Whole-genome detection using multivalent DNA-coated colloids.

To minimize the incorrect use of antibiotics, there is a great need for rapid and inexpensive tests to identify the pathogens that cause an infection. The gold standard of pathogen identification is based on the recognition of DNA sequences that are unique for a given pathogen. Here, we propose and test a strategy to develop simple, fast, and highly sensitive biosensors that make use of multivalency. Our approach uses DNA-functionalized polystyrene colloids that distinguish pathogens on the basis of the frequency of selected short DNA sequences in their genome. Importantly, our method uses entire genomes and does not require nucleic acid amplification. Polystyrene colloids grafted with specially designed surface DNA probes can bind cooperatively to frequently repeated sequences along the entire genome of the target bacteria, resulting in the formation of large and easily detectable colloidal aggregates. Our detection strategy allows "mix and read" detection of the target analyte; it is robust and highly sensitive over a wide concentration range covering, in the case of our test target genome Escherichia coli bl21-de3, 10 orders of magnitude from [Formula: see text] to [Formula: see text] copies/mL. The sensitivity compares well with state-of-the-art sensing techniques and has excellent specificity against nontarget bacteria. When applied to real samples, the proposed technique shows an excellent recovery rate. Our detection strategy opens the way to developing a robust platform for pathogen detection in the fields of food safety, disease control, and environmental monitoring.

The glycocalyx affects the mechanotransductive perception of the topographical microenvironment.

The cell/microenvironment interface is the starting point of integrin-mediated mechanotransduction, but many details of mechanotransductive signal integration remain elusive due to the complexity of the involved (extra)cellular structures, such as the glycocalyx. We used nano-bio-interfaces reproducing the complex nanotopographical features of the extracellular matrix to analyse the glycocalyx impact on PC12 cell mechanosensing at the nanoscale (e.g., by force spectroscopy with functionalised probes). Our data demonstrates that the glycocalyx configuration affects spatio-temporal nanotopography-sensitive mechanotransductive events at the cell/microenvironment interface. Opposing effects of major glycocalyx removal were observed, when comparing flat and specific nanotopographical conditions. The excessive retrograde actin flow speed and force loading are strongly reduced on certain nanotopographies upon strong reduction of the native glycocalyx, while on the flat substrate we observe the opposite trend. Our results highlight the importance of the glycocalyx configuration in a molecular clutch force loading-dependent cellular mechanism for mechanosensing of microenvironmental nanotopographical features.

Large colloidal probes for atomic force microscopy: Fabrication and calibration issues.

Atomic force microscopy (AFM) is a powerful tool to investigate interaction forces at the micro and nanoscale. Cantilever stiffness, dimensions and geometry of the tip can be chosen according to the requirements of the specific application, in terms of spatial resolution and force sensitivity. Colloidal probes (CPs), obtained by attaching a spherical particle to a tipless (TL) cantilever, offer several advantages for accurate force measurements: tunable and well-characterisable radius; higher averaging capabilities (at the expense of spatial resolution) and sensitivity to weak interactions; a well-defined interaction geometry (sphere on flat), which allows accurate and reliable data fitting by means of analytical models. The dynamics of standard AFM probes has been widely investigated, and protocols have been developed for the calibration of the cantilever spring constant. Nevertheless, the dynamics of CPs, and in particular of large CPs, with radius well above 10 µm and mass comparable, or larger, than the cantilever mass, is at present still poorly characterized. Here we describe the fabrication and calibration of (large) CPs. We describe and discuss the peculiar dynamical behaviour of CPs, and present an alternative protocol for the accurate calibration of the spring constant.

Picomolar glyphosate sensitivity of an optical particle-based sensor utilizing biomimetic interaction principles.

The continually growing use of glyphosate and its critically discussed health and biodiversity risks ask for fast, low cost, on-site sensing technologies for food and water. To address this problem, we designed a highly sensitive sensor built on the remarkably specific recognition of glyphosate by its physiological target enzyme 5-enolpyruvyl-shikimate-3-phosphate synthase (EPSPs). This principle is implemented in an interferometric sensor by using the recently established soft colloidal probe (SCP) technique. EPSPs was site-specifically immobilized on a transparent surface utilizing the self-assembling properties of circadian clock gene 2 hydrophobin chimera and homogeneity of the layer was evidenced by atomic force microscopy. Exposure of the enzyme decorated biochip to glyphosate containing samples causes formation of enzyme-analyte complexes and a competitive loss of available binding sites for glyphosate-functionalized poly(ethylene glycol) SCPs. Functionalization of the SCPs with different types of linker molecules and glyphosate was assessed employing confocal laser scanning microscopy as well as confocal Raman microspectroscopy. Overall, reflection interference contrast microscopy analysis of SCP-biochip interactions revealed a strong influence of linker length and glyphosate coupling position on the sensitivity of the sensor. In employing a combination of pentaglycine linker and tethering glyphosate via its secondary amino group, concentrations in aqueous solutions down to 100 pM could be measured by the differential adhesion between SCP and biochip surface, supported by automated image analysis algorithms. This sensing concept could even prove its exceptional pM sensitivity in combination with a superior discrimination against structurally related compounds.

Measuring the Elastic Properties of Living Cells.

Cell's elasticity is an integrative parameter summarizing the biophysical outcome of many known and unknown cellular processes. This includes intracellular signaling, cytoskeletal activity, changes of cell volume and morphology, and many others. Not only intracellular processes defines a cell's elasticity but also environmental factors like their biochemical and biophysical surrounding. Therefore, cell mechanics represents a comprehensive variable of life. A cell in its standard conditions shows variabilities of biochemical and biophysical processes resulting in a certain range of cell's elasticity. Changes of the standard conditions, endogenously or exogenously induced, are frequently paralleled by changes of cell elasticity. Therefore cell elasticity could serve as parameter to characterize different states of a cell. Atomic force microscopy (AFM) combines high spatial resolution with very high force sensitivity and allows investigating mechanical properties of living cells under physiological conditions. However, elastic moduli reported in the literature showed a large variability, sometimes by an order of magnitude (or even more) for the same cell type assessed in different labs. Clearly, a prerequisite for the use of cell elasticity to describe the actual cell status is a standardized procedure that allows obtaining comparable values of a cell independent from the instrument, from the lab and operator. Biologically derived variations of elasticity could not be reduced due to the nature of living cells but technically and methodologically derived variations could be minimized by a standardized procedure.This chapter provides a Standardized Nanomechanical AFM Procedure (SNAP) that reduces strongly the variability of results obtained on soft samples and living cells by a reliable method to calibrate AFM cantilevers.

Calibration of colloidal probes with atomic force microscopy for micromechanical assessment.

Mechanical assessment of biological materials and tissue-engineered scaffolds is increasingly focusing at lower length scale levels. Amongst other techniques, atomic force microscopy (AFM) has gained popularity as an instrument to interrogate material properties, such as the indentation modulus, at the microscale via cantilever-based indentation tests equipped with colloidal probes. Current analysis approaches of the indentation modulus from such tests require the size and shape of the colloidal probe as well as the spring constant of the cantilever. To make this technique reproducible, there still exist the challenge of proper calibration and validation of such mechanical assessment. Here, we present a method to (a) fabricate and characterize cantilevers with colloidal probes and (b) provide a guide for estimating the spring constant and the sphere diameter that should be used for a given sample to achieve the highest possible measurement sensitivity. We validated our method by testing agarose samples with indentation moduli ranging over three orders of magnitude via AFM and compared these results with bulk compression tests. Our results show that quantitative measurements of indentation modulus is achieved over three orders of magnitude ranging from 1 kPa to 1000 kPa via AFM cantilever-based microindentation experiments. Therefore, our approach could be used for quantitative micromechanical measurements without the need to perform further validation via bulk compression experiments.

Characterizing gelatin hydrogel viscoelasticity with diffusing colloidal probe microscopy.

In this study, we investigate viscoelasticity in gelatin hydrogels using diffusing colloidal probe microscopy (DCPM) to directly measure the elastic potential energy interaction between colloidal probes and the underlying viscoelastic media. Gelatin samples are prepared in four different concentrations between 0.3wt% and 0.6wt% to examine changes in viscoelasticity with concentration. A force balance describing the interaction between the colloidal probes and the hydrogel as a spring-damper system lead to a simple model for mean square displacement. A histogram of locations sampled by the colloidal probes is directly related to the elastic potential energy and the effective spring constant of the gelatin hydrogels. The effective spring constant is a fixed parameter used in the mean square displacement model to find effective viscosity. These parameters are comparable to viscoelastic parameters obtain by a microrheology analysis of two-dimensional mean square displacements. These results can serve as a guide for assessing hydrogel systems where viscoelastic properties are an important factor in biomaterial design.

Morphological and nanomechanical changes in mechanosensitive endothelial cells induced by colloidal AFM probes.

Mechanotransduction is one of the main properties of endothelial cells (ECs) phenotype. Hemodynamic forces like flow-generated endothelial shear stress play a fundamental role in ECs cytoskeletal remodeling and activate signaling cascades in ECs. AFM methods are widely used to characterize morphology as well as mechanical properties of cells. In both cases AFM probes directly interact with cell surface exerting mechanical forces on the cellular membrane, which in turn may stimulate mechanosensitive receptors present in EC. This article presents examples of how the colloidal AFM probes influence ECs during multiple scans. The results revealed that multiple scans of the ECs significantly influenced the morphology and elasticity of cells. Moreover, changes in the cell shape and mechanical properties were dependent on the scan direction (across or along the main axis of the cell). Multiple scans with a colloidal probe leaded to reorientation of the cell main axis and this effect was similar to the action of the shear stress induced by flow conditions. Furthermore, the correlation between the tip-induced modification of the cell properties and the remodeling of the cell's glycocalyx was observed. SCANNING 38:654-664, 2016. © 2016 Wiley Periodicals, Inc.

Identifying polyvinylidene fluoride ultrafiltration membrane fouling behavior of different effluent organic matter fractions using colloidal probes.

The interaction forces between effluent organic matter (EfOM) fractions and membrane were measured by atomic force microscopy in conjunction with self-made membrane material colloidal probes. The inter-EfOM-fraction and intra-EfOM-fraction interactions were investigated using corresponding EfOM-fraction-coated colloidal probe. We combined this analysis with corresponding fouling experiments to identify the EfOM fractions responsible for polyvinylidene fluoride (PVDF) ultrafiltration membrane fouling. Results show that hydrophilic and hydrophobic fractions were the dominant fractions responsible for membrane fouling and flux decline in the initial and later filtration stages, respectively, which was mainly attributed to the stronger PVDF-hydrophilic fraction and intra-hydrophobic-fraction interaction forces. This phenomenon, in conjunction with the fact that each interaction force of PVDF-EfOM fraction was stronger than corresponding intra-EfOM-fraction force, suggests that the elimination of the PVDF-hydrophilic fraction interaction force is the best strategy for controlling EfOM fouling. Moreover, the inter-EfOM-fraction interaction force was mainly controlled by the corresponding intra-EfOM-fraction interaction forces. And, while the membrane-EfOM fraction and intra-EfOM-fraction interactions for each type of EfOM fraction are equivalent, the EfOM fractions with the molecular weight smaller than the molecular weight cutoff of the membranes used were mainly responsible for membrane fouling rather than the relatively high-molecular-weight fractions.