The capability of microfluidic networks to pattern substrates with proteins is extended to create density gradients of proteins on surfaces. The networks are micromachined in Si, and the substrates are hydrophobic poly(dimethylsiloxane) (PDMS) elastomers. The gradients result from the progressive depletion of proteins in the fluids due to their adsorption onto the PDMS substrate as the solution travels along the microchannel. Forming gradients of rhodamine-tagged antigens on PDMS and binding the antigens with fluorescein-tagged antibodies from solution enable us to study the binding behavior of these partners on a surface:  Detection of the fluorescence associated with either partner suggests that recognition of the surface-immobilized antigens by an antibody from solution is more effective for a low density of antigens on the surface.

Gradients of biologically active proteins can be obtained by applying photochemical reactions. A photosensitive polysaccharide-based polymer (OptoDex) is used to covalently immobilize proteins on surfaces. Gradients of proteins are generated by varying the dose of light during the photoimmobilization. Probe proteins conserve their catalytic activity or immunological binding characteristics when linked to surfaces exemplified by silicon nitride or polystyrene. Heterogeneous immunoreactions between photoimmobilized antigens and antibodies showed an optimum binding efficiency at an antigen density of approximatly 1.3 ng/mm2.

We describe the microfabrication and use of elastomeric and rigid two-level microfluidic networks (µFNs), made of poly(dimethylsiloxane) (PDMS) or Si, for patterning surfaces. The first level corresponds to microchannels and the second to via holes through the µFNs serving as filling and venting ports. µFNs in PDMS are manufactured using a 'sandwich' replication from a microfabricated four inch mold structured with SU-8 photoresist, which is planarized by mechanical polishing. µFNs in Si are microfabricated using deep reactive ion etching. Both types of µFNs can be positioned onto a substrate, creating sealed microchannels, filled with different liquids, flushed, removed and reused. These two-level µFNs allow us to access the ports from the rear, minimize interchannel crosstalk, and are economic of solutions. The channels are made wettable so that the liquids can flow spontaneously into the conduits, but stop at the venting ports. The sealing of the conduits usually requires that either the µFN or the substrate be soft. A strategy for using hard two-level µFNs, in Si, for patterning hard substrates is presented: despite voids in-between the µFN and the substrate, a water-based solution can be guided by hydrophilic microchannels over a hydrophobic surface. Adjusting the wetting properties of the various surfaces is key to preventing undesired spreading of solutions. We illustrate our concepts by micromolding colored photocurable polymers on glass and patterning proteins as lines on a polystyrene surface.

An assay for quantification of riboflavin (Rf) in milk-based products has been developed using the principle of surface plasmon resonance with on-chip measurement. The quantification was done indirectly by measuring excess of Rf binding protein (RBP) that remains free after complexation with Rf molecules originally present in the sample solution. The chip was modified with covalently immobilized Rf in order to bind the RBP in excess. A chemical modification was performed to introduce a reactive ester group at the N-3 position of the natural Rf to bind amino groups present on the chip surface. Calibration solutions were prepared by mixing a range of Rf standard solutions with an optimized concentration of RBP. The Rf content in the milk-based products was then measured by comparison of the response against the calibration. Results obtained were very close to those from an official HPLC-fluorescence procedure. The limit of quantification was determined to 234 microg/L and the limit of detection to 70 microg/L by an injection volume of 160 microL.