New detection system for toxic agents based on continuous spectroscopic monitoring of living cells.

In this study, we show the feasibility of a new type of cell based biosensor which uses spectroscopic in situ real time detection of biochemical changes in living cells exposed to toxic chemical agents. We used a high power 785 nm laser to measure the time dependent changes in the Raman spectrum of individual living human lung cells (A549 cell line) treated with a toxic agent (Triton X-100, 250 microM solution). Individual cells were monitored by Raman spectroscopy over a total time span of 420 min, with 30 min sampling intervals. During this period of time, the A549 cells were maintained in a purpose designed temperature controlled cell chamber, which allowed the cells to be maintained in physiological conditions. The time dependent changes in the Raman spectra were correlated with the sequences of events that occur during cell death. The molecular mechanisms involved in cell death are indicated by the decrease in the magnitude of Raman peaks corresponding to proteins (1322, 1342 and 1005 cm(-1)) and DNA (decrease by 80-90% in the 786 cm(-1) phosphodiester bonds C'5-O-P-O-C'3). To support these conclusions, viability tests and Western blotting analysis of PARP protein were carried out. This technique could overcome the limitations of other detection systems available, since the specific time dependent biochemical changes in the living cells can be used for the identification and quantification of a large range of toxic agents. This technique could also be used with cellular microarrays for high throughput in vitro toxicological testing of pharmaceuticals and in situ monitoring of the growth of engineered tissues.