Christof Fattinger

High-sensitivity miniaturized immunoassays for tumor necrosis factor α using microfluidic systems

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).

Bidiffractive grating coupler: universal transducer for optical interface analytics

We report on a novel transducer devised for sensitive detection of the binding of molecules in the immediate vicinity of an optical surface. The transducer consists of a chip with a surface coated by an extremely thin waveguide film of amorphous TiO2 structured with a submicron grating relief. This microrelief is composed of two superimposed, uniform diffraction gratings of different periodicities, forming a bidiffractive grating with a frequency spectrum composed of two fundamental spatial harmonics. This bidiffractive grating serves as both an input and an output port for coupling and decoupling light beams to and from the planar waveguide. The bidiffractive coupler has translation-invariant coupling efficiency and allows background-free detection of the light decoupled from the waveguide. We outline the fabrication of the transducer and discuss the material properties of the waveguide film, which are very important for achieving optimum detection sensitivity. We assess the key factors affecting the detection limits of the transducer and comment on the resolution and the dynamic range for the measurement of changes in interfacial mass loading.

Instrument for Label-Free Detection of Noncoding RNAs

We set up a label-free direct binding assay for the detection of noncoding RNAs. The assay is based on nanomechanical cantilever arrays for the detection of surface stress induced by immobilized biomolecules and their interaction partners. We used various means to significantly reduce the drift of the cantilever readout that was a prominent feature in experiments with readout in stationary fluid before and after sample injection. Major improvements were achieved by focusing on a faster system equilibration (for instance temperature control and diffusion independence). Experimental protocols were improved to provide user-friendly and less time-consuming measurements. Further enhancements were achieved by, for example, using pre-gold-coated cantilever array wafers compared to individually prepared ones and a directly implemented data analysis tool as real-time feature of the measurement software. We have demonstrated picomolar specific biomarker target detection and can easily distinguish modified targets with single-nucleotide mismatches that hybridize with lower affinity.

Exploiting Molecular Biology by Time-Resolved Fluorescence Imaging

Many contemporary biological investigations rely on highly sensitive in vitro assays for the analysis of specific molecules in biological specimens, and the main part of these assays depends on high-sensitivity fluorescence detection techniques for the final readout. The analyzed molecules and molecular interactions in the specimen need to be detected in the presence of other highly abundant biomolecules, while the analyzed molecules themselves are only present at nano-, pico-, or even femtomolar concentration.A short scientific rationale of fluorescence is presented. It emphasizes the use of fluorescent labels for sensitive assays in life sciences and specifies the main properties of an ideal fluorophore. With fluorescence lifetimes in the microsecond range and fluorescence quantum yield of 0.4 some water soluble complexes of Ruthenium like modified Ru(sulfobathophenanthroline) complexes fulfill these properties. They are outstanding fluorescent labels for ultrasensitive assays as illustrated in two examples, in drug discovery and in point of care testing.We discuss the fundamentals and the state-of-the-art of the most sensitive time-gated fluorescence assays. We reflect on how the imaging devices currently employed for readout of these assays might evolve in the future. Many contemporary biological investigations rely on highly sensitive in vitro assays for the analysis of specific molecules in biological specimens, and the main part of these assays depends on high-sensitivity fluorescence detection techniques for the final readout. The analyzed molecules and molecular interactions in the specimen need to be detected in the presence of other highly abundant biomolecules, while the analyzed molecules themselves are only present at nano-, pico-, or even femtomolar concentration.A short scientific rationale of fluorescence is presented. It emphasizes the use of fluorescent labels for sensitive assays in life sciences and specifies the main properties of an ideal fluorophore. With fluorescence lifetimes in the microsecond range and fluorescence quantum yield of 0.4 some water soluble complexes of Ruthenium like modified Ru(sulfobathophenanthroline) complexes fulfil these properties. They are outstanding fluorescent labels for ultrasensitive assays as illustrated in two examples, in drug discovery and in point of care testing.We discuss the fundamentals and the state-of-the-art of the most sensitive time-gated fluorescence assays. We reflect on how the imaging devices currently employed for readout of these assays might evolve in the future.