title: High-sensitivity miniaturized immunoassays for tumor necrosis factor alpha using microfluidic systems.
abstract: We use microfluidic chips to detect the biologically important cytokine tumor necrosis factor alpha (TNF- alpha) with picomolar sensitivity using sub-microliter volumes of samples and reagents. The chips comprise a number of independent capillary systems (CSs), each of which is composed of a filling port, an appended microchannel, and a capillary pump. Each CS fills spontaneously by capillary forces and includes a self-regulating mechanism that prevents adventitious drainage of the microchannels. Thus, interactive control of the flow in each CS is easily achieved via collective control of the evaporation in all CSs by means of two Peltier elements that can independently heat and cool. Long incubation times are crucial for high sensitivity assays and can be conveniently obtained by adjusting the evaporation rate to have low flow rates of approximately 30 nL min(-1). The assay is a sandwich fluorescence immunoassay and takes place on the surface of a poly(dimethylsiloxane)(PDMS) slab placed across the microchannels. We precoat PDMS with capture antibodies (Abs), localize the capture of analyte molecules using a chip, then bind the captured analyte molecules with fluorescently-tagged detection Abs using a second chip. The assay results in a mosaic of fluorescence signals on the PDMS surface which are measured using a fluorescence scanner. We show that PDMS is a compatible material for high sensitivity fluorescence assays, provided that detection antibodies with long excitation wavelength fluorophores ( > or =580 nm) are employed. The chip design, long incubation times, proper choice of fluorophores, and optimization of the detection Ab concentration all combine to achieve high-sensitivity assays. This is exemplified by an experiment with 170 assay sites, occupying an area of approximately 0.6 mm(2) on PDMS to detect TNF-alpha in 600 nL of a dendritic cell (DC) culture medium with a sensitivity of approximately 20 pg mL(-1)(1.14 pM).
authors.affiliation: IBM Research GmbH, Zurich Research Laboratory, Saumerstrasse 4, CH-8803 Ruschlikon, Switzerland.
authors.forename: Sandro
authors.initials: S
authors.lastname: Cesaro-Tadic
authors.forename: Gregor
authors.initials: G
authors.lastname: Dernick
authors.forename: David
authors.initials: D
authors.lastname: Juncker
authors.forename: Gerrit
authors.initials: G
authors.lastname: Buurman
authors.forename: Harald
authors.initials: H
authors.lastname: Kropshofer
authors.forename: Bruno
authors.initials: B
authors.lastname: Michel
authors.forename: Christof
authors.initials: C
authors.lastname: Fattinger
authors.forename: Emmanuel
authors.initials: E
authors.lastname: Delamarche
mesh_terms: D000906:Antibodies / Q000276:immunology; D003713:Dendritic Cells / Q000166:cytology* / Q000378:metabolism; D004867:Equipment Design; D019544:Equipment Failure Analysis; D017022:Flow Injection Analysis / Q000295:instrumentation* / Q000379:methods; D016231:Fluorescence Polarization Immunoassay / Q000295:instrumentation* / Q000379:methods; D006801:Humans; D008832:Microchemistry / Q000295:instrumentation* / Q000379:methods; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation* / Q000379:methods; D008904:Miniaturization; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D014409:Tumor Necrosis Factor-alpha / Q000032:analysis* / Q000276:immunology / Q000378:metabolism*
chemical_list: D000906:Antibodies; D014409:Tumor Necrosis Factor-alpha