Quantum Dot Bioconjugates in Molecular Detection

J.M. Mauro, G.P. Anderson, and E.R. Goldman
Center for Bio/Molecular Science and Engineering

H. Mattoussi and B.L. Justus
Optical Sciences Division

Introduction: Fluorescent labeling of biological materials using organic dyes, especially tagging of purified antibodies, is critically important in a wide variety of diagnostic and biological imaging applications. Organic fluorophores, however, have characteristics, such as narrow excitation bands and broad red-tailing emission bands, that often limit their effectiveness. This makes concurrent resolution of multiple light-emitting probes problematic due to spectral overlap. Also, many organic dyes exhibit low resistance to photodegradation.

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 particular, CdSe-ZnS core-shell QDs exhibit size-dependent tunable photoluminescence (PL) with narrow emission bandwidths (FWHM ~ 30 to 45 nm) that span the visible spectrum and broad absorption bands. These allow simultaneous excitation of several particle sizes (colors) at a common wavelength.¹ This, in turn, allows simultaneous resolution of several colors using standard instrumentation (Fig. 9, top) . CdSe-ZnS QDs also have high quantum yields, are resistant to photodegradation, and can be detected optically at concentrations comparable to organic dyes.² Our effort at NRL aims to take advantage of the molecular recognition properties of antibodies in combination with the unique photophysical characteristics of QDs to provide new bioinorganic materials that can be used to detect dissolved chemicals and toxins-both natural and human- derived-within marine environments.

FIGURE 9
(Top) Emission spectra of a several sizes of CdSe-ZnS quantum dots, with excitation at 350 nm in all cases. (Bottom) Idealized mixed-surface QD-protein conjugate. Antibodies labeled with biotin bind efficiently to QD surfaces due to the great strength of their interaction with bridging avidin molecules.

Meeting Our Goal: To make these novel materials, we have developed a conjugation strategy based on electrostatic interactions between negatively charged (acid-capped) CdSe-ZnS core-shell QDs and positively charged proteins.² Both naturally occurring and genetically engineered proteins are useful in this method, which involves simply mixing together the two charged substances (QDs and proteins) to result in self-assembled bioinorganic complexes ready for defined uses. For fluoroimmunoassays, QD-antibody conjugates can be prepared by using an adaptor protein that bridges the inorganic QD fluorophores and antibodies. Figure 9 (bottom) illustrates a mixed surface QD-conjugate consisting of two types of protein molecules surrounding a luminescent nanocrystal. Initially prepared conjugates contain both naturally occuring positively charged hen egg avidin and a variant of E. coli maltose binding protein (MBP-zb) engineered to have a strongly positively charged "tail" or "QD interaction domain." Biotinylated antibodies added subsequently bind tightly to the vacant biotinbinding sites of the immobilized avidin bridges, while the MBP-zb protein functions as a purification tool by allowing conjugate binding to, and elution from, solid-phase polysaccharide affinity media (this process removes any excess unbound antibodies).^3,4 Purified conjugates eluted from affinity columns can be used directly in many fluoroimmunoassays.

Figure 10 is an example of the use of the new bioconjugate materials. In this work, anti-RDX antibodies conjugated to QDs via a bridging generic antibody binding protein (protein G from staphylococcus) have been used to quantitate amounts of the explosive RDX (hexahydro-1,3,5-trinitro-1,3,5-triazine) dissolved in water. In the microtiter-plate based assay scheme shown (Fig. 10, top), competition between surface-immobilized and free RDX for binding to luminescent bioconjugates provides a convenient and sensitive way to monitor low levels of the explosive. Concentrations of RDX as low as 2 micrograms per liter can be detected using the present luminescent QD-conjugates.³ Similarly sensitive assays have been developed for the explosive TNT as well as for protein toxins such as staphylococcal enterotoxin B (SEB).⁴

FIGURE 10
(Top) Schematic of competition-based assay for detection of low levels of dissolved RDX explosive using QD-anti- RDX antibody conjugates. (Bottom) Graphed data from competition assay, showing systematically lowered signal as increasing levels of free RDX compete with surface-immobilized RDX derivative for conjugate binding.

Many Challenges and Opportunities Remain: We have successfully taken advantage of the luminescence properties of water-soluble CdSe-ZnS quantum dots to develop simple fluorimmunoassays with detection sensitivities similar to those obtained using organic dyes. Additional assays that take advantage of the unique properties of the QDs are under development. Ongoing work will exploit the novel photophysics of these semiconductor-based materials to expand the repertoire of their uses. These include simultaneous assay of several substances using multiple QD colors (multiplexed assays) as well as unique analysis methods based on quenching phenomena. Finally, using advanced microscopy methods, we are beginning to conduct experiments aimed at understanding the behavior of these nanocrystals and their bioconjugates at the single-particle level. We anticipate that fuller understanding of single-dot phenomena such as intermittent blinking, spectral shifts, effects of crystal lattice defects and surface traps, etc., and the development of other semiconductor nanocrystals with additional emission wavelengths that are not accessible with CdSe QDs will lead to development of new types of nanosensors with novel applications.

[Sponsored by ONR]

References
¹B.O. Dabbousi, J. Rodriguez-Viejo, F.V. Mikulec, J.R. Heine, H. Mattoussi, R. Ober, K.F. Jensen, and M.G. Bawendi, "(CdSe)ZnS Core-Shell Quantum Dots: Synthesis and Characterization of a Size Series of Highly Luminescent Nanocrystallites," J. Phys. Chem. B 101(46), 9463-9475 (1997).
² H. Mattoussi, J.M. Mauro, E.R. Goldman, G.P. Anderson, V.C. Sundar, F. Mikulec, and M.G. Bawendi, "Self-Assembly of CdSe- ZnS Quantum Dot Bioconjugates Using an Engineered Recombinant Protein," J. Am. Chem. Soc. 122(49), 12142- 12150 (2000).
³ E.R. Goldman, E.D. Balighian, M.K. Kuno, S. LaBrenz, P.T. Tran, G.P. Anderson, J.M. Mauro, and H. Mattoussi, "Luminescent Quantum Dot-Adaptor Protein-Antibody Conjugates for Use in Fluoroimmunoassays," Phys. Stat. Sol. (in press) Jan. 2002.
⁴ E.R. Goldman, G.P. Anderson, P.T. Tran, H. Mattoussi, P.T. Charles, and J.M. Mauro, "Conjugation of Luminescent Quantum Dots with Antibodies Using an Engineered Adaptor Protein Provides New Reagents for Fluoroimmunoassays," Anal. Chem. (in press) Jan. 2002.