Dramatically enhancing electrochemiluminescence performance in the aqueous phase using naphthalene diimides with excellent electron-transfer capability and water solubility

Abstract

Luminescent substances with superior electron transfer capability and excellent water solubility are ideal candidates for constructing high-performance electrochemiluminescence (ECL) sensors. In this work, we designed and synthesized a series of naphthalene diimide (NDI) derivatives from the perspective of structural regulation. Among them, the luminophore NDI-Cl exhibited outstanding ECL performance in the aqueous phase, benefiting from its high water solubility and favorable electron transfer properties. Furthermore, based on electrostatic interactions, the newly developed NDI-Cl-based ECL system showed an ultrasensitive and selective response to ATP, with a limit of detection (LOD) as low as 11.8 μM. This study not only reveals the crucial role of electron transfer in ECL at the molecular level but also provides a new strategy for designing high-performance ECL sensors.

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Introduction

The rapid advancement of energy storage technologies has spotlighted electroactive materials due to their diverse applications, including electrocatalysis,¹ photoluminescence, and electrochemiluminescence (ECL).² ECL, a redox-induced luminescence process, involves the generation of reactive radicals at electrode surfaces, which undergo electron transfer to form luminescent excited states.³ Owing to its low background signal and high sensitivity, ECL is widely utilized in biosensing and diagnostics.⁴ In general, ECL mechanisms are broadly categorized into electron transfer-based systems (e.g., Ru(bpy)₃²⁺, 9,10-diphenylanthracene, and silole)⁵ and chemical bond cleavage systems (e.g., luminol and lucigenin).⁶ Electron transfer pathways, which avoid generating new chemical species, offer reduced interference with analytes. However, their luminescence efficiency is often limited by suboptimal redox properties and poor water solubility of luminophores, necessitating the development of emitters with enhanced redox activity and aqueous compatibility to advance ECL system performance.

Among electroactive materials, naphthalene diimide (NDI) derivatives are distinguished by their structural tunability, solubility across diverse media, high specific capacity, rapid electrochemical reactivity, cost-effectiveness, and environmental sustainability.⁷ Notably, the two carbonyl groups in their molecular structure form a conjugated system with the aromatic ring through imide bonds, constructing a potent electron-capturing center. This structural characteristic enables exceptional electron transfer capabilities, allowing sequential two-electron reductions to form dianions and tetra-anions (Scheme 1), which underpins their potential in organic optoelectronics and as cathode materials in rechargeable metal-ion batteries.⁸ The electron transfer capacity of NDIs is a critical determinant of ECL luminophore performance, particularly for electron transfer-based ECL systems.⁹ Despite this potential, NDI-based ECL research remains underexplored, likely due to the scarcity of appropriately functionalized NDI emitters and optimized electrochemical excitation methods. Water solubility further influences ECL efficacy, as it enhances luminophore dispersion and electrochemical reactivity, thereby amplifying ECL signals.

Scheme 1 The redox process of naphthalene diimede (NDI).

Inspired by the abovementioned results, in this study, we designed and synthesized three NDI derivatives: 1,8,4,5-naphthalenetetracarboxylic dianhydride (NCD), 2,7-bis[3-(dimethylamino)propyl]-1,8,4,5-naphthalenetetraformyl diimide (NDI-N), and ammonium-functionalized, water-soluble 2,7-bis[3-(trimethylamino)propyl]-1,8,4,5-naphthalenetetracarbodiimide (NDI-Cl) (Scheme 2). Among these, NCD represents a rigid planar structure anticipated to demonstrate high luminescence efficiency and stability, while NDI-N possesses a push–pull electronic structure that may enable red-shifted emission and efficient electron injection. Through comparative analysis of the three compounds, we elucidated the structure–ECL performance relationship. Our findings reveal that NDI-Cl, with its excellent water solubility and superior electron transfer properties, exhibits the strongest ECL emission. We developed an ultrasensitive ECL-based sensor for adenosine triphosphate (ATP) detection, achieving a limit of detection of 11.8 μM, thus demonstrating the potential of NDI derivatives for high-performance ECL applications.

Scheme 2 Chemical structures of 1,8,4,5-naphthalenetetracarboxylic dianhydride (NCD), 2,7-bis[3-(dimethylamino)propyl]-1,8,4,5-naphthalenetetraformyl diimide (NDI-N), and 2,7-bis[3-(trimethylamino)propyl]-1,8,4,5-naphthalene tetracarbodiimide (NDI-Cl).

Experimental

Chemicals and materials

In the experiment, all oxygen- or moisture-sensitive reactions were conducted under anhydrous conditions and a nitrogen atmosphere. Analytical-grade reagents were procured from Sinopharm Chemical Reagent Co., Ltd (Shanghai). Hydrogen nuclear magnetic resonance (¹H NMR) spectra were acquired using a Bruker Avance 400 MHz spectrometer with tetramethylsilane (Me₄Si) as the internal standard. Electrospray ionization (ESI) mass spectrometry data were obtained on a Shimadzu LCMS-2010EV instrument. pH measurements were performed using a Mettler Toledo SevenMulti S40 pH meter (Mettler Toledo, Switzerland). The compounds 1,8,4,5-naphthalenetetracarboxylic dianhydride (NCD), 2,7-bis[3-(dimethylamino)propyl]-1,8,4,5-naphthalenetetraformyl diimide (NDI-N), and 2,7-bis[3-(trimethylamino)propyl]-1,8,4,5-naphthalenetetracarbodiimide (NDI-Cl) were synthesized following established protocols with minor modifications.

Electrochemical and ECL experiments

ECL and cyclic voltammetry (CV) measurements were conducted using an MPI-A ECL detection system (Remex Electronic Instrument Co., Ltd, Xi'an, China) with a custom glass electrolytic cell (diameter 2 cm). A conventional three-electrode system was employed, comprising a glassy carbon working electrode, a platinum wire counter electrode, and an Ag/AgCl reference electrode with saturated KCl solution. All electrochemical experiments were performed under a nitrogen atmosphere to minimize interference. The photomultiplier tube (PMT) voltage was set to 800 V. The electrolyte pH was adjusted using a Metrohm 827 pH meter (Metrohm, Switzerland). A 0.1 M phosphate-buffered saline (PBS) solution, containing 0.1 M K₂HPO₄, 0.1 M KH₂PO₄, and 0.1 M KCl, served as the supporting electrolyte, with pH adjusted by varying the K₂HPO₄/KH₂PO₄ ratio.

Calculation

All density functional theory (DFT) calculations were performed to optimize the geometry, molecular orbitals, and three-dimensional structures of the synthesized ECL compounds using the Gaussian 03 (revision 01) software package at the B3LYP/6-311G(d,p) level. The highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) energy levels were calculated to support the analysis and interpretation of experimental data.

Detection of ATP

ECL-based detection of adenosine triphosphate (ATP) was performed by adding ATP (0, 20, 50, 100, 200, 500, 1000, 2000, 4000, and 5000 μM) to a 5 mL custom ECL electrolytic cell containing 0.1 M NDI-Cl in 0.1 M PBS solution (0.1 M KCl and 0.1 mM K₂S₂O₈, pH 7.5). Measurements were conducted with a potential window of 0.0 to −1.8 V (vs. Ag/AgCl) at a scan rate of 0.1 V s⁻¹. The relationship between the ECL intensity and ATP concentration was recorded for each measurement.

Results and discussion

The NDI derivatives NCD, NDI-N, and NDI-Cl were synthesized with high yields following established protocols with minor modifications (SI Schemes S1–S3 and Fig. S1–S6).¹⁰ Their molecular structures and purities were confirmed using ¹H NMR spectroscopy and electrospray ionization mass spectrometry (ESI-MS), with analytical data consistent with the expected chemical structures. Stability assessments under ambient conditions for one month revealed no structural degradation, indicating robust resistance to oxygen and moisture, which underpins their suitability for optoelectronic applications. To elucidate their molecular properties, density functional theory (DFT) calculations were performed at the B3LYP/6-311G(d,p) level using Gaussian 03 (revision 01) to optimize geometric configurations and vibrational frequencies.¹¹ As shown in Fig. 1, all three compounds exhibit conjugated rigid planar structures, minimizing energy losses from molecular rotations and positioning NCD as a high-performance luminescent material.

Fig. 1 The calculated molecular orbital plots of NCD, NDI-N, and NDI-Cl.

Structural analysis of NDI-Cl reveals that its introduced steric side chains effectively suppress π–π stacking and inhibit non-radiative transitions. Simultaneously, the positively charged groups induce a reduction in the LUMO energy level, thereby stabilizing the excited state. Furthermore, the hydrophilic chains create a locally rigid microenvironment that restricts molecular vibrations. These three factors act synergistically to minimize energy dissipation and significantly enhance radiative transition efficiency, ultimately leading to improved luminescence performance. Frontier molecular orbital (FMO) analysis revealed that the lowest unoccupied molecular orbitals (LUMOs) of NCD, NDI-N, and NDI-Cl arise from interactions between amide segments and the naphthalene core (Fig. 1). However, their highest occupied molecular orbitals (HOMOs) differ markedly: NCD and NDI-Cl HOMOs are primarily localized on the naphthalene ring, whereas NDI-N's HOMO is delocalized across the amine and amide moieties. Notably, NDI-Cl exhibits the lowest LUMO energy (−1.72 eV) and the smallest HOMO–LUMO bandgap (3.38 eV), indicating superior electron injection and transport properties, making it a promising candidate for ECL applications.

Photoluminescence and ECL properties

The optical properties of NCD, NDI-N, and NDI-Cl were characterized by UV–Vis absorption spectroscopy in dichloromethane (0.1 mM). As shown in Fig. 2A, the compounds displayed distinct absorption maxima at 348 and 366 nm (NCD), 358 and 378 nm (NDI-N), and 362 and 382 nm (NDI-Cl), attributed to n–π* transitions. Photoluminescence (PL) measurements under 365 nm excitation revealed emission maxima at 395 nm (NCD), 403 nm (NDI-N), and 415 nm (NDI-Cl) (Fig. 2B). The pronounced red shift in NDI-Cl's emission relative to NCD is likely due to strong intermolecular electrostatic interactions arising from its ionic structure.

Fig. 2 (A) UV-Vis and (B) PL spectra of NCD, NDI-N, and NDI-Cl in dichloromethane (0.1 mM). The corresponding excitation wavelength was 365 nm.

Electrochemical impedance spectroscopy (EIS) serves as a powerful technique for probing the electrochemical behavior of materials, offering insights into solution resistance and electron transfer resistance at the electrode–electrolyte interface. To assess the electron transfer characteristics of the three compounds NCD, NDI-N, and NDI-Cl, we performed EIS measurements in dichloromethane solution (0.01 mM) under conditions where faradaic processes were suppressed. As illustrated in Fig. 3, the plots of NDI-N and NCD displayed larger semicircle diameters, corresponding to resistance values of 105 and 141 Ω, respectively, indicative of relatively hindered charge transfer. By contrast, NDI-Cl exhibited the smallest semicircle and lowest resistance (63 Ω), underscoring its superior charge and electron transport properties, which facilitate electrochemical processes during its ECL activity.

Fig. 3 Electrochemical impedance spectroscopy (EIS) data of NCD, NDI-N and NDI-Cl at potentials free of faradaic processes. Test conditions: the frequency range was set as 0.01–10 000 Hz at +0.2 V. The electrolyte was [Fe (CN)₆]^3−/4− (1.0 mM) with KCl (0.1 M).

Subsequently, we investigated the electroluminescence properties of the compounds. Currently, ECL mechanisms are generally classified into two main types: annihilation and co-reactant-assisted pathways. Given that annihilation ECL reflects the intrinsic luminescence characteristics of the luminophores and facilitates mechanistic elucidation, we carried out a detailed examination of the annihilation ECL behavior of NCD, NDI-N, and NDI-Cl in acetonitrile (MeCN, 1.0 mM). As shown in Fig. 4A, the compound NCD exhibited a weak ECL response (∼40 a.u.) at −0.91 V, corresponding to its reduction peak at −1.08 V as observed in the cyclic voltammetry (CV) analysis. Although NDI-N displayed favorable redox characteristics, with an oxidation peak at +1.34 V and a reduction peak at −0.95 V, its ECL intensity remained relatively low (∼60 a.u.) at −0.98 V (Fig. 4B). In the case of NDI-Cl, two reversible reduction peaks (−0.68 V and −1.13 V) and an irreversible oxidation peak (+1.22 V) were recorded, and NDI-Cl generated a noticeable ECL signal at −1.13 V (Fig. 4C). This can be primarily attributed to the positively charged side chains of NDI-Cl, which promote molecular enrichment on the electrode surface through electrostatic interactions, significantly accelerating electron transfer kinetics, while the hydrophilic chains ensure molecular dispersion, preventing electrode passivation and mass transfer resistance caused by aggregation. The synergistic effect of these factors enables efficient and stable electrochemical responses, demonstrating superior redox properties. Nevertheless, the annihilation ECL emissions of all three compounds were relatively weak, which may be attributed to the absence of suitable excitation conditions and a lack of electroactive intermediates.

Fig. 4 CV waves and annihilation ECL–voltage curves of (A) NCD, (B) NDI-N, and (C) NDI-Cl in a mixed solution of H₂O and acetonitrile (MeCN, 1.0 mM). The optimal experimental conditions: a potential window of −1.8 V to +1.8 V (vs. Ag/AgCl), pH = 7.5, and 100 mV s⁻¹ scan rate, with tetrabutylammonium hexafluorophosphate (TBAPF₆) as a supporting electrolyte.

A growing body of evidence has demonstrated that the addition of co-reactants with strong redox activity can markedly enhance ECL signals.^3a,b Guided by this understanding, we explored the ECL performance of the compounds in the presence of a series of representative co-reactants, including tripropylamine (TPrA), potassium persulfate (K₂S₂O₈), oxalate (C₂O₄²⁻), and hydrogen peroxide (H₂O₂). Among these, all three compounds exhibited pronounced ECL responses when K₂S₂O₈ was employed as the co-reactant under optimized experimental conditions (potential window: 0 to −1.8 V vs. Ag/AgCl; 1.0 mM K₂S₂O₈; pH = 7; 0.1 M TBAPF₆ as the supporting electrolyte; and scan rate: 0.10 V s⁻¹). As shown in Fig. 5A, the ECL intensities followed the order NDI-Cl > NDI-N > NCD, with NDI-Cl displaying the highest signal, reaching approximately 17 000 a.u. Concurrent cyclic voltammetry (CV) measurements revealed a sharp increase in ECL intensity beyond −0.98 V in the presence of the co-reactant (Fig. 5B). The corresponding reduction peaks appeared at closely spaced potentials (NCD: −1.08 V, NDI-N: −1.04 V, and NDI-Cl: −0.99 V; Fig. 5C), suggesting that the interaction between K₂S₂O₈ and NDI-Cl under negative potential scans led to the generation of reactive intermediates. These radicals, produced within a relatively narrow potential window, efficiently participated in the ECL process, thereby significantly enhancing emission. Notably, NDI-Cl exhibited the most negative peak current (−1.4 V), indicating superior electron transfer capability for ECL generation. When benchmarked against the standard Ru(bpy)₃²⁺/K₂S₂O₈ system (assigned an ECL efficiency of 1), the calculated ECL efficiencies for NCD, NDI-N, and NDI-Cl were 176%, 278%, and 359%, respectively (Table S2, SI). These values represent some of the highest reported for organic ECL systems, highlighting the exceptional performance of NCD-based luminophores.

Fig. 5 (A) ECL intensities vs. three emitters (NCD, NDI-N, and NDI-Cl); (B) CV waves of three emitters; and (C) synchronous ECL intensity vs. potential. The three substances in MeCN (1.0 mM): a potential window of 0 V to −1.8 V (vs. Ag/AgCl), 1.0 mM K₂S₂O₈, pH = 7.5, and 0.1 V s⁻¹ scan rate under the same experimental conditions.

Given the critical significance of ECL stability for practical applications, we assessed the long-term stability of these three novel ECL systems across 20 consecutive scans. As depicted in Fig. 6, all three emitters demonstrated superior stability with low relative standard deviation (RSD) values (2.86% for NCD, 3.59% for NDI-N, and 1.92% for NDI-Cl), thereby establishing a robust foundation for their subsequent application-oriented research.

Fig. 6 ECL intensity vs. time curve of (A) NCD, (B) NCD-N, and (C) NCD-Cl under optimal conditions for 20 continuous cycles. The optimal experimental conditions: a potential window from 0 to −1.8 V (vs. Ag/AgCl), 0.1 mM K₂S₂O₈, pH = 7.5, 0.1 M TBAPF₆ as a supporting electrolyte, and 0.1 V s⁻¹ scan rate.

To further elucidate the underlying emission mechanism, we recorded the ECL spectra of the three luminophores under optimized experimental conditions using a CHI 650D electrochemical workstation in conjunction with a fluorescence spectrophotometer (F97XP, Shanghai Cold Light Technology Co., Ltd). As shown in Fig. 7, the maximum ECL emission wavelengths for NCD, NDI-N, and NDI-Cl were observed at 415 nm, 443 nm, and 449 nm, respectively. These emission maxima exhibited slight red-shifts relative to their corresponding PL spectra (Fig. 2B), suggesting that ECL and PL processes likely originate from the same excited-state relaxation pathway.

Fig. 7 ECL spectra of (A) NCD (415 nm), (B) NCD-N (443 nm), and (C) NCD-Cl (449 nm) recorded using a CHI 650D electrochemical workstation and a fluorescence spectrophotometer (F97XP, Shanghai Cold Light Technology Co., Ltd).

Based on the experimental results and relevant literature, we propose a plausible ECL mechanism as illustrated in Fig. 8. Initially, the co-reactant K₂S₂O₈ undergoes electrochemical reduction to generate the sulfate radical anion (SO₄˙⁻). Concurrently, the emitter NDI is oxidized by the highly oxidizing SO₄˙⁻ to form the radical cation NDI˙⁺, which is corroborated by the CV peak observed at −0.99 V (Fig. 5B). Additionally, the luminophore NDI can accept an electron to form the corresponding radical anion NDI˙⁻. Subsequently, the generated NDI˙⁺ and NDI˙⁻ undergo a specific redox reaction, leading to the formation of the excited-state species NDI*. Finally, the excited NDI* returns to its ground state, thereby producing the observed intense ECL emission. Overall, this ECL, like most organic-based ECL systems, undergoes a similar electron transfer process.

Fig. 8 The proposed mechanism of the NCD-based ECL system.

Adenosine triphosphate (ATP), as one of the most ubiquitously distributed extracellular signaling molecules, exerts a pivotal role in regulating a multitude of life activities.¹² Its concentration is closely associated with various pathological conditions and serves as an indispensable metabolite for viable microorganisms to maintain their physiological processes. Consequently, ATP is regarded as a hallmark biomarker of cellular viability and a critical indicator for microbial contamination. To date, a range of analytical techniques have been established for ATP detection, such as bioluminescence assay, high-performance liquid chromatography (HPLC), and fluorescence spectrophotometry.¹³

Despite substantial advancements in ATP detection over recent years, several challenges remain to be addressed, including inadequate sensitivity, cumbersome operational procedures, bulky instrumentation, and time-intensive analysis. Thus, the development of a more efficient ATP detection method is of particular urgency. The ECL approach holds promise for overcoming these limitations, owing to its intrinsic ultrahigh sensitivity and selectivity. Furthermore, we considered that under neutral conditions, ATP molecules carry multiple negative charges (primarily derived from their γ-, β-, and α-phosphate groups), while the molecular structure of NDI-Cl contains positively charged quaternary ammonium groups. The two interact through strong electrostatic forces, and as the ATP concentration increases, this interaction leads to a corresponding decrease in the ECL signal. This establishes a quantitative relationship between the change in ECL signal and ATP concentration, enabling detection. Based on this principle, we propose that this NDI-based ECL system can be used for the detection of trace amounts of ATP.

Fig. 9B illustrates the linear relationship between the decrease in ECL intensity and the increase in ATP concentration, with the linear equation expressed as ln(I₀/I) = 2.80581 × 10⁻⁴C − 0.00408, where I₀ and I denote the ECL intensity in the absence and presence of ATP, respectively. Moreover, an excellent correlation coefficient (R² = 0.9982 and n = 3) was obtained within the concentration range of 0 to 5000 μM, confirming a robust linear relationship between the ECL intensity and ATP concentration. The limit of detection (LOD) was calculated to be as low as 11.8 μM at a signal-to-noise ratio of 3, fully demonstrating the high sensitivity of this ATP detection method. These analytical results collectively provide strong technical support for applications in fields such as environmental monitoring, food safety, and health evaluation.

Fig. 9 (A) The variations in the ECL intensity of the NDI-Cl-based system upon introduction of ATP at varying concentrations (0, 20, 50, 100, 200, 500, 1000, 2000, 4000, and 5000 μM) under the optimized conditions; (B) the linear relationship between the ECL intensity and ATP concentration.

To further validate the specificity of the ECL sensor for ATP, we conducted interference tests to evaluate potential confounding factors. Selectivity experiments were performed using various interfering species, including ions (K⁺, Na⁺, and Cl⁻) and nucleotides (adenosine diphosphate [ADP], adenosine monophosphate [AMP], guanosine triphosphate [GTP], cytidine triphosphate [CTP], and uridine triphosphate [UTP]). These species were tested individually and in mixtures with 500 μM ATP. As shown in Fig. S7 (SI), the negligible interference effects of these species, compared to the significant ECL quenching induced by ATP, confirm the high selectivity of the NDI-Cl-based ECL method for ATP detection.

Subsequently, ¹H NMR spectroscopy was employed to investigate the underlying detection mechanism (Fig. S8 and S9, SI). Upon mixing ATP with NDI-Cl at room temperature, distinct changes were observed in the NMR spectrum relative to that of pure NDI-Cl: the proton peak corresponding to the quaternary ammonium group (–N⁺-CH₃) in NDI-Cl (originally at 3.04 ppm) exhibited a significant intensity reduction and a blue shift to 2.97 ppm (highlighted in red and blue boxes). This phenomenon indicates electrostatic interactions between the triphosphate moiety of ATP and the quaternary ammonium groups of NDI-Cl, which ultimately induce the observed regular changes in the ECL signal.

Lastly, to validate the practical utility of this method, we conducted recovery experiments to assess the performance of the sensing strategy in detecting ATP within 10% diluted human serum samples (Table 1). ATP solutions at concentrations of 0 μM, 20 μM, and 100 μM were separately spiked into 10-fold diluted human serum, and the resulting samples were analyzed using our ECL sensor. As presented in Table 1, the sensor achieved recovery rates ranging from 99.7% to 102.5%, accompanied by acceptable relative standard deviations (RSDs) of 2.1% to 3.5%. These analytical outcomes confirm that the sensor exhibits satisfactory capability for detecting the target ATP in real samples. Overall, our research demonstrates the superiority of the NDI-Cl-based ECL method over conventional ATP detection techniques (Table S3),¹⁴ while also highlighting its potential for application in clinical testing.

Table 1 Recovery tests for ATP in diluted human serum samples (n = 3)

Sample	Added	Found	Rate of recovery (%)	RSD (%)
1	10.0 μM	10.3 μM	101.5	3.5
2	20.0 μM	20.8 μM	102.5	3.6
3	50.0 μM	50.4 μM	99.7	2.1

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Conclusions

In summary, this study underscores the critical roles of electron transfer efficiency and aqueous solubility in the development of high-performance ECL systems. By rationally designing and synthesizing a series of NDI derivatives, we identified NDI-Cl as a particularly promising emitter, exhibiting superior ECL activity owing to its favorable redox properties and water solubility. The NDI-Cl-based ECL platform enabled highly sensitive and selective detection of adenosine triphosphate (ATP), achieving a detection limit as low as 11.8 μM. These findings not only establish a molecular design principle for next-generation organic ECL luminophores but also introduce a robust sensing platform for biologically relevant analytes. Ongoing efforts in our laboratory aim to extend this strategy to broader applications in bioanalysis and optoelectronic device development.

Conflicts of interest

The authors declare no competing financial interest.

Data availability

The authors confirm that the data supporting the findings of this study are available within the article (and/or its supplementary materials). Supplementary information: detailed synthesis procedures, nuclear magnetic hydrogen resonance (¹H NMR), mass spectrometry (ESI), UV-Vis, PL, the ECL test process and the detailed detection method of adenosine triphosphate (ATP). See DOI: https://doi.org/10.1039/d5an01074h.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (Grant No. 22174111, 22127803, and 22174110).

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