Systematic Investigation of Insulin Fibrillation on a Chip.

Henk-Willem Veltkamp,Hoon Suk Rho,Christian Breukers,Marcel Ottens,Pamela Habibović,Han Gardeniers,Alexander Thomas Hanke

doi:10.3390/molecules25061380

Abstract

A microfluidic protein aggregation device (microPAD) that allows the user to perform a series of protein incubations with various concentrations of two reagents is demonstrated. The microfluidic device consists of 64 incubation chambers to perform individual incubations of the protein at 64 specific conditions. Parallel processes of metering reagents, stepwise concentration gradient generation, and mixing are achieved simultaneously by pneumatic valves. Fibrillation of bovine insulin was selected to test the device. The effect of insulin and sodium chloride (NaCl) concentration on the formation of fibrillar structures was studied by observing the growth rate of partially folded protein, using the fluorescent marker Thioflavin-T. Moreover, dual gradients of different NaCl and hydrochloric acid (HCl) concentrations were formed, to investigate their interactive roles in the formation of insulin fibrils and spherulites. The chip-system provides a bird’s eye view on protein aggregation, including an overview of the factors that affect the process and their interactions. This microfluidic platform is potentially useful for rapid analysis of the fibrillation of proteins associated with many misfolding-based diseases, such as quantitative and qualitative studies on amyloid growth.

Highlights

Several common neurodegenerative disorders, such as Parkinson’s disease, type II diabetes, and Alzheimer’s disease, are known to be related to amyloidosis, in which innoxious proteins change into amyloid fibrils [1,2,3,4]
We developed a microfluidic protein aggregation device that enabled a series of protein incubations under various conditions
We demonstrated nanoliter-scale bovine insulin aggregations, to evaluate the combined effect of concentrations of sodium chloride (NaCl) and hydrochloric acid (HCl) on the formation of insulin fibrils and fibrillar superstructures

Summary

Introduction

Several common neurodegenerative disorders, such as Parkinson’s disease, type II diabetes, and Alzheimer’s disease, are known to be related to amyloidosis, in which innoxious proteins change into amyloid fibrils [1,2,3,4]. Insulin is commonly used as a model system to evaluate the mechanism of amyloid aggregation because the structural properties of insulin fibrils are similar to those of other amyloidogenic proteins [11,12,13]. Even though the traditional incubation method successfully identified the critical parameters affecting the formation of insulin fibrils and the growth of insulin fibrillar structures, multiple sample preparation steps and long incubation times are required to systematically evaluate the (intertwined) effects of a large number of factors. The conventional method for the kinetic study of protein fibrillation phenomena is limited to the observations of the early stage growth of fibrils only due to the large sample volume, which requires additional processes to analyze the number of fibrils or fibrillar structures

Methods

Results

Conclusion