The document discusses the necessity of increasing food production to combat pest issues and highlights India's significant role in the global pesticide market. It reviews the environmental and health risks associated with pesticide use, including high mortality rates from poisoning, and details advanced detection methods like biosensors for monitoring pesticide residues. Emphasis is placed on the development of enzyme-based biosensors due to their specificity, sensitivity, and quick response times for detecting harmful pesticide levels.

2. Food production has to be
increase to feed Increasing
population
Increase productivity by means
of pest control
Environmental Contamination

3. During the past five decades, the use of agrochemicals (pesticides) has contributed to significant
increase in the crop production by checking the growth of pests.
76%
13%
10% 1%
30%
48%
18%
4%
0%
10%
20%
30%
40%
50%
60%
70%
80%
Insecticide Herbicide Fungicide Others
Comparison of Pesticide usage in India and
Worldwide
India World
• India is the largest producer of
pesticides in Asia and ranks
12th in the world in the use of
pesticides
• Out of the Global Consumption,
India shares only 3.75%
• Impregnable usage of pesticides is a great matter of concern as they impart many toxicological
impacts on living creatures by direct and indirect exposure to pesticides and their residues.
Mortality and chronic illness caused by pesticide and their residues poisoning numbers about 1
million per year.

4. Regarding pesticide residues detection, GC and LC are commonly utilized, These
chromatographic techniques have been coupled to mass spectrometry such as GC–MS and
LC–MS.
Although these methods could realize the quantitative analysis at the same time, they
have their own advantages and shortcomings.
Advantages Shortcomings
• Automated and accurate, with high
specificity
• High cost & being time consuming
• Simultaneous Detection • Need for sample pre treatment &
slow response time
• Requirement for skilled personnel
Moreover they were complicated to operate and were not suitable for on-site and in-field
Detection

5. Therefore, the research focused on
finding fast and reliable devices, to
realize the rapid detection.
The development of biosensor-based instruments for
pesticide residues rapid detection is probably one of the
most promising ways to solve these problems
mentioned previously, which exhibit exceptional
performance and capabilities
 high specificity and
sensitivity
 rapid response, low cost
 portable & relatively compact size
 user-friendly operation and continuous
real time analysis

6. Enzyme based Biosensor for Pesticide Detection
Subhasis Sarkar
Roll No. – 20913 M.Sc. 1st yr. Student
Division of Agricultural Chemicals, ICAR- I.A.R.I
New Delhi - 110012

7. What is Biosensor?
Self-contained integrated device
that is capable of providing
specific qualitative or semi-
quantitative analytical information
using a biological recognition
element which is in direct-spatial
contact with a transduction
element.
(IUPAC,1998)

8. FATHER OF BIOSENSORS
Professor Leland Clark (1918–2005)
The first and the most wide spready used commercial biosensor: the blood
glucose biosensor–developed by Leland C. Clark in 1962

9. History of
Biosensor
Development

10. ELEMENTS OF BIOSENSOR

11. Principle of Biosensor
 Interaction between analyte and biorecognition element
Produce physicochemical change
 This changes were
measured and detected
 The biochemical signal would be converted
into analog Or digital electronic signal
 Signal Processing
 Amplification
Analyte concentration is proportional to signal strength, Based on this principle,
Quantitative detection of pesticide residue could be realized.

12. Classification of Biosensor

13. Detection Instruments Based on Electrochemical Biosensors
Between biological recognition
element and analyte
From the immunoreaction between an
antigen and antibody (Which immobilized
on working electrode)
Antigen specific antibody
immune response

14. Detection Instruments Based on Optical Biosensors
Fluorescence detection
Fluorescence is an emission phenomenon in which
A fluorophore absorbs light or electromagnetic
radiation and emits light at visible range.
Surface Plasmon Resonance
Instruments
SPR occurs when polarized light
illuminates, under conditions of total
reflection, a thin conducting film at the
interface between two transparent
media with different refractive index.
SPR response is a measure of changes in the resonance angle (θ)

15. Detection Instruments Based on Mass-based Biosensors
• Piezoelectric method
The piezoelectric effect occurs in crystals without a center of symmetry.
When pressure was applied to the crystal, the dipole moment arises in
the molecules of the crystal.
• Magnetoelastic method
This magnetoelastic thick-film coupled with a chemical or
biochemical sensing film.

16. Immobilization Techniques
 The most important step in the development of an enzyme sensor is the firm attachment of the enzyme onto
the surface of the working electrode.

17. Indirect Approach Direct approach
Based on the measurement of Enzyme Inhibition Direct measurement of compounds involved in the
enzymatic reaction
 AChE, BChE
 Alkaline Phosphatase
 Tyrosinase
 Peroxidase
 Lipase
 OP Hydrolase

18. Different biosensors configurations for detection of pesticide
Biosensor based on
Enzyme
AcetylCholineEsterase
(AChE)
Alkaline Phosphatase
Organophosphate
hydrolase
Urease
Detected Pesticide Method of Detection
Aldicarb, Carbaryl Electrochemical ( amperometric )
Paraoxon, Chlorpyrifos – methyl oxon Piezoelectric (Quartz crystal microbalance)
Carbofuran, Paraoxon, Carbaryl, Malaoxon Electrochemical ( amperometric )
Dichlorvos Electrochemical ( amperometric )
Coumaphos, Trichlorfon, Methiocarb Electrochemical ( potentiometric )
Metham-sodium Tetradifon Fenitrothion Fluorimetry
2,4 – D , Malathion Amperometry
Paraoxon Chemiluminescence
Chlorpyrifos voltametry
Paraoxon Fluorescence
Atrazine Enzyme field effect Capacitive system
Glyphosate Potentiometric

19. Biosensor based on
Enzyme
Tyrosinase
Laccase
Heme-containing
Enzyme
Detected Pesticide Method of Detection
2,4 -D Amperometry
Parathion, Carbaryl Amperometry
Atrazine ( LOD – 0.1ppt) Amperometry
Atrazine ( LOD – 0.3ppm) Amperometry
Atrazine ( LOD – 10ppb) Amperometry
Mehomyl Square wave voltammetry
Carbofuran, Carbaryl, Ziram Square wave voltammetry
Primicarb Square wave voltammetry
Formetanate Square wave voltammetry
Aldrin, Heptachlor Amperometry
Glyphosate SWV
AminomethylPhosphonic acid Amperometry
Dichlofenthion Amperometry

20. Acetylcholinesterase inhibition-based biosensors for pesticide detection
• Acetylcholinesterases (AChE) are a class of enzymes that catalyse the hydrolysis of
acetylcholine, an ester which is a neurotransmitter (Fukuto, 1990; Stenersen, 2004).
• AChE belongs to the family of hydrolases and at its active site three amino acids:
histidine, serine, and aspartic acid are present. When the binding site attracts the
positively charged quaternary ammonium group of ACh, serine hydroxyl group attacks
and hydrolyzes the compound by deprotonation
• Mode of Reaction :
Following reaction shows the degradation of
acetylcholine.
CH3
Acetyl choline Choline Betain+H2O2
AChE ChOx

21. • The inhibition of AChE by
organophosphates
(Fukuto, 1990).
 Carbamate pesticides are
cholinesterase inhibitors
with a similar mechanism
as of organophosphate
pesticides (Fukuto,1990).
 The hydroxyl of the serine
residue within the active
site of the enzyme is
carbamylated instead of
phosphorylated.

22. A simple, fast, convenient and sensitive method for determination of organophosphorus
pesticides in real samples based on inhibition mechanism of acetylcholinesterase (AChE).
Furthermore, a possible mechanism is put forward to explain the fluorescence quenching
of QDs in the presence of H2O2.
The Biosensor is composed of enzymes (AChE and ChOx (choline oxidase)), QDs and
acetylcholine (Ach)
After the experimental conditions are optimized, the limit of detection (LOD) for
dichlorvos (DDVP) is found to be 4.49 nM

23. a) Schematic principle
for the detection of
OPs
b) The process for the
detection of Ops
c) The colour change
with different conc.
(0,0.45, 6.78µ

24. Fluorescence switch mechanism for the detection of DDVP based on QDs
Fig.(a) Typical time- dependent fluroscence intensity of
QDs in the presence of ChOx, AChE and ACh
Fig.(b) The quenching kinetics of fluroscence intensity
without DDVP and different conc. Of DDVP
Kt= F0/Ft , Kt : Retained
% of fluorescence
Intensity of AChE
P

25. Effect of Enzyme Conc. (a), Influence of Incubating temperature (b), influence of incubating time (c)
on It value
It= Influence coefficient of OPs as a
signal for the detection of OP
compound
It= Kt with DDVP/ Kt without DDVP
Influence of
Enzyme Conc.
On Itvalue
Effect of Incubating
temperature
Influence of Incubating time
Optimum: 0.5 U/ml
3.26
Max. It for 37C
Max. It for 15 min

26. Study of Quenching mechanism of Enzyme reaction on QDs
A. Pristine QDs
B. QDs quenched by 1mM H2O2
C. QDs quenched by 4mM H2O2

27. • Sample pre treatment is a complex multiple-step procedure for the
pesticide determination in real samples for chromatographic
method.
• Liu et al. proposed a new method for fruits pre treatment without
using organic solvents and tedious preconcentration steps.
• Using this method, the apple was first chopped and extracted with
20ml phosphate buffer (pH=8)
• The apple with a low It (1.04±0.04) was used as a reference sample
• Further experiments were carried out with the intentional addition
of DDVP
So, It can be concluded that this method has good performance in
detection of Dichlorvos in fruit sample

28. Alkaline Phosphatase inhibition based biosensor
for Pesticide Detection
• Alkaline phosphatase (ALP) known also as basic phosphatase has a
broad substrate specificity and exhibits maximum activity in alkaline
pH solutions. It is a metalloenzyme that has in its active centre
Mg2+ and Zn2+ ions.
• Mode of Reaction : The enzyme catalyses the reaction of
numerous inorganic and organic compound
• Detect for Pesticides : Organochlorine, Organophosphorus,
Carbamate, a series of heavy metals

29. Free and sol–gel immobilized alkaline phosphatase-based
biosensor for the determination of pesticides
and inorganic compounds
 Alkaline-phosphatase (ALP) catalyses the hydrolysis of 1-naphthyl phosphate to
fluorescent 1-naphthol (λex = 346 nm, λem = 463 nm).
 This enzymatic reaction was investigated in presence of inhibitors: organochlorine
(tetradifon), carbamate (metham-sodium) and organophosphorus pesticides
(fenitrothion), heavy metal (Ag+) and CN−.
 The fluorescent signal, which is inversely dependent on the inhibitor concentration, is
related to the amount of the inhibitor.
 Detection limits between 4.1µM for tetradifon and 91.2µM for metham-sodium were
found.
Garcia Sanchez F et al. 2003

30. Procedure:
• For measurements with free ALP in solution, the appropriate volumes of 0.1M NaHCO3 buffer at pH 9.5
0.43 U/ml alkaline-phosphatase, inhibitors when necessary
0.01M 1-naphthyl phosphate up to 2ml final volume
Added, in the indicated order, to a disposable Cuvette
The variation of the fluorescence intensity, Δ(RFI), per unit of time, Δ(RFI)/Δt (Δt = 0–300 s) was used to
determine the initial rate (V).
• For measurements with immobilized ALP in solution, the appropriate volumes of 0.1M NaHCO3 buffer at pH 9.5
0.43 U/ml alkaline-phosphatase, inhibitors when necessary
0.01M 1-naphthyl phosphate up to 2ml final volume
Added, Cuvette containing gel with ALP immobilized
The percentage inhibition was calculated as follows:
Where, RFIo is the fluorescence without inhibitor, and
RFI is the fluorescence with inhibitor

31. Km= 345.9µM
Parameter determine
the enzymatic Catalysis
Vmax= 1.16, at which the
product were formed
Without inhibitor
Metham sodium
tetradifon
fenitrothion
silver
Kinetic Curve ( 1-napthyl-phosphate/alkaline-phosphatase/inhibitor system in
solution )
cyanide

32. • Separate experiments were conducted to measure the activity of ALP in different
conditions, in aqueous solution, immobilized in gel without inhibitor and
immobilized in gel in the presence of an inhibitor.
RFI of Immobilized ALP 5min 10min 120min
Without inhibitor 21% 47% 96%
50ppm of CN 12% 27% 95%
Immobilization affect to the initial
rate with significant decrease in
fluorescence intensity in presence of
inhibitor

33. Enzymatic and analytical parameters with
ALP in solution
Inhibitor Type of Inhibition Inhibitor Conc. (µM) Inhibition Cont.
(µM)
Detection Limit
(µM)
Metham-sodium Non-competitive 77.40 81.2 36.5
Tetradifon Non-competitive 14.05 5.3 4.1
Fenitrothion Non-competitive 108.30 90.2 45.5
CN- Competitive 307.20 252.4 91.2
Ag+ Non-competitive 53.51 24.6 10.1

34. Organophosphorus hydrolase (OPH) inhibition based
biosensor for Pesticide Detection
• Organophosphorus hydrolase (OPH), first isolated from Pseudomonas diminuta, is a well-
characterized metalloenzyme.
• Mode of Reaction : It has the ability to hydrolyze a large variety of organophosphate
pesticides, and the resulting hydrolysis products change the pH of the solution. The
change of the solution pH is due to the generation of two protons during the
organophosphate hydrolysis, which takes place with the cleavage of the P-X bonds.
• Detect for Pesticides : Organophosphorus

35. • Recently, it was reported that a change in fluorescence properties of a fluorophore in
the vicinity of gold nanoparticles might be used for detection of nanomolar
concentrations of DNA oligonucleotides. The detection strategy was based on the fact
that an enhancement or quenching of fluorescence intensity is a function of the
distances between the gold nanoparticle and fluorophore.
• While these report demonstrate, use of nanoparticle- based sensor for detection of
target DNA, the specificity of enzyme-substrate interaction could be exploited in
similar system.
• To test the feasibility, OPH-gold nanoparticle conjugates were prepared, incubated
with fluorescent enzyme inhibitor or decoy
• Then different paraoxon concentrations were introduced to the OPH–nanoparticle–
conjugate–decoy mixtures, and normalized ratio of fluorescence intensities were
measured.

36. Target Analyte Fluorophore Structures
7-hydroxy-9H-(1,3-dichloro-9,9-
dimethylacridin-2-one) (DDAO
phosphate)
Difluorinated
methylumbelliferyl
phosphate (DiFMUP)
ELF 97 phosphate
Enzyme-nanoparticle
Conjugate

37. Fluorescence Intensity of
DDAO was measured and
used as background
signal (IF1)
OPH/gold nanoparticle
conjugate was added
intensity of fluorescence
of Conjugate-Decoy
complex(IF2) measured
Paraoxon was added in
different concentrations and
fluorescence intensities,(IF3)
were measured
Relative fluorescence intensity change ΔIF was calculated as:
 This represents the ratio of enhancement of fluorescence in the presence of paraoxon
to the enhancement of fluorescence in the absence of paraoxon
 Control experiments were performed to check any fluorescence intensity changes in the absence of
OPH/gold conjugate.
Detection Procedure

38. PrincipleofEnzyme/nanoparticlesensing
(10-40 nm)
(>40 nm)
Schematic of Decoy-Enzyme interaction for
enhancement in the absence of substrate.
Schematic of analyte (S) displacement of decoy
(D) from OPH–gold complex (OPH), leading to
decrease fluorescence signal from the decoy
 The change in fluorescence intensity is related to the concentration of analyte present in
the solution

39. DDAO alone
DDAO+Au+OPH
Decrease in the
fluorescence
intensity
Fig. : System response on different paraoxon concentrations. Relative fluorescence intensity of gold–OPH–
decoy system as a function of location/chemistry of gold nanoparticles attachment : A. monomaleimido
nanogold, B. sulfo-N-hydroxy-succinimido nanogold.
{A} {B}
Effect of Different nanogold attachment Chemistry

40. The minimum paraoxon concentration detected was 20 µM
which is near the KM of the enzyme for this substrate. Good
linearity was observed at paraoxon concentrations up to
240µM
Calibration curve. Relative fluorescence intensity change (ΔIF) is
plotted as a function of added paraoxon (PX) concentration
Advantages of this approach:
 A pH measurement is excluded from the assay scheme that makes analysis easier
 Ability to control sensor performance via both KM of the enzyme for the OP
compound of interest and the Ki of the decoy used in the sensor

41. Tyrosinase inhibition based Biosensor
for Pesticide detection
Tyrosinase is a type of catechol oxidase, found in many
species of bacteria and are copper-containing.
It has two binding sites, the substrate binding site which
has an affinity for aromatic compounds, and the oxygen
site which has an affinity for coordinating agents that bind
to the metal.
• Mode of Reaction : The enzyme catalyses the
hydroxylation of monophenols to o-phenols and the
oxidation of o-phenols to o-quinones
• Used to Detect : carbamate and dithiocarbamate
pesticides, atrazines, chlorophenols and thioureas.

42. A novel pesticide biosensor has been developed using single-layered enzyme-membrane using BPPO
film which was pore-filled with cross-linked PVA containing TYR, with an increased stability.
The prepared enzyme-membrane was assembled on a glassy carbon electrode to detect pesticides.
The voltammetric measurements revealed a coupled reaction, an enzymatic oxidation and an
electrochemical reduction of catechol transported through the prepared membrane.
The activity in the enzyme-membrane was maintained for 1 month due to favourable aqueous
environment of PVA for enzyme activity while BPPO film provided structural stability.
A detection range of parathion and carbaryl was 0.01–1 ppb and 0.01–10 ppb, respectively.

43. Finger-typed
channels
Pore filled with PVA
Including Tyrosinase
Fig. : SEM images obtained from the prepared pBPPO film ((a), (b) side view,
Morphology and asymmetric structure of the base pBPPO film

44.  While an inhibitor such as pesticide exists, the enzymatic reaction is retarded so the less amount of quinone
is generated showing a decreased current.
Fig. : Illustration of GC/single layered enzyme membrane system

45. Anodic Peak
Cathodic Peak
Cont. current response
After 3 min.
 The diffusion rate of catechol is depending on not only thickness of the enzyme-membrane but also water content
of the PVA layer in the membrane
 The analyte can pass through the PVA layer since BPPO is hydrophobic. Thus, it is expected that the response time
of the prepared electrode could be decreased by controlling the membrane thickness and the viscosity of PVA
layer in the prepared membrane.
Electrochemical characterization of the enzyme-membrane
The cyclic voltammograms of the prepared BPPO-tyrosinase-PVA
membrane based pesticide biosensor for 0.5mM of catechol.
Time-dependence of the current response of the
BPPO-tyrosinase-PVA membrane based pesticide
biosensor.

46. AC impedance spectrum of GC electrode with different
membranes ((i) bare GC, (ii) GC/single-layered, and (iii)
GC/three-layered)
 In the impedance measurements, the semicircle
diameter is the electron transfer resistance (Ret), which
controls the electron transfer kinetics of the redox probe
at the electrode interface
 It indicates that the presence of film obstructs the electron
transfer and the increase of Ret is upon the increase of layer
due to an additional resistance.
Electrochemical characterization of the enzyme-membrane

47. Inhibition% = (1- Ai/A0)*100
Ai= Response after pesticide exposure
A0= Response prior pesticide exposure
Inhibition curve for pesticides (Parathion, Carbaryl)
LOD(PPB)
P- 0.01-1
C- 0.01-10
Maintained
Current response
The change of current response and
moisture in the membrane during 50 days
Detection of pesticide and stability of Enzyme electrode

48. Conclusion:
By comparing the EIS measurements of the conventional three-layered TYR-electrode
and the single-layered BPPO-TYR-PVA electrode, the much lower electron transfer
resistance was observed for the prepared enzyme membrane based electrode.
The prepared enzyme-membrane based electrode exhibited feasibility in detecting
parathion, carbaryl, and similar pesticides.
A long-term operational stability of the electrode was expected, represented from the
good stability of the enzyme membrane

49. Other Enzymes
Name of the Enzyme Mode of Reaction Used for Detection
1. Urease Catalyzes the decomposition of urea in
ammonia and carbon dioxide
Heavy metals, Pesticides such
as Atrazine
2. Aldehyde dehydrogenase
(ADH)
Catalyses the oxidation of various aldehydes
using β-nicotinamide adenine dinucleotide
(NAD+) as a cofactor
Dithiocarbamate fungicide
3. Acid phosphatase (AP) Catalyse the reaction:
Orthophosphoric monoester + H2O2
Alcohol+ H3PO4
Malathion, Methyl parathion,
Paraoxon
4. Glutathione-S-transferase
(GST)
Nucleophilic attack of GST on atrazine releases
H+ which can be detected as a pH change which
directly correlates with the concentration of
analyte.
Atrazine
5. Carboxylesterase (CBE) Hydrolyse Esters OP & Carbamate Pesticides

50. Advantages
• Biosensors based on enzyme exhibit exceptional performance capabilities, which include
simplicity, high specificity and sensitivity, rapid response, low cost, portability,
relatively compact size, user-friendly operation and continuous real time analysis.
• The biosensors based on enzymatic inhibition are useful as an alarm or general toxicity
indicator for the fast identification of the samples contaminated with pesticides.
• After further design and improvement, they can be used for the field detection and data
sharing can also be possible.

51. Disadvantages
Enzymatic methods, particularly inhibition-based one have been criticized extensively
due to lack of selectivity of biosensors in the pesticides detection.
Inhibition-based methods can be prone to false positives as handling and storage could
cause loss of enzymatic activity.
As many pesticides irreversibly inhibit an enzyme AChE and therefore regeneration of
the sensor is required after each sample leads to further extended testing time.
Most of the enzyme based biosensors still at laboratory research stage and need to
improve accuracy and stability in actual detection.

52. Conclusion
With development and application of nanomaterials, enzyme-biosensors showed high
sensitivity, low detect limits, super selectivity, and fast responses.
Development of miniaturized, multi-biosensors continues to draw much research effort,
continuing the trends dedicated to biosensors for pesticides.
The goal is to reduce the gap between standard methods and accelerate the path
towards commercial implementation.
However, the number of applications involving real environmental or food samples and
their variety remains limited and toxic metabolites of pesticides have rarely been studied.
Development of novel biosensors relying on enzymes such as aldehyde dehydrogenase or
heme-containing enzymes appears to have stagnated and lost interest in the last decade,
probably for reasons including the unavailability of commercial enzymes, difficulties
related to price, cofactor addition, or unfavourable equilibrium of the enzymatic
reaction or low selectivity