Microfluidic Array Chip for Parallel Detection of Waterborne Bacteria.

Lena Gorgannezhad,Gregor Kijanka,Helen Stratton,Jun Zhang,Nam-Trung Nguyen,Kamalalayam Rajan Sreejith,Melody Christie

doi:10.3390/mi10120883

Lena Gorgannezhad, Gregor Kijanka + Show 5 more

Open Access

https://doi.org/10.3390/mi10120883

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Abstract

The polymerase chain reaction (PCR) is a robust technique used to make multiple copies of a segment of DNA. However, the available PCR platforms require elaborate and time-consuming operations or costly instruments, hindering their application. Herein, we introduce a sandwiched glass–polydimethylsiloxane (PDMS)–glass microchip containing an array of reactors for the real-time PCR-based detection of multiple waterborne bacteria. The PCR solution was loaded into the array of reactors in a single step utilising capillary filling, eliminating the need for pumps, valves, and liquid handling instruments. Issues of generating and trapping bubbles during the loading chip step were addressed by creating smooth internal reactor surfaces. Triton X-100 was used to enhance PCR compatibility in the chip by minimising the nonspecific adsorption of enzymes. A custom-made real-time PCR instrument was also fabricated to provide thermal cycling to the array chip. The microfluidic device was successfully demonstrated for microbial faecal source tracking (MST) in water.

Highlights

The faecal pollution of water is one of the major threats to human health
The two bacteria of Escherichia coli (E. coli) and Bacteroidales were selected to demonstrate the function of the chip
E. coli and Bacteroidales are gram-negative bacteria flourished in the intestine with properties that both positively and negatively affect the host [23]

Summary

Introduction

The faecal pollution of water is one of the major threats to human health. Understanding the origin of faecal pollution is necessary for identifying public health risks, devising effective management, and preventing further pollution [1]. Multiplex PCRs requires accurate primer designs and an optimal reaction mixture to avoid the probability of primer dimer formation that may result in the preferential amplification of certain targets [10]. Microarrays are another alternative method for analysing the pathogen content in wastewater. Recent advances in microfluidics have enabled the development of new cost-effective array devices for the multiplex detection of contaminants. Most PCR-based microfluidic devices need to load the PCR mixture into reaction wells using costly liquid-dispensing robots or pneumatic pressure sources, or the immobilization of primers in a solid matrix [15,16,17]. The capability of the array chip was verified by the simultaneous detection of three human-associated MST markers: Escherichia coli (E.coli) (H8), human-specific bacteroidals (Gen bac III), E.coli (UidA)

Chip Design

Chip Fabrication

Bacterial Samples and PCR Experiment

Results

Validation of Real-Time PCR Using Gel Electrophoresis