KR20230160593A - Microfluidic chip for quantitative analysis using magnetic separation - Google Patents

Abstract

In the present invention, an upper substrate for performing immunohistochemical staining, immunofluorescence, and FISH (fluorescence in situ hybridization), etc., and a lower substrate for magnetic separation of substances to be detected in the sample are stacked vertically, and a plate interposed between them. It relates to a microfluidic chip having a structure including a microfluidic chip, a method of manufacturing the same, and a method of detecting cells or biomolecules using the microfluidic chip.
According to one aspect, the microfluidic chip of the present invention can image or quantify cells over time by magnetically separating cells such as bacteria bound to magnetic particles in a sample and performing FISH, etc., and use this as an indicator for diagnosing bacterial infection. Alternatively, it can be used to predict antibiotic susceptibility.

Description

Microfluidic chip for quantitative analysis using magnetic separation}

In the present invention, an upper substrate for performing immunohistochemical staining, immunofluorescence, and FISH (fluorescence in situ hybridization), etc., and a lower substrate for magnetic separation of substances to be detected in the sample are stacked vertically, and a plate interposed between them. It relates to a microfluidic chip having a structure including a microfluidic chip, a method of manufacturing the same, and a method of detecting cells or biomolecules using the microfluidic chip.

FISH (fluorescence in situ hybridization) is a molecular cytogenetic testing technique that combines a probe that is attached with fluorescence and is capable of complementary binding to a specific nucleic acid sequence for the purpose of confirming the presence or absence of a specific nucleic acid sequence in the chromosome. am. In addition, immunohistochemistry, which stains using an antigen-antibody reaction for the purpose of confirming the presence or absence of a specific protein in cells or tissues, and immunofluorescence, which uses antigens or antibodies labeled with various fluorescent dyes ) are also widely used.

Meanwhile, PNA (peptide nucleic acid) is a polymer chemical similar to the structure of nucleic acids (DNA, RNA). Unlike DNA, it has a neutral structure that does not carry a negative charge by replacing the sugar-phosphate backbone of DNA with a peptide conjugate. Because of this, unlike DNA or RNA, PNA does not experience electrical repulsion against specific complementary base sequences, and thus has stronger binding force and specificity. In addition, it has the advantage of showing high stability against nucleic acid degrading enzymes and temperature in the body, and that secondary modification is easy through the combination of a fluorescent label or a specific functional group. Based on these advantages, PNA can be used in various molecular biology techniques such as genetic diagnosis, in situ hybridization, PCR clamping, and plasmid vector labeling.

When performing FISH, using PNA as a probe has advantages such as short hybridization time, low background binding, excellent reproducibility, and stability of the reagent due to its characteristics such as high affinity and specificity for the target nucleic acid. In addition, PNA probes are efficient at tissue penetration due to their small size, and since they are short single-stranded oligonucleotides, there is no need to denature the probe itself.

Meanwhile, magnetic separation technology is used in various biotechnology fields, such as biological analysis and disease diagnosis and treatment. In general, most known microfluidic chips use lateral fluid flow and use a method of separating magnetic nanoparticles by placing a magnet on the bottom or side of the chip. However, in this case, there was a problem that magnetic nanoparticles could be stacked within the chip and magnetically separated, making accurate detection difficult when performing analysis such as fluorescence detection of particles.

Against this background, the present inventors developed a microfluidic chip for efficiently performing immunohistochemical staining, immunofluorescence, FISH, etc., which has a structure that combines a detection chip with a magnetic separator that places a magnet under the sample injection portion of the detection chip. The microfluidic chip was completed. In the microfluidic chip of the present invention, since the magnetic nanoparticles injected into the sample injection unit are separated in the vertical direction rather than the lateral direction, the magnetic nanoparticles are not stacked within the chip but are uniformly distributed on the bottom surface of the chip, and are therefore more accurate. Efficient FISH fluorescence detection is possible. Through this, cells or biomolecules bound to magnetic nanoparticles in the sample are magnetically separated and FISH is performed on the concentrated target sample, thereby increasing the detection efficiency and accuracy of the target sample, and repeating it by replacing only the used detection chip. Since this becomes possible, it is expected that the time it takes to detect the target sample will also be shortened.

One aspect provides a microfluidic chip including upper and lower substrates stacked vertically, and a plate interposed between the upper and lower substrates.

Another aspect includes preparing an upper substrate and a lower substrate made of a polymer material;

Preparing plates;

Attaching the plate to the lower surface of the upper substrate by plasma treatment;

recessing a magnet into the upper surface of the lower substrate without protruding; and

It provides a method of manufacturing a microfluidic chip, including the step of stacking the upper and lower substrates so that the plate is interposed between the upper and lower substrates.

Another aspect includes preparing a sample containing a substance to be detected;

Forming a detection target material-magnetic particle complex by treating the sample with magnetic particles;

Injecting a sample containing the complex into the microfluidic chip sample injection unit of claim 1;

Discharging unnecessary samples through the microfluidic chip discharge unit of claim 1;

Processing a sample containing the complex with a reagent for immobilization and permeabilization;

A method for detecting cells or biomolecules is provided, comprising treating the immobilized and permeabilized sample with a reagent for labeling or staining.

One aspect is to provide a microfluidic chip including upper and lower substrates stacked vertically, and a plate interposed between the upper and lower substrates.

The microfluidic chip 30 of the present invention injects a sample containing cells combined with magnetic particles into the injection unit 11, removes unnecessary solutions, etc. through the discharge unit 13, and immobilizes and permeabilizes the sample. Then, using magnetism, only the target sample combined with the magnetic particles is separated and concentrated at the bottom of the injection section, and immunohistochemistry, immunofluorescence, and FISH (fluorescence in situ hybridization) are performed on the injection section. This makes it possible to image or quantify it. Additionally, based on these quantified results, they can be used as an indicator for diagnosing bacterial infections or used to predict antibiotic susceptibility.

The upper substrate 14 is for performing a detection method such as FISH, and may include a sample injection unit 11 and a microfluidic channel 12 through which the sample moves, and communicates with the injection unit through the microfluidic channel. It may include one discharge portion (13). Specifically, the injection unit 11 and the microfluidic channel 12 may be provided in at least one number, but are not particularly limited thereto, and may be provided in an appropriate number depending on the method and needs of those skilled in the art. According to one embodiment, eight sample injection parts 11 may be provided at equal intervals on the upper surface of a PDMS chip measuring 24 mm in width, 50 mm in length, and 3 mm in height.

Each sample injection unit 11 may communicate with one discharge unit 13 through a microfluidic channel 12. According to one embodiment, eight sample injection units have a microfluidic channel designed as shown in FIG. 1. It may be connected to the discharge unit through, but is not particularly limited to.

As used herein, the term “channel” refers to a passage through which fluids (eg, samples, reagents, buffers, etc.) move. Specifically, channels extending along a planar flow path (e.g., channels in a tortuous or spiral planar pattern), non-planar flow paths (e.g., helical three-dimensional channels), or microscopic channels It may be a fluid channel (microfluidic channel), and more specifically, it may mean a microfluidic channel.

At the bottom of the sample injection unit 11, target samples magnetically separated by the magnet 21 provided on the lower substrate 22 are concentrated, and detection is performed on the target samples separated and concentrated at the bottom of each injection unit. can do. Therefore, the sample injection unit 11 is a space containing samples and reagents for performing FISH, etc., and can serve as a reaction chamber. The diameter and depth of the sample injection unit 11 may be appropriately selected by a person skilled in the art in consideration of the area and height of the upper substrate 14, the ease of injection of samples, reagents, buffers, etc., and in one embodiment, the diameter is 3 mm. The sample injection parts were manufactured at equal intervals. Meanwhile, the reagent is for immobilization, permeabilization, hybridization of the sample, and staining of the substance to be detected in the sample, and can be appropriately selected according to the characteristics of the sample and the substance to be detected and the needs of those skilled in the art.

The discharge unit 13 is a place where the remaining samples that have not been magnetically separated, unreacted reagents, buffers, etc. move through the microfluidic channel 12 and are discharged, and may be located in the center of the upper substrate 14. , It is not particularly limited thereto, and according to one embodiment, the remaining sample that has not been magnetically separated, unreacted reagents, buffers, etc. can be removed through the discharge unit using a syringe pump. The remaining sample that has not been magnetically separated is discharged through the discharge section, and by continuously injecting the sample containing cells combined with magnetic particles through the injection section, target samples contained in a large amount of samples can be effectively collected. According to one embodiment, an E. coli sample bound to magnetic particles is injected into the injection unit 11 and magnetically separated for 10 minutes, and then unnecessary solution is injected into the discharge unit (11) using a syringe pump at a flow rate of 10 μl/min. 13) was discharged through.

The upper substrate 14 is a thermoplastic resin such as poly(dimethylsiloxane) (PDMS), polycarbonate (PC), poly(methyl methacrylate) (PMMA), cyclic olefin copolymer (COC), polystyrene, polysulfones, poly Vinyl chlorite (PVC), polyethylene (PE), polypropylene (PP), polytetrafluoroethylene (PTFE), poly(L-lactic acid) (PLLA), poly(D, L-lactic acid (PDLLA), poly(glycol) acid) (PGA), poly(caprolactone) (PCL), poly(hydroxyalkanoate), polydioxanone (PDS), polytrimethylene carbonate, poly(lactic-co-glycolic acid) (PLGA), and poly It may be a polymer material selected from the group consisting of (L-lactic acid-co-caprolactone) (PLCL) and poly(glycolic acid-co-caprolactone) (PGCL), but is not particularly limited thereto, and may be used for optical measurement and imaging ( One side of the upper substrate may provide optical transparency for imaging. More specifically, a chip made of PDMS may be used as the upper substrate.

The plate 15 is attached to the lower surface of the upper substrate 14 to facilitate optical measurement, and may be a slide glass, crystal, or cover glass, but is not particularly limited thereto. According to one embodiment, a cover glass (Coverslip) of the same size as the PDMS chip measuring 24 mm in width, 50 mm in height, and 3 mm in height is plasma treated (80 W, 1 min) and attached to the lower surface of the upper substrate 14 for detection. A chip 10 was manufactured.

The term “quantitative detection chip” or “detection chip” in this specification refers to a structure in which the plate 15 is attached to the lower surface of the upper substrate 14, and includes immunohistochemical staining, immunofluorescence, and color development in situ hybridization. Or refers to the part where FISH, etc. is performed.

The term "PNA FISH" in this specification refers to FISH (fluorescence in situ hybridization) using electrically neutral PNA (pentose nucleic acid) as a probe, and exhibits high substrate specificity during in situ hybridization. It has the advantage of enabling rapid hybridization with only a small concentration of PNA probe, thereby reducing background binding and showing a high signal-to-noise ratio.

The lower substrate 22 is for separating the target sample combined with magnetic particles, and the magnet 21 is recessed without protruding on its upper surface, and the magnet is arranged to correspond to the vertical bottom of the sample injection unit 11. do. According to one embodiment, a hole (Biopunch, 1 mm in diameter) is made in the upper surface of the PDMS chip of the same size as the upper substrate 14, and then the prepared cylindrical magnet (N52, 1.6 mm in diameter) is planted to manufacture the lower substrate 22. did.

The lower substrate 22 is made of poly(dimethylsiloxane) (PDMS), polycarbonate (PC), poly(methyl methacrylate) (PMMA), cyclic olefin copolymer (COC), polystyrene, polysulfones, and polyvinyl chlorite. (PVC), polyethylene (PE), polypropylene (PP), polytetrafluoroethylene (PTFE), poly(L-lactic acid) (PLLA), poly(D, L-lactic acid (PDLLA), poly(glycolic acid) ( PGA), poly(caprolactone) (PCL), poly(hydroxyalkanoate), polydioxanone (PDS), polytrimethylene carbonate, poly(lactic-co-glycolic acid) (PLGA), and poly(L- It may be a polymer material selected from the group consisting of lactic acid-co-caprolactone) (PLCL) and poly(glycolic acid-co-caprolactone) (PGCL), but is not particularly limited thereto, and more specifically, a chip made of PDMS. can be used.

The term "magnetic separator" in this specification is a structure in which a magnet 21 is recessed into the upper surface of the lower substrate 22, and magnetic particles and substances to be detected in the sample (biomolecules, nucleic acids, enzymes, cells, proteins, polysaccharides) , organic substances, bacteria, archaea, eukaryotes, viruses and viroids, or any combination thereof) refers to a part for magnetic separation of a combined complex. The complex may be formed by the bond between a sugar structure (e.g., glycan, etc.) that appears specifically on the surface of a specific bacterium and a magnetic particle coated with an immune protein that recognizes it, or by the electrostatic properties of the bacteria and the magnetic particle. , the method or principle of forming the complex is not particularly limited thereto.

The term “sample” as used herein may be a biological sample, and specifically refers to body fluids (e.g., blood, plasma, serum, saliva, sputum or urine), organs, or body fluids isolated from an individual (mammals, including humans). It may be a tissue, fraction, or cell, but is not particularly limited thereto. More specifically, the sample is selected from the group consisting of blood, urine, feces, saliva, lymph, cerebrospinal fluid, synovial fluid, cystic fluid, ascites, interstitial fluid, and ocular fluid isolated from an individual. It may contain substances to be detected, such as cells or biomolecules. According to one example, FISH was performed on blood samples collected from a pig infection model infected with ESBL (+)- E. coli .

The term "magnetic particles (magnetic beads)" in this specification may include any particle that has magnetism and can bind to a substance to be detected (cells or biomolecules, etc.) in a sample, for example This includes magnetic particles that can bind to opsonized bacteria and magnetic particles coated with immune proteins that bind to specific bacteria. Additionally, in this specification, the term “magnetic particle” may be used interchangeably with the term “magnetic nanoparticle.” Specifically, the magnetic particles include iron (Fe), nickel (Ni), cobalt (Co), manganese (Mn), bismuth (Bi), zinc (Zn), strontium (Sr), lanthanum (La), and cerium ( Ce), Prasheodymium (Pr), Neodymium (Nd), Promethium (Pm), Samarium (Sm), Europium (Eu), Gadolinium (Gd), Terbium (Tb), Dysprosium (Dy), Holmium (Ho) , erbium (Er), thulium (Tm), ytterbium (Yb), ruthenium (Lu), copper (Cu), silver (Ag), gold (Au), cadmium (Cd), mercury (Hg), aluminum (Al) , one or more magnets selected from the group consisting of gallium (Ga), indium (In), thallium (Tl), calcium (Ca), barium (Ba), radium (Ra), platinum (Pt), and lead (Pd). May contain elements. Additionally, the magnetic particles can be manufactured and used through known methods, or they can be purchased and used commercially.

Another aspect includes preparing an upper substrate and a lower substrate made of a polymer material; Preparing plates; Attaching the plate to the lower surface of the upper substrate by plasma treatment; recessing a magnet into the upper surface of the lower substrate without protruding; and stacking the upper and lower substrates so that the plate is interposed between the upper and lower substrates. The same parts as described above also apply to the manufacturing method.

The microfluidic chip 30 of the present invention has a structure in which the detection chip 10 and the magnetic separator 20 are stacked up and down, and the magnet 21 of the magnetic separator holds the detection chip with the plate 15 in between. It is located below each injection part 11, and when a sample is injected into the injection part, detection can be performed with cells combined with magnetic particles separated and concentrated at the bottom of the injection part, and detection of the target sample is possible. Efficiency and accuracy can be improved.

In addition, after performing FISH, etc. on a specific sample, detection of another sample can be performed more quickly by leaving the magnetic separator 20 as is, replacing only the detection chip 10, and stacking it again on the magnetic separator. It has the advantage of dramatically shortening the time required when repeated target sample detection is required for the same or different samples.

Another aspect includes preparing a sample containing a substance to be detected; Processing the sample with magnetic particles to form a detection target material-magnetic particle complex; Injecting a sample containing the complex into the microfluidic chip sample injection unit of claim 1; Discharging unnecessary samples through the microfluidic chip discharge unit of claim 1; Processing a sample containing the complex with a reagent for immobilization and permeabilization; To provide a method for detecting cells or biomolecules, including the step of treating the immobilized and permeabilized sample with a reagent for labeling or staining. The same parts as described above also apply to the detection method.

The labeling may be performed using an optical label, an electrical label, a radioactive label, an enzyme label, or a combination thereof. Specifically, it may be fluorescent labeling using an optical label. The optical label is a substance that generates fluorescence or phosphorescence. For example, the substance that generates fluorescence may be fluorescein, rhodamine, cyanine, metal porphyrin complex, Cy-5, or Cy-3. , but is not particularly limited thereto.

In addition, the staining includes DAPI (4', 6-diamidino-2-phenylindole), methylene blue, acetocarmine, toluidine blue, and hematoxylin for cell nucleus staining. It may be Hoechst or any combination thereof, and may be eosin, crystal violet, orange G, or any combination thereof for cytoplasmic staining, but is specifically limited thereto. That is not the case.

According to one aspect, the microfluidic chip of the present invention can image or quantify cells over time by magnetically separating cells such as bacteria bound to magnetic particles in a sample and performing FISH, etc., and use this as an indicator for diagnosing bacterial infection. Alternatively, it can be used to predict antibiotic susceptibility.

Figure 1 shows a top view of a microfluidic chip according to one aspect.
Figure 2 shows a perspective view of the detection chip of the present invention according to one aspect.
Figure 3 shows a perspective view of the magnetic separator of the present invention according to one aspect.
Figure 4 shows a side view of the microfluidic chip of the present invention according to one aspect.
Figure 5 shows a microfluidic chip of the present invention with a detection chip and a magnetic separator stacked on top of each other.
Figure 6 shows the results of FISH using an E. coli sample bound to magnetic beads.
Figure 7 shows changes in Bacterial (Universal bacteria and E. coli ) FISH count in pig blood according to infection time in a pig infection model infected with ESBL(+)- E. coli .
Figure 8 shows the results of comparing E. coli FISH counts in the blood of E. coli infected patients (Infected) and non-infected controls (Non-infected).
Figure 9 shows the results of comparing changes in Universal bacteria and E. coli FISH counts in the blood of sepsis patients before and after antibiotic treatment.

This will be described in more detail through examples below. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples.

Example 1: Production of quantitative detection chip

The device of the present invention includes a detection chip including at least one sample injection unit (Inlet, diameter 3 mm), one outlet, and a microfluidic channel connecting them to each other, and a magnet to correspond to the vertical bottom of each sample injection unit. As a structure including the arranged magnetic separator, first, a PDMS chip designed as shown in Figure 1 was manufactured with a size of 24 mm in width, 50 mm in length, and 3 mm in height, and the same as the PDMS chip was produced through plasma treatment (80 W, 1 min). A detection chip was manufactured by attaching a sized cover glass to the bottom of the PDMS chip (Figure 2).

Example 2: Fabrication of magnetic separator and microfluidic chip

In order to place a cylindrical magnet at a position corresponding to the vertical bottom of each injection part on the detection chip of Example 1, a cylindrical magnet was placed on a PDMS chip of the same size as the detection chip at a position corresponding to the injection part. After making a hole (Biopunch, 1 mm in diameter), a prepared cylindrical magnet (N52, 1.6 mm in diameter) was planted to produce a magnetic separator (FIG. 3). Afterwards, the detection chip of Example 1 was placed on top of the magnetic separator to finally complete the microfluidic chip (FIGS. 4 and 5).

Example 3: E. coli Perform FISH using

E. coli (K-12 W3110, Korean Collection for Type Culture (KCTC) 2223) bound to magnetic particles was prepared as a sample and FISH was performed. First, the E. coli sample was injected into the inlet well and magnetically separated for 10 minutes, and then unnecessary solution was discharged through the outlet at a flow rate of 10 μl/min using a syringe pump. For subsequent immobilization and _{permeabilization} of the samples, ₂₄ % (v/v) ethanol in 1 The reagents were treated with 1X TBST with 5 mM CaCl ₂ (3 minutes). For fluorescent probe hybridization of the sample, the E. coli PNA FISH probe (5'-CTTTACTCCCTTCCT-3') in hybridization solution (1 μM) was treated at 45°C for 1 hour. In addition, for counterstaining of cell nuclei, DAPI (10 ㎍/mL in 2X SSC) was treated at room temperature for 30 minutes, followed by washing using 2X SSC buffer and imaging through a fluorescence microscope.

As a result, it was confirmed that the E. coli PNA FISH probe was successfully bound to the E. coli bound to the magnetic particles and detected fluorescence (FIG. 6).

Example 4: FISH performed using a pig infection model

The amount of E. coli in the blood was quantified using a pig infection model (n = 7) infected with ESBL(+)- E. coli . Blood samples from the control group (CTL) were collected through a catheter connected to the artery, E. coli (10 ¹⁰ CFU per one porcine model) was injected into the blood vessel, and blood was collected at 1-hour intervals (0 h to 11 h). . The antibiotic Ceftriaxone was administered immediately after blood collection 1 hour after infection (1 h), and the ESBL (+)- E. coli used in the experiment is known to be resistant to this. Blood samples collected over time (DNA/RNA ShieldTM blood collection tube; R1150, Zymo Research, CA, USA) contained an immune protein that binds to E. coli (Human recombinant mannose binding lectin; 10405-HNAS, Sino Biological, China). Coated magnetic beads (diameter 200 nm; 03121, Ademtech, France) were treated to magnetically separate and concentrate only E. coli in the blood, and a concentrated sample was prepared, and then the E. coli in the sample was quantified using the manufactured microfluidic chip. proceeded.

As a result, the total amount of bacteria (universal bacteria) and the amount of E. coli in the blood of the pig infection model were quantified by time (Figure 7), and the FISH count of bacteria in the blood did not decrease until 8 hours after infection. It was confirmed that the infection was not recovering.

Example 5: FISH using infected patient blood

Example 5-1: Using Quantitative FISH E. coli infection diagnosis

FISH quantification comparison was performed using blood samples from E. coli- infected patients (where E. coli was cultured from specimens such as blood, urine, sputum, and bile) and blood samples from non-infected control groups. As a result, it was confirmed that a higher E. coli FISH signal was detected in the blood of patients with E. coli infection compared to the control group, proving that the results using the present invention can be used as a diagnostic indicator for bacterial infection. (Figure 8).

Example 5-2: Antibiotic susceptibility prediction using quantitative FISH

We attempted to predict antibiotic susceptibility through changes in bacterial FISH counts in the blood of sepsis patients following antibiotic treatment. As a result, there were cases where the bacterial FISH count in the blood significantly changed (increased or decreased) due to antibiotic treatment (#46, 47, 50, 51, 54, 58, 59, 64, 65, 70, 71) and cases where it did not change. Patients were classified into cases (#44, 52, 55, 56, 57, 60, 62, 68), and it was confirmed that antibiotic susceptibility could be predicted from the degree of change in FISH count before and after antibiotic treatment (Figure 9 ).

The description of the present invention described above is for illustrative purposes, and those skilled in the art will understand that the present invention can be easily modified into other specific forms without changing the technical idea or essential features of the present invention. will be. Therefore, the embodiments described above should be understood in all respects as illustrative and not restrictive.

10: Detection chip
11: Sample injection part
12: Microfluidic channel
13: discharge part
14: upper substrate
15: plate
20: magnetic separator
21: magnet
22: lower substrate
30: Microfluidic chip

Claims (10)

A microfluidic chip including upper and lower substrates stacked up and down, and a plate interposed between the upper and lower substrates,
The upper substrate includes a sample injection unit;
discharge part; and
It includes a microfluidic channel in communication with the sample injection unit and discharge unit,
A microfluidic chip wherein a magnet for magnetic separation of the sample is recessed on the upper surface of the lower substrate, and the magnet is arranged to correspond to the vertical bottom of the sample injection unit.
The microfluidic chip according to claim 1, wherein the sample injection unit, the microfluidic channel, and the magnet are provided as one or more.
The microfluidic chip according to claim 1, wherein the plate is any one selected from the group consisting of slide glass, crystal, and cover glass.
The method of claim 1, wherein the upper and lower substrates include poly(dimethylsiloxane) (PDMS), polycarbonate (PC), poly(methyl methacrylate) (PMMA), cyclic olefin copolymer (COC), polystyrene, and polysulfones. , polyvinyl chlorite (PVC), polyethylene (PE), polypropylene (PP), polytetrafluoroethylene (PTFE), poly(L-lactic acid) (PLLA), poly(D, L-lactic acid (PDLLA), poly (Glycolic acid) (PGA), poly(caprolactone) (PCL), poly(hydroxyalkanoate), polydioxanone (PDS), polytrimethylene carbonate, poly(lactic-co-glycolic acid) (PLGA) and poly(L-lactic acid-co-caprolactone) (PLCL) and poly(glycolic acid-co-caprolactone) (PGCL).
The microfluidic chip according to claim 1, wherein the sample interacts with magnetic particles to form a complex of the detection target material and magnetic particles in the sample.
The method of claim 5, wherein the substance to be detected is selected from the group consisting of biomolecules, nucleic acids, enzymes, cells, proteins, polysaccharides, organic substances, bacteria, archaea, eukaryotes, viruses and viroids, or any combination thereof. This is a microfluidic chip.
The method of claim 5, wherein the sample is selected from the group consisting of blood, urine, feces, saliva, lymph, cerebrospinal fluid, synovial fluid, cystic fluid, ascites, interstitial fluid, and ocular fluid isolated from the individual. The one chosen is the microfluidic chip.
The microfluidic chip of claim 1, wherein the upper substrate is for performing immunohistochemistry, immunofluorescence, chromogenic in situ hybridization, or FISH (fluorescence in situ hybridization).
Preparing an upper substrate and a lower substrate made of polymer material;
Preparing plates;
Attaching the plate to the lower surface of the upper substrate by plasma treatment;
recessing a magnet into the upper surface of the lower substrate without protruding; and
A method of manufacturing a microfluidic chip, comprising the step of stacking the upper and lower substrates so that the plate is interposed between the upper and lower substrates.
Preparing a sample containing a substance to be detected;
Forming a detection target material-magnetic particle complex by treating the sample with magnetic particles;
Injecting a sample containing the complex into the microfluidic chip sample injection unit of claim 1;
Discharging unnecessary samples through the microfluidic chip discharge unit of claim 1;
Processing a sample containing the complex with a reagent for immobilization and permeabilization;
A method for detecting cells or biomolecules, comprising treating the immobilized and permeabilized sample with a reagent for labeling or staining.