Band-broadening suppressed effect in long turned geometry channel and high-sensitive analysis of DNA sample by using floating electrokinetic supercharging on a microchip

Abstract

A featured microchip owning three big reservoirs and long turned geometry channel was designed to improve the detection limit of DNA fragments by using floating electrokinetic supercharging (FEKS) method. The novel design matches the FEKS preconcentration needs of a large sample volume introduction with electrokinetic injection (EKI), as well as long duration of isotachophoresis (ITP) process to enrich low concentration sample. In the curved channel [ approximately 45.6 mm long between port 1 (P1) and the intersection point of two channels], EKI and ITP were performed while the side port 3 (P3) was electrically floated. The turn-induced band broadening with or without ITP process was investigated by a computer simulation (using CFD-ACE+ software) when the analytes traveling through the U-shaped geometry. It was found that the channel curvature determined the extent of band broadening, however, which could be effectively eliminated by the way of ITP. After the ITP-stacked zones passed the intersection point from P1, they were rapidly destacked for separation and detection from ITP to zone electrophoresis by using leading ions from P3. The FEKS carried on the novel chip successfully contributed to higher sensitivities of DNA fragments in comparison with our previous results realized on either a single channel or a cross microchip. The analysis of low concentration 50 bp DNA step ladders (0.23 mugml after 1500-fold diluted) was achieved with normal UV detection at 260 nm. The obtained limit of detections (LODs) were on average 100 times better than using conventional pinched injection, down to several ngml for individual DNA fragment.

Figure 1

(a) Picture and (b) schematic of the designed microchip. Functional distribution of three reservoirs for FEKS method: port 1 is the sample reservoir; ports 2 and 3 are the reservoirs for BGE. See text for detailed dimension.

Figure 2

Frame of a turn geometry microchip for 2D computer simulation. [(a) and (d) segments] Quarter-circle pipe for 90° turn. [(b) and (c) segments] Two quarter-circle pipes connected with a short capillary (100×100 μm²) for 180° turn.

Figure 3

Simulated profile for the band broadening of m50⁻ in the turn. [(a) and (b)] ITP-stacked zone; (c) without ITP process. The inside turn radius r is at (a) 20 μm and [(b) and (c)] 100 μm. The channel width is 100 μm.

Figure 4

The changing in zone length related to ITP effect: (a) with ITP; (b) without ITP in normal electrophoretic migration. The zone length was measured pre- and post-traveling through 180° turn during 0.7 s. The turn radius of channel was all at 100 μm.

Figure 5

Observation of methylene blue and safranine O in U-shaped turn in ITP mode by CCD camera. Sample concentration: 50 μM. LE and TE: 50 mM NH₄OH and 10 mM HCl, respectively, at pH 4.8 adjusted by 2-ethylbutyric acid. Applied voltages: P1=800 V and P2=0 V while P3 was floating.

Figure 6

ITP-stacked zone for differently diluted 50 bp DNA stepladders. (a) 1:100, (b) 1:600, (c) 1:1200, and (d) 1:2500 (original concentration, 340 μg∕ml). BGE (LE), 2% (w∕v) HEC, 50 mM HCl (at pH 8.1 adjusted by adding tris). TE, 20 mM glycine (at pH 8.1 adjusted by tris). Sample and TE injection were 150 and 320 s at P1=0 V and P2=1200 V, respectively, while P3 was floating. UV detection at 260 nm.

Figure 7

Electropherograms of 1:100 diluted DNA sample (3.4 μg∕ml) obtained by using FEKS on (a) three-port chip with long curved channel and (b) Shimadzu cross microchip. Optimized condition for (a): EKI time was 50 s and TE injection was 300 s (P1=0 V, P2=1200 V, and P3 was floating). ZE separation time was 70 s (P3=0 V, P2=430 V, and P1 was floating). Optimized voltages for (b) was described in Ref. , and times for EKI, TE, and ZE separation were 50, 30, and 70 s, respectively. Buffers as described in Fig. 6.

Figure 8

Electropherograms of highly diluted DNA samples (a) 1:600 and (b) 1:1500, preconcentrated by FEKS on a three-port chip. The times of sample injection, TE injection, and ZE separation were 150, 320, and 70 s, respectively. The voltages and buffers as described in Fig. 6.