You are on page 1of 33


Prog. Polym. Sci. 32 (2007) 12051237

Smart polymers: Physical forms and bioengineering applications

Ashok Kumara,b,, Akshay Srivastavaa, Igor Yu Galaevb, Bo Mattiassonb,

Department of Biological Sciences and Bioengineering, Indian Institute of Technology Kanpur, 208016-Kanpur, India
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

Smart polymers (SP) have become one important class of polymers and their applications have been increasing
signicantly. Last two to three decades have witnessed explosive growth in the subject. SP which are also known as stimuliresponsive solubleinsoluble 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 specic application areas
of bioseparations, protein folding, microuidics 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; Microuidics and


Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1206
Polymers as linear free chains in solution. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1208
2.1. Bioseparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1208
2.1.1. Aqueous two-phase polymer system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1208
2.1.2. Afnity precipitation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1211

Abbreviations: AA, acrylic acid; AML, afnity 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 -dimethylacrylamide); PMAA, poly(methacrylic acid); PNiPAAm, poly(N-isopropylacrylamide); PVCL, poly(vinylcaprolactam); poly(VDF),
poly(vinylidene uoride); SP, smart polymers; SPP, 3-[N-(3-methacrylamidopropyl)-N, N-dimethyl] ammonio-propane sulfonate
Also to be corresponded to. Tel.: +91 512 2594010; fax: +91 512 2594051.
Corresponding author. Tel.: +46 46 2228264; fax: +46 46 2224713.
E-mail addresses: (A. Kumar), (B. Mattiasson).
0079-6700/$ - see front matter r 2007 Elsevier Ltd. All rights reserved.

A. Kumar et al. / Prog. Polym. Sci. 32 (2007) 12051237




2.2. Protein folding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1214

Covalently cross-linked, reversible and physical gels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1216
3.1. Microuidics and actuators . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1217
3.2. Smart polymer hydrogels as sensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1219
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 scientic applications. The
synthetic polymers can be classied 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-sensitive 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 followingshape, surface characteristics, 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 polycationpolyanion
complex formation [10]. More recently, changes in
electric [11] and magnetic eld [12], light or
radiation forces [13] have also been reported as
stimuli for these polymers. The physical stimuli,
such as temperature, electric or magnetic elds, 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 hydrogenbonded 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 capabilities 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 classied 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-vinylalkylamides) e.g. poly(N-vinylcaprolactam) with a LCST
of about 3235 1C according to molecular mass of
polymer [18]. There are other types of temperatureresponsive polymers such as poly(ethylene oxide)106poly(propylene oxide)70-poly (ethylene oxide)106
co-polymer [19], which has the trade name Pluronics

A. Kumar et al. / Prog. Polym. Sci. 32 (2007) 12051237

F127 and poly lactic acid-copoly ethylene glycol

poly lactic acid (PLLA)/PEG/PLLA triblock copolymers [20]. Another interesting class of temperature-responsive polymers have recently emerged
which involves elastin like polymers (ELPs) [21].
The specic LCST of all these different polymeric
systems show potential applications in bioengineering 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 transition of pH-sensitive polymer tends to be very sharp
and usually switches within 0.20.3 unit of pH. Copolymers of methylmethacrylate and methacrylic
acid undergo sharp conformational transition and
collapse at low pH around 5, while co-polymers of
methacrylate are soluble at low pH but collapse
and aggregate under slightly alkaline conditions.
Other types of responsive polymers involve electric
eld [11] and magnetic eld [12], the gels of which
can shrink/swell in response to external electric or
magnetic eld stimuli. Polythiophene or sulphonated-polystyrene-based conducting polymers have
shown bending in response to external eld. The
magnetic eld-responsive gel which can be obtained
by dispersing magnetic colloidal particle in poly
(N-isopropylacrylamide-co-poly vinylalcohol) hydrogel matrix and get aggregated in external nonuniform magnetic eld [12].
These responses of polymer systems show usefulness 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 stimuliresponsive mechanisms into one polymer system.
For instance, temperature-sensitive polymers may
also respond to pH changes [2628]. Two or more
signals could be simultaneously applied in order to
induce response in so called dual-responsive polymer 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 reversible or physical gels, which can be either microscopic 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 specic external
parameter is modied. SPs in all the three formsin
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 polymerbiomolecule hybrid system is capable of responding to
biological, physical and chemical stimuli. Hoffman
and colleagues have pioneered the work in combining SPs with a wide variety of biomolecules [3538].
The SPs can be conjugated randomly or sitespecically 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


Fig. 1. Classication 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) 12051237

different applications [33]. This review focuses on

the various potential applications of SPs within the
above three dened 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, microuidics and actuators, chemical valves and tissue
engineering applications.
2. Polymers as linear free chains in solution
In aqueous solution, the delicate balance between
hydrophobichydrophilic conditions controls phase
transition of the polymer. As hydrophobic conditions 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 behavior, their isobaric phase diagrams presenting a
LCST [3942]. The solutions are homogenous at
low temperature and a phase separation appears
when the temperature exceeds a denite 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 sharpness 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
[4446], 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 completely soluble because of hydrophilic state and
forms a clear solution. Water-soluble block copolymers were prepared from the non-ionic monomer of N-isopropylacrylamide (NiPAAm) and the
zwitterionic monomer 3-[N-(3-methacrylamidopropyl)-N,N-dimethyl] ammonio-propane sulfonate
(SPP) by sequential free radical polymerization via
the reversible additionfragmentation 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 copolymers 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 temperatures, 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 reversibly can be prepared in water [49]. Another type of
soluble SPs which respond to microchanges in pH
are the pH-responsive polymerssuch as Eudragit 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 acidication 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 separation of biomolecules in puried 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), afnity precipitation or
thermoresponsive chromatography. The thermoresponsive chromatography comes under smart surfaces and membranes section and will be discussed
2.1.1. Aqueous two-phase polymer system
ATPS is an aqueous, liquidliquid, biphasic
system which is obtained by mixing of aqueous
solution of two polymers, or a polymer and a salt at

A. Kumar et al. / Prog. Polym. Sci. 32 (2007) 12051237

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 afnity. 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 modied
starch, e.g. hydroxypropyl starch (Reppal PES 200)
as a cost-effective alternative. But the major bottleneck 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 afnity ligands for specic
binding to target biomolecule. Application of SP as
stimuli-responsive solubleinsoluble polymers for
ligand carriers in ATPS has shown promising
potential [5254]. The polymerligand complex is
specically 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 thermoseparated aqueous two-phase system. The thermoresponsive polymers used for ATPS include
PNiPAAm, polyvinylcaprolactam (PVCL), cellulose ethers such as ethyl(hydroxyethyl)cellulose

Fig. 2. Type-specic 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) 12051237

(EHEC), ethylene oxidepropylene oxide (EOPO)

random co-polymer and EOPO block co-polymer
[55,56]. There are also other examples wherein
thermoresponsive polymers such as EOPO copolymers [57,58] or poly(N-vinylcaprolactam-covinyl 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 thermoresponsive and it is possible to recycle both
polymers by temperature-induced phase separation
[59]. This is a modied 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 modied random co-polymer of
EO and PO with aliphatic C14H29groups coupled
to each end of the polymer (HMEOPO). In
aqueous solution both polymers will phase separate
above a critical temperature (cloud point for
EO50%PO50% 50 1C, HMEOPO, 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 HMEOPO are mixed
in water, the solution will separate in two phases
above a certain concentration i.e. an aqueous twophase 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 purication factor 5.5 of apolipoprotein A-1
can be achieved [59]. Aqueous two-phase partitioning of endo-polygalacturonase (endo-PG) produced
by Kluyveromyces marxianus strains was carried out
on systems containing the thermoseparating polymer 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 efciency
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 efcient 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
purication 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 amyloglucosidase as well as cell-free extract of a hyperthermostable a-amylase in different ATPSs has demonstrated the potential for partitioning of enzymes
used in extractive bioconversion of starch. The
partition behavior of pure a-amylase and amyloglucosidase in four ATPSs, namely, PEOPPO/
(NH4)2SO4, PEOPPO/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 thermoseparated 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
6098% [64]. A new type of ATPS has recently
been established which uses modied starch derivative and thermoresponsive polymer of VCL as phase
forming polymers [52]. It is also reported that
thermoseparating ATPS for extraction of recombinant cutinase fusion protein from E. coli homogenate can be scaled up to pilot scale [65]. The
application of pH-responsive polymers like polyethyleneoxidemaleic acid co-polymer [66] as phaseforming polymers in ATPS has also been reported.
The polymers, just like proteins, contain two pH
triggerable functionalities (NH+
3 and COO ) that
make them exhibit pH-responsive behavior. Polydiallylaminoethanoate-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 precipitation. Furthermore, in applications such as protein

A. Kumar et al. / Prog. Polym. Sci. 32 (2007) 12051237

partitioning, the protein-like structure of the polymer is expected to enhance proteinpolymer interactions [67].
More interesting application has been shown for
the separation of animal cells by coupling an
antibody (against a cell surface protein) to a temperature-sensitive SP such as PNiPAAm (Fig. 2). The
ATPS composed of PEG and dextran was developed where PNiPAAm was used as a ligand carrier
for specic 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 specically 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 specically 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 purication 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 selectivity 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.
Afnity precipitation of proteins using SP emerged
in the early 1980s. Since then it has evolved as a
technique capable of simple, fast, and efcient
purication of a variety of proteins [6971]. As a
general rule, there are ve basic steps in afnity
precipitation: (i) carrying out afnity interactions in
free solution, (ii) precipitation of the afnity
reagenttarget protein complex from the solution,
(iii) recovery of the precipitate, (iv) dissociation and
recovery of the target molecule from the complex,
and nally, (v) recovery of the afnity reagent.
Afnity 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 afnity precipitation does not

utilize SP, it thus falls beyond the scope of this
review. Hetero-bifunctional format of afnity precipitation is a more general approach, wherein
afnity 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
afnity precipitation must contain reactive groups
for ligand coupling, show moderate interaction with
the ligand or impurities to prevent non-specic coprecipitation of impurities, give complete phase
separation of the polymer upon a change of medium
property, form polymer precipitates that are compact, to allow easy separation and to exclude
trapping of impurities into a gel structure, be easily
solubilized after the precipitate is formed, the
precipitationsolubilization cycle must be repeatable many times with good recovery, be available and
cost effective.
The polymerligand conjugate rstly forms a
complex with the target protein and phase separation 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 macroligandprotein
complex or the precipitate is dissolved, the protein
gets dissociated from the macroligand and the
ligandpolymer conjugate is reprecipitated without
the protein which remains in the supernatant in a
puried form (Fig. 3). Various ligands, such as
triazine dyes, sugars, protease inhibitors, antibodies,
and nucleotides have been successfully used for
afnity precipitation.
There is a range of different proteins/enzymes
which have been puried successfully by afnity
precipitation using pH-responsive polymers [72]. In
general, a specic ligand is chemically coupled to the
polymer backbone which latter binds to the target
protein in solution and the proteinpolymer complex is precipitated by change of pH as it renders the
polymer backbone insoluble. But in some cases the
polymer itself has the afnity 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-responsive SP have been used successfully in afnity


A. Kumar et al. / Prog. Polym. Sci. 32 (2007) 12051237

Fig. 3. Scheme of metal chelate afnity precipitation of proteins. Reproduced from [79] with permission.

precipitation of many proteins, but because of the

charged character of the polymer which shows
some non-specic interactions with other proteins
a more general use has not taken place for these
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 afnity ligand has gained
usefulness by adopting the technique in a nonchromatographic format like metal chelating affinity
precipitation [7578]. By combining the versatile
properties of metal afnity with afnity precipitation, 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 establishing thermosensitive polymers, PNiPAAm or PVCL
[79] as effective SPs in metal-chelate afnity
precipitation. In metal chelating afnity precipitation, metal ligands like imidazole are covalently
coupled to the reversible solubleinsoluble SP by
radical co-polymerization [80]. The co-polymers
carrying metal-chelating ligands are charged with

metal ions and the target protein binds the metalloaded co-polymer in solution via the interaction
between the histidine on the protein and the metal
ion. Many proteins both containing natural metalion binding residues and recombinant proteins
containing His-tag residues have been puried using
metal chelate afnity precipitation [78]. Therefore,
His-tagged protein or cells or bioparticles (with
surface accessible co-ordinating groups) can be
puried through the precipitation of target moleculemetal 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, purication
of extracellularly expressed six histidine-tagged
single chain Fv-antibody fragments (His6-scFv
fragments), from recombinant Escherichia coli cell
culture broth was performed. Quantitative precipitation 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

A. Kumar et al. / Prog. Polym. Sci. 32 (2007) 12051237

(VI) and NiPAAm [Cu(II)-poly(VI-NiPAAm) and

Ni(II)-poly(VI-NiPAAm)]. The precipitation efciency 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 purication, 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 afnity 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 afnity conditions. The AML in
this case is a conjugate between oligomeric AML
precursor and a single-stranded oligonucleotide,
whose sequence is complementary to a specic
afnity motif in the plasmid DNA (triple helix
afnity interaction). By increasing the temperature
the AML-plasmid DNA complex is precipitated.
After ltration and washing, the precipitate is redissolved and the specically bound plasmid DNA
is released [83].
Similarly lectins, concanavalin A (ConA) and
wheat germ lectin (WGL) when conjugated to
PNiPAAm, these lectinpolymer conjugates were
used in the purication of various polysaccharides
or polysaccharide-containing compounds such as
glucan [84]. The thermally reversible solubleinsoluble PNiPAAmdextran derivative (DD) conjugate
has been synthesized by conjugating amino-terminated PNiPAAm to a DD via ethyl-3-(3-dimethylaminopropyl)-carbodiimide and the conjugate was
used as a tool to purify polyclonal antibodies in
serum samples from rabbits subcutaneously immunized 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 transitions within a wide range of conditions [85,86].
These, ELPs have been used as terminal tags in
recombinant systems to facilitate recombinant
protein purication [87,88] and have recently been
used for conjugating to metal binding ligands for
afnity purication via temperature-triggered precipitation [89]. ELPs with repeating sequences of


[(VPGVG)2(VPGKG) (VPGVG)2]21 were synthesized and the free amino groups on the lysine
residues were modied by reacting with imidazole2-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. Purications of two
His-tagged enzymes, b-D-galactosidase and chloramphenicol acetyltransferase, were used to demonstrate the application of metal afnity precipitation
using this new type of afnity 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
purication performance. This has been the rst
report exploiting the features of ELPs for protein
purication based on metal-afnity purication.
The capability of modulating purication conditions by simple temperature triggers and their low
cost of preparation will probably make the ELPbased metal-afnity precipitation a useful method in
future, not only for protein purication but also for
diverse applications in bioseparation such as DNA
purication and environmental remediation [86].
Another interesting example has been a one-pot
afnity precipitation purication of carbohydratebinding 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-afnity precipitation purication of carbohydrate-binding protein
was demonstrated.
Other types of metal-chelating polymers for afnity
precipitation of proteins were reported by synthesizing
highly branched co-polymers of NiPAAm and 1,2propandiol-3-methacrylate (GMA: glycerol monomethacrylate), 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 18 1C
and were successfully used to purify a His-tagged
BRCA-1 protein fragment (a protein implicated in
breast cancer) by afnity precipitation [91,92]. An
interesting example of the use of poly(N-acryloylpiperidine) terminally modied with maltose for afnity precipitation of thermolabile a-glucosidase was


A. Kumar et al. / Prog. Polym. Sci. 32 (2007) 12051237

demonstrated [93]. The use of the polymer with

extremely low LCST (soluble below at 4 1C and
completely insoluble above 8 1C) made it possible
to use the technique for purication of thermolabile
a-glucosidase from cell-free extract of Saccharomyces
cerevisiae achieving 206-fold purication with 68%
Afnity precipitation is readily combined with
other protein isolation techniques, e.g. partitioning
in ATPSs [94]. Partitioning of protein complexed
with ligandpolymer 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 proteinpolymer complex is promoted by changing pH. Trypsin
was puried 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 immunoglobulin G [91,92], respectively. The approach was
also successfully demonstrated by the purication
of microbial xylanases, pullulanases, wheat germ
a-amylase, and sweet potato a-amylase [97,98] and
purication 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 afnity precipitation improves yield and purication 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 bioseparation of proteins using smart afnity ligands
[100,101]. In this method, a water-soluble polymer is oated as an interfacial precipitate by
adding ammonium sulfate and tertiary-butanol.
The polymer (appropriately chosen) in the presence of a protein for which it shows afnity,
selectively binds to the protein and oats as a
polymerprotein complex. By using this approach
wheat germ agglutinin (99% activity recovery
and 40-fold purication) and wheat germ lipase
(94% activity recovery and 27-fold purication)
have been puried using chitosan as a macroafnity
ligand [102].

2.2. Protein folding

Protein refolding is an important step in the
production of many functional recombinant proteins. Modest changes in the proteins 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 important 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 appreciation of varying hydrophobicity of the SP, these
polymers have found potentially interesting applications in the eld 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
modied PEG bound to functional ligand. PEG
was bound to a thermoreactive hydrophobic head
(poly (propylene oxide)phenyl group (PPOPh).
Refolding of bovine carbonic anhydrase was examined in the presence of PPOPhPEG at various
temperatures. The refolding yield of carbonic
anhydrase was strongly enhanced and aggregate
formation was suppressed by addition of PPOPh
PEG at a specic temperature of 5055 1C [103].
An articial 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 articial chaperons have been used to assist
the refolding of bovine carbonic anhydrase using

A. Kumar et al. / Prog. Polym. Sci. 32 (2007) 12051237

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 refolding. The mechanism of PNiPAAm-assisted protein
folding on bovine carbonic anhydrase B as model
protein was investigated and the results of uorescence analysis and equilibrium studies indicate that
PNiPAAm enhances protein refolding by the formation of complexes with aggregation-prone folding 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 denatured proteins and polymer gels could prevent the
aggregation of refolding intermediates, thus enhancing protein renaturation [107]. Recently, Liu et al
[108], prepared an articial chaperon, composed of
temperature-responsive PNiPAAm grafted with
b-cyclodextrin (a weakly hydrophobic stripper)
for protein refolding, where cetyltrimethylammonium 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 uorescence 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 prepared and characterized for its use as articial
chaperon to assist protein refolding of model
proteins like lysozyme and bovine carbonic anhydrase. In this case, the function of tunable hydrophobic segment helps to form the complex with
protein being refolded and the presence of hydrophilic 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 nonspecically. 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
purication of xylanase. It has been found that
microwave-treated Eudragit S-100 also gave better

Fig. 4. Schematic view of the mechanism of temperature-stimuli-articial chaperone assisted protein refolding in vitro.


A. Kumar et al. / Prog. Polym. Sci. 32 (2007) 12051237

results as compared to untreated Eudragit S-100 in

terms of refolding/purication 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 rectied the
protein refolding process with better outcome and
high yield. Furthermore, ongoing research is carving the new applications of SPs in solution form
and investigating different biological and nonbiological systems.
3. Covalently cross-linked, reversible and physical
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 reviewed in recent papers [111113]. Recently hydrogel of large pore size synthesized in moderately
frozen conditions and providing them with interesting properties have emerged which are called
cryogels [114]. Cryogels are obtained at temperatures below the melting temperature of the solvent.
At subzero temperatures most of the solvent is
frozen, while the dissolved substances are concentrated 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
efcient 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 elds
[1113]. Some gels also have been engineered to
respond to specic 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 RTfln1  ^ ^ w^2
V s ne =V 0 ^1=3  ^=2g.
Here, Vs is the molar volume of the solvent, 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 rst 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 equilibrium 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 networks (e.g. hydrogen-bonded networks) have
gained attention only recently. The physical crosslinks 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 scientic interest

A. Kumar et al. / Prog. Polym. Sci. 32 (2007) 12051237

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 uctuations in PNiPAAm gels as a
function of the degree of swelling has been
extensively studied [123]. Late stage spinodal
decomposition kinetics [124] and turbidity measurements 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 microcalorimetry, the thermal volume phase transition in
PNiPAAm hydrogels can be investigated [128].
The pH- and temperature-sensitive interpenetrating 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 polymerization method. These hydrogels exhibited a
combined pH- and temperature-sensitivity at a
temperature range of 3132 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 ow control in


microuidics, sensors in biological and chemical

applications. These are further discussed in the
following sections.
3.1. Microfluidics and actuators
Flow control in integrated multifunctional microuidic devices still remains a major challenge and a
fully functional valve is a key component in
microuidic systems. In recent years, developing
microuidic systems for biological and chemical
applications has been a major challenge [131133].
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 signicant advantage over conventional
microuidic 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
microuidics and actuator systems [134]. Thus, the
use of responsive hydrogel materials to regulate ow
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 microuidics
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 temperature-responsive hydrogel valves have been reported [135137]. The monolithic plugs PNiPAAm
cross-linked with 5% methylenebisacrylamide have

Fig. 5. Illustration of hydrogel volume changes as the gels 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) 12051237

been prepared by photoinitiated polymerization

within the channel of a microuidic device. Thermoelectric elements were used to actuate the valve and
ow through the device was monitored by uorescence measurements via laser-triggered photobleaching of a dye contained in liquid phase [138].
PNiPAAm hydrogel can also be used as valve for
ow 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 photopatterning and used as valves within the channels of
microuidic devices. No changes in performance
were observed even after repeated open-close
cycling of valves [140]. The manipulation of living
biological cells in microuidic channels by a
combination of negative dielectrophoretic barriers
and pressure driven ows is widely employed in labon-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 microuidic devices
as part of switchable supports. Separation schemes
with dynamic temperature control are most likely to
increase efciency of high throughput DNA analysis [142]. The response of hydrogel actuator should
be in order of seconds for a microuidics 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 microuidic 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 ow of different uids, and (d) an isotropic
swelling of the hydrogel and the response to
different stimuli [143].
Temperature-responsive polymers have been used
to construct smart afnity beads that can be
reversibly immobilized on microuidic channel
walls above the LCST to capture the target
biomolecules through its afnity moiety. The smart
afnity beads along with the target biomolecule are
then released from the channel wall on lowering the
temperature of microuidic channel below the
LCST of smart afnity bead [144]. Active thermoresponsive polymer has been integrated into a
microuidic 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 separation of soluble proteins on an integrated uidics
chip [145].
Apart from the temperature-responsive polymers,
pH-responsive polymers have also shown potential
to act as actuators in microuidics. 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 photopolymerization. The device uses a poly(hydroxyethyl
methacrylateacrylic acid) (poly(HEMAAA)),
pH-responsive hydrogel as the actuator in a PDMS
microuidic device [147]. A biomimetic check valve
fabricated by in situ photopolymerization of poly
(HEMAAA) hydrogel inside a glass microchannel
has been applied for directional control of uid ow
[148]. Combination of different types of pHresponsive hydrogels has been incorporated into
ow 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 acidtriethylene glycol dimethacrylate (PMAAEG) has been used to regulate drug
release by deforming a membrane which then
occludes an orice 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 membrane and a exible silicone rubber membrane,
creating a small and efcient 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 uorescein that can be used to construct
entirely polymeric microuidic 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 elds in aqueous NaCl solution for its application in biometric sensors and actuators, which
demonstrate rapid response under the inuence of
external electric eld [154]. A weakly cross-linked

A. Kumar et al. / Prog. Polym. Sci. 32 (2007) 12051237

anionic poly (2-acrylamido-2-methylpropane sulfonic 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 mechanical energy from the chemical energy of the
BelousovZhabotinsky 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 PVApoly(acrylic acid), which expands 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
articial organs [157,158].
The triggered control of interfacial properties
provided by immobilized SP at the solid water
interface has applications in designing of microuidics 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 exible membrane
above a uid reservoir chamber. When the microvalve is open, deformation of the membrane reduces
the volume of the reservoir chamber and pushes
uid through the microvalve. When the microvalve
is closed, expansion of hydrogel array generates a
storable pressure source that will result in uid
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 eld. The contraction
and expansion behavior was similar to that of a
natural muscle. These electroactive polymeric hydrogels can be utilized as biosensors and as articial
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 intriguing 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 composite membranes are prepared with the amine
containing dimethylaminoethyl methacrylate monomer, this results in a polymeric device that swelled in
response to pH changes (neutral to acidic medium).
Enhanced biosensing capabilities of these membranes 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 articial pancreas [163]. The conjugation of these polymers to different recognition proteins, including antibodies, protein A, streptavidin
and enzymes can be done in a random or sitespecic manner. Different SP, including temperature, pH and light-sensitive polymers have been
conjugated to these proteins. Once the analyte is
bound to these recognition proteins, the environmental change triggers the release of these molecules. This triggered release could also be used to
remove inhibitors, toxins or fouling agents from


A. Kumar et al. / Prog. Polym. Sci. 32 (2007) 12051237

the recognition sites of immobilized or free enzymes

and afnity molecules, such as those used in biosensors [164].
The amphiphilic co-polymer consisting of oligodeoxyribonucleotide 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 aggregated 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 uorescent benzofurazans were obtained by co-polymerization 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 homopolymers of two acrylamides [166]. The uorescence
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 uorescent
molecular thermosensors based on thermoresponsive polymers was achieved. Co-polymers of two

kinds of acrylamide derivative (N-n-propylacrylamide, N-isopropylacrylamide, and/or N-isopropyl

methacrylamide) labeled with 4-N-(2-acryloyloxyethyl)-N-methylamino-7-N,N-dimethylaminosulfonyl-2,1,3-benzoxadiazole (DBD-AE) have the
benzofurazan structure as a uorophore. These
uorescent 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 lm. The principle is well suited for a
precise investigation of the behavior of thin hydrogel lms. 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-dened crosslinked co-polymeric hydrogel of poly(methacrylic acid) (PMAA)
and PEG dimethacrylate works on polymer swelling

Fig. 6. Single nucleotide polymorphisms assay using DNA-linked colloidal nanoparticles.

A. Kumar et al. / Prog. Polym. Sci. 32 (2007) 12051237

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 methacrylate (HEMA) and N,N-dimethylaminoethyl methacrylate (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 articial biosystems utilizing stimuli-responsive hydrogels are of great interest due to
wide range of their biochemical and biotechnological applications. Such articial 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 rst 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 bioengineering applications like microuidics, actuator systems
and biosensors. However, these responsive hydrogels are also serving other bioengineering areas
like immobilized biocatalysts [174] and chromatography [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 macromolecule accompanied with a drastic increase
in hydrophobicity triggered by a small change


in environmental conditions. The hydrophobic

collapsed macromolecules aggregate and nally
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 surface 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 example, when PNiPAAm was end-grafted to mercaptopropyl 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 measurements. 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 equilibrium structure of tethered PNiPAAm chains
immersed in water were modeled employing a
numerical self-consistent eld theory where the
experimental phase diagram was used as input to
the theory. At low temperatures, the composition
proles are approximately parabolic and extend
into the solvent. In contrast, at temperatures above
the LCST of the bulk solution, the polymer proles
are collapsed near the surface. The layer thickness
and the effective monomer fraction within the layer
undergo what appears to be a rst-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


A. Kumar et al. / Prog. Polym. Sci. 32 (2007) 12051237

with experiment [178]. The model explains reasonably well the experimental data for the temperaturedependent conformational changes of PNiPAAm
grafted on gold and on silicon oxide over a range of
surface densities and molecular weights obtained
using neutron reection in D2O. The largest
conformational changes were observed for intermediate 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) [180182]. The obvious advantage here is that
one can beforehand determine the properties
(molecular mass, MW distribution) of the to-begrafted 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 difcult 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 efciency of grafting to methods is
pretty low resulting in pronounced losses of the
terminally modied 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 ne 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
[186190]. 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-nbutyl methacrylate) with different transition temperature 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 polyacrylamide 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 grafting techniques, the reader could nd an extensive
review [196] published recently in the same journal.
4.1. Smart surfaces for tissue engineering
The change of surface properties from hydrophobic above critical temperature of the polymer

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) 12051237

grafted to hydrophilic below it has been successfully

used for detachment of mammalian cells. Mammalian 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 [197199].
Since then the technology has been signicantly
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, respectively. 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. Cellcell junctions are retained
intact in the recovered cell sheets which could be
easily transferred to fresh culture dishes with the aid
of hydrophilically modied polyvinylideneuoride
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 coculture of epithelial and mesenchymal cells of lung
[203] or bovine aortic endothelial cells [204].
Microglia [205] or human monocytes and monocyte-derived macrophages [206] were also successfully 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 nanometer-scale range. PNiPAAm-grafted surfaces
with 1520-nm-thick layers exhibit temperaturedependent 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 surfacegrafted polymer chains as a function of grafting,
hydration, and temperature [207]. Lateral regulation of the cell adhesion on the smart surface is
achieved by nano-patterning of surface chemistry.
On a chemically patterned surface (Fig. 7), siteselective adhesion and growth of rat primary
hepatocytes and bovine carotid endothelial cells
allowed for patterned co-culture, exploiting hydrophobic/hydrophilic surface chemistry regulated by
culture temperature as the sole variable. At 27 1C,
seeded hepatocytes adhered exclusively onto hydrophobic, dehydrated poly(N-isopropylacrylamide-con-butyl methacrylate) co-grafted domains (1-mm
area), but not onto neighboring hydrated PNiPAAm domains. Sequentially seeded endothelial
cells then adhered exclusively to PNiPAAm domains which become hydrophobic upon increasing
temperature to 37 1C, achieving patterned cocultures. Reducing culture temperature to 20 1C
promoted hydration of both polymer-grafted domains, permitting release of the co-cultured, patterned cell monolayers as continuous cell sheets
with heterotypic cell interactions. Recovered cocultured cell sheets can be manipulated, moved and
sandwiched with other structures [192]. A wellcoordinated co-culture of three or more cell types
might also be realized since the transition temperature 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 copolymerization. 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 cellcell 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


A. Kumar et al. / Prog. Polym. Sci. 32 (2007) 12051237

achieved by placing a contiguous cell sheet of

conuent 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 PNiPAAmgrafted surfaces were layered into tissue-like laminate 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-modication 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-carboxyisopropylacrylamide) modied with cell adhesion peptide
RGDS (Arg-Gly-Asp-Ser) [213]. A similar behavior
was observed for bovine carotid artery endothelial
cells i.e. the modication with RGDS. RGDS
facilitated initial cell adhesion, while modication
with insulin induced cell proliferation. A more
pronounced cell growth was achieved by coimmobilization of appropriate amount of RGDS
and insulin [214].
On the other hand, co-polymerization with acrylic 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 reduced 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 sufcient to
detach cell sheets from PNiPAAm-co-PEG-grafted

porous membranes. Grafted PEG chains are believed to accelerate the diffusion of water molecules
to PNiPAAm grafts, showing more rapid detachment of cell sheet compared to only PNiPAAmgrafted 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% efciency at reduced temperature [217].
The change in the pattern of attachment of common
oral bacteria Streptococcus mutans following cycling 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, nonmotile 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 mammalian cells requires cell metabolic activity requiring
ATP consumption, signal transduction and cytoskeleton reorganization. Precoating PNiPAAmgrafted surfaces with bronectin improves spreading of less adhesive cultured hepatocytes and
reduces the temperature at which cultured cells are
released from bronectin-adsorbed grafted surfaces.
Immunostaining with anti-bronectin antibodies
revealed that only bronectin located beneath the
cultured cells is removed from culture surfaces after
reducing temperature. Fibronectin adsorbed to
surface areas lacking direct cell attachment remained surface-bound after reducing temperature
[219]. Principal component analysis using time-ofight 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 detachment. 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 surface as compared to the enzymatic methods [220].
At present, the low-temperature liftoff of cell
sheets from surfaces grafted with SP presents a

A. Kumar et al. / Prog. Polym. Sci. 32 (2007) 12051237

mature technique, which constitutes an important

step on the way to the cultivation of functional 3-D
4.2. Smart surfaces for temperature controlled
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
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 happened after 2001.
A modern tendency when separating low-molecular weight compound in an HPLC mode is to use
cross-linked layer of the gel of thermoresponsive
polymer rather than a brush. A new temperatureresponsive method of HPLC using packing materials modied 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 crosslinked co-polymerization of NiPAAm, acrylic acid
and N-tert-butylacrylamide, the stationary phase
changes in surface charge density and hydrophobicity. Alterations of properties of the polymer layer
were conrmed by temperature-responsive phase
transition and shift in apparent pKa values. Analyte
(catecholamine derivatives) retention was primarily
due to the electrostatic interaction. The temperature-induced phase transition of the hydrogel layer
on the stationary phases was evidenced by the
apparent inection point in vant Hoff plots around
36 1C suggesting that solute interactions are changed below and above the transition temperature,
reducing electrostatic interaction above the transition temperature [192,222].


Alternatively, PNiPAAm and its co-polymers

with n-butyl methacrylate and dimethylaminopropylacrylamide, which have reactive terminal functional groups, were synthesized via radical
polymerization using 3-mercaptopropionic acid as
a chain-transfer agent. Terminal carboxyl groups
were esteried by N-hydroxysuccinimide and N,N0 dicyclohexylcarbodiimide prior to modication
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 polypeptide [159], poly(acryloyl-L-proline methyl ester)
[226] or co-polymer of acryloyl-L-valine N-methylamide 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 modied
with poly(acryloyl-L-proline methyl ester) are controlled 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-modied surface, a
poly(acryloyl-L-proline methyl ester)-modied surface has a higher afnity 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 enhanced with temperature increase until the particular 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. Afnity 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 temperature was signicantly 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


A. Kumar et al. / Prog. Polym. Sci. 32 (2007) 12051237

Fig. 8. Chromatograms of steroids on a PNiPAAm terminally-modied 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: owrate, 1.0 mL min1; monitoring, UV at 254 nm. Reproduced from [225] with permission.

lowering the temperature to below the transition

temperature while maintaining other factors such as
pH and ion strength [229].
Afnity 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 releases 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
afnity-bound asislotransferrin from the immobilized agglutinin [230].
At present, one of the main challenges in
bioseparation is the specic separation of mixed
cell cultures into cell sub-populations. It is not
surprising because the cell separation entails great
difculties. 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 viewpoint, 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 difculty in disrupting multivalent interactions is one of the main
problems in designing afnity techniques for cell
separation. The conformational changes of SPs
allow for a nely tuned modication of the surface
properties and hence give a good tool to control
cellsurface 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 CD80or 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 afnity bound cells. When
bearing Cu(II)-iminodiacetate ligands, PNiPAAm
cryogel monoliths bound E. coli cells. The bound
cells were eluted with only 65% efciency 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

A. Kumar et al. / Prog. Polym. Sci. 32 (2007) 12051237

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 afnity 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 hydrophobic state could change the interfacial tension at
the gelliquid 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 solidliquid interface of their
own. This detachment strategy along with continuous porous structure makes these adsorbents very
attractive for application of afnity interactions for
cell separations.
4.3. Smart membranes with controlled porosity:
chemical valve
The environmentally controlled change in macromolecule 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, crosslinking polymerization of PNiPAAm inside the pores
of a sponge allows for reversible controlling water
ux through the composite gel from 0 to


660 L m2 h1, with a temperature change from 23

to 40 1C [235]. Alternatively, grafting PNiPAAm on
polypropylene microltration membranes using plasma-induced polymerization [236,237] or on polyethylene terephtalate and polypropylene membranes
using radiation-induced polymerization [238] allows
for the variation of water ux from depending on the
temperature. PNiPAAm has been grafted both on
at as well as on hollow ber membranes [239].
Apart from PNiPAAm, another thermosensitive
polymer, poly(vinylcaprolactam) (PVCL)) has been
photochemically immobilized on poly(ethylene terephthalate) track etched membranes allowing for
temperature-controlled permittivity of dextran tracers [240]. Thermosensitive membranes have also
been prepared by the phase inversion method from
PNiPAAm-g-poly(vinylidene uoride) (PNiPAAmg-poly(VDF)) co-polymers [241] or by adding
PNiPAAm [242,243] or thermosensitive elastin-like
peptides [244] to tetraethyl orthosilicate solution
prior to starting solgel 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 polycarbonate track-etched membrane [247], by the phase
inversion method from poly(AA)-g-poly(VDF) copolymers [248], as organic-inorganic composite prepared from tetra ethyl orthosilicate and chitosan

Increase in

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

Fig. 9. Schematic presentation of the mechanism of detachment of captured cells by heat-induced shrinkage of macroporous PNiPAAm


A. Kumar et al. / Prog. Polym. Sci. 32 (2007) 12051237

[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 50 nm thick gold
layer followed by self assembly of poly(acrylic acid)
(poly(AA)) conjugated with cystamines on the gold
surface [251]. Polyelectrolyte multilayers with pHresponsive permeability have been assembled from
poly(acrylic acid) (PAA), poly(allylamine hydrochloride) (PAH), and poly(sodium 4-styrenesulfonate) (SPS) in appropriate combinations [252],
from poly(allylamine hydrochloride) and sodium
poly(styrene sulfonate) [253].
The membranes with dual, pH- and thermosensitivity 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)-graftpoly(4-vinylpyridine) co-polymer [255]. The permeability and ux of aqueous solutions through the
membranes prepared by grafting 4-vinylbenzyl
chloride on poly(VDF) followed by modication
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-polymer 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 eld applied when the
composite membrane is produced, the mutual interaction between the magnetic gel beads results in a
pearl chain structures. Thermoresponsive composite-gel membranes capable of regulating permeability 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 coreshell hydrogels. Through appropriate design, the
hydrogel shell can sense chemical environments
such as pH, specic ions, or molecules and allows

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) 12051237

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 eld [258].
When using a specic recognition element, which
could recognize the presence of a specic substance
or a specic 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-isopropylacrylamide, 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 specically 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 ux through the
membrane with plasma graft polymerized co-polymer in the absence of Ba2+ decreased by about two
orders of magnitude as compared to a solution ux
containing Ba2+-ions during polymerization. The
pore size was estimated by the ltration 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 permeability 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 0.1 M 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
(0.1 M) have no signicant 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 microcapsules increases dramatically in the presence of 0.2 M
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-coMAA) hydrogel membrane and the enzymatic oxidation 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 4 h.
These changes in pH are sufcient 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 applications. Many research groups are involved in characterizing these surfaces and engage in nding novel
bioengineering applications. New techniques are also
expected for a more controlled modication of surfaces with polymers allowing the design of different
architecture of polymer chains at the surface.
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 Technology, Government of India and faculty grant of IIT
Kanpur is acknowledged. AS acknowledges the Sr.
Research Fellowship from University Grants Commission, India. The authors also acknowledge the
support from the Swedish Foundation for Strategic
Research (area of Chemistry an Life sciences).
[1] Jeong B, Gutowska A. Lessons from nature: stimuliresponsive polymers and their biomedical applications.
Trends Biotechnol 2002;20:30511.


A. Kumar et al. / Prog. Polym. Sci. 32 (2007) 12051237

[2] Hoffman AS, Stayton PS, Bulmus V, Chen G, Jinping C,

Chueng C, et al. Really smart bioconjugates of smart
polymers and receptor proteins. J Biomed Mater Res 2000;
[3] Galaev IYu, Mattiasson B. Smart polymers and what they
could do in biotechnology and medicine. Trends Biotechnol
[4] Kikuchi A, Okano T. Intelligent thermoresponsive polymeric stationary phases for aqueous chromatography of
biological compounds. Prog Polym Sci 2002;27:116593.
[5] Qiu Y, Park K. Environment-sensitive hydrogels for drug
delivery. Adv Drug Deliv Rev 2001;53:32139.
[6] Okano T. Molecular design of temperature-responsive
polymers as intelligent materials. In: Dusek K, editor.
Responsive gels: volume transitions, vol. II. Berlin: Springer; 1993. p. 18097.
[7] Twaites BR, Alarcon CH, Cunliffe D, Lavigne M,
Pennadam S, Smith JR, et al. Thermo and pH responsive
polymers as gene delivery vectors: effect of polymer
architecture on DNA complexation in vitro. J Control
Release 2004;97:55166.
[8] Lomadze N, Schneider H-J. Ternary complex formation
inducing large expansions of chemomechanical polymers
by metal chelators, aminoacids and peptides as effectors.
Tetrahedron Lett 2005;46:7514.
[9] Kabanov VA. Physicochemical basis and the prospects of
using soluble interpolyelectrolyte complex. Polym Sci
[10] Leclercq L, Boustta M, Vert M. A physico-chemical
approach of polyanion-polycation interactions aimed at
better understanding the in vivo behaviour of polyelectrolyte-based drug delivery and gene transfection. J Drug
Target 2003;11:12938.
[11] Filipcsei G, Feher J, Zrinyi M. Electric eld sensitive
neutral polymer gels. J Mol Struct 2000;554:10917.
[12] Zrinyi M. Intelligent polymer gels controlled by magnetic
elds. Colloid Polym Sci 2000;278:98103.
[13] Juodkazis S, Mukai N, Wakaki R, Yamaguchi A, Matsuo
S, Misawa H. Reversible phase transitions in polymer gels
induced by radiation forces. Nature 2000;408:17881.
[14] Galaev IYu, Gupta MN, Mattiasson B. Use smart
polymers for bioseparations. CHEMTECH 1996;26:1925.
[15] Taylor LD, Gerankowski LD. Preparation of lms
exhibiting balanced temperature dependence to permeation
by aqueous solutionsa study of lower consolute behavior.
J Polymer Sci Polymer Chem Ed 1975;13:255170.
[16] Yan L, Zhu Q, Kenkare PU. Lower critical solution
temperature of linear PNIPA obtained from a Yukawa
potential of polymer chains. J Appl Polym Sci 2000;78:
[17] Heskins M, Guillet JE. Solution properties of poly
(N-isopropylacrylamide). J Macromol Sci Chem 1968;A2:
[18] Suwa K, Morishita K, Kishida A, Akashi M. Synthesis and
functionalities of poly(N-vinylalkylamide). V. Control of a
lower critical solution temperature of poly (N-vinylalkylamide). J Polym Sci Part A: Polym Chem 1997;35:
[19] Wanka G, Hoffmann H, Ulbricht W. Phase diagrams and
aggregation behavior of poly (oxyethylene)-poly (oxypropylene)poly(oxyethylene) triblock copolymers in aqueous
solutions. Macromolecules 1994;27:414559.

[20] Na K, Lee KH, Lee DH, Bae YH. Biodegradable thermosensitive nanoparticles from poly(L-lactic acid)/poly(ethylene glycol) alternating multi-block copolymer for potential
anti-cancer drug carrier. Eur J Pharm Sci 2006;27:11522.
[21] Herrero-Vanrella R, Rincon AC, Alonso M, Reboto V,
Molina-Martineza IT, Rodr guez-Cabelloc JC. Self-assembled particles of an elastin-like polymer as vehicles
for controlled drug release. J Control Release 2005;102:
[22] Chilkoti A, Dreher MR, Meyer DE, Raucher D. Targeted
drug delivery by thermally responsive polymers. Adv Drug
Deliv Rev 2002;54:61330.
[23] Kobayashi J, Kikuchi A, Sakai K, Okano T. Aqueous
chromatography utilizing hydrophobicity-modied anionic
temperature-responsive hydrogel for stationary phases.
J Chromatogr A 2002;958:10919.
[24] Anastase-Ravion S, Ding Z, Pelle A, Hoffman AS,
Letourneur D. New antibody purication procedure
using a thermally responsive poly(N-isopropylacrylamide)
dextran derivative conjugate. J Chromatogr B 2001;761:
[25] Kushida A, Yamato M, Isoi Y, Kikuchi A, Okano T. A
noninvasive transfer system for polarized renal tubule
epithelial cell sheets using temperature-responsive culture
dishes. Eur Cells Mater 2005;10:2330.
[26] Pinkrah VT, Snowden MJ, Mitchell JC, Seidel J,
Chowdhry BZ, Fern GR. Physicochemical properties of
poly (N-isopropylacrylamide-co-4-vinylpyridine) cationic
polyelectrolyte colloidal microgels. Langmuir 2003;19:
[27] Peng T, Cheng YL. PNIPAAm and PMAA co-grafted
porous PE membranes: living radical co-grafting mechanism and multi-stimuli responsive permeability. Polymer
[28] Bignotti F, Penco M, Sartore L, Peroni I, Mendichi R,
Casolaro M, et al. Synthesis, characterisation and solution
behaviour of thermo- and pH-responsive polymers bearing
L-leucine residues in the side chains. Polymer 2000;41:
[29] Kurisawa M, Yui N. Dual-stimuli-responsive drug release
from interpenetrating polymer network-structured hydrogels of gelatin and dextran. J Control Release 1998;54:
[30] Miyata T, Asami N, Uragami T. A reversibly antigenresponsive hydrogel. Nature 1999;399:7669.
[31] Ulijn RV. Enzyme-responsive materials: a new class of
smart biomaterials. J Mater Chem 2006;16:221725.
[32] Kang S, Bae YH. A sulfonamide based glucose-responsive
hydrogel with covalently immobilized glucose oxidase and
catalase. J Control Release 2003;86:11521.
[33] Gil ES, Hudson SM. Stimuli-responsive polymers and their
bioconjugates. Prog Polym Sci 2004;29:1173222.
[34] Galaev IYu, Mattiasson B, editors. Smart polymers for
bioseparation and bioengineering. London: Taylor &
Francis; 2002.
[35] Stayton PS, Hoffman AS. Immobilization of smart
polymerprotein conjugates. In: Cass T, Ligler FS, editors.
Immobilized biomolecules in analysis. Oxford: Oxford
University Press; 1998. p. 13547.
[36] Monji N, Hoffman AS. A novel immunoassay system and
bioseparation process based on thermal phase separating
polymers. Appl Biochem Biotechnol 1987;14:10720.

A. Kumar et al. / Prog. Polym. Sci. 32 (2007) 12051237
[37] Lackey CA, Murthy N, Press OW, Tirrell DA, Hoffman
AS, Stayton PS. Hemolytic activity of pH-responsive
polymerstreptavidin bioconjugates. Bioconjugate Chem
[38] Fong RB, Ding Z, Long CJ, Hoffman AS, Stayton PS.
Thermoprecipitation of streptavidin via oligonucleotidemediated self-assembly with poly(NIPAAm). Bioconjugate
Chem 1999;10:7205.
[39] Eeckman F, Moes AJ, Amighi K. Synthesis and characterization of thermosensitive copolymers for oral controlled
drug delivery. Eur Polym J 2004;40:87381.
[40] Durand A, Hourdet D. Thermoassociative graft copolymers based on poly(N-isopropylacrylamide): effect of
added co-solutes on the rheological behaviour. Polymer
[41] Schild HG. Poly(N-isopropylacrylamide): experiment, theory and application. Prog Polym Sci 1992;17:163249.
[42] Feil H, Bae YH, Jan FJ, Kim SW. Effect of comonomer
hydrophilicity and ionization on the lower critical solution
temperature of N-isopropylacrylamide copolymers. Macromolecules 1993;26:2496500.
[43] Jones MS. Effect of pH on the lower critical solution
temperatures of random copolymers of N-isopropylacrylamide and acrylic acid. Eur Polym J 1999;35:795801.
[44] Eeckman F, Amighi K, Moes AJ. Effect of some
physiological and non-physiological compounds on the
phase transition temperature of thermoresponsive polymers
intended for oral controlled-drug delivery. Int J Pharm
[45] Huang J, Wu XY. Effects of pH, salt, surfactant and
composition on phase transition of poly(NIPAm/MAA)
nanoparticles. J Polym Sci Part A: Polym Chem 1999;37:
[46] Schild HG, Tirrell DA. Microcalorimetric detection of
lower critical solution temperatures in aqueous polymersolutions. J Phys Chem 1990;94:43526.
[47] Kokufuta E, Zhang YQ, Tanaka T, Mamada A. Effects of
surfactants on the phase-transition of poly(N-isopropylacrylamide) gel. Macromolecules 1993;26:10539.
[48] Schild HG, Tirrell DA. Interaction of poly(N-isopropylacrylamide) with sodium normal-alkyl sulfates in aqueoussolution. Langmuir 1991;7:66571.
[49] Arotcarena M, Heise B, Ishaya S, Laschewsky A. Switching the inside and the outside of aggregates of water-soluble
block copolymers with double thermoresponsivity. J Am
Chem Soc 2002;124:378793.
[50] Palomares MR. Practical application of aqueous two-phase
partition to process development for the recovery of
biological products. J Chromatogr B 2004;807:311.
[51] Diamond AD, Hsu JT. Aqueous two phase systems for
biomolecule separation. Adv Biochem Eng Biotechnol
[52] Pietruszka N, Galaev IYu, Kumar A, Brzowski ZK,
Mattiasson B. New polymers forming aqueous two phase
polymer systems. Biotechnol Prog 2000;16:40815.
[53] Kumar A, Kamihira M, Galaev IYu, Mattiasson B,
Iijima S. Type-specic separation of animal cells in aqueous
two-phase systems using antibody conjugates with temperature sensitive polymers. Biotechnol Bioeng 2001;75:
[54] Franko TT, Galaev IYu, Hatti-Kaul R, Holmberg N,
Bulow L, Mattiasson B. Aqueous two-phase system formed
















by thermoreactive vinyl imidazole/vinyl caprolactam copolymer and dextran for partitioning of a protein with a
polyhistidine tail. Biotechnol Technol 1997;11:2315.
Persson J, Johansson HO, Galaev IYu, Mattiasson B,
Tjerneld F. Aqueous polymer two-phase systems formed by
new thermoseparating polymers. Bioseparation 2000;9:
Kishida A, Kikunaga Y, Akashi M. Synthesis and
functionality of poly(N-vinylalkylamides). X. A novel
aqueous two-phase system based on thermosensitive
polymers and dextran. J Appl Polym Sci 1999;73:25458.
Frakas T, Stalbrand H, Tjerneld F. Partitioning of bmannanase and a-galactosidase from Aspergillus niger in
Ucon/Reppal aqueous two phase system. Bioseparation
Lu M, Albertsson PA, Johansson G, Tjerneld F. Uconbenzoyl dextran aqueous two-phase systems: protein
purication with phase component recycling. J Chromatogr
B 1996;680:6570.
Persson J, Johansson H-O, Tjerneld F. Purication of
protein and recycling of polymers in a new aqueous twophase system using two thermoseparating polymers.
J Chromatogr A 1999;864:3148.
Pereira M, Wu Y-T, Venancio A, Teixeira J. Aqueous twophase extraction using thermoseparating polymer: a new
system for the separation of endo-polygalacturonase.
Biochem Eng J 2003;15:1318.
Li M, Kim J-W, Peeples TL. Amylase partitioning and
extractive bioconversion of starch using thermoseparating
aqueous two-phase systems. J Biotechnol 2002;93:1526.
Johansson H-O, Persson J, Tjerneld F. Thermoseparating
water/polymer system: a novel one-polymer aqueous twophase system for protein purication. Biotechnol Bioeng
Jonsson M, Johansson H-O. Protein partitioning in
thermoseparating systems of a charged hydrophobically
modied ethylene oxide polymer. J Chromatogr A 2003;
Bolognese B, Nerli B, Pico G. Application of the aqueous
two-phase systems of ethylene and propylene oxide
copolymermaltodextrin for protein purication. J Chromatogr B 2005;814:34753.
Kepka C, Collet E, Persson J, Stahl A, Lagerstedt T,
Tjerneld F, et al. Pilot-scale extraction of an intracellular
recombinant cutinase from E. coli cell homogenate using a
thermoseparating aqueous two-phase system. J Biotechnol
Kajiuchi T, Shin-Ya Y, Nagai H, Fujiwara K. Partitioning
of chicken egg white proteins in polyelectrolyte/salt
aqueous two-phase systems composed of polyethyleneoxide-maleic acid copolymer and potassium phosphate.
J Chem Technol Biotechnol 2002;77:4959.
Waziri SM, Abu-Sharkh BF, Ali SA. The effect of pH and
salt concentration on the coexistence curves of aqueous
two-phase systems containing a pH responsive copolymer
and polyethylene glycol. Fluid Phase Equilib 2003;205:
Scopes RK. Protein purication: principles and practice.
New York: Springer; 1994.
Mattiasson B, Kaul R. Afnity precipitation. In: Ngo T,
editor. Molecular interactions in bioseparations. New
York: Plenum Press; 1993. p. 46977.


A. Kumar et al. / Prog. Polym. Sci. 32 (2007) 12051237

[70] Gupta MN, Mattiasson B. Afnity precipitation. In: Street

G, editor. Highly selective separations in biotechnology.
London: Blackie Academic and Professional; 1994. p. 733.
[71] Galaev IYu, Mattiasson B. New methods for afnity
purication of proteins. Afnity precipitation: a review.
Biochemistry (Moscow) 1997;62:5717.
[72] Kumar A, Galaev IYu, Mattiasson B. Smart polymers in
afnity precipitation of proteins. In: Mattiasson B, Galaev
IYu, editors. Smart polymers: production, study and
application in biotechnology and biomedicine. Second ed.
Boca Raton, USA: CRC Press, Taylor & Francis Group;
2007. p. 40236.
[73] Senstad C, Mattiasson B. Afnity precipitation using
chitosan as ligand carrier. Biotechnol Bioeng 2004;33:21620.
[74] Hilbrig F, Freitag R. Protein purication by afnity
precipitation. J Chromatogr B 2003;790:7990.
[75] Kumar A, Galaev IYu, Mattiasson B. Metal chelate
afnity precipitation: a new approach to protein purication. Bioseparation 1999;7:18594.
[76] Kumar A, Khalil AAM, Galaev IYu, Mattiasson B. Metal
chelate afnity precipitation: purication of (His)6-tagged
lactate dehydrogenase using poly(vinylimidazole-co-N-isopropylacrylamide) copolymers. Enzyme Microb Technol
[77] Kumar A, Wahlund P-O, Kepka C, Galaev IYu, Mattiasson B. Purication of histidine-tagged single chain Fvantibody fragments by metal chelate afnity precipitation
using thermo-responsive copolymers. Biotechnol Bioeng
[78] Mattiasson B, Kumar A, Ivanov AE, Galaev IYu. Metalchelate afnity precipitation of proteins using responsive
polymers. Nat Protoc 2007;2:21320.
[79] Galaev IYu, Kumar A, Mattiasson B. Metal copolymer
complexes of N-isopropylacrylamide for afnity precipitation of protreins. J Macromol Sci 1999;36:1093105.
[80] Galaev IYu, Kumar A, Agrawal R, Gupta MN, Mattiasson B. Imidazole- a new ligand for metal afnity precipitation: Precipiation of kunitz soybean trypsin inhibitor
using Cu(II)-loaded coplymers of 1-vinylimidazole with
N-vinylcaprolactam and N-isopropylacrylamide. Appl Biochem Biotechnol 1997;68:12133.
[81] Balan S, Murphy J, Galaev IYu, Kumar A, Fox GE,
Mattiasson B, et al. Metal chelate afnity precipitation of
RNA and purication of plasmid DNA. Biotechnol Lett
[82] Murphy JC, Jewell DL, White KI, Fox GE, Willson RC.
Nucleic acid separations utilizing immobilized metal
afnity chromatography. Biotechnol Prog 2003;19:9826.
[83] Costioli MD, Fisch I, Garret-Flaudy F, Hilbrig F, Freitag
R. DNA purication by triple-helix afnity precipitation.
Biotechnol Bioeng 2002;81:53545.
[84] Pan LC, Chien CC. A novel application of thermoresponsive polymer to afnity precipitation of polysaccharide. J Biochem Biophys Methods 2003;55:8794.
[85] Urry DW, Luan CH, Harris C, Parker TM. Protein-based
materials with a profound range of properties and
applications: the elastin DTt hydrophobic paradigm. In:
McGrath K, Kaplan D, editors. Protein based materials.
Boston: Birkhauser; 1997. p. 13377.
[86] Kostal J, Mulchandani A, Chen W. Tunable biopolymers
for heavy metal removal. Macromolecules 2001;34:

[87] Meyer DE, Chilkoti A. Purication of recombinant

proteins by fusion with thermally responsive polypeptides.
Nat Biotechnol 1999;17:11125.
[88] Shimazu M, Mulchandani A, Chen W. Thermally triggered
purication and immobilization of elastin-OPH fusion.
Biotechnol Bioeng 2003;81:749.
[89] Stiborova H, Kostal J, Mulchandani A, Chen W. One-step
metal-afnity purication of histidine-tagged proteins by
temperature-triggered precipitation. Biotechnol Bioeng
[90] Sun X-L, Haller CA, Wu XY, Conticello VP, Chaikof EL.
One-pot glyco-afnity precipitation purication for enhanced proteomics: the exible alignment of solution-phase
capture/release and solid-phase separation. J Proteome Res
[91] Carter S, Rimmer S, Sturdy A, Webb M. Highly branched
stimuli responsive poly[(N-isopropylacrylamide)-co-(1,
2-propandiol-3-methacrylate)]s with protein binding functionality. Macromol Biosci 2005;5:3738.
[92] Carter S, Hunt B, Rimmer S. Highly branched poly
(N-isopropyl-acrylamide)s with imidazole end groups prepared by radical polymerization in the presence of a styryl
monomer containing a dithioester group. Macromolecules
[93] Hoshino K, Taniguchi M, Kitao T, Morohashi S, Sasakura
T. Preparation of a new thermo-responsive adsorbent with
maltose as a ligand and its application to afnity
precipitation. Biotechnol Bioeng 1998;60:56879.
[94] Teotia S, Lata R, Gupta MN. Chitosan as a macroafnity
ligand. Purcation of chitinases by afnity precipitation
and aqueous two-phase extractions. J Chromatogr A 2004;
[95] Chen J-P, Jang F-L. Purication of trypsin by afnity
precipitation combining with aqueous two-phase extraction. Biotechnol Technol 1995;9:4616.
[96] Guoqiang D, Kaul R, Mattiasson B. Integration of
aqueous two-phase extraction and afnity precipitation
for the purication of lactate dehydrogenase. J Chromatogr
A 1994;668:14552.
[97] Teotia S, Gupta MN. Free polymeric bioligands in aqueous
two phase afnity extractions of microbial xylanases and
pullulanase. Prot Expr Purif 2001;22:4848.
[98] Teotia S, Gupta MN. Reversibly soluble macroafnity
ligand in aqueous two phase separation of enzymes.
J Chromatogr A 2001;923:27580.
[99] Kumar A, Kamihara M, Mattiasson B. Two-phase afnity
partitioning of animal cells: implications of multipoint
interactions. In: Gupta MN, editor. Methods for afnitybased separation of proteins/enzymes. Basel, Switzerland:
Birkhauser Verlag; 2002. p. 16380.
[100] Sharma A, Roy I, Gupta MN. Afnity precipitation and
macroafnity ligand facilitate three-phase partitioning for
refolding and simultaneous purication of urea-denatured
pectinase. Biotechnol Prog 2004;20:12558.
[101] Mondal K, Sharma A, Lata R, Gupta MN. Macroafnity
ligand-facilitated three-phase partitioning (MLFTPP) of
a-amylases using a modied Alginate. Biotechnol Prog
[102] Teotia S, Mondal K, Gupta MN. Integration of afnity
precipitation with partitioning methods for bioseparation
of chitin binding lectins. Food Bioprod Process 2006;84:

A. Kumar et al. / Prog. Polym. Sci. 32 (2007) 12051237
[103] Kuboi R, Morita S, Ota H, Umakoshi H. Protein refolding
using stimuli-responsive polymer-modied aqueous twophase systems. J Chromatogr B Biomed Sci Appl 2000;743:
[104] Yoshimoto N, Hashimoto T, Felix MM, Umakoshi H,
Kuboi R. Articial chaperone-assisted refolding of bovine
carbonic anhydrase using molecular assemblies of stimuliresponsive polymers. Biomacromolecules 2003;4:15308.
[105] Chen YJ, Huang LW, Chiu HC, Lin SC. Temperatureresponsive polymer-assisted protein refolding. Enzyme
Microb Technol 2003;32:12030.
[106] Lin SC, Lin KL, Chiu HC, Lin S. Enhanced protein
renaturation by temperature-responsive polymers. Biotechnol Bioeng 2000;67:50512.
[107] Cui Z, Guan Y, Yao S. A temperature-sensitive hydrogel
refolding system: preparation of poly(N-isopropylacrylamide) and its application in lysozyme refolding. Chin
J Chem Eng 2004;12:55660.
[108] Liu D, Zhang K, Liu Z. Protein refolding assisted by an
articial chaperone using temperature stimuli responsive
polymer as the stripper. Biochem Eng J 2005;25:1419.
[109] Roy I, Gupta MN. pH responsive polymer assisted
refolding of urea and organic solvent-denatured a-chymotrypsin. Prot Eng 2003;16:11537.
[110] Roy I, Mondal K, Sharma A, Gupta MN. Simultaneous refolding/purication of xylanase with a microwave
treated smart polymer. Biochim Biophys Acta 2005;1747:
[111] Kopecek J. Smart and genetically engineered biomaterials
and drug delivery systems. Eur J Pharm Sci 2003;20:116.
[112] Rosiak JM, Ulanski P. Synthesis of hydrogels by irradiation of polymers in aqueous solution. Radiat Phys Chem
[113] Roy I, Gupta MN. Smart polymeric materials:emerging
biochemical applications. Chem Biol 2003;10:116171.
[114] Lozinsky VI, Galaev IYu, Plieva FM, Savina IN, Jungvid
H, Mattiasson B. Polymeric cryogels as promising materials of biotechnological interest. Trends Biotechnol
[115] Traitel T, Cohen Y, Kost J. Characterization of glucosesensitive insulin release systems in simulated in vivo
conditions. Biomaterials 2000;21:167987.
[116] Miyataa T, Uragamia T, Nakamaeb K. Biomoleculesensitive hydrogels. Adv Drug Deliv Rev 2002;54:7998.
[117] Flory PJ. Principals of polymer chemistry. Ithaca, NY:
Cornell University Press; 1953.
[118] Badiger MV, Lele AK, Kulkami MG, Mashelkar RA.
Swelling and phase transitions in deforming polymeric gels.
Ind Eng Chem Res 1994;33:242633.
[119] Zhang XZ, Lewis PJ, Chu CC. Fabrication and characterization of a smart drug delivery system: microsphere in
hydrogel. Biomaterials 2005;26:3299309.
[120] Arca MY, Oketem HA, Oketem Z, Tuncel SA. Immobilization of catalase in poly(isopropylacrylamide-co-hydroxyethylmethacrylate) thermally reversible hydrogels. Polym
Int 1999;48:87984.
[121] Hirotsu S. Critical points of the volume phase transition in
N-isopropylacrylamide gels. J Chem Phys 1988;88:42731.
[122] Rinsdorff H, Simon J, Winnik FM. Hydrophobicallymodied poly(N-isopropylacrylamides) in water: probing
of the microdomain composition by nonradiative energy
transfer. Macromolecules 1992;25:535361.


[123] Shibayama M, Tanaka T, Han CC. Small angle neutron

scattering study on poly(N-isopropylacrylamide) gels near
their volume-phase transition temperature. J Chem Phys
[124] Hirotsu S. Static and time-dependent properties of polymer
gels around the volume phase transition. Phase Trans
[125] Dusek K, Sedlacek B. Structure and properties of hydrophilic polymers and their gels. XI. Microsyneresis in
swollen poly(ethylene glycol methacrylate) gels induced
by changes in temperature. Coll Czech Chem Commun
[126] Shen W, Smith GR, Knobler CM, Scott RL. Turbidity
measurements of binary polystyrene solutions near critical
solution points. J Phys Chem 1991;95:33769.
[127] DaMore LW, Jacobs DT. Turbidity of deuterated isobutyric acid and heavy water in the one-phase region near
the critical solution point. J Chem Phys 1992;97:4649.
[128] Grinberg NV, Dubovik AS, Grinberg VY, Kuznetsov DV,
Makhaeva EE, Grosberg AY, et al. Studies of the thermal
volume transition of poly(N-isopropylacrylamide) hydrogels by high-sensitivity differential scanning microcalorimetry. 1. Dynamic effects. Macromolecules 1999;32:
[129] Lee YM, Kim SH, Cho CS. Synthesis and swelling
characteristics of pH and thermo-responsive interpenetrating polymer network hydrogel composed of poly(vinyl
alcohol) and poly(acrylic acid). J Appl Polym Sci 1996;62:
[130] Zhang J, Peppas NA. Synthesis and characterization of
pH- and temperature-sensitive poly(methacrylic acid)/
poly(N-isopropylacrylamide) interpenetrating polymeric
networks. Macromolecules 2000;33:1027.
[131] Chen ZY, Qian SZ, Abrams WR, Malamud D, Bau HH.
Thermosiphon-based PCR reactor: experiment and modeling. Anal Chem 2004;76:370715.
[132] Lagally ET, Scherer JR, Blazej RG, Toriello NM, Diep
BA, Ramchandani M, et al. Integrated portable genetic
analysis microsystem for pathogen/infectious disease detection. Anal Chem 2004;76:316270.
[133] Zangmeister RA, Tarlov MJ. DNA displacement assay
integrated into microuidic channels. Anal Chem 2004;76:
[134] Eddington DT, Beebe DJ. Flow control with hydrogels.
Adv Drug Deliv Rev 2004;56:199210.
[135] Pelton R. Temperature-sensitive aqueous microgels. Adv
Colloid Interface Sci 2000;85:133.
[136] Richter A, Kuckling A, Howitz S, Gehring T, Arndt KF.
Electronically controllable microvalves based on smart
hydrogels: magnitudes and potential applications. J Microelectromech S 2003;12:74853.
[137] Richter A, Howitz S, Kuckling D, Arndt KF. Inuence of
volume phase transition phenomena on the behavior of
hydrogel-based valves. Sens Actuat B 2004;99:4518.
[138] Yu C, Mutlu S, Selvaganapathy P, Mastrangelo CH, Svec
F, Frechett JMJ. Flow control valves for analytical
microuidic chips without mechanical parts based on
thermally responsive monolithic polymers. Anal Chem
[139] Wang J, Chen Z, Mauk M, Hong KS, Li M, Yang S, et al.
Self-actuated, thermo-responsive hydrogel valves for lab on
a chip. Biomed Microdev 2005;7:31322.


A. Kumar et al. / Prog. Polym. Sci. 32 (2007) 12051237

[140] Luo Q, Mutlu S, Gianchandani YB, Svec F, Frechet JM.

Monolithic valves for microuidic chips based on thermoresponsive polymer gels. Electrophoresis 2003;24:3694702.
[141] Seger-Sauli U, Panayiotou M, Schnydrig S, Jordan M,
Renaud P. Temperature measurements in microuidic
systems: heat dissipation of negative dielectrophoresis
barriers. Electrophoresis 2005;26:223946.
[142] Buchholz BA, Doherty AS, Albarghouthi MN, Bogdan
FM, Zahn JM, Barron AE. Microchannel DNA sequencing matrices with a thermally controlled viscosity
switch. Anal Chem 2001;73:15764.
[143] Harmon ME, Tang M, Frank CW. A microuidic actuator
based on thermoresponsive hydrogel. Polymer 2003;44:
[144] Malmstadt N, Yager P, Hoffman AS, Stayton PS. A smart
microuidic afnity chromatography matrix composed of
poly(N-isopropylacrylamide)-coated beads. Anal Chem
[145] Huber DL, Manginell RP, Samara MA, Kim B, Bunker
BC. Programmed adsorption and release of proteins in a
microuidic device. Science 2003;301:3524.
[146] Johnson BD, Beebe DJ, Crone WC. Effects of swelling on
the mechanical properties of a pH-sensitive hydrogel for use
in microuidic devices. Mater Sci Eng C 2004;24:57581.
[147] Liu RH, Yu Q, Beebe DJ. Fabrication and characterization
of hydrogel-based microvalves. J Microelectromech Syst
[148] Yu Q, Bauer JM, Moore JS, Beebe DJ. Responsive
biomimetic hydrogel valve for microuidics. Appl Phys
Lett 2001;78:258991.
[149] Beebe DJ, Moore J, Bauer J, Yu Q, Liu RH, Devadoss C,
et al. Functional hydrogel structures for autonomous ow
control inside microuidic channels. Nature 2000;404:
[150] Cao X, Lai S, Lee LJ. Design of a self-regulated drug
delivery device. Biomed Microdev 2001;3:10918.
[151] Park JY, Oh HJ, Kim DJ, Baek JY, Lee SH. A polymeric
microuidic valve employing a pH-responsive hydrogel
microsphere as an actuating source. J Micromech Microeng
[152] Gu Y, Baldi A, Ziaie B, Siegel RA. Modulation of drug
delivery rate by hydrogel incorporating MEMS devices.
IEEEEMBS special topic conference on microtechnologies in medicine and biology, Madison, WI, 2002. p. 4069.
[153] Sebra RP, Kasko AM, Anseth KS, Bowman CN. Synthesis
and photografting of highly pH-responsive polymer chains.
Sens Actuat B 2006;119:12734.
[154] Kim SJ, Park SJ, Yoon SG, Kim IY, Kim SI. Characterization of smart hydrogels for biometric sensors and
actuators. In: Biomedical engineering 2003. IEEE EMBS
AsianPacic conference, 2003. p. 1145.
[155] Okuzaki H, Osada Y. Electro-driven polymer gels with
biomimetic motility. Polym Gels Networks 1994;2:26777.
[156] Yoshida R, Sakai T, Tambata O, Yamaguchi T. Design of
novel biomimetic polymer gels with self oscillating function. Sci Technol Adv Mater 2002;3:95102.
[157] Marra SP, Ramesh KT, Douglas AS. The actuation of a
biomimetic poly(vinyl alcohol)poly(acrylic acid) gel. Phil
Trans R Soc Lond A 2002;360:17598.
[158] Marra SP, Ramesh KT, Douglas AS. Mechanical characterization of active poly(vinyl alcohol)poly(acrylic acid)
gel. Mater Sci Eng C 2001;14:2534.

[159] Nath N, Chilkoti A. Creating smart surfaces using

stimuli responsive polymers. Adv Mater 2002;14:12437.
[160] Eddington DT, Beebe DJ. A valved responsive hydrogel
microdispensing device with integrated pressure source.
J Microelectromech S 2004;13:58693.
[161] Baldi A, Gu Y, Loftness PE, Siegel RA, Ziaie B. A
hydrogel-actuated environmentally sensitive microvalve for
active ow control. J Microelectromech S 2003;12:61321.
[162] Kim SJ, Park SJ, Kim SI. Properties of smart hydrogel
composed of poly(acrylic acid)/poly(vinyl sulfonic acid)
response to external stimuli. Smart Mater Struct 2004;13:
[163] Brahim S, Narinesingh D, Guiseppi-Elie A. Bio-smart
hydrogels: co-joined molecular recognition and signal
transduction in biosensor fabrication and drug delivery.
Biosens Bioelectron 2002;17:97381.
[164] Hoffman AS. Bioconjugation of intelligent polymers and
recognition proteins for use in diagnostics and afnity
separations. Clin Chem 2000;46:147886.
[165] Tang Z, Mori T, Takarada T, Maeda M. Single nucleotide
polymorphisms (SNPs) assay using reversible association
and dispersion of DNA-linked colloidal nanoparticles.
Nucl Acids Res Suppl 2001;1:1656.
[166] Liu HY, Zhu XX. Lower critical solution temperatures of
N-substituted acrylamide copolymers in aqueous solutions.
Polymer 1999;40:698590.
[167] Uchiyama S, Matsumura Y, de Silva AP, Iwai K.
Modulation of the sensitive temperature range of uorescent molecular thermometers based on thermoresponsive
polymers. Anal Chem 2004;76:17938.
[168] Richter A, Bundb A, Keller M, Arndt K. Characterization
of a microgravimetric sensor based on pH sensitive
hydrogels. Sens Actuat B 2004;99:57985.
[169] Bashira R, Hilt JZ, Elibol O, Gupta A, Peppas NA.
Micromechanical cantilever as an ultrasensitive pH microsensor. Appl Phys Lett 2002;81:30913.
[170] Sheppard NF, Lesho MJ, McNally P, Shaun Francomacaro A. Microfabricated conductimetric pH sensor. Sens
Actuat B 1995;28:95102.
[171] Ruan C, Zeng K, Grimes CA. A mass-sensitive pH sensor
based on a stimuli-responsive polymer. Anal Chim Acta
[172] Eremeev NL, Kukhtin AV, Belyaeva EA, Kazanskaya NF.
Effect of thermosensitive matrix-phase transition on
urease-catalyse urea hydrolysis. Appl Biochem Biotechnol
[173] Cai Q, Zeng K, Ruan C, Desai TA, Grimes CA. A wireless,
remote query glucose biosensor based on pH-sensitive
polymer. Anal Chem 2004;76:403843.
[174] Ivanov AE, Edink E, Kumar A, Galaev IYu, Arendsen AF,
Bruggink A, et al. Conjugation of penicillin acylase with
the reactive copolymer of N- isopropylacrylamide: a step
toward a thermosensitive industrial biocatalyst. Biotechnol
Prog 2003;19:116775.
[175] Kanazawa H, Matsushima Y. Temperature-responsive
chromatography. Trends Anal Chem 1998;17:43540.
[176] Ivanov AE, Ekeroth J, Nilsson L, Galaev IYu, Bergenstahl B,
Mattiasson B. Variations of wettability and protein adsorption on solid siliceous carriers grafted with poly(N-isopropylacrylamide). J Colloid Interface Sci 2006;296:53844.
[177] Balamurugan S, Mendez S, Balamurugan SS, OBrienII MJ,
Lopez GP. Thermal response of poly(N-isopropylacrylamide)

A. Kumar et al. / Prog. Polym. Sci. 32 (2007) 12051237















brushes probed by surface plasmon resonance. Langmuir

Mendez S, Curro JG, McCoy JD, Lopez GP. Computational modeling of the temperature-induced structural
changes of tethered poly(N-isopropylacrylamide) with
self-consistent eld theory. Macromolecules 2005;38:
Yim H, Kent MS, Mendez S, Lopez GP, Satija S, Seo Y.
Effects of grafting density and molecular weight on the
temperature-dependent conformational change of poly(Nisopropylacrylamide) grafted chains in water. Macromolecules 2006;39:34206.
Yuan W, Jiang G, Wang J, Wang G, Song Y, Jiang L.
Temperature/light dual-responsive surface with tunable
wettability created by modication with an azobenzenecontaining copolymer. Macromolecules 2006;39:13003.
Zhang G. Study on conformation change of thermally
sensitive linear grafted poly(N-isopropylacrylamide) chains
by quartz crystal microbalance. Macromolecules
Cunliffe D, Alarcon CH, Peters V, Smith JR, Alexander C.
Thermoresponsive surface-grafted poly(N-isopropylacrylamide) copolymers: effect of phase transitions on protein
and bacterial attachment. Langmuir 2003;19:288899.
Suzuki K, Yumura T, Mizuguchi M, Tanaka Y, Chen
C-W, Akashi M. Poly(N-isopropylacrylamide)-grafted silica as a support of platinum colloids: preparation method,
characterization, and catalytic properties in hydrogenation.
J Appl Polym Sci 2000;77:267884.
Ye M, Zhang D, Han L, Tejada J, Ortiz C. Synthesis,
preparation, and conformation of stimulus-responsive endgrafted poly(methacrylic acid-g-ethylene glycol) layers. Soft
Matter 2006;2:24356.
Pan YV, Wesley RA, Luginbuhl R, Denton DD, Ratner
BD. Plasma polymerized N-isopropylacrylamide: synthesis
and characterization of a smart thermally responsive
coating. Biomacromolecules 2001;2:326.
Xu FJ, Zhong SP, Yung LYL, Tong YW, Kang E-T, Neoh
KG. Thermoresponsive comb-shaped copolymer-Si(1 0 0)
hybrids for accelerated temperature-dependent cell detachment. Biomaterials 2006;27:123645.
Ahn SJ, Kaholek M, Lee W-K, LaMattina B, LaBean TH,
Zauscher S. Surface-initiated polymerization on nanopatterns fabricated by electron-beam lithography. Adv Mater
Kim DJ, Kang SM, Kong B, Kim W-J, Paik H, Choi H,
et al. Formation of thermoresponsive gold nanoparticle/
PNIPAAm hybrids by surface-initiated atom transfer
radical polymerization in aqueous media. Macromol Chem
Phys 2005;206:19416.
Seino M, Yokomachi K, Hayakawa T, Kikuchi R,
Kakimoto M, Horiuchi S. Preparation of poly(N-isopropylacrylamide) grafted silica bead using hyperbranched
polysiloxysilane as polymer brush and application to
temperature-responsive HPLC. Polymer 2006;47:194652.
Lee H-I, Pietrasik J, Matyjaszewski K. Phototunable
temperature-responsive molecular brushes prepared by
ATRP. Macromolecules 2006;39:391420.
Xia F, Feng L, Wang S, Sun T, Song W, Jiang W, et al.
Dual-responsive surfaces that switch between superhydrophilicity and superhydrophobicity. Adv Mater 2006;18:


[192] Tsuda Y, Kikuchi A, Yamato M, Nakao A, Sakurai Y,

Umezu M, et al. The use of patterned dual thermoresponsive surfaces for the collective recovery as co-cultured cell
sheets. Biomaterials 2004;26:188593.
[193] Liu Q, Zhang P, Lu M. Synthesis and swelling behavior of
comb-type grafted hydrogels by reversible additionfragmentation chain transfer polymerization. J Polym Sci Part
A: Polym Chem 2005;43:261524.
[194] Savina IN, Mattiasson B, Galaev IYu. Graft polymerization of vinyl monomers inside macroporous polyacrylamide gel, cryogel, in aqueous and aqueous-organic media
initiated by diperiodatocuprate(III) complexes. J Polym Sci
Part A: Polym Chem 2006;44:195263.
[195] Savina IN, Galaev IYu, Mattiasson B. Graft polymerization of acrylic acid onto macroporous polyacrylamide gel
(cryogel) initiated by potassium diperiodatocuprate. Polymer 2005;46:9596603.
[196] Bhattacharya A, Misra BN. Grafting: a versatile means to
modify polymers. Techniques, factors and applications.
Prog Polym Sci 2004;29:767814.
[197] Okano T, Yamada N, Sakai H, Sakurai Y. A novel
recovery system for cultured cells using plasma-treated
polystyrene dishes grafted with poly(N-isopropylacrylamide). J Biomed Mater Res 1993;27:124351.
[198] Yamada N, Okano T, Sakai H, Karikusa F, Sawasaki Y,
Sakurai Y. Thermo-responsive polymeric surfaces; control
of attachment and detachment of cultured cells. Makromol
Chem Rapid Commun 1990;11:5716.
[199] Takezawa T, Mori Y, Yoshizato K. Cell culture on a
thermoresponsive polymeric surface. Bio/Technology
[200] Hirose M, Kwon OH, Yamato M, Kikuchi A, Okano T.
Creation of designed shape cell sheets that are noninvasively harvested and moved onto another surface. Biomacromolecules 2000;1:37781.
[201] Kushida A, Yamato M, Konno C, Kikuchi A, Sakurai Y,
Okano T. Temperature-responsive culture dishes allow
nonenzymatic harvest of differentiated MadinDarby
canine kidney (MDCK) cell sheets. J Biomed Mater Res
[202] Kushida A, Yamato M, Kikuchi A, Okano T. Twodimensional manipulation of differentiated Madin-Darby
canine kidney (MDCK) cell sheets: the noninvasive harvest
from temperature-responsive culture dishes and transfer to
other surfaces. J Biomed Mater Res 2001;54:3746.
[203] Nandkumar MA, Yamato M, Kushida A, Konno C,
Hirose M, Kikuchi A, et al. Two-dimensional cell sheet
manipulation of heterotypically co-cultured lung cells
utilizing temperature-responsive culture dishes results in
long-term maintenance of differentiated epithelial cell
functions. Biomaterials 2002;23:112130.
[204] Kwon OH, Kikuchi A, Yamato M, Sakurai Y, Okano T.
Rapid cell sheet detachment from poly(N-isopropylacrylamide)-grafted porous cell culture membranes. J Biomed
Mater Res 2000;50:829.
[205] Nakajima K, Honda S, Nakamura Y, Lopez-Redondo F,
Kohsaka S, Yamato M, et al. Intact microglia are cultured
and non-invasively harvested without pathological activation using a novel cultured cell recovery method. Biomaterials 2001;22:121323.
[206] Collier TO, Anderson JM, Kikuchi A, Okano T. Adhesion
behavior of monocytes, macrophages, and foreign body

















A. Kumar et al. / Prog. Polym. Sci. 32 (2007) 12051237

giant cells on poly (N-isopropylacrylamide) temperatureresponsive surfaces. J Biomed Mater Res 2002;59:13643.
Kikuchi A, Okano T. Nanostructured designs of biomedical materials: applications of cell sheet engineering to
functional regenerative tissues and organs. J Control
Release 2005;101:6984.
Yamato M, Konno C, Utsumi M, Kikuchi A, Okano T.
Thermally responsive polymer-grafted surfaces facilitate
patterned cell seeding and co-culture. Biomaterials 2002;23:
Yang J, Yamato M, Kohno C, Nishimoto A, Sekine H,
Fukai F, et al. Cell sheet engineering: recreating tissues
without biodegradable scaffolds. Biomaterials 2005;26:
Harimoto M, Yamato M, Hirose M, Takahashi C, Isoi Y,
Kikuchi A, et al. Novel approach for achieving doublelayered cell sheets co-culture: overlaying endothelial cell sheets
onto monolayer hepatocytes utilizing temperature-responsive
culture dishes. J Biomed Mater Res 2002;62:46470.
Shimizu T, Yamato M, Akutsu T, Shibata T, Isoi Y,
Kikuchi A, et al. Electrically communicating three-dimensional cardiac tissue mimic fabricated by layered cultured
cardiomyocyte sheets. J Biomed Mater Res 2002;60:1107.
Shimizu T, Yamato M, Kikuchi A, Okano T. Cell sheet
engineering for myocardial tissue reconstruction. Biomaterials 2003;24:230916.
Ebara M, Yamato M, Aoyagi T, Kikuchi A, Sakai K,
Okano T. Temperature-responsive cell culture surfaces
enable on-off afnity control between cell integrins and
RGDS ligands. Biomacromolecules 2004;5:50510.
Hatakeyama H, Kikuchi A, Yamato M, Okano T. Biofunctionalized thermoresponsive interfaces facilitating cell
adhesion and proliferation. Biomaterials 2006;27:506978.
Ebara M, Yamato M, Hirose M, Aoyagi T, Kikuchi A,
Sakai K, et al. Copolymerization of 2-carboxyisopropylacrylamide with N-isopropylacrylamide accelerates cell
detachment from grafted surfaces by reducing temperature.
Biomacromolecules 2003;4:3449.
Kwon OH, Kikuchi A, Yamato M, Okano T. Accelerated
cell sheet recovery by co-grafting of PEG with PIPAAm
onto porous cell culture membranes. Biomaterials
Ista LK, Mendez S, Perez-Luna VH, Lopez GP. Synthesis
of poly(N-isopropylacrylamide) on initiator-modied selfassembled monolayers. Langmuir 2001;17:25525.
Alarcon CH, Farhan T, Osborne VL, Huck WTS,
Alexander C. Bioadhesion at micro-patterned stimuliresponsive polymer brushes. J Mater Chem 2005;15:
Yamato M, Konno C, Kushida A, Hirose M, Utsumi M,
Kikuchi A, et al. Release of adsorbed bronectin from
temperature-responsive culture surfaces requires cellular
activity. Biomaterials 2000;21:9816.
Canavan HE, Cheng X, Graham DJ, Ratner BD, Castner
DG. Cell sheet detachment affects the extracellular matrix:
a surface science study comparing thermal liftoff, enzymatic, and mechanical methods. J Biomed Mater Res Part
A 2005;75A:113.
Kanazawa H, Sunamoto T, Ayano E, Matsushima Y,
Kikuchi A, Okano T. Temperature-responsive chromatography using poly(N-isopropylacrylamide) hydrogel-modied silica. Anal Sci 2002;18:458.

[222] Kobayashi J, Kikuchi A, Sakai K, Okano T. Aqueous

chromatography utilizing pH-/temperature-responsive
polymer stationary phases to separate ionic bioactive
compounds. Anal Chem 2001;73:202733.
[223] Sakamoto C, Okada Y, Kanazawa H, Ayano E, Nishimura
T, Ando M, et al. Temperature- and pH-responsive
aminopropyl-silica ion-exchange columns grafted with
copolymers of N-isopropylacrylamide. J Chromatogr A
[224] Ayano E, Nambu K, Sakamoto C, Kanazawa H, Kikuchi
A, Okano T. Aqueous chromatography system using pHand temperature-responsive stationary phase with ionexchange groups. J Chromatogr A 2006;1119:5865.
[225] Ayano E, Okada Y, Sakamoto C, Kanazawa H, Kikuchi
A, Okano T. Study of temperature-responsibility on the
surfaces of a thermo-responsive polymer modied stationary phase. J Chromatogr A 2006;1119:517.
[226] Kanazawa H, Ayano E, Sakamoto C, Yoda R, Kikuchi A,
Okano T. Temperature-responsive stationary phase utilizing a polymer of proline derivative for hydrophobic
interaction chromatography using an aqueous mobile
phase. J Chromatogr A 2006;1106:1528.
[227] Kurata K, Shimoyama T, Dobashi A. Enantiomeric
separation using temperature-responsive chiral polymers
composed of L-valine diamide derivatives in aqueous liquid
chromatography. J Chromatogr A 2003;1012:4756.
[228] Galaev IYu, Warrol C, Mattiasson B. Temperature
induced elution of proteins from dye-afnity columns using
an immobilized polymeric displacer. J Chromatogr A
[229] Yoshizako K, Akiyama Y, Yamanaka H, Shinohara Y,
Hasegawa Y, Carredano E, et al. Regulation of protein
binding toward a ligand on chromatographic matrixes by
masking and forced-releasing effects using thermoresponsive polymer. Anal Chem 2002;74:41606.
[230] Yamanaka H, Yoshizako K, Akiyama Y, Sota H,
Hasegawa Y, Shinohara Y, et al. Afnity chromatography
with collapsibly tethered ligands. Anal Chem 2003;75:
[231] Okamura A, Itayagoshi M, Hagiwaraa T, Yamaguchia M,
Kanamori T, Shinbo T, et al. Poly(N-isopropylacrylamide)graft-polypropylene membranes containing adsorbed antibody for cell separation. Biomaterials 2005;26:128792.
[232] Dainiak MB, Kumar A, Galaev IYu, Mattiasson B.
Detachment of afnity-captured bioparticles by elastic
deformation of a macroporous hydrogel. Proc Nat Acad
Sci USA 2006;103:84954.
[233] Galaev IYu, Dainiak MB, Plieva FM, Mattiasson B. Effect
of matrix elasticity on afnity binding and release of
bioparticles. Elution of bound cells by temperature-induced
shrinkage of the smart macroporous hydrogel. Langmuir
[234] Fu Q, Rao GVR, Ista LK, Wu Y, Andrzejewski BP, Sklar
LA, et al. Control of molecular transport through stimuliresponsive ordered mesoporous materials. Adv Mater
[235] Liang L, Feng X, Martin PFC, Peurrung LM. Temperature-sensitive switch from composite poly(N-isopropylacrylamide) sponge gels. J Appl Polym Sci 2000;75:
[236] Liang L, Shi M, Viswanathan VV, Peurrung LM, Young JS.
Temperature-sensitive polypropylene membranes prepared

A. Kumar et al. / Prog. Polym. Sci. 32 (2007) 12051237













by plasma polymerization. J Membrane Sci 2000;177:

Kim SY, Kanamori T, Shinbo T. Preparation of thermalresponsive poly(propylene) membranes grafted with Nisopropylacrylamide by plasma-induced polymerization
and their water permeation. J Appl Polym Sci 2002;84:
Shtanko NI, Kabanov VY, Apela PY, Yoshida M,
Vilenskii AI. Preparation of permeability-controlled track
membranes on the basis of smart polymers. J Membrane
Sci 2000;179:15561.
Wang WY, Chen L, Yu X. Preparation of temperature
sensitive poly(vinylidene uoride) hollow ber membranes
grafted with N-isopropylacrylamide by a novel approach.
J Appl Polym Sci 2006;101:8337.
Lequieu W, Shtanko NI, Du Prez FE. Track etched
membranes with thermo-adjustable porosity and separation properties by surface immobilization of poly
(N-vinylcaprolactam). J Membrane Sci 2005;256:6471.
Ying L, Kang ET, Neoh KG. Synthesis and characterization of poly(N-isopropylacrylamide)-graft-poly(vinylidene
uoride) copolymers and temperature-sensitive membranes. Langmuir 2002;18:641623.
Rao GVR, Krug ME, Balamurugan S, Xu H, Xu Q, Lopez
GP. Synthesis and characterization of silica-poly(N-isopropylacrylamide) hybrid membranes: switchable molecular lters. Chem Mater 2002;14:507580.
Rao GVR, Lopez GP. Encapsulation of poly(N-isopropylacrylamide) in silica: a stimuli-responsive porous hybrid
material that incorporates molecular nano-valves. Adv
Mater 2000;12:16925.
Rao GVR, Balamurugan S, Meyer DE, Chilkoti A, Lopez
GP. Hybrid bioinorganic smart membranes that incorporate protein-based molecular switches. Langmuir 2002;18:
Peng T, Cheng Y-L. pH-responsive permeability of PEg-PMAA membranes. J Appl Polym Sci 2000;76:77886.
Wang Y, Liu Z, Han B, Dong Z, Wang J, Sun D, et al. pH
sensitive polypropylene porous membrane prepared by
grafting acrylic acid in supercritical carbon dioxide.
Polymer 2004;45:85560.
Smuleac V, Buttereld DA, Bhattacharyya D. Permeability
and separation characteristics of polypeptide-functionalized polycarbonate track-etched membranes. Chem Mater
Ying L, Wang P, Kang ET, Neoh KG. Synthesis and
characterization of poly(acrylic acid)-graft-poly(vinylidene
uoride) copolymers and pH-sensitive membranes. Macromolecules 2002;35:6739.
Park S-B, You J-O, Park H-Y, Haam S-J, Kim W-S. A
novel pH-sensitive membrane from chitosan-TEOS IPN;
preparation and its drug permeation characteristics. Biomaterials 2001;22:32330.


[250] Hester JF, Olugebefola SC, Mayes AM. Preparation of pHresponsive polymer membranes by self-organization.
J Membrane Sci 2002;208:37588.
[251] Zhang H, Ito Y. pH control of transport through a porous
membrane self-assembled with a poly(acrylic acid) loop
brush. Langmuir 2001;17:833640.
[252] Zhai L, Nolte AJ, Cohen RE, Rubner MF. pH-gated
porosity transitions of polyelectrolyte multilayers in conned geometries and their application as tunable Bragg
reectors. Macromolecules 2004;37:611323.
[253] Dejugnat C, Sukhorukov GB. pH-responsive properties of
hollow polyelectrolyte microcapsules templated on various
cores. Langmuir 2004;20:72659.
[254] Nakayama H, Kaetsu I, Uchida K, Sakata S, Tougou K,
Hara T, et al. Radiation curing of intelligent coating for
controlled release and permeation. Radiat Phys Chem
[255] Zhai G. pH- and temperature-sensitive microltration
membranes from blends of poly(vinylideneuoride)-graftpoly(4-vinylpyridine) and poly(N-isopropyl-acrylamide).
J Appl Polym Sci 2006;100:408997.
[256] Liu X, Neoh KG, Kang ET. Redox-sensitive microporous
membranes prepared from poly(vinylidene uoride) grafted
with viologen-containing polymer side chains. Macromolecules 2003;36:83617.
[257] Zhang K, Xiao XY, Wu Y. Temperature and pHresponsive polymeric composite membranes for controlled
delivery of proteins and peptides. Biomaterials 2004;25:
[258] Csetneki I, Filipcsei G, Zrinyi M. Smart nanocomposite
polymer membranes with on/off switching control. Macromolecules 2006;39:193942.
[259] Sumaru K, Ohi K, Takagi T, Kanamori T, Shinbo T.
Photoresponsive properties of poly(N-isopropylacrylamide)
hydrogel partly modied with spirobenzopyran. Langmuir
[260] Ito T, Hioki T, Yamaguchi T, Shinbo T, Nakao S, Kimura
S. Development of a molecular recognition ion gating
membrane and estimation of its pore size control. J Am
Chem Soc 2002;124:78406.
[261] Ito T, Yamaguchi T. Controlled release of model drugs
through a molecular recognition ion gating membrane in
response to a specic ion signal. Langmuir 2006;22:39459.
[262] Tang M, Zhang R, Bowyer A, Eisenthal R, Hubble J. A
reversible hydrogel membrane for controlling the delivery
of macromolecules. Biotechnol Bioeng 2003;82:4753.
[263] Chu L-Y, Liang Y-J, Chen W-M, Ju X-J, Wang H-D.
Preparation of glucose-sensitive microcapsules with a
porous membrane and functional gates. Colloids Surf B
[264] Misra GP, Siegel RA. New mode of drug delivery: long
term autonomous rhythmic hormone release across a
hydrogel membrane. J Control Release 2002;81:16.