(GOD-POD method )

Experiment 6

Measurement of blood glucose

Aim:

1. Learn the principle of GOD-POD method

2. Understand it's clinical significance

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

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.

Why do we choose Glucose

oxidase-peroxidase method

(GOD-POD) to measure blood

glucose ?

GOD-POD

High accuracy,High sensitivity ，

Good stability

Principle

Glucose ﹢ O2 H2O2

Gluconic acid

﹢（ GOD ）

H2O2 ﹢4-NH3-

Antipyrine H2OQuinonimine﹢

（ POD ）﹢ Phenol

The absorption peak of quinonimine is 500nm

The glucose concentration in the sample can be calculated using the standard comparison method

Generation amount of quinone imine is proportional to the content of glucose

Equipment

pipet : 2mL （ ×1 ）

micropipette ： 20μl

Water bath

centrifuge , centrifuge tube 10ml （ ×2 ）

Test tube : 1.5×5cm （ ×3 ）

Reagents

Glucose measurement kit

The glucose standard solution (5.55mmol/L)

Sample ： fresh serum

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

2. Use 3 small test tubes and add reagent according to the table below

NO.

Reagent

Blank tube

Standard tube

Sample tube

dd-Water (μL ) 10 —— ——

Standard glucose (μL )

—— 10 ——

Serum samples (μL )

—— —— 10

Enzyme solution ( mL )

1.5 1.5 1.5

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

Calculation

Write down the data and then calculate them by Lambert-Beer law

Csample =Asample

Astandard

× Cstandard

Cstandard = 5.55 mmol/L

range of normal value ： 3.89 ～ 6.11mmol/L

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.

Question

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

Blood Glucose Meter

Lancing the finger

Apply blood to strip

1-800-DIABETES www.diabetes.org

Applying Blood to Strip

17

Drop, not smear

Cover ALL of test strip window

Some strips wick blood onto the

strip

8. Follow instructions included with the meter when applying blood to strip

1-800-DIABETES www.diabetes.org

Lancing the Finger

7. Hold the lancet device to the side of the finger and press the button to stick the finger.

• Alterative site (per DMMP) the school nurse and/or parent/guardian will give further instructions which sites are appropriate

• Note: In the case of suspected hypoglycemia, only the finger should be used for blood glucose sampling

16

People who have diabetes should be testing their blood glucose regularly at home.

Glucose + GO(FAD) + 2 H+ - -> gluconolactone + GO(FADH2)

GO(FADH2) + 2 Fc+ --> GO(FAD) + 2 Fc + 2 H+

Fc --> Fc+ + 2 e- (at electrode)

Mechanism for catalytic oxidation of glucoseWith Glucose oxidase (GO) and Fc mediator

Signal can also be measured by amperometry:Hold const. E where oxidation occurs, measure I vs

time

Fc = ferrocene carboxylateGluconic acid/gluconolactone ferrocene

Glucose biosensor test strips

MeterRead 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’selectrodes