Label-free electrochemical immunosensor for porcine gelatin using a boron-doped diamond electrode <i>via</i> diazonium salt electrografting
At a Glance
Section titled âAt a Glanceâ| Metadata | Details |
|---|---|
| Publication Date | 2025-06-09 |
| Journal | Journal of Electrochemical Science and Engineering |
| Authors | Irkham Irkham, Fadli Taufik Abdillah, Muhammad Ihda H.L. Zein, Adisyahputra, N. ZAHRA |
| Analysis | Full AI Review Included |
Executive Summary
Section titled âExecutive Summaryâ- Novel Platform: A label-free electrochemical immunosensor was successfully developed using a Boron-Doped Diamond (BDD) electrode for the selective and sensitive detection of porcine gelatin.
- Robust Functionalization: Stable covalent immobilization was achieved via electrografting of aryl diazonium salt (4-aminobenzoic acid, 4-ABA), providing a robust interface for subsequent biomolecule attachment.
- Oriented Antibody Binding: Anti-porcine gelatin antibodies were immobilized via Protein A using EDC/NHS coupling, ensuring optimal orientation and preserving the Fab region for target binding.
- High Sensitivity: The sensor demonstrated excellent analytical performance, achieving a low Limit of Detection (LoD) of 142.15 pg mL-1.
- Label-Free Detection: Detection relies on monitoring the current suppression of the [Fe(CN)6]3-/4- redox probe using Differential Pulse Voltammetry (DPV), simplifying the assay and reducing cost compared to labeled systems.
- High Specificity: Specificity testing confirmed exclusive response to porcine gelatin, showing negligible cross-reactivity (± 7.3% current change) toward bovine gelatin.
- Optimized Protocol: Operational parameters were optimized using the Box-Behnken design, resulting in a rapid detection protocol requiring only 60 min for antibody incubation and 15 min for antigen incubation.
Technical Specifications
Section titled âTechnical Specificationsâ| Parameter | Value | Unit | Context |
|---|---|---|---|
| Sensing Platform | Boron-Doped Diamond (BDD) | N/A | Working electrode material |
| Detection Limit (LoD) | 142.15 | pg mL-1 | Analytical performance |
| Linear Regression (R) | 0.9854 | N/A | Correlation coefficient of calibration curve |
| Coefficient of Determination (R2) | 0.9711 | N/A | Linearity over tested range |
| DPV Potential Range | -0.3 to +0.7 | V | Measurement range (vs. Ag/AgCl) |
| DPV Scan Rate | 0.008 | V s-1 | Measurement parameter |
| CV Scan Rate (Electrografting) | 100 | mV s-1 | BDD modification step |
| Redox Probe System | 0.01 M K3[Fe(CN)6] in 0.1 M KCl | N/A | DPV measurement solution |
| Optimal Antibody Dilution | 1:500 | N/A | Optimized concentration (Box-Behnken) |
| Antibody Incubation Time (Rapid Protocol) | 60 | min | Time used for rapid detection protocol |
| Gelatin Incubation Time (Optimal) | 15 | min | Time used for rapid detection protocol |
| Bovine Gelatin Cross-Reactivity | ± 7.3 | % | Current change upon exposure (indicating high specificity) |
Key Methodologies
Section titled âKey MethodologiesâThe immunosensor fabrication involves a multi-step surface modification process on the BDD electrode:
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BDD Surface Activation (Diazonium Electrografting):
- The BDD electrode was modified by electro-grafting 4-aminobenzoic acid (4-ABA) in 37% HCl solution, cooled to 0 °C.
- Electrografting was performed using 11 consecutive cyclic scans in the potential range of 0 to +0.8 V at a scan rate of 100 mV s-1. This process covalently bonds the diazonium-derived film (containing carboxylic acid groups) to the BDD surface.
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Protein A Immobilization:
- Protein A was immobilized onto the diazonium film using EDC/NHS coupling chemistry to activate the carboxyl groups for stable amide bond formation.
- The electrode was incubated in a solution containing Protein A (0.5 mg mL-1), EDC (4.8 mg mL-1), and NHS (2.87 mg mL-1) for 1 hour at 4 °C.
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Anti-Porcine Gelatin Antibody Immobilization:
- Anti-porcine gelatin antibodies (0.1 g mL-1 stock, 500Ă dilution) were incubated on the Protein A-modified electrode.
- The optimized incubation time for the rapid protocol was set to 60 minutes at 4 °C.
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Surface Blocking:
- The electrode surface was blocked with 1% Bovine Serum Albumin (BSA) solution for 30 minutes at room temperature to prevent non-specific adsorption.
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Antigen Detection (Binding):
- Standard porcine gelatin solution (0.5 ng mL-1) was applied to the electrode.
- The optimized incubation time for antigen binding was 15 minutes at room temperature.
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Electrochemical Readout:
- Detection was performed using Differential Pulse Voltammetry (DPV) with 40 ”L of 0.01 M K3[Fe(CN)6] in 0.1 M KCl as the redox probe.
- The binding of the non-conductive gelatin antigen causes a measurable decrease in the DPV peak current, providing a label-free signal.
Commercial Applications
Section titled âCommercial Applicationsâ- Halal and Kosher Certification: Provides a rapid, highly specific, and reliable method for verifying the absence of prohibited animal-derived ingredients (specifically porcine gelatin) in food, cosmetic, and pharmaceutical products.
- Food Authentication and Fraud Detection: Used by regulatory bodies and food manufacturers for quality control and detecting adulteration in processed foods, such as candies, dairy, and meat substitutes.
- Pharmaceutical Quality Control: Verification of the source material (porcine vs. bovine) used in gelatin capsules and other drug excipients, where source control is critical for regulatory compliance.
- Point-of-Care (POC) Biosensing Devices: The BDD platformâs stability, wide potential window, and the simplicity of the label-free electrochemical detection mechanism are ideal for developing portable, robust, on-site testing kits.
- Advanced Carbon Electrode Functionalization: The aryl diazonium electrografting technique provides a versatile, stable chemical platform for immobilizing various biological recognition elements (antibodies, aptamers) onto inert carbon surfaces for diverse biosensing applications.
View Original Abstract
Porcine gelatin is widely used in the food and pharmaceutical industries due to its favorable functional properties and low cost. However, its presence in consumer products raises serious concerns for individuals with dietary restrictions based on religious, ethical, or health considerations. In this study, a label-free electrochemical immunosensor was developed using a boron-doped diamond electrode modified with aryl diazonium salt for the selective and sensitive detection of porcine gelatin. The diazonium electrografting enabled stable covalent immobilization of anti-porcine gelatin antibodies via protein A, preserving antiÂbody orientation and activity. Experimental parameters were optimized using the Box-Behnken design, yielding ideal conditions of 500Ă antibody concentration, 60 min antibody incubation, and 15 min gelatin incubation. Detection was performed using differential pulse voltammetry with [Fe(CN)â]3-/4- as a redox probe, allowing label-free monitoring of antiÂbody-antigen interactions based on changes in current. The immunosensor demonstrated excellent analytical performance, with a detection limit of 142.15 pg mL-1. Specificity testing confirmed that the sensor responds exclusively to porcine gelatin, showing no cross-reactivity with bovine gelatin. These results demonstrate that the proposed immunosensor provides a rapid, highly sensitive, and specific platform for porcine gelatin detection, offering great potential for food authentication and halal verification.