A highly selective, sensitive, and low-cost hybrid sensing platform is developed based on extraordinary properties of explosives in an ionic liquid and an integrated electrochemical and colorimetric approach.

Self-assembly of surfactant molecules into micelles of various shapes and forms has been extensively used to synthesize soft nanomaterials. Translucent solutions containing rod-like surfactant micelles can self-organize under flow to form viscoelastic gels. This flow-induced structure (FIS) formation has excited much fundamental research and pragmatic interest as a cost-effective manufacturing route for active nanomaterials. However, its practical impact has been very limited because all reported FIS transitions are reversible because the gel disintegrates soon after flow stoppage. We present a new microfluidics-assisted robust laminar-flow process, which allows for the generation of extension rates many orders of magnitude greater than is realizable in conventional devices, to produce purely flow-induced permanent nanogels. Cryogenic transmission electron microscopy imaging of the gel reveals a partially aligned micelle network. The critical flow rate for gel formation is consistent with the Turner-Cates fusion mechanism, proposed originally to explain reversible FIS formation in rod-like micelle solutions.

An integrated lateral flow test strip with an electrochemical sensor (LFTSES) device with rapid, selective, and sensitive response for quantification of exposure to organophosphorus (OP) pesticides and nerve agents has been developed. The principle of this approach is based on parallel measurements of postexposure and baseline acetylcholinesterase (AChE) enzyme activity, where reactivation of the phosphorylated AChE is exploited to enable measurement of the total amount of AChE (including inhibited and active) which is used as a baseline for calculation of AChE inhibition. Quantitative measurement of phosphorylated adduct (OP-AChE) was realized by subtracting the active AChE from the total amount of AChE. The proposed LFTSES device integrates immunochromatographic test strip technology with electrochemical measurement using a disposable screen printed electrode which is located under the test zone. It shows a linear response between AChE enzyme activity and enzyme concentration from 0.05 to 10 nM, with a detection limit of 0.02 nM. On the basis of this reactivation approach, the LFTSES device has been successfully applied for in vitro red blood cells inhibition studies using chlorpyrifos oxon as a model OP agent. This approach not only eliminates the difficulty in screening of low-dose OP exposure because of individual variation of normal AChE values but also avoids the problem in overlapping substrate specificity with cholinesterases and avoids potential interference from other electroactive species in biological samples. It is baseline free and thus provides a rapid, sensitive, selective, and inexpensive tool for in-field and point-of-care assessment of exposures to OP pesticides and nerve agents.

A multiplexed electrochemical immunoassay integrating enzyme amplification and electric field-driven strategy was developed for fast and sensitive quantification of phosphorylated p53 at Ser392 (phospho-p53(392)), Ser15 (phospho-p53(15)), Ser46 (phospho-p53(46)), and total p53 simultaneously. The disposable sensor array has four spatially separated working electrodes, and each of them is modified with different capture antibody, which enables simultaneous immunoassay to be conducted without cross-talk between adjacent electrodes. The enhanced sensitivity was achieved by a multienzyme amplification strategy using gold nanorods (AuNRs) as nanocarrier for coimmobilization of horseradish peroxidase (HRP) and detection antibody (Ab(2)) at a high ratio of HRP/Ab(2), which produced an amplified electrocatalytic response by the reduction of HRP oxidized thionine in the presence of hydrogen peroxide. The immunoreaction processes were accelerated by applying +0.4 V for 3 min and then -0.2 V for 1.5 min; thus, the whole sandwich immunoreactions could be completed in less than 5 min. Under optimal conditions, this method could simultaneously detect phospho-p53(392), phospho-p53(15), phospho-p53(46), and total p53 ranging from 0.01 to 20 nM, 0.05 to 20 nM, 0.1 to 50 nM, and 0.05 to 20 nM with detection limits of 5 pM, 20 pM, 30 pM, and 10 pM, respectively. Accurate determinations of these proteins in human plasma samples were demonstrated by comparison to the standard ELISA method. The disposable immunosensor array shows excellent promise for clinical screening of phosphorylated proteins and convenient point-of-care diagnostics.

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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 disposable electrochemical immunosensor for highly selective and sensitive detection of organophosphorylated butyrylcholinesterase (OP‐BChE), a specific biomarker for exposure to toxic organophosphorus agents, is presented. In this new approach, zirconia nanoparticles (ZrO2) were employed to selectively capture the OP moiety of OP‐BChE adducts, followed by quantum dot (QD)‐tagged anti‐BChE antibodies for amplified quantification. The captured CdSe‐QD tags can be sensitively detected by stripping voltammetry using an in situ bismuth‐plating method. The OP agent, diisopropylfluorophosphate (DFP), was selected to prepare OP‐BChE adducts in various matrices. The formation of OP‐BChE adducts in plasma sample was confirmed using mass spectroscopy. The developed electrochemical immunosensor demonstrates a highly linear voltammetric response over the range of 0.1 to 30 nM OP‐BChE, with a detection limit of 0.03 nM (based on signal/noise = 3), coupled with a good reproducibility (relative standard deviation 4.5%). Moreover, the immunosensor has been validated with biomonitoring of OP‐BChE adducts in the plasma samples. This novel nanoparticle‐based electrochemical immunosensor thus provides an alternative way for designing a sensitive and cost‐effective sensing platform for on‐site screening/evaluating exposure to a variety of OP agents.

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