An integrated microfluidic system using magnetic beads for virus detection

https://doi.org/10.1016/j.diagmicrobio.2007.07.010Get rights and content

Abstract

An integrated system capable of sample pretreatment using antibody-conjugated magnetic beads and one-step reverse transcriptase–polymerase chain reaction (RT-PCR) on a microfluidic system was developed to accelerate the detection of RNA viruses such as dengue virus or enterovirus 71. The targeted virus in the sample was first captured by the specific antibody-conjugated magnetic beads, which were manipulated by micro-electromagnets made of micro-electro-mechanical systems technology. The RNA of the targeted virus then underwent thermal lysis and was reverse-transcripted to cDNA using a μRT-PCR module. The sensitivity to detect dengue virus is around 10–100 PFU, which is equivalent to the commercial RNA extraction kit and a large-scale RT-PCR machine. This microsystem can specifically detect 4 serotypes of dengue virus, as well as enterovirus 71. The specificity was warranted by both antibody and primer. The microfluidic system allows automatic process of sample including mixing, incubation, and reaction. The antibody-conjugated magnetic beads offer sample pretreatment of purification and concentration. The integration of antibody-conjugated magnetic beads into the microfluidic system is promising for fast molecular diagnosis of microorganisms.

Introduction

The micro-electro-mechanical systems (MEMS) technology and micromachining techniques have enabled the miniaturization of biomedical and chemical analysis devices. The technologies of fluid-based microsystems have been realized since the advent of micro-total-analysis systems (Tay, 2002). Various fluidic operations in microfluidic systems such as sample preparation, sample injection, manipulation, filtration, reaction, separation, and detection have been successfully demonstrated. Efficient transportation of fluids in these microsystems is critical for miniaturized biomedical systems (Unger et al., 2000, Fu et al., 2002). For molecular diagnosis, the microfluidic system offers many advantages including smaller sample/reagent requirement (<50 μL), shorter time, lower cost, more precision, and the option of disposability (if dealing with infectious agents, this means improved safety). However, biological samples usually contain a mixture of bioactive substances that may interfere with the subsequent DNA/RNA amplification. The purification and enrichment of the extremely low concentration of biosample become crucial in many biomedical assays (Choi et al., 2001, Choi et al., 2002, Grodzinski et al., 2003). These pretreatment steps can increase the detection limit of the sensing system. For example, the nucleic acids are purified by silica gel binding under the chaotropic agent guanidinium thiocyanate for downstream application (Boom et al., 1990). We have miniaturized the reverse transcriptase–polymerase chain reaction (RT-PCR) system for fast molecular diagnosis utilizing the MEMS technology (Liao et al., 2005a, Liao et al., 2005b). An automatic one-step μRT-PCR system that integrates the sample purification/enrichment devices using superparamagnetic beads into a single chip has also been reported (Lien et al., 2006a). The antibody-conjugated magnetic beads were used to capture the targeted virus. The virus was then subjected to thermolysis, RNA extraction, and RT-PCR on a single chip automatically. In this study, the integrated antibody-conjugated microfluidic system that performs purification, enrichment, and the subsequent RT-PCR was further investigated for RNA virus. The advantages over the traditional RNA extraction kit or manual magnetic bead process are compared and discussed.

Section snippets

Dengue virus and enterovirus 71

All 4 dengue virus serotype strains (serotype 1/766733A, serotype 2/PL046, serotype 3/739079A, and serotype 4/H-241) were obtained from the Center for Disease Control (Taipei, Taiwan). Unless otherwise specified, the dengue virus serotype 2, strain PL046 was used for most experiments. Viruses were propagated in mosquito C6/36 cells in Eagle's minimal essential medium containing 2% heat-inactivated fetal bovine serum at 28 °C for 5 days and quantified by standard plaque assay as previously

An integrated antibody-conjugated microfluidic system for RNA virus detection

We are interested in developing a chip-based RT-PCR system for the amplification of specific nucleic acids and the detection of RNA-based virus. Traditionally, viral samples are incubated with a commercial RNA kit to extract and concentrate the RNA by silica-gel membrane binding. This procedure is labor-intensive and is not amenable to automation. After extraction, the RNA template is loaded onto the reaction chamber of the chip to amplify the RT-PCR. This is therefore a 2-step process. When we

Discussion

To date, miniaturization of the sampling, pretreatment of sample, measurement, and interpretation has been applied to biomedical and chemical analysis devices and systems. Although the microfluidic system offers many advantages of small sample requirement (<50 μL), shorter time, low price, more precision, and disposal, comparing with the large-scale machine, the sample enrichment or purification in the pretreatment procedure is crucially important because of the complexity of composition,

Acknowledgments

This work was supported by grants NSC95-3112-B006-004 and NSC95-2221-E006-012-MY3 from the National Science Council, Taiwan.

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