Potential-Resolved Multicolor Electrochemiluminescence for Multiplex

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Potential-Resolved Multicolor Electrochemiluminescence for Multiplex Immunoassay in a Single Sample Weiliang Guo, Hao Ding, Chaoyue Gu, Yanhuan Liu, Xuecheng Jiang, Bin Su, and Yuanhua Shao J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.8b09422 • Publication Date (Web): 31 Oct 2018 Downloaded from http://pubs.acs.org on November 1, 2018

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Journal of the American Chemical Society

Potential-Resolved Multicolor Electrochemiluminescence for Multiplex Immunoassay in a Single Sample Weiliang Guo,† Hao Ding,† Chaoyue Gu,‡ Yanhuan Liu,† Xuecheng Jiang,§ Bin Su,*,† Yuanhua Shao‡ †

Institute of Analytical Chemistry, Department of Chemistry, Zhejiang University, Hangzhou 310058, China, ‡Beijing National Laboratory for Molecular Sciences, College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, China, §Hangzhou Genesea Biotechnology Limited Company, Hangzhou 315000, China KEYWORDS potential-resolved ECL, spectrum-resolved ECL, multiplex immunoassay, biomarker, simultaneous recognition

ABSTRACT: Electrochemiluminescence (ECL) is a highly successful technique used in commercial immunoassays for clinical diagnosis. Developing ECL-based multiplex immunoassay, with the potential to enable high-throughput detection of multiple biomarkers simultaneously, remains to be one of current research interests yet limited by a narrow choice of ECL luminophores. Herein we report the synthesis, photophysics, electrochemistry and ECL of several new ruthenium(II) and iridium(III) complexes, three of which are eventually used as signal reporters for multiplex immunoassay. The ECL behaviors of individual luminophores and their mixtures were investigated in multiple modes, including light intensity, spectrum and image measurements. The spectral peak separation between Ru(bpy)2(dvbpy)2+ (bpy = 2,2’-bipyridine, dvbpy = 4,4’-bis(4-vinylphenyl)-2,2’-bipyridine) and Ir(dFCF3ppy)2(dtbbpy)+ (dFCF3ppy = 3,5-difluoro-2-[5-(trifluoromethyl)-2-pyridinyl]phenyl, dtbbpy = 4,4’-bis(tert-butyl)-2,2’bipyridine), was up to 145 nm, thus providing the spectrum-resolved possibility of identifying light signals. The potential-resolved ECL signals were achieved for the mixtures of Ir(ppy)3 (ppy = 2-phenylpyridine) with either Ru(bpy)2(dvbpy)2+ or Ir(dFCF3ppy)2(dtbbpy)+, due to the self-annihilation ECL of Ir(ppy)3 at higher potentials as confirmed by electrochemistrycoupled mass spectrometry. A multiplex immunoassay free of spatial spotting antibodies on plates or substrates was ultimately devised by combining luminophore loaded polymer beads with the homogeneous sandwich immunoreaction. Using potential and spectrum dual-resolved ECL as the readout signal, simultaneous recognition of three antigens, namely carcinoembryonic antigen (CEA), alpha-fetoprotein (AFP) and beta-human chorionic gonadotropin (β-HCG), was demonstrated in a single run for a sample volume of 300 L. These results contribute to the understanding of ECL generation by multiple luminophores and devising spotfree multiplex immunoassays with less sample consumption.

munoassays capable of measuring simultaneously multiple analytes in a single run and/or a single volume have become increasingly popular, because they can save both time and precious sample material.13-15 Moreover, multiplex immunoassays are more informative and accurate in diagnosing disease and judging patient status. At present, multiplex immunoassays can be basically classified into two categories, spatially-resolved array assays and microbead-based assays.16 In the former case, different capture antibodies are spotted at defined positions on a plate and share the same reaction volume in a run. A whole array can be read at once and spot coordinates can then be used as an address to determine specific proteins. In the latter case, the capture antibodies are conjugated to microbeads encoded by different dyes, which can be distinguished either by light emitting diode/image-based analysis or by fluorescence intensity using a flow cytometer.8,17 Electrochemiluminescence (ECL) refers to luminescence that is produced by chemical reactions triggered by externally applied potentials.18,19 Due to its intrinsically low background and high sensitivity, ECL has manifested itself to be a highly sensitive analytical tool for the detection of a wide range of analytes, including small molecules,20-23 DNA,24-27 proteins28-34 and cells.35-38 It can serve as a sensitive reporting signal integrated with miniaturized analytical platform, such as microfluidic and paper-based devices.39-42 ECL has also been successful-

INTRODUCTION Immunoassays to measure target analytes within a sample have been developed for almost six decades and will continue to be one of mainstays in diagnosing diseases, estimating prognosis and monitoring medical treatment efficacy.1-3 Numerous efforts have been made recently to develop novel immunosensors in combination with electrochemical,4,5 optical6-8 and mass techniques9,10 for the detection of biomarkers. A large number of immunoassay kits using specific antibody pairs are commercially available, consisting of capture antibodies that are able to pull a target analyte of interest out of the sample and specific detection antibodies that are conjugated with signal generating groups for reading-out. For example, the robust and popular enzyme-linked immunosorbent assay (ELISA), in which the detection antibodies are tagged with enzymes (typically horseradish peroxidase and alkaline phosphatase), performs the measure of target analytes via the formation of immunocomplexes and subsequently the enzyme-catalyzed conversion of substrates to detectable products that are chromogenic, fluorescent or chemiluminescent.11 Typical immunoassays are designed in most cases to measure only one specific target analyte at a time (often termed as singleplex immunoassays), and measuring multiple targets involves time- and sampleconsuming parallel workflows.12 In recent years, multiplex im-

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phenylpyridine)iridium(III) (Ir(ppy)3, 2), is one of the first iridium complexes used in ECL research.46,60-62 Other four luminophores, namely, [4,4’-bis(tert-butyl)-2,2’bipyridine]bis[3,5-difluoro-2-[5-(trifluoromethyl)-2pyridinyl]phenyl]iridium(III) (Ir(dFCF3ppy)2(dtbbpy)+, 1), [4,4’-bis(tert-butyl)-2,2’-bipyridine]bis[2-(2pyridinyl)phenyl]iridium(III) (Ir(ppy)2(dtbbpy)+, 3), [4,4’bis(4-vinylphenyl)-2,2’-bipyridine]bis[(2pyridinyl)phenyl]iridium(III) (Ir(ppy)2(dvbpy)+, 4) and [4,4’-bis(4-vinylphenyl)-2,2’-bipyridine]bis(2,2’bipyridine)ruthenium(II) (Ru(bpy)2(dvbpy)2+, 6) were synthesized in this work according to previous reports.63-66 The full experimental details on the synthesis, NMR and spectroscopic characterizations are provided in Scheme S1, Figures S1 and S2 (see the supporting information, SI, for details). Figure S2 compares UV-Vis absorption and photoluminescence (PL) spectra of six ECL luminophores. These luminophores exhibited PL nearly across the entire visible light range. Their absorption maximum wavelength (max, Abs) and the maximum molar absorptivity (max, Abs), the maximum PL wavelength (max, PL) and the approximate spectroscopic energy of excited state (E0‒0 = 1239.81/max, FL) are summarized in Table 1. The PL quantum yields (ФPL) of luminophores were determined using quinine and Ru(bpy)32+ as the standards,65,70,71 which were 3.79, 0.52, 7.11, 6.18, 1.80 and 2.29, respectively (see calculation details in SI, Figure S3 and Table S1). Figure 1b illustrates the ECL spectra of six luminophores obtained in the presence of tri-n-propylamine (TPrA) as the coreactant in acetonitrile. Intensive ECL was only generated at potentials where the corresponding luminophores were oxidized. Apparently, the ECL spectra are quite similar to PL ones, covering nearly the whole visible light range. The maximum wavelengths (max, ECL) for 16 were 491 nm, 526 nm, 591 nm, 614 nm, 622 nm and 636 nm, respectively (see Table 1). In comparison with PL spectra, the ECL maxima displayed bathochromic shifts, which were most likely associated with an inner-filter effect because of the difference in the concentration used in PL and ECL measurements.67,68 The ECL efficiency, ФECL, of luminophores 1‒6 in the presence of TPrA was measured as photons emitted per oxidation event relative to that of Ru(bpy)32+ (5)/TPrA ECL system.69 ECL-voltage curves and cyclic voltammograms (CVs) of the standard and test samples under the same conditions were used to calculate the amount of photons and electrons (see more details in SI). The calculated ФECL were listed in Table 1. Figure 1c shows CVs of six luminophores obtained in acetonitrile containing 0.1 M tetra-n-butylammonium hexafluoroborate (TBAPF6) as supporting electrolyte at a scan rate of 0.1 V s‒1. 1 displays a reversible one-electron-oxidation process at ca. +1.75 V (E1/2, ox vs. SCE, the same in the following context), which can be formally assigned to Ir(IV)/Ir(III). 2 exhibits a reversible redox peak at a relatively low potential of +0.74 V. 36 show a quite close redox potential at ca. +1.3 V (as compared in Table 1), which can be easily distinguished from that of 1 and 2. In the negative potential range, 1, 3 and 4 show a current wave at ca. ‒1.3 V, which can be assigned to the reduction of Ir(III) center. While 2 displays a current wave at ‒2.26 V. For 5 and 6, they exhibit three waves at ca. ‒1.3 V, ‒1.5 V and ‒1.7 V, corresponding to three successive reduction.

ly applied in the new generation of clinical immunoassays, in which the detection antibodies are directly tagged with ECL molecular luminophores.43 And the light generated heterogeneously at the vicinity of electrode surface under the electrochemical stimulation is capable of quantifying analytes captured in the precedent immunoreactions. Multiplex ECL immunoassays have already been commercialized by using multiarray technology to detect simultaneously different analytes in a sample.44 Although various ECL luminophores have been developed so far, tris(2,2’-bipyridyl)ruthenium(II) (Ru(bpy)32+) and its derivatives remain the best and only ones utilized in commercial ECL systems, owing to their high ECL efficiencies and stable ECL signal output in aqueous solutions. Given most of ruthenium(II) complexes emit light only in the orange-red portion of the visible spectra,45 the current multiplex ECL immunoassays are usually performed in a spatiallyresolved array mode with different capture antibodies spotted at defined positions. Although the proprietary combination with ECL imaging reader results in an exceptionally high sensitivity, broad dynamic range, low sample consumption and high throughput, the assays are expensive since microscopically integrated spot arrays and well-designed conductive multiwall plates are required to spare precious sample materials. If not, macroscopic arrays are essentially equivalent to parallel workflows of a multitude of singleplex assays. The development of ECL-based multiplex immunoassays in a spot-free format remains rather limited at present. The major difficulties lie in the limited choice of ECL luminophores and insufficient control of electrochemical reactions by a potential. Although luminophores with different redox potentials can be facilely synthesized, ECL of each luminophore is persistently generated as long as the electrode potential exceeds the corresponding threshold valve. In this case, one usually obtains a poorly resolved emission for the mixture of multiple luminophores, making the intensity-based analysis infeasible, unless the emission of each luminophore can be spectrally resolved.46,47 Several research groups have previously proposed potential- or spectrum-resolved strategies to explore the possibilities of multiplex immunoassays, using molecular luminophores,48-56 semiconducting quantum dots57 or their combinations.58,59 In this work we attempt to build up a molecular multi-color ECL system by combing the potential- and spectrumresolution to explore the possibility of multiplex immunoassays. Several new iridium(III) and ruthenium(II) complexes were synthesized for this purpose. By ECL intensity, spectrum and imaging measurements, as well as thermodynamic analysis and electrochemistry-coupled mass spectrometry study, we rationalized the ECL generation by single luminophores and their mixtures. A multi-color ECL system including three different luminophores was successfully structured. As a proof-ofconcept experiment, they were loaded into polymeric microbeads to encode three different detection antibodies for simultaneous recognition of carcinoembryonic antigen (CEA), alpha-fetoprotein (AFP) and beta-human chorionic gonadotropin (β-HCG) in a single run.

RESULTS AND DISCUSSION Synthesis and Characterization of ECL Luminophores. Figure 1a shows the molecular structures of six ECL luminophores. Ru(bpy)32+ (5) is recognized as a benchmark ECL luminophore. The neutral luminophore, tris(2-


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Table 1. Photophysical and Electrochemical Properties of Six ECL Luminophores Probes

a

max, Abs / nm

(εmax / M‒1 cm‒1) a

max, PL / nm

a

ФPL (%)

max, ECL / nm a

ФECL d

E1/2, ox /V

E1/2, red /V

E0‒0 / eV f

ΔG / eV g

1 2 3 4 5

211 (25877) 240 (55206) 256 (55360) 275 (60179) 286 (88415)

476 515 555 571 595

3.79b 0.52b 7.11c 6.18c 1.80c

491 526 591 614 622

1.46 0.39e 0.92 0.91 1.00

1.75 0.74 1.28 1.23 1.33

‒1.31 ‒2.26 ‒1.43 ‒1.32 ‒1.29, ‒1.47, ‒1.70

2.60 2.41 2.23 2.17 2.08

0.84 ‒0.03 ‒0.75 ‒0.76 ‒0.94

6

287.5 (112490)

610

2.29c

636

1.82

1.30

‒1.24, ‒1.45, ‒1.69

2.03

‒0.96

max, Abs, max, PL, and max, ECL represent the maximum absorption, PL, and ECL emission wavelengths, respectively. εmax denotes the maximum molar

absorptivity. b Measured at 298 K using quinine sulfate (Ф = 54.6% in 0.5 M H2SO4) as the standard.70 c Measured at 298 K using Ru(bpy)3(PF6)2 as the standard (Ф = 1.8% in aerated acetonitrile).71 d Coreactant ECL efficiencies are given with respect to Ru(bpy)3(PF6)2 in acetonitrile. e ФECL of 2 is 0.33 as cited from ref.46. f E0‒0 is the approximate spectroscopic energy of excited state, which is approximately calculated to be E0‒0 = 1239.81/max, FL (in eV).72 g ΔG is the variation of Gibbs free energy for the co-reactant reaction involved in the generation of excited state. G  E(TPrA ) Eox  E0-0 , where E(TPrA ) is the reduction potential of radical and has been reported previously as 1.70 V.73

Ir(dFCF3ppy)2(dtbbpy)+ (1), were used to design a potential and spectrum dual-resolved system and eventually to develop a multiplex immunosensor. ECL Generation by Single Luminophores. Figure 2 shows the ECL intensity-potential curves overlaid with CVs (a, c, e) and spooling ECL spectra (b, d, f) of Ru(bpy)2(dvbpy)2+ (6), Ir(dFCF3ppy)2(dtbbpy)+ (1) and Ir(ppy)3 (2) dissolved in acetonitrile containing 50 mM TPrA and 0.1 M TBAPF6. The concentrations of luminophores were 2.5 µM, 125 µM and 125 µM, respectively. For Ru(bpy)2(dvbpy)2+ (6)/TPrA (see Figure 2a), a strong oxidation current peak was observed at ca. +1.4 V in the forward scan, while no obvious reduction current was seen in the backward direction because electrogenerated Ru(bpy)2(dvbpy)3+ was reduced by TPrA• to form the excited state [Ru(bpy)2(dvbpy)2+]*. Simultaneously, ECL was detected at ca. +1.3 V and its intensity increased with the potential. Shown in Figure 2b are the spooling ECL spectra captured during a potential scan from +0.8 V to +2.3 V at a scan rate of 0.1 V s‒1. It is clear that one ECL peak centered at 636 nm was displayed and intensity increased with the potential up to +2.3 V. The reaction process of this co-reactant system involves the following fundamental steps (M represents the luminophore), (1) TPrA  TPrA   e

Figure 1. (a) Molecular structures of six ECL luminophores, namely Ir(dFCF3ppy)2(dtbbpy)+ (1), Ir(ppy)3 (2), Ir(ppy)2(dtbbpy)+ (3), Ir(ppy)2(dvbpy)+ (4), Ru(bpy)32+ (5) and Ru(bpy)2(dvbpy)2+ (6). (b) Normalized ECL spectra of six luminophores obtained under their corresponding redox potentials in acetonitrile containing 50 mM TPrA as the co-reactant and 0.1 M TBAPF6 as the supporting electrolyte. (c) Cyclic voltammograms (CVs) of six luminophores (1 mM: 1, 3, 4 and 6; 250 µM: 2 and 5) in acetonitrile containing 0.1 M TBAPF6. The scan rate was 0.1 V s‒1.

TPrA  TPrA  H

(2)



M M +e +

(3) 

TPrA + M  M + P1 +

As seen from Figure 1b and Table 1, the ECL spectral maximum separation between 1 and 2 is only 35 nm, making it difficult to precisely distinguish the spectral signals. Nevertheless, the oxidation potentials of 1 and 2 have a gap up to 1.01 V (Figure 1c), offering the alternative potential-resolved possibility by selective or sequential oxidation of two of them in mixtures via controlling the electrode potential. Meanwhile, other four luminophores, in particular 6, displayed ECL in the longer wavelength range compared to that of 1 and 2. The addition of styryl groups on the parent Ru(bpy)32+ induced a bathochromic shift in max, ECL by lowering the π⁎ (LUMO) energy level. In this case, the separation of ECL spectral maximum between 6 and 2 reached 110 nm, and that between 6 and 1 was 145 nm, thus providing the spectrum-resolved possibility of distinguishing ECL signals generated from their mixtures, in addition to the aforementioned potential-resolved possibility. Herein the red-, green- and cyan-ECL generating luminophores, namely Ru(bpy)2(dvbpy)2+ (6), Ir(ppy)3 (2) and

(4)

M  M  h 

(5) 3+

Eqs 1-3 refer to the generation of Ru(bpy)2(dvbpy) and highly reductive TPrA• at the vicinity of the electrode surface. They further react to yield [Ru(bpy)2(dvbpy)2+]* that relaxes to the ground state and emits light (eqs 4 and 5). Concomitant with the reaction described by eq 4, M* can be also formed via the following route, M + TPrA  M  + P1

(6)

M+ + M  M + M

(7)

This reaction pathway usually occurs simultaneously and cannot be easily discriminated. The excited states generation involved the direct electrooxidation of both luminophores and TPrA at the electrode surface, while a “catalytic route” involving homogeneous reduction of M+• by TPrA could also take place (eq 8),74



M+  TPrA  M  TPrA

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(8)

The contribution of this process to the overall ECL intensity depends upon the concentration of luminophores.75

Figure 3. (a) Schematic illustration of electrochemistry-coupled mass spectrometry (EC-MS) setup. (b) Mass spectra of Ir(ppy)3 (top) and Ir(ppy)3/TPrA (bottom) obtained at +1.0 V (vs. SCE). (c) Dependence of relative MS signal intensity obtained with Ir(ppy)3 (black line) and Ir(ppy)3/TPrA (green line) on the potential. Tetra-n-hexylammonium cation (THA+, m/z 354) served as the internal reference.

To rationalize the annihilation of ECL in this potential range, an electrochemistry-coupled mass spectrometry (EC-MS) technique was used to in situ track and identify intermediate species formed in the ECL process.76,77 Figure 3a depicts the workflow of EC-MS system. The core component is a quartz theta-shaped micropipette with dual barrels, one of which was deposited with carbon by pyrolysis of butane to function as the working electrode. The other one was filled with solutions (in this case is tetrahydrofuran, THF) to serve as a microelectrochemical (micro-EC) cell, in which a silver chloride/silver (AgCl/Ag) wire was inserted to function as the reference electrode (its potential was calibrated using the ferricenium/ferrocene couple as the internal reference). Upon applying a suitable potential to the micro-EC cell via an external DC power supply, the electrochemical reaction takes place on the carbon surface. The micro-EC cell also functioned as the MS nanospray propelled by a piezoelectric pistol, which could inject electrogenerated products and intermediates of ECL reactions directly into MS for analysis. To run this experiment, 1 mM Ir(ppy)3 and 50 mM TPrA were dissolved in THF containing 10 mM NH4Cl as the supporting electrolyte. Tetra-n-hexylammonium cation (THA+, m/z 354) was used as the internal reference to quantitatively address the relationship between the relative MS intensity and applied potentials. Figure 3b compares the mass spectra of Ir(ppy)3 (2) in the absence (top) and presence (bottom) of TPrA at +1.0 V. In the former case, Ir(ppy)3+ (m/z 655) generated from the electrooxidation of Ir(ppy)3 was detected. In contrast, in the presence of TPrA, no significant MS signal of Ir(ppy)3+ (m/z 655) was observed. Instead, the intermediate ions generated from TPrA, namely TPrAH+ (m/z 144), TPrA+• (m/z 142), PrN=CH2+ (m/z 114) and HNPr2+ (m/z 102), were detected. The results indicate that the ECL reaction between 2 and TPrA via eqs. 1-7 is likely dominant at +1.0 V, because they have close oxidation potentials, resulting in a maximum ECL intensity at this potential. Francis and Hogan et al have previously attributed the ECL annihilation in the higher potential range to the quenching of [Ir(ppy)3]* by TPrA+• by eq 10,49,78

Figure 2. ECL intensity-potential curves overlaid with CVs (a, c, e) and spooling ECL spectra (b, d, f) of Ru(bpy)2(dvbpy)2+ (6; a, b), Ir(dFCF3ppy)2(dtbbpy)+ (1; c, d) and Ir(ppy)3 (2; e, f) dissolved in acetonitrile containing 50 mM TPrA and 0.1 M TBAPF6. The concentrations of luminophores were 2.5 µM, 125 µM and 125 µM, respectively. The photomultiplier tube (PMT) was biased at 600 V. The scan rate was 0.1 V s‒1. The spectral integration time was 0.3 s and two adjacent spectra were obtained at potential intervals of 30 mV (the same in the following). Insets show ECL spectra at different potentials.

Similar electrochemical and ECL signals were observed for Ir(dF(CF3)ppy)2(dtbbpy)+ (1)/TPrA (see Figures 2c and 2d). In the forward potential scan, strong ECL was detected when the potential was higher than +1.7 V and its intensity increased with the potential up to +2.3 V. The ECL spectra were dominated by one emission peak centered at 491 nm. Moreover, the low oxidation potential (LOP) ECL proposed by Bard et al.32 was also detected at ca. +1.0 V in the backward scan. The LOP ECL involves the reduction of Ir(dF(CF3)ppy)2(dtbbpy)+ (1) by TPrA• to form Ir(dF(CF3)ppy)2(dtbbpy) by (eq 6), and subsequent oxidation of Ir(dF(CF3)ppy)2(dtbbpy) by TPrA+• to generate [Ir(dF(CF3)ppy)2(dtbbpy)+]* via (eq 9), M + TPrA  M + TPrA

(9)

In the case of Ir(ppy)3 (2), the ECL intensity-potential curve showed two emission maxima at +1.0 V and +1.6 V in the forward potential scan, with the second one being only 10% of the first in the magnitude (see Figure 2e). As shown in Figure 2f, the ECL generated by Ir(ppy)3 (2) exhibited only one emission peak centered at 526 nm and the spectral shape remained unchanged. However, the ECL intensity changed considerably with the potential. The onset potential of ECL generation was at +0.75 V, and the intensity reached the maximum at ca. +0.87 V and then dropped sharply with the potentials. No ECL signal was observed in the higher potential range of +1.05 V  +2.30 V.


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Ir(ppy)3 



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

 TPrA   Ir(ppy)3  TPrA

(10)

As shown in Figure 3c, the MS intensity of Ir(ppy)3+ (m/z 655) in the presence of TPrA (green line) at +1.3 V is 19.6 times larger than that at +1.0 V. At +1.3 V, the concentration of TPrA+• from eq. 2 reached a critical level, allowing eq. 10 to dominate. Thus, the MS signal of Ir(ppy)3+ (m/z 655) increased with increasing the potential. In the control experiment without TPrA (black line), the MS intensity of Ir(ppy)3+ (m/z 655) at +1.3 V is only 1.1 times larger than that at +1.0 V, excluding the influence of potential on the MS intensity. So the results in Figure 3c confirmed the quenching mechanism proposed by Francis and Hogan, namely eq 10. The ECL annihilation behavior of Ir(ppy)3 in the high potential regime offers the opportunity of devising potential-resolved ECL systems. Potential and Spectrum-Resolved ECL Generation. Ir(ppy)3 (2) was mixed with either Ru(bpy)2(dvbpy)2+ (6) or Ir(dFCF3ppy)2(dtbbpy)+ (1) to examine the potentialresolved ECL generation. As seen in Figure 4a for a binary mixture of Ir(ppy)3 (2) and Ru(bpy)2(dvbpy)2+ (6), two distinguishable ECL spectral domains were observed. In the low potential range, the whole spectrum was dominated by Ir(ppy)3 (2) with a maximum at 526 nm, while in the high potential regime by Ru(bpy)2(dvbpy)2+ (6) with a peak at 636 nm. It is clearly illustrated in the inset that ECL generated by Ir(ppy)3 (2) and Ru(bpy)2(dvbpy)2+ (6) can be well separated at a very low or a very high potential. While at an intermediate potential (e.g. +1.01 V) two maxima were displayed, suggesting that both Ir(ppy)3 (2) and Ru(bpy)2(dvbpy)2+ (6) emitted to give rise to a convoluted spectrum. However, as shown in Figures 2a and 2b, in this intermediate potential regime Ru(bpy)2(dvbpy)2+ (6) alone does not generate ECL, meaning that an alternative channel exists to promote the ECL generation by Ru(bpy)2(dvbpy)2+ (6). This hypothesis was also confirmed by ECL images of the glassy carbon working electrode surface captured in the course of potential scan. As shown in Figure 4b, at low potentials (for examples +0.75 V, +0.80 V and +0.85 V) the electrode surface appeared green due to ECL generated by Ir(ppy)3 (2). Upon scanning the potential to positive enough range (e.g., higher than +1.30 V), it turned red since ECL was produced by Ru(bpy)2(dvbpy)2+ (6). While in the interim (+0.90 V  +1.20 V as annotated with the dashed-white box), faint red images were captured. Ir(ppy)3 (2) alone emitted green ECL up to +1.0 V, Ru(bpy)2(dvbpy)2+ (6) alone did not produce obvious red ECL until the potential was more positive than +1.30 V (as shown in Figure 4c, d), suggesting that LOP ECL generation (via eqs 6 and 9) cannot account for the phenomena at intermediate potentials. It is most likely that [Ru(bpy)2(dvbpy)2+]* can be generated to emit in the presence of Ir(ppy)3 (2). Although Ru(bpy)2(dvbpy)2+ (6) cannot be oxidized yet in this intermediate potential range, both Ir(ppy)3+ and TPrA• can be generated in terms of eqs. 1-4. As a strong reductant, TPrA• can reduce Ru(bpy)2(dvbpy)2+ (6) to + Ru(bpy)2(dvbpy) , which can be subsequently oxidized by Ir(ppy)3+ to generate [Ru(bpy)2(dvbpy)2+]*. The reactions can be expressed as, TPrA  Ru(bpy)2(dvbpy)2+  Ru(bpy)2(dvbpy)+  P1 

Ir(ppy)3  Ru(bpy)2 (dvbpy)   Ru(bpy)2 (dvbpy)2+  + Ir(ppy)3

Figure 4. (a, b) Spooling ECL spectra (a) and sequential ECL images (b) generated by Ru(bpy)2(dvbpy)2+ (6) and Ir(ppy)3 (2) in acetonitrile containing 50 mM TPrA and 0.1 M TBAPF6. Their concentrations were 2.5 µM and 125 µM for spectra while doubled for images. Shown in inset are ECL spectra acquired at several different potentials. (c, d) Sequential ECL images of 5 µM Ru(bpy)2(dvbpy)2+ (6; c) and 250 µM Ir(ppy)3 (2; d). The displayed images were obtained with a 3 mm-in-diameter glassy carbon electrode at different applied potentials (shown beneath in volts). The CCD exposure time was 120 s in all cases.

Eq. 12 represents the key step of generating excited state species and the variation of Gibbs free energy (ΔG) of this step is given by,

G  E



2   Ru(bpy)2 (dvbpy)  /Ru(bpy)2 (dvbpy)

0'  EIr(ppy)  /Ir(ppy) 3

3

(13)

The value of ΔG was estimated to be +0.05 eV, indicating this step is unfavorable from pure thermodynamic viewpoint. However, this near-zero value maybe not that accurate because of the uncertainty of excited state potential.54 Here we try to evaluate the influence of this step by chromatic analysis of ECL images. As shown in Figure S4, the red light component expressed as R value was derived from ECL images captured at +0.9 V, +1.0 V, +1.1 V and +1.2 V (see Figure 4b‒d) and compared. Clearly, R values of the binary mixture are much higher than the sum of single luminophores, indicating the existence of interaction. Similar phenomenon was also observed by Swanick et al. when they investigated ECL generation from a heterometallic soft salt, [Ru(dtbubpy)3][Ir(ppy)2(CN)2]2 (dtbubpy = 4,4’-di-tert-butyl-2,2’-bipyridine; ppy = 2phenylpyridine).79 In either co-reactant or annihilation route, no ECL signal was generated from [Ir]‒* moieties, while the emission intensity from [Ru]2+* was obviously enhanced. They attributed this behavior to the electrocatalytic reduction of [Ru]2+ by [Ir]2‒• and insufficient power of TPrA• to reduce [Ir]‒ for [Ir]2‒• generation, thus precluding the formation of

(11)

+

(12)



the iridium(III) emitter and meanwhile improving the generation of ruthenium(II) emitter. It should be noticed that the interaction between luminophores will affect the reading-out of signals in multiplex assays to some extent and reduce the accuracy of analysis. However, the interaction between Ru(bpy)2(dvbpy)2+ (6) and Ir(ppy)3 (2) only takes place in the intermediate potential regime. Thus, by carefully controlling the applied potential, the interaction could be totally avoided. As clearly shown by the plots in the inset of Figure 4a, independent ECL responses of Ir(ppy)3 (2) and Ru(bpy)2(dvbpy)2+ (6) could be acquired from ECL spectra at +0.82 V and +1.24 V, close to their respective oxidation potentials. As for the mixture of Ir(dFCF3ppy)2(dtbbpy)+ (1) and Ir(ppy)3 (2), their ECL spectral peak separation is only 35 nm, making precise quantitative analysis through spectra impossible. However, the sufficient gap up to 1.01 V between their oxidation potentials renders the possibility of sequential oxidation of two luminophores by controlling the applied potential to generate ECL in their respective ranges. In this case, both ECL spectrum and image can be effectively resolved on the potential axis (as illustrated in Figure S5).

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and red images. Note that in the co-reactant ECL systems the classical routes as depicted by eqs. 1‒7 are the main contribution to the overall ECL intensity, although other ECL generation pathways are also possibly involved. At more positive potentials, both Ru(bpy)2(dvbpy)2+ (6) and + Ir(dFCF3ppy)2(dtbbpy) (1) were able to emit, displaying two emission maxima at 526 nm and 636 nm in spectra. Although in this high potential range ECL generated by two luminophores cannot be resolved by potential, it can be easily deconvoluted in a spectrum-resolved manner because the spectral peak separation between two emission band is as large as 145 nm (as shown by the inset of Figure 5a). Moreover, the ECL image appeared pink at a potential higher than +1.70 V, due to the mixed color of red and cyan light generated by two luminophores. Dual-Resolved ECL for Multiplex Immunoassay. As demonstrated above, ECL generation by three luminophores in the ternary mixtures can be resolved by potential modulation and/or their spectra. Figure S6 shows the ECL spectra recorded for the mixture of three luminophores. In comparison with Figure 5a, a new potential resolved ECL domain appears, which is generated by Ir(ppy)3 (2) at low oxidation potentials. Clearly, a potential and spectrum dual-resolved system can be established, which can be eventually applied to multiplex immunosensing for multianalyte identification. Miao et al. has utilized polystyrene beads (PSB) as the carrier of a large number of hydrophobic ECL labels to develop an ultrasensitive DNA hybridization assay24 and C-reactive protein (CRP) immunoassay.28 In this work, we used the similar approach to fabricate a multiplex immunoassay for multiple biomarkers determination. In clinic, a single biomarker is usually not specific enough to meet strict diagnostic criteria.10 The simultaneous analysis of multiple antigens has greater disease specificity and sensitivity than that of any single marker.80 Three luminophores were loaded to carboxylate PSB (267 nm in diameter), respectively, and subsequently modified by specific detection antibodies for AFP, CEA and β-HCG on the basis of biotin-streptavidin complexation to prepare dualcoded PSB (Ab-M@PSB, M = Ru or Ir). The preparation and characterization details can be found in the SI (see Scheme S2, Figure S7 and relevant explanation in the context).24,28,81-83 Taking Ru(bpy)2(dvbpy)2+ (6) as an example, it was loaded to PSB to prepare Ru@PSB, which was then coded with AFP antibodies to prepare Ab(AFP)-Ru@PSB. The multiplex immunoassay of three target markers was carried out in a single run for a sample volume of 300 L, involving three basic steps, namely the homogeneous sandwich immunoreaction, magnetic assisted separation and swelling release of luminophores for recognition (as exemplified in Figure 6a). In the first step, immunomagnetic beads (MB, 1 µm in diameter) coated with three different capture antibodies, designated as Ab-MB, were incubated with the sample containing antigens and subsequently separated with the assistance of magnets, which were then incubated with dual-coded PSB to form the sandwich immunocomplexes. After magnetic separation again, the immunocomplexes were submerged in 100 L of acetonitrile to swell PSB to release multiple luminophores. The solution was finally mixed with another 100 L of acetonitrile containing TPrA and TBAPF6 for ECL measurements, which were performed on an upright microscope connected to an electrochemical workstation (as shown in the left panel of Figure 6b).

Figure 5. ECL spooling spectra (a) and images (b) generated by Ru(bpy)2(dvbpy)2+ (6) and Ir(dFCF3ppy)2(dtbbpy)+ (1) in acetonitrile containing 50 mM TPrA and 0.1 M TBAPF6. Their concentrations were 2.5 µM and 125 µM for spectra while doubled for image. Shown in inset are ECL spectra acquired at several different potentials. (c) ECL images captured with 250 µM Ir(dFCF3ppy)2(dtbbpy)+ (1). The same experiment parameters as in Figure 4 were used.

As shown in Figure 2, both Ir(dFCF3ppy)2(dtbbpy)+ (1) and Ru(bpy)2(dvbpy)2+ (6) produce ECL at very high potentials. Figures 5a and 5b illustrate the spooling ECL spectra and images at different potentials, which can be analyzed in three regimes. Below +1.3 V, no ECL spectrum or image was resolved since neither Ru(bpy)2(dvbpy)2+ (6) nor + Ir(dFCF3ppy)2(dtbbpy) (1) was directly oxidized at the electrode surface. Although LOP ECL could be generated by either of them, but it was not detected due to its low efficiency or low concentration of luminophores used here. In the potential range of +1.30  +1.70 V, Ru(bpy)2(dvbpy)2+ (6) emitted alone to show a single emission maximum at 636 nm in spectra


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Journal of the American Chemical Society confirming the presence of CEA in the sample. When the potential was biased at +1.33 V, a dominating emission peak at 640 nm was acquired (red line), proving the existence of AFP in the sample. In addition, a weak emission band at 500 nm was also observed. And its intensity became stronger at a higher oxidation potential, such as +1.75 V (blue line). This emission band verifies the presence of β-HCG.

Figure 6. (a) The procedure of simultaneous recognition of multiple disease biomarkers. Two incubation steps were carried out at 37 ºC for 30 min, the separation was performed on a magnetic scaffold for 3 min and washed with phosphate buffer saline containing Tween-20 (PBST). 100 µL of acetonitrile was used to swell the PSB to release multiple luminophores from PSB under ultrasonication. (b) Scheme of ECL spectral detection system on the basis of an upright microscopy (Nikon, ECLIPSE LV100ND) equipped with a grating spectrometer (Andor, Shamrock 303i) and an EMCCD camera (Andor, iXon Ultra 897). The constant potential was applied via a CHI 832C potentiostat using a threeelectrode configuration. The ECL spectra were acquired at different potentials by ‘‘step and glue’’ function under conventional acquisition mode. The EMCCD exposure time was set to 2 s.

Figure 7. (a) ECL spectra and (b) normalized spectral patterns obtained at different oxidation potentials for the simultaneous recognition of three types of antigens in an immunoassay. (c) The corresponding spectral peak intensities (left) and the converted digital color map (right). The error bars represented the standard deviations of triplicate measurements. The recognition of CEA, AFP and β-HCG is marked as green, red, and blue colors in the digital colored map.

The left panel of Figure 7c displays the average values of ECL spectral peak intensities at different potentials, which were further converted to a digital color map bearing a unique recognition mode (as shown in the right panel of Figure 7c). This map can be used for simultaneous identification of CEA, AFP and β-HCG. The specificity of recognition with this method was also tested. Each pair of capture antibodies labeled MB and detection antibodies coated PSB was added into the same sample containing three types of antigens. As shown in Figures S8 and S9, the selective recognition of CEA, AFP and β-HCG were successfully accomplished. The results affirmed that the antigens could be efficiently captured to form the sandwich-type immunoconjugates with high binding specificity. Notably, some ECL behaviors in the immunoassay were different from that observed in above experiments. For example, the LOP ECL of Ir(dFCF3ppy)2(dtbbpy)+ (1) can be distinguished at +1.33 V (Figure 7b, blue line), which might be attributed to the use of a more sensitive EMCCD detector and a higher probe concentration employed in the immunoassay (the final volume of each sample was 200 μL, see details in the SI). Furthermore, since the ‘‘step and glue’’ function was run to capture ECL spectra signals using an imaging EMCCD, the spectral resolution was therefore sacrificed inevitably. And this is the reason why the spectrum at above +1.70 V in Figure 5a has two peaks while that in Figure 7a does not. Nevertheless, it would not significantly affect the recognition of corresponding antigens.

A disk-shaped glassy carbon electrode (GCE, 3 mm-indiameter) wrapped up by a plastic tube was used as the electrochemical cell, to which 50 µL of test solution was added to cover the GCE surface. A platinum wire and a silver wire were used as the counter electrode (CE) and quasi-reference electrode (QRE). Prior to ECL measurements, the GCE surface was focused under the dark-field optical mode and maintained at a fixed position on the microscope stage. Finally, three different constant potentials (namely +0.74 V, +1.33 V and +1.75 V) were biased to generate ECL (as exemplified in the right panel of Figure 6b, which was collected by the EMCCD camera equipped with a grating spectrometer. ECL luminophores released from immunocomplexes could be selectively excited at different potentials to emit at characteristic wavelengths for identification and recognition. Figure 7a shows ECL spectra recorded at three different potentials for simultaneous recognition of CEA, AFP and β-HCG in a single modeling sample at a concentration of 5 ng/mL, 25 ng/mL and 5 mIU/mL, respectively, which are the pathological cut-off concentrations. Firstly, the spectral peaks were normalized and analyzed by means of peak-differentiationimitating analysis. As shown in Figure 7b, a single emission peak centered at 520 nm was obtained at +0.74 V (green line),



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TPrA, biotin labeled fluorescein 5(6)-isothiocyanate (bio-FITC), N-hydroxysulfosuccinimide sodium salt (Sulfo-NHS), 1-(3dimethylaminopropyl)-3-ethylcarbodiimide (EDC), 2-(Nmorpholino)ethanesulfonic acid and Ir(ppy)3 were purchased from Aladdin. cis-Dichlorobis(2,2’-bipyridine)ruthenium (cisRu(bpy)2Cl22H2O) was bought from J&K scientific Ltd. Iridium(III) chloride (IrCl3) was obtained from Shaanxi Kaida Chemical Engineering Co., Ltd. 2-(2,4-difluorophenyl)-5(trifluoromethyl)pyridine (dFCF3ppy) was bought from Cochemical Ltd., (Shanghai, China). CEA and AFP standards were purchased from Biocell Biotechnol. Co., Ltd., (Zhengzhou, China). β-HCG standard and antibody pairs for β-HCG were obtained from Genesea Biotechnology Co. Ltd. (Hangzhou, China). Antibody pairs specific for human CEA and AFP were obtained from Keygen Gene Technology Co., Ltd. (Beijing, China). Preparation of capture antibodies conjugated magnetic beads (Ab(CEA)-MB, Ab(AFP)-MB and Ab(β-HCG)-MB) as well as biotinylated detection antibodies for CEA, AFP and β-HCG was accomplished in Genesea Biotechnology Co. Ltd. (Hangzhou, China). Bovine serum albumin (BSA) was obtained from Sigma-Aldrich Chemical Co. (St. Louis, MO). 267 nm and 9.94 µm diameter carboxylate PSB were bought from ACME and Bangs laboratories Inc., respectively. PBS (10 mM, pH 7.2) was obtained from Keyi Biotechnology Co. Ltd. (Hangzhou, China) and it contains 0.005 wt % Tween-20 (PBST). 2-(N-morpholino)ethanesulfonic acid and Tween-20 were used to prepare MES buffer (0.1 M, 0.005 wt % Tween-20) and the solution pH were adjusted to 5.5. Ultrapure water (18.2 MΩ cm) purified with a Millipore system was used throughout the experiments. Spectroscopic Characterization. The spectroscopic measurements were performed in acetonitrile solutions. The UV-Vis absorption spectroscopy measurements were carried out by using a QEpro spectrometer (Ocean Optics). The PL spectra were obtained with a spectrofluorophotometer (RF-5301pc, Shimadzu). ФPL for 1 and 2 was determined using quinine sulfate in 0.5 M H2SO4 as the standard (Ф = 54.6%).70 And that for 3-6 was measured using Ru(bpy)32+ as the standard (Ф = 1.8% in aerated acetonitrile).71 Electrochemistry. Acetonitrile was treated with calcium hydride and then distilled at reduced pressure under a nitrogen atmosphere prior to use. TBAPF6 was used as the electrolyte. A glassy carbon electrode (3 mm in diameter) served as the working electrode. Before experiments, it was polished with a 0.3 µm and 0.05 µm alumina slurry. A platinum wire and a silver wire were used as a counter and a quasi-reference electrode, respectively. At the end of each experiment, its potential was calibrated using the ferricenium/ferrocene couple as the internal reference. In acetonitrile/0.1 M TBAPF6, ferricenium/ferrocene has a formal potential of 0.424 V against the saturated calomel electrode (SCE). All potentials in the context was given against SCE.84 Electrochemical measurements were acquired on CHI 920C electrochemical workstation (CHI Instrument, Shanghai, China). Electrochemistry-Coupled Mass Spectrometry (EC-MS). EC-MS was used to in situ track the ECL reactions. MS was carried out on an Agilent 6300 series ion trap mass spectrometer (Agilent Technologies, Inc, USA). Applied voltage was controlled by a DC power supply (MS-305D, Dongguan Maihao, Ltd., China). The main experimental parameters of MS and the fabrication of the hybrid ultramicroelectrodes were conducted as that reported previously.76 ECL Measurements. ECL signals were captured in three different modes, including intensity-voltage curve, spooling spectrum and electrode image. All ECL measurements were conducted using the classic three-electrode configuration as described above. (i) Intensity. The synchronous acquisition of the ECL intensity-voltage curves overlaid with CVs was realized by placing the glassy-carbon electrode a few millimeters from the photomultiplier tube (PMT)

CONCLUSIONS In summary, we have synthesized several ruthenium and iridium complexes with distinguishable ECL emission wavelengths from 491 nm to 636 nm. The ECL responses from individual luminophores as well as their mixtures have been investigated using multiple ECL readout modes, including intensity, spectrum and imaging measurements. The spectral peak separation is up to 145 nm between the cyan-ECL generating species, Ir(dFCF3ppy)2(dtbbpy)+ (1), and the red one, Ru(bpy)2(dvbpy)2+ (6), making the spectrum-resolved ECL generation achieved in a mixed electrochemiluminescent system. The ECL reaction involving Ir(ppy)3 (2), a green-ECL luminophore with self-annihilation ECL behavior,49 was studied by online EC-MS, with which the self-annihilation at higher potentials was proved experimentally to arise from the quenching of [Ir(ppy)3]* by TPrA+•. So the potential-resolved ECL emissions were achieved by mixing Ir(ppy)3 (2) with either Ru(bpy)2(dvbpy)2+ (6) or Ir(dFCF3ppy)2(dtbbpy)+ (1). Finally, we prepared polystyrene beads loaded with these three luminophores and tagged with three different antibodies, so called dual-coded polymer beads, which were employed to fabricate a multiplex immunoassay for simultaneous recognition of CEA, AFP and β-HCG on the basis of potential and spectrum dual-resolved ECL generated by three luminophores. The multiplex assay is free of spatially spotting antibodies. It also spares the sample volume as the assay can finish in a single measurement. Moreover, this format is compatible with current commercial ECL immunoassay kits by simply replacing the luminophore tagged detection antibodies with corresponding dual-coded polymer beads. Thus the platform can be extended for simultaneous recognition of many other biomarkers. By further designing new ECL luminophores and strictly controlling the multiplex immunoassays, we believe it is possible to carry out quantitative determination of multiple biomarkers simultaneously, although quantitative results in this work is limited. Nevertheless, the current immunoassay format can be adapted to develop diagnostic kits for specific diseases. For example, clinical diagnostic of cervical carcinoma requests CEA as the main biomarker, and AFP, β-HCG and CA125 as three auxiliary ones. The simultaneous recognition of biomarkers above the pathological cut-off level has greater disease specificity. It should be noted that the multiplexed immunoassay format has some limitations, one of which is the narrow choice of ECL luminophores. Synthesis of new molecular and nanoparticle luminophores with potential- and/or wavelength resolved ECL is highly demanded. Moreover, three luminophores used in this work are hydrophobic and lack of conjugation sites to link with antibodies. So they have to be loaded to polymer beads to code specific antibodies and finally released from beads by swelling for ECL detection. The use of water-soluble luminophores with appropriate conjugation sites can significantly simplify the operation of assay, because the swelling release step will be unnecessary.

EXPERIMENTAL SECTION Chemicals and Reagents. Acetonitrile, ethylene glycol, 2methoxyethanol, hexane, acetone, diethyl ether, THF, methanol, ethanol, toluene and potassium hexafluorophosphate (KPF6) were bought from Sinopharm Chem. Co., Ltd. (Shanghai, China). TBAPF6 was obtained from Sigma-Aldrich. Tween-20, 2phenylpyridine (ppy), 4,4’-di-tert-butyl-2,2’-bipyridine (dtbbpy),


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on an MPI-E ECL analytical system (Remex Analysis Instrument, Xi’an, China). The PMT was biased at 600 V and the scan rate was 0.1 V s‒1. (ii) Spooling spectrum (see instrumental details in Figure S10a). ECL spectra were acquired in the period of potential scanning using a custom-built ECL system consisting of an Autolab PGSTAT302N workstation (Metrohm, Netherlands) and the QEpro spectrophotometer. A home-made Teflon cell consisting of a circular quartz window, optic fiber and collimating lens was used. The integration time of individual spectrum was 0.3 s and the scan rate was 0.1 V s‒1. Two adjacent spectra were obtained at potential intervals of 30 mV. The measurement yielded a 3D emission spectral distribution.48,85,86 (iii) Imaging (see instrumental details in Figure S10b). The sequential ECL images at different potentials were obtained with a home-made ECL imaging platform. It consists of a color CCD camera (Q image) equipped with a Model VFA2595H Macro Zoom Iris Megapixel lens (Senko ADL, Japan) and a CHI 832C electrochemical workstation (CHI Instrument, Shanghai, China). In each imaging experiment, the CCD exposure time was set to 120 s while a constant potential was applied at the glassy carbon electrode. The quantitative information of ECL images was further analyzed by MATLAB software.

ASSOCIATED CONTENT Supporting Information. The experimental details on the synthesis, NMR and spectroscopic characterizations of multi-color ECL luminophores; preparation and optical characterization of dualcoded PSB; immunoassay procedure and detection results. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author *

Prof. Bin Su, Email: [email protected]

All authors have given approval to the final version of the manuscript. ORCID Bin Su: 0000-0003-0115-2279 Yuanhua Shao: 0000-0003-3922-6229 Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This work was supported by the Natural Science Foundation of China (21335001 and 21575126), the Zhejiang Provincial Natural Science Foundation (LZ18B050001) and the National Key Research and Development Program of China (No. 2016YFA0201300).

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