Evaluating and Determining Precision and Accuracy for Nanoliter Volumes of Viscous Solutions on an Automated Liquid Handling Instrument

Transcription

1 Evaluating and Determining Precision and Accuracy for Nanoliter Volumes of Viscous Solutions on an Automated Liquid Handling Instrument Application Note 231 Niki Popp, Tim Hegeman, Joan Stevens, Ph.D. and Greg Robinson (Gilson, Inc.) Introduction The Constellation 1200 is a precise, small-volume liquid handler. It consists of a twelve-channel, noncontact dispenser capable of nanoliter to microliter sample delivery. It is capable of spotting to virtually any format or surface, and can be used for applications ranging from microarray and MALDI spotting to crystallography. This study will examine the instrument s precision and accuracy for dispensing nanoliter volumes of solutions used in crystallography applications. The five solutions being evaluated in this application vary in viscosity, composition, concentration and their overall physical characteristics, and include: 1M HEPES, 30% 2-methyl-2,4-pentanediol, 20% PEG 4000/ 20% IPA, 0.1M MgCl 2 and 30% PEG Photo 1. Six 100-nL drops on a dime. June 2005 Page 1 of

2 Materials & Methods Constellation 1200 Hardware Characteristics X/Y/Z robot with a large bed capacity for holding twelve microplates. Aspirates from 96- and 384-well microplates using syringe technology. Dispenses from 50 nl to 500 µl volumes into 96-, 384- and 1536-well plates employing solenoids for small volumes and syringes for large volumes. (Versions of the instrument are available for higher density plates and volumes down to 5 nl.) Contains two syringes and two selector valves. Syringe fluid pathways are independent of solenoids. Dual wash system allows for both flow-through and nonflow-through rinses. External pressure source maintains a constant, low pressure (10 psi) on the solenoids, assuring the longevity of the valves. True volume aspirating and dispensing. Air gap eliminates any sample diffusion. Solenoids can be individually calibrated to a specific application. Noncontact fluid transfer. Sample never passes through the solenoids, eliminating the possibility of contamination and plugging. Photo 2. Constellation 1200 Liquid Handler. June 2005 Page 2 of

3 Smart Tip Technology Ensures precision in the tips by checking the alignment and positioning of each tip in relation to each other and to the bed Accuracy to ±30 µm Precision to ±2 µm Out of position probes will provide an on-screen alert instructing the user to make appropriate adjustments Photo 3. Closeup of the SmartTip Technology. 735 Sampler Software Software allows design of each zone individually. Drag-and-drop tasks allow quick-and-easy method development and optimization changes. Option for incorporating variables into fields increases flexibility. Custom racks allow unique formats and geometries, including nonstandard targets. Figure 1. Example of a plate type with unique zones denoted by the various colored wells. June 2005 Page 3 of

4 Figure 2. Example of a task and its available data fields. Purpose The purpose of this experiment is to determine and evaluate the Constellation s precision and accuracy in dispensing variable volumes of five unique crystallography solutions including: 1M HEPES 30% 2-methyl-2,4-pentanediol 20% PEG 4000/20% IPA 0.1M MgCl 2 30% PEG 8000 Precision and accuracy are defined as follows: Precision = The quality of being reproducible in all replicate transfers. Coefficient of variance will be used to measure precision. Accuracy = The quality of conforming exactly to a requested delivery volume. Average volume will be used to measure accuracy. Method The precision and accuracy of the Constellation 1200 was evaluated by determining the CVs and average volumes of the crystallography solutions for each of the 12 probes at test volumes of 50, 100 and 300 nl. Each volume was tested in triplicate, and all of the solutions were dispensed using a neartouch-off method with the same solenoid calibration. The CVs and averages were calculated based on spectrophotometric analysis of a standard curve (ranging from nl) for each solution. See tables 1-10 for precision and accuracy data for all 12 probes and three plates. June 2005 Page 4 of

5 Photo 4. Near-touch-off method. The probe was placed in very close proximity (~0.1mm) to the plate during the dispense cycle. Upon dispense, the deformation of the fluid at the probe orifice is sufficient to form a drop in the wells of the plate. 735 Sampler Software Task 1) Aspirated sample from source plate utilizing an air gap and three predispenses. 2) Dipped probes in flowing rinse station to eliminate any sample on the outside surface of the probes. 3) Dispensed sample/methylene blue solution into three destination plates and manually brought to volume with water. 4) Performed spectrophotometric analysis on solutions using Molecular Devices Vmax Reader. 5) Calculated precision and accuracy based on solution s standard curve. Photo 5.Closeup of air gaps. June 2005 Page 5 of

6 Table 1: Avg Precision and Accuracy Data for Plates 1 3 Solution CV (%) 50 nl 100 nl 300 nl CV (%) CV (%) 0.1M HEPES % 2-methyl- 2,4-pentanediol 20% PEG 4000/20% IPA M MgCl % PEG Table 2: Precision and Accuracy Data of 12 Probes at 100 nl Solution Probe M HEPES 30% 2- methyl-2,4- pentanediol 20% PEG 4000/ 20% IPA CV (%) CV (%) CV (%) M MgCl CV (%) % PEG 8000 CV (%) June 2005 Page 6 of

7 Summary As a means to determine the precision and accuracy of an automated liquid handling system, it is useful to determine both the CVs and average volumes of the data sets being evaluated. The Constellation 1200 is capable of producing plate CVs (grouping all five solutions as a single data set) that range from % at 50 nl, % at 100 nl and % at 300 nl and average volumes that include nl at 50 nl, nl at 100 nl and nl at 300 nl. Quantitatively, there was no significant difference in either the precision or accuracy among the five solutions. In addition to determining the precision and accuracy of each plate, the CVs and average volumes were also calculated for the 12 probes at each volume. This data is available upon request. Conclusion The results of the experiment demonstrate that the Constellation 1200 is an effective liquid handling instrument for aspirating and dispensing sub microliter volumes of crystallography solutions. The near touch-off method and solenoid calibration that was used in this study proved to produce both accurate and precise results even when the solutions differed in viscosity, composition, concentration and physical properties. These values coupled with hardware characteristics that include independent syringe and solenoid pathways, a dual wash system, ease of solenoid calibration, SmartTip Technology and plate flexibility, make the Constellation 1200 a logical choice for dispensing nanoliter to microliter volumes of solutions for applications that include both microarray and MALDI spotting and crystallography. Gilson, Inc. World Headquarters P.O. Box , Middleton, WI USA Telephone: (1) or (1) Fax: (1) Gilson S.A.S. 19, avenue des Entrepreneurs, F VILLIERS LE BEL France Telephone: (33-1) Fax: (33-1) sales@gilson.com, service@gilson.com, training@gilson.com June 2005 Page 7 of