A Salmonella Microfluidic Chip Combining Non-Contact Eddy Heater and 3D Fan-Shaped Mixer with Recombinase Aided Amplification

Abstract

Foodborne pathogenic bacteria have become a worldwide threat to human health, and rapid and sensitive bacterial detection methods are urgently needed. In this study, a facile microfluidic chip was developed and combined with recombinase-aided amplification (RAA) for rapid and sensitive detection of Salmonella typhimurium using a non-contact eddy heater for dynamic lysis of bacterial cells and a 3D-printed fan-shaped active mixer for continuous-flow mixing. First, the bacterial sample was injected into the chip to flow through the spiral channel coiling around an iron rod under an alternating electromagnetic field, resulting in the dynamic lysis of bacterial cells by this non-contact eddy heater to release their nucleic acids. After cooling to ~75 °C, these nucleic acids were continuous-flow mixed with magnetic silica beads using the fan-shaped mixer and captured in the separation chamber using a magnet. Finally, the captured nucleic acids were eluted by the eluent from the beads to flow into the detection chamber, followed by RAA detection of nucleic acids to determine the bacterial amount. Under the optimal conditions, this microfluidic chip was able to quantitatively detect Salmonella typhimurium from 1.1 × 10² to 1.1 × 10⁵ CFU/mL in 40 min with a detection limit of 89 CFU/mL and might be prospective to offer a simple, low-cost, fast and specific bacterial detection technique for ensuring food safety.

Keywords: 3D fan-shaped mixer; Salmonella detection; eddy heating; microfluidic chip; recombinase-aided amplification.

Figure 1

The schematic of this microfluidic chip for Salmonella typhimurium detection. (A) The procedure of bacterial detection. (B) The structure of the microfluidic chip. (C) The components of the microfluidic chip: ① the spiral lysis channel, ② the ellipsoidal cooling chamber, ③ the fan-shaped mixer, ④ the trapezoid separation chamber, ⑤ the magnet. (D) The photo of the microfluidic chip.

Figure 2

The eddy heating for bacterial cells lysis. (A) The photo of the eddy heater: ① the iron rod, ② the spiral channel, ③ and the alternating electromagnetic field generator. (B) The simulation on the temperature of deionized water in the spiral channel (unit: K). (C) The Ct values of Salmonella cells at 1.1 × 10⁶ CFU/mL for different flow rates (N = 3). (D) The Ct values of Salmonella cells at 1.1 × 10⁷ CFU/mL for different lysis time (N = 3). (E) The Ct values for different concentrations of Salmonella cells at the flow rate of 0.5 mL/min (N = 3). * indicated significance difference, p < 0.05.

Figure 3

The mixing performance of the 3D-shaped mixer. (A) The structure of this 3D-printed fan-shaped mixer: (a) The photo of this mixer; (b) The three-way connector. (B) The simulation results of the fan-shaped mixer. (C) The mixing rate of the chambers with and without the mixer at different flow rates (N = 3). (D) The DNA capture efficiency for different mixing time at 10 mL/min (N = 3).

Figure 4

Performance of this microfluidic chip. (A) The linear relationship between the threshold time and the bacterial concentration (N = 3). (B) The specificity of this microfluidic chip.