Dual-Enhanced Electrochemiluminescence Imaging on a Monolayer Film of Nanoreactors for Probing Cell–Matrix Interaction

Abstract

Cell–matrix interactions play a fundamental role in various biological processes and cellular functions. Although electrochemiluminescence (ECL) microscopy, particularly the negative ECL mode, offers a promising label-free approach for investigating cell–matrix interactions, its broader application is constrained by relatively low luminescence efficiency. In this study, we developed a dual-enhanced negative ECL imaging platform utilizing a monolayer film of gold nano sea-urchin@mesoporous silica (AuNSU@mSiO₂) nanoreactors. This self-assembled monolayer film inherits the nanoconfinement effect and localized surface plasmon resonance properties from individual AuNSU@mSiO₂ nanoreactors, endowing it with a substantial amplification of the ECL signal from the Ru­(bpy)₃ ²⁺/TPrA system. Owing to its flatness and structural uniformity, the monolayer film ensures homogeneous ECL enhancement, thereby enabling high sensitivity and spatially resolved observation of cell adhesion. This platform outperforms the conventional negative ECL systems by providing a superior signal-to-noise ratio and an extended dynamic range, which allows for revealing cell adhesion heterogeneity and strength distribution. This work not only provides a powerful tool for probing cell–matrix interactions but also paves the way for fundamental studies in numerous biological processes.

Introduction

Cell–matrix interactions are critical in numerous biological processes, including cell proliferation, differentiation, migration, and apoptosis. − The binding between adhesion proteins and the extracellular matrix not only mediates physical anchorage but also fundamentally regulates intracellular signaling and microenvironment recognition. Therefore, a comprehensive understanding of cell–matrix interactions is essential for both basic biological research and clinical applications. This underscores the need to develop appropriate analytical methods capable of selective imaging and real-time monitoring of cell–matrix interactions and morphological dynamics on substrates. Currently, fluorescence-based techniques, such as confocal laser scanning microscopy (CLSM) and total internal reflection fluorescence microscopy (TIRFM), serve as the primary tools for observing cell adhesion and morphological changes. − By leveraging fluorescent labeling, these techniques enable the analysis and dynamic tracking of adhesion-related molecules at the single-molecule level. However, the labeling process is often time-consuming, and fluorescence imaging is susceptible to interference from background signals in biological samples, such as autofluorescence. Thus, there is an urgent need to develop simple, rapid, and label-free methods for analyzing cell–matrix interactions.

Owing to high sensitivity, electrochemical techniques have emerged as promising alternatives for cell–matrix interaction studies. The adhesion of cells on electrode surface reduces the effective area due to cellular insulation, thereby suppressing Faradaic currents and altered impedance. − Both responses provide quantitative measures of the adhesion strength. Nevertheless, conventional electrochemical methods are inherently limited in their ability to map the spatial distribution of the cell adhesion.

Electrochemiluminescence (ECL) microscopy, integrating electrochemical excitation with optical detection, has emerged in recent years as a versatile analytical technique and gained widespread adoption in cellular imaging studies. − In a typical ECL process (i.e., Ru­(bpy)₃ ²⁺/TPrA system), an applied voltage induces redox reactions among ECL-active molecules at the electrode surface, ultimately generating excited states (Ru­(bpy)₃ ^2+*) that emit photons. , This mechanism endows ECL with high sensitivity and excellent spatiotemporal controllability with minimal background interference. Among various ECL imaging modalities, negative ECL (also termed shadow ECL) imaging has been established as a rapid and label-free method for analyzing biological entities. , This approach relies on the local obstruction of ECL generation by analytes located at the electrode interface, making it well-suited for investigating interactions between biological entities and the substrate. For instance, Su et al. employed negative ECL imaging to monitor the adhesion behavior of individual cells in real time. − They further demonstrated that by adjusting the type or concentration of coreactants, the thickness of the ECL-emitting layer could be aligned with the location of intercellular junction proteins, thereby enabling spatially selective imaging of cell–cell junctions. Additionally, negative ECL imaging has been extended to the morphological analysis of subcellular structures, such as mitochondria or single bacterial. −

However, the relatively low luminescence efficiency of ECL has limited its broader application in biological studies, largely due to challenges such as an insufficient signal-to-noise ratio and signal stability. To address these issues, several strategies, including super-resolution techniques and nanomaterial-based enhancement, have been developed to improve ECL performance. , Feng et al. achieved super-resolution ECL imaging at single-molecule level, enabling dynamic tracking of adhesion protein movement in live cells. In our previous work, we constructed various porous nanoreactors and demonstrated that nanoconfinement can significantly accelerate ECL reaction rates, leading to substantially enhanced ECL output. − Moreover, the synergy between the nanoconfinement and localized surface plasmon resonance (LSPR) effect has advanced the development of highly efficient nanomaterial-based ECL amplifiers, providing a practical solution to overcome the weak ECL signals at the electrode surface.

Herein, we present a dual-enhanced nanostructure, featuring gold nano sea-urchin (AuNSU) as the core and a mesoporous silica (mSiO₂) shell. Building on previous findings, the AuNSU@mSiO₂ nanoreactor exhibits favorable structural features (i.e., porous channel) and optimal optical properties (i.e., strong spectral overlap with ECL photons), making it highly promising for substantially amplifying the ECL signal from the Ru­(bpy)₃ ²⁺/TPrA system. , Through liquid–liquid interfacial self-assembly, a monolayer film of AuNSU@mSiO₂ nanoreactors was fabricated and transferred onto the ITO electrode surface, serving as an efficient ECL signal-amplifying substrate (Scheme ). This monolayer film inherits the nanoconfinement and LSPR properties from individual nanoreactors, endowing it with an outstanding ECL amplification capability. Furthermore, its structural uniformity, characterized by scanning electron microscopy (SEM), ensures a homogeneous and stable ECL enhancement across the substrate, making the platform ideal for monitoring surface changes and cell–matrix interaction. Once cells adhere to the film, the diffusion of ECL reactants into the mSiO₂ channels is locally hindered, thereby suppressing ECL enhancement in cell-adhered regions and producing negative ECL images that sensitively reflect cell–matrix interactions. Compared with conventional negative ECL imaging of single cells on bare electrode, the ECL signal difference (ΔI) between cell-adhered and cell-free regions was significantly increased through monolayer film modification. This improvement enables a high signal-to-noise ratio and an extended dynamic range in negative ECL imaging. Collectively, the dual-enhanced negative ECL imaging platform developed in this work offers a powerful and reliable tool for probing cell–matrix interactions and shows considerable potential for monitoring fundamental biological processes.

1. Schematic Illustration of Construction of the AuNSU@mSiO₂ Nanoreactor and Dual-Enhanced ECL Platform for Single Cell Imaging.

Experimental Section

Chemical and Reagents

Tris­(2,2′-bipyridyl) ruthenium­(II) chloride hexahydrate (Ru­(bpy)₃Cl₂·6H₂O, 98%), tri-n-propylamine (TPrA), and ascorbic acid were purchased from Sigma-Aldrich. Tetraethoxysilane (TEOS), cetyltrimethylammonium bromide (CTAB), paraformaldehyde, n-hexane, sodium citrate dihydrate, and silver nitrate (AgNO₃) were obtained from Aladdin Industrial Co., Ltd. Acetone, methanol (MeOH), ethanol (EtOH), ammonia solution (NH₃·H₂O, ∼25%), hydrogen peroxide (H₂O₂), and concentrated hydrochloric acid (HCl, 36%–38%) were bought from Sinopharm Chemical Reagent Co., Ltd. Conductive silver paint and carbon cloth strips were provided by Structure Probe Inc. ITO-coated coverslips were supplied by Guluo Glass Co., Ltd. DMEM medium, Opti-MEM reduced-serum medium, fetal bovine serum (FBS), and trypsin were sourced from Gibco. Phosphate buffered solution (PBS, 0.1 M, pH 7.4–7.6) was acquired from Sangon Biotech Co., Ltd. Ultrapure water used in this work was produced through a Millipore Milli-Q water purification system. Before use, ITO glasses were sequentially cleaned in 1 M NaOH, acetone, EtOH, and ultrapure water under ultrasonication. All chemicals were used as received without further purification.

Synthesis of AuNSU and AuNSU@mSiO₂ Nanoreactors

The synthesis of gold nano sea-urchins was carried out using a seed-mediated growth method according to the previous literature with modifications. , The seed solution was prepared by adding 4.5 mL of 1% sodium citrate solution to 30 mL of boiling 1 mM HAuCl₄ solution under vigorous stirring and reacting for 15 min. Prior to use, the seed solution was filtered through a 0.22 μm membrane filter and stored at 4 °C. AuNSUs were synthesized in 50 mL of 0.25 mM HAuCl₄ solution, followed by the addition of 50 μL of concentrated HCl and 500 μL of the seed solution. Under slow stirring, 500 μL of 0.5 mM AgNO₃ and 250 μL of 0.1 M AA were added to initiate the reaction. After 30 s, 5 mL of 0.1 M CTAB was introduced, and stirring was continued for additional 10 min. The resulting mixture was centrifuged at 5000 rpm and washed three times with deionized water to remove the excess reactants. Finally, the purified AuNSUs were redispersed in 5 mL of a 0.5 mM CTAB solution and stored at 4 °C.

The AuNSU@mSiO₂ nanoreactors was synthesized according to the previous method with modifications. , Briefly, 2 mL of AuNSU stock solution was added to 8 mL of 0.5 mM CTAB solution under stirring, followed by the addition of 100 μL of 0.1 M NaOH. Subsequently, 30 μL of 20% (v/v) TEOS in MeOH was added three times at 30 min intervals. After 12 h of the reaction, the AuNSU@mSiO₂ nanoreactors were collected by centrifugation and washed three times with deionized water and EtOH. To remove the CTAB molecules, the obtained product was dispersed in 2 mL of EtOH, mixed with 60 μL of concentrated HCl, and oscillated for 12 h. Finally, the nanoreactors were centrifuged, thoroughly washed with deionized water, and redispersed in 2 mL of deionized water for storage at 4 °C.

Fabrication of the Dual-Enhanced AuNSU@mSiO₂ Monolayer Film

The dual-enhanced monolayer film was fabricated via liquid–liquid interfacial self-assembly of AuNSU@mSiO₂ nanoreactors. − Prior to assembly, the ITO electrode was treated with a mixed solution of water, ammonia solution (25%), and H₂O₂ (8:1:1, v/v) at 80 °C for 10 min. Initially, 300 μL of the AuNSU@mSiO₂ stock solution was dispersed in 6 mL of deionized water and sonicated for 15 min. This dispersion was then transferred to a container, and 1 mL of n-hexane was gently added. Subsequently, 7.2 mL of MeOH was rapidly injected to trigger interfacial self-assembly. After complete evaporation of n-hexane, the resulting monolayer film was transferred onto the pretreated ITO electrode.

Measurements and Instrumentation

The size and morphology of the nanoreactors were characterized by TEM (Tecnai G20, FEI Co., Ltd.) and Cryo-FESEM (Gemini SEM500, Zeiss Co., Ltd.). Hydrodynamic diameter and ζ potential were measured by using a Zetasizer analyzer (ZS90, Malvern Co. Ltd.). UV–vis absorption spectra were recorded on a spectrophotometer (UV–5200PC, Shanghai Metash Instrument Co., Ltd.). Electrochemical measurements were performed on an MPI-M ECL analysis system (Remax Analytical Instrument Co., Ltd.).

All imaging experiments were conducted on a home-built imaging system, which contained a wide-field epi-fluorescence microscope (Olympus) equipped with an oil-immersion objective (60×, NA 1.35) and an EMCCD camera (iXon Life 888, Andor Technology Plc.). For ECL imaging, a two-electrode configuration was employed, consisting of an ITO-coated coverslip as the working electrode and a Pt wire as the counter electrode. A homemade electrochemical cell was constructed by sealing a PDMS chamber onto an ITO coverslip. The ITO coverslip was connected to a carbon cloth strip by using conductive silver paint for voltage application. Typically, the ECL reaction solution containing 0.5–100 μM Ru­(bpy)₃ ²⁺ and 10 mM TPrA was introduced into reaction cell, and a constant potential of 1.35 V was applied through a potentiostat (DG 1021, Rigol Co., Ltd.). All measurements were carried out at room temperature in a dark environment to avoid external light interference.

Cell Culture and ECL Imaging

The SiHa (human cervical squamous cell carcinoma) cell line was obtained from the Cell Bank of the Chinese Academy of Sciences. SiHa cells were maintained in DMEM with 10% FBS at 37 °C in a 5% CO₂ environment. For single-cell ECL imaging, cells in the exponential growth phase were digested using trypsin and then resuspended in 10 mL of Opti-MEM reduced-serum medium. The cells were then seeded into the electrochemical reaction chamber and incubated overnight to allow cell adhesion. After being washed by PBS solution for three times, the cells were fixed with 4% PFA for 10 min. After washing for three times, PBS solution containing 20 μM Ru­(bpy)₃ ²⁺ and 10 mM TPrA was added for imaging. All ECL images were acquired with an exposure time of 5 s.

Results and Discussion

Characterization of AuNSU and AuNSU@mSiO₂ Nanoreactors

Through single-molecule ECL imaging, our previous study revealed that both nanoconfinement and LSPR effects significantly enhanced the ECL intensity by influencing either the electrochemical reaction or photon emission processes. Hence, based on this principle, we design a dual-enhancement nanoreactor that integrates both effects for electrode substrate construction. The nanoreactor features a core–shell structure, consisting of a gold nano sea urchin core and a mesoporous silica shell. As shown in Figure A–C, the AuNSU cores have an average size of 64.6 nm, an approximately spherical shape, and numerous sharp spikes on the surfaces. These multibranched spike structures, formed by the rapid reduction of HAuCl₄ during the seed-mediated growth, act as lightning rods that significantly enhance the local electric field and generate pronounced LSPR effects. After coating with mSiO₂, the resulting AuNSU@mSiO₂ nanoreactors exhibit a uniform spherical morphology with an average size of 110.4 nm (Figure C). Successful silica coating was further confirmed by ζ-potential and hydrodynamic diameter measurements. In detail, the surface potential changed from 37.7 ± 14.9 mV (CTAB-capped AuNSU) to −20.4 ± 14.9 mV (AuNSU@mSiO₂), and the hydrodynamic diameter increased from 68.1 to 105.7 nm, consistent with the TEM results (Figure D,E). These results also indicated good dispersity and stability of the nanoreactors in aqueous solution. The mesoporous shell serves to preconcentrate ECL luminophores and creates a nanoconfined environment that accelerates the electrochemical reaction rate, leading to a significantly enhanced ECL signal. Moreover, the nonconductive shell improves the structural stability of the nanoreactor during the electrochemical process and protects cells from direct electrical stimulation.

1.

Characterization of AuNSU and AuNSU@ mSiO₂. (A,B) Schematic illustration and TEM images. Scale bar: 50 nm. (C) Statistical distribution of diameter. (D) Hydrodynamic diameter. (E) ζ-potential changes during the synthesis process. (F) Absorption spectrum of AuNSU@mSiO₂ and fluorescence spectrum of Ru­(bpy)₃ ²⁺.

As previously reported, the efficacy of LSPR-mediated ECL enhancement critically depends on the spectral overlap between the nanoparticle’s absorption and the ECL luminophore’s emission. In this system, the emitted light from the Ru­(bpy)₃ ²⁺ molecules, which have a spectral peak at approximately 620 nm, serves as the incident light to excite the LSPR effect. Meanwhile, the absorption peak of the AuNSU@mSiO₂ nanoreactor was located at 617 nm, closely matching the emission peak of Ru­(bpy)₃ ²⁺ (Figure F). This strong spectral overlap indicates a high potential resonant coupling, confirming the excellent capability for the local ECL signal via the LSPR effect. Combined with the nanoconfinement effect provided by the mSiO₂ shell, this dual-enhanced nanoreactor demonstrates significant promise for developing the electrode substrate.

Fabrication and Characterization of the Dual-Enhanced AuNSU@mSiO₂ Monolayer Film

To form a uniformly enhanced electrode substrate, the spherical AuNSU@mSiO₂ nanoreactors were assembled into a closely packed monolayer film via the liquid–liquid interfacial self-assembly method. As illustrated in Figure A, driven by the Marangoni effect, the AuNSU@mSiO₂ nanoreactor spontaneously accumulates at the interface between the aqueous solution and n-hexane. Upon complete evaporation of n-hexane, a continuous monolayer film of AuNSU@mSiO₂ forms at the air–aqueous solution interface. After hydrophilic treatment, the ITO electrode was able to transfer the monolayer film onto its surface, forming a uniform electrode substrate. The same transfer process was employed for other substrates for morphological characterization. As shown in Figure B,C, the film consists of a densely packed monolayer of AuNSU@mSiO₂ nanoreactors with minimal interparticle gaps and exhibits large area continuity. This highly ordered and uniform structure ensured a stable and homogeneous ECL enhancement. The side-view SEM image further confirmed that the AuNSU@mSiO₂ nanoreactors assemble into a typical monolayer on the electrode with negligible multilayer stacking, validating the reliability of the self-assembly process (Figure D). In contrast, direct drop-casting of AuNSU@mSiO₂ nanoreactors followed by solvent evaporation resulted in a disordered structure, characterized by severe nanoparticle aggregation and numerous void regions (Figure S1). Such structural inhomogeneity would inevitably lead to nonuniform ECL enhancement across the electrode surface.

2.

(A) Schematic of the self-assembly process of the AuNSU@mSiO₂ monolayer film and its transfer onto the ITO electrode. (B) TEM image of the monolayer film. Scale bar: 100 nm. (C,D) SEM images of the monolayer film. (top- and side-view). Scale bar: 1 μm. (E,F) Cyclic voltammetry and ECL intensity profiles for the Ru­(bpy)₃ ²⁺/TPrA system on the monolayer film-modified ITO and bare ITO electrode with a Pt wire counter electrode and an Ag/AgCl reference electrode at a scan rate of 100 mV/s.

We subsequently assessed the ECL enhancement performance of the electrode substrate. The CV curves of the Ru­(bpy)₃ ²⁺/TPrA system obtained from bare and modified ITO electrodes are shown in Figure E. Despite the nonconductive mSiO₂ shell, the substrate did not impede the electrochemical reaction, as evidenced by the similar current curves observed for both electrodes. The modification shifted the oxidation potential from 1.25 to 1.30 V, consistent with the ECL signal peak shift, suggesting a higher driving potential (Figure F). Crucially, an 8.5-fold enhancement in ECL intensity was achieved with the modified ITO electrode, conclusively verifying the remarkable amplification ability of the AuNSU@mSiO₂ monolayer film.

The designed structure enhanced the ECL signal without hindering the diffusion and reaction of electroactive species from the solution to the electrode surface. This can be largely attributed to the porous architecture of the nanoreactor, which facilitates the transport of reactant molecules. To further investigate the molecular diffusion behavior, we recorded the CV curves at different scan rates and analyzed the relationship between the peak current (i _p) and the square root of the scan rate (v ^1/2). As shown in Figure S2, a linear correlation of i _p on v ^1/2 was observed for both bare and modified electrodes, confirming a diffusion-controlled electrochemical process. Based on the Randles–Ševčík equation, the apparent diffusion coefficients were calculated to be 3.9 × 10^–10 m²/s and 3.5 × 10^–12 m²/s. , The notable decrease in the diffusion coefficient for the modified electrode indicated that the monolayer film effectively restricted the mass transport of reactants near the electrode. This confinement increases the local concentration and molecular collision frequency of the reactive species with the nanoscale reaction layer, thereby amplifying the ECL signal.

ECL Imaging of the Individual AuNSU@mSiO₂ Nanoreactor and Monolayer Film

Direct ECL imaging enables intuitive visualization of the signal enhancement contributed by an individual AuNSU@mSiO₂ nanoreactor and the corresponding film. Upon application of a voltage, the ECL reaction (20 μM Ru­(bpy)₃ ²⁺ and 10 mM TPrA) occurred at the electrode surface and was markedly enhanced at the AuNSU@mSiO₂ sites, resulting in bright areas in the images. As shown in Figures A and S3, distinct and well-dispersed bright spots against the solution background indicated ECL enhancement from an individual AuNSU@mSiO₂ nanoreactor. The ECL enhancement effect was maintained when nanoreactors were assembled into the monolayer film, and the close-packed arrangement formed a continuous ECL enhancement region. In Figure B, the bare ITO region exhibited a weak luminescence signal due to low reaction efficiency, whereas the film region produced intense ECL emission, forming a sharp boundary between the two regions. Single nanoreactors were also present near the boundary, displaying the transition from an individual enhancement site to ean xtended continuous structure. The ECL enhancement factor from the AuNSU@mSiO₂ nanoreactor or film was then quantified using eq

$$\mathrm{E}\mathrm{n}\mathrm{h}\mathrm{a}\mathrm{n}\mathrm{c}\mathrm{e}\mathrm{m}\mathrm{e}\mathrm{n}\mathrm{t}\mathrm{E}\mathrm{C}\mathrm{L}\mathrm{r}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{o}=\frac{I_{\mathrm{p}\mathrm{a}\mathrm{r}\mathrm{t}\mathrm{i}\mathrm{c}\mathrm{l}\mathrm{e}}-I_{\mathrm{b}\mathrm{a}\mathrm{c}\mathrm{k}\mathrm{g}\mathrm{r}\mathrm{o}\mathrm{u}\mathrm{n}\mathrm{d}}}{I_{\mathrm{s}\mathrm{o}\mathrm{l}\mathrm{u}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}}-I_{\mathrm{b}\mathrm{a}\mathrm{c}\mathrm{k}\mathrm{g}\mathrm{r}\mathrm{o}\mathrm{u}\mathrm{n}\mathrm{d}}}$$ 1

where I _particle, I _solution, and I _background represent the A/D counts (ECL intensity) at the nanoparticle or film location and in the solution area and the camera offset background under the same conditions, respectively.

3.

(A) ECL image of the individual AuNSU@mSiO₂ nanoreactor. (B) ECL image of the boundaries between the AuNSU@mSiO₂ monolayer film and bare ITO. Scale bar: 5 μm. (C) ECL enhancement ratio profiles of the red areas in (B). (D) ECL images of the single AuNSU@mSiO₂ nanoreactor and monolayer film under different concentrations of Ru­(bpy)₃ ²⁺ (0.5–100 μM). Scale bar: 2 μm. (E) ECL intensity of the single nanoreactor, monolayer film, and bare ITO electrode under different concentrations of Ru­(bpy)₃ ²⁺ (0.5–100 μM). (F) ECL images and enhancement ratio on nanoreactors and the monolayer film (n = 10). (G) Statistical plot of enhancement ratio.

The profiles of the ECL enhancement ratio across the boundary reveal a substantial signal increase, directly confirming the pronounced enhancement capability of the substrate. The film achieved an average ECL enhancement ratio of 19-fold (Figure C). According to previous reports, ECL intensity depends on the concentration of the luminophore while the coreactant is in excess. As shown in Figure D, the ECL intensity of both the individual nanoreactor and monolayer film initially increased with Ru­(bpy)₃ ²⁺ concentration and plateaued above 20 μM. However, as the ECL signal from bare ITO (I _solution) also increased progressively, the enhancement gradually decreased with increasing concentration (Figure S4). Considering both the absolute ECL intensity and enhancement ratio, a Ru­(bpy)₃ ²⁺ concentration of 20 μM was selected as the optimal condition for cell imaging and subsequent analyses. In addition, the ECL intensity and enhancement ratio of the monolayer film (9.46 ± 0.62) were consistently higher than those of individual nanoreactors (4.83 ± 0.90) across all concentration conditions (Figure F,G). This superior performance may originate from the densely packed architecture, where the nanoscale gaps between adjacent nanoreactors concentrate local Ru­(bpy)₃ ²⁺ molecules and restrict the diffusion of excited-state Ru­(bpy)₃ ²⁺* molecules away from the electrode surface, leading to a significantly amplified signal output. Moreover, the monolayer film exhibited smaller standard deviations, indicating that the assembled structure averages the contributions of individual nanoreactors and reduces intrinsic heterogeneity, thereby yielding uniform enhancement.

Spatial uniformity of the ECL signal is also crucial for reliable negative ECL imaging and single cell analysis. Compared to the disordered structure formed by direct drop-casting, the self-assembled monolayer film offered a flatter surface (Figures C,D and S1). This structural advantage resulted in a uniform ECL signal distribution, in contrast to the spatial heterogeneity of the disordered film (Figure A,B). In the disordered film, dim regions corresponded to uncovered areas, whereas bright signals originated from nanoreactor aggregates. 3D ECL imaging further corroborated that only the uniform enhancement from the monolayer film could reliably reflect surface changes, a requirement unmet by the variable disordered film (Figure C,D). Intensity histograms statistically validated that the disordered film exhibited broad and irregular signal distribution despite some localized enhancement, underscoring the unique suitability of the monolayer film for bioimaging applications.

4.

(A,B) ECL heatmap of the AuNSU@mSiO₂ monolayer film and disordered film. Scale bar: 2 μm. (C,D) Corresponding 3D heatmaps. (E,F) Intensity distribution of ECL images.

Therefore, the AuNSU@mSiO₂ monolayer film, fabricated via liquid–liquid interfacial self-assembly, provided excellent ECL enhancement with high stability and superior spatial uniformity. Notably, when biomolecules of cells cover the mesoporous structure, they can block the pore channels and hinder the diffusion of ECL reactive species. Consequently, this suppression leads to a localized decrease in the ECL enhancement ratio, generating a distinct shadow image. These characteristics form the foundation for negative ECL imaging, which can be leveraged for single-cell morphological analysis and adhesion investigation.

Cell–Matrix Interaction Analysis on the AuNSU@mSiO₂ Monolayer Film

In negative ECL imaging, biological entities, such as cells, organelles, or bacteria, closely contact or firmly adhere to the electrode surface, thereby locally perturbing electrochemical reactions and leading to attenuated ECL signals. The resulting ECL images directly reflect the size, morphology, and interactions with the substrate of these entities. Therefore, the clarity of the negative ECL images is determined by the ECL intensity difference between the regions obscured by the biological entity and the bare electrode. Since the intensity reduction at these biological sites has a definite lower limit (nonluminescent background level), the augmentation of ECL intensity of the electrode substrate effectively improves imaging quality. The AuNSU@mSiO₂ monolayer film, which significantly amplifies the initial ECL signal via nanoconfinement and LSPR effects, thus offers great potential for high-quality negative ECL imaging of biological entities.

Owing to the high biocompatibility of the mSiO₂ shell, cells maintained high viability both in suspension with the AuNSU@mSiO₂ nanoreactor and on the monolayer film substrate, exhibiting rapidly adhesion and sustained proliferation (Figure S5). Additionally, the insulating property of the mSiO₂ shell blocks direct electrical contact between the electrode and the cells, helping maintain the cellular morphology and viability during electrochemical measurements. , Initially, negative ECL images of a single cell on a bare ITO electrode were acquired, in which the cells appeared as distinct shadows (Figure A). The ECL images provided a clear definition of cell morphology, revealing a structure like extended pseudopodia and effectively resolving individual cells within aggregates (Figure S6). However, the inherently low ECL signal from the bare ITO electrode resulted in low image contrast between cells and the background, leading to poorly defined cell boundaries. Furthermore, the ECL signal presented a binary contrast of light and dark, lacking the grayscale variations that encode information on cell–matrix adhesion heterogeneity.

5.

Schematic illustration, ECL images, and corresponding heatmaps of negative ECL imaging on the bare ITO electrode and AuNSU@mSiO₂ monolayer film. Scale bar: 10 μm.

Upon negative ECL imaging of cells on the AuNSU@mSiO₂ monolayer film, a substantially enhanced ECL signal was observed in the bare regions, thereby accentuating the shadow contrast at cell-adhered sites (Figure B). In addition, distinct bright spots, ascribed to incomplete pore blockage, were collected within certain cell regions instead of a homogeneous shadow. These bright spots represent sites of relatively weaker cell–matrix interactions, where partial pore blockage allows ECL luminophores to diffuse into the mesoporous channel and emit photons. These areas appeared in peripheral regions of spreading cells corresponding to focal contacts formed behind the leading edge of the cell. Such adhesion structures exhibit rapid assembly and disassembly, resulting in relatively weak cell–matrix interactions. In contrast, the continuous shadowed regions in the ECL images correspond to stable adhesion structures, consistent with the behavior of focal adhesions and fibrillar adhesions, that establish sustained mechanical coupling. Hence, the spatial distribution of signal intensity in the 3D ECL heatmap directly reveals the characteristics of adhesion strength (Figures and S7). However, it should also be noted that while this method visualizes spatial heterogeneity in interaction strength, it remains fundamentally a qualitative approach. Consequently, negative ECL imaging on the monolayer film provided superior capability for investigating cell–matrix interactions compared to bare ITO electrodes.

This performance originates from the improved intensity contrast and broader dynamic range afforded by the dual-enhancement effect, which reveal subtle intensity variations. As shown in Figure , intensity profiles from negative ECL images on the bare ITO electrodes and monolayer film confirmed this advantage. The signal difference (ΔI) between cell-adhered and bare regions reached 5000 au on the monolayer substrate, a 10-fold increase over the ΔI of 500 au on the bare ITO electrode. This limited ΔI on bare ITO is inadequate for resolving variations arising from differences in the cell–matrix interaction strengths. In contrast, the monolayer film assembled from dual-enhanced nanoreactors significantly amplifies the ΔI, thereby enabling a more refined analysis of the cell–matrix interaction.

6.

(A,B) Negative ECL images of cells on the AuNSU@mSiO₂ monolayer film and corresponding intensity profiles in the selected area. (C,D) Negative ECL images of cells on the ITO electrode and corresponding intensity profiles in the selected area. Scale bar: 10 μm.

Conclusion

In this study, we developed a negative ECL imaging platform utilizing a dual-enhanced monolayer film for highly sensitive, label-free, and direct visualization of cell–matrix interactions. The electrode substrate was fabricated via liquid–liquid interfacial self-assembly of AuNSU@mSiO₂ nanoreactors, integrating nanoconfinement and LSPR effects to yield a remarkable ECL enhancement for the Ru­(bpy)₃ ²⁺/TPrA system. In addition, the flatness and structural uniformity of this monolayer film guaranteed a homogeneous enhancement distribution, which was essential for the reliable investigation of cell–matrix interactions. These properties enabled high-resolution negative ECL imaging that revealed spatial heterogeneity in the cell adhesion strength. Therefore, this developed platform offers a powerful tool for studying cell–matrix interactions and investigating fundamental biological processes, such as cell migration and secretion.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/cbmi.5c00269.

• SEM images of the disordered AuNSU@mSiO₂ film; the maximum current value as a function of the square root of the scan rate on bare GC and AuNSU@mSiO₂; single particle ECL imaging of the AuNSU@mSiO₂ nanoreactor; ECL enhancement ratio of the single AuNSU@mSiO₂ nanoreactor and monolayer film under different concentrations of Ru­(bpy)₃ ²⁺; cell viability under different concentrations of AuNSU@mSiO₂ and on the AuNSU@mSiO₂ monolayer film with different culture times; and single cell ECL imaging and 3D heatmap on the AuNSU@mSiO₂ monolayer film (PDF)

X.H., D.J., D.J., and B.L. designed the experiments. X.H., L.F., Q.S., Z.L., and R.Y. conducted the majority of the experiments. X.H. analyzed the data. X. H., D. J., D.J., and B. L. wrote the manuscript.

The authors declare no competing financial interest.

Published as part of Chemical & Biomedical Imaging special issue “Electrochemistry and Imaging”.