A mi
ro�uidi
 devi
e based on droplet storage
for s
reening solubility diagrams
Philippe Laval,
Ni
olas Lisai, Jean-Baptiste Salmon, and Mathieu Joani
ot
LOF, unité mixte Rhodia�CNRS�Bordeaux 1, 178 avenue du Do
teur S
hweitzer, F�33608 Pessa
 
edex � FRANCE
(Dated: O
tober 25, 2018)
This work des
ribes a new mi
ro�uidi
 devi
e developed for rapid s
reening of solubility diagrams.
In several parallel 
hannels, hundreds of nanoliter-volume droplets of a given solution are �rst stored
with a gradual variation in the solute 
on
entration. Then, the appli
ation of a temperature gradient
along these 
hannels enables us to read dire
tly and quantitatively phase diagrams, 
on
entration
vs. temperature. We show, using a solution of adipi
 a
id, that we 
an measure ten points of the
solubility 
urve in less than 1 hr and with only 250 µL of solution.
I. INTRODUCTION
Chemistry, biology, and pharma
ology, are fa
ing al-
ways more 
omplex systems depending on multiple pa-
rameters. Therefore their 
omplete investigations take
time and require signi�
ant amounts of produ
ts. In this

ontext, roboti
 �uidi
 workstations have already met a
great su

ess and proved their e�
ien
y for instan
e in
the genome sequen
ing and analysis [1℄. However, these
instruments remain very expensive, need important la-
bor, and the volumes involved (≤ mL) are still too large
for some spe
i�
 appli
ations (e.g. proteomi
s) [2, 3℄.
Nowadays, other high throughput te
hniques based on
mi
ro�uidi
s [4, 5℄ 
an o�er suitable alternative solutions
for the development of rapid s
reening tools. Mi
ro�uidi
devi
es are now largely used in biologi
al and 
hemi
al
�elds for multiple appli
ations [6℄ like mole
ular sepa-
rations and 
ells sorting [7℄, polymerase 
hain rea
tion
[8, 9℄, rapid mi
romixing and analysis of 
hemi
al rea
-
tions [10, 11, 12, 13℄. . . Moreover, the development of
mi
rovalves and mi
romixers has made possible the pro-
du
tion of highly integrated systems whi
h 
an be used
to address individually hundreds of rea
tion 
hambers
[14℄. These devi
es are well adapted to 
arry out high
throughput s
reening of phase diagrams, parti
ularly in
the 
ase of protein 
rystallization investigation. However,
their fabri
ation and multiplexing are still 
ompli
ated.
Another possible strategy is the use of droplets playing
the role of nanoliter-sized rea
tion 
ompartments. These
droplets 
an be produ
ed in spe
i�
 mi
ro�uidi
 geome-
tries [15℄, and their volume and 
hemi
al 
omposition 
an
be �xed in a 
ontrolled way. In addition, they also allow
a rapid mixing of the di�erent 
ompounds, prevent from
any hydrodynami
 dispersion and 
ross 
ontamination,
and 
an be stored in mi
ro
hannels (see Ref. [16℄ and
referen
es therein). Su
h a strategy has already proved
to be useful for 
rystallization studies: e.g. s
reening
of protein 
rystallization 
onditions [17, 18℄, or 
rystal
nu
leation kineti
s measurements [19℄.
Figure 1 summarizes the main insights of our work.
Ele
troni
 address: philippe.laval-exterieur�eu.rhodia.
om
We have engineered a new mi
ro�uidi
 
hip that allow
a dire
t and quantitative reading of two-dimensional di-
agrams. Hundreds of nanoliter-sized droplets of di�er-
ent 
hemi
al 
ompositions 
an be stored in parallel mi-

ro
hannels, and a temperature gradient applied along
these 
hannels enables us to obtain a two-dimensional
array of droplets of di�erent 
on
entrations and tempera-
tures. For solubility diagram s
reening, droplets 
ontain-
ing a given solute are �rst stored with a gradual variation
of 
on
entration. Then, 
rystallization in the droplets is
indu
ed by 
ooling, and �nally, the appli
ation of an ad-
equat temperature gradient dissolves 
rystals in droplets
whose temperature is higher than their solubility tem-
perature. As a result, we dire
tly read the limit between
droplets with and without 
rystals as shown on Fig. 1(
),
whi
h gives the solubility temperatures of the solution at
the di�erent 
on
entrations.
In the materials and methods se
tion, we des
ribe the
mi
ro�uidi
 devi
e, the method used to store the droplets
in the 
hannels, and the temperature 
ontrol setup. We
also 
hara
terize the 
on
entration and temperature gra-
dients. In the last se
tion, we present an experimental
proto
ol to measure quantitatively solubility diagrams
using this devi
e. We demonstrate its e�
ien
y by mea-
suring with only 250 µL of solution, the solubility 
urve
of an organi
 
ompound.
II. MATERIALS AND METHODS
A. Mi
rofabri
ation
The mi
ro�uidi
 devi
e is fabri
ated in
poly(dimethylsiloxane) (PDMS) by using soft-
lithographi
 te
hniques [20℄. PDMS (Sili
one Elastomer
Base, Sylgard 184; Dow Corning) is molded on master
fabri
ated on a sili
on wafer (3-In
h-Si-Wafer; Siegert
Consulting e.k.) using a negative photoresist (SU-8 2100;
Mi
roChem). To make molds of 500 µm height, we spin
su

essively two 250 µm thi
k SU-8 layers on the wafer.
After ea
h spin
oating pro
ess, the wafer is soft-baked
(10 min/65
C and 60 min/95
C). Photolithography is
used to de�ne negative images of the mi
ro
hannels.
Eventually, the wafer is hard-baked (25 min/95
C) and
http://arxiv.org/abs/0704.0569v1
mailto:philippe.laval-exterieur@eu.rhodia.com
(a) 1
FIG. 1: (a) Design of the mi
ro�uidi
 devi
e (
hannels width
500 µm). Sili
one oil is inje
ted in inlet 1 and aqueous so-
lutions in inlets 2 and 3. The two dotted areas indi
ate the
positions of the two Peltier modules used to apply tempera-
ture gradients ∇T . The three lines of dots mark the positions
of temperature measurements. (b) Pi
ture of the mi
ro�uidi

hip made of PDMS sealed with a glass slide (76×52 mm
improve 
larity. Droplets 
ontaining a 
olored dye at di�erent

on
entrations are stored in the ten parallel 
hannels. (
) Ex-
ample of dire
t reading of a solubility diagram. The droplets

ontain an organi
 solute. The dotted line bounding droplets

ontaining 
rystals give an estimation of the solubility limit
(see se
tion Results for details).
developed (SU-8 Developer; Mi
roChem). A mixture
10:1 of PDMS is molded on the SU-8 master des
ribed
above (65
C/60 min). The 
rossed linked PDMS layer
is then peeled o� the mold and holes for the inlets
and outlets (1/32 and 1/16 in. o.d.) are pun
hed into
the material. Then, the PDMS surfa
e and a 
lean
sili
on wafer surfa
e (3-In
h-Si-Wafer; 500 µm; Siegert
Consulting e.k.) are a
tivated for 2 min in a UV ozone
apparatus (UVO Cleaner, Model 144AX; Jelight) and
brought together. Finally, the devi
e is pla
ed at 65
for 2 hr to improve the sealing.
B. Droplet storage proto
ol
The devi
e, presented on Fig. 1(a), is 
omposed of
three inlets and ten outlets lo
ated at the extremities
of 
hannels 
1 to 
10. As shown on Fig. 1(b), ea
h out-
let is 
onne
ted to a ≈ 20 
m long rigid tubing (FEP
1/16 in.) ended with a pie
e of soft PVC tubing (Nal-
gene, ≈ 5 
m long) inserted in an automated pin
h ele
-
trovalve (105S�01059P; As
o Jou
omati
). Thanks to
this system, ea
h outlet 
an be independently 
losed or
opened by pin
hing or not the 
orresponding PVC tub-
ing. However, the pin
hing out of a tube leads in a liquid
displa
ement. To minimize the subsequent liquid distur-
ban
e in the mi
ro
hannels, the ele
trovalves are pla
ed

lose to the rigid ones, and the hydrodynami
 resistan
e
after the ele
trovalves is kept as weak as possible using
large tubing.
Sili
one oil (500 
St; Rhodorsil) is inje
ted in inlet 1 at

onstant �ow rate Q1 ≈ 3 mL hr
, and aqueous phases
are inje
ted at �ow rates Q2 and Q3 ranging from 0 to
about 1 mL hr
, in inlets 2 and 3 respe
tively. All
liquids are inje
ted with syringe pumps (PHD 2000 infu-
sion; Harvard Apparatus). At the interse
tion between
the oil and the aqueous streams, monodisperse droplets
of the aqueous phase in oil are 
ontinuously produ
ed
[21℄. Both the droplet volume (about 100�300 nL) and
their produ
tion frequen
y (typi
ally between one to ten
droplets per se
ond) 
an be tuned by the ratio of oil to
aqueous phase �ow rates. The droplet 
omposition is
monitored by the ratio Q2/Q3.
Thanks to the possible opening and 
losing of ea
h out-
let, we 
an store droplets of given aqueous 
ompositions
in the di�erent storage 
hannels 
i. Several steps are ne
-
essary to perform su
h a �lling. First, all the 
hannels
are initially �lled with sili
one oil. Se
ondly, the outlet of

hannel 
1 is opened and all the others are 
losed. In this

on�guration, all the droplets of a given 
omposition �ow
through 
1. Finally, on
e the �ow is stable, the outlet of

1 is suddenly 
losed and simultaneously, the outlet of

hannel 
2 is opened. All the droplets previously present
in 
1 stay immobilized whereas the other droplets, whose

omposition 
an be 
hanged, �ow through 
2. Su

es-
sively, in the same way, we 
an store droplets of various

ompositions in all 
hannels 
i.
C. Chemi
al 
omposition 
ontrol
For solubility investigations, the 
ontrol of the 
on-

entrations in the droplets is 
ru
ial. However, be
ause
of PDMS elasti
ity and syringe pumps pre
ision, an in-
a

ura
y in droplet 
on
entration remains. To estimate
this error, we have performed investigations with a 
on-
fo
al Raman mi
ros
ope (HR800 Horiba; Jobin-Yvon).
A 50× mi
ros
ope obje
tive was used for fo
using a
532 nm wavelength laser beam in the droplets, and for

olle
ting Raman s
attered light, subsequently dispersed
with a grating of 600 lines per millimeter. To minimize
the out-of-fo
us ba
kground signals, we �xed the 
on-
fo
al pinhole at 500 µm. Experiments were performed
on droplets made of two initial aqueous solutions of
K4Fe(CN)6 (0.5 M) and K3Fe(CN)6 (0.5 M) inje
ted in
inlets 2 and 3 respe
tively. These two 
ompounds display
strong and distin
t Raman signals [22℄.
Figure 2 shows three Raman spe
tra measured
in droplets 
ontaining di�erent 
on
entration ratios
RC=[K4Fe(CN)6℄/[K3Fe(CN)6℄. The two bands 
entered
2000 2060 2095 2136 2200
wave number (cm−1)
FIG. 2: Raman spe
tra of droplets 
ontaining di�erent 
on-

entration ratios RC of potassium ferro
yanide K4Fe(CN)6
and potassium ferri
yanide K3Fe(CN)6. (a) RC = 0 (b)
RC = 1 (
) RC = 9.
at 2060 and 2095 
m

orrespond to K3Fe(CN)6 and
the one at 2136 
m

orresponds to K4Fe(CN)6. The

on
entration of ea
h 
ompound 
an be probed from the
area under their spe
i�
 Raman bands by:
Ai = KiCitV , (1)
where Ai is the area under the Raman band of the 
om-
pound i, Ci its 
on
entration, Ki a spe
i�
 
onstant, t
the a
quisition time, and V the analysis volume. As a

onsequen
e, the ratio RA of the Raman bands areas of
K4Fe(CN)6 and K3Fe(CN)6 is proportional to the 
on-

entrations ratio RC , and does not depend on the a
qui-
sition parameters.
In order to optimize the �lling proto
ol, we �rst use Ra-
man mi
ros
opy to follow the kineti
s of the 
on
entra-
tion stabilization in the droplets after a sudden 
hange in
the aqueous phases �ow rates. Indeed, due to the PDMS
elasti
ity and the inje
tion system (syringe pumps), the
�nite response time of the devi
e does not allow instan-
taneous 
hange of the 
on
entrations. To estimate this
response time, we have performed the following experi-
ment: for t < 0 s, Q2 = 0 and Q3 = 500 µL hr
, and
for t > 0 s, Q2 = Q3 = 250 µL hr
. Droplets �rst
�ow through 
hannel 
1 whi
h is 
losed after 30 s. Then,
droplets are stored in �ve other 
hannels after 1, 2, 4,
6, and 10 min. Thus, Raman spe
tra obtained from the
droplets in the di�erent 
hannels enable us to follow the
evolution of RA as a fun
tion of time after the �ow rates

hange. Figure 3(a) shows it rea
hes almost a 
onstant
value after 60 s meaning the 
on
entrations be
ome sta-
ble after this time. Su
h measurements illustrate that
20 min long proto
ols are e�
ient to store droplets of
desired 
ompositions in the ten 
hannels (≈ 2 min per

hannel).
A se
ond series of experiments was performed to es-
timate and 
hara
terize the 
on
entration gradient we

an apply in the devi
e. The storage 
hannels are �lled
with droplets of di�erent 
on
entrations in K3Fe(CN)6
and K4Fe(CN)6 set from the �ow rates. In ea
h 
hannel

i, to rea
h a stable droplets 
omposition, we maintain
the �ow for 90 s before 
losing the outlet to store them
[see Fig. 3(a)℄. By measuring the Raman spe
tra of the
droplets 
omposition in the di�erent 
hannels, we ob-
tain the ratio RA as a fun
tion of the theoreti
al ratio
= Q2/Q3. The error bar 
orresponds to the stan-
dard deviation of the measurements performed on the
droplets in a given 
hannel. As 
an be seen on Fig. 3(b),
a linear relationship between RA and R
is observed as
expe
ted. Deviations of a few per
ents around the linear
law are probably due to the Raman measurements un-

ertainties, to the a

ura
y of the inje
tion system, and
also to the PDMS elasti
ity.
These Raman measurements demonstrate that with
the developed proto
ol, we are able to store hundreds
of droplets in ten 
hannels in about 20 min, and 
on-
suming less than a few hundreds of µL of solution. We
believe that more rigid and smaller mi
rodevi
es 
om-
bined with even more rea
tive inje
tion system would
de
rease signi
antly the amount of liquids used when �ll-
ing the 
hannels. Other strategies involving for instan
e
droplet generation thanks to integrated mi
rovalves [23℄,
may also proved to be useful to de
rease the required
volumes of solution.
0 1 2 3 4
0 60 200 400 600
t (s)
FIG. 3: (a) Evolution of the ratio RA in the droplets after a
sudden 
hange of the aqueous solutions �ow rates Q2 and Q3.
Before t = 0 s, Q2 = 0 and Q3 = 500 µL hr
. For t > 0 s,
Q2 = Q3 = 250 µL hr
. Between 5 and 30 s, RA are obtained
from three single droplets in 
hannel 
1. After t = 60 s, ea
h
point is a mean value 
al
ulated on several droplets in a given

hannel. (b) Con
entration ratio RA in droplets as a fun
tion
of the 
on
entration ratio R
C determined from the aqueous
solutions �ow rates. The dotted line 
orresponds to the linear
�t of the data.
D. Temperature 
ontrol
The temperature �eld of the 
hip is 
ontrolled with two
Peltier modules (30×30×3.3 mm
; CP1.4�71�06L; Mel-

or) pla
ed underneath the wafer at positions marked by
the two dotted areas on Fig. 1(a). Sin
e the two Peltier
modules are independant, we 
an heat or 
ool the de-
vi
e, and also apply important temperature gradients.
We use a sili
on wafer as 
hip support to optimize ther-
mal transfers and thus to 
reate regular temperature gra-
dients along the storage 
hannels. Thin thermo
ouples
(type K, 76 µm o.d., 5SRTC-TTKI-40-1M; Omega) mea-
sure the temperature of the devi
e along three series of
positions parallel to the storage 
hannels. The �rst series
is pla
ed above 
1, the se
ond one between 
5 and 
6, and
the third one below 
10 [see Fig. 1(a)℄. To rea
h the maxi-
mal pre
ision on liquid temperature measurements inside
the 
hannels, the thermo
ouples are inserted in holes pre-
viously pun
hed through the PDMS layer and �lled with
sili
one oil. Thermo
ouples signals are pro
essed with
a data a
quisition instrument (USB�9161; National In-
struments) and LabView software. Figure 4(a) shows
we are able to apply easily temperature gradients up to
C on 5 
m. To estimate the temperature at any po-
0 10 20 30 40
X (mm)
X (mm)C
FIG. 4: Temperature pro�les of the 
hip for a given tem-
perature gradient (a) Temperatures measured along the stor-
age 
hannels with thermo
ouples inserted through the PDMS
layer at di�erent positions shown on Fig. 1(a). (N) measure-
ments series above 
hannel 
1; (�) series between 
5 and 
6;
(H) series below 
10. (b) Interpolated temperature pro�le of
the 
hip.
sitions along the storage 
hannels, we perform a longi-
tudinal and transverse linear interpolation of the three
series of measurements. The �nal pro�le obtained after
su
h interpolation is depi
ted on Fig. 4(b). Note that the
temperature is not perfe
tly homogeneous transversely
to the storage 
hannels. This is due to the size of the
Peltier module as 
ompared to the size of the droplet
storage area: smaller storage area, or larger Peltier mod-
ules, would give homogeneous temperature pro�les along
the transverse dire
tion of the 
hannels.
III. RESULTS
In the previous se
tion we have shown that our mi-

rodevi
e allows us to build a two-dimensional array of
droplets with both 
on
entration and temperature gra-
dients. We now present an appli
ation of this 
hip by
measuring the solubility 
urve of an organi
 solute.
Su
h measurements are 
arried out with an adipi
 a
id
solution previously prepared in a beaker. It is made of
10.14 g of adipi
 a
id (99%; Aldri
h) in 50.66 g of deion-
ized water. The solubility temperature of the solution
is 63
C. To avoid any 
rystallization before the droplets
formation, the syringe 
ontaining the solution and the

orresponding tubing are heated at about 65
C with two
�exible heaters (Min
o) 
ontrolled with temperature 
on-
trollers (Min
o). A stereo mi
ros
ope (SZX12; Olympus)
with an obje
tive (DF PLFL 0.5× PF; Olympus) enables
us to observe the devi
e during the solubility study.
We inje
t the adipi
 a
id solution in inlet 2 and deion-
ized water in inlet 3. By 
hanging the �ow rates ratio
we �ll the storage 
hannels with droplets whose 
on
en-
tration in adipi
 a
id varies from 20 g / 100 g of water
in 
hannel 
1 down to 6 g / 100 g of water in 
10. The
massi
 
on
entration C in the droplets is 
al
ulated a
-

ording to:
1 + (1 + C0)Q3/Q2
where C0 is the massi
 
on
entration of the initial adipi
a
id solution, Q2 and Q3 the respe
tive �ow rates of the
solution and water (we 
he
ked that density variations in-
du
ed by the presen
e of adipi
 a
id are negligible). The
mi
ro�uidi
 
hip is kept at about 65
C using the Peltier
modules to avoid any 
rystallization during the droplet
storage. Before stopping the droplets in a 
hannel, we
maintain it open for 90 s for �ow stabilization. In these

onditions, the total �lling of the ten 
hannels is rea
hed
in less than 20 min and only 250 µL of solution are spent.
After the droplet storage, 
rystallization is indu
ed by

ooling. Note that the mean time of 
rystal nu
leation
is inversely proportional to the rea
tor volume. Indeed,
the nu
leation frequen
y is given by 1/JV where J is the
nu
leation rate that does no depend on the volume V
of the rea
tor (see Refs. [19, 24, 25, 26℄ and referen
es
therein). Crystal nu
leation in a droplet of 100 nL is
thus 10
times longer than in a vial of 1 mL. To redu
e
su
h long indu
tion time, we apply a strong 
ooling to
in
rease signi�
antly the supersaturation. In our 
ase,
down to ≈ −5◦C, 
rystals appear in all the droplets after
a few minutes.
To obtain the solubility 
urve dire
tly on the 
hip, we
then apply a temperature gradient between 32 and 65
after the 
rystallization step. Crystals dissolve in all the
droplets whose temperature is higher than their solubil-
ity temperature. In the other droplets, 
rystals are partly
solubilized but still exist (the equilibrium is rea
hed in
about 20 min). Typi
al images of the storage area are
FIG. 5: Images of a part of the storage area obtained under

rossed polarizers. Droplets of adipi
 a
id solution are stored
in the 
hannels. The 
on
entration in adipi
 a
id was grad-
ually 
hanged between the upper and the bottom 
hannels.
After 
rystallisation of all the droplets, a temperature gradi-
ent is applied (low temperature on the left and high temper-
ature on the right). (a) The dotted line separating droplets

ontaining 
rystals from empty droplets give an estimation
of the solubility limit. (b) Same image but with a di�erent

ontrast displaying the droplets positions.
presented on Fig. 5. Sin
e adipi
 a
id 
rystals have bire-
fringent properties, they are easily dete
ted under 
rossed
polarizers. The smallest dete
table 
rystals size is about
50×50 µm2 at the magni�
ation used. Figure 5(a) en-
ables us to dire
tly observe the limit of 
rystal presen
e.
Using interpolated temperature pro�les su
h as the one
displayed in Fig. 4, allows us to estimate the solubility
temperatures for all the ten 
on
entrations (we 
hoose
them in the middle of the two su
essive droplets with
and without 
rystals). Figure 6 presents su
h solubility
temperatures measured with our mi
ro�uidi
 devi
e. The
error 
orresponds to the temperatures di�eren
e between
the two droplets en
losing the solubility limit positions.
These results are in good agreements with data obtained
from literature [27℄.
Naturally, the errors done on su
h measurements de-
pend on the distan
e between two su

essive droplets,
and on the amplitude of the temperature gradient. In
our 
ase, the temperature gradient of 0.7
a typi
al distan
e of 3 mm between two droplets give an
error of ±1◦C. The appli
ation of smaller temperature
40 45 50 55 60
T (°C)
FIG. 6: (•) Solubility of adipi
 a
id in water measured in the

ase of a temperature gradient of 0.7
. (◦) Solubility
data from literature, the dotted line is a guideline for eyes.
gradients and the redu
tion of the distan
e between two
su

essive droplets would give a better a

ura
y on the
solubility limit.
For the moment, the maximal temperature whi
h 
an
be investigated is limited by the evaporation of water
through the PDMS layer [28℄. Simple measurements
show that the volume of an aqueous droplet stored in
our devi
e at 60
C, de
reases by ≈ 10% in 4 hr. Su
h an
e�e
t is negligible for the experiments des
ribed above
(droplet �lling time 20 min at 65
C), but may explain
the small dis
repan
y observed on Fig. 6 at high temper-
ature. We believe that the use of non-permeable materi-
als su
h as glass, instead of PDMS, 
ould easily broaden
the possibilities o�ered by our system.
IV. CONCLUSION
In this work we have presented a new mi
ro�uidi
tool to perform rapid s
reening of solubility diagrams.
The devi
e enables us to store hundreds of droplets
(≈ 100 nL) of various 
hemi
al 
ompositions in parallel
mi
ro
hannels, and to apply large temperature gradients.
We have demonstrated using a model system (adipi
 a
id
in water), that we 
ould easily and dire
tly a

ess to
ten simultaneous measurements of the solubility 
urve
on a large temperature range, in less than 1 hr, and with
only 250 µL of solution. To 
on
lude, we believe our
devi
e is a suitable tool for solubility diagrams s
reen-
ing, more rapid, with a better temperature 
ontrol, and

heaper than 
lassi
al roboti
 workstations. Su
h a mi-

ro�uidi
 tool may also be useful for many other appli
a-
tions, where two-dimensional s
reening, temperature vs.

omposition, is required.
A
knowledgments
We gratefully thank G. Cristobal, J. Krishnamurti,
J. Leng, and F. Sarrazin for fruitful dis
ussions and 
riti-

al reading of this manus
ript. We also a
knowledge Ré-
gion Aquitaine for funding and support, and the Atelier
Mé
anique of the CRPP for their te
hni
al help.
[1℄ G. H. W. Sanders and A. Manz, Trends Anal. Chem. 19,
364 (2000).
[2℄ J. R. Luft, J. Wol�ey, I. Jurisi
a, J. Glasgow, S. Fortier,
and G. T. DeTitta, J. Cryst. Growth 232, 591 (2001).
[3℄ D. L. Chen and R. F. Ismagilov, Curr. Opin. Chem. Biol.
10, 226 (2006).
[4℄ H. A. Stone, A. D. Stroo
k, and A. Ajdari, Annu. Rev.
Fluid. Me
h. 36, 381 (2004).
[5℄ T. M. Squires and S. R. Quake, Rev. Mod. Phys. 77, 977
(2005).
[6℄ T. Vilkner, D. Janasek, and A. Manz, Anal. Chem. 76,
3373 (2004).
[7℄ N. Min
, C. Futterer, K. D. Dorfman, A. Ban
aud,
C. Gosse, C. Goubault, and J. L. Viovy, Anal. Chem.
76, 3770 (2004).
[8℄ J. Khandurina and A. Guttman, J. Chromatogr. A 943,
159 (2002).
[9℄ M. Chabert, K. D. Dorfman, P. de Cremoux, J. Roer-
aade, and J.-L. Viovy, Anal. Chem. 78, 7722 (2006).
[10℄ A. D. Stroo
k, S. K. Dertinger, A. Ajdari, I. Mezi
, H. A.
Stone, and G. M. Whitesides, S
ien
e 295, 647 (2002).
[11℄ E. M. Chan, A. P. Alivisatos, and R. A. Mathies, J. Am.
Chem. So
. 127, 13854 (2005).
[12℄ J.-B. Salmon, C. Dubro
q, P. Tabeling, S. Charier, D. Al-

or, L. Jullien, and F. Ferrage, Anal. Chem. 77, 3417
(2005).
[13℄ S. A. Khan, A. Gunther, M. A. S
hmidt, and K. F.
Jensen, Langmuir 20, 8604 (2004).
[14℄ T. Thorsen, S. J. Maerkl, and S. R. Quake, S
ien
e 298,
580 (2002).
[15℄ T. Thorsen, R. W. Roberts, F. H. Arnold, and S. R.
Quake, Phys. Rev. Lett. 86, 4163 (2001).
[16℄ H. Song, D. L. Chen, and R. F. Ismagilov, Angew. Chem.
Int. Ed Engl. 45, 7336 (2006).
[17℄ B. Zheng, L. S. Roa
h, and R. F. Ismagilov, J. Am.
Chem. So
. 125, 11170 (2003).
[18℄ J. Shim, G. Cristobal, D. Link, T. Thorsen, and
S. Fraden, Using mi
ro�uidi
s to de
ouple nu
leation and
growth of protein 
rystals, Unpublished Work (2006).
[19℄ P. Laval, J.-B. Salmon, and M. Joani
ot, J. Cryst.
Growth doi:10.1016/j.j
rysgro.2006.12.044 (2007).
[20℄ J. C. M
Donald and G. M. Whitesides, A

. Chem. Res.
35, 491 (2002).
[21℄ S. L. Anna, N. Boutoux, and H. A. Stone, Appl. Phys.
Lett. 82, 364 (2003).
[22℄ G. Cristobal, L. Arbouet, F. Sarrazin, D. Talaga, J.-L.
Bruneel, M. Joani
ot, and L. Servant, Lab Chip 6, 1140
(2006).
[23℄ B. T. Lau, C. A. Baitz, X. P. Dong, and C. L. Hansen,
J. Am. Chem. So
. 129, 454 (2007).
[24℄ A. C. Zettlemoyer, Nu
leation (Mar
el Dekker, New
York, 1969).
[25℄ D. Kash
hiev and G. M. Rosmalen, Cryst. Res. Te
hnol.
38, 555 (2003).
[26℄ J. W. Mullin, Crystallization (Butterworth-Heinemann,
Oxford, 2001), 4th ed.
[27℄ A. Apelblat and E. Manzurola, J. Chem. Thermodynam-
i
s 19, 317 (1986).
[28℄ J. Leng, B. Lonetti, P. Tabeling, M. Joani
ot, and A. Aj-
dari, Physi
al Review Letters 96, 084503 (2006).

  This work describes a new microfluidic device developed for rapid screening
of solubility diagrams. In several parallel channels, hundreds of
nanoliter-volume droplets of a given solution are first stored with a gradual
variation in the solute concentration. Then, the application of a temperature
gradient along these channels enables us to read directly and quantitatively
phase diagrams, concentration vs. temperature. We show, using a solution of
adipic acid, that we can measure ten points of the solubility curve in less
than 1 hr and with only 250 $\mu$L of solution.

A mi
ro�uidi
 devi
e based on droplet storage
for s
reening solubility diagrams
Philippe Laval,
Ni
olas Lisai, Jean-Baptiste Salmon, and Mathieu Joani
ot
LOF, unité mixte Rhodia�CNRS�Bordeaux 1, 178 avenue du Do
teur S
hweitzer, F�33608 Pessa
 
edex � FRANCE
(Dated: O
tober 25, 2018)
This work des
ribes a new mi
ro�uidi
 devi
e developed for rapid s
reening of solubility diagrams.
In several parallel 
hannels, hundreds of nanoliter-volume droplets of a given solution are �rst stored
with a gradual variation in the solute 
on
entration. Then, the appli
ation of a temperature gradient
along these 
hannels enables us to read dire
tly and quantitatively phase diagrams, 
on
entration
vs. temperature. We show, using a solution of adipi
 a
id, that we 
an measure ten points of the
solubility 
urve in less than 1 hr and with only 250 µL of solution.
I. INTRODUCTION
Chemistry, biology, and pharma
ology, are fa
ing al-
ways more 
omplex systems depending on multiple pa-
rameters. Therefore their 
omplete investigations take
time and require signi�
ant amounts of produ
ts. In this

ontext, roboti
 �uidi
 workstations have already met a
great su

ess and proved their e�
ien
y for instan
e in
the genome sequen
ing and analysis [1℄. However, these
instruments remain very expensive, need important la-
bor, and the volumes involved (≤ mL) are still too large
for some spe
i�
 appli
ations (e.g. proteomi
s) [2, 3℄.
Nowadays, other high throughput te
hniques based on
mi
ro�uidi
s [4, 5℄ 
an o�er suitable alternative solutions
for the development of rapid s
reening tools. Mi
ro�uidi
devi
es are now largely used in biologi
al and 
hemi
al
�elds for multiple appli
ations [6℄ like mole
ular sepa-
rations and 
ells sorting [7℄, polymerase 
hain rea
tion
[8, 9℄, rapid mi
romixing and analysis of 
hemi
al rea
-
tions [10, 11, 12, 13℄. . . Moreover, the development of
mi
rovalves and mi
romixers has made possible the pro-
du
tion of highly integrated systems whi
h 
an be used
to address individually hundreds of rea
tion 
hambers
[14℄. These devi
es are well adapted to 
arry out high
throughput s
reening of phase diagrams, parti
ularly in
the 
ase of protein 
rystallization investigation. However,
their fabri
ation and multiplexing are still 
ompli
ated.
Another possible strategy is the use of droplets playing
the role of nanoliter-sized rea
tion 
ompartments. These
droplets 
an be produ
ed in spe
i�
 mi
ro�uidi
 geome-
tries [15℄, and their volume and 
hemi
al 
omposition 
an
be �xed in a 
ontrolled way. In addition, they also allow
a rapid mixing of the di�erent 
ompounds, prevent from
any hydrodynami
 dispersion and 
ross 
ontamination,
and 
an be stored in mi
ro
hannels (see Ref. [16℄ and
referen
es therein). Su
h a strategy has already proved
to be useful for 
rystallization studies: e.g. s
reening
of protein 
rystallization 
onditions [17, 18℄, or 
rystal
nu
leation kineti
s measurements [19℄.
Figure 1 summarizes the main insights of our work.
Ele
troni
 address: philippe.laval-exterieur�eu.rhodia.
om
We have engineered a new mi
ro�uidi
 
hip that allow
a dire
t and quantitative reading of two-dimensional di-
agrams. Hundreds of nanoliter-sized droplets of di�er-
ent 
hemi
al 
ompositions 
an be stored in parallel mi-

ro
hannels, and a temperature gradient applied along
these 
hannels enables us to obtain a two-dimensional
array of droplets of di�erent 
on
entrations and tempera-
tures. For solubility diagram s
reening, droplets 
ontain-
ing a given solute are �rst stored with a gradual variation
of 
on
entration. Then, 
rystallization in the droplets is
indu
ed by 
ooling, and �nally, the appli
ation of an ad-
equat temperature gradient dissolves 
rystals in droplets
whose temperature is higher than their solubility tem-
perature. As a result, we dire
tly read the limit between
droplets with and without 
rystals as shown on Fig. 1(
),
whi
h gives the solubility temperatures of the solution at
the di�erent 
on
entrations.
In the materials and methods se
tion, we des
ribe the
mi
ro�uidi
 devi
e, the method used to store the droplets
in the 
hannels, and the temperature 
ontrol setup. We
also 
hara
terize the 
on
entration and temperature gra-
dients. In the last se
tion, we present an experimental
proto
ol to measure quantitatively solubility diagrams
using this devi
e. We demonstrate its e�
ien
y by mea-
suring with only 250 µL of solution, the solubility 
urve
of an organi
 
ompound.
II. MATERIALS AND METHODS
A. Mi
rofabri
ation
The mi
ro�uidi
 devi
e is fabri
ated in
poly(dimethylsiloxane) (PDMS) by using soft-
lithographi
 te
hniques [20℄. PDMS (Sili
one Elastomer
Base, Sylgard 184; Dow Corning) is molded on master
fabri
ated on a sili
on wafer (3-In
h-Si-Wafer; Siegert
Consulting e.k.) using a negative photoresist (SU-8 2100;
Mi
roChem). To make molds of 500 µm height, we spin
su

essively two 250 µm thi
k SU-8 layers on the wafer.
After ea
h spin
oating pro
ess, the wafer is soft-baked
(10 min/65
C and 60 min/95
C). Photolithography is
used to de�ne negative images of the mi
ro
hannels.
Eventually, the wafer is hard-baked (25 min/95
C) and
http://arxiv.org/abs/0704.0569v1
mailto:philippe.laval-exterieur@eu.rhodia.com
(a) 1
FIG. 1: (a) Design of the mi
ro�uidi
 devi
e (
hannels width
500 µm). Sili
one oil is inje
ted in inlet 1 and aqueous so-
lutions in inlets 2 and 3. The two dotted areas indi
ate the
positions of the two Peltier modules used to apply tempera-
ture gradients ∇T . The three lines of dots mark the positions
of temperature measurements. (b) Pi
ture of the mi
ro�uidi

hip made of PDMS sealed with a glass slide (76×52 mm
improve 
larity. Droplets 
ontaining a 
olored dye at di�erent

on
entrations are stored in the ten parallel 
hannels. (
) Ex-
ample of dire
t reading of a solubility diagram. The droplets

ontain an organi
 solute. The dotted line bounding droplets

ontaining 
rystals give an estimation of the solubility limit
(see se
tion Results for details).
developed (SU-8 Developer; Mi
roChem). A mixture
10:1 of PDMS is molded on the SU-8 master des
ribed
above (65
C/60 min). The 
rossed linked PDMS layer
is then peeled o� the mold and holes for the inlets
and outlets (1/32 and 1/16 in. o.d.) are pun
hed into
the material. Then, the PDMS surfa
e and a 
lean
sili
on wafer surfa
e (3-In
h-Si-Wafer; 500 µm; Siegert
Consulting e.k.) are a
tivated for 2 min in a UV ozone
apparatus (UVO Cleaner, Model 144AX; Jelight) and
brought together. Finally, the devi
e is pla
ed at 65
for 2 hr to improve the sealing.
B. Droplet storage proto
ol
The devi
e, presented on Fig. 1(a), is 
omposed of
three inlets and ten outlets lo
ated at the extremities
of 
hannels 
1 to 
10. As shown on Fig. 1(b), ea
h out-
let is 
onne
ted to a ≈ 20 
m long rigid tubing (FEP
1/16 in.) ended with a pie
e of soft PVC tubing (Nal-
gene, ≈ 5 
m long) inserted in an automated pin
h ele
-
trovalve (105S�01059P; As
o Jou
omati
). Thanks to
this system, ea
h outlet 
an be independently 
losed or
opened by pin
hing or not the 
orresponding PVC tub-
ing. However, the pin
hing out of a tube leads in a liquid
displa
ement. To minimize the subsequent liquid distur-
ban
e in the mi
ro
hannels, the ele
trovalves are pla
ed

lose to the rigid ones, and the hydrodynami
 resistan
e
after the ele
trovalves is kept as weak as possible using
large tubing.
Sili
one oil (500 
St; Rhodorsil) is inje
ted in inlet 1 at

onstant �ow rate Q1 ≈ 3 mL hr
, and aqueous phases
are inje
ted at �ow rates Q2 and Q3 ranging from 0 to
about 1 mL hr
, in inlets 2 and 3 respe
tively. All
liquids are inje
ted with syringe pumps (PHD 2000 infu-
sion; Harvard Apparatus). At the interse
tion between
the oil and the aqueous streams, monodisperse droplets
of the aqueous phase in oil are 
ontinuously produ
ed
[21℄. Both the droplet volume (about 100�300 nL) and
their produ
tion frequen
y (typi
ally between one to ten
droplets per se
ond) 
an be tuned by the ratio of oil to
aqueous phase �ow rates. The droplet 
omposition is
monitored by the ratio Q2/Q3.
Thanks to the possible opening and 
losing of ea
h out-
let, we 
an store droplets of given aqueous 
ompositions
in the di�erent storage 
hannels 
i. Several steps are ne
-
essary to perform su
h a �lling. First, all the 
hannels
are initially �lled with sili
one oil. Se
ondly, the outlet of

hannel 
1 is opened and all the others are 
losed. In this

on�guration, all the droplets of a given 
omposition �ow
through 
1. Finally, on
e the �ow is stable, the outlet of

1 is suddenly 
losed and simultaneously, the outlet of

hannel 
2 is opened. All the droplets previously present
in 
1 stay immobilized whereas the other droplets, whose

omposition 
an be 
hanged, �ow through 
2. Su

es-
sively, in the same way, we 
an store droplets of various

ompositions in all 
hannels 
i.
C. Chemi
al 
omposition 
ontrol
For solubility investigations, the 
ontrol of the 
on-

entrations in the droplets is 
ru
ial. However, be
ause
of PDMS elasti
ity and syringe pumps pre
ision, an in-
a

ura
y in droplet 
on
entration remains. To estimate
this error, we have performed investigations with a 
on-
fo
al Raman mi
ros
ope (HR800 Horiba; Jobin-Yvon).
A 50× mi
ros
ope obje
tive was used for fo
using a
532 nm wavelength laser beam in the droplets, and for

olle
ting Raman s
attered light, subsequently dispersed
with a grating of 600 lines per millimeter. To minimize
the out-of-fo
us ba
kground signals, we �xed the 
on-
fo
al pinhole at 500 µm. Experiments were performed
on droplets made of two initial aqueous solutions of
K4Fe(CN)6 (0.5 M) and K3Fe(CN)6 (0.5 M) inje
ted in
inlets 2 and 3 respe
tively. These two 
ompounds display
strong and distin
t Raman signals [22℄.
Figure 2 shows three Raman spe
tra measured
in droplets 
ontaining di�erent 
on
entration ratios
RC=[K4Fe(CN)6℄/[K3Fe(CN)6℄. The two bands 
entered
2000 2060 2095 2136 2200
wave number (cm−1)
FIG. 2: Raman spe
tra of droplets 
ontaining di�erent 
on-

entration ratios RC of potassium ferro
yanide K4Fe(CN)6
and potassium ferri
yanide K3Fe(CN)6. (a) RC = 0 (b)
RC = 1 (
) RC = 9.
at 2060 and 2095 
m

orrespond to K3Fe(CN)6 and
the one at 2136 
m

orresponds to K4Fe(CN)6. The

on
entration of ea
h 
ompound 
an be probed from the
area under their spe
i�
 Raman bands by:
Ai = KiCitV , (1)
where Ai is the area under the Raman band of the 
om-
pound i, Ci its 
on
entration, Ki a spe
i�
 
onstant, t
the a
quisition time, and V the analysis volume. As a

onsequen
e, the ratio RA of the Raman bands areas of
K4Fe(CN)6 and K3Fe(CN)6 is proportional to the 
on-

entrations ratio RC , and does not depend on the a
qui-
sition parameters.
In order to optimize the �lling proto
ol, we �rst use Ra-
man mi
ros
opy to follow the kineti
s of the 
on
entra-
tion stabilization in the droplets after a sudden 
hange in
the aqueous phases �ow rates. Indeed, due to the PDMS
elasti
ity and the inje
tion system (syringe pumps), the
�nite response time of the devi
e does not allow instan-
taneous 
hange of the 
on
entrations. To estimate this
response time, we have performed the following experi-
ment: for t < 0 s, Q2 = 0 and Q3 = 500 µL hr
, and
for t > 0 s, Q2 = Q3 = 250 µL hr
. Droplets �rst
�ow through 
hannel 
1 whi
h is 
losed after 30 s. Then,
droplets are stored in �ve other 
hannels after 1, 2, 4,
6, and 10 min. Thus, Raman spe
tra obtained from the
droplets in the di�erent 
hannels enable us to follow the
evolution of RA as a fun
tion of time after the �ow rates

hange. Figure 3(a) shows it rea
hes almost a 
onstant
value after 60 s meaning the 
on
entrations be
ome sta-
ble after this time. Su
h measurements illustrate that
20 min long proto
ols are e�
ient to store droplets of
desired 
ompositions in the ten 
hannels (≈ 2 min per

hannel).
A se
ond series of experiments was performed to es-
timate and 
hara
terize the 
on
entration gradient we

an apply in the devi
e. The storage 
hannels are �lled
with droplets of di�erent 
on
entrations in K3Fe(CN)6
and K4Fe(CN)6 set from the �ow rates. In ea
h 
hannel

i, to rea
h a stable droplets 
omposition, we maintain
the �ow for 90 s before 
losing the outlet to store them
[see Fig. 3(a)℄. By measuring the Raman spe
tra of the
droplets 
omposition in the di�erent 
hannels, we ob-
tain the ratio RA as a fun
tion of the theoreti
al ratio
= Q2/Q3. The error bar 
orresponds to the stan-
dard deviation of the measurements performed on the
droplets in a given 
hannel. As 
an be seen on Fig. 3(b),
a linear relationship between RA and R
is observed as
expe
ted. Deviations of a few per
ents around the linear
law are probably due to the Raman measurements un-

ertainties, to the a

ura
y of the inje
tion system, and
also to the PDMS elasti
ity.
These Raman measurements demonstrate that with
the developed proto
ol, we are able to store hundreds
of droplets in ten 
hannels in about 20 min, and 
on-
suming less than a few hundreds of µL of solution. We
believe that more rigid and smaller mi
rodevi
es 
om-
bined with even more rea
tive inje
tion system would
de
rease signi
antly the amount of liquids used when �ll-
ing the 
hannels. Other strategies involving for instan
e
droplet generation thanks to integrated mi
rovalves [23℄,
may also proved to be useful to de
rease the required
volumes of solution.
0 1 2 3 4
0 60 200 400 600
t (s)
FIG. 3: (a) Evolution of the ratio RA in the droplets after a
sudden 
hange of the aqueous solutions �ow rates Q2 and Q3.
Before t = 0 s, Q2 = 0 and Q3 = 500 µL hr
. For t > 0 s,
Q2 = Q3 = 250 µL hr
. Between 5 and 30 s, RA are obtained
from three single droplets in 
hannel 
1. After t = 60 s, ea
h
point is a mean value 
al
ulated on several droplets in a given

hannel. (b) Con
entration ratio RA in droplets as a fun
tion
of the 
on
entration ratio R
C determined from the aqueous
solutions �ow rates. The dotted line 
orresponds to the linear
�t of the data.
D. Temperature 
ontrol
The temperature �eld of the 
hip is 
ontrolled with two
Peltier modules (30×30×3.3 mm
; CP1.4�71�06L; Mel-

or) pla
ed underneath the wafer at positions marked by
the two dotted areas on Fig. 1(a). Sin
e the two Peltier
modules are independant, we 
an heat or 
ool the de-
vi
e, and also apply important temperature gradients.
We use a sili
on wafer as 
hip support to optimize ther-
mal transfers and thus to 
reate regular temperature gra-
dients along the storage 
hannels. Thin thermo
ouples
(type K, 76 µm o.d., 5SRTC-TTKI-40-1M; Omega) mea-
sure the temperature of the devi
e along three series of
positions parallel to the storage 
hannels. The �rst series
is pla
ed above 
1, the se
ond one between 
5 and 
6, and
the third one below 
10 [see Fig. 1(a)℄. To rea
h the maxi-
mal pre
ision on liquid temperature measurements inside
the 
hannels, the thermo
ouples are inserted in holes pre-
viously pun
hed through the PDMS layer and �lled with
sili
one oil. Thermo
ouples signals are pro
essed with
a data a
quisition instrument (USB�9161; National In-
struments) and LabView software. Figure 4(a) shows
we are able to apply easily temperature gradients up to
C on 5 
m. To estimate the temperature at any po-
0 10 20 30 40
X (mm)
X (mm)C
FIG. 4: Temperature pro�les of the 
hip for a given tem-
perature gradient (a) Temperatures measured along the stor-
age 
hannels with thermo
ouples inserted through the PDMS
layer at di�erent positions shown on Fig. 1(a). (N) measure-
ments series above 
hannel 
1; (�) series between 
5 and 
6;
(H) series below 
10. (b) Interpolated temperature pro�le of
the 
hip.
sitions along the storage 
hannels, we perform a longi-
tudinal and transverse linear interpolation of the three
series of measurements. The �nal pro�le obtained after
su
h interpolation is depi
ted on Fig. 4(b). Note that the
temperature is not perfe
tly homogeneous transversely
to the storage 
hannels. This is due to the size of the
Peltier module as 
ompared to the size of the droplet
storage area: smaller storage area, or larger Peltier mod-
ules, would give homogeneous temperature pro�les along
the transverse dire
tion of the 
hannels.
III. RESULTS
In the previous se
tion we have shown that our mi-

rodevi
e allows us to build a two-dimensional array of
droplets with both 
on
entration and temperature gra-
dients. We now present an appli
ation of this 
hip by
measuring the solubility 
urve of an organi
 solute.
Su
h measurements are 
arried out with an adipi
 a
id
solution previously prepared in a beaker. It is made of
10.14 g of adipi
 a
id (99%; Aldri
h) in 50.66 g of deion-
ized water. The solubility temperature of the solution
is 63
C. To avoid any 
rystallization before the droplets
formation, the syringe 
ontaining the solution and the

orresponding tubing are heated at about 65
C with two
�exible heaters (Min
o) 
ontrolled with temperature 
on-
trollers (Min
o). A stereo mi
ros
ope (SZX12; Olympus)
with an obje
tive (DF PLFL 0.5× PF; Olympus) enables
us to observe the devi
e during the solubility study.
We inje
t the adipi
 a
id solution in inlet 2 and deion-
ized water in inlet 3. By 
hanging the �ow rates ratio
we �ll the storage 
hannels with droplets whose 
on
en-
tration in adipi
 a
id varies from 20 g / 100 g of water
in 
hannel 
1 down to 6 g / 100 g of water in 
10. The
massi
 
on
entration C in the droplets is 
al
ulated a
-

ording to:
1 + (1 + C0)Q3/Q2
where C0 is the massi
 
on
entration of the initial adipi
a
id solution, Q2 and Q3 the respe
tive �ow rates of the
solution and water (we 
he
ked that density variations in-
du
ed by the presen
e of adipi
 a
id are negligible). The
mi
ro�uidi
 
hip is kept at about 65
C using the Peltier
modules to avoid any 
rystallization during the droplet
storage. Before stopping the droplets in a 
hannel, we
maintain it open for 90 s for �ow stabilization. In these

onditions, the total �lling of the ten 
hannels is rea
hed
in less than 20 min and only 250 µL of solution are spent.
After the droplet storage, 
rystallization is indu
ed by

ooling. Note that the mean time of 
rystal nu
leation
is inversely proportional to the rea
tor volume. Indeed,
the nu
leation frequen
y is given by 1/JV where J is the
nu
leation rate that does no depend on the volume V
of the rea
tor (see Refs. [19, 24, 25, 26℄ and referen
es
therein). Crystal nu
leation in a droplet of 100 nL is
thus 10
times longer than in a vial of 1 mL. To redu
e
su
h long indu
tion time, we apply a strong 
ooling to
in
rease signi�
antly the supersaturation. In our 
ase,
down to ≈ −5◦C, 
rystals appear in all the droplets after
a few minutes.
To obtain the solubility 
urve dire
tly on the 
hip, we
then apply a temperature gradient between 32 and 65
after the 
rystallization step. Crystals dissolve in all the
droplets whose temperature is higher than their solubil-
ity temperature. In the other droplets, 
rystals are partly
solubilized but still exist (the equilibrium is rea
hed in
about 20 min). Typi
al images of the storage area are
FIG. 5: Images of a part of the storage area obtained under

rossed polarizers. Droplets of adipi
 a
id solution are stored
in the 
hannels. The 
on
entration in adipi
 a
id was grad-
ually 
hanged between the upper and the bottom 
hannels.
After 
rystallisation of all the droplets, a temperature gradi-
ent is applied (low temperature on the left and high temper-
ature on the right). (a) The dotted line separating droplets

ontaining 
rystals from empty droplets give an estimation
of the solubility limit. (b) Same image but with a di�erent

ontrast displaying the droplets positions.
presented on Fig. 5. Sin
e adipi
 a
id 
rystals have bire-
fringent properties, they are easily dete
ted under 
rossed
polarizers. The smallest dete
table 
rystals size is about
50×50 µm2 at the magni�
ation used. Figure 5(a) en-
ables us to dire
tly observe the limit of 
rystal presen
e.
Using interpolated temperature pro�les su
h as the one
displayed in Fig. 4, allows us to estimate the solubility
temperatures for all the ten 
on
entrations (we 
hoose
them in the middle of the two su
essive droplets with
and without 
rystals). Figure 6 presents su
h solubility
temperatures measured with our mi
ro�uidi
 devi
e. The
error 
orresponds to the temperatures di�eren
e between
the two droplets en
losing the solubility limit positions.
These results are in good agreements with data obtained
from literature [27℄.
Naturally, the errors done on su
h measurements de-
pend on the distan
e between two su

essive droplets,
and on the amplitude of the temperature gradient. In
our 
ase, the temperature gradient of 0.7
a typi
al distan
e of 3 mm between two droplets give an
error of ±1◦C. The appli
ation of smaller temperature
40 45 50 55 60
T (°C)
FIG. 6: (•) Solubility of adipi
 a
id in water measured in the

ase of a temperature gradient of 0.7
. (◦) Solubility
data from literature, the dotted line is a guideline for eyes.
gradients and the redu
tion of the distan
e between two
su

essive droplets would give a better a

ura
y on the
solubility limit.
For the moment, the maximal temperature whi
h 
an
be investigated is limited by the evaporation of water
through the PDMS layer [28℄. Simple measurements
show that the volume of an aqueous droplet stored in
our devi
e at 60
C, de
reases by ≈ 10% in 4 hr. Su
h an
e�e
t is negligible for the experiments des
ribed above
(droplet �lling time 20 min at 65
C), but may explain
the small dis
repan
y observed on Fig. 6 at high temper-
ature. We believe that the use of non-permeable materi-
als su
h as glass, instead of PDMS, 
ould easily broaden
the possibilities o�ered by our system.
IV. CONCLUSION
In this work we have presented a new mi
ro�uidi
tool to perform rapid s
reening of solubility diagrams.
The devi
e enables us to store hundreds of droplets
(≈ 100 nL) of various 
hemi
al 
ompositions in parallel
mi
ro
hannels, and to apply large temperature gradients.
We have demonstrated using a model system (adipi
 a
id
in water), that we 
ould easily and dire
tly a

ess to
ten simultaneous measurements of the solubility 
urve
on a large temperature range, in less than 1 hr, and with
only 250 µL of solution. To 
on
lude, we believe our
devi
e is a suitable tool for solubility diagrams s
reen-
ing, more rapid, with a better temperature 
ontrol, and

heaper than 
lassi
al roboti
 workstations. Su
h a mi-

ro�uidi
 tool may also be useful for many other appli
a-
tions, where two-dimensional s
reening, temperature vs.

omposition, is required.
A
knowledgments
We gratefully thank G. Cristobal, J. Krishnamurti,
J. Leng, and F. Sarrazin for fruitful dis
ussions and 
riti-

al reading of this manus
ript. We also a
knowledge Ré-
gion Aquitaine for funding and support, and the Atelier
Mé
anique of the CRPP for their te
hni
al help.
[1℄ G. H. W. Sanders and A. Manz, Trends Anal. Chem. 19,
364 (2000).
[2℄ J. R. Luft, J. Wol�ey, I. Jurisi
a, J. Glasgow, S. Fortier,
and G. T. DeTitta, J. Cryst. Growth 232, 591 (2001).
[3℄ D. L. Chen and R. F. Ismagilov, Curr. Opin. Chem. Biol.
10, 226 (2006).
[4℄ H. A. Stone, A. D. Stroo
k, and A. Ajdari, Annu. Rev.
Fluid. Me
h. 36, 381 (2004).
[5℄ T. M. Squires and S. R. Quake, Rev. Mod. Phys. 77, 977
(2005).
[6℄ T. Vilkner, D. Janasek, and A. Manz, Anal. Chem. 76,
3373 (2004).
[7℄ N. Min
, C. Futterer, K. D. Dorfman, A. Ban
aud,
C. Gosse, C. Goubault, and J. L. Viovy, Anal. Chem.
76, 3770 (2004).
[8℄ J. Khandurina and A. Guttman, J. Chromatogr. A 943,
159 (2002).
[9℄ M. Chabert, K. D. Dorfman, P. de Cremoux, J. Roer-
aade, and J.-L. Viovy, Anal. Chem. 78, 7722 (2006).
[10℄ A. D. Stroo
k, S. K. Dertinger, A. Ajdari, I. Mezi
, H. A.
Stone, and G. M. Whitesides, S
ien
e 295, 647 (2002).
[11℄ E. M. Chan, A. P. Alivisatos, and R. A. Mathies, J. Am.
Chem. So
. 127, 13854 (2005).
[12℄ J.-B. Salmon, C. Dubro
q, P. Tabeling, S. Charier, D. Al-

or, L. Jullien, and F. Ferrage, Anal. Chem. 77, 3417
(2005).
[13℄ S. A. Khan, A. Gunther, M. A. S
hmidt, and K. F.
Jensen, Langmuir 20, 8604 (2004).
[14℄ T. Thorsen, S. J. Maerkl, and S. R. Quake, S
ien
e 298,
580 (2002).
[15℄ T. Thorsen, R. W. Roberts, F. H. Arnold, and S. R.
Quake, Phys. Rev. Lett. 86, 4163 (2001).
[16℄ H. Song, D. L. Chen, and R. F. Ismagilov, Angew. Chem.
Int. Ed Engl. 45, 7336 (2006).
[17℄ B. Zheng, L. S. Roa
h, and R. F. Ismagilov, J. Am.
Chem. So
. 125, 11170 (2003).
[18℄ J. Shim, G. Cristobal, D. Link, T. Thorsen, and
S. Fraden, Using mi
ro�uidi
s to de
ouple nu
leation and
growth of protein 
rystals, Unpublished Work (2006).
[19℄ P. Laval, J.-B. Salmon, and M. Joani
ot, J. Cryst.
Growth doi:10.1016/j.j
rysgro.2006.12.044 (2007).
[20℄ J. C. M
Donald and G. M. Whitesides, A

. Chem. Res.
35, 491 (2002).
[21℄ S. L. Anna, N. Boutoux, and H. A. Stone, Appl. Phys.
Lett. 82, 364 (2003).
[22℄ G. Cristobal, L. Arbouet, F. Sarrazin, D. Talaga, J.-L.
Bruneel, M. Joani
ot, and L. Servant, Lab Chip 6, 1140
(2006).
[23℄ B. T. Lau, C. A. Baitz, X. P. Dong, and C. L. Hansen,
J. Am. Chem. So
. 129, 454 (2007).
[24℄ A. C. Zettlemoyer, Nu
leation (Mar
el Dekker, New
York, 1969).
[25℄ D. Kash
hiev and G. M. Rosmalen, Cryst. Res. Te
hnol.
38, 555 (2003).
[26℄ J. W. Mullin, Crystallization (Butterworth-Heinemann,
Oxford, 2001), 4th ed.
[27℄ A. Apelblat and E. Manzurola, J. Chem. Thermodynam-
i
s 19, 317 (1986).
[28℄ J. Leng, B. Lonetti, P. Tabeling, M. Joani
ot, and A. Aj-
dari, Physi
al Review Letters 96, 084503 (2006).
