Circulating cell-free DNA have been found to freely exist in human body fluids such as blood, urine, and stool. These rare nucleic acids contain a bevy of genetic and epigenetic biomarkers that can be used to reveal hidden pathologies. In cancer, cell-free DNA can be used to determine the status of remote tumors through detecting tumor-associated molecular alterations, such as point mutations and DNA methylation, with great promise for non-invasive monitoring of tumor dynamics, therapeutic response, and disease progression. Since cell-free DNA are present at very low physiological concentrations, PCR-based methodologies, such as mutation allele specific amplification (MASA) and methylation-specific PCR (MSP), are current mainstream methods for their analysis. Unfortunately, the throughput and multiplexing of these methods have been limited. Given that substantial heterogeneity in molecular alterations exists among cancers, analysis of a panel of biomarkers is needed to determine the tissue type and malignant transformation. However, efforts to multiplex PCR have been hampered by issues including mispriming and limited availability of colored florescent dyes. Employing a large number of separate PCRs for each sample is costly and problematic as it requires large amounts of DNA where DNA is the limiting factor for cell-free DNA analysis. We seek to develop a microfluidic single-molecule detection platform to address the unmet need for multiplexed and high-throughput analysis of cell-free DNA biomarkers. Our platform takes advantage of the high sensitivity of single-molecule spectroscopy to enable direct analysis of low-concentration DNA without reliance on PCR.
It aims to achieve highly multiplexed detection of e 48 biomarkers in a single measurement via the use of a molecular coding approach to generate biomarker-specific fluoro-codes that are subsequently decoded by multi-color, single-molecule spectroscopy. In addition, the platform incorporates a microfluidic device for parallel target concentration and arrayed detection, facilitating high-throughput measurements of 50 samples on a chip. The capability of the platform is exemplified by detection of both genetic and epigenetic cancer biomarkers, including point mutations and promoter DNA methylation in serum, sputum and stool samples, collected from patients with ovarian, lung and colon cancer.

Public Health Relevance

The goal of the project is to develop a microfluidic single-molecule detection platform for analysis of cell-free DNA biomarkers in body fluids. This new platform will allow multiplexed detection of e 48 biomarkers in a DNA sample and parallel measurements of 50 samples per chip, unattainable by the current mainstream, PCR-based methods for cell-free DNA detection.

National Institute of Health (NIH)
National Cancer Institute (NCI)
Research Project (R01)
Project #
Application #
Study Section
Instrumentation and Systems Development Study Section (ISD)
Program Officer
Tricoli, James
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
Johns Hopkins University
Engineering (All Types)
Schools of Engineering
United States
Zip Code
Pisanic 2nd, Thomas R; Athamanolap, Pornpat; Wang, Tza-Huei (2017) Defining, distinguishing and detecting the contribution of heterogeneous methylation to cancer heterogeneity. Semin Cell Dev Biol 64:5-17
Friedrich, Sarah M; Burke, Jeffrey M; Liu, Kelvin J et al. (2017) Molecular rheotaxis directs DNA migration and concentration against a pressure-driven flow. Nat Commun 8:1213
Friedrich, Sarah M; Zec, Helena C; Wang, Tza-Huei (2016) Analysis of single nucleic acid molecules in micro- and nano-fluidics. Lab Chip 16:790-811
Stark, Alejandro; Shin, Dong Jin; Pisanic 2nd, Thomas et al. (2016) A parallelized microfluidic DNA bisulfite conversion module for streamlined methylation analysis. Biomed Microdevices 18:5
Zhang, Ye; Zhang, Yi; Burke, Jeffrey M et al. (2016) A Simple Thermoplastic Substrate Containing Hierarchical Silica Lamellae for High-Molecular-Weight DNA Extraction. Adv Mater 28:10630-10636
Friedrich, Sarah M; Liu, Kelvin J; Wang, Tza-Huei (2016) Single Molecule Hydrodynamic Separation Allows Sensitive and Quantitative Analysis of DNA Conformation and Binding Interactions in Free Solution. J Am Chem Soc 138:319-27
Pisanic 2nd, Thomas R; Athamanolap, Pornpat; Poh, Weijie et al. (2015) DREAMing: a simple and ultrasensitive method for assessing intratumor epigenetic heterogeneity directly from liquid biopsies. Nucleic Acids Res 43:e154
Zhang, Yi; Wang, Tza-Huei (2015) High-resolution quantification by charge-dominant electrophoretic mobility shift of quantum dots. Electrophoresis 36:1011-5
Rane, Tushar D; Chen, Liben; Zec, Helena C et al. (2015) Microfluidic continuous flow digital loop-mediated isothermal amplification (LAMP). Lab Chip 15:776-82
Rane, Tushar D; Zec, Helena C; Wang, Tza-Huei (2015) A barcode-free combinatorial screening platform for matrix metalloproteinase screening. Anal Chem 87:1950-6

Showing the most recent 10 out of 32 publications