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Microfluidic Array Chip for Parallel Detection of Waterborne Bacteria

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Microfluidic Array Chip for Parallel Detection of Waterborne Bacteria

Lena Gorgannezhad et al. Micromachines (Basel).

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.

Keywords: array; bacterial nucleic acids; microbial faecal source tracking (MST); microfluidic; polymerase chain reaction (PCR).

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Microfluidic polymerase chain reaction (PCR) chip: (A) three layers of the device; (B) fabricated PCR chip.
Figure 2
Figure 2
Liquid sample loading and isolation of the reactors. (A) Test with food colouring; (B) test with fluorescent dye.
Figure 3
Figure 3
Cross-sectional views of chip operation.
Figure 4
Figure 4
Experimental setup of the PCR thermal cycling platform.
Figure 5
Figure 5
Amplification plot for on-chip detection of Bacteroidales and Escherichia coli (E. coli). Positive controls (PC): Bulk of standard templates of DNA containing GenbacIII (71,800 copies/µL), H8 (52,300 copies/µL), and UidA (13600 copies/µL) sequences. Negative controls (NTC): NTC1 (template DNA from Staphylococcus aureus), NTC2 (no template DNA; water).
Figure 6
Figure 6
(A) Amplification plot for 10-fold serially diluted standard template DNA (Gen bac III sequence); NTC1 (template DNA from Staphylococcus aureus). (B) Standard curve for 10-fold serially diluted standard template DNA.
Figure 7
Figure 7
Gel image of on-chip PCR products. (A) Illustration of GenbacIII, H8, UidA, and negative controls (NTC1, NTC2). (B) Image of serial dilution of GenbacIII sequence.

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