We introduce a detailed design and fabrication process of Silicon microcantilever arrays for biomolecular detection in liquid environment, utilized with laser readout. We present typical fabrication problems and provide related solutions to obtain high quality resonators via a robust, reproducible and high-yield process. Sensors in these arrays are individually functionalized with self-assembled chemical monolayers exposing various pH-active end-groups into solution. Dynamic-mode controlled frequency measurements in varying pH solutions result in stress-induced change of the sensor spring constant. pH changes in the solution lead to deprotonation of exposed functional chemical groups at high pH and the repulsive charges induced strain is proportional to the quantity and confinement of charges at the sensor interface. These built-up strains that affect the mechanical stiffness can be reversibly relaxed when exposed again to low pH environments.

We present a nanomechanical platform for real-time quantitative label-free detection of target biomolecules in a liquid environment with mass sensitivity down to few pg. Newly fabricated arrays of up to 18 cantilevers are integrated in a micromachined fluidic chamber, connected to software-controlled fluidic pumps for automated sample injections. We discuss two functionalization approaches to independently sensitize the interface of different cantilevers. A custom piezo-stack actuator and optical readout system enable the measurement of resonance frequencies up to 2 MHz. We implement a new measurement strategy based on a phase-locked loop (PLL), built via in-house developed software. The PLL allows us to track, within the same experiment, the evolution of resonance frequency over time of up to four modes for all the cantilevers in the array. With respect to the previous measurement technique, based on standard frequency sweep, the PLL enhances the estimated detection limit of the device by a factor of 7 (down to 2 pg in 5 min integration time) and the time resolution by more than threefold (below 15 s), being on par with commercial gold-standard techniques. The detection limit and noise of the new setup are investigated via Allan deviation and standard deviation analysis, considering different resonance modes and interface chemistries. As a proof-of-concept, we show the immobilization and label-free in situ detection of live bacterial cells (E. coli), demonstrating qualitative and quantitative agreement in the mechanical response of three different resonance modes.

Advances in prevention, diagnosis and therapy are coupled to innovation and development of new medical tools, leading to improved patient prognosis. We developed an automatic biosensor platform that could provide a non-invasive, rapid and personalised diagnosis using nanomechanical cantilever sensors. miRNA are involved in gene expression and are extractable biomarkers for multiple diseases. We detected specific expression patterns of miRNA relevant to cancer and adverse drug effects directly in cell lysates or blood based samples using only a few microliters of sample within one hour. Specific miRNA hybridisation to the upper cantilever surface induces physical bending of the sensor which is detected by monitoring the position of a laser that reflects from the sensors surface. Internal reference sensors negate environmental and nonspecific effects. We showed that the sensitivity of label free cantilever nanomechanical sensing of miRNA surpasses that of surface plasmon resonance by more than three orders of magnitude. A cancer associated miRNA expression profile from cell lysates and one associated with hepatocytes derived from necrotic liver tissue in blood-based samples has been successfully detected. Our label free mechanical approach displays the capability to perform in relevant clinical samples while also obtaining comparable results to PCR based techniques. Without the need to individually extend, amplify or label each target allowing multitarget analysis from one sample.

Rheological measurements in biological liquids yield insights into homeostasis and provide information on important molecular processes that affect fluidity. We present a fully automated cantilever-based method for highly precise and sensitive measurements of microliter sample volumes of human blood plasma coagulation (0.009 cP for viscosity range 0.5-3 cP and 0.0012 g/cm3 for density range 0.9-1.1 g/cm3). Microcantilever arrays are driven by a piezoelectric element, and resonance frequencies and quality factors of sensors that change over time are evaluated. A highly accurate approximation of the hydrodynamic function is introduced that correlates resonance frequency and quality factor of cantilever beams immersed in a fluid to the viscosity and density of that fluid. The theoretical model was validated using glycerol reference solutions. We present a surface functionalization protocol that allows minimization of unspecific protein adsorption onto cantilevers. Adsorption leads to measurement distortions and incorrect estimation of the fluid parameters (viscosity and density). Two hydrophilic terminated self-assembled monolayers (SAMs) sensor surfaces are compared to a hydrophobic terminated SAM coating. As expected, the hydrophobic modified surfaces induced the highest mass adsorption and could promote conformational changes of the proteins and subsequent abnormal biological activity. Finally, the activated partial thromboplastin time (aPTT) coagulation assay was performed, and the viscosity, density, and coagulation rate of human blood plasma were measured along with the standard coagulation time. The method could extend and improve current coagulation testing.

Nanoscillations of microcantilevers as a novel strategy to diagnose and monitor blood coagulation disorders, using a few microliters of the sample.

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An understanding of the photoisomerization mechanism of molecules bound to a metal surface at the molecular scale is required for designing photoswitches at surfaces. It has remained a challenge to correlate the surface structure and isomerization of photoswitches at ambient conditions. Herein, the photoisomerization of a self-assembled monolayer of azobenzene-thiol molecules on a Au surface was investigated using scanning tunneling microscopy and tip-enhanced Raman spectroscopy. The unique signature of the cis isomer at 1525 cm(-1) observed in tip-enhanced Raman spectra was clearly distinct from the trans isomer. Furthermore, tip-enhanced Raman images of azobenzene thiols after ultraviolet and blue light irradiation are shown with nanoscale spatial resolution, demonstrating a reversible conformational change. Interestingly, the cis isomers of azobenzene-thiol molecules were preferentially observed at Au grain edges, which is confirmed by density functional theory.

Protein biosynthesis is inherently coupled to cotranslational protein folding. Folding of the nascent chain already occurs during synthesis and is mediated by spatial constraints imposed by the ribosomal exit tunnel as well as self-interactions. The polypeptide's vectorial emergence from the ribosomal tunnel establishes the possible folding pathways leading to its native tertiary structure. How cotranslational protein folding and the rate of synthesis are linked to a protein's amino acid sequence is still not well defined. Here, we follow synthesis by individual ribosomes using dual-trap optical tweezers and observe simultaneous folding of the nascent polypeptide chain in real time. We show that observed stalling during translation correlates with slowed peptide bond formation at successive proline sequence positions and electrostatic interactions between positively charged amino acids and the ribosomal tunnel. We also determine possible cotranslational folding sites initiated by hydrophobic collapse for an unstructured and two globular proteins while directly measuring initial cotranslational folding forces. Our study elucidates the intricate relationship among a protein's amino acid sequence, its cotranslational nascent-chain elongation rate, and folding.

Protein synthesis is directly linked to protein folding. Translation kinetics plays a crucial role in forming secondary structural elements that lead to tertiary structure formation. However, how these two processes are coordinated is still not fully understood. Single-molecule techniques are essential for the study of protein synthesis and subsequent folding and were used in the past (1), since these are asynchronous processes that are extremely difficult to be observed using ensemble methods. Here we show for the first time to our knowledge full synthesis of three distinct proteins (hTau40, DHFR and GFPem) studied with dual-trap optical tweezers at the single-molecule level, continuing our previously published work (2). Our study explicates the intricate relationship between a protein's amino acid sequence, its co-translational elongation rate and folding. We show how hydrophobic collapse, which is the first step in protein folding, correlates with the number of hydrophobic amino acids that emerge from the ribosomal tunnel during synthesis. We also find that translational pauses are caused mainly by consecutive proline residues and positively charged amino acids. Finally, we propose a model that predicts the translation rate at a certain applied force and estimates hydrophobic collapse nucleation sites (manuscript submitted). 1.Katranidis, A., D. Atta, R. Schlesinger, K. H. Nierhaus, T. Choli-Papadopoulou, I. Gregor, M. Gerrits, G. Buldt, and J. Fitter. 2009. Angew. Chem. Intl. ed 48, 1758-1761 2.Katranidis, A., W. Grange, R. Schlesinger, T. Choli-Papadopoulou, D. Bruggemann, M. Hegner, and G. Buldt. 2011. FEBS Lett. 585, 1859-1863

DNA handles are often used as spacers and linkers in single-molecule experiments to isolate and tether RNAs, proteins, enzymes and ribozymes, amongst other biomolecules, between surface-modified beads for nanomechanical investigations. Custom DNA handles with varying lengths and chemical end-modifications are readily and reliably synthesized en masse, enabling force spectroscopic measurements with well-defined and long-lasting mechanical characteristics under physiological conditions over a large range of applied forces. Although these chemically tagged DNA handles are widely used, their further individual modification with protein receptors is less common and would allow for additional flexibility in grabbing biomolecules for mechanical measurements. In-depth information on reliable protocols for the synthesis of these DNA–protein hybrids and on their mechanical characteristics under varying physiological conditions are lacking in literature. Here, optical tweezers are used to investigate different protein-labelled DNA handles in a microfluidic environment under different physiological conditions. Digoxigenin (DIG)-dsDNA-biotin handles of varying sizes (1000, 3034 and 4056 bp) were conjugated with streptavidin or neutravidin proteins. The DIG-modified ends of these hybrids were bound to surface-modified polystyrene (anti-DIG) beads. Using different physiological buffers, optical force measurements showed consistent mechanical characteristics with long dissociation times. These protein-modified DNA hybrids were also interconnected in situ with other tethered biotinylated DNA molecules. Electron-multiplying CCD (EMCCD) imaging control experiments revealed that quantum dot–streptavidin conjugates at the end of DNA handles remain freely accessible. The experiments presented here demonstrate that handles produced with our protein–DNA labelling procedure are excellent candidates for grasping single molecules exposing tags suitable for molecular recognition in time-critical molecular motor studies.

Micro- and nanomechanical analytical devices for diagnostics require small sample volumes, and stable and efficient clamping of the sensors for optimized bioassays. A fully automated device for the readout of the dynamic and static response of cantilevers in a physiological liquid environment including a sample handling system is presented. The device provides sequential readout of the static and dynamic mode providing the best signal to noise ratio for each individual cantilever. In the dynamic mode, it is possible to measure up to the 16 th flexural resonance mode of vibration of a 500 μm long and 1 μm thick cantilever. The automated sample handling system enables the local injection of sub microliter volumes of sample with excellent reproducibility. Data demonstrating the response of the cantilevers to external stimuli highlight the sensitivity of the device and the importance of the use of reference cantilevers to decouple biologically relevant metrics from environmental effects. A specific biomolecular interaction of sensitised nanospheres on cantilever sensors with a mass resolution of ~50 picogram in liquid is shown. PACS numbers: 07.10 Mechanical instruments, 87.85 Biosensors, 47.85. Fluidic

An efficient, high-throughput method for the formation of densely packed molecular films on graphene is reported. The films exhibit high stability and remain intact during a subsequent derivatisation reaction, offering a versatile route for the non-covalent functionalisation of graphene.

A nanomechanical technique for rapid real time detection and monitoring of microorganism growth will significantly reduce costs and diagnosis times in industrial and clinical settings. Owing to their label free detection mechanism and unprecedented sensitivity to the mass and elastic modulus of biological structures, dynamically operated cantilever arrays provide an opportunity to rapidly detect and track the evolution of microbial growth. Here we report the monitoring of the growth of single Aspergillus niger spores via the multimode response of microcantilevers. The fungal hyphal structure affects the cantilevers' nanomechanical properties as it propagates along the sensor. We demonstrate, for the first time, the mapping of cellular events with great accuracy using a cantilever frequency response. Imaging of growth conditions on the cantilever, which is performed in parallel, allows for verification of these results. Theoretical comparison and finite element modelling confirm experimental findings and allow for determination of the hyphal elastic modulus.

We investigate here the nanomechanical response of microcantilever sensors in real-time for detecting a range of ultra-low concentrations of oligonucleotides in a complex background of total cellular RNA extracts from cell lines without labeling or amplification. Cantilever sensor arrays were functionalized with probe single stranded DNA (ssDNA) and reference ssDNA to obtain a differential signal. They were then exposed to complementary target ssDNA strands that were spiked in a fragmented total cellular RNA background in biologically relevant concentrations so as to provide clinically significant analysis. We present a model for prediction of the sensor behavior in competitive backgrounds with parameters that are indicators of the change in nanomechanical response with variation in the target and background concentration. For nanomechanical assays to compete with current technologies it is essential to comprehend such responses with eventual impact on areas like understanding non-coding RNA pharmacokinetics, nucleic acid biomarker assays and miRNA quantification for disease monitoring and diagnosis to mention a few. Additionally, we also achieved a femtomolar sensitivity limit for online oligonucleotide detection in a non-competitive environment with these sensors.

* Correspondence: stephen. buckman@anu.edu.au Australian National University, Canberra, ACT 0200, Australia Full list of author information is available at the end of the article The development of experimental science in the past half century has produced advances in areas such as electroncs and communications, new materials, optical technologies, computer processing techniques and biomedical science, to name just a few. These advances have in turn motivated the design of experiments that are increasingly complex, sophisticated, and ambitious, producing results of unprecedented accuracy, precision, and novelty. As a result, fundamental research results, and the new technologies that may arise from them, are often produced using customised or purpose-built equipment, materials, or software that may exist in only one or two facilities worldwide. The design principles and techniques employed are often ground-breaking, yet results published in traditional journals often do not include the detail necessary for reproducing the work, or applying it in related areas of science. Other helpful information, such as best practices guides, and solutions to common problems, often does not fit within the scope of a traditional academic journal or is brushed over in the focus on the scientific outcome. With the launch of EPJ Techniques and Instrumentation (EPJ TI) we hope to specifically address this need, encouraging the open access dissemination of detailed experimental design descriptions, experimental techniques and practices, and examples of results that arise from such experiments, that go beyond the scope of traditional scientific journal articles. The purpose of EPJ TI is to catalyze the development and adoption of next-generation techniques and technologies. We will do this through the publication of regular articles, openly submitted, and through the use of thematic series where we can stimulate the publication of important papers in contemporary research areas. EPJ TI is an open-access journal, part of the new suite of OA titles published in the EPJ series. As such it is funded by article processing charges (APCs), but a number of initiatives are in place to assist authors if they do not have access to publication funding. These include the SpringerOpen membership scheme (www.springeropen.com/inst/), reduced charges for authors in developing regions, and editorial discretionary waivers. If you are considering submitting to EPJ TI but do not have the necessary funding, please contact us (epjti@edpsciences.org) and we will endeavour to help.

with perylene derivatives for biosensing Nina C. Berner, Sinead Winters, Rohit Mishra, Claudia Backes, Aoife Ryan, Mathias O. Senge, Martin Hegner, Andreas Hirsch, Georg S. Duesberg CRANN and School of Chemistry, Trinity College Dublin, Ireland; School of Physics, Trinity College Dublin, Ireland; SFI Tetrapyrrole Laboratory and School of Chemistry, Trinity College Dublin, Ireland; Department of Chemistry and Pharmacy, University of Erlangen-Nuremberg, Germany

A rapid and reliable multi-mode device capable of the readout of both the static and dynamic response of a micron scale cantilever array in a physiological liquid environment is presented. The resolution of the static mode is on the order of 1–2nm and in the dynamic mode it is possible to measure up to the 19th flexural resonance mode of vibration of a 500μm long and 1μm thick cantilever. Rapid measurement from the tip of the cantilevers or line scan measurement of the cantilever profiles are both possible in dual mode. The device provides sequential readout of both modes to provide the best signal to noise ratio for each mode. Proof of principle measurements are presented demonstrating the capabilities of the device and confirming the readout from both the static bending and multiple dynamic resonance modes.

We present a fibre-optic-based device for the automated readout of microcantilever arrays for fast microorganism growth detection. We determined the ability of our device to track shifts in resonance frequency due to an increase in mass on the cantilever surface or changes in mechanical stiffness. The resonance frequency response of 7 μm thick agarose-functionalised cantilevers was tracked as humidity levels were varied revealing a mass responsivity of ~51±1 pg/Hz. The resonance response of microcantilevers coated with Aspergillus niger (A. niger) spores was monitored for >48 h revealing a growth detection time of >4 h. The growth of mycelium along the cantilevers surface is seen to result in an increase in resonance frequency due to the reinforcement of the cantilever structure. The use of our fibre optic detection technique allows data to be recorded continuously and faster than previously reported.