We report the specific transduction, via surface stress changes, of DNA hybridization and receptor-ligand binding into a direct nanomechanical response of microfabricated cantilevers. Cantilevers in an array were functionalized with a selection of biomolecules. The differential deflection of the cantilevers was found to provide a true molecular recognition signal despite large nonspecific responses of individual cantilevers. Hybridization of complementary oligonucleotides shows that a single base mismatch between two 12-mer oligonucleotides is clearly detectable. Similar experiments on protein A-immunoglobulin interactions demonstrate the wide-ranging applicability of nanomechanical transduction to detect biomolecular recognition.

We report a microarray of cantilevers to detect multiple unlabeled biomolecules simultaneously at nanomolar concentrations within minutes. Ligand-receptor binding interactions such as DNA hybridization or protein recognition occurring on microfabricated silicon cantilevers generate nanomechanical bending, which is detected optically in situ. Differential measurements including reference cantilevers on an array of eight sensors can sequence-specifically detect unlabeled DNA targets in 80-fold excess of nonmatching DNA as a background and discriminate 3 and 5 overhangs. Our experiments suggest that the nanomechanical motion originates from predominantly steric hindrance effects and depends on the concentration of DNA molecules in solution. We show that cantilever arrays can be used to investigate the thermodynamics of biomolecular interactions mechanically, and we have found that the specificity of the reaction on a cantilever is consistent with solution data. Hence cantilever arrays permit multiple binding assays in parallel and can detect femtomoles of DNA on the cantilever at a DNA concentration in solution of 75 nM.

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The availability of entire genome sequences has triggered the development of microarrays for clinical diagnostics that measure the expression levels of specific genes. Methods that involve labelling can achieve picomolar detection sensitivity, but they are costly, labour-intensive and time-consuming. Moreover, target amplification or biochemical labelling can influence the original signal. We have improved the biosensitivity of label-free cantilever-array sensors by orders of magnitude to detect mRNA biomarker candidates in total cellular RNA. Differential gene expression of the gene 1-8U, a potential marker for cancer progression or viral infections, has been observed in a complex background. The measurements provide results within minutes at the picomolar level without target amplification, and are sensitive to base mismatches. This qualifies the technology as a rapid method to validate biomarkers that reveal disease risk, disease progression or therapy response. We foresee cantilever arrays being used as a tool to evaluate treatment response efficacy for personalized medical diagnostics.

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We report a microcantilever-based immunosensor operated in static deflection mode with a performance comparable with surface plasmon resonance, using single-chain Fv (scFv) antibody fragments as receptor molecules. As a model system scFv fragments with specificity to two different antigens were applied. We introduced a cysteine residue at the C terminus of each scFv construct to allow covalent attachment to gold-coated sensor interfaces in directed orientation. Application of an array enabled simultaneous deflection measurements of sensing and reference cantilevers. The differential deflection signal revealed specific antigen binding and was proportional to the antigen concentration in solution. Using small, oriented scFv fragments as receptor molecules we increased the sensitivity of microcantilevers to Ϸ1 nM.cantilever arrays ͉ nanomechanics ͉ proteomics M icrocantilever-based sensors have attracted much interest as devices for fast and reliable detection of small amounts of molecules in air and solution. Over the last few years the application of the cantilever sensor concept was extended to the measurements of biocompounds in solution, resulting in a versatile biosensor (1, 2). Because of its label-free detection principle and small size, this kind of biosensor is advantageous for diagnostic applications, disease monitoring, and research in genomics or proteomics (3, 4). Multicantilever arrays would enable the detection of several analytes simultaneously.The main principle of the cantilever static mode is the transduction of the molecular interaction between analyte and receptors, immobilized as a layer on one surface of a cantilever, into a nanomechanical motion of the cantilever. Biomolecular interactions taking place on a solid-state interface produce a change in surface stress, because of changes in molecular configuration and intermolecular crowding (5). This process results in bending of the cantilever. Microcantilever-based biosensors operated in static mode have been successfully applied for the detection of various molecular interactions such as ssDNA-ssDNA (5-7) or protein-DNA (8, 9). Interactions between proteins were detected with cantilever-based immunosensors, where an antigen was recognized by its cognate antibody randomly immobilized on the sensor surface (10-12).The most critical step in preparation of any immunosensor is the immobilization of capture molecules on the support, a process where the orientation of the antigen-binding sites toward the analyte in solution plays a key role. Immunoglobulins can be either adsorbed on gold directly (10, 12) or attached covalently to the surface modified with hetero-bifunctional self-assembled monolayers of alkylthiols (11). However, these approaches produce a layer of randomly oriented antibody molecules on the cantilever surface, thereby generating conformational heterogeneity and inactive receptor molecules (13,14).As previously shown (13,(15)(16)(17)(18), the sensitivity of immunosensors can be improved by both maximizing the degree of functional ...

Membrane proteins are central to many biological processes, and the interactions between transmembrane protein receptors and their ligands are of fundamental importance in medical research. However, measuring and characterizing these interactions is challenging. Here we report that sensors based on arrays of resonating microcantilevers can measure such interactions under physiological conditions. A protein receptor--the FhuA receptor of Escherichia coli--is crystallized in liposomes, and the proteoliposomes then immobilized on the chemically activated gold-coated surface of the sensor by ink-jet spotting in a humid environment, thus keeping the receptors functional. Quantitative mass-binding measurements of the bacterial virus T5 at subpicomolar concentrations are performed. These experiments demonstrate the potential of resonating microcantilevers for the specific, label-free and time-resolved detection of membrane protein-ligand interactions in a micro-array format.

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