Selection of ssDNA aptamers targeting the serine protease SPSFQ of Acinetobacter baumannii and development of an electrochemical impedance spectroscopy-based ultrasensitive SPSFQ biosensor.

Acinetobacter baumannii (A. baumannii) is a Gram-negative bacterium that creates an increasing burden on the healthcare system due to its ability to develop multidrug resistance. Sensitive, rapid and on-site detection of A. baumannii, which has been declared a critical priority pathogen by the World Health Organization, is of great importance. Today, secretory proteins of pathogenic organisms attract attention not only for their role in invasive processes but also as targets for early diagnosis. In this context, SPSFQ, a recently identified secretory protease, is a valuable target for the detection of A. baumannii at very low initial levels, before significant colonization occurs. In this study, ssDNA aptamers for SPSFQ were selected for the first time using the Systematic Evolution of Ligands by Exponential Enrichment (SELEX) method. The two aptamer sequences identified through SELEX were named Apt1 and Apt2, with Kd values of 42.10 ± 6.7 nM and 26.98 ± 1.35 nM, respectively. An ultrasensitive EIS-based biosensor was developed using Apt2, achieving a detection limit of 5.44 fg/mL and a linear range of 1.0-10,000 fg/mL. The aptasensor exhibited good repeatability (RSD: 2.62%) and reproducibility (RSD: 6.62%), and also maintained satisfactory stability during storage. Furthermore, the developed biosensor demonstrated reliable and remarkable performance in real commercial human serum samples, with recovery values of 110.84% and 104.40% for serum samples spiked with 250 and 6500 fg/mL SPSFQ, respectively.