Enhanced Detection of Klebsiella Pneumoniae Using Electric-Double-Layer FET Sensor: Rapid, Temperature-Boosted Probe-Target Binding Analysis

Abstract

Klebsiella pneumoniae demonstrates versatility in its pathogenicity, leading to diverse infections in humans such as pneumonia, urinary tract infections, bacteremia, wound infections, meningitis, endocarditis, and sepsis. The severity of these infections is contingent upon the patient's immune status, the site of infection, and the existence of underlying medical comorbidities. A sputum or urine culture, on the other hand, typically takes between 24 and 72 hours to produce results. Over recent years, various innovative methodologies have emerged, including optical and electrochemical techniques and molecular detection via PCR. Despite the diversity of available techniques, concerns persist regarding their sensitivity and specificity. Moreover, fluorescence-based methodologies and Surface Plasmon Resonance (SPR)-based sensors have been explored for bacterial detection. However, these existing methods have drawbacks such as elevated costs, prolonged turnaround times, the need for sophisticated instrumentation, and a demand for skilled personnel. Field-effect transistor (FET) sensors have garnered considerable scientific attention since their inception due to their remarkable sensitivity, facile signal readout, and the capability for label-free assays. The aim of this research is to detect the detect Klebsiella pneumoniae bacteria in blood serum by double electric layer (EDL) extended-gate field-effect transistor (FET) sensor. In this study, a preliminary investigation employed a single-step surface functionalization approach to immobilize receptor probes, specifically single-stranded DNA (ssDNA). This strategy aimed to enable the rapid detection of complementary DNA (cDNA) across varying concentrations, leveraging differential gate voltages to modulate the sensitivity of this analytical platform. The designed probes were successfully immobilized onto a disposable chip housing a pair of gold electrodes. A handheld reader, integrating enhancement-mode N-channel MOSFETs, established connectivity with a laptop via a USB port to facilitate the measurement of sensor signals. By applying pulsed gate voltage to the input electrode, which modulates the channel conductivity which behaves as a function of the charge distribution within the EDL structure, consequently influencing the drain current of the MOSFET. The immobilized probe, positioned on the gate electrode, underwent testing using varying concentrations of cDNA introduced into TE buffer. Detection of probe-target binding occurred through fluorescence alterations and electrically by assessing changes in drain current. In order to improve the response time, we elevated the temperature near the Tm enhances sensitivity and binding rates, in shorter time period of 1 minutes. Figure 1