A two-photon excitation fluorescence cross-correlation assay for a model ligand-receptor binding system using quantum dots.

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.

Examination of surface-bound Ku-DNA complexes in an aqueous environment using MAC mode atomic force microscopy.

In the development of biosensors, it is essential to understand how the signal-transducing element may perturb surface-bound proteins and nucleic acids. The tip of the atomic force microscope is such an element in atomic force microscopy. In this paper, we describe the influence of tip-sample interactions on the measured height of the DNA repair protein, Ku, that has been adsorbed onto a mica surface which was submerged in aqueous solution. We find that the measured height of the Ku molecule depends critically on whether or not it is associated with DNA. Additionally, we observed that the conditions (time and concentration) under which Ku is incubated with DNA, affect the appearance (number and type) of the DNA-Ku complexes observed.

Investigation of temperature-induced phase transitions in DOPC and DPPC phospholipid bilayers using temperature-controlled scanning force microscopy.

Under physiological conditions, multicomponent biological membranes undergo structural changes which help define how the membrane functions. An understanding of biomembrane structure-function relations can be based on knowledge of the physical and chemical properties of pure phospholipid bilayers. Here, we have investigated phase transitions in dipalmitoylphosphatidylcholine (DPPC) and dioleoylphosphatidylcholine (DOPC) bilayers. We demonstrated the existence of several phase transitions in DPPC and DOPC mica-supported bilayers by both atomic force microscopy imaging and force measurements. Supported DPPC bilayers show a broad L(beta)-L(alpha) transition. In addition to the main transition we observed structural changes both above and below main transition temperature, which include increase in bilayer coverage and changes in bilayer height. Force measurements provide valuable information on bilayer thickness and phase transitions and are in good agreement with atomic force microscopy imaging data. A De Gennes model was used to characterize the repulsive steric forces as the origin of supported bilayer elastic properties. Both electrostatic and steric forces contribute to the repulsive part of the force plot.

Supported planar bilayer formation by vesicle fusion: the interaction of phospholipid vesicles with surfaces and the effect of gramicidin on bilayer properties using atomic force microscopy.

We have used magnetic alternating current mode atomic force microscopy (MAC-AFM) to investigate the formation of supported phospholipid bilayers (SPB) by the method of vesicle fusion. The systems studied were dioleoylphosphatidylcholine (DOPC) on mica and mica modified with 3-aminopropyl-triethoxy-silane (APTES), and DOPC vesicles with gramicidin incorporated on mica and APTES-modified mica. The AFM images reveal three stages of bilayer formation: localized disklike features that are single bilayer footprints of the vesicles, partial continuous coverage, and finally complete bilayer formation. The mechanism of supported phospholipid bilayers formation is the fusion of proximal vesicles, rather than surface disk migration. This mechanism does not appear to be affected by incorporation of gramicidin or by surface modification. Once formed, the bilayer develops circular defects one bilayer deep. These defects grow in size and number until a dynamic equilibrium is reached.

Near-infrared two-photon excitation of protoporphyrin IX: photodynamics and photoproduct generation.

The spectroscopy and photochemistry of protoporphyrin IX in ethanol and in Triton X-100 micelle solution have been examined using near-infrared two-photon excitation (TPE). TPE will allow photodynamic therapy with highly localized light dosage. We have determined that the photochemistry subsequent to TPE is very similar to that found for one-photon excitation. Moreover, the photoproducts observed possess very intense TPE fluorescence spectra, which allows their detection at low relative concentrations.