Nanostructured transducer surfaces for electrochemical biosensor construction—Interfacing the sensing component with the electrode

For fabrication of effective electrochemical biosensors, interfacing the biomolecular receptor with the underlying transducer represents a critical step. The actual approach taken depends on the tethering layer covering the transducer, which is typically either a conducting polymeric matrix, or a thin film, such as an alkanethiol monolayer. Non-specific immobilisation methods can be either covalent, or non-covalent affinity attachment, with multipoint electrostatic attachment of the sensing biomolecule to either a polyanionic or polycationic layer representing the most common approach. Many specific affinity immobilisation strategies exist, but the majority make use of one of two binding systems. The first relies on the specific and strong affinity between biotin and proteins of the avidin family, with both bioreceptor and transducer bearing pendant biotins and avidin used as the crosslinker. The second approach employs a metal chelating group on the transducer to which can be bound a polyhistidine tag present on the N- or C-terminus of the receptor protein and which can be introduced genetically, when the expression sequence for a recombinant proteins is designed.     

Biomolecule immobilization in biosensor development: tailored strategies based on affinity interactions

The exponential development of biosensors as powerful analytical tools in the last four decades mainly relies on the high sensitivity and selectivity offered when detecting the target analyte. The transducer and the biological receptor are the bases of the biosensor development. Nevertheless, the bioreceptor immobilisation is also important, playing a key role in the retention of the biological activity, and thus affecting the sensitivity. Parameters such as shelf-life and surface regeneration also depend on the biomolecule immobilisation. Researchers are focusing their efforts towards random and oriented immobilisation procedures. Adsorption, entrapment, cross-linking and electrostatic interactions provide randomly immobilised biomolecules, sometimes partially hindering their biological activity. Covalent binding and affinity interactions may enable oriented biomolecule immobilisations, providing controlled, reproducible and highly active modified surfaces. This paper reviews the main immobilisation strategies used in the biosensors development, putting special emphasis on our contribution to mild and oriented immobilisation techniques.     

Amperometric enzyme biosensors based on optimised electron-transfer pathways and non-manual immobilisation procedures

Development of reagentless biosensors implies the tight and functional immobilisation of biological recognition elements on transducer surfaces. Specifically, in the case of amperometric enzyme electrodes, electron-transfer pathways between the immobilised redox protein and the electrode surface have to be established allowing a fast electron transfer concomitantly avoiding free-diffusing redox species. Based on the specific nature of different redox proteins and non-manual immobilisation procedures possible biosensor designs are discussed, namely biosensors based on (i) direct electron transfer between redox proteins and electrodes modified with self-assembled monolayers; (ii) anisotropic orientation of redox proteins at monolayer-modified electrodes; (iii) electron-transfer cascades via redox hydrogels; and (iv) electron-transfer via conducting polymers.     

Structuring of recognizing elements of biosensors. Genetic constructs for the directed immobilization and combination of recognizing and signal generating structures

Data about the genetic-engineering approaches for providing of directed immobilisation of biological molecules on the transducer surface and for indirect revealing the formed specific complexes are analysed in this review. Creation of nanostructures on the transducer surface and a possibility to do directed transfer of biological molecules from one surface on the other one are considered as well.     

Electrochemical DNA biosensor for bovine papillomavirus detection using polymeric film on screen-printed electrode

A new electrochemical DNA biosensor for bovine papillomavirus (BPV) detection that was based on screen-printed electrodes was comprehensively studied by electrochemical methods of cyclic voltammetry (CV) and differential pulse voltammetry (DPV). A BPV probe was immobilised on a working electrode (gold) modified with a polymeric film of poly-L-lysine (PLL) and chitosan. The experimental design was carried out to evaluate the influence of polymers, probe concentration (BPV probe) and immobilisation time on the electrochemical reduction of methylene blue (MB). The polymer poly-L-lysine (PLL), a probe concentration of 1μM and an immobilisation time of 60min showed the best result for the BPV probe immobilisation. With the hybridisation of a complementary target sequence (BPV target), the electrochemical signal decreased compared to a BPV probe immobilised on the modified PLL-gold electrode. Viral DNA that was extracted from cattle with papillomatosis also showed a decrease in the MB electrochemical reduction, which suggested that the decreased electrochemical signal corresponded to a bovine papillomavirus infection. The hybridisation specificity experiments further indicated that the biosensor could discriminate the complementary sequence from the non-complementary sequence. Thus, the results showed that the development of analytical devices, such as a biosensor, could assist in the rapid and efficient detection of bovine papillomavirus DNA and help in the prevention and treatment of papillomatosis in cattle.     

Classical dot–blot format implemented as an amperometric hybridisation genosensor

A new electrochemical hybridisation genosensor has been designed. This genosensor is based on a concept adapted from classical dot–blot DNA analysis, but implemented in an electrochemical biosensor configuration. The use of amperometric transduction and the enzyme label method—that increases the genosensor sensitivity—are the main features of this new approach. The analytical procedure consists of five steps: DNA target immobilisation by adsorption onto a nylon membrane, hybridisation between DNA target and biotin–DNA probe, complexation reaction between biotin-DNA probe and an enzyme (horseradish peroxidase) streptavidin conjugate; integration of the modified membrane onto an electrochemical transducer; and finally, amperometric detection using a suitable substrate for the enzyme labelled duplex. Besides the adapted dot–blot format, a competitive assay in which the target is in solution is reported as well. This procedure, based on amperometric transduction, represents certain advantages with respect to dot–blot analysis: labelled hybrid detection is far simpler, quicker and requires more ordinary or simple reactives; the response obtained is a direct analytical signal via low-cost instrumentation, a nonisotopic labelling is used, and the membranes can be reused. These characteristics are ideal in implementing the procedure developed in kit form.     

New reactive polymer for protein immobilisation on sensor surfaces

Immobilisation of biorecognition elements on transducer surfaces is a key step in the development of biosensors. The immobilisation needs to be fast, cheap and most importantly should not affect the biorecognition activity of the immobilised receptor. A novel protocol for the covalent immobilisation of biomolecules containing primary amines using an inexpensive and simple polymer is presented. This tri-dimensional (3D) network leads to a random immobilisation of antibodies on the polymer and ensures the availability of a high percentage of antibody binding sites. The reactivity of the polymer is based on the reaction between primary amines and thioacetal groups included in the polymer network. These functional groups (thioacetal) do not need any further activation in order to react with proteins, making it attractive for sensor fabrication. The novel polymer also contains thiol derivative groups (disulphide groups or thioethers) that promote self-assembling on a metal transducer surface. For demonstration purposes the polymer was immobilised on Au Biacore chips. The resulting polymer layer was characterised using contact angle meter, atomic force microscopy (AFM) and ellipsometry. A general protocol suitable for the immobilisation of bovine serum albumin (BSA), enzymes and antibodies such as polyclonal anti-microcystin-LR antibody and monoclonal anti-prostate specific antigen (anti-PSA) antibody was then optimised. The affinity characteristics of developed immunosensors were investigated in reaction with microcystin-LR, and PSA. The calculated detection limit for analytes depended on the properties of antibodies. The detection limit for microcystin-LR was 10 ngmL(-1) and for PSA 0.01 ngmL(-1). The non-specific binding of analytes to synthesised polymers was very low. The polymer-coated chips were stored for up to 2 months without any noticeable deterioration in their ability to react with proteins. These findings make this new polymer very promising for the development of low-cost, easy to prepare and sensitive biosensors.     

Towards the protein phosphatase-based biosensor for microcystin detection

A colorimetric test for the detection of microcystins based on immobilised protein phosphatase (PP) has been developed. A PP2A produced by molecular engineering has been used and its performance has been compared to those of commercial PP2A and PP1. Covalent immobilisation of the enzyme using glutaraldehyde, encapsulation by sol-gel and entrapment with photocrosslinkable poly(vinyl alcohol) bearing styrylpyridinium groups (PVA-SbQ) have been compared, the latter method providing the highest immobilisation yields. Screen-printed carbon electrodes (SPEs), Maxisorp microtiter wells and Ultrabind modified polyethersulfone affinity membranes have been used as immobilisation supports. Whilst the highest immobilisation yields were obtained with microtiter wells, the highest operational and storage stabilities were achieved with carbon SPEs and membranes, respectively. The immobilisation of PP by PVA-SbQ provided a means to preserve the enzymatic activity, which decreased at fast rates when the enzyme was kept in solution. The colorimetric test using p-nitrophenyl phosphate has demonstrated that the immobilised enzyme is able to recognise both microcystin variants (MC-LR and MC-RR), although optimisation work should be performed to achieve appropriate limits of detection. With the purpose to develop an electrochemical biosensor, several phosphorylated substrates have been used. Promising results have been achieved with the commercial enzymes and alpha-naphtyl phosphate, p-aminophenol phosphate and catechol monophosphate as enzyme substrates, guaranteeing the viability of the electrochemical approach.     

Oriented coupling of major histocompatibility complex (MHC) to sensor surfaces using light assisted immobilisation technology

Controlled and oriented immobilisation of proteins for biosensor purposes is of extreme interest since this provides more efficient sensors with a larger density of active binding sites per area compared to sensors produced by conventional immobilisation. In this paper oriented coupling of a major histocompatibility complex (MHC class I) to a sensor surface is presented. The coupling was performed using light assisted immobilisation--a novel immobilisation technology which allows specific opening of particular disulphide bridges in proteins which then is used for covalent bonding to thiol-derivatised surfaces via a new disulphide bond. Light assisted immobilisation specifically targets the disulphide bridge in the MHC-I molecule alpha(3)-domain which ensures oriented linking of the complex with the peptide binding site exposed away from the sensor surface. Structural analysis reveals that a similar procedure can be used for covalent immobilisation of MHC class II complexes. The results open for the development of efficient T cell sensors, sensors for recognition of peptides of pathogenic origin, as well as other applications that may benefit from oriented immobilisation of MHC proteins.     

Immobilised enzymes: science or art?

Enzyme immobilisation is experiencing an important transition. Combinatorial approaches are increasingly applied in the design of robust immobilised enzymes by rational combination of fundamental immobilisation techniques (i.e. non-covalent adsorption, covalent binding, entrapment and encapsulation) or with other relevant technologies. The objective is to solve specific problems that cannot be solved by one of these basic immobilisation techniques.     

Electrochemical arrays coupled with magnetic separators for immunochemistry

Electrochemical methods are increasingly applied to immunoassays, because they overcome problems associated with other modes of detection. In particular, with respect to conventional immunoassays, electrochemical immunosensors show versatility, reliability, and fast analysis time. In immunosensor strategy, the antigen or antibody can be immobilized directly onto the surface of the electrochemical transducer that will finally be used to reveal the amount of the affinity reaction. However, the use of the electrode surface as a solid phase as well as an electrochemical transducer presents some problems: a shielding of the surface by biospecifically bound antibody molecules can cause hindrance in the electron transfer, resulting in a reduced voltammetric signal. Thus, as an alternative solid phase, magnetic beads because of their low toxicity and high biocompatibility have gained much attention in chemistry, associated with various analytical techniques, due to their suitability for immobilization of biomolecules. Magnetic micro- or nanobeads can be separated easily and quickly by magnetic forces and will be used together with bioaffine ligands, e.g., antibodies or proteins with a high affinity to the target. The special advantages of magnetic separation techniques are the fast and simple handling of a sample vial and the opportunity to deal with large sample volumes without the need for time-consuming centrifugation steps. This also makes biomagnetic separation ideal for automated assay/analysis systems which will play a very important role in the near future. This review presents some examples of immunochemical assay developed using magnetic beads as a solid phase coupled with electrochemical detection techniques, in particular, using electrochemical arrays as transducers. Applications related to static measurements, together with in-flow detection systems are presented.     

A high-performance glucose biosensor based on monomolecular layer of glucose oxidase covalently immobilised on indium-tin oxide surface

In this paper, a mediatorless amperometric glucose biosensor based on direct covalent immobilisation of monomolecular layer of glucose oxidase (GOx) on a semiconducting indium-tin oxide (ITO) is demonstrated. The abundance of surface hydroxyl functional group of ITO allows it to be used as a suitable platform for direct covalent immobilisation of the enzyme for sensor architecture. The anodic current corresponding to electrochemical oxidation of the enzymatic product, hydrogen peroxide, at a sputtered Pt electrode at 0.500 V (vs. SCE) was obtained as the sensor signal. It was found that the biosensor based on the direct immobilisation scheme shows a fast biosensor response, minimum interference from other common metabolic species and ease of biosensor miniaturisation. A linear range of 0-10 mM of glucose was demonstrated, which exhibits a high sensitivity as far as performance per immobilised GOx molecule is concerned. A detection limit as low as 0.05 mM and long-term stability were observed. Even more important, the biosensor design allows fabrication through a dry process. These characteristics make it possible to achieve mass production of biosensor compatible with the current electronic integrated circuit manufacturing technologies.     

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.     

Electrochemical reduction of cytochrome P450 as an approach to the construction of biosensors and bioreactors

In the present review an attempt was made to present an up-to-date amount of the data on electrochemical reduction of the hemoprotein cytochrome P450. The concept and potentialities of enzyme electrodes--transducers--as the main element for construction of electrochemical biosensors were discussed. Different types of electrodes for bioelectrochemistry were analysed. New nanotechnological approaches to cytochrome P450 immobilisation were reported. It was shown that nanobiotechnology in electrochemistry has potential application in manufacturing biosensors and bioreactors for clinical medicine and pharmacology.     

Rapid electrochemical genosensor assay using a streptavidin carbon-polymer biocomposite electrode

A sensor capable of detecting a specific DNA sequence was designed by bulk modification of a graphite epoxy composite electrode with streptavidin (2% w/w). Streptavidin is used to immobilise a biotinylated capture DNA probe to the surface of the electrode. Simultaneous hybridisation occurs between the biotin DNA capture probe and the target-DNA and between the target-DNA and a digoxigenin modified probe. The rapid binding kinetic of streptavidin-biotin allows a one step immobilisation/hybridisation procedure. Secondly, enzyme labelling of the DNA duplex occurs via an antigen-antibody reaction between the Dig-dsDNA and an anti-Dig-HRP. Finally, electrochemical detection is achieved through a suitable substrate (H2O2) for the enzyme-labelled duplex. Optimisation of the sensor design, the modifier content and the immobilisation and hybridisation times was attained using a simple nucleotide sequence. Regeneration of the surface is achieved with a simple polishing procedure that shows good reproducibility. The generic use of a modified streptavidin carbon-polymer biocomposite electrode capable of surface regeneration and a one step hybridisation/immobilisation procedure are the main advantages of this approach. In DNA analysis, this procedure, if combined with the polymerase chain reaction, would represent certain advantages with respect to classical techniques, which prove to be time consuming in situations where a simple and rapid detection is required. This innovative developed material may be used for the detection of any analyte that can be coupled to the biotin-streptavidin reaction, as is the case of immunoassays.     

A disposable biosensor for urea determination in blood based on an ammonium-sensitive transducer

A potentiometric urea-sensitive biosensor using a NH4(+)-sensitive disposable electrode in double matrix membrane (DMM) technology as transducer is described. The ion-sensitive polymer matrix membrane was formed in the presence of an additional electrochemical inert filter paper matrix to improve the reproducibility in sensor production. The electrodes were prepared from one-side silver-coated filter paper, which is encapsulated for insulation by a heat-sealing film. A defined volume of the NH4(+)-sensitive polymer matrix membrane cocktail was deposited on this filter paper. To obtain the urea-biosensor a layer of urease was cast onto the ion-sensitive membrane. Poly (carbamoylsulfonate) hydrogel, produced from a hydrophilic polyurethane prepolymer blocked with bisulfite, served as immobilisation material. The disposable urea sensitive electrode was combined with a disposable Ag/AgCl reference electrode to obtain the disposable urea biosensor. The sensor responded rapidly and in a stable manner to changes in urea concentrations between 7.2 x 10(-5) and 2.1 x 10(-2)mol/l. The detection limit was 2 x 10(-5) mol/l urea and the slope in the linear range 52 mV/decade. By taking into consideration the influence of the interfering K(+)- and Na(+)-ions the sensor can be used for the determination of urea in human blood and serum samples (diluted or undiluted). A good correlation was found with the data obtained by the spectrophotometric routine method.     

Antibody immobilisation on fibre optic TIRF sensors

A study of antibody immobilisation techniques on quartz and fibre optic surfaces for immunosensors has been carried out. Methods of covalent antibody immobilisation which have not previously been applied to optical fibres were investigated, and compared with classical methods found in the literature. Preliminary experiments on covalent immobilisation methods on planar quartz surfaces were conducted to enable us to choose the most suitable protein immobilisation technique for sensor applications. The immobilisation studies were directed in particular towards obtaining a high density of binding sites for the analyte of interest. Two of the most promising methods, antibody immobilisation on surfaces coated with dextran based hydrogel and F(ab')-SH fragments bound to silanised glass, which resulted in surface densities of active sites of above 0.45 pmol/cm2, were selected for further experiments on a fibre optic total internal reflection fluorescence immunosensor and gave satisfactory responses to changes in analyte concentrations of the order of 10(-8) M. The efficiency of polar organic solvents, such as dimethylsulfoxide, in dissociating the antigen-antibody complex and hence to regenerate the immunosensor surface was also evaluated.     

Introduction to the principles and applications of biosensors

A biosensor is an analytical device that responds to an analyte in an appropriate sample and interprets its concentration as an electrical signal via a suitable combination of a biological recognition system and an electrochemical transducer. As a result of recent scientific and technological progress, such devices are likely to play an increasingly important role in generating analytical information in all sectors of human endeavour, from medicine to the military. In particular, biosensors will form the basis of cheap, simple devices for acquiring chemical information, bringing sophisticated analytical capabilities to the non-specialist and general public alike. The market opportunities for the rapid exploitation of novel developments in this sector are substantial. Biosensor research is also likely to have a significant impact on the development of modern electronics.     

Biosensors for DNA sequence detection

DNA biosensors are being developed as alternatives to conventional DNA microarrays. These devices couple signal transduction directly to sequence recognition. Some of the most sensitive and functional technologies use fibre optics or electrochemical sensors in combination with DNA hybridization. In a shift from sequence recognition by hybridization, two emerging single-molecule techniques read sequence composition using zero-mode waveguides or electrical impedance in nanoscale pores.     

Carbon and gold electrodes as electrochemical transducers for DNA hybridisation sensors

Genosensor technology relying on the use of carbon and gold electrodes is reviewed. The key steps of each analytical procedure, namely DNA-probe immobilisation, hybridisation, labelling and electrochemical investigation of the surface, are discussed in detail with separate sections devoted to label-free and newly emerging magnetic assays. Special emphasis has been given to protocols that have been used with real DNA samples.