Graphene oxide-based electrochemical label-free detection of glycoproteins down to aM level using a lectin biosensor.

A label-free ultrasensitive impedimetric biosensor with lectin immobilised on graphene oxide (GO) for the detection of glycoproteins from 1 aM is shown here. This is the first time a functional lectin biosensor with lectin directly immobilised on a graphene-based interface without any polymer modifier has been described. The study also shows that hydrophilic oxidative debris present on GO has a beneficial effect on the sensitivity of (8.46 ± 0.20)% per decade for the lectin biosensor compared to the sensitivity of (4.52 ± 0.23)% per decade for the lectin biosensor built up from GO with the oxidative debris washed out.

Calcium pectate gel beads for cell entrapment. 6. Morphology of stabilized and hardened calcium pectate gel beads with cells for immobilized biotechnology.

The structure of standard and stabilized calcium pectate gel (CPG) beads has been examined by scanning (SEM) and transmission (TEM) electron microscopy. A two-stage crosslinking procedure with polyethyleneimine (PEI) and glutaraldehyde (GA) led to the formation of a more compact layer on the bead surface. On the other hand, the stabilization procedure did not significantly change either gel bead interior or morphologic properties, vitality and biotransformation activity of immobilized bacterial cells (Nocardia tartaricans) against cis-epoxysuccinate as well as yeast cells (Trigonopsis variabilis) against cephalosporin C. The structure of these cells within the calcium pectate matrix remained unchanged. Moreover, the two-step chemical stabilization of CPG containing T. variabilis or N. tartaricans had a favourable effect on storage and operational stability at semi-continuous and continuous processing in stirred batch and packed-bed reactors. The most valuable effect of stabilization was the fact that the hardened CPG comprising the cells N. tartaricans resisted, for a long time (360 days and more), the destructive effects of the product (such strong sequestering reagent as L-(+)-tartaric acid) at high concentrations (up to 1 M). Non-hardened CPG was destroyed after 21 h. The reference materials, hardened and non-hardened calcium alginate gels (CAG), were destroyed over 3 h or 30 min, respectively.

Examination of bioaffinity immobilization by precipitation of mannan and mannan-containing enzymes with legume lectins.

The interaction of four lectins from crops of the legume family with Saccharomyces cerevisiae alpha-mannan, and also with two glycoenzymes containing mainly alpha-mannan moieties, has been studied. The interaction was characterized by a quantitative precipitation assay. The results of precipitation differ with respect to both quality (the point of maximum precipitation) and of the quantity (the amount of aggregated lectin and saccharide). The lectin concanavalin A [Con A, from jack bean (Canavalia ensiformis)] was observed to form more extensive precipitates with Saccharomyces cerevisiae mannan and glycoenzymes than did lectins from Lens culinaris (lentil) and Pisum sativum (garden pea), while in the case of Vicia faba (broad or fava bean) no interaction was found with either the examined mannans or with glycosylated enzymes. The complete precipitation of invertase and glucoamylase with Con A (enzymes and also Con A; up to 100%) was achieved at a Con A glycoenzyme molar ratio of 20.2 and 2.3 respectively, whereby about 85% of precipitated and also of initial activities of glycoenzymes were determined in the aggregates. More valuable results were achieved by the technique of enzyme immobilization called 'multiple bioaffinity layering' which is based on the stepwise biospecific adsorption of the glycosylated enzymes and Con A on a matrix precoupled with Con A. A 3-fold repetition of the layering procedure afforded up to a 10-fold increase in catalytic activity of the immobilized invertase, in contrast with a 2.1-fold increase in catalytic activity of the immobilized glucoamylase.

Amplification of flow-microcalorimetry signal by means of multiple bioaffinity layering of lectin and glycoenzyme.

This paper demonstrates a positive influence of a special, stepwise technique of enzyme immobilization based on the biospecific adsorption of the glycoenzyme invertase on immobilized concanavalin A (Con A), subsequent adsorption of the free Con A on the immobilized invertase:Con A support and repeated adsorption of invertase on the support. A 3-fold repetition of the same procedure designed preliminarily as bioaffinity layering afforded up to a 10-fold increase in catalytic activity of the immobilized invertase. Reactive hydrogels based on bead cellulose and bead poly(glycidyl methacrylate) were used as immobilization supports for the preparation of these highly active preparations. The enhancement in catalytic activity of immobilized invertase preparations was demonstrated thermometrically, by flow microcalorimetry. Further attractive aspects for utilizing the signal amplification of biosensors with immobilized enzymes are discussed.

The glycosylated enzyme-binding assay for the study of the interaction of free and immobilized lectins with carbohydrates.

The glycosylated enzymes (invertase and glucose oxidase) were used as the competitive markers for a simple and rapid determination of the lectin-saccharide interactions. The method, based on the formation of the conjugate of an appropriate glycoenzyme with the specific carbohydrate-binding lectins and the inhibition of the conjugate formation with a monosaccharide, was described. This method was used to estimate the relative carbohydrate specificity of Concanavalin A for monosaccharides derived from D-mannose. The inhibition effect of the saccharides on the formation of Concanavalin A-glycosylated enzyme precipitate was compared with their influence on the enzyme sorption on conjugate Concanavalin A-bead cellulose support. The amount of the interacting enzyme was estimated either indirectly from its concentration in a supernatant that was determined spectrophotometrically (Con A was in a free or immobilized form) or directly in the immobilized form linked to Con A-sorbent using the flow microcalorimetric method. The results obtained, using different methods, agreed in general.

Lectin-glycoenzyme column chromatography monitored by enzyme flow microcalorimetry.

A method based on the flow microcalorimetric determination of catalytic activity of immobilized enzyme in a so-called enzyme thermistor was used to monitor the process of lectin affinity chromatography of invertase on Concanavalin A-bead cellulose. The strong biospecific interaction between Concanavalin A and invertase was employed to determine the bound enzyme and this principle was used for the investigation of an alternative direct method for monitoring the lectin affinity chromatography of glycoenzymes. The results obtained by flow microcalorimetry showed that the catalytic activity of invertase immobilized on Concanavalin A-bead cellulose can be compared directly with the thermometric value delta Tmax. The validity of the method was also confirmed by the enzyme thermistor post-column method, which is based on the determination of the product from the immobilized invertase enzymatic reaction. The adsorption and desorption in the chromatography column were examined by flow microcalorimetry in small samples withdrawn from the column. Attention has been given to the operating parameters and the storage stability of the affinity sorbent. The binding ability of the affinity matrix decreased with the number of consecutive chromatographic runs, although its storage stability was satisfactory.