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Characterizing Mixed Polyethylene Glycol Monolayers with Surface-Bound Ferrocene for Label-Free Immunosensing.

Emie Marin¹, Brandaise Martinez¹, Tessa Whitaker¹

¹Department of Chemistry, Colorado State University, Fort Collins, Colorado 80523, United States.

Analytical Chemistry

|October 29, 2025

Summary

This study developed a label-free electrochemical immunosensor using a multifunctional monolayer for enhanced sensitivity. The novel sensor design successfully detected SARS-CoV-2 virus nucleocapsid protein with improved detection limits.

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Area of Science:

Electrochemistry
Biosensing
Surface Chemistry

Background:

Label-free electrochemical immunosensors offer direct biomolecular detection but often lack sensitivity for low-abundance analytes.
Current strategies to boost sensitivity typically involve nanomaterial-based electrode modifications.

Purpose of the Study:

To develop a multifunctional monolayer for label-free electrochemical immunosensors to enhance sensitivity.
To incorporate ferrocene for improved electron transfer and biotin for antibody immobilization.

Main Methods:

Assembled a mixed monolayer on thermoplastic electrodes (TPEs) using copper-catalyzed azide-alkyne cycloaddition (CuAAC).
Utilized polyethylene glycol (PEG)-based spacers with ferrocene (Fc) and biotin functionalities.
Characterized monolayer formation using electrochemical methods and X-ray photoelectron spectroscopy (XPS).
Detected inactivated SARS-CoV-2 virus nucleocapsid (N) protein using square wave voltammetry (SWV).

Main Results:

Confirmed successful monolayer formation, ferrocene integration, biotin functionality, and minimized nonspecific adsorption (NSA).
Achieved limits of detection (LOD) of 21.1 ± 10.6 ng/mL (PEG11) and 21.6 ± 10.8 ng/mL (PEG24) using ferrocyanide as the redox probe.
Demonstrated sensor performance was independent of PEG chain length for electron transfer but influenced detection sensitivity.

Conclusions:

Strategic surface chemistry design and redox property integration significantly enhance label-free electrochemical immunosensor sensitivity.
The developed multifunctional monolayer provides a promising platform for sensitive detection of low-abundance analytes.