On-chip integrated hydrolysis, fluorescent labeling, and electrophoretic separation utilized for acetylcholinesterase assay

Heleg-Shabtai, Vered; Gratziany, Natzach; Liron, Zvi

doi:10.1016/j.aca.2006.04.087

Cited by 15 publications

(11 citation statements)

References 34 publications

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“…At the lowest limit, concentrations of 100 p M can be detected, which equates to an astonishing 22 000 molecules of enzyme with an optical probe volume of 360 pL. This detection level approaches and, in many cases, surpasses the sensitivity of recently reported AChE detection methods, such as chemiluminescent dioxetane probes,10b the aggregation‐induced emission (AIE) of tetraphenylethylenes (TPE),10c,d cyano‐substituted poly( p ‐phenylenevinylene) (PPV) probes10e and other fluorescence assays,10f,g and electrochemical detection methods involving gold nanoparticles10h (Table 3). Unlike many of these techniques, BSI does not require substrate analogues, specialized probes, or laborious procedures.…”

Section: Methodsmentioning

confidence: 96%

Back‐Scattering Interferometry: An Ultrasensitive Method for the Unperturbed Detection of Acetylcholinesterase–Inhibitor Interactions

et al. 2012

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A series of inhibitors of acetylcholinesterase (AChE) have been screened by back-scattering interferometry (BSI). Enzyme levels as low as 100 pM (22,000 molecules of AChE) can be detected. This method can be used to screen for mixed AChE inhibitors, agents that have shown high efficacy against Alzheimer's disease, by detecting dual-binding interactions. Keywords detection method; interferometry; acetylcholinesterase; high-throughput screening; enzyme assayIn recent years, the emphasis of drug discovery and optimization has shifted from traditional methods to rapid microfluidic screening to minimize cost and maximize output. Backscattering interferometry (BSI),[1] a technique which quantifies refractive index (RI) changes arising from intermolecular binding interactions, is a novel biosensing platform that may be useful in drug discovery. Acetylcholinesterase (AChE), a widely studied serine hydrolase that plays pivotal roles in Alzheimer's disease (AD), [2] inflammatory processes, [3] and nerve-agent poisoning, [4] is an interesting system with which to study the use of BSI for the rapid detection of drug candidates. The search for potent AChE inhibitors (AChEI) has been driven largely by the need for an effective AD treatment, and is based on the longstanding cholinergic hypothesis [2] and the more recent amyloid hypothesis. [5] It is wellestablished that AChE accelerates amyloid-beta (Ab) peptide deposition, a process which may be mediated by an interaction between plaque precursors and the peripheral anionic site (PAS) of the enzyme.[6] Recent drug discovery efforts have focused on the design of AChEIs that are able to interact with the PAS to target both cholinergic and noncholinergic AD pathologies. [7] Seminal work in this field has led to the discovery of dual-binding inhibitors, such as bisgalantamine [8] and pseudo-irreversible carbamate inhibitors. [9] Given the large number of studies on several diverse classes of AChEIs, this system is a widely accepted benchmark for the development and testing of novel screening techniques.Traditional methods used to detect interactions with AChE focus on the indirect measurement of substrate procedures. [10] To explore the utility of BSI to detect AChEIs, several known and novel inhibitors with diverse potencies and inhibitory mechanisms have been screened. Herein, we show that BSI can: 1) quantify AChE-inhibitor interactions in the We first wanted to probe the utility of BSI to screen AChEIs using a set of known and novel AChEIs (Scheme 1). Edrophonium, a competitive inhibitor, propidium, a noncompetitive inhibitor, and the mixed inhibitors, 1,5-bis(4-allyldimethylammoniumphenyl) pentan-3-one dibromide (BW284c51) and galantamine, were the standards used to validate BSI as a method for the detection of AChEIs. Two separate and distinct saturation curves were observed for inhibitors 4 and 6 (Figure 2 and Supporting Information, Figure S7, respectively). For ligand 4, the dual-binding curve is shown in the graph inset of while the fit of the second bindin...

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Section: Methodsmentioning

confidence: 96%

Back‐Scattering Interferometry: An Ultrasensitive Method for the Unperturbed Detection of Acetylcholinesterase–Inhibitor Interactions

et al. 2012

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“…In the pharmaceutical industry, efforts to increase the throughput of combinatorial chemistry, seeds screening 120–122, and process chemistry 123 are sought after, to improve overall efficiency of the expensive and time‐consuming drug discovery processes 124–126. Kobayashi et al 127 devised an efficient gas–liquid–solid tri‐phase reactions using a microchannel reactor, a reaction normally considered to be very inefficient.…”

Section: Reactionmentioning

confidence: 99%

Microfluidics: Applications for analytical purposes in chemistry and biochemistry

2008

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In this review, we present recent advancements and novel developments in fluidic systems for applied analytical purposes in chemistry, biochemistry, and life science in general that employ and reflect the full benefits of microfluidics. A staggering rise in publications related to integrated, all-in-one microfluidic chips capable of separation, reaction, and detection have been observed, all of which realise the principal of micro total analysis systems or lab-on-a-chip. These integrated chips actively adopt the scaling law concepts, utilising the highly developed fabrication techniques. Their aim is to multi-functionalise and fully automate devices believed to assist the future advancements of point-of-care, clinical, and medical diagnostics.

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“…22 The LOC fluorescence detection of VX and the nerve agent simulant paraoxon was also shown to be feasible, by use of a sensitive enzymatic assay. 23 More specifically, the inhibitor was mixed off-chip with the enzyme acetylcholinesterase (AChE), and then introduced to a microchip with the capability for sequentially introducing first the substrate acetylthiocholine (ATC), and then a thiocholine (TC) reactive fluorophore, eosinmaleimide (Eo-MI), which forms a thioether that can be electrophoretically separated and detected by LIF (Fig. 2).…”

Section: Analysis Of Chemical Threat Agentsmentioning

confidence: 99%

Chemical and biological threat-agent detection using electrophoresis-based lab-on-a-chip devices

Borowsky

Collins

2007

Analyst

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The ability to separate complex mixtures of analytes has made capillary electrophoresis (CE) a powerful analytical tool since its modern configuration was first introduced over 25 years ago. The technique found new utility with its application to the microfluidics based lab-on-a-chip platform (i.e., microchip), which resulted in ever smaller footprints, sample volumes, and analysis times. These features, coupled with the technique's potential for portability, have prompted recent interest in the development of novel analyzers for chemical and biological threat agents. This article will comment on three main areas of microchip CE as applied to the separation and detection of threat agents: detection techniques and their corresponding limits of detection, sampling protocol and preparation time, and system portability. These three areas typify the broad utility of lab-on-a-chip for meeting critical, present-day security, in addition to illustrating areas wherein advances are necessary.

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On-chip integrated hydrolysis, fluorescent labeling, and electrophoretic separation utilized for acetylcholinesterase assay

Cited by 15 publications

References 34 publications

Back‐Scattering Interferometry: An Ultrasensitive Method for the Unperturbed Detection of Acetylcholinesterase–Inhibitor Interactions

Back‐Scattering Interferometry: An Ultrasensitive Method for the Unperturbed Detection of Acetylcholinesterase–Inhibitor Interactions

Microfluidics: Applications for analytical purposes in chemistry and biochemistry

Chemical and biological threat-agent detection using electrophoresis-based lab-on-a-chip devices

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