3× multiplexed detection of antibiotic resistant plasmids with single molecule sensitivity

Abstract

Bacterial pathogens resistant to antibiotics have become a serious health threat. Those species which have developed resistance against multiple drugs such as the carbapenems, are more lethal as these are last line therapy antibiotics. Current diagnostic tests for these resistance traits are based on singleplex target amplification techniques which can be time consuming and prone to errors. Here, we demonstrate a chip based optofluidic system with single molecule sensitivity for amplification-free, multiplexed detection of plasmids with genes corresponding to antibiotic resistance, within one hour. Rotating disks and microfluidic chips with functionalized polymer monoliths provided the upstream sample preparation steps to selectively extract these plasmids from blood spiked with E. coli DH5α cells. Waveguide-based spatial multiplexing using a multi-mode interference waveguide on an optofluidic chip was used for parallel detection of three different carbapenem resistance genes. These results point the way towards rapid, amplification-free, multiplex analysis of antibiotic-resistant pathogens.

Figure 1

Cartoon depicting the process flow of the entire analysis system, (a) Sample: Whole human blood spiked with E. coli cells having antibiotic resistant plasmids. (b) Sample separation hollow disk device which separates bacterial cells from WBC and RBC. (c) Microfluidic chip which elutes selectively captured antibiotic resistant plasmids using a functionalized polymer monolith column. (d) ARROW bio-sensor chip detects the three antibiotic resistant plasmids (KPC, VIM, NDM) simultaneously using an MMI waveguide with single molecule sensitivity.

Figure 2

(a) Top down image of the microfluidic chip used for target specific elution of the antibiotic resistant plasmids (scale: 1 cm). The chip has a serpentine channel in the bottom where a heater denatures the dsDNA. The white shaded part is the polymer monolith column functionalized with target capture probe oligos. (b) SEM image of the cross section of the microfluidic channel with the monolith (scale: 500 μm). (c) SEM image with increased magnification of the polymer monolith shows them to be porous with average nodule size of 1.2 ± 0.2 μm and pore size of 2.0 ± 0.4 μm (scale:5 μm).

Figure 3

(a) Cartoon of ARROW optofluidic chip system for 3X multiplexed detection. Fluorescently labelled targets are introduced in the three LC waveguides. The signals generated by targets from each channel upon excitation by the MMI waveguide are collected simultaneously by a 3x1 Y coupler. (b) Top down image of an actual chip (Scale: 1 mm). (c) Fluorescent image of the MMI patterns in the dye filled LC channels (Ch1 to Ch3: bottom to top) when excited by the MMI waveguide with a 556 nm laser.

Figure 4

(a.i) Fluorescence trace for single plex detection of individual KPC. Fluorescently labelled KPC plasmids are flowed in Ch1 and buffer with dye in Ch2 and Ch3. The MMI waveguide is excited by 556 nm laser ((b) and (c) are signals of NDM and VIM plasmids from similar experiments). (a.ii) Zoomed in signal of a KPC plasmid shows an event with 11 peaks corresponding to the MMI pattern in Ch1. (a.iii) Histogram map of normalized S(t)j of a correctly identified KPC signal.

Figure 5

(a) Fluorescence trace with all three antibiotic resistant plasmids detected simultaneously when the MMI is excited. KPC, NDM and VIM plasmids were flowed in the chip simultaneously in Ch1, Ch2 and Ch3 respectively. Targets were correctly identified by decoding the signals using eq 2. (b) Example of an event analyzed with S(t) clearly identifying the signal to be from channel 1.