A rapid detection microfluidic chip and detection method for prostate cancer markers

Abstract

The present invention provides a rapid detection microfluidic chip and detection method for prostate cancer markers, which belongs to the technical field of rapid screening of prostate cancer. The technical problems of the existing detection methods being cumbersome to operate and having a long detection cycle are solved. The technical solution is as follows: it includes a microarray structure for blood separation and a color development microchannel structure for detecting free prostate-specific antigen F-PSA and total prostate-specific antigen T-PSA respectively; the microarray composed of "V"-shaped grooves forms a blood separation structure, which separates red blood cells, white blood cells, and large platelet particles in the blood by physical separation; the functional fragments of F-PSA and T-PSA are fixed and captured in advance in the color development microchannel structure, and the semi-quantitative detection of F-PSA and T-PSA is achieved by measuring the color development length of the color development microchannel. The beneficial effects of the present invention are: the microfluidic chip of the present invention is simple to operate, the results are easy to read, and the analysis is fast.

Description

Technical Field

[0001] The present invention relates to the technical field of rapid screening of prostate cancer, and in particular to a rapid detection microfluidic chip and a detection method for prostate cancer markers. Background Art

[0002] Prostate cancer is a highly prevalent cancer among men. Due to its subtle early symptoms and lack of specific clinical symptoms, most prostate cancer patients in my country are already in the advanced stages of the disease by the time they are first diagnosed, necessitating the attention of men. Over the past decade, prostate cancer has become a "double-high" malignancy in my country, with both a rapidly increasing incidence and mortality rate. According to the latest data from the my country Cancer Burden Report released by the National Cancer Center, both the incidence and mortality rates of prostate cancer in China have continued to rise, making prostate cancer the sixth most common malignant tumor among Chinese men. Early detection of prostate cancer with radical treatment offers a nearly 100% five-year survival rate. However, for patients who delay treatment until the advanced stages, the five-year survival rate is only 30%. Therefore, strengthening screening, early diagnosis, and treatment for high-risk individuals is crucial for improving the overall survival rate of prostate cancer patients in China.

[0003] Currently, screening for prostate-specific antigen (PSA) is the preferred recommended screening method for prostate cancer. Clinically, the ratio of free prostate-specific antigen (F-PSA) to total prostate-specific antigen (T-PSA) in serum is often used as the basis for the diagnosis of prostate cancer. However, obtaining serum samples requires centrifugation of the collected blood samples using centrifugation equipment in the hospital's central laboratory. Clinical detection methods for F-PSA and T-PSA generally use fluorescent immunoassay, radioimmunoassay, or electrochemiluminescence. These detection methods have disadvantages such as cumbersome operating steps, long detection cycles, and high analysis costs. In addition, many male patients are bound by traditional ideas and are reluctant to seek medical treatment, which also reduces the early screening rate for prostate cancer. Therefore, there is an urgent need to develop a rapid detection device for prostate cancer biomarkers suitable for home self-testing.

[0004] With the rapid development of materials science, microelectromechanical processing technology, and microelectronics, the manufacturing technology of microfluidic chips has also been rapidly improved. They can integrate the basic operating units involved in routine biochemical analysis, such as sample pretreatment, dilution, reagent addition, reaction, and detection, into a chip composed of solid-phase materials such as silicon, glass, plastic, or metal, which is a few square centimeters or even smaller. The chip is composed of a network of various micro-nanochannels, with controllable fluids running through the entire system to realize various functions of conventional chemical or biological laboratories. Currently, microfluidic chips have been widely used in biomedicine, chemical analysis, biosensing, high-throughput screening, environmental monitoring, food safety, and national defense security. They have advantages such as controllable liquid flow, low sample and reagent consumption, and fast analysis speed. In 2017, the Ministry of Science and Technology of China positioned microfluidic chips as a "disruptive technology."

[0005] Therefore, combining the sample separation function of the microfluidic chip and the color change characteristics of metal nanomaterial aggregation is fully suitable for the development of a microfluidic chip for the instant detection of prostate cancer biomarkers. Summary of the Invention

[0006] The present invention aims to provide a rapid microfluidic chip and method for detecting prostate cancer markers. This microfluidic chip can meet the needs of rapid prostate cancer screening and home self-testing. By combining the convenience of the microfluidic chip's physical structure for separating large particles such as red blood cells, white blood cells, and platelets from blood samples with the advantages of nanomaterials' aggregation and color development within the channel, a method for semi-quantitatively calculating the concentrations of F-PSA and T-PSA, as well as their ratio, is proposed by measuring the color development length of the color development channel.

[0007] To achieve the above-mentioned object, the present invention adopts a technical solution specifically comprising: a microfluidic chip for rapid detection of prostate cancer markers, wherein the microfluidic chip is divided into two layers, the lower layer being a substrate with a supporting function; the upper surface of the upper layer is provided with a sample filling port 3, a nanomaterial filling port 4, and a waste liquid discharge port 5 in sequence;

[0008] The lower surface is provided with a sample inflow channel 6, a blood separation area 7, a separated sample outflow channel 8, a sample and nanomaterial mixing pool 9, a nanomaterial inflow channel 10, a mixed sample outflow channel I11, a mixed sample outflow channel II12, a color development microchannel I13 for detecting free prostate-specific antigen F-PSA, a color development channel II14 for detecting total prostate-specific antigen T-PSA, a waste liquid outflow channel I15 and a waste liquid outflow channel II16 in sequence;

[0009] The main part of the blood separation zone 7 is composed of a microarray 17 composed of "V"-shaped grooves. The functional fragments of F-PSA and T-PSA are pre-immobilized and captured in the color development microchannel I13 for detecting F-PSA and the color development channel II14 for detecting T-PSA, respectively. The lower layer 1 and the upper layer 2 are tightly bonded together.

[0010] The sample filling port 3 is connected to one end of the sample inlet channel 6, the other end of the sample inlet channel 6 is connected to the bottom edge of the blood separation area 7, the top edge of the blood separation area 7 is connected to one end of the separated sample outflow channel 8, the other end of which is connected to the sample and nanomaterial mixing pool 9;

[0011] The sample and nanomaterial mixing pool 9 is also connected to one end of the nanomaterial inflow channel 10, one end of the mixed sample outflow channel I11 and one end of the mixed sample outflow channel II12 respectively, and the other end of the nanomaterial inflow channel 10 is connected to the nanomaterial filling port 4;

[0012] The other end of the mixed sample outflow channel I11 is connected to one end of the color development microchannel I13, the other end of the color development microchannel I13 is connected to one end of the waste liquid outflow channel I15, and the other end of the waste liquid outflow channel I15 is connected to the waste liquid outlet 5;

[0013] The other end of the mixed sample outflow channel II12 is connected to one end of the color development channel II14 , the other end of the color development channel II14 is connected to one end of the waste liquid outflow channel II16 , and the other end of the waste liquid outflow channel II16 is connected to the waste liquid outlet 5 .

[0014] Furthermore, the material of the lower layer 1 is polyvinyl chloride PVC, polyethylene PE, polyethylene terephthalate PET, polyvinyl chloride PVC, polymethyl methacrylate PMMA, polyetherimide PEI or glass, and its length is 100mm to 140mm, width is 40mm to 80mm, and height is 2mm to 6mm.

[0015] Furthermore, the material of the upper layer 2 is polydimethylsiloxane PDMS, thermosetting polyester TPE, styrene PS, polycarbonate PC, PMMA, polyethylene glycol diacrylate PEGDA, perfluorinated compound PFEP / PFA / PFPE or polyurethane PU, and its length is 100mm to 140mm, width is 40mm to 80mm, and height is 5mm to 10mm;

[0016] The sample filling port 3 on the upper surface of the upper layer 2 is through-through and circular in shape. The diameter of the sample filling port 3 is 5 mm to 10 mm, and the distance between the circular free end edge of the sample filling port 3 and the upper surface of the upper layer 2 is 5 mm to 10 mm.

[0017] The nanomaterial filling port 4 on the upper surface of the upper layer 2 is through-through and circular in shape. The diameter of the nanomaterial filling port 4 is 5 mm to 10 mm, and the distance between the circular free end edge of the nanomaterial filling port 4 and the upper surface of the upper layer 2 is 60 mm to 84 mm.

[0018] The waste liquid discharge outlet 5 on the upper surface of the upper layer 2 is through-through and circular in shape. The diameter of the waste liquid discharge outlet 5 is 5 mm to 10 mm. The distance between the circular edge of the free end of the waste liquid discharge outlet 5 and the upper surface of the upper layer 2 adjacent to the circular edge of the free end of the waste liquid discharge outlet 5 is 5 mm to 10 mm.

[0019] The sample inflow channel 6 on the lower surface of the upper layer 2 has a length of 2 mm to 8 mm, a width of 40 μm to 400 μm, and a height of 20 μm to 100 μm;

[0020] The blood separation area 7 on the lower surface of the upper layer 2 is an isosceles trapezoidal cavity, the distance between the top and bottom of the isosceles trapezoidal cavity is 18mm to 31mm, the length of the bottom is 30mm to 70mm, the length of the top is 40μm to 400μm, and the height of the blood separation area 7 is 20μm to 100μm;

[0021] The sample outflow channel 8 has a length of 2 mm to 8 mm, a width of 40 μm to 400 μm, and a height of 20 μm to 100 μm;

[0022] The sample and nanomaterial mixing pool 9 has a diameter of 5 mm to 10 mm and a height of 100 μm to 400 μm;

[0023] The sample and nanomaterial flow channel 10 has a length of 10 mm to 20 mm, a width of 40 μm to 400 μm, and a height of 20 μm to 100 μm.

[0024] Furthermore, the mixed sample outflow channel I11 and the mixed sample outflow channel II12 on the lower surface of the upper layer 2 are distributed on both sides of the nanomaterial inflow channel 10 and are symmetrically arranged. The angle between the mixed sample outflow channel I11 and the mixed sample outflow channel II12 is 30° to 60°, the length is 10 mm to 23 mm, the width is 40 μm to 100 μm, and the height is 20 μm to 40 μm.

[0025] The color development microchannel I13 and the color development channel II14 are arranged in parallel. The length of the color development microchannel I13 and the color development channel II14 are both 20 mm to 31 mm, the width is both 40 μm to 100 μm, and the height is both 20 μm to 40 μm.

[0026] The waste liquid outflow channel I15 and the waste liquid outflow channel II16 have lengths of 10 mm to 12 mm, widths of 40 μm to 100 μm, and heights of 20 μm to 40 μm.

[0027] Furthermore, the microarray 17 composed of "V"-shaped grooves constitutes the main part of the blood separation area 7, the opening of the first row of "V"-shaped grooves faces the sample filling port 3, the size of the opening is 30μm~45μm, the angle of the "V"-shaped groove is 30°~60°, the distance between the tops of adjacent sides of adjacent "V"-shaped grooves is 5μm~20μm, the opening direction of the second row of "V"-shaped grooves is consistent with the first row, the distance between the two rows of "V"-shaped grooves is 5μm~15μm, the opening of the second row of "V"-shaped grooves is located between the vertices of the two "V"-shaped grooves in the first row, and the height of the "V"-shaped groove is 20μm~100μm, and so on. The isosceles trapezoidal chamber is covered with the microarray 17 composed of "V"-shaped grooves to constitute the main part of the blood separation area 7.

[0028] Furthermore, the functional fragments of F-PSA and T-PSA respectively fixed in advance in the color development microchannel I13 and the color development channel II14 for capturing are antibodies, aptamers or ligands.

[0029] Furthermore, the substrate of the lower layer 1 and the material of the upper layer 2 are tightly bonded together by thermal bonding, anodic bonding or low-temperature bonding.

[0030] In order to better achieve the above-mentioned purpose of the invention, the present invention also provides a detection method for a rapid detection microfluidic chip of prostate cancer markers, comprising the following steps:

[0031] S1, directly add the blood sample into the sample filling port 3;

[0032] S2, when separated sample is observed to flow out from the end of the blood separation zone 7, a solution containing nanomaterials is added dropwise to the nanomaterial filling port 4;

[0033] S3, after the mixed sample is transported to the color development microchannel I13 for detecting F-PSA and the color development channel II14 for detecting T-PSA, wait for 5 minutes to 10 minutes;

[0034] S4, using a ruler to measure the lengths of the color development microchannel I13 for detecting F-PSA and the color development channel II14 for detecting T-PSA, respectively, and semi-quantitatively calculate the concentrations of F-PSA and T-PSA in the sample and the ratio between them.

[0035] Furthermore: the nanomaterial in step S2 is gold nanoparticles, silver nanoparticles, gold core-silver shell nanoparticles or silver core-gold shell nanoparticles, and its particle size is 25nm to 50nm.

[0036] Compared with the prior art, the present invention has the following beneficial effects:

[0037] 1. The V-groove microarray in the blood separation zone of the prostate cancer marker rapid detection microfluidic chip of the present invention can rapidly separate large particles such as red blood cells, white blood cells, and platelets from a blood sample through physical separation to obtain a serum sample for testing. The sample pretreatment process is simple and efficient, and the V-groove has a stronger fixation effect on the separated substances.

[0038] 2. The prostate cancer marker rapid detection microfluidic chip of the present invention, by combining the aggregation and color development properties of nanomaterials, can semi-quantitatively detect the concentrations of F-PSA and T-PSA, as well as their ratio, by simply measuring the length of the color development channel, providing a new approach for rapid screening of prostate cancer.

[0039] 3. The prostate cancer marker rapid detection microfluidic chip of the present invention effectively integrates the blood sample pretreatment process with the analysis of F-PSA and T-PSA, achieving the unification of sample pretreatment and detection, significantly improving detection efficiency;

[0040] 4. The prostate cancer marker rapid detection microfluidic chip of the present invention is simple to prepare, low in cost, easy to operate, and has rapid analysis, and can fully meet the needs of immediate self-testing at home. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] The accompanying drawings are used to provide further understanding of the present invention and constitute a part of the specification. They are used to explain the present invention together with the embodiments of the present invention and do not constitute a limitation of the present invention.

[0042] Figure 1 Schematic diagram of the microfluidic chip for rapid detection of prostate cancer markers in the present invention.

[0043] Figure 2 for Figure 1 Top view of .

[0044] Among them, the figures are marked as: 1, lower layer; 2, upper layer; 3, sample filling port; 4, nanomaterial filling port; 5, waste liquid discharge port; 6, sample inlet channel; 7, blood separation area; 8, sample outflow channel; 9, sample and nanomaterial mixing pool; 10, nanomaterial inlet channel; 11, mixed sample outflow channel I; 12, mixed sample outflow channel II; 13, color development microchannel I; 14, color development channel II; 15, waste liquid outflow channel I; 16, waste liquid outflow channel II; 17, microarray composed of "V"-shaped grooves. DETAILED DESCRIPTION

[0045] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention will be further described in detail below in conjunction with the accompanying drawings and embodiments. Of course, the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0046] Example 1

[0047] See also Figures 1 to 2 , this embodiment provides a technical solution as follows: the rapid detection microfluidic chip for prostate cancer markers of the present invention is divided into two layers, the lower layer 1 is a substrate with a supporting function; the upper surface of the upper layer 2 is sequentially provided with a sample filling port 3, a nanomaterial filling port 4, and a waste liquid discharge port 5; the lower surface is sequentially provided with a sample inlet channel 6, a blood separation area 7, a separated sample outflow channel 8, a sample and nanomaterial mixing pool 9, a nanomaterial inlet channel 10, a mixed sample outflow channel I11, a mixed sample outflow channel II12, a color development microchannel I13 for detecting free prostate-specific antigen F-PSA, a color development channel II14 for detecting total prostate-specific antigen T-PSA, a waste liquid outflow channel I15 and a waste liquid outflow channel II16, wherein the main part of the blood separation area is composed of a microarray 17 composed of "V"-shaped grooves, the color development microchannel 13 for detecting F-PSA and the color development channel II for detecting T-PSA The functional fragments of F-PSA and T-PSA are pre-fixed in 14 and can be captured respectively, and the lower layer 1 and the upper layer 2 are tightly bonded together.

[0048] The lower layer 1 is made of polyvinyl chloride (PVC), with a length of 100 mm, a width of 40 mm, and a height of 2 mm. The upper layer 2 is made of polydimethylsiloxane (PDMS), with a length of 100 mm, a width of 40 mm, and a height of 5 mm.

[0049] The sample filling port 3 on the upper surface of the upper layer 2 is through-through and circular in shape. The diameter of the sample filling port 3 is 5 mm, and the distance between the circular free end edge of the sample filling port 3 and the upper surface of the upper layer 2 is 5 mm.

[0050] The nano material filling port 4 on the upper surface of the upper layer 2 is through-through and circular in shape. The diameter of the nano material filling port 4 is 5 mm, and the distance between the circular free end edge of the nano material filling port 4 and the upper surface of the upper layer 2 is 60 mm.

[0051] The waste liquid outlet 5 on the upper surface of the upper layer 2 is through-through and circular in shape. The diameter of the waste liquid outlet 5 is 5 mm, and the distance between the circular free end edge of the waste liquid outlet 5 and the upper surface of the upper layer 2 is 5 mm.

[0052] One end of the sample inflow channel 6 on the lower surface of the upper layer 2 is connected to the sample filling port 3 , and has a length of 2 mm, a width of 40 μm, and a height of 20 μm.

[0053] The blood separation area 7 on the lower surface of the upper layer 2 is an isosceles trapezoidal cavity. The bottom edge of the isosceles trapezoidal cavity is connected to the other end of the sample inflow channel 6. The distance between the top and bottom edges of the isosceles trapezoidal cavity is 31 mm, the length of the bottom edge is 30 mm, the length of the top edge is 40 μm, and the height of the blood separation area 7 is 20 μm.

[0054] One end of the separated sample outflow channel 8 on the lower surface of the upper layer 2 is connected to the top edge of the blood separation area 7, and has a length of 2 mm, a width of 40 μm, and a height of 20 μm.

[0055] One end of the sample and nanomaterial mixing pool 9 on the lower surface of the upper layer 2 is connected to the other end of the separated sample outflow channel 8. The sample and nanomaterial mixing pool 9 is circular in shape, with a diameter of 5 mm and a height of 100 μm.

[0056] One end of the nanomaterial inflow channel 10 on the lower surface of the upper layer 2 is connected to the sample and nanomaterial mixing pool 9, and has a length of 10 mm, a width of 40 μm, and a height of 20 μm.

[0057] The mixed sample outflow channel I11 and the mixed sample outflow channel II12 on the lower surface of the upper layer 2 are distributed on both sides of the nanomaterial inflow channel 10 and are arranged symmetrically. One end of the mixed sample outflow channel I11 and the mixed sample outflow channel II12 are both connected to the sample and nanomaterial mixing pool 9. The angle between them is 30°, the length is 10 mm, the width is 40 μm, and the height is 20 μm.

[0058] One end of the color development microchannel I13 for detecting F-PSA and the color development channel II14 for detecting T-PSA on the lower surface of the upper layer 2 are respectively connected to the other end of the mixed sample outflow channel I11 and the mixed sample outflow channel II12, and are arranged in parallel. The length of the color development microchannel I13 for detecting F-PSA and the color development channel II14 for detecting T-PSA are both 20 mm, the width is 40 μm, and the height is 20 μm.

[0059] One end of the waste liquid outflow channel I15 and the waste liquid outflow channel II16 on the lower surface of the upper layer 2 are respectively connected to the other end of the color development microchannel I13 for detecting F-PSA and the color development channel II14 for detecting T-PSA, and the other ends of the waste liquid outflow channel I15 and the waste liquid outflow channel II16 are both connected to the waste liquid outlet 5. The length of the waste liquid outflow channel I15 and the waste liquid outflow channel II16 are both 10 mm, the width is 40 μm, and the height is 20 μm.

[0060] A microarray 17 composed of V-shaped grooves forms the main portion of the blood separation zone 7. The openings of the first row of V-shaped grooves face the sample filling port 3, are 30 μm in size, have a 30° angle, and are 5 μm apart from the tops of adjacent sides. The openings of the second row of V-shaped grooves align with the first row, with a 5 μm distance between the two rows. The opening of the second row of V-shaped grooves is located between the vertices of the first row of V-shaped grooves, and the height of the V-shaped grooves is 20 μm. Similarly, the main portion of the blood separation zone 7 is formed by paving the isosceles trapezoidal chamber with a microarray 1 composed of V-shaped grooves.

[0061] The functional fragments that can capture F-PSA and T-PSA respectively are pre-fixed in the color development microchannel I13 for detecting F-PSA and the color development channel II14 for detecting T-PSA, which are antibodies.

[0062] The substrate of the lower layer 1 and the material of the upper layer 2 are tightly attached together by thermal bonding.

[0063] A method for rapid detection of prostate cancer markers using a microfluidic chip comprises the following steps:

[0064] S1, directly add the blood sample into the sample filling port 3;

[0065] S2, when separated sample is observed to flow out from the end of the blood separation zone 7, a solution containing gold nanoparticles with a particle size of 25 nm is added dropwise to the nanomaterial filling port 4;

[0066] S3, after the mixed sample is transported to the color development microchannel I13 for detecting F-PSA and the color development channel II14 for detecting T-PSA, wait for 5 minutes;

[0067] S4, using a ruler to measure the lengths of the color development microchannel I13 for detecting F-PSA and the color development channel II14 for detecting T-PSA, respectively, and semi-quantitatively calculate the concentrations of F-PSA and T-PSA in the sample and the ratio between them.

[0068] Example 2

[0069] See also Figures 1 to 2This embodiment provides a technical solution, which is that the rapid detection microfluidic chip for prostate cancer markers of the present invention is divided into two layers, the lower layer 1 is a substrate with a supporting function; the upper surface of the upper layer 2 is sequentially provided with a sample filling port 3, a nanomaterial filling port 4, and a waste liquid discharge port 5; the lower surface is sequentially provided with a sample inlet channel 6, a blood separation area 7, a separated sample outflow channel 8, a sample and nanomaterial mixing pool 9, a nanomaterial inlet channel 10, a mixed sample outflow channel I11, a mixed sample outflow channel II12, a color development microchannel I13 for detecting free prostate-specific antigen F-PSA, a color development channel II14 for detecting total prostate-specific antigen T-PSA, a waste liquid outflow channel I15 and a waste liquid outflow channel II16, wherein the main part of the blood separation area is composed of a microarray 17 composed of "V"-shaped grooves, and the color development microchannel I13 for detecting F-PSA and the color development channel II14 for detecting T-PSA are pre-fixed with functional fragments that can capture F-PSA and T-PSA respectively. The lower layer 1 and the upper layer 2 are tightly bonded together.

[0070] The lower layer 1 is made of polyethylene (PE), with a length of 140 mm, a width of 80 mm, and a height of 6 mm. The upper layer 2 is made of polyethylene glycol diacrylate (PEGDA), with a length of 140 mm, a width of 80 mm, and a height of 10 mm.

[0071] The sample filling port 3 on the upper surface of the upper layer 2 is through-through and circular in shape. The diameter of the sample filling port 3 is 10 mm, and the distance between the circular free end edge of the sample filling port 3 and the upper surface of the upper layer 2 is 10 mm.

[0072] The nano material filling port 4 on the upper surface of the upper layer 2 is through-through and circular in shape. The diameter of the nano material filling port 4 is 10 mm, and the distance between the circular free end edge of the nano material filling port 4 and the upper surface of the upper layer 2 is 84 mm.

[0073] The waste liquid outlet 5 on the upper surface of the upper layer 2 is through-through and circular in shape. The diameter of the waste liquid outlet 5 is 10 mm, and the distance between the circular free end edge of the waste liquid outlet 5 and the upper surface of the upper layer 2 is 10 mm.

[0074] One end of the sample inflow channel 6 on the lower surface of the upper layer 2 is connected to the sample filling port 3 , and has a length of 8 mm, a width of 400 μm, and a height of 100 μm.

[0075] The blood separation area 7 on the lower surface of the upper layer 2 is an isosceles trapezoidal cavity. The bottom edge of the isosceles trapezoidal cavity is connected to the other end of the sample inflow channel 6. The distance between the top and bottom edges of the isosceles trapezoidal cavity is 18 mm, the length of the bottom edge is 70 mm, the length of the top edge is 400 μm, and the height of the blood separation area 7 is 100 μm.

[0076] One end of the separated sample outflow channel 8 on the lower surface of the upper layer 2 is connected to the top edge of the blood separation area 7, and has a length of 8 mm, a width of 400 μm, and a height of 100 μm.

[0077] One end of the sample and nanomaterial mixing pool 9 on the lower surface of the upper layer 2 is connected to the other end of the separated sample outflow channel 8. The sample and nanomaterial mixing pool 9 is circular in shape, with a diameter of 10 mm and a height of 400 μm.

[0078] One end of the nanomaterial inflow channel 10 on the lower surface of the upper layer 2 is connected to the sample and nanomaterial mixing pool 9, and has a length of 20 mm, a width of 400 μm, and a height of 100 μm.

[0079] The mixed sample outflow channel I11 and the mixed sample outflow channel II12 on the lower surface of the upper layer 2 are distributed on both sides of the nanomaterial inflow channel 10 and are arranged symmetrically. One end of the mixed sample outflow channel I11 and the mixed sample outflow channel II12 are both connected to the sample and nanomaterial mixing pool 9. The angle between them is 60°, the length is 23 mm, the width is 100 μm, and the height is 40 μm.

[0080] One end of the color development microchannel I13 for detecting F-PSA and the color development channel II14 for detecting T-PSA on the lower surface of the upper layer 2 are respectively connected to the other end of the mixed sample outflow channel I11 and the mixed sample outflow channel II12, and are arranged in parallel. The length of the color development microchannel I13 for detecting F-PSA and the color development channel II 14 for detecting T-PSA are both 31 mm, the width is 100 μm, and the height is 40 μm.

[0081] One end of the waste liquid outflow channel I15 and the waste liquid outflow channel II16 on the lower surface of the upper layer 2 are respectively connected to the other end of the color development microchannel I13 for detecting F-PSA and the color development channel I14 for detecting T-PSA, and the other ends of the waste liquid outflow channel I15 and the waste liquid outflow channel II16 are both connected to the waste liquid outlet 5. The length of the waste liquid outflow channel I15 and the waste liquid outflow channel II16 are both 12 mm, the width is 100 μm, and the height is 40 μm.

[0082] A microarray 17 composed of V-shaped grooves forms the main portion of the blood separation zone 7. The openings of the first row of V-shaped grooves face the sample filling port 3, the openings are 45 μm in size, the angle of the V-shaped grooves is 60°, and the distance between the tops of adjacent sides of adjacent V-shaped grooves is 20 μm. The openings of the second row of V-shaped grooves are oriented in the same direction as the first row, with a distance of 15 μm between the two rows of V-shaped grooves. The opening of the second row of V-shaped grooves is located between the vertices of the two V-shaped grooves in the first row, and the height of the V-shaped grooves is 100 μm. Similarly, the main portion of the blood separation zone 7 is formed by paving the isosceles trapezoidal chamber with a microarray 1 composed of V-shaped grooves.

[0083] The functional fragments that can capture F-PSA and T-PSA respectively are pre-fixed in the color development microchannel I13 for detecting F-PSA and the color development channel I14 for detecting T-PSA, which are aptamers.

[0084] The substrate of the lower layer 1 and the material of the upper layer 2 are tightly adhered together by anodic bonding.

[0085] A method for rapid detection of prostate cancer markers using a microfluidic chip comprises the following steps:

[0086] S1, directly add the blood sample into the sample filling port 3;

[0087] S2, when separated sample is observed to flow out from the end of the blood separation zone 7, a solution containing silver nanoparticles with a particle size of 50 nm is added dropwise to the nanomaterial filling port 4;

[0088] S3, after the mixed sample is transported to the color development microchannel I13 for detecting F-PSA and the color development channel I14 for detecting T-PSA, wait for 10 minutes;

[0089] S4, using a ruler to measure the lengths of the color development microchannel I13 for detecting F-PSA and the color development channel I14 for detecting T-PSA, respectively, and semi-quantitatively calculate the concentrations of F-PSA and T-PSA in the sample and the ratio between them.

[0090] Example 3

[0091] See also Figures 1 to 2The present embodiment provides a technical solution for the rapid detection of prostate cancer markers of the present invention, which is divided into two layers, the lower layer 1 is a substrate with a supporting function; the upper surface of the upper layer 2 is sequentially provided with a sample filling port 3, a nanomaterial filling port 4, and a waste liquid discharge port 5; the lower surface is sequentially provided with a sample inlet channel 6, a blood separation area 7, a separated sample outflow channel 8, a sample and nanomaterial mixing pool 9, a nanomaterial inlet channel 10, a mixed sample outflow channel I11, a mixed sample outflow channel II12, and a sample inlet channel 113 for detecting free prostate. The color development microchannel I13 for detecting the prostate-specific antigen F-PSA, the color development channel II14 for detecting the total prostate-specific antigen T-PSA, the waste liquid outflow channel I15 and the waste liquid outflow channel II16, wherein the main part of the blood separation area is composed of a microarray 17 composed of "V"-shaped grooves, and the color development microchannel I13 for detecting F-PSA and the color development channel II14 for detecting T-PSA are pre-fixed with functional fragments that can capture F-PSA and T-PSA respectively, and the lower layer 1 and the upper layer 2 are tightly bonded together.

[0092] The lower layer 1 is made of polyethylene terephthalate (PET), with a length of 120 mm, a width of 60 mm, and a height of 4 mm. The upper layer 2 is made of polyurethane (PU), with a length of 120 mm, a width of 60 mm, and a height of 7 mm.

[0093] The sample filling port 3 on the upper surface of the upper layer 2 is through-through and circular in shape. The diameter of the sample filling port 3 is 8 mm, and the distance between the circular free end edge of the sample filling port 3 and the upper surface of the upper layer 2 is 8 mm.

[0094] The nano material filling port 4 on the upper surface of the upper layer 2 is through-through and circular in shape. The diameter of the nano material filling port 4 is 8 mm, and the distance between the circular free end edge of the nano material filling port 4 and the upper surface of the upper layer 2 is 72 mm.

[0095] The waste liquid outlet 5 on the upper surface of the upper layer 2 is through-through and circular in shape. The diameter of the waste liquid outlet 5 is 8 mm, and the distance between the circular free end edge of the waste liquid outlet 5 and the upper surface of the upper layer 2 is 8 mm.

[0096] One end of the sample inflow channel 6 on the lower surface of the upper layer 2 is connected to the sample filling port 3 , and has a length of 5 mm, a width of 200 μm, and a height of 60 μm.

[0097] The blood separation area 7 on the lower surface of the upper layer 2 is an isosceles trapezoidal cavity. The bottom edge of the isosceles trapezoidal cavity is connected to the other end of the sample inflow channel 6. The distance between the top and bottom edges of the isosceles trapezoidal cavity is 23 mm, the length of the bottom edge is 50 mm, the length of the top edge is 200 μm, and the height of the blood separation area 7 is 60 μm.

[0098] One end of the separated sample outflow channel 8 on the lower surface of the upper layer 2 is connected to the top edge of the blood separation area 7, and has a length of 5 mm, a width of 200 μm, and a height of 60 μm.

[0099] One end of the sample and nanomaterial mixing pool 9 on the lower surface of the upper layer 2 is connected to the other end of the separated sample outflow channel 8. The sample and nanomaterial mixing pool 9 is circular in shape, with a diameter of 8 mm and a height of 250 μm.

[0100] One end of the nanomaterial inflow channel 10 on the lower surface of the upper layer 2 is connected to the sample and nanomaterial mixing pool 9, and has a length of 15 mm, a width of 200 μm, and a height of 60 μm.

[0101] The mixed sample outflow channel I11 and the mixed sample outflow channel II12 on the lower surface of the upper layer 2 are distributed on both sides of the nanomaterial inflow channel 10 and are arranged symmetrically. One end of the mixed sample outflow channel I11 and the mixed sample outflow channel II12 are both connected to the sample and nanomaterial mixing pool 9. The angle between them is 45°, the length is 16 mm, the width is 70 μm, and the height is 30 μm.

[0102] One end of the color development microchannel I13 for detecting F-PSA and the color development channel I14 for detecting T-PSA on the lower surface of the upper layer 2 are respectively connected to the other end of the mixed sample outflow channel I11 and the mixed sample outflow channel II12, and are arranged in parallel. The length of the color development microchannel I13 for detecting F-PSA and the color development channel II 14 for detecting T-PSA are both 25 mm, the width is 70 μm, and the height is 30 μm.

[0103] One end of the waste liquid outflow channel I15 and the waste liquid outflow channel II16 on the lower surface of the upper layer 2 are respectively connected to the other end of the color development microchannel I13 for detecting F-PSA and the color development channel II 14 for detecting T-PSA, and the other ends of the waste liquid outflow channel I15 and the waste liquid outflow channel II16 are both connected to the waste liquid outlet 5. The length of the waste liquid outflow channel I15 and the waste liquid outflow channel II16 are both 9.2 mm, the width is 70 μm, and the height is 30 μm.

[0104] A microarray 17 composed of V-shaped grooves forms the main portion of the blood separation zone 7. The openings of the first row of V-shaped grooves face the sample filling port 3, the openings are 36 μm in size, the angle of the V-shaped grooves is 45°, and the distance between the tops of adjacent sides of adjacent V-shaped grooves is 12 μm. The openings of the second row of V-shaped grooves are oriented in the same direction as the first row, with a distance of 10 μm between the two rows of V-shaped grooves. The opening of the second row of V-shaped grooves is located between the vertices of the two V-shaped grooves in the first row, and the height of the V-shaped grooves is 60 μm. Similarly, the main portion of the blood separation zone 7 is formed by paving the isosceles trapezoidal chamber with a microarray 1 composed of V-shaped grooves.

[0105] The functional fragments that can capture F-PSA and T-PSA respectively are pre-fixed in the color development microchannel I13 for detecting F-PSA and the color development channel I14 for detecting T-PSA, which are ligands.

[0106] The substrate of the lower layer 1 and the material of the upper layer 2 are tightly attached together by low temperature bonding.

[0107] A method for rapid detection of prostate cancer markers using a microfluidic chip comprises the following steps:

[0108] S1, directly add the blood sample into the sample filling port 3;

[0109] S2, when separated sample is observed to flow out from the end of the blood separation zone 7, a solution containing gold core-silver shell nanoparticles with a particle size of 36 nm is added dropwise to the nanomaterial filling port 4;

[0110] S3, after the mixed sample is transported to the color development microchannel I 13 for detecting F-PSA and the color development channel II 14 for detecting T-PSA, wait for 8 minutes;

[0111] S4, using a ruler to measure the lengths of the color development microchannel I 13 for detecting F-PSA and the color development channel II 14 for detecting T-PSA, respectively, and semi-quantitatively calculate the concentrations of F-PSA and T-PSA in the sample and the ratio therebetween.

[0112] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A microfluidic chip for rapid detection of prostate cancer markers, characterized in that: The microfluidic chip is divided into two layers, the lower layer (1) being a substrate with a supporting function; the upper surface of the upper layer (2) is provided with a sample filling port (3), a nanomaterial filling port (4), and a waste liquid discharge port (5) in sequence; The lower surface is provided with a sample inflow channel (6), a blood separation area (7), a separated sample outflow channel (8), a sample and nanomaterial mixing pool (9), a nanomaterial inflow channel (10), a mixed sample outflow channel I (11), a mixed sample outflow channel II (12), a color development microchannel I (13) for detecting free prostate-specific antigen F-PSA, a color development channel II (14) for detecting total prostate-specific antigen T-PSA, a waste liquid outflow channel I (15) and a waste liquid outflow channel II (16); The main part of the blood separation zone (7) is composed of a microarray (17) composed of "V"-shaped grooves. The functional fragments of F-PSA and T-PSA are respectively fixed and captured in advance in the color development microchannel I (13) for detecting F-PSA and the color development channel II (14) for detecting T-PSA. The lower layer (1) and the upper layer (2) are tightly bonded together. The sample filling port (3) is connected to one end of the sample inflow channel (6), the other end of the sample inflow channel (6) is connected to the bottom edge of the blood separation area (7), the top edge of the blood separation area (7) is connected to one end of the separated sample outflow channel (8), the other end of which is connected to the sample and nanomaterial mixing pool (9); The sample and nanomaterial mixing pool (9) is further connected to one end of the nanomaterial inflow channel (10), one end of the mixed sample outflow channel I (11), and one end of the mixed sample outflow channel II (12), respectively. The other end of the nanomaterial inflow channel (10) is connected to the nanomaterial filling port (4). The other end of the mixed sample outflow channel I (11) is connected to one end of the color development microchannel I (13), the other end of the color development microchannel I (13) is connected to one end of the waste liquid outflow channel I (15), and the other end of the waste liquid outflow channel I (15) is connected to the waste liquid outlet (5); The other end of the mixed sample outflow channel II (12) is connected to one end of the color development channel II (14), the other end of the color development channel II (14) is connected to one end of the waste liquid outflow channel II (16), and the other end of the waste liquid outflow channel II (16) is connected to the waste liquid outlet (5); The mixed sample outflow channel I (11) and the mixed sample outflow channel II (12) on the lower surface of the upper layer (2) are distributed on both sides of the nanomaterial inflow channel (10) and are arranged symmetrically. The angle between the mixed sample outflow channel I (11) and the mixed sample outflow channel II (12) is 30° to 60°, the length is 10 mm to 23 mm, the width is 40 μm to 100 μm, and the height is 20 μm to 40 μm. The color development microchannel I (13) and the color development channel II (14) are arranged in parallel, and the length of the color development microchannel I (13) and the color development channel II (14) are both 20 mm to 31 mm, the width is both 40 μm to 100 μm, and the height is both 20 μm to 40 μm; The waste liquid outflow channel I (15) and the waste liquid outflow channel II (16) are both 10 mm to 12 mm in length, 40 μm to 100 μm in width, and 20 μm to 40 μm in height.

2. The rapid detection microfluidic chip for prostate cancer markers according to claim 1, characterized in that: The material of the lower layer (1) is polyvinyl chloride (PVC), polyethylene (PE), polyethylene terephthalate (PET), polyvinyl chloride (PVC), polymethyl methacrylate (PMMA), polyetherimide (PEI) or glass, and its length is 100 mm to 140 mm, width is 40 mm to 80 mm, and height is 2 mm to 6 mm.

3. The rapid detection microfluidic chip for prostate cancer markers according to claim 1, characterized in that: The material of the upper layer (2) is polydimethylsiloxane (PDMS), thermosetting polyester (TPE), styrene (PS), polycarbonate (PC), PMMA, polyethylene glycol diacrylate (PEGDA), perfluorinated compound (PFEP / PFA / PFPE) or polyurethane (PU), and its length is 100 mm to 140 mm, width is 40 mm to 80 mm, and height is 5 mm to 10 mm; The sample filling port (3) on the upper surface of the upper layer (2) is through-through and circular in shape. The diameter of the sample filling port (3) is 5 mm to 10 mm, and the distance between the circular free end edge of the sample filling port (3) and the upper surface of the upper layer (2) is 5 mm to 10 mm. The nanomaterial filling port (4) on the upper surface of the upper layer (2) is through-through and circular in shape. The diameter of the nanomaterial filling port (4) is 5 mm to 10 mm, and the distance between the circular free end edge of the nanomaterial filling port (4) and the upper surface of the upper layer (2) is 60 mm to 84 mm. The waste liquid discharge outlet (5) on the upper surface of the upper layer (2) is through-through and circular in shape. The diameter of the waste liquid discharge outlet (5) is 5 mm to 10 mm, and the distance between the circular edge of the free end of the waste liquid discharge outlet (5) and the upper surface of the upper layer (2) adjacent to the circular edge of the free end of the waste liquid discharge outlet (5) is 5 mm to 10 mm. The sample inflow channel (6) on the lower surface of the upper layer (2) has a length of 2 mm to 8 mm, a width of 40 μm to 400 μm, and a height of 20 μm to 100 μm; The blood separation area (7) on the lower surface of the upper layer (2) is an isosceles trapezoidal cavity, the distance between the top and bottom of the isosceles trapezoidal cavity is 18 mm to 31 mm, the length of the bottom is 30 mm to 70 mm, the length of the top is 40 μm to 400 μm, and the height of the blood separation area (7) is 20 μm to 100 μm; The sample outflow channel (8) has a length of 2 mm to 8 mm, a width of 40 μm to 400 μm, and a height of 20 μm to 100 μm; The sample and nanomaterial mixing pool (9) has a diameter of 5 mm to 10 mm and a height of 100 μm to 400 μm; The nanomaterial inflow channel (10) has a length of 10 mm to 20 mm, a width of 40 μm to 400 μm, and a height of 20 μm to 100 μm.

4. The rapid detection microfluidic chip for prostate cancer markers according to claim 1, characterized in that: The microarray (17) composed of "V"-shaped grooves constitutes the main part of the blood separation area (7). The opening of the first row of "V"-shaped grooves faces the sample filling port (3), the size of the opening is 30 μm to 45 μm, the angle of the "V"-shaped grooves is 30° to 60°, the distance between the tops of adjacent sides of adjacent "V"-shaped grooves is 5 μm to 20 μm, the opening direction of the second row of "V"-shaped grooves is consistent with the first row, the distance between the two rows of "V"-shaped grooves is 5 μm to 15 μm, the opening of the second row of "V"-shaped grooves is located between the vertices of the two "V"-shaped grooves in the first row, and the height of the "V"-shaped grooves is 20 μm to 100 μm. Similarly, the isosceles trapezoidal chamber is covered with the microarray (17) composed of "V"-shaped grooves to constitute the main part of the blood separation area (7).

5. The rapid detection microfluidic chip for prostate cancer markers according to claim 1, characterized in that: The functional fragments of F-PSA and T-PSA that are respectively fixed and captured in advance in the color development microchannel I (13) and the color development channel II (14) are antibodies, aptamers or ligands.

6. The rapid detection microfluidic chip for prostate cancer markers according to claim 1, characterized in that: The substrate of the lower layer (1) and the material of the upper layer (2) are tightly bonded together by means of thermal bonding, anodic bonding or low-temperature bonding.

7. The method for rapid detection of prostate cancer markers using a microfluidic chip according to any one of claims 1 to 6, characterized in that: The following steps are involved: S1, directly add the blood sample to the sample filling port (3); S2, when separated sample is observed to flow out from the end of the blood separation zone (7), a solution containing nanomaterials is added dropwise to the nanomaterial filling port (4); S3, after the mixed sample is transported to the color development microchannel I (13) for detecting F-PSA and the color development channel II (14) for detecting T-PSA, wait for 5 min to 10 min; S4, using a ruler to measure the lengths of the color development microchannel I (13) for detecting F-PSA and the color development channel II (14) for detecting T-PSA, respectively, and semi-quantitatively calculate the concentrations of F-PSA and T-PSA in the sample and the ratio between them.

8. The method for rapid detection of prostate cancer markers using a microfluidic chip according to claim 7, wherein: The nanomaterial in step S2 is gold nanoparticles, silver nanoparticles, gold core silver shell nanoparticles or silver core gold shell nanoparticles, and its particle size is 25 nm to 50 nm.

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