Luminescence of Nanoparticle-Labeled Antibodies and Antigens


Nicholas A. Kotov Department of Chemical Engineering, University of Michigan

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Bioconjugation of nanoparticles (NPs) is the attachment of specific biological molecules or components to nanoparticles. The resulting structures represent the convolution of biotechnology and nanotechnology, and yield hybrid materials, processes, and devices that can utilize both the unique optical and magnetic properties of NPs and highly selective binding of biological interactions. The combination of these features can potentially make a prominent impact in current biomedical technologies, and possibly in nanoelectronics, microphotonics and related fields. Antigen and antibody interaction is a naturally occurring immunology interaction of the biological defense system, and has been widely used as molecular recognition method for medical diagnostics and detection of biological threat agents. The bioconjugation of NPs with antigens and antibodies are interesting and important as a method for their organization in more complex structures, and as a pathway to new sensing and imaging technologies. In the viewpoint of biologically programmed assembly of nanostructures, the similarity of sizes of NP and proteins limits the overall number of affinity ligands around one semiconductor core to a few proteins leading to, for instance, 1-, 2-, 3-, and 4-valent quantum dots. The practical aspect of NP-antigen/antibody conjugation is related to the further development of immunoluminescence as a technique that affords highly sensitive and specific detection of various biological and nonbiological analytes of military and civilian importance.

Colloid gold nanoparticles have been used in labeling antigens and antibodies as contrasting agents of various immunoassays; however, their sensitivity is limited by the nature of colorimetric detection. Luminescence spectroscopy is more sensitive than absorption spectroscopy because of its substantially better signal-to-noise ratio. We observed the excited-state dipole–dipole coupling between NPs of different sizes in the constructed immunocomplex, which results in the excitation energy transfer from NPs of smaller diameters to those with bigger diameters, i.e., Förster resonance energy transfer (FRET). The detection limits of analytical processes based on FRET can be as low as 10 ppt, with a linear dynamic range of 0.1–1000 ppb, while the utilization of antibodies enables the selective detection of substrates, which may differ only by a few atoms. Highly luminescent semiconductor NP, or quantum dots, are a new class of luminescence materials providing a number of advantages over organic dyes. They can be made in a single-step synthesis, with precisely controlled size distribution and tunable emission spectrum. Compared to organic dyes such as Rhodamine, this new class of luminescent material are 20 times brighter and 100 times more stable against photobleaching, and one-third as wide in spectra linewidth. Therefore luminescence nanoparticles will have tremendous potential in biomedical imaging and diagnostic applications.

Typically, NPs are synthesized by arrested precipitation in the presence of organic molecules strongly coordinating to metal ions, such as thiols or phosphines. The simplified description of the product is a semiconductor core coated with a monolayer of organic molecules attached to surface metal sites. The core of the NPs is highly crystalline, which is a prerequisite for strong luminescence. Many different thiol derivatives may be used for the stabilization of II–VI nanoparticles. The terminal end of the thiol can be conveniently used for further functionalization of nanoparticles and for conjugation to biological molecules by either nonspecific electrostatic absorption or specific conjugation reactions. Most antigens and antibodies are proteins, have a number of amino acids that provide functional groups which can be chemically conjugated, such as –NH2 (lysine, and N-terminal), –COOH (aspartic acid, glutamic acid, and C-terminal), and –SH (cysteine).

The direct NP–protein conjugation method frequently used is the 1-ethyl-3(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC)/N-hydroxysulfo-succinimide (sulfo-NHS) reaction. NHS-conjugated proteins have the highest bioactivity among other conjugates, as established by several comparative studies. The carboxylic acid group of thioglycolic acid-stabilized NPs will form amide bond with the primary amine groups of the protein, or, the amine groups of a cysteine-stabilized NPs can link to the carboxylic acid group of protein.

To explore and demonstrate the potential of NP Bioconjugates, we used EDC/sulfo-NHS reaction to conjugate different-sized CdTe NPs to two antigen/antibody systems: 1) bovine serum albumin (BSA) and anti-BSA IgG (immunoglobulin G) and 2) Brucella suis and its antibody Bru-38 (Anti-GBa-O side chain). Antigens were conjugated to red-emitting CdTe NPs, while green-emitting NPs were attached to the corresponding antibodies. FRET was observed upon the formation of immunocomplex between the complementary antigens/antibodies. the competitive inhibition of FRET by unlabeled antigens was investigated.