A microfluidic-based screening method will be developed to differentiate different disease states of cancer. The screening method is based on high-resolution microchip electrophoresis coupled with laser-induced fluorescence detection. N-Glycan profiles are generated for all samples and compared by statistical analysis. The method relies on the analysis of the entire N-glycan profile, not a single biomarker, to provide sufficien differentiation among control individuals, patients suffering from various stages of cancer, and patients with pre-malignant diseases. Microchip electrophoresis with fluorescence detection provides excellent resolution of glycan structures and their isomers and has tremendous detection sensitivity. Preliminary results suggest that separation channels 20 cm in length and separation field strengths 750 V/cm are able to rapidly and efficiently separate N-glycans derived from clinically relevant serum samples, e.g., from patients with breast cancer, ovarian cancer, and esophageal adenocarcinoma. Efficient separation and sensitive detection on capillary- or chip-based instruments permit both low and high abundance N-glycans to contribute to the statistical analysis for disease-state differentiation. In this application, microfluidic devices will be used as a convenient and reliable platform to screen N-glycans associated with cancer. As microfluidic-based methods are optimized, those N-glycans that contribute most to disease-state differentiation will be identified. Determination of these molecular structures will enable optimization and validation of the screening method through improvements to electrophoretic analysis and detection sensitivity.
The specific aims of this application are to: (1) demonstrate that microchip electrophoresis of N- glycans is a rapid, reliable screening method for various cancers, (2) develop a suite of N-glycan standards to be used as sizing ladders for standard addition in microchip electrophoresis, (3) determine N-glycan structures by mass-spectrometric analysis, and (4) integrate monolithic phases into the microfluidic devices for dialysis, solid-phase extraction, electrochromatography, and enzymatic sequencing.

Public Health Relevance

We will develop a microfluidic-based screening method to differentiate disease states of cancer. Samples are obtained non-invasively from the patients by blood draw, and a glycan profile, not a single biomarker, is used to differentiate between diseased and disease-free samples. The long-term goal of this research is to develop instrumentation that detects the onset and monitors the progression of cancer and related diseases.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM106084-02
Application #
8848840
Study Section
Enabling Bioanalytical and Imaging Technologies Study Section (EBIT)
Program Officer
Sheeley, Douglas
Project Start
2014-05-15
Project End
2018-02-28
Budget Start
2015-03-01
Budget End
2016-02-29
Support Year
2
Fiscal Year
2015
Total Cost
$291,633
Indirect Cost
$91,633
Name
Indiana University Bloomington
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
006046700
City
Bloomington
State
IN
Country
United States
Zip Code
47401
Benktander, John D; Gizaw, Solomon T; Gaunitz, Stefan et al. (2018) Analytical Scheme Leading to Integrated High-Sensitivity Profiling of Glycosphingolipids Together with N- and O-Glycans from One Sample. J Am Soc Mass Spectrom :
Snyder, Christa M; Zhou, Xiaomei; Karty, Jonathan A et al. (2017) Capillary electrophoresis-mass spectrometry for direct structural identification of serum N-glycans. J Chromatogr A 1523:127-139
Novotny, Milos V (2017) Development of capillary liquid chromatography: A personal perspective. J Chromatogr A 1523:3-16
Zou, Guozhang; Benktander, John D; Gizaw, Solomon T et al. (2017) Comprehensive Analytical Approach toward Glycomic Characterization and Profiling in Urinary Exosomes. Anal Chem 89:5364-5372
Gaunitz, Stefan; Nagy, Gabe; Pohl, Nicola L B et al. (2017) Recent Advances in the Analysis of Complex Glycoproteins. Anal Chem 89:389-413
Snyder, Christa M; Alley Jr, William R; Campos, Margit I et al. (2016) Complementary Glycomic Analyses of Sera Derived from Colorectal Cancer Patients by MALDI-TOF-MS and Microchip Electrophoresis. Anal Chem 88:9597-9605
Mitra, Indranil; Snyder, Christa M; Zhou, Xiaomei et al. (2016) Structural Characterization of Serum N-Glycans by Methylamidation, Fluorescent Labeling, and Analysis by Microchip Electrophoresis. Anal Chem 88:8965-71