Protein adsorption at interfaces detected by second harmonic generation

We show that second harmonic (SH) spectroscopy, an intrinsically surface-selective technique, can be used to monitor protein (cytochrome c) adsorption to silica surfaces and negatively charged supported phospholipid bilayers. The origin of the SH signal is due to the effect of the adsorbed protein on the water molecules polarized near the charged interface. Although the protein does not contribute its own SII signal, its binding to the glass surface or the membrane reduces the polarization of the interfacial water molecules and this effect is proportional to the adsorbed protein concentration and can be monitored with high precision, in real time. The free energy of adsorption (Delta G(ads) = -11.8 kcal/mol) of cytochrome c to glass was determined from an adsorption isotherm measurement of the SH signal as a function of the bulk protein concentration. A detection sensitivity of 0.1 pmol/cm(2) of adsorbed cytochrome c protein is readily achieved.