Aleksandar P. Ivanov

DNA tunneling detector embedded in a nanopore.

We report on the fabrication and characterization of a DNA nanopore detector with integrated tunneling electrodes. Functional tunneling devices were identified by tunneling spectroscopy in different solvents and then used in proof-of-principle experiments demonstrating, for the first time, concurrent tunneling detection and ionic current detection of DNA molecules in a nanopore platform. This is an important step toward ultrafast DNA sequencing by tunneling.

Rapid Ultrasensitive Single Particle Surface-Enhanced Raman Spectroscopy Using Metallic Nanopores

Nanopore sensors embedded within thin dielectric membranes have been gaining significant interest due to their single molecule sensitivity and compatibility of detecting a large range of analytes, from DNA and proteins, to small molecules and particles. Building on this concept we utilize a metallic Au solid-state membrane to translocate and rapidly detect single Au nanoparticles (NPs) functionalized with 589 dye molecules using surface-enhanced resonance Raman spectroscopy (SERRS). We show that, due to the plasmonic coupling between the Au metallic nanopore surface and the NP, signal intensities are enhanced when probing analyte molecules bound to the NP surface. Although not single molecule, this nanopore sensing scheme benefits from the ability of SERRS to provide rich vibrational information on the analyte, improving on current nanopore-based electrical and optical detection techniques. We show that the full vibrational spectrum of the analyte can be detected with ultrahigh spectral sensitivity and a rapid temporal resolution of 880 μs.

Nanopore sensing at ultra-low concentrations using single-molecule dielectrophoretic trapping.

Single-molecule techniques are being developed with the exciting prospect of revolutionizing the healthcare industry by generating vast amounts of genetic and proteomic data. One exceptionally promising route is in the use of nanopore sensors. However, a well-known complexity is that detection and capture is predominantly diffusion limited. This problem is compounded when taking into account the capture volume of a nanopore, typically 108–1010 times smaller than the sample volume. To rectify this disproportionate ratio, we demonstrate a simple, yet powerful, method based on coupling single-molecule dielectrophoretic trapping to nanopore sensing. We show that DNA can be captured from a controllable, but typically much larger, volume and concentrated at the tip of a metallic nanopore. This enables the detection of single molecules at concentrations as low as 5 fM, which is approximately a 103 reduction in the limit of detection compared with existing methods, while still maintaining efficient throughput.

Synchronized Optical and Electronic Detection of Biomolecules Using a Low Noise Nanopore Platform

In the past two decades there has been a tremendous amount of research into the use of nanopores as single molecule sensors, which has been inspired by the Coulter counter and molecular transport across biological pores. Recently, the desire to increase structural resolution and analytical throughput has led to the integration of additional detection methods such as fluorescence spectroscopy. For structural information to be probed electronically high bandwidth measurements are crucial due to the high translocation velocity of molecules. The most commonly used solid-state nanopore sensors consist of a silicon nitride membrane and bulk silicon substrate. Unfortunately, the photoinduced noise associated with illumination of these platforms limits their applicability to high-bandwidth, high-laser-power synchronized optical and electronic measurements. Here we present a unique low-noise nanopore platform, composed of a predominately Pyrex substrate and silicon nitride membrane, for synchronized optical and electronic detection of biomolecules. Proof of principle experiments are conducted showing that the Pyrex substrates have substantially lowers ionic current noise arising from both laser illumination and platform capacitance. Furthermore, using confocal microscopy and a partially metallic pore we demonstrate high signal-to-noise synchronized optical and electronic detection of dsDNA.

Precise attoliter temperature control of nanopore sensors using a nanoplasmonic bullseye.

Targeted temperature control in nanopores is greatly important in further understanding biological molecules. Such control would extend the range of examinable molecules and facilitate advanced analysis, including the characterization of temperature-dependent molecule conformations. The work presented within details well-defined plasmonic gold bullseye and silicon nitride nanopore membranes. The bullseye nanoantennae are designed and optimized using simulations and theoretical calculations for interaction with 632.8 nm laser light. Laser heating was monitored experimentally through nanopore conductance measurements. The precise heating of nanopores is demonstrated while minimizing the accumulation of heat in the surrounding membrane material.

Label-free in-flow detection of single DNA molecules using glass nanopipettes.

With the view of enhancing the functionality of label-free single molecule nanopore-based detection, we have designed and developed a highly robust, mechanically stable, integrated nanopipette-microfluidic device which combines the recognized advantages of microfluidic systems and the unique properties/advantages of nanopipettes. Unlike more typical planar solid-state nanopores, which have inherent geometrical constraints, nanopipettes can be easily positioned at any point within a microfluidic channel. This is highly advantageous, especially when taking into account fluid flow properties. We show that we are able to detect and discriminate between DNA molecules of varying lengths when motivated through a microfluidic channel, upon the application of appropriate voltage bias across the nanopipette. The effects of applied voltage and volumetric flow rates have been studied to ascertain translocation event frequency and capture rate. Additionally, by exploiting the advantages associated with microfluidic systems (such as flow control and concomitant control over analyte concentration/presence), we show that the technology offers a new opportunity for single molecule detection and recognition in microfluidic devices.

High Precision Fabrication and Positioning of Nanoelectrodes in a Nanopore

A simple and versatile method for the direct fabrication of tunneling electrodes with controllable gap distance by using electron-beam-induced deposition (EBID) is presented. We show that tunneling nanogaps smaller than the minimum feature size realizable by conventional EBID can be achieved with a standard scanning electron microscope. These gaps can easily be embedded in nanopores with high accuracy. The controllability of this fabrication method and the nanogap geometry was verified by SEM and TEM imaging. Furthermore, tunneling spectroscopy in a group of solvents with different barrier heights was used to determine the nanogap functionality. Ultimately, the presented fabrication method can be further applied for the fabrication of arrays of nanogap/nanopores or nanogap electrodes with tunable electrode materials. Additionally, this method can also offer direct fabrication of nanoscale electrode systems with tunable spacing for redox cycling and plasmonic applications, which represents an important step in the development of tunneling nanopore structures and in enhancing the capabilities of nanopore sensors.

Nanopore extended field-effect transistor for selective single-molecule biosensing

There has been a significant drive to deliver nanotechnological solutions to biosensing, yet there remains an unmet need in the development of biosensors that are affordable, integrated, fast, capable of multiplexed detection, and offer high selectivity for trace analyte detection in biological fluids. Herein, some of these challenges are addressed by designing a new class of nanoscale sensors dubbed nanopore extended field-effect transistor (nexFET) that combine the advantages of nanopore single-molecule sensing, field-effect transistors, and recognition chemistry. We report on a polypyrrole functionalized nexFET, with controllable gate voltage that can be used to switch on/off, and slow down single-molecule DNA transport through a nanopore. This strategy enables higher molecular throughput, enhanced signal-to-noise, and even heightened selectivity via functionalization with an embedded receptor. This is shown for selective sensing of an anti-insulin antibody in the presence of its IgG isotype.Efficient detection of single molecules is vital to many biosensing technologies, which require analytical platforms with high selectivity and sensitivity. Ren et al. combine a nanopore sensor and a field-effect transistor, whereby gate voltage mediates DNA and protein transport through the nanopore.

Double Barrel Nanopores as a New Tool for Controlling Single-Molecule Transport

The ability to control the motion of single biomolecules is key to improving a wide range of biophysical and diagnostic applications. Solid-state nanopores are a promising tool capable of solving this task. However, molecular control and the possibility of slow readouts of long polymer molecules are still limited due to fast analyte transport and low signal-to-noise ratios. Here, we report on a novel approach of actively controlling analyte transport by using a double-nanopore architecture where two nanopores are separated by only a ∼ 20 nm gap. The nanopores can be addressed individually, allowing for two unique modes of operation: (i) pore-to-pore transfer, which can be controlled at near 100% efficiency, and (ii) DNA molecules bridging between the two nanopores, which enables detection with an enhanced temporal resolution (e.g., an increase of more than 2 orders of magnitude in the dwell time) without compromising the signal quality. The simplicity of fabrication and operation of the double-barrel architecture opens a wide range of applications for high-resolution readout of biological molecules.

Single Molecule Trapping and Sensing Using Dual Nanopores Separated by a Zeptoliter Nanobridge

There is a growing realization, especially within the diagnostic and therapeutic community, that the amount of information enclosed in a single molecule can not only enable a better understanding of biophysical pathways, but also offer exceptional value for early stage biomarker detection of disease onset. To this end, numerous single molecule strategies have been proposed, and in terms of label-free routes, nanopore sensing has emerged as one of the most promising methods. However, being able to finely control molecular transport in terms of transport rate, resolution, and signal-to-noise ratio (SNR) is essential to take full advantage of the technology benefits. Here we propose a novel solution to these challenges based on a method that allows biomolecules to be individually confined into a zeptoliter nanoscale droplet bridging two adjacent nanopores (nanobridge) with a 20 nm separation. Molecules that undergo confinement in the nanobridge are slowed down by up to 3 orders of magnitude compared to conventional nanopores. This leads to a dramatic improvement in the SNR, resolution, sensitivity, and limit of detection. The strategy implemented is universal and as highlighted in this manuscript can be used for the detection of dsDNA, RNA, ssDNA, and proteins.