KR20140076212A - Microfluidic Device Having Concentration Gradient and Temperature Gradient at One Time - Google Patents

Abstract

The present invention relates to a microfluidic device in which a concentration gradient and a temperature gradient are simultaneously created in a detection channel of a microchannel and, more specifically, to a microfluidic device comprising a microfluidic channel for a sample and a pair of microfluidic channels for tempreature adjustment. The microfluidic channel for a sample includes: two inlets; a gradient forming region for separating each fluid which is injected into the inlets, into multiple microchannels having a different concentration gradient; a detection channel for joining and moving the fluid separated into the multiple microchannels after passing the gradient forming region; and an outlet for discharging the fluid passed the detection channel. The pair of microfluidic channels for temperature adjustment, wherein each channel is formed in parallel on both sides of the detection channel of the microfluidic channel for a sample by being spaced out in a prefixed interval with the detection channel, and includes: an inlet; an outlet; and a microchannel through which a fluid injected into the inlet moved to the outlet, such that heating mediums having a different temperature are circulated.

Description

(Microfluidic Device Having Concentration Gradient and Temperature Gradient at One Time)

The present invention relates to a microfluidic device in which a concentration gradient and a temperature gradient are simultaneously generated in a detection channel of a microchannel.

Microfluidics deals with fluid flow limited to a few millimeters or less, and has received attention in a variety of fields, including engineering, physics, chemistry, micro-engineering, and biotechnology. Fluids in microsystems are characterized by many variables in the system, such as surface tension, energy dissipation, and resistance, which are different from conventional macroscopic fluids. Microfluidics occurred in the early 1980s and are used in the fields of inkjet printer heads, DNA chips, lab-on-a-chips, and the like.

Recently, microfluidic devices using microfluidics have been used in various fields along with the development of microfabrication technology. The microfluidic device is capable of producing microparticles or performing various reactions and analyzes using the flow of fluid in the microfluidic channel having a width and a depth of several tens to several hundreds of micrometers. It is possible to produce various types of monodisperse microparticles such as fibers, capsules, and beads by modifying the flow rate of the fluid in the microchannel, the combination of the continuous phase and the sample, and the design of the microchannel. Also, in the microchannel, the diffusion rate of the substance, the heat transfer rate is very fast, and the active area per unit volume is very large. Thus, desired product can be obtained at a high yield using a very small amount of catalyst and reactant at a uniform temperature and pressure gradient The fine chemical products using them have recently been commercialized by large chemical companies such as Novartis, Merck and Degussa. Particularly, since there is an advantage that reaction and analysis can be performed using a small amount of materials in the micro channel, many studies have been conducted on applications in biotechnology where expensive reagents are required or analysis is required with a small amount of sample .

The Applicant discloses a method of measuring the sample sensitivity of an biofilm by injecting a sample to form a concentration gradient in a microfluidic channel after forming a biofilm in a microfluidic channel of a microfluidic device, There is one. According to the apparatus, since the concentration gradient is stably formed in the microfluidic device in the microfluidic device, the sensitivity of the biofilm to the concentration of the sample can be easily and reproducibly measured by only one experiment, And can be used efficiently.

In addition to "concentration", "temperature" is one of the important parameters for chemical reactions and biological growth. In chemical reactions, the reaction rate is affected by temperature and the reaction products are also changed. Temperature is also one of the main stressors in the growth of "organisms", which can cause the growth rate to change depending on temperature and also cause death. The applicant of the present application has proposed a device capable of measuring the influence of temperature on the reaction and analysis in the micro flow path in a simple and reproducible manner by generating a temperature gradient in the micro flow path of a microfluidic device, .

In particular, in the case of an organism, the actual growth environment is influenced not only by one condition but also by various conditions. When using the device, it is easy to determine how the concentration or temperature affects the chemical reaction or the growth of an organism (biofilm), but there are limitations to evaluate which factors have a greater impact on competitive conditions .

Patent No. 10-1210590 Patent Application No. 10-2012-38051

SUMMARY OF THE INVENTION It is an object of the present invention to provide a microfluidic device in which a concentration gradient and a temperature gradient are simultaneously generated in a detection channel of a microchannel.

According to an aspect of the present invention, A gradient forming region for separating each fluid injected into the injection port into a plurality of microchannels having different concentration gradients; A detection channel through which a fluid separated into a plurality of microchannels passing through the gradient forming region is joined and moved; And a discharge port through which the fluid that has passed through the detection channel is discharged; and an injection port formed at both sides of the detection channel of the sample microchannel, parallel to the detection channel at a predetermined interval, respectively; outlet; And a pair of temperature-controlling micro-flow paths each of which is composed of a micro-flow path in which the fluid injected into the injection port is moved to the discharge port and the heat of the other temperature is circulated, so that a concentration gradient and a temperature gradient are simultaneously generated in the detection channel To a microfluidic device.

The microfluidic device of the present invention can be applied to various types of materials such as wet etching, dry etching, lithography, molding, milling, turning, drilling, punching, laser micromachining and the like using various kinds of materials such as silicon, glass, metal, And can be manufactured using the same fine processing technology.

The sample microchannel is substantially a microchannel into which a sample is injected, and a concentration gradient and a temperature gradient are concurrently formed in the detection channel of the sample microchannel. Of course, it is of course possible that only a concentration gradient or a temperature gradient may be made as necessary.

The outlet serves to discharge the sample to be injected, but may be used to inject the sample into the detection channel, if necessary. For example, when analyzing the influence of the composition or characteristics of the medium on the formation of biofilm, biofilm formation is first observed by applying a microorganism to the detection channel and then flowing the composition with a gradient and / or temperature. If the microorganisms are injected into the detection channel through the injection port, if microorganisms adhere to the gradient generation area where the flow path is narrow and narrow, the flow of the medium injected into the gradient generation area is influenced and the gradient generation is not smooth . Therefore, it is preferable that microorganisms are injected through the discharge port, not the injection port, while blocking the boundary between the gradient generation region and the detection channel, thereby inhibiting microorganisms from entering the gradient generation region. To this end, it is preferable that the microfluidic device of the present invention be capable of forming a valve at a boundary portion between the gradient forming region and the detection channel so as to cut off the flow path to the gradient generating region when necessary.

The valve may be a conventional valve that can block the flow path by mechanical means, or may be fabricated in the form of a dual chip with a drive channel above or below the fluid channel as shown in FIG. Although FIG. 3 illustrates an example in which the drive channel is formed on the fluid channel, it is of course possible to configure the drive channel to be formed below. The driving channel may have a micro channel at a portion where a valve is to be formed and a portion forming a boundary between the driving channel and the fluid channel may be formed of a material which is easy to swell in a flexible material or solvent, Is injected and pressure is applied or an efficient solvent is caused to flow to swell, thereby blocking the microchannel of the fluid channel so that the valve is locked. The valve can be opened only by removing pressure on the drive channel or by not spilling solvent.

The width of the detection channel may be designed in a range suitable for the purpose of the experiment, but it is preferably 100 to 1000 microns. If the width of the microchannel is too narrow, it is difficult to inject the microchannel in the case of a viscous specimen. If the width of the microchannel is too wide, it is difficult to form a concentration gradient due to stable laminar flow. In addition, since the width of the micro channel is too wide, it is disadvantageous to observe when it is out of the range of the detection window of the microscope.

The gradient region is a region in which a concentration gradient generated by mixing the sample injected from the two injection ports and the moving phase can be linearly formed in the detection channel. More specifically, the sample injected from each injection port and the mobile phase are mixed to divide the sample into a plurality of flow streams having a concentration gradient, and they are merged in the detection channel so that the concentration gradient of the sample in the detection channel is linear do. Since the composition of the gradient forming region can be variously configured by referring to the prior art, detailed descriptions thereof are omitted in the present invention (NL Jeon, H. Baskaran, SKW Dertinger, GM Whitesides, LVD Water and M. Toner, Nat. J. Diao, L. Young, S. Kim, EA Fogarty, SM Heilman, " Biotechnol., 2002,20, 826 .; P. Hung, P. Lee and LP Lee, Biotechnol. , P. Zhou, ML Shuler, M. Wu and MP DeLisa, Lab Chip, 2006, 6, 381.)

The temperature-controlling microchannels are formed parallel to the detection channel at predetermined intervals on both sides of the detection channel, and circulate the heat of different temperatures so that a temperature gradient is formed in the detection channel. The temperature gradient of the detection channel is influenced by the interval between the detection channel and the temperature control microchannel and the temperature and flow rate of the heat of the microchannel through the temperature control microchannel. In the present invention, the term "fruit material " encompasses all of the materials used for temperature control irrespective of the temperature of the medium itself as a heat transfer medium. In other words, it is a concluded 'fruit' which is a medium to raise the temperature by applying heat to the sample, and a 'refrigerant' which is a medium to lower the temperature by taking heat from the sample. In the examples, a temperature gradient of 10 to 30 DEG C was produced in the microfluidic channel by using the fruit of -14 DEG C and 50 DEG C, but it is not limited thereto. That is, using a lower temperature than -14 ° C, or by increasing the circulation rate, a temperature lower than 10 ° C could be achieved, and by using higher temperature berries or lowering the circulation rate, Lt; 0 > C. In order to efficiently perform the heat exchange between the temperature control channel and the detection channel, it is more advantageous to have a wide width of the temperature control channel. However, the detailed width, interval, temperature of the fruit, You will be able to choose.

As the gap between the detection channel and the temperature control microchannel becomes narrower, the heat exchange between the material circulating in the temperature control microchannel and the sample of the detection channel is efficiently performed. However, if the gap is too narrow, . On the other hand, if the distance between the detection channel and the temperature control microchannel is too wide, the heat exchange efficiency becomes low. Therefore, the interval between the detection channel and the temperature control microchannel is preferably 10 to 1,500 microns.

As described above, the concentration gradient and the temperature gradient can be simultaneously generated in the detection channel of the microfluidic device of the microfluidic device of the present invention, and various conditions (nutrients, pH, Antibiotics) and temperature effects can be measured simultaneously and reproducibly. Thus, conditions such as the production of fine chemicals or the biotechnological treatment of environmental contaminants can be used to optimize. In addition, the influence of concentration and temperature on biofilm growth can be competitively measured, and it can be used to analyze under what conditions biofilm growth is more affected.

1 is a schematic view and a photograph of a microfluidic device according to an embodiment of the present invention.
2 is a schematic diagram of a microfluidic device according to another embodiment of the present invention.
FIG. 3 is a schematic view showing the operation principle of the valve in the embodiment of FIG. 2, and a microphotograph showing the operating state of the actual valve. FIG.
FIG. 4 is a graph showing that a temperature gradient is linearly generated in a microfluidic device according to an embodiment of the present invention. FIG.
FIG. 5 is a graph showing that a concentration gradient is linearly generated in a microfluidic device according to an embodiment of the present invention. FIG.

BRIEF DESCRIPTION OF THE DRAWINGS The invention will be described in more detail with reference to the accompanying drawings and examples. However, the drawings and the embodiments are only illustrative of the contents and scope of the technical idea of the present invention, and the technical scope of the present invention is not limited or changed. It will be apparent to those skilled in the art that various changes and modifications can be made within the scope of the technical idea of the present invention based on these examples.

Example 1: Fabrication of microfluidic devices

The microfluidic device is formed by soft lithography, as shown in Fig. 1, one microchannel having two inlets and a gradient generator, a detection channel and one outlet, And two microchannels having microchannels parallel to the detection channels on both sides and having one inlet and one outlet for each microchannel. In FIG. 1, the detection channel of the microchannel formed at the center is a sample microchannel in which a concentration gradient and a temperature gradient of the sample to be injected are formed. The microchannels on both sides are respectively connected to a refrigerant It is a micro flow path for temperature control in which fruit is injected.

Specifically, a negative photoresist (SU-8, Microchem Co., USA) was evenly coated on a silicon wafer and spin coated at 1,000 rpm for 30 seconds to coat the photoresist with a height of 60 탆. UV was irradiated to the photosensitive agent through a mask having a fine channel shape as shown in Fig. 1 to prepare a master mold having an opposite shape to the channel. 1, the width of the detection channel was 0.9 mm, the width of the temperature control channel was 3 mm, and the interval between the detection channel and each temperature control channel was 1.3 mm.

A 10: 1 (w / w) mixture of the base of PDMS (Sylgard 184; Dow Corning, Midland, Mich.) And a curing agent was poured into the master mold, And then cured to prepare a PDMS mold having a microchannel of a desired shape. A microfluidic device was fabricated by adhering a glass substrate to the PDMS mold through oxygen plasma treatment (PDC-002, Harrick, USA).

Example 2: Fabrication of a microfluidic device including a valve

In addition to the structure of FIG. 1, a microfluidic device having the structure of FIG. 2 including a valve at the boundary between the gradient generation region and the detection channel was fabricated.

First, a PDMS mold having a microchannel of a desired shape was fabricated in the same manner as in Example 1 except that the base and the curing agent were cured at 65 ° C for 40 minutes at a ratio of 20: 1 (w / w) Respectively.

In the same manner, a PDMS mold having a valve structure shown in FIG. 2 was prepared by curing at 65 ° C for 1 hour at a base to curing agent ratio of 10: 1 (w / w). At this time, the width of the drive channel for forming the valve was 70 mu m.

A PDMS mold of the drive channel was laminated on the PDMS mold of the fluid channel manufactured by the above method using a microscope so that the drive channel was located at a desired position. At this time, the flow path formed in the PDMS of the fluid channel is positioned in the direction opposite to the coupling surface, and the flow path formed in the PDMS of the drive channel is positioned in the direction of the coupling surface. The laminated PDMS molds were cured for an additional 12 hours at 65 ° C., and the glass substrate was adhered to the PDMS mold surface of the fluid channel through oxygen plasma treatment (PDC-002, Harrick, USA) to prepare a microfluidic device.

FIG. 3 is a schematic view showing the operation principle of the valve and a photomicrograph showing the actual operation. In this embodiment, since the ratio of the curing agent in the fluid channel is lower than that in the driving channel, the PDMS at the interface between the driving channel and the fluid channel has higher flexibility and swelling degree with respect to the solvent as compared with the flow channel constituting the driving channel.

Accordingly, by injecting a gas into the drive channel to increase the pressure or injecting a solvent capable of swelling the channel, the interface is swollen as shown in FIG. 3 (A) to block the fluid channel, thereby closing the valve.

FIG. 3 (B) is a microphotograph before and after injecting nitrogen into the drive channel of the microfluidic device of this embodiment at a pressure of 0.5 MPa. More specifically, the drive channel was self-programmed using the Lab view software (Lab view 8.5, National instruments) and operated manually or automatically on the computer. At this time, a self-assembled solenoid-valve connected to the nitrogen gas to open and close the valve of the drive channel was connected to a tube (Tygon tube, ID 0.020in, OD 0.060in, Saint-gobain PPL Corp.) and syringe needle (Niddle, 21G, Becton dickinson ) Was used to connect the drive channel. Normal nitrogen was used as the nitrogen gas, and the pressure was adjusted to 0.5 MPa using a regulator (GHN-3, CHN-4, Chiyoda). In FIG. 3 (B), it is confirmed that the valve is effectively blocked by the injection of nitrogen.

Example 3: Temperature gradient calibration curve creation

The temperature gradient was observed in the detection channel of the microfluidic device fabricated in Example 1 using a temperature sensitive fluorescent dye, Rhodamine B, and visualized, and a calibration curve for the temperature gradient was obtained. ≪ / RTI >

Using a circulation pump, a coolant of -14 ° C was fed to one side of the temperature control channel and a solution of 50 ° C was fed to the other side at a rate of 0.1 ml / min and 0.4 ml / min, respectively. 10 minutes after the injection of the refrigerant and the fruit, 0.1 mM rhodamine B solution prepared by using 50 mM potassium phosphate buffer (pH 7.5) was added to the detection channel through the both inlet ports of the sample microchannel at 5 μl / min ^-1 .

The fluorescence of rhodamine B was measured using an inverted fluorescence microscope equipped with a CCD camera (Coolsnap cf, Photometrics, USA) in a detection channel, TE2000, Nikon, Japan), and the fluorescence intensity of 0.1 mM rhodamine B at each temperature The temperature gradient in the channel was obtained from the standard curve. 4 (a) is a standard curve of the fluorescence intensity according to the temperature of 0.1 mM Rhodamine B, and (b) is a graph showing the temperature gradient of the detection channel under the above conditions. From FIG. 4, it can be confirmed that a temperature gradient of 10 to 30 ° C in the detection channel is linearly formed.

These calibration curves can be used to calculate the temperature at a particular location in the channel at a particular feed rate, flow rate, and refrigerant conditions.

Example 4: Preparation of concentration gradient curve

A visualization of the chemical gradient occurring in the detection channel of the microfluidic device fabricated in Example 1 was confirmed and a calibration curve for the concentration gradient was obtained.

A 0.1 mM solution of fluorescent dye FITC (fluorescein isothiocyanate) was added to one inlet of the sample microchannel of the microfluidic device prepared in Example 1, and PBS (phosphage buffer saline) was added to the other inlet at 1, 2.5, 5 or 10 Mu] l / min.

The fluorescence of FITC was measured at various positions of the detection channel using an inverted fluorescence microscope equipped with a CCD camera (Coolsnap cf, Photometrics, USA) (TE2000, Nikon, Japan) and the results are shown in Fig. The inflow point of the detection channel where the flow of the nine past flows in the gradient generation area is zero is indicated by 1 to 6 numbered from 1 mm to 6 mm at intervals of 1 mm therefrom. Table 1 shows the linearity (R ² ) at each flow rate and position. FIG. 5 shows the case where the injection rates are (A) 1, (B) 2.5, (C) 5, It is a graph showing the intensity of fluorescence at a representative point.

When the FITC and PBS are injected through the microfluidic device of Embodiment 1, the FITC concentration is divided into the other nine flow flows continuously through the gradient generation region and continuously flows into the detection channel. Therefore, the FITC concentration A gradient is created. As shown in Table 1 and FIG. 5, when the injection rate was 1 to 2.5 μl / min, the linearity was 0.98 or more from the inlet point (0 mm) to the 6 mm point of the detection channel, and the injection rate was 5 μl / min, the linearity was somewhat lowered, but still a high value of 0.96 or more. When the injection rate is 10 ㎕ / min, the concentration gradient in the high concentration region is not effectively achieved in the graph of Fig. 3, and the linearity is further lowered.

Using these calibration curves, the concentration at a specific location in the detection channel can be calculated from the concentration of the incoming compound and the rate of infusion.

Claims (4)

Two inlets; A gradient forming region for separating each fluid injected into the injection port into a plurality of microchannels having different concentration gradients; A detection channel through which a fluid separated into a plurality of microchannels passing through the gradient forming region is joined and moved; And a discharge port through which the fluid having passed through the detection channel is discharged,
A sample inlet channel formed on both sides of the detection channel of the sample microchannel in parallel with a detection channel at a predetermined interval; outlet; And a micro flow path in which the fluid injected into the inlet port is moved to the discharge port, and the heat of the different temperature is circulated;
Wherein a concentration gradient and a temperature gradient are simultaneously generated in the detection channel.
The method according to claim 1,
And a valve is formed at a boundary portion between the gradient forming region and the detection channel.
3. The method according to claim 1 or 2,
Wherein the detection channel has a width of 100 to 1,000 microns.
3. The method according to claim 1 or 2,
Wherein the gap between the detection channel and the temperature control channel is 10 to 1,500 microns.

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