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\DOI{10.5802/crchim.406}
\datereceived{2024-11-23}
\daterevised{2025-03-17}
\dateaccepted{2025-05-20}
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\dateposted{2025-06-26}
\begin{document}

\begin{noXML}

\CDRsetmeta{articletype}{review}

\title{Screen-printed carbon electrodes as a tool for the discovery and
the  characterization of new enzymes active on lignocellulosic biomass}

\alttitle{\'{E}lectrodes de carbone s\'{e}rigraphi\'{e}es comme outil
pour la  d\'{e}couverte et la caract\'{e}risation de nouvelles enzymes
actives sur la  biomasse lignocellulosique}

\author{\firstname{Marjorie} \lastname{Ochs}\CDRorcid{0009-0003-6516-3871}\IsCorresp}
\address{Institut de Chimie et Biochimie Mol\'eculaires et 
Supramol\'eculaires, ICBMS UMR 5246 CNRS, Universit\'{e} de Lyon, 
Universit\'{e} Lyon 1, CNRS, INSA Lyon, CPE Lyon, 1 rue Victor Grignard, 
69622 Villeurbanne Cedex, France}
\email[M. Ochs]{marjorie.ochs@univ-lyon1.fr}

\author{\firstname{Bastien} \lastname{Doum\`{e}che}\CDRorcid{0000-0003-3458-1029}}
\addressSameAs{1}{Institut de Chimie et Biochimie Mol\'eculaires et 
Supramol\'eculaires, ICBMS UMR 5246 CNRS, Universit\'{e} de Lyon, 
Universit\'{e} Lyon 1, CNRS, INSA Lyon, CPE Lyon, 1 rue Victor Grignard, 
69622 Villeurbanne Cedex, France}

\keywords{\kwd{Screen-printed carbon electrodes}
\kwd{Paper-based electrodes}
\kwd{Ligninase}
\kwd{Lytic polysaccharide monooxygenase}
\kwd{Oxidase}
\kwd{Detection of aromatic and hydroxycinnamic compounds}}

\altkeywords{\kwd{\'{E}lectrodes de carbone s\'{e}rigraphi\'{e}es}
\kwd{\'{E}lectrodes \`{a} base de papier}
\kwd{Ligninase}
\kwd{\textit{Lytic polysaccharide monooxigenase}}
\kwd{Oxydase}
\kwd{D\'{e}tection de  compos\'{e}s aromatiques et hydroxycinnamiques}}

\def\thanksname{Note}
\thanks{This article is written at the invitation of the editorial
committee.}

\begin{abstract}
The development of efficient biocatalysts requires the detection and 
quantification of proven enzymatic activities on natural substrates. 
Electrochemical methods are particularly suitable for monitoring redox 
enzymatic activities when the substrates or products are electroactive 
species. These methods also have the advantage of being fast, sensitive
and  suited to heterogeneous environments. In this context,
screen-printed carbon  electrodes are particularly interesting because
of their flexibility and low  cost. 

This article outlines the potential of screen-printed electrodes in the
identification and characterization of new enzymes involved in the 
depolymerization of lignocellulosic biomass. The first part describes
the  interest and the fabrication of screen-printed carbon electrodes
(SPCEs),  with particular emphasis on the use of paper as a support for
screen  printing. The following sections describe examples of
applications of  screen-printed electrodes for the detection of small
aromatic compounds and  for screening peroxidase activities, in
particular their use for the  characterization of a catalase
peroxidase. Finally, the article opens the  way for the use of
paper-based SPCEs to develop a biomimetic plant cell  wall, that can be
applied for the detection of lytic polysaccharide  monooxygenases
(LPMO) and ligninase activities.
\end{abstract}

\begin{altabstract}
Le d\'{e}veloppement de biocatalyseurs efficaces n\'{e}cessite de 
d\'{e}tecter et de quantifier les activit\'{e}s enzymatiques
vis-\`{a}-vis  de leurs substrats naturels. Les m\'{e}thodes
\'{e}lectrochimiques sont  particuli\`{e}rement adapt\'{e}es pour le
suivi des activit\'{e}s  enzymatiques d'oxydo-r\'{e}duction et lorsque
les substrats ou les produits  sont des esp\`{e}ces \'{e}lectroactives.
Ces m\'{e}thodes pr\'{e}sentent  \'{e}galement l'avantage d'\^{e}tre
rapides, sensibles et adapt\'{e}es aux  milieux
h\'{e}t\'{e}rog\`{e}nes. Dans ce contexte, les \'{e}lectrodes de 
carbone s\'{e}rigraphi\'{e}es (SPCE), sont particuli\`{e}rement 
int\'{e}ressantes en raison des divers formats de conceptions possibles
et  de leur faible co\^{u}t.

Cet article expose le potentiel des \'{e}lectrodes
s\'{e}rigraphi\'{e}es  dans l'identification et la caract\'{e}risation
de nouvelles enzymes  impliqu\'{e}es dans la d\'{e}gradation de la
biomasse lignocellulosique. Une  premi\`{e}re partie d\'{e}crit
l'int\'{e}r\^{e}t des \'{e}lectrodes  s\'{e}rigraphi\'{e}es avec une
attention particuli\`{e}re concernant  l'utilisation du papier comme
support pour la s\'{e}rigraphie. Les  paragraphes suivants
d\'{e}crivent des exemples d'applications des  \'{e}lectrodes
s\'{e}rigraphi\'{e}es pour la d\'{e}tection de petits  compos\'{e}s
aromatiques et pour le criblage des activit\'{e}s peroxydases, 
particuli\`{e}rement leur mise en \oe uvre pour la caract\'{e}risation
d'une  catalase-peroxydase. Enfin, l'article ouvre la voie vers
l'utilisation du  papier comme support d'\'{e}lectrodes
s\'{e}rigraphi\'{e}es afin de  d\'{e}velopper un substrat
biomim\'{e}tique de la paroi v\'{e}g\'{e}tale,  utilis\'{e} pour la
d\'{e}tection d'activit\'{e}s \textit{Lytic Polysaccharide
MonoOxigenase} (LPMO) et ligninase.
\end{altabstract}

\maketitle

\vspace*{.5pc}

\twocolumngrid

\end{noXML}

\section{Introduction}\label{sec1}
Lignocellulosic biomass (LCB), the main component of the plant cell
wall, is  obtained from plant biomass like wood, herbaceous plants,
coproducts and  wastes from agro-industries. LCB appears to be the best
promising  alternative to fossil carbon due to its sustainability and
abundance on  Earth. Lignocellulosic biomass is rich in cellulose, a
polymer composed of  linear glucose chains; hemicelluloses, branched
heteropolymers composed of  hexoses and pentoses; and lignin, an
amorphous polymer composed of three  structural units, H
($p$-hydroxyphenyl), G (guaiacyl), and S (syringyl),  resulting from
the condensation of three phenylpropane monomers: coumaryl,  coniferyl,
and sinapyl alcohols~\cite{1}. The composition and amounts of 
polysaccharides and lignins greatly vary between different plant
species,  yielding heterogeneous structures of the LCB. Until now,
polysaccharides  have been the main fraction of interest in biomass
biorefinery processes  while lignin valorization needs further research
efforts. Specific and  selective fractionation of LCB, including lignin
and derivatives, would  enable these compounds to be used in a rational
and sustainable way.  Enzymatic fractionation makes it possible, by
catalyzing specific bond  cleavage under mild and low-energy reaction
conditions. In Nature, the  enzymatic depolymerization of LCB is
efficiently carried out by bacteria,  white- and brown-rot fungi that
decompose wood~\cite{2,3,4}.\looseness=-1 

However, to date, no biotechnological system has been shown to be 
sufficiently efficient to allow their use at industrial scale. The low 
efficiency and stability of the biocatalysts used is the main
limitation of  lignin valorization. Thus, the identification of
efficient and stable  ligninolytic enzymes still presents a
technological challenge. Ligninolytic  enzymes are oxidoreductases and
include mainly laccases, peroxidases  (versatile peroxidases (VP), dye
decolorizing peroxidases (DyP), lignin  peroxidases (LiP) and manganese
peroxidases (MnP))~\cite{5,6,7}. Furthermore, the  enzymatic
fractionation and upgrade of LCB and lignins is improved by the  action
of auxiliary enzymes operating in synergy with the cellulolytic, 
hemicellulolytic and ligninolytic enzymes. Among them, oxidases
generate  hydrogen peroxide, oxygenases convert dioxygen or hydrogen
peroxide into  water, and reductases as well as dehydrogenases reduce
radicals.\looseness=-1

Oxidoreductases involved in lignin degradation are usually identified
and  characterized by methods based on the light absorption properties
of  lignin-like monomers like guaiacol, pyrogallol, sinapic acid,
synthetic  dimers models like guaiacyl glycerol-$\upbeta $-guaiacyl ether
(GGE), veratryl  glycerol-$\upbeta $-guaiacyl ether (VGE) or other
synthetic redox substrates  like syringaldazine or
azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) 
(ABTS)~\cite{3,8,9,10,11}. These methods require clear and uniform
media to perform  accurate enzyme activity measurements. As a result,
the activities monitored  by these methods do not account for the
enzyme interactions with  water-insoluble lignin polymers. Furthermore,
evaluating the impact of  enzymes on polymeric lignin substrates
requires analytical methods like  liquid or gas chromatography combined
with mass spectrometry, nuclear  magnetic resonance~\cite{9,10,12}. The
challenging evaluation of ligninolytic  activities and their effect on
native and technical lignins prevents the  discovery of new efficient
ligninolytic enzymes. 

Electrochemical methods are particularly adapted to monitor redox
enzymatic  activities when substrates or products are electroactive
species. They also  show the advantages of being rapid, sensitive,
adapted to heterogeneous  media and do not require expensive apparatus.
Species to be measured could  be oxidized or reduced at the electrode
resulting in current densities  representative of their concentration.
Consequently, electrochemical methods  have the potential to circumvent
the current lack of convenient methods for  the characterization of
ligninolytic and auxiliary enzymes active on  polymers. 

Among all electrode configurations, screen-printed electrodes (SPEs)
are of  particular interest due to their flexibility and low cost. This
article  outlines the potential of SPEs in identifying and
characterizing novel  enzymes involved in the depolymerization of
lignocellulosic biomass.  Firstly, the choice of screen-printed carbon
electrodes (SPCEs) and  paper-based electrodes is presented; secondly,
an example of the application  of SPCEs for the detection of small
aromatic compounds and for the screening  of peroxidase activities is
outlined. Thirdly, the use of paper-based  electrodes for the detection
of LMPO activity, classified as an auxiliary  activity in the
carbohydrate active enzymes (CAZy) database 
(\url{http://www.cazy.org/})~\cite{13}, is demonstrated. Finally, the
use of  paper-based SPEs as a biomimetic substrate for the detection of
depolymerizing ligninase activity will be presented.


\begin{figure*}
\vspace*{-2pt}
\includegraphics{fig01}
\vspace*{-3pt}
\caption{\label{fig1}(A) Scheme illustrating the principle of screen
printing and (B) commercial and home-made screen-printed electrodes.}
\vspace*{-3pt}
\end{figure*}

\section{Screen-printed electrodes and paper-based electrodes}
\label{sec:screen}
In analytical sciences, electrochemical detection is commonly used
because  it could be conducted in nearly all media (organic or aqueous
media). This  detection is not influenced by media transparency
compared to optical  methods (UV--vis spectroscopy, fluorescence) and
the number of  configurations is nearly infinite from a classical
three-electrode system  (work, reference, counter electrodes) in mL
reactors to microelectrodes for  in vivo sensing. In the field of
biosensors, SPEs are usually preferred  because of their commercial
availability, because they are single-use, a  requirement for diagnosis
and because the geometry of the electrode or its  \mbox{composition} (gold,
silver, carbon) could be easily adapted to the need~\cite{14,15}.
Moreover, screen printing is a mature technology allowing mass 
production. The principle of screen printing is rather simple 
(Figure~\ref{fig1}): 
a  stencil is prepared by photolithography to obtain the electrode
motif  (permeable) on a mesh screen (impermeable). The substrate on
which the  electrodes are to be printed is plated below the mesh
screen. The substrate  could be plastic, paper, ceramics,
printed-circuit board (PCB), fabric, or  any material that could be
obtained in a two-dimensional format. The  electrode material provided
as an ink or paste is applied on the top of the  mesh screen and forced
to pass through the stencil by a squeegee. The  stencil-shaped ink is
recovered on the substrate that is further cured at  medium-to-high
temperature to obtain a solid electrode. The inks and pastes  are
composed of the electrode's conductive material (graphite particle, 
gold, silver, indium thin oxide) and a polymeric binder that ensure the
stability of the electrode after curing, both dissolved in a solvent
that  evaporates during curing. The inks could also be doped with
electrochemical  mediators or nanomaterials, the most common being
Prussian blue particles,  carbon nanotubes, and gold nanoparticles.


Since the key publication of Whitesides et~al.~in 2007 with
paper~\cite{16},  cellulosic material has been proposed as a substrate
for SPEs. Since then,  several reviews on the subject have been
published \mbox{\cite{17,18,19}}. The advantages  of using paper, particularly
\mbox{absorbing} paper such as Whatman {\#}1 are  numerous: (1) it is truly
disposable by incineration whereas plastic is not,  (2) paper and
cellulosic material could be produced everywhere in the world  at low
cost, (3)~paper technology is one of the most ancient in the world 
with numerous types of paper available, (4)~the printing (or drawing) 
technologies on paper are also mature including inkjet, laser, and wax 
printing among others, (5) it is biocompatible and biodegradable
(without  considering the electrode material) and (6)~the porosity of
paper allows to  design hydrophilic microfluidic channels together with
the chromatographic  (separation) properties of the cellulosic
material. Whatman {\#}1 paper for  instance should be seen as a
tridimensional porous material with  interconnected cellulose
microfibrils suitable for chemical modification  close to the electrode
but far enough to prevent passivation.

\begin{figure*}
\vspace*{-3pt}
\includegraphics{fig02}
\vspace*{-3pt}
\caption{\label{fig2}Structures of some (A) hydoxycinnamic acids and
(B) hydroxyphenolic acids.}
\vspace*{-3pt}
\end{figure*}

\vspace*{-2pt}
\section{Application of SPCEs for the detection of small aromatic
compounds and  screening of peroxidase activities}
\vspace*{-2pt}

\subsection{SPCEs used for the detection of aromatic and
phenylpropanoid compounds} 

Screen-printed electrodes (SPEs) are widely used for analytical
purposes.  Commercial electrodes offer the advantage of regular shapes,
composition,  and are ready to use, while home-made SPEs allow
\mbox{customizations,} such as  designing the shape of the electrodes,
choosing the material substrate for  the screen-printing, or adapting
the composition of the inks. One of the  common modifications of SPCEs
is the functionalization of the carbon working  electrode in order to
form nanostructures at the electrode surface. For  example, SPCEs
modified by deposition of thin layers containing bismuth,  gold,
silver, multi-walled carbon nanotubes (MWCNs) or carbon nanofibers 
(CNFs) are used for the detection and the quantification of heavy
metals in  water, beverages, food, and biologic samples~\cite{20}.
Metabolites (e.g.,  glucose, ascorbic acid, uric acid), drugs
(furaltadone, dopamine  methotrexate, erythromycin metronidazole,
estrogens), phenolic and  polyphenolic compounds (bisphenol A, caffeic
acid, tannins, catechins,  flavonoids, etc.) are also usually detected
by using layered SPEs~\cite{21}.  These modifications are known to
increase sensitivity, enabling lower  detection thresholds and broader
detection ranges. 

Hydroxycinnamic acids (Figure~\ref{fig2}A) and hydroxyphenolic 
acids (Figure~\ref{fig2}B)
are  small aromatic and electroactive compounds, allowing their
detection by  electrochemical methods. Caffeic acid is the most
frequently analyzed  hydroxycinnamic acid due to its favorable
electrochemical response and its  prevalence as a common antioxidant in
food and beverages. Its detection and  quantification can be achieved
through the use of SPCEs, with or without  electrode modifications,
including the \mbox{deposition} of CNFs or MWCNs.  Additionally, SPCEs can be
modified with several other materials, including  catechin (CT)
decorated with gold nanoparticles \mbox{(AuNP-CT)} or tungsten  disulfide
supported by carbon black (SPE-CB-WS2/CT), cobalt(II,III) or 
cerium(IV) oxides nanoparticles (Co$_{3}$O$_{4}$/SPCE;  CeO$_{2}$
NPs), Graphene Oxide (GO), and reduced Graphene Oxide  (rGO) itself
decorated with gold (Au@rGO)~\cite{22,23,24,25,26,27,28}. These modifications 
enhance the quantification of hydroxycinnamic acids (Table~\ref{tabI}). The
linear  range for the detection of hydroxycinnamic acids, using
non-functionalized  SPCEs, is comprised between 0.42 and 13.9~$\upmu$M
for caffeic acid between  26 and 515~$\upmu$M for ferulic
acid~\cite{29,30}. Functionalization of the  working electrode allows
broadening the linear range and quantifying higher  concentrations, up
to 2~mM and 1~mM for caffeic and ferulic acids, 
respectively~\cite{23,28}. Moreover, the modification of the carbon
electrode  decreases the oxidation potential, compared to non-modified
carbon  electrodes, improving the specificity of the electrode. This
allows the  detection and quantification of caffeic acid in a mixture
containing ferulic  and gallic acids, or sinapic, $p$-coumaric acids,
present in red wine, rapeseed  oil, or phyto-homeopathic
tablets~\cite{22,23,24}.

\begin{table*} %tab1
\caption{\label{tabI}Electrochemical detection of hydroxycinnamic acids
and gallic acid using SPCEs.\vspace*{-2pt}}
\begin{tabular}{cccccc}
\thead
{Compound}&amp; {Method}&amp; {Electrode modifications}$^{1}$ &amp;  {pH}&amp; 
Linear range ($\upmu$M)&amp;  Ref. \\
\endthead
\morerows{8}{Caffeic acid}&amp;  CV&amp;  None (Dropsens 110)&amp;  ${&lt;}$2&amp;  0.4--13.9&amp;  \cite{29} \\
&amp;  CV&amp;  CNF (Dropsens 110)&amp;  &amp;  0.8--1000&amp;  \cite{22} \\
&amp;  CV&amp;  Au@rGO (EcoBioServices)&amp;  7&amp;  0.5--100&amp;  \cite{25} \\
&amp;  CV&amp;  rGO (EcoBioServices)&amp;  7&amp;  0.5--100&amp;  \cite{25} \\
&amp;  CV&amp;  MWCNT (homemade SPEs)&amp;  ${&lt;}$2&amp;  2.0--50&amp;  \cite{26} \\
&amp;  CV&amp;  CeO$_{2}$ NPs (Dropsens 110)&amp;  7.4&amp;  50--200&amp;  \cite{24} \\
&amp;  DPV&amp;  Co$_{3}$O$_{4}$ (-)&amp;  7&amp;  0.2--272&amp;  \cite{27} \\
&amp;  DPV&amp;  rGO (-)&amp;  7&amp;  0.2--2100&amp;  \cite{28} \\
&amp;  DPV&amp;  CB-WS$_{2}$/AuNP-CT (EcoBioServices)&amp;  7&amp;  0.4--112.5&amp;  \cite{23}\vspace*{3pt} \\
\morerows{1}{Ferulic acid}&amp;  CV&amp;  None (paperbased homemade SPCE)&amp;  5&amp;  26--515&amp; \cite{30} \\
&amp;  CV&amp;  CNF (Dropsens 110)&amp;  &amp;  0.8--1000&amp;  \cite{22}\vspace*{3pt} \\
Sinapic acid&amp; DPV&amp; CB-WS2/AuNP-CT (EcoBioServices)&amp; 7&amp; 0.7--125.0&amp; \cite{23} \\
$p$-Coumaric acid &amp; DPV&amp; CB-WS2/AuNP-CT (EcoBioServices)&amp; 7&amp; 1.4--93.7&amp;  \cite{23} \\
Gallic acid&amp;  CV&amp;  CeO$_{2}$ NPs (Dropsens 110)&amp;  7.4&amp;  2--20&amp; \cite{24}
\botline 
\end{tabular}
\tabnote{CV: cyclic voltammetry; DPV: differential pulsed
voltammetry; CNF: carbon  nanofibers; rGO: reduced graphene oxide;
MWCNT: multi-walled carbon  nanotubes; CeO$_{2}$ NPs: cerium oxide
nanoparticles;  Co$_{3}$O$_{4}$: cobalt oxide; 
CB-WS$_{2}$/AuNP-CT: tungsten disulfide supported by carbon 
black/catechin decorated with gold nanoparticles. 
$^{{1}}$Commercial or homemade SPCEs are indicated.}
\vspace*{-2pt}
\end{table*}

Additionally, caffeic acid oxidation leads to the formation of
electroactive  dimers and contributes to the modification of the
electrode surface.  Furthermore, caffeic acid can also react with other
electroactive compounds  through its quinine equivalent~\cite{31}.

\subsection{Application of SPCEs to the screening of peroxidase substrates}

In a recent work, the electrochemical properties of hydroxycinnamic and
hydroxybenzoic acids are used to develop the first electrochemical
screening  assay for the detection of ligninase activity~\cite{32}. The
assay is applied to  identify small phenolic molecules derived from
lignin as substrates of a  newly discovered bifunctional bacterial
catalase--peroxidase. Peroxidase  activity consists in the oxidation of
a substrate in the presence of  H$_{2}$O$_{2}$ as electron
acceptors.  Peroxidases are often expressed by microorganisms when they
are fed with  lignin-rich substrates. Furthermore, hydroxycinnamic and
hydroxybenzoic  acids have been identified as peroxidase substrates,
which suggests that  these activities contribute to the
depolymerization of lignin. Additionally,  it has been reported that
catalase--peroxidases assist the main  lignin-degrading enzymes and
further oxidize dimers or monomers after the  depolymerization of the
lignin macromolecule~\cite{4}.

The strategy of the screening assay developed in this study is based on
the  use of lignin-derived aromatic substrates as electron donors. The
potential  of hydroxycinnamic and hydroxyphenolic acids, including 
3,4-dihydroxybenzoic, sinapic, vanillic, syringic, $o$-coumaric,
caffeic, and  $p$-coumaric acids as substrates is investigated. These
acids are selected  based on their proven ability to generate anodic
{current} at ${+}$800~mV vs  Ag$|$AgCl. The oxidation of these aromatics
substrates by the enzyme  was monitored by intermittent pulsed
amperometry (IPA) on 96-SPCE plates  (Figure~\ref{fig3}). An oxidation
overpotential of ${+}$800~mV vs Ag$|$AgCl is  applied to detect
electroactive species and to quantify the substrate  depletion after
4~h incubation in the presence of the enzyme. The results  obtained by
the electrochemical method are consistent with those obtained by 
spectrophotometric methods using
azino-bis(3-ethylbenzothiazoline-6-sulfonic  acid) (ABTS) and
Mn$^{{2+}}$ as the positive controls and veratryl  alcohol as
the negative\break one. 

\begin{figure}
{\vspace*{3pt}}
\includegraphics{fig03}
{\vspace*{3pt}}
\caption{\label{fig3}Strategy applied for the screening of small
lignin-derived compounds as substrates of a catalase--peroxidase
($Tx$CP) using SPCEs (adapted from~\cite{32}).}
\end{figure}



The results show that the electrochemical SPCE-based screening is
applicable  to detect the peroxidase activity. It also evidences that
the  catalase--peroxidase from the bacteria \textit{Thermobacillus
xylanilyticus} is able to oxidize  3,4-dihydroxybenzoic (3,4-DHB),
sinapic, vanillic, syringic, $o$-coumaric,  caffeic, and $p$-coumaric
acids, whose current densities differ significantly  (${&gt;}$10{\%}) in the
presence of enzyme, compared to the standard  reaction without enzyme
\mbox{(Table~\ref{tabII})}.

\begin{figure*}
\includegraphics{fig04}
\caption{\label{fig4}(A) Kinetics of ascorbic acid consumption and (B)
Michaelis--Menten plots corresponding to ascorbic acid consumption
rates by \textit{NcL}PMO9C. Reactions are performed in the presence of
1~$\upmu$M \textit{NcL}PMO9C, ascorbic acid (1~mM for (A), 250--1000~$\upmu$M
for (B)), microcrystalline cellulose 1\% (w/v) in sodium phosphate
buffer (20 mM, pH 6) incubated at 45~\textdegree C ($n\geq 3$).}
\end{figure*}

\begin{table*} %tab2
\caption{\label{tabII}Monomers oxidized by the catalase--peroxidase from
Thermobacillus xylanilyticus ($Tx$CP) and screened by intermittent
pulsed amperometry (adapted from~\cite{32}).}
\begin{tabular}{cccc}
\thead
\xmorerows{1}{Compound} &amp; \multicolumn{2}{c}{Current density ($\upmu\mathrm{A}{\cdot}\mathrm{cm}^{-2}$)}&amp; 
\morerows{1}{\parbox[t]{3cm}{\centering Variation ({\%}) of the current density}}
\\\cline{2-3}
 &amp; Without $Tx$CP &amp;  With $Tx$CP &amp;   \\
\endthead
Caffeic acid&amp;  417 ${\pm}$ 18&amp;  620 ${\pm}$ 103&amp;  49 \\
3,4-DHB acid&amp;  498 ${\pm}$ 62&amp;  348 ${\pm}$ 52&amp;  30 \\
Vanillic acid&amp;  90 ${\pm}$ 7&amp;  51 ${\pm}$ 5&amp;  44 \\
Ferulic acid&amp;  50 ${\pm}$ 7&amp;  62 ${\pm}$ 9&amp;  23 \\
Syringic acid&amp;  42 ${\pm}$ 5&amp;  24 ${\pm}$ 4&amp;  42 \\
Sinapic acid&amp;  331 ${\pm}$ 40&amp;  168 ${\pm}$ 22&amp;  49 \\
$o$-coumaric&amp;  68 ${\pm}$ 9 &amp;  28 ${\pm}$ 7&amp;  59 \\
$p$-coumaric&amp;  61 ${\pm}$ 2 &amp;  101 ${\pm}$ 16&amp;  65 \\
MnCl$_2$&amp;  455 ${\pm}$ 27&amp;  321 ${\pm}$ 19&amp;  30 \\
ABTS&amp;  289 ${\pm}$ 48&amp;  205 ${\pm}$ 15&amp;  29 \\
Veratryl alcohol&amp;  22 ${\pm}$ 4 &amp;  20 ${\pm}$ 1&amp;  ${&lt;}$10 (non significant) 
\botline 
\end{tabular}
\end{table*}


\section{Paper-based SPCEs applied to the measurement of LPMO activities}

\subsection{Consumption of ascorbic acid by LPMO activity monitored by
electrochemical methods}

Lytic polysaccharide monooxygenases (LPMO, EC 1.14.99.53-56) are 
copper-dependent oxidative enzymes catalyzing the depolymerization of 
polysaccharides or oligosaccharides in the presence of an oxidant and
an  electron donor. The oxidation of C1 and/or C4 carbon of the
glycosidic  backbone, causing the cleavage of the glycosidic bonds,
results from a  monooxygenase activity in the presence of oxygen or
from a peroxygenase  activity in the presence of hydrogen peroxide 
(Equations~(\ref{eq1}) and (\ref{eq2}), 
respectively)~\cite{33,34,35,36}. In the absence of a glycosidic
substrate, an  oxidase activity is also observed
(Equation~(\ref{eq3}))~\cite{37}.
{\begin{eqnarray}
&amp;&amp;
\mbox{Monooxygenase reaction: }
\nonumber\\&amp;&amp;\quad 
\mathrm{R}\ndash\mathrm{H} + {\mathrm{O}}_{2}  + 
2{\mathrm{e}}^{-}  + 2{\mathrm{H}}^{{+}}  
\stackrel{\mathrm{Cu}(\mathrm{II})}{\longrightarrow}  \mbox{R--OH} + 
{\mathrm{H}}_{2}\mathrm{O} \label{eq1}\Seqnsplit
&amp;&amp;
\mbox{Peroxygenase reaction: }
\nonumber\\&amp;&amp;\quad 
\mathrm{R}\ndash\mathrm{H}+ {\mathrm{H}}_{2}{\mathrm{O}}_{2}  
\stackrel{\mathrm{Cu}(\mathrm{I})}  {\longrightarrow}   \mbox{R--OH} + {\mathrm{H}}_{2}\mathrm{O} \label{eq2}\Seqnsplit
&amp;&amp;
\mbox{Oxidase reaction: }
\nonumber\\&amp;&amp;\quad 
{\mathrm{O}}_{2}  + 2{\mathrm{e}}^{-}  + 2{\mathrm{H}}^{+}
 \stackrel{\mathrm{Cu}(\mathrm{II})}{\longrightarrow} {\mathrm{H}}_{2}{\mathrm{O}}_{2}\label{eq3}
\end{eqnarray}}\unskip

As LPMOs are three-substrate enzymes (oxidant, electron donor, and 
glycosidic substrate), the \mbox{measurement} of their activity is delicate.
Most  of the published studies focus on the quantification and
identification of  some short-length oligosaccharide products using
liquid chromatography  and/or mass
spectrometry~\cite{38,39,40,41,42,43,44,45}. Recently, a method was
developed to  measure the peroxygenase activity using a rotating disc
electrode selective  to hydrogen peroxide through the presence of
Prussian blue as the  electrocatalyst~\cite{46}. This method is adapted
to fundamental studies of the  enzymatic reaction, but is less adapted
to screen applicative conditions in  complex media.

As explained in 1 and 2, SPCEs are adapted to the needs of
high-throughput  screening. In this context, we developed an
electrochemical method to  measure the initial rates of the oxygenase
reaction (equally monooxygenase  and peroxygenase reactions) catalyzed
by LPMOs. The method is based on the  direct detection by IPA of the
remaining electron donor, without further  chemical derivation or
separation steps. Ascorbic acid (1~mM) is used as the  electron donor
(Equation~(\ref{eq4})) in reactions containing a suspension of 
microcrystalline cellulose 1{\%}~(w/v) and LPMO9C isolated from
\textit{Neurospora crassa}  (\textit{Nc}LPMO9C). The reactions are
performed in test tubes at 45~\textdegree C, with  regular sampling. The
remaining ascorbic acid is quantified at ${+}$0.6~V vs  Ag$|$AgCl by IPA
onto SPCEs. 
{\fontsize{9.5}{12}\selectfont
\begin{eqnarray}
&amp;&amp;\hspace*{-1.5pc}
\text{Detection of ascorbic acid: }
\nonumber\\&amp;&amp;\hspace*{-1.5pc}
\text{Ascorbic acid} \longrightarrow 
\text{Dehydroascorbic acid}+ 2{\mathrm{e}}^{-}  + 2\mathrm{H}^{+}\label{eq4}
\end{eqnarray}}\unskip

The consumption of ascorbic acid by LPMOs was linear over at least
70~min  (Figure~\ref{fig4}A). The decrease of the signal, due to
ascorbic acid oxidation at  45~\textdegree C, was less than 20{\%} in
70~min. In the same time, around  80{\%} of the ascorbic acid
disappeared in the presence of LPMO, leading to  a specific activity of
196~nmol${\cdot}$min$^{-1}{\cdot}$mg$^{-1}$. Apparent kinetic constants
for ascorbic acid with  microcrystalline cellulose as co-substrate
obtained from Michaelis--Menten  plots are
$K_{\mathrm{m}}^{\mathrm{App}}  = 601\pm 166$~$\upmu$M  and
$k_{\mathrm{cat}}^{\mathrm{App}}  = 16.1\pm 2.3$~min$^{-1}$
(Figure~\ref{fig4}B).

\begin{table*} %tab3
\caption{\label{tabIII}Electrochemical characteristics of the electron
donors measured using carbon screen-printed electrodes in acetate
buffer (50~mM, pH~5).\vspace*{-1pt}}
\tabcolsep6pt
\begin{tabular}{cccc}
\thead
Electron donor&amp; \parbox[t]{3cm}{\centering Oxidation potential  (V vs Ag/AgCl)}&amp; 
\parbox[t]{3cm}{\centering Reduction potential  (V vs Ag/AgCl)}&amp; 
\parbox[t]{3cm}{\centering Sensitivity at ${+}$0.8~V vs Ag/AgCl 
($\upmu$A${\cdot}$cm$^{-2}{\cdot}\upmu\mathrm{M}^{-1}$)}\vspace*{2pt} \\
\endthead
Ascorbic acid&amp;  ${+}0.46$&amp;  n.d.&amp;  \00.467$^*$ \\
Caffeic acid&amp;  ${+}0.5$\0&amp;  0&amp;  0.796 \\
3,4-DHB acid&amp;  ${+}0.65$&amp;  0&amp;  0.498 \\
Syringic acid&amp;  ${+}0.7$\0&amp;  n.d.&amp;  0.243
\botline 
\end{tabular}
\xxtabnote{$^*$At ${+}$0.6~V vs Ag/AgCl for ascorbic acid; n.d.: not detected.}
\vspace*{-4pt}
\end{table*}

These results highlight that the direct measurement of ascorbic acid 
consumption by electrochemistry appears to be a suitable alternative to 
\mbox{spectrophotometric} methods for the measurement of oxygenase LPMO
activities. 

\subsection{Evaluation of filter paper instead of microcrystalline
cellulose as substrate for the electrochemical detection of LPMO
activity}
The interest in LPMO is increasing since it has been proposed that some
are  active on recalcitrant cellulose and, therefore could play a key
role in the  \mbox{enzymatic} deconstruction of lignocellulosic biomass.
Indeed, these enzymes  are supposed to reduce the crystallinity of
cellulose and operate  synergistically with glycoside hydrolases that
are active on amorphous  cellulose~\cite{39,47,48,49}. 

The three-substrates LPMO is ideal for the development of original
screening  methods for depolymerizing enzymatic activities including
paper-based SPCE  as substrate. Indeed, Whatman paper made of cellulose
can constitute the  polymeric substrate for the reaction. Paper-based
electrodes present  interesting properties that could be exploited for
a screening method. In  this context, we investigated the possibility
of measuring LPMO activity  using Whatman paper instead of
microcrystalline cellulose as the substrate  for \textit{Nc}LPMO.

The rate of ascorbic acid consumption using 1{\%}~(w/v) of both
cellulosic  substrates was measured by IPA with different enzyme
concentrations at  45~\textdegree C. No significant differences between
Whatman paper and  microcrystalline cellulose substrates were observed
regarding ascorbic acid  consumption rates ($15.7\pm 2.2$~min$^{-1}$
and $18.6\pm  3.5$~min$^{-1}$, respectively). This opens the way for
paper-based  SPCEs as a screening tool to detect LPMO activity.

\subsection{Screening of electron donor using SPCEs} 
LPMOs, notably \textit{Nc}LPMO9C, oxidize lignin-derived electroactive
species obtained  from biomass pretreatments~\cite{50}. However, due to
the molecular complexity of  the crude liquid lignin fraction obtained
after lignocellulosic biomass  pretreatment, structural analogs of
lignin monomers are used instead of real  samples. Therefore,
\textit{Nc}LPMO9C's ability to oxidize three putative electron  donors
(caffeic, 3,4-DHB, and syringic acids) was investigated. Cyclic 
\mbox{voltammetry} of these aromatic acids showed oxidation peaks 
\mbox{between} ${+}$0.46  and ${+}$0.7~V vs Ag$|$AgCl in sodium acetate
buffer (50~mM, pH~5)  (Table~\ref{tabIII}). The reduction potential of
caffeic acid and \mbox{3,4-dihydroxybenzoic} acid is ${+}$0~V vs Ag$|$AgCl,
whereas the  oxidation of syringic acid is irreversible. In order to
quantify  simultaneously the remaining substrate after 2~h of enzymatic
reaction, an  overpotential of ${+}$0.8~V vs Ag$|$AgCl was applied on
the\break SPCE. 


In the absence of \textit{Nc}LPMO9C, the current density did not evolve
after 2~h at  45~\textdegree C for caffeic and 3,4-DHB acids 
(946~$\upmu$A${\cdot}$cm$^{-2}$ and 539~$\upmu$A${\cdot}$cm$^{-2}$, 
respectively) while it strongly decreased from
390~$\upmu$A${\cdot}$cm$^{-2}$ to 82~$\upmu$A${\cdot}$cm$^{-2}$ for 
syringic acid, meaning it is unstable in these conditions. The
sensitivities  were also different for the electron donors
(0.243~$\upmu$A${\cdot}$cm$^{-2}{{\cdot}}\upmu$M$^{-1}$ for syringic
acid,  0.498~$\upmu$A${\cdot}$cm$^{-2}{\cdot}\upmu\mathrm{M}^{-1}$ for
3,4-dihydroxybenzoic acid, and 
0.796~$\upmu$A${\cdot}$cm$^{-2}{\cdot}\upmu$M$^{-1}$ for  caffeic acid)
(Table~\ref{tabIII}). \looseness=-1

When \textit{Nc}LPMO9C (1~$\upmu$M) was incubated with ascorbic acid and
Whatman paper  (1{\%}~w/v) for 2~h, the current density was in the
background showing the  full conversion of ascorbic acid. When caffeic,
3,4-DHB or syringic acids  were used as substrates, the current
densities decreased by 60, 56 and  54{\%}, respectively
(Figure~\ref{fig5}), confirming that these compounds are  electron
donors for \textit{Nc}LPMO9C. This also suggests some guidelines
regarding the  redox potential of the electron donor, as it appears
that species having an  oxidation potential up to ${+}$0.7~V vs
Ag$|$AgCl could supply  electrons to \textit{Nc}LPMO9C. The role of
these compounds as electron donor for LPMOs  was also recently shown by
following the apparition of oxidized  oligosaccharide using
HPLC~\cite{40}. While this transformation requires a  20-hour
incubation followed by HPLC analysis, only two~hours are necessary 
with our electrochemical method to \mbox{identify} electron donors as well as
to  estimate the enzymatic conversion rate 
(5~$\upmu$M${\cdot}$min$^{-1}$). 

\begin{figure}
\includegraphics{fig05}
\caption{\label{fig5}Activity of \textit{NcL}PMO9C 1~$\upmu$M using several
electron donors: caffeic acid, 3,4-dihydroxybenzoic acid, and syringic
acid. Reactions are performed with Whatman paper as cellulosic
substrate 1\% (w/v), electron donor 1~mM and incubation for 2~h at
45~\textdegree C. Current density is measured by intermittent pulsed
amperometry at a fixed potential of ${+}$0.8 V vs Ag|AgCl.}
\end{figure}

Using IPA, up to 96 independent samples could be measured at a time
using a  given potential in 1~min~\cite{51,52,53}. The thermolability
of some electron  donors is pointed out, but in this work, all
reactions were performed at  45~\textdegree C (optimal temperature of
\mbox{LPMOs).} However, as the duration of  analysis is short and the
sensitivity of the method is high, the reactions  could also be
performed at room temperature. This enables rapidly  identification of
the electron donor and carbohydrate substrate for one or  more LPMOs,
as well as acquisition of kinetic \mbox{parameters.}

\subsection{Important remarks}
Studying and measuring LPMO activities is challenging because three 
substrates are involved and because several mechanisms are proposed.
The  methods currently applied to monitor the reaction are based on the
quantification of the glycosidic products after hours of incubation,
which  is \mbox{time-consuming} and not representative of the early stage of
the reaction.  In this work, we present the development of a general
electrochemical method  based on monitoring electron donor consumption
by LPMOs, with reduced  analysis time for fast characterization of LPMO
oxygenase activity.

The reaction rates of \textit{Nc}LPMO9C were determined as well as
apparent  Michaelis--Menten constant for ascorbic acid in less than
1~h. The values  obtained are in agreement with published ones.
Activities were equivalent,  either using the cellulosic substrate
(microcrystalline cellulose) or  Whatman paper, confirming the
possibility of using paper-based SPCEs  directly as substrate.
Therefore, this electrochemical method is found to be  suitable to
identify electron donors for LPMOs. This is a proof-of-concept 
highlighting that electrochemical electron donor detection can be used
for  fast monitoring of LPMO oxygenase activity using natural
substrates, i.e.,  without the use of chromophores or fluorophores.
Moreover, the possibility  of acquiring a large amount of data using
IPA together with 96 SPEs open the  way to electrochemical
high-throughput screening assays for \mbox{LPMOs.}

\begin{figure*}
\includegraphics{fig06}
\caption{\label{fig6}(A) Scheme of the biomimetic SPCE configuration
and preparation: First a wax circle is printed on filter paper
(diameter ${=}$ 6 mm); secondly, on the front side, carbon and Ag|AgCl
inks are  screenprinted. Then, lignin (blended with cellulose) is
deposited on the back side of the paper electrode. (B) Detection of
ligninase activity is made possible by recovering the signal
corresponding to a redox probe by cyclic voltammetry after action of
the enzyme (green line) (adapted from~\cite{58}).}
\end{figure*}

\begin{figure*}
\includegraphics{fig07}
\caption{\label{fig7}SEM analysis showing (A) cellulose fibers at the
back side of bare paper-based electrodes, (B)~biomimetic
substrate-electrodes impregnated with lignin showing nanoparticles
coating the cellulose fibers, (C) biomimetic substrate-electrodes
impregnated with lignin after incubation with catalase--peroxidase,
showing degraded cellulose fibers (adapted from~\cite{58}).}
\vspace*{3pt}
\end{figure*}

\section{Paper-based SPCE as a biomimetic substrate for the
detection of lignin depolymerizing enzymes} 
Lignin, a hydrophobic and
amorphous heteropolymer, mainly composed of phenyl  propanoid units, is
one of the three polymers (with cellulose and  hemicellulose) composing
the secondary plant cell wall and the  lignocellulosic biomass. As
lignin constitutes up to 40{\%} of the plant  cell wall, it is
considered to be the most abundant biobased source of  aromatic
compounds such as vanillin, syringaldehyde, acetovanillone, ferulic 
acid or vinyl guaiacol~\cite{4,54}.

The conversion of lignin into smaller aromatic molecules of interest is
challenging owing to its structural heterogeneity. Enzymatic 
depolymerization of lignin is a promising approach~\cite{55}. However,
the  characterization of new ligninolytic enzymes is challenging due to
the lack  of relevant methods. Electrochemical measurements are ideal
for measuring  redox reactions. This non-optical method can measure
different substances in  a few minutes, meaning that electrochemical
methods may be useful to measure  ligninolytic processes more easily.
In \mbox{recent} years, paper-based electrodes  have emerged as tools in the
field of bioelectrochemistry. This is due to a  number of factors,
including their ease of use, biodegradability, and  straightforward
fabrication~\cite{17,56,57}. In a recent work, we described the 
development of a new methodology using paper-based SPCEs to
characterize the  activities of ligninolytic
enzymes~\cite{58}.

The strategy relies on the preparation of an integrated
substrate/detection  system composed of a ``biomimetic'' plant cell
wall on the back side of a  paper-based SPCE (Figure~\ref{fig6}A). The
polymeric substrate is formed by  cellulosic material from Whatman
paper and by lignin coated around the  \mbox{cellulose} microfibrils after
ionic liquid-assisted precipitation.  Characterization of the
substrate/electrode by profilometry and electronic  microscopy showed
that lignin is homogeneously deposited as nanoparticles  around the
cellulosic fibers (Figure~\ref{fig7}A,~B). These nanoparticles are
not  only deposited at the surface but deeply penetrate into the paper,
mimicking  polymer organization in the plant cell wall where lignin
acts as a matrix  engulfing cellulose and hemicellulose.
Electrochemical characterization of  the substrate/electrode
highlighted that, when deposited in sufficient  amount, the coated
lignin constitutes an electrical insulator, preventing  access to the
electrode by soluble compounds (e.g., a redox probe).  Depolymerization
of this biomimetic plant cell wall by the ligninolytic  enzyme
catalase--peroxidase from \textit{Thermobacillus xylanilyticus} 
(\textit{Tx}CP) is evidenced by the recovery of the 
$\mathrm{Fe(CN)}_{{6}}^{3^-/4^-}$ redox signal by cyclic voltammetry 
(Figure~\ref{fig6}B). Detection of the probe is correlated with
physical modification  of the fibers as observed by SEM. Indeed,
aggregates coating the fibers are  no more observed and fibers are
strongly degraded after incubation with  \textit{Tx}CP
(Figure~\ref{fig7}C), \mbox{indicating} that the insulating barrier formed by
the coated  lignin is weakened by the enzyme-catalyzed reaction.

These results highlight the interest of developing paper-based SPCEs to
detect ligninolytic activity and could be used for rapid screening of 
efficient ligninolytic enzymes. This method could be further improved
for  the electrochemical detection of products during oxidative
depolymerization  of lignin. Such a strategy could help solve the
technological challenge of  rapidly identifying robust biocatalysts
with ligninolytic activities.

\section{Conclusion}
The discovery of new enzymes active on plant wall polymers is a major 
challenge for the development of biochemical routes for the
valorization of  lignocellulosic biomass and co-products from
agricultural activities.  Electrochemical methods involving
screen-printed carbon electrodes have  shown their interest in
detecting, characterizing and quantifying small  compounds, and
consequently in screening for enzymatic activities associated  with
these compounds. For example, the detection of small aromatic 
compounds, such as hydroxycinnamic and hydroxyphenolic acids,
potentially  derived from lignin depolymerization, enables the
detection of peroxidase or  lytic polysaccharide monooxygenase
activities by cyclic voltammetry or  intermittent pulsed \mbox{amperometry}.
Modifying or functionalizing the electrodes  could improve the
detection of compounds of interest, thus refining the  determination of
the enzymatic activities. The use of paper as support for 
screen-printed carbon electrodes adds another dimension for screening,
in  particular for cellulose-active enzymes such as lytic
polysaccharide  monooxygenases. In this configuration, a certain
proximity between the  enzyme, its cellulosic substrate and the
electrode used for detection was  achieved. This proximity can help to
overcome substrate accessibility issues  encountered in heterogeneous
media. Paper-based electrodes allow exploiting  the three-dimensional
structure of paper to design biomimetic substrates of  the plant wall,
involving several types of interlocking polymers. Finally,  the
possibility of custom-designing the shape of the electrodes, and in 
particular of miniaturizing the systems, notably to work in
96-electrode  format, opens the way to a wealth of potentialities in
the context of  high-throughput screening of enzymatic activity and
reaction conditions.

\section*{Declaration of interests}

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

\section*{Acknowledgements}
The authors gratefully thank Dr. Varn\'{a}i, Dr. Forsberg and Pr.
Einjsink  from the Norwegian university of life sciences for the
supplying of LPMO.

\back{}

\printbibliography
\refinput{crchim20240961-reference.tex}

\end{document}
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