Formation of Gradients of Proteins on Surfaces with Microfluidic Networks

Abstract

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.

Microfluidic processor allows rapid HER2 immunohistochemistry of breast carcinomas and significantly reduces ambiguous (2+) read-outs

“…The long incubation times in conventional IHC, which are in the range of 30 min to hours, may well result in a marked loss of proportionality between antigen expressions in the tissue and the immunohistochemical signal, as the antibody binding may be hindered by saturation phenomena according to Langmuir's isotherm, by steric hindrance (20), diffusion-limited transport of the antibodies to the surface (19,20,22,(28)(29)(30)(31)(32)(33)(34), or a combination of these factors. Caelen et al (35) performed an experiment in which fluorescently labeled target antigens were immobilized on a surface with different concentrations and incubated with another fluorescent marker-labeled detection antibody in a bulk solution during 30 min, as done in a conventional IHC protocol. They found that the fluorescent signal originating from target antigens immobilized on the surface was not linearly proportional to that of the bound detection antibody, but that the signal of bound detection antibodies saturated after a given target antigen concentration, resulting in overstaining of the midconcentration range and understaining of the high-concentration range of the immobilized target antigen.…”

Microfluidics for Processing Surfaces and Miniaturizing Biological Assays

“…D) Fluorescence micrograph showing short-scale gradients of TRITC-labeled (TRITC: tetramethyl rhodamine isothiocyanate) Abs that were deposited using an MFN in PDMS onto a glass surface. (Adapted with permission from [175,178]. Copyright 2000, American Chemical Society.…”

“…2D). [8,[178][179][180][181] Interestingly, the length scales and diffusion times found in microchannels compare with biochemical processes occurring in living cells. [19,182,183] The ratio between convective and diffusive transport is measured by the (dimensionless) Péclet number:…”

Multilayer poly(vinyl alcohol)-adsorbed coating on poly(dimethylsiloxane) microfluidic chips for biopolymer separation

“…The test mixture of acidic proteins, BSA and b-lactoglobulin, were separated on three-layer PVA(88)-coated PDMS microchip with a good resolution at pH 8 ( Fig. 8) but BSA was strongly adsorbed on original PDMS and used as a protecting coating [38]. Using the low-viscosity separation matrix 2% HPMC (50 cP), FX174 RF/HaeIII fragments can be easily resolved within 400 s (Fig.…”