Ultrasensitive Nonlinear Multi-Photon Laser Wave-Mixing Detection Methods for Environmental and Biomedical Applications

Megan Murphy, Mya Brown, Jean Sebastien Pradel and William G. TongDepartment of Chemistry and Biochemistry, San Diego State University, San Diego, CA 92182

william.tong@sdsu.edu

Abstract

Nonlinear multi-photon laser wave-mixing detection coupled with capillary electrophoresis is presented as ultrasensitive methods for a wide range of biomedical and environmental applications including simultaneous analysis of malachite green, crystal violet, their metabolites leuco-malachite green and leuco-crystal violet. Nonlinear wave mixing offers inherent advantages over conventional laser methods including zepto-mole detection sensitivity, excellent chemical selectivity and specificity levels, and high spatial resolution suitable for single-cell analyses. The wave-mixing signal is a coherent laser-like beam, and hence it can be collected with excellent signal-to-noise ratios and high detection efficiency levels. Chromatic and leuco forms of crystal violet and malachite green absorb in the UV and visible wavelength ranges. We use a 266 nm UV laser to probe label-free analytes in their native form and a visible laser to probe labeled analytes. The wave-mixing signal has a quadratic dependence on analyte concentration, and hence, wave mixing is especially effective for monitoring small changes in analyte properties. In order to further enhance chemical selectivity levels, a capillary (75 µm inside diameter) is used to flow and separate analytes in our custom-built capillary electrophoresis system. The wave-mixing probe volume is small (nL, pL), and hence, it is inherently suitable for interfacing to lab-on-a-chip, microfluidics and microarray systems. Excellent detection sensitivity levels (atto-mole to zepto-mole levels) have been demonstrated using capillary- and chip-based separation systems for different biomarkers and environmental samples. Our nonlinear multi-photon detectors can be easily configured as battery-powered portable devices that are suitable for use in the field where resources are limited. Hence, wave-mixing methods allow application in the field for a wide range of environmental and biomedical applications (biomarkers, viruses and early detection of cancer, etc.).

Acknowledgment: We acknowledge partial support of this work by the NIH (R01), NSF, DoD, Army, DHS, Lockheed Martin and Johnson and Johnson.