Conjugation of Luminescent Quantum Dots with Antibodies Using an

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Anal. Chem. 2002, 74, 841-847

Conjugation of Luminescent Quantum Dots with Antibodies Using an Engineered Adaptor Protein To Provide New Reagents for Fluoroimmunoassays Ellen R. Goldman,*,† George P. Anderson,† Phan T. Tran,† Hedi Mattoussi,*,‡ Paul T. Charles,† and J. Matthew Mauro†

Center for Bio/Molecular Science and Engineering and Division of Optical Sciences, U.S. Naval Research Laboratory, Washington, D.C. 20375

We describe the preparation and characterization of bioinorganic conjugates made with highly luminescent semiconductor CdSe-ZnS core-shell quantum dots (QDs) and antibodies for use in fluoroimmunoassays. The conjugation strategy employs an engineered molecular adaptor protein, attached to the QDs via electrostatic/hydrophobic self-assembly, to link the inorganic fluorophore with antibodies. In this method, the number of antibodies conjugated to a single QD can be varied. In addition, we have developed a simple purification strategy based on mixed-composition conjugates of the molecular adaptor and a second two-domain protein that allows the use of affinity chromatography. QD-antibody conjugates were successfully used in fluoroimmunoassays for detection of both a protein toxin (staphylococcal enterotoxin B) and a small molecule (2,4,6-trinitrotoluene). Fluorescent labeling of biological materials using small organic dyes is widely employed in the life sciences and has been used in a variety of applications that include diagnostics and biological imaging.1 Additionally, sensitive immunoassays have been developed using fluorescent-labeled microspheres.2 Organic fluorophores, however, have characteristics that limit their effectiveness for such applications. These limitations include narrow excitation bands and broad emission bands with red spectral tails, which can make simultaneous evaluation of several light-emitting probes problematic due to spectral overlap. Also, many organic dyes exhibit low resistance to photodegradation.3 Luminescent colloidal semiconductor nanocrystals (quantum dots, QDs) are inorganic fluorophores that have the potential to circumvent some of the functional limitations encountered by organic dyes in biotechnological applications. In particular, CdSeZnS core-shell QDs exhibit size-dependent tunable photolumi* Corresponding authors. E.R.G.: (e-mail) [email protected]; (tel) 202-404-6052; (fax) 202-767-9594. H.M.: (e-mail) [email protected]; (tel) 202-767-9473. † Center for Bio/Molecular Science and Engineering. ‡ Division of Optical Sciences. (1) Schro ¨ck, E.; du Manoir, E.; Veldman, T.; Schoell, B.; Wienberg, J.; FergusonSmith, M. A.; Ning, Y.; Ledbetter, D. H.; Bar-Am, I.; Soenksen, D.; Garini, Y.; Ried, T. Science 1996, 273, 494-497. (2) Hall, M.; Kazakova, I.; Yao, Y. M. Anal. Biochem. 1999, 272, 165-170. (3) Hermanson, G. T. Bioconjugate Techniques; Academic Press: London, 1996; Chapter 8. 10.1021/ac010662m Not subject to U.S. Copyright. Publ. 2002 Am. Chem. Soc.

Published on Web 01/03/2002

nescence (PL) with narrow emission bandwidths (fwhm, of ∼3045 nm) that span the visible spectrum and broad absorption spectra that allow simultaneous excitation of several particle sizes at a single wavelength.4-8 These nanoparticles have a high quantum yield and a high resistance to photodegradation. Photoluminescence from these QDs can be detected at concentrations comparable to organic dyes using conventional fluorescence methods, and individual bioconjugated QDs are easily observable by confocal microscopy.9 We have recently developed a novel conjugation strategy, based on electrostatic interactions between negatively charged dihydrolipoic acid (DHLA)-capped CdSe-ZnS core-shell QDs and a positively charged leucine zipper10 interaction domain appended onto the C-terminus of engineered recombinant proteins.9,11-13 Our electrostatic self-assembly approach represents a departure from previously reported conjugation techniques, e.g., those that use avidin-biotin technology or covalent cross-linking to form QD-antibody bioconjugates.14,15 The electrostatic noncovalent self-assembly approach we have used in this study, and in the work reported previously,9,11 to conjugate luminescent QDs with two-domain recombinant proteins (4) Murray, C. B.; Norris, D. J.; Bawendi, M. G. J. Am. Chem. Soc. 1993, 115, 8706-8715. (5) Mattoussi, H.; Radzilowski, L. H.; Dabbousi, B. O.; Thomas, E. L.; Bawendi, M. G.; Rubner, M. F. J. Appl. Phys. 1998, 83, 7965-7974. (6) Rodriguez-Viejo, J.; Mattoussi, H.; Heine, J. R.; Kuno, M. K.; Michel, J.; Bawendi, M. G.; Jensen, K. F. J. Appl. Phys. 2000, 87, 8526-8534. (7) Hines, M. A.; Guyot-Sionnest, P. J. Phys. Chem. 1996, 100, 468-471. (8) 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. B 1997, 101, 9463-9475. (9) 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. (10) O’Shea, E. K.; Lumb, K. J.; Kim, P. S. Curr. Biol. 1993, 3, 658-667 and references therein. (11) Mattoussi H.; Mauro, J. M.; Goldman, E. R.; Green, T. M.; Anderson, G. P.; Sundar, V. C.; Bawendi, M. G. Phys. Stat. Sol. 2001, 224, 277-283. (12) Goldman, E. R.; Mattoussi, H.; Tran, P. T.; Anderson, G. P.; Mauro, J. M. In Semiconductor Quantum Dots; Fafard, S., Huffaker, D., Leon, R., Noetzel, R., Eds.; Materials Research Society Proceedings, Pittsburgh, 2001; Vol. 642, J2.8.1-J2.8.6. (13) Tran, P. T.; Goldman, E. R.; Mattoussi, H.; Anderson, G. P.; Mauro, J. M. In Nanoparticles and Nanostructured Surfaces: Novel Reporters with Biological Applications; Murphy, C. J., Ed.; Proc. SPIE 2001, 4258, 1-7. (14) Bruchez, M., Jr.; Moronne, M.; Gin, P.; Weiss, S.; Alivisatos, A. P. Science 1998, 281, 2013-2016. (15) Chan, W. C. W.; Nie, S. Science 1998, 281, 2016-2018.

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maltose binding protein (MBP, here employed as a purification tool) as a model protein.9 Using this novel PG-based adaptor protein, QD-IgG bioconjugates can be formed readily, and the conjugates can be used in fluorescence-based assays to detect proteins and small molecules. We show that both direct and sandwich fluoroimmunoassays using these antibody-conjugated QDs can be performed for detection of staphylococcal enterotoxin B (SEB), a causative agent of food poisoning.16 We also describe the use of antibody-conjugated QDs in both plate-based and continuous-flow immunoassays for the detection of low levels of the explosive 2,4,6-trinitrotoluene (TNT) in aqueous samples.

Figure 1. (A) Cartoon of a mixed-surface composition QD-antibody conjugate. The PG-zb (IgG-binding β2 domain of streptococcal protein G modified by genetic fusion with a dimer-forming positively charged tail) acts as a molecular adaptor to connect the inorganic CdSeZnS core-shell nanocrystal quantum dot (capped with a negatively charged dihydrolipoic acid surface) with the Fc region of IgG. The MBP-zb (maltose-binding protein appended with the dimer-forming positively charged tail) serves as a purification tool for separating QDIgG conjugate away from excess IgG through use of affinity chromatography using cross-linked amylose resin. (B) DNA and translated protein sequences of the relevant region of pBadG-zb.

extends and complements existing QD-labeling methods. Conjugate preparation is simple, reproducible, and easily achieved. Other more conventional covalent techniques do not necessarily lead to aggregate-free QD conjugate preparations, and they may require several steps to reach the final conjugate product.9,14,15 Here we describe the preparation of bioconjugates of CdSeZnS QDs with antibodies using mixed-composition conjugates containing both molecular adaptor protein and a second protein used as a purification tool (Figure 1A). The engineered adaptor protein employs the immunoglobulin G (IgG)-binding β2 domain of streptococcal protein G (PG) modified by genetic fusion with the same positively charged leucine zipper interaction domain we previously developed and characterized using Escherichia coli 842

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EXPERIMENTAL SECTION QD Preparation. CdSe-ZnS quantum dots capped with DHLA were prepared and characterized as described previously.9 For most of the studies described, DHLA-capped QDs with emission maximum of 570 nm were used, although in one direct binding experiment, nanocrystals with a slightly smaller core exhibiting an emission maximum of 555 nm were used. Construction of pBadG-zb for Expression of the Molecular Adaptor Protein. The gene for the two-domain protein G-basic leucine zipper (PG-zb) fusion protein was constructed using standard gene assembly and cloning techniques. The coding sequence for the β2 IgG binding domain of streptococcal PG17 and a DNA fragment containing the coding sequences for the polyAsn linker from the pMal plasmid series (New England Biolabs), a dimer-promoting cysteine, the basic leucine zipper,9 and Cterminal hexahistidine tag were cloned into the expression vector pBad/HisB (Invitrogen) to produce the final PbadG-zb construct (Figure 1B). PG-zb Adaptor Protein Expression and Purification. An overnight culture of freshly transformed E. coli Top10 (Invitrogen) was diluted 1/100 into LB media containing 50 µg/mL ampicillin, and the culture was grown with vigorous shaking at 37 °C to an OD600 of ∼0.5. L-(+)-Arabinose (Sigma) was then added to a final concentration of 0.002% (w/v), and the cultures were grown for an additional 2 h before harvesting the cells by centrifugation at 4 °C. The cell pellet from a 500-mL culture was suspended in 12.5 mL of buffer A (100 mM NaH2PO4/10 mM Tris/6 M guanidine hydrochloride, pH 8.0); all soluble material was tumbled for 45 min at room temperature with 3 mL of NiNTA (Qiagen) resin previously equilibrated with buffer A. Resin was loaded into a column, and contaminating proteins were removed by washing with 10 column volumes of buffer A, 10 column volumes of buffer B (10 mM NaH2PO4/10 mM Tris/8 M urea, pH 8.0), and 20 column volumes of buffer C (10 mM NaH2PO4/10 mM Tris/8 M urea, pH 6.3). The PG-zb adaptor protein was refolded while bound to the column by washing with PBS buffer. The renatured protein was eluted with 50 mM NaH2PO4/300 mM NaCl/250 mM imidazole (pH 6.3). SDS polyacrylamide gel electrophoresis under reducing and nonreducing conditions revealed that the purified protein product consisted of the desired disulfide-linked dimer (estimated 75 wt %) together with unlinked monomeric protein (16) Tempelman, L. A.; King, K. D.; Anderson, G. P.; Ligler, F. S. Anal. Biochem. 1996, 233, 50-57. (17) Mauro, J. M.; Cao, L. K.; Kondracki, L. M.; Walz, S. E.; Campbell, J. R. Anal. Biochem. 1996, 235, 61-72.

(25%). The protein was aliquoted and quick frozen for storage at -80 °C. Formation and Purification of Antibody-QD Conjugates. A mixed-surface composition strategy was developed to enable rapid and easy removal of excess unconjugated antibody after coupling of IgG to QDs. Mixed-surface QDs were prepared by incubating DHLA-capped QDs with various molar ratios of purified PG-zb dimer mixed with purified E. coli MBP equipped with the C-terminal peptide linker and the basic leucine zipper interaction domain (MBP-zb) that we had previously characterized.9,11 Adaptor-coated QDs were prepared with molar ratios of MBP-zb/ PG-zb of 1:2.4, 1:1.6, and 1:0.8 as follows: the desired amounts of PG-zb and MBP-zb (constant 2.5 pmol of MBP-zb/pmol of QDs) were combined in buffered solution and this mixture was added to a solution of 1.1 µM initial concentration of QDs. After incubation for 15 min at room temperature, an additional 2.5 pmol of MBP-zb/pmol of QDs was added to the QD-protein mix and incubation was continued for 10 min in order to saturate any remaining protein-binding sites on the nanoparticle surfaces. This procedure yielded self-assembled mixed-surface QD-protein conjugates, free of obvious aggregates. Conjugations were carried out in 10 mM sodium borate (pH 9, borate buffer) or 10 mM HEPES/50 mM NaCl/1 mM EDTA (pH 8, HEPES buffer). Molecular weights of 23 600 for the PG-zb dimer and 110 800 for the MBP-zb dimer were calculated based on their amino acid compositions. To couple PG-zb/MBP-zb modified QDs with IgG, 1.5-6.0 pmol of antibody/ pmol of modified QDs was incubated for at least 1 h at 4 °C. The MBP-zb present on the mixed-surface QDs serves as a purification tool for the removal of excess unbound antibody from the QD-IgG product by affinity chromatography using cross-linked amylose resin (New England Biolabs). In a typical purification, the QD-IgG reaction mixture was applied onto a small (0.5 mL) amylose column previously equilibrated with HEPES buffer. Unbound IgG was removed by washing with 2 mL of HEPES buffer, and the QD-IgG was then eluted with the same buffer containing 10 mM maltose. The binding, washing, and elution of the complex were readily monitored visually using a hand-held UV lamp (exciting at 365 nm). Determination of Antibody/QD Ratios. To quantitate the number of IgG molecules bound per QD, Cy3.5-labeled IgG was used as a tracer in the coupling procedure. Goat IgG was labeled with 1.5 molecules of Cy3.5/IgG according to the manufacturer’s (Pharmacia-Amersham) instructions, except that 3 mg of IgG was labeled instead of 1 mg in order to reduce the final dye-to-protein ratio. QD-IgG was then prepared using dye-labeled antibody (6 pmol of labeled antibody/pmol of QD) followed by removal of unbound antibody as described above. The number of IgG per QD-IgG was calculated using the Cy3.5 absorbance at 581 nm ( )150 000 M-1 cm-1) of the purified product by assuming no loss of QDs during the column purification. The nanocrystal absorption at 581 nm was negligible. Fluoroimmunoassays Using QD-Antibody Conjugates. Staphylococcal enterotoxin B (SEB) and TNT were used as model systems to investigate the use of QD-antibody conjugates in fluoroimmunoassays for protein and small-molecule targets. SEB and polyclonal sheep anti-SEB antibody were purchased from Toxin Technologies, Inc. Mouse monoclonal anti-SEB antibody

Mab 2b ascites was purchased from IGEN International and further purified using MEP-Hypercel (Life Technologies, Inc.). Trinitrobenzenesulfonate-modified ovalbumin (TNB-ovalbumin) was prepared as previously described.18 Monoclonal anti-TNT antibody 11B319 was prepared by IntraCell, Inc. TNT and hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) were purchased from Radian International. TNB-Affi-Gel resin was prepared by reacting 2,4,6trinitrobenzenesulfonic acid (Sigma) in carbonate buffer (pH 8.5) with Affigel 10 (Bio-Rad) modified with a 1,5-diaminopentane spacer arm. For each assay described below, measurements were done in triplicate and mean PL values were corrected by subtracting the mean PL intensity of the appropriate nonspecific binding control. The standard deviation was calculated for each measurement. Mean fluorescence values are reported in arbitrary PL units that are internally consistent within a given experiment but not necessarily directly comparable between experiments. Each set of triplicate measurements was reproduced at least three times. Direct Fluorometric Assay of SEB. Wells of opaque white microtiter plates (Maxisorb, Nunc) were coated overnight (4 °C) with serial dilutions of SEB dissolved in 0.1 M NaHCO3 (pH 8.6) from 2.4 ng/mL to 10 µg/mL toxin using 100 µL per well of each serial dilution of the toxin or using the same volume of buffer containing no antigen to control for nonspecific binding. After removing excess antigen or blank solutions from wells, plates were blocked at 4 °C for 2 h with PBS containing 4% (w/v) powdered nonfat milk. Plates were then washed 2 times with PBST (PBS with 0.1% Tween 20), and QDs conjugated and purified as described above (MBP-zb/PG-zb of 1:1.6) with the desired antiSEB antibody were added to test and control wells and incubated for 1-2 h, gently shaking at room temperature. Unbound QDantibody was removed and discarded; wells were then washed several times with HEPES buffer, followed by automated fluorescence measurement using a SpectraFluor Plus microtiter plate reader (Tecan). A 25-nm band-pass filter was used for excitation at 310 nm and a long-pass filter with a lower cutoff at 530 nm was used for PL data collection. SEB Capture (Sandwich) Assays. Wells of microtiter plates were coated overnight with 10 µg/mL mouse monoclonal antiSEB antibody Mab 2b diluted in 100 µL of 0.1 M NaHCO3 (pH 8.6) and then blocked and washed as described above. Serial dilutions of SEB in PBS were made, and 100 µL of each diluted solution was added to wells coated with the anti-SEB antibody and incubated for 1-1.5 h with gentle shaking at room temperature. After washing 2 times with PBST, QDs conjugated with polyclonal sheep anti-SEB antibody (MBP-zb/PG-zb of 1:1.6; 100 µL/well) were added and incubated for another 1-2 h. Wells were then washed several times with HEPES buffer, and the fluorescence was measured as described above. Control wells used to correct for nonspecific binding were included in each experiment. Plate-Based TNT Competition Assays. Plates were coated overnight with a saturating concentration of TNB-ovalbumin and blocked as described above. In these experiments, the derivatized ovalbumin served as a surface-bound capture agent for QD-anti(18) Good, A. H.; Wolfsy, L.; Henry, C.; Kimura, J. In Selected methods in cellular immunology. Mishell, B. B., Shiigi, S. M., Eds.; W. H. Freeman and Co.: New York, 1980; p 345. (19) Whelan, J. P.; Kusterbeck, A. W.; Wemhoff, G. A.; Bredehorst, R.; Ligler, F. S. Anal. Chem. 1993, 65, 3561-3565.

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TNT antibody conjugate in competition with dissolved TNT. TNT stock solutions were prepared by transferring 10 µL of a 1 mg/ mL TNT solution in acetonitrile into a glass tube, evaporating the volatile solvent by brief passage of nitrogen gas, and redissolving the explosive in 0.5 mL of HEPES buffer to result in a stock solution of 20 µg/mL. Experiments using aqueous TNT were conducted within a few hours of stock sample preparation. Fiftymicroliter portions of TNT dilutions in HEPES buffer (and no TNT controls) were added to appropriate test and control wells, and then 50 µL of QD-anti-TNT antibody conjugate (MBP-zb/PG-zb of 1:1.6; 11B3 anti-TNT antibody) was added to each well. The contents of wells were allowed to come to equilibrium by incubation at room temperature with gentle shaking for 1-2 h. Wells were then washed and fluorescence was measured as described above. TNT Assays Using the Flow Displacement Method. QDanti-TNT antibody conjugate (MBP-zb/PG-zb of 1:0.8; 11B3 antiTNT antibody) was incubated overnight at 4 °C with an equal volume of Affi-gel-diaminopentane-TNB slurry. Unbound conjugate was removed by centrifugation, and the resin was resuspended in HEPES buffer and loaded into a plastic minicolumn. The column was then placed under constant buffer flow (250 µL/min HEPES buffer). A small amount of loosely bound QD-anti-TNT conjugate was eluted under flow until a stable baseline was obtained (2030 min washing). One hundred-microliter aliquots of TNT dissolved in flow buffer were injected immediately upstream of the minicolumn, and the fluorescence intensity profile of displaced conjugate for each injection was recorded downstream using a Jasco 821-FP fluorometer (excitation at 340 nm and detection at 570 nm). Integrated fluorescence peak area (fluorescence vs time) was calculated using Borwin Chromatography software (JMBS Developments). Injections of flow buffer alone as well as injections of dissolved RDX (hexahydro-1,3,5-trinitro-1,3,5-triazine), a highly nitrated explosive with no other structural similarity to TNT, were carried out as controls and resulted in no observable displacement of QD-anti-TNT conjugate from minicolumns. RESULTS AND DISCUSSION Design of PG-zb as a Molecular Bridge between Quantum Dots and Generic IgGs. Several design features of the PG-zb molecular adaptor were incorporated to allow facile coupling between negatively charged DHLA-capped core-shell quantum dots and any IgG having sites in its Fc region able to bind to streptococcal protein G. The IgG-binding domain of protein G was linked to a highly basic leucine zipper domain and expressed as a single polypeptide chain to enable the adaptor protein to form a bridge connecting the QD and antibody. Incorporating a positively charged cysteine-containing leucine zipper at the bridging protein’s C-terminus resulted in spontaneous formation of a high percentage of asymmetrically charged S-S linked homodimers in purified preparations. On the basis of previously published biophysical studies by O’Shea et al.,10 we reasoned that this type of covalently linked homodimer would form permanent parallel helix-helix interactions, resulting in assembly of oriented complexes on QD surfaces that would interact very strongly with any available IgG. Although the binding constant for the β subunit of protein G with antibody Fc is only ∼106 M-1,20 the homodimer construct is designed to provide two protein G subunits positioned to interact simultaneously with both Fc sites of the symmetrical 844

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IgG molecule and is expected to increase greatly the effective strength of the interaction. Meanwhile, at the other end of the molecular complex, the highly positively charged leucine zipper interacts strongly with the QD surface, resulting in very stable QD-antibody conjugates for use in fluoroimmunoassay development. Coupling of Antibodies and QDs with the Engineered Molecular Adaptor PG-zb Protein. Preliminary experiments showed that reacting DHLA-capped CdSe-ZnS QDs with the PGzb molecular adaptor protein yielded QD/PG-zb intermediate preparations containing no macroscopic aggregated material and which retained the QD absorption and photoluminescence properties and the IgG-binding ability of the attached streptococcal IgGbinding β2 subunit (PG).11,12 Preparation of mixed-surface composition QD intermediates by reaction with various ratios of MBPzb/PG-zb provided a means to control the number of antibodies bound per nanocrystal. This method also greatly facilitated purification of the final QD-antibody conjugates through the use of MBP-amylose affinity chromatography to remove excess unbound antibody from stable conjugates. In these experiments, we examined a range of MBP-zb/PG-zb of 1:2.4, 1:1.6, and 1:0.8 in an effort to control the average number of dimeric IgG-binding PG subunits present per QD while filling remaining binding sites on the nanoparticles with an “inert” coating of MBP-zb. As predicted, experiments examining binding of Cy3.5-labeled goat IgG to MBP-zb/PG-zb-coated QDs showed that there is an increase in the average number of conjugated antibodies as the relative proportion of PG-zb to MBP-zb increased. At a MBP-zb/ PG-zb of 1:2.4, 5.5 ( 0.2 IgGs bind per nanocrystal. At a MBPzb/PG-zb of 1:1.6, 3.3 ( 0.3 IgGs bind per dot, while at the highest MBP-zb/PG-zb ratio tested (1:0.8), an average of 2.7 ( 0.1 antibody molecules were bound per QD. These results suggest that it may be possible to titrate the number of bound antibodies downward further in order to approach 1 IgG/nanoparticle, which would be desirable in applications where antibody affinity, rather than overall conjugate avidity, is of importance. Factors that may affect QD-antibody stoichiometry include the size differential between component proteins (e.g., PG and MBP) and the as yet uncharacterized mode of interaction of the leucine zipper interaction domain with charged nanoparticle surfaces. In addition, there may be contributions from monovalent PG-IgG interactions, where each PG in the homodimer can bind the Fc site of a different IgG. Binding of SEB with QD-Anti-SEB Conjugates: Direct Fluorometric Assay. Direct binding assessment showed that QD-anti-SEB conjugate was able to bind to SEB protein toxin adsorbed to the wells of polystyrene microtiter plates (Figure 2A). Effects on fluorescent signals obtained by varying the amount of SEB immobilized in wells were investigated using a QD-Mab2b (anti-SEB) conjugate. In these experiments, the fluorescent signal for bound conjugate was measured over a range of toxin concentrations from 2.4 ng/mL to 10 µg/mL. The lowest concentration of SEB that gave meaningful signal over background was ∼0.010 µg/mL, and signal increased over the range of SEB concentrations applied to wells until saturation was reached at ∼2 µg/mL. (20) Fahnestock, S. R.; Alexander, P.; Nagle, J.; Filpula D. J. Bacteriol. 1986, 167, 870-880.

Figure 3. Capture fluoroimmunoassay for the detection of SEB. Results of a sandwich assay where varying concentrations of SEB (0.49-500 ng/mL), plotted in logarithmic scale, were captured by antiSEB antibody immobilized on wells of a plate. Signal generation was accomplished using QD-Mab2b conjugates.

Figure 2. Direct binding detection of SEB by Mab 2b (an anti-SEB antibody) conjugated QDs. (A) Direct fluoroimmunoassay for the detection of SEB. Wells coated with varying concentrations of SEB (2.4 ng/mL-10 µg/mL) detected by Mab 2b conjugated to QDs. The PL intensity is graphed as a function of log SEB concentration. (B) PL intensity measured for varying concentrations of QD/PG-zb/IgG binding to wells coated with a saturating amount of SEB (10 µg/mL). A comparison of antibody labeled with 555- and 570-nm emitting QDs is also shown.

The effects in microtiter plate assays of increasing QD-Mab2b conjugate concentration while the amount of adsorbed SEB was kept constant at a saturating amount was also investigated in the direct binding mode. Figure 2B shows the relative emission intensity measured as a function of increasing QD-Mab2b conjugate concentration. The fluorescent signal increased relatively linearly through the concentration range up to ∼30 nM QD-Mab2b conjugate per well (3 pmol of total QDs present), approaching saturation above ∼120 nM (12 pmol of total QDs present). This result was expected, since when all available SEB epitope sites are occupied with QD-Mab2b the fluorescent signal should remain constant regardless of the addition of more QD-antibody conjugate. Experiments showed QDs with emission maximums at 555 and 570 nm behave essentially identically in the direct binding assay. SEB Capture Assays in a Sandwich Format. QD-anti-SEB antibody conjugates were also used in a sandwich assay format. A series of dilutions of SEB were applied to microtiter plate wells with adsorbed Mab 2b anti-SEB capture antibody, followed by QD-polyclonal sheep anti-SEB conjugate as the signal-producing reagent. Figure 3 shows an experiment where the fluorescent signal from bound sheep-anti SEB conjugate was measured over a range of concentrations of toxin from 0.49 to 500 ng/mL. The lowest concentration of SEB that gave useful signal over back-

ground was ∼2 ng/mL, and signal increased until saturation was reached at ∼30 ng/mL. A potential complication of the sandwich assay format is high non-antigen-dependent background due to any QD-bound PG-zb not strongly complexed with anti-SEB antibody binding directly to any available Fc regions of plate-adsorbed capture antibodies. When polyclonal sheep-anti-SEB antibody was conjugated to the QDs, we did not see a higher background level for wells with adsorbed Mab 2b compared with control wells having no capture antibody. Should elevated backgrounds be encountered due to this phenomenon, strategies to reduce non-antigen-related signals include addition of excess nonrelevant IgG (i.e., generic goat IgG) with analyte-specific QD-antibody conjugate during the final step or use of avian antibodies that have no affinity for protein G.21 Competitive Fluoroimmunoassays for TNT Using QDAnti-TNT Conjugates. QDs conjugated with the monoclonal antiTNT antibody 11B3 were examined for their ability to bind to adsorbed ovalbumin that had been derivatized by modification with trinitrobenzenesulfonic acid. The trinitrobenzene-derivatized lysine side chains of TNB-ovalbumin serve as structurally similar TNT surrogates for solid-phase binding of anti-TNT antibodies in competition with soluble TNT. In preliminary experiments in the microtiter plate format, increasing concentrations QD-11B3 were incubated in wells that had been coated with an identical and saturating amount of TNB-ovalbumin. After washing away unbound conjugate and quantitation of fluorescence, the results graphed in Figure 4A show that assay response was essentially a linear function of the amount of QD-antibody complex until approaching saturation at ∼40 nM. These results supplied a basis for design of sensitive TNT analyses employing these QD antibody conjugates using competition methods in both microtiter plate and flow injection formats. In the microtiter plate-based competition assay, soluble TNT competed effectively with adsorbed TNB-ovalbumin for binding with QD-11B3 anti-TNT antibody conjugates, as judged by the systematic decrease in fluorescence emission observed in the presence of increasing amounts of TNT (Figure 4B). In these (21) Fischer, M.; Hlinka A. Berl. Munch. Tierarztl. Wochenschr. 2000, 113, 9496.

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Figure 5. PL intensity versus time measured downstream from the analysis column after upstream injection of 100-µL TNT solutions at increasing concentration.

Figure 4. (A) Direct detection of TNB-ovalbumin by QD/PG-zblabeled anti-TNT antibody (11B3). PL intensity was measured for varying concentrations of QD/PG-zb/11B3 binding to wells coated with a saturating amount of TNB-ovalbumin (10 µg/mL). (B) competitive inhibition of QD/PG-zb/11B3 binding to TNB-ovalbumin by TNT. Analyte concentration was plotted on a logarithmic scale; 100% corresponds to PL intensity measured in the absence of competing TNT.

experiments, the lowest detection level based on competition was 0.01 µg/mL. We note that a signal decrease of only ∼50% was observed over a 100-fold range of increasing concentrations of TNT, from 0.01 to 1 µg/mL in each well. Furthermore, even at 10- or 20-fold higher concentrations of TNT, competitive displacement of QD-antibody conjugate was invariably incomplete (data not shown). We hypothesize that this is due to heterogeneity in either our QD-11B3 preparations or in the way they interact with adsorbed TNB-ovalbumin. As discussed above, control experiments have demonstrated that QD-antibody conjugates prepared under the conditions used here have an average of ∼3 antibodies bound per nanoparticle. The present competition results suggest that TNT at low concentrations competes very effectively with adsorbed TNB-ovalbumin for one population of the QD-11B3 conjugate. If these readily displaceable conjugates contain one IgG per QD, then competitive behavior with either adsorbed TNBovalbumin or soluble TNT would tend to be approximated by the inherent antibody binding constant for the free explosive versus the solid-phase surrogate. The remaining conjugate population, which is bound much more tightly to TNB-ovalbumin, carries multiple antibodies resulting in high avidity for the microtiter plate surface, causing dramatically tighter binding than for the single antibody per QD case. Once attached to the plate surface via high avidity interactions, these fluorescent conjugates effectively become irreversibly bound and are impossible to remove by TNT 846 Analytical Chemistry, Vol. 74, No. 4, February 15, 2002

at any reasonable concentration. In future efforts at optimizing this assay format, it will be important to use 1:1 QD-antibody conjugates that are maximally displaceable by soluble TNT. When three different colors of QDs were used in TNT competition assays (emission maximums of 555, 570, and 590 nm), essentially identical results were obtained (data not shown). TNT Assay Using the Flow Displacement Method and QD-Anti-TNT Conjugates. We examined a second assay format based on competition between free TNT and a TNT surrogate bound to agarose beads for binding of QD-anti-TNT antibody conjugate. In preparation for this column-based method, an agarose bead matrix (Affi-gel) was derivatized with a seven-atom spacer (diaminopentane) and then reacted with trinitrobenzenesulfonate (TNBS) to form an affinity matrix able to bind anti-TNT antibody. After presaturation of available binding sites with QD11B3 conjugates, the gel was packed into 250-µL minicolumns and connected to a continuously flowing buffer source. Upstream injections of TNT resulted in elution of QD-11B3 conjugate which was detected fluorometrically immediately downstream of the column. Small peaks above background were observed for 100µL injections of 0.1 µg/mL TNT (10 ng of total TNT) (Figure 5); no signal was observed for injections of buffer alone. Integrated signals obtained from subsequent injections of 1 and 10 µg/mL solutions of TNT were proportionately larger. Repeated injections at higher TNT concentrations resulted in partial depletion of QD-antibody conjugate, as demonstrated by decreased integrated area of the second injections shown in Figure 5. No signal was generated from injections of RDX, another nitrated explosive structurally unrelated to TNT, at concentrations up to 10 µg/mL. Although we refer to this method of assay as flow displacement, we have no evidence that a displacement mechanism is involved in the signal generation that occurs upon injection of TNT. In fact, it is likely that introduction of free analyte into the column acts to inhibit rebinding of QD-antibody conjugates, which are continuously and slowly leaching off of the affinity matrix under nonequilbrium conditions, leading to signal generation as they are relatively rapidly cleared from the column by the flowing buffer stream. Evidence for this mechanism may be seen in the sloping baseline observed for the present system under flowing buffer conditions (Figure 5), where fluorescent material (i.e., QDantibody conjugate) slowly elutes from the column even in the

absence of analyte. These continuous-flow experiments demonstrate that the QD-11B3 conjugate can be useful in this type of rapid and relatively facile detection method. Based on our experience, optimization for sensitivity and reproducibility could involve a number of variables, including increasing the homogeneity of QD-antibody preparations (with 1:1 QD/antibody ratio being ideal), changes in flow buffer composition, pH, or flow rate.19 CONCLUSIONS We have shown QD-IgG conjugates prepared using an engineered molecular adaptor protein can be used for fluoroimmunoassays of both large (proteins) and small chemical (explosives) targets. Development of QD-IgG complexes having a wide (22) Sun, B.; Xie, W.; Yi, G.; Chen, D.; Zhou, Y.; Cheng, J. J. Immunol. Methods 2001, 249, 85-89. (23) Pathak, S.; Choi, S. K.; Arnheim, N.; Thompson, M. E. J. Am. Chem. Soc. 2001, 123, 4103-4104. (24) Willard, D. M.; Carillo, L. L.; Jung, J.; Van Orden, A. Nano Lett. 2001, 1, 469-474.

range of specificities and emission wavelengths should be advantageous in multianalyte assays. The optical properties of these nanoparticles, such as resistance to photobleaching and their ability to be excited over essentially a continuum of wavelengths,8 should make them particularly useful for further development of immunosensor and bioanalytical applications.22-24 ACKNOWLEDGMENT We thank Dr. M. G. Bawendi from MIT and Dr. F. S. Ligler from NRL for the helpful discussions. We also thank Dr. K. Ward at the Office of the Naval Research (ONR) for research support, Grants N0001499WX30470 and 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 June 14, 2001. Accepted October 9, 2001. AC010662M

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