Screen-printed amperometric biosensors for the rapid measurement of L- and D-amino acids

Screen-printed three-electrode amperometric sensors incorporating L- and/or D-amino acid oxidase for the general purpose measurement of L- or D-amino acids is described. The working electrode incorporates rhodinized carbon, to facilitate hydrogen peroxide oxidation at a decreased operating potential, and immobilized enzyme. The devices responded to all 20 common L-amino acids and all of the D-amino acids examined, the exceptions being L- and D-proline. Linear response profiles were observed for L-leucine, L-glycine and L-phenylalanine with limits of detection of 0.47, 0.15 and 0.20 mM respectively. The devices were reproducible and exhibited stability over a 56 d test period. The biosensor compares favourably with a standard photometric amino acid test and was used to monitor milk ageing effects. The assay is cheap, simple to perform and rapid, requiring only buffer-electrolyte and a small sample volume.     

Electrochemical detection of DNA hybridization by using a zirconia modified renewable carbon paste electrode

A simple, polishable and renewable DNA biosensor was fabricated based on a zirconia modified carbon paste electrode. Zirconia was mixed with graphite powder and paraffin wax to produce the paste for the electrode, and response-optimized at 56% graphite powder, 19% ZrO(2) and 25% paraffin wax. An oligonucleotide probe with a terminal 5'-phosphate group was attached to the surface of the electrode via the strong affinity of zirconia for phosphate groups. DNA immobilization and hybridization were characterized by cyclic voltammetry and differential pulse voltammetry, using methylene blue as indicator. Examination of changes in response with complementary or non-complementary DNA sequences showed that the developed biosensor had a high selectivity and sensitivity towards hybridization detection (< or =2x10(-10) M complementary DNA detectable). The surface of the biosensor can be renewed quickly and reproducibly (signal RSD+/-4.6% for five successive renewals) by a simple polishing step.     

Multi-walled Carbon Nanotubes and Gold Nanorod Decorated Biosensor for Detection of microRNA-126

In this article, we introduced a novel electrochemical biosensor for the detection of microRNA-126. The biosensor utilizes a hybridization assay combined with multi-walled carbon nanotubes and gold nanorod-decorated screen-printed carbon electrodes. For electrode preparation, gold nanorods were first immobilized onto the surface of bare and multi-walled carbon nanotube-modified screen-printed carbon electrodes, and the thiol tagged-capture probe was immobilized on the electrode surface through gold and thiol group interaction. After the immobilization, thiol tagged-capture probe hybridized with the target sequence. Under optimum conditions, we determined limit of detection (LOD) and limit of quantification (LOQ) as high as 11 nM and 36 nM, respectively.     

Amperometric screen-printed biosensor arrays with co-immobilised oxidoreductases and cholinesterases

Amperometric screen-printed biosensor arrays for detection of pesticides (organophosphates and carbamates) and phenols have been developed. Cholinesterases (AChE and BChE), tyrosinase (TYR), peroxidases (SBP, soybean and HRP, horseradish) and cellobiose dehydrogenase (CDH) were combined on the same array consisting of one Ag/AgCl reference electrode surrounded by eight radially distributed working electrodes of either carbon or platinum. Mainly cross-linking with glutaraldehyde was employed for enzyme immobilisation. The substrates for the enzymes were acetylthiocholine for cholinesterases (ChEs), cellobiose for CDH and hydrogen peroxide for peroxidases. Hydrogen peroxide was generated in the presence of glucose by co-immobilised glucose oxidase (GOx). All measurements were performed in an electrochemical steady state system specially constructed for eight channel screen-printed electrode arrays. The achieved relative standard deviation values calculated for different enzyme substrates (10 measurements) were typically below 7% and one assay was completed within less than 10 min. The detection limits for pesticides and phenols were in the nanomolar and micromolar ranges, respectively. The developed biosensor array was evaluated on wastewater samples. To simplify interpretation of results, the measured data were treated with multivariate analysis-principal component analysis (PCA).     

DNA electrochemical biosensors

Disposable electrochemical DNA-based biosensors are reviewed; they have been used for the determination of low-molecular weight compounds with affinity for nucleic acids and for the detection of the hybridisation reaction. The first application is related to the molecular interaction between surface-linked DNA and the target pollutants or drugs, in order to develop a simple device for rapid screening of toxic or similar compounds. The determination of such compounds was measured by their effect on the oxidation signal of the guanine peak of calf thymus DNA immobilised on the electrode surface and investigated by chronopotentiometric analysis. The DNA biosensor is able to detect known intercalating compounds, such as daunomycin, polychlorinated biphenyls (PCBs), aflatoxin B1, and aromatic amines. Applicability to river and waste water samples is also demonstrated. Disposable electrochemical sensors for the detection of a specific sequence of DNA were realised by immobilising synthetic single-stranded oligonucleotides onto a graphite screen-printed electrode. The probes became hybridised with different concentrations of complementary sequences present in the sample. The hybrids formed on the electrode surface were evaluated by chronopotentiometric analysis using daunomycin as indicator of the hybridisation reaction. The hybridisation was also performed using real samples. Application to apolipoprotein E (ApoE) is described, in this case samples have to be amplified by PCR and then analysed by DNA biosensor. The extension of such procedures to samples of environmental interest or to contamination of food is discussed.     

Microbial fuel cell-based self-powered biosensing platform for determination of ketamine as an anesthesia drug in clinical serum samples

A novel self-powered DNA biosensor was successfully developed based on a dual-chambered microbial fuel cell (MFC) apparatus as a power supply and ketamine (KET) as a hybridization indicator. A graphite electrode coated with gold nanoparticles (GNP/graphite electrode), which provided larger surface area for immobilization of thiolated single-stranded (ssDNA) probe, was used as biocathode in the MFC system. When KET was used as the hybridization indicator for detection of ssDNA probe, the indicator exhibited excellent selectivity in detecting and discriminating the complementary, single-base mismatched, and noncomplementary target sequences. Furthermore, this self-powered biosensor based on MFC apparatus served as the biosensing platform for determination of KET in clinical serum samples. Under the steady-state operation condition, the difference between power densities of the ssDNA probe-modified GNP/graphite cathode in the absence and presence of accumulated KET (ΔP) served as the detection signal with a detection limit of 0.54 nM. The proposed MFC-based self-powered biosensor, as a low-cost portable device, showed a high sensitivity, stability, and reproducibility. Therefore, it can become a promising platform for determination of KET in clinical researches.     

Application of dual-step potential on single screen-printed modified carbon paste electrodes for detection of amino acids and proteins

A novel screen-printed biosensor for protein and amino acid (AA) estimation is reported. A rhodinised carbon paste working electrode (WE) was used in the three-electrode configuration. Bromine was generated by the electrolysis of an acidic potassium bromide electrolyte on the working electrode when poised at an appropriate potential. The consumption of bromine by the protein and amino acid was then measured at a lower potential. This sensor was used to detect most of the amino acids and some common proteins. The method is highly sensitive and can even detect L- and D- proline which give no response with enzyme electrodes. This sensor has also been used to detect protein/amino acid in fruit juice, milk, and urine. The unique feature of the sensor is that it does not require any reagent/chemical other than buffer. No surface treatment or modification of the WE is necessary and hence the sensor is stable for the lifetime of the basic screen-printed electrode, which is about a year. A very small amount of analyte (10 μL) is required for direct detection. The sensor is inexpensive and disposable and may also be used for continuous-flow systems. This sensor may have useful application in process monitoring and control during the production of amino acids.     

A screen-printed disposable biosensor for selective determination of putrescine

We have developed screen-printed carbon electrodes for the determination of putrescine (Put) via the enzyme monoamine oxidase that was immobilized on the surface of the electrode by cross-linking it with bovine serum albumin using glutaraldehyde. A mixture of 5 % of tetrathiofulvalene (TTF) and carbon ink was used for the fabrication of the screen-printed working electrode. Put was amperometrically detected by measurement of the current due to the oxidation of the mediator TTF. The use of TTF lowers the working potential to +250 mV (vs. a screen-printed Ag/AgCl reference electrode). Response is linear in the range from 16 to 101 μM, and the detection limit is 17.2?±?4.6 μM, with a reproducibility of 9.6 % (n?=?4) in terms of relative standard deviation. The effects of potentially interfering biogenic amines such as cadaverine, histamine, spermine, spermidine and tryptamine were also evaluated. The biosensor was successfully applied to the determination of Put in zucchini and anchovies.

Polychlorinated biphenyls (PCBs) detection in milk samples by an electrochemical magneto-immunosensor (EMI) coupled to solid-phase extraction (SPE) and disposable low-density arrays

An electrochemical immunosensor for polychlorinated biphenyl (PCB) detection based on graphite screen-printed low-density arrays and on magnetic beads is reported.The immunological reaction for the detection of PCBs is based on a direct competitive assay using alkaline phosphatase (AP) as enzymatic label. After the immunochemical recognition, the modified magnetic beads are captured by a magnet on the surface of the graphite working electrode. The electrochemical detection is thus achieved through the addition of the AP substrate (α-naphthyl-phosphate).Two different antibodies (sIgG anti-PCB28 and rIgG anti-PCB77) were tested and compared in terms of sensitivity and ability to recognise different congeners. The developed electrochemical magneto-immunosensor (EMI) was successfully combined with solid-phase extraction (SPE) for the analysis of PCBs in milk samples. In spiked samples a recovery of 80% was obtained. The proposed strategy offers great promise for rapid, simple, cost-effective, and on-site analysis of clinical, food and environmental samples, considering also that low-density arrays allow the simultaneous analysis of different processed samples.     

A biosensor based on graphite epoxy composite electrode for aspartame and ethanol detection

A gelatin membrane with carboxyl esterase and alcohol oxidase was subsequently integrated onto the surface of a graphite epoxy composite electrode (GECE). The developed biosensors showed linearity in the range of 2.5–400 μM for aspartame and 2.5–25 μM for ethanol with response times of 170 and 70 s for each analyte, respectively. The resulting bienzyme biosensor was used for aspartame detection in diet coke samples and ethanol detection in beer and wine samples. From the obtained results, it can be concluded that the developed biosensor is a selective, practical and economic tool for aspartame and ethanol detection in real samples.     

Disposable screen-printed electrode coupled with recombinant Drosophila melanogaster acetylcholinesterase and multiwalled carbon nanotubes for rapid detection of pesticides

Recombinant Drosophila melanogaster acetylcholinesterase (R-DmAChE), multiwalled carbon nanotubes (MWCNTs), and Prussian blue have been combined for development of a three-electrode biosensor with more rapid responses and higher stability than in our previous study. A new disposable screen-printed electrode (SPE) was developed for rapid detection of organophosphate and carbamate pesticides. After optimization, 10 microg MWCNT and 5 microL enzyme immobilization solution consisting of 0.2% glutaraldehyde, 0.1% Nafion, 0.2% bovine serum albumin, 0.1 g/L MWCNT, and 1.5 mU R-DmAChE were fixed on each of the R-DmAChE/MWCNT SPEs. The LOD of this biosensor was 0.5 microg/L for pesticide standards of dichlorvos (DDV) and carbofuran. The performance of this biosensor was tested for vegetable and water samples at various spiked levels, and good stability and sensitivity were found. The obtained recoveries were from 82.6 to 110.5% for DDV at levels of 0.5-5 microg/L and 73.4 to 118.4% for carbofuran at 1-10 microg/L in lake and sea water samples, demonstrating that the proposed approach is an alternative means for rapid detection of pesticide residues and contaminants in food safety and environmental monitoring.     

A Cholinesterase Sensor Based on a Graphite Electrode Modified with 1,3-Disubstituted Calixarenes

New cholinesterase sensors based on screen-printed graphite and graphite/epoxy electrodes modified with 1,3-disubstituted calix[4]arenes were developed for the detection of compounds that form host–guest complexes (using copper(II) and oxalate ions as an example). The effect of calix[4]arenes on the biosensor signal was studied under homogeneous and heterogeneous conditions. It was found that the effect of the studied compounds was due to changes in the electrostatic interactions and mobility of enzyme effectors in the electrode layer. Procedures were developed for the determination of Cu(II) by its activating effect in the concentration range 0.05–4.0 mM and of oxalate ions by their inhibiting effect in the concentration range 0.5–20 mM.     

一次性唾液α-淀粉酶生物传感器的研制

基于丝网印刷技术在PVC薄膜上制备了一次性碳电极,用Nafion固定二茂铁(Fc)作为电子介体,将α-糖苷酶和葡萄糖氧化酶(GOD)滴加于二茂铁修饰的电极上,滴加明胶晾干,戊二醛间接交联固定制成一次性唾液α-淀粉酶生物传感器,用计时电流法测定对α-淀粉酶的响应.实验结果表明,该传感器响应电流与α-淀粉酶活性在60～840 U/L之间呈现良好的线性关系,检出限为17 U/L.该生物传感器响应时间短,达到95%稳态响应时间不超过30 s,具有良好的一致性、准确性和稳定性.探讨了pH、缓冲液、温度及其它干扰物质等对该传感器的影响.该传感器可用于唾液α-淀粉酶浓度的快速、准确检测.     

Sarcosine oxidase composite screen-printed electrode for sarcosine determination in biological samples

As the prostate cancer (PCa) progresses, sarcosine levels increase both in tumor cells and urine samples, suggesting that this metabolite measurements can help in the creation of non-invasive diagnostic methods for this disease. In this work, a biosensor device was developed for the quantification of sarcosine via electrochemical detection of H₂O₂ (at 0.6 V) generated from the catalyzed oxidation of sarcosine. The detection was carried out after the modification of carbon screen printed electrodes (SPEs) by immobilization of sarcosine oxidase (SOX) on the electrode surface. The strategies used herein included the activation of the carbon films by an electrochemical step and the formation of an NHS/EDAC layer to bond the enzyme to the electrode, the use of metallic or semiconductor nanoparticles layer previously or during the enzyme immobilization. In order to improve the sensor stability and selectivity a polymeric layer with extra enzyme content was further added. The proposed methodology for the detection of sarcosine allowed obtaining a limit of detection (LOD) of 16 nM, using a linear concentration range between 10 and 100 nM. The biosensor was successfully applied to the analysis of sarcosine in urine samples.     

可检测有机磷农药残留的丝网印刷酶电极

制备了用于有机磷农药检测的电化学生物酶电极, 并从工作电极上修饰物的选择、测定电压的确定、生物酶电极预活化时间的选定、测定时间的选定、温度的影响、pH值的影响、底物浓度的影响、固定方法的选择、固定酶时温度和时间的影响等多方面对丝网印刷的生物酶电极进行了较为全面的研究, 应用经过优化的生物酶电极对有机磷农药乙基对氧磷进行了检测. 结果表明: 抑制率与乙基对氧磷浓度的常用对数在1.00×10^－7～1.00×10^－5 g•mL^－1范围内成线性关系, 线性回归方程为A/%＝267.2＋37.20lg[c/(g•mL^－1)], 相关系数r＝0.9882. 当信噪比为3时, 检出限为2.10 ng•mL^－1, 低于国家标准所要求的最低残留量.     

Encapsulation of glucose oxidase within poly(ethylene glycol) methyl ether methacrylate microparticles for developing an amperometric glucose biosensor

Poly(ethylene glycol) methyl ether methacrylate (PEGMEM) microparticles were synthesized and glucose oxidase (GOx) was immobilized within the microparticles. An amperometric biosensor was fabricated using the microparticles with GOx as biological component. The enzyme immobilization method was optimized by investigating the influence of monomer concentration and cross-linker content used in the preparation of the microparticles in the response of the biosensor. The best analytical results were obtained with the microparticles prepared with 0.21 M PEGMEM and 0.74% cross-linking. Furthermore, we have investigated the influence on the biosensor behaviour of parameters such as working potential, pH, temperature and enzymatic load. In addition, analytical properties such as sensitivity, linear range, response time and detection limit were determined. The biosensor was used to determine glucose in human serum samples and to avoid common interferents present in human serum such as uric and ascorbic acids. A Nafion layer was deposited on the electrode surface with satisfactory results. The useful lifetime of the biosensor was at least 520 days.     

Fabrication of a Novel Polymeric Scaffold for Amperometric Laccase Biosensor

Present work displays the preparation of an electrochemical biosensor using a conjugated polymer and laccase enzyme for catechol quantification in samples. The biosensing system is based on an enzyme immobilization on polymer modified graphite transducer surface. For that purpose, a random conjugated polymer, thienothiophene‐benzoxadiazole‐alt‐benzodithiophene (BOTT), was coated onto a graphite electrode surface via drop casting method followed by immobilization of a biomolecule (laccase) for sensing experiments. Herein, for the first time, we proposed a BOTT polymer as an inexpensive and effective way to fabricate highly sensitive and fast response biosensors. The proposed sensing system possessed superior properties with 0.38 μM limit of detection and 110.81 μA mM^?1 sensitivity. Furthermore, cyclic voltammetry and scanning electron microscopy techniques were used to examine the surface modifications. The proposed system could be useful for many future studies for catechol quantification in environmental samples.© 2019 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2019, 57, 2333–2339     

Disposable biosensors for determination of biogenic amines

This work reports monoamine oxidase (MAO)/horseradish peroxidase (HRP) and diamine oxidase (DAO)/horseradish peroxidase (HRP) based biosensors using screen-printed carbon electrodes for the determination of biogenic amines (BA). The enzymes have been covalently immobilized onto the carbon working electrode, previously modified by an aryl diazonium salt, using hydroxysuccinimide and carbodiimide. The detection has been performed by measuring the cathodic current due to the reduction of the mediator hydroxymethylferrocene at a low potential, 250 mV vs screen-printed Ag/AgCl reference electrode. The experimental conditions for the enzymes immobilization, as well as for the main variables that can influence the chronoamperometric current have been optimized by the experimental design methodology. Under these optimum conditions, the disposable biosensors have been characterized. A linear response range from 0.2 up to 1.6 μM and from 0.4 to 2.4 μM of histamine was obtained for DAO/HRP and MAO/HRP based biosensors, respectively. The biosensor construction was highly reproducible, yielding relative standard deviations of 10% and 11% in terms of sensitivity for DAO/HRP and MAO/HRP based biosensors, respectively. The capability of detection, 0.18 ± 0.01 μM in the case of DAO/HRP and 0.40 ± 0.04 μM (α = 0.05 and β = 0.005) for MAO/HRP based biosensors, and the biosensor sensitivity towards different BA has also been analyzed. Finally, the developed biosensors have been applied to the determination of the total amine content in fish samples.     

Surface plasmon resonance biosensor for enrofloxacin based on deoxyribonucleic acid

A DNA-based surface plasmon resonance biosensor for enrofloxacin was developed. Heating denatured DNA immobilized on the gold-coated glass surface was exploited. The immobilization was performed by a layer-by-layer co-deposition with a cationic polymer. The sensor performance was tested with real biological probes. Direct and simple determination of enrofloxacin in milk samples was demonstrated. The sensor response obeys Langmuir binding isotherm being almost linear until about 20 μg mL⁻¹. The detection limit in milk samples was estimated to be 3 μg mL⁻¹.     

A comparison study between a disposable electrochemical DNA biosensor and a Vibrio fischeri-based luminescent sensor for the detection of toxicants in water samples

In the present study, a comparison between a disposable electrochemical DNA biosensor and a Vibrio fischeri-based luminescent sensor for the detection of toxicants in water samples was made.In order to realize this study, a disposable electrochemical DNA biosensor has been reported. The DNA biosensor is assembled by immobilizing double stranded Calf Thymus DNA onto the surface of a disposable carbon screen-printed electrode. The oxidation signal of the guanine base, obtained by a square wave voltammetric scan, is used as analytical signal to detect the DNA damage; the presence of low molecular weight compounds with affinity for nucleic acids is measured by their effect on the guanine oxidation peak.Wastewater samples provided during First European Interlaboratory Exercise on water toxicity in the course of the project SWIFT-WFD were analyzed, and biosensor results were compared with a currently used toxicity test ToxAlert^®100 based on the bioluminescence inhibition of Vibrio fischeri. This test have been used because is rapid, easy handling and cost effectively responses for the toxicity assessment in real water samples.The results showed a promising correlation between two tests used for the detection of toxic compounds in water samples.