Ultrasensitive Label-Free Detection of Unamplified Multidrug-Resistance Bacteria Genes with a Bimodal Waveguide Interferometric Biosensor

Abstract

Infections by multidrug-resistant bacteria are becoming a major healthcare emergence with millions of reported cases every year and an increasing incidence of deaths. An advanced diagnostic platform able to directly detect and identify antimicrobial resistance in a faster way than conventional techniques could help in the adoption of early and accurate therapeutic interventions, limiting the actual negative impact on patient outcomes. With this objective, we have developed a new biosensor methodology using an ultrasensitive nanophotonic bimodal waveguide interferometer (BiMW), which allows a rapid and direct detection, without amplification, of two prevalent and clinically relevant Gram-negative antimicrobial resistance encoding sequences: the extended-spectrum betalactamase-encoding gene blaCTX-M-15 and the carbapenemase-encoding gene blaNDM-5 We demonstrate the extreme sensitivity and specificity of our biosensor methodology for the detection of both gene sequences. Our results show that the BiMW biosensor can be employed as an ultrasensitive (attomolar level) and specific diagnostic tool for rapidly (less than 30 min) identifying drug resistance. The BiMW nanobiosensor holds great promise as a powerful tool for the control and management of healthcare-associated infections by multidrug-resistant bacteria.

Keywords: CTX-M; Escherichia coli; NDM; bimodal waveguide interferometer; multidrug-resistance; nanophotonic biosensor.

Figure 1

Picture and scheme of a BiMW chip containing 20 sensors, and scheme of the expected interferometric readout.

Figure 2

(A) Scheme of the biosensor strategy followed for the genes detection. This included DNA extraction, fragmentation, and denaturation to obtain single-stranded fragments, which were then analyzed directly with the BiMW biosensor. (B) Biofunctionalisation of the BiMW sensor surface with amine-modified capture probes.

Figure 3

Evaluation of synthetic DNA target. (A) Real-time interferometric signal obtained for the hybridization of the synthetic target (88 mer) to blaCTX-M-15 probe; (B) Real-time interferometric signals obtained for the binding of 10 nM of target (black line) and negative control (blue line) to the blaCTX-M-15; (C) Comparison of the signal of synthetic blaCTX-M-15 DNA target (10 nM) versus synthetic control target (10 nM). All measurements were done in triplicate, and the data are shown as means + SD.

Figure 4

Detection of blaCTX-M-15 gene by the BiMW biosensor. (A) Schematic representation to detect both DNA strands using forward and reverse probes. (B) Real-time detection of blaCTX-M-15 gene at 4.5 fM (black line), non-specific Pseudomonas aeruginosa at 3.4 fM (blue line), and Escherichia coli without blaCTX-M-15 gene at 2.9 fM (red line). (C) Real-time interferometric signals of blaCTX-M-15 at several concentrations (0.45–4.5 fM). (D) Calibration curve obtained for the range of concentrations analyzed. The curve was fitted to a linear regression (y = a + bx, R² = 0.994). All the measurements were performed in triplicate, and the data are shown as the mean + SD.

Figure 5

Detection of blaNDM-5 gene with two BiMW biosensors (BiMW1 and BiMW2). (A,D) Real-time interferometric signals for different concentrations of blaNDM-5 gene (0.6–3 fM) analyzed over a sensor chip with the blaNDM-5 probe immobilized. (B,E) Comparison of triplicate measurements of blaNDM-5 DNA target (3 fM) versus P. aeruginosa DNA (3.4 fM). (C,F) Calibration curves obtained for the range of concentrations analyzed. All the measurements were performed in triplicate, and the data are shown as mean + SD.