Sensitive Detection of Biomarker in Gingival Crevicular Fluid Based on Enhanced Electrochemiluminescence by Nanochannel-Confined Co3O4 Nanocatalyst

Abstract

The sensitive detection of inflammatory biomarkers in gingival crevicular fluid (GCF) is highly desirable for the evaluation of periodontal disease. Luminol-based electrochemiluminescence (ECL) immunosensors offer a promising approach for the fast and convenient detection of biomarkers. However, luminol's low ECL efficiency under neutral conditions remains a challenge. This study developed an immunosensor by engineering an immunorecognition interface on the outer surface of mesoporous silica nanochannel film (SNF) and confining a Co₃O₄ nanocatalyst within the SNF nanochannels to improve the luminol ECL efficiency. The SNF was grown on an indium tin oxide (ITO) electrode using the simple Stöber solution growth method. A Co₃O₄ nanocatalyst was successfully confined within the SNF nanochannels through in situ electrodeposition, confirmed by X-ray photoelectron spectroscopy (XPS) and electrochemical measurements. The confined Co₃O₄ demonstrated excellent electrocatalytic activity, effectively enhancing luminol and H₂O₂ oxidation and boosting the ECL signal under neutral conditions. Using interleukin-6 (IL-6) as a proof-of-concept demonstration, the epoxy functionalization of the SNF outer surface enabled the covalent immobilization of capture antibodies, forming a specific immunorecognition interface. IL-6 binding induced immunocomplex formation, which reduced the ECL signal and allowed for quantitative detection. The immunosensor showed a linear detection range for IL-6 from 1 fg mL^-1 to 10 ng mL^-1, with a limit of detection (LOD) of 0.64 fg mL^-1. It also demonstrated good selectivity and anti-interference capabilities, enabling the successful detection of IL-6 in artificial GCF samples.

Keywords: Co3O4; electrochemiluminescence; immunosensor; luminol; nanochannel-confined.

Figure 1

Schematic illustration for immunosensor construction and ECL detection of IL-6 through integrating both a specific recognition interface on the outer surface of SNF and Co₃O₄ nanocatalyst confined in SNF nanochannels.

Figure 2

(A) SEM image of the cross-section of SNF/ITO electrode. (B) Top-view TEM image of SNF. (C) Cross-sectional TEM image of SNF. (C–E) CV curves obtained on ITO, SM@SNF/ITO, and SNF/ITO electrodes in 0.05 M KHP (pH 4) containing 0.5 mM of K₃Fe(CN)₆ (D), Ru(NH₃)₆Cl₃, (E) or FcMeOH (F). The scan rate was 50 mV/s.

Figure 3

(A) CV curves obtained on SNF/ITO or Co₃O₄@SNF/ITO in 1 M NaOH. The scanning rate was 100 mV/s, and the scanning potential ranged from –0.1 V to 0.6 V. (B) XPS spectra obtained on the fabricated SNF/ITO or Co₃O₄@SNF/ITO electrode. (C) High-resolution Co 2p spectrum obtained on Co₃O₄@SNF/ITO electrode. (D) SEM image (left image) of Co₃O₄@SNF/ITO electrode after removal of SNF through immersion into a 0.5 M NaOH solution for 3 min and the corresponding O (right and above image) and Co (right and bottom image) element mapping image. (E) Top-view SEM image (left image) of Co₃O₄@/ITO electrode and the corresponding O (right and above image) and Co (right and bottom image) element mapping image.

Figure 4

(A) ECL curves obtained from different electrodes in PBS (0.01 M, pH 7.4) containing luminol (100 μM) and H₂O₂ (100 μM). (B) ECL intensity obtained on Co₃O₄@SNF/ITO or (C) Co₃O₄/ITO from continuously scans in PBS (0.01 M, pH 7.4) containing luminol (100 μM) and H₂O₂ (100 μM). The PMT voltage was set to 750 V. The scanning rate was 100 mV/s, and the scanning potential range was 0 V~0.8 V.

Figure 5

CV curves obtained on SNF/ITO (A) and Co₃O₄@SNF/ITO (B) electrodes in the electrolyte (PBS, 0.01 M, pH 7.4) or electrolyte containing luminol (100 μM) or H₂O₂ (100 μM). (C) ECL intensity obtained at the Co₃O₄@SNF/ITO electrode in PBS (0.01 M, pH 7.4) containing luminol (100 μM) and H₂O₂ (100 μM) in the presence of TBA (100 μg mL⁻¹) or BQ (100 μM). (D) Illustration of possible ECL mechanism for luminol-H₂O₂ system enhanced by Co₃O₄ nanomaterials.

Figure 6

(A) CV curves obtained on Co₃O₄@O-SNF/ITO or Co₃O₄@O-SNF/ITO electrodes in 0.1 M KCl containing 2.5 mM [Fe(CN)₆]^3−/4−. (B) ECL responses obtained on different electrodes in PBS (0.01 M, pH 7.4)) with H₂O₂ (100 μM) and luminol (100 μM). (C) EIS plots obtained on different electrodes in 0.1 M KCl containing 2.5 mM [Fe(CN)₆]^3−/4−.

Figure 7

The effect of deposition time of Co₃O₄ (A), antibody concentration (B), incubation time for antibody immobilization (C), and IL-6 incubation time (D) on the ECL signals of the fabricated immunosensors.

Figure 8

(A) ECL responses of the fabricated immunosensor in presence of various concentrations of IL-6 in PBS (0.01 M, pH 7.4) containing luminol (100 μM) and H₂O₂ (100 μM). (B) The corresponding calibration curves between ECL intensity and the logarithmic concentration of IL-6. (C) Reproducibility of five immunosensors fabricated in parallel for IL-6 detection (10 ng mL⁻¹). (D) The selectivity and anti-interference of ECL immunosensor for the detection of IL-6. The concentration of Na⁺, Cl⁻ was 1 μM, the concentration of K⁺, NO³⁻ was 100 nM, the concentration of glucose was 10 μM, and the concentration of IL-1β, MMP-9, TNF-α was 10 ng mL⁻¹.