Dual Contributions of Analyte Adsorption and Electroosmotic Inhomogeneity to Separation Efficiency in Capillary Electrophoresis of Proteins.

Improving separation efficiency in capillary electrophoresis (CE) requires systematic study of the influence of the electric field (or solute linear velocity) on plate height for a better understanding of the critical parameters controlling peak broadening. Even for poly(diallyldimethylammonium chloride) (PDADMAC)/poly(sodium styrenesulfonate) (PSS) successive multiple ionic-polymer layer (SMIL) coatings, which lead to efficient and reproducible separations of proteins, plate height increases with migration velocity, limiting the use of high electric fields in CE. Solute adsorption onto the capillary wall was generally considered as the main source of peak dispersion, explaining this plate height increase. However, experiments done with Taylor dispersion analysis and CE in the same conditions indicate that other phenomena may come into play. Protein adsorption with slow kinetics and few adsorption sites was established as a source of peak broadening for specific proteins. Surface charge inhomogeneity was also identified as a contribution to plate height due to local electroosmotic fluctuations. A model was proposed and applied to partial PDADMAC/poly(ethylene oxide) capillary coatings as well as PDADMAC/PSS SMIL coatings. Atomic force microscopy with topography and recognition imaging enabled the determination of roughness and charge distribution of the PDADMAC/PSS SMIL surface.

Investigation of Peptides for Molecular Recognition of C-Reactive Protein-Theoretical and Experimental Studies.

We investigate the interactions between C-reactive protein (CRP) and new CRP-binding peptide materials using experimental (biological and physicochemical) methods with the support of theoretical simulations (computational modeling analysis). Three specific CRP-binding peptides (P2, P3, and P9) derived from an M13 bacteriophage have been identified using phage-display technology. The binding efficiency of the peptides exposed on phages toward the CRP protein was demonstrated via biological methods. Fibers of the selected phages/peptides interact differently due to different compositions of amino acid sequences on the exposed peptides, which was confirmed by transmission electron microscopy. Numerical and experimental studies consistently showed that the P3 peptide is the best CRP binder. A combination of theoretical and experimental methods demonstrates that identifying the best binder can be performed simply, cheaply, and fast. Such an approach has not been reported previously for peptide screening and demonstrates a new trend in science where calculations can replace or support laborious experimental techniques. Finally, the best CRP binder─the P3 peptide─was used for CRP recognition on silicate-modified indium tin oxide-coated glass electrodes. The obtained electrodes exhibit a wide range of operation (1.0-100 µg mL-1) with a detection limit (LOD = 3σ/S) of 0.34 µg mL-1. Moreover, the dissociation constant Kd of 4.2 ± 0.144 µg mL-1 (35 ± 1.2 nM) was evaluated from the change in the current. The selectivity of the obtained electrode was demonstrated in the presence of three interfering proteins. These results prove that the presented P3 peptide is a potential candidate as a receptor for CRP, which can replace specific antibodies.

DNA building blocks for AFM tip functionalization: An easy, fast and stable strategy.

Biosensing atomic force microscopy (AFM) offers the unique feature to determine the energy landscape of a bimolecular interaction at the real single molecule level. Furthermore, simultaneous and label-free mapping of molecular recognition and the determination of sample topography at the nanoscale gets possible. A prerequisite and one of the major parts in biosensing AFM are the bio-functionalized AFM tips. In the past decades, different approaches for tip functionalization have been developed. Using these functionalization strategies, several biological highly relevant interactions at the single molecule level have been explored. For the most common approach, the use of a heterobifunctional poly(ethylenglycol) crosslinker, a broad range of linkers for different chemical coupling strategies is available. Nonetheless, the time consuming functionalization protocol as well as the broad distribution of rupture length reduces the possibility of automation and may reduce the accuracy of the results. Here we present a stable and fast forward approach based on tetra-functional DNA tetrahedra. A fast functionalization and a sharp defined distribution of rupture length gets possible with low effort and high success rate. We tested the performance on the classical avidin biotin system by using tetrahedra with three disulfide legs for stable and site directed coupling to gold coated tips and a biotinylated end at the fourth vertex. A special advantage appears when working with a DNA aptamer as sensing molecule. In this case, the fourth strand can be extended by a certain DNA sequence complementary to the linkage part of an aptamer. This AFM tip functionalization protocol was applied on thrombin using DNA aptamers directed against the fibrinogen binding side of human thrombin.

Quantifying biomolecular hydrophobicity: Single molecule force spectroscopy of class II hydrophobins.

Hydrophobins are surface-active proteins produced by filamentous fungi. The amphiphilic structure of hydrophobins is very compact, containing a distinct hydrophobic patch on one side of the molecule, locked by four intramolecular disulfide bridges. Hydrophobins form dimers and multimers in solution to shield these hydrophobic patches from water exposure. Multimer formation in solution is dynamic, and hydrophobin monomers can be exchanged between multimers. Unlike class I hydrophobins, class II hydrophobins assemble into highly ordered films at the air-water interface. In order to increase our understanding of the strength and nature of the interaction between hydrophobins, we used atomic force microscopy for single molecule force spectroscopy to explore the molecular interaction forces between class II hydrophobins from Trichoderma reesei under different environmental conditions. A genetically engineered hydrophobin variant, NCys-HFBI, enabled covalent attachment of proteins to the apex of the atomic force microscopy cantilever tip and sample surfaces in controlled orientation with sufficient freedom of movement to measure molecular forces between hydrophobic patches. The measured rupture force between two assembled hydrophobins was ∼31 pN, at a loading rate of 500 pN/s. The results indicated stronger interaction between hydrophobins and hydrophobic surfaces than between two assembling hydrophobin molecules. Furthermore, this interaction was stable under different environmental conditions, which demonstrates the dominance of hydrophobicity in hydrophobin-hydrophobin interactions. This is the first time that interaction forces between hydrophobin molecules, and also between naturally occurring hydrophobic surfaces, have been measured directly at a single-molecule level.

High-Sensitivity Dual Electrochemical QCM for Reliable Three-Electrode Measurements.

An electrochemical quartz crystal microbalance (EC-QCM) is a versatile gravimetric technique that allows for parallel characterization of mass deposition and electrochemical properties. Despite its broad applicability, simultaneous characterization of two electrodes remains challenging due to practical difficulties posed by the dampening from fixture parasitics and the dissipative medium. In this study, we present a dual electrochemical QCM (dual EC-QCM) that is employed in a three-electrode configuration to enable consequent monitoring of mass deposition and viscous loading on two crystals, the working electrode (WE) and the counter electrode (CE). A novel correction approach, along with a three standard complex impedance calibration, is employed to overcome the effect of dampening while keeping high spectral sensitivity. Separation of viscous loading and rigid mass deposition is achieved by robust characterization of the complex impedance at the resonance frequency. Validation of the presented system is done by cyclic voltammetry characterization of Ag underpotential deposition on gold. The results indicate mass deposition of 412.2 ng for the WE and 345.6 ng for the CE, reflecting a difference of the initially-present Ag adhered to the surface. We also performed higher harmonic measurements that further corroborate the sensitivity and reproducibility of the dual EC-QCM. The demonstrated approach is especially intriguing for electrochemical energy storage applications where mass detection with multiple electrodes is desired.

Control of Ligand-Binding Specificity Using Photocleavable Linkers in AFM Force Spectroscopy.

In recent decades, atomic force microscopy (AFM), in particular the force spectroscopy mode, has become a method of choice to study biomolecular interactions at the single-molecule level. However, grafting procedures as well as determining binding specificity remain challenging. We report here an innovative approach based on a photocleavable group that enables in situ release of the ligands bound to the AFM tip and thus allows direct assessment of the binding specificity. Applicable to a wide variety of molecules, the strategy presented here provides new opportunities to study specific interactions and deliver single molecules with high spatiotemporal resolution in a wide range of applications, including AFM-based cell biology.

Atomic Force Microscopy Imaging in Turbid Liquids: A Promising Tool in Nanomedicine.

Tracking of biological and physiological processes on the nanoscale is a central part of the growing field of nanomedicine. Although atomic force microscopy (AFM) is one of the most appropriate techniques in this area, investigations in non-transparent fluids such as human blood are not possible with conventional AFMs due to limitations caused by the optical readout. Here, we show a promising approach based on self-sensing cantilevers (SSC) as a replacement for optical readout in biological AFM imaging. Piezo-resistors, in the form of a Wheatstone bridge, are embedded into the cantilever, whereas two of them are placed at the bending edge. This enables the deflection of the cantilever to be precisely recorded by measuring the changes in resistance. Furthermore, the conventional acoustic or magnetic vibration excitation in intermittent contact mode can be replaced by a thermal excitation using a heating loop. We show further developments of existing approaches enabling stable measurements in turbid liquids. Different readout and excitation methods are compared under various environmental conditions, ranging from dry state to human blood. To demonstrate the applicability of our laser-free bio-AFM for nanomedical research, we have selected the hemostatic process of blood coagulation as well as ultra-flat red blood cells in different turbid fluids. Furthermore, the effects on noise and scanning speed of different media are compared. The technical realization is shown (1) on a conventional optical beam deflection (OBD)-based AFM, where we replaced the optical part by a new SSC nose cone, and (2) on an all-electric AFM, which we adapted for measurements in turbid liquids.

Characterization of the specific interaction between the DNA aptamer sgc8c and protein tyrosine kinase-7 receptors at the surface of T-cells by biosensing AFM.

We studied the interaction of the specific DNA aptamer sgc8c immobilized at the AFM tip with its corresponding receptor, the protein tyrosine kinase-7 (PTK7) embedded in the membrane of acute lymphoblastic leukemia (ALL) cells (Jurkat T-cells). Performing single molecule force spectroscopy (SMFS) experiments, we showed that the aptamer sgc8c bound with high probability (38.3 ± 7.48%) and high specificity to PTK7, as demonstrated by receptor blocking experiments and through comparison with the binding behavior of a nonspecific aptamer. The determined kinetic off-rate (koff = 5.16 s-1) indicates low dissociation of the sgc8c-PTK7 complex. In addition to the pulling force experiments, simultaneous topography and recognition imaging (TREC) experiments using AFM tips functionalized with sgc8c aptamers were realized on the outer regions surface of surface-immobilized Jurkat cells for the first time. This allowed determination of the distribution of PTK7 without any labeling and at near physiological conditions. As a result, we could show a homogeneous distribution of PTK7 molecules on the outer regions of ALL cells with a surface density of 325 ± 12 PTK7 receptors (or small receptor clusters) per µm2. Graphical Abstract The specific interaction of the DNA aptamer sgc8c and protein tyrosine kinase-7 (PTK7) on acute lymphoblastic leukemia (ALL) cells was characterized. AFM based single molecule force spectroscopy (SMFS) yielded a kinetic off-rate of 5.16 s-1 of the complex. Simultaneous topography and recognition imaging (TREC) revealed a PTK7 density of 325 ± 12 molecules or clusters per µm2 in the cell membrane.

Investigating the binding behaviour of two avidin-based testosterone binders using molecular recognition force spectroscopy.

Molecular recognition force spectroscopy, a biosensing atomic force microscopy technique allows to characterise the dissociation of ligand-receptor complexes at the molecular level. Here, we used molecular recognition force spectroscopy to study the binding capability of recently developed testosterone binders. The two avidin-based proteins called sbAvd-1 and sbAvd-2 are expected to bind both testosterone and biotin but differ in their binding behaviour towards these ligands. To explore the ligand binding and dissociation energy landscape of these proteins, we tethered biotin or testosterone to the atomic force microscopy probe while the testosterone-binding protein was immobilized on the surface. Repeated formation and rupture of the ligand-receptor complex at different pulling velocities allowed determination of the loading rate dependence of the complex-rupturing force. In this way, we obtained the molecular dissociation rate (k(off)) and energy landscape distances (x(ß)) of the four possible complexes: sbAvd-1-biotin, sbAvd-1-testosterone, sbAvd-2-biotin and sbAvd-2-testosterone. It was found that the kinetic off-rates for both proteins and both ligands are similar. In contrast, the x(ß) values, as well as the probability of complex formations, varied considerably. In addition, competitive binding experiments with biotin and testosterone in solution differ significantly for the two testosterone-binding proteins, implying a decreased cross-reactivity of sbAvd-2. Unravelling the binding behaviour of the investigated testosterone-binding proteins is expected to improve their usability for possible sensing applications.

Activation induced morphological changes and integrin αIIbß3 activity of living platelets.

Platelets are essential in hemostasis. Upon activation they undergo a shape-change accompanied with receptor presentation. Atomic force microscopy (AFM) imaging and single molecule force spectroscopy (SMFS) were used as powerful tools for exploring morphological changes as well as receptor activities of platelets. Imaging time series was accomplished with and without fixation steps at the single platelet level. Hereby the response of mechanical stimulation of the platelet by the AFM cantilever tip was directly observed. We demonstrate that living and fixed platelets develop filopodia after a short activation time followed by their disappearance including cellular bleb formation. Thereafter a second filopodia formation (filopodia extrusion) was observed; those filopodia subsequently disappeared again, and finally platelets detached from the support due to cell death. We determined the influence of mechanical stress on the chronology of morphological changes of platelets and demonstrated shear force induced filopodia formation. Through recordings over several hours, topographical AFM images over the full platelet lifetime - from early activation up to apoptosis - are presented. SMFS measurements on living platelets allowed determining the activation state of the most prominent membrane receptor integrin αIIbß3 at all different phases of activation. αIIbß3 was fully activated, independent of the morphological state.

Kinetics of bioconjugate nanoparticle label binding in a sandwich-type immunoassay.

Nanoparticle labels have enhanced the performance of diagnostic, screening, and other measurement applications and hold further promise for more sensitive, precise, and cost-effective assay technologies. Nevertheless, a clear view of the biomolecular interactions on the molecular level is missing. Controlling the ratio of molecular recognition over undesired nonspecific adhesion is the key to improve biosensing with nanoparticles. To improve this ratio with an aim to disallow nonspecific binding, a more detailed perspective into the kinetic differences between the cases is needed. We present the application of two novel methods to determine complex binding kinetics of bioconjugate nanoparticles, interferometry, and force spectroscopy. Force spectroscopy is an atomic force microscopy technique and optical interferometry is a direct method to monitor reaction kinetics in second-hour timescale, both having steadily increasing importance in nanomedicine. The combination is perfectly suited for this purpose, due to the high sensitivity to detect binding events and the ability to investigate biological samples under physiological conditions. We have attached a single biofunctionalized nanoparticle to the outer tip apex and studied the binding behavior of the nanoparticle in a sandwich-type immunoassay using dynamic force spectroscopy in millisecond timescale. Utilization of the two novel methods allowed characterization of binding kinetics in a time range spanning from 50 ms to 4 h. These experiments allowed detection and demonstration of differences between specific and nonspecific binding. Most importantly, nonspecific binding of a nanoparticle was reduced at contact times below 100 ms with the solid-phase surface.

pH-dependent deformations of the energy landscape of avidin-like proteins investigated by single molecule force spectroscopy.

Avidin and avidin-like proteins are widely used in numerous techniques since the avidin-biotin interaction is known to be very robust and reliable. Within this study, we investigated this bond at the molecular level under harsh conditions ranging from very low to very high pH values. We compared avidin with streptavidin and a recently developed avidin-based mutant, chimeric avidin. To gain insights of the energy landscape of these interactions we used a single molecule approach and performed the Single Molecule Force Spectroscopy atomic force microscopy technique. There, the ligand (biotin) is covalently coupled to a sharp AFM tip via a distensible hetero-bi-functional crosslinker, whereas the receptor of interest is immobilized on the probe surface. Receptor-ligand complexes are formed and ruptured by repeatedly approaching and withdrawing the tip from the surface. Varying both pulling velocity and pH value, we could determine changes of the energy landscape of the complexes. Our results clearly demonstrate that avidin, streptavidin and chimeric avidin are stable over a wide pH range although we could identify differences at the outer pH range. Taking this into account, they can be used in a broad range of applications, like surface sensors at extreme pH values.

Mapping the nucleotide binding site of uncoupling protein 1 using atomic force microscopy.

A tight regulation of proton transport in the inner mitochondrial membrane is crucial for physiological processes such as ATP synthesis, heat production, or regulation of the reactive oxygen species as proposed for the uncoupling protein family members (UCP). Specific regulation of proton transport is thus becoming increasingly important in the therapy of obesity and inflammatory, neurodegenerative, and ischemic diseases. We and other research groups have shown previously that UCP1- and UCP2-mediated proton transport is inhibited by purine nucleotides. Several hypotheses have been proposed to explain the inhibitory effect of ATP, although structural details are still lacking. Moreover, the unresolved mystery is how UCP operates in vivo despite the permanent presence of high (millimolar) concentrations of ATP in mitochondria. Here we use the topographic and recognition (TREC) mode of an atomic force microscope to visualize UCP1 reconstituted into lipid bilayers and to analyze the ATP-protein interaction at a single molecule level. The comparison of recognition patterns obtained with anti-UCP1 antibody and ATP led to the conclusion that the ATP binding site can be accessed from both sides of the membrane. Using cantilever tips with different cross-linker lengths, we determined the location of the nucleotide binding site inside the membrane with 1 Å precision. Together with the recently published NMR structure of a UCP family member (Berardi et al. Nature, 2011, 476, 109-113), our data provide a valuable insight into the mechanism of the nucleotide binding and pave the way for new pharmacological approaches against the diseases mentioned above.

Reversible biofunctionalization of surfaces with a switchable mutant of avidin.

Label-free biosensors detect binding of prey molecules (″analytes″) to immobile bait molecules on the sensing surface. Numerous methods are available for immobilization of bait molecules. A convenient option is binding of biotinylated bait molecules to streptavidin-functionalized surfaces, or to biotinylated surfaces via biotin-avidin-biotin bridges. The goal of this study was to find a rapid method for reversible immobilization of biotinylated bait molecules on biotinylated sensor chips. The task was to establish a biotin-avidin-biotin bridge which was easily cleaved when desired, yet perfectly stable under a wide range of measurement conditions. The problem was solved with the avidin mutant M96H which contains extra histidine residues at the subunit-subunit interfaces. This mutant was bound to a mixed self-assembled monolayer (SAM) containing biotin residues on 20% of the oligo(ethylene glycol)-terminated SAM components. Various biotinylated bait molecules were bound on top of the immobilized avidin mutant. The biotin-avidin-biotin bridge was stable at pH ≥3, and it was insensitive to sodium dodecyl sulfate (SDS) at neutral pH. Only the combination of citric acid (2.5%, pH 2) and SDS (0.25%) caused instantaneous cleavage of the biotin-avidin-biotin bridge. As a consequence, the biotinylated bait molecules could be immobilized and removed as often as desired, the only limit being the time span for reproducible chip function when kept in buffer (2-3 weeks at 25 °C). As expected, the high isolectric pH (pI) of the avidin mutant caused nonspecific adsorption of proteins. This problem was solved by acetylation of avidin (to pI < 5), or by optimization of SAM formation and passivation with biotin-BSA and BSA.

Targeted delivery of siRNA into breast cancer cells via phage fusion proteins.

Nucleic acids, including antisense oligonucleotides, small interfering RNA (siRNA), aptamers, and rybozymes, emerged as versatile therapeutics due to their ability to interfere in a well-planned manner with the flow of genetic information from DNA to protein. However, a systemic use of NAs is hindered by their instability in physiological liquids and inability of intracellular accumulation in the site of action. We first evaluated the potential of cancer specific phage fusion proteins as targeting ligands that provide encapsulation, protection, and navigation of siRNA to the target cell. The tumor-specific proteins were isolated from phages that were affinity selected from a landscape phage library against target breast cancer cells. It was found that fusion phage coat protein fpVIII displaying cancer-targeting peptides can effectively encapsulate siRNAs and deliver them into the cells leading to specific silencing of the model gene GAPDH. Complexes of siRNA and phage protein form nanoparticles (nanophages), which were characterized by atomic force microscopy and ELISA, and their stability was demonstrated by resistance of encapsulated siRNA to degradation by serum nucleases. The phage protein/siRNA complexes can make a new type of highly selective, stable, active, and physiologically acceptable cancer nanomedicine.

Nanoscale DNA tetrahedra improve biomolecular recognition on patterned surfaces.

The bottom-up approach of DNA nano-biotechnology can create biomaterials with defined properties relevant for a wide range of applications. This report describes nanoscale DNA tetrahedra that are beneficial to the field of biosensing and the targeted immobilization of biochemical receptors on substrate surfaces. The DNA nanostructures act as immobilization agents that are able to present individual molecules at a defined nanoscale distance to the solvent thereby improving biomolecular recognition of analytes. The tetrahedral display devices are self-assembled from four oligonucleotides. Three of the four tetrahedron vertices are equipped with disulfide groups to enable oriented binding to gold surfaces. The fourth vertex at the top of the bound tetrahedron presents the biomolecular receptor to the solvent. In assays testing the molecular accessibility via DNA hybridization and protein capturing, tetrahedron-tethered receptors outperformed conventional immobilization approaches with regard to specificity and amount of captured polypeptide by a factor of up to seven. The bottom-up strategy of creating DNA tetrahedrons is also compatible with the top-down route of nanopatterning of inorganic substrates, as demonstrated by the specific coating of micro- to nanoscale gold squares amid surrounding blank or poly(ethylene glycol)-passivated glass surfaces. DNA tetrahedra can create biofunctionalized surfaces of rationally designed properties that are of relevance in analytical chemistry, cell biology, and single-molecule biophysics.

Time-resolved chloroquine-induced relaxation of supercoiled plasmid DNA.

Herein, we report on the in vitro change of DNA conformation of plasmids bound to a 3-aminopropyl-modified mica surface and monitoring the events by atomic force microscopy (AFM) imaging under near physiological conditions. In our study, we used an intercalating drug, chloroquine, which is known to decrease the twist of the double helix and thus altered the conformation of the whole DNA. During our experiments, a chloroquine solution was added while imaging a few highly condensed plasmid nanoparticles in solution. AFM images recorded after the drug addition clearly show a time-resolved relaxation of these bionanoparticles into a mixture of loose DNA strands.

Characterization of enhanced monovalent and bivalent thrombin DNA aptamer binding using single molecule force spectroscopy.

Thrombin aptamer binding strength and stability is dependent on sterical parameters when used for atomic force microscopy sensing applications. Sterical improvements on the linker chemistry were developed for high-affinity binding. For this we applied single molecule force spectroscopy using two enhanced biotinylated thrombin aptamers, BFF and BFA immobilized on the atomic force microscopy tip via streptavidin. BFF is a dimer composed of two single-stranded aptamers (aptabody) connected to each other by a complementary sequence close to the biotinylated end. In contrast, BFA consists of a single DNA strand and a complementary strand in the supporting biotinylated part. By varying the pulling velocity in force-distance cycles the formed thrombin-aptamer complexes were ruptured at different force loadings allowing determination of the energy landscape. As a result, BFA aptamer showed a higher binding force at the investigated loading rates and a significantly lower dissociation rate constant, k(off), compared to BFF. Moreover, the potential of the aptabody BFF to form a bivalent complex could clearly be demonstrated.

Modification of the loops in the ligand-binding site turns avidin into a steroid-binding protein.

BACKGROUND: Engineered proteins, with non-immunoglobulin scaffolds, have become an important alternative to antibodies in many biotechnical and therapeutic applications. When compared to antibodies, tailored proteins may provide advantageous properties such as a smaller size or a more stable structure. RESULTS: Avidin is a widely used protein in biomedicine and biotechnology. To tailor the binding properties of avidin, we have designed a sequence-randomized avidin library with mutagenesis focused at the loop area of the binding site. Selection from the generated library led to the isolation of a steroid-binding avidin mutant (sbAvd-1) showing micromolar affinity towards testosterone (Kd ~ 9 µM). Furthermore, a gene library based on the sbAvd-1 gene was created by randomizing the loop area between ß-strands 3 and 4. Phage display selection from this library led to the isolation of a steroid-binding protein with significantly decreased biotin binding affinity compared to sbAvd-1. Importantly, differential scanning calorimetry and analytical gel-filtration revealed that the high stability and the tetrameric structure were preserved in these engineered avidins. CONCLUSIONS: The high stability and structural properties of avidin make it an attractive molecule for the engineering of novel receptors. This methodology may allow the use of avidin as a universal scaffold in the development of novel receptors for small molecules.

Molecular recognition force spectroscopy: a new tool to tailor targeted nanoparticles.

The density of targeting moieties in a nanoparticle-based gene-delivery system has been shown to play a fundamental role in its vectoring performance. Here, molecular recognition force spectroscopy is proposed as a novel screening tool to optimize the density of targeting moieties of functionalized nanoparticles towards attaining cell-specific interaction. By tailoring the nanoparticle formulation, the unbinding event probability between nanoparticles tethered to an atomic force microscopy tip and neuronal cells is directly correlated to the nanoparticle gene-vectoring capacity. Additionally, new insights into protein-receptor interaction are revealed. This novel approach opens new avenues in the field of nanomedicine.