Enzyme sensor for L-lactate with a chitosan-mercury film electrode

Summary An amperometric enzyme sensor composed of a mercury film electrode and an enzyme-immobilized chitosan membrane is developed. This biosensor is based on both a mercury film electrode detecting the consumption of dissolved dioxygen following enzymatic reaction, and a chitosan membrane. The latter provides an excellent permselectivity and excludes electroactive interferents. The detection range of this biosensor was 1.0×10^–5–3.0×10^–4 mol/l and the relative standard deviation, R.S.D. at 5.0×10^–5 mol/l was 1.4% (n=3). This biosensor was applied to the direct determination of L-lactate in human serum without pretreatment.     

Zirconia-based amperometric sensor using ZnO sensing-electrode for selective detection of propene

The sensing characteristics to propene (C₃H₆) were examined at 600 °C under wet condition for the amperometric sensor using a yttria-stabilized zirconia (YSZ) tube and ZnO (+8.5 wt%Pt) sensing-electrode (SE). In order to improve the sensitivity to C₃H₆, the “pulsed-potential method” was adopted here. It was found that the current response varied almost linearly with C₃H₆ concentration in the range of 0–200 ppm when SE was polarized at +1.0 V (vs. Pt/air reference electrode) for a period of 0.3 s. By using the present “pulsed-potential method”, the sensitivity to 100 ppm C₃H₆ was increased about 1000 times, compared with the normal “constant-potential method”. The excellent selectivity to C₃H₆ was also obtained for the present sensor without influence of other hydrocarbons, NO_x, CO, H₂, etc.     

Electrochemical detection of perchloroethylene using differential pulse voltammetry

We demonstrate the application of differential pulse voltammetry (DPV) for the electrochemical detection of perchloroethylene (PCE) on an unmodified glassy carbon electrode surface. Detection sensitivity was substantially improved using DPV, in which dechlorination was denoted by a cathodic peak observed at approximately − 0.6 V (vs Ag/AgCl). Peak current intensity was found to correlate linearly with concentration over a tested range of 0 to 10 μM. The utility of this technique was subsequently evaluated for PCE-spiked environmental samples containing either Methylobacterium adhaesivum (1 × 10⁶ cells/mL) or creek water (10% v/v). In all environmental samples, a linear dynamic range was also observed from approximately 0 to 10 μM. The limit of detection was determined to be 0.3 μM in blank buffer, 0.4 μM in bacteria-containing samples and 1.2 μM in creek water samples.     

Study of a glassy carbon electrode in amperometric detection using d.c., normal and differential pulse techniques

The results of a comparative study on d.c., normal pulse and differential pulse techniques applied to anodic amperometric detection at a glassy carbon electrode in a voltammetric flow-through cell are presented. The important aspects examined are response time, linearity, limit of detection and selectivity. It is shown that the d.c. mode is the most favourable as long as no adsorption of oxidation products takes place. If strong adsorption occurs, normal pulse detection is recommended, although the limit of detection is somewhat larger.     

Sol-gel based amperometric biosensor incorporating an osmium redox polymer as mediator for detection of L-lactate

A novel amperometric biosensor for the determination of lactate was constructed by first immobilizing lactate oxidase and an osmium redox polymer ([Os(bpy)(2)(PVP)(10)Cl]Cl; abbreviated Os-polymer) on the surface of a glassy carbon electrode, followed by coating with a sol-gel film derived from methyltriethoxysilane (MTEOS). The electrooxidation current of this electrode was found to be diffusion controlled. In the presence of lactate, a clear electrocatalytic oxidation wave was observed, and lactate could be determined amperometrically at 400 mV versus Ag AgCl . The concentration range of linear response, slope of linear response and detection limit were 0.1-9 mM, 1.02 microA mM(-1), and 0.05 mM, respectively. Although L-ascorbate was electrooxidized at this potential, uric acid, paracetamol and glucose were found not to interfere.     

Highly sensitive differential pulse amperometric detection of second generation anti-tumor platinum compounds in HPLC effluents

Summary A highly sensitive on-line amperometric detection of platinum compounds in HPLC effluents was possible with the use of a polarographic detector. For TNO-1 (=cis-1,1-di(aminomethyl)-cyclohexane Pt(II)chloride) a linear dynamic range of at least three decades could be obtained (0.034–108g TNO-1/ml) with a detection limit of three times the noise signal at 0.7 ng TNO-1 (20ng Pt/ml). The sensitivity was 0.1 nA/ng TNO-1 (0.2nA/ng Pt). The within-day presicion was 1.1% at a concentration of 10.8g TNO-1/ml (n=10). With this system the compatibility was measured of TNO-6 with infusion fluids. In 0.15 M NaCl, TNO-6 (=cis-1,1-di(aminomethyl)-cyclohexanePt(II)sulphate) was converted into TNO-1 within 40 minutes. Molecular changes of TNO-6 were also observed in a 5% aqueous glucose solution.     

A comparison of differential pulse and d.c. amperometric detection modes for the liquid chromatographic determination of oxalic acid

Oxalic acid determinations are made using two modes of electrochemical detection, namely, classical d.c. and differential pulse, after separation by ion-pair, ion exchange high performance liquid chromatography. Peak height ratio plots are constructed and compared for uric acid and oxalic acid mixtures using both electrochemical detection modes. The enhanced selectivity realized by use of the differential pulse electrochemical detection mode is demonstrated.     

Titanium dioxide nanorod-based amperometric sensor for highly sensitive enzymatic detection of hydrogen peroxide

Titanium dioxide nanorods (TNR) were grown on a titanium electrode by a hydrothermal route and further employed as a supporting matrix for the immobilization of nafion-coated horseradish peroxidase (HRP). The strong electrostatic interaction between HRP and TNR favors the adsorption of HRP and facilitates direct electron transfer on the electrode. The electrocatalytic activity towards hydrogen peroxide (H₂O₂) was investigated via cyclic voltammetry and amperometry. The biosensor exhibits fast response, a high sensitivity (416.9 μA·mM⁻¹), a wide linear response range (2.5 nM to 0.46 mM), a detection limit as low as 12 nM, and a small apparent Michaelis-Menten constant (33.6 μM). The results indicate that this method is a promising technique for enzyme immobilization and for the fabrication of electrochemical biosensors.

A TiO₂ nanorod film was directly grown on Ti substrate by a hydrothermal route, and was further employed for a supporting matrix to immobilize horseradish peroxidase as a biosensor electrode. The as-prepared hydrogen peroxide biosensor based on Nafion/HRP/TNR/Ti electrode exhibited fast response and excellent electrocatalytic activity toward H₂O₂, i.e., a high sensitivity (416.9 μA mM⁻¹), a wide linear range (2.5 × 10⁻⁸ to 4.6 × 10⁻⁴ M) with a low detection limit (0.012 μM) and a small apparent Michaelis-Menten constant (33.6 μM).

     

Titanium dioxide nanorod-based amperometric sensor for highly sensitive enzymatic detection of hydrogen peroxide

Titanium dioxide nanorods (TNR) were grown on a titanium electrode by a hydrothermal route and further employed as a supporting matrix for the immobilization of nafion-coated horseradish peroxidase (HRP). The strong electrostatic interaction between HRP and TNR favors the adsorption of HRP and facilitates direct electron transfer on the electrode. The electrocatalytic activity towards hydrogen peroxide (H₂O₂) was investigated via cyclic voltammetry and amperometry. The biosensor exhibits fast response, a high sensitivity (416.9 μA·mM^?1), a wide linear response range (2.5 nM to 0.46 mM), a detection limit as low as 12 nM, and a small apparent Michaelis-Menten constant (33.6 μM). The results indicate that this method is a promising technique for enzyme immobilization and for the fabrication of electrochemical biosensors.

Enzyme based amperometric biosensors

1. Download : Download high-res image (165KB)
2. Download : Download full-size image

     

Determination of bacterial antigens using a multichannel immunoenzyme amperometric sensor

A procedure for the individual determination of the antigens of the conditionally pathogenic micro-organisms Streptococcus pyogenes and Staphylococcus aureus present in combination using a multichannel immunoenzyme amperometric sensor based on a screen-printed electrode was developed. Conditions for fabricating the biosensing part and for the operation of the sensor (the matrix component, the ratio between biocomponents, and the substrate concentration) were chosen. The determination limits of bacterial antigens obtained using the analytical device were equal to 5 × 10^?9 mg/mL. It was found that bacterial antigens simultaneously present in serums from patients can be determined using the sensor developed.     

Nanostructured platform for the detection of Neisseria gonorrhoeae using electrochemical impedance spectroscopy and differential pulse voltammetry

We report on a nanocomposite based genosensor for the detection of Neisseria gonorrhoeae, a bacterium causing the sexually transmitted disease gonorrhoea. Amino-labeled probe DNA was covalently immobilized on electrochemically prepared polyaniline and iron oxide (PANI-Fe₃O₄) nanocomposite film on an indium tin oxide (ITO) electrode. Scanning electron microscopy, transmission electron microscopy, electrochemical impedance spectroscopy (EIS) and differential pulse voltammetry (DPV) techniques have been employed to characterize surface of the modified electrode. The genosensor has detection limits of 1?×?10^-15 M and 1?×?10^-17 M, respectively, using the EIS and DPV techniques. This biosensor can discriminate a complementary sequence from a single-base mismatch and from non-complementary DNA, and has been utilized for detection of DNA extracted from N. gonorrhoeae culture, and from patient samples with N. gonorrhoeae. It is found to exhibit good specificity for N. gonorrhoeae species and shows no response towards non-gonorrhoeae type of Neisseria species (NgNs) and other gram-negative bacterias (GNBs). The affinity constant for hybridization calculated using the Langmuir adsorption isotherm model is found to be 3.39?×?10⁸ M^-1.

Zirconia-based amperometric sensor using La–Sr-based perovskite-type oxide sensing electrode for detection of NO2

An amperometric zirconia-based sensor attached with perovskite-type oxide sensing electrode was examined for monitoring NO₂ in automobile exhaust. The sensor using La_0.6Sr_0.4Co_0.98Mn_0.02O₃ showed high response to NO₂. The response was almost linear to NO₂ concentration in the range between 50 and 800 ppm, and a 90% reaction time to 400 ppm NO₂ was less than 20 s. Though the NO₂ response of the sensor was slightly affected by the changes in O₂ concentrations, it showed still high response in the examined range of 5–21 vol%.     

In-channel amperometric detection for microchip electrophoresis using a wireless isolated potentiostat

The combination of microchip electrophoresis with amperometric detection leads to a number of analytical challenges that are associated with isolating the detector from the high voltages used for the separation. While methods such as end-channel alignment and the use of decouplers have been employed, they have limitations. A less common method has been to utilize an electrically isolated potentiostat. This approach allows placement of the working electrode directly in the separation channel without using a decoupler. This paper explores the use of microchip electrophoresis and electrochemical detection with an electrically isolated potentiostat for the separation and in-channel detection of several biologically important anions. The separation employed negative polarity voltages and tetradecyltrimethylammonium bromide (as a buffer modifier) for the separation of nitrite (NO??), glutathione, ascorbic acid, and tyrosine. A half-wave potential shift of approximately negative 500 mV was observed for NO?? and H?O? standards in the in-channel configuration compared to end-channel. Higher separation efficiencies were observed for both NO?? and H?O? with the in-channel detection configuration. The limits of detection were approximately two-fold lower and the sensitivity was approximately two-fold higher for in-channel detection of nitrite when compared to end-channel. The application of this microfluidic device for the separation and detection of biomarkers related to oxidative stress is described.     

A microfabricated amperometric moisture sensor

We describe a microfabricated moisture sensor with interdigitated Au or Pt electrodes on a silicon substrate. The sensor active area is covered with a spin-coated, baked-on layer of Nafion(R) perfluorosulfonate ionomer of submicron thickness. The sensor responds to moisture with a 10-90% rise time of 50-100 ms and a 90-10% fall time of 20-30 ms, faster than any other presently available sensor. The logarithm of the sensor current is related to the cube root of the moisture level at a given temperature. At 23 degrees C, the sensor easily measures relative humidities as low as 10%. The sensor response at a given absolute humidity level decreases exponentially with increasing temperature. The film is stable up to a temperature of 150 degrees C, permitting elevated temperature moisture measurement. Since sorbed water is actively decomposed electrolytically, the sensors exhibit negligible hysteresis. Response reproducibility of an individual sensor is <1%, that between identically made sensors is <5%, suggesting mass production techniques without individual calibration will be acceptable for all but the most demanding situation.     

A polypyrrole-based amperometric ammonia sensor

An ammonia sensor is described in this work. The sensing membrane is a thin layer of oxidized polypyrrole (PPy) on a platinum substrate. This sensor is used as the working electrode in a conventional three-electrode system for amperometric measurement of ammonia in aqueous solutions in the potential range of + 0.2 to + 0.4 V (vs. Ag/AgCl). Contact with ammonia causes a current to flow through the electrode. This current is proportional to the concentration of free ammonia in the solution and ammonium ions do not contribute to the measured signal. The signal is due to reduction of PPy by ammonia with subsequent oxidation of PPy by the external voltage source. The sensor is able to detect ammonia reproducibly at the muM level. The main interference is the doping effect of small anions such as Cl(-) and NO(3)(-), also giving a response on PPy at the mM level. This anionic response can, to a certain degree, be reduced by covering the polymer surface with dodecyl sulfate. The sensor gradually loses its activity when exposed to ammonia concentrations greater than 1 mM. The sensor has been tested by the flow injection analysis technique.     

The enzyme-amplified amperometric DNA sensor using an electrodeposited polymer redox mediator

A highly sensitive method for the detection of a breast cancer-associated BRCA-1 gene is reported. The detection is based on a classical sandwich-type assay using horseradish peroxidase (HRP) as a catalytic label and electrodeposited Os^2+/3+ conducting polymer (PAA-PVI-Os) as a redox mediator. Target DNA could be detected by the HRP-catalyzed reduction of H₂O₂, leading to a limit of detection as low as 10 fM. Supported by the National Natural Science Foundation of China (Grant Nos. 20725516, 20704043, 20873175 & 20805055), Shanghai Municipal Commission for Science and Technology (Grant Nos. 0752nm021 & 07ZR14136), Ministry of Science and Technology (Grant Nos. 2006CB933000, 2007CB936000 & 2007AA06A406), Ministry of Health (Grant No. 2009ZX10603), and China Postdoctoral Science Foundation and Shanghai Postdoctoral Scientific Program (Grant No. 07R214160).     

Enzyme sensor for the electrochemical detection of the marine toxin okadaic acid

An enzyme sensor for the electrochemical detection of the marine toxin okadaic acid (OA) has been developed. The strategy was based on the inhibition of immobilised protein phosphatase (PP2A) by this toxin and the electrochemical measurement of the enzyme activity by the use of appropriate enzyme substrates, electrochemically active after dephosphorylation by the enzyme. Colorimetric inhibition assays have demonstrated the PP2A from human red blood cells to be more sensitive and to provide a wider linear range than the one produced by genetic engineering. Catechyl monophosphate (CMP) and p-aminophenyl phosphate (p-APP) have been tested as enzyme substrates, the former providing higher electrochemical currents at convenient working potentials (+450 mV vs. Ag/AgCl). Biosensors with 19.1 and 5.0 U of immobilised enzyme have been applied to the OA detection. Whereas the 19.1-U biosensor has provided higher electrochemical currents and more reliable determinations, the 5.0-U one has attained a lower 50% inhibition coefficient (IC₅₀) value (22.19 in front of 154.84 μg L⁻¹) and a larger working range (2.69-171.87 in front of 42.97-171.87 μg L⁻¹). The analysis of toxicogenic dinoflagellate extracts with both biosensors and the comparison with the colorimetric assay and liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) have demonstrated the applicability of the developed electrochemical devices as screening biotools for the assessment of the toxicity of a sample.