Direct observation of selective protein capturing on molecular imprinting substrates.
A sensing interface for specific protein capture was fabricated using a novel molecular imprinting (MIP) process. Bovine serum albumin (BSA) and ovalbumin (OVA) were imprinted on a quartz substrate with modified alkyl groups, and target protein capture was detected using a deep-UV fluorescence image microscope (UVFLIM). The imprinted protein was immobilized to silica beads (diameter: 15 μm) using a phospholipid polymer containing both active ester groups and silane coupling groups, which were used as protein stamps to prepare the imprinting surface. Protein recognition sites were constructed by integrating sodium dodecyl sulfate (SDS) as the ligand, which was immobilized with a biocompatible photoreactive phospholipid polymer. When BSA solution was added to the BSA-based MIP substrate, strong fluorescence was observed from the tryptophan residue of BSA. In contrast, for the OVA-based MIP substrate and non-MIP substrate, no fluorescence was observed. The surface showed good selectivity of BSA against OVA. The phospholipid polymer layer prevented non-specific protein adsorption, resulting in highly selective protein recognition. Further, when the protein-imprinted substrate was constructed without ligands, neither protein was captured on the substrate. We demonstrated the importance of ligand integration for capturing target proteins at specific positions. UVFLIM can be used to detect biomolecules at the single-molecule level by using intrinsic fluorescence without molecular labeling. Our new protein-imprinted surface used with UVFLIM is a versatile tool for capturing biomolecules.
Department of Materials Engineering, School of Engineering, The University of Tokyo, 7-3-1, Hongo, Tokyo 113-8656, Japan.
Kyoko
K
Fukazawa
Qiang
Q
Li
Stefan
S
Seeger
Kazuhiko
K
Ishihara
D000327:Adsorption; D004867:Equipment Design; D019544:Equipment Failure Analysis; D008856:Microscopy, Fluorescence / Q000295:instrumentation*; D057054:Molecular Imaging / Q000295:instrumentation*; D054802:Molecular Imprinting / Q000295:instrumentation*; D040081:Protein Array Analysis / Q000295:instrumentation*; D011485:Protein Binding; D011506:Proteins / Q000032:analysis* / Q000737:chemistry*
D011506:Proteins