Covalent carbodiimide coupling

The coupling chemistries that have been widely used in organic chemistry for producing chemical bonds have been applied to form irreversible and stable nanoparticle arrays on surfaces. Eychmriller et al. used the carbodiimide coupling chemistry to covalently assemble carboxylate-functionalized CdTe nanocrystals (NCs) onto amino-terminated glass surfaces, which resulted in densely covered nanoparticle films.14 The same principle was also applied to coat Si02 microparticles by CdTe NCs. 

Andres and coworkers demonstrated the iCP of densely packed alkanethiolate-functionalized Au nanoparticle arrays in monolayer and multilayer structures.81,82 Dense and hexagonally packed monolayers of nanoparticles were first assembled on a water surface. By using the Langmuir-Schafer technique, the Au nanoparticle monolayer was transferred to a PDMS stamp, and printed onto a substrate. Multilayers were prepared by repeating the printing process in an LbL scheme, in which subsequent particle layers may be made up of the same or different types of particles. Similarly, the assembly of irregular, densely packed monolayers of polystyrene nanoparticles on iCP substrates via carbodiimide coupling was reported.83 The conformal contact of the carbodiimide-functionalized polystyrene particles resulted in the covalent attachment of the nanoparticles at a carboxylate-functionalized surface. 

Covalent, random, coupled to larger layers SAM, dendrimers or PEG layers with epoxy, aldehyde, carbodiimide groups React with primary amines of lysine and arginine High densihes are available, strong protein attachment, low surface interference/ random orientahon... 

For preparation of the GOD electrode a highly porous graphite foil of 1 mm thickness (Union Carbide, USA) is oxidized in air at 100°C. A 0.1 mol/1 l,l -dimethylferrocene solution in toluene is dropped onto the electrode and the solvent allowed to evaporate. GOD is covalently bound to the oxidized carbon surface by carbodiimide coupling and the surface is covered by a polycarbonate membrane of 0.03 pm thickness. Before use, the electrode is conditioned in 7 mmol/1 glucose solution for 10 h with an applied potential of + 160 mV. 

Laval etal. (1984) bound LDH covalently to electrochemically pretreated carbon. The enzyme was fixed by carbodiimide coupling simultaneously with anodic oxidation of the electrode surface. The total amount of immobilized LDH was determined fluorimetrically after removal from the electrode and hydrolysis. The authors found that at a maximal enzyme loading of 13 pmol/cm2 six enzyme layers are formed. The immobilization yield was about 15%. The kinetic constants, pmax and. Km, were not affected by the immobilization. The obtained enzyme loading factor of 10-3 indicates that diffusion in the enzyme layer was of minor influence on the response of the sensor. The layer behaved like a kinetically controlled enzyme membrane, i.e., the NADH oxidation current was proportional to the substrate concentration only far below Km- With increasing enzyme loading the sensitivity for NADH decreased due to masking of the electrode surface. 

Various approaches were probed to attach the molecule of cytochrome c to the surface of the monolayers. Covalent attachment could be achieved by carbodiimide coupling of the acid groups on the monolayer surface with the amine groups of the protein356,380. Electrostatic adsorption of the protein on the surface of acid-terminated monolayers381,382 was achieved by Tarlov and co workers. Cytochrome c also adsorbs on the surface of a pyridine-containing monolayer383. 

The biomolecules are attached directly to the matrix by chemical/covalent linkage, which is not reversed by pH or changes in ionic strength. Carbodiimide coupling to form peptide bonds has been extensively used for the covalent coupling of the enzyme with polymers [126-128]. Since chemical modification is involved, this method results in the drastic loss of activity of the enzymes/biomolecules. Covalently attached redox biomolecules on polymers have been utilised as highly electron transfer mediators in flavin adenine dinucleotide (FAD) centres of oxidases [73]. 

The protein adsorption on active carbons is an important process contributing to their biocompatibility and the therapeutic effect of hemoperfusion (Arshady 1999). Covalent binding of proteins to a carbon surface allows one to synthesize adsorbents with high selectivity of action similar to that achieved in affinity chromatography. Despite numerous studies of carbon adsorbent interactions with biomolecules in aqueous media, the influence of the adsorbent structure on the characteristics of the interfacial layer of water in the presence of immobilized proteins ranains unclear. Here the relationships between the structural characteristics of activated carbon SCN and the state of the bound water in the presence of BSA and mouse y-globulin (IgG) physically adsorbed or covalently attached to the carbon surface via carbodiimide coupling are analyzed using adsorption and H NMR methods. 

Figure 14.4. Surface functionalization and bioconjugation methods developed for GNRs (a) electrostatic physisorption onto PE-coated GNRs (b) covalent attachment via carbodiimide coupling (c) "click" bioconjugation (d) chemisorption using thiols (e) chemisorption using dithiocarbamates (DTCs).

GOx has been covalently modified with ferrocene electron-relay groups by the carbodiimide coupling of ferrocene... 

Improved stability of tyrosinase-based BDD biosensor was reported by Zhi s group.They combined chemical and electrochemical modifications of BDD film with 4-nitrobenzenediazonium tetrafluoroborate to produce aminophenyl-modified BDD, followed by immobilizing tyrosinase covalently at the BDD surface via carbodiimide coupling. They used this sensor for detection of phenol, p-cresol, and 4-CP and reported 90 % of its original activity after intermittent use for 5 weeks. In all mentioned studies on tyrosinase-based BDD aminosensor no applications on analysis of model or real matrices are presented. 

Biotin-hydrazide also may be used to couple with carboxylate-containing molecules. Hydrazidcs can be coupled with carboxylic acid groups by using the carbodiimide reaction (Chapter 3, Section 1.1). The carbodiimide activates a carboxylate to an o-acylisourea intermediate. Biotin-hydrazide can react with this intermediate via nucleophilic addition to form a stable covalent bond. 

Fig. 5.2 Example of ex situ covalent protein hybridization of CNTs via carbodiimide assisted coupling. Redrawn from [26],...

---