Highly Sensitive Choline Oxidase Enzyme Inhibition Biosensor for Lead Ions Based on Multiwalled Carbon Nanotube Modified Glassy Carbon Electrodes

The determination of lead ions by inhibition of choline oxidase enzyme has been evaluated for the first time using an amperometric choline biosensor. Choline oxidase (ChOx) was immobilized on a glassy carbon electrode (GCE) modified with multiwalled carbon nanotubes (MWCNT) through cross‐linking with glutaraldehyde. In the presence of ChOx, choline was enzymatically oxidized into betaine at –0.3 V versus Ag/AgCl reference electrode, lead ion inhibition of enzyme activity causing a decrease in the choline oxidation current. The experimental conditions were optimised regarding applied potential, buffer pH, enzyme and substrate concentration and incubation time. Under the best conditions for measurement of the lowest concentrations of lead ions, the ChOx/MWCNT/GCE gave a linear response from 0.1 to 1.0 nM Pb²⁺ and a detection limit of 0.04 nM. The inhibition of ChOx by lead ions was also studied by electrochemical impedance spectroscopy, but had a narrower linear response range and low sensitivity. The inhibition biosensor exhibited high selectivity towards lead ions and was successfully applied to their determination in tap water samples.     

Single‐gap Microelectrode Functionalized with Single‐walled Carbon Nanotubes and Pbzyme for the Determination of Pb2+

Lead is a highly toxic metal, which can persist in the natural environment and enrich in biological bodies. It can cause many severe diseases in the human body even at extremely low concentration. Here, we developed a new biosensor using single‐walled carbon nanotubes (SWNTs) modified with a specific Pbzyme (Pbzyme/SWNTs/FET) to detect lead ion (Pb²⁺), which can monitor the lead pollution. This biosensor used 3‐aminopropyltriethoxysilane to immobilize SWNTs on the area between the source and the drain of single‐gap microelectrode (FET), and the duplex DNA (Pbzyme) consisted of DNAzyme (GR‐5) and complementary DNA (CS‐DNA) was functionalized with the SWNTs’ surface through a peptide bond. The use of GR‐5 DANzyme and Pb²⁺ to form a stable complex structure to cleave the CS‐DNA can change radically the Pbzyme's structure on the SWNTs’ surface, which will further affect the number of carriers in SNWTs and the conductivity of the Pbzyme/SWNTs/FET. The change in conductivity can be used to evaluate the Pb²⁺ concentration. Under the optimal conditions, the relative resistances presented a positive correlation with the Pb²⁺ concentrations, showing a good linear relationship in the range of 10 pM to 50 nM, where the linear regression equation was y=10.104log [C_Pb]+5.8656, and the detection limit was 7.4 pM. Finally, the biosensor was applied to measure the Pb²⁺ contents in the soil collected from the forest grass green belt and paint, and the results were compared with the results of atomic fluorescence spectrometry.     

Determination of Lead(II) by a Nitrocellulose Membrane Fluorescent Biosensor Based on G-Quadruplex Conformational Changes

A simple, label-free fluorescence method was developed for the sensitive determination of lead(II) using a nitrocellulose membrane biosensor. The surface of the nitrocellulose membrane was modified by glutaraldehyde to conjugate streptavidin, followed by the immobilization of a DNA probe via a biotin modifier. The biotinylated DNA probe can fold into a G-quadruplex structure in the presence of potassium ion that selectively binds to N-methyl mesoporphyrin IX and yields a strong fluorescence signal. The presence of lead(II) can induce a conformational change of the G-quadruplex to a more compact structure, which results in the release of potassium ion and N-methyl mesoporphyrin IX with a concomitant reduction of the fluorescence signal. The biosensor displayed a detection limit as low as 10 nM with excellent selectivity for lead(II) over other metal ions. The developed biosensor was employed for the determination of lead(II) in spiked river water.     

High‐sensitive Electrochemical Determination of Ethyl Carbamate Using Urethanase and Glutamate Dehydrogenase Modified Electrode

An amperometric biosensor for ethyl carbamate (EC) was developed for the first time through the cascade reactions of urethanase and glutamate dehydrogenase (GLDH). Urethanase decomposes ethyl carbamate to produce ammonia, which converts to L‐glutamate under the catalysis of GLDH in the presence of α‐ketoglutarate and NADH. Then the change of NADH can be detected chronoamperometrically. The two enzymes were entrapped into chitosan/gelatine/γ‐glycidoxy propyl trimethoxy silane sol‐gel and immobilized on the surface of pyrolytic graphite electrode (PGE). The modified electrode was characterized by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). Under the optimized conditions, the amperometric EC biosensor exhibits a linear detection range from 0.5 to 40 μM with a low detection limit of 5.30 nM. The biosensor was successfully used to detect EC in mimic Chinese rice wine samples, and satisfactory recovery and relative standard deviation were achieved.     

Impedimetric DNA‐Based Biosensor for Silver Ions Detection with Hemin/G‐Quadruplex Nanowire as Enhancer

A DNA‐based biosensor was reported for detection of silver ions (Ag⁺) by electrochemical impedance spectroscopy (EIS) with [Fe(CN)₆]^4?/3? as redox probe and hybridization chain reaction (HCR) induced hemin/G‐quadruplex nanowire as enhanced label. In the present of target Ag⁺, Ag⁺ interacted with cytosine‐cytosine (C? C) mismatch to form the stable C? Ag⁺? C complex with the aim of immobilizing the primer DNA on electrode, which thus triggered the HCR to form inert hemin/G‐quadruplex nanowire with an amplified EIS signal. As a result, the DNA biosensor showed a high sensitivity with the concentration range spanning from 0.1 nM to 100 µM and a detection limit of 0.05 nM.     

Impedimetric Aptasensor for Thrombin Recognition Based on CD Support

We report an aptamer biosensing array for thrombin detection by measuring the electrochemical impedance upon aptamer‐protein formation at the surface of CD‐trodes (GCDTs) in the presence of the redox couple [Fe(CN)₆]^3?/4?. GCDTs are fabricated from recordable compact discs that contain a fine gold layer. The biosensor is constructed by self‐assembling of a thiol‐modified thrombin binding aptamer (TBA) onto a GCDT surface. The sensor reveals good ligand specificity, recognition in a wide range of thrombin concentrations from 20 nM to 1 µM with a limit of detection of 5 nM.     

Paper-based scanometric assay for lead ion detection using DNAzyme

A facile and simple paper-based scanometric assay was developed to detect Pb²⁺ using GR5-DNAzyme. Magnetic beads (MBs) and gold nanoparticles (AuNPs) were used as a signal collector and a signal indicator, respectively. They were linked together by GR5-DNAzyme, comprising an enzyme and a substrate strand pairing up with each other. In the presence of Pb²⁺, the substrate strand is cut into two pieces, resulting in the disassembly of AuNPs from the MBs. These AuNPs were spotted on predefined areas on a chromatography paper, where signal is amplified through silver reduction. This sensing platform exhibits high sensitivity and selectivity toward Pb²⁺, giving a detection limit of 0.3 nM and a linear fitting range from 0.1 to 1000 nM. Testing of this biosensor in river water and synthetic urine samples also showed satisfying results. Besides offering simultaneous and multi-sample analysis, this paper-based sensing platform presented here could be potentially applied and served as a general platform for on-site, naked eyes, and low-cost monitoring of other heavy metal ions in environmental and body fluid samples.     

Detection of Adrenaline in Blood Plasma as Biomarker for Adrenal Venous Sampling

An amperometric bi‐enzyme biosensor based on substrate recycling principle for the amplification of the sensor signal has been developed for the detection of adrenaline in blood. Adrenaline can be used as biomarker verifying successful adrenal venous sampling procedure. The adrenaline biosensor has been realized via modification of a galvanic oxygen sensor with a bi‐enzyme membrane combining a genetically modified laccase and a pyrroloquinoline quinone‐dependent glucose dehydrogenase. The measurement conditions such as pH value and temperature were optimized to enhance the sensor performance. A high sensitivity and a low detection limit of about 0.5–1 nM adrenaline have been achieved in phosphate buffer at pH 7.4, relevant for measurements in blood samples. The sensitivity of the biosensor to other catecholamines such as noradrenaline, dopamine and dobutamine has been studied. Finally, the sensor has been successfully applied for the detection of adrenaline in human blood plasma.     

Metallated porphyrin noncovalent interaction with reduced graphene oxide‐modified electrode for amperometric detection of environmental pollutant hydrazine

A reduced graphene oxide/platinum(II) tetraphenylporphyrin nanocomposite (RGO/Pt‐TPP)‐modified glassy carbon electrode was developed for the selective detection of hydrazine. The RGO/Pt‐TPP nanocomposite was successfully prepared via noncovalent π–π stacking interaction. The prepared nanocomposite was characterized using nuclear magnetic resonance, electrochemical impedance, ultraviolet–visible and Raman spectroscopies, scanning electron microscopy and X‐ray diffraction. The electrochemical detection of hydrazine was performed via cyclic voltammetry and amperometry. The RGO/Pt‐TPP nanocomposite exhibited good electrocatalytic activity towards detection of hydrazine with low overpotential and high oxidation peak current. The fabricated sensor exhibited a wide linear range from 13 nM to 232 μM and a detection limit of 5 nM. In addition, the fabricated sensor selectively detected hydrazine even in the presence of 500‐fold excess of common interfering ions. The fabricated electrode exhibited good sensitivity, stability, repeatability and reproducibility. In addition, the practical applicability of the sensor was evaluated in various water samples with acceptable recoveries.     

Amperometric Biosensor Based on Immobilization Acetylcholinesterase on Manganese Porphyrin Nanoparticles for Detection of Trichlorfon with Flow‐Injection Analysis System

A highly sensitive amperometric biosensor for the detection of organophosphate pesticides (OPs) is developed. The biosensor was fabricated by immobilized acetylcholinesterase (AChE) on manganese (III) meso‐tetraphenylporphyrin (MnTPP) nanoparticles (NPs)‐modified glassy carbon (GC) electrode. The MnTPP NPs used in this article were synthesized by mixing solvent techniques. AChE enzyme was immobilized on the MnTPP NPs surface by conjugated with chitosan (CHIT). The electrocatalytic activity of MnTPP NPs led to a greatly improved performance for thiocholine (TCh) product detection. The developed AChE‐CHIT/MnTPPNP/GC biosensor integrated with a flow‐injection analysis (FIA) system was used to monitor trichlorfon (typical OP). A wide linear inhibition response for trichlorfon is observed in the range of 1.0 nM–1.0 mM, corresponding to 10–83% inhibition for AChE with a detection limit of 0.5 nM.     

BisPNA‐assisted Detection of Double‐stranded DNA via Electrochemical Impedance Spectroscopy

A highly effective strategy for quantification of plasmid which was a special dsDNA based on bisPNA by electrochemical impedance spectroscopy was presented in this work. Firstly, through Au?S bond, thiol‐terminated bisPNA probes were immobilized onto the gold electrode surface. Then bisPNA probes directly hybridized with target plasmid DNA pBR322 based on the PNA.DNA‐PNA invasion triplex without denaturation. In the presence of redox electroactive ions [Fe(CN)₆]^3?/4? as hybridization indicator, the charge transfer resistance (R_ct) was produced, and R_ct was measured via electrochemical impedance spectroscopy. Under optimal conditions, this strategy showed a good linear relationship between the ΔR_ct which was the difference of R_ct obtained before and after bisPNA hybridized with plasmid pBR322, and logarithm of the concentration of plasmid pBR322 within the range from 1 nM to 100 nM (R²=0.993), with a limit of detection (LOD) of 0.1 nM. Furthermore, this bisPNA‐assisted biosensor showed good stability and satisfactory analytical reliability. In addition, this novel bisPNA‐assisted biosensor also exhibited excellent analytical results in human serum.     

A Peptide Nucleic Acid (PNA)‐DNA Ferrocenyl Intercalator for Electrochemical Sensing

A ferrocenyl intercalator was investigated to develop an electrochemical DNA biosensor employing a peptide nucleic acid (PNA) sequence as capture probe. After hybridization with single strand DNA sequence, a naphthalene diimide intercalator bearing ferrocene moieties (FND) was introduced to bind with the PNA‐DNA duplex and the electrochemical signal of the ferrocene molecules was used to monitor the DNA recognition. Electrochemical impedance spectroscopy was used to characterize the different modification steps. Differential pulse voltammetry was employed to evaluate the electrochemical signal of the FND intercalator related to its interaction with the complementary PNA‐DNA hybrid. The ferrocene oxidation peaks were utilised for the target DNA quantification. The developed biosensor demonstrated a good linear dependence of FND oxidation peak on DNA concentration in the range 1 fM to 100 nM of target DNA, with a low detection limit of 11.68 fM. Selectivity tests were also investigated with a non‐complementary DNA sequence, indicating that the FND intercalator exhibits a selective response to the target PNA‐DNA duplex.     

Biosensor employing screen‐printed PEDOT:PSS for sensitive detection of phenolic compounds in water

Poly(3,4‐ethylenedioxythiophene) (PEDOT) and its derivatives are relatively new, and unique members of conducting‐polymers family. In this article, we present an approach for simple, reliable and cost‐efficient electrochemical biosensor for real‐time detection and quantification of phenolic compounds (PhCs). The PEDOT:poly(styrene sulfonate) (PSS) polymer, directly screen‐printed on the surface of the working electrode, was shown to act as an effective electrical conductor but also as an efficient redox mediator. It has also been found suitable for the reduction of quinone ions at low reducing potentials, close to 0 V versus Ag/AgCl, thus minimizing interferences due to other electroactive species present in real samples. Based on these properties, a biosensor based on tyrosinase immobilized on PEDOT:PSS‐modified electrodes was developed allowing the detection of PhCs in surface waters. The biosensor displayed very good performance in terms of sensitivity, detection limit and linear range. Assays using surface water previously spiked with bisphenol A showed that the biosensor was able to detect PhCs in real conditions with no matrix effect. © 2012 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2012     

A Novel Electrochemical DNA Biosensor Fabricated with Layer‐by‐Layer Covalent Attachment of Multiwalled Carbon Nanotubes and Gold Nanoparticles

A novel DNA biosensor has been fabricated for the detection of DNA hybridization based on layer‐by‐layer (LBL) covalent assembly of gold nanoparticles (GNPs) and multiwalled carbon nanotubes (MWCNTs). The stepwise LBL assembly process was characterized by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The hybridization events were monitored by differential pulse voltammetry (DPV) measurement of the intercalated doxorubicin, and the factors influencing the performance of the DNA hybridization was investigated in detail. The signal was linearly changed with target DNA concentration increased from 0.5 to 0.01 nM, and had a detection limit of 7.5 pM (signal/noise ratio of 3). In addition, the DNA biosensor showed an excellent reproducibility and stability under the DNA‐hybridization conditions.     

Amperometric Glucose Biosensor Based on Electrochemically Deposited Gold Nanoparticles Covered by Polypyrrole

Graphite rod (GR) modified with electrochemicaly deposited gold nanoparticles (AuNPs) and adsorbed glucose oxidase (GOx) was used in amperometric glucose biosensor design. Enzymatic formation of polypyrrole (Ppy) on the surface of GOx/AuNPs/GR electrode was applied in order to improve analytical characteristics and stability of developed biosensor. The linear glucose detection range for Ppy/GOx/AuNPs/GR electrode was dependent on the duration of Ppy‐layer formation and the linear interval was extended up to 19.9 mmol L⁻¹ after 21 h lasting synthesis of Ppy. The sensitivity of the developed biosensor was determined as 21.7 μA mM⁻¹ cm⁻², the limit of detection – 0.20 mmol L⁻¹. Ppy/GOx/AuNPs/GR electrodes demonstrated advanced good stability (the t _1/2 was 9.8 days), quick detection of glucose (within 5 s) in the wide linear interval. Additionally, formed Ppy layer decreased the influence of electroactive species on the analytical signal. Developed biosensor is suitable for the determination of glucose in human serum samples.     

A Novel Hydrogen Peroxide Sensor Based on the Direct Electron Transfer of Catalase Immobilized on Nano‐Sized NiO/MWCNTs Composite Film

MWCNTs‐nanoNiO composite was used as a glassy carbon electrode modifier for construction of a novel catalase nanobiosensor for hydrogen peroxide. The immobilized catalase exhibited excellent electrocatalytic activity towards the reduction of H₂O₂. The resulting amperometric biosensor exhibited a linear response over a concentration range of 200 µM to 2.53 mM with a low detection limit of 19.0 µM. Electrochemical impedance measurements revealed that the modified electrode can be used for the sensitive detection of H₂O₂. The charge transfer resistance found to decrease significantly after enzymatic reaction of nanobiosensor with H₂O₂. The resulting impedance was highly sensitive to H₂O₂ over a linear range of 19–170 nM with a detection limit of 2.4 nM.     

A Sensitive Electrochemical Biosensor for Detection of Histone Deacetylase Activity Using an Acetylated Peptide

A sensitive electrochemical biosensor was developed for activity detection of histone deacetylase sirtuin2 (SIRT2) using an acetylated peptide substrate. This substrate could be recognized by anti‐acetylated peptide antibody, which could be detected using secondary antibody conjugated alkaline phosphatase which provided an amplified electrochemical signal. In the presence of SIRT2, the substrate was deacetylated, resulting in a decreased electrochemical signal that was correlated to the concentration of SIRT2. Under optimized conditions, the biosensor exhibited a wide linear range from 1 nM to 500 nM with a detection limit of 0.1 nM. The proposed biosensor was also used for detection of SIRT2 inhibitor.     

Highly Sensitive Impedimetric Biosensor Based on Thermolysin Immobilized on a GCE Modified with AuNP-decorated Graphene for the Detection of Ochratoxin A

The fabrication of a thermolysin-based biosensor capable of detecting ochratoxin A (OTA) from food samples is described. The electrochemical deposition of calcium cross-linked cellulose film (CCLC) and gold nanoparticles (AuNPs) on graphene (GR) for modification of a glassy carbon electrode (GCE) is the first step. Then the thermolysin (TLN) enzyme in a polyvinyl alcohol (PVA)/polyethylenimine (PEI) matrix is immobilized. The impedimetric biosensor response is linear from 0.2 nM to 100 nM with a detection limit of 0.2 nM. The obtained stable and reproducible biosensor is then applied for the detection of OTA in spiked extracts from coffee beans.     

An Impedimetric Aptasensor Based on a Novel Line‐Pad‐Line Electrode for the Determination of VEGF165

In this work, a novel, simple and label‐free line‐pad‐line electrode (LPLE) biosensor was developed for detection of vascular endothelial growth factor (VEGF₁₆₅). DNA aptamer was used as a recognition element for high specificity to VEGF₁₆₅, and original LPLE as the substrate electrode for high sensitivity of the biosensor. This sensor was prepared by immobilizing anti‐VEGF₁₆₅ aptamers on the LPLE surface through gold‐sulfur (Au?S) bonding. Upon the addition of VEGF₁₆₅, a large target‐induced conformational change in the surface‐immobilized aptamer was generated and caused variations in the interfacial properties,which led to a corresponding increase in the impedance magnitude of the LPLE. Finally, our results demonstrate that the calibration curve for VEGF₁₆₅determination was linear over the range of 0.026‐31.4 fM with a detection limit as low as 0.017 fM.Additionally, our sensor was fabricated on printed circuit board (PCB) with a new electrode construction, and can potentially be implemented with the advantages of simplicity, low‐cost and easy mass production. Besides, considering its desirable sensitivity and specificity, the proposed use of LPLE provided a promising strategy for a wide variety of sensing applications.     

Sensor Array: Impedimetric Label‐Free Sensing of DNA Hybridization in Real Time for Rapid,PCR‐Based Detection of Microorganisms

A novel, impedance‐based electronic sensor format was used for label‐free, real‐time detection of microbial DNA on oligonucleotide probe arrays. Detection limits of 5–10 nM were achieved for single‐stranded, PCR‐amplified DNA targets. Hybridization selectivity yielded 9‐ to 12‐fold signal increases for specific targets, and the sensor arrays were re‐used multiple times without significant signal degradation. These and other features of the SHARP Laboratories of America (SLA) sensor array, such as its ability to acquire continuous measurements of DNA as it accumulates on the array surface, make it an attractive biosensor platform for field detection and monitoring of sentinel and/or pathogenic microorganisms.