References:

• Yang, V.C. and T.T. Ngo. 2000. Biosensors and Their Applications. Kluwer
Academic/Plenum Publishers, New York, NY. Ligler, F.S. and Rowe Taitt, C.A.
2002.

• Optical Biosensors: Present & Future. Elsevier, The Netherlands

• Bio-sensors: Fundamentals and applications, A.P.F.Turner, I. Karube, and G.S

Wilson, Oxford Science Publications: Oxford, 1987.
 • Enzyme and Microbial bio-sensors: Techniques and Protocols, Ashok
Mulchandani and Kim R Rogers Eds.;Humana Press, Totowa, NJ, 1998.

• Affinity bio-sensors: Techniques and Protocols, Ashok Mulchandani and Kim

R Rogers Eds.;Humana Press, Totowa, NJ, 1998.

Basic subjects must be known:

(A) Physics
(B) Solution Chemistry
(C) Surface chemistry
(D) Electrochemistry
(E) Enzyme Engineering
(F) Instrumental method of Analysis
(G) Organic Chemistry
Sensor

• device that receives a stimulus and responds with an electrical

signal

 Motion, position, displacement • Velocity and acceleration • Force, strain

• Pressure • Flow • Sound • Moisture • Light • Radiation • Temperature
• Chemical presence
 Electrical signal that can be channeled, amplified and modified by
electronic devices

 Voltage
 Current
 Charge

 Voltage, Current or Charge may be described by

 Amplitude
 Frequency
 Phase
 Digital code
 Digital voltmeter LED 3 digit display

Analog signals

Digital signals
Example for How the BP monitoring instrument developed

LCD (Liquid crystal

diode)

Electronic Sphygmomanometer
Aneroid Sphygmomanometer
Pressure gauge:

Mercury pressure monitoring Instrument

 BIOSENSOR

• biosensor is an analytical device which converts a biological response

into an electrical signal

• biosensor is mainly divided into three sections

• Sensor: a sensitive biological element (biological material (eg. tissue,

microorganisms, organelles, cell receptors, enzymes, antibodies, nucleic
acids, etc)
 Transducer: it is the detector element (works in a physicochemical way; optical,
piezoelectric, electrochemical, etc.)

that transforms the signal resulting from the interaction of the analyte with the
biological responsible for the display of the results in a user-friendly way.

Physical or Electronic
Transducers
Chemical changes signals

Electrochemical Potentiometric Ampeometric Conductimetric

Optical Fluorescence absorbance Light Scattering Refractive Index

Selective ion
Ion Field effect transistor determination

Voltage
Mechanical (Potentiometric)

Voltage
Thermal (Potentiometric)
Peizoelectric Pressure sensors
Classification of biosensors based on the transducer element & bio-recognition
element
Transducer Microprocessor

Filter
Amplifier
Digital
Analog

• Electronic filters are circuits which perform signal processing functions,

specifically to remove unwanted frequency components from the signal,
to enhance wanted ones, or both.
 Optical Bio-sensing

Label free mode

based Label based mode

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

• label-free mode, the detected signal is generated directly by the

interaction of the analysed material with the transducer.

E1= hɣ1 or hc/ƛ

Light
source E2= hɣ2 or hc/ƛ
E= hɣ
 Electrochemical sensors

• Converting of chemical energy

Electrochemical cell
to electrical energy
• There is no supply of any
electric current in the system
externally.
Electrolytic cell

Three electrodes connected

the cell
• case the rate of electrochemical reduction of O2 depends on the rate of diffusion
of the oxygen from the bulk solution,

• which in turn is dependent on the concentration gradient and hence the bulk
oxygen concentration
Sensors are devices that register a physical, chemical, or biological change and convert that
into a measurable signal.

The sensor contains a recognition element that enables the selective response to a particular
analyte or a group of analytes, thus minimizing interferences from other sample components
.

Another main component of a sensor is the transducer or the detector device that produces a
signal. A signal processor collects, amplifies, and displays the signal.
• Electrochemical biosensors, a subclass of chemical sensors, combine the sensitivity,

as indicated by low detection limits, of electrochemical transducers with the high

specificity of biological recognition processes.

• Biological recognition element (enzymes, proteins, antibodies, nucleic acids, cells,

tissues or receptors) that selectively reacts with the target analyte and produces an

electrical signal that is related to the concentration of the analyte being studied.
Electrochemical biosensors can be divided into two main categories based on the
nature of the biological recognition process

1. Biocatalytic devices
Biocatalytic devices incorporate enzymes, whole cells or tissue slices that
recognize the target analyte and produce electroactive species. Special emphasis
will be placed on enzyme electrodes for the detection of glucose, lactose, and
xanthine.

2. Affinity sensors:

Affinity sensors rely on a selective binding interaction between the analyte and a
biological component such as an antibody, nucleic acid, or a receptor.

Immunosensors and DNA hybridization biosensors with electrochemical detection

will be discussed as examples of affinity sensors.
The biosensor’s performance is usually experimentally evaluated based on its

sensitivity, limit of detection (LOD), linear and dynamic ranges, reproducibility

or precision of the response, selectivity and its response to interferences

Biocatalytic sensors:

Although many types of biorecognition elements have been used in biosensing

devices, electrochemical biosensors primarily use enzymes due to their high

biocatalytic activity and specificity

• Biocatalytic sensors using enzymes as the recognition element often have relatively

simple designs and do not require expensive instrumentation.

• Such sensors are typically easy to use, compact, and inexpensive devices.

• Different detection configurations can be used such as stationary sample solution

vs. flow conditions or bulk sample solution vs. a micro drop detected using a

microelectrode.
Enzymes, globular proteins composed mainly of the 20 naturally occurring amino acids

that catalyze biochemical reactions, are the oldest and still most commonly used

biorecognition element in biosensors.

Enzymes can increase the rate of a reaction significantly relative to an uncatalyzed

reaction. The enzyme–substrate interactions can be characterized by kinetic studies.

Also, sensitivity of the biorecognition element to experimental conditions such as

pH, temperature, and stirring should be minimal and variation between

measurements should be as low as possible.

Because of their complex molecular structures, enzymes often have exquisite

specificity for their substrate molecule and can detect individual substances in a

complex mixture, such as urine or blood, very selectively.

This removes the need for time-consuming, labor intensive, and interference-prone

sample pretreatment and separation steps used in composite methods.

Researchers also had to find ways to manage the enzyme adsorption that could lead

to electrode fouling as well as denaturation and loss of enzyme’s catalytic activity on

the electrode surface.

Biocatalytic sensors
Introduction to enzyme-based electrodes
Enzyme electrodes are electrochemical probes with a thin layer of immobilized
enzyme on the surface of the working electrode.

The enzyme is the most critical component of the enzyme electrode since it provides
the selectivity for the sensor and catalyzes the formation of the electroactive product
for detection.

The electroactive product can be monitored directly using amperometry, in which the
produced current is measured in response to an applied, constant voltage.

Alternatively, the disappearance of the redox active reactant in an enzyme-catalyzed

reaction can be monitored by the electrode.

The activity of the immobilized enzyme depends on solution parameters and electrode
design. The rapid enzymatic catalysis can also sometimes provide significant signal
amplification in a biosensor.
• Development of biocatalytic sensors for medical applications, primarily blood glucose monitoring
starting in the late 1960s, was the main driving force for this research area.

• Enzyme-based biosensors can be historically divided into three generations.

• First-generation biosensors were oxygen-based whereas second-generation are mediator-based.

• Third-generation biosensors are so-called directly coupled enzyme electrodes.

• Electrodes coated with glucose oxidase (GOx) have been widely used in detection of glucose

• These amperometric sensors became known as the first-generation biosensors or Clark oxygen
electrodes and were soon implemented by Updike and Hicks, who constructed the first functional
biocatalytic sensor for glucose.

• In the first-generation biosensors, an oxidase enzyme is immobilized behind a semipermeable

membrane at the surface of a Pt electrode.
GOx is a readily available, inexpensive, and stable enzyme from Aspergillis niger

that is among the most important enzymes in biosensor applications and industrial

processes. GOx is highly specific for β-D-glucose, which can be detected via the

following reactions.
In eqn (1) the prosthetic group of the enzyme, FAD, is reduced and glucose is
oxidized to d-D-gluconolactone.

Molecular oxygen acts as the oxidizing agent to produce hydrogen peroxide

. During the oxidation of H2O2 at a working electrode two electrons are transferred
directly to the electrode, resulting in the current response of the enzyme electrode.

These first-generation sensors required the ample and constant presence of ambient
oxygen as a co-substrate for the enzyme to function optimally.

However, oxygen is not very soluble in aqueous solutions and can therefore limit the
currents produced in the presence of the analyte.
• Direct redox reactions between enzymes and electrodes are very rare because most proteins tend to
denature at the electrode surface and many direct electron transfer reactions are slow and irreversible.

• However, a limited number of enzymes such as horseradish peroxidase have proven capable of direct
electron transfer between the enzyme active site’s prosthetic group and the electrode.

• The active site of an enzyme that allows the selective targeting of an analyte is usually buried within
the enzyme’s tertiary protein structure, near the centroid of the protein.

• Therefore, the electrons produced in the enzyme-catalyzed reaction cannot always be easily and
rapidly transferred to the electrode surface thereby limiting the electrical communication between the
enzyme and the transducer.

• The widely accepted Marcus theory of electron transfer states that electron transfer decays
exponentially with distance.

• Therefore enzymes often require some assistance with electron transfer to the transducer surface.
• Artificial redox mediators are small, soluble molecules capable of undergoing rapid and reversible
redox reactions, which shuttle electrons between the redox center at the active site of the enzyme and
the electrode surface.

• Mediators have replaced O2 molecules as the electron shuttle in glucose sensors.

• Mediators are re-oxidized at relatively low potentials and generate a current when they come in
contact with the working electrode
• Mediators should ideally be nontoxic, independent of the pH, stable in both the oxidized and
reduced forms, and unreactive with oxygen.

• Although many organic compounds are capable of acting as enzyme mediators, organometallic
redox compounds are the most common.

• Examples of previously used mediators include quinones, organic conducting salts, dyes,
ruthenium complexes, ferrocene, and ferricyanide derivatives.

• Mediated enzyme electrodes had a much better sensor performance than the first-generation
biosensors mainly due to eliminating the O2 dependence and being able to control the
concentration of the oxidizing agent in the biosensor.

• Hand selecting the oxidizing agent for the sensor also allowed more suitable oxidation potentials
to be used for the amperometric sensors. These mediated enzyme electrodes were named second-
generation biosensors.
• Third-generation biosensors have the biorecognition component coupled with the electrode by co-
immobilizing the enzyme and the mediator at an electrode surface.

• This can be achieved by direct electrical contact between the enzyme and the electrode,
immobilizing the enzyme and mediator in a conducting polymer, or ‘wiring’ the enzyme to the
electrode by immobilizing it in a redox polymer.

• The co-immobilization prevents the mediators from diffusing out of the biosensor film.

• The co-immobilized mediators, or the flexible surrounding redox polymer, help to transport
electrons between the enzyme’s active site and the working electrode surface in an array of rapid
electron relays and hence generate high current densities.

• The enzymes immobilized in flexible redox polymers that are covalently attached to the electrode
have been called ‘wired enzymes’.

• The 3rd-generation sensors are ideal for repeated measurements since neither mediator nor enzyme
need to be added.

• This self-contained nature also lowers the cost per measurement and opens up possibilities for
continuously monitoring the analytes.
Voltammetric and amperometric techniques are characterized by applying a potential to a working (or indicator) electrode

versus a reference electrode and measuring the current. The current is a result of electrolysis by means of an

electrochemical reduction or oxidation at the working electrode. The electrolysis current is limited by the mass

transport rate of molecules to the electrode.

The term voltammetry is used for those techniques in which the potential is scanned over a set potential range. The current

response is usually a peak or a plateau that is proportional to the concentration of analyte. Voltammetric methods include

linear sweep voltammetry, cyclic voltammetry, hydrodynamic voltammetry, differential pulse voltammetry, square-wave

voltammetry, ac voltammetry, polarography, and stripping voltammetry. These methods have a wide dynamic range, and

are useful for low level quantitation.

In amperometry, changes in current generated by the electrochemical oxidation or
reduction are monitored directly with time while a constant potential is maintained at the
working electrode with respect to a reference electrode.

It is the absence of a scanning potential that distinguishes amperometry from voltammetry.

The technique is implemented by stepping the potential directly to the desired value and
then measuring the current, or holding the potential at the desired value and flowing
samples across the electrode as in flow injection analysis.

Current is proportional to the concentration of the electroactive species in the sample.

Applications of Electrochemical biosensors

Amperometric method Voltammetry method

Impedance:

Electrochemical impedance spectroscopy (EIS), described by Lorenz and Schulze in

1975,31 measures the resistive and capacitive properties of materials upon
perturbation of a system by a small amplitude sinusoidal ac excitation signal typically
of 2–10 mV.

The frequency is varied over a wide range to obtain the impedance spectrum.

The in-phase and out-of-phase current responses are then determined to obtain the
resistive and capacitive components of impedance, respectively.

Impedance methods are powerful because they are capable of sampling electron
transfer at high frequency and
mass transfer at low frequency. Impedimetric detection is primarily used for affinity
biosensors.
Impedance biosensors
 Affinity biosensors

Most applications of immunoassays (IA) and immunosensors with electrochemical detection were

initially developed at research laboratories due to the level of expertise required, time, and the high initial

cost of developing and optimizing a new immunoassay.

However, the cost of immunological reagents continues to decrease with recent developments in

molecular biology techniques.

Many of the early radio immunoassays were developed for biomedical applications such as detecting

hormones and disease related proteins, but applications in environmental, agricultural, processed food and

beverage areas, and to detect harmful chemical and biological agents in national defense.
Biorecognition and immunochemical reactions. IgG antibodies (Ab), large Y-shaped
glycoproteins of MW E
150 kDa, are produced by a host in response to the presence of a foreign molecule
called antigen (Ag).

Antigens are anything that the body recognizes as foreign such as chemical
compounds, proteins, and particulate matter (dust, pollen, etc.).

Abs are produced by specialized B lymphocyte cells of the immune system and can
usually be found in blood
serum, tissue fluids, and membranes of vertebrates.

Antigens commonly have relatively high molecular weights, are recognized as

nonself or foreign by the immune system and have a certain level of chemical
complexity.
For example, synthetic homopolymers composed of a single sugar or amino acid
tend to lack immunogenicity regardless of their large size due to a lack of structural
complexity.

The production of Abs against low molecular weight analytes (MW o 1000 g mol1)
called haptens is more challenging and often requires coupling the hapten to a
carrier protein with a spacer molecule before an immune response can be provoked
in the host animal.
The four most commonly used types of labeled immunoassays are:
Enzyme Immunoassay (EIA) or Enzyme-linked immunosorbent assay
(ELISA):
• used to visualize and quantify antigens.

• They use an antibody conjugated to an enzyme to bind the antigen, and the
enzyme converts a substrate into an observable end product. The substrate
may be either a chromogen or a fluorogen.

Radioimmunoassay (RIA)
• an immunoassay that uses radiolabeled molecules in a stepwise formation
of immune complexes.
Fluoro immnoassay (FIA)

• In FIAs, antibodies are labeled with fluorescent dyes,

• Traditionally being fluorescein isothiocyanate and lissamine rhodamine which emit

blue-green and orange-red fluorescence.

• In the ultraviolet spectrum. After incubation with the antigen, the antibody-antigen
complexes are isolated, and the fluorescent intensity is measured.

• The intensity read by this type of immunoassay is linearly correlated to the amount
of antigen present.
Chemiluminescence immunoassay (CLIA)

• Luminescence is the release of light due to an electron being kicked up to a higher

energy state (excitation) and emitting a photon as it relaxes down (emission).(400 nm
to 620 nm).

• which is the emission of light based on a particular chemical reaction, i.e the
formation of the antibody-antigen binding complex.
PNPP- p-Nitrophenyl Phosphate, NBT : nitro blue tetrazolium/ 5-Bromo-4-chloro-3-
indolyl phosphate , ABTS: 2,2′-azino-bis(3ethylbenzothiazoline-6-sulfonic acid), TMB :
3,3′,5,5′-Tetramethylbenzidine.
Immunoblots
Immunostaining uses antibodies to detect an antigen in cells
or tissue. The major benefit of immunostaining in
immunohistochemistry (IHC) is the ability to see the desired
target in a tissue sample while maintaining the spatial
context and tissue architecture.
Phalloidin: coupled with fluorophore

DAPI/Hoechst-Ultraviolet (Intercalation with DNA)

 ELISA

• ELISA assay is a widely used biochemical assay to detect in a sample the

presence of and quantity of proteins, such as hormones and antibodies and
bacteria or viruses.

• ELISA assay uses the coupling of antigens and antibodies and relies on the
specificity and affinity of antibodies for antigens.

• Specificity is the ability to discriminate among diverse proteins. Affinity is the

ability to tightly bind to molecules.
• The most commonly used enzyme labels are horseradish peroxidase (HRP) and
alkaline phosphatase (AP). Other enzymes have been used as well; these include β-
galactosidase, acetylcholinesterase, and catalase. A large selection of substrates is
available commercially for performing ELISA with an HRP or AP conjugate.

Direct ELISA method

INDIRECT ELISA method
SANDWICH ELISA method
 Optical sensors

• Optical detection is performed

by exploiting the interaction
of the optical field with a bio-
recognition element.
 Optical Bio-sensing

Label free mode

based Label based mode

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

• label-free mode, the detected signal is generated directly by the

interaction of the analysed material with the transducer.

E1= hɣ1 or hc/ƛ

Light
source E2= hɣ2 or hc/ƛ
E= hɣ
Properties of Light
Behavior of light for determining components
Sources of light

• not only provides the “ medium ” through which information is transferred

• also become a component of the detection circuit

• light source is characterized in terms - emission spectrum, degree of

coherence, radiant intensity, power consumption, lifetime, and all other
parameters decisive for the respective application
 emission spectrum Photons emitted
after absorption
Light
Receives the
complementary color

Wide, narrow, monochromatic

(Selected based on application)

Shape of the emission determines the light colour P= I /Io

perceived by the human eye I- intensity of emitted
light

Io- intensity of incident

light
Perceived colours and corresponding wavelength ranges (approximate
values).
Luminous flux: Light power perceived
by the human eye.

• Luminous energy: Light energy

perceived by the human eye.

• Luminance: Brightness of a light

source.
The smaller a light source, the brighter
it is (at identical luminous intensities).

• Illuminance: Intensity of light incident

on a surface.

• Luminous emittance: Intensity of

light emitted from a surface.
 Light sources Thermal Light sources

IO I
Thermal light sources

Line sources Spectral power density

Light emitting Diodes

Lasers

h = 6.626 × 10 − 34 J s (Planck constant),

c = 2.9979258 × 10 8 m/s (light speed in

vacuum),
λ : wavelength, k = 1.38 × 10 − 23 J/K
(Boltzmann constant)
 • increasing temperatures, the maximum shifts toward smaller
wavelengths.

• that this shift of the wavelength of maximum spectral power density, λmax , is
inversely proportional to the absolute temperature of the glowing body,

λmax ⋅T = 2.898 × 10−3 mK

Incandescent lamps

• an electrically heated, glowing piece of wire or a metal filament. In

the presence of oxygen, such a filament would oxidize almost
immediately.

• The filament is therefore surrounded by a glass vessel filled with an

inert gas.
• for example, with a mixture of nitrogen and argon, or with even
heavier noble gases like krypton or xenon

• maximum temperature of the filament – depends upon the melting

point of the material (usual temperature of about 2800 K)

optical efficiency of a light bulb =

radiant intensity in the visible spectrum

applied electrical power

Typically around 5%

Luminous efficacy = Luminous flux / Electrical power

10 lm/W.
Life time =1000 h
Higher filament temperatures

higher effectivities are achieved with tungsten halogenide lamps

filaments are made of tungsten as well, but the bulb is filled with iodine or
bromine vapor.

• material is evaporated from the

filament, it reacts with the halogen
atoms and forms tungsten –
hexahalogenides (WI6 or WBr6 ,
respectively.
• molecules dissociate again when they
come in contact with the hot filament
surface
• filament material allows higher filament temperatures of up to 3500 K.
• lifetime of around 2000 h
• luminous efficacy of halogen lamps is around 25 lm/W.
Line source

• energy difference is radiated off as a quantum of light with frequency ν

• discharges in a low - pressure gas do generally not emit white

light, but show color effects.
• Neon has several emission lines in the visible spectrum, but also a dominant
line at 632.8 nm. This is why neon tubes emit pink light.

• Sodium (Na) lamps emit yellow light because of the Na D lines, a narrow
-spaced line doublet at 589.0 nm (D2 ) and 589.6 nm (D1 ).

• During power - up, however, they emit red light due to the neon added to
the gas in the bulb.
 • Mercury has a strong emission line at 253.7 nm. Mercury tubes, also called
backlight tubes, emit ultraviolet ( UV ) light.

LED (Light emitting Diodes) P N

Typical parameters of monochromatic LEDs

• free atoms condensate to regular structures and form solid matter

• sharp energy levels become disturbed so far that they form quasicontinuous energy bands.
• bands are still separated by forbidden zones, so - called gaps
• electronic transition from the lower edge of the conduction band to the
upper edge of the valence band will lead to the emission of a light
quantum.
• energy will equal the width of the energy gap, E G :
• h ⋅ν = EG
• Organic Light Emitting Diode s ( OLED s) are based on recombination processes
in thin organic layers with thicknesses between 80 nm and 200 nm.
OLEDs may reach lumi-nous effi cacies of more than 100 lm/W

Laser:
• laser light is strongly concentrated in one beam that hardly changes its diameter over long distances, a
radiant intensity of, for example, 1 mW and a beam diameter of 3 mm will amount to a light intensity (the
radiant power per unit surface) of I ≈ 300 W/m 2 .

Photo detectors

• evaluation of the information that the received light carries can start, it has to be transformed into electrical
signals.

• which light incident onto a surface produces a current or a voltage are therefore processes around which photo
detectors can be designed.

• important of these processes is the photoeffect

Photomultipliers: external photoelectric effect

E kin = h ⋅ν −W

• Multialkali cathodes (Sb –

Na – K–Cs), with increased
sensitivities in the visible
and near – infrared.
• PMTs are characterized by their wavelength - dependent quantum efficiency.
• η ( λ ).
• ratio between the number of released photoelectrons and the number of incident photons.
• When Sλ is the spectral sensitivity of the photocathode in A/W, and with the wavelength given in nm,
• the quantum efficiency can be calculated to

• As the current amplification or gain, g , can reach 107 for a supply voltage of around 2 kV, PMTs must not be
illuminated with high intensities.
• This would result in high-current discharges between the dynodes and to a
destruction of the PMT.
Dark current, Id , is caused by:
• for example, thermionic emission from the electrodes, field emission and by radioactivity from the
environment, cosmic rays, or from the materials used in the PMT.
• This dark current is usually in the range of 1 pA and increases with increasing supply voltage.
• I noise = (2e Id g Δf) 1/2

Photodiodes

• Conversion of light energy to electrical energy

• utilize the effect of photons onto the charge carriers in the depletion zone in the pn - junction of a semiconductor
diode:
• when photons are absorbed, they create electron – hole pairs.
• As the charge carriers remain inside the material, this is called the internal photoeffect.
• An applied reverse voltage forces the charge carriers to drift toward the external electrodes

• produces a photocurrent that is proportional to light intensity.

• This mechanism can be understood as an inversion of the working principle of LEDs and diode lasers.

• bandwidths of pn - diodes do not exceed values of about 10 MHz

• that enable the charge carriers to travel faster across the junction provide higher reverse voltages.

• One of these architectures is a sequence of a p - doped, an undoped (intrinsic), and an n - doped semiconductor
material, a so - called PIN diode.
• proportional to the number of incident photons, that is, to the incident radiant
power, PL . With the reverse current, IS , with UT = kT / e , and with the spectral

sensitivity Sλ , it is

I =IS e (U /UT− 1) -Sλ PL

Photodiodes are typically made of silicon or
germanium.

The spectral sensitivity of Si reaches from

about 300 nm to 1100 nm and peaks at around
950 nm, beyond the visible spectrum, whereas
the maximum spectral sensitivity of Ge is at
around 1450 nm and drops to zero at about
1800 nm.
• are also semiconductor materials with increased sensitivities at larger wavelengths - Iindium -
gallium - arsenide (InGaAs) with a spectral sensitivity range from about 900 nm to about 2500 μ m,
with its maximum at about 1650 nm wavelength.
• Si photodiodes have spectral sensitivities of about 0.5 A/W, while Ge photodiodes can reach values
of 0.9 A/W and InGaAs photodiodes values of more than 1.0 A/W.

Photo resistors

Observe the temperature effects (NTC)

NTC –negative temperature coefficient

 • is a light-controlled variable resistor

• resistance of a photo-resistor decreases

with increasing incident light intensity

• Photo-resistor is made of a high resistance semiconductor

• In the dark, a photo-resistor can have a resistance as high as several

megaohms (MΩ).

• While in the light, a photo-resistor can have a resistance as low as a few

hundred ohms.
• If incident light on a photo-resistor exceeds a certain frequency, photons absorbed by
the semiconductor give bound electrons enough energy to jump into the conduction
band.

• The resulting free electrons (and their hole partners) conduct electricity, thereby
lowering resistance.

• The resistance range and sensitivity of a photo-resistor can substantially differ among
dissimilar devices.

• Moreover, unique photo-resistors may react substantially differently to photons within

certain wavelength bands.
• Photo resistivity of any photo-resistor may vary widely depending on ambient
temperature, making them unsuitable for applications requiring precise
measurement of or sensitivity to light photons.
• Lead sulphide (PbS) and indium antimonite (InSb) LDRs (light-dependent resistors) are used for the mid-
infrared spectral region.

• Ge:Cu photoconductors are among the best far-infrared detectors available, and are used for infrared
astronomy spectroscopy and infrared spectroscopy.
Optical biosensors use the interaction of light (electromagnetic radiation/waves) with matter to measure the
concentration of a species.

The interaction can be measured through adsorption, scattering (absorbing and re-emitting the light), refractive index
changes and fluorescence.

Optical sensors have already been established as a staple of the healthcare industry, ranging from traditional sensors such
as pulse oximeters to modern photoplethysmograms for heart rate monitoring in wearable devices such as the Apple
Watch and Fitbit.

Today, optical biosensors continue to evolve in many applications,

• including food safety,

• pathogen detection

• cancer diagnosis

• environmental monitoring

• DNA sensing

• blood glucose monitoring

Optical biosensors share a common set of working components. Foremost, to capture
the analyte of interest, a bio-recognition element is necessary.

This can be organic such as enzymes, antibodies, aptamers, cells, or tissues (Chen
and Wang, 2020), or inorganic such as molecularly imprinted polymers (MIPs)

Interactions between the bio-recognition element and the analyte lead to a signal
through interactions with light, which can originate from a light source such as a
laser or LED.
These interactions include fluorescence, absorption, refraction index or light
scattering.

The signal produced is subsequently correlated to the concentration of the

measured analyte.

A waveguide, such as a fiber optic is included to guide the light or to produce

evanescent waves.

A detection array, such as a photodiode, light-dependent resistor (LDR),

phototransistor or charged coupled device (CCD), captures and quantifies the
signal.

Finally, a signal processing unit converts the measured signal into readable
information for the users, which in this case is the concentration of the analyte.
 Optical biosensors can be broadly classified as label-free (detection signal is generated
from the interaction of the analyte with the transducer) or label-based (uses a label, and the
optical signal is generated by a colorimetric or luminescent method).

To improve the measured signal, other optical elements, such as lenses for focusing the
beam or filters for removing noise, may also be included.

Some of the prominent technologies that have dominated the field of optical biosensors
include (1) spectrophotometry, (2) fluorimetry, (3) Surface Enhanced Raman Spectroscopy
(SERS), (4) chemiluminescence, and (5) Surface Plasmon Resonance (SPR)
• Spectrophotometry measures the absorption of light as it passes through a solution. Spectrophotometry

• By using the Beer-Lambert law, which states that the absorbance is directly proportional to the concentration of the
analyte (Ball, 2006), the concentration of analyte in a solution can be quantified.

• Spectrophotometry can be sub-divided according to the wavelength of the measured light.

• These include visible spectrometry (also known as colorimetry), UV spectrometry, and infra-red spectrometry .

• Traditionally, colorimetry has been used for qualitative purposes, e.g. in lateral flow immunoassays as
aforementioned.
• However, by measuring the intensity of visible light at a specific wavelength, quantitative applications are also
possible.

• While drug molecules may not inherently absorb nor emit light in the visible spectrum, colorimetric approaches are
made possible by conjugating signal transducers (e.g. gold or silver nanoparticles) to biorecognition elements.
Various research groups have modified the surface of noble metal, such as gold and silver,
nanoparticles with small molecules that interact favourably to the target analyte.

For instance, amidosulfonic acid-capped silver nanoparticles (ASA-AgNPs) were used for
the visible spectrophotometric detection of lamotrigine, an antiepileptic drug.

The amine groups of amidosulfonic acid interact with lamotrigine through cooperative
hydrogen bonding.

These modified AgNPs are normally dispersed and give a yellow color, but in the presence
of lamotrigine, the ASA-AgNPs aggregate and turn red.

Subsequently, further aggregation produces a violet color.

The color change was monitored by measuring the ratio of absorbance at 450 nm–390 nm
after 30 min of incubation of the ASA-AgNPs and sample using a UV-2550
spectrophotometer.
Consequently, biologically derive constructs, such as antibodies, enzymes, and aptamers, have been explored as highly
selective and sensitive bioreceptors.

For example, gold nanoparticles (AuNPs) were employed in conjunction with digoxin aptamers for the quantification of
digoxin.

In the absence of digoxin, the negatively charged

surface of AuNPs adsorbs the positively charged bases of
aptamers via electrostatic interaction.

This stabilizes the AuNPs, protecting them from salt-

induced aggregating and causing the solution to appear
wine-red in color.

When digoxin is added, the aptamers conjugates to

digoxin and detaches from the surface of AuNPs, which
subsequently aggregates upon the addition of sodium
chloride.

This causes the color of the solution to turn blue. Here, the
absorbance at 520 nm was measured using a Synergy H4
microplate reader.
Fluorimetry
Fluorescence occurs when a molecule absorbs electromagnetic radiation and re-emits
light at a different wavelength; the wavelength of the re-emitted light is characteristic of
the molecule.

The intensity of the re-emitted light, measured using a fluorimeter or plate reader, is
subsequently used to determine the concentration of the molecule.

Compounds that exhibit fluorescence are known as fluorophores.

Like visible spectroscopy, most drug molecules are not fluorophores and cannot be
inherently measured through fluorimetry.

Instead, a fluorophore (e.g. safranin) is often conjugated to the biorecognition element.

Other systems may pair the fluorophore with a quenching agent, which is a substance
that reduces the fluorescence intensity of the fluorophore.
Fluorescent energy meter:
Other systems may pair the fluorophore with a quenching agent, which is a substance that reduces the
fluorescence intensity of the fluorophore.

In these, the distance between the fluorophore and the quenching agent is altered as a result of the biorecognition
event. This can occur in several ways, such as a conformational change in the biorecognition element due to
analyte binding, or displacement of the fluorophore/quenching agent by the analyte.

The concentration of the analyte is directly or indirectly proportional to the fluorescence intensity depending on
the system’s native state, i.e. the former if the biorecognition event causes the distance between the fluorophore
and quenching agent to increase.

Occasionally, the analyte of interest may itself act as a quenching agent

Quartz crystal microbalance
 Enzyme Thermistor

• ET - an immobilised enzyme column “immobilised enzyme reactor” or

“IMER”) placed in a flow micro-calorimeter run as a flow-injection analyser.

Working principle of enzyme thermistors

• evolution of heat is a general property accompanying biochemical

transformations.

• total heat evolution is proportional to the molar enthalpy change and to the

total number of moles of product molecules created in the reaction.

 Q=−n Δ(H) Where,
Q – total heat
nP – moles of product
ΔH – molar enthalpy change

• resulting temperature change (ΔT) is dependent on the total heat capacity (CS) of

the system.

• change in temperature recorded by the thermal biosensors can be defined by:

• enthalpy changes for enzymatic catalysis is around −10 to −200 kJ mol −1

• Substrate concentrations at clinically interesting levels for a range of

metabolites including free and esterified cholesterol, ethanol, glucose, lactate ,

oxalate, triglycerides and urea.

• them can be determined in the concentration range down to 10 µM.

 • is especially advantageous when multiple reactions are involved

• the sum of all the reaction enthalpies that determines the sensitivity of
the assay

• In the case of oxidases, it is recommendable to co-immobilise catalase, which

consumes the hydrogen peroxide produced in the oxidase reaction.

• highly exothermic reaction doubles the sensitivity and reduces the deleterious effects
that hydrogen peroxide has on enzyme activity.

• Furthermore, it reduces oxygen consumption thereby increasing the linear range of

the oxidase reaction.
 • the high protonation enthalpy of certain buffers like Tris can be utilised to
enhance the total enthalpy of proton-producing reactions.

• gain in enthalpy change by selecting the most suitable buffer can be as much as
50 kJ mol−1.

• Small amounts of organic solvents (around 5%, v/v) present in the aqueous
buffer increases the temperature (double times the registered temperature
changes by increasing the total enthalpy change in the reaction.
Immuno-biosensors Based on Thermistors

• total heat evolution is proportional to the molar enthalpy change and to the total
number of moles of product molecules created in the reaction.

where ,
Q – total heat,
nP – moles of product,
H – molar enthalpy change.

The resulting temperature change (T) is dependent on the total heat

capacity (CS) of the system including the heat capacity of the solvent.
Bio-recognition elements in thermistors

• several simple, inexpensive devices for the determination of glucose, urea and
other biological compounds.

• generally flow systems based on combining thermometric detection with

immobilised enzymes

• Bio-recognition elements: Enzymes and the effect of reaction evaluated by

measuring temperature.

• Selected enzyme should be - exhibit high substrate specificity- important

property of enzymes is that they can be easily immobilised on a solid support.
• approach consumes less enzyme or highly purified protein and also increases the
enzymatic stability against heat, pH and organic solvents.

• combination with a suitable support immobilised enzymes can be used repeatedly

for as long as they remain active.

• disadvantage of surface-bound enzyme - usually exhibits slightly lower activity

compared to solute state of the same enzyme.

• Choice of coupling technique - the properties of the support material and the

structural characteristics of the enzyme can have a pronounced effect on the

resulting catalytic performance of the surface-bound enzyme.

 Properties of the support material used for immobilisation are also important

• Pores should be large enough for the enzyme to be immobilised within the support
matrix.
• for the substrate to easily access the catalytic site of the surface-bound enzyme.

Structural information about the enzyme:

• such as functional groups available for immobilisation and the size of the enzyme

• can therefore be useful for obtaining the maximum activity of the bound enzyme within
the support matrix.

• each enzyme is unique in its characteristics and an empirical approach is always

required to obtain and maintain the maximum activity of the support-bound enzyme.
Alternative methods:

• Whole cells, organelles, tissues slices

• were less specific than the pure enzyme and responded to a number of interfering
compounds that are usually present in food samples.

• lower specificity - introduction of the split-flow technique with a reference column.

Immobilisation of bio-recognition elements and support materials in ETs

Physical and Chemical property of the support: on which the enzyme is immobilised
has a crucial role for retaining the catalytic activity of the enzyme.
Choosing the support material some basic criteria should be fulfilled:

(1) the support must possess mechanical stability high enough to withstand physical stress

(2) it should give constant and good flow properties

(3) the support material must not have any catalytic activity or tendency to interact with

the sample that would disturb the measurements of the enzymatic reaction .

• controlled pore glass (CPG) which is available is different pore and particle size ranges

and with different ligands.

• CPG offers high enzyme-binding capacity, good mechanical, chemical and microbial

stability and simple immobilisation chemistry

Various supporting material ET process
Nylon tubing crude solutions,
microbiological fermentations,
Waste water

Reticulated vitreous carbon Hybrid biosensors

(RVC)
 Various supporting material ET process
RVC surface using silica sol– immobilisation of glucose
gel oxidase (GOx)/catalase
ceramic hydroxyapatite reversible adsorption and
desorption of enzymes

• the bio-recognition element (such as enzyme) can be covalently + nylon and glut

aldehyde (treated with di-methylsuphate) followed by derivatisation poly-

ethyleneimine.

Reticulated vitreous method

• pore size varying between 100 and 200 m.

• crushed into smaller pieces in order to increase surface area and then coated with
chemically polymerised pyrrole for better electrical conductivity. But the binding
capacity is very less.

• Enzyme is adsorbed onto the conductive matrix (entire column function as an

enzyme reactor)
• RVC (Agrose gel) which makes covalent bonding by using cyanogen bromide
activation.

• immobilisation of glucose oxidase (GOx)/catalase on RVC surface using silica sol–

gel as a binder.
• porous nature of sol–gel increased enzyme loading,
• Hydroxyapatite -accessible and inexpensive chromatographic matrix, which
allows reversible adsorption and desorption of enzymes

• reversible adsorption and desorption of enzymes

• eliminating any need for covalent immobilisation or chemical cross-linking.

Consideration on immobilization of enzyme:

• large excess of enzyme is usually immobilised which leads to almost complete

consumption of the amount of substrate injected.

• enzymes covalently attached to amine-CPG with a pore size in the range 500–
2000˚A and particle size around 80 mesh (0.18 mm) activated with
glutaraldehyde.
• Schiff’s base formation between the aldehyde group and the amine group of the
enzyme
 Cell immobilisation

• entrapped within a three-dimensional lattice of 15% polyacrylamide

• immobilised through the bio-specific interaction -between glycoproteins on

the cell membrane and lectins.

• common to use entrapment in ionotropic hydrogels(Ba/Ca-alginate or -

pectate)
Thermal transducers:
• beads (down to 0.1 mm size) or small
discs of metal oxides with a high
negative temperature coefficient in the
order of −4% degree−1.

• easy to measure (differential)

temperature changes < 10−5

• thermistors are used as sensors in flow

metres for gases and liquids.

• thermopiles that are less flow sensitive

and film thermistors that can be
integrated on chips in lab-on-chip
• transducer is protected from fouling from the solutions passing through the
column, which makes the transduction mechanism drift- and calibration-free.
Construction of Enzyme Thermistor:

• thick-walled aluminium block which can be thermostated at 25, 30 or 37 ◦C

with a stability of at least ±0.01 ◦C

• (0.2–1.0 mL) containing enzyme immobilised on a solid support, such as CPG.

• Connected to a Wheatstone bridge with a maximum sensitivity of 100 mV

for a temperature change of 0.001 ◦C.

• full-scale sensitivities are in the 10–50 m◦C range.

• determination in the 0.01–100 mM range for most reactions

• by peristaltic pump with a flow rate of 0.5–2 mL min −1.

 Mini/micro thermometric devices

• micromachining of liquid filters,

transducers, micro valves and
micro pumps on chips.

• miniaturised ET was scaled down

to about 1/5 times

• plastic devices (50 mm×15 mm)

with 1.5 mm×15 mm GOx/catalase
column
Problem two successful approaches

• small dialysis/filtration unit was included into the construction to remove blood
cells.

• resulted in a linear range of 0–25 mM glucose, which is adequate for diabetes

monitoring.

• Another way: a superporous agarose material was employed as enzyme carrier.

• allows a large number of whole blood samples to be injected without any sign
of clogging.

• showed that these pocket-size biosensors could perform personal monitoring on

1 µL blood samples with 10 times better precision than current electrochemical
personal devices
• 200 whole blood samples could be analysed without need for recalibration.

• Detection of glucose in urine in the concentration range of 1–4 mM

 Microfabricated (micro-) thermometric devices

• 14 mm ×6 mm ×0.4 mm silicon
chip

• the sensitivity and LOD were

reduced due to low enzyme
loading.

• arrangement permits avoiding temp

erature fluctuations and non-specific

heat production due to dilution,

mixing

• Determination of glucose, lactate, urea and penicillin was performed

using an integrated thermal biosensor array
 • It may not be necessary to use reference column or split flow to discriminate
effectively between the desired signal and the deleterious effects.

Integrated thermal biosensor arrays

glucose, lactate, urea and penicillin was performed
using an integrated thermal biosensor array

lactate oxidase, GOx, urease, -lactamase were

separately immobilised on N-hydroxysuccinimide
(NHS)-activated agarose beads, filled into the
individual zones in the micro-channel.

lactate and GOx reactions, catalase was co-

immobilised together with these enzymes to generate
additional heat and recover a portionof the oxygen
which extend their linear ranges.
Hybrid sensors:

• Immobilised cellobiose dehydrogenase

was used in a combined thermometric
and amperometric biosensor to
measure lactose in milk.

• Highly reproducible linear response was

obtained for lactose between 0.05 mM
and 30 mM
• Furthermore applications of the ET have been demonstrated in hyphenation with

optical biosensor techniques for studying mixtures of contaminants such as heavy

metals and pesticides .

• Using the combined setup the mixture of metals such as arsenic and

organophosphate pesticides
 Surface Plasma Resonance

An optical detection process occurs when a

polarized light of certain wavelength and angle
strikes on gold plated prism.

Total internal reflection create and strikes an

electrically conducting gold layer at the interface
between media of different refractive index: the
glass of a sensor surface (high refractive index) and
a buffer (low refractive index).

The free electrons at the surface of the biochip

absorb incident light photons and convert them into
surface plasmon waves.

The effect of the plasmon is to create a very strong

oscillating electric field near the surface, which is
called the evanescent wave
The index of refraction of a surface bound layer is proportional to the concentration of bound
molecules.

It can determine the kinetic on and off rates for the interaction of a biomolecule with
a ligand in real time.

The change of the incident angle required for SPR is defined as SPR response in the unit of response unit
(RU).
The main objectives of SPR are:
i) to quantify proteins and other biological molecules at surfaces.

ii) to detect the interaction of Drug-protein,

iii) to detect the interaction of Hormone-protein,

iv) to detect the interaction of Protein- protein,

v) to detect the interaction of DNA-protein,

vi) to detect the interaction of Carbohydrate-protein,

v) to detect the interaction of Lipid-protein interactions.

vi) to interface with mass spectrometers provides discovery-based research in proteomic studies
Utility of Biacore systems:

Biacore systems are used primarily in pharmaceutical development, quality control and basic life science
research.

Measurements with Biacore systems provide valuable information in a number of application areas:

• To detect the concentration of proteins in body fluids

• To screen the binding partners and ability for binding, for instance in drug discovery and
biopharmaceutical development.

• To determine the interaction capabilities, for example epitope mapping with monoclonal antibodies

• To measure the interaction kinetics and affinity, made possible by realtime detection of interaction events
. Biacore Processing unit contains following components:
Figure 2
i) Two pump
ii) Syringe Needles
iii) Integrated µ-Fluidic Cartridge (IFC),
iv) Optical detector for measuring SPR response.
v) Autosampler
vi) Microprocessor
vii) Two racks for samples for holding samples in vials
viii) Sensor chips holder
Pump: One pump (Eluent) transport running buffer over sensor chip for maintain constant flow
of liquid and another pump (Autosampler) for injecting samples in the auto sampler
One needle is connected to the pump for constant flow of

running buffer and other one is connected to the autosampler

to handle the sample work.

IFC consist of:

Pneumatic valves which control the flow of samples and buffer

through the channels
i) Sample loop which carry the samples of definite volume

ii) Four flow channels directly connected to the optical detector.

iii) Recovery channel collect the sample after binding for

further mass analysis
Auto sample needle acts as a robot for collecting samples from
the vials and injecting to the sensor chip.

Microprocessor: control pumps, auto sampler, IFC and optical

detector
Sensor chips holder place the chip over the IFC. Four flow cells

just placed over the four channels of the IFC.

Immobilization on CM5 chip
Immobilization of ligands containing free thiols by formation of
disulphide bond with sulfur matrix on the sensor chip
 Mass based biosensors

• Mechanical energy (Force) to Electrical signal (Resonance frequency)

Types of biosensors

• Piezoelectric biosensors

• Surface acoustic wave biosensors

• Cantilever biosensors

Piezoelectric effect
• Aluminum phosphate (berlinite)

• Aluminum nitride

• Zinc Oxide

• Crystalized topaz (Al2SiO4(F, OH)2)

• Barium and Lead titanate

• Gallium orthophosphate

• Quartz (SiO2), Tartrate tetrahydrate (Rochelle salt)

• Polyvinylidene fluoride, Polylactic acids.

 there is also a drawback limiting its use for an analytical application because
not only physical or chemical interactions

 but also other properties like viscosity of ambient solution has impact on the
frequency shift.
 • Optimization of desired characteristics of biosensors

• sensitivity,

• selectivity,

• stability,

• detection limit,

• reliability,

Department of • response time,

Click to Edit
Biotechnology
• reproducibility,

• range and linearity,

• safety,

• simplicity
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 • cost, and parameters like operating conditions,
calibration, positive and negative controls.

Sensitivity

The minimum amount of analyte that can be detected by a biosensor defines its
limit of detection (LOD) or sensitivity.

In a number of medical and environmental monitoring applications, a biosensor

is required to detect analyte concentration of as low as ng/ml or even fg/ml to
confirm the presence of traces of analytes in a sample.
Department
Clickof
to Edit
Biotechnolo
For instance, a prostate-specific antigen (PSA) concentration of 4 ng/ml in blood
gy is associated with prostate cancer for which doctors suggest biopsy tests. Hence,
sensitivity is considered to be an important property of a biosensor.

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 Experiment should be carried out by varying the substrate concentration (1M,
0.1 M, 0.01 M, 0.0001 M, 0.00001 M…….

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 Selectivity
• Selectivity is the ability of a bioreceptor to detect a specific analyte in
a sample containing other admixtures and contaminants.

• The best example of selectivity is depicted by the interaction of an

antigen with the antibody.

• Classically, antibodies act as bioreceptors and are immobilized on the

surface of the transducer.

Click to Edit • A solution (usually a buffer containing salts) containing the antigen is
then exposed to the transducer where antibodies interact only with the
antigens.

• To construct a biosensor, selectivity is the main consideration when

choosing bioreceptors.
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Click to Edit

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 Stability is the degree of susceptibility to ambient disturbances in and around the
biosensing system.

These disturbances can cause a drift in the output signals of a biosensor under
measurement.

This can cause an error in the measured concentration and can affect the precision and
accuracy of the biosensor.

Stability is the most crucial feature in applications where a biosensor requires long
incubation steps or continuous monitoring.

Click to Edit The response of transducers and electronics can be temperature-sensitive, which may
influence the stability of a biosensor.

Therefore, appropriate tuning of electronics is required to ensure a stable response of the

sensor.

Another factor that can influence the stability is the affinity of the bioreceptor, which is
the degree to which the analyte binds to the bioreceptor.
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 Bioreceptors with high affinities encourage either strong electrostatic
bonding or covalent linkage of the analyte that fortifies the stability of
a biosensor.

Another factor that affects the stability of a measurement is the

degradation of the bioreceptor over a period of time.

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 Detection limit

Limit of Blank (LoB), Limit of Detection (LoD), and Limit of Quantitation (LoQ) are terms used to
describe the smallest concentration of a measurand that can be reliably measured by an analytical
procedure.

LoB is the highest apparent analyte concentration expected to be found when replicates of a blank
sample containing no analyte are tested.

LoB = mean blank + 1.645(SD blank)

LoD is the lowest analyte concentration likely to be reliably distinguished from the LoB and at which
detection is feasible. LoD is determined by utilising both the measured LoB and test replicates of a
Click to Edit sample known to contain a low concentration of analyte.

LoD = LoB + 1.645(SD low concentration sample)

LoQ is the lowest concentration at which the analyte can not only be reliably detected but at which
some predefined goals for bias and imprecision are met. The LoQ may be equivalent to the LoD or it
could be at a much higher concentration.

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 Reliability

Sensor reliability is the likelihood that a sensor will function as intended without failure or
degradation over a specific time period and under certain conditions. It's influenced by
many factors, including:

Environmental conditions: Sensors can be affected by a variety of environmental

conditions, such as temperature, pressure, humidity, shock, vibration, and noise.

Sensor design: The design of the sensor should consider the required strength.

Manufacturing quality: The quality of manufacturing can impact the reliability of a sensor.

Installation: How the sensor is installed can affect its reliability.

Click to Edit
Calibration: The calibration of the sensor can impact its reliability.

Maintenance: The maintenance of the sensor can impact its reliability.

Operational stress: The operational stress on the sensor can impact its reliability.

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 Response time

Response time is the time taken by the sensor device to respond to a step
concentration change from initial to a definite value of concentration.

Sensor type

The type of sensor affects its response time. For example, a pressure sensor's response
time depends on the media being measured, such as air, water, or oil.

Environmental conditions

The environmental conditions, such as still air, motion, still liquid, or type of liquid, affect the
Click to Edit
sensor's response time.

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 Reproducibility

Reproducibility is the ability of the biosensor to generate identical responses for a

duplicated experimental set-up.

The reproducibility is characterized by the precision and accuracy of the transducer

and electronics in a biosensor.

Precision is the ability of the sensor to provide alike results every time a sample is
measured and accuracy indicates the sensor's capacity to provide a mean value close
to the true value when a sample is measured more than once.
Click to Edit
Reproducible signals provide high reliability and robustness to the inference made on
the response of a biosensor.

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 Range and linearity

Linearity is the attribute that shows the accuracy of the measured response (for a set
of measurements with different concentrations of analyte) to a straight line,
mathematically represented as y=mc, where c is the concentration of the analyte, y is
the output signal, and m is the sensitivity of the biosensor.

Linearity of the biosensor can be associated with the resolution of the biosensor and
range of analyte concentrations under test.

The resolution of the biosensor is defined as the smallest change in the concentration
of an analyte that is required to bring a change in the response of the biosensor.
Click to Edit
Depending on the application, a good resolution is required as most biosensor
applications require not only analyte detection but also measurement of
concentrations of analyte over a wide working range.

Another term associated with linearity is linear range, which is defined as the range of
analyte concentrations for which the biosensor response changes linearly with the
concentration.

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 Safety

Environmental factors:

Temperature,

Reaction nature

Bacterial Interaction

pH

Microbial growth

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 Simplicity

• Fabrication aspect of sensor

• Easy to fabricate

• Not use of huge layers of coating

• Huge layers of coating enhance the diffusion of solution on sensor surface

• Direct measurement

• Less time to preparation of sensors

Click to Edit

Cost: It should be less cost

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 parameters like operating conditions, calibration, positive and negative controls

Ideal response of the sensor

Calibration methods

Different types of sensors require different calibration methods:

Laboratory standards: Some sensors, like pH, dissolved oxygen, and temperature, can
be calibrated against known laboratory standards.

Physical references: For some sensors, like rangefinders, physical references like
rulers and meter sticks can be used for calibration. For temperature sensors, boiling
Click to Edit water and the triple point of pure water can be used.

Intrinsic and extrinsic calibration: In the automotive industry, intrinsic calibration is used
to find parameters for interpreting raw sensor data, while extrinsic calibration is used to
find the offset between a sensor's coordinate system and another coordinate system.

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 Positive controls
Produce the expected result and confirm that the test procedure is working. This
helps to validate the effectiveness of the reagents and kit.

Negative controls
Do not produce a result and are used to confirm that only the antigen of interest is
causing a reaction. This helps to rule out non-specific binding or other

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 1. Write about structure and properties of DNA and Protein

2. Write about structure and properties of peptides and Enzymes

3. Explain about reversible immobilization technique

4. Explain about irreversible immobilization technique

5. Explain about different optimization steps for fabricating biosensors

6. Explain about DNA based biosensors

7. Explain about the whole cell biosensors

Click to Edit
8.Explain about label and label free biosensor.

9. Explain about Nanomaterial used in biosensor technology.

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 DNA based Biosensors

• Nucleic acids have become the ultimate tools in the recognition and monitoring of
many important compounds.

• DNA biosensor technologies are under intense investigation owing to their great
promise for rapid and low-cost detection of specific DNA sequences in human, viral
and bacterial nucleic acids.

• As the sequencing of the human genome continues, the mutations responsible for
numerous inherited human disorders are now mapped.

Click to Edit • Pathogens responsible for disease states, bacteria and viruses are also detectable via
their unique nucleic acid sequences and interest in their detection continues to grow.

• The analysis of nucleic acids has gained broad acceptance in diagnostic testing,
pharmacological research, and numerous other fields including animal husbandry and
detection of transgenes.

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 The Concept of DNA Biosensor

A biosensor is made from a biological sensing element attached to a signal transducer. The
sensing element can be enzymes, antibodies (as in immunosensors), DNA, or
microorganisms; and the transducer may be electrochemical, optical, or acoustic in nature.

Electrochemical transducers measure changes in current or voltage;

optical transducers measure changes in fluorescence, absorbance or reflectance; and

acoustic transducers measure changes in frequency resulting from
small changes in mass bound to their surface.

Click to Edit A signal transducer is an essential component of a biosensor. It converts the recognition
event into a measurable signal.

The transducer can take many forms depending upon the parameters being measured.

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(A) The two half-matching oligos hybridize with the MB to form a nick and may open
the stem slightly. (B) The DNA ligase binds to the nick and catalyzes the ligation of two
short oligos to form a longer oligo. The ligation product then hybridizes with the MB,
which restores the quenched fluorescence of the MB.

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Frequency–time response of a PNA/QCM to additions of the target (T) and mismatch

(M) oligonucleotides. The hybridization event results in decreased frequency,
reflecting the increased mass of the crystal.

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 DNA dendrimers---increase sensitivity

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Schematic drawing showing the hybridization detection at the dendrimer/QCM

biosensor. The 38-mer probe is attached to the core dendrimer by complementary
oligonucleotide (a(-)) binding on one (a(+)) of the outer arms. The probe sequence
for target hybridization is 5¢-GGG GAT CGA AGA CGA TCA GAT ACC GTC
GTA GTC TTA AC-3¢.
 DNA biosensor miniaturization

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 Concept of DNA microarray

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Figure 2
 DNA microarray

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 DNA Microarray (cont’s)

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The fluorescence intensities for each spot is indicative of the

relative aboundance of the corresponding DNA probe in the
Nucleic acid target samples
 Affymetrix’s Gene Chip

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The light directed probe array synthesis process used for the preparation of
Affymetrix’s Gene Chip
 DNA biosensor not limited to DNA detection, but
more…

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