In recent years, with the multi-function microplate readers gradually promoted in various domestic university laboratories, the brand and model of multi-function microplate readers have gradually increased, and the squandering of flowers has become more and more attractive. In addition to the three traditional advantages of PE, MD and TECAN, there are many latecomers to enter the market, such as the acquisition of Thermo in Finland, the Berthold from the light, the BMG for the pharmaceutical screen and the emerging BioTek. Each brand has its own one or more series of product lines with different characteristics. The various technical parameters and technical indicators are dazzling when purchasing.
This article attempts to explore how to look at the various parameters of the pattern from the perspective of the user's actual use, hoping to help you find a truly suitable multi-function microplate reader.
First, the filter Vs grating
There are many classification methods for multi-function microplate readers, but the simplest one is to use their filtering method as a dividing line. In general, it can be divided into two major categories: filter type and grating type. Of course, there are also some models, such as Synergy4 and EnVision, which are equipped with filters and gratings in one machine. However, the filter and the grating cannot complete the same inspection at the same time, or use the grating when using the grating, and use the filter when the filter is used; some experiments are not used, and the other module cannot be realized. Therefore, this type of instrument essentially only puts the filter and the grating together, and does not make the two together to create a new technological breakthrough.
In general, the filter technology has been developed for a long time, and the optical path system such as the dichroic mirror (in fact, another type of filter reflective filter) can realize the needs of most experiments. At present, the highest detection sensitivity in the conventional multi-function microplate reader is made by the filter type. For example, the sensitivity of the fluorescence detection of TECAN Infinite F500 can reach 0.04 fmol/well (fluorescein, 384 holes/80 ul).
However, filter-type instruments are limited by the wavelength and number of filters, and it is impossible to meet the needs of the increasing types of experiments. Sometimes, it is necessary to study the absorption, excitation and emission spectra of substances, so it was born. Grating type instrument.
The first to introduce a grating is MD, whose grating is customarily called a single grating. Due to the lack of light purity, a set of band-stop filters was added behind the grating, and the stray light was filtered again to achieve a stray light ratio of 5×10-4, which was basically consistent with the pure filter system. Later, TECAN developed a double-grating technology. After two gratings, the stray light rate was reduced to 10-6. Later, some of the new instruments from Thermo, BioTek and PE used similar double-grating technology. Since each set of double gratings is used for excitation and emission, such machines are also referred to as four-gray type multi-function microplate readers.
The introduction of the grating type microplate reader makes the user no longer limited in wavelength selection, and surpasses the filter system in terms of stray light rate and bandwidth control. For example, TECAN introduced the M1000 microplate reader using the third-generation four-grating system at the end of 2008. The stray light rate dropped to a new low of 2×10-7, and the bandwidth was continuously adjustable from 2.5 to 20 nm. These are not currently possible or difficult to achieve with a filter-type microplate reader.
Second, the stray light rate & wavelength accuracy
Grating-type filter system has become the mainstream of the current universal multi-function microplate reader. Many manufacturers have worked together to push the grating technology to a new high. Among the many technical parameters of the grating, the most critical is the accuracy of the grating's stray light rate and wavelength selection.
The stray light ratio refers to the ratio of the light of the "unwanted" wavelength to the light of the nominal wavelength in the light obtained after the light source passes through the grating, which characterizes the purity of the filter. Due to the complexity of light interference, diffraction, etc., stray light is inevitable regardless of whether a filter or a grating is used. The essence of various filtering techniques is to find ways to remove stray light as much as possible. In general, the filter type has a stray light ratio of 10-4 to 10-5, and the grating type can achieve 10-6 to 10-7. Since such stray light is non-specific and goes directly to the final detector, how many random errors are introduced by how much stray light is introduced. In the detection process such as fluorescence, due to the amplification effect of the detector, the interference of the stray light rate is also exponentially amplified. Therefore, the stray light rate is the primary performance indicator of a filter system.
Another important indicator of gratings is the accuracy of wavelength selection. Because many tests rely on the characteristic map of a substance at a certain wavelength. Just like the OD260 concentration of DNA/RNA, if the actual detection wavelength deviates from 260 nm by more than a few nm, the mathematical relationship between the OD value and the final concentration will change. Therefore, a set of high performance grating systems, his wavelength selection accuracy should be between ± 0.5 ~ 1nm. Excessive wavelength deviation can sometimes affect the accuracy of the final result.
These two can be described as the most critical technical parameters of the grating and even the filter filter system, which directly affects whether the data is the true measurement result, and is the most basic requirement for the reliability of the experimental results.
Third, certification > parameters
On the basis of ensuring reliable detection, the next difference between different instruments is reflected in the sensitivity of the detection. At this time, people are concerned about how weak the signal can be detected by the instrument.
Sensitivity involves the design and selection of the entire optical path system. It is difficult to say that a certain component will play a decisive role. However, among the various single-function detection items, the non-amplifying photodiode for light absorption detection, the photomultiplier tube for fluorescence detection, and the single-photon counting photomultiplier tube for luminescence detection are three different detections. The device is the preferred choice for each field of testing.
The optimal results for each individual test are performed with the corresponding detector. Of course, some instruments are considered to be compatible with a variety of detection modes due to cost considerations. This can also complete the experimental requirements to a certain extent, but there will be a compromise in performance, and it is impossible to achieve various tests. optimize.
Regarding the definition and testing standards of sensitivity, each manufacturer has its own description, and it will be said how good it is. It is difficult to have an intuitive and objective comparison. Therefore, some reagent manufacturers stand up and provide a third-party certification for the detection performance of some instruments.
More common are Promega's DLReady certification in the field of luminescence detection, Invitrogen's LanthaScreen series certification in the field of fluorescence detection, Cisbio's HTRF certification, and BellBrook's Transcreener certification. These certifications are mainly for a certain type of high-demand kit provided by the company, covering various performance requirements of the test instrument, including: whether the test method can be used, sensitivity, linear range, inter-hole interference, etc. meet the requirements. and many more.
If you need to do related experiments, such as Luciferase luminescence test to see if the instrument has DLReady certification, do TR-FRET, look at HTRF certification, do FP to see Transcreener certification, if the machine has the relevant certification, you can It shows that the machine can better meet the various requirements of similar detection to a certain extent, which is far more telling than comparing a single sensitivity parameter. Because an experiment can be done is not a single sensitivity can be characterized.
It is important to note that LanthaScreen is a series of certifications covering multiple sub-projects such as FI, FRET, TRF, FP, etc. The sub-projects passed by different instruments are different and need to be checked on the Invitrogen website.
Grating-type instruments have only gradually emerged in recent years due to new technologies, limited by technology research and development and instrument costs. The sensitivity and other indicators in the same price range will be slightly worse than the mature filter machines. Therefore, in the field of mid-range multi-function microplate readers, it is rare to obtain various types of certified grating instruments. As a representative of the new generation of grating type microplate readers, TECAN M1000 has obtained all the above four kinds of certifications, and has surpassed many medium and high-end filter type microplate readers in terms of single performance such as sensitivity.
This shows that the development of the grating type microplate reader has entered a new height. In addition to the irreplaceable role in flexibility, it also gradually replaces the traditional filter type microplate reader in terms of detection sensitivity. . The grating type is the main development trend of the multi-function microplate reader in the scientific research field; and the filter type instrument is also improving in the aspect of better single performance.
Fourth, cuvette + micro detection
In addition to the conventional 6-384/1536-well ELISA plate, some instruments are also equipped with compatible functions such as cuvettes and micro-test plates. Whether using vertical cuvettes, horizontal cuvettes or various micro-test plates, the goal is to introduce a standard optical path concept for the detection of unrestricted microplates. Although the ELISA plate can also realize the conversion of the OD value of the 10 mm optical path by means of optical path correction, this is only a mathematical conversion process, and the detection error is accumulated more, and the actual use effect is not good. After the introduction of the cuvette and the micro-test plate, the detection optical path of the light absorption is fixed to a standard length of 10 mm or 0.5 mm, and the OD value conversion is simpler and more accurate. The microplate reader with such detection function is equivalent to the function of the partial spectrophotometer in addition to its own multi-function enzyme label detection, which makes the function more comprehensive and the instrument cost-effective.
Fifth, the brand and after-sales <br> different brands of instruments often have a focus on performance. For example, PE companies have a deeper understanding in the field of reagents, and their microplate readers have been optimized on their TRF series of test kits. Therefore, PE microplate readers generally have a higher TRF field. Many features and relatively high performance indicators. Like TECAN, the focus of research and development is on the research of four-grating. In recent years, the second and third-generation grating multi-function microplate readers (M200, M1000) have been introduced, becoming the field of four-grating microplate readers. Leading sheep. The addition of cuvettes, micro-test plates and other functions to the multi-function microplate reader is also a technological innovation made by TECAN from the user's point of view. Later, such technologies have become the targets of brands such as Thermo and BioTek.
Finally, no matter how good a microplate reader is, it is difficult to operate normally without a good after-sales service system. This year, various manufacturers have set up offices in China and even the Asia Pacific headquarters. Some products have also developed Chinese versions of software and manuals. This shows that manufacturers attach importance to the Chinese market. And because of this emphasis, whether it is factory direct sales or choose agent distribution, they will put forward their own requirements and expectations for domestic after-sales service. The most basic is the network system of application engineers and maintenance engineers who popularize instruments.
It can be said that in the major cities of China, basically, some brands have always had an engineer at your side. Before the purchase, communicate with engineers, fully understand the performance characteristics and after-sales situation of each family, and repeatedly compare, you can always find a multi-function microplate reader that best suits your needs.
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