Carboxymethylated Dextran Layer Research Articles

Ultrasensitive Upconversion Nanoparticle Immunoassay for Human Serum Cardiac Troponin I Detection Achieved with Resonant Waveguide Grating.

Selective detection of biomarkers at low concentrations in blood is crucial for the clinical diagnosis of many diseases but remains challenging. In this work, we aimed to develop an ultrasensitive immunoassay that can detect biomarkers in serum with an attomolar limit of detection (LOD). We proposed a sandwich-type heterogeneous immunosensor in a 3 × 3 well array format by integrating a resonant waveguide grating (RWG) substrate with upconversion nanoparticles (UCNPs). UCNPs were used to label a target biomarker captured by capture antibody molecules immobilized on the surface of the RWG substrate, and the RWG substrate was used to enhance the upconversion luminescence (UCL) of UCNPs through excitation resonance. The LOD of the immunosensor was greatly reduced due to the increased UCL of UCNPs and the reduction of nonspecific adsorption of detection antibody-conjugated UCNPs on the RWG substrate surface by coating the RWG substrate surface with a carboxymethyl dextran layer. The immunosensor exhibited an extremely low LOD [0.24 fg/mL (9.1 aM)] and wide detection range (1 fg/mL to 100 pg/mL) in the detection of cardiac troponin I (cTnI). The cTnI concentrations in human serum samples collected at different times during cyclophosphamide, epirubicin, and 5-fluorouracil (CEF) chemotherapy in a breast cancer patient were measured by an immunosensor, and the results showed that the CEF chemotherapy did cause cardiotoxicity in the patient. Having a higher number of wells in such an array-based biosensor, the sensor can be developed as a high-throughput diagnostic tool for clinically important biomarkers.

Biomimetic Dextran-Based Hydrogel Layers for Cell Micropatterning over Large Areas Using the FluidFM BOT Technology.

Micropatterning of living single cells and cell clusters over millimeter-centimeter scale areas is of high demand in the development of cell-based biosensors. Micropatterning methodologies require both a suitable biomimetic support and a printing technology. In this work, we present the micropatterning of living mammalian cells on carboxymethyl dextran (CMD) hydrogel layers using the FluidFM BOT technology. In contrast to the ultrathin (few nanometers thick in the dry state) CMD films generally used in label-free biosensor applications, we developed CMD layers with thicknesses of several tens of nanometers in order to provide support for the controlled adhesion of living cells. The fabrication method and detailed characterization of the CMD layers are also described. The antifouling ability of the CMD surfaces is demonstrated by in situ optical waveguide lightmode spectroscopy measurements using serum modeling proteins with different electrostatic properties and molecular weights. Cell micropatterning on the CMD surface was obtained by printing cell adhesion mediating cRGDfK peptide molecules (cyclo(Arg-Gly-Asp-d-Phe-Lys)) directly from aqueous solution using microchanneled cantilevers with subsequent incubation of the printed surfaces in the living cell culture. Uniquely, we present cell patterns with different geometries (spot, line, and grid arrays) covering both micrometer and millimeter-centimeter scale areas. The adhered patterns were analyzed by phase contrast microscopy and the adhesion process on the patterns was real-time monitored by digital holographic microscopy, enabling to quantify the survival and migration of cells on the printed cRGDfK arrays.

In situ viscoelastic properties and chain conformations of heavily hydrated carboxymethyl dextran layers: a comparative study using OWLS and QCM-I chips coated with waveguide material

Hydration, viscoelastic properties and dominant structure of thin polymer layers on the surface of waveguide material were evaluated using optical waveguide lightmode spectroscopy (OWLS) and quartz crystal microbalance (QCM) methods. The fundamentally different principles of the two applied label-free biosensors enable to examine analyte layers from complementary aspects, e.g. to determine the amount of bound water in hydrated layers. In this study, a new QCM instrument with impedance measurement (QCM-I) is introduced. Its specially designed sensor chips, covered by thin film of waveguide material, supply identical surface as used in OWLS sensors, thus enabling to perform parallel measurements on the same type of surface. Viscoelastic analysis of the measured data was performed by our evaluation code developed in MATLAB environment, using the Voinova’s Voigt-based model. In situ deposition experiments on the ultrathin films of poly(L-lysine)-graft-poly(ethylene glycol) (PLL-g-PEG) were conducted for instrumental and code validation. Additionally, a novel OWLS-QCM data evaluation methodology has been developed based on the concept of combining hydration and viscoelastic data with optical anisotropy results from OWLS measurements. This methodology provided insight into the time-dependent chain conformation of heavily hydrated nano-scaled layers, resulting in unprecedented structural, hydration and viscoelastic information on covalently grafted ultrathin carboxymethyl dextran (CMD) films. The measured mass values as well as hydration and viscoelastic properties were compared with the characteristics of PLL-g-PEG layers.

Graphene oxide linking layers for highly sensitive SPR biosensing of small molecules

The development of graphene oxide (GO) interfaces can significantly improve the performance of modern surface plasmon resonance (SPR) biosensors. GO linking layers have high binding capacity for a wide range of chemical and biological ligands owing to large surface area of GO and its unique biochemical properties. Here, we compared binding capacities of GO linking layers with different thicknesses and widely-used hydrogel linking layers based on carboxymethylated dextran (CMD). SPR measurements corresponding to the activation of carboxyl groups on the surface of SPR chips showed that the binding capacity of a 50-nm-thick GO linking layer is 30-times higher than for 150-nm-thick CMD layer, which results in enhanced biosensing sensitivity of GO chips and can lead to novel applications for SPR biosensing.

Real-time label-free detection and kinetic analysis of Etanercept—Protein A interactions using quartz crystal microbalance

A quartz crystal microbalance (QCM) was constructed to assess if such a biosensor has value as a complementary real-time label-free analysis platform for the biopharmaceutical industry. This was achieved through modifying QCM crystals with a low-fouling carboxymethyl-dextran layer bearing Protein A, and then injecting solutions containing Etanercept (i.e., Enbrel®) into the QCM chambers. The kinetics of Enbrel® – Protein A interactions was modeled using the Langmuir binding model and Enbrel® concentrations between 0.75–300ngmL−1. The resulting equilibrium dissociation and association constants (KD and KA) were 5.06×10−8M and 1.98×107M−1, respectively. The association and dissociation rate constants (kon and koff) decreased substantially as Enbrel® concentration, [C], increased, despite that the net binding rate, (kon[C]+koff), increased. The decrease in kon and koff was hypothesized to be a consequence of mass transport limitations. To verify this, QCM dissipation measurements were analyzed to provide insight on solution viscosity. As Enbrel® concentration increased, the net change in dissipation, ΔD, became larger. An augmentation of ΔD is associated with a higher solution viscosity, which would result in an increase in mass transport limitations. Therefore, the decrease in kon and koff for increasing Enbrel® concentration can be attributed to mass transport limitations. In conclusion, QCM is a valuable complementary real-time label-free biosensor analysis platform for the biopharmaceutical industry. Unlike the surface plasmon resonance (SPR) platform, QCM allows measuring dissipation, which can provide insight on how mass transport limitations impact interaction kinetics.

Fabrication and characterization of ultrathin dextran layers: Time dependent nanostructure in aqueous environments revealed by OWLS

Surface coatings of the polysaccharide dextran and its derivatives are key ingredients especially in label-free biosensors for the suppression of non-specific binding and for receptor immobilization. Nevertheless, the nanostructure of these ultrathin coatings and its tailoring by the variation of the preparation conditions have not been profoundly characterized and understood. In this work carboxymethylated dextran (CMD) was prepared and used for fabricating ultrathin surface coatings. A grafting method based on covalent coupling to aminosilane- and epoxysilane-functionalized surfaces was applied to obtain thin CMD layers. The carboxyl moiety of the CMD was coupled to the aminated surface by EDC-NHS reagents, while CMD coupling through epoxysilane molecules was performed without any additional reagents. The surface analysis following the grafting procedures consisted of X-ray photoelectron spectroscopy (XPS), attenuated total reflection infrared spectroscopy (ATR-IR), spectroscopic ellipsometry, atomic force microscopy (AFM) and optical waveguide lightmode spectroscopy (OWLS). The XPS and AFM measurements showed that the grafting resulted in a very thin dextran layer of a few nanometers. The OWLS method allowed devising the structure of the interfacial dextran layers by the evaluation of the optogeometrical parameters. The alteration in the nanostructure of the CMD layer with the chemical composition of the silane coverage and the pH of the grafting solution was revealed by in situ OWLS, specifically, lain down chains were found to be prevalent on the surface under neutral and basic conditions on epoxysilylated surfaces. The developed methodologies allowed to design and fabricate nanometer scale CMD layers with well-controlled surface structure, which are very difficult to characterize in aqueous environments using present instrumentations and highly hydrated surface layers.

Comparison of a carboxylated terthiophene surface with carboxymethylated dextran layer for surface plasmon resonance detection of progesterone

Functionalization of a gold surface is usually accomplished by covalent binding via self-assembled monolayers (SAMs) on the gold surface, followed by attachment of flexible polymeric linker layers such as dextran hydrogels. However, these techniques require multiple steps and also have nonspecific interactions and steric problems. In this study, a self-assembled carboxylated terthiophene monolayer was formed onto a gold surface to create a sensitive and stable surface plasmon resonance (SPR) biosensing system. Compared with a commercial carboxymethyl dextran chip (CM5), the terthiophene SAM surface provided more than six times more antibody-binding signals and nearly three times the SPR assay sensitivity for progesterone (P4).

Immobilized carboxymethylated dextran coatings for enhanced ELISA

We here report the development of a new generation of enzyme-linked immunosorbent assay (ELISA) that takes advantage of a low-fouling carboxymethylated dextran (CMD) layer chemically grafted on ELISA wells. In our approach, the overnight capture antibody adsorption step found in classical ELISA was replaced by a covalent attachment step to the CMD layer completed in 15min. As a model, the potential of our approach was highlighted using commercially available anti-human epidermal growth factor (EGF) antibodies to quantify EGF present in various samples. Of interest, the grafted CMD layer was found to be as efficient as the commonly used bovine serum albumine (BSA) to reduce non-specific adsorption, thus eliminating the need of a time-consuming BSA blocking step normally required in classical ELISA. Our results demonstrated similar specificity, affinity, and intra- and inter-assay variations regardless of the diluent used in the assay (BSA-based diluent or protein-free buffer solution) when compared to standard ELISA. Finally, accuracy and precision of the CMD-based ELISA were verified by a spike and recovery test. Dilutions of recombinant human EGF in serum from healthy human volunteers showed almost-perfect linearity and mean recovery rates ranging between 90 and 110%.

Endothelial cell responses towards low-fouling surfaces bearing RGD in a three-dimensional environment

This study reveals that it is possible to obtain a specific cell response towards low-fouling carboxymethyl dextran (CMD) surfaces bearing the RGD adhesive peptide in fibrin. To avoid cell sedimentation on surfaces observed in traditional cell culture systems, CMD surfaces bearing RGD were vertically embedded in fibrin containing human umbilical vein endothelial cells (HUVEC) and their effect over cells was investigated. Compared to the CMD surfaces and to CMD layers bearing the negative control RGE, RGD coatings promoted cell adhesion, induced focal contact formation indicated by co-localization of vinculin and actin fibers, and presented a significant effect over HUVEC net growth during the first 24 h of the culture, as revealed by Ki67 staining and cell counting. The intracellular localization of caveolin-1 combined with the expression of beta 1 integrins was investigated and the orientation of HUVEC towards and on the RGD surfaces was studied. When compared to the negative controls, HUVEC responded to the RGD surface in fibrin resulting in acceleration of morphological changes. RGD surfaces supported fibrin degradation by HUVEC as revealed by fluorescent fibrin experiments as well as multi-cellular structure formation, vacuolation and lumen formation.

Fabrication of Gold Cap-Shaped Nano-Particle Films and Sensing Device for Bioanalytical Application

The nano biosensors based on gold cap-shaped nano-particles like polystyrene and silica nanospheres have been successfully prepared on the substrates of disposable plate (96 wells) and miniplate (72 wells). The sensing film of carboxymethyl dextran layer was modified on the gold surface of the cap-shaped nano-particles. The corresponding micro-device with four channel of optical sensing system has been well constructed and applied to detection of anti-avidin D protein and biotinylated DNA molecules. The DNA hybridization reaction has also been real-time and parallel monitored in four channels, showing that the device possessed a nice reproducibility, which opens a promising bioanalytical application in practice.

Biofouling of dextran-derivative layers investigated by quartz crystal microbalance

This study reports the fouling of carboxymethyl dextran (CMD) layers in cell culture medium, fibronectin, and serum solutions. CMD layers were covalently immobilized onto amine groups available either on an n-heptylamine plasma polymer (HApp) layer or onto a polyethylenimine (PEI) coating grafted to an acetaldehyde plasma polymer (AApp) layer. The successful immobilization of the graft layers was demonstrated by X-ray photoelectron spectroscopy (XPS). Primary amines on HApp and AApp + PEI surfaces were quantified using a colorimetric assay. Quartz crystal microbalance (QCM) was used to investigate in real-time the fouling of the graft layers upon incubation in cell culture medium (RPMI), fibronectin, and foetal bovine serum (FBS) solutions. HApp, AApp and AApp + PEI layers exhibited large fouling in fibronectin and FBS solutions, while fouling in RPMI cell culture medium was not significant. Protein repellent properties of CMD layers in FBS and fibronectin have been demonstrated compared to the other tested surfaces. QCM has shown that both CMD layers were fouled to a small extent in RPMI medium.

Cell adhesion resistance mechanisms using arrays of dextran‐derivative layers

To control interactions at the cell-material interface, low-fouling polymer coatings are used to prevent nonspecific interactions. Subsequent biosignals may be grafted on these low-fouling layers to induce specific biological responses. A polymer array technology was developed to study structural diversity of carboxy-methyl-dextran (CMD) grafted layers in connection with their cell repulsion ability. Arrays of CMD layers were exposed to fibroblasts to screen for cell resistant layers according to the immobilization conditions used to produce these surfaces. Cell cytoskeleton and fibronectin matrix deposition and reorganization were labeled in short-term (4 and 12 h) and long-term (3 days of cell confluence) cell adhesion assays. Results suggest that CMD layers that were resistant to cell adhesion were dense, flexible, and presented a regular (i.e., defect-free) hydrated surface. Cell-resistant CMD layers prevented cell matrix deposition and assembly, affecting cell-substrate adhesion and cytoskeletal organization. Finally, an optimized CMD layer was chosen and proved to be as resistant as a poly(ethylene glycol) layer.

Development of Dextran-Derivative Arrays To Identify Physicochemical Properties Involved in Biofouling from Serum

To control protein adsorption on surfaces, low-fouling polymer coatings such as poly(ethylene oxide) (PEG or PEO) and polysaccharides are used. Their ability to resist protein adsorption is related to the layer structure, hence the immobilization mode. A polymer array technology was developed to study the structural diversity of carboxymethyl dextran (CMD) layers, whose immobilization conditions were varied. CMD arrays were analyzed by X-ray photoelectron spectroscopy (XPS) and by atomic force microscopy (AFM) colloidal probe force measurements. Serum protein adsorption was studied directly on the CMD arrays using surface plasmon resonance (SPR) microscopy. Physicochemical characterization revealed that pinning density regulates surface coverage and the amount of adsorbed molecules, and that salt concentration influences the surface structure of the charged polymer, forming extended or short layers. Protein adsorption experiments from serum showed that repulsive CMD layers are dense, with extended flexible chains. The present study underlines the usefulness of polymer arrays to study structural diversity of thin graft layers and to relate their physicochemical properties to their resistance to nonspecific protein adsorption.

Adsorbed layers of oriented fibronectin: A strategy to control cell–surface interactions

Fibronectin (Fn) is a matrix protein known to induce cell attachment and spreading through its cell binding site and related synergy sites. Fn-coated surfaces are therefore useful in tissue engineering and other cell contacting applications, but a problem with many immobilization strategies is a random distribution of molecular orientations. We sought to control Fn orientation, and thus enhance the availability of its cell binding site, by immobilizing Fn via a carboxymethyl dextran layer onto which are chemically attached monoclonal antibodies specific to a region near to Fn's C terminus (and thus away from the cell binding site). Using optical waveguide lightmode spectroscopy, we show the presence of chemically coupled antibodies to yield a considerably denser and thicker Fn layer, consistent with a more vertically aligned protein. Human umbilical vein endothelial cells spread significantly faster, and in a more spherically symmetric way, on an oriented Fn layer (i.e., in the presence of immobilized monoclonal antibodies) as compared with a control Fn layer (i.e., in the absence of bound antibodies). However, we observe human umbilical vein endothelial cell spreading on the oriented Fn layer to be similar to that on a Fn layer in the absence of a carboxymethyl dextran layer, suggesting that although orienting Fn is a promising strategy, coupling strategies using linkers other than dextran may be needed.

Reversible Hydrophobic Barriers Introduced by Microcontact Printing: Application to Protein Microarrays

Microcontact printing (µCP) has been used to introduce temporary hydrophobic barriers on carboxymethylated dextran (CMD) hydrogels on gold. Among the investigated types of inks, tetraoctadecylammonium bromide (TOAB), electrostatically bound to the CMD layer, provided the most well-defined features both with respect to pattern-definition and reversibility upon exposure to a regeneration solution. The printed patterns were characterized by atomic force microscopy (AFM), scanning electron microscopy (SEM), microscopic wetting and imaging null ellipsometry to explore the influence of concentration of ink solution and contact time on the appearance of the printed layer. AFM revealed that the printed TOAB molecules aggregated into clusters rather than into a homogeneous mono- or multilayer on the CMD hydrogel. It was also observed that printed areas of TOAB that are larger than 25?µm are inhomogeneous most likely because of an edge transfer lithography (ETL) mechanism. A protein model system based on Protein A-rabbit antimouse Fc ? was used to evaluate the potential of the patterned surface as a protein microarray chip by means of surface plasmon microscopy (SPM). Moreover, non-specific adsorption of several proteins onto TOAB barriers was also studied using surface plasmon resonance (SPR), and it is evident that undesired adsorption can be eliminated by removing barriers after ligand immobilization, but prior to analyte exposure, by treating the patterned surface with a simple salt regeneration solution. © Springer-Verlag/Wien 2004.

Sensitive and real-time fiber-optic-based surface plasmon resonance sensors for myoglobin and cardiac troponin I

A sensor to detect markers of cardiac muscle cell death at less than 3 ng ml −1 and in less than 10 min has been achieved. This fiber-optic-based surface plasmon resonance (SPR) sensor is being applied to detect myoglobin (MG) and cardiac troponin I (cTnI) in HEPES buffered saline solution. An in vivo sensor for the early detection of the onset of myocardial infarction (MI) will greatly enhance the patient care. MG and cTnI are two biological markers released from dying cardiac muscle cells during an MI, and their detection at biologically-relevant levels can be diagnostic of MI. Antibodies specific to an antigen of interest are attached to a carboxymethylated dextran layer on a gold SPR surface. With the method developed, the lower limit of detection (LOD) for MG is 2.9 ng ml −1 at 25 °C. The biological level for MG reaches 15–30 ng ml −1 in patient blood after myocardial damage. A Langmuir adsorption isotherm describes the binding well. For cTnI, a lower detection limit of 1.4 ng ml −1 was achieved in preliminary tests. cTnI levels are in the range of 1–3 ng ml −1 in patient blood after myocardial damage. The antibody reaction with the carboxymethylated dextran surface was optimized by modifying the reaction pH, the temperature, and the dextran chain length.

Rapid screening of small ligand affinity to human serum albumin by an optical biosensor

Here we report the use of IAsys biosensor technology for determining the binding parameters of low molecular weight compounds, such as warfarin and bilirubin, to surface immobilized human serum albumin. The protein was covalently immobilized on the surface of the biosensor cuvette, bearing a carboxymethyl dextran layer, through a condensing reaction between the carboxyl groups of the biosensor surface and ε-amine groups of protein lysine residues. This system detects and quantifies the changes in refractive index in the vicinity of the surface of the sensor chip to which the protein is immobilized. The changes in the refractive index are proportional to the change in the absorbed mass, thus the analysis allows the monitoring of the interaction process and the determination of the binding parameters. Optical biosensor analysis, most suited for studying protein/protein or protein/nucleic acid interactions, was sensitive enough to monitor the binding of low molecular weight compounds to human serum albumin and then suitable for a rapid screening of libraries of potential drugs when bioavailability is the research target.

Optical biosensor analysis in studying herpes simplex virus glycoprotein D binding to target nectin1 receptor

Studies on molecular interactions between cellular receptors of herpes simplex virus (HSV) and the viral glycoproteins showing receptor-binding activity are of great relevance for understanding the molecular basis of virus entry. Information on such interactions further provides the basis for a rational design of antiviral drugs. A variety of biochemical and biophysical methodologies are used for determining the binding parameters of interacting biomolecules. Most of them require relatively high amounts of the analyzed compounds, or the use of labeled target molecules. Here, we report the study of the binding of two recombinant forms of HSV glycoprotein D, gD(Δ290-299t) and gD(305t), and a recombinant form of the human cellular receptor for HSV, nectin1-Fc, by using an optical biosensor (IAsys Plus, Affinity Sensors, UK). This device detects and quantifies the changes in refractive index in the vicinity of the surface of sensor chips to which ligands are immobilized. The changes in the refractive index are proportional to the change in the absorbed mass, thus the analysis allows the monitoring of the interaction process in real-time and the determination of the binding parameters. HSV cellular receptor has been immobilized on the surface of the biosensor cuvette, bearing a carboxymethyl dextran layer. The immobilized receptor cuvette was then used for the binding experiments of the two glycoproteins. A significant difference in their dissociation constants was determined, showing for the gD(Δ290-299t) protein a much higher affinity ( K D, 2.8×10 −7 M) with respect to gD(305t) ( K D, 2.8×10 −6 M). The active ligand concentration decreased on time, however the binding properties of the immobilized receptor were maintained over 5 weeks.

Affinity analysis of lectin interaction with immobilized C- and O- gylcosides studied by surface plasmon resonance assay

A biosensor based on the surface plasmon resonance (SPR) principle was used for kinetic analysis of lectin interactions with different immobilized saccharide structures. A novel affinity ligands β- d-glycopyranosylmethylamines derived from common d-aldohexoses linked to the carboxymethyl dextran layer of the SPR sensor surface served for interactions with a wide range of lectins. The method of preparation and use of the β- d-mannopyranosyl glycosylated sensor surface was described. The results of affinity analysis of lectin–ligand interactions were evaluated and compared with data obtained from measurements using commercially available p-aminophenyl α- d-glycopyranosides. Possible applications and advantages of C- and O-glycosylated SPR biosensors are discussed.

Coupling of Antibodies to β-Cyclodextrin-Coated Gold Surfaces via an Intermediate Adamantyl-Modified Carboxymethylated Dextran Layer

β-cyclodextrin-coated sensors chips were obtained by grafting amino-β-cyclodextrin or amino-polymers of β-cyclodextrin (poly-β-CD-NH2) to functionalized gold surfaces. An additional carboxymethylated dextran bearing adamantyl groups (Ad-Dex-COOH) was then immobilized onto the surface by formation of inclusion complexes between β-cyclodextrin cavities and adamantyl groups. Such multilayered structures were stable in aqueous media. However, the initial β-cyclodextrin-coated surface could be recovered by using sodium dodecylsulfate solutions (SDS). After activation withN-hydoxysuccinimide, dextran-coated sensor chips were used to bind antibodies. The immunoreactivity of the resulting biosensors was examined. Moreover, conditions leading to the complete regeneration of the initial surface were investigated. Throughout this study, interfacial adsorption and desorption phenomena were followed in real time by an optical technique, Surface Plasmon Resonance (SPR).