Nanotechnology #3 - Single Molecule Nanopore Spectrometry

There are many problems the human race has to face. Energy, resources, waste, cancer - just to name a few. Scientists are working hard to find new creative approaches to these problems. One of these solutions is to think in very small units. The result lies at the intersection of physics, engineering, chemistry and biology. Nanotechnology has tremendous potential.

Nanotechnology is the idea that we can create devices and machines all the way down to the nanometer scale, which is a billionth of a meter, about half the width of a human DNA molecule.
-Paul McEuen

Background source

Other posts in this series:
Nanotechnology #1 - Introduction
Nanotechnology #2 - Nanotechnology In Cancer: An Overview

Today I want to write about a technology that I have never heard of before, but it sounds pretty interesting. This technology is called Single Molecule Nanopore Spectrometry.

What Is Single Molecule Nanopore Spectrometry?

First of all, what are nanopores? As conveyed in the name, nanopores are pores that have diameters on the nanoscale. They are found in nature, where they form little channels that enable certain substrates to pass through a lipid-bilayer. There are also synthetic nanopores like silicon nitride. Molecules usually require an applied voltage to be forced through the nanopore. By applying a current, the blockade current, these molecules are forced to stay in the pore for a short amount of time. During this time the size of the molecule moving through the nanopore can be determined by measuring the electric conductivity of the nanopore (1). The blockade current can help to identify the molecule moving through the nanopore (2). Thus, a nanopore in itself can serve as a sensor.

I am personally very interested in this technology. Theoretically, this should help to rapidly identify proteins and DNA. Initial studies have shown that it is possible to differentiate very similar linear molecules of poly-ethylene glycol, which differed by a single monomer (3). However, there are several problems that have not yet been overcome. The majority of problems are related to proteomics. It is more difficult to force proteins through a nanopore than, for example, DNA and other naturally linear polymers, which slows down this process (4). Furthermore, it is not yet well understood how the blockade current relates to the amino acid sequence of a protein, which is why purely nanopore-based sensors are not widely used in protein characterization as of now (2). There has been some success, however. A study has shown that it is possible to rapidly discriminate DNA polymers that only differ in sequence by using nanopores (5). Steps are also being taken towards nanopore proteomics. For example, a research group was able to differentiate between proteins that were either not-phosporylated, or single- or double phosphorylated (6). Another study has been shown that is able to read the primary structure of proteines in quadromers (4 amino acids) (7). However, it is desirable to differentiate between individual amino acids. This still is a promising early step. Another problem is that databases are not yet significant, which is why conventional mass spectrometry is still dominating nanopore spectrometry (8). However, possible applications are endless. For example, a group has demonstrated that the surface on the inside of a silicon nitride nanopore can be modified by adsorbing enzymes, which makes the pore very selective (9). Another group "dressed" a nanopore with DNA for complementary sequence detection (10). This seems to be a relatively new technology, which is still in the early stages of its development. In 2016, researchers were very pessimistic about applying nanopore spectrometry to proteomics, but a year later the opinion seems to have changed to a more optimistic outlook (11).

Further reading:

• The evolution of nanopore sequencing
• Single Molecule Nanopore Spectrometry for Peptide Detection
• Single-molecule protein identification by sub-nanopore sensors
• Single-molecule mass spectrometry in solution using a solitary nanopore

My Opinion, Outlook And Hope

When I read about this the first time I felt very happy, very suddenly. I know it is weird to be happy when you read a research article (most people I know would rather jump off a cliff), but this technology just sounds so cool. If the above problems can be overcome, we will be able to characterize individual proteins in very short amounts of time. Additionally, if specificity can be tuned to individual proteins, lipids or peptides we may be able to measure such in complex solutions such as blood or saliva or whatever liquid you may imagine. Another promising aspect of this technology is that due to the nano-size of these pores, it is possible to fix several specific nanopores onto a small chip, which could enable us analyze a complex sample for several analytes (for example measure blood glucose, disease biomarkers and several ions). Something like this has been done at the University of Freiburg. Researchers fixed 16 miniaturized artificial cell membranes on an only one square millimeter large chip (1).
If you are an expert in this area, or just know about it fairly well, please contact me!

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