Automated flow enzyme-linked immunosorbent assay (ELISA) system for analysis of methyl parathion

Sensitive detection of pesticides is of utmost importance in environment and food analysis. Immunological methods are widely used to detect pesticides in agricultural and environmental samples wherein antibodies are employed against the target molecules. Accurate diagnosis depends on the affinity and specificity of the antibody preparation used, and high affinity antibodies are essential for the detection of very small amounts of pesticides. Enzyme linked immuno sorbent assay (ELISA) coupled with flow injection analysis (FIA) technique provides a very high sensitivity with high throughput of analyses. Automation of this analysis scheme ensures precise detection with high accuracy. The present development aims at providing a user-friendly system for achieving this objective. It employs a 8952 microcontroller for precise flow of reagents, samples, substrate and conjugates used for analysis to be passed through an immobilized antibody column at predetermined time. With the sequence and flow control of buffers used, it also provides the option for reuse of the immobilized antibody column. The system is flexible to accommodate multiple sequences up to a maximum of 99 steps. It is customizable for different flow ELISA applications. It can control up to eight solenoid valves (dc 24 V) and two peristaltic pumps and has one 12 bit analog channel for data acquisition. With the serial interface port, the system provides convenient means for data acquisition into the computer. The system has been successfully tested for immuno analysis of organophosphorous pesticide methyl parathion.     

Potentiometric stripping analysis of methyl and ethyl parathion employing carbon nanoparticles and halloysite nanoclay modified carbon paste electrode

Carbon nanoparticles (CNPs) and halloysite nanoclay (HNC) modified carbon paste electrode (HNC–CNP–CPE) was developed for the determination of methyl parathion (MP) and ethyl parathion (EP). The electrochemical behavior of these molecules was investigated employing cyclic voltammetry (CV), chronocoulometry (CC), electrochemical impedance spectroscopy (EIS) and potentiometric stripping analysis (PSA). After optimization of analytical conditions employing this electrode at pH 5.0 in acetate buffer (0.1 M), the peak currents were found to vary linearly with its concentration in the range of 1.55 × 10⁻⁹ to 3.67 × 10⁻⁶ M and 1.21 × 10⁻⁹ to 4.92 × 10⁻⁶ M for MP and EP, respectively. The detection limits (S/N = 3) of 4.70 × 10⁻¹⁰ M and 3.67 × 10⁻¹⁰ M were obtained for MP and EP, respectively, using PSA. The prepared modified electrode showed several advantages such as simple preparation method, high sensitivity, very low detection limits and excellent reproducibility. The proposed method was employed for the determination of MP and EP in fruits, vegetables, water and soil samples.     

Square wave adsorptive cathodic stripping voltammetry automated by sequential injection analysis Potentialities and limitations exemplified by the determination of methyl parathion in water samples

This paper describes the development and evaluation of a sequential injection method to automate the determination of methyl parathion by square wave adsorptive cathodic stripping voltammetry exploiting the concept of monosegmented flow analysis to perform in-line sample conditioning and standard addition. Accumulation and stripping steps are made in the sample medium conditioned with 40 mmol L⁻¹ Britton-Robinson buffer (pH 10) in 0.25 mol L⁻¹ NaNO₃. The homogenized mixture is injected at a flow rate of 10 μL s⁻¹ toward the flow cell, which is adapted to the capillary of a hanging drop mercury electrode. After a suitable deposition time, the flow is stopped and the potential is scanned from −0.3 to −1.0 V versus Ag/AgCl at frequency of 250 Hz and pulse height of 25 mV. The linear dynamic range is observed for methyl parathion concentrations between 0.010 and 0.50 mg L⁻¹, with detection and quantification limits of 2 and 7 μg L⁻¹, respectively. The sampling throughput is 25 h⁻¹ if the in line standard addition and sample conditioning protocols are followed, but this frequency can be increased up to 61 h⁻¹ if the sample is conditioned off-line and quantified using an external calibration curve. The method was applied for determination of methyl parathion in spiked water samples and the accuracy was evaluated either by comparison to high performance liquid chromatography with UV detection, or by the recovery percentages. Although no evidences of statistically significant differences were observed between the expected and obtained concentrations, because of the susceptibility of the method to interference by other pesticides (e.g., parathion, dichlorvos) and natural organic matter (e.g., fulvic and humic acids), isolation of the analyte may be required when more complex sample matrices are encountered.     

A nanosilica/exfoliated graphene composite film-modified electrode for sensitive detection of methyl parathion

Graphene nanosheets (GS) were easily prepared through liquid-phase exfoliation of graphite powder in N,N-dimethylformamide (DMF) with the assistance of sodium citrate. Then, GS was coated onto a glassy carbon electrode (GCE) surface by drop to fabricate a GS/GCE nanointerface. Subsequently, by using tetraethylorthosilicate sol as precursor, nanosilica was electrochemically deposited onto the GS/GCE surface to produce a nanocomposite film electrode (nanosilica/GS/GCE). Electrochemical behaviors of methyl parathion (MP) on the nanosilica/GS/GCE surface were investigated thoroughly. It was found that the nanosilica/GS nanocomposites can improve the redox peak currents of MP significantly due to the synergetic effect. The oxidation peak current was linearly related to MP concentration in the range from 0.0005 μmol/L to 5.6 μmol/L. The detection limit was calculated to be 0.07 nmol/L (S/N=3). The developed method was used to determine MP in real samples. The recoveries were in the range from 95.4% to 104.2%, demonstrating satisfactory results.     

Application of methyl parathion hydrolase (MPH) as a labeling enzyme

Methyl parathion hydrolase (MPH) is an enzyme that catalyzes the degradation of methyl parathion, generating a yellow product with specific absorption at 405 nm. The application of MPH as a new labeling enzyme was illustrated in this study. The key advantages of using MPH as a labeling enzyme are as follows: (1) unlike alkaline phosphatase (AP), horseradish peroxidase (HRP), and glucose oxidase (GOD), MPH is rarely found in animal cells, and it therefore produces less background noise; (2) its active form in solution is the monomer, with a molecular weight of 37 kDa; (3) its turnover number is 114.70 ± 13.19 s⁻¹, which is sufficiently high to yield a significant signal for sensitive detection; and (4) its 3D structure is known and its C-terminal that is exposed to the surface can be easily subjected to the construction of genetic engineering monocloning antibody–enzyme fusion for enzyme-linked immunosorbent assay (ELISA). To demonstrate its utility, MPH was ligated to an single-chain variable fragment (scFv), known as A1E, against a white spot syndrome virus (WSSV) with the insertion of a [–(Gly–Ser)₅–] linker peptide. The resulting fusion protein MPH-A1E possessed both the binding specificity of the scFv segment and the catalytic activity of the MPH segment. When MPH-A1E was used as an ELISA reagent, 25 ng purified WSSV was detected; this was similar to the detection sensitivity obtained using A1E scFv and the HRP/Anti-E Tag Conjugate protocol. The fusion protein also recognized the WSSV in 1 μL hemolymph from an infected shrimp and differentiated it from a healthy shrimp. Figure The 3-D structure of MPH. (a) monomer showing C- and N-terminals; (b) the crystal structure of the dimer W. Yang and Y.-F. Zhou contributed equally to this work.     

Cloud-point extraction of nodularin-R from natural waters

A cloud-point extraction (CPE) technique for the determination of a cyanobacterial hepatotoxin, nodularin-R, in aqueous media using a cationic surfactant, tricaprylmethylammonium chloride (Aliquat-336), was developed. Cloud-point phase separation of Aliquat-336 at ambient temperature was induced by the addition of sodium sulfate. The Aliquat-336/Na₂SO₄ CPE system displayed large preconcentration factor, F_C, for nodularin-R. At the operational CPE conditions, F_C of 709.2 was achieved. Distribution constant, K_D, of the distribution of nodularin-R between the surfactant-rich and aqueous phases of the CPE system was estimated to be (4.94±0.8)×10³. Coupled to liquid chromatography with UV detection, the CPE method offered a detection limit of 330 pg ml⁻¹ (in freshwater)/1.3 ng ml⁻¹ (in seawater) and a repeatability of 6.4% (in freshwater) (n=7, P<0.05) for nodularin-R in a sample of 25 ml. The CPE is a rapid process and no cleanup step is required. Effects of pH, natural abundant anion (chloride) and dissolved organic matters (DOM, humic acid, HA) on the extraction efficiency were evaluated. A double CPE technique was developed to overcome interferences encountered in the analysis of environmental samples. Applicability of the new method to the determination of nodularin-R in real coastal marine water samples has also been demonstrated.     

A new fluorescence probing strategy for the detection of parathion-methyl based on N-doped carbon dots and methyl parathion hydrolase

A new facile fluorescence probing strategy, which was based on N-doped carbon dots(NCDs) and methyl parathion hydrolase(MPH), was developed for the determination of parathion-methyl(PM). The fluorescence intensity of NCDs-MPH system was proportional to PM concentration in the range of 2.38–73.78 mmol/L, with a detection limit of 0.338 mmol/L. Moreover, the present simple and facile method could be used to determine methyl parathion in environmental and agricultural samples successfully.Furthermore, the detection mechanism of this system is inner filter effect and molecular interactions between NCDs and p-nitrophenol, which is the hydrolysis product of PM catalyzed by methyl parathion hydrolase.     

Voltammetric methods for speciation analysis of trace metals in natural waters

Trace metals play an important role in the regulation of primary productivity and phytoplankton community composition. Metal species directly affects the biogeochemical cycling processes, transport, fate, bioavailability and toxicity of trace metals. Therefore, developing powerful methods for metal speciation analysis is very useful for research in a range of fields, including chemical and environmental analysis. Voltammetric methods, such as anodic stripping voltammetry (ASV) and competing ligand exchange-adsorptive cathodic stripping voltammetry (CLE-AdCSV), have been widely adopted for speciation analysis of metals in different natural aquatic systems. This paper provides an overview of the theory of voltammetric methods and their application for metal speciation analysis in natural waters, with a particular focus on current voltammetric methods for the discrimination of labile/inert fractions, redox species and covalently bound species. Speciation analysis of typical trace metals in natural waters including Fe, Cu, Zn, Cd, and Pb are presented and discussed in detail, with future perspectives for metal speciation analysis using voltammetric methods also discussed. This review can elaborate the particular knowledge of theory, merits, application and future challenge of voltammetric methods for speciation analysis of trace metals in natural waters.     

para-Sulfonatocalix[6]arene-modified silver nanoparticles electrodeposited on glassy carbon electrode: Preparation and electrochemical sensing of methyl parathion

In this paper, a new electrochemical sensor, based on modified silver nanoparticles, was fabricated using one-step electrodeposition approach. The para-sulfonatocalix[6]arene-modified silver nanoparticles coated on glassy carbon electrode (pSC₆-Ag NPs/GCE) was characterized by attenuated total reflection IR spectroscopy (ATR-IR), X-ray photoelectron spectroscopy (XPS) and scanning electron microscopy (SEM), etc. The pSC₆ as the host are highly efficient to capture organophosphates (OPs), which dramatically facilitates the enrichment of nitroaromatic OPs onto the electrochemical sensor surface. The combination of the host-guest supramolecular structure and the excellent electrochemical catalytic activities of the pSC₆-Ag NPs/GCE provides a fast, simple, and sensitive electrochemical method for detecting nitroaromatic OPs. In this work, methyl parathion (MP) was used as a nitroaromatic OP model for testing the proposed sensor. In comparison with Ag NPs-modified electrode, the cathodic peak current of MP was amplified significantly. Differential pulse voltammetry was used for the simultaneous determination of MP. Under optimum conditions, the current increased linearly with the increasing concentration of MP in the range of 0.01-80 μM, with a detection limit of 4.0 nM (S/N = 3). The fabrication reproducibility and stability of the sensor is better than that of enzyme-based electrodes. The possible underlying mechanism is discussed.     

Size exclusion chromatography of aluminium species in natural waters

Size exclusion chromatography was used in order to characterize organically bound aluminium in natural water samples. A Superose column was used, with 0.1 M acetate buffer (pH 4.6) as mobile phase. Three detection systems were used; graphite furnace atomic absorption spectrometry, post-column reaction with pyrocatechol violet and UV spectrometry at 254 nm. A single peak was obtained for organic aluminium in natural waters. The results indicated that aluminium binds with a broad size range of humic substances, and that the inorganic aluminium was present in polymeric form.     

蛋白质组学技术筛选与鉴定在甲基对硫磷胁迫下牙鲆脑组织表达的差异蛋白质

以甲基对硫磷(MP)为有机磷农药污染源,采用蛋白质组学技术分离及鉴定在甲基对硫磷胁迫下,牙鲆(Paralichthys olivaceus)脑组织表达的差异蛋白质,从中筛选出潜在的适合于监测甲基对硫磷污染程度的蛋白指示物。实验结果表明:在甲基对硫磷胁迫下,牙鲆脑组织表达出17个差异蛋白质,经肽质量指纹(PMF)图谱技术鉴定后,发现其中部分差异蛋白质为热休克蛋白、细胞色素P450和谷胱甘肽S-转移酶,均是与受甲基对硫磷胁迫有关的蛋白质。     

Determination of phosphorus in natural waters: A historical review

The aim of this paper is to introduce a virtual special issue that reviews the development of analytical approaches to the determination of phosphorus species in natural waters. The focus is on sampling and sample treatment, analytical methods and quality assurance of the data. The export of phosphorus from anthropogenic activities (from diffuse and point sources) can result in increased primary production and eutrophication, and potentially the seasonal development of toxic algal blooms, which can significantly impact on water quality. Therefore the quantification of phosphorus species in natural waters provides important baseline data for studying aquatic phosphorus biogeochemistry, assessing ecosystem health and monitoring compliance with legislation.     

Speciation of chromium in natural waters by micropumping multicommutated light emitting diode photometry

A simple and sensitive multicommutated flow procedure, implemented by employing a homemade light emitting diode (LED) based photometer, has been developed for the determination of chromium (VI) and total chromium in water. The flow system comprised a set of four solenoid micro-pumps, which were assembled to work as fluid propelling and as commutating devices. The core of the detection unit comprised a green LED source, a photodiode and a homemade flow cell of 100 mm length and 2 mm inner diameter. The photometric procedure for the speciation of chromium in natural waters was based on the reaction of Cr (VI) with 1,5-diphenylcarbazide. Cr (III) was previously oxidized to Cr (VI) and determined as the difference between total Cr and Cr (VI). After carrying out the assays to select the best operational conditions the features of the method included: a linear response ranging from 10 to 200 μg l⁻¹ Cr (III) and Cr (VI) (r = 0.999, n = 7); limits of detection of 2.05 and 1.0 μg l⁻¹ for Cr (III) and Cr (VI), respectively; a relative standard deviation lower than 2.0% (n = 20) for a typical solution containing 50 μg l⁻¹ Cr; a sampling throughput of 67 and 105 determinations per hour for total Cr and Cr (VI), respectively, and recovery values within the range of 93-108% for spiked concentrations of the order of 50 μg l⁻¹.     

Multianalyte imaging in one-shot format sensors for natural waters

A one-shot multisensor based on ionophore-chromoionophore chemistry for optical monitoring of potassium, magnesium and hardness in water is presented. The analytical procedure uses a black and white non-cooled CCD camera for image acquisition of the one-shot multisensor after reaction, followed by data treatment for quantitation using the grey value pixel average from a defined region of interest from each sensing area to build the analytical parameter 1 − α. In optimised experimental conditions, the procedure shows a large linear range, up to 6 orders using the linearised model and good detection limits: 9.92 × 10⁻⁵ mM, 1.86 × 10⁻³ mM and 1.30 × 10⁻² mg L⁻¹ of CaCO₃ for potassium, magnesium and hardness, respectively. This analysis system exhibits good precision in terms of relative standard deviation (RSD%) from 2.3 to 3.8 for potassium, from 5.0 to 6.8 for magnesium and from 5.4 to 5.9 for hardness. The trueness of this multisensor procedure was demonstrated comparing it with results obtained by a DAD spectrophotometer used as a reference. Finally, it was satisfactorily applied to the analysis of these analytes in miscellaneous samples, such as water and beverage samples from different origins, validating the results against atomic absorption spectrometry (AAS) as the reference procedure.     

Determination of dissolved organic nitrogen in natural waters using high-temperature catalytic oxidation

Studies on nitrogen in natural waters have generally focussed on dissolved inorganic nitrogen (DIN), primarily because of relative ease of analysis and the important influence of DIN on water quality. Advances in analytical techniques now permit the systematic study of dissolved organic nitrogen (DON), and this work has shown that DON is quantitatively significant in many waters. This article describes the sampling and analytical protocols required for rapid, precise and reliable determinations of DON, involving high-temperature catalytic oxidation (HTCO), coupled to chemiluminescence detection. This approach simultaneously determines dissolved organic carbon (DOC) and total dissolved nitrogen (TDN), and DON is derived by subtraction of DIN measured by colorimetry. The DON determination is simple to perform, exhibits excellent precision (<1% for C and 1.5% for N) and is applicable to a wide range of natural waters.     

Dual‐opposite multi‐walled carbon nanotube modified carbon fiber microelectrode for microfluidic chip‐capillary electrophoresis determination of methyl parathion metabolites in human urine

Methyl parathion (MP) is a highly toxic organophosphate and its exposure may lead to substantial adverse effects to human health. The existence of 4‐nitrophenol (4‐NP) in the form of free phenol, glucuronide (4‐NP‐G) or as a sulfate ester (4‐NP‐S) can be used as biomarkers to assess the duration and extent of MP exposure. In this work, a MC‐CE device incorporating post‐CE amperometric detection using multi‐walled carbon nanotubes (MWNTs) modified carbon fiber microelectrode (CFME) was fabricated and assessed for simultaneous determination of 4‐NP, 4‐NP‐G, and 4‐NP‐S in human urine. The detection sensitivity and stability was greatly enhanced by the modification of MWNTs. The capability of the MC‐CE device with dual MWNTs modified CFME for detecting impurity was assessed and reliability established by high recoveries from 95 to 97% for spiked MP biomarkers. The method developed is shown to provide a simple, sensitive, and reliable means for monitoring 4‐NP, 4‐NP‐G, and 4‐NP‐S in human urine.     

Application of a novel chemometric approach to the determination of aqueous photolysis rates of organic compounds in natural waters

The utility of multivariate optimization methods in the determination of aqueous photolysis rates of organic compounds is examined in this study. A basic pursue was to designate the appropriate experimental design plan that extend the analytical utility of multivariate methods from qualitative data interpretation approaches, as applied thus far, to quantitative estimation methods. A three-level second-order central composite design with parameter concentrations (factor levels) beyond the environmental realistic concentrations was employed for that purpose enabling statistically significant effects to be determined. Method application is demonstrated in the first photodegradation study of two UV absorbing chemicals in natural waters. The results suggest that the proposed approach of enables a good approximation of the real behavior in terms of both qualitative and quantitative data interpretation with minimal loss of information.     

Detection of s-triazine pesticides in natural waters by modified large-volume direct injection HPLC

There is a need for simple and inexpensive methods to quantify potentially harmful persistent pesticides often found in our water-ways and water distribution systems. This paper presents a simple, relatively inexpensive method for the detection of a group of commonly used pesticides (atrazine, simazine and hexazinone) in natural waters using large-volume direct injection high performance liquid chromatography (HPLC) utilizing a monolithic column and a single wavelength ultraviolet-visible light (UV-vis) detector. The best results for this system were obtained with a mobile phase made up of acetonitrile and water in a 30:70 ratio, a flow rate of 2.0 mL min⁻¹, and a detector wavelength of 230 nm. Using this method, we achieved retention times of less than three minutes, and detection limits of 5.7 μg L⁻¹ for atrazine, 4.7 μg L⁻¹ for simazine and 4.0 μg L⁻¹ for hexazinone. The performance of this method was validated with an inter-laboratory trial against a National Association of Testing Authorities (NATA) accredited liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) method commonly used in commercial laboratories.     

Determination of calcium and total hardness in natural waters using a potentiometric sensor array

The determination of calcium and total hardness in natural waters is carried out with a potentiometric sensor array which consists of a series of ion-selective electrodes (ISEs) for Ca²⁺, Mg²⁺, NH₄⁺, K⁺, Na⁺, Li⁺, and H⁺. The selectivity of the calcium and magnesium ISEs is not fully achieved as other cation species may interfere with the analysis. The proposed sensor array device can overcome this drawback since it can take advantage of the cross-selectivities of cation species towards each ISE. In this approach, the multivariate data generated by the sensor array results in a richer source of analytical information which allows the quantification of calcium and total hardness in the water samples by means of chemometric methods. Results obtained are in reasonable concordance with those given by the standard method based on complexometry.