Atomic force microscopy for the characterization of immobilized enzyme molecules on biosensor surfaces

The development of biosensors has been one of the key areas in biotechnology and biomedical studies. Often it is difficult to investigate the immobilized biomolecules on the surfaces for biosensor optimization. Atomic force microscopy (AFM) should provide an ideal means for the visualization of biosensor surface and for the investigation of biomolecule activities. Therefore, AFM has been employed to study the surface topography of immobilized glutamate dehydrogenase (GDH) on two-dimensional glutamate biosensor surfaces. Correlation between the surface topography and the activity of the biosensor was investigated. Surface analysis has revealed that the enzymatic activity of the immobilized GDH molecules on the biosensor surface is linked to surface roughness, as measured by the peak-to-valley distance. Fractal dimension of the immobilization sensor surface was found to be a good parameter for judging the quality of the immobilized biosensors. As enzyme immobilization time increases, the biosensor has its maximum activity with around 18 h of immobilization in 10(-6) M GDH solution. Various biosensors prepared under different experimental conditions have been studied by AFM. This technique is shown to be an effective tool to characterize biosensor surfaces.     

Microscopic and voltammetric characterization of bioanalytical platforms based on lactate oxidase

A microscopic and voltammetric characterization of lactate oxidase- (LOx-) based bioanalytical platforms for biosensor applications is presented. In this context, emphasis is placed on amperometric biosensors based on LOx that have been immobilized by direct absorption on carbon surfaces, in particular, glassy carbon (GC) and highly ordered pyrolytic graphite (HOPG). The immobilized LOx layers have been characterized using atomic force microscopy (AFM) under liquid conditions and cyclic voltammetry. In addition, spatially resolved mapping of enzymatic activity has been carried out using scanning electrochemical microscopy (SECM). In the presence of lactate with hydroxymethylferrocene (HMF) as a redox mediator in solution, biosensors obtained by direct adsorption of LOx onto GC electrodes exhibited a clear electrocatalytic activity, and lactate could be determined amperometrically at 300 mV versus SSCE. The proposed biosensor also exhibits good operating performance in terms of linearity, detection limit, and lifetime.     

Biochemically functionalized silica nanoparticles

In this report, we demonstrate the biochemical modification of silica based nanoparticles. Both pure and dye-doped silica nanoparticles were prepared, and their surfaces were modified with enzymes and biocompatible chemical reagents that allow them to function as biosensors and biomarkers. The nanoparticles produced in this work are uniform in size with a 1.6% relative standard deviation. They have a pure silica surface and can thus be modified easily with many biomolecules for added biochemical functionality. Specifically, we have modified the nanoparticle surfaces with enzyme molecules (glutamate dehydrogenase (GDH) and lactate dehydrogenase (LDH)) and a biocompatible reagent for cell membrane staining. Experimental results show that the silica nanoparticles are a good biocompatible solid support for enzyme immobilization. The immobilized enzyme molecules on the nanoparticle surface have shown excellent enzymatic activity in their respective enzymatic reactions. The nanoparticle surface biochemical functionalization demonstrates the feasibility of using nanoparticles for biosensing and biomarking applications.     

Development of a novel electrochemical biosensor based on catalytic properties of adenosine deaminase immobilized on graphene oxide/carboxymethyl chitosan/multi-wall carbon nanotube platform

A new type of biosensor was designed based on Adenosine deaminase (ADA) immobilized on graphene oxide (GO)/carboxymethyl chitosan (CMC)/multi-wall carbon nanotube (MWCNT) platform nanostructure, fabricated and successfully applied (utilized) in Adenosine detection. Square wave voltammetry was used to study the biosensor catalytic activity. Morphological analysis of the nanostructure was performed by AFM and SEM methods. The results provided here proved that utilizing GO/CMC/MWCNT leads to effective immobilization of ADA which was confirmed by the long term stability of the biosensor during examined intervals. The immobilized ADA activity was examined and the kinetic parameters (K _m and V _max) were found to be 47.5 μM and 5.8 μM min^?1, respectively. Furthermore, benznidazole was introduced as a potent ADA inhibitor using virtual screening. Outstanding inhibition characteristics of benznidazole was observed against ADA. ADA inhibition by benznidazole was non-competitive with the inhibition constant of 0.42 μM. Such an interesting template with an easy preparation process with low cost can provide a novel matrix for developing biosensors and biocatalysts based on enzyme immobilization.     

Application of Different Zeolites for Improvement of the Characteristics of a pH‐FET Biosensor Based on Immobilized Urease

Different modifications of the zeolites Na⁺‐Beta and LTA were applied for improving the working characteristics of a urea biosensor. The bioselective membrane of the biosensor was based on urease and different zeolites co‐immobilized with bovine serum albumin on the surface of a pH‐FET. It was shown that the biosensors modified with the zeolites H⁺‐Beta30 and H⁺‐Beta50 are characterized by increased sensitivity to urea. The influence of the zeolite concentration on the sensitivity of the biosensors was studied. The optimal concentration of the zeolites H⁺‐Beta30 and H⁺‐Beta50 in the bioselective membrane was 15 %. Different variants of co‐immobilization of urease and zeolite H⁺‐Beta30 were studied and the optimal method was selected. Thus, a general conclusion is that the urea biosensor sensitivity can be improved using zeolite H⁺‐Beta30 for urease immobilization in the bioselective membrane.     

Colorimetric multiplexed immunoassay using specific aggregation of antigenic peptide-modified luminous nanoparticles

Two amperometric enzyme biosensor systems, based on glycerol dehydrogenase/diaphorase (GDH/DP) and glycerol kinase/glycerol-3-phosphate oxidase/peroxidase (GK/GPOx/HRP), were developed and used for estimation of glycerol content in a complex biological fluids. Enzymes were immobilized on interchangeable membranes by PCS-prepolymer technique. Buffers containing ferricyanide/NAD⁺ or ferrocyanide/ATP were used for measurements with GDH/DP and GK/GPOx/HRP biosensor, respectively. FIA assay of glycerol biosensor was characterized by a linear range of 0.01-1 or 0.01-1.5 mM glycerol, sensitivity of 6.02 or 1.42 mA/M cm² and with signal loss of 40% after 90 h or 30% after 16 h during continuous operation at a sample throughput of 10 injections/h for GDH/DP or GK/GPOx/HRP biosensors, respectively. Both biosensors were successfully used for off-line monitoring of glycerol during microbial transformation of glycerol to 1,3-propanediol using an automatized flow-through system. The results were consistent with those obtained with HPLC. The stability of described biosensor systems was sufficient for monitoring and control of fermentation process within 24 h. The storage stability of enzyme membranes was several months.     

Biosensors based on direct electron transfer in redox proteins

In biosensors based on direct electron transfer in redox proteins, efficient electron-transfer pathways between the immobilized redox protein and the electrode surface have to be established so to allow a fast electron transfer and concomitantly avoiding free-diffusing redox species. In this review, prerequisites for the direct electron transfer of redox proteins and immobilization of redox proteins on the electrode surfaces are addressed. Based on the specific nature of different proteins and non-manual immobilization procedures, possible biosensor designs are discussed, namely biosensors based on (1) ferritin; (2) cytochrome c; (3) myoglobin; (4) hemoglobin; (5) horseradish peroxidase; (6) catalase; (7) glucose oxidase; and (8) xanthine oxidase.     

Direct Bioelectrocatalysis of PQQ‐Dependent Glucose Dehydrogenase

The direct bioelectrocatalysis was demonstrated for pyrroloquinoline quinone‐dependent glucose dehydrogenase (PQQ‐dependent GDH) covalently attached to single‐walled carbon nanotubes (SWNTs). The homogeneous ink‐like SWNT suspension was used for both creating the SWNT network on the microelectrode carbon surface and for enzyme immobilization. Functionalization of the SWNT surface by forming active ester groups was found to considerably enhance SWNT solubility in water with a range from 0.1 to 1.0 mg/mL. The PQQ‐dependent GDH immobilized on the surface of the SWNTs exhibited fast heterogeneous electron transfer with a rate constant of 3.6 s^?1. Moreover, the immobilized PQQ‐dependent GDH retained its enzymatic activity for glucose oxidation. A fusion of PQQ‐dependent GDH with SWNTs has a great potential for the development of low‐cost and reagentless glucose sensors and biofuel cells.     

Electrochemical glucose biosensor based on silver nanoparticles/multiwalled carbon nanotubes modified electrode

A novel glucose biosensor was fabricated by immobilizing glucose oxidase (GOx) on Ag nanoparticles-decorated multiwalled carbon nanotube (AgNP-MWNT) modified glass carbon electrode (GCE). The AgNP-MWNT composite membrane showed an improving biocompatibility for GOx immobilization and an enhancing electrocatalytic activity toward reduction of oxygen due to decoration of AgNPs on MWNT surfaces. The AgNPs also accelerated the direct electron transfer between redox-active site of GOx and GCE surface because of their excellent conductivity and large capacity for protein loading, leading to direct electrochemistry of GOx. The glucose biosensor of this work showed a lower limit of detection of 0.01 mM (S/N?=?3) and a wide linear range from 0.025 to 1.0 mM, indicating an excellent analytical performance of the obtained biosensor to glucose detection. The resulting biosensor exhibits good stability and excellent reproducibility. Such bionanocomposite provides us good candidate material for fabrication of biosensors based on direct electrochemistry of immobilized enzymes.     

Immobilization of histidine-tagged proteins on electrodes

The development of new enzyme immobilization techniques that do not affect catalytic activity or conformation of a protein is an important research task in biotechnology including biosensor applications and heterogeneous reaction systems. One of the most promising approaches for controlled protein immobilization is based on the immobilized metal ion affinity chromatography (IMAC) principle originally developed for protein purification. Here we describe the current status and future perspectives of immobilization of His-tagged proteins on electrode surfaces. Recombinant proteins comprising histidine-tags or histidine rich native proteins have a strong affinity to transition metal ions. For metal ion immobilization at the electrode surface different matrices can be used such as self-assembled monolayers or conductive polymers. This specific technique allows a reversible immobilization of histidine-tagged proteins at electrodes in a defined orientation which is an important prerequisite for efficient electron transfer between the electrode and the biomolecule. Any application requiring immobilized biocatalysts on electrodes can make use of this immobilization approach, making future biosensors and biocatalytic technologies more sensitive, simpler, reusable and less expensive while only requiring mild enzyme modifications.     

Improved glucose electrochemical biosensor by appropriate immobilization of nano-ZnO

We constructed the transferred ZnO biosensor and the grown ZnO biosensor by two different nano-ZnO immobilization approaches. And the influence of different assembly processes on the biosensor performance has been systematically investigated and compared. An enhanced sensitivity of the grown ZnO biosensor is found to be 52% higher than that of the transferred ZnO biosensor. Correspondingly, the other properties are also better in the grown ZnO biosensor, including the response time, the detection limit and the linear range. These results are well consistent with the fact that more glucose oxidase is immobilized on the well-aligned ZnO arrays, which have higher specific surface area and more direct electron communication path, in the grown sensor than the randomly distributed and stacked ZnO nanorods in the transferred sensor. The nano-ZnO grown directly has been demonstrated more desirable for enzymatic immobilization and signal transduction in the high performance biosensors.     

Comparative Study of Nanostructured Matrices Employed in the Development of Biosensors Based on HRP Enzyme for Determination of Phenolic Compounds

This paper made a comparative study of new matrices of nanostructured materials (multiwall carbon nanotube, fullerene and hydroxylated fullerene) aiming to compare them when employed in the process of immobilization of enzyme horseradish peroxidase (HRP) on the development of amperometric biosensors for the determination phenolic compounds. The results confirm that all the three nanostructured matrices used in the preparation of the biosensor show improvements when acting as a transducer stabilizer and immobilization matrix, comparing to the electrode of carbon paste. Regarding the performance of these matrices it is verified that the developed biosensor employed the multiwall carbon nanotube as matrix immobilized enzyme HRP has shown the best sensitivity for the molecule of phenol (33 nA cm^?2 µmol^?1 L), however, regarding the range of linear response, the elaborated biosensor containing the hydroxylated fullerene has shown the best response (5–200 µmol L^?1). In terms of operational stability, the biosensors maintained their responses around 95 % after more than 200 analyzes. It is also important to mention that in all the cases, the association with the graphite powder improves the answers of the biosensors around 10 to 15 %.     

Immobilization of proteins on carboxylic acid functionalized nanopatterns

The immobilization of proteins on nanopatterned surfaces was investigated using in situ atomic force microscopy (AFM) and ex situ infrared reflectance–absorption spectroscopy (IRAS). The AFM-based lithography technique of nanografting provided control of the size, geometry, and spatial placement of nanopatterns within self-assembled monolayers (SAMs). Square nanopatterns of carboxylate-terminated SAMs were inscribed within methyl-terminated octadecanethiolate SAMs and activated using carbodiimide/succinimide coupling chemistry. Staphylococcal protein A was immobilized on the activated nanopatterns before exposure to rabbit immunoglobulin G. In situ AFM was used to monitor changes in the topography and friction of the nanopatterns in solution upon protein immobilization. Complementary studies with ex situ IRAS confirmed the surface chemistry that occurred during the steps of SAM activation and subsequent protein immobilization on unpatterned samples. Since carbodiimide/succinimide coupling chemistry can be used for surface attachment of different biomolecules, this protocol shows promise for development of other aqueous-based studies for nanopatterned protein immobilization.     

Enhancement of the protein loading density by a pre-cleaning process of a gold substrate: confocal laser scanning microscopic study.

The immobilization of glucose oxidase (GOx), using self assembled monolayers (SAMs) on gold surfaces, was investigated by grazing angle FT-IR spectroscopy, surface plasmon resonance (SPR) spectroscopy, and atomic force microscopy (AFM) in conjunction with confocal laser scanning microscopy (CLSM). To find an optimum condition for the maximum GOx loading density on gold surfaces, different cleaning protocols were examined. The loading density of GOx on surfaces was investigated by AFM and CLSM. In particular, CLSM was more effective for identifying the GOx density than AFM, since its scanning speed is much faster and it covers a larger area. Based on CLSM images of the GOx immobilized on the surfaces, it was concluded that the pre-cleaning process of gold substrates using different solvents, such as acetone, ethanol and 2-propanol, is very important for enhancing the GOx loading density. This result enables us to investigate an effective fabrication process in fabricating biosensors.     

Quantitative Determination of Glycine in Aqueous Solution Using Glutamate Dehydrogenase-Immobilized Glyoxal Agarose Beads

In this study, an enzymatic procedure for the determination of glycine (Gly) was developed by using a column containing immobilized glutamate dehydrogenase (GDH) on glyoxal agarose beads. Ammonia is produced from the enzymatic reactions between Gly and GDH with NAD⁺ in phosphate buffer medium. The indophenol blue method was used for ammonia detection based on the spectrophotometric measurements of blue-colored product absorbing at 640 nm. The calibration graph is linear in the range of 0.1–10 mM of Gly concentrations. The effect of pH, temperature, and time interval was studied to find column stability, and also the interference effects of other amino acids was investigated. The interaction between GDH and glyoxal agarose beads was analyzed by Fourier transform infrared (FTIR) spectroscopy. The morphology of the immobilized and non-immobilized agarose beads were characterized by atomic force microscopy (AFM).     

A Novel Nitric Oxide Cellular Biosensor Based on Red Blood Cells Immobilized on Gold Nanoparticles

Abstract

We have developed a novel nitric oxide (NO) cellular biosensor based upon the immobilization of red blood cells (RBCs) onto nanometer‐size colloidal gold that is attached to an electrochemically pretreated glassy carbon electrode via the bridging of an ethylenediamine monolayer. The biosensor has been characterized by atomic force microscopy (AFM), scanning electron microscopy (SEM), and electrochemistry. The immobilized RBCs display an excellent electrocatalytic response to nitric oxide. The electrocatalytic currents are proportional to the NO concentration in the range from 1.0×10^–8 to 1.0×10^–6 M and the detection limit is as low as 5.0×10^–9 M (S/N=3). Furthermore, the biosensor is very stable and relatively free of potential interference.     

Amperometric Biosensors for Tyramine Determination Based on Graphene Oxide and Polyvinylferrocene Modified Screen‐printed Electrodes

A comparison of the analytical characteristics of two tyramine biosensors, based on graphene oxide (GRO) and polyvinylferrocene (PVF) modified screen‐printed carbon electrodes (SPCE), is reported. Diamine oxidase (DAOx) or monoamine oxidase (MAOx) was immobilized onto the PVF/GRO modified SPCE to fabricate the biosensors. Surface characteristics and electrochemical behaviour of the modified SPCEs were investigated by atomic force microscopy (AFM), scanning electron microscopy (SEM), energy dispersive X‐ray spectroscopy (EDX) and cyclic voltammetry (CV). Electrode surface composition and experimental variables such as pH and working potential were optimized in order to ensure a high performance. Under optimum experimental conditions, both DAOx/PVF/GRO/SPCE and MAOx/PVF/GRO/SPCE biosensors exhibited wide linear dynamic ranges for tyramine from 9.9×10^?7 to 1.2×10^?4 M and from 9.9×10^?7 to 1.1×10^?4 M, respectively. MAOx/PVF/GRO/SPCE biosensor showed higher sensitivity (11.98 μA mM^?1) for tyramine determination than the DAOx/PVF/GRO/SPCE biosensor (7.99 μA mM^?1). The substrate specifity of the biosensors to other biogenic amines namely histamine, putrescine, spermine, spermidine, tryptamine, β‐phenylethylamine and cadaverine was also investigated. The developed biosensors were successfully used for tyramine determination in cheese sample.     

Structure simulation into a lamellar supramolecular network and calculation of the metal ions/ligands ratio

Background

Biosensors have attracted increasing attention as reliable analytical instruments in in situ monitoring of public health and environmental pollution. For enzyme-based biosensors, the stabilization of enzymatic activity on the biological recognition element is of great importance. It is generally acknowledged that an effective immobilization technique is a key step to achieve the construction quality of biosensors.

Results

A novel disposable biosensor was constructed by immobilizing laccase (Lac) with silica spheres on the surface of multi-walled carbon nanotubes (MWCNTs)-doped screen-printed electrode (SPE). Then, it was characterized in morphology and electrochemical properties by scanning electron microscopy (SEM) and cyclic voltammetry (CV). The characterization results indicated that a high loading of Lac and a good electrocatalytic activity could be obtained, attributing to the porous structure, large specific area and good biocompatibility of silica spheres and MWCNTs. Furthermore, the electrochemical sensing properties of the constructed biosensor were investigated by choosing dopamine (DA) as the typical model of phenolic compounds. It was shown that the biosensor displays a good linearity in the range from 1.3 to 85.5 ??M with a detection limit of 0.42 ??M (S/N = 3), and the Michaelis-Menten constant (K_m ^app) was calculated to be 3.78 ??M.

Conclusion

The immobilization of Lac was successfully achieved with silica spheres to construct a disposable biosensor on the MWCNTs-doped SPE (MWCNTs/SPE). This biosensor could determine DA based on a non-oxidative mechanism in a rapid, selective and sensitive way. Besides, the developed biosensor could retain high enzymatic activity and possess good stability without cross-linking reagents. The proposed immobilization approach and the constructed biosensor offer a great potential for the fabrication of the enzyme-based biosensors and the analysis of phenolic compounds.     

Amperometric Study of Au‐Colloid Function on Xanthine Biosensor Based on Xanthine Oxidase Immobilized in Polypyrrole Layer

Four kinds of xanthine oxidase (XOD) based amperometric biosensors were fabricated and their analytical performances were compared. Polypyrrole (PPY)/XOD biosensor was constructed by electrochemical oxidation of pyrrole in the solution containing xanthine oxidase and pyrrole in this paper. Colloidal Au was then immobilized on the biosensor. On the other hand, electron mediator, Prussian Blue (PB), was deposited on the electrode before the immobilization of PPY/XOD to enhance electron‐transfer rate and current response. The results showed that PPY/XOD, PPY/XOD/Au‐colloid, PB/PPY/XOD and PB/PPY/XOD/Au‐colloid biosensors exhibit good response to xanthine in 1×10^?6 M and 2×10^?5 M and Michaelis‐Menten constants (K_m) of these biosensors were 242.2, 113.4, 144.5, 43.2 μmol?L^?1, respectively. The dependence of current responses with applied voltages was discussed, and different mechanisms of these biosensors were discussed. It has been found that colloidal Au can enhance the current response at the same concentration of xanthine solution and decrease the energy‐barrier of electron‐transfer reaction on the electrode.     

Micro- to nanostructured poly(pyrrole-nitrilotriacetic acid) films via nanosphere templates: applications to 3D enzyme attachment by affinity interactions

We report the combination of latex nanosphere lithography with electropolymerization of N-substituted pyrrole monomer bearing a nitrilotriacetic acid (NTA) moiety for the template-assisted nanostructuration of poly(pyrrole-NTA) films and their application for biomolecule immobilization. The electrodes were modified by casting latex beads (100 or 900 nm in diameter) on their surface followed by electropolymerization of the pyrrole-NTA monomer and the subsequent chelation of Cu²⁺ ions. The dissolution of the nanobeads leads then to a nanostructured polymer film with increased surface. Thanks to the versatile affinity interactions between the (NTA)Cu²⁺ complex and histidine- or biotin-tagged proteins, both tyrosinase and glucose oxidase were immobilized on the modified electrode. Nanostructuration of the polypyrrole via nanosphere lithography (NSL) using 900- and 100-nm latex beads allows an increase in surface concentration of enzymes anchored on the functionalized polypyrrole electrode. The nanostructured enzyme electrodes were characterized by fluorescence microscopy, 3D laser scanning confocal microscopy, and scanning electron microscopy. Electrochemical studies demonstrate the increase in the amount of immobilized biomolecules and associated biosensor performances when achieving NSL compared to conventional polymer formation without bead template. In addition, the decrease in nanobead diameter from 900 to 100 nm provides an enhancement in biosensor performance. Between biosensors based on films polymerized without nanobeads and with 100-nm nanobeads, maximum current density values increase from 4 to 56 μA cm^?2 and from 7 to 45 μA cm^?2 for biosensors based on tyrosinase and glucose oxidase, respectively.