experiment _ mesasurement of blood glucose

1. Experiment 6
Measurement of
blood glucose
(GOD-POD method )

2. Aim:
1. Learn the principle of GOD-POD method
2. Understand it's clinical significance

3. What is blood glucose ？
The main sugar that the body makes from the food in the
diet.
Glucose is carried through the bloodstream to provide
energy to all cells in the body.
Cells cannot use glucose without the help of insulin.
The body produces it from protein, fat and, in largest part,
carbohydrate.
Ingested glucose is absorbed directly into the blood from
the intestine and results in a rapid increase in blood
glucose.
Generally speaking, fasting blood glucose level is
constant for healthy people

4.  Serum - serum glucose values decrease
rapidly in samples that have not been
separated from the cellular constituents of
blood. Glucose values decrease by 10% per
hour if serum is left in contact with cells. Note
that the decrease in glucose is enhanced in
patients with increased leukocyte or platelet
counts, even if collected into fluoride oxalate
tubes.

5. GOD-POD
High accuracy,
High sensitivity，
Good stability
Why do we choose Glucose oxidase-peroxidase
method (GOD-POD) to
measure blood glucose ?

6. Principle
Gluconic acid
﹢ （ GOD）
Glucose ﹢ O2 H2O2
H2O2 ﹢ 4-NH3-
（ POD）
﹢ Phenol
Quinonimine﹢ H2O
Antipyrine

7.  The absorption peak of quinonimine
is 500nm
 Generation amount of quinone imine
is proportional to the content of glucose
 The glucose concentration in the sample
can be calculated using the standard
comparison method

8. Equipment
 Test tube : 1.5×5cm（ ×3）
 pipet : 2mL（ ×1）
 micropipette ： 20μl
 centrifuge , centrifuge tube 10ml （ ×2）
 Water bath

9. Reagents
 Glucose measurement kit
 The glucose standard solution (5.55mmol/L)
 Sample： fresh serum

10. Operation Steps
1. Sample processing
Draw blood 3-5
mL
37℃ water bath it
for 15min
Centrifuge the blood
for 10min (3000rpm)
Extract the
supernatant

11. 2. Use 3 small test tubes and add reagent
according to the table below
NO.
Reagent
Blank
tube
Standar
d tube
Sample
tube
dd-Water (μL ) 10 —— ——
Standard
—— 10 ——
glucose (μL )
Serum samples
(μL )
—— —— 10
Enzyme
solution ( mL )
1.5 1.5 1.5

12. 3. After mix the reagents in each tube, 37℃ water bath for 25min，then use 0.5cm cuvette to
measure their UV absorption value At 500nm wavelength

13. Calculation
Write down the data and then
calculate them by Lambert-Beer law
Csample =
Asample
Astandard
× Cstandard

14.  Cstandard = 5.55 mmol/L
 range of normal value ：
3.89 ～ 6.11mmol/L

15. Clinical significance
1. Hyperglycemia
Glucose concentration >7.0mmol / L
e.g. diabetes
2. Hypoglycemia
Glucose concentration <2.8mmol / L
Effects can range from mild dysphoria to more serious
issues such as seizures, unconsciousness, and (rarely)
permanent brain damage or death.

18. Question
Why human can maintain normal serum
glucose at a certain level?

19. Blood Glucose Meter
People who have diabetes should be testing their blood glucose
regularly at home.
 Lancing the finger
 Apply blood to strip
Applying Blood t o St rip
8. Follow instructions included with the meter when
applying blood to strip
1-800-DIABETES www.diabetes.org
17
Drop,
not smear
Cover ALL of
test strip
window
Some strips wick
blood onto the
strip
t he Finger
the side of the finger
stick the finger.
DMMP) the school nurse
will give further
are appropriate
suspect ed hypoglycemia,
be used for blood glucose

20. Mechanism for catalytic oxidation of glucose
With Glucose oxidase (GO) and Fc mediator
Glucose + GO(FAD) + 2 H+ - - > gluconolactone + GO(FADH2)
GO(FADH2) + 2 Fc+ --> GO(FAD) + 2 Fc + 2 H+
Fc --> Fc+ + 2 e- (at electrode)
Fc = ferrocene Gluconic acid/gluconolactone ferrocene carboxylate
Signal can also be measured by amperometry:
Hold const. E where oxidation occurs, measure I vs
time

21. Glucose biosensor test strips
Meter
Read glucose
Dry coating of GO + Fc
Patient adds drop of blood,
then inserts slide into meter
Output:
amperometry
I
t
Patient reads glucose level on meter
e’s
electrodes