To the point presentation on Glucose Biosensor including its working, historical perspective and its generations. It also includes the mechanism of the glucose-sensing method used specifically in hospitals.

1. GLUCOSE BIOSENSOR
Ajeet Pal Singh & Tegbir Singh
Research Interns
CSIR-CSIO, Chandigarh

2. Thank You Dr. Parveen Kumar
(Bio-Nanotechnology Lab & E-
Waste Management Lab,
CSIR-CSIO, Chandigarh) for
your esteemed support

3. TOPICS TO BE COVERED
• Biosensor
• Basic characteristics of a biosensor
• Blood sugar testing
• Requirement for blood sugar testing
• Diabetes and its types
• Glucose biosensor and its mechanism
• Historical perspectives of glucose biosensor
• Blood sugar testing in GMCH-32, Chandigarh
• Field work results

4. BIOSENSOR
 Analytical device which converts biological signal into an electrical
signal.
 Can be used as in both qualitative and quantitative ways, i.e,
determine the type of a bio-analyte and its concentration in a body.
 Biological response is determined by bio-catalytic membrane which
causes the conversion of a reactant to the product.

5. BIOSENSOR
The major components are as follows:-
 a: Biocatalyst
 b: Transducer
 c: Amplifier
 d: Processor
 e: Read out device
 Reference maintains the constant
voltage and is used to compare the
actual results.
http://www1.lsbu.ac.uk/water/enztech/biosensors.html
Block Diagram of a Biosensor

6. BASIC CHARACTERISTICS OF A
BIOSENSOR
 Linearity: Maximum linear value of the standard calibration curve.
Linearity of biosensor should be high for detection of higher
concentration of substrate
 Sensitivity: Value of electrode response per substrate concentration.
 Selectivity: Interference of chemicals must be minimized to obtain
correct results.
 Response time: Minimum response time is appreciated.

7. BLOOD SUGAR TESTING
 Procedure that measures the amount of sugar, or glucose, in your
blood using a glucometer.
 People with diabetes can also use this test to manage their condition.
 Blood sugar tests provide instant results and let you know the
following:
1. your diet or exercise routine needs to change
2. how your diabetes medications or treatment is working
3. if your blood sugar levels are high or low
4. your overall treatment goals for diabetes are manageable

8. BLOOD SUGAR LEVELS
• Depending on your condition and the timing of your test, your blood sugar
levels should be in the target ranges listed below:
• Tracking your blood sugar levels is one way to take control of your diabetes.
Time People without Diabetes People with diabetes
before breakfast under 70-99 mg/dL 80-130 mg/dL
before lunch, dinner and snacks under 70-99 mg/dL 80-130 mg/dL
two hours after eating under 140 mg/dL under 180 mg/dL

9. REQUIREMENT OF BLOOD SUGAR
TESTING
 Blood sugar testing or self-monitoring blood glucose provides useful
information for diabetes management. It can help you:
1. Judge how well you're reaching overall treatment goals
2. Understand how diet and exercise affect blood sugar levels
3. Understand how other factors, such as illness or stress, affect blood
sugar levels
4. Monitor the effect of diabetes medications on blood sugar levels
5. Identify blood sugar levels that are high or low

10. COMPLICATIONS RELATED
TO THE VARYING LEVELS
OF BLOOD SUGAR

11. DIABETES
 Diabetes mellitus, commonly known as diabetes, is a metabolic disease
that causes high blood sugar.
 The hormone insulin moves sugar from the blood into your cells to be
stored or used for energy.
 With diabetes, your body either doesn’t make enough insulin or can’t
effectively use the insulin it does make.
 Untreated high blood sugar from diabetes can damage your nerves, eyes,
kidneys, and other organs.

12. Common symptoms of Diabetes
mellitus
https://www.emedihealth.com/manage-diabetes.html

13. TYPES OF DIABETES
Type 1 Diabetes
 An autoimmune disease
 Immune system attacks the
pancreas where insulin is made.
 It's still unclear what causes it.
 About 10 percent of people with
diabetes have this type.
Type 2 Diabetes
 Chronic medical condition
 Polygenic trait
 Glucose level builds up in the
bloodstream.
 Insulin is either not made or not used
efficiently which does not moves the
sugar from bloodstream to the body
cells.

14. CAUSES OF TYPE 2 DIABETES
 Lifestyle factors
 Combination of genetics
 Being overweight or obese increases your risk too. Carrying extra
weight, especially in your belly, makes your cells more resistant to the
effects of insulin on your blood sugar.
 Family members share genes that make them more likely to get Type
2 diabetes and to be overweight.

15. GESTATIONAL
DIABETES
 Gestational diabetes is high
blood sugar (glucose) that
develops during pregnancy and
usually disappears after giving
birth.
 It can happen at any stage of
pregnancy, but is more common
in the second or third trimester.
 It happens when your body
cannot produce enough insulin
 Gestational diabetes can cause
problems for you and your baby
during pregnancy and after
birth.
https://medlineplus.gov/ency/imagepages/19724.htm

16. HYPOGLYCEMIA
 Condition caused by a very low level of blood sugar (glucose), your body's
main energy source.
 Like fever, hypoglycemia isn't a disease itself — it's an indicator of a health
problem.
 Immediate treatment of hypoglycemia is necessary when blood sugar
levels are at 70 milligrams per deciliter (mg/dL) or 3.9 millimoles per liter
(mM/L) or below.
 Long-term treatment requires identifying and treating the underlying cause
of hypoglycemia.

17. Major symptoms under High and Low
Blood Glucose level
https://www.woundcareinc.com/resources/hyperglycemia-vs-hypoglycemia-what-you-need-to-know

18. DR. LELAND
CLARK
Clark is considered the "father of
biosensors", and the modern-day
glucose sensor used daily by millions of
diabetics is based on his research. He
conducted pioneering research on
heart-lung machines in the 1940s and
'50s and was holder of more than 25
patents.
https://fineartamerica.com/featured/dr-leland-clark-tom-hubbard.html

19. CLARK
ELECTRODE
• The Clark electrode is an electrode that
measures ambient oxygen concentration
in a liquid using a catalytic platinum.
• The electrode has several components: a
platinum cathode (electron receiver),
silver anode (electron donor), electrolyte
solution (typically KCl), impermeable
membrane and a voltage source.
• Reactions are as follows:
1. 2KCl + 2Ag → AgCl + 2K+ + 2e-
2. ½ O2 + 2e- + H2O → 2 OH-
• The overall reactions is as follows:
O2 + 4 e− + 4 H+ → 2 H2O
https://www.openanesthesia.org/aba_clark_electrode/

20. GLUCOSE BIOSENSOR
• Glucose biosensors for SMBG are usually based on the two enzyme
families, Glucose oxidase (GOx) and Glucose dehydrogenase (GDH)
• These enzymes differ in redox potentials, cofactors, turnover rate and
selectivity for glucose.
• GOx is the standard enzyme for biosensors; it has a relatively higher
selectivity for glucose.
• GOx is easy to obtain, cheap, and can withstand greater extremes of pH,
ionic strength, and temperature than many other enzymes.

21. MECHANSIM OF GLUCOSE BIOSENSOR WITH
THE PRESENCE OF GLUCOSE OXIDASE
 Enzyme catalyses the oxidation of glucose to glucolactone and
hydrogen peroxide (H2O2).
 Both glucolactone and H2O2 break down in the presence of Catalase.
 With increased concentration of H2O2, the enzyme activity of GOx is
reduced.
 FAD acts as electron carrier and transfers the electrons from O2 to
form H2O2.
 Ultimaty the concentration of H2O2 is measured which is proportional
to the concentration of glucose in the blood sample.

22. THE REACTION IN GLUCOMETER
WITH THE PRESENCE OF GLUCOSE
OXIDASE
https://www.researchgate.net/figure/The-reaction-calalyzed-by-glucose-oxidase-generation-of-hydrogen-peroxide_fig2_315365846

23. MECHANISM OF GLUCOMETER WITH
THE PRESENCE OF GLUCOSE
DEHYDROGENASE
https://link.springer.com/article/10.1007%2Fs00253-008-1407-4#Sec3

24. FIRST GENERATION OF GLUCOSE
BIOSENSOR
• Glucose estimation was based on the production of H2O2 by GOx utilizing
dissolved oxygen.
• In 1962, Clark and Ann Lyons from the Cincinnati Children’s Hospital
developed the first glucose enzyme electrode. This biosensor was based on
a thin layer of glucose oxidase (GOx) on an oxygen electrode.
• Based on this technology, Yellow Spring Instrument Company, launched the
first commercial glucose biosensor in 1975 for direct glucose
measurement.

25. REACTION AND DRAWBACKS
The drawbacks of the first
generation glucose biosensor are
as follows:
1. Usage of expensive platinum
electrode restricted its usage
to the clinical laboratories.
2. Limited solubility of O2 in
biuological fluids produces
fluctuations in the reaction.
3. Deactivation of GOx due to
production of H2O2.
4. Interference by other redox
species in the blood stream.
https://link.springer.com/article/10.1007%2Fs00253-008-1407-4#Sec3

26. MODEL 23A (YELLOW SPRINGS
INSTRUMENT) BLOOD GLUCOSE
ANALYSER
https://www.lookchem.com/UserFilesUpload/3(270).jpg

27. SECOND GENERATION OF
GLUCOSE BIOSENSOR
 Utilizes the redox mediator to transfer electrons from enzyme to
working electrode surface.
 Redox mediators such as ferricyanide, quinines, methylene blue, etc
were used.
 Used of mediators eliminates the use of O2 for electron transfer.

28. REACTION AND DRAWBACKS
The drawbacks of the second
generation glucose
biosensors were as follows:
1. High competition
between redox mediator
and O2.
2. Interference of other
redox species resulting in
inaccurate results.
https://link.springer.com/article/10.1007%2Fs00253-008-1407-4#Sec3

29. THIRD GENERATION OF GLUCOSE
BIOSENSOR
• These biosensors are based on the direct transfer of the electrons
between ezyme and the electrode surface.
• This type of glucose biosensor involves the immobilization of the
enzyme on the electrode itself.
• These biosenors are reagentless and involves no mediators.

30. REACTION
https://link.springer.com/article/10.1007%2Fs00253-008-1407-4#Sec3

31. BLOOD GLUCOSE MONITORING IN
GMCH-32, CHANDIGARH
 Glucometers are only used in the emergencies to get real time date.
 Otherwise the accurate results are considered from the results of the
chemical analyzer, namely, RX Imola by Randox Laboratories Limited,
U.K
 RX Imola is a clinical benchtop analyzer, commonly used to perform a
number of test on different fluids like blood, urine, etc.
 RX Imola is also used as a back up analyzer.

32. RX IMOLA BY RANDOX
LABORATORIES LTD., U.K

33. WORKING OF RX IMOLA
(COLORIMETRIC METHOD)
 Glucose is determined by enzymatic oxidation by GOx resulting in gluconic
acid and H2O2.
 H2O2 then reacts with the phenol and 4-aminophenazone and forms a red-
violet colored complex called quinoneimine (dye).
 Intensity of the final color is directly proportional to the concentration of
the glucose in the blood sample and is measured at 550 nm.
 RX Imola is internationally accredited and FDA verified.

34. REACTIONS
GOD
Glucose + O2 + H2O → Gluconic acid + H2O2
POD
2 H2O2 + 4-aminophenazone + Phenol → Quinoneimine + 4 H2O

35. REAGENT COMPOSITION IN RX
IMOLA FOR GLUCOSE
ESTIMATION
Contents Initial Concentrations of Solutions
Phosphate Buffer 50 mmol/l, pH 7.0
MOPS Buffer 50 mmol/l, pH 7.0
Phenol 11 mmol/l
4-aminophenazone 0.77 mmol/l
Glucose oxidase
[EC 1.1.3.4, Aspergillus niger, @ 25○C]
≥1.5 kU/l
Peroxidase
[EC 1.11.1.7, Horse radish, @ 20○C]
≥1.5 kU/l

36. REFERENCES
 Review article on Glucose Biosesnsors by Eun-Hyung Yoo and Soo-Youn Lee
(https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3292132/pdf/sensors-10-
04558.pdf)
 http://www1.lsbu.ac.uk/water/enztech/biosensors.html
 https://www.elprocus.com/what-is-a-biosensor-types-of-biosensors-and-
applications/
 https://www.fda.gov/medical-devices/vitro-diagnostics/blood-glucose-
monitoring-devices
 http://www.rafautama.com/index.php?pilih=umum&id=26&ff=rx-
imola.html

37. Thank You