Flow-Through Electrochemical Biosensor for the Detection of Listeria monocytogenes Using Oligonucleotides

Abstract

Consumption of food contaminated by Listeria monocytogenes can result in Listeriosis, an illness with hospitalization rates of 94% and mortality rates up to 30%. As a result, U.S. regulatory agencies governing food safety retain zero-tolerance policies for L. monocytogenes. However, detection at such low concentrations often requires strategies such as increasing sample size or culture enrichment. A novel flow-through immunoelectrochemical biosensor has been developed for Escherichia coli O157:H7 detection in 1 L volumes without enrichment. The current work further augments this biosensor's capabilities to (1) include detection of L. monocytogenes and (2) accommodate genetic detection to help overcome limitations based upon antibody availability and address specificity errors in phenotypic assays. Herein, the conjugation scheme for oligo attachment and the conditions necessary for genetic detection are laid forth while results of the present study demonstrate the sensor's ability to distinguish L. monocytogenes DNA from L. innocua with a limit of detection of ~2 × 10⁴ cells/mL, which agrees with prior studies. Total time for this assay can be constrained to <2.5 h because a timely culture enrichment period is not necessary. Furthermore, the electrochemical detection assay can be performed with hand-held electronics, allowing this platform to be adopted for near-line monitoring systems.

Keywords: Listeria innocua; Listeria monocytogenes; biosensor; flow-through transducer; foodborne pathogen; graphite felt; rapid detection.

Figure 1

Design of the enzyme-amplified electrochemical biosensor using oligos. (A–C) Schematic of the different GFE surfaces utilized, with signal generation ultimately relying upon the presence of the HRP-conjugated oligo to facilitate oxidation of the TMB substrate. (A) The GFE surface utilized to assess blocking performance. Negative sample preparations assessed aspecific oligo adsorption and positive sample preparations defined the maximum signal intensity. (B) The GFE surface utilized for sensor development. The capture oligo binds directly to the detection oligo since it is complementary in sequence. (C) The GFE surface utilized for the detection of L. monocytogenes. The 16S rDNA sequence from L. monocytogenes serves as a bridge binding both the capture oligo and the detection oligo. (D) A nucleotide alignment of the 16S rDNA region from L. ivanovii (Iv), L. innocua (In), and L. monocytogenes (Mo). Nucleotides that differed amongst the species are denoted by bold red letters that are underlined. Oligos utilized and their modifications are also shown. The * denotes modification with HRP whereas the${<}_B^B$ denotes a dual biotin modification.

Figure 2

Effects of blocking agents on assay signal. Blocking agents tested are presented as categorical variables along the x-axis while signal-to-noise ratios for both the electrical currents generated using the flow through electrochemical sensor (top) and the corresponding absorbance readings at 450 (bottom) are presented on the y-axis. The mean of three independent trials is plotted with the error bars representing the standard deviation and statistical differences amongst samples noted by the connecting letters report. Levels not connected by the same letter are significantly different.

Figure 3

Detection limits for the flow-through, enzyme-amplified electrochemical sensor using oligos as biorecognition elements. The normalized current response was measured for a constant amount of F-2 Link (Biotin) oligo conjugated to the surface of the GFE post exposure to various concentrations (10⁻¹³–10⁻¹⁸ M) of the complementary oligo, L-2 (HRP). The mean of three independent trials is plotted with the standard deviations and statistical differences amongst samples noted by the connecting letters report. Levels not connected by the same letter are significantly different.

Figure 4

Specificity of the sensor for L. monocytogenes. Upon exposure of the sensor to a solution containing fragments of 10⁻⁷ M 16S rDNA from either L. innocua (In) or L. monocytogenes (Mo), the resulting normalized responses were determined using both electrochemistry (dark gray bars) and absorbance (light gray bars). Exposure of the sensor to buffer only (no DNA) and the direct application of the detection oligo were used as controls. The mean response of three independent trials is plotted with the standard deviations and statistical differences amongst samples noted by the connecting letters report. Levels not connected by the same letter are significantly different.

Figure 5

Detection of L. monocytogenes from live cells. The normalized response was determined using both electrochemistry (dark gray bars) and absorbance (light gray bars) upon exposure of the sensor to a series of lysed L. monocytogenes cells in a 5 mL sample volume. Exposure of the sensor to buffer only (no cells), sensors that did not contain capture oligo (negative control), and the positive control were also performed. The mean response from three independent trials is plotted with the standard deviations and statistical differences amongst samples noted by the connecting letters report. Levels not connected by the same letter are significantly different.