François Huber

Rapid and label-free nanomechanical detection of biomarker transcripts in human RNA

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 foreseee cantilever arrays being used as a tool to evaluate treatment response efficacy for personalized medical diagnostics.

Sensing surface PEGylation with microcantilevers

Summary 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.

Analyzing Gene Expression Using Combined Nanomechanical Cantilever Sensors

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.

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.

Biosensors: New leverage against superbugs.

As the evolution of new strains of bacteria that are resistant to antibiotics continues, a nanomechanical approach to understanding the interactions between them could help efforts to develop new antibiotics.

Digital processing of multi-mode nano-mechanical cantilever data

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.

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.

Nanosensors for cancer detection

Cancer is a major burden in todays 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 patients condition in a non-invasive manner.