A glassy carbon electrode modified with graphene and tyrosinase immobilized on platinum nanoparticles for sensing organophosphorus pesticides

An amperometric biosensor is described for the detection of organophosphorus pesticides. It is based on the enzyme tyrosinase immobilized on platinum nanoparticles and the use of a glassy carbon electrode modified with graphene. Tyrosinase was immobilized on the electrode surface via electrostatic interaction between a monolayer of cysteamine and the enzyme. In the presence of catechol as a substrate, the pesticides chlorpyrifos, profenofos and malathion can be determined as a result of their inhibition of the enzyme which catalyzes the oxidation of catechol to o-quinone. Platinum nanoparticles and graphene effectively enhance the efficiency of the electrochemical reduction of o-quinone, thus improving sensitivity. Under optimum experimental conditions, the inhibition effect of the pesticides investigated is proportional to their concentrations in the lower ppb-range. The detection limits are 0.2, 0.8 and 3?ppb for chlorpyrifos, profenofos and malathion, respectively. The biosensor displays good repeatability and acceptable stability.

Electrochemical sensor for monitoring the photodegradation of catechol based on DNA-modified graphene oxide

We have immobilized DNA on a glassy carbon electrode (GCE) modified with graphene oxide (GO) to develop an electrochemical biosensor for catechol. Compared to carbon nanotubes, the use of GO dramatically improved the electrooxidative current of the guanine and adenine moieties in DNA but retained the low background current of unmodified GCEs. Factors such as DNA adsorption time, DNA concentration and pH of solution were investigated to optimize experimental conditions. In the presence of catechol, the voltammetric response to DNA was inhibited due to the interaction between DNA and catechol. The response to adenine is linearly proportional to the concentration of catechol in the range from 1.0?×?10^?6 to 1.0?×?10^?4 mol·L^?1. If catechol is degraded by the combined action of UV light and hydrogen peroxide, the response to DNA is restored. Thus, the modified electrode can act as an efficient biosensor for monitoring the degradation of catechol.

Biocentri-voltammetric biosensor for acetylcholine and choline

We report on the determination of choline and acetylcholine via biocentrivoltammetry. This method combines centrifugation and voltammetry and is based on a carbon paste electrode modified with acetylcholinesterase and choline oxidase. The electrode was placed at the bottom of a biocentrivoltammetric cell. Acetylcholine and choline are accumulated on the enzyme electrode via centrifugative forces, upon which a direct voltammetric scan is applied. Reaction time, pH values, quantities of enzyme and centrifugation parameters were optimized. A linear response is obtained in the 0.07 to 10?μM concentration range of acetylcholine, and a limit of detection as low as 0.5?μM. The linear range is between 0.1 and 500?μM for choline. The method was applied to the determination of acetylcholine and choline in spiked serum samples.

Electrochemical sensing of hydrogen peroxide using metal nanoparticles: a review

We are reviewing the state of electrochemical sensing of H₂O₂ based on the use of metal nanoparticles. The article is divided into subsections on sensors based on nanoparticles made from Ag, Pt, Pd, Cu, bimetallic nanoparticles and other metals. Some sensors display high sensitivity, fast response, and good stability. The review is subdivided into sections on sensors based on heme proteins and on nonenzymatic sensors. We also discussed the challenges of nanoscaled sensors and their future aspects.

Synthesis of multi-branched gold nanoparticles by reduction of tetrachloroauric acid with Tris base, and their application to SERS and cellular imaging

A biosensor for hydrogen peroxide was constructed by immobilizing horseradish peroxidase on chitosan-wrapped NiFe₂O₄ nanoparticles on a glassy carbon electrode (GCE). The electron mediator carboxyferrocene was also immobilized on the surface of the GCE. UV?Cvis spectra, Fourier transform IR spectra, scanning electron microscopy, and electrochemical impedance spectra were acquired to characterize the biosensor. The experimental conditions were studied and optimized. The biosensor responds linearly to H₂O₂ in the range from 1.0?×?10^?5 to 2.0?×?10^?3?M and with a detection limit of 2.0?×?10^?6?M (at S/N?=?3).

An innovative strategy for direct electrochemical detection of microRNA biomarkers

We report an electrochemical method for direct, reagentless, and label-free detection of microRNA, based on a conjugated copolymer, poly(5-hydroxy-1,4-naphthoquinone-co-5-hydroxy-2-carboxyethyl-1,4-naphthoquinone), acting as hybridization transducer. Hybridization between the oligonucleotide capture probe and a microRNA target of 22 base pairs generates an increase in the redox current (“signal-on”), which is evidenced by square wave voltammetry. Selectivity is good, with little hybridization for non-complementary targets, and the limit of detection reaches 650 fM. It is also evidenced that this sensitivity benefits from the high affinity of DNA for RNA.

Overcoming the adverse effects of crosslinking in biosensors via addition of PEG: Improved sensing of hydrogen peroxide using immobilized peroxidase

Glutaraldehyde (GA) is widely used as a crosslinker to immobilize enzymes, for examples in biosensors, but often causes partial denaturation. We find that the proper use of poly(ethylene glycol) (PEG) during the crosslinking process can fully preserve the native state and activity of horseradish peroxidase (HRP). An amperometric biosensor was developed based on these findings for the direct determination of hydrogen peroxide. UV-Vis and FTIR spectroscopy reveal that the HRP entrapped in a polypyrrole matrix retains its native structure. The addition of PEG increases the sensitivity and stability of the biosensor and prevents many of effects caused by intra-crosslinking via GA. The biosensor was operated at a potential of ?350?mV (vs Ag/AgCl) without any mediator and gave a linear response to H₂O₂ in the 5 to 190???M concentration range. The apparent Michaelis-Menten constant is 3.37?mM, and maximal current is as high as 3.43???A. The surface of the biosensor was characterized by atomic force microscopy operated in the tapping mode.

A silica-dextran nanocomposite as a novel matrix for immobilization of horseradish peroxidase, and its application to sensing hydrogen peroxide

We report on a novel matrix of solgel organic–inorganic nanocomposite that was fabricated from silica sol gel and dextran. It was used for the immobilization of horseradish peroxidase (HRP) to give a biosensor for hydrogen peroxide (H₂O₂). The sensor film was characterized by Fourier transform infrared and UV–vis spectroscopy with respect to structural features and the conformation of the enzyme. The topographies of the surface of the electrode were investigated by field emission scanning electron microscopy. The biosensor was used to determine H₂O₂ quantitatively in the presence of Methylene blue as a mediator with high electron transfer efficiency. A pair of stable and well defined quasi-reversible redox peaks of the HRP [Fe (III)]/HRP [Fe (II)] redox couple was observed at pH 7.0. The biosensor responds to H₂O₂ in the 0.5 mM to 16.5 mM concentration range, and the limit of detection is 0.5 mM.

Highly sensitive fluorescent sensor for copper (II) based on amplified fluorescence quenching of a water-soluble NIR emitting conjugated polymer

We have synthesized the near-infrared water-soluble conjugated polymer poly[2,5-di(propyloxysulfonate)-1,4-phenylene-ethynylene-9,10-anthrylene (referred to as PPEASO3). Its fluorescence (at wavelengths between 650 and 800?nm following photoexcitation at 550?nm) is efficiently quenched by Cu(II) ions, while other physiologically relevant metal ions do not cause significant quenching at the same concentrations. Under optimum conditions, fluorescence intensity is inversely proportional to the concentration of Cu (II). The calibration curve displays two linear regions over the range of 0–3.2?×?10^?7 mol L^?1 and 3.2?×?10^?7 mol L^?1 to 1.0?×?10^?4 mol L^?1 of Cu(II), respectively. The long-wavelength excitation and emission can substantially reduce interferences by the autofluorescence and light scattering of biological matter under UV excitation. The method was successfully applied to the determination of Cu(II) in synthetic and tea samples.

Ultra-sensitive electrochemical DNA biosensor based on signal amplification using gold nanoparticles modified with molybdenum disulfide,graphene and horseradish peroxidase

We have developed an ultra-sensitive electrochemical DNA biosensor by assembling probe ssDNA on a glassy carbon electrode modified with a composite made from molybdenum disulfide, graphene, chitosan and gold nanoparticles. A thiol-tagged DNA strand coupled to horseradish peroxidase conjugated to AuNP served as a tracer. The nanocomposite on the surface acts as relatively good electrical conductor for accelerating the electron transfer, while the enzyme tagged gold nanoparticles provide signal amplification. Hybridization with the target DNA was studied by measuring the electrochemical signal response of horseradish peroxidase using differential pulse voltammetry. The calibration plot is linear in the 5.0?×?10^?14 and 5.0?×?10^?9 M concentration range, and the limit of detection is 2.2?×?10^?15 M. The biosensor displays high selectivity and can differentiate between single-base mismatched and three-base mismatched sequences of DNA. The approach is deemed to provide a sensitive and reliable tool for highly specific detection of DNA.

Enzyme-Coupled Nanoparticles-Assisted Laser Desorption Ionization Mass Spectrometry for Searching for Low-Mass Inhibitors of Enzymes in Complex Mixtures

In this report, enzyme-coupled magnetic nanoparticles (EMPs) were shown to be an effective affinity-based tool for finding specific interactions between enzymatic targets and the low-mass molecules in complex mixtures using classic MALDI-TOF apparatus. EMPs used in this work act as nonorganic matrix enabling ionization of small molecules without any interference in the low-mass range (enzyme-coupled nanoparticles-assisted laser desorption ionization MS, ENALDI MS) and simultaneously carry the superficial specific binding sites to capture inhibitors present in a studied mixture. We evaluated ENALDI approach in two complementary variations: ‘ion fading’ (IF-ENALDI), based on superficial adsorption of inhibitors and ‘ion hunting’ (IH-ENALDI), based on selective pre-concentration of inhibitors. IF-ENALDI was applied for two sets of enzyme–inhibitor pairs: tyrosinase–glabridin and trypsin–leupeptin and for the real plant sample: Sparrmannia discolor leaf and stem methanol extract. The efficacy of IH-ENALDI was shown for the pair of trypsin–leupeptin. Both ENALDI approaches pose an alternative for bioassay-guided fractionation, the common method for finding inhibitors in the complex mixtures.

Rapid detection of Staphylococcus aureus via a sensitive DNA hybridization assay based on a long-lifetime luminescent europium marker

A two-probe tandem nucleic acid hybridization assay for detection of Staphylococcus aureus is presented. It is based on a europium(III) complex as a marker that has a long fluorescence lifetime, high quantum yield and can be easily conjugated to an oligonucleotide signaling probe. The amino-modified capture probe was associated with the signaling probe to form a two-probe tandem DNA pattern that is complementary to the target DNA. The method was optimized in terms of hybridization temperature, hybridization time and washing time. This resulted in good specificity and sensitivity when detecting such bacteria in food samples.

Glucose biosensor based on glucose oxidase immobilized on unhybridized titanium dioxide nanotube arrays

A glucose biosensor has been fabricated by immobilizing glucose oxidase (GOx) on unhybridized titanium dioxide nanotube arrays using an optimized cross-linking technique. The TiO₂ nanotube arrays were synthesized directly on a titanium substrate by anodic oxidation. The structure and morphology of electrode material were characterized by X-ray diffraction and scanning electron microscopy. The electrochemical performances of the glucose biosensor were conducted by cyclic voltammetry and chronoamperometry measurements. It gives a linear response to glucose in the 0.05 to 0.65 mM concentration range, with a correlation coefficient of 0.9981, a sensitivity of 199.6 μA mM^?1 cm^?2, and a detection limit as low as 3.8 µM. This glucose biosensor exhibited high selectivity for glucose determination in the presence of ascorbic acid, sucrose and other common interfering substances. This glucose biosensor also performed good reproducibility and long-time storage stability. This optimized cross-linking technique could open a new avenue for other enzyme biosensors fabrication.

Magnetic particles–based biosensor for biogenic amines using an optical oxygen sensor as a transducer

We have developed a fibre optic biosensor with incorporated magnetic microparticles for the determination of biogenic amines. The enzyme diamine oxidase from Pisum sativum was immobilized either on chitosan-coated magnetic microparticles or on commercial microbeads modified with a ferrofluid. Both the immobilized enzyme and the ruthenium complex were incorporated into a UV-cured inorganic–organic polymer composite and deposited on a lens that was connected, by optical fibres, to an electro-optical detector. The enzyme catalyzes the oxidation of amines under consumption of oxygen. The latter was determined by measuring the quenched fluorescence lifetime of the ruthenium complex. The limits of detection for the biogenic amines putrescine and cadaverine are 25–30 μmol?L^?1, and responses are linear up to a concentration of 1 mmol L^?1.

Amperometric hydrogen peroxide biosensor based on a glassy carbon electrode modified with polythionine and gold nanoparticles

We report on a novel hydrogen peroxide biosensor that was fabricated by the layer-by-layer deposition method. Thionine was first deposited on a glassy carbon electrode by two-step electropolymerization to form a positively charged surface. The negatively charged gold nanoparticles and positively charged horseradish peroxidase were then immobilized onto the electrode via electrostatic adsorption. The sequential deposition process was characterized using electrochemical impedance spectroscopy by monitoring the impedance change of the electrode surface during the construction process. The electrochemical behaviour of the modified electrode and its response to hydrogen peroxide were studied by cyclic voltammetry. The effects of the experimental variables on the amperometric determination of H₂O₂ such as solution pH and applied potential were investigated for optimum analytical performance. Under the optimized conditions, the biosensor exhibited linear response to H₂O₂ in the concentration ranges from 0.20 to 1.6?mM and 1.6 to 4.0?mM, with a detection limit of 0.067?mM (at an S/N of 3). In addition, the stability and reproducibility of this biosensor was also evaluated and gave satisfactory results.

Dendrimer-based biosensor for chemiluminescent detection of DNA hybridization

We report on a highly sensitive chemiluminescent (CL) biosensor for the sequenc-specific detection of DNA using a novel bio barcode DNA probe modified with gold nanoparticles that were covered with a dendrimer. The modified probe is composed of gold nanoparticles, a dendrimer, the CL reagent, and the DNA. The capture probe DNA was immobilized on magnetic beads covered with gold. It first hybridizes with the target DNA and then with one terminal end of the signal DNA on the barcoded DNA probe. CL was generated by adding H₂O₂ and Co(II) ions as the catalyst. The immobilization of dendrimer onto the gold nanoparticles can significantly enhance sensitivity and gives a detection limit of 6 fmol L^-1 of target DNA.

Aptamer-based molecular recognition for biosensor development

Nucleic acid aptamers are an emerging class of synthetic ligands and have recently attracted significant attention in numerous fields. One is in biosensor development. In principle, nucleic acid aptamers can be discovered to recognize any molecule of interest with high affinity and specificity. In addition, unlike most ligands evolved in nature, synthetic nucleic acid aptamers are usually tolerant of harsh chemical, physical, and biological conditions. These distinguished characteristics make aptamers attractive molecular recognition ligands for biosensing applications. This review first concisely introduces methods for aptamer discovery including upstream selection and downstream truncation, then discusses aptamer-based biosensor development from the viewpoint of signal production.

Solid-phase extraction of mercury(II) with magnetic core-shell nanoparticles, followed by its determination with a rhodamine-based fluorescent probe

Magnetic Fe₃O₄@SiO₂ core shell nanoparticles containing diphenylcarbazide in the shell were utilized for solid phase extraction of Hg(II) from aqueous solutions. The Hg(II) loaded nanoparticles were then separated by applying an external magnetic field. Adsorbed Hg(II) was desorbed and its concentration determined with a rhodamine-based fluorescent probe. The calibration graph for Hg(II) is linear in the 60 nM to 7.0 μM concentration range, and the detection limit is at 23 nM. The method was applied, with satisfying results, to the determination of Hg(II) in industrial waste water.

Development of a stable biosensor based on a SiO2 nanosheet–Nafion–modified glassy carbon electrode for sensitive detection of pesticides

SiO₂ nanosheets (SNS) have been prepared by a chemical method using montmorillonite as raw material and were characterized by scanning electron microscopy and X-ray diffraction. SiO₂ nanosheet–Nafion nanocomposites with excellent conductivity, catalytic activity, and biocompatibility provided an extremely hydrophilic surface for biomolecule adhesion. Chitosan was used as a cross-linker to immobilize acetylcholinesterase (AChE), and Nafion was used as a protective membrane to efficiently improve the stability of the AChE biosensor. The AChE biosensor showed favorable affinity for acetylthiocholine chloride and catalyzed the hydrolysis of acetylthiocholine chloride with an apparent Michaelis–Menten constant of 134 μM to form thiocholine, which was then oxidized to produce a detectable and fast response. Based on the inhibition by pesticides of the enzymatic activity of AChE, detection of the amperometric response from thiocholine on the biosensor is a simple and effective way to biomonitor exposure to pesticides. Under optimum conditions, the biosensor detected methyl parathion, chlorpyrifos, and carbofuran at concentrations ranging from 1.0?×?10^?12 to 1?×?10^?10?M and from 1.0?×?10^?10 to 1?×?10^?8?M. The detection limits for methyl parathion, chlorpyrifos, and carbofuran were 5?×?10^?13?M. The biosensor developed exhibited good sensitivity, stability, reproducibility, and low cost, thus providing a new promising tool for analysis of enzyme inhibitors.

A novel functional imidazole fluorescent ionic liquid: simple and efficient fluorescent probes for superoxide anion radicals

Novel imidazole fluorescent ionic liquids with anthracene groups (ImS-FILA) were synthesized for the first time to act as fluorescent probes. They were developed for the determination of superoxide anion radicals (O₂ ^?-) in an aqueous system. O₂ ^?- was produced by pyrogallol autoxidation. The fluorescence of ImS-FILA was quenched by superoxide anion radicals. The π-bond structure of the fluorescent molecules was oxidized and damaged. This method is very simple and sensitive. The linear range of sensitivity was 1–70 μM ImS-FILA, and the detection limit for reactive oxygen species was 0.1 μM. This method was used to detect superoxide radicals in papaya and garlic, with satisfactory results. Further work is needed to demonstrate the utility of this method in detecting reactive oxygen species in a biological aqueous system.