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.

Video-Rate Switching of High-Reflectivity Hybrid Cavities Spanning All Primary Colors.

Dynamically tunable reflective structural colors are attractive for reflective displays (electronic paper). However, it is challenging to tune a thin layer of structural color across the full red-green-blue (RGB) basis set of colors at video rates and with long-term stability. In this work, this is achieved through a hybrid cavity built from metal-insulator-metal (MIM) "nanocaves" and an electrochromic polymer (PProDOTMe2 ). The reflective colors are modulated by electrochemically doping/dedoping the polymer. Compared with traditional subpixel-based systems, this hybrid structure provides high reflectivity (>40%) due to its "monopixel" nature and switches at video rates. The polymer bistability helps deliver ultralow power consumption (≈2.5 mW cm-2 ) for video display applications and negligible consumption (≈3 µW cm-2 ) for static images, compatible with fully photovoltaic powering. In addition, the color uniformity of the hybrid material is excellent (over cm-2 ) and the scalable fabrication enables large-area production.

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.

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.

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.

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.

Simultaneous, Single-Particle Measurements of Size and Loading Give Insights into the Structure of Drug-Delivery Nanoparticles.

Nanoparticles are a promising solution for delivery of a wide range of medicines and vaccines. Optimizing their design depends on being able to resolve, understand, and predict biophysical and therapeutic properties, as a function of design parameters. While existing tools have made great progress, gaps in understanding remain because of the inability to make detailed measurements of multiple correlated properties. Typically, an average measurement is made across a heterogeneous population, obscuring potentially important information. In this work, we develop and apply a method for characterizing nanoparticles with single-particle resolution. We use convex lens-induced confinement (CLiC) microscopy to isolate and quantify the diffusive trajectories and fluorescent intensities of individual nanoparticles trapped in microwells for long times. First, we benchmark detailed measurements of fluorescent polystyrene nanoparticles against prior data to validate our approach. Second, we apply our method to investigate the size and loading properties of lipid nanoparticle (LNP) vehicles containing silencing RNA (siRNA), as a function of lipid formulation, solution pH, and drug-loading. By taking a comprehensive look at the correlation between the intensity and size measurements, we gain insights into LNP structure and how the siRNA is distributed in the LNP. Beyond introducing an analytic for size and loading, this work allows for future studies of dynamics with single-particle resolution, such as LNP fusion and drug-release kinetics. The prime contribution of this work is to better understand the connections between microscopic and macroscopic properties of drug-delivery vehicles, enabling and accelerating their discovery and development.

Dynamically Tuneable Reflective Structural Coloration with Electroactive Conducting Polymer Nanocavities.

Dynamic control of structural colors across the visible spectrum with high brightness has proven to be a difficult challenge. Here, this is addressed with a tuneable reflective nano-optical cavity that uses an electroactive conducting polymer (poly(thieno[3,4-b]thiophene)) as spacer layer. Electrochemical doping and dedoping of the polymer spacer layer provides reversible tuning of the cavity's structural color throughout the entire visible range and beyond. Furthermore, the cavity provides high peak reflectance that varies only slightly between the reduced and oxidized states of the polymer. The results indicate that the polymer undergoes large reversible thickness changes upon redox tuning, aided by changes in optical properties and low visible absorption. The electroactive cavity concept may find particular use in reflective displays, by opening for tuneable monopixels that eliminate limitations in brightness of traditional subpixel-based systems.

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

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.

Evaluation of the Forsvall biopsy needle in an ex vivo model of transrectal prostate biopsy - a novel needle design with the objective to reduce the risk of post-biopsy infection.

BACKGROUND: Transrectal prostate biopsy (TRbx) transfers colonic bacteria into prostatic tissue, potentially causing infectious complications, including sepsis. Our objective was to determine whether biopsy needle shape, surface properties and sampling mechanism affect the number of bacteria transferred through the colon wall, and evaluate a novel needle with improved properties. METHODS: The standard Tru-Cut biopsy needle used today was evaluated for mechanisms of bacterial transfer in a pilot study. A novel Tru-Cut needle (Forsvall needle prototype) was developed. TRbx was simulated using human colons ex-vivo. Four subtypes of the prototype needle were compared with a standard Tru-Cut needle (BARD 18 G). Prototype and standard needles were used to puncture 4 different colon specimens in 10 randomized sites per colon. Needles were submerged into culture media to capture translocated bacteria. The media was cultured on blood agar and then the total amount of transferred bacteria was calculated for each needle. The primary outcome measure was the percent reduction of bacteria translocated by the prototype needles relative to the standard needle. Secondary outcome measures were the effects of tip design and coating on the percent reduction of translocated bacteria. RESULTS: Prototype needles reduced the number of translocated bacteria by, on average, 96.0% (95% confidence interval 93.0-97.7%; p < 0.001) relative to the standard needle. This percent reduction was not significantly affected by prototype needle tip style or surface coating. CONCLUSIONS: The Forsvall needle significantly reduces colonic bacterial translocation, suggesting that it could reduce infectious complications in prostate biopsy. A clinical trial has been initiated.

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.

A designer FG-Nup that reconstitutes the selective transport barrier of the nuclear pore complex.

Nuclear Pore Complexes (NPCs) regulate bidirectional transport between the nucleus and the cytoplasm. Intrinsically disordered FG-Nups line the NPC lumen and form a selective barrier, where transport of most proteins is inhibited whereas specific transporter proteins freely pass. The mechanism underlying selective transport through the NPC is still debated. Here, we reconstitute the selective behaviour of the NPC bottom-up by introducing a rationally designed artificial FG-Nup that mimics natural Nups. Using QCM-D, we measure selective binding of the artificial FG-Nup brushes to the transport receptor Kap95 over cytosolic proteins such as BSA. Solid-state nanopores with the artificial FG-Nups lining their inner walls support fast translocation of Kap95 while blocking BSA, thus demonstrating selectivity. Coarse-grained molecular dynamics simulations highlight the formation of a selective meshwork with densities comparable to native NPCs. Our findings show that simple design rules can recapitulate the selective behaviour of native FG-Nups and demonstrate that no specific spacer sequence nor a spatial segregation of different FG-motif types are needed to create selective NPCs.

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.

Biochemical Sensing with Nanoplasmonic Architectures: We Know How but Do We Know Why?

Here, the research field of nanoplasmonic sensors is placed under scrutiny, with focus on affinity-based detection using refractive index changes. This review describes how nanostructured plasmonic sensors can deliver unique advantages compared to the established surface plasmon resonance technique, where a planar metal surface is used. At the same time, it shows that these features are actually only useful in quite specific situations. Recent trends in the field are also discussed and some devices that claim extraordinary performance are questioned. It is argued that the most important challenges are related to limited receptor affinity and nonspecific binding rather than instrumental performance. Although some nanoplasmonic sensors may be useful in certain situations, it seems likely that conventional surface plasmon resonance will continue to dominate biomolecular interaction analysis. For detection of analytes in complex samples, plasmonics may be an important tool, but probably not in the form of direct refractometric detection.

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.

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.

Generic high-capacity protein capture and release by pH control.

Techniques for immobilization and release of proteins are of general interest but challenging to develop. Here we show a new method for high-capacity (several µg cm-2) immobilization of proteins in polyelectrolyte brushes by multivalent hydrogen bonds. Upon increasing pH, the proteins are fully released with preserved structure and activity.

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 Sensor Detects Preferential Binding of IRSp53 to Negative Membrane Curvature.

Biosensors based on plasmonic nanostructures are widely used in various applications and benefit from numerous operational advantages. One type of application where nanostructured sensors provide unique value in comparison with, for instance, conventional surface plasmon resonance, is investigations of the influence of nanoscale geometry on biomolecular binding events. In this study, we show that plasmonic "nanowells" conformally coated with a continuous lipid bilayer can be used to detect the preferential binding of the insulin receptor tyrosine kinase substrate protein (IRSp53) I-BAR domain to regions of negative surface curvature, i.e., the interior of the nanowells. Two different sensor architectures with and without an additional niobium oxide layer are compared for this purpose. In both cases, curvature preferential binding of IRSp53 (at around 0.025 nm-1 and higher) can be detected qualitatively. The high refractive index niobium oxide influences the near field distribution and makes the signature for bilayer formation less clear, but the contrast for accumulation at regions of negative curvature is slightly higher. This work shows the first example of analyzing preferential binding of an average-sized and biologically important protein to negative membrane curvature in a label-free manner and in real-time, illustrating a unique application for nanoplasmonic sensors.