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Optical Sensor
Optical sensors are another significant application of optics
technology that has been already studied for 30years in industrial,
medical and engineering applications.
From: Polymer Optical Fibres, 2017

Related terms:

Biosensor, Protein, Ion, Behavior as Electrode, Refractive Index, Wavelength,

Nanomaterial, Luminiscence Type, Fluorescence

Carbon nanotube-based optical platforms for

biomolecular detection
J. Pan, ... J.H. Choi, in Carbon Nanotubes and Graphene for Photonic Applications,
2013

10.1.1 Background
Optical sensors are powerful analytic tools capable of providing analyte information
remotely. Typically, optical sensors for chemical or biological molecules are
composed of molecular recognition elements and signal transducers.1 The
molecular recognition elements interact with the target analytes under study and
provide information about their presence, concentration, and other physical
properties of interest. When target molecules enter the system, the sensors
produce detectable changes in their signals – which are then transduced into easily
measured and quantified optical signals. Compared to other types of sensors that
use electronic,2 electrochemical,3 or mechanical transduction,4,5 optical sensors are
particularly beneficial for remote and multimodal detection, which is preferred in

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chemical and biological applications.6 Optical sensors have utilized various

spectroscopic measurements, including fluorescence,7–9 Raman scattering,10,11 and
surface plasmon resonance (SPR).12
In the development of optical sensors, it is important to evaluate using common
performance indicators such as sensitivity, selectivity, and response time.
Sensitivity can be understood as a change in the measured optical signals
corresponding to a change in the amount of analyte being used. It is closely related
to the detection limit of sensors, which is the minimum resolvable signal in the
system considering the presence of noise interference in the transduction signal.
Selectivity is the degree of a sensor's ability to respond to specific analytes while
resisting interaction with other molecules. Sensor response time is related to the
kinetics of both the molecular recognition and signal transduction processes.
In the design of optical sensors with improved performance indicators, target
molecules13–15 or molecular recognition elements16 are tagged with fluorescent
labels that can be used to generate optical signals. There are a variety of
fluorophores available for use as fluorescent labels, including quantum dots17
fluorescent proteins,18 and organic dyes.19 While organic fluorophores are widely
used, they exhibit several drawbacks, such as fast photobleaching,20 which hinder
their capability to construct more sensitive and robust optical sensor architectures
in complex chemical and biological environments. Consequently, the development
of new types of fluorophores is required. These new fluorophores must have a high
signal-to-noise ratio in biological/chemical environments and high photostability
for long periods of time.
Nanotechnology is now yielding new classes of materials applicable to the
detection of chemical and biological molecules. These materials (some of which
include nanotubes, nanowires, and nanoparticles) offer unique electrical, magnetic,
and optical properties that can be exploited for sensor applications21 Single-walled
carbon nanotubes (SWCNTs) are particularly promising materials for use in novel
optical sensor design because of their structural, electronic, and optical properties.
SWCNTs are especially sensitive to changes in their surface chemistry,22 and will
fluoresce in the near infrared (NIR) range with excellent photostability. These
optical properties enable them to overcome the limitations of other fluorophores,
discussed above. Moreover, SWCNTs can detect target molecules at a single
molecule level based on single SWCNT spectroscopy,23–26 thus enabling the design
of ultrahigh sensitive optical sensors.

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Applications III: Functional Materials, Environmental

and Biological Applications
A.E.G. Cass, in Comprehensive Organometallic Chemistry III, 2007

12.12.2.2 Optical Sensors

Optical sensors based upon luminescence often offer very high limits of detection
combined with versatile sensing formats. Organometallic compounds are usually
employed with either fluorescence or electrogenerated chemiluminescence sensing
and the typical format exploits optical fiber or planar waveguide-based sensors.
Both types of luminescence sensors measure the emission of light from an
electronically excited state, in the former case from photoexcitation and in the
latter via an excited state generated through an electrochemical redox reaction.
Fluorescence and electrogenerated chemiluminescence biosensors have recently
been reviewed in Ref: 18. In this regard coordination complexes of ruthenium or
lanthanides have been widely used.

Smart devices
A.C. Peixoto, A.F. Silva, in Bioinspired Materials for Medical Applications, 2017

11.4.6 Optical sensors

Optical sensors have the capacity to detect and quantify various properties of light
such as intensity, frequency, wavelength, and polarization. These sensors rely on
light detectors that have the ability of converting light into electrical signals. These
detectors are able to sense electromagnetic radiation from the infrared up to the
ultraviolet wavelengths.
Many light detectors such as the photovoltaic and photoconductive, depend on the
interaction between photons and the crystalline lattice of semiconductors. This
interaction is known as the photoelectric effect, and was firstly proposed by
Einstein in 1905. The photoelectric effect explains that when a photon hits the
surface of a conductor it generates a free electron (Fig. 11.17). These free electrons
will affect the material’s electrical properties, therefore allowing the measurement
of the properties of the incident light.

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Fig. 11.17. Photoelectric effect.

A widely used tool for optical sensors is the photodiode. This device is capable of
converting light into electric current and thus being able to sense light intensity.
This device is composed of a p- and n-type semiconductor bonded together
forming a diode. These devices are similar to semiconductor diodes, except that
their packaging allows light to hit the pn-junction. When reversely biased, as light
hits the semiconductor, free electrons will be generated and the current flowing in
the circuit will increase quite noticeably. Even when there is no light, the circuit will
generate a small current known as dark current. This current is independent of
applied voltage and varies only with temperature. On the other hand, when light is
shining over the semiconductor a significantly higher current will flow through the
circuit and is generated by the photocurrent.
One example of an optical sensor used for medical applications is the pulse
oximeter. This device is placed in a thin part of the patient’s body such as a
fingertip or earlobe. Two lights with different wavelengths are passed through the
patient’s body and absorbed in a photodetector on the other side. This system is
able to detect the saturation of oxygen in the blood (Parker, 2004).

Luminescent Optical Sensors Based on Nanoscale

Molecularly Imprinted Polymers
Meiping Zhao, ... Shanshan Wang, in Molecularly Imprinted Sensors, 2012

6 Conclusion

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Optical sensors developed on the basis of MIP at nanoscale exhibit excellent

performance in comparison with their conventional-sized counterparts, typically
represented by the thorough removal of the template, fast response time, and
easiness of combination with other functionalities. Recently published work has
shown many encouraging results of various MIP nanostructures, including
nanoparticles, nanowires, and nanofilms, for miscellaneous templates and
applications. However, some remaining key issues still need to be addressed in the
development of nano-MIP-based optical sensors.
The most urgent issue is how to further enhance the sensitivity of the MIP
optosensing system. A possible solution is to incorporate efficient signal
amplification steps. Another critical problem is to further reduce the nonspecific
binding of undesired components by the polymer matrix. To address this problem,
functional monomers need to be designed more precisely. Catalytic sensors are
often superior to the sensors solely dependent on the binding event itself.
Detection of the product from the analyte conversion may increase the sensitivity
and avoid interference by nonspecific binding. The reports on such exploration are
still very rare. Another important trend in this field goes toward miniaturization,
high-throughput, and multisensing. On-chip MIP biosensors have shown the
advantages of compact size, high sensitivity, high selectivity, low cost, and fast
response. Integration of the latest advanced microfabrication and signal processing
techniques may significantly improve the performance. Nano-MIP-based sensors
have great potential for wide applications in clinical tests, environmental
monitoring, and other analytical areas.

Recycling of Plastics
DrAdrian Merrington, in Applied Plastics Engineering Handbook, 2011

Optical
Optical sensors can be used to separate plastics based on either color or
transparency. This technique is used to separate bottles by color. Developed from
the coffee bean separators that are used to eliminate unripe green beans from the
mix, these computer-controlled systems can rapidly differentiate between the
various hues of the plastic regrind. The color of each piece of plastic is quickly
established using a type of charge coupled device (CCD) camera and is either
allowed to flow downward or ejected with a puff of air into the reject or collection
pile. Satake, for example, produces equipment that is specifically designed to
separate plastics by color but their main business is still from the agricultural

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industry. Satake's ScanMaster IE & DE Optical Sorters are used to differentiate the
colors of HDPE and PET materials in recycling applications [59].

Molecularly Imprinted Polymers for Sensors

Adnan Mujahid, Franz L. Dickert, in Molecularly Imprinted Sensors, 2012

4 Optical Transducers
Optical sensor technology [35] has found significant applications in various fields
such as biotechnology, environmental monitoring, pharmaceuticals, and clinical
analysis. Due to the extraordinary features of MIPs, they can easily be coupled with
optical devices. There are several possible designs for optical sensors, depending
on the optical properties of analyte molecules. If an analyte possesses some optical
features, their recognition can be executed by MIPs. In optical transducers, the
information of binding events on MIP surface is not directly converted into
measurable electronic signals. In fact, the change in optical properties, including
absorption, reflection, or fluorescence of layer material is recorded upon analyte
interactions. Various spectroscopic approaches have been followed for designing
MIP optical sensors.
4.1 UV/Vis Absorption Methods
UV/Vis spectroscopy is one of the most simplified and economical methods for
examining analyte interactions with MIPs where only the change in absorbance is
measured as a function of wavelength. The technique is versatile and gives rapid
response regarding quantitative information on template binding. Besides pure
sensing application, this method is quite suitable for screening [36] MIPs and
choosing the finest polymer composition. With the help of the UV/Vis spectrum,
the thorough mechanism of complexation between templates, monomer, and
cross-linker during polymerization can also be better understood. It has been
observed that after complexation, an absorbance shift toward shorter wavelengths
takes place. The procedure makes it easy to compare the spectrum of free template
and functional monomer with that of the complex formed. This strategy is equally
suitable for monitoring metal polymer complexation in visible regions [37].
Although UV/Vis spectroscopy is not as selective as the fluorescence method, it is
nevertheless quite suitable for designing low-cost MIP sensors with moderate
sensitivity.
4.2 Surface Plasmon Resonance (SPR) Transducers

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The SPR technique was initially used for measuring the change in thickness and
refractive index of thin films upon analyte interaction. Conventionally, it was
adopted for exploring recognition sites in natural antibodies [38,39] for the
adsorption of larger molecules such as proteins, thus designing biosensors. It can
also be combined with imprinted polymers; however, small-size template
molecules do not significantly contribute to a change in the refractive index, and
therefore, the sensitivity is often limited. In contrast to natural receptors, the
primary advantages in using MIPs are their robustness and their excellent storage
over long periods of time. Lotierzo et al. [40] designed homogenized thin MIP
films for the detection of domoic acid and compared the sensor robustness and
sensitivity against natural receptors, that is, monoclonal antibodies. It has been
observed that the recognition properties are not disturbed upon regeneration in
MIPs compared to natural counterparts. Moreover, MIPs can continuously perform
measurements for about two months without losing performance, whereas
antibody can be denatured after some regeneratiuons.
4.3 Fluorescence-based Transducers
The most frequent method for combining optical devices with MIPs is done by
means of fluorescence spectroscopy[41,42]. Enhanced sensitivity, excellent
selectivity, and low-detection limits make them ideally suited to combine with
imprinted layer materials for sensor development. Optical waveguides can be used
as fluorescence transducers as this setup is highly significant for fluorescent
analytes. The working principle is illustrated in Fig. 6.3. The thickness of the layer
material and the large surface size of waveguides drastically contribute to
fluorescence emission

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FIGURE 6.3. Principle of a planar waveguide sensor.

Fluorescence microscopy[43,44] is also a useful way to access the MIP structures,

calculating the number of binding sites and their efficiency for analyte recognition.
There are several ways to design fluorescent sensors using MIPs. One very
prominent method is using fluorescence templates or labeled analytes. This
scheme is quite useful not only for sensor development but also for evaluating MIP
structure, homogeneity, and binding properties. However, this sensing scheme is
no more practical when dealing with nonfluorescence analyte molecules.
The problem can be solved through the formation of fluorescent complexes
between monomers[45,46] possessing inherent fluorescent properties. This actually
allows the use of fluorescence spectroscopy in optical sensing for those analytes
that are not intrinsically fluorescence active. The relative shifts in emission
wavelength can be recorded in the presence of the initial amount of target analyte
molecules; therefore, the information is quantified. This approach has been
extensively studied for synthesizing new functional monomers that are capable of
separating closely related compounds.
Rathbone et al. [47] successfully developed hydrogen bonding monomers and their
use for fluorescence assay of drugs. Another very elegant way of designing MIP
optical sensors is the integration of fluorescent dyes into the polymer matrix. This
method is suitable for those analytes without having any specific optical property.
When the analyte molecules interact with the polymer system, the fluorescence
signal is quenched and therefore the sensing becomes possible. The scheme has
been proven effective in designing chemosensors for essential biological molecules
such as cyclic adenosine monophosphate (cAMP). Chow et al. [48] focused on the

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derivatization of nonfluorescent analytes such as homocysteine by tagging it with

pyrenyl maleimide which is fluorescent active. In general, these approaches are very
promising for detection of no-fluorescent analytes.
Some other optical phenomena are also studied for introducing them with MIPs in
sensor designs, including chemiluminescence [49], reflectometric interference
spectroscopy [50] analogues to SPR, and surface-enhanced Raman scattering
techniques. Besides all presented methods, fluorescence is the most widely
adopted technique and the best choice available for integrating MIPs in optical
sensor devices.

SURFACE PLASMON RESONANCE BIOSENSORS

Jiří Homola Ph.D., ... David Myszka Ph.D., in Optical Biosensors (Second Edition),
2008

4.1.3.2 Optical sensors based on surface plasmon-polaritons

Optical sensors based on resonant excitation of SPWs, often referred to as SPR
sensors, are optical devices that exploit the sensitivity of the propagation constant
of an SPW to refractive index to measure changes in the refractive index or changes
in other quantities that can produce changes in the refractive index. A change in
the refractive index to be measured produces a change in the propagation constant
of the SPW, which results in a change in the characteristics of the LW interacting
with the SPW (see Section 4.1.2.4). Based on which characteristic of the LW
interacting with the SPW is measured, SPR sensors can be classified as follows:
Surface plasmon resonance sensors with angular modulation.
The component of the LW's wavevector parallel to the metal surface matching that
of the SPW is determined by measuring the coupling strength at multiple angles of
incidence of the LW and determining the angle of incidence yielding the strongest
coupling (Figure 4.14a, upper plot). The wavelength of the LW used to excite an
SPW is fixed.

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Figure 4.14. (a) Reflectivity and phase for a TM LW exciting SPWs in the
Kretschmann geometry (SF14 glass prism – 50-nm-thick gold layer – dielectric) as
a function of the angle of incidence for two different refractive indices of the
dielectric; wavelength = 682 nm. (b) Reflectivity for a TM wave in the same

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geometry as a function of the wavelength for two different refractive indices of the
dielectric; angle of incidence = 54°.

Surface plasmon resonance sensors with wavelength modulation.

The component of the LW's wavevector parallel to the metal surface matching that
of the SPW is determined by measuring the coupling strength at multiple
wavelengths and determining the wavelength yielding the strongest coupling
(Figure 4.14b). The angle at which the LW is incident onto the metal film is kept
constant.
Surface plasmon resonance sensors with intensity modulation.
The change in the intensity of the LW interacting with the SPW is measured (Figure
4.14b). Both the angle at which the LW is incident onto the metal film and its
wavelength are kept constant.
Surface plasmon resonance sensors with phase modulation.
The shift in phase of the LW interacting with the SPW is measured (Figure 4.14a,
lower plot). Both the angle at which the LW is made incident onto the interface and
its wavelength are kept constant.
Surface plasmon resonance sensors with polarization modulation.
The amplitude and phase of the TM-polarized wave interacting with the SPW
change if the propagation constant of the SPW changes. Transverse electric-
polarized LW does not interact with SPWs and thus exhibits no resonant amplitude
and phase variations. Therefore, the polarization state of the incident LW consisting
of both the polarizations would also be sensitive to variations in the propagation
constant of the SPW.

Spectrometric Determination of Lanthanides Series

Mohammad Reza Ganjali, ... Parviz Norouzi, in Lanthanides Series Determination
by Various Analytical Methods, 2016

Optical UV–Vis Sensors for Lanthanide Series

Optical sensors form another group of chemical sensors, and the signal used for
detection of the analyte is the changes in the absorption or fluorescence of the
sample. These so-called optical electrodes or optodes are hence devices used for
measuring specific substances through the use of optical signals usually with the
aid of a chemical transducer. The devices are generally composed of some major
parts, including a substance with the tendency to respond to the analyte, a
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polymeric matrix for immobilization of the chemical transducer, an optical fiber, a

light source, a detector, and other electronic parts. These devices can be used
through different optical modes such as reflection, absorption, evanescent wave,
luminescence (fluorescence, phosphorescence, chemiluminescence), and surface
plasmon resonance
Because the measurement of lanthanides is the major focus of the current
document, it is necessary to mention that in the case of these elements, the most
popular modes used for the measurement are UV–vis and luminescence.
These devices, which can be used as alternatives for other analytical instruments,
are rather popular for their low cost, low power requirements, and long lives. The
mechanism of action in the case of lanthanide analytes is generally based on the
selective interaction of a chelating agent which has been immobilized in an organic
or inorganic matrix, with the lanthanide cations, which results in the formation of
complexes that produces an optical signal, which is an indicator of the amount of
the analyte. The common supporting materials include PVC or cellulose
derivatives, which in addition to the desirable physical properties, are transparent
in the spectral area under study. The use of these supports offers several further
advantages, such as freedom from electrical noise, possibility of remote sensing,
and low cost of manufacturing.
Among the different classes of optodes, the colorimetric sensors suffer the
disadvantage of lower sensitivities than electrochemical and potentiometric
sensors, but the fluorescent or luminescentdevices can lead to comparatively lower
detection limits, which make these devices interesting. The lines below shall cover
an overview of the UV–vis optodes reported for the lanthanide ions.
Zare-Dorabei et al., in 2009 [615], developed a selective and sensitive Gd(III) optode
through the immobilization of (Z)-N’-((pyridine-2-yl) methylene)-thiophene-2-
carbohydrazide (PMTC) (Fig. 6.12) on a triacetylcellulose membrane and observed
linear responses in the range of 5.0 × 10–8 to 2.0 × 10–5 M, reaching detection
limits down to 1.1 × 10–8 M. Other characteristics of the device included its short
response time of 1–2 min (depending on the Gd(III) concentration) and rather wide
range of pH independent responses in the pH range of 2.0–9.0. The optode
reportedly showed very good selectivity in the presence of La, Ce, Sm, and Eu ions,
and was successfully regenerated using thiourea solutions.

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Figure 6.12. Structure of (Z)-N′-((pyridine-2-yl) methylene) thiophene-2-

carbohydrazide (PMTC).

The transparent triacetylcellulose membranes used for immobilizing the ligand

were prepared from photographic film tapes, through their chronic treatment with
sodium hypochlorite (for removing the colored gelatinous layers), before treating
them with a clear solution of 0.02% (W = V) PMTC in 10 mL ethylene diamine for
2 min and washing them with water (for removing ethylene diamine and the
loosely trapped indicator). The films were stored under water before use.
Bis(thiophenal) pyridine-2,6-diamine (BPD) (Fig. 6.13) [616] has also been used for
the construction of Gd3+ optodes, which show linear behaviors in the range of
2.5 × 10–6 mol/L to 0.93 × 10–8 mol/L, with the identical response time and pH
behaviors and good selectivity toward the analyte as opposed to La, Ce, Sm, Tm,
Ho, and Eu ions.

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Figure 6.13. Structure of bis(thiophenal) pyridine-2,6-diamine (BPD).

The same group devised Dy3+ optodes through the immobilization of N’-[(2-
hydroxyphenyl)methylene] benzohydrazide (BBH) (Fig. 6.14), on a triacetylcellulose
membrane [617], and achieved linear responses in the range of 5.0 × 10–7 to
8.0 × 10–6 M (but rather shorter response times as compared to the mentioned
Gd3+ sensors (i.e., 30–50 s). The pH range in which the sensor could be applied
without considerable interferences from H+ ions was however rather narrower (i.e.,
3.0–5.0), and the sensor had good selectivity behaviors in the presence of other
lanthanide ions such as La3+, Ce3+, Sm3+, Gd3+, Tm3+, Ho3+, and Eu3+.

Figure 6.14. N′-[(2-Hydroxyphenyl)methylene] benzohydrazide (BBH).

Absorption spectra of the optode film in presence of the Dy(III) ion changed
significantly (Fig. 6.15), which is due to the complex formation between
dysprosium ion and BBH ligand used as a selectophore in the membrane. This
complexation is the basis of determination of this ion.

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Figure 6.15. Absorption spectra of optode film response to Dy(III) in the range of
5.0 × 10−7 to 8 × 10−6 mol/L at pH 5.

The transparent triacetylcellulose membrane films were prepared through

conventional methods, treated with BBH solution (0.003 g) in 10 mL ethylene
diamine for 2 min at ambient temperature. Afterwards, they were washed with
water for the removal of ethylene diamine and the loosely trapped indicator (used
ligand).
The first optical sensor for Sm3+ was reported by Fouladgar and Ensafi [618], who
used 4-(5-bromo-2-pyridylazo)-N,N-diethyl-3-hydroxyanilin (Br-PADAP) as the
ionophore in plasticized PVC membranes. They reported that the interactions
between Sm3+ and Br-PADAP give rise to a new peak at 525 nm, which
corresponds to the resulting complex. The device was reported to have a linear
response range from 2.6 × 10–6 to 2 6 × 10–4 mol/L and a lower detection limit of
1.9 × 10–6, respectively, and rather longer response times of 200 to 300 s
depending on the analyte concentration.
Another optical sensor was devised for La3+ by Ensafi and Fooladgar [619] based on
4-hydroxysalophen (Fig. 6.16) which was immobilized on a hydrolyzed
triacetylcellulose membrane through covalent bonding (Fig. 6.17). The resulting
membrane, on contact with La3+ at pH 4.0, was reported to change color from
white yellow (323–433 nm) to orange and had a linear response from 1.0 × 10–6 to
1.0 × 10–2 mol/L and rather long response times of 5–6 min [619].

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Figure 6.16. Structure of 4-hydroxysalophen.

Figure 6.17. The possible reactions between 4-hydroxy-salophen and the

membrane.

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The researchers hydrolyzed the triacetylcellulose film by hydrolyzing it with a

0.1 mol/L KOH solution for 24 h and next activated through treatment with a
polyvinyl alcohol (0.50% w/v) and thiourea (0.60% w/v) solution at 25°C for 24 h
and next with a 5.7 × 10–4 mol/L 4-hydroxysalophen solution in citrate buffer (pH
6.2) for 6 h at 25°C.
The first Eu3+ sensor was developed in 2012 [620], using chromogenic calix[4]arene
derivatives (Fig. 6.18) and was used for UV–vis absorption spectroscopy
measurements. The device was reported to have good selectivity over Pb2+, Cd2+,
and Mg2+ at pH 6.8.

Figure 6.18. Chemical structure of azo-calix[4]arene: 5,17-bis(4-

nitrophenylazo)-26,28-dihydroxy-25,27-di(ethoxycarbonylmethoxy)-calix[4]arene.

The process for preparing the membranes included dissolving 10 mg of the

ionophore in 1 mL of chloroform. The adhesion of the film to the glass plates was
improved through activating the glass through successive ultrasonic treatments in
acetone and methanol for 20 min before spin-coating the films on the plates at a
controlled speed of 2300 rpm and their annealing at 80°C for an hour.

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Most of the optical membrane sensors are prepared as described above. And a
typical procedure for optical measurement of the amount of analyte by an optical
sensor can be explained as follows.
The prepared optode membrane was soaked into the ions test solution in a suitable
buffer solution for a certain amount of time. Next, the membrane was washed with
distilled water and dried. Then, it was placed vertically inside the sample cuvette
containing 3 mL of buffer solution for about 15 min to reach equilibrium. The
absorbance-measuring system for the optode films is shown in Fig. 6.19. Then, the
spectrum of the membrane is taken. Finally, the sample cell was titrated with ion
solution, and the absorbance of the system was measured over the indicated
wavelength range. Then the absorption changes correlated with the concentration
of the analyte are determined using a calibration graph.

Figure 6.19. Absorbance-measuring system for the film optode.

Fluorescence | Clinical and Drug Applications☆

J.M. Fernández-Romero, M.P. Aguilar-Caballos, in Encyclopedia of Analytical
Science (Third Edition), 2019

Fluorescence Biosensors

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The fluorescent biosensors have characteristics of high sensitivity and selectivity,

and rapid response to the presence of specific biomolecules in the field of
biomedical research. In general, these biosensors can respond through three
general mechanisms: (a) directly through the biomolecule-receptor interaction that
gives rise to a luminescent signal, (b) because the biospecific interaction causes
conformational changes that cause the modification of the luminescent signal and
(c) by changes in the activity of the biomolecule itself in the environment of
biospecific interaction, for example, in the case of enzymatic biosensors.
Fluorescent signals can be monitored well through FRET (Fröster Resonance
Energy Transfer) energy transfer phenomena between a donor agent and a possible
acceptor. The energy transfer occurs when there is a distance between donor and
acceptor less than 10 nm and the dipole moments are aligned. Another way is by
measuring the lifetime of the fluorescent image in tissues in which reactions in
living cells or biospecific interactions can be monitored, called FLIM (fluorescence
lifetime imaging). In these cases, it is possible to monitor the average lifetime of
the fluorescent molecules when they are in their excited state after absorbing a
photon of energy. Using FLIM, it is possible to monitor changes in the half-life of
fluorescent substances in the tissues, or changes in the luminescence of the
fluorophore in response to interactions occurring locally. A third way is using the
denominated FCS (fluorescence correlation spectroscopy) that allows to carry out
the quantification of biomolecules in minimal concentrations. Finally,
measurements of the changes in fluorescence intensity can be carried out in
response to the excitation of the bioreactor sensitive microzone.
Although the fluorimetric biosensors are quite sensitive and selective, in
biomedical research, especially in the early diagnosis of a disease, these analytical
characteristics are quite limiting. Having to resort to previous stages that allow
improving the sensitivity of the biosensor. The urgent need for rapid and
ultrasensitive analytical tools has led research in this field to the development of
biosensors based on the fluorescent monitoring of individual molecules. The
spectacular advances achieved in the last decade in the development of microscopy
and spectroscopy of optics close to the sample (MNFO/SNFO) allows the
monitoring of individual fluorescent molecules, promoting a new generation of
highly sensitive biosensors. These techniques could be applied using in vitro or
in vivo labeling biomolecules with results that provide biomarker concentration
level of single molecule counting (SMC). Compared to conventional techniques,
fluorescent biosensors based on the SMC have an ultra-high sensitivity, an
excellent selectivity, fast analysis time and low sample consumption. Consequently,
single-molecule luminescent monitoring is a promising analytical approach to
quantify low-abundance biomolecules with speed and simplicity.
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In a recent review, these efforts have been summarized by presenting ultrasensitive

biosensors based on the counting of a single luminescent molecule. The individual
monitoring of a molecule can be carried out as a very simple and ultrasensitive
method to quantify the target molecules by merely counting the individual
fluorescent bursts obtained by confocal microscopy integrated in microfluidic
devices or by single-molecule imaging based on reflection fluorescence total
internal Biosensors based on counting individual molecules (with and without
separation) have been developed. In this sense, SMC fluorescent biosensors have
been proposed for the specific detection of diverse target biomolecules, such as
DNA, micro-RNAs, enzyme activity, protein-modifications, HIV-genes, histones-
modified enzymes, and cancer cells. In all cases, these ultrasensitive biosensors can
function as biomarkers related to different diseases. Also, future foundations have
been laid for expanding the practical development of SMC fluorescent biosensors,
more accessible to build and automate, as well as to propose future innovations in
the design of new ultrasensitive biosensors. Due to its already mentioned
characteristics, fluorescent biosensors based on the counting of individual
molecules present a promising future in the field of applications in biological
research, clinical diagnosis and discovery of new drugs.

Trace Determination of Pesticides and their

Degradation Products in Water
J. Richard Aspland, Marie-Claire Hennion, in Techniques and Instrumentation in
Analytical Chemistry, 1997

6.4.1.3 Optical immunosensors

Optical sensors are based on the absorption or emission of light by the immunore-
actants. These types of sensors take advantage of the low cost and flexibility of
optical fibres.
Fibre optic biosensors (FOBs) consist of a fibre-optic strand having an appropriate
sensing layer on the distal tip of the fibre. Light is introduced into the proximal end
and travels to the distal tip by total internal reflection. Changes in the absorbance,
luminescence, polarization, or refractive index can all be used as optical
transduction mechanisms [231,232]. The simplest approach to an optical detection
system uses the measurement of the fluorescence emission from one of the
immunoreactants involved in the reaction. As an example, benzo[a]pyrene bound
to antibodies immobilized on the distal part of a fibre optic can been detected,
owing to its intrinsic fluorescence when excited by laser radiation transmitted into
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the fibre, with a detection limit of a few femtomoles [233]. However, only a few
analytes can be detected directly from their intrinsic fluorescence. Most analytes
have a characteristic wavelength maximum of absorbance, but the absorbed light is
only a small percentage of the total transmitted. This fact explains why competitive
immunoassay configurations using fluorescent labels are preferred.
Evanescent wave (EW) immunosensors have been developed for pesticide
applications in recent years. When light is propagated through a waveguide (n1, see
Fig. 6.24E) by multiple total internal reflections, an electromagnetic wave called an
evanescent wave is generated in the optically rarer external medium (n2 with n2 >
n1). Since the distance of penetration of the evanescent wave is only a few hundred
nanometres, only surface-bound molecules will interact with the light. The
principal advantage of this system is that it avoids possible interferences from the
bulk media. When molecules having an absorption spectrum which includes the
excitation wavelength are located in the evanescent field they absorb energy,
leading to an attenuation in the reflection light of the waveguide. This
measurement is known as attenuated total reflection (ATR). However, the analyte
must have a characteristic absorption band and the absorbed light is usually a very
small percentage of the total transmitted light, leading to poor sensitivity. To
increase the sensitivity, it is necessary to make use of labelled molecules that can
re-emit the absorbed evanescent photons at a longer wavelength, as fluorescence.
Part of this emission is coupled back to the waveguide and is transmitted to the
receptor. This phenomenon is known as total internal reflection fluorescence (TIRF)
and is the basic principle of most immunosensors reported to detect
environmental pollutants with sufficient sensitivity. It was applied to the detection
of atrazine[214,234], terbutryn[235], imazethapir [236,237] and parathion[238], with
detection limits at the low 0.1 μg/l level. Most of the devices make use of
fluorescein as label.
Surface plasmon resonance (SPR) immunosensors contain an optical electronic
transducer. A surface plasmon is an evanescent electromagnetic field generated at
the surface of a metal conductor (usually Ag or Au) when excited by the impact of
light of an appropriate wavelength at a particular angle. Electrons at the metal
surface are excited by the incident light, producing a resonance at a frequency
different from that of the electrons in the bulk of the metal film (see Fig. 6.24 F).
The absorption of light energy during resonance is observed as a sharp minimum
in light reflectance when the varying angle reaches a critical value, which depends
on the wavelength and polarization of the incident light and on the dielectric
properties of the medium adjacent to the metal surface. Therefore, the critical
angle value is affected by analytes binding to the surface and can allow the

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monitoring of biological interactions. A commercial apparatus, BIAcore™

(Pharmacia), available for coating with the desired antibodies or antigens, was used
to develop a SPR-based immunosensor for atrazine detection [239]. Another
system called IAsys™ (Fisons) was developed to monitor binding events in real
time [240]. It exploits a novel form of optical biosensor that combines the
technology of waveguides with the SPR phenomena, the metal layer being replaced
by a dielectric resonant layer of high refractive index (i.e., titania or zirconia) and
separated from the glass prism by a low-refractive index layer of silica.
Reflectometric interference spectroscopy (RIFS) immunosensors have also been
described as optical immunosensors. The basic principle uses the reflected light
produced when a light beam passes through the interface between two media of
different refractive index. A thin transparent film will produce an array of reflected
beams at each of the interfaces, which can be considered as only two reflected
beams when the reflectance of the interfaces is small. The phase difference of these
beams is directly related to the thickness of the layer, and therefore changes in the
film thickness can be determined by changes in the interference spectrum. Brecht
et al. applied this principle to make an immunosensor which can detect atrazine
using a hapten derivatized immunosensor surface, without any label, with
detection limits around 0.25 μg/l [241].

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