Defense Applications of Nanomaterials - American Chemical Society

Chapter 2

Receptor Protein-Based Bioconjugates of Highly Luminescent CdSe-ZnS Quantum Dots: Use in Biosensing Applications 1

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J. M. Mauro , H. Mattoussi ,I.L. Medintz , E. R. Goldman , P. T. Tran , and G. P. Anderson

Downloaded by STANFORD UNIV GREEN LIBR on August 3, 2012 | http://pubs.acs.org Publication Date: January 21, 2005 | doi: 10.1021/bk-2005-0891.ch002

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Center for Bio/Molecular Science and Engineering and Division of Optical Sciences, U.S. Naval Research Laboratory, 4554 Overlook Avenue, SW, Washington, DC 20375

We are developing new and useful ways to prepare bioinorganic conjugates of highly luminescent semiconductor quantum nanocrystals (Quantum Dots; QDs) and proteins for use in biosensing applications. Conjugate assembly is driven by electrostatic interaction of negatively charged QD surfaces with positively charged proteins or protein subdomains. Conjugates retain the properties of both starting materials, i.e., biological activity of die proteins and optical characteristics of the QDs. We have used these hybrid bio-inorganic conjugates as tracking reagents in fluoroimmunoassays.

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© 2005 American Chemical Society

In Defense Applications of Nanomaterials; Miziolek, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2005.


17 Colloidal semiconductor CdSe-ZnS core-shell quantum dots (QDs) are luminescent inorganic nanoparticles that have the potential to obviate some of the functional problems of fluorescent organic dyes in labeling applications. Problems with organic fluorophores include narrow excitation bands and broad emission spectra, which can make multiplex detection difficult due to spectral overlap, as well as low resistance to chemical and photodegradation (1,2). QDs have size-dependent tunable broad excitation and photoluminescence (PL) spectra with narrow emission bandwidths (full width at half maximum of ~ 30-45 nm) that span the visible spectrum (7,2); this allows simultaneous excitation of several particle sizes at a single wavelength. QDs also have exceptional photochemical stability, and their high quantum yield allows luminescence emission to be observed at concentrations comparable to organic dyes using conventional fluorescence methods. Finally, using polar Afunctional compounds such as 1-mercaptoacetic or dihydrolipoic acids to modify QD surfaces allows preparation of stable aqueous nanocrystal dispersions that can subsequently be used for bioconjugation (/-J). In a departure from previous covalent chemistry techniques used for bioconjugate formation (6-8), we have developed a conjugation strategy based on the electrostatic interactions of negatively charged dihydrolipoic acid (DHLA)-capped CdSe-ZnS core-shell QDs with positively charged protein receptors which either occur naturally or are engineered to contain positively charged interaction domains. In this chapter, we describe the formation of water-compatible DHLA-capped quantum dots, methods of making electrostatically-stabilized QD-protein bioconjugates, and some of the properties of these materials. Subsequent preparation of QD-antibody conjugates and their employment in fluoroimmunoassays will also be described (5-5).

CdSe-ZnS Quantum Dot Preparation In a typical preparation, a solution of dimethylcadmium (CdMe ) and trioctylphosphine selenide (TOPSe), diluted in trioctylphosphine (TOP), is rapidly injected into a hot stirring solution of trioctylphosphine oxide (TOPO). (/, and references therein). The rapid introduction of these reagents and concomitant temperature drop result in discrete temporal nucleation of CdSe seeds. After reagent injection, the temperature of the solution is raised to 300350°C in order to grow the particles (Figure 1). The high temperature growth promotes highly crystalline QD cores. Growth is monitored through UV/visible spectroscopy and when the desired size is reached (as monitored by the peak wavelength of the first absorption feature), the temperature is dropped below 100°C to arrest the growth. 2

Passivating the native CdSe QDs with an additional thin layer made of a wider band gap semiconductor (to make core-shell nanocrystals) improves die


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surface quality of the particles by providing better passivation of surface states resulting in dramatic enhancements of thefluorescencequantum yield (Figure 1).

Figure /. High temperature organometallic growth, ZnS overcoating, and DHLA capping of colloidal CdSe quantum dots.

Although the principle was previously known from semiconductor bandgap engineering (l,2\ die optimal set of conditions for creating strongly fluorescent overcoated QDs was not realized until the published work of Hines and Sionnest (P), when they showed that overcoating CdSe QDs with ZnS unproved quantum yields to 30% or greater. This was shortly followed by other studies describing additional characterization of CdSe QDs overcoated with ZnS (JO) and CdS (//).

Cap Exchange for Aqueous Compatibility TOP/TOPO capping groups can be subsequently exchanged with dihydrolipoic acid (DHLA) groups by suspending TOP/TOPO dots in dihydrolipoic acid and heating at ~ 60-70°C for several hours (Figure 1). Following deprotonation of the terminal lipoic acid -COOH groups with potassium-tert-butoxide (K-t-butoxide), the centrifiigally sedimented cap


19 exchanged nanocrystalline material can be dispersed in water (1,3). After removal of excess hydrolyzed K-t-butoxide by repeated dilution/concentration using an Ultra-free centrifugal filtration device (Millipore, M W cut-off of ~ 50,000 daltons), QD suspensions are obtained with the same emission characteristics of the initial nanocrystals and with photoluminescence quantum yields in the range of 10-20% (7,5).


Bioconjugate Formation and Purification Two pioneering studies utilized CdSe nanocrystals bound to biological molecules (6,7). In one study, an avidin-biotin binding scheme was employed to attach CdSe-CdS core-shell nanocrystals to actin fibers. These dots were capped with an additional thin layer of silica in order to render them water compatible. The route to the final bioconjugate was complex, time-consuming and yielded a product with low quantum yield (6). The second study used CdSe-ZnS coreshell nanocrystals capped with mercaptoacetic acid groups. In this case, a conventional covalent cross-linking approach, based on l-ethyl-3-(3dimethylaminopropyl) carbodiimide hydrochloride (EDAC or EDC) condensation, was used to conjugate the nanocrystals to immunoglobulin G (IgG). Successful preparation of QD/IgG-nanocrystal bioconjugates was reported, albeit again with low PL quantum yields (7). Our work in developing bioconjugated QD's has been focused in three principal areas. First, we developed an alternative conjugation strategy, based on self-assembly utilizing electrostatic interactions between negatively charged lipoic acid capped CdSe-ZnS quantum dot surfaces and engineered Afunctional recombinant proteins consisting of highly positively charged attachment domains genetically fused with desired biologically relevant domains, Figure 2A (1,3). Our initial prototype engineered protein consisted of a maltose binding proteinbasic leucine zipper fusion (MBP-zb) which autoassembles into a dimer through an inserted cysteine residue. The highly positively charged attachment domains interact with the capped CdSe-ZnS QD surface while the MBP domain allows purification on amylose columns (5-5). This strategy of combining alkyl-COOH capped CdSe-ZnS nanocrystals and a two-domain recombinant protein cloned with a highly charged leucine zipper tail offers several advantages, (i) The alkyl-COOH terminated capping groups, which permit dispersion of the nanocrystals in water solutions at basic pH, also provide a surface charge distribution that can promote direct self-assembly with other molecules that have a net positive charge, (ii) The synthetic approach used to prepare the QDs can be easily applied to make a number of different sized core-shell nanocrystals,


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resulting in fluorescent probes with tumble emission over a wide range of wavelengths. This contrasts with the need for developing specific chemsitry

Figure 2. A. Cartoon of S-S linked MBP-zb homodimer consisting of the maltose binding protein fused with the dimer-forming positively charged tail (3). B. PGzb (IgG-binding B2 domain ofstreptococcal protein G fused with the dimerforming positively charged C-terminal tail) as molecular linker between QD's and the F region of IgG. MBP-zb is usedfor separating QD-IgG conjugate from excess IgG (4). C. Mixed surface QD-antibody conjugate with avidin bridging the QD and biotinylated antibody (5). c

routes for individual organic fluorescent dyes, (iii) The fusion protein approach provides a general and consistent way to prepare a wide selection of biological macromolecules amenable to alterations in the interaction domain, such as charge, size, stability to pH and temperature. This allows control of the assembly of individual proteins into dimers and tetramers, a property that can be exploited in protein packing around the nanocrystals to form complex bioconjugates. Figure 3 shows the release of QD/MBP-zb bioconjugates from amylose beads after being allowed to self assemble and bind to the beads. Since unconjugated DHLA-QD's do not release from the amylose beads, we are able to routinely use amylose affinity columns in QD-bioconjugate purification.



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Figure 3. Release of QD/MBP-zb bioconjugatesfromamylose beads as a function of concentration of added maltose. Experiments were carried out in 5 mM sodium borate, pH 9 (3). A second research area concerned engineering an adaptor protein to allow conjugation of the QD's to antibodies (Figure 2B) (4). This allows us to prepare bioconjugates of CdSe-ZnS QDs with antibodies using mixed composition conjugates containing both a molecular adaptor protein and a second protein used as a purification tool (Figure 2B). The engineered adaptor protein enqploys the immunoglobulin G (IgG)-binding P2 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 E. coli MBP. 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 (4). We showed 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. We also demonstrated 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 (4). Details of some of these assays will be presented in a later section. A third research effort in QD bioconjugation involves the direct use of avidin (a positively charged tetramer) as a bridge between QD's and biotinylated antibodies to form QD-antibody conjugates (Figure 2C) (J). Avidin, a


22 glycoprotein found in avian egg white, is a homotetramer with a molecular weight of 68,000 Da, which interacts stoichiometrically with biotin, binding one biotin per subunit. Due to the specific nature and high affinity of the interaction (K

Defense Applications of Nanomaterials - American Chemical Society

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