A novel molecularly imprinted graphene-graphite conductive paper electrode for selective electrochemical detection of hyaluronic acid in cosmetic formulations

Highlights

• Developed low-cost graphene–graphite conductive paint-based paper (CPP) electrodes.
• Biodegradable CPP electrode fabricated for stable electrochemical performance.
• MIP layer formed on CPP enabling a sensitive signal-off mechanism for HA sensing.
• Sensor showed wide range (0.1–1000 μM), LOD 0.009 μM, and high selectivity (IF > 3).
• Test on real cosmetic samples yielded∼91% agreement with manufacturers' HA content.

Abstract

Hyaluronic acid (HA), a glycosaminoglycan widely used in cosmetic and healthcare formulations, is valued for its moisturizing, viscoelastic, and regenerative properties. Accurate determination of HA in commercial products has therefore been considered essential for quality control and regulatory compliance. Although several chromatographic and spectroscopic techniques have been employed for HA quantification, their use has been restricted by high operational cost, complex operation, and labour-intensive sample preparation. In this work, a low-cost, flexible, and disposable conductive paint-based paper (CPP) electrode modified with an electropolymerized hyaluronic acid-imprinted poly(p-phenylenediamine) film [HA-MI-P(p-PD)@CPP] has been fabricated for the selective detection of HA. The conductive paint was prepared by dispersing polyacrylate with a graphene nanosheet–graphite (GNS-g) composite in chloroform to yield a mechanically stable and highly conductive substrate. After electropolymerization, the template removal and rebinding processes were investigated using cyclic voltammetry (CV), differential pulse voltammetry (DPV), and electrochemical impedance spectroscopy (EIS) with the [Fe(CN)₆]^3-/[Fe(CN)₆]^4- redox couple. The analytical performance of the imprinted electrode was assessed from DPV signals obtained before and after exposure to varying HA concentrations. The method achieved a low limit of detection (LOD) of 0.009 μM and a limit of quantification (LOQ) of 0.032 μM, exhibiting two linear response ranges (0.1–60 μM and 60–1000 μM). High recoveries (97–104%) and an imprinting factor exceeding 3, confirmed strong selectivity against structurally related interferents. Its affordability, environmental compatibility, and reliable sensing capability highlight its potential for on-site monitoring of HA in cosmetic and healthcare products.

Introduction

Pharmaceutical and cosmetic brands frequently promote products claiming to prevent health issues. One of the most widely highlighted ingredients today is hyaluronan or hyaluronic acid (HA). This biopolymer is a high-molecular-weight glycosaminoglycan consisting of repeating disaccharide units of N-acetyl-d-glucosamine and d-glucuronic acid [1,2]. Synthesized naturally by connective tissue cells, it forms an essential component of the extracellular matrix and plays a vital role in preserving tissue hydration, elasticity, and tissue integrity. Due to its exceptional physicochemical and biological functionalities, HA has found extensive applications in cosmetics, viscosurgery, ophthalmology, orthopaedics, rheumatology, tissue engineering, and cancer therapy [3,4]. Beyond its natural structure, combination-based formulations incorporating HA with bioactive substances have been increasingly investigated for wide range of applications such as facial volume enhancement, osteoarthritis management, wrinkle reduction, cardiovascular and dermatological treatments, urinary tract support, antioxidant and anti-inflammatory therapies, and wound healing [[5], [6], [7]]. In cosmetic formulations, HA is incorporated into serums, creams, and injectable products to enhance hydration, skin plumpness, and anti-aging effects, with sodium hyaluronate being particularly favored for its superior dermal penetration [[8], [9], [10], [11]]. The widespread biomedical and cosmetic acceptance of HA is largely attributed to its excellent biocompatibility, biodegradability, non-toxicity, and non-immunogenicity, although mild side effects such as transient irritation, swelling, or bruising may occasionally be observed [[12], [13], [14], [15], [16]]. In this study, emphasis has been placed on quantifying HA levels in readily available over-the-counter products through electrochemical measurements and comparing the detected HA content with label-declared values.

Several analytical techniques have been reported for determining HA levels in pharmaceutical and cosmetic formulations. Çağlar et al. [17] quantified HA using HPLC within a working range of 5–100 μg/mL and achieved a detection limit (LOD) of 0.226 μg/mL. Kašparová et al. [18] compared HA content in cosmetic products using both HPLC-MS and HPLC-FL, while Suárez-Hernández et al. [19] employed size exclusion chromatography, obtaining a wide linear range of 100–1000 mg/L with an LOD of 12 mg/L. Harmita et al. [20] also utilized HPLC, detecting HA from 5 to 50 μg/mL with an LOD of 3.55 μg/mL. Electrophoretic quantification was reported by Ermolenko et al. [21], and Zhang et al. [22] introduced a pyrene-cored cationic AIEgen system capable of detecting 0–8 μg/mL with an ultra-low LOD of 6.9 ng/mL. Additionally, FTIR spectroscopy has been used for HA analysis in food supplements [23]. Despite offering high sensitivity and accuracy, these techniques typically demand costly instrumentation, lengthy analysis times, and complex sample preparation, restricting their broader application.

Alternatively, eelectrochemical techniques are widely employed in analytical applications owing to their simplicity, rapid response, cost-effectiveness, and ability to achieve very low LODs. For instance, Mobed et al. [24] developed a Pt@Au nano-alloy immunosensor for detecting HA in plasma, achieving a detection range of 0.156–160 ng/mL and an LOD of 0.039 ng. Chen et al. [25] reported an RGO–ZnO nanocomposite-modified GCE biosensor, showing selective detection from 1 to 800 μM with an LOD of 0.42 μM. Niu et al. [26] employed phenosafranine as a cationic probe, achieving a linear range of 0.8–50 mg/L and an LOD of 0.901 mg/L. Another PMMA-based microchip with a gold nanoelectrode ensemble was proposed by Chen et al. [27], offering detection from 0.1 to 10 mg/mL with a 0.1 mg/mL LOD. Perk et al. [28] introduced an AuNP-modified carbon paste electrode for dermatological HA analysis, operating within 0.0075–0.15 mg/100 mL and reaching LODs of 0.0034 mg/100 mL.

To further enhance their LOD, sensitivity and selectivity, the integration of molecularly imprinted polymers (MIPs) has gained significant attention [29]. Owing to their high specificity, selectivity, and superior detection capability, MIPs can efficiently isolate target molecules from complex sample matrices. Hence, MIPs have become a powerful tool for the progress of advanced electrochemical sensors for pharmaceutical and food safety applications [30]. Initially, MIPs were predominantly synthesized through bulk polymerization. In this approach, template molecules are combined with functional monomers and a cross-linker to form a rigid polymer matrix. After polymerization, the monolithic polymer is mechanically ground and sieved to obtain micron-sized particles containing template-specific binding cavities [[31], [32], [33]]. However, this post-polymerization grinding step is inherently inefficient and provides little control over particle size or morphology. Consequently, the resulting MIPs often exhibit irregular structures, random nanoscale features, poor homogeneity, and high levels of non-specific binding, which collectively diminish their affinity and selectivity toward the target analyte [34]. To overcome these drawbacks, surface polymerization techniques have gained popularity owing to their operational simplicity, improved stability, and enhanced mass transfer characteristics. In surface imprinting, recognition sites are formed primarily on the outer layer of a solid substrate, facilitating faster binding–desorption kinetics and higher accessibility of the template-specific cavities. Polymerization of monomers on an electrode or solid support can be achieved using several strategies, including thermal polymerization (TP), photopolymerization (PP), and electropolymerization (EP) [35,36]. Among these, EP is particularly advantageous, as a controlled potential or current is applied to induce polymerization in the presence of the template on the electrode surface. As a result, this technique yields thin, uniform, and strongly adhered MIP films with improved selectivity and reproducibility.

Recently disposable paper-based electrodes made using conductive paints or inks have gained strong attention for medical and environmental sensing, offering good analytical performance. Such paper sensors are inexpensive, easy to fabricate, biodegradable, and suitable for both laboratory and field use [[37], [38], [39]]. Numerous studies have therefore focused on developing inexpensive and straightforward fabrication strategies for paper-based electrochemical sensors. For example, Pradela-Filho and co-workers [40] used glass-varnish conductive ink loaded with graphite to prepare paper electrodes for detecting analytes including dopamine, catechol, hydroquinone, and estriol. Cinti et al. [41] reported a reagent-free, screen-printed paper sensor designed for phosphate determination in river water samples. Likewise, de Araujo Andreotti et al. [42] introduced a graphite-ink formulation using nail polish as a binder for flexible disposable electrodes, while another study by Pradela-Filho et al. [43] developed carbon-powder/nail-polish-based conductive ink for dopamine sensing. Surface modification is widely used to enhance the sensitivity and selectivity of screen-printed carbon electrodes. For example, Buleandra et al. [44] used Prussian blue-modified electrodes to detect hydroquinone and catechol, while Li et al. [45] developed disposable electrodes incorporating multi-walled carbon nanotubes and gold nanoparticles, enabling the differentiation of dihydroxybenzene isomers via DPV. However, no CPP-based MIP has yet been reported in the accessible literature.

This study presents a low-cost, lightweight, and flexible MIP sensor fabricated on a conductive paint-coated paper (CPP) electrode. With slight modification of our previous work [46], the CPP platform was developed by dispersing graphene nanosheets (GNS) and graphite (g) within a polyacrylate binder. The resulting composite offered enhanced conductivity, mechanical stability, and active surface area compared to conventional glass tube electrodes. Once the conductive paper electrode was prepared, it served as the substrate for direct growth of MIP films on its surface. The imprinted layers were formed via surface polymerization of p-Phenylenediamine (p-PD) in the presence of HA template, with radical polymerization initiated electrochemically. Here, p-PD was selected as the functional monomer due to its strong affinity toward HA through hydrogen bonding and electrostatic interactions between amino and carboxyl/hydroxyl groups. Upon electropolymerization, p-PD forms a thin, uniform, and electrically insulating poly(p-PD) layer on the CPP surface, enabling efficient molecular imprinting and signal-off electrochemical detection. The ability to form stable films without chemical cross-linkers further supports its suitability for low-cost, paper-based sensing platforms. This strategy allowed precise control over film formation and efficient incorporation of molecular recognition sites on the CPP electrode, ultimately paving the way for highly selective electrochemical sensing applications.

This study introduces a novel approach by utilizing a low-cost, flexible, and biodegradable conductive paint-based paper substrate that serves as an innovative support for MIP fabrication. By integrating electrochemical techniques with molecular imprinting, highly selective recognition of HA has been achieved with notable precision. The resulting HA-MI-P(p-PD)@CPP sensor demonstrates exceptional sensitivity and selectivity toward HA, while maintaining excellent repeatability, reproducibility, and long-term operational stability. Its practical efficacy was confirmed through successful analysis of cosmetic formulations. Owing to its simplicity, affordability, and eco-friendly nature, this sensing platform holds strong potential for quality control in cosmetic industries and can be further extended for applications in pharmaceuticals and food analysis.