Highly Enhanced Affinity of Multidentate versus Bidentate Zwitterionic Ligands for Long-Term Quantum Dot Bioimaging

High colloidal stability in aqueous conditions is a prerequisite for fluorescent nanocrystals, otherwise known as “quantum dots”, intended to be used in any long-term bioimaging experiment. This essential property implies a strong affinity between the nanoparticles themselves and the ligands they are coated with. To further improve the properties of the bidentate monozwitterionic ligand previously developed in our team, we synthesized a multidentate polyzwitterionic ligand, issued from the copolymerization of a bidentate monomer and a monozwitterionic one. The nanocrystals passivated by this polymeric ligand showed an exceptional colloidal stability, regardless of the medium conditions (pH, salinity, dilution, and biological environment), and we demonstrated the affinity of the polymer exceeded by 3 orders of magnitude that of the bidentate ligand (desorption rates assessed by a competition experiment). The synthesis of the multidentate polyzwitterionic ligand proved also to be easily tunable and allowed facile functionalization of the corresponding quantum dots, which led to successful specific biomolecules targeting

Sulfobetaine–Vinylimidazole Block Copolymers: A Robust Quantum Dot Surface Chemistry Expanding Bioimaging’s Horizons

Long-term inspection of biological phenomena requires probes of elevated intra- and extracellular stability and target biospecificity. The high fluorescence and photostability of quantum dot (QD) nanoparticles contributed to foster their promise as bioimaging tools that could overcome limitations associated with traditional fluorophores. However, QDs’ potential as a bioimaging platform relies upon a precise control over the surface chemistry modifications of these nano-objects. Here, a zwitterion–vinylimidazole block copolymer ligand was synthesized, which regroups all anchoring groups in one compact terminal block, while the rest of the chain is endowed with antifouling and bioconjugation moieties. By further application of an oriented bioconjugation approach with whole IgG antibodies, QD nanobioconjugates were obtained that display outstanding intra- and extracellular stability as well as biorecognition capacity. Imaging the internalization and intracellular dynamics of a transmembrane cell receptor, the CB1 brain cannabinoid receptor, both in HEK293 cells and in neurons, illustrates the breadth of potential applications of these nanoprobes

Real-Time in Situ Probing of High-Temperature Quantum Dots Solution Synthesis

Understanding the formation mechanism of colloidal nanocrystals is of paramount importance in order to design new nanostructures and synthesize them in a predictive fashion. However, reliable data on the pathways leading from molecular precursors to nanocrystals are not available yet. We used synchrotron-based time-resolved <i>in situ</i> small and wide-angle X-ray scattering to experimentally monitor the formation of CdSe quantum dots synthesized in solution through the heating up of precursors in octadecene at 240 °C. Our experiment yields a complete movie of the structure of the solution from the self-assembly of the precursors to the formation of the quantum dots. We show that the initial cadmium precursor lamellar structure melts into small micelles at 100 °C and that the first CdSe nuclei appear at 218.7 °C. The size distributions and concentration in nanocrystals are measured in a quantitative fashion as a function of time. We show that a short nucleation burst lasting 30 s is followed by a slow decrease of nanoparticle concentration. The rate-limiting process of the quantum dot formation is found to be the thermal activation of selenium

Electrolyte-Gated Field Effect Transistor to Probe the Surface Defects and Morphology in Films of Thick CdSe Colloidal Nanoplatelets

The optical and optoelectronic properties of colloidal quantum dots strongly depend on the passivation of their surface. Surface states are however difficult to quantify using optical spectroscopy and techniques based on back gated field effect transistors are limited in the range of carrier density that can be probed, usually significantly below one charge carrier per particle. Here we show that electrolyte gating can be used to quantitatively analyze the increase of defects in a population of nanoparticles with increasing surface irregularities. We illustrate this method using CdSe nanoplatelets that are grown in their thickness using low temperature layer-by-layer method. Spectroscopic analysis of the samples confirm that the nanoplatelet thickness is controlled, on average, with atomic precision, but structural analysis with transmission electron microscopy shows that the number of surface defects increases with the nanoplatelet thickness. The amount of charge defects is probed quantitatively using electrolyte-gated field effect transistor (EFET). We observe that the threshold voltage of the EFET increases with the NPL thickness, in agreement with the structural analysis. All samples displayed n-type conduction with strong current modulation (subthreshold swing slope of 100 mV/decade and on/off ratio close to 10⁷). We also point out that an efficient electrolyte gating of the film requires a fine control of the nanoparticle film morphology

Coupled HgSe Colloidal Quantum Wells through a Tunable Barrier: A Strategy To Uncouple Optical and Transport Band Gap

Among semiconductor nanocrystals (NCs), 2D nanoplatelets (NPLs) are a special class of nanomaterials with well controlled optical features. So far, most of the efforts have been focused on wide band gap materials such as cadmium chalcogenide semiconductors. However, optical absorption can be pushed toward the infrared (IR) range using narrow band gap materials such as mercury chalcogenides. Here we demonstrate the feasibility of a core/shell structure made of a CdSe core with two HgSe external wells. We demonstrate that the optical spectrum of the heterostructure is set by the HgSe wells and this, despite the quasi type II band alignment, makes the band edge energy independent of the inner core thickness. On the other hand, these core/shell NPLs behave, from a transport point of view, as a wide band gap material. We demonstrate that the introduction of a wide band gap CdSe core makes the material less conductive and with a larger photoresponse. Hence, the heterostructure presents an effective electric band gap wider than the optical band gap. This strategy will be of utmost interest to design infrared effective colloidal materials for which the reduction of the carrier density and the associated dark current is a critical property

Engineering Bicolor Emission in 2D Core/Crown CdSe/CdSe_1–<i>x</i>Te_<i>x</i> Nanoplatelet Heterostructures Using Band-Offset Tuning

Colloidal 2D nanoplatelets (NPLs) are a class of nanoparticles that offer the possibility of forming two types of heterostructures, by growing either in the confined direction or perpendicular to the confined direction, called core/crown NPLs. Here, we demonstrate that bicolor emission can be obtained from 2D NPLs with a core/crown geometry. To date, for CdSe/CdTe NPLs with type-II band alignment, only charge transfer emission has been observed due to the very fast (1–<i>x</i>Te_<i>x</i> alloys crowned with the right composition and lateral extension enables the observation of bicolor emission at the single-particle level. One source of emission originates from recombination at the core/crown interface (<i>X</i>_int), and the other emission source originates from direct recombination in the crown (<i>X</i>_crown). This crown emission, which is nonvisible in pure CdSe/CdTe core crown NPLs, results from the large binding energy compared to the reduced conduction band offset existing in the alloy with intermediate (60%) Te content. These observations are only made possible by the 2D geometry of the NPLs

Oriented Bioconjugation of Unmodified Antibodies to Quantum Dots Capped with Copolymeric Ligands as Versatile Cellular Imaging Tools

Distinctive optical properties of inorganic quantum dot (QD) nanoparticles promise highly valuable probes for fluorescence-based detection methods, particularly for in vivo diagnostics, cell phenotyping via multiple markers or single molecule tracking. However, despite high hopes, this promise has not been fully realized yet, mainly due to difficulties at producing stable, nontoxic QD bioconjugates of negligible nonspecific binding. Here, a universal platform for antibody binding to QDs is presented that builds upon the controlled functionalization of CdSe/CdS/ZnS nanoparticles capped with a multidentate dithiol/zwitterion copolymer ligand. In a change-of-paradigm approach, thiol groups are concomitantly used as anchoring and bioconjugation units to covalently bind up to 10 protein A molecules per QD while preserving their long-term colloidal stability. Protein A conjugated to QDs then enables the oriented, stoichiometrically controlled immobilization of whole, unmodified antibodies by simple incubation. This QD–protein A immobilization platform displays remarkable antibody functionality retention after binding, usually a compromised property in antibody conjugation to surfaces. Typical QD–protein A–antibody assemblies contain about three fully functional antibodies. Validation experiments show that these nanobioconjugates overcome current limitations since they retain their colloidal stability and antibody functionality over 6 months, exhibit low nonspecific interactions with live cells and have very low toxicity: after 48 h incubation with 1 μM QD bioconjugates, HeLa cells retain more than 80% of their cellular metabolism. Finally, these QD nanobioconjugates possess a high specificity for extra- and intracellular targets in live and fixed cells. The dithiol/zwitterion QD–protein A nanoconjugates have thus a latent potential to become an off-the-shelf tool destined to unresolved biological questions

Clickable-Zwitterionic Copolymer Capped-Quantum Dots for in Vivo Fluorescence Tumor Imaging

In the last decades, fluorescent quantum dots (QDs) have appeared as high-performance biological fluorescent nanoprobes and have been explored for a variety of biomedical optical imaging applications. However, many central challenges still exist concerning the control of the surface chemistry to ensure high biocompatibility, low toxicity, antifouling, and specific active targeting properties. Regarding in vivo applications, circulation time and clearance of the nanoprobe are also key parameters to control the design and characterization of new optical imaging agents. Herein, the complete design and characterization of a peptide-near-infrared-QD-based nanoprobe for biomedical optical imaging is presented from the synthesis of the QDs and the zwitterionic-azide copolymer ligand, enabling a bio-orthogonal coupling, till the final in vivo test through all the characterization steps. The developed nanoprobes show high fluorescence emission, controlled grafting rate, low toxicity, in vitro active specific targeting, and in vivo long circulating blood time. This is, to our knowledge, the first report characterizing the in vivo circulation kinetics and tumor accumulation of targeted zwitterionic QDs

Influence of Luminescence Quantum Yield, Surface Coating, and Functionalization of Quantum Dots on the Sensitivity of Time-Resolved FRET Bioassays

In clinical diagnostics, homogeneous time-resolved (TR) FRET immunoassays are used for fast and highly sensitive detection of biomarkers in serum samples. The most common immunoassay format is based on europium chelate or cryptate donors and allophycocyanin acceptors. Replacing europium donors with terbium complexes and the acceptors with QDs offers large photophysical advantages for multiplexed diagnostics, because the Tb-complex can be used as FRET donor for QD acceptors of different colors. Water-soluble and biocompatible QDs are commercially available or can be synthesized in the laboratory using many available recipes from the literature. Apart from the semiconductor material composition, an important aspect of choosing the right QD for TR-FRET assays is the thickness of the QD coating, which will influence the photophysical properties and long-term stability as well as the donor–acceptor distance and FRET efficiency. Here we present a detailed time-resolved spectroscopic study of three different QDs with an emission maximum around 605 nm for their application as FRET acceptors (using a common Tb donor) in TR-bioassays: (i) Invitrogen/Life Technologies Qdot605, (ii) eBioscience eFluorNC605 and iii) ter-polymer stabilized CdSe/CdS/ZnS QDs synthesized in our laboratories. All FRET systems are very stable and possess large Förster distances (7.4–9.1 nm), high FRET efficiencies (0.63–0.80) and low detection limits (0.06–2.0 pM) within the FRET-bioassays. Shapes, sizes and the biotin/QD ratio of the biocompatible QDs could be determined directly in the solution phase bioassays at subnanomolar concentrations. Both commercial amphiphilic polymer/lipid encapsulated QDs and self-made ligand-exchanged QDs provide extremely low detection limits for highly sensitive TR-FRET bioassays