Direct Immobilization of Fab' in Nanocapillaries for Manipulating Mass-Limited Samples
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- 作者：Bo Young Kim ; Carla B. Swearingen ; Ja-an A. Ho ; Elena V. Romanova ; Paul W. Bohn ; Jonathan V. Sweedler
- 刊名：Journal of the American Chemical Society
- 出版年：2007
- 出版时间：June 20, 2007
- 年：2007
- 卷：129
- 期：24
- 页码：7620 - 7626
- 全文大小：395K
- 年卷期：v.129,no.24(June 20, 2007)
- ISSN：1520-5126
文摘
Interfacing nanoscale elements into a microfluidic device enables a new range of fluidicmanipulations. Nanocapillary array membranes (NCAMs), consisting of thin (5 
m < d < 20 m) membranescontaining arrays of nanometer diameter (10 nm < a < 500 nm) pores, are a convenient method ofinterfacing vertically separated microchannels in microfluidic devices that allow the external control of analytetransport between microfluidic channels. To add functionality to these nanopores beyond simple fluidtransport, here we incorporate an antibody-based molecular recognition element onto the pore surfacethat allows selective capture, purification, and release of specific analytes from a mixture. The pores arefabricated by electroless plating of gold into the nanopores of an NCAM (Au-NCAM). An antibody is thenimmobilized on the Au-NCAM via gold-thiol chemistry as a thiolated fragment of antigen-binding (Fab')prepared by direct digestion of the antibody followed by reduction of the disulfide linkage on the hingeregion. The successful immobilization and biological activity of the resultant Fab' through this protocol isverified on planar gold by fluorescence microscopy, scanning electron microscopy, and atomic forcemicroscopy. Selective capture and release of human insulin is verified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The relative mass spectral peak intensities for insulin versusnonantigenic peptides increase more than 20-fold after passing through the Fab'-Au-NCAM relative to thecontrol Au-NCAM. The affinity-tagged Au-NCAM can be incorporated into microfluidic devices to allow theconcentration, capture, and characterization of analytes in complex mixtures with high specificity.
m < d < 20 m) membranescontaining arrays of nanometer diameter (10 nm < a < 500 nm) pores, are a convenient method ofinterfacing vertically separated microchannels in microfluidic devices that allow the external control of analytetransport between microfluidic channels. To add functionality to these nanopores beyond simple fluidtransport, here we incorporate an antibody-based molecular recognition element onto the pore surfacethat allows selective capture, purification, and release of specific analytes from a mixture. The pores arefabricated by electroless plating of gold into the nanopores of an NCAM (Au-NCAM). An antibody is thenimmobilized on the Au-NCAM via gold-thiol chemistry as a thiolated fragment of antigen-binding (Fab')prepared by direct digestion of the antibody followed by reduction of the disulfide linkage on the hingeregion. The successful immobilization and biological activity of the resultant Fab' through this protocol isverified on planar gold by fluorescence microscopy, scanning electron microscopy, and atomic forcemicroscopy. Selective capture and release of human insulin is verified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The relative mass spectral peak intensities for insulin versusnonantigenic peptides increase more than 20-fold after passing through the Fab'-Au-NCAM relative to thecontrol Au-NCAM. The affinity-tagged Au-NCAM can be incorporated into microfluidic devices to allow theconcentration, capture, and characterization of analytes in complex mixtures with high specificity.