KR101667149B1 - Microfluidic device for detecting target protein or target peptide, method for preparing the same, and method for detecting target protein or target peptide using the same - Google Patents
Microfluidic device for detecting target protein or target peptide, method for preparing the same, and method for detecting target protein or target peptide using the same Download PDFInfo
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Abstract
Description
The present invention relates to a microfluidic device for detecting a target protein or a target peptide, a method for producing the same, and a method for detecting a target protein or a target peptide using the same.
A peptide is defined as a protein in which two or more amino acids are connected by a peptide bond, and one or two or more peptides form a macromolecule. Proteins and peptides play a vital variety of important roles in organisms and catalyze biological reactions.
A variety of methods for detecting proteins or peptides have been studied and developed, one of which is an aptamer. Aptamers are single-stranded nucleic acid constructs with high specificity and affinity for certain target substances and can be usefully used to detect specific target substances. Aptamers are about 20 to 26 nt molecules long, short, easy to deform, and very stable. Aptamers have been developed for a variety of target substances, and a number of aptamers have been developed that target proteins and peptides. For example, registration number 10-1351647 discloses an aptamer that specifically binds to troponin I, a protein that plays a role in contracting myocardium, a protein known as a biomarker of acute cardiovascular disease.
A microfluidic device is a device in which each component is connected through a microfluidic channel on a substrate, and the entire process is automated until a detection signal is obtained when a sample solution is injected. It is advantageous because it can detect even small amount of sample, use few reagents, and automate the entire process.
Publication No. 10-2014-0032094 discloses a microfluidic device that can be combined with an aptamer. However, the microfluidic device disclosed in the above document has a two-layer structure and flows the sample by adjusting the angle of the flow path, so that the manufacturing process and the use method are complicated. In addition, since the pump is used to inject the sample, it must be powered externally and only one substance can be detected at a time.
The present invention provides a microfluidic device for detecting a target protein or a target peptide, a method for producing the same, and a method for detecting a target protein or a target peptide using the same.
The present invention
Board;
An inlet through which the sample solution formed on the substrate flows from the outside;
A first flow path connected to the inlet port to receive the introduced sample solution;
A second flow path connected to the first flow path;
And an outlet connected to the second flow path, the microfluidic device for detecting a target protein or a target peptide,
The second flow path surface is coated;
A primer is immobilized on the coating;
The immobilized primer is bound to a template for target protein or target peptide detection,
Wherein the target protein or target peptide detection template comprises a primer binding portion complementarily binding to the primer, a target protein bound to one end of the primer binding portion or a portion of an aptamer for detecting a target peptide, Lt; RTI ID = 0.0 > and / or < / RTI > other portions of the target protein or target peptide-
A microfluidic device for detecting a target protein or a target peptide is provided.
In one embodiment of the present invention, the second flow path may be one to twenty.
In another embodiment of the present invention, the second flow path is 2 to 20, and each of the second flow paths is branched at the end of the first flow path, and the target protein bound to the primer fixed on each second flow path or Templates for target peptide detection may be those that bind to the same or different proteins.
In one embodiment of the present invention, the coating is selected from the group consisting of dopamine, 5-hydroxydopamine hydrochloride, norepinephrine, epinephrine, pyrogallol amine, 3,4-Dihydroxyphenylalanine (DOPA), catechin, tannins, pyrogallol, Polyethylene glycol-catechol, polyethyleneimine-catechol, polymethylmethacrylate-catechol, hyaluronic acid-catechol, polylysine-catechol, , And polylysine. ≪ / RTI >
In one embodiment of the present invention, the primer is selected from the group consisting of thiol, amine, hydroxyl, carboxyl, isothiocyanate, NHS ester, aldehyde, epoxide, carbonate, HOBt ester, glutaraldehyde, carbamate, imidazole carbamate, The terminal may be modified at least one terminal group selected from the group consisting of maleimide, aziridine, sulfone, vinyl sulfone, hydrazine, phenylazide, benzophenone, anthraquinone, and a diene group.
In one embodiment of the present invention, the coating is 5-hydroxydopamine hydrochloric acid, and the primer may have a terminal modified to a thiol or amine group.
In one embodiment of the present invention, the primer-binding portion of the template for detecting the target protein or the target peptide may comprise the nucleotide sequence shown in SEQ ID NO: 2.
Proteins and peptides that can be detected by the microfluidic device for detecting target proteins or target peptides of the present invention can be protein antigens, antibodies, growth factors, receptors, interferons, enzymes, cancer-specific antigens, prions and constituent peptides thereof Platelet-derived growth factor (PDGF), Vascular Endothelial Growth Factor (VEGF), and vascular endothelial growth factor (VEGF) Factor, VEGF), interferon-gamma (IFN-y). Progesterone receptor, estrogen receptor, HER2 / neu, epidermal growth factor receptor (EGFR), carbonic anhydrase IX (CAIX), epithelial cell adhesion molecule endothelial cell adhesion molecule, EpCAM).
The present invention also relates to a microfluidic device for detecting the target protein or the target peptide;
dNTP;
Ligase;
And a microfluidic device kit for detecting a target protein or a target peptide, which comprises an isothermal nucleic acid polymerase.
In one embodiment of the present invention, the ligase may be an DNA ligase such as T7 ligase, or CircLigase (TM). The isothermal nucleic acid polymerase may also be a phi 29 polymerase.
In another embodiment of the present invention, the kit may further comprise dithiothreitol (DTT) or pyrophosphatase.
The kit may further comprise a dyeing reagent, a high salt solution, or a fluorescent reagent.
The present invention provides a method for producing a microfluidic device for detecting a target protein or a target peptide, comprising the steps of:
1) A microfluidic device comprising a substrate, an inlet formed on the substrate, a first flow path connected to the inlet and containing the introduced sample solution, a second flow path connected to the first flow path, and a discharge port connected to the second flow path ;
2) coating the second flow path surface of the microfluidic device;
3) fixing the primer to the coating; and
4) A step of binding a target protein or a template for detecting a target peptide to the fixed primer, wherein the target protein or template for detecting a target peptide comprises a primer binding portion complementarily binding to the primer, A portion of the aptamer for detecting the bound target protein or target peptide and another portion of the target protein bound to the other end of the primer binding portion or the aptamer for target peptide detection.
The present invention also provides a method for detecting a target protein or a target peptide using the microfluidic device for detecting a target protein or a target peptide according to claim 1, comprising the following steps.
a) providing a microfluidic device for detecting the target protein or target peptide of claim 1;
b) injecting a sample solution into the inlet of the microfluidic device for detection of the target protein or target peptide; and
c) adding dNTP, ligase, and isothermal nucleic acid polymerase to the second flow path of the microfluidic device.
In one embodiment of the present invention,
d) injecting a dyeing reagent, a high salt solution or a fluorescent reagent into the inlet of the microfluidic device.
The present invention eliminates the need to change the temperature for nucleic acid amplification and allows detection of target proteins or target peptides without external power supply. Therefore, it is not necessary to carry complicated equipment, and it can be manufactured simply, inexpensively, is easy to operate, and is easy to carry. It can also be used for the detection of various proteins and can be used for simultaneous detection of two or more proteins. It can be used for the detection of socially problematic proteins such as prions or the detection of cancer-related antigens, antibodies, interferons and growth factors in biological samples Widely available. Therefore, it can be very useful for field diagnosis.
1 is a plan view (left) and a perspective view (right) of a microfluidic device for detecting a target protein or a target peptide of the present invention.
Description of the Related Art
100: substrate
101: inlet
102: First Euro
103: the second euro
104: Outlet
2 is a view showing the structure of a target protein or a template for detecting a target peptide of the present invention. FIG. 2A shows a linear structure, FIG. 2B schematically shows a form of a template when it is bound to a primer immobilized on a second flow path of a target protein or target peptide detection microfluidic device of the present invention, and FIG. 2C Is a schematic representation of a form when bound to a target protein or target peptide.
3A is a schematic view showing a primer immobilized on a second channel of a microfluidic device for detecting a target protein or a target peptide of the present invention and a template for detecting a target protein or a target peptide bound to the primer, FIG. 3C is a schematic view showing a state in which the template is closed by a ligase, FIG. 3D is a schematic diagram showing a state in which the template is closed by isothermal nucleic acid polymerase, And the nucleic acid is synthesized by the method of the present invention.
FIG. 4A is a graph showing the results of observing the flow of a sample solution using a staining reagent when a target substance detected only in the leftmost second flow path exists in the sample solution in the microfluidic device for detecting a target protein or a target peptide of the present invention And FIG. 4B is a view of the result of FIG. 4A observed using Imager.
Hereinafter, the present invention will be described in detail with reference to the accompanying drawings in order to facilitate understanding of the present invention. It is to be understood, however, that the invention is not limited to the disclosed embodiments, but, on the contrary, is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the invention. Also, in the drawings, the width, length, thickness, etc. of components may be exaggerated for convenience. Like reference numerals designate like elements throughout the specification.
Also, when an element is referred to as being on another element, it includes not only the element just above another element, but also the case where there is another element in between.
Basic Structure of Microfluidic Device for Detection of Target Protein or Target Peptide
Hereinafter, the basic structure of a microfluidic device for detecting a target protein or a target peptide of the present invention will be schematically described.
1 is a schematic diagram of a microfluidic device for detecting a target protein or a target peptide of the present invention. The microfluidic device for detecting a target protein or a target peptide of the present invention is characterized in that an
When the sample solution is injected into the
The primer immobilized on each second flow path and the template for target protein or target peptide detection may be the same or different from each other. A schematic diagram of the basic structure of a target protein or target peptide detection template of the present invention is shown in Fig. Fig. 2A shows a linear structure of the template. A template for detecting a target protein or target peptide of the present invention is a single-stranded nucleic acid, and at the center thereof, there is a portion (primer binding portion) complementarily binding to the primer (indicated by blue in the figure). At the one end of the primer binding site, a part of the aptamer for detecting the target protein or the target peptide (a part indicated by gray in the figure) is bonded, and at the other end, another part of the aptamer ) Are combined with each other.
Aptamer is a single-stranded nucleic acid that selectively binds to a specific target substance. When bound to a target substance, the stereostructure of the nucleic acid changes. In the present invention, a target protein or aptamer for detecting a target peptide is cleaved to attach a moiety to one end of a primer binding site and another moiety to another end of a primer binding site to prepare a template for target protein or target peptide detection.
In the microfluidic device of the present invention, since the primer-bonded portion of the template is complementarily bonded to the primer fixed on the second flow path, the template exists in a state as schematically shown in Fig. 2B. When a target substance is present, as shown in FIG. 2C, a part of the aptamer for detecting a target protein or a target peptide and another part selectively bind to the target substance and the three-dimensional structure changes to bring the 5'- and 3'- Leaving only a small nick in between.
In the microfluidic device of the present invention, the target protein or the target peptide may be any protein, peptide or a part thereof, and there is no limitation on its kind.
The protein or peptide may be a substance requiring rapid detection depending on the situation, for example, prion (PrPc). The protein or peptide may be a protein antigen, an antibody, a growth factor, an interferon, an enzyme, a cancer antigen, or the like. The protein may be a protein that can be a biomarker of any disease. For example, the protein may be a prostate-specific antigen (PSA), a platelet-derived growth factor (PDGF), a vascular endothelial growth factor (VEGF), an interferon- -γ), Fibroblast Growth Factor, S100 calcium binding protein A9, Keratin 10, lactoferrin, calpain-like protease CAPN10b CAPN10b), fibrinogen, fibrin, transferrin, actin, cyclophilin B, myosin, heat shock protein (HSP), zinc finger protein, amylase, trypsin, lipase , Actin binding protein, lipoprotein, apolipoprotein, cytochrome P450, hemoglobin, embigin, p53 protein, progestrone receptor, estrogen, (EGFR), carbonic anhydrase IX (CAIX), endothelial cell adhesion molecule (EpCAM), and the like, as well as the receptor (estrogen receptor), Herceptin 2 receptor (HER2 / neu) . However, the protein that can be detected by the present invention is not limited thereto.
In the microfluidic device of the present invention, the materials of the substrate, the first flow path, the second flow path, the inlet and the outlet are not limited unless they react with the sample solution. For example, materials such as polymer, glass, silicon, and ceramic can be used. Among them, the polymer material is advantageous in consideration of ease of molding and cost. For example, one selected from the group consisting of polypropylene (PP), polycarbonate (PC), polyethylene (PE), polyethylene terephthalate (PET), polymethylmethacrylate (PMMA), polystyrene (PS), and cyclic olefin copolymer Can be used. In addition, it may be made of a material such as polydimethylsiloxane, gold (Au), metal oxide (TiO 2 , Al 2 O 3 , indium-tin oxide).
In the microfluidic device of the present invention, the number of the second flow paths may be one or more, preferably 1 to 20, and more preferably 2 to 5. In the present invention, the number of the second flow paths may be varied according to the number of target substances to be detected. For example, when three substances are simultaneously detected, 4 < / RTI > However, the number of the second flow path is not limited.
In the microfluidic device of the present invention, the diameter of the second flow path may be 1 to 5 mm, and in another embodiment, the diameter of the second flow path may be 50 to 5 mm. Also, in one embodiment, the diameter of the second flow path may be between 0.25 mm and 2 mm, and in another embodiment between 0.5 mm and 1.5 mm. However, the diameter of the second flow path may be suitably adjusted, but is not limited thereto.
In the microfluidic device of the present invention, the coating of the second flow path may be a monomer or a polymer coating of hydroxybenzenes. The coating may also include, for example, a catecholamine polymer coating. Monomers or polymers of hydroxybenzenes can be easily coated on a wide range of materials including noble metals, metal oxides, ceramics, and synthetic polymers due to their excellent surface properties. Specific examples of the monomers and polymers of the hydroxybenzenes include, but are not limited to, dopamine, 5-hydroxydopamine HCl, norepinephrine, epinephrine, pyrogallol amine ( pyrogallol amine, DOPA (3,4-Dihydroxyphenylalanine), catechin, tannins, pyrogallol, pyrocatechol, heparin-catechol, Catechol, polyethylene glycol-catechol, poly (ethyleneimine) -catechol, poly (methyl (meth) acrylate-catechol) methacrylate-cathechol, hyaluronic acid-catechol, polylysine-catechol, polylysine, and the like. In one embodiment of the present invention, when a hydrophilic material is used as a coating material, flow of the sample solution on the second flow path can be assisted.
In the microfluidic device of the present invention, the primer may be any single-stranded nucleic acid having a length of, for example, 10 to 40 mers. There are no limitations on the length and sequence of the primers that can be used in the present invention. The primer may have a functional group suitable for the end so that it can be easily fixed to the coating on the second flow path. For example, it is possible to use one or more compounds selected from the group consisting of thiol, amine, hydroxyl, carboxyl, isothiocyanate, NHS ester, aldehyde, epoxide, ), HOBt esters, glutaraldehyde, carbamate, imidazole carbamate, maleimide, aziridine, sulfone, vinylsulfone, And may have hydrazine, phenyl azide, benzophenone, anthraquinone, diene group and the like, preferably with a thiol group or an amine group. However, the type of the functional group is not limited thereto, and various functional groups may be used depending on the coating on the second flow path.
In the microfluidic device of the present invention, the target protein or target peptide detection template may be designed in various ways in consideration of stability and conversion amplification efficiency depending on the length of aptamer and primer for detection of target protein or target peptide used . For example, a template for protein detection can be a single-stranded nucleic acid having a total length of 40 to 160 m.
In the target protein or template for detection of the target peptide, the primer binding portion may be a single-stranded nucleic acid having any sequence complementary to the primer used in the present invention, which may be, for example, 10- to 40- . There are no restrictions on the length and sequence of the primer binding sites that can be used in the present invention.
In the microfluidic device of the present invention, a portion of the aptamer for detecting the target protein or the target peptide bound to the primer binding portion in the template for detecting the target protein or the target peptide and the other portion of the aptamer for the target protein or target peptide detection One of the target proteins or aptamers for the detection of the target peptide. The aptamer for detecting the target protein or the target peptide means any app tamer that can be used for protein or peptide detection, and can be a known aptamer. A person skilled in the art can, by repeated experimentation, cut a known aptamer at a point suitable for application to the target protein or target peptide detection template of the present invention.
In the microfluidic device of the present invention, the sample solution can flow through the channel by the capillary phenomenon without external power supply. However, it is possible to regulate the solution flow by supplying external power by using a pump, current or the like as needed, and to control the solution flow by adjusting the angle of the flow path.
Method for producing microfluidic device for detection of target protein or target peptide
Hereinafter, a method for producing a microfluidic device for detecting a target protein or a target peptide of the present invention will be schematically described.
Step 1: Step of manufacturing microfluidic device
An
It is also possible to use a commercially available microfluidic device. For example it can be used for a 1 μ-Slide Ⅲ 3in1 uncoated Microscopy Chamber products manufactured by ibidi used.
Step 2: Coating the second flow path of the microfluidic device
Each second flow path of the microfluidic device is coated with a coating material.
Step 3: Step of attaching the primer to the second flow path of the microfluidic device
A primer is attached to the coating of the second flow path of the microfluidic device.
The primer can be prepared in a form suitable for use in the microfluidic device of the present invention. For example, although not essential, it is possible to prepare a linear form of primer in which disulfide bonds produced in the primer have been removed by treating DTT (dithiothreitol). The primer ends can also be modified into functional groups that facilitate the reaction with the coating. For example, a thiol group or an amine group may be bonded to one end of the primer.
The primer can be attached to the coating by reaction between the primer and the coating.
Step 4: Step of immobilizing a template for target protein or target peptide detection in a second flow path of the microfluidic device
As described above, a template for detecting a target protein or a target peptide having a structure as shown in Fig. 2A is prepared. Wherein the primer binding moiety can be any single-stranded nucleic acid that binds complementarily to the primer attached to the second flow path in step 3 above.
When the template for detecting the target protein or the target peptide is placed in the second flow path, complementary binding occurs between the primer binding portion of the target protein or target peptide detection template and the primer immobilized to the second flow path, Or a template for target peptide detection.
Detection method using microfluidic device for detection of target protein or target peptide
Hereinafter, a detection method using a microfluidic device for detecting a target protein or a target peptide of the present invention will be schematically described.
Step 1: introducing the sample solution into the microfluidic device for detecting the target protein or the target peptide
As described above, the microfluidic device for detecting the target protein or target peptide of the present invention is prepared, and the sample solution to be confirmed is injected into the inlet port.
The sample solution injected into the inlet is moved by the capillary phenomenon and flows into the second flow path along the first flow path.
The sample solution can be a variety of solutions in which the protein or peptide is dissolved, for example, a solution of water or soil samples to analyze water pollution, soil contamination, and the like. The sample solution may also be a biological sample. Examples of biological samples include whole blood, serum, plasma, saliva, sputum, ascites, fluid, and urine. However, the kind of the sample solution that can be used in the present invention is not limited thereto.
Step 2: Conversion amplification step
When the sample solution flows into the respective second flow paths, reagents and buffers necessary for the reaction of dNTPs (ATP, GTP, TTP, CTP), ligase, isothermal nucleic acid polymerase, and other enzymes are added to the second flow path. These reagents may be those commercially available with enzymes. Further, DTT may be added in order to break the disulfide bond, and pyrophosphatase may be further added so that the nucleic acid polymerization reaction proceeds stably and quickly.
In the presence of the target substance in the sample solution, a part of the aptamer and the other part of the aptamer bind to the protein in the target protein or target peptide detection template, so that the three-dimensional structure changes and the two ends become close to each other, , That is, only a nick exists. The nicks are ligated to each other by a ligase which is an enzyme that connects the 5'-terminal and the 3'-terminal of the adjacent nicks, so that the target protein or target peptide detection template is in a completely closed form (see FIG. 3C).
The isothermal nucleic acid polymerase is subjected to Rolling Circle Replication (RCA) in which the template for detecting the target protein or target peptide in the closed form is used as a template and the nucleic acid is replicated indefinitely repeatedly until the dNTP is exhausted (see FIG. 3D). Since the replicated portion is detached from the template for target protein or target peptide detection, a linear nucleic acid is generated in which the sequence of the template for target protein or target peptide is repeated. A portion of the aptamer present in the linear nucleic acid and the other portion of the aptamer bind to the target protein or target peptide and a roundtable amplification reaction takes place to produce a long and tangled nucleic acid mass which is coagulated with the coating of the second flow, Thereby forming a hydrogel lump.
The resulting mass of nucleic acid may have a size of, for example, from about 0.5 [mu] m to about 50 [mu] m in diameter. In one embodiment of the present invention, the hydrogel mass in which the nucleic acid mass and the coating are coalesced together may have a size of about 50 μm to 5 mm in diameter, for example, about 0.25 mm to 2 mm in diameter. However, the size of the nucleic acid mass or the hydrogel mass produced in the present invention is not limited thereto.
The amplified nucleic acid mass may be a tangled single strand in the form of a hook-shaped rounded portion and a velcro having a constant length. The length of each strand may be between 0.5 μm and 50 μm. Overall, the mass of the nucleic acid may be in the form of a ternary bulb having a round circular shape and a leg having a certain length.
The ligase can be used without limitation as long as it can ligate adjacent 5 'and 3' ends of the nucleic acid. For example, DNA ligase can be used. The DNA ligase includes enzymes such as T7 ligase, CircLigase (TM), and the like.
The isothermal nucleic acid polymerase can be used without limitation as long as it is a nucleic acid polymerase capable of amplifying the nucleic acid without changing the temperature. For example, phi 29 polymerase can be used. Since the present invention uses an isothermal nucleic acid polymerase, nucleic acid amplification can be performed without adjusting the temperature by using a device such as a thermocycler used for PCR. However, if necessary, a nucleic acid polymerase other than isothermal nucleic acid polymerase may be used. In this case, the enzyme may be used in combination with a device for changing temperature depending on the nature of the enzyme.
Conversion amplification reactions can be performed for 10 minutes to 5 hours, for example 3 hours. The temperature at the time of the reaction need not be adjusted unless the temperature is such that the enzyme is inactivated. For example, the reaction may be carried out at a temperature of from 0 to 50 ° C, for example, from 20 to 40 ° C, for example, at a temperature of 30 ° C.
Depending on the environment in which the reaction is performed, appropriate conditions may be provided to prevent water from evaporating in the flow path. For example, the reaction can be carried out under sealed conditions. As an example, a microfluidic device for detecting a target protein or a target peptide of the present invention may be placed in a plastic container sealed with a parafilm, and the inside of the plastic can be filled with water and dirt.
Step 3: Detection phase
The hydrogel mass in which the resulting nucleic acid and the coating are entangled together blocks the outlet of the second flow path. It is easy to visually confirm that the outlet is clogged by the lumps with high viscosity and the sample solution can not flow. From the above results, it can be seen that the target protein or the target peptide is present in the sample solution.
It is possible to simultaneously detect the presence or absence of two or more proteins by using a microfluidic device having two or more second flow paths since it is possible to attach different target proteins or templates for target peptide detection per second flow path.
The flow of the sample solution can be more easily observed by flowing a substance showing color or a substance increasing turbidity in the flow path.
For example, Gel red ™, trypane blue dye, Evans Blue dye, hematoxylin-eosin stain, crystal violet, methylene blue a dyeing reagent such as methylene blue may be diluted to an appropriate concentration and introduced into the sample inlet. Then, the flow of the sample solution in the flow path can be more easily observed as shown in FIG. 4A. It is also possible to observe more clearly through Imager (Gel Doc ™ EZ (Bio-rad)) as exemplified in FIG. 4B.
Fluorescent reagents may also be used. For example, a substance such as streptavidin beads showing fluorescence, such as Streptavidin Fluoresbrite YG Microspheres (TM), may be diluted to an appropriate concentration and flowed into the sample inlet to observe the flow of the sample solution.
A high salt solution can be used to increase the turbidity by increasing the precipitation. An example of a high salt solution of magnesium chloride (MgCl 2) aqueous solution of ammonium chloride (NH 4 Cl) aqueous solution, ammonium acetate (NH 4 OA C) aqueous solution of sodium chloride (NaCl) aqueous solution of ammonium sulfate ((NH 4) 2 SO 4 ) aqueous solution , Or a neutral amino acid solution.
The microfluidic device for detecting a target protein or target peptide of the present invention can sufficiently discriminate the presence of a target protein or a target peptide visually, but a person of ordinary skill in the art to which the present invention belongs, And can be carried out in various specific forms without being changed. In order to increase the detection sensitivity of the microfluidic device for detecting a target protein or a target peptide of the present invention, a person skilled in the art may further include an electrochemical sensor for measuring the current, voltage, potential difference, An optical sensor using various light sources such as ultraviolet rays, visible light, fluorescence, infrared rays and Raman, a nanosensor using materials such as metals, ceramics and polymers, or a biosensor using biosensors such as enzymes, antigens and antibodies .
Example
Hereinafter, embodiments are described to help understand the present invention. The following examples are provided to further understand the present invention, but the present invention is not limited by the examples.
Example 1. Microfluidic device for detecting platelet-derived growth factor
Platelet derived growth factor (PDGF) is one of the growth factors, and plays an important role in angiogenesis, and is involved in various processes such as embryo development and fibroblast dissociation. Recombinant PDGF is also used as a treatment for chronic ulcers and bone loss.
The present invention provides a microfluidic device for PDGF detection and a method for detecting PDGF using the microfluidic device as described below.
1-1. PDGF Manufacture of microfluidic device for detection
1 μ-Slide Ⅲ 3in1 uncoated Microscopy Chamber manufactured by ibidi Inc. was purchased and prepared. The product has a structure as shown in Fig. 1, and is manufactured for the purpose of observing a microscope of original cells. It is judged that it is inexpensive and has a structure suitable for use in the microfluidic device of the present invention, and the product is used.
5-Hydroxydopamine HCl was used as the coating material of the second flow path. Specifically, 5-hydroxydopamine hydrochloride (Sigma Aldrich) was dissolved in 10 mM Tris buffer (1M UltraPure 1M Tris-HCl pH 8.0 / Invitrogen) at a concentration of 1 mg / ml. Thereafter, the pH was adjusted to 8 to prepare a coating composition. Wherein in the coating composition for a 1 μ-Slide Ⅲ 3in1 uncoated Microscopy Chamber filled the each second flow path of the product. After 2 hours, the cells were washed with DDW (Water Purification System / LABOGENE).
Further, a primer to be adhered to the coating of the second flow path was prepared. Primer-5SS-polyA9 (BIONEER) having the following sequences was purchased and used. The 5 'end of the following primer sequence is bound to a thiol group.
5'-Thiol-AAA AAA AAA GGG ACG TCG ATA CTA GCA TGC TA-3 '(SEQ ID NO: 1)
100 pmol of the above primer and 5M DTT (DL-Dithiothreitol / Sigma Aldrich) were prepared, and 5 pmol of 5M DTT was added to 100 pmol of the primer. DDW (Water Purification System / LABOGENE) . The mixture was allowed to stand for 4 hours so that the disulfide bond that might have formed in the primer was cut off by DTT, and then immersed in a 3K Amicon tube (Amicon Ultra Centrifugal Filters 3K / MILLIPORE) at 132,000 rpm, After primary centrifugation, 40 DD DDW was added and secondary centrifugation was performed to remove residual DTT after the reaction. 5 [mu] L of the primer composition from which the DTT was removed was added to each of the second flow channels and allowed to stand for 2 hours to allow the primer to adhere to the coating on the second flow path, followed by washing with DDW (Water Purification System / LABOGENE).
A template for PDGF detection was also prepared as follows. The primer binding site in the template was prepared so as to have a sequence complementary to the primer shown in SEQ ID NO: 1.
5'-TA GCA TGC TAG TAT CGA CGT-3 '(SEQ ID NO: 2)
Zhao et al. CAGGCTACGGCACGTAG AGCATCACCATGATCCTG-3 'as described in Nature Nanotechnology, Vol 6, August 11, and cleavage between the 17th base and the 18th base at the 5' To the underlined part as a part of the PDGF aptamer and the remaining part as another part of the PDGF aptamer. A part of the PDGF aptamer and another part of the PDGF aptamer were attached to both ends of the primer binding part to prepare a PDGF detection template.
Adding (® lifetechnoligies's gibco) 1X PBS to 100μM PDGF detection template 0.2㎕ (= 20pmole) to prepare a template for a composition for the detection of PDGF 5㎕ and placed in the second flow path. After standing for 2 hours to allow complementary binding between the primer binding portion of the PDGF detection template and the primer immobilized in the second flow path, DDW (Water Purification System / LABOGENE) was washed.
1-2. PDGF detection using microfluidic device for PDGF detection
A solution containing PDGF was prepared as a sample solution. 60 占 퐇 of the sample solution was introduced into the first flow path through the inlet of the microfluidic device for single target gene detection. The sample solution flowing into the first flow path of the single target gene detection microfluidic device moved to the second flow path through the first flow path by the capillary phenomenon. After standing for 2 hours, DDW (Water Purification System / LABOGENE) was washed.
To the second channel, 30 μl of T7 ligase, 0.2 μl of 100 mM DTT, 2 μl of 25 mM dNTP, 50 μl of phi 29 polymerase (10 units / μl), 1 μl of pyrophosphatase, DDW was added. And placed in a plastic container sealed with a parafilm to prevent moisture in the second flow path from flying. The plastic was filled with water dampened with water and 30 ° C water. Then, the plate was allowed to stand in a non-shaking, 30 ° C, and 3-hour condition to allow nick ligation and round-trip amplification of the template for PDGF detection.
The template for PDGF detection is amplified to be visually confirmed to clog the discharge port of the second flow channel by forming a large hydrogel lump with the hydroxy-dopamine hydrochloric acid coated with the second flow channel.
Gel flow (GelRed ™ / Biotium), a dyeing reagent, is diluted to 1: 10000, and the flow of the sample solution can be more easily confirmed.
Example 2: Microfluidic device for prion detection
Prion (PrPc) is a protein particle that has the potential to transmit like a virus. It is a cause of scrapie disease of sheep or goat, mad cow disease, and Creutzfeldt-Jakob disease.
The present invention provides a microfluidic device for detecting a prion and a method for detecting a prion using the microfluidic device.
2-1. Preparation of Microfluidic Device for Prion Detection
In the microfluidic device for detecting a prion of the present invention, the rest of the template for prion detection, except for the aptamer part, can be produced in the same manner as in 1-1.
A template for prion detection can be prepared as follows. The primer binding site in the template was prepared so as to have the sequence of SEQ ID NO: 2 so as to be complementary to the primer represented by SEQ ID NO:
In addition, Lei Zhan et al. CTT ACG GT G GGG CAA TT-3 'as described in ( Analyst , 2013, 138, 825-830), and a 5'- Was cut to make a part of the sequence from the sequence to the underlined part as a part of the prion aptamer and the remaining part as the other part of the prion aptamer. A part of the prion aptamer and another part of the prion aptamer were attached to both ends of the primer binding part to prepare a template for prion detection.
2-2. Detection of prions using microfluidic device for detecting prions
A solution containing a prion was prepared as a sample solution. The prions were detected in the same manner as 1-2 below.
Example 3: Microfluidic device for detecting prostate-specific antigen
Prostate specific antigen (PSA) is a proteolytic enzyme synthesized in the epithelium of the prostate. It is a protein used as a biomarker for prostate cancer because it is rarely expressed in tissues other than the prostate (cancer specific antigen).
The present invention provides a microfluidic device for detecting PSA and a method for detecting a prion using the microfluidic device.
3-1. Preparation of microfluidic device for PSA detection
In the microfluidic device for detecting PSA of the present invention, the rest of the template for PSA detection, except for the aptamer part, can be prepared in the same manner as 1-1.
A template for PSA detection can be prepared as follows. The primer binding site in the template was prepared so as to have the sequence of SEQ ID NO: 2 so as to be complementary to the primer represented by SEQ ID NO: Zhanguang Chen et al. ATT AAA GCT CG C CAT CAA ATA As described in Biosensors and Bioelectronics 36 (2012) 35-40, an aptamer having the sequence of ATA GC-30 was prepared, and the 5 'end was substituted with the 11th base and the 12th base The sections from the above sequence to the underlined part were used as a part of the PSA aptamer and the remaining part was used as another part of the PSA aptamer. A part of the PSA aptamer and another part of the PSA aptamer were attached to both ends of the primer binding part to prepare a PSA detection template.
3-2. PSA detection using microfluidic device for PSA detection
A solution containing PSA was prepared as a sample solution. The PSA was detected in the same manner as 1-2 below.
Example 4. Microfluidic device for detecting vascular endothelial growth factor
Vascular Endothelial Growth Factor (VEGF) is a signaling protein that stimulates angiogenesis and is involved in fetal development. In addition, overexpression of VEGF is known to induce breast cancer and ovarian cancer.
The present invention provides a microfluidic device for VEGF detection and a method for detecting VEGF using the microfluidic device as described below.
4-1. Preparation of Microfluidic Device for VEGF Detection
In the microfluidic device for VEGF detection of the present invention, the remaining portion of the template for VEGF detection except for the aptamer portion can be produced in the same manner as in 1-1.
A template for VEGF detection can be prepared as follows. The primer binding site in the template was prepared so as to have the sequence of SEQ ID NO: 2 so as to be complementary to the primer represented by SEQ ID NO: In addition, Hansang Cho et al. ACG CAG UUU G AG AAG UCG CGC GU-3 ', as described in ( ACS Nano. 2012 September 25; 6 (9): 7607-7614) The 10 th base and the 11 th base were cleaved to make a portion of the VEGF aptamer from the above sequence to the underlined portion and the remaining portion to the other portion of the VEGF aptamer. A part of the VEGF aptamer and another part of the VEGF aptamer were attached to both ends of the primer binding part to prepare a template for VEGF detection.
4-2. VEGF detection using microfluidic device for VEGF detection
A solution containing VEGF was prepared as a sample solution. VEGF was detected in the same manner as 1-2 below.
Example 5: Microfluidic device for interferon-gamma detection
Interferon-γ (Interferon-γ, IFN-γ) is a cytokine produced by T cells and macrophages, and has an immune function against viral infection, bacterial infection and cancer. Abnormal interferon-gamma is known to be associated with excessive inflammation and autoimmune disease.
The present invention provides a microfluidic device for interferon-? Detection as described below and a method for detecting interferon-? Using the same.
5-1. Preparation of microfluidic device for interferon-γ detection
In the microfluidic device for interferon-gamma detection of the present invention, the remaining portion of the template for interferon-y detection except for the aptamer portion can be produced in the same manner as in 1-1.
A template for interferon-gamma detection can be prepared as follows. The primer binding site in the template was prepared so as to have the sequence of SEQ ID NO: 2 so as to be complementary to the primer represented by SEQ ID NO: Ying Liu et al. (. Anal Chem 2010 October 1; 82 (19): 8131-8136) produced an aptamer having the sequence of 5'-C6- GGGGTTGGTTGTG TTGGGTGTTGTGTCCAACCCC-C3-3 'as described and indicated by underlining in the sequence To a portion of the VEGF aptamer, and the rest to another portion of the VEGF aptamer. A portion of the interferon-gamma aptamer and the other portion of the interferon-gamma aptamer were attached to both ends of the primer binding portion to prepare a template for interferon-gamma detection.
5-2. VEGF detection using microfluidic device for interferon-γ detection
A solution containing interferon-y was prepared as a sample solution. Interferon-y was detected by the same method as 1-2 below.
<110> Ewha University - Industry Collaboration Foundation <120> Microfluidic device for detecting target protein or target peptide, method for preparing the same, and method for detecting target protein or target peptide using the same <130> P15-076-EWHA <160> 2 <170> Kopatentin 2.0 <210> 1 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 1 aaaaaaaaag ggacgtcgat actagcatgc ta 32 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer binding region of template <400> 2 tagcatgcta gtatcgacgt 20
Claims (17)
An inlet through which the sample solution formed on the substrate flows from the outside;
A first flow path connected to the inlet port to receive the introduced sample solution;
A second flow path connected to the first flow path;
And an outlet connected to the second flow path, the microfluidic device for detecting a target protein or a target peptide,
The second flow path surface is coated;
A primer is immobilized on the coating;
The immobilized primer is bound to a template for target protein or target peptide detection,
Wherein the target protein or the template for detecting a target peptide comprises a primer binding portion complementarily binding to the primer, a target protein bound to one end of the primer binding portion or a portion of an aptamer for detecting a target peptide, Lt; RTI ID = 0.0 > and / or < / RTI > other portions of the target protein or target peptide-
Microfluidic device for the detection of target proteins or target peptides.
Wherein the target protein bound to the primer immobilized on each second flow path or the template for detecting the target peptide binds to the same or different proteins.
dNTP;
Ligase;
And an isothermal nucleic acid polymerase, for detecting a target protein or a target peptide.
1) A microfluidic device comprising a substrate, an inlet formed on the substrate, a first flow path connected to the inlet and containing the introduced sample solution, a second flow path connected to the first flow path, and a discharge port connected to the second flow path ;
2) coating the second flow path surface of the microfluidic device;
3) fixing the primer to the coating; and
4) A step of binding a target protein or a template for detecting a target peptide to the fixed primer, wherein the target protein or template for detecting a target peptide comprises a primer binding portion complementarily binding to the primer, A portion of the aptamer for detecting the bound target protein or target peptide and another portion of the target protein bound to the other end of the primer binding portion or the aptamer for target peptide detection.
a) providing a microfluidic device for detecting the target protein or target peptide of claim 1;
b) injecting a sample solution into the inlet of the microfluidic device for detection of the target protein or target peptide; and
c) adding dNTP, ligase, and isothermal nucleic acid polymerase to the second flow path of the microfluidic device.
d) injecting a dyeing reagent, a high salt solution or a fluorescent reagent into the inlet of the microfluidic device.
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