Single-molecule sensing of peptides and nucleic acids by engineered aerolysin nanopores

Nanopore sensing is a powerful single-molecule approach for the detection of biomolecules. Recent studies have demonstrated that aerolysin is a promising candidate to improve the accuracy of DNA sequencing and to develop novel single-molecule proteomic strategies. However, the structure-function relationship between the aerolysin nanopore and its molecular sensing properties remains insufficiently explored. Herein, a set of mutated pores were rationally designed and evaluated in silico by molecular simulations and in vitro by single-channel recording and molecular translocation experiments to study the pore structural variation, ion selectivity, ionic conductance and capabilities for sensing several biomolecules. Our results show that the ion selectivity and sensing ability of aerolysin are mostly controlled by electrostatics and the narrow diameter of the double beta-barrel cap. By engineering single-site mutants, a more accurate molecular detection of nucleic acids and peptides has been achieved. These findings open avenues for developing aerolysin nanopores into powerful sensing devices.

Electrochemical and morphological engineering of 2D materials for nanopore sensing

Martina Lihter

Nanopores are nanometer-sized holes that were initially proposed for DNA sequencing. Several years ago sequencing was made possible with biological nanopores. However, solid-state nanopores have plenty of advantages to offer compared to their biological counterparts. This thesis focuses on nanopores made in 2D materials, and their sensing capabilities. They provide an outstanding spatial resolution and a good signal-to-noise ratio (SNR) for sensing. As a 2D semiconductor, molybdenum disulfide (MoS2) exhibits unique (opto)electronic and electrochemical properties. The electronic band structure of a semiconducting MoS2 results in highly sensitivity to its chemical environment and external electric field. Therefore, monitoring the transverse current through the MoS2 during analyte translocation can provide a powerful sensing mechanism. As an n-type semiconductor, MoS2 exhibits a characteristic electrochemical behavior: it shows electrochemical activity when polarized cathodically, while in the anodic region becomes electrochemically inactive. This thesis covers five topics in which I demonstrate how the properties of 2D materials can be used for designing advanced sensing platforms for biosensing. Fabrication of MoS2 Nanopores. Here, we give an overview of a reliable fabrication process optimized for the production of high-quality devices. We discuss the main issues, how to solve and avoid them. In the end, we demonstrate applications of MoS2 nanopores for sensing and for osmotic power generation. Geometrical Effect in 2D Nanopores. We compare two different pore geometries: triangular pores in hexagonal boron-nitride (h-BN) vs. approximately circular pores in MoS2. In h-BN nanopores, we observe a lower conductance drop caused by DNA translocation, than expected from a conventional conductance model. As an explanation, we propose a reduced ion-concentration and ion-mobility inside the pore, supported by molecular dynamics simulations. Transverse Detection of DNA in MoS2 Nanopore. This chapter presents the realization of a hybrid nanopore-FET device, consisting of an MoS2 ribbon and a nanopore drilled in it. Such a device acts as a field-effect transistor (FET), where the transverse current through the MoS2 is modulated by the translocating molecule and the ionic voltage applied across the pore. We show that the transverse current is more sensitive to DNA translocation than the ionic current. Passivation of Electrodes Contacting MoS2. The method is based on the electrochemical deposition of a polymer, which blocks the electron transfer from the surface of the electrodes to ions in solution. To avoid the deposition on MoS2, we used poly(phenylene oxide) (PPO) that polymerizes in the potential window where MoS2 is inactive. Deposition is compatible with MoS2 and highly area-selective, even at the nanoscale, as we demonstrate on the nanoelectrodes of a nanopore-FET device. Electrochemical Modification of MoS2. By polarizing MoS2 cathodically, it is possible to electrochemically deposit a thin layer of aryl-diazonium compounds on MoS2 surface. This approach allows to introduce specific chemical groups on MoS2 and nanopore rim, which could be used for specific recognition of analytes as they translocate through the pore. Furthermore, since the deposition occurs only on the MoS2 to which the voltage was applied, this could enable specific functionalization of adjacent nanoribbons in nanopore-FET devices.

EPFL2020

Interdigitated electrode DNA biosensor for detection of food-borne pathogens by impedance spectroscopy

Daniel Berdat

The commercial application of biosensors has had a significant impact in a number of areas, particularly in the field of medical diagnostics. But in spite of the great number of publications, only a few systems are commercially available in food analysis. We propose an innovative approach to achieve safer food products by creating a user-oriented device for the detection of food-borne pathogens. Our aim is to perform quality control along the food-chain distribution to protect the consumer's health against contaminations; the measurement is based on the detection by impedance spectroscopy of DNA extracted from bacteria. Our work focuses on the conception of a DNA biosensor able to detect the presence of specific DNA sequences from Salmonella bacteria. Within the European project GoodFood, the choice was made to use dairy products and seafood as starting materials, from which cell growth and DNA amplification were performed by our partners, to finally be measured by impedance spectroscopy with the DNA biosensor. The major advantages of this approach are the portability, ease-of-use and rapidity of the system. The sensor fabrication was performed in several steps: At first, the design and the clean-room fabrication was optimized for platinum interdigitated structures with 5 µm spacing. Once the production of the metal electrodes was standardized, the immobilization of the probe DNA (capturing the target DNA) on the sensor surface was performed by silanization. The selectivity of the sensor towards the target DNA was evaluated by fluorescence microscopy using fluorescently-labelled DNA target sequences. The biochip, consisting of 4 interdigitated electrodes, was connected to an impedance analyzer and a first series of measurements was done without DNA, to characterize the sensor in high ionic strength solutions. A next series was performed with probe DNA on the sensor surface, with decreasing concentrations of synthetic target DNA to reach the nanomolar detection limit of the system. Finally, a series of measurements was carried out with DNA target sequences extracted from food samples spiked with salmonella, to apply the biosensor within a "real" biological DNA sample.

EPFL2009

Hybrid Small Animal Imaging System Combining Magnetic Resonance Imaging with Fluorescence Tomography Using Single Photon Avalanche Diode Detectors

Christof Baltes, Lucio Carrara, Edoardo Charbon, Markus Rudin

The high sensitivity of fluorescence imaging enables the detection of molecular processes in living organisms. However, diffuse light propagation in tissue prevents accurate recovery of tomographic information on fluorophore distribution for structures embedded deeper than 0.5 mm. Combining optical with magnetic resonance imaging (MRI) provides an accurate anatomical reference for fluorescence imaging data and thereby enables the correlation of molecular with high quality structural/ functional information. We describe an integrated system for small animal imaging incorporating a noncontact fluorescence molecular tomography (FMT) system into an MRI detector. By adopting a free laser beam design geometrical constraints imposed by the use of optical fibers could be avoided allowing for flexible fluorescence excitation schemes. Photon detection based on a single-photon avalanche diode array enabled simultaneous FMT/MRI measurements without interference between modalities. In vitro characterization revealed good spatial accuracy of FMT data and accurate quantification of dye concentrations. Feasibility of FMT/MRI was demonstrated in vivo by simultaneous assessment of protease activity and tumor morphology in murine colon cancer xenografts.