Fast protein separation using sub-μm particles
The FDA approved PSA test is fraught with false positives and negatives, and does not distinguish aggressive vs. nonaggressive cancer. PSA has dozens of glycoforms that are potentially of diagnostic value. We are developing a new gel-free material for protein electrophoresis that will enable sensitive, high-throughput western blotting to detect the PSA glycoforms in serum. The new technology will provide the needed throughput for validation testing of these glycoforms as biomarkers for aggressive prostate cancer. The new technology can be applied to glycoform analysis for virtually any biomarker candidate. Isoelectric focusing (IEF) is a powerful tool of separating protein isoforms based on slight isoelectric point (PI) changes. We describe the use of packed capillaries IEF (cIEF) show high resolution of PSA glycoforms separation fellow by on-column western bloting. Silica colloidal crystals can replace gels in cIEF for PSA separation. The separated proteins are immobilized by polymer with NHS functional group and fellow by antibody probing. Ultra-homogeneous packing of nonporous silica nano-particles reduces protein diffusion and peak broadening. Unwanted electroosmotic flow is eliminated by brush layer poly acrylamide. The charge heterogeneity on the surface of PSA glycoforms can be separated by different isoelectric point. The cyanine dye label PSA glycoforms (cy3) and protein pI markers (cy5) can be distinguished by different excitation wavelength. Previous study shows the PSA glycoforms will be possible biomarker by compare the sample of cancer patients and health donor. We showed higher resolution of PSA glycoforms separation using new cIEF in the same scale. This is the evidence that our technology can be using for biomarker screening and diagnose.
Wirth, Purdue University.
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