Technical Note UV-Vis spectrophotometry: monochromators vs photodiode arrays Comparing absorbance measurements between the Quad4 Monochromators™-based Infinite® M200 PRO and a multimode reader using photodiode array technology Introduction Materials and methods The most widely established technology for UV-Vis Infinite M200 PRO multimode microplate reader absorbance measurement is a monochromator-based Multimode microplate reader with a PDA microplate reader. Spectrophotometers have undergone a Herring sperm DNA standard great deal of development since their introduction in the Tris-EDTA early 1950s (1) and, in recent years, multimode readers using Orange G (OG) linear photodiode array (PDA) technology for absorbance ddH2O measurements have become available. PDA-based readers 96-well, transparent UV-Star® plates incorporate an optical grating and a solid state array detector, enabling measurement of light intensity throughout the UV and Experiment 1 visible regions of the spectrum. Similar to a monochromator, Linearity in the visible spectrum using Orange G but much faster, they allow the entire UV-Vis spectrum of a In the first experiment, the OD (optical density) linearity of the sample to be captured within a few seconds per well. PDA spectrophotometer in the visible wavelength range was However, this technology suffers from a number of drawbacks, compared with the OD linearity of the monochromator-based mainly due to high levels of stray light. This results in a Infinite M200 PRO. An OG dilution series was prepared in dramatically limited dynamic measurement range (2). ddH20 (200, 150, 112.5, 84.4, 63.3, 47.5 and 35.6 mg/ml) and This technical note compares the results of basic absorbance 200 µl of each concentration was pipetted into a 96-well measurements performed on an Infinite M200 PRO multimode UV-Star plate in triplicate. The plate was then measured on reader equipped with Quad4 Monochromators with those from the Infinite M200 PRO (492 nm, 25 flashes, 100 ms settle a multimode reader equipped with a PDA. time) and on the PDA-based reader (492 nm, 25 flashes, 1 Technical Note 100 ms settle time). The results were plotted in Microsoft With the Infinite M200 PRO, all values that are outside the Excel®, and the R2 values were calculated to estimate the measurement range are displayed as ‘OVER’ values, avoiding signal linearity (dynamic measurement range). any misinterpretation. Experiment 2 OG Infinite M200 MMR with PDA DNA absorbance spectrum concentration PRO spectrophotometer (mg/ml) (OD @ 492 nm) (OD @ 492 nm) A sample containing 3.125 ng/µl of herring sperm DNA Blank 0.030 0.025 standard was prepared in Tris-EDTA and a 200 µl aliquot was 35.6 0.939 0.924 pipetted into one well of a 96-well UV-Star plate. For the blank, 47.5 1.187 1.170 200 µl of Tris-EDTA buffer was pipetted into a second well of 63.3 1.601 1.585 the same plate. The absorbance spectrum of both wells was 84.4 2.100 2.088 recorded on the Infinite M200 PRO (230 to 350 nm, 1 nm 112.5 2.753 2.77 steps, 25 flashes) and the PDA reader (220 to 350 nm, 150 3.706 3.34 1 nm steps, 25 flashes). Blank values were subtracted from 200 OVER 3.5 the sample values, and the spectrum was plotted in Microsoft Table 1: OD values at 492 nm (average of triplicate values). Excel. Figure 1 compares the OD linearity in the visible range for Experiment 3 the Infinite M200 PRO and the PDA reader. It is clear that the DNA A260/A280 reproducibility and UV linearity linearity of the PDA reader is limited above an OD of 2.5. Even The absorbance of a low concentration DNA sample (200 µl when the highest value – which should be indicated as OVER of 0.8 µg/ml solution) was measured ten times on the – is removed, the R2 of the PDA reader is 0.9914 (data not Infinite M200 PRO (260/280 nm, 25 flashes, 100 ms settle shown), a value that is still not acceptable for a well time) and the PDA reader (260/280 nm, 25 flashes, 100 ms performing absorbance spectrophotometer (R2 ≥0.999 is a settle time). This demonstrates the OD reproducibility (% CV) widely-accepted performance value). at the instrument’s detection limit, and is a good indicator of the overall performance in absorbance mode for both instruments. To estimate the UV linearity, a DNA dilution curve (0.8, 1.56, 3.125, 6.25, 12.5, 25, 50, 62.5, 125, 250 and 500 µg/ml; 200 µl/well) was measured in a 96-well UV-Star plate on the Infinite M200 PRO (260/280 nm, 25 flashes, 100 ms settle time) and the PDA reader (260/280 nm, 25 flashes, 100 ms settle time). Results and discussion Experiment 1 Linearity in the visible spectrum using Orange G Table 1 shows the OD values for the Orange G dilution curve measured on the Infinite M200 PRO and the PDA reader. A critical issue with the PDA reader is that there is no indication that values with an OD above 3.5 are outside the instrument’s measurement range. Consequently, incorrect values are Figure 1: Linearity in the visible spectrum using Orange G. displayed that could easily be misinterpreted by the user. 2 Technical Note Experiment 2 quantification and purity checks. The PDA reader is less DNA absorbance spectrum reproducible, with all CVs greater than 10 %, bringing into question whether this reader should be used for DNA quantification. MMR with PDA Infinite M200 PRO DNA (µg/ml) spectrophotometer (OD @ 260 nm) (OD @ 260 nm) 0.8 0.0105 0.0095 1.56 0.0260 0.0255 3.125 0.0574 0.0593 6.25 0.1209 0.1181 12.5 0.2354 0.2322 25 0.4818 0.4744 50 0.9937 0.9702 Figure 2: Spectrum of 3.125 µg/ml herring sperm DNA. 62.5 1.2690 1.236 125 2.4289 2.3315 Figure 2 shows the DNA spectrum recorded with both the 250 OVER 3.3651 Infinite M200 PRO and with the PDA reader. For the PDA 500 OVER 3.4419 reader, even after subtraction of the blank value, the values between 220 and 240 nm are clearly incorrect, with some Table 3: OD values for various DNA concentrations at 260 nm. negative OD values. This demonstrates that the results for the PDA reader are not reliable below 240 nm, and a correct A230/A260 ratio cannot be determined. Experiment 3 DNA A260/A280 reproducibility and UV linearity Infinite M200 MMR with PDA PRO spectrophotometer Mean – A260 0.0105 0.00955 SD – A260 0.00049 0.00101 CV (%) – A260 4.7 10.6 Mean – A280 0.00436 0.00288 SD – A280 0.00025 0.00096 CV (%) – A280 5.7 33.3 Mean – A260/A280 2.4 3.46 SD – A260/A280 0.047 0.7 CV (%) – A260/A280 2 20.3 Table 2: A260/A280 reproducibility using a low concentration DNA sample (0.8 µg/ml herring sperm DNA). Figure 3: UV linearity for DNA quantification. Table 2 shows the reproducibility of measurements at 260 Table 3 and Figure 3 show the OD values for the DNA dilution and 280 nm, as well as the A260/A280 ratios. The Infinite M200 curve measured on the Infinite M200 PRO and the PDA PRO reader shows exceptional reproducibility at both 260 multimode reader. Again, there is no indication that values are and 280 nm, indicating very good performance for DNA 3 Technical Note out of the measurement range for the PDA-based instrument Literature (not even ‘3.5’ is displayed), whereas the two highest concentrations are clearly displayed as ‘OVER’ on the Infinite (1) J.C. Miller, S.A, George and B.G. Willis, Science, 218, M200 PRO. This critically influences the outcome of the 241 (1982) experiment for UV linearity, as the user would include these (2) Yair Talmi and R. W. Simpson, Applied Optics, Vol. 19, erroneous values in the dilution curve, leading to poor linearity. Issue 9, pp. 1401-1414 (1980) Excluding these two values results in an acceptable R2 value of 0.9991 (data not shown). List of abbreviations Conclusion CV Coefficient of variation PDA-based spectrophotometry is a well-established MMR Multimode microplate reader technology for absorbance measurements, offering ultra-fast OD Optical density spectral scanning capabilities. However, it has significant OG Orange G drawbacks, particularly due to increased stray light, resulting PDA Photodiode array in a limited dynamic measurement range. Although fast scanning capabilities are important for some applications, such as full spectral scans of low volume DNA samples, this feature is not required for most absorbance-based measurements. In this study, we show that the monochromator-based Infinite M200 PRO multimode reader offers significant advantages over a PDA-based reader for many applications. These benefits include an extended dynamic measurement range, better OD linearity in the visible and the UV range, and greater measurement reproducibility at 260 and 280 nm. For the majority of applications, these characteristics are of greater importance than high speed spectral scanning, making a monochromator-based multimode reader a better choice for most users. 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