Multi-photon nonlinear laser wave-mixing spectroscopy is a novel absorption-based technique that offers excellent detection sensitivity for biomedical applications, including early diagnosis and investigation of neurodegenerative diseases. α-Synuclein is linked to Parkinson's disease (PD), and characterization of its oligomers and quantification of the protein may contribute to understanding PD. The laser wave-mixing signal has a quadratic dependence on analyte concentration, and hence the technique is effective in monitoring small changes in concentration within biofluids. A wide variety of labels can be employed for laser wave-mixing detection due to its ability to detect both chromophores and fluorophores. In this investigation, two fluorophores and a chromophore are studied and used as labels for the detection of α-synuclein. Wave-mixing detection limits of PD-related protein conjugated with fluorescein isothiocyanate, QSY 35 acetic acid, succinimidyl ester, and Chromeo P503 were determined to be 1.4 × 10(-13) M, 1.4 × 10(-10) M, and 1.9 × 10(-13) M, respectively. Based on the laser probe volume used, the corresponding mass detection limits were determined to be 1.1 × 10(-23) mol, 1.1 × 10(-20) mol, and 1.5 × 10(-23) mol. This study also presents molecular-based separation and quantification of α-synuclein by laser wave mixing coupled with capillary electrophoresis.

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Laser wave-mixing detectors for microfluidics are presented as compact systems for parts-per-quadrillion-level detection of biomedical and environmental samples. Wave mixing offers unique advantages including absorption-based detection of micrometer-thin samples, generation of coherent laser-like signal beams, and sensitive detection of both fluorescing and non-fluorescing samples. Biomarkers could be detected in their native forms without labels, or if labels are desired, one could use both fluorophores and chromophores as labels. By coupling low-power (mW) solid-state lasers and microfluidics and microarrays, one can design compact optical systems that can be battery powered and used in the field for real-time fingerprinting of chemicals.

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