Nanoparticle-based electrochemical immunosensor for the detection of phosphorylated acetylcholinesterase: an exposure biomarker of organophosphate pesticides and nerve agents

Abstract

A nanoparticle-based electrochemical immunosensor has been developed for the detection of phosphorylated acetylcholinesterase (AChE), which is a potential biomarker of exposure to organophosphate (OP) pesticides and chemical warfare nerve agents. Zirconia nanoparticles (ZrO(2) NPs) were used as selective sorbents to capture the phosphorylated AChE adduct, and quantum dots (ZnS@CdS, QDs) were used as tags to label monoclonal anti-AChE antibody to quantify the immunorecognition events. The sandwich-like immunoreactions were performed among the ZrO(2) NPs, which were pre-coated on a screen printed electrode (SPE) by electrodeposition, phosphorylated AChE and QD-anti-AChE. The captured QD tags were determined on the SPE by electrochemical stripping analysis of its metallic component (cadmium) after an acid-dissolution step. Paraoxon was used as the model OP insecticide to prepare the phosphorylated AChE adducts to demonstrate proof of principle for the sensor. The phosphorylated AChE adduct was characterized by Fourier transform infrared spectroscopy (FTIR) and mass spectroscopy. The binding affinity of anti-AChE to the phosphorylated AChE was validated with an enzyme-linked immunosorbent assay. The parameters (e.g., amount of ZrO(2) NP, QD-anti-AChE concentration,) that govern the electrochemical response of immunosensors were optimized. The voltammetric response of the immunosensor is highly linear over the range of 10 pM to 4 nM phosphorylated AChE, and the limit of detection is estimated to be 8.0 pM. The immunosensor also successfully detected phosphorylated AChE in human plasma. This new nanoparticle-based electrochemical immunosensor provides an opportunity to develop field-deployable, sensitive, and quantitative biosensors for monitoring exposure to a variety of OP pesticides and nerve agents.

Figure 1

Various reaction paths of paraoxon after systemic exposure

Figure 2

Typical FTIR spectrums of paraoxon, AChE, and paraoxon-AChE adduct

Figure 3

Mass spectrum of collision-induced dissociation of the 4+ ion, 497.76, from LC-MS/MS analysis of alkyphosphorylated AChE, retention time 4.6 min. Assignment of the resulting fragments peaks (daughter ions) can be confidently assigned to b- and y-ions and internal fragmentation of a subpeptide of the 21 amino acid sequence GGDPTSVTLFGESAGAASVGMh (peptide 222–242) with a homoserine C-terminus (Mh, due to incubation with CNBr). Assignments made from the amino terminus of the parent peptide (far left, G) begin with b1 through b21 (left to right), while those that begin from at the carboxyl terminus (far right, Mh) are numbered y1 and through y21 (right to left). Each experimental peak mass that can be matched to a subsection of the parent peptide is labeled in the spectrum and the corresponding amino acid sequence for the fragment is shown in the upper left of the figure. For example, b7 has a mass of 614.28 (Table S1) and is assigned to GGDPTSV. Using the MASCOT sequence mapping algorithm, an ion score greater than13 indicates identity or extensive homology at p<0.05; a score of 24 was obtained here. The match is further confirmed by detection of internal fragments 4–10 (In4–10, PTSVTLF). Most importantly, a monoethyl-phosphoryl mass adduct of 107.998 Da is included in the assignment of y9, the doubly charged y10++, doubly charged a16++, and y9–126 (neutral loss of monoethylphosphate, MW 126.01 from y9); the overlap of these sequences pinpoints the OP adduct to Ser 13 within the parent peptide (from left to right). In addition, only modification of active site serine S234 (amino acid S13 of this peptide) by alkyphosphorylation is known to yield inactive AChE as demonstrated by our Ellman assay.

Figure 4

The principle of electrochemical immunosensing of phosphorylated AChE, (A) ZrO₂ nanoparticle modified SPE; (B) selective capturing phosphorylated AChE adducts; (C) Immunoreaction between bound phosphorylated AChE adducts and QD-labeled anti-AChE antibody; (D) dissolution of nanoparticle with acid following an electrochemical stripping analysis.

Figure 5

(A) The effect of the amount of ZrO2 on the response of the immunosensor; the amount of ZrO2 was controlled by the cycles of cyclic potential scanning; B) BSA block effect on the immunosensor responses; (C) the effect of QD-anti-AChE concentration; concentration of phosphorylated AChE: 0.5 nM. The conditions of electrochemical measurement are the same as in Table 1.

Figure 6

Typical electrochemical responses of the immunosensor with the increasing phosphorylated AChE concentration (10 pM to 4 nM from a to g). The insets show the resulting calibration plot (bottom) and the electrochemical responses of 10 pM and 0 pM (top) paraoxon-AChE. Each concentration was measured three times with three different immunosensors. The immunoreaction time was 1 hr; 10 µL of QD-Ab conjugate (1/50, v/v) was used during the incubation. Electrochemical measurement conditions were the same as in Table 1.