Ratiometric Normalization of Near-Infrared Fluorescence in Defect-Engineered Single-Walled Carbon Nanotubes for Cholesterol Detection

Ratiometric probing of analytes presents a substantial advancement in molecular recognition, offering self-calibrating signals that enhance the measurement accuracy and reliability. We present a dual-emitting probe based on (6,5) chirality-enriched single-walled carbon nanotubes (SWCNTs) with oxygen defects for cholesterol (Chol) detection using ratiometric fluorescence readouts. The interaction with Chol induced significant intensity variations in the <i>E</i>₁₁ and <i>E</i>₁₁^* emission peaks of oxygen defect-induced SWCNTs, giving rise to ratiometric fluorescence changes. The sensitivity of these probes toward Chol in water and serum was 0.28 ± 0.01 and 0.72 ± 0.05 μM, respectively, which is comparable to that of common gold standards for cholesterol detection used in clinical samples. By utilizing ratiometric readouts, our approach enhanced selectivity over numerous competing analytes, including amino acids, sugars, cations, anions, proteins, steroid hormones, surfactants, and phospholipids. Mechanistic investigations revealed that Chol detection by defect-integrated SWCNTs was facilitated by Chol incorporation within micelles formed by sodium cholate, the surfactant dispersant used for the SWCNT suspension. Oxygen defects played a crucial role by directly interacting with Chol. This strategy employing defect-integrated dual-peak NIR-emitting SWCNTs as sensors for Chol in aqueous and serum environments not only enables background-free detection of biologically relevant analytes but also advances biosensing using SWCNTs through tailored surface functionalization and advanced read-out concepts.