DNA Separation Chips Using Temporally Asymmetric Ratchet Effect in Nonuniform Electric Fields

We present DNA separation chips using the temporally asymmetric ratchet effect in nonuniform electric fi elds. The present separation chip redistributes DNA within a specifi c area in asymmetrically switched nonuniform electric fi elds based on the sizeand fi eld-dependent nonlinearities of DNA drift velocity. Compared with the conventional electrophoresis chips, the present separation chip can be easily integrated into automated DNA analysis systems because of its simple structure and starting-point-independent DNA separation. On the basis of the drift velocity of three different DNA molecules (11.1, 15.6 and 48.5 kbp), we extract the asymmetric alternating electric fi eld conditions (E1 = 4E2 and 23T1 = 3T2), where Phagemid DNA (15.6 kbp) shows zero net velocity while EM3 DNA (11.1 kbp) and λ DNA (48.5 kbp) migrate to the –x and +x directions, respectively. The present chip is composed of a tapered channel to generate nonuniform electric fi elds, a DNA loading slit, and a pair of electrodes to apply the electric fi eld. We focus on the design of the DNA separation chips to identify the nonlinearity of DNA drift velocity using three different DNA molecules in the chips. It is demonstrated that different sizes of DNA show different net migration velocities under the nonuniformly distributed asymmetric alternating electric fi elds. Phagemid DNA moved to its own specifi c location, –1.5 mm from the starting point (+2 mm from the loading slit), then showed zero net migration velocity. Other sample DNA molecules, EM3 and λ DNA, migrated 2.2 mm in the –x direction and 1 mm in the +x direction, respectively, under the alternating asymmetric electric fi eld, toward their own specifi c locations where they show net zero velocity. Also discussed are the potentials of the present DNA chips for the miniaturization of DNA analysis systems and the potentials of the present DNA chips having tunable capability of the target DNA size to be separated. Sensors and Materials, Vol. 21, No. 2 (2009) 105–115 MYU Tokyo

We present DNA separation chips using the temporally asymmetric ratchet effect in nonuniform electric fi elds. The present separation chip redistributes DNA within a specifi c area in asymmetrically switched nonuniform electric fi elds based on the size-and fi eld-dependent nonlinearities of DNA drift velocity. Compared with the conventional electrophoresis chips, the present separation chip can be easily integrated into automated DNA analysis systems because of its simple structure and starting-point-independent DNA separation. On the basis of the drift velocity of three different DNA molecules (11.1, 15.6 and 48.5 kbp), we extract the asymmetric alternating electric fi eld conditions (E 1 = 4E 2 and 23T 1 = 3T 2 ), where Phagemid DNA (15.6 kbp) shows zero net velocity while EM3 DNA (11.1 kbp) and λ DNA (48.5 kbp) migrate to the -x and +x directions, respectively. The present chip is composed of a tapered channel to generate nonuniform electric fi elds, a DNA loading slit, and a pair of electrodes to apply the electric fi eld. We focus on the design of the DNA separation chips to identify the nonlinearity of DNA drift velocity using three different DNA molecules in the chips. It is demonstrated that different sizes of DNA show different net migration velocities under the nonuniformly distributed asymmetric alternating electric fi elds. Phagemid DNA moved to its own specifi c location, -1.5 mm from the starting point (+2 mm from the loading slit), then showed zero net migration velocity. Other sample DNA molecules, EM3 and λ DNA, migrated 2.2 mm in the -x direction and 1 mm in the +x direction, respectively, under the alternating asymmetric electric fi eld, toward their own specifi c locations where they show net zero velocity. Also discussed are the potentials of the present DNA chips for the miniaturization of DNA analysis systems and the potentials of the present DNA chips having tunable capability of the target DNA size to be separated.

Introduction
The DNA separation device is one of the key components of micro DNA analysis systems, (1) since effi cient methods to separate and analyze the different components of a mixture of DNA molecules are of paramount importance, both for research and in biomedical applications. To meet this need, a concerted effort is being made to develop miniaturized bioanalytical devices using the microfabrication technology. As a result of these concerted efforts, several microelectrophoresis chips (2)(3)(4)(5)(6) use microlithographic arrays as an artifi cial gel instead of gel matrix to realize a uniform sieving matrix. To improve the separation effi ciency, the electric fi eld directions (2,5,6) or design-specifi c patterns and geometry of microarrays (4) are changed as occasion demands. Compared with the conventional electrophoresis chips, the present separation chip can be easily integrated into automated DNA analysis systems because of its simple structure and starting-point-independent DNA separation.
Recently, some remarkable DNA properties, (7)(8)(9) such as the nonlinear nature of the DNA drift velocity, have been discovered in the electric fi elds relevant to the physics of macromolecules and biopolymers. Interestingly, these new phenomena in the behavior of DNA in the electric fi elds may also be observed using the common gel electrophoresis and pulsed fi eld gel electrophoresis (PFGE) techniques. To improve the separation capability of DNA PFGE, an asymmetric periodic electric fi eld with a zero average value (10) is introduced. This electric fi eld offers possibilities to manipulate the total drift velocity at will, such as macromolecules of different sizes can be made to move in opposite directions in PFGE. By comparing with the ratcheting of the DNA molecules in modifi ed conventional gel electrophoresis systems, (10) through the miniaturization of the chip, we may also possibly shorten the process time and minimize the disadvantages of the nonuniform electric fi elds, temperature fi eld in the gel, and curvilinear shape of the separated fractions.
The present separation chip ( Fig. 1) redistributes DNA molecules within a specifi c area based on the ratchet effect (11) using asymmetric alternating electric fi elds due to size-and fi eld-dependent nonlinearities of the DNA drift velocity. Determination of the asymmetric alternating electric fi eld condition has been deduced on the basis of the measured DNA migration velocities of the DNA molecules, without being limited by a zero average value. (10) This DNA ratchet effect allows the separation chip to be automated for integration in DNA analysis systems.
The proposed separation chip is to be applied in a rapid, inexpensive restriction fi ngerprinting process, which is utilized in restricted fragment length polymorphism (RFLP), (12,13) screening of embryonic stem cell clones for gene targeting (14) or screening of transgenic animals. (15) Thanks to the temporally asymmetric ratchet effect in this chip, DNA fragments in a wide size range could be separable in a short channel instead of long fi laments that are usually used in RFLP techniques for genetic linkage studies, such as paternity testing and genetic fi ngerprinting. In addition, the proposed chip can be applicable to the screening process for building bacterial artifi cial chromosome (BAC) (16) libraries by identifying DNA molecules of specifi c sizes. Since DNA molecules of specifi c sizes migrate to their own specifi c locations under asymmetric alternating electric fi elds, it is easy to check the existence of DNA molecules of specifi c sizes. Figure 2 shows the principle of DNA ratcheting and separation using the temporally asymmetric ratchet effect due to size-and fi eld-dependent nonlinearities of the DNA drift velocity. When a forward electric fi eld, E 1 is applied for time, T 1 , and then switched periodically to the backward fi eld, E 2 , for T 2 as shown in Fig. 2(a), in the horizontal direction of the channel, the DNA molecules in the channel ( Fig. 2(b)) move forward and backward with the specifi c velocities depending on their molecular sizes. Given the values of E 1 / E 2 and T 1 / T 2 , for example, the DNA of mass M 2 ( Fig. 2(b)) simply oscillates at a specifi c location with zero net velocity, while the others of masses, M 1 and M 3 move to the -x and +x directions, respectively. This phenomenon is based on the drift velocity of the DNA molecules, which is not linear to the electric fi eld intensity and to the size of the DNA molecules. Whatever is the source of this nonlinearity, we can use such a system to build a correlative ratchet that rectifi es temporally biased fl uctuations. (11)

Nonuniform electric fi eld distribution
According to the temporally asymmetric ratchet effect, we can make a DNA molecule of a specifi c size oscillate at a specifi c location with zero net velocity. To make DNA molecules of other sizes migrate to their own specifi c locations with zero net velocity, we need to distribute the electric fi elds nonuniformly along the channel. For the nonunifrom electric fi eld distribution, we vary the channel width ( Fig. 1)  nonuniform electric fi eld zones can be generated in the channel fi lled with agarose gel. By varying the channel width, we can distribute the asymmetric alternating electric fi eld train ( Fig. 2(a)) having a gradient of the intensity along the channel, keeping the same ratio and period. Therefore, by applying the asymmetric alternating electric fi elds in the width-variable channel, M 1 and M 3 can also oscillate as well as M 2 at the specifi c location with net zero velocity. Consequently, DNA molecules of different sizes migrate with different velocities toward their own specifi c locations. After reaching their own specifi c locations, they would have a zero net velocity and then just oscillate near these locations in response to the applied asymmetric electric fi eld train.

Design and Fabrication
To realize the DNA separation in a chip, the asymmetric alternating nonuniform electric fi eld with intensity variation through one channel should be generated. Then, each DNA molecule can have its own specifi c location where the migration velocity becomes zero. To estimate the DNA migration and design the DNA separation chip using agarose gel, we measured the DNA migration velocity in agarose gel with the applied voltage. We measured the migration velocities of the three types of DNA molecules, DNA I (EM3), II (Phargemid) and III (λ DNA) having different sizes of 11.1, 15.6 and 48.5 kbp, respectively. In this process, we used 0.5X Tris-Acetate-EDTA (TAE) buffer for the DNA electrophoresis. To detect the DNA molecules after electrophoresis, the DNA molecules were stained with ethidium bromide (EtBr) by mixing EtBr with the agarose gel before electrophoresis and/or dipping the agarose gel in the EtBr solution after the electrophoresis, as occasion demands. We varied the applied voltage from 5 to 240 V in the conventional gel electrophoresis kit to measure the drift velocity of DNA molecules according to the applied voltage with the fl uorescence image of the gel and the process time. Through the Maxwell simulation of the gel bath of the conventional gel electrophoresis kit, we estimated the magnitude of the electric fi eld of each applied voltage.

Electric fi eld condition
On the basis of the measured DNA migration velocities in Fig. 3, we estimated the asymmetric alternating electric fi eld condition. First, we estimated the condition that will make DNA II oscillate in the channel. When E 1 is 640 V/m, E 2 is 160 V/m (Fig. 3), and 23T 1 is the same as 3T 2 , DNA II shows behavior like that of M 2 in Fig. 2(b), while DNA I and DNA III migrate to the -x and +x directions, respectively. We calculated the net migration velocities of the sample DNA molecules under the conditions of E 1 is 4E 2 and 23T 1 is 3T 2 , and estimated the migration velocities of the sample DNA molecules along the channel.

Chip design and estimation of DNA migration
From the asymmetric alternating electric fi eld condition and the migration velocities of the three different DNA molecules, we designed the DNA separation chip as shown in   Fig. 1. The DNA separation chip has one channel that has a width variation to make the electric fi eld gradient from x = -L to x = +L. Since the electric fi eld difference between the specifi c locations of DNA I and DNA II is less than between that of DNA II and DNA III, we varied the channel width gradient to shorten the distance between the specifi c locations of DNA II and DNA III; then, the electric fi eld gradient between the specifi c locations of DNA II and DNA III is higher than between those of DNA I and DNA II as shown in Fig. 4(a). Through the Maxwell simulation of the designed DNA separation chip with the applied voltages of 40 and -10 V across two electrodes, we could obtain the electric fi eld intensity along the channel as shown in Fig. 4(b).
Using the measured DNA migration velocity graph (Fig. 3) and the electric fi eld intensity( Fig. 4(b)), we estimated the migration of the sample DNA molecules along the channel under the asymmetric alternative electric fi eld (E 1 is 4E 2 and 23T 1 is 3T 2 ). Figure 5 shows the simulated migration of the DNA molecules through the simulation program, which calculates the position of the DNA molecules every second using the velocity information with respect to the electric fi eld values at the position of the DNA molecules in the channel. In the simulation, each DNA molecule migrated with a different net velocity according to its size; DNA I migrated backward and oscillated at x = -1.02 mm, DNA II and DNA III migrated forward and oscillated at x = +2.30 mm and x = +14.8 mm, respectively, as shown in Fig. 6. Therefore, we can predict that all three sample DNA molecules could reach the specifi c locations where they show net zero migration velocity in 1200 min.

Fabrication
The fabrication of the DNA separation chip can be classified into three steps: fabrication of PDMS (PolyDiMethylSiloxane) channel, bonding the PDMS channel to the slide glass, and fi lling agarose gel in the PDMS channel. A channel for fi lling agarose gel was fabricated by the PDMS molding technique. A PDMS mixture (curing agent:PDMS = 1:10) was poured onto the molding jig and cured for 2 h at 85°C. We defi ned the PDMS channel by cutting the PDMS replica. Before bonding a PDMS channel to the slide glass substrates, we treated the former with plasma generated by a high-frequency generator (BD-10AS, Electro-technic Products, Inc.) for the surface activation. The plasma-treated PDMS channel and slide glass substrates were dipped in methanol for cleaning and bonding. After they were bonded, they were cured for 2 h at 120°C. Finally, we fi lled the PDMS channel with the agarose I and TAE buffer mixture (agarose:buffer = 0.5 g:100 ml). To form the congealed agarose gel in the channel, we boiled and fi lled the agarose I and buffer mixture in the PDMS channel, defi ning the slit for loading the DNA sample. Figure 6 shows the fabricated DNA separation chip fi lled with agarose gel.

Experimental Results and Discussion
In order to observe the DNA migration in the DNA separation chip, we used the test apparatus composed of UV lamp, camera, power supply, PC, and circuit for applying asymmetric alternating electric fi eld train. Electric potential (40 V/-10 V) was applied through the platinum electrodes from the circuit. Through the PC using visual C++ program, we regulated the period of the asymmetric alternating electric fi eld train and regulated the amplitude of the electric fi eld E 1 and E 2 through the 2 outputs of the power supply. Three kinds of DNA molecules were used to characterize the DNA separation chip. In order to detect the migration of the DNA molecules in the DNA separation chip on the UV-lamp, we stained the DNA molecules with the fl uorescence dye, YOYO-1 (Molecular Probes Inc. Y-3601) at 5:1 ratio of base pairs to dye molecules. Since we used less DNA sample and the process time is longer compared with the conventional electrophoresis process, we stained the DNA molecules with YOYO-1 instead of EtBr.
When the DNA molecules migrated with the external electric fi eld, we photographed the DNA migration at fi xed time intervals using the CCD camera set above the DNA separation chip. Figure 7 shows the images captured from the fabricated DNA chip every 80 min of the separation process, demonstrating the size-dependent DNA separation in three different bands within the channel. In Fig. 8   According to the measured specifi c location of DNA II, the actual applied electric fi eld gradient is higher than the estimated one in Fig. 8. Since we manually placed an electrode in the DNA separation chip, the distance between 2 electrodes could be closer than in the model shown in Fig. 4. In the future, we should integrate electrodes on the substrate of the DNA separation chip to obtain a more stable result. Although asymmetric alternating electric fi elds were applied for 240 min, all the DNA molecules could not move to their own specifi c locations within the bleaching of the fl uorescence dye, owing to an insuffi cient net migration velocity of the DNA molecules. To make all DNA molecules reach their own specifi c locations and have a zero net migration velocity, it is necessary to design a separation chip with a shorter channel to shorten the migration distance of the DNA molecules. The total time of DNA separation in the present chip (240 min) is still longer than that of recently developed DNA separation chips using artifi cial gel structure (less than 1 h (4)(5)(6) ). In this study, we were trying to verify the feasibility of DNA separation chip using the temporally asymmetric ratchet effect in nonuniform electric fi elds. By decreasing the separation chip sizes, we can decrease the total time of DNA separation time. If the channel shape is made hyperbolic, we could minimize the curvilinear shape of the separation tractions (a smiling effect). In addition, fi lling the sieving matrix that has more uniform pores can clarify the band of the DNA molecules in the channel.

Conclusions
We present single-channel chips for ratcheting and separating DNA molecules using nonuniformly distributed asymmetric alternating electric fi elds in a PDMS microchip. We measured the drift velocities of three types of DNA molecules by varying the electric fi elds. On the basis of the drift velocity data, we found the proper conditions for the separation and trapping of DNA molecules and verifi ed that DNA molecules migrate with the different velocities depending on their size under specifi c asymmetric alternating electric fi elds. Finally, one of the sample DNA molecules, DNA II, reached its own specifi c location, then showed zero net migration velocity, while DNA I and III migrated to the -x and +x directions, respectively. The proposed separation chips have potentials not only for the miniaturization of DNA analysis systems, but also for the tunable capability of the target DNA size to be separated and ratcheted by varying the amplitude and period of the asymmetric alternating electric fi elds and changing the gel property.