Jordan N. Smith

Quantum dot-based immunochromatographic fluorescent biosensor for biomonitoring trichloropyridinol, a biomarker of exposure to chlorpyrifos.

A novel and portable fluorescent sensor that integrates an immunochromatographic test strip assay (ITSA) with a quantum dot (QD) label and a test strip reader was described in this study for simple, rapid, and sensitive biomonitoring of an organophosphorus pesticide metabolite. The principle of this sensor is based on a competitive immunoreaction that was performed on an immunochromatographic test strip, where analytes compete with competitors (QD-conjugated analogs) to bind to antibodies on a test zone. Captured QDs serve as signal vehicles for fluorescent readout. In this work, 3,5,6-trichloropyridinol (TCP) is used as a model analyte to demonstrate the performance of the immunosensor. QD-TCP conjugates were synthesized and characterized with X-ray photoelectron spectroscopy (XPS) and fluorescence spectroscopy. Some parameters (e.g., the amount of QD-modified TCP and immunoreaction time) that govern sensitivity and reproducibility of ITSA were optimized. Under optimal conditions, the sensor has a wide dynamic range and is capable of detecting a minimum 1.0 ng/mL TCP standard analyte in 15 min. The sensor has been successfully applied for detection of TCP spiked in rat plasma with average recovery of 102.0%. Results demonstrate that this sensor provides a rapid, clinically accurate, and quantitative tool for TCP detection and shows great promise for in-field and point-of-care (POC) quantitative testing and screening for metabolite biomarkers, e.g., TCP, for humans exposed to 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.

Imaging nicotine in rat brain tissue by use of nanospray desorption electrospray ionization mass spectrometry.

Imaging mass spectrometry offers simultaneous spatially resolved detection of drugs, drug metabolites, and endogenous substances in a single experiment. This is important when evaluating effects of a drug on a complex organ system such as the brain, where there is a need to understand how regional drug distribution impacts function. Nanospray desorption electrospray ionization, nano-DESI, is a new ambient technique that enables spatially resolved analysis of a variety of samples without special sample pretreatment. This study introduces an experimental approach for accurate spatial mapping of drugs and metabolites in tissue sections by nano-DESI imaging. In this approach, an isotopically labeled standard is added to the nano-DESI solvent to compensate for matrix effects and ion suppression. The analyte image is obtained by normalizing the analyte signal to the signal of the standard in each pixel. We demonstrate that the presence of internal standard enables online quantification of analyte molecules extracted from tissue sections. Ion images are subsequently mapped to the anatomical brain regions in the analyzed section by use of an atlas mesh deformed to match the optical image of the section. Atlas-based registration accounts for the physical variability between animals, which is important for data interpretation. The new approach was used for mapping the distribution of nicotine in rat brain tissue sections following in vivo drug administration. We demonstrate the utility of nano-DESI imaging for sensitive detection of the drug in tissue sections with subfemtomole sensitivity in each pixel of a 27 μm × 150 μm area. Such sensitivity is necessary for spatially resolved detection of low-abundance molecules in complex matrices.

Magnetic Electrochemical Sensing Platform for Biomonitoring of Exposure to Organophosphorus Pesticides and Nerve Agents Based on Simultaneous Measurement of Total Enzyme Amount and Enzyme Activity

We report a new approach for electrochemical quantification of enzymatic inhibition and phosphorylation for biomonitoring of exposure to organophosphorus (OP) pesticides and nerve agents based on a magnetic bead (MB) immunosensing platform. The principle of this approach is based on the combination of MB immunocapture-based enzyme activity assay and competitive immunoassay of the total amount of enzyme for simultaneous detection of enzyme inhibition and phosphorylation in biological fluids. Butyrylcholinesterase (BChE) was chosen as a model enzyme. In competitive immunoassay, the target BChE in a sample competes with the BChE immobilized on the MBs to bind to the limited sites of anti-BChE antibody labeled with quantum dots (QD-anti-BChE), followed by stripping voltammetric analysis of the bound QD conjugate on the MBs. This assay shows a linear response over the total BChE concentration range of 0.1-20 nM. Simultaneous real time BChE activity was measured on an electrochemical carbon nanotube-based sensor coupled with a microflow injection system after immunocapture by the MB-anti-BChE conjugate. Therefore, the formed phosphorylated BChE adduct (OP-BChE) can be estimated by the difference values of the total amount of BChE (including active and OP-inhibited) and active BChE from established calibration curves. This approach not only eliminates the difficulty in screening of low-dose OP exposure (less than 20% inhibition of BChE) because of individual variation of BChE values but also avoids the drawback of the scarce availability of OP-BChE antibody. It is sensitive enough to detect 0.5 nM OP-BChE, which is less than 2% BChE inhibition. This method offers a new method for rapid, accurate, selective, and inexpensive quantification of OP-BChE and enzyme inhibition for biomonitoring of OP and nerve agent exposures.

A novel immunochromatographic electrochemical biosensor for highly sensitive and selective detection of trichloropyridinol, a biomarker of exposure to chlorpyrifos.

We present a novel portable immunochromatographic electrochemical biosensor (IEB) for simple, rapid, and sensitive biomonitoring of trichloropyridinol (TCP), a metabolite biomarker of exposure to organophosphorus insecticides. Our new approach takes the advantage of immunochromatographic test strip for a rapid competitive immunoreaction and a disposable screen-printed carbon electrode for a rapid and sensitive electrochemical analysis of captured HRP labeling. Several key experimental parameters (e.g. immunoreaction time, the amount of HRP labeled TCP, concentration of the substrate for electrochemical measurements, and the blocking agents for the nitrocellulose membrane) were optimized to achieve a high sensitivity, selectivity and stability. Under optimal conditions, the IEB has demonstrated a wide linear range (0.1-100 ng/ml) with a detection limit as low as 0.1 ng/ml TCP. Furthermore, the IEB has been successfully applied for biomonitoring of TCP in the rat plasma samples with in vivo exposure to organophosphorus insecticides like Chlorpyrifos-oxon (CPF-oxon). The IEB thus opens up new pathways for designing a simple, rapid, clinically accurate, and quantitative tool for TCP detection, as well as holds a great promise for in-field screening of metabolite biomarkers, e.g., TCP, for humans exposed to organophosphorus insecticides.

Comparative chlorpyrifos pharmacokinetics via multiple routes of exposure and vehicles of administration in the adult rat

Chlorpyrifos (CPF) is a commonly used organophosphorus pesticide. A number of toxicity and mechanistic studies have been conducted in animals, where CPF has been administered via a variety of different exposure routes and dosing vehicles. This study compared chlorpyrifos (CPF) pharmacokinetics using oral, intravenous (IV), and subcutaneous (SC) exposure routes and corn oil, saline/Tween 20, and dimethyl sulfoxide (DMSO) as dosing vehicles. Two groups of rats were co-administered target doses (5 mg/kg) of CPF and isotopically labeled CPF (L-CPF). One group was exposed by both oral (CPF) and IV (L-CPF) routes using saline/Tween 20 vehicle; whereas, the second group was exposed by the SC route using two vehicles, corn oil (CPF) and DMSO (L-CPF). A third group was only administered CPF by the oral route in corn oil. For all treatments, blood and urine time course samples were collected and analyzed for 3,5,6-trichloro-2-pyridinol (TCPy), and isotopically labeled 3,5,6-trichloro-2-pyridinol (L-TCPy). Peak TCPy/L-TCPy concentrations in blood (20.2 micromol/l), TCPy/L-TCPy blood AUC (94.9 micromol/lh), and percent of dose excreted in urine (100%) were all highest in rats dosed orally with CPF in saline/Tween 20 and second highest in rats dosed orally with CPF in corn oil. Peak TCPy concentrations in blood were more rapidly obtained after oral administration of CPF in saline/Tween 20 compared to all other dosing scenarios (>1.5 h). These results indicate that orally administered CPF is more extensively metabolized than systemic exposures of CPF (SC and IV), and vehicle of administration also has an effect on absorption rates. Thus, equivalent doses via different routes and/or vehicles of administration could potentially lead to different body burdens of CPF, different rates of bioactivation to CPF-oxon, and different toxic responses. Simulations using a physiologically based pharmacokinetic and pharmacodynamic (PBPK/PD) model for CPF are consistent with these possibilities. These results suggest that exposure route and dosing vehicle can substantially impact target tissue dosimetry. This is of particular importance when comparing studies that use varying exposure paradigms, which are then used for extrapolation of risk to humans.

Intracellular accumulation dynamics and fate of zinc ions in alveolar epithelial cells exposed to airborne ZnO nanoparticles at the air–liquid interface

Abstract Airborne nanoparticles (NPs) that enter the respiratory tract are likely to reach the alveolar region. Accumulating observations support a role for zinc oxide (ZnO) NP dissolution in toxicity, but the majority of in-vitro studies were conducted in cells exposed to NPs in growth media, where large doses of dissolved ions are shed into the exposure solution. To determine the precise intracellular accumulation dynamics and fate of zinc ions (Zn2+) shed by airborne NPs in the cellular environment, we exposed alveolar epithelial cells to aerosolized NPs at the air–liquid interface (ALI). Using a fluorescent indicator for Zn2+, together with organelle-specific fluorescent proteins, we quantified Zn2+ in single cells and organelles over time. We found that at the ALI, intracellular Zn2+ values peaked 3 h post exposure and decayed to normal values by 12 h, while in submerged cultures, intracellular Zn2+ values continued to increase over time. The lowest toxic NP dose at the ALI generated peak intracellular Zn2+ values that were nearly three-folds lower than the peak values generated by the lowest toxic dose of NPs in submerged cultures, and eight-folds lower than the peak values generated by the lowest toxic dose of ZnSO4 or Zn2+. At the ALI, the majority of intracellular Zn2+ was found in endosomes and lysosomes as early as 1 h post exposure. In contrast, the majority of intracellular Zn2+ following exposures to ZnSO4 was found in other larger vesicles, with less than 10% in endosomes and lysosomes. Together, our observations indicate that low but critical levels of intracellular Zn2+ have to be reached, concentrated specifically in endosomes and lysosomes, for toxicity to occur, and point to the focal dissolution of the NPs in the cellular environment and the accumulation of the ions specifically in endosomes and lysosomes as the processes underlying the potent toxicity of airborne ZnO NPs.

Direct analysis of trichloropyridinol in human saliva using an Au nanoparticles-based immunochromatographic test strip for biomonitoring of exposure to chlorpyrifos

A portable immunochromatographic strip-based biosensor for direct detection of trichloropyridinol (TCP), a specific biomarker of exposure to chlorpyrifos, in human saliva sample is presented. In this approach, a series of immunoreactions was performed on the test strip, where the targeted analytes (TCP) bound to the Au nanoparticles-labeled antibodies on the conjugate pad to form analyte-Au-antibody conjugates, and then free Au-labeled antibodies were captured by TCP-BSA in the test zone. Captured Au nanoparticles, increased with decreased levels of analytes, can be observed visibly without any equipment and later quantified by a colorimetric reader. Several experimental parameters were optimized including Au nanoparticle-to-TCP antibody coupling ratio, the amount of Au-labeled TCP antibody, immunoreaction time, the pretreatment of sample pad and the preparation of stock solution of Au-TCP antibody that realize sensitivity, selectivity and direct detection of TCP. Under optimal conditions, this biosensor displays a highly linear range of 0.625-20 ng/mL TCP, with a detection limit of 0.47 ng/mL. Moreover, the immunosensor was successfully used for direct analysis of human saliva sample without any pretreatment. These results demonstrate that this Au nanoparticles-based immunochromatographic test strip (ITS) provides a simple, accurate, and quantitative tool for TCP detection and holds a great promise for point-of-care and in-field analysis of other biomarkers.

Enzyme-linked immunosorbent assay for detection of organophosphorylated butyrylcholinesterase: A biomarker of exposure to organophosphate agents

A sandwich enzyme-linked immunosorbent assay (sELISA) has been developed for detection of organophosphorylated butyrylcholinesterase (OP-BChE), a potential biomarker for human exposure to organophosphate insecticides and nerve agents. A pair of antibodies specific to OP-BChE adduct were identified through systematic screening of several anti BChE antibodies (anti-BChE) and anti-phosphoserine antibodies (anti-P(ser)) from different sources. The selected anti-BChE (set as capture antibody) antibodies recognize both phosphorylated and nonphosphorylated BChE. These antibodies can therefore be used to capture both BChE and OP-BChE from the sample matrices. The anti-P(ser) (set as detecting antibody) was used to recognize the OP moiety of OP-BChE adducts. With the combination of the selected antibody pair, several key parameters (such as the concentration of anti-BChE and anti-P(ser), and the blocking agent) were optimized to enhance the sensitivity and selectivity of the sELISA. Under the optimal conditions, the sELISA has shown a wide linear range from 0.03 nM to 30 nM, with a detection limit of 0.03 nM. Furthermore, the sELISA was successfully applied to detect OP-BChE using in vitro biological samples such as rat plasma spiked with OP-BChE with excellent adduct recovery (z>99%). These results demonstrate that this novel approach holds great promise to develop an ELISA kit and offers a simple and cost-effective tool for screening/evaluating exposure to organophosphate insecticides and nerve agents.

Comparative pharmacokinetics of chlorpyrifos versus its major metabolites following oral administration in the rat.

Chlorpyrifos (CPF) is a commonly used diethylphosphorothionate organophosphorus (OP) insecticide. Diethylphosphate (DEP), diethylthiophosphate (DETP) and 3,5,6-trichloro-2-pyridinol (TCPy) are products of both in vivo metabolism and environmental degradation of CPF and are routinely measured in urine as biomarkers of exposure. Hence, urinary biomonitoring of TCPy, DEP and DETP may be reflective of an individuals contact with both the parent pesticide and exposure to these metabolites in the environment. In the current study, simultaneous dosing of 13C- or 2H-isotopically labeled CPF (13C-labeled CPF, 5 13C on the TCPy ring; or 2H-labeled CPF, diethyl-D10 (deuterium labeled) on the side chain) were exploited to directly compare the pharmacokinetics and metabolism of CPF with TCPy, and DETP. The key objective in the current study was to quantitatively evaluate the pharmacokinetics of the individual metabolites relative to their formation following a dose of CPF. Individual metabolites were co-administered (oral gavage) with the parent compound at equal molar doses (14 micromol/kg; approximately 5 mg/kg CPF). Major differences in the pharmacokinetics between CPF and metabolite doses were observed within the first 3h of exposure, due to the required metabolism of CPF to initially form TCPy and DETP. Nonetheless, once a substantial amount of CPF has been metabolized (> or =3h post-dosing) pharmacokinetics for both treatment groups and metabolites were very comparable. Urinary excretion rates for orally administered TCPy and DETP relative to 13C-CPF or (2)H-CPF derived 13C-TCPy and 2H-DETP were consistent with blood pharmacokinetics, and the urinary clearance of metabolite dosed groups were comparable with the results for the 13C- and 2H-CPF groups. Since the pharmacokinetics of the individual metabolites were not modified by co-exposure to CPF; it suggests that environmental exposure to low dose mixtures of pesticides and metabolites will not impact their pharmacokinetics.