(587e) Quartz Crystal Microbalance Analysis of Growth Kinetics for Aggregation Intermediates of the Amyloid-β Protein

Kotarek, J. A. - Presenter, University of South Carolina
Moss, M. A. - Presenter, University of South Carolina
Johnson, K. C. - Presenter, University of Minnesota

Alzheimer's disease (AD) is a neurodegenerative disorder that effects approximately 4.5 million Americans over the age of 65?a statistic that is set to triple by 2050. The widely accepted amyloid hypothesis proposes that the disease is caused by an accumulation of aggregating amyloid-beta protein, which eventually forms insoluble fibrils that deposit in the brain. This theory is supported by several genetic mutations linked to AD which increase the aggregation potential of the protein. Furthermore, experimental studies show that aggregated amyloid-beta is toxic in neuronal cultures, while its monomeric form causes no detrimental effects (Tickler et al. 2005).

While ample evidence supports this amyloid cascade hypothesis, the transformation of benign monomeric amyloid-beta into these toxic aggregates is not completely understood. The kinetics governing the nucleation driven reaction that produces these aggregates are also complex and their quantification has many associated difficulties. Predominant hurdles include both the isolation of specific mechanistic steps as well as the accurate quantification of aggregation in real time. The quartz crystal microbalance (QCM) is well suited as a biosensor for this study, as it provides a heterogeneous system that can measure mass changes in the nanogram range. QCM utilizes the piezoelectric effect in quartz crystals to detect changes in bound elastic mass as a variation in the frequency of oscillation. The methodology developed here excludes nucleation from the analysis and is based on the principle that only amyloid-beta aggregates, and not amyloid-beta monomers, are capable of supporting further aggregate growth. An avidin monolayer is deposited onto the gold surface of a crystal electrode via well established amino-coupling chemistry (Jung et al. 2006), upon which biotinylated amyloid-beta monomer is bound. This surface selectively recognizes aggregated forms of amyloid-beta and immobilizes these unlabeled aggregates, whereas monomer-monomer interactions are virtually non-existent in the system. Subsequent exposure to unlabeled monomer will initiate aggregate growth via aggregate-monomer interactions.

We show that this biosensor can selectively detect aggregates created in vitro and quantify their subsequent growth with sensitivity approaching the nanomolar range. Further quantification of soluble aggregate growth rates revealed a first order dependence on both monomer concentration adn immobilized aggregate density. The kinetic mechanisms isolated within this system compare well with previously developed models. An aggregate dissociation constant of 220 nm was determined, which is in agreement with data from studies of mature insoluble aggregates and indicates that the mechanisms of growth for soluble and insoluble aggregates are similar.

These parameters will allow for future optimization of the biosensor, as well as provide a better understanding of the aggregation process. Future work will explore the ability of the QCM sensor to selectively detect amyloid-beta aggregates in plasma and cerebral spinal fluid isolated from AD patients. Successful implementation of this technique could provide an effective means for the early diagnosis of AD.

Jung, H., Kim, J., Park, J., Lee, S., Lee, H., Kuboi, R., and Kawai, T. (2006). "Atomic force microscopy observation of highly arrayed phospholipid bilayer vesicle on a gold surface." Journal of Bioscience and Bioengineering, 102(1), 28-33.

Tickler, A. K., Wade, J. D., and Separovic, F. (2005). "The Role of Amyloid-beta Peptides in Alzheimer's Disease." Protein & Peptide Letters, 12(6), 513-519.