Nanopore detection of DNA molecules in magnesium chloride solutions
© Zhang et al.; licensee Springer. 2013
Received: 3 April 2013
Accepted: 7 May 2013
Published: 20 May 2013
High translocation speed of a DNA strand through a nanopore is a major bottleneck fornanopore detection of DNA molecules. Here, we choose MgCl2 electrolyte assalt solution to control DNA mobility. Experimental results demonstrate that theduration time for straight state translocation events in 1 M MgCl2solution is about 1.3 ms which is about three times longer than that for thesame DNA in 1 M KCl solution. This is because Mg2+ ions caneffectively reduce the surface charge density of the negative DNA strands and thenlead to the decrease of the DNA electrophoretic speed. It is also found that theMg2+ ions can induce the DNA molecules binding together and reduce theprobability of straight DNA translocation events. The nanopore with small diametercan break off the bound DNA strands and increase the occurrence probability ofstraight DNA translocation events.
KeywordsNanopore DNA sequencing Translocation speed
Nanopore sensor, which is derived from the Coulter counter , has been utilized for detection and analysis of various single chargedmolecules [2–9]. Now, it is a widespread concern as a potential candidate to achieve the‘$1,000 genome’ goal set by the US National Institutes of Health due to itshigh speed and low cost performance. In a typical nanopore-sensing experiment, ions andbiomolecules are driven by an external transmembrane electric field. Biomolecule passagethrough the nanopore can cause a characteristic temporary blockade in the trans-poreionic current. Information of the biomolecules such as length, composition, andinteractions with other biomolecules can be extracted from the blockade ionic current.In order to get the structural information of a DNA strand at the single base level, abottleneck to break through is to control the DNA translocation speed through ananopore. Intuitively, we can change the applied voltage, salt concentration, viscosity,and electrolyte temperature to reduce the translocation speed . The side effect of this method is the reduction of the signal amplitude,which leads to more difficulties in capturing the very weak ionic current change . Another method is to apply a salt gradient on the electrolyte solutionacross the pore, which can be used not only to prolong the translocation time but alsoto enhance the capture rate . Recently, some groups tried introducing positive charges into nanopores asmolecular ‘brakes’, which is proved to be an effective approach to increasethe attractive force between the negative DNA molecule and the positive nanopore innerwall, thus increasing the duration time more than 2 orders of magnitude . The shortcoming of this method is that the residual ionic current during theDNA translocation is insufficient for direct base identification. Aside from an electricfield applied along the nanopore axis direction, Tsutsui et al. added a transverseelectric field to slow down the translocation speed of DNA across the nanopore . It is reported that adding a transverse field of 10 mV/nm in a goldelectrode embedded silicon dioxide channel can make 400-fold decrease in the DNAtranslocation speed. Similarly, He et al. reported a method to control the DNAtranslocation speed by gate modulation of the nanopore wall surface charges. It is foundthat native surface-charge-induced counterions in the electro-osmotic layersubstantially enhance advection flow of fluid, which exerts stronger dragging forces ontranslocating DNA and thereby lowering the DNA translocation speed. Based on thisphenomenon, they regulate DNA translocation by modulating the effective wall surfacecharge density through lateral gate voltages. The DNA translocation speed can be reducedat a rate of about 55 μm/s per 1 mV/nm through this method [15, 16]. Yen et al.  and Ai et al.  reported that applying positive gate voltage could also induce DNA-nanoporeelectrostatic interaction, which can regulate the DNA translocation speed. Lately, afunctionalized soft nanopore composed of a solid-state nanopore and a functionalizedsoft layer was demonstrated that can not only increase DNA capture rate by counterionconcentration polarization occurring at the nanopore mouth but can also decrease DNAtranslocation speed in the nanopore through electro-osmotic flow . Stolovitzky's group designed a nanopore with a metal-dielectric sandwichstructure capable of controlling the DNA translocation process with a single-baseaccuracy by tuning the trapping electric fields inside the nanopore [20–22]. This design is verified by molecular dynamics (MD) simulations, but there isno device reported so far due to its difficulty in fabrication. Applying an externalforce in the opposite direction of the electric field force on DNA could control a DNAstrand through a nanopore at a very slow speed. It can be achieved using optical tweezer  or magnetic tweezer  technologies. However, it is hard to extend these methods to sequence DNA inparallel , such as employing thousands of nanopores on a chip concurrently .
As we know, counterions in solutions can bind to DNA molecules, which may provide a dragforce on the DNA and reduce the translocation speed. Dekker's group found that DNAtranslocation time in LiCl salt solution is longer than that in KCl or NaCl solutions.Through MD simulation, they elucidated that the root of this effect is attributed to thestronger Li+ ion binding DNA than that of K+ and Na+. The DNA electrophoretic mobility depends on its surface charge density andthe applied voltage. If we can adjust the DNA surface charge density, it is possible toactively control the DNA translocation through a nanopore. It has been found thatMg2+ could reduce electrophoretic mobility of DNA molecule more thanNa+ at the same concentration without worrying about changing the DNAmolecule charge to a positive value . It is also known that Mg2+ is regularly used in adhering the DNAto inorganic surfaces, which may also reduce the DNA mobility. Inspired by the processof reducing effective surface charge density of a DNA molecule and that increasing theattractive force between DNA molecule and nanopore inner surface can retard DNA moleculetranslocation, we employed bivalent salt solution such as MgCl2 to observethe DNA translocation event through nanopores. We hope the two kinds of phenomena occurat the same time, thus extending the translocation time further more.
Results and discussion
In Figure 3, some outliers we call as ‘trappedevents’ have been observed in 1 M MgCl2 experiments. Although theprobability is small, the duration time of these events is 22 ms, about 17 times ofthe other events in 1 M MgCl2 experiments. As we know,Si3N4 surface in aqueous solution at pH 8.0 is negativelycharged. The correlations between Mg2+ ions on both the negatively chargedDNA and the Si3N4 surface can generate a net attraction force andthen help stick the DNA into the nanopore, but the phenomenon only obviously occurredfor the 7-nm diameter nanopore experiments. This is because the gap between the DNA andthe inner surface of the nanopore is also increased with the increasing nanoporediameter. With the increase of the gap, the net attraction force is not strong enough tostick the DNA, which leads to the trapped events unremarkable in the 22-nm diameternanopore.
In summary, the duration time for DNA translocation through a nanopore can be extendedwith the use of MgCl2 electrolyte. The side effect is that Mg2+ions may induce more DNA strands binding together, which is harmful to do DNA sequencingin MgCl2 electrolyte. Reducing the nanopore diameter can effectively reducethe occurrence number of the folded DNA translocation events. So, we can say thattheMgCl2 solution is a good choice for nanopore DNA sequencing experimentsif nanopore diameter can be reduced further.
YZ is a PhD candidate of Mechanical Design and Theory at the School of MechanicalEngineering, Southeast University, Nanjing, P.R. China. He is interested in nanoporefabrication and nanopore biosensing. LL is an assistant professor of Mechanical Designand Theory at the School of Mechanical Engineering, Southeast University, Nanjing, P.R.China. His research interests are biomolecule sensing and biodegradable materialsdesign. JS is an assistant professor of Mechanical Design and Theory at the School ofMechanical Engineering, Southeast University, Nanjing, P.R. China. Her research interestis micro-nano fluidic device design. ZN is a professor of Mechanical Manufacture andAutomation at the School of Mechanical Engineering, Southeast University, Nanjing, P.R.China. His research interests are minimally invasive medical devices and microfluidicdiagnostic device design and manufacture. HY is a professor of Mechanical Manufactureand Automation at the School of Mechanical Engineering, Southeast University, Nanjing,P.R. China. His research interest is advanced manufacturing technology. YC is aprofessor of Mechanical Design and Theory at the School of Mechanical Engineering,Southeast University, Nanjing, P.R. China. His research interests cover heat transfer,tribology, micro-nano fluidics, and micro-nano biomedical instrument.
The authors thank the financial support from the National Basic Research Program ofChina (2011CB707601 and 2011CB707605), the Natural Science Foundation of China(grantno.50925519), and the research funding for the Doctorate Program from ChinaEducational Ministry (20100092110051).
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