Atomically Fe‐anchored MOF‐on‐MOF nanozyme with differential signal amplification for ultrasensitive cathodic electrochemiluminescence immunoassay

Abstract The successful application of electrochemiluminescence (ECL) in immunoassays for clinical diagnosis requires stable electrodes and high‐efficient ECL signal amplification strategies. Herein, the authors discovered a new class of atomically dispersed peroxidase‐like nanozymes with multiple active sites (CoNi‐MOF@PCN‐224/Fe), which significantly improved the catalytic performance and uncovered the underlying mechanism. Experimental studies and theoretical calculation results revealed that the nanozyme introduced a Fenton‐like reaction into the catalytic system and the crucial synergistic effects of definite active moieties endow CoNi‐MOF@PCN‐224/Fe strong electron‐withdrawing effect and low thermodynamic activation energy toward H2O2. Benefiting from the high peroxidase‐like activity of the hybrid system, the resultant ECL electrode exhibited superior catalytic activity in the luminol‐H2O2 system and resulted in an ≈17‐fold increase in the ECL intensity. In addition, plasmonic Ag/Au core‐satellite nanocubes (Ag/AuNCs) were designed as high‐efficient co‐reactant quenchers to improve the performance of the ECL immunoassay. On the basis of the differential signal amplification strategy (DSAS) proposed, the immunoassay displayed superior detection ability, with a low limit of detection (LOD) of 0.13 pg mL−1 for prostate‐specific antigen (PSA). The designed atomically anchored MOF‐on‐MOF nanozyme and DSAS strategy provides more possibilities for the ultrasensitive detection of disease markers in clinical diagnosis.


INTRODUCTION
Electrochemiluminescence (ECL), inheriting the advantages of chemiluminescence and electrochemistry, is regarded as a promising analytical technique owing to its low background, wide dynamic range, and high sensitivity. [1][2][3] Since the first report of the ECL phenomenon by Bard et al., [4] numerous ECL luminophores, such as metal-organic frameworks (MOFs), [5,6] luminol, [7,8] Ru(bpy) 3 2+ [9,10] and nanomaterials [11,12] have been extensively investigated. Among them, luminol has attracted considerable interest on account of its acceptable price, low oxidation potential and high quantum yield. [13] So far studies of luminol-based ECL applications are mostly focused on its anodic emission. Nevertheless, anodic ECL is commonly suffered from numerous interference reactions due to the coexisting reductive components. [14] Theoretically, cathodic ECL possesses simpler emission mechanisms and more negative excitation potential, which is beneficial for ECL detection applications. However, luminol can hardly be activated at a negative potential, which led to relatively weak cathodic ECL signals and resulted in limited applications. [15]  of  In the classical luminol system, H 2 O 2 is commonly used as a co-reactant to generate reactive oxygen species (e.g., •O 2 − and •OH) to promote luminol oxidation and enhance ECL intensity. [15] In this process, the co-reactant introduced plays a key role in generating reactive intermediate radicals from coreactants and promoting the ECL intensity of luminophores. Recently, Nanozymes with peroxidase-mimic activity have been discovered to be able to catalyze the H 2 O 2 and produce •O 2 − and •OH with high efficiency. [16][17][18] Thereinto, metalorganic framework (MOF)-based nanozymes constructed with inorganic metal ions (cluster) and organic linkers are promising candidates with excellent characteristics of a well-defined coordination network, tunable porosity and high surface area. [19] To date, only a few MOF nanozymes exhibited peroxidase activity, enabling cascade reactions with high catalytic activity. Nevertheless, the reported MOF nanozymes still suffer from poor catalytic activity due to weak electron transfer kinetics underlying the heterogeneous reactions of the co-reactant and limited accessible active sites of individual MOF. [20,21] MOF-on-MOF nanozymes, which integrate multiple anisotropic MOFs into one unit, are ideal alternatives to increase the active sites and promote catalytic activity on account of their heterogeneous components and diverse structures. [22] However, few attempts have been made to design and apply MOF-on-MOF nanozymes for ECL applications.
To achieve ultrasensitive biodetection, nanoengineering of ECL nanoprobe and the development of signal-enhanced detection strategies are highly desired. At present, resonance energy transfer (ECL-RET) is the most commonly used tactic to quench ECL signal and achieve the detection of analytes. [23,24] However, in the ECL-RET scheme, it is quite difficult to achieve a complete overlap between the emission spectrum of the donor and the absorption spectrum of the acceptor. The spectral mismatch seriously affects the energy transfer and ECL signal amplification. Besides, the intermolecular ECL-RET may result in massive energy loss in the energy transfer process, which limits the signal amplification intensity and detection sensitivity.
Considering the technical bottlenecks mentioned above, herein, we developed a highly-efficient cathodic ECL detection platform by synchronously introducing MOF-on-MOF nanozymes and a differential signal amplification strategy (DSAS). Firstly, an MOF-on-MOF nanozyme (CoNi-MOF@PCN-224/Fe) was designed and nano-engineered to introduce a Fenton-like reaction via an atom-anchoring strategy. The designed CoNi-MOF@PCN-224/Fe exhibited excellent peroxidase-like activity in the luminol-H 2 O 2 system due to the multiple active sites (both PCN-224/Fe and CoNi-MOF) and low activation energy barriers confirmed by DFT calculations. Besides, the Ag/Au core-satellite nanocubes (Ag/AuNCs) as efficient co-reactant (H 2 O 2 ) quenchers were also designed and prepared. In the presence of antigen (e.g., PSA), the co-reactant (H 2 O 2 ) will be consumed by etching the antigen-labeled plasmonic Ag/AuNCs and thus decrease the ECL intensity (Scheme 1). The synergy of ECL intensity enhancement derived from the Fe atom-anchored MOF-on-MOF nanozyme catalysis and efficient ECL signal quenching via co-reactant consumption (called differential signal amplification strategy, DSAS) is proved to be an efficient strategy to achieve ultrasensitive biodetection, by using prostate-specific antigen (PSA) as a specific model. This work provides a new signal amplification strategy for ECL bioassay and will promote practical clinical applications of MOFs in ultrasensitive immunoassays.

. Theoretical calculations of the activity of the Fenton-like nanozyme
Recently, the Fenton reaction has been widely exploited for applications in many fields, such as photodynamic therapy of cancer, pollutant degradation, etc. [25,26] In the reaction, with the participation of iron ions, H 2 O 2 can be efficiently decomposed and converted to reactive oxygen species (ROS, e.g., •OH and •O 2 − ). Thus, introducing the Fenton reaction into porous MOFs to construct highly-efficient nanozymes with multiple active sites is expected to be an ideal strategy to achieve a high ECL signal in the luminol-H 2 O 2 system. Inspired by the metalloporphyrin of natural hemin, PCN-224, which is constructed by Zr 6 cluster and Tetrakis(4carboxyphenyl)porphyrin (TCPP), would be an ideal MOF structure as the Fe 3+ can reside in the center of porphyrin ligand via FeN 4 to afford hemin-like nanozyme activity, as shown in Figure 1A. The atomically Fe-anchored PCN-224 (PCN-224/Fe) is expected to provide an optimized electronic state and behave with superior activity to generate various ROS to further enhance the ECL signal. To confirm this conjecture, density functional theory (DFT) calculation was first conducted. Figure 1B,C is the charge-density diagram, which describes the spatial charge distribution of the nanozymes. After Fe 3+ anchoring, the electron density distribution changes greatly, in which an electron-deficient center is obviously formed around Fe 3+ . Moreover, the PCN-224/Fe shows a strong electron-withdrawing effect (the yellow electron clouds represent electron enrichment) toward H 2 O 2 , as shown in the differential charge density distribution ( Figure 1D). Such structure and electron transfer are expected to be in favor of regulating the activation energy for the reaction intermediates and therefore changing the catalytic reaction rate. Figure 1E,F, Figure S1 and Equation (S1) are the free energy schemes and chemical mechanism during the H 2 O 2 decomposition. The activation energy barriers of H 2 O 2 decomposition to produce •OH and suffered from limited catalytic activity due to the restricted accessible active sites. [27,19] Recently, multi-metal-site catalysts were found to exhibit superior catalytic activity compared with single-metal ones, and this property is assumed to be the result of the synergetic effect of the coexistence of multimetal sites on optimizing the interaction between active sites and reactants. [28,29] In recent year, Cobalt-or nickel-based metal-organic frameworks were found to have peroxidase-like property. By precisely controlling the composition, structure and morphology, the Cobalt-or nickel-based MOFs could exhibit high-efficient catalytic activities in biosensing, cancer treatment, environmental protection etc. [27,30] Inspired by this, we tried to further introduce highly active CoNi-MOF nanosheet into the PCN-224/Fe systems and constructed MOF-on-MOF nanozymes with multi-metal sites (CoNi-MOF@PCN-224/Fe, Scheme 1 and Figure 2A). The asprepared nanozymes are expected to integrate the merits of each active site and will obtain high-performance ECL sensing applications in immunoassay.

.
Morphology and structure characterizations of the nanozymes Figure 2A shows the chemical preparation procedures of the CoNi-MOF@PCN-224/Fe. First, PCN-224 was synthesized by using a facile hydrothermal method. As displayed in Figure 2B, the PCN-224 exhibits a uniform cubic morphology. The rich carboxylic and conjugate benzene groups of PCN-224 were revealed by Fourier Transform Infrared (FTIR) analysis in Figure S2. [31] Moreover, the powder X-ray diffraction (PXRD) pattern of the nanocubes indicated the successful preparation of PCN-224 ( Figure S3). Then the Fe 3+ was implanted into PCN-224 via a solvothermal process. With the insertion of Fe 3+ , the morphology and structure remain intact without any visible changes ( Figure 2C). Subsequently, CoNi-MOF was in-situ covered on the surface of PCN-224/Fe, showing an ultrathin nanosheet structure ( Figure 2D). These dense ultrathin nanosheets further increased the exposed active sites and hence the catalytic activity of the nanozymes. As seen from scanning electron microscope (SEM) and transmission electron microscopy (TEM) images in Figure 2D,E, the typical size of the as-prepared CoNi-MOF@PCN-224/Fe is ≈2 μm. Energy dispersive spectrum (EDS) analysis ( Figure  S4 Figure S5A). The result is consistent with the EDS analysis in Figure S4. For PCN-224, the peak of Pyridinic N is centered at 396.3 eV. After the Fe 3+ anchoring, the peak shifted positively to 396.7 eV due to the coordination between N and Fe 3+ and the charge-oriented transfer from N to Fe 3+ . [32] This effect was also identified by fluorescence quenching of the system after the introduction of  Figure 3C and Figure S6). Moreover, the PCN-224/Fe showed obvious Fe─N at the wavelength of 997 cm −1 and the N─H at 3310 cm −1 disappeared in comparison with PCN-224 and TCPP, demonstrating the atom anchoring of Fe with PCN-224 by Fe─N bonds ( Figure 3D and Figure S2). [32] The peak at 1050 cm −1 is attributed to deformation peak of pyrrole. After the formation of PCN-224 and anchoring of Fe with PCN-224 by Fe─N bonds, the variation of charge density distribution may cause a change in the dipole moment of C─N, which resulted in the increased peak at 1050 cm −1 . [31] Nitrogen sorption isotherms measured at 77 K ( Figure 3E) display a typical type I isotherm with a Brunauer-Emmett-Teller (BET) surface area as high as 790.97 m 2 g −1 . The large surface area and well-ordered porous structures of the nanozymes guarantee sufficient catalytic active sites and high-flux mass transfer, which are essential to boost the catalytic reaction. After the assembly of CoNi-MOF onto PCN-224/Fe, high-resolution XPS of Pyridinic N positively shifted ( Figure 3A) while Fe 2p and Zr 3d peaks negatively shifted ( Figure 3B and Figure S5B), suggesting strong interactions between PCN-224/Fe and CoNi-MOF and the successful preparation of CoNi-MOF@PCN-224/Fe nanozymes. Besides, the CoNi-MOF@PCN-224/Fe showed excellent thermal stability and the primary structure remained intact below 390 • C ( Figure 3F), which is beneficial for practical ECL detection applications.

. Evaluation of the nanozyme-enhanced ECL performance
We then investigated the cathodic ECL response and performance of the MOF-on-MOF nanozymes, the results of which are shown in Figure 4A,B and Figures S7, S8. The optimal pH and concentration of cocatalysts were set at 9.0 and 10 μmol L −1 , respectively. This result is consistent with previous reports as luminol commonly exhibits the strongest luminescence efficiency at alkaline conditions. [13,33,8] There was almost no ECL response on bare glassy carbon electrodes (GCE). Further modifications with the pristine PCN-224 exhibited moderately enhanced ECL intensity due to the weak peroxidase-like activity. However, once the iron atoms were anchored on PCN-224, the cathodic ECL of PCN-224/Fe was remarkably enhanced, demonstrating the high efficiency of the hemin-like nanozymes in promoting ECL emission. This result is consistent with the DFT results that the atomically Fe act as the major catalytic activity sites and is capable of lowering the activation energy of the nanosystem to generate more ROS (Figure 1). After the in situ assembly of CoNi-MOF, the cathodic ECL signal of the resulting nanosystem was further increased, which is about ≈12-fold enhancement compared with the pristine PCN-224 or control CoNi-MOF, respectively ( Figure 4A and Figure S9), proving the excellent ECL performance of CoNi-MOF@PCN-224/Fe. To calculate the electrochemically active surface area (ECSA) of the individual cathode, cyclic voltammetry (CV) curves with different scan rates were recorded to obtain the double-layer capacitance (C dl ). The comparison of C dl value of different samples displays that the CoNi-MOF@PCN-224/Fe had a larger ECSA (51.16 μF cm −2 ) than those of contrast cathodic materials ( Figure 4C and Figures S10−12). The high active surface area was attributed to the unique lamellar surface morphology and hierarchical MOF-on-MOF nanostructures, which increase the contact area with the electrolyte and in accordance with the ECL results. The ECL spectra are shown in Figure 4D with an emission wavelength centered at ≈460 nm, which corresponded to the emission spectrum of the excited state oxidation product of luminol, demonstrating that the ECL signals were derived from luminol. [33] Moreover, all the samples showed low ECL signals in nitrogen-saturated luminol solution (without H 2 O 2 , Figure S13), suggesting the crucial role of H 2 O 2 as a high-efficient cocatalyst in the ECL system. The results indicate that the synergy of the Fenton-like reaction of PCN-224/Fe and CoNi-MOF endows MOF-on-MOF nanozymes with higher peroxidase activity to produce ROS and hence the enhancement of ECL in the luminol-H 2 O 2 system. To prove this, electron paramagnetic resonance (EPR) was conducted as shown in Figure 4E,F and Figure S14 Figure S15, after the addition of isopropanol or benzoquinone, the ECL quenching was obviously observed, which is consistent with the EPR results and identified the peroxidaselike property of CoNi-MOF@PCN-224/Fe. According to the standardized method for enzyme activity evaluation, [34] the Michaelis-Menten constant (K m ) and maximal reaction rate (ν max ) were measured as displayed in Figure S16. The K m value of the nanozyme was calculated to be 1.23 mM for TMB with the ν max of 15.96 μm min −1 , which is higher or comparable with other nanozymes reported (Table S1). On the basis of the above experimental results, a possible ECL mechanism of the luminol-H 2 O 2 system on CoNi-MOF@PCN-224/Fe catalysts is proposed as follows: According to the proposed mechanism, the luminol firstly deprotonates in an alkaline solution (Equation (1)) to generate L − and the H 2 O 2 was catalyzed by CoNi-MOF@PCN-224/Fe nanozymes to produce ROS (Equation (2)). Then the L − reacts with the electrogenerated ROS and forms the excited-state intermediates (3-aminophthalate 2-*). Finally, the excited 3aminophthalate 2-* emits light as they return to the ground states (3-aminophthalate 2-).

. Construction of DSAS system and its application in PSA detection
As shown in Scheme 1, the differential signal amplification strategy (DSAS) based ECL system was established by using Ag/AuNCs as high-efficiency ECL label quenchers and PSA as a model disease biomarker. In the presence of PSA, the Ab 2 -labeled Ag/AuNCs will bond to the nanozyme decorated ECL electrode specifically. In the luminol-H 2 O 2 ECL system, the more Ag/AuNCs bound, the faster the H 2 O 2 will be consumed by etching Ag nanocubes. [35] Thus, the ECL signal is inversely proportional to the concentration of PSA in the test solution. Figure S17 shows the SEM and TEM images of the label quencher (Ag/AuNCs) which exhibited uniform nanocubes with a side length of ≈150 nm. Then, the DSASbased ECL system for immunoassay application was checked by using PSA as a target. As shown in Figure 5A, the resistance of the system decreased gradually with the modification of antibodies due to the insulating property of the biomolecular, which confirmed the successful preparation of the sandwichtype ECL immunoassays. [36] The ECL intensity reduced with the increase of PSA content, which displays a good linear relationship between the PSA concentration and ECL signal in the range from 10 −4 to 10 −13 g mL −1 (Figure 5B,C). The sensitivity and limit of detection (LOD) are calculated to be 7740 mol −1 L and 0.13 pg mL −1 , respectively. The LOD is lower or comparable with those previously reported (Table  S2). The ultrasensitive detection ability of the ECL system is attributed to the favorable catalytic kinetics and thermodynamic behavior of the CoNi-MOF@PCN-224/Fe nanozyme and the high-efficiency ECL quencher of Ag/AuNCs. When Ab 2 -labeled Ag/Au nanocubes are presented in the luminol-H 2 O 2 system, the cocatalyst (H 2 O 2 ) will be directly consumed by etching the Ag/AuNCs ( Figure S18). Moreover, the absorption spectrum of Ag/AuNCs overlaps with the emission spectrum of luminol ( Figure S19), and the subsequent surface plasmon coupling can generate enhanced electromagnetic field hot spots around Ag/AuNCs ( Figure 5D) to accelerate the depletion of H 2 O 2 as identified by the photoelectrochemical H 2 O 2 decomposition experiments shown in Figure S20. [37] By contrast, the ECL cathode only displayed a sensitivity of 4980 mol −1 L while using traditional reducing agents (ascorbic acid, AA) as H 2 O 2 consumption agents, suggesting the superiority of the designed Ag/AuNCs ECL quencher ( Figure S21). To investigate the specificity of the immunoassays for PSA detection, several antigen analogs containing AFP, CEA, BSA were employed to assess the detection selectivity ( Figure 5E). No obvious ECL signal changes were observed when the detection targets were AFP, CEA and BSA (10 −8 g mL −1 ), while for PSA and the mixture containing PSA and the above interferents, the ECL intensity decreases remarkably, which indicates the excellent specificity of the DSAS-based immunoassay for PSA. Moreover, the immunoassay exhibits good repeatability as displayed in Figure 5F. These results suggested that the developed DSASbased immunoassay was very suitable for the ultrasensitive detection of PSA with excellent sensitivity, selectivity, and stability. Finally, the real application of the immunosensor was assessed by detecting the concentration of PSA in human serum. As shown in Table S3, the developed immunosensors showed good recovery, suggesting the validity of the DSAS-based ECL immunoassays in the detection of disease biomarkers. Moreover, the immunoassay exhibited good detection performance in detecting real clinical samples (276.3 ng mL −1 ), consistent with the result by using ELISA kit (251.8 ng mL −1 ), with an error of 9.7%.

 CONCLUSION
In summary, atomically Fe-anchored MOF-on-MOF nanozymes (CoNi-MOF@PCN-224/Fe) were designed and exploited as high-efficiency ECL electrode materials in the luminol-H 2 O 2 system. Mechanistic insights proved that the remarkably enhanced ECL was attributed to the Fenton-like catalytic reaction and multi-metal-sites of the CoNi-MOF@PCN-224/Fe, which obviously decrease the Gibbs free energy of the reaction and promote the generation of the reactive oxygen species. Moreover, a novel signal amplification strategy, DSAS, was proposed and successfully developed by designing novel plasmonic Ag/AuNCs as highefficient co-reactant quenchers in the ECL immunoassay. The developed nanozyme immunoassay exhibited excellent detection ability for PSA with an LOD of 0.13 pg mL −1 . The proposed signal amplification strategy opens a new pathway in designing high-efficiency ECL sensing platforms, which could be widely used in biomedical and chemical analysis, especially in the ultrasensitive detection of target disease biomarkers in clinical diagnosis.

A C K N O W L E D G E M E N T S
Financial support from the National Natural Science Foundation of China (22004002), Anhui Provincial Natural Science Foundation (2008085QB80) and the Open Funds of the State Key Laboratory of Electroanalytical Chemistry (SKLEAC202103) is gratefully acknowledged.

C O N F L I C T O F I N T E R E S T S TAT E M E N T
The authors declare no conflicts of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
All data of this work are present in the article and Supporting Information. The other data that support the findings of this work are available from the corresponding author upon reasonable request.