RESUMO
The products of the SuFEx reaction between sulfonimidoyl fluorides and phenols, sulfonimidates, are shown to display dynamic covalent chemistry with other phenols. This reaction was shown to be enantiospecific, finished in minutes at room temperature in high yields, and useful for both asymmetric synthesis and sustainable polymer production. Its wide scope further extends the usefulness of SuFEx and related click chemistries.
Assuntos
Polímeros , Enxofre , Química Click , Estrutura Molecular , FenóisRESUMO
Laccase-mediated grafting on lignocelluloses has gained considerable attention as an environmentally benign method to covalently modify wood, paper and cork. In recent decades this technique has also been employed to modify fibres with a polysaccharide backbone, such as cellulose or chitosan, to infer colouration, antimicrobial activity or antioxidant activity to the material. The scope of this approach has been further widened by researchers, who apply mediators or high redox potential laccases and those that modify synthetic polymers and proteins. In all cases, the methodology relies on one- or two-electron oxidation of the surface functional groups or of the graftable molecule in solution. However, similar results can very often be achieved through simple deposition, even after extensive washing. This unintended adsorption of the active substance could have an adverse effect on the durability of the applied coating. Differentiating between actual covalent binding and adsorption is therefore essential, but proves to be challenging. This review not only covers excellent research on the topic of laccase-mediated grafting over the last five to ten years, but also provides a critical comparison to highlight either the lack or presence of compelling evidence for covalent grafting.
Assuntos
Lacase/metabolismo , Polímeros/metabolismo , Biopolímeros/química , Biopolímeros/metabolismo , Lacase/química , Estrutura Molecular , Oxirredução , Polímeros/química , Rhus/enzimologia , Trametes/enzimologiaRESUMO
Superhydrophobic surfaces with micro/nanostructures are widely used to prevent nonspecific adsorption of commercial polymeric and/or biological materials. Herein, a self-healing superhydrophobic and highly protein-repellent fluoropolymer brush was grafted onto nanostructured silicon by surface-initiated atom transfer radical polymerization (ATRP). Both the superhydrophobicity and antifouling properties (as indicated for isolated protein solutions and for 10% blood plasma) are well repaired upon serious chemical degradation (by e.g. air plasma). This brush still maintains excellent superhydrophobicity and good antifouling properties even after 5 damage-repair cycles, which opens a new door to fabricate long-term antifouling coatings on various substrates that can be used in harsh environments.
Assuntos
Polímeros/química , Proteínas/química , Adsorção , Animais , Bovinos , Interações Hidrofóbicas e Hidrofílicas , Plasma/química , Polimerização , Soroalbumina Bovina/química , Propriedades de SuperfícieRESUMO
Oils, fats, carbohydrates, lignin, and amino acids are all important raw materials for the production of biorenewables. These compounds already play an important role in everyday life in the form of wood, fabrics, starch, paper and rubber. Enzymatic reactions do, in principle, allow the transformation of these raw materials into biorenewables under mild and sustainable conditions. There are a few examples of processes using immobilised enzymes that are already applied on an industrial scale, such as the production of High-Fructose Corn Syrup, but these are still rather rare. Fortunately, there is a rapid expansion in the research efforts that try to improve this, driven by a combination of economic and ecological reasons. This review focusses on those efforts, by looking at attempts to use fatty acids, carbohydrates, proteins and lignin (and their building blocks), as substrates in the synthesis of biorenewables using immobilised enzymes. Therefore, many examples (390 references) from the recent literature are discussed, in which we look both at the specific reactions as well as to the methods of immobilisation of the enzymes, as the latter are shown to be a crucial factor with respect to stability and reuse. The applications of the renewables produced in this way range from building blocks for the pharmaceutical and polymer industry, transport fuels, to additives for the food industry. A critical evaluation of the relevant factors that need to be improved for large-scale use of these examples is presented in the outlook of this review.
Assuntos
Carboidratos/biossíntese , Enzimas Imobilizadas/metabolismo , Ácidos Graxos/biossíntese , Lignina/biossíntese , Proteínas/metabolismo , Carboidratos/química , Enzimas Imobilizadas/química , Ácidos Graxos/química , Lignina/química , Proteínas/químicaRESUMO
This paper presents a novel surface engineering approach that combines photochemical grafting and surface-initiated atom transfer radical polymerization (SI-ATRP) to attach zwitterionic polymer brushes onto indium tin oxide (ITO) substrates. The photochemically grafted hydroxyl-terminated organic layer serves as an excellent platform for initiator attachment, and the zwitterionic polymer generated via subsequent SI-ATRP exhibits very good antifouling properties. Patterned polymer coatings can be obtained when the surface with covalently attached initiator was subjected to photomasked UV-irradiation, in which the C-Br bond that is present in the initiator was broken upon exposure to UV light. A further, highly versatile top-functionalization of the zwitterionic polymer brush was achieved by a strain-promoted alkyne-azide cycloaddition, without compromising its antifouling property. The attached bioligand (here: biotin) enables the specific immobilization of target proteins in a spatially confined fashion, pointing to future applications of this approach in the design of micropatterned sensing platforms on ITO substrates.
Assuntos
Incrustação Biológica/prevenção & controle , Polímeros/química , Polímeros/farmacologia , Compostos de Estanho/química , Alcinos/química , Animais , Azidas/química , Biotina/química , Bovinos , Reação de Cicloadição , Processos Fotoquímicos , Polimerização , Estreptavidina/química , Propriedades de SuperfícieRESUMO
A new method for attaching antibodies to protein-repellent zwitterionic polymer brushes aimed at recognizing microorganisms while preventing the nonspecific adsorption of proteins is presented. The poly(sulfobetaine methacrylate) (SBMA) brushes were grafted from α-bromo isobutyryl initiator-functionalized silicon nitride (Si(x)N(4), x ≥ 3) surfaces via controlled atom-transfer radical polymerization (ATRP). A trifunctional tris(2-aminoethyl)amine linker was reacted with the terminal alkylbromide of polySBMA chains. N-Hydroxysuccinimide (NHS) functionalization was achieved by reacting the resultant amine-terminated polySBMA brush with bifunctional suberic acid bis(N-hydroxysuccinimide ester). Anti-Salmonella antibodies were subsequently immobilized onto polySBMA-grafted Si(x)N(4) surfaces through these NHS linkers. The protein-repellent properties of the polySBMA-grafted surface after antibody attachment were evaluated by exposing the surfaces to Alexa Fluor 488-labeled fibrinogen (FIB) solution (0.1 g·L(-1)) for 1 h at room temperature. Confocal laser scanning microscopy (CLSM) images revealed the minimal adsorption of FIB onto the antibody-coated polySBMA in comparison with that of antibody-coated epoxide monolayers and also bare Si(x)N(4) surfaces. Subsequently, the interaction of antibodies immobilized onto polySBMA with SYTO9-stained Salmonella solution without using blocking solution was examined by CLSM. The fluorescent images showed that antibody-coated polySBMA efficiently captured Salmonella with only low background noise as compared to antibody-coated monolayers lacking the polymer brush. Finally, the antibody-coated polySBMA surfaces were exposed to a mixture of Alexa Fluor 647-labeled FIB and Salmonella without the prior use of a blocking solution to evaluate the ability of the surfaces to capture bacteria while simultaneously repelling proteins. The fluorescent images showed the capture of Salmonella with no adsorption of FIB as compared to antibody-coated epoxide surfaces, demonstrating the potential of the zwitterionic layer in preventing the nonspecific adsorption of the proteins during the detection of bacteria in complex matrices.
Assuntos
Polímeros/química , Proteínas/química , Compostos de Silício/química , Microscopia ConfocalRESUMO
BACKGROUND: Polymer nanoparticles (PNP) are becoming increasingly important in nanomedicine and food-based applications. Size and surface characteristics are often considered to be important factors in the cellular interactions of these PNP, although systematic investigations on the role of surface properties on cellular interactions and toxicity of PNP are scarce. RESULTS: Fluorescent, monodisperse tri-block copolymer nanoparticles with different sizes (45 and 90 nm) and surface charges (positive and negative) were synthesized, characterized and studied for uptake and cytotoxicity in NR8383 and Caco-2 cells. All types of PNP were taken up by the cells. The positive smaller PNP45 (45 nm) showed a higher cytotoxicity compared to the positive bigger PNP(90) (90 nm) particles including reduction in mitochondrial membrane potential (ΔΨ(m)), induction of reactive oxygen species (ROS) production, ATP depletion and TNF-α release. The negative PNP did not show any cytotoxic effect. Reduction in mitochondrial membrane potential (ΔΨ(m)), uncoupling of the electron transfer chain in mitochondria and the resulting ATP depletion, induction of ROS and oxidative stress may all play a role in the possible mode of action for the cytotoxicity of these PNP. The role of receptor-mediated endocytosis in the intracellular uptake of different PNP was studied by confocal laser scanning microscopy (CLSM). Involvement of size and charge in the cellular uptake of PNP by clathrin (for positive PNP), caveolin (for negative PNP) and mannose receptors (for hydroxylated PNP) were found with smaller PNP45 showing stronger interactions with the receptors than bigger PNP(90). CONCLUSIONS: The size and surface characteristics of polymer nanoparticles (PNP; 45 and 90 nm with different surface charges) play a crucial role in cellular uptake. Specific interactions with cell membrane-bound receptors (clathrin, caveolin and mannose) leading to cellular internalization were observed to depend on size and surface properties of the different PNP. These properties of the nanoparticles also dominate their cytotoxicity, which was analyzed for many factors. The effective reduction in the mitochondrial membrane potential (ΔΨ(m)), uncoupling of the electron transfer chain in mitochondria and resulting ATP depletion, induction of ROS and oxidative stress likely all play a role in the mechanisms behind the cytotoxicity of these PNP.
Assuntos
Enterócitos/efeitos dos fármacos , Nanopartículas/toxicidade , Polímeros/toxicidade , Células CACO-2 , Sobrevivência Celular/efeitos dos fármacos , Enterócitos/metabolismo , Enterócitos/patologia , Fluorescência , Humanos , Macrófagos , Macrófagos Alveolares , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Nanopartículas/química , Estresse Oxidativo/efeitos dos fármacos , Tamanho da Partícula , Fagocitose/efeitos dos fármacos , Polímeros/química , Polímeros/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Propriedades de SuperfícieRESUMO
Zwitterionic poly(sulfobetaine acrylamide) (SBMAA) brushes were grafted from silicon-rich silicon nitride (SixN4, x > 3) surfaces by atom transfer radical polymerization (ATRP) and studied in protein adsorption experiments. To this aim ATRP initiators were immobilized onto SixN4 through stable Si-C linkages via three consecutive reactions. A UV-induced reaction of 1,2-epoxy-9-decene with hydrogen-terminated SixN4 surfaces was followed by conversion of the epoxide with 1,2-ethylenediamine resulting in primary and secondary amine-terminated surfaces. A reaction with 2-bromoisobutyryl bromide led to ATRP initiator-covered surfaces. Zwitterionic polymer brushes of SBMAA were grown from these initiator-coated surfaces (thickness â¼30 nm), and the polymer-coated surfaces were characterized in detail by static water contact angle measurements, X-ray photoelectron spectroscopy (XPS), and an atomic force microscope (AFM). The adsorption of proteins onto zwitterionic polymer coated surfaces was evaluated by in situ reflectometry, using a fibrinogen (FIB) solution of 0.1 g·L(-1), and compared to hexadecyl-coated SixN4 surfaces (C16-SixN4), uncoated air-based plasma oxidized SixN4 surfaces (SiOy-SixN4), and hexa(ethylene oxide)-coated SixN4 surfaces (EO6-SixN4). Excellent protein repellence (>99%) was observed for these zwitterionic polymer-coated SixN4 surfaces during exposure to FIB solution as compared to C16-SixN4 surfaces. Furthermore, the stability of these zwitterionic polymer-coated SixN4 surfaces was surveyed by exposing the surfaces for 1 week to phosphate buffered saline (PBS) solution at room temperature. The zwitterionic polymer-coated SixN4 surfaces before and after exposure to PBS solution were characterized by XPS, AFM, and water contact angle measurements, and their protein-repelling properties were evaluated by reflectometry. After exposure to PBS solution, the zwitterionic polymer coating remained intact, and its thickness was unchanged within experimental error. No hydrolysis was observed for the zwitterionic polymer after 1 week exposure to PBS solution, and the surfaces still repelled 98% FIB as compared to C16-SixN4 surfaces, demonstrating the long-term efficiency of these easily prepared surface coatings.
Assuntos
Betaína/análogos & derivados , Proteínas/química , Compostos de Silício/química , Resinas Acrílicas , Adsorção , Betaína/química , Estrutura Molecular , Estabilidade Proteica , Propriedades de SuperfícieRESUMO
The preparation and characterization of self-assembled monolayers on copper with n-alkyl and functional thiols was investigated. Well-ordered monolayers were obtained, while the copper remained oxide-free. Direct attachment of N-succinimidyl mercaptoundecanoate (NHS-MUA) onto the copper surface allowed for the successful attachment of biomolecules, such as ß-d-glucosamine, the tripeptide glutathione, and biotin. Notably, the copper surfaces remained oxide-free even after two reaction steps. All monolayers were characterized by static water contact angle measurements, X-ray photoelectron spectroscopy, and infrared reflection absorption spectroscopy. In addition, the biotinylated copper surfaces were employed in the immobilization of biomolecules such as streptavidin.
Assuntos
Álcoois/química , Ácidos Carboxílicos/química , Cobre/química , Membranas Artificiais , Succinimidas/química , Compostos de Sulfidrila/química , Estrutura Molecular , Tamanho da Partícula , Estreptavidina/química , Propriedades de SuperfícieRESUMO
A new method is presented for the fast and reproducible functionalization of silicon and silicon nitride surfaces coated with covalently attached alkyl monolayers. After formation of a methyl-terminated 1-hexadecyl monolayer on H-terminated Si(100) and Si(111) surfaces, short plasma treatments (1-3 s) are sufficient to create oxidized functionalities without damaging the underlying oxide-free silicon. The new functional groups can, e.g., be derivatized using the reaction of surface aldehyde groups with primary amines to form imine bonds. In this way, plasma-treated monolayers on silicon or silicon nitride surfaces were successfully coated with nanoparticles, or proteins such as avidin. In addition, we demonstrate the possibility of micropatterning, using a soft contact mask during the plasma treatment. Using water contact angle measurements, ellipsometry, XPS, IRRAS, AFM, and reflectometry, proof of principle is demonstrated of a yet unexplored way to form patterned alkyl monolayers on oxide-free silicon surfaces.
Assuntos
Materiais Revestidos Biocompatíveis/química , Plasma/química , Compostos de Silício/química , Silício/química , Avidina/química , Microscopia de Força Atômica , Modelos Teóricos , Nanopartículas/química , Oxirredução , Espectroscopia Fotoeletrônica , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
In this work a novel room-temperature bonding technique based on chemically activated Fluorinated Ethylene Propylene (FEP) sheet as an intermediate between chemically activated substrates is presented. Surfaces of silicon and glass substrates are chemically modified with APTES bearing amine terminal groups, while FEP sheet surfaces are treated to form carboxyl groups and subsequently activated by means of EDC-NHS chemistry. The activation procedures of silicon, glass and FEP sheet are characterized by contact angle measurements and XPS. Robust bonds are created at room-temperature by simply pressing two amine-terminated substrates together with activated FEP sheet in between. Average tensile strengths of 5.9 MPa and 5.2 MPa are achieved for silicon-silicon and glass-glass bonds, respectively, and the average fluidic pressure that can be operated is 10.2 bar. Moreover, it is demonstrated that FEP-bonded microfluidic chips can handle mild organic solvents at elevated pressures without leakage problems. This versatile room-temperature intermediate layer bonding technique has a high potential for bonding, packaging, and assembly of various (bio-) chemical microfluidic systems and MEMS devices.
Assuntos
Técnicas Analíticas Microfluídicas/métodos , Temperatura , Aminas/química , Vidro/química , Fenômenos Mecânicos , Politetrafluoretileno/análogos & derivados , Politetrafluoretileno/química , Pressão , Silício/químicaRESUMO
Tumor-derived extracellular vesicles (tdEVs) are promising blood biomarkers for cancer disease management. However, blood is a highly complex fluid that contains multiple objects in the same size range as tdEVs (30 nm-1 µm), which obscures an unimpeded analysis of tdEVs. Here, we report a multi-modal analysis platform for the specific capture of tdEVs on antibody-functionalized stainless steel substrates, followed by their analysis using SEM, Raman spectroscopy and AFM, at the single EV level in terms of size and size distribution, and chemical fingerprint. After covalent attachment of anti-EpCAM (epithelial cell adhesion molecule) antibodies on stainless steel substrates, EV samples derived from a prostate cancer cell line (LnCAP) were flushed into a microfluidic device assembled with this stainless steel substrate for capture. To track the captured objects between the different analytical instruments and subsequent correlative analysis, navigation markers were fabricated onto the substrate from a cyanoacrylate glue. Specific capture of tdEVs on the antibody-functionalized surface was demonstrated using SEM, AFM and Raman imaging, with excellent correlation between the data acquired by the individual techniques. The particle distribution was visualized with SEM. Furthermore, a characteristic lipid-protein band at 2850-2950 cm-1 was observed with Raman spectroscopy, and with AFM the size distribution and surface density of the captured EVs was assessed. Finally, correlation of SEM and Raman images enabled discrimination of tdEVs from cyanoacrylate glue particles, highlighting the capability of this multi-modal analysis platform for distinguishing tdEVs from contamination. The trans-instrumental compatibility of the stainless steel substrate and the possibility to spatially correlate the images of the different modalities with the help of the navigation markers open new avenues to a wide spectrum of combinations of different analytical and imaging techniques for the study of more complex EV samples.
Assuntos
Fracionamento Celular/métodos , Vesículas Extracelulares/metabolismo , Microscopia de Força Atômica , Microscopia Eletrônica de Varredura , Análise Espectral Raman , Anticorpos Imobilizados/química , Linhagem Celular Tumoral , Dimetilpolisiloxanos , Humanos , Nylons , Aço Inoxidável/químicaRESUMO
An amphiphilic silk-like protein polymer was efficiently produced in the yeast Pichia pastoris. The secreted product was fully intact and was purified by solubilization in formic acid and subsequent precipitation of denatured host proteins upon dilution with water. In aqueous alkaline solution, the negatively charged acidic polymer assumed extended helical (silk III-like) and unordered conformations. Upon subsequent drying, it assumed a conformation rich in beta-turns. In water at low pH, the uncharged polymer aggregated and the solution became turbid. Concentrated solutions in 70% (v/v) formic acid slowly formed gels. Replacement of the formic acid-water mixture with methanol and subsequent drying resulted in beta-sheets, which stacked into fibril-like structures. The novel polymer instantaneously lowered the air-water interfacial tension under neutral to alkaline conditions and reversed the polarity of hydrophobic and hydrophilic solid surfaces upon adsorption.
Assuntos
Biopolímeros/química , Proteínas Fúngicas/química , Proteínas/química , Adsorção , Biopolímeros/biossíntese , Biopolímeros/isolamento & purificação , Proteínas Fúngicas/biossíntese , Proteínas Fúngicas/isolamento & purificação , Concentração de Íons de Hidrogênio , Transição de Fase , Pichia , Conformação Proteica , Estrutura Secundária de Proteína , Proteínas/isolamento & purificação , Proteínas/metabolismo , SolventesRESUMO
Cyclic siloxanes with pendent naphthalene diimide groups were synthesized via hydrosilylation to form amorphous electron-accepting compounds. Photophysical measurements and >99.9% fluorescence quenching of well-known p-type polymers by the siloxanes demonstrate that these siloxanes form a new class of highly efficient n-type materials that provide some control over intermolecular interactions.
Assuntos
Fenantrolinas/química , Siloxanas/síntese química , Imidas , Estrutura Molecular , Naftalenos , Sensibilidade e Especificidade , Siloxanas/química , Espectrometria de Fluorescência/métodosAssuntos
Barreira Hematoencefálica/metabolismo , Peptídeos/metabolismo , Polímeros/metabolismo , Animais , Transporte Biológico Ativo , Barreira Hematoencefálica/química , Células Cultivadas , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Nanopartículas/administração & dosagem , Nanopartículas/química , Nanotecnologia , Peptídeos/química , Polímeros/administração & dosagem , Polímeros/químicaRESUMO
Micron- and nano-sized particles are extensively used in various biomedical applications. However, their performance is often drastically hampered by the nonspecific adsorption of biomolecules, a process called biofouling, which can cause false-positive and false-negative outcomes in diagnostic tests. Although antifouling coatings have been extensively studied on flat surfaces, their use on micro- and nanoparticles remains largely unexplored, despite the widespread experimental (specifically, clinical) uncertainties that arise because of biofouling. Here, we describe the preparation of magnetic micron-sized beads coated with zwitterionic sulfobetaine polymer brushes that display strong antifouling characteristics. These coated beads can then be equipped with recognition elements of choice, to enable the specific binding of target molecules. First, we present a proof of principle with biotin-functionalized beads that are able to specifically bind fluorescently labeled streptavidin from a complex mixture of serum proteins. Moreover, we show the versatility of the method by demonstrating that it is also possible to functionalize the beads with mannose moieties to specifically bind the carbohydrate-binding protein concanavalin A. Flow cytometry was used to show that thus-modified beads only bind specifically targeted proteins, with minimal/near-zero nonspecific protein adsorption from other proteins that are present. These antifouling zwitterionic polymer-coated beads, therefore, provide a significant advancement for the many bead-based diagnostic and other biosensing applications that require stringent antifouling conditions.
Assuntos
Microesferas , Adsorção , Incrustação Biológica , Citometria de Fluxo , Íons , PolímerosRESUMO
Sensitivity of biosensors depends on the orientation of bio-receptors on the sensor surface. The objective of this study was to organize bio-receptors on surfaces in a way that their analyte binding site is exposed to the analyte solution. VHH proteins recognizing foot-and-mouth disease virus (FMDV) were used for making biosensors, and azides were introduced in the VHH to function as bioorthogonal reactive groups. The importance of the orientation of bio-receptors was addressed by comparing sensors with randomly oriented VHH (with multiple exposed azide groups) to sensors with uniformly oriented VHH (with only a single azide group). A surface plasmon resonance (SPR) chip exposing cyclooctyne was reacted to azide functionalized VHH domains, using click chemistry. Comparison between randomly and uniformly oriented bio-receptors showed up to 800-fold increase in biosensor sensitivity. This technique may increase the containment of infectious diseases such as FMDV as its strongly enhanced sensitivity may facilitate early diagnostics.
Assuntos
Anticorpos Antivirais/imunologia , Técnicas Biossensoriais/instrumentação , Camelídeos Americanos/imunologia , Vírus da Febre Aftosa/imunologia , Vírus da Febre Aftosa/isolamento & purificação , Imunoensaio/instrumentação , Ressonância de Plasmônio de Superfície/instrumentação , Animais , Desenho de Equipamento , Análise de Falha de Equipamento , Conformação Proteica , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
Sensitivity of immune cells (coelomocytes) of Lumbricus rubellus earthworms was investigated for exposure to selected nanoparticles, in order to obtain further insight in mechanisms of effects observed after in vivo C60 exposure. In the in vivo study, tissue damage appeared to occur without accompanying increased immune responses. Coelomocytes exposed in vitro to C60 showed no decrease of their cellular viability, but demonstrated a decrease in gene expression of the cytokine-like protein CCF-1, indicating immunosuppression. Experiments with NR8383 rat macrophage cells and tri-block copolymer nanoparticles were used to compare sensitivity and to demonstrate the usefulness of coelomocytes as a test system for nano-immunotoxicity, respectively. Overall, the results imply that sensitivity towards nanoparticles differs between cell types and nanoparticles. Moreover, this study indicates that injuries in absence of an immune response, observed after in vivo C60 exposure in our earlier work, are caused by immunosuppression rather than coelomocyte mortality.
Assuntos
Sobrevivência Celular/efeitos dos fármacos , Macrófagos Alveolares/efeitos dos fármacos , Nanopartículas/toxicidade , Oligoquetos/citologia , Oligoquetos/efeitos dos fármacos , Animais , Linhagem Celular , Citocinas/análise , Citocinas/metabolismo , Fulerenos/química , Fulerenos/toxicidade , Macrófagos Alveolares/metabolismo , Nanopartículas/química , Fagocitose/efeitos dos fármacos , Polímeros/química , Polímeros/toxicidade , RatosRESUMO
Uniform orientation of capture molecules on biosensors has been reported to increase sensitivity. Here it is investigated which analyte properties contribute to sensitivity by orientation. Orientation of capture molecules on biosensors was investigated using variable domains of llama heavy-chain antibodies (VHHs) as capture molecule, and a surface plasmon resonance (SPR) chip as biosensor. Two VHHs were tested in this study: one recognizing foot-and-mouth disease virus (FMDV) and another recognizing the 16 kDa heat-shock protein of Mycobacterium tuberculosis. SPR chips with randomly immobilized biotinylated VHHs were compared to streptavidin-coated SPR chips, on which similar quantities of oriented biotinylated VHHs were non-covalently immobilized. Analytes that differ in molecular weight, epitope number and epitope affinity were compared using the FMDV-recognizing VHH. When binding of intact FMDV particles (146 S; 8200 kDa) or pentameric FMDV coat protein aggregates (12 S; 282 kDa) was detected, a modest (1-2-fold) increase in sensitivity was observed. When a 26-residue peptide (3 kDa) containing the epitope for VHH recognition was tested, much larger effects of capture molecule orientation (14-fold) on signal were observed. A 20-227-fold improvement was also observed when the epitope peptide was covalently linked to bovine serum albumin (67 kDa) or R-phycoerythrin (240 kDa). The results indicate that orientation of the capture molecule hardly affects high-affinity interactions, while it leads to strong improvements in sensitivity for lower-affinity interactions.