We are pursuing technology developments in the following areas in support of our biodosimetry work.
"Front-End" Sample Collection
We are developing front-end sample-collection / pre-processing technologies in support of all three projects. These are well established for low-throughput assays but currently, represent significant bottlenecks to the use of our corresponding high-throughput biodosimetric or predictive assays. While commercial high-throughput "back-end" screening hardware technologies are increasingly available in university, industry, and clinical testing labs, without efficient front-end interfaces linking these devices to sample collection and pre-processing, they will not be useful for high-throughput application after a large-scale radiological event. Blood collection represents a significant bottleneck for high throughput biodosimetry. To address this neglected need, we are developing a self-contained, self-administered blood collection cartridge to enable sample collection for all projects.
A) The disassembled three-part blood collector system showing internal components B) The blood collector system assembled held in one hand as for use.
We are also developing a sample collection device integrating a fabric-based test card suitable for high throughput MS. Using new technology, Fabric Phase Sorptive Extraction (FPSE) for preprocessing, we have been able to analyze radiation metabolites directly from whole blood without the need for protein precipitation or other pre-extraction sample manipulation prior to direct injection of the sample to the analytical instrument.
For sample collection and shipment in support of Project 1, we are developing a smart transportation system. This is essentially a shipping box that uses control and heating microcontroller boards to maintain a 37oC environment to allow sample culture during shipment, thus saving valuable incubation time for cytogenetic assays.
Assay Development for Commercial High-Throughput Cell Screening
In Project 1 we will continue development of the high-throughput "RABiT2" approach; here we will optimize biodosimetry assays/protocols that can be directly used in commercial robotically-based high-content cell-screening platforms - which are now increasingly available in many settings, nationwide.
Variable Dose Rate External Irradiator (VADER)
Development of a variable-dose-rate external 137Cs irradiator (VADER) to simulate exposure to 137Cs as an internal emitter. In large-scale radiation exposure scenarios, internal exposure to 137Cs is often a major source of radiation exposure. We are building a programmable low-dose-rate external 137Cs irradiator that can simulate the kinetics of 137Cs exposures corresponding to those from internal 137Cs (Figure). The design is based on “recycling” of old 137Cs brachytherapy seeds that are mounted above and below a custom mouse cage. The seed assemblies can be moved away from the mice at a speed that produces the same bio-kinetic dose-rate profile as internal 137Cs. This irradiator will dramatically increase the practicality and cost-effectiveness of 137Cs-based internal emitter research throughout our (and other) CMCR programs. In addition, used in static mode, the system will also provide a range of extremely low dose rates.
Sample Pre-Processing Development
Our technology development aim is focused on automation of sample pre-processing for mass spectrometry-based metabolomics. This will allow for integration of sample pre-processing platform into field-deployable sample collection devices. Currently, metabolomics sample pre-processing is time-consuming and prone to human error due to sample handling procedures. Our goal is to take advantage of fabric phase solid extraction (FPSE) techniques, which are compatible with downstream ultra-performance liquid chromatography-time of flight mass spectrometry (UPLC-ToFMS) metabolomics. To this end, we have tested different fabric phase extraction protocols in collaboration with the Sample Engineering Core to capture a full metabolomic and lipidomic profile in blood. Our initial experiment identified a material, which is efficient in extracting polar metabolites and lipid species from different sample types; whole blood, serum, and diluted serum. Current efforts are focused on refining our protocols to optimize extraction efficiency while using just 50 uL of the sample each time.