Nonlinear wave-mixing spectroscopic methods for bioanalytical and biophysical applications with sensitive detection at the single cell level
- Author(s): Atherton, Adrian Ashley;
- et al.
Nonlinear laser wave-mixing spectroscopy is presented as an absorption-based detection method for single cells and cellular components. Compared to other laser-based techniques, wave mixing offers several advantages including excellent detection sensitivity, high spatial resolution and small probe volumes suitable for detection of biomolecules. The analyte travels through the laser probe volume created by two focused input beams. Laser- induced dynamic gratings are generated in the presence of absorbing analytes. The resulting wave-mixing signal beams are coherent laser-like beams, and hence, they can be collected conveniently and efficiently. Laser wave mixing is demonstrated as a sensitive detection method for proteins. An optical absorption shift is monitored upon chromophore protein complexation. Ultrasensitive detection limits of 4.7 x 1̄0⁻¹⁹ M (i.e., 2.4 x 10⁻²² mol) and 9.3 x 10⁻¹⁴ M (i.e., 3.7 x 10⁻¹⁷ mol) are determined BSA and HPV antibodies, respectively. Wave mixing is used to investigate and detect heavy metals entrapped in a sol-gel optical sensor. Preliminary detection limits of 1.7 x 10⁻¹⁴ M (0.2ppq) and 1.15 x 10⁻⁹ M (59 ppt) are determined for Fe(II) and Cr(VI) ions, respectively. Further development of this sol-gel immobilization technique may lead to sol-gel biosensors for proteins. Nonlinear wave mixing offers detection of biospecific interactions on antibody or DNA microarrays. Microarray spots have specific cytokines or oligonucleotide sequences that bind to their corresponding target molecules. The wave-mixing laser probe volume traverses microarray spots for sensitive mapping and profiling of biospecific interactions with intra-spot spatial resolution. A preliminary detection limit of 6.4 x 10⁻¹³ M or 0.01 pg/mL is determined for cytokines. For the first time to our knowledge, cellular components within a single cell are detected and imaged by nonlinear wave-mixing spectroscopy. Wave mixing profiles and images intact cells and detects cellular proteins that are lysed and separated by capillary electrophoresis. A preliminary mass detection limit of 10 fg/mL is determined for proteins