A Two-Photon Excitation Fluorescence Cross-Correlation Assay for a Model Ligand-Receptor Binding System Using Quantum Dots

Abstract

Two-photon excitation fluorescence cross-correlation spectroscopy (TPE-XCS) is a very suitable method for studying interactions of two distinctly labeled fluorescent molecules. As such, it lends itself nicely to the study of ligand-receptor interactions. By labeling the ligand with one color of fluorescent dye and the receptor with another, it is possible to directly monitor ligand binding rather than inferring binding by monitoring downstream effects. One challenge of the TPE-XCS approach is that of separating the signal due to the receptor from that of the ligand. Using standard organic fluorescent labels there is almost inevitably spectral cross talk between the detection channels, which must be accounted for in TPE-XCS data analysis. However, using quantum dots as labels for both ligand and receptor this limitation can be alleviated, because of the dot's narrower emission spectra. Using solely quantum dots as fluorescent labels is a novel approach to TPE-XCS, which may be generalizable to many pairs of interacting biomolecules after the proof of principle and the assessment of limitations presented here. Moreover, it is essential that relevant pharmacological parameters such as the equilibrium dissociation constant, K(d), can be easily extracted from the XCS data with minimal processing. Herein, we present a modified expression for fractional occupancy based on the auto- and cross-correlation decays obtained from a well-defined ligand-receptor system. Nanocrystalline semiconductor quantum dots functionalized with biotin (lambda(em) = 605 nm) and streptavidin (lambda(em) = 525 nm) were used for which an average K(d) value of 0.30 +/- 0.04 x 10(-9) M was obtained (cf. native system approximately 10(-15)). Additionally, the off-rate coefficient (k(off)) for dissociation of the two quantum dots was determined as 5 x 10(-5) s(-1). This off-rate is slightly larger than for native biotin-streptavidin (5 x 10(-6) s(-1)); the bulky nature of the quantum dots and restricted motion/orientation of functionalized dots in solution can account for differences in the streptavidin-biotin mediated dot-dot binding compared with those for native streptavidin-biotin.

The Impact of Diffusion on Biochemical Assay Kinetics in 3d-Hydrogel Microarrays

“…Name: Streptavidin, cy5 conjugate Supplier: GE Healthcare UK Ltd, England Molar mass: 60 kDa Concentration: 1 mg/ml Diffusion coefficient: (Zhang et al, 2007) Hydrodynamic radius: 2.5 nm (Swift et al, 2006) A corresponding biotin labeled oligonucleotide was used as a capture molecule, the oligonucleotide acts as a spacer for the immobilized biotin molecules Name: Biotinylated-oligonucleotide Supplier: TIB MOLBIOL, Berlin, Germany Size: 24-mer Sequence: BIOTEG-TgCgTCAAAggTgTTTTTTTTTTT Concentration: 100 µM 4.1.2. MICROCHIP SUBSTRATES The standard microchip substrate used throughout this thesis is polymethylmetacrylamide (PMMA) supplied by form.in displays Laser-Center GmbH, Heitersheim, Germany.…”

Shuttle–Cargo Fusion Molecules of Transport Peptides and the hD2/3Receptor Antagonist Fallypride: A Feasible Approach To Preserve Ligand–Receptor Binding?

“…Similar effects of drastically decreased binding properties (up to 6 orders of magnitude) upon conjugation of large constructs have been shown exemplarily on the biotin−streptavidin system recently. 34,35 Thus, we intended to investigate if the conjugation of a small neuroreceptor-affine drug to a carrier peptide can, in principle, result in a hybrid compound that is potentially able to transport the small molecule while at the same time preserving its affinity to the neuroreceptor. We therefore synthesized a palette of fusion drugs, each consisting of a peptide carrier and a derivative of the hD 2/3 receptor-binding drug fallypride in order to test the feasibility of this approach.…”

Cross-talk-free Fluorescence Cross-Correlation Spectroscopy by the Switching Method

“…This method can detect the coincidence of two molecules labeled with fluorophores of different colors in a small detection area on which two laser beams are focused. FCCS has been applied to studies of molecular interactions in homogeneous solutions (Kettling et al, 1998;Doi et al, 2002;Collini et al, 2005;Swift et al, 2006) and in living cells (Saito et al, 2004;Baudendistel et al, 2005;Kim et al, 2005). Unique features of FCCS technology have recently been reviewed by Bacia et al (2006) with emphasis on its application to living cells.…”

“…In this case it is hard to eliminate cross-talk signals at the second channel for the fluorophore that has a longer wavelength. Recent modern FCCS methods such as 2 photon-FCCS (2P-FCCS) (Kim et al, 2005;Swift et al, 2006) and single wavelength excitation FCCS (SW-FCCS) (Liu et al, 2007) require only one wavelength excitation system. These methods have the advantages of simple equipment and perfect matching of the confocal volume for two distinct fluorophore excitations.…”