Abigail E. Miller

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.

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

The adsorption and aggregation of β-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 β-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 β-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.

Prion Aptamer, Free and Bound States

Aptamers are short single strands of DNA or RNA that bind to proteins, peptides, and small molecules. They are likely to fold into different structures when free in solution than when they are bound to a molecular target. The free structures are difficult to determine experimentally, though they can be modeled by calculating the minimum thermodynamic states. We test the validity of the thermodynamic models of a prion aptamer using single molecule pair Forster resonance energy transfer (spFRET) as a structural reporter. The FRET states of the unbound aptamers, the hybridized aptamers and the aptamers bound to PrP peptides are characterized. The DNA aptamers to PrP has a pair of thermodynamic states of roughly the same energy at 25 oC. Their presence in solution is characterized by comparing the single stranded aptamers to its hybridized configuration, thereby removing any internal structure of the aptamers. We demonstrate the existence of both unbound thermodynamic states as well as different interactions between the aptamer and each PrP peptide from static data measuring spFRET in solution.

Fabrication of luminescent nanostructures by dip-pen nanolithography

We used a combination of dip-pen nanolithography and scanning optical confocal microscopy to fabricate and visualize luminescent nanoscale patterns of various materials on glass substrates. We show that this method can be used successfully to push the limits of dip-pen nanolithography down to controlled deposition of single molecules. We also demonstrate that this method is able to create and visualize protein patterns on surfaces. Finally, we show that our method can be used to fabricate polymer nanowires of controlled size using conductive polymers. We also present a kinetic model that accurately describes the deposition process.