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.

Sensitive detection and glycoprofiling of a prostate specific antigen using impedimetric assays.

This study presents a proof-of-concept for the development of an impedimetric biosensor for ultra-sensitive glycoprofiling of prostate specific antigen (PSA). The biosensor exhibits three unique characteristics: (1) analysis of PSA with limit of detection (LOD) down to 4 aM; (2) analysis of the glycan part of PSA with LOD down to 4 aM level and; (3) both assays (i.e., PSA quantification and PSA glycoprofiling) can be performed on the same interface due to label-free analysis.

An ultrasensitive impedimetric glycan biosensor with controlled glycan density for detection of lectins and influenza hemagglutinins.

An impedimetric glycan biosensor with optimised glycan density was applied for the detection of lectins and influenza hemagglutinins down to attomolar concentrations (aM).

Nanoscale controlled architecture for development of ultrasensitive lectin biosensors applicable in glycomics.

In this Minireview the most advanced patterning protocols and transducing schemes for development of ultrasensitive label-free and label-based lectin biosensors for glycoprofiling of disease markers and some cancerous cells are described. Performance of such lectin biosensors with interfacial properties tuned at a nanoscale are critically compared to the most sensitive immunoassay format of analysis and challenges ahead in the field are discussed. Moreover, key elements for future advances of such devices on the way to enhance robustness and practical applicability of lectin biosensors are revealed.

Are glycan biosensors an alternative to glycan microarrays?

Complex carbohydrates (glycans) play an important role in nature and study of their interaction with proteins or intact cells can be useful for understanding many physiological and pathological processes. Such interactions have been successfully interrogated in a highly parallel way using glycan microarrays, but this technique has some limitations. Thus, in recent years glycan biosensors in numerous progressive configurations have been developed offering distinct advantages compared to glycan microarrays. Thus, in this review advances achieved in the field of label-free glycan biosensors are discussed.

Comparison of three distinct ELLA protocols for determination of apparent affinity constants between Con A and glycoproteins.

A procedure for determination of apparent affinity constants K(D)(app) between Concanavalin A (Con A) and naturally d-mannose containing glycoproteins using enzyme-linked lectin assay (ELLA) is reported. Three distinct ELLA protocols are compared to each other with 3 different fitting models used (Liliom, Hill with and without a cooperativity factor). The glycoproteins were physisorbed on a highly charged polystyrene solid surface of immunoassay plates and the amount of lectin bound to the glycoproteins was determined by photometry. The interactions of Con A with five mannose-containing glycoproteins, invertase (INV), glucoamylase (GA), glucose oxidase (GOx), ovalbumin (OVA), and transferrin (TRF) were quantified with apparent affinity constant being in the range 2×10(-7) to 9×10(-6)M. The strength of interaction between Con A and glycoproteins is discussed on the basis of glycan structure/exposure on the protein backbone for each glycoprotein.

Monitoring of dihydroxyacetone production during oxidation of glycerol by immobilized Gluconobacter oxydans cells with an enzyme biosensor.

A bi-enzymatic biosensor for monitoring of dihydroxyacetone production during oxidation of glycerol by bacterial cells of Gluconobacter oxydans is presented. Galactose oxidase oxidizes dihydroxyacetone efficiently producing hydrogen peroxide, which reacts with co-immobilized peroxidase and ferrocene pre-adsorbed on graphite electrode. This mediator-based bi-enzymatic biosensor possesses very high sensitivity (4.7 µA/mM in phosphate buffer), low detection limit (0.8 µM, signal/noise = 3), short response time (22 s, 95% of steady-state) and broad linear range (0.002-0.55 mM in phosphate buffer). The effect of pH, temperature, type of buffer, as well as different stabilizers (combinations of a polyelectrolyte and a polyol) on the sensor performance were carefully optimized and discussed. Dihydroxyacetone produced during a batch conversion of glycerol by the pectate-immobilized bacteria in an air-lift reactor was determined by the biosensor and by reference spectrophotometric method. Both methods were compared and were in a very good correlation. The main advantage of the biosensor is a very short time needed for sample analysis (less than 1 min).

Novel glucose non-interference biosensor for lactose detection based on galactose oxidase-peroxidase with and without co-immobilised beta-galactosidase.

Two types of amperometric biosensors for lactose detection based either on co-immobilisation of two enzymes (galactose oxidase with peroxidase) or co-immobilisation of three enzymes (beta-galactosidase, galactose oxidase and peroxidase) were constructed. A graphite rod with pre-adsorbed ferrocene was used as a working electrode. The use of galactose oxidase instead of the frequently used glucose oxidase resulted in the construction of a glucose-non-interfering lactose sensor. Co-immobilisation of peroxidase with galactose oxidase allowed the effect of borate on the extension of the linear range and the effect of the working potential on galactose oxidase activation to be studied. The presence of beta-galactosidase greatly enhances the sensor's sensitivity, but its linear range is narrower than that of the sensor without beta-galactosidase. Addition of DEAE-dextran and inositol to the enzyme layer improved the half-life more than 16-fold compared with the sensor without stabilisers. A response time between 60 and 75 s (90% of the steady-state value) and a detection limit for lactose determination from 44 to 339 microM (signal-to-noise ratio = 3) were observed depending on the conditions. The precision of measurements of standard lactose solution for the trienzymatic and bienzymatic sensors was 2.19 and 2.02%, respectively. The precision of analysis of dairy products varied from 0.24 to 5.24%. Analyses of real samples showed good correlation with HPLC analysis; eight samples and 10 standard lactose solutions without pre-treatment were analysed in 1 h.

Microbial cell-based biosensor for sensing glucose, sucrose or lactose.

Biosensors for the determination of glucose, sucrose and lactose were based on a Clark-type oxygen electrode covered with a membrane containing microbial cells. The glucose-sensing membrane was prepared with intact cells of Gluconobacter oxydans immobilized in gelatin cross-linked with glutardialdehyde. The disaccharide-sensing membranes were prepared by co-immobilization of G. oxydans with cells of Saccharomyces cerevisiae containing invertase for sucrose determination and with permeabilized cells of Kluyveromyces marxianus containing beta-galactosidase for lactose determination. The strain of G. oxydans that we used was able to oxidize both anomers of glucose at the same rate; there was therefore no need for mutarotase co-immobilization in disaccharide-sensing membranes. The sensitivity of glucose sensor was 50 nA/mM, the range of the calibration curve was 0-0.8 mM, the response time was 2 min, and the response after 1 week of storage was 62% of the initial response. The parameters of the disaccharide sensors were similar: linear range of calibration curve up to 4 mM, response time 5 min. The activities of the sensors after 1 week of storage at ambient temperature were in the range 50-65% of the initial activity.