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Multi-technique Characterization of DNA-Modified Surfaces for Biosensing and Diagnostic Applications

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Surface Analysis and Techniques in Biology

Abstract

Complementary surface analysis techniques—X-ray photoelectron spectroscopy (XPS), near-edge X-ray absorption fine structure (NEXAFS), time-of-flight secondary ion mass spectrometry (ToF-SIMS), and surface plasmon resonance (SPR)—were combined to characterize the structure and composition of DNA-modified surfaces. Both model systems [thiolated single-stranded DNA (ssDNA) on gold surfaces] and commercial systems (ssDNA spotted onto microarray slides) were investigated. Pure thiolated 20-mer ssDNA assembles onto gold, with the ssDNA binding spontaneously to the surface via the thiol groups and nitrogen atoms in the DNA bases, resulting in a monolayer with limited ssDNA chain order. XPS, NEXAFS, SPR, and radiotracer studies showed that when the pure ssDNA monolayers were exposed to short-chain functionalized alkyl thiol diluents [either 11-mercapto-1-undecanol (MCU) or oligo(ethylene glycol) (OEG)], the diluents initially displaced the weaker gold–nitrogen DNA interactions, reorienting the ssDNA chains to a more upright configuration. After longer exposures to diluent thiols, some ssDNA chains were displaced from the gold surface. The efficiency of the target hybridization to complementary DNA from solution depended on the structure and composition of the immobilized probe ssDNA surface. As the upright orientation of the ssDNA chains increased, the amount of hybridization increased. As ssDNA chains were displaced from the surface, the amount of hybridization decreased. Incorporating the diluent thiol eliminated the small amount of nonspecific binding from noncomplementary target DNA observed on the pure ssDNA monolayers. The DNA hybridization kinetics were significantly more rapid on the mixed ssDNA/MCU and ssDNA/OEG surfaces compared to the pure ssDNA surface. The OEG diluent was more effective than the MCU diluent at reducing nonspecific protein adsorption during DNA hybridization from blood serum. Nanoliter drops of amine-terminated ssDNA were robotically spotted onto a glass microscope slide with an amine-reactive microarray polymer coating; the resulting 150-μm spots were imaged with XPS and ToF-SIMS. Imaging XPS provided single-spot phosphorous, nitrogen, sodium, and silicon elemental images. Small-spot XPS data were then used to quantify DNA hybridization efficiencies in each microspot as a function of the ssDNA concentration in the probe printing solution. The DNA microspots were also readily visualized in the negative ToF-SIMS images of key fragments from the DNA backbone (e.g., PO x ), DNA bases (A–H, T–H, G–H, C–H, etc.), and the substrate (e.g., Si). Principal component analysis (PCA) of the ToF-SIMS images was used to distinguish heterogeneities within the DNA microspots due to the variations in printing process and solution additives (salts, sodium dodecyl sulfate, etc.). The different types of data available from combining these complementary surface analytical methods provide new information essential to understanding aspects of DNA on surfaces. Such information is important for designing and improving new technologies that employ nucleic acids on surfaces, including bioassays, diagnostics, molecular computing, self-assembling materials, and miniaturized separations.

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Acknowledgments

We gratefully acknowledge support from NESAC/BIO (NIH grant no. EB-002027) and NIH grant no. EB-001473. NEXAFS studies were performed at the NSLS, Brookhaven National Laboratory, which is supported by the U.S. Department of Energy, Division of Materials Science and Division of Chemical Sciences. Dr. D. A. Fischer is thanked for his expert technical assistance with the NEXAFS experiments. Dr. D. Graham is thanked for assistance with PCA. We also thank Dr. S. Golledge for expert technical assistance with the ToF-SIMS experiments performed at the Center for Advanced Materials Characterization in Oregon.

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Authors and Affiliations

  1. National ESCA and Surface Analysis Center for Biomedical Problems, University of Washington, 351653, Seattle, WA, 98195-1653, USA

    Chi-Ying Lee, Lara J. Gamble & David G. Castner

  2. Department of Chemical Engineering, University of Washington, 351653, Seattle, WA, 98195-1653, USA

    Chi-Ying Lee & David G. Castner

  3. 3M Health Care Business Group, Infection Prevention Division, 3M China R&D Center, 222 Tian Lin Road, Shanghai, 200233, China

    Chi-Ying Lee

  4. Department of Bioengineering, University of Washington, 351653, Seattle, WA, 98195-1653, USA

    Lara J. Gamble & David G. Castner

  5. Replenish, Inc., 2645 Nina St., Pasadena, CA, 91107, USA

    Gregory M. Harbers

  6. Department of Pharmaceutics and Pharmaceutical Chemistry, University of Utah, Salt Lake City, UT, 84112-5820, USA

    Gregory M. Harbers, Ping Gong & David W. Grainger

  7. Seventh Sense Biosystems, 286 Cardinal Medeiros Ave, Cambridge, MA, 02141, USA

    Ping Gong

  8. Department of Bioengineering, University of Utah, Salt Lake City, UT, 84112-5820, USA

    David W. Grainger

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  1. Chi-Ying Lee
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  1. Nanostructures and Surfaces Laboratory GE Global Research, Niskayuna, New York, USA

    Vincent S. Smentkowski

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Lee, CY., Gamble, L.J., Harbers, G.M., Gong, P., Grainger, D.W., Castner, D.G. (2014). Multi-technique Characterization of DNA-Modified Surfaces for Biosensing and Diagnostic Applications. In: Smentkowski, V. (eds) Surface Analysis and Techniques in Biology. Springer, Cham. https://doi.org/10.1007/978-3-319-01360-2_11

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