ANALYTICAL TECHNIQUES

BIOSENSORS
MADE BY: DEEPANSH MODY
U101113FBT259
NIIT UNIVERSITY

INTRODUCTION
A BIOSENSOR IS AN ANALYTICAL DEVICE WHICH IS USED TO DETERMINE THE
PRESENCE AND CONCENTRATION OF A SPECIFIC SUBSTANCE IN A BIOLOGICAL
ANALYTE.
THEY HAVE APPLICATIONS IN DIVERSE FIELDS LIKE DIAGNOSTICS, DISEASE
MONITORING, FOOD INDUSTRY AND ENVIRONMENTAL MONITORING ETC.
PROF. LELAND C CLARK JR. IS KNOWN AS THE FATHER OF BIOSENSORS.

COMPONENTS OF A BIOSENSOR
Desired molecule

Bio receptor
Bio sample

Transducer

Signal

Processing

Biosensor

Compone
nts
Physical
Compone
nts
Transduc
er

Signal
processor

Biological
Compone
nts
Biorecept
or

Display

Bioreceptor
IT IS THE RECOGNITION COMPONENT. IT IS DESIGNED TO INTERACT WITH THE
SPECIFIC ANALYTE OF INTEREST AND PRODUCE AN EFFECT MEASURABLE BY
THE TRANSDUCER.
IT NEEDS TO BE IMMOBILIZED IN THE VICINITY OF THE TRANSDUCER, SO AS TO
OBTAIN PROPER PROBE ORIENTATION, AND INCREASE ACCESSIBILITY TO
TARGET ENZYME.
IT IS DONE EITHER BY PHYSICAL ENTRAPMENT OR CHEMICAL ATTACHMENT.
CHEMICAL ATTACHMENT OFTEN INVOLVES COVALENT BONDING TO
TRANSDUCER SURFACE BY SUITABLE REAGENTS.
IT IS TO BE NOTED THAT ONLY MINUTE QUANTITIES OF BIORECEPTOR
MOLECULES ARE NEEDED, AND THEY ARE USED REPEATEDLY FOR
MEASUREMENTS.

Transducer
A TRANSDUCER IS CAPABLE OF CONVERTING THE BIO-RECOGNITION EVENT
INTO A MEASURABLE SIGNAL.
IT ACTS AS AN INTERFACE, MEASURING THE PHYSICAL CHANGE THAT OCCURS
WITH THE REACTION AT THE BIORECEPTOR, AND THEN TRANSFORMING THAT
ENERGY INTO MEASURABLE ELECTRICAL OUTPUT.
DEPENDING ON THE TYPE OF TRANSDUCER, A BIOSENSOR IS DIVIDED INTO
FOLLOWING CATEGORIES:

WORKING OF A BIOSENSOR
ANALYTE DIFFUSES FROM THE SOLUTION TO THE SURFACE OF THE
BIOSENSOR.
ANALYTE REACTS SPECIFICALLY AND EFFICIENTLY WITH THE BIORECEPTOR.
THIS REACTION CHANGES THE PHYSIO-CHEMICAL PROPERTIES OF THE
TRANSDUCER SURFACE.
THE TRANSDUCER THEN TRANSFORMS THE BIOLOGICAL SIGNAL INTO AN
ELECTRICAL SIGNAL.
THIS SIGNAL IS PASSED ON TO THE MICROPROCESSOR, WHERE IT
UNDERGOES AMPLIFICATION.
IT THEN REACHES THE DISPLAY, OR THE STORAGE DEVICE.

BREAKTHROUGH !!
GLUCOSE MONITORING BIOSENSORS:
THE GLUCOSE CONCENTRATION IN A BLOOD SAMPLE CAN BE MEASURED
DIRECTLY
GLUCOSE + O2 GLUCOSE OXIDASE GLUCONIC ACID + H2O2
BIORECEPTOR: GLUCOSE OXIDASE
TRANSDUCER:
1) OXYGEN SENSOR- MEASURES OXYGEN CONCENTRATION
2) PH SENSOR- MEASURES THE ACID (GLUCONIC ACID)
3) PEROXIDASE SENSOR- MEASURES H2O2 CONCENTRATION

APPLICATIONS OF BIOSENSORS
BIOSENSORS HAVE APPLICATIONS IN A VARIETY OF FIELDS, RANGING FROM
FOOD QUALITY TO DNA SEQUENCING. SOME OF THE APPLICATIONS ARE:
DNA SEQUENCING
MUTATION DETECTION
CRIME DETECTION
FOOD ANALYSIS
DRUG DEVELOPMENT
QUALITY CONTROL
MEDICAL DIAGNOSIS
INDUSTRIAL PROCESS CONTROLS

DNA BASED BIOSENSORS

THE FUNDAMENTAL PRINCIPLE BEHIND DNA BIOSENSORS DEPENDS ON
SEQUENCE COMPLEMENTARITY AS PER CHARGAFF'S RULES OF BASE PAIRING
(FOR DNA, A-T, G- C).
THIS SYSTEM USUALLY RELIES ON THE IMMOBILIZATION OF A SINGLESTRANDED DNA (SSDNA) PROBE ON A SURFACE TO RECOGNIZE ITS
COMPLEMENTARY DNA TARGET SEQUENCE BY HYBRIDIZATION. THUS, BIORECEPTOR IS DNA PROBE.
DEPENDING ON THE TRANSDUCER, A DNA BIOSENSOR IS CATEGORIZED ASOPTICAL DNA BIOSENSORS
ELECTROCHEMICAL DNA BIOSENSORS
PIEZOELECTRIC DNA BIOSENSORS

Optical DNA Biosensors

THEY ARE BASED ON THE OPTICAL TRANSDUCTION OF A BIOLOGICAL SIGNAL

TO AN OPTICAL SIGNAL.
THE DEGREE OF TRANSDUCTION FOLLOWS BEER LAMBERTS LAW.
OPTICAL BIOSENSORS CAN BE MINIATURIZED BY USING FIBRE OPTICS, WHICH
CONVERT EMISSION SIGNAL INTO A DETECTABLE FLORESCENT SIGNAL.
DNA PROBE AND TARGET HYBRIDISATION IS DETECTED BY A FLUORESCENCE
MARKER, WHICH RESULTS IN TOTAL INTERNAL REFLECTION IN THE FIBRE,
WHICH IS MEASURED BY THE DETECTOR.
THE FLUORESCENCE MARKER INTERCALATES WITH THE HELICAL STRUCTURES,
AND IN CASE OF SSDNA, MOLECULAR BEACONS ACT LIKE FALSE HELIX.
A MOLECULAR BEACON IS A SINGLE-STRANDED NUCLEIC ACID PROBE
COMPRISING THREE FUNCTIONAL DOMAINS A STEM, A LOOP, AND A
FLUOROPHORE/QUENCHER PAIR

Electrochemical DNA Biosensors

DNA PROBE IS IMMOBILIZED ONTO AN ELECTRODE , AND A CHANGE IN
ELECTRICAL PARAMETERS (EG: CURRENT, CONDUCTANCE, IMPEDANCE, ETC.)
GENERATED BY THE HYBRIDISATION REACTION IS MEASURED.

Label based indirect

Indicators such as
intercalating dyes or
electrochemiluminiscent
compounds are attached
with the dna probe,
which react with the
target dna and produce
electrical charges.

Without label direct

There is an alteration in
the intrinsic electrical
properties of DNA after
hybridisation, which is
detected and monitored.

Piezoelectric DNA Biosensors

A STRETCH OF NUCLEOTIDE SEQUENCE WITH A FEW HUNDRED BASE PAIRS
POSSESSES A MEASURABLE AMOUNT OF MOLECULAR WEIGHT. WHEN THE
DNA PROBE HYBRIDISES WITH THE TARGET SEQUENCE, THERE IS AN
INCREASE IN MOLECULAR WEIGHT, WHICH FURTHER LEADS TO A DECREASE
IN THE RESONANT FREQUENCY OF THE PIEZOELECTRIC BIOSENSOR.

SIGNAL PROCESSING
THE TRANSDUCED SIGNAL NEEDS TO BE PROCESSED BEFORE IT IS
MONITORED.
THIS IS DONE WITH THE HELP OF DNA-FET (DNA FIELD EFFECT TRANSISTOR).
IT LEADS TO AN AMPLIFICATION IN THE SIGNAL PRODUCED.
HYBRIDISATION OF STRANDS LEADS TO CURRENT TRANSPORT THROUGH
TRANSDUCER, WHICH IS FED INTO THE GATE TERMINAL OF THE FIELD EFFECT
TRANSISTOR.
THE NEGATIVE CHARGES RELEASED VARY THE GATE POTENTIAL, LEADING TO
A VARIED RESPONSE.
THE PROCESSING OF SIGNAL IS DONE USING THE FOLLOWING PROCESSOR:

 THE SOURCE TERMINAL IS GROUNDED, AND OUTPUT IS OBTAINED AT THE

DRAIN TERMINAL.

DNA SEQUENCING
IT IS DONE SO AS TO DETERMINE THE CORRECT ORDER OF BASES IN THE DNA OF A
SPECIES.
IT IS DONE USING PHOTOCLEAVABLE FLUORESCENT NUCLEOTIDES.
IT FOLLOWS SEQUENCING-BY-SYNTHESIS (SBS) METHOD.
THE BIORECEPTOR HERE IS THE SSDNA SEQUENCE WHICH HAS TO BE SEQUENCED.
4 DIFFERENT PHOTOCLEAVABLE FLUORESCENT NUCLEOTIDE ANALOGUES WERE USED:
DGTP-PC-BODIPY-FL-510
DTTP-PC-R6G,
DATP-PC-ROX,
DCTP-PC-BODIPY-650
FLUOROPHORE WAS ATTACHED TO THE 5 POSITION OF THE PYRIMIDINES AND 7
POSITION OF THE PURINES.

 AFTER IMMOBILISATION OF PROBE, PCR WAS CARRIED OUT ONE BASE AT A TIME, I.E,
PCR BEGINS AT THE FIRST NUCLEOTIDE OF THE PROBE STRAND AND GROWS A
COMPLEMENTARY BASE, TO WHICH ONLY ONE OF THE 4 FLUOROPHORES CAN BIND.

THE PCR COULD BE TERMINATED BY THE PHOTOCLEAVABLE

2-NITROBENZYL LINKER ATTACHED TO EACH FLUOROPHORE.
IT WAS DONE USING THE UV NITROGEN LASER WITH 4 DISTINCT
WAVELENGTHS, WHICH FACILITATED EACH FLUOROPHORE TO EMIT
LIGHT, WHICH WAS THEN DETECTED BY THE SCANNER.
MEANWHILE, THE NEXT STEP OF PCR WAS CARRIED BY THE
AZIDO-LABELED PRIMER, WHICH IS A SELF PRIMING COMPLEX ATTACHED TO THE
IMMOBILIZED DNA TEMPLATE BY ENZYMATIC LIGATION.

MUTATION DETECTION
THE POINT MUTATIONS IN GENES RESULT IN VARIOUS DISORDERS,
INCLUDING CANCERS.
THE SNP CAN BE DETECTED USING PIEZOELECTRIC DNA BIOSENSORS.
AS THE HYBRIDISATION OF TARGET DNA STARTS, SIGNALS ARE
TRANSFERRED TO QUARTZ CRYSTAL, WHOSE RESONANT FREQUENCY
CHANGES ACCEDING TO THE RECEIVED SIGNAL. DURING THIS PROCESS, IF
THERE IS A MISMATCH BETWEEN THE PROBES SEQUENCE AND THE TARGET
SEQUENCE, THERE IS A DIFFERENCE IN THE RESONANT FREQUENCY OF THE
QUARTZ CRYSTAL, AND HENCE, MUTATION IS DETECTED.

REFERENCES
WEBSITE LINKS:
HTTP://WWW.DOCSTOC.COM/DOCS/138865457/PPT---BIOSENSORS-DNA-_PROTEIN-BIOSENSORS
HTTP://EN.WIKIPEDIA.ORG/WIKI/DNA_FIELD-EFFECT_TRANSISTOR
HTTP://WWW.IUE.TUWIEN.AC.AT/PHD/WINDBACHER/NODE66.HTML
HTTP://WWW.ACADEMIA.EDU/1715857/PRINCIPLE_AND_REVIEW_ON_DNA_BIOSE
NSOR
HTTP://WWW.RESEARCHGATE.NET/POST/HOW_DOES_ETHIDIUM_BROMIDE_ETBR_INTERCALATE_WITH_THE_SINGLE_STRANDED_DNA_OR_RNA

RESEARCH PAPERS:
OPTICAL AND ELECTROCHEMICAL DNA NANOBIOSENSORS,
HTTP://WWW.MOLECULAR-BEACONS.ORG/DOWNLOAD/LI,AS01(17)1149.PDF
RECENT ADVANCES IN FIBER-OPTIC DNA BIOSENSORS YI-MING WANG1,2*,
XIAO-FENG PANG1 , YU-YU ZHANG2
FOUR-COLOR DNA SEQUENCING BY SYNTHESIS ON A CHIP USING PHOTO
CLEAVABLE FLUORESCENT NUCLEOTIDES
OPTICAL, ELECTROCHEMICAL, AND MAGNETIC DNA BIOSENSORS AN
OVERVIEW, NORTHERN ILLINOIS UNIVERSITY
NUCLEIC ACID BASED BIOSENSORS FOR CLINICAL APPLICATIONS UTPAL
BORA1,2*, ARGHYA SETT1 AND DEEPIKA SINGH1

THANK YOU