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UNIVERSITY OF AGRICULTURAL SCIENCES,

DHARWAD

BIOSENSOR TECHNOLOGIES FOR EARLY DETECTION

AND QUANTIFICATION OF PLANT PATHOGEN

DOCTORAL SEMINAR - I

Chairman
Dr. Shamarao Jahagirdar
Professor
Department of Plant Pathology

Presented by:
Nandhini B
PGS22AGR8908
I Ph.D (Plant Pathology)
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 1 Introduction

Components and Working

2 principle

3 Applications

Outline 4 Various types of biosensors

5 Case Studies

Challenges and Future

6 Prospects

7 Conclusion
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✓ An analytical device used

for the detection of a
chemical substance, that
combines a biological
component with a
physiochemical detector.

Khater et al., 2017

 BIOSENSORS

FATHER OF BIOSENSOR

LELAND C. CLARK

1918-2005

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M. Cramer observed Guilbault &
Montalvo - Poncharal et al.
electric potential
1st potentiometric demonstrated the
arising between parts
enzyme electrode 1st nanobiosensor
of the fluid Leland C. Clark Suzuki et al.
invented the first based sensor for 1st demonstrated
1906 oxygen electrode detecting urea microbe-based 1999
immunosensor

1956 1969 1975

TIMELINE IN DEVELOPMENT OF BIOSENSOR

1922 1962 1983 2018

1975
Clark et al. Girbi et al.
[Link]- experimentally Liedberg et al. demonstrated nerve-
1st glass pH demonstrated an First observed surface on-chip type
electrode amperometric commercial plasmon biosensor for
biosensor for assessment of nerve
enzyme electrode resonance (SPR) impulse conduction
for detecting glucose detection immunosensor
glucose by Yellow Spring
Instruments
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COMPONENTS OF BIOSENSOR

Biological components Physical components

Polysaccharides, Transducers,
Microorganisms, A/D Converter,
Nucleic acid, Amplifier,
Tissue, Display
Enzyme,
Antibody.

Ali et al., 2017

WORKING PRINCIPLE

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Dyussembayev et al., 2021

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MECHANISM

Singh et al., 2013

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COVALENT FABRICATION MATRIX IMMOBILIZATION

MEMBRANE PHYSICAL ADSORPTION

ENCAPSULATION FABRICATION

Ali et al., 2017

APPLICATIONS OF BIOSENSOR

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 CHARACTERISTICS OF BIOSENSOR

Selectivity - Bioreceptor -
STABILITY SELECTIVITY Analyte

Sensitivity - ng/μL or fg/μL

Linearity - Accuracy

Reproducibility - Precision
SENSITIVITY REPRODUCIBILITY and Accuracy - Every time

Stability - Environmental
disturbance

LINEARITY

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TYPES OF BIOSENSORS BASED ON TRANSDUCERS

Rani et al., 2019

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ELECTROCHEMICAL BIOSENSOR

Molecular recognition layer

Core components
Electrochemical transducer

Biological information

Signal in
quantitative
analysis

Electric signal

Dyussembayev et al., 2021

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CLASSIFICATION OF ELECTROCHEMICAL
BIOSENSOR

EIS

QCM
Antibody
based
Microfluidic

Electrochemical ECEIA
Biosensor

Voltametric
DNA based
Impedimetric

Dyussembayev et al., 2021

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A- Antibody based biosensor

B- DNA/RNA based biosensor for analyte detection

Fang and Ramasamy, 2015

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QUARTZ CRYSTAL MICROBALANCE

Working principle:
Quartz layer installed between
working electrodes that oscillates at
a resonance frequency generated by
the change in mass due to surface
binding of the target analyte and
biorecognition molecules
(antibodies or nucleic acids)

▪ Detection of Maize chlorotic

mottle virus
▪ Higher selectivity against
Wheat streak mosaic virus.

Oscillation frequency based detection

Dyussembayev et al., 2021
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Working principle : Measure

of the opposition to the flow of
the current that results from
redox reactions and molecular
interactions at the electrode
surface when voltage is applied
to the cell membrane

▪ Detection of Plum pox virus

(PPV) on gold electrodes.
▪ Detection of Prunus necrotic
ringspot virus (PNRSV)
using carbon electrodes.
▪ Detection of Pseudomonas
syringae pv. lachrymans on
Equivalent electrical circuit Nyquist plot antibody modified gold
electrode.

Dyussembayev et al., 2021

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MICROFLUIDIC IMMUNOSENSOR

▪ Introduction of solutions in the sensor – syringe pump system.

▪ Example:
Early detection of Xanthomonas arabicola in walnut samples
Detection limit: 1.5 × 102 CFU mL-1
Detection time: 30 mins

ELECTROCHEMICAL ENZYME-LINKED
IMMUNOASSAY (ECEIA)

▪ Sensitivity - 10 times higher than that of standard ELISA.

▪ Measurement of current - Linear sweep voltammetry using a hanging mercury
electrode.
▪ Example:
Detection of Cucumber mosaic virus, Tobacco mosaic virus, Potato virus Y,
Southern bean mosaic virus, Turnip mosaic virus, Cercospora leaf spot on rice,
Black spot of crucifer.

Khater et al., 2017

 National Institute of Molecular Biology and Biotechnology,
University of Philippines

OBJECTIVE: Electrochemical detection of Phytophthora palmivora, a notorious

pathogen of cacao causing severe crop loss, using nanobiosensor and to study the
specificity and selectivity.

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➢ Isolation DNA extraction PCR Amplification Sequencing
➢ The detector and the capture probes for the nano-biosensor were designed
➢ Oligonucleotide probe labelling with Electrically-active magnetic (EAM) nanoparticles
➢ Sandwich hybrids between two oligonucleotide probes & genomic DNA of P. palmivora were
prepared & electrochemically detected.
➢ Oligonucleotide probes- designed based on ITS sequence of the P. palmivora field isolates
➢ Working electrode: Screen-printed Ag/AgCl electrode (SPE) with graphite.

Electrochemical detection of sandwich hybrid:

➢ Carried out by subjecting the hybrids to cyclic

voltammetry.
➢ Hybrids obtained from the sandwich hybridization step
were placed on the surface-modified electrode surface &
incubated for 15 min at room temp.
➢ Wash electrode with nuclease-free water.
➢ Dry at room temp. (15 min), add 100μL of 0.1M HCl
solution & allow to equilibrate (5 min)
➢ Cyclic voltammetry scans were performed from −0.40 to Electrochemical response of EAM in
+0.80 V with a scan rate of 50 mV s−1. 0.1M HCl with increasing scan rate

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 (a) Artificially-infected cacao pod at three days after inoculation
(b) Field infected cacao pods.

Electrochemical response of the biosensor on Anodic peak height at different P. palmivora

P. palmivora genomic DNA concentrations genomic DNA concentrations ranging from
ranging from 0 to 5.0 ng μL-1 at 50 mV/s 0 to 5.0 ng μL-1
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Anodic peak height of cacao-associated Anodic peak height of cacao pod

fungal pathogens samples

Output

▪ Concentration of P. palmivora genomic DNA and the mean anodic peak height were
positively correlated.
▪ Limit of detection LOD - 0.30 ng DNA μL−1.
▪ Selectivity of the nano-biosensor towards genomic DNA was shown against several cacao-
associated fungal species - Colletotrichum gloeosporioides, Fusarium equiseti, and
Lasiodiplodia theobromae.
PLANT PATHOGENS DETECTED BY
ELECTROCHEMICAL BIOSENSOR

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OPTICAL BIOSENSOR

➢ Measures the interaction between a target analyte and ligand using a light
source, an optical transmission medium, an immobilized biorecognition element,
and a signal detection system.
➢ Change in amplitude, phase, and frequency of the given light in response to
physicochemical changes generated by biorecognition process is measured.

Colorimetric Biosensor

Fluorescence based Biosensor

Plasmon resonance based Biosensor

Ray et al., 2017

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CLASSIFICATION OF OPTICAL BIOSENSOR

Lateral flow
immunoassay

SPR
Antibody
based Microsphere
immunoassay

Optical Biosensor
Bridging flocculation

Electro
DNA based chemiluminescence

Microfluidic
microarray

Dyussembayev et al., 2021

PLANT PATHOGENS DETECTED BY OPTICAL
BIOSENSOR

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LATERAL FLOW SYSTEMS

➢ Principle is based on antigen- A – Phytophthora sp.

antibody specific interaction B – Erwinia amylovora
➢ 1st LFIA - Tobacco mosaic virus C – Potato virus Y
(Tsyda et al., 1992) Khater et al., 2017
 National Research Centre for Banana,
Tiruchirapalli, India

Objective: To develop a practical, rapid, sensitive, specific and user-friendly lateral

flow immunoassay (LFIA) test for the on-site detection of BBrMV

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Selvarajan et al., 2020 31
Detection of BBrMV in various parts of the infected banana plants using LFIA strips

Strip 1: Bract
Strip 2: Young leaf
Strip 3: Leaf sheath
Strip 4: Flower
Strip 5: Seed coat
Strip 6: Endosperm
Strip 7: Embryo
Strip 8: Healthy leaf control

✓ Symptomatic tissues from young leaf, leaf sheath and bract always gave
BBrMV +ve results quickly whereas floral parts & older leaf samples of
infected plants produced faint bands on both the control and test lines.

✓ Consistent with PTA-ELISA studies -10-month-old infected banana plant - virus

conc. was highest in bracts followed by the youngest leaf and leaf sheath, while the
older leaves had less virus.

✓ Weak signals observed with floral parts might be due to either a low conc. of virus or
a high conc. of inhibitors in these parts which might react with the conjugate.

✓ High quantity of polyphenols and pigments in floral parts might hamper virus
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detection in banana.
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 DETECTION USING DETECTION USING
CONVENTIONAL METHOD COMMERCIAL BIOSENSOR

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PIEZOELECTRIC SENSORS

Working principle:
▪ Piezoelectricity (core of a piezoelectric sensor is
composed of piezoelectric crystals which are direct
converters of mechanical stress to electric charge)
▪ Used to record substrate borne vibrational
signals(SBVSs)

E-NOSE BIOSENSORS

Working principle:
▪ Detection – Changes in VOC
▪ High stability and Easy processing of data.
▪ Post harvest pathogens

Ali et al., 2017

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BACTERIOPHAGE SENSORS

ANALYTE Infected Sample

Phage-based Magnoelastic
biosensors for the detection
of Salmonella typhimurium
on the surface of tomato
and spinach leaves.

Phage based biosensors for

the detection of
Pseudomonas cannabina
pv. alisalensis, causing
bacterial blight of crucifers
(Schofeild et al., 2013)

➢ Effective for detecting bacterial pathogens at different temperatures.

➢ Can separate live and dead bacteria pathogens, which influence the false-
positive results during detection.
➢ Interaction between phage and the targeted bacterial component.
➢ Very sensitive, cost effective approach.
Singh et al., 2020
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NANOMATERIAL-BASED BIOSENSORS

➢ Nanoparticle-based biosensors include all sensors that

employ metallic NPs as enhancers of biochemical signals.
➢ The NPs include metal and noble metal NPs, Au, Ag, Pt,
Pd, Co, Fe, Cu, and metal oxide NPs.
➢ NP coating with various matrices, such as metal oxides,
silica network, polymers, graphene, fibers and
dendrimers.
➢ The most often employed metal oxide nanoparticles include
CuO, NiO, Fe2O3, Co3O4, MnO2, ZnO, TiO2, SnO2,
CdO, MoO3, and CeO2
➢ Nanotube-based biosensors are used as enhancers of reaction
specificity and efficiency.

Vinay Kumar et al., 2019

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MICROSPHERE IMMUNOASSAY

A-Specific antibody-
coated microspheres-
mixed with samples &
incubated

B-Unbound antigens
were washed & removed
using magnetic separator.

C-The cocktail of RPE-

labeled antibodies was
added & incubated.

D-The unbound RPE-

labeled antibodies were
washed & removed by
magnetic separator
before signals acquired
by Luminex machine
Charlermroj et al., 2013
 National Centre for Genetic Engineering
and Biotechnology, Thailand

Objective: To employ a microsphere immunoassay (MIA) to simultaneously detect

multiple plant pathogens (Potyviruses, Watermelon silver mottle virus, Melon yellow
spot virus, and Acidovorax avenae subsp. citrulli) in actual plant samples of various
cucurbit varieties
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Field plant samples - 146
Inoculated samples
PRSV-W - Datura stramonium
WSMoV - Physalis minima Microsphere immunoassay - MIA:
MYSV - Cucumis sativus ▪ Antibodies coupling to the magnetic
beads
Pathogen Antibody RPE ▪ Conjugation of secondary antibodies
Detection coated Labelled with R-Phycoerythrin
microsphere antibody ▪ Assay condition for the microsphere
Papaya ringspot immunoassay
virus type W 1G8 1B4
(PRSV- W)
Watermelon silver Relative accuracy = (PA + NA)/N× 100%
mottle virus TOsIV 2D6 Relative specificity =(NA/(PD + NA)) × 100%
(WSMoV) Relative sensitivity = (PA/(PA +ND)) ×100%
where,
Melon yellow spot PA - positive agreement or true positive.
virus (MYSV) 5E7 Gen Tos
PD - positive deviation or false positive.
Acidovorax NA - negative agreement or true negative.
avenae sub sp. MPC MPC ND - negative deviation or false negative.
citruli (Aac) N -total number of samples (PA + PD+ ND+ NA)

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 Specific Plant sample was A cocktail of Each bead set was
antibody extracted using R- Phycoerythrin identified using a red
coated optimized extraction (RPE)-labeled laser and RPE signals
magnetic buffer & incubated antibodies was were acquired using a
microspheres with antibody-coated added and green laser by a
were mixed microspheres incubated Luminex machine.

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Buffer

1 - Phosphate
buffer saline
containing
Tween 20
2 - Carbonate-
based extraction
buffer pH 9.6
3 - Sulfate-based
extraction buffer
pH 8.5
4 - General
extraction buffer
from Agdia
5 - General
extraction buffer 2
from an Agdia Aac
detection kit
6 - Extraction
buffer pH 9.6 from
an Agdia potyvirus
detection kit

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Specificity

Result

▪ MIA allows highly accurate multiplex detection for four plant pathogen groups in cucurbit samples.
▪ Employs single sample extraction method & single assay format to simultaneously detect four
pathogens.
▪ Simplifies sample preparation & reduces the amount of samples required for the detection.
▪ Assay time - 2.5 hours
▪ Can be expanded to detect up to 50 pathogens if their specific antibodies are available.
▪ High relative accuracy, specificity and sensitivity

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PROPERTIES OF A GOOD BIOSENSOR

Highly specific for the analyte

Response should be linear over a broad spectrum range

Small, compatible, rapid and accurate, sterilizable

Low cost and easy to use

Assay costs should be lower than the conventional tests

Assay should be fast, reliable and repeatable

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CHALLENGES AND FUTURE PERSPECTIVES

Need of careful validation within a particular pathosystem.

Pathogen detection and quantification should be used in conjugation

with other multiple factors of farming systems.

Cultural, cropping and climatic factors should be considered.

Futureproofing of IDM strategies.

Validated biosensors improve the accuracy of epidemiological models.

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