Single cell lysis and DNA extending using electroporation microfluidic ...

BioChip J. (2012) 6(1): 84-90 DOI 10.1007/s13206-012-6111-x

Original Research

Single cell lysis and DNA extending using electroporation microfluidic device Min-Sheng Hung1 & Ya-Tun Chang2

Received: 2 December 2011 / Accepted: 27 January 2012 / Published online: 20 March 2012 � The Korean BioChip Society and Springer 2012

Abstract The purpose of cell lysis is to obtain intracellular substances such as DNA and proteins for analysis. Commonly used methods include chemical (chemical solution decomposition) and physical (electricity or mechanical force). This study proposes an integrated system using an electroporation and microfluidic device made by micro-photolithography to lyse a single cell and stretch its DNA. The PDMS, as the manufacturing material of the microfluidic device, consists of 2 parts: the cell lytic zone, in which the immobilized cells trapped within the dense microstructure are lysed at a single-cell level, and the DNA stretching and recovery zone. This study showed that in a hypotonic environment (75 mM glucose solution), when electric field conditions were 100 Vpp and 1 kHz, the target cell was lysed and its DNA was released into the solution. When injected with proteinase K, the DNA flowed along the rectangular microstructure and was stretched to a length exceeding 840 μm. Keywords: Electroporation, Cell lysis, DNA extending, Microfluidic device, PDMS

Introduction Microfluidic devices made using Microelectromechanical Systems (MEMS) technology have the features of precise measurement, improved analytical capacity 1

Department of Biomechatronic Engineering, National Chiayi University, Chiayi 60004, Taiwan 2 Wecon Automation Corporation, Hsinchu 30072, Taiwan Correspondence and requests for materials should be addressed to M.-S. Hung ( [email protected])

for biological and chemical samples, shortened reaction times, and reduced drugs and cost. Microfabrication technology has been exploited on the chip level for cell lysis and DNA extraction and recovery. Because DNA recovery is completed on the chip, it is not subject to loss by centrifugation, separation, precipitation, or other operations. Electroporation is a commonly used method for cell lysis on a chip, mainly because it requires only a simple microchannel design and the construction of an electric field. Several researchers have used electroporation to lyse cells with transmembrane voltage. Kotnik et al.1 demonstrated cell lysis using an electric field. They compressed the membrane structure with an electric field, causing the cell membrane to disrupt, to obtain nuclei. Previous studies have developed microfluidic chips to demonstrate electroporation for high throughput screening of a large number of chemical and biological samples.2-5 Electroporation was carried out in a microfluidic platform combined with an AC voltage field2,3 or a direct current (DC) field.4,5 Lu et al.2 fabricated a chip for cellular lysis with AC voltage by micro-photolithography. The challenge of electroporation in a DC field is the generation of bubbles associated with the high field strength needed. Therefore, previous work used a narrow microchannel section to increase the intensity of the electric field.4,5 Wang and Lu4 designed microfluidic channels with geometric variation to lyse cells by intensifying the electric field at narrow sections of the microchannels. Their results show that at a higher electric field, cell lysis takes only a few milliseconds. Lee and Cho5 used a DC electric field generated in a narrow section of the flow channel, where a higher intensity of an electric field and electroosmotic flow was created to cause cell lysis. To

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achieve complete cell lysis (irreversible electroporation), Lin and Lee6 created an optically induced cell lysis device that can selectively lyse a single cell within a group of cells. Irreversible electroporation is a precursor to cell lysis when the transmembrane potential exceeds 1 V. The DNA released into the solution following cell lysis can be recovered in microsystem. Chung et al.7 designed a highly efficient DNA extraction microchip made of PMMA (Poly-methylmethacrylate) to extract DNA from lysed cells. Liu et al.8 designed a device consisting of a Printed Circuit Board (PCB) and the microfluidic chip for sample preparation, polymerase chain reaction amplification and DNA microarray detection. This approach begins with mixing a blood sample from Escherichia coli K12 cells with magnetic beads and capturing target cells within microchannels, and ends with cell lysis following heating. Lee et al.9 combined PDMS (Polydimethylsiloxane) with glass using MEMS technology to form a system capable of performing cell lysis and DNA amplification for Streptococcus Pneumoniae testing. Electroporation technology is suitable for microfluidic systems because it does not damage microchannels, and because of its short reaction time and easy-touse properties. This study investigates cell lysis at a single-cell level and extracting DNA in a micro-fluid system for subsequent analysis. An immobilized cell in the microchannel is lysed by electroporation because of an electric field effect.

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Results and Discussion This study determines the osmotic environment of cell lysis based on various concentrations of glucose solution. Figure 1 shows the cells placed in varied osmotic environments. In a hypertonic environment (300 mM glucose solution), the cells’ projected diameter is as small as 13.3±2.2 μm (Figure 1(a)). In an isotonic environment (150 mM glucose solution), their projected diameter is approximately 16.0±1.6 μm (Figure 1(b)). In a hypotonic environment (75 mM glucose solution), the cells’ projected diameter increases to 19.8±2.3 μm because of swelling caused by water entry (Figure 1(c)). The cell-capturing experiment in this study uses 35 of the same type of (but differently sized) cells at various glucose concentrations. Figure 2 shows the results of cells captured between the hexagonal microstructure and cell lysis in varied osmotic conditions, with an electric field preconditioned as 100 Vpp and 1 kHz. When the cells flowed and were captured between the hexagonal microstructure, surplus cells flowed to the

10 μm

Figure 1. Cell images under varied glucose concentrations, (a) 300 mM, (b) 150 mM, (c) 75 mM.

waste outlet (Figure 2(a)). Before an electric field is applied, the cell shape remained intact. Figure 2(b), 2(c), and 2(d) show how cells were disrupted under the electric field and in 300 mM, 150 mM, and 75 mM glucose solutions, respectively. Projected diameter increases after pulsation of 3 s and DNA fluorescence appears, regardless of the glucose concentrations, as indicated by the white arrow in the center of Figure 2. The data at other frequencies (data not shown) are similar. These results are consistent with those in previous studies4; that is, the cell diameter increased under the field intensity of cell lysis. In addition, the intensity of DNA fluorescence increased and the fluorescing range decreased after the pulsation of 30 s in 150 mM and

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Cells captured in capturing zone

Large view of cells captured

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Figure 2. Cells captured and cells lysis under varied concentration glucose solution at electric field frequency of 1 kHz, (a) cells captured in-between the hexagonal microstructure, (b) 300 mM, (c) 150 mM, (d) 75 mM.

300 mM glucose solutions. These results were also observed at the lower frequencies (between 125 Hz and 500 Hz), regardless of the glucose concentrations (data not shown). However, the fluorescing range of post-lysis DNA was larger than the pre-lysis projected range of the cell in a hypotonic environment (75 mM glucose solution) at 1 kHz, despite a pulsation duration of 48 s (the right side of Figure 2(d)). The larger fluorescing range of post-lysis DNA signifies that more DNA was released following lysis.

This result is good for the goal of this work to recover DNA from a single cell. In addition, the post-lytic DNA fluorescing range decrease may be because the DNA-protein complex was condensed after the pulsation because of heating protein (e.g., histones). At low frequency (‹500 Hz), the protein may be heated because of Joule heating with a high field strength (¤3.0 ×105 V/m) in the cell-capturing zone. At a frequency of 1 kHz, the Joule heating still causes the DNA-protein complex to condense except under a hypotonic

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Figure 3. Cell lysis at single-cell level and DNA stretching (75 mM glucose, 100 Vpp, 1 kHz), (a) before the pulsation, (b) cell lysis, (c) injection of proteinase K, (d) DNA fluorescence range increases, (e) DNA flows within the solution, (f) DNA is gradually stretched (The white dotted lines indicate the edges of PDMS).

environment. In a 75 mM glucose solution, the increase in cell volume (swelling) results in more complete disruption of the cell after pulsation (consistent with previous research10), and DNA can be more fully released in solution (the center of Figure 2(d)). Because the post-lytic DNA-protein complex is away from the zone of high field strength (meanwhile lowering the effect of Joule heating at higher frequency), the influence of Joule heating on protein is reduced in the solution. The post-lytic DNA can be more fully released in solution, facilitating follow-up stretch and post-lytic DNA extending and recovery. This experiment concludes that cells release DNA completely after lysis when placed in an environment of 75 mM glucose

solution with an electric field of 100 Vpp and 1 kHz. In addition, the cell capturing efficiency in this study reached 78% (seven cells captured at the nine capturing zone except two capturing zones on the both sides of the microchannel) based on the cell size and morphology under the laminar flow. Figure 3 shows the results of cell lysis and DNA stretching in a 75 mM glucose solution with an electric field of 100 Vpp and 1 kHz. Prior to applying the voltage, the cell was intact and free from YO-PRO-1 staining under fluorescence (Figure 3(a)). The cell membrane began to disrupt upon application of the electric field, and its DNA was stained with fluorescent dye, as Figure 3(b) shows. The cell disrupted completely and

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the DNA fluorescence range increased after proteinase K (98.5 μg/mL) was added and stirred in the flow by a peristaltic pump, as indicated by the white arrow in Figure 3(c). The DNA fluorescence scope became larger when the histone was degraded by proteinase K, as Figure 3(d) shows. The DNA in the solution flowed along the rectangular microstructure (Figure 3(e) and 3(f)), and DNA stretched up to a length larger than 840 μm.

Conclusions This study demonstrates cell lysis at the single-cell level and DNA stretching in a microfluidic device. This study uses electroporation to investigate the effects of varied osmotic environments and electric field frequencies on cell lysis and the subsequently released DNA. The results of this study show that the hexagonal structure in the microchannels facilitates the immobilization of cells (cell capturing efficiency of 78%); at a voltage of 100 Vpp, a higher intensity of electric field (membrane potential greater than 1 V) is produced in the microstructure; electroporation occurs and lyses the cells. Under a hypotonic solution (75 mM glucose) at the electric field frequency of 1 kHz, the lysed cells have relatively larger projected areas because of the increase in cell volume caused by inflow of water. This results in more complete disruption of the cell and greater release of DNA. The data collected from the study signify the most favorable conditions for lysis and DNA release: 75 mM glucose solution, 100 Vpp, and 1 kHz. The DNA fluorescence scope expands following lysis. The addition of histone degraded enzyme and solution flow force contributed to the elongation of the DNA, which stretched to a length exceeding 840 μm. The extended DNA fibers reported in prior studies were only about one hundred micrometers long.11,12 In this study, when the cell is completely disrupted, the DNA in solution flows along the rectangular microstructure without disintegration, as Figure 3 shows. This suggests a way to directly recover and analyze the DNA from a single cell in microchannels. This method can be further applied to genes and chemical cytometry analysis at the single-cell level. Additionally, the stretched DNA segment, which is only about 0.1% of the cell’s complete length, eventually exits the observable zone because it flows along the solution. A suggested improvement is to modify the microchannel surface property so that the negatively charged DNA in the suspension can be fixed, enabling the DNA to elongate further for real-time analysis in a microfluidic device.

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Materials and Methods Electroporation was performed by applying pulsed electric fields to transiently and reversibly permeabilize cells and irreversibly disrupt cell membrane. The voltage level determines the degree of damage to the cell and its internal compartments. The principle of electroporation is that when the electric field is large enough, the inner and outer layers of the membrane compress due to the mutual attraction formed by opposite electrical charges. This ultimately thins the membrane tapers to the point of disruption.13 The transmembrane potential equation is14 ΔVm=-1.5Er cos©

(1)

Where ΔVm is transmembrane potential; E is the applied electric field; r is the radius of the cell; © is the angle between an external electric field and a line normal to the point of interest in the cell membrane. Figure 4 shows the experiment system proposed in this study. The circuit system includes a pair of rod platinum electrodes (with a 1 mm diameter), a function generator (Agilent 33220A, USA) and a power amplifier (NF HSA4012, Japan). The equipment that comprises the image processing system includes an inverted fluorescent microscope (IX71, Olympus, Japan), a TV screen, and a CCD camera (SSC-DC80, Sony, Japan). The cell sample used for this study was U937 (Human Histiocytic Lymphoma cells). Before applying a voltage pulse, fluorescent dyes YO-PRO-1 (491 nm/ 509 nm, Invitrogen) was added to the cell media for the cell’s DNA stain. Proteinase K (Fluka, Japan) was used for histone degradation and the glucose solution was used to adjust the cell osmotic pressure. U937 cells were grown in medium (RPMI 1640) supplemented with 10% (v/v) fetal bovine serum (FBS, Sigma) at 37� C in a humidified 5% CO2 incubator. The final cell

Pt Peristaltic Pump Microfluidic PDMS Device

Microscope Monitor CCD

Figure 4. Schematic diagram of experimental system set up.

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Large view

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V =mmmmmmmmmmmmmm E= 2L1(9W2/W1)+L2

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Figure 5. Schematic diagram of the microfluidic device.

solution for the experiment was 10 μM YO-PRO-1, 1% β-ME (2-Mercaptethanol, to inhibit the oxidation of YO-PRO-1) in varied concentrations of glucose solution. The microfluidic device for the experiment consisted of PDMS (Polydimethylsiloxane, Dow Corning). PDMS is an important material for microfluidic devices because it allows simple soft-lithographic fabrication and has good chemical physical properties.15 Figure 5 shows a schematic diagram of the microfluidic device. The microfluidic device’s hexagonal microstructure array fixes cells at two tips where a high density electric field triggers electroporation. The proposed design includes eight hexagonal microstructures for cell capture in (not shown in Figure 5). The device used in this study had the following dimensions: L1=1.5 mm, L2=60 μm, W1=1 mm, and W2=10 μm. It was manufactured using micro-photolithography to produce a negative photoresist mask pattern SU-8 2025 (MicroChem Corp., USA) microstructure approximately 40 μm thick. The hexagonal microstructure spaces were approximately 10 μm of manufactured PDMS microfluidic device (not shown). The potential drop at individual sections of the microfluidic device is proportional to its resistance within the section. The field strength E can be calculated using the following equations.4

(2)

Where E is the field strength in the narrow section. There are nine cell capturing zones (the gaps between two hexagonal microstructures) in our design. In previous literature, cell lysis occurs when transmembrane potential is greater than 1 V.4,6 To investigate the effects of varied osmotic conditions and electric field frequencies on cell lysis, the experiments in this study used a voltage (V) of 100 Vpp as a preset condition (calculated E ~303 V/mm), with the transmembrane potential greater than 1 V from Eq.(1). The pulse duration of electric field was 100 μsec and the shape of the voltage pulse was square-wave. The frequency of the electric field was 125 Hz, 250 Hz, 500 Hz, or 1 kHz. This study also simulates the electric field using COMSOL Multiphysics 3.3 software based on the following conditions and equation: the uniform external electric field equation, insulation of PDMS microfluidics for the boundary condition, 100 Vpp for the applied field, and 2.1×10-4 S/m for the 150 mM glucose conductivity. The simulation result of the electric field is approximately 3.0×105-3.5×105 V/m (300-350 V/ mm) in the cell capturing zone (data not shown). The electric field results presented by simulated and calculated by Eq.(2) indicate that the field is the highest in the cell capturing zone. The experiment began by placing the microfluidic device on the inverted fluorescent microscope platform, setting up the circuit system and a peristaltic pump (ISMATEC, 11446), and injecting cell solution (flow rate of 10 μL/min) into the entry of the microfluidic device. First, cells were moved by the pump in the microfluidic device. When the cells flowed and were captured between the hexagonal microstructure, surplus cells flowed to the waste outlet, electric field is applied to produce electroporation for cell lysis while fluorescence is used to stain the released DNA. Finally, the post-lytic DNA was stretched by the flowing movement of the solution. Acknowledgements The authors deeply appreciate the financial support by NSC, Taiwan, under the grant number NSC 99-2313-B-415-008-MY2.

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