Journal of Laboratory Automation

A significant difference between the reported volumes delivered by a fluid-filled automated liquid-handling system with eight stainless steel fixed tips was observed when a gravimetric method and a dual dye photometric method were used to measure delivered volumes between 20 and 200 μL. A series of experiments, using the default pipetting parameters, was performed which demonstrated that the difference in the reported volume was due to a dilution effect by the system liquid of the liquid handler and did not indicate an error in the delivered volumes. This dilution effect led to a decrease in the reported volume by the dual dye method, which alerted the user to the problem of sample dilution. In contrast, the gravimetric method provided the expected volume, and therefore did not alert the user to the problem. Without optimization of pipetting parameters, the dilution issue can be significant, because the resultant change in the concentration of the compound(s) of interest in the sample could lead to an unidentified error in the sample assay.

In recent years, rapid advancements have been made in the biomedical applications of microtechnology and nanotechnology. While the focus of such technologies have been primarily on in vitro analytical and diagnostic tools, more recently in vivo therapeutic and sensing applications have gained attention. The long-term integration of cells with inorganic materials provides the basis for novel sensing platforms. The work presented here focuses on the ability to maintain cells long-term in nanoporous silicon-based microenvironments. This paper describes the creation of nanoporous, biocompatible, alumina membranes as a platform for incorporation into a cell-based device targeted for in situ recording of cellular electrical activity variations due to the changes associated with the surrounding microenvironments. Studies described herein focus on the interaction of nanoporous alumina substrates embedded in silicon patterned with cells of interest. The fidelity of such a system is demonstrated in terms of viability, proliferation, and functionality. The capability of such microfabricated nanoporous membranes, as in vitro for cell-based assays for sensing and drug delivery applications, is also demonstrated. It has potential in vivo application for therapeutic immunoisolation.

Optimizing the choice of polymorphs of an active pharmaceutical ingredient (API) has become a significant step in the drug development process. High-throughput combinatorial techniques have been developed to reduce the time required to identify and select the best form of an API. A very important part of the high-throughput crystallization (HTC) workflow is reliable integration of information produced by various analytical instruments and easy access to the information by multiple scientists. Raman spectroscopy has become one of the key analytic techniques used to differentiate between the polymorphs and salts of the API. A Raman microscope has been integrated into an HTC workflow analyzing samples in a 96-well microtiter plate. The control software on the Raman instrument has been modified to open a text file prepared by the HTC software suite to initiate semiautomated analysis of the samples in a specified well plate. All of the data acquisition and spectral analysis is performed by the Raman instrument including various chemometric techniques for classifying and clustering the samples based on their Raman spectra. The final analytical results and spectra are then formatted and saved for easy entry into the central database (SQL LIMS). An HTC software suite has been developed in-house for the HTC laboratory, which includes routines for display and manipulation of the combined information.

In the sunflower (Helianthus annuus L.) cotyledons, two distinguishable thiolase activities have been identified: acetoacetyl CoA thiolase (AACT), specifically active during oxidation of short chain acetoacetyl CoA, and 3-oxoacyl CoA thiolase (OACT), active towards short, medium, and long-chain acyl CoAs. The purpose of this research was to optimize the purification of the AACT expressed in bacteria. Escherischia coli (E. coli) with the full-length sunflower AACT cDNA cloned into the expression vector pBAD-His B was induced for production of the AACT using 0.2% arabinose. Optimizing the conditions for protein purification is often an extremely empirical process requiring several iterations of a basic protocol each with specific changes. This iterative process was simplified using the programmable Äkta chromatography system from Amersham Pharmacia (now GE Healthcare). Programming this system for automatic sample injection and variation of elution conditions allows for optimization of the protein purification protocol to be achieved overnight as opposed to taking a day for each iteration done manually. Once optimized, soluble AACT was purified from E. coli bacteria in one step using a His HiTrap HP column containing Ni-Sepharose resin in conjunction with the Äkta chromatography system. AACT activity was detected in the induced bacteria, whereas no activity was observed with the uninduced control.

In the modern drug discovery, clinical, and forensic laboratories, there is an ever-increasing requirement for validation of every aspect of laboratory operations. The proper functioning of the liquid handlers, which are at the center of many of these operations, is crucial because a single mistake may lead to missing a compound that is a potentially important drug candidate. Two new instruments have been developed, which overcome this shortcoming and have other attractive features. The Sciclone inLI0 and Sciclone i 1000 liquid handlers incorporate microflowmeters in each dispensing channel giving them the unique ability to validate each and every liquid handling function, pinpointing problems when they happen. The inclusion of the microflowmeters provides closed-loop control over all liquid handling functions and data on all executed functions. Each of the 96 fluidic channels is essentially an independent liquid handling channel meaning that 96 different volumes can be aspirated/ dispensed simultaneously enabling one-step plate normalizations and other variable volume assays. The Sciclone inLIO covers a volume range from 20 nL to 10 μL with fixed cannula whereas the Sciclone i 1000 uses disposable tips across a range from 5 to 1000 μL. Coefficient of variances have been shown to be below 3% at the upper volume range and approach 8% at 20 nL.

A seamless workflow from the medicinal chemist to the screening collections and then to project team assays for new chemical entities has been achieved, by using 1.4-ml 2D bar-coded tubes, an automated tube store system, and a database built in-house. A process of sample collecting, logging, shipping, and sample reformatting has been established with a ComPOUND/ ComMOTION automated tube store system at the heart of the process.

Creating an automated assay team with multiple skills that can support diverse screening responsibilities is a key challenge for drug discovery research sites. Development of ultra high throughput screening (uHTS) screens, development of lead identification and lead optimization assays within medicinal chemistry supported projects, and automation and miniaturization of assays are best performed by a dedicated team of varying backgrounds, experience, and skill sets. This article discusses how integration with therapeutic franchises, regular communication processes, and formal and informal cross-training facilitate the establishment of a streamlined and efficient model at our site for supporting multiple projects at relatively modest financial outlay and recruitment levels.