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This journal is cThe Royal Society of Chemistry 2012 Chem. Soc. Rev., 2012, 41, 1363–1402 1363
Cite this:
Chem. Soc. Rev
., 2012, 41, 1363–1402
Fundamentals and application of ordered molecular assemblies to
affinity biosensing
Zimple Matharu,w
ac
Amay Jairaj Bandodkar,wz
b
Vinay Gupta
c
and
Bansi Dhar Malhotra*
ade
Received 28th May 2011
DOI: 10.1039/c1cs15145b
Organization of biomolecules in two/three dimensional assemblies has recently aroused much
interest in nanobiotechnology. In this context, the development of techniques for controlling spatial
arrangement and orientation of the desired molecules to generate highly-ordered nanostructures in the
form of a mono/multi layer is considered highly significant. The studies of monolayer films to date
have focused on three distinct methods of preparation: (i) the Langmuir–Blodgett (LB) technique,
involving the transfer of a monolayer assembled at the gas–liquid interface; (ii) self-assembly at the
liquid–solid interface, based on spontaneous adsorption of desired molecules from a solution directly
onto a solid surface; and (iii) Layer-by-layer (LBL) self-assembly at a liquid–solid interface, based on
inter-layer electrostatic attractions for fabrication of multilayers. A variety of monolayers have been
utilized to fabricate biomolecular electronic devices including biosensors. The composition of a
monolayer based matrix has been found to influence the activity(ies) of biomolecule(s). We present
comprehensive and critical analysis of ordered molecular assemblies formed by LB and self-assembly
with potential applications to affinity biosensing. This critical review on fundamentals and application
of ordered molecular assemblies to affinity biosensing is likely to benefit researchers working in this as
well as related fields of research (401 references).
1. Introduction
The last century has seen the manufacture of giant machines.
Today we recognize a considerable need for developing devices
that are as small as possible with the capability of doing
myriads of tasks. The 21st century is all about understanding
and manipulating the properties of the infinitesimally small
building blocks that build these tiny devices.
The continued efforts toward search for techniques for
fabricating such machines have led researchers to the footsteps
of Mother Nature. Nature is full of examples of such machines
viz. the smallest bacteria, virus etc. These intricate bio-machines
are capable of performing tremendously complex tasks. Develop-
ing such machines is a highly skilled job and requires an in-depth
understanding of the laws of nature. A basic, but crucial tech-
nique followed by nature is the ordered molecular assembly of
chemical species at the molecular level to form nanostructures.
In the beginning, this process may perhaps appear to be simple.
But beware, looks are deceptive! Understanding this pheno-
menon requires sophisticated instruments that have only been
developed in the 20th and 21th centuries. The simplicity of
molecular assembly belies the profound effect this molecular-
level process has on the overall properties of our macroscopic
world.
Self-assembly may perhaps be considered as the un-sung
hero of the complex molecular designs that one observes in
nature. Understanding self-assembly requires knowledge from
almost every imaginable discipline of engineering and science.
Biologists are busy understanding how nature effortlessly
produces intricate structures from simple building blocks.
Chemists are producing molecules to develop ever larger and
more complex systems. Engineers are inventing manufacturing
methods, pushing boundaries of the engineered systems to
the nanoscale. Thus, self-assembly is truly a multi-disciplinary
endeavor.
Several research groups have come up with different definitions
of self-assembly.
1–3
Based on these definitions self-assembly may
a
Department of Science and Technology Centre on Biomolecular
Electronics, Biomedical Instrumentation Section, Materials Physics
& Engineering Division, National Physical Laboratory (Council of
Scientific & Industrial Research) Dr K. S. Krishnan Marg,
New Delhi-110012, India. E-mail: bansi.malhotra@gmail.com;
Tel: +91-11-45609152
b
Department of Applied Chemistry, Institute of Technology,
Banaras Hindu University, Varanasi-221005, India
c
Department of Physics & Astrophysics, University of Delhi,
Delhi-110007, India
d
Centre for Nano Bioengineering & Spin Tronics, Chungnam National
University, 220 Gung-Dong, Yuseong-GU, Daejeon, 305-764, Korea
e
Department of Biotechnology, Delhi Technological University,
Shabad Daulatpur, Main Bawana Road, Delhi 110042, India
wBoth authors have contributed equally to this article.
zPresent address: Department of NanoEngineering, University of
California, San Diego, USA.
Chem Soc Rev Dynamic Article Links
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1364 Chem. Soc. Rev., 2012, 41, 1363–1402 This journal is cThe Royal Society of Chemistry 2012
be best defined as follows: ‘‘Self-assembly refers to the spontaneous
formation of organized structures through a stochastic process that
involves pre-existing components,is reversible,and can be controlled
by appropriate design of the components,the environment,and the
driving force’’.
A variant of self-assembly that is important to nanotech-
nology is the assembly occurring at the gas–liquid interface,
and the monolayer thus formed is called a Langmuir mono-
layer (LM). The LM can then be transferred onto a solid
substrate to form a Langmuir–Blodgett (LB) film as a first step
to fabricate nano-devices. In the LB technique the molecular
organization precedes adsorption.
4
Another widely used fabri-
cation process in nanotechnology is the chemisorption of
molecules at a liquid–solid interface to form a self-assembled
monolayer (SAM). SAMs can be formed by simple immersion
of a suitable solid support into a solution containing self-
assembling molecules. During SAM growth adsorption precedes
the molecular organization.
4
Another method that is closely related to SAM process is
layer-by-layer (LBL) assembly. In general, the LBL technique
is used to prepare multilayers comprising of alternate layers of
oppositely charged species at the liquid–solid interface.
Both LB films and SAMs provide a large spectrum of nano-
functionalized surfaces and have been the driving force for
fabrication of many molecular electronic devices including
detectors, displays, electronic circuit components and bio-
molecular electronic devices such as biosensors.
5–8
Among
these, biosensors have aroused much interest because of their
potential application in healthcare. The high degree of struc-
tural order, combined with the ability to fine-tune the structure
makes these ordered assemblies interesting systems for appli-
cation to biosensors.
1.1. Importance of monolayers in biosensing
Healthcare is presently one of the fastest growing sectors in
both developed and developing countries. The heart of this
Zimple Matharu
Dr Zimple Matharu has
received her BSc and MSc
degrees from University of
Lucknow, India. She has com-
pleted her PhD in Physics
(2011) from University of
Delhi, India. She is the recipi-
ent of seven gold medals for
securing top rank in MSc and
the prestigious fellowship from
Council of Scientific and
Industrial Research, India,
for pursuing doctoral studies.
She has worked as a research
scholar at National Physical
Laboratory, New Delhi, India.
Her primary research interests are the study of Langmuir–
Blodgett monolayers, self-assembled monolayers, protein inter-
actions and functionalization of nanostructures for biosensing
applications.
Amay Jairaj Bandodkar
Mr Amay Jairaj Bandodkar
completed his Integrated
Masters in Technology in
Industrial Chemistry (2011)
from the Institute of Tech-
nology, Banaras Hindu Univer-
sity, India. He developed a
penchant for the area of Bio-
sensors during his sophomoric
year and has been involved
in projects from NPL
(New Delhi), IIT-Bombay,
Fraunhofer IBMT (Potsdam,
Germany) and GE Healthcare
(India). Presently he is a
Doctoral student in the
Department of NanoEngineering, University of California,
San Diego (USA).
Vinay Gupta
Prof. Vinay Gupta received his
BSc, MSc, and PhD degrees
in physics 1987, 1989 and
1995, respectively from the
University of Delhi, India.
Presently he is Professor in
the Department of Physics
and Astrophysics, University
of Delhi. He is a recipient of
BOYSCAST fellowship
(2003). He is a senior member
of IEEE and has more than
100 publications in reputable
international journals. His
current research interests are
in piezoelectric thin films for
sensor applications, oxide thin films for biosensing applications,
ferroelectric films for electro-optic applications, oxide nano-
structures for multifunctional applications.
Bansi Dhar Malhotra
Dr B. D. Malhotra received
his PhD degree in physics
from University of Delhi,
Delhi, India, in 1980. He has
published 212 papers, filed 9
patents and edited/co-edited
books on biosensors and polymer
electronics. He has recently
moved to the Delhi Technologi-
cal University, India, after his
stint as Scientist G and Head of
the DST Centre on Bio-
molecular Electronics at the
National Physical Laboratory,
New Delhi, India. He has
research experience of about
25 years in the field of biomolecular electronics and has guided
18 PhD students till date. His current activities include bio-
sensors, nanobiomaterials, conducting polymers, Langmuir–Blodgett
films and self-assembled monolayers etc.
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sector is diagnosis of the clinically relevant analytes. A little
delay in accurate diagnosis of a particular disease may in some
cases be fatal. The current conventional techniques necessitate
the sample to be sent to a laboratory for ‘‘off-site’’ analysis
and testing. Moreover, these techniques are expensive, time-
consuming, and require expertise of trained personnel. The
recent trends in clinical diagnostics have led to the develop-
ment of high performance biosensors as innovative analytical
tools that can provide fast, reliable, and sensitive measure-
ments with lower cost and can be used with minimal training.
9
Fabrication of a typical biosensor requires integration of
biorecognition elements with a support matrix that in turn is
coupled to a transducer that converts a biochemical reaction
into a physically measurable signal (Fig. 1). Biosensors can be
broadly categorized into enzymatic and affinity biosensors.
Enzyme based biosensors utilize enzymes as biorecognition
elements to detect the desired bioanalytes. An enzymatic reac-
tion transforms an analyte into a reaction product dectectable
by electrochemical/optical/piezoelectric/magnetic transducers.
On the other hand, affinity biosensors make use of specific
capability of a biorecognition element to bind with a desired
analyte resulting in the formation of a complex. This binding
event is recorded by a suitable transducer. Affinity biosensors
include immunosensors, DNA sensors, aptasensors, peptide
nucleic acid sensors, whole cell biosensors and molecularly
imprinted polymer based biosensors.
A crucial step involved in developing a biosensor is the
immobilization of desired biomolecules at a sensor surface.
Biomolecules are highly susceptible to their environment and
are quickly denatured by changes in their surrounding condi-
tions. To fabricate a sensitive, selective biosensor with a long
shelf-life, it is essential for the biomolecules to maintain their
bioactivities for a long time. For this purpose, proper orienta-
tion of the biomolecules with negligible conformational changes
plays an important role. The direct physisorption of biomolecules
onto hard sensor surfaces may lead to conformational changes
and may result in decreased bioactivities with time. Thus, modifi-
cation of the sensor surface with an appropriate matrix is essential
since it may provide an environment that is conducive to the
immobilized biomolecules. In this context, self-assembly has
proved to be an excellent choice. The highly dense molecularly
ordered architectures in the form of LB films, SAMs or layer-
by-layer (LBL) films can be used to bio-functionalize the surface
for development of highly specific and sensitive sensors. Due
to almost complete monolayer obtained by these techniques,
loading of the biomolecules is quite high (useful for developing
ultrasensitive biosensors). Additionally, by tuning the proper-
ties of the self-assembling species, one can obtain novel inter-
faces for biosensing purposes as demonstrated by Nagel et al.
10
Thus, careful tailoring of sensor surfaces by LB/SAM/LBL
offers promising possibilities to the growing field of biosensors.
1.2. Importance of monolayers in immunosensing and
DNA sensing
An immunosensor can be used to detect a desired analyte
(antigen) based on the antibody–antigen binding that results in
the formation of an immuno-complex. Immunosensor fabri-
cation mostly involves use of the immunoglobulin G (IgG)
antibody due to its high affinity and specificity towards its
antigen (other immunoglobulins like IgM, IgE, IgA and IgD
can also be used). By using LB films and SAMs, researchers
have developed a whole new array of novel ultrasensitive
immunosensors. For example, immunosensors can be fabri-
cated by using LB films of IgGs, composite LB films of IgGs
and amphiphiles or by immobilizing IgGs on LB films of different
materials (e.g. fatty acids, polymers, nanoparticles etc.). Similarly,
SAMs on solid surfaces have been extensively investigated
for developing a wide range of analyte detection protocols like
sandwich, competitive assays etc. The unification of nano-
materials and self-assembly processes have empowered researchers
to develop highly sensitive and robust immunosensors.
In case of immunosensors, the biological event is a mere
adsorption of antigens onto the immobilized antibodies via
hydrogen-bonding, van der Waals forces and electrostatic
interactions. The complex samples contain a large number of
Fig. 1 Schematic of a typical biosensor.
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non-specific proteins.
11
Since proteins have mass and charge,
the non-specific adsorption of such proteins is difficult to
distinguish from the binding of an antigen to the immuno-
sensor surface. This makes immunosensors a weak contender
when it comes to precise and selective sensing in a complex
sample.
To overcome the shortcomings of the immunosensors,
researchers have come up with the use of DNA sensors. The
main principle of a DNA sensor is that the single stranded
DNA (ssDNA) is initially in a coiled state. However, on
hybridization with its complementary or target DNA (tDNA)
a rigid, well-structured double helix, called double stranded
DNA (dsDNA), is formed. The specificity of ssDNA probe to
its tDNA is quite high, and can be used to distinguish tDNA
from even those ssDNAs having a single mismatch. Similar to
the immunosensors, a wide variety of DNA sensing devices
can be fabricated using LB films, SAMs and LBL assemblies.
Of all the self-assembling systems at the liquid–solid interface,
the process of thiol self-assembly on an Au surface to form a
dense SAM has been of great help to researchers. By attaching
a thiol linker to one end of ssDNA, several research groups
have directly tethered ssDNA probes onto Au surfaces. Conju-
gation of nanomaterials and self-assembly has further led to
the development of exciting new DNA sensors.
In spite of continued competition between immuno and DNA
sensors, both are being widely used for detection of desired
analytes.
1.3. Scope and organization of the review
This review provides a critical appraisal of the ordered mole-
cular architectures formed by self-organization of molecules in
the form of SAMs and LB films along with their application in
the field of affinity biosensors. The first part preludes detailed
analysis of the LB technique, including Langmuir monolayer
formation, deposition and the factors affecting it, followed by
manipulation of these ordered architectures for incorporation
of biomolecules for development of affinity biosensors. The
succeeding part of the review is devoted to an astute study on
the mechanism of SAM formation and the parameters affect-
ing the process. We also discuss the various protocols using
SAMs for developing novel affinity sensors.
In order to compose a succinct and focused review article,
the most widely studied SAM viz., organosulfur SAM on an
Au surface has been discussed. The applications sections of the
two self-assembling techniques (LB and organosulfur SAM
onto Au surface) include detailed discussion on immuno-
sensors and DNA sensors with a brief discussion on PNA
and aptamer based biosensors. Furthermore, with a view to
provide a complete picture of the various self-assembling
techniques used in affinity biosensing, a brief discussion on
fundamentals and the applications of the LBL assembly
technique to DNA and immunosensing is given at the end of
this article. Finally, future prospects pertaining to these orga-
nized molecular assemblies for affinity biosensing have been
discussed.
2. Langmuir monolayer and Langmuir–Blodgett
(LB) films
The assembly of molecules at the gas–liquid (generally air–water
(A–W)) interface in the form of a monomolecular layer is known
as the Langmuir monolayer (LM). The transfer of this floating
LM on a solid surface is known as the LB technique.
The LB film deposition technique provides several ways of
organizing molecules in 2-D arrays that have been investigated
expansively over the past nine decades. A methodical study on
a floating monolayer on the water surface was first performed
by Irving Langmuir in the late 1910s and early 1920s.
12
Langmuir later reported transfer of the fatty acid molecules
from water surfaces onto solid supports. The detailed descrip-
tion of sequential monolayer transfer onto solid substrates was
later given by Katherine Blodgett.
13,14
Hence these mono-
molecular assemblies are referred to as LB films with deference
to these researchers for their pioneering contributions.
2.1. Molecules at the gas–liquid interface
Some organic molecules (having amphiphilic character (Fig. 2);
consisting of long hydrophobic chains with hydrophilic head
groups) can organize themselves at a gas–liquid interface in
order to minimize their free energy.
14
These amphiphilic mole-
cules get trapped at the interface due to the exact balance
between the two opposing forces, viz., hydrophobic–hydrophobic
interactions between the hydrocarbon chains and interaction
between the hydrophilic part (of amphiphiles) and the liquid.
Thus if the hydrophobic part is too short, the molecules sink into
the subphase as the latter interactive force subjugates the former.
Fatty acids and phospholipids are the most common examples of
amphiphiles. If the amphiphilic molecules are electrically neutral,
Fig. 2 Structure of amphiphilic stearic acid (SA) and octadecylamine (ODA).
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then the forces between them are short-ranged and the surface
layer will have a thickness of one or two molecular diameters.
However, if the molecules are charged then the associated
coulombic forces may result in multilayer formation.
2.2. LM preparation and its characteristics at the A–W
interface
To prepare an LM, a dilute solution of an amphiphile in a
volatile organic solvent (the spreading solvent) is prepared
and is gradually spread over a surface of an immiscible liquid
(usually water), known as the subphase (Fig. 3). The immisci-
bility of the two media leads to a well-defined bilayer of
solvents. Due to their amphiphilic nature, the hydrophobic
parts of the molecules remain in the spreading solvent, while
the hydrophilic head-groups enter the subphase, causing their
assembly at the organic solvent–water interface. Only dilute
solutions of amphiphiles must be used when highly ordered
LMs are desired. Higher concentrations may lead to unordered
multilayers at the interface. The volatile organic solvent evapo-
rates leaving the molecules randomly oriented at the A–W
interface. The randomly oriented amphiphiles are then com-
pressed with a surface barrier to form a floating two-dimensional
solid monolayer known as LM. This LM formation is achieved
at a particular surface pressure.
Introduction of a surface active material (like an amphiphile)
at the surface of liquid results in decreased surface tension leading
to expansion of the interface. Hence, presence of a LM on a liquid
surface affects the surface tension of a pure liquid. This pheno-
menon can be used to study the characteristics of LM by monitor-
ing the surface pressure (P) on compressing the monolayer as given
by eqn (1).
P=g
o
g(1)
where g
o
is surface tension of the pure liquid and gis surface
tension of the film-covered surface.
When a solution containing the amphiphilic molecules is
first spread on the subphase, the molecules are initially far apart
and randomly oriented. In this state, no external pressure is
applied to the monolayer and the molecules behave as a 2D gas
(shown by point ‘‘A’’ in Fig. 4), that can be described by eqn (2).
Pa=kT (2)
where ‘‘a’’ is the molecular area, ‘‘k’’ is the Boltzmann con-
stant and ‘‘T’’ is the temperature. The 2D gas phase passes
through different ordered phases on compression of the
monolayer with the help of a barrier. The phase change of a
monolayer may be identified by monitoring a ‘‘Pvs a’’ plot.
(Fig. 4) The shape of an isotherm or phases of a monolayer
depends on the physical and chemical properties of the mono-
layer material, the subphase temperature and composition.
As the gaseous monolayer (point ‘‘A’’ Fig. 4) is compressed,
an expanded liquid-like state appears (point ‘‘B’’ Fig. 4) due to
increased interaction of hydrophobic chains of the amphi-
philes. This region is accompanied by a constant pressure
region in which transition of the monolayer occurs to complete
2D liquid phase (point ‘‘C’’ Fig. 4). In the region ‘‘B–C’’, the
monolayer comprises of a mixture of two phases (liquid +
gas). The region ‘‘C–D’’ shows compression of the 2D liquid
phase. Compression of the monolayer provides additional
ordering and close-packing of the molecules to form a con-
densed liquid phase (point ‘‘D’’ Fig. 4). On further reduction
of ‘‘a’’, the monolayer compresses at constant ‘‘P’’ until it
reaches point ‘‘E’’ where it transforms into a 2D solid phase.
Continuing the compression leads to sudden increase in ‘‘P’’
(region ‘‘E–F’’). This region of isotherm is characterized by a
steep slope which usually exhibits a linear relationship between
‘‘P’’ and ‘‘a’’. At point ‘‘F’’, ‘‘P’’ is the highest and the
amphiphiles are as closely packed as possible. This point
represents a stable, complete and homogeneous 2D monolayer
popularly known as LM. Horizontal regions of constant ‘‘P’’
(B–C and D–E) are characteristic of first order thermodynamic
transition. However, isotherms of many long chain organic com-
pounds do not exhibit such regions indicating existence of higher
order transitions. Impurities can also be a reason for deviation
from the ideal case.
15
Beyond point ‘‘F’’ it is not possible to further increase ‘‘P’’
and area of the film decreases if the pressure is kept constant,
or ‘‘P’’ falls if the film is held at constant area. This is referred
to as ‘‘collapse’’. The pressure exerted on the molecules at this
point becomes too strong for confining them at the A–W
interface, and the molecules are ejected out of the monolayer
plane into either the subphase or on the air side (Fig. 5a and b).
At collapse pressure the film may, reversibly or irreversibly, lose
its monolayer form. The collapse pressure can be defined as the
maximum pressure to which a LM can be compressed without
detectable expulsion of the molecules from it. The onset of
collapse depends upon the nature of amphiphiles, temperature
and the rate at which the LM is compressed.
At the stage of collapse, destabilization of the 2D structure
of floating LM occurs, leading to formation of different 3D
Fig. 3 LM formation at A–W interface in a LB Trough.
Fig. 4 Pressure–area isotherm of a long chain amphiphile.
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structures depending on properties of the monolayer material.
Rigid LMs exhibit high collapse pressures while relatively
flexible LMs show low collapse pressures. In general, rigid
LMs fracture to form multilayered (bi-layer or tri-layer)
aggregates at the air side of the interface (Fig. 5a).
16,17
Con-
versely, flexible LMs exhibit buckling of the monolayer leading
to formation of bi-layer folds followed by vesicle formation into
the subphase (Fig. 5b).
18
In case of mixed LM of two amphi-
philes with vast difference in collapse pressures, the amphiphile
with low collapse pressure squeezes out of the LM (which
finally contains only the high collapse pressure amphiphiles)
into the subphase and forms vesicles. In this case the collapse
is said to be irreversible i.e. the aggregates cannot be re-spread
in the form of LM upon decrease of the compression pressure or
monolayer surface density. However, addition of a surfactant
protein into the mixed LM can avoid detachment of the vesicle
from the LM and may make the buckling collapse reversible.
16
2.3. LB deposition
Prior to the LB film formation, a stable LM of an amphiphile
is prepared
12,13
and later transferred onto a solid substrate to
form an LB film by vertically dipping it through the inter-
face at controlled ‘‘P’’ (Fig. 6). Further layers are deposited
on each subsequent pass of the substrate through the A–W
interface. Multilayers can, therefore, be deposited by upward
and downward strokes to produce a film, the thickness of which
is a product of the individual molecular chain length and the
number of strokes through the A–W interface. A LM can also
be transferred by horizontal lifting method known as the
Langmuir–Schaefer method.
Fig. 5 (a) Formation of bi-layer and tri-layer structures in a LM upon increasing lateral pressure (b) Formation of buckle, bi-layer fold and
vesicle in a lipid LM upon increasing lateral pressure.
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The arrangement of LMs on a substrate depends on its
wetting property and the attractive forces between the amphi-
philes. If the substrate is hydrophilic, deposition takes place
during the upstroke. On the other hand, for hydrophobic
surface the monolayer is always deposited on the downward
path. Three types of deposition modes e.g. Y-type, X-type and
Z-type can be obtained depending on the interactive forces e.g.
amphiphile–amphiphile and amphiphile–substrate (Fig. 7). In
the Y-type films, interactions between the successive layers are
head-to-head and tail-to-tail, these films thus have alternate
hydrophobic/hydrophilic regions. However, in Z or X-type
deposition, the monolayer deposits only in the up or down
direction. To date, a cogent reason for obtaining X-type and
Z-type LB films has not been found. Mixed deposition modes
may also be obtained for some materials.
Composite LB films containing more than one type of amphi-
phile can also be built by alternate layer deposition (Fig. 8a) and
by preparing mixed LM of two or more compounds (Fig. 8b).
The latter approach has been widely used for preparing LB films
of polymers and other organic/inorganic materials that are of
great importance in the field of biomolecular electronics.
2.3.1. Important factors governing LB deposition
a. Transfer ratio. This parameter decides the efficiency
of transferring a LM onto a solid substrate and the quality
Fig. 6 Vertical dipping of a solid substrate for LM transfer.
Fig. 7 Different types of LB deposition.
Fig. 8 (a) Compsite LB of different amphiphiles obtained by alter-
nate LM deposition. (b) Composite LM of different amphiphiles.
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of deposition. Transfer ratio, ‘‘t’’ is the decrease in the area of
LM (held at constant pressure) on the subphase (A
L
) divided
by the coated area of the substrate (A
S
),
19
i.e.
t¼AL
AS
ð3Þ
‘‘t’’ is measured for each pass of the substrate through the
A–W interface. Ideally tB1 suggests 100% LM transfer. ‘‘t’’
depends on the type of substrate used and its affinity towards
the amphiphiles. It also varies with ‘‘P’’ and speed of transfer.
Usually high deposition speed results in decreased ‘‘t’’.
19
Also,
too low ‘‘P’’ leads to poor values of ‘‘t’’ as in this case molecules
cannot be packed closely enough to form a good condensed
LM.
20
It is thus an excellent parameter to evaluate surface
coverage of amphiphilic species. However, in case of non-
amphiphilic molecules, ‘‘t’’ value cannot be completely trusted
as observed by Rubinger et al..
21
b. Subphase. Although a variety of liquids (water, mercury,
hydrocarbons, glycerol) can be used as subphase for the LB
technique, the majority of the work reported in literature has
been conducted using aqueous subphases. The quality of water
used for LB film formation is of utmost importance. As only a
few micrograms of amphiphile is spread on a large volume of
subphase (usually in litres), even a minute impurity can be a
problem for LB deposition. It is therefore, a good practice to
use highly pure water with specific resistance 418 MOcm.
The presence of large surfactants has minor effect on resistivity
of the subphase. Hence a shake test must be performed to
check for any amphiphilic impurities. In this test, bubbles are
generated in the subphase bulk by shaking. If these bubbles
instantly break when they reach the surface, the subphase
contains no amphiphilic impurities. Otherwise, the subphase
must be further purified for removing such impurities.
c. Subphase temperature and pH. Temperature and pH of
the subphase largely affect organization of the amphiphiles at
the A–W interface. Dhanabalan et al.
20
have studied pressure–
area isotherms of polyaniline (PANI) under different subphase
conditions such as temperature, pH, and salt concentration.
A shift in the pressure–area isotherm for PANI LM is obtained
toward higher mean molecular area on lowering the subphase
(pH 4.0) temperature from 25 to 10 1C, indicating lower com-
pressibility of LM at lower temperature. This may be attributed
to the fact that the polymer chains are comparatively more rigid
at lower temperature, thereby imposing restriction on close
packing leading to increased mean molecular area. However,
at a given subphase pH, the increase of subphase temperature
decreases rigidity of the polymer chains. As a result, the polymer
chains are packed closely as indicated by a lower mean molecular
area. Besides this, on lowering the subphase pH, the isotherm
is found to shift toward a higher mean molecular area and a
decrease in overall compressibility is observed.
d. Dipping and compression speed. As LM is transferred
onto a substrate, the water gradually drains out of the trans-
ferred monolayer. This drainage occurs due to adhesion that
acts along the line of contact between the monolayer molecules
being transferred and the monolayer already on the substrate,
and drives out the water. The rate at which a film can be built-up
is limited by the rate at which the ascending monolayer sheds
water. Thus the substrate should not be raised faster than the rate
at which water drains out. To improve the film quality it is often
appropriate to temporarily halt the dipping process after an
upstroke and wait until the transferred monolayer is completely
dry before the downward movement. The transfer of the first
monolayer should be done at relatively lower dipping speed
(mms
1
to mm s
1
). The best results are achieved for deposi-
tion made at dipping speeds (o1 mm min
1
). Nevertheless,
3–5 mm min
1
dipping speed has been found to yield good
quality LB films of polypyrrole (PPY) and PPY–cadmium
stearate
22
and PANI–gold nanoparticles (AuNPs).
23
In some
cases much higher speeds (30 mm min
1
) also produce excellent
films.
19
The compression speed of the barrier also affects the quality
of a LB film as it controls molecular packing of the monolayer
molecules at the interface. The effect of compression speed is
more prominent for complex molecules than for simple ones as
observed by Rubinger et al.
21
The results obtained demon-
strate better molecular packing of polymer molecules at lower
compression speeds. The collapse pressure is found to decrease
by reducing the compression speed. This behavior is expected
due to higher rigidity of the polymer layers when compared to
simple amphiphilic molecules.
3. LB films of different materials
Amphiphilic materials (Fig. 2) such as fatty acids (e.g. stearic
acid (SA), behenic acid (BA), arachidic acid, linoleic acid etc.),
amines (octadecylamine (ODA)), alcohols and phospholipids
have been widely used for preparing stable LB films.
24–29
Longer chain fatty acids form more closely packed LMs
as compared to their shorter counterparts.
28
Furthermore,
unsaturated fatty acids are less closely packed than saturated
fatty acids of the same carbon chain length.
28
The incorpora-
tion of divalent cations into the subphase can augment the
stability of the fatty acid LM since these divalent ions reduce
repulsion between the adjacent ionized groups and make the
monolayer more stable (Fig. 9). Similarly, addition of negatively
charged ions in the subphase of long chain amine LM provides
stability to the monolayer. Although LB films of fatty acids have
excellent monolayer forming qualities, these materials possess
low melting points and have poor mechanical properties and
hence are not the best choice for device fabrication.
Phospholipids are naturally occurring amphiphiles having
one head and two tails. The heads are charged and are attracted
to water, and the tails (not necessarily of the same length) are
non-polar and repel water. The hydrophilic head contains the
negatively charged phosphate group, and may contain other
polar groups. The hydrophobic tail usually consists of long
Fig. 9 Interaction of divalent cations (Cd
2+
) with monolayer of fatty
acid at A–W interface.
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fatty acid hydrocarbon chains. Depending on their specific
properties, phospholipids form a variety of structures at the
A–W interface. Dipalmitoyl phosphatidic acid (DPPA)
29
and
dipalmitoyl phosphatidyl glycerol (DPPG)
30
are the most
common examples of phospholipids (Fig. 10). The multilayer
Y-type films of these phospholipids may be built-up from
either a pure water subphase or the one containing divalent
cations (Ca
2+
,Cd
2+
,Mn
2+
etc.). Phospholipids may also be
‘‘zwitterionic’’ (i.e. containing both positive and negative charges)
e.g. phosphatidylethanolamine,
31
phosphatidylcholine
32,33
etc.
The LMs of phospholipids mimic the living cell membranes
and may provide an excellent method for development of bio-
mimetic membranes. However, the LB films of these amphi-
philes suffer from many defects that may perhaps limit their
use in device fabrication.
34
The preparation and applications of LB
films of these materials have been discussed in the literature.
34,35
Some of the other difficulties faced by the phospholipid LB
films relate to the stronger affinity of their head groups with
the water subphase than for the hydrophilic substrate, that
may lead to slipping of the monolayer from the substrate.
36
Thus the forces applied during the transfer process are not
sufficient to pull up the monolayer from the water surface
resulting in transfer of up to a few monolayers (B5). Only a
few phospholipids can form LB multilayers of more than five
layers. This predicament may be circumvented by using mixed
LB films. In this context, Girard-Egrot et al. have achieved
transfer of [DPPA–dipalmitoylphosphatidylcholine)] mixed
monolayers (molar ratio 2: 1) up to 21 layers from a pure water
subphase onto different hydrophilic substrates.
37
In another
study, Lee et al.
29
have deposited 21 composite layers of DPPA
and dipalmitoylphosphatidylcholine using a subphase contain-
ing Ca
2+
ions.
Keeping in view the current technological needs, there is a
considerable demand for LB films of materials like polymers,
nanoparticles (both metals and semiconductors) etc. The
following sections deal with the LB film fabrication of these
important materials.
3.1. Protocols for preparing LB films of organic polymers
There are three most commonly used procedures for preparing
LB films of polymers as discussed below.
3.1.1. Polymerization of monomer LM or monomer LB film.
This method utilizes either (i) preparation of LB film of mono-
mers followed by polymerization using UV radiation
38–40
or
(ii) polymerization of monomer at the A–W interface follo-
wed by its transfer onto the solid substrate.
41
The former
approach has been extensively used to prepare polydiacetylene
LB films. These thin films have been prepared by deposit-
ing amphiphilic diacetylenes, having general formula C
m
H
2m+1
–CRC–CRC–C–(CH
2
)
n
–COOH, onto solid sub-
strates by the LB technique followed by polymerization using
UV radiation
38
and a high-pressure Hg lamp.
39
On the other
hand, Day and Lando’s pioneering work reports protocols to
polymerize amphiphilic diacetylene LM for preparing poly-
diacetylene at the A–W interface and its subsequent transfer
on a suitable substrate.
42,43
Thin films of this material are tech-
nologically important as the color of this polymer is sensitive
to the external stimuli such as temperature, pH, and other
mechanical stresses due to changes in the effective conjugation
length of the polydiacetylene backbone.
44,45
Similarily PPY
films have been prepared by polymerization of the monomer
either at the A–W interface
41
or in the LB film structure.
46
3.1.2. LB films of polymers. Amphiphilicity is the most
essential factor for achieving a stable and uniform LM. LB
films of various amphiphilic polymers (long hydrophobic
chain with a hydrophilic side group) can be easily prepared.
47
Tredgold et al. have reported results of their studies on
preparation of LB films of different amphiphilic polymers
and copolymers.
48,49
LB manipulation of a large variety of
technologically important electroactive polymers need further
processing for obtaining stable LB films due to their poor
amphiphilicity and low solubility in organic solvents. These
drawbacks can be circumvented by either derivatizing the
polymer backbone with long alkyl chains
50,51
and hydrophilic
groups
52
or doping it with functionalized acids to enhance
processability.
53
Both these approaches have been successfully
employed for preparing LB films of polythiophenes,
52
PANI,
54
PPY
55
etc.
Among the many different conducting polymers, PANI LB
films have been most studied as PANI is environmentally
stable, easy to prepare and has a broad range of tunable
properties.
56
Different strategies have been employed to make
PANI processable for LB formation. The emeraldine-base
form of PANI is made processable by dissolving it in either
N-methyl-pyrrolidone
57
or in N-methyl-pyrrolidone–chloroform
solution.
58
However, LB films processed with N-methyl-
pyrrolidone generally give rise to an irregular surface after a
certain number of layers due to non-volatility and miscibility
Fig. 10 Structure of dipalmitoyl phosphatidyl glycerol (DPPG) phospholipid.
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of N-methyl-pyrrolidone in water. Another approach to make
PANI processable in organic solvents without significant loss
of its main characteristics is to substitute alkyl or alkoxy
groups either in monomer or in the polymeric chains.
59,60
Substituted PANI like poly(o-anisidine) and poly(ethoxy-
aniline) are found to be soluble in chloroform. LB film of
poly(o-anisidine) has excellent quality and conductivity
(0.1 S cm
1
) compared to that of the PANI.
61
Further it has
been shown that its conductivity can be enhanced by doping.
Efforts by Ram et al. have led to a better understanding of
the effect of doping on the properties of poly (o-anisidine)
Langmuir films.
62
Although these studies have suggested that
association of the counter ion (Cl
) with the poly (o-anisidine)
backbone distorts the microstructure of the polymer to accom-
modate the extra mass, the presence of counter ions results in
improved stability of the LM.
The dissolution of parent PANI in organic solvent can be
accomplished by doping with functionalized acids.
63,64
Riul
et al. have fabricated LB films of PANI by dissolving it in ten
different combinations of chloroform solutions using camphor
sulfonic acid, dodecyl benzene sulfonic acid, and toluene sulfonic
acid as dopant and m-cresol as processing agent.
65
The authors
suggest that m-cresol leads to conformation change in the PANI
chains from their coil-like structure to rod-like structure (con-
sidered as a memory effect of m-cresol) and improves crystallinity
of the LB film. In a recent study, Aoki et al.
66
have recently
discussed the advantages of dodecyl benzene sulfonic acid and
have reported that ion complex of PANI with dodecyl benzene
sulfonic acid enables PANI not only to be soluble in organic
solvents but also makes it amphiphilic.
3.1.3. Composite LB films of polymers and amphiphiles.
The excellent spreading properties and good alignment of
amphiphiles like fatty acids at the A–W interface have led to
the idea of fabrication of fatty acid assisted conducting polymer
LB films. In the composite LB film of conducting polymers and
amphiphiles the latter provides a better molecular organization
to the desired polymer molecules and supports its deposition on
a solid substrate.
54
To form a composite or a mixed monolayer,
a suitable proportion of polymer and amphiphile is dissolved in
an organic solvent and spread over the subphase. The stable
and well-aligned composite LM can then be transferred onto a
solid substrate. Several researchers have explored composite LB
films comprising of PANI and fatty acids.
19,20,67
A crucial point
relating to the preparation of such a film is the weight fraction
of the polymer that should be dissolved in the organic solvent.
Some researchers have experimented on different weight frac-
tions of the PANI oligomers in the spreading solution for
suitable composite LB deposition and have found that the
monolayer with a 30–55% weight fraction of PANI is required
for stable film preparation.
19,68
If a higher PANI amount is used,
the PANI molecules are most likely to be squeezed out of the
air–liquid interface
68
leading to increased surface roughness and
lower conductivity of the composite film.
69
The composite LB
films have also been successfully prepared using other important
conducting polymers like polythiophenes,
70
PPY
22
etc. Apart
from fatty acids, researchers have employed suitable amphiphilic
polymers with an excellent capability to form condensed LM for
preparing composite LB films with conducting polymers.
71
3.2. Protocols for preparing LB films of nanomaterials
An important topical problem in nanoscience is the organiza-
tion of nanoparticles as a thin film. One of the routes being
intensely pursued uses colloidal chemistry for synthesis of the
nanoparticles followed by their arrangement in thin films by
methods such as self-assembly at liquid–solid interface and
organization at the A–W interface to prepare LB thin films.
The LB technique has an edge over self-assembly as the former
allows full control of arrangement of the nanoparticles in a
monolayer as well as the number of monolayers transferred
onto a solid substrate. Typically there are three ways in which
Langmuir films of nano-materials can be prepared: (a) by
utilizing electrostatic complexation between nanoparticles in
the aqueous subphase and an amphiphile template monolayer
at the interface (Fig. 11a);
72,73
(b) by direct spreading of
surfactant capped or hydrophobized nanoparticles at the
A–W interface (Fig. 11b);
74,75
and (c) spontaneous reduction
of a precursor at the A–W interface to form LM of hydro-
phobic nanoparticles.
76
3.2.1. Electrostatic complexation at the A–W interface.
This method generally comprises of spontaneous immo-
bilization of negatively charged colloidal nanoparticles present
in the subphase onto a positively charged template LM
(Fig. 11a).
72,73
The strong attractive electrostatic interaction
drives the complexation process. LM of ODA is commonly
employed as a template for this purpose.
77,78
Immobilization
of nanoparticles causes increased rigidity of the LM with
increase in the nanoparticles density at the interface
79
along
with increase in the area/molecule.
80
The complexation pro-
cess is found to be time-dependent and can be monitored
in situ by studying pressure–time isotherms (Fig. 11a).
79
Since
complexation is a function of the charge present on the
amphiphiles and nanoparticles, the intensity of electrostatic
attraction between these species can be modulated by varying
the pH of the subphase. For example, in case of carboxy-
functionalized silver or AuNPs, a subphase pH of B9 is found
to be appropriate since both the amine and carboxylic acid
groups are fully charged at this pH, thereby leading to maximum
electrostatic interaction between nanoparticles and the ODA
Langmuir film.
80
Several other LMs based on octadecanethiol,
81
PANI
23
etc.
have been used as templates for preparing nanoparticles based
LB films.
3.2.2. LM of hydrophobic nanoparticles. The second approach
of fabricating a LM of nanoparticles is to spread the hydro-
phobically stabilized nanoparticles at the A–W interface
75
(Fig. 11b). In this method, hydrophobized nanoparticles are
dissolved in an organic solvent and then spread onto aqueous
medium to form LM of these particles. There are essentially
two ways of preparing hydrophobized nanoparticles: (i) synthesis
of hydrophobic ligand capped nanoparticles;
82
and (ii) synthesis
of hydrophilic ligand capped nanoparticles followed by hydro-
phobizing them by assembling amphiphiles over the hydrophilic
nanoparticles using electrostatic interaction between the hydro-
philic group present on the nanoparticles and that of the
amphiphile.
83,84
The LB films of a number of nanoparticles
can be prepared using this technique as shown in Table 1.
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3.2.3. Reduction of precursor at the A–W interface. The
reduction of precursors of the nanoparticles in the subphase
by the template LM followed by their LB deposition is an
excellent route to obtain highly organized nanoparticles assem-
blies. Swami et al. have demonstrated the utility of this method
by spontaneously reducing AuCl
4
ions present in the subphase
(at low pH) by 4-hexadecylaniline LM.
76
The 4-hexadecyl-
aniline reduces AuCl
4
ions and simultaneously forms a complex
at the interface thus forming the LM of nanoparticles. These
authors have observed that the AuNPs assemble in a ribbon-like
pattern at the LM. Later these authors prepared silver nano-
particle LB films by reducing silver ions (present in the subphase
at high pH) with 3-pentadecylphenol LM.
91
Table 2 contains
details of several techniques used for characterizing the LM and
LB films.
Table 1 Various nanoparticles with capping agents used for LB preparation
Nanoparticle Capping agent (ligand) Ref.
Pt Poly(vinylpyrrolidone) 82
Pt 4-Mercaptoaniline hydrophobized by 2-thiophenecarbonyl chloride 85
Au 4-CTP hydrophobized by ODA 83, 84
Fe
3
O
4
Oleic acid stabilized 86
Au Laurylamine 72
Au (nanocage) Poly(vinyl) pyrrolidone 87
Silver ODA 88
Silver nanocubes Poly(vinyl) pyrrolidone 87, 89
Silica — 90
Fig. 11 (a) Preparation of AuNPs LB film by electrostatic interaction. (b) Hydrophobized AuNPs LM at the A–W interface.
Table 2 Various techniques used for characterizing LM and LB films
Different techniques to characterize LM Ref.
Pressure–area isotherm 90
Brewster angle microscopy (BAM) 19
Transmission electron microscopy (TEM) 91
Different techniques to characterize LB film
UV–visible spectroscopy 90
Quartz crystal microbalance (QCM) 84
Cyclic voltammetry 23, 67
Scanning electron microscopy 67, 90
X-Ray photoelectron spectroscopy (XPS) 85
Surface-enhanced Raman scattering (SERS) 89
FT-IR 67
Atomicforce microscopy (AFM) 23
X-Ray diffraction (XRD) 92
Fluorescence spectroscopy 51
Ellipsometry 93
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4. LB film based affinity biosensors
Several strategies and materials have been used for developing
LB based immunosensors and DNA sensors. Some of the
extensively used protocols are discussed below.
4.1. LB film based immunosensors
4.1.1. Antibody LB film based immunosensors. The formation
of LM of only IgGs is difficult as they do not have marked
amphiphilic properties and most of them are soluble in water.
Thus only a fraction of the biomolecules remains at the A–W
interface in dynamic equilibrium with those present in the
bulk of the subphase.
94
Still many attempts have been made
to prepare stable LM of antibodies by adjusting pH of the
subphase to the isoelectric point of these proteins as at this pH
the IgGs have no net charge and are hence less soluble in water.
95
The antibody LMs transferred onto different substrates by this
method lead to LB film in which adhesion of the antibody
molecules is poor. To overcome this problem, researchers have
utilized functionalized substrates for covalent binding of the
transferred antibody molecules.
96
LB films of antibodies
covalently bound to salinized surfaces have been employed to
fabricate immunosensors for detecting IgG,
97,98
hemoproteins,
99
Salmonella typhimurium
100
etc. using fluorescence spectroscopy,
spectrophotometry, magnetoelastic resonance etc. Some authors
have also modified glass substrates by functionalized polymers
for covalent binding of the antibodies on the LB film.
98
4.1.2. Antibody–amphiphile composite LB film based immuno-
sensors. These composite LMs are generally prepared by adsorp-
tion of antibody molecules onto a template LM under specific
conditions of subphase electrolyte concentration. The protocol
for fabrication of these sensors involves dissolving antibodies
in the subphase and preparing an LM of a suitable amphiphile.
Due to charge of the hydrophilic head groups of amphiphiles,
antibodies are electrostatically adsorbed onto the LM. Sub-
sequently the antibody-amphiphile LM can be transferred onto
the sensor surface and used for analyte detection.
101
Covalent
binding of the antibody fragments (Fab) (present in the sub-
phase) to the template LM has also been utilized to fabricate
composite LB film based immunosensors. This is done by
preparing LMs of lipids such as (N-(e-male-imidocaproyl)-
dipalmitoylphosphatidylethanolamine and N-[3-(2-pyridyldithio)-
propionyl]dipalmitoylphosphatidylethanolamine) that can bind
with free thiols of the Fab.
102
The antibodies can also be tagged
with lipids to make them amphiphilic in nature. These con-
jugated IgGs can then be directly used to prepare LB films for
immunosensing.
103
The pH of subphase plays a key role for electrostatic com-
plexation of an antibody with the template LM. For example,
BA has pK
a
of around 5.4, thus it is nearly neutrally charged
at pH 5.5.
104
When pH of the subphase is close to pK
a
value of
BA, the strong ion–dipole interaction among the carboxyl
groups of BA molecules decreases the intermolecular distance
at the A–W interface
105
making it more difficult for the large IgGs
to be entrapped into the BA LM and thus no IgG is incorporated
into the LM. When the pH reaches 7.3, the ionic repulsion
between polar groups of BA molecules increases the inter-
molecular distance, and makes it easier for IgGs to be entrapped
into the BA LM as reported from the pressure–area isotherm.
4.1.3. Antibody tethered preformed LB film based immuno-
sensors. The analysis of LMs of IgG by using techniques like
circular dichroism and fluorescence measurements have shown
that IgGs are partially denatured in the LM
106
and partial
desorption of protein molecules into water solution frequently
occurs.
107
This has led to the idea of immobilization of anti-
body molecules on the preformed LB films that may provide
stability to the LB based immunosensors. Owaku et al. first
prepared LB film of protein A and then immobilized anti-
human IgG via the specific binding of F
c
part of IgGs to
protein A. The immunosensor thus developed has been used
to detect human IgG.
108
In another study, mixed LB films of
1,2-dipalmitoyl-sn-glycero-3-phosphoglycolipoate and 1-palmitoyl-
2-(16-(S-methyldithio)hexadecanoyl)-sn-glycero-3-phosphocholine
phospholipids have been prepared for covalent immobilization
of Fab fragments.
109
In a recent study, polyaniline–SA com-
posite LB films have been utilized for covalent immobilization
of anti-apolipoprotein B antibody for low density lipoprotein
(LDL) detection.
110
4.2. LB film based DNA sensors
LMs of amphiphiles have been commonly used as templates
for development of DNA-LMs. The formation of DNA-LMs
is based on electrostatic complexation of DNA molecules
(present in the subphase) with charged LM. The complexation
of ssDNA with the monolayer is time dependent and can be
in situ characterized by increased area/molecule in the isotherm.
The DNA-LB film is featured by bilayers of amphiphile with
lamellarly ordered DNAs adsorbed parallel to the dipping
direction between them.
111
The amphiphile LM could be
cationic,
112
anionic
113
or zwitterionic.
114,115
In case of cationic amphiphile,
112
the negatively charged DNA
can be directly adsorbed from the subphase to the positive
amphiphile LM without the need of additional agents. How-
ever, while using anionic amphiphiles such as fatty acids,
116,117
agents like ZnSO
4
need to be added in the subphase. Zn
++
ion
has a dual role, viz. (i) stabilization of LM of anionic amphi-
philes by charge interaction and (ii) affinity towards DNA.
118
Thus in case of anionic amphiphiles, DNA binds to LM via
Zn
++
ions. LMs of zwitterionic molecules can also be used as
templates for adsorption of DNA.
114,115
Since zwitterions have no
net electric charge, agents like bivalent cations e.g. Mg
2+
,Ca
2+
etc. are found to be essential for preparation of DNA-LMs.
There are three parameters that perhaps control the DNA–
Langmuir film structure, namely—(1) amphiphile–DNA inter-
action
112
(2) amphiphile–amphiphile interaction
119
and (3)
surface pressure.
120
Stronger amphiphile–DNA (in case of
ODA) interaction leads to splitting of dsDNA while weaker
forces, e.g. hexadecymdimethylammonium bromide
112
and
dioctadecyldimethylammonium
111
with DNA, maintain the native
dsDNA structure. The hydrophobic amphiphile–amphiphile
interaction also affects the overall structure of DNA assembly.
The gemini surfactants are excellent choice for such studies.
These studies have revealed that by varying the spacer length
of gemini surfactants a variety of 2D DNA-LB topographies
can be obtained.
119
The research has also shown that DNA-
LM structure can be modified by varying the surface pressure.
120
Another interesting method is to use LMs of a nucleo-
base amphiphile to recognize and assemble cDNA at the
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A–W interface.
121,122
The superiority of this technique lies in
the fact that the DNA assembly at the LM is dependent on the
specific Watson–Crick DNA base pairing instead of universal
non-specific electrostatic attraction.
Two possible methods can be used to fabricate DNA
sensors as discussed below.
4.2.1. ssDNA-amphiphile composite LB based DNA sensors.
The strategy of tethering ssDNA probes at the A–W interface
has been widely used for preparing DNA sensors. The modus
operandi involves preparing an LM of a cationic amphiphile
followed by adsorbing negatively ssDNA (dissolved in the
subphase along with an intercalator) to the LM. cDNA is then
introduced in the subphase that selectively hybridizes with the
2D self-assembled ssDNA. Subsequently an LB film of the
dsDNA is prepared on a substrate and a suitable transduction
technique is used to estimate the amount of cDNA.
123,124
4.2.2. ssDNA tethered preformed LB based DNA sensors.
In the technique discussed above, ssDNA is dissolved in the
subphase. Due to large volume of the subphase, higher amount
of ssDNA is required to achieve homogeneous coverage. This
leads to squandering of the expensive ssDNA. In order to
overcome this drawback researchers have immobilized ssDNA
on preformed LB films. With this protocol only the required
amount of ssDNA is used. The ssDNA can be adsorbed directly
onto the LB film with certain modification if required
125
or it
can be covalently attached to the LB film. Another way is to
first immobilize avidin to the LB film followed by adsorption of
biotinylated ssDNA probes.
126
The organization of molecules at the A–W interface using
LB technique is extremely promising for development of the
high performance affinity biosensors and may provide a pro-
mising template for fabrication of biochips. However, few
efforts have so far been made in this direction. There are some
reports on LB based DNA sensors and immunosensors as
discussed in the preceding sections. To the best of our know-
ledge very few efforts have been made to develop LB based
aptasensors
127
and PNA sensors. Therefore, attempts should be
made in this direction. Table 3 describes some of the recent
results obtained relating to the LB film based biosensing.
LB technique is a versatile method to obtain a variety of
molecular assemblies by varying factors such as distance,
orientation, and extent of interaction.
131
Though LB films are
being utilized for biosensing applications, yet from a funda-
mental point of view, they are thermodynamically unstable
and consequently, in some cases temperature changes or
exposure to harsh solvent may ruin their structure.
132,133
As
a result, there has recently been a steady shift from the LB
technique towards self-assembly methods at the liquid–solid
interface for fabrication of ordered monolayers for biosensing.
Two reasons for this shift are (i) greater robustness of self-
assembled materials relative to most LB films (due to the
presence of strong interactions between the material and
substrate molecules), and (ii) ease of preparation. In contrast
to the LB method, that requires a film balance and careful
control over surface pressure during dipping and transfer, self-
assembly at liquid–solid interface is carried out by simple
immersion of a suitable substrate into a solution containing
excess of self-assembling molecules. The bio-functionalization
of SAMs can be attained by attaching desired biomolecules
to an organic SAM via physisorption, cross-linking or by
modifying the terminal groups of the biomolecule for direct
self-assembly onto a substrate. Though, SAMs have several
advantages over LB films, there are many fields of technology
where LB films have higher applicability than SAMs. For
example, by LB technique, packing density of the amphiphiles
can be tailored to obtain desired thin films. Similarly, a com-
parison of the absolute electrical properties of the LB films and
the SAMs suggests that the LB deposition process (in which
molecular organization precedes chemisorption) produces
films that are more highly organized than those produced by
the self-assembly process, in which chemisorption precedes
molecular organization.
4
Thus, depending on these require-
ments, either of the two techniques can be adopted to form
highly organized molecular architectures.
5. SAM at liquid–solid interface
5.1. Thiol SAM on Au
As mentioned earlier (section 1) self-assembly is a process of
spontaneous organization of molecules without an external
Table 3 LB based affinity biosensors
Type of affinity
sensor LB matrix
Biorecognition
element
Method of biomolecule
incorporation Analyte Transducer Ref.
Immunosensor ODA/IgG,
BA/IgG
IgG Electrostatic interaction
at the subphase
Antigen Impedimetric 101
Immunosensor IgG Anti-human IgG Direct spreading on the
subphase
Human IgG — 98
Immunosensor Protein A Anti-human IgG Self-assembly on
protein A LB films
Human IgG Fluorescence
spectroscopy
108
Immunosensor Polyaniline/SA Anti-apolipoprotein B Covalent binding via
EDC-NHS
LDL Impedimetric 110
DNA sensor dsDNA/ODA
mixed LB
dsDNA Electrostatic interaction
at the subphase
methotrexate Square wave
voltammetry
128
DNA sensor PPY/SA/DNA ssDNA Electrochemical entrapment DNA hybridization Electrochemical 125
DNA sensor Zn-arachidate ssDNA Electrostatic interaction
at the subphase
DNA hybridization Fluorescence
spectroscopy
129
DNA sensor Escherichia coli
RNA polymerase
T7 DNA Electrostatic interaction
at the subphase
RNA–DNA
interaction
LB and SPR 130
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1376 Chem. Soc. Rev., 2012, 41, 1363–1402 This journal is cThe Royal Society of Chemistry 2012
driving force. Of the many self-assembly systems known to
date, a special system has caught the attention of several
researchers—SAMs of organosulfur compounds especially
thiols on an Au surface.
134,135
Easy preparation of a SAM
from the gas phase or from a solution, and its relatively high
stability mediated by the strength of the Au–S bond and by
van der Waals interactions
136
are quite rewarding attributes.
And, the availability of various head-groups in thiols that can
be further used for binding/adsorbing other chemical species
for ‘‘bottom-up’’ fabrication provides thiol SAM a special
importance in the area of nanotechnology. Some of the other
reasons for the popularity of this system include (i) biocom-
patibility of Au and (ii) ease of obtaining thin films as well as
colloidal NPs of Au. Some researchers have also investigated
thiol SAM on other substrates viz. Ag,
137
Cu,
138
Pd,
139,140
Pt,
141
Ni
142
and Fe.
143
In the next sections we discuss the
fundamentals of self-assembly process of thiols on Au surface
and the various parameters that affect it.
5.2. Steps involved in thiol SAM growth on Au
After almost two decades of incisive effort, researchers have
concluded that adsorption of thiol on Au is not a mere one
step process but comprises of two distinct steps,
144,145
although
in some rare cases a single fast step has also been observed.
146
The entire process of self-assembly can be described by a steady-
state model involving an initial physisorption, followed by a
chemisorption step, ultimately leading to a complete monolayer
formation. The first step usually commences within a few minutes
and almost 90% of the film thickness is formed.
147
In this
physisorption step, small monolayer islands of disoriented thiols
in the ‘‘laying’’ position on the Au substrate are generated
148,149
and in some cases even multilayers may be formed.
150
The second
chemisorption step that follows generally takes from a few hours
to a few days to get completed.
151
It includes gradual growth of
the thiol islands and ordered orientation on the Au surface
(Fig. 12). The relative rates of adsorption in the fast regime
depend both on the population of the physisorbed state and the
barrier to chemisorption. Based on analysis of the experimental
data and the kinetic steady-state model, it has been reported that
relative rates of adsorption in the slow ordering regime are
perhaps additionally influenced by the rates of diffusion of the
adsorbates through the partially formed monolayer films.
152
Although a two step route is commonly observed during growth
of thiol SAM on Au, a three step course has also been detected
by Yamada and Uosaki.
153
The duo first observed patches of
adsorbed molecules with no periodic structures on molecular-
length scales. The authors have suggested that these patches may
correspond to a disordered phase. In this stage of growth, pits (or
vacancy islands) are formed on the Au surface. The subsequent
step involves appearance of the patches in which ‘‘striped’’
patterns are observed. Although several periodic length scales
have been found, all are greater than the molecular length (and
increase systematically with alkyl chain length). These observa-
tions and the structural similarities of the features observed on
vapor-phase deposited thiol films suggest that these striped phase
domains are composed of thiol molecules lying down on the
surface in various ordered epitaxial arrangements. In the third
and final stage of growth, islands of apparently greater film
thickness are formed and grow to cover the surface. Such a three
step process has also been observed by other groups.
154
5.2.1. Thiol SAM structure and Au–S interface. Keeping in
view the technological importance of thiol SAMs, it is impera-
tive to have a sound knowledge about the various aspects of this
system right from the initial step involving the Au–S bonding to
the final step of molecular ordering of the adsorbed thiols.
Though studying the Au–S bonding is quite important to
fathom the SAM formation process, it is indeed quite difficult
because the self-assembly continues till saturation. And thus
studying the Au–S bond that is deeply buried into the structure
becomes a complicated task. In order to investigate the Au–S
interface various techniques have been used as shown in Table 4.
This section discusses the overall structure of the thiol SAM
and then focuses on the Au–S interface to understand how the
structure of the thiol SAM evolves from the various processes
occurring at this interface.
5.2.2. Lattice structure and phases. One of the first trans-
mission electron microscopy and diffraction studies
165
of this
Fig. 12 Formation of a thiol SAM on an Au surface.
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system showed that the adsorbed thiols form a hexagonal O3
O3R301lattice structure indicating that all the thiol molecules
are adsorbed at equivalent sites. Consideration of the hexagonal
O3O3R301lattice structure gives only a partial picture of the
thiol Au system. This structure cannot cogently explain the
close packing observed for thiol SAMs.
166,168
It was only later
that higher mess structures of the adsorbed thiols were observed
that could provide the missing link between the experimental
properties and the SAM structure. Nuzzo et al. were the first to
insinuate the presence of a superstructure of thiol SAM based
on their observation of splittings in the IR spectra.
169
Klein and
Hautman also made similar predictions based on their MD
simulations.
170
The presence of a superlattice called c(4 2)
was later confirmed by helium diffraction,
167
GIXD
171
and
Scanning Tunneling Microscopy (STM).
172
The c(4 2)
superstructure results due to a patterned arrangement and
rotations of the hydrocarbon chains about their molecular
axes and unlike the O3O3R301lattice, it is characterized
by thiol molecules adsorbed at two or more non-equivalent
sites.
The SAMs of thiols display various phases depending on
their molecular packing density. In the preliminary stages, the
alkanethiol molecules form a liquid-like phase on Au(111).
The liquid phase gradually transmutes into a series of unstable
striped-like phases called (pO3) striped phases prior to the
formation of the final close-packed phase.
173,174
Here, pdenotes
the period of the stripe in units of the lattice spacing of Au(111).
It should be noted that during transition between the various
phases, a disordered phase is usually found to coexist with the
ordered phases. According to recent studies six different phases
of thiol SAMs have been detected (Table 5). The a-phase is a
universal phase and is found to be independent of the thiol
chain length. The c(4 2) supperlattice is formed only when the
thiols are adsorbed at nonequivalent sites. This can be clearly
seen in Table 5.
It has been observed that orientation of the individual
molecule changes continuously with time until it becomes
stable with the chain uniformly tilted from the surface normal.
179
The orientation of the thiol molecule undergoes three important
changes. Preliminarily the thiols are known to have a highly
kinked orientation when physisorbed on the Au substrate. It is
during the second step that transition of the hydrocarbon chains
from a highly kinked to an all-trans conformation takes place.
The final stage consists of the reorientation of the terminal
methyl groups from a state in which methyl groups are dis-
ordered relative to one another to a state where they are all
aligned. This sequence implies that the ordering process can be
viewed as consecutive steps originating at the Au–S interface
and moving outwards. At the end of the ordering process, both
computer simulation and experimental (X-ray absorption near
edge spectroscopy, diffraction studies, IR spectroscopy)
180,181
studies have demonstrated that the thiols have a tilt angle
(y)E301,twistangle(b)E551and azimuthal angle (w)E141.
5.2.3. Role of Au adatoms. Initially the thiol SAM was
believed to exhibit only the O3O3R301lattice structure and
hence it was assumed that the Au surface is a mere spectator
in the SAM formation process i.e. adsorption of thiol on
Au surface has no effect on the structure of Au. However,
discovery of the c(4 2) superlattice has forced researchers
to doubt this notion. Some groups have tried to explain the
formation of the superlattice on flat and defect-free Au surface
by considering nonequivalent chain torsion angles,
172
sulfur
dimerization,
168
or differences in adsorption sites.
182,183
This
reasoning is, however, not completely acceptable. It is only
recently, on the basis of several studies, majority of the researchers
believe that reconstruction of Au surface to produce Au-adatoms
occurs during SAM growth.
182
During the assembly process,
the Au-adatoms are supplied by lifting the herringbone
structure of the Au(111) surface,
184
as well as etching of the
monatomic steps and terraces
185
and this reconstruction is
independent of the thiol chain length.
186
The Au-adatoms
significantly affect energetics of the Au–S bond formation
and thus play an important role in the entire self-assembly
process
187,188
as discussed later. To further bolster the concept
of adatom formation, recent STM, GIXRD and density
functional theory (DFT) simulation results have revealed that
out of the six prototypical packing structures for thiol SAM
on Au, five are incompatible with flat Au surface conditions
but can be fitted only to a reconstructed Au surface containing
Au-adatoms.
189
The stoichiometry of thiol adsorption also
plays a vital role in determination of properties of the SAMs.
Two stoichiometric ratios (Au ad-atom : S = 1 : 2 or 1 : 1) have
been considered. For the system with ratio equal to 1 : 2, both
theoretical and experimental studies have pointed that the
sulfur atom bonds on one side to the Au ad-atom and to the
Table 4 Various techniques used for characterizing Au–S bond based SAMs
Technique Ref. Technique Ref.
Scanning tunnelling microscopy 155, 156 Grazing incidence X-ray diffraction 157
Low-energy electron diffraction 158 Normal incidence X-ray standing waves 159
Helium atom scattering 160, 161 Infrared spectroscopy 162
X-Ray photoelectron spectroscopy (XPS) 163 Temperature programmed desorption 164
Table 5 Different phases of thiol SAM
Lattice Phase Structure Ref.
O3O3R301aAll thiols have equal height 175
c(4 2) b,gand dTwo different heights for the adsorbed thiols 176
eThree different heights of thiols 177
xFour different molecular heights 178
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1378 Chem. Soc. Rev., 2012, 41, 1363–1402 This journal is cThe Royal Society of Chemistry 2012
underlying lattice atom on the other side.
184
Such a system
was first proved for methanethiol at both low
190
as well as
high
188,191
coverage using simulation studies, GIXRD and
photoelectron diffraction. Later, studies have shown that
such a stoichiometry exists even for longer chain thiols like
hexanethiol
191
octanethiol, dodecanethiol
186
and even benzen-
thiols
192
and arenthiols.
187
On the other hand there are almost
an equal number of groups with equally convincing results
indicating that the thiols adsorb to Au ad-atoms in a 1 : 1
ratio.
182,193
Thus this elusive aspect of thiol SAMs requires
further study.
5.2.4. Adsorption site. Another area in thiol SAMs that is
highly debatable is the site of adsorption of a thiol molecule.
Several theoretical research groups have reported that the
most suitable binding site is the three-fold hollow sites
194
or
the fcc-bridge site (bridge with sulfur slightly displaced toward
the three-fold hollow fcc site)
195,196
with the atop site being
least favorable. This is inconsistent with the results of experi-
mental research groups who believe that thiols adsorb at atop
sites. Studies conducted at low
197
and high coverage
159
have
revealed that thiols occupy the singly coordinated atop surface
sites. Some of the plausible reasons for this conflict between
theoretical and experimental results are the use of inappro-
priate computational models, presence of local energy minima
and the fact that most of the simulation studies consider
adsorption of methanethiol on Au surface where van der
Waals interaction is negligible, though these forces have been
proven to greatly affect the final structure of SAM.
175,183,191
However, according to Woodruff’s group,
182
the interpreta-
tion of results by both these opposing groups is correct but
incomplete. The excellent work by this group cites two main
aspects on thiol SAMs viz., (i) inconsistency between the
theoretical and experimental results (ii) facile transition
between ordered structures. The researchers suggest that during
ordering of adsorbed thiols on Au surface, it is not the thiols
that undergo ordering but rather the Au adatom-thiolate
moiety (formed by the adsorption of the thiolate at the atop
site of Au adatom). It is proposed that it is this moiety that
binds to the three-fold fcc hollow site and can easily diffuse to a
non-equivalent three-fold hcp hollow site as the energy barrier
for such a transition is quite low. By considering such a process,
one can cogently explain both the experimental and theoretical
results. A recent study by another group
175
has supported
this concept by showing that the moiety at the fcc and hcp
domains have almost the same appearance in the STM images.
A characteristic fcc-hcp translational shift of this moiety can
be observed between an fcc domain and its neighboring hcp
domain but within each domain, either the fcc or the hcp
hollow site is occupied.
198
5.2.5. Fate of S–H bond and hybridization state of sulfur.
The high stability of thiol SAM is a result of strong bonding
between thiol molecule and the Au surface. As mentioned
earlier the Au surface undergoes reconstruction during the self-
assembly process that helps in increasing the bond strength.
Similarly it is also necessary to understand as to whether –SH
group of the thiol also undergoes changes during adsorption in
order to improve bonding. The adsorption of methanethiol and
dimethyldisulfide on Au surface has been intensely investigated
to discern the elusive attacking species. In case of disulfides the
case is simple. Both theoretical
199
and experimental
200
studies
have revealed that the adsorption of ‘‘intact’’ disulfides is not
energetically favorable and the adsorption involves dissociation
of the disulfides into thiolates followed by their assembly on the
Au surface. The situation is, however, complicated in case of
alkanethiols. Nuzzo and coworkers have reported adsorption of
‘‘intact’’ methanethiols on Au surface.
200
However, several
theoretical
158
and experimental
201
studies suggest that dissocia-
tive adsorption of methanethiol takes place. One group even
suggests the co-existence of both ‘‘intact’’ thiols and thiolates
adsorbed on Au surface.
202
On the other hand, a recent study
has revealed that no cleavage of the S–H bond occurs.
203
Zhou
et al. have conducted detailed investigations of the Au–S inter-
face as a function of coverage and found that at low coverage
adsorption of ‘‘intact’’ thiols is favorable on a defect-free Au
surface.
204
However, at higher coverage dissociation of thiols to
form thiolates at surface defects like vacancies and adatoms
followed by their adsorption has been found to be favorable.
The authors have demonstrated that a defect-free Au surface
cannot support dissociation of thiols but surface defects act as
catalysts for this process, leading to the formation of a more
stable thiolate SAM. Similar result has been obtained by other
research groups.
205,206
In an independent study, Bencini and
coworkers
207
have carried out mixed DFT calculations to ascer-
tain the attacking species. The authors consider the adsorption of
CH
3
–SH, CH
3
–S
and CH
3
–Son both flat and reconstructed
Au surfaces, i.e. with Au adatoms. For a flat Au surface, CH
3
–S
species are most strongly bound species at the bridge-fcc site. The
studies by other groups also reveal similar results.
199
However,
when the authors consider a reconstructed Au surface, the
simulation results suggest that CH
3
–S
is the most stable
species to be bound to the Au surface at atop sites. Adsorption
of CH
3
–SH is the least stable at both flat and reconstructed Au
surfaces. Hence thiolates are considered to be the attacking
species during the SAM formation process. Efforts must be
made to further attest this point.
As chemical bonding is involved in the Au–S bond for-
mation, it is imperative to find the type of hybridization the
sulfur atom undergoes during bonding. It has been suggested
208
that the carbon–sulfur–Au bond can have two configurations
of almost equal energy, viz. sp
3
or sp hybridization. Although
the energy difference between the two states is quite low
(1.72 kJ mol
1
), theoretical calculations have shown that
sp
3
-hybridization of sulfur is slightly more favorable than
sp-hybridization. Thus the sulfur atom can either be
sp-hybridized or sp
3
-hybridized and this depends on the inter-
molecular van der Waals forces. This implies that the thiol moiety
adsorbed to the Au surface is flexible at the Au–S bond. Such a
behavior is also reflected in the simulation studies.
208
Another
group
209
has concluded that state of hybridization depends on
the flexibility of the thiol chain. These authors suggest that in
case of flexible thiols like n-alkanethiols, both sp and sp
3
hybridized sulfur can co-exist. However, rigidity of the thiol
chain causes the sulphur atom to exist in the sp-hybridized state.
SAMs generated from linear n-alkanethiols on Au have been
found to have some shortcomings. Firstly, they are some-
what fragile,
210
decomposing upon exposure to moderate heat
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(e.g.,801C in hexadecane).
147
Secondly, it is difficult to
generate well-defined multicomponent interfaces by adsorp-
tion of the mixtures of two or more alkanethiols.
211,212
These
shortcomings can be overcome by using arylthiol SAMs, as the
p-stacking of the aromatic rings results in additional stability
to the SAMs and the aromatic ring can be used as a spectro-
scopic tag for structural and orientational characterization of
the films.
213–215
Unlike their aliphatic counterparts, studies on
adsorption of arythiols have not attracted much interest.
However some attempts have been made to discuss the
structure of arylthiol SAMs on Au.
216,217
6. Parameters affecting the SAM structure
6.1. Effect of chain branching and chain length
The structure of the alkyl region of the thiol is responsible
for van der Waals interaction. Thus, any change in the alkyl
structure is bound to affect these forces and ultimately the
structure of the SAM. Hence, studying the effect of branching
on SAM properties is important. In this regard, the tert-
butanelthiol (tBT) SAM has been extensively studied. STM
images of the tBT SAM have shown that branching signifi-
cantly affects its structure since instead of forming the classical
O3O3R301lattice observed for n-alkanethiol SAMs, tBT
forms a 2O7O7)R19.11lattice in which all adsorbate
molecules are at equivalent sites.
218
The reason for such an
unusual lattice structure has been provided by DFT simula-
tions.
219
These simulations have demonstrated that tBT SAM,
having a O3O3R301lattice, is unstable due to steric
repulsion between the adsorbates while in the observed 2O7
O7)R19.11lattice, the nearest tBT molecules are quite far
apart, resulting in a decreased steric effect. The study
219
also
points out that though STM images reveal the existence of a
2O7O7)R19.11lattice in which the all tBT molecules are
expected to be adsorbed at the fcc-bridge position, in reality
they may bind at two energetically equivalent fcc-bridge and fcc
sites giving rise to a slightly different lattice structure ((2O7
O7)R19.11-2) having STM images similar to 2O7O7)R19.11.
Another important feature of the tBT SAM that differs from
linear thiol SAMs is the absence of Au ad-atoms (proved by
computer simulation). The large difference in the SAM structure
and formation process due to a subtle difference in thiol structure
is beautifully demonstrated when one considers SAMs of sec-
butanethiol (an isomer of tBT). Various studies
220
have shown
that sec-butanethiol (sBT) forms a complex (8 O3)-4 super-
lattice instead of the expected (2O7O7)R19.11-2 obtained
for a tBT SAM. Also, DFT simulations have indicated that
unlike its isomeric form (tBT), the sBT SAM undergoes an
ad-atom mediated growth. Thus, by comparing the closely
related propanethiol, tBT and sBT SAMs, one can clearly see
that branching of thiols has a significant effect on the struc-
tural properties as well as growth processes of SAMs.
As mentioned earlier, the adsorbate–adsorbate van der Waals
interactions play an important role in deciding the density and
arrangement of the thiols on Au surface. The interaction
between the aliphatic chains and the Au substrate is an oppos-
ing force and decreases the adsorbate–adsorbate van der Waals
interactions causing the monolayer to be less densely packed
with higher degree of disorder (Fig. 13).
147
This increased
disorder modulates the charge permeability across the SAM
since charge can easily penetrate through the less densely
packed SAM of shorter thiols.
221
Not only the overall density
of the SAM, but also the tilt angle of the adsorbed thiol varies
with chain length as shown by Alexiadis et al.
222
These authors
are of the opinion that the tilt direction on the Au surface
exhibits a transition from NN to the NNN direction as the
chain length increases from 10 or 16 carbon atoms to 22 carbon
atoms. The length of the aliphatic chain also affects the kinetics
of SAM formation, with shorter chain thiols forming saturated
SAMs faster than their longer chain counterparts.
223
The inter-
actions between thiol molecules and solvents are also known to
affect the structure of a SAM. Shorter thiols are more soluble in
the solvent than the higher-chain thiols, and as a result bulk
adsorption is observed in the latter case.
224
6.2. Effect of solvent and thiol concentration
In practice, SAMs formed by immersion in solutions with very
low concentrations do not exhibit the same physical properties
as those formed from more concentrated solutions. In case of
extremely dilute thiol solutions, impurities (especially sulfur-
containing compounds) can greatly affect the SAM structure.
The displacement of already bound solvent molecules from Au
Fig. 13 Effect of chain length and chain interactions on SAM formation.
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surface is important during the SAM formation (Fig. 14). The
effect of this process becomes prominent at low thiol concen-
trations, as observed in the adsorption isotherm. At moderate
thiol concentrations the adsorption kinetics are more precursor-
like, while a Langmuir-like adsorption isotherm is observed at
high micro-molar concentration of thiols.
225
Kawasaki et al.
226
have found that at lower concentrations of thiols a two-step
process occurs while for higher concentrations the process
constitutes of just a single fast step. Since the rate of SAM
growth process is directly proportional to the thiol concen-
tration, it can thus be concluded that saturated SAMs of thiols
are formed much faster in higher concentrations of thiols as
opposed to lower concentrations. This has also been experi-
mentally verified.
227
The selection of a solvent for self-assembly of thiols on Au is
a crucial step that many researchers generally take for granted.
The solvent in which the SAMs are grown greatly affects the
structure as well as the properties of the SAM. For a thiol to
be adsorbed on the Au substrate it has to first displace the
already bound solvent molecules from the Au surface and then
bind itself to the substrate, i.e. the solvent–Au interaction must
not be strong. In addition, solubility of the thiols (solvent–
thiol interaction) is important, as highly soluble thiols tend to
remain soluble and thus their tendency to bind to the Au
substrate is lowered (Fig. 15). Care should, however, be taken
that the thiol forms a homogenous solution with the solvent.
From these conditions, it can be concluded that the SAM quality
is inversely proportional to the solubility of thiol in the solvent.
This conclusion has been reached by Schneider and Buttry.
150
This duo have shown that in dimethylformamide solution a
complete monolayer is never formed. In acetonitrile solution
on the other hand, the initially physisorbed formed film is slowly
converted to a densely packed monolayer, indicating the prodi-
gious role of solvent in the self-assembly. The kinetics of SAM
formation may be affected by the two interactions mentioned
above. Researchers have demonstrated that non-polar organic
solvents (e.g. heptane, hexane) enhance the SAM growth rate as
compared to polar organic solvents like ethanol, but this increase
in rate of SAM formation comes with a drawback of poor
molecular structure.
147,228,229
Not only does the solvent affect the
SAM structure during its formation, but also after the complete
SAM is formed as shown by Anderson et al.
230
6.3. Effect of head groups
The self-assembly of thiols on an Au surface is usually the first
step towards fabrication of the more complex molecular
assemblies. The subsequent development of these intricate
designs depends on the head groups of the thiols. It is these
head groups that decide the future course of the techniques to
be used for developing molecular structures. It is thus impor-
tant to analyze the consequence of the head groups on the
overall structure of the SAMs. Nuzzo et al.
231
have analyzed
–CH
3
, COOH, CONH
2
, COOCH
3
and CH
2
OH terminal
groups and have found that they have relatively little effect
on structure of the film in the region of hydrocarbon chains
(Fig. 16a) and propose specific orientations for the functional
groups. The lateral density of these groups is high, suggesting
that steric screening effects may play an important role in
wetting and reactivity patterns. The vibrational data obtained
by the authors has revealed that the head groups reside in
chemically and physically distinct environments that are
poorly modelled by the bulk phases. The STM analysis of
–CHQCH
2
terminated SAM has shown that it has a structure
similar to that of alkanethiols, while the –COOH terminated
thiol displays double-row adlayers arising due to hydrogen
bonding between the adjacent –COOH groups
232
(Fig. 16b).
However, in some cases the environment may affect the head
group thus modulating the structure of the SAM. The best
example is the SAM formed by –CN terminated thiols on an
Au surface. The STM images of these SAMs reveal a structure
similar to that of –COOH terminated thiols, i.e. double-row
Fig. 14 Schematic showing displacement of solvent molecules by thiol.
Fig. 15 Effect of solubility of thiol in the solvent.
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adlayers indicating hydrolysis of the nitrile groups due to
moisture in the surrounding to produce amido- or carboxylic
acid-terminated SAM, and it is the image of this SAM that is
observed in the STM analysis
232
(Fig. 16b). Another example of
thiols where interaction of the head groups manipulates the
properties of the SAMs is dithiols. It has been found that amount
of the lying-down phase decreases sharply from shorter dithiols
to longer dithiols irrespective of the self-assembly procedure. This
may perhaps be due to a chemical reaction involving hydrogen
exchange between an incoming free dithiol molecule with a
chemisorbed lying-down dithiolate on Au for the transition
process from the lying-down phase to standing-up phase. Inter-
action between free –SH terminal groups also occurs for longer
chain dithiols leading to formation of disulfide bridges. How-
ever, no such disulfides are observed for shorter chain dithiol
SAMs.
233
In an independent work, Qu and coworkers
234
have
extensively studied the structure of the 1,6-hexanedithiol
(HSC
6
SH) SAMs on Au(111) and compared it with that of
the hexanethiol (C
6
SH). XPS studies have revealed that both
thiolate and thiol moieties exist for HSC
6
SH SAMs. The
Sum Frequency Generation Spectroscopy and electrochemical
measurements have shown that alkyl chains within the
HSC
6
SH SAMs are in an all-trans conformation. MMC study
reveals that HSC
6
SH SAMs are more stable than C
6
SH ones
because of the additional end–end interaction among the free
thiol groups, complementary to the results of Millone et al.
233
7. SAM based affinity biosensing
A wide variety of protocols and configurations have been utilized
for fabricating SAM based affinity biosensors. The manufacture
of specially engineered SAMs of different organic materials
and biomolecules based on Au–S bonding for the fabrication
of efficient affinity biosensing devices are discussed in the
following subsections.
7.1. SAM based immunosensors
7.1.1. Unary and binary SAM based immunosensors. The
fabrication of the simplest thiol SAM based immunosensor
includes growth of unary or single organosulfur compound
SAM on an Au substrate followed by immobilization of anti-
bodies either by covalent attachment or by non-covalent inter-
action (Fig. 17). Covalent immobilization is a more widely used
technique than physisorption, as the latter method may lead
to an irreproducible response.
235
Several cross-linking agents
such as EDC/NHS,
236
glutaraldehyde
237
etc. have been used
for covalent immobilization of the antibodies. However, the
extra step of using a cross-linker for modifying thiol SAM can
be circumvented by using SAMs of compounds that have a
succinimidyl head group such as dithiobissuccinimidyl propionate
(DTSP).
238
The strong affinity between biotin and avidin/streptavidin
is widely used by researchers for development of high perfor-
mance immunosensors. Here the antibody is biotinylated
followed by its selective binding to a streptavidin-modified
thiol SAM surface.
239
Although this method involves extra
steps to functionalize the sensor surface with avidin/strepravidin
and antibodies with biotin, yet it is a preferred method since the
immobilization strategy produces uniform bio-functionalization
and this helps in achieving an excellent sensor response. Guest–
host interactions can also be used for antibody functionalization
of SAMs as shown by Park et al.
240
Fig. 16 (a) Schematic presentation of thiol SAMs having different head groups. (b) Schematic presentation of thiol SAMs with different head
groups and their behavior.
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In the preparation of immunosensors, the immobilization
step is followed by tethering of blocking agents like BSA for
minimizing false positives arising due to non-specific binding
of proteins to the sensor surface. The antibody–antigen inter-
action can be investigated using several electroanalytical,
241
optical,
242
electrochemiluminescence
239
and piezoelectric tech-
niques.
236
There are various parameters that one needs to
consider while developing immunosensors. These include
concentration of antibodies to be used and the optimum time
for their immobilization,
242
sample exposure time and shelf-
life.
235
The sensors developed using unary SAMs have been
successfully utilized for estimation of important analytes like
penicillin G in milk,
243
Sudan I dyes
244
and for detection of
tuberculosis
245
using EIS, low density lipoprotein using SPR
and QCM,
236
cortisol,
238
prostate specific antigen by micro-
cantilevers
246
etc.
Though Unary SAM based immunosensors have been
successfully used, yet their sensor response can be greatly
hampered due to over-crowding of the antibodies immobilized
on the sensor surface. Antibody–antigen complex is efficiently
formed when paratopes of the antibodies are free to bind with
the epitopes of respective antigen. However, the steric hindrance
caused due to the close proximity of an immobilized antibody
with neighbouring antibodies reduces the effectiveness of the
antibody–antigen interaction. To avoid this problem, one needs
to introduce an appropriate distance between the immobilized
antibodies. This can be done by preparing binary or mixed
SAMs of spacer thiols and functionalized thiols. The latter is
used for covalent binding of antibodies while the former does not
participate in immobilization and its only use is to provide a
sufficient distance between the antibodies immobilized onto the
functionalized thiols. The hydroxy-terminated alkanethiols,
247
thiolated polyethylene glycols (PEG),
246
etc. have been extensively
used as spacer thiols. The molar ratios of the two thiols used have
substantial effects on the sensor response.
247
Very diluted binary
SAMs lead to poor antibody loading, thus reducing the response
characteristics. Meanwhile, using a low spacer thiol concentration
makes the use of a binary SAM futile. The majority of immuno-
sensors discussed henceforth utilize binary SAMs.
The steps involving first the thiol SAM growth on Au surface
followed by immobilization of the antibodies can be merged
together by using recombinant thiolated antibodies. Thiolation
of antibodies for direct SAM formation on Au substrates can be
performed to achieve better orientation of the antibodies
248
without the need for any underlying matrix. This approach has
a number of advantages, such as immobilization based on
covalent attachment and efficient immobilization with uniform
surface coverage. The thiolation of antibody generally relies on
the use of amine- or sulfhydryl-reactive heterobifunctional
cross-linkers like N-succinimidyl-3-(2-pyridyldithio)propionate,
succinimidyloxycarbonyl-a-methyl-a-(2-pyridyldithio)toluene
etc. Zhang et al. have used sulfosuccinimidyl 6-[30-(2-pyridyl-
dithio) propionamido] hexanoate as cross-linker for thiolation
of antibodies and have demonstrated that using these recom-
binant antibodies is an excellent method for fabrication of
insulin immunosensor.
249
The high performance sandwich
immunosensors can also be developed using thiolated antibodies
as shown by Charathi and coworkers to detect staphylococcal
enterotoxin B using enzyme tagged sandwich immunosensor.
250
For the fabrication, the researchers have tested several cross-
linkers like N-succinimidyl 3-(2-pyridyldithio) propionate, sulfo-
succinimidyl 6-[3-(2-pyridyldithio)-propionamido]hexanoate, and
3,30-dithiobis[sulfosuccinimidylpropionate.
7.1.2. Nanostructured surface based immunosensors. The
primary event occurring at the immunosensor surface is the
binding of antigen to the immobilized antibody. Hence
the sensitivity, detection response and range increase with
increase of surface concentration of the immobilized anti-
bodies. A widely used technique for increasing surface load-
ing of the antibodies is nanostructuring the sensor surface,
as nanostructures have a high surface-to-volume ratio.
Furthermore, nanostructured surfaces have distinct optical
and/or electrical properties that can be harnessed for fabricat-
ing ultrasensitive immunosensors. He et al. have prepared a
nanostructured surface by self-assembly of AuNPs onto a
cysteamine-modified Au electrode. The authors have used this
electrode for detection of hepatitis B by further functionalizing
the electrode by antibodies.
251
Similarly, Lin and coworkers
have developed a sensitive SPR-based fiber optic immuno-
sensor for Pb
+2
ions by functionalizing the fiber surface with
AuNPs followed by immobilization of the antibodies. Tang
et al. have developed a QCM-based nanostructured immuno-
sensor for carcinoma antigen 125 detection.
252
Here the authors
prepare layer-by-layer self-assembly of AuNP nanowires using
2-aminoethanethiol as the monolayer spacer onto the QCM
sensor surface. Similarly researchers have developed novel
nanostructured surface based immunosensors for detection
of desired analytes.
253
Lee et al. have demonstrated utility of
Au-bipyramid-modified substrates for immunosensing.
254
Fig. 17 Simplest immunosensing format.
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7.1.3. Sandwich immunosensors. There are several impor-
tant antigens whose concentrations are extremely low. In such
cases, the simple immunosensor (discussed above) cannot be
used as it fails to achieve the required low detection limits due
to a weak signal generated at low antigen concentration. Thus
there is a need to develop protocols for signal amplification.
This can be achieved by using sandwich immunosensors.
Here the antigen is sandwiched between the two antibodies
(Fig. 18). Firstly, one set of antibodies is immobilized onto the
sensor surface followed by the capture of the antigens from
the analyte solution. Subsequently, another set of antibodies
(directed against the antigen) is passed over the captured anti-
gens. Thus for every antigen bound there is at least one extra
antibody on top of it leading to signal amplification. It can be
noticed that for developing successful sandwich immunosensors,
the antigen should have at least two epitopes present far away
from each other so that the two set of antibodies can effectively
bind with the antigen. The second set of antibodies can either be
unlabeled or labeled with enzymes/nanoparticles. For higher
amplification, several sets of antibodies can be used. In case of
label-free, the transducers used are usually SPR, QCM or EIS.
Labeled sandwich immunosensors developed using thiol SAMs
on Au are discussed in detail below.
a. Enzyme-tagged sandwich immunosensors. Detection of
low concentrations of antigen demands a protocol wherein
even a single antibody–antigen binding event may trigger a
large number of reactions leading to large number of products
that can be easily quantified. Enzymes have high turnover
number (10
3
–10
7
s
1
). A single enzyme can convert a large
number of substrates into products. This property of enzymes
has led to their use as labels for developing sandwich immuno-
sensors wherein the antigen is quantified by the amount of
the enzymatic product formed. Typically, antibodies are first
immobilized on the sensor surface, followed by capture of the
antigens from the analyte solution. Thereafter, a second set of
enzyme tagged antibodies is exposed to the sensor causing the
tagged antibodies to bind with the already bound antigens.
Later a fixed amount of substrate is passed over the sensor for
the enzymatic reaction to occur. For developing a colorimetric
sandwich immunosensor, a dye is introduced that results in
colour change due to the enzymatic reaction. If the product of
the enzymatic reaction is electroactive, then an electrochemical
sandwich immunosensor can be developed. Enzymes such
as alkaline phosphatase, HRP etc. have been extensively used
for fabricating thiol SAM based sandwich immunosensors for
important analytes like prostate-specific antigen,
255
urinary
tract infection
256
carcinoembryonic antigen
257
etc.
b. Nanoparticle-tagged sandwich immunosensors. Enzyme-
tagged sandwich immunosensors face an inherent drawback
of low shelf-life due to deactivation of the labeled enzyme with
time. This problem can be solved by using fluorophores as
labels.
258
However, fluorophores too lose their fluorescence
with time and under long exposure to light. In this context,
nanoparticles have proved to be of great help. The absorbance
of even a single nanoparticle is thousand-fold higher than
the cross-section of a similarly sized nanosphere filled with
fluorescein to the self-quenching limit.
259
Also, its absorbance
does not degrade with time. These characteristics have led
to the prolific use of nanoparticles as labels. The technique is
similar to that of enzyme labeled sandwich immunosensors
with the difference that nanoparticles are conjugated with the
second set of antibodies instead of enzyme. These immuno-
sensors have been developed for the detection of the various
analytes.
260,261
7.1.4. Competitive immunosensors. In the immunosensors
discussed so far, sensitivity of the device depends on ability of the
transducer to detect the antibody–antigen binding. Thus binding
Fig. 18 Various sandwich based immunosensing configurations.
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of large antigens can be detected with high sensitivities using
these immunosensors. Unfortunately detection of small analytes
(a few Daltons) cannot be done with such sensors. Due to their
low molecular weight, the signal generated by the transducer
is quite low compared to the background signal, and hence
possibility of false positives is very high. This problem can be
circumvented by using a new type of immunosensor based on
a competitive format (Fig. 19). These sensors are fabricated by
immobilizing the antigens (usually a conjugate of BSA and the
antigen) on the sensor surface. For sensing, a fixed amount of
the antibodies is mixed in the analyte solution and is passed
over the antigen-immobilized sensor surface. Consequently,
the free antibodies present in the solution bind with the immo-
bilized antigens and can be quantified by the transducer. The
amount of free antibodies is a function of the amount of
antigens present in the analyte solution and hence these sensors
are indirect immunosensors. The effectiveness of the competi-
tive immunosensors in detecting small antigens lies in the fact
that these sensors do not directly detect the small antigens but
rather act indirectly by detecting the large antibodies against the
small antigens. Several groups have developed label-free com-
petitive immunosensors on thiol SAM modified gold surfaces.
Kumbhat et al. have fabricated a competitive SPR-based
immunosensor for dengue virus;
262
March and co-workers
have developed a piezoelectric competitive immunosensor
for the determination of pesticide residues. Singh et al. have
prepared an SPR-based TNT immunosensor.
263
Competitive
immunosensors wherein the antibodies are labeled with either
enzymes
264
or nanoparticles
265
have also been fabricated.
Instead of labeling the primary antibody (against the antigen),
a labeled secondary antibody (specific to the primary anti-
body) can be used
265
after the free primary antibodies have
been bound to the antigen.
A slight modification of these sensors involves immobiliza-
tion of antibodies instead of the antigens. Here the analyte
solution is mixed with a fixed amount of tracer molecules
(labeled analyte or labeled analogs of the antigen).
266
In this
case, the tracer molecules and the analyte compete to bind
with the immobilized antibodies. Thus as the concentration of
analyte increases fewer tracer molecules are able to bind with
the antibodies, leading to a decreased signal. Hence, in such
immunosensors, the signal decreases with increase in analyte
concentration.
7.1.5. Oriented antibody based immunosensors
a. Protein A/G based immunosensors. Covalent immobiliza-
tion is the most preferred methodology for antibody immobi-
lization. Due to their abundance, the amino/carboxylic groups
present on the antibodies are mostly used for this process.
Antibodies have these groups present almost uniformly over
their surface leading to their random orientation after immo-
bilization (Fig. 20a). This may hinder the antibody–antigen
binding, resulting in poor sensor performance. This problem
can be overcome by using protein A/G, found on the surface
of a variety of staphylococci and streptococci
267
for immobili-
zation of the antibodies. These proteins bind specifically with
the non-antigenic F
c
region of antibodies (Fig. 20b) leading
to better antigen binding capacity
268
and decrease in non-
specific binding.
269
Protein G shows a broad range of binding
to IgG subclasses and higher-affinity constants as compared to
protein A.
270
Typically, protein A/G is immobilized on a thiol
SAM modified Au surface followed by oriented immobiliza-
tion of the antibodies.
271
The use of recombinant protein A/G
for a better sensing response has been discussed in many research
papers. Ren et al. have recently demonstrated that recombinant
protein A, synthesized by introduction of a –SH group to the
fused B domains of protein A, leads to enhanced stability of the
interface for orderly immobilization of the antibodies.
272
Fowler
and group
273
have clearly shown the superiority of directly
assembling a recombinant thiolated protein G over the conven-
tional technique of covalently binding the native protein G.
274
The same group has developed a sandwich immunosensor using
Fig. 19 General competitive immunosensing configurations.
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recombinant protein G that is thiolated with succinimidyl-6-
[30-(2-pyridyldithio)-propionamido] hexanoate for capturing
the antibodies against human chorionic gonadotrophin. Patrie
et al. have used nickel–histidine chemistry for immobilization
of protein A/G.
275
The authors first prepared a SAM of Ni
2+
chelates and subsequently bound His-tagged protein A/G for
oriented antibody immobilization. They used matrix-assisted
laser desorption/ionization time-of-flight mass spectrometry
for diagnosing multiple sclerosis.
b. F(ab0)
2
, Fab and scFv fragments based immunosensors.
A typical antibody comprises of a common structure of four
peptide chains with two identical light (each B25 kDa) and
heavy chains (B55 kDa), respectively (Fig. 21). The disulfide
bonds and other covalent interactions (salt linkages, hydrogen
bonds and hydrophobic bonds) bind the light and heavy
chains to form a heterodimer structure. The first few hundred
amino acids, starting from the amino terminal of the light and
heavy chains (VL and VH), vary among antibodies for devel-
oping specificity towards respective antigens and comprise the
complementarity-determining region (CDR) or paratopes (Fig. 21).
The remaining region (F
c
), however, is almost similar for all
the antibodies and plays no role in the antibody binding with
antigen. So, from the immunosensor point of view only the
small CDR of antibodies is important. Immunosensors with
high specificity, detection range and signal have been devel-
oped by using whole antibodies. There is a considerable scope
to improve the characteristics of immunosensors. Antibody
fragments [F(ab0)
2
, Fab and scFv] that contain important
CDRs and exclude the trivial F
c
region of antibodies (Table 6)
can be conveniently prepared (Fig. 21)
276,277
and directly
immobilized in the form of a monolayer onto the Au surface
by an Au–S bond
278
(Fig. 22). These fragments are small as
compared to the whole antibody, thus by using the former for
fabricating immunosensors, higher loading of the antigen
binding sites per unit area of sensor surface can be achieved.
This leads to an improved detection range and sensitivity. Also,
by using these fragments, redundant peptide chains (belonging
to the F
c
regions) are excluded. Consequently, non-specific
protein binding is reduced leading to higher specificity. Further-
more, the disulfide groups present at the edge of the two Fab
fragments can be used for orientational tethering resulting in
highly sensitive sensors (Fig. 22).
The superiority of F(ab0)
2
and Fab fragments over whole
antibodies has been elucidated by Tsai et al.
279
The authors
Fig. 20 Antibody attached with (a) a simple thiol SAM; (b) a protein
A immobilized thiol SAM.
Fig. 21 Fragmenation of IgG molecule.
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have developed and compared F(ab0)
2
, Fab and whole anti-
body based thiol SPR sensor for staphylococcal enterotoxin B
and have found that response of the whole antibody based
sensor is lowest while that of the Fab fragment based immuno-
sensor is the highest.
279
The authors attribute this to reduced
molecular size of the biosensing element leading to a decrease in
the steric hindrance and increased accessible binding sites. The
authors have further compared the random and oriented Fab
fragments immobilized via the NH
2
groups using EDC/NHS
and via thiol group (present away from the CDRs) using a thiol-
reactive crosslinker, 2-(2-pyridinyldithio)ethaneamine hydro-
chloride in combination with EDC/NHS. An increase of 30%
in the binding efficiency has been observed for binding of the
Fab fragment using thiol group due to better orientation of
CDRs. Using Fab has been found to be better than recombi-
nant thiolated whole antibodies as displayed by Nassef et al.
280
Between F(ab0)
2
and Fab fragments, the latter has proved to be
a better immunosensing element because of the smaller size and
excellent packing density achieved after immobilization.
281
The single chain antibody fragment variables (scFv) are also
attractive alternatives to monoclonal antibodies as once the
VH and VL segments of the monoclonal antibodies gene are
obtained, the maintenance of hybridoma lines is not necessary;
there is an unlimited supply of scFv through production in
bacterial culture; and it is more cost-effective to maintain
bacteria than mice (required for antibody production). The
scFv fragments can be easily modified in the peptide linker
region for introducing thiol groups (Fig. 23a). Through these
thiol groups, dense SAMs of scFv fragments can be formed
easily on an Au surface for fabricating novel immunosensors
whose efficiency is greater than that obtained from Fab
fragments.
282
Alternatively, immunosensors can be fabricated
by immobilizing scFv fragments onto Ni
+2
chelate-modified Au
surface through histidines introduced in their peptide linkers.
283
Researchers have also shown the utility of incorporating arginine
within the scFv linker for their electrostatic immobilization on
anionic thiol SAMs for developing immunosensors.
284
scFv
fragments can also be biotinylated and then immobilized on
a neutravidin modified thiol SAM surface as shown by Shen
et al. for developing a Cytochrome P450 1B1 piezoelectric
immunosensor.
285
7.1.6. Specially engineered variable heavy chain antibodies
(VHH) and bispecific diabodies based immunosensors. VHH is a
functional class of antibodies with only a heavy chain, origi-
nating from camelid species (Fig. 23b).
286
The antigen bind-
ing site of these antibodies is limited to the single variable
domain of the heavy chain. The VHH fragments are easier
to produce by molecular engineering techniques than conven-
tional antibodies.
287
These VHHs are interesting antigen binding
structures and offer advantages in terms of size, stability, and
ease of generation as compared to the other antibody frag-
ments. Besides these, VHHs retain their activity even under
harsh conditions, like presence of detergents,
288
low pH and
elevated temperatures. These conditions are usually required
during regeneration of immunosensors and hence VHHs are
excellent candidates as compared to conventional antibodies
for fabricating reusable immunosensors.
289
In their studies,
Saerens and co-workers have immobilized VHH, monoclonal
antibodies and scFv onto mixed thiol SAMs on Au surface
and have convincingly demonstrated the superiority of VHH
over monoclonal antibodies and their scFv fragments for ultra-
sensitive detection of prostate-specific antigen.
290
Similarly, thiol
SAM modified Au sensor surfaces can be used for tethering
VHHs for piezoelectric detection of HIV1 virion infectivity
factor.
291
It has also been reported that highly sensitive detection
of analytes is possible by enlarging the F
c
part of VHHs with
higher molecular mass.
Diabodies are another class of engineered antibodies formed
by cross-pairing of two scFv fragments each from two different
antibodies
292
(Fig. 23c). Each scFv fragment is specific to its
respective antigen and hence a single diabody can bind to two
different antigens. Due to their small size care must be taken while
immobilizing diabodies on a sensor surface as any negligence
may lead to their immobilization via their CDRs causing loss
of sensitivity. Research has shown that cysteine modification
of diabodies is a good protocol for their self-assembly on Au
sensor surfaces. Using this protocol Sirk et al. have created
cysteine modified bispecific diabodies specific for simulta-
neously detecting two cancer targets CD20 (lymphoma) and
Table 6 Table describing widely used antibody fragments
Antibody fragment Description
Fab Fragment containing an antigen binding site
F(ab0)
2
Dimeric structures of two Fab units linked together by a disulfide bridge
Single chain antibody fragment
variables (scFv)
Small heterodimers consisting of the variable heavy and light chain parts of the antibody (domains that are
responsible for antigen binding) linked with peptide linkers produced by genetically engineered cloning
vectors in bacterial hosts
Fig. 22 (a) Randomly oriented full length antibody on Au surface;
(b) SAM of F(ab0)
2
fragments on Au; (c) SAM of Fab fragments
on Au.
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HER2 (breast cancer).
293
Some of the recent examples of SAM
based immunosensors are recapitulated in Table 7.
7.2. SAM based DNA sensors
7.2.1. Unary and binary SAM based DNA sensors. The
simplest DNA sensor comprises of direct immobilization of
thiolated ssDNA probes on an Au surface followed by hybri-
dization with tDNA (Fig. 24). The DNA capture probe consists
of a thiolated alkyl chain specifically attached to one end. As a
standard protocol, a strand of DNA is attached to a HS(CH
2
)
6
linker molecule at the 50end phosphate group. The linker keeps
the DNA away from the Au surface and makes the binding
event sterically more favorable. The length of the alkyl chain
plays an important role in the response of a sensor.
294
These
sensors can be fabricated using a single step i.e. growth of thiolated
ssDNA probe SAM on the Au substrate at the liquid–solid
interface. The electrochemical detection technique has been
widely used for this genre of sensor with methylene blue (MB),
[Ru(NH
3
)
6
]
3+
or [Co(phen)
3
]
3+
as redox probes. MB has affinity
toward the guanine base of the ssDNA,
295
while [Co(phen)
3
]
3+
and [Ru(NH
3
)
6
]
3+
have affinity toward dsDNA. The current
decreases (MB) or increases ([Co(phen)
3
]
3+
and [Ru(NH
3
)
6
]
3+
)
upon hybridization of tDNA with probe ssDNA and it is this
variation in current that is used to detect and estimate the
amount of tDNA. Instead of using current variation due to the
redox probe, some researchers have detected tDNA by analyzing
the electrochemical current obtained due to formation of free
adenine and guanine upon acid treatment of the sensor surface
after hybridization of probes with tDNA.
296
Such sensors have
been developed for detection of meningitis,
297
human papilloma
virus
296
etc.
Though these DNA sensors are quite easy to fabricate, they
face serious deficiencies. The ssDNA probes present in the
SAM are highly coiled and thus easy hybridization with tDNA
is not possible as first uncoiling of the probe is essential.
Secondly, the probes are quite close to each other, thus hinder-
ing hybridization with tDNA. Also, non-specific adsorption at
the sites where ssDNA probes fail to adsorb on the Au surface
may give a false positive.
The drawbacks faced by the unary SAMs of thiolated ssDNA
can perhaps be overcome by preparing a binary SAM of thiolated
DNA probe and spacer thiol. The spacer thiols used extensively
are PEG
298
and 6-mercaptohexenol (MCH) (Fig. 25).
299,300
The
reasons for preparing binary SAMs are: (1) to orient the ssDNA
probes via electrostatic interations;
301
(2) to form a passivation
layer and minimize non-specific adsoption; (3) to act as spacers
and thus reduce steric hindrance to hybridization.
These SAMs can be prepared in two ways: (i) a one-step
method, involving simultaneous co-adsorption of both thiol-
ated ssDNA and spacer thiol;
302
or (ii) a two step method,
involving self-assembly of ssDNA followed by the spacer
thiol.
294
However, a recent study reveals that the one-step
method produces better results.
295
The ratio of ssDNA to the
spacer thiol used to fabricate the sensor plays a crucial role in
the observed response. O’Sullivan’s group has studied the
effect of this ratio to the hybridization efficiency with tDNA
295
and found that 1 : 100 ratio gives the best results. The surface
density of ssDNA probes in binary SAMs of ssDNA and
MCH can be controlled using MgCl
2.303
Recent pioneering
work by Wang’s group reveals superiority of using ternary
SAMs over binary SAMs for developing DNA sensors. By
using two spacer thiols, this group has successfully fabricated
highly sensitive DNA sensors.
304,305
Almost all the DNA sensors (discussed henceforth) are a
manifestation of this class of sensor. This protocol for devel-
oping DNA sensors has been used to prepare QCM,
306
SPR,
307
evanescent field
308
and electrochemical
309
based DNA sensors.
Fig. 23 Schematic representation of (a) scFv. (b) VHH in heavy chain antibody. (c) Diabodies.
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Table 7 SAM based immunosensors
Matrix Antibody Immobilization technique Analyte Transduction method Sensing format Ref.
4-Aminothio phenol Anti-apolipoprotein B Covalent binding Low density
lipoprotein
SPR and QCM Unary and binary
SAM based
236
DTSP Anti-PSA Covalent binding PSA Microcantilevers Unary and binary
SAM based
246
AuNP self-assembled on cysteamine SAM Anti-hepatitis B Physisorption Hepatitis B Electrochemical Nanostructured
surface based
251
40,4-Dithiodibutyric acid SAM on Au coated Silica particles
assembled on Au surface
Anti-casein Covalent binding Casein SPR Nanostructured
surface based
253
2,2-(3,5-Bis((6-mercaptohexyl)oxy)phenyl)-3,6,9,12,15,18,21-
heptaoxadocosanoic acid and 22-(3,5-bis((6-mercaptohexyl)-
oxy)phenyl)-3,6,9,12,15,18,21-heptaoxadocosanoic acid
N-hydroxysuccinimide ester mixed SAM
Anti-carcino
embryonic antigen
Covalent binding Carcinoembryonic
antigen
Electrochemical Enzyme tagged
sandwich based
257
Mixed SAM of 16-mercapto-1-hexadecanoic acid and
11-mercapto-1-undecanol
Anti-PSA Covalent binding Prostate-specific
antigen
SPR Nanoparticle tagged
sandwich based
260
3-Mercaptopropionic acid (MPA) Anti-aflatoxin B
1
Covalent binding Aflatoxin B
1
QCM Competitive based 265
DTSP Anti-dioxin Covalent binding Polychlorinated
dibenzo-p-dioxins
QCM Competitive based 266
Thiolated protein G Anti-alkaline phosphatase Protein G–IgG affinity Alkaline phosphatase Electrochemical Protein G based 273
Thiolated Fab Anti-staphylococcal
enterotoxin B fragments
Self-assembly by
gold–thiol chemistry
Staphylococcal
enterotoxin B (SEB)
SPR Fab fragment based 279
Thiolated Fab and F(ab0)2 fragments Anti-C-reactive protein
Fab and F(ab0)2 fragments
Self-assembly by
gold–thiol chemistry
C-Reactive protein SPR Fab and F(ab0)
2
fragment based
281
11-Mercaptoundecanoic acid and poly(sodium
4-styrenesulfonate
Arginine tagged A10B
scFv-RG3 fragments
Electrostatic Rabbit IgG QCM scFv fragment based 284
Thiolated diabodies Anti-CD20 and
anti-HER2 scFv fragments
Self-assembly by
gold–thiol chemistry
Lymphoma,
breast cancer
Fluorescence based
optical detection
Diabody based 293
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For fabrication of electrochemical DNA (E-DNA) sensors,
generally redox probes are introduced in the electrolyte for
detecting hybridization. However, one may avoid this by
covalently binding the redox probe to the free terminal of the
ssDNA probe. In such sensors, the bound redox moiety is held
close to the electrode surface in a single stranded form. How-
ever, hybridization leads to the formation of a rigid rod-like
dsDNA pushing the redox moiety away from the vicinity of the
electrode (Fig. 26).
310
Ferrocene and MB are widely used for
fabrication of such sensors with former showing better signal
response but lower shelf-life.
311
In several E-DNA sensors, the
thiol SAMs act as matrices to which ssDNA can be covalently
immobilized. Thus for efficient E-DNA sensors, a densely
packed matrix with low electrical resistance is desired. Longer
thiols form dense SAMs but have high resistance, while shorter
thiols form less dense SAMs with low resistance. To overcome
this problem, researchers have proposed the use of short flexible
tri-thiol SAMs that exhibit better stability than that of thiol
SAMs.
312
The strong affinity of biotin towards streptavidin is exten-
sively being used by researchers for developing DNA sensors.
The modus operandi of such sensors is that a monolayer of
avidin is first grown on a thiol SAM modified Au sensor
surface followed by binding of the biotinylated ssDNA probes
(Fig. 27).
313
Though it is an additional step while construct-
ing DNA as compared to direct immobilization of thiolated
ssDNA probes, it has been shown that immobilization of
DNA probes via use of biotin–avidin affinity is superior to
direct immobilization of thiolated DNA probes.
314
The direct
immobilization of a thiolated ssDNA probe leads to higher
densities of oligonucleotide probes on the sensor surface but
leads to lower hybridization efficiency due to steric hindrance
in comparison to streptavidin SAM architectures. The differences
in interfacial architecture have an impact on the detection limits.
Studies reveal that the streptavidin SAM based sensors have a
lower detection limit as opposed to the thiolated ssDNA SAM
based sensors. Additionally, streptavidin confers anti-fouling
characteristics to the biosensing platform. Several QCM,
315
SPR,
315
surface plasmon diffraction
316
etc. based DNA sensors
have been developed utilizing biotin–streptavidin affinity to
immobilize ssDNA probes onto thiol SAM modified Au surface.
7.2.2. Nanostructured material based DNA sensors. Many
nanomaterials viz, nanotubes, nanoparticles, nanomagnetic
beads, and nanocomposites, are being used to develop highly
sensitive and robust DNA sensors. Unique size-dependent
electrical and optical properties help researchers develop novel
ultrasensitive DNA sensors. In this section, we discuss appli-
cation of the optical, electrical and magnetic properties of
nanoparticles for developing novel and highly sensitive DNA
sensors.
a. Nanostructured surface based DNA sensors. Nanostruc-
tured sensor surfaces are known to be superior to atomically-
flat surfaces since they have higher surface area and thus lead
to increased surface density of ssDNA probes. Also the excep-
tional optical and electrical properties of nanostructures greatly
help to increase both the sensitivity as well as the detection limit
of the DNA sensors.
317
There are various ways to fabricate a nanostructured sensor
surface. The most common technique is self-assembly of AuNPs
on sensor surface followed by immobilization of thiolated
ssDNA probes (Fig. 28).
318
Apart from solid AuNPs, hollow AuNPs can be used, as
their properties are different than those of solid AuNPs.
Fig. 25 Binary thiolated ssDNA SAM based DNA sensor.
Fig. 26 Working principle of E-DNA sensor with a redox probe
attached to ssDNA.
Fig. 24 Unary thiolated ssDNA SAM based DNA sensor.
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A recent study shows that hollow AuNP modified electrodes,
especially the hollow AuNPs with an outer surface surrounded
by dense spike-like nanocrystallites, can greatly enhance the
DNA hybridization ability with high stability and reusability.
319
Another protocol for preparing a nanostructured sensor surface
with hollow AuNPs is to first self-assemble nanoparticles of
a different material followed by electro-deposition of Au on
the nanostructured sensor surface to obtain a hollow AuNPs
coated surface that can be further used to immobilize the
ssDNA probes to develop the desired DNA sensor.
320
Besides
this, AuNPs can be grown on nanowires and by doing so one
can use the virtues of both nanowires and AuNPs while
fabricating DNA sensors.
321
A replacement for first fabricat-
ing a nanostructured Au sensor surface, is to prepare a AuNP–
thiolated-ssDNA composite and then cast the material on
the sensor surface.
322
As discussed earlier, spacer thiols can
be used to reduce steric hindrance during hybridization of
tDNA with immobilized ssDNA probes. By using a nano-
structured Au surface, one may obviate the need of using
spacer thiols as shown by Marques et al.
323
The group prepared
AuNP–graphite–epoxy composite films for immobilization of
thiolated DNA (Fig. 29). The rigid, non-chemisorbing, con-
ducting graphite–epoxy acts as a spacer for the ssDNA probes
tethered to the AuNPs islands resulting in an improved limit of
detection.
b. Nanoparticle conjugation based DNA sensors. Apart from
preparing a nanostructured sensor surface for development of
DNA sensors, scientists are making efforts to enhance the
sensing characteristics of DNA sensors by preparing nano-
particles conjugated DNA.
b.i. Nanoparticle tagged tDNA based solid support DNA
sensors. This genre of sensors involves tethering probe ssDNA
to the solid sensor surface followed by its hybridization to
tDNA that is conjugated with the nanoparticles for signal
accretion. For example, Ito and group
324
demonstrate through
Maxwell–Garnett theory that such AuNP-conjugation with
tDNA enhances the SPR signal.
Other nanoparticles can also be used for fabricating such
sensors. Fu et al. have developed a competitive type DNA
sensor by preparing a binary SAM of thiolated ssDNA and
MCH on an Au surface followed by competitive hybridization
between AgNP conjugated tDNA and free tDNA. This step
is followed by immobilization of HRP (HRP selectively binds
to silver nanoparticles and not to untagged tDNA or free
ssDNA probes) and flow of H
2
O
2
to generate an electrochemical
signal.
325
Similarly, semiconducting nanoparticles, quantum dots
and magnetic nanoparticles are also used. Fang’s group have
Fig. 27 DNA sensors based on streptavidin–biotin affinity.
Fig. 28 Fabrication process of a typical nanostructured surface
based DNA sensor and comparison of signal response with unary
SAM based DNA sensors.
Fig. 29 AuNP embedded GEC composite for DNA immobilization.
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shown that CdS nanoparticles can be utilized for signal ampli-
fication in Faradaic-impedance based DNA sensors
326
due to
their high density of negative charge, space blocking, and
semiconducting properties. Niu and Knoll
327
have described a
protocol for fabrication of a multi-analyte surface plasmon
fluorescence DNA sensor based on hybridization of quantum
dot conjugated tDNA with different ssDNA probes tethered to
the fingers of the Au array.
b.ii. Nanoparticle tagged reporter DNA (rDNA) based solid
support DNA sensors. A slight variation of the above men-
tioned category of DNA sensors consists of using another
DNA that is allowed to hybridize with the tDNA after it has
hybridized with the ssDNA probe. This extra DNA used is
generally called reporter or detector (rDNA) and is tagged
with nanoparticles rather than the tDNA (Fig. 30). This
technique has an edge over the tagged tDNA sensors as one
does not waste time tagging the tDNA, but can directly
analyze the sample with rDNA. In these sensors, use of an
extra DNA (ie. rDNA) and nanoparticles leads to extremely
low LOD, in the range of femtomoles.
317
Fluorescent tagged
rDNA may also be used. However, research reveals that
nanoparticle tagged rDNA has 1000 times more sensitivity
than their fluoroscent analogs.
328
AuNPs have received immense attention for the preparation
of rDNA. The reasons are quite obvious, since AuNPs are
highly biocompatible, show excellent electrical and optical
properties that come in handy for detecting tDNA. Fan’s
group has developed an ultrasensitive DNA sensor by devel-
oping a binary SAM of ssDNA and MCH for capturing
tDNA, followed by hybridization with AuNP conjugated
rDNA.
317
DNA–nanoparticle conjugates have high mass
and can be used for developing mass-based sensors, as shown
for detection of E. coli O157:H7 tDNA.
329
Dong et al.
330
have
developed a carbon-nanotube FET DNA sensor with signal
amplification by AuNP tagged rDNA. Using AuNP’s size-
dependent optical property, Mirkin’s team
331
has fabricated a
multi-analyte optical DNA sensor. The researchers prepared a
binary SAM of two probes directed towards two different
tDNAs. AuNPs having different sizes are separately tagged
with two different rDNA (each one selective to its respective
tDNA). After co-hybridization of the probe and rDNA with
their respective tDNA, the two tDNAs are detected via
absorbance studies.
b.iii. Nanoparticle tagged DNA for solution based DNA
sensors. In this type of DNA sensor, the DNA hybridization
occurs in solution. Optical properties of several nanoparticles
(AuNPs, quantum dots) are known to vary on aggregation.
This property is central to the principle of detection of solution
based DNA sensors. The probe ssDNAs are conjugated with
suitable nanoparticles and then introduced in the tDNA
sample solution. Due to hybridization with tDNA, the nano-
particles aggregate and produce an optical signal in the form
of absorbance shift. An SERS based DNA sensor can be
developed using this principle as shown by Thuy et al.
332
The
sensor consists of AuNP tagged ssDNA probes which on
hybridization with tDNA leads to agglomeration causing
generation of ‘‘hot-spots’’.
In high salt concentration analyte solutions, the hybridiza-
tion of tDNA can stop aggregation of ssDNA tagged nano-
particles. This phenomenon can be used to develop novel
sensors for tDNA in high salt concentration samples as shown
by Baptista et al.
333
These authors have shown that the ssDNA
tagged AuNPs aggregate in high salt solutions without tDNA.
However, in the presence of tDNA, hybridization hinders
aggregation and hence can be used for its detection. Instead
of just tagging the ssDNA probes, tDNA can also be tagged
with nanoparticles. Thus in such cases, hybridization causes the
two nanoparticles (conjugated to ssDNA and tDNA) to come
close, resulting in a change in some of the physical properties of
the nanoparticles. For example, close proximity of quantum
dots with AuNPs causes quenching and/or enhancing of the
former’s photoluminescence intensity.
334
Liu’s group has devel-
oped a novel DNA sensor by tagging the ssDNA probes with
CdSe@ZnS core/shell quantum dots while tDNA with
AuNPs.
334
Upon hybridization, the photoluminescence intensity
of the quantum dots is quenched due to AuNPs. Zhang et al.
Fig. 30 Fabrication of a typical nanoparticle tagged rDNA based solid support DNA sensor.
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have fabricated a sandwich-type solution based DNA sensor for
the Salmonella enteritidis gene.
335
For this purpose, these authors
have tagged AuNP to rDNA and a fluorescein to barcode DNA,
while the ssDNA is conjugated with magnetic nanoparticles.
Upon hybridization, the sandwich assembly is separated by
magnetic separation, followed by heat treatment to release the
barcode DNA. The tDNA is indirectly quantified depending on
the fluorescence of the barcode DNA.
Some researchers have made efforts to use this category of
DNA sensors to develop E-DNA sensors. Willner’s group
336
have developed an E-DNA sensor using a sandwich analog of
solution based DNA sensors. The authors conjugated both the
ssDNA probe and rDNA with AuNPs. After hybridization,
the dense DNA–AuNP aggregate is self-assembled on a dithiol
SAM modified Au electrode for quantifying tDNA with MB
as redox probe. Similarly, magnetic nanoparticles have also
been used for preparing such E-DNA sensors.
337
7.2.3. Specially engineered DNA probes based DNA sensors
a. Need for hairpin DNA (hDNA) probes. Specificity of
probe-target hybridization is a crucial factor that determines
the efficiency of DNA sensors. A probe ssDNA is highly
specific to tDNA if the free energy penalty for hybridization
with mismatched DNA is high as compared to that with
tDNA. The thermodynamic analysis of the hybridization
process proves that it is a general feature of structurally con-
strained probes to distinguish mismatches over a larger range of
temperatures or other experimental parameters compared to
unstructured probes.
338
The thermodynamic studies point out
that the oligonucleotide which folds onto itself, forming a
hairpin-like structure must be more specific to the tDNA as
compared to the linear ssDNA. hDNA probes are specially
engineered ssDNA containing a sequence complementary to the
tDNA that is flanked by self-complementary target-unrelated
termini (Fig. 31). Hairpin structures do not remain static. They
fluctuate between different conformations. All the conforma-
tions can be divided into two main states: the open state and the
closed one (Fig. 32).
The tDNA hybridizes to the loop portion of the hDNA
probes resulting in stem opening and subsequent formation of
a rigid rod-like double helix. As opposed to the conventional
linear probe ssDNA, such a deliberate secondary structure
enhances sequence selectivity since the intermolecular probe–
target hybridization needs to overcome the intramolecular
unzipping of the stem base pairs.
339
This added competing
factor renders more discernible the rates and equilibrium
amounts of captured targets between perfectly matched and
singly mismatched cases.
340
Thus, hDNA probes allow for a
wider window of stringency conditions that provide better
match/mismatch discrimination.
b. Molecular beacon (MBE) based DNA sensors. A MBE is
a hDNA which is conjugated with a fluorophore and a
quencher at the 30- and 50-ends (Fig. 33)
341
and acts like a
switch that is initially closed—or ‘off’. The stem structure
holds the fluorophore and the quencher in close proximity to
one another, preventing the fluorophore from emitting a
fluorescence signal due to strong intramolecular dipole–dipole
coupling.
342
On hybridization with the tDNA, the stem melts
and the MBE opens causing spatial separation of the fluoro-
phore from the quencher causing enhancement in fluorescence
i.e. signal ‘on’ (Fig. 34). Because of the unique structural and
thermodynamic properties of MBEs, these probes offer several
advantages like the ability to differentiate single mismatched
DNA with tDNA.
343
With appropriate fluorophore and quencher
pairs and MBE designs,
344
very high signal-to-background ratios
of more than two orders upon target hybridization can be
achieved. Organic quenchers have been widely used to develop
MBEs. However, their quenching efficiencies usually vary
significantly from one dye to another.
345
Experimental and
theoretical studies show that AuNPs are ultra-high fluoro-
scence quenchers.
346
This unique property of AuNPs has
enabled researchers to use them as quenchers while designing
MBEs instead of traditional organic quenchers.
345,347–349
The
modus operandi of this type of MBE optical DNA sensor is
that in the ‘off’ state the fluorophore is in close proximity to
the AuNP (quencher) and hence its fluorescence is quenched.
When the tDNA is exposed to the AuNP-conjugated MBEs,
Fig. 31 Structure of hairpin DNA (hDNA).
Fig. 32 Closed and open state of hairpin DNA structure.
Fig. 33 Schematic of a molecular beacon (MBE).
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opening of the stem occurs causing pushing of the fluorophore
away from AuNPs leading to generation of fluorescent signal
(Fig. 35).
c. MBE based lab-on-chip DNA sensors. In conventional
MBE DNA sensors, hybridization takes place in solution
phase. Hence to construct a chip-based MBE sensor, some
researchers have made an attempt to develop a lab-on-chip
device for hybridizing tDNA and MBE.
350
In these devices, a
Y-shaped microfluidic channel is prepared. From one arm,
MBEs are passed while (simultaneously) through other arm
the tDNAs are flowed. The two meet and subsequently
hybridize in the stem part of the Y-shaped channel to give
fluorescence (Fig. 36). MBE probes can also be tethered on Au
substrate via a thiol linker bound to one end of MBE while a
fluorophore is attached to the other. Here macroscopic Au
acts as a quencher, thus leading to the elimination of the step
of attaching a separate quencher to MBEs.
351
The working
principle of such DNA sensors is similar to that of AuNP-
conjugated MBE DNA sensors with the difference that instead
of AuNPs, here macroscopic Au (an Au substrate) is used.
Fig. 37 shows a schematic of this genre of optical MBE DNA
sensors. Instead of using fluorescence, other transducing tech-
niques like electrogenerated chemiluminescence can also be
used.
352
d. Electrochemical hDNA sensors: signal ‘OFF’ design. Optical
MBEs face an inherent drawback. Fluorescent and strongly
absorbing interferents are common in clinical and environmental
samples that may impede the utility of such optical approaches
for detection in complex sample solutions.
353
Apart from this,
optical sensors need expensive instruments for optical imaging,
laser light and labeled probes. Furthermore, the detection limit of
optical biosensor array depends on the thickness of the test
solution. On the other hand, electroactive interferants are com-
paratively rare; the advantages of electrochemical approach
include speed, sensitivity, and low cost/mass/power require-
ments
354
with non-overlapping redox potentials for simultaneous
detection of multiple analytes;
355
availability of various electro-
active labels having high stability. The superiority of electro-
chemical transduction over optical, encouraged Fan et al.
356
to
develop an electrochemical analog of optical MBE DNA sensor.
In their work, the researchers self-assembled hDNA probes on
Fig. 34 Working principle of a MBE based DNA sensor.
Fig. 35 Methodology of tDNA detection by an AuNP conjugated MBE DNA sensor.
Fig. 36 Detection strategy of a microfluidic based MBE DNA
sensor.
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Au electrode that contained a thiol linker at the 30position while
a ferrocene moiety was bound to the NH
2
group at the 50position.
The redox-tagged hDNA probes works similar to their sister
probes (optical MBEs). In the ‘off’ state, the redox moiety is held
close to the electrode surface resulting in enhanced current.
However, upon hybridization with tDNA, the redox species is
thrown away from the electrode causing decrease in current
(Fig. 38). Plaxco’s team has worked extensively in the field of
electrochemical hDNA sensors and has thrown light on the effect
of nature of the co-adsorbate forming the SAM,
357
probe length,
probe geometry and placement of redox-tag on the performance
of electrochemical hairpin probes.
358
e. Electrochemical hDNA sensors: signal ‘ON’ design.
Though electrochemical hDNA probes are superior to their
optical counterparts, they have a limitation due to their method
of detection. Hybridization causes a decrease in electrochemical
current. Thus detection beyond complete current suppression is
not possible. This limits the application of such electrochemical
hDNA probes. A way to solve this problem is to design an
electrochemical hDNA probe that causes a gain in current upon
hybridization with tDNA instead of a decrease i.e., the probe
should be in the ‘on’ state after hybridization. It has been found
that these signal ‘on’ designed probes show a much better
sensing response as compared to the conventional signal ‘off’
designed probes. In this context, Immoos et al. first designed a
DNA sensor consisting of a DNA PEG DNA triblock probe
immobilized onto an electrode and labeled at its distal terminus
with a redox reporter. Hybridization with a target that is
complementary to both of the flanking DNA elements brings
the labeled end of the probe into proximity with the electrode,
increasing electron transfer.
359
Another way to develop such
sensors is to use a specially designed DNA probe that, on
binding with its target, displaces a flexible single-stranded
element containing a redox moiety. The displaced strand brings
the redox species close to the electrode surface causing a current
Fig. 37 Schematic of an optical MBE based lab-on-chip DNA sensor.
Fig. 38 Pictorial representation of an electrochemical hDNA sensor: signal ‘OFF’ design.
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increment (Fig. 39).
360
A complication of this technique is that
the labeled probe which is partially displaced upon target
binding is only held onto the sensor via hybridization with the
surface attached strand and thus stability and reusability of this
sensor is poor. In response, a new form of signal ‘on’ probes
that adopt a double-stem–loop pseudo-knot structure to hold the
redox tag away from the surface has recently been developed.
361
Target binding disrupts the pseudo-knot, liberating a flexible,
single-stranded element that more readily collides with the
electrode surface and produces a strong signal increase. Table 8
describes some of the recently reported DNA biosensors of
different genre based on thiol SAMs.
7.3. SAM based aptasensors
DNA sensors have been found to be more selective towards
their target as compared to immunosensors. However, DNA
sensors cannot detect various analytes ranging from low mole-
cular weight organic/inorganic molecules to large molecules like
Fig. 39 Pictorial representation of an electrochemical hDNA sensor: signal ‘ON’ design.
Table 8 SAM based DNA biosensors
Matrix
Probe tethering
technique tDNA
Transducing
method Genre Ref.
Thiolated-ssDNA Self-assembly by
gold–thiol chemistry
Human papilloma
virus
Square wave
voltammetry
Unary SAM based 296
Thiolated ssDNA Self-assembly by
gold–thiol chemistry
Meningitis Voltammetric Unary SAM based 297
HS-ssDNA /AuNP
modified mercapto-
diazoaminobenzene
monolayer
Self-assembly by
gold–thiol chemistry
Complementary
DNA
Differential pulse
voltametry
Unary SAM based 362
ssDNA + 4-mercapto-
1-butanol
Self-assembly by
gold–thiol chemistry
25-mer and
37-mer oligonucleotide
Impedimetric Binary SAM based 363
Thiolated-C
11
(ethylene
glycol)
2
–C
16
COOH (3 : 1)
Streptavidin–biotinylated
ssDNA affinity
23-mer oligonucleotide SPR Biotin–streptavidin
affinity based
313
Thiolated DNA SAM on
nanostructured Au surface
Self-assembly by
gold–thiol chemistry
Complementary DNA Optical Nanostructured
surface based
320
Thiolated ssDNA+MCH Self-assembly by
gold–thiol chemistry
Complementary DNA Amperometric Nanoparticle tagged
target DNA based
solid support
325
ssDNA Self-assembly by
gold–thiol chemistry
Escherichia coli O157:H7 Piezoelectric Nanoparticle tagged
reporter DNA based
solid support DNA
sensors
329
ssDNA conjugated AuNPs Self-assembly by
gold–thiol chemistry
25-mer oligonucleotide Surface-enhanced
Raman scattering
Nanoparticle tagged
DNA for solution based
332
Hairpin ssDNA conjugated
AuNPs
Self-assembly by
gold–thiol chemistry
Complementary DNA Fo
¨ster-type fluorescence
resonance energy transfer
MBE based 347
Hairpin ssDNA +
mercapto-propanol
Self-assembly by
gold–thiol chemistry
Complementary DNA Fo
¨ster-type fluorescence
resonance energy transfer
MBE based lab-on-chip 364
Hairpin ssDNA + MCH Self-assembly by
gold–thiol chemistry
Complementary DNA Chronocoulometric Electrochemical hDNA
sensors: signal ‘OFF’
design
359
Hairpin ssDNA + PEG Self-assembly by
gold–thiol chemistry
Complementary DNA Electrochemical
impedance spectroscopy
Electrochemical hDNA
sensors: signal ‘ON’ design
365
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proteins and cells, as immunosensors do. This dilemma has led
researchers to develop signaling agents, known as aptamers,
whose binding affinities are as strong as IgGs and are highly
selective like the DNA.
366
Aptamers are artificial short nucleic
acids (DNA and RNA) having high specificity and selecti-
vity towards amino acids, drugs, proteins etc. As opposed to
synthesis of antibodies which involve in vivo immunization
of animals, aptamers can be easily synthesized in vitro by a
process called SELEX (systematic evolution of ligands by
exponential enrichment).
367,368
Besides this, they are more
cost-effective and show good stability in terms of temperature,
non-toxicity and biological acivity.
366
Thus aptasensors have
an edge over both immunosensors and DNA sensors.
For fabrication of desired aptasensors, the aptamers can
either be immobilized on thiol SAM via crosslinkers
369
or can
be modified to have a thiol group for SAM formation on Au
and AuNP surfaces.
370,371
Different architectures of these short
oligonucleotides such as molecular beacons,
369
nanoparticle-
conjugated aptamers,
372–375
redox moiety tagged aptamers
376
and enzyme labeled
366
aptamers have been utilized for applica-
tion to biosensing.
7.4. SAM based peptide nucleic acid (PNA) sensors
DNA sensors are quite popular in the bio-analytical sector
for their high specificity. However, their selectivity is currently
not of the highest level as in some cases they are unable to
distinguish single-base mismatches from the exact complemen-
tary targets. Additionally, the DNA probes are less stable
at higher temperatures. When the negatively-charged sugar–
phosphate backbone of a DNA molecule is replaced by its
neutral pseudo-peptide counterpart containing repeated units
of N-(2-aminoethyl) glycine, the DNA strand is transmuted
into PNA.
377,378
This type of modification in the structure
prevents it from degradation by nucleases and makes it more
biologically stable.
379
The neutral character of PNA results
in increased affinity with tDNA during hybridization thus
enhancing the stability of the PNA/DNA duplex. These attri-
butes of PNA have made it superior to DNA for fabrication
of highly specific biosensors.
380,381
PNA probes are usually
tethered to the Au surface via self-assembly of the thiolated
PNA probes.
377,378,382,383
7.5. LBL self-assembly and its application to affinity
biosensing
LBL assembly is a versatile and inexpensive technique by
which thin films of controlled thickness and composition can
be prepared. In general, the LBL process is achieved by
alternately exposing a substrate to positively and negatively
charged polymers or particles. This step is carried out until a
desired number of layers are obtained. It has been observed
that during the LBL assembly process, an exponential growth
is found for first several layers, followed by a linear growth
for subsequent layers evolving toward a steady state.
384
The
study has demonstrated that abutting layers are highly inter-
penetrating, while stratification is seen for every four or more
layers. There are several factors that affect the quality of the
LBL films, viz., charge density, molecular weight, temperature,
deposition time, concentration and pH.
385
Conventional LBL methods depend on electrostatic inter-
actions, hydrogen bonds, step-by-step reactions, sol–gel processes,
molecular recognition, charge-transfer, stepwise stereo-complex
assembly, and electrochemistry. However, these methods are
not suitable for fabricating multilayers of single charged,
electrically neutral, or water-insoluble species. LBL assemblies
of such species can be formed by unconventional techniques
involving complexes, non-covalent modification, co-ordination
polyelectrolytes, electrostatic complex formation, block copolymer
micelles and electrochemically triggered ‘‘click’’ coupling.
386,387
LBL has been successfully used for developing technologi-
cally important multilayers of polymers,
388
polyelectrolytes,
389
nanomaterials,
390
proteins,
391
etc. for developing magnetic
resonance imaging contrast agents,
392
electrochromic devices,
393
etc. The ability of the LBL technique to (i) incorporate bio-
molecules and nanomaterials within the multilayers with high
loading and (ii) easily tailor the composition of each layer has led
to its immense use in the field of medical diagnostics. Bountiful
research is being carried out to harness the capabilities of this
method to develop exciting immunosensors
394–396
and DNA
sensors.
397,398
For more details on this technique and its application to
biosensing please refer to excellent comprehensive review
articles.
399–401
8. Future scope and challenges
Ordered molecular assembly has truly come-of-age and has
pushed the limits of miniaturization of bioelectronic devices.
Through this review article, an attempt has been made to
analyze critically the interesting field of ordered molecular self-
assemblies for affinity biosensing. As shown in this article,
control over properties at the molecular level has empowered
researchers to develop diagnostic tools with felicitous char-
acteristics. The future prospects of this technique appear quite
bright. However, the complete potential of this technique has
not yet been realized. Researchers should channel their studies
to increase the stability of molecularly assembled architectures.
This is highly desired in case of LB films as the present films are
quite fragile and hence fabrication of robust biosensors invol-
ving this technique is currently a dream. Realization of this
dream will decisively help researchers to capture the full power
of LB-films that in turn may open up new vistas in the field of
bio-molecular electronics. Also, one needs to juxtapose similar
related fields with the area of ordered molecular assembly. For
example, by coupling techniques like lithography, one can open
up the possibility of developing unconventional self-organized
molecular structures with unique surface properties.
‘‘Click’’ chemistry is still a rudimentary technique but its
tremendous scope in nanotechnology can be easily perceived.
Coupling of this technique with ordered molecular assembly is
likely to provide new facet to surface chemistry. On a wider
aspect, chemists can help to synthesize new breed of highly
biocompatible molecules that can form excellent molecular
self-assemblies for developing novel biosensors. A better
integration of bio-conjugate chemistry with ordered molecular
self-assembly techniques, if achieved, will help nano-
bio-technologists to develop superior bioelectronic devices.
Hence, it is advisable that biochemists should perhaps direct
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This journal is cThe Royal Society of Chemistry 2012 Chem. Soc. Rev., 2012, 41, 1363–1402 1397
their research on producing novel non-destructive ways of
immobilizing biomolecules to the molecular structures formed
by assembly. Biologists can significantly contribute by devel-
oping or modifying biomolecules that can be easily integrated
with molecular self-assembled systems without losing their
inherent bio-activity. Similarly, researchers from other fields
of science and engineering can contribute toward further
spreading the use of molecular self-assembly to the field of
clinical diagnostics. It is abundantly clear that ordered mole-
cular assemblies are presently in the forefront nanotechnology.
There is, however, a considerable need towards up-scaling the
inter-disciplinary efforts that would not only unravel many
hitherto hidden laws of nature but will also lead to many new
biosensing applications that are likely to result in decisive
improvement in the quality of our day-to-day lives.
Acknowledgements
We thank the Director, National Physical Laboratory, New Delhi,
India for the use of its facilities. Z. M. is thankful to the
Council of Scientific & Industrial Research (CSIR), India, for
the award of a Senior Research Fellowship. We acknowledge
financial support received from the Department of Science &
Technology (DST), Govt. of India [DST/TSG/ME/2008/18
and GAP- 070932], in-house project (OLP-070632D), India
Japan project (DST/INT/JAP/P-21/07) and the Department
of Biotechnology, Govt. of India (DBT/GAP 070832). One of
us (BDM) thanks National Research Foundation of Korea,
the Ministry of Education, Science and Technology for giving
the opportunity to visit the Centre for NanoBioengineering &
SpinTronics under the WCU (World Class University) program
(R32-20026) during August 2011.
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