Terbium to Quantum Dot FRET Bioconjugates for Clinical Diagnostics: Influence of Human Plasma on Optical and Assembly Properties

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.

Interfacing Engineered Nanoparticles with Biological Systems: Anticipating Adverse Nano–Bio Interactions

“…compared to buffer solutions, the photophysical performances of each component of the FRET pair are likely to be modified. Indeed, Hildebrandt and co‐workers observed both dynamic and static quenching of QDs upon introduction into various protein‐rich media, in agreement with the formation of a protein corona 46. Importantly, the extent of dynamic and static quenching varied depending on the composition of the medium, on the size, composition and surface coating of the QD.…”

“…The reverse situation can also be observed, namely, uncontrolled NP aggregation through interparticle bridging by proteins, possibly triggered upon protein denaturation 38, 45. Finally, adsorption of a protein corona can affect the physical properties of the individual NP itself, for instance by shifting its plasmon resonance41 or altering its luminescence quantum yield 46…”

A time-resolved luminescent competitive assay to detect L-selectin using aptamers as recognition elements

“…Moreover, the presence of metal cations such as Pb 2þ and Hg 2þ can disturb analytical signal. Nevertheless, similar FRET systems with similar LOD which fully functioned in human serum have already been demonstrated [44].…”

“…When combining those properties with time-gated time-resolved luminescence signal acquisition, the background signal resulting from undesired effects, such as biosample autofluorescence, short-living fluorescence of fluorophores as well as scattered light are reduced significantly. In recent years, FRET systems based on the lanthanide complexes as donors have been used for multiplexed diagnostics [40,41], as molecular ruler [42,43], for investigations of the plasma influence on FRET signal [44], and in biotin-streptavidin assays [17,16] as well as for the determination of macromolecular biomarkers, such as prostate specific antigen (PSA) [47,48], carcinoembryonic antigen (CEA) [49], CARM1 activators [50], and total protein concentration [51].…”

Biophotonic logic devices based on quantum dots and temporally-staggered Förster energy transfer relays

“…77 Caspase-catalyzed cleavage of A647 and/or Tb from the central QD would change the overall photonic logic and therefore could be used to monitor molecules that activate caspase enzymes as part of the pathways initiated during apoptosis or programmed cell death, which is associated with diseases such as cancer and Alzheimer's disease. 82 The excellent multiplexing suitability of Tb and QD-based FRET systems 83,84 further suggests that simultaneous use of multiple differentially colored photonic gates within a single sample is also possible. 80 When used alone or in conjunction with other biomolecular logic platforms (e.g., oligonucleotides or enzymes), these QD-based logic gates could potentially be useful for small chemical/biomolecular identication as part of a theranostic device.…”