Electrostatic Interaction-Induced Formation of Enzyme-on-MOF as Chemo-Biocatalyst for Cascade Reaction with Unexpectedly Acid-Stable Catalytic Performance.

Combining biocatalytic and chemocatalytic reactions in a one-pot reaction not only avoids the tedious isolation of intermediates during the reactions but also provides a desirable alternative to extend the range of catalytic reactions. Here, we report a facile strategy to immobilize an enzyme, glucose oxidase (GOx), on PCN-222(Fe) induced by electrostatic interaction in which PCN-222(Fe) serves as both a support and chemocatalyst. The immobilization was confirmed through ζ potential measurement, confocal laser scanning microscopy, Fourier transform infrared spectrometry, and UV-vis spectroscopy. This chemo-biocatalyst was applied to a cascade reaction to catalyze glucose oxidation and ABTS (ABTS = 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (or pyrogallol) oxidation. The catalytic kinetics studies show that these chemo-biocatalytic cascade reactions obey the Michaelis-Menten equation, which indicates that the cascade reactions follow the typical enzymatic dynamic regulation process. Interestingly, GOx/PCN-222(Fe) exhibits an exceptional acid-stable catalytic performance as evidenced by circular dichroism spectroscopy where no significant structure change was observed toward acidic solutions with different pH values. GOx/PCN-222(Fe) also displays desirable recyclability since no significant loss of conversion rates was found after six repeated reactions. This work presents a convenient strategy to construct metal-organic framework based chemo-biocatalysts, which may find potential applications in sensing and nanomachines.

Advanced Cellulose Fibers for Efficient Immobilization of Enzymes.

Biocatalytic pulp fibers were prepared using surface functionalization of bleached kraft pulp with amino groups (F) and further immobilization of a cross-linked glucose oxidase (G*) from Aspergillus niger. The cross-linked enzymes (G*) were characterized using X-ray spectroscopy, Fourier transform infrared spectroscopy, dynamic scanning calorimetry, and dynamic light scattering. According to standard assays, the G* content on the resulting fibers (FG*) was of 11 mg/g of fiber, and enzyme activity was of 215 U/g. The results from confocal- and stimulated emission depletion microscopy techniques demonstrated that glucose oxidase do not penetrate the interlayers of fibers. The benefit of pulp fiber functionalization was evident in the present case, as the introduction of amino groups allowed the immobilization of larger amount of enzymes and rendered more efficient systems. Using the approach described on this paper, several advanced materials from wood pulp fibers and new bioprocesses might be developed by selecting the correct enzyme for the target applications.

Combining an optical resonance biosensor with enzyme activity kinetics to understand protein adsorption and denaturation.

Understanding protein adsorption and resultant conformation changes on modified and unmodified silicon dioxide surfaces is a subject of keen interest in biosensors, microfluidic systems and for medical diagnostics. However, it has been proven difficult to investigate the kinetics of the adsorption process on these surfaces as well as understand the topic of the denaturation of proteins and its effect on enzyme activity. A highly sensitive optical whispering gallery mode (WGM) resonator was used to study a catalytic enzyme's adsorption processes on different silane modified glass substrates (plain glass control, DETA, 13 F, and SiPEG). The WGM sensor was able to obtain high resolution kinetic data of glucose oxidase (GO) adsorption with sensitivity of adsorption better than that possible with SPR. The kinetic data, in combination with a functional assay of the enzyme activity, was used to test hypotheses on adsorption mechanisms. By fitting numerical models to the WGM sensograms for protein adsorption, and by confirming numerical predictions of enzyme activity in a separate assay, we were able to identify mechanisms for GO adsorption on different alkylsilanes and infer information about the adsorption of protein on nanostructured surfaces.

Enzyme adsorption, precipitation and crosslinking of glucose oxidase and laccase on polyaniline nanofibers for highly stable enzymatic biofuel cells.

Enzymatic biofuel cells have many great features as a small power source for medical, environmental and military applications. Both glucose oxidase (GOx) and laccase (LAC) are widely used anode and cathode enzymes for enzymatic biofuel cells, respectively. In this paper, we employed three different approaches to immobilize GOx and LAC on polyaniline nanofibers (PANFs): enzyme adsorption (EA), enzyme adsorption and crosslinking (EAC) and enzyme adsorption, precipitation and crosslinking (EAPC) approaches. The activity of EAPC-LAC was 32 and 25 times higher than that of EA-LAC and EAC-LAC, respectively. The half-life of EAPC-LAC was 53 days, while those of EA-LAC and EAC-LAC were 6 and 21 days, respectively. Similar to LAC, EAPC-GOx also showed higher activity and stability than EA-GOx and EAC-GOx. For the biofuel cell application, EAPC-GOx and EAPC-LAC were applied over the carbon papers to form enzyme anode and cathode, respectively. In order to improve the power density output of enzymatic biofuel cell, 1,4-benzoquinone (BQ) and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) were introduced as the electron transfer mediators on the enzyme anode and enzyme cathode, respectively. BQ- and ABTS-mediated enzymatic biofuel cells fabricated by EAPC-GOx and EAPC-LAC showed the maximum power density output of 37.4 µW/cm(2), while the power density output of 3.1 µW/cm(2) was shown without mediators. Under room temperature and 4°C for 28 days, enzymatic biofuel cells maintained 54 and 70% of its initial power density, respectively.

Ultrasound-radiated synthesis of PAMAM-Au nanocomposites and its application on glucose biosensor.

Hybrid nanocomposites of carboxyl-terminated generation 4 (G 4) poly(amidoamine) dendrimers (PAMAM) with gold nanoparticles (AuNPs) encapsulated inside them were synthesized under ultrasound irradiation. The obtained nanocomposites were used to fabricate highly sensitive amperometric glucose biosensor which exhibited a high and reproducible sensitivity of 2.9 mA/mM/cm(2), response time less than 5 s, linear dynamic range from 0.1 to 15.8 microM, correlation coefficient of R(2)=0.9988, and limit of detection (LOD), based on S/N ratio (S/N=3) of 0.05 microM. A value of 2.7 mM for the apparent Michaelis-Menten constant K(M)(app) was obtained. The high sensitivity, wider linear range, good reproducibility and stability make this biosensor a promising candidate for portable amperometric glucose biosensor.

Bienzyme system for the biocatalyzed deposition of polyaniline templated by multiwalled carbon nanotubes: a biosensor design.

A novel method based on covalent attachment of two enzymes, glucose oxidase (GOD) and horseradish peroxide (HRP), onto carboxylic-derived multiwalled carbon nanotubes (MWNTs) for the deposition of electroactive polyaniline (PANI) under ambient conditions is described. Ultraviolet-visible spectroscopy, Fourier-transform infrared (FTIR) spectroscopy, and transmission electron microscopy were used to characterize the assembling of bienzyme and the morphology of PANI|MWNTs. Under the bienzyme biocatalytic condition, a head-to-tail structure of PANI templated by MWNTs was formed. The voltammetric characteristics of the resulting biosensor were investigated by cyclic voltammetry in the presence of glucose. The current response of PANI was linearly related to glucose concentration between 0.05 and 12.0mM with a correlation coefficient of 0.994. The synergistic performance of bienzyme, highly efficient polymerization, and templated deposition provide a general platform for the synthesis of nanowires and nanocircuits, the construction of bioelectronic devices, and the design of novel biosensors.

AFM nanometer surface morphological study of in situ electropolymerized neutral red redox mediator oxysilane sol-gel encapsulated glucose oxidase electrochemical biosensors.

Four different silica sol-gel films: methyltrimethoxysilane (MTMOS), tetraethoxysilane (TEOS), 3-aminopropyltriethoxysilane (APTOS) and 3-glycidoxypropyl-trimethoxysilane (GOPMOS) assembled onto highly oriented pyrolytic graphite (HOPG) were characterized using atomic force microscopy (AFM), due to their use in the development of glucose biosensors. The chemical structure of the oxysilane precursor and the composition of the sol-gel mixture both influenced the roughness, the size and the distribution of pores in the sol-gel films, which is relevant for enzyme encapsulation. The GOPMOS sol-gel film fulfils all the morphological characteristics required for good encapsulation of the enzyme, due to a smooth topography with very dense and uniform distribution of only small, 50 nm diameter, pores at the surface. APTOS and MTMOS sol-gel films developed small pores together with large ones of 300-400 nm that allow the leakage of enzymes, while the TEOS film formed a rough and incomplete network on the electrode, less suitable for enzyme immobilisation. GOPMOS sol-gel film with encapsulated glucose oxidase and poly(neutral red) redox mediator, prepared by in situ electropolymerization, were also morphologically characterized by AFM. The AFM results explain the variation of the stability in time, sensitivity and limit of detection obtained with different oxysilane sol-gel encapsulated glucose oxidase biosensors with redox mediator.

Kinetic studies of glucose oxidase in polyelectrolyte multilayer films by means of scanning electrochemical microscopy (SECM).

Multilayer films of glucose oxidase (GOx) and poly(dimethyl diallyl ammonium chloride) (PDDA) prepared by layer-by-layer deposition were studied using scanning electrochemical microscopy (SECM). Aminated glass slides were coated with five bilayers of poly(styrene sulfonate) (PSS) and PDDA and used as substrates onto which GOx/PDDA multilayers were deposited. UV-Vis experiments confirmed multilayer growth, scanning force microscopic images provided morphological information about the films. SECM current-distance curves enabled the determination of kinetic information about GOx in GOx/PDDA multilayers as a function of layer number, film termination, inert covering layers, and enzyme substrate concentration after fitting to numerical models. The results indicate that only the topmost layers contributed significantly to the conversion. An odd-even pattern was observed for PDDA-terminated films or GOx-terminated films that correlated with morphological changes.

SP1 as a novel scaffold building block for self-assembly nanofabrication of submicron enzymatic structures.

In this study, SP1, a ring-shaped highly stable homododecamer protein complex was utilized for the self-assembly of multiple domains in a predefined manner. Glucose oxidase (GOx) was fused in-frame to SP1 and expressed in Escherichia coli. Complexes where GOx encircled SP1 dodecamer were observed, and moreover, the enzymatic monomers self-assembled into active multienzyme nanotube particles containing hundreds of GOx molecules per tube. This work demonstrates the value of SP1 as a self-assembly scaffold.

Carbon nanotube-templated assembly of protein.

This paper describes a novel general strategy for fabricating protein-polyion multilayers by electrostatic layer-by-layer (LBL) self-assembly on carbon nanotube templates. Such a noncovalent functionalization method is important for preserving the activity of biomolecular, the mechanical, and electrical properties of carbon nanotubes. Glucose oxidase and poly(diallyldimethylammonium) chloride polymer (PDDA) were used as models to investigate the LBL process on CNT templates. High-resolution TEM and electrochemical characterization confirm the formation of LBL nanostructures on carboxyl functionalized carbon nanotubes. We have also demonstrated the applications of these nanoshell bioreactors to direct electrochemistry of protein and biosensing. This strategy can be applied to assemble other biological molecules such as antibody, antigen, and DNA for wide bioassay applications.

Glucose oxidase immobilization on a novel cellulose acetate-polymethylmethacrylate membrane.

Glucose oxidase (GOD) was immobilized on cellulose acetate-polymethylmethacrylate (CA-PMMA) membrane. The immobilized GOD showed better performance as compared to the free enzyme in terms of thermal stability retaining 46% of the original activity at 70 degrees C where the original activity corresponded to that obtained at 20 degrees C. FT-IR and SEM were employed to study the membrane morphology and structure after treatment at 70 degrees C. The pH profile of the immobilized and the free enzyme was found to be similar. A 2.4-fold increase in Km value was observed after immobilization whereas Vmax value was lower for the immobilized GOD. Immobilized glucose oxidase showed improved operational stability by maintaining 33% of the initial activity after 35 cycles of repeated use and was found to retain 94% of activity after 1 month storage period. Improved resistance against urea denaturation was achieved and the immobilized glucose oxidase retained 50% of the activity without urea in the presence of 5M urea whereas free enzyme retained only 8% activity.

SPR and AFM study of engineered biomolecule immobilisation techniques.

A comparative study into two novel and diverse schemes designed to improve immobilization of biomolecules for biosensing purposes is presented. In the first method a silicon rich matrix is created using PECVD. The second method involves creating nano-patterns on the sensor surface to create a large number of surface discontinuities to which the proteins will bind preferentially. The basic theory of SPR is provided to show the importance of the surface sensitive nature of this optical transduction technique. The present work suggests that both may prove both for SPR and other biosensing applications. Of the two schemes proposed, the results for nano-patterning seem to suggest that it is promoting better surface attachment of biomolecules. The results of SPR and AFM studies are presented that have shown that each of these schemes promotes improved binding of various proteins.

Template-synthesized protein nanotubes.

A layer-by-layer deposition strategy for preparing protein nanotubes within the pores of a nanopore alumina template membrane is described. This method entails alternately exposing the template membrane to a solution of the desired protein and then to a solution of glutaraldehyde, which acts as cross-linking agent to hold the protein layers together. The number of layers of protein that make up the nanotube walls can be controlled at will by varying the number of alternate protein/glutaraldehyde cycles. After the desired number of layers have been deposited on the pore walls, the alumina template can be dissolved to liberate the protein nanotubes. We show here that glucose oxidase nanotubes prepared in this way catalyze glucose oxidation and that hemoglobin nanotubes retain their heme electroactivity. Furthermore, for the glucose oxidase nanotubes, the enzymatic activity increases with the nanotube wall thickness.

Application of integrated SECM ultra-micro-electrode and AFM force probe to biosensor surfaces.

The integration of scanning electrochemical ultra-micro-electrode (UME) with atomic force microscope cantilever probe have been achieved by using a homemade photolithography system. A gold-film-coated AFM cantilever was insulated with photo resist coating and a pointed end of the AFM probe was opened by illuminating with maskless arbitrary optical micro-pattern generator. To realize precise control of probe sample distance constantly, the resulting scanning electrochemical microscopy (SECM)-AFM probe was operated using a dynamic force microscopy (DFM) technique with magnetic field excitation. From a steady-state voltammetric experiment, the effective electrode diameters of the probes thus prepared were estimated to be from 0.050 to 6.2 microm. The capability of this SECM-AFM probe have been tested using gold comb in the presence of Fe(CN)(6)(3-). The simultaneous imaging of the topography and electrochemical activity of the strip electrode was successfully obtained. We also used the SECM-AFM to examine in situ topography and enzymatic activity measurement. Comparison of topography and oxidation current profiles above enzyme-modified electrode showed active parts distribution of biosensor surface.

Properties of mixed alkanethiol-dendrimer layers and their applications in biosensing.

We studied the properties of mixed alkanethiol-dendrimer layers on a gold support and their application in biosensing. We showed that properties of glucose sensor can be modified using a different ratio of 1-hexadecanethiol (HDT) and poly(amidoamine) dendrimer of first generation (G1). The cyclic voltammetry in the presence of the redox couple, Fe(CN)(6)(3-)/Fe(CN)(6)(4-), was used for estimating how effectively the layer blocks the redox probe's access to the electrode surface. A scanning electrochemical microscope (SECM) was used to image the resulting distribution of the organic compounds. We found that with increasing content of dendrimers, the integrity of the layers was improved.

Glucose sensor for flow injection analysis of serum glucose based on immobilization of glucose oxidase in titania sol-gel membrane.

A novel amperometric glucose sensor was constructed by immobilizing glucose oxidase (GOD) in a titania sol-gel film, which was prepared with a vapor deposition method. The sol-gel film was uniform, porous and showed a very low mass transport barrier and a regular dense distribution of GOD. Titania sol-gel matrix retained the native structure and activity of entrapped enzyme and prevented the cracking of conventional sol-gel glasses and the leaking of enzyme out of the film. With ferrocenium as a mediator the glucose sensor exhibited a fast response, a wide linear range from 0.07 to 15 mM. It showed a good accuracy and high sensitivity as 7.2 microA cm(-2) mM(-1). The general interferences coexisted in blood except ascorbic acid did not affect glucose determination, and coating Nafion film on the sol-gel film could eliminate the interference from ascorbic acid. The serum glucose determination results obtained with a flow injection analysis (FIA) system showed an acceptable accuracy, a good reproducibility and stability and indicated the sensor could be used in FIA determination of glucose. The vapor deposition method could fabricate glucose sensor in batches with a very small amount of enzyme.

Encapsulation of glucose oxidase microparticles within a nanoscale layer-by-layer film: immobilization and biosensor applications.

We report on an immobilization strategy utilizing layer-by-layer encapsulated microparticles of enzymes within a nanoscale polyelectrolyte film. Encapsulation of glucose oxidase (GOD) microparticles was achieved by the sequential adsorption of oppositely charged polyelectrolytes onto the GOD biocrystal surface. The polyelectrolyte system polyallylamine/polystyrene sulfonate was used under high salt conditions to preserve the solid state of the highly water soluble GOD biocrystals during the encapsulation process. The resulting polymer multilayer capsule of about 15 nm wall thickness is permeable for small molecules (glucose), but non-permeable for macromolecules thus preventing the enzyme from leakage and at the same time shielding it from the outer environment e.g., from protease or microbial activity. Decrease of the buffer salt concentration leads to the dissolution of the enzyme under formation of mu-bioreactors. The spherical mu-bioreactors are bearing an extremely high loading of biocompound per volume. Encapsulated GOD was subsequently used to construct a biosensor by nanoengineered immobilisation of mu-bioreactor capsules onto an electrode surface. The presented approach demonstrates a general method to encapsulate highly soluble solid biomaterials and an immobilization strategy with the potential to create highly active thin and stable films of biomaterial.