Description and Discussion on the Sensitivity of Multi-function Microplate Reader

Now, the multi-function microplate reader is basically the basic research equipment necessary for every biological laboratory. The brands of multi-function microplate readers on the market mainly include Bio-Tek, BMG, MD, PE, Tecan and so on. Each brand has its own characteristics and advantages. However, in addition to characterizing the OD value of the light absorption value, such as the fluorescence detection sensitivity and the luminescence detection sensitivity, the brand manufacturers often do not have a unified way and definition (name). Wait). This will cause some confusion when the user compares the performance of the instrument when purchasing the microplate reader. Let's take a look at the different descriptions of the dominant brands in the market for the sensitivity of fluorescence detection:

Table 1:

As can be seen from Table 1 above, for the description of sensitivity, there are mainly two statements of “Sensitivity” and “Detection Limit”. Let us look at their definitions separately:
A: Detection Limit: detection limit, here are two understandings: analysis method detection limit and instrument detection limit

1. Detection limit of analytical methods: International Association of Theoretical and Applied Chemistry (IUPAC) regulations for detection limits: For various optical analysis methods, the minimum analytical signal XL that can be measured is expressed by:

• XL=Xb+kSb
• The average value of Xb-space multiple determination in the formula (n≥20)
• Standard deviation of multiple determinations of Sb-blank
• k - coefficient determined according to a certain confidence level (IUPAC specifies k = 3, the confidence level at this time is 90%). In order to improve the reliability of the detection limit, the concept of Limit of Guaranteed Detection is also proposed, which is the detection limit when k=6 in the above DL formula.

2. Instrument detection limit: used to evaluate the detection energy of the instrument, that is: the corresponding background, the minimum signal that the instrument reliably detects, usually expressed by the signal-to-noise ratio S/N, which is S/N ≥ K, according to the concept of confidence. , K may take 2 or 3, which is a reasonable value.

B: Sensitivity: Sensitivity. For the concept of sensitivity, the definition of IUPAC is: the sensitivity m of the method indicates the change of the analytical signal caused by the concentration or content of the measured component changing by one unit, ie m=dX/dc. Where X is the analytical signal and c is the concentration or content. On the calibration curve of the analytical method, the sensitivity is generally considered to be the slope of the analytical calibration curve. The greater the slope of the curve, the higher the sensitivity. However, the "Sensitivity" mentioned in the instrument parameters does not mean this concept, but specifically refers to "DL (Detection Limit) or LOD (Limit Of Detection)". For example, BMG clearly states in the annotation of Sensitivity. It is "LOD". If the "Detection Limit" (Tecan/MD/PE) described in the above (in the table) parameter refers to the "method detection limit" and "Sensitivity" (BMG/Bio-Tek/Thermo) refers to "LOD", Then Sensitivity=LOD=DL=Detection Limit. Although the descriptors are different, the concepts are the same and their parameters can be directly compared.

At present, from the officially published information of various manufacturers, only BMG clearly states that the International Association of Theoretical and Applied Chemistry (IUPAC) testing standards are used to clarify the parameters. Only BMG has clearly stated its definition of Sensitivity in the parameters, including definitions, detailed measurement conditions, and so on. For example, in BMG's latest high-performance gradient filter-type grating multi-function microplate reader CLARIOstar parameter description, the sensitivity of FI (fluorescence) top reading is FI Filters (top) < 0.4 pM (< 8 amol/well Fluorescein, 384sv, 20 μL), the method of testing sensitivity is clearly stated after the parameters: Fluorescein is used as the standard curve of 6 points for the substrate, using 384 Small Volume microplate, 20 μL detection system . It is important to know here that the type of microplate used to determine the parameters and the volume of the detection system are very important. The sensitivity calculation results are different because of different conditions. Table 2 below is the parameter data of the official website of each microplate reader manufacturer:

Table 2:

As can be seen from Table 2, each manufacturer has different standards to determine its detection sensitivity. Generally, the detection concentration and quality used will be written out, but some manufacturers or models only include concentration units, such as Bio-Tek Neo, MD SpectraMax M5/M3, etc. To fully compare the detection sensitivity, it is necessary to consider the concentration, quality and detection volume . If there is only the concentration (M), what is the actual volume detected? 200 μl? 100 μl or 20 μl? Different volumes will make the actual quality of the test different, which can be ten times or more.

In addition, uniform testing standards are also important. For example, the top read fluorescence of all BMG models is measured with a 384 small volume microplate each time and in a volume of 20 μl. It can be seen from Table 2 that the FLUOstar Omega from the mid-end research type to the highest-end PHERAstar FS measure and display the sensitivity using a uniform detection standard. If the test standards vary between models, such as the Tecan entry model F200 and the high-end model F500, the former is made with 100μl; the latter is made with 10μl. This way the user will find an interesting phenomenon – just look at the concentration, the F200 (0.85pM) entry type seems to be more sensitive than the F500 (1pM) high-end model! Of course, users will also have questions, why is the lower-end F200 10 times more volume than the high-end F500? Everyone is also a 384-well plate. Why is the volume standard for testing different? Is that the same sensitivity (85 amol) that can be made with 20 μl of the parameters made by 100 μl? This is believed to be worth thinking about.

All parameters of BMG are detected according to the IUPAC standard, and the methods, volume, microplate, concentration and quality used are clearly illustrated. A volume of 20 μl was consistently used in 384-well plates because this is the closest to actual experiments. Some manufacturers use a different detection volume than the actual situation. For example, Biotek's Synergy 2 fluorescence parameter is labeled: “Fluorescein 1 pM typical (0.1 fmol/well 384-well plate) – Top”, although the manufacturer did not write it, but as long as Careful calculations can be made with a 100 μl volume for the 384-well plate. In fact, the volume of each well of a 384-well plate on the market is generally only about 110 μl (for example, CORNING), and the microplate manufacturer recommends a volume of 20 μl - 80 μl. If 100μl is used for testing, it is a bit unrealistic, because the volume is too full, it will make the liquid in the hole easier to detect during the moving process. This also makes the hole plate unable to use the vibration function to shake the hole during the experiment. The sample affects the test results. Furthermore, the reason for using 384-well plates is that, in addition to high throughput, it is more important to use smaller samples and reagent volumes. If you want to use 100μl of reagent sample on a 384-well plate, it runs counter to the original intention of saving money. Of course, the smaller the detection volume, the higher the sensitivity of the detection sensitivity of the instrument, and the greater the challenge to the manufacturer. However, BMG would rather give up the beautiful parameters made with larger volumes, but also be loyal to the actual operation, and make parameters for customers with small volume. In fact, even BMG's most popular Optima series of chemiluminescence and fluorescence microplate readers have been used by customers to publish more than 110 articles by Nature or Nature Publishing Group, and its excellent performance is actually beyond doubt.

In all the above aspects, when comparing the parameters of the microplate reader, the unit uniformity is very important, and the conditions used when measuring the parameters are equally important. For many manufacturers' instruments, it is difficult to compare them in exactly the same way, because the conditions used by different manufacturers are different. After all, the performance and performance of the instrument is to be used by the user, not just to write the parameters beautifully.

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