Protection-free fabrication of glycopolymer-decorated electrodes for label-free electrochemical detection of pathogenic bacteria

Abstract

We present a novel interfacial glycosylation method via biomimetic adhesion and π–π stacking without protecting groups. The resulting glycan-coated electrodes detect pathogenic bacteria down to 10² CFU mL⁻¹ in real food samples, demonstrating strong anti-interference ability and potential for food safety monitoring.

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To effectively address the increasing challenges posed by bacterial infections in biomedical, food, and environmental sectors, the identification and quantification of pathogenic microorganisms are critical for safeguarding health and ensuring safety.¹ Traditional detection methods, such as culture-based techniques and molecular approaches like polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA), are cumbersome.² These gold standard methods not only require several days for culture growth and involve multiple steps but also demand skilled technicians, rendering them less suitable for urgent situations.³ In this context, electrochemical biosensors offer a transformative solution for the rapid detection of pathogenic bacteria. They provide high sensitivity, quick response times, user-friendly operation, and the potential for miniaturization, qualities that traditional methods often lack.⁴ In particular, glycan-based electrochemical biosensors excel due to their exceptional sensitivity compared to conventional electrochemical techniques, such as direct cyclic voltammetry. This enhanced sensitivity arises from the specificity of carbohydrate recognition, which effectively minimizes background interference caused by non-specific adsorption. Many pathogenic bacteria possess surface lectins that can selectively recognize and bind to specific glycan bioreceptors, such as mannose and galactose.⁵ By mimicking and studying these interactions through the printing of glycans on various surfaces, scientists create an impactful “sweet decoy” mechanism for efficiently capturing pathogens.⁶ When bacteria or proteins adhere to the electrode surface, they induce changes in interfacial charge, resulting in increased interfacial resistance or impedance. By monitoring these electrical signals, typically correlating impedance variations with bacterial concentrations, accurate and rapid detection of bacteria can be achieved using an electrochemical workstation.⁷

Glycan-based electrochemical sensors primarily depend on interfacial glycosylation modifications on the electrode surface.⁸ In recent years, researchers have developed various synthetic strategies to create glycosylated electrodes.⁹ A common method for modifying a gold electrode involves the formation of a self-assembled monolayer (SAM).¹⁰ This process uses a difunctionalized linker with a thiol group at one end for immobilization on the gold surface, while the other terminal groups facilitate further glycan modification. Based on this strategy, Lazar et al. introduced a technique for creating glycan-coated gold electrodes through Surface-Initiated Atom Transfer Radical Polymerization (SI-ATRP). They used these electrodes as a highly effective electrochemical impedance spectroscopy (EIS)-based biosensing platform to study lectin-binding kinetics.¹¹ However, the synthesis of glycosyl monomers involves multi-step protection–deprotection processes, complicating the interfacial glycan modification procedure (Fig. 1A). Additionally, a sugar molecule containing an amino linker can be directly immobilized onto the sensor surface using the SAM strategy, with the help of 11-mercaptoundecanoic acid, which provides a terminal carboxylic acid group for glycan attachment.¹² Yet, the chemical glycosylation process still required multi-step protective chemistry for the synthesis of glycosyl derivatives. To address the issues of slow SAM formation and unstable gold–sulfur (Au–S) bonds, Ma and colleagues developed a novel polythiophene interface containing fused quinone moieties. These quinones were then glycosylated to create a glycan-based electrode for bacterial detection.¹³ This work represents a notable advancement in the efficient and rapid fabrication of glycan-based biosensors. However, the preparation of key thiol-modified mannosyl derivatives remains a significant challenge, as it requires the use of orthogonal protecting chemistry. In addition to gold electrodes, surface glycosylation modifications have also been applied to other types of electrodes for label-free detection. Lai et al. reported an electrografting technique that covalently attaches amine-functionalized mannosyl derivatives directly to a carbon electrode surface.¹⁴ Meanwhile, Szunerits et al. fabricated a carbohydrate-coated boron-doped diamond electrode using interfacial click chemistry with azide-functionalized carbohydrates.¹⁵ These glycosylated electrochemical sensors show significant potential for applications in the biomedical field. However, the key glycosyl derivatives used for interfacial glycosylation modifications, such as glycosyl amines and glycosyl azides, require preparation through protection–deprotection steps and column chromatography for separation.

Fig. 1 Schematic representation of the construction steps of glycan-coated electrodes: (A) traditional method and (B) protection-free strategy.

In this study, we aim to provide a simple and versatile surface-glycosylation strategy, which was applied to create glycopolymer-coated electrodes. It combines N-hydroxysuccinimide (NHS) ester conjugation with mucus-inspired and supramolecular adhesion (Fig. 1B). This new method offers several advantages over traditional approaches, including shorter reaction times, greener conditions, and greater applicability to different types of electrodes. Both gold and graphite electrodes can be easily functionalized with glycans for the rapid and sensitive detection of E. coli and S. aureus. To validate their effectiveness in practical applications, these glyco-electrochemical sensors have been used to detect pathogenic bacteria in real samples such as milk, juice, coffee, and cola. This work contributes to the advancement of glycan-based biosensors, particularly in developing prevention strategies against microbial infections in food safety.

Our initial efforts focused on the preparation of a glycan-coated gold electrode. We have developed an innovative two-step, one-pot strategy to create an easy-to-use glycan coating using a dopamine-containing glycopolymer. This glycopolymer is synthesized through NHS-ester-mediated conjugation, involving reactive polymers, lactose-NH₂, and dopamine (Scheme S1). The resulting polymers bearing catechol pendants can be effectively modified on the gold surface under alkaline conditions. This approach offers a hassle-free and efficient method for achieving a glycan-coated electrode. The prepared glycan-based electrochemical sensors were then utilized to detect lectins and bacteria (Fig. 2A). The lectin model substrate was first used to test the detection performance of this glycopolymer-modified gold electrode. The adsorption of ConA onto the electrode surface was examined using the EIS method, and the changes in the electrochemical signal at each concentration were recorded. When the glycan receptors capture ConA, a large insulating layer forms on the electrode surface. This layer significantly increases the electron transfer resistance, allowing the lectin concentration to be quantified indirectly by monitoring the change in impedance. The results shown in Fig. 2B indicate that as the concentration of ConA increases, the impedance value measured on the electrode surface also increases gradually. The charge transfer resistance, revised from the Randles Circuit model, exhibited a clearer dependence on substrate concentration (Fig. 2C).¹⁶ When the concentration of the ConA solution was 1.0 mg mL⁻¹, the resistance increased significantly. The resistance signal decreased accordingly with the reduction of the detected protein concentration, exhibiting a good linear relationship within the range of 10⁻¹ to 10⁻⁵ mg mL⁻¹. The sensor response showed a deviation from linearity over 0.1 mg mL⁻¹ for EIS measurements. When the lectin concentration was reduced to 10⁻⁶ mg mL⁻¹, the change in resistance was not significantly different from that of the blank control group (PBS solution), indicating that the LOD of this glycosylated gold electrode for the ConA solution is 10 ng mL⁻¹.

Fig. 2 Fabrication of a glycan-coated gold electrode for the label-free electrochemical detection of pathogenic bacteria. (A) Schematic illustration of sensor preparation and bacterial detection. (B) EIS measurements of the sensor against ConA concentrations of 10⁰–10⁻⁶ mg mL⁻¹. (C) Charge transfer resistance against ConA with varied concentrations. (D) EIS measurements of the sensor against S. aureus concentrations of 10²–10⁸ CFU mL⁻¹. (E) Charge transfer resistance against S. aureus with varied concentrations. (F) EIS measurements of the sensor against E. coli concentrations of 10²–10⁸ CFU mL⁻¹. (G) Charge transfer resistance against E. coli with varied concentrations.

The lectin test results confirm the feasibility of using this glycan-coated gold electrode for the detection of carbohydrate recognition receptors. Subsequently, the electroanalytical performance was evaluated for bacterial samples, and the results are exhibited in Fig. 2D–G. As observed, the increase in the S. aureus and E. coli concentrations from 10² to 10⁸ CFU mL⁻¹ resulted in an increase in the sensor response for EIS testing. Furthermore, the detection of S. aureus demonstrated a high sensitivity, achieving an LOD of 10² CFU mL⁻¹ (Fig. 2D and E). In contrast, E. coli exhibited a better linear response within the concentration range of 10³ to 10⁶ CFU mL⁻¹, with an LOD of 10² CFU mL⁻¹ (Fig. 2F and G). These results indicated that the developed glycan-based biosensors can effectively detect pathogenic bacteria across a wide range, with a low limit of detection.

We then applied this interfacial glycan modification strategy to the modification and application of graphite electrodes (Fig. 3A). Through an NHS-active ester-mediated coupling reaction, amino-lactose and amino-pyrene units were introduced into the polymer side chains, respectively. The glycan modification on the carbon electrode surface was achieved via π–π stacking interactions.¹⁷ Successful modification was confirmed by the observed fluorescence of the electrode after rinsing with water (Fig. S10). Next, different samples were subjected to electrochemical detection experiments using the differential pulse voltammetry (DPV) method. Herein, the DPV mode served as an effective complement to the EIS method, allowing direct measurement of the faradaic current of the electroactive marker. By constructing a calibration curve of electron transfer resistance against bacterial concentration, quantitative detection was gained.¹⁸

Fig. 3 Fabrication of the glycan-coated graphite electrode for the label-free electrochemical detection of pathogenic bacteria. (A) Schematic illustration of sensor preparation and bacterial detection. (B) DPV measurements of the sensor against ConA concentrations of 10⁰–10⁻⁶ mg mL⁻¹. (C) Data on anodic peak current change (ΔI) obtained from CV analysis against ConA with different concentrations. (D) DPV measurements of the sensor against S. aureus concentrations of 10²–10⁸ CFU mL⁻¹. (E) ΔI analysis against S. aureus with different concentrations. (F) DPV measurements of the sensor against E. coli concentrations of 10²–10⁸ CFU mL⁻¹. (G) ΔI analysis against E. coli with different concentrations.

Detection of the lectin (ConA) is shown in Fig. 3B. Throughout the DPV measurements, all electrodes exhibited a consistent trend of diminishing current response with increasing substrate concentration. Here, ΔI refers to the change in current, determined by the difference between the peak current of the blank and the peak current observed after recognizing the target (Fig. 3C). This metric provides a clearer way to compare the suppression of the signal during the interaction with the target sample since it results in a positive value.¹⁹ The detection sensitivity of the graphite electrode against ConA is 10⁻⁴ mg mL⁻¹, which is worse than that of the gold electrode. The testing of bacterial samples produced similar results: the LODs were 10⁵ CFU mL⁻¹ for S. aureus and 10³ CFU mL⁻¹ for E. coli (Fig. 3D–G). The results are 1–3 orders of magnitude higher than those achieved with the gold electrode. These results indicate that the detection sensitivity of the glycosylated graphite electrode in DPV mode is significantly lower than that of the gold electrode when using the EIS method. However, the graphite electrode shows a better linear correlation between substrate concentration and current signal within a specific concentration range (Fig. S13).

E. coli is one of the main pathogenic microbial contaminants found in food, leading to foodborne illnesses. Raw milk with a coliform concentration exceeding 10³ CFU mL⁻¹ is considered unsafe for human consumption.²⁰ Consequently, detecting E. coli through fast and cost-effective methods is a primary goal for the food industry to ensure food safety.²¹ To evaluate the practical application of glycan-coated electrodes in food safety, we prepared gradient dilutions of E. coli in four representative beverages: milk, coffee, cola, and orange juice. We tested these samples using both gold and graphite electrodes. The results are summarized in Table 1. The glycosylated gold electrode demonstrated a LOD of 10² CFU mL⁻¹ across all four beverage types in EIS mode. This matched the performance observed in the PBS dilution group, indicating that components such as proteins, caffeine, carbonic acid, and vitamins present in these beverages did not interfere with the detection method. The good performance in complex environments also underscores the sensor's high selectivity for the target bacteria over other food components. More importantly, the detection sensitivity met the critical threshold required for food safety testing, confirming its suitability for real-world applications. In contrast, the glycosylated graphite electrode showed significant variability across different samples, with LOD values consistently higher than those in the PBS control group. This suggests that components such as proteins in complex environments significantly influence the detection performance when using DPV mode. In addition, the glyco-electrode can be effectively recycled through a gentle polishing and cleaning procedure. After that, the electrode is re-modified with glycopolymer and used for other detection experiments. Electrodes can be reused multiple times without significant loss in performance, underscoring their practical reproducibility and potential cost-effectiveness.

Table 1 Determined limits of detection for E. coli (CFU mL⁻¹) in real samples

	Milk	Coffee	Cola	Juice
Glycan-coated gold electrodes	10^2	10^2	10^2	10^2
Glycan-coated graphite electrodes	10^5	10^3	10^5	10^4

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In conclusion, this study presents a glycan-coated electrochemical biosensing platform for rapid detection of infectious bacteria. Compared to conventional glycosylated electrode preparation, the proposed method enables protection-free synthesis and rapid surface modification. Gold electrodes are modified via biomimetic adhesion using catechol-bearing block glycopolymers, while graphite electrodes are functionalized through π–π stacking with pyrene-modified glycopolymers. Specific binding of surface-anchored glycans to target bacteria induces measurable changes in current and impedance, enabling label-free and sensitive pathogen detection. The glycan-coated gold electrode demonstrated excellent performance in beverage samples, achieving a LOD of 10² CFU mL⁻¹ and strong anti-interference ability, thus meeting practical food safety needs. This protection-free approach is not merely a synthetic shortcut; it represents a paradigm shift toward more practical, sustainable, and versatile methodologies for constructing advanced bio-interfaces. As a result, it significantly lowers the barrier to developing next-generation glycan-based electrochemical biosensors.

Author contributions

L. L., Q. C., and A. H.: investigation and writing – original draft. Q. G. and M. W.: investigation. W. S. and J. L.: methodology, conceptualization, and investigation. G. L.: conceptualization, supervision, funding acquisition, and writing – review & editing. All authors have given approval to the final version.

Conflicts of interest

There are no conflicts to declare.

Data availability

The data supporting the findings of this study are available within the article and its supplementary information (SI). The Supplementary Information file associated with this article contains experimental section, NMR spectra, GPC data, as well as the calculated datasets of electrochemical testing. See DOI: https://doi.org/10.1039/d5an01136a.

Acknowledgements

This work is funded by the National Natural Science Foundation of China (22377056).

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