A Lignin Molecular Brace Controls Precision Processing of Cell Walls Critical for Surface Integrity in Arabidopsis.

The cell wall, a defining feature of plants, provides a rigid structure critical for bonding cells together. To overcome this physical constraint, plants must process cell wall linkages during growth and development. However, little is known about the mechanism guiding cell-cell detachment and cell wall remodeling. Here, we identify two neighboring cell types in Arabidopsis that coordinate their activities to control cell wall processing, thereby ensuring precise abscission to discard organs. One cell type produces a honeycomb structure of lignin, which acts as a mechanical "brace" to localize cell wall breakdown and spatially limit abscising cells. The second cell type undergoes transdifferentiation into epidermal cells, forming protective cuticle, demonstrating de novo specification of epidermal cells, previously thought to be restricted to embryogenesis. Loss of the lignin brace leads to inadequate cuticle formation, resulting in surface barrier defects and susceptible to infection. Together, we show how plants precisely accomplish abscission.

Advanced surface passivation for high-sensitivity studies of biomolecular condensates.

Biomolecular condensates are cellular compartments that concentrate biomolecules without an encapsulating membrane. In recent years, significant advances have been made in the understanding of condensates through biochemical reconstitution and microscopic detection of these structures. Quantitative visualization and biochemical assays of biomolecular condensates rely on surface passivation to minimize background and artifacts due to condensate adhesion. However, the challenge of undesired interactions between condensates and glass surfaces, which can alter material properties and impair observational accuracy, remains a critical hurdle. Here, we introduce an efficient, broadly applicable, and simple passivation method employing self-assembly of the surfactant Pluronic F127 (PF127). The method greatly reduces nonspecific binding across a range of condensates systems for both phase-separated droplets and biomolecules in dilute phase. Additionally, by integrating PF127 passivation with the Biotin-NeutrAvidin system, we achieve controlled multipoint attachment of condensates to surfaces. This not only preserves condensate properties but also facilitates long-time fluorescence recovery after photobleaching imaging and high-precision single-molecule analyses. Using this method, we have explored the dynamics of polySIM molecules within polySUMO/polySIM condensates at the single-molecule level. Our observations suggest a potential heterogeneity in the distribution of available polySIM-binding sites within the condensates.

Improving the Biocompatibility and Functionality of Neural Interface Devices with Silica Nanoparticles.

ConspectusNeural interface technologies enable bidirectional communication between the nervous system and external instrumentation. Advancements in neural interface devices not only open new frontiers for neuroscience research, but also hold great promise for clinical diagnosis, therapy, and rehabilitation for various neurological disorders. However, the performance of current neural electrode devices, often termed neural probes, is far from satisfactory. Glial scarring, neuronal degeneration, and electrode degradation eventually cause the devices to lose their connection with the brain. To improve the chronic performance of neural probes, efforts need to be made on two fronts: enhancing the physiochemical properties of the electrode materials and mitigating the undesired host tissue response.In this Account, we discuss our efforts in developing silica-nanoparticle-based (SiNP) coatings aimed at enhancing neural probe electrochemical properties and promoting device-tissue integration. Our work focuses on three approaches:(1) SiNPs' surface texturization to enhance biomimetic protein coatings for promoting neural integration. Through covalent immobilization, SiNP introduces biologically relevant nanotopography to neural probe surfaces, enhancing neuronal cell attachments and inhibiting microglia. The SiNP base coating further increases the binding density and stability of bioactive molecules such as L1CAM and facilitates the widespread dissemination of biomimetic coatings. (2) Doping SiNPs into conductive polymer electrode coatings improves the electrochemical properties and stability. As neural interface devices are moving to subcellular sizes to escape the immune response and high electrode site density to increase spatial resolution, the electrode sites need to be very small. The smaller electrode size comes at the cost of a high electrode impedance, elevated thermal noise, and insufficient charge injection capacity. Electrochemically deposited conductive polymer films reduce electrode impedance but do not endure prolonged electrical cycling. When incorporated into conductive polymer coatings as a dopant, the SiNP provides structural support for the polymer thin films, significantly increasing their stability and durability. Low interfacial impedance maintained by the conducting polymer/SiNP composite is critical for extended electrode longevity and effective charge injection in chronic neural stimulation applications. (3) Porous nanoparticles are used as drug carriers in conductive polymer coatings for local drug/neurochemical delivery. When triggered by external electrical stimuli, drug molecules and neurochemicals can be released in a controlled manner. Such precise focal manipulation of cellular and vascular behavior enables us to probe brain circuitry and develop therapeutic applications.We foresee tremendous opportunities for further advancing the functionality of SiNP coatings by incorporating new nanoscale components and integrating the coating with other design strategies. With an enriched nanoscale toolbox and optimized design strategies, we can create customizable multifunctional and multimodal neural interfaces that can operate at multiple spatial levels and seamlessly integrate with the host tissue for extended applications.

Nanochemistry of gold: from surface engineering to dental healthcare applications.

Advancements in nanochemistry have led to the development of engineered gold nanostructures (GNSs) with remarkable potential for a variety of dental healthcare applications. These innovative nanomaterials offer unique properties and functionalities that can significantly improve dental diagnostics, treatment, and overall oral healthcare applications. This review provides an overview of the latest advancements in the design, synthesis, and application of GNSs for dental healthcare applications. Engineered GNSs have emerged as versatile tools, demonstrating immense potential across different aspects of dentistry, including enhanced imaging and diagnosis, prevention, bioactive coatings, and targeted treatment of oral diseases. Key highlights encompass the precise control over GNSs' size, crystal structure, shape, and surface functionalization, enabling their integration into sensing, imaging diagnostics, drug delivery systems, and regenerative therapies. GNSs, with their exceptional biocompatibility and antimicrobial properties, have demonstrated efficacy in combating dental caries, periodontitis, peri-implantitis, and oral mucosal diseases. Additionally, they show great promise in the development of advanced sensing techniques for early diagnosis, such as nanobiosensor technology, while their role in targeted drug delivery, photothermal therapy, and immunomodulatory approaches has opened new avenues for oral cancer therapy. Challenges including long-term toxicity, biosafety, immune recognition, and personalized treatment are under rigorous investigation. As research at the intersection of nanotechnology and dentistry continues to thrive, this review highlights the transformative potential of engineered GNSs in revolutionizing dental healthcare, offering accurate, personalized, and minimally invasive solutions to address the oral health challenges of the modern era.

New Avenues for Capturing Mineralization Events at Biomaterial Interfaces with Liquid-Transmission Electron Microscopy.

Liquid-transmission electron microscopy (liquid-TEM) provides exciting potential for capturing mineralization events at biomaterial interfaces, though it is largely unexplored. To address this, we established a unique approach to visualize calcium phosphate (CaP)-titanium (Ti) interfacial mineralization events by combining the nanofabrication of Ti lamellae by focused ion beam with in situ liquid-TEM. Multiphasic CaP particles were observed to nucleate, adhere, and form different assemblies onto and adjacent to Ti lamellae. Here, we discuss new approaches for exploring the interaction between biomaterials and liquids at the nanoscale. Driving this technology is crucial for understanding and controlling biomineralization to improve implant osseointegration and direct new pathways for mineralized tissue disease treatment in the future.

Lifting the Concentration Limit of Mass Photometry by PEG Nanopatterning.

Mass photometry (MP) is a rapidly growing optical technique for label-free mass measurement of single biomolecules in solution. The underlying measurement principle provides numerous advantages over ensemble-based methods but has been limited to low analyte concentrations due to the need to uniquely and accurately quantify the binding of individual molecules to the measurement surface, which results in diffraction-limited spots. Here, we combine nanoparticle lithography with surface PEGylation to substantially lower surface binding, resulting in a 2 orders of magnitude improvement in the upper concentration limit associated with mass photometry. We demonstrate the facile tunability of degree of passivation, enabling measurements at increased analyte concentrations. These advances provide access to protein-protein interactions in the high nanomolar to low micromolar range, substantially expanding the application space of mass photometry.

Surface Passivation of Norland Optical Adhesive Improves the Guiding Efficiency of Gliding Microtubules in Microfluidic Devices.

The microtubule-kinesin biomolecular motor system, which is vital for cellular function, holds significant promise for nanotechnological applications. In vitro gliding assays have demonstrated the ability to transport microcargo by propelling microtubules across kinesin-coated surfaces. However, the uncontrolled directional motion of microtubules has posed significant challenges, limiting the system's application for precise cargo delivery. Microfluidic devices provide a means to direct microtubule movement through their geometric features. Norland Optical Adhesive (NOA) is valued for its mold-free application in microfluidic device fabrication; however, microtubules often climb up channel walls, limiting controlled movement. In this study, a surface passivation method for NOA is introduced, using polyethylene glycol via a thiol-ene click reaction. This technique significantly improved the directional control and concentration of microtubules within NOA microchannels. This approach presents new possibilities for the precise application of biomolecular motors in nanotechnology, enabling advancements in the design of microfluidic systems for complex biomolecular manipulations.

Synergistic Influence of Fibrous Pattern Orientation and Modulus on Cellular Mechanoresponse.

The fibrous extracellular matrix (ECM) is vital for tissue regeneration and impacts implanted device treatments. Previous research on fibrous biomaterials shows varying cellular reactions to surface orientation, often due to unclear interactions between surface topography and substrate elasticity. Our study addresses this gap by achieving the rapid creation of hydrogels with diverse fibrous topographies and varying substrate moduli through a surface printing strategy. Cells exhibit heightened traction force on nanopatterned soft hydrogels, particularly with randomly distributed patterns compared with regular soft hydrogels. Meanwhile, on stiff hydrogels featuring an aligned topography, optimal cellular mechanosensing is observed compared to random topography. Mechanistic investigations highlight that cellular force-sensing and adhesion are influenced by the interplay of pattern deformability and focal adhesion orientation, subsequently mediating stem cell differentiation. Our findings highlight the importance of combining substrate modulus and topography to guide cellular behavior in designing advanced tissue engineering biomaterials.

Manipulation of Macrophage Uptake by Controlling the Aspect Ratio of Graft Copolymer Micelles.

Nanostructures of drug carriers play a crucial role in nanomedicine due to their ability to influence drug delivery. There is yet no clear consensus regarding the optimal size and shape (e.g., aspect ratio) of nanoparticles for minimizing macrophage uptake, given the difficulties in controlling the shape and size of nanoparticles while maintaining identical surface properties. Here, we employed graft copolymer self-assembly to prepare polymer micelles with aspect ratios ranging from 1.0 (spherical) to 10.8 (cylindrical) and closely matched interfacial properties. Notably, our findings emphasize that cylindrical micelles with an aspect ratio of 2.4 are the least susceptible to macrophage uptake compared with both their longer counterparts and spherical micelles. This reduced uptake of the short cylindrical micelles results in a 3.3-fold increase in blood circulation time compared with their spherical counterparts. Controlling the aspect ratio of nanoparticles is crucial for improving drug delivery efficacy through better nanoparticle design.

Facile Fabrication of Multifunctional Hydrogel Nanoweb Coating Using Carboxymethyl Chitosan-Based Short Nanofibers for Blood-Contacting Medical Devices.

Blood-contacting medical devices (BCDs) require antithrombotic, antibacterial, and low-friction surfaces. Incorporating a nanostructured surface with the functional hydrogel onto BCD surfaces can enhance the performances; however, their fabrication remains challenging. Here, we introduce a straightforward method to fabricate a multifunctional hydrogel-based nanostructure on BCD surfaces using O-carboxymethyl chitosan-based short nanofibers (CMC-SNFs). CMC-SNFs, fabricated via electrospinning and cutting processes, are easily sprayed and entangled onto the BCD surface. The deposited CMC-SNFs form a robust nanoweb layer via fusion at the contact area of the nanofiber interfaces. The superhydrophilic CMC-SNF nanoweb surface creates a water-bound layer that effectively prevents the nonspecific adhesion of bacteria and blood cells, thereby enhancing both antimicrobial and antithrombotic performances. Furthermore, the CMC-SNF nanoweb exhibits excellent lubricity and durability on the bovine aorta. The demonstration results of the CMC-SNF coating on catheters and sheaths provide evidence of its capability to apply multifunctional surfaces simply for diverse BCDs.

Cellulose Surface Nanoengineering for Visualizing Food Safety.

Food safety is vital to human health, necessitating the development of nondestructive, convenient, and highly sensitive methods for detecting harmful substances. This study integrates cellulose dissolution, aligned regeneration, in situ nanoparticle synthesis, and structural reconstitution to create flexible, transparent, customizable, and nanowrinkled cellulose/Ag nanoparticle membranes (NWCM-Ag). These three-dimensional nanowrinkled structures considerably improve the spatial-electromagnetic-coupling effect of metal nanoparticles on the membrane surface, providing a 2.3 × 108 enhancement factor for the surface-enhanced Raman scattering (SERS) effect for trace detection of pesticides in foods. Notably, the distribution of pesticides in the apple peel and pulp layers is visualized through Raman imaging, confirming that the pesticides penetrate the peel layer into the pulp layer (∼30 µm depth). Thus, the risk of pesticide ingestion from fruits cannot be avoided by simple washing other than peeling. This study provides a new idea for designing nanowrinkled structures and broadening cellulose utilization in food safety.

Droplet Polymer Bilayers for Bioelectronic Membrane Interfacing.

Model membranes interfaced with bioelectronics allow for the exploration of fundamental cell processes and the design of biomimetic sensors. Organic conducting polymers are an attractive surface on which to study the electrical properties of membranes because of their low impedance, high biocompatibility, and hygroscopic nature. However, establishing supported lipid bilayers (SLBs) on conducting polymers has lagged significantly behind other substrate materials, namely, for challenges in membrane electrical sealing and stability. Unlike SLBs that are highly dependent on surface interactions, droplet interface bilayers (DIBs) and droplet hydrogel bilayers (DHBs) leverage the energetically favorable organization of phospholipids at atomically smooth liquid interfaces to build high-integrity membranes. For the first time, we report the formation of droplet polymer bilayers (DPBs) between a lipid-coated aqueous droplet and the high-performing conducting polymer poly(3,4-ethylenedioxythiophene) polystyrenesulfonate (PEDOT:PSS). The resulting bilayers can be produced from a range of lipid compositions and demonstrate strong electrical sealing that outcompetes SLBs. DPBs are subsequently translated to patterned and planar microelectrode arrays to ease barriers to implementation and improve the reliability of membrane formation. This platform enables more reproducible and robust membranes on conducting polymers to further the mission of merging bioelectronics and synthetic, natural, or hybrid bilayer membranes.

Coacervate Dense Phase Displaces Surface-Established Pseudomonas aeruginosa Biofilms.

For millions of years, barnacles and mussels have successfully adhered to wet rocks near tide-swept seashores. While the chemistry and mechanics of their underwater adhesives are being thoroughly investigated, an overlooked aspect of marine organismal adhesion is their ability to remove underlying biofilms from rocks and prepare clean surfaces before the deposition of adhesive anchors. Herein, we demonstrate that nonionic, coacervating synthetic polymers that mimic the physicochemical features of marine underwater adhesives remove ∼99% of Pseudomonas aeruginosa (P. aeruginosa) biofilm biomass from underwater surfaces. The efficiency of biofilm removal appears to align with the compositional differences between various bacterial biofilms. In addition, the surface energy influences the ability of the polymer to displace the biofilm, with biofilm removal efficiency decreasing for surfaces with lower surface energies. These synthetic polymers weaken the biofilm-surface interactions and exert shear stress to fracture the biofilms grown on surfaces with diverse surface energies. Since bacterial biofilms are 1000-fold more tolerant to common antimicrobial agents and pose immense health and economic risks, we anticipate that our unconventional approach inspired by marine underwater adhesion will open a new paradigm in creating antibiofilm agents that target the interfacial and viscoelastic properties of established bacterial biofilms.

Synthesis of Mesoporous Catechin Nanoparticles as Biocompatible Drug-Free Antibacterial Mesoformulation.

While polyphenolic substances stand as excellent antibacterial agents, their antimicrobial properties rely on the auxiliary support of micro-/nanostructures. Despite offering a novel avenue for enhancing polymer performance, controllable fabrication of mesoporous polymeric nanomaterials encounters significant challenges due to intricate intermolecular forces. In this article, mesoporous catechin nanoparticles have been successfully fabricated using a balanced multivariate interaction approach. The harmonization of the water-ethanol ratio and ionic strength effectively balances the forces of hydrogen bonding and π-π stacking, facilitating the controlled assembly of mesostructures. The mesoporous catechin nanoparticles exhibit a uniform spherical structure (∼100 nm), open mesopores with a diameter of ∼15 nm, and a high surface area of ∼106 m2 g-1. While exhibiting a good biocompatibility and negative surface charge, the mesoporous catechins possess outstanding antibacterial ability and function as an antibiotic mesoformulation without the necessity of loading any drugs. This mesoformulation inhibits 50% in vitro Staphylococcus aureus growth with a low concentration of ∼10 µg mL-1 and achieves complete inhibition at ∼25 µg mL-1. In a mouse wound model, accelerated wound healing and complete closure within 6-8 days are achieved. Proteomics of bacteria reveals that the excellent antibacterial property is attributed to the synergetic effect of mesoformulation's mesostructure and the catechin molecule intervening in bacterial metabolism. Overall, this work may pave a novel way for the future exploration of polymer nanomaterials and antibiotic formulations.

Orientation of Antibody Modified and Reacted on Carboxy-Functionalized Polystyrene Particle Revealed by Zeta Potential Measurement.

The comprehensive understanding of the orientation of antibodies on a solid surface is crucial for affinity-based sensing mechanisms. In this study, we demonstrated that the orientation of primary antibodies modified on carboxy-functionalized polystyrene (PS) particles can be analyzed using zeta potential behavior at different pH based on the combined Gouy-Chapman-Stern model and the acid dissociation of carboxy groups and antibodies. We observed that at low surface concentrations of the primary antibody, a side-on orientation was predominant. However, at higher concentrations (approximately 30000 antibodies per PS particle), the orientation shifted to an end-on type due to steric hindrance. Furthermore, the reaction mechanism of the secondary antibody exhibited pH-dependent behavior. At pH > 7, the zeta potential changes were attributed to the antibody-antibody reaction, whereas at pH < 7, adsorption of secondary antibody onto the PS particle was observed, leading to a change in the orientation of the primary antibody modified on the PS particle to an end-on type. The change in zeta potential due to secondary antibody binding indicated a detection limit of 37000 antibodies per PS particle. As a result, we revealed that the analysis of zeta potential behavior enables the evaluation of antibody orientation and the detection of zeptomole order antibodies. This study represents the first demonstration of this capability. We anticipate that the present concept and results will broaden the quantitative application of zeta potential measurements and have significant implications for research areas, including physical chemistry and analytical chemistry.

Surface Oxygen Deficiency Enabled Spontaneous Antiprotein Fouling in WO3 Nanosheets for Biosensing in Biological Fluids.

Biofouling deteriorates the performance of sensors operated in biofluids. Protein adsorption is believed to be the first step of biofouling, which also reduces biocompatibility by further inducing cell adhesion, platelet activation, and even inflammation. Current studies of antifouling coatings are focused on polymers and hydrogels, which have succeeded in remaining resistant to protein adsorption, but their application on sensor electrodes is limited due to low conductivity and biocompatibility. Here, we report a spontaneous antibiofouling strategy for sensor electrodes by controlling oxygen vacancies in WO3 nanosheets. Irreversible adsorption of proteins was reduced by 76% in unprocessed human plasma when electrodes were coated with WO3 rich in surface oxygen vacancy. These electrodes maintained 91% of the initial current density after 1 month of incubation in human plasma.

Fabricating Ultrathin Imprinting Layer for Fast Capture of Valsartan via a Metal Affinity-Oriented Surface Imprinting Method.

Rapid separation and enrichment of targets in biological matrixes are of significant interest in multiple life sciences disciplines. Molecularly imprinted polymers (MIPs) have vital applications in extraction and sample cleanup owing to their excellent specificity and selectivity. However, the low mass transfer rate, caused by the heterogeneity of imprinted cavities in polymer networks and strong driving forces, significantly limits its application in high-throughput analysis. Herein, one novel metal affinity-oriented surface imprinting method was proposed to fabricate an MIP with an ultrathin imprinting layer. MIPs were prepared by immobilized template molecules on magnetic nanoparticles (NPs) with metal ions as bridges via coordination, and then polymerization was done. Under the optimized conditions, the thickness of the imprinting layer was merely 1 nm, and the adsorption toward VAL well matched the Langmuir model. Moreover, it took just 5 min to achieve adsorption equilibrium significantly faster than other reported MIPs toward VAL. Adsorption capacity still can reach 25.3 mg/g ascribed to the high imprinting efficiency of the method (the imprinting factor was as high as 5). All evidence proved that recognition sites were all external cavities and were evenly distributed on the surface of the NPs. The obtained MIP NPs exhibited excellent selectivity and specificity toward VAL, with good dispersibility and stability. Coupled with high-performance liquid chromatography, it was successfully used as a dispersed solid phase extraction material to determine VAL in serum. Average recoveries are over 90.0% with relative standard deviations less than 2.14% at three spiked levels (n = 3). All evidence testified that the MIPs fabricated with the proposed method showed a fast trans mass rate and a large rebinding capacity. The method can potentially use high-throughput separation and enrichment of target molecules in batch samples to meet practical applications.

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.

Surface Sialic Acid Detection of Small Extracellular Vesicles at the Single-Particle Level by Nano-Flow Cytometry.

Glycans, particularly sialic acids (SAs), play crucial roles in diverse biological processes. Despite their significance, analyzing specific glycans, such as sialic acids, on individual small extracellular vesicles (sEVs) has remained challenging due to the limited glycan capacity and substantial heterogeneity of sEVs. To tackle this issue, we introduce a chemical modification method of surface SAs on sEVs named PALEV-nFCM, which involves periodate oxidation and aniline-catalyzed oxime ligation (PAL), in conjunction with single-particle analysis using a laboratory-built nano-flow cytometer (nFCM). The specificity of the PALEV labeling method was validated using SA-decorated liposomes, enzymatic removal of terminal SA residues, lectin preblocking, and cellular treatment with an endogenous sialyltransferase inhibitor. Comprehensive mapping of SA distributions was conducted for sEVs derived from different sources, including conditioned cell culture medium (CCCM) of various cell lines, human saliva, and human red blood cells (RBCs). Notably, treatment with the calcium ionophore substantially increases the population of SA-positive RBC sEVs and enhances the SA content on individual RBC sEVs as well. nFCM provides a sensitive and versatile platform for mapping SAs of individual sEVs, which could significantly contribute to resolving the heterogeneity of sEVs and advancing the understanding of their glycosignature.

Early-Stage Protein Adsorption Sequence on Blood-Contacting Surfaces: Answer to Vroman's Question.

Plasma protein adsorption on blood-contacting surfaces is the initiating significant event and modulates the subsequent coagulation response. Despite decades of research in this area, Vroman's questions in 1986 "Who gets there first?" and "When does the next protein arrive?" remain unanswered due to the lack of detection techniques with sufficient temporal resolution. In this work, we develop a droplet microfluidic technology to detect protein adsorption sequences on six typical blood-contacting surfaces in milliseconds. Apolipoproteins (Apo) are found to be the first proteins to adsorb onto the surfaces in a plasma droplet, and the specific type of apolipoprotein depends on the surface. Apo CI is the first protein adsorbed on gold, platinum, graphene, stainless steel, and polyvinyl chloride with the adsorption time varying from 0.01 to 1 s, while Apo CIII preferentially reaches the titanium alloy surface within 1 s. Subsequent to the initial adsorption, Apo AI, AII, and other proteins continue to adsorb until albumin arrives. Thus, the adsorption sequence is revealed, and Vroman's questions are answered. Moreover, this finding demonstrates the influence of the initial protein adsorption on subsequent coagulation at the surface, and it offers new insights into the development of anticoagulant surfaces.