Elsevier

Biosensors and Bioelectronics

Volume 24, Issue 5, 1 January 2009, Pages 1195-1200
Biosensors and Bioelectronics

Particle flow assays for fluorescent protein microarray applications

https://doi.org/10.1016/j.bios.2008.07.005Get rights and content

Abstract

Microarray technology has brought a paradigmatic change in bioanalytics. However, highly sensitive and accurate assays are still needed for a real breakthrough. We present a simple and generic approach for fluorescent signal amplification with fluorescent microparticle labels. The assay principle was demonstrated using a reverse array model consisting of spots of bovine serum albumin with a small fraction of the proteins biotinylated. Specific binding of streptavidin coated fluorescent microparticles to the spots was promoted by applying a controlled continuous microparticle flow. The surface bound beads were visualized and quantified with confocal microscopy images.

Comparison with standard fluorescent and flow discrimination assays has revealed several advantages of our approach. First, non-specific particle binding could be reduced to less than 1 particle/spot making therefore the visualization of single biomolecular bonds possible. Second, the amplification scheme presented here is generic and can be applied to any fluorescent microarray. Furthermore, this assay makes use of a biotin–streptavidin linkage and can therefore be applied to all kind of assays. Finally, single fluorescent microbeads can be easily visualized with standard optical equipments, so that no high performance equipment is required.

Introduction

Over the last decades considerable effort has been put into the development of novel biosensing platforms for the detection of specific biological components such as proteins or nucleic acids in a complex analyte. Applications range from the medical and diagnostics field to food technology and environmental monitoring (Eggins, 2002).

More recently, technologies utilizing arrays of immobilized biomolecules on planar surfaces have been developed as powerful tools for bioanalytical measurements. These so-called microarrays make it possible to study, in a single experiment, thousands of biomolecular interactions in parallel and with high-throughput. They are therefore becoming essential tools in basic life science, medical research, clinical diagnostics and drug discovery (Kambhampati, 2004, MacBeath, 2002, Müller and Nicolau, 2005, Templin et al., 2002). While DNA microarrays are now a standard for high-throughput analysis of nucleic acids (Debouck and Goodfellow, 1999, DeRisi et al., 1997, Lockhart and Winzeler, 2000, Ramsay, 1998), highly sensitive and accurate assays are still needed for many protein microarray applications (Templin et al., 2002, Wilson and Nock, 2003).

Many different protein microarray formats have been developed, all of which can be classified into two major types.

Type 1: Capture arrays consist of spots of antibodies that can catch their target antigens from a given sample. Thus, many measurements are being performed in parallel on the same sample. A second labeled antibody is needed for the quantitative assessment; and multiplexing of more than 10 assays requires extensive optimization (LaBaer and Ramachandran, 2005, Liotta et al., 2003, Templin et al., 2002).

Type 2: Reverse arrays are preferred when the presence of a few analytes should be measured and compared across a large variety of samples (Liotta et al., 2003). Cell lysate arrays, for example, have a tremendous potential for protein expression profiling and signaling pathways studies, since they are much faster than conventional proteomics techniques such as 2D gel electrophoresis and mass spectrometry analysis or Western blotting (Charboneau et al., 2002, Paweletz et al., 2001). In this assay format, a complex sample is spotted directly onto the chip surface, usually without any purification step or “fishing out” from solution as it is typically the case for a capture array format. Therefore, only a few copies of the protein of interest might be present on the spot (LaBaer and Ramachandran, 2005), especially since most of the proteome species are low abundance ones (Miklos and Maleszka, 2001). As such, extremely sensitive assays are even more important for the implementation of reverse phase arrays; and signal amplification schemes are urgently needed to facilitate the assessment of low abundance analytes.

Currently, fluorescently labeled reporter molecules are the most common in microarray technology (Bally et al., 2006, Kambhampati, 2004). Other labeling approaches include the use of enzymes for the generation of luminescent or chromogenic signals (Espina et al., 2004, Joos et al., 2000) as well as isotopic labeling (Ge, 2000, Zhu et al., 2001).

More recently, the use of nano- and microparticle labels has also been reported. Micro and nanoparticles include metallic (Francois et al., 2003, Storhoff et al., 2004, Taton et al., 2000, Wang et al., 2005) and magnetic particles (Ferreira et al., 2005, Graham et al., 2004, Osaka et al., 2006, Richardson et al., 2001) as well as quantum dots (Liotta et al., 2003, Shingyoji et al., 2005). Although bigger particles limit the dynamic range, microparticles have the advantage that they can be individually visualized with a standard microscope and that they can generate a strong fluorescent signal since they carry a high amount of fluorophores. Furthermore, micron-sized beads can be easily manipulated by external forces such as magnetic (Edelstein et al., 2000, Lee et al., 2000, Rife et al., 2003) or fluid forces (Cozens-Roberts et al., 1990, Zocchi, 2001). Recently, a biosensor based on magnetoelectronic detection of magnetic microparticles was presented (Mulvaney et al., 2007). In this approach by Mulvaney and co-workers, fluid forces were used to discriminate between non-specifically and specifically bound particles.

Nevertheless, today conventional microarray readers do not meet the requirements in sensitivity for the detection of a variety of low abundant proteins and new high-performance devices are needed to overcome this limitation. As highlighted above, sensitivity is more a limiting factor for reverse assay formats where no pre-fractioning is performed. In the latter case even the most sensitive fluorescence array readers are not sufficient for the detection of low abundance proteins as, for example, cytokines which can be present in as little as 20 copies per nanoliter of sample. New signal fluorescent amplification schemes are therefore urgently needed to overcome these limitations and to increase the dynamic range also for less sensitive detection setups.

In this work, we present a simple approach for signal amplification using fluorescent microparticle labels. This amplification is generic and can be used to enhance the performance of any fluorescent microarray assay format. We demonstrate signal amplification on a model reverse array consisting of spots of unlabeled and biotinylated BSA compared to a standard assay performed with fluorescently labeled streptavidin. In addition, we present the lowest reported background values of less than 1 particle/spot using streptavidin coated fluorescent microparticles in flow assays.

Section snippets

Microarray preparation

Biotinylated bovine serum albumin (BSA-biotin) was purchased from Sigma–Aldrich (Switzerland) and diluted to the right concentration for spotting in 50% HEPES buffer (10 mM 4-(2-hydroxyethyl)piperazine-1-ethane-sulfonic acid and 150 mM NaCl, pH 7.4) and 50% ZeptoMARK spotting buffer (Zeptosens, a division of Bayer (Schweiz) AG, Switzerland). The biotinylated protein was spotted onto Ta2O5 slides coated with dodecylphosphate (Zeptosens) using a pin and ring spotter (GMS 417 arrayer, Affymetrix,

Constant bead flow approach

A microarray of BSA-biotin spots was exposed to a flow of fluorescent microspheres coated with streptavidin (Fig. 1). The bead flow rate was kept constant during the whole assay and different flow rates were investigated.

The number of beads bound to the BSA-biotin spots was evaluated using confocal laser scanning microscopy images. The bead densities found on spots with different BSA-biotin concentrations ranging from 250 μg/ml to 25 ng/ml (1:10 dilution steps) as well as on the background

Discussion

In the study presented here, streptavidin coated microparticles were used as labels to visualize biomolecular interactions. A model reverse protein array was produced by spotting BSA-biotin in different concentrations and blocking with non-biotinylated BSA. Micron-sized fluorescent particles have the advantage that they can be individually visualized using, for example, a standard microscope. Therefore, the assay performance does not depend on the device sensitivity but rather on the ability of

Conclusions

We have presented a new fluorescent signal amplification scheme which makes use of fluorescent micron-sized particles for sensitive detection of biomolecular interactions on a microarray. A continuous particle flow was used to reduce non-specific binding on the background surface and to specifically immobilize the fluorescent particles on the protein spots. The performance of the flow assay was superior to a “sediment-and-rinse approach” allowing for the detection of single specific bonds.

Acknowledgements

We would like to thank Dr. Markus Ehrat and Dr. Ekkehard Kaufmann for valuable discussions as well as Paul Lüthi for manufacturing the flow cell. Funding contributions for this work were provided by the Swiss Commission for Technology and Innovation CTI (Project No. 7241.1 NMPP-NM) and ETH Zurich.

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