# Dynamics of DNA Through Solid-state Nanopores Fabricated by Controlled Dielectric Breakdown

Izadora Mayumi Fujinami Tanimoto, Jiayi Zhang, Benjamin Cressiot, Bruno

Le Pioufle, Laurent Bacri, Juan Pelta

► To cite this version:

Izadora Mayumi Fujinami Tanimoto, Jiayi Zhang, Benjamin Cressiot, Bruno Le Pioufle, Laurent Bacri, et al.. Dynamics of DNA Through Solid-state Nanopores Fabricated by Controlled Dielectric Breakdown. Chemistry - An Asian Journal, 2022, 17 (24), pp.e202200888. 10.1002/asia.202200888. hal-04068157

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<figure><img src="image_1.png" /></figure>
# Dynamics of DNA through solid-state nanopores fabricated by controlled dielectric breakdown

M.S. Izadora Mayumi Fujinami Tanimoto*[a,b], Jiayi Zhang[a], Associate professor Dr. Benjamin Cressiot*[c], Prof. Dr. Bruno Le Pioufle[b], Associate professor Dr. Laurent Bacri*[a] and Prof. Dr. Juan Pelta*[a]

**Abstract:** Controlled dielectric breakdown (CDB) is gaining popularity for fabricating solid-state nanopores *in situ* with size control in a simple, low-cost, and scalable way. This technique could be used for a broad type of applications in the field of nucleic acid analysis and even for protein studies. In this work, we studied the entry and transport of double-stranded DNAs using a solid-state nanopore fabricated by CDB as a function of applied voltage for two different DNA lengths. We showed that the blockade rate increases exponentially with voltage up to 120 mV. The energy barrier depends on the chain length, and the dwell times decrease with applied voltage up to 120 mV. Moreover, no matter the chain length, it is possible to differentiate two families of blockade amplitudes, high and low ones, due to DNA folding.

## Introduction

Transport in a confined nanoscale environment has been used to address emerging challenges in biotechnology [1-5] and biological processes [1,6-10]. The nanopore technology relies on the modulation of ionic current as a single molecule or particle passes through a nanometer-scale pore. Since the first demonstration of nucleic acid detection using a protein nanopore [11]¹, intensive work has been done to understand the physics governing its translocation, such as driving force, particle-pore interaction, and translocation time (τ) [12]. Recently, it has been shown that nanopore fabrication by Controlled Dielectric Breakdown (CDB) in a scalable and low-cost way could be used for nucleic acid analysis [13].

Nanopores have proven their potential for DNA sequencing [14-17]. The scaled-down portable device Minlon from Oxford Nanopore Technologies allowed measurements *in situ* at remote locations being capable of surveilling outbreaks, such as the Ebola in Africa [18] and, more recently, the pandemic SARS-CoV-2 [19,20].

For this study, we will now focus on DNA dynamics through solid-state nanopores. DNA or RNA molecules are uniformly charged and move through a nanopore mainly by electrical forces [14]. A direct proof of DNA translocation was shown by the fluorescence associated with DNA transport through the nanopore [21]. Studies using solid-state nanopores have been used to analyze DNA conformations [12,22-26]. Li *et al*. observed individual molecules of double-stranded DNA and their molecular folding behavior using nanopores of 3 nm, which do not allow the passage of folded molecules, and with 10 nm, in a non-confined state. Discrimination between single- and double-stranded nucleic-acid molecules and separation of purine-based homopolymers from pyrimidine-based ones could also be achieved [24]. Later, another work using double-stranded DNA (dsDNA) with structural motifs revealed a two-stage translocation behavior through synthetic nanopores, showing that the translocation initially slows with time before accelerating close to the end of the process [12]. An alkaline pH leads to the denaturation of dsDNA to single-stranded DNA (ssDNA); this conformational transition is associated with a different electrical signature [27]. The various forms of DNA (linear, relaxed, and supercoiled) are related to specific current blockages in terms of dwell time and current drop [22].

DNA translocation has been investigated as a function of chain length and nanopore diameter. If the radius of gyration of the DNA is larger than the pore radius, a power law is found for the translocation versus DNA length, with an exponent of 1.26 ± 0.07 when the gyration radius ($R_G$) is bigger than the persistence length ($L_p$) in a good solvent and an exponent of 1 in the case of an ideal solvent [25]. This law expresses that the dynamics are determined by a balance between an electrical driving force that pulls the chain through the nanopore and an opposite viscous drag force (hydrodynamic) that depends on the blob size or chain length. Another group also confirmed this behavior [22]. If the pore radius decreases even more, two exponents are found, 1.40 and 2.28, respectively, for short and medium length (< 3.5 kbp) and longer molecules. The last regime is due to DNA interaction

<footnote>[a] I.M.F.T, J.Z., L.B.*, J.P.*
Laboratoire Analyse, Modélisation, Matériaux pour la Biologie et l'Environnement, CNRS UMR8587
Université Paris-Saclay, Université d'Evry Val d'Essonne
Boulevard François Mitterrand, 91025 Evry CEDEX, France
E-mail: lzadora.fujinamitanimoto@univ-evry.fr, laurent.bacri@univ-evry.fr, juan.pelta@univ-evry.fr</footnote>

<footnote>[b] B.L.P.
Institut d'Alembert, CNRS, FR3234
Université Paris-Saclay, Ecole Normale Supérieure Paris-Saclay
4 avenue des sciences, 91190 Gif-sur-Yvette, France</footnote>

<footnote>[c] B.C.*
Laboratoire Analyse, Modélisation, Matériaux pour la Biologie et l'Environnement, CNRS UMR8587
CY Cergy Paris Université
33 Boulevard du port, 95011 Cergy-Pontoise CEDEX, France
E-mail: benjamin.cressiot@cyu.fr</footnote>
between the chain part located outside the nanopore and the membrane during chain transport [28].

The dynamics of entry or transport of DNA have been studied as a function of applied voltage (V) or temperature, when the chain size is larger than the pore diameter. An exponential behavior is found for the event's frequency as a function of V or 1T for the short and medium DNA lengths due to the free energy barrier [29]. The fit of the Arrhenius plot leads to an energy barrier in the order of 12 k_b T. For long DNA length (48.5 kbp), a linear behavior is observed probably due to a diffusion regime [29]. The dwell time decreases exponentially with V. During the transport, the DNA chain passes through energy barriers due to chain-pore interaction or chain deformation [29,30]. An electrophoretic behavior is also found for the translocation time [30].

Other interesting studies have been performed to identify epigenetic modification of DNA [31]. The methylation of DNA cytosine leads to an electrical signal change in current drop and dwell time. miRNA are implied in gene regulation, and in human diseases, a specific protein was used to tag miRNA to form a duplex. The detection and identification could be quantified down to the picogram level [29].

After discovering that a beam of energetic particles could create a nanometer-sized hole in dielectric materials [26,32], solid-state nanopores have been at the forefront of single-molecule or particle research. The main fabrication methods consist of focused beams of electrons or ions. However, these techniques are costly, and their use remains confined to laboratories equipped with such expensive research equipment and highly-trained personnel. Therefore, a new method named controlled breakdown has been gaining interest in the nanopore community [33-36]. It is a low-cost, scalable fabrication of precisely sized solid-state nanopores [13,37]. One could pierce a single nanopore down to 2 nm in size with sub-nm precision directly in an aqueous solution [37]. The method relies on applying a voltage across a thin (10 - 30 nm) insulating membrane to generate a high electric field while monitoring the induced leakage current. Electrical stress randomly introduces defects in the insulating membrane, and

eventually, these defects form a nanopore through the membrane [38]. The nanopore can be grown in the same order as the membrane thickness. To verify nanopore formation, DNA has been used as a molecular ruler to measure membrane thickness [13,39]. Nevertheless, we could focus on the interactions between DNA and the nanopore. Previously we have shown that the drilling method, whether by focused ion beam (FIB) [40] or by transmission electron microscopy (TEM) [41], led to different interactions between analytes and the inner side of the nanopore. In this publication, we study the DNA dynamics at low voltages to evaluate the activation energy in CDB nanopore and compare this behavior with the previously observed TEM-based nanopore. We study the blockade rate and dwell time as a function of applied voltage and chain length.

Moreover, these CDB nanopores also discriminate between single- and double-stranded DNA with ~ 2 nm pores. A slightly larger nanopore could be used to investigate the overstretching transition in short dsDNA fragments [42].

## Results and Discussion

In this work, we focus on showing dsDNA chain transport's voltage and length dependence through nanopores fabricated by the CDB technique. CDB was carried out in a two-step protocol using 1 M KCl and then 3.6 M LiCl, both buffered with 10 mM HEPES and pH 8, described in [38]. Figure 1a-b shows the experimental set-up in which the CDB was performed. An 8.5 ± 0.2 nm nanopore diameter was formed with an Ohmic conductance in 1 M KCl, where a biased voltage from - 200 mV to 200 mV was applied over the membrane (Figure 1c). Once the nanopore was formed, we started the dsDNA experiments by adding DNA molecules of (30 ng/µL) 1 kbp DNA or 5 kbp DNA in the cis-chamber and applying a positive voltage (Figure 1d). Then, the ionic current was recorded to extract characteristic signals shown in figure 1e.

<figure><img src="image_2.png" /><figcaption>Figure 1 - Characterization and fabrication of solid-state nanopores using a controlled breakdown approach for single molecule sensing. a) Cross section view of the flow cell system. b) Sketch of the solid-state nanopore fabrication set-up using the controlled breakdown approach. c) Characteristic I-V curve in 1M KCl 10mM Tris, 1mM EDTA pH 7.4. d) Sketch of dsDNA sensing method using a nanopore drilled into a SiNₓ-based membrane. The DNA conformation could be linear or</figcaption></figure>
partially folded. e) Characteristic signal showing transient ionic current blockades. 1 kbp DNA, ΔV = 80 mV, I₀ is the open pore current, I_b is the ionic blockade, ΔI_b is the amplitude blockade, t_d is the dwell time, and t_i is the inter-event time between the starting points of two following events. Created with BioRender.com.

To determine the nanopore size, a current vs. voltage (I-V) curve was used to infer the ionic conductance, following Kowalczyk et al. [43]:

$$G = \sigma \left[ \left( \frac{4L}{\pi d^2} \right) + \left( \frac{1}{d} \right) \right]^{-1} \quad (1)$$

where σ is the solution conductivity, L the membrane thickness, d the pore diameter, and G the nanopore conductance.

In figure 2, we can observe the evolution of characteristic ionic current traces in the presence of dsDNA as a function of applied voltage. We showed that the blockade rate increases with voltage up to 120 mV. After this threshold voltage, the rate decreases. This could be explained by the 10 kHz cutoff frequency of the amplifier (see below) [44]. Briefly, above 120 mV, the number of blockades characterized by a very short duration and a low amplitude increase. They could not be discriminated against the noise because the DNA chain velocity becomes too high to be detected. Then, the apparent rate seems to decrease above 120 mV.

By comparing the two sizes of dsDNA, we observe that the blockade rate of 1 kbp DNA is greater than the 5 kbp DNA, shown in figures 2b-c, with a lower inter-event time (figure 2d).

The blockade rate shown in figure 2c is the blockade frequency normalized by the DNA concentration. From this graph, we calculate the molecule's energy barrier to enter the pore [40,45-47]. This blockade rate corresponds to the capture rate R, described by a Van't Hoff-Arrhenius law or transition-state relation R = R₀ exp([V]/V₀), where R₀ ∝ R* exp(-U*/(K₀T)) is the zero-voltage capture rate that is controlled by an activation barrier U*, with R* as a rate factor. The ratio [V]/V₀ = (zeV)/(K₀T) is a barrier reduction due to the applied voltage V, z is the effective total

number of elementary charges, e is the elementary charge and K₀T is the thermal energy.

The voltage V₀ = (K₀T)/(zeV) is the potential the particle needs to overcome the Brownian motion. The normalized blockade of capture rate (Figure 2c) is well-described by an exponential fit of R, where R₀ = (3.1 ± 0.6) x10⁻¹⁹ m³ s⁻¹ and V₀ = (42.8 ± 3.7) mV for 49.4 nM 1 kbp DNA and R₀ = (6 ± 0.5)² m³ s⁻¹ and V₀ = (62.3 ± 3.3) mV for 9.9 nM 5 kbp DNA.

The activation energy U* = - ln (R₀/R*) K₀T is estimated by the rate factor R* = (Ddiff Apore)/Lpore where Apore is the nanopore cross-sectional area, Lpore is the membrane thickness and Ddiff is the diffusion coefficient.

$$D_{\text{diff 1kbp}} = (7.15 \pm 0.2) \times 10^{-12} \text{ m}^2\text{s}^{-1} \text{ [48]}, \quad D_{\text{diff 5kbp}} = (1.75 \pm 0.1) \times 10^{-12} \text{ m}^2\text{s}^{-1} \text{ extrapolated from [49]}, \quad A_{\text{pore}} = 5.35 \times 10^{-17} \text{ m}^2 \text{ and } L_{\text{pore}} = 12 \times 10^{-9} \text{ m is the pore length. We calculate } R^*_{1\text{kbp}} = 3.4 \times 10^{-20} \text{ m}^3\text{s}^{-1}, R^*_{5\text{kbp}} = 8.3 \times 10^{-21} \text{ m}^3\text{s}^{-1}, |U^*_{1\text{kbp DNA}}| = (2.2 \pm 0.2) K_0 T \text{ and } |U^*_{5\text{kbp DNA}}| = (4.3 \pm 0.1) K_0 T.$$

As expected for longer chains, the activation barrier of 5 kbp DNA is higher than the 1 kbp DNA.

In figure 3, we represent the density of the scatter plot of the minimum current level Ib of each blockade according to its duration td for 3 voltages 60 mV, 100 mV, and 140 mV for each DNA length. The density of events is higher for 1 kbp than for 5 kbp for the same applied voltage. We explain it by a larger 1 kbp molar concentration and lower activation energy for the 1 kbp. When we increase the voltage, we observe that these two populations are better discriminated against, except for the 1 kbp at 140 mV. In both DNA sizes, we can differentiate two types of blockade amplitudes “high blockade” and “low blockade”. They refer to high and low current blockade I₀-I_b, respectively.

<figure><img src="image_3.png" /></figure>
RESEARCH ARTICLE

Figure 2 – dsDNA transport characteristic dependence with DNA length and applied voltage. a) Voltage dependence of 1 kbp DNA translocation. b) Comparison between 1 kbp and 5 kbp DNA at 100 mV. 1M KCl, 10 mM Tris, 1 mM EDTA pH 7.4. c) Blockade rate normalized by 1 kbp and 5 kbp DNA concentration. d) Histogram of inter-event time of 1 kbp and 5 kbp DNA.

<figure><img src="image_4.png" /><figcaption>Figure 3 - Events density over dwell time for 1 kbp and 5 kbp DNA passing through an 8.45 nm diameter pore. a and b) Blockade density over dwell time at 60, 100, and 140 mV for 1 kbp and 5 kbp DNA translocation, respectively. We represent here the minimum current level.</figcaption></figure>

We remark that the low blockade family is due to the passage of linear chains, with a single step while the high blockade presents folded ones (in a “loop” form), a complex step, depicted in Figure 4e [50,51].

Figure 4a-b shows that the translocation time decreases with the voltage independent of the DNA length and state, linear or partially folded, proving that electrical forces govern the dynamics. As expected, the longer 5kbp DNA chains take longer to pass the pore. Surprisingly, we observe that the two populations present similar dwell time regarding each DNA length. The experimental data of Fig. 4a-b was fitted by an exponential f(V) = A exp (V/Vc). Linear form: $VC_{1kbpDNA} = 55.3 \pm 3.5$ mV; $VC_{5kbpDNA} = 150.5 \pm 6.5$ mV. Partially folded form: $VC_{1kbpDNA} = 36.7 \pm 4.8$ mV; $VC_{5kbpDNA} = 112.7 \pm 13.6$ mV. Showing that longer DNA chains need more energy to cross the nanopore and that Vc is higher in a linear form no matter the DNA length. This could be due to the higher friction of linear and longer chains.

The ratio $VC_{loop}/VC_{linear}$ for 1 kbp and 5 kbp DNA is 0.64 and 0.75, respectively. The transport energy $E \approx ze \cdot Vc$, where z is the net charge of the portion of the DNA chain inside the nanopore [52]. Analyzing this ratio, we observe that the energy barrier is independent of the linear or partially folded conformations due to the low confinement of the chain in the nanopore. Nevertheless, Vc increases with DNA chain. A similar regime was observed by Wanunu et al. in a 4 nm large nanopore drilled by TEM [52], where this regime is attributed to the complex interactions between the part of the chain located outside the nanopore and the external surface of the membrane.”

However, we can notice that for the 1 kbp DNA, voltages greater than 100 - 120 mV lead to translocation time below 35 µs. This duration is a threshold time below which the blockade shapes are deformed by the cutoff frequency $f_c$ of the amplifier [53]. This duration corresponds to the rise time $T_r$ of the amplifier ($T_r = 0.33/f_c$) [44,54]. By working with a non-confined media, 1 kbp chains pass too fast through the pore. This effect can also be seen in the blockade rate graph (Figure 2c), where there is a rate decrease for higher voltages. Takeda's group proposed an idea to decrease the translocation speed [55], in which CDB with a divalent metal cation provides a silicon nitride nanopore with the ability to decelerate ssDNA speed around 100 µs.

The relative blockade, that is normalized by the open pore current, is shown in figure 4c-d. As mentioned before, the transport of 1 kbp chains is too fast. Therefore, for voltages > 100 mV, the amplifier cannot resolve the events. Nevertheless, we can focus on the 5 kbp DNA transport until 180 mV. Then, we could observe that the relative blockade reaches a steady value of 9.4 ± 0.1 % for single blockades and 14.8 ± 0.3 % for complex blockades. This behavior shows that the voltage does not modify the 5 kbp DNA conformation. According to previous publications, the relative blockade ratio of 1.6 agrees with the assumption of the linear and loop conformation [50,51]. For low blockades (Figure 4d), as the DNA chains are in a linear form, there is almost no change in the chain's conformation. We also notice that for 60 - 80 mV, both 1 kbp and 5 kbp DNA present the same relative blockade because they have the same apparent diameter in the linear conformation. In the case of high blockades (Figure 4c), where the DNA is in a loop form, we can observe two regimes. The first one, up to 120 mV, there is a deformation of the DNA chains because they are
being pulled up by the electric field. After 120 mV, the relative blockade seems to reach a plateau, indicating that the DNA does not change its conformation anymore. For low blockades (Figure 4d), as the DNA chains are in a linear form, there is almost no

change in the chain's conformation. We also notice that for 60 - 80 mV, both 1 kbp and 5 kbp present the same relative blockade, because they have the same apparent diameter in this form.

<figure><img src="image_5.png" /><figcaption>Figure 4 - a-b) Translocation time vs. voltage of partially folded and low linear blockades, respectively, for 1 kbp and 5 kbp DNA. The black dashed line is a guide to the eyes showing the acquisition limitation. The continuous line corresponds to a fit $f(V) = A\exp(-V/V_c)$. Linear form: $A_{1kbpDNA} = 399.2 \pm 148 \ \mu\text{s}$, $VC_{1kbpDNA} = 55.3 \pm 3.5 \ \text{mV}$; $A_{5kbpDNA} = 294 \pm 38.8 \ \mu\text{s}$, $VC_{5kbpDNA} = 150.5 \pm 6.5 \ \text{mV}$. Partially folded form: $A_{1kbpDNA} = 686.8 \pm 280 \ \mu\text{s}$, $VC_{1kbpDNA} = 36.7 \pm 4.8 \ \text{mV}$; $A_{5kbpDNA} = 361.4 \pm 25.9 \ \mu\text{s}$, $VC_{5kbpDNA} = 112.7 \pm 13.6 \ \text{mV}$. c-d) Normalized current blockade ($I_o/I_b$)/$I_o$ of high and low blockades, respectively. $I_b$ is the minimum current level. Dashed red lines correspond to a linear fit of 5 kbp DNA values: 9.4 ± 0.1% for linear blockades and 14.8 ± 0.3% for partially folded blockades. e) Example of 5 kbp DNA distribution event in 100 mV of $I_b$ as a function of the $t_d$, pink circles are the blockade current of linear chains, and the green ones are the blockade current of partially folded. On top right is the dwell time histogram and on the bottom left is the histogram of $I_b$. On the top left, it depicts examples of the current blockade signals (events) assigned to linear (pink) and partially folded (green) DNA molecules.</figcaption></figure>

## Conclusion

We used a CDB technique to fabricate solid-state nanopores in a simple, accessible, and reproducible way. We have discussed the dynamics of double-stranded nucleic acids where the chain's size, $R_G$, is larger than the nanopore size. The blockade rate increases exponentially up to 120 mV. We also described two types of blockades in the ionic current distribution associated with different DNA conformation, linear and partially folded ones. We measure a higher activation energy from 5 kbp DNA ($|U^*_{5 kbp DNA}| = 4.3 K_B T$) compared with the case where a shorter DNA length of 1 kbp dsDNA is translocated ($|U^*_{1 kbp DNA}| = 2.2 K_B T$).

Further work with solid-state nanopores will be the control of surface properties to avoid pore clogging due to analyte-membrane interactions. Bandara et al., demonstrated that adding sodium hypochlorite during CDB changed the surface charge and increased the frequency rate; they could sense dsDNA, human serum transferrin, and maltodextrin [56]50. The same group also demonstrated several membrane functionalization after CDB via photohydrosilylation [57], decreasing the clogging of the nanopore and adding chemical functions to the membrane.

Another limitation with CDB is to fabricate pores larger than 20 nm. Some groups have proposed different techniques to make larger pores to solve this limit. One is to do a laser-assisted breakdown and controlled pore enlargement by limiting the applied voltage [58]. Another strategy developed by Takeda's group, called "two-step breakdown" could also yield pores larger than 20 nm. In this technique, they first did a local conductive thin

portion (not a nanopore) in the membrane by dielectric breakdown. Then, the created thin portion is penetrated by voltage pulses to create the nanopore and enlarge it [59]. The same group also worked with high alkaline pH (≥11.3) to fabricate pores about 40 nm within one min [60]. These works pave the way to fabricate nanopores larger than 20 nm, allowing the future analysis of a wider variety of analytes.

## Experimental Section

### Materials

Ethylene diamine tetraacetic acid (EDTA, ref. A10713), lithium chloride (LiCl, ref. 36217) were purchased from Alfa Aesar. N-(2-hydroxyethyl) piperazine-N'-(2-ethanesulfonic acid) (HEPES, ref. H3375), Tris (hydroxymethyl) aminomethane (Tris, ref. T87602) and sulfuric acid ($H_2SO_4$, ref. 07208) were purchased from Merck. Potassium chloride (KCl, ref. AAA1166201) and sodium chloride (NaCl, ref. AC447300010) were purchased from Fisher Scientific. Hydrogen peroxide 30% ($H_2O_2$, ref. EM1.07209.0500) was purchased from VWR chemicals. Milli-Q filtered water (Millipore). NoLimits 1000 bp DNA fragment (ref. SM1671) and NoLimits 5000 bp DNA fragment (ref. SM1731) were purchased from ThermoScientific.

### Buffer fabrication

The nanopore fabrication was performed in 1 M KCl 10 mM HEPES pH 8 and 3.6 M LiCl 10 mM HEPES pH 8. The DNA experiments were performed in 1 M KCl 10 mM Tris 1mM EDTA pH 7.4. The final pH was
adjusted with HCl, NaOH, or KOH solutions using a pH meter (WTWTM pH meter inoLabTM 720, Xylem Analytics, Germany).

## Nanopore fabrication and characterization

Nanopores were fabricated in 40 µm x 40 µm membranes of SiNₓ with 12 ± 2 nm thickness (NXDB-50H405V122, Norcada). Each membrane was supported on silicon chips of 5 mm x 5 mm and 200 µm thick. Before nanopore fabrication, the SiNx membranes were piranha cleaned using a 3:1 solution of H₂SO₄:H₂O₂ around 80°C for 1 hour. The cleaned chips were then rinsed with Milli-Q filtered water to remove piranha solution and then mounted into 3D printed fluidic cells (Northern Nanopore Instruments). The set-up was rinsed with Propan-2-ol and degassed Milli-Q water to remove air from the flow cell channels and wet the SiNx membrane. Then, the cell was filled with a solution of 1 M KCl, 10 mM HEPES (pH 8), and it was electrically connected to a current amplifier by two Ag/AgCl electrodes. Before fabrication, we check that no current (<pA) is measured across a membrane at low voltages (≤-1 V) with Axopatch 200B (Molecular devices, U.S.A.). The entire set-up is placed inside a Faraday cage to isolate it from electromagnetic noise. Afterward, the nanopore was fabricated using the controlled dielectric breakdown method ³⁸ with the Spark-E2 hardware (Northern Nanopore Instruments) in a two steps protocol: i) fabrication process in 1M KCl, 10mM HEPES (pH 8), in which a potential ramp from 0 to -5V followed by a smother ramp from -5 to -17.5 V was applied across the SiNₓ membrane until breakdown event, where the ionic current should spike, and the applied voltage should go to zero by the software. ii) conditioning process in 3.6 M LiCl 10mM HEPES (pH 8). A pulse wave of ±3V (4 cycles) is applied several times to enlarge the nanopore until an apparent target diameter of 8.5 ± 0.2 nm is achieved. After the nanopore fabrication, it was characterized by I-V curves from -200 mV to +200 mV in 1 M KCl, 10 mM Tris, 1 mM EDTA (pH 7.4).

## DNA measurements

DNA transports were performed by adding in the *cis*-chamber 1.2 µL of dsDNA fragments of 1 kbp or 5 kbp, for a final concentration of 49.4 nM and 9.9 nM, respectively in 1 M KCl, 10 mM Tris, 1 mM EDTA (pH 7.4).

## Instrumentation and Data Acquisition

The flow cell was filled up with the electrolyte solution. Ag/AgCl electrodes were inserted in the cell device's *cis* (ground) and *trans* (working electrode) channels to measure the ionic current passing through the nanopore. The current was recorded using an Axopatch 200B with a 4-pole Bessel filter set at $f_c$=10 kHz, with a sampling rate of 250 kHz. A PCIe-6251 (National Instruments, U.S.A.) board interfaced with a custom .Net-based program was used for data acquisition. Data analysis was carried out using a custom-designed IGOR Pro (WaveMetrics, U.S.A.) software to measure the duration and amplitude of each current blockade event. The I-V curves were obtained by applying 60 s extended voltage steps $\Delta$V = 20 mV varying from -200 mV to +200 mV. The final set-up, consisting of the microfluidic device, the nanopore membrane, and the amplifier headstage, was placed inside a Faraday cage to avoid electromagnetic interference. All the measurements were carried out at room temperature 23 ± 2 °C.

## Acknowledgements

We acknowledge financial support from DIM Respore, Region Ile de France, and University Paris-Saclay EOBE Doctorate school (PhD grants), ANR Epsilomics (17-CE09-0044-02), CY Initiative of Excellence (grant "Investissements d'Avenir" ANR-16-IDEX-0008), Réseau sur le Stockage Electrochimique de l'Energie (RS2E), Jean Roman the predecessor of my work with solid-state nanopores for all the support and advice, and Matthew Waugh

from Northern Nanopore Instruments for all the help with the nanopore fabrication.

**Keywords:** DNA translocation • solid-state nanopore • controlled dielectric breakdown • nanopore sensing

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