Ul asonics Sonochemis y 88 (2022) 106096
A ailable online 18 July 2022
1350-4177/© 2022 The Au ho s. Published by Else ie B.V. This is an open access a icle unde he CC BY license (h p://c ea i ecommons.o g/licenses/by/4.0/).
Lysozyme c ys alliza ion in hyd ogel media unde ul asound i adia ion
Ma iia Sa chenko
a
,
c
,
d
, Manuel Hu ado
b
,
e
,
,
g
, Modes o T. Lopez-Lopez
c
,
g
, Guille mo Rus
e
,
,
g
,
Luis ´
Al a ez de Cien uegos
a
,
g
,
*
, Juan Melcho
b
,
,
g
,
*
, Jos´
e A. Ga i a
d
,
*
a
Uni e sidad de G anada (UGR), Depa amen o de Química O g´
anica, Unidad de Excelencia Química Aplicada a Biomedicina y Medioambien e (UEQ), C. U.
Fuen enue a, A da. Se e o Ochoa s/n, E-18071 G anada, Spain
b
Uni e sidad de G anada (UGR), Depa amen o de Es adís ica e In es igaci´
on Ope a i a, Spain
c
Uni e sidad de G anada (UGR), Depa amen o de Física Aplicada, C. U. Fuen enue a, A da. Se e o Ochoa s/n, E-18071 G anada, Spain
d
Labo a o io de Es udios C is alog ´
a icos, Ins i u o Andaluz de Ciencias de la Tie a (Consejo Supe io de In es igaciones Cien í icas-UGR), UEQ, A enida de las
Palme as 4, 18100 A milla, G anada, Spain
e
Depa amen o de Mec´
anica de Es uc u as e Ingenie ía Hid ´
aulica, Ul asonics Lab TEP-959, Uni e sidad de G anada, Spain
Unidad de Excelencia Modeling Na u e MNAT, Uni e sidad de G anada, Spain
g
Ins i u o de In es igaci´
on Biosani a ia Ibs, GRANADA, G anada, Spain
ARTICLE INFO
Keywo ds:
P o ein c ys alliza ion
Nuclea ion
Ul asound
Hyd ogels
Lysozyme
ABSTRACT
Sonoc ys alliza ion implies he applica ion o ul asound adia ion o con ol he nuclea ion and c ys al g ow h
depending on he ac ua ion ime and in ensi y. I s applica ion allows o induce nuclea ion a lowe supe sa u-
a ions han equi ed unde s anda d condi ions. Al hough ex ended in ino ganic and o ganic c ys alliza ion, i
has been sca cely explo ed in p o ein c ys alliza ion. Now, ha indus ial p o ein c ys alliza ion is gaining
momen um, he in e es on new ways o con ol p o ein nuclea ion and c ys al g ow h is ad ancing. In his wo k
we p esen he de elopmen o a no el ul asound bio eac o o s udy i s in luence on p o ein c ys alliza ion in
aga ose gel. Gel media minimize con en ion cu en s and sedimen a ion, a o ing a mo e homogeneous and
s able condi ions o s udy he e ec o an ex e nally gene a ed low ene gy ul asonic i adia ion on p o ein
c ys alliza ion a oiding o he undesi ed e ec s such as empe a u e inc ease, in oduc ion o su aces which
induce nuclea ion, des uc i e ca i a ion phenomena, e c. In-dep h s a is ical analysis o he esul s has shown
ha he impac o ul asound in gel media on c ys al size popula ions a e s a is ically signi ican and
ep oducible.
1. In oduc ion
P o ein c ys alliza ion is an essen ial ool o deciphe he h ee-
dimensional s uc u e o a p o ein in i s c ys alline s a e by means o
X- ay di ac ion, as well as he ca aly ic cen e o an enzyme, allowing
us o unde s and i s mechanism o ac ion [1]. The e o e, p o ein c ys-
alliza ion is needed, among o he s, in ields o s uc u al biology and
d ug de elopmen and disco e y. Mo eo e , p o ein c ys alliza ion can
also be use ul in p o ein pu i ica ion and isola ion being used in bio-
pha maceu ical indus ies ou inely [2,3]. Ne e heless, due o he high
molecula weigh s and dynamic na u e o p o eins in solu ion; p o ein
c ys alliza ion is a complex p ocess ha equi es high supe sa u a ion
a ios and usually p oceeds by slow nuclea ion and c ys al g ow h ki-
ne ics [4,5]. In his ega d, i is undamen al o de elop no el echniques
o p o ocols ha allow a mo e e icien and eliable c ys alliza ion
p ocess. P o ein c ys alliza ion in gel media has u ned ou o be an
excellen s a egy o ob ain p o ein c ys als o high quali y and size,
ideal o X- ay di ac ion, mainly due o he educ ion o con ec ion
cu en s, a oiding c ys als sedimen a ion and empe a u e o concen-
a ion g adien s [6]. A such, c ys alliza ion in gels is simila o c ys-
alliza ion unde educed g a i y condi ion p oducing c ys als o
excellen quali y [7]. Mo eo e , gel ibe s ge inco po a ed wi hin he
c ys als ans o ming hem in o new composi e ma e ials [8,9].
P o ein c ys alliza ion is also highly a ec ed by ex e nal s imuli,
such as elec ic [10–14] and magne ic ields [15–18], ligh [19,20],
audible sounds [21], mic owa e [22] and ul asound (US) i adia ion
[23–26], ha ing a di ec in luence on nuclea ion [14]. Among hese
s a egies, in he las decade, sonoc ys alliza ion, ha is, he applica ion
o US in c ys alliza ion p ocess, is ecei ing a g owing in e es since i
has shown o: inc ease nuclea ion a e, educe he induc ion ime and
* Co esponding au ho s a : Ins i u o de In es igaci´
on Biosani a ia Ibs, GRANADA, G anada, Spain (L.´
A. Cien uegos and J. Melcho ).
E-mail add esses: [email p o ec ed] (L. ´
Al a ez de Cien uegos), [email p o ec ed] (J. Melcho ), [email p o ec ed] (J.A. Ga i a).
Con en s lis s a ailable a ScienceDi ec
Ul asonics Sonochemis y
jou nal homepage: www.else ie .com/loca e/ul son
h ps://doi.o g/10.1016/j.ul sonch.2022.106096
Recei ed 29 Ap il 2022; Recei ed in e ised o m 4 July 2022; Accep ed 14 July 2022
Ul asonics Sonochemis y 88 (2022) 106096
2
he me as able zone wid h, inc eased c ys al g ow h a e, educed
agglome a ion, and imp o e c ys al quali y and size dis ibu ion
[27–30]. The e ec o ul asounds on he kine ic o c ys alliza ion has
been known om mo e han 80 yea s wi h an ea ly e iew al eady
published in 1967 and in which he mul iple possible mechanisms i.e.
ca i a ion, agi a ion, cooling e ec o mechanical ib a ion we e hy-
po hesized [31]. Besides an ex ended numbe o i s e ec s on he
nuclea ion and g ow h o ino ganic/o ganic c ys als and e en a [32],
only ecen ly he e ec o e p o ein c ys allogenesis has been epo ed
[25,26]. Ne e heless, he applica ion o US in p o ein c ys alliza ion
has no been sys ema ically s udied and sonica ion ac o s such as,
sou ce, powe , ime, di ec ion and ampli ude o he ul asound wa es,
a e s ill o be in es iga ed. Mo eo e , any s udy ca ies ou unde
s anda d c ys alliza ion se -ups will be a ec ed by sedimen a ion and
con ec ion mass anspo hinde ing he in e p e a ion o he esul s.
Only ecen ly, Fe ei a and co-wo ke s ha e explo ed he e ec o US
pulse o e lysozyme mic od ople s [25]. In o de o ex end he po en ial
use o sonoc ys alliza ion o bigge olumes and o explo e he easi-
bili y o his echnique in combina ion wi h o he c ys alliza ion p o-
ocols ele an o indus ial c ys alliza ion, we ha e chosen o include
aga ose o minimize con ec ion and c ys al sedimen a ion.
Aga ose is one o he mos used media o gel a c ys allizing solu ion
o ino ganic and o ganic compounds [33], coo dina ion polyme s [34]
o p o eins [35], o p oduce new polymo phs [36] o o emula e in i o
media in biomine aliza ion s udies [37]. Aga ose gels a e composed o
in e connec ed uncha ged linea polysaccha ide chains easily ob ained
by cooling he sol below i s gelling empe a u e and classi ied as phys-
ical gel since he in e ac ions be ween polysaccha ide chains a e non-
co alen . I uses is widely sp ead in biochemis y labs as suppo ing
ma e ial o ho izon al elec opho esis. The physical p ope ies o he
gel ha e been well cha ac e ized and i is well known ha below he
c i ical gel concen a ion o 0.12 % (w/ ) aga ose solu ions beha e like
non-New onian luids, while abo e his concen a ion i beha es as a
egula iscoelas ic gel [38]. Mo eo e , a a concen a ion as low as
0.04 % (w/ ) aga ose gels a e able o o e come buoyancy and c ys al
sedimen a ion [39]. Also, aga ose gels ha e been p e iously used o
s udy he in luence o a magne ic ield while a oiding sedimen a ion and
he associa e e ec s o con ec i e mass anspo on nuclea ion [18,40].
In he p esen wo k, we ha e s udied he in luence o US in p o ein
c ys alliza ion a di e en aga ose concen a ion. To ca y ou his s udy
a no el US bio eac o has been speci ically designed o ha e a mo e
p ecise con ol o e he wa e pa ame e s o i adia ion. In-dep h
mul i a ia e s a is ical analysis ha e shown ha , as al eady published,
US induced he nuclea ion o lysozyme o e he induc o e ec o
aga ose [41,42]. Ou esul s also shown ha abo e a h eshold con-
cen a ion o aga ose he US e ec is de lec ed p obably due o he
damping e ec o aga ose ibe s. The esul s ob ained by his se -up a e
s a is ically signi ican allowing us o s ablished a clea e ec o he
ul asound on he nuclea ion o lysozyme unde s udied condi ions.
2. Ma e ials and me hods
2.1. Reagen s and ma e ials
Lysozyme (62971, HEWL, h ee- imes c ys allized powde ) and so-
dium ace a e (AcONa 99 %) we e pu chased om Sigma-Ald ich
(Mad id, Spain). Lysozyme was dissol ed in 50 mM AcONa, dialyzed
(24 h) agains 50 mM AcONa (pH 4.5) in a a io 1:1000 a 4 ◦C and
concen a ed by cen i uga ion a 4 ◦C (g =*5000/25 min) using 10-kDa
cu o Cen icon concen a o s (Amicon) o ≈150 mg mL
−1
de e mined
spec opho ome ically a 280 nm using a heo e ical alue o he
ex inc ion coe icien o 2.56 mL mg
−1
. Then he solu ion was il e ed
h ough a 0.45
μ
m po e-size il e memb ane sys em (Millipo e). Sodium
chlo ide (Sigma-Ald ich, Mad id, Spain) was p epa ed a 20 % (w/ ) in
50 mM AcONa (pH 4.5) and used as s ock solu ion. Solu ions o NaCl a
desi ed concen a ion we e p epa ed by dilu ing wi h 50 mM AcONa
and il e ed h ough a 0.45
μ
m po e-size il e memb ane sys em (Mil-
lipo e) p io using i .
Aga ose D5 wi h a mel ing poin o 92 ◦C and gelling poin o 37 ◦C
was supplied by Hispanaga (Mad id, Spain). Aga ose sols wi h desi able
concen a ion we e ob ained by dissol ing aga ose in 50 mM AcONa (pH
4.5) and hea ed a 90 ◦C o ge a homogeneous anspa en solu ion.
Then he solu ion was cooled down o 50 ◦C and kep a his empe a u e
un il inally mixed wi h he p o ein and p ecipi an solu ion.
2.2. C ys alliza ion expe imen s
Ba ch me hod was selec ed o s udy he in luence o ul asonic wa es
on lysozyme c ys alliza ion in solu ion (Fig. 1.A). Fo he expe imen s in
solu ion (Fig. 1.A1), lysozyme, NaCl and AcONa we e mixed oge he in
Fig. 1. The expe imen al scheme: A) P epa a ion o he samples A1: Expe imen s in solu ion, A2: Expe imen s in gels made wi h aga ose; B) US-Bio eac o design
and soni ica ion p ocedu e and illus a ion o he se -up; C) Obse a ion o he samples and da a analysis.
M. Sa chenko e al.
Ul asonics Sonochemis y 88 (2022) 106096
3
one Eppendo ube, homogenized and di ided in h ee aliquo s o 100
µL using mic o spec opho ome e isible-cu e es (B and, GMBH, CO-
KG, Ge many) and kep a 20 ◦C.
To selec he app op ia e condi ions, we sc eened lysozyme (25–50
mg mL
−1
) and NaCl (3.5–5 % w/ ). We ha e selec ed he inal con-
cen a ion o lysozyme 40 mg mL
−1
and NaCl 4.3 % (w/ ) o he ex-
pe imen s in un-gelled solu ion and 40 mg mL
−1
and NaCl 4.0 % (w/ )
o he expe imen in aga ose gels. The e olu ion o he expe imen s was
ollowed by s anda d op ical mic oscopy (Nikon AZ100 zoom 2x2x0.6)
obse ing he o ma ion o iny c ys als in all he samples (un-gelled
solu ion as well as in samples wi h aga ose gels) a e 1 h.
The in luence o aga ose (Fig. 1A2) was s udied by a ying he inal
amoun o aga ose in he sys em: 0.010 %, 0.025 %, 0.050 %, 0.100 %
and 0.200 % (w/ ). We se he uppe limi a 0.200 % (w/ ) o c oss he
c i ical concen a ion alue o 0.120 % w/ de ining he ansi ion o he
iscoelas ic beha io [43]. Fo each expe imen h ee aliquo s we e
p epa ed and di ided as “silen ” o he sample wi hou ul asonic in-
luence, “P o ocol US1” o he aliquo immedia ely i adia ed o 30
min long and “P o ocol US2” o he aliquo exposed o he ul asonic
in luence 30 min a e p epa a ion and i adia ed o 30 min (Fig. 1B).
All he expe imen s we e pe o med h ee old o s a is ical signi icance.
Numbe and size o c ys als we e e alua ed a e 24 h by op ical
mic oscopy using he Image-Focus-Alpha so wa e o he Nikon AZ100
mic oscope (zoom 2x2x0.6). C ys als numbe we e coun ed manually
and size measu ed along he c axes om c ys als clea ly iden i ied. Each
image was di ided in 25 equal egions (5 columns ×5 ows) a oiding
zones nea cu e es bo de s whe e i was no possible o see he c ys als
clea ly, and all he c ys als we e coun ed in all he egions. C ys als size
we e analyzed by measu ing a minimum o 100 c ys als o each epli-
ca e hus, a minimum o 300 measu emen s we e analyzed o each
condi ion. Taking in o accoun all he con ols and di e en expe i-
men al condi ions a o al o 5400 measu emen s we e done.
2.3. US-Bio eac o design
To a oid a di ec con ac o he US emi e wi h he gel/c ys alliza-
ion media ha could cause gel dis up ion o se e as he e ogeneous
nuclean , a US-bio eac o was designed, manu ac u ed and p o o yped
in he Ul asonics lab a he Uni e si y o G anada. I consis s o a
con aine o hold he cu e e, a polyme hylme hac yla e (PMMA)
chambe ha was chosen due o i s mechanical and low-densi y p op-
e ies and ul asonic ansduce s (Fig. 2). In pa allel, he p opaga ion o
he wa es was allowed and maximized wi hou loss o ampli ude
because o impedance be ween mediums.
The suppo o he ansduce designed in Fig. 2C has he unc ion o
holding he ansduce s, o be s able and pe pendicula o he block wi h
a speci ic geome y showed in Fig. 2A-B. The decision was ocused a
allowing he whole wa e incidence in o he chambe o he samples
whe e he cu e e is in eg a ed, o ensu e maximum i adia ion. A
p essu e on he block is also equi ed o ha e a di ec ansmission
a oiding possible ai bubbles. Mo eo e , he bo om o he cu e e mus
coincide wi h he cen al axis o he ansduce o achie e op imum
in ensi y due o he maximiza ion o he esolu ion o beam in he
cen al axis. The p opaga ion on o he mechanical ul asonic wa es is
ansmi ed pe pendicula ly o he su ace o he ansduce in con ac
wi h he con aine .
The ela ionship be ween wa e p opaga ion, acous ic impedance o
he ma e ials, nea ield, he possible empe a u e ise o he ansduce s
and mechanical na u e o he sample ha e been also conside ed o he
design o he con aine and con igu a ion o he ul asonic ansduce s.
The impedance is cha ac e ized as a p oduc o densi y and eloci y o
sound o he ma e ial as ollows:
Fig. 2. A and B) Shows he la e al and op 1–2 ans e sal sec ion iews, espec i ely. The chambe Type 1 was designed o hold he cu e e and i is loca ed a 3.5
mm in on o he block. The dimensions o his chambe had 15 mm wid h, 12 mm o e ec i e high and 91.4 mm o hickness (app oxima ed olume o 16.4 cm
3
).
The chambe s Type 2 and Type 3 we e in oduced o hold se e al cu e es in he u u e conside ing di e en p essu es changing he main con igu a ion. C) Exhibi s
he 3D p in ed suppo o main ain he ul asonic ansduce (100 KHz) in a co ec posi ion o ensu e he alignmen o he p opaga ing wa es. D) 3D schema ic ull
con igu a ion o he US-Bio eac o . E) Pic u e o he US-Bio eac o p epa ed o he expe imen al i adia ion o gels.
Table 1
Cha ac e iza ion o PMMA, Wa e and PS in e ms o impedance o quan i y he
e ec o he ul asonic p opaga ion in he sample.
Ma e ial (
ρ
) Densi y Kg/
m
3
(c) Veloci y (m/
s)
(Z) Impedance (Pa.s/
m)
PMMA 1180 2765 3,263,183
Wa e (27◦) 993 1525 1,515,584
Polys y ene
(PS)
612 2340 1,432,080
M. Sa chenko e al.
Ul asonics Sonochemis y 88 (2022) 106096
4
Z=
ρ
•c(1)
whe e (Z) is he impedance, (
ρ
) is he densi y and (c) is he eloci y o
he wa e h ough ma e ial.
The mechanical pa ame e s o he US-bio eac o ma e ials we e
cha ac e ized o analyze he p opaga ion h ough he sample, he im-
pedances o PMMA, wa e and he cu e e-polys y ene (PS) we e also
conside ed as is shown in Table 1 acco ding o Equa ion (1) and (2).
Table 1 desc ibes densi ies, eloci ies o sound and impedances o hese
ma e ials.
The ansmission coe icien a he di e en in e aces is also
necessa y o de e mine he p essu e in he sample a e p opaga ion
h ough he di e en laye s. I is explici ly o mula ed as:
D=4Z1Z2
[Z1+Z2]2(2)
whe e (D) is he ansmission coe icien , (Z
1
) is he impedance o ma-
e ial 1 and (Z
2
) is he impedance o ma e ial 2. The calcula ed alues
a e summa ized in Table 2.
T ansmi ed p essu e has been also de i ed o he ansi ion be-
ween wo media acco ding o equa ion (2) (Fig. 2B). While, he wa e is
oscilla ing a 100 kHz o equency om he ul asonic ansduce , i
a els h ough he PMMA, a e he wa e and inally he polys y ene
un il ge in con ac wi h he gel o solu ion sample. Fo his calcula ion,
we ha e app oxima ed he densi y and he eloci y o sound o he gel o
solu ion equal o wa e , because hey ha e almos he same alue. Thus,
acous ic p essu e in wa e has been quan i ied as 618 Pa, conside ed
well below he ca i a ion limi a his equency, and heo e ical de i-
a ion o acous ic p essu e inside he gel o solu ion has been ex ac ed
acco ding i s ansmission coe icien D,
F om wa e o polys y ene → P =618 Pa ⋅ 0,99 =611,82 Pa
F om polys y ene o gel o solu ion → P =611,82 Pa ⋅ 0,99 =605,7
Pa
Se e al epo s in he li e a u e show ha he esponse o US is
igge ed by empe a u e ac ua ion [44,45]. The e o e, o e i y his
poin , empe a u e is also measu ed in wo in e als, a 5 min and a 15
min. The empe a u e ac ua ion is conside ed as , so i is no necessa y
o measu e i o e a longe pe iod o ime. We ha e used a classical
he mome e o con ol his e ec , whose accu acy is 0.01 ◦C. Fo each
case, in 100 kHz o equency, he measu emen s on nea es chambe s o
ul asonic ansduce a e lowe han <0.01 ◦C, i.e., he hea ing e ec is
negligible wi h ou con igu a ion.
O he wise, he samples we e placed in he cu e e (Fig. 2D) o
enhance he wa e p opaga ion. So ha he chosen dis ance was
app oxima ely 8 cm om he ansduce s o a oid he nea ield a ea (in
his egion he sound p essu e le els a y conside ably in e ms andom
posi ions o ene gy and i is di icul o con ol he sound p essu e ho-
mogenei y), he h eshold dis ance is calcula ed acco ding o Equa ion
(3) as ollows,
N=D2
4c(3)
whe e (N) is he nea ield, (D) is he diame e , ( ) is he equency and
(c) is he eloci y o sound o he inciden he wa e ha cha ac e ize he
media o p opaga ion (Table 3).
In his esea ch, aseline was used as coupling gel due o he du a ion
p ope ies o his se o expe imen s, he common coupling gels a e d ied
in a ew hou s. Addi ionally, con en ional ubbe s bands ha e been
added o he US-bio eac o suppo o ix he ansduce o ensu e an
op imal p essu e o con ac and no displacemen du ing he i adia ion
expe imen s.
2.4. Expe imen al US con igu a ion
The ul asonic se -up o he expe imen was designed o analyze he
di e ences be ween he e ec on c ys alliza ion o he solu ion and
aga ose gels a di e en concen a ions (Fig. 1). In consequence o he
limi a ion o he wa e gene a o o 10 V, an ampli ie has been needed o
each 180 peak- o-peak V as op imum scale le el o gene a ed he
desi ed e ec . The wa e o m used was con igu ed wi h a 5 % du y cycle
and 50 ms o bu s pe iod simula ing a con inuous p opaga ion o he
wa e. The ampli ied wa e signal was emi ed a 100 kHz o cen al
equency acco ding o a compa ible wa eleng h wi h he sample di-
mensions [23].
Fo his design equi emen s, con ac ansduce s ha e been selec ed
due o hei p ope ies as non- uned de ices. They p o ide a damped
b oadband ha minimizes he undesi ed noise. This ype o ansduce s
a e usually adequa e in Non-Des uc i e Tes ing (NDT) applica ions and
Table 2
T ansmission coe icien s be ween wa e , PMMA and PS.
D PMMA Wa e /gel PS
PMMA 1 0,86 –
Wa e /gel 0,86 1 0,992
PS – 0,9992 1
Table 3
Nea ield calcula ion in e ms o equency, eloci y and diame e o he
ansduce .
( ) F equency
(kHz)
(D) Diame e
(cm)
(c) Veloci y (m/
s)
(N) Nea ield
(cm)
100 4,5 1525 3,32
Fig. 3. 3D-CAD illus a ion o he p o o yped o US-Bio eac o wi h a ec ea ion o mul ilaye media p opaga ion h ough aseline, PMMA, wa e , polys y ene and
gel sample.
M. Sa chenko e al.
Ul asonics Sonochemis y 88 (2022) 106096
5
biomedical enginee ing [46]. Then, he eason o choose piezo-elec ic
ansduce s is ha hey a e made o a single PZT ce amic elemen .
They ypically gene a e a longi udinal monoch oma ic wa e in con ac
wi h he sample, he e o e, hey mee he speci ica ions o gene a e a
monoch oma ic wa e o be p opaga ed in mul ilaye media (Fig. 3).
2.5. Rheological cha ac e iza ion o hyd ogels
Fo he heological cha ac e iza ion we used a Bohlin CS10
con olled s ess heome e , p o ided wi h a measu ing geome y o
concen ic cylinde s wi h g oo ed su aces.
(a) Kine ics o gela ion
We measu ed he gela ion kine ics o he aga ose solu ions used o
he c ys alliza ion du ing 1 h ( ha includes he ime applied o bo h
P o ocol US1 and P o ocol US2) by subjec ing hem o oscilla o y s ain
o 1 Hz o equency and 1 Pa o he s ess. The measu emen s s a ed 10
s a e he mix u e o he componen s ( he delay esul ed om he ime
equi ed o lowe he inne cylinde o he measu ing posi ion and o
s a he measu emen ). Th ee di e en samples we e measu ed o
ensu e s a is ical signi icance o he esul s. The mean alues and s an-
da d de ia ions o each magni ude we e p o ided in his wo k.
(b) Mechanical p ope ies
We cha ac e ized he mechanical p ope ies o 0.100 % and 0.200 %
(w/ ) aga ose gels be o e and a e he i adia ion o US (using bo h
P o ocol US1 and US2). Fo his aim, we ob ained he s o age (G’) and
loss (G’’) moduli o he gels as unc ions o equency ( om 0.1 o 10 Hz)
a a cons an s ess o 1 Pa. Th ee di e en samples we e measu ed o
ensu e s a is ical signi icance o he esul s. The mean alues and s an-
da d de ia ions o each magni ude we e p o ided in his wo k.
2.6. S a is ical analysis
To explo e an in e ence abou he di e ences be ween he samples
wi h and wi hou US a wo le els o exposi ion, a mul iple eg ession
analysis ia ANOVA was conside ed. The i s s ep was o check he
p oo o no mali y ia he Kolmogo o -Smi no and Shapi o-Wilk es s.
They we e calcula ed o de e mine he no mali y o he size and numbe
o obse ed c ys als, espec i ely. In he cases whe e he dis ibu ion o
he a iable was non-no mal, K uskal-Wallis es was pe o med o
ob ain he simul aneous mean di e ences by expe imen and g oup o
ea men o le el (Con ol, US1 and US2). Fu he mo e, hey ha e been
co obo a ed wi h Dunn’s es o assess he p- alue signi icance in a-
g oup. Fo he pa ame ic dis ibu ed a iables, we ha e applied clas-
sical one-way ANOVA s a is ical me hodology o desc ibe he di e -
ences o means by g oup o ea men .
The p- alues ha e been compa ed wi h he signi icance le el o
e alua e he null hypo hesis whe e he e we e no di e ences be ween
means o when he null hypo hesis indica es ha he popula ion means
a e all equal. A signi icance le el ype I e o o 0,05 was conside ed o
be he minimum accep ed le el ha deno es a di e ence be ween
means. The no a ion ha we ha e included he ea e is * p <0,05, ** p
<0,001 and *** p <0,0001 when he di e ences be ween means a e
s a is ically signi ican . Fo he eco d he indi idual analysis o each
expe imen in numbe and c ys als size a e included in he supplemen-
a y ma e ial Figu es S1 and S2.
3. Resul s and discussion
Nuclea ion is a s ochas ic phenomenon ha equi es o o e come an
ene gy ba ie . This ene gy ba ie can be educed by inc easing he
supe sa u a ion bu , on he o he hand, an excess o supe sa u a ion
educes he abili y o con ol he nuclea ion ime and densi y [4]. The e
a e di e en ways o o e come he longes induc ion ime imposed by
he hickness o he me as able zone. To slim down he me as able zone,
nuclea ion has been p omo ed by using di e en su aces ([47] and
e e ences he e in), gels [41][42], and ex e nal ields such as ligh
i adia ion, elec ic and magne ic ield o US ([14] and e e ences he e
in), all o hem p omo ing he nuclea ion and he e o e educing he
nuclea ion induc ion ime and inc easing he numbe o c ys als [14].
The ul asonic ac i a ion o me as able solu ions o induce
Fig. 4. Row A) Shows lysozyme c ys als ob ained in solu ion unde silen condi ion (Con ol) and ul asonic i adia ion o 30 min immedia ely a e p epa a ion o
he expe imen (US1) and 30 min a e p epa a ion (US2). The s a is ical analysis o he numbe (B) and size (C) o he c ys als a e shown o he h ee eplica es. The
scale ba in he op ical mic oscopy images is 500 µm in all he pic u es.
M. Sa chenko e al.

Ul asonics Sonochemis y 88 (2022) 106096
6
nuclea ion is he s anda d applica ion o sononuclea ion echnique o
con ol c ys al size dis ibu ion, mo phology o polymo ph selec ion
[48,49]. In o de o s udy he in luence o US a a ixed supe sa u a ion
wi h an unce ainly me as able zone, he applica ion o US a di e en
imes, a e he se ing up o he c ys alliza ion expe imen s, seems o be
he simple way.
We p oposed a se -up in which US is gene a ed ex e nally o he bulk
c ys alliza ion solu ion and which includes a hyd ogel media o a oid
c ys als sedimen a ion and con ec ion so ha he US e ec could be un-
couple om any mass anspo e ec s. To es he e iciency o ou se -
up we ca ied ou a i s se o expe imen s in an un-gelled solu ion. We
es ed a ange o supe sa u a ion alues by changing he lysozyme
concen a ion, om 25 o 50 mg⋅mL
−1
, NaCl concen a ion om 3.5 %
o 5.0 % (w/ ) and he i adia ion ime se a 10 s, 10 min and 30 min.
F om his ini ial sc eening we de e mined ha using 40 mg⋅mL
−1
o
lysozyme and 4.3 % (w/ ) o NaCl he nuclea ion induc ion ime mo ed
in he ange o he ini ial 60 min and he e o e i ed ou expe imen al
equi emen s. We also de e mined ha sho i adia ion pe iods, 10 s o
10 min, a e p epa a ion did no in luence he nuclea ion beha io and
he e o e a minimum o 30 min o i adia ion was equi ed which could
be loca ed wi hin he 60 min o induc ion ime, i.e. a he beginning
(P o ocol US1) and 30 min a e he p epa a ion (P o ocol US2). Numbe
and size o c ys als we e e alua ed a e 24 h by op ical mic oscopy
using he Image-Focus-Alpha so wa e o he Nikon AZ100 mic oscope
(zoom 2×2×0.6).
As expec ed om p e ious esul s unde simila condi ions [26], he
i adia ed sample in solu ion showed an inc ease o he numbe o
c ys als, o smalle sizes, independen ly o he used p o ocol (Fig. 4 and
Figu e S3). Wi hou i adia ion, 50 % o measu ed c ys als we e
comp ised be ween 150 and 200 µm while nea 70 % o he immedia ely
i adia ed c ys als (US1) showed sizes anging 50–100 µm and when
i adia ed 30 min a e sample p epa a ion (US2) almos 90 % o he
c ys als showed sizes anging 50–100 µm. No ably his no el bio eac o
seems o be ideal o exe a con ol o e he nuclea ion p ocess. Mo e-
o e , he highe con ol o e he c ys al size obse ed in p o ocol US2
could be explained as ollowing: i) since US2 is applied la e , c ys alline
ma e ial is al eady o med; ii) he applica ion o he US o e his al eady
o med nuclei may o could disagg ega e he c ys alline ma e ial and iii)
he disagg ega ed agmen s ha e now he oppo uni y o g ow gi ing
ise o a na owe c ys al size dis ibu ion.
To a oid c ys al sedimen a ion, aga ose, a di e en concen a ions,
was used as a media. Aga ose is a well-known nuclea ion induc ion
media ully cha ac e ized unde egula condi ion bu no unde he
in luence o US. The e o e, i s ly, we ully cha ac e ized he in luence o
ou selec ed p o ocol o e he gel/gela ion beha io o aga ose.
In o de o in es iga e how he ul asound may a ec he kine ic o
gel o ma ion and he mechanical p ope ies o he gel we s ick o he
concen a ion ange in which aga ose ansi om non-New onian luid
(0.100 % w/ ) o a egula gel (0.200 % w/ ) [38].
As expec ed, a aga ose concen a ion below 0.100 %, no gel
beha io was ob ained i.e. s o age modulus (G) being always smalle
han loss modulus (G′′), e en a e moni o ing o 1 h. Fo 0.100 % and
0.200 % (w/ ) aga ose concen a ion G′was highe han G′′ om he
e y beginning o he measu emen s. G′>G′′ is ypical o gel-like
samples and he e o e, i can be concluded ha gel poin was eached
e y quickly a e he solu ions we e p epa ed. Ne e heless, as
Fig. 5. Gela ion kine ic o 0.100 % (A) and 0.200 % (B) solu ions o aga ose. Da ke lines ep esen he mean alues, whe eas he ligh e bands a ound hem
ep esen he s anda d de ia ions. The bo om pa showing he iscoelas ic moduli as a unc ion o equency a a cons an s ess o 1 Pa o 0.100 % (C) and 0.200 %
(D) aga ose gels be o e and a e applying he p o ocol US1. Da ke lines ep esen he mean alues, whe eas he ligh e bands a ound hem ep esen he s anda d
de ia ions. No e ha he nonsymme ic appea ance o s anda d de ia ions is due o he loga i hmic scale.
M. Sa chenko e al.
Ul asonics Sonochemis y 88 (2022) 106096
7
obse ed in Fig. 5A-B, G′inc eased o e ime, which indica es ha
gela ion p oceeded a e he gel poin was eached, and no s eady s a e
was eached a e 3500 s o 0.100 %. Fo 0.200 % he ini ial
enhancemen o G′was e y ab up , bu a e 2500 s a end owa ds a
s eady s a e was obse ed indica ing he gel o ma ion. Fig. 5C-D, shows
he iscoelas ic moduli as a unc ion o equency o a cons an shea
s ess o 1 Pa a e he gela ion was comple ed. These cu es seem o
co obo a e ha bo h samples con aining 0.100 % and 0.200 % o
aga ose demons a ed a gel-like beha io , howe e , alues o G′′/G′a e
in he ange 0.1–1, mos ly o sample 0.100 %, ypical o weak gels [50].
Al hough a i s i looks like he e is some di e ences on he iscoelas ic
moduli be ween i adia ed and non-i adia ed samples, he s anda d
de ia ion clea ly o e laps indica ing ha he e a e no signi ican
di e ences.
Also no e ha in he double loga i hmic scale, G′and G′′ a e weakly
dependen on equency, which is ypical o gels. We also in es iga ed i
he applica ion o US2 could a ec he gel beha io . As i is illus a ed in
he supplemen ma e ial (Figu e S4) we did no obse e any e ec .
The e o e, om ou esul s i could be concluded ha bo h samples
a e gels, wi h sample 0.100 % aga ose p esen ing weake mechanical
p ope ies han 0.200 % aga ose, which a e no a ec ed by he US
i adia ion.
Sonoc ys allyza ion s udies we e e alua ed a di e en aga ose gel
concen a ions om 0.010 %, o 0.200 % (w/ ) by quan i ying he
numbe o c ys als and c ys al size 24 h a e he applica ion o he US
i adia ion p o ocols (US1 & US2) (Figs. 6 and 7). A he lowes aga ose
concen a ion (0.010 % w/ ) esul s showed a signi ican inc ease in he
numbe o c ys als and a consequen educ ion o c ys als size inde-
penden ly o he applied p o ocol, al hough in US2 c ys als size
dis ibu ion was na owe (Fig. 7 and Figu e S5). A aga ose concen-
a ion o 0.025 % and 0.050 % (w/ ) he numbe o c ys als is simila
bu hey show a s a is ically signi ican smalle sizes (Fig. 7) and na -
owe size dis ibu ion (Figu e S5) han in solu ion. A 0.100 % and
0.200 % (w/ ) aga ose concen a ion he e ec in he numbe o c ys als
and c ys al sizes a e no signi ican ly di e en om he silen condi ions
(Con ol) meaning ha he induc ion e ec o aga ose o e come he
po en ial e ec o US. This can be clea ly obse ed in he numbe o
c ys als ha a e highe and o smalle size. (Figu e S5 and S6).
Taking in o accoun size dis ibu ion (Figu e S5) i was clea ha
bo h expe imen al se -ups (US1 and US2) e ol ed simila ly as he
aga ose concen a ion inc eased and became almos iden ical o he si-
len con ol expe imen s when aga ose concen a ion was 0.100 % (w/
).
4. Conclusions
Sonoc ys alliza ion al hough e y p omising in p o ein c ys alliza-
ion has no been s udied in-dep h and con adic o y esul s ha e been
epo ed [25]. To shed ligh o e his ield and alida ed p e ious wo ks,
we ha e compa ed he e ec o US in lysozyme c ys alliza ion in solu-
ion and in gel media, bo h expe imen s ca ied ou unde he same US
condi ions.
We ha e obse ed an induc ion e ec o an US i adia ion on he
nuclea ion o lysozyme when c ys allized in he absence o he p esence
o aga ose a a concen a ion below 0.100 % w/ . In bo h media he
e ec o ul asound is simila , he induc ion o he nuclea ion gi ing ise
o a highe numbe o c ys als o smalle size. Abo e 0.100 % w/
aga ose concen a ion he e ec o US is hinde ed maybe due o he
Fig. 6. Lysozyme c ys als ob ained in solu ion unde silen condi ion (Con ol) and ul asonic i adia ion o 30 min immedia ely a e p epa ing he expe imen
(US1) and 30 min a e p epa a ion, i adia ed o 30 min (US2). F om le o igh i is shown he esul in solu ion and inc easing concen a ion o aga ose. The scale
ba in he op ical mic oscopy images is 500 µm in all he pic u es.
M. Sa chenko e al.
Ul asonics Sonochemis y 88 (2022) 106096
8
enhancemen o he mechanical p ope ies o he gel.
The esul s ob ained in solu ion and in aga ose gels a e s a is ically
signi ican and co obo a es he e ec o US in c ys alliza ion. We ha e
also demons a ed ha he combina ion o bo h echniques, he use o
hyd ogel and US, a e compa ible in p o ein c ys alliza ion.
CRediT au ho ship con ibu ion s a emen
Ma iia Sa chenko: Fo mal analysis, In es iga ion, Valida ion.
Manuel Hu ado: Fo mal analysis, In es iga ion, Valida ion. Modes o
T. Lopez-Lopez: Me hodology, Funding acquisi ion, Fo mal analysis,
W i ing – o iginal d a . Guille mo Rus: . Luis ´
Al a ez de Cien uegos:
Concep ualiza ion, Funding acquisi ion, Me hodology, P ojec admin-
is a ion, Supe ision, W i ing – o iginal d a , W i ing – e iew &
edi ing. Juan Melcho : Funding acquisi ion, Me hodology, P ojec
adminis a ion, Supe ision, W i ing – o iginal d a , W i ing – e iew &
edi ing. Jos´
e A. Ga i a: Concep ualiza ion, Funding acquisi ion,
Me hodology, P ojec adminis a ion, Supe ision, W i ing – o iginal
d a , W i ing – e iew & edi ing.
Decla a ion o Compe ing In e es
The au ho s decla e ha hey ha e no known compe ing inancial
in e es s o pe sonal ela ionships ha could ha e appea ed o in luence
he wo k epo ed in his pape .
Acknowledgemen s
This s udy was suppo ed by p ojec PID2020-118498GB-I00,
PID2020-116261GB-I00 and PID2020-115372RB-I00 unded by
MCIN/AEI/ 10.13039/501100011033/ and PID2019-106947RA-C22
unded by MCIN/AEI/ 10.13039/501100011033/FEDER “Una mane a
de hace Eu opa”, Spain, and by FEDER/Jun a de Andalucía-Conseje ía
de T ans o maci´
on Econ´
omica, Indus ia, Conocimien o y Uni e sidades
(Spain) p ojec s A-FQM-340-UGR20, P18-FR-3533, P18-RT-1653, B-
TEP-026-UGR18. We would like o acknowledge Ra a Ma qu´
es who
illus a ed he expe imen al p ocedu e in Fig. 1.
Appendix A. Supplemen a y da a
Supplemen a y da a o his a icle can be ound online a h ps://doi.
o g/10.1016/j.ul sonch.2022.106096.
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