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Continuous-Flow Ogston Sieving-Based Separation of Short DNA and SDS-Protein Complexes and Entropic Trapping-Based Separation of Long DNA Through the ANA

Jianping Fu, Jongyoon Han

1. Ogston sieving-based separation of short DNA and SDS-protein complexes

To explicitly demonstrate the steric sieving effect of the ANA, we first injected a low molecular weight DNA ladder sample (the PCR marker) at TBE 5X buffer under a broad range of field conditions (Fig. 1 ). Since TBE 5X buffer has an equivalent ionic strength about 130 mM with a corresponding Debye length λD of about 0.84 nm (much smaller than the nanofilter shallow region depth ds ~55 nm), steric interactions dominate molecular jump dynamics across the nanofilter. The PCR marker contains 5 different DNA fragments of sizes ranging from 50- to 766 bp. Since the persistence length of DNA is about 50 nm (about the contour length of 150 bp DNA), the PCR marker fragments appear relatively straight, and recognizable as rigid, rod-like molecules with an end-to-end distance of about 16 nm to 150 nm. The entry into the confining nanofilter can only be realized if the rod-like DNA molecules are properly positioned and oriented without overlapping the wall, which limits the configurational freedom and creates an entropic barrier (i.e., Ogston sieving).

Figure 1 show 6 fluorescence photographs of the PCR marker stream pattern in the ANA when horizontal and vertical fields of different values were applied. Figure 1A-C shows the PCR marker stream pattern as  Ex was raised from 0 V/cm to 60 V/cm at fixed Ey = 25 V/cm. In the experiment of Fig. 1A, only the vertical field Ey= 25 V/cm was applied, and the PCR marker sample formed a vertical stream without any separation. The initial stream width W of 30 µm gradually widened to about 50 µm at the end of the ANA after drifting for about 210 s (less than 4 min). The applied horizontal field Ex quickly deflected DNA fragments according to their molecular weights (size), with the stream deflection angle θ and the stream width W depending on the exact field conditions. Increasing the horizontal field Ex resulted in larger deflection angles as well as wider spreading of the streams. Please note that, for all the electropherograms measured at the bottom of the ANA, we have used Gaussian functions for fitting to determine the means (the maximum intensity) as well as the stream widths.

Figure 1 Ogston sieving of short DNA (the PCR marker) through the ANA. For A, only Ey applied and Ey = 25 V/cm; for B, Ex = 35 V/cm, Ey = 25 V/cm; for C, Ex = 60 V/cm, Ey = 25 V/cm; for D, Ex = 35 V/cm, Ey = 12.5 V/cm; for E, Ex = 35 V/cm, Ey = 50 V/cm; for F, Ex = 35 V/cm, Ey = 75 V/cm. Band assignment: (1) 50 bp; (2) 150 bp; (3) 300 bp; (4) 500 bp; (5) 766 bp. Fluorescence intensity profiles (of arbitrary units) were measured at the ANA bottom edge. The bars underneath the peaks are centered at the means and label the stream widths (standard deviation, ±s.d.).

The ANA is also capable of separating mixtures of denatured proteins based on their molecular weights (MW). As proof of concept, we first prepared two Alexa Fluor 488-conjugated protein complexes: cholera toxin subunit B (MW ~ 11.4 kDa) and β-galactosidase (MW ~ 116.3 kDa), and denatured them by addition of sodium dodecyl sulfate (SDS) and dithiothreitol (DTT). With the horizontal field Ex = 75 V/cm and the vertical field Ey = 50 V/cm, the SDS-protein complexes were base-line separated into 2 streams within 2 min. The protein stream widths at 1 mm, 3 mm, and 5 mm from the injection point corresponded to separation resolutions Rs of 0.57, 0.94 and 1.47, respectively (Fig. 2A). Cholera toxin subunit B was deflected more than β-galactosidase in all the experiments, suggesting Ogston sieving to account for the jump dynamics of these linear protein complexes (Fig. 2B, top). Further increasing Ex resulted in larger lateral separation between the two streams. However, resolution Rs was compromised due to broader lateral dispersion, as evidenced by the decrease in the resolution curve (Fig. 2B, bottom).

Figure 2 Continuous-flow separation of proteins under denaturing conditions through the ANA. A, Composite fluorescent photograph showing separation of Alexa Fluor 488-conjugated cholera toxin subunit B (band 1, MW ~ 11.4 kDa) and β-galactosidase (band 2, MW ~ 116.3 kDa) with Ex = 75 V/cm and Ey = 50 V/cm. The protein stream widths at 1 mm, 3 mm, and 5 mm from the injection point corresponded to resolutions Rs of 0.57, 0.94 and 1.47, respectively. B, Measured deflection angle θ (top) of cholera toxin subunit B and β-galactosidase (™) as a function of Ex when Ey = 50 V/cm. The bottom shows the corresponding separation resolutions. The ±s.d. of θ are indicated as error bars (drawn if larger than the symbol).

2. Entropic trapping for size-based continuous-flow separation of long DNA

The ANA can separate long DNA molecules based on the entropic trapping mechanism. We prepared a mixture of long DNA molecules (the λ DNA-Hind III digest) in TBE 5× buffer, which contains 6 DNA fragments with sizes ranging from 2,027- to 23,130-bp and corresponding equilibrium (unconfined) radii of gyration Rg of about 140 nm to 520 nm. These equilibrium radii of gyration are useful estimates of the spherical DNA size, and they are all greater than the nanofilter constriction depth ds. Therefore, the nanofilter jump dynamics involves necessarily the deformation and hernia nucleation (i.e., entropic trapping). With application of the horizontal field Ex = 185 V/cm and the vertical field Ey = 100 V/cm, λ DNA-Hind III digest was separated in less than 1 min with base-line resolution (Fig.  3A-B; note that the shortest 2,027 bp fragment was too dim for clear visualization in Fig. 3, but with higher gain setting and longer exposure time of the charge-coupled device (CCD), the 2,027 bp fragment was identified to be base-line separated with the others). A closer look at the fluorescence photographs further revealed that, as expected, longer DNA fragments followed more deflected migration trajectories than shorter ones, a clear distinction of entropic trapping from Ogston sieving. The streams of λ DNA-Hind III digest followed more deflected and resolved trajectories as Ex was increased (Fig. 3C-F). This observation is consistent with the argument that increased horizontal field Ex lowers the activation energy barrier height leading to a higher jump passage rate Px.

Figure 3 Entropic trapping of long DNA (the λ DNA-Hind III digest) through the ANA. Fluorescent photographs show separation of λ DNA-Hind III digest with different electric field conditions. A, B, F, Ex = 185 V/cm and Ey = 100 V/cm. C, Ex = 50 V/cm and Ey = 100 V/cm. D, Ex = 145 V/cm and Ey = 100 V/cm. E, Ex = 170 V/cm and Ey = 100 V/cm. Band assignments are 2,322 bp (1), 4,361 bp (2), 6,557 bp (3), 9,416 bp (4), 23,130 bp (5). Fluorescence intensity profiles were measured at the ANA bottom edge. The bars underneath the peaks are centered at the means and label the stream widths (±s.d.).

MOVIE LINKS

• Continuous-flow separation of short DNA (the PCR marker, 50 - 1,000 bp) through two-dimensional periodic array of nanofilters (Anisotropic Nanofilter Array: ANA), based on the Ogston sieving mechanism. (This video was taken with exposure time of 1300 ms/image and image size of 1300 µm X 1620 µm. The time scale in this movie has been compressed by a factor of 20. At the beginning of the movie, only Ey = 25 V/cm was applied. The orthogonal field Ex = 35 V/cm was applied at 3 sec in the movie, and the separation was finished at about 12 sec in the movie. Video by Jianping Fu.)

• Continuous-flow separation of long DNA (the λ DNA-Hind III digest, 2,000 - 23,000 bp) through ANA, based on the entropic trapping mechanism. (This video was taken with exposure time of 600 ms/image and image size of 3270 µm X 4080 µm. The time scale in this movie has been compressed by a factor of 20. At the beginning of the movie, only Ey = 100 V/cm was applied. The orthogonal field Ex = 185 V/cm was applied at 1 sec in the movie, and the separation was finished at about 5 sec in the movie. Video by Jianping Fu.)

• Continuous-flow separation of denatured proteins through ANA. (This video was taken with exposure time of 1000 ms/image and image size of 3270 µm X 4080 µm. The time scale in this movie has been compressed by a factor of 20. At the beginning of the movie, only the vertical field Ey = 50 V/cm was applied. The horizontal field Ex = 75 V/cm was applied at 1 sec in the movie, and the separation was finished at about 6 sec in the movie. Video by Jianping Fu.)

1. Fu, J. & Han, J. "A nanofilter array chip for fast gel-free biomolecule separation," Proceedings of the MicroTAS 2005 Symposium, Boston, MA, vol. 2, pp. 1531-1533. (pdf)
2. Fu, J. & Han, J. “Continuous biomolecule separation in a nanofilter,” United States Provisional Patent, MIT, Oct. 2005.
3. Fu, J. & Han. J. “Patterned periodic nanofilter array for continuous-flow biomolecule separation,” American Physical Society National March Meeting, Baltimore, Maryland USA, March 2006. (pdf)
4. Fu, J. & Han, J. "Continuous-flow biomolecule separation through patterned anisotropic nanofluidic sieving structure," Proceedings of the MicroTAS 2006 Symposium, Tokyo, Japan, vol. 1, pp. 519-521. (pdf)
5. Fu, J., Schoch, R. B., Stevens, A. L., Tannenbaum, S. R. & Han, J. "A patterned anisotropic nanofluidic sieving structure for continuous-flow separation of DNA and proteins,” Nature Nanotechnology 2, 121-128 (2007). (pdf)