The prolific worldwide misuse of organophosphate (OP) pesticides, coupled with a grave homeland security concern that OP-based nerve agents may be deployed either against our troops or civilians on our home soil, has stimulated the search for rapid, miniaturized and inexpensive point-of-care (POC) diagnostic devices for first responders and medical personnel to detect the degree of OP exposure. Identification of the OP agent during an exposure is challenging as it relies on acid hydrolysis products in urine that are rapidly excreted. In contrast, acetylcholinesterase (AChE) and butylcholinesterase (BChE) enzyme activity are established biomarkers for risk assessment because they substantiate exposure (as OPs bind directly to AChE and BChE), directly provide a quantitative biochemical effect of the exposure (i.e., enzyme inhibition), and several weeks post-exposure. Measurement of cholinesterase (ChE) is complicated by inter- and intra- individual variability of ChE activity and relationship to clinical signs and symptoms. The variability of BChE activity between individuals based simply on genetics, sex, race, or age is estimated as high as 65% deviation of the mean. The variation in BChE levels in the population underscores the importance of having individual baseline values of ChE activity to interpret results when assessing exposures to BChE-inhibitors. In the absence of a baseline (which is likely the case in the event of a mass casualty scenario), BChE inhibition measurements are often based on a population average, which introduces a large degree of uncertainty in the reported result. These uncertainties reduce the sensitivity of BChE activity as a biomarker and thus may provide ambiguous results for subclinical, low-dose OP exposure (<20% enzyme inhibition). It is critical to note that low cholinesterase inhibition may be subclinical in appearance only, as there is evidence that even a low dose exposure of OP neurotoxicants can cause long-term, significant chronic neurological deficits. Numerous biochemical methods have been developed to measure ChE levels, including the Ellman assay, fluorescence assays, electrochemical assays, the Michel (?pH) ChE assay, radioactive assays, and the Walter Reed Army Institute of Research (WRAIR) assay. However, all of these methods are based on detecting a meaningful decrease in enzyme activity from a previously established baseline. Recent advances in the application of nanomaterials as transducers, recognition agents or labels, and advances in immunoassay techniques are now being exploited in the design of improved devices for the biomedical detection of non-metabolized OP pesticides and nerve agents, metabolites, and protein adducts. Our primary objective is to develop a baseline free, broad-spectrum for all OPs, field-deployable point of care (POC) device that confirms and quantitates the degree of exposure to OP neurotoxicants by measuring a ratio of OP-bound to unbound BChE.
The overall objective of the work proposed herein is to complete development and preliminary optimization of a prototype point of care device that semi-quantitates a ratio of OP-adducted BChE to free BChE from human plasma containing titrations of OP to produce a baseline-free readout. Completion of this project will significantly advance the development of a sensitive, rapid, baseline free, OP-broad-spectrum, rugged, field-deployable point of care (POC) device that quantitates the degree of exposure to OP neurotoxicants by measuring a ratio of OP-bound to unbound BChE as a diagnostic tool for medical personnel administering antidotal therapy.
