Bidirectional Electrochemiluminescence Color Switch: An Application

Feb 8, 2018 - On the basis of BPE arrays coupled with the ECL switch, the detection of three biomarkers of prostate cancer, PSA, microRNA-141, and ...
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Cite This: Anal. Chem. XXXX, XXX, XXX−XXX

Bidirectional Electrochemiluminescence Color Switch: An Application in Detecting Multimarkers of Prostate Cancer Yin-Zhu Wang, Si-Yuan Ji, Heng-Yu Xu, Wei Zhao,* Jing-Juan Xu,* and Hong-Yuan Chen 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, P. R. China S Supporting Information *

ABSTRACT: A selective excitation of [Ir(df-ppy)2(pic)] and [Ru(bpy)3]2+ through tuning the electrode potential is reported in this work. Bidirectional color change from bluegreen to red could be observed along with increase and decrease of the potential, which was ascribed to the dualpotential excitation property of [Ir(df-ppy)2(pic)]. Similar to the three-electrode system, selective excitation of ECL could be achieved at the anode of the bipolar electrode (BPE). Both increase and decrease of the faradic reactions at the cathode of the BPE could induce ECL reporting color at the other pole switched from blue-green to red. We applied a closed BPE device for the bioanalysis of multicolor ECL since the organic solvent containing electrochemiluminophores could be separated from the bioanalytes. On the basis of BPE arrays coupled with the ECL switch, the detection of three biomarkers of prostate cancer, PSA, microRNA-141, and sarcosine were integrated in a same device. The cutoff values of the biomarkers could be recognized directly by the naked eye. Such a device holds great potential in the early diagnosis of prostate cancer.

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which promotes electrochemical reactions at its extremities. [Ru(bpy)3]2+ and [Ir(ppy)3] mixture in organic solvent was added in the anodic cell. Similar to a three-electrode system, selective excitation of the electrochemiluminophores could be achieved by tuning the interfacial potential (Δϕa) at the poles of BPE. By decreasing the resistance of BPE, [Ru(bpy)3]2+ and [Ir(ppy)3] with distinct oxidation potentials were successively excited with unidirectional change of color from green to yellow and red at the anode. As the first multicolor ECL device in biological analysis, the BPE based sensor was utilized to determine prostate-specific antigen (PSA) with remarkable color variation. In addition to the BPE resistance-resolved multicolor ECL, reactions at the cathode could be readout through the emission color at the reporting pole. Herein, bis(3,5-difluoro-2-(2pyridyl) phenyl-(2-carboxypyridyl) iridium(III), commonly abbreviated as FIrpic or [Ir(df-ppy)2(pic)] was adopted to mix with [Ru(bpy)3]2+ for multicolor ECL generation. As a blue phosphorescent material, [Ir(df-ppy)2(pic)], showed ECL with distinguished emission spectrum from [Ru(bpy)3]2+.9,10 The electrochemical property of [Ir(df-ppy)2(pic)] was carefully studied.11,12 In this work, we found that different from most reported Ru(II) and Ir(III) luminophores, Ir(dfppy)2(pic) exhibited two distinct ECL excitation potentials

lectrochemiluminescence (ECL) is a potential initiated form of chemiluminescence, where luminophores are oxidized or reduced at the surface of electrode and create emissive excited-state products.1 The Ru (II) complex, [Ru(bpy)3]2+ with red emission centered at 620 nm, is the most commonly used electrochemiluminophore, because of its high ECL efficiency at low voltage, long excited-state lifetime and easy synthesis.2 In recent years, iridium(III) complexes have been investigated as new ECL reagents and have attracted great attention, since they cover a much broader spectral distribution from blue to red through tuning the ligands, and show high emission quantum yields by controlling the HOMO and LUMO levels, which could reach ten-fold compared to that of [Ru(bpy)3]2+.3,4 Owning to the high ECL efficiencies of Ru(II) and Ir(III) luminophores with tri-n-propylamine (TPrA) as a coreactant and their multiple variation in emission wavelengths, considerable efforts have been devoted in tuning ECL emission color of the concomitant electrochemiluminophores via controlling the applied potential, which showed perspective in luminescent devices.5,6 However, although the selective excitation of electrochemiluminophores was highly expected to open new avenues for multianalyte ECL detection, over the years, the application in this area has remained limited. The major drawback is the weak solubility of Ir complexes in water, which limits their biological application.7 Recently, we reported a multicolor ECL device based on a closed bipolar electrode (BPE).8 In a closed BPE system, two electrochemical cells are physically separated from one another, and the BPE serves as the only current path between the cells © XXXX American Chemical Society

Received: January 2, 2018 Accepted: February 8, 2018 Published: February 8, 2018 A

DOI: 10.1021/acs.analchem.8b00014 Anal. Chem. XXXX, XXX, XXX−XXX

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Modification of the BPEs Arrays. Three BPEs were parallel connected in the system. For PSA determination, sensing interface was first incubated with 60 μL peptide by Au NPs via cysteine (C) at the end of the peptide, and incubated 4 °C for overnight. Then, it was washed thoroughly with washing buffer. Second, the electrode of peptide was further incubated with 80 μL 1.0 mg/mL EDC and 0.25 mg/mL NHS to activate its terminal carboxylic acid groups for 30 min at 4 °C, followed by washing with 10 mM PBS (pH 7.4) three times. The activated peptide was incubated with 60 μL Fc-PAMAM dendrimers at 4 °C for 2 h with further careful washing to remove unbounded residue. For miRNA-141 analysis, the hairpin DNA was denatured for 2 min at 95 °C and slowly cooled to room temperature to form stem-loop structures prior to use. Prior to probe immobilization, the thiol-modified hairpin capture probes (20 μL, 50 μM SH−CP-141) were mixed with 10 μL of 100 mM TCEP in a microcentrifuge tube and incubated for 60 min at room temperature to reduce the disulfide bonds of the SH−CP. The mixture was then diluted to a total volume of 100 μL with 0.1 M PBS, and 30 μL of the diluted mixture was incubated with BPE cathode for 2 h at 37 °C in the dark. After washing by 0.1 M PBS, the electrode surface was blocked with 1 mM MCH solution for 1 h and rinsed with the water. For sarcosine determination, 60 μL 0.175 M L-cysteine was incubated at the surface of AuNPs modified ITO for approximately 4 h allowing the formation of carboxyl group on Au surface. 2.0 mg EDC and 1.0 mg NHS were then added and kept for 2 h. Then 60 μL of SOD solution (1 mg/mL) were deposited on the surface of BPE cathode at 4 °C during 20 h and rinsed with PBS. Procedure of ECL Measurement. The PSA sensing surface (first BPE) was incubated with 60 μL PSA solutions for 30 min at 37 °C; followed by a thorough washing with 10 mM PBS (pH 7.4); meanwhile, the cathode of second BPE was incubated with 60 μL solution containing miRNA-141 and 0.05 U DSN in 10× DSN master buffer (500 mM Tris-HCl, 50 mM MgCl2, 10 mM DTT, pH 8.0) for 30 min at 37 °C. The electrode was then carefully washed to remove unreacted residue. Finally, before detection, 60 μL sarcosine solution (5 μM-3 mM) was directly injected in the cathodic reservoir of the third BPE. Subsequently, the cathode reservoirs of the BPEs were filled with 1× PBS (first BPE), 1× Tris-HCl (second BPE) and 0.1× PBS solution (third BPE), and the anode reservoirs were filled with acetonitrile containing 0.1 M TBAPF6 with a mixture of 2.5 mM [Ir(df-ppy)2(pic)], 0.125 mM [Ru(bpy)3]2+ and 37.5 mM TPrA. RGB Analysis. ImageJ was used to automate the cropping and analysis of the images. Images were cropped to the smallest circular area containing the emissive electrode areas, this resulted in a 100 × 100 pixel image. The mean RGB values were measured for the circular area of the electrode using the “Measure-RGB Values” function built into ImageJ.

using TPrA as a coreactant, which followed different ECL generation pathways. As the excitation potential of [Ru(bpy)3]2+ is in between those two of [Ir(df-ppy)2(pic)], fascinatingly, along with the increasing potential, the emission color presented bidirectional change (blue-green to red to bluegreen). Therefore, either decrease or increase of faradaic current in the system could be read out by the color switching from blue-green to red, which enabled us to detect multiple prostate cancer biomarkers on a BPE array with different strategies. With a single DC power supply, prostate-specific antigen (PSA), circulating microRNA-141 (miRNA-141), and small molecular marker, sarcosine, were simultaneously detected by the array. The proposed method with intuitive observation mode may greatly improve the accuracy of prostate cancer diagnosis.

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EXPERIMENTAL SECTION Synthesis of Ferrocene-PAMAM Dendrimers (FcPAMAM). Ferrocenyl-tethered PAMAM dendrimer was synthesized by an imine formation reaction between partial primary amines of NH2-terminated G4 PAMAM dendrimer and ferrocenecarboxylic acid, as previously described. 2.0 mg of imidazole was first added into 200 μL of 1 mM Fc, and the solution was adjusted to pH 7.4 by 1.0 M HCl. Next, 20 mM EDC and 10 mM NHS was then added into this prepared solution to activate those carboxylic groups on Fc. The reaction mixture was slowly stirred at 37 °C for 2 h. A solution of PAMAM (50 μL, 4 × 10−6 M) was slowly added, and the resulting solution was stirred for 2 h. The Fc-PAMAM solution was dialyzed with a cellulose dialysis sack having a molecular weight cutoff of 12 000 to remove impurities. Finally, the collected products were stored at 4 °C for later use. BPEs Array Design and Fabrication. In brief, a piece of ITO was first cut into small slices (6 × 7 cm2). For screenprinting, oil ink (essential ingredient of acrylic resin) was transmitted through the silk-screen mold onto the ITO layer by brush. Once the planar electrodes were completed, a wet chemical etching procedure was carried out with chemical solution (HF/NH4F/HNO3 = 1:0.5:0.5), which produced the desired ITO electrodes. Those ITO electrode slices were cleaned by immersion in a boiling solution of 2 M KOH in 2propanol for 20 min, followed by washing with milli-Q water. To obtain the PDMS molds with one or more predesigned channels, SG-2506 borosilicate glass was fabricated by traditional photolithography and wet chemical etching techniques. SG-2506 borosilicate glass plates were exposed to UV radiation under a mask with the designed patterns, followed by developing with a 0.5% NaOH solution, the Cr layer was then removed by a 0.2 M ammonium cerium(IV) nitrate solution. Etching of these glass plates was carried out in 1 M HF-NH4F solution (40 °C) with a water bath for 28 min. After that, designed channel structure molds were obtained. Then degassed PDMS was cast on these glass masks for 1 h in 80 °C. After cooling at room temperature, the PDMS was stripped from the masks, producing the PDMS molds. The PDMS molds were drilled with circular holes as reservoirs. Finally, to protect the cathode of the electrodes (both driving electrode and BPE) from damage at negative potential, gold films were deposited in situ. All of the reservoirs were filled with 30 μL HAuCl4 (2.4 mM), then a driving voltage (5.0 V) was applied for 300 s, finally the electrode was rinsed with water and dried in the air.

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RESULTS AND DISCUSSION Preliminary Experiments of [Ir(df-ppy)2(pic)] and [Ru(bpy)3]2+. As a phosphorescent material, [Ir(df-ppy)2(pic)] was mostly adopted as one of the efficient organic light emitting diodes (OLEDs),13 and its ECL behavior was barely reported. We first recorded the cyclic voltammogram and ECL of [Ir(dfppy)2(pic)] in acetonitrile (containing 0.1 M TBAPF6). With 2.5 mM [Ir(df-ppy)2(pic)] and 7.5 mM TPrA, two ECL emission waves centered at ca. 0.54 and 0.99 V vs Fc0/+ were B

DOI: 10.1021/acs.analchem.8b00014 Anal. Chem. XXXX, XXX, XXX−XXX

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observed (Figure 1A), consistent with the oxidation potentials of TPrA and [Ir(df-ppy)2(pic)], respectively. At high potential

TPrA• + H+

→

(7)

[Ir(df‐ppy)2 (pic)] + TPrA• → [Ir(df‐ppy)2 (pic)]•− + P (8) •−

[Ir(df‐ppy)2 (pic)]

•+

+ TPrA → [Ir(df‐ppy)2 (pic)]* + P

[Ir(df‐ppy)2 (pic)]*

→

(9)

[Ir(df‐ppy)2 (pic)] + hv• (10)

Although the mechanism had been proven, excitation at the oxidation potential of TPrA was not a general phenomenon for most reported Ir(III) or Ru(II) complexes. Lee’s group reported high ECL efficiency of Ir (III) complexes by controlling their relative positions of HOMO and LUMO levels (oxidation potential and reduction potential),4 and it was found that the well matched reduction potential (Ered) of the metal complex and TPrA• made the donation of the electron more efficient. We recorded the CV of [Ir(df-ppy)2(pic)] in deoxygenated acetonitrile. The first reduction is ca. −2.29 V vs Fc0/+ (Figure S1 of the Supporting Information, SI), which is close to its reported electrochemical property,11,12 and matched the reduction potential (Ered) of TPrA•, −1.7 ± 0.1 V vs SCE (ca. −2.15 ± 0.1 V vs Fc0/+). Increasing the concentration of TPrA to reach visualized ECL emission, the quasi-reversible peaks of [Ir(df-ppy)2(pic)] were drowning in the oxidation current of TPrA, but we still observed strong ECL emissions of [Ir(df-ppy)2(pic)] at two potentials (Figure S2A). As for [Ru(bpy)3]2+, a notable ECL wave was only observed at 0.87 V vs Fc0/+ (Figure 1B), which followed the classic pathway.4 Although TPrA is a sufficiently powerful reductant to donate an electron to [Ru(bpy)3]2+ (Ered = −1.78 V vs Fc0/+),4 at the oxidation potential of TPrA, only a small wave showed, suggesting inefficient ECL emission via the TPrA oxidation pathway. With increased concentration of TPrA, the first wave was hard to be recognized and only one ECL emission peak was observed (Figure S2B). Similar to Bard’s report,10 with high concentration (∼mM) of [Ru(bpy)3 ]2+, a classic mechanism is the major pathway of its ECL emission. Since the ECL excitation potential of [Ru(bpy)3]2+ is in between the two of [Ir(df-ppy)2(pic)], we would expect more abundant color changes. Tuning ECL Emission Color by Electrode Potential. As a blue phosphorescent material, the PL spectrum of [Ir(dfppy)2(pic)] shows emission maxima at 465 nm (Figure S3). We examined its ECL spectrum at 0.6 V vs Fc0/+, the maximum of emission (λmax) located at 480 nm (Figure S3). Therefore, its ECL emission color is blue-green instead of pure blue. Spooling ECL spectra of [Ir(df-ppy)2(pic)] and [Ru(bpy)3]2+ were collected (Figure 2A). The blue-green emitter [Ir(dfppy)2(pic)] (λmax = 480 nm) started to be excited at 0.5 V vs Fc0/+, after a slow growth, suddenly increased at 0.95 V and reached maximum at 1.05 V. The red emission of [Ru(bpy)3]2+ (λmax = 620 nm) was increased from 0.65 V and reached platform at 0.85 V. The differences of the excitation potentials and emission wavelengths (Δλ ≈ 140 nm) of the two emitters are both distinguished. The ECL emission of [Ir(df-ppy)2(pic)] and [Ru(bpy)3]2+ over wide ranges of potential and emission wavelength were quickly recorded using a CCD camera. ECL images of [Ir(dfppy)2(pic)] and [Ru(bpy)3]2+ from the electrode surface by pulsing to different potentials are shown in Figure S4. The intensities of the ECL varied along with the increasing potential. The trends were consistent with ECL intensities

Figure 1. Cyclic voltammograms and their corresponding ECL signals of 2.5 mM [Ir(df-ppy)2(pic)] (A) and 0.125 mM [Ru(bpy)3]2+ (B) with 7.5 mM TPrA in acetonitrile containing 0.1 M TBAPF6. (scan rate, 100 mV/s; voltage of PMT, 200 V). Molecular structures of the two species are inserted.

of 0.99 V, it is generally accepted the mechanism follows the classic route:14 [Ir(df‐ppy)2 (pic)] TPrA TPrA•+

→

[Ir(df‐ppy)2 (pic)]+ + e−

TPrA•+ + e−

→ →

(2)

TPrA• + H+

[Ir(df‐ppy)2 (pic)]+ + TPrA•

[Ir(df‐ppy)2 (pic)]*

→

(1)

(3) [Ir(df‐ppy)2 (pic)]* + P

(4)

[Ir(df‐ppy)2 (pic)] + hv

(5)

→

At low potential of 0.54 V, the pathway of ECL could be explained by the mechanism proposed by Bard et al.,15 involving the injection of an electron from neutral radical TPrA• into the lowest unoccupied molecular orbital (LUMO) of [Ir(df-ppy)2(pic)] to form [Ir(df-ppy)2(pic)]−, which reacted with TPrA•+ and produced excited-state species [Ir(df-ppy)2(pic)]*. TPrA

→

TPrA•+ + e−

(6) C

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Figure 2. Spooling ECL spectra of 2.5 mM [Ir(df-ppy)2(pic)] (A) and 0.125 mM [Ru(bpy)3]2+ (B) generated at potentials from 0.4 to 1.15 V vs Fc0/+ in acetonitrile containing 37.5 mM TPrA and 0.1 M TBAPF6. (C) ECL images at the electrode surface at potentials from 0.55 to 1.05 V vs Fc0/+ for the mixture of the two emitters.

(Figure 2A, B), which could be easily observed with naked eyes. The mixture of the two emitters showed interesting bidirectional ECL color trends (Figure 2C), which changed from deep blue-green to red, then turned to light blue-green. The bidirectional variations were immediate and reversible, requiring only the selection of appropriate applied potentials. It should be noted that high concentration (>75 mM) of TPrA induced quenching of [Ir(df-ppy)2(pic)]* (Figure S5A), called as the ECL “switch-off” phenomenon, which was similar to that of [Ir(ppy)3] under coreactant ECL mode.8 Correspondingly, the ECL emission of [Ir(df-ppy)2(pic)] and [Ru(bpy)3]2+ mixture showed unidirectional change from blue-green to red along with the increasing potential (Figure S5B). To achieve more abundant color changes, the concentration of TPrA was optimized 37.5 mM. Bidirectional ECL Color Switch at BPE. Since the closed BPE could physically separate the organic solvent from analytes, it is a perfect choice for biological analysis.16 Similar to a three-electrode system, selective excitation of ECL could be achieved by tuning the interfacial potential (Δϕa) at the poles of BPE. Here we applied a closed BPE made of ITO glass and PDMS molds, and added different concentrations (10 nM to 0.1 mM) of ferrocene in the cathodic reservior, and the ECL emitters (2.5 mM [Ir(df-ppy)2(pic)] and 0.125 mM [Ru(bpy)3]2+) in the anodic reservoir (Figure 3A). After pulsing a potential of 5.0 V to oxidize Fc to Fc+, a reversed potential of 7.5 V was immediately applied. Photographs of the anode of BPE were taken with CCD and the total current (itot) of the system was recorded simultaneously. As shown in Figure 3B and C, along with the increasing concentration of ferrocene, the current flowing in this system increased, meanwhile, the ECL color changed from dark blue-green to dark pink, red, light pink, greyish, and light blue-green, which was close to the phenomenon achieved at working electrode by increasing the potential. It is a successful demonstration of the currentresolved multicolor ECL-BPE device. Since arrays of BPEs could be controlled with a single DC power,17 it is possible to integrate a batch of tests in one device and directly observe the results with naked eyes. In addition, with the bidirectional color change mixture, both increase and decrease of the current could be utilized as the color switch for semiquantitative analysis, which make the design of the sensing interface much easier. Analysis of Multimarkers of Prostate Cancer. On the basis of such concept, we developed a three channel BPE array to determine multiple tumor markers of prostate cancer. As is

Figure 3. (A) Schematic illustration of a closed BPE system. (B) ECL images and (C) total current of the closed BPE device with different concentrations of ferrocene.

well-known, prostate-specific antigen (PSA) is the most widely used biomarker for prostate cancer diagnosis.18 However, since it is often elevated in the presence of prostate cancer or other prostate disorders, only relying on the value of PSA may result in “overdiagnosis” and “overtreatment”. In 2009, Sreekumar published a work to discuss the potential of sarcosine in the evaluation of prostate cancer progression.19 Also circulating tumor cell and circulating microRNA have been reported for the diagnosis of cancer.20,21 Here, we chose three important prostate cancer biomarkers, serum markers PSA and circulating microRNA (miR-141), and urine marker sarcosine as the target analytes, and tried to do simultaneous detection of these three with the visualized ECL-BPE array. Principles of the detections are shown in Figure 4. Three of BPEs were parallel connected in the system. For the first BPE (BPE1), Fc-PAMAM labeled specific peptide (CHSSKLQK) was modified at the cathode. After incubation with PSA for 30 min, the peptide was cleaved, leading to the leaving of ferrocene from the surface of the BPE cathode and a decrease of the faradic current. Regarding the second BPE (BPE2), Fc labeled hairpin probes were selfassembled at the cathode. With addition of miR-141, the hairpin was opened, leading to the formation of RNA/DNA duplexes, and DSN subsequently cleaved duplexes to recycle the target of miRNA-141, which induced a decrease of current. As for the last BPE (BPE3), sarcosine oxidase (SOD) was modified at its cathode as a catalyst. Once sarcosine was added in the reservoir, it could be oxidized to hydrogen peroxide, which was reduced at the cathode and promoted the current flow through the BPE. Since the mixture of [Ir(df-ppy)2(pic)] and [Ru(bpy)3]2+ could exhibit color change from blue-green to red with both increase and decrease of the potential, we used them to observe the cutoff values of these biomarkers. As reported, the cutoff values of PSA, miRNA-141 in blood serum, and sarcosine in urine are 4.0 ng/mL,22−24 ∼ 10−14 M,25 and ∼10−6 M, respectively.26 After carefully optimizing the experimental conditions including driving voltage, incubation D

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Figure 4. Schematic illustration of closed BPE arrays and the sensing principles for three biomarkers.

miRNA-141 at 10−14 M, and sarcosine at 10−6 M yielded the relative standard deviations of 6.5%, 5.9%, and 4.0%, respectively (Figure S12), indicating good reproducibility of the method. We used real samples to test the feasibility of proposed multicolor ECL-BPE sensor. Human blood serums (from the Jiangsu Tumor Hospital) were adopted for the test of PSA. Since we did not have blood serum samples with clinical reference values of miRNA-141, we did standard addition of the microRNA in horse serum. And sarcosine was added in healthy human urine sample kindly donated by a PhD student. As shown in Table 1, for all these samples with different

time of miRNA-141, and concentration of the PBS buffer (Figures S6−S8), we got perfect color palettes for the semiquantitative analysis of multiple biomarkers with the naked eye. As shown in Figure 5, after addition of 4.0 ng/mL

Table 1. ECL Images for Determination of PSA in Human Blood Serum, miRNA-141 in Horse Serum, and Sarcosine in Human Urine Samples Figure 5. ECL images on BPE array with increased concentrations of PSA (0 to 25 ng mL−1); miRNA-141(0 to 1.0 nM); and sarcosine (0 M to 10−3 M).

PSA, 20 fM miRNA-141, and 1.0 M sarcosine in the cathodic reservoirs of the BPE arrays, the color of ECL emission turned from blue-green to pink. Along with the increasing concentration, the color turned darker. It should be noted that after full removal of the electroactive Fc from the surface, the ECL at BPE1 and BPE2 were set as red emission, therefore, with extremely high concentration of PSA and microRNA, the ECL emission switch will not be reversed. Similarly, for BPE3, under optimized condition, after adding 1.0 mM sarcosine (an unreachable high concentration in urine), the ECL emission was red. For quantitative analysis, we applied an R/G value based on the ECL images. Good linear relationships between the R/G value and the target concentration were achieved in the ranges of 1.0−25 ng/mL for PSA (RSD = 0.990) (Figure S9), 10−15 M to 10−10 M for miRNA-141 (RSD = 0.992) (Figure S10), and 5 × 10−7 M to 5 × 10−4 M for sarcosine (RSD = 0.995) (Figure S11). The reproducibility of the multiple biomarker sensor was also examined with RGB analysis. Six repetitive measurements of PSA at 4.0 ng/mL,

biomarkers, colors of ECL emission fit well with the reference values. The proposed BPE-ECL sensor showed good potential for fast and intuitive judgment of the status of prostate disease.

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CONCLUSIONS In summary, we reported the potential-resolved ECL excitation of the mixing [Ir(df-ppy)2(pic)] and [Ru(bpy)3]2+. It was interesting to find that [Ir(df-ppy)2(pic)] exhibited two distinct potentials with TPrA as the coreactant, which resulted in bidirectional color change from deep blue-green to red to light blue-green. Such phenomenon was observed in both the threeE

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electrode and the closed BPE systems. The color of ECL emission of [Ru(bpy)3]2+ and [Ir(df-ppy)2(pic)] complexes mixture at the anode of the BPE was influenced by the cathodic reactions. The proposed BPE-ECL sensor could be applied for multiple targets analysis, for example, the fast and intuitive judgment of the status of prostate disease via detection of different biomarkers, which is of great potential application in clinical diagnostics.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.8b00014. Regents and apparatus, electrochemical behavior of [Ir(df-ppy)2(pic)] in acetonitrile, ECL and CV characterizations of [Ir(df-ppy)2(pic)] and [Ru(bpy)3]2+ with visualized ECL emission, phosphorescent spectrum and ECL spectrum of [Ir(df-ppy)2(pic)], RGB analysis of the ECL emissions of [Ir(df-ppy)2(pic)] and [Ru(bpy)3]2+at different potentials, and optimization of multiple markers assay and stability of the device (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*Tel/Fax: +86-25-89687294. E-mail: [email protected] (W.Z.). *Tel/Fax: +86-25-89687294. E-mail: [email protected] (J.J.X.). ORCID

Jing-Juan Xu: 0000-0001-9579-9318 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the Science and Technology Ministry of China (Grant No. 2016YFA0201200), the National Natural Science Foundation (Grants 21327902, 21535003) of China. This work was also supported by a Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions.

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DOI: 10.1021/acs.analchem.8b00014 Anal. Chem. XXXX, XXX, XXX−XXX