Enhanced Visual Wireless Electrochemiluminescence

Apr 16, 2019 - Finally, the resulting ECL of luminol and H2O2 at the anodic poles was monitored ... Materials and apparatus, synthesis of MIL-53(Fe)-N...
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Enhanced visual wireless electrochemiluminescence immunosensing of prostate-specific antigen based on the luminol loaded into MIL-53(Fe)-NH2 accelerator and hydrogen evolution reaction mediation Seyyed Mehdi Khoshfetrat, Hosein Khoshsafar, Abbas Afkhami, Masoud Ayatollahi Mehrgardi, and Hasan Bagheri Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.9b01506 • Publication Date (Web): 16 Apr 2019 Downloaded from http://pubs.acs.org on April 17, 2019

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Analytical Chemistry

Enhanced visual wireless electrochemiluminescence immunosensing of prostate-specific antigen based on the luminol loaded into MIL53(Fe)-NH2 accelerator and hydrogen evolution reaction mediation Seyyed Mehdi Khoshfetrata, Hosein Khoshsafarb, Abbas Afkhamic, Masoud A. Mehrgardid, Hasan Bagherie,* a

Biosensor Research Center, Endocrinology and Metabolism Molecular-Cellular Sciences Institute, Tehran University of Medical Sciences 14117-13137, Tehran, Iran. b Research and Development Department, Farin Behbood Tashkhis LTD, Tehran, Iran. c Faculty of Chemistry, Bu-Ali Sina University, Hamedan, Iran. d Department of Chemistry, University of Isfahan, Isfahan 81746-73441, Iran e Chemical Injuries Research Center, Systems Biology and Poisonings Institute, Baqiyatallah University of Medical Sciences, Tehran, Iran. E-mail addresses: [email protected], [email protected] ABSTRACT: A sensitive PSA detection method using a visual-readout closed bipolar electrode (BPE) system has been introduced by integration of hydrogen evolution reaction (HER) in cathodic pole and electrochemiluminescence (ECL) of luminol-loaded within the MIL-53(Fe)-NH2 (L@MIL-53(Fe)-NH2) in the anodic pole. The cathode of the BPE was electrochemically synthesized by 3D porous copper foam, followed by decorating with nitrogen-doped graphene nanosheet and ruthenium nanoparticles. The alternative, we employed carboxylate-modified magnetic nanoparticles (MNPs) for immobilization of the primary antibody (Ab1) and utilized the L@MIL-53(Fe)-NH2 conjugated to secondary antibody (Ab2) as a signaling probe and co-reaction accelerator. After sandwiching the target PSA between Ab1 and Ab2, the MNP/Ab1-PSA-Ab2/L@MIL-53(Fe) were introduced to a gold anodic BPE. Finally, the resulting ECL of luminol and H2O2 at the anodic poles is monitored using a photomultiplier tube (PMT) or digital camera. The PMT and visual (camera)-based detection showed a linear response from 1 pg mL-1 to 300 ng mL-1 (limit of detection 0.2 pg mL-1) and 5 pg mL-1 to 200 ng mL-1 (limit of detection 0.1 pg mL-1), respectively. This strategy provides an effective method for high-performance bioanalysis and opens a new door towards the development of the high sensitive and user-friendly device.

INTRODUCTION Sensitive and selective detection of low-abundance diseaserelated biomarkers provides a great promise for the early diagnosis of cancer.1 To achieve sensitive detection of cancer biomarkers, signal enhancing strategies such as applications of biocatalyst-enzymes as in classical enzyme-linked immunosorbent assay (ELISA)2-3 and metallic nanomaterials4, have been developed. Natural enzyme-based sensors suffer some intrinsic drawbacks such as susceptibility to enzyme denaturation, laborious separation, and time-consuming purification, as well as low stability and sensitivity of catalytic activity in temperature and pH conditions greatly restrained its applications. Among the various electrochemical5, 6-7 8-9 and colorimetric sensors, fluorescence electrochemiluminescence (ECL) immonosensors have garnered considerable attention owing to its remarkable advantages of high detection sensitivity, easy to control, low background intensity and no need for excitation light source1011 . In the classic luminescence system, luminophore was put in the detection solution12 instead of immobilizing on the

nanomaterials13 or electrode surface14. In contrast, immobilizing luminophore on nanomaterials plays a key role in ECL signal amplification of biosensors because of the catalytic properties of and high loading amount of ECL labels on the nanomaterials15. Luminol or its derivatives, N-(aminobutyl)-Nethylisoluminol (ABEI),-functionalized nanoparticles were also as labels for ECL DNA sensor16-17 and multiplexed genotyping of various single nucleotide polymorphisms (SNPs)18. Further signal amplifications were achieved using graphene’s19-20 or metal-organic framework’s (MOF’s) high-surface-area carriers21 and intercalation Dox-ABEI complex into tetrahedron DNA dendrimers22. However, weak π-π interactions luminol or its derivatives with graphene and conjugated only through covalent attachment of nitrogen atoms to nanoparticles leading to limit high loading capacity for luminol/ABEI23. Besides, large steric hindrances of tetrahedron DNA dendrimers cause to restrict the electron transfer. Alternatively, the tunable pore sizes, abundant large pore volumes, and open cavities of MOFs, a broad class of porous crystals with self-assembled from the organic ligand and metal ions, make them become an ideal

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candidate for MOF-based ECL21. In addition, different biomedical applications of MOFs have been suggested, including drug delivery24-25 and sensing26. Various methods to synthesize MOF-based ECL including27 i) post-synthetic anchoring of luminophore, ii) luminophore as a ligand and iii) encapsulation of luminophore into the pores of the MOFs have been developed. Post-synthetic anchoring of luminophore needs for additional fluorescent labels which is synthetically demanding, costly, time-consuming and providing a low efficient for luminophore immobilization. Furthermore, luminophore as a ligand limits the load capacity for luminophore due to its large steric hindrance23. Thanks to the merit of MOFs, the encapsulation methods could increase the high quantities of luminophore inside of MOFs, which leads to high-sensitivity detection of biomarkers and inorganic components. Chi et al.28 developed a sensitive fluorescent amine-capped carbon quantum dot into the ZIF-8 for detection of Cu2+. Ruthenium-based encapsulation in the pores of MOFs has been used for the ECL detection of Hg2+, NT-proBNP, and cocaine29-31. However, the large steric hindrance of Ru(bpy)32+ derivatives limited the loading amount of Ru(bpy)32+ derivatives into the MOF. To overcome the above shortcoming, Xiao group reported mesoporous zirconium-based MOF, PCN777, as a carrier to immobilize Ru(bpy)2(mcpbpy)2+ 32. Nevertheless, this single function for signal amplification might not meet the requirement for trace bioanalysis. One strategy that aims to address this limitation is to use a co-reaction accelerator which leads to higher concentration of active intermediates, therefore, boosted overall efficiency between luminophore and co-reactant for ECL signal amplification33-34. However, the function of separating luminophore and co-reaction accelerator decrease the efficiency of the accelerator. In light of the demand for highly sensitive detection, integration luminophore and coreaction accelerator within a nanostructure could help to circumvent this disadvantage35. Despite their generally advanced detection strategies and improved detection limits, the ECL-based measurements are made using external electronic readers and are not usable in resource-limited environments and emergency situations. In order to address these disadvantages, ECL approaches based on bipolar electrochemistry (BE) are involved in the construction of ECL biosensors36. In bipolar electrochemistry (BE), when a sufficient voltage is applied between the driving electrodes, electrochemical reactions generate at the end of anodic and cathodic poles of the bipolar electrode (BPE) in the wireless mode37. With the aim of improving the sensitivity of BPE-ECL, many efforts have been devoted to highly sensitive detection in BPE-ECL biosensors. These signal amplifications are mainly based on the anode and cathode amplification technologies on BPE. The amplification approaches for sensitive anodic detection involve the presence of a large number of luminophore onto the nanomaterials18, 38. On the other hand, some noble nanoparticles and molecular reporters have also been used into the cathodic poles of BPEs to reduce the potential difference across the electrode (ΔEelec)19, 39 . Open or closed bipolar configurations can be applied for BE. Compared with the open bipolar system, closed bipolar configuration has drawn more interests since the sensing and reporting poles are inserted in two separated solutions and the only current flow path is through BPE. Therefore, the closed BPE approach provides high current efficiency with higher sensitivities.

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Herein, a triple signal amplification strategy based on high current efficiency of closed BPE-ECL was achieved for ultrasensitive immunosensing of prostate specific antigen (PSA), using high loading amount of luminol inside of MIL53(Fe)-NH2 (L@MIL-53(Fe)-NH2) as a signaling probe and coreaction accelerator to label signal antibody (Ab2), along with carboxylate-modified magnetic nanoparticles (MNPs) as both the separation/enrichment tool and the immobilization support for the capture antibody (Ab1) and ruthenium nanoparticles electrodeposited on nitrogen-doped graphene-coated Cu foam (fCu/N-GN/RuNPs) for decreasing of overvoltage HER at cathodic pole. Thanks to the high load capacity of luminol in MIL-53(Fe)-NH2 and co-reaction accelerator of MIL-53(Fe)NH2, which promote the conversion of co-reactant H2O2 into the hydroxyl radicals (●OH), the obtained L@MIL-53(Fe)-NH2 exhibited superior ECL property. Besides, the enhanced cathodic current as well as low overpotential for the reduction of hydrogen, followed by the reduction of ΔEelec on fCu/NGN/RuNPs, results in an increased ECL of luminol/H2O2 on anode BPE.

EXPERIMENTAL SECTION Materials and Apparatus The details of materials, reagents, and instrumentation for physicochemical characterization are given in Supporting Information.

Loading of luminol into MIL-53(Fe)-NH2 (L@MIL53(Fe)-NH2) MIL-53(Fe)-NH2 was synthesized by a solvothermal method according to the literature40. To load the luminol into MIL53(Fe)-NH2, 1 mg mL-1 of MIL-53(Fe)-NH2 were incubated with luminol (1 mg mL-1) in PB (0.1 M, pH = 8.5). The L@MIL-53(Fe)-NH2 was collected by centrifugation and washed extensively with PB.

Preparation of Ab2-Functionalized L@MIL-53(Fe)-NH2 Functionalization of MIL-53(Fe)-NH2 using Ab2 (Ab2/L@MIL-53(Fe)) was undertaken by conjugation of surface amines of MIL-53(Fe)-NH2 to Ab2 via EDC-NHS crosslinking reaction. First, the carboxylic groups of Ab2 (10 ng mL-1) were activated by EDC: NHS (1:1 mass ratio). Then, 5 mg mL-1 L@MIL-53(Fe)-NH2 was incubated with activated Ab2 at room temperature.

Immobilization of Ab1 on magnetic nanoparticles (MNPs)

carboxylate-modified

The carboxylate-modified MNPs were prepared based on the procedure described in the literature with slight modifications41. In order to prepare MNPs/Ab1, Ab1 were covalently attached to MNPs by using EDC/NHS chemistry. For this purpose, primarily an aliquot of 10 mg MNPs (500 µL) was activated by using EDC:NHS (1:1 mass ratio) for 15 min at room temperature. After washing the resulting product with PBS 1x and resuspended in PBS 1x, 500 µL of 10 ng mL-1 Ab1 was added to the solution and incubated under shaking condition for 30 min. Subsequently, the magnetically separated MNP/Ab1 was washed with PBS1x to remove the unbound Ab1. Finally, the magnetic surface was then blocked using 2% BSA to attenuate unspecific adsorption of the PSA on the sensor surface.

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Analytical Chemistry

Sandwich immunocomplex formation The prepared MNPs/Ab1 was incubated with different concentrations of PSA for 30 min. The MNPs/Ab1 nanoconjugates were magnetically separated, washed with PBS 1x to remove the unbound PSA and resuspended them in 500 µL PB containing L@MIL-53(Fe)-NH2 for 30 min. Thereafter, the immunocomplex (MNP/Ab1-PSA-Ab2/L@MIL-53(Fe)) was washed with a PB and followed resuspended in PB.

Fabrication of ruthenium nanoparticles electrodeposited on nitrogen-doped graphene-coated Cu foam (fCu/NGN/RuNPs) Synthesis of Cu foam (fCu) was conducted on a small piece of Cu sheet according to the following method reported in the literature42. fCu formation was proceeded by applying a constant current to Cu sheet in a cell containing 0.4 M CuSO4 and 1.5 M H2SO4 solution. The nitrogen-doped graphene nanosheet (N-GN) was prepared base on the literature.43 The fCu/N-GN electrode was prepared by drop casting 50 μL of NGN (0.5 mg mL-1), on the fCu according to our previously reported43. The fCu/N-GN was modified further by electrodeposition of Ru nanoparticles (fCu/N-GN/RuNPs) in 0.1 M H2SO4 aqueous solution containing 0.2 mM RuCl3 at −0.25 V for 50 s.

Closed split BPE-ECL Cell A closed bipolar cell has been designed as shown in scheme 1. The anodic gold BPE was prepared using small pieces of the archival compact disk (CD) with the gold reflective layer as previously our reported in the literature44-45. 10 μL of MNPs/Ab1-PSA-Ab2/L@MIL-53(Fe) was pipetted onto the anodic pole of the gold BPE under a magnetic field below the BPE. The ECL signal of the luminol/H2O2 was monitored as an analytical signal using a digital camera or PMT in a dark room. Alternatively, the fCu/N-GN/RuNPs was used as a cathodic pole and two electrodes are connected using a conducting wire. Two driving plate electrodes (304 stainless steel sheets) were situated at the ends of the cell, and a potential of 4.5 V was applied to these electrodes using a regulated DC power supply. The ECL signals were recorded using a PMT detector at a set potential of 800 V. In both the visual imaging and PMT detectors, the anodic and cathodic chambers were filled with 0.1 M PB (pH=8.5) solution containing 2 mM H2O2 and 0.5 M H2SO4 solution, respectively. During the BPE-ECL measurement in PMT-based detection manner, the cell was covered with a lid and an optical fiber with 1.2 mm diameter was inserted in a hole on the top of the lid in anodic section to transfer the ECL signal to the detector.

Results and Discussion Early stage detection of cancer biomarkers plays significant roles in clinical analysis. In particular, prostate cancer has been recognized as the second cancer mortality in men and the sixth most common cancer worldwide over the past decades46. High sensitive, as well as cost-effective, accurate and easy-tointerpret, are major technological challenges in the early-stage diagnosis and treatment of prostate cancer. Herein, to address these challenges, a wireless ultrasensitive detection of the PSA, a biomarker for the diagnosis of possible prostate cancer, visual-based closed split BPE-ECL has been introduced (Scheme 1).

Scheme 1. Schematic illustration of the amplified visual ECL detection of PSA.

Characterization of the L@MIL-53(Fe)-NH2 The phase purity and structural identity of constructed nanocomposites are determined using XRD (Figure 1A). The XRD pattern of MIL-53(Fe)-NH2 exhibits well-defined diffraction peaks that is consistent with the simulated pattern, which suggested its good crystallinity40. Compared to MIL53(Fe)-NH2, the positions and corresponding diffraction peaks intensities of L@MIL-53(Fe)-NH2 shows no significant changes, confirming the L@MIL-53(Fe)-NH2 nanocomposites are highly crystallized even after encapsulation of luminol. In order to investigate the molecular structure and incorporation of luminol molecules into MIL-53(Fe)-NH2, the FTIR spectroscopy was performed and the results were presented in Figure 1B. The characteristic bending vibrations bonds of C-O (1373 and 1403 cm-1), Fe-O (540 cm-1), C-H (760 cm-1) and C=O (1650 cm-1) were observed for MIL-53(Fe)-NH2. The stretching asymmetric and symmetric absorptions of the N-H in primary groups positioned at 3416 cm-1 and 3310 cm-1 are observed for NH2-BDC ligands Moreover, the spectra of L@MIL-53(Fe)-NH2 indicate the presence of characteristic sorption bonds of luminol18 (showed with black dash rectangle), which verified the successful the incorporation of luminol molecules into MIL-53(Fe)-NH2. TGA was also performed to quantitatively determine the composition of the nanocomposite (Figure 1C). For pure luminol, a distinct weight loss started at 280 ℃ and ended at 360 ℃ with nearly total weight loss over the 700 oC which consistent with decomposition of luminol molecules. The weight loss around 200 ℃ in MIL-53(Fe)-NH2 was attributed to the loss of solvent molecules, DMF, from the cavities. The L@MIL-53(Fe)-NH2 nanocomposite displays an approximate 20 % weight losses in the range of 270−640℃ that can be ascribed to the weight loss of luminol in the nanocomposite. Using an appropriate calibration curve of the fluorescence intensity, the loading amount of luminol corresponded to 64 nmol mg-1, demonstrating the high loading ability of MOFs (Figure S1). The morphology of the assynthesized L@MIL-53(Fe)-NH2 and MIL-53(Fe)-NH2 was observed by SEM and TEM. The observed morphology of the MIL-53(Fe)-NH2 is spindle shape with mean sizes of about 300 nm in length and a width of about 100 nm (Figure 1D). The L@MIL-53(Fe)-NH2 (Figure 1E) nanocomposite displays the same morphology of MIL-53(Fe)-NH2, indicating that

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L@MIL-53(Fe)-NH2 maintained the crystalline integrity after encapsulation of luminol, which was in accordance with the XRD results. However, the surface of L@MIL-53(Fe)-NH2 became rough, demonstrating the formation of the L@MIL53(Fe)-NH2 crystals with an amorphous structure. Moreover, The TEM (Figure 1F) images of L@MIL-53(Fe)-NH2 shows transparent flakes on its edge, consistent with the SEM results, validating the presence of luminol molecules in the MIL53(Fe)-NH2.

Figure 1. (A) The XRD patterns, (B) FTIR spectra, and (C) TGA of (a) luminol, (b) MIL-53(Fe)-NH2, and (c) L@MIL-53(Fe)-NH2. (D and E) SEM images of MIL-53(Fe)-NH2 and L@MIL-53(Fe)NH2, respectively. (F) TEM image of L@MIL-53(Fe)-NH2.

Characterization of fCu/N-GN/RuNPs Electrodeposition technique is a cost-effective and convenient method for the synthesis of 3D metal foam and has drawn great attention as an effective tool because it is fast and involves green and non-toxic solvents. The SEM images of the fCu (Figure 2A-C) show 3D foam structures of copper with interesting porous micro-nanostructures with ramified and nanodendrite walls. The SEM images of the fCu/N-GN (Figure 2D-F) depict many interlaced walls conjugated nanostructures with highly developed porous structures. The fCu/RuNPs exhibits (Figure 2G-I) rectangular cuboid morphology of RuNPs that were uniformly dispersed on the fCu with an average size of 200 nm. The SEM image obtained on the fCu/NGN/RuNPs (Figure 2J-L) electrode shows the many interlaced walls along with uniformly nucleated and well-dispersed of a rectangular cuboid. The XRD patterns of fCu, fCu/RuNPs and fCu/N-GN/RuNPs foams (Figure S2) demonstrated the presence of RuNPs and GN on the fCu (For details discussion pleases see the Supporting Information).

Electrocatalytic activity of the nanocomposite for HER The hydrogen evolution catalytic efficiency of fCu/NGN/RuNPs was evaluated. The Pt or Pt-based materials is considered as the best-known catalyst for electrochemical HER due to their negligible overpotential and excellent kinetics47. However, prohibitively high cost, inadequacy, and poor stability limit their widespread use 48. Therefore, the design and development of low cost, non-precious and non-noble metal electrocatalysts to reduce large overpotential is highly desirable. While the fCu electrode exhibits low electrocatalytic

Figure 2. SEM images of (A-C) fCu, (D-F) fCu/N-GN, (G-I) fCu/RuNPs, and (J-L) fCu/N-GN/RuNPs in different magnification.

activity with an overpotential of -377 mV at -10 mA cm-2, the fCu/N-GN shows high electrocatalytic activity with -213 mV overpotential to drive catalytic current of -10 mA cm-2 (Figure 3A). Two reasons could be suggested for a significant increase in the HER activity of fCu/N-GN49. (i) The pyridinic nitrogen (in accordance to the previous study using the XPS to determine the surface chemical composition of N-GN, nitrogen bonding configurations and the content of nitrogen43) in N-GN can enhance electron transfer efficiency with low resistance metallic properties of N-GN via contributing two p electrons into the π system of the graphene. (ii) The increased surface roughness can enlarge the electrode/electrolyte interface area and reduce electrode polarization. Furthermore, the nanostructure characteristics also bring favorable effects for catalytic performance. As can be seen, the fCu/RuNPs electrode shows the activity of the electrodes is significantly enhanced by the presence of RuNPs. Most importantly, fCu/N-GN/RuNPs shows the most impressive HER activity with small overpotentials of -114 mV to reach a current density of 10 mA cm-2. This potential is 263, 99, and 76 mV less than that of the fCu, fCu/N-GN, and fCu/RuNPs respectively. Also, the onset potential observed for the HER on the fCu/N-GN/RuNPs is about 260 mV, 140 mV and 112 less negative values (more facile direction) than those observed on fCu, fCu/N-GN, and fCu/RuNPs electrodes, respectively. The results demonstrate that the fCu/N-GN/RuNPs has the lowest onset potential and highest current density for HER, close to reported for commercial Pt/C50. We also evaluated the kinetics of the catalytic activities of the electrocatalysts from Tafel plots. The Tafel plots derived from Figure 3A is shown in Figure 3B, where their linear parts were fitted to the Tafel equation, η =-b

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log(j0) +b log(j) where b is Tafel slope, and j0 is exchanged current density. Interestingly, the fCu/N-GN/RuNPs gives a small Tafel slope of 81 mV/dec, which is much smaller than those observed for fCu (164 mV/dec), fCu/N-GN (130 mV/dec), and fCu/RuNPs (84 mV/dec), indicating a facilitated mass transportation between the catalyst and the electrolyte as well as a fast electron transfer with the increasing overpotentials. Moreover, the j0 fCu/N-GN/RuNPs electrode is about 9, 3.5, and 2.2 times higher than those observed for the fCu, fCu/N-GN, and fCu/RuNPs electrodes, respectively. Overall, the porous 3D structure of fCu with open pores and numerous dendritic morphologies and the formation of an efficient electrical network using direct incorporation of RuNPs and the N-GN with no need for any polymeric binder or additive leads to considerably easier accessibility of electrolytes to active electrode surface and a considerable boost in electrochemical performance as well.

Electrochemical behavior of different platforms

about 0.2 V vs Ag/AgCl (Figure 4C). Moreover, the onset potential observed for HER on fCu/N-GN/RuNPs is about -0.3 mV vs. Ag/AgCl (Figure 4D). Therefore, the ΔEelec is ∼0.5 V. The ΔEelec of the obtained from the onsets of oxidation and reduction is significantly lower than the previously reported for BE. Consequently, as aforementioned, the reduction of ΔEelec resulted in ultrasensitive detection of PSA using PBE-ECL.

BPE-ECL PSA detection using PMT detector To explore the role of MIL-53(Fe)-NH2 in luminol/H2O2 system, the ECL of different solution including (a) luminol, (b) luminol+MIL-53(Fe)-NH2, (c) luminol+H2O2 and (d) luminol+MIL-53(Fe)-NH2+H2O2 was studied to evaluate the possible reaction of luminol, H2O2 and MIL-53(Fe)-NH2. (where the concentrations of luminol, H2O2, and MIL-53(Fe)NH2 were 64 μM, 2 mM, and 1 mg mL-1 in 0.1 M PB (pH 8.5), respectively). As shown in Figure 5A, the ECL intensity of luminol is negligible and exhibited no remarkable changes in

Electrochemical properties of the gold electrode at each modification step was investigated in a 0.5 mM Fe[(CN)6]3-/4solution, containing 0.1 M KCl using a classical three-electrode cell (Figure 4A and B). Compared with the bare gold electrode (curve a, ΔEpeak=85 mV, Rct=205 Ω), a coating of the MNP/Ab1 on the gold surface increased the ΔEpeak and Rct to 120 mV and 1420 Ω, respectively. This is in accordance with hindrance introduced by the adsorption of insulating MNPs and Ab1. Further increase in ΔEpeak and Rct was shown upon the

Figure 3. Steady-state voltammograms obtain in 0.5 M H2SO4 solution at room temperature and potential scan rate of 5 mV s−1. (A) on (a) Cu sheet, (b) fCu, (c) fCu/N-GN, (d) fCu/RuNPs, and (e) fCu/N-GN/RuNPs. (B) The steady-state polarization curves of (Tafel plots) extracted from the panels (A).

modification of the gold electrode with the MNP/Ab1-PSA. Coating of the gold electrode by the MNP/Ab1-PSA-Ab2/MIL53(Fe) sandwich considerably increased the ΔEpeak and Rct to 359 mV and 11200 Ω, respectively. This observation can be attributed to the very effective coverage of the electrode surface and the blocking effect of the modifiers to the redox probe. In order to investigate the voltage difference between the onset of luminol oxidation and hydrogen reduction, ΔEelec, the CVs obtained using a conventional three-electrode cell. While, the voltammograms of different modification steps of the gold electrode in PB (0.1 M, pH=8.5)+2 mM H2O2, i.e., bare gold, MNP/Ab1, and MNP/Ab1-PSA, are featureless, the MNP/Ab1PSA-Ab2/L@MIL-53(Fe) modified gold electrode shows a well-defined voltammetric response, attributable to the encapsulated luminol into the pore size of the MIL-53(Fe)-NH2. As can be seen the onset potential of luminol in this electrode is

Figure 4. (A) Cyclic voltammograms and (B) complex plane plots of the EIS obtained in the presence of 0.5 mM Fe[(CN)6]3-/4solution, containing 0.1 M KCl on (a) bare gold electrode, (b) MNP/Ab1/gold electrode, (c) MNP/Ab1-PSA/gold electrode, (d) MNP/Ab1-PSA-Ab2/L@MIL-53(Fe)/gold electrode. (C) Cyclic voltammograms of (a) bare gold electrode, (b) MNP/Ab1/gold electrode, (c) MNP/Ab1-PSA/gold electrode, (d) MNP/Ab1-PSAAb2/L@MIL-53(Fe)/gold electrode in 0.1 PB (pH=8.5). (D) Cyclic voltammograms of Cu sheet and fCu/N-GN/RuNPs electrode in 0.5 M H2SO4 solution at scan rate 5 mV s−1.

comparison with that in the presence of MIL-53(Fe)-NH2 solution, revealing that MIL-53(Fe)-NH2 had no direct effect on luminol ECL emission. However, the ECL intensity of the luminol+H2O2 was enhanced, which indicated that H2O2 was the co-reactant of luminol to improve the ECL response. Interestingly, when MIL-53(Fe)-NH2 was introduced in luminol+H2O2 solution, great enhancement of the ECL signal appeared. This was primarily attributed to the reason that the MIL-53(Fe)-NH2 which can accelerate the decomposition of H2O2 to increase the concentration of ●OH, consequently, promote the ECL reaction rate of luminom/H2O2 system for significantly boosting the ECL signal. In order to demonstrate

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the generation of ●OH by the MIL-53(Fe)-NH2 accelerator, 100 µL isopropanol as a scavenger for ●OH was added into the 0.1 M PB (pH=8.5) and 2 mM H2O2 in the anodic pole. As illustrated in Figure 5A, trace e, the ECL intensity was diminished in the presence of isopropanol. These results obtained confirmed that MIL-53(Fe)-NH2 could accelerate H2O2 to produce more oxidant mediators of ●OH. The catalytic mechanism of MIL-53(Fe)-NH2 as the co-reaction accelerator was further assessed by the detection of fluorescence intensity of (2-hydroxyterephthalic acid) TAOH from the oxidation of the terephthalic acid (TA) under ●OH-generating reaction40. As shown in Figure S3, the fluorescence intensity of TA, TA+H2O2, MIL-53(Fe)-NH2, was weak and approximately similar to MIL-53(Fe)-NH2+H2O2 solution. However, the fluorescence intensity of the mixture of MIL-53(Fe)-NH2+H2O2 and TA was significantly enhanced. This result demonstrated that the MIL-53(Fe)-NH2 could catalyze the H2O2 to produce ● OH radicals, which could then react with the weakly fluorescent TA to high fluorescent TAOH. To better understand the electrochemical behavior of the fCu/N-GN/RuNPs electrode regulated the ECL performance of luminol/H2O2 at the anodic pole of the BPE, the ECL intensity of luminol in the absence and presence of fCu/N-GN/RuNPs electrode (Figure 5B) was investigated using a PMT detector. In the absence of electrocatalyst of fCu/N-GN/RuNPs electrode, luminol/H2O2 system and hydrogen were oxidized at the anode of gold BPE and the cathodic pole of the Cu sheet BPE, respectively, when the external voltage was 4.5 V. As shown in Figure 5B, trace a, exhibits a rather weak ECL intensity. However, when we used the fCu/N-GN/RuNPs electrode as a cathodic pole of BPE, the lowest voltage for driving the ECL reaction was decreased. It displays a dramatically increased ECL signal which is approximately 3.7 times brighter than that in the absence of fCu/N-GN/RuNPs electrode in closed BPE (trace b). These results indicate that the fCu/N-GN/RuNPs electrode could be used as an electrocatalyst for HER to facilitate the electron transfer flow through BPE and realize the sensitive ECL detection. Besides, confined a large amount of luminol considerably boost the overall efficiency of ECL emission by minimizing the decomposition of highly active ●OH by reducing the diffusion barrier. To verify the advantages of the L@MIL-53(Fe)-NH2 signal probe, the ECL intensity of the immunosensor was conducted using Ab2/MIL-53(Fe) with the solution of luminol, instead of luminol-loaded MIL-53(Fe)NH2. The comparison between the Figure 5B, trace b and c shows the integrated L@MIL-53(Fe)-NH2 (trace b) exhibited more than 2.2-fold ECL intensity compared to the mixture of luminol and MIL-53(Fe)-NH2 (trace c). The generated ●OH would readily interact with the adjacent luminol before the disappearance of the radical species for the integrated L@MIL53(Fe)-NH2. In contrast, for the mixture, the formed ●OH had a long diffusion distance from MIL-53(Fe)-NH2 to luminol, may be decomposed during the diffusion, resulting in dramatically lower the reaction efficiency. The ECL intensities of the anodic pole of BPE at different steps in the presence of 0.1 M PB (pH=8.5) and 2 mM H2O2 were investigated under the optimized electrode potential of driving electrodes (Etot) (details of which are presented in Supporting Information and Figure S4). As Figure 5C reveals, no ECL emissions are observed on the bare gold electrode (trace a), MNP/Ab1 (trace b), and MNP/Ab1-PSA (trace c). However, the enhanced ECL is

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observed with MNP/Ab1-PSA-Ab2/L@MIL-53(Fe) (trace d). To prove the ECL emission is directly correlated to sandwich immunocomplex rather than to nonspecific adsorption of the Ab2/L@MIL-53(Fe) on the MNP/Ab1, without PSA, 100 μL of MNP/Ab1 (1 mg mL-1) were mixed with 100 μL of Ab2/L@MIL-53(Fe) (1 mg mL-1) for 30 min. As shown in Figure 5C, trace e, the ECL signal of the luminol/H2O2 is negligible. It suggests that the nonspecific adsorptions of Ab2/L@MIL-53(Fe) are not significant and the conjugation takes place just through the specific binding between PSA and Ab2/L@MIL-53(Fe). To demonstrate that the ECL signal of the anodic pole of gold BPE only originates from the presence of luminol into the MIL-53(Fe), the ECL signal of MNP/Ab1PSA-Ab2/MIL-53(Fe), without luminol, was recorded in 0.1 M PB (pH=8.5) containing 2 mM H2O2. After magnetically separated and washing with a PBS 1x, the resulting MNP/Ab1PSA-Ab2/MIL-53(Fe) was dropped on the anodic poles of the BPE-ECL. The resulting ECL (trace f) indicates no emission light is observed. To evaluate the selectivity of the BPE-ECL immunosensor, the sandwich immunocomplex was prepared in the presence of HSA as interfering protein. In the presence of HAS, 10 ng mL-1, (Figure 5D, trace a), no significant ECL response was observed, similar to that of the blank sample (Figure 5C, trace e). Also, the specificity of the sandwich immunocomplex was confirmed with a mixture solution of PSA (0.05 ng mL-1 ) and HAS (10 ng mL-1) (Figure 5D, trace b). No obvious ECL signal change is observed, indicating a similar ECL signal of the luminol/H2O2 system with that 0.05 ng mL-1 of PSA only (trace c). These results demonstrate the designed immunosensor possessed high selectivity. The quantitative behavior of the ECL immunosensor was performed in response to various concentrations of PSA. As shown in Figure 5E the ECL peak intensities increase with the increase in the concentration of PSA. The corresponding calibration plot (Figure 5F) was linearly related to the logarithmic values of the PSA concentration over the range of 1 pg mL-1 to 300 ng mL-1 with an extremely low detection limit of 0.2 pg mL-1 (S/N = 3). We quantified PSA with our developed BPE-ECL immunosensor in real samples using a standard addition method. To this end, spiked human serum samples were prepared with different amounts of PSA, (0.01, 1, and 100 ng mL-1). Based on the calibration curve, the calculated recovery percentages were 98.2% (0.01 ng mL-1), 100.4% (1 ng mL-1) and 99.5% (100 ng mL-1). The stability of the MNP/Ab1-PSA-Ab2/L@MIL-53(Fe)modified anodic BP gold electrode was also followed after the immunosensor incubated with 1 ng mL-1 PSA. As shown in Figure S5, the ten experiments on a BPE exhibited no significant changes in ECL, with acceptable relative standard deviation (RSD) of