Rapid antimicrobial susceptibility testing with electrokinetics enhanced biosensors for diagnosis of acute bacterial infections

Abstract

Rapid pathogen detection and antimicrobial susceptibility testing (AST) are required in diagnosis of acute bacterial infections to determine the appropriate antibiotic treatment. Molecular approaches for AST are often based on the detection of known antibiotic resistance genes. Phenotypic culture analysis requires several days from sample collection to result reporting. Toward rapid diagnosis of bacterial infection in non-traditional healthcare settings, we have developed a rapid AST approach that combines phenotypic culture of bacterial pathogens in physiological samples and electrochemical sensing of bacterial 16S rRNA. The assay determines the susceptibility of pathogens by detecting bacterial growth under various antibiotic conditions. AC electrokinetic fluid motion and Joule heating induced temperature elevation are optimized to enhance the sensor signal and minimize the matrix effect, which improve the overall sensitivity of the assay. The electrokinetics enhanced biosensor directly detects the bacterial pathogens in blood culture without prior purification. Rapid determination of the antibiotic resistance profile of Escherichia coli clinical isolates is demonstrated.

Figure 1

(a–b) The electrode design for AC electrokinetics enhanced electrochemical biosensing. R, W and A represent reference, working and auxiliary electrodes, respectively. (c) The detection strategy for the electrochemical assay for bacterial 16S rRNA. The gold electrode is pre-coated with an alkanethoil self-assembled monolayer. The capture probe for hybridizing to the target molecular is immobilized on the electrode surface by biotin-streptavidin interaction. The target is also hybridized to a detector probe for signal generation.

Figure 2

(a) A schematic diagram of the AC electrokinetics enhanced hybridization process. (b) Visualization of the fluid motion by particle tracing. Tracer particles were seeded in hybridization buffer and a 200 kHz square wave with 5 volts peak to peak was applied. (c) 3D representation of electrothermal fluid motion in the sensor well.

Figure 3

The dependence of the electrode thickness on the temperature distribution in the sensor well. The experiments were performed in buffer solution containing 2.5% BSA and 1M phosphate. Data were collected using an infrared camera after the temperature reached equilibrium. A 200 kHz square wave with 6 volts peak to peak was applied.

Figure 4

(a) A schematic diagram of the electric field distribution in the sensor well. Heating are concentrated near the connection region of the work and auxiliary electrodes (red color). (b) The maximum temperature in the sensor well as a function of the electrode thickness. The experiments were performed using Tris buffer with 2.5% BSA. A 200 kHz square wave with 6 volts peak to peak was applied.

Figure 5

Antimicrobial susceptibility testing in blood culture and MH broth. EC132 with a starting concentration of 5 × 10⁶ CFU/ml were cultured in (a) blood-MH broth for 5 hours (1:1) and (b) MH broth for 3 hours. The antibiotic resistance profile was determined. X-axis represent different growth conditions: Control = no antibiotics; AMP = ampicillin; CIP = ciprofloxacin; and SXT (trimethoprim/sulfamethoxazole).

Figure 6

The signal-to-noise (S/N) ratios of the electrochemical assay with electrokinetic enhancement and by diffusion during the hybridization step. EC132 samples with different starting concentrations were cultured in blood-MH broth (1:9) for 3 hours.

Figure 7

The signal-to-noise (S/N) ratios of the electrochemical assay with electrokinetic enhancement and by diffusion during the hybridization step. EC132 with the same starting concentration of 1 × 10⁴ CFU/ml were cultured in blood-MH broth (1:9) for 3 hours with or without antibiotics (CIP and SXT).