One-step separation-free detection of carcinoembryonic antigen in whole serum: Combination of two-photon excitation fluorescence and optical trapping

Direct analysis of biomolecules in complex biological samples remains a major challenge for fluorescence-based approaches due to the interference of background signals. Herein, we report an analytical methodology by exploiting a single low-cost near-infrared sub-nanosecond pulse laser to synchronously actualize optical trapping and two-photon excitation fluorescence for senstive detection of carcinoembryonic antigen (CEA) in buffer solution and human whole serum with no separation steps. The assay is performed by simultaneously trapping and exciting the same immune-conjugated microsphere fabricated with a sandwich immunization strategy. Since the signal is strictly limited in the region of a three-dimensional focal volume where the microsphere is trapped, no obvious background signal is found to contribute the detected signals and thus high signal-to-background data are obtained. As a proof-of-concept study, the constructed platform exhibits good specificity for CEA and the detection limit reaches as low as 8 pg/mL (45 fM) with a wide linear range from 0.01 to 60 ng/mL in the both cases. To investigate the potential application of this platform in clinical diagnosis, 15 cases of serum samples were analyzed with satisfactory results, which further confirm the applicability of this method.