Metabolic reactions in living cells are limited by diffusion of reagents in the cytoplasm. Any attempt to quantify the kinetics of biochemical reactions in the cytosol should be preceded by careful measurements of the physical properties of the cellular interior. The cytoplasm is a complex, crowded fluid characterized by effective viscosity dependent on its structure at a nanoscopic length scale. In this work, we present and validate the model describing the cytoplasmic nanoviscosity, based on measurements in seven human cell lines, for nanoprobes ranging in diameters from 1 to 150 nm. Irrespective of cell line origin (epithelial–mesenchymal, cancerous–noncancerous, male–female, young–adult), we obtained a similar dependence of the viscosity on the size of the nanoprobes, with characteristic length-scales of 20 ± 11 nm (hydrodynamic radii of major crowders in the cytoplasm) and 4.6 ± 0.7 nm (radii of intercrowder gaps). Moreover, we revealed that the cytoplasm behaves as a liquid for length scales smaller than 100 nm and as a physical gel for larger length scales.

Biochemistry in living cells is an emerging field of science. Current quantitative bioassays are performed ex vivo , thus equilibrium constants and reaction rates of reactions occurring in human cells are still unknown. To address this issue, we present a non-invasive method to quantitatively characterize interactions (equilibrium constants, K D ) directly within the cytosol of living cells. We reveal that cytosolic hydrodynamic drag depends exponentially on a probe’s size, and provide a model for its determination for different protein sizes (1–70 nm). We analysed oligomerization of dynamin-related protein 1 (Drp1, wild type and mutants: K668E, G363D, C505A) in HeLa cells. We detected the coexistence of wt-Drp1 dimers and tetramers in cytosol, and determined that K D for tetramers was 0.7 ± 0.5 μM. Drp1 kinetics was modelled by independent simulations, giving computational results which matched experimental data. This robust method can be applied to in vivo determination of K D for other protein-protein complexes, or drug-target interactions.

We present synthesis of a free-standing monolayer film of gold nanoparticles (AuNPs) which are linked by covalent bonds. In the method developed, the free-standing film is obtained by chemical cross-linking of the AuNPs of the core diameter of 5.6 nm that form a dense monolayer at the oil–liquid interface. As the cross-linking agent, naphthalene dianhydride derivative, which forms amide bonds with the ligand molecules, is used. The AuNPs are coated with aminothiolate ligands that can change their character from a hydrophilic to a hydrophobic one via the reversible protonation/deprotonation mechanism. When adsorbed at the oil–water interface, such functionalized AuNPs display amphiphilic (Janus-like) structure and self-assemble into a highly stable monolayer. To bring the AuNPs at the oil–water interface, an excitation of the system that leads to the formation of the oil-in-water emulsion is required. After the excitation, the AuNPs are transported onto the oil–water interface on the surface of the oil droplets that carry them as their “cargo”. A thermodynamic mechanism explaining this cargo effect is put forward. The as-synthesized free-standing film can be easily transferred from the oil–water interface onto solid support, as well as porous grids, and it is found to be stable in air.