Protein analysis at the single cell level by nonlinear laser wave-mixing spectroscopy for high throughput capillary electrophoresis applications .

摘要

Nonlinear degenerate four-wave mixing is presented as an ultrasensitive optical absorption-based method for detection and measurement of biological samples. Wave-mixing imaging detection technique can localize and quantify biomolecules in single cells and tissue sections with excellent spatial distribution of light absorbed by a target sample. Cellular components can be label-free or labeled with a chromophore or a fluorophore and imaged by wave mixing using a CCD camera. In a 2-D forward-scattering wave-mixing geometry, two overlapping laser beams form interference gratings and transfer their energy to an absorbing medium, creating thermal gratings followed by changes in the refractive index. The probe beam diffracts off these laser-induced gratings to produce the signal beam, which is detected by a CCD camera or a photodiode. A single bio cell can be placed in a glass slide and as the laser beams probe the labeled cellular component, the CCD camera captures wave-mixing signals corresponding to the absorbing cellular components. This nonlinear imaging technique can be used for both live and fixed cells in real time to obtain information on sequential changes in the number, morphology and distribution of cellular components in a single cell.;Nonlinear laser wave-mixing spectroscopy coupled with capillary electrophoresis provides a novel ultrasensitive method for single-cell protein analysis. This method is used to detect proteins separated within a single cell. Nonlinear wave mixing has many advantages including quadratic dependency on analyte concentration, high spatial resolution and small sample requirements. Furthermore, wave mixing offers excellent detection sensitivity levels even when using very short optical path lengths, and hence, it can be easily interfaced to capillary electrophoresis separation systems. A single cell is injected into a coated capillary, lysed and labeled inside the capillary with a chromophore. Labeled proteins are separated in a sieving matrix under applied voltage through the capillary based on their mass-to-charge ratio differences.;To further improve protein separation, a random amphiphilic copolymer, poly(n-dodecylacrylate-3-sulfopropylmethylacrylate) or C12SO 3H (25/75%), is synthesized and used for protein separation in capillary electrophoresis. Amphiphilic polymers offer many benefits for protein separation. They can be used in both hydrophobic and hydrophilic moieties, they are easy to synthesize, they require less polymer percentage, and they are cost effective. Shorter analysis times have been obtained with this polymer for standard proteins. Amphiphilic polymers can be used for the analysis of hydrophobic proteins to obtain higher separation efficiency and resolution. Nonlinear laser wave mixing coupled with this new polymer can enhance CE separation of proteins.;High throughput CE is performed for the analysis of many samples in a short time scale. Using wave mixing, chromophore-labeled proteins passing through the capillary windows can be detected in an array of capillaries lined up tightly. Signal spots are collected by a photodiode array with an NMOS image sensor. The wave-mixing signal is a collimated coherent laser-like beam, and hence, it can be collected with nearly 100% optical collection efficiency against a dark background. Unique features include short optical path lengths, quadratic dependency on sample absorption coefficient and concentration, and cubic dependency on laser power.;Deep UV 266-nm laser wave-mixing detection is presented as a novel sensitive method for proteins in their native form without using labels. We demonstrate absorption-based wave mixing as an inherently effective detection method for several viable proteins in their native form using capillary electrophoresis. Excellent detection sensitivity levels are obtained using this unusually sensitive absorption-based method.