Sensitive detection of enzyme activity by multi-photon nonlinear laser spectroscopy

摘要

Multi-photon laser wave mixing is an unusually sensitive nonlinear spectroscopic method that offers excellent sensitivity levels in the detection of enzyme activity levels. Coherent interactions between two crossed laser beams inside an absorbing medium generate dynamic gratings which in turn diffract off incoming photons to create a coherent laser-like signal beam. Since the signal beam is generated only in the presence of an absorbing medium, it yields very low background noise levels. Enzyme activity is measured by monitoring the change in the signal intensity as the absorbing compound is consumed or produced as a result of the enzyme catalyzed reactions. Optical setup for this one-color multi-photon method is simple compared to other multi-photon methods. Unlike iluorescence methods, wave mixing offers virtually 100% signal collection efficiency. Wave mixing is also less susceptibility to quenching and it allows analysis of both fluorescing and non-fluorescing labels. Since the laser wave-mixing signal has a quadratic dependence on analyte absorptivity, this method is more sensitive to small changes in the composition of the solution as compared to conventional detection methods. In addition, the use of nanoliter or picoliter probe volumes allows sensitive detection of small samples with high spatial resolution. Thermal and other physical properties of condensed-phase analytes can be advantageously used to enhance the sharpness of the laser-induced gratings and, hence, the analytical signal.