Media 2: Three dimensional multi-molecule tracking in thick samples with extended depth-of-field

Originally published in Optics Express on 26 January 2015 (oe-23-2-787

Media 1: Three dimensional multi-molecule tracking in thick samples with extended depth-of-field

Originally published in Optics Express on 26 January 2015 (oe-23-2-787

Molecular Insights into the Improved Bioactivity of Interferon Conjugates Attached to a Helical Polyglutamate

Attaching polymers, especially polyethylene glycol (PEG), to protein drugs has emerged as a successful strategy to prolong circulation time in the bloodstream. The hypothesis is that the flexible chain wobbles on the protein’s surface, thus resisting potential nonspecific adsorption. Such a theoretical framework may be challenged when a helical polyglutamate is used to conjugate with target proteins. In this study, we investigated the structure–activity relationships of polyglutamate-interferon conjugates P(EG3Glu)-IFN using molecular simulations. Our results show that the local crowding effect induced by oligoethylene glycols (i.e., EG3) is the primary driving force for helix formation in P(EG3Glu), and its helicity can be effectively increased by reducing the free volume of the two termini. Furthermore, it was found that the steric hindrance induced by IFN is not conductive to the helicity of P(EG3Glu) but contributes to its dominant orientation relative to interferon. The orientation of IFN relative to the helical P(EG3Glu) can help to protect the protein drug from neutralizing antibodies while maintaining its bioactivity. These findings suggest that the helical structure and its orientation are critical factors to consider when updating the theoretical framework for protein–polymer conjugates

Analysis of Pirlimycin Residues in Beef Muscle, Milk, and Honey by a Biotin–Streptavidin-Amplified Enzyme-Linked Immunosorbent Assay

Food contamination by veterinary drug residues is a worldwide public health concern and requires continuous monitoring. In this study, we developed a biotin–streptavidin-amplified ELISA (BA-ELISA) using a produced monoclonal antibody for detecting pirlimycin residues in beef muscle, milk, and honey. The IC₅₀ value of the BA-ELISA was 1.6 ng/mL for pirlimycin in buffer, and the sensitivity was improved 3 times compared to traditional ELISAs. The optimized BA-ELISA can be used to quantitate trace amounts of pirlimycin residues in beef muscle, milk, and honey. This method had limits of detection (LODs) of 4.45 μg/kg in beef muscle, 1.65 μg/L in milk, and 2.75 μg/kg in honey. The average recovery of the BA-ELISA ranged from 78 to 97%, and the coefficient of variation ranged from 5.3 to 13.5%. The developed BA-ELISA method was validated using LC-MS/MS, and the BA-ELISA can be used for routine screening analysis of pirlimycin residues

Image_1_Cytotoxicity of InP/ZnS Quantum Dots With Different Surface Functional Groups Toward Two Lung-Derived Cell Lines.TIF

<p>Although InP/ZnS quantum dots (QDs) have emerged as a presumably less hazardous alternative to cadmium-based QDs, their toxicity has not been fully understood. In this work, we report the cytotoxicity of InP/ZnS QDs with different surface groups (NH₂, COOH, OH) toward two lung-derived cell lines. The diameter and the spectra of InP/ZnS QDs were characterized and the hydrodynamic size of QDs in aqueous solution was compared. The confocal laser scanning microscopy was applied to visualize the labeling of QDs for human lung cancer cell HCC-15 and Alveolar type II epithelial cell RLE-6TN. The flow cytometry was used to confirm qualitatively the uptake efficiency of QDs, the cell apoptosis and ROS generation, respectively. The results showed that in deionized water, InP/ZnS-OH QDs were easier to aggregate, and the hydrodynamic size was much greater than the other InP/ZnS QDs. All these InP/ZnS QDs were able to enter the cells, with higher uptake efficiency for InP/ZnS-COOH and InP/ZnS-NH₂ at low concentration. High doses of InP/ZnS QDs caused the cell viability to decrease, and InP/ZnS-COOH QDs and InP/ZnS-NH₂ QDs appeared to be more toxic than InP/ZnS-OH QDs. In addition, all these InP/ZnS QDs promoted cell apoptosis and intracellular ROS generation after co-cultured with cells. These results suggested that appropriate concentration and surface functional groups should be optimized when InP/ZnS QDs are utilized for biological imaging and therapeutic purpose in the future.</p

Size-Dependent Property and Cell Labeling of Semiconducting Polymer Dots

Semiconducting polymer dots (Pdots) represent a new class of fluorescent nanoparticles for biological applications. In this study, we investigated their size-dependent fluorescence and cellular labeling properties. We demonstrate that the polymer conformation in solution phase largely affects the polymer folding and packing during the nanoparticle preparation process, resulting in solution-phase control over the fluorescence properties of semiconducting polymer nanoparticles. The resulting Pdots exhibit apparent size dependent absorption and emission, a characteristic feature of different chain packing behaviors due to the preparation conditions. Single-particle fluorescence imaging was employed to perform a side-by-side comparison on the Pdot brightness, indicating a quadratic dependence of single-particle brightness on particle size. Upon introducing a positively charged dye Nile blue, all the three type of Pdots were quenched very efficiently (<i>K</i>_sv > 1 × 10⁷ M^–1) in an applied quenching process at low dye concentrations, but exhibit apparent difference in quenching efficiency with increasing dye concentration. Furthermore, Pdots of different sizes were used for cell uptake and cellular labeling involving biotin–streptavidin interactions. Fluorescence imaging together with flow cytometry studies clearly showed size dependent labeling brightness. Small-sized Pdots appear to be more effective for immunolabeling of cell surface, whereas medium-sized Pdots exhibit the highest uptake efficiency. This study provides a concrete guidance for selecting appropriate particle size for biological imaging and sensing applications