Niko Hildebrandt

A homogeneous time-resolved fluoroimmunoassay (TR-FIA) using antibody mediated luminescence quenching

The determination of low-molecular weight substances (haptens) is demonstrated with a homogeneous time-resolved immunoassay using antibody-induced luminescence quenching. Our novel assay technology uses the newly developed monoclonal antibody (G24-BA9) to quench the luminescence of europium trisbipyridine (EuTBP). We performed a competitive biotin immunoassay including an EuTBP–biotin conjugate, the anti-EuTBP antibody G24-BA9 and streptavidin as assay components. Steric hindrance allows only the binding of either G24-BA9 (to the EuTBP moiety) or streptavidin (to the biotin moiety) to the EuTBP–biotin conjugate. Addition of the analyte biotin resulted in the binding of streptavidin to biotin and a concomitant preferred binding of G24-BA9 to EuTBP–biotin. Since G24-BA9 quenches the luminescence of EuTBP within the conjugate, the luminescence signal could be used to indicate and quantify the presence of free biotin in the system. All experiments were carried out in solution in the presence of 5% serum demonstrating the possibility of using our novel assay for a very fast determination of low molecular weight substances in biological fluids.

Multiplexed diagnostics and spectroscopic ruler applications with terbium to quantum dots FRET

In this contribution we present the application of five different commercially available semiconductor core/shell quantum dots (Qdot® Nanocrystals - Invitrogen Corp.) as multiplexing FRET acceptors together with a commercial supramolecular terbium complex (Lumi4®-Tb - Lumiphore Inc.) as donor in a homogeneous immunoassay format. To realize the molecular recognition necessary for a FRET assay, the terbium complex was labeled to streptavidin (sAv- Lumi4-Tb) and the QDs were surface functionalized with biotin (Biot-QD). The biotin-streptavidin binding serves as a proof-of-principle representative for many biological interactions taking place on the nanometer scale (e.g. immunoassays). The presented FRET system can be efficiently used for the detection of inter- and intramolecular processes for clinical diagnostics and biomedical spectroscopy as well as molecular ruler applications and microscopy.

Time-resolved and steady-state FRET spectroscopy on commercial biocompatible quantum dots

Semiconductor nanocrystals (quantum dots - QDs) possess unique photophysical properties that make them highly interesting for many biochemical applications. Besides their common use as fluorophores in conventional spectroscopy and microscopy, QDs are well-suited for studying Förster resonance energy transfer (FRET). Size-dependent broadband absorption and narrow emission bands offer several advantages for the use of QDs both as FRET donors and acceptors. QD-based FRET pairs can be efficiently used as biological and chemical sensors for highly sensitive multiplexed detection. In this contribution we present the use of several commercially available QDs (Qdot® Nanocrystals - Invitrogen) as FRET donors in combination with commercial organic dyes as FRET acceptors. In order to investigate the FRET process within our donor-acceptor pairs, we used biotinylated QDs and streptavidin-labeled dyes. The well-known biotinstreptavidin molecular recognition enables effective FRET from QDs to dye molecules and provides defined distances between donor and acceptor. Steady-state and time-resolved fluorescence measurements were performed in order to investigate QD-to-dye FRET. Despite a thick polymer shell around the QDs, our results demonstrate the potential of these QDs as efficient donors both for steady-state and time-resolved FRET applications in nano-biotechnology.

Optical transmission gratings tuned by electro active polymers

Dielectric elastomer actuators (DEA) of poly-styrene-ethylene-butadiene-styrene (SEBS) and poly-acrylic VHB4910 were studied for voltage tunable optical transmission gratings. A new geometry is proposed, in which the grating is placed in an area without electrodes, permitting for light transmission through the device. Experiments were performed to implement surface relief gratings on DEA films from pattern masters made from holographic recorded gratings. Since the actuation strain of the DEA depends strongly on the boundary conditions, the desired voltage-controllable deformation of the grating can be achieved by choosing suitable manufacturing parameters. Conditions were found permitting a relative change up to 8.5 % in a 1 µm grating. A model based on independently measured material parameters is shown to describe the optical behavior.

Optical size determination of quantum dots using FRET with terbium complexes as donors

We present the use of luminescent terbium complexes (LTCs) as FRET donors and luminescent semiconductor quantum dots (QDs) as FRET acceptors for spectroscopic ruler measurements. The LTCs were labeled to polyhistidine-appended peptides which self-assembled onto three different QDs allowing optical multiplexed measurements. Forster distances were in the range of 60-75 Å and FRET efficiencies of up to 97 % were realized. Time-resolved analysis allowed the determination of donor-acceptor separation distances. The results suggests the efficient use of our LTC-to-QD FRET systems for multiplexed optical size determination, molecular ruler measurements and multi-parameter diagnostics.

Energy transfer from terbium complexes to quantum dots: the advantage of independent donor and acceptor decay time analysis for investigations on FRET distance dependence

The efficient use of luminescent semiconductor quantum dots (QDs) as Förster Resonance Energy Transfer (FRET) acceptors can be accomplished with terbium complexes (TCs) as donors. TCs exhibit long excited state lifetimes (in the millisecond range) up to 105 times longer than typical QD lifetimes. When FRET occurs from TCs to QDs the measured TC luminescence decay times decrease (FRET quenching), whereas the QD decay times increase (FRET sensitization). Due to the large difference between the TC and QD excited state lifetimes the FRET formalism can be applied to both the TC donors as well as the QD acceptors. This is a big advantage because the FRET information from one experiment can be received from both sides of the FRET pair allowing for the use of different detection channels and wavelengths for donor and acceptor. Thus, a multiplexing format becomes possible with one single donor (TC) and several different acceptors (different QDs). In this contribution we show the theoretical background for simultaneously applying FRET to donor and acceptor and give an example with a commercially available TC-QD donor-acceptor pair.

Tumor specific lung cancer diagnostics with multiplexed FRET immunoassays

An optical multiplexed homogeneous (liquid phase) immunoassay based on FRET from a terbium complex to eight different fluorescent dyes is presented. We achieved highly sensitive parallel detection of four different lung cancer specific tumor markers (CEA, NSE, SCC and CYFRA21-1) within a single assay and show a proof-of-principle for 5- fold multiplexing. The method is well suited for fast and low-cost miniaturized point-of-care testing as well as for highthroughput screening in a broad range of in-vitro diagnostic applications.

Quantum Dots as Acceptors in FRET-Assays Containing Serum *

Quantum dots (QDs) are common as luminescing markers for imaging in biological applications because their optical properties seem to be inert against their surrounding solvent. This, together with broad and strong absorption bands and intense, sharp tuneable luminescence bands, makes them interesting candidates for methods utilizing Forster Resonance Energy Transfer (FRET), e. g. for sensitive homogeneous fluoroimmunoassays (FIA). In this work we demonstrate energy transfer from Eu3+-trisbipyridin (Eu-TBP) donors to CdSe-ZnS-QD acceptors in solutions with and without serum. The QDs are commercially available CdSe-ZnS core-shell particles emitting at 655 nm (QD655). The FRET system was achieved by the binding of the streptavidin conjugated donors with the biotin conjugated acceptors. After excitation of Eu-TBP and as result of the energy transfer, the luminescence of the QD655 acceptors also showed lengthened decay times like the donors. The energy transfer efficiency, as calculated from the decay times of the bound and the unbound components, amounted to 37%. The Forster-radius, estimated from the absorption and emission bands, was ca. 77Å. The effective binding ratio, which not only depends on the ratio of binding pairs but also on unspecific binding, was obtained from the donor emission dependent on the concentration. As serum promotes unspecific binding, the overall FRET efficiency of the assay was reduced. We conclude that QDs are good substitutes for acceptors in FRET if combined with slow decay donors like Europium. The investigation of the influence of the serum provides guidance towards improving binding properties of QD assays.