Diimide-activated Amidation Research Articles

Covalent Attachment of Sulfur-doped Graphene Quantum Dots to Glucose Oxidase via Diimide-activated Amidation

The conjugation of biomolecules to quantum dots has recently gained attention in the field of bionanotechnology. Graphene quantum dots (GQDs) have been employed in various sensing applications due to their tunable fluorescence, controllable size, low cytotoxicity, and high biocompatibility. In this work, sulfur-doped graphene quantum dots (S-GQDs) were conjugated to glucose oxidase (GOx) for the potential detection of glucose. Fluorescent S-GQDs were synthesized via one-step pyrolysis of citric acid and thioglycolic acid at 200 °C. S-GQDs were conjugated to GOx using the carbodiimide/succinimide (EDC/NHS) coupling reaction, followed by purification via centrifugal ultrafiltration. Fluorescence spectroscopy, UV-Vis absorption spectroscopy, and transmission electron microscopy were employed to characterize the S-GQDs and S-GQD-GOx conjugates. The S-GQDs obtained had an average diameter of 49.8 ± 7.4 nm (n = 12) and emitted strong blue fluorescence (λem = 460 nm) when excited with 365-nm UV light. Trinder’s assay was used to assess the catalytic activity of free GOx and the S-GQD-GOx conjugates. The S-GQD-GOx conjugates consistently showed significantly greater catalytic activity compared to free GOx. The results obtained open avenues for the fabrication of a sensitive fluorescent GQD-based glucose probe in the future.

A Novel Method for Carbon Nanotube Functionalization Using Immobilized Candida antarctica Lipase.

Carbon nanotubes (CNTs) have been proposed as nanovehicles for drug or antigen delivery since they can be functionalized with different biomolecules. For this purpose, different types of molecules have been chemically bonded to CNTs; however, this method has low efficiency and generates solvent waste. Candida antarctica lipase is an enzyme that, in an organic solvent, can bind a carboxylic to a hydroxyl group by esterase activity. The objective of this work was to functionalize purified CNTs with insulin as a protein model using an immobilized lipase of Candida antarctica to develop a sustainable functionalization method with high protein attachment. The functionalized CNTs were characterized by scanning electron microscope (SEM), Raman spectroscopy, Fourier-transform infrared spectroscopy (FTIR), thermogravimetric analysis (TGA), and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS–PAGE). The enzymatic functionalization of insulin on the surface of the CNTs was found to have an efficiency of 21%, which is higher in conversion and greener than previously reported by the diimide-activated amidation method. These results suggest that enzymatic esterification is a convenient and efficient method for CNT functionalization with proteins. Moreover, this functionalization method can be used to enhance the cellular-specific release of proteins by lysosomal esterases.

Design and In-silico study of bioimaging fluorescence Graphene quantum dot-Bovine serum albumin complex synthesized by diimide-activated amidation

Graphene quantum dot possesses advantageous characteristics like tunable fluorescence, nanometer size, low cytotoxicity, high biocompatibility enabling them as an ideal material for fluorescence bio-imaging. It exhibits a unique characteristic of DNA cleavage activity enhancer, gene/drug carrier, and anticancer targeting applications. In this article, we discussed the preparation of graphene quantum dot through the bottom-up method. Carbodiimide-activated amidation reactions were used for the functionalization of graphene quantum dot with Bovine Serum Albumin. Fluorescence spectroscopy data showed that the graphene quantum dot has size-dependent fluorescence emission. TEM and AFM studies showed that the size of graphene quantum dot was around 20 nm with narrow size distribution. Carbodiimide-activated amidation conjugation was successful in binding the protein onto graphene quantum dot and these conjugates were characterized by DLS, FTIR, fluorescence spectroscopy, and agarose gel electrophoresis. We also studied the structural-based in-silico molecular dynamic simulation by AutoDock, PyRx, and Discovery Studio Visualizer. Based on the virtual screening analysis and higher negative energy incorporation, it is observed that graphene quantum dot conjugated with bovine serum albumin quickly and formed is highly stable complex, which makes them a potential candidate for future applications in the field of bio-imaging, bio-sensing, gene/drug delivery, and tumor theragnostic.

Adamantane-Modified Graphene Oxide for Cyanate Ester Resin Composites with Improved Properties

The conjugation of graphene and polymers has attracted great attention for the fabrication of functional hybrid nanomaterials. Here, we demonstrate the modification of graphene oxide (GO) with adamantane (AMT) through the diimide-activated amidation reaction. The modification of GO with AMT improves the dispersion and decreases the interfacial polarization of GO, causing a lower dielectric constant for the fabricated GO/AMT hybrid materials. The structures of GO/AMT were studied by Fourier transform infrared spectroscopy and Raman spectroscopy. Furthermore, the mechanical properties, thermal stability, and dielectric constant of GO/AMT composites were measured at a low cured temperature using various techniques, such as differential scanning calorimetry, thermogravimetric analysis, and dynamic mechanical thermal analysis. It was found that the synthesized GO/AMT materials with different contents were blended into cyanate ester (CE) resins, resulting in a lower cure temperature, smaller dielectric constant, higher thermal stability, and stronger water resistance. It is expected that this novel GO/AMT-CE material will have potential applications for replacing traditional thermosetting resins.

Pinewood nanobiochar: A unique carrier for the immobilization of crude laccase by covalent bonding

Nanotechnology-inspired biocatalytic systems attracted attention for many applications since nanosized supports for enzyme immobilization can improve efficiency-determining factors e.g. enhancing the surface area and loading capacity and reducing the mass transfer resistance. Among the nanomaterials, nanobiochar has unique features as a support for enzyme immobilization i.e. high surface to volume ratio, porous structure, and presence of functional groups on its surface. However, the performance of the immobilization is highly dependent on the immobilization conditions and the properties of the enzyme and the support material. In this research, crude laccase was covalently immobilized onto functionalized nanobiochar using a two-step method of diimide-activated amidation. The effect of different parameters was investigated. The optimal conditions were found to be 14 mg/mL of laccase concentration, 5 mg/mL of nanobiochar, 8.2 mM of cross-linker and 3 h of contact time. For investigating the pH, thermal, storage, and operational stability, the sample obtained from the optimized conditions was used. The results showed the higher stability of immobilized laccase against temperature and pH variation compared to free laccase. In addition, immobilized laccase maintained its catalytic performance up to seven cycles of utilization and showed more than 50% of initial activity after two months of room temperature storage.

Covalent immobilization of phytase on the multi-walled carbon nanotubes via diimide-activated amidation: structural and stability study

Phytase is the most widely used feed enzyme in the world. This enzyme inactivates under pelleting conditions such as temperatures above 80 °C. The present study reported covalent attachment of phytase onto functionalized multi-walled carbon nanotubes (F-MWNT). The optimum enzyme loading and immobilization efficiency were 110 µg/mg of functionalized multi-walled carbon nanotubes and 62%, respectively. Characterization, structural changes and immobilization of phytase were studied by scanning electron microscopy (SEM), circular dichroism (CD), zeta potential measurement and Fourier transform infrared (FTIR) spectroscopy. Immobilized phytase exhibited improved stability towards temperature than the free phytase. The free phytase retain 3% and 27% of relative activity at 90 and 80 °C, respectively after 2 min of incubations. While immobilized phytase retained about 33% and 51% at the same condition. Also the results showed that the presence of small quantities of NaCl (<1 M) in the reaction media increased enzyme activity of immobilized phytase up to 78% but free phytase activity was not significantly changed until 1 M NaCl which increased by 30%. Higher concentrations of 1 M NaCl drastically reduced both free and immobilized phytase activities.

Graphene-oxide-supported Pt nanoparticles with high activity and stability for hydrazine electro-oxidation in a strong acidic solution

Graphene-oxide-supported Pt (GO-Pt) nanoparticles were prepared by performing diimide-activated amidation and used in an electrocatalyst for hydrazine electro-oxidation in 0.5M H2SO4 solution. The physico-chemical properties of the GO-Pt nanoparticles were characterized with various techniques, which revealed that highly dispersed Pt nanoparticles with an average size of 2.6nm were densely deposited on the amidated GO due to their strong adhesion. Cyclic voltammograms were obtained and demonstrate that the GO-Pt catalyst exhibits significantly improved catalytic activity and long-term stability in hydrazine electro-oxidation in a strong acidic solution when compared to commercial Pt/C and Pt metal electrodes. These enhanced electrochemical properties are attributed to the large electrochemically active surface area that results from the smaller size and excellent dispersion of the Pt nanoparticles on amidated GO.

Multi-walled carbon nanotubes functionalized with recombinant Dengue virus 3 envelope proteins induce significant and specific immune responses in mice

BackgroundDengue is the most prevalent arthropod-borne viral disease in the world. In this article we present results on the development, characterization and immunogenic evaluation of an alternative vaccine candidate against Dengue.MethodsThe MWNT-DENV3E nanoconjugate was developed by covalent functionalization of carboxylated multi-walled carbon nanotubes (MWNT) with recombinant dengue envelope (DENV3E) proteins. The recombinant antigens were bound to the MWNT using a diimide-activated amidation process and the immunogen was characterized by TEM, AFM and Raman Spectroscopy. Furthermore, the immunogenicity of this vaccine candidate was evaluated in a murine model.ResultsImmunization with MWNT-DENV3E induced comparable IgG responses in relation to the immunization with non-conjugated proteins; however, the inoculation of the nanoconjugate into mice generated higher titers of neutralizing antibodies. Cell-mediated responses were also evaluated, and higher dengue-specific splenocyte proliferation was observed in cell cultures derived from mice immunized with MWNT-DENV3E when compared to animals immunized with the non-conjugated DENV3E.ConclusionsDespite the recent licensure of the CYD-TDV dengue vaccine in some countries, results from the vaccine’s phase III trial have cast doubts about its overall efficacy and global applicability. While questions about the effectiveness of the CYD-TDV vaccine still lingers, it is wise to keep at hand an array of vaccine candidates, including alternative non-classical approaches like the one presented here.

Conjugation of insulin onto the sidewalls&amp;nbsp;of single-walled carbon nanotubes through functionalization and diimide-activated amidation

PurposeThe high aspect ratio of carbon nanotubes (CNTs) allows the attachment of compounds that enhance the functionality of the drug vehicle. Considering this, use of CNTs as a multifunctional insulin carrier may be an interesting prospect to explore.Materials and methodsThe carboxylic acid groups were functionalized on the sidewalls of single-walled CNTs (SWCNTs) followed by diimidation to form amide bonds with the amine groups of the insulin.ResultsScanning transmission electron microscopy and transmission electron microscopy establish clear conjugation of insulin onto the surface of nanotube sidewalls. The incorporation of insulin further increased the solubility of SWCNTs in biological solution for the tested period of 5 months. Bicinchoninic acid assay confirms that 0.42 mg of insulin could be attached to every 1 mg of carboxylated SWCNTs.ConclusionWith the successful conjugation of insulin to SWCNTs, it opens up the potential use of SWCNTs as an insulin carrier which in need of further biological studies.

Carbon nanotube-based lateral flow biosensor for sensitive and rapid detection of DNA sequence

In this article, we describe a carbon nanotube (CNT)-based lateral flow biosensor (LFB) for rapid and sensitive detection of DNA sequence. Amine-modified DNA detection probe was covalently immobilized on the shortened multi-walled carbon nanotubes (MWCNTs) via diimide-activated amidation between the carboxyl groups on the CNT surface and amine groups on the detection DNA probes. Sandwich-type DNA hybridization reactions were performed on the LFB and the captured MWCNTs on test zone and control zone of LFB produced the characteristic black bands, enabling visual detection of DNA sequences. Combining the advantages of lateral flow chromatographic separation with unique physical properties of MWCNT (large surface area), the optimized LFB was capable of detecting of 0.1nM target DNA without instrumentation. Quantitative detection could be realized by recording the intensity of the test line with the Image J software, and the detection limit of 40pM was obtained. This detection limit is 12.5 times lower than that of gold nanoparticle (GNP)-based LFB (0.5nM, Mao et al. Anal. Chem. 2009, 81, 1660–1668). Another important feature is that the preparation of MWCNT–DNA conjugates was robust and the use of MWCNT labels avoided the aggregation of conjugates and tedious preparation time, which were often met in the traditional GNP-based nucleic acid LFB. The applications of MWCNT-based LFB can be extended to visually detect protein biomarkers using MWCNT–antibody conjugates. The MWCNT-based LFB thus open a new door to prepare a new generation of LFB, and shows great promise for in-field and point-of-care diagnosis of genetic diseases and for the detection of infectious agents.

Immobilization of Trypsin on Graphene Oxide Nanosheets for Increased Proteolytic Stability

Abstract To increase the thermal and chemical stabilities of trypsin-proteolysis activity, trypsin was immobilized on graphene oxide nanosheets (GONSs) by a one-step diimide-activated amidation. The trypsin-immobilized GONS (1.9 mg per mg-GONS) showed significantly higher thermal and chemical stabilities than the free form. After heat treatment at 50 °C for 6 h, the trypsin–GONS retained 80% of its initial activity, but free trypsin retained only 4% of its initial activity.

Preparation of Pt- and Pd-decorated CNTs by DCC-activated amidation and investigation of their electrocatalytic activities

Pt- and Pd-decorated carbon nanotubes (CNTs) have been prepared by diimide-activated amidation, an approach that avoids the harmful products of conventional SOCl 2 treatment. In this study, N, N′-dicyclohexylcarbodiimide (DCC) was used as a coupling agent for the formation of an amide linkage. According to ICP measurements, the resulting materials consist of 7.1 wt.% loaded Pt and 8.6 wt.% loaded Pd. The TEM images show that size-similar and quasi-spherical nanoparticles are highly dispersed along the entire CNT walls. The prepared Pt-CNT exhibited an especially high oxidation current toward methanol, formic acid, and formaldehyde compared with that of the pristine CNT and a Pt/CNT mixture (10 wt.% Pt). Furthermore, the Pd-CNT displayed higher activity toward H 2O 2 reduction than that of the reference systems. The better catalytic activities can be explained by the high electrochemically active surface area that resulted from the smaller size and excellent dispersion of metal nanoparticles on CNT. These results demonstrate the DCC-activated amidation to be a very effective and useful way to tune size and disperse metal species in metal-decorated CNTs, markedly improving their catalytic activity.

Synthesis of graphene oxide-based biocomposites through diimide-activated amidation

In this work, a novel and facile method for covalent attachment of biomaterials to graphene oxide sheets (GOS) was developed. Four conjugates were obtained via the diimide-activated amidation reaction under ambient conditions. Final products were characterized by FT-IR spectroscopy, atomic force microscopy and transmission electron microscopy. Electrochemical characterization of the composite showed that the covalently bonded biomaterial retained its bioactivity. This method may provide a way for further preparation of graphene-based biodevices.

Synthesis of hydrophilic and organophilic chemically modified graphene oxide sheets

In this work, hydrophilic and organophilic chemically modified graphene oxide (CMGO) sheets were prepared through a two-step diimide-activated amidation. The hydrophilic and organophilic products were characterized by atomic force microscopy, transmission electron microscopy and ultraviolet-visible spectroscopy. The resulted dispersions are homogeneous and exhibit long-term stability, which will facilitate the combination of CMGO sheets with polymers to yield homogeneous composites.

Covalent attaching protein to graphene oxide via diimide-activated amidation

In this paper, graphene oxide nanosheets (GOS) are functionalized by bovine serum albumin (BSA) via diimide-activated amidation under ambient conditions. The obtained GOS–BSA conjugate is highly water-soluble. Results of atomic force microscopy (AFM), Raman spectra and Fourier transform infrared spectroscopy analysis confirm that GOS–BSA conjugate contains both GOS and BSA protein. AFM image shows that GOS are fully exfoliated. Results of cyclic volatammograms show that the protein in the GOS–BSA conjugate retains its bioactivity. The present method may also provide a way to synthesize graphene-based composites with other biomolecules.

Functionalization of carbon encapsulated iron nanoparticles

Carbon-encapsulated magnetic nanoparticles are a new class of materials where the core magnetic nanoparticle is protected from reactions with its environment by graphite shells. Having a structure similar to carbon nanotubes, these nanoparticles could be potentially functionalized using methods which are already applied to those structures. We present the effects of acidic treatments based on HCl, HNO3, and H2SO4 on these nanoparticles highlighting the impact on their magnetic and surface properties. We show that acidic treatments based on HNO3 can be successfully applied for the generation of carboxylic groups on the surface of the nanoparticles. Using methylamine as a model, we demonstrate that these functional groups can be used for further functionalization with amino-containing biomolecules via diimide-activated amidation.

Bioconjugation of Interferon-alpha Molecules to Lysine-Capped Gold Nanoparticles for Further Drug Delivery Applications

Gold nanoparticles are potentially very attractive components for therapeutic delivery since they can be synthesized with any diameter from 1 to 200 nm to carry a payload of therapeutic molecules into a cell without triggering an immune response. Gold nanoparticles must undergo surface transformations before coupling to therapeutic molecules to become eligible for this purpose. It is now more understood that amine groups can bind to gold nanoparticles strongly, which has enabled surface modification of gold nanoparticles with amino acid lysine through its amine group. These lysine capped gold nanoparticles can further be coupled to therapeutic molecules for delivery purposes. In this study gold nanoparticles were first synthesized and capped with lysine molecules. TEM and FTIR measurements demonstrated the synthesis of lysine-capped gold nanoparticles with an average diameter of 10 nanometers. Interferon alpha molecules-one of the most important therapeutic protein were then chemically bound to lysine-capped gold nanoparticles through a two-step process of diimide-activated amidation. The conjugation of interferon molecules to lysine capped gold nanoparticles was carried out via the reaction between the free amine group of lysine and carboxyl groups of interferon using N-ethyl-N′-13-dimethyl-aminopropyl (EDAC) as a coupling agent. The process of conjugation has also been studied by transmission electron microscopy.

Synthesis of aligned carbon nanotubes by microwave chemical vapour deposition and investigation of their covalent bonding with antibodies for bioapplications

Synthesis of aligned carbon nanotubes arrays and their covalent bonding with antibodies for potential antibody-antigen immunosensing are presented. Aligned carbon nanotubes were prepared by a template assisted microwave chemical vapour deposition. This method will produce less amorphous carbon due to the existing temperature gradient. The effect of catalysts for carbon nanotubes growth was studied. A post treatment process was developed to remove the amorphous carbon layer and partially etch the template so that nanotubes tips were exposed. After functionalisation of carbon nanotubes arrays, fluorescence-labelled IgG antibodies were attached to their surface by a two-step process of diimide-activated amidation. The covalent bonding between antibodies and carbon nanotubes arrays was evaluated by a fluorescent microscope. A stronger bonding was observed from functionalised carbon nanotubes than unfunctionalised ones. It is believed that both the alignment of carbon nanotubes and their covalent bonding with antibodies play important roles for immunosensors with high sensitivity and selectivity.

Functionalized Carbon Nanotubes for Detecting Viral Proteins

We investigated the biocompatibility, specificity, and activity of a ligand-receptor-protein system covalently bound to oxidized single-walled carbon nanotubes (SWNTs) as a model proof-of-concept for employing such SWNTs as biosensors. SWNTs were functionalized under ambient conditions with either the Knob protein domain from adenovirus serotype 12 (Ad 12 Knob) or its human cellular receptor, the CAR protein, via diimide-activated amidation. We confirmed the biological activity of Knob protein immobilized on the nanotube surfaces by using its labeled conjugate antibody and evaluated the activity and specificity of bound CAR on SWNTs, first, in the presence of fluorescently labeled Knob, which interacts specifically with CAR, and second, with a negative control protein, YieF, which is not recognized by biologically active CAR proteins. In addition, current-gate voltage (I-V(g)) measurements on a dozen nanotube devices explored the effect of protein binding on the intrinsic electronic properties of the SWNTs, and also demonstrated the devices' high sensitivity in detecting protein activity. All data showed that both Knob and CAR immobilized on SWNT surfaces fully retained their biological activities, suggesting that SWNT-CAR complexes can serve as biosensors for detecting environmental adenoviruses.

Biological functionalization and fluorescent imaging of carbon nanotubes

Biofunctionalization and manipulating of carbon nanotubes (CNTs) is important for biomedical research and application. Cy5 labeled goat anti-rabbit IgG (anti-IgG-Cy5) is chemically bonded to CNTs via a two-step process of diimide-activated amidation. This process can avoid the intermolecular connection of proteins. Fluorescent imaging of CNTs in aqueous solution has been demonstrated using anti-IgG-Cy5 immobilized CNTs (IgG-CNTs) as the model. The biologically functionalized carbon nanotubes (f-CNTs) in solution have been observed successfully using fluorescence microscopy. The fluorescent image of highly oriented f-CNTs is obtained at first time. The influencing factors on fluorescent imaging including oxidation duration, background noise and reactant concentration are discussed.