Development of a method to evaluate caspase-3 activity in a single cell using a nanoneedle and a fluorescent probe.
A method to detect an enzymatic reaction in a single living cell using an atomic force microscope equipped with an ultra-thin needle (a nanoneedle) and a fluorescent probe molecule was developed. The nanoneedle enables the low-invasive delivery of molecules attached onto its surface directly into a single cell. We hypothesized that an enzymatic reaction in a cell could be profiled by monitoring a probe immobilized on a nanoneedle introduced into the cell. In this study, a new probe substrate (NHGcas546) for caspase-3 activity based on fluorescent resonance energy transfer (FRET) was constructed and fixed on a nanoneedle. The NHGcas546-modified nanoneedle was inserted into apoptotic cells, in which caspase-3 is activated after apoptosis induction, and a change in the emission spectrum of the immobilized probe could be observed on the surface of the nanoneedle. Thus, we have developed a successful practical method for detecting a biological phenomenon in a single cell. We call the method MOlecular MEter with Nanoneedle Technology (MOMENT).
Kihara
Takanori
T
The Center for NanoBio Integration, The University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo 113-8656, Japan.
Nakamura
Chikashi
C
Suzuki
Miho
M
Han
Sung-Woong
SW
Fukazawa
Kyoko
K
Ishihara
Kazuhiko
K
Miyake
Jun
J
D017209:Apoptosis; D053148:Caspase 3 / Q000032:analysis* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry* / Q000302:isolation & purification; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000302:isolation & purification; D006367:HeLa Cells; D006801:Humans; D010957:Plasmids; D012825:Silicon / Q000737:chemistry; D013499:Surface Properties
D005456:Fluorescent Dyes; D049452:Green Fluorescent Proteins; D053148:Caspase 3; D012825:Silicon