The separation of molecules can be obtained in one or two dimensions on micro-/ nanofluidic devices. Compared to 1D where the separation of samples is obtained as a function of time, 2D allows the separation in space and has the advantages of continuous-flow, unlimited sample amount, and convenient collection of the sample for subsequent analysis. However, until nowadays, regular 2D sieving structures have proven efficacious only for separation of long DNA molecules and microspheres, and their applicability to smaller, physiologically relevant macromolecules remains questionable, which clearly limits progress towards a future integrated bioanalysis system.

1. Sieving mechanisms

The anisotropic nanofilter array (ANA) allows the separation of proteins under native conditions based on their size or charge [1,2]. Proteins are injected into the sieving structure following the direction of electro-osmotic drag flow. Molecular crossings of the nanofilter under the influence of Ex can be described as biased, thermally activated jumps across free energy barriers at the nanofilter threshold, and these free energy barriers depend on both steric and electrostatic interactions between charged molecules an charged nanofilter walls.

At high ionic strength where the Debye length λD is negligible compared to the nanofilter shallow regions depth ds, electrostatic interactions between charged molecules and charged nanofilter walls are largely screened. The energy barriers are therefore solely determined by the configurational or conformational entropy loss within the constriction due to steric exclusion. The steric energy barrier has shown to favor proteins with a smaller size for passage, consistent with Ogston sieving for which separation mechanism molecules with diameters smaller than the nanofilter constriction size is described [3,4]. Therefore, in Ogston sieving, smaller molecules experience a greater jump passage rate Px and a larger stream deflection angle θ (Fig. 1a).

For low-ionic-strength solutions where the Debye length λD becomes comparable to the nanofilter shallow region depth ds, repulsive electrostatic interactions between negatively charged biomolecules and like charged nanofilter walls become prominent and start to dictate jump dynamics across the nanofilter (Fig. 1b). The electric potential remains negative in the entire nanochannel, resulting in an electrostatic exclusion of negatively charged molecules [5,6]. Such electrostatic effects on the partitioning of macromolecules through nanopores have been well studied in membrane science, and have recently been applied for pH-controlled diffusion of proteins across a nanochannel [7]. Therefore, biomolecules bearing a lower net charge are energetically favored for passage through the nanofilter, resulting in a greater jump passage Px rate and a larger deflection angle θ.

Figure 1 Schematic of separation mechanisms of proteins under native conditions through the ANA under the influence of two orthogonal electric fields Ex and Ey. a, When the Debye length λD « ds (Electrical double layer highlighted in yellow), the steric exclusion effect dictates jump dynamics, and smaller-sized proteins (in green) are preferred for nanofilter passage in Ogston sieving, resulting in a greater nanofilter jump passage rate Px.

b, Electrostatic sieving becomes dominant when λD ≈ ds. Proteins with a lower net negative charge (in green) experience lesser electrostatic repulsion when crossing the negatively charged nanofilter, resulting in a greater passage rate Px and a larger stream deflection angle θ, where θ is defined with respect to the positive y-axis.

2. Size- or charge-based separation of proteins with the ANA

Mixtures of proteins under native conditions have been separated by the ANA based on either size or charge, depending on buffer ionic strength. As proof of concept, we investigated the following various standard proteins dissolved in either TBE 5× or TBE 0.05× (4.45 mM Tris-Borate, 0.1 mM EDTA) buffer, both at pH 9.6: lectin from Lens culinaris (lentil) (MW ~49 kDa, pI ~8.0-8.8); streptavidin (MW ~52.8 kDa, pI ~5-6); B-phycoerythrin (MW ~240 kDa, pI ~4.2-4.4); and fibrinogen (MW ~340 kDa, pI ~5.5). Under the horizontal field Ex = 100 V/cm and the vertical field Ey = 50 V/cm, a mixture of lectin, B-phycoerythrin and fibrinogen was driven through the ANA at TBE 5× (Fig. 5a-c) . The three proteins were clearly separated into three distinct streams according to their molecular weight. The stream deflection angles of lectin, B-phycoerythrin and fibrinogen are about 30.21°, 27.88° and 24.04°, respectively. The resolution values Rs for lectin and B-phycoerythrin at 1.5 mm and 5 mm (linearly extrapolated) from the injection point are 0.33 and 0.47, respectively, and for B-phycoerythrin and fibrinogen, the Rs values are 0.95 and 1.24, respectively. In all experiments B-phycoerythrin, lectin was deflected most, followed by B-phycoerythrin and then fibrinogen, suggesting Ogston sieving to account for the jump dynamics. Further increasing Ex resulted in larger lateral separation and broader lateral diffusion of the streams.

Electrostatic sieving in the ANA was demonstrated at TBE 0.05× by separating two proteins, lectin and streptavidin, under native conditions. The proteins have similar molecular weights and a relatively large difference in pI values. No separation of these two proteins was observed at TBE 5× (Fig. 5d), which excludes the possibility of size-based separation. However, at TBE 0.05× (equivalent ionic strength of 1.3 mM and a corresponding Debye length of 8.4 nm), where electrostatic interactions become dominant [6], separation of lectin and streptavidin was clearly achieved, with two distinct streams visible under a horizontal field Ex = 250 V/cm and vertical field Ey = 75 V/cm. Streptavidin is more negatively charged at pH 9.6 compared to lectin due to its lower pI value, and therefore streptavidin experiences greater repulsion during the jump across the nanofilter constriction, leading to a lower jump passage rate Px and a smaller deflection angle θ (Fig. 5e-f). The stream deflection angles of streptavidin and lectin were about 7.44° and 28.50°, respectively, and the resolution values Rs, at 0.9 mm and 5 mm (linearly extrapolated) from the injection point, are 0.32 and 1.96, respectively, indicating baseline resolution at the bottom of the ANA.

Figure 2 Continuous-flow separation of proteins through the ANA. With TBE 5×, the separation time was within a few minutes; with TBE 0.05×, the separation time was of the order of tens of seconds. a,b, Fluorescence photographs showing separation of lectin, B-phycoerythrin and fibrinogen at TBE 5× with Ex = 100 V/cm and Ey = 50 V/cm. Images a and b were taken from the same ANA area (a with a Texas Red filter set, b with a FITC filter set). c, Maximum fluorescence intensity along the streams measured for both a and b as a function of x and y. d, No separation was observed for lectin and streptavidin at TBE 5× with Ex = 150 V/cm and Ey = 75 V/cm.

e, Fluorescence photograph shows separation of lectin and streptavidin at TBE 0.05× with Ex = 250 V/cm and Ey = 75 V/cm. The inset shows the fluorescence intensity profile scanned along the dashed line (at y = 175 µm).

f, Maximum fluorescence intensity along the streams measured for e as a function of x and y.

References

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* These authors contributed equally to this work.

† Copyright by American Institute of Physics. This article may be downloaded for personal use only. Any other use requires prior permission of the author and the American Institute of Physics.

‡ Copyright by the American Physical Society.