An Efficient Electrochemiluminescence Enhancement Strategy on

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An Efficient Electrochemiluminescence Enhancement Strategy on Bipolar Electrode for Bioanalysis Nan Zhang, Hang Gao, Cong-Hui Xu, Yixiang Cheng, Hong-Yuan Chen, and Jing-Juan Xu Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.9b03477 • Publication Date (Web): 29 Aug 2019 Downloaded from pubs.acs.org on August 30, 2019

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Analytical Chemistry

An Efficient Electrochemiluminescence Enhancement Strategy on Bipolar Electrode for Bioanalysis Nan Zhang,† Hang Gao,† Cong-Hui Xu,* Yixiang Cheng, Hong-Yuan Chen, and Jing-Juan Xu* State Key Laboratory of Analytical Chemistry for Life Science and Collaborative Innovation Center of Chemistry for Life Sciences, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210023, China. ABSTRACT: This paper develops an efficient electrochemiluminescence (ECL) enhancement strategy on closed bipolar electrode for the detection of prostate specific antigen (PSA). We first synthesized a cyclometalated iridium(III) complex ((pq)2Irbza) with high ECL efficiency and used as ECL emitter in the anodic cell of BPE. While we introduced a Pt-tipped Au NRs and constructed a sandwich immune structure at the cathodic pole of BPE. Combined the signal amplification strategies of enzyme catalysis and the synergistic catalytic effect of bimetallic structure for the reduction of H2O2, the attached Pt-tipped Au NRs-GOx-Ab2 nanocomplex as both recognition probes and signal amplification units could mediate the ECL signals of (pq)2Irbza/tripropylamine (TPrA) on the anodes of BPE through faradaic reaction due to the charge neutrality of BPE. Therefore, a highly sensitive BPE-ECL sensor for detection of PSA with a detection limit of 0.72 pg/mL and a linear range from 1.0 pg/mL to 10 ng/mL was obtained. This work is expected to broad the application of iridium complex and bimetallic nanocatalyst in biological detection and could be utilized to detect many other biological molecules.

Electrochemiluminescence (ECL) biosensors based on bipolar electrodes (BPEs), have attracted great attention in bioanalysis.1-5 BPE is usually set up in a microchannel with two poles in one (open BPE) or two solutions (closed BPE).6 When an external voltage is applied across the microchannel, oxidation and reduction reactions occur at the same rate on the anodic and cathodic poles of BPE. ECL signals on BPE could be collected to indicate the redox events that occur at both ends of the BPE. The charge balance of BPE makes the redox species on both poles determine its detection performance. Therefore, there are considerable efforts to amplify the signals at its extremities. On one hand, the design strategy of sensing side focused on enhancing electron transfer to accelerate the rate of reduction reaction and reduce the driving voltage.7-10 On the other hand, at the ECL reporting side, some materials as good carriers or bridges were employed to improve the loading number of electrochemiluminophores,11,12 moreover, higher ECL efficiency materials were synthesized and applied.13,14 In terms of the luminophore at the anode, we noted that cyclometalated iridium(III) complexes are becoming one of the most excellent ECL materials owing to their long luminescent lifetimes, high photoluminescence (PL) efficiencies,15 large Stokes shifts, and the tunability of wavelength ranging from the near-infrared to deep blue via ligand changing.16 However, because of the poor solubility in the aqueous solution for most of the iridium complex, its biological application is greatly limited in traditional threeelectrode system. It is worth noting that the closed BPE has a great advantage in solving this problem due to its separated extremities. Then we tracked a series of work using iridium complexes as ECL luminophores. Kim and coworkers17 first reported two different types of neutral Ir(III)

complexes, (pq)2Ir(acac) and (pq)2Ir(tmd) respectively, whose ancillary ligands were OÔ type, with much higher ECL efficiencies compared to that of [Ru(bpy)3]2+. Impressed by the result, considerable work of cyclometalated iridium(III) complexes with ancillary ligands of OÔ type have been conducted. For example, Zhou and co-workers17 synthesized a series of iridium(III) complexes containing OÔ type ancillary ligand. In addition, these (pq)2Ir(OÔ) complexes most demonstrated emissions peak centered at around 600 nm, close to the emission of [Ru(bpy)3]2+, beneficial to practical assays without changing detector equipped in the current commercial ECL measurement. Herein, a sensitive ECL sensor for detection prostate specifc antigen (PSA) based on closed bipolar electrode was fabricated and some efforts were devoted to the two sides. At the anode of BPE, a new cyclometalated iridium(III) complex of (pq)2Irbza was synthesized and used for the first time, where bza was OÔ type ancillary ligand containing benzene ring and alkyl chain, and it displayed excellent performance with a 0.7 μs for photoluminescence (PL) lifetime and 5.16 times that of [Ru(bpy)3]2+ for relative ECL emission efficiency. At the cathode of BPE, in the sandwich immune structure, a Pt-tipped Au NRs was introduced to accelerate the reduction of H2O2 (Scheme 1). This special structure could reduce the amount of platinum under the premise of ensuring good catalytic performance. In the biosensor, the number of attached Pt-tipped Au NRs-GOxAb2 (nanocomplex) was in proportion to the concentration of PSA. Meanwhile, the more nanocomplex, the more H2O2 generated at the cathode. The oxidation rate of luminophore and coreactant at the anode equals to the reduction rate of H2O2 at the cathode due to the charge balance of BPE. Accordingly, PSA concentration could be determined by

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monitoring the ECL intensity at the anode. With this design, a rapid and sensitive detection of PSA was achieved.

Scheme 1. Schematic diagram and mechanism for PSA detection based on closed bipolar electrode. EXPERIMENTAL SECTION

Reagents and Apparatus. Chemical reagents and apparatus are shown in the Supporting Information. Synthesis of (pq)2Irbza. The Ir(III) complex was synthesized following the procedure described in the literature.18 And the synthesis route is shown in Scheme 2. A mixture of 2-phenylquinoline (1.44 g, 7.0 mmol), IrCl3 (1.0 g, 3.4 mmol), 2-ethoxyethanol (15 mL), and distilled water (5 mL) was stirred under argon at 120 °C for 24 h. After the mixture was cooled, the resulting precipitate was collected by filtration and washed with water, ethanol, and hexane successively. The above Ir(III) dimer (269 mg, 0.21 mmol), benzoyl propanone (85.7 mg, 0.53 mmol) and Na2CO3 (248.0 mg, 2.4 mmol) were mixed into 2-ethoxyethanol (15 mL) and heated at 120 °C for 15 hours. After the mixture was cooled, distilled water (30 mL) was added. The resulting precipitate was collected by filtration and washed with water, followed by portions of Et2O and n-hexane. The crude product was purified through flash column chromatography on a silica gel (eluent: dichloromethane: methanol = 50/1-20/1) to afford (pq)2Irbza (red solid, yield: 53%).

Scheme 2. Synthesis route of (pq)2Irbza used in this work. Preparation of Au nanorods (NRs). The Au NRs were synthesized by seed-mediated method according to the literature.19-21 The process is mainly composed of seed preparation and gold rod growth. Preparation of seed solution. The seed solution was prepared by mixing 0.025 mL of 0.1 M HAuCl4·3H2O and 10 mL of 0.1 M CTAB. Then, freshly prepared, ice-cold aqueous solution of NaBH4 (0.6 mL, 0.01 M) was added. The solution

was stirred vigorously (1000 rpm) for 2 min and then aged at room temperature without disturbance for 30 min before use. The growth of Au NRs. The growth solution was prepared by dissolving CTAB (3.6 g) and 5-bromosalicylic acid (0.44 g) in 100 mL of warm water (Millipore, 55 °C). Then, 1.92 mL of 0.01 M AgNO3 was added and the solution was maintained at room temperature for 15 min. After that, 100 mL of 1 mM HAuCl4·3H2O solution was added. After the solution was stirred gently (400 rpm) for 15 min, 0.512 mL of 0.1 M ascorbic acid was added with vigorously stirring for 30 s until the color of the solution changed from yellow to colorless. At this moment, 0.32 mL seed solution was added to the growth solution with stirring for 30 s to make the growth solution homogeneous and left undisturbed at 27 °C for 12 h. After 12 hours of growth, the color of the growth solution changed from colorless to violet indicating the formation of Au NRs. Preparation of Pt-tipped Au NRs. Briefly, mixing 0.568 mL of 0.1 M ascorbic acid and 10 mL of Au NRs solution prepared above, Then, 99 µL of 0.01 M H2PtCl6·6H2O and 0.08 mL of 0.01 M HCl were added to the mixture. The mixture was left undisturbed for 12 h at 27 °C. After 12 hours of reaction, the color of the mixture changed from violet to atropurpureus, then the solution was centrifuged (8000 rpm, 10 min) and washed twice with water. Preparation of Pt-tipped Au NRs-GOx-Ab2. Mixing 400 μL Pt-tipped Au NRs solution and 400 μL PSS (2 g L-1, 1 mM NaCl). After 1 h adsorption time, it was centrifuged (8000 rpm, 10 min) to remove excess poly electrolyte and dispersed in 400 μL Millipore water. Subsequently, 400 μL PDADMAC (2 g L-1, 1 mM NaCl) was added into the above solution and adsorbed for 1 h. Then the NRs was centrifuged (8000 rpm, 10 min) and dispersed in 400 μL Millipore water, then, the pH of solution was adjusted to 7.4. At last, 100 μL GOx (1 mg mL-1, pI: 4.2) and 20 μL Ab2 (20 μg mL-1, pI: 6.5-7.2) were added into the solution and adsorbed for another 1 h, then centrifuged (8000 rpm, 10 min) to discard free GOx and the target bioconjugates were dispersed in 400 μL PBS (10 mM, pH 7.4) and stored at 4 °C. Closed bipolar electrodes design and fabrication. The preparation process mainly includes ITO wet etching, ITO post-treatment, construction of reaction cell by PDMS, and deposition of gold film. Specific steps are detailed in the Supporting Information (SI). Preparation of the closed bipolar electrodes biosensor. A volume of 50 μL L-cysteine (0.01 M) solution into the cathodic reservoir of BPE and incubated at 4 °C for 10 h to allow the formation of the carboxyl group on the Au surface. After the electrodes were rinsed with PBS, 50 μL of a freshly prepared mixture solution containing 20 mg/mL EDC and 10 mg/mL NHS was added to this channel and incubated at 37 °C for 1 h. Then the electrodes were washed with PBS and 30 μL of Ab1 (20 μg/mL) was added into the reservoir and kept overnight. Subsequently, the physically adsorbed antibodies were washed with PBS, and then blocked with 50 L of 1% BSA for 30 min. Next, the electrodes were incubated with different concentrations of PSA solutions (30 L) at 37 °C for 1 h, followed by washing with PBS. Then, it was further incubated with 30 L of the


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Analytical Chemistry prepared Pt-tipped Au NRs-GOx-Ab2 solution at 37 °C for 1 h and rinsed with PBS. Electrochemical and ECL measurements employed with three-electrode system. The cyclic voltammetry of (pq)2Irbza and three-electrode ECL measurements were performed with a glass carbon electrode (GCE, 3 mm in diameter), an Ag/AgNO3 (10 mM) non-aqueous quasireference electrode, and a platinum wire counter electrode. The bare GCE was polished in sequential order with 1.0, 0.3, and 0.05 μm alumina slurry to obtain a mirror-like surface, followed by sonication in the ultrapure water and ethanol, in turn, and finally dried in air. The quasi-reference electrode was separated from the electrolyte by glass tube and was calibrated against a saturated calomel electrode (SCE) by the addition of ferrocene (Fc) as an internal standard at the end of each experiment, taking the redox potential of the ferrocene/ferrocenium (Fc/Fc+) couple as 0.424 V vs. SCE. ECL detection of PSA. The ECL detection process was operated as follows: The sensing cell was filled with 50 L of PBS (pH 7.4, 0.1 M) containing 10 mM glucose and the reporting cell was immersed in 50 L acetonitrile containing 0.1 M TBAPF6 with a mixture of 0.1 mM (pq)2Irbza and 17.5 mM TPrA. The ECL measurements were performed using MPI-E electrochemical and electrochemiluminescence analyzer. The ECL voltage curves were obtained by applying a cyclic scan from 0 V to 6.5 V on the two ends of driving electrodes with the scan rate of 0.5 V/s. The voltage of photomultiplier tube (PMT) was set at 400 V during detection. RESULTS AND DISCUSSION

Characterization of (pq)2Irbza. In this work, we synthesized (pq)2Irbza, a cyclometalated iridium(III) complex with high ECL efficiency and used it as ECL emitter in the anodic cell of BPE (the synthesis method is illustrated in Scheme 2). The 1H NMR and 13C NMR spectra of (pq)2Irbza are shown in Figure S1 and Figure S2. The matching information of the corresponding nuclear magnetic peak is attached to the figures. The result of Mass spectrometry characterization is in Figure S3. These results could confirm that the product was synthesized according to the established design.

/s). (B) Cyclic voltammogram (blue line) and corresponding ECL signals (red line) of 0.1 mM (pq)2Irbza with 17.5 mM TPrA in CH3CN containing 0.1 M TBAPF6 (scan rate, 100 mV/s; voltage of PMT, 200 V). (C)ECL Intensity comparation of 0.1 mM (pq)2Irbza /17.5 mM TPrA and 0.1 mM [Ru(bpy)3]2+ /17.5 mM TPrA. (scan rate, 500 mV /s; voltage of PMT, 500 V). (D) Normalized fluorescence spectrum (blue line) of (pq)2Irbza and normalized ECL spectrum of (pq)2Irbza with TPrA in CH3CN.

Prior to the ECL study, we first performed electrochemical characterization of (pq)2Irbza by cyclic voltammetry (CV) to obtain its redox property. It was investigated in CH3CN solution containing 0.1 M TBAPF6 as the supporting electrolyte and the result was displayed in Figure 1A. The (pq)2Irbza had a reversible oxidation wave at peak potential of +0.871 V with an onset oxidation potential of +0.715 V attributed to the single electron redox reaction of iridium. An irreversible reduction wave at peak potential of -1.662 V was observed which represented the reduction of the ancillary ligand. As for the ECL test, the CV and ECL of 0.1 mM (pq)2Irbza with 17.5 mM TPrA were recorded in CH3CN containing 0.1 M TBAPF6 with a scan rate of 100 mV/s by the voltage of PMT of 200V. As revealed in Figure 1B, in the part of potential experiment, the reversible oxidation wave of (pq)2Irbza was hidden by the strong, inreversible TPrA oxidation wave due to the considerably higher concentration of TPrA and the ECL peak lagged behind the oxidation of TPrA. On the basis of the understanding of the oxidative-reduction mechanism that led to the ECL of the [Ru(bpy)3]2+/TPrA system22,23 and other iridium complex /TPrA systems,17,18,24,25 we proposed that the present (pq)2Irbza /TPrA system followed the classic route, the ECL mechanism could be outlined as the following equations (15).

Furthermore, to intuitively display the ECL performance of (pq)2Irbza, the commercial luminophore [Ru(bpy)3]2+ was used as reference. As Figure S4 shows, the ECL intensity of (pq)2Irbza was about 4 times that of [Ru(bpy)3]2+ at the same experiment condition. Meanwhile, ECL relative quantum efficiency was calculated using the equation (6) 15,26

( )( ) 𝑡

∅𝑥 = ∅𝑠𝑡

Figure 1. (A) Cyclic voltammogram of 2 mM (pq)2Irbza in deoxidizing CH3CN containing 0.1 M TBAPF6 (scan rate, 100 mV

𝑡

∫0𝐼 𝑑𝑡

/

𝑡

∫0𝑖 𝑑𝑡

𝑥

∫0𝐼 𝑑𝑡

(6)

𝑡

∫0𝑖 𝑑𝑡

𝑠𝑡

In the equation, ∅st is the ECL efficiency of [Ru(bpy)3]2+(0.1 mM) with the value of 1 used as the standard in CH3CN containing 0.1 M TBAPF6 and 17.5 mM TPrA, I is ECL intensity, i is current value, st represent [Ru(bpy)3]2+ and x represent the sample, the value of ∅x is calculated to be 5.16. It indicated that the (pq)2Irbza has a relatively higher ECL quantum efficiency which is 5.16



times that of the standard [Ru(bpy)3]2+/TPrA. We also used the prepared BPE to test the ECL performance of (pq)2Irbza, as Figure 1C shows, at the anode, the ECL intensity of (pq)2Irbza is much stronger than [Ru(bpy)3]2+ when the cathode was filled in the same concentration of PBS solution. The UV-vis absorption of the (pq)2Irbza complex is shown in Figure S5. The intense intraligand absorption bands below 380 nm was assigned to be the π−π* transition centered on the C^N main ligand and the OÔ ancillary ligand. The featureless weak absorptions in the visible range of 400-550 nm could be considered as spin-allowed and spin-forbidden singlet and triplet metal-to-ligand charge transfer transitions (MLCT). Since the luminous emission of the excited state back to the ground state is basically the same of PL and ECL, the spectra of the two processes are compared in Figure 1D. The strong PL emission peak at 601 nm was observed, which was nearly identical to that of the corresponding ECL emission centered at 611 nm. And the slight difference between two spectra might be attributed to the existence of other species (such as TPrA and TBAPF6) in the ECL measurement and different photomultiplier tube (PMT) used for the ECL and PL test. Meanwhile, as Figure S6 displayed, the phosphorescent lifetime of (pq)2Irbza was 0.7 μs which illustrated that the complex possess a relative long PL lifetime compared with the literature reported of 0.28 μs for (ppz)2Ir1iq and 0.24 μs for (6tpz)2Ir1iq.27 Characterization of Au NRs and Pt-tipped Au NRs. As we know, the reactions at both ends of the bipolar electrode are coupled to each other. In our design, in addition to the use of high ECL efficiency materials at the anode, we also introduced a bimetallic nanomaterial (Pt-tipped Au NRs) capable of synergistically catalyzing H2O2 at the cathode. Figure 2A displays the TEM image of the prepared Au NRs, it can be observed that the Au NRs is relatively uniform with an average length of 55 nm and an average width of 13 nm. It can be found that the tips of the Au NRs were loaded with some nanoparticles in Figure 2B, we further conducted high-resolution transmission electron microscopy (HRTEM) and energy-dispersive X-ray analysis (EDX) characterizations to further demonstrate the successful deposition of Pt at the tips of Au NRs. The result of HRTEM is shown in Figure 2C, the characteristic spacing of 0.196 nm belonged to the (200) lattice planes of FCC metallic platinum. In addition, the energy-dispersive X-ray analysis (EDX) map of the Au and Pt distributions is presented in Figure 2D. It can be obviously seen that the samples have greater Pt concentration at the ends of NRs, these results indicated that we have successfully prepared the Pt tipped Au NRs nanocomplex. Moreover, a UV-vis absorption spectrometer was used to demonstrate the deposition process, and the results of Au NRs (black line) and Pt-tipped Au NRs (blue line) were recorded in Figure S7. It can be noted that the spectra show two absorbance peaks, which are corresponding to localized surface plasma resonances in the transverse (TSPR) and longitudinal (LSPR) directions, respectively.28 It is clearly that the TSPR peak of the Au NRs is at the wavelength of the 520 nm, and the LSPR peak is at 805 nm. As Pt is deposited onto the tip of Au NRs, the LSPR peak is

red-shifted to about 910 nm, the TSPR peak is less sensitive to the deposition of metals onto Au NRs and has a little red shift to 550 nm, which is consistent with literature reported.29 The inset shows that after Pt deposited onto the tip of Au NRs, the color of the solution changed from wine red to purple-dark, which is corresponding to the UV-vis

results.

Figure 2. TEM image of (A) Au NRs and (B) Pt-tipped Au NRs. (C) HRTEM image of Pt-tipped Au NRs. (D) EDX elemental mapping image of the Au and Pt distributions in the NRs. (All the accelerating voltage was 200 KV)

CTAB-protected Pt-tipped Au NRs are prone to aggregation during washing and centrifugation, so we used PSS (negatively charged) and PDADMAC (positively charged) to improve the stability of the Pt-tipped Au NRs and facilitate the modification of subsequent GOx and Ab2. We used UV-vis, zeta potentials and dynamic light scattering (DLS) to track the modification process and the results were recorded in Figure S8. In the UV-vis spectra of Figure S8A, compared to the LSPR peak of the Pt-tipped Au NRs, there is a little red shift for the NRs with a PSS coating (923nm, curve b) and PDADMAC coating (931 nm, curve c). After introduced GOx and Ab2, the LSPR peak further red shifted to 985 nm. The results of zeta potentials are displayed in Figure S8B. The zeta-potential of the assynthesized Pt-tipped Au NRs is about 37 mV due to the presence of a bilayer of cationic CTAB on the surface. When the negatively charged PSS and the positively charged PDADMAC formed the outermost layer, negative and positive zeta potentials can be observed at -24 mV and 23 mV, respectively. When GOx and Ab2 adsorbed on to the nanocomplex, the zeta potential is about -5 mV. DLS of the modification process were also performed and recorded in Figure S8C. The hydrate particle size of the bioconjugates increased slightly after each step of modification. The results of UV-vis, zeta potentials and DLS declare that the polyelectrolytes, GOx and Ab2 are successfully assembled on to the NRs via electrostatic interactions.


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Analytical Chemistry Comparation of catalytic performances of the Au NRs and Pt-tipped Au NRs for H2O2. In order to intuitively express the catalytic perfomance of the prepared materials, we recorded current response of three kinds of electrodes to H2O2. Bare GCE, GCE modified Au NRs and GCE modified Pt-tipped Au NRs were scanned in PBS and H2O2 from 0 V to -0.8 V, respectively. The corresponding results were displayed in Figure 3, for bare GCE, the current was only a slight increase at -0.8 V in 10 mM H2O2 (curve b) compared in PBS (curve a). Then we tested the catalytic performance of Au NRs, as shown in curve c, its reduction current at -0.8 V was about 3.5 times that of the current of bare GCE in H2O2. Importantly, after modifying the prepared Pt-tipped Au NRs nanocomplex on GCE, there was a large reduction peak at about -0.25 V, and the onset reduction potential was much lower to the formers, which confirmed the Pt-tipped Au NRs possess more outstanding electrocatalytic performance to the reduction of H2O2.

To demonstrate the successful fabrication of the biosensor, EIS was measured in 5 mM [Fe(CN)6]3-/4-solution after each step. The corresponding results are exhibited in Figure S9. The Ret of the modified BPE was increased when the Ab1 (curve c), BSA (curve d), PSA (curve e) and Pt-tipped Au NRs@GOx-Ab2 (curve f) were introduced, which suggested that our proposed ECL biosensor was constructed successfully. Optimization of the Experiment Conditions. For a better performance of ECL in this assay, several experimental conditions containing the concentration of TPrA, the concentration of Glu and the incubation time of Glu were optimized. As shown in Figure S10A, the ECL intensity increased with the concentration of TPrA increasing when the concentration is below 17.5 mM. Then the ECL intensity decreased as the concentration increased. Meanwhile, the concentration and the incubation time of Glu were investigated. As Figure S10B displayed, △ECL became larger in the concentration of glu from 2 mM to 10 mM, and more than 10 mM, the ECL intensity had little change. In Figure S10C, the biggest value of △ECL was achieved when the incubation time of Glu was 40 minutes. Furthermore, we used two nanocomplexes, Au NRs@GOxAb2 and Pt-tipped Au NRs@GOx-Ab2 to construct biosensors, respectively. As Figure 3B presents, the signal amplification performance of the Pt-tipped nanocomplex

Figure 3. (A) Current responses of bare GCE in PBS solution (curve a), bare GCE (curve b), GCE modified Au NRs (curve c), and GCE modified Pt-tipped Au NRs (curve d) scanned in 10 mM H2O2, respectively. (B) ECL intensities comparation of Pttipped Au NRs@GOx-Ab2 (curve a) and Au NRs@GOx-Ab2 (curve b), which the PSA was 10 ng /mL. (scan rate: 500 mV /s, PMT: 400V).

Detection principle, fabrication and characterization of the ECL Biosensor. The fabrication procedure of the closed BPE and the detecting mechanism of PSA are described in detail in Scheme 1. It is clearly that the anode and the cathode were separated by two pieces of PDMS. For the side of anode, (pq)2Irbza and TPrA were added into the anode reservoir as the ECL reporting channel. The cathode side was designed as the sensing channel. First, we electrodeposited a layer of gold film on the surface of ITO to protect ITO from negative voltage damage. The binding amount of the Pt-tipped Au NRs@GOx-Ab2 is positively correlated with the amount of PSA in the system. In addition, the nanocomplex could achieve double amplification of the signal. First, glucose is catalyzed by GOx to form H2O2. Next, the Pt-tipped Au NRs could catalytic the reduction of H2O2 and then increase the Faraday current of the cathode. As literature reported,30 the oxidation reaction at anode and the reduction reaction at cathode are balanced and occur simultaneously due to the electroneutrality within the BPE. Therefore, the reduction of the H2O2 at the cathode can promote the ECL output at the anode. The ECL intensity at the anode increases with the PSA level increases at the cathode. We used the change of ECL intensity (△ECL ) between the stage of Ab1 combined and the stage of Ab2 complex combined to minimize the interference from background signals.

was more excellent than Au NRs-nanocomplex which due to the bimetallic synergistic catalysis effect. Figure 4. (A) ECL intensities in the presence of different concentrations of PSA from 1.0 pg /mL to 10 ng /mL. (B) The relationship between the △ECL intensities and the concentrations of PSA from 1.0 pg /mL to 10 ng /mL. Inset: the linear relationship. (C) The reproducibility of the ECL immunosensor with 1 ng/mL PSA. (D) Selectivity investigation of the proposed sensor for 1 ng /mL PSA by comparing to blank assay and the interfering proteins at 100 ng /mL level: BSA, CEA, cTnI, IgA and P53.

ECL detection of PSA. As displayed in Figure 4A, the ECL intensity increased with the increasing of the PSA concentration. ECL changes (△ ECL) were found to be logarithmically related to the concentration of PSA in the range from 1.0 pg/mL to 10 ng/mL in Figure 4B. And the linear fitting equation was I=5432.0+1664.1×LgC with R2 = 0.998 (inset in Figure 4B). Meanwhile, the PSA



concentration as low as 0.72 pg/mL could be detected (S/N=3). And the reproducibility of the ECL immunosensor with 1.0 ng/mL PSA is shown in Figure 4C. The relative standard deviation (RSD) of five measurements was 3.2%, showing good precision and acceptable reproducibility. Furthermore, the selectivity of the biosensor for the target PSA of 1.0 ng/mL against a blank assay and the interfering proteins at 100 ng/mL levels of BSA, CEA, cTnI, IgA and P53 was also investigated. As shown in Figure 4D, the ECL intensity changes were negligible for the control and blank assays, while the presence of the target PSA led to a signifcant increase. These results demonstrated that our BPE-ECL immunosensor possesses high selectivity. CONCLUSION

In this work, we synthesized a cyclometalated iridium(III) complex with high ECL efficiency. And this complex was applied into an ECL immunosensor for the sensitive detection of PSA based on a closed BPE. Combined the signal amplification strategies of enzyme catalysis and Pt-tipped Au NRs bimetallic structure, the developed sensor displayed high sensitivity for PSA. And the proposed ECL assay also showed good analytical performance in terms of reproducibility and selectivity. The constructed immunosensor is expected to broad the application of iridium complex and bimetallic nanocatalyst in biological detection and could be utilized to detect many other biological molecules. ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Characterization of (pq)2Irbza including 1H NMR, 13C NMR, MS (ESI), UV-vis and PL lifetime spectra. ECL Intensity comparation of (pq)2Irbza and [Ru(bpy)3]2+. UV-vis of Au NRs and Pt-tipped Au NRs. UV-vis, zeta potentials and DLS characterizations of the stepwise preparation of bioconjugates. EIS characterization of the stepwise fabrications of the biosensor. Optimization of the experimental conditions. (PDF)

AUTHOR INFORMATION Corresponding Author * E-mail: [email protected]. * E-mail: [email protected] ORCID Jing-Juan Xu: 0000-0001-9579-9318 Author Contributions †N.Z. and H.G. contributed equally. The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT We gratefully acknowledge the National Key R&D Program of China (Grant No. 2016YFA0201200), and the National Natural Science Foundation (Grants 21535003) of China. This work

was also supported by Excellent Research Program of Nanjing University (ZYJH004).

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