Surface Charge Density-Dependent DNA Capture through Polymer

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Surface Charge Density Dependent DNA Capture Through Polymer Planar Nanopores Zheng Jia, Junseo Choi, and Sunggook Park ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b14423 • Publication Date (Web): 29 Oct 2018 Downloaded from http://pubs.acs.org on November 1, 2018

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ACS Applied Materials & Interfaces

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Surface Charge Density Dependent DNA Capture

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Through Polymer Planar Nanopores

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Zheng Jia‡, Junseo Choi‡ and Sunggook Park*

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Mechanical & Industrial Engineering Department and Center for BioModular Multiscale

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Systems for Precision Medicine, Louisiana State University, USA

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‡These authors contributed equally to this work.

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* CORRESPONDING AUTHOR.

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Prof. Sunggook Park

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Mechanical & Industrial Engineering Department and Center for Bio-Modular Multiscale

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Systems for Precision Medicine

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3290M Patrick F. Taylor Hall, Louisiana State University, Baton Rouge, LA70803, USA

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Fax: +1 225 578 5924; Tel: +1 225 578 0279; E-mail: [email protected]

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KEYWORDS: polymer planar nanopore · nanoimprint lithography (NIL) · DNA translocation

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· surface charge density · effective driving force

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ACS Paragon Plus Environment

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ABSTRACT: Surface charge density of nanopore walls plays a critical role in DNA capture in

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nanopore-based sensing platforms. This paper studied the effect of surface charge density on

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the capture of double-stranded (ds) DNA molecules into a polymer planar nanopore

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numerically and experimentally. First, we simulated the effective driving force (𝐹𝑒𝑓𝑓) for the

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translocation of a dsDNA through a planar nanopore with different sizes and surface charge

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densities. Focus was given on the capture stage from the nanopore mouth into the nanopore

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by placing a rod-like DNA at the nanopore mouth rather than inside the nanopore. For

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negatively charged DNA and nanopore walls, electrophoretic driving force (𝐹𝐸𝑃) under an

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electric field is opposed by the viscous drag force by electroosmotic flow (𝐹𝐸𝑂𝐹). As the surface

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charge density of the nanopore wall becomes more negative, 𝐹𝐸𝑂𝐹 exceeds 𝐹𝐸𝑃 beyond a

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threshold surface charge density, 𝜎𝑡ℎ𝑟𝑒𝑠ℎ𝑜𝑙𝑑, where DNA molecules cannot be driven through

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the nanopore via electrophoretic motion. For a 10 nm diameter nanopore filled with 1× TE

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buffer, 𝜎𝑡ℎ𝑟𝑒𝑠ℎ𝑜𝑙𝑑 was determined to be -50 mC/m2. The simulation results were verified by

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performing dsDNA translocation experiments using a planar nanopore with 10 nm equivalent

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diameter imprinted on three polymer substrates with different surface charge densities. Both

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fluorescence observation and ionic current measurement confirmed that only nanopore

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devices with the surface charge density less negative than 𝜎𝑡ℎ𝑟𝑒𝑠ℎ𝑜𝑙𝑑 allowed DNA

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translocation, indicating that the simulated 𝜎𝑡ℎ𝑟𝑒𝑠ℎ𝑜𝑙𝑑 value can be used as a parameter to

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estimate the translocation of biopolymers in the design of nanopore devices.

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INTRODUCTION

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Nanopore-based fluidic devices are promising tools for DNA sensing due to their label-free,

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high throughput and low cost features.1-2 The sensing mechanism is straightforward: when a

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negatively charged DNA molecule passes a nanopore filled with electrolyte, ion transport

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through the nanopore is temporarily blocked, giving rise to a transient current signal as a

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molecular signature. The biophysical information can be deduced from the electrical signal.

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The nanopore-based DNA sensing platforms can be classified into two categories: biological

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nanopores and solid-state nanopores. Biological nanopores are pore-forming proteins with

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well-defined pore sizes and they can easily be modified by advanced biology technologies.3-4

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Solid-state nanopores are usually fabricated in a thin membrane of an inorganic material (e.g.

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SiO2, Si3N4, and glass) using high energy electron and ion beams.5-7 Compared with biological

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nanopores, solid-state nanopores can have controllable pore sizes ranging from sub-nanometer

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scale to tens of nanometers depending on the types of biomolecules to be interrogated and the

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target applications.4,

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experimental conditions (e.g. electrolyte concentrations, pH and temperature),8 which makes

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solid-state nanopores a good alternative to biological nanopores. However, due to the

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requirement of high-ends nanofabrication tools in the fabrication of solid-state nanopores with

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nanometer precision, it is still challenging and time consuming to produce nanopore devices

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in large scale.

8-9

They are also mechanically robust and stable under various

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Irrespective of the types of nanopore sensing platforms used, efficient capture of DNA

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molecules into nanopores is the basic but challenging operational requirement.10-13 DNA

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capture into a nanopore involves two stages. In the first stage, DNA molecules far from the

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nanopore are driven to nanopore mouth by diffusion and drift under an electric field extending

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out of the nanopore capture radius.14-16 The first stage is dominated by DNA molecule

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diffusion, and several methods have been proposed to facilitate the DNA capture in this stage,

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which include application of higher driving voltage,17-18 use of a buffer system with a salt

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gradient15, 19 and metal layer deposition on the nanopore wall.10 Once a DNA molecule reaches

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the nanopore mouth, in the second stage the coiled DNA molecule has to overcome the

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entropic barrier by putting a portion of its chain, sometimes the chain end or folded chain,

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into the nanopore.14, 16, 20-21 To overcome the entropic barrier, a sufficient driving force needs

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to be provided on the DNA molecule in the direction from the nanopore mouth to the

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nanopore. In order to facilitate DNA translocation at this stage, a tapered gradient inlet22-23 or

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pillar arrays24-25 have been integrated prior to a nanopore or nanochannel. For vertical nanopore

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membranes a threshold driving voltage of less than 100 mV is usually required to overcome the

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entropic barrier26, while for planar nanopore/nanochannel devices, the threshold driving voltage

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varies depending on the fluidic structures formed prior and past the nanopore/nanochannel. It

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should also be noted that, usually, the capture of long chain DNA molecules in vertical nanopores

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is limited by the diffusion in the first capture stage, whereas short chain DNA capture is limited

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by the entropy barrier in the second capture stage.12, 15

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This paper focuses on the study of DNA capture in the second capture stage, i.e. introduction of

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DNA molecules from the nanopore mouth into the nanopore, where the entropic barrier needs to

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be overcome by a sufficient driving force. Commonly used nanopores are negatively charged; for

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example, silicon (Si) based nanopores and most thermoplastics such as polymethyl methacrylate

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(PMMA). Electroosmotic flow (EOF) induced by negative surface charge of the nanopore wall is

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the intrinsic resistance to the electrophoretic motion of DNA for both capture stages.11-12, 27-29 In

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particular, for DNA molecules delivered to the nanopore mouth, a strong EOF leads to a high

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hydrodynamic drag force, 𝐹𝐸𝑂𝐹 against the electrophoretic force, 𝐹𝐸𝑃, on the unscreened DNA

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molecule in electric field. Both 𝐹𝐸𝑂𝐹 and 𝐹𝐸𝑃 correspond to the force in the direction of the

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nanopore axis. Therefore, the effective driving force, 𝐹𝑒𝑓𝑓, on a DNA molecule at the pore mouth

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in a negatively charged nanopore can be expressed as the difference of 𝐹𝐸𝑃 and 𝐹𝐸𝑂𝐹,28, 30-32

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𝐹𝑒𝑓𝑓 = 𝐹𝐸𝑃 ― 𝐹𝐸𝑂𝐹

(1)

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The effect of diffusion on the motion of DNA is negligible because the molecule is already in

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the confined zone of the nanopore mouth. Based on Equation (1), a high 𝐹𝐸𝑂𝐹 results in a low 𝐹𝑒𝑓𝑓,

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which may hinder the DNA molecule from overcoming the entropic barrier. If 𝐹𝑒𝑓𝑓 has a positive

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value, DNA can be pulled into the pore, and vice versa. The threshold surface charge density,

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𝜎𝑡ℎ𝑟𝑒𝑠ℎ𝑜𝑙𝑑, for DNA translocation is defined as the surface charge density of the nanopore wall

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where 𝐹𝐸𝑃 is balanced by 𝐹𝐸𝑂𝐹, namely 𝐹𝑒𝑓𝑓 = 0. It should also be noted that for positively charged

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nanopore devices, the directions of 𝐹𝐸𝑃 and 𝐹𝐸𝑂𝐹 are identical.

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In order to facilitate the design of solid-state nanopore devices and material selection for the

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devices, it is important to gain extensive knowledge on the effect of the surface charge on 𝐹𝑒𝑓𝑓

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for DNA translocation. The inversion of 𝐹𝑒𝑓𝑓 for DNA translocation by controlling the surface 5



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charge density of nanopores was first predicted by Luan et al. by means of all atom molecular

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dynamic simulations with a rod-like stretched poly(dA20)poly(dT20) DNA duplex in a Si3N4

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nanopore.33 𝜎𝑡ℎ𝑟𝑒𝑠ℎ𝑜𝑙𝑑 was also predicted based on continuum model simulation28,

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calculating 𝐹𝑒𝑓𝑓 of a rod-like stretched DNA molecule positioned inside nanopore. However,

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such models are not applicable for studying the capture of DNA for the second capture stage

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from the nanopore mouth to the nanopore because of the impractical assumption of the DNA

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position. Their results only helped understand 𝐹𝑒𝑓𝑓 on the DNA translocating inside the

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nanopore32, and DNA translocation kinetics and interactions within nanopore.36 To study the

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DNA capture at the second capture stage, 𝐹𝑒𝑓𝑓 should be calculated with a partially stretched

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DNA located in front of the nanopore. Moreover, the simulation results were rarely

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demonstrated by translocation experiments due to low throughput and time-consuming device

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fabrication. For example, He et al. simulated that DNA capture can also be manipulated by surface

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charge modulation using a gate electrode.11

34-35

by

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In this work, we systematically studied the effect of surface charge density on 𝐹𝑒𝑓𝑓 for DNA

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capture through polymer planar nanopores by numerical simulation and experimentation.

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Compared to traditional vertical nanopores formed in a thin membrane, planar nanopores are

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ideal platforms for optical observation of the DNA translocation, because no additional

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structure (for example, zero-mode waveguide) is required to enhance the signal-to-noise ratio

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for the observation.37-38 Besides, the entire translocation process can be observed, including

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diffusion, drift and threading.39-40 Another important advantage of the planar design is that 6


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multiple nanopore devices41 can be formed on polymer substrates with various surface

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properties via a single step molding process, for example nanoimprint lithography (NIL),

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which can alleviate the scale-up issue of solid-state nanopore manufacturing.42-44 Using

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COMSOL 5.0 (COMSOL, Inc.), we first simulated 𝐹𝑒𝑓𝑓 on a rod-like double stranded (ds) DNA

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located at the pore mouth of a planar 10 nm pore with different surface charge density values.

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In order to verify the simulation results, optical observation and electrical detection were

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carried out for λ-DNA translocation through a 10 nm equivalent diameter nanopore imprinted

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in polymer substrates with different surface charge densities: poly(ethylene glycol) diacrylate

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(PEGDA), poly(methyl methacrylate) (PMMA) and cyclic olefin copolymer (COC).

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Experimental results were in good agreement with the simulation results in that the DNA

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translocation was observed only for the polymer nanopore with positive 𝐹𝑒𝑓𝑓.

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RESULTS AND DISCUSSION

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Numerical simulation on effective driving force for DNA translocation

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DNA molecules can be driven into a nanopore by electrophoretic motion only when the

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surface charge density of the nanopore wall is less negative than 𝜎𝑡ℎ𝑟𝑒𝑠ℎ𝑜𝑙𝑑 due to a lower

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hydrodynamic drag force caused by weaker EOF.31-33 𝜎𝑡ℎ𝑟𝑒𝑠ℎ𝑜𝑙𝑑 of the nanopore device can be

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predicted by performing COMSOL simulation on the electrokinetic behavior of a DNA

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molecule in the nanopore device with different surface charge density values.31, 7


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Our


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structural model consists of a planar nanopore with 10 nm diameter and 60 nm length

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connected to a tapered inlet and outlet structure. The surface charge density of the entire

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device wall varied from -20 to -80 mC/m2. To simulate 𝐹𝑒𝑓𝑓 of a rod-like, double-stranded λ-

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DNA molecule into a planar nanopore, a cylindrical rod with a diameter of 2 nm and the

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surface charge density of -0.15 C/m2 31, 45 was placed at the nanopore mouth. This initial DNA

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location assumes that one of the DNA chain ends was pre-stretched and approached the

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nanopore mouth. 1× TE buffer was chosen as electrolyte in consideration of the subsequent

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fluorescence observation. Supporting Information (SI) Figure S1 shows the axisymmetric

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structural model and boundary conditions used for the simulation. It should be noted that this

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model is similar to the planar nanopore structure used for experimental verification. With the

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model, 𝐹𝐸𝑃 and 𝐹𝐸𝑂𝐹 were calculated by solving the coupled Poisson-Nernst-Planck (PNP) and

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Navier–Stokes equations, which gives 𝐹𝑒𝑓𝑓 from Equation (1). Details regarding the simulation

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model and parameters used can be found in the method section and in SI Table S1.

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Figure 1 shows 𝐹𝐸𝑂𝐹 and 𝐹𝐸𝑃 exerting on the DNA molecule surface placed in front of a 10

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nm pore as a function of surface charge density of the nanopore wall. It should be reminded

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that 𝐹𝐸𝑂𝐹 and 𝐹𝐸𝑃 correspond to the force in the direction of the nanopore axis. 𝐹𝐸𝑂𝐹 was obtained

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by contour integration of 𝐹𝐸𝑂𝐹, 𝑖𝑧 around the DNA contour surface, 𝐹𝐸𝑂𝐹 = ∮𝐹𝐸𝑂𝐹,𝑖𝑧, where

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𝐹𝐸𝑂𝐹, 𝑖𝑧 is the hydrodynamic drag force at a given DNA surface. As shown in Figure 1a, 𝐹𝐸𝑂𝐹

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increases as the surface charge density of the nanopore wall becomes more negative. Figure 1b

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shows the spatial distribution of the simulated EOF velocity in the direction of the nanopore 8


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axis for three representative surface charge density values of -20 mC/m2, -30 mC/m2 and -80

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mC/m2, respectively. More negative surface charge density leads to a higher EOF velocity

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against the electrophoretic motion of the molecule.

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On the other hand, as shown in Figure 1c, 𝐹𝐸𝑃 initially increases, shows a maximum at -30

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mC/m2 and then decreases as the surface charge density value becomes more negative.

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According to the Coulomb’s law, 𝐹𝐸𝑃 applied on the DNA surface depends on the electric field

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in the nanopore axis direction (i.e. z-axis in simulation model), 𝐸𝑧, by 𝐹𝐸𝑃 = ∮𝑞𝐸𝑧 around the

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DNA surface contour. While a constant external voltage of 1 V is applied between top and

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bottom sides of the nanopore, different surface charges on the device wall modify 𝐸𝑧, leading

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to a dependence of 𝐹𝐸𝑃 on the surface charge density. Figure 1d shows the horizontal

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distribution of 𝐸𝑧 at z = -45 nm, which is 15 nm away from the front end of the DNA molecule.

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The direction of 𝐸𝑧 near the tapered wall was reversed from the direction of the electric field

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by applied voltage due to the negative surface charge of the wall. The reversed electric field

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decays with the distance from the tapered wall. As the surface charge density becomes more

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negative, the magnitude of the reversed 𝐸𝑧 near the tapered wall increases, resulting in a

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reduction of 𝐸𝑧 at and near the DNA molecule and thus 𝐹𝐸𝑃. This accounts for the behavior of

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𝐹𝐸𝑃 for the surface charge density ranging from -30 mC/m2 to -80 mC/m2.

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Figure 1. (a) 𝐹𝐸𝑂𝐹 for a rod-like DNA placed at the mouth of a 10 nm diameter nanopore having

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various surface charge densities; (b) Spatial distribution the simulated EOF velocity in the

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direction of the nanopore axis for three representative surface charge density values of -20

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mC/m2, -30 mC/m2 and -80 mC/m2, respectively; higher surface charge density (absolute value)

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leads to a higher EOF velocity at or near the DNA molecule. (c) 𝐹𝐸𝑃 for a rod-like DNA placed

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at the mouth of a 10 nm diameter nanopore having various surface charge densities; (d)

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Horizontal distribution of electric field in nanopore axis direction, 𝐸𝑧, at z = -45 nm for three

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representative surface charge density values of -20 mC/m2, -30 mC/m2 and -80 mC/m2,

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respectively. Higher surface charge (absolute value) density leads to stronger built up electric field

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near tapered wall and it has negative effect on 𝐸𝑧 at DNA surface. 10


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The behavior of 𝐹𝐸𝑃 for the surface charge density below -30 mC/m2 can be explained by

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the enlarged Debye length (or electric double layer) resulting from the surface charge of the

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device walls. As shown in SI Figure S2, The nanopore device with more negative surface charge

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density has a higher number of charge carriers inside the nanopore. The increased number of

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charge carriers inside the nanopore results in a decreased nanopore resistance and thus a lower

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𝐸𝑧 within nanopore, which subsequently leads to a higher 𝐸𝑧 outside the nanopore. This

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explains the behavior of 𝐹𝐸𝑃 for the surface charge density below -30 mC/m2.

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Figure 2a presents 𝐹𝑒𝑓𝑓 obtained from the simulated 𝐹𝐸𝑃 and 𝐹𝐸𝑂𝐹 using Equation (1) as a

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function of surface charge density for different nanopore diameters. The solid triangle of

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Figure 2a presents 𝐹𝑒𝑓𝑓 for a 10 nm diameter nanopore obtained from Figure 1a and 1c.

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Overall, 𝐹𝑒𝑓𝑓 decreases as the surface charge density becomes more negative. Interestingly, the

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sign of 𝐹𝑒𝑓𝑓 is reversed at a threshold surface charge density of -50 mC/m2, indicating that the

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DNA molecule cannot be driven into the nanopore when the surface charge density is more

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negative than this threshold surface charge density value.

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Figure 2. (a) 𝐹𝑒𝑓𝑓 for a rod-like DNA placed at the mouth nanopores with different pore sizes

3

and various surface charge densities; (b) 𝐹𝑒𝑓𝑓 for a rod-like DNA placed inside nanopores with

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different pore sizes and various surface charge densities. A positive value of 𝐹𝑒𝑓𝑓 indicates a

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favor of DNA entrance into the nanopore, and vice versa. Insets indicate DNA location in

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simulation model.

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We further simulated 𝐹𝑒𝑓𝑓 versus surface charge density for different nanopore diameters of

8

3 nm and 5 nm which is also shown in Figure 2a. For nanopores with the same surface charge

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density, 𝐹𝑒𝑓𝑓 decreases with decreasing the nanopore size, indicating that it is more difficult

10

to drive the DNA molecule through smaller nanopores by electrophoretic motion. The

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negative effect of the tapered inlet structure on 𝐸𝑍 and 𝐹𝑒𝑓𝑓 becomes smaller for larger

12

nanopores because the DNA is further away from the tapered wall. 𝜎𝑡ℎ𝑟𝑒𝑠ℎ𝑜𝑙𝑑 is reduced from

13

-50 mC/m2 to -41 mC/m2 as the pore diameter decreases from 10 nm to 5 nm. For the 3 nm 12


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nanopore, 𝐹𝑒𝑓𝑓 is always negative irrespective of the magnitude of the negative surface charge

2

density, indicating that it is not possible to drive the DNA molecule through the pore (when

3

1× TE buffer is used).

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Our results indicate that the negative effect from the tapered inlet structure on 𝐹𝑒𝑓𝑓 can be

5

reduced with a flat inlet structure. SI Figure S3 shows the simulation model with a flat inlet

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structure, and 𝐹𝐸𝑃, F𝐸𝑂𝐹 and 𝐹𝑒𝑓𝑓 versus surface charge density for a nanopore of 10 nm

7

diameter. Both 𝐸𝐸𝑃 and 𝐸𝐸𝑂𝐹 increase with surface charge density. While 𝐹𝑒𝑓𝑓 decreases with

8

the surface charge density, the 𝐹𝑒𝑓𝑓 values are always positive irrespective of the surface charge

9

density used for the simulation, indicating that DNA molecules reaching at the nanopore

10

mouth are readily introduced into the nanopore. An interesting conclusion can be deduced

11

from the simulation results; a tapered inlet structure built prior to a nanopore or nanochannel

12

has been known to be helpful to reduce the entropic barrier to capture DNA molecules into a

13

nanopore or nanochannel.22-24 However, this merit is compromised by the negative effect of

14

reduced or even inverted 𝐹𝑒𝑓𝑓. Therefore, this trade-off needs to be considered in the design

15

of nanopore or nanochannel devices to improve the capture of the DNA molecules.

16

For comparison, we also simulated 𝐹𝑒𝑓𝑓 for a DNA molecule placed inside the nanopore of

17

different diameters as shown in Figure 2b. The simulation model is shown in SI Figure S4.

18

Similar to the previous case of DNA at nanopore mouth, 𝐹𝑒𝑓𝑓 decreases as the surface charge

19

density becomes more negative and increases along with the nanopore size. Bringing the DNA

20

molecule in the nanopore leads to an increase in both 𝐹𝐸𝑃 and 𝐹𝐸𝑂𝐹 compared to those values for 13



1

the DNA at the nanopore entrance. But the magnitude of this increase for 𝐹𝐸𝑃 and 𝐹𝐸𝑂𝐹 varies with

2

the pore size. As the pore size becomes smaller, the increase of 𝐹𝐸𝑂𝐹 becomes dominant over the

3

increase of 𝐹𝐸𝑃, leading to an increase in the negative slope of the 𝐹𝑒𝑓𝑓 vs 𝜎 curves (Figure 2b).

4

The 𝜎𝑡ℎ𝑟𝑒𝑠ℎ𝑜𝑙𝑑 values for 3 and 5 nm pores are -22 and -61 mC/m2, respectively. For the 10 nm

5

pore, 𝐹𝑒𝑓𝑓 show always positive values within the range of the surface charge density values

6

investigated. Consequently, for a certain combination of 𝜎 and nanopore size, a DNA molecule

7

inside the nanopore may be pulled out of the nanopore easily while the same DNA is hard to enter

8

the nanopore because it is repelled from the nanopore at the pore entrance. This is the case for 10

9

nm nanopore and with the surface charge density less than -50 mC/m2. It is not easy to

10

experimentally verify the easy pull-out of DNA from the nanopore because it is difficult to

11

introduce the DNA into the nanopore. An optical tweezer may help to reveal the forces of DNA at

12

the nanopore entrance and within nanopore.32 Nevertheless, the results clearly show that the

13

location of the DNA molecule is critical to properly understand the capture behavior of the

14

molecule from the nanopore mouth into the nanopore through the numerical simulation.

15

It is worthwhile to compare our results with 𝜎𝑡ℎ𝑟𝑒𝑠ℎ𝑜𝑙𝑑 calculated from the MD simulation by

16

Luan et al. For a rod-like stretched poly(dA20)poly(dT20) DNA duplex placed in the center of a

17

Si3N4 nanopore of 6 nm diameter and 6.4 nm length, the MD simulation resulted in a 𝜎𝑡ℎ𝑟𝑒𝑠ℎ𝑜𝑙𝑑 of

18

-29 mC/m2 (-0.18 e/nm2), which was less negative than the 𝜎𝑡ℎ𝑟𝑒𝑠ℎ𝑜𝑙𝑑 of -41 mC/m2 for 5 nm

19

nanopore in our continuum COMSOL simulation. The different 𝜎𝑡ℎ𝑟𝑒𝑠ℎ𝑜𝑙𝑑 values can be attributed

20

to the different nanopore lengths (6.4 nm vs. 60 nm) and ion concentrations used for the two

21

simulations (1 M KCl vs. 10 mM KCl).

22 14


Page 14 of 42

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1

Experimental verification

2

Fabrication of polymer planar nanopore devices. In order to verify the simulation results,

3

DNA translocation experiments were designed and conducted using polymer nanopore devices

4

with different surface charge densities. Previous studies show that the surface charge density

5

of O2 plasma treated COC substrates is higher than that of O2 plasma treated PMMA.46 Also,

6

poly-ethylene glycol (PEG) is a well-known coating material to suppress EOF on a target

7

surface.47-48 Therefore, in this study we chose PEGDA, PMMA and COC as substrates for

8

device fabrication. We used the procedure developed in our group to produce polymer

9

nanopore and nanochannel devices, as illustrated in SI Figure S5.49-50 First, a Si master mold

10

with microchannel fluidic networks and planar nanopore was fabricated by a combination of

11

photolithography, wet etching of Si, and focused ion beam (FIB) milling. The patterns on the

12

Si mater were then transferred to a UV curable resin mold, which exerts excellent demolding

13

features. Finally, the planar nanopore device patterns were imprinted on target polymer

14

substrates via UV- or thermal-NIL.

15

Figure 3 shows SEM images of imprinted planar nanopores on different polymer substrates.

16

In comparison with the original Si mater mold, imprinted nanopores show good replication

17

fidelity. It should be mentioned that planar nanopore formed by FIB milling has a parabolic

18

shape, not circular. Thus, their equivalent circular pore diameter was calculated with the pore

19

width and depth measured from the SEM images, which amounts to 25 ± 5.3 nm before

20

thermal bonding to a cover plate. Also, tapered inlet /outlet and pillar arrays were built at both 15



1

sides of the nanopore to pre-stretch DNA. It should be mentioned that our numerical

2

simulation model is 2D axisymmetric, therefore representing 3D nature of DNA molecules and

3

nanopore structures. On the other hand, the planar nanopore that were used in our

4

experimental work is a quasi-2D system in that it was machined in a top-down manner.

5 6

Figure 3. SEM images of Si master mold (a, e) and imprinted planar nanopore on PEGDA (b,

7

f), PMMA (c, g) and COC (d, h) substrates. Nanopillar arrays with 300 nm gap and tapered

8

inlet/outlet with an angle of 60 ° are designed to pre-stretch DNA. Nanopore length is 60 nm,

9

defined by AFM and SEM measurements and this value was used for all COMSOL simulation.

10

Images were taken under 5 kV, 5.9 pA by FEI Quanta 3D instrument. Despite deposition of a

11

4 nm Au/Pd layer on polymer chips to avoid charging effect, the nanopore feature on PMMA

12

substrate was deformed (enlarged and bent) under high magnification by electron beams (g).

16


Page 16 of 42

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1

Compared with original Si master mold, all imprinted nanopores have good replication fidelity.

2

Scale bars, 3 µm in white and 100 nm red.

3 4

For all imprinted substrates, we used O2 plasma treated COC sheets as cover plate for bonding

5

at 70°C, 1 MPa for 15 min.51 These hybrid devices are denoted as PEGDA-COC, PMMA-COC

6

and COC-COC, respectively. The thermal fusion bonding process intrinsically utilizes mixing

7

of polymer chains between the imprinted substrate and the cover plate. Thus, we took

8

advantage of the thermal fusion bonding process as a process to reduce the nanopore size

9

further. The pore sizes for the enclosed planar nanopore devices after thermal fusion bonding

10

were estimated by measuring their conductance filled with 1 M KCl solution and comparing

11

the measured conductance values with the simulated conductance values of different nanopore

12

sizes. The equivalent pore diameters estimated for all the polymer devices showed similar

13

values of 10.3 ± 3.3 nm as shown in SI Table S2 and SI Figure S6.

14 15

Surface charge density measurement. Prior to DNA translocation experiments, surface charge

16

densities of PEGDA-COC, PMMA-COC and COC-COC devices were experimentally

17

determined by measuring conductance through the nanochannel devices for different KCl

18

concentration. The surface charges on polymer substrates are determined by several factors

19

including the diploe formation induced by the molecular structure at the surface, the ability

20

to deprotonate from the polymer chains, and dissociation of surface chemical groups such as 17



Page 18 of 42

1

carboxyl group. Also, the oxygen plasma treatment performed prior to the cover plate bonding

2

further modifies the ability to produce surface charges. The polyethylene glycol (PEG) group

3

in PEGDA is known to be close to neutral in aqueous solution while the carboxylic group

4

present in methyl methacrylate monomer makes PMMA highly negatively charged. When the

5

KCl concentration is greater than 10-2 M, measured conductance fits linearly to the theoretical

6

bulk conductance. At lower salt concentration, however, conductance saturates at a value

7

which is dependent on the surface charge density and device geometry. From the transition

8

point on the plot, the effective surface charge density, 𝜎𝑠 , can be calculated through the

9

following equation46, 52

10

𝜎𝑠 =

103𝑁𝐴 ∗ 𝑒 ∗ 𝑤 ∗ ℎ ∗ 2𝜇𝑜𝑝𝑝 ∗ 𝑐𝑡

(2)

(𝑤 + ℎ)(𝜇𝐾 + + 𝜇𝐶𝑙 ― )

11

, where 𝑁𝐴 is the Avogadro constant, 𝑒 is the elementary charge, 𝑤 and ℎ are the width and

12

height (depth) of the nanochannel, 𝜇𝐾 + and 𝜇𝐶𝑙 ― are the ion mobilities of 𝐾 + and 𝐶𝑙 ― ions,

13

𝜇𝑜𝑝𝑝 is the mobility of the counterions, and 𝑐𝑡 is the transition concentration between the two

14

regimes. Details on the derivation of Equation (2) are shown in SI.

15

The hybrid devices with five nanochannels were fabricated for this purpose as shown in

16

Figure 4a-d. Figure 4e shows the nanochannel conductance as a function of salt concentrations

17

for different hybrid devices. The effective surface charge densities obtained from Equation (2)

18

were -24.1 mC/m², -51.7 mC/m², and -73.8 mC/m² for PEGDA-COC, PMMA-COC and COC-

19

COC devices, respectively.52 Surface charge density of O2 plasma treated PMMA and COC are

20

close to the values reported in the previous studies.51,

53

18


It should be reminded that the

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1

simulated 𝜎𝑡ℎ𝑟𝑒𝑠ℎ𝑜𝑙𝑑 of the 10 nm pore (filled with 1× TE) was -50 mC/m2, from which we

2

hypothesize that DNA molecules could only be driven through the nanopore made of PEGDA-

3

COC, not devices made of PMMA-COC and COC-COC.

4

It should be mentioned that different experimental conditions will change surface charge

5

density of polymer substrate. For example, surface charge becomes more negative with an

6

increase in the pH value. The understanding of surface charges on different polymer substrates,

7

i.e. polymer atomic structure-surface charge correlations, under various experimental

8

conditions requires atomistic modeling approaches such as molecular dynamic simulation or

9

quantum mechanical modeling such as density functional theory. But this is beyond the scope

10

of this work.

11 12

Figure 4. SEM images of nanochannel devices for surface charge density calculation and surface

13

charge density calculation results; (a) Si master mold, (b) UV imprinted nanochannels on PEGDA 19



1

substrate, (c) thermal imprinted nanochannels on PMMA substrate, and (d) thermal imprinted

2

nanochannels on COC substrate. (e) Nanochannel conductance for PEGDA-COC, PMMA-

3

COC and COC-COC as a function of salt concentration. At high salt concentration,

4

nanochannel conductance is dependent on bulk solution concentration. At low salt

5

concentration, nanochannel conductance saturates at a value, which is dependent on surface

6

charge density of nanochannel walls. The transition concentration, 𝑐𝑡, between these two

7

regimes is indicated in graph.

8 9

Optical and electrical detection of λ-DNA translocation. In order to verify the surface

10

charge dependent DNA capture behavior in nanopores, we conducted optical observation

11

and electrical measurements for λ-DNA translocation through nanopore devices with tapered

12

inlet/outlet made of different hybrid substrates. We used 1× TE buffer to ensure enough

13

binding strength between YOYO-1 dye and λ-DNA backbones.54 Figure 5 shows

14

fluorescence images when the stained λ-DNA molecules were driven towards the nanopore

15

electrophoretically. For all the devices, fluorescence signals from λ-DNA molecules were

16

seen in the nanopillar array region up the nanopore mouth, indicating that DNA molecules

17

captured from the microchannels were accumulated in the nanopillar array region. However,

18

their translocation behavior through the nanopore was different. For PEGDA-COC device,

19

λ-DNA molecules translocated through the nanopore under bias voltage as low as 100-400

20

mV, which is similar to driving voltages reported for λ-DNA translocation through vertical 20


Page 20 of 42

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1

nanopores.20, 26, 55 We also observed that stretched λ-DNA molecules hesitated for a short

2

time prior to entering into the nanopore, similar to a previous report for DNA translocation

3

through a planar nanochannel.23 When applying high driving voltage, this phenomenon

4

became more obvious as λ-DNA molecules piled up at the nanopore entrance region while

5

the molecule can still enter into the nanopore. For PMMA-COC and COC-COC devices, on

6

the other hand, λ-DNA molecules were stretched by pillar arrays and delivered to the

7

nanopore mouth, but could not enter into the nanopore. We reversed the bias several times

8

and also increased the driving voltage up to 10 V. However, no translocation events were

9

observed while the molecules piled up prior to the nanopore.

21



1 2

Figure 5. Sequential fluorescence images of stained λ-DNA translocation through planar

3

single nanopore devices fabricated on (a) PEGDA (-24.1 mC/m2), (b) PMMA (-51.7 mC/m2)

4

and (c) COC (-73.8 mC/m2). λ-DNA molecules passed through PEGDA nanopore under bias

5

voltage as low as 100 mV but they could not do through PMMA and COC nanopore even

6

under 10 V. The white arrows in Figure 5a indicate translocated λ-DNA molecules through

7

PEGDA nanopore. Scale bar, 5 µm in white.

8

22


Page 22 of 42

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1

Immediately after fluorescence observation, we used the same nanopore devices for

2

electrical detection of DNA translocation by using the same 1× TE buffer as electrolyte. Figure

3

6a shows ionic current traces measured from PEGDA-COC, PMMA-COC and COC-COC

4

devices under a driving voltage of 1 V, which is the maximum value that can be applied with

5

a commercial patch clamp used. Electrical measurement results were in an agreement with the

6

optical observation. Current blockage events from translocated λ-DNA molecules occurred

7

only for PEGDA-COC device, while no current blockage events were observed for PMMA-

8

COC and COC-COC devices. Both the optical and electrical measurements support our

9

hypothesis set based on the simulation results that λ-DNA molecules can be threaded into the

10

planar nanopore only when the surface charge density of the device is less negative than

11

𝜎𝑡ℎ𝑟𝑒𝑠ℎ𝑜𝑙𝑑. In other words, the simulated 𝜎𝑡ℎ𝑟𝑒𝑠ℎ𝑜𝑙𝑑 value may be the parameter that can be used

12

as a guide to estimate the translocation of biopolymers through planar nanopore in the

13

geometrical design and material selection of nanopore devices in consideration of nanopore

14

geometry and surface charge density.

23



1 2

Figure 6. (a) Long duration current trace for stained λ-DNA translocation through PEGDA,

3

PMMA and COC based planar nanopores; (b) Scatter plot of DNA translocation events in

4

PEGDA device; (c) Typical unstretched, partially stretched and fully stretched current

5

blockade events.

6 7

For vertical nanopore membranes, the blockade event of a nanopore by a DNA molecule at

8

low salt concentration usually gives rise to an increased transient current peak due to the

9

enhanced flow of counterions along the DNA molecular chain.56 In our experiments, despite 24


Page 24 of 42

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1

the use of 1× TE buffer with low salt concentration, all the transient current peaks were

2

downward. The reason of decreased transient current peaks at low salt concentration is still

3

not clear, but such opposite results have been reported for DNA translocation through

4

transverse electrodes57-58 and carbon nanotubes.59 One possible explanation is that our planar

5

nanopore is a quasi-2D structure and the DNA molecule may block more efficiently the

6

nanopore entrance, as compared to the 3D nature of vertical nanopores. The tapered inlet may

7

also help the DNA molecule to block the nanopore entrance efficiently. Moreover, the event

8

rate of λ-DNA translocation in PEGDA device (0.15 event/ 10 s for 5 ng/μl λ-DNA under a

9

1000 mV driving voltage) was significantly low compared to that reported for vertical

10

nanopores (> 1 event/ 10 s for 5 ng/μl λ-DNA under a 200 mV driving voltage).10, 15 Possible

11

explanation for the low event rate maybe thicker EDLs at the DNA molecule and the nanopore

12

surfaces that make DNA translocation more difficult.10, 60-61 As presented in SI Figure S7a,

13

𝐹𝑒𝑓𝑓 was simulated with different electrolyte concentration, higher concentration KCl leads to

14

higher 𝐹𝑒𝑓𝑓 due to thinner EDL, especially for nanopore with more negative surface charge

15

density. Ionic current measurement confirmed a higher event rate of 1.6 event/ 10 s for the

16

same PEGDA device filled with 1 M KCl, as shown in SI Figure S7b.

17

We further analyzed the current transient peaks from λ-DNA translocation events in

18

PEGDA-COC device. Figure 6b shows a scattered plot for the current drop and dwell time.

19

Three regimes can be identified: (1) short dwell time regime, (2) short current drop regime,

20

and (3) transition between (1) and (2) regimes. In Figure 6c is shown a representative current 25



1

transient peak corresponding to each regime. Different current transient peaks indicate that

2

λ-DNA molecules translocated through the nanopore in different translocation modes, i.e.

3

unstretched, partially stretched, and fully stretched, which is similar to λ-DNA translocation

4

observed in vertical solid-state nanopores.20-21 Generally speaking, fully stretched

5

translocation events lead to a smaller current drop, longer dwelling time. Partially stretched

6

or unstretched (e.g. single local folded, double local folded or fully folded) events show a larger

7

current drop and shorter dwelling time. In addition to high rate fabrication modalities,

8

polymer planar nanopores are advantageous in that both optical and electrical measurements

9

can be performed in a single chip and that high throughput manufacturing modalities are

10

readily available. Our results indicate that polymer planar nanopores can be an alternative

11

platform to vertical nanopore membranes in obtaining biophysical information of

12

biopolymers.

13

The conformation of a DNA molecule in nanoscale confinement depends on the relative size of

14

the nanoscale confinement to the characteristic molecular scales such as the radius of gyration and

15

persistence length.62 The diameter of the axisymmetric tapered nanopore inlet up to the length of

16

the cylinder DNA used in the numerical simulation varies from 10 to 80 nm while the persistence

17

length of a double stranded DNA at 10 mM salt concentration amounts to 50 - 80 nm. Therefore,

18

the DNA in this region will have the conformation in the classical Odijk regime, meaning that the

19

DNA chain can only store contour through a serious of successive deflections with the wall,

20

characterized by the deflection length63 and that the use of a cylinder to model the DNA in the

21

simulation is justified. On the other hand, both the width and depth of the tapered inlet used for 26


Page 26 of 42

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1

DNA translocation experiments varies from tens of nanometers to several hundred nanometers.

2

Therefore, only the small portion of the -DNA molecule adjacent to the nanopore would have

3

conformation in the Odijk regime while most of the DNA chain in the tail will be in the de Gennes

4

regime with the diameter of the DNA blobs varying along the nanopore axis.

5

It should be noted that DNA transport dynamics depends not only on nanopore material and

6

nanopore design and diameter, but also on other parameters such as electrolyte concentration, pH.

7

temperature, light illumination, voltage, etc. This is a topic of research that needs to be performed

8

to fully develop planar nanopore devices. We performed the verification experiments with the

9

planar nanopore of 10 nm diameter because it is still challenging to manufacture sub-10 nm planar

10

nanopores in a controllable manner in different polymer substrates. Despite such a shortcoming,

11

we believe that our work provides a new insight on the capture of DNA in planar polymer nanopore

12

devices.

13 14

CONCLUSIONS

15

The effect of the surface charge density of planar nanopore devices with tapered inlet/outlet

16

on the capture of λ-DNA molecules was studied numerically and experimentally. In our

17

continuum model, DNA was positioned in front of nanopore rather than inside nanopore. The

18

simulation results indicate that 𝐹𝑒𝑓𝑓 can be lowered or even reversed when the nanopore

19

device is made of materials with highly negative surface charge, hindering DNA translocation

20

by electrophoretic motion. This effect becomes more significant for smaller nanopores and

21

nanopores with a tapered inlet structure. The simulated 𝜎𝑡ℎ𝑟𝑒𝑠ℎ𝑜𝑙𝑑 at which the sign of 𝐹𝑒𝑓𝑓 is

22

reversed can be used as an indicator to determine the molecular translocation through 27



1

nanopore devices, as verified by DNA translocation experiments where devices with the

2

measured surface charge density more negative than the simulated 𝜎𝑡ℎ𝑟𝑒𝑠ℎ𝑜𝑙𝑑 did not allow

3

translocation of λ-DNA translocation. Compared with PMMA and COC based devices, PEGDA

4

based nanopore fabricated by UV-NIL has the potential to replace Si based solid-state nanopore

5

in the future due to its low surface charge, hydrophilic nature and large-scale fabrication

6

possibility.

7 8

EXPERIMENTAL SECTION

9

Effective driving forces simulation. A 2-D axisymmetric dimensional model was built in

10

COMSOL 5.0 (COMSOL, Inc.) to investigate the effect of surface charge density on 𝐹𝑒𝑓𝑓 for

11

DNA capture.64-65 Electrostatic module, transport of diluted species module and

12

incompressible Navier–Stokes module were used to solve the coupled Poisson-Nernst-Planck

13

(PNP) and Navier–Stokes equations. We assume that a dsDNA is partially pre-stretched by a

14

nanopillar array and reaches the nanopore mouth with no initial velocity. Thus, the DNA

15

molecule can be modeled as a 2 nm diameter cylinder. Since the electric field is strong near

16

nanopore mouth, the end of the cylinder near the nanopore makes a major contribution to

17

𝐹𝑒𝑓𝑓.28 In our model, we placed a 30 nm long cylinder (which is shorter than dsDNA

18

persistence length, 50-80 nm) with no velocity in front of the nanopore to calculate 𝐹𝐸𝑂𝐹 and

19

𝐹𝐸𝑃. 𝐹𝑒𝑓𝑓 was obtained by Equation (1). For the negatively charged DNA, we used the DNA’s

20

bare surface charge density of -0.15 C/m2 (2e per base pair).31, 45 28


Page 28 of 42

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1


For most DNA translocation simulation, KCl solution was the common electrolyte

65-67.

In

2

our experiment, we used 1× TE buffer (Sigma-Aldrich, containing 10 mM Tris-HCl, 1mM

3

EDTA, pH 8.0) as electrolyte for both fluorescence observation and electrical measurement.

4

For COMSOL simulation, therefore, we used 10 mM Tris-HCl concentration as the KCl

5

concentration, making sure that they have the same Debye length (ionic strength). All

6

parameters for COMSOL simulation are shown in SI Table S1.

7 8

Polymer device fabrication. Fluidic devices with a planar nanopore was produced by UV- or

9

thermal-NIL into three different polymer substrates: PEGDA (MW = 200, Sigma-Aldrich),

10

PMMA (ePlastics) and COC (COC6013, Tg = 142 °C, TOPAS) as shown in Figure 3. In order to

11

cure, PEGDA was exposed to flash-type UV light (250-400 nm) for 1 min at an intensity of

12

~1.8 W/cm2 after adding 1 wt% of the UV initiator (2,2-dimethoxy-2-phenylacetophenone,

13

Sigma-Aldrich). PMMA and COC were thermally imprinted at 135 °C, 3.5 MPa and 160 °C, 5

14

MPa for 15 min, respectively. For bonding at 70 °C, 1 MPa for 15 min, a low Tg thin COC sheet

15

(COC8007, Tg = 78 °C, TOPAS) was used as the cover plate for all three nanopore substrates.

16

The use of a low Tg cover plate minimize the deformation of the nanostructured substrate.51

17

Details on the device fabrication can be found in SI and our previous work.22, 50-51

18

In order to enhance wetting of the nanopore device and reduce hydrophobic interaction

19

between DNA and the nanopore wall, the patterned substrates (except for PEGDA) and cover

20

sheets were modified to become hydrophilic by O2 plasma prior to bonding.68 Water contact 29



1

angles of these materials before and after O2 plasma treatment are shown in SI Figure S8. The

2

bare PEGDA substrate has a similar water contact angle to O2 plasma treated PMMA and COC

3

substrates.

4 5

Measurement of surface charge density. Nanofluidic devices with an array of nanochannels

6

with the width and height of 154 and 203 nm, respectively, were used to measure surface

7

charge density of different polymer substrates. SEM images of the Si master and imprinted

8

polymer nanochannel substrates used for the surface charge density measurements are shown

9

in Figure 4a-d. Conductance through these nanochannel arrays at each KCl concentration was

10

determined by measuring I-V curves using Axopatch 200B (Molecular Devices). The

11

measurement was repeated for different salt concentrations ranging from 10-6 M to 1 M. The

12

current-voltage measurements were performed after the baseline current became stable during

13

current-time measurement. 10 devices were measured for each substrate material. At high salt

14

concentration the nanochannel conductance is dependent on the salt concentration while at

15

low salt concentration the conductance is governed by surface charge density on the wall,46,

16

52, 69

17

be obtained from the transition point between these two regimes. Details on the determination

18

of the surface charge density can be found in SI.

as shown in Figure 4e. The effective surface charge density of a nanochannel substrate can

19

30


Page 30 of 42

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1

Optical and electrical detection of λ-DNA translocation. Prior to introducing DNA molecules,

2

all devices were filled with 1 M KCl solution (Sigma-Aldrich) containing 1× TE (Sigma-

3

Aldrich), pH 8.0 at room temperature and ionic current (conductance) across the nanopore

4

was measured via Axopatch 200B (Molecular Devices) to make sure that all testing devices

5

have similar pore sizes. The nanopore size was determined by comparing the measured

6

conductance through the nanopore with the simulated conductance for a given nanopore

7

size.56, 70 The average ionic current prior to introducing DNA molecules was 150 ± 31 nA

8

under 1 V, which equals to 150 ± 31 nS in conductance. The estimated equivalent nanopore

9

diameter was 10.3 ± 3.3 nm (See SI Figure S6).

10

Then, 1 M KCl solution containing 1× TE was replaced to 1× TE buffer (Sigma-Aldrich) and a

11

solution of 5 ng/μL double-strand λ-DNA (New England BioLabs) stained with YOYO-1 dye

12

(ThermoFisher Scientific) was added to the cis side of the microchannel. Pt electrodes were

13

used to drive DNA molecules with a commercial power supply (BK Precision DC power supply

14

1735). The DNA movement was observed under a fluorescence microscope (Olympus IX70)

15

with a 100× oil immersion objective (Olympus). Fluorescence images and videos were captured

16

by a CCD camera (Photon Max, Princeton Instruments).

17

Immediately after fluorescence observation, we used the same nanopore device for electrical

18

detection of DNA translocation by using same 1× TE buffer as electrolyte. In order to reduce

19

the noise level, the device was kept in a homemade Faraday cage during electrical

20

measurement. Ag/AgCl electrodes were placed in inlet/outlet reservoirs to drive stained DNA 31



1

molecules. Ionic transient current signal was recorded at a sampling rate of 250 kHz and low-

2

pass filtered at 10 kHz.

3 4

ASSOCIATED CONTENT

5

Supporting Information. Additional results including COMSOL simulation, polymer

6

nanofluidic device fabrication, nanopore size estimation, surface charge density measurement

7

and DNA translocation videos are supplied as Supporting Information.

8 9

AUTHOR INFORMATION

10

Corresponding Author

11

*E-mail: [email protected]

12

ORCID

13

Zheng Jia: 0000-0003-2476-2969

14

Junseo Choi: 0000-0002-3461-3820

15

Sunggook Park: 0000-0003-0424-6318

16 17

Notes

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The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was supported by the P41 Center for BioModular Multiscale Systems for Precision Medicine (P41EB020594) from the National Institutes of Health.

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Graphical Abstract

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Surface Charge Density-Dependent DNA Capture through Polymer

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