Anal. Chem. 2002, 74, 6114-6120
Nine-Analyte Detection Using an Array-Based Biosensor Chris Rowe Taitt,*,† George P. Anderson,† Brian M. Lingerfelt,‡ Mark J. Feldstein,† and Frances S. Ligler†
Center for Bio/Molecular Science and Engineering, U.S. Naval Research Laboratory, Washington, D.C. 20375, and George Mason University, Fairfax, Virginia 22030
A fluorescence-based multianalyte immunosensor has been developed for simultaneous analysis of multiple samples. While the standard 6 × 6 format of the array sensor has been used to analyze six samples for six different analytes, this same format has the potential to allow a single sample to be tested for 36 different agents. The method described herein demonstrates proof of principle that the number of analytes detectable using a single array can be increased simply by using complementary mixtures of capture and tracer antibodies. Mixtures were optimized to allow detection of closely related analytes without significant cross-reactivity. Following this facile modification of patterning and assay procedures, the following nine targets could be detected in a single 3 × 3 array: Staphylococcal enterotoxin B, ricin, cholera toxin, Bacillus anthracis Sterne, Bacillus globigii, Francisella tularensis LVS, Yersinia pestis F1 antigen, MS2 coliphage, and Salmonella typhimurium. This work maximizes the efficiency and utility of the described array technology, increasing only reagent usage and cost; production and fabrication costs are not affected. Recently, the field of array-based technology for multianalyte detection has exploded. These techniques take advantage of the two-dimensional layout of recognition elements to allow simultaneous detection and quantification of multiple analytes. While the vast majority (up to 95%) of microarray-based research utilizes nucleic acids for analyte recognition,1 descriptions of protein-, peptide-, or small molecule-based arrays are increasing. These latter arrays have been used for such diverse purposes as antibody screening,2-4 monitoring of enzyme reactions,5-7 protein-ligand * To whom correspondence should be addressed: Code 6900, U.S. Naval Research Laboratory, Washington, D.C. 20375; (phone) (202)-404-4208; (fax) (202)-404-8688; (e-mail)
[email protected]. † U.S. Naval Research Laboratory. ‡ George Mason University. (1) Grills, G.; Griffin, C.; Massimi, A.; Lilley, K.; Knudtson, K.; VanEe, J. J. Biomol. Tech. 2000, 11, 22. (2) Lueking, A.; Horn, M.; Eickhoff, H.; Bussow, K.; Lehrach, H.; Walter, G. Anal. Biochem. 1999, 270, 103-111. (3) Joos, T. O.; Schrenk, M.; Hopfl, P.; Kroger, K.; Chowdhury, U.; Stoll, D.; Schorner, D.; Durr, M.; Herick, K.; Rupp, S.; Sohn, K.; Hammerle, H. Electrophoresis 2000, 21, 2641-2650. (4) Avseenko, N. V.; Morozova, T. Y.; Ataullakhanov, F. I.; Morozov, V. N. Anal. Chem. 2002, 74, 927-933. (5) Arenkov, P.; Kukhtin, A.; Gemmell, A.; Voloshchuk, S.; Chupeeva, V.; Mirzabekov, A. Anal. Biochem. 2000, 278, 123-131.
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or protein-protein interactions,6-8 ligand fishing,9 and diagnostics/ proteomics.2,10-13 In a particularly notable study, Brown’s group produced 1100-element microarrays and demonstrated binding of 115 antibody-antigen pairs.10 In this, as well as other studies, detection of binding was accomplished by labeling all components of the sample with a marker. The marker was then detected either directly, as with a fluorophore or radioactive tag,5-7,9,10 or indirectly, as with a biotin label.12 Where the samples of interest were related species (e.g., antibody screening), a single tracer antibody with broad specificity was used.3-5,14,15 A niche distinct from those described above is detection of multiple unrelated analytes in complex samples without significant sample pretreatment. Several array-based systems fitting within this last niche have been described.11,13,16-20 While most of these systems employ sandwich immunoassays for multianalyte detection and quantification, one system has been developed that utilized competitive assays performed on microarrays of haptens to detect several contaminants simultaneously.16 The array biosensor developed at the Naval Research Laboratory (NRL) has the additional advantage of rapid ( 0.5), independent of whether capture antibodies were immobilized individually or in combination (Table 1). On the other hand, with the exception of F1 antigen, we observed a significant decrease (P < 0.001) in the fluorescent signals generated by array elements patterned with mixtures of capture antibodies when compared to the individual controls. Decreases in signals ranged from 15% to 75% of control values. This is presumably due to competition for the limited number of potential binding sites on the surface;32 the patterning mixes contained the same concentration of each antibody as in the control lanes (10-20 µg/mL), but the relative abundance of each antibody was decreased by two-thirds in the mixes. It was expected that the fluorescence decrease would correspond to the respective contribution of each component in the patterning mixture, provided each species was present at subsaturating densities. However, it appeared that several capture antibodies retained a larger percentage of their control activities than expected: anti-SEB (mix A, 21% loss in activity), anti-F1 (mix B, no loss), and anti-MS2 (mix C, 25% loss in activity). This effect is most probably due to differences in the titers, affinities, or avidities of the IgG preparations. This effective “dilution” of each capture antibody immobilized on the surface, and the resultant decrease in fluorescence intensities, demonstrate a potentially serious limitation of this method. Depending of the affinity/avidity of each antibody component of the patterning mixes, we anticipate that limits of detection will be higher in the modified protocol than in control experiments (capture antibodies not mixed). This loss in sensitivity may be especially exaggerated in situations where larger numbers of capture antibodies are mixed (e.g., a 6 × 6 array) or when complex sample matrixes may cause some decrease in sensitivity (unpublished). A series of dose-response curves for each component of the patterning mixes and further optimization of the mixes’ compositions should improve, or at least, minimize the loss in signals and, hence, the decrease in sensitivity. However, despite the decreased signals in loci patterned with the antibody patterning mixtures, at the concentrations tested, the antigen-specific loci were still clearly distinguishable from the nonspecific loci. Figure 3 shows the pattern of fluorescence obtained with each of the nine analytes. In all cases, fluorescence from the antigen-specific spot was significantly higher than the other eight spots in the 3 × 3 array (P < 0.001). Furthermore, optimization of the patterning mixtures and tracer cocktails (e.g., using anti-B. globigii and anti-B. anthracis antibodies in different patterning and tracer mixes) minimized fluorescence from crossreactive loci. Thus, cross-reactive spots evident in the control lanes depicted in Figure 2 were not observed in the “mixed” assays (P > 0.1). CONCLUSION Automated arrayer systems are becoming popular tools to deposit large numbers of recognition elements on a single surface in reproducible patterns. While the majority of research done with Analytical Chemistry, Vol. 74, No. 23, December 1, 2002
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microarray technology is based on nucleic acid interactions,1 reports of protein-, receptor-, and antibody-based microarrays are becoming more frequent. Protein microchips have been used for antibody screening;2,44 detection of multiple immunoglobulins or immunoglobulin subtypes;3,4,14,15,45,46 studies of protein-protein,6,47 protein-lipid,47 protein-ligand, or antibody-antigen interactions;10,44,46 detection and quantification of multiple cytokines,11,19 plasma proteins,5,6,10,13 markers of disease,3,4,15 toxins and bacteria,20 or environmental contaminants;16 and enzymatic analyses.5,48 While the system described here utilizes significantly smaller numbers of recognition loci than high-resolution microarrays, the NRL array sensor occupies a unique nichesnamely, rapid detection of multiple unrelated analytes without significant sample pretreatment. (44) Hanes, J.; Schaffitzel, C.; Knappik, A.; Pluckthun, A. Nat. Biotechnol. 2000, 18, 1287-1292. (45) Mendoza, L. G.; McQuary, P.; Mongan, A.; Gangadharan, R.; Brignac, S.; Eggers, M. Biotechniques 1999, 27, 778-788. (46) De Wildt, R. M.; Mundy, T.; Gorick, B. D.; Tomlinson, I. M. Nat. Biotechnol. 2000, 18, 989-994. (47) Zhu, H.; Bilgin, M.; Bangham, R.; Hall, D.; Casamayor, A.; Bertone, P.; Lan, N.; Jansen, R.; Bidlingmaier, S.; Houfek, T.; Mitchell, T.; Miller, P.; Dean, R. A.; Gerstein, M.; Snyder, M. Science 2001, 293, 2101-2105. (48) Zhu, H.; Klemic, J. F.; Chang, S.; Bertone, P.; Casamayor, A.; Klemic, K. G.; Smith, D.; Gerstein, M.; Reed, M. A.; Snyder, M. Nat. Genet. 2000, 26, 283-289.
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The current study demonstrates the proof of concept of a lowtech, inexpensive method for increasing the number of analytes that can be detected on a single microscope slide, without the requirement for costly equipment and lengthy or complex patterning procedures. By using complementary mixtures of capture and tracer antibodies, we were able to detect nine analytes, including proteins, a glycoprotein, Gram-negative and Grampositive bacteria, and a virus, using a simple 3 × 3 array. This facile modification of existing protocols has resulted in a significant increase in the number of analytes unambiguously detected in a single sample, using a rapid (15-min) immunoassay format. ACKNOWLEDGMENT The authors thank Mr. Joel Golden, Dr. Kim Sapsford, Dr. James Delehanty, Mr. Yura Shubin, and Ms. Lisa Shriver-Lake for their input and insightful discussions. Financial support for this research was provided by NASA and Naval Surface Warfare Center. The views expressed here are those of the authors and do not represent those of the U.S. Navy, the Department of Defense, or the U.S. government. Received for review August 1, 2002. Accepted October 7, 2002. AC0260185
