Evaluating Quantum Dot Performance in Homogeneous FRET Immunoassays for Prostate Specific Antigen.

Shashi Bhuckory,Niko Hildebrandt,Olivier Lefebvre,Karl Wegner,Xue Qiu

doi:10.3390/s16020197

Abstract

The integration of semiconductor quantum dots (QDs) into homogeneous Förster resonance energy transfer (FRET) immunoassay kits for clinical diagnostics can provide significant advantages concerning multiplexing and sensitivity. Here we present a facile and functional QD-antibody conjugation method using three commercially available QDs with different photoluminescence (PL) maxima (605 nm, 655 nm, and 705 nm). The QD-antibody conjugates were successfully applied for FRET immunoassays against prostate specific antigen (PSA) in 50 µL serum samples using Lumi4-Tb (Tb) antibody conjugates as FRET donors and time-gated PL detection on a KRYPTOR clinical plate reader. Förster distance and Tb donor background PL were directly related to the analytical sensitivity for PSA, which resulted in the lowest limits of detection for Tb-QD705 (2 ng/mL), followed by Tb-QD655 (4 ng/mL), and Tb-QD605 (23 ng/mL). Duplexed PSA detection using the Tb-QD655 and Tb-QD705 FRET-pairs demonstrated the multiplexing ability of our immunoassays. Our results show that FRET based on QD acceptors is suitable for multiplexed and sensitive biomarker detection in clinical diagnostics.

Highlights

Over the last two decades semiconductor quantum dots (QDs) have become one of the most attractive nanomaterials for fluorescence-based biosensing [1,2]
Technologies) possessing a thick polymer polyethylene glycol (PEG) coating and surface amino functionalization was possessing a thick polymer PEG coating and surface amino functionalization was tested in tested in homogeneous Förster resonance energy transfer (FRET) immunoassays
QDs were successfully labeled with fragmented F(ab) ABs against prostate specific antigen (PSA), which were used in combination with Lumi4-Tb-AB conjugates for Tb-to-QD FRET assays

Summary

Introduction

Over the last two decades semiconductor quantum dots (QDs) have become one of the most attractive nanomaterials for fluorescence-based biosensing [1,2]. Their application in biosensors provides advantages such as photostability, high brightness, and size-dependent color tunability, which are unique compared to other commonly used fluorophores such as organic dyes or fluorescent proteins [3,4]. The photophysical and nanometric properties of QDs are ideal for their application in Förster resonance energy transfer (FRET), a non-radiative r6 distance dependent energy transfer between two fluorophores (a donor and an acceptor) in ca. One of the main problems remains a widely applicable, reproducible and stable bioconjugation that allows a full exploitation of both the photophysical advantages of the QDs and Sensors 2016, 16, 197; doi:10.3390/s16020197 www.mdpi.com/journal/sensors

Methods

Discussion

Conclusion