A Microfluidic Biosensor Based on Magnetic Nanoparticle Separation, Quantum Dots Labeling and MnO2 Nanoflower Amplification for Rapid and Sensitive Detection of Salmonella Typhimurium.

Li Hao,Zengli Wang,Miao Zhang,Fengchun Huang,Jianhan Lin,Li Xue,Gaozhe Cai,Qing'An Han,Wuzhen Qi

doi:10.3390/mi11030281

Abstract

Screening of foodborne pathogens is an effective way to prevent microbial food poisoning. A microfluidic biosensor was developed for rapid and sensitive detection of Salmonella Typhimurium using quantum dots (QDs) as fluorescent probes for sensor readout and manganese dioxide nanoflowers (MnO2 NFs) and as QDs nanocarriers for signal amplification. Prior to testing, amino-modified MnO2 nanoflowers (MnO2-NH2 NFs) were conjugated with carboxyl-modified QDs through EDC/NHSS method to form MnO2-QD NFs, and MnO2-QD NFs were functionalized with polyclonal antibodies (pAbs) to form MnO2-QD-pAb NFs. First, the mixture of target Salmonella Typhimurium cells and magnetic nanoparticles (MNPs) modified with monoclonal antibodies (mAbs) was injected with MnO2-QD-pAb NFs into a microfluidic chip to form MNP-bacteria-QD-MnO2 complexes. Then, glutathione (GSH) was injected to dissolve MnO2 on the complexes into Mn2+, resulting in the release of QDs. Finally, fluorescent intensity of the released QDs was measured using the fluorescent detector to determine the amount of Salmonella. A linear relationship between fluorescent intensity and bacterial concentration from 1.0 × 102 to 1.0 × 107 CFU/mL was found with a low detection limit of 43 CFU/mL and mean recovery of 99.7% for Salmonella in spiked chicken meats, indicating the feasibility of this biosensor for practical applications.

Highlights

Food safety has become one of the most important issues in the world
Foodborne pathogenic bacteria are the main cause of foodborne illnesses, including Escherichia coli O157:H7, Listeria monocytogenes, and Salmonella etc
After 100 μL of GSH (20 mM) was injected to the chamber through inlet 3 and incubated for 15 min to release the quantum dots (QDs) from the MnO2 NFs, the released QDs were flushed out and measured by the optical detector to determine the amount of target bacteria

Summary

Introduction

Food safety has become one of the most important issues in the world. Foodborne pathogenic bacteria are the main cause of foodborne illnesses, including Escherichia coli O157:H7, Listeria monocytogenes, and Salmonella etc. Available methods for detecting these bacteria include culture plating, enzyme-linked immunosorbent assay (ELISA), and polymerase chain reaction (PCR), etc. They either require a long time, need professional operation, or lack sensitivity. Simple, fast and sensitive methods for pathogenic bacteria detection are needed to ensure food safety. The design of the microfluidic chip with the convergence-divergence mixing channel was inspired by a pTrheveioduessirgenpoortf [t3h7e]. The prepolymer of PDMS and the curing agent were mixed at the ratio of 10:1 and poured into the mold, followed by curing at 65 ◦C for 12 h after degassing for 15 min in vacuum to remove bubbles. The PDMS replica was peeled off and bonded onto the glass slide to form the microfluidic chip after surface plasmon treatment (Harrick Plasma, Ithaca, NY, USA) and baked at 65 ◦C for aging to obtain the microfluidic chip

Synthesis of the MnO2-QD-pAb NFs

Detection of the Target Bacteria in Pure Cultures

Detection of the Target Bacteria in Spiked Samples

Simulation of the Microfluidic Chip

Calibration Model of this Biosensor

Conclusions