A Microfluidic Biosensor for Quantitative Detection of Salmonella in Traditional Chinese Medicine

Abstract

Microbial contamination is an important factor threatening the safety of Chinese medicine preparations, and microfluidic detection methods have demonstrated excellent advantages in the application of rapid bacterial detection. In our study, a novel optical biosensor was developed for the rapid and sensitive detection of Salmonella in traditional Chinese medicine on a microfluidic chip. Immune gold@platinum nanocatalysts (Au@PtNCs) were utilized for specific bacterial labeling, while magnetic nano-beads (MNBs) with a novel high-gradient magnetic field were employed for the specific capture of bacteria. The immune MNBs, immune Au@PtNCs, and bacterial samples were introduced into a novel passive microfluidic micromixer for full mixing, resulting in the formation of a double-antibody sandwich structure due to antigen-antibody immune reactions. Subsequently, the mixture flowed into the reaction cell, where the MNBs-Salmonella-Au@PtNCs complex was captured by the magnetic field. After washing, hydrogen peroxide-tetramethylbenzidine substrate (H₂O₂-TMB) was added, reacting with the Au@PtNCs peroxidase to produce a blue reaction product. This entire process was automated using a portable device, and Salmonella concentration was analyzed via a phone application. This simple biosensor has good specificity with a detection range of 9 × 10¹-9 × 10⁵ CFU/mL and can detect Salmonella concentrations as low as 90 CFU/mL within 74 min. The average recoveries of the spiked samples ranged from 76.8% to 109.5.

Keywords: biosensor; microbial contamination; microfluidics; nanocatalysts; traditional Chinese medicine.

Scheme 1

Microfluidic biosensor for quantitative detection of Salmonella in traditional Chinese medicine.

Figure 1

Schematic of the microfluidic biosensor for Salmonella detection. (A) The principle and the whole procedure of the biosensor. (B) Structure of the microfluidic chip. (C) The portable device and the smartphone app. (D) Color change of the catalysate for different amounts of Au@PtNCs.

Figure 2

Characterization of materials. (A) DLS images of Au@PtNCs. (B) XRD pattern of Au@PtNCs. (C) EDS elemental mapping of Au@PtNCs. (D) TEM images of AuNPs and Au@PtNCs.

Figure 3

Validation of the performance of microfluidic chip. (A) Comparison round chambers with diamond-shaped chambers. (B) Comparison of flow rate effects (N = 3). (C) Simulation of the micro-mixing zone. (D) Simulated concentration schematic of the concentration in the micro-mixing zone. (E) Ink mixing experiment.

Figure 4

Performance of magnetic field. (A) The structure of the magnetic field. (B) Photo of the magnetic field. (C) Magnetic induction line of the magnetic field. (D) The strength of the magnetic field and the location of the reaction chamber. (E) Comparison of the magnetic field density of this magnetic field with that of a circular magnet. (F) Magnetic recovery results for this magnetic field (N = 3).

Figure 5

Optimization of microfluidic biosensor (N = 3). (A) Optimization of the amount of immune MNBs. (B) Optimization of the amount of immune Au@PtNCs. (C) Optimization of the incubation time. (D) Optimization of the catalytic time.

Figure 6

Performance of microfluidic biosensor. (A) TEM image of MNBs-Salmonella-Au@PtNCs complex. (B) Saturation for Salmonella at different concentrations (N = 3). (C) Calibration curve of the biosensor (N = 3). (D) Specificity of the biosensor. (E) Pre-treatment process of the Niu Huang Qing Xin Wan (N = 3). (F) Recovery rate of spiked Salmonella typhimurium samples (N = 3).