Thermoplastic nanofluidic devices for identifying abasic sites in single DNA molecules

Swarnagowri Vaidyanathan, Bioengineering Program, The University of Kansas, Lawrence, KS 66045, USA and Center of BioModular Multiscale Systems for Precision Medicine, Lawrence, KS 66047, USA.
Kumuditha M. Weerakoon-Ratnayake, Center of BioModular Multiscale Systems for Precision Medicine, Lawrence, KS 66047, USA and Department of Chemistry, The University of Kansas, Lawrence, KS 66045, USA.
Franklin I. Uba, Department of Chemistry, University of North Carolina at Chapel Hill, NC 27599, USA.
Bo Hu, Department of Biomedical Engineering, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.
David Kaufman, Center of BioModular Multiscale Systems for Precision Medicine, Lawrence, KS 66047, USA and Department of Pathology and Laboratory Medicine, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.
Junseo Choi, Center of BioModular Multiscale Systems for Precision Medicine, Lawrence, KS 66047, USA and Mechanical & Industrial Engineering, Louisiana State University, Baton Rouge, LA 70803, USA.
Sunggook Park, Center of BioModular Multiscale Systems for Precision Medicine, Lawrence, KS 66047, USA and Mechanical & Industrial Engineering, Louisiana State University, Baton Rouge, LA 70803, USA.
Steven A. Soper, Bioengineering Program, The University of Kansas, Lawrence, KS 66045, USA and Center of BioModular Multiscale Systems for Precision Medicine, Lawrence, KS 66047, USA and Department of Chemistry, The University of Kansas, Lawrence, KS 66045, USA and Department of Cancer Biology and KU Cancer Center, The University of Kansas Medical Center, Kansas City, KS 66106, USA. ssoper@ku.edu and Department of Mechanical Engineering, The University of Kansas, Lawrence, KS 66045, USA.

Document Type

Article

Publication Date

4-20-2021

Abstract

DNA damage can take many forms such as double-strand breaks and/or the formation of abasic (apurinic/apyrimidinic; AP) sites. The presence of AP sites can be used to determine therapeutic efficacy of many drugs, such as doxorubicin. While there are different assays to search for DNA damage, they are fraught with limitations, such as the need for large amounts of DNA secured from millions of cells. This is challenging due to the growing importance of using liquid biopsies as a source of biomarkers for many in vitro diagnostic assays. To accommodate the mass limits imposed by the use of liquid biopsies, we report a single-molecule DNA damage assay that uses plastic nanofluidic chips to stretch DNA to near its full contour length when the channel dimensions (width and depth) are near the persistence length (∼50 nm) of double-stranded (ds) DNA. The nanofluidic chip consisted of input funnels for high loading efficiency of single DNA molecules, entropic traps to store the DNA and simultaneously load a series of nanochannels for high throughput processing, and an array of stretching nanochannels to read the AP sites. Single dsDNA molecules, which were labeled with an intercalating dye and a biotinylated aldehyde reactive probe (bARP), could be parked in the stretching nanochannels, where the AP sites were read directly using a dual-color fluorescence microscope equipped with an EMCCD camera. One color of the microscope was used to read the DNA length and the second color detected the AP sites. The nanofluidic chip was made from thermoplastics via nanoimprint lithography, which obviated the need for direct writing the devices in glass or quartz using focused ion beam milling. We show that we can read the frequency of AP sites in single dsDNA molecules with the frequency of AP sites determined by associating fluorescently-labeled streptavidin with bARP through a biotin/streptavidin complex.

Publication Source (Journal or Book title)

Lab on a chip

First Page

1579

Last Page

1589

This document is currently not available here.

Share

COinS