Detection of membrane-bound enzymes in cells using immunoassay and Raman microspectroscopy

The method of surface-enhanced Raman microspectroscopy was developed for direct detection of membrane-bound enzymes in cells. Cells were cultured, fixed, and incubated with specific primary antibodies and their corresponding labeled secondary antibodies, and surface-enhanced Raman scattering (SERS) was detected directly in the wells of a multiwell plate. First, specific primary antibodies were separately bound to enzymes in cells. Then, the peroxidase-labeled secondary antibodies were added to bind these primary antibodies. Peroxidase substrates, o-phenylenediamine and hydrogen peroxide, were added and reacted for 15 min at room temperature to form azoaniline, a compound with strong Raman scattering. Then, Raman scattering of this enzymatic product was enhanced by silver colloids. Samples were excited with a He/Ne laser at 632.8 nm and SERS was detected by a CCD camera. The SERS spectrum of this product showed an intense peak at 1370 cm-1 and its intensity was used for assessment of cellular enzymes. The observed amount of enzyme was normalized to protein content in each well. The method was successfully used to detect prostaglandin H synthase-1 and -2 (PGHS-1 and -2) in normal human hepatocytes and human hepatocellular carcinoma (HepG2) cells. The detection limit of these PGHS enzymes by this method was about 0.1 pg per well. An immunohistochemical staining was also used to detect the expression of both PGHS isozymes in these cells.