Xu Zhang

Zhipeng Sun

Nicolas Richy

Olivier Mongin

Frédéric Paul
email: frederic.paul@univ-rennes1.fr

Mireille Blanchard-Desce
email: mireille.blanchard-desce@u-bordeaux.fr

Christine O Paul-Roth
email: christine.paul@insa-rennes.fr

Prof B Todd

Marder

Synthesis, characterization and optical properties of new tetrafluorenyl-porphyrins peripherally functionalized with conjugated 2-fluorenone groups

Keywords:

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Introduction

Porphyrin are attractive because their properties can be fine-tuned by synthetic modifications of the peripheral substituents. [1][2][3] In particular, we focus hereafter on a set of three new starshaped porphyrins decorated with extra peripheral fluorenone substituents.

Scheme 1. Molecular structures of meso-tetrafluorenylporphyrin (TFP), the corresponding alkynyl-substituted compound tetra(7-ethynyl-2-fluorenyl)porphyrin (TEFP) and tetrafluorenonylporphyrin (TFOP).

Accordingly, in line with independent findings of Bo and coworkers, [4] some of us had identified 5, 10,15,20-tetrafluorenylporphyrin (TFP) as an appealing luminophore (Scheme 1). [5] As a meso-tetraarylporphyrin, this compound presents a remarkable quantum yield (24%) which can be ascribed to the 2-fluorenyl units. The same year we also found that a slightly higher quantum yield (ФF = 26%) could be obtained with the 2-fluorenone analogue (TFOP) [6] and later on that introduction of four 2-fluorenyl units at the periphery of a mesotetraphenylporphyrin core allows increasing further the light harvesting properties and luminescence quantum yield, provided that the fluorenyl units are conjugated with the central porphyrin. [7] For instance, whereas TPP has an emission quantum yield around 11%, TPP1 exhibits an improved emission quantum yield of 20%. This yield can however not be further improved by attaching additional fluorenyl units at the periphery since neither TPP2 (20%) nor TPP3 (19%) present improved fluorescence quantum yields (Scheme 2). [7] A similar statement was also made for the dendrimer series based on a meso-tetrafluorenylporphyrin core (TFP1-3 in scheme 3; see F values). [8] In contrast, when TFP-Bu was functionalized with pendant ethynyl groups at its periphery, as in TEFP (Scheme 1), the fluorescence quantum yield dropped to 15%. This observation points to the strong influence of apparently slight structural modifications at the periphery of the TFP core on its emission properties. [8b] Important two-photon absorption (2PA) cross-sections were also evidenced for all these new dendritic systems in addition to their well-known oxygen-photosensitizing capability of the tetrapyrrolic core. [9] Thus, besides applications related to OLEDs or color displays, [5b-c] we also realized that, after proper functionalization, molecular analogues of TFP, series TPP1-3 or TFP1-3 might give rise to efficient biphotonic photosensitizers for photodynamic therapy (PDT). [9,10] Due to the practical advantages of two-photon excitation, this field is rapidly expanding and many porphyrin-based two-photon photosensitizers have been reported to date. [10,11] Such innovative developments rest on the rapid intersystem crossing taking place between the singlet and triplet excited manifolds in porphyrins (which is the first reason for their limited luminescence) and the large 2PA cross-section that related dendrimeric systems can present. [7,8,12] In our case, the intrinsic luminescence would even allow to perform simultaneous imaging, allowing an entry into so-called "two-photon theranostics". [10b] For these applications, in addition to the optimization of their luminescence quantum yields, simultaneous optimization of their two-photon cross-section and oxygen photosensitization quantum yields are also required. In this respect, systematic structure-property studies need to be conducted on new systems. Previous such studies pointed to star-shaped structures, such as TFP1, for possessing the most promising potential. [8a,11a] Accordingly, we have now decided Accordingly, (i) the synthesis and characterization of two new star-shaped derivatives, TFOP2', a deoxidized analogue of TFOP2, and TFOP2, and of their precursor TFP1' will be described first. (ii) Their electronic absorption (1PA) and emission spectra, their oxygen photosensitization properties and their 2PA spectra will next be determined. (iii) Finally, the impact of peripheral 9-fluorenyl oxidation on the photophysical properties of TFP1' as well as the potential interest of TFOP2' and TFOP2 for PDT will be briefly discussed.

Results and discussion

Porphyrin synthesis

To synthesize the new star-shaped porphyrin TFOP2 featuring four fluorenone units at its periphery, a synthetic protocol adapted from that previously used to isolate TFOP [6] was envisioned. Oxidation of TFP1' was expected to keep internal 2,7-fluorenyl units untouched and to form 2-fluorenone units specifically on external positions. During the course of this synthesis, one di-oxidized porphyrin isomer was isolated as a side-product "en route" toward TFOP2 and also characterized. Facile synthetic access to the required precursor TFP1' was expected using the Lindsey method, [13] meaning that the corresponding linear difluorenyl aldehyde had to be synthesized first. This last compound (3) was obtained with fair yield (63%) from a Sonogashira coupling reaction [14] between the known aldehyde 1 [8a] and 2ethynylfluorenyl 2 [7a] (Scheme 5).

Scheme 5. Synthesis of the new linear difluorenyl aldehyde 3.

From this new aldehyde 3, the new TFP1' porphyrin was obtained using classic Lindsey conditions (Scheme 6). [13] and isolated in correct yield (18%), as a dark red powder after chromatographic separation. Likewise to the synthesis of TFOP previously obtained by oxidation of TFP [6] , the desired porphyrins were obtained from TFP1' (Scheme 7) by aerobic oxidation in a basic biphasic (organic/aqueous) medium using Aliquat 336 as the phase transfer agent. Each time, the disappearance of the starting compound TFP1' was monitored by TLC to ensure completion of the reaction. The partially oxidized symmetric porphyrin TFOP2' was isolated in 37% as a dark red powder after chromatographic purification of the crude reaction mixture. These compounds were obtained after running the reaction in pure DMF for 59 h, while the symmetrical A4 porphyrin TFOP2 was obtained similarly in 38%, after running the reaction in a DMF/THF (1:1) mixture for 72 h. During the formation of TFOP2', we noticed that the reaction medium contained many insolubles at the start of the reaction, whereas it is totally homogeneous in the DMF/THF solvent mixture. We believe that the formation of partially oxidized intermediates is favored when the synthesis of TFOP2 is attempted in DMF, because this solvent does not dissolve all the precursor TFP1' at the start of the reaction and thus will contribute to slow down the overall oxidation reaction. As a result, only TFOP2' could be separated by chromatography from the reaction mixture after 59 h in DMF, while other partially oxidized intermediates that must also have been formed were not isolated. The purity of the various porphyrin sample is established by combined mass spectrometry and elemental analysis. The new aldehyde 3 and porphyrins TFP1', TFOP2' and TFOP2 were characterized by 1 H NMR (see ESI). Thus, for the various porphyrin derivatives, in addition to the (i) β-pyrrolic protons of the porphyrin core which come out as a singlet around 9 ppm and (ii) the NH protons of porphyrin cavity which come out as a second singlet around -2.5 ppm, some of the diagnostic features of the aldehyde 3 are retrieved, i.e. (iii) aromatic protons located as multiplets in region 8.0-7.3 ppm and (iv) alkyl protons of the various butyl chains around 2.2-0.5 ppm. In addition, for TFP1' and TFOP2', the methylene protons of nonsubstituted fluorenyl groups are also observed as diagnostic singlets at 4.1 ppm. These signals progressively disappear, upon proceeding from TFP1' to TFOP2. The 1 H NMR spectrum of the compound TFOP2' appears as a one-to-one superposition of the two previous spectra in the aromatic region. The cis (5,10-isomer) or trans (5,15-isomer) nature of the TFOP2' isomer isolated could not be ascertained from available data.

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Photophysical properties

In order to investigate the applied potential of these new porphyrin derivatives, we examined the absorption and emission behaviors of TFP1' and TFOP2-2' (Table 1). Their capability to photosensitize oxygen and, finally, their 2PA properties will also be subsequently measured (Table 2). During these studies, TFP, TEFP (Scheme 1), TFP-Bu and extended TFP1 [8a] (Scheme 3) were chosen as references compound to help analyzing the influence of the fluorenone groups at periphery of these star-shaped derivatives.

Absorption spectra

The UV-visible absorption spectra of the new para-substituted porphyrins TFP1' and TFOP2-2' are typical for these free base porphyrins (Figure 1) with (i) an intense Soret-band around 430 nm and four Q-bands from 520-650 nm, and (ii) an extra absorption, around 310-400 nm, which corresponds to *← transitions in the conjugated arms. [7,8] This UV extra absorption, largely fluorenyl-based, is absent for TEFP, possibly because the less-conjugated meso-fluorenyl groups of TEFP absorb around 290 nm, whereas those of the porphyrins TFP1' and TFOP2-2', conjugated with the porphyrin core through the ethynyl bridges, are strongly red-shifted (330-350 nm) and more intense. [8a] Related differences can also be observed between TFP1' (or TFOP2-2') and TFP. [8] Table 1. Photophysical properties data of the new series of fluorenone porphyrins: reference After normalizing the spectra on the Soret-band intensity, the dendron-based absorption clearly separates in two parts when going from TFP1' to TFOP2, i.e. with increasing fluorenone units (Figure 1), while the porphyrin-based transitions (Soret-band and Q-bands) remain constant. It can also be noticed that the intensity of the first dendron-based absorption gets weaker and larger with the increasing number of fluorenone units, suggesting that fluorenone sub-chromophores in TFOP2 and TFOP2' behave as weaker absorbers than do terminal fluorene units.

Emission spectra

Upon excitation in their Soret-band, TFP1', TFOP2-2' and the reference compound TEFP exhibit the characteristic porphyrin emission peaks Q(0,0) and Q(0,1). [7,8] After normalizing the emission intensities of the various compounds on their Q(0,0) peaks, all four compounds exhibit similar emission spectra (Figure 2a). Then, for TFP1' and TFOP2-2', the intensity ratios between Q(0,0) and Q(0,1) remain constant, as previously observed for the porphyrincored dendrimers. [7,8] All three compounds TFP1' and TFOP2-2' show slightly lower quantum yields (ΦF ~22%) than TFP-cored porphyrin dendrimers TFP1-3 (~24%). [8a] However, when compared to the TPP-cored porphyrins TPP1-3 (~20%), [7] their luminescence TEFP TFP1' TFOP2' TFOP2

quantum yields are slightly higher of ca. 2%, in contrast to the tetra-alkynyl porphyrin TEFP (Table1). Energy Transfer from conjugated arms to porphyrin core

The existence of an energy transfer (ET) between the peripheral arms and the central porphyrin core was also probed. Figure 2b presents the emission spectra of TFP1' and TFOP2-2' upon UV excitation in the arm-based absorption (~380 nm). The resulting emission spectra show only the red emission (at 660 and 725 nm), characteristic of the porphyrin core, and no residual blue emission, characteristic of the arm, meaning that the dendron emission is completely quenched through an efficient process most likely corresponding to a through-bond ET (TBET) [15] from the peripheral fluorenone moieties to the porphyrin core via the fluorene perpendicular connectors.

Oxygen Sensitization

We next wondered about the oxygen-sensitizing capabilities of TFP1' and TFOP2-2'. The quantum yields of singlet oxygen generation were therefore determined for these compounds and compared to those of TFP1 [8a] (Table 2). All the new compounds exhibit values (~ 65%) higher than that of TFP1 ( = 62%). Interestingly, these measurements reveal that the high fluorescence efficiency of the porphyrins (especially in comparison with model TPP) is not obtained at the expense of the singlet oxygen production.

Two-Photon Absorption

Given the important 2PA cross-sections previously evidenced TPP1 and TFP1 (Schemes 2 and 3), [7,8a] we finally turned our attention to the 2PA properties of TFP1' and TFOP2-2'

(which exhibit quite similar two-photon absorptivities). Taking advantage of their good fluorescence, the 2PA cross-sections of these TFP-cored compounds were determined by twophoton excited fluorescence (TPEF). Measurements were performed with 10 -4 M solutions, using a mode-locked Ti:sapphire laser delivering femtosecond pulses, following the experimental protocol described by Xu and Webb. [16] A fully quadratic dependence of the fluorescence intensity on the excitation power was observed for each sample at all the wavelengths of the spectra shown in Figure 3, indicating that the cross-sections determined are only due to 2PA. A significant increase of the 2PA cross-sections compared to that of TPP (12 GM at 790 nm) was observed for all the new porphyrins (Table 2). We have shown earlier that replacing the four phenyl groups at meso-positions by four fluorenyl groups leads to a clear improvement in the 2PA properties. [7,8] When comparing

TFP1 TFP1' TFOP2' TFOP2

TFP-cored porphyrins together, it appears that butyl chains on the meso-fluorenyl groups of the porphyrin in TFP-Bu (140 GM) favor TPA over bare fluorenyl arms in TFP (90 GM).

Similarly, 2-fluorenone units appears more efficient for promoting 2PA than bare 2ethynylfluorenyl groups at 7-positions of meso-fluorenyl groups in TFP1' (530 GM). Thus, a TFP-Bu core decorated with such units (TFP1') is a worst two-photon absorber than TFOP2-2' (610-640 GM) or TFP1 (770 GM).

Conclusions

The synthesis and characterization of two conjugated meso-tetrafluorenylporphyrin-cored compounds peripherally functionalized with additional ethynyl-2-fluorenyl/fluorenon-2-yl units (TFOP2-2') has been reported here. These new starshaped derivatives were easily obtained by aerobic oxidation of the non-oxidized porphyrin precursor TFP1'. In terms of photophysical properties, all three new porphyrins exhibit remarkably high luminescence quantum yields (ΦF ~21-22%) for meso-tetraarylporphyrins and present also a potentially large brightness, thanks to the very efficient energy transfer from the peripheral arms units toward the central porphyrin core (TBET). While these high luminescence quantum yields make them potentially interesting for organic light emitting devices, these compounds behave also as efficient photosensitizers for oxygen, with quantum yields of singlet oxygen generation around 65% i.e. higher to that previously reported for TFP1 ( = 62%). Interestingly, their high fluorescence quantum yields (especially in comparison with the TPP model compound) are not obtained at the expense of the singlet oxygen production. Finally, their 2PA cross-sections of TFP1' and TFOP2-2', when compared to that of TFP (90 GM), are much larger (~600 GM) pointing to the beneficial impact of increasing the size of the -manifold in the peripheral arms. Actually, TFOP2 and TFOP2' exhibit quite similar two-photon absorptivities (640-610 GM), which are significantly higher than for the non-oxidized compound TFP1' (530 GM), in line with a beneficial role of 2-fluorene for 2-fluorenone replacement on nonlinear absorption properties.

Thus, combined to their remarkable fluorescence quantum yields and oxygen-photosensitizing properties, their rather large 2PA cross-sections make them potentially interesting for applications in theranostics. After proper functionalization, this type of structure should, likewise to related systems recently developed for such uses, [10b,11] give rise to original porphyrin-based molecular assemblies combining two-photon PDT and two-photon fluorescence imaging. In connection with previous results, [11b] the replacement of peripheral 9,9-dibutyl-9H-fluoren-2-yl units by units by 9H-fluoren-9-one-2-yl units in the corresponding water-soluble star-shaped analogues should therefore not drastically affect their 2PA cross-section, nor their relevant linear optical properties. However, other important properties such as their solubility, self-aggregation or bio-distribution might be more profoundly modified. As such, oxidation of the peripheral 2-fluorenyl units should be envisioned as a potential mean to improve the overall efficiency of two-photon photosensitizers related to TFP1' or TFP1.

Experimental Section

General Unless otherwise stated, all solvents used in reactions were distilled using common purification protocols, [START_REF] Perrin | Purification of Laboratory Chemicals[END_REF] except DMF and i Pr2NH which were dried on molecular sieves ( 3Å). Compounds were purified by chromatography on silica gel using different mixtures of eluents as specified. 1 H and 13 C NMR spectra were recorded on BRUKER Ascend 400 and 500 at 298 K. The chemical shifts are referenced to internal tetramethylsilane. High-resolution mass spectra were recorded on different spectrometers: a Bruker MicrOTOF-Q II, a Thermo Fisher Scientific Q-Exactive in ESI positive mode and a Bruker Ultraflex III MALDI Spectrometer at CRMPO (centre regional de mesures physiques de l'Ouest) in Rennes.

Reagents were purchased from commercial suppliers and used as received. Compounds 7bromo-9,9-dibutyl-fluorene-2-carboxaldehyde (1) [8a] and 2-ethynyl-fluorene (2), [7b] were synthesized as described earlier, using an adapted approach for 2 (ESI).

Synthesis of organic precursors

Difluorenyl aldehyde 3. In a Schlenk tube, a mixture of 7-bromo-9,9-dibutylfluorene-2-carboxaldehyde (1) (492 mg, 1.28 mmol, 1 equiv), 2-ethynyl-fluorene (2) (365 mg, 1.92 mmol, 1.5 equiv), Pd(PPh3)2Cl2 (5.4 mg, 0.008 mmol, 0.6% equiv.) and CuI (excess)

were dissolved in DMF (5 mL) and then i Pr2NH (5 mL) was added under argon. The reaction medium was degassed by freeze-pump-thaw twice and heated for 48h at 95 o C. After evaporation of the volatiles, residue was further purified by silica chromatography using heptane as eluent; the desired aldehyde 3 (398 mg, 63% yield) was obtained as a yellow powder. 1 H NMR (400 MHz, CDCl3): δ = 10.07 (s, 1H, HCHO), 7.89-7.76 (m, 7H, Hfluorenyl), 7.63-7.57 (m, 3H, Hfluorenyl), 7.43-7.31 (m, 3H, Hfluorenyl), 3.94 (s, 2H, H9-fluorenyl), 2.10-1.99 (m, 4H, Ha), 1.16-1.04 (m, 4H, Hc), 0.70-0.44 (m, 10H, Hb, Hd). 13 C NMR (100 MHz, CDCl3, ppm): δ = 186. 5, 161.0, 157.6, 156.2, 155.2, 150.9, 150.6, 149.0, 143.1, 139.5, 139.1, 138.3, 137.8, 133.6, 131.2, 130.7, 128.3, 105.1, 99.5, 99.4, 41.5, 40.4, 33.2, 23.9, 11.1. HRMS-ESI for C37H35O: m/z = 495.2680 [M+H] + (calcd: 495.26824).

Porphyrin TFP1'. In a two-neck flask, a mixture of previously prepared difluorenyl aldehyde 3 (564 mg, 1.14 mmol, 1 equiv.) and pyrrole (0.08 mL, 1.14 mmol, 1 equiv.) were dissolved in distilled chloroform (250 mL) under argon. After degassing the mixture with argon bubbling for 30 min, BF3•OEt2 (0.04 mL, 0.29 mmol, 0.25 equiv.) was injected and the reaction was stirred in dark for 3 h under argon at room temperature. Then oxidant p-chloranil (210 mg, 0.86 mmol, 0.75 equiv.) was added, and the reaction was heated at 45 o C for another 1 h without any protection. After cooling the reaction to room temperature, NEt3 (2 mL) was injected, and then keep stirring for several minutes. After evaporation of the volatiles, purification was done by silica gel chromatography using THF/heptane (1:4) mixture as eluents, the porphyrin TFP1' was collected as violet powder (110 mg, 18% yield). MP: 216-218°C (dec). Rf: 0.72 (THF/Heptane [1:1]). 2, 149.4, 143.7, 143.4, 141.9, 141.5, 141.3, 141.0, 140.4, 133.5, 130.7, 130.2, 129.3, 128.6, 127.9, 127.0, 126.7, 125.9, 124.8, 122.5, 121.6, 120.6, 120.1, 119.9, 119.7, 118.2, 90.4, 90.2, 55.2, 40.1, 36.2, 29.6, 26.4, 13.4

Spectroscopic Measurements

All photophysical measurements have been performed with freshly-prepared air-equilibrated solutions at room temperature (298 K). UV-Vis absorption spectra were recorded on a Jasco V-570 spectrophotometer. Steady-state fluorescence measurements were performed on dilute solutions (ca. 10 6 M, optical density < 0.1) contained in standard 1 cm quartz cuvettes using an Edinburgh Instrument (FLS920) spectrometer in photon-counting mode. Fully corrected emission spectra were obtained, for each compound, after excitation at the wavelength of the absorption maximum, with Aex < 0.1 to minimize internal absorption.

Measurements of singlet oxygen quantum yield ()

Measurements were performed on a Fluorolog-3 (Horiba Jobin Yvon), using a 450 W Xenon lamp. The emission at 1272 nm was detected using a liquid nitrogen-cooled Ge-detector model (EO-817L, North Coast Scientific Co). Singlet oxygen quantum yields  were determined in dichloromethane solutions, using tetraphenylporphyrin (TPP) in dichloromethane as reference solution ([TPP] = 0.60) and were estimated from 1 O2 luminescence at 1272 nm.

Two-Photon Absorption Experiments

To span the 790-920 nm range, a Nd:YLF-pumped Ti:sapphire oscillator (Chameleon Ultra, TPEF cross sections of 10 -4 M dichloromethane solutions were measured relative to fluorescein in 0.01 M aqueous NaOH using the well-established method described by Xu and

Webb [16] and the appropriate solvent-related refractive index corrections. [START_REF] Werts | [END_REF] The quadratic dependence of the fluorescence intensity on the excitation power was checked for each sample and all wavelengths.

Supporting Informations. Synthetic details, 1 H NMR and mass spectra of all new compounds. This material is available free of charge via the Internet at http://pubs.acs.org.

Scheme 2 .

 2 Scheme 2. Molecular structures of TPP-cored porphyrin dendrimers TPP1-TPP3 and their reference TPP.

  to explore the impact of the replacement of the four terminal 9H-fluoren-2-yl (2-fluorenyl) groups by 9H-fluoren-9-one-2-yl (2-fluorenonyl) ones on the optical properties of interest (Scheme 4).

Scheme 4 .

 4 Scheme 4. Molecular structures of the targeted molecule TFOP2 vs. the reference compound TFP1'.

Scheme 6 .

 6 Scheme 6. Synthesis of non-oxidized porphyrin TFP1' under Lindsey conditions.

  Scheme 7. Synthesis of TFOP2 and TFOP2' from precursor TFP1'.

Figure 1 .

 1 Figure 1. UV-visible spectra of porphyrins TFP1', TFOP2-2' and reference TEFP in CH2Cl2.

Figure 2 .

 2 Figure 2. a) Normalized emission spectra of TEFP, TFP1' and TFOP2-2' in CH2Cl2 (500-820 nm) after excitation in their Soret band; b) Complete normalized emission spectra of TFP1' and TFOP2-2' excited in their arm-based absorption (380 nm). Corresponding non-normalized spectra are provided in the ESI.

Figure 3 .

 3 Figure 3. Two-Photon Absorption Spectra for TFP-cored compounds TFP1' and TFOP2-2' compared to TFP1 in CH2Cl2.

  Coherent) was used generating 140 fs pulses at a 80 MHz rate. The excitation power is controlled using neutral density filters of varying optical density mounted in a computercontrolled filter wheel. After five-fold expansion through two achromatic doublets, the laser beam is focused by a microscope objective (10×, NA 0.25, Olympus, Japan) into a standard 1 cm absorption cuvette containing the sample. The applied average laser power arriving at the sample is typically between 0.5 and 40 mW, leading to a time-averaged light flux in the focal volume on the order of 0.1-10 mW/mm 2 . The fluorescence from the sample is collected in epifluorescence mode, through the microscope objective, and reflected by a dichroic mirror (Chroma Technology Corporation, USA; ''blue'' filter set: 675dcxru; ''red'' filter set: 780dxcrr). This makes it possible to avoid the inner filter effects related to the high dye concentrations used(10 -4 M) by focusing the laser near the cuvette window. Residual excitation light is removed using a barrier filter (Chroma Technology; ''blue'': e650-2p, ''red'': e750sp-2p). The fluorescence is coupled into a 600 µm multimode fiber by an achromatic doublet. The fiber is connected to a compact CCD-based spectrometer (BTC112-E, B&WTek, USA), which measures the two-photon excited emission spectrum. The emission spectra are corrected for the wavelength-dependence of the detection efficiency using correction factors established through the measurement of reference compounds having known fluorescence emission spectra. Briefly, the set-up allows for the recording of corrected fluorescence emission spectra under multiphoton excitation at variable excitation power and wavelength. TPA cross sections (σ2) were determined from the two-photon excited fluorescence (TPEF) cross sections (σ2.ΦF) and the fluorescence emission quantum yield (ΦF).

Table 2 .

 2 Photophysical properties of TFP-cored porphyrins and relevant reference compounds.

	Compound	abs (Soret)		em	F a 	F b 	 c	2 d
		(nm)	(M -1 cm -1 )	(nm)				(GM)
	TFP	425	_	659, 724	0.24	0.24	0.60	90
	TFP-Bu	426	_	660, 724	0.18	0.20	0.64	140
	TFP1	432	669 000	660, 726	0.23	0.24	0.62	770
	TFP1'	431	477 000	660, 726	0.21	0.22	0.66	530
	TFOP2'	431	578 000	660, 726	0.21	0.21	0.65	640
	TFOP2	431	509 000	660, 726	0.21	0.22	0.64	610

a Fluorescence quantum yield in dichloromethane determined relative to TPP in toluene.

b

Fluorescence quantum yield in toluene.

c

Singlet oxygen formation quantum yield determined relative to tetraphenylporphyrin in dichloromethane (Δ[TPP] = 0.60). d Intrinsic 2PA cross-sections at 790 nm in dichloromethane, measured by TPEF in the femtosecond regime; a fully quadratic dependence of the fluorescence intensity on the excitation power is observed and 2PA responses are fully non-resonant (exc ≥ 790 nm).

Acknowledgements

The authors acknowledge CNRS for their financial support and China Scholarship Council (CSC) for PhD funding (XZ and ZS). This project was supported by the departmental committees CD35, CD28 and CD29 of the "Ligue contre le Cancer du Grand-Ouest". We also thank Guillaume Clermont (ISM) for his help in the two-photon and singlet oxygen measurements.

Conflict of Interest


Li-Ping Zong

Ling-Yu Ruan

Junji Li
email: junjili@njust.edu.cn

Robert S Marks

Jun-Song Wang

Serge Cosnier

Xue-Ji Zhang

Dan Shan
email: danshan@njust.edu.cn

Electrochemiluminescence for in situ Detection of H2O2 Released from Hela Cells

Keywords: 1, 10-phenanthroline-2, 9-dicarboxylic acid (PDA), enzyme mimetic, electrochemiluminescence (ECL), hydrogen peroxide, Hela cells

des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Fe-MOGs-based enzyme mimetic and its mediated electrochemiluminescence for in situ detection of H2O2 released from Hela cells

Li-Ping Zong, Ling-Yu Ruan, Junji Li, Robert S Marks, Jun-Song Wang, Serge Cosnier, Xue-Ji Zhang, Dan Shan

To cite this version:

Li-Ping Zong, Ling-Yu Ruan, Junji Li, Robert S Marks, Jun-Song Wang, et al.. Fe-MOGs-based enzyme mimetic and its mediated electrochemiluminescence for in situ detection of H2O2 released from Hela cells. Biosensors and Bioelectronics, 2021, 184, pp.113216. 10.1016/j.bios.2021.113216. hal-03382950

Introduction

Natural enzymes are exquisite biocatalysts that mediate every life process in an organism and are widely applicated in virous fields due to remarkable catalytic efficiency and substrate specificity (Harrigan et al. 2018;Piazza et al. 2018;Zhang 2017). But due to their inherent defects hindering the practical application of enzymes, such as low operational stability, high cost of preparation and purification, developing enzyme substitutes has remained an outstanding challenge.

Enzyme mimetics, nanomaterials with enzyme-like characteristics, have attracted broad interest into their application and mechanisms of action (Wei and Wang 2013).

The reactivity of enzyme mimetics bears clear resemblance to that of natural enzymes, and show advantages such as low cost, high stability and commercial accessibility (Wu et al. 2019). Several approaches have been undertaken to attain high reactivity of enzyme mimetics, such as controlling the size, morphology, and composition, forming hybrids, using external stimuli, etc. (Bete et al. 2019;Chen et al. 2018;Ding et al. 2019;Hu et al. 2017;Kluenker et al. 2017;Liu et al. 2019;Singh et al. 2017;Sun et al. 2013).

With the in-depth study of the physicochemical properties and catalytic mechanism of natural enzymes, simulating the structure of active center of enzyme can be a promising method (Bete et al. 2019;Wang et al. 2019a).

Similar to metal organic frameworks (MOFs), metal organic gels (MOGs) are one kind of supramolecular assemblies driven by metal-ligand interactions. MOGs feature tunable-soft porosity, open metal sites for catalysis and rapid mass transfer or permeability, rendering convenience in imitating the active center of natural enzymes.

MOGs can be synthesized under mild conditions (e.g., room temperature, atmospheric pressure, neutral condition, short reaction time, routine solvent, etc.). Jiao et al. used potassium chloride (KCl) as template, dimethylimidazole (MI) as ligand to obtain enzyme mimetic. Compared with other metals Zn, Co, less electron transfer was observed between Fe atom and the adsorbed hydroxyl group, which is beneficial to reduce the energy barrier for the formation of hydroxyl radicals, thereby improving peroxidase-like activity (Jiao et al. 2020). The active center of horseradish peroxidase (HRP) is the axial coordination of heme iron with a histidine residue at the N-terminus.

Herein, we took HRP as the simulation object. To mimic the planar Fe-N structure of HRP, 1,10-phenanthroline-2,9-dicarboxylic acid (PDA) was chosen as ligand to coordinate with iron. As an organic ligand rich in aromatic nitrogen heterocycles, PDA is hallmarked by general chelating and bridging capabilities and a large conjugated aromatic plane resulting from a synergistic combination of donor groups with multichelating sites (Miao et al. 2015;Mirzaei et al. 2019). These properties contribute to construct metal clusters with high preorganization and thermodynamic stability.

In this work, a kind of two-dimensional sheet-like Fe-MOGs were synthesized by a one-step rapid reaction at room temperature. Structural information and electrochemical performance were systematically evaluated to reveal structure-activity relationship of the enzyme mimetic. The resultant Fe-MOGs possessed Fe-NX catalytic sites similar to HRP, and exhibited exceptional activity for the decomposition of H2O2.

A Fe-MOGs based luminol-H2O2 ECL platform was further applied for in-situ detection of H2O2 generated from Hela cells. The excellent performance of the proposed method indicated promising application of MOGs in the medical detection and monitoring of biological small molecules.

Experimental section

Materials and Reagents

All reagents were commercially available and used without further pretreatments or purifications.

Apparatus

Ultraviolet-vis (UV-Vis) spectra were recorded on the Shimadzu UV-3600 spectrophotometer. Fourier-transformation infrared (FT-IR) spectra were obtained with an IR-Prestige-21 FT-IR spectrometer (Shimadzu Co., Japan). Photoluminescence (PL) spectra were recorded by an Edinburgh FLS920 fluorescence spectrometer (Livingston, UK). The morphology was investigated with a XL-30E scanning electron microscope (SEM) and a Tecnai G2 20 LaB6 high resolution transmission electron microscopy (HRTEM, FEI Co., USA). The X-ray photoelectron spectroscopy (XPS) experiments were carried out on K-Alpha (Thermo Fisher Scientific Co., USA). Cyclic voltammetry (CV) measurements were carried out on a CHI 660D electrochemical workstation (Chenhua, China) at room temperature. ECL studies were performed on an MPI-EII multifunctional electrochemical and chemiluminescent analytical system (Xi'an Remex Analytical Instrument Co., Ltd, China), and the photomultiplier tube was set at 1000 V, the magnification level was 3. An Autolab PGSTAT30 (Eco Chemie) which was controlled by NOVA 1.10 software was applied for Electrochemical impedance spectroscopy (EIS) measurement. All electrochemical studies were performed with a conventional three-electrode system, a bare or modified glassy carbon electrode (GCE, 5 mm in diameter) as working electrode, a saturated calomel electrode as reference electrode and a Pt wire as counter electrode.

Methods

Synthesis of the Fe-MOGs

The detailed preparation process of Fe-MOGs was according to the literature with a slight modification (He et al. 2017). In a typical one-step synthesis, 10 mM Fe(OAc)2 (10.0 mL) aqueous solution was added dropwise into the DMSO solution of 10 mM PDA (10.0 mL). The above mixed light yellow solution was stirred vigorously for 30 s at room temperature. The light yellow powder denoted as Fe-MOGs was then collected by centrifugation (8000 rpm), washed with ethanol three times and freeze-dried overnight in a vacuum.

Peroxidase activity assay of the Fe-MOGs

The GCE was respectively polished with 0.3 and 0.05 μm alumina powder and rinsed by deionized water for further use. For the cyclic voltammetry test, 10 μL of the Fe-MOGs solution (2 mg mL -1 ) and 10 μL of HRP in 0.01 M, pH 7.4 PBS solution (2 mg mL -1 ) was dripped on the surface of GCE and dried at 37 °C to obtain Fe-MOGs/GCE and HRP/GCE, respectively.

Hela cells culture and in vitro cytotoxicity

Hela cells were grown in Dulbecco's modified Eagle's medium (DMEM, 10%)

containing FBS (10%), L-glutamine (4 mM), penicillin (100 units mL -1 ), and streptomycin (100 mg mL -1 ) under oxidative stress conditions (1 μg mL -1 lipopolysaccharide (LPS)), and then incubated in a humidified atmosphere of 5% CO2 at 37 °C. Hela cells were collected and suspended into sterilized PBS solution (0.01 M, pH 7.4) with specific concentration (cells mL -1 ).

To investigate in vitro cytotoxicity of the Fe-MOGs, CCK-8 assay for Hela cells was performed. 100 μL of cells, at a density of 2 × 10 4 cells/well, were seeded into a 96well plate, and incubated for 24 h at 37 °C in a humidified 5% CO2-containing atmosphere. Then various concentrations of mixed Fe-MOGs/luminol were added. The cells were incubated for an additional 24 h at 37 °C. 10 μL of CCK-8 solution was then added and incubated for 0.5 h. Untreated cells were used as controls. The absorbance was measured at a wavelength of 450 nm.

In situ detection for H2O2 released from Hela cells

For ECL detection of H2O2, 15 μL 2 mg mL -1 Fe-MOGs aqueous solution and 15 μL 0.01 M luminol in 0.1 M NaOH aqueous solution were pre-mixed prior to modifying the GCE and then dried at 37 °C to obtain Fe-MOGs/luminol/GCE. Different concentrations of H2O2 were added into 4 mL of PBS working buffer. For ECL detection of Hela cells, different amounts of Hela cells and chitosan aqueous solution (0.1% wt) were pre-mixed and dripped on the Fe-MOGs/luminol/GCE to obtain Hela cells/chitosan/Fe-MOGs/luminol/GCE. Hela cells were adhered on the surface of Fe-MOGs/luminol/GCE by chitosan aqueous solution. ECL detection was studied at a scan rate of 30 mV s -1 within the scan range from 0 to 0.8 V.

Results and discussions

Synthesis and characterization of the Fe-MOGs

The procedures for the enzyme mimetic synthesis and the ECL immunosensor fabrication were illustrated in Scheme 1. In order to obtain the Fe-NX structure containing HRP-like enzyme mimetic, Fe(OAc)2 and PDA were selected as the metal precursor and source of nitrogen. PDA can effectively chelate iron ions to achieve gel in a short time owing to the possible interactions between oxygen-containing groups, nitrogen-containing groups and metallic irons.

[Scheme 1]

In order to verify the synthesis a success or not, spectral data of the as-prepared sample were collected first. The UV-vis absorption spectra (Figure 1A) of the Fe-MOGs 1I) (Hou et al. 2020). Thus, on the basis of the preceding surveys, it is rational to come to the conclusion that the coordination between iron ions, bidentate nitrogen heterocycles can endow the Fe-MOGs a structure similar to the active center of natural HRP.

[Figure 1]

Electrochemical performance of the Fe-MOGs

Due to the similar Fe-NX active sites to the natural HRP, the Fe-MOGs with Fe-NX active sites are expected to possess the intrinsic peroxidase activity. It is well known that the active center of peroxidase such as HRP is located at the metal-N site.

Considering the presence of Fe-N sites in the structure, we suspected that the Fe-MOGs may have catalytic properties similar to those of peroxidase. The HRP-like activity of Fe-MOGs was confirmed via the reaction of 3, 5, 3', 5'-tetramethylbenzidine (TMB), and the absorbance band at 652 nm, originating from the oxidation product of TMB, is observed in the presence of Fe-MOGs and H2O2 (Figure S2A). Furthermore, the absorbance at 652 nm increases with the increasing concentration of Fe-MOGs (Figure S2B). Electrochemical behaviors of the Fe-MOGs were investigated by cyclic voltammetry at 25℃ in nitrogen saturated PBS solution (0.01 M, pH 7.4). As shown in Figure 2A, the electrochemical response of the Fe-MOGs (curve e) exhibited a redox peak attributed to the redox reaction of Fe(Ⅱ)/Fe(Ⅲ). Electric potential of anode peak and cathodic peak (Epa, Epc) are -0.053 V, -0.176 V at the scan rate of 10 mV s -1 , respectively. The anode peak and cathodic peak currents (Ipa, Ipc) vary linearly with the square root of scan rate (Figure S3), indicating characteristic behavior of diffusion controlled redox species. The linear regression equation is Ipa (μA) = 0.35ν 1/2 + 3.05, R 2 = 0.9344; Ipc (μA) = -0.26ν 1/2 -1.52, R 2 = 0.9969, respectively. Electrocatalytic properties of the Fe-MOGs were characterized by the comparative CV curves of Fe-MOGs/GCE in 0.01 M PBS solution containing different concentration of H2O2 (Figure 2B). The reduction peak current increases with the increase of H2O2 concentration, which is consistent with that of HRP (Figure S4), reflecting the HRP-like decomposition activity of Fe-MOGs on H2O2. And it can be implied that the catalytically active site Fe-NX may participate in the electron transfer reaction by changing the valence state of the center iron ions. Figure S5A shows the current-time recordings of Fe-MOGs modified GCE to the successive addition of H2O2 at the potential of -0.3 V in 0.01 M, pH 7.4 PBS at room temperature. In addition, Michaelis-Menten kinetics of Fe-MOGs were investigated. The values obtained for the Michaelis-Menten constant (Km) and maximum initial velocity (Vmax) from the Lineweaver-Burk plot (Figure S5B inset) are 217.9 μM and 2.818 μM s -1 , respectively. The Km value is much lower than that of HRP, 437.6 μM (Jiang et al. 2018a), indicating that a lower concentration of H2O2 was needed to reach the maximal catalytic activity of Fe-MOGs.

[Figure 2]

Mechanism research of ECL signal amplification

In the light of the mimic peroxidase activity of the Fe-MOGs, it is highly possible to mediate enhanced ECL emission in the luminol-H2O2 system. To study the role of Fe-MOGs in the luminol-H2O2 system, the ECL behaviors of Fe-MOGs/luminol/GCE and luminol/GCE were investigated in 4.0 mL of PBS solution (0.01 M, pH 7.4) containing 15 μM H2O2 respectively. It is shown in Figure 3A system. In this case, 0 V was selected as initial potential to achieve a strong ECL intensity in a comparatively narrow potential scanning window.

In order to reveal the mechanism of ECL signal amplification, comparative cyclic

𝐹𝑒 2+ +• 𝑂𝐻 → 𝐹𝑒 3+ + 𝑂𝐻 - (7) 𝐹𝑒 3+ + 𝐻 2 𝑂 2 → 𝐹𝑒 2+ + 𝐻𝑂 2 • + 𝐻 + (8) 𝐻 2 𝑂 2 +• 𝑂𝐻 → 𝐻𝑂 2 • + 𝐻 2 𝑂 (9) ECL emission Ⅱ: 𝐿 •-+ 𝐻𝑂 2 • → 𝐻𝐿𝑂 2 2- (10) 𝐻𝐿𝑂 2 2-→ 𝐴𝑃 2- * + 𝑁 2 (11) 𝐴𝑃 2- * → 𝐴𝑃 2-+ ℎ𝑣 (12)
First, luminol anions (LH -) are electrochemically oxidized to luminol anionic radicals (L •-). In Route I [Eq. ( 2)], H2O2 is reduced by the FeNX active sites to produce

•OH, and it is the decisive step. In Route II [Eq. ( 6)], followed by the oxidization of Fe 2+ upon anodic potential scanning, H2O2 is catalyzed by the pre-oxidized FeNX active sites to generate HO2 • . Finally, rapidly accumulated ROS react with L •-to form excitedstate 3-aminophthalate anion (AP 2-*), thus achieving enhanced ECL emission.

Biosensor for in situ detection of H2O2 released from Hela cells

H2O2 mediated inhibition of protein phosphatases contributes to oncogene overexpression, a hallmark of many cancers (Fruehauf and Meyskens 2007). The excessive levels of H2O2 cause oxidative damage to cellular protein, nucleic acids, and lipid molecules, thereby contributing to aging and age-related diseases including cancer.

H2O2 content at the cellular level diverses slightly from cancer cell to normal cell. The number of extracellular released H2O2 molecules is around 10 11 per cell (Guo et al. 2010).

Therefore, developing highly sensitive biosensor for trace detection of H2O2 at the cell level is of great reference significance for early cancer screening and population mixed sample detection. Relationship between ECL intensity and H2O2 concentrations was determined in the next step for further fabrication of Hela cell biosensor. The material capacity of electrode and the luminol concentration have been optimized. A capacity of 0.15 μg cm -2 Fe-MOGs demonstrates the best ECL performance and the optimized luminol concentration turns out to be 5 mM (Figure S9). As illustrated in Figure 3C, the ECL peak intensity is proportional to the concentration of H2O2 (0.01, 0.05, 0.1, 0.5, , 5, 10, 15, 20, 25, 30, 35, 40 μM), and two good linear segments were observed. As shown in Figure 3D and 3E, I (a.u.) = 188.89 + 788.77C (0.01-1 μM), R 2 = 0.993 and I (a.u.) = 784.70 + 415.32C (1-40 μM), R 2 = 0.998, respectively. The limit of detection (LOD) was calculated to be 2.2 nM (signal-to-noise ratio of 3). As shown in Table 1, compared with most of the reported electrochemical (EC), fluorescent (FL) and ECL sensors for H2O2 detection, our proposed ECL sensor displays high sensitivity with a low LOD of 2.2 nM. But further work is still necessitated to improve the sensitivity and simplify the fabrication of the sensor.

1
Furthermore, ECL potential cycling stability and long-term stability of the asprepared sensor were tested. The ECL response of Fe-MOGs/luminol/GCE is observed with relative standard deviations (RSD) of 0.172% when tested in 0.01 M, pH 7.4 PBS containing 10 μM H2O2 upon 20 cycles of continuous cyclic scans between 0 ~ +0.8 V (Figure S10A), indicating the excellent ECL potential cycling stability for H2O2 assay.

As shown in Figure S10B, the ECL response of Fe-MOGs/luminol/GCE in 0.01 M, pH 7.4 PBS containing 15 μM H2O2 was detected at one-day intervals for 9 days, which only slightly decreases after five tests with RSD 3.8%, reflecting a good long-term stability of this sensor. Selectivity was also investigated in Figure S11, compared with 10 μM H2O2, the introduction of 1 mM Na + , Cl -, NO2 -, ascorbic acid (AA), glutamine (Gln) and glucose (Glu) had a negligible effect on the ECL signal. The ECL responses upon Ca 2+ was obviously fluctuated, which may be affected by the chelation of the metal with the ligand. Overall, the survey results indicate that the prepared ECL sensor has a considerate stability and selectivity for H2O2.

To study general applicability of the proposed sensor in real biological samples, ECL platform based on Fe-MOGs/luminol was exploited to evaluate the H2O2 generated from Hela cells. To investigate the biocompatibility of the biosensor based on the Fe-MOGs, CCK-8 assay of Hela cells that incubated with various concentrations of mixed Fe-MOGs/luminol was conducted. The initial concentrations of Fe-MOGs and luminol in the medium are 2 mg mL -2 and 10 mM, respectively. The concentration of the mixed Fe-MOGs/luminol was diluted 10, 40, 160, 640 and 2560 times, respectively. As shown in Figure S12A, Fe-MOGs/luminol displayed relatively low cytotoxicity for Hela cells, as the viability remained above 69% after 24 h incubation time (Shamsipur et al. 2018).

Hela cells dispersed in 0.01 M, pH 7.4 PBS were prepared in advance. Since chitosan film is not only favorable for cell immobilization but also increases the space between cells and the electrode (Liu et al. 2018) The ECL peak intensity is proportional to the number of Hela cells in a range of 4000 to 300000. The resulting calibration curve is illustrated in Figure 3F and can be represented as I (a.u.) = 1012.2 + 0.0555 n (10 4 ) with a square of correlation coefficient of 0.994. The limit of detection (LOD) was estimated to be 42 cells (signal-to-noise ratio of 3). According to the calibration equations obtained from the constructed ECL biosensor, and knowing the cell density, the electrode surface area, the concentration of released H2O2, and volume of electrolyte (4 mL), the amount of H2O2 released from Hela cells under the cell density of 2 × 10 5 / (π × 0.25 × 0.25) cells cm -2 was calculated to be around 1 × 10 -7 mol. The average number of extracellular H2O2 molecules released from per cell (N0) was calculated according to the Avogadro equation (n = N0/NA, NA = 6.02×10 23 mol -1 ) (Guo et al. 2010), which agreed with other reported methods, as shown in Table S1. Therefore, the analysis result can be used as an accurate representation of H2O2 production under oxidative stress.

[Figure 3]

[Table 1]

Conclusions

In summary, we present an efficient enzyme mimetic with excellent peroxidase-like activity derived from the inspiration of natural enzyme HRP. The Fe-MOGs possesses similar catalytic activity and planar Fe-NX active sites to HRP. We confirmed that the

(

  curve c ) shows an absorption at 230 nm standing for K-E combined band, which is caused by the conjugation effect of carboxyl group to benzene ring and an absorption at 254 nm standing for B band(Xu et al. 2019), demonstrating the existence of PDA in the prepared samples. A certain blue shift of Fe-MOGs/luminol (curve d) occurred in the absorption peaks at 290 nm compared with luminol (curve e) due to the interaction with the Fe-MOGs. Therefore, luminol was demonstrated to be successfully functionalized with the Fe-MOGs.In FTIR spectra (Figure1B), the C=N skeleton vibration of Fe-MOGs (1467 cm -1 ) (curve a) is blue-shifted relative to the C=N skeleton vibration of PDA (1448 cm -1 ) (curve b)(Mirzaei et al. 2019), which is most likely due to the increased electronegativity of pyridine N in PDA after donating a lone electron pair to iron ion, resulting in an enhanced polarity of the chemical bond between N and C on the pyridine ring. Also the attenuation of ν(C=O) absorption around 1719 cm -1 in the Fe-MOGs can be ascribed to deprotonation of carboxyl groups in the ligands, implying the complete deprotonation of the carboxylic groups in the PDA 2-ligands(Mirzaei et al. 2019;Ramezanpour et al. 2019). Fluorescent spectroscopic measurements were also conducted for more detailed structural information. As depicted in Figure1C, the Fe-MOGs (curve a) shows little fluorescent emission in comparation with PDA (curve b), which might be assigned to the decrease in the density of π electron clouds on the ring offset around the nitrogen atom due to the formation of Fe-N bonds even though the lone electron pair of N does not participate in conjugation.The morphological data were characterized by scanning electron microscopy (SEM) and transmission electron microscope (TEM). As shown in Figure1D, 1E and 1F, the observed morphology of the Fe-MOGs distributed in thin layer sheet form, which may be beneficial for the mass transfer of Fe-NX active sites HRP. The surface compositions and electronic states were further investigated by X-ray photo-electron spectroscopy (XPS), confirming the presence of C, N, O and Fe (FigureS1). As shown in Figure1G, the Fe 2p1/2 and 2p3/2 regions are fitted with Fe(Ⅱ), Fe(Ⅲ) and their respective satellite peaks. The two main peaks in the Fe 2p3/2 region have positions of 709.9 and 711.7 eV and can be assigned to Fe 2+ and Fe 3+ oxidation state respectively(Sun et al. 2019).These results indicate that iron on the Fe-MOGs is mainly in the Fe 2+ state, which is consistent with the light yellow colour of samples. The high-resolution N 1s spectrum of the Fe-MOGs displays three deconvoluted peaks ascribed to Fe-NX (399.5 eV), and pyridinic N (398.6 eV) as well as π-π* satellite (406.1 eV) (Figure1H)(Hou et al. 2020;Jiao et al. 2020;Lou et al. 2020), further demonstrating the formation of Fe-NX active sites. Fitted high-resolution C 1s spectrum evidences the presence of indefinite C-C (284.8 eV), C-N (285.7 eV), O-C=O (288.2 eV) (Figure

  that the Fe-MOGs/GCE (curve d) showed no ECL performance confirming luminol the only luminophore and Fe-MOGs/luminol/GCE (curve a) and luminol/GCE (curve b) both had a cathode ECL emission at about 0.5 V attributed to luminol. The ECL intensity in the presence of the Fe-MOGs reached around 7000 a.u., about two times the intensity in the absence of the Fe-MOGs, which achieved approximately 3000 a.u., while PDA had negligible influence on the ECL intensity (curve c). And in the inset of Figure3B, the decrease in the initial oxidation potential of Fe-MOGs/luminol (curve a) also illustrates that the Fe-MOGs can effectively enhance the ECL signal of luminol. On the other hand, in consideration of the redox potential of Fe(Ⅱ)/Fe(Ⅲ), the initial potential was optimized to improve accuracy of ECL detection. As shown in FigureS6A, in the absence of H2O2, the ECL of luminol/GCE under air (curve a) saturated atmosphere boosted compared with nitrogen saturated condition when the initial potential reached -0.4 V, which is the reduction potential of dissolved oxygen. While in the presence of 15 μM H2O2 in FigureS6B, the ECL of Fe-MOGs/luminol/GCE increased simultaneously under air (curve a) and nitrogen (curve b) saturated atmosphere as the initial potential decreased, implying that the Fe-MOGs are not sensitive to dissolved oxygen and H2O2 is the only available co-reactant source in Fe-MOGs/luminol-H2O2

  , chitosan (low viscosity: < 200 mPa. s) 0.1% wt aqueous solution was used as an intermediate binder for cell adhesion to the surface of the Fe-MOGs/luminol modified GCE. As shown in Figure S12B, the ECL response of Fe-MOGs/luminol/GCE modified with Hela cells coated with chitosan is approximately three times that of Fe-MOGs/luminol/GCE modified with Hela cells without chitosan. And the cell-free Fe-MOGs/luminol/GCE modified by chitosan has almost no ECL response, indicating that chitosan facilitates cell immobilization without interfering with ECL detection. EIS was applied for characterizing the successful immobilization of Hela cells onto the Fe-MOGs/luminol/GCE. The Nyquist plot of the Fe-MOGs/luminol and the chitosan-coated Hela cells modified Fe-MOGs/luminol were displayed in Figure S12C. Compared with the semicircular diameter obtained from Fe-MOGs/luminol (curve a), the semicircular diameter of chitosan-coated Hela cells modified Fe-MOGs/luminol is significantly increased (curve b), indicating the successful immobilization of chitosan-coated Hela cells onto Fe-MOGs/luminol. In addition, similar to the literature, LPS was used to stimulate the production of H2O2 in cells (Jiang et al. 2018b). Control experiments were conducted to confirm the selectivity to H2O2 in this practical situation. As shown in Figure S12D, no ECL response was observed in the control experiment of LPS/chitosan/Fe-MOGs/luminol/GCE without Hela cells (curve a), eliminating the interference of LPS itself to ECL response. The oxidative stress induced by LPS can be confirmed by comparing the ECL response of Hela cells with (curve c) and without (curve b) LPS treatment, indicating that inducedROS radicals (excluding H2O2) affected little on ECL, which is consistent with the reported literature(Jiang et al. 2018b). On the other hand, when catalase (a selective scavenger of H2O2) was added (curve e), the ECL response caused by LPS-treated Hela cells/chitosan/Fe-MOGs/luminol/GCE (curve d) decreased significantly, referring to the method from literature(Guo et al. 2010). It can be concluded that ECL response is mainly ascribed to the Fe-MOGs-catalyzed H2O2, which is released from Hela cells through LPS stimulation. Eventually, ECL responses of the as-fabricated biosensor to various concentrations of Hela cells were recorded, as illustrated in Figure3FInset.

Figure 1

 1 Figure 1 (A) UV-vis absorption spectra of (a) PDA, (b) Fe(Ac)2, (c) Fe-MOGs, (d) Fe-

Figure 2

 2 Figure 2 (A) CVs of bare GCE (a), PDA/GCE (b), Fe-MOGs/GCE (e) in 0.01 M, pH

Figure 3

 3 Figure 3 (A) ECL-time responses of (a) Fe-MOGs/luminol/GCE, (b)

Table 1 .

 1 Different Methods for H2O2 Detection

	Electrode material	Method	Linearity (μM)	LOD (nM)	Advantages	Disadvantages	ref
	Cu-MOF/SPE	EC	10-1000, 1400-6800	4100	High flexibility	Time-consuming fabrication	(Ling et al. 2019)
	PAN-PNMThH HRP	EC	5-60000	3200	Easy operation	Temperature and pH sensitive (Chen et al. 2016)
	PEP-Npb1-Cy3	FL	0-40	260	High specificity	Complex fabrication	(Zhao et al. 2018)
	Fe/N-C	ECL	0.001-0.3	0.93	High sensitivity	Dynamic range limited	(Tian et al. 2019)
					Fast electron transfer	Time-consuming fabrication
	L012@PAni-PAAm	ECL	0.01-50	2.9	High sensitivity	Complex fabrication	(Xiaojin et al. 2020)
	ABEI-Ag@PAni-ATMP	ECL	0.01-200	9.6	Excellent conductivity	Interference	(Xu et al. 2019)
	PMT array chip	ECL	1-1000	170	Visualized detection	Software dependent	(Qi et al. 2016)
	ABEI-Ag@PAni-PA	ECL	0.01-40	3.3	Good biocompatibility	Complex fabrication	(Jiang et al. 2018b)

Fe-MOGs/luminol ECL 0.01-40 2.2 High sensitivity Complex fabrication This work

Acknowledgements

This research was supported by the National Natural Science Foundation of China (Grant Nos. 21675086 and 62001224), the Natural Science Foundation of Jiangsu Province (No. BK20190457), the Fundamental Research Funds for the Central Universities (Nos. 30918012202 and 30919011213), "Overseas Academic Partnership Program" of Nanjing University of Technology (2019), and a project founded by the priority academic program development of Jiangsu Higher Education Institutions (PAPD). The authors wish also to acknowledge the support from the Sino-French international research network "New nanostructured materials and biomaterials for

voltammetry of Fe-MOGs/luminol modified GCE in 0.01 M, pH 7.4 PBS solution in the (a) absence and (b) presence of 15 μM H2O2 was conducted (Figure S7). An oxidization peak at about 0.49 V can be observed in both curves while stronger in the presence of 15 μM H2O2. It corresponds to the oxidation of luminol which can be ascribed to the reduction of H2O2 (Wang et al. 2019b). To further clarify the proposed mechanism, the active intermediates formed in the ECL system were determined. As shown in Figure S8, the effects of different reactive oxygen species (ROS) scavengers on the ECL signal of Fe-MOGs/luminol-H2O2 and luminol-H2O2 system were studied.

Isopropanol (IPA), benzoquinone (BQ) and L-Cysteine (L-Cys) were used to scavenge •OH, O2 •-and almost all ROS, respectively. The ECL intensity was obviously inhibited when 1.0 mM IPA and BQ was added to the electrolyte respectively and the ECL intensity was almost completely suppressed when 1.0 mM L-Cys was added, insinuating ROS like •OH and HO2 •-might be produced in the system. And in the presence of Fe-MOGs, IPA achieved stronger quenching effect, indicating more •OH was generated in Fe-MOGs/luminol-H2O2 system. On this basis, a possible mechanism for luminol-H2O2 ECL systems based on Fe-NX catalysts is described as follows.

𝐿𝐻 --𝑒 -→ 𝐿 •-+ 𝐻 +

(1)

Notes

The authors declare no competing financial interest.

renewable electrical energy sources" for providing facilities.