1 Reaction curve.
Firstly,
let's take a look at the time and absorbance of a chemical reaction curve
figure. It can be divided into four regions: the delay region, the constant
velocity region, the transition region and the equilibrium region, as
shown in the following figure:
2 Reaction principle.
Delay region is
senseless to test indicator and without any law.
The indicator
detection method in the constant velocity zone is the rate method. The rate
method, also known as the continuous monitoring method, is to continuously
select four or more photometric points in the linear period of the
time-absorbance curve as the reading point and calculate the value via the
absorbance change value per unit time. When measuring the enzyme
activity or determining the metabolite by enzymatic method. The linear
phase is the difference in absorbance between the various photometric points
during this period, the substrate of the enzymatic reaction is a zero-order
reaction. The photometric point is generally set after the start-up factor is
added and incubated for a period, last for 1-3 min, the continuous
monitoring method is generally applied to the enzyme project, and the factor
method can be used to calculate the concentration.
Schematic
diagram of metabolism assay detection principle
The transition
region corresponding to the fixed-time method. A special case in the
endpoint method, refers to choose two reading points on the time-absorbance
curve. These two points are neither the reaction initial absorbance nor the
equilibrium spot absorbance. The difference of the absorbance of
these two points is used to calculate the result. The goal is to solve
certain non-specific problems about chemical
reactions and improve accuracy.
According to the
number of light-measuring points, the balance region corresponding to
the end point method can be divided into one-point termination method and
two-point termination method.
The one-point
termination method is to select a light-measuring point to calculate the
concentration of the substance to be tested when the reaction reaches the
equilibrium point, that is, the absorbance does not change on the
time-absorbance curve.
The two-point
termination method is often used in the two-reagent assay. Most of the first
reagents usually contain only buffers and other components. They generally do
not react specifically with the sample; therefore, before the second reagent is
added, select a light-measuring as the first reading point, the absorbance at
this time is equivalent to the sample blank. After the second reagent is added,
the substance to be tested reacts and reaches an equilibrium point after a
certain period. At this time, select the second reading point , and calculate
the concentration of the substance by the difference between the absorbance of
the two reading points .The two-point method can effectively eliminate the
light absorption interference caused by hemolysis, jaundice, and turbidity of
the sample. Such as enzymatic determination of glucose, total cholesterol,
triglycerides, uric acid, creatinine, and chemical methods for determination of
total protein, total bilirubin, direct bilirubin, calcium, phosphorus,
magnesium, iron, etc.; and reach the end of the reaction within 2-5 min
after adding the second reagent.