A tumor marker detection chip and its preparation method

Abstract

The present invention relates to a tumor marker detection chip, which comprises an immune reaction chip and a sensor chip. Among them, the immune reaction chip includes a reaction layer substrate and a sample addition layer on the upper layer. The sensor chip includes a flexible sensing layer and a reaction chamber. The reaction chamber is vertically through, the upper end of the reaction chamber is sealed by the flexible sensing layer, the reaction chamber is filled with a reaction solution, and the lower end of the reaction chamber is sealed by the sample addition layer of the immune reaction chip. In this technical solution, the immune reaction chip is combined with the flexible sensing chip, with less reagent and sample consumption. The capture antibody is immobilized by using graphene oxide quantum dot nanomaterials, which can bind the capture antibody in a large area and with high uniformity. By using a sensitive flexible sensing chip, the high-sensitive flexible sensing chip is triggered by the gas pressure generated by the immune reaction and the reaction of the reaction solution, realizing the detection of high-sensitive tumor markers.

Description

Technical Field

[0001] The present invention relates to the field of biological detection technology, and in particular to a tumor marker detection chip and a preparation method thereof. Background Art

[0002] With the continuous development of various new technologies in the field of biotechnology, high-throughput, accurate and rapid detection methods have become important research methods in the fields of biological science and medicine. Most of the traditional high-throughput biomolecule detection technologies are based on biochip technology. At present, the biochip technologies commonly used for biomacromolecule detection are gene chips and protein chips. However, due to the high production cost and long cycle of biochips and the need for expensive detection equipment, the application of biochip technology has certain limitations. So far, the screening methods for tumors are mainly self-examination by patients, clinical breast examination (CBE), ultrasonography (US) and X-ray CT (X-CT). Generally, tumors can only be found after they are larger than 2 cm, and US can only find masses larger than 0.5 cm, which is very unfavorable for the treatment of tumors. Early screening of tumors can greatly improve the survival rate of patients. It has been found that 30% of patients with gastric cancer, ovarian cancer, lung cancer and breast cancer have elevated CEA levels in their blood. The increase in tumor marker levels is a signal sent by our body to us. How to capture these signals quickly, efficiently and simply is a hot topic of research now.

[0003] Currently, many detection methods based on enzyme-linked immunosorbent assay, radioimmunoassay, Roche electrochemistry, mass spectrometry, etc. have been established, but most of them have large sample consumption, expensive equipment and complex operation, which limit their application in rapid detection and daily life detection. There is an urgent need for a new tumor marker chip and its preparation method. Summary of the invention

[0004] In view of the shortcomings of the prior art, the present invention provides a tumor marker detection chip and a preparation method thereof to solve the problems of reagent and sample consumption, complex detection, and expensive detection equipment in the prior art biological detection technology. The present invention can detect the content of tumor markers by combining the immune response chip with the flexible sensor chip, and judge the possibility of cancer by detecting the change of the flexible sensor signal generated by the deformation of the sensor caused by the generation of non-toxic and harmless gas.

[0005] Terminology explanation:

[0006] 1. APTES: 3-aminopropyltriethoxysilane;

[0007] 2. PDMS: polydimethylsiloxane;

[0008] 3. BSA: Bovine Serum Albumin;

[0009] 4. DPBS: Dulbecco's Phosphate Buffered Saline.

[0010] The technical solution of the present invention is as follows:

[0011] A tumor marker detection chip, comprising:

[0012] An immunoreaction chip and a sensor chip, wherein the immunoreaction chip includes a reaction layer substrate and a sample addition layer on the upper layer, the sensor chip includes a flexible sensing layer and a reaction chamber, the reaction chamber is vertically through, the upper end of the reaction chamber is sealed by the flexible sensing layer, the reaction chamber is filled with a reaction solution, and the lower end of the reaction chamber is sealed by the sample addition layer of the immunoreaction chip.

[0013] Further, the reaction layer substrate includes a substrate functionalized with graphene quantum dot materials and capture antibodies immobilized on the substrate.

[0014] Further, a sample addition hole is provided on the sample addition layer, the sample addition hole is vertically connected, the lower end of the sample addition hole is bonded to the reaction layer substrate as a whole, and a sample is added to the upper end of the sample addition hole.

[0015] Further, the reaction solution is a hydrogen peroxide solution with a concentration of 0.5 - 35 wt%.

[0016] Further, the reaction chamber is an acrylic plate or a PDMS sheet with a through-hole array.

[0017] A method for preparing a tumor marker detection chip, comprising:

[0018] Preparing the sample addition layer: Mixing polydimethylsiloxane and a curing agent and then drying to prepare the sample addition layer, and making a sample addition hole on the sample addition layer;

[0019] Preparing the reaction layer substrate: Performing plasma treatment on the substrate and then soaking it in a nanomaterial solution to grow nanomaterials, and then bonding the substrate to the sample addition layer to form the reaction layer substrate;

[0020] Preparing the flexible sensing layer: Bonding a patterned graphene layer and a PDMS sheet to form the flexible sensing layer;

[0021] Preparing the reaction chamber: Mixing polydimethylsiloxane and a curing agent to prepare a PDMS layer, and making a reaction chamber on the PDMS layer;

[0022] Sealing the flexible sensing layer and the reaction chamber to form the sensor chip;

[0023] Sealing the sensor chip and the immunoreaction chip to form the tumor marker detection chip.

[0024] Further, forming the flexible sensing layer by laminating the patterned graphene layer and the PDMS sheet includes preparing a patterned graphene layer on a polyimide (PI) substrate through a laser engraving process, then using a sticky PDMS sheet to adhere to the PI of the patterned graphene layer, transferring the graphene layer onto the PDMS sheet by mechanical peeling, connecting leads to the electrode positions of the graphene layer, and finally closely laminating the PDMS sheet with the patterned graphene layer under the condition of aligning the graphene layer patterns to form the flexible sensing layer.

[0025] Further, plasma treating the substrate includes plasma treating a glass or acrylic substrate for 5 - 20 min and then soaking it in a coupling agent APTES for 40 - 60 min.

[0026] Further, preparing the sample addition layer by mixing polydimethylsiloxane and a curing agent and then drying includes mixing according to the ratio of polydimethylsiloxane:curing agent = 5:1 - 20:1, and preparing the PDMS layer by drying at 80 - 110 °C for 1 - 2 h.

[0027] Further, bonding the substrate and the sample addition layer to form the immunoreaction substrate includes adding a capture antibody solution to the sample addition holes and incubating for 2 - 15 h, fixing the capture antibody to the substrate at the sample addition hole positions, and finally performing a blocking treatment on the sample addition hole positions with BSA containing a concentration of 0.5 - 10 wt% for 5 - 30 min to form the reaction layer substrate.

[0028] The beneficial effects of the present invention are as follows:

[0029] Compared with the prior art, in the technical solution of the present application, combining the immunoreaction chip and the flexible sensing chip provides a small and convenient tumor marker detection solution; using graphene oxide quantum dot nanomaterials to immobilize capture antibodies can bind capture antibodies in a large area and with high uniformity; the through-hole array on the chip can simultaneously detect multiple samples; using a sensitive flexible sensing chip, triggering the highly sensitive flexible sensing chip through the gas pressure generated by the immunoreaction and the reaction solution, realizing highly sensitive tumor marker detection, no longer requiring expensive detection instrument equipment, and can achieve tumor chip detection anytime and anywhere. The structure of the present invention is simple, the performance is stable, and it has obvious progressiveness and good popularization and application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] The specification drawings forming a part of this application are used to provide a further understanding of this application. The schematic embodiments of this application and their descriptions are used to explain this application and do not constitute an improper limitation to this application.

[0031] Figure 1A flowchart for fabricating an immunoresponse chip of a tumor marker detection chip according to an embodiment of the present invention;

[0032] Figure 2 A flowchart for fabricating a sensing chip of a tumor marker detection chip according to an embodiment of the present invention;

[0033] Figure 3 A structural diagram of an immunoresponse chip according to an embodiment of the present invention;

[0034] Figure 4 A structural diagram of a sensing chip according to an embodiment of the present invention;

[0035] Figure 5 A bonding diagram of an immunoresponse chip and a sensing chip according to an embodiment of the present invention.

[0036] In the figure: 1. Sample loading layer; 2. Sample loading hole; 3. Graphene oxide quantum dot material; 4. Reaction layer substrate; 5. Capture antibody; 6. Flexible sensor; 7. Reaction chamber. Detailed implementation manners

[0037] The present invention will be further limited below in conjunction with the description of the drawings and embodiments, but not limited thereto.

[0038] Embodiment 1

[0039] As Figure 1 shown, a chip for tumor marker detection includes Figure 3 an immunoresponse chip, Figure 4 a sensor chip. The immunoresponse chip includes a reaction layer substrate 4, an upper sample loading layer 1, a substrate functionalized with graphene oxide quantum dot material 3, and a capture antibody 5 fixed thereon.

[0040] In the above solution, the sample loading holes on the sample loading layer 1 are vertically communicated, and its lower end is bonded to the reaction layer substrate. The sample is added from the upper end, that is, the sample loading layer and the reaction layer substrate are bonded together. The sample is added into the sample loading hole of the sample loading layer, and the sample loading layer plays a role in fixing the sample loading position.

[0041] In the above solution, the sensor chip includes a flexible sensing layer 6 and a reaction chamber 7. The reaction chamber is vertically penetrated, and its upper end is sealed by the flexible sensing chip, and its lower end is aligned and sealed with the sample loading hole of the immunoresponse chip to form a sealed space.

[0042] In the above solution, the flexible sensing layer includes a flexible sensor. The sample addition hole of the immune chip is used to load the sample. After the sample is loaded into the reaction chamber of the sensing chip and undergoes an immune reaction, it reacts with the reaction solution in the reaction chamber to generate gas. The generated gas will cause a pressure change, thereby triggering the response of the flexible sensor. According to the proportional relationship between the pressure change caused by the gas generation and the content of the tumor marker, the content of the tumor marker is obtained after the flexible sensor detects the pressure data.

[0043] The reaction solution is a hydrogen peroxide solution with a concentration of 0.5 - 35 wt%.

[0044] In the above solution, the reaction chamber is an acrylic plate with a through-hole array. Through a carbon dioxide laser engraving machine, holes are cut in the middle of the acrylic plate chamber.

[0045] In the above solution, after bonding the substrate and the sample addition layer, a reaction layer substrate is formed, including functionalizing the capture antibody on the substrate coated with nanomaterials at the sample addition port position. Specifically, on the substrate after growing the nanomaterials, the capture antibody is laid at the sample addition port position and incubated for 2 - 15 h, and then the excess detection antibody is drained, and the capture antibody is fixed on the substrate.

[0046] In the above solution, the middle part of the reaction chamber is hollowed out to form a state of being through from top to bottom. The upper end is sealed by the flexible sensing layer, and the lower end forms an overall sealed environment with the sample addition layer of the processed immune reaction chip.

[0047] In the above solution, during the process of punching the sample addition hole, the solidified PDMS is processed by a 100W laser engraving machine with a cutting power of 20%, a stepping speed of 10 mm / s, and a hole size of 3 mm * 3 mm.

[0048] In the above solution, the functional material in the material functionalization refers to a material with specific functions after the action of light, electricity, magnetism, heat, chemistry, biochemistry, etc. The surface of this chip is formed with amino groups by soaking in APTES.

[0049] Example 2

[0050] A method for preparing a tumor marker detection chip includes the following steps:

[0051] Step 1: Clean the substrate and grow nanomaterials on the substrate.

[0052] 1.1. First, ultrasonically clean the substrate to remove surface impurities.

[0053] 1.2. Then, perform oxygen plasma treatment for 10 min to remove surface organic pollution and form a layer of hydroxyl groups on its surface;

[0054] 1.3. Prepare an APTES solution using 95% alcohol as the solvent. Select an APTES concentration of 2 wt% and soak for 40 min. Then, perform ultrasonic cleaning and dry with nitrogen to form a nanomaterial coupling layer.

[0055] 1.4. Dilute the nanomaterial concentration to 1 mg / ml using ultrapure water as the solvent. Then, soak the substrate dried in 1.3 for 40 min, followed by ultrasonic cleaning and drying with nitrogen to form a nanomaterial layer.

[0056] 1.5. Repeat the above steps 5 times to form a reaction substrate functionalized with nanomaterials and store it in a 4°C refrigerator.

[0057] Step 2: Prepare the sample addition layer 1 and punch the sample addition holes 2.

[0058] 2.1. Prepare the sample addition layer 1 according to the ratio of polydimethylsiloxane:curing agent = 10:1. First, evacuate the uniformly mixed PDMS solution until there are no obvious air bubbles, then invert it in a mold of a specific thickness and place it in an 80°C oven for 1 h of drying.

[0059] 2.2. Take out the prepared sample addition layer 1 and use a puncher or laser engraver to design and process 3 mm * 3 mm sample addition holes 2 to form a sample addition hole array.

[0060] Step 3: Bond the substrate and the sample addition layer, and add the capture antibody at the position of the sample addition hole and incubate for 2 - 15 h to immobilize the capture antibody on the substrate for reacting with tumor markers.

[0061] 3.1. Bond the sample addition layer 1 to the substrate grown with nanomaterials.

[0062] 3.2. Add the capture antibody into the sample addition port and incubate for 2 - 15 h.

[0063] 3.3. Drain the excess capture antibody.

[0064] Step 4: Add a 3 wt% BSA blocking agent to block the blank sites.

[0065] 4.1. Prepare a 3 wt% BSA blocking agent using a DPBS buffer solution.

[0066] 4.2. Add the 3 wt% BSA blocking agent and react for 20 min, then drain the excess solution to form an immunoreaction chip.

[0067] The fabricated immunoreaction chip is as Figure 3 shown.

[0068] Step 5: Fabricate a flexible sensing chip.

[0069] 5.1. Engrave a specific pattern on the polyimide film under the action of a carbon dioxide laser. The pattern size is 8 mm * 8 mm, as shown in Figure 4 Figure 6.

[0070] 5.2. Bond the sticky PDMS sheet on the engraved pattern, and peel off the laser-induced graphene layer from the PI layer by mechanical peeling and transfer it to the sticky PDMS.

[0071] 5.3. For the encapsulation of the flexible sensor, select two PDMS sheets transferred with the laser-induced graphene layer, lay wires on one of them, and then closely fit the two together to form a flexible sensing layer. Due to the stickiness of PDMS itself, it can well encapsulate the conductive layer.

[0072] 5.4. On an acrylic plate or PDMS layer with a certain thickness, prepare through holes with a size of 10 mm * 10 mm and a thickness of 3 mm by a carbon dioxide laser engraving machine or a punching machine to prepare a reaction chamber layer.

[0073] 5.5. Seal and fix the flexible sensing layer on the through hole of the reaction chamber through a sealing ring or other sealing materials to form a sensing chip.

[0074] The production of the sensing chip is completed as shown in Figure 4 Figure.

[0075] Align and seal the sample loading hole and the reaction chamber of the immune reaction chip with the sensing chip to form a tumor marker detection chip.

[0076] The above are only the preferred embodiments of the present application and are not used to limit the present application. For those skilled in the art, various changes and modifications can be made to the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

[0077] Although the specific implementation manners of the present invention are described above in conjunction with the accompanying drawings, it is not a limitation to the protection scope of the present invention. Those skilled in the art should understand that based on the technical solutions of the present invention, various modifications or deformations that can be made by those skilled in the art without creative efforts are still within the protection scope of the present invention.

Claims

1. A tumor marker detection chip, characterized in that, Comprising: An immune reaction chip and a sensor chip, wherein the immune reaction chip includes a reaction layer substrate and an upper sample addition layer, the sensor chip includes a flexible sensing layer and a reaction chamber, the reaction chamber is vertically through, the upper end of the reaction chamber is sealed by the flexible sensing layer, a reaction solution is contained in the reaction chamber, and the lower end of the reaction chamber is sealed by the sample addition layer of the immune reaction chip; The reaction layer substrate includes a substrate functionalized with graphene oxide quantum dot material and capture antibodies immobilized on the substrate; A sample addition hole is provided on the sample addition layer, the sample addition hole is vertically connected, the lower end of the sample addition hole is bonded to the reaction layer substrate as a whole, and the upper end of the sample addition hole is used for adding samples; The flexible sensing layer includes a flexible sensor. After the reaction chamber of the sensor chip is used to load the sample for immune reaction, it reacts with the reaction solution in the reaction chamber to generate gas, causing a pressure change to trigger the response of the flexible sensor. According to the proportional relationship between the pressure change caused by the gas generation and the content of the tumor marker, the content of the tumor marker is obtained after the flexible sensor detects the pressure data.

2. The tumor marker detection chip according to claim 1, characterized in that, The reaction solution is a hydrogen peroxide solution with a concentration of 0.5-35 wt%.

3. The tumor marker detection chip according to claim 1, characterized in that, The reaction chamber is an acrylic plate or PDMS of a through-hole array.

4. A method for preparing the tumor marker detection chip according to any one of claims 1-3, characterized in that, Comprising: Preparing the sample addition layer: Mix polydimethylsiloxane and a curing agent and then prepare the sample addition layer by drying, and make a sample addition hole on the sample addition layer; Preparing the reaction layer substrate: Grow nanomaterials by subjecting the substrate to plasma treatment and soaking it in a nanomaterial solution, and then bond the substrate to the sample addition layer to form the reaction layer substrate; Preparing the flexible sensing layer: Form a flexible sensing layer by laminating a patterned graphene layer and a PDMS thin sheet; Preparing the reaction chamber: Mix polydimethylsiloxane and a curing agent to prepare a PDMS layer, and make a reaction chamber on the PDMS layer; Form a sensor chip by sealing the flexible sensing layer and the reaction chamber; Form a tumor marker detection chip by sealing the sensor chip and the immune reaction chip.

5. The method for preparing the tumor marker detection chip according to claim 4, characterized in that, The forming of the flexible sensing layer by laminating the patterned graphene layer and the PDMS thin sheet includes preparing a patterned graphene layer on a polyimide substrate by a laser engraving process, then using a sticky PDMS thin sheet to stick on the polyimide of the patterned graphene layer, transferring the graphene layer to the PDMS thin sheet by mechanical peeling, connecting leads at the electrode positions of the graphene layer, and finally tightly laminating the PDMS thin sheet with the patterned graphene layer with the graphene layer pattern aligned to form the flexible sensing layer.

6. The method for preparing the tumor marker detection chip according to claim 4, characterized in that, The plasma treatment of the substrate includes subjecting a glass or acrylic substrate to plasma treatment for 5-20 min and then soaking it in a coupling agent APTES for 40-60 min.

7. The method for preparing the tumor marker detection chip according to claim 4, characterized in that, The preparation of the sample addition layer by mixing polydimethylsiloxane and a curing agent and then drying includes mixing according to the ratio of polydimethylsiloxane:curing agent = 5:1-20:1, and preparing a PDMS layer by drying at 80-110 °C for 1-2 h.

8. The method for preparing the tumor marker detection chip according to claim 4, characterized in that, After bonding the substrate with the sample application layer to form an immunoreaction substrate, it includes adding a capture antibody solution into the sample application well and incubating for 2 - 15 h, fixing the capture antibody onto the substrate at the position of the sample application well, and finally performing a blocking treatment on the position of the sample application well with BSA containing a concentration of 0.5 - 10 wt% for 5 - 30 min to form a reaction layer substrate.

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