\makeatletter
\@ifundefined{HCode}
{\documentclass[screen,CRCHIM,Unicode,biblatex]{cedram}
\addbibresource{crchim20250948.bib}
\newenvironment{noXML}{}{}
\def\tsup#1{$^{{#1}}$}
\def\tsub#1{$_{{#1}}$}
\def\ndash{\text{--}}
\usepackage{amssymb}
\usepackage[T1]{fontenc}
\def\thead{\noalign{\relax}\hline}
\def\tbody{\noalign{\relax}\hline}
\def\endthead{\noalign{\relax}\hline}
\def\tabnote#1{\vskip4pt\parbox{.55\linewidth}{#1}}
\RequirePackage{etoolbox}
\def\jobid{crchim20250948}
%\graphicspath{{/tmp/\jobid_figs/web/}}
\graphicspath{{./figures/}}
\newcounter{runlevel}
\let\MakeYrStrItalic\relax
\csdef{Seqnsplit}{\\}
\def\refinput#1{}
\def\back#1{}
\let\ubreak\break
\def\botline{\\\hline}
\def\dollar{\$}
\def\og{\guillemotleft}
\def\fg{\guillemotright}
\def\mn{\phantom{$-$}}
\def\0{\phantom{0}}
\def\xmorerows#1#2{#2}
\usepackage{multirow} 
\def\nrow#1{\@tempcnta #1\relax%
\advance\@tempcnta by 1\relax%
\xdef\lenrow{\the\@tempcnta}}
\def\morerows#1#2{\nrow{#1}\multirow{\lenrow}{*}{#2}}
\usepackage{hyperref}
\def\Lbreak{\newline}
\makeatletter
\g@addto@macro{\UrlBreaks}{\UrlOrds}
\gappto{\UrlBreaks}{\UrlOrds}
\DOI{10.5802/crchim.443}
\datereceived{2025-11-24}
\daterevised{2026-01-08}
\dateaccepted{2026-01-28}
\ItHasTeXPublished
}
{\documentclass[crchim]{article}
\usepackage[T1]{fontenc}
\def\CDRdoi{10.5802/crchim.443}
\let\refinput\input
\def\href#1#2{\url[#1]{#2}}
\let\ubreak\relax
\makeatletter
}
\makeatother

\usepackage{upgreek}

\begin{document}

\begin{noXML}

\CDRsetmeta{articletype}{review}

\title{Coreactant radical intermediates in electrochemiluminescence:
mechanisms, detection strategies, and bioanalytical performance}

\alttitle{Interm\'{e}diaires radicalaires des cor\'{e}actifs en
\'{e}lectrochimiluminescence : m\'{e}canismes, strat\'{e}gies de
d\'{e}tection et performances bioanalytiques}

\author{\firstname{Yifan} \lastname{He}}
\address{College of Materials and Environmental Engineering, Hangzhou
Dianzi University, Hangzhou, 310018, PR China}
\email[Y. He]{221200038@hdu.edu.cn}

\author{\firstname{Yanfei} \lastname{Lv}}
\addressSameAs{1}{College of Materials and Environmental Engineering, Hangzhou
Dianzi University, Hangzhou, 310018, PR China}
\email[Y. Lv]{lvyanfei@hdu.edu.cn}

\author{\firstname{Li} \lastname{Fu}\CDRorcid{0000-0002-5957-7790}\IsCorresp}
\addressSameAs{1}{College of Materials and Environmental Engineering,
Hangzhou Dianzi University, Hangzhou, 310018, PR China}
\email[L. Fu]{fuli@hdu.edu.cn}

\author{\firstname{Shichao} \lastname{Zhao}}
\addressSameAs{1}{College of Materials and Environmental Engineering,
Hangzhou Dianzi University, Hangzhou, 310018, PR China}
\email[S. Zhao]{zhaoshichao@hdu.edu.cn}

\author{\firstname{Hassan} \lastname{Karimi-Maleh}\IsCorresp}
\address{The Quzhou Affiliated Hospital of Wenzhou Medical University,
Quzhou People's Hospital, Quzhou, 324000, China}
\address{School of Chemistry, Damghan University, Damghan, 36716-45667, Iran}
\email[H. Karimi-Maleh]{hassan@wmu.edu.cn}

\shortrunauthors

\keywords{\kwd{Radical intermediates}
\kwd{Mechanistic pathways}
\kwd{Detection techniques}
\kwd{Coreactant design}
\kwd{Electrochemical microscopy}}

\altkeywords{\kwd{Interm\'{e}diaires radicalaires}
\kwd{Voies m\'{e}canistiques}
\kwd{Techniques de d\'{e}tection}
\kwd{Conception de cor\'{e}actifs}
\kwd{Microscopie \'{e}lectrochimique}}

\begin{abstract}
Electrochemiluminescence (ECL) has evolved into a cornerstone of modern
bioanalytical science, primarily due to the development of
coreactant-based systems that enable highly sensitive detection in
aqueous media. The performance of these systems is fundamentally
governed by the transient radical intermediates generated from the
coreactants themselves. This review provides a critical examination of
these pivotal species, focusing on the two most significant and
mechanistically distinct coreactant classes: tertiary amines,
exemplified by tri-\textit{n}-propylamine (TPrA), and persulfate. We
delve into the complex and often contentious mechanistic pathways of
radical generation, dissecting the long-standing debate between direct
oxidation and catalytic routes for the TPrA system and exploring the
emerging evidence for non-radical pathways in persulfate chemistry. A
comprehensive overview of advanced analytical techniques is presented,
highlighting how methods such as electron paramagnetic resonance (EPR)
spectroscopy, real-time mass spectrometry, and high-resolution ECL
microscopy have been instrumental in detecting and characterizing these
fleeting radicals, thereby providing the experimental foundation for
mechanistic elucidation. Finally, we establish a direct link between
the fundamental physicochemical properties of these radical
intermediates and the ultimate performance of ECL-based bioanalytical
platforms, including sensitivity, selectivity, and robustness. By
synthesizing the current state of knowledge and identifying key
unresolved questions, this review aims to provide a nuanced
understanding of coreactant radical intermediates and to chart a course
for the future rational design of next-generation ECL systems.
\end{abstract}

\begin{altabstract}
L'\'{e}lectrochimiluminescence (ECL) est devenue un pilier de la
bioanalyse moderne, principalement gr\^{a}ce au d\'{e}veloppement de
syst\`{e}mes \`{a} cor\'{e}actif permettant une d\'{e}tection hautement
sensible en milieu aqueux. Les performances de ces syst\`{e}mes sont
fondamentalement gouvern\'{e}es par les interm\'{e}diaires radicalaires
transitoires g\'{e}n\'{e}r\'{e}s \`{a} partir des cor\'{e}actifs
eux-m\^{e}mes. La pr\'{e}sente revue propose un examen critique de ces
esp\`{e}ces cl\'{e}s, en se concentrant sur les deux classes de
cor\'{e}actifs les plus importantes et les plus distinctes sur le plan
m\'{e}canistique : les amines tertiaires, illustr\'{e}es par la
tri-\textit{n}-propylamine (TPrA), et le persulfate. Nous analysons en
profondeur les voies m\'{e}canistiques complexes --- et
souvent controvers\'{e}es --- de g\'{e}n\'{e}ration des
radicaux, en examinant le d\'{e}bat de longue date entre oxydation
directe et voies catalytiques dans le syst\`{e}me TPrA, et en discutant
les preuves \'{e}mergentes de voies non radicalaires en chimie des
persulfates. Une vue d'ensemble compl\`{e}te des techniques analytiques
avanc\'{e}es est ensuite pr\'{e}sent\'{e}e, en soulignant comment la
spectroscopie de r\'{e}sonance paramagn\'{e}tique \'{e}lectronique
(EPR), la spectrom\'{e}trie de masse en temps r\'{e}el et la
microscopie ECL \`{a} haute r\'{e}solution ont \'{e}t\'{e}
d\'{e}terminantes pour d\'{e}tecter et caract\'{e}riser ces radicaux
fugaces, fournissant ainsi la base exp\'{e}rimentale de
l'\'{e}lucidation m\'{e}canistique. Enfin, nous \'{e}tablissons un lien
direct entre les propri\'{e}t\'{e}s physico-chimiques fondamentales de
ces interm\'{e}diaires radicalaires et les performances finales des
plateformes bioanalytiques bas\'{e}es sur l'ECL, notamment la
sensibilit\'{e}, la s\'{e}lectivit\'{e} et la robustesse. En
synth\'{e}tisant l'\'{e}tat actuel des connaissances et en identifiant
les principales questions non r\'{e}solues, cette revue vise \`{a}
offrir une compr\'{e}hension nuanc\'{e}e des interm\'{e}diaires
radicalaires des cor\'{e}actifs et \`{a} tracer une trajectoire pour la
conception rationnelle de syst\`{e}mes ECL de nouvelle
g\'{e}n\'{e}ration.
\end{altabstract}

%\input{CR-pagedemetas}

\thanks{Hassan Research Initiation Fund (Grant number KYQD2024-046),
Quzhou Affiliated Hospital of Wenzhou Medical University (Grant
Number 2025K064).}

\maketitle

\twocolumngrid

\end{noXML}

\vspace*{3pc}
\section{Introduction}\label{sec1}

Electrochemiluminescence (ECL), the production of light from
electrochemically generated species, has transitioned from a laboratory
curiosity to a powerful analytical technique with widespread \mbox{commercial}
success, particularly in clinical diagnostics and  bioassays~\cite{1}.
This remarkable evolution was largely contingent on a pivotal
mechanistic shift away from the so-called ``annihilation''
pathway~\cite{2}. In the annihilation mechanism, radical cations and
anions of a luminophore, generated by applying alternating oxidative
and reductive potentials, react to produce an excited state. While
effective in aprotic organic solvents with wide potential windows, this
approach is largely unfeasible in the aqueous environments required for
most biological applications, where the electrochemical splitting of
water severely restricts the accessible potential range~\cite{3}.

The introduction of coreactants elegantly circumvented this limitation,
enabling robust ECL generation from a single potential step or scan in
one direction~\cite{4}. A coreactant is a species that, upon
electrochemical activation, initiates a cascade of chemical reactions
that culminates in the formation of the luminophore's excited state.
This innovation unlocked the full potential of ECL for bioanalysis,
offering near-zero background signal, high sensitivity, and excellent
spatiotemporal control~\cite{5}. At the heart of this process lie the
highly reactive transient radical intermediates generated from the
coreactants. The properties, reactivity, and fate of these radicals are
the ultimate determinants of the efficiency, spatial distribution, and
analytical performance of the entire ECL system~\cite{6}.

Coreactant pathways are broadly categorized into two dominant
mechanisms. The ``oxidative-reduction'' pathway is typified by tertiary
amines, most notably TPrA. In this mechanism, the coreactant is
electrochemically oxidized, and through subsequent chemical steps,
generates a powerful reducing radical intermediate that drives the
formation of the luminophore's excited state~\cite{7}. Conversely, the
``reductive-oxidation'' pathway, exemplified by the persulfate anion
($\mathrm{S}_{2}\mathrm{O}_{8}^{2-}$), involves the
electrochemical reduction of the coreactant to produce a potent
oxidizing radical intermediate, which then reacts with the reduced form
of the luminophore to generate light~\cite{8}.

A deep and nuanced understanding of the generation, stability,
diffusion, and reactivity of these coreactant-derived radical
intermediates, such as the TPrA radical cation (TPrA${\bullet}^{+}$), the
${\upalpha}$-aminoalkyl radical (TPrA$\bullet$), and the sulfate
radical anion (SO$_{4}{\bullet}^-$), is therefore not merely an academic
exercise. It is the fundamental prerequisite for rationally designing
more efficient luminophores~\cite{9}, developing novel coreactants with
tailored properties, and optimizing assay conditions to push the
boundaries of analytical sensitivity. This review posits that these
transient species are the linchpin of coreactant ECL~\cite{10}. We will
critically examine the intricate and often-debated mechanistic pathways
that govern their formation, survey the sophisticated analytical
strategies employed to detect and characterize these transient species,
and analyze how their fundamental chemical properties directly
translate into the bioanalytical performance that has made ECL an
indispensable tool in modern science~\cite{11}.

\section{Mechanistic pathways and controversies in radical intermediate
generation}\label{sec2}

The apparent simplicity of coreactant ECL, where applying a potential
to a solution of luminophore and coreactant produces light, belies a
sophisticated and multifaceted reaction network. The generation of
radical intermediates is not a monolithic process but a series of
competing and often condition-dependent pathways. This section dissects
the mechanistic details and ongoing controversies surrounding the two
most important coreactant systems, TPrA and persulfate, providing a
critical analysis of the evidence that shapes our current
understanding.

\begin{figure*}
\includegraphics{fig01}
\caption{\label{fig1}The mechanism of coreactant ECL for
[Ru(bpy)$_{3}$]$^{2+}$~and TPrA (reproduced
from Ref.~\cite{14} with permission from ACS,
copyright 2002).}
\end{figure*}

\subsection{The tri-\emph{n}-propylamine (TPrA) system}\label{sec21}

The tris(2,2$^{\prime}$-bipyridine)ruthenium(II)
[Ru(bpy)$_{3}$]$^{2+}$/{\ubreak}TPrA system (Figure~\ref{fig1}) is the
undisputed workhorse of ECL, forming the basis of virtually all
commercial immunoassay platforms. Its widespread use, however, has only
intensified the scrutiny of its complex mechanism, which remains a
subject of active research and debate~\cite{12}. The process begins
with two foundational steps: the one-electron oxidation of TPrA at the
electrode surface to form its radical cation, TPrA${\bullet}^{+}$,
followed by the rapid deprotonation of this species to yield the
neutral, highly reducing ${\upalpha}$-aminoalkyl radical,
TPrA$\bullet$. The reactions are as follows:
{\begin{eqnarray}
&\mathrm{TPrA} - \mathrm{e}^- {\rightarrow} \mathrm{TPrA}{\bullet}^+ \label{eq1}\Seqnsplit
&\mathrm{TPrA}{\bullet}^+ {\rightarrow} \mathrm{TPrA}\bullet + \mathrm{H}^{+} \label{eq2}
\end{eqnarray}}\unskip

The TPrA${\bullet}^+$ intermediate is notably short-lived, with an
estimated half-life of approximately 0.2 ms in neutral aqueous
solution. This transient stability is a critical parameter, as it
dictates that subsequent reactions involving this species are confined
to a thin diffusion layer, typically on the micrometer scale, near the
electrode surface~\cite{11,13}. From this common starting point,
several competing pathways can lead to the generation of the excited
state.

A central and long-standing controversy in the field revolves around
the initial oxidation events: the ``direct oxidation'' versus the
``catalytic'' route. The direct oxidation route, also known as the
oxidative-reduction pathway, posits that at a sufficiently high anodic
potential, both the luminophore and TPrA are oxidized directly at the
electrode surface~\cite{15}. The resulting oxidized luminophore, then
reacts with the highly reducing TPrA$\bullet$ radical to produce the
excited state:
{\begin{eqnarray}
&[\mathrm{Ru}(\mathrm{bpy})_{3}]^{2+}- 
\mathrm{e}^- \rightarrow [\mathrm{Ru}(\mathrm{bpy})_{3}]^{3+} 
\label{eq3}\Seqnsplit
&{}[\mathrm{Ru}(\mathrm{bpy})_{3}]^{3+}  + \mathrm{TPrA}
\bullet \rightarrow {[\mathrm{Ru}(\mathrm{bpy})_{3}]^{2+}}^\ast 
+ \mbox{ Products} \nonumber\\
\label{eq4}
\end{eqnarray}}\unskip

In contrast, the catalytic route proposes that only the luminophore is
electrochemically oxidized. This species then acts as a homogeneous
oxidant, diffusing into the solution to react with a neutral TPrA
molecule, thereby generating the TPrA${\bullet}^+$ radical cation in the
diffusion layer, which then proceeds through deprotonation
(Equation~(\ref{eq2})) as usual:
{\begin{equation}\label{eq5}
[\mathrm{Ru}(\mathrm{bpy})_{3}]^{3+} + \mathrm{TPrA} \rightarrow 
[\mathrm{Ru}(\mathrm{bpy})_{3}]^{2+} + \mathrm{TPrA}{\bullet}^{+}
\end{equation}}\unskip

%tab1
\begin{table*}
\caption{\label{tab1}Key physicochemical properties of primary ECL
radical intermediates}
\begin{tabular}{lllllc}
\thead
\parbox[t]{1cm}{\raggedright Radical species} & 
\parbox[t]{1.2cm}{\raggedright Generation method} & 
\parbox[t]{4.5cm}{\raggedright Approximate formal/estimated redox potential} & 
\parbox[t]{2cm}{\raggedright Estimated half-life/{\ubreak}stability} & 
\parbox[t]{1.8cm}{\raggedright Primary role in ECL}\vspace*{2pt} & References\\
\endthead
TPrA${\bullet}^+$ & \parbox[t]{2.5cm}{\raggedright Electrochemical oxidation of TPrA$\bullet$} & 
\parbox[t]{4.5cm}{\raggedright E\textdegree$_{\mathrm{est}}$(TPrA${\bullet}^{+}$/TPrA$\bullet$) 
${\approx}-1.7$ V vs.\ SCE (aqueous; commonly used estimate for the reducing power of TPrA$\bullet$)}\vspace*{2pt}
& \parbox[t]{1.5cm}{\raggedright${\sim}$0.2 ms (aqueous)} & 
Oxidant & \cite{14,24,25} \\
SO$_{4}{\bullet}^-$ & \parbox[t]{2.3cm}{\raggedright Electrochemical
reduction of $\mathrm{S}_{2}\mathrm{O}_{8}^{2-}$}&
\parbox[t]{4.5cm}{\raggedright E\textdegree(SO$_{4}{\bullet}^-$/$\mathrm{SO}_{4}^{2-}$) $\approx +2.43$ V vs.\ 
NHE (often quoted ${\sim}+2.5$--3.1 V in reviews; condition/{\ubreak}speciation dependent)}\vspace*{2pt}
& 30--40 ${\upmu}$s & \parbox[t]{1.3cm}{\raggedright Strong oxidant} & \cite{26,27,28}
\botline
\end{tabular}
\end{table*}


Crucially, the dominance of one pathway over the other is not an
intrinsic property of the system but is dictated by the experimental
conditions~\cite{16}. A wealth of evidence demonstrates that the
electrode material acts as a primary ``mechanistic switch''. At glassy
carbon (GC) electrodes, the direct oxidation of TPrA is kinetically
favored, and the direct oxidation route is generally considered to be
predominant~\cite{17}. However, at noble metal electrodes like platinum
(Pt) and gold (Au), the formation of passivating surface oxide layers
under the positive potentials required for ECL significantly inhibits
the direct heterogeneous oxidation of TPrA~\cite{18}. Under these
conditions, the catalytic route becomes more significant, particularly
at higher concentrations of the luminophore, which can more effectively
act as a mediator for TPrA oxidation~\cite{19}. This mechanistic
difference directly explains the frequently observed phenomenon of
lower ECL intensities on Pt and Au electrodes compared to carbon-based
electrodes for the same system~\cite{20}. Furthermore, the
concentration ratio of luminophore to coreactant is a key modulator;
high [$[\mathrm{Ru}(\mathrm{bpy})_{3}]^{2+}$]/[TPrA] ratios strongly favor the
catalytic pathway, as the abundance of electrogenerated
[Ru(bpy)$_{3}$]$^{3+}$ provides more opportunities for homogeneous
oxidation of TPrA~\cite{21}. The physicochemical characteristics of
these radical intermediates are summarized in  Table~\ref{tab1},
highlighting their formation pathways, redox potentials, and stability
regimes. These parameters dictate not only the efficiency but also the
spatial and temporal domains of light generation in ECL~\cite{22}. For
instance, the sub-millisecond lifetime of TPrA${\bullet}^{+}$ confines
its reactivity to the near-electrode diffusion layer, whereas the
strong oxidizing potential of SO$_{4}{\bullet}^-$ in persulfate systems
extends reaction zones farther into solution~\cite{23}. Comparing these
radicals underscores the delicate kinetic balance between electron
transfer, diffusion, and radical decay that governs overall ECL
performance.


The mechanistic picture was further refined by a seminal 2002 paper
from Bard and coworkers, which reconciled several anomalous
observations by proposing a ``new route''. This pathway involves the
reaction between the TPrA radical cation, TPrA${\bullet}^+$, and the
reduced form of the luminophore, $[\mathrm{Ru}(\mathrm{bpy})_{3}]^{+}$. The
reduced luminophore is generated via a competing reaction pathway where
the potent TPrA$\bullet$ radical reduces a ground-state
[Ru(bpy)$_{3}$]$^{2+}$ molecule:
{\begin{eqnarray}
&[\mathrm{Ru}(\mathrm{bpy})_{3}]^{2+} + \mathrm{TPrA}\bullet\rightarrow 
[\mathrm{Ru}(\mathrm{bpy})_{3}]^{+} + \mbox{Products}\qquad \label{eq6}\Seqnsplit
&[\mathrm{Ru}(\mathrm{bpy})_{3}]^{+} + \mathrm{TPrA}{\bullet}^+ 
\rightarrow [\mathrm{Ru}(\mathrm{bpy})_{3}]^{2+*} + \mathrm{TPrA}\qquad \label{eq7}
\end{eqnarray}}\unskip

This mechanism is particularly vital to explain the oxidative
(TPrA${\bullet}^+$) excitation ECL phenomenon, where light is generated
at potentials positive enough to oxidize TPrA but insufficient to
directly oxidize [{Ru(bpy)}$_{3}$]$^{2+}$ (typically $<$1.0~V  vs.\ 
saturated calomel electrode [SCE])~\cite{25}. In this scenario, direct
generation of [Ru(bpy)$_{3}$]$^{3+}$  at the electrode is impossible.
Instead, only TPrA is oxidized, creating a flux of both TPrA${\bullet}^+$
and TPrA$\bullet$ radicals diffusing away from the electrode. The
subsequent homogeneous reactions~(\ref{eq6})  and~(\ref{eq7}) are then
solely responsible for generating the excited state. This oxidative
(TPrA${\bullet}^+$) excitation mechanism is of paramount importance in
the context of modern bioassays, especially bead-based
immunoassays~\cite{29}. In these formats, the luminophore is conjugated
to an antibody and immobilized on the surface of a microbead, which is
spatially separated from the electrode surface. The ability of the
electrogenerated TPrA radicals to diffuse over micrometer-scale
distances to reach and react with the luminophore labels on the bead is
the fundamental principle that enables these highly sensitive assays to
function~\cite{30}.

%tab2
\begin{table*}
\caption{\label{tab2}Comparison of mechanistic pathways in the
[Ru(bpy)$_{3}$]$^{2+}$/TPrA system}
\begin{tabular}{llllll}
\thead
\parbox[t]{1.5cm}{\raggedright Pathway name} &
\parbox[t]{3cm}{\raggedright Required electrode potential (E$_{\mathrm{app}}$)} 
& \parbox[t]{2.5cm}{\raggedright Key reactants at electrode} & 
\parbox[t]{3.3cm}{\raggedright Critical homogeneous reactions} & 
\parbox[t]{3.3cm}{\raggedright Dominant conditions/applications}\vspace*{2pt} \\
\endthead
\parbox[t]{1.5cm}{\raggedright Direct oxidation} &
$\mathrm{E}_{\mathrm{app}}>$ E\textdegree(Ru$^{2+/3+}$) & 
\parbox[t]{2.5cm}{\raggedright [Ru(bpy)$_{3}$]$^{2+}$, TPrA} &
\parbox[t]{3.3cm}{\raggedright[Ru(bpy)$_{3}$]$^{3+}  +$ 
TPrA$\bullet \rightarrow [\mathrm{Ru}(\mathrm{bpy})_{3}]^{2+\ast}$} & 
\parbox[t]{3.3cm}{\raggedright GC electrodes; Low 
[[Ru(bpy)$_{3}$]$^{2+}$]/[TPrA] ratio~\cite{35}} \\

Catalytic & $\mathrm{E}_{\mathrm{app}}>$ E\textdegree(Ru$^{2+/3+}$) & [Ru(bpy)$_{3}$]$^{2+}$ & 
\parbox[t]{3.3cm}{\raggedright[Ru(bpy)$_{3}$]$^{3+} +$ TPrA $\rightarrow$ [Ru(bpy)$_{3}$]$^{2+} +$ TPrA${\bullet}^+$}
& \parbox[t]{3.3cm}{\raggedright Pt, Au electrodes; High [[Ru(bpy)$_{3}$]$^{2+}$]/[TPrA] ratio~\cite{36,37,38}} \\

\parbox[t]{1.5cm}{\raggedright Oxidative (TPrA${\bullet}^+$) excitation}
& \parbox[t]{3cm}{\raggedright E\textdegree(TPrA/TPrA${\bullet}^+$)
$<\mathrm{E}_{\mathrm{app}}<$
E\textdegree(Ru$^{2+/3+}$)} & TPrA only 
&\parbox[t]{3.3cm}{\raggedright [Ru(bpy)$_{3}$]$^{+} +$ TPrA${\bullet}^+\rightarrow$ [Ru(bpy)$_{3}$]$^{2+\ast}$} & 
\parbox[t]{3.3cm}{\raggedright Bead-based immunoassays; Spatially separated luminophore~\cite{39}}\vspace*{2pt}
\botline
\end{tabular}
\end{table*}

The comparative features of the three principal mechanistic routes are
summarized in  Table~\ref{tab2}, which delineates their operative
potentials, key reactants, and dominant experimental regimes. This
tabulation underscores that the boundary between ``direct'',
``catalytic'', and ``oxidative (TPrA${\bullet}^+$) excitation''
mechanisms is not rigid but dynamically dictated by the electrode
surface chemistry and the redox landscape~\cite{31}. For instance,
while both the direct and catalytic pathways operate above the
Ru$^{2+}$/$^{3+}$ oxidation potential, they diverge sharply
in the identity of the electroactive species and in the locus of
radical generation (heterogeneous vs.\ homogeneous)~\cite{32}. The
oxidative (TPrA${\bullet}^+$) excitation regime, operating at potentials
below the Ru oxidation potential, introduces a spatial decoupling
between charge injection and light emission, enabling photon generation
far from the electrode interface~\cite{33}. This mechanistic diversity
provides an essential framework for rational assay design: selecting
electrode materials that favor TPrA oxidation enhances ECL efficiency,
while exploiting the oxidative (TPrA${\bullet}^+$) excitation pathway is
indispensable for bead- and nanoparticle-based formats where
diffusion-mediated radical transfer drives excitation~\cite{34}.


\subsection{The persulfate ($\mathrm{S}_2\mathrm{O}_8^{2-}$) system: radical vs.\ 
non-radical pathways}\label{sec22}

Persulfate, specifically the peroxydisulfate anion
($\mathrm{S}_{2}\mathrm{O}_{8}^{2-}$), is the archetypal coreactant for the
reductive-oxidation pathway~\cite{40}. This pathway is particularly
useful for luminophores that are more readily reduced than oxidized and
has found extensive use not only in ECL but also in the broader field
of advanced oxidation processes (AOPs) for environmental remediation,
providing a rich literature from which to draw mechanistic insights.

The canonical mechanism involves the one-electron electrochemical
reduction of $\mathrm{S}_{2}\mathrm{O}_{8}^{2-}$ at the cathode. This reaction cleaves
the peroxide bond to generate the highly oxidizing sulfate radical
anion (SO$_{4}{\bullet}^-$) and a stable sulfate ion $\mathrm{SO}_{4}^{2-}$.
{\begin{equation}\label{eq8}
\mathrm{S}_{2}\mathrm{O}_{8}^{2-} + 
\mathrm{e}^- \rightarrow \mathrm{SO}_{4}^{2-} + \mathrm{SO}_{4}{\bullet}^- 
\end{equation}}\unskip

The SO$_{4}{\bullet}^-$ radical is a powerful one-electron oxidant, with
a standard redox potential reported in the range of ${+}$2.5 to ${+}$3.1 V vs.\ 
normal hydrogen electrode (NHE), comparable to that of the hydroxyl
\mbox{radical~\cite{26}.} In the context of ECL, this potent radical reacts
with the electrochemically generated reduced form of the luminophore
(L${\bullet}^-$) in a highly exergonic electron
transfer reaction to produce the light-emitting excited state,
$\mathrm{L}^*$:
{\begin{eqnarray}
&\mathrm{L} + \mathrm{e}^- \rightarrow \mathrm{L}{\bullet}^- \label{eq9}\Seqnsplit
&\mathrm{L}{\bullet}^-  + \mathrm{SO}_{4}{\bullet}^- \rightarrow \mathrm{L}^*  + 
\mathrm{SO}_{4}^{2-} \label{eq10}
\end{eqnarray}}\unskip

An important distinction from the TPrA system, highlighted by the work
of Paolucci and Valenti, is that a heterogeneous ECL mechanism
analogous to the oxidative (TPrA${\bullet}^+$) excitation route is not
feasible with persulfate~\cite{41}. Because the SO$_{4}{\bullet}^-$
radical acts as an oxidant, the luminophore must first be reduced to
L${\bullet}^-$ for the light-producing reaction to occur~\cite{42}.
Therefore, in persulfate systems, both the luminophore and the
coreactant must be able to interact with the electrode surface, unlike
in bead-based TPrA assays where the luminophore can be spatially
separated from the electrode.

While the SO$_{4}{\bullet}^-$-mediated pathway is well-established, a
significant controversy has emerged, largely from the AOPs field,
regarding the potential role of non-radical pathways in persulfate
activation. Proponents of non-radical mechanisms suggest that oxidation
can occur via pathways that do not involve free sulfate
radicals~\cite{43}. These proposed mechanisms include direct electron
transfer from a substrate to the persulfate molecule, often mediated by
a catalyst surface, or the generation of other reactive oxygen species
such as singlet oxygen ($^1$O$_{2}$)~\cite{44}. Evidence for these
pathways often relies on chemical quenching experiments, where the
addition of common radical scavengers like methanol or
\textit{tert}-butanol (which react rapidly with SO$_{4}{\bullet}^-$ and
$\bullet$OH) fails to completely inhibit the degradation of a target
pollutant.

The focus of this controversy lies in the difficulty of unequivocally
proving the absence of radicals and the potential for misinterpretation
of indirect evidence. The boundary between radical and non-radical
processes can be ambiguous, and it is likely that multiple pathways
coexist, with their relative contributions being highly dependent on
factors such as pH, the nature of the catalyst, and the electronic
properties of the substrate~\cite{45}. For example, at high pH,
SO$_{4}{\bullet}^-$ can react with OH$^-$ {to generate} the hydroxyl
radical ($\bullet$OH), adding another layer of complexity to the
system. While this debate is most active in the context of pollutant
degradation, its implications for persulfate-based ECL are
significant~\cite{46}. If non-radical pathways or alternative radical
species are major contributors to the oxidative chemistry, they could
represent untapped avenues for designing new and more efficient ECL\break
systems.

\subsection{A broader perspective: alternative and bio-derived
coreactants}\label{sec23}

The search for coreactants beyond TPrA and persulfate is a vibrant area
of research, driven by the desire for lower toxicity, improved
biocompatibility, higher efficiency, and novel mechanistic pathways. A
variety of aliphatic amines and related compounds have been explored as
alternatives to TPrA. For example, 2-(dibutylamino)ethanol (DBAE) was
introduced as a less toxic and less volatile alternative. Biological
buffers containing tertiary amine moieties, such as HEPES and BIS-TRIS,
have also been shown to function as effective coreactants, offering the
potential for simplified assay formulations where the buffer,
electrolyte, and coreactant are the same molecule~\cite{47}.
Comparative studies of these analogs reveal a complex interplay between
the coreactant's structure, the stability of its radical intermediates,
and the resulting ECL efficiency~\cite{48}. Wang and coworkers
demonstrated that the ECL distance (related to radical lifetime) and
reactivity of the coreactant co-determine the sensitivity of bead-based
immunoassays, and found that BIS-TRIS offered a superior balance of
these properties compared to TPrA, leading to a significant enhancement
in sensitivity~\cite{49}. In addition, reduced nicotinamide adenine
dinucleotide (NADH), a ubiquitous endogenous redox cofactor, should be
considered a biocompatible coreactant in
[Ru(bpy)$_{3}$]$^{2+}$-based ECL: it is recognized among
established [Ru(bpy)$_{3}$]$^{2+}$ coreactants, can be
generated enzymatically in situ from NAD$^{+}$ in dehydrogenase-coupled
assays to drive ECL without adding exogenous amine reagents, and has
recently been exploited as an efficient endogenous coreactant for ECL
sensing and imaging in biological systems~\cite{50,51,52}. 

\begin{figure*}
\vspace*{1pt}
\includegraphics{fig02}
\vspace*{1pt}
\caption{\label{fig2}Reaction mechanism of electrochemiluminescence
generation from [Ru(bpy)$_{3}$]$^{2+}$  on BDD electrode
(platinum spiral and Ag/AgCl as working and counter/reference
electrodes) with sulfate ions (reproduced from Ref.~\cite{53} with
permission from ACS, copyright 2016).}
\vspace*{2pt}
\end{figure*}

\begin{table*}
\caption{\label{tab3}Selected alternative coreactants and their radical
intermediates\vspace*{1pt}}
\tabcolsep3pt\fontsize{9.5}{11.5}\selectfont
\begin{tabular}{lllllc}
\thead
\parbox[t]{2.3cm}{\raggedright Coreactant name} & Type & 
\parbox[t]{2cm}{\raggedright Key radical(s) generated} & 
\parbox[t]{3cm}{\raggedright Relative ECL efficiency (vs.\ TPrA)} & 
\parbox[t]{3cm}{\raggedright Notable advantages/{\ubreak}disadvantages} & 
References\vspace*{2pt} \\
\endthead
\parbox[t]{2.7cm}{\raggedright 2-(dibutylamino)\unskip\break ethanol (DBAE)} & 
\parbox[t]{1.2cm}{\raggedright  Oxidative-reduction} & 
\parbox[t]{2cm}{\raggedright  Amine radical cation, ${\upalpha}$-aminoalkyl radical}\vspace*{2pt} & 
\parbox[t]{3cm}{\raggedright Variable; can be higher at low concentrations} & 
\parbox[t]{3cm}{\raggedright Lower toxicity and volatility than TPrA} & \cite{55} \\

 BIS-TRIS & \parbox[t]{1.2cm}{\raggedright Oxidative-reduction} & 
 \parbox[t]{2cm}{\raggedright  Amine radical cation, ${\upalpha}$-aminoalkyl radical} & 
 \parbox[t]{3cm}{\raggedright  Can be ${>}$200\% higher in bead assays} & 
 \parbox[t]{3cm}{\raggedright  Biocompatible buffer; balances reactivity and diffusion}\vspace*{2pt} & \cite{56} \\
 
Oxalate (C$_{2}$O$_{4}{}^{2-}$) & 
\parbox[t]{1.2cm}{\raggedright Oxidative-reduction} & 
CO$_{2}{\bullet}^-$ & 
\parbox[t]{3cm}{\raggedright Generally lower than TPrA} & 
\parbox[t]{3cm}{\raggedright Classic coreactant; less efficient in many systems}\vspace*{2pt} &\cite{57,56} \\

\parbox[t]{2.7cm}{\raggedright In-situ-generated $\mathrm{S}_{2}\mathrm{O}_{8}^{2-}$} & 
\parbox[t]{1.2cm}{\raggedright Reductive-oxidation} & SO$_{4}{\bullet}^-$ & 
\parbox[t]{3cm}{\raggedright Potentially high} & 
\parbox[t]{3cm}{\raggedright``Coreactant-free'' system; requires BDD electrode}\vspace*{2pt} & \cite{58} \\

Carbohydrazide & Intramolecular & CON$_{4}$H$_{6}\bullet$ & 
\parbox[t]{3cm}{\raggedright High in intramolecular systems} & 
\parbox[t]{3cm}{\raggedright Covalently linked to luminophore; improves efficiency}\vspace*{2pt} & \cite{59}

\botline
\end{tabular}
\vspace*{1pt}
\end{table*}

A particularly innovative strategy involves generating the coreactant
in situ from an inert and benign precursor present in the bulk
solution. A prime example is the work by Irkham and colleagues, who
utilized the unique properties of boron-doped diamond (BDD)
electrodes~\cite{53}. BDD electrodes can withstand extremely high
anodic potentials in aqueous solution without significant water
oxidation. This allows for the direct oxidation of inert sulfate anions
($\mathrm{SO}_{4}^{2-}$), a common component of many buffers, into the highly
reactive peroxydisulfate ($\mathrm{S}_{2}\mathrm{O}_{8}^{2-}$) coreactant directly at
the electrode surface. This in-situ generated $\mathrm{S}_{2}\mathrm{O}_{8}^{2-}$ then
participates in a conventional reductive-oxidation ECL mechanism with
[Ru(bpy)$_{3}$]$^{2+}$ (Figure~\ref{fig2}). This
``coreactant-free'' approach is highly attractive for bioanalysis as it
eliminates the need to add potentially interfering or toxic coreactants
to the sample, thereby improving the biocompatibility and simplifying
the assay design. Other novel approaches include the use of
metal--organic gels (MOGs) with amino-rich groups as solid-state
coreactants, which can enhance ECL through both chemical functionality
and electrocatalytic effects~\cite{54}. The representative examples
summarized in Table~\ref{tab3} highlight how structural diversity among
alternative and bio-derived coreactants translates into distinct
radical intermediates and performance outcomes. The contrast between
oxidative-reduction and reductive-oxidation types emphasizes how
radical lifetime, diffusion distance, and redox balance jointly dictate
ECL efficiency. Particularly, the high activity of BIS-TRIS and
in-situ-generated $\mathrm{S}_{2}\mathrm{O}_{8}^{2-}$ \mbox{illustrates} two emerging
paradigms: exploiting biocompatible multifunctional buffers and
electrode-driven radical generation. These findings collectively
redefine the design space for next-generation, sustainable, and
low-toxicity ECL systems.


\section{Advanced methodologies for the detection and characterization
of radical intermediates}\label{sec3}

The transient nature and low concentration of coreactant radical
intermediates make their detection and characterization a formidable
analytical challenge. Elucidating the complex mechanisms discussed in
the previous section has only been possible through the development and
application of a suite of highly specialized, sensitive, and often
coupled analytical techniques. No single method provides a complete
picture; rather, robust mechanistic conclusions are built upon a
convergence of \mbox{evidence} from complementary approaches that probe
different aspects of the radical's identity, reactivity, and spatial
behavior.

\subsection{Spectroscopic and spectroelectrochemical detection}\label{sec31}

Spectroscopic methods provide some of the most direct and unambiguous
evidence for the existence and structure of radical intermediates.
Among these, electron paramagnetic resonance (EPR) spectroscopy stands
out as the definitive technique for studying species with unpaired
electrons. EPR is analogous to NMR but probes the spins of electrons
rather than nuclei, yielding spectra with characteristic hyperfine
splitting patterns that serve as a ``fingerprint'' for a specific
radical. A landmark achievement in ECL mechanism research was the
direct EPR detection of the TPrA radical cation. By using a flow-cell
setup to rapidly transport the electrochemically generated species from
the electrode into the EPR cavity before it could decompose, they
captured its spectrum providing incontrovertible proof of its formation
as a key intermediate in the ECL pathway.

For radicals that are too short-lived or exist at concentrations below
the limit of direct EPR detection, the technique of spin trapping is
indispensable~\cite{60}. In this method, a diamagnetic ``spin trap''
molecule, such as 5,5-dimethyl-1-pyrroline N-oxide (DMPO), is added to
the system. The trap reacts rapidly with the transient radical to form
a much more stable and persistent nitroxide radical adduct, which
accumulates to a concentration sufficient for EPR detection~\cite{61}.
The hyperfine splitting constants of the resulting adduct's spectrum
are characteristic of the trapped radical, allowing its identification.
Spin trapping has been crucial for identifying the {formation} of both
sulfate radicals and hydroxyl radicals in persulfate-based
systems~\cite{62}. However, careful control experiments are essential,
as it has been shown that spin traps can sometimes react directly with
the persulfate precursor, leading to the formation of artifactual
signals that could be misinterpreted as evidence for a specific radical
pathway.

\begin{figure*}
\includegraphics{fig03}
\vspace*{-6pt}
\caption{\label{fig3}Schematic of the RT-Triplex setup for
electricity-luminescence-mass synchronization. Insets show the sideview
schematic diagrams of the EC reaction occurring at the two ends of the
capillary ({reproduced from ref}~\cite{71} with permission from RSC,
copyright 2022).}
\vspace*{-6pt}
\end{figure*}

Spectroelectrochemistry (SEC) provides a powerful complementary view by
coupling an electrochemical experiment with a spectroscopic
measurement, typically UV--Visible absorption spectroscopy, in the same
cell~\cite{63}. By monitoring the absorbance spectrum at the electrode
surface as a function of the applied potential, SEC can track the
depletion of reactants and the formation of stable or semi-stable
products and intermediates in real time~\cite{64}. While it generally
lacks the specificity to identify transient radicals directly, it
provides invaluable kinetic data and helps to correlate electrochemical
events (e.g., current peaks) with specific chemical transformations,
thereby helping to piece together the overall reaction
sequence~\cite{65}. To illustrate a quantitative-use case that
dovetails with coreactant ECL chemistry, thin-layer UV--Vis SEC has
been applied to the [{Ru(bpy)}$_{3}$]$^{2+}$ system under oxidative
bias to follow the disappearance of the MLCT band (${\sim}$450--460 nm)
and concomitant growth of the oxidized species' signatures in real
time~\cite{66}. In practice, stepping the potential to the
Ru$^{\mathrm{II/III}}$ region produces an immediate approximately
linear drop of $A_{450}$ within the first hundreds of
milliseconds~\cite{67,68,69}, followed by a slower exponential phase;
fitting the time-resolved $A(t)$ traces yields pseudo-first-order rate
constants for consumption of [{Ru(bpy)}$_{3}$]$^{2+}$ that increase
with overpotential, and the absorbance change scales with the
electrolysis charge in a Nernstian manner. These SEC-derived kinetics
have been used side by side with ECL readouts in the canonical
[{Ru(bpy)}$_{3}$]$^{2+}$/amine coreactant platform to rationalize how
the build-up of [{Ru(bpy)}$_{3}$]$^{3+}$ during the anodic half-cycle
gates the downstream radical chemistry of the amine; e.g., studies that
combine SEC with mechanistic probes on the
[{Ru(bpy)}$_{3}$]$^{2+}$/TPrA \mbox{couple} demonstrate that the spectroscopic
depletion of Ru$^{\mathrm{II}}$ (${\Delta}A_{450} \gtrsim 10$--30\% in
thin cells) coincides with the potential window where ECL turns on,
providing a quantitative bridge between the electrochemical generation
of Ru$^{\mathrm{III}}$ and the chemical step that yields the emissive
*Ru$^{\mathrm{II}}$ state~\cite{70}.

\begin{figure*}
\includegraphics{fig04}
\vspace*{-5pt}
\caption{\label{fig4}Computed relative free energy profile for the
electrocatalytic oxidation of TPrA (reproduced from Ref.~\cite{79}
with permission from Wiley, copyright 2025).}
\vspace*{-5pt}
\end{figure*}

\subsection{Mass spectrometry and computational approaches}\label{sec32}

The direct identification of intermediates by their mass-to-charge
ratio offers an orthogonal and highly specific detection method.
Electrochemical mass spectrometry (EC--MS) achieves this by interfacing
an electrochemical flow cell with a mass spectrometer, allowing the
continuous, real-time analysis of species generated at the electrode.
Recent innovations in ambient ionization techniques have led to the
development of platforms capable of capturing even very short-lived
intermediates. To illustrate the quantitative power of EC--MS in ECL
studies, Zhang and coworkers~\cite{71} built a real-time, triply
synchronized platform (RT-Triplex) that couples a capillary
electrochemical microreactor directly to a Venturi easy ambient
sonic-spray MS while recording the photomultiplier tube (PMT) signal
(Figure~\ref{fig3}). In this geometry, fleeting intermediates with
sub-millisecond lifetimes were transferred from the
electrode--electrolyte interface to the MS inlet at a volumetric flow
of 152~$\upmu \mathrm{L}{\cdot}\mathrm{min}^{-1}$ (linear velocity 
$\approx 0.108~\mathrm{m}{\cdot}\mathrm{s}^{-1}$), enabling
time-aligned molecular and optical readouts. Using a BODIPY/TPrA ECL
system, they detected the iminium ion [Pr$_{2}$N ${=}$ CHEt]$^{+}$ (m/z
142.1592) from TPrA electrooxidation and observed that its
extracted-ion chronogram turned on at 0.76~V, essentially coincident
with the ECL onset at 0.79~V measured by the PMT. Moreover,
post-electrolysis infusion experiments showed that introducing TPrA
collapsed the ``tailing'' of the BODIPY$^{{\cdot}+}$ (m/z 318.2081)
signal to ${\sim}$4~s, quantitatively linking coreactant availability to
radical persistence. Together with direct capture of characteristic m/z
features for other key intermediates (e.g., luminol-derived L--OOH at
m/z 208.0358 under alkaline conditions, \unskip\break{concomitant} with intensified
ECL), these measurements provide potential-resolved, number-anchored
evidence that ties specific radical/proton-transfer steps to the light
output in real time~\cite{72}. Such synchronized datasets---onset
potentials, transport rates, and decay times---move EC--MS beyond
qualitative assignment and into kinetic discrimination of competing ECL
pathways at the electrode interface~\cite{73}.\looseness=-1

\begin{figure*}
\vspace*{3pt}
\includegraphics{fig05}
\vspace*{-5pt}
\caption{\label{fig5}ECL images of a single bead labeled with
[{Ru(bpy)}$_{3}$]$^{2+}$ in a 0.3 M phosphate buffer with 180 mM TPrA
(Ru@Beads) (a)~without and (b) with 100~${\upmu}$M of
[Ir(dfppy)$_{2}$(pt-TEG)]$^{+}$. (c) Comparison between the single-bead
ECL intensity profiles of Ru@Beads (grey line) and
Ru@Beads/[Ir(dfppy)$_{2}$(pt-TEG)]$^{+}$ (red line). Inset: histogram of
the comparison between the respective averaged maximum values of ECL
intensity (reproduced from Ref.~\cite{83} with permission from RSC,
copyright 2024).}
\end{figure*}


Computational chemistry, particularly density functional theory (DFT),
has become an essential tool for augmenting and interpreting
experimental findings~\cite{74}. DFT calculations can provide
invaluable insights into the electronic structure, geometry, and
energetic properties of proposed radical intermediates~\cite{75}. For
example, theoretical calculations can be used to predict the standard
redox potentials of radical couples, which are often difficult to
measure experimentally but are critical for assessing the thermodynamic
feasibility of a proposed ECL pathway~\cite{76}. Furthermore, DFT can
be used to simulate EPR spectra, helping to confirm the assignment of
an experimentally observed signal to a specific radical
structure~\cite{77}. By modeling reaction pathways and calculating
activation energies for transition states, computational approaches can
help to rationalize kinetic observations and explain why certain
coreactants or pathways are more efficient than others~\cite{78}. A
recent study \mbox{illustrates} how such calculations \mbox{deliver} quantitative,
mechanism-level insight for coreactant ECL~\cite{79}. Using nortropine
N-oxyl (NNO) as an organic redox mediator in the archetypal
[{Ru(bpy)}$_{3}$]$^{2+}$/amine system, DFT was employed to map the
free-energy profile of the key oxidation step that generates the
reducing amine-derived species (Figure~\ref{fig4})~\cite{80}. The
computed activation free energy for hydride transfer from TPrA to the
oxidized mediator (NNO$^{+}$) is only $\Delta G =
7.4~\mathrm{kcal}{\cdot}\mathrm{mol}^{-1}$ in acetonitrile, implying
very fast kinetics and, consequently, efficient population of the
coreactant radical manifold under mild anodic bias. This low barrier
rationalizes the experimentally observed facilitation of ECL at lower
oxidation overpotentials when NNO is present and provides a
thermodynamic/kinetic basis for the enhanced emission efficiency
reported for the redox-mediated route. By quantitatively connecting a
specific transition state to macroscopic observables (onset and
brightness), the DFT analysis does not simply ``support'' the mechanism
but predicts its feasibility and impact in numerical terms, thereby
guiding the design of alternative mediators and coreactants with
comparably accessible\break barriers.

\subsection{Microscopy and spatially resolved techniques}\label{sec33}

While the aforementioned techniques identify \textit{what} the
intermediates are, ECL microscopy (ECLM) \mbox{reveals} \textit{where} they
react. By replacing the conventional photomultiplier tube (PMT) with a
sensitive camera (e.g., an EMCCD), ECLM captures a two-dimensional
image of the light emission originating from the electrode surface.
Although ECLM does not detect the radicals directly, it provides
powerful, spatially resolved information about the reaction zone, which
is intimately linked to the diffusion and lifetime of the radical
intermediates. The thickness of the ECL emission layer~\cite{81}, for
instance, can be measured and correlated with the diffusion length of
the key radicals, offering a method to distinguish between different
mechanistic pathways~\cite{82}.



Fracassa \etal~\cite{83} using ECLM to study bead-based assays has been
particularly illuminating. By imaging single luminophore-labeled
microbeads on an electrode surface, they observed that ECL emission
originates from the entire surface of the bead, even regions several
micrometers away from the electrode (Figure~\ref{fig5}). This
observation provides compelling visual evidence for the oxidative
(TPrA${\bullet}^+$) excitation mechanism, demonstrating that TPrA-derived
radicals are indeed generated at the electrode and must diffuse through
the solution to reach and react with the distal luminophore labels.
This ability to map reactivity in space makes ECLM an indispensable
tool for understanding the mechanisms of heterogeneous and spatially
confined ECL systems, which are the most relevant for modern
bioanalytical applications~\cite{84}. The analytical landscape
summarized in Table~\ref{tab4} highlights how diverse techniques
collectively unravel the fleeting existence of coreactant radicals.
Each method contributes a distinct layer of mechanistic insight---EPR
offering structural fingerprints, SEC providing kinetic correlations,
EC--MS yielding molecular confirmation, and ECLM visualizing spatial
dynamics. Together, these complementary tools bridge temporal and
spatial scales from nanoseconds to micrometers. Notably, the
integration of in-situ and \textit{operando} approaches, combining
spectroscopic and imaging modalities, is transforming ECL research
toward quantitative mapping of radical generation, transport, and
reaction efficiency in real time.

%tab4
\begin{table*}
\caption{\label{tab4}Advanced analytical techniques for radical
intermediate detection}
\tabcolsep4pt
\begin{tabular}{lllll}
\thead
 Technique & Principle & \parbox[t]{3.3cm}{\raggedright Information obtained} & 
\parbox[t]{3cm}{\raggedright Key advantages} & \parbox[t]{3cm}{\raggedright Key limitations}\vspace*{2pt} \\
\endthead
\parbox[t]{2cm}{\raggedright EPR---direct detection} & \parbox[t]{3cm}{\raggedright Absorption of microwave radiation by unpaired electrons in a magnetic field.} &  
\parbox[t]{3cm}{\raggedright Unambiguous radical structure; Concentration}
& \parbox[t]{3cm}{\raggedright Unsurpassed specificity for radicals} & 
\parbox[t]{3cm}{\raggedright Requires relatively stable radicals at sufficient concentration}\vspace*{2pt} \\

\parbox[t]{2cm}{\raggedright EPR---spin trapping} & 
\parbox[t]{3cm}{\raggedright Chemical reaction of a transient radical with a ``trap'' 
to form a persistent radical adduct} & 
\parbox[t]{3cm}{\raggedright Identification of ultra-short-lived or low-concentration radicals}
& \parbox[t]{3cm}{\raggedright Greatly extends the range of detectable radicals} & 
\parbox[t]{3cm}{\raggedright Susceptible to artifacts; indirect detection~\cite{85}}\vspace*{2pt} \\

 SEC & \parbox[t]{3cm}{\raggedright Simultaneous electrochemical control and UV--Vis spectroscopic monitoring at the electrode} & 
 \parbox[t]{3cm}{\raggedright  Concentration profiles of chromophore species vs.\ potential; Reaction kinetics}
 & \parbox[t]{3cm}{\raggedright Provides real-time kinetic data} & 
 \parbox[t]{3cm}{\raggedright  Generally not specific for transient radical species}\vspace*{2pt}\\
 
 EC--MS & \parbox[t]{3cm}{\raggedright Coupling of an electrochemical cell to a mass spectrometer for real-time analysis}
 &\parbox[t]{3cm}{\raggedright  Molecular weight of intermediates and products; Reaction pathways} & 
 \parbox[t]{3cm}{\raggedright  High sensitivity and molecular specificity} & 
 \parbox[t]{3cm}{\raggedright  Can be complex to implement; ionization efficiency varies}\vspace*{2pt} \\
 
 ECLM & \parbox[t]{3cm}{\raggedright Imaging of light emission from the electrode surface with a camera}
 & \parbox[t]{3cm}{\raggedright Spatial distribution of ECL reaction; Thickness of the emission layer}
 &\parbox[t]{3cm}{\raggedright  Provides powerful spatial information on reaction zones} & 
 \parbox[t]{3cm}{\raggedright  Indirect detection of radicals; provides information on light emission only}\vspace*{2pt}
\botline
\end{tabular}
\vspace*{-3pt}
\end{table*}

\section{Bioanalytical performance: linking radical properties to assay
sensitivity and selectivity}\label{sec4}

The profound commercial and scientific success of ECL, particularly in
the form of electrochemiluminescence immunoassays (ECLIA), stems from
its exceptional analytical performance, including high sensitivity,
wide dynamic range, and robustness. This performance is not an emergent
property of the \mbox{system} as a whole but is a direct consequence of the
fundamental physicochemical properties of the coreactant radical
intermediates~\cite{86}. Bridging the gap between the mechanistic
details discussed previously and their practical impact on bioassay
function is crucial for the rational improvement of existing
technologies and the development of\break new ones.

%tab5
\begin{table*}
\caption{\label{tab5}Influence of electrode material on TPrA oxidation
and ECL emission}
\tabcolsep4pt
\begin{tabular}{lllllc}
\thead
\parbox[t]{1.5cm}{\raggedright Electrode material} & 
\parbox[t]{2.5cm}{\raggedright TPrA oxidation behavior} & 
\parbox[t]{2.5cm}{\raggedright Dominant ECL pathway} & 
\parbox[t]{2cm}{\raggedright Relative ECL intensity} & 
\parbox[t]{3.5cm}{\raggedright Implications for bioanalysis} & 
References \vspace*{2pt}\\
\endthead
GC & \parbox[t]{2.5cm}{\raggedright Easy, broad irreversible oxidation peak starting at ${\sim}$0.6~V vs.\ SCE}
&Direct oxidation & High &
\parbox[t]{3.5cm}{\raggedright  Preferred for fundamental studies and
disposable sensors due to high efficiency}\vspace*{2pt} & \cite{35,94} \\

Au & \parbox[t]{2.5cm}{\raggedright Inhibited by surface oxide formation; catalytic oxidation at low potentials}
&\parbox[t]{2.8cm}{\raggedright Catalytic (at high potentials) or oxidative (TPrA${\bullet}^+$) excitation}
& \parbox[t]{2cm}{\raggedright  Moderate to low} & 
\parbox[t]{3.5cm}{\raggedright Requires surface modification or additives to improve performance; widely used in reusable sensors}\vspace*{2pt}
& \cite{35,36,95} \\

Pt & \parbox[t]{2.5cm}{\raggedright Strongly inhibited by surface oxide formation} &
Catalytic & Low & \parbox[t]{3.5cm}{\raggedright  Generally yields weak ECL with TPrA, limiting its use in sensitive assays}\vspace*{2pt}
& \cite{35,94} \\

BDD&\parbox[t]{2.5cm}{\raggedright Sluggish kinetics for TPrA oxidation} & 
\parbox[t]{2.5cm}{\raggedright Direct oxidation} & 
Moderate & \parbox[t]{3.5cm}{\raggedright  Can enhance ECL of labeled beads; enables in-situ coreactant generation}
& \cite{58,96}\vspace*{2pt}
\botline
\end{tabular}
\end{table*}

\subsection{The direct impact of radical intermediates on biosensor function}\label{sec41}

The stability, diffusion distance, and reactivity of these
intermediates are the key parameters that \mbox{govern} this
process~\cite{87}. A critical insight is the \mbox{operation} of a ``Goldilocks
Principle'' for radical stability in the context of heterogeneous
bioassays, such as the widely used magnetic bead-based format. In these
systems, the luminophore is immobilized on a microbead surface,
physically separated from the electrode. For an ECL signal to be
generated, the coreactant radicals must be stable enough to survive
diffusion across the micrometer-scale gap from the electrode to the
bead surface~\cite{88}. However, they must also remain highly reactive
to participate in an efficient, thermodynamically favorable
electron-transfer reaction upon arrival to generate the luminophore's
excited state. A radical that is too reactive might decompose before
reaching the bead, while a radical that is too stable may lack the
necessary thermodynamic driving force for efficient light
generation~\cite{89}. The remarkable success of TPrA in these assays is
largely because its radical intermediates, particularly
TPrA${\bullet}^+$, possess a near-optimal balance: its half-life of
${\sim}$0.2~ms allows diffusion over the required length scale, while the
resulting TPrA$\bullet$ remains a potent enough reductant to drive the
ECL reaction efficiently.


The intrinsic reactivity of the radical, governed by its standard redox
potential, directly determines the ECL efficiency and, by extension,
the analytical \mbox{sensitivity} of a biosensor. The highly negative redox
potential of the TPrA$\bullet$ radical (ca.\ ${-}$1.7~V vs.\ SCE) ensures
that its reaction with oxidized or ground-state luminophores is highly
exergonic, leading to a high probability of forming the excited state
and thus of a strong light signal~\cite{90}. Any factor that increases
the effective concentration or generation rate of these key radicals,
such as using an electrode material that facilitates TPrA oxidation
(Table~\ref{tab5}), tends to directly increase the ECL signal and
improve the limit of detection (LOD)~\cite{91}.

However, the high reactivity that makes these radicals effective can
also be a source of limitations. Radical intermediates can engage in
unwanted side reactions with components of the biological sample
matrix, such as proteins or endogenous antioxidants. These ``dark''
pathways consume the radicals without producing light, effectively
quenching the ECL signal and potentially compromising assay accuracy.
Furthermore, the stability of the radicals themselves can be influenced
by the sample matrix; for instance, pH changes can alter the rate of
the critical \mbox{deprotonation} step of TPrA${\bullet}^+$~\cite{92}.
Therefore, the robustness of an ECL immunoassay depends not only on the
specificity of the antibody--antigen interaction but also on the
stability and predictable reactivity of the coreactant radical
intermediates within complex biological fluids like serum or
plasma~\cite{93}.\looseness=1

\subsection{Rational design of coreactants and assay formats}\label{sec42}

A deep mechanistic understanding of radical intermediates enables the
rational design of superior ECL systems. Instead of relying on
empirical screening, researchers can now tailor coreactant structures
and assay configurations to optimize radical generation and reactivity.
One promising strategy is to overcome the limitations of intermolecular
diffusion by physically linking the coreactant and luminophore. For
example, covalently integrating coreactant moieties into the structure
of a porous emitter, such as a covalent organic framework (COF), has
been shown to enhance ECL intensity by over three orders of 
magnitude~\cite{97}. This dramatic improvement is attributed to the
facilitation of rapid intra-framework charge transfer between the
luminophore and coreactant radicals, maximizing the efficiency of the
light-generation step.

Another advanced approach involves the use of synergistic coreactant
systems or ``co-reaction accelerators''. In these systems, a second
chemical species is added to catalyze the generation of the primary
coreactant radicals. For instance, Ding and Su~\cite{96}
demonstrated that adding a small amount of triethanolamine (TEOA), a
relatively weak ECL coreactant, to the classic
[Ru(bpy)$_{3}$]$^{2+}$/TPrA
system resulted in a more than tenfold amplification of the ECL signal.
They proposed a ``chemical oxidation mechanism'' where the
electrochemically generated TEOA radical cation acts as a homogeneous
oxidant for TPrA in the solution phase~\cite{98}. This opens an
additional, highly efficient channel for the generation of TPrA
radicals, effectively accelerating the overall reaction kinetics and
boosting the light output~\cite{99}. These examples illustrate a
clear trend in the field: moving from simply using coreactants to
actively engineering the generation, environment, and reactivity of
their radical intermediates to achieve unprecedented analytical
performance.

\section{Conclusions and future perspectives}\label{sec5}

The study of coreactant radical intermediates has been central to the
transformation of ECL from a physical phenomenon into a dominant
bioanalytical technology. This review has traced the critical role of
these transient species, from their generation at the electrode surface
to their ultimate impact on assay performance. It is clear that the
chemical identity, redox properties, stability, and transport dynamics
of radicals derived from coreactants like TPrA and persulfate are the
fundamental parameters that dictate the efficiency and applicability of
ECL systems. The intricate web of competing mechanistic pathways, such
as the direct oxidation, catalytic, and oxidative (TPrA${\bullet}^+$)
excitation routes in the TPrA system, is not merely of academic
interest but has profound practical consequences, determining which
electrode materials and assay formats yield optimal results. Similarly,
the ongoing investigation into radical versus non-radical pathways in
persulfate chemistry highlights a frontier where fundamental
understanding continues to evolve. The development of a sophisticated
analytical arsenal, including EPR spectroscopy, real-time mass
spectrometry, and ECL microscopy, has been indispensable in providing
the experimental evidence needed to construct and validate these
complex mechanistic models.

Despite significant progress, several key questions and challenges
remain, representing fertile ground for future research. The precise
nature and kinetics of ``dark'' side reactions involving coreactant
radicals, especially within complex biological matrices, are still not
fully understood and represent a major factor in assay stability and
reproducibility. While the dominant conditions for different TPrA
pathways are qualitatively known, a comprehensive, \mbox{quantitative} model
that can predict the relative contributions of each pathway under any
given set of experimental parameters has yet to be developed.
Furthermore, the role of non-radical pathways in persulfate-based ECL
remains an open and intriguing question that warrants further
investigation.

Looking forward, the field is poised for exciting advancements driven
by a continued focus on the chemistry of radical intermediates. A major
direction will be the rational design of novel coreactants. This will
move beyond simple structural analogs to ``smart'' molecules with
tailored radical stabilities, enhanced biocompatibility, or even
built-in responsiveness to biological analytes. The integration of
coreactant functionalities into nanostructured materials, such as MOFs
(metal--organic frameworks) and COFs, represents a powerful strategy to
control the local radical environment and enhance charge-transfer
efficiency. Progress will also be heavily reliant on the development of
advanced \textit{operando} characterization techniques. The future lies
in multi-modal platforms that can simultaneously capture
electrochemical, spectroscopic, and spatial information in real time to
provide a complete, dynamic picture of the reaction layer. Finally,
computational and machine learning approaches will play an increasingly
vital role. High-throughput DFT screening could rapidly identify
promising new coreactant structures, while machine learning models
could be trained on multi-parameter experimental data to predict
optimal assay conditions and even assist in deciphering complex
mechanistic data. By \mbox{continuing} to focus on the fundamental chemistry
of these pivotal radical intermediates, the scientific community can
ensure that ECL remains at the forefront of analytical and
bioanalytical\break innovation.

\section*{CRediT authorship contribution statement}

\textbf{Yifan He:} Conceptualization, Investigation, Formal analysis,
Writing---original draft.

\noindent\textbf{Yanfei Lv:} Formal analysis, Visualization.

\noindent\textbf{Li Fu:} Supervision, Writing---review \& editing.

\noindent\textbf{Shichao Zhao:} Conceptualization, Writing---review \&
editing.

\noindent\textbf{Hassan Karimi-Maleh:} Supervision, Writing---review \&
editing.

\section*{Acknowledgements} 
This work was supported by the Hassan Research Initiation Fund Grant
Number KYQD2024-046 and Theoretical and Experimental Development of an
MXene/GoldAmplified Electrochemical Biosensor for Monitoring Lung
Cancer Drugs: Adagrasib and Afatinib Dimaleate Grant Number 2025K064,
The Quzhou Affiliated Hospital of Wenzhou Medical University.

\CDRGrant[KYQD]{KYQD2024-046}
\CDRGrant[KYQD]{2025K064}

\section*{Declaration of interests}

The authors do not work for, advise, own shares in, or receive funds
from any organization that could \mbox{benefit} from this article, and
have declared no affiliations other than their research organizations.

\back{}

\printbibliography
\refinput{crchim20250948-reference.tex}

\end{document}
