Highly Sensitive and Selective Detection of L-Tryptophan by ECL Using Boron-Doped Diamond Electrodes

At a Glance

Metadata	Details
Publication Date	2024-06-04
Journal	Sensors
Authors	Emmanuel Scorsone, Samuel Stewart, Matthieu Hamel
Institutions	Commissariat à l’Énergie Atomique et aux Énergies Alternatives, CEA LIST
Citations	1
Analysis	Full AI Review Included

Executive Summary

This analysis summarizes a novel electrochemiluminescence (ECL) method for the highly sensitive and selective detection of L-Tryptophan (L-Trp) utilizing Boron-Doped Diamond (BDD) electrodes.

Core Innovation: The method relies on the in situ electrogenerated production of hydrogen peroxide (H₂O₂) at the BDD surface during cathodic polarization, eliminating the need for external coreactants in the analytical solution.
Performance Benchmark: A record-low limit of detection (LOD) of 0.4 nM and a limit of quantification (LOQ) of 1.4 nM were achieved in phosphate buffer saline (PBS, pH 7.4).
Analytical Range: The ECL response showed excellent linearity (R² = 0.99%) across the concentration range of 0.005 to 1 µM L-Trp.
Selectivity: The BDD-based system demonstrated high selectivity, showing no significant ECL signal from common indolic interferences such as tryptamine, indole, skatole (3-methylindole), and serotonin.
Mechanism Confirmation: Spectroscopic data confirmed that the ECL emission peaks at approximately 425 nm, suggesting the reaction proceeds via the formation of a dioxetane intermediate, which is characteristic of ROS-enhanced ECL.
Material Advantage: BDD electrodes are uniquely suited for this reaction due to their wide potential window (allowing high cathodic overpotential for efficient H₂O₂ generation) and low adsorption properties (making H₂O₂ available in solution for the ECL reaction).

Technical Specifications

Parameter	Value	Unit	Context
Limit of Detection (LOD)	0.4	nM	L-Tryptophan in PBS (pH 7.4)
Limit of Quantification (LOQ)	1.4	nM	L-Tryptophan in PBS (pH 7.4)
Linear Range	0.005 to 1	µM	Calibration curve (R² = 0.99%)
Optimal CV Scan Rate	0.25	V.s^-1	Optimized for stable ECL signal
Optimal Cathodic Potential	-3.0	V	vs. Ag/AgCl (Maximum H₂O₂ generation)
Optimal Anodic Potential	1.5	V	vs. Ag/AgCl (Maximum ECL emission)
BDD Film Thickness	ca. 800	nm	Grown by PE-CVD
BDD Doping Level	2 x 10²¹	boron atom.cm^-3	Determined by SIMS measurements
Substrate	4-inch <100>	Silicon Wafer	Highly conductive
Microwave Power (Growth)	3.5	kW	PE-CVD reactor
Growth Pressure	40	Torr	1% CH₄ in H₂ gas phase
ECL Emission Peak	ca. 425	nm	Associated with dioxetane intermediate

Key Methodologies

The detection relies on a three-step electrochemical process (BDD fabrication, H₂O₂ generation, and L-Trp oxidation/ECL emission) using a three-electrode cell (BDD working/counter, Pt pseudo-reference).

1. Boron-Doped Diamond (BDD) Electrode Fabrication

Method: Plasma Enhanced Chemical Vapor Deposition (PE-CVD).
Substrate: Highly conductive 4-inch <100> silicon wafer.
Gas Mixture: 1% methane (CH₄) in hydrogen (H₂).
Dopant Source: Trimethylboron added to the gas phase.
Growth Conditions: 40 Torr pressure, 3.5 kW microwave power.
Resulting Film: ca. 800 nm thickness with a doping level of 2 x 10²¹ boron atom.cm^-3.

2. Electrochemiluminescence (ECL) Measurement Cycle

Electrolyte: 0.1 M Phosphate Buffer Saline (PBS, pH 7.4).
Polarization Sequence (Cyclic Voltammetry): Successive cathodic reduction followed by anodic oxidation (0 V → -3 V → 1.5 V → 0 V).
Step 1: Electro-Reduction (H₂O₂ Generation):
- Potential is swept cathodically down to -3.0 V vs. Ag/AgCl.
- Dissolved oxygen (O₂) undergoes reduction and protonation steps on the BDD surface, efficiently generating the superoxide anion radical (O₂^-) and subsequently hydrogen peroxide (H₂O₂).
Step 2: Electro-Oxidation (ECL Emission):
- Potential is swept anodically up to 1.5 V vs. Ag/AgCl.
- L-Tryptophan is electro-oxidized (starting at approx. 0.9 V).
- The oxidized L-Trp intermediate interacts with the electrogenerated H₂O₂ to form a hydroperoxide intermediate.
- This intermediate rearranges to form a dioxetane group, which then decomposes to an excited state, resulting in light emission (ECL peak at ca. 425 nm).

Commercial Applications

The high sensitivity and selectivity achieved by this BDD-based ECL sensor make it highly relevant for rapid, low-cost analytical screening in several critical sectors:

Clinical Diagnostics and Neuroscience: Highly sensitive quantification of L-tryptophan in biological fluids (blood, urine, saliva) for monitoring neurological disorders (e.g., depression, Parkinson’s disease) and oncology studies, where L-Trp metabolism is a key biomarker.
Food Safety and Quality Control: Rapid and accurate determination of L-tryptophan content in food products and dietary supplements, ensuring compliance with nutritional standards.
Pharmaceutical Quality Control (QC): Use as a robust, high-throughput sensor for quantifying L-Trp in drug formulations.
Advanced Electrochemical Sensing: Leveraging the unique properties of BDD (wide potential window, low fouling, high stability) for developing highly selective sensors for other complex organic molecules in aqueous matrices.
Water Treatment/ROS Generation: The demonstrated efficiency of BDD in generating Reactive Oxygen Species (ROS), specifically H₂O₂, at high overpotentials reinforces its utility in advanced oxidation processes (AOPs) for environmental remediation.

View Original Abstract

L-tryptophan is an amino acid that is essential to the metabolism of humans. Therefore, there is a high interest for its detection in biological fluids including blood, urine, and saliva for medical studies, but also in food products. Towards this goal, we report on a new electrochemiluminescence (ECL) method for L-tryptophan detection involving the in situ production of hydrogen peroxide at the surface of boron-doped diamond (BDD) electrodes. We demonstrate that the ECL response efficiency is directly related to H2O2 production at the electrode surface and propose a mechanism for the ECL emission of L-tryptophan. After optimizing the analytical conditions, we show that the ECL response to L-tryptophan is directly linear with concentration in the range of 0.005 to 1 µM. We achieved a limit of detection of 0.4 nM and limit of quantification of 1.4 nM in phosphate buffer saline (PBS, pH 7.4). Good selectivity against other indolic compounds (serotonin, 3-methylindole, tryptamine, indole) potentially found in biological fluids was observed, thus making this approach highly promising for quantifying L-tryptophan in a broad range of aqueous matrices of interest.

Tech Support

Original Source

DOI: https://doi.org/10.3390/s24113627

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