission intensity and medium viscosity (R2 = 0.9936), whereas TPE-MZ showed comparable viscosity responsiveness (R2 = 0.9835). In acidic aqueous media, the emission intensities of both probes increased with the concentrations of collagen types I and II, enabling quantitative collagen determination. However, direct spectral inspection alone was insufficient for robust subtype identification. Therefore, principal component analysis (PCA) was applied to the fluorescence datasets which resulted in clear separation of collagen I and collagen II signatures. TPE-NN produced tighter clustering and more distinct interclass separation than TPE-MZ. Molecular docking provided mechanistic insight into the differential binding behavior underlying subtype discrimination. These results establish a fluorescence-chemometrics strategy for collagen subtype identification and identify TPE-NN as a promising AIE probe for collagen discrimination in complex microenvironments.