DNA molecule manipulation chip based on coupling driving of electric field and bending microchannel and control method thereof

Abstract

The application discloses a DNA molecule manipulation chip based on electric field and bending micro-channel coupling driving, which comprises a glass substrate and a structure layer, the structure layer is arranged on the glass substrate, at least one group of micro-fluid systems are arranged in the structure layer, two ends of the micro-fluid system are respectively communicated with a liquid inlet hole and a liquid outlet hole, the liquid inlet hole and the liquid outlet hole are vertically arranged through the structure layer, electrodes are respectively inserted into the liquid inlet hole and the liquid outlet hole, and the electrodes are used for applying direct current voltage to generate a spatial non-uniform electric field; a control method applied to the chip comprises the following steps: chip pretreatment, application of driving electric field, DNA molecule acceleration and polarization, DNA molecule positive dielectrophoresis focusing and migration; the application realizes high-efficiency mechanical-free focusing of the DNA molecules, and greatly improves the precision of subsequent optical detection and separation.

Description

Technical Field

[0001] This invention belongs to the field of microfluidic chip technology, specifically relating to a DNA molecule manipulation chip and its control method based on the coupling of electric field and curved microchannel. Background Technology

[0002] Microfluidic chip technology, hailed as a laboratory on a chip, has shown great promise in fields such as biochemical analysis, disease diagnosis, and drug screening due to its outstanding advantages, including low sample consumption, fast analysis speed, and high integration. Among the many microfluidic operations, the precise manipulation and transport of biological macromolecules (such as DNA and proteins) is one of the key technologies for achieving efficient separation and accurate detection.

[0003] Electric field manipulation, as an efficient, flexible, and mechanically component-free method, is widely used for the transport control of biomolecules at the micro- and nano-scale. Under the influence of a DC or AC electric field, charged molecules exhibit directional motion through electrophoresis or dielectrophoresis. For flexible chain-like biomolecules like DNA, their dynamic behavior in microchannels is far superior to that of rigid particles. Under the influence of an electric field, DNA molecules undergo complex deformation, orientation, and stretching processes, and their motion characteristics are strongly dependent on intrinsic molecular properties (such as length, charge density, and conformation) as well as external environmental factors (such as electric field strength, solution ionic strength, and channel geometry). Therefore, in-depth research into the dynamic behavior of DNA molecules under electric field manipulation is of significant theoretical guiding importance for the development of next-generation high-performance biomolecular manipulation technologies.

[0004] Combining single-molecule imaging with micro / nanofluidics allows for direct, real-time study of the static and dynamic behavior of individual biomolecules. Currently, most research focuses on the migration behavior of DNA in linear micro / nanochannels. However, to achieve higher integration and more complex functions (such as mixing, separation, and reaction), practical microfluidic chips inevitably incorporate various curved structures (such as semi-circular channels, serpentine channels, and helical channels). When DNA molecules traverse these curved regions, their motion is influenced by a more complex coupling of flow and electric field distributions. On one hand, the centrifugal effect and secondary flow caused by channel curvature significantly alter the flow environment around the molecule; on the other hand, the curved structure also causes a redistribution of electric field lines, generating a non-uniform electric field, which may exert additional dielectrophoretic forces on the DNA molecule or change its electrophoretic mobility. This multi-physics coupling interaction between the physical field and the flexible molecule results in unique and yet-to-be-fully-understood dynamic properties of DNA in curved channels (such as migration speed, deformation, orientation, and transit time). Existing work has mostly focused on the observation of macroscopic transport efficiency, while there is a lack of in-depth research and effective engineering devices on the detailed dynamics of a single DNA molecule passing through a bend, and on how to optimize the migration and focusing efficiency of DNA molecules by precisely designing the geometric features of the bend channel and the conical port.

[0005] For example, the invention patent application with application number 201110430811.1, entitled "Microfluidic Chip Device for Immunoassay," has the following defects: 1. The reaction zone has curved microchannels that are 6 to 18 cm long and filled with arrow-shaped, comb-shaped, and other microstructures. In actual chip manufacturing processes, such extremely long and complex channels generate huge fluid resistance. To drive liquid flow through such a high-resistance channel, the sample pump at the front end must provide high driving pressure. In common soft lithography bonding systems such as PDMS and glass, the high back pressure inside can easily lead to microchannel deformation or irreversible bonding failure and leakage. 2. A core innovation of this device is to illuminate the light source along the extension direction of the detection channel to obtain an optical path of 0.5 to 2 cm. However, the width and height of the detection channel are only 0.3 mm to 2 mm. This means that in actual operation, the external light source and detector must be perfectly coaxially aligned with the extremely narrow cross-section of this microchannel. Even the slightest mechanical vibration or external detector assembly tolerances can cause severe refraction and scattering of the light beam hitting the microchannel sidewalls, resulting in inaccurate absorbance readings. 3. This method relies on generating larger latex immune complex particles to increase turbidity. Combined with the "comb-like microstructures" and "arrow-like microstructures" mentioned in the patent, these passive mixing structures inevitably create localized flow stagnation zones or dead volumes within the channel. As the reaction proceeds, the ever-growing aggregated particles are highly susceptible to non-specific adsorption and physical interception at the corners of these microstructures, leading to channel blockage, altering the preset flow field distribution, and fatally impacting chip cleaning and reuse (e.g., continuous detection of multiple samples). 4. Although the patent claims to avoid purchasing bulky instruments and is portable, according to its claims, the system is highly dependent on external sample pumps or hydraulic sample introduction components and external light source detection systems. It merely achieves miniaturization of the "reaction vessel," while the surrounding fluid drive and optical detection equipment are not highly integrated, significantly reducing the overall portability of the system.

[0006] Based on this, this application will investigate the dynamic characteristics of DNA molecules passing through sinusoidal curved microchannels driven by electric field force. Summary of the Invention

[0007] One of the technical problems to be solved by the present invention is to overcome the shortcomings of the prior art and provide a DNA molecule manipulation chip based on the coupling of electric field and curved microchannel.

[0008] Another technical problem to be solved by the present invention is to provide a control method for DNA molecule manipulation chips driven by coupling of electric field and curved microchannel.

[0009] The technical solution adopted to solve the above technical problems is: a DNA molecule manipulation chip driven by coupling electric field and curved microchannel, including a glass substrate and a structural layer. The structural layer is disposed on the glass substrate, and at least one set of microfluidic system is disposed inside the structural layer. The two ends of the microfluidic system are respectively connected to a liquid inlet and a liquid outlet. The liquid inlet and the liquid outlet are vertically disposed through the structural layer. Electrodes are inserted into the liquid inlet and the liquid outlet, respectively. The electrodes are used to apply DC voltage to generate a spatial non-uniform electric field.

[0010] The microfluidic system of the present invention includes a straight channel and a curved channel. One end of the straight channel is connected to the inlet and the other end is connected to the outlet. The curved channel is connected to the middle of the straight channel through a conical channel and a straight buffer channel. The DNA sample solution enters the straight channel from the inlet and then enters the curved channel through the conical channel. Driven by the positive mesophoresis effect, the DNA molecules are extremely focused into a thin particle beam on the inner wall of the channel with the highest electric field strength, pass through the straight channel and enter the outlet.

[0011] The curved channel of the present invention includes at least two microchannels, each of which is connected to the straight channel through a tapered channel and a straight buffer channel.

[0012] The shape of the microchannel in this invention can be any one of a sine curve, a cosine curve, or a tangent curve.

[0013] The conical channel of the present invention gradually converges from the straight channel to the microchannel, with a semi-cone angle of 15° to 45°.

[0014] The width of the straight channel and the height of the curved channel in this invention are 2 to 8 μm and 2 to 8 μm respectively.

[0015] The structural layer of the present invention is made of polydimethylsiloxane and is tightly bonded to the glass substrate by oxygen plasma activation treatment.

[0016] The present invention has several vertically arranged columns in the straight and curved channels, and the liquid inlet and outlet are provided with a cross-shaped keel frame.

[0017] This invention relates to a control method for DNA molecule manipulation chips driven by coupling between an electric field and a curved microchannel, comprising the following steps:

[0018] Step 1, Chip Pretreatment: Inject buffer solution into the inlet well to fill the microfluidic system, and then inject the DNA sample solution through the inlet well;

[0019] Step 2: Apply driving electric field: Insert the platinum wire electrode into the positive and negative electrode sample cells, connect an external high voltage power supply, and apply a 0-50V DC voltage to form a spatial non-uniform electric field.

[0020] Step 3, DNA molecule acceleration and polarization: When DNA molecules flow through the cone-shaped channel, they undergo a sharp change in the electric field gradient, resulting in rapid polarization and accelerated migration, quickly passing through the cone-shaped channel into the straight buffer channel;

[0021] Step 4: DNA molecules focus and migrate via mesophoresis: DNA molecules enter the sinusoidal microchannel. At the inflection point, the geometric curvature induces a non-uniform distribution of electric field lines, which are denser inside and sparser outside. Driven by the mesophoresis effect, the DNA molecules are focused into a thin beam of particles on the inner wall of the channel where the electric field intensity is highest.

[0022] Compared with the prior art, the present invention has the following advantages:

[0023] 1. The conical channel designed in this invention balances the acceleration effect with the reliability of chip packaging. It enables the electric field strength to increase sharply within the conical channel, achieving rapid polarization and acceleration of DNA molecules, while effectively avoiding the problem of excessive pressure difference causing chip explosion due to large-angle designs.

[0024] 2. The microchannel of the present invention employs multiple sinusoidal waveform channels, utilizing the direct modulation effect of the sinusoidal waveform channel geometry on the migration rate. The sin2x type channel is adopted, which has the shortest geometric period and the most frequent curvature changes, inducing higher frequency field strength spatial oscillations. This significantly increases the frequency of DNA molecules capturing high-energy electrophoretic flux per unit time, and its migration efficiency is much higher than that of conventional channels.

[0025] 3. This invention utilizes the extremely non-uniform electric field distribution generated at the inflection point of the curved channel, combined with the strong orthogonal mesophoretic properties of DNA molecules, to generate a transverse mesophoretic driving force. This forcefully pulls the DNA molecules scattered throughout the full width of the channel toward the inner side of the channel, forming an extremely fine, extremely bright particle beam with clear boundaries. This achieves efficient, non-mechanical focusing of DNA molecules, greatly improving the accuracy of subsequent optical detection and separation. Attached Figure Description

[0026] Figure 1 This is a schematic diagram of the structure of the present invention.

[0027] Figure 2 yes Figure 1 A cross-sectional view of the structural layers. (a) is... Figure 1 (a) is a cross-sectional view of the microfluidic system 4; (b) is a structural schematic diagram of the liquid inlet 5; (c) is an enlarged schematic diagram of the tapered channel 4-2; and (d) is an enlarged schematic diagram of the microchannel.

[0028] Figure 3 This is a diagram of the experimental apparatus of the present invention.

[0029] Figure 4This is a diagram showing the movement of DNA molecules at the cone-shaped inlet and outlet. (a) Actual image of DNA movement at the inlet, (b) Velocity of DNA molecules at the cone-shaped inlet under different voltages, (c) Actual image of DNA movement at the outlet, (d) Velocity of DNA molecules at the cone-shaped outlet under different voltages.

[0030] Figure 5 These are schematic diagrams of curved microchannels of different function types and velocity diagrams of DNA molecules in different types of channels. (a) Motion of DNA molecules within the channels, (b) 2sinx channel, (c) sin2x channel, (d) sinx channel.

[0031] Figure 6 This is a velocity distribution map. (a) The microchannel is divided into three regions: region K, region R and region P; (b) The average velocity in different regions.

[0032] Figure 7 It is a migration trajectory map of DNA molecules under a low electric field, located once every 100 milliseconds.

[0033] Figure 8 This is a diagram showing the aggregation of DNA molecules. (a) Aggregating on the inner channel wall, (b) Aggregating in the center of the channel, (c) Aggregating on the inner channel wall.

[0034] Figure 9 These are dynamic conformation diagrams of DNA molecules at different velocities. (a) Low field strength, (b) Medium field strength, (c) High field strength.

[0035] Figure 10 These are simulation diagrams of electric field variation at a conical opening. (a) Schematic diagram of a 30° conical channel opening; (b) Electric field variation at the central axis of a 30° conical channel opening with voltage; (c) Electric field variation at the central axis of conical openings at different angles under 5V voltage.

[0036] Figure 11 These are simulation diagrams showing the changes in field strength at different angles of a conical aperture. (a) 45° conical aperture, (b) 30° conical aperture, (c) 15° conical aperture.

[0037] Figure 12 This is a field strength diagram inside the 2sinx channel. (a) Field strength change inside the channel, (a1) Field strength change to the left of the inflection point, (a2) Field strength change to the right of the inflection point, (a3) ​​Field strength change of the left straight channel, (a4) Field strength change of the right straight channel, (a5) Field strength change at the inflection point.

[0038] Figure 13 This is a field strength diagram inside the channel. (a) Radial field strength variation at the inflection point and on both sides of the inflection point, (b) Maximum and minimum field strength inside the channel.

[0039] In the figure: 1. Glass substrate; 2. Structural layer; 3. Electrode; 4. Microfluidic system; 5. Liquid inlet; 6. Liquid outlet; 4-1. Straight channel; 4-2. Conical channel; 4-3. Curved channel; 4-4. Straight buffer channel. Detailed Implementation

[0040] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments, but the present invention is not limited to these embodiments.

[0041] Example 1

[0042] exist Figure 1 , 2 This invention relates to a DNA molecule manipulation chip driven by coupling an electric field with a curved microchannel. The chip comprises a glass substrate 1 and a structural layer 2, the structural layer 2 being made of polydimethylsiloxane. The polymer and curing agent are mixed in a 10:1 ratio. Glue A should be added first, followed by Glue B; otherwise, reversing the order may lead to poor cross-linking. The mixed Glue A and Glue B are thoroughly stirred in a centrifuge. The stirring is divided into two stages: the first stage at 2000 rpm for 60 seconds; the second stage at 2200 rpm for 120 seconds. The structural layer 2 is tightly bonded to the glass substrate 1 through oxygen plasma activation treatment. At least one set of microfluidic systems 4 is provided inside the structural layer 2. In this embodiment, three sets of microfluidic systems 4 are provided. The length of the microparticle system 4 is 10 mm. The two ends of the microfluidic system 4 are respectively connected to the liquid inlet 5 and the liquid outlet 6. The liquid inlet 5 and the liquid outlet 6 are vertically inserted through the structural layer 2. Electrodes 3 are respectively inserted into the liquid inlet 5 and the liquid outlet 6. Electrodes 3 refer to positive and negative electrode pairs. Electrodes 3 are used to apply DC voltage to generate a spatial non-uniform electric field.

[0043] The microfluidic system 4 of this embodiment includes a straight channel 4-1 and a curved channel 4-3. The width and height of the straight channel 4-1 and the curved channel 4-3 are 1-10 μm and 1-10 μm respectively. One end of the straight channel 4-1 is connected to the inlet port 5 and the other end is connected to the outlet port 6. The curved channel 4-3 is connected to the middle of the straight channel 4-1 through a tapered channel 4-2 and a straight buffer channel 4-4. The curved channel 4-3 includes at least two microchannels. Each microchannel is connected to the straight channel 4-1 through the tapered channel 4-2 and the straight buffer channel 4-4. The shape of the microchannel is any one of a sine curve, cosine curve, or tangent curve. This embodiment has five microchannels. The shape of the microchannels is a sine curve. The tapered channel 4-2 gradually converges from the straight channel 4-1 to the microchannels, with a half-cone angle of 30°. The width and height of the straight channel 4-1 and the curved channel 4-3 are 5 μm and 5 μm respectively.

[0044] The DNA sample solution enters the straight channel 4-1 through the inlet 5, then passes through the conical channel 4-2 into the curved channel 4-3. Driven by the positive mesophoresis effect, the DNA molecules are extremely focused into a thin beam of particles on the inner wall of the channel with the highest electric field strength, passing through the straight channel 4-1 and entering the outlet 6. To prevent the structural layer from collapsing, several vertical pillars are installed in the straight channel 4-1 and the curved channel 4-3, and a cross-shaped framework is installed in the inlet 5 and the outlet 6.

[0045] The control method for the DNA molecule manipulation chip based on the coupling of electric field and curved microchannel includes the following steps:

[0046] Step 1, Chip Pretreatment: Inject buffer solution into inlet 5 to fill microfluidic system 4, and inject DNA sample solution through inlet 5;

[0047] Step 2: Apply driving electric field: Insert the platinum wire electrode into the positive and negative electrode sample cells, connect an external high voltage power supply, and apply a 0-50V DC voltage to form a spatial non-uniform electric field.

[0048] Step 3, DNA molecule acceleration and polarization: When DNA molecules flow through the cone channel 4-2, they undergo a rapid change in electric field gradient, resulting in rapid polarization and accelerated migration, and quickly pass through the cone channel 4-2 into the straight buffer channel 4-4;

[0049] Step 4: DNA molecules focus and migrate via mesophoresis: DNA molecules enter the sinusoidal microchannel. At the inflection point, the geometric curvature induces a non-uniform distribution of electric field lines, which are denser inside and sparser outside. Driven by the mesophoresis effect, the DNA molecules are focused into a thin beam of particles on the inner wall of the channel where the electric field intensity is highest.

[0050] Experiment 1

[0051] The following experiments were conducted using the DNA molecule manipulation chip and its control method based on electric field and curved microchannel coupling driven in Example 1 above:

[0052] 1. Solution preparation

[0053] The experiment used λ-DNA molecules (Fuxintes Biotechnology Co., Ltd., Shenzhen, China) and labeled them with the dye YOYO-1 molecules (Invitrogen, New York, USA). To balance fluorescence intensity and molecular structural integrity, the ratio of YOYO-1 dye molecules to base pairs was 10:1.

[0054] The preparation process of the λ-DNA and YOYO-1 fluorescent dye solution is as follows: (1) Take 100 μL of Bis-Tris (pH=8) and 1000 μL of Tris-HCl (pH=8) and add them to two tubes A and B respectively; (2) Take 2.055 μL of DNA stock solution and 0.5 μL of YOYO-1 stock solution and add them to tubes A and B respectively, and incubate them in the dark for 60 min; (3) Take 200 μL of YOYO-1 dilution from B and add it to A and mix thoroughly. Incubate in the dark for 60 min, and then place it in a freezer at -4℃ for later use. The concentration of λ-DNA in the DNA / YOYO-1 mixed solution is 2.275 mg / L.

[0055] 2. Fabrication of Bending Microfluidic Chips

[0056] like Figure 1 As shown, the microfluidic chip consists of two layers: the upper layer is the structural layer, which includes the microfluidic system 4, the liquid inlet 5, and the liquid outlet 6; the lower layer is the glass substrate 2, used to encapsulate the microchannels. The microfluidic system 4 is fabricated using soft photolithography. The fabrication process mainly includes three key steps: ultraviolet exposure, molding, and drilling and bonding.

[0057] UV Exposure: Photoresist negatives (SU-8, Microchem, California, USA) are spin-coated onto a clean, flat wafer. After baking, UV exposure is performed using appropriate exposure parameters (MIDASMDA400LJ, Daejeon, Republic of Korea). Unactivated photoresist is then removed with a developer, transferring the photomask pattern to the wafer. Molding: PDMS is used to replicate the microfluidic chip's structural layer 2. During this process, the ratio of PDMS A-resin to B-resin is adjusted from 10:1 to 9:1 to increase the PDMS's flexibility and reduce debris generation. Drilling and Bonding: Using a 1mm diameter drill bit, inlet holes 5 and outlet holes 6 are fabricated at the edges of the large channels on both sides. Oxygen plasma activation treatment is then used to achieve tight bonding between the upper and lower layers. Each microfluidic chip has three sets of microchannel fluid systems, increasing chip utilization.

[0058] The self-made microfluidic system 4 in this experiment has a total length of 10 mm and contains five curved channels with bending functions (sinx, sin2x, 2sinx) and a width of 5 μm. Five sets of conical channels (length: 86 μm, inner diameter: gradually decreasing from 105 μm to 5 μm) are connected in parallel at their inlet ends to converge into a straight channel 4-1 with a width of 560 μm. The outlet ends connect to a 5 μm microchannel, and the other end has the same design. All channels have a uniform height of 5 μm to avoid DNA molecules accumulating and clogging at the junctions due to abrupt changes in channel height, thus improving DNA throughput. The inlet and electrode inlet share a single hole to reduce the number of repeated drilling operations. The hole is located at both ends of the chip, 10 mm apart.

[0059] 3. Experimental Procedure

[0060] Experimental observation and analysis apparatus such as Figure 3 As shown, the main components included: an inverted fluorescence microscope (IX-70, Olympus, Japan); an EMCCD camera (iXon+885, Andor, USA); a microfluidic chip; a syringe pump (PUMP33, HARVARD Apparatus, Massachusetts, USA); a sample injector (50μL, Hamilton, Nevada, USA); platinum wire electrodes (Shanghai Jieyu Electronic Technology Co., Ltd.); and an external high-voltage power supply (PS 8000 2U, EPS, Germany). The voltage adjustment range during the experiment was 0~50V. Image data acquisition, analysis, and processing were all performed using Image software. The entire experiment was conducted in a darkroom with the laboratory temperature controlled at 23℃.

[0061] The experimental procedure is summarized as follows: Before the experiment, the microfluidic chip was pre-degassed. First, Tris-HCl buffer was injected into inlet 5 at a rate of 5 μL / h using a syringe pump to wet the channel. After 20 minutes, the channel was completely filled with buffer and allowed to stand for 30 minutes to ensure sufficient contact between the channel and the buffer. Next, λ-DNA / YOYO-1 sample solution was injected into inlet 5 at a rate of 5 μL / h for approximately 20 minutes to ensure sufficient DNA molecules were injected into the channel. Then, the injector was removed to release the pressure in the inlet, almost stopping the flow of DNA molecules and preventing this flow from affecting the experimental results. Finally, a pair of platinum electrodes were inserted into inlet 5 and outlet 6. The electric field strength was changed by adjusting the voltage (0~50V) to drive the movement of λ-DNA molecules in the chip. During the experiment, the movement of DNA molecules in the microchannel was recorded in real time using an EMCCD with the following parameters: exposure time 50 ms and image interval 100 ms.

[0062] 4. Use COMSOL Multiphysics 6.1 software to simulate the electric field distribution within the conical microfluidic channel system to aid in the analysis of the phenomena monitored in the experiment.

[0063] Table 1 lists the physical parameters used in the analysis and numerical simulation. It is assumed that all fluid properties are constant and homogeneous throughout the microchannel.

[0064] Table 1 Physical parameters used in numerical simulation

[0065]

[0066] 5. Experimental Results

[0067] The laboratory experiments were conducted in a darkroom. This study used a self-made microfluidic chip to guide DNA molecules through sinusoidal (sinx, sin2x, 2sinx) curved microchannels driven by an external electric field. The motion characteristics of DNA molecules traversing these curved microchannels under different applied electric fields were analyzed in detail. The velocity changes of DNA molecules through the three different function types of microchannels were compared. The experiments showed that as the voltage increased from 0V to 50V, the velocity of DNA molecules within the curved channels changed significantly. Due to the unique design of the conical channel opening, the electric field intensity was unevenly distributed within the conical channel, causing the DNA molecules to accelerate at the conical entrance. The electric field distribution inside the channel tended to be uniform, resulting in approximately uniform velocity movement of the DNA molecules within the microchannel, although the velocity decreased at the channel inflection points. Furthermore, the velocity change was greatest in the sin2x microchannel, followed by the sinx channel, and smallest in the 2sinx channel.

[0068] 5.1 Movement of DNA molecules within the cone-shaped opening

[0069] By observing the movement of DNA at the cone-shaped end, such as Figure 4 As shown, the analysis reveals the variation in migration speed of DNA molecules passing through the cone-shaped entrance and exit near the central axis. Figure 4 (a)(b)(c)(d) are physical diagrams and velocity diagrams of DNA molecules passing through the cone-shaped inlet and outlet. For example... Figure 4 As shown in (b), regardless of the applied voltage, the velocity of DNA molecules at the inlet increases as the conical channel narrows. Furthermore, the higher the applied voltage, the faster the DNA molecules move. Between 10 μm and 60 μm, in this region, the channel is relatively wide, the rate of change in cross-sectional area is small, the effect of voltage changes is minimal, and the velocity increases slowly with position, exhibiting an approximately linear relationship. Between 60 μm and 90 μm, near the tip of the channel, the cross-sectional area decreases, and the velocity of DNA molecules increases at all voltages. Between 90 μm and 100 μm, corresponding to the tip of the channel, DNA molecules undergo rapid polarization and accelerated migration due to the drastic change in the electric field gradient, quickly passing through the conical port. Simulations verify that a 30° half-cone angle is the best choice to balance the "acceleration effect" and "chip packaging reliability (avoiding excessive pressure differential causing chip explosion)." Figure 4 As shown in (d), at the exit, the speed of DNA molecules is exactly the opposite of that at the exit.

[0070] 5.2 Movement of DNA molecules inside curved channels

[0071] like Figure 5As shown in (a), when DNA molecules enter the channel, we analyzed the channel center region at nine locations on both sides of the two straight buffer zones, three inflection points, and four inflection points to study the movement of DNA molecules inside the microfluidic chip. Figure 5 (b)(c)(d) The relationship between the molecular migration rate within the microchannel and the voltage across its terminals was statistically analyzed. The results show that after DNA molecules enter the microchannel from the cone-shaped port, they initially accelerate in the straight buffer region, and their speed stabilizes as they enter the curved channel. We also found that the speed of DNA molecules on both sides of the inflection point is greater than that at the inflection point itself. As DNA molecules approach the cone-shaped exit, their speed gradually decreases in the straight buffer region, eventually becoming approximately the same as their speed at the cone-shaped entrance. Between 0 and 50 V, the DNA molecule migration rate increases with increasing voltage. At 5 V, the change in DNA molecule speed is minimal because the voltage is low, and the driving force of dielectrophoresis on the DNA molecules is small. At 25 V, the DNA molecule speed increases significantly. At 50 V, the increase in DNA molecule speed is several times greater than the increase between 0 and 5 V, indicating that dielectrophoresis has a strong influence on DNA molecules.

[0072] Experimental results show that DNA molecules exhibit significant dynamic differences in three curved channels with different sinusoidal function characteristics. Their migration efficiencies, in descending order, are: sinx > sin²x > 2sinx. The physical essence of this velocity modulation effect stems from the determining effect of channel geometry on the global path length. The sinx channel, due to its minimal fluctuations, shortest effective physical path, and lowest internal voltage loss, provides the highest global driving electric field for the molecule, thus achieving the largest absolute migration rate.

[0073] However, from the perspective of acceleration and deformation, the sin2x channel exhibits the strongest modulation effect. Due to its shortest geometric period and most frequent curvature changes, it induces higher frequency spatial oscillations of the electric field intensity within the same straight-line distance. This extremely steep spatial electric field gradient (▽|E|) 2 This causes DNA molecules to undergo a series of high-intensity "rapid acceleration-deceleration" processes as they pass through the channel. This high-frequency mechanical impact greatly overcomes the molecule's entropic elastic restoring force, leading to the most dramatic remodeling within the channel. In contrast, the 2sinx channel, due to its longer path and gentler curvature transition, exhibits the lowest absolute velocity and driving force gradient.

[0074] 5.3 The movement of DNA molecules within microchannels

[0075] 5.3.1 Velocity Distribution Pattern in Microchannels

[0076] To compare the radial velocity within the same region with velocities in different regions, we divided the curved channel into three parts: the left side of the inflection point (regions K1, K2, and K3), the inflection point itself (regions R1, R2, and R3), and the right side of the inflection point (regions P1, P2, and P3). Figure 6 As shown in (a). Figure 6 (b) shows the velocity of each region of the sin2x microchannel at 5V (the average velocity of 10 DNA molecules passing through this region was statistically analyzed). From the data graph, we found that: (1) For DNA molecules in the same region, the velocity of DNA molecules at the channel inflection point shows a great difference in the radial direction. The velocity of molecules near the outer curved wall (R1 region) is the lowest, only 8.2µm / s; while the velocity of molecules near the inner curved wall (R3 region) increases to 16.9µm / s. The velocity in the central region (R2) is 15.2µm / s. This indicates that in the curved region, the driving force of the electric field on DNA molecules also varies due to the different radial positions of the molecules; on the left and right sides of the inflection point, the velocity difference of DNA molecules at different radial positions (K1 region, K2 region and K3 region) and (P1 region, P2 region and P3 region) is small, but the velocity of DNA molecules located on the central axis is greater than that of DNA molecules located near the channel wall. (2) The velocity of DNA molecules in different regions is greater in the region on both sides of the microchannel (K / P) than in the R region at the inflection point. (3) For the radial velocity difference in the same region, this phenomenon is mainly determined by the non-uniformity of the electric field distribution and the different dominant roles of near-wall hydrodynamic effects in different geometric segments. First, in curved geometries, according to the electric field theory model... (where θ is the central angle and r is the radius of curvature), the electric field intensity is distributed in a gradient along the radial direction, which leads to a huge difference in the longitudinal electrophoretic driving force of molecules at different radial positions, thus producing the "racetrack effect" in the bend (i.e., faster on the inside and slower on the outside); secondly, in the straight regions on both sides of the inflection point, although the electric field distribution on the cross section is uniform, molecules near the wall (such as K1 / K3, P1 / P3 regions) are significantly affected by the no-slip boundary conditions and the near-wall viscous drag of the fluid. Compared with molecules on the central axis, the electrophoretic mobility is further suppressed. In summary, the electric field distortion in the curved section and the near-wall drag effect in the straight section alternately dominate, jointly leading to the difference in the radial velocity of DNA molecules. (4) For the velocity difference between different regions, the overall migration behavior of DNA molecules in the microchannel maps the topological structure of the spatial electric field. In the flat regions on both sides of the channel, the geometric configuration is symmetrical, and the electric field lines are uniformly distributed and parallel to the channel axis. Therefore, the longitudinal driving force obtained by molecules in this region is basically consistent, resulting in a stable migration velocity. However, at the channel inflection point, the topological structure of the electric field is distorted by curvature: when the current passes through the channel inflection point, the electric field lines exhibit a non-uniform distribution in space. This local electric field change caused by geometric curvature alters the relatively constant driving state in the flat region, causing changes in the effective downstream driving electric field and the fluid hindrance environment faced by DNA molecules in this region. This electrical topological evolution process explains, from a physical mechanism perspective, the phenomenon of velocity differentiation of DNA molecules between the sides and the inflection point observed in the experiment. We will further analyze this mechanism in subsequent physical field numerical simulations.

[0077] 5.3.2 The migration trajectory of DNA within microchannels

[0078] 5.3.2.1 Migration trajectory under low flow velocity

[0079] Figure 7 (a)~(f) are images taken every 100ms of four independent DNA molecules near the central channel and channel wall, indicated by green, red, purple and yellow diamonds respectively. The continuous stroboscopic images show that when the DNA molecule speed is low, at the inflection point and on both sides of the inflection point, the migration trajectory of the molecule at the microscopic scale continues to migrate forward along the original radial position.

[0080] 5.3.2.2 Migration Trajectory under High Flow Velocity

[0081] Under the influence of an electric field, a unique physical phenomenon occurs within the curved microchannel. As the electric field strength increases, the movement of DNA molecules accelerates. At the inflection point, the DNA molecules scattered throughout the channel space seem to be subjected to an invisible compression, converging from the full width of the channel towards the inner wall. Ultimately, these high-speed moving DNA molecules are highly compressed at the bend into an extremely thin, bright, and clearly defined beam of particles that travels along the vicinity of the channel wall, as... Figure 8 As shown in (a) and (c). In the straight sections on either side of the inflection point, DNA molecules scattered throughout the channel converge towards the center, forming a high-brightness particle beam at the center, such as... Figure 8 As shown in (b).

[0082] At the inflection point, the electric field gradient formed by the curved channel results in a stronger electric field on the inner channel wall and a weaker electric field on the outer channel wall. Due to the strong counter-ion polarization effect of DNA molecules, they exhibit positive dielectrophoresis (pDEP) characteristics. As the electric field strength increases, the dielectrophoretic force gradually increases, strongly pulling the DNA molecules towards the inner side of the channel, forming a very fine bundle of DNA particles. In the straight, flat regions on either side of the inflection point, the lateral electric field gradient (▽|E|)... 2 The electrophoretic force is almost zero, and the positive electrophoretic force disappears. At this point, the lateral constraint of the molecules disappears, and they are mainly driven by the uniform longitudinal electric field. Molecules gather towards the center here, mainly due to the steric hindrance effect and electrostatic repulsion near the channel wall, causing DNA molecules to spontaneously migrate towards the central region of the channel.

[0083] 5.3.2.3. Movement patterns of DNA molecules within the channel

[0084] like Figure 9 As shown in (a), when the voltage is 5V, the electric field strength is relatively small, and DNA molecules appear as small bright spots moving within the channel. As the voltage increases to 25V, the electric field force increases, and the molecules experience spatially uneven electric field forces within the curved channel. The electric field forces at the two ends of the molecule and on different segments differ. This localized pulling force begins to overcome the entropic elastic restoring force of the molecule, causing the DNA coil to unwind and exhibit partially stretched shapes such as long strips, crescent shapes, or dumbbell shapes, as shown in [example image]. Figure 9 As shown in (b), under a high electric field (50V), the stretching force generated by the flow field or electric field gradient is much greater than the entropic elasticity of the molecules. The DNA molecules are stretched violently, almost completely unfolding along their outline length, exhibiting bright and elongated linear trajectories in the channels, such as... Figure 9 As shown in (c).

[0085] 5.4 Mechanism Analysis of DNA Molecules Crossing Bending Microchannels

[0086] 5.4.1 Pure electrodynamic flow driving mechanism and mesoelectrophoresis effect of DNA molecules

[0087] In this experimental system, the microchannel inner wall was modified with poloxamer to block silanol groups on the channel wall surface, and the high ionic strength PBS buffer compressed the Debye length (κ) of the electric double layer (EDL). -1 The synergistic effect of these two factors suppressed electroosmotic flow (EOF≈0). Furthermore, in the low Reynolds number (Re≪1) fluid environment of the microfluidic system, the inertial effect of DNA molecules is negligible; therefore, the motion mechanism of λ-DNA molecules within the sinusoidal microchannel can be attributed to pure electrokinetics. Its overall velocity U... P This can be expressed as the longitudinal electrophoresis velocity U. EP and transverse dielectrophoresis velocity U DEP The vector sum, i.e.

[0088]

[0089] In the formula, E is the local electric field intensity vector, ▽|E| 2 μ represents the squared gradient of the electric field intensity. EP and μ DEP These represent the electrophoretic mobility and dielectrophoretic mobility of DNA, respectively.

[0090] On the one hand, λ-DNA, as a highly negatively charged flexible polymeric anion, undergoes electrophoretic motion driven by Coulomb force under the action of a DC electric field. This velocity is linearly proportional to the local electric field strength E, constituting the main downstream driving force for molecular migration towards the positive electrode along the channel.

[0091] On the other hand, when DNA molecules flow through the curved sections of the microchannel, the channel's geometric curvature causes a non-uniform spatial electric field distribution, resulting in a radial electric field gradient ▽|E|. 2 A non-uniform electric field exerts a dielectrophoretic force on polarized molecules; the direction and strength of this force depend on the molecular electrophoresis fp. CM Clausius-Mosotti Factor (Clausius-Mosotti Factor):

[0092]

[0093] In the formula, σ p It is the particle conductivity, σ f It is the fluid conductivity. According to σ... p and σ f The relative size between them, the CM factor can be negative or positive. Traditional polymer particles typically exhibit negative dielectrophoretic properties (nDEP, f) in high-conductivity buffers (such as PBS). CM<0). However, due to the high density of negative charges attached to the DNA backbone, its surface is tightly bound with a high concentration of counterions. Under an applied DC electric field, this counterion cloud undergoes strong counterion polarization, resulting in a decrease in the effective low-frequency conductivity (σ) of the DNA-counterion complex. p The conductivity (σ) is significantly higher than that of the buffer solution. f ), thus satisfying f CM >0.

[0094] Based on the aforementioned positive mesoelectrophoresis (pDEP) effect, DNA molecules, when traversing the tortuous microchannel, are driven by transverse mesoelectrophoretic forces to migrate radially towards the region of maximum electric field gradient, moving towards the inner wall of the tortuous section of the channel.

[41] This purely electrodynamic theoretical model fully explains, from the physical foundation of mechanical equilibrium and interfacial polarization mechanisms, the dynamic behavior observed in the experiment where DNA molecules tend to focus towards the inner wall and induce local velocity differentiation.

[0095] 5.4.2 Microchannel Field Strength Distribution Analysis

[0096] We used COMSOL Multiphysics 6.1 software to simulate the field intensity distribution inside the microchannel, and analyzed the field intensity extrema at the conical channel opening, the channel inflection point, and both sides of the inflection point, as well as compared the field intensity extrema of three types of sinusoidal functions, to explain the motion mechanism of DNA molecules in the sinusoidal function-type curved microfluidic channel system.

[0097] 5.4.2.1 Field Intensity Analysis at the Conical Channel Opening

[0098] In the physics simulation, we selected a 2sinx type channel to analyze the electric field intensity variation at the conical channel opening, where different types of channels share the same conical channel design. For example... Figure 10 As shown in (a), we established a coordinate system with point O(0,0) at the entrance of the conical channel as the origin and statistically analyzed the changes in field strength along the centerline.

[0099] like Figure 10 (b) As shown in the data graph, when the voltage across the channel is 5V, the electric field strength increases from 3.22 × 10² V / m to 5.62 × 10³ V / m. At a voltage of 25V, the electric field strength increases from 1.5 × 10³ V / m to 2.73 × 10³ V / m. 4 V / m. At a voltage of 50V, the electric field strength increases from 3.18×10³V / m to 5.54×10⁻⁶V / m. 4V / m. These data show that the electric field strength increases with increasing voltage, and the change is particularly rapid at the cone-shaped channel opening. This indicates that DNA molecules undergo accelerated motion and rapid polarization as they pass through these paths. This change in electric field gradient causes the electric force on the DNA molecules to gradually increase as they pass through the cone-shaped channel opening, leading to rapid polarization and accelerated motion.

[0100] like Figure 11 As shown, we simulated the electric field changes at different angles (15°, 30°, 45°) of the conical channel opening under a 5V voltage. Figure 9 (c) The field strength data along the central axis of the conical opening at different angles shows that the field strength changes fastest at the 45° conical channel opening, followed by the 30° conical channel, and slowest at the 15° conical channel. Therefore, the 45° conical channel is beneficial for the accelerated movement of DNA at the conical channel opening. However, in actual experiments, the maximum size of the 45° conical channel is 146. The difference between the 5μm and m dimensions of the microchannel is too large, resulting in excessive solution throughput and pressure difference, which could easily cause the chip to explode. Secondly, even at low voltage, DNA molecules move too quickly within the 45° conical channel, hindering experimental data measurement. Therefore, we chose a 30° conical channel in our experiment.

[0101] 5.4.2.2 Electric field conditions at and around the inflection point

[0102] When the voltage across the channel is 5V Figure 12 The electric field intensity variations at and around the inflection point are described. It can be seen that the electric field intensity exhibits significant radial non-uniformity at the inflection point of the channel. The inner side of the channel corresponds to the maximum electric field intensity, and the outer side corresponds to the minimum electric field intensity. The color distribution is uniform on both sides of the inflection point, and the electric field intensity is radially uniform in the straight sections entering and leaving the inflection point. This indicates that the maximum electric field gradient occurs at the inflection point, where the electric field distortion is most significant, and the resulting dielectrophoretic force is also the strongest. Figure 13 (a) Describes the radial electric field intensity variation at the inflection point and at the same horizontal position on both sides of the inflection point. The red dotted curve (representing the inflection point) shows a clear linear increasing trend (with a large slope). This indicates that at the inflection point cross-section, as the radial position X moves from the outer side (low field strength, approximately 4000 V / m) to the inner side (high field strength, approximately 4740 V / m), the electric field intensity increases sharply. The blue triangular / gray square curve (representing both sides of the inflection point / straight path) is approximately a horizontal straight line. This indicates that in the straight path region far from the inflection point, the electric field intensity remains constant (approximately 4400 V / m) and does not change with radial position.

[0103] First, the non-uniformity of the electric field distribution stems from the geometric characteristics of the curved sections of the microchannel. Under a constant voltage difference, the magnitude of the electric field strength is inversely proportional to the arc length along the current path. For example... Figure 12As shown in (a5), the inner side of the curved channel at the inflection point has a smaller radius of curvature and a shorter current path, resulting in a steep potential drop gradient and forming a high field strength region (R3 region); conversely, the outer path is longer, resulting in a gentler potential drop and forming a low field strength region (R1 region). For both sides of the inflection point, as... Figure 12 As shown in (a3)(a4), the electric field distribution is uniform, and the electric field strength of the inner channel wall is almost the same as that of the outer channel wall. Figure 13 The quantitative data in (a) further confirm this pattern: at the inflection point (red curve), the electric field strength shows a significant linear decrease from the inside to the outside with radial position, while the straight areas on both sides of the inflection point (blue / gray curve) maintain a uniform distribution.

[0104] Secondly, the velocity of DNA molecules is primarily determined by the electric driving force they experience. In the straight, gentle sections on either side of the inflection point, due to the uniform electric field distribution across the cross-section, DNA molecules experience almost the same downstream electrophoretic driving force regardless of their radial position, thus exhibiting stable high-speed motion. However, upon entering the curved section, the electric field strength in regions R1 and R2 is lower than in the straight sections on either side (regions P and K), thus the electrophoretic force on particles in these regions is relatively weakened; while the electric field strength in region R3 is significantly greater than in the straight sections on either side, theoretically, particles in this region should experience a stronger longitudinal electrophoretic acceleration force. Although the strongest downstream electrophoretic driving force exists globally near the inner wall of the inflection point (region R3), experiments have observed that the overall velocity of DNA molecules at the inflection point is slightly lower than in the straight sections on either side. The fundamental physical mechanism of this phenomenon lies in the near-wall hydrodynamic hindrance effect induced by normal mesoelectrophoresis (pDEP). In a purely electrically driven system, there is no macroscopic bulk fluid flow within the channel, eliminating interference from fluid inertia and Dean's flow. At the inflection point, the spatial electric field gradient generates a strong positive mesoelectrophoretic force (pDEP), forcing DNA molecules to undergo significant radial migration. This pushes the DNA molecules towards the inner wall (R3 region) where the electric field strength is highest, causing them to focus. The focused DNA molecules then travel along a path of "highest energy density." Although the DNA molecules experience significant electrophoretic acceleration at this point, their proximity to the wall due to the mesoelectrophoretic force increases localized fluid viscous friction, slowing their movement and offsetting the acceleration effect of the high electric field. In summary, the positive mesoelectrophoretic focusing caused by the high electric field gradient due to the curved geometry, coupled with the strong negative coupling between the near-wall viscous drag resulting from the molecules' extreme proximity to the inner wall and the electrophoretic driving force, is the fundamental mechanism causing the instantaneous decrease in the velocity of DNA molecules at the inflection point.

[0105] 5.4.2.3 Electric Field Inside Curved Channels of Three Types of Sine Functions

[0106] Figure 13(b) Numerical simulation results of the electric field intensity distribution within the microchannel under three different sinusoidal function configurations are presented. The simulation results show that, under the same 5V voltage condition, the sinx channel has the shortest physical path and the smallest overall electric field loss, thus exhibiting the highest local field strength peak (corresponding to the highest absolute velocity observed in the experiment). The sin2x channel, on the other hand, exhibits the highest frequency of spatial oscillations in field strength and the most intense gradient fluctuations. Due to its significantly shortened geometric period, the electric field undergoes a more concentrated "compression-relaxation" cycle within a unit displacement, forming a high-density energy level step in space. This extremely large abrupt change in electric field gradient significantly increases the frequency of strong positive mesoelectrophoresis (pDEP) forces and instantaneous high accelerations experienced by DNA molecules per unit time. Therefore, although DNA molecules have the fastest translational velocity in the sinx channel, they experience the most intense dynamic perturbations and pulling effects in the sin2x channel.

[0107] Therefore, this application systematically investigated the motion characteristics of λ-DNA molecules in microchannels driven by an electric field by using a sinusoidal bending microfluidic chip, combined with single-molecule fluorescence imaging technology and physical field simulation. The main conclusions are as follows:

[0108] 1. Decoupling Modulation of Global Velocity and Local Acceleration by Channel Geometry: Experiments and simulations show that the geometric characteristics of a tortuous channel simultaneously determine the magnitude and gradient of the absolute electric field. The sinx-type channel, with its shortest effective current path, imparts the highest absolute translational velocity to DNA molecules. While the sin2x-type channel has a moderate absolute velocity, its extremely high curvature change frequency creates the steepest radial electric field gradient at the microscopic scale, causing DNA molecules to experience the most extreme instantaneous acceleration abrupt changes and the strongest positive mesoelectrophoretic pull when crossing inflection points. This indicates that by rationally modulating the channel's geometric period and curvature, precise decoupling control of macromolecular transport velocity and conformational reconstruction can be achieved.

[0109] 2. Localized electric field distortion-induced mesophoretic aggregation: At the bends of the channel, the electric field lines exhibit an extremely non-uniform distribution, with a denser inner layer and a sparser outer layer. Due to the strong polarizability of the DNA-counterion complex, it exhibits significant mesophoretic (pDEP) characteristics. This transverse mesophoretic driving force, dominated by the electric field gradient, causes high-speed DNA molecules to precisely aggregate and move close to the inner wall of the channel where the electric field energy density is highest.

[0110] 3. Kinetic hysteresis dominated by near-wall viscous drag: Although there is the strongest downstream electrophoretic driving electric field on the inner wall of the inflection point, the acceleration effect of the high electric field is partially offset by the near-wall fluid viscous drag due to the mesophoretic force that brings the DNA molecules close to the inner channel wall. As a result, the speed of the DNA molecules when crossing the inflection point is slightly less than that of the straight regions on both sides.

Claims

1. A DNA molecule manipulation chip driven by coupling between an electric field and a curved microchannel, characterized in that: The structure includes a glass substrate (1) and a structural layer (2). The structural layer (2) is disposed on the glass substrate (1). At least one set of microfluidic systems (4) is disposed inside the structural layer (2). The two ends of the microfluidic system (4) are respectively connected to a liquid inlet (5) and a liquid outlet (6). The liquid inlet (5) and the liquid outlet (6) are vertically disposed through the structural layer (2). Electrodes (3) are inserted into the liquid inlet (5) and the liquid outlet (6) respectively. The electrodes (3) are used to apply a DC voltage to generate a spatial non-uniform electric field.

2. The DNA molecule manipulation chip based on electric field and curved microchannel coupling drive according to claim 1, characterized in that: The microfluidic system (4) includes a straight channel (4-1) and a curved channel (4-3). One end of the straight channel (4-1) is connected to the inlet hole (5) and the other end is connected to the outlet hole (6). The curved channel (4-3) is connected to the middle of the straight channel (4-1) through a conical channel (4-2) and a straight buffer channel (4-4). The DNA sample solution enters the straight channel (4-1) from the inlet hole (5) and enters the curved channel (4-3) through the conical channel (4-2). Driven by the positive mesophoresis effect, the DNA molecules are extremely focused into a thin particle beam on the inner wall of the channel with the highest electric field strength and pass through the straight channel (4-1) before entering the outlet hole (6).

3. The DNA molecule manipulation chip based on electric field and curved microchannel coupling drive according to claim 2, characterized in that: The curved channel (4-3) includes at least two microchannels, each of which is connected to the straight channel (4-1) through a tapered channel (4-2) and a straight buffer channel (4-4).

4. The DNA molecule manipulation chip based on electric field and curved microchannel coupling drive according to claim 3, characterized in that: The shape of the microchannel can be any one of a sine curve, a cosine curve, or a tangent curve.

5. The DNA molecule manipulation chip based on electric field and curved microchannel coupling drive according to claim 3, characterized in that: The conical channel (4-2) gradually converges from the straight channel (4-1) to the microchannel, with a semi-cone angle of 15° to 45°.

6. The DNA molecule manipulation chip based on electric field and curved microchannel coupling drive according to claim 2, characterized in that: The width of the straight channel (4-1) and the curved channel (4-3) is 1-10 μm and the height is 1-10 μm.

7. The DNA molecule manipulation chip based on electric field and curved microchannel coupling drive according to claim 1, characterized in that: The structural layer (2) is made of polydimethylsiloxane and is tightly bonded to the glass substrate (1) by oxygen plasma activation treatment.

8. The DNA molecule manipulation chip based on electric field and curved microchannel coupling drive according to claim 2, characterized in that: Several vertical columns are arranged in the straight channel (4-1) and the curved channel (4-3), and a cross-shaped keel frame is arranged in the liquid inlet (5) and the liquid outlet (6).

9. A DNA molecule manipulation chip driven by coupling of electric field and curved microchannel, applied to the control method of the DNA molecule manipulation chip driven by coupling of electric field and curved microchannel as described in any one of claims 1 to 8, characterized in that... Includes the following steps: Step 1, chip pretreatment: Inject buffer solution into the inlet hole (5) to fill the microfluidic system (4), and inject DNA sample solution through the inlet hole (5); Step 2: Apply driving electric field: Insert the platinum wire electrode into the positive and negative electrode sample cells, connect an external high voltage power supply, and apply a 0-50V DC voltage to form a spatial non-uniform electric field. Step 3, DNA molecule acceleration and polarization: When DNA molecules flow through the cone-shaped channel (4-2), they undergo a rapid change in electric field gradient, resulting in rapid polarization and accelerated migration, and quickly pass through the cone-shaped channel (4-2) into the straight buffer channel (4-4). Step 4: DNA molecules focus and migrate via mesophoresis: DNA molecules enter the sinusoidal microchannel. At the inflection point, the geometric curvature induces a non-uniform distribution of electric field lines, which are denser inside and sparser outside. Driven by the mesophoresis effect, the DNA molecules are focused into a thin beam of particles on the inner wall of the channel where the electric field intensity is highest.

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