Lab on a Chip
PAPER
Ci e his: Lab Chip,2024,24,2669
Recei ed 3 d Janua y 2024,
Accep ed 4 h Ap il 2024
DOI: 10.1039/d4lc00009a
sc.li/loc
Ve sa ile hyb id echnique o passi e s aigh
mic omixe manu ac u ing by combining pulsed
lase abla ion, s e eoli hog aphic 3D p in ing and
compu a ional luid dynamics
Bas ián Ca ne o,
ab
Yago Radziunas-Salinas,
a
B uno K. Rodiño-Janei o,
b
Syl ana Va ela Balles a
bc
and M. Te esa Flo es-A ias *
a
The e is a need o de elop new and e sa ile ab ica ion me hods o achie e e icien mixing o luids in
mic o luidic channels using mic os uc u es. This wo k p esen s a new echnique ha combines
s e eoli hog aphy (SLA) and pulsed lase abla ion (PLA) o manu ac u e a s aigh mic omixe o uni o m
mixing o wo samples. Compu a ional luid dynamics (CFD) simula ion is pe o med o deeply unde s and
he physical mechanisms o he p ocess. The esul s sugges ha his new op ical echnique holds he
po en ial o become a e sa ile hyb id echnique o manu ac u ing ema kable mixing mic o luidic de ices.
1. In oduc ion
Mic o luidics is one o he mos p omising ields in applied
science. This in e disciplina y ield in eg a es p inciples om
physics, chemis y, and enginee ing o achie e p ecise
manipula ion o luid dynamics on a minia u e scale, anging
om mic oli e s o millili es.
1
Mic o luidics o e s a huge
numbe o ad an ages, including educed esou ce
consump ion, enhanced eac ion e iciency, and minimized
was e p oduc ion. Due o he high con ol o e luid
beha iou , one o he mos ele an applica ions o
mic o luidics is he con olled mixing and homogeniza ion o
chemical eac ions.
2
Howe e , mixing in mic o luidics is a complex issue, due
o he p e alence o low Reynolds numbe (Re) in hese
sys ems. A low Reynolds numbe s, he ine ia o luid
mo ion is negligible compa ed o iscous o ces, leading o
lamina low p o iles. In such lamina lows, he inhe en
mixing o luids ia di usion alone is o en insu icien o
achie e he desi ed le el o homogenei y o he solu ion o
he comple eness o he eac ion.
To add ess his limi a ion, mic o luidic esea che s ha e
inco po a ed mic omixing s a egies inside o mic o eac o s,
which play a c ucial ole in a b oad ange o applica ions
including syn hesis o nanopa icles,
3,4
selec i e
polyme iza ion,
5,6
chemical analysis,
7
molecula biology
8,9
and poin -o -ca e applica ions.
10
Consequen ly, mic omixe s
all in o wo main ca ego ies: ac i e and passi e. On he one
hand, ac i e mic omixe s use ex e nal o ces o ene gy
sou ces, such as elec ic ields, acous ic wa es,
9
o magne ic
ields,
11
o ac i ely induce mixing in he mic o luidic
channels. On he o he hand, passi e mic omixe s ely on he
s uc u al cha ac e is ics o he mic ochannel design o
p omo e mixing wi hou in ol ing ex e nal sou ces. They
equen ly inco po a e mic os uc u es such as idges,
12
g oo es, o se pen ines
13
capable o dis up ing he
cha ac e is ic lamina low o mic oscale luids. In ou case
o s udy, we will ocus on channels ea u ing g oo es on he
bo om, which a e classi ied as slan ed mic omixe s.
14
These passi e slan ed mic omixe s a e o g ea in e es :
hey consume minimal powe , making hem ideal o
po able applica ions; hey exhibi obus ness, no depending
on con inuous ope a ion o mechanical o elec ical
componen s; and hey o e cos -e iciency and easy
in eg a ion in o complex mic o luidic sys ems.
To his end, op ical echnologies ha e been essen ial o
accu a ely manu ac u e his wide a ie y o 2D and 3D
mic os uc u es capable o c ea ing u bulen lows and
enhancing di usion.
15,16
Un il now, pho oli hog aphy has
been he mos used op ical echnology in mic o luidics,
17
cha ac e ised by complex and ime-consuming p o ocols o
mic opa e n pho o esis s in 2D, which also p oduce highly
pollu ing chemical was e. The eby, in ecen yea s, he apid
de elopmen s in lase s ha e a ou ed he appea ance o
di e en echnologies ha can be used o mic os uc u e
Lab Chip,2024,24,2669–2682 | 2669This jou nal is © The Royal Socie y o Chemis y 2024
a
Pho onics4Li e Resea ch G oup, Applied Physics Depa men , Facul ade de Física,
iMATUS, Uni e sidade de San iago de Compos ela, Campus Vida, E-15782
San iago de Compos ela, Spain. E-mail: mai e. lo [email protected]
b
BFlow SL, Edi icio Emp endia, Campus Vida, San iago de Compos ela, E-15706, Spain
c
Depa amen d'Enginye ia Mecànica, Uni e si a Ro i a i Vi gili, Ta agona, E-
43007, Spain
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di e en subs a es in a mo e use - iendly way. This is he
case o pulsed lase abla ion (PLA) ha allows us o achie e
ou s anding esolu ions when mic opa e ning 2D su aces,
enabling he c ea ion o mic o
18–20
and nano
21
s uc u es
wi h p omising esul s. 3D p in e s based on he selec i e
s e eoli hog aphy (SLA) o liquid esins ha e made i easie o
manu ac u e 3D objec s wi h signi ican s uc u al complexi y
( anging be ween hund eds o mic ome e s and millime e s),
demons a ing subs an ial po en ial in he ield o
mic o luidics
22–24
and, in pa icula , mic omixe s.
9,25,26
Bo h
echnologies ha e he po en ial o o e come he complexi ies
and limi a ions associa ed wi h adi ional pho oli hog aphic
manu ac u ing me hods, in e ms o ime consump ion,
scalabili y, cos and pollu ion.
Mic omixe s such as he ones ha will be p esen ed in
his wo k ha e been used o pe o m chemical eac ions in
he li e a u e. Fo ins ance, Koo e al.
12
p oposed he
ab ica ion o a mic o eac o using a use deposi ion
modelling 3D p in e . In his wo k, a ious p in ing angles
we e used o o m s uc u es ha will be eplica ed by so
li hog aphy o PDMS c ea ing a g oo e pa e n on he
eplicas. These au ho s u ilized channels wi h s uc u es in a
simila dimensional ange o he one ha will be p esen ed
in ou s udy (800 μm diame e channels wi h g oo es anging
om 200 o 300 μm), imp o ing he mixing pe o mance o
se e al iodide–ioda e eac ions (Ville maux–Dushman
me hod
27
), as he angle and low a e inc eased.
O he s udies ha e highligh ed he ad an ages o he
slan ed g oo e mic omixe posi ioned on he bo om side
o he channel in di e se applica ions. Fo ins ance, Xu
e al.
28
demons a ed he syn hesis o a s a is ical-
copolyme -b ush composi ion g adien o wo monome s
ollowing he manu ac u ing p o ocol o Cab al e al.
29
This echnique based on on al pho opolyme iza ion o a
pho o esis sandwiched be ween wo glass slides allows
ob aining ou wa d g oo es o 190 and 340 μm on he
bo om o a 600 μm channel, which is close o he esul s
p esen ed in ou wo k.
In ano he case, Abonnenc e al.
30
ab ica ed a
mic o eac o h ough UV-pho oabla ion o a mic omachined
channel (o 100 μm in wid h and 35 μm in dep h) on a
polye hylene e eph hala e (PET) shee , ob aining 45°
o ien ed inwa d g oo es o 35 μm in dep h. This de ice was
used o conduc a agging eac ion o enable p o ein agging
o applica ions in liquid ch oma og aphy-mass
spec ome y wo k lows.
Beyond di ec chemical eac ions, Moon e al. de eloped
a slan ed g oo e mic omixe , ob ained by pho oli hog aphy
and eplica ion in PDMS, ea u ing g oo es o 50 μmin
wid h and 10 μm in diame e .
31
This eac o was applied in
hei nex wo k o c ea e an enzyma ic mic o eac o o
con inuous glucose le el moni o ing ia subcu aneous
mic odialysis in a s.
32
In ligh o he in e es ga ne ed by hese de ices and hei
applica ions, his wo k p esen s o he i s ime, o ou
knowledge, a no el hyb id echnique ha combines PLA and
SLA o simply manu ac u e and es s aigh mic omixe s
ea u ing di e en pa e ns.
2. Ma e ials and me hods
2.1 Manu ac u ing
The p oposed manu ac u ing p ocess o mic omixe s consis s
o wo s eps, in which each echnique (SLA and PLA) is used
in i s mos sui able dimensional ange (millime es and
mic ome es, espec i ely).
2.1.1 S e eoli hog aphic 3D p in ing. A Fo m 3B
(Fo mlabs, Some ille, Massachuse s, USA) SLA 3D p in e
was used o p oduce he subs a e (Fig. 1a). This p in e
ea u es a UV lase (λ= 405 nm, P= 250 mW) and o e s an
XY esolu ion o 25 μm and a lase spo size o 85 μm. The
esolu ion in he Zaxis depends on he selec ed esin.
P in ed subs a es ea u ed inwa d and ou wa d squa e
sec ion (0.55 ×0.55 mm
2
) channels on hei su ace. These
channels, 25 mm long, inco po a e a bi u ca ion a he
beginning, in ended o in oduce wo samples o be mixed
(Fig. 1b). The eby, Y-shaped
33
mic omixe s we e designed
using he Compu e Aided Design (CAD) so wa e Fusion 360
Fig. 1 a) SLA 3D p in ing p inciple. b) CAD images o he p in ed
pla es ea u ing Y-shaped mic ochannels (inwa d and ou wa d) on he
su ace. c) Lase se up used o PLA mic opa e ning o he bo om o
he inwa d channels ( he same p inciple applies o he op o ou wa d
channels). d) PDMS so li hog aphy p ocess o eplica ing ou wa d
channels.
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(Au odesk, San F ancisco, Cali o nia, USA) and hen managed
wi h he p in e so wa e P e o m.
Comme cial Model V2 esin om Fo mlabs was selec ed
as he p in ing esin gi en he p ecision i o e s (25 μmin
Z)
22
and i s pe o mance when eplica ing polyme s. In he
pos cu ing p ocess, he piece was washed in 2-p opanol and
cu ed in o a UV chambe o 60 minu es a 60 °C. Mo eo e ,
he selec ed esin o e s a e y good esponse o he lase
abla ion in he in a ed (IR) egime.
2.1.2 Pulsed lase abla ion. Nex , PLA mic os uc u a ion
o he bo om ( op) a ea o he inwa d (ou wa d)
mic ochannel was pe o med (Fig. 1c) using he San iago
TE awa Lase (STELA) o he Lase Labo a o y o
Accele a ion and Applica ions (L2A2) acili y in San iago de
Compos ela. This lase o e s an ene gy o 1 mJ pe pulse, a
pulse du a ion o 35 s, a ixed epe i ion a e o 1 kHz and a
wa eleng h o 800 nm wi h a bandwid h o 75 nm. The beam
was ocused h ough a mic oscope objec i e M Plan APO
NIR20X (Mi u oyo, Sakado, Japan) on he 3D p in ed a ge .
This a ge was held by a high p ecision mic op ocessing
sys em made o wo ILS200LM-S s ages (Newpo , I ine,
Cali o nia, USA) wi h an accu acy o 1.5 μm in he Xand Y
axes and 100 nm in he Zaxis.
The design o he pa e ns was made using Fusion 360
CAD so wa e (Au odesk, San F ancisco, Cali o nia, USA). The
a ge was main ained always a he ocal leng h o he
mic oscope hanks o a collinea lase eedback wi h a
esolu ion o 0.4 μm. Du ing i adia ion o he a ge , he
ocal posi ion was au oma ically eadjus ed.
2.1.3 So li hog aphy o PDMS. Ou wa d channels we e
employed as he mas e s uc u es, c ea ing inwa d channels
by eplica ion wi h so li hog aphy o polydime hylsiloxane
(PDMS). This is one o he mos used polyme s o ab ica e
mic o luidic de ices gi en i s op ical anspa ency,
pe meabili y o gases, elas ici y, and biocompa ibili y.
34,35
PDMS was p epa ed om Sylga d 184 elas ome (Dow
Chemical Company, Midland, Michigan, USA). The
eplica ion p ocess consis ed o he ollowing s eps: he
uncu ed (1:10 a io) polyme was deposi ed on he mas e
ab ica ed by SLA and mic opa e ned by PLA and in oduced
in a acuum chambe (40 min a 400 mba ) o emo e
bubbles p oduced du ing he mixing p ocess. Finally, he
mas e wi h he degassed PDMS was cu ed in an o en o 12
ha 60°C and easily peeled o (Fig. 1d). A e ha , PDMS
eplicas can be easily demolded om he esin mas e .
2.1.4 Sealing o he channels. Two di e en app oaches
we e used o seal he channels o in oduce low h ough
hem. These wo echniques depend on whe he he channel
is 3D p in ed (made o esin) o eplica ed (made o PDMS).
3D p in ed channels we e sealed using anspa en PET/
ac ylic adhesi e ilms (The moFishe , Wal ham,
Massachuse s, USA). The s icke s a e easy o apply, ensu e
eliable con ainmen o luids, p e en con amina ion, and
show op ical anspa ency.
Replica ed PDMS channels we e punched ( o c ea e he
luid en ies) and plasma bonded o a mic oscope glass slide.
Fo his, a Zep o plasma cleane (Diene Elec onics,
Ebhausen, Ge many) was employed, exposing he su aces o
oxygen plasma. The powe was ixed a 20 W, and he pieces
we e exposed o 20 s. Bonded de ices we e hen he mally
ea ed o 30 min a 90 °C.
2.2 Mic omixe es ing
Unde s anding he low beha iou and mixing e iciency
wi hin mic omixe s is c ucial o op imizing hei
pe o mance and applica ion in a ious ields. In his sec ion,
we del e in o he in es iga ion o mic omixe lows h ough
wo di e en app oaches: pe is al ic pump luid pe usion
and CFD simula ion.
2.2.1 Flow assays. Fo es ing he eal pe o mance o he
mic omixe s, a pe is al ic pump and dyes we e used. A ou
channel Minipuls 3 pe is al ic pump (Gilson, Middle on,
Wisconsin, USA) was employed, allowing us o p ecisely
con ol he low a e and ensu e consis en low condi ions
o accu a e measu emen s and obse a ions. To assess he
mixing capabili ies o he mic omixe s, di e en colou dyes
(blue and yellow) we e in oduced simul aneously in o he
sys em. The selec ion o mul iple dyes o e s he ad an age
o isualizing and quan i ying he mixing p ocess, as a
dis inc colou pa e n eme ges because o e icien o
ine icien mixing. The low was es ablished h ough 3-s op
pump pla inum-cu ed silicone ubing (inne diame e (ID) 1
mm and ou e diame e (OD) 3 mm) (Da win, Pa is,
F ance), connec ed wi h s oppe connec o s (BFlow SL,
San iago de Compos ela, Spain) and silicon ubing (ID 1
mm and OD 3 mm). S aigh connec o s (BFlow SL,
San iago de Compos ela, Spain) we e used o in oduce low
in he mic omixe . The used caudal was Q=1mlmin
−1
,
which co esponds o Re = 30.16.
2.2.2 Compu e luid dynamics simula ion. Compu a ional
luid dynamics (CFD) allows he analysis o a ious low
cha ac e is ics, such as luid eloci y,
36
p essu e dis ibu ion,
and mixing pa e ns
37
employing nume ical modelling and
simula ion o he incomp essible Na ie –S okes equa ions.
I s use has been epo ed in medical disciplines
38
and, in
pa icula , mic omixe s.
39
In his wo k, CFD simula ions we e
used o es he geome ies and co esponding pe o mance
o he manu ac u ed mic omixe s, employing ANSYS Fluen
(Ansys, Canonsbu g, Pennsyl ania, USA) so wa e. Nume ical
simula ions we e de eloped conside ing incomp essible
luids, no ic ion, double p ecision, lamina low, ansien ,
he p essu e-based sol e , implici olume o luid, implici
body o ce, dispe sed in e ace modelling, and wo-phase
olume o he luid model wi h dispe sed in e ace
modelling. Fo he p ima y phase, we conside ed liquid wa e
(ρ
1
= 998.2 kg m
−3
and μ
1
= 0.001003 kg ms
−1
) and o he
seconda y phase, we selec ed a hypo he ical luid wi h ρ
2
=
997.2 kg m
−3
and μ
2
= 0.001002 kg ms
−1
( he physical
p ope ies di e by 0.1% om he wa e p ope ies) wi hou
a su ace ension coe icien be ween he wo phases. Ano he
sample was conside ed o es he adap abili y o he
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mic omixe s, in ha case, an aqueous solu ion o wa e wi h
glyce ine a 20% was chosen as he i s phase (ρ
3
= 1047 kg
m
−3
and μ
3
= 0.00184 kg ms
−1
), and a hypo he ical luid
di e ing by 0.1% om he i s one (ρ
4
= 1046 kg m
−3
and μ
4
= 0.00183 kg ms
−1
) was selec ed as he second phase.
The inle s 1 and 2 we e se a Q=1mlmin
−1
and
he ou le was se a a mosphe ic p essu e (101325 Pa).
The o he su aces we e se as walls wi h no slip
condi ions. A e ahed al mesh e ined nea he walls was
employed o enhance accu acy in egions cha ac e ized by
high g adien s o eloci y. Mix u es we e conside ed
iso he mal and isoba ic.
Th ee meshes o a ying g id densi y and size we e
conside ed, comp ising 99668 elemen s o g id 1, 358813
elemen s o g id 2, and 509 873 elemen s o g id 3. The
simula ion o a mic omixe ea u ing 20°o ien ed g oo es
was conduc ed on all h ee g ids. When compa ing he
maximum eloci y alues o he heo e ical alue ( = 0.2204
ms
−1
), g id 2 exhibi ed a de ia ion o app oxima ely 5%
(g id 1 o e ed a de ia ion o 10%), p o iding good accu acy
while equi ing less compu a ional e o compa ed o g id
3. Consequen ly, all simula ion cases we e conduc ed using
g ids wi h a numbe o elemen s o he o de o g id 2 and
Q= 0.5, 1, and 2 ml min
−1
, which co espond o Re = 15,
30, and 60.
Simula ions we e un un il c i ical con e gence o
esiduals o 1 ×10
−6
unde a SIMPLE solu ion me hod wi h
200 s eps o 0.01 seconds and maximum in e ac ion/ ime
s eps o 20. The spa ial disc e iza ion includes a leas squa es
cell-based g adien , PRESTO! disc e iza ion o p essu es,
second o de upwind momen um, i s o de upwind olume
ac ion and i s o de implici ansien o mula ion.
2.3 Da a collec ion
Con ocal images o he mic os uc u es manu ac u ed on he
pla es we e aken using a 3D op ical p o ilome e S Neox
(Senso a Me ology, Te assa, Spain). This p o ilome e
allowed us o measu e he dimensions (measu emen s we e
aken 10 imes, ob aining a mean alue and i s
co esponding s anda d de ia ion) and o analyse he
mo phology o he channels. A Nikon MM-400 me allu gic
mic oscope (Nikon Ins umen s Eu ope B.V., Ams e dam,
The Ne he lands) was used o acqui e he op ical images,
using an LU Plan Fluo objec i e (Nikon Ins umen s,
Mel ille, USA) wi h a 5×magni ica ion and a CCD came a
Nikon DS-FI2 (Nikon Ins umen s, Mel ille, USA).
3. Resul s and discussion
3.1 Lase pa ame e s o manu ac u ing
The i s s ep in he expe imen al sec ion consis ed o a
comp ehensi e s udy ocused on he op imiza ion o lase
abla ion pa ame e s. Speci ically, we conduc ed expe imen s
in ol ing he mic opa e ning o g oo es by PLA on a la
esin su ace. Du ing hese expe imen s, we sys ema ically
a ied he lase powe (P) and he numbe o passes (N) o
ind he op imal channel esul s, analysing he mal damage,
homogenei y, egula p o iles, and dimensions by con ocal/
op ical images (Fig. 2). The eloci y o 2D pla o ms (whe e
he esin pla e was placed) was ixed a 1 mm s
−1
.
Fi s , geome ic da a we e collec ed using a con ocal
mic oscope, which allowed us o measu e he wid h (Fig. 2a)
and dep h (Fig. 2b) o he ab ica ed channels. Since hese
a e age measu emen s a e no su icien o desc ibe he eal
aspec o he channels, he esul s we e c oss-checked wi h
con ocal images (Fig. 2c–j).
In e ms o channel wid h (W), a clea co ela ion is
obse ed be ween his wid h and he powe o he pulses
(Fig. 2a): he channels, o a ixed powe , a e wide han
hose ob ained o lowe powe s. This is e i ied o 1, 2
and 3 passes o he lase . Howe e , o each o he powe s
s udied, he numbe o passes does no always imply an
inc ease in he wid h o he channel. This beha iou is
obse ed in he esul s o highe powe s (110, 130 and 150
mW) and lowe ones (10 mW), and occu s o di e en
easons: in he case o highe powe s, o example 150 mW
(Fig. 2c), a deepe channel is ob ained o 1 pass, while an
inhomogeneous esul is obse ed o 2 and 3 passes. This
phenomenon has i s o igin in he immedia e a i al o e y
ene ge ic second and hi d pulses, which p e en he
ejec ion and adequa e e acua ion o he ma e ial ha is
ins an ly ecas in he bo de s o he channels, educing i s
Fig. 2 A e age a) wid h and b) dep h o he mic ochannels abla ed by
PLA on he su ace o he esin o di e en powe s (10–130 mW) and
lase passes (1–3). Top (le ) and 3D ( igh ) con ocal images o
mic ochannels ob ained o c) and d) 130, e) and ) 90, g) and h) 50
and i) and j) 10 mW, espec i ely. Scale ba s: 100 μm.
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wid h. Fo lowe powe s, o example 10 mW (Fig. 2i),
manu ac u ed mic ochannels p esen a egula s uc u e o
only 1 lase pass. When he numbe o passes is inc eased,
his low ene gy alue o he pulses is unable o abla e he
p e iously abla ed a eas in a cons an manne , c ea ing
small i egula g oo es along he channel. In his scena io,
he concep o a e age wid h does no adequa ely desc ibe
he esul , as i a ies locally ( he same applies o dep hs,
as can be seen in Fig. 2j). Fo in e media e powe s, o
example 90 (Fig. 2e) and 50 mW (Fig. 2g), in gene al e ms,
a g ea e numbe o passes inc eases he wid h o he
channel, since he ene gy is enough o allow apo iza ion
and p ope elimina ion o he ma e ial a e he i s lase
pass and no ecas ing is obse ed.
Rega ding he dep h (D) o he channels (Fig. 2b), we
obse e wo egimes wi h e y di e en beha iou s. Fo high
powe s (150, 130 and 110 mW), as men ioned be o e, he
a i al o he second and hi d pulses a ou s he collapse o
he walls and he accumula ion o he ma e ial in he
emaining space, which is immedia ely ecas and deposi ed
again inside he channel (Fig. 2d). This a ou s ha o hese
h ee powe s, when h ee passes a e pe o med, he dep hs
all o app oxima ely 20 μm. Fo wo passes, some ma e ial
can escape, so o 130 and 110 mW, we each mic ochannels
wi h a dep h o a ound 100 μm. Fo lowe powe s (10–90
mW), we ob ain a mo e e ec i e esul , pulses do no block
he emo al o he ma e ial and he dep h inc eases wi h he
numbe o passes. In his egime, he highe he powe , he
deepe he channels will be (compa ing he same numbe o
passes). The uni o mi y o channel dep hs is e i ied, as
e idenced by 3D con ocal images o P= 90, 50 mW
(Fig. 2 and h) and wo passes.
A e an ex ended analysis o he esul s ob ained o
di e en alues o powe o he abla ion, he selec ed one
was 90 mW, combined wi h 2 passes, wi h which we can
ob ain channels wi h a wid h o W= 39.6 ± 1.9 μm and a
diame e o D= 129.1 ± 2.3 μm. This ene gy and passes we e
selec ed because hey a e a he in e media e poin be ween
high and low powe s, p oducing op imal abla ion esul s
(Fig. 2e) and allowing he pa e ning o egula ,
homogeneous, and ep oducible supe icial channels.
To his poin , he ob ained channels a e wi hin he
dimensional ange o some epo ed in he li e a u e,
40,41
sugges ing ha he luid pene a ion in o hese channels is
expec ed o be su icien . Howe e , conce ning he demolding
o PDMS om he mas e (see sec ion 2.1.3), i is necessa y
o sligh ly inc ease he channel wid h o p e en any polyme
e en ion wi hin he mas e . To each his goal, wide
channels a e needed, which leads o he ollowing app oach:
ob aining a wide channel h ough he abla ion o wo o
mo e con iguous lines on a la esin su ace. The abla ion
esul s o se e al con iguous lines canno be di ec ly
ex apola ed om he esul s ob ained o a single line, so a
new s udy and op imiza ion o pa ame e s we e ca ied ou
e alua ing he abla ion esul s o 2 and 3 con iguous lines
depending on he sepa a ion dis ance be ween hem (d) and
main aining he p e ious abla ion pa ame e s: 90 mW, 2
passes and 1 mm s
−1
.
The esul s o his s udy a e shown in Fig. 3. In his igu e,
se e al con ocal images a e p esen ed, in he op ow, hose
co esponding o he s udy o wo con iguous lines and in
he middle ow, hose co esponding o h ee lines. Fo 2
lines, he ene gy is e y spa ially con ined, and a big quan i y
o ma e ial is emo ed, p omo ing he c ea ion o he deepes
channels (D= 330.0 ± 4.2 μm, W= 67.2 ± 3.2 μm) o d=20
μm and N= 3. These dimensions a e signi ican ly g ea e
han hose ob ained o he s udy pe o med o a single line.
This is logical since widening he channel allows he ma e ial
o be elimina ed in a la ge amoun . In his case, he
s uc u es ha e a W/D a io ha has o be inc eased in o de
o p omo e liquid inse ion and non- apping o PDMS
du ing eplica ion, as we men ioned be o e. Fo g ea e
sepa a ion dis ances, he ene gy ails o adequa ely emo e
he ma e ial in he in e media e egion, being especially
no iceable in Fig. 3c, whe e a aised, no abla ed egion
(highligh ed wi h a ows) is obse ed be ween he wo
con iguous lines.
Mo e adequa e esul s a e ob ained when using h ee
con iguous lines, since he in e media e line g an s p ope
abla ion o he in e media e egion, hus ob aining a wide
channel wi h a egula bo om. Fo he lowes sepa a ion
s udied, 25 μm (Fig. 3d), he ene gy densi y pe a ea is e y
high, so he edges a e i egula , and some b eaks can be
obse ed, especially o N= 3. These esul s will no g an
p ope sealing o he channels when using an adhesi e ilm
o plasma bonding, and some leakages o luids can occu . A
simila scena io is obse ed o d=30μm. Rega ding d=35
μm (Fig. 3 ), we conside ha he esul s ob ained o N=2
a e he mos app op ia e since hey allow ob aining a
channel wi h an app op ia e size (D= 230.9 ± 4.7 μm, W=
95.5 ± 5.2 μm) o he sample o en e , ea u ing a
homogeneous channel bo om and a egula edge ha will
Fig. 3 Con ocal images o he ob ained lase abla ed mic ochannels
on he su ace o he esin. The uppe ow shows he esul s when 2
lines we e abla ed con iguously a di e en sepa a ions: a) 30, b) 40
and c) 50 μm. The middle ow shows he esul s when 3 lines we e
con iguously abla ed wi h sepa a ions o d) 25, e) 30 and ) 35 μm,
espec i ely. g) 3D con ocal p o ile and h) c oss sec ion o he
mic ochannels ob ained in ).
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a ou sealing wi hou leakage. A 3D con ocal image o hese
esul s can be seen in Fig. 3g, in addi ion o a c oss sec ion
p o ile (Fig. 3h), whe e egula e ical walls o he channels
can be obse ed.
3.2 Mic opa e ning esul s and cha ac e iza ion
Two di e en p o ocols we e used in he manu ac u ing
p ocess. In p o ocol 1, PLA was used o mic opa e n he
bo om o inwa d channels, which we e sealed di ec ly using
an adhesi e ilm o immedia e use. In p o ocol 2, we
employed PLA o mic opa e n he op o ou wa d channels,
wi h he in en ion o la e eplica ing hem h ough PDMS
so li hog aphy o ob ain he inal mic omixe s. I is wo h
men ioning ha , in his case, walls we e added o he
mas e s in o de o ill hem wi h PDMS. This p o ocol makes
possible he p oduc ion o mul iple eplicas o he
mic omixe s, a ea u e o g ea in e es o scaling up he
p oduc ion p ocess. In bo h cases, he ollowing PLA
pa ame e s we e used: an a e age powe o 90 mW, 3
con iguous lines sepa a ed by 35 μm, 2 passes pe line, a
epe i ion a e o 1 kHz and a pla o m eloci y o 1 mm s
−1
.
3.2.1 Inwa d channels. Va ious images illus a ing he
esul s o p o ocol 1 a e displayed in Fig. 4. One channel was
in en ionally le unpa e ned as a con ol.
In Fig. 4a and e, he p in ing quali y is no o ious, as
e idenced by he consis en edge egula i y and channel
s aigh ness. The cha ac e is ic oughness o he su aces
p in ed by SLA was s udied in de ail in ou p e ious wo k,
22
in which a alue o S
a
= 1.444 ± 0.247 μm was measu ed o
he employed esin. Subsequen ly, ollowing he applica ion
o PLA wi h he speci ied pa ame e s on he bo om o he
channel, we can obse e he o ma ion o o ien ed g oo es.
In hese g oo es, a se o dis inc i e pa ame e s (wid h (W),
leng h (L), dep h (D), and pe iod (p)) can be iden i ied and
measu ed using p o iles de i ed om he con ocal images.
The alues ob ained a e p esen ed in Table 1. In he case o
90°o ien ed s uc u es, he op ical image (Fig. 4b) exhibi s
no he mal damage and consis en esul s in e ms o
s uc u e o ma ion a he bo om o he channel. When
assessing hei dimensions, we no e ha he wid h o hese
g oo es (94.3 ± 8.3 μm) ma ched wha was ob ained in he
pa ame e s udy (95.5 ± 5.2 μm).
In e ms o g oo e leng h, i is somewha sho e han
an icipa ed (designed channels had a 550 ×550 μm
2
sec ion).
This disc epancy a ises om he manne in which he p in e
cons uc s squa e-sec ion s uc u es, esul ing in a shape
close o a apezoid,
22
wi h a na owe base wid h compa ed
o he op. Gi en ha abla ion akes place in his speci ic
egion, he g oo es end up measu ing less in leng h.
Addi ionally, a mino accumula ion o esidues is isible
along he sides o he channel. This accumula ion can be
seen in he con ocal image (Fig. 4 ) and can be a ibu ed o
he challenge o e ec i ely emo ing ola ilized deb is. The
highligh ed a ea in his image is p esen ed in a 3D iew in
Fig. 4i, helping us o be e unde s and he abla ion esul s
and he achie ed dep hs o his o ien a ion (243.5 ± 15.5
μm), which ag ee wi h he p e iously ob ained esul s on a
la su ace (230.9 ± 4.7 μm). The di e ence be ween he
wid h and he pe iod allows us o de e mine he wid h o he
idges, which a e s uc u es o med by he esin ha is no
subjec ed o abla ion. These s uc u es, measu ing 140 μmin
wid h, a e sligh ly wide han he g oo es and exhibi a
squa e sec ion.
Rega ding he 20°o ien ed s uc u es, Fig. 4c displays
well-de ined g oo es ha a e sligh ly na owe (86.6 ± 4.0
μm) han hose achie ed a 90°. This di e ence is due o he
o e lapping o he h ee con iguous lines used in PLA when
abla ion is pe o med a a ce ain angle. No ably, he
accumula ion o esidues in hese inwa d channels is he
mos signi ican among all he obse ed, as e iden in he
con ocal image (Fig. 4g). This can be a ibu ed o he g ea e
leng h o hese s uc u es (1429.3 ± 14.8 μm) in compa ison
wi h he o he s, which a ou s ha a la ge amoun o
ma e ial is ola ilized and ca ied owa ds he edges o he
Fig. 4 Op ical ( op) and con ocal (bo om) mic oscopy images o he
mic opa e ned s uc u es on he bo om o 3D p in ed
mic ochannels, ea u ing: a) and e) none, b) and ) 90°, c) and g) 20°,
and d and h) 45°o ien ed g oo es, espec i ely. Scale ba : 300 μm.
Magni ica ion: 5×. i) Con ocal image o he highligh ed a ea in pic u e
). j) Pic u e o he 3D p in ed esin pla e whe e all s uc u es we e
mic opa e ned. An enla ged iew o he inne pa o one mic omixe
is shown. The measu ed g oo e pa ame e s (W,L,D,p) a e also
deno ed in he image.
Table 1 Measu ed pa ame e s o he abla ed s uc u es a he bo om o
he channel
Angle (°) Wid h (μm) Leng h (μm) Dep h (μm) Pe iod (μm)
90 94.3 ± 8.3 488.3 ± 8.1 243.5 ± 15.5 235.3 ± 3.3
20 86.6 ± 4.0 1429.3 ± 14.8 253.3 ± 11.1 167.0 ± 4.0
45 89.3 ± 2.5 631.8 ± 14.4 248.3 ± 9.8 189.7 ± 4.0
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channel du ing PLA. The pe iod allows us o es ima e a close
dimensional simila i y be ween he g oo es and idges,
which a e app oxima ely 80 μm in wid h in his case. Finally,
hese channels show he g ea es o he dep hs ob ained
(253.3 ± 11.1 μm), due o he g ea e o e lapping o he lines
men ioned abo e.
Fo channels wi h 45°o ien ed s uc u es (Fig. 4d), he
wid h o he idges (89.3 ± 2.5 μm) is an in e media e poin
be ween 0°and 20°, jus as happens o he o ien a ion
angle. The same applies o he dep h o he idges (248.3 ±
9.8 μm). The con ocal image (Fig. 4h) shows a sub le
accumula ion o deb is in ce ain a eas, somewha dis an
om he edges o he channel. Howe e , no he mal damage
is obse ed in any case and he esul s ag ee wi h he ones
ob ained in sec ion 3.1. The pe iod indica es a sui able
ma ch be ween he g oo es and idges, o app oxima ely 90
μm in wid h. The e sa ili y o he p oposed manu ac u ing
echnique has also enabled he c ea ion o h ee
mic opa e ned channels (and wo con ols) e o lessly and
ep oducibly on a single mul ichannel esin pla e (Fig. 4j),
whe e i e unpa e ned channels we e p in ed a i s . The
o al manu ac u ing ime o he h ee s uc u es has no
exceeded i een minu es. The in eg a ion o hese
mic os uc u es wi hin he same pla o m allows o
con enien expe imen a ion and di ec compa ison o hei
pe o mance. These de ices can be used di ec ly by sealing
hem using an adhesi e ilm.
3.2.2 Ou wa d channels. The ou comes o p o ocol 2
(co esponding o ou wa d channels) a e shown in Fig. 5
and 6, while Table 2 p esen s he alues o he pa ame e s
o he s uc u es. Jus as in he p e ious sec ion, we
delibe a ely le he i s channel wi hou pa e ning, as
shown in he con ocal images (Fig. 5a), which is p ope ly
ansla ed o he PDMS eplica (Fig. 5e). The p e iously
men ioned su ace oughness o he employed esin is
e iden in he eplica. This unde sco es he ema kable le el
o de ail ha PDMS can ai h ully eplica e, ex ending down
o he mic ome e scale.
Rega ding he channels wi h 90°o ien ed s uc u es, he
i s s ep consis ed o mic opa e ning he op o ou wa d
channels by PLA. Abla ion and so li hog aphy esul s a e
shown in Fig. 5b and and allow us o obse e he co ec
o ma ion o he g oo es, ea u ing W= 98.3 ± 6.3 μm in he
mas e o esin and W= 123.0 ± 3.8 μm in he eplica. I is
wo h men ioning ha his di e ence in size is since i is a
eplica ion p ocess, he idges in mas e a e he s uc u es
ha o m he g oo es in he eplicas and ice e sa. A e y
simple e i ica ion o hese ou comes is ha he sum o bo h
wid hs mus be equal o he pe iod, which i ually happens
wi hin he s anda d de ia ions. Fu he mo e, he pe iods
coincide o bo h he mas e and he eplica, demons a ing
he p ecision o he so li hog aphy echnique used in his
p ocess. Rega ding he leng h, he measu ed alue o
ou wa d channels is g ea e han ha o he inwa d ones.
This is because du ing SLA p in ing, he ou wa d channels
elease a la ge amoun o uncu ed esin, allowing hem o
achie e a less apezoidal shape, close o a squa e. The mos
signi ican di e ence compa ed o he p e ious case lies in
he dep h measu emen (301.3 ± 9.9 μm), which is 70 μm
g ea e han wha was ob ained du ing he pa ame e s udy
on a la su ace. This inc ease o igina es om he abla ion
Fig. 5 Con ocal ( op) and op ical (bo om) mic oscopy images o he
mic opa e ned s uc u es on he op o p in ed mic ochannels ( op)
(a–d) and hei co esponding PDMS eplicas (bo om) (e–h), ea u ing:
a) and e) none, b) and ) 90°, c) and g) 20°, and d) and h) 45°o ien ed
g oo es. The measu ed g oo e pa ame e s (W,L,p) a e also shown in
he image. Scale ba : 300 μm. Magni ica ion: 5×.
Fig. 6 3D con ocal iew o he mic opa e ned s uc u es on he op
o p in ed mic ochannels (a–c) and hei co esponding PDMS (d– )
eplicas ea u ing: a) and d) 90°, b) and e) 20°and c) and ) 45°
o ien ed g oo es.
Table 2 Measu ed dimensions o he abla ed s uc u es a he op o he
channel and he co esponding dimensions o he PDMS eplicas
Angle (°)Wid h(μm) Leng h (μm) Dep h (μm) Pe iod (μm)
Resin
mas e
90 98.3 ± 6.3 502.8 ± 9.5 301.3 ± 9.9 234.0 ± 7.0
20 87.7 ± 2.1 1438.5 ± 30.7 299.3 ± 17.2 179.0 ± 3.0
45 88.8 ± 5.6 648.3 ± 14.4 306.5 ± 12.9 182.3 ± 6.6
PDMS
eplica
90 123.0 ± 4.8 495.0 ± 4.8 293.3 ± 12.4 227.3 ± 4.6
20 98.0 ± 4.6 1318.5 ± 36.1 299.0 ± 25.0 177.7 ± 16.5
45 99.8 ± 5.1 690.8 ± 20.8 306.3 ± 39.5 188.3 ± 9.2
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being ca ied ou on a s uc u e ha is aised abo e i s
su oundings (Fig. 6a). As a esul , he esin can be
ola ilized and elimina ed wi hou es ic ions when
su ounded by ai . Finally, a mo e comp ehensi e 3D
con ocal iew o he p ope eplica ion o he mas e
s uc u e in o he PDMS eplica can be obse ed in
Fig. 6a and d.
Fo channels ea u ing s uc u es o ien ed a 20°, he
mas e g oo es exhibi simila dimensions (W= 87.7 ± 2.1
μm and L= 1438.5 ± 30.7 μm) o he PDMS eplicas (W= 98.0
± 4.6 μm and L= 1318.5 ± 36.1 μm) and bo h a e consis en
wi h he s udy o pa ame e s. Meanwhile, he dep h ollows
he same end as ha ob ained o 45°, being signi ican ly
g ea e han ha achie ed on a la su ace due o inc eased
deb is elease, so deepe s uc u es a e ob ained (Fig. 6b) and
ansla ed o eplicas (Fig. 6e). A e y illus a i e esul o he
eplica ion p ocess explo ed in his sec ion is he p oduc ion
o specula images o he o iginal pa e n, so ha i he
mas e s uc u e is o ien ed a 20°(Fig. 5c), wi h e e ence o
he e ical axis in he pho o, he eplica will be o ien ed a
−20°(Fig. 5g). A e he s uc u es we e ab ica ed and
cha ac e ized h ough con ocal mic oscopy, hey we e
ec ea ed using CAD so wa e (Fig. 6g–i) o conduc u he
simula ions.
3.3 Flow assays
In o de o demons a e he e iciency o he mic omixe
ab ica ed, low expe imen s we e ca ied ou using a
pe is al ic pump o assess hei mixing pe o mance (Fig. 7).
Two di e en dyes (yellow and blue) we e simul aneously
in oduced in o he mic omixe s, ep esen ing dis inc luid
s eams (Fig. 7a). By obse ing he esul ing colou pa e ns,
he e iciency and e ec i eness o he mixing p ocess can be
e alua ed in e ms o he homogenei y o he g een colou
ob ained a he ou le . PDMS eplica ed mic omixe s we e
u ilized due o hei comple e anspa ency. Resin mic omixe s
a e expec ed o yield simila esul s, bu hei opaqueness
makes i challenging o cap u e images by op ical mic oscopy.
In he i s column (Fig. 7b), i is e iden ha he blue
and yellow dyes emain seg ega ed and do no mix e ec i ely
wi hin he 25 mm mic ochannel. This is p ima ily due o
lamina low, cha ac e ized by low s eams mo ing in
pa allel laye s wi h minimal in e ac ion, caused by he
absence o mic os uc u es designed o induce u bulen
mixing. Mo ing o he second case wi h s uc u es o ien ed
a 90°, as depic ed in Fig. 7c, no able di e ences om he
i s image become appa en .
Ini ially, apping is obse ed when he luid in e ac s wi h
he i s se o s uc u es. Howe e , his issue seems o be
localized, and, o e all, he colou s signi ican ly mix a he
de ice's ou le . This sugges s ha he 90°o ien ed s uc u es
do enhance he e iciency o dye mixing compa ed o he
p e ious con igu a ion wi hou mic os uc u es. Ne e heless,
i is wo h no ing ha he p esence o blue colou dominance
on he igh side could be a ibu ed o speci ic low pa e ns
o une en dis ibu ion o s uc u es wi hin he de ice. In he
nex column, co esponding o s uc u es o ien ed a 20°
(Fig. 7d), e ec i e mixing is also obse ed a he de ice's
ou le . Ne e heless, isible s eams o yellow colou ,
eminiscen o he blue s eams in he p e ious mic omixe ,
indica e ha he e a e s ill a eas whe e mixing is no en i ely
uni o m. Finally, he mic omixe ea u ing 45°o ien ed
g oo es, as illus a ed in Fig. 7e, demons a es he mos
p omising esul s, wi h an ex emely homogeneous mix u e
a he ou le . In his case, he g een colou a he ou pu
exhibi s he g ea es uni o mi y obse ed, su passing all
o he low con igu a ions.
In summa y, he 45°con igu a ion appea s o o e come
he limi a ions obse ed in he p e ious images, o e ing a
high deg ee o mix u e homogenei y. This makes i a
p omising choice o luid mixing applica ions in
mic omixe s. Howe e , i 's impo an o no e ha hese
expe imen al esul s a e la gely quali a i e, making i
challenging o d aw de ini i e conclusions. As such,
quan i a i e me hods a e needed o gi e us a be e
unde s anding o he sys em beha iou .
3.4 Nume ical simula ions
3.4.1 Simula ion o manu ac u ed mic omixe s. To
quan i a i ely assess he e ec i eness o he mic omixe s,
simula ions we e conduc ed on he manu ac u ed
mic omixe s (Fig. 8) and mixing his og ams we e ob ained.
The wa e olume ac ion is ep esen ed in he x-axis o wo
Fig. 7 a) Expe imen al se up used in he low assays whe e wo dyes
(blue and yellow) we e mixed in he mic omixe s unde an op ical
mic oscope. Op ical mic oscopy images (5×) o he mixing es inside
manu ac u ed mic omixe s ea u ing b) none, c) 90°,d)20°, and e) 45°
o ien ed g oo es. Scale ba : 500 μm.
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c oss sec ions loca ed a wo dis ances, s a ing om he
junc ion: x
1
=−5.1 mm ( ed his og am) and x
2
=−9.5 mm
(blue his og am), a bi a ily chosen. On he o he hand, he
y-axis illus a es he pe cen age o coun s eco ded a hese
wo sec ions. Fo he sec ions, hei colou scaleba
ep esen s he olume ac ion o wa e in each pixel, which
can be in e p e ed as he deg ee o mixing. Volume ac ions
close o g een colou ed pixels indica e a balanced coexis ence
o bo h liquids, while eddish o bluish pixels ep esen he
sole p esence o one phase.
To quan i a i ely assess he deg ee o mixing in each
his og am, we de ine he e icien mixing index (EMI) as
EMI
¼
numbe o coun s wi h wa e olume ac ion be ween 0:4–0:6
o al numbe o coun s
Hence o h, he EMI will be calcula ed a he x
2
c oss sec ion,
as a highe deg ee o mix u e is expec ed gi en he highe
dis ance om he luids' mee ing poin .
Fig. 8a shows a ans e sal sec ion o he mic omixe
wi hou s uc u al elemen s a he bo om (ac ing as a
con ol). The c oss sec ions e eal poo liquid mixing
pe o mance, which ag ees wi h he absence o g een pixels.
The his og am shows nea ly iden ical la e al dis ibu ions o
bo h c oss sec ions ac oss he mic ochannel, sepa a ed wi h
a gap co esponding o he ange o 0.4–0.5 wa e olume
ac ion. An EMI = 0.100 was ob ained, implying ha
comple e mixing is no achie ed.
Fig. 8b illus a es he ini ially p oposed mic omixe
ea u ing angled s uc u es a a 90°o ien a ion, designed o
enhance he mixing p ocess. In his con igu a ion, he
his og ams display a mo e cen alized pa e n, signi ying ha
a no o ious po ion o he sample is in a s a e o mixing. A
posi ion x
1
, he majo i y o da a poin s all wi hin he ange
o 0.2 o 0.8 wa e olume ac ion, while a posi ion x
2
, he
deg ee o mix u e inc eases eaching an EMI = 0.588. I 's
wo h no ing ha he his og ams show a subs an ial po ion
o he sample composed o pu e wa e (be ween 0.9 and 1),
accoun ing o 15% a x
1
and almos 20% a x
2
. This
phenomenon is pa icula ly no iceable in he c oss-sec ion,
whe e i becomes e iden ha in he lowe egion o he
mixe , co esponding o he g oo es, wa e becomes apped.
This can be explained by he highe densi y o he wa e used
in he simula ions (ρ
1
= 998.2 kg m
−3
) compa ed o he o he
luid (ρ
2
= 997.2 kg m
−3
), causing he luid dynamics o
p omo e downwa d low and p e en ing e ec i e in e ac ion
be ween he samples. Ne e heless, i we dis ega d he egion
con aining he g oo es, he mix u e is highly a ou able a
posi ion x
2
, as indica ed by he g een colou in he op
egion, co esponding o 0.5 wa e olume ac ion. In he
case o he mic omixe ea u ing 20°angled s uc u es
(Fig. 8c), a signi ican imp o emen in mixing pe o mance
was obse ed compa ed o he wo p e ious cases discussed.
A posi ion x
1
, he his og am dis ibu ion shows a wide
p o ile, and he la ges ba o he his og am (27%)
co esponds o poin s clus e ing a ound 0.6 and 0.8 wa e
olume ac ion.
Howe e , he mic omixe demons a es i s mos
p omising ou comes, showcasing an EMI = 0.846 a posi ion
x
2
, which is closes o achie ing comple e mixing. This
subs an ial imp o emen is isually e iden in he c oss-
sec ional iews, whe e colou a ia ions pe sis a x
1
bu
disappea a x
2
, p esen ing a ully homogeneous dis ibu ion.
Ne e heless, a phenomenon like he p e ious case is
obse ed, wi h he lowe s uc u es ending o ap wa e .
None heless, i his lowe egion is excluded, he esul s a e
o o al mix u e.
Fig. 8 Top iew, ans e sal sec ion, and his og ams o he olume ac ion ( om 0 o 1) o wo low samples inside a) an unpa e ned
mic ochannel and manu ac u ed mic omixe s ea u ing b) 90°,c)20°and d) 45°mic opa e ned g oo es. The his og ams co espond o wo
di e en poin s o he mic omixe , x
1
=−5.1 mm ( ed) and x
2
=−9.5 mm (blue). The co esponding e icien mixing index (EMI) is p esen ed o
each his og am.
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