Carboxymethyl Dextran Surface Research Articles

A surface plasmon resonance immunoassay for the rapid analysis of methamphetamine in forensic oral fluid.

BackgroundCurrent chromatographic methods applied for the forensic analysis of methamphetamine are costly, time‐consuming, and require complicated pretreatment procedures. Thus, the rapid detection of methamphetamine is a critical and unmet need. In this study, a surface plasmon resonance (SPR) system based on indirect inhibitive immunoassay was designed for the analysis of methamphetamine in forensic oral fluid samples.MethodsFor the inhibition immunoassay, the diluted oral fluid was mixed with methamphetamine antibody and then injected into the SPR sensor chip. The biosensor chip was constructed by covalently immobilizing of methamphetamine‐bovine serum albumin conjugate onto a carboxymethyl dextran surface at an optimized pH. The concentration of antibody was also optimized.ResultsThe SPR biosensor showed good sensitivity with a limit of detection of 0.44 ng/mL and was comparable or lower than the pre‐existing methods. The method was finally tested using oral fluid samples from 20 suspected drug abusers in forensic cases, and it provided an acceptable recovery of 113.2%, indicating good anti‐interference capability of the SPR sensor.ConclusionThe SPR biosensor was rapid, reproducible, and had a great potential approach for the forensic detection of methamphetamine.

Controlled immobilization of IgG1 on carboxymethyl and amino-dextran SPR chips under external vertical electric fields

Aiming at producing surface plasmon resonance (SPR) sensor chips of improved performance, a novel approach for controlled immobilization of antibody (Ab) molecules has been developed through the implementation of surface functionalization and external vertical electric field (EVEF) assisted immobilization techniques. Carboxymethyl dextran (CMD) chips were reacted with ethylenediamine to create surface amino groups and subsequently reacted with the activated carboxyl group of the fragment crystallisable (Fc) region of Ab. An external vertical electric field was applied to control the movement of antibody molecules toward the chip and their orientation on the surface. The EVEF imparted a current density of 4.17 μA cm−2 through the solution. Immobilization was achieved via a carbodiimide reaction as the antibody molecules reached the functionalized surface. Physical characterization was performed through ATR-FTIR, AFM and surface plasmon resonance (SPR) analyses. Results show that the orientation of antibody molecule on the amino dextran (AmDex) surface is better than on the CMD surface in terms of antibody accessibility for interaction with toxin molecule. Moreover, applying EVEF during the antibody immobilization stage leads to an increased density of the antibody layer and enhanced accessibility of antibody binding domain due to improved orientation of antibody in the direction of the electric field. Discrete-Time-Fourier-Transformation analysis of the chip surface line profiles obtained by AFM analysis clearly show an increased order of Ab molecules after implementation of EVEF. The new findings obtained in this study are expected to be useful for controlling Ab molecule orientation on the immobilizing surface.

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.

Influence of the SPR Experimental Conditions on the G-Quadruplex DNA Recognition by Porphyrin Derivatives.

Surface plasmon resonance (SPR) is a powerful technique to study the interactions of ligands with analytes and therefore a number of biosensor surfaces and injection methods have been developed so far. However, many experimental parameters can affect the interactions and consequently the affinity measurements. In particular, the interactions of positively charged analytes (often used for anionic nucleic acids targets) can be influenced by the sensing surfaces (e.g., negatively charged), leading to significant nonspecific interactions as well as regeneration problems. The aim of the present work is to investigate the effect of different parameters, including ionic strength, SPR biosensor (i.e., nature of the surfaces), and the injection method on the recognition of porphyrin G-quadruplex ligands. We demonstrate that the injection method does not influence the affinity whereas the ionic strength and the nature of the surface impact the recognition properties of the porphyrin for the G-quadruplex DNA. We also found that self-assembled monolayer coating surface presents many advantages in comparison with carboxymethylated dextran surface for SPR studies of G-quadruplex DNA/ligand interactions: (i) the electrostatic interaction with charged analytes is less important, (ii) its structure/composition is less sensitive to the ionic concentration and less prone to unspecific adsorption, (iii) it is easily homemade, and (iv) the cost is approximately 10 times cheaper.

Optimization of 3D Surfaces of Dextran with Different Molecule Weights for Real-Time Detection of Biomolecular Interactions by a QCM Biosensor.

Quartz crystal microbalance (QCM) has been extensively applied in real-time and label-free biomolecular interaction studies. However, the sensitive detection by QCM technology remains challenging, mainly due to the limited surface immobilization capacity. Here, a three-dimensional (3D) carboxymethyl dextran coated gold sensor chip surface was successfully fabricated with dextran of different molecular weight (100, 500 and 2000 kDa, respectively). To evaluate the 3D carboxymethyl dextran surface immobilization capacity, the 3D surface was used for studying antigen–antibody interactions on the QCM biosensor. The results showed that the protein immobilization capacity of the 3D carboxymethyl dextran (2000 kDa) surface exceeded more than 4 times the capacity of the 2D carboxyl surface, and 2 times the capacity of the traditional 3D carboxymethyl dextran (500 kDa) surface. Furthermore, the kinetic and affinity properties of antigen–antibody interactions were performed. Most notably, the optimized 3D carboxymethyl dextran (2000 kDa) surface could be used for small molecule detection, where the binding of biotinylated oligo (0.67 kDa) reached 8.1 Hz. The results confirmed that a 3D carboxymethyl dextran (2000 kDa) surface can be exploited for sensitive detection of low molecular weight analytes, which have great potential applications for characterizing the interactions between small molecule drugs and proteins.

A novel enzyme based SPR-biosensor to detect bromocriptine as an ergoline derivative drug

In the modern biomedical and therapeutic drug monitoring technology, development of high performance sensing methods for ergoline derivative drug, bromocriptine (BC), is a critical issue due to its plasma low concentration and important function in Parkinson’s disease. The results of the protein-ligand docking simulation consistent with competitive inhibition experimental study strongly support the binding of BC to the laccase active site. Therefore, a new biosensor for direct label-free detection of BC is presented based on surface plasmon resonance (SPR) using laccase from Bacillus sp. HR03 as a recognition element. The biosensor chip surface was constructed by covalent immobilization of the laccase on a SPR carboxymethyl dextran surface. The calculated kinetic affinity (KD) was 5.4nM, which indicated the high affinity between BC and the immobilized laccase. The biosensor could determine BC in a linear detection range from 0.001ng/ml to 1000ng/ml with a lower detection limit of 0.001ng/ml. The biosensor provided acceptable specificity with suitable recovery in simulated blood samples. This new biosensor represents a novel, fast, sensitive, economic cost effective and accurate method for the therapeutic monitoring and detection of the BC.

Immobilization of flavan-3-ols onto sensor chips to study their interactions with proteins and pectins by SPR

Interactions between plant polyphenols and biomacromolecules such as proteins and pectins have been studied by several methods in solution (e.g. isothermal titration calorimetry, dynamic light scattering, nuclear magnetic resonance and spectrophotometry). Herein, these interactions were investigated in real time by Surface Plasmon Resonance (SPR) analysis after immobilization of flavan-3-ols onto a sensor chip surface. (−)-epicatechin, (+)-catechin and flavan-3-ol oligomers with an average degree of polymerization of 2 and 8 were chemically modified using N-(2-(tritylthio)ethyl)propiolamide in order to introduce a spacer unit onto the catecholic B ring. Modified flavan-3-ols were then immobilized onto a carboxymethylated dextran surface (CM5). Immobilization was validated and further verified by evaluating flavan-3-ol interaction with bovine serum albumin (BSA), poly-l-proline or commercial pectins. BSA was found to have a stronger association with monomeric flavan-3-ols than oligomers. SPR analysis of selected flavan-3-ols immobilized onto CM5 sensor chips showed a stronger association for citrus pectins than apple pectins, regardless of flavan-3-ol degree of polymerization.

Serum ABO Blood Typing by Surface Plasmon Resonance Technique

Aim of this work is to develop a surface plasmon resonance (SPR) technique for detection of anti-A and anti-B which are the antibodies specific to ABO blood group in serum based on solid phase immobilization of antigens on the surface. Synthetic antigens A and B were immobilized on the carboxymethyldextran (CMD) surface to measure the antibodies. The immobilized synthetic antigen surface were tested function by injection of monoclonal anti-A and anti-B. Total 20 plasma samples at 1:10 dilution were measured by SPR techniuqe and the result were concordant to standard agglutination technique. The cost of blood typing could be economized by high throughput and surface regeneration on SPR technique.

Highly Sensitive and Selective Sensor Chips with Graphene-Oxide Linking Layer.

The development of sensing interfaces can significantly improve the performance of biological sensors. Graphene oxide provides a remarkable immobilization platform for surface plasmon resonance (SPR) biosensors due to its excellent optical and biochemical properties. Here, we describe a novel sensor chip for SPR biosensors based on graphene-oxide linking layers. The biosensing assay model was based on a graphene oxide film containing streptavidin. The proposed sensor chip has three times higher sensitivity than the carboxymethylated dextran surface of a commercial sensor chip. Moreover, the demonstrated sensor chips are bioselective with more than 25 times reduced binding for nonspecific interaction and can be used multiple times. We consider the results presented here of importance for any future applications of highly sensitive SPR biosensing.

Quantitative analysis of histamine- and agmatine–DNA interactions using surface plasmon resonance

To exploit the binding affinity for efficient plasmid purification, agmatine and histamine were immobilized on carboxymethylated dextran surface. Their binding strength is evaluated with oligonucleotides and pUC19 (2.69kbp), pVAX1-LacZ (6.05kbp) and pcDNA3-myc-FLNa S2152A (14kbp) isoforms by measuring the KD using SPR-biosensor. The oligonucleotides and plasmid isoforms bind more strongly to histamine than to agmatine. Concerning the oligonucleotides, the highest affinities are found for poly 30, and polyT and polyG series showed lowest affinity with both ligands. These results are corroborating with the hetero-oligonucleotides since the lowest binding affinity is found for GGGTTT, indicating that thymidine and guanosine together decrease the binding strength. The largest plasmid has the highest binding, while pUC19 shows the weaker binding. The linear isoform exhibit the highest binding affinity to both amino acid-derivatives.Decreasing the pH above 6, the interaction strength of linear isoform increases possibly due to positively charged of the surface, which enhances electrostatic interactions. However, no binding response is detected with high salt concentrations (1M NaCl) and for different buffers. The affinity information accessible with the biosensor offers new insight into nucleic acids–ligand interactions, as well as, new criteria leading the optimization of the novel ligands for preparative plasmid purification.

Pyruvate Formate-lyase and Its Activation by Pyruvate Formate-lyase Activating Enzyme

The activation of pyruvate formate-lyase (PFL) by pyruvate formate-lyase activating enzyme (PFL-AE) involves formation of a specific glycyl radical on PFL by the PFL-AE in a reaction requiring S-adenosylmethionine (AdoMet). Surface plasmon resonance experiments were performed under anaerobic conditions on the oxygen-sensitive PFL-AE to determine the kinetics and equilibrium constant for its interaction with PFL. These experiments show that the interaction is very slow and rate-limited by large conformational changes. A novel AdoMet binding assay was used to accurately determine the equilibrium constants for AdoMet binding to PFL-AE alone and in complex with PFL. The PFL-AE bound AdoMet with the same affinity (∼6 μM) regardless of the presence or absence of PFL. Activation of PFL in the presence of its substrate pyruvate or the analog oxamate resulted in stoichiometric conversion of the [4Fe-4S](1+) cluster to the glycyl radical on PFL; however, 3.7-fold less activation was achieved in the absence of these small molecules, demonstrating that pyruvate or oxamate are required for optimal activation. Finally, in vivo concentrations of the entire PFL system were calculated to estimate the amount of bound protein in the cell. PFL, PFL-AE, and AdoMet are essentially fully bound in vivo, whereas electron donor proteins are partially bound.

INS-1 cell glucose-stimulated insulin secretion is reduced by the downregulation of the 67 kDa laminin receptor

Understanding β cell-extracellular matrix (ECM) interactions can advance our knowledge of the mechanisms that control glucose homeostasis and improve culture methods used in islet transplantation for the treatment of diabetes. Laminin is the main constituent of the basement membrane and is involved in pancreatic β cell survival and function, even enhancing glucose-stimulated insulin secretion. Most of the studies on cell responses towards laminin have focused on integrin-mediated interactions, while much less attention has been paid on non-integrin receptors, such as the 67 kDa laminin receptor (67LR). The specificity of the receptor-ligand interaction through the adhesion of INS-1 cells (a rat insulinoma cell line) to CDPGYIGSR-, GRGDSPC- or CDPGYIGSR + GRGDSPC-covered surfaces was evaluated. Also, the effects of the 67LR knocking down over glucose-stimulated insulin secretion were investigated. Culture of the INS-1 cells on the bioactive surfaces was improved compared to the low-fouling carboxymethyl dextran (CMD) surfaces, while downregulation of the 67LR resulted in reduced cell adhesion to surfaces bearing the CDPGYIGSR peptide. Glucose-stimulated insulin secretion was hindered by downregulation of the 67LR, regardless of the biological motif available on the biomimetic surfaces on which the cells were cultured. This finding illustrates the importance of the 67LR in glucose-stimulated insulin secretion and points to a possible role of the 67LR in the mechanisms of insulin secretion.

Solution composition impacts fibronectin immobilization on carboxymethyl-dextran surfaces and INS-1 insulin secretion

It is shown that solution composition during immobilization plays a critical role in the properties of fibronectin (FN) surfaces and their bioactivity towards insulinoma (INS-1) cell function. X-ray photoelectron spectroscopy revealed FN grafting onto low-fouling carboxymethyl-dextran (CMD) surfaces was successful with solutions composed of 10μM CaCl2, 10μM MgCl2, 10μM MnCl2, and 10μM and 1mM NaCl, but unsuccessful with those made of 150mM NaCl or 1× PBS. Circular dichroism and photon correlation spectroscopy revealed that regardless of solution composition, no measurable differences in free FN conformation prevail. AFM imaging of FN-CMD revealed, while there are no quantitative differences in surface roughness, there are some subtle qualitative differences in topography. FN surface immobilization scheme does not influence INS-1 cell growth after 3 and 7 days regardless of the underlying substrate or solution composition. INS-1 cell insulin secretion in response to glucose is affected by the substrate and solution composition during FN immobilization.

Culturing INS-1 cells on CDPGYIGSR-, RGD- and fibronectin surfaces improves insulin secretion and cell proliferation

Rat insulinoma cells (INS-1), an immortalized pancreatic beta cell line, were cultured on low-fouling carboxymethyl-dextran (CMD) layers bearing fibronectin, the tripeptide Arg–Gly–Asp (RGD) or CDPGYIGSR, a laminin nonapeptide. INS-1 cells were non-adherent on CMD and RGE but adhered to fibronectin- and peptide-coated CMD surfaces and to tissue culture polystyrene (TCPS). On CMD bearing fibronectin and the peptides, INS-1 cells showed higher glucose-stimulated insulin secretion compared to those on TCPS, bare CMD and RGE. INS-1 cells experienced a net cell growth, with the lowest found after 7 days on CMD and the highest on fibronectin. Similarly, cells on RGD and CDPGYIGSR showed lower net growth rates than those on fibronectin. Expression of E-cadherin and integrins α vβ 3 and α 5 were similar between the conditions, except for α 5 expression on fibronectin, RGD and CDPGYIGSR. Larger numbers of Ki-67-positive cells were found on CDPGYIGSR, TCPS, fibronectin and RGD. Cells in all conditions expressed Pdx1.

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.

Assessment of glycoprotein interactions with 4-[(2-aminoethyl)carbamoyl]phenylboronic acid surfaces using surface plasmon resonance spectroscopy.

Reported here are analyses of the interactions between a select group of solution-phase glycoproteins and a unique boronic acid capture surface. The boronic acid derivative, 4-[(2-aminoethyl)carbamoyl]phenylboronic acid, AECPBA, was synthesized and then immobilized on carboxymethyl dextran surfaces using simple coupling methods. From surface plasmon resonance spectroscopy responses, it is found that model glycoproteins interact strongly with the AECPBA surface and subsequently can be readily released from the AECPBA surface using borate buffer. A striking difference between the glycoproteins fetuin and asialofetuin (desialylated fetuin), in terms of glycoprotein binding to the AECPBA surface, indicates that the interaction of glycoproteins with the immobilized AECPBA is dictated by the terminal saccharide of the heteroglycan chain. Surprisingly, secondary interactions of glycosylated and nonglycosylated proteins with the carboxymethyl dextran hydrogel matrix are observed. Importantly, it is demonstrated that use of tris(hydroxymethyl)aminomethane buffer allows for decreased secondary interactions of nonglycosylated proteins on the AECPBA/dextran surface, as noted with the model protein ExtrAvidin.

Immobilization of carbohydrate epitopes for surface plasmon resonance using the Staudinger ligation

The detection of carbohydrate–protein interactions is often performed using techniques that require surface immobilization of the lectin or the glycan. A commonly used assay for lectin binding is surface plasmon resonance (SPR). We describe an implementation of the Staudinger ligation as a method to immobilize carbohydrate epitopes to a biosensor surface. This was accomplished by first introducing an azide functionality to a carboxymethyldextran surface, followed by reaction with a phosphane-modified carbohydrate ligand. The chemistry employed is extremely mild and was easily adapted to a commercial biosensor system. Using this approach, we investigated the binding of jacalin and wheat germ agglutinin (WGA) to galactose, lactose, and N-acetyl-lactosamine. We observed that WGA binding shows evidence of multivalent interaction with the surface. Additionally, we found that jacalin binding was influenced by the presence of a flexible and hydrophobic galactosyl aglycone.

PB1 Domain Interaction of p62/Sequestosome 1 and MEKK3 Regulates NF-κB Activation

p62/Sequestosome 1 is a scaffold protein involved in the regulation of autophagy, trafficking of proteins to the proteasome, and activation of NF-kappaB. p62 encodes an N-terminal PB1 domain in addition to the ZZ domain, TRAF6-binding domain, LC3 interaction region, and ubiquitin-associated domain, each critical for the physiological function of p62. PB1 domains have a beta-grasp topology where the front end of one PB1 domain binds the back end of a second PB1 domain. The p62 PB1 domain homodimerizes as well as heterodimerizes with other PB1 domains. The front end of the PB1 domain in p62 binds the PB1 domain of atypical protein kinases C, the MAPK kinase, MEK5, and the NBR1 protein. Other than its role in homodimerization, the rear end acidic cluster region of the p62 PB1 domain had no previous defined binding partners. Herein, we demonstrate that the rear end acidic cluster region of the p62 PB1 domain binds the front end basic region of the MAPK kinase kinase, MEKK3. p62 and MEKK3 co-localize in speckles or aggregates that are centers for organizing TRAF6-regulated NF-kappaB signaling and the assembly of polyubiquinated proteins sorting to sequestosomes and proteasomes. The p62-MEKK3 complex binds TRAF6, which regulates the ubiquitination of the IKK complex and NF-kappaB activation. p62 is required for the association of MEKK3 with TRAF6 and short hairpin RNA knockdown of p62 inhibits IL-1 and MEKK3 activation of NF-kappaB. The rear end acidic cluster of the p62 PB1 domain is used to organize cytosolic aggregates or speckles-associated TRAF6-p62-MEKK3 complex for control of NF-kappaB activation.

Rapid Optimization of a Peptide Inhibitor of Malaria Parasite Invasion by Comprehensive N-Methyl Scanning

Apical membrane antigen 1 (AMA1) of the malaria parasite Plasmodium falciparum has been implicated in the invasion of host erythrocytes and is an important vaccine candidate. We have previously described a 20-residue peptide, R1, that binds to AMA1 and subsequently blocks parasite invasion. Because this peptide appears to target a site critical for AMA1 function, it represents an important lead compound for anti-malarial drug development. However, the effectiveness of this peptide inhibitor was limited to a subset of parasite isolates, indicating a requirement for broader strain specificity. Furthermore, a barrier to the utility of any peptide as a potential therapeutic is its susceptibility to rapid proteolytic degradation. In this study, we sought to improve the proteolytic stability and AMA1 binding properties of the R1 peptide by systematic methylation of backbone amides (N-methylation). The inclusion of a single N-methyl group in the R1 peptide backbone dramatically increased AMA1 affinity, bioactivity, and proteolytic stability without introducing global structural alterations. In addition, N-methylation of multiple R1 residues further improved these properties. Therefore, we have shown that modifications to a biologically active peptide can dramatically enhance activity. This approach could be applied to many lead peptides or peptide therapeutics to simultaneously optimize a number of parameters.

The optical biosensor study of the redox partner interaction with the cytochrome P450 2B4-containing monooxygenase system under hydroxylation conditions

Interactions between cytochrome P450 2B4, NADPH:cytochrome P450 reductase and cytochrome b5 have been investigated in the presence of a substrate (7-pentoxyresorufin) and an electron donor, NADPH, in the monomeric reconstituted P450 2B4-contained monooxygenase system. Each partner was immobilized via its amino groups on the carboxymethyldextran biochip surface of an optical biosensor IAsys+. It was shown that, despite immobilization of any of the partners (via their respective amino groups) onto the carboxymethyldextran surface of the IAsys+ optical biosensor, its activity didn't loss. The formation of binary d-Fp/d-2B4 complexes was registered. The association/dissociation rate constants (kon/koff) were (0,013 +/- 0,005) x 10(6) M(-1) x s(-1)/0,05 +/- 0,02 s(-1), equilibrium dissociation constant (K(D)) was (0,26 +/- 0,13) x 10(-6) M. Comparison of kon, koff and K(D) for d-Fp/d-2B4 complexes in oxidation conditions with corresponding constants for the oxidized protein forms--(0,10 +/- 0,03) x 10(6) M(-1) x s(-1)/(0,14 +/- 0,06) s(-1), (0,71 +/- 0,37) x 10(-6) M--shows that the decrease in kon and K(D) occurs due to the increase in lifetime during transition from oxidized to hydroxylation conditions. Complex formation between d-Fp and d-b5 was not registered in oxidation and hydroxylation conditions. The ternary d-Fp/d-2B4/d-b5 complexes formation was shown in hydroxylation and oxidation conditions.