Stroke is the third leading killer in the US and the main cause for over 795,000 cases of adult disability each year. The two major types of stroke, ischemic and hemorrhagic, cannot be clinically differentiated;30% of patients presenting stroke-like symptoms do not have a stroke at all. It is imperative that a stroke diagnosis be made quickly and accurately because ischemic and hemorrhagic strokes require different treatments that have only a small window of time to be effective (3-6 hours). Computed tomography (CT) or magnetic resonance imaging (MRI) is commonly used for the diagnosis, which delays therapeutic intervention. Unfortunately, a molecular diagnostic test does not currently exist for stroke due primarily to the fact that potential molecular marker panels are waiting to be clinically validated. This validation process is, in part, hampered by the tools used for their analysis, such as reverse-transcription PCR (RT-PCR) for nucleic acid-based markers or immunoassays for serum protein markers, due to the lack of sample process automation and the lengthy processing steps used. In this application, a highly innovative, fully-automated system will be developed that has exquisite analytical sensitivity to monitor minute changes in the expression levels of different molecular markers with a turn-around-time (TAT) of less than 15 minutes. The system will facilitate the identification and validation of new molecular markers for the rapid, specific, and sensitive diagnosis of stroke. The molecular assay and novel hardware will be used to evaluate the potential of a messenger RNA marker panel as an unprecedented blood-based test (using peripheral blood mononucleated cells, PBMC) for the diagnosis of stroke. Two technologies will form the core of the system: (1) modular fluidic bio-processor made from polymers via replication that contains all of the sample processing steps;and (2) single pair fluorescence resonance energy transfer (spFRET) that eliminates several sample processing steps. Micro-replication will be used for the fabrication of the bio-processor to keep cost low and make this consumable appropriate for single- use applications, as demanded by clinical diagnostics. The bio-processor will contain a fluidic motherboard with task-specific modules interconnected on it to provide design flexibility. The molecular processing steps poised on the processor will be: (1) PBMC isolation;(2) PBMC lysis;(3) mRNA solid-phase purification;(4) reverse transcription to cDNA;(5) ligase detection reactions (LDRs) to generate molecular beacons with donor/acceptor fluorescent pairs and;(6) spFRET digital detection. The molecular (i.e., digital) counting using spFRET will provide the ability to eliminate several sample pre-processing steps shortening the assay TAT and generate the necessary analytical sensitivity to detect subtle changes in expression levels of mRNA markers, which may be critical during early times following a stroke event. The system can potentially be used for any application requiring mRNA expression profiling, but will be used in the current project for validating molecular markers for the diagnosis of stroke.

Public Health Relevance

Stroke is the third leading killer in the US and causes over 795,000 cases of adult disability each year. Unfortunately, in vitro diagnostic tests for stroke that can be administered to patients for effective and timely treatment are not currently available. The goal of this project is to develop an innovative, discovery-based technology for securing highly quantitative results quickly through full-process automation. The technology will assist in the validation of different molecular markers for stroke to deliver an unprecedented in vitro diagnostic blood-based test.

Agency
National Institute of Health (NIH)
Institute
National Institute of Biomedical Imaging and Bioengineering (NIBIB)
Type
Research Project (R01)
Project #
5R01EB010087-04
Application #
8606462
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Lash, Tiffani Bailey
Project Start
2011-02-15
Project End
2015-01-31
Budget Start
2014-02-01
Budget End
2015-01-31
Support Year
4
Fiscal Year
2014
Total Cost
Indirect Cost
Name
Louisiana State University A&M Col Baton Rouge
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
City
Baton Rouge
State
LA
Country
United States
Zip Code
70803
Lee, Tae Yoon; Han, Kyudong; Barrett, Dwhyte O et al. (2018) Accurate, predictable, repeatable micro-assembly technology for polymer, microfluidic modules. Sens Actuators B Chem 254:1249-1258
Baird, Alison E; Soper, Steven A; Pullagurla, Swathi R et al. (2015) Recent and near-future advances in nucleic acid-based diagnosis of stroke. Expert Rev Mol Diagn 15:665-79
Janket, Sok-Ja; Baird, Alison E; Jones, Judith A et al. (2014) Number of teeth, C-reactive protein, fibrinogen and cardiovascular mortality: a 15-year follow-up study in a Finnish cohort. J Clin Periodontol 41:131-40
Adamski, Mateusz G; Li, Yan; Wagner, Erin et al. (2014) Expression profile based gene clusters for ischemic stroke detection. Genomics 104:163-9
Adamski, Mateusz G; Golenia, Aleksandra; Turaj, Wojciech et al. (2014) The AGTR1 gene A1166C polymorphism as a risk factor and outcome predictor of primary intracerebral and aneurysmal subarachnoid hemorrhages. Neurol Neurochir Pol 48:242-7
Adamski, Mateusz G; Gumann, Patryk; Baird, Alison E (2014) A method for quantitative analysis of standard and high-throughput qPCR expression data based on input sample quantity. PLoS One 9:e103917
Hupert, Mateusz L; Jackson, Joshua M; Wang, Hong et al. (2014) Arrays of High-Aspect Ratio Microchannels for High-Throughput Isolation of Circulating Tumor Cells (CTCs). Microsyst Technol 20:1815-1825
Torphy, Robert J; Tignanelli, Christopher J; Kamande, Joyce W et al. (2014) Circulating tumor cells as a biomarker of response to treatment in patient-derived xenograft mouse models of pancreatic adenocarcinoma. PLoS One 9:e89474
Adamski, Mateusz G; Li, Yan; Wagner, Erin et al. (2014) Pre-existing hypertension dominates ??T cell reduction in human ischemic stroke. PLoS One 9:e97755
Pullagurla, Swathi R; Witek, Ma?gorzata A; Jackson, Joshua M et al. (2014) Parallel affinity-based isolation of leukocyte subsets using microfluidics: application for stroke diagnosis. Anal Chem 86:4058-65

Showing the most recent 10 out of 16 publications