Selective Detection of Single-Stranded DNA Molecules Using a Glass

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Selective Detection of Single-Stranded DNA Molecules Using a Glass Nanocapillary Functionalized with DNA Yeoan Youn, Choongman Lee, Joo Hyoung Kim, Young Wook Chang, Dug Young Kim, and Kyung-Hwa Yoo Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.5b02540 • Publication Date (Web): 26 Nov 2015 Downloaded from http://pubs.acs.org on November 26, 2015

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Analytical Chemistry

Selective Detection of Single-Stranded DNA Molecules Using a Glass Nanocapillary Functionalized with DNA Yeoan Youn†, Choongman Lee†, Joo Hyoung Kim†, Young Wook Chang§, Dug Young Kim†, and Kyung-Hwa Yoo†*

† §

Department of Physics, Yonsei University, Seoul, 120-749, Republic of Korea

Department of Materials Science and Engineering, Yonsei University, Seoul, 120-749, Republic of Korea

ABSTRACT: We describe glass nanocapillaries with single-stranded DNA molecules (ssDNA) covalently attached to the capillary surface. These DNA-functionalized nanocapillaries selectively facilitate the translocation of target ssDNA that is complementary to the probe ssDNA. In addition, the complementary target ssDNA exhibits a longer event duration time than the non-complementary target ssDNA. The temperature dependence measurements of translocation events show that the longer duration time is a result of an interaction between probe and target ssDNA, and is dependent on the basepair binding strength. These results demonstrate that single-base mismatch transport selectivity can be achieved using the DNA-functionalized nanocapillaries.

Since Kasianowicz and coworkers demonstrated translocation of single-stranded DNA molecules (ssDNA) through α-hemolysin nanopores in a lipid membrane,1 numerous studies have focused on the detection and analysis of single biomolecules using protein-based nanopores2-5 or solid-state nanopores.6-9 When charged single molecules are transported through a voltage-biased nanopore, resistive ionic current pulses are recorded, which allow the study of the size, conformation, and activity of single biomolecules. To distinguish ssDNA with different base sequences, the nanopores have been functionalized with probe ssDNA. For example, α-hemolysin nanopores were functionalized by covalently attaching the probe ssDNA within the lumen of α-hemolysin.10 When a target ssDNA complementary to the probe ssDNA was electrophoretically driven through this functionalized nanopore, a longer translocation time or event duration was observed as compared to a target ssDNA that differed by only a single base. Gold nanotube membranes11 or nanopores in a silicon oxide membrane12 have been modified with hair-pin loop DNA, and a higher frequency of translocation events or flux was found for the target ssDNA complementary to the probe, as compared to the single-base mismatch target. In addition, nanocapillaries combined with DNA origami have also been reported, where the event duration increased for target ssDNA with

segments that were complementary to the binding sequence of the overhangs of the DNA origami nanopore.13 Here, we describe glass nanocapillaries covalently functionalized with probe ssDNA (Figure 1a). Although nanopores in silicon oxide or nitride membranes can be fabricated using an electron beam from a transmission electron microscope14 or a focused ion beam,6 their fabrication is expensive and time-consuming. In contrast, glass nanocapillaries have a conical shape and a very small orifice at their tip made by laser-assisted pipet pulling, so they are easily fabricated at low cost and high throughput. However, there have been few reports to investigate translocation of ssDNA through DNA-functionalized nanocapillaries. We have therefore investigated the selectivity of DNA-functionalized nanocapillaries. If a target ssDNA is selectively recognized by the DNAfunctionalized nanocapillary, just as for the silicon oxide nanopore, the glass nanocapillaries could be regarded as an inexpensive alternative to biological nanopores or nanopores in silicon membranes. Furthermore, these nanocapillaries may find more varied applications since they are user-friendly substrates for nanopores in combination with optical tools, such as optical tweezers15,16 and optical imaging.17 Indeed, compared to target ssDNA that is not complementary to the probe or a target with a single-base mismatch, the complementary target ssDNA

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Figure 1. (a) Schematic of the experimental setup. The inset shows a schematic cross-section of the glass nanocapillary functionalized with the probe ssDNA (green). The target ssDNA added into the negative electrode chamber is represented in red. (b) Current (I) – voltage (V) curves measured for the bare nanocapillaries with different pore sizes. The inset shows the ionic conductance versus the nanopore diameter. Red solid line is given by Eq. (1). (c) Real-time ionic current representative data of the bare nanocapillary for different target ssDNA: no DNA (control, black), poly(dC)100 (blue), poly(dA)100 (red). (d) Scatter plot of the translocation values for the bare nanocapillary for poly(dC)100 (blue) and poly(dA)100 (red). Each dot indicates a single translocation event. The upper inset shows the histogram of event duration (τ) and the right inset shows the histogram of ∆I/I0.

urements, the nanocapillaries with a diameter of about 40 nm were usually used.

exhibited higher frequency of translocation events and longer translocation time, demonstrating the single-base mismatch transport selectivity. In addition, we have also investigated the temperature dependence of translocation events, and found that the translocation duration time was affected by an interaction potential between the probe and the target ssDNA.

Functionalization of glass nanocapillaries. To functionalize the nanocapillary, the probe ssDNA with the amine group at the 5′ terminal was attached to the surface of the nanocapillary via a covalent bond between an amine group of the DNA and a carboxyl group on the glass surface (Supporting Information, Figure S2). Prior to functionalization, oxygen plasma treatments were performed on the glass nanocapillary for 30 s to improve the hydrophilicity of the nanocapillary wall. Then, the nanocapillary was immersed in 10% (3aminopropyl)triethoxysilane (APTES, Sigma Aldrich) in ethanol solvent, and dried in a vacuum desiccator for 1 h, followed by soaking in a solution of 0.1 M succinic anhydride in dimethyl sulfoxide (DMSO) for 1 h to react with succinic anhydride. Finally, the nanocapillary was immersed in 100 nM amine-modified probe DNA solution with 1 mg/ml N-hydroxysuccinimide (NHS) and 2 mg/ml of ethyl(dimethylaminopropyl) carbodiimide (EDC) for 24 h, resulting in the binding of the amine group of the probe DNA to a carboxyl group on the surface of the nanocapillary. Between each step, the capillary was rinsed with pure ethanol. Figure S2b illustrates the fluorescent optical images of the nanocapillary measured before and after functionalization, where the DNA probe was labeled

EXPERIMENTAL SECTION Fabrication of glass nanocapillaries. For all experiments in this study, commercial quartz glass capillaries with an outer diameter of 1.0 mm and an inner diameter of 0.5 mm (Sutter Instrument, USA) were used. Before pulling the quartz glass capillary, the capillary was cleansed by sonication in acetone for 10 min and then rinsed using ethanol. The rinsed capillary was dried by nitrogen gas and heated on a hot plate at 80 °C for 30 min to evaporate residual ethanol. Next, the capillary was heated and pulled by using a micropipette puller (P-2000, Sutter Instrument Co., USA) under controlled parameters, and it was separated into two capillaries, producing a conical nanopore at the tip of both divided pieces. The diameter of the fabricated nanocapillaries was measured to be 40 ~ 80 nm using a field emission scanning microscope (JEOL, JSM-6500F, Japan), and their taper length was about 4 mm, as determined using an optical microscope (Supporting Information, Figure S1). For transport meas-


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by fluorescent ssDNA staining dye, TOTO®-1 Iodide (Molecular Probes, USA). The images confirmed that the probe DNA was attached on the surface of the nanocapillary.

the negative electrode(cis) chamber (Figure 1a) and the real-time ionic current was measured with a bias voltage

DNA translocation measurements. The functionalized capillary was assembled into a PDMS cell that consisted of two reservoirs connected only by a capillary. The reservoirs were filled with 0.5 M KCl solution and Tris-EDTA buffer (pH 7.6, 10 mM Tris-HCl, 1 mM EDTA, Bioneer), and the whole cell was degassed in the desiccator to remove air bubbles in the nanocapillary. Then, the Ag/AgCl electrode was inserted into the capillary (trans chamber) and another Ag/AgCl electrode was in contact with the solution in the reservoir of the tip side (cis chamber) (Figure 1a). The target ssDNA was added into the cis chamber, diluted to a final concentration of 10 nM. Then, the ionic current was measured at room temperature with V=0.5 V using Axopatch 200 B amplifier (Axon Instruments, USA) in voltage clamp mode. Data were recorded by Clampex 10.3 (Molecular Devices, USA) with a sampling rate of 250 kHz and 4-pole Bessel low-pass filter at 10 kHz bandwidth, and translocation events of DNA molecules were detected by Opennanopore v1.2.18 The frequency of translocation events was estimated by dividing the total number of events for each target ssDNA by the total time of measurement. The successful rate of translocation measurements was about 30%. So, we repeated measurements with 10-15 nanocapillaries for each condition, chose three experimental results and analyzed them.

Figure 2. (a) Real-time ionic current representative data of the poly(dT)20-functionalized nanocapillary for the target poly(dC)20 (navy) and poly(dA)20 (magenta). Histograms of the logarithm of event duration time (log(τ)) at 27 °C for target poly(dC)20 (b) and poly(dA)20 (c). (d) Histogram of logarithm of event duration time (log(τ)) at 40 °C for target poly(dA)20. The log(τ) histograms show two normal populations, described by a sum of two Gaussian functions (black curves). Each Gaussian function (red curve) was used to estimate the most frequent duration time of fast (τp1, green) and slow (τp2, magenta) translocations. Histograms of relative blockade current (ΔI/I0) at 27 °C for target poly(dC)20 (e) and poly(dA)20 (f). (g) Histogram of relative blockade current (ΔI/I0) at 40 °C for target poly(dA)20.

Temperature dependence measurements. To measure DNA translocation at different temperatures, a commercial Peltier device was used. The whole PDMS cell was placed on the Peltier device, and a direct current voltage was applied using a battery to raise the temperature. The PDMS cell was covered to prevent evaporation of the solution and the temperature was measured by a Non-contact Infrared Thermometer (CEM, China). RESULTS AND DISCUSSION We fabricated glass nanocapillaries with a diameter of 40 ~ 80 nm and a taper length of 4 mm using a laser-assisted capillary pipet puller (Supporting Information, Figure S1). All nanocapillaries showed nearly linear current (I) – voltage (V) curves in 0.5 M KCl solution (Figure 1b). The ionic conductance estimated from the I-V curves increased with increasing pore diameter and was well fitted to Eq. (1) which describes the conductance (G) of a conical nanocapillary (inset of Figure 1b).19 G = σ

of 0.5 V. As reported by others,1-10 blockade current pulses were measured due to the electrophoretic force that is exerted on the negatively charged DNA when applying a positive voltage (Figure 1c). Translocation events were observed as a decrease in the ionic current because the translocating DNA excludes ions from the nanopore. Figure 1d is the scatter plot of normalized blockade current change (∆I/I0) versus event duration (τ) obtained from the measured real-time ionic current (Figure 1c), where I0 and IDNA are the current measured before and after adding DNA molecules, respectively, and the values of ∆I=I0 − IDNA are normalized with respect to I0 since different nanocapillaries may have different ionic conductance in 0.5 M KCl because of their different pore sizes and/or geometries. Each point represents a single translocation event. In Figure 1d, poly(dA)100 and poly(dC)100 exhibited similar distribution for τ and ∆I/I0, indicating that they cannot be distinguished using the bare nanocapillary.

(1)

where t is the taper length (4 mm), d the diameter of the nanopore at the tip, σ the specific conductance of the solution (6.3 S/m at 0.5 M KCl), and D is the diameter of the nanocapillary at the shaft (0.5 mm). For transport experiments, ssDNA containing adenines (poly(dA)100) or cytosines (poly(dC)100) were added into



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Similar measurements were also conducted with poly(dA)20 or poly(dC)20 (Supporting Information, Figure S3a). Compared to poly(dC)100 or poly(dA)100, poly(dC)20 or

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poly(dA)20, which was complementary to the probe poly(dT)20, was inserted in the same chamber (lower panel of Figure 2a). Unlike the target poly(dC)20, the target poly(dA)20 yielded more distinct and frequent current pulses. These observations suggest that translocation events occur more frequently for the complementary target ssDNA than for the non-complementary target ssDNA (Table 1), as reported for DNA-functionalized gold nanotube membranes11 or nanopores in silicon oxide membranes.12 To determine whether a decrease in ionic current was really caused by the translocation of target DNA molecules, we measured real-time ionic currents with various bias voltages (Supporting Information, Figure S4). As the bias voltage increased, ∆I increased and the frequency of translocation events increased, supporting that current pulses were ascribed to the translocation of poly(dA)20. The scatter plots of τ versus ∆I/I0 obtained from the realtime ionic current of a poly(dT)20-functionalized nanocapillary at T=27 °C are shown in Figure S5 for the target poly(dC)20 and poly(dA)20. The τ distribution of poly(dC)20 was approximated by a single Gaussian function with the most frequent time (or peak time)τp of about 0.09 ms (Figure 2b), while the τ distribution for poly(dA)20 was fitted to a double Gaussian function (Figure 2c). From the curve fits, τp1 and τp2 were estimated to be about 0.096 and 0.45 ms, respectively. Accordingly, translocation events were divided into fast translocation (peak at τp1) and slow translocation (peak at τp2), denoting the individual normal distributions for the first (τp1) and the second most frequent times (τp2). The population in fast and slow translocation was estimated to be about 15.4 and 84.6% from the area under the respective curves, implying that the slow translocation was dominant for poly(dA)20. Furthermore, the estimated value of τp1 for poly(dA)20 was noted to be comparable to τp of poly(dC)20. From these findings, we conjectured that fast translocation corresponded to translocation without interaction with the probe DNA, whereas slow translocation might be ascribed to an interaction of the target poly(dA)20 with the probe poly(dT)20 immobilized on the surface of nanocapillary. To test our idea, we also measured translocation of poly(dA)20 at T=40 °C, a temperature that is slightly lower than the melting temperature of 43.8 °C.22 As observed at T = 27 °C, the τ distribution of poly(dA)20 measured at 40°C was fitted to the double Gaussian function (Figure 2d), although more translocation events were detected at 40 °C due to decreased fluid viscosity or an increased diffusion constant at higher temperatures (Table 1), as reported by others.23 However, compared to the data measured at 27 °C, the most frequent times shifted to shorter times (τp1 ≈ 0.077 ms and τp2 ≈ 0.27 ms). Moreover, the population in fast translocation increased, whereas the population in slow translocation decreased at 40 °C. These results support that slow translocation was resulted from the interaction between the target and the probe ssDNA since the weakened base-pair interaction at high temperatures possibly shortens the duration time and decreases the population in slow translocation.

Figure 3. (a) Real-time ionic current representative data of hDNA-functionalized nanocapillary at 25 °C(black) and 65 °C (red) for PC-DNA. (b) Real-time ionic current representative data of hDNA-functionalized nanocapillary at 25 °C(black) and 65 °C (red) for SM-DNA.

poly(dA)20 exhibited the shorter τ because of the shorter length (Supporting Information, Figure S3b). To confer selectivity to the glass nanocapillary, the nanocapillary was functionalized with probe ssDNA. The amine-modified ssDNA containing thymines (5′-NH2(T)20-3′) were attached to the surface of the nanocapillary via covalent bonding of the amine group to a carboxyl group on the glass surface (Supporting Information, Figure S2a and S2b). After the functionalization, the ionic conductance decreased due to the reduced effective pore size (Supporting Information, Figure S2c). According to Eq. (1), the effective nanopore radius decreased from ~ 20 to 9 nm. This reduction was comparable to the length of poly(dT)20 ( ~10 nm). To investigate whether target ssDNA with different base sequences were selectively recognized by the DNAfunctionalized nanocapillary, target poly(dC)20 was added into the negative electrode chamber of a poly(dT)20functionalized nanocapillary, and then the real-time ionic current was measured at T=27 °C with the bias voltage of 0.5 V (upper panel of Figure 2a). Few current pulses were detected for the target poly(dC)20 which was not complementary to the probe poly(dT)20. Subsequently, the target


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Analytical Chemistry poly(dA)20 and poly(dC)20 using the poly(dT)20-

The ∆I/I0 distributions measured at 27 °C for target

Figure 4. Histograms of the logarithm of event duration time (log(τ)) for PC-DNA (red) and SM-DNA(blue) at 25 °C (a) and 65 °C (b). The log(τ) histograms show two normal populations, described by the sum of two Gaussian functions (black curves). Histograms of relative blockade current (ΔI/I0) for PC-DNA (red) and SM-DNA (blue) at 25 °C (c) and 65 °C (d).

translocation events for single base mismatched targets were also reported by Iqbal, et al,12 who measured translocation pulses using silicon oxide membrane-based nanopores functionalized with a probe of hair-pin loop DNA, and explained their results in terms of interaction between the probe and the target ssDNA. The distributions of τ and ∆I/I0 measured at 25 °C for SM-DNA and PC-DNA are shown in Figures 4a and 4c, respectively (Supporting Information, Figure S7). The τ distributions were fitted to a double Gaussian function for both PC-DNA and SM-DNA. However, the estimated values of τp1 and τp2 for the PC-DNA(τp1 ≈ 0.09 ms and τp2 ≈ 0.51 ms) were longer than τp1 and τp2 of SM-DNA (τp1 ≈ 0.07 ms and τp2 ≈ 0.31 ms) and the number of slow translocation events was very small for SM-DNA, indicating that slow translocation was ascribed to the interaction between hDNA and PC-DNA. In addition, compared to the poly(dT)20-functionalized nanocapillary in the presence of poly(dA)20, PC-DNA exhibited a longer τp2. This longer τp2 was probably ascribed to the stronger interaction between hDNA and PC-DNA than between poly(dT)20 and poly(dA)20 since the Gibbs free energy values are calculated to be -17.8 and -27.4 kcal/mol with melting temperature of 43.8 and 57°C in 0.5 M NaCl at 25 °C and pH 7 for the respective pairs poly(dT)20 and poly(dA)20, and hDNA and PC-DNA.22 Figures 4b and 4d show the distributions of τ and ∆I/I0 measured at 65 °C for SM-DNA and PC-DNA, respectively. Compared to the data at 27 °C, the frequency of translocation events increased, and τp1 and τp2 shifted to the shorter times due to the increased diffusion con-

functionalized nanocapillary are shown in Figures 2e and 2f, respectively. Unlike the τ distribution, the ∆I/I0 distribution was approximated by a single Gaussian function for both target molecules. However, the peak value (∆I/I0)peak for the target poly(dA)20 was slightly larger than (∆I/I0)peak of the target poly(dC)20 (Table I). Figure 2g shows the ∆I/I0 distribution of the target poly(dA)20 measured at 40 °C. I0 increased with increasing temperature (Supporting Information, Figure S6). Interestingly, (∆I/I0)peak of the target poly(dA)20 at 40 °C was similar to (∆I/I0)peak of the target poly(dC)20 at 27 °C, although further studies will be necessary to explain the relationship between (∆I/I0)peak and the base-pair interaction. To confirm the selectivity of the DNA-functionalized nanocapillary, we conducted similar experiments at 25 and 65 °C with another nanocapillary functionalized with the probe ssDNA containing the heterogeneous base sequences (5′-NH2-GGT TGG TTG TGT GTT GGT TGG-3′, hDNA). For the hDNA-functionalized nanocapillary, we used the perfect complement (5′-CCA ACC AAC ACA CAA CCA ACC-3′, PC-DNA) and the singlebase mismatched (5′-CCA ATC AAC ACA CAA CCA ACC-3′, SM-DNA) ssDNA as the target molecules. As for the poly(dT)20-functionalized nanocapillary, the target PC-DNA that was complementary to the probe hDNA resulted in clear and frequent current pulses (Figure 3a), while the target SM-DNA that was not complementary to the probe showed few current pulses (Figure 3b), although the frequency of translocation events increased with increasing temperature (Table 1). Similar reduced



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stant at high temperatures for both SM-DNA and PCDNA (Table 1). In particular, τp2 was more significantly Table 1. Summary of the translocation events of target ssDNA through bare and probe ssDNA-functionalized nanocapillaries. Probe ssDNA Bare

Target ssDNA

Temperature (℃)

Translocation Events (min-1)

Count

(ms)a

(ms)b

/

poly(dA)100

27

13.3

456

0.14±0.02

-

0.020±0.011

poly(dC)100

27

12.3

428

0.17±0.03

-

0.018±0.009

27

32.8

164

0.096±0.015

0.45±0.04

0.013±0.011

40

949

949

0.077±0.026

0.27±0.03

0.011±0.008

27

3.8

141

0.093±0.0032

-

0.010±0.010

25

166.7

500

0.089±0.025

0.51±0.02

0.028±0.011

65

560

560

0.055±0.019

0.21±0.02

0.013±0.009

25

29.3

88

0.066±0.036

0.31±0.03

0.017±0.012

65

108.3

325

0.055±0.024

0.15±0.02

0.010±0.007

poly(dA)20 poly(dT)20 poly(dC)20 PC-DNA hDNA SM-DNA a

b

τp1 is the event duration time of fast translocation represented by most frequent value. τp2 is the event duration time of slow c translocation represented by most frequent value. ∆I/I0 is the ionic current change normalized by the ionic current without DNA molecules represented by most frequent value. Data represent mean±standard deviations (n = 3). shortened than τp1. In addition, PC-DNA and SM-DNA exhibited similar distributions of fast translocation at 65 °C. Since 65 °C is above the melting temperature for hDNA and PC-DNA, these results confirmed that the translocation of ssDNA through the DNA-functionalized nanocapillary was governed by the interaction between the probe and the target DNA molecules, so the target ssDNA could be selectively recognized by the DNAfunctionalized nanocapillary with sensitivity for a single-base mismatch.

ASSOCIATED CONTENT Supporting Information Additional figures and information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author *Corresponding author e-mail: K-H Yoo, [email protected]

CONCLUSION In summary, we have investigated the translocation of the target ssDNA with different base sequences through nanocapillaries functionalized with poly(dT)20 or hDNA. Compared to the target ssDNA that is not complementary to the probe ssDNA, the complementary target ssDNA yielded the slower and more frequent translocation events through the DNA-functionalized capillary, which we ascribed to the interaction between the target and probe ssDNA covalently attached on the channel. In particular, the scatter plots of the hDNA-functionalized nanocapillary measured at different temperatures showed different distributions of τ or ∆I/I0 for PC-DNA and SM-DNA, demonstrating that the single-base mismatch DNA molecules could be discriminated using the functionalized nanocapillary. The glass nanocapillaries are produced at low cost and high throughput, so these selective nanocapillaries could provide a simple approach to investigate molecular interactions, such as hybridization of nucleic acids, DNA or RNA-protein, and protein-protein interactions.

Present Addresses †Department of Physics, Yonsei University, Seoul, 120-749, Republic of Korea §Department of Materials Science and Engineering, Yonsei University, Seoul, 120-749, Republic of Korea

ACKNOWLEDGMENT This work has been financially supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT and Future Planning (Grant No. 2011-0017486 and 2012R1A4A1029061)

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