Ethylcarbodiimide Hydrochloride Research Articles

Synthesis and evaluation of hyaluronic acid–poly(ethylene oxide) hydrogel via Michael-type addition reaction

Abstract Hyaluronic acid-based hydrogel was synthesized by reacting hyaluronic acid with aminopropyl methacrylate and then with poly(ethylene oxide). Gelation was observed within 5.5 h with a rheometer by employing viscous and elastic modulus. While the hydrogel was analyzed by light and electron microscopy for morphological observation, other analysis was performed by Fourier-transformed infrared and electron spectroscopy for chemical analysis. Understanding of a putative reaction scheme was outlined by reference to N -(3-diethylpropyl)- N -ethylcarbodiimide hydrochloride for acrylation of hyaluronic acid and by a Michael-type reaction between methacrylate in aminopropyl methacrylate and thiol arms in PEO end groups.

Electrochemical Behaviors of Brilliant Cresyl Blue on DNA Modified Gold Electrode

DNA modified electrode was prepared by covalent immobilization of ssDNA or dsDNA on a mercaptoethanol monolayer self-assembled gold electrode with the help of N-(3-dimethylaminopropyl)-N′-ethylcarbodi- imidehydrochloride (EDC) and N-hydroxysuccinimide (NHS). The electrochemical behaviors of brilliant cresyl blue (BCB) on ssDNA or dsDNA electrodes were studied. BCB had different binding models with ssDNA or dsDNA and the adsorptive constants were 1.67×10~4 L·mol~(-1) for ssDNA/Au electrode and 3.22×10~4 L·mol~(-1) for dsDNA/Au electrode. BCB bound to ssDNA via electrostatic interaction while bound to dsDNA through both the electrostatic and intercalative models. The results showed that dsDNA had a stronger appetency for BCB, which indicated that BCB was a suitable electroactive indicator.

Electrochemical DNA biosensor for the detection of short DNA species of Chronic Myelogenous Leukemia by using methylene blue

A novel assay for the voltammetric detection of 18-bases DNA sequences relating to Chronic Myelogenous Leukemia (CML, Type b3a2) using methylene blue (MB) as the hybridization indicator was reported. DNA was covalently attached onto a glassy carbon electrode (GCE) through amines of the DNA bases using N-hydroxysulfosuccinimide (NHS) and N-(3-dimethylamion)propyl- N′-ethyl carbodiimidehydrochloride (EDC). The covalently immobilized single-stranded DNA (ssDNA) could selectively hybridize with its complementary DNA (cDNA) in solution to form double-stranded DNA (dsDNA) on the surface. A significant increase of the peak current for methylene blue upon the hybridization of immobilized ssDNA with cDNA in the solution was observed. This peak current change was used to monitor the recognition of CML DNA sequence. This electrochemical approach is sequence specific as indicated by the control experiments in which no peak current change was observed if a non-complementary DNA sequence was used. Factors, such as DNA target concentration and hybridization conditions determining the sensitivity of the electrochemical assay were investigated. Under optimal conditions, this sensor has a good calibration range between 1.25 × 10 −7 and 6.75 × 10 −7 M, with CML DNA sequence detection limit of 5.9 × 10 −8 M.

Carbodiimide-Based Benzimidazole Library Method

Using carbodiimide reagents [1,3-diisopropylcarbodiimide or N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC)], we have developed a mild, generalized, one-pot method that delivers N-2-arylaminobenzimidazole esters from commercially available aryl isothiocyanates and o-phenylenediamines. Following saponification and acidifying, the benzimidazole acids were isolated in overall yields ranging from 75 to 88% from the starting aryl isothiocyanates. Nine benzimidazole acids were converted into a library consisting of 180 benzimidazole amides following EDC coupling with commercially available amines. The National Institute of General Medical Science will dispense these benzimidazole amides to academia groups for pilot scale biomedical studies. Using these mild conditions and environmentally safe reagents, we demonstrated that these pharmaceutically ornate heterocycles can also be constructed on solid support.

N-Glycosylation of Fibroblast Growth Factor Receptor 1 Regulates Ligand and Heparan Sulfate Co-receptor Binding

The regulation of cell function by fibroblast growth factors (FGF) occurs through a dual receptor system consisting of a receptor-tyrosine kinase, FGFR and the glycosaminoglycan heparan sulfate (HS). Mutations of some potential N-glycosylation sites in human fgfr lead to phenotypes characteristic of receptor overactivation. To establish how N-glycosylation may affect FGFR function, soluble- and membrane-bound recombinant receptors corresponding to the extracellular ligand binding domain of FGFR1-IIIc were produced in Chinese Hamster Ovary cells. Both forms of FGFR1-IIIc were observed to be heavily N-glycosylated and migrated on SDS-PAGE as a series of multiple bands between 50 and 75 kDa, whereas the deglycosylated receptors migrated at 32 kDa, corresponding to the expected molecular weight of the polypeptides. Optical biosensor and quartz crystal microbalance-dissipation binding assays show that the removal of the N-glycans from FGFR1-IIIc caused an increase in the binding of the receptor to FGF-2 and to heparin-derived oligosaccharides, a proxy for cellular HS. This effect is mediated by N-glycosylation reducing the association rate constant of the receptor for FGF-2 and heparin oligosaccharides. N-Glycans were analyzed by mass spectrometry, which demonstrates a predominance of bi- and tri-antennary core-fucosylated complex type structures carrying one, two, and/or three sialic acids. Modeling of such glycan structures on the receptor protein suggests that at least some may be strategically positioned to interfere with interactions of the receptor with FGF ligand and/or the HS co-receptor. Thus, the N-glycans of the receptor represent an additional pathway for the regulation of the activity of FGFs.

Analysis of freeze-gelation and cross-linking processes for preparing porous chitosan scaffolds

A newly developed freeze-gelation method for fabricating porous chitosan scaffolds for tissue engineering applications was analyzed. The influences of three process variables of freeze-gelation method on the tensile properties of the scaffolds were investigated. It was found that a higher freezing temperature (−20 °C) and concentration of acetic acid (1–2 M) significantly increased the tensile stress and strain of the scaffolds at maximum load, whereas a higher ethanol concentration (95%) in the rinse buffer slightly increased the tensile stress of the scaffolds at maximum load. Further, we successfully established a correlation to quantify the influence of these process variables of the freeze-gelation method on the tensile properties of the scaffolds. Hence, the correlation can be used for quantitative predictions of the tensile properties of the scaffolds. To enhance the tensile strength, the scaffolds were cross-linked using glutaraldehyde (GTA), N-(3-dimethyl-aminopropyl)- N′-ethylcarbodiimide hydrochloride (EDC), or tripolyphosphate (TPP). We found that both the tensile stress and strain at maximum load significantly and dose-dependently increased with the addition of the cross-linking agent, GTA. However, the addition of the cross-linking agents, EDC and TPP, only moderately increased the tensile stress and strain at maximum load, but no dose response was observed. In summary, our study analyzed the freeze-gelation and cross-linking processes for fabricating porous chitosan tissue engineering scaffolds with different tensile properties.

Chondroitin sulfate-based anti-inflammatory macromolecular prodrugs

Macromolecular prodrugs of three non-steroidal anti-inflammatory drugs (NSAIDs), ibuprofen, ketoprofen, and naproxen, were prepared by the covalent attachment of the drugs onto chondroitin sulfate (ChS) using PEG 1000 as a spacer. Drug–PEG adducts were synthesized using 1,1′-carbonyl diimidazole as a coupling agent in dimethyl sulfoxide, followed by the reaction with ChS in highly dilute aqueous solution at pH 6.8 via N-(3-dimethylaminopropyl)- N′-ethylcarbodiimide hydrochloride (EDAC) as a conjugation agent. The drug–ChS conjugates were confirmed by FTIR, 1H NMR and 13C NMR and the molar percent of drug substitution onto ChS was characterized by 1H NMR using the peak areas of the three protons of –ΦCHC H 3 on the drugs to those of –NHCOC H 3 on ChS. All drug–ChS conjugates are water-soluble. The release amounts of the free drugs from their corresponding drug–ChS conjugates were evaluated in the presence or absence of either esterase or chondroitinase, and the both enzymes in pH 7.4 Tris-buffer solutions at 37 °C by high performance liquid chromatography (HPLC). Keto–ChS conjugates released ∼100% ketoprofen within 12 h in the presence of esterase, but the combination with chondroitinase did not accelerate the release rate. The degradation of Keto–ChS conjugates by chondroitinase was confirmed by gel permeation chromatography (GPC). The Keto–ChS conjugates still retained the enzymatic recognition even at the substitution of ketoprofen as high as 56 mol%. The inhibition percent of carrageenan-induced edema of Keto–ChS-56 was comparable to that of a simple blend of ChS and ketoprofen, suggesting that biologically active ChS and ketoprofen could be liberated from the conjugate.

Sample Preparation for Headspace GC Analysis of Residual Solvents in Hyaluronic Acid Derivative Fiber

Hyaluronic acid (HA) is a linear polysaccharide built from repeating disaccharides of alternating D-glucuronic acid and N-acetyl-D-glucosamine. HA can be isolated from biological sources such as vitreous humor, human umbilical cord and rooster and hen comb. HA has biocompatibility and its average molecular weight is usually several million. HA is very viscous in aqueous solution because of its molecular conformation. Due to its viscoelastic properties, HA is used in the medical applications as ophthalmology, dermatology, osteoarthritis, urology and wound healing. HA derivative has an improved rheological property without losing natural biocompatibility of HA. Because HA derivative is water-insoluble or slowly dispersible in human body, it lasts much longer than HA in human body. Therefore, HA derivatives have been widely developed as post-operative adhesion-preventing films or gels, materials for wrinkle treatment, materials for soft tissue augmentation, materials for the arthritis treatment, vehicles for drug delivery, etc. In this study, a HA derivative fiber is obtained from amide reaction of chitosan and HA using N(3-Dimethylaminopropyl)-N'-ethylcarbodiimide (EDC) hydrochloride and N-hydroxysuccinimide (NHS). HA derivative fiber is turned into HA derivative gel in water. Headspace gas chromatography (HS-GC) method has been used for the determination of residual solvents in pharmaceutical compounds. Direct injection of analytes evaporated through equilibration between liquid (or solid) phase and gas phase to GC system minimized the contamination of GC system and the deterioration of GC column. In addition, the automation of equilibrium and injection procedure reduced analysis time and improved reproducibility in injection procedure. In this study, HA derivative fiber was hydrolyzed by hyaluronidase (HAse) in buffer solution for the minimization of variation in the viscosity of HA derivative fiber in water resulting in the higher extraction of residual solvents from the gel matrix. HAse is an enzyme that randomly hydrolyzes the linkage between the N-acetyl-D-glucosamine and the D-glucuronate residues in HA. The hydrolysis rate is dependent on pH and concentration of HAse. In this study viscosimetric assay was applied because it was a simple and rapid method for the measurement of activity of HAse. The aim of this study is to develop efficient sample preparation method for HS-GC analysis of residual solvents in HA derivative fiber. Compared to direct extraction of residual solvents from HA derivative fiber, the extraction through the hydrolysis of HA derivative fiber by HAse gave more complete and higher reproducible quantification of residual solvent. To validate HS-GC analysis method of residual solvents, specificity, limits of detection and quantification, linearity, accuracy and precision are investigated in the study.

Urease immobilization on an ion-exchange textile for urea hydrolysis

Ion-exchange textiles are used as organic supports for urease immobilization with the aim of developing reactive fibrous materials able to promote urea removal. A non-woven, polypropylene-based cation-exchange textile was prepared using UV-induced graft polymerization. Urease was covalently immobilized onto the cation-exchange textile using three different coupling agents: N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC), N-cyclohexyl-N′-(b-[N-methylmorpholino]ethyl)carbodiimide p-toluenesulfonate (CMC), and glutaraldehyde (GA). The immobilized biocatalyst was characterized by means of FT-IR spectrometry, SEM micrographs, dependence of the enzyme activity on pH and temperature, and according to the kinetic constants of the free and immobilized ureases. The biotextile prepared with EDC in the presence of N-hydroxysuccinimide performs best. The optimum pH was 7.2 for the free urease and 7.6 for the immobilized ureases. The reactivity was maximal at 45 °C for free urease, 50 °C for biotextiles prepared using EDC or CMC, and 55 °C for biotextiles prepared with GA. The activation energy for the immobilized ureases was 4.73–5.67 kcal mol−1, which is somewhat higher than 4.3 kcal mol−1 for free urease. The urea conversion for a continuous-flow immobilized urease reactor is nearly as good as a continuously stirred tank reactor having a much longer residence time, suggesting that the packed bed reactor had sufficient diffusive mixing and residence time to reach nearly optimal results. Urease immobilized on a biotextile using EDC has good storage and operational stability. Copyright © 2006 Society of Chemical Industry

First steps towards tissue engineering of small‐diameter blood vessels: Preparation of flat scaffolds of collagen and elastin by means of freeze drying

Porous scaffolds composed of collagen or collagen and elastin were prepared by freeze drying at temperatures between -18 and -196 degrees C. All scaffolds had a porosity of 90-98% and a homogeneous distribution of pores. Freeze drying at -18 degrees C afforded collagen and collagen/elastin matrices with average pore sizes of 340 and 130 mum, respectively. After 20 successive cycles up to 10% of strain, collagen/elastin dense films had a total degree of strain recovery of 70% +/- 5%, which was higher than that of collagen films (42% +/- 6%). Crosslinking of collagen/elastin matrices either in water or ethanol/water (40% v/v) was carried out using a carbodiimide (N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride, EDC) in combination with a succinimide (N-hydroxysuccinimide, NHS) in the presence or absence of a diamine (J230) or by reaction with butanediol diglycidylether (BDGE), followed by EDC/NHS. Crosslinking with EDC/NHS or EDC/NHS/J230 resulted in matrices with increased stiffness as compared to noncrosslinked matrices, whereas sequential crosslinking with the diglycidylether and EDC/NHS yielded very brittle scaffolds. Ethanol/water was the preferred solvent in the crosslinking process because of its ability to preserve the open porous structure during crosslinking. Smooth muscle cells were seeded on the (crosslinked) scaffolds and could be expanded during 14 days of culturing.

Physical characterization of vascular grafts cultured in a bioreactor

Tubular scaffolds of collagen and elastin (weight ratio 1:1) with interconnected pores were prepared by freeze drying and crosslinked with N-(3-dimethylaminopropyl)- N′-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) in the presence or absence of a Jeffamine spacer (poly(propylene glycol)-bis-(2-aminopropyl ether), J230). The crosslinked and uncrosslinked matrices had porosities of 90% and average pore sizes of 131–151 μm. Smooth muscle cells (SMC) were cultured in the crosslinked and uncrosslinked tubular scaffolds under pulsatile flow conditions (mean flow rate 9.6 ml/min, 120 beats/min, pressure 80–120 mmHg). All the constructs could withstand cyclic mechanical strain in the absence of any mechanical support without cracking or suffering permanent deformation. After 7 d, SMC were homogeneously distributed throughout the uncrosslinked and EDC/NHS crosslinked constructs, whereas hardly any cell was observed on the luminal side of J230/EDC/NHS crosslinked matrices. Considering the better mechanical performance of EDC/NHS crosslinked matrices compared to non-crosslinked constructs after 7 d of culture, SMC were dynamically cultured in the former scaffolds for 14 d. During this period, the high strain stiffness of the constructs increased more than two-fold to 38±2 kPa, whereas the low strain stiffness doubled to 8±2 kPa. The yield stress and yield strain were 30±10 kPa and 120±20%, respectively. SMC were homogeneously distributed throughout the EDC/NHS crosslinked collagen/elastin constructs and collagen fibres tended to orient in the circumferential direction.

A Three-dimensional Working Model of the Multienzyme Complex of Aminoacyl-tRNA Synthetases Based on Electron Microscopic Placements of tRNA and Proteins

It has become evident that the process of protein synthesis is performed by many cellular polypeptides acting in concert within the structural confines of protein complexes. In multicellular eukaryotes, one of these assemblies is a multienzyme complex composed of eight proteins that have aminoacyl-tRNA synthetase activities as well as three non-synthetase proteins (p43, p38, and p18) with diverse functions. This study uses electron microscopy and three-dimensional reconstruction to explore the arrangement of proteins and tRNA substrates within this "core" multisynthetase complex. Binding of unfractionated tRNA establishes that these molecules are widely distributed on the exterior of the structure. Binding of gold-labeled tRNA(Leu) places leucyl-tRNA synthetase and the bifunctional glutamyl-/prolyl-tRNA synthetase at the base of this asymmetric "V"-shaped particle. A stable cell line has been produced that incorporates hexahistidine-labeled p43 into the multisynthetase complex. Using a gold-labeled nickel-nitrilotriacetic acid probe, the polypeptides of the p43 dimer have been located along one face of the particle. The results of this and previous studies are combined into an initial three-dimensional working model of the multisynthetase complex. This is the first conceptualization of how the protein constituents and tRNA substrates are arrayed within the structural confines of this multiprotein assembly.

Electrospinning of collagen and elastin for tissue engineering applications

Meshes of collagen and/or elastin were successfully prepared by means of electrospinning from aqueous solutions. Flow rate, applied electric field, collecting distance and composition of the starting solutions determined the morphology of the obtained fibres. Addition of PEO ( M w = 8 × 1 0 6 ) and NaCl was always necessary to spin continuous and homogeneous fibres. Spinning a mixture of collagen and elastin resulted in fibres in which the single components could not be distinguished by SEM. Increasing the elastin content determined an increase in fibres diameters from 220 to 600 nm. The voltage necessary for a continuous production of fibres was dependent on the composition of the starting solution, but always between 10 and 25 kV. Under these conditions, non-woven meshes could be formed and a partial orientation of the fibres constituting the mesh was obtained by using a rotating tubular mandrel as collector. Collagen/elastin (1:1) meshes were stabilized by crosslinking with N-(3-dimethylaminopropyl)- N′-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS). This treatment afforded materials with a high thermal stability ( T d = 79 ° C ) without altering their original morphology. Upon crosslinking PEO and NaCl were fully leached out. Smooth muscle cells grew as a confluent layer on top of the crosslinked meshes after 14 d of culture.

Electrochemical detection of enzyme labeled DNA based on disposable pencil graphite electrode

Electrochemical biosensor for the detection of DNA hybridization using the reduction signal of α-naphthol is described. A pencil graphite electrode was used as a working electrode. Capture probes were covalently attached on to the pencil graphite electrode surface (PGE) at the 5′ end amino group by using N-(dimethylamino)propyl- N′-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysulfosuccinimide (NHS) as a coupling agent on to PGE. After capture probe immobilization on to PGE surface; probe was hybridized with complementary biotinylated oligonucleotide. Alkaline phosphatase labeled with extravidin (Ex-AP) binds to biotinylated hybrid via biotin–avidin interaction. α-Naphthyl phosphate (α-NAP) was added and the reaction between alkaline phosphatase (AP) and α-NAP was occurred consequently as a substrate of AP, α-NAP reduction signal was obtained from this reaction, at −0.100 V by using differential pulse voltammetry (DPV). Other experimental parameters were studied such as; optimizations of hybridization time, and the concentrations of capture probe, biotinylated oligonucleotide and enzyme.

Electrochemical genosensor based on colloidal gold nanoparticles for the detection of Factor V Leiden mutation using disposable pencil graphite electrodes.

Electrochemical genosensors for the detection of the Factor V Leiden mutation from polymerase chain reaction (PCR) amplicons using the oxidation signal of colloidal gold (Au) is described. A pencil graphite electrode (PGE) modified with target DNA, when hybridized with complementary probes conjugated to Au nanoparticles, responded with the appearance of a Au oxide wave at approximately +1.20 V. Specific probes were immobilized onto the Au nanoparticles in two different modes: (a) Inosine-substituted probes were covalently attached from their amino groups at the 5' end using N-(3-dimethylamino)propyl)-N'-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysulfosuccinimide (NHS) as a coupling agent onto a carboxylate-terminated l-cysteine self-assembled monolayer (SAM) preformed on the Au nanoparticles, and (b) probes with a hexanethiol group at their 5' phosphate end formed a SAM on Au nanoparticles. The genosensor relies on the hybridization of the probes with their complementary targets, which are covalently immobilized at the PGE surface. Au-tagged 23-mer capture probes were challenged with the synthetic 23-mer target, 131-base single-stranded DNA or denatured 256-base polymerase chain reaction (PCR) amplicon. The appearance of the Au oxidation signal shortened the assay time and simplified the detection of the Factor V Leiden mutation from PCR amplified real samples. The discrimination between the homozygous and heterozygous mutations was also established by comparing the peak currents of the Au signals. Numerous factors affecting the hybridization and nonspecific binding events were optimized. The detection limit for the PCR amplicons was found to be as low as 0.78 fmol; thus, it is suitable for point-of-care applications.

Generation of Periodic Enzyme Patterns by Soft Lithography and Activity Imaging by Scanning Electrochemical Microscopy

Micropatterns of the enzymes glucose oxidase (GOx) and horseradish peroxidase (HRP) have been formed on polycrystalline gold and glass surfaces using different soft-lithographic approaches. The patterns have been analyzed by noncontact scanning force microscopy. The activities of the immobilized patterns were probed with local resolution by scanning electrochemical microscopy. The following approaches have been tested: (i) microcontact printing of octadecanethiol on gold, followed by chemisorption of cystaminium chloride and incubation of a mixture of HRP and glutaraldehyde to form a patterned cross-linked and grafted enzyme gel; (ii) microcontact printing of octadecanethiol on gold followed by chemisorption of HRP into which an thiol group had been introduced by modification with S-acetylthioglycolic acid-N-succinimidyl ester; (iii) application of a mixture of GOx with N-(3-dimethylaminopropyl)-N‘-ethylcarbodiimide hydrochloride (EDAC) to a stamp and contact with an aminated glass surface; (iv) delivery...

Electrochemical detection of hybridization using peptide nucleic acids and methylene blue on self-assembled alkanethiol monolayer modified gold electrodes

An electrochemical hybridization biosensor based on peptide nucleic acid (PNA) probes is presented. PNA probes were attached covalently through a competition of free amines on the guanine bases and also at the 5 ′ end of the probe, using N-(3-dimethylamino)propyl)- N ′-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) onto a carboxylate terminated alkanethiol self-assembled monolayer (SAM) preformed on a gold electrode (AuE). The covalently immobilized probe could selectively hybridize with the target DNA to form a hybrid on the surface despite the bases being attached to the SAM. The changes in the peak currents of methylene blue (3,7-bis(dimethylamino)phenothiazin-5-ium chloride, MB), an electroactive label, were observed upon hybridization of probe with the target. Effective discrimination against point mutation was also obtained. Performance characteristics of the sensor are described, along with future prospects.

DNA Sensor for Recognition of Native Yeast DNA Sequence with Methylene Blue as an Electrochemical Hybridization Indicator

An electrochemical DNA sensor based on the recognition of single-stranded yeast DNA (ssDNA) immobilized on gold electrode to its complementary ssDNA (cDNA) is presented for hybridization detection. The yeast single-stranded DNA was covalently bound on a self-assembled 3-mercaptopropionic acid monolayer by using water soluble N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC) as a linker. The self-assembled monolayer prevented the nonspecific adsorption of long DNA chain on the electrode surface. The covalently immobilized ssDNA could selectively hybridize with cDNA in solution to form double-stranded DNA (dsDNA) on the surface. The increase of peak currents of methylene blue (MB), an electroactive indicator, upon the hybridization of immobilized ssDNA with cDNA in the solution was observed, which was used to monitor the recognition of yeast DNA sequence. The interaction of immobilized ssDNA and dsDNA with MB also resulted in a transition of electrode process from diffusion-controlled to surface-controlled process. The adsorption constants of methylene blue on ssDNA and dsDNA modified gold electrode surface were found to be (3.3±0.3)×103 M−1 and (6.6±0.4)×103 M−1, respectively, indicating a higher affinity of dsDNA to MB.

Voltammetric determination of DNA hybridization using methylene blue and self-assembled alkanethiol monolayer on gold electrodes

An electrochemical DNA biosensor based on the recognition of single stranded DNA (ssDNA) by hybridization detection with immobilized complementary DNA oligonucleotides is presented. DNA and oligonucleotides were covalently attached through free amines on the DNA bases using N-hydroxysulfosuccinimide (NHS) and N-(3-dimethylamino)propyl- N′-ethylcarbodiimide hydrochloride (EDC) onto a carboxylate terminated alkanethiol self-assembled monolayers (SAM) preformed on a gold electrode (AuE). Differential pulse voltammetry (DPV) was used to investigate the surface coverage and molecular orientation of the immobilized DNA molecules. The covalently immobilized probe could selectively hybridize with the target DNA to form a hybrid on the surface despite the bases being attached to the SAM. The changes in the peak currents of methylene blue (MB), an electroactive label, were observed upon hybridization of probe with the target. Peak currents were found to increase in the following order: hybrid-modified AuE, mismatched hybrid-modified AuE, and the probe-modified AuE which indicates the MB signal is determined by the extent of exposed bases. Control experiments were performed using a non-complementary DNA sequence. The effect of the DNA target concentration on the hybridization signal was also studied. The interaction of MB with inosine substituted probes was investigated. Performance characteristics of the sensor are described.

Cross-linking and characterisation of gelatin matrices for biomedical applications

Cross-linking of gelatin A and B with N,N-(3-dimethylaminopropyl)-N′-ethyl-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) was optimised by varying the NHS/ EDC molar ratio at constant EDC concentration. Native and cross-linked gelatin gels were characterised using the degree of swelling, the number of free amine groups, the phase transition temperature, and titration of the carboxylic acid residues. The cross-linking reaction was most efficient at a NHS to EDC molar ratio of 0.2. At higher NHS/EDC molar ratios, the reaction of EDC with NHS becomes more pronounced, thereby reducing the effective amount of EDC for cross-linking. Swelling measurements of cross-linked gelatin gels gave deviating results when no NHS was used, which was explained by heterogeneous localisation of cross-links in the gelatin gel. The incorporation of undesired compounds into the gelatin gels during the cross-linking reaction was not observed. At optimal NHS to EDC molar ratio, gelatin A and B were cross-linked using increasing EDC/COOHgelatin molar ratios. A range of samples varying from very low cross-link density to very high cross-link density (at high EDC/COOHgelatin) was obtained. Stability of the gels is enhanced with increasing cross-link density, but a minimal cross-link density is required to obtain gelatin gels which are stable at 40°C.