Quantitation of reduced IL-33 levels in human serum: mitigating interference from endogenous binding partners.

Aim: IL-33 is a potential therapeutic target but commercially available assays for the quantitation of systemic IL-33 have poor reliability. Results: In commercial IL-33 kits, interference from endogenous binding partners (e.g., soluble ST2) causes under-quantitation. Mitigating this required acid dissociation and addition of the detection reagent simultaneously with the capture step. This enabled detection of total, reduced (active) levels of IL-33 in human serum (LLOQ 6.25 pg/ml). Conclusion: Acid treatment of serum samples dissociates IL-33 from endogenous binding partners, increasing soluble ST2 tolerance to >1000 ng/ml. The modified method was specific for reduced endogenous IL-33. Analysis of over 300 samples from individuals with and without asthma and with different smoking status revealed no difference in serum IL-33.

Near infrared bioluminescence resonance energy transfer from firefly luciferase--quantum dot bionanoconjugates.

The bioluminescence resonance energy transfer (BRET) between firefly luciferase enzymes and semiconductive quantum dots (QDs) with near infrared emission is described. The QD were phase transferred to aqueous buffers using a histidine mediated phase transfer route, and incubated with a hexahistidine tagged, green emitting variant of firefly luciferase from Photinus pyralis (PPyGRTS). The PPyGRTS were bound to the QD interface via the hexahistidine tag, which effectively displaces the histidine layer and binds directly to the QD interfaces, allowing for short donor-acceptor distances (∼5.5 nm). Due to this, high BRET efficiency ratios of ∼5 were obtained. These PPyGRTS-QD bio-nano conjugates were characterized by transmission electron microscopy, thermal gravimetric analysis, Fourier transform infrared spectroscopy and BRET emission studies. The final optimized conjugate was easily observable by night vision imaging, demonstrating the potential of these materials in imaging and signaling/sensing applications.

Novel multistep BRET-FRET energy transfer using nanoconjugates of firefly proteins, quantum dots, and red fluorescent proteins.

Sequential bioluminescence resonance energy transfer (BRET) and fluorescence resonance energy transfer (FRET) from firefly luciferase to red fluorescent proteins using quantum dot or rod acceptor/donor linkers is described. The effect of morphology and tuned optical properties on the efficiency of this unique BRET-FRET system was evaluated.

A modular phase transfer and ligand exchange protocol for quantum dots.

In this paper, we describe a quantum dot (qdot) phase transfer protocol using ligand exchange and the amino acid histidine. The phase transfer from nonpolar solvents to aqueous buffers is homogeneous, and no appreciable precipitation occurs. The molecule histidine was chosen in order to first displace the organic encapsulation and second to provide a weakly chemisorbing intermediate at the qdot ionic interface. This allows the histidine to act as an intermediate shell upon which further direct ligand exchange can occur. Since this intermediate encapsulation is easily displaced by an assortment of different molecules while in aqueous buffers, we refer to this approach as modular. Characterization via FTIR and NMR revealed the extent of ligand exchange, and provides insights into the interfacial binding mechanism. The colloidal stability and photostability of the qdots was probed via UV-vis and steady state fluorescence, which revealed promising quantum yield stability of greater than 1 year. The qdots have hydrodynamic diameters of <12 nm and surface charges dependent upon ligand type and coverage. The modularity of this approach is shown by tailoring the qdot surface charge via sequential ligand exchange using mixed monolayers of carboxylic acid and poly(ethylene glycol)-terminated thiols.