OPTICAL

BIOSENSORS
 INTRODUCTION
 Optical Biosensors as the name suggests are based on the involvement of light.

 The basic objective of an optical biosensor is to produce a signal which is proportionate to the concentration of a
measured substance (analyte).

 Measures changes in optical properties of substances –

o Absorbance
o Fluorescence
o Reflectance
o Refractive index
o Luminescence
o Surface Plasmon Resonance
DESIGN OF OPTICAL BIOSENSOR
The components of an optical biosensor are:

(i) a light source (may or may not be present)

(ii) an optical transmission medium (fiber, waveguide, etc.)

(iii) immobilized biological recognition element (enzymes, antibodies or microbes)

(iv) optical probes (such as a fluorescent marker) for transduction,

(v) an optical detection system.

CLASSIFICATION
 Optical
Biosensor

Label-
Label-free
based
 In a label-free mode, the detected signal is generated directly by the interaction of the analysed material with the
transducer.

 In contrast, label-based sensing involves the use of a label and the optical signal is then generated by a colorimetric,
fluorescent or luminescent method.

 Simple molecules such as glucose can be detected by enzymatic oxidation using label-assisted sensing. The glucose
analysis of blood is the most commercially successful (so far) application of a biosensor, i.e. the handheld glucose
meter used by diabetics.

 However, in some situations, e.g. antibody–antigen interaction where a label is conjugated with one of the
bioreactants, labelling can alter the binding properties and therefore introduce systematic error to the biosensor
analysis.
 SPECTROSCOPY
It is the study of the interaction between matter and
electromagnetic radiation. Historically, spectroscopy originated through the
study of visible light dispersed according to its wavelength, by a prism. Later
the concept was expanded greatly to include any interaction with radioactive
energy as a function of its wavelength or frequency. Spectroscopic data are
often represented by an emission spectrum, a plot of the response of interest as
a function of wavelength or frequency.
 PRINCIPLE
• The central concepts in spectroscopy is a resonance and its corresponding resonant frequency.
• In quantum mechanical systems, the analogous resonance is a coupling of two quantum
mechanical stationary states of one system, such as an atom, via an oscillatory source of energy such as
a photon. The coupling of the two states is strongest when the energy of the source matches the energy
difference between the two states. The energy (E) of a photon is related to its frequency
(v) by E=h\v where (h) is Planck's constant, and so a spectrum of the system response vs. photon
frequency will peak at the resonant frequency or energy. Particles such as electrons and neutrons have a
comparable relationship, the de Broglie relations, between their kinetic energy and their wavelength and
frequency and therefore can also excite resonant interactions.
• Spectra of atoms and molecules often consist of a series of spectral lines, each one representing a
resonance between two different quantum states
• The hydrogen spectral series in particular was first successfully explained by the Rutherford-Bohr
quantum model.
 TYPES

• Absorption spectroscopy: Absorption occurs when energy from the radioactive

source is absorbed by the material
• Emission spectroscopy: Emission indicates that radioactive energy is released by
the material
• Elastic Scattering: Determines how incident radiation is reflected or scattered
by a material
• Impedance spectroscopy: Ability of a medium to slow the transmittance of
energy
• Inelastic Scattering (Raman): Involve an exchange of energy between the
radiation and the matter that shifts the wavelength of the scattered radiation
 OPTICAL FIBERS
 It is used for the transmission of light from one point to another.

 Transmits light on the basis of the principle of total internal reflection (TIR).

 The materials used for making optical fibers are glass, plastic, silicon, etc.

 Advantages of optical fibers:

• Excellent light delivery
• Long interaction length
• Low cost
• Ability not only to excite the target molecules but also to capture the emitted light from the targets

 Optical biosensors based on the use of fiber optics can be classified into two different categories:

i. Intrinsic sensors, where interaction with the analyte occurs within an element of the optical fiber

ii. Extrinsic sensors, in which the optical fiber is used to couple light, usually to and from the region where the light
beam is influenced by the measurand.
 Basic fiber has a core with refractive index n1
surrounded by cladding layer with refractive
index n2, n1 > n2
 The difference between n1 and n2 is less than
0.5%.
 The cladding layer is surrounded by one or more
protective coating.
 The materials for making waveguide varies for the
wavelength of light being used
X. > 450 nm plastic (such as polyacrylamide)
X. > 350 nm glass
X. < 350 nm fused silica
X. > 1000 nm germanium crystal guides
 TOTAL INTERNAL REFLECTION
Total internal reflection is the phenomenon which occurs when a propagated wave strikes a medium boundary at an
angle larger than a particular critical angle with respect to the normal to the surface.

If the refractive index is lower on the other side of the boundary and the incident angle is greater than the critical
angle, the wave cannot pass through and is entirely reflected.

The critical angle is the angle of incidence for which the angle of refraction is 90°. Above this angle total internal
reflection occurs
 ABSORPTION BASED
 These are simplest type of Biosensor.

 It is a phenomenon to determine changes in the concerntration of analayte.

 It is based on the Beer-Lambert Law. (log(I/lo) = A = έCl)

 Each molecule has an absorption maxima at a particular wavelength of light.

 The sensor works by sending light through an optical fiber to the bio sample.

 The amount of light absorbed by the analyte is determined by measuring the

light coupled out via the optical fiber.

 Example of such biosensor is- pH meter.

 Example- pH Meter

Since, the absorption is usually wavelength dependent and different species have different absorption spectra, by
measuring the absorption spectra via fiber optical sensor, different species and concentration levels can be
determined.
 FLUORESCENCE BIOSENSOR
 Fluorescence is the property of some atoms and molecules to absorb
light at a particular wavelength and to subsequently emit light of
longer wavelength after a brief interval.

 PROCESS:
i. Electrons are excited by a light source
ii. Electrons enter into an excited state.
iii. Energy loss occurs in nanoseconds.
iv. Electrons are returned to ground state emitting light of a greater
wavelength.

Example of fluorescence:
Substrate+ NAD(P)+dehydrogenase product+ NAD(P)H

NADH or NADPH is produced, which can be detected using fibre optics

through its fluorescence at λex = 350 nm, λem = 450 nm
 If the analyte-reagent complex does not show fluorescence we can use a fluorophore in the
reagent.

 A fluorophore (or fluorochrome) is a fluorescent chemical compound that can re-emit light upon
light excitation.

 Fluorophores can be divided into three general groups:

I. Organic dyes - Synthetic organic dyes, such as fluorescein, were the first fluorescent compounds
used in biological research. Other historically common fluorophores are derivatives of rhodamine
(TRITC), coumarin, and cyanine.

II. Biological fluorophores- Green fluorescent protein (GFP) was cloned from the jellyfish Aequorea
victoria and used as a gene expression reporter. Since that time, derivatives of the original GFP,
phycobiliproteins (allophycocyanin), phycocyanin, phycoerythrin, and phycoerythrocyanin) and
many other proteins have been designed for use in biological expression systems,

III. Quantum dots- These are nanoscale-sized (2-50 nm) semiconductors that, when excited, emit
fluorescence at a wavelength based on the size of the particle; smaller quantum dots emit higher
energy than large quantum dots, and therefore the emitted light shifts from blue to red as the
size of the nanocrystal increases.
 CHEMILUMINESCENCE BIOSENSOR
Chemiluminescence is similar to fluorescence. The difference is that chemiluminescence occurs by exciting molecules with a
chemical reaction (usually occurring by the oxidation of certain substances such as oxygen or hydrogen peroxide), whereas
fluorescence occurs by exciting molecules via light.

In the case of chemiluminescence, no external source of light is required to initiate the reaction.

For example, if [A] is luminol and [A’] is hydrogen peroxide in the presence of a suitable catalyst we have:
luminol + H2O2 →3-APA[◊] →3-APA + light
Where: 3-APA is 3-aminophthalate and 3-APA[◊] is the excited state producing light as it decays to a lower energy level.
USE OF CHEMILUMINESCENCE:
Food analysis-
 Organophosphorous compounds are most popular pesticides.
 Most commonly used organophosphorous: QUINALPHOS (O,O diethyl-o-quinoxalinly phosphorothioate)
• Quinalphos +H2O2 peroxophosphonate
• Peroxophosphonate+ luminol 3aminophthalate anion*
• 3-aminophthalate* 3-aminophthalate + observed emission

ADVANTAGES:
 Fast dynamic response
 Simple instrumentation
 A wide calibration range

DISADVANTAGES:
 Light leaks from assay reagent & reaction vessels
 Ultra sensitive – stringent controls on purity of reagents
 High intensity light emission leads to pulse pileup in photomultiplier tubes leads to underestimation
 The probe includes the HMDNAzyme domain (blue) for stimulating the generation of chemiluminescence(CL) in the
presence of luminol and H2O2, and a nucleic acid domain (green) that is complementary and hybridizes with the target
DNA (pink). When the free probe and GO are mixed in an aqueous buffer solution, little CL emission is observed.

 But in the presence of the target DNA, the free probe first hybridizes with the target DNA resulting in the formation of
double-stranded DNA (dsDNA), causing the release of the probe from GO and thus a significant CL emission increase will
be observed. The increased CL emission intensity will be positively related to the concentration of the target added in the
assay solution.
 BIOLUMINESCENCE BIOSENSOR

Bioluminescence is the production and emission of light by a living organism. It is a form of chemiluminescence.
ADVANTAGES:
 Quick response time
 Not sensitive to environmental changes
 Easy to operate and control

DISADVANTAGES:
 Difficult to remain the cell alive and viable
 Not very stable during the sensing time
 Less specific comparing to other types of biosensors

EXAMPLE
 Bioluminescence can be used for detection of pollutant like pesticides and the some of the bacterial species
tested includes Vibrio fischeri, Vibrio harveyi and Pseudomonas fluorescens.

 Natural or genetically modified bacteria can be used for preparation of biosensor probes which helps in
screening and detection of toxicity.

 The main principal involved behind this is very simple; in case of optimal condition the bacteria emit light
normally but in presence of toxic substances their luminescence decreases. Thus presence of toxic molecules
can be evaluated.
Bioluminescence biosensor for detection of pathogen
 SURFACE PLASMON RESONANCE
 Surface plasmon resonance is an optical transduction method,
which has been commercially employed for optical biosensors.
 SPR biosensors measures the change in refractive index directly in
contact with sensor surface (Au)
 SPR biosensors are used to monitor binding events between
molecules, ranging from ions to viruses in a label free manner.
 To effectively generate SPR, proper wavelength is needed.
 SPR biosensors has their applications in the field of life sciences,
pharmaceutical research, drug discovery and clinical diagnostics.
 A major application area includes detection of low levels of
biological analytes and study of biomolecular interactions.
 SPR instrumentation uses a detection scheme called Kretschmann
configuration.
 PRINCIPLE
 Incident light wave performs total internal reflection inside the
propagation medium.
 Some light wave leaves the propagation medium and is known as
evanescent wave. This wave can excite the electrons of the
metal sheet (Au of 10 nm).
 When the electrons resonate together between the propagation
medium and the metal sheet, it leads to the formation of surface
plasmon wave.
 The angle at which the resonance occurs is sensitive to any
change in the refractive index of the medium adjacent to the
metal surface. It changes as the analyte binds to the biomaterial.
 The surface plasmon resonance angle mainly depends on the
properties of the metal film, the wavelength of the incident light
and the refractive index of the media on either side of the metal
film.
SPR SETUP AND MECHANISM
SENSOGRAM
 THANK YOU
1.NOUMI SARKAR – BTB/15/107
2.ARSHDEEP KAUR- BTB/15/118
3.SHIVANGI SAHAY- BTB/15/120
4.NATASHA NADEEM KHAN- BTB/15/137
5.PALLAVI TRIPATHY- BTB/15/154
6.DRISHTI GUPTA- BTB/15/155
7.GARIMA- BTB/15/162
8.MONIS ATHAR KHAN- BTB/15/165
9.ROHAN KUMAR BHAGAT- BTB/15/168