BK21 Seminar

 

▶Subject :  In vivo Single Cell Analysis in Nanoscales

▶Speaker: Prof. Chang Ming Li (Nanyang Technological University)

▶Date : 4:00PM/May.17(Thu)/2012

▶Place : Room 104, Life Science Bldg

 

*Abstract
To understand the myriad of cellular processes going on in a single cell, highly sensitive methods are required to monitor and thus decipher these important cellular signaling pathways. Classical analytical techniques require large amount of cells (>1000cells) for analysis and only report the population averaged values thus masking the cell-to-cell variations. Heterogeneity in cellular compositions is an important parameter in understanding tumor progression which may lead to the discovery of biomarkers for early diagnosis of cancer. Single cell analysis is also critical when rare cell types such as primary cells are being investigated as these cells can not be propagated. Furthermore, fixation and lysis steps usually performed with conventional biochemical analysis destroy cell viability and may distort the native molecular conformations. Due to the ultra small size of single-cell, ultra trace amount of component, and ultra rapid biochemical reaction, single-cell analysis in nanoscales presents a great challenge to develop a methodology with high sensitivity, high selectivity, high temporal resolution and ultrasmall sampling-volume.
A unique optoelectronic nanoprobe is used to analyze single cells in nanoscales to achieve high temporal resolution for dynamic analysis, in which excitation light from the monochromator is launched into the delivery optical fiber that is coupled to the nanoprobe for optical detection while its surrounding nanoelectrode is used to sensing biologically interesting biomolecules. With these approach, early developed cancerous cells (MCF-7) gave significantly higher fluorescence intensity than the control nanoprobes (hMSC) in neuclus thus confirming the detection of endogeneous telomerases.
The nanoprobes are also used to monitor the extracellular lactate concentrations of cancer cells by immobilizing its nanotip with lactate dehydrogenases, which could catalyze lactate conversion to generate NADH for sensitive fluorescence detection]. The results demonstrate that the fabricated nanosensor can successfully detect the extracellular lactate concentrations for single HeLa, MCF-7 and human fetal osteoblast (hFOB) cells, showing that the cancer cells have distinctly higher extracellular lactate concentrations than normal cells as that predicted by Warburg effect. The nanosensor was also employed to investigate the effect of a monocarboxylate transporter inhibitor on the lactate efflux from cancer cells. Different lactate efflux inhibition profiles were obtained for HeLa and MCF-7 cell lines.
A bifunctional electro-optical nanoprobe with integrated nanoring electrode and optical nanotip was fabricated and investigated to simultaneously detect both electrical and optical signals in real-time with high spatial resolution. Concurrent measurements of the oxidant generation and the intracellular antioxidant levels in single cells correlate the stronger oxidant generation with an altered initial antioxidant response in the breast cancer cells in comparison to the normal ones suggesting that the cell malignancy is associated with the strength of oxidative stress, and the higher antioxidant level may be the cause of the drug resistance. While the optical detection indicates the fluctuation of the intracellular redox homeostasis, the chronoamperometric signals allow quantitatively real-time detection of the H2O2 release and decay. This method promises applications in study of the dynamics of important physiological processes, and provides the opportunity to unravel the interplay of various signalling pathways.
We have investigated the drug-cell interaction at a subcellular level by using an rGO functionalized optical fiber nanoprobe. Distinct ROS generation, the main medical effecter and rise in Ca2+ level, an important mediator of cancer cell death, observed at different subcellular locations pinpoint that the perinuclear region is the subcellular site of action. The developed method has the potential to characterize other new drugs. This work provides not only scientific insights of subcellular drug-cell interaction but also valuable knowledge for rational design of subcellular targeted delivery or spatially resolved signal intervention.

 

☎ Inquiry :Prof. Kim Kyong Tai (279-2297)

*This Seminar will be given in English