High-sensitivity miniaturized immunoassays for tumor necrosis factor ? 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).

Microfluidic ELISA on non-passivated PDMS chip using magnetic bead transfer inside dual networks of channels

“…Moreover, improving bead dispersion and compromising on the incubation times would increase the sensitivity of the assay significantly. Nevertheless, even with the current protocol, the dual network system is among the most sensitive microfluidic immunoassays reported to date, 3,4,[27][28][29][30][31][32][33][34][35][36][37][38] without the use of costly, complex and time-consuming pre-treatments.…”

Restraining non-specific adsorption of protein using Parylene C-caulked polydimethylsiloxane

“…[3][4][5] Meanwhile, in a heterogeneous sensing such as the DNA microarray 6 or the immunoassay 7 biosensor, the NSA of labelled molecules (dyes) may also limit the detection effectiveness. A typical biosensor consists of probe molecules on the surface and a detectable signal will be generated upon the binding between the probes and target molecules.…”

“…NSA of dyes on PDMS surface causes a high background (noise) signal and degrades the sensing performance. 6,7 Efforts have been made to passivate the PDMS surface and restrain the NSA on top. The commonly used surface passivation could be classified into two types.…”

Silicon-on-quartz bonding based SPR chip

“…air-gap distance) in microfluidic systems is an important trend, motivated by the need to use expensive reagents and precious samples sparingly. Also a number of researchers have proposed microfluidic systems with miniaturized channel height [12][13]. But, in the Kretschmann coupling configuration, the channel height cannot 3 be miniaturized because it has to be much larger than the wavelength of the incident light, to avoid interference with the upper wall or substrate [7].…”