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Measurement of kinetics using microarrays requires adapted experimental analysis for data evaluation and normalization. Here we present a new algorithm based on alignment of data, which solves inconsistencies of current normalization methods utilizing baseline correction. Our results show that this method leads to better data consistency and relative errors four times smaller on average.

Nanomechanical sensors based on cantilever technology allow the measurement of various physical properties. Here we present a software for the comprehensive analysis of such data. An example for the combined measurement of mass and surface stress is presented.

In diseases such as cancer or during viral infections gene expression is greatly altered. These changes in gene activity can be analysed at different levels of cellular activity, like transcription activation, transcription and translation. Currently, no simple method is available to detect all these biochemical signals simultaneously and rapidly. Micromechanical cantilever sensor array technology is applied, because it has the advantage that sample preconditioning like labelling and amplification is not required. Furthermore, DNA, RNA, protein or combinations thereof could be detected in parallel on a single cantilever array. With such a device, diagnosis and therefore treatment of diseases can be improved. Here we present successive detection of DNA hybridization and antigen using the same micromechanical cantilever sensor array.

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 ≈1 nM.

Nanobodies are the smallest fragments of naturally occurring single-domain antibodies that have evolved to be fully functional in the absence of a light chain. Nanobodies are strictly monomeric, very stable, and highly soluble entities. We identified a nanobody with subnanomolar affinity for the human tumor-associated carcinoembryonic antigen. This nanobody was conjugated to Enterobacter cloacae β-lactamase, and its site-selective anticancer prodrug activation capacity was evaluated. The conjugate was readily purified in high yields without aggregation or loss of functionality of the constituents. In vitro experiments showed that the nanobody–enzyme conjugate effectively activated the release of phenylenediamine mustard from the cephalosporin nitrogen mustard prodrug 7-(4-carboxybutanamido) cephalosporin mustard at the surface of carcinoembryonic antigen-expressing LS174T cancer cells. In vivo studies demonstrated that the conjugate had an excellent biodistribution profile and induced regressions and cures of established tumor xenografts. The easy generation and manufacturing yield of nanobody-based conjugates together with their potent antitumor activity make nanobodies promising vehicles for new generation cancer therapeutics.

A cantilever-based protein biosensor has been developed providing a customizable multilayer platform for the detection of antibodies. It consists of a biotin-terminated PEG layer pre-functionalized on the gold-coated cantilever surface, onto which NeutrAvidin is adsorbed through biotin/NeutrAvidin specific binding. NeutrAvidin is used as a bridge layer between the biotin-coated surface and the biotinylated biomolecules, such as biotinylated bovine serum albumin (biotinylated BSA), forming a multilayer sensor for direct antibody capture. The cantilever biosensor has been successfully applied to the detection of mouse anti-BSA (m-IgG) and sheep anti-BSA(s-IgG) antibodies. As expected, the average differential surface stress signals of about 5.7 +/- 0.8 x 10(-3) N/m are very similar for BSA/m-IgG and BSA/s-IgG binding, i.e., they are independent of the origin of the antibody. A statistic evaluation of 112 response curves confirms that the multilayer protein cantilever biosensor shows high reproducibility. As a control test, a biotinylated maltose binding protein was used for detecting specificity of IgG, the result shows a signal of bBSA layer in response to antibody is 5.8 x 10(-3) N/m compared to bMBP. The pre-functionalized biotin/PEG cantilever surface is found to show a long shelf-life of at least 40 days and retains its responsivity of above 70% of the signal when stored in PBS buffer at 4 degrees C. The protein cantilever biosensor represents a rapid, label-free, sensitive and reliable detection technique for a real-time protein assay.

Malignant melanoma, the deadliest form of skin cancer, is characterized by a predominant mutation in the BRAF gene(1-3). Drugs that target tumours carrying this mutation have recently entered the clinic(4-7). Accordingly, patients are routinely screened for mutations in this gene to determine whether they can benefit from this type of treatment. The current gold standard for mutation screening uses real-time polymerase chain reaction and sequencing methods(8). Here we show that an assay based on microcantilever arrays can detect the mutation nanomechanically without amplification in total RNA samples isolated from melanoma cells. The assay is based on a BRAF-specific oligonucleotide probe. We detected mutant BRAF at a concentration of 500 pM in a 50-fold excess of the wild-type sequence. The method was able to distinguish melanoma cells carrying the mutation from wild-type cells using as little as 20 ng mu l(-1) of RNA material, without prior PCR amplification and use of labels.

Polymers are often used to modify surface properties to control interfacial processes. Their sensitivity to solvent conditions and ability to undergo conformational transitions makes polymers attractive in tailoring surface properties with specific functionalities leading to applications in diverse areas ranging from tribology to colloidal stability and medicine. A key example is polyethylene glycol (PEG), which is widely used as a protein-resistant coating given its low toxicity and biocompatibility. We report here a microcantilever-based sensor for the in situ characterization of PEG monolayer formation on Au using the “grafting to” approach. Moreover, we demonstrate how microcantilevers can be used to monitor conformational changes in the grafted PEG layer in different solvent conditions. This is supported by atomic force microscope (AFM) images and force–distance curve measurements of the microcantilever chip surface, which show that the grafted PEG undergoes a reversible collapse when switching between good and poor solvent conditions, respectively.

Microfabricated silicon cantilever sensor arrays represent a powerful platform for sensing applications in physics, chemistry, material science, biology and medicine. The sensor response is mechanical bending due to absorption of molecules. In gaseous environment, polymer-coated microcantilevers are used as electronic nose for characterization of vapors, resulting in cantilever bending due to polymer swelling upon exposure. Medical applications involve fast characterization of exhaled patient's breath samples for detection of diseases, based on the presence of certain chemicals in breath. We present a portable, compact, modular microcantilever setup, which uses a micropump for aspiration and a bluetooth interface for remote data acquisition.

We fused the epitope-recognizing fragment of heavy-chain antibodies from Camelidae sp. with fluorescent proteins to generate fluorescent, antigen-binding nanobodies (chromobodies) that can be expressed in living cells. We demonstrate that chromobodies can recognize and trace antigens in different subcellular compartments throughout S phase and mitosis. Chromobodies should enable new functional studies, as potentially any antigenic structure can be targeted and traced in living cells in this fashion.

Over the last years there has been a growing interest in the use of antibodies and antibody fragments as therapeutic entities to treat cancer. Identification of the smallest antibody fragment still capable of binding to antigen has progressed from full antibody molecules to Fab and recombinant single chain Fv fragments. A further reduction to single-domain binding proteins based upon immuno-globulin VH and VH-like domains is being pursued since they offer exciting prospects in the development of novel immunotherapeutics. Unfortunately, the poor solubility and reduced antigen affinity requires additional engineering. By serendipity, it was discovered that this engineering is already performed in nature. Part of the humoral immune response of camels and llamas is based largely on heavy-chain antibodies where the light chain is totally absent. These unique antibody isotypes interact with the antigen by virtue of only one single variable domain, referred to as VHH. Here we describe the properties of camel VHH that offer added value over conventional antibody fragments as treatment modality for tumour patients.