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Streamline your Analysis Workflow

Analyzing Nanopore Data with Nanolyzer™

Learn how to use Nanolyzer™ to use the software to extract every bit of useful information from your nanopore data and provide an opportunity for you to tell us how we can further develop the tools to address your specific analysis needs. 

During our next webinar, Applications of Solid-state Nanopores: Exploring Potential Impact Learn, we will discuss the broad potential impact of solid-state nanopores being explored across multiple fields, including genomics, proteomics, metabolomics, digital information storage, and even the search for extraterrestrial life.  Sign up here!

  • What range of pore sizes can be made using Northern Nanopore tools? What is the ideal nanopore diameter for dsDNA translocation?
    The pore size range accessible to NNi’s Spark-E2 depends somewhat on the details of the membrane used. For example, it is able to reliably fabricate nanopores between 2-30 nm in diameter in 10 nm thick silicon nitride membranes. For other membrane materials in this thickness range you can expect similar performance and range, and we suggest reaching out to us to discuss protocol optimization in those cases. The ideal size for molecular translocation is also context-dependent, but a general rule of thumb that can be applied is that your nanopore should be slightly larger than your target molecule. For dsDNA with a diameter of 2.2 nm, this suggests a pore size of 3-5 nm, which will allow for free translocation while still providing a strong signal to noise ratio. You can find some additional discussion here.
  • Is it possible to localize the created nanopore on a particular region of the membrane?
    While the Spark-E2 does not provide pore localization directly, there are several ways that pores made using controlled breakdown can be localized. The papers below provide several methods by which this can be achieved (note: this is not an exhaustive list). If you have an application for which this capability is required and you need assistance in implementation, contact us to discuss how we can make this possible for your specific use case.
  • What is a “rectifying” nanopore?
    A rectifying nanopore is one which conducts preferentially in one voltage polarity. Usually this behavior indicates some asymmetry in the system, whether in the pore geometry or the surface charge in and around the pore. It can usually be fixed by enlarging the pore, or by using a higher concentration of salt in your measurement buffer, which will in turn better shield any asymmetries.
  • How long can I store my pores for and how can I store them to keep them stable? How long can pores last in storage?
    Generally, we see the best and most consistent nanopore performance when nanopores are used immediately after pore fabrication and conditioning. For this reason, we do not recommend storage of pre-fabricated nanopores, and instead strongly encourage a workflow that involves making and optimizing a nanopore immediately prior to use. If storage is needed, then optimal methods depend on the timescale. Nanopores made in silicon nitride membranes can usually be stored safely overnight in 3.6M LiCl pH 8 with minimal growth, though the actual degree of growth is highly dependent on the batch of membranes used. For storage over longer timescales (days to weeks), a solution of 50% Ethanol and deionized water works well. For pores that require storage longer than a few weeks, or for storage of different membrane materials, feel free to reach out to discuss your specific requirements.
  • Have you all had any success with base calling DNA on the 2 nm pores?
    The spatial sensitivity of a nanopore is set by the length of its narrowest constriction (where the electric field is strongest and most of the signal is generated). For a solid-state nanopore in a silicon nitride membrane, this usually corresponds to a cylindrical region up to 10 nm in length (which spans about 30 nucleotides), and as such the signal is a convolution of many bases that precludes direct reading. Strategies to overcome this limitation are one of the major challenges that we will discuss in detail in a later webinar, for which you can sign up here.
  • What other strategies do you suggest to control DNA translocation speed other than voltage gradient?
    Molecular translocation speed is highly dependent on the experimental details, including the salt concentration and type. Generally speaking, a higher concentration of a lower molecular weight salt will slow down translocation, as discussed here. Pore size and membrane type can also have an impact. Controlled breakdown can also be used to create unique nanostructures that can strongly impact translocation speed and characteristics, for example using local nanofilters to stretch DNA prior to translocation, or create pores through complex metal-dielectric film stacks. These structures are often not possible to fabricate by any other method. If you are interested in the unique possibilities offered by NNi’s nanopore fabrication methods for molecular control, please reach out to discuss the details.
  • How can you tell if you have only one pore of the correct size and not two smaller pores? Can this be confirmed during fabrication or conditioning?
    With proper protocol optimization, the chance of fabricating two nanopores instead of one is much less than 1%, and so this is a non-issue if you are following Northern Nanopore’s recommended protocols. You can read more about how to ensure single pore formation here. Northern Nanopore tools measure the size of the nanopore using the IV characteristics of the nanopore, a process you can learn more about here. From this static measurement it is not possible to tell the difference between one and two nanopores. A method was developed that can use changes in pore size over time to obtain this information, but it is not supported by Northern Nanopore software. If you are finding that you are seeing multiple nanopores regularly, please reach out to discuss protocol optimization to avoid this issue.
  • Is it possible to monitor pore formation in real-time other than checking ionic current?
    While Northern Nanopore’s Spark-E2 does not natively support other methods of monitoring, it is possible to implement controlled breakdown in a microscope and monitor the process optically instead. We are able to provide custom microscopy solutions for this purpose for many commonly used microscopes. If you are in need of a custom setup for optical monitoring, get in touch with the details.
  • How long can we use a nanopore? How do we decide when the pore's lifetime is over?
    Pore lifetime is usually determined by either geometric stability, or pore clogging. In the former case, an unstable membrane type or batch can lead to slow pore growth over time, which eventually results in a pore that is too large to be useful, though where this line is drawn depends strongly on the experimental details and the information being sought. In the latter case, molecules, especially reactive ones such as single-stranded DNA or proteins, can often stick in or near the pore. While it is often possible to remove these clogs by reversing the voltage used to drive translocation, this is not always the case. Generally speaking a pore should be used until enough data has been collected to reach statistical significance for the hypothesis being tested. If you are having issues with pore lifetime and need assistance, please contact us.
  • Are there any alternatives (even if less effective) to Piranha cleaning? How does piranha solution compare to plasma cleaning?
    Plasma cleaning is a reasonable alternative to Piranha as a means to make a membrane surface hydrophilic, which in turn often improves yield of pores with reduced levels of low-frequency noise. However, Piranha cleaning remains by far the most effective cleaning method. We understand that Piranha cleaning is a dangerous process. To that end, one of the items in the Nanopore Lab Starter Pack is a custom jig that facilitates cleaning nanopore chips with the smallest possible volume of Piranha solution.
  • What is the optimal method for removing analytes after each experiment? Is it possible to have continuous flow of liquid during the nanopore experiment?
    Northern Nanopore flow cells are designed for laminar flow, which makes removing analytes very simple. A flush with ~200 microliters of clean salt solution using a standard pipette is usually enough to remove dsDNA completely. For other molecules such as proteins, the process can be more difficult as these molecules often stick to surfaces and resist removal by flow. Cleaning methods are molecule-dependent in these cases. Please reach out with the details of your experiment if you are in need of assistance.
  • Can the nanopore be functionalized to improve the selectivity during DNA and protein sequencing?
    Yes! Several protocols have been published that describe methods to functionalize nanopore surfaces for improved translocation characteristics. Note that this is not an exhaustive list.
  • We have observed passive growth of glass nanocapillaries in KCl but not LiCl. Do silicon nitride pores exhibit the same behavior?
    Yes, silicon nitride pores are usually more stable in LiCl, as discussed in one of our early works. Care should be taken when making the comparison that fresh solution be used when measuring, however. KCl evaporates and loses its water content faster than LiCl and subsequently increases the solution conductivity faster, which in turn can lead to overestimates of the pore size.
  • How stable are the flow cells in solvents other than water?
    The 3D printer resins we use for our flow cells are generally compatible with aqueous solutions and some organic solvents over short timescales, but are not exhaustively tested for chemical compatibility. In cases where more resistant materials are needed, we are able to provide flow cells with high chemical resistivity as well. Contact us with the details of your chemical compatibility requirements for details.
  • What would be the mechanistic reason for conductance increase without pore formation?
    The mechanistic details of the process are complex, but generally the act of forcing leakage current through a dielectric below the dielectric strength causes damage to the membrane that locally reinforces the current, leading to a runaway process which you can read about in more detail here, and in references therein. Care should be taken in interpreting the leakage current during fabrication, however. Depending on the details of the chip architecture, it is possible that the majority of the leakage current observed reflects current through the support chip rather than the specific site on the membrane in which the pore forms. The details of the leakage current during fabrication are therefore generally not useful as a physical indicator of the details of the physics at the site of the nanopore.
  • Can we form more than 1 SS nanopore (for multiplex detection) or the system is designed for a singleton pore?
    The NNi Spark-E2 is designed to fabricate a single nanopore (in each channel). Parallelization of nanopores can be achieved in a variety of ways, and is an area of active development for Northern Nanopore. If you are interested in multiplexed detection of nanopores and are in need of assistance with implementation, please reach out to discuss the specifics of your requirements.

Learn more about nanopore fabrication by controlled breakdown


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