Journal: ACS Applied Materials & Interfaces

Article Title: Nanoscale Cellular Traction Force Quantification: CRISPR-Cas12a Supercharged DNA Tension Sensors in Nanoclustered Ligand Patterns

doi: 10.1021/acsami.4c18358

Figure Lengend Snippet: Fabrication steps for nanopatterning 2 ligands on a fully PEGylated topography-free background (a) Assembly of negatively charged colloidal particles on the surface. (b) Left over organic polyelectrolyte removal using a short O 2 plasma reactive ion etching (RIE). (c) Deposition of a thin sacrificial metallic Cr layer to generate the patterning mask, and removal of the colloidal particles by tape stripping. After removal of the particles the generated apertures are cleaned by a short O 2 RIE. (d) An antifouling polymer terminated with a biotin tag [PAcrAm- g -PEG-Biotin (NH 2 , Si)] forms a monolayer. (e) 2 1/2 min Cr wet etching removes the Cr layer, and the PEG-Biotin bound to it. (f) The deposition of biospecific PAcrAm- g -PEG-N 3 polymer on the surface allows for the orthogonal binding of two bioligands while remaining antifouling. At this stage, the multiwell-sized nanopatterned thin glass substrate (#1.5H) is attached to a sticky 96-well plate. (g) A schematic representing the top view of the patterned substrate after protein incubation. The inserts show the antifouling polymer’s covalent siloxane and electrostatic attachment to the glass. The biorthogonal tag is visualized to attach the biomolecule of interest to a DBCO-labeled protein (protein 1) or a biotin-binding protein (protein 2).

Article Snippet: The purified DBCO conjugated DNA strands were then reacted overnight at 37 °C with 2× molar excess of cyclo-RGD-Azide (Vivitide, RGD-3749-PI), and once again purified by RP-HPLC as above.

Techniques: Clinical Proteomics, Stripping Membranes, Generated, Polymer, Binding Assay, Incubation, Labeling

Journal: ACS Applied Materials & Interfaces

Article Title: Nanoscale Cellular Traction Force Quantification: CRISPR-Cas12a Supercharged DNA Tension Sensors in Nanoclustered Ligand Patterns

doi: 10.1021/acsami.4c18358

Figure Lengend Snippet: Nanoparticle assembly and protein patterns across several size ranges. (a) SEM images of particles assembled on the surface. Features: (i) 80 nm, (ii) 300 nm, and (iii) 800 nm particles. (b) Verification of protein nanopattern formation through confocal laser scanning fluorescence microscopy (CLSM) and DNA-PAINT super-resolution microscopy. (i) CLSM image of 800 nm protein patterns and (ii) 300 nm and (iii) 800 nm protein patterns with reverse tags on the surface. (iv) A DNA-PAINT super-resolution image of 80 nm of streptavidin. The top left inserts are magnifications of the same surfaces. Green indicates streptavidin, and red DBCO-BSA was used to demonstrate the patterning system.

Article Snippet: The purified DBCO conjugated DNA strands were then reacted overnight at 37 °C with 2× molar excess of cyclo-RGD-Azide (Vivitide, RGD-3749-PI), and once again purified by RP-HPLC as above.

Techniques: Fluorescence, Microscopy, Super-Resolution Microscopy