Acetylcholinesterase Modified Porous Silicon for Electrochemical Measurement of Total Active Immobilized Enzyme Amount and Effective Malathion Detection

At a Glance

Metadata	Details
Publication Date	2020-03-20
Journal	Instrumentation Mesure Métrologie
Authors	Khadidja Khaldi, S. Sam, N. Gabouze
Institutions	Centre de Recherche en Technologie des Semi-conducteurs pour l’Energétique, Centre de Recherche sur l’Information Scientifique et Technique
Citations	1
Analysis	Full AI Review Included

Executive Summary

This research details the development and characterization of a high-performance amperometric biosensor for detecting organophosphate (OP) inhibitors, specifically Malathion, utilizing a hybrid Porous Silicon (PSi) and Boron Doped Polycrystalline Diamond (BDPD) architecture.

Hybrid Architecture: The biosensor employs a PSi matrix, functionalized with Gold Nanoparticles (AuNP), for high-density covalent immobilization of Acetylcholinesterase (AChE). The electrochemical signal is recorded using a highly stable BDPD working electrode.
Enhanced Immobilization: The use of AuNP on the PSi surface significantly reduced hydrophobicity, leading to a favorable orientation of the AChE active sites and achieving a high active enzyme surface concentration (Γ ≈ 1.8 x 10¹² AChE cm^-2).
Superior Detection Stability: BDPD was confirmed to be the optimal transducer, exhibiting robust stability and high electrochemical activity for thiocholine oxidation (the enzymatic product), overcoming the inherent low conductivity and stability issues of PSi.
High Sensitivity: The method demonstrated effective quantification of Malathion with a good linear calibration curve in the environmentally relevant ultra-trace range of 2 to 6 nM.
Kinetic Characterization: The Michaelis constant (K_m) for the immobilized enzyme was determined to be 0.43 mM, confirming the viability of the enzymatic reaction on the modified surface.
Industrial Potential: The proposed method is simple, rapid, sensitive, and offers flexibility in electrode design (using separate support and working electrodes), making it promising for various industrial and environmental monitoring applications.

Technical Specifications

Parameter	Value	Unit	Context
PSi Layer Thickness	3.2	µm	Determined by SEM cleavage observation.
PSi Average Pore Size	20-30	nm	Confirmed by SEM micrograph (surface view).
PSi Etching Current Density	80	mA cm^-2	Wet electrochemical etching process.
BDPD Boron Doping Level	10¹⁹-10²⁰	B cm^-3	Assessed by SIMS measurements.
Active AChE Surface Concentration (Γ)	1.8 x 10¹²	AChE cm^-2	Estimated via free AChE calibration curve.
Michaelis Constant (K_m)	0.43	mM	Assessed via Lineweaver-Burk plot.
Malathion Linear Detection Range	2 to 6	nM	Inhibition measured within 6 minutes.
Thiocholine Oxidation Peak Potential (BDPD)	1.0	V	Anodic peak potential (vs Ag/AgCl).
Immobilized AChE Oxidation Peak Potential (BDPD)	0.7	V	Anodic peak potential (vs Ag/AgCl) after incubation.
Cyclic Voltammetry Scan Rate	50	mV/s	Used for all electrochemical measurements.
APTES Curing Temperature	80	°C	Used to stabilize the amine functionalization.

Key Methodologies

The fabrication of the PSi-AuNP-NH-AChE matrix and the electrochemical detection protocol using the BDPD electrode followed these key steps:

PSi Substrate Preparation:
- P-type, (100)-oriented silicon wafers (0.08-0.12 Ω cm resistivity) were cleaned using Piranha solution (1/3 H₂O₂/H₂SO₄) at 100 °C.
- Native oxide was removed using 47% aqueous HF for 30 seconds.
- PSi layers were formed by wet electrochemical etching in a 1:1 (v/v) 47% HF/absolute ethanol mixture at a current density of 80 mA cm^-2 for 30 seconds.
Gold Nanoparticle (AuNP) Decoration:
- AuNP were deposited via electroless plating by dropping 50 µl of 1% HAuCl₄ solution (in HF) onto the fresh PSi surface for 1 minute.
Surface Functionalization:
- The PSi-AuNP surface was oxidized at 200 °C for 30 minutes to form -OH bonds.
- Amine functionalization was achieved by immersing the substrate in 3% (v/v) 3-aminopropyltriethoxysilane (APTES) in 95% ethanol for 2 hours, followed by curing at 80 °C for 20 minutes.
AChE Covalent Immobilization:
- The amine-functionalized PSi-AuNP-NH₂ surface was introduced into a solution mixture containing 0.08 µM AChE, 5 mM EDC, and 5 mM NHS in PBS.
- The reaction was allowed to proceed overnight at room temperature to ensure covalent linking.
Electrochemical Detection:
- All measurements were performed using a BDPD working electrode, a Pt counter electrode, and an Ag/AgCl reference electrode.
- The detection relies on the anodic oxidation of thiocholine (product of AChE hydrolysis of ATCI) at the BDPD surface.
Malathion Inhibition Assay:
- The PSi-AuNP-NH-AChE surface was incubated in Malathion solutions (2 to 6 nM) for 6 minutes.
- The inhibited surface was then incubated in 0.55 mM ATCI solution for 20 minutes, and the resulting decrease in thiocholine oxidation current (at the BDPD electrode) was measured to quantify inhibition.

Commercial Applications

This technology, leveraging the stability of BDPD and the high surface area of PSi, is highly relevant for several high-value engineering and commercial sectors:

Environmental and Water Quality Monitoring:
- Development of portable, rapid, and highly sensitive biosensors for field testing of OP pesticides in agricultural runoff, drinking water sources, and food processing facilities.
- Creating robust, reusable sensor platforms that can withstand harsh environmental conditions due to the chemical stability of the BDPD electrode.
Chemical and Biological Warfare Agent Detection:
- The high sensitivity (nM range) makes this platform suitable for detecting ultra-trace levels of nerve agents (which are OP compounds) in air or water, crucial for defense and security applications.
Pharmaceutical and Clinical Diagnostics:
- Screening for drugs that inhibit or modulate AChE activity, relevant for research into neurodegenerative diseases like Alzheimer’s.
- Biomonitoring platforms for assessing human exposure levels to pesticides or related toxins.
Advanced Materials and Microelectronics (Relevant to Diamond Technology):
- The use of BDPD highlights the commercial viability of diamond electrodes in complex electrochemical systems where traditional electrodes (Au, Pt) fail due to fouling or limited potential windows.
- Integration of high surface area PSi matrices into microfluidic or lab-on-a-chip devices for miniaturized biosensing applications.

View Original Abstract

An amperometric method to measure the total amount of active and inhibited immobilized Acetylcholinesterase enzyme (AChE) and the quantification of AChE inhibitors was realized.In this approach porous silicon (PSi) surface was used as a matrix for AChE immobilization and Boron doped polycrystalline diamant (BDPD) was used as working electrode for the electrochemical measurements.The covalent immobilization of AChE from electric eel was achieved on amine functionalized PSi surface previously decorated with Au particles.This surface is suitable for a stable attachment of AChE enzyme.The amperometric detection of AChE activity at Boron doped polycrystalline diamond electrode is based on the oxidation of thiocholine, the enzymatic reaction product of immobilized AChE in the presence of the substrate acetylthiocholine chlorid (ATCl) and this without the need to further modify the BDPD surface or the use of other reagents.The concentration of immobilized active AChE enzymes was estimated to Γ≈ 1.8 10 12 AChE cm -2 by means of a calibration curve.Michaelis constant was assessed with a Km of 4.3 10 -4 M. Finally, the electrochemical quantification and detection of Malathion shows a good linear calibration curve where the concentration of Malathion range is from 2 to 6 nM within 6 minutes after inhibitor addition.

Tech Support

Original Source

DOI: https://doi.org/10.18280/i2m.190107