Urease immobilization on biotinylated polypyrrole coated ChemFEC devices for urea biosensor development

In this report, we describe a novel strategy for the design of a clinical urea biosensor using a process based on assembled multilayer systems. Biotinylated enzyme (urease–subiotin) was immobilized on the biotinylated polypyrrole coated Chemical field effect capacitance (ChemFEC) devices using the high avidin–biotin affinity. The immobilized enzyme activity was checked by its catalytic conversion of urea into carbon dioxide and ammonia. Electrochemical response of the bridge system constructed on Si/SiO₂/Si₃N₄ electrodes to urea addition was evaluated using the capacity–potential measurements. In addition, contact-angle measurements were carried out to control the change of surface energy and their components before and after each layer formation. The developed urea biosensor demonstrates high performances: a good sensitivity of 50 mV/pUrea in the linear urea concentration range from 10⁻⁴ to 10⁻¹ M and an excellent operational stability after three weeks of continuous use. The immobilized urease was characterised through its apparent Michaelis–Menten constant (5 mM) and the activation energy of the enzymatic reaction (20 kJ mol⁻¹). It was also shown that capacitive measurements can be used to quantify the interaction between molecular systems, based on avidin and biotin molecules.     

Amperometric biosensor based on a site-specific immobilization of acetylcholinesterase via affinity bonds on a nanostructured polymer membrane with integrated multiwall carbon nanotubes

Acetylcholinesterase (AChE) was immobilized on chemically modified poly-(acrylonitrile-methyl-methacrylate-sodium vinylsulfonate) membranes in accordance with three different methods, the first of which involved random enzyme immobilization via glutaraldehyde, the second one—site-specific enzyme immobilization via glutaraldehyde and Concanavalin A (Con A) and the third method—modified site-specific enzyme immobilization via glutaraldehyde in the presence of a mixture of multiwall carbon nanotubes and albumin (MWCNs + BSA), glutaraldehyde and Con A. Preliminary tests for the activity of immobilized AChE were carried out using these three methods. The third method was selected as the most efficient one for the immobilization of AChE and the prepared enzyme carriers were used for the construction of amperometric biosensors for the detection of acetylthiocholine (ATCh).A five level three factorial central composite design was chosen to determine the optimal conditions for the enzyme immobilization with three critical variables: concentration of enzyme, Concanavalin A and MWCNs. The design illustrated that the optimum values of the factors influencing the amperometric current were C_E: 70 U mL⁻¹; C_{Con A}: 1.5 mg mL⁻¹ and C_MWCN: 11 mg mL⁻¹, with an amperometric current 0.418 μA. The basic amperometric characteristics of the constructed biosensor were investigated. A calibration plot was obtained for a series of ATCh concentrations ranging from 5 to 400 μM. A linear interval was detected along the calibration curve from 5 to 200 μM. The correlation coefficient for this concentration range was 0.995. The biosensor sensitivity was calculated to be 0.065 μA μM⁻¹ cm⁻². The detection limit with regard to ATCh was calculated to be 0.34 μM. The potential application of the biosensor for detection and quantification of organophosphate pesticides was investigated as well. It was tested against sample solutions of Paraoxon. The biosensor detection limit was determined to be 1.39 × 10⁻¹² g L⁻¹ of Paraoxon, as well as the interval (10⁻¹¹ to 10⁻⁸ g L⁻¹) within which the biosensor response was linearly dependant on the Paraoxon concentration. Finally the storage stability of the enzyme carrier was traced for a period of 120 days. After 30-day storage the sensor retained 76% of its initial current response, after 60 days—68% and after 120 days—61%.     

Resonant mirror biosensor detection method based on yessotoxin-phosphodiesterase interactions

Yessotoxin (YTX) is a generic name for a group of lipophilic compounds recently discovered and chemically characterized. Association measurements were done in a resonant mirror biosensor. The instrument detects changes in the refractive index and/or thickness occurring within a few hundred nanometers form the sensor surface where a molecule is attached. We used aminosilane surfaces where phosphodiesterase 3',5'-cyclic-nucleotide-specific from bovine brain (PDEs) was immobilized. Over this immobilized ligand different amounts of YTX were added and typical association curve profiles were observed. These association curves fit a pseudo-first-order kinetic equation where the apparent association rate constant (k(on)) can be calculated. The value of this constant increases with YTX concentration. From the representation of k(on) versus YTX concentration we obtained the association rate constant (k(ass)) 248+/-40 M(-1)s(-1) and the dissociation rate constant (k(diss)) 9.36 x 10(-4)+/-1.72 x 10(-4)s(-1). From these values the kinetic equilibrium dissociation constant (K(D)) for YTX-PDEs association can be calculated. The value of this last constant is 3.74 x 10(-6)+/-8.25 x 10(-8)M YTX. The PDE-YTX association was used as a method suitable for determination of the toxin concentration in a shellfish sample. The assay had sufficient sensitivity and can be used on simple shellfish extracts.     

Recent developments in downstream processing based on affinity interactions

Novel purification processes have been developed, based on the interaction between complementary biomolecules, to circumvent the difficulties encountered by conventional affinity chromatography. Depending upon the procedure used for isolating the ligand—binder complex, the process can be termed affinity cross-flow filtration, affinity partition or affinity precipitation. This review describes the developments and potentials of such purification techniques.     

Laccase immobilization on the tailored cellulose-based Granocel carriers

Extracellular laccase produced by Cerrena unicolor was immobilized by adsorption or covalent bonds formation on the cellulose-based carrier Granocel. Immobilization was optimized by changing the anchor groups and the methods of activation/immobilization. On the base of measured activity and stability of immobilized preparations, the covalent method was selected. It was shown that coupling of the enzyme to the carrier via divinyl sulfone or glutaraldehyde yielded an enzyme-carrier preparation of high activity and storage stability. Further optimization of the carrier's superstructure consisted in changing pore diameters and amount of functional groups on the carriers surface. Three-fold higher activity was noted when the enzyme was immobilized on NH2-modified Granocel with the highest size exclusion limit and amino group content. Relatively low products sorption was observed on the carrier surface. The effects of protein concentration and pH-value of the coupling mixture on immobilization efficiency were evaluated also.     

Piezoelectric urea biosensor based on immobilization of urease onto nanoporous alumina membranes

The urease was immobilized onto nanoporous alumina membranes prepared by the two-step anodization method, and a novel piezoelectric urea sensing system with separated porous alumina/urease electrode has been developed through measuring the conductivity change of immobilized urease/urea reaction. The process of urease immobilization was optimized and the performance of the developed urea biosensor was evaluated. The obtained urea biosensor presented high-selectivity monitoring of urea, better reproducibility (S.D. = 0.02, n = 6), shorter response time (30 s), wider linear range (0.5 μM to 3 mM), lower detection limit (0.2 μM) and good long-term storage stability (with about 76% of the enzymatic activity retained after 30 days). The clinical analysis of the urea biosensor confirmed the feasibility of urea detection in urine samples.     

Potential of biosensor technology for the characterization of interactions by quantitative affinity chromatography

This review places the characterization of interactions by biosensor technology in the broader context of their study by quantitative affinity chromatography. The general reluctance to consider biosensor-based characterization as a form of quantitative affinity chromatography on the grounds of a difference in aims of the two techniques reflects a mistaken belief that BIAcore and IAsys studies characterize the kinetics of the chemical reaction responsible for biospecific adsorption of a soluble reactant to an immobilized form of its affinity partner. It now transpires that the association and dissociation rate constants thereby determined refer to thermodynamic characterization of biospecific adsorption in terms of a single-phase model in which affinity sites are distributed uniformly throughout the liquid-phase volume accessible to the partitioning reactant—the model used for characterization of biospecific adsorption by quantitative affinity chromatography. In that light the most important attribute of biosensor technology is its potential for thermodynamic characterization of biospecific adsorption by virtue of its ability to monitor complex formation directly; and hence its potential for the characterization of interactions with affinities that are too strong for study by forms of quantitative affinity chromatography that monitor complex formation on the basis of reactant depletion from the liquid phase. Kinetic as well as thermodynamic analyses of biosensor data are described for attainment of that potential.     

Glutamate optical biosensor based on the immobilization of glutamate dehydrogenase in titanium dioxide sol-gel matrix

A simple and novel titania sol-gel derived optical biosensor coupled with carboxy seminaphthorhodamine-1-dextran (SNARF-1-dextran) as the fluorescent dye was fabricated for the determination of glutamate in water and biological samples. The NADH-dependent glutamate dehydrogenase (GLDH) was trapped in titania sol-gel derived matrix prepared by vapor deposition method. In addition, scanning electron microscopy (SEM) and atomic force microscopy (AFM) were used to characterize the surface morphology of the spots. SEM and AFM images showed that the deposition of titania precursor at 27 degrees C for 6.5h was found to be suitable to form transparent titania sol-gel matrix to encapsulate GLDH and fluorescent probe. AFM images showed that the roughness of TiO(2) surface increased from 2.16 nm in the absence of GLDH and SNARF to 37.8 nm after the immobilization. The developed titania biosensor has good analytical performance with water samples. A dynamic range between 0.04 and 10mM with the detection limit of 5.5 microM were observed. The responses to glutamate in biological samples also showed good performances, and the dynamic range and detection limit were 0.02-10mM and 6.7 microM, respectively. High precision with relative standard deviations of 4.2 and 10.7% in water and biological samples, respectively, were also demonstrated. In addition, the biosensor showed a relatively high storage stability over more than 1 month. Results obtained in this study clearly demonstrate that this simple vapor deposition method can be successfully used to form transparent titania sol-gel film for the fabrication of glutamate biosensors that are suitable for optical detection of glutamate in water and biological samples.     

Development of amperometric biosensor for glucose based on a novel attractive enzyme immobilization matrix: calcium carbonate nanoparticles

Calcium carbonate nanoparticles (nano-CaCO3) may be a promising material for enzyme immobilization owing to their high biocompatibility, large specific surface area and their aggregation properties. This attractive material was exploited for the mild immobilization of glucose oxidase (GOD) in order to develop glucose amperometric biosensor. The GOD/nano-CaCO3-based sensor exhibited a marked improvement in thermal stability compared to other glucose biosensors based on inorganic host matrixes. Amperometric detection of glucose was evaluated by holding the modified electrode at 0.60 V (versus SCE) in order to oxidize the hydrogen peroxide generated by the enzymatic reaction. The biosensor exhibited a rapid response (6s), a low detection limit (0.1 microM), a wide linear range of 0.001-12 mM, a high sensitivity (58.1 mAcm-2M-1), as well as a good operational and storage stability. In addition, optimization of the biosensor construction, the effects of the applied potential as well as common interfering compounds on the amperometric response of the sensor were investigated and discussed herein.     

Multi-wall carbon nanotube-polyaniline biosensor based on lectin-carbohydrate affinity for ultrasensitive detection of Con A

Surface plasmon resonance (SPR) biosensor detects intracellular signaling events as a change of the angle of resonance (AR). We previously reported that the activation of epidermal growth factor receptor (EGFR) on keratinocytes causes a unique triphasic change of AR, whereas the activation of other receptors, such as IgE receptor and adenosine A3 receptor on mast cells, causes a transient monophasic increase of AR. To study the mechanism of AR changes induced by EGFR activation, we introduced wild and mutated EGFR cDNAs into Chinese hamster ovary (CHO) cells and analyzed changes of AR in response to EGF. CHO cells expressing wild-type EGFR showed a triphasic change of AR, whereas cells expressing kinase-dead EGFR (K721M) showed minimum change of AR. A phosphatidylinositol 3-kinase inhibitor, wortmannin, attenuated the third phase of AR change in CHO cells expressing wild-type EGFR. The pattern of AR change was independent on the concentration of EGF. We also analyzed changes of AR with a nontumorigenic keratinocyte cell line, HaCaT, and several cell lines of carcinoma to explore the feasibility of SPR biosensor as a tool for clinical diagnosis. The activation of HaCaT cells and one out of six carcinoma cell lines showed a full triphasic change of AR. In contrast, five out of the six cell lines showed mono- or bi-phasic change of AR. These results suggest that EGF induces the SPR signals via the phosphorylation of EGFR, and provide a possibility that the SPR biosensor could be applied to the real-time detection and diagnosis of malignant tumors.     

A rapid in situ immobilization of d-amino acid oxidase based on immobilized metal affinity chromatography

A rapid in situ immobilization process was developed based on conventional separation technique of immobilized metal affinity chromatography (IMAC) and was studied in the case of d-amino acid oxidase (DAAO) with binding–enhancing Heli-tag (His-Arg-Asn-Tyr-Gly-Gly-Cys-Cys-Gly). A recombinant Escherichia coli strain JM105 (Δase)/pGEMK-R-DAAO-Heli was successfully constructed to synthesize chimeric protein DAAO-Heli. Without additional purification procedure, the tagged enzyme DAAO-Heli could be directly immobilized to EP-IDA-Ni²⁺ support with purity of 90 % and DAAO activity of over 70 U/g support. Experimental results showed that the immobilized DAAO-Heli was 73 times more thermally stable than free enzyme. Besides, it remained 67 % of initial activity after 100 cycles of batch catalysis and its operational stability was improved 36 times than that of the previously IMAC-immobilized DAAO-His. Furthermore, the epoxy (EP) support could be easily recovered and repeatedly used with simple steps, which could reduce the immobilization costs significantly.     

Reagentless biosensor for hydrogen peroxide based on immobilization of protein in zirconia nanoparticles enhanced grafted collagen matrix

A novel matrix, zirconia nanoparticles enhanced grafted collagen (ZrO2-grafted collagen) hybrid composite, for immobilization of protein and biosensing was developed. The scanning electron microscopy, UV-vis and Fourier transform infrared spectra, and electrochemical measurements showed that the matrix was well biocompatible and could retain the bioactivity of immobilized protein to a large extent. The direct electron transfer of the immobilized myoglobin (Mb) exhibited a couple of stable and well-defined redox peaks with the formal potential of -336 mV (versus SCE) in 0.1M pH 7.0 PBS. This matrix could accelerate the electron transfer between Mb and the electrode with a surface-controlled process and an electron transfer rate constant of 3.58+/-0.35s-1 at 10-500 mVs-1. The Mb immobilized in the matrix showed a high thermal stability up to 70 degrees C and an electrocatalytic activity to the reduction of hydrogen peroxide (H2O2) without the help of an electron mediator. The linear response range of the biosensor to H2O2 concentration was from 1.0 to 85.0 microM with the limit of detection of 0.63 microM at a signal-to-noise ratio of 3sigma. The biosensor exhibited high sensitivity, acceptable stability and reproducibility. This work opened a way for the further study on the direct electron transfer and biosensing application of the immobilized protein in collagen-related matrices.     

A novel glucose biosensor based on the immobilization of glucose oxidase onto gold nanoparticles-modified Pb nanowires

A novel glucose biosensor was developed, based on the immobilization of glucose oxidase (GOD) with cross-linking in the matrix of bovine serum albumin (BSA) on a Pt electrode, which was modified with gold nanoparticles decorated Pb nanowires (GNPs-Pb NWs). Pb nanowires (Pb NWs) were synthesized by an l-cysteine-assisted self-assembly route, and then gold nanoparticles (GNPs) were attached onto the nanowire surface through –SH–Au specific interaction. The morphological characterization of GNPs-Pb NWs was examined by transmission electron microscopy (TEM). Cyclic voltammetry and chronoamperometry were used to study and to optimize the electrochemical performance of the resulting biosensor. The synergistic effect of Pb NWs and GNPs made the biosensor exhibit excellent electrocatalytic activity and good response performance to glucose. The effects of pH and applied potential on the amperometric response of the biosensor have been systemically studied. In pH 7.0, the biosensor showed the sensitivity of 135.5 μA mM⁻¹ cm⁻², the detection limit of 2 μM (S/N = 3), and the response time <5 s with a linear range of 5–2200 μM. Furthermore, the biosensor exhibits good reproducibility, long-term stability and relative good anti-interference.     

Physical polymer matrices based on affinity interactions between peptides and polysaccharides

A rapidly forming polymer matrix with affinity-based controlled release properties was developed based upon interactions between heparin-binding peptides and heparin. Dynamic mechanical testing of 10% (w/v) compositions consisting of a 3:1 molar ratio of poly(ethylene glycol)-co-peptide (approximately 18,000 g/mol) to heparin (approximately 18,000 g/mol) revealed a viscoelastic profile similar to that of concentrated, large molecular weight polymer solutions and melts. In addition, the biopolymer mixtures recovered quickly following thermal denaturation and mechanical insult. These gel-like materials were able to sequester exogenous heparin-binding peptides and could release these peptides over several days at rates dependent on relative heparin affinity. The initial release rates ranged from 3.3% per hour for a peptide with low heparin affinity to 0.025% per hour for a peptide with strong heparin affinity. By altering the affinity of peptides to heparin, a series of peptides can be developed to yield a range of release profiles useful for controlled in vivo delivery of therapeutics.     

Control of protein immobilization: coupling immobilization and site-directed mutagenesis to improve biocatalyst or biosensor performance

Mutagenesis and immobilization are usually considered to be unrelated techniques with potential applications to improve protein properties. However, there are several reports showing that the use of site-directed mutagenesis to improve enzyme properties directly, but also how enzymes are immobilized on a support, can be a powerful tool to improve the properties of immobilized biomolecules for use as biosensors or biocatalysts. Standard immobilizations are not fully random processes, but the protein orientation may be difficult to alter. Initially, most efforts using this idea were addressed towards controlling the orientation of the enzyme on the immobilization support, in many cases to facilitate electron transfer from the support to the enzyme in redox biosensors. Usually, Cys residues are used to directly immobilize the protein on a support that contains disulfide groups or that is made from gold. There are also some examples using His in the target areas of the protein and using supports modified with immobilized metal chelates and other tags (e.g., using immobilized antibodies). Furthermore, site-directed mutagenesis to control immobilization is useful for improving the activity, the stability and even the selectivity of the immobilized protein, for example, via site-directed rigidification of selected areas of the protein. Initially, only Cys and disulfide supports were employed, but other supports with higher potential to give multipoint covalent attachment are being employed (e.g., glyoxyl or epoxy-disulfide supports). The advances in support design and the deeper knowledge of the mechanisms of enzyme-support interactions have permitted exploration of the possibilities of the coupled use of site-directed mutagenesis and immobilization in a new way. This paper intends to review some of the advances and possibilities that these coupled strategies permit.     

Frontal affinity chromatography: sugar-protein interactions

Frontal affinity chromatography using fluorescence detection (FAC-FD) is a versatile technique for the precise determination of dissociation constants (Kd) between glycan-binding proteins (lectins) and fluorescent-labeled glycans. A series of glycan-containing solutions is applied to a lectin-immobilized column, and the elution profile of each glycan (termed the 'elution front', V) is compared with that (V0) for an appropriate control. Here we describe our standard protocol using an automated FAC system (FAC-1), consisting of two isocratic pumps, an autosampler, a column oven and two miniature columns connected to a fluorescence detector. Analysis time for 100 sugar-protein interactions is approximately 10 h, using as little as 2.5 pmol of pyridylaminated (PA) oligosaccharide per analysis. Using FAC-FD, we have so far obtained quantitative interaction data of >100 lectins for >100 PA oligosaccharides.     

A novel approach to determining the affinity of protein-carbohydrate interactions employing adherent cancer cells grown on a biosensor surface

The development of biological agents for the treatment of solid tumours is an area of considerable activity. We are pursuing carbohydrate-binding proteins (lectins) in a strategy aimed at targeting cancer-associated changes in glycosylation. To evaluate lectin-cancer cell interactions we developed a novel cell biosensor in which binding events take place at the cell surface, more closely mimicking an in vivo system. Metastatic, SW620, and non-metastatic, SW480, colorectal cancer cells were grown on the surface of a tissue-culture compatible polystyrene coated biosensor chip and housed in a quartz crystal microbalance (QCM) apparatus, the kinetics of binding of a diverse range of lectins was evaluated. The lectin Helix pomatia agglutinin (HPA) has been shown to bind aggressive metastatic cancer and was produced in recombinant form (His- and RFP-tagged). The affinity of HPA was in the nanomolar range to the metastatic SW620 cells but was only in the micromolar range to the non-metastatic SW480. Overall, the dissociation constant (K(D)) of the lectins tested in the new cell biosensor system was an order of magnitude lower (nanomolar range) than has generally been reported with systems such as QCM/SPR. This new cell-biosensor enables molecular interactions to be studied in a more relevant environment. An intrinsic problem with developing new biological therapies is the difficulty in determining the affinity with which proteins will interact with intact cell surfaces. This methodology will be of interest to researchers developing new biological approaches for targeting cell surfaces in a wide range of diseases, including cancer.     

Study of immobilization and properties of urease for creation of a biosensor based on semiconductor structures]

Many-sided investigations of urease immobilization methods were carried out to create the biosensor devices on the base of semiconductor structures. Special attention was concentrated on the biomembrane formation by means of urease and bovine serum albumin (BSA) cross-linking by gaseous glutaraldehyde. Optimal conditions for the formation process were selected which preserve about 20% of total urease activity after the cross-linking. The properties of enzyme immobilized by the above-mentioned method have been comprehensively studied. They included the urease activity dependence on pH, ionic strength, incubation buffer capacity as well as the enzyme stability during its functioning, storing and thermoinactivation. As was shown, for immobilized ureas Km value for urea at pH 7.0 and 20 degrees C is 1.65 time less than for free enzyme. In the presence of EDTA (1 mM) the enzyme activity in the biomembrane is practically unchanged under a month storing. Biomembrane possesses good adhesion to silicon surface and its swelling level under different conditions does not exceed 35%. The conclusion is made about the prospects of the used method of biomembrane formation for biosensor technology based on semiconductor structures.     

A novel glucose biosensor based on immobilization of glucose oxidase into multiwall carbon nanotubes-polyelectrolyte-loaded electrospun nanofibrous membrane

Nanofibrous glucose electrodes were fabricated by the immobilization of glucose oxidase (GOx) into an electrospun composite membrane consisting of polymethylmethacrylate (PMMA) dispersed with multiwall carbon nanotubes (MWCNTs) wrapped by a cationic polymer (poly(diallyldimethylammonium chloride) (PDDA)) and this nanofibrous electrode (NFE) is abbreviated as PMMA-MWCNT(PDDA)/GOx-NFE. The NFE was characterized for morphology and electroactivity by using electron microscopy and cyclic voltammetry, respectively. Field emission transmission electron microscopy (FETEM) image reveals the dispersion of MWCNT(PDDA) within the matrix of PMMA. Cyclic voltammetry informs that NFE is suitable for performing surface-confined electrochemical reactions. PMMA-MWCNT(PDDA)/GOx-NFE exhibits excellent electrocatalytic activity towards hydrogen peroxide (H(2)O(2)) with a pronounced oxidation current at +100 mV. Glucose is amperometrically detected at +100 mV (vs. Ag/AgCl) in 0.1M phosphate buffer solution (PBS, pH 7). The linear response for glucose detection is in the range of 20 microM to 15 mM with a detection limit of 1 microM and a shorter response time of approximately 4 s. The superior performance of PMMA-MWCNT(PDDA)/GOx-NFE is due to the wrapping of PDDA over MWCNTs that binds GOx through electrostatic interactions. As a result, an effective electron mediation is achieved. A layer of nafion is made over PMMA-MWCNT(PDDA)/GOx-NFE that significantly suppressed the electrochemical interference from ascorbic acid or uric acid. In all, PMMA-MWCNT(PDDA)/GOx-nafion-NFE has exhibited excellent properties for the sensitive determination of glucose like high selectivity, good reproducibility, remarkable stability and without interference from other co-existing electroactive species.