Quantum Dot/Carrier–Protein/Haptens Conjugate as a Detection Nanobioprobe for FRET-Based Immunoassay of Small Analytes with All-Fiber Microfluidic Biosensing Platform

Abstract

This study demonstrates the use of carrier-protein/haptens conjugate (e.g., BSA/2,4-dichlorophenoxyacetic acid, 2,4-D-BSA) for biological modification of quantum dots (QDs) for the detection of small analytes. Bioconjugated QDs, which are used as a detection nanoimmunoprobe, were prepared through conjugating carboxyl QDs with 2,4-D-BSA conjugate. Based on the principle of quantum dot-fluorescence resonance energy transfer (QD-FRET), an all-fiber microfluidic biosensing platform has been developed for investigating FRET efficiency, immunoassay mechanism and format, and binding kinetics between QD immunoprobe and fluorescence labeled anti-2,4-D monoclonal antibody. The structure of multiplex-haptens/BSA conjugate coupling to QD greatly improves the FRET efficiency and the sensitivity of the nanosensor. With a competitive detection mode, samples containing different concentrations of 2,4-D were incubated with a given concentration of QD immunoprobe and fluorescence-labeled antibody, and then detected by the all-fiber microfluidic biosensing platform. A higher concentration of 2,4-D led to less fluorescence-labeled anti-2,4-D antibody bound to the QD immunoprobe surface and, thus, a lower fluorescence signal. The quantification of 2,4-D over concentration ranges from 0.5 nM to 3 μM with a detection limit determined as 0.5 nM. The performance of the nanosensor with spiked real water samples showed good recovery, precision, and accuracy, indicating that it was less suspectable to water matrix effects. With the use of different QD nanobioprobes modified by other carrier-protein/haptens conjugates, this biosensing protocol based on QD-FRET can be potentially applied for on-site, real-time, inexpensive, and easy-to-use monitoring of other trace analytes.