Standardized Nanomechanical Atomic Force Microscopy Procedure (SNAP) for Measuring Soft and Biological Samples.

We present a procedure that allows a reliable determination of the elastic (Young's) modulus of soft samples, including living cells, by atomic force microscopy (AFM). The standardized nanomechanical AFM procedure (SNAP) ensures the precise adjustment of the AFM optical lever system, a prerequisite for all kinds of force spectroscopy methods, to obtain reliable values independent of the instrument, laboratory and operator. Measurements of soft hydrogel samples with a well-defined elastic modulus using different AFMs revealed that the uncertainties in the determination of the deflection sensitivity and subsequently cantilever's spring constant were the main sources of error. SNAP eliminates those errors by calculating the correct deflection sensitivity based on spring constants determined with a vibrometer. The procedure was validated within a large network of European laboratories by measuring the elastic properties of gels and living cells, showing that its application reduces the variability in elastic moduli of hydrogels down to 1%, and increased the consistency of living cells elasticity measurements by a factor of two. The high reproducibility of elasticity measurements provided by SNAP could improve significantly the applicability of cell mechanics as a quantitative marker to discriminate between cell types and conditions.

Adsorption orientations and immunological recognition of antibodies on graphene.

Large-scale molecular dynamics (MD) simulations and atomic force microscopy (AFM) in liquid are combined to characterize the adsorption of Immunoglobulin G (IgG) antibodies over a hydrophobic surface modeled with a three-layer graphene slab. We consider explicitly the water solvent, simulating systems with massive sizes (up to 770 000 atoms), for four different adsorption orientations. Protocols based on steered MD to speed up the protein diffusion stage and to enhance the dehydration process are combined with long simulation times (>150 ns) in order to make sure that the final adsorption states correspond to actual stable configurations. Our MD results and the AFM images demonstrate that the IgG antibodies are strongly adsorbed, do not unfold, and retain their secondary and tertiary structures upon deposition. Statistical analysis of the AFM images shows that many of the antibodies adopt vertical orientations, even at very small coverages, which expose at least one Fab binding site for recognition events. Single molecule force spectroscopy experiments demonstrate the immunological response of the deposited antibodies by recognizing its specific antigens. The above properties together with the strong anchoring and preservation of the secondary structure, make graphene an excellent candidate for the development of immunosensors.

Unbinding forces and energies between a siRNA molecule and a dendrimer measured by force spectroscopy.

We have measured the intermolecular forces between small interference RNA (siRNA) and polyamidoamine dendrimers at the single molecular level. A single molecule force spectroscopy approach has been developed to measure the unbinding forces and energies between a siRNA molecule and polyamidoamine dendrimers deposited on a mica surface in a buffer solution. We report three types of unbinding events which are characterized by forces and free unbinding energies, respectively, of 28 pN, 0.709 eV; 38 pN, 0.722 eV; and 50 pN, 0.724 eV. These events reflect different possible electrostatic interactions between the positive charges of one or two dendrimers and the negatively charged phosphate groups of a single siRNA. We have evidence of a high binding affinity of siRNA towards polyamidoamine dendrimers that leads to a 45% probability of measuring specific unbinding events.

Multiscale sensing of antibody-antigen interactions by organic transistors and single-molecule force spectroscopy.

Antibody-antigen (Ab-Ag) recognition is the primary event at the basis of many biosensing platforms. In label-free biosensors, these events occurring at solid-liquid interfaces are complex and often difficult to control technologically across the smallest length scales down to the molecular scale. Here a molecular-scale technique, such as single-molecule force spectroscopy, is performed across areas of a real electrode functionalized for the immunodetection of an inflammatory cytokine, viz. interleukin-4 (IL4). The statistical analysis of force-distance curves allows us to quantify the probability, the characteristic length scales, the adhesion energy, and the time scales of specific recognition. These results enable us to rationalize the response of an electrolyte-gated organic field-effect transistor (EGOFET) operated as an IL4 immunosensor. Two different strategies for the immobilization of IL4 antibodies on the Au gate electrode have been compared: antibodies are bound to (i) a smooth film of His-tagged protein G (PG)/Au; (ii) a 6-aminohexanethiol (HSC6NH2) self-assembled monolayer on Au through glutaraldehyde. The most sensitive EGOFET (concentration minimum detection level down to 5 nM of IL4) is obtained with the first functionalization strategy. This result is correlated to the highest probability (30%) of specific binding events detected by force spectroscopy on Ab/PG/Au electrodes, compared to 10% probability on electrodes with the second functionalization. Specifically, this demonstrates that Ab/PG/Au yields the largest areal density of oriented antibodies available for recognition. More in general, this work shows that specific recognition events in multiscale biosensors can be assessed, quantified, and optimized by means of a nanoscale technique.

Fast nanomechanical spectroscopy of soft matter.

A method that combines high spatial resolution, quantitative and non-destructive mapping of surfaces and interfaces is a long standing goal in nanoscale microscopy. The method would facilitate the development of hybrid devices and materials made up of nanostructures of different properties. Here we develop a multifrequency force microscopy method that enables simultaneous mapping of nanomechanical spectra of soft matter surfaces with nanoscale spatial resolution. The properties include the Young's modulus and the viscous or damping coefficients. In addition, it provides the peak force and the indentation. The method does not limit the data acquisition speed nor the spatial resolution of the force microscope. It is non-invasive and minimizes the influence of the tip radius on the measurements. The same tip is used to measure in air heterogeneous interfaces with near four orders of magnitude variations in the elastic modulus, from 1 MPa to 3 GPa.

Three-dimensional quantitative force maps in liquid with 10 piconewton, angstrom and sub-minute resolutions.

We develop a bimodal force microscopy method to map the three-dimensional force fields and their time-evolution on a variety of solid-water interfaces. The force maps show an oscillatory decaying force perpendicular to the solid surface with a 0.3 nm periodicity. The technique enables the three-dimensional imaging and mapping of the hydration layers and forces on mica and protein GroEL surfaces with 10 piconewton, 2 angstrom and 40 second (whole volume) resolutions. We record the existence and evolution of nanoscale perturbations involving thousands of water molecules of the protein-liquid interface. Those instabilities are not found in stiff and atomically flat interfaces.

Theoretical study of the frequency shift in bimodal FM-AFM by fractional calculus.

Bimodal atomic force microscopy is a force-microscopy method that requires the simultaneous excitation of two eigenmodes of the cantilever. This method enables the simultaneous recording of several material properties and, at the same time, it also increases the sensitivity of the microscope. Here we apply fractional calculus to express the frequency shift of the second eigenmode in terms of the fractional derivative of the interaction force. We show that this approximation is valid for situations in which the amplitude of the first mode is larger than the length of scale of the force, corresponding to the most common experimental case. We also show that this approximation is valid for very different types of tip-surface forces such as the Lennard-Jones and Derjaguin-Muller-Toporov forces.

The emergence of multifrequency force microscopy.

In atomic force microscopy a cantilever with a sharp tip attached to it is scanned over the surface of a sample, and information about the surface is extracted by measuring how the deflection of the cantilever - which is caused by interactions between the tip and the surface - varies with position. In the most common form of atomic force microscopy, dynamic force microscopy, the cantilever is made to vibrate at a specific frequency, and the deflection of the tip is measured at this frequency. But the motion of the cantilever is highly nonlinear, and in conventional dynamic force microscopy, information about the sample that is encoded in the deflection at frequencies other than the excitation frequency is irreversibly lost. Multifrequency force microscopy involves the excitation and/or detection of the deflection at two or more frequencies, and it has the potential to overcome limitations in the spatial resolution and acquisition times of conventional force microscopes. Here we review the development of five different modes of multifrequency force microscopy and examine its application in studies of proteins, the imaging of vibrating nanostructures, measurements of ion diffusion and subsurface imaging in cells.

Nanomechanical coupling enables detection and imaging of 5 nm superparamagnetic particles in liquid.

We demonstrate that a force microscope operated in a bimodal mode enables the imaging and detection of superparamagnetic particles down to 5 nm. The bimodal method exploits the nanomechanical coupling of the excited modes to enhance the sensitivity of the higher mode to detect changes in material properties. The coupling requires the presence of nonlinear forces. Remarkably, bimodal operation enables us to identify changes of slowly varying forces (quasi-linear) in the presence of a stronger nonlinear force. Thus, unambiguous identification of single apoferritin (non-magnetic) and ferritin (magnetic) molecules in air and liquid is accomplished.