Conformational Change of Bacteriorhodopsin Quantitatively Monitored by Microcantilever Sensors

Braun, Thomas; Backmann, Natalija; Vögtli, Manuel; Bietsch, Alexander; Engel, Andréas; Lang, H.P.; Gerber, Christoph; Hegner, Martin

doi:10.1529/biophysj.105.072934

Cited by 65 publications

(56 citation statements)

References 41 publications

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“…In recent years a versatile platform for biodetection has been developed based on microfabricated arrays of silicon cantilevers 14,15,16,17,18,19,20,21 , each coated with a sensitive layer for molecular recognition, e.g. a gene specific oligonucleotide.…”

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confidence: 99%

Direct detection of a BRAF mutation in total RNA from melanoma cells using cantilever arrays

et al. 2013

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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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Direct detection of a BRAF mutation in total RNA from melanoma cells using cantilever arrays

et al. 2013

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“…tensiometric biosensors to label-free study of molecular collective surface nanomechanics [33][34][35][36], opening new insights in surfaces confined molecular transformations and machines [32,[37][38][39][40], up to single cell investigation [41][42][43]. Experiments exploiting static deflection of MC beams are reported in literature to study the chemi-or physisorption of synthetic lipid bilayer on a cantilever surface, on either gold (Au) or silicon dioxide (SiO 2 ) surfaces [17,18].…”

Section: Interaction Between Extracellular Vesicles and Microcantileversmentioning

confidence: 99%

Interaction of Extracellular Vesicles with Si Surface Studied by Nanomechanical Microcantilever Sensors

et al. 2018

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Abstract:We report on the interaction of small (<150 nm) extracellular vesicles (EVs) with silicon surface. The study is conducted by leveraging Si nanomechanical microcantilever sensors actuated in static and dynamic modes, that allow tracking of EV collective adsorption energy and adsorbed mass. Upon incubation for 30 min at about 10 nM concentration, EVs isolated from human vascular endothelial cell (HVEC) lines form a patchy layer that partially covers the Si total surface. Formation of this layer releases a surface energy equal to (8 ± 1) mJ/m 2 , typical of weak electrostatic interactions. These findings give a first insight into the EV-Si interface and proof the possibility to realize new hybrid biointerphases that can be exploited as advanced models to investigate properties of biological membranes and/or biosensing platforms that take advantage of biomolecules embedded/supported in membranes.

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“…Where there is a protein in the crystal lattice with a changed conformation, the surface symmetry is altered. Such an event was observed by the bleaching of the BR (Stahlberg et al, 2001;Braun et al, 2006). A drawback of the method is that the fast reversible processes of the conformational changes cannot be observed (Braun et al, 2006).…”

Section: Effect Of Small Peptides On Supported Membranesmentioning

confidence: 99%

Artificial and Natural Membranes

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Szegletes²

2012

Atomic Force Microscopy Investigations Into Biology - From Cell to Protein

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Conformational Change of Bacteriorhodopsin Quantitatively Monitored by Microcantilever Sensors

Cited by 65 publications

References 41 publications

Direct detection of a BRAF mutation in total RNA from melanoma cells using cantilever arrays

Direct detection of a BRAF mutation in total RNA from melanoma cells using cantilever arrays

Interaction of Extracellular Vesicles with Si Surface Studied by Nanomechanical Microcantilever Sensors

Artificial and Natural Membranes

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