Nanoscale
PAPER
Ci e his: Nanoscale, 2023, 15, 14399
Recei ed 2nd May 2023,
Accep ed 29 h July 2023
DOI: 10.1039/d3n 02026
sc.li/nanoscale
Pla inum-based nanodend i es as glucose
oxidase-mimicking su oga es†
Jose I. Ga cia-Pei o,‡
a,b,c,d
Ja ie Bone -Ale a, ‡
a,b,c,d
Ma ia L. Tamayo-F aile,
a,b
Jose L. Hueso *
a,b,c,d
and Jesus San ama ia*
a,b,c,d
Ca aly ic con e sion o glucose ep esen s an in e es ing field o esea ch wi h mul iple applica ions. F om
he bio echnology poin o iew, glucose con e sion leads o he ab ica ion o diffe en added- alue by-
p oduc s. In he field o nanoca aly ic medicine, he educ ion o glucose le els wi hin he umo mic o-
en i onmen (TME) ep esen s an appealing app oach based on he s a a ion o cance cells. Glucose
ypically achie es high con e sion a es wi h he aid o glucose oxidase (GOx) enzymes o by e men a-
ion. GOx is subjec ed o deg ada ion, possesses poo ecyclabili y and ope a es unde e y specific eac-
ion condi ions. Gold-based ma e ials ha e been ypically explo ed as ino ganic ca aly ic al e na i es o
GOx in o de o con e glucose in o building block chemicals o in e es . S ill, he lack o sufficien
selec i i y owa ds ce ain p oduc s such as gluconolac one, he equi emen o high fluxes o oxygen o
he c i ical size dependency hinde hei ull po en ial, especially in liquid phase eac ions. The p esen
wo k desc ibes he syn hesis o pla inum-based nanodend i es as no el enzyme-mimicking ino ganic
su oga es able o con e glucose in o gluconolac one wi h ou s anding selec i i y alues abo e 85%. We
ha e also s udied he enzyma ic beha io o hese P -based nanozymes using he Michaelis–Men en and
Linewea e –Bu k models and used he main calcula ion app oaches a ailable in he li e a u e o de e -
mine highly compe i i e glucose u no e a es o P o P –Au nanodend i es.
In oduc ion
The alo iza ion o glucose, ca bohyd a es and biomass in o
chemical building blocks o in e es ep esen s one o he
mos cu en ly explo ed g een s a egies in bio echnology. Gas
phase eac ions (oxida ion, educ ion, e o ming) a mild o
mode a ed eac ion empe a u es lead o he gene a ion o
complex oxidized by-p oduc s and g een hyd ogen ene gy
ec o s. Liquid phase eac ions also lead o mul iple added-
alue p oduc s in he o m o gluconic acid, hyd ogen pe -
oxide, glucona es o complex es e s.
1
Na u al enzymes such as
glucose oxidase (GOx) may yield a highly selec i e con e sion
o glucose in o hyd ogen pe oxide and gluconolac one. Fi s ,
he glucose molecule binds o he ac i e si e o he enzyme,
which con ains a la in adenine dinucleo ide (FAD) co ac o .
This binding causes a con o ma ional change in he ac i e
si e, which p omo es he ans e o elec ons om he glucose
molecule o he FAD co ac o and o ms FADH
2
. Glucose oxi-
da ion leads o an aldehyde g oup a he C1 posi ion, o ming
glucono-1,5-lac one o gluconolac one. Then, he elec ons a e
ans e ed o a bound molecula oxygen molecule, which
causes i o be educed o H
2
O
2
and he FADH
2
is oxidized
back o FAD (Fig. 1a). Finally, he H
2
O
2
p oduced in he
second s ep is eleased om he enzyme and he gluconolac-
one p oduc is usually u he hyd olyzed o gluconic acid.
2,3
The main d awbacks o na u al enzymes such as GOx a e
ela ed o hei ins abili y and cos , hei difficul e-
usabili y and hei highly speci ic ope a ion window con-
di ions ha hinde hei po en ial applica ion in biomedical
applica ions.
4–8
In ecen yea s, nanozymes ha e eme ged as a diffe en
kind o nanoma e ial wi h a ca aly ic esponse mimicking he
ac i i y o enzymes.
9–13
Mo eo e , nanozymes ha e been classi-
ied in o wo diffe en ypes. Type 1 nanozymes we e mo e
dominan in he i s decade o his cen u y and e e o
immobilized ca alys s in nanoma e ials. They we e in ensely
s udied by academic esea che s such as Ro ello and co-
wo ke s,
14
showing po en ial applica ions in biomedicine and
†Elec onic supplemen a y in o ma ion (ESI) a ailable. See DOI: h ps://doi.o g/
10.1039/d3n 02026
‡These au ho s con ibu ed equally o his wo k.
a
Ins i u o de Nanociencia y Ma e iales de A agon (INMA); CSIC-Uni e sidad de
Za agoza, Campus Rio Eb o, Edi icio I+D, C/Poe a Ma iano Esquillo , s/n, 50018,
Za agoza, Spain. E-mail: jlhueso@uniza .es, jesus.san ama ia@uniza .es
b
Depa men o Chemical and En i onmen al Enginee ing; Uni e si y o Za agoza,
Spain, Campus Rio Eb o, C/Ma ía de Luna, 3, 50018 Za agoza, Spain
c
Ne wo king Resea ch Cen e in Bioma e ials, Bioenginee ing and Nanomedicine
(CIBER-BBN), Ins i u o de Salud Ca los III, 28029 Mad id, Spain
d
Ins i u o de In es igación Sani a ia (IIS) de A agón, A enida San Juan Bosco, 13,
50009 Za agoza, Spain
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in acellula ca alysis.
15
Type 2 nanozymes a e based on in-
o ganic nanoma e ials wi h su ace ca aly ic p ope ies and
was he dominan ype in he las decade.
4
The e m nanozyme e e s o e y diffe en ca aly ic en i ies:
om molecula ca alys s en apped in polyme ic scaffolds o
en i e nanopa icles. This wide ange o possibili ies makes i
challenging o es ablish a s anda dized me hodology o quan-
i y and eliably compa e he ca aly ic pe o mance among
diffe en nanozymes.
5,7,16
Al hough i is gene ally accep ed
ha a nanozyme implies he en i e nanopa icle, a nanozyme
may ha e mo e han one ac i e si e on he su ace. In he li -
e a u e, diffe en no maliza ion en i ies such as he numbe o
su ace si es, he numbe o pa icles, o al mass, o o al
me al a om concen a ion ha e been used o de ine he nano-
zyme uni and o calcula e o compa e he u no e a e
(k
ca
).
17
The lack o a clea de ini ion o a nanozyme uni can
be misleading when a emp ing o p ope ly e alua e he diffe -
ences in ac i i y ha may a y o e 10
6
- old o he same
sys em.
7,16–19
These ou ways o de ining a nanozyme uni can
all be use ul depending on he applica ion scena ios and all o
hem should be p o ided in o de o acili a e compa ison o
u he s udies in he li e a u e.
17
Mos o he s udies ha deal wi h his issue use pe oxidase-
like nanozymes as examples o ca aly ic compa ison.
Howe e , much less a en ion has been paid o GOx-mimick-
ing eac ions.
20,21
GOx nanozymes offe g ea po en ial in
indus ial p ocesses, including he ood and be e age,
pha maceu ical, and biosensing sec o s. Since he ea ly 2000s,
Au NPs ha e been ex ensi ely e alua ed as ino ganic enzyme
mimics o he speci ic pu pose o ca alyzing he oxida ion o
glucose o ende gluconic acid and hyd ogen pe oxide
(Fig. 1b).
22
Ini ially, Pd
23
and P
24–26
nanopa icles we e also
e alua ed as ca alys s in he ca aly ic oxida ion o glucose wi h
O
2
. Howe e , he bu s o Au sys ems bu ied he po en ial o P
and Pd un il ecen ly, whe e a se ies o ecen ad ances ha e
made hem a ac i e again, especially o po en ial cance
Fig. 1 Compa ison o glucose oxidase mechanisms: (a) schema ic illus a ion o glucose oxida ion ca alyzed by he glucose oxidase (GOx) enzyme.
The enzyma ic eac ion in ol es he FADH co ac o whe e glucose in e ac s wi h he ac i e si e o o m δ-gluconolac one and ans e s he elec-
ons o FAD o o m FADH. O
2
is used o ans e elec ons om FADH and o m H
2
O
2
; (b) mos epo ed mechanism o glucose oxida ion in he
p esence o nanozymes such as noble me als o Mn. In his case, he eac ion in ol es molecula oxygen and o ms H
2
O
2
and gluconic acid ins ead;
(c) p oposed mechanism o P -based nanodend i es desc ibed in his wo k whe e O
2
leads o glucose oxida ion owa ds δ-gluconolac one as he
main glucose oxida ion p oduc .
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he apy applica ions.
8
Noble me als such as Pd and especially
P ha e been in he spo ligh in he las ew yea s due o hei
appealing physicochemical p ope ies, including hose wi h a
dend i ic and po ous s uc u e.
27–31
In pa icula , he ca alase-
like ac i i y o P -based nanozymes may help in alle ia ing
umo hypoxia in he umo mic oen i onmen (TME) using
o e exp essed hyd ogen pe oxide as he oxygen sou ce. This
in si u oxygen supply acili a es glucose oxida ion a low O
2
concen a ions.
8
Mo eo e , P nanozymes also hold g ea
po en ial o he apeu ic syne gy due o hei esponse o elec-
omagne ic adia ion such as NIR ligh hea ing,
32
X- ay
abso p ion
33
and elec ic ields.
8,34
A good unde s anding o GOx enzyme-mimicking su o-
ga es in compa ison wi h na u al enzymes is necessa y.
Unde s anding glucose adso p ion o oxygen ac i a ion is key
o u he op imiza ion o glucose oxida ion ca alysis.
35
In
addi ion, moni o ing he selec i i y in enzyma ic eac ions
may also help in elucida ing he in ol ed mechanism in he
nanozyme-mimicking al e na i e pa hway. Nanozymes ca alyze
eac ions by p o iding su ace si es o subs a e binding and
p omo ing he ans e o elec ons, he eby acili a ing he o -
ma ion o eac ion in e media es. I has been commonly
epo ed how eac i e oxygen species (ROS) such as
•
Oo
•
OH
in he p esence o O
2
o H
2
O
2
a e key o pe o m oxidase-like
eac ions on o ganic subs a es such as TMB o OPD.
8
Howe e , in he las ew yea s, i has also been epo ed how
he oxida ion o glucose does no solely ely on he oxida ion
abili y o O
2
.
20
Dong’s g oup s udied he mechanism o
glucose oxida ion by noble me als. They s udied he oxida ion
eac ion o glucose ha in ol es he dehyd ogena ion o he
hyd oxyl g oups on glucose o o m aldehyde g oups. In
addi ion, hey epo ed ha he eac ion migh p oceed by
abs ac ing hyd ogen om glucose o o m an M–H in e medi-
a e and u he ans e ing H o diffe en subs a es o gene-
a e sub-H adduc s. The a e-de e mining s ep in ol ed he
b eaking o he C–H bond. Mo eo e , in Au-ca alyzed oxygen
educ ion, he ene gy ba ie o b eaking he O O double
bond is highe , leading o a p edominan 2e
−
pa hway.
Howe e , o he noble me al nanopa icles unde go a simila
ca aly ic p ocess, al hough O
2
is ypically educed o wa e
ins ead by using he 4e
−
pa hway. In summa y, bo h GOx and
noble me al NPs can ca alyze elec on ans e om glucose o
o he elec on accep o s such as O
2
.
20
In his wo k, we p esen a no el ype 2 P nanozyme wi h
GOx-like ac i i y. To e alua e he ca aly ic ac i i y o he nano-
zyme, we de ined he nanozyme uni using diffe en app oxi-
ma ions epo ed in he ecen li e a u e and compa ed
hem. We also e alua ed he ac i i y o co e–shell bime allic
NPs (Au and P ) o compa e he ac i i y o diffe en , hough
analogous, sys ems. Rega dless o he me hodology o calcu-
la e he ca aly ic u no e s, we ound ha P nanodend i es
exhibi a GOx-like beha io ha can be i ed o he
Michaelis–Men en and Linewea e –Bu k models, wi h ou -
s anding selec i i y owa ds gluconolac one (Fig. 1c) and
highly compe i i e k
ca
alues in compa ison wi h he exis ing
Au coun e pa s.
Resul s and discussion
Syn hesis and cha ac e iza ion o P -based nanozymes
A empla ed polyme -assis ed me hod was employed o syn-
hesize co e–shell Au–P nanodend i es and P nanodend i es
(NDs), espec i ely, as depic ed by ansmission elec on
mic oscopy (TEMs) image (Fig. 2a and b). The p ocess began
by comple ely dissol ing Plu onic F-127, and hen mixing in
me al p ecu so s and asco bic acid (A.A.). The eac ion was
pe o med o 4 days. X- ay diff ac ion (XRD) analysis displayed
pa e ns ha co espond o he cubic s uc u e o P and/o Au
(Fig. 2c).
36,37
XPS analysis (Fig. 2d and e) o he P 4 egion
u he con i med he P - ich composi ion o he su ace o
bo h he AuP hyb id and P -NDs. Fo he AuP -NDs, he Au a %
inc eased owa ds he inne pa only a e an e ching ea men
(da a no shown) and mic owa e plasma a omic emission spec-
oscopy (MP-AES) e ealed an o e all composi ion o 43% and
57% o Au and P , espec i ely. Finally, he op ical p ope ies o
he P -NDs we e cha ac e ized using a UV- is spec opho o-
me e . B oad abso p ion bands sp eading owa ds he isible-
nea in a ed window we e iden i ied, especially when Au NPs
we e p esen in he nanoma e ials
38,39
(Fig. 2 ).
Glucose oxidase-mimicking ac i i y o P -based nanozymes:
calcula ion o K
M
and V
max
We e alua ed he glucose oxidase-like p ope ies o bo h AuP -
NDs and P -NDs. To ha e a meaning ul compa ison o he
na u al enzyme GOx, we ca ied ou he glucose oxida ion eac-
ion unde mild condi ions, i.e. pH close o 7 and oom emp-
e a u e (Fig. 3a) and analyzed he supe na an by UPLC-mass
spec oscopy (MS). Ou main goal was o quan i y how much
glucose was oxidized a ea ly eac ion imes and iden i y he
by-p oduc s gene a ed. Then, we e alua ed he ini ial eac ion
a es (V
0
,mMs
−1
)a diffe en glucose le els o build up
Michaelis–Men en and Linewea e –Bu k plo s o de e mine
wo key pa ame e s o he nanozyme: he maximum ini ial a e
(V
max
,mMs
−1
) ela ed o he maximum subs a e concen-
a ion a nanozyme can affo d, and he Michaelis–Men en con-
s an (K
M
, mM), which is a well-es ablished desc ip o o he
affini y be ween he subs a e and he nanozyme.
A e 15 minu es o eac ion be ween glucose and AuP
NDs, we could de ec he gene a ion o wo MS adduc s by MS:
[M −H]
−
= 195 and [M + Cl]
−
= 213.15, oge he wi h he
un eac ed glucose ([M + Cl]
−
= 215.15) (Fig. 3b). The de ec ed
ions sugges ed he gene a ion o (i) gluconic acid, one o he
mos epo ed by-p oduc s o glucose oxidase-like
nanozymes
40,41
and (ii) δ-gluconolac one, he esul ing
p oduc om he dehyd ogena ion eac ion o glucose. Wi h
he aid o s anda ds, we could con i m hei gene a ion unde
ou eac ion condi ions (Fig. ESI-3†). The quan i ica ion o
bo h glucose and δ-gluconolac one by UPLC-MS e ealed a as
consump ion a ea ly eac ion imes (Fig. ESI-4a and b†), bu
he δ-gluconolac one le els we e much highe han hose o
he gene a ed gluconic acid (Fig. 3c). We calcula ed a selec i-
i y owa ds δ-gluconolac one o abou 86.0%, and only 8.2%
owa ds gluconic acid. Inc easing he ini ial glucose concen-
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a ion led o a linea inc ease o V
0
un il he alue le eled off
a ound a glucose concen a ion o 60 mM. Da a we e co ec ly
adjus ed o he Michaelis–Men en plo , indica ing an enzyme-
like beha io o AuP -NDs (Fig. 3d). The Linewea e –Bu k plo
yielded a good linea i ing (R
2
> 0.99), which allowed us o
de e mine V
max
and K
M
o be 0.044 mM s
−1
and 262.10 mM,
espec i ely (Fig. 3e).
Analogous esul s we e ound o he P -NDs wi h bo h glu-
conic acid and δ-gluconolac one de ec ed a e 15 minu es o
eac ion (Fig. 3 ) wi h simila pa e ns o glucose consump ion
and gluconolac one gene a ion (Fig. 3g and Fig. ESI-4c, d†). In
his case, selec i i y owa ds δ-gluconolac one was 70.1% and
ha owa ds gluconic acid eached 13.1%. This indica ed ha
he p esence o P on wo diffe en ca alys s, AuP -NDs and P -
NDs d i e he eac ion owa ds dehyd ogena ion p e e en ially
a he han he di ec gene a ion o gluconic acid. This was
ini ially unexpec ed, since mos o he epo ed GOx-like nano-
ma e ials ypically epo gluconic acid as he main eac ion
p oduc .
40,41
We sea ched he li e a u e o p e ious s udies on glucose
oxidase-mimicking nanozymes, wi h special a en ion o he
me hods used o de ec eac ion p oduc s and he calcula ed
kine ic pa ame e s (Table 1). Mos wo ks used he as-gene a ed
H
2
O
2
using HRP o ABTS o indi ec ly con i m he glucose
oxidase-like ac i i y. In he case o de ec ion o gluconic acid,
he mos widely employed me hod was indi ec de ec ion
h ough de i a iza ion wi h NH
2
OH and subsequen complexa-
ion wi h Fe
3+
o o m a ed-colo ed p oduc , which was inally
de ec ed by UV- is spec oscopy (Fig. ESI-5a†). We pe o med a
con ol expe imen using δ-gluconolac one in he same de i a-
iza ion eac ion, which yielded an iden ical signal co es-
ponding o gluconic acid (Fig. ESI-5b†), indica ing ha his
de i a iza ion canno be eliably used o dis inguish among
he possible eac ion p oduc s. In his wo k ins ead, we p o ide
a di ec de ec ion o δ-gluconolac one as a p oduc o glucose
oxida ion when using P -based nanopla o ms. Howe e , since
he de i a iza ion does no allow o disc imina e gluconolac one
om gluconic acid, i is possible ha o he nanozymes may ha e
also yielded δ-gluconolac one bu has gone unde ec ed due o
he lack o a p o ocol such as he one epo ed in his wo k.
In e ms o enzyme-like beha io , he absence o Au in he
nanopa icle co e did no seem o ha e a de imen al effec on
he ac i i y o P -NDs (Fig. 3h), sugges ing ha he ca aly ic be-
ha io is mainly con olled by he dominan p esence o P on
he ex e nal su ace o he pa icles. O e all, he ca aly ic
ac i i y o P -NDs ou pe o med hei bime allic AuP coun e -
pa s when compa ing bo h ca alys s a hei maximum con-
e sion pe o mance. The Linewea e –Bu k plo o P -NDs
Fig. 2 Cha ac e iza ion o AuP and P nanodend i es: (a) TEM image o AuP -NDs, e ealing he solid Au co e and dend i ic P shell o he nano-
pa icles, inse : schema ic illus a ion o AuP -NDs; scale ba = 50 nm; (b) TEM image o P -NDs wi h a dend i ic shape, inse : schema ic illus a ion
o P -NDs; scale ba = 100 nm; (c) X- ay diff ac ion pa e ns o AuP -NDs and P -NDs and hei compa ison wi h hose o P , Au and P ; (d) X- ay
pho oemission spec a o he P 4 egion, e ealing he P
0
and P
2+
species in AuP NDs; he inse shows he limi ed p esence o Au in he Au 4
egion; (e) X- ay pho oemission spec a o P
0
in P NDs, ( ) UV- is spec a o AuP and P -NDs. We obse e a con inuous dec easing abso p ion
band ela ed o P NPs. AuP -NDs also possess an in ense abso p ion peak a 650 nm ela ed o he plasmonic p ope ies o he sphe ical Au co e.
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(R
2
= 0.99) ga e alues o he enzyma ic pa ame e s K
M
and
V
max
as 249.11 mM and 0.093 mM s
−1
, espec i ely (Fig. 3i).
This means ha he V
max
alues ob ained o Pd-NDs we e
oughly doubled in compa ison wi h hose ob ained o hei
Pd–Au coun e pa s. In his ega d, i is in e es ing o conside
he eac ion mechanism p oposed by Chen e al.
20
whe e a
i s H a om is ans e ed om glucose o he noble me al-
based nanopa icle su ace, hen o an accep o molecule. In
ou case, molecula O
2
dissol ed in solu ion may ac as an
elec on sink as we could moni o i s consump ion du ing he
eac ion o bo h nanozymes (Fig. ESI-6†). Unde his scena io,
he P su ace will abs ac an H om glucose o o m
δ-gluconolac one, and hen will ans e i o a dissol ed O
2
molecule. The p esence o Au in he co e o he pa icles could
po en ially limi he a e o his la e H ans e since he
elec onega i i y o Au is highe han ha o P , hus ac ing as
an elec on sink and making he H ans e mo e ene ge ically
un a o able; u he s udies and de ailed modeling (mainly
DFT) will be needed o elucida e his end.
Finally, when a emp ing o compa e he ob ained V
max
and
K
M
alues wi h he exis ing li e a u e, we ound la ge diffe -
ences in he case o P -NDs (Table 1). Howe e , i mus be
no ed ha V
max
alues a e dependen on he amoun o he
nanozyme employed in each expe imen . The e o e, o p o ide
a mo e accu a e compa ison, we p oceeded o no malize V
max
by he numbe o ac i e cen e s employed, using k
ca
.
Calcula ion o k
ca
assuming he ND concen a ion de e mined
by diffe en me hods
k
ca
is a key pa ame e used o compa e he ca aly ic efficien-
cies o diffe en enzymes o nanozymes. I ep esen s he
maximum numbe o subs a e molecules ha can be con-
e ed o p oduc s by each ac i e si e o he enzyme/nanozyme
pe uni o ime, unde sa u a ing subs a e concen a ion con-
di ions. k
ca
was calcula ed by di iding V
max
, ob ained om he
Linewea e –Bu k plo , by he concen a ion o ac i e enzyme
si es ([E]) o a gi en enzyme-ca alyzed eac ion (eqn (1)).
Fig. 3 Glucose oxidase-mimicking esponse o AuP and P nanodend i es: (a) schema ic illus a ion o he glucose oxida ion eac ion wi h P -
based NDs. Oxygen is used as an elec on accep o , leading o δ-gluconolac one as he main eac ion p oduc ; (b) ca aly ic eac ion o AuP -NDs
moni o ed by MS analysis a e 0 and 15 minu es o eac ion. [Glu-Cl] adduc wi h an m/z= 215.0, [δ-glu-Cl] adduc wi h an m/z= 214.0, [G.A.-H]
wi h an m/z= 195.0; (c) e olu ion o glucose and δ-gluconolac one wi h he eac ion ime o AuP -NDs; (d) Michaelis–Men en cu e o he AuP -
NDs o GOx-like ac i i y; (e) Linewea e –Bu k plo ob ained om he glucose oxida ion o AuP -NDs used o de e mine enzyma ic cons an s (V
max
,
K
M
); ( ) ca aly ic eac ion o P -NDs moni o ed by MS analysis a e 0 and 15 minu es o eac ion. [Glu-Cl] adduc wi h an m/z= 215.0, [δ-glu-Cl]
adduc wi h an m/z= 214.0, [G.A.-H] wi h an m/z= 195.0; (g) e olu ion o glucose and δ-gluconolac one wi h he eac ion ime o P -NDs; (h)
Michaelis–Men en cu e o he P -NDs o GOx-like ac i i y; (i) Linewea e –Bu k plo ob ained om he glucose oxida ion o P -NDs used o de e -
mine enzyma ic cons an s (V
max
,K
M
). Resul s a e exp essed as he a e age ± s anda d e o measu emen (S.E.M. –n= 2).
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Calcula ion o k
ca
o a gi en eac ion ca alyzed by an
enzyme/nanozyme:
kca ¼Vmax
½Eð1Þ
Al hough he de e mina ion o V
max
using he Linewea e –
Bu k plo is well es ablished in he nanozyme- ela ed li e a-
u e, assigning a alue o he concen a ion o ac i e enzyme
si es is he subjec o conside able con o e sy, bu canno be
a oided since i is necessa y o de e mine p ope ly he alue o
k
ca
. In a ecen e iew published by Zandieh e al.
16
in 2021,
diffe en app oaches o calcula e he nanozyme concen a ion
we e p esen ed, hey a e schema ized in Fig. 4.
The cen al issue is o cou se es ablishing a clea de ini ion
o ac i e si es. I is also necessa y o ind a consensus in he
way [E] is calcula ed, since diffe en assump ions can yield
diffe ences in ac i i y alues up o 8–10 o de s o
magni ude.
7,16
Success in such s anda diza ion effo s would
Table 1 Compila ion o epo ed V
max
and K
M
alues om glucose oxida ion epo ed in he li e a u e, oge he wi h he me hodology employed o
de ec eac ion by-p oduc s
Nanozyme V
max
(M s
−1
)K
M
(mM) De ec ion me hodology Re .
AuNPs 6.3 × 10
−7
7.0 G.A.: i a ion wi h NaOH 41
H
2
O
2
: colo ime ic eac ion (HRP and ABTS)
AuNP-SBA15 5.3 × 10
−7
26.2 G.A.: complex wi h Fe
3+
40
H
2
O
2
: colo ime ic eac ion (TMB and low pH)
Au-MIP 2–6×10
−5
0.18 O
2
decay measu emen using an oxime e 42
Au-MIP-PFOB 3.6 × 10
−5
0.11 H
2
O
2
: colo ime ic eac ion (HRP and ABTS)
Au-APBA 2.9 × 10
−6
0.25 G.A.: complex wi h Fe
3+
AuNPs 5.2 × 10
−7
0.42
AuNPs 8.7 × 10
−9
0.42 H
2
O
2
: colo ime ic eac ion (HRP and ABTS) 43
AuP 1.8 × 10
−8
0.33
AuP -MIP 4.6 × 10
−7
0.11
Au-Fe-MPSN 2.5 × 10
−5
0.16 G.A.: complex wi h Fe
3+
and i a ion wi h NaOH 44
H
2
O
2
: colo ime ic eac ion (TMB, low pH and Fe
3
O
4
)
Au-MCM41 1.8 × 10
−5
55 G.A.: complex wi h Fe
3+
45
H
2
O
2
: colo ime ic eac ion (HRP and ABTS)
UCNPs_MOF_Au 1.3 × 10
−6
44.3 H
2
O
2
: assay ki (Abcam) 46
G.A.: pH alues
AuNPs 5.7 × 10
−7
24.6 H
2
O
2
: colo ime ic eac ion (HRP and DPD) 47
HMSN_Au 4.0 × 10
−7
50.7 G.A.: complex wi h Fe
3+
48
H
2
O
2
: colo ime ic eac ion (HRP and TMB)
BiogenAuNPs 1.3 × 10
−7
0.089 H
2
O
2
: colo ime ic eac ion (TMB and low pH) 49
AuP -Silk 1.3 × 10
−7
0.25 Glucose: colo ime ic eac ion (DNS eagen ) 50
Au-Hyd ogel 8.0 × 10
−7
4.98 H
2
O
2
: colo ime ic eac ion (HRP and TMB) 51
G.A.: colo ime ic pH indica o (me hyl ed)
AuPd-Ae ogel 13 × 10
−8
0.19 G.A.: complex wi h Fe
3+
and a pH me e 52
H
2
O
2
: colo ime ic eac ion (HRP and TMB)
AuP -NDs 4.5 × 10
−5
262.1 UPLC-MS
P -NDs 9.3 × 10
−5
249.1 O
2
: oxime e
Fig. 4 Diffe en app oaches o define he nanozyme ac i e uni s o calcula e k
ca
: (a) o al numbe o P a oms used as a e e ence. Each P a om is
conside ed a specific ac i e si e independen ly o whe he i is on he su ace o P -NDs o in inne laye s; (b) o al numbe o P -NDs in solu ion
used as a e e ence. Each P -ND is conside ed a specific ac i e si e independen ly o he numbe o ac i e cen e s on i s su ace; (c) o al numbe o
su ace P a oms used as a e e ence. Each su ace P a om is conside ed a specific ac i e si e independen ly o i s ac i i y o he eac ion wi h
glucose.
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be in aluable, p o iding a p oduc i e way o link nanozymes
wo k wi h na u al enzymes and he e ogeneous ca alysis. In
his case we ha e de e mined [E] employed in he ca aly ic
glucose oxida ion ollowing he diffe en assump ions p o-
posed by Zandieh e al.
16
The mos s aigh o wa d me hodology o calcula e [E] con-
sis s o conside ing he o al ca alys mass pe uni olume
(mg L
−1
) o ob ain k
ca
. Howe e , his app oach can lead o
huge diffe ences among nanopa icles wi h no subs an ial
a ia ions in he size o shape. Mo eo e , his me hod can lead
o a po en ial o e es ima ion o [E] since i conside s e e y
a om o he nanopa icles as ac i e, e en hose in he inne
laye s o he s uc u e ha p obably emain una ailable o
he ca aly ic eac ion. In ou nanosys ems, his me hod-
ology would be equi alen o conside he o al mass o P
in bo h AuP -NDs and P -NDs assuming P as he ac i e
elemen in he glucose oxida ion eac ion. Ob iously, he
u no e alues ob ained by his me hod a e conside ably
lowe compa ed o hose o na u al enzymes and also
makes i ha d o compa e u no e a es among diffe en
nanozymes ound in he li e a u e, since ac o s such as he
shape o aspec a io ha e a s ong in luence on he ac ion
o exposed me al a oms. To ollow his app oach, he o al
P amoun o AuP -NDs and P -NDs was de e mined using
MP-AES, and he calcula ed [P ] alue o AuP -NDs and P -
NDs was 0.292 and 0.512 mM, espec i ely (see also Table 2,
ide in a).
Ano he common app oach is o conside he whole nano-
pa icles as a nanozyme uni (Fig. 4). This me hod is equi-
alen o assuming ha each pa icle p o ides only one ac i e
si e and he e o e leads o an o e es ima ion o he ca aly ic
ac i i y o nanozymes wi h esul s conside ably highe han
hose o na u al enzymes. The numbe o nanopa icles pe
uni olume was de e mined by NTA o AuP -NDs and P -NDs.
Fo a gi en concen a ion o 0.1 mg mL
−1
, he nanopa icle
concen a ion o AuP -NDs and P -NDs was 5.33 × 10
10
and
1.31 × 10
10
pa icles pe mL o 8.80 × 10
−8
and 2.17 × 10
−8
i
exp essed in mM, espec i ely (see Table 2).
The hi d me hod no malizes ac i i y by he numbe o
su ace ac i e si es (Fig. 4). This seems he mos easonable
app oach as i would no conside in e nal a oms ha a e gen-
e ally no a ailable o he ca aly ic p ocess bu accoun s o all
he su ace a oms po en ially in con ac wi h he subs a e.
This me hodology would also allow o compa e be ween nano-
zymes wi h diffe en mo phologies and shapes by aking in o
accoun he exposed a oms. Depending on he nanopa icle
s uc u e, he numbe o su ace ac i e si es can be de e mined
by wo me hods: (i) calcula ion o he su ace pe pa icle
using he nanopa icle adii, de e mined by di ec measu e-
men using TEM o NTA
16
o (ii) measu emen o he o al
su ace pe g am o ma e ial using BET (see he ESI† o
de ails and Fig. 4). We belie e he i s me hod can only be
applied o non-po ous and sphe ical nanopa icles as i could
o he wise unde es ima e he numbe o ac i e si es exposed o
he eac ion. We calcula ed [E] wi h e e y me hod o p o ide
an app op ia e compa ison o he ob ained esul s (see he
ESI†and Fig. 4). The a e age adius de e mined by TEM o
AuP -NDs and P -NDs was 27 and 40 nm (Fig. 1) and yielded a
concen a ion o P o ca. 1.08 × 10
−2
and 6.7 × 10
−3
mM,
espec i ely. Howe e , NTA adii inc ease up o 44 and 96 nm,
espec i ely, yielding [P ] = 2.88 × 10
−2
mM o AuP -NDs and
3.50 × 10
−3
mM o P -NDs, espec i ely (no malized by he
numbe o pa icles, see Fig. ESI-1†). NTA employs a lase o
i adia e pho ons on he pa icles and measu es he sca e ed
ligh , which can be ela ed o he pa icle size. Using pa icles
wi h high ex inc ion coefficien in he ange o he employed
lase , as noble me al-based nanos uc u es like AuP -NDs o
P -NDs, can a i icially dec ease he ligh ha inally eaches
he de ec o and affec he inal ou come.
53
In addi ion, NTA
measu es he hyd odynamic size o agg ega es bu hese agg e-
ga es a e no dense s uc u es, bu an agglome a ion o indi-
idual pa icles, whose su aces a e s ill a leas pa ly a ail-
able. Fo hese easons we belie e ha TEM is mo e app op i-
a e, especially when low-po osi y noble-me al nanopa icles
a e conside ed.
Finally, calcula ions o su ace a oms om he BET da a
can be mo e app op ia e when using po ous o su ace i egu-
la ma e ials as hey p o ide a di ec measu emen o he
nanopa icle su ace. In con as , TEM o NTA analysis is mo e
p one o e o when acing nanos uc u es wi h ough su aces
o a high polydispe si y index. The ma gin o unce ain y will
depend on how much he eal pa icle su ace de ia es om
an ideal sphe ical su ace. On he o he hand, BET analysis
equi es a highe amoun o sample, in con as o he low
quan i y necessa y o TEM. Calcula ed [P ] si es o AuP and
P using BET we e 4.73 × 10
−2
and 5.93 × 10
−3
mM, espec i ely
(see Table 2 o compa ison and he ESI† o de ailed calcu-
la ions). We belie e ha his me hod is mo e app op ia e o
Table 2 Concen a ion o su ace P si es and he co esponding k
ca
alues calcula ed using he pa icle size de e mined by TEM and NTA o he
di ec su ace a ea pe g am o ma e ial measu ed by BET
Calcula ion me hod
AuP -NDs P -NDs
[P ] (mM) k
ca
(s
−1
) [P ] (mM) k
ca
(s
−1
)
To al P mass 0.292 0.15 0.512 0.18
To al numbe o NPs 8.80 × 10
−8
5.1 × 10
5
2.17 × 10
−8
4.3 × 10
6
To al numbe o su ace P a oms (TEM) 1.08 × 10
−2
4.2 6.70 × 10
−3
13.8
To al numbe o su ace P a oms (NTA) 2.88 × 10
−2
1.6 3.50 × 10
−2
2.7
To al numbe o su ace P a oms (BET) 4.73 × 10
−2
0.94 5.93 × 10
−3
15.6
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ou P -NDs and AuP -NDs since N
2
adso p ion gi es a mo e
eliable es ima ion o he numbe o su ace a oms o ex u ed
su aces, such as he dend i es p epa ed in his wo k.
Ano he app oach p oposed by Zandieh e al.
16
e ines he
calcula ions by disc imina ing he non-ac i e su ace a oms.
This is especially impo an in mixed-elemen nanopa icles
such as Fe
3
O
4
, whe e oxygen su ace a oms do no pa icipa e
in ca alysis as ac i e si es. Howe e , his me hod will no be
conside ed in his wo k as we assume bo h pa icles only
con ain P on hei ou e su ace laye s. The inal [P ] and k
ca
(calcula ed by eqn (1)) alues de e mined by he desc ibed
me hod a e summa ized in Table 2 ( ide in a).
Fo he sake o compa ison, we ha e summa ized ep esen-
a i e alues o k
ca
o Au-based nanozymes ound in he li -
e a u e in Table 3. The i s example o Au as a GOx-like
ma e ial epo ed by Como i e al.
54
ound a k
ca
alue o 13.9
s
−1
, calcula ed conside ing [E] as he o al numbe o Au
a oms. Howe e , in hei wo k he pH o he eac ion was ixed
o 9.5. This inc eases he eac ion a e, since alkaline pH
alues a o he eac ion, acco ding o he mechanism
epo ed ecen ly by Chen e al.
20
The k
ca
alues calcula ed o
ou AuP -NDs ollowing he same me hodology a e compa able
(0.94–16.2 s
−1
) e en hough hey we e ob ained a pH = 6.5,
which is likely affec ed by lowe kine ics, gi en he impo ance
o
–
OH ions in he eac ion.
20
Fo P -NDs, he k
ca
alues no -
malized pe P su ace a om a e la ge , indica ing an e en
be e pe o mance (Table 2). Howe e , in o he cases, a mean-
ing ul compa ison o ou esul s is challenging. As explained
abo e, es ima ing [E] as he o al numbe o NPs gi es ise o
ex emely high k
ca
alues, which a e p obably o e es ima ed.
This is o example he case o Luo e al.,
41
who epo ed a k
ca
alue o 18.52 s
−1
assuming each AuNP as one enzyme uni .
In summa y, he P -NDs p epa ed in his wo k p esen high
k
ca
alues ha can be compa ed o he bes alues epo ed o
nanozymes in he li e a u e. Howe e , a ealis ic compa ison
among wo ks ca ied ou unde diffe en condi ions is highly
challenging. I seems necessa y o each a consensus no only
ega ding he way [E] is calcula ed, bu also unde he con-
di ions (e.g. pH) o glucose oxida ion expe imen s and e en on
he echniques employed o quan i y he eac ion p oduc s.
Expe imen al
Chemicals and ma e ials
Pla inum(IV) chlo ide H
2
P Cl
6
acid solu ion, gold(III) chlo ide
hyd a e (50% Au basis), L-asco bic acid (99%), Plu onic F-127
(F-127), glucose (98%), and PBS pelle s we e pu chased om
Sigma-Ald ich (Da ms ad , Ge many) and used wi hou u he
pu i ica ion.
Syn hesis o P nanozymes
P NPs we e syn he ized ollowing a no el p o ocol. In a ypical
syn hesis, 60 mg o Plu onic F-127 we e comple ely dissol ed
in 2 mL o dis illed wa e . Then, H
2
P Cl
6
(1 mL, 100 mM) was
added o he p e ious solu ion o a o al olume o 4 mL. The
mix u e was ul asonica ed o 1 min. Then, L-asco bic acid
(2 mL, 0.25 M) was added o he eac ion mix u e. The mix u e
was allowed o se le a oom empe a u e o 3 days wi hou
u he s i ing o a o he g ow h o P dend i es. The solu-
ion colo u ned om yellow o in ensely black, indica ing he
educ ion o he P
4+
p ecu so o P
0
. The inal p oduc was
hen pu i ied by cen i uga ion (7500 pm o 7 min, wo
cycles, oom empe a u e), and inally esuspended in 1 mL o
mili-Q H
2
O. Au@P NPs we e syn he ized mixing H
2
P Cl
6
(0.5 mL, 100 mM) and HAuCl
3
(0.5 mL, 100 mM) and allowed
o se le a oom empe a u e o 1 day wi hou u he s i ing.
The syn hesis o hese ma e ials has been pe o med a he
Pla o m o P oduc ion o Bioma e ials and Nanopa icles o
he NANBIOSIS ICTS, mo e speci ically by he Nanopa icle
Syn hesis Uni o he CIBER in BioEnginee ing, Bioma e ials
and Nanomedicine (CIBER-BBN, Mad id, Spain).
Cha ac e iza ion echniques
T ansmission elec on mic oscopy (TEM) was pe o med using
a FEI TECNAI T20 mic oscope (Tecnai, Eindho en, The
Ne he lands) ope a ed a 200 keV. Samples we e p epa ed by
d op-cas ing 3–5 µL o he NP suspension on o a holey ca bon
TEM g id. The speci ic su ace a ea was calcula ed based on
he B unaue –Emme –Telle (BET) me hod. NTA analysis
(Nanosigh NS200, Mal e n Panaly ical) was used o de e mine
he diame e and concen a ion (exp essed in pa icles pe
mL) o P NZ. The amoun o P in he samples was quan i ied
by using a quad upole ICP mass spec ome e (4100 MP-AES,
Agilen Technologies, USA). Pa icle size dis ibu ion was
measu ed wi h ImageJ and using TEM images. X- ay diff ac-
ion pa e ns we e ob ained on an Empy ean ins umen
(Mal e n-PANaly ical, Mal e n, UK) in B agg–B en ano con-
igu a ion using CuKα adia ion and equipped wi h a PIXcel1D
de ec o . The su ace composi ion o he samples was analyzed
by X- ay pho oelec on spec oscopy (XPS) wi h an Axis Ul a
DLD (K a os Tech.). Spec a we e exci ed wi h a monoch o-
ma ic Al Kαsou ce (1486.6 eV) ope a ing a 12 kV and 10 mA
and a s ep ene gy o 20 eV was used. The binding ene gies
we e e e enced o he in e nal C 1s s anda d (284.3 eV). Peak
analyses we e pe o med wi h CasaXPS so wa e using a
weigh ed sum o Lo en zian and Gaussian componen cu es
a e he Shi ley backg ound sub ac ion.
Table 3 Rep esen a i e examples o k
ca
alues ound in he li e a u e
o glucose oxidase-mimicking nanozymes
Nanozyme k
ca
(s
−1
) Re .
Na u al GOx 9.71 16
AuNPs 13.9 54
AuNPs 18.52 41
Au-MIP 34.2 42
Au-MIP-PFOB 47.41
Au-APBA 3.749
AuNPs 0.6858
AuNPs 0.686 43
AuP 1.597
AuP -MIP 36.381
Au-MCM41 14.2 45
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Glucose oxidase-mimicking eac ions
P NPs we e used in o de o mimic GOx. The glucose oxi-
da ion eac ion was exposed o ai and moni o ed o e ime
using UPLC-MS. B ie ly, 2 mL o he eac ion mix u e wi h a
a iable amoun o glucose was s i ed in a ial a oom emp-
e a u e a pH = 6.5 (CH
3
COO
−
/CH
3
COOH 0.05 M).
Subsequen ly, 20 µL o he nanozyme s ock solu ion was
added o he eac ion mix u e and he solu ion was incuba ed
o 5, 10 and 15 minu es. Then, 20 µL we e sampled om he
eac ion mix u e o he acquisi ion o samples and mixed
wi h 980 µL o a H
2
O : ACN mix u e. The dilu ed solu ion was
il e ed and analyzed by UPLC-MS. Ch oma og aphic sepa -
a ion was pe o med using a BEH AMIDE® UPLC column a
85 °C and 0.5 mL min
−1
low consis ing o a s a ing compo-
si ion o 90% ace oni ile and 10% milli Q wa e . Then, wa e
concen a ion inc eased o 3 min un il 65% ACN was eached.
To modi y he mobile phase, 0.1% / o 10 mM ammonium
chlo ide in ammonium solu ion was added o bo h sol en s.
Cl
−
ions in he sol en solu ion in e ac ed wi h glucose and
δ-gluconolac one o o m an [M −Cl] –adduc , which was
de ec ed using a coupled ACQUITY QDa mass de ec o (MS).
In he case o gluconic acid, he de ec ed ion was a m/z= 195
[M −H]
−
.
Conclusions
AuP and P nanodend i es syn hesized wi h Plu onic
F-127 ha e demons a ed a s ong GOx-mimicking esponse.
These ca alys s displayed a high selec i i y owa ds gluconolac-
one as he main by-p oduc and we e able o ope a e unde
close o neu al pH condi ions. In his wo k, we ha e also
add essed an impo an issue ha is s ill unde discussion:
he lack o consensus ega ding he way o calcula e ca aly ic
u no e s in enzyme-mimicking sys ems, a ques ion ha boils
down o he e y de ini ion o ac i e si es o hese en i ies.
Finally, we also ad oca e di ec ch oma og aphy analysis as he
mos accu a e de ec ion me hod o epo ue eac ion selec-
i i ies, a oiding po en ially misleading con usions be ween
gluconolac one and gluconic acid due o he lack o disc imi-
na ion o colo ime ic p obes o en used o indi ec analyses.
All hese ques ions need o be ully add essed in o de o
es ablish a clea compa ison be ween diffe en nanozyme
sys ems. Finally, we ha e been able o es ablish he excellen
pe o mance o P -based nanosys ems as GOx su oga es able
o p o ide glucose con e sion wi h high selec i i y o glucono-
lac one, while a he same ime p o iding ca alase-like ac i i y,
an ex emely use ul ea u e in oxygen-dep i ed, H
2
O
2
- ich
en i onmen s.
Au ho con ibu ions
Concep ualiza ion: J. I. G. P., J. B., M. T., J. S. and J. L. H.; da a
cu a ion: J. I. G. P., M. T., J. B. and J. L. H.; o mal analysis:
J. I. G. P., J. B. and J. L. H.; me hodology: J. I. G. P., J. B., and
M. T.; alida ion: J. I. G. P., J. B. and J. L. H.; in es iga ion:
J. I. G. P., J. B., M. T., J. S. and J. L. H.; unding acquisi ion:
J. S.; supe ision: J. L. H. and J. S.; w i ing –o iginal d a :
J. B., J. I. G. P. and J. L. H.; w i ing – e iew and edi ing: J. B.,
J. I. G. P., J. S. and J. L. H.
Conflic s o in e es
The e a e no con lic s o decla e.
Acknowledgemen s
Financial suppo om he Eu opean Resea ch Council
(ERC-Ad anced G an CADENCE numbe 742684) and he
Spanish Resea ch Agency (LAERTES- PID2020-114926RB-I00) is
acknowledged. The TEM measu emen s we e conduc ed a he
Labo a o io de Mic oscopias A anzadas, ICTS ELECMI, Spain.
The syn hesis o ma e ials has been pe o med by he Pla o m
o P oduc ion o Bioma e ials and Nanopa icles o he
NANBIOSIS ICTS, mo e speci ically by he Nanopa icle
Syn hesis Uni o he CIBER in BioEnginee ing, Bioma e ials
and Nanomedicine (CIBER-BBN). J. I. G.-P. and J. B-A. acknowl-
edge he A agon Regional Go e nmen and he Spanish
Go e nmen , espec i ely, o hei p edoc o al con ac s.
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