life Review Biological Nanopores: Engineering on Demand Ana Crnkovi´c*, Marija Srnko and Gregor Anderluh National Institute of Chemistry, Hajdrihova 19, 1000 Ljubljana, Slovenia; [email protected] (M.S.); [email protected] (G.A.) * Correspondence: [email protected] Abstract: Nanopore-based sensing is a powerful technique for the detection of diverse organic and inorganic molecules, long-read sequencing of nucleic acids, and single-molecule analyses of enzymatic reactions. Selected from natural sources, protein-based nanopores enable rapid, label-free detection of analytes. Furthermore, these proteins are easy to produce, form pores with defined sizes, and can be easily manipulated with standard molecular biology techniques. The range of possible analytes can be extended by using externally added adapter molecules. Here, we provide an overview of current nanopore applications with a focus on engineering strategies and solutions. Keywords: nanopores; pore-forming toxins; sensing; aptamers; oligomerization 1. Introduction Nanopore-based sensing is an emerging technology with great potential for the de- tection of diverse organic molecules, sequencing of nucleic acids, and single-molecule analyses of enzymatic reactions and protein folding. Conceptually, nanopore biosensing belongs to the so-called resistive-pulse methods. A classic example of such a method is the Coulter counter, originally developed in the 1950s to count blood cells [1]. The instrument contains a capillary, which is divided into two parts by a wall containing a 20 µm–2 mm aperture. The capillary is filled with an electrolyte solution and an applied electric field causes ions to move through the opening, creating a constant ionic current. As the blood Citation: Crnkovi´c,A.; Srnko, M.; cells move through the narrow aperture, they partially block the aperture, causing a de- Anderluh, G. Biological Nanopores: crease in solution conductivity. This decrease in conductivity (also called resistive pulse) Engineering on Demand. Life 2021, 11, 27. https://doi.org/10.3390/ occurs at a frequency equal to the cell number, while its amplitude depends on the cell size. life11010027 As with other resistive-pulse methods, the particles to be analyzed must have a diameter close to the diameter of the aperture. Thus, the size of the aperture dictates the detection Received: 30 November 2020 limit. Using the Coulter counter, particles with a size of ~10 µm could be detected [1]. An Accepted: 31 December 2020 increase in the detection limit came in the 1970s when it was shown that particles ~100 nm Published: 5 January 2021 in size could be detected using nuclear-track etched pores with a diameter of 500 nm [2]. While the sensitivity of this device was sufficient to detect particles as small as viruses [3], Publisher’s Note: MDPI stays neu- increasing the limit enough to detect molecules requires molecule-sized apertures, such as tral with regard to jurisdictional clai- those found in natural membrane channels and pores. ms in published maps and institutio- The use of biological nanopores for resistive-pulse detection of molecular analytes nal affiliations. was made possible by developments in the field of ion-channel physiology in the 1960s and 1970s, namely the development of planar bilayer recording [4] and the development of single-channel current measurements [5]. However, endeavors to combine the principles of the resistive-pulse method with single-channel measurements, thus laying the foundation Copyright: © 2021 by the authors. Li- censee MDPI, Basel, Switzerland. for nanopore biosensing were not made until the late 1980s [6]. Neutral polymers, origi- This article is an open access article nally used to investigate the volume of an ion channel [7], were the first to be recognized as distributed under the terms and con- potential analytes [8]. Early studies used channels generated by alamethicin, an antibiotic ditions of the Creative Commons At- peptide [8], Staphylococcus aureus α-hemolysin, and cholera toxins [9,10]. Possibly the tribution (CC BY) license (https:// biggest push for the development of nanopore biosensing came after it was found that creativecommons.org/licenses/by/ single-stranded DNA and RNA could be threaded through a nanopore, making it a poten- 4.0/). tial technology for nucleic acid sequencing [11]. Since then, the number of applications for Life 2021, 11, 27. https://doi.org/10.3390/life11010027 https://www.mdpi.com/journal/life Life 2021, 11, x FOR PEER REVIEW 2 of 30 Life 2021, 11, 27 2 of 30 found that single-stranded DNA and RNA could be threaded through a nanopore, mak- ing it a potential technology for nucleic acid sequencing [11]. Since then, the number of applicationsnanopore biosensing for nanopore has grown biosensing immensely, has grown along immensely, with improvements along with in improvements instrumentation in instrumentationand data processing and methods.data processing methods. Nanopore biosensing biosensing is is based based on on naturally naturally occurring occurring protein protein pores. pores. In a In typical a typical ex- periment,experiment, the the pores pores are are embedded embedded in in a alipid lipid bilayer, bilayer, which which separates separates two two chambers, cis and transtrans,, filledfilled with with electrolyte electrolyte solution solution (Figure (Figure1). An1). appliedAn applied voltage voltage causes causes ions to ions move to movethrough through the pore the and pore create and create an electrical an electrical field. Anfield. analyte An analyte can be can captured be captured and transported and trans- portedacross theacross pore the by pore different by different mechanisms mechanisms [12]. For [12]. charged For charged analytes, analytes, electrophoresis electrophoresis may maybe the be dominant the dominant form form of transport, of transport, carrying carrying the analyte the analyte toward toward the electrode the electrode of opposite of op- positepolarity polarity [13]. [13]. For neutralFor neutral or less-chargedor less-charged molecules, molecules, electroosmotic electroosmotic flow flow may may bebe the dominant force force directing directing capture capture and/or and/or transp transportort [14,15]. [14,15 Furthermore,]. Furthermore, an analyte an analyte of ap- of propriateappropriate size size can can enter enter the the pore pore and and alter alter the the ionic ionic current. current. Figure 1. AnAn illustration illustration of of a abiological biological nanopore nanopore and and the the principle principle of ofnanopore-based nanopore-based biosensing. biosensing. Direct Direct sensing sensing relies relies on the on thedistinct distinct current current signatures signatures produced produced by the by thetranslocation translocation of individual of individual anal analytesytes (top (top right). right). Indirect Indirect sensing sensing employs employs an ad- an additional,ditional, adapter adapter molecule molecule that that specifically specifically recognizes recognizes the the an analytealyte (bottom (bottom right). right). The The adapter adapter (shown (shown in in red) specificallyspecifically interacts with with the the analyte analyte (yellow) (yellow) and and the the translocation translocation of of th thee adapter•analyte adapter•analyte complex complex results results in in a aunique unique current current signal. signal. An analyte analyte may may change change the the ionic ionic current current by by(i) producing (i) producing a change a change in the in electric the electric field withinfield within the pore the [16] pore or [through16] or through (ii) volume (ii) volumeexclusion exclusion and binding and of binding ions to ofthe ions traversing to the analyte,traversing which analyte, reduce which the ionic reduce current the ionic [17]. current Importantly, [17]. Importantly, because numerous because ions numerous accom- panyions accompanythe passage of the a passagesingle analyte, of a single a large analyte, electrical a largeamplification electrical occurs amplification during a single- occurs moleculeduring a single-moleculetranslocation [18]. translocation In a simplified [18]. scenario, In a simplified the duration scenario, and theamplitude duration of andthe currentamplitude alteration of the current is unique alteration to each is uniqueanalyte to or each monomeric analyte or unit, monomeric in the case unit, of in polymer the case analytesof polymer (Figure analytes 1). (Figure1). Nanopore-based sensing sensing can employ both naturally occurring, protein pores and manufactured, solid-state solid-state nanopores nanopores [19]. [19 ].Fu Furthermore,rthermore, nanopores nanopores composed composed of DNA of DNA ori- gamiorigami [20] [ 20as] well as well as hybrid as hybrid pores pores that thatcombine combine several several types types of pores of pores[21–23] [21 are–23 also] are being also developed.being developed. Specific Specific advantages advantages may be mayfound be fo foundr all systems. for all systems.Due to their Due durability to their dura- over ability wide over range a of wide conditions range ofand conditions relative ease and of relative integration ease with of integration microfluidic with devices, microflu- cer- tainidic applications devices, certain can applicationsonly be realized can onlythrough be realizedthe use throughof solid-state the use nanopores of solid-state (e.g., [24,25]).nanopores However, (e.g., [24 biological,25]). However, nanopores, biological althou nanopores,gh not as robust although as solid-state not