UNIVERSIDAD JAUME I
NANOBIOSENSORS
Vera Beltrán Pitarch, Luisa Hernández Górriz, Nacho Prada Barceló
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Index
1. - Introduction. .................................................................................................................... 2
1.1. - Nanobiosensor structure........................................................................................... 3
1.2. - Nanobiosensor functioning. ...................................................................................... 4
1.3. - Selection and Optimization of Nanomaterials for Sensor Technology. ...................... 4
1.4. - Types of nanobiosensors. ........................................................................................ 8
1.5. - Applications of nanobiosensors. ............................................................................. 10
2. - Glucose nanobiosensors............................................................................................... 10
2.1 Brief History of Electrochemical glucose biosensors. ................................................. 11
2.2 Electrochemical biosensors. ...................................................................................... 12
2.3 Nanobiosensors......................................................................................................... 13
2.3.1 Nanoparticles. ..................................................................................................... 13
2.3.2 Nanocables, Nanofilms and nanofibers. .............................................................. 13
2.3.3 Carbon nanotubes............................................................................................... 14
2.3.4 Fluorescent polymeric nanosensors. ................................................................... 15
2.3.5 Quantum dots in glucose sensors. ...................................................................... 15
2.6 Conclusion................................................................................................................. 16
3. - Antibody-antigen recognition. ........................................................................................ 17
3.1. - Antibody structure. ................................................................................................. 17
3.2. - Detection of individual antibody-antigen recognition events by atomic force
microscopy (AFM) ........................................................................................................... 18
3.3. Antibody´s recognition introduction ........................................................................... 19
3.4. Method ..................................................................................................................... 20
3.5. Results and discussion ............................................................................................. 21
3.6. Conclusions .............................................................................................................. 24
4. - Bibliography. ................................................................................................................. 26
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1. - Introduction.
A biosensor can be defined like an analytical device which incorporates a biologically active
element with an appropriate physical transducer to generate a measurable signal
proportional to the concentration of chemical species in any type of sample.
It can detect a wide range of targets from small protein molecules to large pathogens.
Some characteristics of biosensors are:
● Selectivity. Probably the most important feature of biosensors. Selectivity means
that sensor detects a certain analyte and does not react to admixtures and
contaminants. Antigen-antibody interaction has the highest selectivity, it is analytespecific.
● Accuracy. Is a characteristic of any scientific device that makes quantitative
measurements. It is usually characterized in terms of the standard deviation of
measurements. Signal error in measured concentration. Signal stability influences
the accuracy of sensor. It is an important characteristic of a sensor that performs
continuous monitoring.
● Sensitivity. It shows the minimal amount or concentration of analyte that can be
detected.
● Working range. Is the range of analyte concentrations in which the sensor can
operate. Working range of sensor should correlate with the range of possible
concentrations analyte in the assay.
● Response time. Is the time required to analyze the assay.
● Regeneration time. The time required to return the sensor to working state after
interaction with the sample.
● Number of cycles is the number of times the sensor can be operated. Degradation of
biological material is inevitable and it needs to be replaced.
Besides this, to be commercially successful, a biosensor has to meet the general
requirements of commercial sensors that include specificity, accuracy, sensitivity, ease of
use, reproducibility, near real-time assay, robustness, speed of response, running costs and
life, and it has to be insensitivity to temperature and to electrical and other environmental
interference.
The number of false positive and false negative results of a biosensor should be very low,
ideally zero, for it to be an acceptable practical device.
A nanobiosensor is just a nanoscale biosensors that have exhibits rapid responses combined
with very high sensitivities.
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1.1. - Nanobiosensor structure.
To work, the biosensor consists of three main parts:
1. Biological recognition element. It is a biologically derived material or biomimetic
component that interacts (binds or recognizes) the analyte under study. It can be a
tissue, microorganisms, organelles, cell receptors, enzymes, antibodies, nucleic acids,
etc...
2. Transducer or detector element. Transforms the signal resulting from the interaction
of the analyte with the biological element into another signal that can be more easily
measured and quantified. It works in a physicochemical way: optical, piezoelectric,
electrochemical, etc.
3. Signal processing electronics. Responsible for the display of the results in a userfriendly way.
Figure 1. Nanobiosensor components.
Figure 2. Elements of a biosensor.
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1.2. - Nanobiosensor functioning.
Broadly speaking, the nanobiosensor works as follows:
In first place, bioreceptor recognizes the analyte. Then the biological material is immobilized
and a contact is made between the immobilized biological material and the transducer. The
analyte binds to the biological material to form a bound analyte which in turn produces the
electronic response that can be measured. Following, the transducer converts the product
linked changes into electrical signals which can be amplified and measured. And finally, the
output from the transducer is amplified, processed and displayed.
Figure 3. Nanobiosensor functioning.
1.3. - Selection and Optimization of Nanomaterials for Sensor Technology.
There is a multitude, factors which govern or decide the use of a particular kind of
nanomaterials for biosensing applications. These factors are the chief ingredients of their
physical and chemical properties along with their energy sensitive and selective responses.
Before exactly implementing or adding a nanomaterial for the sensing applications, we first
focus on their desired manufacturing which is a part of experimental design known as
“Nanofabrication.” The technique of nanofabrication targets two vital operations, namely,
the manufacturing and design of nanoscale adhesive surfaces via the technology of
integrated circuits and the engineering of nanomaterial surfaces through the process of
micromachining. This technique, thus developed for biosensing, uses the variations of four
basic processes, namely, photolithography, thin film etching/growth, surface etching
strategies, and chemical bonding parameters.
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Nanoscale electrodes which have come into picture as a result of lithography technique
have enhanced the biosensing accuracy by providing much better and greater surface areas
that in turn enable the immobilization to be achieved with greater precision. Glucose
biosensors, making use of enzyme glucose oxidase, have been developed using these
innovations. The strategies involving the use of active nanoparticles of platinum over the
sheets of carbon nanotubes have significantly enhanced the immobilization of enzyme
systems required for the detection of the analyte materials. These systems have significantly
much wider applications to biosensing technology, enabling the detection of glucose from
several sources other than blood. In a similar manner, couples of immunosensors have also
been developed which involve coating of thin films over the sensing surface that enables
faster and better detection of the corresponding analytes.
Highly sensitive electrical and electromechanical properties are incorporated into several
materials by engineering them with nanoelectromechanical systems (NEMS), which have
enabled the generation of complex electrical, mechanical, fluidic, thermal, optical,and
magnetic properties of the materials with sizes down to the nanometer level. NEMS
technology has thus provided many materials with novel properties due to their nanoscale
functionalization. NEMS and MEMS devices have enabled better and better sophisticated
performance of the mechanical materials as the mechanical properties of a material are a
critical function of its size. In addition, these devices have been coupled with biological
systems and molecules to improve their bio adhesion characteristics and the response to a
wide range of stimuli. With the implementation of NEMS and MEMS, surface forces like
friction, adhesion, cohesive forces, and viscous drag forces can be controlled in a very
precise manner that enables the best modeling of the biochemical interactions taking part in
the biosensing technology.
Another important factor considered while using nanomaterials for sensing application is
the monitoring and optimization of their optical properties. The phenomena like surface
plasmon resonance are very interesting and in particular expected from nanoparticles so as
to maximize the sharp and precise scale optical response of the sensing materials with the
incident light. The surface plasmon resonance effect is concerned with the excitation of
particle surface with the ionic species and charged particles which create ions and result in
excitation of the fluidic state of charged particles. This property is highly suitable in case of
nanoparticles due to their unique optical properties which give them photonic character and
excellent ability to be used as fluorophores. This phenomenon makes use of total internal
reflection which takes place for angle of incidence reaching beyond a critical value. Here,
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the reflection of light through a thin film of metallic nanoparticles coated over a surface is
optimized by the corresponding adjustment of the critical angle of reflection. In case of
nanomaterials, this phenomenon is highly logical and is especially named as localized
surface plasmon resonance. Surface plasmon resonance effect is also dependent upon the
refractive index of a medium and it is the most fundamental property governing the flow of
light through a medium. Due to the phenomenon of surface plasmon resonance, a
nanobiosensor is better equipped to detect the minutest interacting phenomenon, which
enables a far greater and much reliable degree of estimation of biological interactions
through a nanobiosensor in comparison with a biosensor.
In this way, nanomaterials, irrespective of their nature, need to be optimized for their
performance and effect as per the desired goal before being actually implemented for the
biosensing purpose. Nanostructured semiconductor crystals can be efficiently used to
improve the detection of neurological responses via coupling through the sensing molecule
of biological nature. These can be coupled with peptide assembly of a range of
nanomaterials so that efficient interaction can be generated by means of self-assembly and
this also saves a lot of time that is being involved in the currently available technologies and
methods. Moreover, these can rapidly detect the biological stimulus such as that of a DNA
segment or a characteristic nucleotide sequence pertaining to proteins or even RNA.
Moreover a key strategy into shaping-up of nanomaterials for desired applications involves
the tuning and engineering of their surface by sophisticated inroads collectively termed as
micromachining procedures. Factors like aspect ratios, functionalization with other
materials and compatibility issues with respect to the material being analyzed for are highly
critical for the use of nanomaterials in biosensing applications.
There is a table of most common nanomaterials used in nanobiosensors:
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Carbon nanotubes
Carbon nanotubes were discovered in 1992 and they are cylindrical structures rolled up
graphene sheets. Carbon nanotubes can be formed by one sheet with a diameter form 0,4
to 2nm or by a couple of sheets, with a diameter from 2 and 100nm.
Figure 3: Dimensions of carbon nanotubes.
These nanostructures have a good application in the construction of nanodevices, they
permit the biocomponent to interact with a higher superficial area, providing higher
conductivity and better electrical communication between surfaces and immobilized
biocomponents. The combination of nanotubes with redox active enzymes has promoted
the development of more reactive platforms in nanobiosensors. We can find various
electrochemical biosensors systems, with an improvement in the catalytic signal compared
with the one in the macrocarbon electrode.
Nanoparticles
The most widely used nanomaterial in industry overall to date, however, is the silver
nanoparticle. These have also been harnessed as a simple electrochemical label in a highly
sensitive amperimetric immunoassay intended for distributed diagnostics and as an
inexpensive solution for immunoassays performed in developing countries.
Most recently, nanostructured materials have been used to deliver label-free
electrochemical immunoassays.
Quantum dots
Enzymes are essential in the human body, and the disorder of enzymatic activities has been
associated with many different diseases and stages of disease. Luminescent semiconductor
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nanocrystals, also known as quantum dots (QDs), have garnered great attention in
molecular diagnostics. Owing to their superior optical properties, tunable and narrow
emissions, stable brightness and long lifetime, QD-based enzyme activity measurement has
demonstrated improved detection sensitivity, which is considered particularly valuable for
early disease diagnosis. Recent studies have also shown that QD-based nanosensors are
capable of probing multiple enzyme activities simultaneously.
Nanowires
Nanowire (NW)-based FETs are promising devices with potential applications ranging from
health monitoring to drug discovery. In fact, these devices have demonstrated the ability to
detect a variety of analytes such as particular DNA sequences, cancer biomarkers, and larger
entities such as viruses. These sensor devices have also been used to monitor enzymatic
activities and study the behavior of potential drug molecules. The detection of the analytes
occurs with high specificity and sensitivity in reasonably short time.
1.4. - Types of nanobiosensors.
Biosensors can be categorized according to the basic principles of signal transduction and
biorecognition elements.
In the general scheme of a biosensor (Figure 2), the biorecognition element responds to the
target compound and the transducer converts the biological response to a detectable signal,
which can be measured electrochemically, optically, acoustically, mechanically,
calorimetrically, or electronically, and then correlated with the analyte concentration.
Then we will go to introduce the most popular types of biosensing devices in use today.
Micro-Biosensors
The major progress in microsystem technologies for creating small, integrated and reliable
microtransducers devices in combination with biological sensing elements has
revolutionized the field of biosensors during the last decade. Such micro-biosensor systems
raised the expectation to get a comprehensive insight into dynamic cellular metabolic
events and subsequently a complete understanding of the metabolism of human biology.
Currently, cancer can be detected by monitoring the concentration of certain antigens
present in the bloodstream or other bodily fluids, or through tissue examinations.
Correspondingly, diabetes is monitored by determining the glucose concentrations in the
blood over time. However, despite their widespread clinical use, these techniques have a
number of potential limitations. For example, a number of diagnostic devices have slow
response times and are burdensome to patients. Furthermore, these assays are expensive
and cost the health care industry billions of dollars every year. Therefore, there is a need to
develop more efficient and reliable sensing and detection technologies.
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Within this classification we can find: Electrochemical Biosensors, Potentiometric
Biosensors, Amperometric Biosensors and Voltammetric Biosensors.
Optical biosensors.
Optical detection biosensors are the most diverse class of biosensors because they can be
used for any different types of spectroscopy, such as absorption, fluorescence,
phosphorescence, Raman, SERS, refraction, and dispersion spectrometry. In addition, these
spectroscopic methods can all measure different properties, such as energy, polarization,
amplitude, decay time, and/or phase. Amplitude is the most commonly measured as it can
easily be correlated to the concentration of the analyte of interest [28]. In optical
biosensors, the optical fibers allow detection of analytes on the basis of absorption,
fluorescence or light scattering. Since they are non-electrical, optical biosensors have the
advantages of lending themselves to in vivo applications and allowing multiple analytes to
be detected by using different monitoring wavelengths. The versatility of fiber optics probes
is due to their capacity to transmit signals that reports on changes in wavelength, wave
propagation, time, intensity, distribution of the spectrum, or polarity of the light. In general,
acquisition of the signal from these devices is accomplished through flexible cables, which
can transmit light to the biological component.
The most common optical biosensors are: Fluorescence-based Biosensors, Surface Plasmon
Resonance Biosensors and Optical fiber based biosensors.
Acoustic biosensors.
Electroacoustic devices used in biosensors are based on the detection of a change of mass
density, elastic, viscoelastic, electric, or dielectric properties of a membrane made of
chemically interactive materials in contact with a piezoelectric material. Bulk acoustic wave
(BAW) and surface acoustic wave (SAW) propagation transducers are commonly used. In the
first, a crystal resonator, usually quartz, is connected to an amplifier to form an oscillator
whose resonant frequency is a function of the properties of two membranes attached to it.
The latter is based on the propagation of SAWs along a layer of a substrate covered by the
membrane whose properties affect the propagation loss and phase velocity of the wave.
SAWs are produced and measured by metal interdigital transducers deposited on the
piezoelectric substrate. Even though SAW-based biosensor systems have been the focus of
academic and industrial research for a number of years, most of these approaches only
feature laboratory setups that are suitable for proof-of-principle evaluation and first
experimental tests. For real commercial success, two crucial issues need to be solved: an
appropriate production process is required, as is an applicable handling process for future
SAW based biosensors.
Since 1959, the most used acoustic device for sensor has been The Quartz Crystal
Microbalance (QCM).
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1.5. - Applications of nanobiosensors.
Biosensors have been intensively studied and extensively utilized in various applications
such as:
● DNA sensors: Genetic monitoring, disease.
● Immunosensors: HIV, Hepatitis, other viral diseas, drug testing, environmental
monitoring…
● Cell-based sensors: functional sensors, drug testing…
● Point-of-care sensors: blood, urine, electrolytes, gases, steroids, drugs, hormones,
proteins, …
● Bacteria sensors: food industry, medicine, environmental, …
● Enzyme sensors: diabetics, drug testing, ...
In this document we will focus on nanobiosensors dedicated to the detection of glucose in
the blood (enzyme sensor) and antigen-antibody recognition.
2. - Glucose nanobiosensors.
One of the most common application of the nanobiosensors is used to detect the glucose
levels in the blood. Diabetes is a health problem that affects millions of people all over the
world and part of the treatment involves monitoring the glucose levels in blood with the
nanobiosensors and the enzyme glucose oxidase, so that there can be action taken if these
levels go very high. Glucose oxidase is a small and stable enzyme that oxidizes the glucose
into gluconolactone, converting oxygen into hydrogen peroxide, which is sensed by a
platinum electrode. The hydrogen peroxide is a toxic compound that can be used to kill
bacteria. The more peroxide is formed; the stronger the signal is at the electrode. The
glucose oxidase converts something that is difficult to measure, the glucose, into something
easy to measure, the hydrogen peroxide.
In the last decades it has been a very fast development in the investigation of the
nanobiosensors for controlling the levels of the glucose in blood. The nanobiosensors
nowadays are one of the most important achievements in nanotechnology, due to the fact
that their biocomponents increase sensibility, stability, miniaturization and exchange of
microfluidics with different parts of the body. In particular, the investigation of
nanobiosensors for monitoring the glucose levels has promoted also the design of new
nanomaterials and the integration of nanostructured areas or nanomaterials useful for
developing the construction of biocompatible devices.
Glucose is the main source of energy for the cells, it is transported to the cells through the
insulin in blood. The human body regulates the levels of glucose in the blood in a
concentration of 4-8mM (70-120mgdL-1). Diabetes is a metabolic disease that produces
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anormal levels of glucose in blood and consequently inflammation and apoptosis events in
cells. There are very important problems associated to this disease, like problems in the
retina, circulatory system or kidneys. It is estimated that there will be an increase of 69% of
diabetes patients in the developing countries and a 20% in the developed countries.
In order to control the glucose levels in blood and reduce complications of hyper and
hypoglycemia, the scientific community has devoted important resources on the
development of intelligent diagnostic tools to fight against diabetes. Patients need to
control the glucose levels in blood very frequently with a painful measurement and also
huge variations in the monitoring, consequently the increasing demand of new technologies
to satisfy a continuous and noninvasive monitoring of glucose with high precision and low
cost. Traditional monitoring of blood glucose uses discrete blood sampling time points
during the course of a day, however continuous monitoring allows the patient to intercede
and avoid complications, highlighting the advantages of an increased frequency of
measurement. Another advantage is the estimation of future blood glucose levels and the
possibility to monitor without the patient intervention. On the other hand it has some
disadvantages as all the provided devices are implanted sensors which have a maximum
useful lifetime of several days to a week and as furthermore they have a time lag in
measurements taken during periods of rapid concentration changes, estimated from several
minutes to nearly 30 minutes. Finally, current sensors are expensive and not always covered
by health insurance plans so the technology has not been widely adopted.
2.1 Brief History of Electrochemical glucose biosensors.
The history of glucose enzyme electrodes began in 1962 with the development of the first
device by Clark and Lyons of the Cincinnati Children’s Hospital. Their first glucose enzyme
electrode depended on a thin film of GOx (glucose oxidase) catched by an oxygen electrode
with a semipermeable dialysis membrane. The oxygen consumed by the enzyme-catalyzed
reaction was monitorized. A negative potential was applied to the platinum cathode for a
reductive detection of the oxygen consumption. The entire field of biosensors can trace its
origin to this original glucose enzyme electrode.
In 1973, Guilbault and Lubrano described an enzyme electrode for the measurement of
blood glucose based on amperometric monitoring of the hydrogen peroxide product. The
resulting biosensor offered good accuracy and precision. Use of electron acceptors for
replacing oxygen in GOx-based blood glucose measurements was demonstrated in 1974.
In 1974 was proposed the ex-vivo monitoring of blood glucose and in 1982 was
demonstrated the in-vivo glucose monitoring.
During the decade of the 1980 considerable efforts were made focused on the development
of mediator-based ‘second-generation’ glucose biosensors, introduction of commercial
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screen-printed strips for selfmonitoring of blood glucose, and use of modified electrodes
and tailored membranes for enhancing sensor performance. In the 1990s, there was
extensive activity directed toward the establishment of electrical communication between
the redox center of GOx and the electrode surface.
2.2 Electrochemical biosensors.
The biosensors combine the selectivity of the enzymes with the simplicity of the
amperometric transductors. Figure 1 shows the working process of the device. The analyte
spreads through the solution and throw the membrane until it arrives at the active center of
the enzyme where it reacts creating the product, generally with redox properties. This is
oxidized on the electrode and creates a new product spread onto the solution again.
Figure 1: Glucose detector working process.
We can find three different types of amperometric biosensors with different methods to
transfer electrons between the enzyme and the amperometric transduction.
Figure 2: Three generations of biosensors based on oxidoreductases.
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2.3 Nanobiosensors.
Nanobiosensors are a big advance in order to measure the glucose levels in blood, they have
numerous advantages that can contribute in the improvement of the attention to the
diabetic patients. Nanobiosensors increase the sensibility in the quantification limits and
provide a better analysis and the use of nanomaterials with biocomponents improves the
stability and specificity of the system detection and the reliability. Nanotechnology allows
the miniaturization and integration of biocomponents in complex nanobiosensors, capable
to monitor continuously the glucose with implantable devices like the lab-on -chip for the
rapid detection and low cost. These characteristics have motivated the investigators to
explore alternative strategies based on the different nanotechnologies of nanomaterials or
nanostructures, to develop the optical or electrochemical biosensors. Numerous kinds of
nanomaterials have been used to develop biosensors, related to particles, nanotubes,
nanocables, nanowires, nanofibers, nanocompounds, nanofilms, nanopolímers and
nanoplates. These materials are capable of improving the performance of the detection
systems due to the physic, chemical, mechanical, optical and magnetic characteristics.
The development of nanotechnology has contributed to the efforts to make real the use of
nanomaterials to detect glucose, in a faster response time, operativity stability, selectivity
and easy measurement.
2.3.1 Nanoparticles.
Metallic nanoparticles (NP) have been used in the electrochemical biosensors showing great
advantages in the sensibility of the concentration limits. Gold nanoparticles (AUNPs) are
used to fix the biocomponents in the established platforms. They have excellent
biocompatibility between proteins and gold and also good electroanalytical applications. It
has been demonstrated that the immobilization of the enzyme glucose oxidase in the gold
nanoparticles provides better stability in the biosensors for a long period of time and also
improves the analytical performance of the biosensors.
Nanoparticles of iron oxide (Fe3O4) have also good properties to immobilize the
biocomponents, due to the biocompatibility, paramagnetic properties and low toxicity.
2.3.2 Nanocables, Nanofilms and nanofibers.
Nanocables, nanofilms and nanofibers have exclusive characteristics for the electrons
transfer and superficial area. These materials can be incorporated in high density molds
providing a higher superficial area of high current density and producing an increase on the
transference of electrons and a better sensibility.
Biosensors based in nanocables were developed in 2010 by immobilization of glucose
oxidase with nanocables what made an increase on the enzyme absorption and electrons
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transfer with the area. It produced high sensibility, low detection limits, fast response and
good sensor stability.
Nanofibers together with other polymeric conductor nanostructures have been extensively
used like materials for the biosensors. They have special properties and have been used for
different applications since the electrodes, capacitors and molecular conductor wires
development for the immobilization of the biologic material in the sensors.
Nanoparticles of polietilendioxitofen (PEDOT) are used for the deposition of aladio and
glucose oxidase nanoparticles and nanofibers of Co3O4 for the construction of a no
enzymatic sensor that detects glucose and shows high sensibility, reproducibility and
selectivity.
2.3.3 Carbon nanotubes.
The incorporation of nanomaterials in the sensors, as we have described before, provides a
variety of advantages including increased surface, more efficient electron transfer from
enzyme to electrode and the ability to include additional catalytic steps.
The most common modification in the enzymatic electrode detection of glucose is the
carbon nanotube incorporation. Another approach is to modify the nanotubes with an
electrochemical mediator such as ferrocene to improve the electron transfer between the
enzyme and the electrode. Combining nanotubes with additional nanomaterials (silver,
platinum, gold nanoparticles, etc.) improves aspects such as catalytic activity.
Nanostructured polymers can improve the development of glucose sensors. Hollow spheres
of conductive polymer can be used to transfer electrons from GOx to the electrode;
Conductive polymer electrodes can be used in a method similar to other nanostructured
surfaces, where GOx is immobilized directly in the modified electrode. The use of polymers
introduces the operation at varying potentials.
However, the sensors based on biological recognition have several disadvantages including a
poor stability compared with the nonbiological systems. As a result of this limitation many
research groups have focused on the development of glucose detection assays that do not
rely on a protein for recognition and, as a result, could have longer storage lifetimes. The
most developed research area in nonenzymatic glucose sensors is detection of glucose
oxidation directly at an electrode. Glucose detection has been demonstrated using copper
and copper oxide nanowires, porous films as well as nanoflowers and nanorods. Several of
the direct glucose oxidation sensors perform in biological samples; however they will not
see much utility in clinical settings without significant work to improve their ability to work
in undiluted samples, those obtained routinely by patients.
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2.3.4 Fluorescent polymeric nanosensors.
For in-vivo continuous monitoring, fluorescence based sensors offer the advantage to
optically interrogate the sensors through the skin rather than having an electrode system
implanted. This approach often involves a “smart tatoo” for the patient, as sensors would be
implanted into the skin of the patient. That would be only temporary and would need to be
replaced on weeks or months. These sensors would change fluorescence properties in
response to blood glucose, and this change could be read out using optical interrogation
through the skin. This method would eliminate or reduce the need for patients to take blood
samples while allowing data to be collected in a more continuous manner. A variety of
nanosensor technologies have been developed using fluorescence signals.
2.3.5 Quantum dots in glucose sensors.
Semiconductor quantum dots (QDs) have excellent optical properties to use in sensors such
as narrow fluorescence peaks and minimal photobleaching. However they do not interact
with glucose, and consequently have no inherent recognition ability and must be coupled to
a recognition element for successful implementation. To fabricate sensing systems it is used
the cadmium telluride QDs with the GOx. They allow rapid optical detection of glucose
2.4. Home Testing of Blood Glucose
Since blood glucose home testing devices are used daily to diagnose life-threatening events
they must be of extremely high quality. The majority of personal blood glucose monitors
rely on disposable screen-printed enzyme electrode test strips. Each strip contains the
printed working and reference electrodes, with the working one coated with the necessary
reagents and membranes.
Despite these remarkable technological advances, home testing of blood glucose often
suffers from low and irregular testing frequency or inadequate interpretation of the results
by the patient and requires compliance by patients. More integrated devices offering
multifunctional capability and convenient monitoring of changes in the glucose level are
expected in the near future.
Although self-testing is considered a major advance in glucose monitoring, it is limited by
the number of tests per day it permits and results in poor approximation of blood glucose
variations. Tighter glycemic control, through continuous monitoring, is desired for detecting
changes in the glucose level and to activate an alarm in cases of hypo- and hyperglycemia.
Continuous glucose monitoring provides maximal information about changing blood glucose
levels throughout the day, including the magnitude, duration, and frequency of that
fluctuations, and provides the opportunity of making fast and optimal therapeutic
interventions.
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The ideal sensor would be one that provides a reliable real-time continuous monitoring of
all blood glucose variations throughout the day with high selectivity and speed over
extended periods under hard conditions. An undesirable interaction between the surface of
the implanted device and biological medium cause deterioration of the sensor performance
and it is a barrier to the development of reliable in-vivo glucose probes.
In blood, a complication arises from surface fouling of the electrode by proteins and
coagulation composites and the risk of thromboembolism consequently most glucose
biosensors don’t have the biocompatibility necessary for reliable prolonged operation in
blood. Accordingly, the majority of the sensors being developed for continuous glucose
monitoring do not measure blood glucose directly. Alternative locations, particularly the
subcutaneous tissue, have received growing attention. It is minimally invasive, and its
glucose level reflects the blood glucose concentration. However, such subcutaneous
implantation generates a wound location that experiences an intense local inflammatory
reaction. Although major advances have been made and several short-term in-vivo glucose
sensors are approaching the commercial stage, major efforts are required before a reliable
long-term minimally invasive or noninvasive sensing becomes a reality.
2.5. Subcutaneous Monitoring
Most of the recent attention regarding real-time in-vivo monitoring has been given to the
development of subcutaneously implantable needle-type electrodes.
Such devices track blood glucose levels by measuring the glucose concentration in the
interstitial fluid of the subcutaneous tissue. Subcutaneously implantable devices are
commonly designed to operate for a few days, after which they are replaced by the patient.
They are commonly inserted into the subcutaneous tissue in the abdomen or upper arm.
Success in this direction has reached the level of short-term human implantation;
continuously functioning devices, possessing adequate stability, are expected in the very
near future.
Such devices would enable a swift and appropriate corrective action through use of a
closed-loop insulin delivery system, i.e., an artificial pancreas. Computer algorithms
correcting for the transient difference (short time lag) between blood and tissue glucose
concentrations have been developed. Subcutaneously implantable glucose sensors have
moved from the purely experimental stage to commercially available products.
2.6 Conclusion.
The success of glucose blood meters has stimulated considerable interest in in-vitro and invivo devices for monitoring other physiologically important compounds and new materials
and concepts, developed originally for improving glucose biosensors, now benefit a wide
range of sensing applications.
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Fundamental progress has been made in this area to improve the reliability of glucose
measuring devices but despite the impressive progress in the development of glucose
biosensors, the promise of tight diabetes management has not been accomplished, and
there are still many challenges related to the achievement of a highly stable and reliable
continuous monitoring. In order to achieve this goal the future work should emphasize in
testing with realistic, clinical samples and the cost and effort of the new sensor approaches
should provide also improvements in the patient quality life but minimal additional new
cost.
Nanoscale sensors have the potential to improve continuous glucose monitoring capabilities
and improve patient quality of life, allowing more long-term monitoring and reaching the
goal of closed-loop artificial pancreas. The closed-loop pancreas will eliminate the need for
the patient to perform the steps to inject the insulin, as it would perform all the tasks,
similar to a natural pancreas.
3. - Antibody-antigen recognition.
Antigen-antibody interaction, or antigen-antibody reaction, is a specific chemical interaction
between antibodies produced by B cells of the white blood cellsand antigens during immune
reaction. It is the fundamental reaction in the body by which the body is protected from
complex foreign molecules, such as pathogens and their chemical toxins. In the blood, the
antigens are specifically and with high affinity bound by antibodies to form an antigenantibody complex. The immune complex is then transported to cellular systems where it can
be destroyed or deactivated.
There are several types of antibodies and antigens, and each antibody is capable of binding
only to a specific antigen. The specificity of the binding is due to specific chemical
constitution of each antibody. The antigenic determinant or epitope is recognized by the
paratope of antibody, situated at the variable region of the polypeptide chain. The variable
region in turn has hypervariable regions which are unique amino acid sequences in each
antibody. Antigens are bound to antibodies through weak and noncovalent bonds such as
electrostatic interactions, hydrogen bonds, Van der Waals forces, and hydrophobic
interactions.
The principles of specificity and cross-reactivity of the antigen-antibody interaction are
useful in clinical laboratory for diagnositic purposes. One basic application is determination
of ABO blood group. It is also used as a molecular technique for infection with different
pathogens, such as HIV, microbes, and helminth parasites.
3.1. - Antibody structure.
In an antibody, the FAB (fragment, antigen-binding) region terminates into an aminoterminal end of both the light and heavy chains of the immunoglobulin polypeptide. This
region called V (variable) domain is composed of amnio acid sequences that define each
type of antibody and their binding affinity to an antigen. The combined sequence of variable
light chain (VL) and variable heavy chain (VH) creates three hypervariable regions (HV1, HV2,
17
and HV3). In VL these are roughly from residues 28 to 35, from 49 to 59, and from 92 to 103,
respectively. HV3 is the most variable part. Thus these regions are the paratope, the binding
site of antigen. The rest of the V region between the hypervariable regions are called
framework regions. Each V domain has four framework domains, namely FR1, FR2, FR3, and
FR4.
3.2. - Detection of individual antibody-antigen recognition events by atomic force
microscopy (AFM)
A methodology has been developed for the study of molecular recognition at the level of
single events and for the localization of sites on biosurfaces, in combining forcé microscopy
with molecular recognition by specific ligands. For this goal, a sensor was designed by
covalently linking an antibody (anti-human serum albumin, polyclonal) via a flexible spacer
to the tip of a force microscope. This sensor
permitted detection of single antibody-antigen recognition events by force signals of unique
shape with an unbinding force of 244 + 22 pN. Analysis revealed that observed unbinding
forces originate from the dissociation of individual Fab fragments from a human serum
albumin molecule. The two Fab fragments of the antibody were found to bind
independently and with equal probability. The flexible linkage provided the antibody with a
6-nm dynamical reach for binding, rendering binding probability high, 0.5 for encounter
times of 60 ms. This permitted fast and reliable detection of antigenic sites during lateral
scans with a positional accuracy of 1.5 nm. It is indicated that this methodology has promise
for characterizing rate constants and kinetics of molecular recognition complexes and for
molecular mapping of biosurfaces such as membranes.
18
3.3. Antibody´s recognition introduction
The invention of Scanning Force Microscopy (SFM) and its modification to optical detection
of forces has opened the exciting perspective of imaging the surface of living biological
specimens. The additional potential of SFM for the study of molecular recognition, using a
measuring tip with ligands bound, has recently gained much attention.
The idea is to detect and study the binding of ligands on tips to surface-bound receptors
by applying an increasing force to the complex that reduces its lifetime until it dissociates
at a measurable unbinding force.
So far, interaction forces were reported for the ligand-receptor pair biotin-avidin and for
complementary DNA nucleotides. For these studies, SFM tips were covered with
immobilized ligands. This strategy failed for antibody-antigen recognition (1), and the failure
was attributed to the lack of molecular mobility and to unspecific tip-probe adhesion forces,
obscuring specific interactions.
Apart from detection and study of single recognition events, the concept of using SFM tips
with ligands ("sensors") has further perspectives:
(i)
(ii)
(iii)
for localizing individual recognition sites.
for imaging their distribution at surfaces.
for combining recognition imaging by ligands with structural imaging by the tip as
a method for molecular mapping of the topography of biosurfaces.
Advances toward this goal are expected from the realization of an appropriately designed
sensor. The ideal sensor configuration appears to be a single ligand on a tip that is free to
orient and move for unconditioned recognition during surface imaging by the tip. We
approached this configuration by covalently coupling ligands to tips via a long flexible spacer
19
molecule at a sufficiently low ligand concentration so that about one ligand is expected to
have access to surface-bound receptors.
We are going to use an antibody as the sensor molecule trying to demonstrate the
suitability of this sensor design for detection and characterization of single antibodyantigen recognition events and for the localization of antigenic sites. Perspectives of the
method are discussed.
3.4. Method
Preparation of Sensor and Probe. A newly synthesized 8nm long polyethylene glycol (PEG)
derivative with an aminereactive end and a thiol-reactive end was used as spacer for
covalent linkage of the antibody to silicon nitride tips and of the antigen to mica surfaces.
Tips were cleaned by a standard procedure for 10 min in chloroform and for 30 min in
H2SO4/H202, 70:30 (vol/vol), and then extensively rinsed with deionized water.
Surface-bound water was removed by drying freshly cleaved mica and cleaned tips in an
oven for 2 hours at 180°C. For functionalization with amine-containing groups, tips and mica
sheets were then immediately esterified in a solution of ethanolamine chloride in dimethyl
sulfoxide, overnight at 100°C with 0.3-nm molecular sieve beads while applying an aspirator
vacuum and trapping H20 in a CaCl2 tower. Binding of PEG to amine-containing substrates
and consecutive coupling of antibody and antigen to the thiol-reactive end of the spacer
was done as described.
Determination of Surface Density: Sensitive high-resolution fluorescence imaging with
accurate calibration was employed for the determination of the surface density of HSA and
antibody, which had been fluorescence-labeled prior to surface linkage.
Probes with high HSA densities showed considerable clustering in fluorescence images.
Force Microscopy. Cantilevers of sensors had spring constants between 0.11 and 0.27 N/m.
Briefly, a silicon cantilever with a spring constant of kr = 0.18 ± 0.02 N/m was taken as
reference lever. The silicon nitride cantilevers used in the study were calibrated by
comparing their repulsive force-distance slopes on a solid support with slopes found in
contact with the reference lever. All silicon nitride cantilevers were calibrated with the same
reference lever and calibrations were carried out in buffer.
20
Adhesion forces between sensor tip and probe were generally absent. For applications of
the sensor to biosurfaces, sensors showed no adhesion to a cell membrane (mast cell) or to
a lipid membrane (dimyristoylphosphatidylcholine) on mica in aqueous solution with 150
mM NaCl.
3.5. Results and discussion
For a sensor molecule on the SFM tip, we chose a polyclonal anti-HSA antibody. The
antibody was covalently linked to the tip. The density of antibodies on tips was adjusted to
best meet the expectation that only one antibody may interact with the probe. More
specifically, the SFM tips carried many antibodies. Their surface density was, however,
chosen sufficiently low so that, on the average, only about one of the flexibly linked
antibodies is expected to be bound to the tip end that will reach to HSA molecules on the
probe surface. Forces between sensors and probes with high HSA surface density were
monitored during force-distance cycles (figure A) by moving the probe continuously up
("trace") and down ("retrace") at constant lateral positions. It presents evidence that the
sensor permits detection of single antibody-antigen binding events.
(A) Effect of antibody-antigen binding on the force between tip and probe, illustrated for a
typical record of a force-distance cycle. Binding of the antibody to the antigen during
approach connects tip to probe. This causes an extra force signal of particular shape during
tip retraction ("retrace"), reflecting extensión of the flexible connection to its full length. The
force increases until unbinding occurs at the unbinding force. The length of the connection at
the moment of unbinding was determined by subtracting tip deflection from the distance
between contact and unbinding.
Retraces of force-distance cycles showed attractive force signals of unique shape,
interpreted to reflect antibody-antigen recognition. The attractive force develops
nonlinearly with a significant delay as expected from stretching the long and flexible tipprobe connection after antibodyantigen association. The connection sustains the increasing
force until the complex dissociates at a characteristic "unbinding force,". Such signals were
repeatedly observed in consecutive force-distance cycles. Typically, 700 cycles were
recorded at one fixed lateral position for which the 50 retraces shown in figure B are
21
representative. Twenty five of the retraces show one or two unbinding events. In the other
cycles, there was no event detectable and retraces were identical to traces.
(B) Force records of consecutive force-distance cycles. Unbinding events, shown here as
upward signals, occurred in 25 out of the 50 records (only retraces are shown).
One experiment is shown in figure C. The binding probability, Pb, which is the probability for
observing an unbinding event in a forcedistance cycle, was followed before and after
addition of HSA and after its removal (wash). The high initial value of Pb is immediately
reduced to about 10% upon addition of free HSA and fully recovered about 27 min after
perfusion with buffer. The few events detected after addition of free HSA are attributed to
adsorption ofHSA molecules from solution to the probe surface. In additional controls using
tips and probes with the PEG spacer bound but not conjugated with antibody and HSA,
retraces were generally devoid of unspecific sensorprobe adhesion forces (retraces and
traces were identical). Small adhesion forces were resolvable in less than 1% of the retraces
that were easily discernible from unbinding events due to their clear separation from the
delayed occurrence of unbinding. From controls and from observing unique signals of
expected shape in the absence of adhesion forces, it appears safe to conclude that the
signals are specifically due to antibody-antigen recognition. The ability of the antibody to
bind, tested in more than 8000 force-distance cycles with four sensors, was found not to
deteriorate, even after 2 month of storage in buffer. The force signal for unbinding contains
information about antibody-antigen recognition at the level of single molecule
interaction.
(C) Time profile of antibody-antigen unbinding events for block by free HSA and for its
recovery upon HSA removal. The probability per force-distance cycle for antibody binding to
surface-bound HSA was Pb = 0.49 (0-30 min). Injection of 0.15 tpM free HSA resulted in a
sudden and effective decrease of Pb to 0.06. After 30 min, the probe was washed for 1 min
22
with buffer after which unbinding events fully recovered within 27 min. Bars indicate times of
continuous recordings of force-distance cycles at 1 Hz and their heights show the mean Pb
values observed.
In the figure 2 shows unbinding force and unbinding length distributions, determined from
201 consecutive forcedistance cycles at one position on a probe at high HSA density This
unitary force is attributed to the recognition of single HSA molecules by one of the two
binding sites of the antibody.
The distribution of sensor length lu in figure 2B extends to values up to 30 nm. Irrespective of
the apparent difference in sensor configuration during unbinding, the two Fab fragments
showed virtually identical unbinding force distributions (Figure 2A). These findings indicate
that the configuration of the molecular link and its momentary length during unbinding does
not influence the unbinding force, attributed to the flexibility introduced by the spacer
molecule.
The described analysis was used to select sensors with effectively one antibody having
excess to surface-bound antigens, selected by criterion of no more than two events per
retrace. This applied to about 30% of the sensors in preparations at appropriate antibody
surface density. For the other sensors up to six events were found in force-distance cycles,
indicating that up to three antibodies were able to bind to HSA on the surface.
The flexibly linked antibody was not found to be entrapped between tip and probe since the
points of tip-probe contact in force-distance cycles were as expected for a plain surface. This
also indicates that the imaging capability of the tip is not significantly perturbed by the
chemical treatment for antibody linkage. This opens the perspective for a microscopy
capable of simultaneously imaging surface topography and the distribution of recognition
sites or of mapping the molecular topography of biosurfaces. While the sensor appears apt
for such uses, realization will require software extension of measuring modes to area scans
during force-distance cycles at an appropriate feedback control that is currently being
developed.t Potentials for Studying Kinetics of Antibody-Antigen Interaction. From the
dynamical reach reff and the vertical scan velocity, it is possible to estimate the antibodyantigen association rate constant kass.
23
This confirms the conclusion that the antibody is quite free to move and orient for binding
within the constraints set by its dynamic reach, which compares with the length of the PEG
spacer. The recovery time from block by free HSA of 1500 s is a direct measure for the
lifetime of the antibody-antigecomplex in the absence of force or for its dissociation rate
constant.
Figure 3. Localization of antigen sites by a scanning tip-antibody sensor. (A) Histogram of
unbinding events for the sensor passing one HSA molecule. The probe was laterally moved at
0.6 nm/s during force cycles at 3 Hz with a 100-nm amplitude. The number of events was
sampled every 2.6 nm, corresponding to 13 cycles. The recognition profile represents 23
unbinding events in total. Mean distance between HSA molecules was 100 nm, as
determined by fluorescence microscopy.
Statistical analysis showed that the peak position reflects the true mean of the distribution
within an uncertainty of 1.5 nm. (B) Overlay of binding profiles as seen inA. The distribution
contains the data from six profiles, normalized to the average binding probability of 0.38, at
maximum. It has a width of 6 nm, determined from the standard deviation of the Gaussian
fit shown. All events occurred singly in force-distance cycles, indicating recognition of single
HSA molecules.
The mean fu value was 270 pN for the chosen cycle rate of 3 Hz and cantilever spring
constant of 0.22 N/m.
3.6. Conclusions
Flexible linkage of an antibody to an SFM tip has allowed thedetection of single recognition
events between an antibody and an antigen. Analysis of force profiles revealed insight into
the process of antibody-antigen binding and unbinding at the level of single molecular
events, unconstrained by the linkage used. Antigenic sites were reliably detected during
lateral scans. This was rendered possible by realization of a sensor design with effectively
one antibody covalently coupled to the tip via a sufficiently long flexible spacer molecule.
The functional groups of the spacer used are applicable to coupling of ligands and proteins
in general, which provides the method with a broad perspective in the study of molecular
recognition. With the possibility of combining molecular recognition by ligands with
structural resolution by the tip, a first tool comes into sight for molecular mapping the
topography of biosurfaces.
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Schematic diagram for metal oxide nanostructure-based biosensing devices.
The generation of output potential for the fabricated sensor device could result from the
reaction mechanism, which creates a charged environment around the working electrode,
and the resulting potential difference can be measured using a pH meter. It has been shown
that CuO itself exhibits catalytic properties that can improve the efficiency of cholesterol
oxidase and create a rapid and direct electron transfer rate between the active sites of
cholesterol oxidase and its own surface. Moreover, the fabricated cholesterol biosensor
using a bundle of CuO nanowires was found to be highly reproducible, repeatable, stable
and selective.
TEM images of nanowire bundles; (d) calibration curve of cholesterol biosensor with
detection limit; and (e) response time of the cholesterol biosensor in the different cholesterol
concentrations [98].
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4. - Bibliography.
• NANOBIOSENSORS. Nano Science & Technology
http://es.slideshare.net/tabirsir/nanobiosensors-5734391
Consortium.
Disponible
en:
• NANO-TECHNOLOGY. DEVELOPMENT OF NANO-BIOSENSORS. Thompson Research Group.
The University of Southern California. Disponible en: http://met.usc.edu/projects/nano.php
• Florinel-Gabriel Banica. CHEMICAL SENSORS AND BIOSENSORS: FUNDAMENTALS AND
APPLICATIONS.
• Ahmed Touhami. BIOSENSORS AND NANOBIOSENSORS: DESIGN AND APPLICATIONS.
Physics & Astronomy Department, University of Texas at Brownsville
• RECENT TRENDS IN NANOBIOSENSORS AND THEIR APPLICATIONS - A REVIEW. Department
of Physics, Sree Sastha Institute of Engineering and Technology, Chembarambakkam,
Chennai-600 123, India. Department of Biotechnology, Sree Sastha Institute of Engineering
and Technology, Chembarambakkam, Chennai-600 123, India.
• NANOSENSORS WITH APPLICATIONS. Prathamesh V. Kolekar Dept. Instrumentation Engg.
Vishwakarma Institute of Technology, Pune.
• THE GRAMICIDIN-BASED BIOSENSOR: A FUNCTIONING NANO-MACHINE. Novartis Found
Symp. 1999;225:231-49; discussion 249-54.
• NANOBIOSENSORS: CONCEPTS AND VARIATIONS. Parth Malik,1 Varun Katyal,2 Vibhuti
Malik,3 Archana Asatkar,4 Gajendra Inwati,1 and Tapan K. Mukherjee5. ISRN Nanomaterials
Volume 2013 (2013), Article ID 327435
• QUANTUM DOT-BASED NANOSENSORS FOR DIAGNOSIS VIA ENZYME ACTIVITY
MEASUREMENT. Expert Rev Mol Diagn. 2013 May. Knudsen BR1, Jepsen ML, Ho YP.
• REAL-TIME, LABEL-FREE DETECTION OF BIOLOGICAL ENTITIES USING NANOWIRE-BASED
FETS. Curreli, M. ; Dept. of Chem., Univ. of Southern California, Los Angeles, CA ; Rui Zhang ;
Ishikawa, F.N. ; Chang, Hsiao-Kang.
• Peter hinterdorfer, werner baumgartner, hermann j. Gruber, kurt schilcher, and hansgeorg
schindler. Institute for biophysics, university of linz, a-4040 linz, Austria.
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