Fast Detection of Biomolecules in Diffusion-Limited Regime Using Micromechanical Pillars

We have developed a micromechanical sensor based on vertically oriented oscillating beams, in which contrary to what is normally done (for example with oscillating cantilevers) the sensitive area is located at the free end of the oscillator. In the micropillar geometry used here, analyte adsorption is confined only to the tip of the micropillar, thus reducing the volume from which the analyte molecules must diffuse to saturate the surface to a sphere of radius more than 2 orders of magnitude smaller than the corresponding linear distance valid for adsorption on a macroscopic surface. Hence the absorption rate is 3 orders of magnitude faster than on a typical 200 × 20 square micrometer cantilever. Pillar oscillations are detected by means of an optical lever method, but the geometry is suitable for multiplexing with compact integrated detection. We demonstrate our technology by investigating the formation of a single-strand DNA self-assembled monolayer (SAM) consisting of less than 10⁶ DNA molecules and by measuring their hybridization efficiency. We show that the binding rate is 1000 times faster than on a “macroscopic” surface. We also show that the hybridization of a SAM of maximum density DNA is 40% or 4 times the value reported in the literature. These results suggest that the lower values previously reported in the literature can be attributed to incomplete saturation of the surface due to the slower adsorption rate on the “macroscopic” surfaces used

A Revertible, Autonomous, Self-Assembled DNA-Origami Nanoactuator

A DNA-origami actuator capable of autonomous internal motion in accord to an external chemical signal was designed, built, operated and imaged. The functional DNA nanostructure consists of a disk connected to an external ring in two, diametrically opposite points. A single stranded DNA, named probe, was connected to two edges of the disk perpendicularly to the axis of constrain. In the presence of a hybridizing target molecule, the probe coiled into a double helix that stretched the inner disk forcing the edges to move toward each other. The addition of a third single stranded molecule that displaced the target from the probe restored the initial state of the origami. Operation, dimension and shape were carefully characterized by combining microscopy and fluorescence techniques

Investigation of Adhesion and Mechanical Properties of Human Glioma Cells by Single Cell Force Spectroscopy and Atomic Force Microscopy

<div><p>Active cell migration and invasion is a peculiar feature of glioma that makes this tumor able to rapidly infiltrate into the surrounding brain tissue. In our recent work, we identified a novel class of glioma-associated-stem cells (defined as GASC for high-grade glioma -HG- and Gasc for low-grade glioma -LG-) that, although not tumorigenic, act supporting the biological aggressiveness of glioma-initiating stem cells (defined as GSC for HG and Gsc for LG) favoring also their motility. Migrating cancer cells undergo considerable molecular and cellular changes by remodeling their cytoskeleton and cell interactions with surrounding environment. To get a better understanding about the role of the glioma-associated-stem cells in tumor progression, cell deformability and interactions between glioma-initiating stem cells and glioma-associated-stem cells were investigated. Adhesion of HG/LG-cancer cells on HG/LG-glioma-associated stem cells was studied by time-lapse microscopy, while cell deformability and cell-cell adhesion strengths were quantified by indentation measurements by atomic force microscopy and single cell force spectroscopy. Our results demonstrate that for both HG and LG glioma, cancer-initiating-stem cells are softer than glioma-associated-stem cells, in agreement with their neoplastic features. The adhesion strength of GSC on GASC appears to be significantly lower than that observed for Gsc on Gasc. Whereas, GSC spread and firmly adhere on Gasc with an adhesion strength increased as compared to that obtained on GASC. These findings highlight that the grade of glioma-associated-stem cells plays an important role in modulating cancer cell adhesion, which could affect glioma cell migration, invasion and thus cancer aggressiveness. Moreover this work provides evidence about the importance of investigating cell adhesion and elasticity for new developments in disease diagnostics and therapeutics.</p></div

Co-culture of GSC on GASC, Gsc on Gasc and GSC on Gasc.

<p>(<b>A</b>) Fluorescence images of Hoechst-labeled glioma-initiating cells (red color) on CFSE-labeled glioma associated cells (green color) at different time point (scale bar 200 µm). (<b>B</b>) Quantitative analysis of the number of GSC adherent to GASC (red line), Gsc adherent to Gasc (black line) and GSC adherent to Gasc (blue line), respectively. Cell type: p<0.0001, time: p = 0.09. Data are presented as mean ± standard error. *, p<0.05 of GSC-GASC vs Gsc-Gasc; **, p<0.05 of GSC-GASC vs GSC-Gasc.</p

Interpopulation adhesion measurements.

<p>Comparison of time dependent detachment force (<b>A</b>) and work of detachment (<b>B</b>) evaluated for Gsc-Gasc, GSC-Gasc and GSC-GASC; (*) p value<0.05 is considered statistically significant. (<b>A</b>) The detachment force of GSC-GASC is higher than that of Gsc-Gasc (p<0.0001 at 10 sec and 40 sec, p = 0.0003 at 160 sec). Detachment force of GSC-Gasc increases significantly as compared to HGG (10 sec p = 0.0267; 40 sec p<0.0001; 160 sec p = 0.0014). (<b>B</b>) For work of detachment at 10 sec contact time no significant differences are detected; for higher contact time (40 sec) Gsc-Gasc is significantly higher than GSC-GASC (p = 0.0068) as also the increment of GSC-Gasc as compared to GSC-GASC (p<0.0001); for 160 sec Gsc-Gasc is significantly higher than GSC-GASC (p = 0.0006) as also the increment for GSC-Gasc as compared to GSC-GASC (p<0.0001). For a better visualization and comparison of the data, Y scale is reported as Log scale.</p

Cell-cell adhesion measurements.

<p>(<b>A</b>) Differential interference contrast (DIC) optical image of a GSC immobilized on a tipless cantilever brought into contact with a GASC cultured on glass coverslip coated with fibronectin (scale bar 20 µm); representative F-D retraction traces acquired for GSC-GASC (<b>B</b>) and Gsc-Gasc (<b>C</b>) for 10 sec contact time; features of the curves that enables to quantify adhesion properties are highlited: the maximum force exerted to detach the cell (F_detachment); the area, included within the retraction curve and the dot line, represents the work done by the cantilever to completely detach the cell from the substrate (work of detachment).</p

Time dependent analysis of detachment force and work.

<p>(<b>A–B</b>) Data obtained for GSC-GASC compared with GSC-laminin and GSC-fibronectin and (<b>C–D</b>) Gsc-Gasc compared with Gsc-laminin and Gsc-fibronectin; the values inside the box represent the first (25%) and third quartile (75%), while the line within the box represents the median value (50%); the (–) indicate the maximum and minimum observations; while outliers are indicated by (•); the mean value is indicated in the plot as (+); (*) p value<0.05 is considered statistically significant. (<b>A–B</b>) Detachment force and work of GSC-laminin are significantly higher than that obtained for GSC-GASC or GSC-fibronectin for each contact time investigated (p<0.0001); No significant differences are obtained between GSC-GASC and GSC-fibronectin, except for work of detachment at 160 sec (p = 0.0097). (<b>C–D</b>) The detachment force of Gsc-laminin is higher than that obtained for Gsc-Gasc (p<0.00001 for 10 sec and 40 sec, p = 0.0118 for 160 sec) and Gsc-fibronectin (p<0.00001 for 10 sec and 40 sec, p = 0.0005 for 160 sec). The work of detachment of Gsc-laminin is significantly higher than that obtained for Gsc-Gasc (p = 0.0042) at 10 sec and (p = 0.0006) at 40 sec contact time, while at 160 sec they are no significantly different (p = 0.1167); the work of detachment of Gsc-laminin is higher than that obtained for Gsc-fibronectin for all the contact time investigated (p = 0.0004 for 10 sec, p<0.0001 for 40 sec, p = 0.0006 for 160 sec). No significant differences were obtained between Gsc-Gasc and Gsc-fibronectin, except for work of detachment at 160 sec (p = 0.0004). For a better visualization and comparison of the data, Y scale is reported as Log scale.</p

AFM indentation measurements and analysis of each cell subpopulation.

<p>(<b>A–D</b>) Examples of F-D approaching curves converted into a dependence of load force versus indentations for each cell populations. (<b>E–H</b>) Young’s modulus distribution obtained for each cell population. A p value<0.05 is considered statistically significant. GSC and Gsc are significantly softer than GASC and Gasc (p<0.0001); GSC appear also significantly softer than Gsc (p<0.01), while GASC and Gasc do not show significant difference.</p