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 ∼5 times higher. The Pt sensors exhibit a longer extension of the evanescent field and ∼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.

Control of Polymer Brush Morphology, Rheology, and Protein Repulsion by Hydrogen Bond Complexation.

Polymer brushes are widely used to alter the properties of interfaces. In particular, poly(ethylene glycol) (PEG) and similar polymers can make surfaces inert toward biomolecular adsorption. Neutral hydrophilic brushes are normally considered to have static properties at a given temperature. As an example, PEG is not responsive to pH or ionic strength. Here we show that, by simply introducing a polymeric acid such as poly(methacrylic acid) (PMAA), the highly hydrated brush barrier can change its properties entirely. This is caused by multivalent hydrogen bonds in an extremely pH-sensitive process. Remarkably, it is sufficient to reduce the pH to 5 for complexation to occur at the interface, which is two units higher than in the corresponding bulk systems. Below this critical pH, PMAA starts to bind to PEG in large amounts (comparable to the PEG amount), causing the brush to gradually compact and dehydrate. The brush also undergoes major rheology changes, from viscoelastic to rigid. Furthermore, the protein repelling ability of PEG is lost after reaching a threshold in the amount of PMAA bound. The changes in brush properties are tunable and become more pronounced when more PMAA is bound. The initial brush state is fully recovered when releasing PMAA by returning to physiological pH. Our findings are relevant for many applications involving functional interfaces, such as capture-release of biomolecules.

Large Changes in Protonation of Weak Polyelectrolyte Brushes with Salt Concentration-Implications for Protein Immobilization.

We report for the first time that the protonation behavior of weak polyelectrolyte brushes depends very strongly on ionic strength. The pKa changes by one pH step per order of magnitude in salt concentration. For low salt concentrations (∼1 mM), a very high pH is required to deprotonate a polyacidic brush and a very low pH is required to protonate a polybasic brush. This has major consequences for interactions with other macromolecules, as the brushes are actually almost fully neutral when believed to be charged. We propose that many previous studies on electrostatic interactions between polyelectrolytes and proteins have, in fact, looked at other types of intermolecular forces, in particular, hydrophobic interactions and hydrogen bonds.

Enzyme Immobilization in Polyelectrolyte Brushes: High Loading and Enhanced Activity Compared to Monolayers.

Catalysis by enzymes on surfaces has many applications. However, strategies for efficient enzyme immobilization with preserved activity are still in need of further development. In this work, we investigate polyelectrolyte brushes prepared by both grafting-to and grafting-from with the aim to achieve high catalytic activity. For comparison, self-assembled monolayers that bind enzymes with the same chemical interactions are included. We use the model enzyme glucose oxidase and two kinds of polymers: anionic poly(acrylic acid) and cationic poly(diethylamino)methyl methacrylate. Surface plasmon resonance and spectroscopic ellipsometry are used for accurate quantification of surface coverage. Besides binding more enzymes, the "3D-like" brush environment enhances the specific activity compared to immobilization on self-assembled monolayers. For grafting-from brushes, multilayers of enzymes were spontaneously and irreversibly immobilized without conjugation chemistry. When the pH was between the pI of the enzyme and the p Ka of the polymer, binding was considerable (thousands of ng/cm2 or up to 50% of the polymer mass), even at physiological ionic strength. However, binding was observed also when the brushes were neutrally charged. For acidic brushes (both grafting-to and grafting-from), the activity was higher for covalent immobilization compared to noncovalent. For grafting-from brushes, a fully preserved specific activity compared to enzymes in the liquid bulk was achieved, both with covalent (acidic brush) and noncovalent (basic brush) immobilization. Catalytic activity of hundreds of pmol cm-2 s-1 was easily obtained for polybasic brushes only tens of nanometers in dry thickness. This study provides new insights for designing functional interfaces based on enzymatic catalysis.

Detecting Selective Protein Binding Inside Plasmonic Nanopores: Toward a Mimic of the Nuclear Pore Complex.

Biosensors based on plasmonic nanostructures offer label-free and real-time monitoring of biomolecular interactions. However, so do many other surface sensitive techniques with equal or better resolution in terms of surface coverage. Yet, plasmonic nanostructures offer unique possibilities to study effects associated with nanoscale geometry. In this work we use plasmonic nanopores with double gold films and detect binding of proteins inside them. By thiol and trietoxysilane chemistry, receptors are selectively positioned on the silicon nitride interior walls. Larger (~150 nm) nanopores are used detect binding of averaged sized proteins (~60 kg/mol) with high signal to noise (>100). Further, we fabricate pores that approach the size of the nuclear pore complex (diameter down to 50 nm) and graft disordered phenylalanine-glycine nucleoporin domains to the walls, followed by titration of karyopherinß1 transport receptors. The interactions are shown to occur with similar affinity as determined by conventional surface plasmon resonance on planar surfaces. Our work illustrates another unique application of plasmonic nanostructures, namely the possibility to mimic the geometry of a biological nanomachine with integrated optical sensing capabilities.