Development Of Amperometric Biosensor Research Articles

One-step electrodeposited hybrid nanofilms in amperometric biosensor development.

This review summarizes recent developments in amperometric biosensors, based on one-step electrodeposited organic-inorganic hybrid layers, used for analysis of low molecular weight compounds. The factors affecting self-assembly of one-step electrodeposited films, methods for verifying their composition, advantages, limitations and approaches affecting the electroanalytical performance of amperometric biosensors based on organic-inorganic hybrid layers were systemized. Moreover, issues related to the formation of one-step organic-inorganic hybrid functional layers with different structures in biosensors produced under the same electrodeposition parameters are discussed. The systemized dependencies can support the preliminary choice of functional sensing layers with architectures tuned for specific biotechnology and life science applications. Finally, the capabilities of one-step electrodeposition of organic-inorganic hybrid functional films beyond amperometric biosensors were highlighted.

Nanomaterials as Redox Mediators in Laccase-Based Amperometric Biosensors for Catechol Assay.

Laccase is a copper-containing enzyme that does not require hydrogen peroxide as a co-substrate or additional cofactors for an enzymatic reaction. Nanomaterials of various chemical structures are usually applied to the construction of enzyme-based biosensors. Metals, metal oxides, semiconductors, and composite NPs perform various functions in electrochemical transformation schemes as a platform for the enzyme immobilization, a mediator of an electron transfer, and a signal amplifier. We describe here the development of amperometric biosensors (ABSs) based on laccase and redox-active micro/nanoparticles (hereafter—NPs), which were immobilized on a graphite electrode (GE). For this purpose, we isolated a highly purified enzyme from the fungus Trametes zonatus, and then synthesized bi- and trimetallic NPs of noble and transition metals, as well as hexacyanoferrates (HCF) of noble metals; these were layered onto the surfaces of GEs. The electroactivity of many of the NPs immobilized on the GEs was characterized by cyclic voltammetry (CV) experiments. The most effective mediators of electron transfer were selected as the platform for the development of laccase-based ABSs. As a result, a number of catechol-sensitive ABSs were constructed and characterized. The laccase/CuCo/GE was demonstrated to possess the highest sensitivity to catechol (4523 A·M−1·m−2) among the tested ABSs. The proposed ABSs may be promising for the analysis of phenolic derivatives in real samples of drinking water, wastewater, and food products.

Highly Porous 3D Gold Enhances Sensitivity of Amperometric Biosensors Based on Oxidases and CuCe Nanoparticles.

Metallic nanoparticles potentially have wide practical applications in various fields of science and industry. In biosensorics, they usually act as catalysts or nanozymes (NZs) and as mediators of electron transfer. We describe here the development of amperometric biosensors (ABSs) based on purified oxidases, synthesized nanoparticles of CuCe (nCuCe), and micro/nanoporous gold (pAu), which were electro-deposited on a graphite electrode (GE). As an effective peroxidase (PO)-like NZ, nCuCe was used here as a hydrogen-peroxide-sensing platform in ABSs that were based on glucose oxidase, alcohol oxidase, methylamine oxidase, and L-arginine oxidase. At the same time, nCuCe is an electroactive mediator and has been used in laccase-based ABSs. As a result, the ABSs we constructed and characterized were based on glucose, methanol, methyl amine, L-arginine, and catechol, respectively. The developed nCuCe-based ABSs exhibited improved analytical characteristics in comparison with the corresponding PO-based ABSs. Additionally, the presence of pAu, with its extremely advanced chemo-sensing surface layer, was shown to significantly increase the sensitivities of all constructed ABSs. As an example, the bioelectrodes containing laccase/GE, laccase/nCuCe/GE, and laccase/nCuCe/pAu/GE exhibited sensitivities to catechol at 2300, 5055, and 9280 A·M−1·m−2, respectively. We demonstrate here that pAu is an effective carrier of electroactive nanomaterials coupled with oxidases, which may be promising in biosensors.

Development of amperometric biosensor based on cloned hemagglutinin gene of H1N1 (swine flu) virus.

The recent emergence of respiratory viruses especially COVID-19 and swine flu has underscored the need for robust and bedside detection methods. Swine flu virus is a very infectious virus of the respiratory system. Timely detection of this virus with high specificity and sensitivity is crucial for reducing morbidity as well as mortality. Cloning of gene segments into a non-infectious agent helps in the development of detection methods, vaccine development, and other studies. In this study, cloning was used to develop a biosensor for H1N1 pdm09 detection. A segment of the hemaglutinin gene was cloned in a vector and characterized with the help of colony touch PCR and blue–white screening. The recombinant plasmid was extracted, and the gene segment was confirmed with the help of HA-specific primers. A 5′ amine group-attached hemagglutinin (HA) gene-specific DNA probe was immobilized on the working gold electrode surface to make a quick, specific, reliable, and sensitive detection method for H1N1pdm09 virus in human nasal swab samples. The HA probe was immobilized on the cysteine applied gold electrode of the screen-printed electrode through 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS). Differential pulse voltammetry was performed with the help of methylene blue, which is a redox indicator for the detection of single-stranded cloned HA gene segment. The developed sensor depicted high sensitivity for the H1N1 influenza virus with a detection limit of 0.6 ng ssDNA/6 µl of the cloned HA sample. Specificity was also checked using H3N2 virus, N. meningitides, influenza A and positive H1N1pdm09 samples.

LDI-MS scanner: Laser desorption ionization mass spectrometry-based biosensor standardization

Amperometric biosensors have been widely utilized for the cost-effective and rapid analysis of various bioanalytes, for example glucose. However, a lack of standardization and validation procedures remains a major limitation in biosensor development. Therefore, despite rapid advances in material science driving the development of amperometric biosensors, to date only a few biosensors, detecting a limited range of analytes, are available on the market. It is believed, once this issue is addressed, it can significantly facilitate the next step in the overall concept “go to the market” production and implementation of amprerometric biosensors for a large industrial scale.Herein, we report on the use of laser desorption ionization mass spectrometry (LDI-MS) for the standardization of amperometric biosensors, based upon a complete and non-destructive characterization and validation of layer-by-layer (LbL) biosensors at each fabrication step. We reveal that specific ionization pathways of mediators, polymers and enzymes from the biosensor surface allows for robust quality control during LbL biosensor manufacture. Furthermore, this LDI-MS approach can also be used to monitor, and therefore ensure, the encapsulation of enzymes in one-step nanobiosensors. Specifically, we show that LDI-MS can be used for the rapid chemical profiling of LbL biosensors and one-step synthesized nanobiosensors, as well as to assess their synthesis quality and to monitor for batch-to-batch and intra- and inter-day changes in their function and behavior. Our novel approach will thus contribute to the future development, improved design and fine tuning of both conventional LbL-fabricated amperometric biosensors and one-step designed nanobiosensors.

Development of amperometric biosensors using VO2/GOx films for detection of glucose

Vanadium dioxide (VO2) film was successfully synthesized by hydrothermal treatment as well as the immobilization with glucose oxidase (GOx) onto the film. The VO2/GOx film was characterized by electrochemical studies in different condition of scan rate, pH and glucose concentration. The result of immobilization of GOx onto VO2 showed a change of voltammetric profile. The VO2/GOx film was investigated as a biosensor in the pH range of 4–10 and presented a sensitivity of 33.9 mV/pH. The roughness factor of VO2/GOx was calculated as being 1.7 × 10−4. This value of sensitivity suggests that the material can be a candidate for applications as disposable biosensor.

The Synergy of Thermally Reduced Graphene Oxide in Amperometric Urea Biosensor: Application for Medical Technologies.

Thermally reduced graphene oxide (TRGO) is a graphene-based nanomaterial that has been identified as promising for the development of amperometric biosensors. Urease, in combination with TRGO, allowed us to create a mediator-free amperometric biosensor with the intention of precise detection of urea in clinical trials. Beyond simplicity of the technology, the biosensor exhibited high sensitivity (2.3 ± 0.1 µA cm−2 mM−1), great operational and storage stabilities (up to seven months), and appropriate reproducibility (relative standard deviation (RSD) about 2%). The analytical recovery of the TRGO-based biosensor in urine of 101 ÷ 104% with RSD of 1.2 ÷ 1.7% and in blood of 92.7 ÷ 96.4%, RSD of 1.0 ÷ 2.5%, confirmed that the biosensor is acceptable and reliable. These properties allowed us to apply the biosensor in the monitoring of urea levels in samples of urine, blood, and spent dialysate collected during hemodialysis. Accuracy of the biosensor was validated by good correlation (R = 0.9898 and R = 0.9982) for dialysate and blood, utilizing approved methods. The advantages of the proposed biosensing technology could benefit the development of point-of-care and non-invasive medical instruments.

Development of amperometric biosensor in modified carbon paste with enzymatic preparation based on lactase immobilized on carbon nanotubes.

The variety of products derived from milk, without or with lactose, encourages the development of more effective analytical techniques that can be applied to the quality control of both the production line and the final products. Thus, in this work an efficient and minimally invasive method for the detection of lactose was proposed, using a biosensor containing the enzyme lactase (LAC) immobilised on carbon nanotubes (CNTs) that, when reacting with lactose, emit an electrochemical signal. This biosensor was connected to a potentiostat, and its electrochemical cell was composed of the following three electrodes: reference electrode (Ag/AgCl), auxiliary electrode (platinum wire), and working electrode (biosensor) on which graphite (carbon) paste (CP), CNTs, and LAC were deposited. The transmission electron microscopy and scanning electron microscopy were used in the characterisation of the composite morphology, indicating excellent interactions between the CNTs and LAC. The sensitivity of the CP/LAC/CNT biosensor was determined as 5.67μAcm-2.mmol-1L and detection limits around 100 × 10-6molL-1 (electrode area = 0.12cm2) and an increase in the stability of the system was observed with the introduction of CNTs because, with about 12h of use, there was no variation in the signal (current). The results indicate that the association between the CNTs and LAC favoured the electrochemical system.

The Presence of Polysaccharides, Glycerol, and Polyethyleneimine in Hydrogel Enhances the Performance of the Glucose Biosensor.

The use of amperometric biosensors has attracted particular attention in recent years, both from researchers and from companies, as they have proven to be low-cost, reliable, and very sensitive devices, with a wide range of uses in different matrices. The continuous development of amperometric biosensors, since their use involves an enzyme, is specifically aimed at keeping and increasing the catalytic properties of the loaded protein, so as to be able to use the same device over time. The present study aimed to investigate the impact of glycerol and polysaccharides, in the presence of polycationic substances to constitute a hydrogel, in enhancing the enzymatic and analytic performance of a glucose biosensor. Initially, it was possible to verify how the deposition of the starch-based hydrogel, in addition to allowing the electropolymerization of the poly(p-phenylenediamine) polymer and the maintenance of its ability to shield the ascorbic acid, did not substantially limit the permeability towards hydrogen peroxide. Moreover, different biosensor designs, loading a mixture containing all the components (alone or in combination) and the enzyme, were tested in order to evaluate the changes of the apparent enzyme kinetic parameters, such as VMAX and KM, and analytical response in terms of Linear Region Slope, highlighting how the presence of all components (starch, glycerol, and polyethyleneimine) were able to substantially enhance the performance of the biosensors. The surface analysis of the biosensors was performed by scanning electron microscope (SEM). More, it was shown that the same performances were kept unchanged for seven days, proving the suitability of this biosensor design for short- and mid-term use.

Utilization of enzyme extract self-encapsulated within polypyrrole in sensitive detection of catechol

Utilization of polyphenol oxidases (laccase, tyrosinase) in biosensor technology is an efficient approach towards phenol detection, which is significant in numerous fields such as environmental monitoring, food industry etc. The use of crude extract instead of pure enzyme eliminates the need for costly and laborious processes of enzyme separation and purification. This study employs polyphenol oxidase extract, biosynthesized by white-rot fungi Trametes pubescens (TP) for the development of amperometric biosensors for catechol detection. The catalytic activity of the crude extract was firstly used to induce the bio-synthesis of conducting polymer - polypyrrole (Ppy), resulting in the self-encapsulation of the enzyme extract within the conducting material. The viability and biological integrity of the enzyme extract was preserved after the synthesis and was able to efficiently detect phenolic compounds such as catechol. Comparative evaluations between the biosynthesized Ppy based biosensor (bio-Ppy) and the biosensor based on bio-PPy with additional enzyme extract (bio-Ppy-TP) were performed. Lastly, the performance of these two biosensors was compared with that of a third one, based on chemically synthesized Ppy with enzyme extract (chem-Ppy-TP). All three types of biosensors proved high efficiency for catechol detection at low concentration (1–60 μM) and were employed for real sample detection in fruit wines showing linear correlation with the spectrophotometric results obtained with the Folin-Ciocalteau standard test.

DEVELOPMENT OF AN AMPEROMETRIC BIOSENSOR FOR THE ACETYLCHOLINE DETERMINATION IN BIOLOGICAL SAMPLES

The work is devoted to the development of amperometric biosensor for the determination of acetylcholine in biological samples. In the work, two different variants of co-immobilization of choline oxidase and acetylcholinesterase were compared, and two-layer method of immobilization was selected (acetylcholinesterase-based membrane was deposited as the first layer, and choline oxidase-based membrane – as the second layer). Next, influence of the solution properties (buffer capacity, ionic strength, pH) on the biosensor analytical characteristics was studied. It was shown that the biosensor is characterized by good signal reproducibility and operational stability. Selectivity of the biosensor towards substances, which can be present in biological samples, was evaluated. Main analytical characteristics of the biosensor (sensitivity, linear range, limit of detection, noise, drift, reproducibility of responses) were determined. It was proven that the developed biosensor could be used for the determination of acetylcholine concentrations in biological samples.

Development of amperometric biosensors using screen-printed carbon electrodes modified with conducting polymer and nanomaterials for the analysis of ethanol, methanol and their mixtures

Amperometric biosensors based on alcohol dehydrogenase (ADH) and alcohol oxidase (AOx) were developed for analysis of ethanol, methanol and their mixtures. The first biosensor based on ADH responds only to ethanol, the second biosensor based on AOx responds to both methanol and ethanol. Screen printed carbon electrodes (SPCEs) were used as the base electrodes. They were first modified with nanoparticles (multiwalled carbon nanotubes and gold nanoparticles) and polyneutral red film. Then the enzyme ADH was immobilized on them to prepare ADH based biosensor for ethanol analysis or the enzyme AOx was immobilized on them to prepare AOx based biosensor for methanol analysis. The analytical characterization parameters sensitivity, linear range, limits of detection and quantification were found as 0.432 μA·mM−1, 178.5–1000 μM, 53.5 μM and 178.5 μM, respectively, for ethanol analysis with AOx based biosensor, as 0.509 μA·mM−1, 335.9–1000 μM, 100.8 μM and 335.9 μM, respectively, for methanol analysis with AOx based biosensor and 0.490 μA·mM−1, 320–1000 μM, 96.1 μM and 320.2 μM, respectively for ethanol analysis with ADH based biosensor. The same parameters were also determined for the analysis of different mixtures with AOx based biosensor. The developed biosensors have been tested on a commercial alcoholic drink for the analysis of its ethanol and methanol contents.

Fabrication of AChE/SnO2-cMWCNTs/Cu Nanocomposite-Based Sensor Electrode for Detection of Methyl Parathion in Water

The work highlights inhibition-based Acetylcholinesterase (AChE) fabrication using composite nanomaterial comprising tin oxide nanoparticles (SnO2) and carboxylated multiwalled carbon nanotubes (cMWCNTs) for detection of pesticide methyl parathion (MP) in water samples. Working electrode AChE/SnO2-cMWCNTs/Cu exhibited high sensitivity with a linearity range of 1.0 μM to 160 μM and a minimum detection limit of 0.1 μM for MP in water. The fabricated electrode was found biocompatible and nontoxic which can be used to detect low concentrations of pesticide in water samples. The synergistic and facilitated electron transferring properties of SnO2-cMWCNTs/Cu made it an excellent support for immobilization of enzyme in sensing technology. The enzyme AChE was covalently immobilized with cMWCNTs using glutaraldehyde as crosslinking agent which has enhanced the storage stability and reusability of the method. The reusability attained was 30 times for 40 days when AChE/SnO2-cMWCNTs/Cu was stored at low temperature of 4°C. Developed sensor showed excellent analytical recovery of pesticide in water sample with negligible effect of interfering species. Also, AChE/SnO2-cMWCNTs/Cu was easily reactivated simply by varying pH of phosphate buffer. This method is fast, reliable, and accurate showing successful development of amperometric biosensor for detection of MP in water sample.

Comparative Study of Poly(4-vinylpyridine) and Polylactic Acid-block-poly (2-vinylpyridine) Nanocomposites on Structural, Morphological and Electrochemical Properties

Polymer-based nanocomposites have attracted a lot of attention for amperometric biosensor development due to their general physical and chemical properties including biocompatibility, film-forming ability, stability and different functional groups that can be bonded with other biomolecues. In this study, poly-4-vinlyridine homopolymer (P4VP) and polylactic acid-block-poly(2-vinylpyridine) block copolymer (PLA-b-P2VP) were used to hybridize with gold precursors (Au3+) based on the association between the nitrogen of the pyridine group of P4VP or P2VP block with gold precursors. P4VP/Au3+ and PLA-b-P2VP/Au3+ nanocomposites were prepared with ratio of gold to P2VP or P4VP (10:1). The Au3+ in both polymers was reduced to gold nanoparticles (AuNPs) via in-situ approach by using hydrazine. Fourier transform infrared spectroscopy (FTIR), ultraviolet-visible spectroscopy (UV-vis), transmission electron microscopy (TEM) and cyclic voltammetry (CV) were used to characterize the structural, morphological and electrochemical properties of the nanocomposites. The peak currents of P4VP/AuNPs and PLA-b-P2VP/AuNPs nanocomposites modified electrode were 6.685 nA and 69.432 nA, respectively, which are much lower than bare electrode (205.019 nA) due to the non-conductivity of P4VP and PLA-b-P2VP. In order to improve the electron transfer capability of electrode, graphene oxide (GO) was blended and electrochemically reduced to obtain P4VP/AuNPs/rGO and PLA-b-P2VP/AuNPs/rGO nanocomposites. After immobilization of these two nanocomposites on electrode through drop casting method, the peak currents of P4VP/AuNPs/rGO and PLA-b-P2VP/AuNPs/rGO nanocomposites modified electrode were 871.172 nA and 663.947 nA, respectively, which are much higher than bare electrode (205.019 nA) and shown good capability to accelerate electron transfer. Based on these characterizations, P4VP/AuNPs/rGO has potential as the nanocomposite to modify the electrode for enzymatic biosensor development.

Design and development of amperometric biosensor for the detection of lead and mercury ions in water matrix-a permeability approach.

Intake ofwater contaminated with lead (Pb2+) and mercury (Hg2+) ions leads to various toxic effects and health issues. In this context, an amperometric urease inhibition-based biosensor was developed to detect Pb2+ and Hg2+ ions in water matrix. The modified Pt/CeO2/urease electrode was fabricated by immobilizing CeO2 nanoparticles and urease using a semi-permeable adsorption layer of nafion. With urea as a substrate, urease catalytic activity was examined through cyclic voltammetry. Further, maximum amperometric inhibitive response of the modified Pt/CeO2/urease electrode was observed in the presence of Pb2+ and Hg2+ ions due to the urease inhibition at specific potentials of -0.03 and 0V, respectively. The developed sensor exhibited a detection limit of 0.019±0.001μM with a sensitivity of 89.2×10-3μAμM-1 for Pb2+ ions. A detection limit of 0.018±0.003 with a sensitivity of 94.1×10-3μAμM-1 was achieved in detecting Hg2+ ions. The developed biosensor showed a fast response time (<1s) with a linear range of 0.5-2.2 and 0.02-0.8μM for Pb2+ and Hg2+ ions, respectively. The modified electrode offered a good stability for 20days with a good repeatability and reproducibility. The developed sensor was used to detect Pb2+ and Hg2+ ions contaminating Cauvery river water and the observed results were in good co-ordination with atomic absorption spectroscopic data.

Development of amperometric biosensor for choline determination

Development of amperometric biosensor for choline determination

Layer-by-layer design and optimization of xerogel-based amperometric first generation biosensors for uric acid

A layer-by-layer (LbL) strategy of modifying an electrode with a specific combination of polymeric and xerogel materials is used to create an effective first generation biosensor for uric acid (UA) - a clinically relevant molecule implemented in pregnancy induced hypertension (PIH) diagnosis, a condition that can lead to a serious disorder called preeclampsia. In addition to offering a new, promising sensor for UA, this study represents significant progress for amperometric biosensor development since the strategy and materials employed were successfully and readily adapted from a glucose biosensor model scheme and used for the effective detection of UA, a fundamentally different molecule compared to glucose. Specifically, each of four, functional modifying layers (outer polyurethane (PU) selective membrane, the inner selective electropolymer, and the xerogel bi-layer) are systematically investigated and tailored for UA permeability and interferent discrimination. The role of PU hydrophobicity and its UA permeability are established while the enzyme-doped and outer diffusional xerogel layers are evaluated for uricase (UOx) species/loading and silane precursor dependence, respectively. LbL systematic evaluation reveals the specific combination of hydroxyl-methyl triethoxy silane (HMTES) xerogels, a polyluminol-aniline electropolymer, and 100% hydrophilic polyurethane yields impressive uric acid sensing performance: effective sensitivity (0.8nA/μM), linear response across physiologically relevant UA concentrations (100–700μM), fast response times (~10s), low limits of detection (<10μM), and selectivity against most common interferents. Toward the specific application of PIH risk assessment, the optimized sensor exhibited 10day stability as well as effective shelf-life exceeding 35days. The presented system rivals or exceeds UA biosensor performance found in the literature and offers the possibility of miniaturization for in situ or in vivo remote diagnostic sensing. The successful adaptation suggests that the strategy and materials may be applicable to detecting/monitoring other medically significant molecules via sensor development.

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

AbstractThis 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 %.

Mathematical analysis of an enzyme-entrapped conducting polymer modified electrode

A mathematical model for the enzyme-entrapped conducting polymer modified electrode is discussed. Approximate expressions for the steady state concentration of the various species in the film and the current response of the enzyme electrode are derived. In this work we employ the Homotopy analysis method (HAM) to solve the nonlinear equations. These approximate analytical results are found to be in good agreement with the previously reported limiting case results. The obtained results are valid for the whole solution domain. Also, the redox enzyme in conducting polymer electrode is the most fundamental requirement for the development of amperometric biosensor and biofuel cell elements.

Improvement of the detection limit for biosensors: Advances on the optimization of biocomposite composition

In this work the application of advanced characterization techniques in the development of amperometric biosensors based on biocomposites is described. The optimization of the conductive particle distribution and the amount of the biological material inside the biomaterial have allowed an improvement of the electrochemical properties, regarding the electroanalytical properties such as signal stability and limit of detection. The high signal-to-noise ratio obtained in the electrochemical transduction has allowed enhancing the limit of detection of the biosensor. In the present study, it has been demonstrated the feasibility of electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) for the characterization and optimization of biosensors based on graphite–epoxy–enzyme, using an enzyme model. The optimum biocomposite proportion based on graphite–epoxy which incorporates the enzyme glucose oxidase (GOD) on the matrix ranges between 16% and 17% of graphite using 1% and 2% of enzyme. This range provides the optimal electroanalytical properties. Low limit of detection and good sensitivity have been achieved. Furthermore, confocal laser scanning microscopy was used to visualize the enzyme distribution onto the surface electrode.