Most microplate reader specifications will express its sensitivity as an amount of a substance, ATP or Luciferase for Luminescence and Fluorescein for Fluorescence. For other detection technologies it could be any other substance that is detected by a kit using that technology. They will also use terms such as Femtomole, Attomole and Zeptomole. So, what do these terms mean?
As you know, Avogadro's number is the number of molecules in a mole of substance. It has a value of 6.022 x 1023. Thus, a mole of a substance consists of 602,250,000,000,000,000,000,000 molecules:
- A Femtomole is 1 x 10-15 or one quadrillionth of a mole (0.000000000000001). This is ~602,200,000 molecules (Avogadro number X (1 x 10-15).
- An Attomole is 1 x 10-18 which is one quintillionth of a mole (0.000000000000000001) and is ~ 602,200 molecules.
- A Zeptomole is 1 x 10-21 which is one sextillionth of a mole (0.000000000000000000001) and is ~602 molecules.
As you can see from the above we are dealing with extremely small numbers, especially at the Zeptomole level. So, how can we use all of this to evaluate the performance of any instrument? There are two key criteria we need to take into consideration: the lower limit of detection (sensitivity) and the coefficient of variation.
- The lower limit of detection is the minimum sample quantity that generates an instrument response above the blank noise and is referred to as sensitivity.
- The coefficient of variation is the percent of change over a set of readings over time - this implies the consistency of the instrument performance.
The coefficient of variation and the lower limit of detection should be obtained using only known standards.
Calculating the lower limit of detection
We at Berthold use only known standards and the following formula to calculate the detection limit:
|ConcentrationS||concentration of standard* signal in pg/mL|
|StdevB||standard deviation of wells filled with blank|
|meanS||average of wells with standard*|
|meanB||average of wells filled with blank|
* Any standard point with average readings at least 100 times above the average readings of the blank.
Because of the formula above a stable background becomes extremely important. We take 3 standard deviations of the mean value of the background which is > 99% of the normal distribution. A sensitive instrument will have a clear distinction between the blank noise and the sample signal. Look at the example below to see the impact of higher background variation:
|Instrument 1||Instrument 2|
|Calculated detection limit||0.030||0.045|
Although instrument 2 has a lower background signal it is less sensitive due to its higher background standard deviation. A lower limit of detection (sensitivity) can help you save both time and money especially when detecting signals that are close to the detection limit. This can help reduce the consumption of expensive reagents or valuable cells. This can also significantly reduce the reading time per sample and save total operation time. Stability of the detector used in your instrument is extremely important when looking at sensitivity. This is the reason why we at Berthold hand select all of our Photo Multiplier Tubes (PMT's) and test them using various parameters and at various temperatures to ensure the best PMT goes into Berthold plate readers.
- Low noise, stable background
- Blue/green/red efficiencies
- Long term stability
- High detection efficiencies
Only PMTs with low noise, stable background, high efficiencies and long-term stability at the usual laboratory temperatures are used for our microplate readers. This guarantees the performance you need for a successful research. This makes the Centro XS LB 963 Microplate Luminometer and the Tristar 3 Multimode Reader the most sensitive instruments in their class.