When the complexes were imaged by fluorescence microscopy, anti-RNAP-pcQDs (Figure. stability, overall size, efficient and straightforward conjugation of functional biomolecules, and easy QDs targeting to cellular components all have to be simultaneously optimized. We have previously developed a compact surface coating chemistry that employs synthetic peptides for solubilization, biofunctionalization and long-term stabilization of colloidal QD solutions (> 6 month)(1,2). These peptide coated QDs (pcQDs) have been targeted in cells and animals using various small functional groups such as biotin(2,3), FITC(4)or DOTA(5,6). The results have provided ample proof of pcQDs advantages for both live cell single molecule imaging(7)and whole animal imaging applications. However, as recently demonstrated by the monofunctional conjugation of avidin to pcQDs(7)conjugation of large molecules to QDs is still challenging because steric hindrance, geometry, stoichiometry of the conjugate, and its final functional activity have to be addressed. For that reason, we have here focused our efforts on developing conjugation-ready pcQDs that are stable at 4 C for >46 months and provide a flexible approach for rapid bioconjugation to antibodies and other biomolecules in aqueous buffers and with precise and reproducible control over the conjugate stoichiometry. Previously, commercial linkers and activators have been used for various types of pcQD conjugation reactions with mixed success. For instance, cetuximab, a clinical anti-EGFR (Epidermal Growth Factor Receptor) antibody conjugated to QDs via EDC (1-ethyl-3-(3 dimethylaminopropyl) carbodiimide hydrochloride) was observed to have low reactivity and the QD conjugates were prone to aggregation(8). Alternative QD-cetuximab conjugation strategies using Succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) or N-Succinimidyl- 6-(3-(2-PyridylDithio)-Propionamido)-hexanoate LC-(SPDP)/SMCC linkers were more successful but required multiple preparative steps and reduction of the antibody which could compromise its activity(8). To overcome some of these limitations, we developed a bis-aryl hydrazone linkage strategy for the coupling of pcQDs DHCR24 to antibodies. We demonstrate the utility of this approach in live cells labeling and for invitroapplications. This reaction is based on the bio-orthogonal stable Schiff base mediated conjugation between aromatic aldehydes and aromatic hydrazines(911). In this scheme, UV traceable bifunctional linkers, succinimidyl-4-formylbenzamide (NHS-FB) or pentafluorophenyl-polyethyleneglycol-4-formylbenzamide (PF-PEG-FB) and succinimidyl-6-hydrazinonicotinate acetone hydrazone (NHS-HyNic) were conjugated to pcQD and antibodies respectively and further reacted together to yield stable QD conjugates via the formation of a covalent bis-aryl hydrazone bond with virtually no competing side reactions (Scheme 1). These linkers can be used interchangeably on pcQDs and antibodies or other biomolecules without a loss of generality, yield, or stability. Bis-Aryl hydrazone bond formation can also be catalyzed and reaction kinetics improved 10100 fold in the presence of aniline in the pH range of 58 in the presence of aniline(1214). The reaction of aromatic aldehyde with aromatic hydrazine is also completely bioorthogonal as the functional linkers do not cross-react with other side groups present in the side chains of natural amino acids or oligonucleotides. To provide additional flexibility and control for conjugation, QDs were also coated with peptides directly synthesized with an N-terminal HyNic group. This approach saves an extra step for the preparation of reactive pcQDs and permits a better control of the number of HyNic reactive groups available at the surface of QDs using mixture of peptides with different functionalities 1,2-Dipalmitoyl-sn-glycerol 3-phosphate during the coating procedure(1,2). == Scheme 1. == Preparation of aromatic benzaldehyde capped CdSe/ZnS peptide coated QDs and aromatic hydrazine functionalized antibodies. (Not to scale). Peptide coated quantum dots (pc- QDs) are modified with PEG-4FB,1Avia their primary amines to yield PEG-4FB-pcQDs,1B; Antibodies are modified with C6-SANH,2Avia surface exposed primary amines to yield C6- SANH-Abs,2B(nucleophilic aromatic hydrazine is protected as an acetone hydrazone);1Bis conjugated to2Bto form a pc-QD-Abs,3bioconjugate via stable uv-traceable bis-aryl hydrazone linkage. CdSe/CdS/ZnS QDs emitting at 630 nm (Supporting Information Figure S.1) were synthesized as described previously(15). CdSe/ZnS QDs emitting at 605 nm were obtained in their hydrophobic form from commercial 1,2-Dipalmitoyl-sn-glycerol 3-phosphate vendors. Both types of QDs were coated with glycine or lysine N-terminated synthetic peptides which resulted in available primary amines(1,2)for NHS-FB or PF-PEG-FB modifications of pcQD (Seesupporting information methodsfor peptide sequences). For the production of hydrazine functionalized QDs, QDs were directly coated with HyNic N-terminated synthetic peptides (HyNic-pcQD). The mean core/shell diameter of these 630 nm or 605 nm red emitting QDs was ~7.5 nm and ~5.0 nm respectively based on TEM measurements(16)(Supporting Information Figure S.1) and their hydrodynamic diameter increased to ~12.0 nm and ~9.5 nm respectively once coated with peptides(17). Scheme 1illustrates one approach for the conjugation of pcQDs to antibodies via bis-aryl hydrazone linkage. NHS-FB or 1,2-Dipalmitoyl-sn-glycerol 3-phosphate PF-PEG-FB (1A) were mixed with amine terminated pcQDs either in DMSO or directly in aqueous buffer at pH 7.2 to obtain pcQDs activated with FB or with PEG-FB which further enhances the solubility pcQDs. The resulting FB-activated pcQDs (1B) were purified via gel filtration using water as mobile phase and rapidly exchanged into a 100.