Magnetic electrochemical immunoassays with quantum dot labels for detection of phosphorylated acetylcholinesterase in plasma

A new magnetic electrochemical immunoassay has been developed as a tool for biomonitoring exposures to organophosphate (OP) compounds, e.g., insecticides and chemical nerve agents, by directly detecting organophosphorylated acetylcholinesterase (OP-AChE). This immunoassay uniquely incorporates highly efficient magnetic separation with ultrasensitive square wave voltammetry (SWV) analysis with quantum dots (QDs) as labels. A pair of antibodies was used to achieve the specific recognition of OP-AChE that was prepared with paraoxon as an OP model agent. Antiphosphoserine polyclonal antibodies were anchored on amorphous magnetic particles preferably chosen to capture OP-AChE from the sample matrixes by binding their phosphoserine moieties that were exposed through unfolding the protein adducts. This was validated by electrochemical examinations and enzyme-linked immunosorbent assays. Furthermore, antihuman AChE monoclonal antibodies were labeled with cadmium-source QDs to selectively recognize the captured OP-AChE, as characterized by transmission electron microscopy. The subsequent electrochemical SWV analysis of the cadmium component released by acid from the coupled QDs was conducted on disposable screen-printed electrodes. Experimental results indicated that the SWV-based immunoassays could yield a linear response over a broad concentration range of 0.3-300 ng/mL OP-AChE in human plasma with a detection limit of 0.15 ng/mL. Such a novel electrochemical immunoassay holds great promise as a simple, selective, sensitive, and field-deployable tool for the effective biomonitoring and diagnosis of potential exposures to nerve agents and pesticides

Biomonitoring of organophosphorus agent exposure by reactivation of cholinesterase enzyme based on carbon nanotube-enhanced flow-injection amperometric detection

A portable, rapid, and sensitive assessment of subclinical organophosphorus (OP) agent exposure based on reactivation of cholinesterase (ChE) from OP-inhibited ChE using rat saliva (in vitro) was developed using an electrochemical sensor coupled with a microflow-injection system. The sensor was based on a carbon nanotube (CNT)-modified screen printed carbon electrode (SPE), which was integrated into a flow cell. Because of the extent of interindividual ChE activity variability, ChE biomonitoring often requires an initial baseline determination (noninhibited) of enzyme activity which is then directly compared with activity after OP exposure. This manuscript describes an alternative strategy where reactivation of the phosphorylated enzyme was exploited to enable measurement of both inhibited and baseline ChE activity (after reactivation by an oxime, i.e., pralidoxime iodide) in the same sample. The use of CNT makes the electrochemical detection of the products from enzymatic reactions more feasible with extremely high sensitivity (5% ChE inhibition) and selectivity. Paraoxon was selected as a model OP compound for in vitro inhibition studies. Some experimental parameters, e.g., inhibition and reactivation time, have been optimized such that 92-95% of ChE reactivation can be achieved over a broad range of ChE inhibition (5-94%) with paraoxon. The extent of enzyme inhibition using this electrochemical sensor correlates well with conventional enzyme activity measurements. On the basis of the double determinations of enzyme activity, this flow-injection device has been successfully used to detect paraoxon inhibition efficiency in saliva samples (95% of ChE activity is due to butyrylcholinesterase), which demonstrated its promise as a sensitive monitor of OP exposure in biological fluids. Since it excludes inter- or intraindividual variation in the normal levels of ChE, this new CNT-based electrochemical sensor thus provides a sensitive and quantitative tool for point-of-care assessment and noninvasive biomonitoring of the exposure to OP pesticides and chemical nerve agents

Nanotechnology-Based Electrochemical Sensors for Biomonitoring Chemical Exposures

The coupling of dosimetry measurements and modeling represents a promising strategy for deciphering the relationship between chemical exposure and disease outcome. To support the development and implementation of biological monitoring programs, quantitative technologies for measuring xenobiotic exposure are needed. The development of portable nanotechnology-based electrochemical sensors has the potential to meet the needs for low cost, rapid, high-throughput and ultrasensitive detectors for biomonitoring an array of chemical markers. Highly selective electrochemical (EC) sensors capable of pM sensitivity, high-throughput and low sample requirements (<50uL) are discussed. These portable analytical systems have many advantages over currently available technologies, thus potentially representing the next-generation of biomonitoring analyzers. This manuscript highlights research focused on the development of field-deployable analytical instruments based on EC detection. Background information and a general overview of EC detection methods and integrated use of nanomaterials in the development of these sensors are provided. New developments in EC sensors using various types of screen-printed electrodes, integrated nanomaterials, and immunoassays are presented. Recent applications of EC sensors for assessing exposure to pesticides or detecting biomarkers of disease are highlighted to demonstrate the ability to monitor chemical metabolites, enzyme activity, or protein biomarkers of disease. In addition, future considerations and opportunities for advancing the use of EC platforms for dosimetric studies are discussed

Pharmacokinetics and pharmacodynamics of chlorpyrifos and 3,5,6-trichloro-2-pyridinol in rat saliva after chlorpyrifos administration

Sensors have been developed for noninvasive biomonitoring of the organophosphate pesticide chlorpyrifos (CPF), and previous studies have suggested consistent partitioning of 3,5,6-trichloro-2-pyridinol (TCPy), a metabolite of CPF, into saliva after exposure to TCPy. The objective of this study was to quantitatively evaluate in vivo pharmacokinetics and pharmacodynamics of CPF and TCPy in saliva after CPF administration. Rats were coadministered CPF (0.5-5mg/kg) and pilocarpine (~13 mg/kg) iv. Saliva and blood were collected, and levels of CPF, TCPy, and cholinesterase (ChE) activity were quantified. Experimental results suggest that CPF is rapidly metabolized after iv administration. Formation of TCPy from administered CPF at the low dose (0.5 mg/kg) was slower than from higher CPF doses, potentially due to differences in plasma protein binding to CPF. CPF was measured in saliva only at the first time point sampled (0-15 min), indicating low partitioning and rapid metabolism. After formation, TCPy pharmacokinetics were very similar in blood and saliva. Saliva/blood TCPy concentration ratios were not affected by TCPy concentration in blood, saliva flow rate, or salivary pH and were consistent with previous studies. ChE activity in plasma demonstrated a dose-dependent decrease, and ChE activity in saliva was extremely variable and demonstrated no dose relationship. A physiologically based pharmacokinetic and pharmacodynamic model for CPF was modified and predicted the data reasonably well. It is envisioned that a combination of biomonitoring compounds like TCPy in saliva coupled with computational modeling will form an approach to measure pesticide exposure to susceptible human populations such as agricultural workers

Detection of Cd, Pb, and Cu in non-pretreated natural waters and urine with thiol functionalized mesoporous silica and Nafion composite electrodes

Electrochemical sensors have great potential for environmental monitoring of toxic metal ions in waters due to their portability, field-deployability and excellent detection limits. However, electrochemical sensors employing mercury-free approaches typically suffer from binding competition for metal ions and fouling by organic substances and surfactants in natural waters, making sample pretreatments such as wet ashing necessary. In this work, we have developed mercury-free sensors by coating a composite of thiol self-assembled monolayers on mesoporous supports (SH-SAMMS(™)) and Nafion on glassy carbon electrodes. With the combined benefit of SH-SAMMS(™) as an outstanding metal preconcentrator and Nafion as an antifouling binder, the sensors could detect 0.5 ppb of Pb and 2.5 ppb of Cd in river water, Hanford groundwater, and seawater with a minimal amount of preconcentration time (few minutes) and without any sample pretreatment. The sensor could also detect 2.5 ppb of Cd, Pb, and Cu simultaneously. The electrodes have long service times and excellent single and inter-electrode reproducibility (5% RSD after 8 consecutive measurements). Unlike SAMMS(™)-carbon paste electrodes, the SAMMS(™)-Nafion electrodes were not fouled in samples containing albumin and successfully detected Cd in human urine. Other potentially confounding factors affecting metal detection at SAMMS(™)-Nafion electrodes were studied, including pH effect, transport resistance of metal ions, and detection interference. With the ability to reliably detect low metal concentration ranges without sample pretreatment and fouling, SAMMS(™)-Nafion composite sensors have the potential to become the next generation metal analyzers for environmental and bio-monitoring of toxic metals