|Send to printer »|
Tutorials : Mar 1, 2006 ( )
Achieving Low-Volume Liquid Handling
Utilizing the mosquito to Keep Pace with Demands for Lower Cost and High Throughput!--h2>
Incorporating new liquid-handling technologies into the drug discovery process has been key to keeping pace with the demands for ever-increasing throughput and lower costs. Systems must be designed to cope with the sheer number of assays and novel chemical entities being developed. Furthermore, the screening process can be costly as the testing of valuable compounds can result in substantial waste. Additionally, manual pipetting techniques can, over time, present health and safety risks. As a result there has been an increasing demand for automated liquid-handling systems, such as the mosquito, an automated nanoliter pipettor from TTP LabTech (www.ttplabtech.com).
The mosquito was recently integrated into the pain research and neuroscience drug discovery program at Merck (www. merck.com) Research Laboratories. Here mosquito has supported a number of assays used to investigate an array of molecular targets involved in pain signal-transduction pathways.
The mosquito has several unique features and applications for the drug discovery process. For example, it uses miniature, disposable micropipettes to eliminate carry-over, can accommodate varying plate types in one protocol, and has the ability to facilitate the miniaturization of assays, through precise serial dilutions, on a microliter scale. In addition, mosquito has been used to provide low-volume, assay-ready drug plates and daughter plates for further analysis. This, in turn, allowed us to develop an integrated screening platform for use in our ligand binding and functional cell-based assays.
Nanoliter Liquid Handling
In our laboratories, the Genesis Freedom from Tecan (www.tecan.com) is used primarily to set up ten-point dose titrations, doing threefold serial dilutions (10 uL in 20 uL). Reagents Freedom or titration plates are passed to mosquito to generate other assay types. Using a Freedom-generated titration plate (20 uL/well), mosquito can spawn up to four daughter plates with volumes ranging from 0.05 - 1.2 uL.
Alternatively, mosquito can be set up to perform ten threefold serial dilutions across a 96-well plate (0.6 uL in 1.2 uL). However, prior to the installation of mosquito, extensive testing was carried out alongside Freedom. Thus, mosquito was trialed rigorously over a period of one month to ensure its precision and accuracy. Several methods were designed to accomplish this goal.
Method 1: Examination of mosquito-generated Daughter Plates
To analyze the accuracy of mosquito-generated daughter plates, dilutions of fluorescein, dispensed by mosquito, were compared against a standard. This was completed by plotting the absorbance of the mosquito-plated fluorescein (500 nL of stock fluorescein dye diluted 400-fold in buffer) and normalizing data to the gold standard for all 96 wells (100 uL of stock fluorescein dye diluted 400-fold in buffer; Figure 1). All the values for the 96-well gold standard plate were averaged to get one number and that was the number used to normalize the test plate done by the mosquito. The accuracy of pipetting measured at 500 nL with mosquito was within 2% (SD <2%).
Method 2: Serial Dilution by mosquito on a 1.2-uL Level
A study of mosquito and the Freedom was developed to test the liquid-handling capabilities of mosquito. Fluorescein (10 mM) was dissolved in dimethyl sulfoxide (DMSO) to mimic a typical drug solution and was used to analyze the accuracy and reproducibility of solution transfer. Threefold serial dilutions in 100% DMSO were carried out by mosquito at the 1.2-uL level and by the Freedom at the 20-uL level. Each protocol used the same eight tips across the plate from column 2 to column 11.
Finally, 20-mM HEPES (pH 7.5) was added to the serial dilutions and fluorescence signals measured using Molecular Devices (www.moleculardevices.com) Spectra Max Gemini XS (excitation 485nm, emission 520nm). Ratios of the mosquito and Freedom generated fluorescence were calculated to yield relative concentrationsthe ratio of 1 demonstrating 100% accuracy. Scatter graphs and box plots were generated to demonstrate accuracy (ratio of 1) and reproducibility (variation in each column) of mosquito.
This method was applied to two different plate types, Costar and Nunc (Figures 2a and 2b), to analyze the relative concentrations derived from mosquito-generated threefold dilutions. Figures generated from measuring the fluorescence for the Costar and Nunc plate fell within our accepted range of specifications for reproducibility and accuracy. Nonetheless, it is clear from our data that the accuracy of serial dilutions, carried out by mosquito can vary according to well type and plate material. This, however, does not pose a problem. The advanced pipetting parameters of mosquito allow the user to dial in better precision parameters to achieve improved CVs.
Method 3: Serial Dilution Using Radio-ligand Binding
In addition to the previous method, a radio-ligand binding assay was employed to test pipetting accuracy. Here, ten point, threefold serial dilutions in 100% DMSO were carried out with mosquito (0.6 uL into 1.2 uL) and the Freedom (10 uL into 20 uL) to provide dose-titration curves for analysis. Two types of 96-well conical, v-bottom plates from Nunc and Costar were used for these dilutions.
An illustrative radio-ligand binding assay was run to judge the ability of mosquito to generate inhibitor binding constants (Ki values) similar to those generated by the Freedom. Preparation of ligand, membranes, and buffers were as previously described1 and the Ki values were determined using a Kd 0.18nM of the radiolabel ([3H]-Prazosin) and a nominal radiolabel concentration 0.4 nM. The data demonstrated that mosquito provided Ki values within an acceptable range of the Freedom.
Protocols for the generation of daughter plates and threefold serial dilutions were set up using mosquitos software. By specifying the volume and number of tip changes alongside the more advanced functions of modifying dispense heights and mixing cycles, the user can maximize efficiency of the pipetting process. Additionally, there is a test function to allow review of the protocol prior to initiation.
These studies demonstrate that the mosquito can accurately and reproducibly pipette nanoliter volumes from variable plate formats. Before integrating mosquito into the workflow, all pipetting for this project was carried out using multichannel pipettors or a 96-well pipetting instrument. Our research group subsequently used the Freedom to make serial dilutions (10 uL into 20 uL) and relied on mosquito to then transfer these ten-point dilutions into other plates for further use. This data demonstrated that mosquito is capable of carrying out both dilution steps with improved pipetting times of roughly two minutes per plate.
The provision of low-volume assay-ready plates and the improved accuracy of dose-inhibition curves have been accompanied by a significant decrease in unnecessary workload. Additionally, it reduced the volume of DMSO waste and decreased the amount of valuable compound used by up to 16-fold. mosquitos ability to miniaturize assays means much smaller volumes, resulting in saving on resources such as buffers, assay kits, radio-ligands, and enzymes. The use of this automated multichannel pipettor has also reduced the risk of human error by eliminating manual pipetting and the associated health and safety risks.
The mosquito enabled the screening of up to nine assay plates, or 72 compounds per run, and has supported a variety of assays, including viral, radio-ligand, enzyme, fluorometric imaging plate reader assays, and bioluminescent assays. In summary, mosquito is a cost-effective solution to optimize workflow and efficiency within the screening laboratory. Moreover, it has potential for additional applications in the drug discovery sector, including confirmation, specificity, cytotoxicity, and P450 assays. We have demonstrated that mosquito has the ability to make assay-ready plates and daughter plates for further analysis and we are now looking to integrate the use of mosquito into our crystallography projects.
© 2013 Genetic Engineering & Biotechnology News, All Rights Reserved