Direct immobilization of Fab′ in nanocapillaries for manipulating mass-limited samples

Interfacing nanoscale elements into a microfluidic device enables a new range of fluidic manipulations. Nanocapillary array membranes (NCAMs), consisting of thin (5 mu m < d < 20 mu m) membranes containing arrays of nanometer diameter (10 nm < a < 500 nm) pores, are a convenient method of interfacing vertically separated microchannels in microfluidic devices that allow the external control of analyte transport between microfluidic channels. To add functionality to these nanopores beyond simple fluid transport, here we incorporate an antibody-based molecular recognition element onto the pore surface that allows selective capture, purification, and release of specific analytes from a mixture. The pores are fabricated by electroless plating of gold into the nanopores of an NCAM (Au-NCAM). An antibody is then immobilized 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 hinge region. The successful immobilization and biological activity of the resultant Fab' through this protocol is verified on planar gold by fluorescence microscopy, scanning electron microscopy, and atomic force microscopy. 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 versus nonantigenic peptides increase more than 20-fold after passing through the Fab'-Au-NCAM relative to the control Au-NCAM. The affinity-tagged Au-NCAM can be incorporated into microfluidic devices to allow the concentration, capture, and characterization of analytes in complex mixtures with high specificity.