Construction of an Acetylcholinesterase Sensor Based on Synthesized Paramagnetic Nanoparticles, a Simple Tool for Neurotoxic Compounds Assay

Abstract

Magnetic particles (MPs) have been widely used in biological applications in recent years as a carrier for various molecules. Their big advantage is in repeated use of immobilized molecules including enzymes. Acetylcholinesterase (AChE) is an enzyme playing crucial role in neurotransmission and the enzyme is targeted by various molecules like Alzheimer's drugs, pesticides and warfare agents. In this work, an electrochemical biosensor having AChE immobilized onto MPs and stabilized through glutaraldehyde (GA) molecule was proposed for assay of the neurotoxic compounds. The prepared nanoparticles were modified by pure AChE and they were used for the measurement anti-Alzheimer's drug galantamine and carbamate pesticide carbofuran with limit of detection 1.5 µM and 20 nM, respectively. All measurements were carried out using screen-printed sensor with carbon working, silver reference, and carbon auxiliary electrode. Standard Ellman's assay was used for validation measurement of both inhibitors. Part of this work was the elimination of reversible inhibitors represented by galantamine from the active site of AChE. For this purpose, we used a lower pH to get the original activity of AChE after inhibition by galantamine. We also observed decarbamylation of the AChE-carbofuran adduct. Influence of organic solvents to AChE as well as repeatability of measurement with MPs with AChE was also established.

Keywords: acetylcholinesterase; carbofuran; electrochemistry; galantamine; magnetic particles; nanomaterial; nanoparticles; screen-printed sensor.

Figure 1

Immobilization principle of enzyme using glutaraldehyde.

Figure 2

Comparision of binding process of AChE to different magnetic particles measured by Ellman’s assay. MPs with GA = magnetic particles prepared with glutaraldehyde, MPs without GA magnetic particles prepared without glutaraldehyde. Error bars indicate standard error of the mean for n = 3.

Figure 3

Saturation curve of AChE and acetylthiocholine as a substrate performed in PBS 7.4. Hill funciton with a coefficient of cooperativity n = 1 was used for fitting. Error bars indicate standard deviation for n = 3.

Figure 4

SWV curves of acetylthiocholine performed in PBS 7.4.

Figure 5

Galantamine calibration curve performed in PBS 7.4. Error bars indicate standard deviation for n = 3.

Figure 6

SWV curves for galantamine performed in PBS 7.4.

Figure 7

Carbofuran calibration performed in PBS 7.4. Error bars indicate standard deviation for n = 3.

Figure 8

SWV curves of carbofuran performed in PBS 7.4.

Figure 9

Validation of galantamine measurement compared to standard Ellman’s assay. Error bars indicate standard deviation for n = 3.

Figure 10

Validation of carbofuran measurement compared to standard Ellman’s assay. Error bars indicate standard deviation for n = 3.

Figure 11

Wash out of galanthamine from galanthamine-inhibited magnetic particles. Error bars indicate standard deviation for n = 3.

Figure 12

Spontaneous decarbamylation performed in PBS 7.4. Error bars indicate standard deviation for n = 3.

Figure 13

Organic solvents performed in PBS 7.4.

Figure 14

Repeatability of magnetic particles with AChE performed in PBS 7.4. Error bars indicate standard deviation for n = 3.