Poly(N-Isopropylacrylamide) Polymer Brush Functionalized Nanostructures as Thermo-Responsive Gating Systems for Protein Transportation

Controlling molecule translocation through nanosized gaps is of great interest in novel systems for single molecule analysis and biomolecular membranes. The molecular gating property of thermo-responsive end-grafted poly(N-isopropylacrylamide) (PNIPAM) polymer brushes on well-shaped gold-silica nanostructures is intended to be investigated for controlled protein transportation via extinction spectroscopy and fluorescence microscopy methods below and above PNIPAM lower critical solution temperature (LCST; 32 \ub0C in water). Polymer brushes are prepared via Activators Regenerated by Electron Transfer Atom Transfer Radical Polymerization (ARGET-ATRP) by employing Bis[2-(2- bromoisobutyryloxy)undecyl] disulfide (DTBU) and its thiol (TBU) equivalent as initiators for the reaction. Variation of PNIPAMreaction time/solvent constituency during the polymerization, results in different swollen/collapsed polymer brush thicknesses, indicated by the plasmonic shifts in extinction spectroscopy and surface plasmon resonance experiments. By having sufficient polymer thickness and grafting density for, e.g. 80-90 nm, nanowells, polymer conformational change below and above LCST, allows controlled gating of these nanostructures. This feature was employed for protein transportation through the polymer brush interface in and out of the fabricated nanowell according to its plasmonic activity. In addition, we investigated molecular gating of fluorescently labelled protein transportation by complimentary fluorescence microscopy measurements

Biomolecule Trapping With Stimuli-Responsive Polymer Coated Nanostructures

Trapping biomolecules in nanosized gaps is of great interest in novel systemsfor single molecule analysis and membranes, which filter biomolecules. Currentplatforms are lacking in full functionality to facilitate biomolecule trapping andtransport in their native environment and without covalent tethering to surfaces.Thus, we propose a system of thermo-responsive polymer poly(N-isopropylacrylamide)(PNIPAM) coated nanostructures, which are suited to controllablytrap and release proteins, and overcome such challenges. PNIPAM polymerbrushes (i.e. the barrier for proteins) on nanostructures were prepared via ActivatorsRegenerated by Electron Transfer Atom Transfer Radical Polymerization(ARGET-ATRP) by employing a self-assembled monolayer of initiator moleculesfor the reaction. Variation of PNIPAM reaction time and/or solvent constituencyduring the polymerization results in different swollen/collapsed polymer brushthicknesses, indicated by the plasmonic shifts in extinction spectroscopy and surfaceplasmon resonance experiments. By having sufficient polymer film thicknessand grafting density for nanowells, e.g. 120 nm, polymer conformational changebelow and above LCST allowed for controlled gating of these nanostructures.This feature was used to allow or block proteins from entering the interior ofthe nanostructures (small molecules diffuse freely in both states) as investigatedby nanostructure plasmonic activity (extinction spectroscopy) and fluorescencemicroscopy below and above PNIPAM lower critical solution temperature (32 \ub0Cin water). In addition, with fluorescence microscopy experiments we showed thatit is possible to trap and release many proteins with single nanowell resolution

Accurate Correction of the "bulk Response" in Surface Plasmon Resonance Sensing Provides New Insights on Interactions Involving Lysozyme and Poly(ethylene glycol)

Surface plasmon resonance is a very well-established surface sensitive technique for label-free analysis of biomolecular interactions, generating thousands of publications each year. An inconvenient effect that complicates interpretation of SPR results is the "bulk response"from molecules in solution, which generate signals without really binding to the surface. Here we present a physical model for determining the bulk response contribution and verify its accuracy. Our method does not require a reference channel or a separate surface region. We show that proper subtraction of the bulk response reveals an interaction between poly(ethylene glycol) brushes and the protein lysozyme at physiological conditions. Importantly, we also show that the bulk response correction method implemented in commercial instruments is not generally accurate. Using our method, the equilibrium affinity between polymer and protein is determined to be KD = 200 μM. One reason for the weak affinity is that the interaction is relatively short-lived (1/koff < 30 s). Furthermore, we show that the bulk response correction also reveals the dynamics of self-interactions between lysozyme molecules on surfaces. Besides providing new insights on important biomolecular interactions, our method can be widely applied to improve the accuracy of SPR data generated by instruments worldwide

Surface plasmon resonance sensing with thin films of palladium and platinum - quantitative and real-time analysis

Surface plasmon resonance (SPR) is a highly useful technique in biology and is gradually becoming useful also for materials science. However, measurements to date have been performed almost exclusively on gold, which limits the possibility to probe chemical modifications of other metals. In this work we show that 20 nm Pd and Pt films work "fairly well" for quantitative SPR sensing of organic films despite the high light absorption. In the interval between total reflection and the SPR angle, high intensity changes occur when a film is formed on the surface. Fresnel models accurately describe the full angular spectra and our data analysis provides good resolution of surface coverage in air (a few ng cm(-2)). Overall, the Pd sensors behave quite similarly to 50 nm gold in terms of sensitivity and field extension, although the noise level in real-time measurements is similar to 5 times higher. The Pt sensors exhibit a longer extension of the evanescent field and similar to 10 times higher noise compared to gold. Yet, formation of organic layers a few nm in thickness can still be monitored in real-time. As a model system, we use thiolated poly(ethylene glycol) to make Pd and Pt protein repelling. Our findings show how SPR can be used for studying chemical modifications of two metals that are important in several contexts, for instance within heterogeneous catalysis. We emphasize the advantages of simple sample preparation and accurate quantitative analysis in the planar geometry by Fresnel models

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

Video Speed Switching of Plasmonic Structural Colors with High Contrast and Superior Lifetime

Abstract: Reflective displays or “electronic paper” technologies provide a solution to the high energy consumption of emissive displays by simply utilizing ambient light. However, it has proven challenging to develop electronic paper with competitive image quality and video speed capabilities. Here, the first technology that provides video speed switching of structural colors with high contrast over the whole visible is shown. Importantly, this is achieved with a broadband‐absorbing polarization‐insensitive electrochromic polymer instead of liquid crystals, which makes it possible to maintain high reflectivity. It is shown that promoting electrophoretic ion transport (drift motion) improves the switch speed. In combination with new nanostructures that have high surface curvature, this enables video speed switching (20 ms) at high contrast (50% reflectivity change). A detailed analysis of the optical signal during switching shows that the polaron formation starts to obey first order reaction kinetics in the video speed regime. Additionally, the system still operates at ultralow power consumption during video speed switching (<1 mW cm−2) and has negligible power consumption (<1 µW cm−2) in bistability mode. Finally, the fast switching increases device lifetime to at least 107 cycles, an order of magnitude more than state‐of‐the‐art

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