Bioelectrochemical Magnetic Immunosensing of Trichloropyridinol: A Potential Insecticide Biomarker

A fast, simple and sensitive bioelectrochemical magnetic immunosensing method is developed to monitor a potential insecticide biomarker, trichloropyridinol (TCP), in environmental sample. A magnet/glassy carbon (MGC) working electrode was used to accumulate immunocomplex associated magnetic beads and separate free and unbound reagents after liquid phase competitive immunoreaction among TCP antibody coated magnetic beads, TCP analyte and horseradish peroxidase (HRP) labeled TCP. The activity of HRP tracers was monitored by square‐wave voltammetry (SWV) by scanning electrocactive enzymatic product in the presence of 3,3′,5,5′‐tetramethylbenzidine dihydrochloride and hydrogen peroxide (TMB‐H₂O₂) substrate solution. The electrochemical signal of enzymatic product was greatly enhanced by dual accumulation events: magnetic accumulation of enzyme tracers bound magnetic beads and constant potential accumulation of enzymatic product. The voltammetric characteristics of substrate and enzymatic product were investigated, and the parameters of SWV analysis and immunoassay were optimized. Under the optimal conditions the immunosensor was used to measure as low as 5 ng L^?1 (ppt) TCP, which is 50‐fold lower than the value indicated by the manufacture of the TCP RaPID Assay kit (0.25 μg/L, colorimetric detection). The performance of the developed immunosensing system was successfully evaluated with river water samples spiked with TCP, indicating this convenient and sensitive technique offers great promise for decentralized environmental application. This technique could be readily used for detection of other environmental contaminants by developing specific antibodies against the contaminants and are expected to open new opportunities for environmental monitoring and public health.     

Robotic sample pretreatment-immunoassay determination of chlorpyrifos metabolite (TCP) in soil and fruit

A fully automated method for leaching of TCP (3,5,6-trichloro-2-pyridinol, the major degradation product of the widely used chlorpyrifos insecticides) from soil and fruit with subsequent determination by ELISA is reported. The automation of the weighing and leaching steps enables unattended development of sample preparation. The determination of the target analyte, traditionally performed by liquid chromatography, has been substituted by specific immunoassay using a non-commercial monoclonal antibody which enables the determination of TCP within the range 0.01-7 ng 1(-1) with an R.S.D. of 8.6%.     

Determination of 3,5,6-trichloro-2-pyridinol (TCP) in water by a continuous competitive immunoassay system based on the streptavidin-biotin interaction

A competitive continuous immunoassay system for the determination of 3,5,6-trichloro-2-pyridinol (TCP), the major degradation product of the insecticide chlorpyrifos, in water is described. The immunoassay system is based on the transient retention of the specific LIB-MC2 monoclonal antibody anti-TCP as a biotinylated derivative using the streptavidin-biotin interaction. The permanent immobilization of streptavidin on controlled-pore-glass provides an adequate active support for the transient retention of the biotinylated monoclonal antibody anti-TCP. In a subsequent step, the immuno-competitive reaction between the biotinylated LIB-MC2 and the TCP/hapten-POD mixture takes place. This competitive assay relies on the determination of the biocatalytic action of peroxidase, retained in the active support, on a derivatization reaction which yields a fluorescent product. The method exhibits a determination range of 0.01-200 microg L(-1) of TCP (r2=0.9919, n=9) with a precision, expressed as RSD, lower than 4.2% and a sampling frequency of 3 h(-1). The approach has been applied to the determination of TCP in water with recoveries of 89.7-105.6%.     

Modified carbon paste electrode for the electrochemical sensing of 3,5,6-trichloro-2-pyridinol

An electrochemical sensor for the determination of 3,5,6-trichloro-2-pyridinol (TCP), the main metabolite of the pesticide chlorpyrifos, was herein developed. TCP has greater solubility than the source pesticide, and its occurrence in ground and surface water is more frequent and more dangerous. The sensor was fabricated using carbon paste modified with the inorganic complex chloro-5,10,15,20-tetrakis-(pentafluorophenyl)-21 H,23 H-porphyrin iron(III) (FeTPPCl); this metallic complex has a chemical core structure similar to the heme cofactor of the cytochrome P450 (CYPs). Measurements were performed with square-wave voltammetry. Using the optimised voltammetric parameters and without any sample preparation, the sensor showed a limit of detection of 2.8 mg L^?1 (14 μmol L^?1), recoveries ca. 102%, suitable selectivity and long durability (over 1 month).     

A piezoelectric immunosensor for the determination of pesticide residues and metabolites in fruit juices

A quartz crystal microbalance (QCM) immunosensor was developed for the determination of the insecticide carbaryl and 3,5,6-trichloro-2-pyridinol (TCP), the main metabolite of the insecticide chlorpyrifos and of the herbicide triclopyr. The detection was based on a competitive conjugate-immobilized immunoassay format using monoclonal antibodies (MAbs). Hapten conjugates were covalently immobilized, via thioctic acid self-assembled monolayer (SAM), onto the gold electrode sensitive surface of the quartz crystal. This covalent immobilization allowed the reusability of the modified electrode surface for at least one hundred and fifty assays without significant loss of sensitivity. The piezoimmunosensor showed detection limits (analyte concentrations producing 10% inhibition of the maximum signal) of 11 and 7 μg l⁻¹ for carbaryl and TCP, respectively. The sensitivity attained (I₅₀ value) was around 30 μg l⁻¹ for both compounds. Linear working ranges were 15-53 μg l⁻¹ for carbaryl and 13-83 μg l⁻¹ for TCP. Each complete assay cycle took 20 min. The good sensitivity, specificity, and reusability achieved, together with the short response time, allowed the application of this immunosensor to the determination of carbaryl and TCP in fruits and vegetables at European regulatory levels, with high precision and accuracy.     

2-氨基-3-羟基吡啶-过氧化氢-辣根过氧化物酶伏安酶联免疫体系分析人血清癌胚抗原

以氮杂环化合物为电化学分析底物的2-氨基-3-羟基吡啶-H2O2-辣根过氧化物酶(HRP)伏安酶联免疫体系测定人血清癌胚抗原(CEA).HRP催化H2O2氧化2-氨基-3-羟基吡啶的酶促反应产物,在缓冲液中-0.36 V处产生一个灵敏的伏安还原峰,借助此峰可以测定游离的HRP,进而可用于以HRP为标记物的酶联免疫分析.对酶促反应条件和测定条件的优化反应条件为:以B-R缓冲液(pH 6.0)为反应介质,在10 mL总反应液中含有1.0 mL 0.2 mol/L B-R缓冲液、3.0 mL 8.0 mmol/L 2-氨基-3-羟基吡啶溶液以及1.5 mL 0.5 mmol/L H2O2溶液,反应温度37 ℃,反应时间30 min.最佳测定条件为:B-R缓冲液(pH 7.0)为支持电解质,在10 mL总测定溶液中含有5 mL上述总反应液、1.0 mL 0.2 mol/L B-R缓冲液.测定仪器条件:起始电位0.00 V,终止电位-0.80 V,电位扫描速度400 mV/s,滴汞静止时间7 s.在最佳的反应条件和测定条件下,新体系测定游离HRP的线性范围为4.0×10-4～1.0 μg/L; 对HRP的检出限为0.12 ng/L.新体系对CEA测定的线性范围为0.50～80.0 μg/L; 检出限为0.50 μg/L.为经典ELISA法的检出限的1/10.     

Sensitive Electrochemical Detection of Horseradish Peroxidase at Disposable Screen‐Printed Carbon Electrode

A rapid, simple and sensitive electrochemical assay of horseradish peroxidase (HRP) performed on disposable screen‐printed carbon electrode was developed. HRP activities were monitored by square‐wave voltammetric (SWV) measuring the electroactive enzymatic product in the presence of o‐aminophenol and hydrogen peroxide substrate solution. SWV analysis demonstrated a greater sensitivity and shorter analysis time than the widely used amperometric and differential‐pulsed voltammetric methods. The voltammetric characteristics of substrate and enzymatic product as well as the parameters of SWV analysis were optimized. Under optimized conditions, a linear response for HRP from 0.003 to 0.1 U/mL and a detection limit of 0.002 U/mL (1.25×10^?15 mol in 25 μL) were obtained with a good precision (RSD=8%; n=6). This rapid and sensitive HRP assay with microliter‐assay volume could be readily integrated to portable devices and point‐of‐care (POC) diagnosis applications.     

An infrared study of the tautomerism of chlorinated 2-pyridinols isolated in an argon matrix

The infrared spectra of 3-, 4-, 5- and 6-monochloro-, 3,5,6-trichloro-2-pyridinols (pyridones), and of the unsubstituted 2-pyridinol isolated in a low temperature (20°K) argon matrix were studied. The results indicate that the enol/keto ratio for the isolated pyridinols decreases according to the position of the chlorine atom(s) on the pyridine ring; i.e., 3,5,6-trichloro >-6 > 5 > 4 > 3 monochloro-2-pyridinols. 3,5,6-Trichloro-2-pyridinol exists predominantly in the enol form.     

On-line determination of 3,5,6-trichloro-2-pyridinol in human urine samples by surface plasmon resonance immunosensing

An immunochemical method for the analysis of 3,5,6-trichloro-2-pyridinol (TCP), a major urinary metabolite of chlorpyrifos, is developed using a surface plasmon resonance (SPR)-based biosensor. The stability of the assay was assessed by covalently linking the analyte derivative to a thin, gold-modified sensor surface. For optimization of analyte derivative immobilization, sensor chips were activated via alkanethiol monolayers with terminal amine or carboxyl groups. Binding inhibition tests were performed in untreated urine samples and compared to those obtained in distilled water and PBS was used as control. In all cases, similar detection limits, at the micrograms per litre level (0.1–0.24 μg L⁻¹), were attained for TCP assays independently of the dilution buffer. Reproducibility of measurements was studied throughout more than 130 regeneration cycles, which allowed the repeated use of the same immunosensor surface without significant variation of the SPR signal. All measurements were developed in real-time in only 10 min, using a SPR portable system. The device could be applied as a valuable analytical method to both environmental screening and clinic diagnostics.     

Investigation of voltammetric enzyme-linked immunoassay based on a new system of HAP-H2O2-HRP

By introducing heterocyclic compound to immunoassay system as an electrochemical substrate for the fist time, a new voltammetric enzyme-linked immunoassay system of 3-hydroxyl-2-aminopyridine (HAP)-H(2)O(2)-horseradish peroxidase (HRP) has been developed. HAP was oxidized with H(2)O(2) catalyzed by HRP, and the resulting electroactive product produced a sensitive voltammetric peak at potential of -0.36 V (vs. SCE) in Britton-Robinson (BR) buffer solution. The process of the enzyme-catalyzed reaction and the electro-reduction of the product have been investigated in detail. The linear range for detection of free HRP was from 4.0x10(-13) to 1.0x10(-9) g/mL with a detection limit of 1.2x10(-13) g/mL. The new system has been successfully applied for the assay of alpha-fetoprotein (alphaFP) in human serum ranging from 0.1 to 200 ng/mL with a detection limit of 0.1 ng/mL, which was 10 times lower than that of traditional spectrophotometric enzyme-linked immunosorbent assay (ELISA) method. HAP-H(2)O(2)-HRP voltammetric enzyme-linked immunoassay showed a promising alternative approach in the detection of alphaFP in clinical diagnosis.     

Simultaneous determination of chlorpyrifos and 3,5,6-trichloro-2-pyridinol in duck muscle by modified QuEChERS coupled to gas chromatography tandem mass spectrometry (GC-MS/MS)

A rapid, specific, and sensitive method based on modified quick, easy, cheap, effective, rugged, and safe (QuEChERS) coupled to gas chromatography tandem mass spectrometry (GC-MS/MS) was developed and validated for simultaneous determination of chlorpyrifos (CP) and its metabolite 3,5,6-trichloro-2-pyridinol (TCP) in duck muscle. The residues of CP and TCP were extracted by acidified acetonitrile. The fat layer of the extract was removed under ?20 °C, then the organic layer was evaporated. The analytes were derivatized by N-(tert-butyldimethylsilyl)-N-methyltrifluoroacetamide (MTBSTFA) and cleaned up by a mixture of 150 mg MgSO₄, 25 mg graphitized carbon black (GCB), and 50 mg N-propylethylenediamine (PSA) to remove interference. The final extract was analyzed by GC-MS/MS. Recovery values at the spiking concentrations ranged from 86.2 to 92.3 % for CP and from 74.8 to 81.8 % for TCP, with relative standard deviations (RSDs) lower than 9.5 and 12.3, respectively. The correlation coefficients of CP (from 2 to 2,000 μg/kg) and TCP (from 1 to 1,000 μg/kg) were equal to or higher than 0.998. The limits of detection (LODs) were 0.3 and 0.15 μg/kg, and the limits of quantification (LOQs) were 1.0 and 0.5 μg/kg for CP and TCP in duck muscle, respectively. The average intra- and inter-day accuracy ranged from 84.6 to 91.2 % for CP and 75.6 to 82.3 % for TCP, and the intra- and inter-day precisions were from 5.8 to 8.2 % for CP and 6.5 to 11.9 % for TCP. Furthermore, the CP and TCP residues in duck muscle samples were detected for dietary risk assessment using the validated method.

Determination of chlorpyrifos and its metabolites in rat brain tissue using coupled-column liquid chromatography/electrospray ionization tandem mass spectrometry

A method has been developed to quantify chlorpyrifos (O,O-diethyl-O-[3,5,6,-trichloro-2-pyridyl] phosphorothionate) and its metabolites chlorpyrifos-oxon (O,O-diethyl-O-[3,5,6,trichloro-2-pyridinyl] phosphate) and TCP (3,5,6,-trichloro-2-pyridinol) in rat brain tissue by coupled-column liquid chromatography/electrospray ionization tandem mass spectrometry (LC/LC/ESI-MS/MS). Rat brains were homogenized and treated by protein precipitation using ice-cold acetonitrile. The supernatant was directly injected onto the coupled-column system. Sample clean-up was achieved on a Zorbax Extend-C(18) column (2.1 x 50 mm, 5 microm) using a mobile phase of acetonitrile/water with 0.0025% formic acid (40:60, v/v). The compounds were separated isocratically on a Zorbax Eclipse XDB C(8) column (2.0 x 150 mm, 5 microm) using a mobile phase of acetonitrile/water with 0.0025% formic acid (75:25, v/v). Chlorpyrifos and chlorpyrifos-oxon were detected in positive ion mode using multiple reaction monitoring (MRM). TCP was detected in negative ion mode using precursor-to-precursor transition monitoring. The method was validated and the specificity, linearity, limit of quantitation (LOQ), precision, accuracy, stability, and recoveries were determined. Calibration curves for all three analytes yielded correlation coefficients of 0.993 or greater. The LOQs were 25.3 ng/g for chlorpyrifos and 6.3 ng/g for chlorpyrifos-oxon and TCP. All precision relative standard deviations (RSDs) were less than 16% for the LOQ and less than 11% for the other QC samples. This method was successfully applied to six rats that were injected subcutaneously with chlorpyrifos.     

Supercritical fluid extraction of chlorpyrifos and 3,5,6-trichloro-2-pyridinol from garden compost

A method was developed for the simultaneous supercritical carbon dioxide extraction of chlorpyrifos and its primary degradate, 3,5,6-trichloro-2-pyridinol (TCP), from garden compost. In situ derivatization with N,O-bis(trimethylsilyl)trifluoracetamide was necessary for extraction of TCP. Recoveries for TCP and chlorpyrifos were quantitative for spiked compost samples. Sodium chloride was used as the packing material in extractions with in situ derivatization. Optimum results were obtained for air-dried samples containing 4-7% moisture. No sample cleanup was required prior to analysis by GC-flame ionization detection. The effects of compost moisture content and ageing were investigated for chlorpyrifos recovery. No significantly negative effect on recovery for up to 20% (w/w) moisture for chlorpyrifos was observed. Effects of ageing showed a decrease in extraction efficiency over time with 52% recovery after 10 days.     

An improved ELISA for the determination of tobacco mosaic virus with linear sweep voltammetry detection based on a new system of PAP-H2O2-HRP

An improved ELISA for the determination of tobacco mosaic virus (TMV) with linear sweep voltammetry based on a new system of p-aminophenol (PAP)- H₂O₂- horseradish peroxidase (HRP) has been developed. The enzymatic product 3-[(4-hydroxyphenyl)amino]-4-(2-amino-5-hydroxyphenyl)-6-[(4-hydroxyphenyl)imino]-2,4-cyclohexadiene-1-one, produced from HRP catalyzing the oxidation of PAP with H₂O₂, yields a sensitive linear sweep voltammetric response at a potential of –0.45 V (vs. SCE) in Britton-Robinson (BR) buffer solution. By using this voltammetric peak, HRP can be measured with a detection limit of 0.4 mU/L and a linear range of 1.0 ∼ 1.0 × 10² mU/ L. The detection limit for the clarified TMV is 4.0 ng/mL and the highest dilution ratio detected for the infected leaf sap is 1 : 3.9 × 10⁶. The processes of the enzyme-catalyzed reaction and the electro-reduction of the product of the enzyme-catalyzed reaction have been investigated. Received: 17 November 1998 / Revised: 17 March 1999 / Accepted: 20 March 1999     

Enzyme-catalyzed reaction of OPD-H202-HRP voltammetric enzyme-linked immunoassay system

Enzyme-catalyzed reaction of o-phenylenediamine (OPD)-H_z0₂-horseradish peroxidase (HRP) voltammetric enzyme-linked immunoassay system has been studied in detail with electrochemical analysis, high performance liquid chromatography (HPLC), ultraviolet/visible (UV/Vis) spectroscopy, infrared (IR) spectroscopy and nuclear magnetic resonance (NMR) spectroscopy. The pure product of H₂0₂ oxidizing OPD catalyzed by HRP was prepared with chemical method. The experimental results of voltammetry and HPLC indicate that only one product of enzyme-catalyzed reaction has been obtained under the selected enzyme-catalyzed reaction conditions. Identifications by UV/ Vis spectrum, IR spectrum and¹³C NMR spectrum show that the product is 2,3-diaminophenazine. The processes of the enzyme-catalyzed reaction and the electroreduction of the product of the enzyme-catalyzed reaction are described. Project supported by the National Natural Science Foundation of China     

Determination of diazinon, chlorpyrifos, and their metabolites in rat plasma and urine by high-performance liquid chromatography

This study reports a simple and rapid high-performance liquid chromatographic (HPLC) method for the determination of the insecticide diazinon (O,O-diethyl-O[2-isopropyl-6-methylpyridimidinyl] phosphorothioate), its metabolites diazoxon (O,O-diethyl-O-2-isopropyl-6-methylpyridimidinyl phosphate) and 2-isopropyl-6-methyl-4-pyrimidinol, the insecticide chlorpyrifos (O,O-diethyl-O[3,5,6-trichloro-2-pyridinyl] phosphorothioate) and its metabolites chlorpyrifos-oxon (O,O-diethyl-O[3,5,6-trichloro-2-pyridinyl] phosphate), and TCP (3,5,6-trichloro-2-pyridinol) in rat plasma and urine samples. The method is based on using C18 Sep-Pak cartridges for solid-phase extraction and HPLC with a reversed-phase C18 column and programmed UV detection ranging between 254 and 280 nm. The compounds are separated using a gradient of 1% to 80% acetonitrile in water (pH 3.0) at a flow rate ranging between 1 and 1.5 mL/min in a period of 16 min. The limits of detection ranged between 50 and 150 ng/mL, and the limits of quantitation were 100 to 200 ng/mL. The average percentage recovery of five spiked plasma samples were 86.3 +/- 8.6, 77.4 +/- 7.0, 82.1 +/- 8.2, 81.8 +/- 8.7, 73.1 +/- 7.4, and 80.3 +/- 8.0 and from urine were 81.8 +/- 7.6, 76.6 +/- 7.1, 81.5 +/- 7.9, 81.8 +/- 7.1, 73.7 +/- 8.6, and 80.7 +/- 7.7 for diazinon, diazoxon, 2-isopropyl-6-methyl-4-pyrimidinol, chlorpyrifos, chlorpyrifos-oxon, and TCP, respectively. The relationship between the peak area and concentration was linear over a range of 200 to 2,000 ng/mL. This method was applied in order to analyze these chemicals and metabolites following dermal administration in rats.     

白藜芦醇作辣根过氧化物酶底物的布氏杆菌抗体酶联免疫传感器研究

一种纯天然产物白藜芦醇用作辣根过氧化物酶(HRP)底物。对其化学性质的研究证实,白藜芦醇在空气中较稳定,对HRP、H2O2的电化学响应性能优于传统HRP底物,对人体无毒害。白藜芦醇在HRP催化下可被H2O2氧化成醌,产物醌在电极上于-376 mV处可被还原,其电流的大小与HRP的浓试在一定浓度范围内呈线性相关。将兔布氏杆菌抗原包埋在石墨-石蜡基质中制备了测定兔布氏杆菌抗体的电化学酶联免疫传感器,该传感器测定兔布氏杆菌抗体的线性范围为3×10-4~1.65×10-2g/L;检出限为1×10-4g/L;RSD为4.6%。本方法制备免疫传感器的电化学性能稳定,抗原活性保持良好。     

Enzyme-catalyzed reaction of voltammetric enzyme-linked immunoassay system based on OAP as substrate

The o-aminophenol (OAP)-H_2O_2-horseradish peroxidase (HRP) voltammetric enzyme-linked immunoassay new system has extremely high sensitivity. HRP can be measured with a detection limit of 6.0×10~-(10) g/L and a linear range of 1.0×10~(-9)—4.0×10~(-6) g/L. The pure product of H_2O_2 oxidizing OAP catalyzed by HRP was prepared with chemical method. The enzyme-catalyzed reaction has been investigated with electroanalytical chemistry, UV/Vis spectrum, IR spectrum, ~(13)C NMR, ~1H NMR, mass spectrum, elemental analysis, etc. Under the selected enzyme-catalyzed reaction conditions, the oxidation product of OAP with H_2_O2 catalyzed by HRP is 2-aminophe-noxazine-3-one. The processes of the enzyme-catalyzed reaction and the electroreduction of the product of the enzymecatalyzed reaction have been described.     

Reference values of urinary 3,5,6-trichloro-2-pyridinol in the Italian population--validation of analytical method and preliminary results (multicentric study)

The interlaboratory validation of analytical procedures for the assay of urinary 3,5,6-trichloro-2-pyridinol (TCP) in the general Italian population is reported. The determinations were performed by high-resolution gas chromatography (HRGS) with electron capture detection and HRGS with mass spectrometry (MS) in 2 laboratories. The urine samples were from 42 participants from 3 regions of Italy. The results were evaluated by interlaboratory quality control. Urinary TCP concentrations were above the detection limit (1.2 micrograms/L) in 88% of the population, with a mean detectable concentration [GM (GSD)] of 2.8 (1.9) micrograms/g creatinine (creat). (GM, geometric mean; GSD, geometric standard deviation.) The Mann-Whitney U test showed that wine consumption was a statistically significant variable (p < 0.05) for urinary concentrations of TCP. Analysis of variance of the logarithm of urinary TCP versus wine consumption and diet showed a statistically significant fit. The model used explained 30% of the total variance: wine consumption and diet accounted for 37 and 17% respectively of the explained variance.     

Electrochemical studies of 1,5-dihydroxynaphthalene as substrate for voltammetric enzyme immunoassay

Immunoassay is one of the biochemical analytical techniques using the specific antigen antibody com-plexation for analytical purposes. It has extensive ap-plication in clinical diagnostics, prevention and cure of diseases, and virus diagnostics. The presentation and progress of immunoassay methodology are one of the greatest achievements of bioanalytical chemistry. It is estimated that several-hundred millions of immuno-analytical determinations are carried out every year all over the world. E…