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The fluorescence-based detection and counting of single protein molecules after specific binding to antibodies at interfaces is presented. A diode laser was used as the excitation source. The unspecific binding at the interface has been reduced to a level of only 0.1% of the maximum signal level. At present, the detection limit of this molecule-counting process is in the range of [10.sup.-17] mol/L, and the dynamic range of the signal corresponds to 7 orders of magnitude of antigen concentration, but these values are not limiting. As a preliminary application in early-stage diagnosis, we have investigated the detection of a single cardiac actin molecule in human plasma, which is of interest in myocardial infarction diagnosis.

Region-selective chemical modification of nano- and microstructures can unlock a world of novel functional surfaces. However, this small scale makes region selectivity challenging, especially on homogeneous and chemically inert synthetic structures. Here, we report the one-step in situ dynamic synthesis of heterogeneous multicomponent hybrid silicone nanorods (MCH-SNRs). These nanorods bear specific modifiable regions that can be assigned to different positions on-demand and selectively functionalized via a photoinitiated, radical-based thiol-ene click reaction. The distribution of different constituent components with desired properties hinges on the independent growth of the individual segment of the bamboo-shaped structure, which can be tailored by using different precursors under specific reaction conditions. Region selectivity of the functionalization is validated by exploiting wetting transitions along bamboo segments, visualized by confocal microscopy.

The knowledge of droplet friction on liquid-infused surfaces (LIS) is of paramount importance for applications involving liquid manipulation. While the possible dissipation mechanisms are well-understood, the effect of surface texture has thus far been mainly investigated on LIS with highly regular solid topographies. In this work, we aim to address this experimental gap by studying the friction experienced by water droplets on LIS based on both random and regular polysilsesquioxane nanostructures. We show that the available models apply to the tested surfaces, but we observe a previously unreported droplet memory effect: as consecutive droplets travel along the same path, their velocity increases up to a plateau value before returning to the original state after a sufficiently long time. We study the features of this phenomenon by evaluating the motion of droplets when they cross the path of a previous sequence of droplets, discovering that moving droplets create a low-friction trace in their wake, whose size matches their base diameter. Finally, we attribute this to the temporary smoothing out of an initially conformal lubricant layer by means of a Landau-Levich-Derjaguin liquid film deposition behind the moving droplet. The proposed mechanism might apply to any LIS with a conformal lubricant layer.

(Super)antiwetting shielding around engineering materials and protecting them against harsh environmental conditions have been achieved via growing various geometry polysiloxane (or silicone) patterns around them by using a droplet-assisted growth method, where the polymerization takes place inside of the water droplets acting as reaction vessels. The size and distribution of these reaction vessels are the main factors in making different geometry silicone patterns; however, very little is known about the fate of these droplets throughout the polymerization. Here, we propose keeping the relative humidity (% RH) inside the reactor stable throughout the polymerization as a new coating parameter to force the size of the reaction vessel water droplets to be the same for building simply shaped silicone rods with controlled geometry and distribution. In this manner, we grew simple geometry cylindric microrods on surfaces and could tune their length, diameter, inter-rod spacing, and thus the (super)hydrophobicity. Here, we also demonstrate that with changes in the amplitude and stability of the % RH, it is possible to fabricate different (super)hydrophobic nanograsses, conical silicone microrods, and isotropic silicone nanofilaments. The proposed new way of tuning initial and in situ reaction vessel droplet size can be used as a single factor to formulate different geometry silicone patterns with tunable dimensions, leading to different roughness and hydrophobicity. To a certain extent, the droplet size-assisted silicone shaping in this work provides a new way to control the length, diameter, morphology, inter-rod spacing, and thus the (super)hydrophobicity of the silicone patterns, especially those in the shape of simple cylindrical microrods. This control over silicone architecting will help to prepare new (super)hydrophobic coatings with more controlled morphology and thus wettability; on the contrary, it will support surface scientists modeling the connection between surface geometry and (super)antiwetting of such irregular pillared surfaces that remain elusive.

In this work, silicone nanofilament (SNF) coatings were prepared via a droplet-assisted growth and shaping (DAGS) approach, where the preparation of the coatings is allowed under ambient conditions. The application of SNF coatings as catalyst supports for amino moieties from (3-aminopropyl)triethoxysilane (APTES) was investigated. With the optimized coating conditions identified, the Brunauer-Emmett-Teller surface areas of a bare glass filter substrate and bare glass beads after the coating have increased by 5-fold and 16-fold, respectively. The SNF-coated filters were readily functionalized with amino groups via a liquid-phase deposition process, and their catalytic activities for a Knoevenagel reaction were evaluated using a batch reactor and a packed bed reactor. In both reactors, the as-prepared filters demonstrated superior catalytic performance over the functionalized filters without SNF coatings. Notably, the unique flexibility of the SNF coatings allowed the facile preparation of a packed bed reactor and a scalable catalytic system. It is expected that the packed bed system established in this study will support the development and the use of various SNF-supported organocatalysts and catalytic materials., Competing Interests: The authors declare no competing financial interest., (© 2022 The Authors. Published by American Chemical Society.)

Sand is one of the most fundamental construction materials that is of significant importance and widely used for making concrete, plasters, and mortars, and also for filling under floor and basements. Sand-derived functional materials, for instance superhydrophobic sand, which can be used to prepare liquid marble, separate oil-water mixtures, and transport liquids, have recently been a highly topical and promising research field. However, such materials are mainly prepared using valuable surface modification agents via complicated procedures that are difficult for mass-production, which restricted their true applications. Here, we developed a simple, low-cost, and efficient method for the development of sand-based hierarchical micro/nanostructured composite materials with diverse applications. Briefly, micro/nanostructured superhydrophobic sand was synthesized by one-step in situ growth of a network layer of silicone nanofilaments on the surface of sand microparticles, using only one cheap chemical of small molecules of silanes. The as-prepared superhydrophobic sand displays excellent performance in waterproofing, water storage, soil moisturizing, and oil-water separation. Furthermore, sand-supported micro/nanocomposite catalysts were obtained through covalent attachment of polyamines on the surface of silicone nanofilaments. Such composites, packed in a glass column, were used as a simple flow reactor for Knoevenagel condensation reactions. Quantitative amounts of pure products without further purification can be obtained in such a simple way that just allowing the reactants solution flows through the composite catalysts driven by gravity. These results pave the way toward the development of sand-based multifunctional materials with great potential for industrial use, given their versatile functions and excellent performances but easy-to-fabricate, low-cost preparation procedure.

Despite great scientific and industrial interest in waterproof cellulosic paper, its real world application is hindered by complicated and costly fabrication processes, limitations in scale-up production, and use of organic solvents. Furthermore, simultaneously achieving nonwetting properties and printability on paper surfaces still remains a technical and chemical challenge. Herein, we demonstrate a nonsolvent strategy for scalable and fast fabrication of waterproofing paper through in situ surface engineering with polysilsesquioxane nanorods (PSNRs). Excellent superhydrophobicity is attained on the functionalized paper surface with a water contact angle greater than 160°. Notably, the engineered paper features outstanding printability and writability, as well as greatly enhanced strength and integrity upon prolonged exposure to water (tensile strength ≈ 9.0 MPa). Additionally, the PSNRs concurrently armor paper-based printed items and artwork with waterproofing, self-cleaning, and antimicrobial functionalities without compromising their appearance, readability, and mechanical properties. We also demonstrate that the engineered paper holds the additional advantages of easy processing, low cost, and mechanochemical robustness, which makes it particularly promising for real world applications.

Performance of engineered surfaces can be enhanced by making them hydrophobic or superhydrophobic via coating them with low-surface-energy micro- and nanopatterns. However, the wetting phenomena of particularly irregular shape and spacing (super)hydrophobic patterns such as polysiloxane coatings are not yet fully understood from a microscopic perspective. Here, we show a new method to collect 3D confocal images from irregular polysiloxane micro- and nanorods from a single rod resolution to discuss their wetting response over long liquid/solid interaction times and quantify the length and diameter of these rods. To collect such 3D confocal images, fluorescent dye containing water droplets were left on our superhydrophobic and hydrophobic polysiloxane coated surfaces. Then their liquid/solid interfaces were imaged at different staining scenarios: (i) using different fluorescent dyes, (ii) when the droplets were in contact with surfaces, or (iii) after the droplets were taken away from the surface at the end of staining. Using such staining strategies, we could resolve the micro- and nanorods from root to top and determine their length and diameter, which were then found to be in good agreement with those obtained from their electron microscopy images. 3D confocal images in this paper, for the first time, present the long-time existence of more than one wetting state under the same droplet in contact with surfaces, as well as external and internal three-phase contact lines shifting and pinning. In the end, these findings were used to explain the time-dependent wetting kinetics of our surfaces. We believe that the proposed imaging strategy here will, in the future, be used to study many other irregular patterned (super)antiwetting surfaces to describe their wetting theory, which is today impossible due to the complicated surface geometry of these irregular patterns.

The amyloid-β peptide is correlated with Alzheimer's disease and is assumed to cause toxicity by its interaction with the neuron membrane. A custom-made microscope objective based on the supercritical angle technique was developed by our group, which allows investigation of interfacial events by performing surface-sensitive and low-invasive spectroscopy. Applied to Raman spectroscopy, this technique was used to collect information about the structure of polypeptides that interact with a supported lipid bilayer. Notably, the conformation used by amyloid-β(1-40) and amyloid-β(1-42) when interacting directly with or next to the supported lipid bilayer was characterized. We observed two distinct secondary structures, α-helix and β-sheet, which were exhibited by the peptide. These two structures were detected simultaneously. The propensity of the peptide to fold into these structures seemed dependent on both their number of amino acids and their proximity with the supported lipid bilayer. The α-helix structure was observed for amyloid-β(1-42) fragments that were closer to the lipid bilayer. Peptides that were located further away from the bilayer favored the β-sheet structure. Amyloid-β(1-40) was less prone to adopt the α-helix secondary structure.

The understanding of the interaction between the membrane of neurons and amyloid-β peptides is of crucial importance to shed light on the mechanism of toxicity in Alzheimer's disease. This paper describes how supercritical angle fluorescence spectroscopy was applied to monitor in real-time the interaction between a supported lipid bilayer (SLB) and the peptide. Different forms of amyloid-β (40 and 42 amino acids composition) were tested, and the interfacial fluorescence was measured to get information about the lipid integrity and mobility. The results show a concentration-dependent damaging process of the lipid bilayer. Prolonged interaction with the peptide up to 48 h lead to an extraction and clustering of lipid molecules from the surface and a potential disruption of the bilayer, correlated with the formation of peptide aggregates. The natural diffusion of the lipid was slightly hindered by the interaction with amyloid-β(1-42) and closely related to the oligomerization of the peptide. The adsorption and desorption of Amyloid-β was also characterized in terms of affinity. Amyloid-β(1-42) exhibited a slightly higher affinity than amyloid-β(1-40). The former was also more prone to aggregate and to adsorb on the bilayer as oligomer.

Superhydrophobic surfaces hold tremendous potential in a wide range of applications owing to their multifaced functionalities. However, the mechanochemical susceptibility of such materials hinders their widespread usage in practical applications. Here, we present a simple, solvent-free, and environmentally friendly approach to fabricate flexible and robust superhydrophobic composite films with durable self-cleaning functionality. The obtained composite film features unexpected but surprising hierarchical micro/nanoscopic structures as well as robust superhydrophobicity with a water contact angle of ∼170° and a sliding angle below 4°. Notably, the composite film exhibits mechanical robustness under cyclic abrasion, tape peeling, flexing, intensive finger wiping, and knife cutting; maintains excellent superhydrophobicity after long-time exposure to a high-humidity environment; and sustains exposure to highly corrosive species, such as strong acid/base solutions and organic solvents. The robust superhydrophobicity is ascribed to the induced micro/nanohierarchical surface structures, resulting in the trapped dual-scale air pockets, which could largely reduce the solid/liquid interface. In addition, even after oil contamination, the composite film maintains its water repellency and self-cleaning functionality. The robust superhydrophobic composite film developed here is expected to extend the application scope of superhydrophobic materials and should find potential usage in various industries and daily life.

We present here a new eco-efficiency process-improvement method to highlight combined environmental and costs hotspots of the production process of new material at a very early development stage. Production-specific and scaled-up results for life cycle assessment (LCA) and production costs are combined in a new analysis to identify synergetic improvement potentials and trade-offs, setting goals for the eco-design of new processes. The identified hotspots and bottlenecks will help users to focus on the relevant steps for improvements from an eco-efficiency perspective and potentially reduce their associated environmental impacts and production costs. Our method is illustrated with a case study of nanocellulose. The results indicate that the production route should start with carrot pomace, use heat and solvent recovery, and deactivate the enzymes with bleach instead of heat. To further improve the process, the results show that focus should be laid on the carrier polymer, sodium alginate, and the production of the GripX coating. Overall, the method shows that the underlying LCA scale-up framework is valuable for purposes beyond conventional LCA studies and is applicable at a very early stage to provide researchers with a better understanding of their production process.

Large aggregates of misfolded α-synuclein inside neuronal cells are the hallmarks of Parkinson's disease. The protein's natural function and its supposed toxicity, however, are believed to be closely related to its interaction with cell and vesicle membranes. Upon this interaction, the protein folds into an α-helical structure and intercalates into the membrane. In this study, we focus on the changes in the lipid bilayer caused by this intrusion. In situ X-ray reflectivity was applied to determine the vertical density structure of the bilayer before and after exposure to α-synuclein. It was found that the α-synuclein insertion, wild type and E57K variant, caused a reduction in bilayer thickness. This effect may be one factor in the membrane pore formation ability of α-synuclein.

The newly developed Nepenthes pitcher (NP)-inspired slippery surfaces, formed by immobilizing fluoroliquids on lotus leaf (LL)-inspired superoleophobic surfaces, are of great general interest, whereas there are many interesting phenomena and fundamental scientific issues remaining to be unveiled. Here we present our findings of the effects of evaporation of the fluoroliquid, an inevitable process in most cases, -induced transition from NP-inspired to LL-inspired surfaces on the wettability, transparency, and self-cleaning property of the surfaces. The transition is controlled by regulating the evaporation temperature of the model fluoroliquid, Krytox100. The evaporation of Krytox100 has great a influence on the wettability, transparency, and self-cleaning property. An intermediate "sticky" state is observed in the transition process. We believe that our findings in the transition process are helpful in understanding the similarities and differences between the NP-inspired and LL-inspired surfaces and in designing new bioinspired antiwetting surfaces and exploring their potential applications.

Mesocrystals with the symmetry defying morphologies and highly ordered superstructures composed of primary units are of particular interest, but the fabrication has proved extremely challenging. A novel strategy based on biomineralization approach for the synthesis of hematite mesocrystals is developed by using silk fibroin as a biotemplate. The resultant hematite mesocrystals are uniform, highly crystalline, and porous nanostructures with tunable size and morphologies by simply varying the concentration of the silk fibroin and iron(III) chloride in this biomineralization system. In particular, we demonstrate a complex mesoscale biomineralization process induced by the silk fibroin for the formation of hematite mesocrystals. This biomimetic strategy features precisely tunable, high efficiency, and low-cost and opens up an avenue to access new novel functional mesocrystals with hierarchical structures in various practical applications.

We report new types of silicone nanostructures by a gas-phase reaction of trichloromethylsilane: 1-D silicone nanofilaments with a raveled end and silicone nanoteeth. Filaments with a raveled end are obtained on poly(vinyl chloride), which is superficially doped with the detergent Span 20. Silicone nanoteeth grow on sodium chloride using dibutyl phthalate as superficial dopant. Without dopants, no structures are observed. The dopants are identified by mass spectroscopy and the silicone nanostructures are analyzed by infrared spectroscopy and energy-dispersive analysis of X-rays. The growth of silicone nanostructures on a hydrophobic substrate (poly(vinyl chloride)/Span 20) and a substrate free of hydroxyl groups (sodium chloride/dibutyl phthalate) questions the currently discussed mechanisms for the growth of 1-D silicone nanofilaments, which is discussed. We suggest superficial doping as an alternative pretreatment method to oxidizing activation and prove this principle by the successful coating of copper, which is superficially doped with Span 20.

Inspired by the mussel adhesive protein and the lotus leaf, Ag-based double-layer polyester (DL-PET) textiles were fabricated for effective removal of organic pollutants in water. The DL-PET textiles are composed of a top superamphiphilic layer and a bottom superhydrophobic/superoleophilic layer. First, the PET textiles were modified with a layer of polydopamine (PDA) and deposited with Ag nanoparticles to form the PET@PDA@Ag textiles. The top superamphiphilic layer, formed by immobilizing Ag3PO4 nanoparticles on the PET@PDA@Ag textile, shows excellent visible-light photocatalytic activity. The bottom superhydrophobic/superoleophilic layer, formed by modifying the PET@PDA@Ag textile using dodecyl mercaptan, is mechanically, environmentally, and chemically very stable. The water-insoluble oils with low surface tension can penetrate both layers of the DL-PET textiles, while the water with soluble organic dyes can only selectively wet the top layer owing to their unique wettability. Consequently, the water-soluble organic contaminants in the collected water can be decomposed by the Ag3PO4 nanoparticles of the top layer under visible-light irradiation or even sunlight in room conditions. Thus, the DL-PET textiles can remove various kinds of organic pollutants in water including both insoluble oils and soluble dyes. The DL-PET textiles feature unique wettability, high oil/water separation efficiency, and visible-light photocatalytic activity.

The aggregation of α-synuclein (α-Syn) is believed to be one of the key steps driving the pathology of Parkinson's disease and related neurodegenerative disorders. One of the present hypotheses is that the onset of such pathologies is related to the rise of α-Syn levels above a critical concentration at which toxic oligomers or mature fibrils are formed. In the present study, we find that α-Syn aggregation in vitro is a spontaneous process arising at bulk concentrations as low as 1 nM and below in the presence of both hydrophilic glass surfaces and cell membrane mimicking supported lipid bilayers (SLBs). Using three-dimensional supercritical angle fluorescence (3D-SAF) microscopy, we observed the process of α-Syn aggregation in situ. As soon as α-Syn monomers were exposed to the surface, they started to adsorb and aggregate along the surface plane without a prior lag phase. However, at a later stage of the aggregation process, a second type of aggregate was observed. In contrast to the first type, these aggregates showed an extended structure being tethered with one end to the surface and being mobile at the other end, which protruded into the solution. While both types of α-Syn aggregates were found to contain amyloid structures, their growing mechanisms turned out to be significantly different. Given the clear evidence that surface-induced α-Syn aggregation in vitro can be triggered at bulk concentrations far below physiological concentrations, the concept of a critical concentration initiating aggregation in vivo needs to be reconsidered.

Parkinson's disease is a common progressive neurodegenerative condition, characterized by the deposition of amyloid fibrils as Lewy bodies in the substantia nigra of affected individuals. These insoluble aggregates predominantly consist of the protein α-synuclein. There is increasing evidence suggesting that the aggregation of α-synuclein is influenced by lipid membranes and, vice versa, the membrane integrity is severely affected by the presence of bound aggregates. Here, using the surface-sensitive imaging technique supercritical angle fluorescence microscopy and Förster resonance energy transfer, we report the direct observation of α-synuclein aggregation on supported lipid bilayers. Both the wild-type and the two mutant forms of α-synuclein studied, namely, the familiar variant A53T and the designed highly toxic variant E57K, were found to follow the same mechanism of polymerization and membrane damage. This mechanism involved the extraction of lipids from the bilayer and their clustering around growing α-synuclein aggregates. Despite all three isoforms following the same pathway, the extent of aggregation and their effect on the bilayers was seen to be variant and concentration dependent. Both A53T and E57K formed cross-β-sheet aggregates and damaged the membrane at submicromolar concentrations. The wild-type also formed aggregates in this range; however, the extent of membrane disruption was greatly reduced. The process of membrane damage could resemble part of the yet poorly understood cellular toxicity phenomenon in vivo., (© 2011 American Chemical Society)

Fluorescence resonance energy transfer (FRET) using biotinylated β-galactosidase (βGAL) as a donor and Alexa Fluor 350 (AF350) labeled avidin as an acceptor has been investigated by means of steady-state fluorescence and time-resolved fluorescence spectroscopy. The donors are readily paired with acceptors through the well-established binding affinity of biotin and avidin. The fluorescence energy transfer efficiency was determined by the donor fluorescence emission and lifetime changes in the presence and absence of acceptor. The theoretical energy transfer efficiency and theoretical average distance between donor and acceptor after noncovalent binding was calculated by taking the distribution of tryptophan residues in βGAL and avidin as well as the location of AF350 in avidin into account, which agree with the experimental data. It is shown how information of the location of the acceptor can be obtained. Further, the fluorescence intensity image of AF350 on a biotinylated βGAL-coated quartz surface through UV FRET has been recorded using deep UV laser-based fluorescence lifetime microscopy. The results demonstrate that (a) deep UV laser-based fluorescence lifetime microscopy is a simple and useful method to study UV FRET of proteins using intrinsic fluorescence, (b) structural information even in complex multidonor systems can be obtained, and (c) FRET signals can be obtained to detect binding events using the native fluorescence of proteins as multidonor systems., (© 2011 American Chemical Society)

In this work, the elemental composition of fine and ultrafine particles emitted by ten different laser printing devices (LPD) is examined. The particle number concentration time series was measured as well as the particle size distributions. In parallel, emitted particles were size-selectively sampled with a cascade impactor and subsequently analyzed by the means of XRF. In order to identify potential sources for the aerosol's elemental composition, materials involved in the printing process such as toner, paper, and structural components of the printer were also analyzed. While the majority of particle emissions from laser printers are known to consist of recondensated semi volatile organic compounds, elemental analysis identifies Si, S, Cl, Ca, Ti, Cr, and Fe as well as traces of Ni and Zn in different size fractions of the aerosols. These elements can mainly be assigned to contributions from toner and paper. The detection of elements that are likely to be present in inorganic compounds is in good agreement with the measurement of nonvolatile particles. Quantitative measurements of solid particles at 400 °C resulted in residues of 1.6 × 10(9) and 1.5 × 10(10) particles per print job, representing fractions of 0.2% and 1.9% of the total number of emitted particles at room temperature. In combination with the XRF results it is concluded that solid inorganic particles contribute to LPD emissions in measurable quantities. Furthermore, for the first time Br was detected in significant concentrations in the aerosol emitted from two LPD. The analysis of several possible sources identified the plastic housings of the fuser units as main sources due to substantial Br concentrations related to brominated flame retardants.

An inexpensive and easy-to-use immunoassay platform for the sensitive detection of analytes is presented. It comprises single-use polymer test tubes and a compact fluorescence reader. The optics for the capture of supercritical angle fluorescence (SAF) has been built into the tubes allowing for the extremely sensitive readout of solid phase immunoassays in real time and without washing steps. One-step sandwich immunoassays with interleukin 2 (IL-2) were carried out with capture antibodies immobilized in the tubes. At a turn around time of 12 min, the limit of detection for IL-2 was 0.27 pM (4.5 pg/mL) and the linear range covered 3 orders of magnitude. The developed technology is also adaptable to well plates and has great potential of replacing the work-intensive and time-consuming enzyme-linked immunosorbant assay (ELISA).

Cooperative effects play a vital role in protein adsorption events on biological interfaces. Despite a number of studies in this field molecular adsorption mechanisms that include cooperativity are still under debate. In this work we use a Monte Carlo-type simulation to explore the microscopic details behind cooperative protein adsorption. The simulation was designed to implement our previously proposed mechanism through which proteins are not necessarily rejected if they approach the surface to an occupied region. Instead, we suggest that proteins can be tracked laterally for a certain distance due to the influence of preadsorbed proteins in order to reach the nearest available binding site. The simulation results were compared with experimental data obtained by using the supercritical angle fluorescence (SAF) microscopy technique. It was found that the tracking distance may be up to 2.5 times the protein's diameter depending on the investigated system. The general validity of this tracking mechanism is supported by a number of linear or upward concave adsorption kinetics reported in the literature which are consistent with our simulation results. Furthermore, the self-organization of proteins adsorbing under cooperative conditions on the surface is shown to necessarily cause density inhomogeneities in the surface distribution of proteins which is also in agreement with experimental observations.

Despite many experimental studies on cooperative effects during protein adsorption events, this phenomenon is still poorly characterized and subject of much controversy. In this study, we address the topic of cooperativity using two distinct experimental approaches, namely, kinetic analysis and surface imaging, both based on supercritical angle fluorescence (SAF) microscopy. Several model systems comprising the two proteins BSA and fibrinogen, two different ionic strength conditions and varying pH environments were investigated. The combination of the experimental information obtained from kinetic analysis and from real-time in situ scan images unravel a clear correlation between cooperative adsorption and a heterogeneous protein layer build-up. We propose a mechanistic model of protein adsorption based on an overlap of classical Langmuir-type adsorption on unoccupied surface areas and an additional cooperative adsorption pathway near preadsorbed proteins which is consistent with the experimental observations. Moreover, the growth of two-dimensional surface clusters as an often assumed element of cooperativity could be excluded for the studied systems. The model includes the often observed phenomenon that the adsorption rate decelerates abruptly above a certain coverage limit. Furthermore, the observed evolution of the heterogeneous protein distribution on the surface is in good agreement with the proposed model.

We present a simple and versatile technique of tailoring functionalized surface structures for protein enrichment and purification applications based on a superhydrophobic silicone nanofilament coating. Using amino and carboxyl group containing silanes, silicone nanofilament templates were chemically modified to mimic anionic and cationic exchange resins. Investigations on the selectivity of the functionalized surfaces toward adsorption of charged model proteins were carried out by means of fluorescence techniques. Due to a high contact area resulting from the nanoroughness of the coating, excellent protein retention characteristics under various conditions were found. The surfaces were shown to be highly stable and reusable over several retention-elution cycles. Especially the full optical transparency and the possibility to use glass substrates as support material open new opportunities for the development of optical biosensors, open geometry microfluidics, or lab-on-a-chip devices.

We present the detection of single beta-galactosidase molecules from Escherichia coli (Ecbeta Gal) using deep UV laser-based fluorescence lifetime microscopy. The native fluorescence from intrinsic tryptophan emission has been observed after one-photon excitation at 266 nm. Applying the time-resolved single-photon counting method, we investigated the fluorescence lifetime distribution and the bursts of autofluorescence photons from tryptophan residues in Ecbeta Gal protein as well as fluorescence correlation spectroscopy of Ecbeta Gal. The results demonstrate that deep UV laser-based fluorescence lifetime microscopy is useful for identification of biological macromolecules at the single-molecule level using intrinsic fluorescence.

We present a new concept for ultrasensitive detection of surface-generated fluorescence which is made possible by a new optical module. The detection method leads to an enhancement in fluorescence collection efficiency to more than 65% of the total of emitted light, whereas high-aperture microscope objectives are able to collect 44% at best. Moreover, by employing this new optical module, the detection volume can be restricted to approximately 10(-17) L. This allows for an exceptional discrimination of bulk-generated against surface-generated fluorescence, which may be of great value when surface-binding processes are monitored. We demonstrate the performance of the new detection system by detecting single fluorescent molecules and by determining antigen concentrations down to 5 fmol.