Detection of specific haptene–protein interactions. (A) The transfer of two oligonucleotides coupled with different haptenes (biotin or digoxigenin) and one oligonucleotide without haptene label (top surface) onto spots containing the respective binding partners, as well as proteins not specific for the haptene (bottom surface), is determined. (B) Diagram showing the fluorescence intensities measured on (left to right) anti-digoxigenin, streptavidin, anti-biotin, and anti-antitrypsin spots on the bottom surface. Red bars correspond to biotin on the top surface, green bars correspond to digoxigenin, and yellow bars correspond to DNA without haptene. The ratio of specific to nonspecific transfer is always better than 50:1 for the two haptenes and their respective negative controls (transfer onto a specific binding partner versus transfer of the same haptene onto another “nonspecific” molecule).

Detection of specific antibody–antigen interactions. (A) The antibodies were coupled to the DNA force sensor (top surface) via biotin and streptavidin. Their corresponding antigens were immobilized on the bottom surface. Each antibody was tested against all antigens in the respective series (PDMS 1, monoclonal anti-β-galactosidase; PDMS 2, monoclonal anti-GFP; PDMS 3, monoclonal anti-HSA; PDMS 4, polyclonal anti-rabbit). (B) Fluorescence intensities measured on the bottom surface on spots containing (left to right) immobilized β-galactosidase, GFP, HSA, and rabbit antibodies. Red bars correspond to anti-β-galactosidase antibodies on the top surface, green bars correspond to anti-GFP, blue bars correspond to anti-HSA, and yellow bars correspond to anti-rabbit antibodies. The ratio of specific to nonspecific transfer is always better than 7:1 for the four antibodies and their respective negative controls (transfer onto a specific binding partner versus transfer of the same antibody onto another “nonspecific” molecule).

An antibody sandwich assay for the detection of a hepatitis C virus antigen, based on the differential force test. (A) The detection antibodies are connected to the top surface via a DNA force sensor and a PEG spacer (PDMS 1, D1; PDMS 2, D2; PDMS 3, mixture of D1 and D2). Specific capture antibodies (C1, C2, and C1/C2), as well as one antibody binding human serum albumin as a negative control, are immobilized on the bottom surface. The antigen is bound by shaking the bottom surface in an antigen-containing solution. (B) Fluorescence intensities on the bottom surface on spots with (left to right) C1 capture antibodies, C2 capture antibodies, a mixture of C1 and C2 antibodies, and the negative control. Green bars represent D1 detection antibodies on the top chip surface, blue bars represent D2 detection antibodies, and striped bars represent a mixture of both. Specific to nonspecific ratios vary between 2.4:1 and 10.1:1 depending on the particular combination of sandwich antibodies, which were compared with the negative control.

Experimental realization of the differential force test. (A) DNA duplexes are connected to a microstructured silicone elastomer surface (top surface) via PEG spacers. The spacers are covalently bound to the silicone and in the next step are covalently attached to the 5′ end of one of the DNA strands. The complementary DNA strand contains a fluorescence label at the 5′ end and a 3′-digoxigenin label attached at the end of a poly(A) spacer sequence. (B) The PDMS surface is brought into contact with a second chip surface (bottom surface) containing spots of immobilized anti-digoxigenin antibodies, streptavidin proteins, or just the PEG passivation layer. (C) On separation of the two chip surfaces, the PEG spacers are extended and a force is built up in the molecular chains between the two surfaces. (D) As the two surfaces are further separated, the weakest molecular bond in each chain breaks, and the fluorescence label remains connected to the stronger bond. (E) After separation of the two surfaces, a fluorescence image of the bottom surface reveals strong fluorescence intensity on the spot carrying the anti-digoxigenin antibodies (Left), no fluorescence on the streptavidin spot (Center), and very little fluorescence on the PEG-coated control area (Right). The dark grids in the fluorescence images represent grooves of the microstructured PDMS. Note that in Left image the spots from the top and the bottom surface do not overlap entirely. Areas where the two spots do not overlap can be used as additional controls.