Planar Interdigitated Aptasensor for Flow-Through Detection of Listeria spp. in Hydroponic Lettuce Growth Media

Abstract

Irrigation water is a primary source of fresh produce contamination by bacteria during the preharvest, particularly in hydroponic systems where the control of pests and pathogens is a major challenge. In this work, we demonstrate the development of a Listeria biosensor using platinum interdigitated microelectrodes (Pt-IME). The sensor is incorporated into a particle/sediment trap for the real-time analysis of irrigation water in a hydroponic lettuce system. We demonstrate the application of this system using a smartphone-based potentiostat for rapid on-site analysis of water quality. A detailed characterization of the electrochemical behavior was conducted in the presence/absence of DNA and Listeria spp., which was followed by calibration in various solutions with and without flow. In flow conditions (100 mL samples), the aptasensor had a sensitivity of 3.37 ± 0.21 k log-CFU^-1 mL, and the LOD was 48 ± 12 CFU mL^-1 with a linear range of 10² to 10⁴ CFU mL^-1. In stagnant solution with no flow, the aptasensor performance was significantly improved in buffer, vegetable broth, and hydroponic media. Sensor hysteresis ranged from 2 to 16% after rinsing in a strong basic solution (direct reuse) and was insignificant after removing the aptamer via washing in Piranha solution (reuse after adsorption with fresh aptamer). This is the first demonstration of an aptasensor used to monitor microbial water quality for hydroponic lettuce in real time using a smartphone-based acquisition system for volumes that conform with the regulatory standards. The aptasensor demonstrated a recovery of 90% and may be reused a limited number of times with minor washing steps.

Keywords: SNAPS; electrochemical sensing; food safety; foodborne pathogen; fresh produce; interdigitated electrodes; sensor analytic point solution.

Figure 1

Microfabrication process for platinum interdigitated microelectrodes (Pt-IME) on SiO₂ wafers. (A) Photoresist deposition, (B) UV exposure with IME mask, (C) Development of resist, (D) Ti/Pt deposition by chemical vapor deposition (CVD), and (E) Pattern lift off. (F) Photographs of Pt-IME array with eight electrodes.

Figure 2

Electrochemical characterization of Pt-IME for gap spacing of 50 μm. (A) Representative cyclic voltammograms in 4 mM K₄Fe(CN)₆ + 1 M KNO₃ at room temperature. (B) Randles–Sevcik plots for oxidative and reductive peak current indicate diffusion-limited transport to the Pt-IME. (C) Nicholson plots for the determination of heterogenous electron transfer (HET) constant (k⁰). Full analysis of all Pt-IME electrode gap spacing (with replicates) can be found in the supplemental section.

Figure 3

Adsorption of 47-mer on Pt-IME (50 µm gap spacing). (A) Representative cyclic voltammetry (CV) at different aptamer concentrations shows an increase in oxidative current after aptamer adsorption in 4 mM K₄Fe(CN)₆ + 1 M KNO₃ (pH = 7.1) at room temperature. (B) Average peak oxidative current from CV. (C) Representative Bode plots at different aptamer concentrations shows an increase in impedance after aptamer adsorption. (D) Average net impedance at a cutoff frequency of 1 Hz from Bode plots. (E) Representative Nyquist plots at different aptamer concentrations shows an increase in impedance after aptamer adsorption. (F) Average charge transfer resistance from Nyquist plots. All error bars represent standard deviation of the arithmetic mean (n = 6 replicate electrodes). Exponential models were based on Langmuir kinetics, Frenudlich kinetics, or log normal modeling, a 4-parameter empirical model was developed for analyzing data from Bode and Nyquist plots.

Figure 4

(A) Representative Bode plot for L. innocua in phosphate buffer saline (PBS) using a laboratory potentiostat under controlled conditions (inset shows linear region between 1 to 1.2 Hz). (B) Calibration plot using change in impedance at a cutoff frequency of 1 Hz. (C) Representative Bode plot for L. monocytogenes in in vegetable broth using a laboratory potentiostat under controlled conditions (inset shows linear region between 1 and 1.2 Hz). (D) Calibration plot in vegetable broth using a change in impedance at a cutoff frequency of 1 Hz. (E) Representative Bode plot for L. innocua in PBS using a smartphone-based potentiostat in hydroponic media (inset shows linear region between 10 and 100 MHz). (F) Calibration plot using change in impedance at a cutoff frequency of 0.03 Hz. All error bars represent standard deviation of the arithmetic mean (n = 3).

Figure 5

Reusability of 47-mer aptasensor after washing with NaOH at 25 °C (pH = 9.4) and rinsed in PBS (pH = 7.1). The average hysteresis after NaOH washing was 2.1 ± 2.0% for up to three reuse cycles, and 15.6 ± 6.5% after five reuse cycles. Error bars represent standard deviation of the arithmetic mean.

Figure 6

Calibration of Pt-IME biosensor in hydroponic growth media for Listeria innocua in the particle flow trap. (A) Representative Nyquist plot for increasing concentrations of Listeria innocua in growth media. (B) Calibration plot using ΔR_ct (Ω) and logistic regression curve. Dashed lines show the 99% confidence interval for the logistic curve. Error bars represent standard deviation of the arithmetic mean (n = 3). (C) Schematic of flow-through system with aptasensor (red) in particle trap connected to a smartphone potentiostat. See Supplementary Figure S11 for photograph.