Organophosphates (OP) including chemical nerve agents and pesticides represent a diverse group of highly toxic compounds.1, 2
The acute toxicity resulting from OP exposure stems from the fact that they readily inhibit important cholinergic enzymes, such as acetylcholinesterase (AChE) through an attack on the serine residue of AChE, forming a phosphorylated adduct. When the function of AChE is inhibited, accumulation of acetylcholine will result in overstimulation of corresponding muscarinic and nicotinic cholinergic receptors, which may lead to serious health problems or even death.3, 4
Due to the widespread use of OPs as pesticides across the world and the increased threat to peoples’ health resulting from the potential use of chemical nerve agents in terrorist attacks or military activities, there is a need to develop fast, sensitive and field-deployable screening technology for quick response to exposure to OPs to facilitate triage. Secondly, a rapid screening technology can also be exploited for real-time biological monitoring of workers that are involved in the manufacture and/or application of OP insecticides. At present, detection and evaluation of OP exposure is generally performed at dedicated centralized laboratories using large, automated analyzers such as liquid or gas chromatography coupled with mass spectrometry (HPLC/GC-MS),5-8
requiring sample transportation and processing which ultimately increases the waiting time for results. Rapid near-patient or field biomonitoring tools for the first responders is highly desirable to facilitate rapid screening to initiate appropriate treatments.
Currently, three approaches have been developed for biomonitoring of OP exposure: (1) assay of enzyme activity9, 10
(2) measurement of metabolites11-13
and (3) detection of phosphorylated adducts.14-16
Although measurement of metabolites is a sensitive and accurate method for detection and identification of OPs, it is not suitable for rapid detection or field biomonitoring because this approach generally involves a laboratory based analysis, such as GC/LC-MS. Phosphorylated complex (i.e. adducts) may also serve as an indicator of OP exposures; however, a challenge may lie in the current unavailability of recognition elements or appropriate receptors, i.e., antibodies, for specifically targeting phosphorylated enzyme. Enzyme activity assays have been extensively used for screening of OP exposure, in a number of applications including: occupational and environmental health and military screening. Cholinesterase (ChE) enzyme activity measurements from blood (and potentially saliva) are a good biomarker to evaluate OP exposure. The assays are relatively simple, as numerous biomonitoring methods have been developed to measure the enzyme activity of cholinesterase including, but not limited to, Ellman Assay
,17 Fluorescence Assay
,18 Michel (ΔpH) ChE Assay
,19 Radioactive Assay
and Walter Reed Army Institute of Research (WRAIR) Assay.21, 22
Most recently, an enzyme activity kits (Test-Mate Assay
) for fast screening OP exposure has been developed and is commercially available.23
However, this kit and the other available screening methods have an inherent disadvantage since a control or baseline is needed because the measured value of enzyme activity has to be compared with the unexposed normal (baseline) values. To circumvent this problem, a statistically derived value of enzyme activity measured from a large sample size of population generally serves as the control. However, these methods are not accurate because of large variability in baseline values derived from the variation of enzyme activity between individuals (e.g. sex, age, ethnic, etc) and the deviation of measurement methods from different laboratories.24
In general, considering inter- and intra-individual variations in the normal levels of ChE, the exposure that results in inhibition of less than about 20% (especially if clinical symptoms are absent) may not be easily detectable or provide reliable evidence for current available screening methods unless recent control values of that particular individual are available.24, 25
In a worse case situation, these methods may provide ambiguous results. Moreover, biomonitoring of individuals with a low level of OP exposure (< 10% inhibition) may be problematic without a pre-exposure base-line enzyme determination with these methods.
It was found that some compounds such as nucleophiles 26-30
or fluoride ions 31-34
can reactivate the phosphorylated complex yielding a restored enzyme activity. This process is called “reactivation”. Based on the reactivation, a number of assay methods such as measuring released OP from phosphorylated enzyme (fluoride ions-induced reactivation) using MS have been developed for detection and identification of exposure to OPs.31, 32
In parallel, oxime-induced reactivation has been used to evaluate drug efficacy and for therapeutic intervention as a treatment for OP poisoning. The most commonly used pyridinium oxime is pralidoxime iodide (2-PAM).27
Current biomonitoring methods for OP exposures ignore evaluation of the restored enzyme activity via reactivation. However, if the activity of phosphorylated enzyme could be regenerated completely to its original state and activity determined, this restored enzyme could serve as its own control or baseline. Therefore, a double test of enzyme activity in biological samples before and after reactivation could be exploited to quantify the extent of enzyme activity/inhibition.
In this paper, we report a novel approach combining the advantage of enzyme reactivation and signal enhancement of the nanomaterials for fast, inexpensive, non-invasive, baseline-free, and field-deployable biomonitoring of OPs using saliva samples. Rat saliva was utilized as an initial biological matrix for evaluation, since our research group has previously characterized rat saliva ChE activity, it is readily obtainable (i.e. non-invasive) and under well controlled experimental conditions appears to parallel the response of plasma ChE.35, 36
Different from available optical method, this approach is based on a flow-injection amperometric sensor, of which the carbon nanotube-screen printed electrode is integrated in the flow cell. In this approach a double-test mode detects the variation of enzyme activity before and after reactivation. This method provides advantages since a control sample is not needed and it excludes inter- or intra-individual variation in the normal levels of ChE. Therefore, this measurement is accurate and reliable. Furthermore, this assay is highly sensitive and selective because the use of carbon nanotubes makes the electrochemical detection of the products from enzymatic reactions more feasible at low potentials. In general, this method is inexpensive, sensitive, portable, non-invasive, and provides real-time results. It is anticipated that this novel sensor method will open up a new avenue for rapid point-of-care (POC) screening and early assessment of OP exposures providing for early clinical intervention in the case of severe exposures and rapid identification of victims in a terrorist attack.