Fabrication of solid-state Nanopores for DNA analysis

One of revolutionary technologies to sequence single molecules, such as DNA, is using nanopore devices. A single-channel with nanometer-scale aperture allows an extremely rapid electric analysis of such single molecules. The objective of this work is to fabricate nanopores into robust, solid-state membranes to achieve better performances. The technological challenge is in the small required dimension of the nanopore, around 50-100 nm in diameter and depth. To reach this objective, we used two different techniques: the ion track technique and a microfabrication method associated with Focused Ion Beam (FIB). A literature review has been done on the ion track technique. Threshold values of the ion track formation have been found for different insulator materials (polymers, SiO2, SiN). We did experiments with the linear accelerator UNILAC (Universal Linear Accelerator) at the GSI (Darmstadt, Germany) where we irradiated our samples with heavy gold ions (11.4 MeV/nucleon). On very thin membranes, we expect that a direct perforation would occur, and we chose SiN membranes and SU-8 thin films as samples. However, the experiment of ion bombardment was not possible to be done early enough, thus TEM analysis have not been done yet. If the direct perforation by ion bombardment is not possible, a chemical etching step would be necessary. About the microfabrication method associated with Focused Ion Beam (FIB) perforation, we did experiments at the Center of Micro Nano Technology (CMI) in EPFL. First trial with FIB was done on SiN membranes. In order to obtain required aperture size, reduction of membrane thickness before perforation by FIB was attempted, but this process was difficult to be achieved because of charging. To overcome this dimension limitation we chose a silicon membrane. With silicon membranes, oxidation would be performed after perforation by FIB, since channel opening will be reduced by SiO2 growth. Moreover, this oxide layer allows a functionalisation of the inner surface of the nanopore by silane chemistry. That should improve the resolution of DNA analysis. Though the aperture size reduction by oxidation has not been done yet, refining and perforation by FIB will be done according to this idea. We should reach ca. 100 nm-aperture size. Even if validation measurements are not easy, our specifications should be reached with the oxidation. More experiments and analysis have to be performed for further improvement.

Stimuli-responsive nanostructured surfaces

Ana Maria Popa

The coupling of stimuli-responsive macromolecules to nanostructured surfaces opens the perspective for the fabrication and integration of "smart" micro- and nano-electro-mechanical systems (MEMS&NEMS) in which the smallest motile unit is the polymeric chain. The goal of this work was to develop thermoresponsive, durable nanoporous surfaces as a first step toward the fabrication of functional nanocontainers and nanovalves. The proposed strategy involves in a first step the design and implementation of a clean-room compatible, reproducible and up-scalable method for defining organized nanoporous arrays in MEMS compatible surfaces, such as silicon, silicon oxide and silicon nitride. The main objective of the second step was the covalent immobilization of responsive macromolecules on the obtained structures, as to provide the later with a "smart" behavior. The nanostructuring step was realized by block-copolymer assisted lithography. This newly developed technique enables the definition of nanoscale features on semiconductor substrates over large surface areas and in a cost effective manner. A novel method for patterning thin metal films by a lift-off approach, using spin coated block-copolymer micelles monolayers as a template, was investigated. The obtained nanoporous mask was used as resist for the structuring of hard substrates in plasma-assisted processes and structures with aspect ratios as high as 1:10 were be obtained. By using different block-copolymer micelles and spin-coating conditions, the proposed approach enables the variation of the nanopores' diameter from 40 to 80 nm and a surface coverage between 11.7% and 25.5 % in an independent manner. This technique was successfully integrated in a standard microfabrication process for the batch production of thin nanoporous silicon nitride membranes. The 100 nm thick membranes span over thousands of square micrometers and present porosities higher than 109 pores/cm2. They are thus competitive in terms of porosity with the commercially available polymeric membranes. In addition they are two orders of magnitude thinner than polymeric membranes, this making them extremely interesting for applications where permeation rate or response time is important. The rapid flow of water soluble small molecular species through the nanopores was demonstrated in a qualitative manner in this work. Another series of investigations targeted the functionalization of the available nanoporous surfaces with responsive molecules. Poly(N-isopropyl acrylamide) (PNIPAAM) was chosen as a model system. Its coupling to silicon surfaces by a "grafting-to" approach from melt, using an epoxy-functionalized silane as anchoring layer was studied. The analysis of the yielded grafting density showed results superior to those obtained by classic grafting-to approaches. The relation between the polymer's weight and its responsiveness was investigated in liquid media by atomic force microscopy and by dynamic force spectroscopy. Polymers with more than 500 monomers in the back bone were shown to exhibit enhanced responsiveness. Additionally, two bioengineered polymers from the class of elastin-like polymers (ELPs) were investigated as possible candidates for the fabrication of stimuli responsive nanovalves. The molecules were synthesized in collaboration with the Bioforge group (Universidad de Valladolid, Spain) and details of the bioproduction process are presented. A method for the covalent photoattachment of macromolecules using a benzophenone-functionalized silane, was tested in this work. First the capacity of the benzophenone silane to graft synthetic thermo-responsive polymers such as PNIPAAM was demonstrated. The temperature-induced morphology change of the photografted PNIPAAM was not affected by this anchoring strategy and was monitored by tapping AFM in liquid media. The investigations were then extended to the photo-immobilization of ELPs on nanoporous silicon surfaces and the XPS measurements revealed the success of this step. However no conformation modification of the proteins could be evidenced by tapping-mode AFM in liquid media under different ionic strengths.

EPFL2009

Nanopore sensing of single molecules application to RNAP-DNA complexes, fabrication of graphene-nanopore devices and translocation algorithm analysis

Camille Alice Raillon

Nanopore sensing is a single-molecule technique that allows to probe the nano-bio-world with accuracy. It is a non-invasive technique, which means that the molecule of interest does not need to be labeled to be detected by the nanopore. Most of the studies done with nanopores use electrophoresis. With an electric field applied across the nanopore, charged molecules move through this tiny hole. When a molecule passes through the nanopore it creates a dip in current, called current blockage, because it partially blocks the ions from going through and so it increases the nanopore resistance. This phenomenon is observed using a refined ammeter that detects changes in current in the range of pA. At the Laboratory of Nanoscale Biology (LBEN), we are interested in very small biological units, and this thesis is focused on the RNA polymerase, the enzyme that catalyzes the transcription reaction. Transcription is the first step in gene expression where DNA is copied into RNA. It is extensively studied at the bulk level especially the regulation mechanism, which in cancerous cells is impaired. We were interested in studying this enzyme at the single-molecule level for its functional as well as molecular motor properties. This work was done in close collaboration with Pascal Cousin at the Center for Integrative Genomics (CIG) in the group of Nouria Hernandez who helped to design the protein-DNA complexes and analyze the data. With nanopore sensing we were able to observe RNA polymerase-DNA complexes translocate through nanopores and capable to distinguish between individual complexes and bare RNA polymerase. We were also able to observe orientation of RNA polymerase in the nanopore whether flow or electric field predominates. The complexity of the signals from the protein-DNA complexes experiment motivated the development of a level detection software. This software was written in collaboration with Pierre Granjon from GIPSA-lab in Grenoble who had the expertise on a change detection method called the CUSUM algorithm. Our software was designed to analyze in details current blockages in nanopore signals with very little prior knowledge on the signal. With this work one can separate events according to their number of levels and study those sub-populations separately. This method also allows a global statistical view of all levels by plotting what we call a level histogram. We demonstrate that with such a technique one can identify populations that cannot be seen otherwise. As the theoretical literature has shown, it should be possible to sequence a ssDNA with a nanopore using transverse electronics for tunneling current measurement. In this thesis, we will discuss the fabrication work done on the first generation of graphene-nanopore devices and how we overcame the fabrication issues to produce reliable devices.

EPFL2012

Pressure-Induced Enlargement and Ionic Current Rectification in Symmetric Nanopores

Andrey Chernev, Sebastian James Davis, Michal Daniel Macha, Sanjin Marion, Aleksandra Radenovic

Nanopores in solid state membranes are a tool able to probe nanofluidic phenomena or can act as a single molecular sensor. They also have diverse applications in filtration, desalination, or osmotic power generation. Many of these applications involve chemical, or hydrostatic pressure differences which act on both the supporting membrane, and the ion transport through the pore. By using pressure differences between the sides of the membrane and an alternating current approach to probe ion transport, we investigate two distinct physical phenomena: the elastic deformation of the membrane through the measurement of strain at the nanopore, and the growth of ionic current rectification with pressure due to pore entrance effects. These measurements are a significant step toward the understanding of the role of elastic membrane deformation or fluid flow on linear and nonlinear transport properties of nanopores.