TWM581591U - Microchannel chip having slack flow block with small aperture, and microchannel structure - Google Patents

Abstract

This creation is a microchannel structure with a small-aperture slow-flow section, including: a detection section with a first end and a second end, wherein the first end is used to receive the microfluid sample and rest in the detection area Test or process the microfluidic sample with a first aperture at the second end; and a slow-flow section connected to the second end with a second aperture, wherein the second aperture is smaller than the first aperture to delay the micro The velocity of the fluid sample flowing through the detection zone.

Description

Micro-flow channel wafer with small-aperture slow-flow section and micro-flow channel structure

This creation is about a micro-channel chip and a micro-channel structure that increases the capture rate of biological substances, especially a micro-channel chip and a micro-channel structure with a small-aperture slow flow section.

Detecting blood circulating tumor cells (Circulating tumor cells, CTC) is a potential way to judge the occurrence and spread of cancer, so the severity of cancer often depends on accurate counting of CTCs or molecular markers. According to research, for the prognostic evaluation of tumor patients, cancer cell metastasis in blood is the cause of patient death, so cancer detection is closely related to CTCs.

The number of CTCs will change dynamically, depending on the changes in the tumor itself and the response to treatment, so it can be used for early diagnosis in vitro, rapid evaluation of drugs, personalized treatment and other applications. However, in patients with metastatic cancer, CTCs are rare cells, with one CTC per ¹⁰⁹ blood cells, making it technically difficult to detect and isolate CTCs. Therefore, a centralized collection method must be used to effectively detect and isolate CTCs.

One example of the current centralized collection method is the use of highly overexpressing cell surface biomarkers, such as epithelial cell adhesion molecules (EpCAM), that are highly specific and sensitive to CTC. Nagrath Et al. (Nature 2007, 450: 1235-9) developed anti-EpCAM antibody-coated microfluidic wafers for CTC detection and collection. However, the drawback of the above technique is the low detection rate of pure CTC, which is attributed to the non-specific binding of blood cells to anti-EpCAM antibodies.

Despite advances in the technology for detecting and isolating CTCs, more specific and effective methods are needed to detect, purify, and release CTCs and other biological substances for further cultivation and characterization.

The reason for this is that the applicant, in view of the lack of known technology, has carefully studied and researched, and has persevered in the spirit, and finally created this case, "Microchannel Chips and Microchannels with Small Aperture Slow Flow Section Structure "to improve the shortcomings of the conventional techniques.

This creation is a new type of microfluidic system, which includes a microfluidic wafer and beads located in the microfluidic wafer and capable of grasping circulating tumor cells to separate circulating tumor cells from blood cells. The created microchannel structure and microchannel chip especially include a small-aperture slow-flow section, which can slow down the flow rate of microfluid samples in the microchannel structure and the microchannel chip, and prevent beads from entering the slow-flow Section.

The principle of the created microfluidic system is to use the characteristics of circulating tumor cell surface antigens and antibodies implanted on the bead surface to capture the bead structure. The bead structure leads to the largest contact area per unit volume, followed by the fluid resistance of the microfluidic structure. The curved structure causes turbulence, which causes the circulating tumor cells to rotate or roll and increase the chance of contact with the beads to enhance the grasping effect, and the special design of the microfluidic channel structure reduces the non-EpCAM antibody Specific binding.

One aspect of this creation is to provide a micro-flow channel wafer carrying a bead having a particle size, including: a substrate; a body having a first surface and a second surface, wherein the second surface is closely adhered Covering the substrate; and a microfluidic channel structure embedded in the second surface, so that the microfluidic channel structure forms a microfluidic channel between the body and the substrate for a blood sample to be in the microfluidic channel structure Medium flow, wherein the microchannel structure includes: a detection section, the bead is disposed in the detection section; and a slow-flow section, connected to the detection section, with a depth, wherein the The particle size is greater than the depth to retard the speed at which the blood sample flows through the detection zone and prevent the beads from entering the flow resistance restriction zone.

Another aspect of this creation is to provide a microfluidic channel structure including a structural body for passing a microfluid sample through the microfluidic channel structure to be inspected or processed. The structural body includes: a detection area The segment has a first end and a second end, wherein the first end is used to receive the microfluid sample, and the microfluid sample is tested or processed in the detection section, and the second end is provided with a first An aperture; and a slow flow section connected to the second end and having a second aperture, wherein the second aperture is smaller than the first aperture to retard the velocity of the microfluidic sample flowing through the detection section.

In order to make the above and other objects, features, and advantages of this creation more comprehensible, the preferred embodiments are described below in conjunction with the accompanying drawings for a detailed description.

10‧‧‧Micro channel chip

100‧‧‧ substrate

200‧‧‧ Ontology

210‧‧‧first surface

220‧‧‧ second surface

300‧‧‧Micro channel structure

310‧‧‧ Blood sample entrance

320‧‧‧ Expansion Section

330‧‧‧Resistance section

340‧‧‧ Detection section

341‧‧‧ the first end

342‧‧‧Detect main area

343‧‧‧second end

350‧‧‧ Slow stream section

360‧‧‧ blood sample export

40‧‧‧ beads

50‧‧‧Micro channel structure

500‧‧‧ structure ontology

510‧‧‧Microfluid sample inlet

520‧‧‧Increase resistance section

530‧‧‧ Detection section

540‧‧‧ Slow section

550‧‧‧Microfluidic sample outlet

60‧‧‧ Beads

FIG. 1 is a schematic top view of a creative microchannel wafer; Figure 2 is a schematic diagram of the longitudinal section of AA 'in Figure 1; Figure 3 is a schematic diagram of the beads set in the detection section of the creative microchannel chip; Figure 4 is a microchannel chip Schematic diagram of the depth of the middle slow flow section smaller than the particle size of the beads; Figure 5 is a schematic diagram of the microchannel structure in another embodiment of the creation; Figure 6 is the result of the recovery efficiency and detection limit of the microfluid-free system Figure; Figure 7 is the result of the recovery efficiency and detection limit of the creative microchannel chip; Figures 8 (A) -8 (C) are the separation results of the blood sample passing through the creative microchannel chip Illustration.

The following describes the embodiments of the "microchannel chip and microchannel structure with a small-aperture slow-flow section" in this case. Please refer to the drawings, but the actual configuration and the method adopted do not have to be completely consistent. The content described, those skilled in the art can make various changes and modifications without departing from the actual spirit and scope of the case.

An example of this creation is the separation of circulating tumor cells from the blood. The microfluidic chip has a plurality of transparent beads inside. When the beads capture the circulating tumor cells, the circulating tumor cells will be separated from the blood and positioned in the detection section, and the remaining normal blood cells will flow out from the outlet. Instead, it flows into the waste storage tank. In order to capture and isolate circulating tumor cells in the blood, the substance coated on the surface of the beads is preferably an antibody against epithelial cell adhesion molecules (Epithelial Cell Adhesion Molecule, EpCAM).

The bead carried on the microchannel wafer of this creation is especially a large bead with a particle size of 100-200 μm. The surface of the bead is coated with a reactive substance, and the reactive substance package Contains (1) a releasable composition that releases or removes non-specific blood cells and other blood components (such as proteins); (2) a biologically active composition that captures biological substances; or (3) a link to a releasable composition and a biologically active composition Linker. When the microfluidic sample flows through the bead, the bead can adsorb the biological material in the microfluidic sample that can react with the reactive material on the surface of the bead, and can release the adsorbed biological material for further research and detection. The material of the beads is transparent plastic or transparent resin. The microfluidic sample may be a body fluid or a bacterial fluid, and the body fluid includes blood, cerebrospinal fluid, various digestive fluids, semen, saliva, sweat, urine, vaginal fluid, or a solution containing biological substances. Biological substances include CTC, CTC circulating stem cells (such as tumor stem cells, liver stem cells, and bone marrow stem cells), fetal cells, bacteria, viruses, epithelial cells, endothelial cells, or other biological substances. Therefore, depending on the object (biological substance) to be grasped, the reactive substance applied on the bead surface is also different.

Please refer to FIGS. 1, 2 and 3, which are a schematic plan view of the microfluidic wafer and a vertical cross section along A-A '. The created microfluidic wafer 10 includes a substrate 100, a body 200 and a microfluidic structure 300. The body 200 has a first surface 210 and a second surface 220 opposite to the first surface 210. The microchannel structure 300 is embedded in the second surface 220 of the body 200, and the second surface 220 is closely covered on the substrate 100. The microfluidic channel structure 300 is formed between the body 200 and the substrate 100.

The created microfluidic channel structure 300 includes a blood sample inlet 310, an expansion section 320, a resistance increasing section 330, a detection section 340, a slow flow section 350, and a blood sample outlet 360 in order from the entrance to the exit.

The author creates a blood sample inlet 310 from the first surface 210 of the body 200 Extends to the second surface 220 for blood samples to enter the flow channel. The blood sample inlet 310 may be a circular hole or a polygonal hole, preferably a circular hole. The diameter of the blood sample inlet 310 of this creation is between 0.8-1.2mm, and it can accommodate a syringe with an 18-21G needle (about 0.7-0.9mm).

One end of the creative expansion section 320 is connected to the blood sample inlet 310 and the other end is connected to the resistance increasing section 330. The aperture of the expansion section 320 may be circular or polygonal, preferably square. The width of this creative expansion section 320 is between 0.8-1.5mm, and the depth is 1mm.

One end of the creative resistance increasing section 330 is connected to the expansion section 320 and the other end is connected to the detecting section 340. The aperture of the resistance increasing section 330 may be circular or polygonal, preferably square. The width of the resistance-increasing section 330 is 250-500 μm, and the depth is 1 mm.

The creation detection section 340 includes a first end 341, a detection main area 342, and a second end 343. The first end 341 is connected to the resistance increasing section 330, and the second end 343 is connected to the slow flow section 350. The main measurement region 342 is located between the first end 341 and the second end 343, and a bead 40 (as shown in FIG. 3) capable of adsorbing circulating tumor cells in the blood is provided. The aperture of the detection section 340 may be circular or polygonal, preferably square. In the embodiment of the present invention, the aperture of the detection section 340 is square. The depth of the detection section 340 is the particle size of the bead 40 plus 20-50 μm. Therefore, the depth of the detection section 340 is between 120-250 μm. The widths of the first end 341 and the main detection area 342 of the detection section 340 are sufficient for the beads 40 to pass through, and therefore are between 250 μm and 1.5 mm. The width of the first end 341 of the detection section 340 may be the same as the width of the resistance increasing section 330 or from the width of the resistance increasing section 330. The degree gradually increases to the width of the first end 341. The width of the second end 343 of the detection section 340 of the present creation is between 150-250 μm.

One end of the creation slow flow section 350 is connected to the second end 343 of the detection section 340, and the other end is connected to the blood sample outlet 360. The aperture of the slow-flow section 350 may be circular or polygonal, preferably square. In the present creative embodiment, the aperture of the slow-flow section 350 is square. The width of the slow-flow section 350 may be equal to or smaller than the width of the second end 343 of the detection section 340, and the depth of the slow-flow section 350 is smaller than the depth of the detection section 340. The width of the slow-flow section 350 is between 150-250 μm, and the depth of the slow-flow section 350 is between 50-100 μm.

In order to prevent the bead 40 from entering the slow-flow section 350, the particle diameter of the bead 40 is larger than the depth of the slow-flow section 350 (as shown in FIG. 4), so that the bead 40 can resist the second part of the detection section 340. End 343.

In order to stabilize the flow velocity of the blood sample in the micro-channel wafer 10, the author specially designed a slow-flow section 350 with a small aperture. The small-aperture slow-flow section 350 includes: (1) the pore size of the slow-flow section 350 is smaller than the pore size of the detection section 340 (as shown in Figures 1 and 3), and the pore diameter of the 340 section of the detection area may be a slow-flow 1.2-50 times the pore diameter of section 350; and (2) the particle diameter of bead 40 is greater than the depth of the slow-flow section (as shown in Figure 4). The small-aperture slow-flow section 350 can increase the fluid resistance and slow down the flow rate of the blood sample in the microchannel wafer 10, so that the blood sample will not be caused by the increased pressure or unstable force when injected from the blood sample inlet 310. The uneven flow rate can ensure that the circulating tumor cells always have the same flow rate when detecting the main region 342, thereby increasing the probability of the circulating tumor cells adsorbing to the beads 40.

The slower the flow rate of the blood sample, the higher the adsorption efficiency of the beads 40. Table 1 shows the effect of the depth of the slow flow section 350 on the adsorption of biological substances in the blood sample by the beads 40.

It can be known from Table 1 that when the depth of the slow-flow section 350 is 100 μm, the adsorption efficiency of the beads 40 is 10-20%, and when the depth of the slow-flow section 350 is 50 μm, the The adsorption efficiency is 88%. Therefore, the smaller the depth of the slow-flow section 350 is, the smaller the cross-sectional area of the slow-flow section 350 is, and the lower the flow velocity and the flow rate of the blood sample in the micro-flow channel structure 300 is. The higher the adsorption efficiency of the beads 40 is. .

One end of the creative blood sample outlet 360 is connected to the slow-flow section 350, and the other end extends from the second surface 220 to the first surface 210 of the body 200. Blood cells not captured by the bead 40 will flow to the waste liquid recovery area (not shown) through the blood sample outlet 360. The blood sample outlet 360 may be a round hole or a square hole, preferably a round hole, and its diameter is between 0.8-1.2 mm.

The following table 2 is a preferred embodiment of the particle diameter of the bead 40 and the pore diameter of each section in the microchannel structure 300.

Table 2

The material of the substrate 100 can be acrylic (polymethylmethacrylate, PMMA), polyethylene terephthalate (PET), polycarbonate (PC), or polydimethylsiloxane ( polydimethylsilicon (PDMS), silicone, rubber, plastic or glass. The material of the body 200 may be acrylic (polymethylmethacrylate (PMMA)), polyethylene terephthalate (PET), polycarbonate (PC), polydimethylsilicon (PDMS), silicone, rubber or plastic. When selecting materials for the substrate 100 and the body 200, the material characteristics between the substrate 100 and the body 200 must be considered. In the embodiment of the present invention, the substrate 100 is glass, and the body 200 is polydimethylsiloxane.

The present invention further provides another embodiment of the microchannel structure 50, as shown in FIG. The microchannel structure 50 contains beads 60 and has a structure body 500. The structure body 500 includes a microfluid sample inlet 510, a resistance increasing section 520, a detection section 530, a slow flow section 540, and The fluid sample outlet 550, in which the bead 60 is located in the detection section 530, and the pore diameter of the slow flow section 540 is smaller than the pore diameter of the detection section 530 to retard the flow velocity of the microfluid sample. After the microfluidic sample enters from the microfluidic sample inlet 510, it can directly enter the detection section 530 through the resistance increasing section 520, and the beads 60 in the detection section 530 capture the biological material in the microfluidic sample to After the microfluidic sample is inspected or processed, it enters the slow flow section 540, and finally flows out of the microfluidic channel structure 50 from the microfluidic sample outlet 550.

The method of preparing the micro-fluidic wafer of this creation is to first use a 3D printer to print a master mold. The master mold is a light-curing resin, which is rinsed with 95% alcohol, and then cured by UV light for 2 minutes. Bake for 10 minutes. The food-grade material PDMS liquid was poured into the master mold in proportion to the curing process for 50 minutes and 80 degrees, and then bonded to the glass substrate with an oxygen plasma machine.

Experimental example

Study on the Circulating Tumor Cell Beads in Cultured Circulating Tumor Cells

1. Recovery efficiency and detection limit of large beads (200 microns in diameter) in microfluid-free systems

Put 10, 1,000, and 100,000 circulating tumor cells and beads and 1 mL of physiological saline buffer solution (simulating blood environment) into a centrifuge tube, and fully circulate the tumor cells and beads in physiological saline buffer solution. After mixing, observe the grasping efficiency of the beads. According to Figure 6, the experimental results show that only 1.5% of the cells (about 1500) were captured by the beads in the experimental group with 100,000 circulating tumor cells, but the experiments with 10 and 1000 circulating tumor cells In the group, the beads did not capture any circulating tumor cells, which means that the blood environment of less than 1000 circulating tumor cells, the beads could not capture any circulating tumor cells.

2.Recycling efficiency and detection limit of large beads (200 microns in diameter) in the microchannel wafer

10, 50, 100, 500, and 1000 circulating tumor cells were mixed with 1 mL of physiological saline buffer solution, and the mixed liquid sample was passed through the beads in the microchannel wafer of this creation, and the beads were observed Body grabbing efficiency. According to Figure 7, the experimental results show that using the microfluidic wafer of this creation, a liquid sample containing more than 50 circulating tumor cells can be grasped, compared with the results obtained without a microfluidic system (requiring 100,000 cells to (Captured, as shown in Figure 7), the detection limit is significantly reduced by 2000 times, and the recycling efficiency of the micro-fluidic wafers using this creation is on average higher than 5%, which is about 3 times higher than the recycling efficiency of microfluid-free systems .

When an average of about 50 circulating tumor cells in the blood of a human body per 10 mL, it means that the person has a high risk of cancer. Therefore, this experiment proves that only large beads (200 micrometers in diameter) cannot distinguish the risk of human cancer. However, large beads combined with the microchannel chip created by this method can effectively and accurately capture circulating tumor cells in the blood, which can Find out faster if you have cancer.

Please refer to Figures 8 (A) -8 (C), which are the separation results of the microfluidic wafer created by the actual staining of blood samples of cancer patients, where green is circulating tumor cells and red is white blood cells. Figure 8 (A) shows that circulating tumor cells (green dots) are captured on transparent beads, and Figure 8 (B) is white blood cells (red dots). After incorrect capture, Figure 8 (C) is Figures 8 (A) and 8 (B) are synthesized to obtain the positions of all captured cells. This experiment demonstrates the results exhibited by patients with stage II cancer in the microfluidic chip of this creation. The results show that about 13 circulating tumor cells were captured, and only 3 white blood cells were captured (because of 1mL of blood in the human body) The number of white blood cells is between about 10 ⁶ and 10 ^7. According to strict definition, it is estimated by 10 ⁶ white blood cells, that is, only 3 white blood cells are captured by one million white blood cells, which is far lower than the current FDA proof. Cellsearch instrument's catch rate (10 ⁶ white blood cells caught about 3000 ~ 4000)). In addition, the results of this experiment took only 30 minutes from the blood sample acquisition to the imaging results display, compared with the previous pre-processing and circulating tumor cell separation to read the imaging results in 6-9 hours, which is much shorter in time. Therefore, using the microfluidic chip of this creation can effectively capture a small amount of circulating tumor cells in the blood, has a very low error rate, and the result can be obtained in only 30 minutes, so the microfluidic chip of this creation It can be used as a quick-screen biochip for preliminary detection of cancer.

Other embodiments

1. A micro-flow channel wafer carrying a bead having a particle size, comprising: a substrate; a body having a first surface and a second surface, wherein the second surface is closely covered on the substrate And a microchannel structure embedded in the second surface, so that the microchannel structure forms a microchannel between the body and the substrate for a blood sample to flow in the microchannel structure, wherein the The micro-channel structure includes: a detection section in which the beads are arranged; and a slow-flow section connected to the detection section with a depth, wherein the particle diameter is greater than the depth To retard the speed at which the blood sample flows through the detection zone and prevent the beads from entering the flow resistance restriction zone.

2. The micro-channel wafer according to embodiment 1, wherein the particle diameter is 100-200 μm, and the depth is 50-100 μm.

3. The micro-channel wafer according to embodiment 2, wherein when the particle diameter is 100 μm, the depth is 50 μm, and when the particle diameter is 200 μm, the depth is 100 μm.

4. The micro-flow channel wafer according to embodiment 1, wherein the material of the substrate is polymethylmethacrylate (PMMA), polyethylene terephthalate (PET), and polycarbonate (polycarbonate, PC), polydimethylsilicon (PDMS), silicone, rubber, plastic or glass, and the material of the body is polymethylmethacrylate (PMMA), polyethylene terephthalate , PET), polycarbonate (PC), polydimethylsilicon (PDMS), silicone, rubber or plastic.

5. The micro-channel wafer according to embodiment 4, wherein the first surface is disposed opposite to the second surface.

6. A microfluidic channel structure comprising a structural body for passing a microfluid sample through the microfluidic channel structure to be inspected or processed, wherein the structural body includes: a detection section with a first And a second end, wherein the first end is used to receive the microfluid sample, and the microfluid sample is tested or processed in the detection section, and the second end has a first aperture; and a slow flow The segment is connected to the second end and has a second aperture, wherein the second aperture is smaller than the first aperture to delay the velocity of the microfluidic sample flowing through the detection segment.

7. The microchannel structure according to embodiment 6, wherein the detection section includes a detection main area between the first end and the second end, the detection main area has a third aperture, and The third aperture is 1.2 to 50 times the first aperture.

8. The micro-channel structure according to embodiment 7, wherein the first aperture has a first width, the second aperture has a second width, and the first width is the same as the second width.

9. The microchannel structure according to embodiment 6, wherein the detection section has a bead, the bead has a bead pore size, the second pore size has a depth, and the bead pore size is greater than the depth .

10. The microchannel structure according to embodiment 6, wherein the microfluidic sample is a body fluid or a bacterial fluid.

To sum up, the new model can indeed use a novel concept to match the micro-fluidic chip created by this creation with large beads, which can effectively capture the micro- The amount of circulating tumor cells and reduce the rate of false positives to determine the occurrence of cancer early. In addition, the microfluidic chip created by this invention has a small-aperture slow-flow section with a smaller pore diameter than the detection section and a smaller depth than the bead to increase the fluid resistance in the microfluidic chip to slow the microfluidic sample in the microfluidic flow. The flow velocity in the channel structure and prevents beads from entering the slow flow section. Therefore, those who are familiar with this technique can be modified by various techniques, but they are not inferior to those who want to protect the scope of patent application.

Claims (10)

A micro-flow channel wafer carrying a bead having a particle size, comprising: a substrate; a body having a first surface and a second surface, wherein the second surface is closely covered on the substrate; and A micro-channel structure is embedded on the second surface, so that the micro-channel structure forms a micro-channel between the body and the substrate for a blood sample to flow in the micro-channel structure, wherein the micro-flow The channel structure includes: a detection section in which the beads are arranged; and a slow flow section connected to the detection section with a depth, wherein the particle diameter is greater than the depth, and Delaying the speed at which the blood sample flows through the detection zone and preventing the beads from entering the flow restriction zone. The micro-channel wafer according to item 1 of the patent application range, wherein the particle diameter is 100-200 μm, and the depth is 50-100 μm. The micro-channel wafer according to item 2 of the scope of patent application, wherein when the particle diameter is 100 μm, the depth is 50 μm, and when the particle diameter is 200 μm, the depth is 100 μm. The micro-fluidic wafer as described in item 1 of the patent application scope, wherein the material of the substrate is polymethylmethacrylate (PMMA), polyethylene terephthalate (PET), and polycarbonate (PC), polydimethylsilicon (PDMS), silicone, rubber, plastic or glass, and the material of the body is polymethylmethacrylate (PMMA), polyethylene terephthalate (polyethylene terephthalate (PET), polycarbonate (PC), polydimethylsilicon (PDMS), silicone, rubber or plastic. The micro-flow channel wafer according to item 1 of the patent application scope, wherein the first surface is disposed opposite to the second surface. A microfluidic channel structure includes a structural body for passing a microfluid sample through the microfluidic channel structure to be inspected or processed, wherein the structural body includes: a detection section having a first end and A second end, wherein the first end is used for receiving the microfluid sample, and the microfluid sample is tested or processed in the detection section, and the second end has a first aperture; and a slow-flow section Is connected to the second end and has a second aperture, wherein the second aperture is smaller than the first aperture to delay the velocity of the microfluidic sample flowing through the detection section. According to the micro channel structure described in the patent application item 6, wherein the detection section includes a detection main area between the first end and the second end, the detection main area has a third aperture, And the third aperture is 1.2 to 50 times the first aperture. The micro-channel structure according to item 7 of the scope of patent application, wherein the first aperture has a first width, the second aperture has a second width, and the first width is the same as the second width. The microchannel structure according to item 6 of the patent application scope, wherein the detection section has a bead, the bead has a bead pore size, the second pore size has a depth, and the bead pore size is larger than the depth. The microfluidic channel structure described in item 6 of the patent application scope, wherein the microfluidic sample is a body fluid or a bacterial fluid.