Potential-Resolved Multicolor Electrochemiluminescence for Multiplex Immunoassay in a Single Sample

Article abstract of DOI:10.1021/jacs.8b09422

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(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 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.

Full text of DOI:10.1021/jacs.8b09422

Subscriber access provided by The University of Texas at El Paso (UTEP)
Article
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
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a service to the research community to expedite the dissemination
of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in
full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully
peer reviewed, but should not be considered the official version of record. They are citable by the
Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore,
the “Just Accepted” Web site may not include all articles that will be published in the journal. After
a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web
site and published as an ASAP article. Note that technical editing may introduce minor changes
to the manuscript text and/or graphics which could affect content, and all legal disclaimers and
ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or
consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W.,
Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 12
Journal of the American Chemical Society
1
2
3
4
5
6
7
8
9
Potential-Resolved Multicolor Electrochemiluminescence for Multi-
plex Immunoassay in a Single Sample
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
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 Na-
tional 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 diag-
nosis. Developing ECL-based multiplex immunoassay, with the potential to enable high-throughput detection of multiple bi-
omarkers 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 electrochemistry-
coupled mass spectrometry. A multiplex immunoassay free of spatial spotting antibodies on plates or substrates was ultimately de-
vised 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 spot-
free 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 pre-
cious 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 en-
coded 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
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 ef-
forts have been made recently to develop novel immunosen-
sors 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 commer-
cially 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 horse-
radish peroxidase and alkaline phosphatase), performs the
measure of target analytes via the formation of immunocom-
plexes and subsequently the enzyme-catalyzed conversion of
substrates to detectable products that are chromogenic, fluo-
rescent or chemiluminescent.11 Typical immunoassays are de-
signed in most cases to measure only one specific target ana-
lyte at a time (often termed as singleplex immunoassays), and
measuring multiple targets involves time- and sample-
consuming parallel workflows.12 In recent years, multiplex im-
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 inte-
grated with miniaturized analytical platform, such as microflu-
idic and paper-based devices.39-42 ECL has also been successful-
ACS Paragon Plus Environment
1

Journal of the American Chemical Society
Page 2 of 12
ly applied in the new generation of clinical immunoassays, in
phenylpyridine)iridium(III) (Ir(ppy)3, 2), is one of the first
iridium complexes used in ECL research.46,60-62 Other four lu-
minophores, namely, [4,4’-bis(tert-butyl)-2,2’-
1
2
3
4
5
6
7
8
which the detection antibodies are directly tagged with ECL
molecular luminophores.43 And the light generated heteroge-
neously at the vicinity of electrode surface under the electro-
chemical stimulation is capable of quantifying analytes cap-
tured in the precedent immunoreactions. Multiplex ECL im-
munoassays have already been commercialized by using multi-
array technology to detect simultaneously different analytes in
a sample.44 Although various ECL luminophores have been
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’-
developed
so
far,
tris(2,2’-bipyridyl)ruthenium(II)
9
(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 multi-
plex ECL immunoassays are usually performed in a spatially-
resolved array mode with different capture antibodies spotted
at defined positions. Although the proprietary combination
with ECL imaging reader results in an exceptionally high sensi-
tivity, 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 work-
flows of a multitude of singleplex assays.
bipyridine)ruthenium(II) (Ru(bpy)2(dvbpy)2+, 6) were syn-
thesized 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 exhib-
ited PL nearly across the entire visible light range. Their ab-
sorption maximum wavelength (max, Abs) and the maximum
molar absorptivity (max, Abs), the maximum PL wavelength
(max, PL) and the approximate spectroscopic energy of excited
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
state (E0 0 = 1239.81/max, FL) are summarized in Table 1. The
‒
PL quantum yields (ФPL) of luminophores were determined
2+
using quinine and Ru(bpy)3 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).
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 corre-
sponding threshold valve. In this case, one usually obtains a
poorly resolved emission for the mixture of multiple lumino-
phores, 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 possi-
bilities of multiplex immunoassays, using molecular lumino-
phores,48-56 semiconducting quantum dots57 or their combina-
tions.58,59 In this work we attempt to build up a molecular mul-
ti-color ECL system by combing the potential- 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 ration-
alized the ECL generation by single luminophores and their
mixtures. A multi-color ECL system including three different
luminophores was successfully structured. As a proof-of-
concept experiment, they were loaded into polymeric mi-
crobeads to encode three different detection antibodies for
simultaneous recognition of carcinoembryonic antigen (CEA),
alpha-fetoprotein (AFP) and beta-human chorionic gonado-
tropin (β-HCG) in a single run.
Figure 1b illustrates the ECL spectra of six luminophores ob-
tained 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 oxi-
dized. Apparently, the ECL spectra are quite similar to PL
ones, covering nearly the whole visible light range. The maxi-
mum 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 bath-
ochromic shifts, which were most likely associated with an
inner-filter effect because of the difference in the concentra-
tion used in PL and ECL measurements.67,68 The ECL efficien-
cy, ФECL, of luminophores 1‒6 in the presence of TPrA was
measured as photons emitted per oxidation event relative to
2+
that of Ru(bpy)3 (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 acetoni-
trile containing 0.1 M tetra-n-butylammonium hexafluorobo-
rate (TBAPF6) as supporting electrolyte at a scan rate of 0.1 V
‒
1
s . 1 displays a reversible one-electron-oxidation process at ca.
+1.75 V (E1/2, vs. SCE, the same in the following context),
ox
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 com-
pared 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 reduc-
tion 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.
RESULTS AND DISCUSSION
Synthesis and Characterization of ECL Luminophores. Figure 1a
shows the molecular structures of six ECL luminophores.
2+
Ru(bpy)3 (5) is recognized as a benchmark ECL lumino-
phore.
The
neutral
luminophore,
tris(2-
ACS Paragon Plus Environment
2

Page 3 of 12
Journal of the American Chemical Society
Table 1. Photophysical and Electrochemical Properties of Six ECL Luminophores
1
ФPL
(%)
E1/2, ox
/ V
E1/2, red
/ V
ΔG
2
3
4
5
6
7
8
9

max, Abs / nm
max, PL
/ nm
max, ECL
E0
0
‒
d
Probes
ФECL
/ eV g
‒
‒
1
/ eV f
a
/ nm a
a
(εmax / M 1 cm
)
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
2.60
2.41
2.23
2.17
2.08
‒1.31
‒2.26
‒1.43
‒1.32
0.84
‒0.03
‒0.75
‒0.76
‒0.94
‒1.29, ‒1.47, ‒1.70
6
287.5 (112490)
610
2.29c
636
1.82
1.30
2.03
‒1.24, ‒1.45, ‒1.69
‒0.96
a max, Abs, max, PL, and max, ECL represent the maximum absorption, PL, and ECL emission wavelengths, respectively. εmax denotes the maximum molar
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
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. ФECL of 2 is 0.33
e
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 )E E0-0 , where
ox
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 Fig-
ure 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 detect-
ed 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 . 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),
TPrA TPrA e
TPrA TPrA H
‒
1
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)3 (5) and Ru(bpy)2(dvbpy)2+ (6). (b)
2+
Normalized ECL spectra of six luminophores obtained under their corre-
sponding 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
(1)
(2)
MM+ +e
TPrA + M+  M+ P
(3)
‒
1
s
.
(4)
1
As seen from Figure 1b and Table 1, the ECL spectral maxi-
M M h
mum separation between 1 and 2 is only 35 nm, making it dif-
ficult 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 possibil-
ity by selective or sequential oxidation of two of them in mix-
tures 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
(5)
Eqs 1-3 refer to the generation of Ru(bpy)2(dvbpy)3+ and high-
ly 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 + P
(6)
2+
styryl groups on the parent Ru(bpy)3 induced a batho-
1
chromic 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 lumino-
phores, namely Ru(bpy)2(dvbpy)2+ (6), Ir(ppy)3 (2) and
M+ + M M+ M
(7)
This reaction pathway usually occurs simultaneously and can-
not be easily discriminated. The excited states generation in-
volved the direct electrooxidation of both luminophores and
TPrA at the electrode surface, while a “catalytic route” involv-
ing homogeneous reduction of M+• by TPrA could also take
place (eq 8),74
ACS Paragon Plus Environment
3

Journal of the American Chemical Society
Page 4 of 12
M+  TPrA M  TPrA
(8)
1
2
3
The contribution of this process to the overall ECL intensity
depends upon the concentration of luminophores.75
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
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 po-
tential. 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 micro-
electrochemical (micro-EC) cell, in which a silver chlo-
ride/silver (AgCl/Ag) wire was inserted to function as the
reference electrode (its potential was calibrated using the fer-
ricenium/ferrocene couple as the internal reference). Upon
applying a suitable potential to the micro-EC cell via an exter-
nal 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 exper-
iment, 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. Fig-
ure 3b compares the mass spectra of Ir(ppy)3 (2) in the ab-
sence (top) and presence (bottom) of TPrA at +1.0 V. In the
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 concen-
trations of luminophores were 2.5 µM, 125 µM and 125 µM, respec-
tively. The photomultiplier tube (PMT) was biased at 600 V. The
scan rate was 0.1 V s . 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.
‒
1
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 domi-
nated 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)
+
former case, Ir(ppy)3 (m/z 655) generated from the electro-
oxidation of Ir(ppy)3 was detected. In contrast, in the presence
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 for-
ward 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 emis-
sion 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.
+
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 poten-
tial.
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
ACS Paragon Plus Environment
4

Page 5 of 12
1
Journal of the American Chemical Society

3 
Ir(ppy)  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) in-
creased with increasing the potential. In the control experi-
2
3
4
5
6
7
+
+
ment without TPrA (black line), the MS intensity of Ir(ppy)3
8
(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 anni-
hilation behavior of Ir(ppy)3 in the high potential regime offers
the opportunity of devising potential-resolved ECL systems.
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
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 potential-
resolved ECL generation. As seen in Figure 4a for a binary mix-
ture of Ir(ppy)3 (2) and Ru(bpy)2(dvbpy)2+ (6), two distin-
guishable 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 po-
tential 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, suggest-
ing 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 genera-
tion by Ru(bpy)2(dvbpy)2+ (6). This hypothesis was also con-
firmed 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), suggest-
ing 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).
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 dis-
played images were obtained with a 3 mm-in-diameter glassy car-
bon 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 spe-
cies and the variation of Gibbs free energy (ΔG) of this step is
given by,
0'
G  E
E
/Ru(bpy)2 (dvbpy)
(13)
Ir(ppy)3 /Ir(ppy)3
2 

Ru(bpy)2 (dvbpy)

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 com-
pared. 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
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,
(dtbubpy
= 4,4’-di-tert-butyl-2,2’-bipyridine; ppy = 2-
phenylpyridine).79 In either co-reactant or annihilation route,
no ECL signal was generated from [Ir]‒ moieties, while the
*
TPrA Ru(bpy)2(dvbpy)2+ Ru(bpy)2(dvbpy)+ P (11)
1
emission intensity from [Ru]2+* was obviously enhanced. They
attributed this behavior to the electrocatalytic reduction of
Ir(ppy)3  Ru(bpy)2(dvbpy)+ 
Ru(bpy)2(dvbpy)2+  + Ir(ppy)3
(12)
[Ru]2+ by [Ir]2 and insufficient power of TPrA• to reduce
‒•


[Ir]‒ for [Ir]2 generation, thus precluding the formation of
‒•
ACS Paragon Plus Environment
5

Journal of the American Chemical Society
Page 6 of 12
the iridium(III) emitter and meanwhile improving the genera- and red images. Note that in the co-reactant ECL systems the
1
2
3
4
5
6
7
8
tion of ruthenium(II) emitter.
classical routes as depicted by eqs. 1‒7 are the main contribu-
tion to the overall ECL intensity, although other ECL genera-
tion pathways are also possibly involved. At more positive po-
It should be noticed that the interaction between lumino-
phores 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 spec-
tra at +0.82 V and +1.24 V, close to their respective oxidation
potentials.
tentials,
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 lumino-
phores cannot be resolved by potential, it can be easily decon-
voluted 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 lumi-
nophores.
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
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 impossi-
ble. However, the sufficient gap up to 1.01 V between their
oxidation potentials renders the possibility of sequential oxida-
tion 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).
Dual-Resolved ECL for Multiplex Immunoassay. As demon-
strated 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. Clear-
ly, a potential and spectrum dual-resolved system can be estab-
lished, which can be eventually applied to multiplex immuno-
sensing for multianalyte identification.
Miao et al. has utilized polystyrene beads (PSB) as the carri-
er of a large number of hydrophobic ECL labels to develop an
ultrasensitive DNA hybridization assay24 and C-reactive pro-
tein (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 usu-
ally 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 spe-
cific detection antibodies for AFP, CEA and β-HCG on the
basis of biotin-streptavidin complexation to prepare dual-
coded 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 Tak-
ing 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 immu-
noassay 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, magnet-
ic assisted separation and swelling release of luminophores for
recognition (as exemplified in Figure 6a). In the first step, im-
munomagnetic beads (MB, 1 µm in diameter) coated with
three different capture antibodies, designated as Ab-MB, were
incubated with the sample containing antigens and subse-
quently separated with the assistance of magnets, which were
then incubated with dual-coded PSB to form the sandwich
immunocomplexes. After magnetic separation again, the im-
munocomplexes 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 per-
formed on an upright microscope connected to an electro-
chemical 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 ace-
tonitrile containing 50 mM TPrA and 0.1 M TBAPF6. Their con-
centrations were 2.5 µM and 125 µM for spectra while doubled for
image. Shown in inset are ECL spectra acquired at several differ-
ent 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 imag-
es at different potentials, which can be analyzed in three re-
gimes. 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 elec-
trode surface. Although LOP ECL could be generated by ei-
ther of them, but it was not detected due to its low efficiency
or low concentration of luminophores used here. In the poten-
tial 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
ACS Paragon Plus Environment
6

Page 7 of 12
Journal of the American Chemical Society
confirming the presence of CEA in the sample. When the po-
1
2
3
4
5
6
tential 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 high-
er oxidation potential, such as +1.75 V (blue line). This emis-
sion band verifies the presence of β-HCG.
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
Figure 6. (a) The procedure of simultaneous recognition of multi-
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
ple 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 con-
taining 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 three-
electrode configuration. The ECL spectra were acquired at differ-
ent 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 la-
beled 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 specifici-
ty. Notably, some ECL behaviors in the immunoassay were
different from that observed in above experiments. For exam-
ple, the LOP ECL of Ir(dFCF3ppy)2(dtbbpy)+ (1) can be dis-
tinguished 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-in-
diameter) wrapped up by a plastic tube was used as the elec-
trochemical 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 elec-
trode (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 dif-
ferent 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 lumino-
phores released from immunocomplexes could be selectively
excited at different potentials to emit at characteristic wave-
lengths for identification and recognition.
Figure 7a shows ECL spectra recorded at three different po-
tentials 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 pathologi-
cal cut-off concentrations. Firstly, 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),
ACS Paragon Plus Environment
7

Journal of the American Chemical Society
Page 8 of 12
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. Iridi-
um(III) chloride (IrCl3) was obtained from Shaanxi Kaida Chem-
CONCLUSIONS
1
2
3
4
5
6
7
8
In summary, we have synthesized several ruthenium and iridi-
um 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, spec-
trum and imaging measurements. The spectral peak separation
is up to 145 nm between the cyan-ECL generating species,
ical
Engineering
Co.,
Ltd.
2-(2,4-difluorophenyl)-5-
(trifluoromethyl)pyridine (dFCF3ppy) was bought from Cochem-
ical Ltd., (Shanghai, China). CEA and AFP standards were pur-
chased 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). Anti-
body 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 Gene-
sea 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 atmos-
phere prior to use. TBAPF6 was used as the electrolyte. A glassy
carbon electrode (3 mm in diameter) served as the working elec-
trode. 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 ferrice-
nium/ferrocene couple as the internal reference. In acetoni-
trile/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 work-
station (CHI Instrument, Shanghai, China).
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 sys-
tem. The ECL reaction involving Ir(ppy)3 (2), a green-ECL
luminophore with self-annihilation ECL behavior,49 was stud-
ied by online EC-MS, with which the self-annihilation at high-
er 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 recogni-
tion 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.
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
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 simul-
taneous recognition of many other biomarkers. By further de-
signing new ECL luminophores and strictly controlling the
multiplex immunoassays, we believe it is possible to carry out
quantitative determination of multiple biomarkers simultane-
ously, 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 auxilia-
ry 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 lumino-
phores with appropriate conjugation sites can significantly
simplify the operation of assay, because the swelling release
step will be unnecessary.
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 Tech-
nologies, 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 pre-
viously.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) Inten-
sity. 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)
EXPERIMENTAL SECTION
Chemicals and Reagents. Acetonitrile, ethylene glycol, 2-
methoxyethanol, 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),
ACS Paragon Plus Environment
8

Page 9 of 12
Journal of the American Chemical Society
(5) Chikkaveeraiah, B. V.; Bhirde, A. A.; Morgan, N. Y.; Eden, H. S.;
on an MPI-E ECL analytical system (Remex Analysis Instrument,
Chen, X., Electrochemical immunosensors for detection of cancer
protein biomarkers. ACS Nano 2012, 6, 6546.
1
2
3
4
5
6
7
8
Xi’an, China). The PMT was biased at 600 V and the scan rate was
‒
1
0.1 V s . (ii) Spooling spectrum (see instrumental details in Fig-
ure S10a). ECL spectra were acquired in the period of potential
scanning using a custom-built ECL system consisting of an Au-
tolab PGSTAT302N workstation (Metrohm, Netherlands) and
the QEpro spectrophotometer. A home-made Teflon cell consist-
ing of a circular quartz window, optic fiber and collimating lens
was used. The integration time of individual spectrum was 0.3 s
(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.
‒
1
(8) Bake, K. D.; Walt, D. R., Multiplexed spectroscopic detections. Ann.
Rev. Anal. Chem. 2008, 1, 515.
and the scan rate was 0.1 V s . Two adjacent spectra were ob-
tained at potential intervals of 30 mV. The measurement yielded a
3D emission spectral distribution.48,85,86 (iii) Imaging (see instru-
mental details in Figure S10b). The sequential ECL images at dif-
ferent 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 infor-
mation of ECL images was further analyzed by MATLAB software.
9
(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.
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
(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
ASSOCIATED CONTENT
electrochemiluminescence imaging resolved at the single bead level. J.
Am. Chem. Soc. 2009, 131, 6088.
(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 multi-analyte
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.
Supporting Information. The experimental details on the synthesis,
NMR and spectroscopic characterizations of multi-color ECL
luminophores; preparation and optical characterization of dual-
coded 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: subin@zju.edu.cn
(18) Richter, M. M., Electrochemiluminescence (ECL). Chem. Rev. 2004,
104, 3003.
(19) Hu, L. Z.; Xu, G. B., Applications and trends in
All authors have given approval to the final version of the manu-
script.
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 chemiluminescence-based
analyses. Anal. Chem. 2016, 88, 4751.
ORCID
Bin Su: 0000-0003-0115-2279
Yuanhua Shao: 0000-0003-3922-6229
Notes
The authors declare no competing financial interest.
(22) Kitte, S. A.; Gao, W.; Zholudov, Y. T.; Qi, L.; Nsabimana, A.; Liu, Z.;
Xu, G., Stainless steel electrode for sensitive luminol
ACKNOWLEDGMENT
electrochemiluminescent detection of H2O2, glucose, and glucose
oxidase activity. Anal. Chem. 2017, 89, 9864.
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 Re-
search and Development Program of China (No.
2016YFA0201300).
(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.,
REFERENCES
RuSi@Ru(bpy)32+/Au@Ag2S nanoparticles
(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.
Discov. 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.
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.
ACS Paragon Plus Environment
9

Journal of the American Chemical Society
Page 10 of 12
(29) Sardesai, N. P.; Barron, J. C.; Rusling, J. F., Carbon nanotube
(52) Kerr, E.; Doeven, E. H.; Barbante, G. J.; Hogan, C. F.; Bower, D. J.;
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.,
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.
1
2
3
4
5
6
7
8
Electrochemiluminescence on bipolar electrodes for visual
bioanalysis. Chem. Sci. 2013, 4, 1182.
(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
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., Potential-
resolved 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, 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.
(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.
9
(33) Wang, N.; Feng, Y.; Wang, Y.; Ju, H.; Yan, F.,
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
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. 2015, 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-of-concept 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., Three-dimensional
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, 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.
(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-n-
ACS Paragon Plus Environment
10

Page 11 of 12
1
2
3
Journal of the American Chemical Society
propylamine, its radical cation, and intermediate free radical in
MeCN/benzene solutions. J. Phys. Chem. A 2003, 107, 3335.
ruthenium(II)–iridium(III) complex soft salt. Chem. Eur. J. 2015, 21,
7435.
(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.
(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.
4
5
6
7
8
(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.
(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.
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
(79) Swanick, K. N.; Sandroni, M.; Ding, Z.; Zysman-Colman, E.,
Enhanced electrochemiluminescence from a stoichiometric
ACS Paragon Plus Environment
11

Journal of the American Chemical Society
Page 12 of 12
For Table of Contents Only
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
12
ACS Paragon Plus Environment