Conformational Changes of Immobilized Polythymine due to External Stressors Studied with Temperature-Controlled Electrochemical Microdevices

Abstract

Conformational changes of single-stranded DNA (ssDNA) play an important role in a DNA strand's ability to bind to target ligands. A variety of factors can influence conformation, including temperature, ionic strength, pH, buffer cation valency, strand length, and sequence. To better understand the effects of these factors on immobilized DNA structures, we employ temperature-controlled electrochemical microsensors to study the effects of salt concentration and temperature variation on the conformation and motion of polythymine (polyT) strands of varying lengths (10, 20, 50 nucleotides). PolyT strands were tethered to a gold working electrode at the proximal end through a thiol linker via covalent bonding between the Au electrode and sulfur link, which can tend to decompose between a temperature range of 60 and 90 °C. The strands were also modified with an electrochemically active methylene blue (MB) moiety at the distal end. Electron transfer (eT) was measured by square wave voltammetry (SWV) and used to infer information pertaining to the average distance between the MB and the working electrode. We observe changes in DNA flexibility due to varying ionic strength, while the effects of increased DNA thermal motion are tracked for elevated temperatures. This work elucidates the behavior of ssDNA in the presence of a phosphate-buffered saline at NaCl concentrations ranging from 20 to 1000 mmol/L through a temperature range of 10-50 °C in 1° increments, well below the decomposition temperature range. The results lay the groundwork for studies on more complex DNA strands in conjunction with different chemical and physical conditions.

Figure 1

(a) Microscope image of an electrochemical device as seen from above, with an embedded Pt serpentine PRT insulated from a Pt counter electrode (CE), Pt quasi-reference electrode (RE), and a central Au working electrode (WE); (b) cross-sectional schematic of a DNA polyT strand bound to the gold working electrode of the device through thiol-linking and including a methylene blue moiety at the distal end.

Figure 2

Top image (left) and side schematic (right) of the device mounted with epoxy on a PCB and a Peltier unit underneath to heat/cool the EC sensing interface. A poly(dimethylsiloxane) (PDMS) well and cover on top of the device is used to contain the sample and prevent evaporation. The drawing on the right (not to scale) shows a representative schematic of the device configuration.

Figure 3

Factors affecting the measured current level (a) rigidity of the strand, wherein the moiety is far from the Au electrode, represented as d^rig; (b) minimal rigidity/maximum flexibility of the strand, bringing the moiety closer to Au, and represented as d^flex; (c) stretching of the strand (high rigidity), moving the MB farther away, represented as d^str; (d) thermal motion of the strand, shown here as bringing the average position for the MB closer to the Au in multiple directions, represented as d_avg; The current in all cases is influenced by 1/d, where d is the distance between the moiety and the Au electrode. We suggest that the profile for a strand’s flexibility due to ionic concentration can range from rigid to moderately flexible, to overcharged, the last of which is extremely rigid. The profile for a strand’s thermal motion due to heating can range from minimal motion to moderate motion, to stretching, the last of which also results in minimal motion.

Figure 4

(a) Peak current versus temperature profile of a single run of 10-mer polyT in varying salt concentrations; (b) schematics of effects that may explain the behavior of 10-mers as a function of both temperature and ion concentration. The electron transfer (eT) level descriptors (low to high) give qualitative comparative magnitudes for the measurements of this strand under different conditions.

Figure 5

(a) Peak current versus temperature profile of a single run of 20-mer polyT in varying salt concentrations; (b) schematics of effects that may explain the behavior of 20-mers as a function of both temperature and ion concentration. The electron transfer (eT) level descriptors (low, moderate, high) give qualitative comparative magnitudes for the measurements of this strand under different conditions.

Figure 6

(a) Peak current versus temperature profile of a single run of 50-mer polyT in varying salt concentrations; (b) schematics of effects that may explain the behavior of 50-mers as a function of both temperature and ion concentration. The electron transfer (eT) level descriptors (low, moderate, high) give qualitative comparative magnitudes for the measurements of this strand under different conditions.

Figure 7

Trends for the temperature of maximum observed current (within the studied range of 10–50 °C) versus salt concentration for 10-mer polyT (red squares), 20-mer polyT (green circles), and 50-mer polyT (blue triangles). The plots shown here were obtained using data from three separate trial runs (beginning with Piranha and CV cleaning, followed by DNA attachment, and SAM attachment) at each salt concentration and for each strand length and show an increase in temperature of maximum current as the strand length increases. Error bars are the standard error of the mean for a population with a 95% confidence interval.

Figure 8

Peak current versus NaCl concentration for all three polyT strand lengths (left, middle, and right) plotted for fixed temperatures, taken from the data in Figures 4–6. In each case, the hypothesized effect that dominates the observed trend is indicated: (a) 10-mer at 10 °C, dominated by overcharging from excess cations; (b) 10-mer at 50 °C, still dominated by overcharging from excess cations; (c) 20-mer at 10 °C dominated by increased flexibility; (d) 20-mer at 50 °C dominated by thermal motion of the strand; (e) 50-mer at 10 °C dominated by flexibility; and (f) 50-mer at 50 °C, dominated by thermal motion.