Applications of Enzyme Biosensors in

Food Industry
Structure of a Typical Biosensor
Impurities

Target Analyte
Enzyme/whole cell

Pic source: https://www.slideshare.net/SonikaSingh9/biosensors-in-food-industry-presentation

https://www.researchgate.net/figure/The-working-mechanism-of-the-biosensor-for-the-detection-of-
vitamin-B-12_fig1_280964104
Types of Biosensors
Based on Biological Recognition Element Based on Transducers

Affinity
Enzyme based Whole Cell Based Optical Electrochemical Others
biomolecule based
• Catalytic • General inhibition • Antibodies • Fluorescence • Conductometric • Magnetic
transformation of of cellular
specific substrates respiration • Nucleic Acids • Bioluminescence • Amperometric • Piezoelectric

• Specific inhibition • Analyte acting as • Receptors • Fiber Optic • Potentiometric • Thermal Sensors
of enzyme activity inducer of specific
by the target catalytic protein
• UV-visible • Impedimetric
analyte
absorbance
• Effect of enzyme • Calorimetric
• Surface Plasmon
activity by the
target analyte Resonance
which acts as a
modulator of
cofactor of
enzyme
A Quick Glance At Some Enzymatic Biosensors

Importance of Enzyme Types of Mechanism Analyte Sensor

based Biosensors : Biosensor
☞ High specificity and sensitivity Electrochemical Enzyme electrode Glucose Glucose oxidase
☞ Rapid response
Potentiometric Enzyme electrode Urea urease
☞ Low cost
☞ Robustness Electrochemical Screen printed Peanut allergens AP-labeled mAb
electrode
☞ Easiness of immobilization
☞ Relatively compact size Amperometric Dual enzyme Organophosphorous Cholinesterase &
electrode pesticides Choline oxidase
☞ User friendly operation

optical bioluminescence paraquat cyanobacteria

Photoelectro- photobiocatalysis Alcohol Alcohol oxidase

chemical

Electrochemical Carbon paste Acrylamide hemoglobin

electrode

Pic Source: https://antsceramics.com/screen-printed-electrode

Application of Biosensors in Foods Industry
Quality Control in Foods Industry - Features of Biosensors used in Foods Industry -

☛ Detection of heavy metals ☛ Target specificity

☛ Detection of residual agrochemicals ☛ Small size

☛ Food borne pathogen detection ☛ Fast response time

☛ Detection of toxic metabolites Acr ☛ Stability

ylam
ide
!!
☛ Assurance of food safety ☛ Mass producibility

☛ Indicators of product acceptability ☛ Continuous signal

☛ Detection of unpermitted chemicals

☛ Sensory analysis
Global Scenario in Usage of Biosensors

An Optical Sensor With E-Nose E-Tongue (Handy) Toxin detector using

Smart Phone Display smartphone App

Pathogen Detector

Pic Source: https://www.sciencedirect.com/science/article/pii/S2214180417301137

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6022933/
https://www.slideshare.net/SonikaSingh9/biosensors-in-food-industry-presentation
Application of Enzyme-Based Biosensors in Foods Industries

☞ Dairy industry
☞ Brewing industry
☞ Fruit and Vegetable industries
☞ Fish and Meat products
☞ Tea industry
☞ Organic Acids
☞ Food Processing industry
Why Glucose Sensors Are Required?

• Glucose is the source of energy

• Increase in glucose concentration is an early indication of contamination
• Glucose causes browning during dehydration and long term storage (Maillard’s reaction)

First glucose sensor was developed by Yellow Springs Instrument Company (1975) using Clark’s technology.

Amperometric enzyme-based sensors are a common choice for glucose

sensing because of its good selectivity, high sensitivity, rapid response and
reproducibility
Glucose Biosensors

Glucose oxidase (GOx): Glucose + O2 → Gluconolactone + H2O2

• Inexpensive
• Requires O2 as co-substrate

Glucose Dehydrogenase (GDH): Glucose + NAD+ → Gluconolactone + NADH

• NAD+ dependent and O2 independent
• Well established probe

Pyrroloquinoline-quinone GDH (PQQ-GDH): Glucose + PQQ(ox) → Gluconolactone + PQQ(red)

• Very efficient and rapid electron transfer
• Costly
Schematic Diagram of Amperometric Glucose Sensors

Cathode
Anode

Source: derangedphysiology.com
Source: derangedphysiology.com
Source: derangedphysiology.com
Response of the Sensors

Source: derangedphysiology.com
Modern Glucose Sensing

Sensing by direct electron transfer (DET) of multi-factor enzymes

• Closest to the actual reduction potential of the enzyme
• Orientation of the enzyme matters

↓e-

Fig.1. Direct Electron Transfer Fig.2. Enzyme immobilized on both

Freire et. al, J. Braz. Chem. Soc. Vol 3. (2003) electrodes Source: Bme240.com
Examples of Immobilization
. Systems Used for Designing Glucose Sensors

Fig.3. Multipoint covalent binding

Zhang H. et al, Bio. & Bio. Engg. (2020)

Fig.4. Immobilization by polyaniline microtube

Zhang L. et al, J. Met. Chem. (2015)
Debate on DET ability of Glucose Oxidase:

• Bratlett et al (2018) showed DET of GOx is almost unlikely

• Distance between FAD to electrode is 17-22Å
• Moreover, there is glycosylation
• Homodimer structure of Gox

Fig.5. Orientation and distance from the cofactor matter

Fig.6. 3D structure of GOx

Fig.7. Approach to tackle DET
Bartlett et al, J. Electroanal. Chem. (2018)
 Biosensors To Analyze Freshness Of Food

Why are they required?

P. K. Prabhakar, S. Vatsa, Prem P. Srivastav, S. S. Pathak, Food Research International, Volume 133, 2020, 109157
HYPOXANTHINE
 HYPOXANTHINE
Recorder

AlP reactor
Screen-Printed
Electrode

Peristaltic Pump
To Waste

NP-reactor XO-reactor
Injection Port
Flow Cell

Buffer Tank

K =100(HxR + Hx)/(IMP + HxR + Hx)

Reproduced from:
 OCTOPINE

N. Os, H. J. Smits, L. Schmitt, K. Grieshaber Journal of Experimental Biology

Octopine Pyruvate
Oxidase
Dehydrogenase Acetyl
Octopine Pyruvic Acid Phosphate +
O2 H2O2
OCTOPINE
 BIOGENIC AMINES
Freshness of packaged fruits could be determined by electrochemical biosensors based on immobilized diamine oxidase (DAO)
and polyamine oxidase (PAO) onto polymeric membranes.

M. Esti, G. Volpe, L. Massignan, D. Compagnone, E. LaNotte, G. César A. Lázarode la Torre, Carlos A.Conte-Junior
Palleschi, J. Agric. Food Chem. 1998, 46, 4233
Pesticide Biosensors

• Why do we need pesticide biosensors?

Enzymes Used in Pesticide Biosensors

• Which enzyme?

• Where is it present?

• What is its function?

• Why this enzyme?

How Does the Inhibition Takes Place?
How Does the Inhibition Takes Place?
Cholinesterase based biosensors

 
Figure 1. Scheme depicting the functional principle of biosensor.

AChE
Acetylthiocholine + H2O Thiocholine + Acetic acid
Anodic oxidation
2ThioCholine 2 Thiocholine +2H++ 2 e–
Reduced state
Oxidated state

Fig source: Sarka et al.,  J Enzyme Inhib Med Chem, 2016; 31(S3): 180–193
Figure 2: Strategy for measurement of activity of immobilized AChE in pesticide
solution
Fig source: Everlyn et al., Talanta 155 (2016) 289–304
How to Overcome the Irreversible inhibition

• Reactivation of the enzyme can be done with oxime type reactivation

agents (2-PAM)

• Use of replaceable enzymatic membranes.

• Cheap disposable Biosensors

Amperometric Biosensors Used for Pesticides

Electrode System Analyte

Platinum working electrode containing ChE enzymes. Organophosphorus
Reference electrode:Ag/AgCl and carbamate
insecticides
Clark oxygen electrode or platinum electrode Organophosphorus
containing ChOD enzymes and carbamate
Reference electrode:Ag/AgCl insecticides

CoPC-modified graphite ChE-based electrodes Carbamate insecticides

Reference electrode: Ag/AgI
H2O2-based electrodes containing immobilized Herbicides
thylakoids
Aldehyde dehydrogenase-based electrodes Dithiocarbamate
fungicides
Hexacyanoferrate(III) modified aldehyde Dithiocarbamate
dehydrogenase-based electrodes fungicides
Conclusions

•  Biosensors with their applications, are widely used to tackle challenges in

food production and its sustainability

• With stringent food regulations, quality assurance will increase therefore

increasing the usage of biosensors

• Global market of food analysis needs reliable inexpensive methods for

evaluating food quality and safely
References:
• Lopes, F. M., Batista, K. D. A., Batista, G. L. A., & Fernandes, K. F. (2012). Biosensor for determination of glucose in real samples of beverages. Food Science
and Technology, 32(1), 65-69.
• Freire, R. S., Pessoa, C. A., Mello, L. D., & Kubota, L. T. (2003). Direct electron transfer: an approach for electrochemical biosensors with higher selectivity
and sensitivity. Journal of the Brazilian chemical society, 14(2), 230-243.
• Cao, Ling, Panpan Wang, Li Chen, Ying Wu, and Junwei Di. "A photoelectrochemical glucose sensor based on gold nanoparticles as a mimic enzyme of
glucose oxidase." RSC advances 9, no. 27 (2019): 15307-15313.
• Zhang, H., Hua, S. F., Li, C. Q., Zhang, L., Fan, Y. C., & Duan, P. (2020). Effect of graphene oxide with different morphological characteristics on properties of
immobilized enzyme in the covalent method. Bioprocess and Biosystems Engineering, 1-12.
• Nunes, G. S., & Barcelo, D. (1998). Electrochemical biosensors for pesticide determination in food samples.  Analusis, 26(6), 156-159.
• La Rosa, C., Pariente, F., Hernandez, L., & Lorenzo, E. (1995). Amperometric flow-through biosensor for the determination of pesticides.  Analytica Chimica
Acta, 308(1-3), 129-136.
• Marty, J. L., Leca, B., & Noguer, T. (1998). Biosensors for the detection of pesticides. Analusis, 26(6), 144-148.
• Skládal, P., Nunes, G. S., Yamanaka, H., & Ribeiro, M. L. (1997). Detection of carbamate pesticides in vegetable samples using cholinesterase ‐based
biosensors. Electroanalysis, 9(14), 1083-1087.
• Songa, E. A., & Okonkwo, J. O. (2016). Recent approaches to improving selectivity and sensitivity of enzyme-based biosensors for organophosphorus
pesticides: A review. Talanta, 155, 289-304.
• Nilay Gazel and Huseyin B. Yildiz. Enzyme-Based Biosensors in Food Industry via Surface Modifications. ISBN: 9783527698813
• Aniruddha M. Vaidya, Uday Annapure. Enzymes in biotechnology; chapter 38- enzymes in biosensors for food quality assessment; Food engineering and
technology department, institute of chemical technology, Mumbai, india
• Aldo Roda, Elisa Michelini, Martina Zangheri, Massimo Di Fusco, Donato Calabria, Patrizia Simoni (2015). Smartphone-based biosensors: A critical review
and perspectives. Trends in Analytical Chemistry 79 (2016) 317–325
• Sunita Adhikari, Application of immobilized enzymes in the food industry. Chapter 41.Page 711-720
• B.D. Meshram, A.K. Agrawal, Shaikh Adil, Suvartan Ranvir and K.K. Sande. Review Article: Biosensor and its Application in Food and Dairy Industry..
International Journal of Current Microbiology and Applied Sciences. Volume 7 Number 02 (2018)
 Thank You!
Chandreyee Sarkar (20BT60R10)
Sudipta Dash (20BT60R01)
Anwesha Chatterjee (20BT60R22)
Deeksha Saripalla (20BT60R08)