Detection of lipopolysaccharide binding peptides by the use of a lipopolysaccharide-coated piezoelectric crystal biosensor

A method has been developed to monitor the interaction between a lipopolysaccharide (LPS) and LPS-binding peptides using a piezoelectric quartz crystal (PQC). Different pH conditions were evaluated to coat LPS onto AT-cut crystals that had been sputtered with gold and carboxylated with a 4,4-dithiodi(n-butyric acid). The optimal pH for LPS coating onto the crystal was 4–5. Synthetic peptides that represent different regions of human bactericidal/permeability-increasing protein, BPI (BPI 85–99, BPI 90–101, BPI 157–167) and polymyxin B (PmB) as well as negative control peptide (HBsAg 139–147) were utilized to compare their binding ability to this LPS-coated PQC sensor. The results showed that PmB gave the greatest decrease to the resonant frequency indicating greatest binding ability. BPI 85–299, considered the main part of the LPS binding domain of BPI, was the next greatest, while BPI 157–167 and HBsAg 139–147 showed little response. In addition, BPI 90–101 and PmB-mimicking peptide showed intermediate LPS-binding ability, which was less than that of BPI 85–99, but was higher than that of BPI 157–167. These results suggest the PQC biosensor is potentially useful for the detection and comparison of the LPS-binding ability of different peptides by using an LPS-coated piezoelectric crystal.     

Frequency characteristic of series piezoelectric sensor and application to the determination of water in organic solvents

Theoretical consideration was proposed for the frequency response of a series piezoelectric sensor to the properties of liquid and verified experimentally. This kind of sensor has sensitive and selective frequency response to the permittivity and specific conductivity of solution with excellent stability, and was applied to the determination of micro-content of water in organic solvents. The detection limit is 0.01 g/L in dioxane.     

Development of a portable biosensor for screening neurotoxic agents in water samples

A high sensitive portable biosensor system capable of determining the presence of neurotoxic agents in water has been developed. The system consists of (i) a screen-printed electrode with acetylcholinesterase (AChE) immobilized on it, (ii) a self-developed portable potentiostat with an analog to digital (A/D) converter and a serial interface for transferring data to a portable PC and (iii) an own designed software, developed with Lab-Windows CVI, used to record and process the measurements. The system has been developed to perform high precision amperometrical measurements with low drifts, low noise and a good reproducibility. In the configuration depicted, the percentage of AChE inhibition is proportional to the content of neurotoxic agents in a sample. This type of measurement is performed by the steady-state method from the first steady current (by a phosphate buffer solution) and the second steady current (by an enzymatic reaction produced by the addition of acetylthiocholine chloride to the solution). Validation was performed by analyzing spiked water samples containing pesticides. The design is specially suited for screening purposes, does not need sample preconcentration, is totally autonomous and suitable for the field detection of neurotoxic agents in water.     

Adsorption of metal ions from solution onto a piezoelectric quartz crystal

Many metal ions are spontaneously adsorbed onto a piezoelectric quartz crystal and change the oscillation frequency. The pH ranges in which the metal ions adsorbed were just below that of precipitate formation as the ‘hydroxides’; frequency changes caused by adsorption were not observed in these pH ranges where the ‘hydroxides’ formed. Cationic organic reagents, such as crystal violet and methylene blue, were also adsorbed on the piezoelectric quartz crystal but non-ionic and anionic organic compounds were not. These results showed that the crystal was negatively charged on the surface.     

Determination of carbon monoxide in ambient air using piezoelectric crystal sorption detection

A new piezoelectric quartz crystal (PQC) sorption detector was developed to monitor carbon monoxide (CO) at sub-ppm level in ambient air. Out of the 28 coating materials studied, the palladium(II) acetamide complex with a 1:10 mole ratio of Pd(II) to acetamide was found to be the best. The detection is based on a non-reversible gas/coating interaction with sensitivity depending on gas flowrate. For 5-15 min exposure at a flowrate of 50 ml/min, the working ranges were found to vary from 0.7 to 40 ppm (total exposure from 8 to 160 μg CO) and detection limits (S/N=2) from 0.7 to 2 ppm CO (total exposure to 8 μg CO). The repeatability at 10 ppm CO was 11.8% (R.S.D. for n=3). The sensor lifetime was found dependent on exposure up to 160 μg CO or not exceeding 1000 Hz accumulative shift of frequency to avoid saturation of active sites at the crystal surface. No interference to CO detection was found for H₂, H₂S, SO₂, NO₂, CO₂, HCHO, gasoline and water vapors at concentrations much higher than ambient air. Compared to existing CO monitor, the PQC detector developed has advantages of adequate selectivity, high sensitivity, fast response and a much lower detection limit for detecting CO at sub-ppm levels. However, it is limited by the total exposure to a maximum of 160 μg CO that restricts its application to intermittent monitoring of low CO concentration. The present work has demonstrated the advantages of using strong non-reversible interaction to enhance PQC sensitivity, as the total exposure can be adjusted easily by a suitable control of the gas flowrate.     

Development of a chemical vapor sensor using piezoelectric quartz crystals with coated unusual lipids

Piezoelectric quartz crystal sensors were developed using lipids with various properties for highly sensitive detection of chemical vapors. Lipids with varying lengths of alkyl chains were coated onto 10 MHz AT-cut quartz crystal resonators and the response of these modified crystals to chemical vapors were measured. It was shown that hydrophilic compounds, such as ethanol and methanol, could be recognized efficiently by lipids having shorter alkyl chains, whereas lipids with longer alkyl chains showed affinity to more hydrophobic vapors, such as toluene, hexane and cyclohexane. Frequency changes caused by adsorption of alcohols could be enhanced when cholesterol was co-immobilized in the lipid layer. To confirm the assumption that the sensor-response might be affected by the properties of lipids derived from acyl chains, we have examined the effects of two types of newly synthesized unusual lipids on sensor response. When lipids having one triple bond each at different positions on their alkyl chains were coated onto quartz crystals separately, lower responses were observed compared to responses obtained for a sensor with immobilized, saturated phosphatidylcholine. Lipids containing -branched acyl chains, however, showed good affinity for organic vapors, and sensor responses improved 4–5-fold. Moreover, these sensors were shown to have sensitivity of the same order as the humans' sense of smell (10⁻⁵–10⁻⁶ w/w in liquid paraffin) when measured using standard odorants (isovaleric acid, skatole, etc.) for an olfactometry established in Japan.     

Detection of β-thalassemia by a DNA piezoelectric biosensor coupled with polymerase chain reaction

β-Thalassemia is an inherited disorder mainly caused by mutations in the gene of the β-globin chain of adult haemoglobin (HbA). Clinically, β-thalassemia can be a mild or silent condition, or it can cause severe diseases, leading to transfusion dependence. Studies at the gene level have identified a large number of variations in the β-globin gene in different populations. In the Mediterranean area one of the most common mutation is the C → T substitution in the codon 39 of the gene.A new procedure for detecting codon 39 mutation in the β-globin gene is reported, based on a DNA piezoelectric biosensor. An oligonucletidic probe (25-mer), specific for the region around codon 39, is immobilised on the gold surface of a piezoelectric quartz crystal. The hybridisation between the immobilised probe and the complementary strand in solution is detected recording the variations of the crystal frequency.Experiments with synthetic oligonucleotides were initially performed. Distinguishable frequency shifts were obtained from the interaction between the immobilised probe and the complementary and the mismatch oligonucleotides. A solution containing 50% of both the oligonucleotides has been also tested and distinguished from the others evaluating the resulting signals. Experiments with non-complementary oligonucleotides gave no signal variation. The biosensor was able to distinguish between sequences differing in only one base also using polymerase chain reaction-amplified samples [771 base pairs (bp)] of DNA extracted from human blood of thalassemic and healthy (normal) patients or patients with β-thalassemia traits.The optimised DNA piezoelectric biosensor has been successfully applied to the determination of one of the most frequent mutation characteristic of β-thalassemia in the Mediterranean population.     

A novel piezoelectric immunosensor for the detection of malarial Plasmodium falciparum histidine rich protein-2 antigen

A novel piezoelectric (PZ) immunosensor for the direct detection of malarial Plasmodium falciparum histidine rich protein-2 (PfHRP-2) antigen was developed. The mixed self-assembled monolayers (SAMs) of thioctic acid and 1-dodecanethiol were formed on gold surface of quartz crystal. Cyclic voltammetry, electrochemical impedance spectroscopy and surface Raman spectroscopy techniques were used to characterize the mixed SAMs. The rabbit anti-PfHRP-2 antibodies were coupled on mixed SAM modified gold surface of quartz crystal via NHS/EDC activation method. The PZ immunosensor was applied to detect PfHRP-2 in the linear range of 15-60 ng/ml with a detection limit of 12 ng/ml. It was also found that even after 14 days of storage, 50% of the activity still remained. Clinical human serum samples were tested with this method, and the results were in agreement with those obtained from commercially available ICT kit (NOW^® Malaria).     

Sensitive determination of aliphatic amines in water by high-performance liquid chromatography with chemiluminescence detection

A sensitive method has been developed for liquid chromatographic determination of short aliphatic amines in water samples. Analytes are preconcentrated and dansylated on solid sorbents (C18 solid-phase extraction cartridges). The dansyl derivatives are chromatographed and post-column mixed with peroxyoxalate (TCPO) and H2O2 in order to perform chemiluminescence detection. Optimal results have been obtained using a sample volume of 5 ml. The method has been applied to the quantification or screening of several aliphatic amines: methylamine, ethylamine, butylamine, diethylamine, pentylamine and hexylamine. The screening procedure has been developed including also polyamines (putrescine, cadaverine, spermidine and spermine). The results obtained by using chemiluminescence (CL) detection have been compared with other detection systems (fluorescence and UV). The sensitivity can increase from 3 to 75 times respect UV detection and from 2 to 10 times respect fluorescence detection depending on the amine. The detection limits achieved were between 0.15 and 0.9 microg/l.     

An improved sealed quartz‐tube method for the determination of hydrogen isotopes in water

Hydrogen stable isotope analysis has been a valuable tool in the fields of geochemistry and ecological research as well as many other research fields. The methods are mainly divided into the dual‐inlet method (off‐line method) and continuous flow method. The dual‐inlet method is complicated and inefficient, but it is still important because of its high precision and wide application range. Although the continuous flow method improves the experimental efficiency, the memory effect is noticeable and the accuracy is reduced. An improved sealed quartz‐tube method is proposed in this paper. The sample is sealed in a capillary tube and placed in a quartz tube containing chromium powder. It is then packaged, evacuated, reacted at a high temperature, and analyzed for hydrogen isotope ratio. Excellent data accuracy, good reproducibility (<1‰), and no memory effect occurred in the method. The process is relatively simple, and the experimental efficiency is greatly improved, which provides an effective method for the analysis of hydrogen isotopes in complex liquid samples.     

Determination of 2-mercaptobenzothiazole in river water by HPLC

Summary A method is proposed for the determination of 2-mercaptobenzothiazole in river waters using multi-electrode electrochemical detection HPLC. 2-mercaptobenzothiazole determination is unsatisfactory by gas chromatography as it degrades readily on the column. Multielectrode electrochemical detection HPLC combines sensitivity and the ability to screen out other electrochemically active species. The development work leading up to the proposed method is discussed. The method has a limit of detection of 0.798gl^–1 2-mercaptobenzothiazole and a total standard deviation of 2.06gl^–1 2-mercaptobenzothiazole at a concentration of 7.97gl^–1 2-mercaptobenzothiazole in river water.     

Amperometric detection of Enterobacteriaceae in river water by measuring β-galactosidase activity at interdigitated microelectrode arrays

Two simple methodologies are compared for the detection of faecal contamination in water using amperometry at gold interdigitated microelectrodes. They rely on the detection of β-galactosidase (β-gal) by redox cycling amperometry of the p-aminophenol (PAP) produced by the enzyme from the 4-aminophenyl β-d-galactopyranoside (PAPG) substrate. The use of phages as specific agents for the release of the bacteria-enclosed enzyme allowed the detection of 6 × 10⁵ CFU mL⁻¹Escherichia coli in 2 h without any pre-enrichment or preconcentration steps. Better limits of detection were achieved for the second strategy in the absence of phages. In this case, bacteria were enriched in the presence of both β-d-1-thiogalactopyranoside (IPTG) and substrate but in the absence of phages. Under such experimental conditions, 5 × 10⁴ CFU mL⁻¹E. coli could be detected after 2 h of incubation, while 7 h of incubation were enough to detect down to 10 CFU mL⁻¹ in river water samples. This represents a straightforward one-step method for the detection of faecal contamination that can be conducted in a single working day with minimal sample manipulation by the user.     

Analysis of acidic photosystem II—Interacting herbicides in lake and river water by capillary electrophoresis

Summary The separation of cyanophenolic (ioxynil and bromoxynil) photosystem II-interacting herbicides and mecoprop in surface water extracts was obtained on a poly(vinyl alcohol)—coated capillary in 75 mM acetate buffer, pH 6, at 16 kV applied voltage. Styrene-divinyl benzene packed cartridges were used for solid phase extraction of the real surface water samples at neutral pH. To increase method sensitivity different injection techniques, namely hydrodynamic, electrophoretic and field—amplified sample injection (FASI), were compared.     

Direct determination of total carbonate salts in soil samples by continuous-flow piezoelectric detection

Two new methods for the determination of total carbonate salts, as CaCO₃, in soil by continuous-flow piezoelectric (PZ) detection are proposed. Both use a piezoelectric flow cell and a manifold including a dynamic gas extraction device to purge gaseous CO₂ released by the sample solution upon acidification. One of the methods involves monitoring the pressure generated by the CO₂ produced upon addition of hydrochloric acid; in the other, the CO₂ is quantified by using a quartz crystal coated with tetramethylammonium fluoride tetrahydrate (TMAF). The precision of both methods is compared with that of the officially endorsed method. The proposed methods allow calcium carbonate amounts over the ranges 10-100 mg and 2-15 mg, respectively, to be determined. Both were applied to the determination of CaCO₃ in soil samples. The standard deviation and throughput achieved were 2.7% and 30 samples per hour, respectively, with the pressure-based method; and 6.0% and six samples per hour, respectively, with the mass-based method.     

Toxicological study of pesticides in air and precipitations of Paris by means of a bioluminescence method

A detailed toxicological study on several pesticides, including chlorothalonil, cyprodynil, dichlobénil, pendimethaline, trifluraline, and α-endosulfan, present at trace levels in air and total atmospheric precipitations of Paris is presented. The pesticides contained in the atmospheric samples, collected during sampling campaigns in February–March 2007, are identified and quantified by a high-performance liquid chromatographic (HPLC)-UV detection method. The toxicity measurements are performed by means of the Microtox® bioluminescence method, based on the evaluation of the bioluminescence inhibition of the Vibrio fischeri marine bacteria at two exposure times to the pesticide solutions. The specific toxicity, corresponding to the particular toxicity of the compound under study and represented by the EC₅₀ parameter, is determined for these pesticides. Also, the global toxicity, which is the toxicity of all micro-pollutants present in the sample under study, is estimated for the extracts of air and atmospheric precipitation (rainwater) samples. The specific toxicities strongly vary with the nature of the pesticide, the EC₅₀ parameter values being comprised between 0.17 and 0.83 mg/mL and 0.15 and 0.66 mg/mL, respectively, for exposure times of 5 and 15 min. The importance of the atmospheric samples’ global toxicity and the respective contribution of the toxic potency of the various pesticides contained in these samples are discussed.

Determination of gadolinium in river water by SPE preconcentration and ICP-MS

An analytical scheme was developed for the determination of Gd-diethylenetriaminepentaacetate (Gd-DTPA), Gd and the other rare earth elements (REE) in river water by inductively coupled plasma (quadrupole) mass spectrometry (ICP-Q-MS). The preconcentration step was essential, since the limits of detection of this multielemental analytical technique are higher than the trace concentrations of the interesting elements in river water.Solid phase extraction (SPE) with different commercially available complexing agents (Chelex 100, Toyopearl and ethylhexylphosphates) was employed for the preconcentration of REE. The investigations revealed that complex stability (varying in dependence of the pH value) has a strong influence on the degree of the enrichment of Gd-DTPA. Based on acidified water samples (pH<3) a procedure using ethylhexylphosphates was proposed for the preconcentration of Gd and REE from surface water samples. For this purpose C₁₈-cartridges loaded with ethylhexylphosphates were used, resulting in an enrichment factor of 40.     

Ultraviolet laser-induced fluorescence detection strategies in capillary electrophoresis: determination of naphthalene sulphonates in river water

Various UV-laser-induced fluorescence detection strategies for capillary electrophoresis (CE) are compared, i.e. two UV-laser systems (a pulsed laser providing up to 25 mW of tunable emission, applied at 280, 290 and 325 nm, and a continuous wave (cw) laser providing up to 100 mW of 257 nm emission) and different methods to collect the fluorescence emission signal and to reduce the background. Attention is focused on the determination of amino- and hydroxy-substituted naphthalene sulphonates (NS) in river water; these analytes exhibit native fluorescence upon UV excitation. Optimum results were obtained by applying only a minor portion of the available (average) laser powers, viz. 0.7 mW at 280 nm for the pulsed laser, and 5 mW for the cw laser. For emission collection, the most favourable results were obtained with a mirror-based microscope objective, which facilitates efficient spatial filtering and does not produce impurity fluorescence upon UV-laser irradiation. For standard solutions, the cw laser gave around 20-fold better detection limits (10⁻⁹–10⁻¹⁰ M) than the pulsed laser. For river water, excitation of interferences (presumably humic acids, which exhibit native fluorescence) could be much better suppressed if the pulsed laser was used with selective excitation at 280 nm. Therefore, for real-sample analysis the latter combination is to be preferred. The set-up was used for the identification and quantification (at the 1–35 μg l⁻¹ level) of NS in a river Elbe sample.     

Determination of bisphenols A and F and their diglycidyl ethers in wastewater and river water by coacervative extraction and liquid chromatography-fluorimetry

A simple, rapid and sensitive analytical method was developed for the simultaneous determination of bisphenol A (BPA), bisphenol F (BPF) and their corresponding diglycidyl ethers (BADGE and BFDGE) in wastewater and river water, in order to have a useful tool for evaluating their fate and distribution in aquatic environments. It was based on their extraction with coacervates made up of decanoic acid reverse micelles and subsequent determination by liquid chromatography-fluorimetry. The procedure involved the extraction of 10.8 mL of water sample for 5 min, its centrifugation for 10 min to accelerate phase separation and then the chromatographic analysis of the target compounds. Clean-up or solvent evaporation steps were not necessary to get the required sensitivity and selectivity. Extraction efficiencies and concentration factors mainly depended on the amount of decanoic acid and tetrahydrofuran making up the coacervate. A general equation for the prediction of the volume of the coacervate as a function of its components has been proposed and fitted by nonlinear regression. This equation permits to know a priori the maximum concentration factors that can be achieved under given experimental conditions. Extractions were independent of salt addition (up to 1 M), the temperature (up to 60 °C) and the pH (below 4) rendering the method robust. Recoveries in samples ranged between about 80 and 92% and the actual concentrations factors were between 87 and 102, which resulted in practical detection limits around 30-35 ng L⁻¹. The method was successfully applied to the determination of the target pollutants in raw and treated sewage from four mechanical-biological treatment plants and three rivers. Bisphenols and their diglycidyl ethers were present in wastewater influents at concentrations in the range 0.96 to 1.6 μg L⁻¹. The biological treatment at the wastewater treatment plants (WWTP_s) studied reduced the concentration of BPA and BPF in a percentage above 75%, while diglycidyl ethers were not detected in most of the effluents investigated. Only BPA was detected in surface waters.     

Sensitive determination of Fenamiphos in water samples by flow injection photoinduced chemiluminescence

In this work, a sensitive flow injection chemiluminescence (FI-CL) method for the determination of nematicide Fenamiphos in a rapid and simple way is proposed. Fenamiphos is first photodegraded in basic medium. These photofragments react with Ce(IV) providing the chemiluminescence signal. To the authors’ knowledge, no chemiluminescence method has been described in the literature for the determination of the nematicide Fenamiphos. All physical and chemical parameters in the flow injection chemiluminescence system were optimized in order to obtain the best sensitivity, selectivity and sample throughput. Before the injection of the sample in the FI-CL system, a preconcentration step with solid phase extraction C18 cartridges was performed. By applying solid phase extraction (SPE) to 250?mL of standard (final volume 10?mL), the linear dynamic range was between 3.4 and 60?µg?L^?1, and the detection limit was 1?µg?L^?1. When SPE was applied to 500?mL of standard (final volume 10?mL), the detection limit was 0.5?µg?L^?1. These detection limits are below the emission limit value established by the Spanish Regulations of the Hydraulic Public Domain for pesticides (50?µg?L^?1) and of the same order as the limit established for total pesticides (0.5?µg?L^?1) at European Directive on the quality of water for human consumption. The sample throughput was 126 hour^?1. Intraday and interday coefficients of variation were below 10% in all cases. No interference was registered in presence of usual concentrations of anions, cations and other organophosphorus pesticides. The method was successfully applied to the analysis of environmental water samples, obtaining recoveries between 96 and 107.5%.     

TIRF-based biosensor for sensitive detection of progesterone in milk based on ultra-sensitive progesterone detection in water

We report on recent advances of our immunoassay for the hormone progesterone in cows milk. Detection is based on total internal reflectance fluorescence (TIRF), the binding-inhibition assay with an immobilized progesterone derivative, and a commercially available monoclonal antibody to progesterone as biological recognition element. The fully automated River Analyzer (RIANA) biosensor for unattended, cost-effective, and continuous monitoring of environmental pollution therefore was adapted for sensitive determination of progesterone in milk. First, the sensitivity and robustness of the existing progesterone assay for water analysis were improved, resulting in a detection limit (LOD) of only 0.2 pg mL^–1 and a quantification limit (LOQ) of only 2.0 pg mL^–1. These extraordinary results are the lowest detection and quantification limits for progesterone determination using biosensors yet reported in the literature. Second, the accurate indicator of ovulation was calibrated and detected in three different types of milk (UHT milk, fresh milk, and raw milk). For commercial milk and randomly procured raw milk nominal levels of progesterone are typically in the range 5–15 ng mL^–1. Limits of detection (LOD) achieved for added progesterone (i.e. spiked samples) were between 45.5 and 56.1 pg mL^–1 depending on milk type. Having in mind the 1:10 dilution factor, these results are still a success. For the first time a commercially available antibody was incorporated into an immunoassay for progesterone detection in bovine milk, giving a detection limit below 1 ng mL^–1 for a fully automated biosensor. Thus the outstanding progress made with this biosensor in environmental monitoring and water analysis has now been successfully adapted to milk analysis for use in the field of reproduction management.Dedicated to the memory of Wilhelm Fresenius.