"Smart" mobile affinity matrix for microfluidic immunoassays.
There is a current need for simple methods for immobilizing biomolecules within microfluidic channels. Here, a technique is reported for reversibly immobilizing immunoassay components in a channel zone that can be simply controlled by integrated heating elements. Latex beads were modified with the temperature-responsive polymer poly(N-isopropylacrylamide)(PNIPAAm) and co-modified with biotinylated poly(ethylene glycol)(PEG). PNIPAAm undergoes a hydrophilic-to-hydrophobic transition when the temperature is raised above the lower critical solution temperature (LCST)( approximately 28 degrees C in the solutions used here). This reversible transition drives the aggregation and dis-aggregation of the modified beads in heated zones within poly(ethylene terephthalate)(PET) microchannels. Biotinylated monoclonal antibodies for the drug digoxin were bound via streptavidin to the biotin-PEG-coated beads. These antibody-functionalized beads were then reversibly immobilized by aggregation and hydrophobic adhesion to the surface of PET microfluidic channels in response to a thermal stimulus. The antibodies on the beads immobilized in the channel were shown to bind digoxin and a competitor fluorescent ligand from a flow stream in a quantitative competitive assay format that reported the digoxin concentration. The antibodies could be replenished for each immunoassay trial, using the reversible, temperature-controlled immobilization process. This technique allows reagent immobilization immediately prior to an analytical procedure, following the removal of previously utilized beads, guaranteeing fresh and active immobilized biomolecules. Furthermore, it provides a simple approach to multiplexing through the simultaneous or sequential injection of different antibody-coated bead species, potentially at multiple sites in the integrated device channels.
Department of Bioengineering, University of Washington, Seattle, WA 98195, USA.
Noah
N
Malmstadt
Allan S
AS
Hoffman
Patrick S
PS
Stayton
D001710:Biotin / Q000737:chemistry; D019921:Biotinylation / Q000379:methods; D002846:Chromatography, Affinity / Q000379:methods*; D004077:Digoxin / Q000737:chemistry; D007118:Immunoassay / Q000295:instrumentation* / Q000379:methods*; D044085:Microfluidics / Q000295:instrumentation / Q000379:methods*; D011108:Polymers / Q000737:chemistry; D013696:Temperature
D011108:Polymers; D001710:Biotin; D004077:Digoxin