Effects of Physical and Chemical Stressors on DNA Conformation Investigated By Temperature-Controlled Electrochemical Microdevices.

Abstract

Introduction Biosensors offer rapid, scalable, and inexpensive biological/chemical measurements compared to some more traditional detection techniques. For example, electrochemical biosensors can sense the presence of drugs for therapeutic drug monitoring or biomarkers for early disease diagnostics. DNA, which is frequently used as a strong recognition element for biomarkers, can exist in single-stranded or double-stranded form, or as an aptamer: a series of oligonucleotides selected to bind to a specific target, so that aptamer-functionalized sensors can detect biomarkers such as varied nucleic acids or proteins [1]. However, because aptamers are complex, and each aptamer has unique properties, more needs to be understood about the general nature of their behavior in varied electrochemical environments to ensure reliability for aptamer-functionalized sensors for disease diagnostics (including at the point-of-care). In model studies, a microscale electrochemical platform and several characterization methods have been employed to explore the fundamental behavior of a simple polythymine (poly-T) strand and two aptamers (PDGF, StrepApt2) immobilized on Au electrodes, and their response to various physical and chemical stressors. Surface Functionalization and Characterization The poly-T strand, functionalized with a thiol at the 5’ end to bind to the Au, and a methylene blue (MB) tag at the 3’ end, was incubated in a reducing agent (TCEP) to reduce the thiol group. Next, the strand was diluted in a PBS solution and pipetted onto the electrochemical microdevice to allow the now-reduced thiol group to bind to the Au working electrode. Then, a self-assembled monolayer (6-mercaptohexanol) was pipetted to prevent non-specific adsorption of the DNA to the Au electrode. After the completion of this step, the devices were ready for electrochemical monitoring. In addition, DNA-functionalized surfaces were characterized with x-ray photoemission spectroscopy (XPS) to determine packing density and atomic force microscopy (AFM) to determine surface roughness. Devices and Experimental Setup The reported studies use custom temperature-controlled electrochemical microdevices [2] with a built-in platinum resistance thermometer (PRT) and which are attached to a commercially purchased Peltier unit (Fig.1). Sensing was done in different working buffers, which included varying concentrations/types of salt as well as varying pH in the buffering range of 5.8-7.4. During electrochemical sensing, a PDMS well was placed atop the electrodes, and ~10 µL of the working buffer solution was pipetted into the well and covered to prevent evaporation due to temperature variation. The PRT is used in regulating the temperature of the Peltier, which heats and cools (from 10 ⁰C to 60 ⁰C) to alter the temperature of the immobilized species, and thereby affect their folding and conformation. Physical Environment and DNA Variations We have used our devices to study the effects of physical environment and length on a system with single-stranded polythymine. Our aim is to understand the ability of electrons to transfer between a methylene blue (MB) redox label and the Au working electrode of the device as a function of how different stressors alter conformation. Variations in and effects from temperature, working buffer salt concentration, working buffer pH, and length of the DNA strand are monitored, first, for poly-T ssDNA with a thiol linker at the 5’ end and a methylene blue redox reporter tag at the 3’. Additionally, a second type of poly-T strand was studied with both a thiol and an anthraquinone (AQ) reference tag at the 5’ end, and a MB tag at the 3’ end. Each tag offers a means to track the conformation of the poly-T strand via current measurements at characteristic potentials, V, that depend on the tag-Au distance. The surface in both the single-tag and double-tag instances was backfilled with 6-mercaptohexanol. Sensing was carried out through square wave voltammetry and analyzed by plotting the peak current versus temperature. Results and Conclusions Currents at the tag-feature potentials for MB and AQ are recorded to provide information reflecting on the distances between the tags and the Au electrode. In this way, the electrochemical micro-platform is used to monitor conformational changes of the immobilized poly-T, as well as inform on other phenomena, such as fragmentation or desorption. A collection of current profiles was obtained for poly-T under different buffer conditions (salt and pH variations). The single-stranded poly-T with a MB redox label exhibits a high current for low temperature and a low current for high temperature, regardless of pH, if it is within the working range of the phosphate buffer solution (5.8-7.4), as seen in Fig 2a.Similar methods were then extended towards studying more complex DNA, such as streptavidin aptamer and platelet-derived growth factor aptamers. These two aptamers, in their folded state, are in a stem-loop type configuration, and exhibit a parabolic curve when the working buffer is PBS (250 mM NaCl), and sigmoidal behavior when the working buffer is Tris HCl (100 mM NaCl, 1mM CaCl2, 2mM MgCl2, 5mM KCl). Results were collected for the streptavidin aptamer (StrepApt2) on its own, along with the aptamer binding to streptavidin, as seen in Fig 2b.Such studies can serve as an initial, proof-of-concept step towards eventual disease diagnostics at the point of care by elucidating the true nature of DNA’s behavior under various stressors.