Integrating Nanosensors into Macroporous Hydrogels for Implantation.

Macroporous hydrogels are an attractive platform for implantable sensors because the network of interconnected macropores facilitates tissue integration. Embedded sensing elements, in our case, plasmonic gold nanoparticles, can transduce the presence, absence, and concentration of biochemical markers to the outside. We present here how to integrate such nanosensors into a macroporous hydrogel while preserving the nanosensor functionality in order to produce implantable sensors. We demonstrate that out of four different polymers, the poly(2-hydroxyethyl methacrylate-poly(ethylene glycole)diacrylate copolymer (pHEMA-PEGDA) results in a working sensor. Our approach of incorporating nanosized sensor elements into a hydrogel matrix generally identifies suitable polymers for implantable sensor systems.

Implantable Sensors Based on Gold Nanoparticles for Continuous Long-Term Concentration Monitoring in the Body.

Implantable sensors continuously transmit information on vital values or biomarker concentrations in bodily fluids, enabling physicians to survey disease progression and monitor therapeutic success. However, currently available technologies still face difficulties with long-term operation and transferability to different analytes. We show the potential of a generalizable platform based on gold nanoparticles embedded in a hydrogel for long-term implanted biosensing. Using optical imaging and an intelligent sensor/reference-design, we assess the tissue concentration of kanamycin in anesthetized rats by interrogating our implanted sensor noninvasively through the skin. Combining a tissue-integrating matrix, robust aptamer receptors, and photostable gold nanoparticles, our technology has strong potential to extend the lifetime of implanted sensors. Because of the easy adaptability of gold nanoparticles toward different analytes, our concept will find versatile applications in personalized medicine or pharmaceutical development.

Warhead Reactivity Limits the Speed of Inhibition of the Cysteine Protease Rhodesain.

Viral and parasitic pathogens rely critically on cysteine proteases for host invasion, replication, and infectivity. Their inhibition by synthetic inhibitors, such as vinyl sulfone compounds, has emerged as a promising treatment strategy. However, the individual reaction steps of protease inhibition are not fully understood. Using the trypanosomal cysteine protease rhodesain as a medically relevant target, we design photoinduced electron transfer (PET) fluorescence probes to detect kinetics of binding of reversible and irreversible vinyl sulfones directly in solution. Intriguingly, the irreversible inhibitor, apart from its unlimited residence time in the enzyme, reacts 5 times faster than the reversible one. Results show that the reactivity of the warhead, and not binding of the peptidic recognition unit, limits the rate constant of protease inhibition. The use of a reversible inhibitor decreases the risk of off-target side effects not only by allowing its release from an off-target but also by reducing the rate constant of binding.

Single Out-of-Resonance Dielectric Nanoparticles as Molecular Sensors.

Light scattering from single nanoparticles and nanostructures is a commonly used readout method for nanosensors. Increasing the spectral sensitivity of resonant nanosensors to changes in their local surrounding has been the focus of many studies. Switching from spectral to intensity monitoring allows one to investigate nonresonant or out-of-resonance dielectric nanoparticles. Here, we systematically compared such dielectric silica nanoparticles with plasmonic gold nanorods by deriving analytical expressions and by performing experiments. The experiments show a similar sensitivity for the detection of an adsorbate layer for both particle types, which is in good agreement with theory. The flat spectral response of dielectric silica nanoparticles simplifies the choice of illumination wavelength. Furthermore, such dielectric nanoparticles can be made from many oxides, polymers, and even biological assemblies, broadening the choice of materials for the nanosensor.

Intensity-Based Single Particle Plasmon Sensing.

Plasmon sensors respond to local changes of their surrounding environment with a shift in their resonance wavelength. This response is usually detected by measuring light scattering spectra to determine the resonance wavelength. However, single wavelength detection has become increasingly important because it simplifies the setup, increases speed, and improves statistics. Therefore, we investigated theoretically how the sensitivity toward such single wavelength scattering intensity changes depend on the material and shape of the plasmonic sensor. Surprisingly, simple equations describe this intensity sensitivity very accurately and allow us to distinguish the various contributions: Rayleigh scattering, dielectric contrast, plasmon shift, and frequency-dependent plasmon bulk damping. We find very good agreement of theoretical predictions and experimental data obtained by single particle spectroscopy.

Plasmonic Nanosensors for the Label-Free Imaging of Dynamic Protein Patterns.

We introduce a new approach to monitor the dynamics and spatial patterns of biological molecular assemblies. Our molecular imaging method relies on plasmonic gold nanoparticles as point-like detectors and requires no labeling of the molecules. We show spatial resolution of up to 5 µm and 30 ms temporal resolution, which is comparable to wide-field fluorescence microscopy, while requiring only readily available gold nanoparticles and a dark-field optical microscope. We demonstrate the method on MinDE proteins attaching to and detaching from lipid membranes of different composition for 24 h. We foresee our new imaging method as an indispensable tool in advanced molecular biology and biophysics laboratories around the world.

Interfacial States Cause Equal Decay of Plasmons and Hot Electrons at Gold-Metal Oxide Interfaces.

We compare the decay of plasmons and "conventional" hot electrons within the same series of gold/metal oxide interfaces. We found an accelerated decay of hot electrons at gold-metal oxide interfaces with decreasing band gap of the oxide material. The decay is accelerated by the increased phase space for electron scattering caused by additional interfacial states. Since plasmons decay faster within the same series of gold-metal oxide interfaces, we propose plasmons are able to decay into the same interfacial states as hot electrons. The similarity of plasmon damping to conventional hot electron decay implies that many classical surface analysis techniques and theoretical concepts are transferable to plasmonic systems. Our results support the mechanism of direct decay of plasmons into interfacial hot electron pairs but the utility of these interfacial states for charge transfer reactions remains to be investigated.

Structural and mechanistic insights into the interaction of the circadian transcription factor BMAL1 with the KIX domain of the CREB-binding protein.

The mammalian CLOCK:BMAL1 transcription factor complex and its coactivators CREB-binding protein (CBP)/p300 and mixed-lineage leukemia 1 (MLL1) critically regulate circadian transcription and chromatin modification. Circadian oscillations are regulated by interactions of BMAL1's C-terminal transactivation domain (TAD) with the KIX domain of CBP/p300 (activating) and with the clock protein CRY1 (repressing) as well as by the BMAL1 G-region preceding the TAD. Circadian acetylation of Lys537 within the G-region enhances repressive BMAL1-TAD-CRY1 interactions. Here, we characterized the interaction of the CBP-KIX domain with BMAL1 proteins, including the BMAL1-TAD, parts of the G-region, and Lys537 Tethering the small compound 1-10 in the MLL-binding pocket of the CBP-KIX domain weakened BMAL1 binding, and MLL1-bound KIX did not form a ternary complex with BMAL1, indicating that the MLL-binding pocket is important for KIX-BMAL1 interactions. Small-angle X-ray scattering (SAXS) models of BMAL1 and BMAL1:KIX complexes revealed that the N-terminal BMAL1 G-region including Lys537 forms elongated extensions emerging from the bulkier BMAL1-TAD:KIX core complex. Fitting high-resolution KIX domain structures into the SAXS-derived envelopes suggested that the G-region emerges near the MLL-binding pocket, further supporting a role of this pocket in BMAL1 binding. Additionally, mutations in the second CREB-pKID/c-Myb-binding pocket of the KIX domain moderately impacted BMAL1 binding. The BMAL1(K537Q) mutation mimicking Lys537 acetylation, however, did not affect the KIX-binding affinity, in contrast to its enhancing effect on CRY1 binding. Our results significantly advance the mechanistic understanding of the protein interaction networks controlling CLOCK:BMAL1- and CBP-dependent gene regulation in the mammalian circadian clock.

Plasmon damping depends on the chemical nature of the nanoparticle interface.

The chemical nature of surface adsorbates affects the localized surface plasmon resonance of metal nanoparticles. However, classical electromagnetic simulations are blind to this effect, whereas experiments are typically plagued by ensemble averaging that also includes size and shape variations. In this work, we are able to isolate the contribution of surface adsorbates to the plasmon resonance by carefully selecting adsorbate isomers, using single-particle spectroscopy to obtain homogeneous linewidths, and comparing experimental results to high-level quantum mechanical calculations based on embedded correlated wavefunction theory. Our approach allows us to indisputably show that nanoparticle plasmons are influenced by the chemical nature of the adsorbates 1,7-dicarbadodecaborane(12)-1-thiol (M1) and 1,7-dicarbadodecaborane(12)-9-thiol (M9). These surface adsorbates induce inside the metal electric dipoles that act as additional scattering centers for plasmon dephasing. In contrast, charge transfer from the plasmon to adsorbates-the most widely suggested mechanism to date-does not play a role here.

Plasmonic Nanosensors Reveal a Height Dependence of MinDE Protein Oscillations on Membrane Features.

Single-particle plasmon spectroscopy has become a standard technique to detect and quantify the presence of unlabeled macromolecules. Here, we extend this method to determine their exact distance from the plasmon sensors with sub-nanometer resolution by systematically varying the sensing range into the surrounding by adjusting the size of the plasmonic nanoparticles. We improved current single-particle plasmon spectroscopy to record continuously for hours the scattering spectra of thousands of nanoparticles of different sizes simultaneously with 1.8 s time resolution. We apply this technique to study the interaction dynamics of bacterial Min proteins with supported lipid membranes of different composition. Our experiments reveal a surprisingly flexible operating mode of the Min proteins: In the presence of cardiolipin and membrane curvature induced by nanoparticles, the protein oscillation occurs on top of a stationary MinD patch. Our results reveal the need to consider membrane composition and local curvature as important parameters to quantitatively understand the Min protein system and could be extrapolated to other macromolecular systems. Our label-free method is generally easily implementable and well suited to measure distances of interacting biological macromolecules.

Conformational Dynamics of a Single Protein Monitored for 24 h at Video Rate.

We use plasmon rulers to follow the conformational dynamics of a single protein for up to 24 h at a video rate. The plasmon ruler consists of two gold nanospheres connected by a single protein linker. In our experiment, we follow the dynamics of the molecular chaperone heat shock protein 90 (Hsp90), which is known to show "open" and "closed" conformations. Our measurements confirm the previously known conformational dynamics with transition times in the second to minute time scale and reveals new dynamics on the time scale of minutes to hours. Plasmon rulers thus extend the observation bandwidth 3-4 orders of magnitude with respect to single-molecule fluorescence resonance energy transfer and enable the study of molecular dynamics with unprecedented precision.

Synthesis of Au-CdS@CdSe Hybrid Nanoparticles with a Highly Reactive Gold Domain.

We propose a novel route to synthesize semiconductor-gold hybrid nanoparticles directly in water, resulting in much larger gold domains than previous protocols (up to 50 nm) with very reactive surfaces which allow further functionalization. This method advances the possibility of self-assembly into complex structures with catalytic activity toward the reduction of nitro compounds by hydrides. The large size of these gold domains in hybrid particles supports efficient light scattering at the plasmon resonance frequency, making such structures attractive for single-particle studies.

Momentum Distribution of Electrons Emitted from Resonantly Excited Individual Gold Nanorods.

Electron emission by femtosecond laser pulses from individual Au nanorods is studied with a time-of-flight momentum resolving photoemission electron microscope (ToF k-PEEM). The Au nanorods adhere to a transparent indium-tin oxide substrate, allowing for illumination from the rear side at normal incidence. Localized plasmon polaritons are resonantly excited at 800 nm with 100 fs long pulses. The momentum distribution of emitted electrons reveals two distinct emission mechanisms: a coherent multiphoton photoemission process from the optically heated electron gas leads to an isotropic emission distribution. In contrast, an additional emission process resulting from the optical field enhancement at both ends of the nanorod leads to a strongly directional emission parallel to the nanorod's long axis. The relative intensity of both contributions can be controlled by the peak intensity of the incident light.

Chemical Interface Damping Depends on Electrons Reaching the Surface.

Metallic nanoparticles show extraordinary strong light absorption near their plasmon resonance, orders of magnitude larger compared to nonmetallic nanoparticles. This "antenna" effect has recently been exploited to transfer electrons into empty states of an attached material, for example to create electric currents in photovoltaic devices or to induce chemical reactions. It is generally assumed that plasmons decay into hot electrons, which then transfer to the attached material. Ultrafast electron-electron scattering reduces the lifetime of hot electrons drastically in metals and therefore strongly limits the efficiency of plasmon induced hot electron transfer. However, recent work has revived the concept of plasmons decaying directly into an interfacial charge transfer state, thus avoiding the intermediate creation of hot electrons. This direct decay mechanism has mostly been neglected, and has been termed chemical interface damping (CID). CID manifests itself as an additional damping contribution to the homogeneous plasmon line width. In this study, we investigate the size dependence of CID by following the plasmon line width of gold nanorods during the adsorption process of thiols on the gold surface with single particle spectroscopy. We show that CID scales inversely with the effective path length of electrons, i.e., the average distance of electrons to the surface. Moreover, we compare the contribution of CID to other competing plasmon decay channels and predict that CID becomes the dominating plasmon energy decay mechanism for very small gold nanorods.

Plasmonic Nanosensors for the Determination of Drug Effectiveness on Membrane Receptors.

We demonstrate the potential of the NanoSPR (nanoscale surface plasmon resonance sensors) method as a simple and cheap tool for the quantitative study of membrane protein-protein interactions. We use NanoSPR to determine the effectiveness of two potential drug candidates that inhibit the protein complex formation between FtsA and ZipA at initial stages of bacterial division. As the NanoSPR method relies on individual gold nanorods as sensing elements, there is no need for fluorescent labels or organic cosolvents, and it provides intrinsically high statistics. NanoSPR could become a powerful tool in drug development, drug delivery, and membrane studies.

Gold Nanorods as Plasmonic Sensors for Particle Diffusion.

Plasmonic gold nanoparticles are normally used as sensor to detect analytes permanently bound to their surface. If the interaction between the analyte and the nanosensor surface is negligible, it only diffuses through the sensor's sensing volume, causing a small temporal shift of the plasmon resonance position. By using a very sensitive and fast detection scheme, we are able to detect these small fluctuations in the plasmon resonance. With the help of a theoretical model consistent with our detection geometry, we determine the analyte's diffusion coefficient. The method is verified by observing the trends upon changing diffusor size and medium viscosity, and the diffusion coefficients obtained were found to reflect reduced diffusion close to a solid interface. Our method, which we refer to as NanoPCS (for nanoscale plasmon correlation spectroscopy), is of practical importance for any application involving the diffusion of analytes close to nanoparticles.

Organization into Higher Ordered Ring Structures Counteracts Membrane Binding of IM30, a Protein Associated with Inner Membranes in Chloroplasts and Cyanobacteria.

The IM30 (inner membrane-associated protein of 30 kDa), also known as the Vipp1 (vesicle-inducing protein in plastids 1), has a crucial role in thylakoid membrane biogenesis and maintenance. Recent results suggest that the protein binds peripherally to membranes containing negatively charged lipids. However, although IM30 monomers interact and assemble into large oligomeric ring complexes with different numbers of monomers, it is still an open question whether ring formation is crucial for membrane interaction. Here we show that binding of IM30 rings to negatively charged phosphatidylglycerol membrane surfaces results in a higher ordered membrane state, both in the head group and in the inner core region of the lipid bilayer. Furthermore, by using gold nanorods covered with phosphatidylglycerol layers and single particle spectroscopy, we show that not only IM30 rings but also lower oligomeric IM30 structures interact with membranes, although with higher affinity. Thus, ring formation is not crucial for, and even counteracts, membrane interaction of IM30.

Single Particle Plasmon Sensors as Label-Free Technique To Monitor MinDE Protein Wave Propagation on Membranes.

We use individual gold nanorods as pointlike detectors for the intrinsic dynamics of an oscillating biological system. We chose the pattern forming MinDE protein system from Escherichia coli (E. coli), a prominent example for self-organized chemical oscillations of membrane-associated proteins that are involved in the bacterial cell division process. Similar to surface plasmon resonance (SPR), the gold nanorods report changes in their protein surface coverage without the need for fluorescence labeling, a technique we refer to as NanoSPR. Comparing the dynamics for fluorescence labeled and unlabeled proteins, we find a reduction of the oscillation period by about 20%. The absence of photobleaching allows us to investigate Min proteins attaching and detaching from lipid coated gold nanorods with an unprecedented bandwidth of 100 ms time resolution and 1 h observation time. The long observation reveals small changes of the oscillation period over time. Averaging many cycles yields the precise wave profile that exhibits the four phases suggested in previous reports. Unexpected from previous fluorescence-based studies, we found an immobile static protein layer not dissociating during the oscillation cycle. Hence, NanoSPR is an attractive label-free real-time technique for the local investigation of molecular dynamics with high observation bandwidth. It gives access to systems, which cannot be fluorescently labeled, and resolves local dynamics that would average out over the sensor area used in conventional SPR.

The role of halide ions in the anisotropic growth of gold nanoparticles: a microscopic, atomistic perspective.

We provide a microscopic view of the role of halides in controlling the anisotropic growth of gold nanorods through a combined computational and experimental study. Atomistic molecular dynamics simulations unveil that Br(-) adsorption is not only responsible for surface passivation, but also acts as the driving force for CTAB micelle adsorption and stabilization on the gold surface in a facet-dependent way. The partial replacement of Br(-) by Cl(-) decreases the difference between facets and the surfactant density. Finally, in the CTAC solution, no halides or micellar structures protect the gold surface and further gold reduction should be uniformly possible. Experimentally observed nanoparticle's growth in different CTAB/CTAC mixtures is more uniform and faster as the amount of Cl(-) increases, confirming the picture from the simulations. In addition, the surfactant layer thickness measured on nanorods exposed to CTAB and CTAC quantitatively agrees with the simulation results.

Comparative evaluation of the impact on endothelial cells induced by different nanoparticle structures and functionalization.

In the research field of nanoparticles, many studies demonstrated a high impact of the shape, size and surface charge, which is determined by the functionalization, of nanoparticles on cell viability and internalization into cells. This work focused on the comparison of three different nanoparticle types to give a better insight into general rules determining the biocompatibility of gold, Janus and semiconductor (quantum dot) nanoparticles. Endothelial cells were subject of this study, since blood is the first barrier after intravenous nanoparticle application. In particular, stronger effects on the viability of endothelial cells were found for nanoparticles with an elongated shape in comparison to spherical ones. Furthermore, a positively charged nanoparticle surface (NH2, CyA) leads to the strongest reduction in cell viability, whereas neutral and negatively charged nanoparticles are highly biocompatible to endothelial cells. These findings are attributed to a rapid internalization of the NH2-functionalized nanoparticles in combination with the damage of intracellular membranes. Interestingly, the endocytotic pathway seems to be a size-dependent process whereas nanoparticles with a size of 20 nm are internalized by caveolae-mediated endocytosis and nanoparticles with a size of 40 nm are taken up by clathrin-mediated internalization and macropinocytosis. Our results can be summarized to formulate five general rules, which are further specified in the text and which determine the biocompatibility of nanoparticles on endothelial cells. Our findings will help to design new nanoparticles with optimized properties concerning biocompatibility and uptake behavior with respect to the respective intended application.