Fast Diagnostics of BRAF Mutations in Biopsies from Malignant Melanoma.

According to the American skin cancer foundation, there are more new cases of skin cancer than the combined incidence of cancers of the breast, prostate, lung, and colon each year, and malignant melanoma represents its deadliest form. About 50% of all cases are characterized by a particular mutation BRAF(V600E) in the BRAF (Rapid Acceleration of Fibrosarcoma gene B) gene. Recently developed highly specific drugs are able to fight BRAF(V600E) mutated tumors but require diagnostic tools for fast and reliable mutation detection to warrant treatment efficiency. We completed a preliminary clinical trial applying cantilever array sensors to demonstrate identification of a BRAF(V600E) single-point mutation using total RNA obtained from biopsies of metastatic melanoma of diverse sources (surgical material either frozen or fixated with formalin and embedded in paraffin). The method is faster than the standard Sanger or pyrosequencing methods and comparably sensitive as next-generation sequencing. Processing time from biopsy to diagnosis is below 1 day and does not require PCR amplification, sequencing, and labels.

Development of robust and standardized cantilever sensors based on biotin/NeutrAvidin coupling for antibody detection.

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 × 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 × 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 °C. The protein cantilever biosensor represents a rapid, label-free, sensitive and reliable detection technique for a real-time protein assay.

Rapid Bacteria Detection from Patients' Blood Bypassing Classical Bacterial Culturing.

Sepsis is a life-threatening condition mostly caused by a bacterial infection resulting in inflammatory reaction and organ dysfunction if not treated effectively. Rapid identification of the causing bacterial pathogen already in the early stage of bacteremia is therefore vital. Current technologies still rely on time-consuming procedures including bacterial culturing up to 72 h. Our approach is based on ultra-rapid and highly sensitive nanomechanical sensor arrays. In measurements we observe two clearly distinguishable distributions consisting of samples with bacteria and without bacteria respectively. Compressive surface stress indicates the presence of bacteria. For this proof-of-concept, we extracted total RNA from EDTA whole blood samples from patients with blood-culture-confirmed bacteremia, which is the reference standard in diagnostics. We determined the presence or absence of bacterial RNA in the sample through 16S-rRNA hybridization and species-specific probes using nanomechanical sensor arrays. Via both probes, we identified two clinically highly-relevant bacterial species i.e., Escherichia coli and Staphylococcus aureus down to an equivalent of 20 CFU per milliliter EDTA whole blood. The dynamic range of three orders of magnitude covers most clinical cases. We correctly identified all patient samples regarding the presence or absence of bacteria. We envision our technology as an important contribution to early and sensitive sepsis diagnosis directly from blood without requirement for cultivation. This would be a game changer in diagnostics, as no commercial PCR or POCT device currently exists who can do this.

Rapid and Ultrasensitive Detection of Mutations and Genes Relevant to Antimicrobial Resistance in Bacteria.

The worldwide emergence of multidrug-resistant (MDR) bacteria is associated with significant morbidity, mortality, and healthcare costs. Rapid and accurate diagnostic methods to detect antibiotic resistance are critical for antibiotic stewardship and infection control measurements. Here a cantilever nanosensor-based diagnostic assay is shown to detect single nucleotide polymorphisms (SNPs) and genes associated with antibiotic resistance in Gram negative (Pseudomonas aeruginosa) and positive (Enterococcus faecium) bacteria, representing frequent causes for MDR infections. Highly specific RNA capture probes for SNPs (ampRD135G or ampRG154R ) or resistance genes (vanA, vanB, and vanD) allow to detect the binding of bacterial RNA within less than 5 min. Serial dilutions of bacterial RNA indicate an unprecedented sensitivity of 10 fg µL-1 total RNA corresponding to less than ten bacterial cells for SNPs and 1 fg µL-1 total RNA for vanD detection equivalent to single bacterial cell sensitivity.

Label free analysis of transcription factors using microcantilever arrays.

We report the measurement of protein interaction with double-stranded DNA oligonucleotides using cantilever microarray technology. We investigated two different DNA-binding proteins, the transcription factors SP1 and NF-kappaB, using cantilever arrays as they allow label-free measurement of different biomolecular interactions in parallel. Double-stranded DNA oligonucleotides containing a specific binding site for a transcription factor were sensitized on gold-coated cantilevers. The binding of the transcription factor creates a surface stress, resulting in a bending of the cantilevers. Both transcription factors could be detected independently at concentrations of 80-100 nM. A concentration dependence of the bending signal was measured using concentrations from 100 to 400 nM of NF-kappaB. The experiments show that the recognition sequence of one transcription factor can serve as a reference for the other, highlighting the sequence specificity of transcription factor binding.

Nanosensors for cancer detection.

Cancer is a major burden in today's society and one of the leading causes of death in industrialised countries. Various avenues for the detection of cancer exist, most of which rely on standard methods, such as histology, ELISA, and PCR. Here we put the focus on nanomechanical biosensors derived from atomic force microscopy cantilevers. The versatility of this novel technology has been demonstrated in different applications and in some ways surpasses current technologies, such as microarray, quartz crystal microbalance and surface plasmon resonance. The technology enables label free biomarker detection without the necessity of target amplification in a total cellular background, such as BRAF mutation analysis in malignant melanoma. A unique application of the cantilever array format is the analysis of conformational dynamics of membrane proteins associated to surface stress changes. Another development is characterisation of exhaled breath which allows assessment of a patient's condition in a non-invasive manner.

How do fibroblasts translate mechanical signals into changes in extracellular matrix production?

Mechanical forces are important regulators of connective tissue homeostasis. Our recent experiments in vivo indicate that externally applied mechanical load can lead to the rapid and sequential induction of distinct extracellular matrix (ECM) components in fibroblasts, rather than to a generalized hypertrophic response. Thus, ECM composition seems to be adapted specifically to changes in load. Mechanical stress can regulate the production of ECM proteins indirectly, by stimulating the release of a paracrine growth factor, or directly, by triggering an intracellular signalling pathway that activates the gene. We have evidence that tenascin-C is an ECM component directly regulated by mechanical stress: induction of its mRNA in stretched fibroblasts is rapid both in vivo and in vitro, does not depend on prior protein synthesis, and is not mediated by factors released into the medium. Fibroblasts sense force-induced deformations (strains) in their ECM. Findings by other researchers indicate that integrins within cell-matrix adhesions can act as 'strain gauges', triggering MAPK and NF-kappaB pathways in response to changes in mechanical stress. Our results indicate that cytoskeletal 'pre-stress' is important for mechanotransduction to work: relaxation of the cytoskeleton (e.g. by inhibiting Rho-dependent kinase) suppresses induction of the tenascin-C gene by cyclic stretch, and hence desensitizes the fibroblasts to mechanical signals. On the level of the ECM genes, we identified related enhancer sequences that respond to static stretch in both the tenascin-C and the collagen XII promoter. In the case of the tenascin-C gene, different promoter elements might be involved in induction by cyclic stretch. Thus, different mechanical signals seem to regulate distinct ECM genes in complex ways.

Sensing surface PEGylation with microcantilevers.

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.

Analyzing refractive index changes and differential bending in microcantilever arrays.

A new microcantilever array design is investigated comprising eight flexible microcantilevers introducing two solid bars, enabling to subtract contributions from differences in refractive index in an optical laser read out system. Changes in the refractive index do not contribute undesirably to bending signals at picomolar to micromolar DNA or protein concentrations. However, measurements of samples with high salt concentrations or serum are affected, requiring corrections for refractive index artifacts. Moreover, to obtain a deeper understanding of molecular stress formation, the differential curvature of cantilevers is analyzed by positioning the laser spots along the surface of the levers during pH experiments.

Label-free detection of amyloid growth with microcantilever sensors.

We present an approach for sensing protein aggregation using microcantilever systems. Results from both single cantilever experiments with internal reference and multicantilever array measurements with dedicated reference cantilevers are discussed. We show that in both cases protein aggregation on the sensor can be detected through associated changes in surface stress.

A label-free immunosensor array using single-chain antibody fragments.

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 approximately 1 nM.

Phosphorylation of beta3 integrin controls ligand binding strength.

The cytoplasmic domain of beta(3) integrin contains tyrosines at positions 747 and 759 in domains that have been implicated in regulation of alpha(v)beta(3) function and that serve as potential substrates for Src family kinases. The phosphorylation level of beta(3) integrin was modulated using a temperature-sensitive v-Src kinase. Increased beta(3) phosphorylation abolished alpha(v)beta(3)- but not alpha(5)beta(1)-mediated adhesion to fibronectin. alpha(v)beta(3)-Mediated cell adhesion was restored by the expression of beta(3) containing Y747F or Y759F mutations but not by wild type beta(3) integrin. Thus, phosphorylation of the cytoplasmic domain of beta(3) is a negative regulator of alpha(v)beta(3)-fibronectin binding strength.