Hydrogels in Sensing Applications

Progress in Polymer Science 37 (2012) 16781719
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Progress in Polymer Science
j our nal home page: www. el sevi er . com/ l ocat e/ ppol ysci
Hydrogels in sensing applications
Daniel Buenger
a,1
, Fuat Topuz
a,1
, Juergen Groll
b,
a
DWI e.V. and Institute of Technical and Macromolecular Chemistry, RWTH Aachen University, Forckenbeckstr. 50, 52074 Aachen, Germany
b
Department and Chair of Functional Materials in Medicine and Dentistry, University of Wrzburg, Pleicherwall 2, 97070 Wrzburg, Germany
a r t i c l e i n f o
Article history:
Received 10 January 2012
Received in revised form29 August 2012
Accepted 7 September 2012
Available online 14 September 2012
Keywords:
Hydrogels
Sensors
Biosensors
Stimuli responsive
Phase transitions
a b s t r a c t
Hydrogels are hydrophilic, highly water swellable polymer networks capable of convert-
ing chemical energy into mechanical energy and vice versa. They can be tailored regarding
their chemical nature and physical structure, sensitiveness to external stimuli and biocom-
patibility; they can be formed in various structures and integrated into (micro-)systems.
Accordingly, over the last decade, these materials have gained considerable recognition as
valuable tool for sensors and in diagnostics. This article reviews the use of hydrogels in
sensor development with focus on recent efforts in the application of stimuli responsive
hydrogels as sensors, hydrogels as suitable matrices in which the sensing (bio-)molecules
are embedded and hydrogels for modication and protection of sensor surfaces. In the rst
part of the review, both sensors and hydrogels are dened and a basic theoretical overview
of hydrogels and their behavior is given. Subsequent chapters focus on hydrogels in physico-
chemical and biochemical sensing mechanisms with a primary emphasis on the hydrogels
as such and the applied sensing mechanism. Finally, the review is concluded by a sum-
mary and discussion including an outlook on future perspectives for hydrogels in sensing
applications.
2012 Elsevier Ltd. All rights reserved.
Contents
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1679
1.1. Sensors: denition and classication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1679
1.2. Biosensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1680
1.3. Hydrogels: general overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1681
1.3.1. Theory of swelling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1682
1.3.2. Mechanisms of response for stimuli sensitive hydrogels. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1683
1.4. Hydrogels for sensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1684
1.4.1. Hydrogels as sensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1684
1.4.2. Hydrogels for biosensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1685
1.5. Scope and structure of the review . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1685
2. Physicochemical sensing mechanisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1686
2.1. Temperature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1686
2.2. Ions, ionic strength and pH . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1688
Corresponding author. Tel.: +49 (0) 931 201 73510;

fax: +49 (0) 931 201 73500.
E-mail address: juergen.groll@fmz.uni-wuerzburg.de (J. Groll).
1
These authors contributed equally.
0079-6700/$ see front matter 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.progpolymsci.2012.09.001
D. Buenger et al. / Progress in Polymer Science 37 (2012) 16781719 1679
2.3. Gas and humidity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1691
2.3.1. Gas . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1691
2.3.2. Humidity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1692
3. Biochemical sensing mechanisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1694
3.1. Molecular interactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1694
3.1.1. Enzymesubstrate interaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1694
3.1.2. Antibodyantigen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1698
3.1.3. Nucleotide, oligonucleotide and DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1701
3.1.4. Other binding mechanisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1703
3.2. Living sensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1707
3.2.1. Bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1708
3.2.2. Cells. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1708
4. Summary and perspective . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1712
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1712
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1712
1. Introduction
1.1. Sensors: denition and classication
In general, the term sensor is derived from the Latin
word sensus, which directly translated means sense, or
to sense something. If one thinks about a sense the rst
idea that comes up are the traditional ve human senses:
sight, hearing, taste, smell and touch. The senses give the
human body the ability to receive signals or stimuli from
the environment and react or respond to them. If a sense
gives the ability to receive and respond to a signal, we can
transfer this to the technical level and dene a sensor as
followed:
A sensor is a device that receives and responds to sig-
nals and stimuli fromthe environment.
Furthermore, the human senses give a rst hint how
sensors can be classied. Sight, hearing and touch have in
common that they sense physical stimuli, namely light, in
form of electro-magnetically waves, acoustic waves and
pressure. Smell and taste respond to chemical stimuli,
like odor and taste molecules. Accordingly, sensors can
be divided into two big classes: physical and chemical
sensors. The IUPAC dened in 1991 the terms physi-
cal and chemical sensors with the following denitions
[1]:
A physical sensor is a device that provides informa-
tion about a physical property of the system and A
chemical sensor is a device that transforms chemical
information, ranging from the concentration of a spe-
cic sample component to total composition analysis,
into an analytically useful signal.
Now that the term sensor is dened and a rst classi-
cation, corresponding to the sensed stimulus, is made a
closer look at the general structure of sensors can be taken.
Again the human senses lead the way to this structure:
whenever receptor cells in the human body receive certain
stimuli, e.g. an electro-magnetically wave hits a photore-
ceptor cell in the retina, a chemical reaction is triggered,
and then transduced, in formof an electrical signal, to the
brain. Deductive sensors consist of two basic parts, namely
a receptor and a transducer part. Technically spoken the
receptor-part of a sensor senses a chemical or physical
stimulus and transforms it into a formof energy. The trans-
ducer part is than capable of transducing this energy into
a useful analytical signal which can be processed and dis-
played. Fig. 1 illustrates the general sensor structure.
On the technical level there are many ways to clas-
sify sensors e.g. according to the measuring effect, given
analyte, receptor principle, eld of application, mode of
application and so on. The most established way is the one
following the principles of signal transduction, committed
by the IUPAC 1991 for chemical sensors [1].
1. Optical sensors, based on absorbance, reectance, lumi-
nescence, uorescence, index of refraction, optothermal
effect, and light scattering effects.
2. Electrochemical sensors, based on voltammetric, incl.
amperometric, and potentiometric effects, chemically
sensitized eld effect transistors (CHEMFET) and poten-
tiometric solid electrolyte gas sensors.
3. Electrical sensors, based on metal oxide semiconductors
and organic semiconductors.
4. Mass-sensitive sensors, based on piezoelectric and sur-
face acoustic wave effects.
5. Magnetic sensors, basedonparamagnetic gas properties.
6. Thermometric sensors, based on heat effects of specic
chemical reaction or adsorption.
7. Radiation sensitive sensors, based on absorbance, or
emission of X-, -, or I-radiation that is used for the
determination of a chemical composition.
As the classication shows the IUPAC draws no clear
line between chemical and physical sensors and counts
physical sensors that measure e.g. a chemical state, reac-
tion, or composition as chemical sensors. This shows that
a general approach to classify is difcult as chemical and
physical sensing almost always go hand in hand. Hence,
more generally, sensors can be dened as devices that
determine selectively and reversibly the concentration
of analyte molecules by amplifying the signal resulting
from interaction between analyte and receptor without
need of any other instrument and additional reagents
[2].
1680 D. Buenger et al. / Progress in Polymer Science 37 (2012) 16781719
Fig. 1. General assembly of a sensor comprising a receptor that interacts with the analyte and a transducer. Beyond that core sensing unit, data processing
and display are important parts of complete sensor systems.
1.2. Biosensors
If a physical sensor is a device that provides informa-
tion about a physical property of a system and a chemical
sensor is a device that transforms chemical information
into an analytically useful signal, a biosensor consequently
must work in a similar way. Actually the class of biosensors
can be counted as a cross of both, physical and chemi-
cal sensors. They can be devices that provide information
about a bio-physical property of a systemand/or transform
bio-chemical information into an analytically useful signal.
What is more important is the fact that biosensors have a
biological recognition element that enables the analysis of
relevant biological information. This concept corresponds
with the denition for biosensors given by the IUPAC in
1999 [3]:
A biosensor is an integrated receptor-transducer
device, which is capable of providing selective quanti-
tative or semi-quantitative analytical information using
a biological recognition element.
A classication of biosensors can be done for example
after one of the following principles:
1. Following the principle of signal transduction, e.g. elec-
trochemical, optical, mass sensitive and so on.
2. Depending on the biological recognition element, e.g.
biological ionophores, enzymes, antigens/antibodies,
whole cells, membrane receptors, plant and animal
tissue, protein receptors and channels, oligonucleotides,
specic ligands etc.
3. Classied after the sensed analyte, e.g. glucose, DNA,
enzymes, toxins, certain drugs and so on.
From the rst biosensor invention led by Leland C.
Clark in 1962 that concerned a potentiometric enzyme
electrode developed to measure the concentration of
glucose [4], numerous biosensors with different specici-
ties have been developed. During the last decade, much
work was devoted to downsize the laboratory workspace
towards small and versatile biosensor devices. However,
indispensable requirements including fast responsibility,
reproducibility, reliability and stability remained the same.
Crucial parameters in designing biosensors with high per-
formance are (i) immobilization of biomolecules in their
native conformation, (ii) accessibility of receptors to the
targets, and (iii) specic adsorption to the support [57].
Especial regarding these demands, hydrogels offer specic
advantages for biosensors.
Biosensors are becoming increasingly important and
practical tools to suit a vast pool of application areas
including point-of-care testing, home diagnostics, and
environmental monitoring (Fig. 2) [8]. As already men-
tioned, a biosensor consists of a bioelement for recognizing
a specic analyte, and a sensor element that transduces the
specic recognition into an electrical signal.
Enzymes, antibodies, living cells, or tissues may
serve as bioelements and ideally should recognize one
Fig. 2. Types and components of biosensors as well as their elds of application. Figure is partly redrawn from[8].
Fig. 3. Overviewon the four types of bioelementsensor coupling methods. Figure is partly redrawn from[8].
analyte specically while being insensitive to other
potentially interfering molecules. These bioelements can
be coupled with sensing elements through membrane
entrapment, physical adsorption, matrix entrapment, or
covalent incorporation(Fig. 3). Inmembrane entrapment, a
semipermeable membrane is applied between the analyte
and the bioelement, with the sensor attached to the bioele-
ment [8]. For physical adsorption van der Waals forces,
hydrophobic forces, hydrogen bonds, and/or ionic forces
may be used to attach the bioelement to the surface of
the sensor. In case of covalent binding, the bioelement is
directly linked to the sensor surface by a covalent bond
through a chemical reaction. Finally, porous entrapment is
based on forming a porous encapsulation matrix around
the bioelement that ensures its presence close to the sen-
sor.
Biosensors can produce signals tuned by the concen-
tration of analytes of interest either directly or indirectly.
In a direct case, a signal that comes fromthe production of
heat, light or chemical products is sensedwithsignal inten-
sity proportional to the analyte concentration. For indirect
measurements, the analyte inhibits the productionof a sec-
ondary chemical product (e.g. by blocking active enzyme
sites) which concentration is sensed. For both ways, sen-
sitivity of the sensor can be given by the change in its
response as a function of the corresponding change in
quantity being monitored. Biosensors become convenient
to use if they exhibit a linear relationship between the vari-
ation in the amplitude of the output, ^a, and the input,
^m:
zu = s zm (1)
where s corresponds to the sensitivity of the biosensor and
determines the suitability of the sensor for use in a particu-
lar application. It is possible that mis not an actual quantity
to be measured, but a function of that quantity. This is the
case for potentiometric biosensors in which the amplitude
of the signal is proportional to the logarithmof the analyte
concentration, c, according to Nernst Law:
zu = s z(logc) (2)
When a variation in the bioelement recognition stops
to yield an appreciable signal variation, then the detection
limit has been reached. The detection limit usually corre-
sponds to the limit of linear dependence range between
bioelement recognition and signal intensity at low con-
centrations. The linear region of a biosensor is derived
from a calibration curve of its response to different ana-
lyte concentrations. The curve is only meaningful if the
calibration is made for increasing and decreasing series
of analyte concentration, and it is important that the
biosensor shows no memory effect and hysteresis. A good
calibration curve is required to indicate the stability of
the response of the biosensor, which should neither drift
nor oscillate with time. In some cases, corrective devices
are available that can go beyond the linear zone and
make it possible to exploit the non-linear responses at
low concentrations, thus lowering the detection limits of
biosensors.
An important parameter for biosensors especially
regarding their practicability and applicability is the
response time of the setup. This is the time the system
needs to reach a steady state fromthe instant of the varia-
tion in analyte concentration. Theoretically, the response
time is innitely long; however, a reasonable value can
be found by xing an interval between the instantaneous
value of the response and the theoretical nal value, as a
function of the required precision. A steady state is then
considered as obtained when the predetermined inter-
val has been covered. The response time of the biosensor
indicates howquickly it responds to a variation in concen-
tration and it also denes a minimum delay, after which
data can be collected with a given precision. It is generally
quicker to obtain the same information fromthe derivative
of the response curve.
1.3. Hydrogels: general overview
Hydrogels are three-dimensionally cross-linked
hydrophilic polymer networks. They swell without disso-
lution up to 99% (w/w) water of their dry weights [911].
Depending on the type of cross-linking, hydrogels can
be divided into two classes: (i) chemically cross-linked
Fig. 4. A cross-linked hydrogel structure with the mesh size and the average molecular weight between the cross-linking points M
C
, respectively.
hydrogels, and (ii) physically cross-linked hydrogels.
Chemical hydrogels are formed by covalent networks and
do not dissolve in water without breakage of covalent
bonds [1215]. Physical hydrogels are, however, formed
by dynamic cross-linking of synthetic or natural build-
ing blocks based on non-covalent interactions such as
hydrophobic and electrostatic interaction or hydrogen-
bridges [1618]. The hydrophilic and mostly inert nature
of hydrogels often leads to minimized non-specic inter-
action with proteins and cells which makes hydrogels ideal
candidates for numerous bio-related applications [19,20].
If the polymer network of a hydrogel is endowed with
functional groups, hydrogels become responsive to some
physical, chemical or biochemical stimuli [2131]. Expo-
sure to the respective stimuli causes reversible changes in
swelling of the network. This response is determined by
the hydrogel composition, cross-linking type and degree
of cross-linking which is the density of junctions joining
the chains into a permanent form. All these properties can
be tailored to create hydrogels with adjusted properties
[32]. For example, a higher cross-linking degree leads
to increase of the mechanical strength and decrease of
the diffusion rate, thereby reducing the mesh size of the
network [33]. Mesh size () is used to dene the diffusional
space available for the transfer of molecules or particles
through the matrix of a hydrogel (Fig. 4) [34].
When hydrogels are used in sensing, it is usually one of
the three following main characteristics that is exploited:
(i) their semiwet and inert structure that predetermines
hydrogels as host-network, (ii) the amplication effect
of their sensitiveness on a molecular level that is trans-
lated into macroscopic effects such as a change in swelling
degree, and (iii) the ability to control the diffusion behav-
ior of molecules through the polymer matrix [3537]. In
the following paragraph, the theory of hydrogel swelling
and the mechanism of response will be outlined in more
detail as basis for the sensing applications described in the
following chapters.
1.3.1. Theory of swelling
Hydrophilicity of the polymers that constitute the net-
work creates an osmotic pressure within hydrogels that
leads to swelling of the matrix upon exposure to water
[38]. The swelling process occurs in three steps: (i) diffu-
sion of water molecules through the matrix, (ii) relaxation
of polymer chains via hydration, and(iii) expansionof poly-
mer network upon relaxation [39,40]. Hydrogels absorb
water to a maximumdegree possible, the so-calledequilib-
rium water content. This degree is dened as the balance
between osmotic pressure and the elastic retractive forces
of the polymer chains in the three-dimensional network.
The stretching of polymer chains increases elastic retrac-
tive forces as a counteraction for the network expansion.
When these forces are balanced, the network expansion
stops and comes to equilibrium. When either the osmotic
pressure changes, e.g. due to deprotonation of carboxylic
acid groups in the network due to a shift in pH, or the
cross-linking density changes, e.g. due to degradation of
the network and thus decreasing number of cross-linking
points, this balance is brokenanda result of that, the hydro-
gel exhibits a change in the degree of swelling.
The most widely used theory to explain the swelling
in neutral hydrogels is the equilibrium swelling theory of
Flory and Rehner [41,42]. This assumes a Gaussian distri-
bution of polymer chains and the average cross-links as
tetrafunctional branching units. Swelling of the polymer
networkis a functionof elastic forces of the polymer chains,
and the thermodynamical compatibility between polymer
and water molecules. Accordingly, the free energy of a neu-
tral hydrogel (^G) can be expressed as:
zC = zC
el
+zC
mix
(3)
where^G
el
represents thecontributionof elastic refractive
forces, and ^G
mix
is the thermodynamic compatibility of
polymer and solvent.
This equation can be rewritten in terms of chemical
potentials. At theequilibriumconditions, thetotal chemical
potential () has to be equal to zero:
=
el
+
mix
= 0 (4)
When the total chemical potential is equal to zero, the
change in chemical potential due to mixing is balanced by
elastic retractive forces. The change in chemical potential
due to elastic forces can be expressed by using the theory
of rubber elasticity [43], while the contribution of mixing
to chemical potential change is determined using the heat
and the entropy of mixing [11]. With equaling these two
contributions, the molecular weight between two cross-
links (M
C
) in the absence of a solvent can be expressed as:
1
M
C
=
2
M
N
( v]v
1
)[ln(1 v
2,s
) +v
2,s
+y
1
v
2
2,s
]
v
1]3
2,s

v
2,s
]2
(5)
where M
N
is the average molecular weight of the polymer
chains preparedintheabsenceof across-linker, v andV
1
are
the specic and the molar volume of water, respectively.
v
2,s
is the polymer volume fraction in the fully swollen
state, andy
1
is the polymersolvent interactionparameter.
The presence of water changes the chemical potential,
so that a new term is required for the volume fraction
density of the polymer chains. Thus, the above original
FloryRehner model canbe rewrittenby incorporating v
2,r
,
a termdescribing the polymer fraction in the relaxed state
according to the theory of Peppas and Merrill [44].
1
M
C
=
2
M
N
( v]v
1
)[ln(1 v
2,s
) +v
2,s
+y
1
v
2
2,s
]
v
2,r
[(v
2,s
]v
2,r
)
1]3
(v
2,s
]2v
2,r
)]
(6)
When ionic groups are present in the network, the
swelling equilibriumof the matrix becomes more compli-
cated. New terms concerning the ionic strength, I and the
dissociation constants, K
a
, and K
b
have to be added to Eq.
(6). Equivalent expressions for anionic and cationic gels are
formulated as shown in Eqs. (7) and (8), respectively.
v
1
4l
v
2
2,s
v
K
u
10
pH
K
u
2
= [ln(1 v
2,s
) +v
2,s
+y
1
v
2
2,s
]
+
v
1
vM
c
1
2M
c
M
N
v
2,r
v
2,s
v
2,r
1]3
v
2,s
2v
2,r
(7)
v
1
4l
v
2
2,s
v
K
b
10
pH14
K
u
2
= [ln(1 v
2,s
) +v
2,s
+y
1
v
2
2,s
]
+
v
1
vM
c
1
2M
c
M
N
v
2,r
v
2,s
v
2,r
1]3
v
2,s
2v
2,r
(8)
Hydrogel mesh size (i.e. correlation length), based on
values for the cross-linking density or molecular weight
between cross-links can be described by the equation:
= D
1]3
2,s
( r
2
c
)
1]2
= ( r
2
c
)
1]2
(9)
where is the elongation of the polymer chains in any
direction, and( r
2
c
)
1]2
, theunperturbedend-to-enddistance
of the polymer chains between the cross-linking points.
Assuming an isotropic swelling, the end-to-end distance
and the pore size of a swollen polymeric network can be
calculated using the equation:
= D
1]3
2,s
2C
N
M
C
M
r
1]2
l (10)
where l is the length of the bond along the backbone chain,
and C
N
, M
r
are the Flory characteristic ratio and the molec-
ular weight of the repeating units, respectively.
Swelling is affected by numerous physicochemical con-
ditions or structural factors [4549]. The nature of the
polymer and the cross-linking degree are crucial ones that
determine the swelling ratio. For instance, highly cross-
linked hydrogels have a smaller mesh size and swell less
in comparison to loosely cross-linked ones. The swelling
kinetics depends on the mechanism of solvent penetra-
tionintothe matrix, andis either diffusion-controlled(Case
I) or relaxation-controlled (Case II) [40,50,51]. In Case I,
also known as Fickian type behavior, diffusion of water
molecules through hydrogel matrix occurs much faster
than the relaxation of the polymer chains, and the swelling
is controlled by a concentration gradient. However, in
Case II (also called relaxation-controlled), the swelling is
controlled by the rate of polymer relaxation. A detailed
mathematical treatment of kinetics is beyond the scope
of this article and can be found in a paper by Peppas and
Colombo [52].
It becomes clear that analyte-induced changes in the
characteristics of the network such as the cross-linking
density, content of ionic species or chemical nature of the
polymer backbone will result in macroscopic effects such
as altered swelling behavior. Such changes can result from
physicochemical stimuli such as changes in temperature,
pHor ion strength, or they may originate frombiochemical
recognition events as described below.
1.3.2. Mechanisms of response for stimuli sensitive
hydrogels
Stimuli responsive hydrogels undergo a volume-phase
transitions upon exposure to the respective stimulus due
to molecular interactions resulting in abrupt changes in
the network such as swelling, collapse or solution-to-gel
transitions. Thus, the stimulus as such plays key role in
the mechanism of hydrogel response. In this section, we
describe hydrogel systems coupled withdifferent response
mechanisms that dene their behavior and application.
Hydrogels that are responsive to changes in pH are the
most studied stimuli responsive hydrogels. Either acidic or
basic pendant groups on the polymer network lead to pH-
induced volume-phase transitions. In the case of anionic
networks, when the environmental pH is above the acid
groups characteristic pK
a
, ionization occurs, leading to
increases of (i) thenumber of xedcharges, (ii) hydrophilic-
ity of the network, and (iii) electrostatic repulsion between
the chains [53]. Hence, the mesh size becomes sensi-
tive to pH variations. However, the protonation of the
ionized acidic groups upon lowering pH of an aqueous
solutioncauses adecreaseinthecontent of mobilecounter-
ions inside the matrix and in the strength of electrostatic
repulsions of the chain segments. So, the network gains
hydrophobic character, and the swollen form shrinks into
a compact state. Cationic networks (e.g. having amino
pendant groups) exhibit a similar behavior but with oppo-
site trend. When pK
b
of the cationic pendant groups is
lower thantheenvironmental pH, hydrophilicityof thenet-
work increases, and the hydrogel starts to swell [54,55].
So, the hydrogels display pH-dependent swelling behavior
with opposite swelling/deswelling response to pHchanges
as stated for anionic networks.
Some hydrogels are responsive to temperature vari-
ations and display reversible temperature-dependent
swelling behavior. These hydrogels are subdivided into
three main groups: (i) positive, (ii) negative, and (iii)
thermally reversible gels [56,57]. Positive thermosensi-
tive hydrogels (such as poly(acrylic acid), polyacrylamide,
and poly(acrylamide-co-butyl methacrylate)) have a char-
acteristic upper critical solution temperature (UCST).
These gels shrink if the temperature sinks below the
UCST [58]. Negative temperature-sensitive hydrogels
(like N-methylacrylamide, N,N-dimethylacrylamide or N-
isopropylacrylamide) are characterized by a lower critical
solution temperature (LCST). These gels are highly swollen
below the LCST while the network collapses when the
temperature increases above the LCST [59]. Several the-
ories have been proposed to explain this LCST behavior
in temperature-sensitive polymers [6064]. According to
these theories, hydrogen bonding and hydrophobic inter-
actions between non-polar components in the polymer
network are crucial factors. Belowthe LCST, the hydropho-
bic parts are surrounded by water molecules that form
a cage around the group with only weak interaction
with the non-polar center. With increasing temperature,
molecules become more mobile, and this cage of immo-
bile water molecules is partially lost and the protection
of the hydrophobic groups gets weaker. At the LCST,
the entropy loss of water molecules that are completely
released from the network is small enough to be counter
balanced by the energy win of the hydrophobic inter-
action between clustering non-polar components of the
network, so that the gel releases water and the network
collapses.
Certain hydrogels can undergo volume-phase transi-
tions in the presence of biomolecules. These so-called
biomolecule-sensitive gels have attracted considerable
attention as intelligent materials, since they can sense
biochemical changes through structural changes [6568].
Glucose- and protein-sensitive hydrogels are most promi-
nent examples in this family. For instance, glucose
responsivity can be achieved by incorporating boronic
based-ligands as recognition agents in the network. Com-
plexation of glucose with these ligands induces a release of
protons that can be sensed by conductance measurements
andusedfor thedeterminationof glucosecontent [69]. Also
boronic acid functional thermo-responsive hydrogels are
also used to sense glucose. When glucose binds to these
networks, the hydrophilic/hydrophobic balance is changed
so that the LCST of these networks shifts as a function
of glucose content what can be exploited for sensing the
concentration of glucose [70]. Alternatively, biomolecule
responsive hydrogels can be prepared by immobilization
of specic enzymes in the hydrogel matrix. For instance,
glucose oxidase (GOX) functional hydrogels are used to
sense glucose through the glucose oxidase mediated enzy-
matic oxidation fromglucose to gluconic acid [71]. Antigen
responsive hydrogels are prepared either by chemically
grafting antibodies to polymeric chains or by afnity bind-
ing of antibodies to polymer-modied with corresponding
antigen [72]. In the presence of free antigens, competi-
tive binding occurs betweenfree andimmobilizedantigens
and results in volume-phase transitions. Following the
same principle, ion-responsive gels are built by endow-
ing the network with appropriate ion-sensing molecules
[73]. For example, calciumions can be detected by hydro-
gels endowed with calmodulin (CaM), a calcium binding
protein capable of switching its swelling degree as func-
tion of Ca
2+
ion content [74]. Alternatively, a network can
be built by imprinting the sensing molecules within the
matrix [75]. This approach however requires a high cross-
linking density to x the structure molecular recognition
sites (molecular cavities) in the network that are intended
for subsequent rebinding of the target molecules with high
specicity.
These examples for different mechanisms of response
in stimuli sensitive hydrogels underline the versatility of
hydrogels that are ina permanent dialogue withtheir envi-
ronments and thus ideal tools for (bio-)chemical sensors.
High water content and minimal unspecic interaction
with biological molecules predetermine hydrogels espe-
cially for sensing based on biochemical recognition. Hence,
thefollowingparagraphis dedicatedtotheuseof hydrogels
in biosensing.
1.4. Hydrogels for sensors
Hydrogels may be prepared in aqueous solutions by UV
[76] or thermo-initiated radical polymerization [77], addi-
tion reaction [78], self-assembly of recognition motifs such
as coiled-coils [79,80], peptides [8183], hydrogen-bridges
[84] or DNA [12,29,85,86]. These methods can be applied
in a number of more specialized techniques for the pro-
duction of peculiar hydrogel structures that are especially
useful for sensing. One example is the vapor-phase induced
generationof well-orderedarrays of hydrogel microworms
withcylindrical shape andhighsurface tovolume ratiothat
may be used for different sensing applications, e.g. as u-
orescence sodium sensors [8789]. Hydrogels can also be
formed by in situ gelation fromlow-viscosity solutions and
thus be injected into small voids or complex uidic and
microuidic devices. This predisposition to miniaturiza-
tion and integration into microsystemin desired geometry
broadens their analytical applications. Hydrogels may thus
beformedbyinsitupatterningondesireddevices for possi-
ble sensing applications [90]. All these production-related
advantages support the exploitation of hydrogels for ana-
lytical purposes.
1.4.1. Hydrogels as sensors
Stimulus-sensitive hydrogels may act as active sensing
material according to the mechanisms introduced in Sec-
tion 1.3.2. Such gels are sensitive to small changes in the
environment and give the response to physical stimuli
(temperature [91], light, pressure [92], electric eld [93],
ionic strength [94], and magnetic eld [95]), chemical
stimuli (pH [96], ions [97]) or biological stimuli (glucose
[98], enzyme [99] and antigen [100]) through volume
changes. The response rate is dependent on the hydrogel
composition, shape and size and can be increased by sev-
eral proposed techniques such as reducing the size [101],
decreasing the cross-linking density [102], and increasing
boththe content of ionic groups [103], andpore size [15]. In
the presence of these stimuli, the hydrogels undergo phase
transition by the sensing molecules and simultaneously
translate this sense into a macroscopic event. In this way,
the conversion of hydrogel swelling to an electrical output
is possible with various techniques like light transmission
measurement [104], conductometry [105] and pressure
generated by the gel swelling [106].
1.4.2. Hydrogels for biosensors
The high water content and hydrophilic nature of
hydrogel is similar to the void-lling component of the
extracellular matrix, the natural environment of mam-
malian cells, and renders themintrinsically biocompatible.
Hydrogels are thus used for a wide range of biomed-
ical applications such as soft contact lenses [107] and
controlled drug delivery systems [108]. Hence, a straight-
forward application of hydrogels in biosensors is a
protection and coating function of sensor parts to prevent
undesired interaction with biological molecules or cells.
Their open porous structure and hydrophilic environment
allows diffusion of analytes through the hydrogel matrix.
However, diffusion of larger molecules such as proteins
may be limited and even prevented by an increased cross-
linking density, and cells are usually not able to penetrate
unless the gel structure is biodegradable.
Beyond simple protection, hydrogels can be used as
immobilization matrix for the biosensing elements. Essen-
tial criteria in designing biosensors with high selectivity
and sensitivity are receptors that have to be immobilized
in their native conformation to be able to specically inter-
act with the analyte, while support material should be
resistant to unspecic adsorption. Furthermore, the qual-
ity of sensing and the sensitivity level mainly depend
on accessibility and activity of the immobilized sensing
molecules. Hydrogels provide excellent environments for
enzymes and other biomolecules to preserve their active
and functional structure [109]. Also cells are commonly
immobilized in hydrogel matrices for various purposes
[110]. As hydrogels may easily be tailored in their prop-
erties, they form an ideal platform for this purpose,
and extensive work has been carried out with a vari-
ety of analytes such as proteins, ions, carbohydrates, and
nucleic acids [111]. Recognition in hydrogel-based sen-
sors is provided by interaction between an analyte and
sensing element by volume changes in response to tar-
get molecules. This recognition induced volumetric change
creates a newkind of sensing systemas alternative to clas-
sical biosensors based on electrochemical sensing.
1.5. Scope and structure of the review
Hydrogels are a broad and extensive eld of
research, especially for applications in cell culture,
tissue engineering, drug delivery and also in microtech-
nology. Accordingly, a number of comprehensive review
articles have been published over the last 5 years. In 2006,
Peppas et al. gave an excellent overviewabout hydrogels in
biology and medicine [112]. Beside a detailed description
of synthetic, biological and biohybrid hydrogel systems,
the review intensively describes the use of hydrogels in
therapeutics and also covers aspects of diagnostic devices.
In 2007 Ulijn et al. published a review concerning biore-
sponsive hydrogel systems that change their properties
in response to selective biological recognition events
[36]. They highlight the utilization of such hydrogels for
drug delivery, sensing and tissue engineering. In 2010,
Deligkaris et al. published a comprehensive review about
hydrogel-based devices for biomedical applications [113].
They survey cross-linking methods, operation principles
and transduction mechanisms. Further applications of
hydrogel devices in specic elds of interest such as
uid control, drug delivery, nerve regeneration and other
biomedical applications constitute the main focus. Also
in 2010, Wandera et al. published a review about stimuli
responsive membranes [114]. Their article gives some
interesting examples of hydrogel-based membrane sys-
tems that react to changes in e.g. pH, temperature, ionic
strength and chemical cues. All these excellent review
articles are either focused on a special class of hydrogels
or a specic area of application. So far, no comprehensive
review concerns the general use of hydrogels in sensing
applications disregarding the physicochemical nature
of the gel, the structure and composition of the device,
the mechanism for sensing and detection and nally the
analyte.
In the last decade, remarkable efforts have been
achieved to manifold sensitivity and selectivity of
hydrogel-based sensors and biosensors. By using different
gel combinations, the systems with a variety of specici-
ties andimprovedsensor performance have beenprepared.
We have recently summarized the use of hydrogels for
biosensing [115]. This review gives an overview on the
use of hydrogels in sensing applications, not only actively
acting as sensing material themselves but also used for
immobilization of molecules that cause the sensing stim-
ulus upon presence of the analyte. We have structured
the following detailed discussion strictly according to the
sensing mechanism. Chapter 2 comprises examples for
physicochemical sensing where the signal is generated
as response to changes in parameters such as temper-
ature, pH, ion strength, gas and humidity. Importantly,
no biochemical recognition process is involved in these
sensing mechanisms. Studies where the sensing mecha-
nismis based on such processes, either through molecular
interaction or by reaction of bacteria or cells towards
the analyte, are presented and discussed in Chapter 3.
Molecular interactions may be based on enzymesubstrate
interaction, where a chemical reaction is catalyzed and
causes the effect that may be sensed, or on bind-
ing/debinding events caused by presence of the analyte
that cause changes in the hydrogel network. Anti-
body/analyte interaction, peptidepeptide bonding or
streptavidin/biotin are examples for such selective binding
systems with high analyte afnity. Also chemical moieties
Fig. 5. Structure and optical reectance of 3Dhydrogel PCs formed through holographic lithography. (a) Simulated 3Dholographic interferential structure.
The front side of the image is the (111) plane. (b) SEM image of the (111) plane of FCC lattices and (c) SEM image of the 408 tilted cross-section of a
fractured sample. The inset image shows windows between lattices on the (110) plane. Scale bar: 500nm. (d) Reectance spectrumof a nitrogen-dried
hydrogel structure measured by Fourier transformIR spectrometry at roomtemperature. Figure partly redrawn from[123].
that mimick biological ligand binding, such as the binding
of glucose to phenylboronic acids, are included in Chapter
3. The article is concluded with a short summary and brief
discussion on trends and future perspectives for hydrogels
in sensing applications.
2. Physicochemical sensing mechanisms
An excellent and detailed overview of stimulus-
sensitive hydrogels used in sensor and actuators has been
published by van der Linden et al. in 2003 [116]. In
2010, the use of hydrogels in photonic crystal sensors
has been reviewed in detail by Nair and Vijaya [117].
Hence, this chapter will briey introduce the underlying
mechanisms that build the basis for sensing and sub-
sequently focus on the advances achieved in the last
decade.
2.1. Temperature
Temperature sensors are important in a wide range
of science and technology elds such as marine research,
food processing, underground geochemical studies, and in
biotechnology [118,119]. Hence, sensors are needed that
precisely detect the exact temperature in a variety of dif-
ferent environmental conditions. Despite the high number
of temperature sensors that are available, the sharp and
tunable entropy driven collapse of LCST polymeric systems
at a given temperature is therefore attracting consider-
able attention [120]. Thus, thermo-responsive polymers
are widely used in temperature sensing as bulk hydrogel,
in patterns, as photonic crystal gels and in other physical
forms and structures [121126]. Accordingly, this chapter
concerns hydrogel-based temperature sensing systems in
whichhydrogels were usedas 3Dsensing networks formed
by temperature-sensitive polymers (as bulk hydrogel form,
hydrogel photonic crystals (PCs), intelligent polymerized
crystalline colloidal arrays (IPCCA)) and as appropriate
matrices for temperature sensing probes.
PCs are materials in which the dielectric constant is
varying periodically. This creates a photonic band struc-
tureinwhichelectromagnetic waves canor cannot proceed
depending on their wavelength. A number of interest-
ing effects result from this such as tunable photonic stop
bands that give rise to numerous sensing applications
[117,127]. Sensing applications of PC usually exploit a
change in the periodic structure due to external stimu-
lus or presence of the analyte. With the incorporation of
a thermo-responsive material into the structure, PC can
be used for the detection of temperature. In a typical
example, thermo-responsive hydroxyethyl methacrylate
(HEMA)-based hydrogels PCs were used for temperature
sensing [123]. 3D hydrogel PCs were constructed with a
combination of prismholographic lithography and hydro-
gel photoresists (Fig. 5). With change in temperature,
the swelling of PCs occurred resulting in morphological
changes. The changes were explored by inversionof hydro-
gel PCs into silica structure. During the swelling, the lattice
distance increases in the (111) direction and the swollen
PCs are deformed and recovered. These changes result in
Fig. 6. Typical changes in the diffraction fromIPCCA particle dispersions upon analyte recognition by the hydrogel matrix. Figure is redrawn from[130].
shifting of the photonic stop band with proportional to
temperature change (Fig. 5).
Similar to PCs, IPCCA can be used for the detection of
temperature. IPCCA consists of a crystal colloidal array
of polymer spheres polymerized in a hydrogel matrix
[128,129]. Thearrayof colloidal spheres acts similar topho-
tonic crystal and diffracts the light giving rise to intense
color. Following this principle, with incorporation of a
temperature-sensitive polymer, IPCCA structure becomes
an ideal candidate for temperature sensing. With change
in temperature, the volume transition of hydrogel induces
color changes by the means of change indiffractionproper-
ties. The diffraction through PCCA particles follows Braggs
law, which is formalized below[130].
mz = 2ndsin0 (11)
where d is the diffraction order, z, the wavelength of light
in vacuum, and n and d refer to refractive index of the sys-
temand the diffracting phase spacing, respectively. Here, 0
shows the Bragg glancing angle. Thus, when the hydrogel
volume increases upontemperature variation, the distance
between the colloidal spheres increases and so the Bragg
peak of the diffracted light shifts to longer wavelengths as
illustrated in Fig. 6 [130].
Recently, a temperature sensing construction based on
IPCCA particles was designed by cross-linking of acryl-
amide (AAm) or N-isopropylacrylamide (NIPAM) with
bisacrylamide (BAAm) in the presence of UV photoinitia-
tor 2,2-diethoxyacetophenone (DEAP) around polystyrene
colloidal particles [131]. These particles diffract the visi-
ble light because the (111) planes of the face-centered
cubic (fcc) polystyrene colloidal particle array have an
200nm lattice constant. This NIPAM-IPCCA is swollen
in cold water, but collapses with increasing temperature
due to the phase transition behavior, resulting in a blue
shift of the diffraction. The detection of temperature is
succeeded via monitoring the diffraction wavelength at
a dened angle relative to the incident light. The sys-
tem works well in the temperature range of 060
C.
Phase transition behavior of NIPAM gels can also be
controlledbyincorporatingmorehydrophilic or hydropho-
bic monomers. For instance, incorporation of hydrophilic
AAm as a co-monomer reduces polymer hydrophobic-
ity and increases the interaction between water and
hydrophilic groups. Thus, the LCST is increased, since the
hydrophobic interactions are compensated for up to higher
temperature by the stronger polymerwater interactions
[132].
Both methods described above are based on hydro-
gel sensing network. However, hydrogels are also used as
3D environments to prolong the lifetime of sensing ele-
ments, especially in luminescence and uorescent-based
temperature sensing probes. These probes are sensitive
to temperature and exhibit temperature-dependent emis-
sion. The advantages of this systemover other temperature
sensing schemes are (i) high sensitivity, (ii) contactless
operation, and (iii) inertness to even strong electrical elds
[133]. As luminescence probes, europium(III) complexes
are widely used for their strong temperature-dependent
luminescence and rather narrow emission bands peaking
at around 616nm [119,134,135]. These complexes have
been designed for wide temperature ranges and have been
immobilized into polymer matrix to form thin lms for
sensing applications.
In one study, dipyrazolyltriazine tris(-diketonate)
europium(III) complexes are used as luminescence tem-
perature sensor [119]. Incorporation of these probes into a
polyurethane hydrogel matrix provided sufcient stability
of the probes. Temperature-sensitive microbeads are
prepared by mixing of palladium(II) 5,10,15,20-tetrakis
(2,3,4,5,6-pentauorophenyl) porphyrin/poly(styreneco-
acrylonitril (Pd-TFPP/ PSAN) microbeads and europium(III)
tris(thenoyltriuoroacetonate) trihydrate/poly(4-tert-
butyl styrene) (Eu(tta)
3
L/PTBS) microbeads with a solution
of polyurethane hydrogel (type D4) in ethanol/water
followed by stirring for a certain time and subsequent
drying at ambient air. When the resulting beads are excited
with a light of 405nm, the bright luminescence exhibits
Fig. 7. (a) Chemical structures of the indicators of the europium(III) complexes used for sensing temperature in [119] and (b) temperature dependence of
the decay time. Figure is partly redrawn [119].
temperature-dependent decay times that can be used to
sense the temperature between 0 and 70
C (Fig. 7).
In a similar attempt, polyurethane hydrogel stabilized
terbium-tris[(2-hydroxy-bezoyl)-2-aminoethyl]amine
complexes (Tb-THBA) were studied as luminescence based
temperature sensor by Sun et al. [133]. When the probe
photo-excited at 341nm, it displays typical Tb
3+
ion emis-
sion bands with the strongest peak at 546nmand a typical
decay time of 1.15ms at 15
C. The emission intensity and

lifetime decrease with increasing temperature, probably as
a consequence of thermal deactivation of the excited state.
The proposed system is appropriate for the temperature
sensing over the range from15 to 65
C and, possibly even

outside this range with further optimization. Another
similar luminescent-based temperature sensing system
was constructed by Borisov et al. who used the highly
temperature-dependent luminescence of ruthenium tris-
1,10-phenathroline embedded in a polyurethane hydrogel
[136]. Fromchange in luminescence decay time, they could
determine the exact temperature in the range between 0
and 60
C.
Also uorescence-based temperature sensing systems
have been reported, for example based on surfactant-
free poly(vinyl alcohol)/borax/2-naphthol hydrogels [137].
2-naphtol acts as uorescence indicator that exhibits
a decrease of uorescence intensity upon rising tem-
perature when it is embedded in aqueous PVA/borax
gel network with excitation at 365nm. In contrast, the
blue color emission intensity (photoluminescence (PL):
z
max
=426nm) of 2-naphthol in a basic hydrogel changes
gradually to strong from 30 to 80
C, when excited at
365nm.
2.2. Ions, ionic strength and pH
pH-sensitive hydrogels change their volume, mass and
elasticity reversibly in response to a change in the pHvalue
[138]. The pH-sensitive character of hydrogels [139145]
makes thempromising materials for a broadrange of appli-
cations as microsensors [138141] and microactuators in
MEMS devices. The principles for swelling dependent pH
value detection can be various: changes of the holographic
diffraction wavelength in optical Bragg grating sensors
[144] shifts of the resonance frequency of a quartz crystal
microbalance in microgravimetric sensors [146] a bending
of micromechanical bilayer cantilevers [145], as well as a
deection of silicon membranes in piezoresistive pressure
sensors [143,145]. Before we give some detailed exam-
ples for pH sensors based on hydrogels, we would like to
advisethereader tohaveacloser lookat thecomprehensive
reviewabout hydrogel-basedpHsensors and microsensors
published by Richter et al. [147]. The review introduces
the physical background of the special properties of pH-
responsive hydrogels and gives a comprehensive overview
about transducers which are able to convert the changes
of the physical properties of stimuli responsive hydrogels
into electrical signals.
An example for the use of pH-sensitive hydrogels based
on an IPCCA was developed by Reese et al. [131]. The
hydrogel was constructed by dissolving of acrylamide,
bisacrylamide, and 2,2-diethoxyacetophenone in a disper-
sion of diffracting polystyrene colloids. This CCA mixture
was injected into a quartz cell with a 250m spacer and
photopolymerized. Through partial hydrolysis of an acryl-
amide hydrogel with NaOH, the hydrogel network was
endowedwithcarboxylate groups. As describedabove, this
hydrogel was polymerized around a face-centered cubic
(fcc) array of monodisperse, 100-m diameter highly
negatively charged polystyrene colloidal particles. Hence,
change in pH-induced swelling/deswelling of the hydrogel
due to protonation/deprotonation of the particle surface
andthe hydrogel. The resulting color change was measured
according to the temperature-sensitive IPCCA described
above. They could determine pH with a 0.05 pH unit reso-
lution in deionized water.
The group of Gerald Gerlach used the strong swelling
ability of pH-responsive hydrogels [145] to develop a sen-
sor which combines piezoresistive-responsive elements as
mechano-electrical transducers and the phase transition
behavior of hydrogels as a chemo-mechanical transducer.
They demonstrated the operational principle of chemical
and pH sensors based on the swelling behavior of hydro-
gels [146]. For the realization of pH sensors poly(vinyl
alcohol)/poly(acrylic acid) (PVA/PAA) hydrogels with a pH
Fig. 8. Microlens sensor in which the wateroil interface forms the liquid microlens that is pinned in a construction on a hydrogel ring (a). Expansion and
contraction of the hydrogel regulates the shape of the liquid meniscus by changing the angle v of the pinned wateroil interface (b). The blue dashed lines
show the expanded state of the hydrogel ring (IIh) and the corresponding divergent microlens (Im) at 0 =0
. The red dashed lines show the contracted

state of the hydrogel ring (Iih) and the corresponding convergent microlens (IIm) at 0 =(90
).(For interpretation of the references to color in this

gure legend, the reader is referred to the web version of the article.) Figure reproduced from[152].
value dependent swelling behavior were used as chemo-
mechanical transducers for a piezoresistive sensor setup.
A similar setup but with poly(N-isopropylacrylamide) has
been applied and investigated for pH sensors as well.
From the same group, Trinh et al. reported a simula-
tion and design study of the system [148]. To optimize
the sensor design, Sorber et al. investigated the swelling
effect of the mentioned (PAA/PVA) hydrogel system by
Fourier transform infrared (FT-IR) attenuated total reec-
tion spectroscopic imaging under in situ conditions [149].
The results of the FT-IR spectroscopic images rendered
an improved chemical sensor possible and demonstrated
that in situ FT-IR imaging is a powerful method for the
characterization of molecular processes within chemical-
sensitive materials.
Lee et al. observed the transformations of pH-sensitive
hydrogel-based inverse opal photonic sensors froma face-
centered cubic (fcc) to a L1
1
-like crystal structure [150].
By directly imaging a Rhodamine B (RhoB)-labeled inverse
opal hydrogel, using two-photon laser scanning uores-
cence microscopy, it was possible to characterize the
mesostructure evolution of the pH-sensitive sensors dur-
ing swelling. Along with the expected swelling normal to
the substrate, they found that the template inverse opal
structure undergoes a number of unique structural trans-
formations upon swelling, including a transformation from
FCC to an L1
1
-like crystal structure.
Shin et al. [151] utilized inverse opal hydrogel struc-
tures to fabricate a fast responding photonic crystal pH
sensor. The sensor was fabricated by templated photo-
polymerization of 2-hydroxyethyl methacrylate (HEMA)
and ethylene glycol dimethacrylate (EGDM) within the
interstitial space of a self-assembled polystyrene (PS) col-
loidal photonic crystal. After removal of the PS colloidal
photonic crystal structure by chloroform, pH-dependent
reectance measurements were performed using a ber-
optic UVvis spectrometer coupled with a reected light
microscope. For calculating reectance, a silver mirror was
assumed to have a 100% reectance. The pH sensor exhib-
ited response times of about 12s for pHvariations between
5 and 6 and had a lifetime of over 5 months without loss of
reproducibility or iridescent color.
A quite interesting optical sensing system was devel-
oped by Dong et al. who utilized the stimuli responsive
swelling character of a hydrogel lens [152]. As central
component of their system, a stimuli responsive hydro-
gel was integrated into a microuidic system that served
as the container for a liquid droplet (Fig. 8). The hydrogel
simultaneously sensed the presence of stimuli and actu-
ated adjustments to the shape and hence focal length
of the droplet. The group demonstrated two systems: a
pH-sensitive liquid microlens, using an acrylic acid based
hydrogel, and a temperature-sensitive liquid microlens,
using a N-isopropylacrylamide hydrogel.
An easy and biologically inspired method for sensing
a pH value was presented by Kim and Beebe [153]. The
method is based on elastic instabilities of bi-polymer
swelling hydrogels: two different bi-polymer gel stripes
were xed in such a way together that their differential
response in swelling, induced by a designated stimulus,
caused the strip to bend. Due to the xation of the bi-
polymer gel stripes the swelling energy released in an
explosive elastic instability. One example was a setup
with an acid responsive dimethylaminoethyl methacrylate
(DMAEMA)/hydroxyethyl methacrylate (HEMA)-based
hydrogel on one side and an acrylic acid (AA)/HEMA-based
hydrogel on the other side. In contact with a base, or
buffer the bi-polymer stripe delaminated in a rapid motion
(Fig. 9).
Sannino et al. used a quartz crystal microbalance (QCM)
to recognize even very small changes in pH [154]. They
established a spin coating method to coat QCM plates
with cellulose based superabsorbent hydrogels to obtain
hydrogel-based fast sensors. The hydrogel network was
obtainedbycross-linkinghydroxyethyl cellulose(HEC) and
carboxymethyl cellulose(CMC) bydi-functional divinylsul-
fone (DVS). Due to the polyelectrolyte CMC the hydrogels
displayed sensitivity to variations of ionic strength and
pH of the water solution in which they were immersed.
The change in mass resulted in a change of the oscillating
Fig. 9. A self-destructive hydrogel sandwich sensor composed of acid and base responsive hydrogels. The layers are bond together in a way so that an
outward bending is created by stimuli like base and buffer. Once the elastic force overcomes the bonding force the gels delaminate in a rapid motion [153].
frequencyof thequartzplatewithafast responseandanal
accuracyonthe weight variationof the order of nanograms.
Ahydrogel-based method to recognize a local pHdistri-
bution by a change in color was developed by Maruyama
et al. [155]. They found a novel and easy technique to
investigate local pH distribution on a chip using hydro-
gel lms made of UV sensitive resin (SU-8) patterned by
photolithography. To make the hydrogels sensitive to pH
changes they were functionalized with a pH-indicator, e.g.
Bromocresol Green(BCG). The local pHwas measuredfrom
the color of the hydrogel impregnated with BCG based on
the calibrated color information in YCrCb color space.
Ion sensitive hydrogels generally work in a very similar
fashion to pH-sensitive systems. Guenther et al. pro-
posed an on-line analytical system to monitor metal
ions using gels as chemo-mechanical transducers [156].
In their study, they improved a rheochemical sensor
based on piezoresistive pressure sensor chips. This sen-
sor was constructed from two piezoresistive sensor chips
(chemical and viscosity sensors) that were mounted on
a socket with an integrated capillary. A piezoresistive
Wheatstone bridge was used as mechano-electrical trans-
ducer for the transformation of the plate deection
into an electrical output signal U
out
. For ion-sensing,
a thin layer of N-isopropylacrylamide (NIPAAm), 2-
(dimethyl maleimide)-N-ethylacrylamide (DMIAAm) as
chromophore and poly(2-vinylpyridine) (P2VP) or N,N-
dimethyl acrylamide (DMAAm), respectively, as chemo-
mechanical transducer, was photo cross-linked onto the
backside of the bending plate. Immersion into an ion con-
taining aqueous solution has induced a volume change
of this hydrogel which was monitored by the integrated
Wheatstone bridge inside a rectangular silicon plate. The
plate deection caused a stress state change inside the
plate, and therefore a resistivity change of the resistors
affecting proportionally the output voltage of the sensor.
An increase of this output voltage corresponded to the
hydrogel swelling whereas a reduction of electrical output
voltage corresponded to the shrinkage of the gel. The sen-
sor has beentestedinaqueous solutions of alcohol andsalts
of different concentrations and showed proof of principle
for this sensor design.
Also the already introduced IPCCA based sensors can
be modied for ion-sensing, for example for the detection
of Pb
2+
over a broad range of ion strengths ranging from
high ionic-strength solutions to detection in body uids.
Therefore, crown ether groups that chelate Pb
2+
with high
specicity where incorporated in the hydrogel network
between the spheres, either by functionalization of the
hydrogel with crown ether or by direct copolymerization
of vinyl-crown ether into the hydrogel matrix [157]. When
chelation of Pb
2+
with crown ether occurred, the hydro-
gel started to swell driven by osmotic pressure. Due to this
swelling, the distance between the spheres increased and
red-shift of the diffractedlight occurredproportional to the
Pb
2+
concentration. The detection limit of Pb
2+
was lower
than 0.5mol/L.
Hydrogel modied microcantilevers have been used
for ion-sensing. Microcantilevers are small rectangular
monocrystalline silicon tips of less than 1m thickness
that provide an excellent dynamic response even to small
amounts of analyte by bending upon analyte adsorption
on one side of the cantilever due to change in surface
tension. CrO
4
2
ions can be sensed using microcantilever
Fig. 10. (A) Optical image of perforated diaphragm pressure sensor array. Inset shows a scanning electron microscope (SEM) micrograph of one of the
1 mm1mm sensors. (B) SEM micrograph showing the pores (d=40m) etched into one quarter of the 1mm1mm sensor diaphragm. The gure is
taken from[160].
based on self-assembled monolayer (SAM) of a triethyl-
12-mercaptododecylammonium[158]. The detection limit
of this sensor is as low as 10
9
M, but this sensor gradu-
ally loses its sensitivity over 1 week because of instability
of the layer. Hence, to enhance long term stability, Zhang
et al. used a hydrogel layer based on acrylamide and (3-
acrylamidopropyl) triethyl ammoniumchloride instead of
the SAM [159]. When this microcantilever is exposed to
CrO
4
2
ions, deection occurs as a function of CrO
4
2
con-
centration. This systemshows higher specicity to CrO
4
2
compared to other ions like Br
, HPO
4
2
and NO
3
2
with a
sensitivity as lowas 10
11
M.
Following a different methodology, hydrogel arrays
combined with perforated piezoresistive diaphragms were
used for sensing of ionic strength and pH. The sen-
sor was constructed with three components: (i) the
piezoresistive sensors, (ii) the pH-sensitive hydrogel
(composed of hydroxypropyl methacrylate (HPMA), N,N-
dmethylaminoethyl methacrylate (DMA) and the cross-
linker tetra-ethylene glycol dimethacrylate (TEGMA)), and
(iii) a backing plate (Fig. 10) [160]. Diffusion pores
were comprised to allow analyte ow to the hydro-
gel. Alternatively, the backing plate was perforated.
Swelling/deswelling of the hydrogel due to changes in ion
strength induced pressure changes onto the piezoresistive
sensors that are directly converted into electric signals. The
hydrogel quickly and reversibly swelled when placed envi-
ronments of physiological buffer solutions (PBS) with ionic
strengths ranging from0.025 to 0.15M, making it ideal for
proof-of-concept testing and initial characterizations with
higher sensitivity.
Very recently, a DNA hydrogel systemwas used for the
detection of Hg (II) ions [161]. The selective binding of
Hg
2+
between two thymine bases of DNA induces a hairpin
structure. Upon addition of SYBR Green I dye, green u-
orescence is observed only when this hairpin structure is
present. Inthe absence of the Hg
2+
ion, additionof dye leads
toyellowuorescence. Athymine-richDNA-functionalized
polyacrylamide hydrogel was used that allowed sensitive
and selective detection of Hg
2+
via a visual uorescence
change. The proposed system can be regenerated using a
simple acid treatment to remove Hg
2+
fromsamples. Using
the naked eye, the detection limit in a 50mL water sample
is 10nMHg
2+
.
2.3. Gas and humidity
2.3.1. Gas
In the eld of gas-sensing, hydrogels are mainly used
for three purposes. Due to their anti-adhesive and protein-
repellent character, hydrogels can be used just as a
passive protection coating for a sensor/electrode [162].
Alternatively, hydrogels can be modied by gas sensitive
molecules, e.g. special uorescent dyes [163] which ren-
ders them sensitive for certain gases. Finally, the stimuli
responsive swelling character can be used for sensing,
mainly for sensing carbon dioxide after the Severing-
haus principle [164,165]. This strategy utilizes the pH
change resulting from CO
2
diffusion into an electrolyte
solution which can then be sensed by a pH-sensitive
hydrogel.
One example for the use as protective layer is the
internal hydrogel separation layer of the planar ultrami-
croelectrode nitric oxide (NO) sensor developedby Oh et al.
to measure local NOsurface concentrations [166]. The sen-
sor array consists of a platinized working electrode and a
silver paint reference electrode coated with a thin silicone
rubber gas permeable membrane. The working electrode
and the gas permeable membrane were separated by an
internal hydrogel layer. They could demonstrate that the
NO-selective ultramicroelectrode is an appropriate tool for
determining accurate steady state surface NO concentra-
tions.
A hydrogel gas sensor from the second sub class was
described by Zguris and Pishko [167]. They developed a
possible nitric oxide, hydrogel biosensor that utilized 4-
amino-5-methylamino-2
,7
-diuorouorescein (DAF-FM)
entrapped in poly(ethylene glycol) (PEG) hydrogel micro-
structures. By utilizing the NO-sensitive uorescence of the
dye and bio-inert character of the hydrogel matrix it was
possible to create a disposable biosensor that is applicable
for the nitric oxide production in certain mammalian cells.
The sensor has a lower detection range of 0.5Mof nitric
Fig. 11. Exploded viewof all parts and the assembly drawing of the nal hydrogel-based CO
2
sensor. Figure reproduced from[170].
oxide in solution. However, due to the irreversibility of the
dye it is a non-dynamic systemthat may only be used once.
An example for a hydrogel sensor for the detection
of carbon dioxide based on the Severinghaus princi-
ple was developed by Herber et al. [168]. Therefore
a pH-sensitive hydroxyethyl methacrylate (HEMA)-co-
dimethylaminoethyl methacrylate (DMAEMA) hydrogel
disk, which swelled and de-swelled in response to pH
changes, was clamped between a silicon pressure sensor
chip and a porous metal screen together with a bicar-
bonate solution. CO
2
reacts with the bicarbonate solution
resulting in a pH change, which was converted into a
pressure by the enclosed hydrogel. This pressure was a
measure for the partial pressure of CO
2
. A more sophis-
ticated hydrogel biosensor based on the Severinghaus
principle was described by ter Steege et al. [169]. They
developed the prototype of a continuous hydrogel CO
2
sensor as an alternative and improvement to standard air
tonometry. The work follows up on the previous work
of Herber et al. [170]. The sensor consisted of a pH-
sensitive, dimethylaminoethyl methacrylate (DMAEMA)
and co-monomer 2-hydroxyethyl methacrylate (HEMA),
based hydrogel in a bicarbonate solution mounted on a
catheter-tip pressure sensor. It is covered by a gas per-
meable membrane (Fig. 11). The hydrogel swells/shrinks
dependent on the CO
2
concentration, which leads to a
change in volume and pressure reected by the pressure
sensor.
The sensor enabled continuous measurement of lumi-
nal CO
2
and fast detection of sudden and gradual
changes in pCO
2
in a clinical signicant range. Due to
the hand-made assembly, the sensor lacked the tem-
perature stability to meet clinical demands, but the
authors gave a positive outlook for automated assembly
system.
A cross-over of the principles was followed by
Kocincova et al. [171]. They presented an interesting
method for non-invasive, simultaneous optical mon-
itoring of oxygen and pH during bacterial cultivation
in 24-well microplates by using an integrated dual
sensor for dissolved oxygen and pH values. The dual
sensor was based on oxygen-sensitive microparticles
ruthenium-tris-4,7-diphenyl-1,10-phenanthrolinedi-
(trimethylsilylpropane-sulfonate) (Ru(dpp)
3
(TMS)
2
)
in organosilica and methacrylate-based pH-sensitive
microbeads (HQ-N-1-HP1), stained with the pH probe
8-hydroxypyrene-1,3,6-trisulfonate (HPTS) embedded
into a polyurethane hydrogel. The readout was based on a
phase-domain uorescence lifetime. The sensor was used
for monitoring the growth of Pseudomonas putida bacterial
cultures (Fig. 12).
2.3.2. Humidity
Humidity sensing is important in numerous industries
such as food, petroleum, textiles, ceramics and many
others [172]. Optical-based detection via color change
offers the most attractive way for detection of humidity
without complex system conguration and tuning in
comparison to other sensing mechanisms [173]. Photonic
crystal (PC) hydrogels have been introduced in Section
2.1 and are widely used in optical-based sensing devices
[128,174,175]. The color change feature of PC hydrogels
due to swelling upon contact with water predetermines
themto act as a humidity sensor.
A humidity sensitive PC hydrogel was prepared by
inltrating acrylamide (AAm) solution into a poly(styrene-
co-methylmethacrylate-co-acrylic acid) PC template and
subsequent photo-polymerization [174]. Due to the intrin-
sic humidity sensitive property of acrylamide, a broad
range of humidity (from20%to100%) couldbe identiedby
Fig. 12. 24-Channel SensorDish
Reader on the shaker (a) and cross-section of a well of a microplate with included sensor at its bottom(b). Excitation and
detection of the emission is performed through the bottomof the plate [171].
obvious color changes. This was attributed to the humid-
ity sensitivity of the samples stopband. The color changes
and optical properties were also investigated by UVvis
spectroscopy. The stopbands of the PC hydrogels shift to
different values with regard to exposed humidity (Fig. 13).
A different photonic crystal based humidity sensing
system was reported by Kang et al. [123]. They pre-
pared a responsive photonic crystal hydrogel-based on
amphiphilic poly(styrene-b-quaternized 2-vinyl pyridine)
block-copolymers. This structure forms a single one-
dimensional periodic lamellar structure resulting in a
responsive photonic crystal gel structure. The glassy
hydrophobic layer forces expansionof thehydrophilic layer
along the layer normal, yielding extremely large optical
tunability through the changes in both layer thickness and
index of refractions. The wavelength for this system, was
changed 575% (z
peak
=3501600nm) in the position of the
stopband depending on degree of humidity.
Barry and Wiltzius reported the detection of humid-
ity via polyacrylamide-based inverse opal hydrogels (IOH)
[176]. These smart materials have the capacity to respond
to humidity changes by shifting its optical reection
peak noticeably within the visible wavelength range due
to interlayer swelling and shrinking of the structures.
With increasing humidity, equilibrium-swollen hydrogels
absorb more water from air to restore equilibrium condi-
tions. This volume change results in shifting the refractive
spectrum. These IOHstructures exhibited reectance spec-
tra with highly differentiable peaks due to their layered
structure and Bragg reection properties.
Galindo et al. reported another optical-based systemfor
humidity detection [177]. They encapsulated uorescent
avylium salts in a poly(2-hydroxylethyl methacrylate)
(PHEMA) network matrix. Flavylium salts are strongly
colored and display a uorescence emission that is
quenched by water for avyliumsalts possessing hydroxyl
substituents. This is due to the presence of efcient inter-
molecular excitedstate protontransfer (ESPT) to water and
opens the use of these molecules for humidity sensing.
By this, swelling and deswelling of hydrogels at relative
humidities between 7% and 100% could be measured by
uorescence decay times of the avyliumsalts.
A long period grating (LPG) coated photopolymerized
poly (acrylic acid-co-vinylpyridine) hydrogel with N,N
-
dimethyl bisacrylamide cross-linker has been used for
monitoring relative humidity levels by Wang et al. [178].
With increasing humidity, the response wavelength direc-
tion, and the resonance dip increases, resulting in an
increase in the coupling strength. This sensor construction
is efcient in a range from38.9% to 100% relative humidity
Fig. 13. Photographs of PAA-P(St-MMA-AA) hydrogels corresponding to relative humidity (a) and color/stopband changes of PC hydrogel as a function of
humidity (b). Figure redrawn from[174].
(RH) with a sensitivity of 0.2nm/% RH and accuracy of
2.3% RH.
Arregui et al. [179] used hydrogel-coated optical bers
for the detection of humidity. Four different hydrogels;
poly (hydroxyethyl methacrylate), poly (acrylamide), poly
(N-vinyl pyrrolidinone) and agarose were deposited on
optical ber by means of direct polymerization on the
optical ber surface. All of these hydrogels were suitable
as humidity sensors. As a generic rule, the bigger pore
size, the higher dynamic range and shorter response times
were obtained. Optical humidity sensors were basedonthe
refractive index changes of hydrogels with humidity. Best
performance was obtained by poly-HEMA hydrogel with a
response time of 90s and with a range of application from
10% to 100% of RH.
3. Biochemical sensing mechanisms
So far, studies and approaches were presented and dis-
cussed where sensing was based on swelling or deswelling
of the hydrogel network based on physical or chemical
stimuli. This paragraph concerns sensing based on more
complex and also more specic biochemical mechanisms.
At rst, sensing based on molecular interactions is dis-
cussed, followed by a paragraph about living sensors
where bacteria or cells that are immobilizedinor onhydro-
gels generate the signal that is used for sensing.
3.1. Molecular interactions
3.1.1. Enzymesubstrate interaction
Enzymes are highly specic and efcient in their reac-
tions with substrates. They act as catalysts by lowering the
activation energy of chemical reactions. Therefore, in rst
approximation, the substrate is in a rst step forming a
complex with the enzyme by docking into the highly selec-
tive active pocket of the enzyme. This enzymesubstrate
complex represents the reactive transition state of the
reaction in which the substrate is activated. After the sub-
sequent chemical reaction the product is released and the
enzyme is restored in its functional conformation. If the
concentration of the substrate is sufciently high, the cat-
alyst is saturated with substrate and the reaction is only
determined by the rate of conversion from substrate to
product. This results in a pseudo-rst order kinetic of the
reaction with regard to the concentration of the enzyme
according to Eq. (10) (MichaelisMenton equation), where
v is the rate of the reaction, k
cat
the rate constant of the
catalyzed reaction, K
m
a constant that comprises k
cat
and
the rate constant for the enzymesubstrate complex for-
mation, [E
0
] the starting concentration of the enzyme and
[S] the concentration of the substrate.
v =
k
cat
[
0
] [S]
K
m
+[S]
(12)
In reality often more complex reaction schemes such
as multiple binding and allosteric rearrangements occur,
in which the afnity and enzymatic activity is different
for the consecutive binding events. However, the math-
ematical description of these processes is comparable. In
any case, this brief introduction into enzyme activity and
enzyme kinetic shows that enzymatic activity may be used
for precise determination of analyte concentrations. In the
following sections, a variety of different sensors based on
enzyme substrate interaction in hydrogel matrices are pre-
sented and discussed ordered by the analytes that have
been detected.
3.1.1.1. Alcohol. Wu et al. developed an organic-phase
alcohol biosensor by co-entrapping alcohol oxidase and
horseradish peroxidase (HRP) within an ionotropic poly-
mer hydrogel matrix fabricated from silica gel particles
[180]. By entrapping the enzymes into the hydrogel matrix
it was possible to maintain a bio-catalytic reaction at high
and stable rates for an on-line detection of methanol in n-
hexane under owoperationmode. The analytical working
range of the sensor was 2.390mM methanol in n-hexane
with an operational biosensor-lifetime of more than 45
assays and a shelf lifetime of longer than 2 weeks.
Jang and Koh [181] employed a multiplexed enzyme-
basedassaywithinamicrouidic deviceusingshape-coded
poly(ethylene glycol) (PEG) hydrogel microparticles. The
microuidic device was constructed by serially connect-
ing two patterning chambers and a microlter-integrated
detection chamber through a Y-shaped microchannel.
Different shapes and sizes of hydrogel microparticles
with entrapped enzymes were fabricated in the pattern-
ing chamber by photolithography and collected in the
detection chamber by pressure-driven ow. As a model
experiment for multiple sensing applications, simulta-
neous detection of glucose and ethanol was investigated.
Therefore glucose oxidase (GOX) and alcohol oxidase
(AOX) were entrapped into differently shaped hydrogel
microparticles. Each enzyme-catalyzed reaction was eas-
ily identied and a simultaneous detection of glucose and
ethanol was possible by uorescence imaging in a con-
centration range from 1.0 to 10.0mM without cross-talk
by using same uorescence indicator in a single detection
chamber (Fig. 14).
3.1.1.2. Amino acids. Castillo et al. developed a bi-enzyme
redox poly(1-viylimidazole) (PVI) complexed with Os-
(4,4
-diethylbpy)
2
Cl (termed PVI
19
-dmeOs)/Poly(ethylene
glycol)(400)diglycidyl ether (PEGDGE) hydrogel for detec-
tion of l-glutamate [182]. The sensor was capable of
detecting the release of this excitatory neurotransmitter
from adherently growing HN10 and C6 cell cultures upon
stimulation. Monitoring was utilized using an electrode
modied with the bi-enzyme redox hydrogel at an applied
potential of 50mV versus the internal Ag reference elec-
trode. The detection limit, 0.5M, and the response time
of the sensor, 35s, satised the experimental requirements
for application in cell culture.
3.1.1.3. Ammonia. A different bi-enzyme sensor was con-
structed for the amperometric determination of ammo-
nium (NH
4
+
) by immobilizing glutamate oxidase and
glutamatedehydrogenaseona clark-typeoxygenelectrode
by Kwan et al. [183]. To guarantee the catalytic activity
the enzymes were entrapped in a poly(carbamoyl) sul-
fonate hydrogel matrix. The sensor has fast response (2s)
and short recovery times (2min), a linear detection range
Fig. 14. Simultaneous detection of glucose and ethanol using shape-coded hydrogel microparticles within microuidic device. (a) Detection logic, (b)
optical and uorescence image depending on sample composition, (c) uorescence intensity of circular and square hydrogel microparticles which were
reacted with ve different samples. (Each sample was composed of glucose and ethanol at different molar ratio). Figure was taken from[181].
between 10 and 300Mammoniumand with a detection
limit of 2.06M.
Urea is an important parameter in clinical analysis
because its increased concentration in blood and reduced
level in urine is a strong indication for renal dysfunction.
Due to the fact that one equivalent urea is hydrolyzed
by urease to two equivalents ammonia according to
the reaction (NH
2
)
2
CO+2H
2
O+H
+
2NH
4
+
+HCO
3
it is
possible to determine the urea level depending on the
NH
4
+
concentration. Eggenstein et al. developed a double
matrix membrane (DMM) potentiometric urea biosen-
sor. A NH
4
+
-sensitive disposable electrode was coated
by a poly(carbamoylsulfonate) (PCS) hydrogel layer with
immobilized urease and a disposable Ag/AgCl electrode as
reference [184] (Fig. 15).
The group tested different polymer materials like
polyvinylchloride, polyvinylpyrrolidone, polyestersulfonic
acid and photo cross-linkable prepolymers as gel matrix
for entrapping the urease. Best results concerning adhesion
capability, limit of detection, slope, linear range and life-
time were obtained with PCS produced froma hydrophilic
polyurethane prepolymer blocked with bisulte and cross-
linked with polyethyleneimine (PEI) as gel material. With
the biosensor setup, it was possible to detect urea con-
centrations between 7.210
5
and 2.110
2
mol/L. The
detection limit was 210
5
mol/L urea and the slope in
the linear range 52mV/decade.
3.1.1.4. Glucose. Due to increasing numbers of diabetes
patients, the demand for smart glucose-sensing systems
has grown and with it the effort to utilize hydrogels for
glucose-sensing applications. Before concentrating inmore
detail on hydrogel-biosensor systems that are used for glu-
cose sensing we would like to draw the readers attention
on the review Chemically controlled closed-loop insulin
delivery written by Ravaine et al. in 2008 [185]. The
reviewfocuses on glucose-responsive hydrogels and gives
a great overview about hydrogel systems that are either
Fig. 15. Schematic view of the disposable urea biosensor consisting a NH
4
+
-sensitive disposable electrode coated by a poly(carbamoylsulfonate) (PCS)
hydrogel layer with immobilized urease and a disposable Ag/AgCl electrode as reference [184].
modied, e.g. by glucose oxidase, lectin and phenylboronic
acid moiety, release insulin, or swell/shrink in the pres-
ence of glucose. Accordingly, this chapter focuses on the
major strategies for enzyme-based glucose sensing and on
recently published studies.
One of the most conventional methods to detect glu-
cose levels is the use of enzyme-systems like glucose
oxidase (GOX) [186,187], or glucose dehydrogenase [188].
When for example glucose oxidase is immobilized in a
pH-sensitive hydrogel matrix the oxidation reactions from
glucose to gluconic acid will force the hydrogel to swell.
The swelling of the hydrogel can then be sensed e.g. by
a mass-sensitive magneto-elastic sensors [189]. Kim et al.
described a micropatterning technique to immobilize pro-
teins on the surface of three-dimensional poly(ethylene
glycol) (PEG)-based hydrogel microstructures that could
be used for hydrogel-biosensor applications [190]. There-
fore hydrogel microstructures were fabricated by replica
molding using a poly(dimethylsiloxane) (PDMS) replica
as a molding insert and photolithography. Bovine serum
albumin (BSA), a model protein, was covalently immo-
bilized to the otherwise protein-repellant surface of the
hydrogel structures. The Empty space inside hydrogel
microstructures could be occupied by different proteins
andsequential bienzymatic reactionof glucose oxidase and
peroxidase (POD) could be demonstrated in a hydrogel
microstructure by immobilizing the GOX on the surface of
POD-entrapping hydrogel microstructure. The activity of
immobilized glucose oxidase was 16.5Umg
1
and a detec-
tion of different glucose concentrations ranged from0.1 to
20mM was shown.
Another biosensor utilizing GOX entrapped into a
poly(ethylene glycol) diacrylate (PEG-DA) hydrogel matrix
was developed by Mugweru et al. [191]. In comparison
to the already mentioned swelling dependent sensors,
the group succeeded in fabricating glucose sensor arrays
on gold electrodes on exible polyimide sheets by
photo-polymerization that can be analyzed by cyclic
voltammetry. The sensor principle is based on the reaction
of avinadenine dinucleotide of glucose oxidase GOX(FAD)
with -d-glucose, where a reduced formGOX(FADH
2
), glu-
conic acid and hydrogen peroxide are formed inside the
hydrogel. The reduced formof GOX(FADH
2
) is in turn oxi-
dized by the electrochemically generated Os
3+
form of
the redox polymer, setting up a catalytic pathway that
produces an enhanced oxidation peak. The electrons are
transferred from the enzyme to the redox polymer, shut-
tled between the redox sites in a self-exchange reaction
until being transferred to the electrode surface. The cat-
alytic current produced is proportional to the glucose
concentration. The sensor has a linear dependency for
concentrations of 0360mg/dL glucose with a calculated
sensitivity of 1.782A/(cm
2
mM).
In 2009 Merchant et al. built a high-sensitivity
reagentless amperometric double function biosensor for
glucose and hydrogen peroxide by immobilizing GOX
and horseradish peroxidase (HRP) in cross-linked lms of
ferrocene-modied linear poly(ethylenimine) [192]. Fer-
rocene was involved as mediator of electron transfer to the
electrode surface. Current densities 4 times higher than
those obtainedwithother ferrocene-basedredox polymers
could be achieved. This sensor generated a limiting cat-
alytic density of 1.2mA/cm
2
which is the highest known
density ever reported for redox polymers with GOX at pH
7. Due to the very high sensitivity (73nA/cm
2
M) of the
systemit was possible to detect glucose concentrations in
the micromolar range.
In 2011 Kim et al. [193] developed a process to
create self-assembled peptide hydrogel networks con-
sisting of Fmoc-diphenylalanine nanobers. By physically
encapsulating enzyme bio receptors, like GOX or HRP,
in combination with uorescent reporters, like CdTe and
CdSe quantum dots (QDs), the group was able to create a
newoptical biosensing platform. Encapsulation of QDs and
enzymes within the hydrogel matrix was achieved in situ
in a single step by simply mixing aqueous solutions of QDs
and enzymes with the monomeric Fmoc-diphenylalanine
solution. The group successfully tested the system for the
Fig. 16. Measurement setup used for uorescence excitation and data collection of continuous glucose sensing by a glucose sensitive hydrogel attached to
a ber-optic hardware system. Various ber-optic sensors were tested by attachment to the permanent hardware system[194].
detection glucose and toxic phenolic compounds by using
a photoluminescence quenching of the hybridized QDs.
For glucose the degree of quenching was well correlated
in the range from 1 to 10mM of glucose concentration,
which fully covers the range of 3.56.5mM required for
the diagnosis of diabetes. For phenol and hydroquinone the
response was linear up to 3mM. Leakage of encapsulated
compounds fromthe hydrogel matrix was assessed. While
the release of QDs was negligible and only trace amounts
could be found in the supernatant, the release test for GOX
showedareleaseof 3%after 0.5h, 0.06%after 1hand0.002%
after 2h. Aninitial release rate of 3%is signicant andneeds
tobereduced, as GOXmay insolutioninteract withtheana-
lyte and interfere with correct concentration detection of
the sensor.
In 2010 Siegrist et al. presented rst promising steps
towards an enzyme-based electrochemical glucose sen-
sor for in vivo operation [194]. The group reports the
use of a novel polypeptide-based uorescent glucose-
sensing system that could overcome many drawbacks of
an enzyme-based system while showing the potential for
high accuracy, especially at hypoglycemic levels. Fluores-
cently labeled glucose recognition polypeptide elements
were derived from glucose binding proteins (GBP) and
genetically modiedwitha unique cysteine near the ligand
binding pocket. This facilitates a conjugation reaction for
site-specic labeling with N-[2-(1-maleimidyl)ethyl]-7-
(diethylamino)coumarin-3-carboxamide (MDCC), an envi-
ronmentallysensitive, thiol-reactiveuorophore. TheGBPs
wereimmobilizedina polyacrylamidehydrogel matrixand
placed on the tip of an optical ber to realize a continuous
glucose-sensing device (Fig. 16).
The group performed an in vitro validation in buffered
solutions and whole blood. Performance tests in the
hypoglycemic levels demonstrated that the reagentless
polypeptide-based glucose-sensing systemhas an approx-
imate precision of 3.6mg/dL (0.2mM) in a glucose range
displayed between 90mg/dL (5mM) and 36mg/dL (2mM).
It was further possible to operate the sensor in blood at
physiologic temperature (namely, 37
C) for 1.5h with a

precision of 9.5mg/dL in a range of 36mg/dL (2mM) and
180mg/dL (10mM) of glucose, without interference from
other sugars (namely, fructose).
It is important to mention the impact of oxygen concen-
tration uctuations on GOX-based sensing systems. Since
oxidase-based devices rely on the use of oxygen as the
physiological electronacceptor, uctuations inoxygenten-
sion and the stoichiometric limitation of oxygen, so-called
oxygen decit, which reects the normal oxygen concen-
trations, are about 1 order of magnitude lower than the
physiological level of glucose and thus induce changes in
sensor response and reduced upper limit of linearity [195].
Possibilities toaddress this problemarefor exampletheuse
of mass-transport-limitinglms (e.g. polyurethaneor poly-
carbonate) for tailoring the ux of glucose and oxygen by
increasing the oxygen/glucose permeability ratio. Alterna-
tively, applicationof materials that act as aninternal source
of oxygen such as a uoro-carbon pasting liquid (e.g. Kel-F
oil) generates an internal ux of oxygen that can thus sup-
port the enzymatic reaction, even in oxygen-free glucose
solutions [196199]. Situations of oxygendecit canalsobe
counterbalanced by using glucose dehydrogenase (GDH),
which does not require an oxygen cofactor, instead of GOx.
3.1.1.5. Hydrogenperoxide. Hydrogenperoxide is clinically
relevant as reaction product of the oxidative metabolism
catalyzed by oxidases. It is also used in many industrial
processes as oxidizing, bleaching and sterilizing agent, so
that its detection is prominent in environmental, clini-
cal and biological studies and industries [200]. Various
methods have been developed for the determination of
hydrogen peroxide via different methods like titrimetry,
electrochemistry and UV-spectrophotometry [201]. Per-
oxidases (POD) are generally used in these constructions
to convert hydrogen peroxide into radicals. The sensitiv-
ity of the determination of hydrogen peroxide via POD
depends on the ability of the immobilizing matrix to retain
the functional conformation of the enzyme for a long time.
Through the last decade, an impressive number of inven-
tive designs for hydrogen peroxide determination have
appeared. Here we will focus on some illustrative example
studies of hydrogel-based hydrogen peroxide detection,
generally based on electrochemical sensing.
The detection of H
2
O
2
based on an intracellular opti-
cal nanosensor utilizing poly(ethylene glycol) hydrogel
spheres containing HRP was reported by Kim et al. [202].
HRP was encapsulated in PEG hydrogel spheres by reverse
emulsion photo-polymerization without losing activity.
Afterwards, the uorescence emission response of these
hydrogel spheres changed as a function of H
2
O
2
concen-
tration in the presence of Amplex Red, and no leaching
of HRP was observed from the spheres. The HRP-loaded
hydrogel spheres were introduced via phagocytosis inside
macrophages and were found to respond to both exoge-
nous and endogenous sources of oxidative stress. This
approach may guide the way to use cells of the innate
immune system as real-time in vivo sensors for oxidative
stress situations.
Varma and Mattiasson developed a simple amperomet-
ric biosensor for the detection of hydrogen peroxide in
water and organic solvents [203]. They entrapped catalase
in 30% polyacrylamide gels and created a two electrode
system with Pt electrodes and the hydrogel in a teon
holder. By studying the kinetics of the catalase modied
electrode by cyclic voltammetry (CV) and coupling it to
a ow injection analysis setup it was possible to create
a sensing system that could monitor a broad range of
hydrogen peroxide concentration (0.5100mM) in differ-
ent solvents. As mentioned above, amperometric detection
of H
2
O
2
has also been achieved via immobilization of
HRP in cross-linked lms of ferrocene-modied linear
poly(ethylenimine) [192]. When small amounts of H
2
O
2
were added to the solution, oxidation peaks disappeared
andreductionpeaks increase due to increase inredox poly-
mer mediationof the HRP-catalyzedreductionof H
2
O
2
. The
system showed high sensitivity (500nA/cm
2
3M H
2
O
2
)
and a linear increase of the current with the H
2
O
2
concen-
tration up to 1.5mM.
A new-type solgel/hydrogel composite based on sil-
ica sol and a graft copolymer of poly vinyl alcohol with
4-vinyl pyridine was fabricated for hydrogen peroxide
sensing by Wang et al. [204]. The lmwas characterized by
Fourier-transform infrared (FT-IR) spectroscopy and opti-
mum analytical performance was obtained dependent on
pH and electrochemical behavior of the biosensor using
potassiumhexacyanoferrate(II) as a mediator. This system
exhibited high sensitivity (15AmM
1
) and a detection
limit of 510
7
M.
Optical-based local detection of H
2
O
2
secreted by stim-
ulated macrophages was achieved through enzyme-based
biosensor by Yan et al. [205]. Photolithographic pattern-
ing of a PEG-based hydrogel with incorporation of HRP was
applied to construct microstructures that served as sensing
agent (Fig. 17). Amplex Red, a nonuorescent compound
that is oxidized to the uorescent molecule resorun as a
result of the HRP-catalyzed breakdown of H
2
O
2
, was either
immobilized inside hydrogel elements alongside enzyme
molecules or added into the cell culture media during cell
activation. Theproductionof H
2
O
2
after mitogenic stimula-
tionof macrophages resultedinappearance of uorescence
in the HRP-containing hydrogel microstructures.
Another hydrogen peroxide sensing hydrogel
microstructure was constructed by Lee et al. [206].
They also used PEG hydrogel as matrix for the sensing
enzyme, in this case hydrogen peroxidase. The hydro-
gel was micropatterned on a glass substrate as shown
in Fig. 18. Cells were cultured ob such substrates, and
micropatterning of co-cultures of hepatocytes and non-
parenchymal cells onto non-fouling PEG structures was
achieved, thus offering a technique for precisely control-
ling physical and endocrine interactions between two
types of cells. Also here, Amplex Red served as detection
method for the HRP-catalyzed oxidation of hydrogen
peroxide.
3.1.2. Antibodyantigen
Antibodyantigen based sensors are afnity based
devices with a coupling of immunochemical reactions
[207]. The general working principle of this sensor is based
on the specic immunochemical recognition of antibod-
ies (or antigens) immobilized on a transducer to antigen
(or antibodies), that produce signals which depend on
the concentration of the analyte. Antibodyantigen based
detection of target molecules have gained attention in last
three decades due to its high specicity and lowdetection
limit [208].
Detection in antibodyantigen sensing system can be
performed in different ways. One of the widely used tech-
niques is quartz crystal microgravimetry with dissipation
(QCM-D) which provides real-time immunoassay capabil-
ity. QCMis a detection method that quanties the decrease
of the resonant frequency of the quartz crystal when the
mass increases, e.g. when antigen binds to immobilized
antibodies. Dissipation measurement provides additional
information about viscoelasticity of the surface which can
increase with antigen binding. This technique is widely
used in immunoassay applications because of high sensi-
tivity.
Carrigan et al. used low molecular weight
polyethyleneimine (PEI) and carboxymethylcellulose
(CMC), and covalently immobilized antibody by cross-
linking between carboxymethyl groups of CMC and
succinimide esters generated on the antibody [209]. This
hydrogel-interface has been evaluated with detection
of cytokines rhIL-1ra, rhIL-6 and rhiL-18BPa antigens.
Increasing the CMC concentration leads to higher antibody
immobilization with decreasing non-specic binding. At
optimum conditions, this system gives a detection limit
as low as 400ng/mL for a 44kDa antigen. Another study
was performed by the same group using specic antibody
capture ligands such as protein A, protein G, anti-IgG Fc for
oriented antibody immobilization and higher frequency
QCM-D crystals to increase the detection limit [210]. In
this case, detection limit with new technique increased
to 25ng/mL on 5MHz crystals. Reducing of non-specic
adhesion for this method was shown with incorporation of
Fig. 17. (Up)Fluorescence images of hydrogel microstructures with embedded HRP after 5min incubation with 5M H
2
O
2
(A) and 20M H
2
O
2
(B) in
the presence of Amplex Red. (Down) HRP-containing PEG hydrogel micropatterns are fabricated on glass substrate (C) followed by seeding and culture of
macrophages that only attach on the glass next to sensing hydrogel structures (D). Release of H
2
O
2
by macrophages upon mitogenic stimulation can thus
be sensed (E) [205].
PEG into this biointerface [211]. A different methodology
used by Lu et al. is based on periodate-oxidized dextran
layer [212]. Staphylococcus aureus protein A (SpA) was
immobilized on poly-l-lysine modied piezoelectric crys-
tal surface to improve their stability, activity, and binding
specicity with human immunoglobulin G (IgG) in ow
injection assays. The prepared sensing crystal shows best
sensitivity and reusability at a ow rate of 140L/min
with a detection limit as low as 0.2nM with proposed
system.
Fig. 18. Schematic diagramdescribing surface microfabrication procedure (Steps 15) to forma micropatterned co-culture assembly of hepatozytes and
stromal cells such as broblasts (Steps 6 and 7). Hydrogel microstructures remain free of cells and can contain enzyme molecules such as HRP for sensing
cellular microenvironment [206].
Similar in function to QCM, surface plasmon resonance
(SPR) is an established method for the label-free detection
of biomacromolecular interactions. SPR uses evanescent
waves to examine the surface. Analyte binding leads to
change in refractive index at the sensing surface inuenc-
ing resonant angle and shifting to generate real-time signal
[213]. SPR allows measurements in real-time with high
precision and repeatability and provides some advantages
over other techniques. These are: (i) the amount of com-
plex at equilibriumis measuredinthe presence of unbound
reactant without disturbing the reaction equilibrium, (ii)
the stability of the immobilized ligand can be monitored
by tracing the surface binding capacity and the baseline
stability, and (iii) the low amount of material for analysis
[214]. As for QCM, the surface of SPRsensors shouldbe anti-
adhesive tounwantedcompounds andinteract specically,
and hydrogels have been used for this purpose.
Guidi et al. studied the detection of insulin-like growth
factor-1 (IGF-1), a suspected carcinogen, in cow milk via
SPR based sensing system[215]. Hyperimmune polyclonal
anti-IGF-1 antibodies were immobilized to detect IGF-1.
The amount of native IGF-1 in fresh rawmilk was detected
down to 4ng/mL. A similar study was performed by Col-
lier et al. [216]. Another SPR based sensing was used for
the detection of prostate-specic antigen [217]. Anti-PSA
antibodies were coupled covalently to gel sensor disc that
can detect PSA molecules with a concentration of 0.15ng
in 1mL, which is sufcient detection of physiological
(<4ng/mL) and clinical (410ng/mL) PSA levels. A differ-
ent study was reported for the detection of apolipoprotein
B100 sensitivity to 10 different monoclonal antibodies
(mAbs, all IgG
1
). Sensitivity was evaluated in terms of
different structure recognition. The detection of recombi-
nant human interferon- (IFN-) with SPR in plasma was
reported by Stigter et al. [218]. To test non-specic adsorp-
tion of plasma components, numbers of SPR coatings were
evaluated. Non-modied dextran coated SPR disks were
the best candidate for the detectionof IFN-inplasma with
a lower detection limit of 150ng/mL.
SPR based sensors are also used for the detection
of steroids. In a typical sensor, real-time detection of a
pair steroids, cortisol and cortisone in saliva and urine
was carried out using surface plasmon resonance-based
immunosensor [219]. A polycarboxylate based hydro-
gel layer was used for immobilization of the antibodies
anticortisol and anticortisone. These antibodies are very
specic for cortisol and cortisone and contribute to high
specicity of the sensor without binding other steroids like
testosterone, epitestosterone and corticosterone. The layer
exhibitedhighdegreeof stabilityover timeduringrepeated
regenerations. Saliva and urine sources were selected due
to presence of lowamount of cortisol andcortisone incom-
parison to the 100-fold higher concentration in blood. The
detection limit of both steroids in saliva and urine samples
was less than10g/L whichis sufcient sensitivity for both
clinical and forensic use.
A novel immunosensing system for determination
of human -ferroprotein (AFP) was established using
a boronate immunoafnity column via ow injection
chemoluminescence. Interaction between carbohydrates
and boronic acid was used to immobilize the AFP antigen
through the sepharose matrix. For assessment of the
usability for sensing, a mixture of the analyte AFP and HRP-
labeled AFP antibody (HRP-anti-AFP) was injected into the
column. The antigen bound to the column which leads to
subsequent trapping of the free HRP-anti-AFP. Detection
was performedviachemoluminescenceduetothesensitiv-
ity effects of HRP on the reaction of luminol with hydrogen
peroxide [220].
Also three-dimensional (3D) hydrogel thin lms were
used for the detection of protein binding (antigenicity) and
antibodyfunctionalityinamicroarrayformat [221]. Protein
antigenicity was evaluated using the protein toxin staphy-
lococcal enterotoxin B (SEB). Cy3-labeled anti-mouse IgG
(capture antibody) was microarrayed onto the hydrogel
surfaces and interrogated with the corresponding Cy5-
labeled mouse IgG (antigen). Lee et al. introduced an
elastic and hydrophilic composite mold, which enables the
transfer of hydrophilic polar ink such as polyelectrolyte
multilayer [222]. The mold is composed of an urethane-
related commercial polymer and poly(ethylene glycol)
diacrylate (PEGDA) which forms a hydrogel upon UV-
polymerization. The group established a simple strategy
for immobilizing antibodies exclusively inside the micro-
reservoirs of the patterned composite mold, which made
the system suitable for biosensing applications. A rst try
regarding the detection of Escherichia coli (E. coli) O157:H7
was explored.
Electrochemical based sensing is also used in
antibodyantigen sensors for the detection of IgG. In
one case, a redox hydrogel-coated carbon electrode with
avidin incorporated in the hydrogel was employed [223].
Redox hydrogel lmwas prepared by electrodeposition of
solutions containing polyacrylamide-poly(4-vinylpyride)-
[Os(bpy)
2
Cl]
2+/3+
(PAA-PVP-Os) and phosphate buffer
onto the screen-printed carbon disks (SPE) with a
potential of 1.0V (vs Ag/AgCl) for 2min. Avidin was
conjugated with biotin labeled anti-rabbit IgG. After
exposure the tested solution to capture of IgG, the
sandwich-assay was completed by binding of HRP-labeled
anti-labeled IgG. Electrical contact between the HRP and
the electrode bound hydrogel resulted in formation of an
electrocatalyst for the electroreduction of H
2
O
2
to water.
The detection limit with this method was as low as
3ng/mL. Another electrochemical based sensing was
performed by using a three-dimensional (3D) porous
chitosan network as a matrix for hepatitis B surface
antibody (HBsAb) [224]. Porous network was prepared
by electrodepositing a designer nanocomposite chitosan
solution with encapsulated silica nanoparticle as hybrid
lmon an ITO electrode, followed by removal of the silica
nanoparticles with HF solution. HBsAb were covalently
immobilized to the hydrogel network for the detection
of hepatitis B surface antigen. A novel label-free electro-
chemical detection method of ractomine was developed
by Shen and He [225]. Ractopamine-bovine thyroglobulin
antigen was incorporated into agorose hydrogel lm
on a glassy carbon electrode. The K
3
Fe(CN)
6
/K
4
Fe(CN)
6
redox probe system was used to translate the binding
to an electrochemical signal with addition of polyclonal
antibody against ractopamine. Using similar mechanism
and same hydrogel layer with different antibody, the
detection of ferritin was performed by immobilizing the
ferritin antibody (FeAb) on the electrode [226]. Cyclic
voltammetry (CV) and differential pulse voltammetry
(DPV) were used for the conversion of signal to electrical
output. The detection limit of ferritin with this method was
1.510
5
g/L and the linear range was 55010
5
g/L.
3.1.3. Nucleotide, oligonucleotide and DNA
Advances in nucleic acid detection have become
increasingly important in diagnostics systems by virtue of
importance in genetic diagnostics, forensic analysis, dis-
ease diagnosis and basic research areas [227229]. Beside
the simplicity combined with low cost, an effective DNA-
sensing systems require high selectivity to eventually be
able to detect single-nucleotide polymorphism(SNPs) and
high sensitivity to enable sensing of few hybridization
events of corresponding small amounts of target DNA frag-
ments [230,231]. These high demands have led to the
development of different methods, generally via electro-
chemical based sensing systems due to rapid and direct
detection combining high sensitivity, small dimensions,
low cost, and compatibility with microfabrication tech-
nology of transducers [232236]. Generally, DNA sensors
comprise three main steps: (i) immobilizing the oligonu-
cleotide probes on the substrate, (ii) hybridization with
the target sequences through base-pairing interactions
and (iii) read-out by converting DNA hybridization into a
useful analytical signal by a transducer [237,238]. Fluores-
cence, electrochemical [239,240], optical [241], electrical
[242] andmicrogravimetric basedsystems [241] arewidely
used for the detection of DNA in concentrations down to
10
18
M.
Efciency of nucleic acid fragments immobilization
on the substrate is the key factor of high-throughput
DNA analysis technologies as it is crucial for accuracy
and sensitivity of the detection. Most studies indicate
that the hybridization is dramatically hindered on solid
surfaces [243], whereas nucleic acid hybridization in
hydrogels was reported to closely resemble hybridization
in solution [244]. Moreover, hydrogel-based sensor sys-
tems result in 100-fold in signal density due to higher
immobilization capacity of sensing moieties within the
matrix [245]. Due to the highly water swollen, hydrophilic
nature and exible structure, hydrogels do not disturb
the thermodynamically stable form of nucleic acid frag-
ments and improve the immobilization capacity due
to the three-dimensional structure. These solution-like
characteristics offer important advantages for capturing
probes in comparison to two-dimensional rigid substrates
and self-assembled monolayers providing. In this sec-
tion, we present currently used hydrogel-based techniques
for the detection of oligonucleotides. For general DNA-
sensing methods including non-hydrogel-based systems,
we wouldlike todrawthe readers attentiononthe reviews
Recent advances in DNA sensors by Cosnier and Mail-
ley [246], Electrochemical DNA sensors by Drummond
et al. [247] and Target-responsive structural switching for
nucleic acid-based sensors by Li et al. [248].
One widely applied tool for DNA sensors are microcan-
tilevers. As alreadyintroducedinChapter 2.2, a cantilever is
a small rectangular monocrystalline silicon tip of less than
1m thickness and can transduce changes of mass, tem-
perature, or stress, into bending (static mode) and change
in resonance frequency (dynamic mode) [249]. Accord-
ingly, hydrogel-coatedcantilevers combine the advantages
of hydrogels, minimal denaturation of immobilized probes
and unspecic interaction, with high sensitivity and direct
transduction mechanism without need of any labeling of
the microstructure-based sensing system. The immobiliza-
tion of target-selective receptors on the cantilever makes it
possible to identify analyte molecules by means of changes
in surface tension. The change in surface tension will lead
to a bending of the cantilever and can be sensed by opti-
cal laser detection or capacitance changes [250]. In one
case, cantilevers with electrodeposited chitosan lms were
used for the detection of nucleic acid hybridization [251].
Electrodeposition was performed by immersing the chip
in an acidic chitosan solution and then applying a negative
potential to the electrode in the solution. Subsequent net
hydrogel consumption at the cathode leads to a pH gra-
dient that, at pH values above 6.5, results in deposition of
chitosan on the cathode. Oligonucleotides were bound to
thechitosanandtheresultingbiofunctional cantilever used
for detection of DNA hybridization by static mode (in solu-
tion) and dynamic mode (resonant frequency shift in air)
as illustrated in Fig. 19.
For the dynamic mode, the resonant frequency shift
was measured in air before and after the bimolecular
detection event and in the presence of analyte. In static
mode the corresponding measurements were performed
regarding bending of the cantilever in solution. Static
measurements in solution conrmed successful hybridiza-
tion and yielded a surface tension value that, together
with the results of the dynamic measurements in air
conrmed surface density of oligonucleotides two orders
of magnitude larger than comparable results obtained
by probes tethered to SAMs. This direct measurement
result demonstrates the higher probe concentration in the
three-dimensional (3D) hydrogel network in comparison
to the two-dimensional (2D) self-assembled monolayer
substrates.
DNAmicroarrays, integrated into optouidic hybridiza-
tion systems, are also often used in the eld of DNA
detection. In one case, a DNA hybridization construct was
designed based on microchannels in combination with
UV-cross-linked hydrogels and photolithography dened
weirs [252]. The PEG-based hydrogel microstructures were
prepared by UV exposure of PEG-DA through a photomask
and used for dening the DNA detection microcham-
ber. Single-stranded (ss) DNA probes modied microbeads
were immobilized in the microchambers and used to
capture the complemantry target sequences. The trans-
port of complementary DNA target solution was provided
by an electrophoretic transport (hydrogel switch open).
Subsequently, hybridized DNA targets could be isolated
via weirs using pump-driven pressure (hydrogel switch
closed). Biotin labeled ssDNA probes were immobilized on
the microbeads and coated with strepavidin. Hybridization
reactions were conducted by moving the targets across the
arrayof probe-containingmicrochambers byelectrophore-
sis. The hybridization of ourescein-labeled ssDNA targets
to complementary probes was observed by uorescence
Fig. 19. Cartoon illustration (a), SEM (b), and optical microscopy (c) photos of microcantilever construction after chitosan electrodeposition and contact
proler scan of chitosan lmalong dashed line in panel c (d) [251].
microscopy. In 1min 90% of the DNA were captured and
could sequently be released and recovered.
Another strategy based on a multi-analyte sensor array
platform consisting of analyte specic features which
were indexed by shape was followed by Meiring et al.
[237]. Soft-lithography was used for array fabrication with
a poly(ethylene glycol) acrylate hydrogel. Fluorescently
tagged methacrylamide-modied oligonucleotides were
copolymerized into the hydrogel matrix and their cova-
lent immobilization conrmed. Each target sequence was
labeled with a different uorophore and retained the
ability to hybridize selectively to the designated targets.
Different DNA probes were incubated in the solution of
three-different target sequence mix. After the hybridiza-
tion the hydrogels were rinsed with buffer to remove
the unbound samples. One bright-eld and three uores-
cence micrographs were captured; each micrograph was
obtained using a specic optical lter set for one of the
three different target uorophores (Fig. 20).
By using surface-immobilizedDNA-basedafnity struc-
tures such as oligonucleotides, aptamers or ssDNA with
appropriate redox labels, it is possible to monitor their tar-
get inducedstructural switching. This is done by measuring
electrochemical currents which are directly associated to
the distance between the redox label and the electrode
[227]. In one case, an enzyme-amplied amperomet-
ric DNA hybridization with ssDNA probes immobilized
in a carboxymethylated dextran layer on the electrode
was performed by Hajdukiewicz et al. [253]. Using a
ferrocenemethanol electron mediator, the hybridization
of ssDNA probes to biotinylated-ssDNA and addition of
glucose oxidase-avidin conjugate lead to a signal current.
To analyze the signal current the group use cyclic voltam-
metryandachieveda detectionlimit of 0.2nmol ina 500L
sample utilizing a graphite electrode.
DNA sensors can also be used to detect gene mutations.
One example for this application is the highly sensitive
detection of a K-ras point mutation in codon 12 based
on DNA carrying hydrogel microspheres [254]. The G-
G mismatch in codon 12 of the K-ras gene leads to a
mutation that is suspected to be a reason for pancreatic
cancer. This example underlines the crucial importance
of single mismatch detection in early diagnosis of genetic
disorders. The hydrogel microspheres used in this study
were synthesized by precipitation polymerization. To
detect the mutation in the sequence a hydrogel-enhanced
sandwich method of a SPR assay was used. The use of the
DNA carrying microspheres enhanced the SPR response as
a result of an increased dielectric constant. This method
improved detection limit from500 to 5nM, corresponding
to a 100-fold improved in sensitivity and 14-fold SPR angle
shift compared to that of non-amplied detection.
DNA-functionalized monolithic hydrogels were suc-
cessfully used for highly sensitive and selective colorimet-
ric DNA detection [161]. DNA bearing methacrylamide-
groups at the 5
-end were covalently incorporated into

polyacrylamide hydrogels through copolymerization dur-
ing gel formation. IncombinationwithDNA-functionalized
gold nanoparticles that selectively bind to immobilized
probe-target pairs, high optical transparency of the hydro-
gels allowed direct visual detection of down to 0.1nM
target DNA. By using the attached gold nanoparticles to
Fig. 20. Micrographs of the DNA detection assay demonstrating the successful detection of each of the complementary target sequences with high signal
and negligible cross-hybridizational noise [237].
catalyze the reduction of Ag
+
to metallic silver, 1pM tar-
get DNA could be detected. The gels can be regenerated
by a thermal treatment, and the regenerated gels perform
similarly to freshly prepared ones.
Gu and colleagues designed a systemthat combines the
advantages of quantum-dot-encoded technology, biore-
sponsive hydrogels, andphotonic crystal sensor [255]. DNA
responsive hydrogel bead with highly ordered 3D inverse
opal structures were fabricated by template replication of
silica colloidal crystal beads (SCCBs). Hybridization of tar-
get DNA with the cross-linked ssDNA in the hydrogel grid
caused hydrogel shrinking, which could be detected as a
corresponding blue shift in the Bragg diffraction peak posi-
tion of the beads with a detection limit of 10
9
M.
A novel methodology for uorometric DNA hybridiza-
tion detection based on non-covalent coupling of DNA to
a water soluble zwitterionic polythiophenylene derivative
was developped by Nilsson and Inganas [256]. Therefore,
3-[(S)-5-amino-5-carboxyl-3-oxapentyl]-2,5-
thiophenylene hydrochloride (POWT) hydrogel has
been cross-linked with ssDNA on a polystyrene surface.
The zwitterionic side chain of the polythiophene forms a
complex with single-stranded oligonucleotide inducing
a planar polymer and aggregation of the polymer chains.
This change detected as a decrease of the intensity and
a red-shift of the uorescence. On addition of a comple-
mentary oligonucleotide, the intensity of the emitted light
increased and blue-shifted. This method is highly sequence
specic, and a single-nucleotide mismatch can be detected
within 5min without using any denaturation steps, with
an overall lower detection limit for DNA hybridization of
10
11
M.
3.1.4. Other binding mechanisms
3.1.4.1. Biotinstreptavidin. Responsive hydrogel-based
microlenses can change their optical properties upon
volume-phase transitions in the presence of stimuli. They
can be easily constructed in simple and rapid fashion
with high sensitivity [257]. In hydrogel microlenses,
the stimulus induced phase transition changes the focal
lengths what causes a focusing or defocusing of the
projected image [258]. Most studies in the literature
concerning hydrogel microlenses for biosensing have
been performed by Lyon and co-workers. In one case,
a bioresponsive hydrogel construct was developed to
investigate specic and non-specic adsorption [259].
Poly (N-isopropylacrylamide-co-acrylic acid) (pNIPAM-
co-AAc) based bioresponsive microgels were prepared
by incorporation of both biotin and aminobenzophenone
(ABP) as a photoafnity label via carbodiimide chemistry.
Functionalization with ABP allows for photochemi-
cal tethering of anti-biotin after it is associated to the
microlens through native antibodyantigen association.
Hydrogel microlenses were generated by electrostatic
assembly of anionic microgels on positively charged,
silane modied glass substrates. Two different routes were
used for determination of the specic and unspecic bind-
ing. In the rst route, the hydrogel microparticles were
functionalized with biotin with EDC coupling, whereas
in the second route, these particles were functionalized
with biotin-ABP via EDC and DCC coupling. With a sec-
ondary IgG exposure, specic/ancillary binding effects
were investigated via brighteld and uorescencence
microscopy. Biotin and polyclonal anti-biotin sensitive
hydrogel microlenses were constructed using the same
microlens approach as shown in Fig. 21 [257].
First, pNIPAm-co-AAc hydrogel particles were pre-
pared by free-radical precipitation polymerization method
(Fig. 21(A)). Then, biotin was incorporated to hydrogel
particles via EDC coupling (Fig. 21(B)), followed by forma-
tion of cross-links in the hydrogel by multivalent binding
of avidin to biotin and anti-biotin antibodies to biotin
providedonthehydrogel microlenses (Fig. 21(C, D), respec-
tively). Multivalent binding of both avidin and polyclonal
Fig. 21. Cartoon representation of the hydrogel microlens assay developed in [257] composed of biotinylatedmicrogels. Binding of either streptavidin or
biotin-antibody led to volume changes that altered the focal lengths of the microlenses [257].
anti-biotin antibody was determined by monitoring the
optical properties via a brighteld optical microscopy. The
same group developed a new method label-free biosens-
ing by combining antibodyantigen cross-linked hydrogel
microlenses to use for the detection of small molecules
with a tunable sensitivity [260].
Piezoelectric biosensors have widely been used for
afnity interaction [261]. In one case, hydrogel-coated
streptavidin piezochips were used for the physical and
chemical immobilization of streptavidin molecules. Sub-
sequent rinsing with biotinylated bovin serum albumin
(BSA) revealed a higher binding capacity for the covalently
immobilized streptavidin than on the afnity-prepared
one, resulting in a higher subsequent BSA binding. Another
PZ sensor was used for the detection of poly His-tagged
proteins using nitrilotriacetic acid (NTA) captured oxidized
dextran hydrogel layer [262]. The binding of chelating
ions, Co
2+
, Cu
2+
and Ni
2+
was investigated with regard to
binding to poly His-tag proteins, and Ni
2+
exhibited the
highest binding capability. The system was assessed by
using glutathione-S-transferase C-terminally tagged with
a 6-His tag coated NTA PZ chip to determine the cel-
lular glutathione level that is normally in the range of
110mM.
A hydrogel-based sensor for monitoring protease
activity has been constructed by combining a versatile
solid-phase synthesis (SPS) with the simplicity of liquid
crystal display (LCD) [263]. In this study, polyethylene
glycol acrylamide (PEGA) was used as SPS support for
immobilization of peptide sequences that are specically
recognized and cleaved by proteases. In the presence of
proteases, the respective peptides were cleaved with high
specicity, resulting in their release fromthe hydrogel and
interaction with the LC surface. This leads to a realignment
in the liquid crystalline layer and thus to optical changes
frombright todark. Hence, this methodoffers thescreening
of protease specicity by eye without need of special
complex instrumentation (Fig. 22). Due to irreversibility
of the cleavage, the system can however only be used
once.
Lee et al. created micropatterns on PEG hydrogels
through graft polymerization and photolithography for the
detection of streptavidin binding to an avidin modied
hydrogel layer [264]. Surface modication of the anti-
adhesive PEG hydrogel layer was performed by grating
poly acrylic acid (PAA) via photoinduced surface-initiated
polymerization with benzophenone as photoinitator.
Biotinyl-3,6,9-dioxaoctanediamine was coupled to the
PAA micropattern via EDC/NHS-coupling. Specic binding
between biotin and streptavidin was used for evaluation of
this new PAA modied PEG surface and thus showed that
potential biosensor application of these micropatterned
PEG hydrogels are possible.
3.1.4.2. Calmodulin based sensing. Daunert and co-workers
developed a new hydrogel microlens system that can
change its optical properties via ligand exchange [265].
Protein calmodulin (CaM) was used as a model hinge-
motion binding protein for the chemical tunability of the
microlenses. CaM was chemically bound to the hydrogel
network as well as its low afnity ligand phenothiazine.
The recognition between CaM and phenothiazine led to
a further increase in cross-linking density. However, in
the presence of a high afnity CaMligand like chlorprom-
azine (CPZ), an exchange between phenothiazine ligand
and CPZ occurs and thus leads to a decrease in cross-
linking. In consequence, the hydrogel is swelling and the
refractive index decreases. This ability of volume change
in response to a (bio)chemical stimulus allows its uses as
chemically tunable microlenses. The swelling response of
these stimulus-responsive hydrogel microlenses is quite
rapid, reaching 74% of the total response within 120s,
which makes this setup interesting for rapid sensing appli-
cations.
Fig. 22. Schematic representation of the protease sensitive sensor chamber developed in [263]. Fmoc-protected protease sensitive peptides are specically
split upon presence of the respective protease, leading to interference of the release Fmoc with the LCD and color change from white to black. Figure
redrawn from[263].
3.1.4.3. Carbohydrate recognition by boronic acids. Besides
the already mentioned enzyme receptors also synthetic
glucose receptors like phenylboronic acids have been
intensively studied for glucose sensing [129,266268]. The
sensingeffect of phenylboronic acidis basedonthefact that
themoleculebecomes negativelychargeduponsugar bind-
ing, whichthencangenerateanosmotic pressureduetothe
resulting Donnan potential [269]. This results in a swelling
of thehydrogel that canbeeasilysensed. Thegroupof Asher
worked intensively on this eld and did some pioneering
work by applying a crystalline colloidal readout between
glucose and phenylboronic acid [129,270]. Due to the
analyte-induced swelling of the hydrogel described above,
the separation between the colloidal spheres increases and
the Bragg peak of the diffracted light will be shifted to
longer wavelengths. Same group developed a fast respon-
sive crystalline colloidal array photonic crystal glucose
sensor that composed of hydrogel network embed an array
of 100nm diameter monodisperse polystyrene colloids.
This network diffracts light in the visible spectral region as
theglucoseconcentrationvaries [271]. Thesensor hada full
response rate within 90s to the average glucose concentra-
tions foundinblood(5mM) andwithin300s to the average
glucose concentrations found in tear uid (0.15mM). They
demonstrated that the sensor is responsive to 0.15mM
glucose concentrations in articial tear uid solution.
Another sensing method based on phenylboronic acid
was presented by Worsley et al. in their work about
continues blood glucose monitoring using a thin-lm
optical sensor [272]. The sensor was based on thin-lm
polymer hydrogel containing phenylboronic acid recep-
tors. The hydrogel system is composed of acrylamide
cross-linked by N,N
-methylenebisacrylamide and phenyl-

boronic acid receptors (3-acrylamidophenylboronicacid,
and (3-acrylamidopropyl)trimethylammonium chloride).
When the system is illuminated with ordinary white
light, the holographic fringes within the gel collectively
diffract a narrow band of wavelengths in a manner
governed by the Bragg equation (z
max
=2ndsin^). The
end result is a characteristic spectral reection peak,
the wavelength of which can be correlated with glu-
cose concentration. Utilizing the sensor it was possible
to measure glucose concentrations of 333mmol/L in
human blood plasma in static mode in vitro. In a ow
cell experiment variations in the glucose concentra-
tion of approximately 0.170.28mmol/L min could be
monitored.
Lapeyre et al. [273] reported the synthesis of close to
monodispersepoly(N-isopropylacrylamide) submicromet-
ric microgels, modied with a phenylboronic acid (PBA)
derivative. The PBA-modied particles were sensitive to
the presence of glucose at pH8.5, i.e., at pHconditions close
to the pK
a
of the PBA derivative (pK
a
=8.2), with a swelling
degree proportional to the concentration of glucose. Due
to their characteristics the particles might serve as build-
ing blocks for the design of colorimetric sensors based on
the light diffraction of colloidal crystals.
3.1.4.4. Fluorescence-based glucose sensing. Glucose
sensing based on uorescence is usually based on uo-
rescence (or Frster) resonance energy transfer (FRET).
FRET involves the nonradiative energy transfer from a
uorescent donor molecule to a uorescent acceptor
molecule in close proximity, and is usually brought about
by dipoledipole interactions. The efciency of this pro-
cess is strongly dependent on distance between donor
and acceptor dye, and the signal in FRET is a decrease
in uorescence intensity and lifetime of the donor. For
glucose sensing, concanavalin A (Con A), a plant lectin
which has four binding sites for glucose per molecule, and
uorescently labeled dextran, usually with the uores-
cence dye uorescein isocyanate, are applied. Sensing is
based on the competitive binding of either glucose or the
labeled dextran to Con A. A more detailed description of
this process as well as some information about the use
of PEG-based hydrogels for improved biocompatibility of
implanted sensors is given in the review of Pickup et al.
[274] about uorescence-based glucose sensors.
Fig. 23. Array patterning template for the immobilization of capture antibodies and application of assay reagents. (A) When the PDMS patterning module
placed in contact with hydrogel, cross-linkers and capture antibodies are applied into each of the six lanes and allowed to incubate. (B) For application of
toxin and tracer antibody, the six channels of the PDMS assay module are placed parallel to the patterned capture antibodies on the hydrogel slide with
different concentrations in each lane to determine detection limits [279].
One recent example for a hydrogel-based uorescence
biosensor for glucose is the work from Chaudhary et al.
[275]. The group investigated the feasibility of dissolved-
core alginate-templated uorescent microspheres as
glucose biosensors in simulated interstitial uid (SIF). The
sensor worked on the principle of competitive binding and
uorescence resonance energy transfer and consisted of
multilayer thin lm coated alginate microspheres incor-
porating dye-labeled glucose receptor and competing
ligand within the partially dissolved alginate core. It was
further possible to combine the visible sensing assay (FITC-
dextran-TRITC-Con A/FITC-dextran-TRITC-apo-GOx) with
a near infrared sensing assay (AF-647-dextran amino-
QSY-21-apo-GOx), co-encapsulated inside the polymeric
hydrogel microspheres. The incorporation of long wave-
length NIR dyes enables more efcient excitation through
scattering tissue, which effectively improves the odds of
successful use in vivo. The system exhibited a maximum
response time of 120s and a sensitivity of 0.94%/mM glu-
cose with a response in range of 050mMglucose.
3.1.4.5. Peptide modied hydrogels for toxin screening.
Immobilization of peptides and especially proteins on pla-
nar sensor substrates often results in (partial) loss of
activity or in the case of antibodies reduced afnity to
antigens [276278]. This effect originates from interfa-
cial energy driven structural deformations in the binding
region or even the protein structure itself, with negative
impact on the activity, binding kinetics, or even inhi-
bition of the complete binding process. As highlighted
before, the solution-like environment of water swollen 3D
hydrogel networks can preserve the protein structure
while also granting multidirectional access for ligand bind-
ing to the active binding sites. Charles et al. made use of
these benets that 3D hydrogels offer when they devel-
oped a galactose polyacrylate-based hydrogel scaffold for
the detection of cholera toxin (CT) and staphylococcal
B (SEB) in a sandwich immunoassay format [279]. They
succeeded in creating a hydrogel system that provided
enhancedsensitivity withlownon-specic binding charac-
teristics. By utilizing a PDMS microchannel template they
patterned arrays of the immobilized antibodies for the
development of a sandwich immunoassay (Fig. 23). Flu-
orescence and 3D imaging by confocal microscopy and
laser scanning confocal microscopy of Cy3-labeled anti-CT
and/or Cy3-anti-SEB tracer molecules provided qualita-
tive and quantitative measurements on the efciency of
protein immobilization, detection sensitivity and signal-
to-noise ratios. Withthe established systemthey were able
toachieve lowlimits of detectionfor SEBandcholera toxins
(1.0ng/mL).
Beside the integration of active biomolecules it is also
possible to create hydrogels that are cross-linked by pep-
tides, which dissolve in the presence of certain stimuli, like
reducing agents or enzymes [280,281]. The cross-linking
system can be designed in such a way that the hydrogel
can act as a self-sacricing biosensor for enzymatic active
analytes. Such sacricing hydrogels can e.g. be used for
sensing toxins that possess enzymatic activity, such as
botulinumneurotoxin type A (BoNT/A). Frisk et al. used so-
called SNAP (synaptosome-associated protein) modied
hydrogels in combination with microuidics for sensing
Fig. 24. Fifteen micrograms per milliliter botulinumneurotoxin type A (BoNT/A) degrade synaptasome-associated protein (SNAP) modied hydrogel post
sensors. (a) Initial 425mSNAP peptide post in 1 PBS, t =0h; (b) after 48h exposure to BoNT/A; (c) 325mcontrol post with BoNT/A insensitive cross-
linker after 48h toxin exposure. Corresponding images directly below ac are edge-outlined using ImageJ software for better visualization of degraded
hydrogels. Scale bars =150m[282].
enzymatic activity of BoNT/A [282]. They were able to
generate autonomous sensors that simply required visual
readout (Fig. 24). Introductionof ca. 20Ltoxin-ladensolu-
tioninto the microuidic device allowed observationof the
degradation without the necessity for detectors, pumps, or
valves, thus making microuidic-based biosensors promis-
ing as toxin-screening vehicles. Due to the fact that the
analysis of the toxin level is done by an optical comparison
of non-degraded posts to degraded posts, this system can
be used as a great indicator for toxins in a medium, but not
as an accurate analytical method. To improve the readout
and analytical process it could be interesting to integrate
such a peptide cross-linked hydrogel systeminto a hydro-
gel biosensor based on photonic crystals. In this case even
the smallest degradation could be sensed.
3.2. Living sensors
This chapter gives an overview about the combina-
tion of hydrogels with living cells and microorganisms
to form living cellpolymer composites for biosensing
application. The use of microorganisms and cells as bio-
logical sensing elements in biosensors is well established.
Microorganisms offer advantages of ability to detect a
wide range of chemical substances, amenability to genetic
modication, and broad operating pH and temperature
range, making them ideal as biological sensing mate-
rials. For in-depth overviews on this eld we want to
draw the readers attention to the short but comprehen-
sive reviews about Microbial Biosensors by Lei et al.
[283] and Whole Cell Biosensors by Bousse [284]. The
reviewof Lei et al. concentrates on the advantages of using
microorganisms as biosensing elements, the immobiliza-
tion of microorganisms, electrochemical, amperometric,
potentiometric, conductimetric, optical, bioluminescence,
uorescence and colorimetric microbial biosensors as
well as Microbial fuel cell type biosensors, uorescence
protein-based biosensors and O
2
-sensitive uorescent
material-based biosensors. Luc Bousse concentrates on
the eld of biosensors in which the biological component
consists of living cells and points out some of the most
important reasons for using living cells, i.e. to obtain func-
tional information about the effects of a stimuli on a living
system.
This chapter focuses on advances in the use of liv-
ing cells and microorganisms in biosensors utilizing
hydrogels in the last decade. The immobilization of
cells in different hydrogel types for biosensing appli-
cations has been reported for polyacrylamide [285,286]
polyurethane-based hydrogels [287], photo cross-linkable
resins [287,288] and polyvinyl alcohol [289293], poly-
acrylonitrile membranes [294] and poly(ethylene glycol)
hydrogel [295], alginate [296298], carrageenan [286],
low-melting agarose [299], chitosan [300] and proteinic
matrix [301]. One of the main benets hydrogels have is
their high water content and the three-dimensional poly-
mer network, which allows a high gas exchange rate and
transport of nutritions to cells immobilized in the polymer
network. These characteristics, in combination with their
biocompatibility, make it possible to even cultivate cells or
Fig. 25. Schematic diagram of the A. adeninivorans LS3 microbial sensor. It illustrates the microbial consumption of dissolved oxygen (a) before and (b)
after the addition of biodegradable pollutants. Figure redrawn from[302].
bacteria inside of hydrogels and to use the cellpolymer
composites for biosensing applications.
3.2.1. Bacteria
Bacterial cells as biological recognition elements have
for example been used in a biosensor for the rapid determi-
nation of the concentration of biodegradable pollutants in
wastewater by Renneberg et al. [302]. The sensor consisted
of salt-tolerant yeast Arxula adeninivorans LS3 cells immo-
bilized in a Poly(carbamoyl)sulfonate (PCS) hydrogel, on
a Clark-type oxygen electrode. The group claimed that
the sensor made it possible to get comparable results for
the so-called standard 5-day Biochemical Oxygen Demand
(BOD5) method, employed as a pollution index, in just
5min (Fig. 25).
Another example for yeast cells in a hydrogel matrix
are the ber-optic biosensors based on luminescent yeast
cells entrapped in calcium alginate, or polyvinyl alco-
hol (PVA) hydrogels, constructed by Fine et al. [303].
The group entrapped genetically modied Saccharomyces
cerevisiae cells, which contained the estrogen receptor
alpha-mediated expression of the luc reporter gene and
enabled a novel approach for estrogenic endocrine disrup-
ting chemical (EDC) bio-detection. Due to the protective
hydrogel matrix it was possible to operate even under
non-sterile conditions. Martinez et al. described a fast
method to study cell metabolic characteristics immobiliz-
ing yeast cells (S. cerevisiae) in a microbial biosensor-like
device [304]. Main components of the sensor where S.
cerevisiae, e.g. entrapped in a poly(carbamoylsulphonate)
hydrogel membrane, and a carbon dioxide electrode. The
sensor device allowedthe calculationof MichaelisMenten
parameters related to the kinetics of transport and degra-
dation of several carbohydrates (i.e., glucose, fructose,
galactose, sucrose and xylose, with K
m(app)
of 6.0, 5.8, 0.9,
2.0, and147mM, respectively), andthestudyof thekinetics
of expression of nonconstitutive proteins related to the
transport and degradation of galactose.
Koster et al. made use of an immobilized Staphy-
lococcus type to create an amperometric, microbial
respiration-basedmicrobiosensors for the quanticationof
available dissolved organic carbon (ADOC) instantaneously
respired by microorganisms [305]. Therefore they immo-
bilized aerobic seawater microorganisms, closely related
to Staphylococcus warneri, in a polyurethane hydrogel
sensing membrane. The group focused on biochemical and
physiological properties of respiration-based ADOC micro-
biosensors that measure all of the dissolved organic carbon
compounds that areinstantaneouslyrespiredbytheimmo-
bilized cells. The sensor had a detection limit equivalent to
about 610Mglucose in a 90% response time of 15min.
The shelf lives of individual sensors were up to 2 weeks.
With E. coli Fesenko et al. used one of the most
popular bacteria strains for their hydrogel microchipdevel-
opment [306]. They developed a cellhydrogel system
for biosensing and monitoring of cell populations using
a polyacrylamide-based hydrogel bacterial microchip
(HBMChip). The microchip represented an array of hemi-
spherical gel elements that contained live immobilized
E. coli cells. They were able to measure and monitor
intracellular metabolism using vital uorescent stains,
engineeredconstructs encoding bioluminescent or uores-
cent reporters. It was possible to monitor the dynamics of
nucleic acids in growing E. coli cells using vital uorescent
stain SYTO 9. Further they made use of the biosens-
ing opportunities the cell-chip offered and illustrated the
detectionof antibiotics andsodiummeta-arsenite byquan-
titative analysis.
3.2.2. Cells
The same characteristics that make hydrogels prefer-
able candidates for bacterial biosensors count as well
for cell-based biosensors. Besides the already mentioned
biocompatibility and the high diffusion rate for nutri-
tions, gas etc. hydrogels have structural similarity to the
macromolecular-based cellular microenvironment in the
human body, the so-called extracellular matrix, which
offers applications in vivo and in vitro. To get a bet-
ter overview about human cellhydrogel composites we
advise the reader to have a closer look at the review
Hydrogels for Tissue Engineering written by Lee and
Mooney [307]. Further Hynd et al. published a compre-
hensive review about hydrogels in applications for neural
cell engineering [308]. The review gives a nice overview
about acrylate-based polymers and their derivatives, used
in biomedical applications, including drug delivery, tis-
sue engineering, cell encapsulation, and as templates for
directedcell growth, attachment andproliferation. Herewe
want to focus on hydrogels used for cell-based biosensors.
3.2.2.1. Hydrogel-coatings and inuence of the third dimen-
sion. In the eld of cell-based biosensors hydrogels are
preferably used in two different shapes: either as coat-
ing material for recognition elements like electrodes,
implantable sensors etc., or as 3D-Matrix for cells that are
utilized as biological recognition element themself. Hydro-
gels that are used as sensor coatings in most cases play
a passive role; they e.g. support gas transportation, ion-
exchange etc., or make it possible to immobilize cells on
sensor surfaces. An example for the immobilization of cells
on a sensor surface is the work of Ding et al. [309]. They
prepared a nanocomposite by neutralizing a solution of
chitosanencapsulatedgoldnanoparticles formedbyreduc-
ing in situ tetrachloroauric acid in chitosan. The gel was
designed for immobilization and electrochemical study of
cells, monitoring their adhesion, proliferation, and apopto-
sis onelectrodes. The nanocomposite gel showedimproved
immobilizationcapacityfor K562leukemiacells, as amodel
cell line, and good biocompatibility for preserving their
activity on glassy carbon electrodes. Another example
came from the same group when Hao et al. combined
the biocompatibility of chitosan (CS) and conductivity of
carbon nanober (CNF) [310]. By an electrodeposition of
soluble CNF-doped CS colloidal solution they were able to
forma robust CNFCS nanocomposite lmwith good bio-
compatibility for the immobilization and cytosensing of
K562 cells on an electrode.
The biocompatibility of hydrogels can be increased by
modifying them with certain biomolecules that support
the immobilization of cells, or decrease the inamma-
tion of tissue in case the sensor is implanted. Norton
et al. examined the release of vascular endothelial growth
factor (VEGF) and dexamethasone (DX) from non-fouling
hydrogel-based sensor coatings and their effect on the for-
eign body response [311]. The hydrogels were prepared
from 2-hydroxyethyl methacrylate, N-vinyl pyrrolidi-
none, and polyethylene glycol. The study revealed that
VEGF releasing hydrogel-coated bers increased vas-
cularity and inammation in the surrounding tissue
after 2 weeks of implantation compared to hydrogel-
coated bers. DX releasing hydrogel-coated bers reduced
inammation compared to hydrogel-coated bers and
had reduced capsule vascularity. Due to the fact that
after 6 weeks, there were no detectable differences
between drug-releasing hydrogel-coated bers and con-
trol bers it could be concluded that the drug depot was
exhausted.
Aside of immobilizing cells on hydrogel-coatings, they
can be embedded in the coating as well. In this case the
hydrogel structurally supports the cells by forming a cell
friendly3Denvironment andcells canbecultivatedas close
as possible to the sensor surface. Klueh et al. for example
reported the use of tissue-interactive bio-hydrogels, such
as brin, toenhance gene delivery at sites of sensor implan-
tation [312]. Therefore cells, genetically engineered to
over-express the angiogenic factor (AF) vascular endothe-
lial cell growth factor (VEGF), were incorporated into an
ex ova chicken embryo chorioallantoic membrane (CAM)-
glucose sensor model. The VEGF-producing cells were
delivered to sites of glucose sensor implantation on the
CAM using a tissue-interactive brin bio-hydrogel as a
cell support and activation matrix. This VEGFcellbrin
system induced signicant neovascularization surround-
ing the implanted sensor, and signicantly enhanced the
glucose sensor function in vivo.
Desai et al. demonstrated different behavior of cells
cultured on a two-dimensional coating or in a 3D net-
work [313]. They used SH-SY5Y human neuroblastoma
cells, cultured in 3D collagen hydrogel, and applied confo-
cal microscopy and immunouorescence staining to assess
the merit of the systemas a functional, cell-based biosen-
sor. They were able to show the depolarization-induced
differences in intracellular calcium release when cultured
using a 2D versus a 3D matrix. Similar results regarding
the difference in on 2D, or in 3Dsubstrates could be shown
by Wu et al. [314]. They fabricated a packed Cytodex
3 microbead array as a simple 3D cell-based biosensing
format. Resting membrane potentials and voltage-gated
calciumchannel (VGCC) function of SH-SY5Y human neu-
roblastoma cells cultured on the microbead array versus
collagen-coated at (2D) substrates were evaluated by
confocal microscopy with a potentiometric dye and a
calciumuorescent indicator. The exaggerated 2D cell cal-
cium dynamics, in comparison with those of 3D cells,
were consistent with previous 2D/3Dcomparative studies.
The study established the rationale and feasibility of the
microbead array format for 3D cell-based biosensing.
3.2.2.2. Cell preservation in 3D hydrogels. This paragraph
highlights some recent results regarding the inuence of
entrapping cells into hydrogels and possibilities to pre-
serve them over longer times in in vitro. Here we would
like to draw the readers attention to the review about
preservation methods to keep biosensor microorganisms
alive and active, published by Bjerketorp et al. [315]. It is
known from literature that cells can survive the entrap-
ment into hydrogels. Zguris et al. demonstrated that it
is possible to immobilize cells in a hydrogel matrix by
photo-polymerization without a loss of activity [316]. They
demonstrated the ability to entrap human microsomes in a
poly(ethylene) glycol hydrogel microstructures by photo-
polymerization without loss of activity, as monitored by
reference to the activity of cytochrome P450. They fur-
ther developed two photo-polymerization methods for the
Fig. 26. Schematic illustration of poly(ethylene glycol) dimethacrylate (PEGDMA) hydrogel microwell array fabrication and T-cell arraying. PAH:
poly(allylamine hydrochloride); PAA: poly(acrylic acid); PEO: poly(ethylene oxide) [321].
fabrication of heterogeneous PEG hydrogel structures con-
taining multiple phenotypes of mammalian cells [317].
Beside a two-step photoreaction injection molding process
they described a novel method for one-step fabrications
of stacked hydrogel microstructures using a microuidic
mold. In addition to hydrogels containing uorescent dyes
they were able to create differing extracellular matrixes,
unmodied or modied with the cell adhesion promoter
RGD, within one hydrogel microstructure and to gener-
ate patterns of hydrogels containing multiple phenotypes.
The studies revealed newpromising opportunities for cell-
based hydrogel biosensors.
Preservation of cells for certain time periods in a
hydrogel matrix has been shown by Itle et al. when
they entrapped cells into PEG hydrogel microstructures
for biosensing applications [318]. In their work about
Hepatocyte viability and protein expression within hydro-
gel microstructures they reported the entrapment of
mammalian hepatocytes in hydrogels of varying PEG
compositions. They were able to maintain cell viability for
7 days in free structures and in structures contained in
microuidic channels. The production of protein by cells
in hydrogels decreased with increasing PEG concentra-
tion, suggestingmass transfer limitations inhydrogels with
higher PEG compositions. Following up on this topic they
reportedthe fabricationof mammaliancell-containing PEG
hydrogel microarrays by a stage-down freezing process
for potential applications in drug screening and pathogen
detection [319]. The work revealed possibilities of fabricat-
ing cell-containing microdevices and cryopreserving them
for later use. Their work resultedinthe fabricationof whole
mammalian cell biosensors for the optical monitoring of
cell viability and response to two model chemotoxins:
sodium hypochlorite and sodium azide, and one model
biotoxin, concanavalin A [320]. Therefore the cells were
entrapped either in PEG microspheres, or in array format.
It was possible to detect sodiumazide in micromolar quan-
tities in fewer than 30min.
Fig. 27. (a) Representation of the array of electrode employed in [323]: i, ii and iii are working electrodes; iv, v and vi are counter electrodes; and vii is
the reference electrode. (b) Photograph and, (c) schematic representation of the well plate with and without the membrane insert positioned over the
integrated electrodes on the bottom. Figure redrawn from[323].
3.2.2.3. Pathogen sensing. One example for pathogen
detection with hydrogel biosensors is the work of Kim
et al. who examined the potential of a sensing strategy
based on live T-cell/B-cell interactions in a biosensor chip
format [321]. They developed an approach to fabricate
patterned poly(ethylene glycol) dimethacrylate (PEGDMA)
hydrogel microwells functionalized at their bases with
antibodies to promote specic immobilization of lym-
phocytes. The microwells were used to array single T
cells in a regular pattern on a substrate. A sensing plat-
form was created by overlaying arrayed T cells with
a conuent layer of antigen-capturing B cells. Utilizing
the hydrogel/lymphocyte sensing chip it was possible to
detect a model peptide analyte within minutes, with dose-
dependent linear signals over the concentration range
0.055M(Fig. 26).
Another example for the detection of pathogens was
published by Banerjee et al. [322]. They developed a multi-
well plate-based biosensor containing B-cell hybridoma,
Ped-2E9, encapsulated in type I collagen matrix, for rapid
detection of pathogenic Listeria, the toxin listeriolysin O,
and the enterotoxin from Bacillus species. This sensor
measured the alkaline phosphatase release from infected
Ped-2E9 cells colorimetrically and gave an example of
a cell-based sensing system using collagen-encapsulated
mammalian cells for rapid detection of pathogenic bacteria
or toxin.
3.2.2.4. Nitric oxide/glutamate. Castillo et al. presented the
simultaneous detection of nitric oxide and glutamate using
anarrayof individuallyaddressable electrodes, whichwere
modied with a sensitive nitric oxide sensing chemistry
and a glutamate oxidase/redox hydrogel-based glutamate
biosensor [323]. The rst working electrode was covered
with a positively charged Ni porphyrin, entrapped into
an negatively charged electrodeposition paint, the sec-
ond working electrode by a bi-enzyme sensor architecture
based on cross-linked redox poly(ethylene glycol) digly-
cidyl ether (PEGDGE) hydrogels withentrappedperoxidase
and glutamate oxidase. Adherently growing C6-glioma
cells were grown on membrane inserts and placed in close
distance to the modied sensor surfaces. After the estab-
lishment of a stable background current at both electrodes,
20L of a mixture containing 10L (160nM) bradykinin
and 10L KCl solution (2M) were carefully injected into
the buffer solution around the cells, and changes in cur-
rent signals at both sensors were monitored. The current
responses recorded at each electrode after stimulation of
glutamate and NO release by means of K
+
and bradykinin
demonstrated the ability of the individual electrodes in the
array to detect the analyte without interference from the
neighboring electrode or other analytes present in the test
mixture (Fig. 27).
From the same group Wartelle et al. reported test
results from a compact, biocompatible, hydrogel-based,
electrochemical sensing device for the detection of nitric
oxide released formC6-glioma cells [324]. Due to the use of
anionicallyconductingkappa-carrageenanhydrogel mem-
brane, in combination with the integrated electrode setup,
used by Castillo et al. as well, it was possible to create a
living cell-based device for NO-detection.
4. Summary and perspective
Especially in the last decade, hydrogels have gained
tremendous interest in the eld of (bio-)sensing. Many
intrinsic properties of hydrogels predetermine their use for
this application, most importantly their high water con-
tent that renders thembiocompatible and allows diffusion
of water soluble compound through the polymer network
as well as their chemical diversity and the ability to tune
their chemical andphysical properties. This allows the gen-
eration and design of stimuli sensitive hydrogels that may
act as sensors themselves, for example by introduction of
hydrophobic groups for temperature sensitivity or protic
groups for pH sensitivity. It also enables functionalization
of the hydrogel with moieties that act as sensors. The range
of molecules that can be and has been used for this purpose
is very broad and comprises (uorescence) dyes, lumines-
cent compounds, enzymes, biochemical recognition sites
such as oligonucleotides, antibodies, lectins and other very
specic recognition mechanisms. Due to accessibility of
the complete hydrogel volume high sensitivities can be
achievedwhat makes the use of hydrogels for sensingespe-
cially interesting.
Hydrogels may be prepared in aqueous solutions
througha variety of mechanisms. Most prominently, either
UV or temperature initiated radical polymerization tech-
niques are used. The gelation from low viscous solutions
to gels allows application specic preparation of the gels in
desired geometries and is compatible with miniaturization
and microuidic applications. Moreover, hydrogels can be
prepared by preparation methods that are compatible with
livingcells or bacteriasothat thesecanbeencapsulatedand
may serve as sensors in the hydrogel network.
Recently, especially multifunctionalization of the gels,
the combination of hydrogels and other intelligent mate-
rials and advances in sensor design has led to advances.
Examples are combinations of responsive hydrogels that
react with volume changes to stimuli and piezoelectric
readout. Also optical readout through microsphere arrays
embedded in a responsive hydrogel matrix is an elegant
approach. Double modication of hydrogels with enzymes
such as peroxidase and glucose oxidase allows sequential
bienzymatic reactions in the hydrogel. Aside of applica-
tion specic sensor design, microfabrication techniques
are more commonly used to miniaturize and parallelize
sensing, and hydrogels offer multiple application points
in the combination with miniaturization. Future develop-
ments of hydrogels for sensing will majorly benet from
thegrowingprecisioninmacromolecular chemistrytocon-
trol size, shape, structure and functionality of polymers as
basic building blocks for hydrogels. Moreover, the combi-
nation with modern biotechnological tools will allow for
the design and preparation of biohybrid-hydrogels with
unsurpassed specicity and sensitivity in sensing.
Acknowledgments
The authors gratefully acknowledge the DFG funded
SPP 1257 for nancial support and stimulating discussions.
F. T. thanks to Marie Curie ITN project Hierarchy (con-
tract: PITN-2007-215851) for PhD fellowship. This review
evolved from the book chapter cited in reference [115],
coauthored by the authors of this review, together with
D. Tanaka. This review, which presents an updated, more
detailed and broadened version of the topic in reference
[115], reproduces segments fromthat source, with the per-
mission of the publisher, Elsevier Ltd.
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C. The emission intensity and

C and, possibly even

. The red dashed lines show the contracted

).(For interpretation of the references to color in this

C) for 1.5h with a

-end were covalently incorporated into

-methylenebisacrylamide and phenyl-

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