Enzyme-based electrochemical biosensors

There has been an increased interest in the use of electrochemical sensors in clinical diagnostics because of the recent trend toward testing in satellite laboratories and the physician's office. The coupling of enzymes with electrochemical sensors permits the simple determination of metabolites, therapeutic drugs, antigens, and antibodies.     

Graphene electrochemistry: fabricating amperometric biosensors

The electrochemical sensing of hydrogen peroxide is of substantial interest to the operation of oxidase-based amperometric biosensors. We explore the fabrication of a novel and highly sensitive electro-analytical biosensor using well characterised commercially available graphene and compare and contrast responses using Nafion -graphene and -graphite modified electrodes. Interestingly we observe that graphite exhibits a superior electrochemical response due to its enhanced percentage of edge plane sites when compared to graphene. However, when Nafion, routinely used in amperometric biosensors, is introduced onto graphene and graphite modified electrodes, re-orientation occurs in both cases which is beneficial in the former and detrimental in the latter; insights into this contrasting behaviour are consequently presented providing acuity into sensor design and development where graphene is utilised in biosensors.     

Enzyme based amperometric biosensors

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Electron-transfer mechanisms in amperometric biosensors

The function of amperometric biosensors is related to electron-transfer processes between the active site of an (immobilized) enzyme and an electrode surface which is poised to an appropriate working potential. Problems and specific features of architectures for amperometric biosensors using different electron-transfer pathways such as mediated electron transfer, electron-hopping in redox polymers, electron transfer using mediator-modified enzymes and carbon-paste electrodes, direct electron transfer by means of self-assembled monolayers or via conducting-polymer chains are discussed.     

Prussian-Blue-based amperometric biosensors in flow-injection analysis

Optimisation of the electrodeposition of Prussian Blue onto mirrored glassy carbon electrodes yielded a modified electrode practically insensitive to oxygen reduction. At the same time the electrode activity towards hydrogen peroxide reduction was extremely high. This allowed the detection of hydrogen peroxide by electroreduction over a wide potential range. Flow-injection investigations of this electrode inserted into a flowthrough electrochemical cell of the confined wall-jet type showed that the response for hydrogen peroxide is limited by diffusion. Glucose and alcohol biosensors were made by immobilisation of glucose oxidase and alcohol oxidase respectively, within a Nafion layer, onto the top of the Prussian-Blue-modified electrodes. By increasing the density of Nafion and decreasing the measuring potential the glucose biosensor was made completely insensitive to both ascorbate and acetominophes.     

Surface acoustic wave biosensors: a review

This review presents an overview of 20 years of worldwide development in the field of biosensors based on special types of surface acoustic wave (SAW) devices that permit the highly sensitive detection of biorelevant molecules in liquid media (such as water or aqueous buffer solutions). 1987 saw the first approaches, which used either horizontally polarized shear waves (HPSW) in a delay line configuration on lithium tantalate (LiTaO₃) substrates or SAW resonator structures on quartz or LiTaO₃ with periodic mass gratings. The latter are termed “surface transverse waves” (STW), and they have comparatively low attenuation values when operated in liquids. Later Love wave devices were developed, which used a film resonance effect to significantly reduce attenuation. All of these sensor approaches were accompanied by the development of appropriate sensing films. First attempts used simple layers of adsorbed antibodies. Later approaches used various types of covalently bound layers, for example those utilizing intermediate hydrogel layers. Recent approaches involve SAW biosensor devices inserted into compact systems with integrated fluidics for sample handling. To achieve this, the SAW biosensors can be embedded into micromachined polymer housings. Combining these two features will extend the system to create versatile biosensor arrays for generic lab use or for diagnostic purposes. SAW based biosensor immersed in a sample flow. Analyte molecules binding to the immobilized antibodies on the sensor surface will influence the velocity of the SAW and hence the output signal generated by the driving electronics.     

Catalytic electrooxidation of NADH for dehydrogenase amperometric biosensors

The developments in the techniques of NADH catalytic oxidation relevant for incorporation in amperometric biosensors with dehydrogenase enzymes are reviewed with special emphasis in the years following 1990. The review stresses the direct electro-catalytic methods of NAD⁺ recycling as opposed to enzymatic regeneration of the coenzyme. These developments are viewed and evaluated from a mechanistic perspective of recycling of NADH to enzymatically active NAD⁺, and from the point of view of development of technologically useful reagentless dehydrogenase biosensors. An effort is made to propose a method for the standardization of evaluation of new mediating and direct coenzyme recycling schemes. A perspective is given for the requirements that have to be met for successful biosensor development incorporating dehydrogenase enzymes that open the analytical possibilities to a number of new analytes. The intrinsic limitations of the system are finally discussed and a view of the future of the field is presented.     

Modified graphites: application to the development of enzyme-based amperometric biosensors

For a series of graphite electrodes, modified with microquantities of Pd+Pt mixture in varied proportions, surface morphology of the catalytically active phase was studied with scanning electron microscopy (SEM), while the catalytic activity was examined at electrochemical reduction of hydrogen peroxide by means of steady-state polarization curves and constant potential amperometry. It was proven that the graphite, exhibiting the highest electrocatalytic activity (modified with Pd+Pt mixed in the ratio 70%:30% at t(deposit)=10 s) is distinguished with the smallest average size of the microformations. The operational characteristics of the same electrode and graphite modified with microquantities of Pd+Au mixed in the same ratio (70%:30%; t(deposit)=10 s) were compared. The application of these electrodes as basic transducers in highly selective biosensors for glucose and xanthine was demonstrated.     

Microneedle array-based carbon paste amperometric sensors and biosensors

The design and characterization of a microneedle array-based carbon paste electrode towards minimally invasive electrochemical sensing are described. Arrays consisting of 3 × 3 pyramidal microneedle structures, each with an opening of 425 μm, were loaded with a metallized carbon paste transducer. The renewable nature of carbon paste electrodes enables the convenient packing of hollow non-planar microneedles with pastes that contain assorted catalysts and biocatalysts. Smoothing the surface results in good microelectrode-to-microelectrode uniformity. Optical and scanning electron micrographs shed useful insights into the surface morphology at the microneedle apertures. The attractive performance of the novel microneedle electrode arrays is illustrated in vitro for the low-potential detection of hydrogen peroxide at rhodium-dispersed carbon paste microneedles and for lactate biosensing by the inclusion of lactate oxidase in the metallized carbon paste matrix. Highly repeatable sensing is observed following consecutive cycles of packing/unpacking the carbon paste. The operational stability of the array is demonstrated as well as the interference-free detection of lactate in the presence of physiologically relevant levels of ascorbic acid, uric acid, and acetaminophen. Upon addressing the biofouling effects associated with on-body sensing, the microneedle carbon paste platform would be attractive for the subcutaneous electrochemical monitoring of a number of physiologically relevant analytes.     

Carbon nanomaterials for salivary-based biosensors: a review

In recent years, saliva has been introduced as an alternate to conventional biofluid assays owing to both accessibility and reliability with regard to the assessment of different biomarkers. The capability of immediate online collection and analysis of salivary biomarkers offers myriad benefits for clinical applications, resulting in the demands for quantifying salivary biomarkers rapidly and reliably with the help of biosensing technology. Carbon–nanomaterial based biosensors provide potential instruments for a non-aggressive pain-free style of saliva-dependent recognition to diagnoses, monitor, and formulate a therapeutic modality and for managing patients. This review covers the importance of carbon nanomaterial in fabricating salivary-based detectors applied clinically for diagnostics and therapeutics. The utilization of carbon nanomaterials comprising carbon dots (CDs), carbon nanotubes (CNTs), graphene, graphene oxide (GO), reduced graphene oxide (rGO) and graphitic carbon nitride (g-C₃N₄) in salivary-based detection has been highlighted here with up-to-date instances. These sensing systems are capable of detecting a vast range of molecules with clinical relevance, including glucose, hormones, amino acids, viruses, bacteria, cancer antigens, cancer biomarkers, dopamine, sialic acid, uric acid, etc. which were discussed in this paper.     

Simultaneous determination of creatine and creatinine using amperometric biosensors

In order to determine creatine and creatinine amperometric biosensors were proposed. A bienzymatic biosensor based on creatinase (CI) and sarcosine oxidase (SO) was used for the assay of creatine and a trienzymatic biosensor based on CI, SO and creatininase (CA) for the assay of creatinine. The linear concentration ranges are of pmol l⁻¹ to nmol l⁻¹ magnitude order, with very low limits of detection. The biosensors proved high reliability for determination of creatine and creatinine as raw material, and in the pharmaceutical formulation.     

The application of boron-doped diamond electrodes in amperometric biosensors

Boron-doped diamond (BDD) electrodes outperform conventional electrodes in terms of high stability, chemical inertness, wide potential window and low background current. Combining the superior properties of BDD electrodes with the merits of biosensors, such as specificity, sensitivity, and fast response, amperometric biosensors based on BDD electrodes have attracted the interests of many researchers. In this review, the latest advances of BDD electrodes with different surfaces including hydrogen-terminated, oxygen-terminated, metal nanoparticles-modified, amine-terminated, and carboxyl-terminated thin films, and microelectrodes, for the construction of various biosensors or the direct detection of biomolecules were demonstrated. The future trends of BDD electrodes in biosensing were also discussed.     

Phenols monitoring and Hill coefficient evaluation using tyrosinase-based amperometric biosensors

Sensitive amperometric biosensors for phenols compounds, based on tyrosinase (polyphenoloxidase, PPO) immobilized on a Pt electrode in an electropolymerized poly-amphiphilic pyrrole matrix or cross-linked with glutaraldehyde, were constructed and compared. Steady-state amperometric measurements, performed at -50 mV vs. SCE in aqueous phosphate buffer containing LiClO(4) 0.1 M (pH 7) as well as in a chloroform solution containing 0.1 M C(6)H(5)CH(2)N(CH(3))(3)Cl, were used in order to compare the electroanalytical and kinetic parameters of the investigated amperometric biosensors in aqueous and nonaqueous media. It was established that the polypyrrole matrix has a higher efficiency for enzyme retention resulting in higher bioelectrode sensitivity, both in aqueous buffer (690 microA M(-1)) and in chloroform (149 microA M(-1)).     

Developmental aspects of amperometric ATP biosensors based on entrapped enzymes

A novel concept for a dual-enzyme-based microbiosensor for the detection of adenosine-5′-triphosphate (ATP) was developed. The employed enzymes pyrroloquinoline quinone-dependent glucose dehydrogenase (PQQ-GDH) and hexokinase were entrapped, using pH-shift-induced precipitation of electrodeposition paint (EDP) at platinum microelectrodes (diameter of 25 µm). PQQ-GDH is known showing a superior activity for glucose conversion at the relevant conditions (low oxygen concentration) for ATP detection in targeted biomedical studies. For immobilizing the two enzymes PQQ-GDH and hexokinase, the deposition conditions of EDP Resydrol AY498w/35WA were adapted to ensure high immobilization rates. Prior to ATP sensing, the conversion of glucose, which is the co-substrate for both enzymatic reactions, was optimized. Optimization was targeted towards ATP measurements in biomedical environments by optimizing the PQQ-GDH sensor for glucose. Therefore, different mediators were tested regarding their electron transfer rate and their compatibility with the enzyme: free-diffusing N-methylphenazonium methyl sulfate (PMS) and ferrocenemethanol, and an immobilized chromium hexacyanoferrate layer at platinum electrode. Free-diffusing ferrocenemethanol reveals high sensitivity towards glucose of 1.5?±?0.4 nA/mM. In a next step, hexokinase was co-entrapped in the polymer film resulting in a sensitivity of up to 290 pA/µM.     

Compact amperometric algal biosensors for the evaluation of water toxicity

Unicellular microalga Chlorella vulgaris was entrapped in an alginate gel or a polyion complex membrane immobilized directly on the surface of a transparent indium tin oxide electrode. Photosynthetically generated oxygen of the immobilized algae was monitored amperometically. Responses of the algal biosensor to four toxic compounds, 6-chloro-N-ethyl-N-isopropyl-1,3,5-triazine-2,4-diamine (atrazine), 3-(3,4-dichlorophenyl)-1,1-diethylurea (DCMU), toluene and benzene, were evaluated as inhibition ratios of the reduction current. The concentrations that give 50% inhibition of the oxygen reduction current (IC^′₅₀) for atrazine, DCMU, toluene and benzene were 2.0, 0.05, 1550 and 3000 μmol dm⁻³, respectively. There was a good correlation between these data and those of the conventional standard growth test. In comparison with the conventional algal biosensors based on the Clark-type oxygen electrode, the present sensor is much smaller and less expensive, and its assay time is much shorter (≤200 s).     

Determination of L- and D-enantiomers of methotrexate using amperometric biosensors

In order to determine the enantiopurity of methotrexate (Mtx), seven biosensors were proposed for the assay of l-Mtx and three biosensors for the assay of d-Mtx. The biosensors were designed using physical and chemical immobilization of glutamate oxidase and/or l-amino acid oxidase (l-AAOD) and/or horseradish peroxidase (HRP) for the assay of l-methotherexate, and d-amino acid oxidase (d-AAOD) and HRP for the assay of d-Mtx. Electrode characteristics were obtained and compared for the different carbon paste based biosensors. The linear concentration ranges for the proposed biosensors were in the ranges of fmol l⁻¹ to pmol l⁻¹, magnitude order with limits of detection in the fmol l⁻¹ to nmol l⁻¹ concentration range. All biosensors were successful for the determination of the enantiopurity of Mtx as raw material, and in its pharmaceutical formulations (tablets and injections).     

Enzyme/semiconductor nanoclusters combined systems for novel amperometric biosensors

In this work quantum-sized CdS nanocrystals were synthesized using a quaternary water-in-oil microemulsion and immobilized onto gold working electrode by self-assembled monolayers techniques. Formaldehyde dehydrogenase was covalently immobilized onto a protecting membrane, which was stratified on part of the semiconductor nanoparticles modified electrode. The covalent enzyme immobilization has been required to improve the stability of the catalytic oxidation of formaldehyde, which occurs after light stimulation of the semiconductor through the electron/hole recombination. A study about the best electrochemical oxidation potentials under different flow conditions was performed. Preliminary sensor stability and interferences tests were also carried out, for a sensitive and selective detection of formaldehyde. A detection limit of 41 ppb of formaldehyde was calculated and an operational stability of 6 h was achieved under flow conditions by means of this novel amperometric biosensor based on FDH-semiconductor hybrid systems, not requiring NAD⁺/NADH as charge transfer in the enzymatic reaction.