Quantum Dots: Heralding a Brighter Future for Clinical Diagnostics

Tamer M Samir; Mai MH Mansour; Steven C Kazmierczak; Hassan ME Azzazy


Nanomedicine. 2012;7(11):1755-1769. 

In This Article

Bioconjugation of QDs

Their unique optical and physical properties have made QDs appealing tools for applications in bioimaging, medical diagnostics and therapeutics.[15] These applications require the conjugation of different biomolecules to achieve biomolecule specificity or targeting. Linking biomolecules to QDs has been successfully achieved through different approaches including adsorption, electrostatic interaction, mercapto (-SH) exchange and covalent linkage.

Conjugation of QDs to Proteins

A single QD can be conjugated to multiple protein molecules, which can be similar or different depending on the intended application. Approximately 15–20 maltose binding proteins, a 44-kDa protein measuring 3 × 4 × 6.5 nm, can be attached to a single 6-nm QD.[1] Table 1 summarizes different approaches for conjugation of QDs to proteins.

Proteins can be conjugated to QDs by crosslinkers such as EDC or succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC). Zero-length crosslinkers such as EDC mediate conjugation of two molecules by forming covalent bonds between an atom in the first molecule and an atom in the second, without adding any additional atoms or spacers.[23] Carbodiimides such as EDC are among the most common crosslinkers used for conjugating QDs to proteins or peptides. They mediate formation of an amide bond by condensation of a primary amine with a carboxylic acid, or a phosphoramidate linkage by reacting a primary amine with an organic phosphate group. Chan and Nie used EDC to conjugate transferrin to a CdSe/ZnS QD surface.[19] The QDs were solubilized with mercaptoacetic acid, which provided a free carboxylic group. Transferrin was then covalently coupled to QDs by crosslinking to the free carboxyl group. In another study, Xue et al. used EDC to conjugate TAT peptide to thiol-capped CdTe QDs to enhance their intracellular delivery into hepatocellular carcinoma (QGY) cells.[24]

Another commonly used class of crosslinkers is heterobifunctional crosslinkers, for example SMCC. These crosslinkers contain two different reactive groups that can couple proteins to macromolecules through two different functional groups, so the crosslinker binds the protein from one side and a macromolecule from the other. Unlike zero-length crosslinkers, heterobifunctional crosslinkers add an additional spacer between the protein and the macromolecule. SMCC is used for crosslinking molecules with -SH and/or -NH2 groups (Figure 2A). Pathak et al. used a commercially available direct conjugation kit to conjugate antibodies to QDs.[25] Antibodies were reduced by dithiothreitol then conjugated to QDs by SMCC linkage.[25]

Figure 2.

Quantum dots' bioconjugation strategies. (A) Protein conjugated to QDs by SMCC crosslinker. (B) QDs are conjugated to a two-domain peptide: phytochelatin-derived C-terminal domain rich in cysteine for the thiolation of QDs, and a flexible hydrophilic linker domain, which increases QD solubility. (C) QDs conjugated to MBP–basic zipper fusion protein. (D) MBP with pentahistidine segment attached to its C-terminal conjugated to DHLA-capped QDs. (E) Thiolated DNA conjugated to the QDs.
DHLA: Dihydrolipoic acid; MBP: Maltose binding protein; QD: Quantum dot; SMCC: Succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate.
Adapted with permission from [13,20,22,25,27].

Peptides can also be conjugated to QDs in a direct approach by linking to thiol-rich domains. A direct binding approach was used by Pinaud et al. to bioactivate and solubilize QDs with phytochelatin-related peptides.[13] Synthetic two-domain phytochelatin-related peptides, composed of phytochelatin-derived C-terminal domains rich in cysteine, were used to increase the solubility of QDs. Next, a flexible hydrophilic linker domain was used for attachment of various bioaffinity tags (Figure 2B).[13]

For conjugation of QDs to antibodies the orientation of the antibody on the QD is important for its functionality as a targeting moiety. The conjugation strategy contributes to the control of antibody orientation. For example, the use of biotinylated antibodies and streptavidin-coated QDs provides no control over the orientation of the antibody on the surface of the QD owing to the presence of multiple biotinylation sites on the antibody. The same applies when using carbodiimide chemistry crosslinkers such as EDC. However, direct conjugation of an antibody through crosslinking of the QD's surface amine to the antibody's sulfhydryl group, developed by reduction of the antibody's disulfide bond, has led to a more uniform orientation of antibodies on the surface of QDs.[26]

Electrostatic self-assembly is another common approach for direct protein/peptide conjugation to the surface of QDs capped with DHLA. In this approach, proteins are fused to positively charged domains, such as basic leucine zipper domains, which allow for self-assembly on the negatively charged QD surface through electrostatic attraction. Mattoussi et al. constructed a fusion protein of maltose-binding protein and basic leucine zipper.[20] The purified protein was conjugated to DHLA-capped QDs (Figure 2C).[20]

The third approach for direct protein/peptide conjugation to QDs depends on the metal-affinity coordination of histidine residues and Zn in the ZnS shell of QDs. Medintz et al. used this approach to conjugate proteins to DHLA-capped CdSe/ZnS QDs.[27] A variant of maltose binding protein with a pentahistidine segment attached to its C-terminal was used for coordinative conjugation of the protein to the surface of QDs (Figure 2D).[27] The same approach was also used for peptide conjugation, by attaching a hexahistidine sequence to the peptide amino-terminal to facilitate self-assembly on DHLAcapped QDs.[28]

Conjugation of QDs to Oligonucleotides

Different schemes have been developed to conjugate ssDNA and dsDNA to the surface of QDs. DNA–QD conjugates retain the selectivity of DNA and the photophysical properties of QDs ,allowing detection of single or multiple DNA targets. DNA–QD conjugates require solubility in water, stability under physiological conditions and minimal nonspecific DNA binding to the QD surface. The thiol-modified oligonucleotide can be conjugated to QDs in a direct ligand exchange approach (native cap exchange) where the oligonucleotide displaces the surface-bound mercaptopropionic acid and yields aqueous stable and strongly fluorescent oligonucleotidebound QDs (Figure 2E).[22]

The use of mercaptoacetic acid to solubilize QDs and surface carboxylic acid groups to attach amine-modified biomolecules resulted in inefficient oligonucleotide loading on the QDs and poor long-term stability.[29] The free carboxylic acid groups on the QD surface caused nonspecific binding to the oligonucleotide backbone as well as target cells. To overcome these problems, CdSe/ZnS QDs were reacted with EDC and imidazole and conjugated to 5'-aminated oligonucleotides.[23,30]

Thiol-bearing silanized QDs stabilized by PEG and phosphonate can be conjugated to amino-modified oligonucleotides through a two-step procedure using the heterobifunctional crosslinker sulfo-SMCC.[31] Sulfo-SMCC contains an N-hydroxy-succinimide ester that reacts with primary amines to form an amide bond at one end, while its maleimide group reacts with thiol to form a thioester at the other end.