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 sur l’Information Scientifique et Technique, Centre de Recherche en Technologie des Semi-conducteurs pour l’Energétique
Citations	1
Analysis	Full AI Review Included

Technical Documentation & Analysis: Boron-Doped Diamond for High-Performance Electrochemical Biosensors

This document analyzes the research paper “Acetylcholinesterase Modified Porous Silicon for Electrochemical Measurement of Total Active Immobilized Enzyme Amount and Effective Malathion Detection” to highlight the critical role of Boron-Doped Polycrystalline Diamond (BDPD) and align the findings with 6CCVD’s advanced MPCVD diamond capabilities.

Executive Summary

The research successfully developed a highly sensitive and stable amperometric biosensor for quantifying active immobilized Acetylcholinesterase (AChE) and detecting its inhibitors, specifically Malathion (an organophosphate pesticide).

Core Material Success: Boron-Doped Polycrystalline Diamond (BDPD) was utilized as the robust working electrode, demonstrating superior electrochemical stability and activity compared to the porous silicon (PSi) enzyme matrix.
High Sensitivity: The BDPD electrode enabled linear Malathion detection in the ultra-low concentration range of 2 to 6 nM within 6 minutes of inhibitor exposure.
Quantifiable Results: The study accurately estimated the surface concentration of active immobilized AChE at Γ ≈ 1.8 x 10¹² AChE cm^-2.
Electrochemical Stability: BDPD maintained consistent current intensity and shape over successive cyclic voltammograms, confirming its suitability for long-term, reliable biosensing applications.
Methodology: The approach combined the high surface area of AuNP-decorated PSi for enzyme immobilization with the exceptional electrochemical properties of BDPD for signal transduction.
Commercial Relevance: The resulting device is simple, rapid, and sensitive, offering a promising solution for industrial and environmental detection of organophosphate compounds.

Technical Specifications

The following hard data points were extracted from the research detailing the materials and performance metrics.

Parameter	Value	Unit	Context
Working Electrode Material	BDPD (Polycrystalline Diamond)	N/A	Used for electrochemical measurements
BDPD Film Thickness	1.5 - 2.0	µm	Deposited on Silicon substrates
Boron Doping Level	10¹⁹ - 10²⁰	B cm^-3	Assessed by SIMS measurements
Substrate Resistivity (PSi precursor)	0.08 - 0.12	Ω cm	Boron-doped p-type Silicon
Anodic Peak Potential	1	V	Oxidation of thiocholine on BDPD
Scan Rate (Voltammetry)	50	mV/s	Used for cyclic voltammograms
Immobilized Enzyme Concentration (Γ)	1.8 x 10¹²	AChE cm^-2	Estimated using calibration curve
Michaelis Constant (K_m)	0.43 x 10^-3	M	Kinetic parameter of enzymatic reaction
Malathion Detection Range	2 to 6	nM	Linear calibration curve range
Porous Silicon Layer Thickness	3.2	µm	Determined by SEM observation

Key Methodologies

The experimental procedure involved precise material synthesis and surface functionalization steps:

Substrate Preparation: Silicon wafers (15 x 15 mm²) were cleaned using Piranha solution (H₂O₂/H₂SO₄) at 100 °C, followed by native oxide removal using 47% aqueous HF.
Porous Silicon (PSi) Formation: Wet electrochemical etching of H-terminated surfaces was performed in a 1/1 (v/v) 47% HF/absolute ethanol mixture for 30 s at a current density of 80 mA cm^-2.
Gold Nanoparticle (AuNP) Decoration: Electroless plating using 1% HAuCl₄ solution (in HF) was deposited onto the freshly prepared PSi surface for 1 min.
Oxidation and Amine Functionalization: The PSi-AuNP surface was oxidized at 200 °C for 30 min, followed by immersion in a 3% (v/v) APTES solution (in 95% ethanol) for 2h at room temperature, and cured at 80 °C for 20 min.
AChE Covalent Immobilization: The functionalized PSi-AuNP-NH₂ surface was introduced to a mixture containing 0.08 µM AChE, 5 mM EDC, and 5 mM NHS in PBS solution, reacting overnight at room temperature.
Electrochemical Measurement: Cyclic voltammograms were recorded using a three-electrode cell configuration, employing a Platinum counter electrode, Ag/AgCl reference electrode, and the BDPD working electrode.

6CCVD Solutions & Capabilities

The success of this high-sensitivity biosensor relies fundamentally on the stability and wide electrochemical window provided by the Boron-Doped Diamond (BDD) electrode. 6CCVD is uniquely positioned to supply and customize the necessary diamond materials to replicate and advance this research.

Applicable Materials

To replicate the high-performance working electrode used in this study, researchers require:

Heavy Boron-Doped Polycrystalline Diamond (PCD) Wafers: 6CCVD specializes in MPCVD growth of BDD films with precise doping control (targeting the 10¹⁹-10²⁰ B cm^-3 range) necessary to achieve the low residual current and exceptional stability demonstrated in the paper.
Custom Thickness BDD Films: We offer BDD films ranging from 0.1 µm to 500 µm, allowing engineers to optimize the electrode thickness (1.5-2.0 µm used here) for specific device integration and conductivity requirements.

Customization Potential

6CCVD’s in-house capabilities directly address the complex fabrication needs of advanced biosensors:

Research Requirement	6CCVD Solution & Capability	Technical Advantage
Electrode Stability & Performance	High-Quality BDD Wafers (up to 125 mm)	Provides the robust, chemically inert, and electrochemically active platform essential for reliable, long-duration biosensing, far surpassing the stability of traditional carbon or metal electrodes.
Custom Film Thickness	Thickness Control (0.1 µm - 500 µm)	We supply BDD films tailored to specific device architectures, whether thin films on silicon substrates (as used here) or thick, free-standing substrates (up to 10 mm).
Surface Functionalization Preparation	Internal Metalization Services (Au, Pt, Ti, etc.)	While the paper used AuNP decoration on PSi, 6CCVD can apply custom metal contacts (Au, Pt, Pd, Ti, W, Cu) directly to the diamond surface, simplifying integration and providing stable contact points for complex sensor designs.
Surface Finish for Immobilization	Advanced Polishing (Ra < 5 nm for PCD)	We ensure ultra-smooth surfaces, critical for uniform chemical functionalization (APTES grafting) and stable enzyme immobilization (AChE), maximizing the active enzyme concentration (Γ).

Engineering Support

The successful integration of BDD into complex electrochemical systems, such as the Malathion biosensor, requires specialized knowledge.

6CCVD’s in-house PhD team can assist with material selection and optimization for similar electrochemical biosensor and environmental monitoring projects. We provide consultation on:

Optimizing boron doping levels for specific redox reactions (like thiocholine oxidation at 1 V).
Selecting appropriate surface termination (e.g., hydrogen or oxygen) to enhance subsequent chemical grafting steps.
Designing custom electrode geometries and dimensions for high-throughput sensor arrays.

For custom specifications or material consultation, visit 6ccvd.com or contact our engineering team directly.

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