Identification for a Large-volume Food-borne Bacteria on a Fully

Integrated Portable Centrifugal Disc

Hau Van Nguyen, Van Dan Nguyen, Eun Yeol Lee and Tae Seok Seo

Department of Chemical Engineering, Kyung Hee University, Yongin 17104, South Korea

Keywords: Centrifugal Microdevice, Portable Genetic Analyser, Loop-mediated Isothermal Amplification, Colorimetric

Detection, Super Absorbent Polymer.

Abstract: Herein, we present a fully integrated portable centrifugal microsystem for multiplex detection of food

poisoning bacteria with a large volume of sample up to 1 mL. The microsystem consists of a portable rotary

genetic analyzer and a fully integrated lab-on-a-disc device. The portable rotary genetic analyzer is equipped

with a couple of heating blocks, a motor and a UV-Vis optical detector. The device was designed with two

units: a 3D printed solution-loading cartridge and a centrifugal microfluidic disc. All the essential solutions

for the LAMP reaction (a sample, a washing, an elution and a LAMP cocktail solution) are stored inside the

cartridge, and orderly released into centrifugal microdevice by a rotation program. Each unit of the device

was designed with 20 reaction chambers for simultaneously detecting 19 kinds of food poisoning bacteria in

one test. To increase the amount of a sample to 1 mL, we incorporated the super absorbent polymer (SAP) in

the waste chamber to absorb the sample and washing solution during the device operation. The whole process

was automatically conducted from bead-based DNA extraction to isothermal DNA amplification by EBT-

mediated LAMP reaction to colorimetric and UV-vis detection of amplicons in 60 min to identify three kinds

of bacteria (Escherichia coli O157:H7, Salmonella Typhimurium, and Vibrio parahaemolyticus).

1 INTRODUCTION

Point-of-care testing (POCT) is recently blooming up

and plays a vital role in supporting immediate

treatment. The central laboratories are equipped with

high cost and automatic diagnostic platform for

highly sensitive, precise and accurate testing.

However, due to the bulkiness of the analytical

instruments, they are not adequate for on-site

diagnostics. On the contrary, the POC testing allows

simple and rapid analysis, which can help the doctor

to make a timely decision on the treatment method for

the patients. Also, the POC testing offers a user-

friendly prototype suitable for the un-trained worker,

minimal operation steps to reduce analysis time,

minimizing sample storage and transportation, and

cost-effective treatment in resource-limited

environments. The POC testing system includes a

paper-based microfluidic device (PPM) (Choi, 2015;

Ye, 2018), a lateral flow assay (LFA) (Deng, 2018;

Takalkar, 2017), a microfluidic device (DuVall,

2017; Zhang, 2017), a miniaturized PCR platform

(Guarnaccia, 2017; Liu, 2017), a smartphone-based

device (Berg, 2015; Priye, 2016; Stedtfeld, 2012;

Wang, 2017). Among these POCT approaches, a

microfluidic device has attracted huge attention over

decades, and, in particular, a centrifugal microfluidic

was considered as a promising candidate for complex

diagnostic purposes. Centrifugal microfluidics have

demonstrated the high fidelity for the unit operation

and integration on a single device such as sample

loading and reagent storage (Stumpf, 2016; van

Oordt, 2013), serial dilution (Kim, 2018), metering,

aliquoting, mixing, and incubation (Jung, 2015; Oh,

2016; Park, 2017), and detection (Andreasen, 2015;

Martin, 2017; Schwemmer, 2016). However, the

major challenge in the centrifugal microdevice is the

low volume of sample pretreatment, which could

affect the limit-of-detection level. In this study, we

proposed a prototype of a fully integrated centrifugal

device for the POC testing, which is capable of

multiplex bacteria detection with a large volume of

the sample up to 1mL. developed for multiplex

bacteria detection in a large volume of the sample up

to 1mL. In addition, this sample-to-answer platform

can fulfill all the requirements for on-site nucleic acid

analysis since the DNA solid-phase extraction by

glass bead, isothermal amplification by LAMP

114

Van Nguyen, H., Nguyen, V., Lee, E. and Seo, T.

Identiﬁcation for a Large-volume Food-borne Bacteria on a Fully Integrated Portable Centrifugal Disc.

DOI: 10.5220/0007690401140118

In Proceedings of the 12th International Joint Conference on Biomedical Engineering Systems and Technologies (BIOSTEC 2019), pages 114-118

ISBN: 978-989-758-353-7

reaction, and amplicon quantification by a UV-Vis

detector can be performed on the integrated

centrifugal microdevice in a portable genetic analyser

system.

2 EXPERIMENTAL

2.1 Design of the Centrifugal Device

The disc was designed to perform the DNA solid-

phase extraction and high-throughput LAMP assay

with 20 reaction chambers. We proposed the device

with two units: a centrifugal microdevice for DNA

extraction and LAMP reaction, and a 3D-printed

cartridge for solution loading. The centrifugal

microdevice was designed with AutoCAD, and

etched in a 3.0 mm thick poly(methylmethacrylate)

(PMMA) plate using a CNC machine. All the siphon

channels were coated with a hydrophobic reagent,

Vistex 111-50. The positive reaction chamber was

coated with a primer set of the target bacteria. The

waste chamber was integrated with super absorption

polymer (SAP) from baby diaper for utterly absorbing

1 mL sample solution. A pressure sensitive adhesive

(PSA) foil layer was applied to seal the disc. The acid

wash glass bead (150-212 μm, Sigma) was then

packed into the DNA extraction channel. Finally, the

bead-packed channel was incubated in 6M Gu-HCl

for 30 min to enhance the DNA capture capacity.

2.2 Portable Rotary Platform with

UV-Vis Detector

To adapt our system for POC testing, we also

proposed a portable compact and small size rotary

platform for operating the disc. The rotary platform

consists of: (1) a spindle motor, (2) a couple of Minco

heater and (3) a UV-Vis optical detector. The UV-Vis

optical system is composed of a yellow LED 570 nm

and a red LED 650 nm directed toward LAMP

reaction chambers through an optical fiber. A filter

was used for eliminating the interference from excited

light and an aspheric lens for reducing optical

aberrations. The transmittance light intensity was

then measured by a CMOS camera sensor and

converted into relative absorbance.

2.3 Procedure for the on-Chip Genetic

Analysis

Firstly, 1 mL of sample lysis mixture was prepared

containing 500 µL of bacteria sample, 250 µL of AL

buffer (Qiagen, Netherlands), and 250 µL of 6 mM

Gu-HCl (Thermo Fisher Scientific, USA). Sample

lysis mixture, a washing solution (70% Ethanol), an

elution solution (DNase/RNase water), and a

LAMP/EBT cocktail solution were then injected into

the cartridge at the injection hole. An in-house

program automatically performed all the operation

steps including spinning for solution transferring,

Figure 1: (a) Schematic illustration of the integrated centrifugal microdevice. (b) Digital images of the disc. (c) Components

of the microdevice, (i) Aliquoting structure and LAMP reaction chamber, (ii) Siphon channel coated with Vistex, (iii)

Glassbead-packed channel for DNA extraction, and (iv) Super Absorption Polymer (SAP) integrated waste chamber.

Identiﬁcation for a Large-volume Food-borne Bacteria on a Fully Integrated Portable Centrifugal Disc

115

Figure 2: (a) Digital images of the portable rotary platform. (b) UV-Vis detector for measuring absorbance of reaction

chamber. (c) A couple of Minco heater. (d) Digital images of the portable rotary platform with closed lib. (e) Schematic

illustration of the rotary platform with a centrifugal motor, a couple of Minco heater, and a UV-Vis detector with two light

emitting diode (LED) light source at 640 and 570 nm.

shaking for mixing, heating for proceeding LAMP

reaction, measuring solution absorbance in real-time,

and data production. The absorbance at 640 nm

(Abs640) and 570 nm (Abs570) were recorded at a 5

min interval time during 60 min of LAMP reaction.

The ratio of Abs640 to Abs570 (Abs640/Abs570) was

then calculated. The real-time curve was plotted

between Abs640/Abs570 ratio and time.

3 RESULTS AND DISCUSSION

3.1 Optical Real-Time Sensing LAMP

Reaction Amplicon by in-House

Building System

We recorded the UV-Vis absorption spectrum of the

LAMP mixture before and after LAMP reaction. The

color of LAMP mixture changed from violet to sky

blue during the process of a LAMP reaction with the

change of maximum absorption wavelength from 570

nm to 640 nm, respectively. Therefore, we designed

the UV-Vis detector on the portable rotary platform

with the two LED light source at 570 and 640 nm. We

measured the relative absorbance at 640 nm and 570

nm of negative (NC) and positive (PC) chamber with

5 min interval time during the LAMP reaction. The

NC chamber has no change in the Abs640/Abs570

ratio. In contrast, for the PC chamber, the

Abs640/Abs570 ratio has a change when the LAMP

reaction occurs at 40-50 min and became saturated at

55-60 min. These results are in a good agree-ment

with UV-vis absorption spectrum of the NC and PC.

Therefore, the Abs640/Abs570 ratio could be used as

the criteria to identify a positive result of which

Abs640/Abs570 ratio is higher than 1.0.

3.2 Singleplex and Multiplex Detection

on the Integrated Portable System

The disc was designed for processing parallel 2

samples in one run and in 20 reaction chambers for

each sample. Theoretically, up to 20 kinds of

foodborne pathogens can be simultaneously detected.

In this experiment, we targeted three kinds of bacteria

(E. coli O157:H7, S. Typhimurium and V.

parahaemolyticus) as a model. While no color change

was observed in negative control chambers

(chambers from left 1,5,9,13,17), the rest chambers

with the coated primer for targeting E. coli O157:H7,

S. Typhimurium and V. parahaemolyticus exhibited

color change from purple into sky blue.

The absorbance of 20 chambers was also measured

during the LAMP reaction. The Abs640/570 ratio

after the LAMP reaction shows that all the PC

chambers with sky blue color had an Abs640/570

ratio higher than 1.0, and all the NC chamber with

violet color had an Abs640/570 ratio lower than 1.0.

Consequently, we demonstrated that the proposed

microdevice could simultaneously detect multiple

pathogen targets in 20 reaction chambers.

BIODEVICES 2019 - 12th International Conference on Biomedical Electronics and Devices

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Figure 3: Multiplex detection of foodborne pathogens in

samples based on the prototype device. (a) Colorimetric

detection of single pathogen (E. coli O157:H7). (b)

Colorimetric detection of triple pathogens (E. coli O157:H7,

S. Typhimurium and V. parahaemolyticus). (c) The graph

of Abs640/570 ratio of negative and positive chamber.

4 CONCLUSIONS

We have developed a sample-to-answer disc for

multiplex food poisoning bacteria screening with a

large volume of sample (up to 1 mL). The system was

automatic and small suitable for POC testing. The

disc was designed with the solution-loading

cartridges to accomplish a full automation, and a

specific SAP integrated waste chamber for large

sample volume handling. All experimental processes

of the molecular diagnostics were integrated in a

single device including extraction, amplification,

detection, and data analyzing/reporting. The sample

and other essential solutions for LAMP assay are

loaded into the 3D printed cartridge, and orderly

released into the centrifugal microdevice by a specific

channel design and spinning program. The portable

genetic analyzer provides a user-friendly interface

and a simple operation protocol which is affordable

for less technical training staff.

ACKNOWLEDGEMENTS

This work was supported by the Engineering

Research Center of Excellence Program of Korea

Ministry of Science, ICT & Future Planning

(MSIP)/National Research Foundation of Korea

(NRF) (2014R1A5A1009799) and by a grant of the

Korean Health Technology R&D Project, Ministry of

Health & Welfare, Republic of Korea (grant no.

HI13C1232).

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