Day 1-FN-Dr.SBB.pdf msrit college bengaluru

engineering

1. Biosensors – A Conceptual Overview

Dr. S.B. Bhanu Prashanth
Professor, Medical Electronics
BMSCE
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2. Presentation Outline
1.Small Molecules
2.Biosensors : Define, Components, Classification, Applications
3.Bioreceptors – 1 example
4.Evolution Glucose sensor as a case study : 3 Gs
5.Measurement Techniques (3 considered here)
i.Potentiometric
ii.Voltammetric
iii.Photometric (SPR)
6.Biochips
7.Conclusions
8.Acknowledgements
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3. Small Molecules ?
•1000 Da in size (1 Dalton = 1.6605e-18 µg !!)
•Wide variety of different chemical compounds (either natural or
pharmaceutical origin)
•Relevant - Biological, Pharmacologically, or environmentally
•Detection and quantification - important in many disciplines
•Naturally, nearly every cell contains a collection of 100 to 200
different low molecular weight organic molecules, including the
common amino acids, nucleotides, sugars, and their phosphorylated derivatives.
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Small Molecules : Good / Bad ? BOTH !!!

5. Detection of small molecules
Chromatographic methods
•High sensitivity and specificity
•High cost & bulky instrumentation
•Require expertise
•Less suitable for every purpose

Biosensors can offer a cheaper and faster alternative.
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Real-time monitoring
High specificity
Fast response times
Reduced consumption of organic solvents
Portability
No need of skilled personnel

6. •Sensor / Transducer - a device, capable of being actuated by a
measurand, converts one form of energy into another MORE
useable form.


•Bio – Anything connected with life and living things



•Biosensor - a device, which converts a Biological signal into a more
useful electrical signal.




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Back to Basics

7. Biosensor
A biosensor is a class of sensor devices or systems detecting
the presence or level of a known analyte
A biochemical interaction is exploited as the basis
This analyte may be an
•inorganic compound such as a salt
•a small biological molecule such as a vitamin or sugar molecule
•Larger biological macromolecules such as proteins or nucleic acids.
•Pathogen like a Virus or Bacteria

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Analyte: a substance whose chemical constituents
are being identified and measured.
The initial impetus for advancing sensor technology came from health care area :
Measurements of blood gases, ions and metabolites are often essential and allow a
better estimation of the metabolic state of a patient.

9. The level of glucose in the droplet is inferred through a biochemical reaction
that occurs between the glucose in the sample and an enzyme (glucose
oxidase or hexokinase) in the test strips.

The progress of this reaction is measured either optically, as a change in the
color of the strip, or electrically, by measuring the electrical current produced
by the reaction.
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11. Classification of Biosensors ?

= Not Easy
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13. Environmental quality, Medicine and Industry
(mainly by identifying material and the degree of concentration present)
Applications of Biosensors
• Clinical diagnosis and biomedicine
• Farm, garden and veterinary analysis
• Process control :
fermentation control and analysis
food and drink production and analysis
• Microbiology: bacterial and viral analysis
• Pharmaceutical and drug analysis
• Industrial effluent control
• Pollution control and monitoring
• Mining, industrial and toxic gases
• Military applications
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14. Bioreceptors – Most important
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The choice of the biological recognition element is the
crucial decision
while developing a novel biosensor

15. Most common forms of bioreceptors
1)Antibody-antigen interactions
2)Protein-protein interactions
3)Nucleic acid interactions
4)Enzyme interactions
5)Cellular interactions
6)Interactions using biomimetic materials
7)Synthetic bioreceptor, aptamers, peptides

Aptamers are single-stranded DNA or RNA (ssDNA / ssRNA) molecules that can bind

to pre-selected targets including proteins and peptides with high affinity & specificity.
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The choice of the biological
recognition element is the
crucial
decision while developing a
novel biosensor design

16. •When infected with SARS-CoV-2, the body produces
antibodies that bind specifically to the spike proteins and
other antigens to help eliminate the virus.
•This binding can be harnessed to develop antibody
and antigen-based diagnostic tests.
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Antibody-antigen interactions based Biosensor – An Example

17. An antibody test
•Detects antibodies in their blood or serum.
•Laboratory test such as an ELISA (enzyme-linked immunosorbent assay) or
•CIA (chemiluminescent immunoassay) or
•POC test based on lateral flow technology.
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Data suggests that in the case of SARS-CoV-2, the IgM
antibody response peaks around two weeks after infection,
followed by the IgG antibody peak at three weeks.
https://www.technologynetworks.com/immunology/articles/antibody-vs-antigen-testing-
for-covid-19-336486

18. 1. A high degree of specificity / selectivity

2. A good stability to operating conditions
(Temperature, pH, ionic strength etc.,)

3. No undesirable sample contamination
4.Retention of biological activity in the immobilized state
5.Good Sensitivity
6.Long term stability (6 months- 1 year)
7.Possibility of chemical modification with redox mediators, binding, or
crosslinking with the immobilization matrix


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Desirable Properties of Biosensors

19. Brief History of Biosensors
1916 First report on immobilization of proteins (adsorption of invertase)

1922 First glass electrode (pH)

1956 Clark published his definitive paper on the oxygen electrode.

1962 An amperometric enzyme electrodre for glucose

1969 First potentiometric biosensor (Urea)

1970 Ion Selective Field Effect Transistor (ISFET)

1975 Fibre-optic sensor with immobilised indicator (CO2 / O2)

1975 First commercial biosensor (Yellow springs Instruments)


1998 Blood glucose biosensor launch (LifeScan FastTake)

……………………………………………………………… .

 Currently : nanoparticles, nanowires, nanotube, Graphene, Biochips …..

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21. A Case study on Glucose Biosensor
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The simplest of the carbohydrates - monosaccharide (has one sugar)
Along with fat, glucose is one of the body’s preferred sources of
fuel in the form of carbohydrates.

22. Origin of Biosensor
The biosensor was first described by Clark and Lyons in 1962,
when the term enzyme-electrode was adopted.

In this first enzyme electrode (glucose oxidase) was held next
to a platinum electrode in a membrane sandwich.




The major interference to the
peroxide measurement is
ascorbic acid.
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24. Birth & Growth of Glucose-Biosensors
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25. Evolution of Biosensors / Bio – Sensor Union:
3 Generations of Biosensors
Based on
 The degree of integration of the separate components
 Mediators (natural/synthetic) used to better the measurement process
 Improved transduction techniques (Advances in silicon & nanotechnology,
polymer fabrication, optics and processing have revealed new materials and
methods suitable for exploitation as biosensors. )
Photometric, Potentiometric, Amperometric

TO

PZTs, SAW, Plasmons, conductimetric,
Thermodynamic , ChemFET, Nanorods, CNTs
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26. Birth & Growth of Glucose-Biosensors
The first generation glucose biosensors
The rate of reduction of oxygen is directly proportional to
the glucose concentration. Measure :
> the reduced oxygen concentration OR
> the increased concentration of hydrogen peroxide.

The electrons transferred are recognized by electrode
and thus the number is directly proportional to the
number of glucose molecules present.
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27. Major drawbacks of first generation glucose biosensor:-

•Interference from electroactive species present in blood, such as uric
acid, ascorbic acid and other constituents of blood
•Required for amperometric measurement of hydrogen peroxide.

•Limited selectivity of the analyzer and results in inaccurate
measurements of glucose concentration.

•Oxygen deficit – fluctuations in oxygen tension due to the limited
solubility of oxygen in biological fluids. This reduces the linear range of
the biosensor.
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28. Second Generation glucose biosensor
Search for an alternative for the natural oxygen acting as an electron acceptor
Use of synthetic electron mediators opened up new horizons.
The synthetic electron mediators eliminated the need of oxygen for recording the
electron transfer at the electrode surface overcoming the drawbacks of
x Limited oxygen pressure observed in first generation biosensor
x High redox potential 0.6V --- ( reduced to 0.0V to 0.2V )
No interference from other electro active species such as ascorbic acid and uric
acid
Also ensured faster rate of shuttling of electrons from the redox center of the
enzyme to the surface of the electrode.

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29. FAD: flavin adenine dinucleotiede
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oHigh competition between synthetic
mediator and dissolved oxygen
oInterference
oStability of synthetic mediator near
electrode surface

30. Third-Generation Glucose Biosensors
•To avoid complications offered by the mediators in 2G biosensors
•Research to find new strategies for direct electron transfer (DET)
between the electrode and active center of enzyme
•The intrinsic barrier to electron flow is the globular structure of GOx
is the major hindrance for DET in case of thin film or hydrogel based
electrodes.
•Led to exploration of new electrode materials (Gold Nanowires,
nanoparticles, CNTS)
•Development of highly selective and sensitive third-generation
biosensors.
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32. (1) Potentiometric Techniques
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Transduction / Measurement
Techniques
(3 of the many types)

33. Glass type pH Electrode (Potentiometric Type)
•The glass electrode consists of a glass membrane which is selectively permeable
only to H+ and an internal Ag/AgCl electrode immersed in 0.1 Molar KCl solution.
•The measured voltage follows Nernst equation:-
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‘Selectivity’ of ISEs

•Selectivity of membrane for an ion ‘a’ in the presence of interfering ‘b’




•Kab the Selectivity constant << 1 for good selectivity of ‘a’

•In well-behaved commercial probes, such errors are reduced to mere constants.

Other Performance characteristics of ISEs

Slope (Sensitivity)
Range of response
Stability & Reproducibility
Response time
Sensitivity to ambient conditions
Ease of use
Availability
Cost & Lifetime




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37. Semiconductor Electrodes
 Chemical sensitive FET (CHEMFET)
 Gas -sensitive Metal Gate (IGFET)
 Suspended gate field effect transistor(SGFET)
 Ion selective FET (ISFET)
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CHEMFET

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NANOFET

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NANOFET
with
Specificity

41. (2) Voltammetry & Amperometry
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42. Voltammetry
• Sweep Voltage - Measure Current
•Active technique (the applied potential forces a change in the concentration of an electro
active species at the electrode surface by electrochemically reducing or oxidizing it.)
[ Amperometry : Current proportional to analyte concentration is monitored at a fixed potential ]
as the reduction proceeds, a diffusion
layer is formed and the rate of the
electrode reduction becomes diffusion
limited.
At this point, the current slowly
declines.
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Cyclic voltammetry makes possible
the elucidation of the kinetics of
electrochemical
reactions taking place at electrode
surfaces
From the sweep-rate dependence
of the peak amplitudes, widths and
potentials of the peaks observed in
the voltammogram, it is possible to
investigate the role of adsorption,
diffusion, & coupled homogeneous
chemical reaction mechanisms.

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45. (3) Optical Biosensor
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46. Changes measured
- Intensity
- Frequency
- Phase shift
- Polarization
Types of measurements
- Absorbance
- Fluorescence
- Refractive index
- Light scattering
Types of components
- Wave guides
- Photodiode
- Spectroscopy
- Charge coupled device (CCD)
- Single photon APD
- Interferometers

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Optical Biosensors

47. 8/3/2020 MSRIT_FDP_BS_Overview_Bhanu 47
Review, Optical Biosensors for Label-Free Detection of Small Molecules, Riikka Peltomaa, Bettina Glahn-Martínez, Elena Benito-Peña and
María C. Moreno-Bondi , Sensors 2018, 18, 4126; doi:10.3390/s18124126

48. Surface Plasmon Resonance (SPR) – based Biosensors
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T I R
When the electrical field energy of the photon
is just right, it can interact with the free
electron constellations (outer shell and
conduction-band electrons) in the gold
surface.

The incident light photons are absorbed and
the energy is transferred to the electrons,
which convert into surface plasmons.
Like all conversions, the photon to
plasmon transformation must conserve
both momentum and energy in the
process.

Plasmons have a characteristic momentum defined
by factors that include the nature of the conducting
film and the properties of the medium on either side
of the film.

Resonance occurs when the momentum of
incoming light is equal to the momentum of
the plasmons (momentum resonance).
Gold film

50. SPR Measurements
1.Angular SPR, also known as resonant angle SPR
2.Resonant wavelength SPR or spectral SPR : At a fixed angle of incident
light, the wavelength can be varied until resonance occurs (not widely used)
3.The phase shift of the light can be detected when the coupling
between light and plasmons occur. (Has a high sensitivity : Needs a reference
beam : More complex setup than other methods.)
4.Measurement of the intensity of the reflected light by SPR imaging
5.Measurement of changes in RI (in biosensing aplications)
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51. (1) The SPR-angle
•The SPR-angle depends on : properties of the metal film, the wavelength of
the incident light & the RI of the media on either side of the metal film

•The metal must have conduction band electrons capable of resonating with
the incoming light at a suitable wavelength.

•Metals that satisfy this condition are silver, gold, copper, aluminium,
sodium and indium.

•In addition, the metal on the sensor surface must be free of oxides,
sulphides and it must not react with other molecules on exposure to the
atmosphere or liquid
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Heavy Metal Ion detection Protein Concentration Variations

53. •Gold is very resistant to oxidation and other
atmospheric contaminants but is compatible
with many chemical modification systems.


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Indium is too expensive
Sodium too reactive
Copper and Aluminium too broad in their SPR response
Silver too susceptible to oxidation
This leaves gold as the most practical metal

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The SPR detects and measures changes in
RI due to the binding and dissociation of
interacting molecules at or in proximity to
the gold surface.

This change of the RI is proportional to the
concentration of the interacting molecule &
causes a shift in the angle of incidence at
which the SPR phenomenon occurs.
Fortunately, the change in refractive index on the surface is linear to the
number of molecules bound to the sensor surface (mainly for protein-protein
interactions that have a refractive index change of about 0.18-.019)
5. Measurement of changes in RI (in biosensing aplications)

55. Biochips
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Biochip is a collection of miniaturized test sites (microarrays) arranged on a
solid substrate that allows many tests to be performed at the same time in
order to achieve higher throughput and pace.

56. •A computer chip that can perform millions of operations/second
•A biochip can perform thousands of biological reactions, such as decoding
genes in few seconds.

•A genetic biochip is designed to freeze into place the structures of many
short strands of DNA - the basic chemical instruction that establishes the
characteristics of an organism.

•Effectively, it is used as a test tube for real chemical samples. A specially
designed microscope can determine where the sample hybridized with DNA
strands in the biochip.

•Biochips would help dramatically in accelerating the identification of the
estimated 80,000 genes in human DNA
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57. •In addition to genetic applications, the biochip is being used in
toxicological, protein, and biochemical research.

•Biochips can also be used to rapidly detect chemical agents used in
biological warfare so that defensive measures can be taken.
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Technical Segmentation
(Patent Categorization)

59. Summary
Small Molecules - Sensors
Biosensor basics, Classification
Bioreceptors
Evolution of Glucose sensors
Techniques
Potentiometric
Voltammetry
Optical (SPR)
Biochips
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60. … Thank You ….
([email protected])
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