Surfing the Nano Wave: Dramatic Leap in DNA Sensing with Surfactant-Coated Nanopores

Significance 

Solid-state nanopores (ssNPs) are nanoscopic apertures, fabricated in ultrathin solid membranes made of silicon or graphene. These devices can be fabricated with an adjustable size, enabling the selective passage of individual molecules one at a time. This capability makes them useful in various technological applications, particularly in biosensing, where ssNPs can detect and analyze individual molecules, such as DNA, RNA, proteins, or other biological molecules. Nanopore plays a crucial role in high-precision biomedical research, diagnostics and emerging single proteins molecule sensing applications. The underlying working principle of these nano devices involves time-dependent measurement of the ion flux through the nanopore during the electro-kinetic passage of bio-analyte molecules. For example, when a single DNA strand passes through the narrow orifice, changes in ionic current can provide information about the sequence of bases DNA is composed of. Another advantage for ssNPs is that they can perform real-time analysis which is much faster than traditional methods. In all nanopore sensing applications, the speed at which the bio-analytes translocate is crucial in determining the method’s sensitivity and resolution. To that end, many efforts have been invested to engineer the nanopores’ specific physical and chemical properties, such as their size, shape, or surface chemistry, allowing them to be tailored for specific sensing applications.

The ability to modulate the characteristics of these nanopores, especially their surface charges, has opened new avenues in biopolymer analysis. The study in question hinges on this principle, using ssNPs modified with an anionic surfactant coating to significantly alter the DNA translocation dynamics. In a new study published in ACS’s Nano Letters led by Professor Amit Meller and conducted by PhD candidate Neeraj Soni, Dr. Navneet Chandra Verma and Ms. Noam Talor from the Faculty of Biomedical Engineering at Technion – Israel Institute of Technology, the researchers focused on enhancing the capabilities of ssNPs in DNA translocation dynamics. The team used silicon nitride (SiNx) nanopores as the base in their experiments. The nanopores were then coated with the anionic surfactant, sodium dodecyl sulfate (SDS), which greatly enhances the negative surface charge of the nanopores. This modification aimed to manipulate the Electro-Osmotic Flow (EOF) within the nanopores, creating movement of liquid in the opposite direction to the motion of the DNA molecules inside the nanopore, hence slowing down their overall speed. This results in a much-improved sensing resolution.

To evaluate the effects of this surfactant coating, the researchers used double-stranded DNA (dsDNA) of varying lengths. These molecules were passed through the modified nanopores to observe changes in translocation dynamics. In a novel approach, the team also used uncharged analytes (small zwitterion organic dyes) for real-time visualization of voltage-dependent Electro-osmotic flow in custom electro-optical apparatus capable of sensing single fluorophores flow in the ssNP. This was done to de-couple the effects of EOF from electrophoretic forces, thereby providing a clearer and quantitative understanding of EOF’s role in DNA translocation by a direct electro-optical measurement.

The authors found over 30-fold increase in the translocation dwell-times (tD) of dsDNA through the SDS-coated nanopores. This meant that the DNA molecules spent a much longer time within the nanopores compared to uncoated nanopores. Importantly, this enhancement in dwell-time did not come at the cost of increased nanopore noise. Maintaining a low noise level is crucial for the accuracy and reliability of measurements in nanopore sensing. The researchers also observed that the SDS coating did not negatively impact the capture rates of shorter DNA fragments (unlike, for example increasing the medium viscosity). This is important for applications in biomedical research where short DNA sequences are often the subject of analysis.

The ability to slow down DNA translocation significantly enhances the potential for detailed analysis of DNA sequences. This is particularly valuable for applications such as detecting and characterizing short DNA fragments, which are important in various biomedical contexts, such as cell-free genomic DNA. Moreover, the study provided valuable insights into the behavior of EOF within planar ssNPs. Understanding the mechanics of EOF in such systems is essential for both theoretical knowledge and practical applications in biosensing. In summary, Meller’s research team successfully manipulated the EOF in nanopores by coating them with an anionic surfactant, leading to a significant improvement in the capabilities of these nanopores for DNA sensing. Indeed, the enhanced functionality of ssNPs due to surfactant coating opens up new possibilities in nanopore technology. The authors’ findings could pave the way for more sensitive and accurate biosensors, capable of detecting a wide range of biomolecules with high precision. In statement to Advances in Engineering, Neeraj said: ‘’These SDS coated nanopores represent the advanced iteration of solid-state nanopores, providing precise control and manipulation of small molecule dynamics through these minute apertures with higher accuracy’’. Co-author Navneet told Advances in Engineering: “The integration of nanopores with single molecule optical methods hold promise for investigating nanoscale dynamics, including the visualization of electro-osmotic flow via individual fluorophores”.

Surfing the Nano Wave: Dramatic Leap in DNA Sensing with Surfactant-Coated Nanopores - Advances in Engineering

About the author

Mr. Neeraj Soni is a PhD candidate in the Nanoscience and Nanotechnology program at the Technion – Israel Institute of Technology. Neeraj’s academic journey includes the successful completion of his master’s degree in Biophysical Chemistry at the Indian Institute of Technology Mandi in 2019, prior to which he studied in Delhi University and graduated in 2017 with a major in chemistry. The primary objective of Neeraj’s ongoing research is to decipher the translocation processes of full-length proteins through ultra-small solid-state nanopore (sub 5 nm regime). For his studies, he uses electro-optical sensing methodologies. His work is dedicated to the development of a universal protocol aimed at achieving single file translocations of the entire proteome, thus enabling the identification of each individual protein sequence. This approach holds immense promise, not just for advancing our understanding of protein behavior and structure, but also for unraveling potential relationships between biomarker expression and disease diagnosis and/or prognosis.

About the author

Dr. Navneet Chandra Verma is a postdoctoral fellow in the Technion-Israel Institute of Technology’s Faculty of Biomedical Engineering. He obtained his M. Tech Degree in Computational and Systems Biology from JNU, New Delhi, and his M.Sc. in Physics from D.D.U. Gorakhpur University, India. He obtained his PhD from Indian Institute of Technology (IIT) Mandi, India. He secured a prestigious fellowship from CSIR India during the Ph.D. and an IASH Excellence Fellowship for the postdoc. He constructed an optical system for super resolution microscopy and single-molecule measurements while pursuing his doctorate in IIT Mandi. His postdoctoral work at Technion under Prof. Meller focuses on creating single-molecule techniques for protein fingerprinting that make use of fluorescence detection and nanopores. In particular, he is working on an on-chip protein separation-identification-fingerprinting system that would enable high-resolution protein categorization, visualization, and counting of biological samples at the single molecule level—and eventually open doors to the testing of clinical samples and medical diagnostics.

About the author

Ms. Noam Talor is conducting her MSc studies in the Meller Lab in the Faculty of Biomedical Engineering at the Technion – Israel Institute of Technology. She completed her undergraduate degree in Biomedical Engineering at the Technion in 2021. Noam’s research aims to control the translocation speed of protein passing through solid-state nanopores. She applies different strategies to achieve labelling of full-length denatured proteins with high efficiency; this often involves the use of different fluorescent markers. These efficient protein labelling techniques will be further used to discriminate biomarkers which are of very similar length, but different in amino acid composition, by electro-optical sensing. Noam has also helped to establish a nanopore coating protocol with an anionic surfactant to utilize the electroosmotic force to slow down the translocation of biomolecules.

About the author

Dr Amit Meller is a Full Professor in the Faculty of Biomedical Engineering at the Technion – Israel Institute of Technology. Dr Meller’s passion lies at the intersection of state-of-the-art technologies, materials, nanoscale sensing and advanced computer analysis (including AI) to solve unmet biomedical sensing challenges. He received his PhD degree in Physics from Weizmann Institute, Israel in 1997 working in soft-condensed matter physics. From 1998 – 2000 he held a position as a Post-Doctoral Fellow at Harvard University working with Daniel Branton and Jene Golovchenko, publishing some of the earliest works in the nanopore sensing field. In 2000, Dr Meller was recruited to the Rowland Institute at Harvard University as a senior fellow, where he established his single-molecule biophysics group. His group developed several single-molecule methodologies including nanopore sensors and single molecule Förster Energy Transfer (smFRET), applying them to develop DNA sequencing techniques and for the study of protein translation biophysics. In 2006, Dr Meller joined the faculty of Boston University as an Associate Professor at the department of Biomedical Engineering and department of Physics.  Dr Meller’s current research interests include single-molecule sensing and single molecule biophysics, bio-optics, nano-sciences and nano-biotechnology. Particularly, the convergence of multiple bio and nano-scale technologies (referred to as “Bio-convergence”) is a major focus in Dr Meller’s lab, applied to the field of proteomics, metabolomics and other “omics”, as well as to the pharmaceutical and medical diagnostical industries.

Reference

Soni N, Chandra Verma N, Talor N, Meller A. Over 30-Fold Enhancement in DNA Translocation Dynamics through Nanoscale Pores Coated with an Anionic Surfactant. Nano Lett. 2023;23(10):4609-4616. doi: 10.1021/acs.nanolett.3c01096.

Go to Nano Lett.

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