Achievement and assessment of direct electron transfer of glucose oxidase in electrochemical biosensing using carbon nanotubes,graphene, and their nanocomposites

Carbon nanotubes, graphenes, and their hybridized composites with nanoparticles have been attempted to establish a direct electrical communication between the recognition biomolecule and its underlying electrode surface. This review (with 133 refs.) focuses on advances, strategies and technical challenges in the development of reagentless electrochemical biosensors for glucose with enhanced detection sensitivity, selectivity, and simplicity. Specifically, the review commences with a discussion of the relevance of direct electron transfer (DET) in biosensing together with the fundamental of electro-enzymology and kinetics. General aspects of glucose oxidase (GOx), the most popular enzyme with a flavin cofactor, are discussed in view of its historical and important role in the development of electrical biosensors for blood glucose. The next section assesses DET of GOx based on the Marcus theory and the Laviron formalism. The reorganizational energy of the Marcus model and the overpotential play an important role in reaction kinetics and affect the rate of electron transfer significantly. The presence of nanomaterials, particularly for graphene oxide, decreases the electron transfer distance between the enzyme redox center and the underlying electrode surface well beyond 15 Å. The improper Marcus-Hush-Chidsey integral is now simplified to estimate the rate of electron transfer with very good accuracy. Critiques, technical challenges, and future possibilities of glucose electrodes with respect to DET are also presented and discussed.

Open image in new window

Graphical abstract This review (with 133 refs.) focuses on advances, strategies and technical challenges in the development of reagentless electrochemical biosensors for glucose with enhanced detection sensitivity, selectivity, and simplicity.

     

Evaluation of apparent non-specific protein loss due to adsorption on sample tube surfaces and/or altered immunogenicity

The non-specific loss of protein analytes can have a major effect on assay results particularly where the concentrations of such analytes are extremely low and the matrix is complex. This report assesses how the protein incubated in sample tubes may be lost due to adsorption. Use of proteins, such as bovine serum albumin (BSA), may be used to pre-treat tubes to reduce such losses. However, such losses may also be associated with structural perturbations leading to changes in immunogenicity (as a result of alterations in specific epitope-related conformations). This can lead to erroneous results or lack of comparability with a range of methodologies such as the bicinchoninic protein assay and immunoassays or when surface plasmon resonance (SPR)-based approaches are used. A model system to evaluate these phenomena is proposed.     

Rapid and simple preparation of a reagentless glucose electrochemical biosensor

A rapid and simple procedure was developed for the preparation of a highly stable and leach-proof glucose oxidase (GOx)-bound glassy carbon electrode (GCE). Crosslinked GOx via glutaraldehyde was drop-cast on a KOH-pretreated GCE followed by drop-casting of 3-aminopropyltriethoxysilane (APTES) to form a stable bioactive layer. At -0.45 V, the biosensor exhibited a wide dynamic detection range of 0.5-48 mM for commercial glucose and 1.3-28.2 mM for Sugar-Chex blood glucose linearity standards. Several endogenous electroactive substances and drug metabolites commonly found in blood were tested and provoked no signal response. To our knowledge, the developed procedure is the most rapid method for preparing a glucose biosensor. The biosensor suffered no biofouling after 7 days of immersion in Sugar-Chex blood glucose. With excellent production reproducibility, GOx-bound electrodes stored dry at room temperature retained their initial activity after several weeks.     

Solid phase inclusive immunoradiometric assay for human chorionic gonadotropin using monoclonal antibodies

Summary A simple, one step, inclusive immunoradiometric assay for human chorionic gonadotropin (hCG) employing monoclonal antibodies is described. Commercially available monoclonal antibodies from various commercial sources were screened. Identified “detection” antibody was radiolabeled with ¹²⁵I and the selected “capture” antibody was chemically coupled to magnetizable cellulose to form a solid phase. In the procedure developed, standard/sample, radiolabeled antibody and capture antibody were incubated together for 3 hours at room temperature with shaking. After incubation, the bound complex was quantitated for the associated radioactivity. The analytical sensitivity observed was 1.0 mIU/ml with a wide concentration range up to 1000 mIU/ml of hCG. “High dose hook” of the developed assay was observed beyond 2000 mIU/ml. Results showed that the developed assay had a good precision: intra-assay CV less than 8%, inter-assay CV less than 10% and good analytical recovery of 97-109%. The clinical samples analyzed by the developed procedure showed a good correlation with that of the commercial kit (r = 0.92; y = 0.99x+0.51).     

Monte carlo simulations of gamma images: application to plutonium rich mixed carbide fuel

A novel approach for quantitave characterisation of nuclear fuel for homogeneity based on comparison of autoradiographs with simulated standards is proposed. Autoradiographs which are self generated radiation images are ususally only qualitatively evaluated due to the absence of calibration standards for reference which are not practically possible to synthesize in all possible variations owing to the difficulty in handling plutonium. Monte Carlo simulation for radiation transport has been attempted for the first time to estimate the minimum detectable limit (MDL) of pure PuC agglomerate and generate virtual autoradiographic images of plutonium rich mixed carbide fuel for visual comparison reference purpose.

     

Solid phase immunoradiometric assay for CA125 antigen levels in blood using monoclonal antibodies

We have developed a simple one step ‘sandwich’ immunoradiometric assay for CA125 using monoclonal antibodies directed against two different epitopes of the antigen. The detection antibody was radiolabeled with I-125 and the selected capture antibody was chemically coupled to magnetizable cellulose to form immobilized solid support. In the developed inclusive assay procedure, 200 μL of standard or sample was incubated with 100 μL of radiolabeled and capture antibody suspension for 18 h at room temperature with shaking. At the end of the incubation, the sandwich complex attached to solid phase is separated and counted for associated radioactivity. The analytical sensitivity for the developed assay procedure was observed to be 3.0 U/mL with an assay range up to 500 U/mL of CA125. The developed assay displayed acceptable precision; expressed in terms of percentage Coefficient of Variation (CV) estimated by repeated analyses of the quality control samples. Intra-assay CV was observed to be less than 5% whereas inter-assay CV was also less than 6%. The analytical recovery of the developed assay observed to be in the range of 88–107%. The clinical samples analyzed by the developed procedure showed a good correlation with that of a commercial kit (r = 0.99; y = 1.0052x − 38.942).     

Effect of antibody immobilization strategies on the analytical performance of a surface plasmon resonance-based immunoassay

Antibody immobilization strategies (random, covalent, orientated and combinations of each) were examined to determine their performance in a surface plasmon resonance-based immunoassay using human fetuin A (HFA) as the model antigen system. The random antibody immobilization strategy selected was based on passive adsorption of anti-HFA antibody on 3-aminopropyltriethoxysilane (APTES)-functionalized gold (Au) chips. The covalent strategy employed covalent crosslinking of anti-HFA antibody on APTES-functionalized chips using 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide (EDC) and sulfo-N-hydroxysuccinimide (SNHS). The orientation strategy used passive adsorption of protein A (PrA) on Au chips, with subsequent binding of the anti-HFA antibody in an orientated fashion via its fragment crystallisable (Fc) region. In the covalent-orientated strategy, PrA was first bound covalently, to the surface, which in turn, then binds the anti-HFA antibody in an orientated manner. Finally, in the most widely used strategy, covalent binding of anti-HFA antibody to carboxymethyldextran (CM5-dextran) was employed. This immobilization strategy gave the highest anti-HFA antibody immobilization density, whereas the highest HFA response was obtained with the covalent-orientated immobilization strategy. Therefore, the covalent-orientated strategy was the best for SPR-based HFA immunoassay and can detect 0.6-20.0 ng/mL of HFA in less than 10 min.     

Technology behind commercial devices for blood glucose monitoring in diabetes management: a review

The blood glucose monitoring devices (BGMDs) are an integral part of diabetes management now-a-days. They have evolved tremendously within the last four decades in terms of miniaturization, rapid response, greater specificity, simplicity, minute sample requirement, painless sample uptake, sophisticated software and data management. This article aims to review the developments in the technologies behind commercial BGMD, especially those in the areas of chemistries, mediators and other components. The technology concerns, on-going developments and future trends in blood glucose monitoring (BGM) are also discussed.     

Selection and optimization of monoclonal antibody immobilization procedure for its use in IRMA

Immobilization of monoclonal antibody (McAb) on polystyrene support is an integral part of any immunoassay (IA) procedure. Two different immobilization approaches (direct and indirect) were evaluated for their effectiveness. In case of direct immobilization, mouse monoclonal antibody was adsorbed ‘passively’ on polystyrene tubes. The same antibody was immobilized as ‘pre-formed complex’ in case of indirect approach via mouse IgG as linker. Both the approaches displayed comparable Bmax using different buffer systems. However, NSB observed via mouse IgG linker was always on the higher side (0.8–4.0 %). This could be significantly reduced (up to <0.4 %) by controlling the concentration of mouse IgG.Theoretical advantages envisaged via indirect approach viz. economy and stability of immobilized antibody were similar to that of simple passive adsorption. Hence, we have selected simple passive adsorption method using bicarbonate buffer (pH 8.4). Our study also confirmed the need for ‘tube maturation’ in order to retain the immunological integrity of the immobilized antibody. The performance of the tubes prepared by direct immobilization method was evaluated in an immunoradiometric assay for human Luteinizing hormone (LH).The developed method is simple, convenient and amenable for large scale production of antibody immobilized polystyrene tubes.