Identification of Microorganisms Using an EWOD System

Abstract

Among the advantages of an electrowetting-on-dielectric (EWOD) chip are its uncomplicated fabrication and low cost; one of its greatest strengths that might be applied in the field of biomedical technology is that it can accurately control volume and reduces the amount of samples and reagents. We present an EWOD for the biochemical identification of microorganisms, which is required to confirm the source of microbial contamination or quality inspection of product-added bacteria, etc. The traditional kit we used existed in the market; the detection results are judged by the pattern of color change after incubation. After a preliminary study, we confirmed that an image-processing tool (ImageJ) provides a suitable method of analysis, and that, when the concentration of the sugar reagent is 38 µg/µL, the best operating parameters for the EWOD chip in silicone oil are 40 V and 1.5 kHz. Additionally, we completed the biochemical identification of five bacterial species on the EWOD chip at the required concentration of the kit. Next, we found a decreased duration of reaction and that the least number of bacteria that were identifiable on the chip lies between 100 and 1000 CFU per droplet. Because the number of bacteria required on the chip is much smaller than for the kit, we tested whether a single colony can be used for identification, which provided a positive result. Finally, we designed an experimental flow to simulate an actual sample in an unclean environment, in which we divided the various processed samples into four groups to conduct experiments on the chip.

Keywords: digital microfluidics system; electrowetting on dielectric system; microorganism identification.

Figure 1

Advantage of integrating an EWOD with a commercial kit based on biochemical principles.

Figure 2

Design of an EWOD chip to detect two strains simultaneously. (a) schematic diagram of a parallel-plate EWOD chip; (b) path design for bacterial identification on the chip.

Figure 2

Design of an EWOD chip to detect two strains simultaneously. (a) schematic diagram of a parallel-plate EWOD chip; (b) path design for bacterial identification on the chip.

Figure 3

Image of EWOD chip under experimental stage.

Figure 4

Results of cultivating selected Enterobacteriaceae at concentration 10⁵ CFU/µL. (a) Comparing E. coli images before and after culturing. (b–f) Changes of hue after 18 h indicate that different bacteria use different sugars. (Red dotted line indicated the threshold line at Hue value equal to 60).

Figure 5

Advantages of combining API with an EWOD technique. (a) Reaction duration required on the kit and the chip. (b,c) Data of number of E. coli for identification. Dotted line indicates that the Hue value of these three mixtures, which supposed be positive reaction, did not approach 60. (d) Number of bacteria in a single colony. (e) Results of E. coli detection with single colony.

Figure 6

Results of two isolated colonies, (a) Colony-A and (b) Colony-B, in the first group with experiments on chips. The solid triangle symbol indicates that this unknown strain does not use the corresponding sugar in large quantities, so the hue would not change to 60. (Red dotted line indicated the threshold line at Hue value equal to 60).

Figure 7

Results of five isolated colonies in the second group, (a) Analyte-1, (b) Analyte-2 and Analyte-5, and (c) Analyte-3 and Analyte-4, which was in contact with filth, from the experiments on the chips. (Hollow circles means Negative; filled triangles means Positive; and Red dotted line indicated the threshold line at Hue value equal to 60).

Figure 8

Illustration of the duration difference between doing the bacterial detection in a traditional kit and on the chip.