Novel Nonlinear Laser Wave-Mixing Spectroscopy Coupled with Capillary Electrophoresis for the Sensitive Detection of Sars-Cov-2 Biomarkers.

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We present a highly sensitive detection method for the multivalent SARS-CoV-2 nucleocapsid protein, a crucial RNA-binding protein involved in virus assembly and genome packaging. The nucleocapsid protein's structure facilitates the recognition of antiviral drug targets, making it essential for viral RNA production and antiviral drug development. Traditional nucleic acid amplification assays like PCR, gene sequencing, CRISPR, CT-scan, ELISA, LFA, and LAMP are currently used for COVID-19 identification. However, these techniques have limitations such as false results, template quality issues, cost, limited specificity, and sensitivity. To overcome these challenges, we introduce our patented nonlinear laser wave-mixing technique, offering numerous advantages including label-free native detection, enhanced sensitivity, minimal sample requirements, short optical path length, and high spatial resolution. Our approach utilizes four-wave mixing (FWM), a nonlinear optics phenomenon, to generate coherent signals by interacting two or three wavelengths and producing new wavelengths. Phase- matched four-wave mixing is achieved using collinear incommensurate waves, resulting in efficient signal generation within the analyte. The small probe volume, achieved by overlapping two laser beams, makes our technique compatible with microfluidics and capillary-based electrophoresis systems. Furthermore, wave-mixing, being an absorption-based method, enables the detection of small amino acids and proteins using common labeling methods like fluorophores and chromophores. In our study, we successfully detected the SARS-CoV-2 nucleocapsid protein labeled with the Chromeo P540 tag using a 532 nm solid-state laser as the excitation source. Our technique exhibited high sensitivity, with a detection concentration limit of 0.26 nM, corresponding to a mass detection limit of 19.3 zeptomoles. This innovative detection method holds great promise for advancing COVID-19 diagnostics by providing superior sensitivity, compatibility with various labeling techniques, and the ability to detect infected carriers who may not exhibit clinical symptoms.