An Aptamer-Based Biosensor for the Azole Class of Antifungal Drugs.

David S Perlin,Gregory R Wiedman,Ramya Vishnubhotla,Mariana Castanheira,A T Charlie Johnson,Jinglei Ping,Arkady Mustaev,Yanan Zhao

doi:10.1128/msphere.00274-17

Abstract

This technical report describes the development of an aptamer for sensing azole antifungal drugs during therapeutic drug monitoring. Modified synthetic evolution of ligands through exponential enrichment (SELEX) was used to discover a DNA aptamer recognizing azole class antifungal drugs. This aptamer undergoes a secondary structural change upon binding to its target molecule, as shown through fluorescence anisotropy-based binding measurements. Experiments using circular dichroism spectroscopy revealed a unique G-quadruplex structure that was essential and specific for binding to the azole antifungal target. Aptamer-functionalized graphene field effect transistor (GFET) devices were created and used to measure the strength of binding of azole antifungals to this surface. In total, this aptamer and the supporting sensing platform provide a valuable tool for therapeutic drug monitoring of patients with invasive fungal infections. IMPORTANCE We have developed the first aptamer directed toward the azole class of antifungal drugs and a functional biosensor for these drugs. This aptamer has a unique secondary structure that allows it to bind to highly hydrophobic drugs. The aptamer works as a capture component of a graphene field effect transistor device. These devices can provide a quick and easy assay for determining drug concentrations. These will be useful for therapeutic drug monitoring of azole antifungal drugs, which is necessary to deal with the complex drug dosage profiles.

Highlights

This technical report describes the development of an aptamer for sensing azole antifungal drugs during therapeutic drug monitoring
This study included several control sequences to further investigate the importance of the structure of the round 13 aptamer (Rd 13): Rd 13 Scrambled (S), Rd 13 T6, and Rd 13 T1 were created with various middle lengths
This report highlights the development of an azole drug-capturing oligonucleotide using the synthetic evolution of ligands through exponential enrichment (SELEX) process and the discovery of a unique structure that allows it to bind azole antifungal drugs

Summary

Introduction

This technical report describes the development of an aptamer for sensing azole antifungal drugs during therapeutic drug monitoring. Aptamer-functionalized graphene field effect transistor (GFET) devices were created and used to measure the strength of binding of azole antifungals to this surface This aptamer and the supporting sensing platform provide a valuable tool for therapeutic drug monitoring of patients with invasive fungal infections. The best therapeutic management can be achieved by maintaining a therapeutic level in a patient’s bloodstream and by optimizing individual dosage regimens These analyses generally rely upon trough and peak monitoring and real-time kinetic drug modeling. TDM requires blood to be drawn from patients and drug levels in blood to be evaluated by analytical instrumentation at some later point in time Analytical techniques such as liquid chromatography (LC) and mass spectrometry (MS) require skilled staff and resources that are not found in all hospitals [8]. Effective methods for sensing small drug molecules in blood samples would make it easier to determine drug concentrations

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