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Preparation of Quantum Dot-Biotin Conjugates and Their Use in Immunochromatography Assays Brian M. Lingerfelt,† Hedi Mattoussi,‡ Ellen R. Goldman,§ J. Matthew Mauro,§ and George P. Anderson*,§
George Mason University, Fairfax, Virginia 22030, and Optical Sciences Division and Center for Bio/Molecular Science and Engineering, U.S. Naval Research Laboratory, Washington, DC 20375
Biotinylated, highly luminescent CdSe-ZnS quantum dot (QD) conjugates were prepared and used in immunofiltration assays. Water-soluble quantum dot surfaces having a homogeneous negative charge density at basic pH were initially coated with a two-domain recombinant maltose-binding protein appended with a positively charged leucine zipper. Biotin functionalization of these electrostatically stabilized QD-protein complexes was then carried out using amine-reactive NHS biotin. These proteincoated biotin-functionalized quantum dot conjugates were incorporated into flow immunofiltration/displacement assays employing Affi-gel agarose resin for antibody immobilization, analyte capture, and immune complex formation with a second biotinylated antibody. A key component of the assay was the use of tetranitromethanemodified NeutrAvidin, used to link the biotinylated QDs to the immune complexes and facilitate their release at basic pH for subsequent quantification. This assay methodology was used to detect as little as 10 ng/mL staphylococcal enterotoxin type-B. Colloidal semiconductor quantum dots (QDs), such as those made of CdSe and CdTe, synthesized using high-temperature solution chemistry techniques, are nanoscale spherical particles with size-dependent tunable luminescent emission.1,2 The sizedependence of their properties results from quantum confinement of electron and hole carriers at dimensions smaller than the bulk Bohr exciton radius.2-4 QDs can be prepared with adequate homogeneity in size and shape to allow emission with narrow bandwidths [full width at half-maximum (fwhm) of ∼30-45 nm].1,4 In addition, when their inorganic core is over-coated with a thin layer of a wider band-gap semiconducting material, a substantial * To whom correspondence should be addressed at CBMSE, Code 6900, Naval Research Laboratory. Phone (202) 404-6033. Fax (202) 767-9594. E-mail:
[email protected]. † George Mason University. ‡ Optical Sciences Division, U.S. Naval Research Laboratory. § Center for Bio/Molecular Science and Engineering, U.S. Naval Research Laboratory. (1) Murray, C. B.; Norris, D. J.; Bawendi, M. G. J. Am. Chem. Soc. 1993, 115, 8706-8715. (2) Gaponenko, S. V. Optical Properties of Semiconductor Nanocrystals; Cambridge University Press: Cambridge, 1999. (3) Efros, A. L.; Rosen, M. Annu. Rev. Mater. Sci. 2000, 30, 475-521. (4) Murray, C. B.; Kagan, C. R.; Bawendi, M. G. Annu. Rev. Mater. Sci. 2000, 30, 545-610. 10.1021/ac034139e CCC: $25.00 Published on Web 06/28/2003
© 2003 American Chemical Society
enhancement in the QD photoluminescence quantum yield (PL QY) occurs.5-7 Finally, use of appropriate surface-capping ligands permits their dispersion in a variety of solvents, including water, which provides a surface suitable for bioconjugation.1,4,8 Colloidal QDs made of CdS, CdSe, and CdTe cores have broad excitation bands that span a wide range of the UV and visible regions of the optical spectrum, which permits simultaneous excitation of several particle sizes at a single wavelength, yet their emission depends only on the nanocrystal radius and the type of core material used.2-4 This feature makes luminescent QDs naturally suitable for multiplexing applications to simultaneously screen for several target analytes. The exceptional resistance of CdSe-ZnS core-shell QDs to photodegradation, combined with the advantages described above, make colloidal luminescent QDs attractive for use in biological tagging studies in which they can offer significant advantages over conventional organic molecular labels in applications such as cellular and biomedical imaging and fluoroimmunoassays. These unique properties have resulted in intense efforts aimed at designing various methods for water-solubilization and stabilization of luminescent colloidal QDs and at developing approaches for forming bioactive conjugates of QDs that can be used in place of, or in addition to, conventional organic dyes.9-18 Our group has developed conjugation strategies based on electrostatic attraction between surface-charged water-soluble CdSe-ZnS core-shell nanocrystals and recombinant proteins appended with an electrostatic interaction domain.13,16-18 In this technique, negatively (5) Hines, M. A.; Guyot-Sionnest, P. J. Phys. Chem. 1996, 100, 468-471. (6) Dabbousi, B. O.; Rodrigez-Viejo, J.; Mikulec, F. V.; Heine, J. R.; Mattoussi, H.; Ober, R.; Jensen, K. F.; Bawendi, M. G. J. Phys. Chem. 1997, 101, 94639475. (7) Peng, X.; Schlamp, M. C.; Kadavanich, A. V.; Banin, U.; Alivisatos, A. P. J. Am. Chem. Soc. 1997, 119, 7019-7029. (8) Mattoussi, H.; Cumming, A. W.; Murray, C. B.; Bawendi, M. G.; Ober, R. Phys. Rev. B 1998, 58, 7850-7863. (9) Mattoussi, H.; Kuno, M. K.; Goldman, E. R.; Anderson, G. P.; Mauro, J. M. In Optical Biosensors: Present and Future; Ligler, F. S., Rowe, C. A., Eds.; Elsevier: The Netherlands, 2002; pp 537-569. (10) Bruchez, M., Jr.; Moronne, M.; Gin, P.; Weiss, S.; Alivisatos, A. P. Science 1998, 281, 2013-2016. (11) Chan, W. C. W.; Nie, S. M. Science 1998, 281, 2016-2018. (12) Mitchell, G. P.; Mirkin, C. A.; Letsinger, R. L. J. Am. Chem. Soc. 1999, 121, 8122-8123. (13) Mattoussi, H.; Mauro, J. M.; Goldman, E. R.; Anderson, G. P.; Sundar, V. C.; Mikulec, F. V.; Bawendi, M. G. J. Am. Chem. Soc. 2000, 122, 1214212150. (14) Pathak, S.; Choi, S. K.; Arnheim, N.; Thompson, M. E. J. Am. Chem. Soc. 2001, 123, 4103-4104.
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charged CdSe-ZnS QDs are first prepared having have stable bidentate dihydrolipoic acid (DHLA) surface capping ligands terminated with carboxylic acid groups to enhance aqueous solubility.13 QD-bioconjugate formation is achieved via electrostatic self-assembly of DHLA-capped QDs with two-domain recombinant proteins genetically engineered with a positively charged basic leucine zipper (zb) interaction domain appended onto the protein functional domain or with naturally occurring avidin (a positively charged glycoprotein tetramer found in avian egg white).13,16-18 We designed and have used extensively maltose-binding protein (MBP)-zb, a two domain recombinant protein, which was found to bind tightly to the DHLA coating on QD surfaces and to provide surface passivation while maintaining its maltose-binding capability.13 Furthermore, conjugation to MBP-zb resulted in a dramatic increase in the nanocrystal PL quantum yield.13 In subsequent work, we have made considerable use of QDs that are at least partially coated with MBP-zb (in mixed surface QDprotein conjugates), since the presence of this functionality allows facile chemical manipulation and affinity purification of bioconjugates.16-18 To develop immunoassays, QD bioconjugates were prepared that included either a Protein G-zb or avidin, which facilitated the binding of antibodies or biotinylated-antibodies.16-18 Although these QD-conjugate preparations performed well in direct and sandwich immunoassays carried out in microtiter plate wells, we were interested in developing a method not so constrained by surface area and providing an enhanced antigen-capture efficiency. In the present work, we used QD-MBP-zb bioconjugates as starting materials for preparing biotinylated quantum dot reagents. The biotinylated QD-MBP-zb (Bt-QD-MBP-zb) conjugates were used as the tracer reagent in fluoroimmunoassays in conjunction with a linker protein, nitroavidin or nitro-NeutrAvidin, for attachment to immune complexes, formed using biotinylated antibodies (IgGs). These two reagents used in combination make an ideal pair for the development of a novel flow immunofiltration/ displacement assay. The efficacy of the new Bt-QD-MBP-zb’s conjugates as signal-generating agents in tandem with nitroNeutrAvidin was demonstrated. EXPERIMENTAL METHODS Materials. Affi-gel active NHS ester agarose, micro Bio-Spin chromatography columns, and P-10 Bio-Gel were obtained from Bio-Rad (Hercules, CA). Amylose resin was purchased from New England BioLabs, Inc (Beverly, MA). NHS-LC-biotin, NHSiminobiotin-HBr, and NeutrAvidin were purchased from Pierce (Rockland, IL). Tetranitromethane, avidin, casein, and bovine serum albumin (BSA) were obtained from Sigma (St. Louis, MO). Goat IgG and rabbit anti-goat IgG were purchased from Rockland (Gilbertsville, PA). Staphylococcal enterotoxin B (SEB), sheep antiSEB IgG, and affinity-purified rabbit anti-SEB IgG were obtained from Toxin Technologies (Sarasota, FL). Triton X-100 was (15) Mamedova, N. N.; Kotov, N. A.; Rogach, A. L.; Studer, J. Nano Lett. 2001, 1, 281-286. (16) Goldman, E. R.; Anderson, G. P.; Tran, P. T.; Mattoussi, H.; Charles, P. T.; Mauro, J. M. Anal. Chem. 2002, 74, 841-847. (17) Goldman, E. R.; Balighian, E. D.; Kuno, M. K.; Labrenz, S.; Tran, P. T.; Anderson, G. P.; Mauro, J. M.; Mattoussi, H. Phys. Status Solidi B 2002, 229, 407-414. (18) Goldman, E. R.; Balighian, E. D.; Mattoussi, H.; Kuno, M. K.; Mauro, J. M.; Tran, P. T.; Anderson, G. P. J. Am. Chem. Soc. 2002, 124, 6378-6382.
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purchased from Calbiochem (La Jolla, CA). Photoluminescence was measured using a microtiter plate reader from Tecan U.S. (Durham, NC). The PBT wash buffer used for SEB sandwich assays was 8.3 mM phosphate pH 7.2 + 0.05% Triton X-100 (v/v) + 0.02% sodium azide (w/v). Capture Antibody Immobilization onto Affi-gel Beads. Affigel agarose beads (10/15) were thoroughly washed with cold Milli-Q water in order to remove the stabilizing solvent. The beads were then derivatized with antibodies (goat IgG and sheep antiSEB IgG) by overnight incubation at 4 °C in 50 mM borate buffer (pH 9), 40 mM NaCl. Any unbound antibody was removed from the modified beads by washing with 40 mL of PBS (phosphate buffered saline), pH 7.4, and the capture beads were stored at 4 °C. Concentrations of stock suspensions of Affi-gel beads reacted with goat IgG and sheep anti-SEB IgG (as prepared) were ∼8 mg/mL and ∼1 mg/mL, respectively. Preparation of Bt-QD-MBP-zb. The quantum dots and the MBP-zb used in the present study were prepared in our laboratory, as described previously.13,16,18 QD-MBP-zb conjugates were first prepared by combining the QDs (4.50 µL of 24 µM stock solutions in borate buffer and MBP-zb protein (54 µL of a 1.8 mg/mL stock solution) in 100 µL of 10 mM sodium tetraborate/10 mM NaCl buffer, pH 9. The protein-to-nanocrystal nominal molar ratio of ∼16:1 was high enough to permit effective shielding of the QD surfaces from any undesired electrostatically driven interactions with the surrounding environment during subsequent steps. After reaction for 1 h at 25 °C, the QD-MBPzb conjugates were loaded, and consequently bound, onto amylose resin in a small column equilibrated with 2 mL of borate buffer.13 In parallel, 50 µL of NHS-LC-biotin (from a 1.42 mg/mL stock solution in DMSO) was diluted into 2 mL of borate buffer.19 The NHS-LC-biotin solution was immediately loaded onto the amylose column containing the immobilized QD-MBP-zb conjugates and allowed to flow freely until replenishing the full length of the column, whereupon the column was capped and allowed to equilibrate for 15 min to allow biotinylation of accessible lysine -amino groups of the MBP-zb. After washing the column with 1 mL of borate buffer, the Bt-QD-MBP-zb conjugates were eluted with 1 mL of 10 mM maltose solution in borate buffer. Preparation of iminobiotinylated QD-MBP-zb conjugates was performed identically, except that NHS-iminobiotin was substituted for NHS-LC-biotin.19 Preparation of Biotinylated Antibodies. Biotinylated capture antibodies were prepared at pH 9 by reaction with NHS-LC-biotin ester at a 1:5 antibody/NHS-LC-biotin molar ratio for at least 30 min, followed by gel filtration purification using Bio-Gel P10 column chromatography. Preparation of Nitro-Avidin and Nitro-NeutrAvidin. Tetranitromethane adds a nitro group to the ortho position of accessible tyrosine residues.20,21 In avidin and related biotinbinding proteins, formation of nitrotyrosine in contact with the biotin-binding site allows pH-dependent control of protein-ligand binding.21,22 To prepare nitroavidin, 20 µL of tetranitromethane (19) Hermanson, G. T. Bioconjugate Techniques; Academic Press: London (U.K.), 1996; Chapter 8. (20) Means, G. E., Feeney, R. E., Eds.; Chemical Modifications of Proteins; HoldenDay: San Francisco, CA, 1971. (21) Morag, E.; Bayer, E. A.; Wilchek, M. Biochem. J. 1996, 316, 193-199. (22) Morag, E.; Bayer, E. A.; Wilchek, M. Anal. Biochem. 1996, 243, 257-263.
Figure 1. Sketch of the two types of assays, direct (a) and sandwich (b), performed on the Affi-gel bead surfaces employing biotinylated QD-MBP-zb reagents for signal generation. “Bt” designates biotin (either LC-biotin or iminobiotin) groups attached to IgG or MBP-zb. QDs emitting at 555 nm were used in all experiments.
(in ∼6-fold molar excess) was added to avidin present at 1.1 mg/ mL in borate buffer. After reacting for 45 min at room temperature, the nitro-modified avidin was separated from excess tetranitromethane using Bio-Gel P-10; the first eluted yellow band contained the nitroavidin. Nitro-NeutrAvidin was prepared using the same protocol but substituting NeutrAvidin for avidin. The amount of labeling was determined spectrophotometrically using the nitrotyrosine absorption at 428 nm (428 ) 4200 M‚cm-1). Binding of Bt-QD-MBP-zb Conjugates to Affi-gelAntibody BeadssA Direct Binding Demonstration. Slurries of goat IgG-derivatized Affi-gel beads (150 µL) were loaded into Bio-Spin centrifuge columns (100-µL packed-bead volume), followed by addition of 100-µL aliquots of biotinylated rabbit antigoat IgG solutions at various concentrations, plus a control column prepared without addition of biotinylated rabbit anti-goat; antibody stock solutions were diluted into PBS containing 1 mg/mL BSA and 1 mg/mL casein. Capped columns were shaken briefly, equilibrated for 1 h, and washed three times with 0.25 mL of PBS. Subsequently, 100-µL aliquots of nitroavidin (∼50 µg/mL in PBS)
were applied, and the columns were shaken briefly and allowed to equilibrate for 1 h. After washing with PBS as before, 100 µL of the Bt-QD-MBP-zb complex (prepared as described above and diluted in borate buffer to a concentration of 10.8 pM) was applied to each column (Figure 1A). After addition of the BtQD-MBP-zb conjugates, the columns were equilibrated for 90 min and then washed with PBS. Finally, the bound QDs were eluted by three (150-µL) additions of 5 mM biotin solution in 10 mM borate, 10 mM NaCl buffer, pH 10.8, with 5 min incubations between washes. Assays that employ avidin or NeutrAvidin as the bridging proteins involve simply substituting one of these proteins for nitroavidin in the above description. Then a fraction of each sample was subdivided into three wells (100 µL/well) in a white 96-well plate. Fluorescence was read using a Tecan SpectraFluor microplate reader equipped with a 310-nm excitation filter and a 530-nm long-pass emission filter. The average (SEM minus the control was plotted. SEB Sandwich Assay with Nitroavidin. Affi-gel beads (150 µL) activated with sheep anti-SEB IgG were loaded into Bio-Spin Analytical Chemistry, Vol. 75, No. 16, August 15, 2003
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centrifuge columns (100-µL packed volume) followed by the addition of 100-µL aliquots of SEB at various concentrations, as well as one control column lacking SEB. SEB was diluted using PBT containing 1 mg/mL BSA and 1 mg/mL casein. The columns were shaken, equilibrated for 1 h, and washed with PBT. Biotinylated rabbit anti-SEB IgG (100 µL at 10 µg/mL, diluted from stock solution using PBT containing BSA and casein) was added to the beads. The columns were again briefly shaken and allowed to equilibrate for 1 h, followed by washing with PBT. Binding of nitroavidin to the last layer of biotinylated rabbit antiSEB antibodies was carried out by adding 100 µL of nitroavidin (diluted to ∼50 µg/mL in BSA/casein/PBT buffer). After equilibration for 1 h and subsequent washing, 100 µL of biotinylated QD-MBP-zb conjugate (at a concentration of 10.8 pM as described above) was added to each column and reacted for an additional 1 h (Figure 1B). At that time, the beads were washed with PBT, followed by elution of specifically bound QDs using three washes (150 µL each) of 5 mM biotin in 10 mM sodium borate/10 mM NaCl (pH 10.8) with 5 min of incubation/shaking between washes. The protocol for the SEB sandwich assay with nitro-NeutrAvidin was identical, with the exception that nitroNeutrAvidin was used in place of nitroavidin. The eluted samples were analyzed as above. RESULTS AND DISCUSSION A new type of fluoroimmunoassay reagent based on luminescent quantum dots coated with a biotinylated surface-passivating protein was prepared and tested in an immunochromatographic format. In this immunochromatographic format, Affi-gel resin was used as the assay support surface for antigen capture because of its high binding capacity. In the present work, the packed volume of capture beads used in Bio-Spin columns was 100 µL. This corresponds to ∼800 µg and 100 µg of goat IgG and sheep antiSEB IgG immobilized onto the Affi-gel beads in the columns for each assay, respectively. Immunoassays using 100-µL columns of Affi-gel beads can bind a substantially larger amount of fluorescent QD conjugate and with higher efficiency than can bind to individual microtiter plate wells, each of which has a capacity of only ∼100 ng IgG.23 The following sections describe assays in which the conditions for conjugate formation, type of bridging units employed, and assay format were varied, with the ultimate goal of enhancing detection sensitivity. Both direct binding and sandwich assays were performed; SEB was the analyte employed in the sandwich assays. Elution of Bound QD Complexes in a Direct Binding Assay. In all experiments, the immobilized Bt-QD-MBP-zb conjugates were displaced from the Affi-gel resin once the assay was complete, and the fluorescence signal from those QD complexes was measured; this allowed measurements of a “positive” signal. The binding between avidin and biotin is for all practical purposes irreversible; thus, we used chemically modified reagents with reversible binding behavior. To this end, we first tested NHS-iminobiotin for forming the biotin-labeled QD-MBPzb conjugates, instead of NHS-LC-Biotin, with avidin being the bridging protein.19 A pH-dependent binding and releasing assay (23) Harlow, E.; Lane, D. Antibodies, A Laboratory Manual; Cold Spring Harbor Laboratory: New York, 1988.
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was utilized in which iminobiotin on the QD-MBP-zb was bound by avidin at high pH (∼pH 10.5). In an ealier step, the avidin was allowed to bind to biotinylated rabbit anti-goat IgG that had been immobilized on the Affi-gel beads. The amount of QDs bound is quantified after their release from the column at lower pH (∼pH 7) in the presence of extra free biotin. The excess biotin increases the efficiency of iminobiotin-QD-MBP-zb dissociation from the avidin bridges and their release from the gel matrix, as shown in Figure 2a. This assay format did not improve the sensitivity, however, because only a limit of detection of 5 µg/mL of biotinylated rabbit anti-goat IgG was measured (Figure 2b). The lack of improvement in the sensitivity could be attributed to the fact that during immune complex formation, the column was exposed to a pH higher than optimal conditions for antibody binding, while the iminobiotin-QD-MBP-zb conjugates were eluted using a lower pH range, a pH where the QDs are less stable.13 Direct Binding Assay Using Nitroavidin as the Bridging Protein. In a study performed by Morag et al., nitroavidin was used as the solid support from which biotinylated BSA was released as the pH was raised above 10.21 We extended this methodology to the QD-MBP-zb-biotin conjugate binding and subsequent release by modifying the avidin-bridging unit with tetranitromethane, which reacts with the critical binding site tyrosine on the avidin protein and alters its affinity for biotin.20,21 On average, 3.4 tyrosine residues per avidin tetramer appeared to be nitrated. This assay format permits more favorable pH conditions for both antibody binding (neutral pH) and the QDs, which are better suited in basic pH conditions.13 Nitrotyrosinemodified avidin still bound biotin tightly at neutral pH, but at basic pH, the nitrotyrosine deprotonates, thus decreasing the nitroavidin’s affinity for biotin on both the biotinylated rabbit anti-goat IgG and Bt-QD-MBP-zb conjugates, resulting, most likely, in elution of a mixture of free Bt-QD-MBP-zb and Bt-QDMBP-zb complexed with nitroavidin. After binding the biotinylated rabbit anti-goat IgG onto the gel-activated resin, addition of nitroavidin and subsequently Bt-QD-MBP-zb conjugates were performed in PBS at pH 7.4 (see Figure 3a). Once assembled, the QD complexes were released by raising the pH to 10.8, and the fluorescence of the eluent was measured (Figure 3b). This binding and release format using modified avidin bridges improved the sensitivity as lower limits of detection were reached (500 ng/mL of biotinylated rabbit anti-goat IgG). Nonetheless, we found that nonspecific binding of Bt-QD-MBP-zb complexes to the resin interfered with attempts to reach better sensitivity. Sandwich Assay for SEB. After demonstrating increased sensitivity for the direct binding assay using nitroavidin as the bridging protein between biotinylated QD-MBP-zb conjugates and rabbit anti-goat-IgG immobilized onto the Affi-gel, the immunofiltration assay was further applied to a sandwich immunoassay for SEB utilizing nitroavidin. A limit of detection determined using this format was at least 500 ng/mL of SEB (data not shown), but sensitivity could not be further improved because of a rather high background signal, a problem attributed to nonspecific binding of the QD-MBP-zb-biotin conjugates to the Affi-gel beads. To identify the origins of the nonspecific binding, a modified assay was performed in which the setup described above was repeated with the omission of one of the three
Figure 2. (a) Sketch of the direct binding assay performed on the Affi-gel bead surfaces using goat IgG and rabbit anti-goat IgG, followed by elution of the iminobiotinylated QD-MBP-zb reagents at neutral pH. (b) Relative fluorescence for various concentrations of biotin-rabbit antigoat IgG (N ) 3) is shown. “Blk” indicates the signal of a buffer blank of equal volume added to the wells containing QD conjugates. Excitation was at 310 nm; photoluminescence signal collection employed a cutoff emission filter at 530 nm. QDs emitting at 555 nm were used.
components (SEB, biotinylated rabbit anti-SEB IgG, or nitroavidin), with the control not lacking any component. We concluded that the nitroavidin was binding nonspecifically to the Affi-gel beads, thereby immobilizing the QD-MBP-zb-biotin conjugates, which would account for the measured high background signal mentioned above. This rather substantial nonspecific binding of nitroavidin may be driven by electrostatic adsorption of the avidin proteins directly onto the Affi-gel beads. Avidin has a basic isoelectric point (pI of 10), which implies that it has a nonnegligible charge density on its surface in PBS buffer and could bind (via electrostatic attractions) to the Affi-gel beads. To circumvent this problem, nitrotyrosine-modified NeutrAvidin (nitro-NeutrAvidin) was employed as the bridging protein between QD-MBP-zb-biotin conjugates and biotinylated rabbitanti-SEB antibody. Nitro-NeutrAvidin has a more acidic isoelectric point (pI ) 6.3) and lower nonspecific interaction with the Affigel matrix. This, in turn, reduces nonspecific adsorption of biotinylated QD-MBP-zb-biotin conjugates on the beads. This modification increased the signal-to-background ratios substantially, resulting in improved sensitivity of the assay, as shown by the data reported in Figure 4. In particular, a substantially lower limit of detection of 10 ng/mL SEB concentration was measured following the above modification in the assay design.
Despite this substantial improvement in the detection sensitivity reached using the present setup (e.g., biotinylated anti-SEB antibodies, nitro-NeutrAvidin bridging protein, and luminescent biotinylated QD-MBP-zb transducer complexes, combined with a larger capture surface assay format), further improvements are necessary to meet the need for a rapid and sensitive immunoassay. Although these properties have been achieved by other flow immunofiltrations assays,24-26 QDs may one day improve upon these assays by making single column multianalyte tests possible. Although there are currently numerous diagnostics methods for laboratory immunoassays,27-30 few techniques are suitable for field (24) Shah, J.; Wilkins, E. Electroanalysis 2003, 15, 157-167. (25) Usleber, E.; Dietrich, R.; Burk, C.; Schneider, E.; Martlbauer, E. J. AOAC Int. 2001, 84, 1649-1656. (26) Abdel-Hamid, I.; Ivnitski, D.; Atanasov, P.; Wilkins, E. Anal. Chem. ACTA 1999, 399, 99-108. (27) Uehara, M.; Lapcik, O.; Hampl, R.; Al-Maharik, N.; Makela, T.; Wahala, K.; Mikola, H. J. Steroid Biochem. 2000, 72, 273-282. (28) McBride, M. T.; Gammon, S.; Pitesky, M.; O’Brien, T. W.; Smith, T.; Aldrich, J.; Langlois, R. G.; Colston, B.; Venkateswaran, K. S. Anal. Chem. 2003, 75, 1924-1930. (29) Hale, M. L.; Campbell, T. A.; Campbell, Y. G.; Fong, S. E.; Stiles, B. G. J. Immunol. Methods 2001, 257, 83-92. (30) Kijek, T. M.; Rossi, C. A.; Moss, D.; Parker, R. W.; Henchal, E. A. Immunol. Methods 2000, 236, 9-17.
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Figure 3. (a) Sketch of the direct binding assay performed on the Affi-gel bead surfaces using goat IgG and rabbit anti-goat IgG. The assay was constructed at pH 7.4 using nitroavidin as the bridging protein. Elution of the biotinylated QD-MBP-zb reagents along with a fraction of avidin-bound QD-MBP-zb-biotin complexes was achieved by increasing the pH of the medium to 10.8. (b) Relative fluorescence (over the control) for various concentrations of biotin-rabbit anti-goat IgG added to goat IgG Affi-gel beads (N ) 3) is shown. Excitation was at 310 nm; cut-off emission filter at 530 nm was used for signal collection. QDs emitting at 555 nm were used.
testing.31-35 Field assays are important for providing rapid information when time is critical, whether that be for first responders dealing with a potential biohazard or medical personnel providing treatment in remote locations. A multianalyte QD flow immunofiltration assay could provide the sensitivity and flexibility required to be a useful field analysis method. CONCLUSIONS In the present investigation, novel biotinylated QD conjugates were designed by chemically attaching biotin groups onto the functional domain of two-domain recombinant maltose binding protein, MBP-zb, in a preformed QD-MBP-zb conjugate away
Figure 4. Sandwich assay for SEB performed using sheep antiSEB and biotinylated rabbit anti-SEB IgGs. The assay was carried out at pH 7.4 with nitro-NeutrAvidin as the bridging protein. Elution of the biotinylated QD-MBP-zb reagents was achieved by increasing the pH of the medium to 10.8. Relative fluorescence (over the control) for various concentrations of SEB (N ) 3) is shown. Excitation was at 310 nm; cut-off emission filter at 530 nm was used for signal collection. QDs emitting at 555 nm were used. 4048 Analytical Chemistry, Vol. 75, No. 16, August 15, 2003
(31) Mya, M. M.; Saxena, R. K.; Roy, A.; Roy, K. B. Parasitol. Res. 2003, 89, 371-374. (32) Rowe-Taitt, C. A.; Hazzard, J. W.; Hoffman, K. E.; Cras, J. J.; Golden, J. P.; Ligler, F. S. Biosens. Bioelectron. 2000, 15, 579-589. (33) Anderson, G. P.; Nerurkar, N. L. J. Immunol. Methods 2002, 271, 17-24. (34) Chanteau, S.; Rahalison, L.; Ratsitorahina, M.; Mahafaly, M.; Rasolomaharo, M.; Boisier, P.; O’Brien, T.; Aldrich, J.; Keleher, A.; Morgan, C.; Burans, J. Int. J. Med. Microbiol. 2000, 290, 279-283. (35) Ballesteros, B.; Barcelo, D.; Dankwardt, A.; Schneider, P.; Marco, M. P. Anal. Chem. Acta 2003, 475, 105-115.
from the QD surface. This approach builds on previous success with self-assembling, stable, aggregate-free, and functional QDMBP-zb conjugates.13 The above biotinylated QD-MBP-zb enabled the formation of QD immunocomplexes via avidin and NeutrAvidin bridges, thus utilizing the well-characterized and highly specific biotin-avidin interactions. Additionally, these QD complexes were utilized in a flow immunofiltration assay that, owing to the large capture surface, has improved capture efficiency and capacity. We also took advantage of the pH-sensitive binding that tetranitromethane-modified avidin and NeutrAvidin exhibits for biotin to optimize the assay conditions and its sensitivity. In particular, the substitution of nitro-NeutrAvidin for nitroavidin, in a sandwich assay screening for SEB, reduced interference from nonspecific interactions with the bead surfaces and lowered the limit of detection to 10 ng/mL of SEB. The present results promise to expand the range of applications in which biotinylated QD conjugates could be easily employed. This work brings QD bioconjugates a step closer to use in conventional diagnostic assays and implies that simple modifica-
tion of QD conjugates can allow them to be tailored to a variety of purposes. Although further optimization of the reagents and the parameters involved in the assay design is still needed, QD labels have the potential to improve the sensitivity of immunoassays. ACKNOWLEDGMENT We thank Dr. Peter Sveshnikov of the Russian Research Center of Molecular Diagnostics and Therapies, Moscow, for his helpful discussions. We also thank Dr. K. Ward at the Office of the Naval Research (ONR) for financial support, Grants no. N0001499WX30470 and no. N0001400WX20094. The views, opinions, and findings described in this report are those of the authors and should not be construed as official Department of the Navy positions, policies, or decisions. Received for review February 12, 2003. Accepted May 27, 2003. AC034139E
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