Thomas Moore

Evaluation of Liquid Handling Conditions in Microplates

Liquid handling in higher density microplates (e.g., 1536-well microplates) for more efficient drug screening necessitates carefully selected and optimized parameters. The quality of a liquid handling procedure is dependent on the carryover rate of residual liquids during the pipetting process, the mixing behavior in the wells, foam and bubble formation, and evaporation. We compared and optimized these parameters in 96-, 384-, and 1536-well microplates, and herein we critically evaluate the performance of the CyBi™-Well 96/384/1536 automated micropipetting device, which formed the basis of our evaluation studies.

An Improved Method for Checking HTS/uHTS Liquid-Handling Systems

An efficient method is presented to determine precision and accuracy of multichannel liquid-handling systems under conditions near to application. Themethod consists of gravimetrical determination of accuracy and optical determination of precision based on the dilution of absorbing and fluorescent dye solutions in microplates. Mean delivery volume per well can be determined with precision better than a 0.04% coefficient of variation (CV). Optical signal precision, CV(S), is improved by multiwavelength measurements. Precision of absorbance measurement yields a better resolution than precision of fluorescence measurement (0.3% and 1.5%, respectively), indicating that absorbance measurements should be preferred. From CV(S), an upper bound of the precision of the volumes delivered is derived. Method performance is demonstrated with the dispenser CyBi™-Drop and the pipettor CyBi™-Well using different ejection principles; with commonly used fluids; with 96-, 384-, and 1536-well microplates; and with photometric and fluorometric indicators. Precision of the volumes delivered, as obtained with optimized methods, all plate formats, and both devices, is better than 2% CV with 2 µ L set volume and about 1% CV with higher set volumes.

Miniaturization of a Functional Transcription Assay in Yeast (Human Progesterone Receptor) in the 384- and 1536-Well Plate Format

Miniaturization of high throughput screening assays to high-density microplate formats (384 or 1536 wells) is currently the focus of research activity in modern drug discovery facilities. In this article, we describe the adaptation of a fluorescence-based functional transcription assay in yeast for assessing modulators of human progesterone receptor to the 384- and 1536-well microplate format, comparing the experimental results to those obtained in the well-established 96-well format. The experiences gained from the optimization of the liquid-handling procedures and the miniaturization of an enzyme assay (β-galactosidase) were implemented. Thus optimized pipetting protocols were developed to perform a reporter gene assay in yeast in microplate formats of higher density. In the functional transcription assay in yeast, the reporter gene expression showed the expected dependence on the ligands dose and affinity in principle in all three microplate formats. For the first time, this assay system has been established in the 1536-well microplate format using CyBi™-Well 96/384/1536 as the liquid-handling unit. The comparison of the signal:background ratios showed a lower sensitivity of the assay in the microplate formats of higher density. This study is an example of a successful miniaturization of a yeast cell-based assay to high-density plate formats on the basis of a careful adaptation procedure and optimized liquid-handling conditions.

Searching biomarker candidates in serum using multidimensional native chromatography I. Enhanced separation method

The microplate-based method developed by our group for non-denaturing multidimensional proteome separation was improved on using improved column arrays and a newly developed robot. Currently size exclusion, anion exchange and lectin affinity chromatography are combined orthogonally. Different samples run simultaneously to enhance reliability of intercomparison. LC-ESI (electro-spray ionization) MS/MS analysis of selected fractions identified 32,288 peptides matching 2,669 serum proteins. The present contribution (I) shows the characteristics of the method, whereas prove of principle by applying it to search for biomarker candidates with model diseases is reported in an accompanying paper (II).

Miniaturization of an Enzyme Assay (β-Galactosidase) in the 384- and 1536-Well Plate Format

Miniaturization is one way to realize todays demands in the drug discovery process by moving from the standard 96-well plate to higher density microplate formats. In this article we describe the adaptation of a fluorescence-based enzyme assay to the challenges of the 384- and 1536-well plate format. The liquid-handling was realized by the automated micropipettor CyBi-Well™ 96/384/1536* (CyBio AG — formerly JENOPTIK Bioinstruments Gmbh — Jena, Germany). On the basis of optimized liquid-handling parameters pipetting routines were established to perform an enzyme assay (β-galactosidase) in the microplate formats of higher density. Finally, the experimental results were compared to those obtained in the well-established 96-well format. In the enzyme assay, the bioconversion of the substrate Fluorescein-di-(β-D-galactopyranoside) (FDG), occurred as a linear function of the β-galactosidase concentration comparably in all three assay formats. We conclude that miniaturization using the higher density 384- and 1536-well plate formats is advantageous as the next evolutionary step in HTS, especially using enzyme assays. A careful individual adaptation procedure for each microplate format and assay at the basis of the optimized liquid-handling parameters is essential. CyBi-Well™ 96/384/1536 proves to be a powerful tool for a careful adaptation of the liquid-handling procedures of biological assays especially also in the 384- and 1536-well formats.