DNA aggregation controlled by super-hydrophobic devices

Since the discovery of the “molecule of life” [1][2], fundamental advances have been made in the characterization of the DNA molecule. Despite the continuous technical achievements, there is still need for improvements toward the elucidation of nucleic acids and proteins structures and their interaction with other analytes at the sub-nanoscale level.

In recent works [3][4][5][6], we propose a new method for the direct imaging and the structural characterization of biomolecules such as nucleic acids, proteins, cell membrane pores, and phospholipid bilayer. The technique relies on the manipulation, concentration, self-organization, and suspension of biomaterials on micro-patterned devices with super-hydrophobic properties. Briefly, the molecules of interest are diluted at a properly tuned concentration in a physiologically compatible buffer, to ensure the best conditions and stability. Afterward, a small drop of the solution 5–10?μl in volume is pipetted on top of the microstructures and is let to evaporate until it is completely dry. The droplet maintains its quasi-spherical shape during all the processes and shows a contact angle higher than 150° with the device due to the super-hydrophobicity of the substrate used. Due to solution evaporation, the droplet reduces its volume, and, while shrinking, its contact line with the substrate surface jumps from one pillar to the neighbor one. In this process, the molecules dispersed in the solution are forced in the same direction. Some of the molecules statistically can link to a pillar head and, following the droplet regression, are extended across the inter-pillar gap. Those that remain dispersed, along with the salts and all other chemicals in the original solution, become more and more concentrated toward the final point of the evaporation in the center of the device. On the other hand, some of the molecules in solution spontaneously self-organize and stretch between and over the micro-pillars top leading to formation of bundles of growing sizes, while the rest concentrate in a final solid residual in the center of the device.

The buffer requirements, biomolecule preparation, micro-structure patterns and dimension, temperature, and humidity necessary throughout all the process, can be fine adjusted time by time on the basis of the moiety studied.

In the case herein reported, the biomolecule investigated is the double-stranded form of the lambda phage DNA, diluted in a saline buffer containing silver ions. The DNA was chosen on the basis of its widely known structural characteristics and due to its length of approximately 50?kb, suitable to cover the pillar–pillar distance with one molecule only. A short thermal ramp to ensure the correct base pairing of the hemi-helices was followed by the deposition of a 5-μl droplet of the DNA solution (concentration of 50?ng/μl) on a super-hydrophobic device. Silicon micro-pillars are distributed in a concentric pattern and are characterized by a regular height of approximately 10?μm, a diameter of 6?μm, and an inter-distance of 12?μm. The image was acquired with a Scanning Electron Microscope (SEM, Quanta 200, FEI) at the Imaging and Characterization Core Labs of the King Abdullah University of Science and Technology (KAUST) working at an acceleration voltage of 3?kV, a current of 21?pA, and a magnification of 688×. It shows the result of droplet evaporation: DNA molecules self-assemble into clearly visible bundles whose diameters vary between a few nanometers to approximately 200?nm in the proximity of the droplet residual. The non-suspended materials accumulate and dry with a final structure unambiguously appreciable in the picture; this occurs in a confined area of approximately 300?μm in diameter.

This approach has been used for the study of biomolecules such as proteins, DNA, and the complex systems of the neuronal cell membrane phospholipid bilayer with its embedded proteins. The characterization techniques that can be applied to such devices and the related suspended molecules span from Raman and Surface-Enhanced Raman (SERS) Spectroscopies [7][8] to electron microscopy.

Similar super-hydrophobic devices have been modified with the fabrication of holes between the micro-pillars. Such devices can be used in Transmission Electron Microscope (TEM) and High-Resolution TEM (HRTEM) to provide a background-free platform for biomolecules direct imaging. In addition, the suspended molecules do not need any additional staining or coating before any measurement. With this approach, we imaged Rad51 protein, cell membranes, and membrane ion channels (K channel, Ca gap junction, and GABAAreceptor) by TEM with a resolution of 3.3?Å [5]DNA has been investigated by HRTEM with an unprecedented resolution of 1.5?Å [3]. For the first time, the DNA bases and the phosphate backbone were resolved and directly measured.

We are confident that in the near future this approach will shed light on several phenomena that are still under debate. The DNA structural characterization will be of fundamental importance in the study and sensing of molecules strongly interacting with DNA bases and backbone. For example, we can mention the cases of heavy metals’ contamination and adducts obtained by the exposure to platinum-based chemotherapeutic agents. In both cases, the double helix undergoes a strong perturbation of its pristine form, due to unwinding, backbone bending, and hydrogen bonds’ disruption [9]. This novel technique will be further extended to proteins studies, especially, in those cases in which the molecule cannot undergo the crystallization processes.

Further reading

[1] R.E. Franklin, R.G. Gosling
Nature, 171 (1953), pp. 740-741

[2] J.D. Watson, F.H. Crick
Nature, 171 (1953), pp. 964-967

[3] M. Marini, et al.
Sci. Adv., 1 (2015)

[4] F. Gentile, et al.
Nano Lett., 12 (2012), pp. 6453-6458
CrossRefView Record in Scopus

[5] M. Marini, et al.
Nanoscale (2017)

[6] M. Marini, et al.
La Riv. Del Nuovo Cim., 40 (2017), pp. 241-277

[7] M. Marini, et al.
Microelectron. Eng., 119 (2014), pp. 151-154

[8] M. Marini, et al.
Microelectron. Eng., 175 (2017), pp. 38-42

[9] A. Eastman
Pharmacol. Ther., 34 (1987), pp. 155-166

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DOI: 10.1016/j.mattod.2018.03.024