Faster Diagnosis of Alpha-1-Antitrypsin Deficiency through Electrochemical and Quartz Crystal Microbalance Testing

Abstract

Alpha-1-Antitrypsin (A1AT) Deficiency (A1AD) is a potentially debilitating inherited condition that can lead to reduced levels of A1AT in the lungs and increased levels in the liver. In the lungs, the condition is routinely associated with an imbalance in levels of Human Neutrophil Elastase (HNE) and its native inhibitor A1AT (predisposing those affected to early-onset emphysema). In the liver, certain genetic mutations lead to an accumulation of A1AT. No cure exists, and current approaches for diagnosis are insufficient as illustrated by the fact that A1AD is underdiagnosed and diagnosis is often delayed.1New methods for diagnosis are needed, and one possible route is the use of electrochemical techniques combined with quartz crystal microbalance (QCM) approaches. Often, QCM and electrochemistry are combined using a gold working electrode that has been functionalized with a particular molecule. In our case, Porcine Pancreatic elastase (PPE), a surrogate of HNE, has been used as an enzymatic marker. When a potentiostat was combined in parallel to the apparatus, measurements of both the frequency and the current responses were simultaneously performed. PPE was immobilized to the surface of the gold electrode through the use of a crosslinker. The immobilized PPE was then incubated with various concentrations of N-Succinyl-Ala-Ala-Ala-p-Nitroanalide (SApNA), a chromogenic substrate for elastase, allowed to react, and the absorbance measured to determine the activity of the enzyme. This same test was also completed with the A1AT inhibitor in the solution with the SApNA and the absorbance measured again. From these results, the frequency was measured and converted to mass to determine the extent of inhibition. This data was then examined via kinetics-based models to estimate the kinetic parameters of inhibition. Electrochemical measurements have been performed concurrently with the frequency measurements. Using phosphate buffered saline (PBS) in the presence of ferrocyanide/ferricyanide as the redox species, cyclic voltammetry was performed to determine the extent that the electrode surface is covered. As the SApNA and A1AT was added to the solution, the change in the ferrocyanide/ferricyanide peaks were observed. It is the ultimate goal of this work to develop a test that can be used to determine the inhibition kinetics of HNE via A1AT, and thus shed light on new approaches to diagnose A1AD. Such an approach could also serve as a platform that could be useful in the early detection and diagnosis of many different diseases that could allow for preemptive treatment and could potentially improve life expectancy outcomes for those afflicted. American Thoracic Society/European Respiratory Society Statement: Standards for the Diagnosis and Management of Individuals with Alpha-1-Antitrypsin Deficiency. (2003). Am. J. Respir. Crit. Care Med. 168(7): 818-900.