Nanometer-scale fluidic devices offer an alternative to gels for separating biomolecules with better control and accuracy. Here we demonstrate the quantitative analysis of disease-marker proteins by continuously separating the antibody-protein immunocomplexes from the unbound antibodies, utilizing the anisotropically-patterned nano-sieve array (ANA) structures. The ANA structures, composed of periodically-patterned deep channels and shallow regions, allow the small antibodies to pass through the shallow regions easier than the large immunocomplex, when the flow-field is applied in an oblique direction. We examined two proteins used as disease markers, human C-reactive protein (CRP) and human chorionic gonadotropin (hCG), by using fluorescent-labeled polyclonal antibodies. We showed that the size of the immunocomplex and the field strength are the critical factors for the separation, and we successfully demonstrated the quantification of the proteins in the range of 0.05 to 10 μg/mL. Additionally, this device allows a convenient measurement of homogeneous binding kinetics, without the need for repeated binding experiments and immobilizing the molecules. The presented nanofluidic device will be a useful tool for the rapid quantification and the preparative immunoseparation of the target proteins.
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