Complex Sample Analysis
A biotechnology research and development effort to modify the chemical content of wood in commercially growth tree species required a rapid and quantitative method for evaluation of several chemical metabolites. This R&D pipeline is part of a product development process to identify genes that will lead to improved industrial processing of the resulting trees in chemical properties, growth rate, and overall tree form.
The assay specs required that accurate results be obtained from very small amounts of tissue and be capable of analysis of hundreds of samples within a given budget and time frame. A rapid turn-around time was required for results in order to facilitate selection of superior candidates from the test set for a next round of testing that needed to be initiated within tight time constraints.
No existing technology was present that could be readily integrated into the R&D process that would meet the specs for timely and inexpensive analysis of the desired metabolites. The project started with evaluation of the existing technologies for chemical metabolite testing with consideration given for the sensitivity of detection for a wide range of substances, amount of starting material required, and the cost per sample. Techniques considered included standard wet chemistry methods, near infrared spectroscopy, Raman spectroscopy, high performance liquid chromatography, gas chromatography, mass spectroscopy, and pyrolysis molecular beam mass spectroscopy (pyMBMS).
The technique best meeting the specifications was pyMBMS but there were no established techniques for accurate detection of the desired metabolites with very small sample sizes. Desired metabolites included precursor molecules for the major components of wood: lignin, cellulose, and hemicellulose. A recognized expert in pyMBMS analysis was located at the National Renewable Energy Laboratory (NREL) Golden, CO, USA and a collaborative research project was entered to develop an effective high throughput process for chemical metabolite analysis that could be seamlessly integrated into the existing R&D process using only small amounts of sample tissue.
The project required coordination of two different research groups in different physical locations. One group was devoted to wood sample collection and handling, interpretation of the results from the pyMBMS analysis, and integration of the analysis into the appropriate point in the research and development process. The second group streamlined the pyMBMS techniques and performed the analysis according to a set a strict target guidelines.
The project was successful in developing a routine method for detection of metabolites. This process was used to map the distribution of lignin within poplar trees as described in Sykes et al. 2008 (Wood Science and Technology) and is now be routinely used in-house and as service for hire. The improved process is capable of using milligram quantities of wood obtained at several key stages of the R&D process. Turn-around time for the analysis is within two to three weeks and is sufficient to allow rapid decisions for selection of materials that meet specifications for improved chemical profile. The new process can detect variants in a number of different chemical precursors of lignin, cellulose, and hemicelluloses that are indicative of changes in the overall quantity of these wood components and can also be used to identify unique structural variants in lignin structure. This technique is especially suited for rapid analysis of large numbers of milligram-quantity samples for screening studies.
The significance of the modified lignin, cellulose, and hemicelluloses is that these compounds affect the yield of wood pulp in papermaking operations and in conversion of wood to ethanol. These chemical modifications are also important in designing feedstock for improved digestion of grasses in grazing livestock and in carbon sequestration to combat global warming.
Other relevant experience in complex sample analysis include :
Developing techniques for isolation and analysis of DNA, RNA and protein from biological tissues and their subsequent analysis using a variety of molecular biology methods such as microarray analysis, polymerase chain reaction PCR, DNA and RNA blotting, polyacrylamide gel electrophoresis, liquid chromatography, and spectroscopy. These analysis methods were directed toward tissues that are considered difficult to work with because of the high concentrations of carbohydrates and/or phenolic compounds. A special application for these techniques was the ability to obtain workable amounts of target molecules from very small amounts of starting material in order to avoid sacrificing the organism where possible.
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