Nonlinear multi-photon laser wave-mixing detection coupled with capillary electrophoresis provides an ultrasensitive method for analyzing malachite green, crystal violet, and their metabolites. This method offers zepto-mole detection sensitivity, excellent chemical selectivity and specificity, and high spatial resolution suitable for single-cell analysis. A 266 nm UV laser probes analytes in their native form while a visible laser probes labeled analytes. Coupling with capillary electrophoresis further enhances chemical selectivity. This portable, battery-powered method is suitable for a wide range of biomedical and environmental applications.

1. Ultrasensitive Nonlinear Multi-Photon Laser Wave-Mixing Detection Methods for
Environmental and Biomedical Applications
Megan Murphy, Mya Brown, Jean Sebastien Pradel and William G. Tong
Department 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.