by Rachel Parshley, John Thomas Bradshaw, and Karl Olson


Chemistry


Development of a 100% Dimethyl Sulfoxide (DMSO) Sample Solution for Liquid Handler Performance Verification


imethyl sulfoxide (DMSO) has become one of the most important solvents used for high-throughput screening (HTS) and compound management applica- tions due to its ability to readily dissolve a wide range of compounds. It is also the solvent of choice for the new breed of acoustic droplet ejection (ADE) liquid handling instruments, developed by companies such as Labcyte (Sunnyvale, CA) and EDC Biosystems (Fre- mont, CA), which have revolutionized HTS and compound management.


D


Because DMSO has very different physical prop- erties than water, the liquid most commonly used for calibration and performance verifica- tion, it behaves differently when handled by air-displacement pipettors or acoustic droplet ejectors, creating the need for a solvent-specific volume verification method.


Artel (Westbrook, ME) has developed and characterized a 100% DMSO-based dye solu- tion for use with the Artel MVS® Multichannel Verification System. This article discusses the challenges and performance capabilities of a DMSO-based dual-dye solution developed for the MVS. The DMSO-based solution is capable of measuring dispensed volumes as small as 10 nL. A comparison of the performance of this new dye versus gravimetric measurements is presented.


Introduction Nearly all assays performed within a microwell


plate are concentration-, and therefore volume-, dependent. Ensuring that volumes transferred during the preparation of assay plates are ac- curate is therefore of critical importance.1


Research conducted by Nathaniel Hentz, Ph.D., of North Carolina State University (Raleigh) has


shown that the volumes dispensed during drug screening programs can have a significant ef- fect on the results obtained by assay screens.2 Any error made during these deliveries affects the concentration of the drug candidate and can lead to misleading data about compound activity being generated. Data were recently presented that suggest that if two successive liquid transfers are inaccurate, critical reagent concentrations could be affected by as much as 50%.3


Traditionally, liquid delivery performance has been measured using water, or aqueous-based test solutions. This approach gives a clear pic- ture of how automated liquid handlers (ALH) function when water is the test solvent. The dual-dye Ratiometric Photometry™ method used by the Artel MVS was initially developed to test the performance of liquid handling devices when dispensing aqueous-based MVS dyes that were made to mimic water. This absorbance-based method employs two dif- ferent solutions:


1. MVS Sample Solution, which contains a known concentration of red dye (and in some sample solutions a known, fixed concentration of blue dye); and


2. MVS Diluent, which contains a known, fixed concentration of blue dye. The red and blue dyes utilized within the sample solution and diluent both have distinct absorbance maxima at two different wavelengths, 520 nm (red) and 730 nm (blue).


The MVS Sample Solutions are shown in Figure 1.


When using the MVS, the desired volume of red sample solution is dispensed into 96- or 384-well microtiter plates, after which diluent is added to increase the total working volume


AMERICAN LABORATORY • 31 • JUNE/JULY 2013


Figure 1 – MVS Sample Solutions.


to either 200 µL (96-well plates) or 55 µL (384- well plates). Photometric measurements are then collected for both the red and blue dyes at their respective wavelengths. The system simultaneously calculates accuracy and preci- sion with no need to prepare standard curves or solutions. Measurement results are traceable to international standards, which allows for stan- dardization between methods, instruments, and locations.


However, it is commonly understood that this approach does not truly represent ALH per- formance when dispensing organic solutions. While an aqueous-based test can be used to predict a change in instrument performance, pipetting performance with the actual solvent to be used needs to be measured if assay vari- ability is to be minimized.


An application was developed for the MVS which allowed for limited DMSO testing down to 100 nL in a 384-well microtiter plate. This ap- plication required the user to prepare a custom


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