Nano-biosensors for rapid detection of antibiotic resistance genes blaCTX-M in Escherichia coli and blaKPC in Klebsiella pneumoniae.

The wide spread of antibiotic resistance genes in clinical isolates of Escherichia coli (E. coli) and Klebsiella pneumoniae (K. pneumoniae) has become a worrying issue worldwide. Thus, electrochemical DNA-based biosensors were developed and applied for the rapid detection of antibiotic resistance genes (blaCTX-M extended-spectrum β-lactamase (ESBL) in E. coli and blaKPC carbapenemase in Klebsiella pneumoniae). Selective ssDNA probes specific to the targeting complementary genes were designed and individually immobilized onto disposable sensor chips that were functionalized with nanocomposite (ssDNA-probe@ATP@AuNPs-PPY-V2O5). In this regard, the sensor surfaces were first modified with vanadium oxide (V2O5) and polypyrrole (PPy) then gold nanoparticles (AuNPs) to effectively promote the chemical immobilization of each of the designed bio-receptor (ssDNA probe) onto the sensor matrix using the 4-aminothiophenol (ATP) as the chemical cross-linker. Different parameters that affect the biosensing performance were studied and optimized. Accordingly, high sensitivity to both E. coli blaCTX-M and K. pneumoniae blaKPC genes were obtained with a dynamic linear range from 10-6 to 0.1 ng/μL with correlation coefficient (R2) of 0.991 and 0.988, limit of detection (LODs) of 0.5 × 10-7 ng/μL and 1 × 10-7 ng/μL, and limit of quantification (LOQs) of 1.2 × 10-7 and 3 × 10-7 ng/μL, respectively. High selectivity was provided since no electrochemical signals were obtained from various competitors of common bacteria. Finally, different types of clinical samples were analyzed and validated.