Elsevier

Ultramicroscopy

Volume 108, Issue 10, September 2008, Pages 1384-1389
Ultramicroscopy

Superporous agarose beads as a solid support for microfluidic immunoassay

https://doi.org/10.1016/j.ultramic.2008.04.044Get rights and content

Abstract

We demonstrate here with the feasibility of superporous agarose (SA) beads as a solid support in microfluidic immunoassay by detecting goat IgG. In our procedure, SA beads containing superpores were covalently conjugated to protein A. The conjugated beads were introduced into a polydimethyl siloxane microfluidic device. The sandwich immunoassay was performed in the microfluidic device by subsequently introducing anti-goat IgG as the primary antibodies, goat IgG as analytes, alkaline phosphatase-conjugated F(ab′)2 anti-goat IgG as detection antibodies, and 5-bromo-4-chloro-3-indolylphosphate/nitroblue tetrazolium as substrate in a flow. Depending on the goat IgG concentration, dark and pinky precipitates appeared inside the microchannel immediately after the introduction of all the reagents. The minimum detection limit, 100 pg goat IgG/mL in PBS, was achieved with the naked eye. This enhanced sensitivity is mainly because analytical reagents were allowed to access the outer surface as well as the inner matrices of the beads. This is supported by the facts that the binding of fluorescein isothiocyanate IgG happened throughout the inside matrices of protein A-conjugated SA beads but was limited to the outer surface of protein A-conjugated homogeneous agarose beads. These results suggest that SA beads are highly suitable as a solid support for microfluidic immunoassays.

Introduction

Microfluidic devices possess many features that enhance bioanalyses, such as high throughput, short analysis time, the ability to operate with small sample volume and high sensitivity. For example, the diffusion distance between interacting molecules in a microwell of a microtiter plate is in the range of a few millimeters compared with tens of microns in a microchannel, which reduces incubation and mixing times [1]. Also, the option of a tailor-made integrated system enables researchers to easily adopt microfluidic devices for specific analyses of different biological analytes [2]. These features have led to the development of microfluidic immunoassays. However, some problems still exist with this technology. For example, the surface-to-volume (S/V) ratio of a typical microchannel is higher than that of a standard microtiter plate [1] but still smaller than that of membranes used in immunochromatography.

Thus, different types of solid supports have been exploited to increase the S/V ratio in the microchannels of microfluidic devices. The most popular solid support is microbeads because the S/V ratio can be significantly increased by packing them into microchannels [3]. This is much easier than other methods, such as fabricating macroporous structures [4] or incorporating porous polymers [5] in microchannels. Another advantage of bead-based microfluidic immunoassay is that affinity molecules can easily be immobilized in a certain region of a microfluidic device in which beads previously sensitized with affinity molecules are trapped in either a tapered microchannel or a fabricated filter. So far, however, the immobilization of affinity molecules has been limited to the outer surface of beads because of the lack of beads having pores large enough for the free transport of antibodies or antigens.

Recently, superporous agarose (SA) beads have been successfully used as matrices in separation techniques such as chromatography and electrophoresis [6]. SA beads contain two sets of pores; typical diffusion pores and so-called superpores (average diameter of 28 μm) [7]. Superpores constitute a significant portion of the bead, up to 1/10–1/3 of the bead particle diameter. SA beads have been used successfully as matrices in immobilizing human red blood cells [8], three-dimensional biofilm formation [9], and high-speed protein chromatography [10]. Their physical features can also offer several advantages for bead-based microfluidic immunoassays. First, the surface in microchannels can be increased by packing SA beads as opposed to normal homogeneous beads. Second, mass transfer inside microchannels can be improved because superpores allow a considerable fraction of the bulk chromatographic flow to penetrate individual beads. This also enables bead-packed microfluidic devices to be operated at a high flow rate without sudden backpressure buildup even with tightly packed beads in microchannels, thus resulting in an improved performance for microfluidic immunoassays.

In the present work, we introduce SA beads as a solid support for microfluidic immunoassay. The SA beads were prepared by a double emulsification procedure [6], [7]. Anti-goat IgG as capture antibodies were then immobilized to the SA beads and the sensitized beads were introduced and trapped into a filter structure of a microfluidic device fabricated by soft lithography [11], [12]. Later, a sample solution containing goat IgG (analyte), a solution containing anti-goat IgG F(ab′)2 conjugated with alkaline phosphatase (AP), and a solution containing substrate (5-bromo-4-chloro-3-indolylphosphate (BCIP)/nitroblue tetrazolium (NBT)) were allowed to flow in sequence into the SA bead-packed microfluidic device. As a result, goat IgG was detected by a color-producing enzymatic reaction. The density of color in the detection zone was proportional to the amount of goat IgG present in a sample.

Section snippets

Materials

SU-8 2035 was purchased from MicroChem Corporation (Newton, MA, USA). Polydimethyl siloxane (PDMS) prepolymer and curing agent (Sylgard 184) was purchased from Dow Corning (Midland, MI, USA). Agarose was purchased from Cambrex Bio Science Rockland, Inc. (Rockland, ME, USA). Sorbitane trioleate (Span® 85), polyoxyethylene sorbitanmonooleate (Tween 80), anti-goat IgG (Rabbit serum), goat IgG, BCIP/NBT, and fluorescein isothiocyanate (FITC)-rabbit IgG were purchased from Sigma (St. Louis, MO,

Results and discussion

The physical features of HA and SA beads were observed by optical microscopy, as shown in Fig. 2. HA beads contained only diffusion pores (100–300 nm), which were not visible in the micrograph, whereas SA beads contained diffusion pores as well as superpores with diameters from 10 to 80 μm, which were clearly visible in the micrograph. Superpores in the beads can offer advantages over conventional microbeads for bead-based immunoassays in terms of flow control and antibody immobilization.

Conclusion

SA beads as a solid support brought two advantages to microfluidic immunoassays. The macroporous structure of SA beads enabled solutions to flow easily through both the inner and outer space of the beads, thereby relieving back pressure buildup, which often occurs while flowing a solution through bead-packed microfluidic devices. Another advantage is the increase in the S/V ratio inside the microchannel, thereby enhancing the sensitivity of the microfluidic immunoassay. Our results successfully

Acknowledgments

Funding for this study was provided by the SRC program of the Korea Science and Engineering Foundation (KOSEF) through the Center for intelligent Nano-Bio Materials at Ewha Womans University (Grant R11-2005-008-02003-0) and by the Nano/Bio Science & Technology Program (M1053-6090002-05N3609-00210) of the Ministry of Science and Technology (MOST) of Korea. The authors received additional support from the Brain Korea 21 (BK21) fellowship of the Ministry of Education of Korea.

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