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.

Electrochromic Inorganic Nanostructures with High Chromaticity and Superior Brightness.

The possibility of actively controlling structural colors has recently attracted a lot of attention, in particular for new types of reflective displays (electronic paper). However, it has proven challenging to achieve good image quality in such devices, mainly because many subpixels are necessary and the semitransparent counter electrodes lower the total reflectance. Here we present an inorganic electrochromic nanostructure based on tungsten trioxide, gold, and a thin platinum mirror. The platinum reflector provides a wide color range and makes it possible to "reverse" the device design so that electrolyte and counter electrode can be placed behind the nanostructures with respect to the viewer. Importantly, this makes it possible to maintain high reflectance regardless of how the electrochemical cell is constructed. We show that our nanostructures clearly outperform the latest commercial color e-reader in terms of both color range and brightness.

Electrically Switchable Polymer Brushes for Protein Capture and Release in Biological Environments.

Interfaces functionalized with polymers are known for providing excellent resistance towards biomolecular adsorption and for their ability to bind high amounts of protein while preserving their structure. However, making an interface that switches between these two states has proven challenging and concepts to date rely on changes in the physiochemical environment, which is static in biological systems. Here we present the first interface that can be electrically switched between a high-capacity (>1 µg cm-2 ) multilayer protein binding state and a completely non-fouling state (no detectable adsorption). Switching is possible over multiple cycles without any regeneration. Importantly, switching works even when the interface is in direct contact with biological fluids and a buffered environment. The technology offers many applications such as zero fouling on demand, patterning or separation of proteins as well as controlled release of biologics in a physiological environment, showing high potential for future drug delivery in vivo.

Tuning the Thermoresponsive Behavior of Surface-Attached PNIPAM Networks: Varying the Crosslinker Content in SI-ATRP.

The synthesis and thermoresponsive properties of surface-attached poly(N-isopropylacrylamide)-co-N,N'-methylene bisacrylamide (PNIPAM-co-MBAM) networks are investigated. The networks are formed via SI-ARGET-ATRP ("grafting-from") on thiol-based initiator-functionalized gold films. This method is reliable, well controlled, fast, and applicable to patterned surfaces (e.g., nanopores) for networks with dry thicknesses >20 nm. Surface-attached PNIPAM-co-MBAM gels are swollen below their volume phase transition temperature but above collapse without complete expulsion of water (retain ∼50 vol %). The swelling/collapse transition is studied using complementary SPR and QCMD techniques. The ratio between swollen and collapsed heights characterizes the thermoresponsive behavior and is shown to not depend on network height but to vary with MBAM content. The higher the proportion of the crosslinker, the lower the magnitude of the phase transition, until all responsiveness is lost at 5 mol % MBAM. The temperature range of the transition is broadened for more crosslinked PNIPAM-co-MBAM gels but remains centered around 32 °C. Upon reswelling, less crosslinked networks display sharp transitions, while for those containing ≥3 mol % MBAM, transitions remain broad. This tunable behavior persists for gels on nanostructured gold surfaces. Investigating PNIPAM-co-MBAM networks on gold plasmonic nanowell arrays is a starting point for expanding their scope as thermo-controlled nanoactuators.

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.

Active control of plasmonic colors: emerging display technologies.

In recent years there has been a growing interest in the use of plasmonic nanostructures for color generation, a technology that dates back to ancient times. Plasmonic structural colors have several attractive features but once the structures are prepared the colors are normally fixed. Lately, several concepts have emerged for actively tuning the colors, which opens up for many new potential applications, the most obvious being novel color displays. In this review we summarize recent progress in active control of plasmonic colors and evaluate them with respect to performance criteria for color displays. It is suggested that actively controlled plasmonic colors are generally less interesting for emissive displays but could be useful for new types of electrochromic devices relying on ambient light (electronic paper). Furthermore, there are several other potential applications such as images to be revealed on demand and colorimetric sensors.

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.

Nanoplasmonic Sensing Architectures for Decoding Membrane Curvature-Dependent Biomacromolecular Interactions.

Nanoplasmonic sensors have emerged as a promising measurement approach to track biomacromolecular interactions involving lipid membrane interfaces. By taking advantage of nanoscale fabrication capabilities, it is possible to design sensing platforms with various architectural configurations. Such capabilities open the door to fabricating lipid membrane-coated nanoplasmonic sensors with varying degrees of membrane curvature in order to understand how biomacromolecular interaction processes are influenced by membrane curvature. Herein, we employed an indirect nanoplasmonic sensing approach to characterize the fabrication of supported lipid bilayers (SLBs) on silica-coated nanowell and nanodisk sensing platforms and to investigate how membrane curvature influences membrane-peptide interactions by evaluating the corresponding measurement responses from different spectral signatures that are sensitive to specific regions of the sensor geometries. SLBs were prepared by the vesicle fusion method, as monitored in real-time by nanoplasmonic sensing measurements and further characterized by fluorescence recovery after photobleaching (FRAP) experiments. By resolving different spectral signatures in the nanoplasmonic sensing measurements, it was determined that peptide binding induces membrane disruption at positively curved membrane regions, while peptide binding without subsequent disruption was observed at planar and negatively curved regions. These findings are consistent with the peptide's known preference to selectively form pores in positively curved membranes, providing validation to the nanoplasmonic sensing approach and highlighting how the integration of nanoplasmonic sensors with different nanoscale architectures can be utilized to study the influence of membrane curvature on biomacromolecular interaction processes.

Switchable Plasmonic Metasurfaces with High Chromaticity Containing Only Abundant Metals.

Plasmonic color generation offers several advantages but is also limited by the cost and availability of noble metals like gold. In this work, we present color-tunable metasurfaces with high chromaticity and reflectivity consisting of an aluminum mirror, a dielectric spacer, and a plasmonic nanohole array in copper. Copper is shown to be an excellent alternative to gold when properly protected from oxidation and makes it possible to generate a wide RGB gamut covering 27% of the standard RGB. By patterning the metasurfaces into microscale pixel triplets, color photos can be well reproduced with high resolution over wafer-sized areas. Further, we demonstrate active modulation of the reflected intensity using an electrochromic conductive polymer deposited on top of the nanostructures by screen printing. This technology opens up for ultrathin and flexible reflective displays in full color, that is, plasmonic electronic paper, compatible with large-scale sustainable production.

Fabrication and Characterization of Plasmonic Nanopores with Cavities in the Solid Support.

Plasmonic nanostructures are widely used for various sensing applications by monitoring changes in refractive index through optical spectroscopy or as substrates for surface enhanced Raman spectroscopy. However, in most practical situations conventional surface plasmon resonance is preferred for biomolecular interaction analysis because of its high resolution in surface coverage and the simple single-material planar interface. Still, plasmonic nanostructures may find unique sensing applications, for instance when the nanoscale geometry itself is of interest. This calls for new methods to prepare nanoscale particles and cavities with controllable dimensions and curvature. In this work, we present two types of plasmonic nanopores where the solid support underneath a nanohole array has been etched, thereby creating cavities denoted as 'nanowells' or 'nanocaves' depending on the degree of anisotropy (dry or wet etch). The refractometric sensitivity is shown to be enhanced upon removing the solid support because of an increased probing volume and a shift of the asymmetric plasmonic field towards the liquid side of the finite gold film. Furthermore, the structures exhibit different spectral changes upon binding inside the cavities compared to the gold surface, which means that the structures can be used for location-specific detection. Other sensing applications are also suggested.

Dual-Wavelength Surface Plasmon Resonance for Determining the Size and Concentration of Sub-Populations of Extracellular Vesicles.

Accurate concentration determination of subpopulations of extracellular vesicles (EVs), such as exosomes, is of importance both in the context of understanding their fundamental biological role and of potentially using them as disease biomarkers. In principle, this can be achieved by measuring the rate of diffusion-limited mass uptake to a sensor surface modified with a receptor designed to only bind the subpopulation of interest. However, a significant error is introduced if the targeted EV subpopulation has a size, and thus hydrodynamic diffusion coefficient, that differs from the mean size and diffusion coefficient of the whole EV population and/or if the EVs become deformed upon binding to the surface. We here demonstrate a new approach to determine the mean size (or effective film thickness) of bound nanoparticles, in general, and EV subpopulation carrying a marker of interest, in particular. The method is based on operating surface plasmon resonance simultaneously at two wavelengths with different sensing depths and using the ratio of the corresponding responses to extract the particle size on the surface. By estimating in this way the degree of deformation of adsorbed EVs, we markedly improved their bulk concentration determination and showed that EVs carrying the exosomal marker CD63 correspond to not more than around 10% of the EV sample.

Biosensing using plasmonic nanohole arrays with small, homogenous and tunable aperture diameters.

Plasmonic nanohole arrays are widely used for optical label-free molecular detection. An important factor for many applications is the diameter of the apertures. So far nanohole arrays with controllable diameters below 100 nm have not been demonstrated and it has not been systematically investigated how the diameter influences the optical properties. In this work we fine-tune the diameter in short range ordered nanohole arrays down to 50 nm. The experimental far field spectra show how the wavelength of maximum extinction remains unaffected while the transmission maximum blue shifts with smaller diameters. The near field is visualized by numerical simulations, showing a homogenous enhancement throughout the cylindrical void at the transmission maximum for diameters between 50 and 100 nm. For diameters below 50 nm plasmon excitation is no longer possible experimentally or by simulations. Further, we investigate the refractive index sensing capabilities of the smaller holes. As the diameter was reduced, the sensitivity in terms of resonance shift with bulk liquid refractive index was found to be unaltered. However, for the transmission maximum the sensitivity becomes more strongly localized to the hole interior. By directing molecular binding to the bottom of the holes we demonstrate how smaller holes enhance the sensitivity in terms of signal per molecule. A real-time detection limit well below one protein per nanohole is demonstrated. The smaller plasmonic nanoholes should be suitable for studies of molecules confined in small volumes and as mimics of biological nanopores.

Mechanistic investigation of the on-surface enzymatic digestion (oSED) protein adsorption detection method using targeted mass spectrometry.

This study describes our efforts to study some of the mechanistic aspects of the earlier established on-surface enzymatic digestion (oSED) method. In a multitude of application areas, it has become important to be able to fully characterize and understand selective protein adsorption to biomaterial surfaces for various applications, including biomedicine (implants), nanotechnology (microchip surfaces and sensors) and materials sciences. Herein, the investigation of the mechanistic aspects was based on microdialysis catheter tubes that were flushed with controlled protein solutions mimicking the extracellular fluid of the brain. The protein adsorption properties were monitored using high-resolution liquid chromatography tandem mass spectrometry (LC-MS/MS) with a targeted method. The temporally resolved results show that most proteins stay adsorbed onto the surface during the entire digestion process and are only cut away piece by piece, whereas smaller proteins and peptides seem to desorb rather easily from the surface. This information will simplify the interpretation of data generated using the oSED method and can also be used for the characterization of the physicochemical properties controlling the adsorption of individual proteins to specific surfaces.

Plasmon Enhanced Internal Photoemission in Antenna-Spacer-Mirror Based Au/TiO2 Nanostructures.

Emission of photoexcited hot electrons from plasmonic metal nanostructures to semiconductors is key to a number of proposed nanophotonics technologies for solar harvesting, water splitting, photocatalysis, and a variety of optical sensing and photodetector applications. Favorable materials and catalytic properties make systems based on gold and TiO2 particularly interesting, but the internal photoemission efficiency for visible light is low because of the wide bandgap of the semiconductor. We investigated the incident photon-to-electron conversion efficiency of thin TiO2 films decorated with Au nanodisk antennas in an electrochemical circuit and found that incorporation of a Au mirror beneath the semiconductor amplified the photoresponse for light with wavelength λ = 500-950 nm by a factor 2-10 compared to identical structures lacking the mirror component. Classical electrodynamics simulations showed that the enhancement effect is caused by a favorable interplay between localized surface plasmon excitations and cavity modes that together amplify the light absorption in the Au/TiO2 interface. The experimentally determined internal quantum efficiency for hot electron transfer decreases monotonically with wavelength, similar to the probability for interband excitations with energy higher than the Schottky barrier obtained from a density functional theory band structure simulation of a thin Au/TiO2 slab.

Influence of surface modification and static pressure on microdialysis protein extraction efficiency.

There is growing interest in using microdialysis (MD) for monitoring larger and more complex molecules such as neuropeptides and proteins. This promotes the use of MD membranes with molecular weight cut off (MWCO) of 100 kDa or above. The hydrodynamic property of the membrane goes to ultrafiltration or beyond, making the MD catheters more sensitive to pressure. In the meantime, despite the large pore size, studies have shown that membrane biofouling still lead to unstable catheter performance. The objective is to study in vitro how 500 kDa dextran and Poloxamer 407 surface modification affect the fluid recovery (FR) and extraction efficiency (EE) of 100 kDa MWCO MD catheters. A pressure chamber was designed to facilitate the tests, using as MD sample a protein standard with similar concentrations as in human cerebral spinal fluid, comparing native and Poloxamer 407 modified MD catheters. The collected dialysate fractions were examined for FR and protein EE, employing Dot-it Spot-it Protein Assay for total protein EE and targeted mass spectrometry (MS) for EE of individual proteins and peptides. The FR results suggested that the surface modified catheters were less sensitive to the pressure and provide higher precision, and provided a FR closer to 100%. The surface modification did not show a significant effect on the protein EE. The average total protein EE of surface modified catheters was slightly higher than that of the native ones. The MS EE data of individual proteins showed a clear trend of complex response in EE with pressure.

Sensing applications based on plasmonic nanopores: The hole story.

A review of sensing applications based on plasmonic nanopores is given. Many new types of plasmonic nanopores have recently been fabricated, including pores penetrating multilayers of thin films, using a great variety of fabrication techniques based on either serial nanolithography or self-assembly. One unique advantage with nanopores compared to other plasmonic sensors is that sample liquids can flow through the surface, which increases the rate of binding and improves the detection limit under certain conditions. Also, by utilizing the continuous metal films, electrical control can be implemented for electrochemistry, dielectrophoresis and resistive heating. Much effort is still spent on trying to improve sensor performance in various ways, but the literature uses inconsistent benchmark parameters. Recently plasmonic nanopores have been used to analyse targets of high clinical or academic interest. Although this is an important step forward, one should probably reflect upon whether the same results could have been achieved with another optical technique. Overall, this critical review suggests that the research field would benefit by focusing on applications where plasmonic nanopores truly can offer unique advantages over similar techniques.

A thermal plasmonic sensor platform: resistive heating of nanohole arrays.

We have created a simple and efficient thermal plasmonic sensor platform by letting a DC current heat plasmonic nanohole arrays. The sensor can be used to determine thermodynamic parameters in addition to monitoring molecular reactions in real-time. As an application example, we use the thermal sensor to determine the kinetics and activation energy for desorption of thiol monolayers on gold. Further, the temperature of the metal can be measured optically by the spectral shift of the bonding surface plasmon mode (0.015 nm/K). We show that this resonance shift is caused by thermal lattice expansion, which reduces the plasma frequency of the metal. The sensor is also used to determine the thin film thermal expansion coefficient through a theoretical model for the expected resonance shift.

Refined microdialysis method for protein biomarker sampling in acute brain injury in the neurointensive care setting.

There is growing interest in cerebral microdialysis (MD) for sampling of protein biomarkers in neurointensive care (NIC) patients. Published data point to inherent problems with this methodology including protein interaction and biofouling leading to unstable catheter performance. This study tested the in vivo performance of a refined MD method including catheter surface modification, for protein biomarker sampling in a clinically relevant porcine brain injury model. Seven pigs of both sexes (10-12 weeks old; 22.2-27.3 kg) were included. Mean arterial blood pressure, heart rate, intracranial pressure (ICP) and cerebral perfusion pressure was recorded during the stepwise elevation of intracranial pressure by inflation of an epidural balloon catheter with saline (1 mL/20 min) until brain death. One naïve MD catheter and one surface modified with Pluronic F-127 (10 mm membrane, 100 kDa molecular weight cutoff MD catheter) were inserted into the right frontal cortex and perfused with mock CSF with 3% Dextran 500 at a flow rate of 1.0 µL/min and 20 min sample collection. Naïve catheters showed unstable fluid recovery, sensitive to ICP changes, which was significantly stabilized by surface modification. Three of seven naïve catheters failed to deliver a stable fluid recovery. MD levels of glucose, lactate, pyruvate, glutamate, glycerol and urea measured enzymatically showed an expected gradual ischemic and cellular distress response to the intervention without differences between naïve and surface modified catheters. The 17 most common proteins quantified by iTRAQ and nanoflow LC-MS/MS were used as biomarker models. These proteins showed a significantly more homogeneous response to the ICP intervention in surface modified compared to naïve MD catheters with improved extraction efficiency for most of the proteins. The refined MD method appears to improve the accuracy and precision of protein biomarker sampling in the NIC setting.

Impact of static pressure on transmembrane fluid exchange in high molecular weight cut off microdialysis.

With the interest of studying larger biomolecules by microdialysis (MD), this sampling technique has reached into the ultrafiltration region of fluid exchange, where fluid recovery (FR) has a strong dependence on pressure. Hence in this study, we focus on the fluid exchange across the high molecular weight cut off MD membrane under the influence of the static pressure in the sampling environment. A theoretical model is presented for MD with such membranes, where FR has a linear dependence upon the static pressure of the sample. Transmembrane (TM) osmotic pressure difference and MD perfusion rate decide how fast FR increases with increased static pressure. A test chamber for in vitro MD under static pressure was constructed and validated. It can hold four MD probes under controlled pressurized conditions. Comparison showed good agreement between experiment and theory. Moreover, test results showed that the fluid recovery of the test chamber MD can be set accurately via the chamber pressure, which is controlled by sample injection into the chamber at precise rate. This in vitro system is designed for modelling in vivo MD in cerebrospinal fluid and studies with biological samples in this system may be good models for in vivo MD.

Fluorescence imaging of macromolecule transport in high molecular weight cut-off microdialysis.

When microdialysis (MD) membrane exceeds molecular weight cut-off (MWCO) of 100 kDa, the fluid mechanics are in the ultrafiltration regime. Consequently, fluidic mass transport of macromolecules in the perfusate over the membrane may reduce the biological relevance of the sampling and cause an inflammatory response in the test subject. Therefore, a method to investigate the molecular transport of high MWCO MD is presented. An in vitro test chamber was fabricated to facilitate the fluorescent imaging of the MD sampling process, using fluoresceinylisothiocyanate (FITC) dextran and fluorescence microscopy. Qualitative studies on dextran behavior inside and outside the membrane were performed. Semiquantitative results showed clear dextran leakage from both 40 and 250 kDa dextran when 100 kDa MWCO membranes were used. Dextran 40 kDa leaked out with an order of magnitude higher concentration and the leakage pattern resembled more of a convective flow pattern compared with dextran 250 kDa, where the leakage pattern was more diffusion based. No leakage was observed when dextran 500 kDa was used as a colloid osmotic agent. The results in this study suggest that fluorescence imaging could be used as a method for qualitative and semiquantitative molecular transport and fluid dynamics studies of MD membranes and other hollow fiber catheter membranes.