DNA molecules and configurations in a solid-state nanopore microscope

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Entry by Haifei Zhang, AP 225, Fall 2009

Second Entry by Hsin-I Lu, AP 225, Fall 2009

Third Entry by Xu Zhang, AP 225, Fall 2009

Soft matter keywords

Nanopore, Biopolymer, DNA, Viscosity

Summary

The authors examined the properties of single DNA molecules using a solid-state nanopore membrane. Ultimately, the technique could provide rapid DNA sequencing.

To carry out the procedure, the authors used ion-beam sculpting to create 3 or 10 nm pores in a 5-10 nm thick silicon nitride membrane. Then they positioned the membrane between two chambers containing conducting electrolyte solution and applied a 120 mV bias across the nanopore to set up open-pore ionic conduction. Adding dsDNA to the negatively charged chamber resulted in momentary reductions in current flow as individual DNA molecules moved through the pore. The expected current blockage for a single molecule is linearly dependent on the cross-sectional area of the molecule.

For the 3 nm pore, all the current blockage events appeared relatively straightforward. In contrast, around 40% of the blockages for the 10 nm pore showed more complex patterns, an observation that the authors attributed to folding of the molecules. The DNA molecules were about 2 nm wide, and so were unable to pass through the 3 nm pore whilst folded.

Experimental setup

Fig. 1. Details of the experimental setup.
Fig. 2. Distribution of events as a function of td and <ΔIb> for 10-kb dsDNA.

At its heart the microscope consists of a voltage-biased nanopore, fabricated in a silicon nitride membrane. The membrane separates two chambers of conducting electrolyte solution. The only electrical conduction path from one chamber to the other passes through the nanopore. To resolve interesting molecular structure, the nanopore dimensions must be small enough to avoid averaging over continuous single-molecule configurations induced by thermal fluctuations and large enough to pass the smallest dimensions of the molecule to be probed. For double-stranded DNA (dsDNA), this means a pore diameter and membrane thickness smaller than the molecule persistence length, 50 nm for dsDNA, and a pore diameter larger than the ~2 nm cross-sectional size of the molecule.The recent discovery of ion-beam sculpting11 allows structures that meet these criteria to be fabricated with desired nanometre-scale dimensions from solid-state materials such as silicon nitride.A transmission electron micrograph (TEM) of an ion-sculpted 3-nm nanopore in a membrane 5–10 nm thick is shown Fig. 1a, and a schematic of the experimental setup is shown in Fig. 1b. In Fig. 1b,Solid-state nanopore microscope used to obtain electrical signals from single DNA molecules.

Figure 2 shows two plots of the density distribution from many transient molecular events over the parameters <ΔIb> and td. <ΔIb> is defined as the average value of a current blockade over td (regardless of the signal’s shape).Figure 2a is obtained from experiments using 10-kb dsDNA with a 3-nm pore, and Fig. 2b from experiments with 10-kb dsDNA and a 10-nm pore.The voltage bias across the pore in both cases was 120 mV. The colour coding is keyed to the local density of events normalized by the total number of events for each case.Although both distributions peak at td ~ 300–400 μs, the distribution in td is quite broad for the 3-nm pore experiment and much sharper for the 10-nm pore experiment. On the other hand, the distribution of events in <ΔIb> for the 10-nm pore is much broader than for the 3-nm pore,with larger <ΔIb> events showing a definite trend towards having smaller values of td. In Fig. 2a, it shows a 3-nm pore,2,674 events; Fig. 2b, it shows a 10-nm pore,9,477 events.The bias voltage was 120mV. The colour scale represents event-fraction density normalized as a probability distribution so that the integral of the density over all td and <ΔIb> is equal to 1.

Translocation speed

Fig. 3. Translocation time distribution function for 3-kb and 10-kb dsDNA molecules in a 10-nm nanopore at 120-mV bias,and for 10-kb dsDNA at 60-mV bias.

The average speed of dsDNA molecules translocating through 10- nm pores biased at 120 mV is ~1cms−1.A quantitative understanding of this result can involve many complex issues like hydrodynamic interactions and screenings, electro-osmotic flow in and near the pore, and non-equilibrium statistical considerations13–16.Here we note that a simple combination of likely relevant parameters, derived by equating the electric force on the charged polymer in the pore to a viscous drag on an effective sphere of radius a on either side of the pore,gives an average translocation speed of

<math>v_t=C \frac {\sigma V_{bias}} {(2a) (6 \pi \eta) }</math>

where <math>\sigma</math> is the linear charge density on the molecule,<math>\eta</math> the viscosity of the solution,<math>V_{bias}</math> the pore voltage bias, and C is a factor of order unity accounting for the complex issues such as hydrodynamic interactions and electro-osmotic flow. Setting <math>a</math> to the persistence length of DNA and assuming a charge of e/3 per phosphate, they found that C ~ 1/2 brings <math>v_t</math> in the model close to experimental observations.

Results

Fig. 4. Density of events over td and <ΔIb> for 10-kb dsDNA passing through a 10-nm pore.

Figure 4a shows the density plot of the simple translocation events for 10-kb dsDNA passing through the 10-nm pore, whereas figure 4b shows a density plot for more complex 'multi-level' events that remain after the simple ones are substracted. Characteristic event time recordings are shown in the inset.These remarkable additional features are attributed to DNA molecules that are folded on themselves as they pass through the pore. This assumption is also confirmed by a study of the distribution of instantaneous blockade current magnitude over all events. Assuming the instantaneous magnitude of the blocked current is in proportion to the instantaneous number of strands of dsDNA in the nanopore, a quantization of local instantaneous ΔIb values corresponding to zero,one two,... is expected. A histogram of ΔIb over ~9,500 events is shown in fig5, where the expected quantization of sampled ΔIb values is clearly seen corresponding to zero, one and two molecule strands occupying the nanopore.

Soft matter details

DNA.GIF

DNA is a polymer, or rather biopolyer. The monomer units of DNA are nucleotides, and the polymer is known as a "polynucleotide." Each nucleotide consists of a 5-carbon sugar (deoxyribose), a nitrogen containing base attached to the sugar, and a phosphate group. There are four different types of nucleotides found in DNA, differing only in the nitrogenous base. The four nucleotides are given one letter abbreviations as shorthand for the four bases. A is for adenine; G is for guanine; C is for cytosine; and T is for thymine.

Although earlier studies have used biopore detectors, solid-state nanopores have the advantage that scientists can choose their diameters, enabling the study of molecules such as RNA, hybridized DNA and proteins. Solid-state nanopores are also more physically robust, and could be used at high or low temperatures and under voltage and pH conditions that would destroy biopore-membrane systems.

According to the authors, the solid-state pores provide a new way of studying the folding and pairing configurations of single long-chain molecules, the differences between chemically identical molecules in a statistical ensemble, and induced changes in molecular structure.

The authors plan to add electrical contacts to the nanopores, a feature that should enable techniques such as electronic tunnelling and near-field optical studies of molecules as they pass through the nanopore. Such local single-molecule spectroscopy could increase longitudinal resolution, perhaps even up to the single-base level for DNA, allowing extremely rapid sequencing of long molecules.

References

[1] J. Li, M. Gershow, D. Stein, E. Brandin, and J. A. Golovchenko, DNA molecules and configurations in a solid-state nanopore microscope, Nature Materials, 2: 611-615 (2003).

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DNA molecules and configurations in a solid-state nanopore microscope (by Hsin-I Lu)

Summary

This article used a solid-state nanopore fabicrated from silicon nitride to observe individual double-stranded DNA (dsDNA) and it's folding behavior. In the absence of dsDNA molecules, open-pore ionic current develops with some biased electric field across the nanopore. With dsDNA molecules added to the negative cis chamber, current blockades appeared in the form of isolated transient reductions in current flow through the pore. By studying the amount and duration of current blocades with different biased field, the authors found the force dragging dsDNA through nanopore is due to electric force. They also identified the folding of dsDNA in the pore.

Soft matter keywords

atomic-force microscopes, double-stranded DNA, electro-osmotic flow, viscous drag, molecular folding

Soft Matter

Fig. 1: Experimental setup
Fig. 2: Translocation time distribution function
Fig. 3: Instantaneous time distribution of blockade current
  • Experiments:

The dimension of nanopore (Fig. 1a) plays an important role in this experiment. It's diameter has to be large enough (~3-10 nm) to allow single dsDNA pass through. It's thickness (5-10 nm) has to be small enough to contributions on signal from continuous single-molecule configurations induced by thermal fluctuations. Fig. 1b shows the experimental setup. Characteristic signals showing transient molecular current blockades is shown in Fig. 1c. We can learn two important pieces of information from current blockades: translocation time, <math>t_d</math>, and current blockage, <math>\Delta I_b</math>. Since <math>t_d</math> is measuring how long dsDNA is passing through nanopore, it should be related to the length of DNA and how fast DNA is dragging through the nanopore.

Fig. 2 shows the <math>t_D</math> distribution function for 3-kb and 10-kb dsDNA molecules in a 10-nm nanopore at 120-mV bias,and for 10-kb dsDNA at 60-mV bias. 10-kb DNA takes about three times longer to move through the pore than 3-kb DNA at the same bias. A factor of two increase in biased field boubles <math>t_d</math> for 10-kb dsDNA.

  • Model of translocation speed:

The average speed of dsDNA molecules translocating through 10-nm pores biased at 120 mV is ~1 <math>cms^{-1}</math>. The authors proposed a simple model to descirbe translocaiton speed by equating the electric force on the charged polymer in the pore to a viscous drag on an effective sphere of radius a on either side of the pore. Translocation speed is as follows,

<math>v_t=C \frac {\sigma V_{bias}} {(2a) (6 \pi \eta) }</math>

where <math>\sigma</math> is the linear charge density on the molecule,<math>\eta</math> the viscosity of the solution,<math>V_{bias}</math> the pore voltage bias, and C is a factor of order unity accounting for the complex issues such as hydrodynamic interactions and electro-osmotic flow. Setting <math>a</math> to the persistence length of DNA and assuming a charge of e/3 per phosphate, they found that C ~ 1/2 brings <math>v_t</math> in the model close to experimental observations.

  • Quantized blockade currents:

In the absence of DNA molecules, the ionic current should be constant and set by the biased field, concentration of KCL, and the size of nanopore. In the other extreme case, there should be no ionic current signal if the nanoport is completely 'plugged' by DNA molecules. Therefore the ionic current should depend linearly on the uncovered area of nanopore. Fig. 3 demonstrates how current blockades vary with zero, one and two strands of 10-kb DNA molecules in 10 nm pore under a, 120-mV bias and b, 60-mV bias.

  • Discussion:

Nanopore microscopy reported in this paper provided a way to study the folding dynamics of single DNA or polymer. If folding of DNA happens at the same time when it translocates through nanopore, the ionic current signal should reveal the singature of two quantized blockade currents in a single event. The authors also point out a major advantage of this technique. When long DNA or polymer pass thourgh nanopore, they are essentially localized and linearized in the nanopore. With additionally applying electronic tunnelling and near-field optical methods, local single molecule spectroscopies are possible. Increasing longitudinal resolution, possibly to the single-base level for DNA, allows for extremely rapid sequencing of long molecules.

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DNA molecules and configurations in a solid-state nanopore microscope (summarized by Xu Zhang)

Reference

J. Li, M. Gershow, D. Stein, E. Brandin, and J. A. Golovchenko, DNA molecules and configurations in a solid-state nanopore microscope, Nature Materials, 2: 611-615 (2003).

Key Words

nanopore, polymer, DNA folding

Summary

This paper shows

1. A solid-state nanopore 'microscope' which can electronically characterize single long-chain polymers such as DNA molecules.

2.The first observation of molecule-induced quantized current blockades that reveal the folding configuration of single molecules as they pass through the nanopore.

Mechanism: Each Translocating molecule blocks the open pore ionic current, providing an electrical signal that depends on the characteristics of the molecule.

Nanopore dimension

Nanopore dimensions must be small enough to avoid averaging fluctuations and large enough to pass the smallest dimensions of the molecule to be probed.

In this paper, a transmission electron micrograph(TEM)of an ion-sculpted 3-nm nanopore in a membrane 5-10nm thick is shown in Fig.1a.

Fig. 1a: TEM image of a 3nm silicon nitride nanopore

Microscope set up

Solid-state nanopore microscope used to obtain electrical signals from single DNA molecules is shown in Fig.1b.

Fig. 1b: Solid-state nanopore microscope used to obtain electrical signals from single DNA molecules

Characteristic signals

Open-poreionic conduction was first established with 120-mV bias across the nanopore. Then 3-kilobase(kb)dsDNA was added to the negative cis chamber and the Fig.1c shows the current blockades in the form of isolated transient reductions in current flow through the pore.

Fig1c.jpg

The expected current blockage from a single molecule blocking the pore is linearly dependent on the cross-sectional area of the molecule and independent of the area of the pore.

Signal analysis

1. The dependence of <math><\Delta I_b></math> (average current blockade)and<math> t_d</math> (translocation time) on DNA size, pore size and the bias voltage. Figure 2 and 3 show the dependent relationship. From these two figures, we can conclude that with all the other parameter unchanged

(1)Translocation time is longer with a shorter nanopore.

(2)The average current blockade is bigger with bigger nanopore and shorter tranlocation time.

(3)Translocation time is approximately proportional to the length of dsDNA.

(4)Translocation time is approximately inversely proportional to the bias voltage.

Fig22.jpg

These observations provide strong evidence that each simple single-level event corresponds to a DNA molecule translocating in single-file order through the nanopore under the influence of electrophoretic forces.

2. Single-level and multi-level events Figure 4a shows the density plot of the simple translocation events for 10-kb dsDNA passing through the 10-nm pore, whereas figure 4b shows a density plot for more complex 'multi-level' events that remain after the simple ones are substracted. Characteristic event time recordings are shown in the inset.These remarkable additional features are attributed to DNA molecules that are folded on themselves as they pass through the pore. This assumption is also confirmed by a study of the distribution of instantaneous blockade current magnitude over all events. Assuming the instantaneous magnitude of the blocked current is in proportion to the instantaneous number of strands of dsDNA in the nanopore, a quantization of local instantaneous <math>\Delta I_b</math> values corresponding to zero,one two,... is expected. A histogram of <math>\Delta I_b</math> over ~9,500 events is shown in fig5, where the expected quantization of sampled <math>\Delta I_b</math> values is clearly seen corresponding to zero, one and two molecule strands occupying the nanopore.

Fig4&5.jpg

3.Translocation speed

Here the expression for the translocation speed is derived by equating the electric force on the charged polymer in the pore to a viscous drag on an effective sphere of radius a on either side of the pore,

<math>\nu=D\frac{\sigma V_{bias}}{(2a)(6\pi \eta)}</math>

<math>\sigma</math>:the linear charge density on the molecule.

<math>\eta</math>: the viscosity of the solution.

Connection to Soft Matter

The solid-state pores provide a new way of studying the folding and pairing configurations of single long-chain molecules, the differences between chemically identical molecules in a statistical ensemble, and induced changes in molecular structure that, because of energy restrictions, do not occur naturally in solution. These nanopores can allow electronic tunneling and near-field optical studies of translocating molecules that are linearized and confined in a nanopore of the microscope. Applying these new physical local interactions to molecules translocating through nanopores can provide local single molecule spectroscopies not afforded by measurement of ionic current alone, and offer a means of increasing longitudinal resolution, possibly to the single-base level for DNA, allowing for extremely rapid sequencing of long molecules.