Digoxin detection for therapeutic drug monitoring using target-triggered aptamer hairpin switch and nicking enzyme-assisted signal amplification

Abstract

Digoxin, a cardiac glycoside drug, is commonly used to treat heart failure and arrhythmias. The therapeutic concentration range of digoxin, with a narrow therapeutic index, is between 0.5 and 2.0 ng mL⁻¹. Hence, it is important for patients to monitor their blood levels after taking medication to achieve effective treatment and reduce the likelihood of experiencing drug side effects. Due to the complex steps and high cost of immunoassays, aptasensors that use aptamers to recognize the targets offer the advantages of low cost and good stability over other analysis methods. Nicking enzyme-assisted signal amplification is a novel isothermal signal amplification technology that relies on nicking enzymes to recognize and cleave restriction sites on one oligonucleotide strand. In this study, we develop a fluorescent aptasensor coupled with target-triggered aptamer hairpin switch and nicking enzyme-assisted signal amplification for digoxin detection in plasma for therapeutic drug monitoring. After optimizing the experimental parameters, we design hairpin probes with ten base pairs of the aptamer sequence and extended sequence complement to react with digoxin in a 10 mM Tris buffer containing 150 mM NaCl and 50 mM MgCl₂ (pH 7.4). The signal amplification reactions were performed for 3 hours. The fluorescent aptasensor exhibited high sensitivity with a detection limit of 88 pg mL⁻¹ for detecting digoxin in plasma and a linear range from 0.1 ng mL⁻¹ to 5 ng mL⁻¹. This technology was successfully used for digoxin detection to improve treatment effectiveness and minimize the risk of adverse side effects.