Microbial detection in microfluidic devices through dual staining of quantum dots-labeled immunoassay and RNA hybridization

doi:10.1016/j.aca.2005.07.003

Analytica Chimica Acta

Volume 556, Issue 1, 18 January 2006, Pages 171-177

https://doi.org/10.1016/j.aca.2005.07.003 Get rights and content

Abstract

This paper reported the development of a microfludic device for the rapid detection of viable and nonviable microbial cells through dual labeling by fluorescent in situ hybridization (FISH) and quantum dots (QDs)-labeled immunofluorescent assay (IFA). The coin sized device consists of a microchannel and filtering pillars (gap = 1–2 μm) and was demonstrated to effectively trap and concentrate microbial cells (i.e. Giardia lamblia). After sample injection, FISH probe solution and QDs-labeled antibody solution were sequentially pumped into the device to accelerate the fluorescent labeling reactions at optimized flow rates (i.e. 1 and 20 μL/min, respectively). After 2 min washing for each assay, the whole process could be finished within 30 min, with minimum consumption of labeling reagents and superior fluorescent signal intensity. The choice of QDs 525 for IFA resulted in bright and stable fluorescent signal, with minimum interference with the Cy3 signal from FISH detection.

Introduction

Combined immunofluorescent assay (IFA) and florescent in situ hybridization (FISH) have been successfully demonstrated for the detection of microbial cells [1]. IFA staining provides strong signal intensity, while FISH staining is highly specific and could be used to differentiate viable and nonviable cells [2]. However, normal practices performed on glass slide or in test tube often require multiple reactions and washing steps, which are considered to be time consuming, reagent consuming and labor intensive [3], [4].

Alternatively, rapid IFA could be achieved by performing this assay on microfluidic or lab-on-a-chip devices [6], [7]. One simple approach is to create sample inlet and outlet, microfluidic channels and weir-type trapping region on a silicon-based device [7]. After injecting sample solution containing the target cells in the microchannel, these cells can be mechanically trapped at the weir region, and be labeled by flushing the microchannel with a solution containing fluorescent-conjugated antibody specific to the target cells. After brief washing, these fluorescently labeled target cells can be easily detected under a fluorescent microscopy at single cell level. Since all the reactions were performed in micro-scales, sample and reagent consumption could be reduced significantly. By employing a flow through format, assaying time could also be reduced from hours to minutes. Further integration, automation and parallel processing of multiple samples are also possible based on these microfluidic platforms [8], [9].

Based on the concept described above, this study has further demonstrated that IFA and FISH could be performed in sequence on a microfluidic filter-based device to achieve rapid detection of viable and nonviable microbial cells. The device consisted of a microchannel and filtering pillars to trap and concentrate the model microbial cells (i.e. Giardia lamblia). To further increase the signal intensity and reproducibility of IFA, semiconductor quantum dots (QDs) have been recently introduced as a novel inorganic fluorescence dye. QDs are small nano-particles (ca., 2–50 nm in size) with narrow, symmetrical and tunable emission spectra and can be excited by a wide spectrum of wavelength [10]. With the excellent emission property, signal intensity and photostability, QD label can be easily differentiated from autofluorescence particles present in environments, thereby appearing to be a preferred fluorescence dye in the IFA detection of protozoa cells [11]. It is expected that these microfluidic devices can be potentially used in the rapid diagnostic of clinical samples, and in the monitoring of water quality and public health in environments.

Section snippets

Design, fabrication and simulation of microfluidic device

Fig. 1 illustrates the design and features of the microfluidic device used. The device (20 mm × 10 mm) consists of a 650 μm thick silicon base and a 500 μm thick Pyrex glass cover (corning 7740, Dow corning corporation, Midland, MI). The base plate contains a reaction chamber (50 μm in depth), an inlet (1 mm in width), an outlet (1 mm in width), a coarse filter region and a trapping filter region for microbial cells. The coarse screen which is designed for pre-filtrating large impurities from the sample

Microfluidic device

Fig. 3a and c show the scanning electron microscope (SEM) images for the cell-trapping filter region and the coarse filter region. The two pillar-type trapping regions were fabricated toward to the end of the microchannels (Fig. 3a). The distance between any two given pillars (Fig. 3b) was approximately 1.5 μm and the depth of the pillars was 50 μm. The coarse filter region (Fig. 3c) was made of arrays of diamond-shape pillars (30 μm × 30 μm) with a space of 20, 30 or 50 μm between any two given

Discussion

This study has clearly demonstrated that the use of microfluidic device as a platform could achieve rapid biochemical reactions or microbial cells detection. For IFA, rapid detection was likely achieved by improving the mass transfer rate for the labeling reagent (antibodies) in the solution to bind onto the target sites (i.e. antigens) on the surface of microbial cells. Thus, the time required to achieve a high S/N ratio could be shortened by increasing either the concentrations of the

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Microbial detection in microfluidic devices through dual staining of quantum dots-labeled immunoassay and RNA hybridization

Abstract

Introduction

Section snippets

Design, fabrication and simulation of microfluidic device

Microfluidic device

Discussion

J. Microbiol. Meth.

J. Appl. Microbiol.

Am. J. Trop. Med. Hyg.

Appl. Environ. Microb.

Cytometry

Anal. Chem.