Neurotoxic organophosphates (OPs) have been widely used as pesticides in agricultural industry and as chemical warfare agents.1, 2
As a result, OP contaminations have been widespread in air, water, soil, and food, such that there is a potential for human exposures. Therefore, public concern about the development of effective detection devices for effective monitoring OPs and evaluation of human health risk of OP exposure has grown steadily in recent years.3–5
Moreover, after the Tokyo subway attack in 1995,6
the needs for feasible OP detection methods have become increasingly urgent for purpose of early warning of potential terrorist attack and diagnostic mitigation of the effects from alleged nerve-agent exposures.2, 3, 5–7
In recent decades, numerous analysis methods have been developed for the assessment of OP exposures, and the relevance of end points to human health is of utmost concern.3, 8–10
In this regard, biomonitoring of OP exposures is recognized to be one of the best approaches.8
The internal dosages of OP agents or their metabolites are quantitatively and / or qualitatively measured on the basis of our knowledge of the metabolic fate of the toxicants, thus providing the accurate evaluation of the health risk of integrated OP exposure. Unfortunately, the majority of current biomonitoring protocols for OP exposures, 3, 8, 11, 12
may still suffer from some intrinsic disadvantages of either low detection specificity and sensitivity (i.e., Ellman colorimetric assays13, 14
), or expensive analysis settings entailing well-trained personnel and inconvenience for field applications (i.e., gas or liquid chromatography coupled with mass spectrometry (GC- or LC-MS) 4, 15–17
). Hence, simple, sensitive, selective and field-deployable tools are still highly desired for biomonitoring and diagnostic evaluation of OP exposures at present, especially for the enhancement of our response to a sudden emergency and the improvement of our ability to medically counteract the effects.
It is generally recognized that selection of suitable biomarkers for OP detections is of central importance for developing a biomonitoring strategy.8, 12
Biomarkers that are currently used include: free OPs in blood, or their metabolites in urine, and cholinesterase (ChE) inhibition in blood.3, 8, 9, 12
OPs can stoichiometrically bind with ChE and inhibit the enzyme activity, at the same time, they are metabolized by organophosphorus hydrolase to form inactive phosphonic acids that are then renally excreted.8
The high reactivity of OPs with these enzymes suggests that the levels of free OPs will be inherently low (typically in the range of nanogram per liter or parts per trillion in blood 12
), such that ultra-sensitive detection methodologies are thereby required; yet, the formidable false positive signals might be difficult to avoid. Moreover, while OP metabolite level in urine is also considered a sensitive indicator of OP exposure,8, 18
the fact that not all toxicant specific metabolites are derived solely from OPs is a real concern.12
Also, ChE inhibition as a biomarker of OP exposure effect has historically been an important strategy, but the inhibition-based quantification can also be problematic.12, 19
For example, for a quantitative assessment a baseline enzyme level is required to accommodate the individual fluctuations in enzyme levels. All of these factors thereby make the blood ChE measurements less viable for assessing some OP exposures.12
Therefore, exploring selective, sensitive and reliable alternative biomarkers is an important consideration for biomonitoring of OP exposures.
Electrochemical immunoassays with high selectivity and sensitivity have evolved rapidly over the past decades.20–23
Their detection sensitivity may be enhanced by using various nano-scale materials newly emerged, i.e., quantum dots,24
for electrochemical signal amplifications.23, 25
Such a versatile analysis tool can possess some advantages over the present standard methods for assessment of OP exposures that are based on GC-MS and LC-MS.20, 21, 26
More importantly, their simple operation and miniaturized analysis instruments can meet the requirements of decentralized point–of-care tests or field detections.21, 22, 27
Moreover, according to the biochemical mechanism widely accepted for ChE phosphorylation,28, 29
the inhibition event may produce very stable enzyme complexes with structurally precise phosphoserine esters,30
suggesting that these products may serve as selective indicators directly correlated to the severity 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 ChE. Although some specific antibodies against OP agents, i.e., paraoxon, have been recently developed and commercially available for immunoassays,31–33
they might be unable to selectively recognize modified or aged OP moieties of OP-AChE., which is addressed in this study.
In this work, we present preliminary studies to establish a novel electrochemical immunoassay of phosphorylated AChE as a biomarker for biomonitoring and diagnosis of OP exposures. OP-AChE, which was prepared by incubating human AChE with paraoxon, was used as the model targets. In order to circumvent the current limitations of OP-AChE recognition, two kinds of antibodies, anti-phosphoserine polyclonal antibodies (termed as Ab1) and anti-human AChE monoclonal antibodies (termed as Ab2), were employed to facilitate the specific recognitions of OP-AChE. Amorphous magnetic particles (MPs) with large surface-to-volume ratio were chosen to load Ab1 to capture the OP-AChE from the sample matrixes through binding the phosphoserine moieties, which were disclosed through reductively unfolding the protein adducts via dithiothreitol (DDT), followed by the second recognition of Ab2 labeled with QDs serving as the signal-amplifying tags. The sandwich immunoreaction events were subsequently quantified by square wave voltammetric (SWV) analysis. Main parameters governing the SWV responses were optimized including the MP-Ab1 conjugate dosage, QD-Ab2 label concentration, and reaction time. Moreover, a blocking agent consisting of 3 % BSA and 1 % PEG was introduced for effective minimization of nonspecific adsorptions in the immunoassays. The magnetic-aided immunoassay was overall proofed with simple, selective and sensitive analysis features by spiking human plasma with known OP-AChE concentrations.