A Quantum‐Dot‐Based Molecular Ruler for Multiplexed Optical Analysis

Abstract

In memory of Theodor Förster on the centenary of his birth on May 15th 2010Applications based on Förster resonance energy transfer (FRET) play an important role in the determination of concentrations and distances within nanometer-scale systems in vitro and in vivo in the fields of biology, biochemistry, medicine, and other life sciences. [1][2][3] Due to the r À6 distance dependence of FRET, structural changes of molecular systems in the 1-10 nm range can be measured with high accuracy far below the light diffraction limit. Stryer et al. [4,5] demonstrated the spectroscopic ruler FRET technique more than 40 years ago, and it is still frequently used for in-and exvivo studies of inter-and intramolecular interactions by spectroscopy and microscopy down to the single-molecule level.[6-9] Several FRET-based biosensors for functional intracellular investigations have been developed. [10][11][12][13][14] Although most of these applications use single sensors, there have been some recent developments of dual FRET pairs for cellular imaging using fluorescent proteins, [15][16][17] and even with a single excitation wavelength.[18] Using a multiplexed FRET technique allows the simultaneous measurement of multiple distances or conformational changes, thereby decreasing time and effort whilst increasing bioanalytical information due to the possible correlation of simultaneous events.The FRET pair combination of luminescent terbium complexes (LTCs) as donors and semiconductor quantum dots (QDs) as acceptors holds significant advantages concerning sensitivity, distance, and multiparametric analysis compared to other donor-acceptor pairs. [19,20] Due to large overlap integral values, exceptionally long Förster radii (R 0 , the donor-acceptor distance at which the FRET efficiency is 50 %) of up to 11 nm can be achieved, [21][22][23] whereas conventional donor-acceptor pairs have much smaller R 0 values that rarely exceed 6 nm.[24] Although nanoplasmonic molecular rulers have been developed for which distances of up to about 70 nm can be measured, [25,26] these applications use relatively large noble metal nanoparticles (up to 40 nm) and are restricted in their multiplexed use of simultaneously measuring variable distances of different systems (for example, several different intracellular functional events within one measurement). The pioneering work of Weiss et al. demonstrated multiplexed optical rulers using quantum dots and ultrahigh-resolution colocalization (UHRC).[27] Although FRET has advantages concerning resolution accuracy and dynamic measurements, [28] UHRC is well-suited to measuring distances in the range of few nanometers to tens of micrometers. [29] Two very important aspects for intracellular studies with QDs are the shape and the size of these nanosensors, which can be crucial, for example, for cell penetration and for evaluation of the nanoparticle impact on the targeted biomolecules. Measuring the core/shell dimensions of the semiconductor material with TEM is possible with relatively good accuracy. However, ...

Controlling resonance energy transfer in nanostructure emitters by positioning near a mirror

“…Furthermore, the drastic upturn shown in Fig.3(c)opens up potential applications for highly sensitive positioning tools. [51][52][53] In Sec.III B, similar results exhibit by the coupling between NW-NW near a single mirror. Nevertheless, in contrast to the QD case, a more than 120% increment in the energy transfer rate can be gained as shown in the plots of Fig.5(a).…”

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

“…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].…”

Extending Förster resonance energy transfer measurements beyond 100 Å using common organic fluorophores: enhanced transfer in the presence of multiple acceptors

“…Using acceptors such as quantum dots (QD) and phycobiliproteins (PE), which possess significantly higher absorption and molar extinction coefficients, R 0 values of 90 to 100 Å have been measured when using lanthanide donors. [18][19][20] Therefore, in principle, it should be possible to monitor FRET at distances less than 150 Å under practical experimental conditions when using acceptable donor-acceptor pairs. However, it should be noted that both chelated lanthanide donors and acceptors, such as QD and PE molecules, are significantly larger than conventional organic acceptor molecules.…”

“…This will result in limiting the effective observation distance to around 100 Å under most experimental conditions. 20 Morgner et al used this disadvantage in an imaginative application to develop a simple and inexpensive FRET method to monitor the size of QDs, the results of which compare favorably with the results of more demanding transmission electron microscopy. 20 The molar extinction coefficients of the organic sensitizer molecules used in lanthanide-based probes are typically less than 20; 000 M −1 cm −1 .…”

“…20 Morgner et al used this disadvantage in an imaginative application to develop a simple and inexpensive FRET method to monitor the size of QDs, the results of which compare favorably with the results of more demanding transmission electron microscopy. 20 The molar extinction coefficients of the organic sensitizer molecules used in lanthanide-based probes are typically less than 20; 000 M −1 cm −1 . This, together with the intrinsic ms lanthanide lifetimes, results in a significantly smaller photon flux that limits the applications of cellular microscopy-based FRET.…”