Journal of Colloid and Interface Science

Nanoparticles are promising alternatives to antibiotics since nanoparticles are easy to manufacture, non-toxic, and do not promote resistance. Nanoparticles act via physical disruption of the bacterial membrane and/or the generation of high concentrations of reactive-oxygen species locally. Potential for physical disruption of the bacterial membrane may be quantified by free energy methods, such as the extended Derjuan-Landau-Verwey-Overbeek theory, which predicts the initial surface-material interactions. The generation of reactive-oxygen species may be quantified using enthalpies of formation to predict minimum inhibitory concentrations. Neither of these two quantitative structure-activity values describes the dynamic, in situ behavioral changes in the bacterias struggle to survive. In this paper, borrowing parameters from logistic, oscillatory, and diauxic growth models, we use principal component analysis and agglomerative hierarchical clustering to classify survival modes across nanoparticle types and concentrations. We compare the growth parameters of 170 experimental interactions between nanoparticles and bacteria. The bacteria studied include Escherichia coli, Staphylococcus aureus, Methicillin-Resistant Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, and Helicobacter pylori, and were tested across multiple concentrations of liposomal drug delivery systems, amphiphilic peptide, and silver and selenium nanoparticles. Clustering reveals specific pairs of bacteria and nanoparticles where the nanoparticle induced growth dynamics could potentially spread the infection through the development of resistance and tolerance. This rapid screening also shows that bacteria generated nanoparticles do not induce growth modes indicative of the development of resistance. This methodology can be used to rapidly screen for novel therapeutics that do not induce resistance before using more robust intracellular content screening. This methodology can also be used as a quality check on batch manufactured nanoparticles.

The top layer of skin, the stratum corneum, provides a formidable barrier to the skin. Nanoparticles are utilized and further explored for personal and health care applications related to the skin. In past years several researchers have studied the translocation and permeation of nanoparticles of various shapes, sizes, and surface chemistry through the cell membranes. Most of these studies focused on a single nanoparticle and a simple bilayer system, whereas skin has a highly complex lipid membrane architecture. Moreover, it is highly unlikely that a nanoparticle formulation applied on the skin will not have multiple nanoparticle-nanoparticle and skin-nanoparticle interactions. In this study, we have utilized coarse-grained MARTINI molecular dynamics simulations to assess the interactions of two types (bare and dodecane-thiol coated) of nanoparticles with two models (single bilayer and double bilayer) of skin lipid membranes. The nanoparticles were found to be partitioned from the water layer to the lipid membrane as an individual entity as well as in the cluster form. It was discovered that each nanoparticle reached the interior of both single bilayer and double bilayer membrane irrespective of nanoparticle type and concentration, though coated particles were observed to efficiently traverse across bilayer when compared with bare particles. The coated nanoparticles also created a single large cluster inside the membrane, whereas bare nanoparticles were found in small clusters. Both the nanoparticles exhibited preferential interactions with cholesterol molecules present in the lipid membrane as compared to other lipid components of the membrane. We have also observed that the single membrane model exhibited unrealistic instability at moderate to the higher concentration of nanoparticles, and hence for translocation study, at minimum double bilayer model should be employed.

Microplastics and nanoplastics (MNPs), originating from plastic degradation, have arisen to be a threat to ecology and human health. Alarmingly, the penetration of MNPs across the highly selective blood-brain barrier (BBB) poses an emerging and urgent risk, yet its molecular mechanism remains unexplored. In this work, using long-time-scale (over 27 s) all-atom explicit solvent steered molecular dynamics, we examine the free energy of the passive permeation of four polymer nanoparticles: polyethylene, polypropylene, polystyrene, and polyethylene terephthalate. Polyethylene and polypropylene nanoparticles exhibited a remarkable preference for entering the BBB, attributed to their high hydrophobicity. Our study reveals that polymers can enter the BBB as polymerized nanoplastics and exit as dispersed polymer chains as the nanoparticles dissolve within the BBB. Further, the crystalline structure of polyethylene nanoparticles is found to adopt varying orientations. Our work advances the knowledge about the mechanism of nanoplastic penetration across the BBB, which could aid in the rational design of therapeutics for nanoplastic penetration inhibitors. TOC Graphics O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=151 SRC="FIGDIR/small/675462v1_ufig1.gif" ALT="Figure 1"> View larger version (71K): org.highwire.dtl.DTLVardef@1b20796org.highwire.dtl.DTLVardef@1102985org.highwire.dtl.DTLVardef@15fc22org.highwire.dtl.DTLVardef@15c2260_HPS_FORMAT_FIGEXP M_FIG C_FIG

A thick layer of mucus covering the gastrointestinal tract acts as an innate barrier guarding the epithelial surface. The high molecular weight and cross-linked glycoproteins (mucins), the major building blocks of mucus, can effectively obstruct or trap invading noxious substances, such as detrimental bacteria and virus. The mucus layer as well as any trapped material can be regularly removed by the friction force from food flow and gastrointestinal peristalsis, the process of which primarily relies on the viscoelastic and shear-thinning properties. Conversely, the process by which beneficial substances, such as drug nanoparticles, cross the mucus layer and contact the epithelium is also influenced by the chemical and rheological properties of the mucus layer. Gastrointestinal disorders, most notably colitis, are often accompanied by changes to the mucosal structure. In this study, we experimentally characterized the viscoelasticity and dynamic viscosity of mucus collected from human intestinal cells. In addition, we developed a bi-component mesoscopic-scale mucus model that contained Muc2, the dominant mucin secreted in healthy individuals, and Muc5AC, which is secreted by intestinal goblet cells in certain intestinal disorders. This model enabled us to study the effects of cross-linking and mucin concentration on rheological properties of mucus. Furthermore, we quantified changes in the diffusion dynamics of nanoparticles in mucus networks caused by factors such as the size of nanoparticles, nanoparticle-mucin interactions, and the degree of mucin cross-linking.

Biomolecular condensates driven by liquid-liquid phase separation (LLPS) have received attention as novel activity regulators of living organisms. In intracellular LLPS, an important question is what type of biomolecules form condensates under what conditions. In this regard, possible interactions between biomolecules have been investigated. Recently, LLPS condensates have been reported to regulate the membrane structure upon wetting. However, the possibility of membrane wetting, in which the membrane conversely regulates the LLPS, remains unexplored. Using droplets of short polyethylene glycol and long dextran blends encapsulated with a lipid membrane, we demonstrate that membrane wetting regulates LLPS in cell-size spaces and alters the equilibrium state. In smaller droplets, the two-phase region expands beyond the bulk system, and the fractionation degree increases, particularly during the separation between short PEG and long dextran. We explain the space-size dependent LLPS based on the competitive membrane wetting between the polymers. Smaller droplets promote the membrane wetting of short PEG, which enhances the depletion force between long dextran molecules and finally induces LLPS. This shows that competition for membrane wettability among various molecules can regulate LLPS in cell-size spaces, rendering this LLPS principle feasible in living cells.

As the central organelles of lipid and energy homeostasis in plant seeds, lipid droplets (LDs) consist of a neutral lipid core, decorated by phospholipids (prominently phosphatidylcholines, PCs) and surfactant-like proteins (mostly oleosins, OLs). So far, the dynamic interplay between PCs and OLs at the LD interface remains unclear. The presented work addresses this knowledge gap by reconstituting oil-in-water emulsions stabilized by OLs and PCs using microfluidic systems. Our results show that the resistance to droplet coalescence is primarily provided by OLs. We further reveal that the addition of PCs alters the assembly of OLs at the interface, reducing the OL network density and interfacial elasticity, thereby rendering a weaker interface. In conclusion, our study suggests complementary roles, with OLs acting as the primary stabilizers while PCs playing a destabilizing role. This contrast likely contributes to the observed metastability of LDs and can be exploited to design stimuli-responsive emulsions. HIGHLIGHTSO_LIOleosins form an interfacial network that stabilizes the oil-water interface. C_LIO_LIPhosphatidylcholines globally weaken the oleosin network and promote droplet coalescence. C_LIO_LIMicrofluidics enables controlled reconstitution and real-time analysis of lipid droplets. C_LIO_LIOleosin-phospholipid interplay explains lipid droplet metastability and can guide the design of bio-inspired emulsions. C_LI

This study provides the first comprehensive investigation into how pathogen-laden respiratory droplets transfer diseases via inanimate surfaces. Respiratory fluid ejections containing pathogens pose a significant health threat, especially in high-traffic areas like hospitals, public transport, restaurants, and schools. When these droplets dry on surfaces, they form deposits that can transfer pathogens to healthy individuals through contact and can be ingested via the oral or nasal route. The study examined the effects of varying salt and mucin concentrations in respiratory fluid droplets containing Pseudomonas aeruginosa (PA). Results showed that PA viability increased tenfold at elevated mucin concentrations, while changes in salt concentration had minimal impact. Adhesive properties of the deposits were analysed using atomic force spectroscopy and scotch tape test. Pathogen transfer from the deposit to a fingerprint patterned model thumb at different relative humidity (RH) levels was assessed using confocal microscopy, showing significant pathogen transfer at elevated RH. Out of 106 CFU/ml pathogens in deposits, 17% to 38% are potentially transferable, with most of the transfer occurring from the droplets edge deposits. The study also explored evaporation, internal flow, and precipitation dynamics, with deposit and pathogen distribution characterized by optical profilometry, scanning electron microscopy, and confocal microscopy.

Reference materials (RM)-assisted Rayleigh-Gans-Debye approximation (rm-RGDA) has been developed and used to in situ determine the size and thickness of the adlayer on the particles in solution. The particle size determined by rm-RGDA is quite close to that measured by electron microscopy but significantly smaller than that measured by DLS. The BSA adlayer absorbed on PS50, PS100 and SiO2 NPs is 3.3, 0.9 and 1.2 nm, respectively, and close to those observed by SEM, which is 4.6, 1.3 and 3.8 nm, respectively. The FTIR analysis results show that the BSA absorbed on larger particles or hydroxyl-abundant surface, e.g. PS100 and SiO2 NPs can lose its secondary structure, e.g. -helix, to a great extent and that absorbed on a more curve surface, e.g. smaller PS50 particles can largely preserve its secondary structure as its free state. The measurement results show the curvature of the NPs is closely related to the structure change of the adsorbed protein. This method provide a facile and new approach to measure the size and its adlayer change of the hybrid and core-shell structured nanoparticles in a wide range of wavelength. SIGNIFICANCEQuantitative study on the adsorption of the protein on colloidal nanoparticles is an important approach to understand the biophysical effect, compared with other ex situ methods such as TEM and SEM, where the specimen are undergone pre-processing and no longer the original state in measurement. It is, therefore, a big challenge. In order to cope with this challenge, UV-vis based RGDA has been developed and applied to in situ measure the size of the dispersed colloidal nanoparticles and their protein adlayer thickness, where the protein adlayer thickness on the colloidal nanoparticles can be easily determined. We believe this method provide a facile and sensitive way to in situ measure the dimension change of hybrid colloidal nanoparticles.

Underwater adhesion research increasingly draws on bioinspired systems to uncover the molecular mechanisms that enable strong interfacial binding in aqueous environments. The biofilm adhesin Bap1 from Vibrio cholerae contains a short peptide motif, SYWFFGWHTK (CP), which exhibits exceptional adhesive performance, surpassing mussel foot protein mfp5 under comparable conditions. Despite its promise, the roles of ionic environments and aggregation behavior in governing CP adhesion remain unclear. In this study, we investigate how ion identity influences CP aggregation, film formation, and interfacial properties. Using dynamic light scattering, we identify the formation of micron-scale assemblies of aggregated molecular clusters (AAMCs), with size distributions modulated by salt type. Quartz crystal microbalance with dissipation and liquid atomic force microscopy reveal that CP film formation is both surface- and ion-dependent. On gold substrates, AAMCs preferentially adsorb and collapse into rigid, smooth nanofilms, consistent with hydrophobic-driven compaction. In contrast, silicate surfaces inhibit such collapse, yielding distinct morphologies and interfacial energetics. These findings demonstrate that surface chemistry and ionic conditions jointly regulate peptide aggregation and adhesion. This work provides mechanistic insight into hydrophobic-rich peptide systems and informs the rational design of next-generation wet adhesives, with broader implications for biomaterials and peptide-based formulations. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=130 SRC="FIGDIR/small/733527v1_ufig1.gif" ALT="Figure 1"> View larger version (40K): org.highwire.dtl.DTLVardef@1bd012aorg.highwire.dtl.DTLVardef@1977892org.highwire.dtl.DTLVardef@16cf79borg.highwire.dtl.DTLVardef@f405bf_HPS_FORMAT_FIGEXP M_FIG C_FIG

In this letter, the pull-off forces of adsorbed films of four Bap1-inspired peptides in various solvents were investigated on negatively charged mica substrates using the surface forces apparatus (SFA), complemented with dynamic light scattering (DLS) for characterizing the aggregation behavior of peptides in solution. Bap1-inspired peptides consisted of the 57 amino acid wild-type sequence (WT); a scrambled version of the WT used to investigate the impact of the primary amino acid sequence in pull-off forces (Scr); a ten amino acid sequence rich in hydrophobic content (CP) of the WT sequence, and an eight amino acid sequence (Sh1) that corresponds to the pseudo-repeating sequence in the 57 AA. SFA results showed remarkable pull-off forces for CP, particularly in the presence of salts: measured pull-off forces were 26.0 {+/-} 7.0 mN/m for no dwell-time and up to 42.0 {+/-} 8.8 mN/m when surfaces were left in contact for 30 minutes. DLS observations indicate that salts favor large peptide aggregation for all constructs (Hz > 1 {micro}m), as compared to milliQ (Hz {approx} 100-500 nm) water and DMSO (Hz {approx} 100 nm), resulting in heterogeneous peptide film thicknesses. This letter concludes with a comparison to the pull-off forces of mussel foot protein-inspired peptides reported in the literature.

Plastic materials are widely used in engineered systems and increasingly accumulate in natural environments, where their surfaces interact with colloids, microorganisms, and dissolved organic matter. However, the relative roles of plastic surface properties versus particle-specific characteristics in governing organic matter retention remain poorly constrained. Here, attachment efficiency () was used to quantify intrinsic particle-collector affinity on three common thermoplastics (ABS, HDPE, HIPS) and glass beads as an inorganic reference. Surface chemistry, hydrophobicity, roughness, and charge were characterized, and interactions with submicron carbon particles (SCPs) and Escherichia coli were evaluated using column experiments. Extended DLVO (XDLVO) theory was applied to predict interaction energy barriers, and humic acid (HA) adsorption was quantified through batch isotherms. XDLVO modeling predicted higher affinity of particles for plastics relative to glass; however, experimentally measured attachment efficiencies were uniformly low ( < 0.05) across all materials. Attachment was primarily governed by particle size and surface charge rather than collector hydrophobicity, roughness, or surface chemistry. SCP consistently exhibited higher than bacteria, while differences among plastics were minor. Similarly, HA adsorption was weak and near-linear, with uptake following ABS {approx} HIPS > HDPE > glass, indicating reversible, partitioning-like association dominated by polymer-specific functionality rather than electrostatics. The absence of correlation between and XDLVO-predicted energy barriers further demonstrates limitations of classical physicochemical models in describing particle- plastic interactions. Collectively, these results indicate that pristine thermoplastic surfaces exhibit intrinsically low affinity for organic matter and that particle-specific properties dominate retention under low ionic strength conditions. Enhanced accumulation in environmental systems likely requires surface aging or conditioning processes not captured by classical interaction theory.

Biofilms in the built environment (BE) can harbor pathogens and have been linked with negative health outcomes, particularly in hospital environments. The formation of biofilms requires bacterial cell attachment on surfaces, such as hospital plumbing, which can have varying properties, including roughness, wettability, chemistry, and charge. Despite the importance of bacterial attachment to surfaces, the role of multiple surface properties has been minimally investigated. Using seven materials with differing surface characteristics, this work considers the initial attachment of Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis, and Staphylococcus aureus to investigate the impact of several surface characteristics. Initial attachment was evaluated using column experiments and compared to batch experiments in which bacterial growth on coupons was monitored. The attachment of all bacterial species was not influenced by material surface properties, with similar attachment seen across materials tested. Bacterial cell envelope morphology affected attachment, with gram-negative species displaying greater attachment than gram-positive species. Attachment efficiency () was found to be a good predictor of bacterial attachment, with greater sensitivity than batch tests. Establishment of commensal communities should be the focus for limiting pathogens in the BE, as engineering surfaces to reduce microbial attachment appears to offer limited benefit.

Self-assembling protein subunits hold great potential as biomaterials with improved functions. Among the self-assembled protein structures functional amyloids are promising unique properties such as resistance to harsh physical and chemical conditions their mechanical strength, and ease of functionalization. Curli proteins, which are functional amyloids of bacterial biofilms can be programmed as intelligent biomaterials. In order to obtain controllable curli based biomaterials for biomedical applications, and to understand role of each of the curli forming monomeric proteins (namely CsgA and CsgB from Escherichia coli) we characterized their binding kinetics to gold, hydroxyapatite, and silica surfaces. We demonstrated that CsgA, CsgB, and their equimolar mixture have different binding strengths for different surfaces. On hydroxyapatite and silica surfaces, CsgB is the crucial element that determines the final adhesiveness of the CsgA-CsgB mixture. On the gold surface, on the other hand, CsgA controls the behavior of the mixture. Those findings uncover the binding behavior of curli proteins CsgA and CsgB on different biomedically valuable surfaces to obtain a more precise control on their adhesion to a targeted surface.

Reentrant condensation (RC) is a phase behavior of protein solution comprising at least two components. In RC, a protein state varies from one phase to two phases and then back to one phase as the concentration of one component monotonically increases. To understand the phase behavior of multicomponent complex solutions of biomolecules, it is worth constructing an experimental multicomponent system that exhibits RC behavior. Here, we used a cola/milk mixture to investigate RC of a multicomponent complex system and explained the RC mechanism by reducing the system to two pure components, polyphosphate (polyP) and casein. In the multicomponent complex system, RC was observed with 20-60% cola and 1% milk. In the pure system, RC occurred with 0.01-2 mM tetraphosphate and 0.5 mg/ml casein. Moreover, the phase diagram showed that the condensation of casein depended on the chain length of the polyP. The present study succeeded in experimentally inducing RC in a multicomponent system and reproducing RC even when the system was reduced to its pure components. The fact that RC can be experimentally induced using common materials will provide important insights into the understanding of phase-separation behavior of biomolecules.

An instrument-free particle-templated droplet formation can be achieved upon simple mixing of amphiphilic particles with aqueous and oil phases in a well plate by using a common lab pipette. Here, a two-dimensional, two-phase flow model was established using a finite element method to mimic the droplet formation within a concentric amphiphilic particle, which consisted of an outer hydrophobic layer and an inner hydrophilic layer. Immiscible water and oil phases selectively interacted with the hydrophilic and hydrophobic layers of the particle, respectively, to form an isolated aqueous compartment within a cavity. Three extreme models were also simulated, including completely hydrophilic, completely hydrophobic, and oppositely amphiphilic particle, which indicated that a right order of the particle layers was necessary to capture the droplet inside the cavity. Moreover, we performed a systematic study of particle-templated droplet formation by varying the individual layer thicknesses of particle, particle height, interfacial tension between water and oil, contact angle of interface with different surfaces, velocity of incoming oil media, and distance between neighboring particles. The volume fraction of water droplet trapped within the target cavity region was calculated to characterize the droplet formation. Our work will help to optimize the particle fabrication process, predict the experiment droplet formation, and explain the physical mechanism underlying compartmentalization phenomena.

Surface functionalization of inorganic quantum dot nanoparticles is of great interest in the application of these materials toward a wide range of biological applications where membrane interactions are critical. The use of amphiphilic lipids to functionalize the surfaces of quantum dots represents a promising alternative to produce water-soluble and membrane-active materials with facile tuning of the quantum dots surface properties. Here, we demonstrate an experimental approach that yields lipid-coated quantum dots with highly tunable surface charge by controlling the concentration of cationic lipids during preparation. Through fluorescence-activated cell sorting assays, we show that these cationic lipid-coated quantum dots can enhance membrane interactions and increase membrane labeling density in live HEK293 cells. We further employed coarse-grained molecular dynamics simulations to model the lipid self-assembly process using an implicit solvent force field and subsequently model the adsorption of lipid-coated quantum dots to model membranes. Our simulations show that we can control the effective surface charge of lipid-coated quantum dots and influence the strength of adsorption to oppositely charged lipid membranes, a process that is mediated by the release of counterions at the quantum dot-membrane interface. This work supports the future development of biocompatible and water-soluble inorganic nanoparticles with highly tunable surfaces, and provides mechanistic insight into how different lipids can influence nanoparticle-membrane interactions at a molecular scale.

Airborne transmission of pathogens occurs via aerosol particles, whose morphology provides insights into the microenvironments that pathogens experience. Aerosol morphology includes particle size, shape, phase state, and chemical homogeneity, yet systematic studies remain limited. Here, we characterized model bioaerosol morphologies generated from (1) NaCl-organic two-component mixtures, (2) common cell culture media, and (3) artificial respiratory fluids. Particles were collected using a virtual impactor and Andersen cascade impactor and analyzed by scanning electron microscopy (SEM) and energy-dispersive X-ray spectroscopy (EDX). Results show that organic components modulate the morphology: dipalmitoylphosphatidylcholine (DPPC) promotes organic-inorganic phase separation while proteins prohibit formation of large crystals and leads to better mixing among components. At 30% RH with a drying period of 10 seconds, most aerosols appeared desiccated, though NaCl-glucose, DMEM-complete-media and artificial saliva with mucin remained semi-solid or gel-like. Among all formulations examined EMEM-complete-media and artificial saliva (non mucin) show a size-dependent morphology. Our study demonstrates how chemical composition and size alters surrogate bioaerosol phase (semi-solid or solid) and morphology and provides new insights into the microenvironment of aerosol particles for aerovirology investigations.

Respiratory infections by RNA viruses are one of the major burdens upon global health and economy. Viruses like influenza or coronaviruses can be transmitted through respiratory droplets or contaminated surfaces. An effective antiviral coating can decrease the viability of the virus particles in the outside environment significantly, hence reducing their transmission rate. In this work, we have screened a series of nanoparticles and their composites for antiviral activity using Nano Luciferase based highly sensitive influenza A reporter virus. Using this screening system, we have identified copper-graphene (Cu-Gr) nanocomposite shows strong antiviral activity. Extensive material and biological characterization of the nanocomposite suggested a unique metal oxide embedded graphene sheet architecture that can inactivate the virion particles only within 30 minutes of pre-incubation and subsequently interferes with the entry of these virion particles into the host cell. This ultimately results in reduced viral gene expression, replication and production of progeny virus particles, slowing down the overall pace of progression of infection. Using PVA as a capping agent, we have been able to generate a Cu-Gr nanocomposite based highly transparent coating that retains its original antiviral activity in the solid form.

The rapid emergence of drug-resistant bacteria and fungi poses a threat for healthcare worldwide. The development of novel effective small molecule therapeutic strategies in this space has remained challenging. An orthogonal approach, therefore, is to explore biomaterials with physical modes of action which have the potential to generate antimicrobial activity and in some cases even prevent antimicrobial resistance. Here, to this effect, we describe an approach for forming silk-based films that contain embedded selenium nanoparticles. We show that these materials exhibit both antibacterial and antifungal properties while crucially also remaining highly biocompatible and non-cytotoxic towards mammalian cells. By incorporating the nanoparticles into silk films, the protein scaffold acts in a two-fold manner; it protects the mammalian cells from the cytotoxic effects of the bare nanoparticles, while also providing a template for bacterial and fungal eradication. A range of hybrid inorganic/organic films were produced and an optimum concentration was found, which allowed for both high bacterial and fungal death while also exhibiting low mammalian cell cytotoxicity. Such films can thus pave the way for next generation antimicrobial materials for applications such as wound healing and as agents against topical infections, with the added benefit that bacteria and fungi are unlikely to develop antimicrobial resistance to these hybrid materials.

We introduce a method for conjugating antigens to gold nanoparticles (GNPs) while synthesizing them using gas plasma, which eliminates the need for chemical linkers intended to facilitate the conjugation procedure for immunotherapy purposes. We report a physical approach to conjugate antigen Nestin (NES) as a marker in malignant tumors to GNPs. Two approaches were used to perform the conjugation of GNPs and NES. The first method involved using citrate to synthesize GNPs, and then NES was conjugated onto the GNPs surface by plasma. In the second method, GNPs were simultaneously synthesized and linker-freely conjugated to NES by plasma treatment. Enzyme-linked immunosorbent assay with the protocol defined in this study, Zeta-sizer, Ultraviolet-visible spectroscopy, and Transmission Electron Microscopy results confirmed NES conjugation to GNPs. In addition, the toxicity of the prepared samples was investigated in vitro using peripheral blood mononuclear cells (PBMCs) and flow cytometry, which proved the non-toxicity of the samples. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=113 SRC="FIGDIR/small/570950v2_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@1bffa43org.highwire.dtl.DTLVardef@c75811org.highwire.dtl.DTLVardef@166f4b3org.highwire.dtl.DTLVardef@4b4def_HPS_FORMAT_FIGEXP M_FIG C_FIG