Nanopore Analysis

About Nanopores

Nanopore real image

In practice, a human can wield a single protein nanopore molecule as a tool by forming a lipid bilayer (basically a film as thin as a bubble) over a tiny hole poked in a teflon film. The tiny hole is known as the aperture, and can be even smaller than a human hair! The aperture and teflon film is situated between two compartments of essentially salt water. It is common to use 0.88% salt (150 mM NaCl) that is similar bodily salt. Some experiments use as much as 1 M salt, which is even higher than the amount of salt in salt-water (3.5%)! Nanopores are molecular machines found in nature, that are repurposed for our human goals of developing miniature sensing elements. Shown as an example here is a Mycobacterium Smegmatis molecular machine (MspA) that is involved in transporting nutrients across the bacterial cellular boundaries. About 10 years ago, some folks discovered this bacterial molecular machine could be used by humans as a miniature DNA sequencer. Recently, I have been using it to analyze other types of molecules. First I had to obtain the molecular machine by purifying it from bacteria, after which I had some electron microscope pictures taken, shown above. Then I started to use the nanopore as shown above. The one of my works similar to above was recently published in ChemBioChem DOI:10.1002/cbic.202100092, and can also be found here and here and was also a cover feature shown below! I also have some larger projects and collaborations nearing completion and preprint/submission soon and much in the works. And there are many other interesting nanopore works by my awesome colleagues in the Min Chen group at UMass Amherst (though that site needs an update)!

Keepin it real (time)

Sometimes I like to think about the scales of things, and shown below is an illustration as to how amazingly small this nanopore sensor tool is. A human in control of a single nanopore is be similar in scale to the entire planet Earth being in control of a large powertool.

Nanopore as a Tool

I also 3D printed a bunch of nanopore structures and protein analytes, some of my favorite nanopore structures are shown below, and nanopores were also recently features as PDB's molecule of month!

Nanopore as a Tool

Another thing to think about is the scales and concentrations of molecules and physical supports within the actual nanopore device. If the aperture shown above was scaled to the size of the UMass campus pond, an MspA nanopore would be about the size of a small bug, illustrated below.

Nanopore real image

A typical nanopore experiment is conducted by forming a lipid membrane over the aperture, followed by adding nanopores.

A concentration of 1 pM is a typical amount of nanopore to add, which equals 6x10^8, or 600 million nanopores in the ~1 mL volume (cube of 0.01 m sides). Relative to the aperature comparison to UMass pond scenario, in a 25m*(0.01m/2.5x10^-5m)=10,000 m sided cube, that would be 6x10^8 ladybugs in 1x10^12 m^3, or 1 ladybug per 2000 m^3, or 1 ladybud in the 3 m of space extending above the area of the UMass pond.

Ladybug velocity is about 5 m/s. The mean diffusion velocity of a nanopore could be roughly 3 microns/s, or 3 m/s if adjusted to the UMass pond scenario. In a nanopore experiment, a pore may insert within 10-30 minutes, which would likely correspond to thousands of non-insertion collisions with the membrane for every inserted pore. However, increasing the size of the aperture would greatly enhance all colisions and thus speed insertion.