Research Program
Biomolecular Devices and Analysis

Objectives
The goal of this research project is to develop quantitative instrumentation and nanoscale devices to accurately measure the early aggregation state of proteins such as α-synuclein and amyloid-beta (Ab).  While the historical point of view has been that the large protein aggregates of α-synuclein found in the Lewy Bodies of Parkinson’s Disease or the amyloid-beta plagues found in Alzheimer’s disease are the cause of disease, more recent evidence suggests that smaller soluble species - misfolded and aggregated into low copy number oligomers - are actually the toxic moiety. We will employ micro- and nano-fluidic devices to quantify low copy number fluorescently labeled α-synuclein and amyloid-beta (Ab) oligomers, and determine how monomer concentration, pH, ionic strength and the presence of interacting species such as protein chaperones or pharmaceuticals changes the oligomer size distribution.

Methods
Aim 1.  Our first aim involves the use of micron-scale fluidic systems for the general characterization of protein aggregate size based on fluorescence per particle.  Zeonex microchannels are fabricated from a silicon master into a 1 mm thick Zeonex slide to produce channels 2µm wide and 8µm deep.  Micron scale devices have the advantage of minimal plugging and ease of fabrication, and provide an immediate tool for obtaining estimates of aggregate size distributions while the more quantitative system described in Goal 2 is fabricated.  We also are developing optimized hardware, software and methodologies to quantify the broad range of oligomers detected using our micro-channels.  We are currently applying these flow devices in a study of the effect of co-expression of HSP70 on GFP-α-synuclein aggregation in vivo by measuring the oligomer size distribution of cytosolic extracts at different times after transfection (Figures 1 and 2).

Aim 2.   In this aim we are fabricating nanoscale channel devices (Figure 3) to quantify low copy number Ab oligomers (1 to ~20 proteins/aggregate) formed during in vitro aggregation of Ab-42.  Based on Poisson statistics, roughly 50 photons per fluorophore need to be detected during oligomer passage through the focal volume to differentiate between monomers and dimers, dimers and trimers etc.  In previous single fluorophore collection experiments using similar nanochannels only 2 to 5 photons were collected per ~100µs fluorophore passage time.  At saturation a typical fluorophore can produce about 50,000 photons in 100 µs, so there is ample room for improvement. By combining our nanochannel devices with zero-dead time, high sensitivity photodetectors, high speed photon counting circuitry and a slightly higher numerical aperture, we estimate we can increase the collection efficiency by a factor of 10-20 to achieve integer fluorophore discrimination.

Summary
Using nanofabrication techniques and novel optoelectronics developed we are constructing a highly sensitive nanochannel based fluorescence monitoring device that will enable exact numbers of the fluorophores to be counted as labeled species pass through a tightly focused laser beam.   The system will be used to study the aggregation dynamics and kinetics of fluorescently labeled α-synuclein and amyloid-beta peptides under various in vitro aggregation conditions.  This device will advance biomedical research by providing a new tool to study and screen at the single molecule level.  This device development could also have significant clinical potential in future applications that require highly sensitive detection of any type of rare molecular species that can be fluorescently labeled.