Photosensitive Hydrogel with Temperature-Controlled Reversible Nano-Apertures for Single-Cell Protein Analysis.

Single cell western blot (scWB) is one of the most important methods for cellular heterogeneity profiling. However, current scWB based on conventional photoactive polyacrylamide hydrogel material suffers from the tradeoff between in-gel probing and separation resolution. Here, a highly sensitive temperature-controlled single-cell western blotting (tc-scWB) method is introduced, which is based on a thermo/photo-dualistic-sensitive polyacrylamide hydrogel, namely acrylic acid-functionalized graphene oxide (AFGO) assisted, N-isopropylacrylamide modified polyacrylamide (ANP) hydrogel. The ANP hydrogel is contracted at high-temperature to constrain protein band diffusion during microchip electrophoretic separation, while the gel aperture is expanded under low-temperature for better antibody penetration into the hydrogel. The tc-scWB method enables the separation and profiling of small-molecule-weight proteins with highly crosslinked gel (12% T) in SDS-PAGE. The tc-scWB is demonstrated on three metabolic and ER stress-specific proteins (CHOP, MDH2 and FH) in four pancreatic cell subtypes, revealing the expression of key enzymes in the Krebs cycle is upregulated with enhanced ER stress. It is found that ER stress can regulate crucial enzyme (MDH2 and FH) activities of metabolic cascade in cancer cells, boosting aerobic respiration to attenuate the Warburg effect and promote cell apoptosis. The tc-scWB is a general toolbox for the analysis of low-abundance small-molecular functional proteins at the single-cell level.

para-Sulfonatocalix[4]arene stabilized gold nanoparticles multilayers interfaced to electrodes through host-guest interaction for sensitive ErbB2 detection.

Nanoparticle (NP) structure, compositing and the nature of the NP-functionalized electrode interface have a strong influence upon electrochemical properties that are critical in applications such as sensing, photocatalysis and electrocatalysis. Existing methods to fabricate NP-functionalized electrodes do not allow or precise control over all these variables, especially the NP-electrode interface, making it difficult to understand and predict how structural changes influence electrode activity and consequently limit the application. To conquer this problem, in this study, we fabricated a stepwise construction of a novel supramolecular stabilized gold nanoparticles (AuNPs) multilayer mediated by guest molecules, yielding 3D AuNPs assembly at the electrode interface. para-Sulfonatocalix[4]arene (pSC4), a water soluble macrocyclic synthetic receptor, has been served as a stabilizing ligand for preparation and gaining new insights into pSC4 stabilized gold nanoparticles (pSC4-AuNPs) tethered on the electrode interface through host-guest interaction. We investigated the electrochemical properties of multilayer pSC4-AuNPs modified gold electrode using different core size of AuNPs with varying layer number. The electron transfer ability was characterized by electrochemical impedance spectroscopy (EIS). Electrochemical signals are significantly enhanced through the layer-by-layer assembly of pSC4-AuNPs due to its high conductivity and high effective area. With this innovative method, by taking the assay of a tumor marker as an example, human epidermal growth factor receptor 2 (ErbB2) was successfully measured with a detection limit of 0.5ng/mL. Taking the advantage of the pSC4-AuNPs multilayer's good biocompatibility, high effective area and high electronic transmission, 3D AuNPs multilayer produced on the electrode interface suggests a portable synthetic pathway for the application into sensitive electrochemical biosensor.