Studies of Polythymine Conformational Changes with Electrochemical Microdevices
Authors: Vishnubhotla Ramya , Montgomery Christopher , Steffens Kristen , Semancik Steve 
Published: July 2021

Introduction

Electrochemical biosensors can offer rapid, scalable, and inexpensive biological and chemical detection compared to more traditional measurement techniques. These devices can sense the presence of drugs for therapeutic drug monitoring or detect biomarkers for early disease diagnostics. DNA, which is frequently used as a recognition element for a biomarker, can exist in single-stranded or double-stranded form, or as an aptamer: a series of oligonucleotides selected to bind to a specific biomolecular species or chemical. Aptamers can be selected for different targets, allowing aptamer-functionalized sensors to detect biomarkers such as varied nucleic acids or proteins1. However, to ensure reliability for aptamer-functionalized sensors, more needs to be understood about the general nature of DNA’s behavior under a variety of chemical and physical conditions. This study employs a microscale electrochemical platform (Fig. 1a) to explore the conformational behavior of immobilized polythymine (polyT) strands of differing lengths and their responses to various physical and chemical stressors (Fig. 1b), as the influence of these factors can have an effect on target binding.

Methods

The commercially-purchased polyT strands consisting of 10, 20 and 50 thymines (10-mer, 20-mer and 50-mer, respectively), with a thiol on one end and tagged with a methylene blue (MB) moiety at the other (distal) end, were incubated in a TCEP solution to reduce the thiol, and then pipetted onto the electrochemical microdevice to immobilize the DNA on the Au working electrode. Next, a self-assembled monolayer solution of 6-mercaptohexanol was pipetted onto the working electrode and left to incubate for one hour in the dark (as a means of preventing nonspecific binding). After completion of this step, the devices were ready for electrochemical sensing. DNA-functionalized surfaces of the 20-mer were also characterized with x-ray photoemission spectroscopy (XPS) to determine packing density.
The reported studies involved custom temperature-controlled electrochemical microdevices with built-in platinum resistive thermometers (PRT) which are used in conjunction with a commercial Peltier unit2. All sensing was done in phosphate buffer saline (PBS) working buffer, which includes varying concentrations of NaCl at a biologically-relevant pH of 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. The PRT was monitored to provide the approximate temperature of the electrochemical interface (10 ⁰C to 50 ⁰C), which was cooled and heated under voltage control of the Peltier module.

Results

Conformational changes occurring for the immobilized polyT strands under the influence of varied chemical and thermal environments were signaled by current changes measured using square-wave voltammetry (SWV). Generally, the currents are inversely proportional to the distance between the MB and working electrode. Figure 2 shows thermal profiles (current vs temperature) reflective of varying polyT conformations that were measured for 20-mer polyT at seven different working buffer salt concentrations. Similar plots were acquired for the shorter 10-mer polyT as well as the longer 50-mer polyT, and all of those data were utilized to produce the results in Figure 3, representing how the temperature of maximum current in the range of 10 ⁰C to 50 ⁰C depends on strand length and salt concentration.

Conclusion

Observations made in these electrochemical studies of the polythymine strands indicated that the conformational behavior of the model system was actually rather complex, with dependencies on the polyT length as well as on the thermal and chemical stressors. The conformational responses can be explained primarily by salt-induced conversion of rigidity to flexibility and thermally driven motion. However, other factors such as strand stretching and overcharging effects also appear to be necessary to understand the acquired profiles. These findings suggest that care must be taken for certain cases of DNA- and aptamer-based electrochemical biosensing, as the functioning of receptors can be quite sensitive to even small changes in the operating conditions.

References

[1] White, R. J. & Plaxco, K. W. Engineering New Aptamer Geometries for Electrochemical Aptamer-Based Sensors. Proc SPIE Int Soc Opt Eng
732105, 820419 (2009).
[2] Robinson, S. M. et al. Ligand-Based Stability Changes in Duplex DNA Measured with a Microscale Electrochemical Platform. Biosensors (Basel)
9, 54, (2019).

Figure 1