Glycopolymer-substituted gold nanoparticles were prepared via living radical polymerization with a reversible addition-fragmentation chain transfer (RAFT) reagent. Polyacrylamide derivatives with a-mannose (a-Man) and N-acetyl-bglucosamine (b-GlcNAc) were synthesized and hydrogenated to obtain thiol-terminated polymer. The thiol-terminated glycopolymers were mixed with gold nanoparticles to yield the polymer substituted gold nanoparticles with various diameters, which aggregated on addition of saccharide-recognition proteins (lectins). The aggregation properties were analyzed using transmission electron microscopy and UV spectra. Molecular recognition was studied with E. coli, which induced aggregation of the nanoparticles at the cell periphery.

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A novel recognition element has been synthesized using glycopolymer-modified gold nanoparticles (GNPs) in sugar ratios of 0, 6, 12, and 50%. The as-synthesized glycopolymer-modified GNPs have been employed in a novel lateral flow assay for the detection of proteins. A test solution of concanavalin A (ConA) at 0.01–100 µg mL−1 was readily detectable with the glycopolymer-modified GNPs with a sugar ratio of 6%. The results were clear enough to be visible with the naked eye, which demonstrates the convenience of this lateral flow assay for performing the detection of Shiga toxins and influenza viruses. Therefore, this lateral flow assay provides an attractive biosensor for the detection of proteins without the handling of toxic reagents, while allowing an easy and rapid procedure.

Glycopolymers with a-galactose (a-Gal) and a-mannose (a-Man) were synthesized by means of living radical polymerization with a reversible addition-fragment chain transfer reagent, and the thin-layer formation of glycopolymers was investigated in terms of protein recognition abilities. Thiol-terminated glycopolymers formed a thin layer of about 2.5 nm in thickness on a gold substrate, and the glycopolymer thin layer showed specific interaction with sugar recognition proteins (lectins and Shiga toxins (Stxs)). The interactions were highly specific, and the signal-to-noise ratio of protein recognition was greater than 16. Glycopolymer-substituted gold nanoparticles (GNPs) also showed biorecognition abilities and protein-specific aggregation. The protein recognition abilities of the GNPs were also analyzed. The glycopolymer-substituted GNPs were utilized for signal amplification of surface plasmon resonance (SPR) to detect protein-saccharide recognition. The glycopolymer with a-Gal showed a strong interaction with Stxs according to SPR measurements, suggesting a possible application of a-Gal-substituted GNPs in Stx-1 biosensing.

In macroautophagy, de novo formation of the double membrane-bound organelles, termed autophagosomes, is essential for engulfing and sequestering the cytoplasmic contents to be degraded in the lytic compartments such as vacuoles and lysosomes. Atg8-family proteins have been known to be responsible for autophagosome formation via membrane tethering and fusion events of precursor membrane structures. Nevertheless, how Atg8 proteins act directly upon autophagosome formation still remains enigmatic. Here, to further gain molecular insights into Atg8-mediated autophagic membrane dynamics, we study the two representative human Atg8 orthologs, LC3B and GATE-16, by quantitatively evaluating their intrinsic potency to physically tether lipid membranes in a chemically defined reconstitution system using purified Atg8 proteins and synthetic liposomes. Both LC3B and GATE-16 retained the capacities to trigger efficient membrane tethering at the protein-to-lipid molar ratios ranging from 1:100 to 1:5,000. These human Atg8-mediated membranetethering reactions require trans-assembly between the membrane-anchored forms of LC3B and GATE-16 and can be reversibly and strictly controlled by the membrane attachment and detachment cycles. Strikingly, we further uncovered distinct membrane curvature dependences of LC3B-and GATE-16-mediated membrane tethering reactions: LC3B can drive tethering more efficiently than GATE-16 for highly curved small vesicles (e.g., 50 nm in diameter), although GATE-16 turns out to be a more potent tether than LC3B for flatter large vesicles (e.g., 200 and 400 nm in diameter). Our findings establish curvature-sensitive trans-assembly of human Atg8-family proteins in reconstituted membrane tethering, which recapitulates an essential subreaction of the biogenesis of autophagosomes in vivo.