Design and 3D printing of an electrochemical sensor for Listeria monocytogenes detection based on loop mediated isothermal amplification

Abstract

The aim of this work is the design and 3D printing of a new electrochemical sensor for the detection of Listeria monocytogenes based on loop mediated isothermal amplification (LAMP). The food related diseases involve a serious health issue all over the world. Listeria monocytogenes is one of the major problems of contaminated food, this pathogen causes a disease called listeriosis with a high rate of hospitalization and mortality. Having a fast, sensitive and specific detection method for food quality control is a must in the food industry to avoid the presence of this pathogen in the food chain (raw materials, facilities and products). A point-of-care biosensor based in LAMP and electrochemical detection is one of the best options to detect the bacteria on site and in a very short period of time. With the numerical analysis of different geometries and flow rates during sample injection in order to avoid bubbles, an optimized design of the microfluidic biosensor chamber was selected for 3D-printing and experimental analysis. For the electrochemical detection, a novel custom gold concentric-3-electrode consisting in a working electrode, reference electrode and a counter electrode was designed and placed in the bottom of the chamber. The LAMP reaction was optimized specifically for a primers set with a limit of detection of 1.25 pg of genomic DNA per reaction and 100% specific for detecting all 12 Listeria monocytogenes serotypes and no other Listeria species or food-related bacteria. The methylene blue redox-active molecule was tested as the electrochemical transducer and shown to be compatible with the LAMP reaction and very clearly distinguished negative from positive food samples when the reaction is measured at the end-point inside the biosensor.

Keywords: 3D printing; Biosensor; Electrochemical detection; Listeria monocytogenes; Loop-mediated isothermal AMPlification (LAMP); Numerical analysis.

Graphical abstract

Figure 1

Preliminary design of the microdevice.

Figure 2

Gold electrodes unit fabricated by photolithography (a) on a 4″ silicon dioxide wafer (b). Each electrode unit comprises of a working electrode, a counter and a reference electrode.

Figure 3

Analysis and comparison of the thermophysical properties of samples M1 and M2: density (a), thermal expansion coefficient (b), viscosity (c), thermal conductivity (d), and refractive index (e–f) variations.

Figure 4

Designed electrochemical sensors geometries for numerical simulations with (a) and without (b) 90° corners.

Figure 5

Numerical results of the microdevice filling process analyzing the effect of the designed geometries (a) and the used injection flow rates (b).

Figure 6

Temperature distribution over the reaction chamber together with different graphs representing the variation along the height and length of the electrochemical sensor.

Figure 7

Experimental set up (a) used to test the fabricate devices: 1) Infusion pump to fill the reaction chamber, 2) heater and 3) printed chip. An example of filled sensor without bubbles and leakages (b).

Figure 8

Specificity results for the LM19v2 primer set. a) Colorimetric results; L. monocytogenes serotypes in green (Table 1: 1–12) and other Listeria species (Table 1: 13–19) and the negative control in orange (CN). b) Fluorescence emission results for L. monocytogenes serotypes represented in blue and other listeria in orange (same samples of image a), c) Colorimetric results; L. monocytogenes in green (CP) and exclusive species (Table 1: 20–27) and the negative control in orange (CN). d) Fluorescence emission results for L. monocytogenes serotypes represented in blue and other Listeria species in orange (same samples of image c); Time To Detection (TTD) represented at the bottom (min) and RFU (Relative Fluorescens Units) on the left side.

Figure 9

Detection limit results obtained for L. monocytogenes CECT 935 (serotype 4b) a) Colorimetric results; different amounts of L. monocytogenes genomic DNA, positive samples in green and negative samples in orange. NC: Negative control. b) Real-time monitorization of the fluorescence, TTD represented at the bottom (min) and RFU (Relative Fluorescens Units) side emitted by 25pg in blue, 2.5pg in light blue and 1.25pg in violet of L. monocytogenes DNA, in the left. c) Correlation between DNA amount represented at the bottom (pg) and TTD on the left side (min).

Figure 10

Validation of the assay for the electrochemical detection of L. monocytogenes in the off-chip configuration (a). Validation of the assay for the electrochemical detection of L. monocytogenes on-chip configuration. Voltammograms and peak currents for the positive and negative samples (b). Food sample validation with the on-chip configuration, dairy milk results in blue, fresh cheese in orange and smoked sample in grey. Current peak represented on the left and L.monocytogenes cfu/25 g of food in the bottom (c).