Development of Nano-Micro Fused LSPR Chip for In Situ Single-Cell Secretion Analysis.

Single-cell analysis has become increasingly important in uncovering cell heterogeneity, which has great implications in medicine and biology for a deep understanding of cell characteristics. Owing to its significance, it is vital to create novel devices that can reveal special or unique cells. In this work, we developed a single-cell secretion detection chip consisting of microwells that can trap single cells. Each well is surrounded by Au nanopillars capable of localized surface plasmon resonance (LSPR) measurement. Using microfabrication and nanofabrication techniques, Au nanopillar and microwell structures were fabricated on a COP film. The Au nanopillar was modified with IL-6 antibodies for the direct detection of single-cell secreted IL-6 via LSPR absorbance peak shift. Specific IL-6 detection was successfully demonstrated using a null and IL-6 oversecreting Jurkat cell. A high single-cell trapping efficiency of over 80% was also achieved. Overall, the development of this single-cell secretion detection chip with a simple LSPR measurement setup represents a significant development in the field of cell biology and immunology, providing researchers with a powerful tool for studying individual cells and their secreted cytokines, and is useful for point-of-care testing (POCT) diagnostics.

Au-Capped Nanopillar Immobilized with a Length-Controlled Glycopolymer for Immune-Related Protein Detection.

A Au-capped nanopillar chip was prepared using nanoimprint lithography (NIL) and Au sputtering onto a cyclo-olefin polymer film. The Au surface of the chip exerting localized surface plasmon resonance (LSPR) phenomena was immobilized with a glycopolymer for the detection of cytokines. The glycopolymers were synthesized by reversible addition-fragmentation chain transfer (RAFT) polymerization for controlled polymer chain length, and thiol-terminated glycopolymers with chain lengths of 20-, 100-, and 200-mers were designed. The thickness of the biomolecular layer on the Au surface was controlled by changing the polymer chain length of the immobilized glycopolymer, and the absorption of proteins onto the Au surface was detected by the shift of absorbance peak wavelength. The value of absorbance peak wavelength shift by protein adsorption increased as the glycopolymer layer thickness became thinner. This difference in LSPR signal response was remarkable for cytokine recognition compared to larger proteins. It was shown that controlling the biomolecular layer thickness was effective for the detection of small proteins, and our research suggested the usefulness of the controlled glycopolymer surface as a molecular recognition material for cytokine detection.

Screening of a Glycopolymer Library of GM1 Mimics Containing Hydrophobic Units Using Surface Plasmon Resonance Imaging.

Effective screening methods for the development of glycopolymers as molecular recognition materials are desirable for the discovery of novel biofunctional materials. A glycopolymer library was prepared to obtain guidelines for the design of glycopolymers for the recognition of cholera toxin B subunits (CTB). Glycopolymers with varying ratios of hydrophobic and sugar units were synthesized by reversible addition fragmentation chain transfer polymerization. N-tert-Butylacrylamide, N-phenylacrylamide, and N-cyclohexylacrylamide as hydrophobic units were copolymerized in the polymer backbone, and galactose, which contributes to CTB recognition, was introduced into the side chains by "post-click" chemistry. The thiol-terminated glycopolymers were immobilized on a gold surface. The polymer immobilization substrate was analyzed in terms of interaction with galactose recognition proteins (CTB, peanut agglutinin, and Ricinus communis agglutinin I) using surface plasmon resonance imaging. The polymers with high ratios of sugar and hydrophobic units had the strongest interactions with the CTB, which was different from the trend with peanut agglutinin and Ricinus communis agglutinin I. The binding constant of the CTB with the glycopolymer with hydrophobic units was 4.1 × 106 M-1, which was approximately eight times larger than that of the polymer without hydrophobic units. A correlation was observed between the log P value and the binding constant, indicating that the hydrophobic interaction played an important role in binding. New guidelines for the design of recognition materials were obtained by our screening method.

Glycopolymers Mimicking GM1 Gangliosides: Cooperativity of Galactose and Neuraminic Acid for Cholera Toxin Recognition.

Glycopolymers mimicking GM1 gangliosides were synthesized by incorporating multiple types of carbohydrates into the polymer backbone. The glycopolymers were immobilized onto gold surfaces, and the interactions with the cholera toxin B subunit (CTB) were analyzed using surface plasmon resonance imaging. The glycopolymer containing both galactose and neuraminic acid showed enhanced recognition of CTB. The interaction was enhanced mainly because of an improvement in the dissociation process by the binding of the neuraminic acid group in the GM1 binding pocket. This cooperativity of galactose and neuraminic acid was achieved by incorporation into the same flexible polymer backbone, and the importance of the close placement of galactose and neuraminic acid groups was revealed. These results will be valuable in medical fields and also for the development of biofunctional materials.

Signal amplified two-dimensional photonic crystal biosensor immobilized with glyco-nanoparticles.

A two-dimensional, glycopolymer-immobilized, photonic crystal (PhC) biosensor was developed for the detection of proteins. Glycopolymers with different conformations, homopolymers and sugar-incorporating nanoparticles were immobilized on the PhC using intermediate succinimide-containing polymers and proteins. The surface modification was analyzed in detail, and the sugar-protein interaction was detected by monitoring changes in the reflection intensity that was expressed by the two-dimensional PhC. The surface modifications were performed successfully, and specific interactions were detected between the glycopolymers and the proteins. Stronger bonds were present between the glycopolymers and the target proteins than between the glycopolymers and the monovalent sugar, because of a clustering effect. The sugar-incorporating nanoparticles showed a larger binding capacity compared with the homopolymers, and low protein concentrations (with a detection limit of 6.0 ng mL-1) were detected using the sugar-incorporating nanoparticle-immobilized PhC. The detection limit of the developed biosensor was lower than that of surface plasmon resonance sensor (1.43 µg mL-1). The results of this study indicated the potential of the developed biosensor for the detection of a variety of biomolecules.