nA μM Research Articles

RNA Aptamer-Based Electrochemical Biosensor for Selective and Label-Free Analysis of Dopamine

The inherent redox activity of dopamine enables its direct electrochemical in vivo analysis ( Venton , B. J.; Wightman, M. R. Anal. Chem. 2003, 75, 414A). However, dopamine analysis is complicated by the interference from other electrochemically active endogenous compounds present in the brain, including dopamine precursors and metabolites and other neurotransmitters (NT). Here we report an electrochemical RNA aptamer-based biosensor for analysis of dopamine in the presence of other NT. The biosensor exploits a specific binding of dopamine by the RNA aptamer, immobilized at a cysteamine-modified Au electrode, and further electrochemical oxidation of dopamine. Specific recognition of dopamine by the aptamer allowed a selective amperometric detection of dopamine within the physiologically relevant 100 nM to 5 μM range in the presence of competitive concentrations of catechol, epinephrine, norepinephrine, 3,4-dihydroxy-phenylalanine (L-DOPA), 3,4-dihydroxyphenylacetic acid (DOPAC), methyldopamine, and tyramine, which gave negligible signals under conditions of experiments (electroanalysis at 0.185 V vs Ag/AgCl). The interference from ascorbic and uric acids was eliminated by application of a Nafion-coated membrane. The aptasensor response time was <1 s, and the sensitivity of analysis was 62 nA μM(-1) cm(-2). The proposed design of the aptasensor, based on electrostatic interactions between the positively charged cysteamine-modified electrode and the negatively charged aptamer, may be used as a general strategy not to restrict the conformational freedom and binding properties of surface-bound aptamers and, thus, be applicable for the development of other aptasensors.

Highly Sensitive and Selective Amperometric Detection of Periodate at Glassy Carbon Electrode Modified with a Cyclometalated Iridium(III) Complex and Single‐Wall Carbon Nanotubes

AbstractSingle‐wall carbon nanotubes (SWCNTs) were used as an immobilization matrix to incorporate [Ir(ppy)2(phen‐dione)](PF6) complex onto a glassy carbon electrode for the study of electrocatalytic reduction of periodate ion. Detailed preliminary electrochemical data for the Ir(III)‐complex in acetonitrile solution and for the modified GCE/SWCNTs/[Ir(ppy)2(phen‐dione)](PF6)/CGE are presented. The modified electrode was applied to selective amperometric detection of periodate through its electrocatalytic reduction to iodide at 0.200 V and pH 2.0. The use of amperometry resulted in two calibration plots over the concentration ranges of 1‐20 μM and 20‐450 μM, with a detection limit of 0.6 μM and sensitivity of 198 nA μM−1.

A polydimethylsiloxane electrophoresis microchip with a thickness controllable insulating layer for capacitatively coupled contactless conductivity detection

A polydimethylsiloxane (PDMS) electrophoresis microchip with a thickness-controllable insulating layer for capacitatively coupled contactless conductivity detection is presented. A PDMS film is spin-coated on a glass slide with Pt microelectrodes to be the insulating layer, and then permanently bonded with a PDMS channel plate to make the whole microchip. The thickness of PDMS films can be precisely controlled down to submicrometers. With a microchip with a 0.6μm-thick PDMS insulating layer, a superior limit of detection (LOD) of 0.07μM for Na+ was obtained. For comparison, another two microchips with the same design but different insulating layer thicknesses (15μm and 50μm) were tested, and LODs were 1μM and 3μM respectively, which are almost two orders of magnitude higher. Moreover, the microchip detector also exhibited excellent linearity, reproducibilities and separation efficiencies.

High sensitivity amperometric and voltammetric determination of persulfate with neutral red/nickel oxide nanowires modified carbon paste electrodes

Abstract A novel, highly sensitive sensor for detection of persulfate (S2O82−) is demonstrated by modification of carbon paste electrode (CPE) with nickel oxide nanowires (NiOx NWs) and neutral red (NR). Ni NWs are prepared by DC electrodeposition of Ni in the cylindrical pores of anodized aluminum oxide (AAO) templates. NWs are released, oxidized and immobilized with NR on the surface of CPE. Cyclic voltammetry studies demonstrate a pair of well-defined nearly reversible redox couples (E°′ = −0.2 V in a buffer solution pH 2.0) at a wide pH range of 1–12. The surface coverage and heterogeneous electron transfer rate constant of the adsorbed redox couple are 7.368 × 10−12 mol cm−2 and 7.9 s−1, respectively, indicating a high NR loading efficiency for NiOx NWs and efficient electron transfer between redox couple and the electrode. The modified CPE exhibits an excellent electrocatalytic activity for a S2O82− reduction, with a measured kcat of ∼7.14 × 103 M−1 s−1. This catalytic reduction allows an amperometric detection of S2O82− at a potential of −0.2 V with detection limit of 30 nM, concentration calibration range of 0.1 μM to 12 mM and a linear sensitivity of 647.33 nA μM−1, respectively.

Electrochemical determination of hydrogen peroxide production by isolated mitochondria: A novel nanocomposite carbon–maghemite nanoparticle electrode

Abstract A simple carbon paste electrode modified with novel maghemite (γ-Fe 2 O 3 ) nanoparticles, characterized by a mean diameter of about 10 nm and specific surface properties (called surface active maghemite nanoparticles, SAMNs) has been developed. The electrode is able to catalyze the electro-reduction of hydrogen peroxide at low applied potentials (−0.1 V vs. SCE). In order to improve the electrocatalytic properties of the modified electrode and to protect electrode surface from fouling due to the presence of biological materials, a surfactant, namely cetyltrimethylammonium bromide, was introduced, as monomolecular layer, on electrode surface. At −0.1 V, the sensitivity of the modified nanocomposite electrode was 58 nA μM −1 cm −2 , with a detection limit of 2.78 μM, in the 0.01–1.5 mM H 2 O 2 concentration range, revealing to be one of the most sensitive electrodes described in literature. The resulting electrode was used to determine native monoamine oxidase activity on intact mitochondria suspensions, allowing the determination of catalytic parameters of the enzyme. Following this new electrochemical methodology, we implemented a novel electrochemical assay for the investigation of aerobic metabolism in preparations of isolated mitochondria revealing active H 2 O 2 production by respiring rat liver mitochondria. The present work demonstrates the feasibility of these novel maghemite nanoparticles modified electrode as efficient hydrogen peroxide electro-catalyst, for the application on biological samples at low applied potentials.

Amprometric detection of Glycine, l-Serine, and l-Alanine using glassy carbon electrode modified by NiO nanoparticles

Glassy carbon electrode modified with nickel oxide nanoparticles has been used to investigate the electrochemical oxidation of Glycine, l-Serine, and l-Alanine in an alkaloid solution. The electrochemical behavior of the modified electrode was characterized by cyclic voltammetry in detail. The electrocatalytic behavior is further exploited as a sensitive detection scheme for the above amino acids by hydrodynamic amperometry. Under optimized conditions, the calibration curves are linear in the concentration ranges of 1–200 μM for Glycine, 1–400 μM for l-Serine, and 30–200 μM for l-Alanine, respectively. The respective detection limit (S/N = 3) and sensitivity are 0.9 μM and 24.3 nA μM−1 for Glycine, 0.85 μM and 12.4 nA μM−1 for l-Serine, and 29.67 μM and 0.4 nA μM−1 for l-Alanine. The prepared electrode exhibits a satisfactory stability and long life-time, while it is stored at ambient conditions.

A Biosensor Utilizing l-Glutamate Dehydrogenase and Diaphorase Immobilized on Nanocomposite Electrode for Determination of l-Glutamate in Food Samples

Amperometric biosensor utilizing bienzymatic composition consisting of l-glutamate dehydrogenase and diaphorase for the determination of l-glutamate has been developed. Enzymes were immobilized between chitosan layers onto the surface of planar nanocomposite electrodes consisting of multi-walled carbon nanotubes (diameter = 60–100 nm; length = 5–15 μm, 95+ % purity). Linear response was obtained from 10 to 3,495 μM in phosphate buffer solution of pH 9.0 and in the presence of enzyme cofactor NAD+ (2 mM) and mediator ferricyanide (5 mM). The limit of detection was 5.4 μM, and sensitivity was found to be 28 nA μM−1 cm−2. The biosensor showed a short response time (within 60 s), good storage (no loss of activity for at least 3 months), and operational (response ability above 90 % after 7 days since its first use) stability. Finally, the results obtained from measurements of the food samples were compared with those obtained with an enzymatic–spectrophotometric method and correlated well. Analytical performance of the biosensor indicated that the bienzyme system utilizing diaphorase as a secondary enzyme could be a general basis for other biosensors based on NAD+-dependent dehydrogenases.

Electroanalytical Behavior of Poly-l-Lysine Dendrigrafts at the Interface between Two Immiscible Electrolyte Solutions

In this work, the electrochemical behavior of nonredox-active poly-L-lysine dendrigraft molecules of four different generations was investigated at the interface between two immiscible electrolyte solutions (ITIES). The influence of the dendrigraft generation on the electrochemical response, sensitivity of the calibration curves, and limit of detection was studied. Cyclic voltammetry at the ITIES revealed that the sensitivity increased (1840 to 25 800 nA μM(-1)) and the limit of detection decreased (11.10 to 0.65 μM) as the dendrigraft generation increased from generation G2 through to generation G5, respectively. The results are compared to those for protein voltammetry at the ITIES. Our studies suggest that the sensitivity expected for a synthetic ionized macromolecule can be predicted on the basis of its net charge and its diffusion coefficient. However, electrochemistry at the ITIES demonstrates a greater sensitivity toward proteins, which is attributed to their tertiary structure.

A novel dinuclear Schiff-base copper(ii) complex modified electrode for ascorbic acid catalytic oxidation and determination

A new dinuclear copper salicylaldehyde-glycine Schiff-base complex [Cu(2)(Sal-Gly)(2)(H(2)O)(2)] was synthesized and structurally characterized. [Cu(2)(Sal-Gly)(2)(H(2)O)(2)] crystallized in the monoclinic system in the P2(1)/c space group. The molecule is a dinuclear complex, formed by two [Cu(Sal-Gly)(H(2)O)] units. The electropolymerization properties of the copper complex on a glass carbon electrode were studied at different potential ranges. The electropolymerization occurred when the high scan potential reached 1.4 V. The modified electrode exhibited good electrocatalytic oxidation properties to ascorbic acid and showed a sensitivity of 22.9 nA μM(-1) (r(2) = 0.9998) and detection limit of 0.39 μM (S/N = 3) in the amperometric determination of ascorbic acid. The designed determination method can be used to analyze vitamin C tablets.

Nanomolar detection of p-nitrophenol via in situ generation of p-aminophenol at nanostructured microelectrodes

A new method and a new buffer medium, expected to be practical for miniaturized electrochemical immunoassays, were developed for rapid detection of nanomolar levels of p-nitrophenol (p-NP). The method exploits rapid reduction of p-NP to p-aminophenol (p-AP) by fast scan cyclic voltammetry at 500 V s(-1) at nanostructured carbon fiber microdisk electrodes (~7 μm dia.) fabricated from polyacrylonitrile (PAN) fibers. Large surfaces of the nanostructured microdisks facilitate the rapid reduction of p-NP to p-aminophenol (p-AP), as confirmed by the overlap with the analytical signals of the standards, which is then rapidly detected by fast scan cyclic voltammetry. A new 2-amino-2-(hydroxymethyl)propane-1,3-diol (Tris)-HAc buffer medium was developed in order to allow adaptation of this detection strategy to alkaline phosphatase (ALP)-based immunoassays. Tris-HAc is a stable medium, while the traditional Tris-HCl buffer medium produces large residual faradaic currents attributed to chloride oxidation. Addition of sodium acetate to a Tris-HAc buffer medium allows sensitivity enhancement by a factor of 2 to 0.85 nA μM(-1), similar to the best sensitivity reported at the nanostructured PAN carbon fiber microdisk sensors.

A hydrogen peroxide sensor based on the nanocomposites of poly(brilliant cresyl blue) and single walled-carbon nanotubes

A poly(brilliant cresyl blue) (PBCB) functionalized single walled-carbon nanotube (SWCNT) modified glassy carbon electrode (PBCB/SWCNT/GCE) is fabricated by electropolymerization. The electrocatalytic activity of the PBCB/SWCNT/GCE towards the reduction of hydrogen peroxide is then investigated. The electrode shows high sensitivity [4.1 nA (μM) −1] to the electrocatalytic reduction of H2O2 at an applied potential of −0.30 V (vs. SCE). Important practical advantages such as a fast response time (within 5 s), a low detection limit (0.12 μM), wide linear dynamic range (4.9 × 10−7 to 2.3 × 10−4 M, R2 = 0.994) and stability (remains ∼93% of its original activity after 30 days) are achieved with the PBCB/SWCNT/GCE. Further, the presence of probable interferences such as glucose, ascorbic acid, dopamine and uric acid does not interfere during the detection of H2O2. The application of the sensor to determine H2O2 in tap water with recovery values of 96% to 102% shows the sensor is suitable for routine analysis.

Visible Photolysis and Amperometric Detection of S-Nitrosothiols

The concentration of S-nitrosothiols (RSNOs), endogenous transporters of the signaling molecule nitric oxide (NO), fluctuate greatly in physiology often as a function of disease state. RSNOs may be measured indirectly by cleaving the S-N bond and monitoring the liberated NO. While ultraviolet photolysis and reductive-based cleavage both decompose RSNOs to NO, poor selectivity and the need for additional reagents preclude their utility clinically. Herein, we report the coupling of visible photolysis (i.e., 500-550 nm) and amperometric NO detection to quantify RSNOs with greater selectivity and sensitivity. Enhanced sensitivity (up to 1.56 nA μM(-1)) and lowered theoretical detection limits (down to 30 nM) were achieved for low molecular weight RSNOs (i.e., S-nitrosoglutathione, S-nitrosocysteine) by tuning the irradiation exposure. Detection of nitrosated proteins (i.e., S-nitrosoalbumin) was also possible, albeit at a decreased sensitivity (0.11 nA μM(-1)). This detection scheme was used to measure RSNOs in plasma and illustrate the potential of this method for future physiological studies.

Multienzymatic amperometric biosensor based on gold and nanocomposite planar electrodes for glycerol determination in wine

Amperometric biosensors based on gold planar or nanocomposite electrode containing multiwalled carbon nanotubes for determination of glycerol were developed. The biosensors were constructed by immobilization of a novel multienzyme cascade consisting of glycerol kinase/creatine kinase/creatinase/sarcosine oxidase/peroxidase between a chitosan “sandwich.” A measuring buffer contained adenosine 5′-triphosphate (ATP), creatine phosphate, and an artificial electrochemical mediator ferrocyanide. The currents proportional to glycerol concentration were measured at working potential of −50 mV against Ag/AgCl reference electrode. The biosensors showed linearity over the ranges of 5–640 μM and 5–566 μM with detection limits of 1.96 and 2.24 μM and sensitivities of 0.80 and 0.81 nA μM −1, respectively. Both types of biosensors had a response time of 70 s. The biosensors demonstrated satisfactory operational stability (no loss of sensitivity after 90 consecutive measurements) and excellent storage stability (90% of the initial sensitivity after 15 months of storage at room temperature). The results obtained from measurements of wines correlated well with those obtained with an enzymatic–spectrophotometric assay. The presented multienzyme cascade can be used also for determination of triglycerides or various kinase substrates when glycerol kinase is replaced by other kinases.

Sensitive amperometric detection of omeprazole and pantoperazole at electrodeposited nickel oxide nanoparticles modified glassy carbon electrode

Glassy carbon electrode modified with electrodeposited nickel oxide nanoparticles (NiOxNPs) was used as electrocatalyst for oxidation of omeprazole and pentoperazole in alkaline solution. The modified electrode exhibited efficient electrocatalytic activity for the oxidation of omeprazole and pentoperazole with relatively high sensitivity, excellent stability, and long lifetime. Hydrodynamic amperometric method is used for determination of selected analytes. Under optimized condition, the linear concentration range, detection limit, and sensitivity of modified electrode toward omeprazole detection are 4.5–120 μM, 0.4 μM (at signal to noise 3), and 40.1 nA μM−1 cm−2, respectively. For pantoperazole, hydrodynamic amperometric determination yielded calibration curve with linear range of 2.5–180 μM, detection limit of 0.2 μM, and sensitivity of 39.2 nA μM−1 cm−2, respectively. The proposed method was successfully applied to pentoperazole and omeprazole determination in drug samples.

Immobilization of lutetium bisphthalocyanine in nanostructured biomimetic sensors using the LbL technique for phenol detection

This study describes the development of amperometric sensors based on poly(allylamine hydrochloride) (PAH) and lutetium bisphthalocyanine (LuPc 2) films assembled using the Layer-by-Layer (LbL) technique. The films have been used as modified electrodes for catechol quantification. Electrochemical measurements have been employed to investigate the catalytic properties of the LuPc 2 immobilized in the LbL films. By chronoamperometry, the sensors present excellent sensitivity (20 nA μM −1) in a wide linear range ( R 2 = 0.994) up to 900 μM and limit of detection ( s/ n = 3) of 37.5 × 10 −8 M for catechol. The sensors have good reproducibility and can be used at least for ten times. The work potential is +0.3 V vs. saturated calomel electrode (SCE). In voltammetry measurements, the calibration curve shows a good linearity ( R 2 = 0.992) in the range of catechol up to 500 μM with a sensitivity of 90 nA μM −1 and LD of 8 μM.

Azure A Mediated Polypyrrole‐Based Amperometric Nitrate Biosensor

AbstractThe co‐entrapment of Aspergillus niger‐nitrate reductase (NaR) and azure A (AzA), as a redox mediator has been investigated for fabrication of a polypyrrole (PPy)‐based electron mediated amperometric nitrate biosensor. Optimum formation of PPy‐NaR‐AzA film was achieved in a monomer solution which contained 0.3 M Py, 0.1 M KCl, 1U/mL NaR, 50 µM AzA with an applied current density of 0.5 mA/cm2 for 240 s. The optimum applied potential of −0.25 V for amperometric detection of nitrate with PPy‐NaR‐AzA biosensor was 50, 100 and 150 mV more positive than those achieved with the use of safranin O (Saf), methyl viologen (MV) and bromophenol blue (BrB) or red (BrR), respectively, as mediators. Similar optimum applied of −0.25 V was achieved with thionin acetate (ThAc) which was in the same phenothiazine group of mediators as AzA. The electron mediated amperometric detection of nitrate with the PPy‐NaR‐AzA biosensor also gave the best performance with a sensitivity of 40.8 nA µM−1 cm−2, detection limit of 0.5 µM, a response time of 18–20 s and a linear concentration range of 20–500 µM.

Multiwalled Carbon‐Nanotube‐Functionalized Microelectrode Arrays Fabricated by Microcontact Printing: Platform for Studying Chemical and Electrical Neuronal Signaling

A facile method is proposed for the deposition of multiwalled carbon nanotube (MWCNT) layers onto microelectrode arrays by means of a microcontact printing technique, leading to the fabrication of MEAs characterized by well defined electrical and morphological properties. Using polydimethyl siloxane stamps, produced from different mold designs, a flexibility of printing is achieved that provides access to microscale, nanostructured electrodes. The thickness of MWCNT layers can be exactly predetermined by evaluating the concentration of the MWCNT solution employed in the process. The electrode morphology is further characterized using laser scanning and scanning electron microscopy. Next, by means of impedance spectroscopy analysis, the MWCNT-electrode contact resistance and MWCNT film resistance is measured, while electrochemical impedance spectroscopy is used to estimate the obtained electrode-electrolyte interface. Structural and electrochemical properties make these electrodes suitable for electrical stimulation and recording of neurons and electrochemical detection of dopamine. MWCNT-functionalized electrodes show the ability to detect micromolar amounts of dopamine with a sensitivity of 19 nA μm(-1) . In combination with their biosensing properties, preliminary electrophysiological measurements show that MWCNT microelectrodes have recording properties superior to those of commercial TiN microelectrodes when detecting neuronal electrical activity under long-term cell-culture conditions. MWCNT-functionalized microelectrode arrays fabricated by microcontact printing represent a versatile and multipurpose platform for cell-culture monitoring.

Direct Voltammetry of Copper, Zinc‐Superoxide Dismutase Immobilized onto Electrodeposited Nickel Oxide Nanoparticles: Fabrication of Amperometric Superoxide Biosensor

AbstractDirect electron transfer of immobilized copper, zinc‐superoxide dismutase (SOD) onto electrodeposited nickel‐oxide (NiOx) nanoparticle modified glassy carbon (GC) electrode displays a well defined redox process with formal potential of −0.03 V in pH 7.4. Cyclic voltammetry was used for deposition of (NiOx) nanoparticles and immobilization of SOD onto GC electrode. The surface coverage (Γ) and heterogeneous electron transfer rate constant (ks) of immobilized SOD are 1.75×10−11 mol cm−2 and 7.5±0.5 s−1, respectively. The biosensor shows a fast amperometric response (3 s) toward superoxide at a wide concentration range from 10 µM to 0.25 mM with sensitivity of 13.40 nA µM−1 cm−2 and 12.40 nA µM−1 cm−2, detection limit of 2.66 and 3.1 µM based on anodically and cathodically detection. This biosensor exhibits excellent stability, reproducibility and long life time.

Electrocatalytic activity of carbon spheres towards NADH oxidation at low overpotential and its applications in biosensors and biofuel cells

The excellent electrocatalytic activity of a micro-structured carbon material, carbon hollow spheres (CS), to the oxidation of dihydronicotinamide adenine dinucleotide (NADH) is demonstrated here. Compared to conventional bare glassy carbon electrodes, a substantial decrease by 450 mV in the overpotential of NADH electrooxidation was observed using CS coatings, with oxidation starting at ca. −0.10 V (vs.Ag/AgCl, pH 7.0). The CS-coated glassy carbon electrode (CS/GC) thus allows highly sensitive and direct amperometric detection of NADH at lower potential, ranging from 0.20 to 100 μM with a high sensitivity of 7.3 ± 0.2 nA μM−1 (i.e., 103.3 ± 2.8 nA μM−1 cm−2), low detection limit of 0.08 ± 0.03 μM, and minimization of surface fouling. With lactate dehydrogenase (LDH) as a model, a lactate biosensor with the LDH-CS/GC electrode was constructed and the biosensor shows rapid and highly sensitive amperometric response to lactate ranging from 0.5 to 12 μM with a detection limit of 3.7 ± 0.2 μM, a sensitivity of 4.1 ± 0.2 nA μM−1 (i.e., 57.9 ± 2.8 nA μM−1 cm−2), good reproducibility and excellent stability. Furthermore, the promoted direct electron transfer (DET) of bilirubin oxidase (BOD) on CS/GC electrode was investigated, and thus a membrane-less lactate/oxygen biofuel cell was assembled by using LDH-CS/GC as bioanode for lactate oxidation and BOD-CS/GC electrode for oxygen reduction, with a high open-circuit potential of 0.60 V. Such ability of CS to decrease the NADH oxidation overpotential and promote DET of blue-copper oxidases suggests great promise for dehydrogenase-based amperometric biosensors and biofuel cells.

Label-free Electrochemical Biosensor Based on Graphene/Ionic Liquid Nanocomposite for the Detection of Organophosphate Pesticides

ABSTRACTThe advanced electrodes for detecting organophosphate pesticides were prepared by modification of the gold (Au) electrode with the reduced graphene oxide/ionic liquid (RGO/IL) nanohybrids. Due to the cationic and anionic parts, the ILs on RGO sheets provide the amount of functional groups for dispersion of hybrids and immobilization of organophosphorus hydrolase (OPH) enzymes. After the immobilization of OPH on the RGO/IL-modified Au electrodes, the modified electrodes represent faster electron transfer than that of Au electrode, resulting in high performance of biosensor with response time (~ 10 s) and sensitivity (4.56 nA μM−1). In addition, the OPH/RGO/IL-modified Au electrode displayed good stability and reproducibility.