Single biomolecule sensing using solid-state nanopore membranes
Nanopores are narrow channels or holes in a material with dimensions in nanometer range. Organic nanopores exist in nature and are also fabricated artificially using different methods. The natural nanopores are found in cell membranes, neurons, and lipid bilayers and are often formed by pore-forming proteins. These proteins are also used by the immune system to attack bacteria, viruses, and protozoa. Although such protein-based nanopores are easily reproducible and can be used for some exciting translocation experiments, they do not have many practical applications as they are easily susceptible to external conditions. Any change in pH, temperature, stress, concentration of salts etc. can make the biological nanopore membranes completely unstable. On the other hand, artificial nanopores whereas are hard to fabricate, have distinct advantages over their biological counterparts owing to their superior mechanical, thermal, and chemical stability, robustness, complete control of dimensions and structure of nanopores, adjustable surface properties, and easy integration in solid-state devices. Since the discovery of natural nanopores and the development of methods for their artificial fabrication, nanopore membranes have been used and studied for numerous applications in the areas of bio-sensing, chemical-sensing, nano-electronics, water desalination, nano-fluidics, bioelectronics, ion-current rectification, size-selective transport of ions and many more. While all these applications are very interesting, we will focus on the use of solid-state nanopores for bio-sensing in this article.
Let us now try to understand how the whole sensing process works. In my research, I have been using silicon oxide, silicon oxynitride and silicon nitride membranes for single-biomolecule sensing. The first step is the fabrication of very thin free-standing membranes of these materials. Over the past decade, research efforts have focused on making very thin (1nm-10nm) free-standing membranes. Usually, a thin layer of these materials is deposited on a Silicon wafer using chemical-vapor deposition technique and then silicon is removed in definite shape and size using electron-beam lithography and chemical etching.
Figure: (a) Measurement principle for the single nanopore sensor. (b) Example of real-translocation trace of lambda-DNA.
Once, we have fabricated a thin free-standing membrane, the next step is the fabrication of nanopores (small holes) in the membranes. I have been using two different methods for the fabrication of nanopores. The first method known as the track-etch technique employs very fast-moving ions (ions of different species such as Au, Xe, Ag, Pt, Pd etc. with energies of few tens of MeVs to few GeVs traveling at velocities near to 100,000 km/s) to create nanopores. These fast-moving ions when are bombarded on these membranes, create very small holes in the membranes. The number of ions determines the number of nanopores created and often only a single nanopore is used for bio-sensing applications. The other method known as the controlled breakdown technique applies a variable controlled voltage across the membrane which eventually leads to dielectric breakdown of the material and that leads to the fabrication of a nanopore in the membrane.
Now, that we have fabricated a solid-state nanopore membrane, let’s use this membrane for some bio-sensing purposes. Although a lot of complex electronics is underplaying to reduce noise and have enough bandwidth for sensing, the theory behind the bio-sensing using a nanopore membrane is quite simple and can easily be understood using part(a) of the figure. It is based on the blockage of ion-current through the nanopore when a molecule translocates through it. For instance, when a single-stranded DNA is passed through the nanopore, all the base pairs on the DNA will block the nanopore differently which leads to different current drops for different base pairs. The magnitude and duration of the current drop are characteristic of the analyte molecule and the current drop can then be evaluated to know the characteristics of the biomolecule. Part(b) of the figure shows real measurements of lambda-DNA. Each spike in the trace is characteristic of passage of different parts of the DNA. We are further using machine learning and making a library of characteristics of relevant molecules which will then allow us to quickly analyze the composition and concentration of biomarkers in bodily fluids.
With firm foundations from decades of solid-state physics, materials physics, biophysics, and nuclear physics, the possibilities of future solid-state nanopore research seem endless. Researchers are now trying to mimic and study fundamental biological processes under controlled conditions as well as working towards the fabrication of nanofluidic-logic devices and fabricating artificial neurons using these nanopore membranes. In a few years, I see these nanopore membranes surpassing the organic membranes in aspects of inventiveness and performance, blurring the lines between organic and inorganic, natural and lab-made.