Semiconductor Quantum Dot Composite Colloids for Biomedical Applications

Abstract

Quantum dot composite colloid (QDCC) can be defined as tens of nanometer to micron-sized colloidal particles that incorporate multiple QDs using polymer matrices such as pre-formed polymer beads, silica template, lipid, and polymersome. QDCCs can have high QD loading capacity, robustness against leakage, and flexible functionalization capability, that are suited for multiplexed imaging and bioanalytical applications. Herein, we report amphiphilic polyethylenimine derivative (amPEI) and CdSe/CdS/ZnS core/shell/shell QDs were used to form highly fluorescent QDCCs, named as QD-amPEI. We synthesized amPEI by partially alkylating the amine functionalities of PEI. As-synthesized bright QDs (with the quantum efficiency over 60%) with fatty acids on the surfaces were introduced to the hydrophobic pocket of amPEI without losing the bright fluorescence. QD-amPEI could have average 20 QDs inside per QDCC as determined by fluorescence correlation spectroscopy. The inside hydrophobic compartment was utilized for co-hosting QDs and nitric oxide (NO)-sensitive motifs for NO sensing. We report two kinds of ratiometric NO sensing platforms using amPEI-based QDCCs via irreversible or reversible methods. The flexible amPEI shells of the QDCCs can permeate NO. For ratiometric/irreversible NO sensors, 1,2-diaminoanthraquinone (DAQ, NO sensing dye molecules) and QDs (insensitive fluorophore for NO) were co-encapsulated in the QDCCs. It could detect the oxidized NO products (e.g. N2O3) to record the maximum exposure levels. For ratiometric/reversible NO sensor, ferric center complexes with a tetra-amido macrocyclic ligand (Fe-TAML) and two kinds of QDs (emits at around either 470 nm (QD420) or 720 nm (QD720)) were co-incorporated in the QDCCs. In an abundant NO environment, NO could immediately coordinate to the metal center of Fe-TAML (Fe-TAML-NO). Fe-TAML-NO showed the appearance of strong absorption in visible (500~650 nm) and NIR (700~1000 nm) ranges, which could effectively modulate the FL intensity of QD720, whereas the original FL intensity of QD470 was maintained as an internal standard. It could reversibly monitor the transient concentration of NO. The flexibility of QD-amPEI polymer shells is beneficial for some applications such as endocytosis or biosensor along with QDs inside of amPEI matrices. However, QD-amPEI needs to have robust shells maintaining the structural rigidity for other applications such as in vitro bioassay applications. To sophisticatedly control the plasticity of the QDCCs, layer-by-layer (LbL) decorated QD-amPEIs up to by four polyelectrolyte layers were prepared using poly(sodium 4-styrene sulfonate) and polyethylenimine. As changing the number of LbL layers on QDCC shells, the permeation/intrusion and payload mixing/exchange properties were investigated. QDCC fluorescence (FL) quenching by permeation and intrusion of copper ions was studied. For another type of quenching experiment, a quenching polymersome was prepared using hydrophobic dark quencher molecules in amPEI and payload exchange rate was studied by mixing with QDCCs and monitoring the FL quenching. For both types of quenching experiments, LbL multiple layered QDCCs could retain the bright FL over days, whereas the FL of as-is QD-amPEI quenched in hours. These results indicate that the LbL decoration effectively strengthens the structural integrity of QDCCs. With the enhanced structural integrity of QDCCs via the LbL decoration, the outermost layer of QDCC can be easily extended to other functional groups such as amines, carboxylic acid, zwitterionic moieties (ZW), or a combination of those. Especially, ZW functionalized QDCCs (ZW-QDCCs) were colloidally very stable over a broad pH range and even in saturated NaCl solution. To extend the applicability of our QDCCs to specific labeling, bioconjugations were performed using biotin and streptavidin (SA). The specific to non-specific ratio (SNSR) of ZW-QDCC was 14.1 times larger than the case of ZW-QD due to the high multivalency of SA or biotin and FL intensity of QDCCs. As a proof-of-concept, we performed a signal amplification via biological self-assembly using biotin-tethered and SA tethered ZW multi-shell QD-amPEIs as the FL signal reporter. Typical sandwich-type immunoassay procedures were adopted, and the targeted protein-binding events were effectively transduced and amplified by the fluorescence of the biotin- and SA-conjugated ZW-QDCC pair. The detection limit of our enzyme-free FL immunoassay can reach 15.9 fM (1.91 ng/L) after 6 amplification rounds by alternative treatment of the streptavidin/biotin tethered ZW-QDCC pair, which is almost more than 500,000 times more sensitive than conventional ELISA (8.8 nM, 1.06 mg/L). Also, our method took a short amount of processing time (6 min for 6 amplification rounds of self-assembly vs 60 min required for the conventional ELISA method). Simultaneous control over the robustness, minimal non-specific binding level, and flexible functionalization of these bright QDCCs will be further applied in many ultrasensitive detection applications.