Spectroscopic and separation methods for bioanalysis
Quiroz, Michelle M
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Several analytical methods have been developed to study apoptosis (programmed cell death), many of which rely on fluorescence processes. Apoptosis is a highly regulated biological event and is a vital process that helps regulate tissue growth, normal cell turnover, immune response, and tissue development. However, diseases such as cancer and heart disease are associated with malfunctions in the apoptosis machinery. There is therefore a need to elucidate the processes of apoptosis induction and inhibition. Fluorescence assays continue to play a major role in apoptosis assays, and probe and method development are ongoing. There are several standard techniques such as flow cytometry and confocal microscopy for apoptosis study in cells. In addition, new techniques such as super-resolution microscopy, multi-photon excitation, and single cell-single molecule spectroscopy are quickly emerging. In this work, detection of apoptosis was studied by single molecule fluorescence correlation spectroscopy (FCS) in living cells. Caspase activity was assayed using a new red fluorogenic probe that avoids the spectral overlap of green fluorescent probes and cell autofluorescence. This new probe, 2SBPO-Casp, was synthesized by coupling a water-soluble Nile Blue derivative (2SBPO) to an aspartic acid residue. Upon apoptosis induction and caspase activation, free 2SBPO dye is shown to accumulate inside the cell after probe cleavage. In previous work in our lab, single molecule fluorescence in single apoptotic cells was detected 45 min after induction using a rhodamine 110-based probe. However, significant statistical analysis was needed to exclude false positives. The use of 2SBPO-Casp overcomes the autofluorescence problem and offers a steady fluorescence signal. In our single molecule FCS measurements, Ramos cells were determined apoptotic on the basis of their correlation coefficient value (R2). Cells that contain an R2= 0.65 were identified as highly correlated and therefore determined to be apoptotic. Single apoptotic cells identified in this manner were found as early as 30 min after induction and the number of apoptotic cells reached a peak value at the 3rd hour, which is consistent with other techniques. Using single molecule techniques and a new apoptosis probe, the temporal dynamics were elucidated with better sensitivity and resolution than in previous studies. Finally, to increase the throughput of cellular analyses, three different types of polymer based microdevices were fabricated and demonstrated. Poly(dimethyl silxane), (PDMS), and poly(methyl methacrylate), (PMMA), were used to fabricate T-shaped chips. The separation resolution, and efficiency was analyzed for native PDMS, PMMA, sol-gel modified PDMS, and native PDMS with a dynamic coating of sodium dodecyl sulfate (SDS). With an electric field of 512 V/cm, PDMS coated with SDS showed to give high electroosmotic flow (EOF) and was able to separate fluorescence dyes in Ramos cells.