title: Mechanical force-based probing of intracellular proteins from living cells using antibody-immobilized nanoneedles.
abstract: We developed a method combining atomic force microscopy (AFM) and antibody-immobilized nanoneedles to discriminate living cells by probing intracellular cytoskeletal proteins without the need for cell labeling. The nanoneedles are ultra-thin AFM probes sharpened to 200 nm in diameter. While retracting a nanoneedle inserted into a cell, we measured the mechanical force needed to unbind the antibody-target protein complex. Using this method, the intermediate filament protein, nestin and neurofilament were successfully detected in mouse embryonic carcinoma P19 cells and rat primary hippocampal cells within a minute for a single cell and cell differentiation states could be determined. Additionally, the measured magnitude of the force detecting nestin was indicative of the malignancy of breast cancer cells. This method was shown to affect neither the doubling time of cells nor does it leave extrinsic antibodies within the examined cells, allowing to be used in subsequent analyses in their native state.
authors.affiliation: Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, Koganei, Tokyo 184-8588, Japan.
authors.forename: Shingo
authors.initials: S
authors.lastname: Mieda
authors.forename: Yosuke
authors.initials: Y
authors.lastname: Amemiya
authors.forename: Takanori
authors.initials: T
authors.lastname: Kihara
authors.forename: Tomoko
authors.initials: T
authors.lastname: Okada
authors.forename: Toshiya
authors.initials: T
authors.lastname: Sato
authors.forename: Kyoko
authors.initials: K
authors.lastname: Fukazawa
authors.forename: Kazuhiko
authors.initials: K
authors.lastname: Ishihara
authors.forename: Noriyuki
authors.initials: N
authors.lastname: Nakamura
authors.forename: Jun
authors.initials: J
authors.lastname: Miyake
authors.forename: Chikashi
authors.initials: C
authors.lastname: Nakamura
mesh_terms: D000936:Antigen-Antibody Complex / Q000276:immunology*; D004867:Equipment Design; D019544:Equipment Failure Analysis; D007118:Immunoassay / Q000295:instrumentation*; D055617:Micro-Electrical-Mechanical Systems / Q000295:instrumentation*; D008846:Micromanipulation / Q000295:instrumentation*; D015336:Molecular Probe Techniques / Q000295:instrumentation*; D036103:Nanotechnology / Q000295:instrumentation*; D009339:Needles; D025941:Protein Interaction Mapping / Q000295:instrumentation*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013314:Stress, Mechanical
chemical_list: D000936:Antigen-Antibody Complex