Optical sensor for the detection of caspase-9 activity in a single cell.
We demonstrate for the first time, the application and utility of a unique optical sensor having a nanoprobe for monitoring the onset of the mitochondrial pathway of apoptosis in a single living cell by detecting enzymatic activities of caspase-9. Minimally invasive analysis of single live MCF-7 cells for caspase-9 activity is demonstrated using the optical sensor which employs a modification of an immunochemical assay format for the immobilization of nonfluorescent enzyme substrate, Leucine-GlutamicAcid-Histidine-AsparticAcid-7-amino-4-methylcoumarin (LEHD-AMC). LEHD-AMC covalently attached on the nanoprobe tip of an optical sensor is cleaved during apoptosis by caspase-9 generating free AMC. An evanescent field is used to excite cleaved AMC and the resulting fluorescence signal is detected. By quantitatively monitoring the changes in fluorescence signals, caspase-9 activity within a single living MCF-7 cell was detected. By comparing of the fluorescence signals from apoptotic cells induced by photodynamic treatment and nonapoptotic cells, we successfully detected caspase-9 activity, which indicates the onset of apoptosis in the cells.
Kasili
Paul M
PM
Advanced Biomedical Science and Technology Group, Life Sciences Division, Oak Ridge National Laboratory, P.O. Box 2008, Oak Ridge, Tennessee 37831-6101, USA. kasilipm@ornl.gov
Song
Joon Myong
JM
Vo-Dinh
Tuan
T
D000622:Aminolevulinic Acid / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology; D015374:Biosensing Techniques / Q000295:instrumentation / Q000379:methods*; D001943:Breast Neoplasms / Q000188:drug therapy / Q000201:enzymology / Q000473:pathology; D053453:Caspase 9; D020169:Caspases / Q000378:metabolism*; D045744:Cell Line, Tumor; D003374:Coumarins / Q000737:chemistry; D006801:Humans; D008856:Microscopy, Fluorescence; D036103:Nanotechnology / Q000295:instrumentation / Q000379:methods*; D009842:Oligopeptides / Q000737:chemistry / Q000378:metabolism; D055095:Optics and Photonics; D010778:Photochemotherapy
D003374:Coumarins; D009842:Oligopeptides; D000622:Aminolevulinic Acid; C506433:CASP9 protein, human; D053453:Caspase 9; D020169:Caspases; C028743:7-amino-4-methylcoumarin