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This article describes a paper-based low cost single cell HaloChip assay that can be used to assess drug- and radiation-induced DNA damage at point-of-care. Printing ink on paper effectively blocks fluorescence of paper materials, provides high affinity to charged polyelectrolytes, and prevents penetration of water in paper. After exposure to drug or ionizing radiation, cells are patterned on paper to create discrete and ordered single cell arrays, embedded inside an agarose gel, lysed with alkaline solution to allow damaged DNA fragments to diffuse out of nucleus cores, and form diffusing halos in the gel matrix. After staining DNA with a fluorescent dye, characteristic halos formed around cells, and the level of DNA damage can be quantified by determining sizes of halos and nucleus with an image processing program based on MATLAB. With its low fabrication cost and easy operation, this HaloChip on paper platform will be attractive to rapidly and accurately determine DNA damage for point-of-care evaluation of drug efficacy and radiation condition. Graphical Abstract Single cell HaloChip on paper.

Background: Single-cell gel electrophoresis, or the comet assay, a technique widely used for DNA damage analysis, has been used recently for detecting DNA fragmentation in cells undergoing apoptosis. However, the number of variants of this assay used thus far primarily detected the late stages of DNA fragmentation. Therefore, monitoring the progression of DNA fragmentation, which could greatly improve the analysis of cell death induction and progression at the single-cell level, has not been possible with this assay., Methods: In the present study, a modification of the original neutral comet assay developed by Ostling and Johanson (Biochem Biophys Res Commun 123:291-298, 1984) was used to detect various stages of DNA fragmentation. This assay involves cell lysis with anionic detergents at nearly neutral pH (9.5) and does not include high salt concentration, unlike most other published methods. BMG-1 human glioma cells were induced to undergo programmed cell death by treating with a large dose (100 microM) of etoposide, and comets were prepared after different durations (1-24 h) of treatment., Results: In contrast to results of previously published studies, comets with different shapes reflecting progressive stages of DNA fragmentation were observed. Of these, six distinct shapes were identified and divided into three different categories based on the extent of fragmentation. Type A comets had a large head separated by a narrow "neck" region from an oval bulging tail that indicated initiation of fragmentation. Type B and C comets had a constantly diminishing head associated with a corresponding expansion of the tail and reflected intermediate and late stages of fragmentation, respectively. Type A and B comets appeared at a high frequency during early time points (1-6 h), whereas type C comets that indicated late stages of fragmentation were observed only after extended treatment (24 h). As a result, an elaborate kinetics of the progression of DNA fragmentation could be obtained., Conclusion: The present single-cell gel electrophoresis assay offers a significant improvement in monitoring the kinetics of DNA fragmentation induced during programmed cell death. Coupled with its simplicity and the ability to detect responses of small cell subpopulations, this method can be used for a reliable and sensitive analysis of the progression of cell death in different cell types and treatment conditions., (Copyright 2004 Wiley-Liss, Inc.)