Abstract

Förster resonance energy transfer (FRET) from luminescent terbium complexes (LTC) as donors to semiconductor quantum dots (QDs) as acceptors allows extraordinary large FRET efficiencies due to the long Förster distances afforded. Moreover, time-gated detection permits an efficient suppression of autofluorescent background leading to sub-picomolar detection limits even within multiplexed detection formats. These characteristics make FRET-systems with LTC and QDs excellent candidates for clinical diagnostics. So far, such proofs of principle for highly sensitive multiplexed biosensing have only been performed under optimized buffer conditions and interactions between real-life clinical media such as human serum or plasma and LTC-QD-FRET-systems have not yet been taken into account. Here we present an extensive spectroscopic analysis of absorption, excitation and emission spectra along with the luminescence decay times of both the single components as well as the assembled FRET-systems in TRIS-buffer, TRIS-buffer with 2% bovine serum albumin, and fresh human plasma. Moreover, we evaluated homogeneous LTC-QD FRET assays in QD conjugates assembled with either the well-known, specific biotin-streptavidin biological interaction or, alternatively, the metal-affinity coordination of histidine to zinc. In the case of conjugates assembled with biotin-streptavidin no significant interference with the optical and binding properties occurs whereas the histidine-zinc system appears to be affected by human plasma.

Highlights

  • Over the last decades, applications based on Förster Resonance Energy Transfer (FRET) have become very valuable tools for innumerable applications in the fields of medicine and biology [1,2,3].Due to the r−6 distance dependence of Förster resonance energy transfer (FRET) it is possible to gain access to small structural changes in biological processes such as protein folding and to kinetic data of reactions and binding events

  • luminescent terbium complexes (LTC)-conjugated sAv was binding to biotinylated quantum dots (QDs), whereas peptides functionalized with LTCs on one end and displaying a hexahistidine tag on the other end were assembled directly to the zinc-rich surface of dihydrolipoic acid (DHLA)-capped QDs

  • Biot-sAv and His-Zn conjugate assays to show the influence of plasma on QD-based FRET for clinical assays

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Summary

Introduction

Applications based on Förster Resonance Energy Transfer (FRET) have become very valuable tools for innumerable applications in the fields of medicine and biology [1,2,3]. Using the well-characterized organic dye family as FRET probes has the advantage of a small size, which alleviates some bioconjugation issues and guarantees a small impact on the biomolecule They exhibit long-term storage stability in a wide range of media and facile use [8]. Brightness and luminescence quantum yields, large Förster distances and an excellent temporal as well as spectral separation make this FRET-pair a powerful tool for multiplexed FRET measurements in a wide range of applications in research and diagnostics [13,14]. In this study we compare conjugation of LTCs to biocompatible QDs in buffer and plasma to evaluate the influence of plasma on photophysical properties and the sensing performance of several different LTC-QD-systems. LTC-conjugated sAv was binding to biotinylated QDs, whereas peptides functionalized with LTCs on one end and displaying a hexahistidine tag on the other end were assembled directly to the zinc-rich surface of DHLA-capped QDs

Experimental Section and Methods
Results and Discussion
Photophysical Characterization of FRET-Donors and -Acceptors
FRET-Assays
Specific Binding Assays with Biotin and Streptavidin
Metal Affinity Coordination Assays with Zinc and Polyhistidine
Conclusions
Full Text
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