1. BIOSENSORS

2. CONTENTS Introduction Basic components of Biosensor
Working of Biosensor
Types of Biosensor
Applications of Biosensor
Conclusion

3. DEFFINITION
A device that uses specific biochemical reactions mediated by isolated enzymes, immunosystems, tissues, organelles or whole cells to detect chemical compounds usually by electrical, thermal or optical signals. 0r
The term biosensor is defined as a sensor incorporating biological elements such as enzymes, antibodies, receptors proteins, nucleic acids, cells, or tissue sections - as the recognition element, coupled to a transducer.

4. Block Diagram of a Biosensor
Sample (Analyte or Substrate)
Signal Processing Device
BiorecognitionElement
Transducer

5. PRINCIPLE
The biological material is immobilized and a contact is made between the immobilized biological material and the transducer
The analyte binds to the biological material to form a bound analyte which in turn produces the electronic response that can be measured.
Sometimes the analyte is converted to a product which could be associated with the release of heat, gas (oxygen), electrons or hydrogen ions. The transducer then converts the product linked changes into electrical signals which can be amplified and measured

6. WORKING PRINCIPLE
Biosensors basically involve the quantitative analysis of various substances by converting their biological actions into measurable signals.
Generally the performance of the biosensors is mostly dependent on the specificity and sensitivity of the biological reaction, besides the stability of the enzyme.

7. WORKING PRINCIPLE
Analyte diffuses from the solution to the surface of the Biosensor.
Analyte reacts specifically & efficiently with the Biological Component of the Biosensor.
This reaction changes the physicochemical properties of the Transducer surface.
This leads to a change in the optical/electronic properties of the Transducer Surface.
The change in the optical/electronic properties is measured/converted into electrical signal, which is detected.

8. Analyte
Response
Detection
Sample handling/preparation
Signal
Analysis

13. (f)
(e)
(d)
(a)
(c)
(b)
The interaction of the analyte (a) with the bioreceptor, which identifies the stimulus (b) is designed to produce an effect measured by the transducer (c), which converts it to an electrical signal. The output from the transducer is amplified (d), processed (e) and displayed (f).

14. IDEAL BIOSENSORS
The output signal must be relevant to measurement environment.
The functional surface must be compatible with the transducer.
High specificity and selectivity (low interference).
Sufficient sensitivity and resolution .

15. Sufficient accuracy and repeatability
Sufficient speed of response
Sufficient dynamic range.
Insensitivity to environmental interference or their effects must be compensated

16. Biosensor components Analyte Biological elements Transducer
(processor)
(monitor)

17. ANALYTE

18. THE ANALYTE
(What do you want to detect?) Molecule Protein, toxin, peptide, vitamin, sugar, metal ion
Cholera toxin
Glucose

19. BIOLOGICAL ELEMENTS

20. BIOLOGICAL ELEMENTS Fab Active site Membrane receptors
Competitive binding Fc
Antibody
Enzyme
Cell
Polymer/Hydrogel

21. Biological recognition element-
It is the sensitive biological element or biological material (tissue ,microorganisms, organelles,cell receptors , enzymes, antibodies, nucleic acids,etc.) or bio- mimetic component that interacts (binds or recognises) the analyte under study.
The biologically sensitive elements can also be created by biological engineering.

22. Enzymes Antibodies Hormone receptors Cells Cell organelle Tissues
Biological elements
Enzymes
Antibodies
Hormone receptors
Cells
Cell organelle
Tissues
Membranes
Micro organisms
Nucleic acids
Biomimetic materials

24. 1.Enzymes-
Enzyme-based biosensors use their catalytic activity and binding capabilities for specific detection .
The catalytic activity of the enzymes provides these types of biosensors with
the ability to detect much lower limits than with normal binding techniques.
This catalytic activity is related to the integrity of the native protein structure.

25. Most common – Glucose oxidase, Urease, Alcohol oxidase etc.
Commercial example is GLUCOSE SENSOR using oxidase (GOD) .
Clarks (1962)
Glucose + O2 -- glusoce oxidase  Gluconic acid + H2O2 (1)
H2O2  O2 + 2H+ + 2e- (3) at anode
O2 + 4H+ + 4 e-  2H2O (2) at cathode
-and current flows.
There are three measurement routes-
-pH change (acid production) O2 consumption (fluorophore monitor)
- H2O2 production (electrochemical)

26. 2. Micro-organisms –
Microorganisms such as bacteria and fungi can be used as biosensors to detect specific molecules or the overall state of the surrounding environment.
Cell behaviour such as cell metabolism,
cell viability, cell respiration, and bio- luminescence can be used as indicators for the detection.
Furthermore, proteins that are present in cells can also be used as bio-receptors for the detection of specific analytes.
Rhodococcus erythropolis (in collagen) used to measure BOD.

27. 4. Organelles – Mitochondra , Cell walls etc. 5. Membranes .
3. Cell / Tissue samples.
Use of cell as a biosensor occurred in 1977 by Rechnitz .
Rechnitz coupled Streptococcus faecium on the surface of an ammonia gas sensing membrane electrode .
This Rechnitz electrode was capable of detecting the amino acid ARGININE.
4. Organelles – Mitochondra , Cell walls etc.
5. Membranes .

28. 6. Bio-mimetic materials -
A bio mimetic biosensor is an artificial or synthetic sensor that mimics the function of a natural biosensor .
These can include aptasensors , where apta -sensors use aptamers as the biocomponent.
Aptamers are synthetic single stranded nucleic acid that can be designed to identify or recognize amino-acids, oligosaccharides , peptides , and proteins . Aptamers have high affinity , high selectivity,
cheaper & easy to synthesize .

29. 1. Antibody / Antigen (Immunosensor) -
B. Affinity biosensors:
device in which receptor molecules bind analyte molecules causing physicochemical change that is detected by a transducer.
Receptors used are-
1. Antibody / Antigen (Immunosensor) -
The high specificity between an antibody and an antigen can be utilized in this type of sensor technology. Biosensors utilizing this specificity must ensure that binding occurs under conditions where nonspecific interactions are minimized . Binding can be detected either through fluorescent labelling or by observing a refractive index or reflectivity changes.
2. Hormone receptor/Antagonist-

30. 3. Nucleic acid --
The complementary relationships between nucleic acid ‘bases’ in the DNA form the basis of specificity in nucleic acid based biosensors.
These sensors are capable of detecting trace amounts of microorganism DNA by comparing it to a complementary strand of known DNA.
By unwinding the target DNA strand, adding the DNA probe, and annealing the two strands , the probe will hydrolyze to the complementary sequence on the adjacent strand . If the probe is tagged with a fluorescent compound , then this annealing can be visualized under a microscope.
eg. DNA Chip.

31. (Target probe ) (Capture probe)
General DNA biosensor scheme--Target DNA is captured at the recognition layer (A),
and the resulting hybridization is transduced into a measurable electronic signal (B).
It is used for genome mutation detection & clinical diagnosis.

32. BIOLOGICAL ELEMENTS CONSTRUCTION
The principle of detection is the specific binding of the analyte of interest to the complementary biorecognition element immobilised on a suitable support medium.

33. BIOLOGICAL ELEMENTS CONSTRUCTION
Recognition element immobilization (Immobilization of biological receptor)-
Biological receptors, i.e. enzymes, antibodies, cells or tissues with high biological activity, can be immobilized in a thin layer at the transducer surface by using different procedures.
Entrapment behind a membrane - viscous aqueous solution
trapped by membrane permeable to analyte.
(b) Entrapment of biological receptors within a polymeric matrix-
Membranes—
Cellophane, Cellulose acetate, Polyurethane membranes .
Gel entrapment–
Agarose, Gelatin, Agar gel , Polyacrylamide gel.
Microencapsulation-
Encapsulation inside Liposomes, or absorbed in fine carbon
particles that are incorporated in a gel or membrane .

34. (c) Entrapment of biological receptors within
self-assembled monolayers (SAMs)
or bilayer lipid membranes (BLMs).
(d) Adsorption:
direct adsorption onto membrane or transducer;
can also be adsorbed onto pre -adsorbed proteins .
(e) Covalent binding (via –COOH, -NH2, -OH ) , or cross linking (via glutaraldehyde ) to transducer or
membrane surface.
Receptors are immobilized either alone or are mixed with other proteins, such as bovine serum albumin (BSA), either directly on the transducer surface, or on a polymer membrane covering it .

35. (How do you know there was a detection?)
SIGNAL
(How do you know there was a detection?)
Specific recognition?

36. PRINCIPLE OF DETECTION
The principle of detection is the specific binding of the analyte of interest to the complementary biorecognition element immobilised on a suitable support medium.
The specific interaction results in a change in one or more physico-chemical properties (pH change, electron transfer, mass change, heat transfer, uptake or release of gases or specific ions) which are detected and may be measured by the transducer.

37. TRANSDUCER-
The transducer or the detector element transforms the signal resulting from the interaction of the analyte with the biological recognition element into another signal that can be more easily measured and quantified.
AMPLIFIER, MICROPROCESSOR AND DISPLAY-
Biosensor reader device with the associated electronics or signal processors that are primarily responsible for the display of the results.

39. TYPES OF BIOSENSOR I) On the basis of biological element
Biosensor is broadly classified into two classes:
I) On the basis of biological element
a) Enzyme Biosensor
b) Microbial Biosensor
c) Antibody Based.

40. II) ON THE BASIS OF TRANSDUCING ELEMENT
Calorimetric/Thermal Detection Biosensors.
Optical Biosensors.
Resonant Biosensors.
Piezoelectric Biosensors.
Ion Sensitive Biosensors.
Electrochemical Biosensors.
Conductimetric Sensors.
Amperometric Sensors.
Potentiometric Sensors.

41. Calorimetric / Thermal Detection Biosensors.
Uses Absorption / Production of Heat.
Total heat produced/absorbed is ᾶ Molar Enthalpy/Total No. of molecules in the rn.
Temp. measured by Enzyme Thermistors.
Advantages:
No need of Frequent recalibration.
Insensitive to the Optical & Electrochemical Properties of the sample.
Uses:
Detection of: (1) Pesticides .
(2) Pathogenic Bacteria.

42. Optical Biosensors. Resonant Biosensors.
Colorimetric for colour - Measures change in Light Adsorption.
Photometric for Light Intensity - Detects the Photon output.
Resonant Biosensors.
An Acoustic Wave Transducer is coupled with Bioelement.
Measures the change in Resonant Frequency.

43. Piezoelectric Biosensors.
Uses Gold - To detect specific angle at which ȇ waves are emitted when the substance is exposed to laser light/crystals like quartz, which vibrates under the influence of an electric field.
Change in Frequency ᾶ Mass of Absorbed material.
Ion Sensitive Biosensors.
Are semiconductor with ion-sensitive surface.
Surface Electrical Potential changes when the ions & semiconductors interact.
Measures the Change in Potential.
Uses:
pH Detection.

44. Measures Electrical Conductance/Resistance of the solution.
Electrochemical Biosensors.
Underlying Principle – Many chem.rns produce or consume ions or ȇs causing some change in the elctrical properties of the solution that can be sensed out & used as a measuring parameter.
Uses:
Detection of :
Hybridized DNA
DNA- binding Drugs &
Glucose Concentration.
Conductimetric Sensors.
Measures Electrical Conductance/Resistance of the solution.

45. Conductance Measurements have relatively Low Sensitivity.
Amperometric Biosensors.
High Sensitivity Biosensor.
Detects electroactive species present in the biological test samples.
Measured Parameter – Current.

46. Potentiometric Sensors.
Working Principle – When ramp voltage is applied to an electrode in solution, a current flow occurs because of electrochemical reactions.
Measured Parameter – Oxidation / reduction Potential of an Electrochemical rn.

47. APPLICATIONS

48. 1. MEDICAL
Biosensors are used in both clinical and laboratory use in medical care.
Glucose monitoring in diabetes patients .
Medtronic glucose sensor - implants in major vein of heart.
b. Tumor cells are used as biosensors to monitor the susceptibility of
chemotherapeutic drugs.
c. Routine analytical measurement of folic acid, biotin, vitamin B12 and
pentothenic acid .
d. Micro- and nanoscale biosensors—
Genome mutation detection , cancer detection & clinical diagnosis.
Bacterial-UTI , Human Immunodeficiency Virus (HIV) Detection, Hepatitis and Anthrax detection.

49. 2. BIO / PHARMACEUTICAL RESEARCH
Quality assurance
b. Study of biomolecules and their interaction
c. Protein engineering .
Drug discovery , evaluation monitor the manufacturing
of biological activity of new compounds (research field).
e. Biosensors are used for measuring concentration of various metal
ions by specific protein concentration or by using genetically
modified organisms.
Aptamers are used for the detection of proteins –Thrombin , IgE ,
HIV – tat protein , Lysozyme , toxin.

50. 2. BIO / PHARMACEUTICAL RESEARCH
g. Biosensors are used in monitoring of the glutamate and acetyl
choline , which is the main cause in neurodegenerative diseases.
h. Microbiology: bacterial and viral analysis .
i. Biosensors are used in analysing micro dialysis samples.
j. Biosensors are used in biotechnological process such as to
determine proteins or peptides.
k. Biosensors are also used in determining intracellular proteins and
also plasmids.
l. Detection of cancer biomarkers – CEA , PSA , CA-125 &
Tumour necrosis factor .

51. 3. INDUSTRIAL / AGRICULTURAL
Biosensors used in process control will be able to measure materials present in the process.
Use of biosensors in industry will improve manufacturing techniques, this will allow for usage of wider variety of sensing molecules.
Biosensors are used in controlling the industrial processes.
Microbial sensor measure Ammonia & Methane.

52. 4. ENVIRONMENTAL
a. Biosensors are used in detecting environmental pollutants and monitoring of Mines, Industries and toxic gases.
b. Biosensors are used in the BOD measurement during waste
water treatment.
c. Biosensors are used in the detection of poly aromatic
hydrocarbons present in water.
d. Environmental applications e.g. the detection of pesticides
and river water contaminants such as heavy metal ions.
e. Detection and determination of organophosphates .

53. 5. FOOD INDUSTRY
Quality assurance in food industries , ex. E. Coli, Salmonella.
b. Food & drink production analysis.
Biosensors are used for detection of food freshness marker determining parameters in wine industry.
d. Determination of drug residues in food, such as antibiotics
and growth promoters, particularly meat and honey.
e. Detection of toxic metabolites such as mycotoxins.

55. Example of biosensors
Pregnancy test - Detects the hCG(human chorionic gonadotropin) protein in urine.

56. Glucose monitoring device (for diabetes patients)
Monitors the glucose level in the blood.

57. Infectous disease biosensor from RBS
Old time coal miners’ biosensor

58. 6. BIODEFENCE a. Detection of pathogens.
b. Remote sensing of airborne bacteria / virus ,
e.g. In counter bio-terrorist activities.
c. Detection system for biological welfare agent
eg. Bacillus anthracis (anthrax) spores.
d. Determining levels of toxic substances before and
after bioremediation.
e. Crime detection.

59. GLUCOSE OXIDASE SENSOR
A common example of a commercial biosensor is the blood glucose biosensor, which uses the enzyme glucose oxidase to break blood glucose down.
In doing so it first oxidizes glucose and uses two electrons to reduce the FAD (a component of the enzyme) to FADH2.
This in turn is oxidized by the electrode (accepting two electrons from the electrode) in a number of steps.
The resulting current is a measure of the concentration of glucose. In this case, the electrode is the transducer and the enzyme is the biologically active component.

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61. THANK YOU
-PHARMA STREET