KR100922232B1 - Method for Direct Patterning of live Cells using Electrohydrodynamic Printing Method and Device therefor - Google Patents

Abstract

The present invention relates to a micropatterning technique of living biological cells. The present invention provides a method for micropatterning bacteria directly, quickly and efficiently using electrohydrodynamic printing, and a substrate including micropatterned biological cells formed thereby. It also provides optimal conditions for the patterning of living bacterial cells. It was confirmed through experiments that the cells patterned by the present invention survive. The present invention provides a bio-ink which can maximize the resolution of the pattern while maximizing the survival rate of living cells, and also provides a biopaper that can be used in tissue engineering, bio-electronic devices, biochips, etc. by culturing live cells by patterning them. do.

EHD, bacterial cells, patterning

Description

Method for direct patterning of live cells using electrohydrodynamic printing method and device therefor}

The present invention relates to a method for patterning live cells and an apparatus therefor, and an article comprising the cells patterned by the method.

Live cell patterning technology is an important technology that can be used to regulate cell growth, develop cell-based high-precision analytical methods, tissue engineering, and develop bio-electronic devices. Micropatterning and local spatial arrangements have been used in cell and tissue engineering to handle and arrange various types of cells on culture plates or scaffolds.

Much research has been done on micropatterning techniques for biomaterials, and PDMS stamping, microfluidic channeling, laser guided direct writing and inkjet printing are emerging technologies in cell patterning and tissue engineering. .

PDMS stamping microchannels patterned (patterning microfluidic channel) method uses a flow path structure of a micro unit for the patterning of cells or biological material [such as PD Noytmer J Aero Sci 2000; 31: 1165-1172; Dertinger SKW et al. Anal Chem 2001; 73 : 1240-1246; Takayama S, etc. Proc Natl Acad Sci 1999; 96: 5545-5548. These methods have the disadvantage of requiring a powerful method for obtaining high resolution capable of single cell patterning or a photolithography process. The use of a photolithography process means that the manufacturing process takes a long time, and a complicated and sophisticated manufacturing process is required.

The laser guided direct writing (LWDW) method also has the advantage of controlling single cells, but the disadvantages are that the process is too slow and the device is too expensive.

Inkjet printing uses commercially available inkjet printers, which is easy to apply, inexpensive, fast and easy to operate. Thus, attempts have been made to pattern bacterial colony arrays using inkjet printers [Roth EA et al. Biomaterials 2004; 25 : 3707-3715; Xu T et al. Biomaterials 2005; 26 : 93-99], printer nozzles are so small that nozzle clogging often occurs, and the heat treatment process for bubble spray causes problems with heat-sensitive biomaterials. The effect of heat on biomaterials is particularly acute when patterning live cells. Therefore, there is an urgent need for the development of a technique capable of quickly and easily micropatterning at low cost without affecting the survival of biological cells that are weak in heat.

On the other hand, one of the direct writing methods is an electro-hydrodynamic (EHD) method, which has some advantages [Regero JD et al. J Aero Sci 2002; 33: 1471-1479. Firstly, this method can produce finely sprayed thin patterns by controlling the supply voltage, feed flow rate, and the physical and electrical properties of the spray. Secondly, since this method is driven by an electrokinetic force induced by a high strength electric field, the nozzle size need not be small if the strength of the surface tension of the injection liquid is appropriate. Therefore, even with a nozzle diameter (100 μm) larger than the ink jet method (about 20 μm), patterning with a line width of about 50 to 200 μm is possible. Large nozzle diameters reduce nozzle clogging, eliminate thermal issues, and allow for the patterning of various biomaterials. Thirdly, this method can create a pattern directly on the substrate without using any lithography process.

The EHD method has been proposed as a core technology for the composition of electronic circuits of non-biomaterials [ Appl such as Lee DY Phys Lett 2007; 90: 081905], 20 nm diameter SiO2 particles were used to pattern line widths of about 60 μm thickness on quarts substrates (Wang DZ et al. J Nanoparticle Research 2005; 3: 301-306). In addition, by using the EHD method has been reported to be made in a pattern with a line width of several tens of micrometers nanoparticles bar Appl etc. [Lee DY Phys A 2006; 82: 671-674. In the case of biomaterials, the present inventors have succeeded in patterning the cell-fixed collagen onto a substrate [Kim HS et al. Experimental Techniques 2007; online publication; Korean Patent Publication No. 10-0740228], Lee JG et al. Have reported that can produce a DNA micropattern [Lee JG et al. Biosensors Bioelectronics 2006; 21: 2240-2247.

However, no research has been reported on the direct patterning of live bacterial cells using the EHD method.

It is a problem to be solved in the present invention to develop a method for micropatterning live bacterial cells directly, quickly and efficiently, and to find an optimal condition for them.

As a result of continuous research in order to solve the above problems, the present inventors have developed a method of efficiently and directly printing living bacterial cells on a membrane filter using a electro-hydrodynamic (EHD) technique in a desired pattern, thereby leading to the present invention. The patterned bacterial cells grow on nutrient agar. The present invention also provides optimum conditions for efficient live cell patterning.

The method using the EHD technique provided by the present invention allows living cells to be kept alive and to produce finely sprayed thin patterns. In addition, even with a nozzle diameter larger than the inkjet method, it is possible to realize patterning of a biomaterial having a line width of about 50 to 200 μm, and to generate a pattern directly on a substrate without using a lithography process. Therefore, the EHD printing method provided by the present invention enables the formation of high resolution patterns of living cells quickly, directly and immediately at low cost.

In addition, since the EHD method according to the present invention is a non-contact method, it can be applied to three-dimensional patterning of living cells.

The present invention provides a bio-ink which can maximize the resolution of the pattern while maximizing the survival rate of living cells, and also provides a biopaper that can be used in tissue engineering, bio-electronic devices, biochips, etc. by culturing live cells by patterning them. do.

The EHD technique according to the present invention can be applied to biosensors of bacterial cells used for gene expression research, new drug screening, environmental monitoring research such as toxicity measurement, and can be used in various fields such as cell engineering or tissue engineering.

In accordance with one aspect of the present invention, there is provided a method of patterning a living cell on a substrate, the method comprising: preparing a solution of the living cell; Providing a needle for injecting the prepared cell solution at a constant hydraulic pressure, and a pin-shaped ground electrode at a distance spaced at a predetermined distance in a position corresponding to the needle; Positioning a membrane filter substrate between the needle and the ground electrode; Applying a voltage between the needle and the ground electrode when the solution is injected from the needle; Attaching the solution to the substrate while moving the substrate in a predetermined pattern shape while the solution is sprayed; And it provides a patterning method of living cells comprising the step of culturing the patterned substrate in contact with nutrient agar media (nutrient agar media).

In addition, the cell solution provides a method for patterning living cells comprising one or more selected from the group consisting of ethylene glycol, phosphate buffer, sodium chloride.

In addition, the ethylene glycol content in the cell solution is 40% or less provides a method for patterning live cells.

In addition, the concentration of the phosphate buffer (phosphate buffer) in the cell solution provides a method for patterning live cells.

In addition, the concentration of the sodium chloride in the cell solution provides a method for patterning live cells that is less than 10mM.

In the cell patterning method, there is provided a method for patterning living cells, wherein a voltage to be applied is 4 to 8.5 kV.

In addition, in the cell patterning method, the living cell is a bacterial cell provides a patterning method of living cells.

The invention also provides a substrate comprising live cells patterned by the method according to the invention.

The invention also provides an article comprising a substrate comprising live cells patterned by the method according to the invention. The product may include a biosensor for detecting reagents for antibiotic development, a biosensor for disease identification, and a product for tissue engineering.

In another aspect, the present invention provides a bio-ink that can maximize the resolution of the pattern while maximizing the survival rate of living cells.

In addition, it provides a biopaper that can be used in tissue engineering, bio-electronic devices, biochips and the like by culturing live cells by patterning.

The inventors of the present invention were able to form the first surviving bacterial colony pattern using EHD printing techniques. Bacterial colony patterns were formed by spraying E. coli cells in broth media using an EHD device controlled by a computer and controller. Bacterial colony patterns were generated by patterning on membrane filters and incubating on agar media. The width of the generated pattern was about 150 um, and it was confirmed that the bacterial cells in the generated pattern were alive. The EHD printing method according to the present invention has been successful in enabling live cells to be patterned at high resolution quickly and inexpensively. In addition, direct, efficient and fast patterning was possible using stable spray droplets with thin line widths through nozzles with larger diameters than conventional methods. Injection solution provided in the present invention can be used as a bio-ink to maximize the resolution of the pattern while maximizing the survival rate of living cells.

In the case of EHD spraying, the spraying pattern is changed according to the applied voltage and the change of the ground shape, so that it is possible to control a series of spraying modes such as dripping mode, micro dripping mode, conical liquid pouring mode, unstable liquid pouring mode, and spray mode. Will be. This spray mode can be variously applied depending on the application. Cone-jet mode is preferable in order to form a predetermined pattern at a desired position without being short and short.

As a substrate for EHD printing, a cover glass used for a polyamide film, a silicon wafer, and a microscope is usually used. In the case of inkjet patterning of live cells, the agar medium may be coated on the cover glass and sprayed, but the EHD method may not be used because of the risk of discharge. Therefore, the present invention attempted to absorb agar medium essential for bacterial culture using a membrane filter. Using a membrane filter, it was confirmed that absorption was performed immediately after injection, and the pattern was formed thinner without spreading sideways. The substrate of the present invention can also be used as a biopaper that can be used for tissue engineering, bio-electronic devices, biochips and the like by patterning and culturing living cells.

Hereinafter, an Example is given and this invention is demonstrated in detail. However, the embodiments of the present invention may be modified in many different forms, and the embodiments of the present invention are provided to more completely explain the present invention to those skilled in the art, and thus the scope of the present invention. Should not be construed as being limited below by them.

< Example  1> Living cell Fine patterning  for Printing  system

The EHD printing system for micropatterning live cells according to the present invention consists of four important parts: a solution injection port, an electrode control unit, a substrate moving unit, and a computer and a power supply for operating the system (FIG. 1).

The injection nozzle consists of a micro syringe pump, a 5ml syringe and a needle. The droplet from which the solution is injected can be varied by the supply flow rate of the solution supplied to the needle tip, which is directly related to the operation of the injection pump. The injection pump (KDscientific, USA) has a feed flow rate as low as 0.06 μl / min. It is possible until. The injection needle is made of stainless steel with 0.15 mm and 0.35 mm outer diameter. The scan pump is mounted to be adjusted in a vertical direction on the substrate surface to adjust the distance between the scan needle, the electrode and the substrate.

The electrode regulator consists of a triaxial translational stage and an acrylic structure as in FIG. The electrode is mounted at the end of this acrylic structure and is adjustable using a three-axis translational stage to align with the tip of the injection needle. The electrode is made of awl-like aluminum and has a diameter of 2 mm. The distance between the needle and the electrode is an important factor that must be adjusted for the optimization of EHD printing.

The substrate moving part is composed of an acrylic frame to fix the substrate and a two-axis translational motor stage (DCT, Korea) modified with a linear motor. The two-axis translational motor stage can move a distance of at least 2㎛ and the moving speed is Adjustable from 125mm / min down to 500mm / mim. The substrate moving part is fixed to the substrate to reflect the movement of the motorized stage, and the two-axis translational motorized stage is automatically controlled by a computer and a control program (DCT, Korea).

In the present invention, the substrate uses a membrane filter. Available membrane filters include Durapore membranes ( Milipore , USA ), and the like . In the inkjet technique, cover glass coated with nutrient agar was used as a substrate for bacterial cell patterning. However, the inventors of the present invention determined that the EHD printing technique using a high DC voltage is not suitable as a material of the substrate positioned between the needle and the electrode because corona discharge is generated by the high electrical conductivity of the nutrient agar. HVLP 04700, Durapore membrane, Millipore, USA) was used as the substrate material. The membrane filter acts as a nonconductor, has hydrophilic and biocompatible properties, and is made of millions of holes with a diameter of about 0.45 μm. This perforation structure facilitates the uptake of agar media for bacterial cell growth during incubation. Commercially available membrane filters were circular in shape with a diameter of 47 mm and a thickness of 125 µm, but were cut to fit the cover glass (24 mm x 60 mm x 1 mm).

The power supply unit (KSC, Korea) can supply a current voltage of up to 30kV. In addition, the current from this device is fixed at a current of several uA to prevent corona discharge and to generate attraction by a nearly pure electric field. Under these high voltage environments, even the slightest current can be harmful to bacterial cells, and the supply voltage is one of the most important factors that determine the formation of spray droplets.

< Example  2> Living cells Patterning  Solution

As an example of a solution for patterning live cells by EHD spraying, a solution containing the bacterium E. coli was prepared. E. coli (ATCC 47013: antibiotic resistant strain) was applied to Difco ^TM Broth (BD, USA) was incubated at 37 ° C. for 24 hours, and 1 μl of ampicillin (ampicillin; Sigma Aldrich cat. A0166) was added per mL. The concentration of all bacterial solutions was fixed at 2.5 × 10 ⁸ cells / ml using a spectrophotometer. This solution may include glycerin, ethylene glycol, phosphate buffer, sodium chloride, and the like.

EHD printing methods are affected by the viscosity of the solution, the electrical conductivity of the solution, the flow rate of the solution and the applied voltage. Higher viscosities allow spraying in cone-jet mode with smaller diameter stable jets. For this purpose, gelatin, glycerin, ethylene glycol and the like can be added. In addition, sodium chloride may be added to impart a predetermined electrical conductivity to the patterning solution. In addition, phosphate buffer or sodium chloride may be added to alleviate cell survival and toxicity of ethylene glycol .

< Example  3> Living cell EHD Printing  By technique Patterning  process

The process of patterning live cells on a substrate using an EHD printing apparatus is as follows. An appropriate voltage is applied to the needle through the electrode, and the live cell solution to be patterned is sprayed from the needle using a micro injection pump while moving the substrate according to the previously input pattern shape.

Prior to filling the syringe with the solution, the syringe and needle were sterilized and dried with 70% alcohol. The distance between the needle and the electrode is closely related to the generation of electric and electric fields. By Coulomb's law, the amount of electrostatic force between two electrodes is inversely proportional to the square of the distance between charged electrodes. However, as a result of several experiments, the droplet injection tended to become unstable when the distance between the two electrodes was too close. This may be due to strong polarization on the cover glass surface, which occurs when the two electrodes are too close to the cover glass. In this experiment, the distance between the needle and the electrode was 3mm, and the substrate was placed in the center of these two electrodes. The distance between the needle and the substrate was 1mm, and the distance between the electrode and the electrode was 1mm. By adjusting the biaxial translational vibration stage, droplets of E. coli mixed solution were sprayed onto the membrane filter surface along a path. A Motion Control Unit (MCU) program was used to set the path. The supply flow rate was 1.5 µl / min and the stage moving speed was 500 mm / min. The applied voltage was 6.5kV.

< Example  4> Patterned  Bacteria Colony  culture

Nutrient agar media (Difco, USA) was used for the cultivation of the patterned bacteria on the membrane filter. Ampicillin was treated on the surface after the medium was solidified. Printed membrane filters were attached onto the solid batch and incubated at 37 ° C. for 24 hours. Because membrane filters can absorb nutrient agar, bacterial cells can survive during culture if the patterned bacteria survive intact.

E. coli after bacterial colonies are formed Colonoys were observed with a confocal laser scanning microscope and a wide-field optical microscope. All images were stored and the length, line width and thickness were measured.

< Example  5> Patterning  Determination of optimal applied voltage in the process

The optimum condition for making minimal droplets when varying the applied voltage was found. The electrokinetic force is too weak when the applied voltage is too low and becomes stronger as the voltage is increased to overcome the surface tension of the droplet at the nozzle tip. However, if the applied voltage exceeds the optimum condition, the dripping motion becomes unstable and the droplets become large.

Figure 2 is the average size of the droplets when changing the applied voltage from 3kV to 8.5kV when using a glass substrate or Kapton® polyimide film substrate. The average size of the droplets was 120-950 um. From the above results, it can be seen that 4 to 8.5 kV is preferable and 4 kV -6.5 kV is more preferable to form the smallest stable droplet.

< Example  7> Living cells  Optimal concentration of viscous adducts in solution

Viscous material of viable cell solution, ethylene glycol (EG) had higher resolution in terms of droplet size than other viscous materials such as glycerin. EG is used as an antifreeze for long-term preservation of cells, but is generally known to be toxic to live cells. Cell survival under antifreeze decreases over time. The survival time of the cells was checked before starting the spray experiment. Ethylene glycol (EG) was added to the bacterial solution prepared in Example 2 to prepare a mixture of E. coli, distilled water (DW) and ethylene glycol ( E. coli- EG-DW). The volume ratio of EG of the E. coli -EG-DW mixture was prepared in six steps (0, 20, 40, 60, 80, and 100 vol%). Observations were performed to determine cell survival time under E. coli-EG-DW solution. Normally, cells begin to clump together in a harsh and toxic environment. To identify the aggregation of cells, fluorescent staining (SYTO9, Molecular Probe) was treated with E. coli before making the solution. This observation confirmed that aggregation of cells occurred as soon as the solution was mixed under the conditions of 100 vol% EG mixed solution. Aggregation of cells was not observed in a solution containing less than 60 vol% EG until 4 hours after solution mixing. This result confirmed that cells survived without killing under EG solution conditions within at least 1 hour.

In order to find the optimal EG mixing ratio for EHD printing, the printing results according to the volume ratio of EG were compared.

Printed E. coli could be incubated with linear or circular colonies by changing the composition of EG. The pattern was designed by controlling the two-axis translational motor stage through the MCU program. When implementing the ideally designed linear pattern in Figure 3 can be seen that the colony pattern is changed according to different EG composition changes.

In (A) of FIG. 3, when the EG composition was injected with a solution containing 0 vol%, that is, EG, it was observed that coarse colonies were formed after the culture. In this pattern, there are often unpatterned portions and the line width is thicker than the EHD pattern that forms the thinnest line width. This incomplete pattern may be due to the low viscosity of the bacterial solution containing only DW and not containing EG.

At 20 vol% (B of FIG. 3) and 40 vol% (C of FIG. 3), the composition of EG can be confirmed that a linear pattern of thin and dense bacterial colonies was formed. At 60 vol% EG composition (FIG. 3D), the colony linewidth was thicker than in B and C. Therefore, it can be predicted that optimal conditions exist at less than 60 vol% of EG composition.

At an EG composition of 80 vol% (FIG. 3E), the colony pattern was very disturbed and formed into large droplets, far from the intended linear pattern, and the cells may have blocked the needle. Because the aggregated cell mass may be larger than 150 μm, the inner diameter of the needle.

The pattern height was used through a confocal laser scanning microscope to analyze the performance of the EHD printing technique. In FIG. 4G, a three-dimensional photograph of a colony pattern can be seen, and a graph below the three-dimensional photograph shows a cross-sectional height measured along the red line indicated by G. FIG. These three-dimensional images confirmed that the printed E. coli was successfully incubated on membrane filters and grew well into colonies approximately 50 μm high.

In addition, the length and height of the linear pattern changed according to the EG composition was measured under a microscope. Measurement data were analyzed from eight 15 mm bacterial colony linear patterns (total 120 mm). In the graph (A) of FIG. 5, the length of the linear colony pattern is expressed as the ratio of the path created by the EHD technique to the actual implemented colony region. In this result, the highest implementation ratio was shown when the EG composition was about 20 and 40 vol%, and the thinnest line width was realized when the EG composition was 40 vol% (Fig. 5- (B)). Therefore, it was found that the linear colony pattern with 40 vol% EG composition was the best result. Under these conditions, the average line width was about 160 μm.

The width of the pattern varied with the EG concentration. As the EG concentration increases, the viscosity increases. But the conductivity decreases. This condition is advantageous for a stable cone-jet mode for producing clear patterns of bacterial cells. However, higher concentrations of EG result in cell aggregation.

< Example  8> Living cells  Sodium chloride in solution or Of phosphate buffer  Determination of Optimal Concentration

In live cell solutions, sodium chloride or phosphate buffer may be added to create a viable environment for the cells and to reduce the toxicity of ethylene glycol. However, they can affect the conductivity of the solution. The electrical conductivity and viscosity of the printing solution are two important parameters that determine the stability of the spray droplets. The lower the conductivity and the higher the viscosity, the more stable the cone-jet tends to be produced [Master Res Inno 2002, et al. 92 -95]. Therefore, in the present invention, the optimum concentration of sodium chloride and phosphate buffer solution was investigated. Viscosity and electrical conductivity were measured by viscosity meter (SV-10, AND) and conductivity meter (F-54W, Horiba), respectively.

The bacterial solution of Example 2 was treated with phosphate buffer (PB), sodium chloride (NaCl), and E. coli , distilled water, phosphate buffer mixture ( E. coli- EG-PB-DW), E. coli , Distilled water and sodium chloride mixture ( E. coli -EG-NaCl-DW) were made.

Phosphate buffer of 0.1M, pH 7.4 was prepared by dissolving 0.9 g of Na ₂ HPO ₄ (anhydrous, Phosphate dibasic) and 3.2 g of NaH ₂ PO ₄ (anhydrous, Phosphate monobasic) in distilled water (DW).

Another mixture of PB and NaCl in six different concentrations (0, 10, 20, 40, 80, and 160 mM) ( E. coli -EG-PB-DW, and E. coli -EG-NaCl- DW) was also prepared. The E.coli -EG-PB-DW, with the E. coli -EG-NaCl-DW viscosity of the mixture and the electrical conductivity are shown in Table 1, 2, 3, 4.

Comparing the properties of this mixture at a certain ratio, it can be seen that increasing the concentration of EG causes a decrease in the conductivity of the solution and an increase in the viscosity.

The inventors of the present invention compared the pattern formation according to different EG and PB composition ratio. 6 is a photograph taken of the result of the optical microscope. Photos classified into three types in the horizontal direction represent 20, 40, and 60 vol% of EG composition in order from the left. Six types classified in the vertical direction show P, 0, 20, 40, 80, and 160mM in order from the top. Added. No significant change was observed with the EG composition, and the pattern was severely disturbed as the amount of PB added increased. This result shows that the addition of PB increases the electrical conductivity, making the spray droplets unstable, which in turn disrupted colony pattern formation. In FIG. 7, the line width and the implementation ratio according to each condition are represented as a graph.

Similar to the E. coli -EG-PB-DW solution spray experiment,

Sodium chloride (NaCl) was added instead of PB to improve. Similarly, three kinds of EG

A total of 18 kinds of mixtures were prepared using the composition ratio and the amount of sodium chloride added in six steps. 8 is a photograph of the pattern results according to different EG composition ratio and the amount of sodium chloride added. Horizontal direction means EG composition ratio of 20, 40, 60 vol% and vertical direction means sodium chloride addition amount of 0, 20, 40, 80, 160mM. As with the experiment of PB addition amount change, colony pattern formation by the EG composition ratio was insignificant and the instability of the pattern increased as the amount of sodium chloride added increased. In FIG. 9, the line width and the implementation ratio of each composition are represented in a graph.

In this experiment, solutions of all compositions were realized as bacterial colony patterns that survived after printing and incubation, but colony patterns of solutions with relatively high PB and sodium chloride content were embodied in unstable or overlapping bold circular shapes (FIGS. 6 and 8). . These results indicate that the addition of PB and sodium chloride increases the electrical conductivity of the solution itself, although it helps to create an environment favorable for the survival of bacterial cells. This increase in electrical conductivity leads to instability of the spray droplets. However, the addition of PB and sodium chloride does not appear to affect the viscosity of the solution (Figures 6 and 8). Because EG is the most important substance that determines the viscosity characteristics of a solution.

The results of Figures 6-9 are very closely related to the electrical conductivity and the measurements of the viscosity of Tables 1-4. In the table, it can be seen that as the amount of PB and sodium chloride added to the printing solution increases, the electrical conductivity increases rapidly, and the line width and the implementation cost change are closely related to the change of the content.

As a result, the optimal E. coli-EG-PB-NaCl-DW printing solution composition ratio was determined by this experimental method. We found that the optimal results were obtained when the EG composition was less than 40 vol% and the minimum amount of PB and sodium chloride in the patterning.

< Example  9> Living cell  Intricate patterns Printing  Broken heart

A spiral linear pattern was formed as an example of complex pattern printing of living cells (FIG. 10). Immediately after the E. coli solution was sprayed on the membrane filter by EHD, the solution was absorbed and the agar media was absorbed. Therefore, it was difficult to visually identify the pattern. The bright spots are fluorescently stained bacterial cells. Immediately after being patterned on the membrane filter surface via EHD equipment, the array of bacterial cells formed a pattern with the intended design. Bacterial cells injected without fluorescence staining were grown for visual identification by forming colonies through incubation at 37 ° C. for 24 hours. 10 is an optical photomicrograph of the spraying result of 40 vol% E. coli-EG-DW solution of EG composition by EHD direct patterning.

The general accuracy of the EHD printing technique was analyzed with experimental intent. Based on the experimental conditions, a linear colony pattern with a minimum line width of about 165 μm was created using a flow rate of 1.5 μl / min, a substrate transfer rate of 500 mm / min, and an E. coli-EG-DW solution with an EG composition of 40 vol%. made. The formation of this pattern affects the supply flow rate and supply voltage of the injection pump, the thickness of the injection needle, the viscosity and the electrical conductivity of the solution.

The EHD device of the present invention can produce a linear pattern of 10 mm per second. Within 12 minutes, a linear pattern of 145 line widths of 165 μm and a length of 60 mm can be created in the microscope cover glass. If a dot pattern is formed at the same speed, 2175 colony points with a diameter of 165 μm can be made in one minute. Existing equipment (genetix Qbot) can generate 100,000 colonies per hour, and inkjet systems can produce 50,000 colonies of 500 μm diameter per minute [Xu T. et al., Biotechnology Bioengineering 2004; 85 (1): 29 -33]. EHD can produce faster colony patterns than conventional equipment, and can be somewhat slower than inkjet, but can make the diameter of the colony smaller, despite the larger nozzle diameter than inkjet.

1 is a schematic diagram of an EHD printing system of the present invention.

2 is an average size of droplets sprayed when the applied voltage is changed.

Figure 3 is a E. coli with E. coli -EG DW-solution composition was changed EG Picture of colony pattern. (A) 0 vol% EG, 100 vol% DW; (B) 20 vol% EG; (C) 40 vol% EG; (D) 60 vol% EG; (E) 80 vol% EG; (F) 100 vol% EG

4 is a confocal laser scanning micrograph of (G) 40 vol% EG solution pattern.

Figure 5 shows the results of EHD printing using E. coli -EG-DW solution (A) the ratio of the intended pattern to the actual implemented pattern, (B) the average line width of the implemented pattern. The error range represents the standard deviation (n = 8).

     6 is a linear colony pattern photograph of an E. coli solution containing ethylene glycol and phosphate buffer.

(A) 20 vol% EG, 0 mM PB; (B) 20 vol% EG, 10 mM PB; (C) 20 vol% EG, 20 mM PB; (D) 20 vol% EG, 40 mM PB; (E) 20 vol% EG, 80 mM PB; (F) 20 vol% EG, 160 mM PB; (G) 40 vol% EG, 0 mM PB; (H) 40 vol% EG, 10 mM PB; (I) 40 vol% EG, 20 mM PB; (J) 40 vol% EG, 40 mM PB; (K) 40 vol% EG, 80 mM PB; (L) 40 vol% EG, 160 mM PB; (M) 60 vol% EG, 0 mM PB; (N) 60 vol% EG, 10 mM PB; (O) 60 vol% EG, 20 mM PB; (P) 60 vol% EG, 40 mM PB; (Q) 60 vol% EG, 80 mM PB; (R) 60 vol% EG, 160 mM PB; Black line means 2 mm.

7 shows EHD printing results using E. coli -EG-PB-DW solution (A) the ratio of the intended pattern to the actual implemented pattern, (B) the average line width of the implemented pattern. The error range represents the standard deviation (n = 8).

8 is a linear colony pattern photograph of an E. coli solution containing ethylene glycol and sodium chloride.

(A) 20 vol% EG, 0 mM NaCl; (B) 20 vol% EG, 10 mM NaCl; (C) 20 vol% EG, 20 mM NaCl; (D) 20 vol% EG, 40 mM NaCl; (E) 20 vol% EG, 80 mM NaCl; (F) 20 vol% EG, 160 mM NaCl; (G) 40 vol% EG, 0 mM NaCl; (H) 40 vol% EG, 10 mM NaCl; (I) 40 vol% EG, 20 mM NaCl; (J) 40 vol% EG, 40 mM NaCl; (K) 40 vol% EG, 80 mM NaCl; (L) 40 vol% EG, 160 mM NaCl;

(M) 60 vol% EG, 0 mM NaCl; (N) 60 vol% EG, 10 mM NaCl; (O) 60 vol% EG, 20 mM NaCl; (P) 60 vol% EG, 40 mM NaCl; (Q) 60 vol% EG, 80 mM PB; (R) 60 vol% EG, 160 mM NaCl; black line means 2 mm.

9 shows the results of EHD printing using E. coli -EG-NaCl-DW solution. The implementation ratio of (A) the intended pattern to the actual implemented pattern, (B) the average line width of the implemented pattern. The error range represents the standard deviation (n = 8).

10 shows a pattern implementation using an E. coli -EG-DW solution having an EG composition of 40 vol%. (A) Fluorescently stained bacterial photos before injection, (B) After culture of patterned bacteria in the same shape. Black line means 2mm.

Claims (10)

In a method for patterning live cells on a substrate, Preparing a solution of the living cells; Providing a needle for injecting the prepared cell solution at a constant hydraulic pressure, and a pin-shaped ground electrode at a distance spaced at a predetermined distance in a position corresponding to the needle; Positioning a membrane filter substrate between the needle and the ground electrode; Applying a voltage between the needle and the ground electrode when the solution is injected from the needle; Attaching the solution to the substrate while moving the substrate in a predetermined pattern shape while the solution is sprayed; And And contacting the substrate on which the pattern is formed with nutrient agar media to incubate. The method of claim 1, wherein the cell solution comprises one or more selected from the group consisting of ethylene glycol, phosphate buffer, sodium chloride. The method of claim 2, wherein the ethylene glycol content in the cell solution is 40% or less. The method of claim 2, wherein the concentration of phosphate buffer in the cell solution is 10 mM or less. The method of claim 2, wherein the concentration of sodium chloride in the cell solution is 10 mM or less. The method of claim 1, wherein the applied voltage is between 4 and 8.5 kV. The method of claim 1, wherein the living cells are bacterial cells. A substrate comprising live cells patterned by the method of claim 1. A biosensor comprising the substrate of claim 8. Viable cell patterning solution containing living cells, up to 40% by volume of ethylene glycol, up to 10 mM phosphate buffer, up to 10 mM sodium chloride

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