Reagentless Measurement of Aminoglycoside Antibiotics in Blood Serum via an Electrochemical, Ribonucleic Acid Aptamer-Based Biosensor

摘要

Biosensors built using ribonucleic acid (RNA) aptamersnshow promise as tools for point-of-care medical diagnostics,nbut they remain vulnerable to nuclease degradationnwhen deployed in clinical samples. To explore methodsnfor protecting RNA-based biosensors from such degradationnwe have constructed and characterized an electrochemical,naptamer-based sensor for the detection ofnaminoglycosidic antibiotics. We find that while this sensornachieves low micromolar detection limits and subminutenequilibration times when challenged in buffer, it deterioratesnrapidly when immersed directly in blood serum. Innorder to circumvent this problem, we have developed andntested sensors employing modified versions of the samenaptamer. Our first effort to this end entailed the methylationnof all of the 2′-hydroxyl groups outside of thenaptamer’s antibiotic binding pocket. However, while devicesnemploying this modified aptamer are as sensitivenas those employing an unmodified parent, the modificationnfails to confer greater stability when the sensor isnchallenged directly in blood serum. As a second potentiallynnaive alternative, we replaced the RNA bases in thenaptamer with their more degradation-resistant deoxyribonucleicnacid (DNA) equivalents. Surprisingly and unlikencontrol DNA-stem loops employing other sequences, thisnDNA aptamer retains the ability to bind aminoglycosides,nalbeit with poorer affinity than the parent RNA aptamer.nUnfortunately, however, while sensors fabricated usingnthis DNA aptamer are stable in blood serum, its lowernaffinity pushes their detection limits above the therapeuticallynrelevant range. Finally, we find that ultrafiltrationnthrough a low-molecular-weight-cutoff spin column rapidlynand efficiently removes the relevant nucleases from serumnsamples spiked with gentamicin, allowing the convenientndetection of this aminoglycoside at clinically relevantnconcentrations using the original RNA-based sensor.