Stable trapping of multiple proteins at physiological conditions using nanoscale chambers with macromolecular gates.

The possibility to detect and analyze single or few biological molecules is very important for understanding interactions and reaction mechanisms. Ideally, the molecules should be confined to a nanoscale volume so that the observation time by optical methods can be extended. However, it has proven difficult to develop reliable, non-invasive trapping techniques for biomolecules under physiological conditions. Here we present a platform for long-term tether-free (solution phase) trapping of proteins without exposing them to any field gradient forces. We show that a responsive polymer brush can make solid state nanopores switch between a fully open and a fully closed state with respect to proteins, while always allowing the passage of solvent, ions and small molecules. This makes it possible to trap a very high number of proteins (500-1000) inside nanoscale chambers as small as one attoliter, reaching concentrations up to 60 gL-1. Our method is fully compatible with parallelization by imaging arrays of nanochambers. Additionally, we show that enzymatic cascade reactions can be performed with multiple native enzymes under full nanoscale confinement and steady supply of reactants. This platform will greatly extend the possibilities to optically analyze interactions involving multiple proteins, such as the dynamics of oligomerization events.

Polymer Brushes on Silica Nanostructures Prepared by Aminopropylsilatrane Click Chemistry: Superior Antifouling and Biofunctionality.

In nanobiotechnology, the importance of controlling interactions between biological molecules and surfaces is paramount. In recent years, many devices based on nanostructured silicon materials have been presented, such as nanopores and nanochannels. However, there is still a clear lack of simple, reliable, and efficient protocols for preventing and controlling biomolecule adsorption in such structures. In this work, we show a simple method for passivation or selective biofunctionalization of silica, without the need for polymerization reactions or vapor-phase deposition. The surface is simply exposed stepwise to three different chemicals over the course of ∼1 h. First, the use of aminopropylsilatrane is used to create a monolayer of amines, yielding more uniform layers than conventional silanization protocols. Second, a cross-linker layer and click chemistry are used to make the surface reactive toward thiols. In the third step, thick and dense poly(ethylene glycol) brushes are prepared by a grafting-to approach. The modified surfaces are shown to be superior to existing options for silica modification, exhibiting ultralow fouling (a few ng/cm2) after exposure to crude serum. In addition, by including a fraction of biotinylated polymer end groups, the surface can be functionalized further. We show that avidin can be detected label-free from a serum solution with a selectivity (compared to nonspecific binding) of more than 98% without the need for a reference channel. Furthermore, we show that our method can passivate the interior of 150 nm × 100 nm nanochannels in silica, showing complete elimination of adsorption of a sticky fluorescent protein. Additionally, our method is shown to be compatible with modifications of solid-state nanopores in 20 nm thin silicon nitride membranes and reduces the noise in the ion current. We consider these findings highly important for the broad field of nanobiotechnology, and we believe that our method will be very useful for a great variety of surface-based sensors and analytical devices.

Pore performance: artificial nanoscale constructs that mimic the biomolecular transport of the nuclear pore complex.

The nuclear pore complex is a nanoscale assembly that achieves shuttle-cargo transport of biomolecules: a certain cargo molecule can only pass the barrier if it is attached to a shuttle molecule. In this review we summarize the most important efforts aiming to reproduce this feature in artificial settings. This can be achieved by solid state nanopores that have been functionalized with the most important proteins found in the biological system. Alternatively, the nanopores are chemically modified with synthetic polymers. However, only a few studies have demonstrated a shuttle-cargo transport mechanism and due to cargo leakage, the selectivity is not comparable to that of the biological system. Other recent approaches are based on DNA origami, though biomolecule transport has not yet been studied with these. The highest selectivity has been achieved with macroscopic gels, but they are yet to be scaled down to nano-dimensions. It is concluded that although several interesting studies exist, we are still far from achieving selective and efficient artificial shuttle-cargo transport of biomolecules. Besides being of fundamental interest, such a system could be potentially useful in bioanalytical devices.