Nanoelectrode Arrays Fabricated by Thermal Nanoimprint Lithography for Biosensing Application

At a Glance

Metadata	Details
Publication Date	2020-08-05
Journal	Biosensors
Authors	Alessandra Zanut, Alessandro Cian, Nicola Cefarin, Alessandro Pozzato, Massimo Tormen
Institutions	Fondazione Bruno Kessler, University of Trieste
Citations	22
Analysis	Full AI Review Included

Executive Summary

This analysis details the fabrication and performance of Nanoelectrode Arrays (NEA) developed for electrochemical biosensing, leveraging Thermal Nanoimprint Lithography (TNIL) for scalable production.

Core Value Proposition: The platform utilizes TNIL to fabricate NEAs on Boron-Doped Diamond (BDD) macroelectrodes, offering a highly reliable, reproducible, and low-cost alternative to traditional electron beam lithography (EBL) for manufacturing nanostructured sensors.
Enhanced Performance: The NEA geometry effectively suppresses the capacitive background signal, a characteristic of nanoelectrodes that significantly improves the signal-to-noise ratio and detection limits.
Material System: Polycarbonate (PC) film acts as the dielectric layer, which is chemically activated (5 M NaOH) to enable robust physisorption and immobilization of protein antigens (gliadin).
Demonstrated Application: The system successfully detected gliadin protein fragments using an immuno-indirect assay format (primary antibody, HRP-labeled secondary antibody, Methylene Blue/H₂O₂ electrocatalysis).
Analytical Results: Successful gliadin detection was achieved in the concentration range of 0.5-10 µg/mL, exhibiting a linear response (R² = 0.86) up to 0.5 µg/mL.
Scalability: The use of TNIL, capable of high-resolution structures down to the 10 nm scale, positions this technology for industrial scale-up in both food and biomedical applications.

Technical Specifications

Parameter	Value	Unit	Context
BDD Layer Thickness	400	nm	Conductive substrate layer
Polycarbonate (PC) Film Thickness	220	nm	Dielectric/Resist layer
NEA Dot Diameter (Average)	~260	nm	Size of exposed BDD nanoelectrode
NEA Interelectrode Spacing (Period)	800	nm	Center-to-center distance
TNIL Imprint Pressure	10	MPa	Fabrication process parameter
TNIL Imprint Temperature	180	°C	Fabrication process parameter
Gliadin Detection Range	0.5-10	µg/mL	Demonstrated operational range
Linear Response Range (R²)	≤0.5 (0.86)	µg/mL	Analytical performance
Optimal H₂O₂ Substrate Conc.	1.5	mM	Electrocatalytic optimization plateau
Mediator (MB) Concentration	0.1	mM	Cyclic Voltammetry electrolyte
Supporting Electrolyte	0.01	M	PBS (pH 7.2)
CV Scan Rate	50	mV s^-1	Electrochemical measurement speed
Sensitivity Loss (Day 7 Storage)	57	%	Stability assessment (stored at 4 °C)

Key Methodologies

The NEA fabrication relies on a two-step TNIL process: stamp creation followed by pattern transfer onto the BDD substrate.

1. Stamp Fabrication (Silicon Master)

Resist Coating: Si <100> substrate spin-coated with mr-I 7010E resist (115 nm thickness), annealed at 140 °C for 2 min.
TNIL Replication: Commercial master (800 nm period, 400 nm diameter/depth holes) replicated at 10 MPa pressure for 15 min at 100 °C.
Pattern Transfer (ICP-RIE):
- Residual Layer Removal (O₂ Plasma): 15 s duration, 200 W coil power, 10 W platen power, 40 sccm O₂ flow, 4 mT pressure.
- Silicon Etching (Fluorine Plasma): 15 s duration, 400 W coil power, 20 W platen power, SF₆/C₄F₈/Ar gas mixture (30/60/10 sccm), 8 mT pressure.
- Resist Ashing (O₂ Plasma): 15 s duration, 800 W coil power, 20 W platen power, 50 sccm O₂ flow, 20 mT pressure.
Release Layer: Stamp functionalized with octyl-trichlorosilane monolayer in vapor phase.

2. NEA Fabrication on BDD

PC Film Preparation: 4% w/v PC solution spin-coated onto 400 nm BDD/Si substrate (resulting thickness 220 nm). Film annealed at 180 °C for 30 min.
TNIL Imprinting: Stamp applied to PC film at 10 MPa pressure for 10 min at 180 °C (release temperature 80 °C).
Residual Layer Removal: O₂ plasma cleaning (4 s duration, 4 mT pressure, 200 W coil, 10 W platen) to expose the BDD surface at the bottom of the nanoholes.

3. Surface Functionalization and Detection

PC Activation: NEA dipped in 5 M NaOH solution for 1 min (to deprotonate carboxylic groups and increase hydrophilicity), followed by Milli-Q rinse.
Antigen Immobilization: 10 µL of 10 µg/mL gliadin fragments physisorbed onto the PC surface for 2 h at 25 °C.
Immunoassay Sequence:
1. Incubation with primary anti-gliadin antibody (10 µg/mL, 60 min).
2. Blocking with 1% BSA (30 min).
3. Incubation with HRP-labeled secondary antibody (10 µg/mL, 60 min).
Electrochemical Detection: Cyclic voltammetry (CV) performed in 0.1 mM Methylene Blue (MB) mediator solution, with the addition of H₂O₂ substrate (optimized at 1.5 mM) to initiate the HRP electrocatalytic cycle.

Commercial Applications

The combination of high-sensitivity NEA architecture and low-cost, high-throughput TNIL fabrication makes this technology highly relevant for mass-market sensing platforms.

Food Safety and Allergen Testing:
- High-volume, low-cost monitoring of specific allergens (e.g., gluten/gliadin, peanuts, dairy proteins) in packaged and processed foods to ensure regulatory compliance (e.g., <20 ppm gluten).
- Development of portable, rapid testing kits for on-site quality control in manufacturing and distribution.
Clinical and Biomedical Diagnostics (Point-of-Care):
- Creation of disposable immunosensors for rapid, sensitive detection of disease biomarkers (proteins, antibodies, hormones) in blood or saliva samples, suitable for POC settings.
- The BDD substrate offers excellent electrochemical stability, crucial for use in complex biological matrices.
Environmental Monitoring:
- Utilizing the robust BDD/PC platform for sensitive detection of trace contaminants, such as pesticides, heavy metals, or pharmaceutical residues, in water sources.
High-Throughput Manufacturing:
- TNIL enables the transition from expensive, small-batch lithography (EBL) to industrial-scale production of nanostructured electrochemical chips, significantly reducing unit costs for biosensor components.

View Original Abstract

Electrochemical sensors are devices capable of detecting molecules and biomolecules in solutions and determining the concentration through direct electrical measurements. These systems can be miniaturized to a size less than 1 µm through the creation of small-size arrays of nanoelectrodes (NEA), offering advantages in terms of increased sensitivity and compactness. In this work, we present the fabrication of an electrochemical platform based on an array of nanoelectrodes (NEA) and its possible use for the detection of antigens of interest. NEAs were fabricated by forming arrays of nanoholes on a thin film of polycarbonate (PC) deposited on boron-doped diamond (BDD) macroelectrodes by thermal nanoimprint lithography (TNIL), which demonstrated to be a highly reliable and reproducible process. As proof of principle, gliadin protein fragments were physisorbed on the polycarbonate surface of NEAs and detected by immuno-indirect assay using a secondary antibody labelled with horseradish peroxidase (HRP). This method allows a successful detection of gliadin, in the range of concentration of 0.5-10 μg/mL, by cyclic voltammetry taking advantage from the properties of NEAs to strongly suppress the capacitive background signal. We demonstrate that the characteristics of the TNIL technology in the fabrication of high-resolution nanostructures together with their low-cost production, may allow to scale up the production of NEAs-based electrochemical sensing platform to monitor biochemical molecules for both food and biomedical applications.

Tech Support

Original Source

DOI: https://doi.org/10.3390/bios10080090

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