Array biosensor for detection of biohazards

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Abstract

A fluorescence-based biosensor has been developed for simultaneous analysis of multiple samples for multiple biohazardous agents. A patterned array of antibodies immobilized on the surface of a planar waveguide is used to capture antigen present in samples; bound analyte is then quantified by means of fluorescent tracer antibodies. Upon excitation of the fluorophore by a small diode laser, a CCD camera detects the pattern of fluorescent antibody:antigen complexes on the waveguide surface. Image analysis software correlates the position of fluorescent signals with the identity of the analyte. This array biosensor has been used to detect toxins, toxoids, and killed or non-pathogenic (vaccine) strains of pathogenic bacteria. Limits of detection in the mid-ng/ml range (toxins and toxoids) and in the 103–106 cfu/ml range (bacterial analytes) were achieved with a facile 14-min off-line assay. In addition, a fluidics and imaging system has been developed which allows automated detection of staphylococcal enterotoxin B (SEB) in the low ng/ml range.

Introduction

A number of investigators have described optical biosensors capable of simultaneous analysis of samples for multiple analytes. However, most of the published work describes only detection of a single analyte (Ekins et al., 1990, Ekins and Chu, 1993, Abel et al., 1996, Herron et al., 1997, Blawas et al., 1998, Brecht et al., 1998). The majority of experiments actually demonstrating the simultaneous detection of multiple analytes accomplished this by putting a single sample over multiple, discrete sensing elements (Kakabakos et al., 1992, Parsons et al., 1993, Bakaltcheva et al., 1998, Narang et al., 1998). Berger et al. (1998), on the other hand, utilized several discrete regions of a single sensing surface to monitor four simultaneous reactions on a four-channel surface plasmon resonance system. However, for monitoring complex samples, the label-free methods continue to be susceptible to problems such as low sensitivity and increased backgrounds due to non-specific binding. Wadkins et al., 1997, Wadkins et al., 1998 and Silzel et al. (1998) avoided such problems by using fluorescent tracer antibodies and performing a measurement insensitive to non-specifically bound proteins (other than the tracer antibody).

The antibody array biosensor described here is composed of three parts: an array of immobilized capture antibodies acting as molecular recognition elements, an image capture and processing system, and an automated fluidics unit (Fig. 1A). Antibodies specific for hazardous analytes are immobilized in discrete regions on an avidin-coated waveguide by flowing solutions of biotinylated antibodies through a network of polymer channels that confine the solutions to separate regions (Ligler et al., 1998a). Unknown sample is subsequently flowed over the substrate in an orientation perpendicular to the stripes of immobilized antibodies and any antigens present in the sample bind to the appropriate analyte-specific loci in the array. Bound antigens are then incubated with a mixture of fluorescently-labeled tracer antibodies. The resultant antibody/antigen/ fluorescent-antibody complex is detected using a CCD camera upon excitation by a small diode laser. Automated image analysis software correlates the position of the fluorescence with the identity of the hazardous component, with results displayed directly to the user. Each antibody-coated substrate is a fully disposable unit designed to be used repeatedly until one or more analytes are detected.

The central element of the array biosensor is the planar waveguide used to direct evanescent excitation light to fluorophores which are bound (by immune complex) to the waveguide surface. A major problem originally encountered with this sensing element was the stripping of light when a flow cell was attached to the waveguide. To solve this problem, a unique patterned reflective cladding was developed to optically insulate the waveguide from the flow cell (Feldstein et al., 1999). The pattern of this silver-based cladding covers the area where a six-channel flow cell contacts the waveguide. The rest of the waveguide surface is left unclad and is suitable for performing optical immunoassays.

In order to reduce the size and weight of the sensor, the potential for sample carryover, and the possibility of system contamination, a modular fluidics system has been developed (Fig. 1B; Feldstein et al., 1999). This system consists of permanent elements (pump, valves), replaceable subsystems (sample manifold, inlets and outlets), and a disposable unit (waveguide and flow chamber module). Samples do not pass through any valves, thus avoiding the substantial problem of valve clogging by complex or particulate samples. Further, the system has been designed to include an easily replaceable sample manifold in order to eliminate the problem of cross-contamination from sequential introduction of samples. The valves and pump are computer controlled to operate the assay protocols in an automated fashion. A unique feature of the fluidics system is the ability to assay up to six samples simultaneously using a single multi-channel pump and a single switching valve.

Fluorescent images are analyzed using data acquisition software developed at NRL (Feldstein et al., 1999). The signal from each antigen-specific spot is automatically corrected for non-specific binding of the fluorescent reagent and for the slight variations in excitation intensity across the surface of the waveguide.

To date, work performed using the array biosensor has demonstrated that mixtures of fluorescent tracer antibodies can be used in rapid assays for protein, bacterial, and viral analytes with sensitivities similar to standard ELISAs (Fig. 2; Wadkins et al., 1998, Rowe et al., 1999a, Rowe et al., 1999b). Results obtained using mixtures of antibodies were not significantly different from those obtained from parallel assays utilizing individual tracers (Rowe et al., 1999b). Furthermore, mixtures of analytes could also be detected and identified. Moreover, analytes present in complex sample matrices such as blood and urine could be detected and quantified using the automated data analysis program, provided a suitable ‘clean’ sample was assayed on the same slide (Rowe et al., 1999a).

These previous experiments were performed using a non-automated version of the array biosensor, which requires user manipulation of samples, flow chamber modules, and optical waveguides. While the strength of this sensor is its ability to detect multiple analytes simultaneously (Wadkins et al., 1998, Ligler et al., 1998a, Ligler et al., 1998b, Rowe et al., 1999a, Rowe et al., 1999b), the purpose of this report is to document further development of rapid assays for potentially hazardous analytes at concentrations similar to competing technologies. In addition, we describe in Section 3.2 testing of a fully automated system utilizing a computer-controlled fluidics system (Fig. 1), the data analysis program, and flow guides which have been permanently mounted onto patterned waveguides. This demonstration of full automation is the first description of an array sensor which requires no user intervention after samples are loaded.

Section snippets

Antibodies and analytes

Antibodies and the majority of antigens used in this work were generous gifts of Woody Johnson and Jennifer Aldrich at Naval Medical Research Center (NMRC, Bethesda, MD). The NMRC antibody preparations were provided after purification with Protein A or Protein G chromatography. The anti-Bacillus anthracis antibody had also been affinity purified by J. Aldrich and W. Johnson. Individual components in the array assays and their sources are listed in Table 1.

None of the bacterial analytes used in

Off-line, non-automated assays

Assays were developed for six analytes potentially capable of causing illness or disease. These assays consisted of simple sandwich immunoassays performed on the surface of planar waveguides using biotinylated capture antibodies for antigen recognition; these platforms were prepared in advance of the assays and incubation of sample with the prepared substrates required only 7 min. Bound antigens were then detected by a 3 min incubation with fluorescent tracer molecules. Following this 14-min

Conclusion

The array biosensor assays multiple samples simultaneously for multiple analytes. Not only does it exhibit sensitivity comparable with other antibody-based methods that require sample aliquots to be assayed individually for different agents, but the multianalyte assays have been automated. The off-line assays took an average of 15–18 min to complete the biochemical assays, image the slide, and collect the data. Limits of detection for bacterial analytes ranged from 624 cfu/ml (B. anthracis) to

Acknowledgements

The authors would like to thank Woody Johnson and Jennifer Aldrich at NRMC for the generous gift of antibodies and antigens which were used in much of this research. Dr Feldstein was supported by a postdoctoral fellowship from the National Research Council. This work was funded by the Office of Naval Research and the US Department of Defense. The views expressed here are those of the authors and do not represent those of the US Navy, the US Department of Defense, or the US government.

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