Behavior of ß-amyloid 1-16 at the air-water interface at varying pH by nonlinear spectroscopy and molecular dynamics simulations.

The adsorption and aggregation of ß-amyloid (1-16) fragment at the air-water interface was investigated by the combination of second harmonic generation (SHG) spectroscopy, Brewster angle microscopy (BAM), and molecular dynamics simulations (MD). The Gibbs free energy of surface adsorption was measured to be -10.3 kcal/mol for bulk pHs of 7.4 and 3, but no adsorption was observed for pH 10-11. The 1-16 fragment is believed not to be involved in fibril formation of the ß-amyloid protein, but it exhibits interesting behavior at the air-water interface, as manifested in two time scales for the observed SHG response. The shorter time scale (minutes) reflects the surface adsorption, and the longer time scale (hours) reflects rearrangement and aggregation of the peptide at the air-water interface. Both of these processes are also evidenced by BAM measurements. MD simulations confirm the pH dependence of surface behavior of the ß-amyloid, with largest surface affinity found at pH = 7. It also follows from the simulations that phenylalanine is the most surface exposed residue, followed by tyrosine and histidine in their neutral form.

Improving nanoparticle dispersion and charge transfer in cadmium telluride tetrapod and conjugated polymer blends.

The dispersion of CdTe tetrapods in a conducting polymer and the resulting charge transfer is studied using a combination of confocal fluorescence microscopy and atomic force microscopy (AFM). The results of this work show that both the tetrapod dispersion and charge transfer between the CdTe and conducting polymer (P3HT) are greatly enhanced by exchanging the ligands on the surface of the CdTe and by choosing proper solvent mixtures. The ability to experimentally probe the relationship between particle dispersion and charge transfer through the combination of AFM and fluorescence microscopy provides another avenue to assess the performance of polymer/semiconductor nanoparticle composites.

Fluorescence cross-correlation spectroscopy as a universal method for protein detection with low false positives.

Specific, quantitative, and sensitive protein detection with minimal sample preparation is an enduring need in biology and medicine. Protein detection assays ideally provide quick, definitive measurements that use only small amounts of material. Fluorescence cross-correlation spectroscopy (FCCS) has been proposed and developed as a protein detection assay for several years. Here, we combine several recent advances in FCCS apparatus and analysis to demonstrate it as an important method for sensitive, quantitative, information-rich protein detection with low false positives. The addition of alternating laser excitation (ALEX) to FCCS along with a method to exclude signals from occasional aggregates leads to a very low rate of false positives, allowing the detection and quantification of the concentrations of a wide variety of proteins. We detect human chorionic gonadotropin (hCG) using an antibody-based sandwich assay and quantitatively compare our results with calculations based on binding equilibrium equations. Furthermore, using our aggregate exclusion method, we detect smaller oligomers of the prion protein PrP by excluding bright signals from large aggregates.

Raman spectroscopy of DNA packaging in individual human sperm cells distinguishes normal from abnormal cells.

Healthy human males produce sperm cells of which about 25-40% have abnormal head shapes. Increases in the percentage of sperm exhibiting aberrant sperm head morphologies have been correlated with male infertility, and biochemical studies of pooled sperm have suggested that sperm with abnormal shape may contain DNA that has not been properly repackaged by protamine during spermatid development. We have used micro-Raman spectroscopy to obtain Raman spectra from individual human sperm cells and examined how differences in the Raman spectra of sperm chromatin correlate with cell shape. We show that Raman spectra of individual sperm cells contain vibrational marker modes that can be used to assess the efficiency of DNA-packaging for each cell. Raman spectra obtained from sperm cells with normal shape provide evidence that DNA in these sperm is very efficiently packaged. We find, however, that the relative protein content per cell and DNA packaging efficiencies are distributed over a relatively wide range for sperm cells with both normal and abnormal shape. These findings indicate that single cell Raman spectroscopy should be a valuable tool in assessing the quality of sperm cells for in-vitro fertilization.

Motion of a DNA sliding clamp observed by single molecule fluorescence spectroscopy.

DNA sliding clamps attach to polymerases and slide along DNA to allow rapid, processive replication of DNA. These clamps contain many positively charged residues that could curtail the sliding due to attractive interactions with the negatively charged DNA. By single-molecule spectroscopy we have observed a fluorescently labeled sliding clamp (polymerase III beta subunit or beta clamp) loaded onto freely diffusing, single-stranded M13 circular DNA annealed with fluorescently labeled DNA oligomers of up to 90 bases. We find that the diffusion constant for the beta clamp diffusing along DNA is on the order of 10(-14) m(2)/s, at least 3 orders of magnitude less than that for diffusion through water alone. We also find evidence that the beta clamp remains at the 3' end in the presence of Escherichia coli single-stranded-binding protein. These results may imply that the clamp not only acts to hold the polymerase on the DNA but also prevents excessive drifting along the DNA.

Simultaneous time- and wavelength-resolved fluorescence spectroscopy for near real-time tissue diagnosis.

A novel fiber-optic-based method for simultaneous time- and wavelength-resolved fluorescence spectroscopy for the rapid diagnosis of diseased tissue is demonstrated. By combining multiple bandpass and dichroic filters (405/40, 460/50, and 550/50) with different lengths of optical fiber (1, 10, and 19 m) acting as an optical delay this system enables the near real-time acquisition and characterization of time-resolved fluorescence spectra using a single detector and excitation input. The recording of multiple fluorescence response pulses at selected wavelengths can be completed in hundreds of nanoseconds, which provides the capability of a real-time characterization of biological systems.

Correlation spectroscopy of minor fluorescent species: signal purification and distribution analysis.

We are performing experiments that use fluorescence resonance energy transfer (FRET) and fluorescence correlation spectroscopy (FCS) to monitor the movement of an individual donor-labeled sliding clamp protein molecule along acceptor-labeled DNA. In addition to the FRET signal sought from the sliding clamp-DNA complexes, the detection channel for FRET contains undesirable signal from free sliding clamp and free DNA. When multiple fluorescent species contribute to a correlation signal, it is difficult or impossible to distinguish between contributions from individual species. As a remedy, we introduce "purified FCS", which uses single molecule burst analysis to select a species of interest and extract the correlation signal for further analysis. We show that by expanding the correlation region around a burst, the correlated signal is retained and the functional forms of FCS fitting equations remain valid. We demonstrate the use of purified FCS in experiments with DNA sliding clamps. We also introduce "single-molecule FCS", which obtains diffusion time estimates for each burst using expanded correlation regions. By monitoring the detachment of weakly-bound 30-mer DNA oligomers from a single-stranded DNA plasmid, we show that single-molecule FCS can distinguish between bursts from species that differ by a factor of 5 in diffusion constant.

Single-molecule dynamics of phytochrome-bound fluorophores probed by fluorescence correlation spectroscopy.

Fluorescence correlation spectroscopy (FCS) was used to investigate the hydrodynamic and photophysical properties of PR1 (phytofluor red 1), an intensely red fluorescent biliprotein variant of the truncated cyanobacterial phytochrome 1 (Cph1Delta, which consists of the N-terminal 514 amino acids). Single-molecule diffusion measurements showed that PR1 has excellent fluorescence properties at the single-molecule level, making it an interesting candidate for red fluorescent protein fusions. FCS measurements for probing dimer formation in solution over a range of protein concentrations were enabled by addition of Cph1Delta apoprotein (apoCph1Delta) to nanomolar solutions of PR1. FCS brightness analysis showed that heterodimerization of PR1 with apoCph1Delta altered the chemical environment of the PR1 chromophore to further enhance its fluorescence emission. Fluorescence correlation measurements also revealed interactions between apoCph1Delta and the red fluorescent dyes Cy5.18 and Atto 655 but not Alexa Fluor 660. The concentration dependence of protein:dye complex formation indicated that Atto 655 interacted with, or influenced the formation of, the apoCph1 dimer. These studies presage the utility of phytofluor tags for probing single-molecule dynamics in living cells in which the fluorescence signal can be controlled by the addition of various chromophores that have different structures and photophysical properties, thereby imparting different types of information, such as dimer formation or the presence of open binding faces on a protein.

Bio-assay based on single molecule fluorescence detection in microfluidic channels.

A rapid bioassay is described based on the detection of colocalized fluorescent DNA probes bound to DNA targets in a pressure-driven solution flowing through a planar microfluidic channel. By employing total internal reflection excitation of the fluorescent probes and illumination of almost the entire flow channel, single fluorescent molecules can be efficiently detected leading to the rapid analysis of nearly the entire solution flowed through the device. Cross-correlation between images obtained from two spectrally distinct probes is used to determine the target concentration and efficiently reduces the number of false positives. The rapid analysis of DNA targets in the low pM range in less than a minute is demonstrated.

Surface-enhanced Raman scattering from individual au nanoparticles and nanoparticle dimer substrates.

Surface-enhanced Raman scattering (SERS) intensities for individual Au nanospheres, nanoshells, and nanosphere and nanoshell dimers coated with nonresonant molecules are measured, where the precise nanoscale geometry of each monomer and dimer is identified through in situ atomic force microscopy. The observed intensities correlate with the integrated quartic local electromagnetic field calculated for each specific nanostructure geometry. In this study, we find that suitably fabricated nanoshells can provide SERS enhancements comparable to nanosphere dimers.

Intracellular pH sensors based on surface-enhanced raman scattering.

We present the development of nanoscale pH sensors based on functionalized silver nanoparticles and surface-enhanced Raman scattering (SERS). The SERS spectrum from individual silver nanoparticle (50-80 nm in diameter) clusters functionalized with 4-mercaptobenzoic acid shows a characteristic response to the pH of the surrounding solution and is sensitive to pH changes in the range of 6-8. Measurements from nanoparticles incorporated in living Chinese hamster ovary cells demonstrate that the nanoparticle sensors retain their robust signal and sensitivity to pH when incorporated into a cell.