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.

Nanobodies are the smallest fragments of naturally occurring singledomain 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 ␤-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.

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.

Malignant melanoma, the deadliest form of skin cancer, is characterised by a predominant mutation in the BRAF gene. Drugs that target tumours carrying this mutation have recently entered the clinic. Therefore 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 (PCR) and sequencing methods. 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/µl of RNA material, without prior PCR amplification and use of labels.The identification of alterations in specific signalling pathways and recurrent oncogenic mutations in particular types of cancers has led in the past decade to the explosion of targeted therapy approaches. In cutaneous melanoma, a significant improvement in overall survival has been achieved by vemurafenib and similar drugs that selectively inhibit tumours carrying a mutated BRAF 2 gene 1,2 . Additional drugs for combination therapies with higher efficacies and fewer side effects are in clinical trials 3 . BRAF is one of three RAF genes (rapidly accelerated fibrosarcoma A, B and C) encoding cytoplasmic protein serine/threonine kinases belonging to the mitogen-activated protein kinase (MAPK) signal transduction cascade, a pathway controlling various cellular processes such as proliferation, migration and survival 4,5 . BRAF somatic mutations are present in half of cutaneous melanomas. Over 90% of the mutations are a single T to A transversion at position 1799 in the BRAF coding sequence (cT1799A), which converts a valine amino acid residue at position 600 in the protein to a glutamic acid (V600E). This mutation renders the protein constitutively active, resulting in a deregulated MAPK pathway 6 and thus uncontrolled cell growth and cancer. BRAF mutations are also present in other neoplasms, including hairy cell leukemias, thyroid and colon carcinomas 7,8,9 . As the presence of the cT1799A/V600E BRAF (hereafter BRAF V600E ) mutation determines eligibility to BRAF inhibitor treatment, molecular screening of tumour biopsies is now carried out routinely. with sensitivity comparable to COBAS TM , silicon nanowire field-effect transistors 12 and a threedimensional gold nanowire platform 13 . The latter technologies have only been shown to work using synthetic oligonucleotide targets, larger gene fragments or still rely on an initial PCR amplification. In particular, they have not been applied to the direct identification of a mutated messenger RNA (mRNA) sequence in total RNA (constituted primarily by ribosomal RNA and containing all mRNAs transcribed from gen...

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Bacteriorhodopsin proteoliposomes were used as a model system to explore the applicability of micromechanical cantilever arrays to detect conformational changes in membrane protein patches. The three main results of our study concern: 1), reliable functionalization of micromechanical cantilever arrays with proteoliposomes using ink jet spotting; 2), successful detection of the prosthetic retinal removal (bleaching) from the bacteriorhodopsin protein by measuring the induced nanomechanical surface stress change; and 3), the quantitative response thereof, which depends linearly on the amount of removed retinal. Our results show this technique to be a potential tool to measure membrane protein-based receptor-ligand interactions and conformational changes.

SummaryPolymers 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.