Measurements of forces in the piconewton range are very important for the study of molecular adhesion and mechanics. Recently, a micropipet-based force transducer for this type of experiment was presented (E. Evans, K. Ritchie, and R. Merkel, 1995, Biophys. J., 68:2580-2587). In the present article we give a detailed mechanical analysis of this transducer, including nonlinear effects. An analytical expression for the transducer stiffness at small elongations is given. Using magnetic tweezers (F. Ziemann, J. Rädler, and E. Sackmann, 1994, Biophys. J., 66:2210-2216), we were able to determine the force displacement relation of this transducer experimentally. Forces from approximately 10 pN to 500 pN were applied. Theoretical predictions and experimental results coincide remarkably well.

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We investigated the force-induced dissociation of single specific bonds between biomolecules. For these experiments we used a micropipet-based picoforce transducer. It consisted of a red blood cell tensed by micropipet suction. The stiffness of this "spring" was tuned by suction pressure. The proteins under investigation were immobilized onto microbeads. One type of bead carried the adhesion proteins and was biochemically attached to the red blood cell membrane. The second type, carrying the respective ligand, was held in a second micropipet which was moved by a piezoelectric transducer. Complementary beads were repeatedly brought into contact in order to form and break bonds. Yield forces exceeding 3 pN could be detected. For protein binding, the microbeads were covalently coated with a hydrogel of several nanometer thickness onto which the proteins were bound. This preparation resulted in a low frequency of nonspecific interactions between the beads. Linkages between beads and proteins were of sufficient strength for mechanical experiments on single molecules. We controlled the number of available binding sites by competitive blocking with soluble ligand so that only single molecular bonds were present between the microbeads. Protein A-IgG bonds were studied. Here we found a marked dependence of bond strength on the rate of force application. This effect is due to a force dependent rate of bond dissociation.

We collected test reactions which can be applied in an easy and reproducible way to the chemical composition of the abalone organic matrix. Several chemical and biochemical test reactions were applied to the interlamellar organic matrix of abalone nacre to study the content and nature of polysaccharides. The preparation of the polysaccharide matrix was examined in parallel by light microscopy. The polysaccharide core is covered by a honeycomblike structure, which can be completely removed by the protease subtilisin under release of the hydrophobic amino acid proline. The honeycomb pattern is in its size and its shape exactly the inverse matrix of the aragonite tablets, which are well-known to build up the nacreous layer of abalone shells. With this protocol we proved and verified in a straightforward and simple way the polysaccharides in abalone to be chitin. In addition, 1 H and 13 C NMR analysis of the interlamellar organic matrix of abalone nacre confirmed that it consists to a high extent of the polysaccharide chitosan or its partially/completely N-acetylated derivative chitin (β-(1f4)-2-acetamido-2-deoxy-D-glucose or N-acetyl-D-glucosamine).