Direct Observation of Oxidation Reaction via Closed Bipolar Electrode

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Direct Observation of Oxidation Reaction via Closed Bipolar Electrode-Anodic Electrochemiluminescence Protocol#Structural Property and Sensing Applications Jia-Dong Zhang, Lei Lu, Xiu-Fang Zhu, Li-Jing Zhang, Shan Yun, Chuan-Song Duanmu, and Lei He ACS Sens., Just Accepted Manuscript • DOI: 10.1021/acssensors.8b00736 • Publication Date (Web): 16 Oct 2018 Downloaded from http://pubs.acs.org on October 23, 2018

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Direct Observation of Oxidation Reaction via Closed Bipolar ElectrodeAnodic Electrochemiluminescence Protocol ： Structural Property and Sensing Applications Jia-Dong Zhang *, Lei Lu, Xiu-Fang Zhu, Li-Jing Zhang, Shan Yun, Chuan-Song Duanmu, Lei He * National & Local Joint Engineering Research Center for Deep Utilization Technology of Rock-salt Resource, School of Chemical Engineering, Huaiyin Institute of Technology, Huai’an, Jiangsu,223003, China *Corresponding author: Ph.D. Jia-Dong Zhang, Ph.D. Lei He E-mail address: [email protected]; [email protected]. Tel/Fax: +86 517 83559061

Abstract In this work, we developed an innovative closed bipolar electrode (BPE)electrochemiluminescence (ECL) sensing strategy with generality for target detection. Based on charge balance and 100% current efficiency between the closed BPE poles and the driving electrodes, one of the driving electrodes in one cell of the closed BPE system was employed as ECL sensing surface to reflect the target on the BPE pole in the opposite cell. Compared with traditional BPE-ECL sensing method, which in general adopted the anodic ECL reagents such as Ru(bpy)32+ and its co-reactant on one pole (anode) to reflect the target (occurring reduction reaction) on the other pole (cathode), the difference was that the targets occurring oxidation reaction could be detected by the anodic ECL reagents based on this strategy. To verify the feasibility of this strategy, the detection principle was stated firstly, and Fe(CN)64- as model target at anodic BPE pole were detected by anodic ECL reagents (Ru(bpy)32+ and TprA) on the driving electrode firstly. The ECL signals showed good performance for target detection. By changing the size and the material of the BPE pole where the targets were located, the detection of L-ascorbic acid (AA) uric 1 / 19

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acid (UA) and dopamine (DA) as other model targets with higher detection limit were accomplished. And visual and high-throughput detection of AA, UA, DA were also successfully realized by an array of the closed BPE system. This closed BPE (array) system is an effective supplement of traditional BPE-ECL sensing and could greatly expand the scope of the detection target. Key words: closed bipolar electrode; anodic electrochemiluminescence; driving electrode; oxidized targets; visual detection; high-throughput detection; universal platform Over the past two decades, bipolar electrode (BPE) has been adopted to be an effective tool for chemical and biological analysis1-6, materials synthesis7-9, and catalysts screening10-12, etc. In the field of chemical and biological analysis and catalysts screening, BPE is usually combined with electrochemiluminescence (ECL)13-16, electrochromism11, 17-19,

electro-fluorescence12, 20 as signal readout. In all of the signal readout manners, ECL

is the first and still the most popular reporting method for its overwhelming advantages, such as low background interference, simple operation process, high throughput and visual imaging capability1, 21. ECL is a kind of chemiluminescence at electrode surfaces generated by electrochemical excitation. Manz and co-workers first introduced the ECL reagent Ru(bpy)32+ as signal reporter at an open BPE1. Later Crooks and co-workers confirmed the strict quantitative relationship between the ECL signal on one pole of the open BPE and the reaction occurring on the other BPE pole2. Since then, BPE combining with ECL (BPEECL) has been developed for various purposes by many researchers3-4, 6, 14, 22-29. Most of the BPE-ECL applications, whether the structure is the open BPE or the closed BPE, are mainly based on two basic ways: target like co-reactants and quenchers at the same BPE pole with the ECL reagents16, or target at the opposite pole which is based on the charge balance between the two BPE pole30. And the latter first originated from the work of Crooks et al. is more universal. So far, Ru(bpy)32+ and luminol are the two ideal ECL reporting reagents in BPE application due to their high luminous efficacy and enough luminous intensity especially for visual imaging31. Nevertheless, both of the two ECL 2 / 19


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reporting reagents are excited at positive potential and thus occur on the anodic pole of BPE. It means that based on the charge balance of the BPE poles, the targets would be at the cathodic pole and occur reduction reaction. For the targets occur oxidation reaction, it is hard to get directly detection based on the above BPE-ECL sensing strategy. This undoubtedly limits the further application of BPE-ECL sensing strategy. Recently, Xu’s group developed a visual ECL ratiometry for the detection of prostate specific antigen (PSA) on a closed bipolar electrode (BPE), of which CdTe QDs with high efficient was synthesized at BPE cathode15. It is a very good try but due to the particle characteristic of CdTe QDs, the light intensity seems not to reach good uniformity. Wang’s group developed a full-featured ECL sensing platform based on a multichannel closed bipolar system, which also could detect the target occurred oxidation reaction but need complicated structure32 . Herein, a new type of closed BPE system was constructed, whose driving electrode was used for ECL signal readout for target detection. Based on charge balance and 100% current efficiency between the two closed BPE poles and the driving electrodes, one of the driving electrode was employed as ECL sensing surface to reflect the target at the BPE anodic pole in opposite cell. The mechanism of the closed-BPE using driving electrode as ECL signal reporting readout is stated detailedly. Fe(CN)64-, L-ascorbic acid (AA), uric acid (UA) and dopamine (DA) as model targets were detected on the anodic BPE pole by the anodic ECL signal on the driving electrode. And based on the same detection strategy, high-throughput visual detection of AA, UA and DA was also successfully carried out by ECL imaging with a closed BPE array system. This innovative and universal closed-BPE (array) system could use anodic ECL reagents to detect the target occurring oxidation process, which will certainly expand the application of the BPE-ECL sensing. Experimental Section Materials and reagents Potassium ferrocyanide (K4Fe(CN)6•3H2O) , L(+)-Ascorbic acid (AA), uric acid (UA) 3 / 19


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and dopamine (DA) were obtained from Aladdin (Shanghai, China). Tris(2,2’-bipyridyl) ruthenium(II) hexahydrate (Ru(bpy)3Cl2·6H2O) and tripropylamine (TPrA) were received from Sigma-Aldrich (St. Louis, MO, USA). Ag-AgCl paste was purchased from Shanghai Poly Long Electronic Technology Co. Ltd (Shanghai, China). Grapheme oxide (1 mg/mL) was got from XFNANO (Nanjing, China). Polydimethylsiloxane (PDMS) Sylgard 184 was from Dow Corning (Midland, MI, USA). ITO-coated glass (thickness≈100 nm, resistance < 17 W/m2) was obtained from Zhuhai Kaivo Electronic components (Zhuhai, China). HAc-NaAc (0.1 M, pH=5.0) was used for the solvent for AA, UA and DA. And PBS buffers (0.1 M, pH 6.0) was applied for the buffer of ECL reagents. All chemicals of analytical reagent grade were used as received without further purification. Instruments Electrochemical experiments were performed with a CHI660E electrochemical workstation (Shanghai Chenhua, China). ECL signals were measured by a home-made electrochemical and chemiluminescent analytical system (Nanjing University, China). In the home-made system, the photomultiplier tube (PMT, model C105, Beijing Hamamatsu photon techniques Inc., Beijing, China) was connected to an analog to digital converter and the electropherograms were recorded. A normal camera with the resolution of twenty million pixels on the mobile phone, Oppo R11, (China) was applied for ECL imaging, and the results were analyzed by Image-Pro Plus (IPP) 6.0 software. Fabrication of the closed-BPE system and the closed-BPE array system The closed-BPE and its driving electrodes system were fabricated by microchip manufacturing technology as mentioned in the previous work33-34 . The diagrammatic sketch of the fabricated system connecting with a potentiostat (CHI660E) is shown in Scheme 1c. And closed-BPE systems with different materials and sizes of electrodes were fabricated; in each system, cell 1 and cell 2 are both in the same sizes with 12 mm in length, 5 mm in width and 2 mm in depth. The materials and sizes of the electrodes in these systems were showed in Table 1. The Ag/AgCl electrodes were prepared by coating Ag/AgCl paste 4 / 19


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on the ITO substrates. The closed-BPE array system for ECL imaging as shown in Fig 4A was the array of closed-BPE systems and adopted the same fabrication method. Electrochemical behavior comparison of the closed-BPE system with the conventional three-electrode system As Ag/AgCl material was used as pole 1 in scheme 1, for electrochemical behavior comparison, 200 μL Ru(bpy)32+/TprA (5mM/25mM) solution was added to cell 1 of the closed BPE system and 200 μL Fe(CN)64- (5 mM, 1 KCl) solution was added to cell 2, electrochemical behaviors of the closed BPE system were recorded via cyclic voltammetry (CV) from 0 V to 1.5 V (0.05V/s). A conventional three-electrode system in cell 2 (Scheme 1d) was also constructed by welding the WE wire onto the middle of the BPE. By this change, the original CE, RE and WE wires became connecting with 3 electrodes of the three-electrode system in cell 2. Electrochemical behaviors of 5 mM Fe(CN)64- in the threeelectrode system were recorded via CV. The CVs with Fe(CN)64- (1mM and 5mM) in cell 2 and Ru(bpy)32+/TprA (5mM/25mM) in cell 1 in the closed BPE system under different material and size of pole 1 in table 1 were also recorded. ECL measurements of the closed-BPE system employing the driving electrode as the ECL reporting interface For ECL measurements, Fe(CN)64- (200 μL, 1M KCl) and AA, UA, DA in NaAc-HAc solutions (200 μ L, 0.1 M, pH 5.0) were used respectively as targets. During measurements, targets in different concentrations were added to the sensing cell (cell 2) and the ECL reporting reagents (Ru(bpy)32+/TprA, 5 mM/25 mM for detection of Fe(CN)64and AA, 2.5 mM/12.5 mM for detection of UA and DA) in PBS buffer (200 μl, 0.1 M, pH 6.0) was added to the reporting cell (cell 1). The size of pole 2 for the detection of Fe(CN)64- was 1 mm while that for AA UA and DA were 3 mm. CVs from 0.25 to 1.75V (0.05V/s) for Fe(CN)64-, from 0.2 V to 1.7 V (0.05V/s) for AA, from 0.1 to 1.6 V for UA, and from -0.3 V to 1.2 V for DA were applied respectively by the potentiostat (CHI 660E). Besides, for the detection of DA, grapheme oxide (10 uL, 1 mg/mL) was covered on the 5 / 19


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surface of ITO pole 2 and then reduced to reduced grapheme oxide (rGO) by the threeelectrode system in cell 2 (scheme 1d) with 5 times cyclic voltammetry (0 ～ -1.2V). During the electrochemical process, emission intensities of the ECL signals on the driving electrode (electrode 1 in scheme 1c) were also recorded by the PMT. For the measuring accuracy, cell 2 was rinsed with the target solution before each measurement. ECL imaging for the detection by the closed-BPE array system A closed-BPE array system with 5 units was constructed as shown in Fig.3. AA with different concentrations (1 μM, 2μM, 5μM, 8μM, 10μM) in NaAc-HAc solutions (200 μL, pH=5.0) were added to the sensing cell (cell 2) of each unit, while the ECL reporting reagents (Ru(bpy)32+/TprA, 5mM/25mM) in PBS buffers (200 μL, 0.1 M, pH 6.0) were injected into the reporting cell (cell 1) of each unit. Optimizing the driving voltage by increasing the driving voltage 0.1 V per time and then recording the ECL imagings from the driving electrodes array by the digital camera of the mobile phone (Oppo R11, China). Analogously, UA with different concentrations

in NaAc-HAc solutions (200 μL,

pH=5.0), and DA with different concentrations in NaAc-HAc solutions (200 μL, pH=5.0) were detected by the closed BPE array system based on the same strategy. Results and Discussion Working principle for the closed-BPE system using driving electrode as ECL reporting interface

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Scheme 1. The mechanism of BPE-ECL sensing with a typical closed-BPE system based on charge balance (a), a new-style closed-BPE system using driving electrode as reporting interface (b), and a separated CE and RE (b) in order to compare electrochemical signals with three-electrode system (d) .

Scheme 1a illustrates the most common detection principle for closed BPE-ECL sensing using Ru(bpy)32+ and TprA as ECL reagents which have be employed by both open and closed BPEs in many studies31, 35-37. In these systems, nonpolarized electrode materials such as Ag/AgCl or conventional electrode materials with large areas (Pt, Au, ITO) were employed as the two driving electrodes to supply stable potential for decreasing the impact from the driving electrodes. By adding the ECL sensing reagents in cell 1 and the target in cell 2, the target on pole 2 can be reflected by the ECL signals from pole 1 under a proper driving voltage based on the charge balance between the two poles2, 22. When high ionic strength electrolyte is existed in the solution of cell 1 and cell 2, the solution resistance in cell 1 and cell 2 could be ignored and the driving voltage (Etot) in scheme 1a can be expressed as23: Etot (scheme1a) = E1 (Ag/AgCl, Cl-) - Epole1 (Ru(bpy)32+/TPrA+, Ru(bpy)32+/TPrA) + Epole2 (target reduced ) - E2 (Ag/AgCl, Cl-) =

(E1 (Ag/ AgCl, Cl-) - E2 (Ag/AgCl, Cl-)) + (Epole2 (target reduced ) - Epole1 (Ru(bpy)32+/ TPrA +,

Ru(bpy)32+/ TPrA))

≈ K + (Epole2 (target reduced ) - Epole1 (Ru(bpy)32+/ TPrA +, Ru(bpy)32+/ TPrA))

(1)

Due to Ag/AgCl electrode can offer stable potential , their difference value is also equal to a constant number (K) approximatively. The driving voltage mainly refer to the potential difference of pole 1 and pole 2. The ECL signal on pole 1 is applied to reflect the target based on charge balance. Unlike the type of open BPE, only electronic current (no ionic current, scheme 1a) can path through two poles of the closed BPE, which means the absolute value of current on the BPE poles are also equal to those of the two driving electrodes in the closed-loop circuit. That is to say, the charge balance not only exists in the two BPE poles, but also exists in the whole closed loop circuit theoretically. The current should be equal everywhere, including the two driving electrodes (electrode 1, electrode 2) 37. Scheme 1b is similar to scheme 1a, the only difference is that nonpolarized Ag/AgCl electrode is not employed for 7 / 19


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electrode 1 but for pole 1. By this change, when the anodic ECL signals by Ru(bpy)32+/TprA occur on electrode 1, the detection target on pole 2 should be occur the process of oxidation. The driving voltage here is: Etot (scheme 1b) = E1 (Ru(bpy)32+/ TPrA +, Ru(bpy)32+/ TPrA) - Epole1 (Ag/AgCl, Cl-) + Epole2 (target oxidized) - E2 (Ag/AgCl, Cl-) (Ag/AgCl, Cl-))

= (E1 (Ru(bpy)32+/ TPrA +, Ru(bpy)32+/ TPrA) + Epole2(target oxidized)) - (Epole1 (Ag/AgCl, , Cl-) + E2 ≈ (E1 (Ru(bpy)32+/ TPrA +, Ru(bpy)32+/ TPrA) + Epole2(target oxidized)) - K’

(2)

Their sum value of Ag/AgCl electrode is also equal to a constant number (K’) approximatively, the driving voltage mainly refers to the sum of potentials on driving electrode 1 and pole 2. Theoretically, the oxidized process of the target on pole 2 can also be reflected by the ECL reagents on driving electrode 1 like that of Scheme 1a. For the convenience of comparing the electrochemical behavior with the conventional three-electrode system that we are used to in the following work, CE and RE in scheme 1b is separated as shown in scheme 1c. The driving voltage is similar to that of scheme 1b and can be expressed as33: Etot (scheme 1c) = E1 (Ru(bpy)32+/ TPrA +, Ru(bpy)32+/ TPrA) - Epole1 (Ag/AgCl, Cl-) + Epole2 (target oxidized) = (E1 (Ru(bpy)32+/ TPrA +, Ru(bpy)32+/ TPrA) Ru(bpy)32+/ TPrA)

+ Epole2(target oxidized)) - Epole1 (Ag/AgCl, , Cl-) ≈ (E1 (Ru(bpy)32+/ TPrA +,

+ Epole2 (target oxidized)) - K’’ = Epole2 (target oxidized) + (E1 (Ru(bpy)32+/ TPrA +, Ru(bpy)32+/ TPrA)-

K’’)

(3)

The potential on pole 2 could also be treated as constant (K’’), and the driving voltage also mainly refers to the sum of potentials on electrode 1 and pole 2. Similarly

to

traditional BPE-ECL sensing strategy, based on the charge balance between the BPE pole 1 and the driving electrode 1, the anodic ECL signal by Ru(bpy)32+/TprA on driving electrode 1 in theory also could reflect the target on pole 2 which occurs oxidation reaction. And all the data below were based on connection manner in the scheme 1c. Advantage of using pole 1 with Ag/AgCl material for the BPE-ECL sensing

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Fig 1. The voltammetric responses of the closed-BPE system for the oxidation of 5 mM Fe(CN)64- (1 M KCl) in cell 2 while Ru(bpy)32+ / TprA (5mM/ 25mM) in cell 1(red line). And the corresponding responses of 5 mM Fe(CN)64- (1 M KCl) in the conventional three-electrode system of scheme 1d (black line) (A). The CVs of 1mM (green line) and 5 mM (red line) Fe(CN)64- oxidation on pole 2 and Ru(bpy)32+/ TPrA (5 mM/25 mM) oxidation on electrode 1 with different electrode sizes and materials of pole 1 (B)(C)(D) (see Table 1).

The oxidations of Fe(CN)64- on pole 2 and anodic ECL reagents on driving electrode 1 was used as an example to study the electrochemical behavior of the closed BPE system. As shown in Fig 1A, the voltammetric response (CV, red line) is shifted to more positive potential although the shape is some similar to that of oxidation of Fe(CN)64- (black line) in the compared three-electrode system in scheme 1d. This was mainly due to existence of the reactions in cell 1, extra driving voltage ((E1

(Ru(bpy)32+/ TPrA +, Ru(bpy)32+/ TPrA)-

K’’) in

equation 3, that is 1.15 V in Table 1) was needed before the oxidation of Fe(CN)64- on pole 2. The potential peak is lower while the width of CV is wider than that of the compared three-electrode system (Fig 1A), which might be derived from the limitation on the transferring speed of ions and electrons, etc. The above results is some similar to reported researches by Zhang’s group25, 38-39 and Xu’s group33, where they had confirmed that under a certain condition, the voltammetric response of the bipolar electrode system has a similar sigmoidal shape as a conventional two or three electrode system. 9 / 19


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Table 1. The diameters (D.) and materials for the electrodes in the closed BPE system.

The impacts of difference material and size of pole 1 was investigated (Fig 1B-D) by studying the shift of peak potentials under different concentration of target in cell 2. Table 1 is the diameters and materials of pole 1 in the closed BPE system. Other things being equal, we could intuitively find out that the difference value of the peak potential using Ag/AgCl as pole 1 (Fig 1B) was smaller than those with ITO as pole 1 (Fig 1C-D) under different concentration of Fe(CN)64- in cell 2. The difference value shown in Table 1 was only 0.06 V for Ag/AgCl as pole 1 while up to 0.25 V for the same size of ITO. As the diameter of ITO was decreased to 1 mm, the difference value would further increase to 0.28 V. The results clearly indicate that using Ag/AgCl material as pole 1 own stable potential in the face of the changing of current. For BPE-ECL sensing especially for visual detection, the detection is usually under the same driving voltage for different concentration of the detection target. Thus , smaller potential shift is conducive to get a better linear range. From this result, it can be concluded that Ag/AgCl is a more competent material employed in pole 1 to avoid potential drift. BPE-ECL sensing using driving electrode as ECL reporting interface

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Fig 2. ECL intensity on the driving electrode (electrode 1) for different concentrations of Fe(CN)64- (1～ 5mM) in cell 2 (A) and AA (0.5 ～ 100 μM) in cell 2 (C). The calibration curve for ECL intensity and concentration of Fe(CN)64- (B). And the relationship between the concentration of AA and ECL signals (D). Inset: Calibration curve for AA detection. Error bars were obtained from three experiments and the RSD of each point was less than 6.2%. scan rate: 0.05 V/s.

Fig 2A is the corresponding ECL intensities of the anodic ECL reagents (Ru(bpy)32+ /TprA, 5mM/ 25mM) on the electrode 1 as different concentration of Fe(CN)64- was oxidized on pole 2. The range of the driving potential (0 ～ 1.6V) depended on the corresponding electrochemical signal of the closed BPE system. As shown in Fig S1, due to the existence of potential difference in cell 1 (E1 (Ru(bpy)32+/

TPrA +, Ru(bpy)32+/ TPrA)-

K’’ in

equation 3), the peak potentials about Fe(CN)64- move to the more positive potentials but still hold the similar shapes to that in the three-electrode system (Fig 1A) 25, 38-39. We could find that the change of ECL intensity on electrode 1 (Fig 2A) is very similar to that of current (Fig S1), and increases with the increase of the concentration of Fe(CN)64-. Though the peak potentials in Fig S1 and ECL intensities in Fig 2A have a little bit of inconsistency (about 0.1V) due to the complex reaction between Ru(bpy)32+ and TPrA25 , the peak 11 / 19


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potentials are the desired driving potentials and the peak ECL intensities are those we need to record. The increment of ECL signals at the each peak intensity are approximately linear (Fig 2B). These results confirm that the driving electrode of the closed BPE system was successfully employed for ECL sensing. And more importantly, anodic ECL reagents such as Ru(bpy)32+ /TprA is applicable for targets oxidized during the detection in the closedBPE system. The detection concentration in Fig 2A is the level of mM, which is not sensitive enough for many targets detection. Something must be done if we want to detect lower detection range (such as the level of μM or even lower). As is known to all, based on derivation of Fick’s laws, current is proportional to the area of electrode and the concentration of reactant. And because of the charge balance between pole 2 and electrode 1, and the lower concentration of the targets on pole 2 compared with the ECL reagents (Ru(byp)32+/TPrA, mM/mM) on the electrode 1, the current of the closed BPE system would be mainly limited by pole 2 rather than electrode 133, 38-39. That is to say, pole 2 is the limited pole. Thus the size and material of pole 2 would heavily affect the current of the whole system25, which in turn determines the ECL detection range of the targets due to their positive correlation. AA, UA and DA with lower concentrations were used as the representatives. When we simply increased the size of the pole 2 (Table 1) to 3 mm in diameter, the current of the closed BPE system increased heavily for the same concentration of UA (50 μM) in cell 2 (Fig S2). As shown in Fig 2C, AA was detected by ECL intensity on the driving electrode 1. As low as 0.5μM AA on pole 2 could be reflected by ECL signal on driving electrode 1. And the detection limit was 0.3 μM. The ECL intensity increases along with the increase of AA concentration. Although some deviation occurs during the increasing, linear increments are still kept in the range of 0.5～10 μM and 50～100 μM as shown in inset of Fig 2D.

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Fig 3. ECL intensities on electrode 1 with fixed concentration of ECL reagents (2.5 mM/ 12.5 mM Ru(bpy)32+ /TprA) in cell 1 and different concentrations of UA (2～150μM) (A) and DA (0.5 ～ 200 μ M) (C) in cell 2. The relationships between the concentration of UA and ECL signals (B), DA and ECL signals (D). Insets of (B) and (D) are the the calibration curve for ECL intensity and corresponding concentration. Error bars were obtained from three experiments..

Similar to the detection of AA, as shown in Fig 3A, the ECL intensity on the driving electrode 1 also increased with the increasing concentration of UA. Fig 3B is the relationship between the peak ECL intensities and the concentrations of UA in cell 1. The inset shows that a good linear increment between 2～60μM. The RSD of all the points was less than 9.8%. Besides by increasing the size of pole 2 to increase the current of closed BPE system, another strategy was also employed to increase the ECL detection sensitivity. As shown in Fig S3, when the naked ITO electrode of pole 2 was covered with reduced graphene oxide (rGO), the current of the closed BPE system would increase and the peak potential would decrease due to the catalytic action of rGO to DA. Fig 3C is the ECL intensities on electrode 1 for different concentrations of DA in cell 2. The peak ECL intensities have positive 13 / 19


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correlation with the concentration of DA (Fig 3D) and have linear increment between 0.5～ 20μM and 60～200μM. The RSD of all the points was less than 7.6%. Visual detection with the closed-BPE array system

Fig 4. The schematic diagram for visual detection with the closed-BPE array system using driving electrodes as ECL reporting interface (A). The ECL images on the driving electrodes for the detection of AA (1, 2, 5, 8, 10μM) on pole 2 (B) and the calibration curve (C), UA (2, 10, 20, 30, 40μM) on pole 2 (D) and the calibration curve (E), DA (1, 10, 20, 30, 40 μM) on pole 2 (F) and the calibration curve (G).

An advantage of the BPE-ECL sensing is that it could realize high throughput detection by visual imaging techniques. A closed BPE array system with 5 parallel unit were constructed and Fig 4 A is the schematic diagram. Various concentrations of AA were added to cell 2 and its 4 parallel cells and would be oxidized on pole 2 and its parallel poles in the array at proper driving voltage. The diameter of pole 2 in Table 1 was further increased to 4 mm in order to increase detection sensitivity. During the experiment, the driving voltage started at 0 V and when the voltage reached 1.3 V, the ECL imaging from 14 / 19


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the ECL reagents (Ru(bpy)32+/TprA, 5mM/25mM) on electrode 1 corresponding to the minimum concentration of AA (1 μM) began to be visible. 1.4 V was chosen as the driving voltage for the need for visual detection. The ECL images on 5 driving array electrodes were recorded by the camera (twenty million pixel) of a common smartphone (Oppo R11, China). As shown in Fig 4B, we can see the changing of ECL lightness corresponding to the different concentrations of AA. The relationship between the ECL lightness and the AA concentration is shown in Fig 4C, which exhibits a linear relationship from 1 μM to 10 μM. The RSD for three experiments was less than 14.2%. Likewise, UA and DA were also detected by the same strategy. Fig 4D is the ECL images by the ECL reagents (Ru(bpy)32+/TprA, 2.5mM/12.5mM) on the driving array electrodes corresponding to the different concentrations of UA on pole 2 and its parallel poles. The ECL lightness captured by a smartphone (vivoX20, China) at the driving voltage of 1.4 V shows increasing intensity with the increase of the UA concentration (Fig 4D), and has acceptable linear relation with the UA concentrations between 2～40μM (Fig 4E). For the detection of DA, the ECL lightness at the driving voltage of 1.5 V increases with the increase of the DA concentration (Fig 4F), and has positive linear relation with the DA concentrations between 1～40μM (Fig 4G). In a word, high-throughput and visual detection for the oxidation process of targets could also be realized by anodic ECL reagents with the closed BPE array system. Conclusions In summary, we developed an innovative closed bipolar electrode (BPE)electrochemiluminescence (ECL) sensing strategy for target detection, in which the anodic ECL reagents on the driving electrode of the closed BPE system could detect the target oxidized on the BPE pole in the opposite cell. Theory and experimental evidence both proved the feasibility of the sensing strategy. Ag/AgCl material as pole 1 of the closed BPE system could effectively decrease the potential drift and showed better performance than the common ITO material. Fe(CN)64- , AA, UA and DA oxidized in the detection process 15 / 19


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as model targets, were successfully detected by the ECL reporting reagents (Ru(bpy)3Cl2·6H2O and TprA) on the driving electrode. Furthermore, high throughput and visual detection was also achieved by the array of closed-BPE system. This closed BPE (array) system provided an innovative and universal detection method based on BPE-ECL sensing and could greatly expand the scope of the detection target. ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssensors…… The corresponding electrochemical signals of the closed-BPE system with different concentrations of Fe(CN)64-, sizes of pole 2 and materials of pole 2. AUTHOR INFORMATION Corresponding Authors *E-mail: [email protected] *E-mail: [email protected] ORCID Jia-Dong Zhang:0000-0001-5231-347X Notes The authors declare no competing financial interest. Acknowledgements This work was supported by the National Natural Science Foundation of China (Grant No. 21605055, Grant No. 51704123); the Natural Science Foundation of Jiangsu Province (Grant No. BK20160424, BK20160425).

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For TOC only:

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Direct Observation of Oxidation Reaction via Closed Bipolar Electrode

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