Biochemical detection system for matrix metalloproteinase-1

Abstract

The utility model relates to the technical field of blood detection systems, and provides a biochemical detection system for matrix metalloproteinase-1. The biochemical detection system comprises a gold membrane incubation unit, a microfluidic unit and an optical fiber biochemical sensing unit, the gold membrane incubation unit and the microfluidic unit respectively comprise a glass tube, a first capillary hose, a water suction pump and a second capillary hose; the glass tube is communicated with a water suction pump through a first capillary hose; the glass tube is also communicated with a second capillary hose; a second capillary hose of the gold membrane incubation unit is communicated with a beaker; a second capillary hose of the microfluidic unit is communicated with a test tube; the optical fiber biochemical sensing unit comprises a light source, an optical fiber biochemical sensor and an optical fiber grating demodulator; the light source is connected with the fiber bragg grating demodulator through an optical fiber; an optical fiber biochemical sensor is arranged on the optical fiber and is positioned in the reaction tank. According to the utility model, the quantitative analysis of the matrix metalloproteinase-1 can be realized, the detection accuracy is improved, and the use cost is reduced.

Description

Technical Field

[0001] The utility model relates to the technical field of blood detection systems, in particular to a biochemical detection system for matrix metalloproteinase-1. Background Art

[0002] Glioblastoma is a heterogeneous brain tumor with different biological and clinical characteristics. According to the fifth edition of the World Health Organization (WHO) Classification of Tumors of the Central Nervous System (CNS) (WHO CNS5) in 2021, glioblastomas are classified into grades 1-4. Grades 1-2 are low-grade glioblastomas, and grades 3-4 are high-grade glioblastomas. Among them, grades 1-2 are low-grade glioblastomas, and grades 3-4 are high-grade glioblastomas. The higher the tumor grade, the higher the malignancy and the worse the prognosis. Among primary malignant central nervous system (CNS) tumors, glioblastoma multiforme (GBM, WHO grade 4) has the highest incidence, accounting for 46.6%, approximately 3.20 / 100,000.

[0003] Glioblastomas usually grow deep in the brain or in functional areas. The preoperative diagnosis of glioblastomas mainly relies on the symptoms, signs of patients and imaging examinations such as CT and magnetic resonance imaging (MRI). However, no single imaging technique is sufficient to completely display the structure, nature and molecular information of the tumor, often resulting in clinical "misdiagnosis". During surgical resection, it is necessary to minimize damage to normal brain tissue, which increases the difficulty and risk of the surgery.

[0004] In recent years, studies have shown that matrix metalloproteinases (MMPs) are overexpressed in many cancers, and matrix metalloproteinase-1 is an important cause of enhanced tumor invasive ability. There are also studies showing that matrix metalloproteinase-1 should not be present in a healthy brain.

[0005] Currently, for the quantitative analysis of matrix metalloproteinase-1, fluorescence quantitative PCR, immunoblotting and immunohistochemistry techniques are mostly used. Fluorescence quantitative PCR can only indirectly reflect the protein content by quantitatively analyzing mRNA. There are many regulatory factors during transcription, resulting in fluorescence quantitative PCR being unable to accurately reflect the exact content of matrix metalloproteinase-1. Due to many subjective factors interfering in the experimental process, the immunohistochemistry technique has poor experimental stability, so it cannot be accurately measured, limiting the widespread application of this technique. Immunoblotting requires the purchase of matrix metalloproteinase antibodies, internal reference antibodies and secondary antibodies, with a high cost. At the same time, the detection is time-consuming and laborious, and the throughput of a single detection is limited. Content of the Utility Model

[0006] The utility model mainly solves the technical problems that the current fluorescence quantitative PCR cannot accurately reflect the exact content of matrix metalloproteinase-1, and the immunoblotting method and immunohistochemistry technology respectively have problems such as high cost and inability to popularize and inaccurate measurement. A biochemical detection system for matrix metalloproteinase-1 is proposed to achieve quantitative analysis of matrix metalloproteinase-1, improve detection accuracy, and reduce the use cost.

[0007] The utility model provides a biochemical detection system for matrix metalloproteinase-1, comprising: a gold film incubation unit, a microfluidic unit, and an optical fiber biochemical sensing unit;

[0008] The gold film incubation unit and the microfluidic unit respectively comprise: a glass tube, a first capillary hose, a water pump, and a second capillary hose;

[0009] A reaction pool is formed inside the glass tube; the glass tube is communicated with the water pump through the first capillary hose; the glass tube is also communicated with a second capillary hose;

[0010] The second capillary hose of the gold film incubation unit is communicated with a beaker, and the beaker is communicated with the water pump through a conduit;

[0011] The second capillary hose of the microfluidic unit is communicated with a test tube;

[0012] The optical fiber biochemical sensing unit comprises: a light source, an optical fiber biochemical sensor, and an optical fiber grating demodulator;

[0013] The light source is connected to the optical fiber grating demodulator through an optical fiber;

[0014] An optical fiber biochemical sensor is arranged on the optical fiber, and the optical fiber biochemical sensor is located inside the reaction pool;

[0015] The optical fiber biochemical sensor comprises: an optical fiber core, an optical fiber cladding, an optical fiber gold film, and a TFBG grating region;

[0016] The optical fiber core has a TFBG grating region; an optical fiber cladding is arranged on the outer periphery of the optical fiber core, and an optical fiber gold film is plated on the outer surface of the optical fiber cladding.

[0017] Preferably, a cysteamine layer connected with a matrix metalloproteinase-1 antibody is arranged on the surface of the optical fiber gold film.

[0018] Preferably, plugging devices are arranged at both ends of the glass tube;

[0019] The optical fiber passes through the plugging device, so that the optical fiber biochemical sensor is located inside the reaction pool.

[0020] Preferably, a first valve is arranged on the first capillary hose;

[0021] A second valve is arranged on the second capillary hose.

[0022] Preferably, the thickness of the optical fiber gold film is 50 nm.

[0023] Preferably, the angle of the TFBG grating region is 8°.

[0024] Preferably, the test tube is used to hold the blood sample to be detected.

[0025] Preferably, a polarization controller is provided on the optical fiber between the light source and the glass tube.

[0026] A biochemical detection system for matrix metalloproteinase-1 provided by the present utility model adopts a capture system for matrix metalloproteinase-1 of nano-gold film-cysteamine-matrix metalloproteinase-1 antibody, which can generate SPR effect on the surface of the optical fiber, thereby realizing the quantitative analysis of matrix metalloproteinase-1 and improving the detection accuracy. It has high sensitivity and specificity, and solves the problem of inaccurate detection results of fluorescence quantitative PCR; the system has strong shielding ability against external changes, so a strict laboratory environment is not required, and the whole process is simple to operate, solving the problem of poor experimental stability of immunohistochemistry technology; compared with immunoblotting, the number of antibodies required for this system is reduced from three to one, only matrix metalloproteinase-1 antibody is needed, and the operation is simple and no laboratory conditions are required, greatly reducing the use cost and popularization cost.

[0027] The present utility model adopts a detachable optical fiber design. The optical fiber has an optical fiber biochemical sensor, and two units, namely a gold film incubation unit and a microfluidic unit, are provided, which can make the two processes of incubating the optical fiber gold film and detecting the blood sample proceed simultaneously, or multiple optical fibers with optical fiber biochemical sensors can be prepared in advance, further shortening the detection time and improving the detection efficiency. Moreover, it avoids the problem of mRNA contamination of fluorescence quantitative PCR and does not require the laboratory environment required by other detection means.

[0028] Generally speaking, the present utility model has the characteristics of high sensitivity, high specificity, short detection time, low cost and no need for laboratory environment, making point-of-care testing possible. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 is a schematic structural diagram of the biochemical detection system for matrix metalloproteinase-1 provided by the present utility model;

[0030] Figure 2 is a schematic structural diagram of the gold film incubation unit provided by the present utility model;

[0031] Figure 3 is a schematic structural diagram of the optical fiber biochemical sensor provided by the present utility model;

[0032] Figure 4It is a schematic diagram of the surface layer of the optical fiber gold film provided by the present utility model.

[0033] Reference numerals: 1, light source; 2, polarization controller; 3, optical fiber; 4, glass tube; 5, test tube; 6, first capillary hose; 7, reaction cell; 8, water pump; 9, fiber Bragg grating demodulator; 10, sealing device; 11, optical fiber gold film; 12, TFBG grating region; 13, optical fiber cladding; 14, optical fiber core; 15, matrix metalloproteinase-1; 16, cysteamine layer; 17, second capillary hose; 18, first valve; 19, beaker; 20, conduit; 21, second valve. Detailed implementation manners

[0034] To make the technical problems solved by the present utility model, the technical solutions adopted and the achieved technical effects clearer, the present utility model will be further described in detail below with reference to the accompanying drawings and embodiments. It can be understood that the specific embodiments described herein are only used to explain the present utility model, rather than limiting the present utility model. In addition, it should be noted that for the sake of description, only parts related to the present utility model are shown in the accompanying drawings rather than all the content.

[0035] As Figure 1 shown, a biochemical detection system for matrix metalloproteinase-1 provided by an embodiment of the present utility model includes: a gold film incubation unit, a microfluidic unit, and an optical fiber biochemical sensing unit.

[0036] As Figure 2 shown, the gold film incubation unit and the microfluidic unit respectively include: a glass tube 4, a first capillary hose 6, a water pump 8, and a second capillary hose 17.

[0037] A reaction cell 7 is formed inside the glass tube 4. The optical fiber 3 passes through the sealing device 10 so that the optical fiber biochemical sensor is located inside the reaction cell 7. Sealing devices 10 are provided at both ends of the glass tube 4. The sealing devices 10 are detachable, and the glass tube 4 and the sealing devices 10 can be used for the convenient operation of disassembling and installing the optical fiber biochemical sensor.

[0038] The glass tube 4 is communicated with the water pump 8 through the first capillary hose 6; a first valve 18 is provided on the first capillary hose 6; the glass tube 4 is also communicated with a second capillary hose 17. A second valve 21 is provided on the second capillary hose 17.

[0039] The second capillary hose 17 of the gold film incubation unit is communicated with a beaker 19, and the beaker 19 is communicated with the water pump 8 through a conduit 20. The beaker 19 is used to hold various liquids required during the gold film incubation process, which are cysteamine ethanol solution, ethanol solution, water, NHS solution, EDC solution, PBS buffer solution, matrix metalloproteinase-1 antibody solution mixture, HBS-ES buffer solution, and distilled water in sequence.

[0040] The second capillary hose 17 of the microfluidic unit is connected to the test tube 5. The test tube 5 is used to hold the blood sample to be detected.

[0041] In the present utility model, the main structures of the gold film incubation unit and the microfluidic unit are the same, and the difference is that: the second capillary hose 17 of the gold film incubation unit is connected to the beaker 19, and a conduit 20 is connected between the beaker 19 and the water pump 8; the second capillary hose 17 of the microfluidic unit is connected to the test tube 5. The gold film incubation unit is used to incubate the cysteamine layer 16 of the optical fiber gold film 11, and the microfluidic unit is used to detect the blood sample. In actual application, a set of the gold film incubation unit and the microfluidic unit can be set, and the beaker 19 and the test tube 5 can be replaced for use, but the incubation of the optical fiber gold film 11 and the detection of the blood sample cannot be carried out simultaneously, and the efficiency is low; in order to improve the test efficiency, the gold film incubation unit and the microfluidic unit can be separately set, and the incubation of the optical fiber gold film 11 and the detection of the blood sample can be carried out simultaneously, and the efficiency is high.

[0042] The optical fiber biochemical sensing unit includes: a light source 1, a polarization controller 2, an optical fiber biochemical sensor, and an optical fiber grating demodulator 9. The light source 1 uses a broadband light source. The light source 1 is connected to the optical fiber grating demodulator 9 through an optical fiber 3; an optical fiber biochemical sensor is arranged on the optical fiber 3, and the optical fiber biochemical sensor is located in the reaction cell 7. A polarization controller 2 is arranged on the optical fiber 3 between the light source 1 and the glass tube 4.

[0043] As Figure 3 shown, the optical fiber biochemical sensor includes: an optical fiber core 14, an optical fiber cladding 13, an optical fiber gold film 11, and a TFBG grating region 12. The optical fiber core 14 has a TFBG grating region 12; an optical fiber cladding 13 is arranged on the outer periphery of the optical fiber core 14, and an optical fiber gold film 11 is plated on the outer surface of the optical fiber cladding 13. The thickness of the optical fiber gold film 11 is 50 nm. The TFBG grating region is written by using a phase mask template, and the angle of the TFBG grating region 12 is 8°.

[0044] As Figure 4 shown, the surface of the optical fiber gold film 11 has a cysteamine layer 16 connected with matrix metalloproteinase-1 antibody, and the cysteamine layer 16 is formed by the combination of matrix metalloproteinase-1 antibody and cysteamine through an amide bond (covalent immobilization). The matrix metalloproteinase-1 antibody can specifically bind to matrix metalloproteinase-1, and the combination of the two causes a change in the effective refractive index of the optical fiber gold film 11, resulting in a shift in the wavelength of the SPR resonance peak and the TFBG cladding resonance peak.

[0045] The fiber optic biochemical sensor of the present utility model has a TFBG grating region 12. As an innovative passive fiber optic device, the tilted fiber Bragg grating (TFBG) exhibits its unique advantages in the communication band. The TFBG not only inherits the characteristics of the traditional fiber Bragg grating (FBG) such as low cost, compact size, and high sensitivity, but also has excellent cladding mode coupling ability. This ability enables the TFBG to flexibly excite high-order cladding modes and effectively transfer the optical energy from the fiber core to the fiber surface, thereby generating diverse physical field coupling mechanisms when interacting with external materials, and achieving highly sensitive detection of external perturbations. Although the evanescent field of the TFBG in the bare fiber state is relatively weak, resulting in limited detection sensitivity to environmental changes, the surface plasmon resonance (SPR) technology provides a solution with its high sensitivity and fast real-time monitoring ability. With the integration and development of the SPR technology and the fiber optic sensing technology, the sensor design combining the TFBG and the SPR can significantly improve the detection sensitivity and reduce the detection limit. Therefore, the application of the TFBG in the field of biochemical detection has been extended to multiple sensing technology fields.

[0046] The operation process of a biochemical detection system for matrix metalloproteinase-1 of the present utility model:

[0047] First is the gold film incubation process:

[0048] Place the optical fiber 3 coated with a 50-nm fiber optic gold film 11 in the glass tube 4 of the gold film incubation unit. Place an appropriate amount of 20 mM cysteamine ethanol solution in the beaker 19. Open the first valve 18 of the gold film incubation unit, close the second valve 21 of the gold film incubation unit, and turn on the water pump 8 of the gold film incubation unit. After the cysteamine ethanol solution fills the glass tube 4 of the gold film incubation unit through the conduit 20, turn off the water pump 8 of the gold film incubation unit, and soak the optical fiber in the 20 mM cysteamine ethanol solution for at least 1 hour. After forming the cysteamine layer 16, open the second valve 21 of the gold film incubation unit to allow the waste liquid to flow into the beaker 19.

[0049] Next, perform the cleaning operation. After washing the beaker 19, add ethanol solution. Open the first valve 18 and the second valve 21 of the gold film incubation unit, and turn on the water pump of the gold film incubation unit to make the ethanol solution continuously flow on the fiber optic gold film 11 for one minute. Then replace the ethanol solution with water and repeat the cleaning operation.

[0050] After the beaker 19 is washed, an NHS solution, an EDC solution, a PBS buffer solution, and a matrix metalloproteinase-1 antibody solution mixture are added, and the mixing ratio is NHS: EDC: pBS: antibody solution = 1:1:1:10. Open the first valve 18 and the second valve 21 of the gold film incubation unit, turn on the water pump 8 of the gold film incubation unit, and keep it running for 5 minutes to activate the antibody. After this process is completed, turn off the water pump 8 and drain the waste liquid. Among them, carbodiimide (EDC) forms derivatives by activating carboxyl groups and catalyzes the formation of amide bonds between amines and carboxyl groups. In the presence of EDC, N-hydroxysuccinimide (NHS) is used for the coupling reaction of carbodiimide. This reaction includes the formation of an intermediate active ester, which further reacts with amine groups to generate covalent amine bonds.

[0051] Next, the cleaning operation is performed again. Repeat the above cleaning operations with HBS-ES buffer solution and distilled water respectively, with the durations being 1 minute and 2 minutes respectively.

[0052] Through the above operations, an optical fiber biochemical sensor with a gold film 11 and a cysteamine layer 16 is fabricated on the optical fiber 3.

[0053] Next is the detection process:

[0054] Assemble an optical fiber 3 with an optical fiber biochemical sensor prepared into the optical fiber biochemical sensing unit, and make the optical fiber biochemical sensor located in the reaction cell 7 of the microfluidic unit.

[0055] Dilute the blood sample (in PBS buffer solution) so that the analysis signal conforms to the linear range of the calibration curve. Place the blood sample in the test tube 5. Turn on the light source 1, open the first valve 18 and the second valve 21 of the microfluidic unit, turn on the water pump 8 of the microfluidic unit, and make the diluted plasma flow evenly in the reaction cell 7 and combine the corresponding markers with the gold film 11 of the optical fiber biochemical sensor, thereby causing a significant change in the TFBG-SPR spectrum. After the spectrum no longer changes significantly, use the fiber Bragg grating demodulator 9 to collect the spectral changes and perform spectral demodulation in combination with MatLab to judge the effective refractive index of the current solution, so as to determine the concentration of matrix metalloproteinase-1 in the plasma sample.

[0056] Turn off the water pump 8 of the microfluidic unit, close the first valve 18 and the second valve 21 of the microfluidic unit, disassemble and replace the optical fiber 3 (since the gold film incubation process and the detection process are carried out simultaneously, there is a spare optical fiber), and repeat the above operations to complete the detection of blood samples of multiple patients.

[0057] The description of this part only represents an example of a method for quantitatively analyzing matrix metalloproteinase-1 by this device. This fiber optic biochemical sensor is not only applicable to the detection of glioma markers such as matrix metalloproteinase-1, but also to other proteins. If the target (matrix metalloproteinase-1 in this example) needs to be changed, only the cysteamine and matrix metalloproteinase-1 antibody need to be replaced with appropriate linkers and antibodies, and then the gold film incubation process can be redesigned. This utility model realizes a bio-molecule capture, recognition and detection method without PCR, with high specificity and recyclability.

[0058] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present utility model, rather than to limit it; although the present utility model has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that: modifying the technical solutions recorded in the foregoing embodiments, or equivalently replacing some or all of the technical features therein, does not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present utility model.

Claims

1. A biochemical detection system for matrix metalloproteinase-1, characterized in that: include: Gold film incubation unit, microfluidics unit, and fiber optic biochemical sensing unit; The gold film incubation unit and the microfluidic unit respectively comprise: a glass tube (4), a first capillary hose (6), a water pump (8) and a second capillary hose (17); A reaction pool (7) is formed in the glass tube (4); the glass tube (4) is connected to a water pump (8) via a first capillary hose (6); the glass tube (4) is also connected to a second capillary hose (17); The second capillary hose (17) of the gold film incubation unit is connected to a beaker (19), and the beaker (19) is connected to a water pump (8) via a conduit (20); The second capillary hose (17) of the microfluidic unit is connected to the test tube (5); The optical fiber biochemical sensing unit comprises: a light source (1), an optical fiber biochemical sensor and an optical fiber Bragg grating demodulator (9); The light source (1) is connected to a fiber grating demodulator (9) via an optical fiber (3); An optical fiber biochemical sensor is arranged on the optical fiber (3), and the optical fiber biochemical sensor is located in the reaction pool (7); The optical fiber biochemical sensor comprises: an optical fiber core (14), an optical fiber cladding (13), an optical fiber gold film (11) and a TFBG gate region (12); The optical fiber core (14) has a TFBG grating region (12); an optical fiber cladding (13) is arranged on the periphery of the optical fiber core (14), and an outer surface of the optical fiber cladding (13) is plated with an optical fiber gold film (11).

2. The biochemical detection system of matrix metalloproteinase-1 according to claim 1, characterized in that: The surface of the optical fiber gold film (11) has a cysteamine layer (16) connected to a matrix metalloproteinase-1 antibody.

3. The biochemical detection system of matrix metalloproteinase-1 according to claim 1, characterized in that: Sealing devices (10) are provided at both ends of the glass tube (4); The optical fiber (3) passes through the blocking device (10), so that the optical fiber biochemical sensor is located in the reaction pool (7).

4. The biochemical detection system of matrix metalloproteinase-1 according to claim 1, characterized in that: A first valve (18) is provided on the first capillary hose (6); A second valve (21) is provided on the second capillary hose (17).

5. The biochemical detection system of matrix metalloproteinase-1 according to claim 1, characterized in that: The thickness of the optical fiber gold film (11) is 50 nm.

6. The biochemical detection system of matrix metalloproteinase-1 according to claim 1, characterized in that: The angle of the TFBG gate region (12) is 8°.

7. The biochemical detection system of matrix metalloproteinase-1 according to claim 1, characterized in that: The test tube (5) is used to hold a blood sample to be tested.

8. The biochemical detection system of matrix metalloproteinase-1 according to claim 1, characterized in that: A polarization controller (2) is arranged on the optical fiber (3) between the light source (1) and the glass tube (4).

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