Coupling WO3−x dots-encapsulated metal-organic frameworks and template-free branched polymerization for dual signal-amplified electrochemiluminescence biosensing

Coupling WO₃₋_x dots-encapsulated metal-organic frameworks and template-free branched polymerization for dual signal-amplified electrochemiluminescence biosensing

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Fei Yin^a, Erli Yang^a, Xue Ge^a, Qian Sun^a, Fan Mo^a, Guoqiu Wu^b^,^c, Yanfei Shen*^,^a^,^b^,^c

Chinese Chemical Letters | 2024, 35(4) : 108753

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Chinese Chemical Letters | 2024, 35(4): 108753

• Communication •

Coupling WO₃₋_x dots-encapsulated metal-organic frameworks and template-free branched polymerization for dual signal-amplified electrochemiluminescence biosensing

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Fei Yin^a, Erli Yang^a, Xue Ge^a, Qian Sun^a, Fan Mo^a, Guoqiu Wu^b^,^c, Yanfei Shen*^,^a^,^b^,^c

Affiliations

^aMedical School, Southeast University, Nanjing 210009, China

^bCenter of Clinical Laboratory Medicine, Zhongda Hospital, Southeast University, Nanjing 210009, China

^cJiangsu Provincial Key Laboratory of Critical Care Medicine, Southeast University, Nanjing 210009, China

Published: 2024-04-15 doi: 10.1016/j.cclet.2023.108753

Outline

Abstract

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Developing accurate and sensitive DNA methyltransferase (MTase) analysis methods is essential for early clinical diagnosis and development of antimicrobial drug targets. In this work, by coupling WO₃₋_x dots-encapsulated metal-organic frameworks (MOFs) as co-reactants and terminal deoxynucleotidyl transferase (TdT)-mediated template-free branched polymerization, a dual signal-amplified electrochemiluminescent (ECL) biosensor was constructed to detect DNA adenine methylation (Dam) MTase. The employment of WO₃₋_x dots-encapsulated MOFs (i.e., NH₂-UIO66@WO₃₋_x) was not only beneficial for biomolecule conjugation because of the abundant amino groups but also led to a 7-fold enhanced ECL response due to the increased loading of WO₃₋_x. Moreover, TdT-mediated template-free branched polymerization promoted the capture of ECL emitters on the electrode surface, achieving 20-fold enhanced signal amplification. The presented ECL biosensor demonstrated a low detection limit of 2.4 × 10⁻⁴ U/mL, and displayed high reliability for the detection of Dam MTase in both spiked human serum and E. coli cell samples, and for the screening of potential inhibitors. This study opens a new avenue for designing a dual signal amplification-based ECL bioassay for Dam MTase and screening inhibitors in the fields of clinical diagnosis and drug development.

Key words

Dual signal-amplified electrochemiluminescence / WO₃₋_x dots / MOFs / Template-free branched polymerization / DNA methyltransferase

Cite this Article

Fei Yin, Erli Yang, Xue Ge, Qian Sun, Fan Mo, Guoqiu Wu, Yanfei Shen. Coupling WO₃₋_x dots-encapsulated metal-organic frameworks and template-free branched polymerization for dual signal-amplified electrochemiluminescence biosensing[J]. Chinese Chemical Letters, 2024 , 35 (4) : 108753 - . DOI: 10.1016/j.cclet.2023.108753

Full Text

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DNA methyltransferase (MTase) widely exists in biological organisms and can catalyze methylation reactions, which transfer cytosine or adenine bases in specific DNA with S-adenosyl-L-methionine (SAM) as a methyl donor [1,2]. From prokaryotes to eukaryotes, many major physiological processes are involved in the regulation of methyltransferases [3]. In humans, DNA MTase is a crucial component in gene expression, genome maintenance, and parental imprinting and its overexpression may be closely related to the occurrence and progression of various cancers [4–6]. In bacterial species, DNA MTase participates in the regulation of bacterial virulence and motility, and is a critical factor for bacterial colonization of hosts to cause disease [7–9]. Hence, DNA MTase may serve as a typical diagnostic biomarker as well as a target for antimicrobial drugs [10,11]. Recently, many methods have been available for determining DNA MTase activity, including colorimetry [12], fluorescence [13], electrochemistry [14] and photoelectrochemistry [15]. However, the quantitative determination of DNA MTase with high sensitivity remains challenging owing to the instability of detection probes and the complexity of detection procedures. Therefore, it is important to construct develop a highly accurate and sensitive and specific biosensor method for detecting the activity of DNA MTases and screening inhibitors.

Electrochemiluminescence (ECL) is a process that forms high-energy excited states from luminescent substances through electrochemical reactions on the electrode surface and then relaxes to the ground states, emitting light [16–19]. Because ECL fully integrates the merits of electrochemistry and chemiluminescence, it is a promising technology with high sensitivity, controllable luminescence, and easy miniaturization [20–22]. In particular, the co-reactant-type ECL is widely employed in biomedical analysis and clinical diagnosis, where co-reactants play an extremely significant role [17,23–25]. However, current commercial co-reactants such as tripropylamine (TPrA) possess gross dilemmas, such as high biotoxicity and strong background signals in the anodic ECL reaction [26,27]. As a result, it is highly desirable to explore alternative ECL co-reactants.

Semiconductor quantum dots such as WO₃₋_x dots show great potential in the field of ECL because of their low cost, ease of preparation, stable performance, and tunable size [28]. However, the lack of active groups for linking biomolecules hinders their broad application in bioassays [28]. Recently, surface functionalization with polymers or inorganic materials has been regarded as an effective way to improve the ability to couple with biomolecules and accommodate guest-active nanomaterials [29–35]. Metal-organic frameworks (MOFs) are one of the most popular materials with large specific surface areas, high porosities, and tunable chemical properties [36–39], allowing them to encapsulate guest molecules effectively [40–42]. On the other hand, MOFs can be conveniently conjugated with biomolecules by selecting suitable organic ligands with reactive terminal groups [43,44]. Therefore, MOFs can be a desirable candidate to accommodate WO₃₋_x dots for the preparation of highly efficient co-reactants for ECL bioanalysis.

Herein, a dual signal-amplified ECL biosensor was constructed by coupling the co-reactants of WO₃₋_x dots-encapsulated MOFs (NH₂-UIO66@WO₃₋_x) with terminal deoxynucleotidyl transferase (TdT)-mediated template-free branched polymerization for quantitative determination of DNA adenine methylation (Dam) MTase, a type of DNA MTase. The employment of NH₂-UIO66@WO₃₋_x was not only beneficial for biomolecule conjugation because of the abundant amino groups, but also led to an elevated ECL response ascribed to the enhanced loading of WO₃₋_x by NH₂-UIO66. Moreover, the use of branched double-stranded DNA (dsDNA) instead of traditional linear dsDNA for TdT-mediated template-free branched polymerization promoted the capture of ECL emitters, resulting in enhanced signal amplification. The developed biosensor achieved a low detection limit of 2.4 × 10⁻⁴ U/mL, and was successfully applied for screening inhibitors and determining Dam MTase in spiked serum samples and E. coli cells with high reliability. This study offers a new strategy for designing a dual signal-amplified ECL biosensor for the sensitive determination of Dam MTase activity and screening inhibitors for clinical diagnosis and drug development.

The NH₂-UIO66@WO₃₋_x nanocomposites were synthesized via a facile two-step procedure (Fig. 1a). The transmission electron microscopy (TEM) image showed WO₃₋_x dots with excellent monodispersity in aqueous solution exhibited average size of 2.82±0.66 nm (Fig. 1b) and an interplanar d spacing of 0.37 nm. WO₃₋_x dots with hydroxyl groups were immobilized on protonated NH₂-UIO66 with a regular octahedral nanostructure (Fig. 1c) through electrostatic interactions. After complexation with WO₃₋_x, the resulting NH₂-UIO66@WO₃₋_x nanocomposites retained their original shape of NH₂-UIO66 with tiny amount of nanoparticles on the surface (Fig. 1d). The EDS mapping analysis showed that N, Zr, and W were uniformly distributed in the nanocomposites (Fig. S1 in Supporting information). Elemental line scanning analysis (Fig. 1e) demonstrated that W was found throughout the nanostructure, suggesting that WO₃₋_x dots were distributed mainly in the pores of NH₂-UIO66, and the small number of nanoparticles around the NH₂-UIO66 nanostructure were also WO₃₋_x dots.

The X-ray photoelectron spectroscopy (XPS) was applied for investigating the bonding information of NH₂-UIO66@WO₃₋_x nanocomposites. The typical peaks of O 1s, C 1s, N 1s, Zr 3d and W 4f were observed for NH₂-UIO66@WO₃₋_x as illustrated in Fig. 2a. The fitting curve of W 4f spectrum showed two spin-orbit doublets correlating to W⁶⁺ and W⁵⁺ (Fig. 2b), which was consistent with those of individual WO₃₋_x dots (Fig. S2 in Supporting information), indicating the presence of WO₃₋_x dots in the nanocomposites [45,46]. Moreover, the deconvoluted Zr 3d spectrum of the NH₂-UIO66@WO₃₋_x nanocomposites showed two strong peaks at 182.9 and 185.3 eV (Fig. 2c), which were attributed to Zr 3d_5/2 and Zr 3d_3/2, respectively (Fig. S3 in Supporting information) [47–49], suggesting the presence of NH₂-UIO66 in the nanocomposites. Fourier Transform infrared spectroscopy (FTIR) results further verified the presence of both NH₂-UIO66 and WO₃₋_x. As shown in Fig. S4 (Supporting information), the FTIR spectrum of NH₂-UIO66@WO₃₋_x displayed a broad characteristic peak at 3475 cm⁻¹, assigned to N—H symmetric and asymmetric stretching vibrations, and two peaks at 1659 and 1569 cm⁻¹, assigned to the Zr⁴⁺-coordinated COOH [50,51]. Moreover, the peak at 1252 cm⁻¹ was related to the C—N stretching vibration of aromatic amines from NH₂-UIO66 [48,49,52]. Notably, the sharp absorption peaks at 806, 896, and 961 cm⁻¹ were attributed to W-O bending and asymmetric and tensile vibrations of the WO₃₋_x dots, respectively [46]. Thermogravimetric analysis (TGA) demonstrated that the mass percentage of WO₃₋_x dots in NH₂-UIO66@WO₃₋_x was greater than 20% (Fig. S5 in Supporting information). Thus, these results demonstrated the successful synthesis of the NH₂-UIO66@WO₃₋_x nanocomposites.

To obtain more structural information on the NH₂-UIO66@WO₃₋_x nanocomposites, the surface areas of NH₂-UIO66@WO₃₋_x and NH₂-UIO66 were evaluated by N₂ adsorption-desorption isotherms (Fig. 2d). The surface areas of NH₂-UIO66@WO₃₋_x and NH₂-UIO66 were calculated as 922 m²/g and 1011 m²/g, respectively. The pore size distribution results (Fig. S6 in Supporting information) showed two kinds of pores in the range of 0.5–0.8 nm (tetrahedral microporous cages) and 1–1.5 nm (octahedral microporous cages) by density functional theory (DFT) [53,54]. After the complexation with WO₃₋_x dots, the pore volumes reduced slightly, which might be due to the fact the WO₃₋_x dots occupied or blocked the cavities of the NH₂-UIO66 [55].

Ru(phen)₃Cl₂, a typical ECL emitter, which can be embedded in the groove of dsDNA via electrostatic and covalent interactions [56,57], was selected for the biosensor fabrication. As shown in Fig. 3a, the anodic ECL signal of Ru(phen)₃Cl₂/NH₂-UIO66@WO₃₋_x was much higher than that of Ru(phen)₃Cl₂ alone, and NH₂-UIO66@WO₃₋_x alone did not exhibit an evident ECL signal, indicating the co-reactant effect of NH₂-UIO66@WO₃₋_x on the ECL of Ru(phen)₃Cl₂. The mass ratio of NH₂-UIO66: WO₃₋_x dots was further optimized at 1:1 for the preparation of the NH₂-UIO66@WO₃₋_x nanocomposites (Fig. S7 in Supporting information). It is noteworthy that the ECL signal of Ru(phen)₃Cl₂/NH₂-UIO66 was similar to that of Ru(phen)₃Cl₂ alone, and was negligible compared to Ru(phen)₃Cl₂/NH₂-UIO66@WO₃₋_x (Fig. S8 in Supporting information), suggesting a co-reactant effect of WO₃₋_x dots. To explore the mechanism of the Ru(phen)₃Cl₂/NH₂-UIO66@WO₃₋_x ECL system, the oxidation behavior of Ru(phen)₃Cl₂ and NH₂-UIO66@WO₃₋_x in PBS was investigated by cyclic voltammetry (CV), and the ECL-potential curves were recorded. As shown in Fig. 3a, the onset oxidation potentials of Ru(phen)₃Cl₂ and NH₂-UIO66@WO₃₋_x were at approximately 1.07 V and 0.77 V, respectively, indicating that NH₂-UIO66@WO₃₋_x was more easily oxidized than Ru(phen)₃Cl₂. Moreover, the ECL spectra of Ru(phen)₃Cl₂/NH₂-UIO66@WO₃₋_x collected at different potentials (Fig. 3b) suggested an onset potential at 1.1 V and a peak wavelength at approximately 600 nm, attributed to the emission of Ru(phen)₃Cl₂ (Fig. S9 in Supporting information), demonstrating that the generation of an ECL signal was after the time that both Ru(phen)₃Cl₂ and NH₂-UIO66@WO₃₋_x were oxidized. Along this line, it could be concluded that the ECL reaction of Ru(phen)₃Cl₂/NH₂-UIO66@WO₃₋_x was driven by an "oxidation–reduction" co-reaction route [58,59]. A linear relationship between the oxidation peak current and ν^1/2 and the ECL peak intensity and ν^1/2 was observed, respectively, indicating a typical diffusion-controlled process (Fig. S10 in Supporting information) [59]. Thus, it was preliminarily inferred that the ECL reaction mechanism of the Ru(phen)₃Cl₂/NH₂-UIO66@WO₃₋_x system was described in Scheme S1 (Supporting information).

Based on the excellent ECL-promoting performance of NH₂-UIO66@WO₃₋_x for the Ru(phen)₃²⁺-ECL system, a signal-amplified ECL biosensor for Dam MTase was constructed using NH₂-UIO66@WO₃₋_x as a co-reactant. As shown in Fig. 4a, in the presence of Dam MTase, a palindromic probe was methylated at the adenine base and then specifically cleaved by the endonuclease DpnI, releasing DNA fragments with 3′-OH terminals. After the modification of NH₂-UIO66@WO₃₋_x nanocomposites onto GCE (NH₂-UIO66@WO₃₋_x/GCE), the released DNA fragments were covalently linked to NH₂-UIO66@WO₃₋_x on GCE via the -CO—NH- bond. The exposed 3′-OH terminals were then extended with dATP via TdT-mediated template-free polymerization to form poly-A chains [60], which subsequently hybridized with free poly-T fragments. Moreover, all the anchored poly-T fragments were continuously extended via TdT-mediated template-free polymerization to produce generous branched long poly-A chains, which were further hybridized with more free poly-T fragments. After multiple rounds of extension and assembly, a branched dsDNA nanostructure was formed on the NH₂-UIO66@WO₃₋_x modified GCE surface. It should be noted that with template-free branched polymerization, the branched dsDNA nanostructures can capture more ECL emitters of Ru(phen)₃Cl₂ onto the electrode surface to boost the ECL signal, and the ECL intensity of Ru(phen)₃Cl₂ can be correlated to the amount of Dam MTase in the biosensing system.

The feasibility of TdT-mediated branched template-free polymerization was verified using polyacrylamide gel electrophoresis (PAGE) in Fig. S11 (Supporting information). The eletrophoresis band was obtained for the renatured palindromic probe (Lane 2). In the presence of Dam MTase and SAM (Lane 3), the band of the palindromic probe disappeared, and a new band was observed, indicating that the methylated palindromic probe was digested and new DNA fragments were released. Upon the addition of TdT and dATP, a new band with a significantly enhanced molecular weight was obtained (Lane 1), which could be ascribed to TdT-mediated template-free polymerization and the formation of long linear dsDNA. It is worth noting that all the anchored poly-T fragments can be continuously extended by TdT-mediated template-free polymerization, generating generous branched long poly-A chains, which will hybridize with more free poly-T fragments to form branched dsDNA. As a result, after multiple rounds of extension and assembly, as described above, a new band with a higher molecular weight appeared in the TdT-mediated branched polymerization (Lane 4). It should be noted that the TdT-mediated branched polymerization resulted in a 20-fold enhanced ECL response compared to that without polymerization and a 3.2-fold signal enhancement compared to that with linear polymerization (Fig. 4b). On the other hand, compared with individual WO₃₋_x dots, NH₂-UIO66@WO₃₋_x was not only beneficial for biomolecule conjugation because of the abundant amino groups on NH₂-UIO66, but also showed a 7-fold enhanced ECL response due to the increased loading of WO₃₋_x by NH₂-UIO66 (Fig. 4c). Thus, coupling TdT-mediated branched polymerization with NH₂-UIO66@WO₃₋_x would result in dual signal amplification for biosensing.

The assembly of the biosensor was confirmed by CV and electrochemical impedance spectroscopy (EIS). As shown in Fig. S12a (Supporting information), the CV of bare GCE in 0.1 mol/L PBS (pH 7.4) containing 2 mmol/L [Fe(CN)₆]³⁻ displayed a pair of redox peaks with a peak potential difference of less than 85 mV, indicative of a reversible redox reaction. After NH₂-UIO66@WO₃₋_x was modified on the GCE, the peak current decreased obviously, ascribed to the poor electron conductivity of NH₂-UIO66@WO₃₋_x. After the covalent immobilization of DNA and the subsequent TdT-mediated branched polymerization reaction of DNA on the electrode, the peak current further decreased gradually because of the blocked electron transfer of K₃Fe(CN)₆ by DNA. After the intercalation of Ru(phen)₃Cl₂ into dsDNA, the peak current increased slightly, probably because of the mediating role of Ru(phen)₃Cl₂ on the electrode for electron transfer between the electrode and K₃Fe(CN)₆ in the solution. These results demonstrated the successful construction of the biosensor, which was consistent with the EIS results (Fig. S12b in Supporting information).

Under the optimized conditions (Fig. S13 in Supporting information), the ECL response of the dual signal-amplified biosensor for Dam MTase was recorded in the concentration from 1 × 10⁻³ U/mL to 100 U/mL (Fig. 5a). With increasing the Dam MTase concentration (Fig. 5b), a desirable linear relationship between the ECL signal (I) and the logarithm of Dam MTase concentration (lgC_{Dam MTase}) was obtained as I = 1119.7 lgC_{Dam MTase} + 4292.1 (R² = 0.991), and the limit of detection (LOD) was calculated as 2.4 × 10⁻⁴ U/mL. The performance of the established biosensor was comparable to or superior to that of most of the previously reported detection methods (Table S1 in Supporting information).

The specificity of the constructed biosensing system was explored by using the M.SssI MTase as the interference protein and the single component of Dam MTase or DpnI as interference control. As shown in Fig. 5c, the ECL responses to Dam MTase, DpnI and M.SssI + DpnI were negligible compared to those of Dam MTase + DpnI, indicating that this biosensor is highly selective for Dam MTase analysis. Moreover, the ECL signal fluctuation of the proposed biosensor was negligible after 10 cycles of continuous scanning, with a relative standard deviation (RSD) of 0.62% (Fig. 5d). The stability was investigated by collecting the ECL signal of the constructed biosensor after a storage of 1, 3, 7, and 14 days at 4 ℃, and 88.3% of the initial signal was observed after 14 days (Fig. S14 in Supporting information), thereby demonstrating the high stability of the biosensor.

The practical feasibility of the system was evaluated via recovery experiments by adding the Dam MTase standard into 10% diluted human serum samples, and the Dam MTase concentration was calculated using the standard curve shown in Fig. 5b. As shown in Table S2 (Supporting information), the recovery of Dam MTase was in the range of 96.45%−103.41%, with an RSD of less than 6.99%, indicating that the constructed biosensing system was applicable for complex biological samples. In addition, JM110 and DH5α E. coli cells were used as negative and positive samples to determine Dam MTase activity, respectively [61,62]. As shown in Fig. S15 (Supporting information), both the PBS and JM110 cell groups displayed negligible ECL signals, whereas the ECL signal of the DH5α cell group, which contained endogenous Dam MTase, was much higher, further verifying the practical feasibility of the as-constructed biosensing system.

Precise screening of targeted DNA MTase inhibitors opens up new channels for clinical treatment and drug development. The Dam MTase inhibition assay was performed using gentamicin, penicillin, and 5-fluorouracil as inhibitors [63–65]. The results showed that all three inhibitors could inhibit Dam MTase activity (Fig. 6a). It was found that 5-fluorouracil was more effective in inhibiting the Dam MTase activity. As shown in Fig. 6b, Dam MTase activity gradually reduced under 5-fluorouracil treatment, and the half-maximal inhibitory concentration (IC₅₀) of 5-fluorouracil was calculated as 0.47 µmol/L, which was consistent with the previous reports [61,62,65]. These results demonstrated that the proposed system can be widely applied to screen targeted DNA MTase inhibitors.

In summary, we developed a dual signal-amplified ECL biosensor for the quantitative determination of Dam MTase by coupling TdT-mediated template-free branched polymerization for ECL emitter capture and WO₃₋_x dots-encapsulated MOFs (NH₂-UIO66@WO₃₋_x) as co-reactants. By applying NH₂-UIO66@WO₃₋_x as co-reactants for the ECL of Ru(phen)₃Cl₂, a 7-fold higher ECL intensity was observed compared to that using the individual WO₃₋_x dots as co-reactants. Moreover, the ECL biosensor showed a 20-fold enhanced ECL signal using TdT-mediated branched polymerization for the promoted capture of ECL emitters. The constructed ECL biosensor platform was not only suitable for both spiked serum samples and E. coli cells but also could screen potential inhibitors. This study provides a promising analytical platform for Dam MTase activity evaluation and inhibitor screening in the fields of clinical diagnosis and drug development.

Ethical statement

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The human serum specimens were obtained from Zhongda Hospital (Nanjing, China) without any sample pretreatment except a dilution step, the human studies were reviewed and approved by the Independent Ethics Committee (IEC) for Clinical Research of the Affiliated Zhongda Hospital of Southeast University.

Delaration of competing interest

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The authors declare no conflicts of interest.

Acknowledgment

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This study was supported by the National Natural Science Foundation of China (Nos. 22074015 and 22174014).

Supplementary materials

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Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.cclet.2023.108753.

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Year 2024 volume 35 Issue 4

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doi: 10.1016/j.cclet.2023.108753

Receive Date：2023-03-11
Online Date：2025-11-20
Published：2024-04-15

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Received：2023-03-11
Revised：2023-06-08
Accepted：2023-06-28

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^aMedical School, Southeast University, Nanjing 210009, China

^bCenter of Clinical Laboratory Medicine, Zhongda Hospital, Southeast University, Nanjing 210009, China

^cJiangsu Provincial Key Laboratory of Critical Care Medicine, Southeast University, Nanjing 210009, China

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表12种不同金属材料的力学参数

科 Family	属数 Number of genus	种数 Number of species	占总种数比例 Percentage of total species (%)	属 Genus	种数 Number of species	占总种数比例 Percentage of total species (%)
鹅膏菌科Amanitaceae	2	11	5.26	鹅膏菌属 Amanita	10	4.78
小菇科 Mycenaceae	2	12	5.74	丝盖伞属 Inocybe	5	2.39
多孔菌科 Polyporaceae	8	14	6.70	蜡蘑属 Laccaria	5	2.39
红菇科 Russulaceae	3	23	11.00	小皮伞属 Marasmius	6	2.87
				小菇属 Mycena	11	5.26
				光柄菇属 Pluteus	5	2.39
				红菇属 Russula	17	8.13
				栓菌属 Trametes	5	2.39

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Fig. 1 (a) Scheme for the preparation of NH₂-UIO66@WO₃₋_x. TEM images of (b) WO₃₋_x dots, (c) NH₂-UIO66 and (d) NH₂-UIO66@WO₃₋_x. (e) Elemental line scan of a single NH₂-UIO66@WO₃₋_x particle indicated by the dashed line in the inset. Inset in (b): Size distribution of the WO₃₋_x dots.

Fig. 2 (a) XPS survey spectrum of NH₂-UIO66@WO₃₋_x. Narrow XPS scan spectra of (b) W 4f and (c) Zr 3d spectra of NH₂-UIO66@WO₃₋_x. (d) N₂ adsorption-desorption isotherms curves of NH₂-UIO66@WO₃₋_x and NH₂-UIO66.

Fig. 3 (a) ECL-potential and CV curves of GCE in 0.1 mol/L PBS (pH 7.4) containing 0.5 mg/mL NH₂-UIO66@WO₃₋_x, 50 mmol/L Ru(phen)₃Cl₂, and the mixture of 50 mmol/L Ru(phen)₃Cl₂ and 0.5 mg/mL NH₂-UIO66@WO₃₋_x. (b) ECL spectra of Ru(phen)₃Cl₂/NH₂-UIO66@WO₃₋_x in 0.1 mol/L PBS (pH 7.4) collected at different potentials.

Fig. 4 (a) Scheme for the construction of the proposed ECL biosensor. (b) ECL response of the biosensor fabricated with different amplification reactions in 0.1 mol/L PBS (pH 7.4). Black line: without polymerization; red line: linear polymerization; blue line: branched polymerization. (c) ECL response of the sensing system using WO₃₋_x dots (black line) and NH₂-UIO66@WO₃₋_x (red line) as co-reactants in 0.1 mol/L PBS (pH 7.4). Potential scanning: 0 to 1.2 V, scan rate: 0.2 V/s, PMT: 800 V.

Fig. 5 (a) ECL response of the biosensor to different concentrations of Dam MTase in 0.1 mol/L PBS (pH 7.4) containing 80 U/mL DpnI. (b) Calibration curve of the ECL biosensor for Dam MTase. (c) ECL response of the proposed biosensor to Dam MTase, DpnI, M.SssI MTase + DpnI and Dam MTase + DpnI under the same experimental conditions. (d) Signal stability of the biosensor for 10 U/mL of Dam MTase under 10 continuous scanning cycles in 0.1 mol/L PBS (pH 7.4) containing 80 U/mL DpnI. Potential scanning: 0 to 1.2 V, scan rate: 0.2 V/s, PMT: 800 V.

Fig. 6 (a) Inhibitory effect of 1 µmol/L of different inhibitors on Dam MTase activity (100 U/mL). (b) Inhibitory effect of different concentrations of 5-fluorouracil on Dam MTase activity (100 U/mL). Potential scanning: 0 to 1.2 V, scan rate: 0.2 V/s, PMT: 800 V.

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