pubs.acs.org/NanoLett
Detecting DNA Folding with Nanocapillaries Lorenz J. Steinbock, Oliver Otto, Catalin Chimerel, Joanne Gornall, and Ulrich F. Keyser* Cavendish Laboratory, University of Cambridge, J. J. Thomson Avenue, Cambridge CB3 0HE, United Kingdom ABSTRACT We demonstrate for the first time the detection of the folding state of double-stranded DNA in nanocapillaries with the resistive pulse technique. We show that glass capillaries can be pulled into nanocapillaries with diameters down to 45 nm. We study translocation of λ -DNA which is driven by an electrophoretic force through the nanocapillary. The resulting change in ionic current indicates the folding state of single λ -DNA molecules. Our experiments prove that nanocapillaries are suitable for label-free analysis of DNA in aqueous solutions and viable alternatives to solid-state nanopores made by silicon nanotechnology. KEYWORDS Nanocapillary, nanopore, DNA folding, sensing, electrophoretic translocation
T
tion, and applied potential.16 Recently, an external bottomup etching technique to fabricate nanopores in glass capillaries was described. By further constricting the orifice with a circular βCD molecule Gao et al. were able to distinguish enantiomeric molecules.17 Other groups used a long glass nanochannel glued into a glass pipet to detect single DNA strands with a Coulter counter setup.18 In the present work, we demonstrate the single-molecule detection of double-stranded (ds) DNA using single laserpulled quartz glass capillaries with diameters down to a few tens of nanometers. We demonstrate that we can detect the folding state of linear double-stranded λ-DNA of 48.5 kbp. In our experiments, the DNA is driven by electrophoresis through the nanocapillary. We observe numerous levels of blockade current. Each current level can be attributed to a folding state of the translocating DNA.19,20 Our analysis excludes the presence of multiple dsDNA inside the detection volume. Our work paves the way for simple and cheap single molecule characterization experiments which require merely a current amplifier and a pipet laser puller. Methods and Materials. Quartz glass capillaries with an outer diameter of 0.5 mm and an inner diameter of 0.3 mm (Hilgenberg GmbH, Germany) were used for all experiments. Prior to pulling the capillaries, the glass pipettes were thoroughly cleaned by sonicating in acetone and ethanol for a duration of 5 min in each step. This removed any contamination resulting from the production process or dirt acquired during transport, storage, and handling. Residual ethanol from the cleaning process was removed with gaseous nitrogen. Afterward, the capillary ends were attached to the pulling slides in the pipet laser puller. The CO2 laser heated a spot of 0.1 mm in the middle of the capillary. Meanwhile the glass capillary was pulled in opposite directions at both ends. Consequently, the glass shrank in diameter in the heated area. A final stronger pull separated the capillary in the middle resulting in capillary tips of identical shape and with the same diameter. The set of parameters shown in Table 1 gives nanocapillaries with inner diameters as small as 45 nm as shown in the scanning
he resistive pulse principle forms the technical basis for the well-known Coulter counter. It provides a means for the label-free characterization of microand nanoparticles in solution.1-3 Early experiments revealed that it is possible to count and size bacteria cells with a Coulter counter.4 Subsequently, the application range was expanded to particles such as pollen, viruses, or colloids coated with, e.g., antibodies.5,6 The recent miniaturization of the sensing volume, with the help of solid-state or biological nanopores has extended the range of detection to single biomolecules.7-9 The Coulter counter technique is based on two reservoirs, both filled with a particle-laden salt solution and connected by a single orifice. When an electrical potential is applied between the two reservoirs, an ionic current is measured. The ionic current depends on the dimensions of the orifice and decreases when it is partially blocked. Using hydrodynamic pressure or an electrical field, one can transport individual particles or molecules through the pore. By monitoring the changes in the ionic current, a Coulter counter is able to count the number of translocating objects through the pore as well as determine the size of the particles. Glass capillaries have emerged over the past decade as an alternative to nanopores made from channel-like transmembrane proteins10 or solid-state nanopores in silicon membranes.11 Borosilicate or quartz capillaries can be pulled with a laser-equipped pipet puller into micro- or nanocapillaries. Depending on parameters such as glass thickness, temperature, and pulling force, diameters down to nanometer range can be achieved.12,13 First experiments have shown that nanocapillaries allow for the detection of DNAcoated colloids.14 Our recent work revealed that it is possible to differentiate not only colloids of different size but also DNA-coated colloids from uncoated colloids.15 This was supported by a dynamical model taking into account the conical shape and orifice of microcapillaries, ion concentra* To whom correspondence should be addressed,
[email protected]. Received for review: 03/21/2010 Published on Web: 06/01/2010 © 2010 American Chemical Society
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TABLE 1. Parameters for Pulling Nanocapillaries with Inner Diameters down to 45 nm Using the Pipette Puller P-2000 and Quartz Glass with an Outer Diameter of 0.5 mm and a Wall Thickness of 0.1 mma cycle
heat
Fil
Vel
Del
Pull
1 2 3 4
520 520 520 520
0 0 0 0
10 10 10 15
128 128 128 128
0 0 0 200
program (LabVIEW 8.5, National Instruments) determined the baseline ionic current of each event by calculating the average ionic current within the first third of the data points. The end of an event was determined once the ionic current recovered to the baseline level and vice versa for the beginning of the translocation. The time difference between these two points was set to be the duration of the translocation event. We determined the mean event current by integrating the ionic current values and dividing by the event duration. Results. Experiments always begin with a measurement in order to check for stable current-voltage characteristics (see Figure 2a). In addition, we monitor the current noise at different voltage levels. Ideally the rms noise at 5 kHz bandwidth should not exceed 5 pA. It is possible to estimate the inner diameter, di, of the mounted nanocapillary by use of a simplified equation for conical nanocapillaries21
a A successful pull process did not loop with a heat duration of approximately 1.6 s.
electron microscopy (SEM) images (see Figure 1a). Prior to visualization in the SEM, nanocapillaries were coated with a 10 nm thick layer of platinum (Pt). For translocation experiments platinum-free nanocapillaries were glued into a PDMS form, connecting two reservoirs (see Figure 1b). A cover glass, 0.15 mm thick (Menzel-Glaser GmbH, Germany), sealed the reservoirs at the bottom and the top. In each chamber a 50 µm diameter silver electrode was embedded which was chlorinated (Ag/AgCl) before the measurement. One electrode was glued into the left reservoir in front of the nanocapillary tip. The other electrode was passed through the right reservoir into the glass capillary until it touched the conical part of the capillary (see Figure 1c). Then the nanocapillaries were inspected by optical microscopy. Afterward, the two reservoirs and the micropipet were filled with potassium chloride (KCl) solution of 0.5 mol/L. We kept the pH at 8 throughout all experiments by using Tris-EDTA solution added at 10% of the salt concentration. Air was completely removed from the flow cells and nanocapillaries by placing the assembled cells into desiccators and evacuating with a vacuum pump for around 30 min. Before the experiment started, the electrode offset was set to zero, and the nanocapillary was tested for stable currentvoltage characteristics and a root mean square (rms) of the ionic current signal below 5 pA. During the experiment, the ionic current was checked regularly for stable current-voltage characteristics. The λ-DNA (Invitrogen, U.K.) was diluted by 1:10 to a 10 nmol/L concentration and added to the reservoir as close as possible to the tip of the nanocapillary. For all ionic current measurements we used the Axopatch 200B (Axon Instruments, USA) amplifier in voltage-clamp mode with the internal four-pole Bessel filter at 10 kHz bandwidth. The signals were digitized with a NI-PCIe-6251 card (National Instruments, USA). Data recording was performed with a program based on C/C++ and LabVIEW (LabVIEW 8.5, National Instruments). During each recording the specific potential (+0.5 V), KCl concentration (0.5 mol/L), sampling rate (300 kHz), and ionic current (0-500 pA) were monitored. Voltage-driven translocation of λ-DNA showed a characteristic decrease in the ionic current which was recorded whenever a specific threshold was exceeded. For statistical analysis, between 400 and 1000 translocation events were recorded. Each translocation event consisted of 400 data points before and after the threshold was crossed. A custom-made © 2010 American Chemical Society
di )
4Gl πgdb
(1)
with l the length of the conical part (1 mm for this capillary), db the diameter of the capillary before starting to converge (0.3 mm), g the specific conductance (6.3 S/m) for 0.5 mol/L KCl, and G the nanocapillary conductance in the respective KCl solution (30 nS). Equation 1 reveals an inner diameter of ∼19 nm and is in good agreement with the diameter visible in the SEM image of the nanocapillary. The deviation from a linear dependence in the current-voltage graph (see Figure 2a) is caused by the asymmetry of the nanocapillary.22 We always perform control measurements, by applying +0.5 V to the electrode inside the capillary, while having a DNA-free solution in the reservoirs. Figure 2b (Control) shows stable ionic current traces for the control experiment which demonstrate the absence of any translocating molecules. After the negative control measurement, the reservoir containing the nanocapillary is filled with a 10 nmol/L λ-DNA solution. Again, +0.5 V is applied to the electrode inside the capillary, which immediately, causes events showing a typical decrease in the ionic current signal (see Figure 2b (DNA)). These findings are consistent with negatively charged DNA being pulled toward the positive electrode in the capillary, driven by electrophoretic force. The events show a decrease in the ionic current, indicating that translocating DNA is excluding ions from the nanocapillary, as observed by other groups working with biological and solid-state nanopores.10,11 The first two events are unfolded events caused by stretched DNA translocating through the nanocapillary. Analysis of the shown translocation events demonstrate that they last for 0.5-3 ms. The mean ionic current of the events ranges from -20 to -120 pA (see Figure 2b). This is further illustrated in Figure 3 summarizing all 431 events recorded in this experiment. Each point represents a single translocation event, and the position is determined by the 2494
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FIGURE 1. (a) Quartz glass nanocapillary after pulling with a laser-assisted pipet puller. The nanocapillary was coated with a 10 nm thick Pt layer to prevent charging effects. The black scale bar is 100 nm long, the inner diameter of the tip is around 45 nm. (b) Photograph of the PDMS cell with two reservoirs which were connected by the nanocapillary. One electrode was placed in the reservoir, facing the nanocapillary. The second electrode was inserted into the capillary from the opposite reservoir. (c) Schematic of the experimental setup with sample cell, nanocapillary and measuring circuits.
FIGURE 2. (a) Current-voltage dependence for the nanocapillary in 0.5 mol/L KCl. The rectification resulting in a slightly nonlinear current-voltage curve is caused by the asymmetry of the nanocapillary. (b) Control and DNA experiment with a potential of +0.5 V and a baseline ionic current at +15 nA. The control measurements show no translocation events. Once a 10 nmol/L λ-DNA solution is added, clear events are observed showing a decrease of the ionic current.
FIGURE 3. (a) Scatter plot showing the duration and mean event current of 431 recorded events, with a λ-DNA 10 nmol/L buffer solution, an applied potential of +0.5 V, and in buffered 0.5 mol/L KCl solution at pH 8. The black line is calculated with eq 2 using the values dun ) 1.56 ms and Iun ) -43 pA to calculate the current I(d) and was first introduced by Li et al.19 (b) Duration histogram of the 72 unfolded events having a stable mean event current of -43 ( 12 pA with no additional values below -55 pA. Distribution is fitted with a Gauss function giving rise to a peak at 1.56 ( 0.03 ms.
where I(d) is the mean event current of the event and d the duration. Iun and dun are the mean event current and duration for the unfolded events, respectively. A Gauss fit of the mean event current and duration histogram for the unfolded events yields Iun ) -43 ( 1.1 pA (not shown) and dun ) 1.56 ( 0.03 ms (Figure 3b). With these parameters, the black line in Figure 3a is calculated using eq 2. Obviously, this results in a very good describtion of the events in Figure 3 (a). Closer investigation of all current traces in Figure 2b (DNA) reveal that the events can be grouped into plateaus at current levels which are multiples of ∼-40 pA. This is
duration and mean event current. The events show a duration of 0.5-3 ms and a mean event current between -20 and -120 pA. The distribution in the scatter plot is not circularly symmetric. Notably, we observe a trend of higher mean event currents for shorter durations, which was also observed in solid-state nanopore experiments.19,20,23 As described by Li et al., our events in the scatter plot (Figure 3a) obey the following equation19
I(d) )
Iundun d
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(2)
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FIGURE 4. Current histogram for the control and DNA experiment. For each event the mean baseline current is subtracted before generating the histogram. Control experiment without DNA (black area) shows a single peak at 0 pA representing the open pore current. The DNA current histogram (red area) displays several peaks besides the baseline peak at around 0 pA. The baseline and four translocation peaks are fitted with a multipeak Gauss function (gray line) and reveal peaks at -0 ( 0.1, -43 ( 0.5, -81 ( 2, -113 ( 18, and -144 ( 32 pA.
FIGURE 5. Histogram of the total charge excluded from the nanocapillary during translocation events. The area histogram can be fitted with a Gaussian function, which places the peak at about 59 fAs.
nanocapillary once parameters like l, db, G, and g are known. This allows for secondary structure analysis of single DNA molecules with a benchtop technology. We shall now discuss whether these plateaus in Figure 4 are due to a single DNA strand being folded on itself or by the presence of two or more DNA molecules inside the nanocapillary. By looking at the mean event current of unfolded events, we find a value of ∼1.56 ms, which is much shorter than the average time between two subsequent events of 0.14 s. Since the chance to have two molecules passing at the same time is very small and the percentage of events showing -80 pA or more mean event current is 22% of the total translocation events, the folded DNA configuration appears to be the most likely explanation. This assumption is also supported by investigating the area of the translocation events. Since the total excluded charge should be more or less constant per translocating molecule, one can test further the possibility of single or multiple translocations by integrating the event area.20 The area of each event is determined during data analysis with our custom written program. The resulting area histogram shown in Figure 5 can be fitted with a single Gaussian with a maximum at xc ) -59 fAs. A mixed population of single molecule translocation events and simultaneous translocation of multiple DNA strands would lead to several peaks in Figure 5. The observation of a single peak supports our hypothesis of λ-DNA molecules translocating as individual polymers through the nanocapillary while either being completely unfolded or partly folded. The value xc ) -59 fAs is also confirmed by multiplying the mean event current of ∼-43 pA and the duration of ∼1.56 ms for unfolded events resulting in an area of ∼-67 fAs.
further supported by a histogram of all ionic current recordings (see Figure 4). The histograms for the control and the DNA experiment are normalized to allow for a better comparison. The highest peak at 0 pA is the open pore current and remains the only peak for the control experiment and illustrates the constant baseline. The histogram of the DNA experiment shows additional peaks at about ∼-43, ∼-81, and ∼-113 pA, and a fourth less clear peak at ∼-144 pA, respectively. The peaks are identified by fitting a multipeak Gauss function to the current histogram (see Figure 4 gray line). The peak at ∼-43 pA results from a single dsDNA molecule translocating in a unfolded state through the nanocapillary. The second peak at ∼-81 pA is explained either by the presence of two dsDNA molecules inside the nanocapillary or by one partially folded dsDNA molecule. Both scenarios lead to a doubling of the occupied area in the sensing volume in contrast to a single dsDNA strand. This causes a doubling of the blockade current. The peaks at ∼-113 and ∼-144 pA can be the exclusion by up to either three or four DNA molecules. Whether the origin for the quantization is caused by folded dsDNA or multiple dsDNA strands inside the nanocapillary will be ascertained in the discussion. Discussion. A resistive pulse technique based on nanocapillaries offers several advantages over state of the art silicon membrane and biological nanopores. Biological nanopores need a lipid bilayer which sets serious limitations to experiments at both extreme pH and high voltages. In contrast, a quartz nanocapillary is less sensitive to pH changes or high electric fields. However, control of the surface composition on the atomic level is currently not possible. Compared to the silicon membrane nanopores, our nanocapillaries do not require elaborate electron microscopes for fabrication. In addition, the capacitance and, therefore, the noise can be much lower than that for nanopores fabricated in silicon chips. As shown with eq 1, one can already calculate an approximated diameter of the © 2010 American Chemical Society
The minimum detectable cross section size and length of the translocating polymer depends on the signal-to-noise ratio and the sampling frequency. The minimum resolvable event amplitude in Figure 4 lies at approximately ∼25 pA, which corresponds to a minimum polymer cross section of 1.2 nm. This minimum current event resolution is due to the big nanocapillary of more than 45 nm and its conical 2496
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detection and optical tweezers in order to probe translocation of biomolecules.
shape. To allow detection of polymers with smaller cross sections, one possibility would be to use smaller nanocapillaries with shorter tapers. This would increase the signalto-noise ratio when the molecule translocates through the nanocapillary. Further, from the frequency of the current recording of 300 kHz, the Bessel filter at 10 kHz bandwidth, and the duration of ∼1.56 ms for a fully unfolded λ-DNA molecule, we conclude a minimum polymer length of ∼1 µm which could be easily detected. The similarity of the current traces (see Figure 2b (DNA)) with events recorded in silicon membrane nanopores of Li et al., Storm et al., and Smeets et al.19,20,23 provides good evidence that we observe DNA translocations. We obtained mean event current values of ∼-43 pA and durations of ∼1.56 ms for unfolded DNA translocations. These results are in good agreement with -30 pA and 1.2 ms measured by Smeets et al., having the same KCl concentration, pH, and DNA length as in our experiment.23 The quantization of the current histogram shows that one can detect the folding states of translocating dsDNA with a nanocapillary. Furthermore, the correlation of shorter duration with higher event amplitudes and the area histogram with one peak suggest thatweobservethetranslocationofasingledsDNAmolecule.19,20 The length of λ-DNA of 16 µm and the translocation time of ∼1.56 ms results in a translocation velocity of 10.3 mm/s. This is in very good agreement with silicon membrane experiments by Li et al. and Smeets et al. showing a translocation of 10 mm/s for 10 kbp dsDNA.19,20 Another advantage of our nanocapillaries is the distance from the cover slides of less than 0.25 mm. This relatively small distance enables the use of high-resolution objectives with numerical apertures up to 1.2. This will allow the trapping of colloids in front of the nanocapillary with the use of optical tweezers. Thus, DNA translocation could be controlled in a similar way as demonstrated for solid-state nanopores.24,25 Since the nanocapillary is perpendicular to the optical axis of the laser, the displacement can be recorded in the imaging plane. This will increase the force and position resolution as compared to the membrane nanopore setups, where the displacement of the colloid has to be detected along the optical axis.26 Conclusion. We have been discussing a nanocapillarybased Coulter counter for the detection of λ-DNA molecules. Our setup can detect a single DNA molecule. Importantly, the resistive-pulse measurements show the folding state of a single DNA molecule inside the nanocapillary. For the latter finding we investigated the current histogram of the events which revealed the existence of several current levels. Our results establish nanocapillaries as a promising alternative to solid-state nanopores for label-free single molecule analysis. Our findings pave the way for the use of nanocapillaries in combination with optical techniques like fluorescence
© 2010 American Chemical Society
Acknowledgment. This work was supported by the Emmy Noether-Program of the DFG. Further, L. J. Steinbock and O. Otto thank the Deutsche Telekom Foundation and the Boehringer Ingelheim Foundation, respectively, for a PhD scholarship. J. Gornall is supported by a grant from the Nanoscience E+ initiative and the EPSRC. The authors thank Prof. D. Klenerman’s group and L. Hild for experimental help. Note Added after Issue Publication. Reference 13 was added and the citations in the paper changed in the original version published on June 1, 2010. The revised version was published on the Web on June 28, 2013. An Addition/Correction was published in volume 13, issue 7.
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