Affinity Binding-Guided Fluorescent Nanobiosensor for Acetylcholinesterase Inhibitors via Distance Modulation between the Fluorophore and Metallic Nanoparticle

Abstract

The magnitude of fluorescence enhancement was found to depend strongly on the distance between fluorophores and metal nanostructures in metal-enhanced fluorescence (MEF). However, the precise placement of the particle in front of the molecule with nanometer accuracy and distance control is a great challenge. We describe a method using acetylcholinesterase (AChE) to modulate the distance between a gold nanoparticle (AuNP) and the fluorophore 7-hydroxy-9H-(1,3-dichloro-9,9-dimethylacridin-2-one) (DDAO). We found that DDAO is a reversible mixed type-I AChE inhibitor. DDAO binds to the peripheral anionic site and penetrates into the active gorge site of AChE via inhibition kinetics test and molecular docking study. The affinity ligand DDAO bound to AChE which was immobilized onto AuNPs, and its fluorescence was sharply enhanced due to MEF. The fluorescence was reduced by distance variations between the AuNP and DDAO, which resulted from other inhibitors competitively binding with AChE and partly or completely displacing DDAO. Experimental results show that changes in fluorescence intensity are related to the concentration of inhibitors present in the solution. In addition, the nanobiosensor has high sensitivity, with detection limits as low as 0.4 μM for paraoxon and 10 nM for tacrine, and also exhibits different reduction efficiencies for the two types of inhibitor. Thus, instead of an inhibition test, a new type of affinity binding-guided fluorescent nanobiosensor was fabricated to detect AChE inhibitors, determine AChE inhibitor binding mode, and screen more potent AChE inhibitors. The proposed strategy may be applied to other proteins or protein domains via changes in the affinity ligand.

Review: Applications of surface-enhanced fluorescence (SEF) spectroscopy in bio-detection and biosensing

“…and DNA/CeO 2 complex, 360 nm lg(I562/I424) = −0.2713lgc – 2.312 (R2 = 0.995), RSD – 3.8% and 4.6% Thomas Schalkhammer [ 16 ] Fluorescein isothiocyanate NA NA Si-SiFs, 488 nm EF – 14 Samuel S.R. Dasary [ 39 ] Organophosphorus agents 1–25 μM 1 μM AuNPs, 395 nm Rsq 0.99, EF - 5 Fu Tang [ 19 ] PFV NA NA Ag@SiO 2 NPs, 405 nm EF – 1.3 Ying Wang [ 40 ] Adenosine 200 nM - 200 μM 48 nM AgNP aptasensor, 532 nm Rsq = 0.995, y = 0.395× + 0.3668 Yaodong Zhang [ 41 ] Paraoxon and Tacrine 0.5 μM–10 μM Paraoxon, 15 nM to 120 nM Tacrine 0.4 μM – Paraoxon, 10 nM - Tacrine AuNPs modified with DDAO-AChE, 447 nm EF - 6, RSD 1.7% Paraoxon, 2.1% Tacrine Yecang Tang [ 42 ] Glucose 2–52 mM 1,86 mM AgNP-CdSe QD, 400 nm EF – 9, Rsq = 0.991, RSD – 1.4% Eunji Gang [ 43 ] Glucose 1 μM – 1 mM 50 nM Ag@SiO2-entrapped hydrogel microarray, 580 nm EF – 4, Rsq = 0.997, RSD – 6.5% Tian Tian [ 24 ] Ascorbic acid 0.2–60 μM 25 nM Ag-CD nanohybrid, 500 nm Rsq = 0.986, Recoveries 92–106% Ramar Rajamanikandan [ 44 ] Dopamine 20–220 nM 0,35 nM GSH-Ag nanoclusters, 490 nm EF – 100, S/N – 3, Recoveries 96–98% Qiao Cheng [ 45 ] Heparin 0.025 to 2.5 μM 8.2 nM Ag-g-CNQDs, 450 nm ΔF = 133.4C + 25.18 (Rsq = 0.99) ...…”

“…In addition, this aptasensor showed high selectivity towards adenosine in the presence of other nucleosides. In the following year, the group of Dr. Zhang performed a series of experiments to test their newly designed AuNPs-based sensor for the detection of acetylcholinesterase (AChE) inhibitors [ 41 ]. This sensor is based on the combination of gold nanoparticles with a 7-hydroxy-9H-(1,3-dichloro-9,9-dimethylacridin-2-one) (DDAO) fluorophore bound to AChE.…”

Nanomaterials-Based Optical Techniques for the Detection of Acetylcholinesterase and Pesticides

“…One of factors affecting the enhancement magnitude is the distance between fluorophores and metal nanostructures. Interestingly, Zhang et al found that AChE could modulate the distance between AuNPs and fluorophore 7-hydroxy-9H-(1,3-dichloro-9,9-dimethylacridin-2-one) (DDAO) ( Figure 7 ) [ 63 ]. Binding of DDAO to AChE immobilized onto AuNPs lead to the enhancement of DDAO's fluorescence due to MEF ( Figure 7a ).…”

“…( a ) Fabrication of AChE-AuNP conjugate via EDC/NHS coupling chemistry; ( b ) Metal enhanced fluorescence of DDAO bound with the active sites of AChE via distance regulation between the fluorophore and AuNP; ( c ) The fluorescence reduction via distance modulation due to AChE inhibitor binding. Reprinted with permission from [ 63 ]. Copyright 2012 American Chemical Society.…”

Fabrication of an Electrochemical Biosensor Array for Simultaneous Detection of L-Glutamate and Acetylcholine

“…Glutamate is the major excitatory neurotransmitter in the central nervous system, [7][8][9][10][11] if the glutamate is in dysfunction, 12 it would lead to series diseases, such as epilepsy and schizophrenia, etc. Acetylcholinesterase (AChE) widely distributes in the central neural system and is bonded to the cellular membranes of excitable tissues, [13][14][15][16][17] and is a key enzyme playing a significant role in the nerve impulse transduction. Acetylthiocholine iodide (ATChI) is the substrate of acetylcholinesterase and nicotinic acetylcholine receptor agonist.…”