How to use the Thermoplate Reader
How to use the Thermoplate Reader I. Measurement wavelength The labeling enzymes used in the domestic common ELISA kits are all horseradish peroxidase (HRP), and the substrates are usually tetramethylbenzidine (TMB) and o-phenylenediamine (OPD). It is oxidized to 2,2,-diaminoazobenzene (DAB) and biphenyl hydrazine by HRP in the presence of a hydrogen peroxide solution. When the pH value is about 5.0, DAB has maximum absorption at 450nm wavelength. When the pH value drops to L 0, the maximum absorption wavelength shifts to 492 nm, while the molar extinction coefficient becomes larger and the color develops deeper. Therefore, strong acid such as sulfuric acid is commonly used. Or hydrochloric acid to terminate the reaction. The oxidation product of TMB, biphenyl fluorene, has a maximum extinction coefficient at a wavelength of 450 nm. If the amount of HRP is small, when H:O: and TMB are excessive, a blue cation root is formed. By lowering the pH, the blue cation root can be converted to yellow biphenyl hydrazine, and the sulfuric acid can be used as a terminator to stabilize the product for 90 min. Therefore, the two wavelengths of 450 nm and 492 nm are currently the most commonly used for ELISA assays. Various microplate readers are equipped with auto-convertible components for the placement of filters. Six to eight filters can be installed at the same time. The filters included should include the above two wavelengths. Some microplate readers The 490nm filter replaces the 492nm filter, which has little effect. The measurement wavelength of the general microplate reader is between 400~750nm or 800nm, which can fully meet the ELISA color measurement. . In addition to these two basic filters, in consideration of the need for dual-wavelength colorimetry, filters with wavelengths of 620 nm or 630 nm or 650 nm and 405 nm should be available. Other filters can be selected according to their needs. Sometimes, some laboratories want to use the microplate reader for micro biochemical determination. Therefore, the microplate reader manufacturer has extended the ultraviolet detection function to the microplate reader produced by it, and a 340 nm wavelength filter is needed. At this time, the measurement wavelength range of the microplate reader is 340-750 nm or 800 nm. Second, the measured absorbance range Third, the optical system Fourth, the detection speed Fifth, the shock board function Sixth, the incubation function Seven, software features Baby Squid,Baby Octopus,Poulp Squid,Squid Tube GOLD STAR FISHERY ZHOUSHAN CO.,LTD. , https://www.goldstar-aquatic.com
The microplate reader has single-wavelength and dual-wavelength detection functions. Sometimes users do not know under what circumstances to use single or dual-wavelength detection. The so-called "single wavelength" is a colorimetric measurement using a wavelength of 450 nm or 492 nm, which is the maximum absorption of the chromophore; and "dual wavelength" is the wavelength of 450 nm or 492, which is the maximum absorption of the color. In addition to the colorimetric measurement of nm, the wavelength is determined to be insensitive to specific color development, such as 630 nm, and the absorbance finally printed by the microplate reader is the difference between the two. The absorbance at 630 nm is non-specific and comes from absorption on the board such as fingerprints, dust, dirt and the like. Therefore, in the ELISA colorimetric assay, it is preferred to use dual wavelengths without the need for blank wells.
Generally, the absorbance of the microplate reader is measured in the range. Between 0 and 2.5 can meet the measurement requirements of ELISA. The absorbance of the early microplate readers was generally between 0 and 2.5, but now it has been broadened to a maximum of 3.5 and can maintain good precision and linearity. For example, Labsystems' Dragon Wellscan MK-2 has an absorbance measurement range of 0-3.5, while Multiskan Ascent has an absorbance measurement range (Abs) of 0-4.0. In the range of 0-4.0, the linearity error does not exceed ±2.0% at 0.3-3.0. In the absorbance range, the measurement precision CV is less than ±0.2%; in the range of 3.0-4.0 Abs, the measurement precision CV is less than ±0.2%. Therefore, it is not necessary to deliberately pursue a large absorbance range for the absorbance range of the microplate reader, mainly depending on the linearity and precision within a certain absorbance range.
The optical system of the microplate reader uses a multi-channel vertical light path (usually 8 or 12 channels, also has a single channel). It is usually a silicon tube or an optical fiber. In addition to the measurement channel, there is also a microplate reader. The reference channel allows self-calibration for each measurement. The optical system function of the microplate reader can be reflected by the absorbance range, linearity, precision and accuracy measured by the microplate reader. If the optical system is good, the above indicators should also be better. The precision of the measurement is directly related to the uniformity between the measurement channels. Single channel avoids differences due to different channels.
The detection speed of the microplate reader refers to the time required for the completion of the colorimetric measurement. The detection speed is fast, which is beneficial to improve the precision of the detection, that is, to avoid the difference in absorbance between the micropores due to the difference in measurement time during the measurement process. The commonly used microplate readers on the market are very fast, usually within a few seconds. For example, Labsystems' Dragon Wellscan MK-2 and MK-3 detect 96 wells in 2s (continuous mode) or 5s (step mode). Multiskan Ascent is a single channel test and requires only 9s (continuous mode) or 14s (step mode). Another example is BioRad's Benchmark detection speed with a single wavelength of 7s and dual wavelengths of 15s.
The shock plate function of the microplate reader means that the microplate reader oscillates and mixes the ELISA plate holes before the colorimetric measurement, so that the color in the plate holes is uniform. At present, the common microplate readers on the market have a shock plate function. The difference is the vibration plate mode, and some can be adjusted according to the up, down, left and right or rotation modes and the oscillation amplitude, such as Labsystems MK-2; others More fixed, such as BioRad 550. After using the microplate reader with the shock plate function, after the color reaction is completed by ELISA, the addition of the terminator can be carried out without oscillating and mixing, and can be directly measured on a microplate reader.
The incubation function means that the microplate reader itself can automatically and accurately control the temperature inside the instrument as required, so that the incubation process of the microporous strip in the ELISA assay can be completed inside the instrument without the need for an external thermostat. Currently, only a small number of microplate readers have this function.
The software function refers to the function of the ELISA for qualitative and quantitative determination of ELISA and other statistical methods such as enzyme kinetics, UV and agglutination, and reporting the results. The software function is a very important function of the high-end microplate reader. If the hardware is not very different, the software becomes the only indicator to determine the pros and cons of the microplate reader. For the user, a good software function will be of great help to the actual work.