This document summarizes applications of smart polymers over the last decade. Smart polymers, also known as stimuli-responsive polymers, can change their physical form in response to environmental triggers like temperature, pH, ionic strength, and electric or magnetic fields. The review discusses three common physical forms of smart polymers and their bioengineering applications: 1) linear chains in solution that collapse or dissolve in response to stimuli, 2) covalently cross-linked gels that swell or shrink, and 3) surface-grafted polymers that swell or collapse surfaces. Key applications discussed include bioseparations, protein folding, microfluidics, sensors, and smart surfaces.

1. Prog. Polym. Sci. 32 (2007) 1205–1237
Smart polymers: Physical forms and bioengineering applications
Ashok Kumara,b,ÃÃ, Akshay Srivastavaa
, Igor Yu Galaevb
, Bo Mattiassonb,Ã
a
Department of Biological Sciences and Bioengineering, Indian Institute of Technology Kanpur, 208016-Kanpur, India
b
Department of Biotechnology, Center for Chemistry and Chemical Engineering, Lund University, PO Box 124, SE-22100 Lund, Sweden
Received 22 February 2007; received in revised form 22 May 2007; accepted 22 May 2007
Available online 2 June 2007
Abstract
Smart polymers (SP) have become one important class of polymers and their applications have been increasing
significantly. Last two to three decades have witnessed explosive growth in the subject. SP which are also known as stimuli-
responsive soluble–insoluble polymers or environmentally sensitive polymers have been used in the area of biotechnology,
medicine and engineering. The present review is aimed to highlight the applications of SP when these polymers are
presented in three common physical forms (i) linear free chains in solution where polymer undergoes a reversible collapse
after an external stimulus is applied, (ii) covalently cross-linked reversible gels where swelling or shrinking of the gels can be
triggered by environmental change and (iii) chain adsorbed or surface-grafted form, where the polymer reversibly swells or
collapses on surface, once an external parameter is changed. Though there are number of reviews coming up in this area in
recent times, the present review mainly addresses the developments of SP in the last decade with specific application areas
of bioseparations, protein folding, microfluidics and actuators, sensors, smart surfaces and membranes.
r 2007 Elsevier Ltd. All rights reserved.
Keywords: Smart polymer; Stimuli-responsive polymer; Bioseparation; Protein folding; Smart surfaces and membranes; Microfluidics and
actuators
Contents
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1206
2. Polymers as linear free chains in solution. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1208
2.1. Bioseparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1208
2.1.1. Aqueous two-phase polymer system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1208
2.1.2. Affinity precipitation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1211
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www.elsevier.com/locate/ppolysci
0079-6700/$ - see front matter r 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.progpolymsci.2007.05.003
Abbreviations: AA, acrylic acid; AML, affinity macroligand; ATPS, aqueous two-phase system; ATRP, atom transfer radical
polymerizations; CP, critical point; ConA, concanavalin A; EDTA, ethylenediaminetetraacetic acid; EOPO, ethylene oxide propylene
oxide; ELP, elastin like polymer; IPN, interpenetrating network; LCST, lower critical solution temperature; MAA, methacrylic acid;
NiPAAm, N-isopropylacrylamide; PEG, poly(ethylene glycol); poly(AA), poly(acrylic acid); poly(DMAAM), poly(N, N0
-dimethylacry-
lamide); PMAA, poly(methacrylic acid); PNiPAAm, poly(N-isopropylacrylamide); PVCL, poly(vinylcaprolactam); poly(VDF),
poly(vinylidene fluoride); SP, smart polymers; SPP, 3-[N-(3-methacrylamidopropyl)-N, N-dimethyl] ammonio-propane sulfonate
ÃÃAlso to be corresponded to. Tel.: +91 512 2594010; fax: +91 512 2594051.
ÃCorresponding author. Tel.: +46 46 2228264; fax: +46 46 2224713.
E-mail addresses: ashokkum@iitk.ac.in (A. Kumar), Bo.Mattiasson@biotek.lu.se (B. Mattiasson).

2. 2.2. Protein folding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1214
3. Covalently cross-linked, reversible and physical gels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1216
3.1. Microfluidics and actuators . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1217
3.2. Smart polymer hydrogels as sensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1219
4. SP in chain adsorbed or surface-grafted form (smart surfaces and membranes) . . . . . . . . . . . . . . . . . . . . . . 1221
4.1. Smart surfaces for tissue engineering. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1222
4.2. Smart surfaces for temperature controlled separations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1225
4.3. Smart membranes with controlled porosity: ‘‘chemical valve’’ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1227
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1229
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1229
1. Introduction
Polymers such as proteins, polysaccharides and
nucleic acids are present as basic components in
living organic systems. Synthetic polymers, which
are designed to mimic these biopolymers, have been
developed into variety of functional forms to meet
the industrial and scientific applications. The
synthetic polymers can be classified into different
categories based on their chemical properties. Out
of these, some special types of polymers have
emerged as a very useful class of polymers and
have their own special chemical properties and
applications in various areas. These polymers are
coined with different names, based on their physical
or chemical properties like, ‘‘stimuli-responsive
polymers’’ [1] or ‘‘smart polymers (SP)’’ [2,3] or
‘‘intelligent polymers’’ [4] or ‘‘environmental-sensi-
tive’’ polymers [5]. We shall use further on the name
‘‘smart polymers’’ for such polymer systems in this
review. The characteristic feature that actually
makes them ‘‘smart’’ is their ability to respond to
very slight changes in the surrounding environment.
The uniqueness of these materials lies not only in
the fast macroscopic changes occurring in their
structure but also these transitions being reversible.
The responses are manifested as changes in one or
more of the following—shape, surface characteris-
tics, solubility, formation of an intricate molecular
assembly, a sol-to-gel transition and others. The
environmental trigger behind these transitions can
be either change in temperature [6] or pH shift [7],
increase in ionic strength [7], presence of certain
metabolic chemicals [8], addition of an oppositely
charged polymer [9] and polycation–polyanion
complex formation [10]. More recently, changes in
electric [11] and magnetic field [12], light or
radiation forces [13] have also been reported as
stimuli for these polymers. The physical stimuli,
such as temperature, electric or magnetic fields, and
mechanical stress, will affect the level of various
energy sources and alter molecular interactions at
critical onset points. They undergo fast, reversible
changes in microstructure from a hydrophilic to a
hydrophobic state [14]. These changes are apparent
at the macroscopic level as precipitate formation
from a solution or order-of-magnitude changes in
the size and water content of stimuli-responsive
hydrogels [15]. An appropriate proportion of
hydrophobicity and hydrophilicity in the molecular
structure of the polymer is believed to be required
for the phase transition to occur.
Temperature-sensitive polymers exhibit lower
critical solution temperature (LCST) behavior
where phase separation is induced by surpassing a
certain temperature threshold. Polymers of this type
undergo a thermally induced, reversible phase
transition; they are soluble in a solvent (water) at
low temperatures but become insoluble as the
temperature rises above the LCST [16]. The LCST
corresponds to the region in the phase diagram at
which the enthalpy contribution of water hydrogen-
bonded to the polymer chain becomes less than the
entropic gain of the system as a whole and thus is
largely dependent on the hydrogen-bonding cap-
abilities of the constituent monomer units. In
principle, the LCST of a given polymer can be
‘‘tuned’’ as desired by variation in hydrophilic or
hydrophobic co-monomer content. Thermosensitive
polymers can be classified into different groups
depending on the mechanism and chemistry of the
groups. These are (a) poly(N-alkyl substituted
acrylamides) e.g. poly(N-isopropylacrylamide) with
LCST of 32 1C [17] and (b) poly (N-vinylalkyla-
mides) e.g. poly(N-vinylcaprolactam) with a LCST
of about 32–35 1C according to molecular mass of
polymer [18]. There are other types of temperature-
responsive polymers such as poly(ethylene oxide)106-
poly(propylene oxide)70-poly (ethylene oxide)106
co-polymer [19], which has the trade name Pluronics
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3. F127 and poly lactic acid-co–poly ethylene glycol–
poly lactic acid (PLLA)/PEG/PLLA triblock co-
polymers [20]. Another interesting class of tempera-
ture-responsive polymers have recently emerged
which involves elastin like polymers (ELPs) [21].
The specific LCST of all these different polymeric
systems show potential applications in bioengineer-
ing and biotechnology.
On the other hand in a typical pH-sensitive
polymer, protonation/deprotonation events occur
and impart the charge over the molecule (generally
on carboxyl or amino groups), therefore it depends
strongly on the pH. The pH-induced phase transi-
tion of pH-sensitive polymer tends to be very sharp
and usually switches within 0.2–0.3 unit of pH. Co-
polymers of methylmethacrylate and methacrylic
acid undergo sharp conformational transition and
collapse at low pH around 5, while co-polymers of
methylmethacrylate with dimethylaminoethyl
methacrylate are soluble at low pH but collapse
and aggregate under slightly alkaline conditions.
Other types of responsive polymers involve electric
field [11] and magnetic field [12], the gels of which
can shrink/swell in response to external electric or
magnetic field stimuli. Polythiophene or sulpho-
nated-polystyrene-based conducting polymers have
shown bending in response to external field. The
magnetic field-responsive gel which can be obtained
by dispersing magnetic colloidal particle in poly
(N-isopropylacrylamide-co-poly vinylalcohol) hy-
drogel matrix and get aggregated in external non-
uniform magnetic field [12].
These responses of polymer systems show use-
fulness in bio-related applications such as drug
delivery [5,22], bioseparation [3], chromatography
[4,23,24] and cell culture [25]. Some systems have
been developed to combine two or more stimuli-
responsive mechanisms into one polymer system.
For instance, temperature-sensitive polymers may
also respond to pH changes [26–28]. Two or more
signals could be simultaneously applied in order to
induce response in so called dual-responsive poly-
mer systems [29]. Recently, biochemical stimuli
have been considered as another strategy, which
involves the responses to antigen [30], enzyme [31]
and biochemical agents [32]. There is a great
deal of literature available about different forms
of SP, but it is beyond the scope and aim of
the present review to describe it in detail here.
For more details, readers are advised to go
through some of the recent reviews and book
chapters [33,34].
SP can be categorized into three classes according
to their physical forms (Fig. 1). They are (i) linear
free chains in solution, where polymer undergoes a
reversible collapse after an external stimulus is
applied, (ii) covalently cross-linked gels and rever-
sible or physical gels, which can be either micro-
scopic or macroscopic networks and for which
swelling behavior is environmentally triggered and
(iii) chain adsorbed or surface-grafted form, where
the polymer reversibly swells or collapses on a
surface, converting the interface from hydrophilic to
hydrophobic and vice versa, once a specific external
parameter is modified. SPs in all the three forms—in
solution, as hydrogels and on surfaces can be
conjugated with biomolecules, thereby widening
their potential scope of use in many interesting
ways. Biological molecules that may be conjugated
with SPs include proteins and oligopeptides, sugars
and polysaccharides, single- and double-stranded
oligonucleotides and DNA plasmids, simple lipids
and phospholipids, and other recognition ligands
and synthetic drug molecules. The polymer–biomo-
lecule hybrid system is capable of responding to
biological, physical and chemical stimuli. Hoffman
and colleagues have pioneered the work in combin-
ing SPs with a wide variety of biomolecules [35–38].
The SPs can be conjugated randomly or site-
specifically to protein biomolecules. An earlier
review published in the same journal has described
various forms of stimuli-responsive polymers and
their bioconjugates that have been utilized for
ARTICLE IN PRESS
S
T
I
M
U
L
U
S
Fig. 1. Classification of the polymers by their physical form: (i)
linear free chains in solution where polymer undergoes a
reversible collapse after an external stimulus is applied; (ii)
covalently cross-linked reversible gels where swelling or shrinking
of the gels can be triggered by environmental change; and (iii)
chain adsorbed or surface-grafted form, where the polymer
reversibly swells or collapses on surface, once an external
parameter is changed.
A. Kumar et al. / Prog. Polym. Sci. 32 (2007) 1205–1237 1207

4. different applications [33]. This review focuses on
the various potential applications of SPs within the
above three defined categories. The main aim of the
review is to highlight the recent developments
within the last decade of SPs for applications in
areas like bioseparation, protein folding, microflui-
dics and actuators, chemical valves and tissue
engineering applications.
2. Polymers as linear free chains in solution
In aqueous solution, the delicate balance between
hydrophobic–hydrophilic conditions controls phase
transition of the polymer. As hydrophobic condi-
tions increase the polymer precipitates forming an
altogether different phase. This conversion from
soluble to insoluble form can be achieved by either
reducing the number of hydrogen bonds which the
polymer forms with water or by neutralizing the
electric charges present on the polymeric network.
Aqueous solutions of thermoresponsive polymers
are characterized by an inverse dissolution beha-
vior, their isobaric phase diagrams presenting a
LCST [39–42]. The solutions are homogenous at
low temperature and a phase separation appears
when the temperature exceeds a definite value. The
LCST is the minimum of the phase diagram of the
system, and in the practical cases to be treated in
the following, the phase separation temperatures at
which the phase transition occurs, also called
demixtion, will be denoted ‘‘Td’’ or critical point
(CP). Poly-N-isopropylacrylamide (PNiPAAm)
gained its popularity mainly because of the sharp-
ness of its phase transition, LCST of about 32 1C
which is close to the physiological temperature, and
the easiness to vary its phase separation temperature
by co-polymerization [42,43], addition of salts
[44–46], or addition of surfactants [44,47,48] to the
polymer solution. When heated above 32 1C, the
polymer becomes hydrophobic and precipitates out
from solution and below LCST it becomes com-
pletely soluble because of hydrophilic state and
forms a clear solution. Water-soluble block co-
polymers were prepared from the non-ionic mono-
mer of N-isopropylacrylamide (NiPAAm) and the
zwitterionic monomer 3-[N-(3-methacrylamidopro-
pyl)-N,N-dimethyl] ammonio-propane sulfonate
(SPP) by sequential free radical polymerization via
the reversible addition–fragmentation chain transfer
(RAFT) process. Such block co-polymers with two
hydrophilic blocks exhibit double thermoresponsive
behavior in water: the PNiPAAm block shows a
LCST, whereas the poly-SPP block exhibits an
upper critical solution temperature. Appropriate
design of the block lengths leads to block co-
polymers which stay in solution in the full
temperature range between 0 and 100 1C. Both
blocks of these polymers dissolve in water at
intermediate temperatures, whereas at high tem-
peratures, the PNiPAAm block forms colloidal
hydrophobic associates that are kept in solution
by the poly-SPP block, and at low temperatures, the
poly-SPP block forms colloidal polar aggregates
that are kept in solution by the PNiPAAm block. In
this way, colloidal aggregates which switch rever-
sibly can be prepared in water [49]. Another type of
soluble SPs which respond to microchanges in pH
are the ‘‘pH-responsive polymers’’—such as Eu-
dragit S-100 (co-polymer of methylmethacrylate
and methacrylic acid) and the natural polymer,
chitosan (deacetylated chitin). As the pH is lowered,
these polymers become increasingly protonated and
hydrophobic, and eventually precipitate and this
transition can be sharp. For example Eudragit
S-100 precipitates from aqueous solution on acid-
ification to around pH 5.5 whereas chitosan
precipitates at a relatively higher pH of about 7.
Such class of SP in solution phase has various
applications, such as bioseparation of proteins, cells
and bioparticles and it is also investigated that SP
play a role in the new direction like protein folding.
These application areas are discussed here.
2.1. Bioseparation
The production of macromolecules and separa-
tion of biomolecules in purified form, through the
process of bioseparation needs special efforts to
bring down the overall cost of production and
improve the purity of the product. Use of SP may
contribute to the simple and cost-effective processes
to separate target molecules. The separation of
target substance can be performed in different ways
using these polymers, like aqueous two-phase
polymer system (ATPS), affinity precipitation or
thermoresponsive chromatography. The thermore-
sponsive chromatography comes under smart sur-
faces and membranes section and will be discussed
there.
2.1.1. Aqueous two-phase polymer system
ATPS is an aqueous, liquid–liquid, biphasic
system which is obtained by mixing of aqueous
solution of two polymers, or a polymer and a salt at
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5. appropriate concentrations. ATPS has attracted a
great deal of attention for the fractionation of
various biological substances such as proteins, cells
and some low molecular weight substances because
of its gentleness for biological materials and easy
scale-up features [50,51]. ATPS provides aqueous
environment for the partitioning of biomolecules on
the basis of solubility or affinity. An example of the
ATPS system is illustrated in Fig. 2. Polymers
mainly used in ATPS are poly(ethylene glycol)
(PEG) and dextran or hydrophobically modified
starch, e.g. hydroxypropyl starch (Reppal PES 200)
as a cost-effective alternative. But the major bottle-
neck in this technique has been the separation of
target biomolecule from phase-forming polymer.
This is where SP have provided an appropriate
solution. With the help of SP it is possible to
affect the properties of a separation system.
Furthermore, these polymers are water soluble,
inert and do not have denaturing effects towards
biomolecules. They can be derivatized, e.g. with
charged groups and affinity ligands for specific
binding to target biomolecule. Application of SP as
stimuli-responsive soluble–insoluble polymers for
ligand carriers in ATPS has shown promising
potential [52–54]. The polymer–ligand complex is
specifically partitioned to the top phase and can be
easily recovered by changing the medium condition.
Thermoresponsive polymer separates from water
solution above LCST and can be used in thermo-
separated aqueous two-phase system. The thermo-
responsive polymers used for ATPS include
PNiPAAm, polyvinylcaprolactam (PVCL), cellu-
lose ethers such as ethyl(hydroxyethyl)cellulose
ARTICLE IN PRESS
Fig. 2. Type-specific separation of animal cells in aqueous two-phase systems using antibody conjugates with temperature-sensitive
polymers, PNiPAAm (poly(NIPAM)). Adopted from [53] with permission.
A. Kumar et al. / Prog. Polym. Sci. 32 (2007) 1205–1237 1209

6. (EHEC), ethylene oxide–propylene oxide (EOPO)
random co-polymer and EOPO block co-polymer
[55,56]. There are also other examples wherein
thermoresponsive polymers such as EOPO co-
polymers [57,58] or poly(N-vinylcaprolactam-co-
vinyl imidazole) [54] form two-phase systems with
dextran and have been used to purify proteins.
Aqueous two-phase systems have even been formed
with polymers where both polymers are thermo-
responsive and it is possible to recycle both
polymers by temperature-induced phase separation
[59]. This is a modified and improved form of the
ATPS system than the generally used system where
one of the polymers is thermoresponsive and the
other polymer is dextran or a starch derivative. The
polymers mostly used in these works are
EO50PO50, a random co-polymer of 50% ethylene
oxide (EO) and 50% propylene oxide (PO), and a
hydrophobically modified random co-polymer of
EO and PO with aliphatic C14H29–groups coupled
to each end of the polymer (HM–EOPO). In
aqueous solution both polymers will phase separate
above a critical temperature (cloud point for
EO50%PO50% 50 1C, HM–EOPO, 14 1C) and this
will for both polymers lead to formation of an
upper water phase and a lower polymer enriched
phase. When EO50PO50 and HM–EOPO are mixed
in water, the solution will separate in two phases
above a certain concentration i.e. an aqueous two-
phase system is formed analogous to PEG/dextran
system. The partitioning of three proteins, bovine
serum albumin (BSA), lysozyme and apolipoprotein
A-1, has been studied in the EO50PO50/HM–
EOPO system. It was shown that the yield of 78%
and purification factor 5.5 of apolipoprotein A-1
can be achieved [59]. Aqueous two-phase partition-
ing of endo-polygalacturonase (endo-PG) produced
by Kluyveromyces marxianus strains was carried out
on systems containing the thermoseparating poly-
mer Ucon 50-HB-5100 (a random co-polymer of
50% EO and 50% PO) as one of the phase-forming
component. On testing the partitioning efficiency
of the enzyme on different ATPSs comprised of
Ucon 50-HB-5100 (Ucon)/polyvinyl alcohol (PVA
10,000), Ucon 50-HB-5100/hydroxypropyl starch
(Reppal PES100) and Ucon 50-HB-5100/
(NH4)2SO4 it was found that Ucon 50-HB-5100/
(NH4)2SO4 was the most efficient for enzyme
partitioning, in comparison with total protein which
strongly partitioned to the salt-rich phase at 22 1C.
The proposed separation scheme for endo-PG
purification consists of three in series extraction
stages and enables a 10-fold enzyme concentration
while maintaining more than 95% of the initial
enzyme activity. Such system shows cost viability as
compared to many polymer/polymer and polymer/
salt aqueous two-phase extraction systems [60].
Partitioning of pure a-amylase and amyloglucosi-
dase as well as cell-free extract of a hyperthermo-
stable a-amylase in different ATPSs has demon-
strated the potential for partitioning of enzymes
used in extractive bioconversion of starch. The
partition behavior of pure a-amylase and amylo-
glucosidase in four ATPSs, namely, PEO–PPO/
(NH4)2SO4, PEO–PPO/MgSO4, polyethylene glycol
(PEG)/(NH4)2SO4, and PEG/MgSO4 has also been
evaluated [61]. The partitioning behavior of three
proteins (lysozyme, BSA, and apolipoprotein A-1)
in water/HM-EOPO two-phase systems has been
studied and the effect of various ions, pH, and
temperature on protein partitioning was monitored.
This approach has useful potential as it involves
only one polymer for phase formation [62]. BSA
and lysozyme were partitioned in the thermosepa-
rated water/HM-EO two-phase system of the
cationic polymer at different pH, salt and SDS
concentrations [63]. The use of both a low-cost
starch derivative (maltodextrin) as replacement for
dextran and a co-polymer of thermoreactive EOPO
was investigated. The partitioning behavior of three
model proteins: BSA, lysozyme and trypsin was
analyzed in order to evaluate the capability of this
novel ATPS for protein separation and it was found
that the protein recovery was in the range of
60–98% [64]. A new type of ATPS has recently
been established which uses modified starch deriva-
tive and thermoresponsive polymer of VCL as phase
forming polymers [52]. It is also reported that
thermoseparating ATPS for extraction of recombi-
nant cutinase fusion protein from E. coli homo-
genate can be scaled up to pilot scale [65]. The
application of pH-responsive polymers like poly-
ethyleneoxide–maleic acid co-polymer [66] as phase-
forming polymers in ATPS has also been reported.
The polymers, just like proteins, contain two pH-
triggerable functionalities (NH3
+
– and COO–
–) that
make them exhibit pH-responsive behavior. Poly-
diallylaminoethanoate-dimethyl sulfoxide (PAEDS)
co-polymer is a polyelectrolyte that is almost
completely water-insoluble in acidic conditions.
This behavior makes it a potential candidate for
industrial applications since it can be effectively
recovered from solution by pH-controlled precipita-
tion. Furthermore, in applications such as protein
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7. partitioning, the protein-like structure of the poly-
mer is expected to enhance protein–polymer inter-
actions [67].
More interesting application has been shown for
the separation of animal cells by coupling an
antibody (against a cell surface protein) to a tempe-
rature-sensitive SP such as PNiPAAm (Fig. 2). The
ATPS composed of PEG and dextran was devel-
oped where PNiPAAm was used as a ligand carrier
for specific separation of animal cells. Monoclonal
antibodies were conjugated with the carrier and
added to the polyethylene glycol 8000-dextran T500
aqueous two-phase system. About 80% of the
animal cells which specifically bind to the antibody–
polymer conjugate partitioned to the top phase of
ATPS. As a model system, CD34+
-positive human
acute myeloid leukemia cells (KG-1) were specifi-
cally separated from human T lymphoma cells
(Jurkat) by applying anti-CD34 conjugated with
PNiPAAm in the ATPS [53].
2.1.2. Affinity precipitation
The selective precipitation of a target molecule
from a mixture is a very attractive approach in
bioseparations. Precipitation can be highly selective
technique for protein purification or enrichment.
Traditionally precipitation of the target protein is
achieved by the addition of large amounts of salts,
like ammonium sulfate, organic solvents miscible
with water, like acetone or ethanol or by the
addition of polymers, like PEG [68]. It is not
expected to have high selectivity to be achieved by
traditional precipitation techniques, as the selectiv-
ity of precipitation is limited to the differences in
integral surface properties of protein molecules.
Thus, the introduction of high selectivity to the
precipitation techniques is of great importance.
Affinity precipitation of proteins using SP emerged
in the early 1980s. Since then it has evolved as a
technique capable of simple, fast, and efficient
purification of a variety of proteins [69–71]. As a
general rule, there are five basic steps in affinity
precipitation: (i) carrying out affinity interactions in
free solution, (ii) precipitation of the affinity
reagent–target protein complex from the solution,
(iii) recovery of the precipitate, (iv) dissociation and
recovery of the target molecule from the complex,
and finally, (v) recovery of the affinity reagent.
Affinity precipitation methods have two main
approaches which have been described in the
literature [70], as precipitation with homo- and
hetero-bifunctional ligands. However, as the homo-
bifunctional mode of affinity precipitation does not
utilize SP, it thus falls beyond the scope of this
review. Hetero-bifunctional format of affinity pre-
cipitation is a more general approach, wherein
affinity ligands are covalently coupled to soluble–
insoluble polymers to form an affinity macroligand
(AML). The macroligands could be synthesized
either by covalent linking of the ligands (directly or
through a short spacer) or by co-polymerization of
ligands to a water-soluble SP. An ideal polymer for
affinity precipitation must contain reactive groups
for ligand coupling, show moderate interaction with
the ligand or impurities to prevent non-specific co-
precipitation of impurities, give complete phase
separation of the polymer upon a change of medium
property, form polymer precipitates that are com-
pact, to allow easy separation and to exclude
trapping of impurities into a gel structure, be easily
solubilized after the precipitate is formed, the
precipitation–solubilization cycle must be repeata-
ble many times with good recovery, be available and
cost effective.
The polymer–ligand conjugate firstly forms a
complex with the target protein and phase separa-
tion of the complex is triggered by small changes in
environment, resulting in transition of backbone
into an insoluble state. The target protein is then
either eluted from insoluble macroligand–protein
complex or the precipitate is dissolved, the protein
gets dissociated from the macroligand and the
ligand–polymer conjugate is reprecipitated without
the protein which remains in the supernatant in a
purified form (Fig. 3). Various ligands, such as
triazine dyes, sugars, protease inhibitors, antibodies,
and nucleotides have been successfully used for
affinity precipitation.
There is a range of different proteins/enzymes
which have been purified successfully by affinity
precipitation using pH-responsive polymers [72]. In
general, a specific ligand is chemically coupled to the
polymer backbone which latter binds to the target
protein in solution and the protein–polymer com-
plex is precipitated by change of pH as it renders the
polymer backbone insoluble. But in some cases the
polymer itself has the affinity for the target protein
and the polymer acts as a macroligand. Chitosan
was used to precipitate lysozyme or lectins such
as wheat germ agglutinin and similarly Eudragit
S-100 was used as a macroligand for the binding
and precipitating xylanase or lactate dehydrogenase
or endopoly-galacturonase [73,74]. The pH-respon-
sive SP have been used successfully in affinity
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8. precipitation of many proteins, but because of the
charged character of the polymer which shows
some non-specific interactions with other proteins
a more general use has not taken place for these
polymers.
On the other hand, a thermoresponsive polymer
is expected to deliver better performance, because of
its uncharged nature. Their wide spread application
by using metal as affinity ligand has gained
usefulness by adopting the technique in a non-
chromatographic format like metal chelating affinity
precipitation [75–78]. By combining the versatile
properties of metal affinity with affinity precipita-
tion, the technique presents enormous potential as a
selective separation strategy and makes this method
more simple and cost effective when the intended
applications are for large-scale processes. Extensive
efforts are being made in this direction for establish-
ing thermosensitive polymers, PNiPAAm or PVCL
[79] as effective SPs in metal-chelate affinity
precipitation. In metal chelating affinity precipita-
tion, metal ligands like imidazole are covalently
coupled to the reversible soluble–insoluble SP by
radical co-polymerization [80]. The co-polymers
carrying metal-chelating ligands are charged with
metal ions and the target protein binds the metal-
loaded co-polymer in solution via the interaction
between the histidine on the protein and the metal
ion. Many proteins both containing natural metal-
ion binding residues and recombinant proteins
containing His-tag residues have been purified using
metal chelate affinity precipitation [78]. Therefore,
His-tagged protein or cells or bioparticles (with
surface accessible co-ordinating groups) can be
purified through the precipitation of target molecu-
le–metal loaded polymer complex from the mixture.
The precipitated complex is solubilized by reversing
the precipitation conditions and the target molecule
is dissociated from the precipitated polymer by
using imidazole or EDTA as eluting agent. The
biomolecule is recovered from the co-polymer by
precipitating the latter at elevated temperature in
the presence of NaCl. In a recent study, purification
of extracellularly expressed six histidine-tagged
single chain Fv-antibody fragments (His6-scFv
fragments), from recombinant Escherichia coli cell
culture broth was performed. Quantitative precipi-
tation of the His6-scFv fragments was tested at
different loads of the cell supernatant using Cu(II)
and Ni(II) loaded co-polymers of vinylimidazole
ARTICLE IN PRESS
Fig. 3. Scheme of metal chelate affinity precipitation of proteins. Reproduced from [79] with permission.
A. Kumar et al. / Prog. Polym. Sci. 32 (2007) 1205–12371212

9. (VI) and NiPAAm [Cu(II)-poly(VI-NiPAAm) and
Ni(II)-poly(VI-NiPAAm)]. The precipitation effi-
ciency with Ni(II)-poly(VI-NiPAAm) was lower
than for Cu(II)-poly(VI-NiPAAm), but selectivity
was better in the former case. The bound His6-scFv
fragments were recovered almost completely
(495%) by elution with 50 mM EDTA buffer, pH
8.0 [77].
Besides protein purification, the metal-ion
charged co-polymer of poly(VI-NiPAAm) can also
be applied for the separation of single stranded
nucleic acids like RNA from double stranded linear
and plasmid DNA by affinity precipitation [81]. The
separation method utilizes the interaction of metal
ions to the aromatic nitrogens in exposed purines in
single-stranded nucleic acids [82]. Alternatively
plasmid DNA can also be selectively captured at
all scales with an appropriate amount of AML
under appropriate affinity conditions. The AML in
this case is a conjugate between oligomeric AML
precursor and a single-stranded oligonucleotide,
whose sequence is complementary to a specific
affinity motif in the plasmid DNA (triple helix
affinity interaction). By increasing the temperature
the AML-plasmid DNA complex is precipitated.
After filtration and washing, the precipitate is re-
dissolved and the specifically bound plasmid DNA
is released [83].
Similarly lectins, concanavalin A (ConA) and
wheat germ lectin (WGL) when conjugated to
PNiPAAm, these lectin–polymer conjugates were
used in the purification of various polysaccharides
or polysaccharide-containing compounds such as
glucan [84]. The thermally reversible soluble–inso-
luble PNiPAAm–dextran derivative (DD) conjugate
has been synthesized by conjugating amino-termi-
nated PNiPAAm to a DD via ethyl-3-(3-dimethy-
laminopropyl)-carbodiimide and the conjugate was
used as a tool to purify polyclonal antibodies in
serum samples from rabbits subcutaneously immu-
nized with the derivatized dextran [24].
Recently elastin like polymers (ELPs) consisting
of the repeating penta-peptide, VPGVG which
behave very similar to PNiPAAm polymers have
been shown to undergo reversible phase transi-
tions within a wide range of conditions [85,86].
These, ELPs have been used as terminal tags in
recombinant systems to facilitate recombinant
protein purification [87,88] and have recently been
used for conjugating to metal binding ligands for
affinity purification via temperature-triggered pre-
cipitation [89]. ELPs with repeating sequences of
[(VPGVG)2(VPGKG) (VPGVG)2]21 were synthe-
sized and the free amino groups on the lysine
residues were modified by reacting with imidazole-
2-carboxyaldehyde to incorporate the metal-binding
ligands into the ELP biopolymers. Biopolymers
charged with Ni(II) were able to interact with a
His-tag on the target proteins. Purifications of two
His-tagged enzymes, b-D-galactosidase and chlor-
amphenicol acetyltransferase, were used to demon-
strate the application of metal affinity precipitation
using this new type of affinity reagent. The bound
enzymes were easily released by the addition of
either EDTA or imidazole and over 85% recovery
was observed in both cases. The recovered ELPs
were reused with no observable decrease in the
purification performance. This has been the first
report exploiting the features of ELPs for protein
purification based on metal-affinity purification.
The capability of modulating purification condi-
tions by simple temperature triggers and their low
cost of preparation will probably make the ELP-
based metal-affinity precipitation a useful method in
future, not only for protein purification but also for
diverse applications in bioseparation such as DNA
purification and environmental remediation [86].
Another interesting example has been a one-pot
affinity precipitation purification of carbohydrate-
binding protein reported by Sun et al [90]. By
designing thermally responsive glyco-polypeptide
polymers, which were synthesized by selective
coupling of pendant carbohydrate groups to a
recombinant triblock ELP, glyco-affinity precipita-
tion purification of carbohydrate-binding protein
was demonstrated.
Other types of metal-chelating polymers for affinity
precipitation of proteins were reported by synthesizing
highly branched co-polymers of NiPAAm and 1,2-
propandiol-3-methacrylate (GMA: glycerol mono-
methacrylate), poly(NiPAAm-co-GMA) using the
technique of RAFT polymerization using a chain
transfer agent that allows the incorporation of
imidazole functionality in the polymer chain-ends.
The LCST of the co-polymers can be controlled by
the amount of hydrophobic and GMA co-monomers
incorporated during co-polymerization procedures.
The co-polymers demonstrated LCST below 181C
and were successfully used to purify a His-tagged
BRCA-1 protein fragment (a protein implicated in
breast cancer) by affinity precipitation [91,92]. An
interesting example of the use of poly(N-acryloylpi-
peridine) terminally modified with maltose for affi-
nity precipitation of thermolabile a-glucosidase was
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10. demonstrated [93]. The use of the polymer with
extremely low LCST (soluble below at 41C and
completely insoluble above 81C) made it possible
to use the technique for purification of thermolabile
a-glucosidase from cell-free extract of Saccharomyces
cerevisiae achieving 206-fold purification with 68%
recovery.
Affinity precipitation is readily combined with
other protein isolation techniques, e.g. partitioning
in ATPSs [94]. Partitioning of protein complexed
with ligand–polymer conjugate is usually directed to
the upper hydrophobic phase of ATPSs formed by
PEG and dextran/hydroxypropyl starch, whereas
most of the proteins present in crude extracts or cell
homogenates partition into lower hydrophilic
phase. Then the precipitation of the protein–poly-
mer complex is promoted by changing pH. Trypsin
was purified using conjugate of soybean trypsin
inhibitor with hydroxypropylcellulose succinate
acetate [95], lactate dehydrogenase, and protein A
using a conjugate of Eudragit S-100 with the
triazine dye Cibacron Blue [96] and immunoglobu-
lin G [91,92], respectively. The approach was
also successfully demonstrated by the purification
of microbial xylanases, pullulanases, wheat germ
a-amylase, and sweet potato a-amylase [97,98] and
purification of lectins from wheat germ, potato
and tomato. Other attractive extension of this
approach has been to separate animal cells by
crafting the smart AMLs by coupling an antibody
(against a cell surface protein) to a SP [99].
Combination of partitioning with affinity precipita-
tion improves yield and purification factor and
allows easier isolation of protein from particulate
feed streams.
A new concept has recently been introduced
where AML facilitated three-phase partitioning
and develops three-phase partitioning into a more
selective and predictable technique for biosepa-
ration of proteins using smart affinity ligands
[100,101]. In this method, a water-soluble poly-
mer is floated as an interfacial precipitate by
adding ammonium sulfate and tertiary-butanol.
The polymer (appropriately chosen) in the pre-
sence of a protein for which it shows affinity,
selectively binds to the protein and floats as a
polymer–protein complex. By using this approach
wheat germ agglutinin (99% activity recovery
and 40-fold purification) and wheat germ lipase
(94% activity recovery and 27-fold purification)
have been purified using chitosan as a macroaffinity
ligand [102].
2.2. Protein folding
Protein refolding is an important step in the
production of many functional recombinant pro-
teins. Modest changes in the protein’s environment
can bring about structural changes that can affect
its function. Modern DNA cloning techniques have
made possible the over-expression of recombinant
proteins in various host systems. Among the many
systems, the Gram-negative bacterium E. coli has
been the most commonly used system for the
production of heterologous proteins due to ease of
large-scale and high-density cultivation. However,
the use of E. coli for large-scale protein production
is frequently plagued by the formation of insoluble
protein aggregates, in cytoplasm or periplasm, thus
reducing the yield of soluble, active proteins. To
attain the native structure and function of proteins,
the refolding process is a major challenge in
currently ongoing biochemical research. Using
surfactant is a common practice which inhibits
protein aggregation in protein refolding procedure.
The hydrophobicity of the surfactant is the im-
portant factor which facilitates or hinders the
conformational transition of unfolded protein,
depending on the magnitude of the intramolecular
hydrophobic force of the protein. With the appre-
ciation of varying hydrophobicity of the SP, these
polymers have found potentially interesting applica-
tions in the field of protein folding. SP have distinct
advantage over conventional surfactant as their
hydrophobicity can be manipulated simply by
temperature or pH and simple separation of SP
from refolded protein can easily be achieved which
makes them available for the refolding of different
proteins. The utility of SP was studied for protein
refolding in ATPS. The system consisted of
modified PEG bound to functional ligand. PEG
was bound to a thermoreactive hydrophobic head
(poly (propylene oxide)–phenyl group (PPO–Ph).
Refolding of bovine carbonic anhydrase was exam-
ined in the presence of PPO–Ph–PEG at various
temperatures. The refolding yield of carbonic
anhydrase was strongly enhanced and aggregate
formation was suppressed by addition of PPO–Ph–
PEG at a specific temperature of 50–55 1C [103].
An artificial chaperone, which can decrease
protein aggregation and increase reactivation yield
of denatured protein in a fashion similar to that of a
natural chaperone was developed by using SP.
These artificial chaperons have been used to assist
the refolding of bovine carbonic anhydrase using
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A. Kumar et al. / Prog. Polym. Sci. 32 (2007) 1205–12371214

11. molecular assemblies of SP [104]. Since the LCST of
PNiPAAm is only slightly higher than that of the
ambient conditions for protein processing, the
separation and thus recycling of the polymer from
renatured proteins can be easily achieved under
non-denaturing temperature, upon protein refold-
ing. The mechanism of PNiPAAm-assisted protein
folding on bovine carbonic anhydrase B as model
protein was investigated and the results of fluores-
cence analysis and equilibrium studies indicate that
PNiPAAm enhances protein refolding by the for-
mation of complexes with aggregation-prone fold-
ing intermediates via hydrophobic interactions
[105]. Another application of PNiPAAm for the
renaturation of b-lactamase from inclusion bodies
has shown that PNiPAAm was more effective than
PEG in enhancing protein renaturation. At a
concentration of 0.1% (w/v), PNiPAAm improved
the yield of b-lactamase activity by 41% compared
to 26% with PEG [106]. PNiPAAm gels had also
been used in renaturation of lysozyme. With the
addition of fast responsive PNiPAAm gel beads, the
total lysozyme activity recovery was about 70% in
3 h, as compared to about 40% achieved by simple
batch dilution. The mechanism revealed that when
PNiPAAm gels were added into the refolding
buffer, the hydrophobic interactions between dena-
tured proteins and polymer gels could prevent the
aggregation of refolding intermediates, thus enhan-
cing protein renaturation [107]. Recently, Liu et al
[108], prepared an artificial chaperon, composed of
temperature-responsive PNiPAAm grafted with
b-cyclodextrin (a weakly hydrophobic stripper)
for protein refolding, where cetyltrimethylammo-
nium bromide (CTAB) was taken as surfactant.
Lysozyme was used as model protein and the
result showed that b-CD-g-PNiPAAm not only
strips CTAB from the CTAB-denatured lysozyme
complex with the b-CD segment, which was proved
by fluorescence emission spectroscopy, but also
inhibits the formation of protein aggregates during
the following refolding step (Fig. 4). This is due to
the PNiPAAm segment that interacts with the
protein being refolded via hydrophobic interaction.
As a result, an improved refolding yield is obtained,
particularly at a high temperature [108]. In another
work, dextran-grafted-PNiPAAm (DGP) was pre-
pared and characterized for its use as artificial
chaperon to assist protein refolding of model
proteins like lysozyme and bovine carbonic anhy-
drase. In this case, the function of tunable hydro-
phobic segment helps to form the complex with
protein being refolded and the presence of hydro-
philic segment is to accommodate and disperse the
folded or partially folded protein. The result has
shown that DGP favors high refolding yield as
compared to refolding assisted by surfactant. Here
the hydrophobicity can be tuned by varying the
temperature ranges from above the LCST of DGP
to lower temperature, which can be programmed in
such a way to match the protein hydrophobicity
during its refolding process and also the demands of
different protein refolding.
Eudragit S-100, a pH-responsive polymer is
supposed to increase the rate of refolding and
refolding percentage of denatured protein and this
was found to assist refolding of a-chymotrypsin,
which is known to bind to the polymer rather non-
specifically. Complete activity of a-chymotrypsin
could be regained within 10 min during the refolding
study. It has been proposed that Eudragit S-100
could help in reversing protein aggregation in
amyloid based diseases [109]. Eudragit S-100 has
also been exploited for simultaneous refolding and
purification of xylanase. It has been found that
microwave-treated Eudragit S-100 also gave better
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Fig. 4. Schematic view of the mechanism of ‘‘temperature-stimuli-artificial chaperone’’ assisted protein refolding in vitro.
A. Kumar et al. / Prog. Polym. Sci. 32 (2007) 1205–1237 1215

12. results as compared to untreated Eudragit S-100 in
terms of refolding/purification after denaturation
with 8 M urea. It is believed that polymers bind to
hydrophobic sites on the proteins and prevent
aggregation [110].
Hence, the properties of linear free chains of SPs
in solution are well studied and endowed for the
above-discussed applications. These studies advance
the understanding of biochemical processes and
biomolecular interactions of various biomolecules
in basic life sciences. The application of SP and their
bioconjugates in solution have thus shown potential
and cost-effective applications in bioseparation of
proteins and other bioparticles for basic life sciences
research and other industrial applications. The
application of SP in solution form have shown
promise to new advancement of protein folding
procedure as well, as it enhanced and rectified the
protein refolding process with better outcome and
high yield. Furthermore, ongoing research is car-
ving the new applications of SPs in solution form
and investigating different biological and non-
biological systems.
3. Covalently cross-linked, reversible and physical
gels
The most extensive investigations on SPs have
been carried out on hydrogels that swell in aqueous
solutions. These smart gels are synthesized through
conventional procedure wherein synthesis takes
place at room temperature and provides hydrogels
of small pore sizes. Smart macroporous hydrogels
have also been synthesized by various approaches,
which show different applications which are re-
viewed in recent papers [111–113]. Recently hydro-
gel of large pore size synthesized in moderately
frozen conditions and providing them with interest-
ing properties have emerged which are called
cryogels [114]. Cryogels are obtained at tempera-
tures below the melting temperature of the solvent.
At subzero temperatures most of the solvent is
frozen, while the dissolved substances are concen-
trated in small non-frozen regions, so called ‘‘liquid
microphase’’. As the volume of the non-frozen
liquid microphase is much less than that of the solid
phase, the local monomer concentration is much
higher than the monomer concentration in the
initial reaction mixture. The gel formation occurs
in this liquid microphase and the crystals of frozen
solvents perform like porogen. After melting the ice
crystals, a system of large interconnected pores is
formed. Thus, an attractive system for a surface
grafting is formed with large interconnected pores
ensuring high surface area available for grafting and
efficient mass transport of monomers. Currently
most of the work has focused on hydrogels that
respond sharply to small changes in temperature or
pH [5]. But other gels have also been investigated
that respond to changes in ionic strength, solvent,
light intensity, and electric or magnetic fields
[11–13]. Some gels also have been engineered to
respond to specific biomolecule or chemical triggers,
such as glucose [115,116]. This stimulus response of
gels makes it possible to exploit them as smart
materials and numerous applications of these
materials have been established.
The reversible volume phase transition in gels
occurs because of the ‘‘osmotic forces’’ which swell
or collapse the network structure. The basic features
of the osmotic forces are expressed qualitatively by
the Flory equation [117]:
p ¼ RTflnð1 À ^Þ þ ^ þ w^2
þ Vsðne=V0Þð^1=3
À ^=2Þg.
Here, Vs is the molar volume of the solvent, ø the
volume fraction of the network, R the gas constant,
T the absolute temperature, w the interaction
parameter and (ne/V0) is cross-linked density in
prepared gel. In Flory equation, the first three terms
represent a ‘‘swelling force’’ of the network due to
the energetically favorable mixing of polymer chains
with the solvent molecules, while the last term is an
‘‘elastic retractive force’’ which tries to bring the
network back to its unstrained state. The equili-
brium swelling capacity of the gel results from a
balance of these two forces. Thus, for a given
gel-solvent system, the swelling capacity of the gel
is strongly dependent on its cross-link density.
Volume transitions are discontinuous for networks
which have charged polymer chains and/or stiff
chains. Whereas phase transitions in chemically
cross-linked networks are well understood, the
phase transitions in physically cross-linked net-
works (e.g. hydrogen-bonded networks) have
gained attention only recently. The physical cross-
links are weak and temporary and can be disrupted
reversibly by imposing a deformation. Therefore,
deformation is likely to affect the equilibrium
swelling capacity and the phase transitions in such
gels [118].
The thermoresponsive, PNiPAAm gels have
attracted great attention for their scientific interest
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13. as well as for their industrial applications including
drug delivery [5,119] and immobilized-enzyme
reactors [120]. These gels show deswelling at higher
temperatures because of hydrophobic interactions
which lead to the expulsion of the solvent [121,122].
The equilibrium structure factors and dynamics of
concentration fluctuations in PNiPAAm gels as a
function of the degree of swelling has been
extensively studied [123]. Late stage spinodal
decomposition kinetics [124] and turbidity measure-
ments during phase separation [125] have also been
reported. The turbidity measurements have been
used to study the spinodal decomposition and to
measure critical exponents for many systems which
are in equilibrium state at varying temperatures
[126,127]. Using differential scanning microcalori-
metry, the thermal volume phase transition in
PNiPAAm hydrogels can be investigated [128].
The pH- and temperature-sensitive interpenetrat-
ing polymer networks (IPNs) composed of
poly(vinyl alcohol) (PVA) and poly(acrylic acid)
(PAAc) IPN hydrogels can be synthesized by UV
irradiation, followed by a repetitive freezing and
thawing process by which PVA hydrogel networks
are formed inside of cross-linked PAAc chains.
Swelling ratios of all IPNs were relatively high, and
they showed reasonable sensitivity to both pH and
temperature [129]. Similarly hydrogels of IPN
composed of the temperature-sensitive PNiPAAm
and the pH-sensitive poly(methacrylic acid)
(PMAA) were prepared by a sequential UV poly-
merization method. These hydrogels exhibited a
combined pH- and temperature-sensitivity at a
temperature range of 31–32 1C and a pH value of
approximately 5.5 [130]. These responsive hydrogels
have potential application in various biological and
non-biological systems such as flow control in
microfluidics, sensors in biological and chemical
applications. These are further discussed in the
following sections.
3.1. Microfluidics and actuators
Flow control in integrated multifunctional micro-
fluidic devices still remains a major challenge and a
fully functional valve is a key component in
microfluidic systems. In recent years, developing
microfluidic systems for biological and chemical
applications has been a major challenge [131–133].
Conventional microactuators (using, for example,
electromagnetic, electrostatic or thermopneumatic
effects) require external power for operation and are
relatively complex assemblies, which limits their use
in practical systems. Stimuli-responsive hydrogels
have a significant advantage over conventional
microfluidic actuators owing to their ability to
undergo abrupt volume changes in response to the
surrounding environment without the requirement
of an external power source. Existing studies on
responsive hydrogels in bulk suggest that these
materials should be well suited for applications in
microfluidics and actuator systems [134]. Thus, the
use of responsive hydrogel materials to regulate flow
eliminates the need for external power, external
control and complex fabrication schemes. These
valves in the form of responsive hydrogels are
incorporated or fabricated within the microfluidics
channels and can shrink or swell in response to
external stimuli which in turn cause opening or
closing of channels, respectively (Fig. 5). During the
past decade, different fabrication systems of tem-
perature-responsive hydrogel valves have been re-
ported [135–137]. The monolithic plugs PNiPAAm
cross-linked with 5% methylenebisacrylamide have
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Fig. 5. Illustration of hydrogel volume changes as the gel’s temperature varies. The top (a) and bottom (b) rows correspond, respectively,
to temperatures above and below the critical temperature (TC). The left and right columns provide, respectively, top and side views.
A. Kumar et al. / Prog. Polym. Sci. 32 (2007) 1205–1237 1217

14. been prepared by photoinitiated polymerization
within the channel of a microfluidic device. Thermo-
electric elements were used to actuate the valve and
flow through the device was monitored by fluores-
cence measurements via laser-triggered photo-
bleaching of a dye contained in liquid phase [138].
PNiPAAm hydrogel can also be used as valve for
flow switching, distribution, metering and sealing of
a PCR reactor chamber [139]. Thermoresponsive
monolithic co-polymer gel of NiPAAm and
N-ethylacrylamide has been synthesized by photo-
patterning and used as valves within the channels of
microfluidic devices. No changes in performance
were observed even after repeated open-close
cycling of valves [140]. The manipulation of living
biological cells in microfluidic channels by a
combination of negative dielectrophoretic barriers
and pressure driven flows is widely employed in lab-
on-a-chip systems [141]. The study concerned
thermoprecipitating N-alkyl-substituted acrylamide
polymers that can act as threshold probes in
miniaturized systems [141]. Responsive hydrogels
have also been incorporated in microfluidic devices
as part of switchable supports. Separation schemes
with dynamic temperature control are most likely to
increase efficiency of high throughput DNA analy-
sis [142]. The response of hydrogel actuator should
be in order of seconds for a microfluidics system,
and this seems possible for actuators smaller than
100 mm. The rate of response can also be controlled
by forming semi-interpenetrating networks (IPNs)
with NiPAAm as crosslinked component [143].
Responsive hydrogels have been evaluated as
building materials for microfluidic systems using
several criteria: (a) the ease of fabrication of
actuators, (b) the kinetics of the volume phase
transition as a function of gel size and composition,
(c) the ability of the actuators to block and displace
the flow of different fluids, and (d) an isotropic
swelling of the hydrogel and the response to
different stimuli [143].
Temperature-responsive polymers have been used
to construct ‘‘smart’’ affinity beads that can be
reversibly immobilized on microfluidic channel
walls above the LCST to capture the target
biomolecules through its affinity moiety. The smart
affinity beads along with the target biomolecule are
then released from the channel wall on lowering the
temperature of microfluidic channel below the
LCST of smart affinity bead [144]. Active thermo-
responsive polymer has been integrated into a
microfluidic hot plate that is programmed to adsorb
and desorbs protein monolayers in less than 1 s.
This active device can be manipulated for proteomic
functions, including pre-concentration and separa-
tion of soluble proteins on an integrated fluidics
chip [145].
Apart from the temperature-responsive polymers,
pH-responsive polymers have also shown potential
to act as actuators in microfluidics. Studies have
been conducted on the mechanical properties of
pH-sensitive hydrogels, to produce optimal hydrogel
valves and sensors [146]. pH-responsive hydrogels
can be patterned in a microchannel by photopoly-
merization. The device uses a poly(hydroxyethyl
methacrylate–acrylic acid) (poly(HEMA–AA)),
pH-responsive hydrogel as the actuator in a PDMS
microfluidic device [147]. A biomimetic check valve
fabricated by in situ photopolymerization of poly
(HEMA–AA) hydrogel inside a glass microchannel
has been applied for directional control of fluid flow
[148]. Combination of different types of pH-
responsive hydrogels has been incorporated into
flow sorter [149] and each of them behaves
differently at the same pH and this property has
been well utilized in a variety of applications.
A device based on pH-responsive hydrogel disks
of polymethacrylic acid–triethylene glycol dimetha-
crylate (PMAA–EG) has been used to regulate drug
release by deforming a membrane which then
occludes an orifice thus preventing drug release
[150]. An in situ photopolymerization method has
been employed to build micro functional structure
which was employed to generate a pH-responsive
micropsphere that can act as actuating component
of microvalve inside a channel [151]. In another
example, the valve was fabricated of responsive
hydrogel sandwiched between a rigid porous mem-
brane and a flexible silicone rubber membrane,
creating a small and efficient regulatory valve for
diffusion of chemical species through the porous
plate [152]. Another approach involved integration
of photolithographic techniques with living radical
photografting of poly(ethylene glycol) acrylate
succinyl fluorescein that can be used to construct
entirely polymeric microfluidic device for rapid
pH-sensing applications [153].
An electroactive interpenetrating network (IPN)
developed of PVA and PNiPAAm has been studied
for its swelling ratio and bending behavior under
electric fields in aqueous NaCl solution for its appli-
cation in biometric sensors and actuators, which
demonstrate rapid response under the influence of
external electric field [154]. A weakly cross-linked
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15. anionic poly (2-acrylamido-2-methylpropane sulfo-
nic acid) (PAMPS) gel with cationic surfactant
N-alkylpyridinium chloride (C10PyCl) exhibits
greatest and fastest chemo-mechanical movement
under DC current thus exhibiting biomimetic
pulsation. The gel shrinkage was attributed to the
neutralization of negative sulfonate groups of the
gel due to complex formation with surfactant
cations through electrostatic interaction which leads
to decrease in the osmotic pressure difference
between the interior of the gel and the surrounding
solution. When the DC voltage was turned on, the
surfactant molecules move by electrophoresis and
bind with the gel, which cause an isotropic
contraction of the gel [155]. Novel polymeric
biomimetic gels with self-oscillating function have
been developed which generates periodic mechan-
ical energy from the chemical energy of the
Belousov–Zhabotinsky reaction. The self-oscillating
gel may be useful in a number of application like
pulse generator or chemical pacemaker, self-walking
(auto-mobile) actuators or micropumps, etc [156].
When looking into the mechanical characterization
of an electric and pH-responsive gel consisting of a
co-polymer of PVA–poly(acrylic acid), which ex-
pands and contracts in response to external stimuli,
the gel was found to be viscoelastic, mechanically
compressible over a relatively large deformation.
These properties could be useful in developing
them into biomimetic actuators or as scaffolds for
artificial organs [157,158].
The triggered control of interfacial properties
provided by immobilized SP at the solid water
interface has applications in designing of micro-
fluidics bioanalytical devices [159]. SP-hydrogels
provide actuation pressure required for both valving
and dispensing functions in microdispensing device.
The microdispensing device uses an array of
responsive hydrogels to deform a flexible membrane
above a fluid reservoir chamber. When the micro-
valve is open, deformation of the membrane reduces
the volume of the reservoir chamber and pushes
fluid through the microvalve. When the microvalve
is closed, expansion of hydrogel array generates a
storable pressure source that will result in fluid
dispensing once the microvalve is opened [160].
A phenylboronic acid-based hydrogel was used to
construct a smart microvalve that responds to the
change in the glucose and pH concentrations [161].
Random co-polymers composed of poly(acrylic
acid) and poly(vinyl sulfonic acid) have been
analyzed for their swelling ratios to characterize
their response at various temperatures and pH,
while deformation ratio of gels was determined to
see their behavior in electrical field. The contraction
and expansion behavior was similar to that of a
natural muscle. These electroactive polymeric hy-
drogels can be utilized as biosensors and as artificial
muscle when employing an electrical stimulus [162].
3.2. Smart polymer hydrogels as sensors
The term ‘‘biosensor’’ is used to cover sensor
devices in order to determine the concentration of
substances and other analytes of biological interest,
in some cases even where they do not utilize a
biological system directly. The last two decades
witnessed the emergence of polymers as an intri-
guing class of macromolecules that have the
electrical and optical properties and hence show
applications as sensors. SP are used in biosensors in
three main ways: signal detection, transmission of
signal to a measuring electrode or as response
element which controls the feedback response to the
signal. Polymer swelling can lead to physical work,
such as shutting off a valve or making contact
between the sensor and a secondary component.
When cross-linked hydrogel components of compo-
site membranes are prepared with the amine
containing dimethylaminoethyl methacrylate mono-
mer, this results in a polymeric device that swelled in
response to pH changes (neutral to acidic medium).
Enhanced biosensing capabilities of these mem-
branes have been demonstrated in the fabrication of
glucose-, cholesterol- and galactose-amperometric
biosensors. Entrapment of glucose oxidase within
these materials made them glucose-responsive
through the formation of gluconic acid by the
oxidation of surrounding glucose. When insulin was
co-loaded with glucose oxidase into these bio-smart
devices, there is two-fold increase in release rate of
insulin in the presence of glucose. This feature may
hold potential to develop them into chemically
synthesized artificial pancreas [163]. The conjuga-
tion of these polymers to different recognition pro-
teins, including antibodies, protein A, streptavidin
and enzymes can be done in a random or site-
specific manner. Different SP, including tempera-
ture, pH and light-sensitive polymers have been
conjugated to these proteins. Once the analyte is
bound to these recognition proteins, the environ-
mental change triggers the release of these mole-
cules. This triggered release could also be used to
remove inhibitors, toxins or fouling agents from
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16. the recognition sites of immobilized or free enzymes
and affinity molecules, such as those used in bio-
sensors [164].
The amphiphilic co-polymer consisting of oligo-
deoxyribonucleotide as the hydrophilic part and
thermoresponsive, PNiPAAm as the hydrophobic
part have been investigated. The co-polymers
formed DNA-linked colloidal nanoparticles above
the phase transition temperature of PNiPAAm as
the DNA was hybridized to the complimentary
oligodeoxyribonucleotide. The nanoparticles aggre-
gated rapidly when the complementary strand was
added into the dispersion. In contrast, they remain
dispersed in the absence of the complementary
strand and in the presence of the point-mutated
oligonucleotide. These distinct phenomena may be
applied for an oligonucleotide analysis system in
gene diagnosis (Fig. 6) [165].
Fluorescent molecular thermosensors based on
polymers showing a temperature-induced phase
transition and labeled with polarity-sensitive fluor-
escent benzofurazans were obtained by co-polymer-
ization of two kinds of acrylamide derivatives. The
polymer underwent a temperature-induced phase
transition, and its LCST value correlated well to
unit ratio in the co-polymer and the LCST values of
the non-derivatized corresponding homopoly-
mers of two acrylamides [166]. The fluorescence
intensity increases sharply with decreasing solvent
polarity and for these acrylamide-derivatived
polymers, the microenviroment polarity near the
main chain is seen to decrease considerably
with increasing temperature at phase transition.
Utilizing this property, a method for modulating the
sensitive temperature range of the fluorescent
molecular thermosensors based on thermorespon-
sive polymers was achieved. Co-polymers of two
kinds of acrylamide derivative (N-n-propylacryla-
mide, N-isopropylacrylamide, and/or N-isopropyl
methacrylamide) labeled with 4-N-(2-acryloylox-
yethyl)-N-methylamino-7-N,N-dimethylaminosulfo-
nyl-2,1,3-benzoxadiazole (DBD-AE) have the
benzofurazan structure as a fluorophore. These
fluorescent molecular thermosensors differ from
each other in sensitive temperature range between
20 and 49 1C [167].
Quartz crystal microbalance (QCMB) technique
is a general transduction principle for the use of
hydrogels as chemical- and pH-sensor materials in
the liquid medium. QCMB technique measures
mass by measuring the change in frequency of a
piezoelectric quartz crystal when it is disturbed by
the change in small mass such as virus or any other
tiny object intended to be measured and also change
in damping depends on the changes in elasticity of
hydrogel film. The principle is well suited for a
precise investigation of the behavior of thin hydro-
gel films. It is possible to use hydrogel-coated quartz
crystals as liquid sensors to observe special state
values of liquid media in real time. Furthermore, as
smart hydrogels offer a range of sensitivity across
substances and ion concentrations in liquids, they
are bound to play an important role in liquid
sensors in near future. The volume phase changing
behavior of such gels and the short response times
of thin hydrogel structures can allow for the
development of highly sensitive and real-time
measurement devices, e.g. PVA/PAA-coated quartz
crystals were used for pH measurements in the
range up to pH 3.5 [168]. The pH sensor with ultra
high sensitivity based on a microcantilever structure
with a lithographically-defined crosslinked co-poly-
meric hydrogel of poly(methacrylic acid) (PMAA)
and PEG dimethacrylate works on polymer swelling
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Fig. 6. Single nucleotide polymorphisms assay using DNA-linked colloidal nanoparticles.
A. Kumar et al. / Prog. Polym. Sci. 32 (2007) 1205–12371220

17. as a function of pH change [169]. The design,
development and evaluation of a microfabricated
conductimetric pH sensor utilizing a pH-responsive
hydrogel co-polymer of 2-hydroxyethyl methacry-
late (HEMA) and N,N-dimethylaminoethyl metha-
crylate (DMAEMA) as sensing layer has also been
described [170]. The sensitivity of the pH sensor is
enhanced by increasing the acrylic acid fraction in
poly(acrylic acid-co-isooctyl acrylate) co-polymer.
These pH sensors are stable even after repeated
cycling between high and low pH solutions for an
extended period [171].
Similarly the artificial biosystems utilizing stimu-
li-responsive hydrogels are of great interest due to
wide range of their biochemical and biotechnologi-
cal applications. Such artificial biosystems had been
developed which may be used for sensing small
changes in analyte concentrations and for ‘‘smart’’
regulation of activities of co-immobilized enzymes.
For example, the swelling ratio of the collapsed
thermoreversible urease-containing gel increases
after addition of urea. The observed effect is due
to the change in concentration of the products of
urease-catalyzed hydrolysis of urea in the gel [172].
Another example has been a novel wireless glucose
biosensor based on a mass-sensitive magnetoelastic
sensor which consist of magnetoelastic ribbon that
is coated with two layers; the first one is a coat of a
pH-sensitive polymer upon which a second layer of
bienzymatic system containing glucose oxidase
(GOx) and catalase is coated. The enzymatic
oxidation of glucose decreases the pH that is sensed
by the pH-responsive polymer and results in
polymer shrinking. The polymer shrinking decreases
the mass loading on the sensor, and as a result, the
sensor resonance frequency increases [173].
Though this section of the review described the
synthesis, property and applications of smart
hydrogels which mainly focused in the bioengineer-
ing applications like microfluidics, actuator systems
and biosensors. However, these responsive hydro-
gels are also serving other bioengineering areas
like immobilized biocatalysts [174] and chromato-
graphy [175].
4. SP in chain adsorbed or surface-grafted form
(smart surfaces and membranes)
The driving force behind phase separation of
SPs is a sharp conformational change of a macro-
molecule accompanied with a drastic increase
in hydrophobicity triggered by a small change
in environmental conditions. The hydrophobic
‘‘collapsed’’ macromolecules aggregate and finally
the polymer aggregates form a separate phase.
When attached to the surface, SPs could not
aggregate but the conformational transition from
hydrophilic to hydrophobic state endows the sur-
face with regulated hydrophobicity: the surface is
hydrophilic when the SP is in expanded ‘‘soluble’’
conformation and hydrophobic when the polymer is
in collapsed ‘‘insoluble’’ conformation. For exam-
ple, when PNiPAAm was end-grafted to mercapto-
propyl derivatives of silica gel, plane glass sheets
and glass capillary tubing, the polymer monolayer
attached to the glass carriers provided them with
thermally controlled wetability registered by two
independent methods: direct measurements of the
water contact angle and capillary rise. The water
contact angle changed from 54731 to 68731 in the
temperature range from 20 to 50 1C [176]. The
thermally induced transition of surface-grafted
PNiPAAm brushes with a dry thickness of
$50 nm was probed by surface plasmon resonance
spectroscopy (SPR) and contact angle measure-
ments. The results suggest that the polymer
segments in the outermost region of the brush
remain highly solvated until the LCST for the
polymer in solution, while densely packed, less
solvated segments within the brush layer close to the
surface undergo dehydration and collapse over a
broad range of temperatures [177].
Recently, the effects of temperature, degree of
polymerization, and surface coverage on the equili-
brium structure of tethered PNiPAAm chains
immersed in water were modeled employing a
numerical self-consistent field theory where the
experimental phase diagram was used as input to
the theory. At low temperatures, the composition
profiles are approximately parabolic and extend
into the solvent. In contrast, at temperatures above
the LCST of the bulk solution, the polymer profiles
are collapsed near the surface. The layer thickness
and the effective monomer fraction within the layer
undergo what appears to be a first-order change at a
temperature that depends on surface coverage and
chain length. As a result of the tethering constraint,
the phase diagram becomes distorted relative to the
bulk polymer solution and exhibits closed loop
behavior. As a consequence, the relative magnitude
of the layer thickness change at 20 and 40 1C is a
non-monotonic function of surface coverage, with
a maximum that shifts to lower surface coverage
as the chain length increases in qualitative agreement
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18. with experiment [178]. The model explains reason-
ably well the experimental data for the temperature-
dependent conformational changes of PNiPAAm
grafted on gold and on silicon oxide over a range of
surface densities and molecular weights obtained
using neutron reflection in D2O. The largest
conformational changes were observed for inter-
mediate grafting densities and high molecular
weights [179].
The grafting of SP to the surface could be
achieved via chemical bonding between reactive
groups on the gel surface and reactive terminal
groups of the preformed polymer (so called grafting
to) [180–182]. The obvious advantage here is that
one can beforehand determine the properties
(molecular mass, MW distribution) of the to-be-
grafted polymer. The problem is that the surface
should have reactive groups suitable for grafting
and the grafted chain should carry the proper
functionality at the end. It is very difficult to achieve
high grafting densities using the grafting to methods
because of steric crowding of reactive sites at the gel
surface by already bound polymer molecules.
Moreover, the efficiency of grafting to methods is
pretty low resulting in pronounced losses of the
terminally modified polymer. Hence, an alternative
approach also called grafting from has been mainly
used for the production of SP brushes. Attachment
of active precursors, such as initiator, transfer agent
or co-monomer onto the surface followed by radical
polymerization of NiPAAm allows for a fine control
of the density and thickness of polymer brushes
[183,184]. Alternatively, deposition of polymer at
the surface from monomer vapor under plasma
glow discharge was successfully used [185]. Atom
transfer radical polymerization (ATRP) becomes
increasingly popular for the synthesis of SP brushes
[186–190]. During the grafting from polymerization,
the polymer chains ‘‘grow’’ from the surface. The
graft-type surfaces with long chains and high
density of polymer grafted can be prepared. When
a co-polymer was grafted from instead of a
homopolymer, there is a possibility of producing a
brush with dual sensitivity e.g. responding to both
changes in temperature and UV irradiation [180] or
temperature and pH [191]. Moreover, a secondary
smart polymer, poly(N-isopropylacrylamide-co-n-
butyl methacrylate) with different transition tem-
perature could be pattern-grafted as side chains to
PNiPAAm main grafted chains (Fig. 7) resulting in
dual thermoresponsive surfaces [192].
Apart of the solid surfaces like gold, silica,
polystyrene, PNiPAAm was grafted also at the soft
materials, e.g. at the thermoresponsive PNiPAAm
gel using RAFT polymerization. The hydrogels with
more grafted chains and longer chain lengths
allowed higher equilibrium swelling and rapid
shrinking [193]. Alternatively, PNiPAAm was
grafted inside the pores of macroporous polyacry-
lamide hydrogel, so called cryogel [194]. With high
density of the gel phase in the pore walls of cryogels,
the grafting takes place mainly at the gel-liquid
interface [195]. For more details on polymer graft-
ing techniques, the reader could find an extensive
review [196] published recently in the same journal.
4.1. Smart surfaces for tissue engineering
The change of surface properties from hydro-
phobic above critical temperature of the polymer
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Fig. 7. Preparation of patterned dual thermoresponsive polymer-grafted culture dishes. Reproduced from [192] with permission.
A. Kumar et al. / Prog. Polym. Sci. 32 (2007) 1205–12371222

19. grafted to hydrophilic below it has been successfully
used for detachment of mammalian cells. Mamma-
lian cells are normally cultivated on a hydrophobic
solid substrate and detached from the substrate
by protease treatment, which often damages the
cells by hydrolyzing various membrane-associated
protein molecules. PNiPAAm-grafted surface is
hydrophobic at 37 1C because this temperature is
above the critical temperature for the grafted
polymer and cells are well growing on it. Decrease
in temperature results in surface transition to
hydrophilic state and the cells are easily detached
from the solid substrate without any damage. This
technology was pioneered in the 1990s by Teruo
Okano [197–199].
Since then the technology has been significantly
developed allowing for example, cultivation of cell
sheets with designed shape for tissue engineering.
Shaping of cell sheets were achieved by the use of
PNiPAAm and poly(N,N0
-dimethylacrylamide)
[poly(DMAAM)] for temperature-responsive cell
adhesive and cell non-adhesive domains, respec-
tively. These polymers were covalently grafted onto
tissue culture polystyrene dish surfaces by electron
beam irradiation with mask patterns. At 37 1C,
human aortic endothelial cells attach, spread,
and proliferate to make a monolayer only on
PNiPAAm-grafted domains. However, endothelial
cells do not adhere on poly(DMAAM)-grafted
domains even after 1 month cultivation. When the
culture temperature was reduced below 32 1C,
PNiPAAm-grafted chains become hydrophilic and
the sheets of endothelial cells were detached from
the PNiPAAm-grafted surfaces without any need of
protease treatment. Cell–cell junctions are retained
intact in the recovered cell sheets which could be
easily transferred to fresh culture dishes with the aid
of hydrophilically modified polyvinylidenefluoride
membrane as a supporter during the transfer. The
transferred cell sheets adhere rapidly onto the dish
surfaces, and the supporting membrane is easily
peeled off from the cell layers. Endothelial cell
sheets transferred to new dishes revealed the
identical shape and size to those before transfer
[200]. The same approach was used to cultivate cell
sheets of renal epithelial cell [201,202], random co-
culture of epithelial and mesenchymal cells of lung
[203] or bovine aortic endothelial cells [204].
Microglia [205] or human monocytes and mono-
cyte-derived macrophages [206] were also success-
fully cultivated on PNiPAAm-grafted substrates
and released by decreasing temperature.
Cellular interactions with PNiPAAm-grafted
surfaces can be regulated vertically using the
thickness of the grafted polymer layers in nan-
ometer-scale range. PNiPAAm-grafted surfaces
with 15–20-nm-thick layers exhibit temperature-
dependent cell adhesion/detachment, while surfaces
with layer thicker than 30 nm do not support cell
adhesion. These changes in cell adhesion are
explained by the limited mobility of the surface-
grafted polymer chains as a function of grafting,
hydration, and temperature [207]. Lateral regula-
tion of the cell adhesion on the smart surface is
achieved by nano-patterning of surface chemistry.
On a chemically patterned surface (Fig. 7), site-
selective adhesion and growth of rat primary
hepatocytes and bovine carotid endothelial cells
allowed for patterned co-culture, exploiting hydro-
phobic/hydrophilic surface chemistry regulated by
culture temperature as the sole variable. At 27 1C,
seeded hepatocytes adhered exclusively onto hydro-
phobic, dehydrated poly(N-isopropylacrylamide-co-
n-butyl methacrylate) co-grafted domains (1-mm
area), but not onto neighboring hydrated PNi-
PAAm domains. Sequentially seeded endothelial
cells then adhered exclusively to PNiPAAm do-
mains which become hydrophobic upon increasing
temperature to 37 1C, achieving patterned co-
cultures. Reducing culture temperature to 20 1C
promoted hydration of both polymer-grafted do-
mains, permitting release of the co-cultured, pat-
terned cell monolayers as continuous cell sheets
with heterotypic cell interactions. Recovered co-
cultured cell sheets can be manipulated, moved and
sandwiched with other structures [192]. A well-
coordinated co-culture of three or more cell types
might also be realized since the transition tempera-
ture of grafted thermosensitive (co)polymer can be
readily varied, both over successive temperature
regimes and spatially across the culture surface
simultaneously using coordinated masks and co-
polymerization. Combinations of multiple masking
different co-polymers at each mask step would
produce a thermally varied, spatially responsive
surface capable of supporting selective sequential
seeding of multiple cell types, depending on seed
temperature [208].
As the cell–cell contacts are maintained intact in
the cell sheets detached from the PNiPAAm-grafted
surfaces, the cell sheets could be used for an
advanced engineering of 3-D-functional tissues
mimicking the structure of tissues in the living
organisms [209]. The double-layered co-culture was
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20. achieved by placing a contiguous cell sheet of
confluent human aortic endothelial cells recovered
from PNiPAAm-grafted surfaces onto the rat
hepatocyte layer. The double-layered structure of
endothelial cells and hepatocytes remained in tight
contact during culture. Hepatocytes in the layered
co-culture system with the sheet of endothelial cells
maintained the differentiated cell shape and the
albumin expression for over 41 days of culture,
whereas the functions disappeared within 10 days of
culture in control hepatocytes without the sheet of
endothelial cells [210]. Cultured chick embryonic
cardiomyocyte sheets detached from PNiPAAm-
grafted surfaces were layered into tissue-like lami-
nate stacks using hydrophilic support and transfer
membranes. The layered cell sheets rapidly adhered
to each other, establishing cell-to-cell connections
characteristic of heart tissue, including desmosomes
and intercalated disks. Bilayer cell sheets pulsed
spontaneously and synchronously, altering their
characteristic pulsing frequency with applied electric
stimulation transmitted across the sheets. These
results demonstrate that electrically communicative
three-dimensional cardiac constructs can be
achieved by stacking monolayer cardiomyocyte
sheets [211,212].
Co-polymerization of PNiPAAm with functional
co-monomers and/or post-modification of grafted
chains allow for the control of the rate of cell
adhesion and spreading or alternatively the rate of
cell detachment. Human umbilical vein endothelial
cells spread readily on the surface grafted with
poly(N-isopropylacrylamide-co-2-carboxyisopropyl-
acrylamide) modified with cell adhesion peptide
RGDS (Arg-Gly-Asp-Ser) [213]. A similar behavior
was observed for bovine carotid artery endothelial
cells i.e. the modification with RGDS. RGDS
facilitated initial cell adhesion, while modification
with insulin induced cell proliferation. A more
pronounced cell growth was achieved by co-
immobilization of appropriate amount of RGDS
and insulin [214].
On the other hand, co-polymerization with acry-
lic acid or 2-carboxyisopropylacrylamide [215] or
co-grafting of PNiPAAm with PEG onto porous
culture membranes by electron beam irradiation
[216] allow for accelerated cell detachment at redu-
ced temperature. Whereas approximately 35 min
incubation at 20 1C was required to completely
detach cell sheets from PNiPAAm-grafted surface in
static conditions, only 19 min was sufficient to
detach cell sheets from PNiPAAm-co-PEG-grafted
porous membranes. Grafted PEG chains are be-
lieved to accelerate the diffusion of water molecules
to PNiPAAm grafts, showing more rapid detach-
ment of cell sheet compared to only PNiPAAm-
grafted membranes [216].
Microbial cells, like Halomonas marina (ATCC
25374) capable of adhesion to hydrophobic surfaces
can be also bound to PNiPAAm-grafted surfaces
above the transition temperature and released with
about 90% efficiency at reduced temperature [217].
The change in the pattern of attachment of common
oral bacteria Streptococcus mutans following ‘‘cy-
cling’’ of PNiPAAm brushes above and below the
transition temperature was correlated with changes
in the surface properties as a result of the phase
transitions [218]. A similar correlation of short-term
attachment of gram negative and motile bacteria
(Salmonella typhimurium) and gram positive, non-
motile species (Bacillus cereus) with changes in
surface properties was observed for surfaces with
grafted co-polymers of NiPAAm with acrylamide or
N-tert-butylacrylamide [182].
Temperature-regulated detachment of mamma-
lian cells requires cell metabolic activity requiring
ATP consumption, signal transduction and cytos-
keleton reorganization. Precoating PNiPAAm-
grafted surfaces with fibronectin improves spread-
ing of less adhesive cultured hepatocytes and
reduces the temperature at which cultured cells are
released from fibronectin-adsorbed grafted surfaces.
Immunostaining with anti-fibronectin antibodies
revealed that only fibronectin located beneath the
cultured cells is removed from culture surfaces after
reducing temperature. Fibronectin adsorbed to
surface areas lacking direct cell attachment re-
mained surface-bound after reducing temperature
[219]. Principal component analysis using time-of-
flight secondary ion mass spectrometry indicates
that molecular ion fragments of amino acids are
present on the surface after low-temperature liftoff
from PNiPAAm brushes. Seeding new cells on
surfaces from which an initial layer of cells was
removed indicates that liftoff dissociation leaves
behind surfaces that better promote cell adhesion as
compared to cell detachment by enzymatic detach-
ment. It was concluded that the removal of bovine
aortic endothelial cells via low-temperature liftoff
from PNiPAAm brushes is less damaging to the
extracellular matrix proteins remaining at the sur-
face as compared to the enzymatic methods [220].
At present, the low-temperature liftoff of cell
sheets from surfaces grafted with SP presents a
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21. mature technique, which constitutes an important
step on the way to the cultivation of functional 3-D
tissues.
4.2. Smart surfaces for temperature controlled
separations
The change of hydrophobicity of the surface in
response to changing temperature can be exploited
for the separation of substances which interact
differently with the hydrophobic matrix. In 2002,
Kikuchi and Okano [4] reviewed extensively in this
journal the area of chromatography using matrices
with grafted thermosensitive polymers. Several
factors were discussed including the effects of the
temperature-responsive hydrophilic/hydrophobic
changes, co-polymer composition, graft polymer
molecular architecture and the incorporation of
charged groups. The section below is addressing
only the recent developments mainly those hap-
pened after 2001.
A modern tendency when separating low-mole-
cular weight compound in an HPLC mode is to use
cross-linked layer of the gel of thermoresponsive
polymer rather than a brush. A new temperature-
responsive method of HPLC using packing materi-
als modified with cross-linked PNiPAAm hydrogel
has been developed. The surface properties and
functions of the stationary phases changed in
response to the external temperature. Therefore it
was possible to control the interactions of a solute
with the surface with a constant aqueous mobile
phase. A temperature-responsive elution behavior
was demonstrated on the separation of steroids
and phenylthiohydantoin derivatives of amino
acids [221]. When the gel was produced by cross-
linked co-polymerization of NiPAAm, acrylic acid
and N-tert-butylacrylamide, the stationary phase
showed simultaneous temperature-responsive
changes in surface charge density and hydrophobi-
city. Alterations of properties of the polymer layer
were confirmed by temperature-responsive phase
transition and shift in apparent pKa values. Analyte
(catecholamine derivatives) retention was primarily
due to the electrostatic interaction. The tempera-
ture-induced phase transition of the hydrogel layer
on the stationary phases was evidenced by the
apparent inflection point in van’t Hoff plots around
36 1C suggesting that solute interactions are chan-
ged below and above the transition temperature,
reducing electrostatic interaction above the transi-
tion temperature [192,222].
Alternatively, PNiPAAm and its co-polymers
with n-butyl methacrylate and dimethylaminopro-
pylacrylamide, which have reactive terminal func-
tional groups, were synthesized via radical
polymerization using 3-mercaptopropionic acid as
a chain-transfer agent. Terminal carboxyl groups
were esterified by N-hydroxysuccinimide and N,N0
-
dicyclohexylcarbodiimide prior to modification
of aminopropyl silica. The elution behaviors of
organic acids and phenylthiohydantoin-amino acids
on this matrix were controlled by temperature
changes without addition of organic solvents in
the mobile phase [223,224]. A solvent gradient
elution-like effect could be achieved with a single
mobile phase by programmed temperature changes
during chromatographic runs (Fig. 8) [225].
Other than PNiPAAm and its co-polymers
thermoresponsive polymers, like elastin-like poly-
peptide [159], poly(acryloyl-L-proline methyl ester)
[226] or co-polymer of acryloyl-L-valine N-methy-
lamide and its N,N-dimethylamide analog [227]
have been bound on silica gel supports to produce
thermoresponsive chromatographic matrices. The
surface properties of stationary phases modified
with poly(acryloyl-L-proline methyl ester) are con-
trolled by the external temperature allowing steroids
and amino acids with different hydrophobicities
to be separated using a sole aqueous mobile phase.
In contrast to a PNiPAAm-modified surface, a
poly(acryloyl-L-proline methyl ester)-modified sur-
face has a higher affinity for hydrophobic amino
acids [226]. In the latter case, the retention of amino
acid derivatives is prolonged with an increase in
column temperature. Enantioselectivity is also en-
hanced with temperature increase until the parti-
cular critical temperature [227].
In the development of the ideas pioneered in our
laboratory [228], grafted thermosensitive polymers
have been used to modify the access of protein
molecules to the ligands at the chromatographic
stationary phase. Affinity ligand Cibacron Blue
F3G-A was immobilized using two different lengths
of spacer molecules, together with PNiPAAm.
Chromatographic analyses using BSA as a model
protein showed a clear correlation between spacer
length and binding capacity at temperatures lower
than the transition temperature of PNiPAAm. The
protein-binding capacity below the transition tem-
perature was significantly reduced only when the
spacer length was shorter than the mean size of the
extended PNiPAAm chains. The adsorbed protein
could be released from the matrix surface by
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A. Kumar et al. / Prog. Polym. Sci. 32 (2007) 1205–1237 1225

22. lowering the temperature to below the transition
temperature while maintaining other factors such as
pH and ion strength [229].
Affinity ligand, Ricinus communis agglutinin, has
been coupled to PNiPAAm chains along with
lactose and the polymer has then been immobilized
on Sepharose column. At 5 1C, the column retains
the glycoprotein target, asialotransferrin, but re-
leases most (95%) of the asialotransferrin upon
warming to 30 1C. This temperature-induced elution
was much greater than can be explained by
temperature dependency of sugar recognition by
agglutinin. The simplest explanation is that upon
thermally induced dehydration and collapse of the
PNiPAAm chains, co-immobilized agglutinin ligand
and lactose are brought into closer proximity to
each other, enabling immobilized lactose to displace
affinity-bound asislotransferrin from the immobi-
lized agglutinin [230].
At present, one of the main challenges in
bioseparation is the specific separation of mixed
cell cultures into cell sub-populations. It is not
surprising because the cell separation entails great
difficulties. As separation objects cells are relatively
large, their diffusivity is negligible and only
convective transport can be used. The cells,
especially mammalian cells are rather fragile and
sensitive to shear stresses. From a chemical view-
point, the surface of different cell sub-populations is
very much the same and the physico-chemical
differences between sub-populations are very small.
Moreover, the cells interact with a solid matrix via
multipoint interactions and the difficulty in disrupt-
ing multivalent interactions is one of the main
problems in designing affinity techniques for cell
separation. The conformational changes of SPs
allow for a finely tuned modification of the surface
properties and hence give a good tool to control
cell–surface interactions. For example, adsorption
of anti-mouse CD80 monoclonal antibodies onto
PNiPAAm-grafted polypropylene membrane at
37 1C and their desorption at 4 1C was exploited
for the capture of mouse-CD80 transfected cells at
37 1C which facilitated detachment of captured cells
by washing at 4 1C. With this method, mouse CD80-
or mouse CD86-transfected cells were enriched from
a 1:1 cell suspension to 72% or 66%, respectively,
and with high yield [231].
Recently we have exploited the heat-induced
shrinkage of macroporous hydrogel prepared by
cross-linking polymerization of NiPAAm under
semi-frozen conditions (so called cryogel) for the
improved detachment of affinity bound cells. When
bearing Cu(II)-iminodiacetate ligands, PNiPAAm
cryogel monoliths bound E. coli cells. The bound
cells were eluted with only 65% efficiency using
0.2 M imidazole buffer at 25 1C i.e. below the
transition temperature. However, when elution
was carried out with the same buffer at 40 1C, i.e.
above the transition temperature, the PNiPAAm
cryogel shrank almost instantaneously upon contact
with the ‘‘warm’’ buffer, resulting in the release of
85% of the bound cells [232]. Correspondingly,
whereas conventional elution of yeast cells captured
ARTICLE IN PRESS
Fig. 8. Chromatograms of steroids on a PNiPAAm terminally-modified column at 10 and 50 1C using pure water as a mobile phase.
Peaks: (1) hydrocortisone; (2) prednisolone; (3) dexamethasone; (4) hydrocortisone acetate; and (5) testosterone. HPLC conditions: flow-
rate, 1.0 mL minÀ1
; monitoring, UV at 254 nm. Reproduced from [225] with permission.
A. Kumar et al. / Prog. Polym. Sci. 32 (2007) 1205–12371226

23. via ConA ligand with competing low-molecular
weight ligand, a-D-manno-pyranoside, released only
a minor amount of bound cells, the temperature
induced detachment improved cell release up to
37% of bound cells [233]. The possible reasons
for the disruption of affinity bonds can be the
deformation of the pore wall surface (Fig. 9).
Moreover, the elastic deformation of the gel surface
accompanied by the conformational transition of
PNiPAAm chains to a collapsed and more hydro-
phobic state could change the interfacial tension at
the gel–liquid interface. This change will hardly
affect soluble protein molecules bound to the gel,
but could have a dramatic effect on the bound cells,
which have a distinct solid–liquid interface of their
own. This detachment strategy along with contin-
uous porous structure makes these adsorbents very
attractive for application of affinity interactions for
cell separations.
4.3. Smart membranes with controlled porosity:
‘‘chemical valve’’
The environmentally controlled change in macro-
molecule size from a compact hydrophobic globule
to an expanded hydrophilic coil is exploited when SP
are used in the systems with environmentally
controlled porosity, so called ‘‘chemical valves’’.
When a smart polymer is grafted to the surface of
the pores in a porous membrane or chromatographic
matrix, the transition in the macromolecule affects
the total free volume of the pores available for the
solvent and hence presents a means to regulate the
porosity of the system [234]. For example, cross-
linking polymerization of PNiPAAm inside the pores
of a sponge allows for reversible controlling water
flux through the composite gel from 0 to
660 L mÀ2
hÀ1
, with a temperature change from 23
to 401C [235]. Alternatively, grafting PNiPAAm on
polypropylene microfiltration membranes using plas-
ma-induced polymerization [236,237] or on poly-
ethylene terephtalate and polypropylene membranes
using radiation-induced polymerization [238] allows
for the variation of water flux from depending on the
temperature. PNiPAAm has been grafted both on
flat as well as on hollow fiber membranes [239].
Apart from PNiPAAm, another thermosensitive
polymer, poly(vinylcaprolactam) (PVCL)) has been
photochemically immobilized on poly(ethylene ter-
ephthalate) track etched membranes allowing for
temperature-controlled permittivity of dextran tra-
cers [240]. Thermosensitive membranes have also
been prepared by the phase inversion method from
PNiPAAm-g-poly(vinylidene fluoride) (PNiPAAm-
g-poly(VDF)) co-polymers [241] or by adding
PNiPAAm [242,243] or thermosensitive elastin-like
peptides [244] to tetraethyl orthosilicate solution
prior to starting sol–gel process. At temperature
below the LCST for the corresponding soluble
polymers, the silicagel membranes are impermeable
to all of the PEG markers regardless of their
molecular weight whereas above the LCSTs, the
membranes are permeable to PEG markers with
molecular weight below a certain limit.
Membranes with pH-responsive permeability are
produced by grafting (PMAA) within the pores of
porous polyethylene membranes [245], by grafting
acrylic acid (AA) on the porous polypropylene
membrane in supercritical carbon dioxide [246], by
immobilization of poly-L-glutamic acid on a poly-
carbonate track-etched membrane [247], by the phase
inversion method from poly(AA)-g-poly(VDF) co-
polymers [248], as organic-inorganic composite pre-
pared from tetra ethyl orthosilicate and chitosan
ARTICLE IN PRESS
Affinity ligand at the pore
surface of macroporous hydrogel
binds receptor at the cell surface
Detachment of captured cell
by heat-induced shrinkage
of PNiPAAm
Increase in
temperature
Fig. 9. Schematic presentation of the mechanism of detachment of captured cells by heat-induced shrinkage of macroporous PNiPAAm
cryogel.
A. Kumar et al. / Prog. Polym. Sci. 32 (2007) 1205–1237 1227

24. [249], as self-organizing blends of (poly(VDF)) and
an amphiphilic comb polymer having a poly(VDF)
backbone and PMAA [250]; or by covering porous
polycarbonate membrane with 50nm thick gold
layer followed by self assembly of poly(acrylic acid)
(poly(AA)) conjugated with cystamines on the gold
surface [251]. Polyelectrolyte multilayers with pH-
responsive permeability have been assembled from
poly(acrylic acid) (PAA), poly(allylamine hydro-
chloride) (PAH), and poly(sodium 4-styrenesulfo-
nate) (SPS) in appropriate combinations [252],
from poly(allylamine hydrochloride) and sodium
poly(styrene sulfonate) [253].
The membranes with dual, pH- and thermosensi-
tivity are produced by co-grafting of NiPAAm
and methacrylic acid (MAA) [195]; by UV-induced
cross-linking co-polymerization of NiPAAm
and AA deposited on the surface of cross-linked
poly(2-hydroxyethyl methacrylate) matrix [254]; or
by blending PNiPAAm with poly(VDF)-graft-
poly(4-vinylpyridine) co-polymer [255]. The perme-
ability and flux of aqueous solutions through the
membranes prepared by grafting 4-vinylbenzyl
chloride on poly(VDF) followed by modification
with viologen can be reversibly regulated by redox
reactions [256].
A novel concept of producing membranes with
‘‘on-off’’ permeability has been introduced recently
[257,258]. Nanoparticles capable of changing their
size in response to external stimuli are embedded
into the bulk matrix. When shrinking, the latex
particles open the channels and hence increase the
permeability of the membrane. For example,
nanoparticles of poly(NiPAAm-co-MAA) co-poly-
mer were embedded in ethylcellulose membrane.
Permeability of the solutes (N-benzoyl-L-tyrosine
ethyl ester HCl, many peptides, Leuprolide, vitamin
B12, insulin, and lysozyme) across the membranes
increased with increasing temperature or particle
concentration, while decreased with increasing pH
[257]. Alternatively, the channels were designed to
contain an ordered array of magnetic polystyrene
latex particles embedded into a PVA gel. Due to the
static uniform magnetic field applied when the
composite membrane is produced, the mutual inter-
action between the magnetic gel beads results in a
pearl chain structures. Thermoresponsive compo-
site-gel membranes capable of regulating perme-
ability in response to external temperature change
have been produced by embedding PNiPAAm
coated magnetic polystyrene latex particles
(Fig. 10). By varying the thickness of PNiPAAm
shell, it is possible to tune the permeability of
the membranes over a wide range. This approach is
not merely limited to temperature responsive core-
shell hydrogels. Through appropriate design, the
hydrogel shell can sense chemical environments
such as pH, specific ions, or molecules and allows
ARTICLE IN PRESS
Fig. 10. Schematic representation of channels made of magnetic polystyrene latex covered with PNiPAAm) and built in the PVA gel
matrix: (a) ‘‘off’’ state below the collapse transition temperature; (b) ‘‘on’’ state above the collapse transition temperature. Arrows indicate
the diffusive mass transfer in the channels of PVA membrane. Reproduced from [258] with permission.
A. Kumar et al. / Prog. Polym. Sci. 32 (2007) 1205–12371228

25. for self-regulated mass transfer. This concept could
also be used to develop smart membranes with
pores that can open and close by electronically
induced external triggers such as an electric or
magnetic field [258].
When using a specific recognition element, which
could recognize the presence of a specific substance
or a specific stimulus and translate the signal into a
change of physico-chemical properties, e.g. pH, a
smart membrane changing its permeability in
response to the presence of particular substances
could be constructed.
A photoresponsive hydrogel was prepared by
radical co-polymerization of N-isopropylacryla-
mide, cross-linker and a light-recognition element,
namely vinyl monomer having a spirobenzopyran
residue. The hydrogel in an acidic condition
exhibited drastic and rapid volume shrinkage and
proton dissociation when it was irradiated with blue
light. The gel permeability for 1 mM HCl aqueous
solution increased two-fold in response to the blue
light irradiation, and the photoresponse of the
permeability was repeatable [259].
Another example of the recognition element is
benzo crown-ether which traps specifically K+
- or
Ba2+
-ions. When benzo crown-6-acrylamide was
co-polymerized with NiPAAm, the LCST of the
resulting co-polymer depended on the presence of
K+
- or Ba2+
-ions. The solution flux through the
membrane with plasma graft polymerized co-poly-
mer in the absence of Ba2+
decreased by about two
orders of magnitude as compared to a solution flux
containing Ba2+
-ions during polymerization. The
pore size was estimated by the filtration of aqueous
dextran solutions with various solute sizes. This
revealed that the membrane changes its pore size
between 5 and 27 nm in response to the Ba2+
concentration changes. No such change is observed
for Ca2+
solutions [260]. The Ca/Ba-sensitivity of
the membrane allows for controlling the perme-
ability of vitamin B12 and lysozyme through the
membrane [261].
Glucose-sensitive membranes were constructed from
cross-linked dextrans to which ConA was coupled via
a spacer arm. The presence of 0.1M glucose which is
competing with dextran for binding to ConA, increases
the rates of diffusion of both insulin and lysozyme
through the membrane, whereas glycerol or L-glucose
(0.1M) have no significant effect [262]. Alternatively,
poly(AA) has been grafted into the pores of the
microcapsule membrane by plasma-polymerization
and glucose oxidase has been immobilized onto the
poly(AA)-grafted microcapsules. The release rates of
model drug vitamin B12 from the fabricated micro-
capsules increases dramatically in the presence of 0.2M
glucose as grafted poly(AA) chains collapsed due to
the decrease in pH caused by glucose oxidation [263].
Using negative, nonlinear feedback between the
swelling state of a pH-responsive poly(NiPAAm-co-
MAA) hydrogel membrane and the enzymatic oxida-
tion of glucose with glucose oxidase accompanied with
the release of hydrogen ions, a rhythmic system has
been developed with pulsations between pH values
5.05 and 5.1 and with the oscillation period of $4h.
These changes in pH are sufficient to affect the
permeability of the hydrogel membrane allowing for
the release of gonadotropin-releasing hormone in
short, repetitive pulses over 1 week [264].
With the possibility of combining biorecognition
with the other unique features of smart surfaces,
expectations are running high in this area. Only the
time and more experimentation will determine
whether biosensitive smart surfaces will live up to
their generous promises. The smart surfaces are thus
created to alter the surface characteristics of a normal
surface to establish biomaterial interaction. The
interaction of cells and other biomolecules with smart
surfaces are tested and studied for various applica-
tions. Many research groups are involved in char-
acterizing these surfaces and engage in finding novel
bioengineering applications. New techniques are also
expected for a more controlled modification of sur-
faces with polymers allowing the design of different
architecture of polymer chains at the surface.
Acknowledgments
The authors would like to thank all those
scientists whose research contributions of smart
polymers have been referred here. Financial support
to AK from SIDA/VR link project, Department of
Biotechnology, Ministry of Science and Technol-
ogy, Government of India and faculty grant of IIT
Kanpur is acknowledged. AS acknowledges the Sr.
Research Fellowship from University Grants Com-
mission, India. The authors also acknowledge the
support from the Swedish Foundation for Strategic
Research (area of Chemistry an Life sciences).
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