SOLID-STATE NANOPORES

Introduction

Nanometer-scale pores are ubiquitous in nature, from structures that guard the genetic material inside the nucleus to the mechanism found on many viral capsids that eject invading nucleic acids into cells. While protein pores have important biological functions, many can also be used as probes for characterizing biomolecules.

In our lab, we study an artificial analog to protein pores known collectively as solid-state nanopores. It is our goal to use these devices to gain insight into the fine structure of molecules on an individual basis. The concept of this system is simple: a small (nanometer scale) hole is formed in a thin membrane, which is then used as a barrier between two basins of salt solution. When a voltage is applied across the membrane, a strong electrical field is created through the fabricated hole, resulting in a measurable ionic current. By placing charged molecules on one side of the membrane, the electric field is able to pull the molecules through the hole one at a time and on to the opposite side. When this happens, the temporary presence of the molecule inside the hole blocks the measured ionic current by a certain amount that is related to the size of the molecule (right).

Fabrication

Traditionally, solid-state nanopores have been fabricated using a handful of techniques, including transmission electron microscope ablation or focused Gallium ion beam milling. While effective, these approaches tend to be either lower-throughput or lower-resolution than is ideal. Our lab has pioneered a new technique for the production of these devices that uses a highly-focused beam of helium ions to mill individual pores locally in a free-standing solid-state membrane. This approach yields high controllability over pore diameters (reaching as low as 3 nm) and can be performed quickly even on multiple samples through the use of lithographic patterning and a motorized stage.

 

We have also shown that the same ion beam can be used to control the local thickness of the membrane surrounding the pore (an important feature that can determine measurement sensitivity) and reduce the intrinsic fluorescence of the membrane material (for use in optical analysis of translocations).

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Biomedical analysis

For about two decades, solid-state nanopores have been viewed as an emerging tool for biosensing and molecular analysis. But, this potential has gone largely unrealized, partially because of limitations in device availability, and partially because of a lack of measurement approaches that can go beyond what is achievable by conventional technologies. We are aiming to change that by developing new strategies for selective nanopore analysis and by using the platform to probe new molecular targets.

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Nucleic acid biomarkers

We have developed a new, selective strategy for detecting and quantifying diverse nucleic acid biomarkers. It relies on the fact that small molecules will travel through the nanopore rapidly and will not be resolvable in the electrical signal, but as the molecule (or construct) approaches the size of the pore itself, interactions with the sidewalls will slow the translocation process and make the passage detectable. For example, a short DNA (below left) or a small protein (below center) will pass through the pore without being seen, but when combined, the larger construct (below right) will yield electrical events that are easily resolved.

This concept can be applied broadly. For example, by controlling why the protein binds to the DNA, we can detect specific elements within its structure. We have used selective labeling techniques to introduce binding elements at the locations of epigenetic modifications (right), which are important and hard-to-study features in DNA.

We have also found that the structural differences between single-stranded and double-stranded DNA can cause selective signals. As a result, the use of a ssDNA probe sequence enables any short sequence to be identified and quantified from among a mixture (left). We have already applied this to the detection of microRNAs, focusing on cancer specific varieties, and are currently expanding to other sequence biomarkers.

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Glycosaminoglycans

Glycans have diverse roles in biology, and the functions of some glycans like hyaluronan are size dependent. However, unlike DNA, RNA, and proteins, the tools for analyzing glycans have limitations in areas like sensitivity, complexity, dynamic range, and expense. We have applied solid-state nanopore technology to this challenge. Since each molecule traveling through the pore yields a characteristic electrical signal that reports on molecular size (molecular weight), a full size distribution of an entire population of glycans can be determined from a few hundred or thousand individual molecules. 

In our initial experiments, we probed hyaluronan, first showing explicitly with monodisperse synthetic material (right top) that size correlates with nanopore signal (right bottom), and then utilizing the approach to determine the hyaluronan size distribution in physiological synovial fluid. We studied a horse model of osteoarthritis; a disease in which knee joint degradation accompanies a shortening of the lubricating hyaluronan in the synovium.

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Water-insoluble protein detection

When you digest plant proteins (as with other proteins), the enzymes in your gut break them down into amino acids for use in your body. However, water-insoluble proteins (i.e "prolamines", or those rich in the amino acids proline and glutamine) can't be broken down fully, and remain as short peptides instead. These peptides can be transported through a leaky gut wall, where they are recognized by the immune system. This can cause inflammation, and further separation of the gut wall that can amplify the issue.

Using water-alcohol mixtures with soluble ions to carry current, we were able to show detection of water-insoluble proteins with nanopores (right). Our initial experiments focused on the corn protein family called zeins as a demonstration vehicle. This approach may offer a route to food testing to avoid allergens.

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Other molecular biomarkers

More information can be found on our Publications page.

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