Conductimetric immunosensor for atrazine detection based on antibodies labelled with gold nanoparticles

A novel conductimetric immunosensor for atrazine detection has been designed and developed. This immunosensor is mainly based on antibodies labelled with gold nanoparticles. Additionally, the immunosensor consists of an array of two coplanar non-passivated interdigitated metallic μ-electrodes (IDμE) and immunoreagents specifically developed to detect this pesticide. The chemical recognition layer was covalent immobilized on the interdigital space. Immunochemical detection of the concentration of atrazine is achieved by a competitive reaction that occurs before the inclusion of the labelled antibodies. It is shown that the gold nanoparticles provide an amplification of the conductive signal and hence makes possible to detect atrazine by means of simple DC measurements.The conductimetric immunosensor and its biofunctionalization steps have been characterized by chemical affinity methods and impedance spectroscopy.This work describes the immunosensor structure, fabrication, physico-chemical and analytical characterization, and the immunosensor response using conductivity measurements. The immunosensor developed detects atrazine with limits of detection in the order of 0.1–1 μg L⁻¹, far below the maximum residue level (MRL) (100 μg L⁻¹) established by European Union (EU) for residues of this herbicide in the wine.Although in this paper the competitive reaction occurs in buffer, an initial study of the wine matrix effect is also described.     

Determination of reduced glutathione using an amperometric carbon paste electrode chemically modified with TTF–TCNQ

The development of an amperometric sensor for the determination of reduced glutathione (GSH) is described. The sensor is based on tetrathiafulvalene–tetracyanoquinodimethane (TTF–TCNQ) incorporated into the graphite powder/Nujol oil matrix. The electrooxidation of GSH was monitored amperometrically at 200 mV versus SCE (saturated calomel electrode). The amperometric response of the sensor was linearly proportional to the GSH concentration between 20 and 300 μmol l⁻¹, in 0.1 mol l⁻¹ phosphate buffer (pH 8.0), containing 0.1 mol l⁻¹ KCl and 0.5 mmol l⁻¹ Na₂H₂EDTA, as supporting electrolyte.

The detection limit, considering signal/noise ratio equal three, was 4.2 μmol l⁻¹ for GSH and the repeatability obtained as relative standard deviation was of 5.1% for a series of 10 successive measurements.     

Sol–gel derived polycrystalline Cr1.8Ti0.2O3 thick films for alcohols sensing application

The powder sample of Cr_1.8Ti_0.2O₃ (CTO) was obtained by a sol–gel method. The thick films were developed on identical ceramic tubes of 4 mm length comprising of two Au-electrodes and printing an eight-layer film prepared by mixing CTO with glass powder and -terpinol as an organic vehicle. X-ray powder diffraction (XRD) patterns showed the formation of a single phase. The scanning electron microscope (SEM) images of the ceramic sensor treated at 850 °C revealed that the grain size was larger than 400 nm for the individual isolated grains on the surface, and the agglomerated dense spheroidal platelets had the size of 1–4 μm in diameter. The AC impedance measurement in ambient air showed that the resistance decreased nearly by two orders of magnitude with an increase in temperature in the range of 400–600 °C for both the powder sample and the thick film, and the activation energy E_a derived from the measurement was found to be 0.35 and 0.36 eV for the powder and the film, respectively. The films were exposed to various concentrations of alcohols (0.4–1.2 ppm of methanol and 1.0–5.0 ppm of ethanol), followed by determination of sensor response, sensitivity and reversibility and reproducibility. The origin of the gas response was attributed to the surface reaction of R-OH (R = methyl and ethyl group) with O⁻_(ads) to form adsorbed R-CHO, which was desorbed as a gas at 400 °C after the sensor departing from the gas.     

Impedancemetric gas sensor based on Pt and WO3 co-loaded TiO2 and ZrO2 as total NOx sensing materials

Pt-loaded metal oxides [WO₃/ZrO₂, MO_x/TiO₂ (MO_x = WO₃, MoO₃, V₂O₅), WO₃ and TiO₂] equipped with interdigital Au electrodes have been tested as a NO_x (NO and NO₂) gas sensor at 500 °C. The impedance value at 4 Hz was used as a sensing signal. Among the samples tested, Pt-WO₃/TiO₂ showed the highest sensor response magnitude to NO. The sensor was found to respond consistently and rapidly to change in concentration of NO and NO₂ in the oxygen rich and moist gas mixture at 500 °C. The 90% response and 90% recovery times were as short as less than 5–10 s. The impedance at 4 Hz of the present device was found to vary almost linearly with the logarithm of NO_x (NO or NO₂) concentration from 10 to 570 ppm. Pt-WO₃/TiO₂ showed responses to NO and NO₂ of the same algebraic sign and nearly the same magnitude, while Pt/WO₃ and WO₃/TiO₂ showed higher response to NO than NO₂. The impedance at 4 Hz in the presence of NO for Pt-WO₃/TiO₂ was almost equal at any O₂ concentration examined (1–99%), while in the case of Pt/WO₃ and WO₃/TiO₂ the impedance increased with the oxygen concentration. The features of Pt-WO₃/TiO₂ are favorable as a NO_x sensor that can monitor and control the NO_x concentration in automotive exhaust. The effect of WO₃ loading of Pt-WO₃/ZrO₂-based sensor is studied to discuss the role of surface W-OH sites on the NO_x sensing.     

Composites: A novel alternative to construct solid state Ag/AgCl reference electrodes

An Ag/AgCl solid-state reference electrode is developed by means of a graphite–AgCl–Silver dag-epoxy resin composite. The response of the composite reference electrode (CRE) to chloride ions is evaluated; a linear non-nernstian response is observed associated to the following equation E = −15.15 (±1.10) − 44.05 (±0.38) log[Cl⁻]. Comparing the CRE's response potential versus a saturated commercial Ag/AgCl reference in KCl 0.1 M, a mean of 40.7 ± 0.4 mV of the distribution of potential versus time data is observed over a period of 1 h. The performance of the CRE as reference for a glass membrane electrode by means of direct pH measurements and quantitative determination of acids by acid–base titrations is evaluated obtaining statistically stable, precise and exact results compared with those obtained using a combined glass electrode. The typical cylindrical configuration of the CRE is changed to adapt it to a FIA system for the determination of ammonium ion, obtaining a sensitivity 50.30 ± 0.26 mV/log[NH₄⁺] and a linear range 8.5 × 10⁻⁵ to 0.1 M, which are analytical parameters statistically equivalent to those presented by the classical determination system.     

Whole-body vertical biodynamic response characteristics of the seated vehicle driver: Measurement and model development

The vertical driving-point mechanical impedance characteristics applicable to seated vehicle drivers are measured in the 0.625–10 Hz frequency range with excitation amplitudes ranging from 1.0 to 2.0 m s⁻² using a whole-body vehicular vibration simulator. The measurements are performed for seated subjects with feet supported and hands held in a driving position. Variations in the seated posture, backrest angle, and nature and amplitude of the vibration excitation are introduced within a prescribed range of likely conditions to illustrate their influence on the driving-point mechanical impedance of seated vehicle drivers. Within the 0.75–10 Hz frequency range and for excitation amplitudes maintained below 4 m s⁻², a four-degree-of-freedom linear driver model is proposed for which the parameters are estimated to satisfy both the measured driving-point mechanical impedance and the seat-to-head transmissibility characteristics defined from a synthesis of published data for subjects seated erect without backrest support. The parameter identification technique involves the solution of a multivariable optimization function comprising the sum of squared magnitude and phase errors associated with both the mechanical impedance and seat-to-head transmissibility target values, subject to limit constraints identified from the anthropometric and biomechanical data. The model response, however, is found to provide a closer agreement with the mechanical impedance target values than that with the seat-to-head transmissibility. From the model, the main body resonant frequencies computed on the basis of both biodynamic response functions are found to be within close bounds to that expected for the human body.

Relevance to industry

The development of an appropriate analytical seated vehicle driver model should provide means of estimating the forces and motions being transmitted within the body under specific vehicular vibration environments. Furthermore, its use in conjunction with a corresponding model for the vehicle seat should allow the prediction of the driver's vibration exposure levels and the seat's ability to attenuate the vibration in particular vehicles.     

The effect of humidity on the electrical conductivity of mesoporous polythiophene

Commercial samples of bromine terminated polythiophene powder have been compacted to a mesoporous state in order to measure their electrical properties at different relative humidities by impedance spectroscopy, in this case the complex admittance versus frequency. The sample powders were compacted between the electrodes of a co-axial tube capacitor arrangement and connected to a standard impedance bridge with a frequency range of 20–1 MHz. The low humidity conductivity shows a dc limit at about 4 × 10⁻⁶ S/m. When the conductivity measurements were recalculated by subtracting the low humidity response, assumed dry, from the medium or high humidity response a peak was seen at values ranging from 20 to 60 kHz with a sharp fall off at about 100 kHz. Plots of the change in susceptibility did not show peaks, but the excess response vanished at a lower frequency than seen for the conductivity. The log–log plots of the data showed good low frequency fits to a straight line, implying power law dependencies similar to those seen in ionic dielectric materials. I conclude that in humid air there is no significant conduction through the adsorbed water layer itself, but that the presence of surface water molecules affects the polythiophene conduction that occurs through the overlap of adjacent π-bonds. In this model the presence of surface water enhances the π-bond overlap, but has a strong frequency dependence.     

Adsorption studies of carbowax and poly dimethyl siloxane to use as chemical array for nitro aromatic vapour sensing

Trinitro toluene (TNT)-based explosives contain 2,4-dinitro toluene (DNT) and 1,4-dinitro benzene (DNB) as a manufacturing impurity, which form more vapour in the vicinity than TNT itself, and hence form a distinctive ‘chemical signature’ indicative of explosive. The concentration of these compounds over landmines is extremely low and well below the minimum detection limits of most field-portable chemical sensors. Carbowax was found to give good adsorption for 2,4-DNT vapour at higher temperature and reverse was in case of 2,6-DNT vapour. On the other hand, the same polymer film gives reasonably good adsorption for 1,4-DNB, more than 33% adsorption and poor response for 1,3-DNB, which is around only 20%. The adsorption rate was specific for all the four isomers as observed 0.81 ng/(cm² min) for 2,4-DNT, 0.14 ng/(cm² min) for 1,3-DNB, 0.07 ng/(cm² min) for 2,6-DNT, and 0.04 ng/(cm² min) for 1,4-DNB. This polymer was found to give different relative response to these nitro aromatic isomers. PDMS gives very good response for 2,4-DNT (98–100%) at 50 °C appears to be a distinguishing performance. The results of carbowax-1000 and poly dimethyl siloxane (PDMS) indicated that these two polymers could be more suitable for the realisation of polymer-based chemical sensor.     

Micro-piezoelectric immunoassay chip for simultaneous detection of Hepatitis B virus and α-fetoprotein

In this paper, a piezoelectric diaphragm-based immunoassay chip was developed to simultaneously detect anti-Hepatitis B virus (HBV) and anti-alpha-fetoprotein (AFP). The chip was fabricated by micro-machining technology and consists of eight individual circular sensors with a diameter of 800 μm. Hepatitis B surface antigen (HBsAg), Hepatitis C core antigen (HBcAg) and AFP as the probe molecules were immobilized on different sensing spots on the chip. A solution containing anti-HBsAg and anti-AFP was applied into the reaction chambers in all sensors of the chip, and significant frequency shifts were only observed in the sensors with HBsAg and AFP for immunoassay detection. The fluorescence image further confirmed the successful detection of anti-HBsAg and anti-AFP. The total assay time was less than 2 h. The frequency shift-based calibration curves show a detection limit of 0.1 ng/ml and a dynamic detection range of 0.1-10,000 ng/ml for both anti-HBsAg and anti-AFP, respectively, thus demonstrating that the developed piezoelectric immunoassay chip has potential applications for rapid, specific, sensitive, and multiple detections of HBV.     

Characterization of protective coatings for planar automotive gas sensors

Alumina support material suitable for use as a planar automotive gas sensor support was coated in thin films of yttria-stabilised zirconia (YSZ) and titania. The morphology, composition, thickness and homogeneity of the coating was measured. The coating was applied to the ‘green’ form of a tape cast alumina substrate which was subsequently fired at 1500 °C to produce the final form of the coated alumina. The YSZ coating gave a continuous 5 μm thick coating with no evidence of mixed oxide formation between the YSZ and the alumina substrate. XRD indicated a face centred cubic Y doped ZrO₂ or primitive tetragonal Zr_0.9Y_0.1O_1.95 phase. The titania coatings were much thinner (<1 μm) with signs of trace amounts of aluminium titanium oxide (Al₂TiO₅) as well as rutile titania in XRD. Spot analysis using X-ray photoelectron spectroscopy showed a fairly regular titania coverage. Atomic force microscopy analysis showed a particle size of 1–3 μm for the YSZ coating and 0.5 μm for titania.     

Characteristics of a label-free piezoelectric immunosensor detecting Pseudomonas aeruginosa

A label-free immunosensor system detecting a psychrophylic bacterium, Pseudomonas aeruginosa was developed as follows. Four types of anti-P. aeruginosa antibody were individually chemisorbed onto one-side gold electrodes of piezoelectric quartz crystals according to a thiolated antibody coupling procedure initiated with a thiol-cleavable heterobifunctional cross-linker, sulfosuccinimidyl-6-[3-(2-pyridyldithio)propionamido]hexanoate. A flow-type biosensor system was operated optimally at 0.2 M sodium potassium phosphate, pH 7.2 with a minimal matrix effect and the selected flow rate for it was 0.155 ml/min. A biosensor response was detected by measuring a steady-state resonant frequency shift after the response time around 8 min. The frequency shifts obtained were quite specific according to the antibody types and P. aeruginosa strains. The biosensor responses to varying concentrations of the P. aeruginosa cells ranging from 1.3×10⁷ to 1.3×10⁸ CFU/ml were determined as 17–176 Hz and a linear calibration curve (r=0.942) was obtained by plotting the responses in a double-logarithmic scale. The selectivity of the biosensor between P. aeruginosa and Xanthomonas spp. which both belong to the aerobic pseudomonads was, however, not so good owing to the property of the antibody used. The sensor chip could be reused at least seven times without an appreciable decrease in sensitivity.     

Fabrication and characterization of low temperature sintering PMN–PZN–PZT step-down multilayer piezoelectric transformer

For the fabrication as step-down multilayer piezoelectric transformer, piezoelectric properties of Pb(Mg_1/3Nb_2/3)O₃–Pb(Zn_1/3Nb_2/3)O₃–Pb(Zr_0.52Ti_0.48)O₃ (PMN–PZN–PZT) ceramics were optimized by ZnO–Li₂CO₃ (ZL) and Pb₃O₄ content. Effects of the additions on the structure, bulk density and electrical properties of ceramics were investigated. The results revealed that the proper additions of ZL with Pb₃O₄ content could modify the electrical properties of the PMN–PZN–PZT ceramics. The composition sintered at 995 °C with 0. 01 wt.% ZL and 0.10 wt.% Pb₃O₄ content showed higher values, which were listed as follows: d₃₃ = 256 pC/N, K_p = 0.60, Q_m = 1910, _r = 1032, tan δ = 0.0070 and r = 2.09 Ω. In addition, the step-down piezoelectric transformers with optimized PMN–PZN–PZT composites were fabricated and the characteristics as the output power and resistance loads were measured. Meanwhile, the step-down piezoelectric transformers sintered at 995 °C showed the favorable characteristics with a higher gain G of 0.204 and a lower temperature rise of 6 °C when the output power was 5 W, and the driving frequency were approximately constant (≈126 kHz) when the output power was from 5 to 13 W. Moreover, the maximum efficiency (90.2%) was obtained at load resistance of 10 Ω.     

Time-resolved absorption as optical method for herbicide detection

A new sensing system for the detection of photosynthetic herbicides in water has been developed, based on the use of a trans-membrane protein complex, the reaction centre (RC) isolated from Rhodobacter sphaeroides. The stationary and excited state of this protein are characterised by different absorption properties. The path followed by the protein to return to the stationary state is influenced by the presence of photosynthetic herbicides. Therefore the concentration of herbicides could be measured by monitoring the temporal changes of absorption following optical excitation. For this purpose, an optoelectronic system has been realised. It makes use of a 860 nm light emitting diode and a hybrid photodetector and is coupled to a 5 cm-long optical cell containing the RC solution through optical fibres. The system was tested with atrazine and a limit of detection of 10 nM was obtained.     

Mechanical impedance of the human hand-arm system

The mechanical impedance of the human hand-arm system was measured within the frequency range of 20–1500 Hz. A handle, specially designed for such measurements, was used. The studies were carried out on eight healthy male subjects during different experimental conditions defined by three different hard-arm postures, hand grip forces (25–75 N) adopted by the subjects, the amplitude (27–53 mm/ s_rms; 1.4–2.8 g at 80 Hz) and direction of the vibration stimuli. The outcome shows that the mechanical impedance of the hand-arm system depends on the frequency of the vibration stimuli. Above 200 Hz, the impedance, in general, increases quite rapidly, from about 150 Ns/m up to about 500 Ns/m at 1500 Hz, with the frequency. At lower frequencies, however, various shapes of the impedance curves were found which were most pronounced between different hand-arm postures. For the transverse direction, the impedance increased from about 50 Ns/m at 20 Hz to maximum about 100 Hz followed by a slight decrease. For the proximal-distal direction the impedance decreased from about 150 Ns/m at 20 Hz to minimum at about 100 Hz. More firm hand grips, as well, as higher vibration levels, resulted in higher impedance magnitudes for frequencies above about 100 Hz. Remarkably enough, for lower frequencies an almost opposite relationship was found. Furthermore, the results indicate a non-linear relationship between mechanical impedence and the studied experimental variables. Therefore, prior to setting up future standards, the mechanical properties of the hand-arm system should be taken into careful consideration.     

Analysis of dc and ac properties of humidity sensor based on polypyrrole materials

Polypyrrole (PPY) materials were prepared by using chemical polymerization at room temperature for 96 h. A resistive-type humidity sensor was fabricated on a ceramic substrate with Ag–Pd interdigital electrodes. The humidity-sensing mechanism of the polypyrrole materials was discussed in terms of capacitance, direct current, complex impedance and dielectric loss properties. The discussion on the sensing mechanism indicated that not only electrons or ions but also dipoles contributed to the conduction in the whole relative humidity range.     

Development of an SH-SAW sensor for the detection of DNA hybridization

We have developed SH (shear horizontal) surface acoustic wave (SAW) sensors for the detection of DNA (deoxyribonucleic acid) hybridization on the gold-coated delay line of transverse SAW devices. DNA hybridization experiments were performed with 15-mer oligonucleotides (probe and complementary target DNA). The sensor consists of twin SAW delay line oscillators (sensing channel and reference channel) operating at 100 MHz fabricated on 36° rotated Y-cut X-propagation LiTaO₃ piezoelectric single crystals. The relative change in the frequency of the two oscillators was monitored to detect hybridization between the target DNA and probe DNA immobilized on the delay line of the SAW sensor. The measurement results showed a good response of the sensor to the mass loading effects of the DNA hybridization with a sensitivity level up to 1.55 ng/ml/Hz.     

Immunoassay using poly-tetrafluoroethylene microstructure in organic solvent

This paper reports the first application of poly-tetrafluoroethylene (PTFE) microstructure, which fabricated by synchrotron radiation induced photo-evaporation process, to enzyme-linked immunosorbent assay. The advantages of PTFE microstructure for the development of lab-on-a-chip due to the extremely high-aspect ratio microstructure and chemical stability of PTFE is discussed. The results of immunoassay shows the successful detection of analyte (mouse IgG) with detection range with 0–100 ng/ml. This result suggests the successful immobilization of antibody (anti-mouse IgG goat antibody) onto the X-ray exposed surface of PTFE microstructure and successful demonstration of antigen–antibody reaction in the PTFE microstructure. We also demonstrated the detection of polychlorinated biphenyl (PCB). As the result of demonstration, we successfully detected PCB with ranging analyte concentration of 0.1–10 ng/ml.     

A hydrogen peroxide biosensor based on direct electrochemistry of hemoglobin in Hb–Ag sol films

Hemoglobin (Hb) was used as a template to fabricate hemoglobin–silver (Hb–Ag) sol in which the hemoglobin showed direct electrochemistry on a glass carbon (GC) electrode. Ultraviolet–visible (UV–vis) spectra and reflectance absorption infrared (RAIR) spectra suggested that hemoglobin in Hb–Ag sol retained its native secondary structure. Scanning electron microscopy (SEM) demonstrated that the morphology of the Hb film was much different from the Hb–Ag sol film. The Hb–Ag film proved to exhibit a good electrocatalytic activity for the reduction of hydrogen peroxide. Based on this, a novel amperometric hydrogen peroxide biosensor was developed, which showed a sensitive response to the reduction of H₂O₂ without any electron mediator. Under optimum conditions, the biosensor responded linearly to H₂O₂ in the concentration range of 1 × 10⁻⁶ to 2.5 × 10⁻² M with detection limit of 1 × 10⁻⁷ M at 3σ. Moreover, the studied biosensor exhibited high sensibility, good reproducibility, and long-term stability.     

Trace detection of nitroaromatic compounds with layer-by-layer assembled {SBA/PSS}n/PDDA modified electrode

The {SBA/PSS}_n/PDDA films modified electrode was prepared by layer-by-layer (LBL) assembly with mesoporous SiO₂ (SBA), poly(sodium 4-styrene-sulfonate) (PSS) and poly(diallyldimethylammonium chloride) (PDDA) in this paper. SBA is a large pore-size mesoporous material with highly ordered hexagonally arranged mesochannels and high thermal stability etc. The electrochemical characteristics of the {SBA/PSS}_n/PDDA films have been studied by electrochemical impedance spectroscopy in 0.1 M KCl solution containing 5.0 mM Fe(CN)₆³⁻/Fe(CN)₆⁴⁻ at the formal potential of 0.230 V. The ultratrace nitroaromatic compounds (NACs) such as TNT, TNB, DNT and DNB were determined by differential pulse voltammetry (DPV) measurement. The sensitivities for NACs determination with {SBA/PSS}_n/PDDA modified electrode were dependent on the number of layers, pH and ionic strength of electrolyte, based on which a set of optimized conditions for film fabrication was inferred. The current responses were linear with NACs ranging from 10⁻⁹ to 10⁻⁷ mol/l. The results showed that the {SBA/PSS}_n/PDDA modified electrode established a new way for fast, simple and sensitive analysis of NACs.     

Highly sensitive label-free immunosensor for ochratoxin A based on functionalized magnetic nanoparticles and EIS/SPR detection

A label-free immunosensor for the detection of ochratoxin A (OTA) based on use of magnetic nanoparticles (MNPs) was developed. A gold electrode was modified using bovine serum albumin conjugate with a glutaraldehyde-thiolamine linker, creating a layer that prevents non-specific binding of OTA on gold. The OTA antibodies were attached to MNPs using the carbodiimide chemistry and afterwards were immobilized on the modified gold electrode using a strong magnetic field. Cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS) and surface plasmon resonance (SPR) were used to characterize each step in immunosensor development. The impedance variation due to the specific antibody-OTA interaction was correlated with the OTA concentration in the samples. The increase in electron-transfer resistance values was proportional to the concentration of OTA on a linear range between 0.01 and 5 ng/mL, with a detection limit of 0.01 ng/mL. SPR measurements showed a larger response range (1-50 ng/mL) with a detection limit of 0.94 ng/mL. Analytical results were in accordance with standard ELISA test kit.