Biosensors for the evaluation of lipase activity

In the review a comprehensive analysis is given of instrumental analytical approaches to control the activity of lipolytic enzymes and their substrates. A special attention is attached to the methods based on the biosensor technology. A number of electrochemical, optical and mechanical biosensors that were developed in several laboratories are analyzed in detail. In addition, perspectives are evaluated for the development of these biosensors and their practical application. It was concluded that biosensors may provide all practical demands which are in enzymology field at the express determination of lipase activity and triacylglycerol levels in biological objects.     

Fabrication of a miniature CMOS-based optical biosensor

This work presents a novel, miniature optical biosensor by immobilizing horseradish peroxidase (HRP) or the HRP/glucose oxidase (GOx) coupled enzyme pair on a CMOS photosensing chip with a detection area of 0.5 mm × 0.5 mm. A highly transparent TEOS/PDMS Ormosil is used to encapsulate and immobilize enzymes on the surface of the photosensor. Interestingly, HRP-catalyzed luminol luminescence can be detected in real time on optical H₂O₂ and glucose biosensors. The minimum reaction volume of the developed optical biosensors is 10 μL. Both optical H₂O₂ and glucose biosensors have an optimal operation temperature and pH of 20–25 °C and pH 8.4, respectively. The linear dynamic range of optical H₂O₂ and glucose biosensors is 0.05–20 mM H₂O₂ and 0.5–20 mM glucose, respectively. The miniature optical glucose biosensor also exhibits good reproducibility with a relative standard deviation of 4.3%. Additionally, ascorbic acid and uric acid, two major interfering substances in the serum during electrochemical analysis, cause only slight interference with the fabricated optical glucose biosensor. In conclusion, the CMOS-photodiode-based optical biosensors proposed herein have many advantages, such as a short detection time, a small sample volume requirement, high reproducibility and wide dynamic range.     

Amperometric ATP biosensor based on polymer entrapped enzymes

A dual enzyme electrode for the detection of adenosine-5'-triphosphate (ATP) at physiologically relevant pH levels was developed by co-immobilization of the enzymes glucose oxidase (GOD) and hexokinase (HEX) using pH-shift induced deposition of enzyme containing polymer films. Application of a simple electrochemical procedure for the co-immobilization of the enzymes at electrode surfaces exhibits a major improvement of sensitivity, response time, reproducibility, and ease of fabrication of ATP biosensors. Competition between glucose oxidase and hexokinase for the substrate glucose involving ATP as a co-substrate allows the determination of ATP concentrations. Notable control on the immobilization process enables fabrication of micro biosensors with a diameter of 25 microm. The presented concept provides the technological basis for a new generation of fast responding, sensitive, and robust biosensors for the detection of ATP at physiological pH values with a detection limit of 10 nmol l(-1).     

Prussian Blue based screen printed biosensors with improved characteristics of long-term lifetime and pH stability

The promising advantages of Prussian Blue (PB) as catalyst and of the thick film screen printing technology have been combined to assemble sensors with improved characteristics for the amperometric determination of H(2)O(2). PB-modified screen printed electrodes were applied to detect H(2)O(2) at an applied potential of -0.05 V versus the internal screen printed Ag pseudoreference electrode, showing a detection limit of 10(-7) mol l(-1), a linearity range from 10(-7) to 5x10(-5) mol l(-1), a sensitivity of 234 microA mmol l(-1) cm(-2), and a high selectivity. Improved stability at alkaline pH values was also observed, which made possible their use with enzymes having an optimum basic pH. Then, the immobilisation of a single enzyme (glucose oxidase (GOD) or choline oxidase (ChOX)) or of two enzymes, acetylcholinesterase (AchE) coimmobilised with ChOX, has been performed on the surface of PB modified screen-printed electrodes (SPEs) using glutaraldehyde and Nafion. ChOX has been selected as an example of enzyme working at alkaline pH. The choline biosensors showed a detection limit of 5x10(-7) mol l(-1), a wide linearity range (5x10(-7)-10(-4) mol l(-1)), a high selectivity and a remarkable long term stability of 9 months at 4 degrees C, and at least 4 weeks at room temperature. Similar analytical characteristics and stability were observed with the acetylcholine biosensors.     

New redox mediator-modified polysulfone composite films for the development of dehydrogenase-based biosensors

This work presents polysulfone membranes as new materials for the development of compact dehydrogenase-based biosensors. Composite films were prepared by mixing polysulfone with graphite and were deposited on epoxy-graphite composite electrodes. Redox mediators were successfully immobilized in the composite film leading to highly reproducible biosensors, without leakage of the immobilized species. This results in a more reliable analytical system as, at the same time, problems of electrode fouling related to the detection of the coenzyme nicotinamide adenine dinucleotide (NADH) on which is based the amperometric detection of dehydrogenase-based biosensors are avoided. Scanning electron microscopy was used to study the morphological characteristics of the surface and the cross-section of the polysulfone-graphite composite films. Several procedures to immobilize enzymes in these membranes were demonstrated. Glutamate dehydrogenase (GlDH) was immobilized as an example of dehydrogenase enzyme, in this case for the development of an ammonium biosensor. High sensitivity, good selectivity, wide linear ranges and short response times were obtained for the optimized sensors and biosensors. Their good performance combined with the simplicity of the construction method, make the polysulfone-graphite composite films attractive matrices for the development of new enzyme-based biosensors, especially those based on dehydrogenase enzymes.     

Electrodeposited nonconducting polytyramine for the development of glucose biosensors

Biosensors with the composition of carbon/Prussian blue/(glucose oxidase+glutaraldehyde+polytyramine) were constructed. Before tyramine monomers were electropolymerized, glucose oxidase and tyramine monomers were cross-linked with glutaraldehyde onto the surface of Prussian-blue-modified electrodes. The constructed biosensors produced highly reproducible and stable devices. The biosensors exhibited neglectable decrease in current response after 10 repeated uses or after 1 month of dry storage. The resultant biosensors had a linear range of 0.1-1 mM glucose and a detection limit of 0.05 mM. Since the following electrocatalytic process proceeds at a low electrode potential (ca. -0.3 V vs Ag/AgCl), ascorbate and uric acid do not produce observable interfering signal for the determination of glucose.     

Amperometric glucose biosensor based on layer-by-layer assembly of multilayer films composed of chitosan, gold nanoparticles and glucose oxidase modified Pt electrode

A new strategy for fabricating glucose biosensor was presented by layer-by-layer assembled chitosan (CS)/gold nanoparticles (GNp)/glucose oxidase (GOD) multilayer films modified Pt electrode. First, a cleaned Pt electrode was immersed in poly(allylamine) (PAA), and then transferred to GNp, followed by the adsorption of GOD (GOD/GNp/PAA/Pt). Second, the GOD/GNp/PAA/Pt electrode was immersed in CS, and then transferred to GNp, followed by the adsorption of GOD (GOD/GNp/CS/GOD/GNp/PAA/Pt). Third, different layers of multilayer films modified Pt electrodes were assembled by repeating the second process. Film assembling and characterization were studied by quart crystal microbalance, and properties of the resulting glucose biosensors were measured by electrochemical measurements. The results confirmed that the assembling process of multilayer films was simple to operate, the immobilized GOD displayed an excellent catalytic property to glucose, and GNp in the biosensing interface efficiently improved the electron transfer between analyte and electrode surface. The amperometric response of the biosensors uniformly increased from one to six layers of multilayer films, and then reached saturation after the seven layers. Among the resulting biosensors, the biosensor based on the six layers of multilayer films was best. It showed a wide linear range of 0.5-16 mM, with a detection limit of 7.0 microM estimated at a signal-to-noise ratio of 3, fast response time (within 8s). Moreover, it exhibited good reproducibility, long-term stability and interference free. This method can be used for constructing other thin films, which is a universal immobilization method for biosensor fabrication.     

Electrochemical selective determination of ascorbic acid at redox active polymer modified electrode derived from direct blue 71

A simple selective method for determination of ascorbic acid using polymerized direct blue 71 (DB71) is described. Anodic polymerization of the azo dye DB71 on glassy carbon (GC) electrode in 0.1M H(2)SO(4) acidic medium was found to yield thin and stable polymeric films. The poly(DB71) films were electroactive in wide pH range (1-13). A pair of symmetrical redox peaks at a formal redox potential, E('0)=-0.02V vs. Ag/AgCl (pH 7.0) was observed with a Nernstian slope -0.058V, is attributed to a 1:1 proton+electron involving polymer redox reactions at the modified electrode. Scanning electron microscope (SEM), atomic force microscope (AFM) and electrochemical impedance spectroscopy (EIS) measurements were used for surface studies of polymer modified electrode. Poly(DB71) modified GC electrode showed excellent electrocatalytic activity towards ascorbic acid in neutral buffer solution. Using amperometric method, linear range (1x10(-6)-2x10(-3)M), dynamic range (1x10(-6)-0.01M) and detection limit (1x10(-6)M, S/N=3) were estimated for measurement of ascorbic acid in pH 7.0 buffer solution. Major interferences such as dopamine and uric acid are tested at this modified electrode and found that selective detection of ascorbic acid can be achieved. This new method successfully applied for determination of ascorbic acid in commercial tablets with satisfactory results.     

Screen-printed electrodes modified with glucose oxidase immobilized in hybrid organosilicon sol-gel matrix

A new polymer composition was developed based on hybrid sol-gel material for the immobilization of enzymes on the surface of screen-printed carbon electrodes modified with Prussian blue. The existing and developed methods for the glucose oxidase (GO) immobilization are compared. Highly stable bioelectrodes were shown to be obtained by enzyme immobilization on a hybrid composition consisting of solgel/poly vinyl alcohol (PVA) (up to 60 sequential chains) and agar gel (up to 45 sequential chains). The range of glucose concentrations detected during enzyme immobilization in a hybrid sol-gel/PVA composition or agar gel without dilution of a sample was 1.0–5.9 μM and 3.6–6.3 μM, respectively. An analysis of wine products was conducted. The results obtained using the proposed biosensors were shown to differ insignificantly from those obtained by high-performance liquid chromatography (the correlation coefficient was 0.9998).     

Entrapment of biomolecules in sol-gel matrix for applications in biosensors: problems and future prospects

An emerging area that has attracted increased attention in recent years is the development of biosensors based on sol-gel-derived platforms which must be predicated on an understanding of the short and long-term interactions between the biorecognition elements and evolving sol-gel matrix. This review focuses on the growing field of entrapment of biomolecules such as proteins, enzymes and antibodies in sol-gel matrices prepared from alkoxide precursors. Basic aspects of sol-gel, its advantages and disadvantages, factor affecting the sol-gel-derived thin films, strategies for improving entrapment of biomolecules in sol-gel materials and their organic modifications are discussed. Organically modified silane precursors have the ability to tune physical and chemical properties with desired characteristics of sol-gel preparations by simply changing different precursors and their molar ratio. The usefulness of optical method especially time-resolved fluorescence spectroscopy for the characterization of internal environment of sol-gel as well as dynamics of proteins within the sol-gel is highlighted. Significance and designing of new biocompatible sol-gel precursors with the purpose of making the glassy matrix more compatible with entrapped biomolecules has been described. Considerable attention has been drawn on problems and future prospects of sol-gel matrix for entrapment of biomolecules for applications in biosensors.     

Amperometric third-generation hydrogen peroxide biosensor based on the immobilization of hemoglobin on multiwall carbon nanotubes and gold colloidal nanoparticles

A convenient and effective strategy for preparation nanohybrid film of multi-wall carbon nanotubes (MWNT) and gold colloidal nanoparticles (GNPs) by using proteins as linker is proposed. In such a strategy, hemoglobin (Hb) was selected as model protein to fabricate third-generation H2O2 biosensor based on MWNT and GNPs. Acid-pretreated, negatively charged MWNT was first modified on the surface of glassy carbon (GC) electrode, then, positively charged Hb was adsorbed onto MWNT films by electrostatic interaction. The {Hb/GNPs}n multilayer films were finally assembled onto Hb/MWNT film through layer-by-layer assembly technique. The assembly of Hb and GNPs was characterized with cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS) and transmission electron microscopy (TEM). The direct electron transfer of Hb is observed on Hb/GNPs/Hb/MWNT/GC electrode, which exhibits excellent electrocatalytic activity for the reduction of H2O2 to construct a third-generation mediator-free H2O2 biosensor. As compared to those H2O2 biosensors only based on carbon nanotubes, the proposed biosensor modified with MWNT and GNPs displays a broader linear range and a lower detection limit for H2O2 determination. The linear range is from 2.1x10(-7) to 3.0x10(-3) M with a detection limit of 8.0x10(-8) M at 3sigma. The Michaelies-Menten constant KMapp value is estimated to be 0.26 mM. Moreover, this biosensor displays rapid response to H2O2 and possesses good stability and reproducibility.     

Exposing culprit organic pollutants: a review

There is a continuing need for monitoring the health of the environment due to the presence of pollutants. Here, we review the development and attributes of biosensors by which bacteria have been genetically modified to express the luminescence genes, i.e. to glow, in a quantified manner, in response to pollutants. We have concentrated on the detection of organic hydrocarbon pollutants and discussed the molecular mechanisms by which some of these chemicals act as effector molecules on the respective regulatory systems. The future of environmental biosensors is predictably bright. As more knowledge is gathered on the sensing regulatory component, the possibility of developing targeted or pollutant-specific biosensors is promising. Moreover, the repertoire of biosensors for culprit organic pollutants is expected to be enlarged through advances in genomics technology and identification of new sensory or receptor molecules. The need for pollutant detection at concentrations in the parts per trillion range or biosensors configured in a nanoscale is anticipated.     

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.     

A review of the use of genetically engineered enzymes in electrochemical biosensors

This article gives an overview of the electrochemical biosensors that incorporate genetically modified enzymes. Firstly, the improvements on the sensitivity and selectivity of biosensors that integrate mutated enzymes are summarised. Next, new trends focused on the oriented immobilisation of mutated enzymes through specific functional groups located at their surface are reviewed. Finally, the effect of enzyme mutations on the electron transfer distance and kinetics of electrochemical biosensors is described.     

Preparation and optical parameter characterization of two aldehyde derivative thin films for photonic applications by drop casting method

In this study, thin films of polymer poly(methyl methacrylate) were prepared using a drop casting method. Two newly synthesized aldehyde derivatives, 2‐bromomalonaldehyde and 5,6‐dihydroimidazo[2,1‐b]thiazole‐2‐carbaldehyde, were used at different concentrations to dope the films. The prepared films were transparent and therefore studied for application in photonics. Optical characterization of the samples was carried out using different spectroscopy techniques. Absorption spectra for both samples were obtained using a UV–vis light spectrophotometer. Other significant optical parameters, such as refractive index, extinction coefficient, and band gap energies, were calculated from the absorption spectra. The effect of doping concentration on these parameters was studied. Emission spectra were obtained using a fluorescence spectrophotometer and the effect of doping was observed. Fourier transform infrared spectra of the doped films were obtained and compared with the pure compound to note changes in peak values and peak intensity. This present work studied the effect of doping on optical properties and examined the application of the samples for photonics.     

Fabrication of Au/Fe3O4/RGO based aptasensor for measurement of miRNA‐128, a biomarker for acute lymphoblastic leukemia (ALL)

Due to their high sensitivity, simplicity, portability, self‐contained, and low cost, the development of electrochemical biosensors is a beneficial way to diagnose and anticipate many types of cancers. An electrochemical nanocomposite‐based aptasensor is fabricated for the determination of miRNA‐128 concentration as the acute lymphoblastic leukemia (ALL) biomarker for the first time. The aptamer chains were immobilized on the surface of the glassy carbon electrode (GCE) through gold nanoparticles/magnetite/reduced graphene oxide (AuNPs/Fe₃O₄/RGO). Fast Fourier transform infrared (FTIR), X‐ray diffraction (XRD), vibrating sample magnetometer (VSM), and transmission electron microscopy (TEM) were used to characterize synthesized nanomaterials. Cyclic voltammetry (CV), square wave voltammetry (SWV), and electrochemical impedance spectroscopy (EIS) were used to characterize the modified GCE in both label‐free and labeled methods. The results indicate that the modified working electrode has high selectivity and for miRNA‐128 over other biomolecules. The hexacyanoferrate redox system typically operated at around 0.3 V (vs. Ag/AgCl), and the methylene blue redox system ran at about 0 V, were used as an electrochemical probe. The detection limit and linear detection range for hexacyanoferrate and methylene blue are 0.05346 fM, 0.1–0.9 fM, and 0.005483 fM, 0.01–0.09 fM, respectively. The stability and diffusion control analyses were performed as well. In both label‐free and labeled methods, the modified electron showed high selectivity for miRNA‐128. The use of methylene blue as a safer redox mediator caused miRNA‐128 to be detected with greater accuracy at low potentials in PBS media. The findings also show the substantial improvement in detection limit and linearity by using reduced graphene oxide‐magnetite‐gold nanoparticles that can be verified by comparing with previous studies on the detection of other miRNAs.     

Pt based enzyme electrode probes assembled with Prussian Blue and conducting polymer nanostructures

Conductive polymer nanotubules of 1,2-diaminobenzene (1,2-DAB) were prepared using a porous polycarbonate membrane template, placed on a Pt foil and used to support the polymer, then, the electropolymerisation was performed by chronocoulometry. The obtained conductive polymer nanostructures were then placed on Pt electrode and used to support highly dispersed prussian blue (PB), which acts as the active component for H2O2 detection. The observed good stability of PB as catalyst of H2O2 was related to the presence of organic non-conventional conducting polymers in a composite nanostructured film. These nanostructured polymer/PB composite films were also characterised by scanning electron microscopy (SEM) and Raman spectroscopy. The non-conventional conducting polymer nanotubules/PB modified Pt electrodes were tested by cyclic voltammeter for stability at different pH values, then, by amperometry, for hydrogen peroxide, ascorbic acid, acetaminophen, uric acid and acetylcholine. Glucose oxidase (GOD), lactate oxidase (LOD), L-amino acid oxidase (L-AAOD), alcohol oxidase (AOD), glycerol-3-phosphate oxidase (GPO), lysine oxidase (LyOx), and choline oxidase (ChOx) were immobilised on PB layer supported on 1,2-diaminobenzene (1,2-DAB) nanotubules onto the Pt electrodes. Different strategies for enzyme immobilisation were performed and used. Analytical parameters such as reproducibility, interference rejection, response time, storage and operational stability of the sensors have been studied and optimised. Results provide a guide to design high sensitive, stable and interference-free biosensors. The glucose biosensors assembled with nanostructured poly(1,2-DAB) showed a detection limit of 5 x 10(-5) mol l(-1), a wide linearity range (5 x 10(-5) to 5 x 10(-3) mol l(-1)), a high selectivity, a stability of 3 months at 4 degrees C, and at least 4 weeks at room temperature. Similar analytical parameters and stability were also studied for L-(+)-lactic acid, L-leucine, ethanol, glycerol-3-phosphate, lysine, and choline biosensors.     

A new set up for multi-analyte sensing: at-line bio-process monitoring

A multi-analyte sensing device is described, for simultaneous at-line monitoring of glucose, ethanol, pO?-value and cell density. It consists of a dual biosensor, a modified microscope and a fiber optical pO?-sensor that are integrated into a flow analysis (FA) system. The biosensor is based on a conventional thin layer flow-through cell equipped with a gold (Au) dual electrode (serial configuration). The biosensors with no cross-talking were produced by modifying the electrochemical transducers. Each Au surface was initially modified by self-assembled monolayer (SAM) of cysteamine. Alcohol oxidase (AOx) and pyranose oxidase (PyOx) were immobilized each onto a gold surface by means of PAMAM (polyamidoamine) dendrimer via glutaraldehyde cross-linking. The responses for glucose and ethanol were linear up to 0.5 mM. The operational stability of the biosensors was very promising, after 11 h continuous operation, only 6.0% of the initial activity was lost. The potential of the described biosensor was demonstrated by parallel determination of ethanol and glucose in yeast fermentation process. Simultaneously the cell density of the culture was monitored with an in situ microscope (ISM), which was integrated into the FA system. Both the used in situ microscope and the image processing algorithm used for the analysis of the acquired image data are described. Furthermore the pO?-value was monitored using a fiber optical sensor, which was embedded in a flow cell. The multi-sensor device allows the at-line monitoring of several process values without the need for further sampling or time consuming offline measurements.     

Immobilization of a trienzymatic system in a sol-gel matrix: a new fluorescent biosensor for xanthine

In this work we report the development of a highly sensitive fluorescent multienzymatic biosensor for quantitative xanthine detection. This biosensor is built by the simultaneous encapsulation of three enzymes, xanthine oxidase, superoxide dismutase and peroxidase, in a single sol-gel matrix coupled to the Amplex Red probe. The sol-gel chemistry yields a porous, optically transparent matrix that retains the natural conformation and the reactivity of the three co-immobilized proteins. Xanthine determination is based on a sequence of reactions, namely catalytic oxidation of xanthine to uric acid and superoxide radical, and subsequent catalytic dismutation of the radical, resulting in the formation of hydrogen peroxide, which reacts stoichiometrically with non-fluorescent Amplex Red to produce highly fluorescent resorufin. The optimal operational conditions for the biosensor were investigated. Linearity was observed for xanthine concentrations up to 3.5 microM, with a detection limit of 20 nM, which largely improved the sensitivity of the current xanthine biosensors. The developed biosensor is reusable and remains stable for 2 weeks under adequate storage conditions.     

Ultrathin hydrogel films for rapid optical biosensing

Novel biosensors have been designed by reporting an analyte-induced (de)swelling of a stimuli-responsive hydrogel (usually in a form of thin film) with a suitable optical transducer. These simple, inexpensive hydrogel biosensors are highly desirable, however, their practical applications have been hindered, largely because of their slow response. Here we show that quick response hydrogel sensors can be designed from ultrathin hydrogel films. By the adoption of layer-by-layer assembly, a simple but versatile approach, glucose-sensitive hydrogel films with thickness on submicrometer or micrometer scale, which is 2 orders of magnitude thinner than films used in ordinary hydrogel sensors, can be facilely fabricated. The hydrogel films can not only respond to the variation in glucose concentration, but also report the event via the shift of Fabry-Perot fringes using the thin film itself as Fabry-Perot cavity. The response is linear and reversible. More importantly, the response is quite fast, making it possible to be used for continuous glucose monitoring.