Solid-state nanopores are sensors capable of analyzing individual unlabelled DNA molecules in solution. While the critical information obtained from nanopores (e.g., DNA sequence) is the signal collected during DNA translocation, the throughput of the method is determined by the rate at which molecules arrive and thread into the pores. Here we study the process of DNA capture into nanofabricated silicon nitride pores of molecular dimensions. For fixed analyte concentrations we find an increase in capture rate as the DNA length increases from 800 to 8,000 basepairs, a length-independent capture rate for longer molecules, and increasing capture rates when ionic gradients are established across the pore. In addition, we show that application of a 20-fold salt gradient enables detection of picomolar DNA concentrations at high throughput. The salt gradients enhance the electric field, focusing more molecules into the pore, thereby advancing the possibility of analyzing unamplified DNA samples using nanopores.

We present measurements and theoretical modeling of the ionic conductance G of solid-state nanopores with 5-100 nm diameters, with and without DNA inserted into the pore. First, we show that it is essential to include access resistance to describe the conductance, in particular for larger pore diameters. We then present an exact solution for G of an hourglass-shaped pore, which agrees very well with our measurements without any adjustable parameters, and which is an improvement over the cylindrical approximation. Subsequently we discuss the conductance blockade ΔG due to the insertion of a DNA molecule into the pore, which we study experimentally as a function of pore diameter. We find that ΔG decreases with pore diameter, contrary to the predictions of earlier models that forecasted a constant ΔG. We compare three models for ΔG, all of which provide good agreement with our experimental data.

Measurements on protein translocation through solid-state nanopores reveal anomalous (nonSmoluchowski) transport behavior, as evidenced by extremely low detected event rates, i.e., the capture rates are orders of magnitude smaller than what is theoretically expected. Systematic experimental measurements of the event rate dependence on the diffusion constant are performed by translocating proteins ranging in size from 6 kDa to 660 kDa. The discrepancy is observed to be significantly larger for smaller proteins, which move faster and have a lower signal-to-noise ratio. This is further confirmed by measuring the event rate dependence on the pore size and concentration for a large 540 kDa protein and a small 37 kDa protein, where only the large protein follows the expected behavior. We dismiss various possible causes for this phenomenon, and conclude that it is due to a combination of the limited temporal resolution and low signal-to-noise ratio. A one-dimensional first-passage time-distribution model supports this and suggests that the bulk of the proteins translocate on timescales faster than can be detected. We discuss the implications for protein characterization using solid-state nanopores and highlight several possible routes to address this problem. Keywordsprotein; nanopore; translocation; temporal; Smoluchowski; bandwidth Over the past decade, nanopores have become a very popular method to study analytes at the single-molecule level. While the focus has been on nucleic acids 1, 2 , proteins and protein DNA-complexes increasingly are becoming a prime target of investigation [3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18] .There are two notable differences which make proteins more difficult to characterize than DNA by use of nanopore measurements. The well-defined structure of proteins, the source of their biological function, is normally globular, with lengths in all dimensions that are comparable to or smaller than the thickness of the pore. DNA and other polynucleotides, Europe PMC Funders Author ManuscriptsEurope PMC Funders Author Manuscripts however, typically have one dimension much larger than the length of the pore, leading to much longer dwell times inside the nanopore compared to proteins. Secondly, proteins have a variety of different charges, that are distributed unevenly throughout their structure. Again, this contrasts the case for DNA which is highly negatively charged with a uniform charge distribution along its length. These differences make nanopore experiments on proteins more challenging than on DNA. This study focuses at the issues caused by the protein's short translocation times and the implications this has for characterization of proteins with solidstate nanopores. Figure 1 shows the setup and data for a typical nanopore protein measurement. A voltage is applied across the pore, which electrokinetically drives a protein through it, temporarily blocking the ionic current. The dynamics of the translocation process can be split into two stages, the capture step and th...

We have used the nanometer scale alpha-Hemolysin pore to study the unzipping kinetics of individual DNA hairpins under constant force or constant loading rate. Using a dynamic voltage control method, the entry rate of polynucleotides into the pore and the voltage pattern applied to induce hairpin unzipping are independently set. Thus, hundreds of unzipping events can be tested in a short period of time (few minutes), independently of the unzipping voltage amplitude. Because our method does not entail the physical coupling of the molecules under test to a force transducer, very high throughput can be achieved. We used our method to study DNA unzipping kinetics at small forces, which have not been accessed before. We find that in this regime the static unzipping times decrease exponentially with voltage with a characteristic slope that is independent of the duplex region sequence, and that the intercept depends strongly on the duplex region energy. We also present the first nanopore dynamic force measurements (time varying force). Our results are in agreement with the approximately logV dependence at high V (where V is the loading rate) observed by other methods. The extension of these measurements to lower loading rates reveals a much weaker dependence on V.

We argue that in order to maintain the biological function of DNA confined inside the cell nucleus, its spatial structure has to be unknotted, of the so-called <> type. The furation of a particular realization of this non-equilibrium structure by attractive interactions between specific units imposes a connection between the spatial structure of DNA and the statistical distribution of these units along the chain contour. This suggests that both primary sequence and spatial structure of native DNA were formed simultaneously by a self-similar evolution process. The predictions of our model are compared with recent observations of long-range correlations in intron-containing genes and non-transcribed regulatory elements and further experimental tests are proposed.

No abstract

No abstract

We present a detailed analysis of the process of voltage driven capture of DNA molecules by nanopores. We show that ionic current generates a nonuniform electric field that acts on both the DNA and on its counterions and that the response of DNA to the electric field is affected by its electroosmotic coupling to the mobile counterions. We calculate the voltage and molecular mass dependence of the radius of capture and of the capture rate in the diffusion limited regime. We argue that electroosmotic flow through the DNA coil is suppressed in the vicinity of the pore and present a tentative estimate of the capture rate in the barrier limited regime.