KR102405337B1 - A resin for microstructure lab-on-a-chip and lab-on-a-chip using the same - Google Patents

Abstract

The present invention relates to a lab-on-a-chip resin and a lab-on-a-chip using the same, and the lab-on-a-chip resin can be used for 3D printing, so that a lab-on-a-chip tailored to individual experiments can be manufactured, and in particular, microstructures and microchannels A lab-on-a-chip can be manufactured, and the lab-on-a-chip can be manufactured by 3D printing and can have microchannels.

Description

Resin for microstructure 3D printing and lab-on-a-chip using the same {A RESIN FOR MICROSTRUCTURE LAB-ON-A-CHIP AND LAB-ON-A-CHIP USING THE SAME}

The present invention relates to a resin for microstructure 3D printing and a lab-on-a-chip using the same. Specifically, it relates to a lab-on-a-chip capable of extracting/concentrating DNA.

The lab-on-a-chip system using microfluidic control technology implements the functions of bulky and expensive equipment mainly used in laboratories on a chip the size of a finger, so that various pre-processing and analysis required in the laboratory can be efficiently performed within a short time. (Curtis et al., Lab Chip. Vol. 7, pp. 41-57, 2007). However, in order to drive a general lab-on-a-chip system, it is complicated to use and requires an expensive fluid driven pump. For this reason, a lab-on-a-chip system having numerous advantages is not widely used.

In order to solve the problems of the lab-on-a-chip system, the lab-on-a-chip system is implemented by using the capillary phenomenon of paper fibers and microstructures, or by driving a microfluid using the degassing process of polydimethylsiloxane (PDMS), a porous material. did However, there is a limitation in that the analysis time using the lab-on-a-chip system becomes long due to the slow driving speed of the microfluid. In addition, the analysis using large-sized cells is limited due to paper fibers and microstructures, and the analysis of small-sized materials is also limited due to the limitations of materials.

In addition, since the conventional lab-on-a-chip is generally mass-produced for a specific purpose, there was a difficulty in supplying and supplying a lab-on-a-chip tailored to individual experimental purposes at the laboratory level.

Accordingly, the present inventors have researched to solve the above problems, and have developed a material that can be used in a 3D printer while being able to produce a finer channel than in the prior art, and through this, a lab-on-a-chip suitable for individual experimental purposes can be produced. By confirming that there is, the present invention was completed.

1. Curtis et al., Lab Chip. Vol. 7, pp. 41-57, 2007

One aspect of the present invention is a polyethylene glycol-based monomer containing two or more acrylate groups as a monomer, IRG (IRGACURE 819) as a photoinitiator, and 2-isopropyl thioxanthone (ITX) as a photosensitizer An object of the present invention is to provide a resin for chips.

Another aspect of the present invention aims to provide a lab-on-a-chip manufactured using the resin.

One aspect of the present invention is a polyethylene glycol-based monomer containing two or more acrylate groups as a monomer, IRG (IRGACURE 819) as a photoinitiator, and 2-isopropyl thioxanthone (ITX) as a photosensitizer Provides resin for chips.

As used herein, the term "lab-on-a-chip" refers to a chip manufactured to implement various operations such as mixing, reaction, separation, and analysis performed in a laboratory.

In one embodiment of the present invention, the polyethylene glycol-based monomer may contain 2 to 6 of the acrylate groups, and may include at least polyethylene glycol diacrylate (PEG-DA). In addition, the polyethylene glycol diacrylate may have a unit molecular weight of 258 to 1000, specifically, 258, 575, 700 or 1000. In addition, the polyethylene glycol diacrylate may be any one of PEG-DA-258, PEG-DA-575 and PEG-DA-700, and specifically, PEG-DA-258 of Formula I below. The PEG-DA-258 means polyethylene glycol diacrylate having a molecular weight of 258, PEG-DA-575 means polyethylene glycol diacrylate having a molecular weight of 575, and PEG-DA-700 is polyethylene glycol diacrylate having a molecular weight of 700 means rate.

[Formula I]

The term "photoinitiator" used in the present invention refers to a material that induces polymerization and curing of a photosensitive polymer composition, and in the present invention, polyethylene glycol diacrylate.

The term "photosensitizer" used in the present invention is generally referred to as a photoactive compound, PAC (Photo Active Compound), or photosensitive acid generator, and refers to a compound that can be activated by light irradiation or can generate an acid.

As an embodiment of the present invention, the photoinitiator may be IRGACURE 819 of Formula II below, and the photosensitizer may be 2-isopropyl thioxanthone (ITX) of Formula III below.

[Formula II]

[Formula III]

Specifically, the IRG (IRGACURE 819) is 0.2 to 0.6% (W / W), specifically, 0.4 to 0.6% (W / W), the 2-isopropyl thioxanthone (2-isopropyl) based on the total weight of the resin thioxanthone, ITX) may be included in 0.1 to 0.6% (W / W), more specifically, the IRG (IRGACURE 819) is 0.6% (W / W) based on the total weight of the resin, the 2-isopropyl thioxanthone (2-isopropyl thioxanthone, ITX) may be included in 0.6% (W/W).

By using the resin of the present invention in the composition and content as described above, it is possible to form a fine channel.

As an embodiment of the present invention, the resin for the lab-on-a-chip is for a 3D printer. As described above, the conventional lab-on-a-chip was purchased and used mass-produced for a specific purpose designed by the manufacturer, and as a result, there was a problem in that it was difficult to manufacture and use a lab-on-a-chip suitable for individual experimental purposes. The composition of the present invention can be used in a 3D printer, has high light absorption at a specific wavelength and is rapidly cured to produce a lab-on-a-chip with a fine structure and micro-channels, so it is possible to produce a lab-on-a-chip suitable for individual experimental purposes. make it possible

Another aspect of the present invention provides a lab-on-a-chip manufactured using the resin.

In one embodiment of the present invention, the lab-on-a-chip may include a channel at a level through which the sample (solution) does not escape into the sink or source chamber, which may be a channel with a size of several tens to hundreds of μm, specifically , 27 to 150 μm, more specifically 66 to 75 μm. The 'channel' refers to a fine void through which a fluid can flow in the lab-on-a-chip, and specifically, a target or undesired substance is extracted and separated from the sample by an external force that induces the flow of the fluid, such as an electric current. means structure.

Conventional lab-on-a-chip has a problem in manufacturing fine channels due to limitations in materials, but channels of 66 to 75 μm can be formed in the lab-on-a-chip through the composition and content of the above-described resin of the present invention. not.

In one embodiment of the present invention, the lab-on-a-chip is manufactured by a 3D printer. As described above, according to the present invention, a lab-on-a-chip, specifically, a lab-on-a-chip having a microchannel can be manufactured by 3D printing by using the above-described resin for lab-on-a-chip. And, the lab-on-a-chip may be used for DNA extraction.

Since the resin for lab-on-a-chip of the present invention can be used for 3D printing, a lab-on-a-chip tailored to individual experiments can be manufactured, and in particular, a lab-on-a-chip having a microstructure and microchannels can be manufactured.

In addition, the lab-on-a-chip of the present invention can be manufactured by 3D printing and can have microchannels, so that the DNA of the sample can be extracted/concentrated.

1 is a graph showing the results according to Experimental Example 1-1 and shows the degree of absorption according to the wavelength of each component of the resin.
2 is a photograph showing the result according to Experimental Example 1-2 and confirming the possibility of forming a microchannel.
3 is a photograph of a lab-on-a chip of the present invention manufactured according to Example 2.
4 is a photograph confirming the microchannels of the lab-on-a chip of the present invention manufactured according to Silhim Example 2;
5 shows the results of Experimental Example 2-1, and is a photograph confirming the microfluid flow.
6 and 7 show the experimental process and results of Experimental Example 2-2.

Hereinafter, one or more specific examples will be described in more detail through examples. However, these examples are for illustrative purposes of one or more embodiments, and the scope of the present invention is not limited to these examples.

Example 1: Preparation of resin for lab-on-a-chip

A resin for a lab-on-a-chip of the present invention was prepared.

Specifically, as the acrylate macromer, PEG-DA-258 (Sigma Aldrich), a photocurable resin, was used, and Irgacure 819 (IRG) (BASF Corporation) was used as a photoinitiator at 0.6% (w/w), and as a photosensitizer, 2-isopropyl 0.6% (w/w) of thioxanthone (ITX) was used. To prevent photopolymerization by light, the materials were placed in a polypropylene tube wrapped in aluminum foil. After mixing them, it was placed in an oven at 70° C. and heated for 30 minutes to completely dissolve ITX and IRG.

As a result, a resin for a lab-on-a-chip was manufactured.

Experimental Example 1: Confirmation of the possibility of forming microchannels

The following experiment was performed to confirm that a lab-on-a-chip having a mashing channel could be manufactured using the resin of Example 1 above.

Experimental Example 1-1. Confirmation of light absorption capacity

When the resin of Example 1 was used, the light absorption ability of the resin related to curing was checked in order to check whether a structure having a size fine enough to make a microfluid could be formed.

Specifically, 0.6% IRG, 0.6% ITX and 0.6% IRG and 0.6% ITX were dissolved in acetone, respectively, and then UV absorbance was measured using Nanodrop.

As a result, as confirmed in FIG. 1, the composition of the present invention showed overall high light absorption (about 3-fold increase) compared to the resin mixed with IRG or ITX alone, and in particular, the energy absorbed near 385 nm was the highest confirmed that. This is considered to be due to the fact that IRG, a photoinitiator, is suitable for a light source of 385 nm, and ITX, a photosensitizer, has a high light absorption capacity.

In addition, the high light absorption capacity of the resin of the present invention not only means faster photocuring compared to the composition mixed with IRG or ITX alone, but also has a greater ability to absorb light per unit thickness, so that light can penetrate from the surface of the resin. There is a feature that the thickness can be adjusted. This makes it possible to make a fine structure by realizing fast curing and thin resin curing thickness when 3D printing is performed.

Experimental Example 1-2. Confirmation of microchannel formation potential

It was confirmed whether microchannels could be formed using the resin of Example 1.

Specifically, after creating a 3D drawing using Inventor of AutoCAD, a 3D modeling program, it is converted into .stl format and input to the printer, and the printer performs printing by slicing it to the desired thickness through its own program. At this time, a 3D structure was manufactured using the resin developed above.

As a result, as shown in FIG. 2 , it was confirmed that the resin material for lab-on-a-chip of the present invention could form a microfluidic channel with a level of 130 μm even though it was manufactured by 3D printing.

Example 2: Preparation of Lab-on-a-Chip

Using Inventor® (Autodesk, San Rafael, CA) and printed with the DLPSL printer Asiga Pico2 HD (Asiga, Sydney, Australia) as a 3D printer and using the resin of Example 1, a lab-on-a-chip was printed in the following way. prepared.

(i) after preparing the bottom layer (ii) after forming a structure layer forming a porous barrier channel structure that can fill the hydrogel on the bottom layer (iii) source on the structure layer A chamber, a sink chamber, and an inlet for hydrogel injection are formed. And (iv) after forming an inlet into which a sample can be put, (v) an electrode holder into which an electrode can be inserted. After that (vi), it was washed with distilled water and loaded with 2% agarose to prepare a lab-on-a-chip.

For the bottom layer, a glass slide (75 mm (L) Х 50 mm (W) Х 1.0 mm (T)) was used. The glass slides were prepared by washing with ethanol, acetone, washing with distilled water, and drying overnight at 70° C. In addition, the glass slides were treated with oxygen plasma (Deiner Zepto, Thierry Corporation) for 180 seconds at 60 W, 670 mTorr pressure. Then, it was derivatized with 3-(trimethoxysilyl)propyl methacrylate (TMSPMA) (Sigma-Aldrich) at 85°C for 8 hours.

As a result, a lab-on-a-chip was prepared as shown in FIGS. 3 and 4, and the lab-on-a-chip had a porous barrier channel of 274 µm, channels of 66 µm, 71 µm, 72 µm and 75 µm ( capillary chanel) and structures (barriers) having sizes of 223 μm, 228 μm and 231 μm were confirmed.

Experimental Example 2: Confirmation of the action of Lab-on-a-Chip

Experimental Example 2-1. Confirmation of microfluidic flow

In order to form a porous structure in the lab-on-a chip prepared in Example 2, hydrogel (2% agarose) was injected and prepared.

Specifically, as shown in FIG. 5 , it can be seen that the agarose solution dyed red for visibility is filled along the porous barrier channel in the center. It can be seen that it is selectively injected only inside the porous barrier channel without escaping into the chamber. After the agarose solution is injected, when the temperature of the solution drops, it changes to a solid phase and forms a porous wall.

As a result, as shown in FIG. 5 , it was confirmed that the agarose solution injected into the gel inlet filled the porous barrier channel and formed a solid porous wall between the source/sink chamber.

Experimental Example 2-2. DNA extractability confirmation

It was confirmed whether the gene could be extracted from the cell eluate using the prepared lab-on-a chip.

After putting each cell eluate and elution buffer into the source chamber and the sink chamber of the lab-on-a chip manufactured in Example 2, platinum electrodes were mounted on electrode holders in each chamber. After biting the negative electrode connected to the power supply to the electrode of the source chamber and the positive electrode to the electrode of the sink chamber, a voltage of 1 V was applied to induce the DNA to selectively move from the source chamber to the sink chamber (FIG. 6).

In order to check the movement of DNA according to the time when electricity is applied to the electrode, the time for which electricity is applied to each chamber is varied for 5, 10, 15, and 20 minutes to extract DNA, and then use the extracted DNA to target the target through PCR After the gene was amplified, the presence or absence of the target gene was confirmed through electrophoresis.

As a result, as shown in FIG. 7 , it was confirmed that the DNA was moved by electricity, and since the gene amplification signal was generated even in the experiment in which electricity was applied for 5 minutes, the time required for gene extraction using a conventional silica membrane was reduced. It can be seen that it can be significantly shortened.

In addition, when the number of cells used to prepare the cell eluate was changed from 10 ⁶ to 10 ³ , the extraction experiment was performed, and as a result of gene detection through real-time PCR, the more the number of cells used, the faster the Ct value. was able to confirm

So far, the present invention has been looked at with respect to preferred embodiments thereof. Those of ordinary skill in the art to which the present invention pertains will understand that the present invention can be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments are to be considered in an illustrative rather than a restrictive sense. The scope of the present invention is indicated in the claims rather than the foregoing description, and all differences within the scope equivalent thereto should be construed as being included in the present invention.

Claims (11)

As a lab-on-a-chip resin comprising PEG-DA-258 as a monomer, IRG (IRGACURE 819) as a photoinitiator, and 2-isopropyl thioxanthone (ITX) as a photosensitizer
The IRG (IRGACURE 819) is 0.2 to 0.6% (W / W) based on the total weight of the resin,
The 2-isopropyl thioxanthone (2-isopropyl thioxanthone, ITX) is included in 0.1 to 0.6% (W / W) of a resin for lab-on-a-chip.
delete delete delete delete delete The method of claim 1,
The resin for the lab-on-a-chip is a resin for a lab-on-a-chip for a 3D printer.
A lab-on-a-chip manufactured using the resin of claim 1.
The lab-on-a-chip of claim 8 , wherein the lab-on-a-chip includes a channel of 27 to 150 μm.
The lab-on-a-chip of claim 8, wherein the lab-on-a-chip is manufactured by a 3D printer.
The lab-on-a-chip according to claim 8, wherein the lab-on-a-chip is for DNA extraction and concentration.

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