Potential-Resolved Multicolor Electrochemiluminescence for Multiplex

Article pubs.acs.org/JACS

Cite This: J. Am. Chem. Soc. 2018, 140, 15904−15915

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‡ †

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

J. Am. Chem. Soc. 2018.140:15904-15915. Downloaded from pubs.acs.org by UNIV OF LOUISIANA AT LAFAYETTE on 11/29/18. For personal use only.

‡

S Supporting Information *

ABSTRACT: Electrochemiluminescence (ECL) is a highly successful technique used in commercial immunoassays for clinical diagnosis. Developing an ECL-based multiplex immunoassay, with the potential to enable high-throughput detection of multiple biomarkers simultaneously, remains a current research interest yet is 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(4vinylphenyl)-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 = 2phenylpyridine) 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 electrochemistry-coupled 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 μL. These results contribute to the understanding of ECL generation by multiple luminophores and devising spot-free multiplex immunoassays with less sample consumption.

■

INTRODUCTION

phosphatase), measures 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 timeand sample-consuming parallel workflows.12 In recent years, multiplex immunoassays 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

Immunoassays to measure target analytes within a sample have been developed for almost six decades and will continue to be a mainstay 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 optical,6−8 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 © 2018 American Chemical Society

Received: August 31, 2018 Published: October 31, 2018 15904

DOI: 10.1021/jacs.8b09422 J. Am. Chem. Soc. 2018, 140, 15904−15915

Article

Journal of the American Chemical Society

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.

tives 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 that most 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 spatially resolved array mode with different capture antibodies spotted at defined positions. Although the proprietary combination with an 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 value. 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 dots,57 or their combinations.58,59 In this work we attempt to build up a molecular multicolor ECL system by combing the potential-

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 proteins,28−34 and cells.35−38 It can serve as a sensitive reporting signal integrated with a miniaturized analytical platform, such as microfluidic and paper-based devices.39−42 ECL has also been successfully applied in the new generation of clinical immunoassays, in which the detection antibodies are directly tagged with ECL molecular luminophores.43 The light generated heterogeneously at the vicinity of the 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 deriva15905


Article

Journal of the American Chemical Society Table 1. Photophysical and Electrochemical Properties of Six ECL Luminophores probe 1 2 3 4 5 6

λmax, Abs/nm (εmax/M−1 cm−1)a 211 (25 877) 240 (55 206) 256 (55 360) 275 (60 179) 286 (88 415) 287.5 (112 490)

λmax,PL/nma 476 515 555 571 595 610

ΦPL (%)

λmax,ECL/nma

ΦECLd

E1/2 ox/V

E1/2 red/V

E0−0/eVf

ΔG/eVg

491 526 591 614 622 636

1.46 0.39e 0.92 0.91 1.00 1.82

1.75 0.74 1.28 1.23 1.33 1.30

−1.31 −2.26 −1.43 −1.32 −1.29, −1.47, −1.70 −1.24, −1.45, −1.69

2.60 2.41 2.23 2.17 2.08 2.03

−0.84 −0.03 −0.75 −0.76 −0.94 −0.96

b

3.79 0.52b 7.11c 6.18c 1.80c 2.29c

a λmax,Abs, λmax,PL, and λmax,ECL represent the maximum absorption, PL, and ECL emission wavelengths, respectively. εmax denotes the maximum molar absorptivity. bMeasured at 298 K using quinine sulfate (Φ = 54.6% in 0.5 M H2SO4) as the standard.70 cMeasured at 298 K using Ru(bpy)3(PF6)2 as the standard (Φ = 1.8% in aerated acetonitrile).71 dCo-reactant ECL efficiencies are given with respect to Ru(bpy)3(PF6)2 in acetonitrile. eΦECL of 2 is 0.33 as cited from ref 46. fE0−0 is the approximate spectroscopic energy of the excited state, which is calculated to be approximately E0−0 = 1239.81/λmax,FL (in eV).72 gΔG is the variation of Gibbs free energy for the co-reactant reaction involved in the generation of the excited state. ΔG = E(TPrA•) − Eox + E0−0, where E(TPrA•) is the reduction potential of the radical and has been reported previously as −1.70 V.73

Figure 1b illustrates the ECL spectra of six luminophores obtained in the presence of tri-n-propylamine (TPrA) as the co-reactant 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 (λmax,ECL) for 1−6 were 491, 526, 591, 614, 622, 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, ΦECL, of luminophores 1−6 in the presence of TPrA was measured as photons emitted per oxidation event relative to that of the 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 the SI). The calculated ΦECL values are 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 the Ir(III) center. While 2 displays a current wave at −2.26 V, 5 and 6 exhibit three waves at ca. −1.3, −1.5, and −1.7 V, corresponding to three successive reductions. 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, the 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 λmax,ECL by lowering the π*(LUMO) energy level. In this case, the separation of the ECL spectral

and spectrum-resolution 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 multicolor ECL system including three different luminophores was successfully structured. As a proof-of-concept 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-phenylpyridine)iridium(III) (Ir(ppy)3, 2), is one of the first iridium complexes used in ECL research.46,60−62 The other four luminophores, namely, [4,4′-bis(tert-butyl)-2,2′bipyridine]bis[3,5-difluoro-2-[5-(trifluoromethyl)-2-pyridinyl]phenyl]iridium(III) (Ir(dFCF3ppy)2(dtbbpy)+, 1), [4,4′-bis(tert-butyl)-2,2′-bipyridine]bis[2-(2-pyridinyl)phenyl]iridium(III) (Ir(ppy)2(dtbbpy)+, 3), [4,4′-bis(4-vinylphenyl)-2,2′bipyridine]bis[(2-pyridinyl)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 and 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 (λmax,Abs) and the maximum molar absorptivity (εmax,Abs), the maximum PL wavelength (λmax,PL), and the approximate spectroscopic energy of the excited state (E0−0 = 1239.81/λmax, FL) are summarized in Table 1. The PL quantum yields (ΦPL) of the 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). 15906


Article


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, 125, 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.

system involves the following fundamental steps (M represents the luminophore),

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 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, 125, 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 were captured during a potential scan from +0.8 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

TPrA → TPrA+• + e−

(1)

TPrA+• → TPrA• + H+

(2)

M → M+• + e−

(3)

TPrA• + M+• → M* + P1

(4)

M* → M + hν

(5)

Equations 1−3 refer to the generation of Ru(bpy)2(dvbpy)3+ and highly reductive TPrA• at the vicinity of the electrode surface. They further react to yield [Ru(bpy)2(dvbpy)2+]*, which 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-state 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 15907


Article


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.

M+• + TPrA → M + TPrA+•

butane to function as the working electrode. The other one was filled with solutions (in this case, 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 electro-oxidation 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

(8)

The contribution of this process to the overall ECL intensity depends upon the concentration of luminophores.75 Similar electrochemical and ECL signals were observed for Ir(dF(CF3)ppy)2(dtbbpy)+ (1)/TPrA (see Figure 2c and d). 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(CF 3 )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 and +1.6 V in the forward potential scan, with the second one being only 10% of the first in 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 to +2.30 V. To rationalize the annihilation of ECL in this potential range, an electrochemistry-coupled mass spectrometry (ECMS) technique was used to in situ track and identify intermediate species formed in the ECL process.76,77 Figure 3a depicts the workflow of the EC-MS system. The core component is a quartz theta-shaped micropipet with dual barrels, one of which was deposited with carbon by pyrolysis of

[Ir(ppy)3 ]* + TPrA+• → Ir(ppy)3+ + TPrA

(10)

Ir(ppy)3+

As shown in Figure 3c, the MS intensity of (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 15908


Article


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 Figure 2a and b, 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 example, +0.75, + 0.80, 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 to +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, and 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

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 potentialresolved 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

TPrA• + Ru(bpy)2 (dvbpy)2 + → Ru(bpy)2 (dvbpy)+ + P1 (11) +

+

Ir(ppy)3 + Ru(bpy)2 (dvbpy) → [Ru(bpy)2 (dvbpy)2 + ]* + Ir(ppy)3

(12)

Equation 12 represents the key step of generating excited state species, and the variation of the Gibbs free energy (ΔG) of this step is given by 0 ′ 3+ /Ir(ppy)3 ΔG = E[*Ru(bpy) (dvbpy)2+ ]* /Ru(bpy) (dvbpy)+ − E Ir(ppy) 2

2

(13)

The value of ΔG was estimated to be +0.05 eV, indicating this step is unfavorable from a purely thermodynamic viewpoint. However, this near-zero value may not be that accurate because of the uncertainty of the 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, +1.0, +1.1, 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. A 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-butyl2,2′-bipyridine; ppy = 2-phenylpyridine).79 In either coreactant 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 the iridium(III)

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 and 125 μM for spectra, while doubled for images. Shown in the 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.

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, 15909


Article


range of +1.30 to +1.70 V, Ru(bpy)2(dvbpy)2+ (6) emitted alone to show a single emission maximum at 636 nm in spectra 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 and 636 nm in the 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 bands 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 the 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 (PSBs) 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 a similar approach to fabricate a multiplex immunoassay for multiple biomarkers’ determination. In the 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 PSBs (267 nm in diameter), respectively, and subsequently modified by specific detection antibodies for AFP, CEA, and βHCG on the basis of biotin−streptavidin complexation to prepare dual-coded PSBs (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 explanations in the text).24,28,81−83 Taking Ru(bpy)2(dvbpy)2+ (6) as an example, it was loaded to PSBs 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 μL, 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 (MBs, 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 PSBs to form the sandwich immunocomplexes. After magnetic separation again, the immunocomplexes were submerged in 100 μL of acetonitrile to swell the PSBs to release multiple luminophores. The solution was finally mixed with another 100 μL of acetonitrile containing TPrA and TBAPF6 for ECL measurements, which were performed on an upright microscope

emitter and meanwhile improving the generation of the 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 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). As shown in Figure 2, both Ir(dFCF3ppy)2(dtbbpy)+ (1) and Ru(bpy)2(dvbpy)2+ (6) produce ECL at very high potentials. Figure 5a and b illustrate the spooling ECL spectra

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 and 125 μM for spectra, while doubled for image. Shown in the 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.

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, it was not detected due to its low efficiency or low concentration of luminophores used here. In the potential 15910


Article


Figure 6. (a) Procedure of simultaneous recognition of multiple disease biomarkers. Two incubation steps were carried out at 37 °C for 30 min, and the separation was performed on a magnetic scaffold for 3 min and washed with phosphate buffer saline containing Tween-20 (PBST). Acetonitrile (100 μL) was used to swell the PSBs to release multiple luminophores from the PSBs under ultrasonication. (b) Scheme of the ECL spectral detection system on the basis of an upright microscope (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 the “step and glue” function under conventional acquisition mode. The EMCCD exposure time was set to 2 s.

pathological cutoff concentrations. First, the spectral peaks were normalized and analyzed by means of peak-differentiation-imitating analysis. As shown in Figure 7b, a single emission peak centered at 520 nm was obtained at +0.74 V (green line), 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. 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 MBs and detection antibodies-coated PSBs 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 was successfully accomplished. The results affirmed

connected to an electrochemical workstation (as shown in the left panel of Figure 6b). A disk-shaped glassy carbon electrode (GCE, 3 mm in diameter) wrapped in 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 quasireference 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, +1.33, 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 βHCG in a single modeling sample at a concentration of 5 ng/ mL, 25 ng/mL, and 5 mIU/mL, respectively, which are the 15911


Article


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 diagnosis of cervical carcinoma requires CEA as the main biomarker and AFP, β-HCG, and CA125 as three auxiliary ones. The simultaneous recognition of biomarkers above the pathological cutoff 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 conjugation sites to link with antibodies. So they have to be loaded to polymer beads to code specific antibodies and finally released from the beads by swelling for ECL detection. The use of water-soluble luminophores with appropriate conjugation sites can significantly simplify the assay operation, because the swelling release step will be unnecessary.

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) Corresponding spectral peak intensities (left) and the converted digital color map (right). The error bars represent 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.

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 the 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. This is the reason that 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.

■

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, 2-phenylpyridine (ppy), 4,4′-di-tert-butyl-2,2′-bipyridine (dtbbpy), TPrA, biotin-labeled fluorescein 5(6)-isothiocyanate (bio-FITC), N-hydroxysulfosuccinimide sodium salt (sulfo-NHS), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC), 2-(N-morpholino)ethanesulfonic acid, and Ir(ppy)3 were purchased from Aladdin. cis-Dichlorobis(2,2′-bipyridine)ruthenium (cis-Ru(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 at Genesea Biotechnology Co. Ltd. (Hangzhou, China). Bovine serum albumin (BSA) was obtained from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA).

■

CONCLUSIONS In summary, we have synthesized several ruthenium and iridium complexes with distinguishable ECL emission wavelengths from 491 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-ECLgenerating species, Ir(dFCF3ppy)2(dtbbpy)+ (1), and the red one, Ru(bpy)2(dvbpy)2+ (6), achieving the spectrum-resolved ECL generation 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 15912


Article

Journal of the American Chemical Society Carboxylate PSBs (267 nm and 9.94 μm in diameter) 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-(NMorpholino)ethanesulfonic acid and Tween-20 were used to prepare MES buffer (0.1 M, 0.005 wt % Tween-20), and the solution pH was 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). ΦPL for 1 and 2 was determined using quinine sulfate in 0.5 M H2SO4 as the standard (Φ = 54.6%),70 and that for 3−6 was measured using Ru(bpy)32+ as the standard (Φ = 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 and 0.05 μm alumina slurry. A platinum wire and a silver wire were used as a counter and a quasireference 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 text are given against SCE.84 Electrochemical measurements were acquired on a CHI 920C electrochemical workstation (CHI Instruments, Shanghai, China). Electrochemistry-Coupled Mass Spectrometry. 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 (MS305D, Dongguan Maihao, Ltd., China). The main experimental parameters of MS and the fabrication of the hybrid ultramicroelectrodes were conducted as 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) 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, The Netherlands) and the QEpro spectrophotometer. A homemade Teflon cell consisting of a circular quartz window, optic fiber, and collimating lens was used. The integration time of an 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 homemade 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.

■

■

The experimental details on the synthesis, NMR, and spectroscopic characterizations of multicolor ECL luminophores; preparation and optical characterization of dual-coded PSB; immunoassay procedure and detection results (PDF)

AUTHOR INFORMATION

Corresponding Author

*[email protected] ORCID

Weiliang Guo: 0000-0002-4457-2911 Bin Su: 0000-0003-0115-2279 Yuanhua Shao: 0000-0003-3922-6229 Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS 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).

■

REFERENCES

(1) Yalow, R. S.; Berson, S. A. Immunoassay of endogenous plasma insulin in man. J. Clin. Invest. 1960, 39, 1157. (2) Wulfkuhle, J. D.; Liotta, L. A.; Petricoin, E. F. Proteomic applications for the early detection of cancer. Nat. Rev. Cancer 2003, 3, 267. (3) Kingsmore, S. F. Multiplexed protein measurement: technologies and applications of protein and antibody arrays. Nat. Rev. Drug Discovery 2006, 5, 310. (4) Liu, G.; Wang, J.; Kim, J.; Jan, M. R.; Collins, G. E. Electrochemical coding for multiplexed immunoassays of proteins. Anal. Chem. 2004, 76, 7126. (5) Chikkaveeraiah, B. V.; Bhirde, A. A.; Morgan, N. Y.; Eden, H. S.; Chen, X. Electrochemical immunosensors for detection of cancer protein biomarkers. ACS Nano 2012, 6, 6546. (6) Goldman, E. R.; Clapp, A. R.; Anderson, G. P.; Uyeda, H. T.; Mauro, J. M.; Medintz, I. L.; Mattoussi, H. Multiplexed toxin analysis using four colors of quantum dot fluororeagents. Anal. Chem. 2004, 76, 684. (7) Song, E.; Han, W.; Li, J.; Jiang, Y.; Cheng, D.; Song, Y.; Zhang, P.; Tan, W. Magnetic-encoded fluorescent multifunctional nanospheres for simultaneous multicomponent analysis. Anal. Chem. 2014, 86, 9434. (8) Bake, K. D.; Walt, D. R. Multiplexed spectroscopic detections. Annu. Rev. Anal. Chem. 2008, 1, 515. (9) Wang, W.; Zhou, H.; Lin, H.; Roy, S.; Shaler, T. A.; Hill, L. R.; Norton, S.; Kumar, P.; Anderle, M.; Becker, C. H. Quantification of proteins and metabolites by mass spectrometry without isotopic labeling or spiked standards. Anal. Chem. 2003, 75, 4818. (10) Diamandis, E. P. Mass spectrometry as a diagnostic and a cancer biomarker discovery tool: opportunities and potential limitations. Mol. Cell. Proteomics 2004, 3, 367. (11) Wu, A. H. B. A selected history and future of immunoassay development and applications in clinical chemistry. Clin. Chim. Acta 2006, 369, 119. (12) Borrebaeck, C. A. K. Antibodies in diagnostics − from immunoassays to protein chips. Immunol. Today 2000, 21, 379. (13) Deiss, F.; LaFratta, C. N.; Symer, M.; Blicharz, T. M.; Sojic, N.; Walt, D. R. Multiplexed sandwich immunoassays using electrochemiluminescence imaging resolved at the single bead level. J. Am. Chem. Soc. 2009, 131, 6088.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/jacs.8b09422. 15913


Article

Journal of the American Chemical Society (14) Zong, C.; Wu, J.; Wang, C.; Ju, H.; Yan, F. Chemiluminescence imaging immunoassay of multiple tumor markers for cancer screening. Anal. Chem. 2012, 84, 2410. (15) Rissin, D. M.; Kan, C. W.; Song, L.; Rivnak, A. J.; Fishburn, M. W.; Shao, Q.; Piech, T.; Ferrell, E. P.; Meyer, R. E.; Campbell, T. G. Multiplexed single molecule immunoassays. Lab Chip 2013, 13, 2902. (16) Pakchin, P. S.; Nakhjavani, S. A.; Saber, R.; Ghanbari, H.; Omidi, Y. Recent advances in simultaneous electrochemical multianalyte sensing platforms. TrAC, Trends Anal. Chem. 2017, 92, 32. (17) Vignali, D. A. A. Multiplexed particle-based flow cytometric assays. J. Immunol. Methods 2000, 243, 243. (18) Richter, M. M. Electrochemiluminescence (ECL). Chem. Rev. 2004, 104, 3003. (19) Hu, L. Z.; Xu, G. B. Applications and trends in electrochemiluminescence. Chem. Soc. Rev. 2010, 39, 3275. (20) Zhang, X.; Chen, C.; Li, J.; Zhang, L.; Wang, E. New insight into a microfluidic-based bipolar system for an electrochemiluminescence sensing platform. Anal. Chem. 2013, 85, 5335. (21) Lim, H.; Ju, Y.; Kim, J. Tailoring catalytic activity of Pt nanoparticles encapsulated inside dendrimers by tuning nanoparticle sizes with subnanometer accuracy for sensitive chemiluminescencebased analyses. Anal. Chem. 2016, 88, 4751. (22) Kitte, S. A.; Gao, W.; Zholudov, Y. T.; Qi, L.; Nsabimana, A.; Liu, Z.; Xu, G. Stainless steel electrode for sensitive luminol electrochemiluminescent detection of H2O2, glucose, and glucose oxidase activity. Anal. Chem. 2017, 89, 9864. (23) Zhang, X.; Li, J.; Jia, X.; Li, D.; Wang, E. Full-featured electrochemiluminescence sensing platform based on the multichannel closed bipolar system. Anal. Chem. 2014, 86, 5595. (24) Miao, W.; Bard, A. J. Electrogenerated chemiluminescence. 77. DNA hybridization detection at high amplification with [Ru(bpy)3]2+containing microspheres. Anal. Chem. 2004, 76, 5379. (25) Wu, M. S.; He, L. J.; Xu, J. J.; Chen, H. Y. RuSi@Ru(bpy)32+/ Au@Ag2S nanoparticles electrochemiluminescence resonance energy transfer system for sensitive DNA detection. Anal. Chem. 2014, 86, 4559. (26) Mani, V.; Kadimisetty, K.; Malla, S.; Joshi, A. A.; Rusling, J. F. Paper-based electrochemiluminescent screening for genotoxic activity in the environment. Environ. Sci. Technol. 2013, 47, 1937. (27) Wasalathanthri, D. P.; Malla, S.; Bist, I.; Tang, C. K.; Faria, R. C.; Rusling, J. F. High-throughput metabolic genotoxicity screening with a fluidic microwell chip and electrochemiluminescence. Lab Chip 2013, 13, 4554. (28) Miao, W.; Bard, A. J. Electrogenerated chemiluminescence. 80. C-reactive protein determination at high amplification with [Ru(bpy)3]2+-containing microspheres. Anal. Chem. 2004, 76, 7109. (29) Sardesai, N. P.; Barron, J. C.; Rusling, J. F. Carbon nanotube microwell array for sensitive electrochemiluminescent detection of cancer biomarker proteins. Anal. Chem. 2011, 83, 6698. (30) Wu, M. S.; Yuan, D. J.; Xu, J. J.; Chen, H. Y. Electrochemiluminescence on bipolar electrodes for visual bioanalysis. Chem. Sci. 2013, 4, 1182. (31) Wu, M. S.; Liu, Z.; Shi, H. W.; Chen, H. Y.; Xu, J. J. Visual electrochemiluminescence detection of cancer biomarkers on a closed bipolar electrode array chip. Anal. Chem. 2015, 87, 530. (32) Zhang, X.; Tan, X.; Zhang, B.; Miao, W. J.; Zou, G. Z. Spectrum-based electrochemiluminescent immunoassay with ternary CdZnSe nanocrystals as labels. Anal. Chem. 2016, 88, 6947. (33) Wang, N.; Feng, Y.; Wang, Y.; Ju, H.; Yan, F. Electrochemiluminescent imaging for multi-immunoassay sensitized by dual DNA amplification of polymer dot signal. Anal. Chem. 2018, 90, 7708. (34) Zhai, Q.; Zhang, X.; Han, Y.; Zhai, J.; Li, J.; Wang, E. A nanoscale multi-channel closed bipolar electrode array for electrochemiluminescence sensing platform. Anal. Chem. 2016, 88, 945. (35) Zhou, J.; Ma, G.; Chen, Y.; Fang, D.; Jiang, D.; Chen, H. Electrochemiluminescence imaging for parallel single-cell analysis of active membrane cholesterol. Anal. Chem. 2015, 87, 8138.

(36) Xu, J. J.; Huang, P. Y.; Qin, Y.; Jiang, D. C.; Chen, H. Y. Analysis of intracellular glucose at single cells using electrochemiluminescence imaging. Anal. Chem. 2016, 88, 4609. (37) Valenti, G.; Scarabino, S.; Goudeau, B.; Lesch, A.; Jović, M.; Villani, E.; Sentic, M.; Rapino, S.; Arbault, S.; Paolucci, F.; Sojic, N. Single cell electrochemiluminescence imaging: from the proof-ofconcept to disposable device-based analysis. J. Am. Chem. Soc. 2017, 139, 16830. (38) Liu, G.; Ma, C.; Jin, B. K.; Chen, Z.; Zhu, J. J. Direct electrochemiluminescence imaging of a single cell on a chitosan film modified electrode. Anal. Chem. 2018, 90, 4801. (39) Zhan, W.; Alvarez, J.; Crooks, R. M. Electrochemical sensing in microfluldic systems using electrogenerated chemiluminescence as a photonic reporter of redox reactions. J. Am. Chem. Soc. 2002, 124, 13265. (40) Chow, K.-F.; Mavré, F.; Crooks, R. M. Wireless electrochemical DNA microarray sensor. J. Am. Chem. Soc. 2008, 130, 7544. (41) Delaney, J. L.; Hogan, C. F.; Tian, J. F.; Shen, W. Electrogenerated chemiluminescence detection in paper-based microfluidic sensors. Anal. Chem. 2011, 83, 1300. (42) Ge, L.; Yan, J.; Song, X.; Yan, M.; Ge, S.; Yu, J. Threedimensional paper-based electrochemiluminescence immunodevice for multiplexed measurement of biomarkers and point-of-care testing. Biomaterials 2012, 33, 1024. (43) Muzyka, K. Current trends in the development of the electrochemiluminescent immunosensors. Biosens. Bioelectron. 2014, 54, 393. (44) Meso Scale Diagnostics, www.mesoscale.com. (45) Liu, Z.; Qi, W.; Xu, G. Recent advances in electrochemiluminescence. Chem. Soc. Rev. 2015, 44, 3117. (46) Bruce, D.; Richter, M. M. Green electrochemiluminescence from ortho-metalated tris(2-phenylpyridine)iridium(III). Anal. Chem. 2002, 74, 1340. (47) Dennany, L.; Forster, R. J.; White, B.; Smyth, M.; Rusling, J. F. Direct electrochemiluminescence detection of oxidized DNA in ultrathin films containing [Os(bpy)2(PVP)10]2+. J. Am. Chem. Soc. 2004, 126, 8835. (48) Doeven, E. H.; Zammit, E. M.; Barbante, G. J.; Hogan, C. F.; Barnett, N. W.; Francis, P. S. Selective excitation of concomitant electrochemiluminophores: tuning emission color by electrode potential. Angew. Chem., Int. Ed. 2012, 51, 4354. (49) Doeven, E. H.; Zammit, E. M.; Barbante, G. J.; Francis, P. S.; Barnett, N. W.; Hogan, C. F. A potential-controlled switch on/off mechanism for selective excitation in mixed electrochemiluminescent systems. Chem. Sci. 2013, 4, 977. (50) Doeven, E. H.; Barbante, G. J.; Kerr, E.; Hogan, C. F.; Endler, J. A.; Francis, P. S. Red-Green-Blue electrogenerated chemiluminescence utilizing a digital camera as detector. Anal. Chem. 2014, 86, 2727. (51) Han, F.; Jiang, H.; Fang, D.; Jiang, D. Potential-resolved electrochemiluminescence for determination of two antigens at the cell surface. Anal. Chem. 2014, 86, 6896. (52) Kerr, E.; Doeven, E. H.; Barbante, G. J.; Hogan, C. F.; Bower, D. J.; Donnelly, P. S.; Connell, T. U.; Francis, P. S. Annihilation electrogenerated chemiluminescence of mixed metal chelates in solution: modulating emission colour by manipulating the energetics. Chem. Sci. 2015, 6, 472. (53) Haghighatbin, M. A.; Lo, S.; Burn, P.; Hogan, C. F. Electrochemically tuneable multi-colour electrochemiluminescence from a single emitter. Chem. Sci. 2016, 7, 6974. (54) Kerr, E.; Doeven, E. H.; Barbante, G. J.; Hogan, C. F.; Hayne, D. J.; Donnelly, P. S.; Francis, P. S. New perspectives on the annihilation electrogenerated chemiluminescence of mixed metal complexes in solution. Chem. Sci. 2016, 7, 5271. (55) Wang, Y.-Z.; Xu, C.-H.; Zhao, W.; Guan, Q.-Y.; Chen, H.-Y.; Xu, J.-J. Bipolar electrode based multicolor electrochemiluminescence biosensor. Anal. Chem. 2017, 89, 8050. (56) Wang, Y.-Z.; Ji, S.-Y.; Xu, H.-Y.; Zhao, W.; Xu, J.-J.; Chen, H.-Y. Bidirectional electrochemiluminescence color switch: an application 15914


Article

Journal of the American Chemical Society in detecting multimarkers of prostate cancer. Anal. Chem. 2018, 90, 3570. (57) Zou, G.; Tan, X.; Long, X.; He, Y.; Miao, W. Spectrum-resolved dual-color electrochemiluminescence immunoassay for simultaneous detection of two targets with nanocrystals as tags. Anal. Chem. 2017, 89, 13024. (58) Hesari, M.; Swanick, K. N.; Lu, J.-S.; Whyte, R.; Wang, S.; Ding, Z. Highly efficient dual-color electrochemiluminescence from BODIPY-capped PbS nanocrystals. J. Am. Chem. Soc. 2015, 137, 11266. (59) Shu, J.; Han, Z.; Zheng, T.; Du, D.; Zou, G.; Cui, H. Potentialresolved multicolor electrochemiluminescence of N-(4-aminobutyl)N-ethylisoluminol/tetra(4-carboxyphenyl)porphyrin/TiO2 nanoluminophores. Anal. Chem. 2017, 89, 12636. (60) Kapturkiewicz, A. Cyclometalated iridium(III) chelates-a new exceptional class of the electrochemiluminescent luminophores. Anal. Bioanal. Chem. 2016, 408, 7013. (61) Gross, E. M.; Armstrong, N. R.; Wightman, R. M. Electrogenerated chemiluminescence from phosphorescent molecules used in organic light-emitting diodes. J. Electrochem. Soc. 2002, 149, 137. (62) Kapturkiewicz, A.; Angulo, G. Extremely efficient electrochemiluminescence systems based on tris (2-phenylpyridine) iridium (III). Dalton Trans. 2003, No. 20, 3907. (63) Lowry, M. S.; Goldsmith, J. I.; Slinker, J. D.; Rohl, R.; Pascal, R. A.; Malliaras, G. G.; Bernhard, S. Single-layer electroluminescent devices and photoinduced hydrogen production from an ionic iridium(III) complex. Chem. Mater. 2005, 17, 5712. (64) Slinker, J. D.; Gorodetsky, A. A.; Lowry, M. S.; Wang, J. J.; Parker, S.; Rohl, R.; Bernhard, S.; Malliaras, G. G. Efficient yellow electroluminescence from a single layer of a cyclometalated iridium complex. J. Am. Chem. Soc. 2004, 126, 2763. (65) Guo, W.; Cao, Z.; Liu, Y.; Su, B. Electrochemiluminescence of vinyl-functionalized ruthenium complex and its monolayer formed by photoinduced thiol-ene click reaction. ChemElectroChem 2017, 4, 1763. (66) Yang, S.-C.; Chang, G.; Yang, G.-J.; Wang, Y.-J.; Fang, B. Photocatalytic hydrogen generation from water reduction using orchestrated photosensitizers. Catal. Sci. Technol. 2015, 5, 228. (67) Sartin, M. M.; Camerel, F.; Ziessel, R.; Bard, A. J. Electrogenerated chemiluminescence of B8amide: a BODIPY-based molecule with asymmetric ECL transients. J. Phys. Chem. C 2008, 112, 10833. (68) Suk, J.; Natarajan, P.; Moorthy, J. N.; Bard, A. J. Electrochemistry and electrogenerated chemiluminescence of twisted anthracene-functionalized bimesitylenes. J. Am. Chem. Soc. 2012, 134, 3451. (69) Hesari, M.; Lu, J.; Wang, S.; Ding, Z. Efficient electrochemiluminescence of a boron-dipyrromethene (BODIPY) dye. Chem. Commun. 2015, 51, 1081. (70) Melhuish, W. H. Quantum efficiencies of fluorescence organic substances - effect of solvent and concentration of fluorescent solute. J. Phys. Chem. 1961, 65, 229. (71) Ishida, H.; Tobita, S.; Hasegawa, Y.; Katoh, R.; Nozaki, K. Recent advances in instrumentation for absolute emission quantum yield measurements. Coord. Chem. Rev. 2010, 254, 2449. (72) Bard, A. J. Electrogenerated Chemiluminescence; Marcel Dekker: New York, 2004. (73) Lai, R. Y.; Bard, A. J. Electrogenerated chemiluminescence. 70. The application of ECL to determine electrode potentials of tri-npropylamine, its radical cation, and intermediate free radical in MeCN/benzene solutions. J. Phys. Chem. A 2003, 107, 3335. (74) Miao, W. J.; Choi, J. P.; Bard, A. J. Electrogenerated chemiluminescence 69: the tris(2,2’-bipyridine)ruthenium(II), (Ru(bpy)32+)/tri-n-propylamine (TPrA) system revisited-A new route involving TPrA•+ cation radicals. J. Am. Chem. Soc. 2002, 124, 14478. (75) Zu, Y.; Bard, A. J. Electrogenerated chemiluminescence. 66. the role of direct coreactant oxidation in the ruthenium Tris(2,2’)bipyridyl/tripropylamine system and the effect of halide ions on the emission intensity. Anal. Chem. 2000, 72, 3223.

(76) Qiu, R.; Zhang, X.; Luo, H.; Shao, Y. Mass spectrometric snapshots for electrochemical reactions. Chem. Sci. 2016, 7, 6684. (77) Qin, X.; Gu, C.; Wang, M.; Dong, Y.; Nie, X.; Li, M.; Zhu, Z.; Yang, D.; Shao, Y. Triethanolamine-modified gold nanoparticles synthesized by a one-pot method and their application in electrochemiluminescent immunoassy. Anal. Chem. 2018, 90, 2826. (78) Barbante, G. J.; Kebede, N.; Hindson, C. M.; Doeven, E. H.; Zammit, E. M.; Hanson, G. R.; Hogan, C. F.; Francis, P. S. Control of excitation and quenching in multi-colour electrogenerated chemiluminescence systems through choice of co-reactant. Chem. - Eur. J. 2014, 20, 14026. (79) Swanick, K. N.; Sandroni, M.; Ding, Z.; Zysman-Colman, E. Enhanced electrochemiluminescence from a stoichiometric ruthenium(II)−iridium(III) complex soft salt. Chem. - Eur. J. 2015, 21, 7435. (80) Fu, Q.; Zhu, J.; Eyk, J. E. V. Comparison of multiplex immunoassay platforms. Clin. Chem. 2010, 56, 314. (81) Valenti, G.; Rampazzo, E.; Kesarkar, S.; Genovese, D.; Fiorani, A.; Zanut, A.; Palomba, F.; Marcaccio, M.; Paolucci, F.; Prodi, L. Electrogenerated chemiluminescence from metal complexes-based nanoparticles for highly sensitive sensors applications. Coord. Chem. Rev. 2018, 367, 65. (82) Dhiraj, A.; Pradip, B.; Le, L. D.; Paul, A. M.; Estefania, F.; Diamond, M. S.; Miao, W. J.; Bai, F. W. An ultrasensitive electrogenerated chemiluminescence-based immunoassay for specific detection of Zika virus. Sci. Rep. 2016, 6, 32227. (83) Qi, H.; Peng, Y.; Gao, Q.; Zhang, C. Applications of nanomaterials in electrogenerated chemiluminescence biosensors. Sensors 2009, 9, 674. (84) Swanick, K. N.; Ladouceur, S.; Zysman-Colman, E.; Ding, Z. F. Self-enhanced electrochemiluminescence of an iridium(III) complex: mechanistic insight. Angew. Chem., Int. Ed. 2012, 51, 11079. (85) Hesari, M.; Ding, Z. A grand avenue to Au nanocluster electrochemiluminescence. Acc. Chem. Res. 2017, 50, 218. (86) Kim, J. M.; Jeong, S.; Song, J. K.; Kim, J. Near-infrared electrochemiluminescence from orange fluorescent Au nanoclusters in water. Chem. Commun. 2018, 54, 2838.

15915


Potential-Resolved Multicolor Electrochemiluminescence for Multiplex

Recommend Documents