Microfluidic system and method capable of detecting tumor drug concentration and tumor gene mutation

Abstract

The invention discloses a microfluidic system and method capable of detecting tumor drug concentration and tumor gene mutation. The microfluidic system comprises a specimen pretreatment filtering device, a microfluidic HPLC (High Performance Liquid Chromatography) detection device, a protein cracking device and a microfluidic microcavity PCR (Polymerase Chain Reaction) device. The microfluidic HPLC detection device is used for tumor drug concentration detection, the microfluidic microcavity PCR device is used for tumor gene mutation detection, the same sample is adopted for pretreatment, and the drug concentration of a patient and common tumor mutation can be detected by using 3 microliters of treated sample, so that the defects of special visceral organs and the like are overcome; and the detection capability of a small amount of tumor cells in specimens such as cerebrospinal fluid is insufficient.

Description

Technical Field

[0001] The present invention belongs to the technical field of medical devices and relates to a microfluidic system capable of detecting tumor drug concentration and tumor gene mutation and a corresponding detection method. Background Art

[0002] EGFR-TKIs therapy, the full name of which is epidermal growth factor receptor tyrosine kinase inhibitor therapy, has opened the way for precision treatment of advanced lung cancer. By inhibiting the tyrosine kinase activity of EGFR and blocking the EGFR signal transduction pathway, the proliferation, growth and metastasis of tumor cells are inhibited, achieving the purpose of treating advanced lung cancer with EGFR mutations.

[0003] Classic mutations of EGFR include: 1) Exon 19 deletion mutation (19del), which is one of the most common mutation types of EGFR gene, accounting for about 45% of all EGFR mutations. This mutation causes the structure of the tyrosine kinase domain of the EGFR receptor protein to change, making it continuously activated, thereby promoting the proliferation and survival of tumor cells. 2) Exon 21 L858R point mutation, which accounts for about 40-45% of all EGFR mutations. This mutation also causes the tyrosine kinase domain of the EGFR receptor protein to be continuously activated, thereby promoting the growth and spread of tumor cells. EGFR mutations are particularly common in Asian populations. For classic EGFR mutations, EGFR-TKIs treatment is the first choice. Currently, there are three generations of EGFR-TKIs available. The third-generation EGFR-TKIs represented by osimertinib are not only effective for patients with advanced lung cancer with primary EGFR mutations, but are also effective for first- and second-generation drug resistance caused by EGFR exon 20 T790 mutations. Currently, the third-generation EGFR-TKIs are widely used clinically.

[0004] Currently, third-generation EGFR-TKIs are usually taken orally at fixed doses, but there are many reasons that can affect the final drug concentration: 1) differences in drug absorption and metabolism, 2) drug interactions, 3) individual differences among patients (patient drug gene polymorphisms), and 5) special target organ structures.

[0005] In summary, the third-generation EGFR-TKI, such as osimertinib, encountered problems of insignificant efficacy and disease progression during its use. It may be that the concentration of EGFR-TKIs did not reach the effective dose, or that the tumor had EGFR-TKI resistance mutations. Patients with classical mutations who have poor clinical treatment effects generally have the following situations: 1) If the drug concentration is lower than the effective range and the original classical mutation can still be detected in EGFR, the treatment effect can be observed by increasing the drug concentration; 2) If the drug concentration is within the effective range and the original classical mutation can still be detected in EGFR, other third-generation EGFR-TKIs or the same drug escalating dose treatment can be used; 3) If the drug concentration is within the effective range and the original classical mutation of EGFR disappears, it indicates the existence of other resistance mechanisms, and further molecular resistance mechanism detection should be performed to provide a basis for subsequent treatment adjustments. Therefore, simultaneous monitoring of blood drug concentrations and tumor mutation sites has important clinical functions such as monitoring the condition, evaluating the efficacy, and optimizing the treatment plan for individualized treatment. The drug dose is adjusted according to individual differences to ensure the best treatment effect, while reducing unnecessary drug waste and the occurrence of toxic and side effects.

[0006] At present, the equipment used to detect the concentration of osimertinib in clinical practice is HPLC or HPLC-MS. The equipment is large and complex to operate, it is difficult to unify the standard products, the experimental cycle is long, and the amount of specimens and reagents used is large. Especially for specimens that are difficult to obtain or small in quantity, such as cerebrospinal fluid, the amount of specimen required for a single measurement may only be enough to do a single item of drug concentration, resulting in the inability to test other items. For peripheral blood EGFR mutation detection, although there are quite a few kits now, in order to collect a sufficient amount of ctDNA, generally 10ml of whole blood specimens are required, and the detection capacity for a small amount of tumor cells in special organs, such as cerebrospinal fluid specimens, is insufficient.

[0007] The above is only the application of osimertinib in non-small cell lung cancer. There are similar problems and needs in the targeted treatment of other tumor drugs. Based on the above reasons, this invention is proposed. Summary of the invention

[0008] The present invention is aimed at the above-mentioned problems and provides a microfluidic system and method for detecting tumor drug concentration and tumor gene mutation, so as to use a small amount (3 microliters) and the same specimen to simultaneously detect the patient's drug concentration and common tumor mutations, and achieve the possibility of POCT.

[0009] To achieve the above object, the present invention adopts the following technical solutions:

[0010] In one aspect, the present invention provides a microfluidic system for detecting tumor drug concentration and tumor gene mutation, which is composed of a sample pretreatment filtration device, a microfluidic HPLC detection device, a protein lysis device, and a microfluidic microcavity PCR device. The preferred composition structure of the four devices is as follows:

[0011] 1. Specimen pretreatment filtration device

[0012] The specimen pretreatment filtration device is shaped like a syringe barrel, with a total volume of 9mL. The inner part of the barrel is filled with multiple layers of filter particles with gradually decreasing filter particles from top to bottom, and a filter membrane is set at the connection between the barrel and the head; white blood cells, red blood cells, platelets and other particulate impurities are removed through physical action.

[0013] In a preferred embodiment of the present invention, the filter particle layer is filled in the lower half of the cylinder, and a total of three filter layers are provided, which are respectively filled with filter particles of φ 10μm, filter particles of φ 5μm, and particles of φ 0.5μm; the pore size of the filter membrane is 0.22μm, and the head part below the filter membrane is also filled with particles of φ 0.5μm.

[0014] Further preferably, the filter particles are polyvinylidene fluoride particles; the filter particles of the three filter layers are filled in the column in a parabolic shape, which can obtain a larger contact area and filtration efficiency.

[0015] 2. Microfluidic HPLC Detection Device

[0016] The microfluidic HPLC detection device is used to detect drug concentration. It is a microfluidic HPLC chip, including a stator area, a rotor arranged above the stator, an injection area connected to the stator, a mobile phase injection port, a chromatographic column and a waste liquid collection pool, and a detection window connected to the chromatographic column outlet.

[0017] The stator area is provided with six longitudinal microchannels arranged in a regular hexagon, which are connected to the microchannels of related functions on the chip; for example, the channel between the two longitudinal microchannels at the 0 o'clock and 6 o'clock positions is a quantitative microchannel, and the remaining four microchannels are respectively connected to the injection port, waste liquid pool, chromatographic column and mobile phase injection port, and can also be adjusted according to the actual structure.

[0018] The rotor is provided with a handle, by which it can be rotated clockwise or counterclockwise within a range of 60°; there are 6 microholes at positions corresponding to the 6 longitudinal microchannels of the rotor and the stator, and every two adjacent microholes are connected to form 3 microchannels; when the vertical channel of the rotor is connected to the vertical channel on the microfluidic plate, the liquid can form a certain passage.

[0019] The injection area is provided with 5 injection ports, which are connected to a longitudinal microchannel of the stator through a microchannel. The 5 injection ports are respectively a plasma low-concentration standard sample injection port, a test sample injection port, a high-concentration standard sample injection port, a cerebrospinal fluid sample injection port, and a cerebrospinal fluid high-concentration standard sample injection port. The end of each of the 5 injection microchannels is provided with a silicone conical one-way valve device.

[0020] The mobile phase in the mobile phase injection port is a mixed liquid of an inorganic phase and an organic phase in a constant ratio, and downstream thereof is a liquid flow maintaining microchannel, which is connected to a micro high-pressure constant-current plunger pump and finally connected to one of the longitudinal microchannels in the stator area.

[0021] The filling channel of the chromatographic column is connected upstream to one of the longitudinal microchannels in the stator region. The volume of the chromatographic column is about 20 μl and is filled with 1.7 μm C18. The end of the microchannel connected to the chromatographic column channel has a detection window for an ultraviolet spectrophotometer, which can be used for detection.

[0022] 3. Protein Lysis Device

[0023] Used for DNA precipitation from solution. The main structure is an elliptical dish-shaped container made of inert material with a total volume of 10 ml. Its port is provided with a sealable specimen and reagent inlet with a diameter of 50 mm and a lysis product outlet, which matches the sample inlet of the microfluidic micro-cavity PCR device.

[0024] Preferably, the oval dish-shaped container is made of polytetrafluoroethylene material, which has good heat resistance and does not adsorb DNA. The bag can be used in conjunction with a heating mixer for denuclease treatment. The heating mixer can adjust the temperature to 37°C and 60°C and the speed of shaking mixing can be adjusted (2500rpm).

[0025] 4. Microfluidic Micro-chamber PCR Device

[0026] It consists of three parts: ctDNA purification module, sample injection and mixing module, and micro-chamber PCR reaction module.

[0027] The ctDNA purification module includes a conical sampling pool and a ctDNA adsorption membrane (preferably a silica gel adsorption membrane) integrated at the bottom of the pool. There is a bifurcated pipe under the adsorption membrane. A control valve is provided in the bifurcated pipe. The outlet at one end of the pipe is connected to the negative pressure suction device, and the other end is connected to the sampling mixing module.

[0028] The sample injection and mixing module includes a quantitative sample injection turntable and a sample mixing structure. The quantitative sample injection turntable is a turntable that can rotate 180 degrees. The turntable has two parallel microchannels. One of the microchannels is used as a quantitative microchannel to connect the ctDNA purification module, and the second microchannel is connected to the reaction premix injection channel; the reaction premix injection channel is connected to the sample mixing structure, and the sample mixing structure is an equilateral parallelogram with two acute angles and two obtuse angles. The two acute angles are respectively provided with a premix injection port and a mixed liquid outlet. The mixed liquid outlet is in the shape of a flat brush, and the flat brush is suspended above the microcavity reaction plate of the microcavity PCR reaction module in virtual contact.

[0029] The micro-cavity PCR reaction module includes a brush-shaped sample applicator connected to the mixed liquid sample outlet and a micro-cavity PCR chip slidably arranged thereunder. The brush head of the brush-shaped sample applicator is a silicone strip device with a horizontal opening; the micro-cavity PCR chip is a digital PCR chip, which is installed on a base and is provided with 10,000 reaction pools; the base is installed above the micro-cavity reaction plate through a track, and a pull rope is provided at one end, through which the sample is evenly spread in the reaction pool, and after the coating is completed, a glass cover is covered, and the pool is filled with paraffin oil, and then the subsequent PCR reaction and scanning analysis of the PCR chip are carried out.

[0030] When performing PCR, the PCR amplifier, microcavity PCR chip scanner and ultraviolet spectrophotometer are integrated into a small all-in-one machine, and the PCR reaction products are detected using four fluorescence channels: fam, cy5, vic and internal control rox.

[0031] In a second aspect, the present invention provides a method for detecting tumor drug concentration and tumor gene mutation using the microfluidic system described above, comprising the following steps:

[0032] A. Specimen pretreatment

[0033] Take a whole blood or cerebrospinal fluid specimen with a volume of 30% to 40% of the volume of the specimen pretreatment filter device, add it to the filter device, remove the sealing accessories at the head of the device, and provide pressure to the piston by pushing the handle. The air in the cylinder, the specimen to be tested with white blood cells, red blood cells, platelets and cell fragments removed will slowly flow out, including the free drugs to be tested, the drugs bound to proteins and ctDNA;

[0034] B. Microfluidic HPLC Detection

[0035] The rotor is adjusted so that the device is in the injection state, and the plasma low-concentration standard, the sample to be tested, the high-concentration standard or the cerebrospinal fluid sample, the cerebrospinal fluid high-concentration standard enters the corresponding longitudinal microchannel of the stator area through the injection port, a part of it enters the quantitative microchannel, and the excess sample enters the waste liquid pool; the mobile phase liquid reaches the chromatographic column through another stator microchannel, and the excess liquid enters the waste liquid pool;

[0036] After the rotor rotates 60° counterclockwise, the liquid forms another passage, and the mobile phase brings the sample in the quantitative microchannel into the chromatographic column through the stator connected to the chromatographic column, and then performs binding and elution. The range of tumor drug concentration in the sample is analyzed by recording the obtained peaks in the detection window;

[0037] C. Protein cleavage

[0038] After setting aside the amount of the sample used in step B from the pretreated sample obtained in step A, the remaining portion is injected into the oval disc-shaped bag of the protein lysis device, and a lysis solution with the same volume as the sample and a small amount of proteinase K are added; after heating at 56° C. for a certain period of time, external force is applied to mix the sample to obtain a sample that releases free ctDNA;

[0039] D. PCR detection

[0040] D-1 ctDNA purification

[0041] The sample after protein lysis is directly squeezed into the conical injection pool, anhydrous ethanol is added, and it is fully vortexed to precipitate DNA from the solution and can be adsorbed by the silica gel membrane on the chip; the negative pressure suction device is connected to complete the filtration of the sample under the action of negative pressure attraction; when the sample is completely filtered, close the valve and add BufferAW1 to the conical pool, open the valve to absorb the liquid by negative pressure, then close the valve and add bufferAW2 to the conical pool, open the valve and absorb the liquid by negative pressure; repeat this step, then dry it for 5-10 minutes, close the valve, add BufferAVE to the ctDNA adsorption membrane, leave it at room temperature for 5 minutes, and prepare to proceed to the next step;

[0042] D-2 Pre-reaction solution mixing

[0043] The turntable is rotated to connect the quantitative channel. Under the action of negative pressure suction, the ctDNA sample enters the quantitative microchannel through the microchannel below the conical pool. When the quantitative microchannel is filled with the sample, the turntable is rotated 180° to connect another microchannel. The premixed PCR reaction solution is added and pushed into the mixing pool through the injector, and the PCR reaction system in the mixing pool is pushed into the mixing structure again.

[0044] Preparation of D-3 micro-chamber PCR chip

[0045] After the reaction system is mixed, it flows into the brush-shaped sample applicator through the microchannel, and the sample is evenly applied to the reaction pool by pulling the rope on the base; then paraffin oil is dripped on the microcavity PCR chip, and the chip is sealed with a glass cover, and the rope is pulled continuously to slide the prepared microcavity PCR chip out from above the microcavity reaction plate;

[0046] D-4PCR detection

[0047] The microcavity PCR chip is placed in a PCR instrument capable of amplifying the chip for amplification, and the chip is scanned using a fluorescence detection device and analyzed using software; more than 3 points are considered positive as the judgment standard, and the corresponding gene mutation is determined based on the premise that there is a signal in the internal control channel.

[0048] In a third aspect, the present invention provides a method for detecting osimertinib and lung cancer gene mutation using the method, which is used to detect whether the blood concentration of osimertinib is within the effective range of 50 ng / ml to 500 ng / ml and / or whether the cerebrospinal fluid is within the effective concentration range of 2 to 5 ng / ml, which is mainly embodied in step B and step D, as follows:

[0049] In step B, when the test specimen is a blood sample, a mixture of 2 μl of 100 ng / ml osimertinib and 2 μl of 500 ng / ml osimertinib is added to the plasma low concentration standard injection port, and both are dissolved in the mobile phase; a mixture of 2 μl of sample and 2 μl of 500 ng / ml osimertinib is added to the test specimen injection port; 3 μl of 500 ng / ml osimertinib is added to the high concentration standard injection port and dissolved in the mobile phase;

[0050] When the test specimen is a cerebrospinal fluid sample, a mixture of 2 μl of sample and 2 μl of 2 ng / ml osimertinib is added to the cerebrospinal fluid specimen injection port; a mixture of 3 ul of 2 ng / ml osimertinib is added to the cerebrospinal fluid high concentration standard injection port and dissolved with the mobile phase.

[0051] When performing HPLC detection, a mixture of 31% potassium dihydrogen phosphate (pH=3.6) and 69% acetonitrile is used as a fixed component mobile phase to elute the detected osimertinib, the column temperature is maintained at 30°C, the ultraviolet detection wavelength is 251nm, and the elution peak of osimertinib can be obtained in about 6 minutes; if the metabolites of osimertinib are to be detected at the same time, the absorption wavelength can be selected to be 265nm, and the elution peak of its metabolite AZ5104 can be obtained before the elution peak of osimertinib, and the corresponding concentration of AZ5104 is added to the plasma low concentration standard sample injection port and the high concentration standard sample injection port for concentration judgment.

[0052] After passing through the microfluidic HPLC device, when detected by the UV spectrophotometer, if the area under the peak of the sample injection port to be tested is between the area under the peak of the plasma low-concentration standard injection port and the high-concentration standard injection port, it means that the plasma drug concentration is within the effective range; if the area under the peak of the cerebrospinal fluid sample injection port is larger than that of the cerebrospinal fluid high-concentration standard injection port, it means that the cerebrospinal fluid concentration is effective;

[0053] In step D, the reaction conditions of fluorescent PCR were as follows: pre-denaturation at 95°C for 10 min, 45 cycles of denaturation at 94°C for 30 s, annealing and extension at 56°C for 60 s: terminal extension at 72°C for 2 min, and incubation at 4°C;

[0054] The reaction solution preparation system is as follows: 3.0 μl, 2× TaqMan dPCR Master Mix, 1.0 μl, 10 uM of each primer mixture component, 1.0 μl 10 uM of each probe, 1.0 μl sample ctDNA;

[0055] If a FAM signal is read, it indicates the presence of EGFR 19-del mutation; if a CY5 signal is read, it indicates the presence of L858R mutation; if a VIC signal is read, it indicates the presence of T790M mutation, all on the premise that there is a signal in the internal control channel.

[0056] Compared with the prior art, the present invention has the following beneficial effects:

[0057] The system of the present invention is integrated with a sample pretreatment filtration device, a microfluidic HPLC detection device, a protein lysis device, and a microfluidic microcavity PCR device. The same sample is used for pretreatment, and the samples treated with 3 microliters can detect the drug concentration of the patient and the common mutations of the tumor, overcoming the technical problem of insufficient detection ability of a small amount of tumor cells in special organs, such as cerebrospinal fluid and other specimens. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Figure 1 It is a schematic diagram of the structure of the sample pre-treatment filtering device;

[0059] Figure 2 Schematic diagram of the structure of the microfluidic HPLC detection device, a, front view schematic diagram; b, AA direction cross-sectional diagram; c, stator structural diagram;

[0060] Figure 3 It is a schematic diagram of the structure of the protein lysis device;

[0061] Figure 4 Schematic diagram of the structure of the microfluidic microchamber PCR device. DETAILED DESCRIPTION

[0062] In order to make the purpose, technical solution and advantages of the embodiment of the present invention clearer, the technical solution of the embodiment of the present invention will be clearly and completely described below in conjunction with the accompanying drawings of the embodiment of the present invention. Obviously, the described embodiment is a part of the embodiment of the present invention, not all of the embodiments. Based on the described embodiment of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0063] The microfluidic system capable of detecting tumor drug concentration and tumor gene mutation of the present invention is integrated with a specimen pretreatment filtering device, a microfluidic HPLC detection device, a protein cracking device and a microfluidic microcavity PCR device.

[0064] Example 1

[0065] The structure of the specimen pretreatment filtration device is as follows: Figure 1 As shown, the total volume is 9 ml, the outer cavity 10 is shaped like a syringe, and is filled with particles 16 of different sizes as a physical support for the filter membrane 15. The filter membrane 15 is 0.22 μm in diameter, and three different sizes of microparticles are filled above the filter membrane. These three kinds of particles are preferably particles 12 with a diameter of 10 μm per ml, particles 13 with a diameter of 5 μm per ml, and particles 14 with a diameter of 0.5 μm per ml. The microparticles are made of polyvinylidene fluoride (PVDF) material, and the filling particles 13 and 14 are filled in the column according to the shape of the surface being a parabola, which can obtain a larger contact area and filtration efficiency.

[0066] The reason for designing a parabola is that when the specimen flows, the specimen flow basically follows the laminar flow state, and the velocity distribution presents a parabolic shape. The classic formula describing this flow is the Hagen-Poiseuille law. In the parabolic velocity formula, liquid viscosity (η) and pressure difference (ΔP) are the parameters most closely related to the physical properties of the specimen. Although the viscosity of the specimen is affected by anticoagulants, protein concentration, cell components, etc., and the pressure difference is provided by an external drive system, the shape of the parabola will be different, but the filling efficiency is much higher than that of the horizontal line.

[0067] During specimen pretreatment, 3 ml of whole blood or cerebrospinal fluid specimens can be taken and added to the filtration device. The sealing accessories at the head of the device are removed, and the piston is pushed to provide pressure to compress the air in the device 11. The specimen to be tested, which has been freed of white blood cells, red blood cells, platelets and cell fragments, will slowly flow out, including the free drugs to be tested, the drugs bound to proteins and ctDNA.

[0068] Example 2

[0069] Microfluidic HPLC detection device such as Figure 2 As shown, the structures are all based on a microfluidic plate 100. The HPLC microfluidic plate includes a mobile phase injection port 51 required for detection, a pump 52 required for maintaining flow, five injection ports 41-45, stators 31-36 and a rotor 30 constituting an injector, a chromatographic column 53 integrated on the microfluidic plate, a detection window 54 that can be connected to a small ultraviolet spectrophotometer, and a waste liquid collection pool 60.

[0070] The mobile phase injection port 51 is used for the mobile phase of the chromatographic column to enter; the pump 52 uses a micro high-pressure constant-current plunger pump. The injection ports 41-45 are equipped with a filter membrane with a diameter of 0.22 μm, 41 is a low-concentration standard injection port for plasma, 42 is a sample injection port for the sample to be tested, 43 is a high-concentration standard injection port, 44 is a cerebrospinal fluid sample injection port, and 45 is a high-concentration standard injection port for cerebrospinal fluid. Preferably, the injection microchannel 40 has a diameter of 80 μm, and all samples pass through the corresponding microchannels and are collected at the stator 33 of the injector through the single-hole valve 46. The single-hole valve 46 is an elliptical cylinder, which is perpendicular to the injection microchannel and has a microchannel with a diameter of 80 above the channel 41. By pulling 46 outward, each microchannel of 41-45 can become a unique passage. When injecting, use the samples from low to high concentration (44, 45, 41, 42, 43). If there is only cerebrospinal fluid or only plasma, only channels 41-43 can be used.

[0071] The sample injector is composed of 6 vertical microchannels 31-36 and a rotor 30. 31-36 are respectively connected to the functional channels on the HPLC microfluidic plate. The rotor 30 has 6 vertical microchannels at the positions corresponding to 31-36. The rotor can be rotated clockwise or counterclockwise within a range of 60 degrees through a handle 37. Figure 2 As shown in c, the six vertical channels on the rotor are connected in pairs by three arc-shaped microchannels; Figure 2 As shown in FIG. 2 , when the vertical channel of the rotor is connected to the vertical channel on the microfluidic plate, the liquid can form a certain passage. Between stators 31 and 34 is a quantitative microchannel, preferably, the channel has a diameter of 80 μm, a length of 40 cm, and a total volume of 2 μl. Stator 32 is connected to waste liquid pool 60 through a microchannel. A reverse chromatographic column filled with octadecyl silica gel is connected downstream of stator 35, preferably the microchannel φ of the filling column is 100 μm, the total volume is 20 μl, and the C18 particle φ is a chromatographic column of 1.7 μm. There is a microchannel downstream connected to a waste liquid pool, preferably the microchannel φ is 80 μm.

[0072] like Figure 2 As shown in a, when the handle 37 is in the vertical direction (6 o'clock direction), the liquid can form a passage (33-34-36-35), that is, the injection state. At this time, the sample reaches 33 through the injection port, passes through the microchannel of the rotor, and enters the quantitative microchannel after reaching 34. Excessive samples can enter the waste liquid pool; the constant current micro pump allows the mobile phase liquid to pass through 36 and 35 and then reach the chromatographic column and enter the waste liquid pool. At this time, after the handle 37 is turned counterclockwise by 60°, the liquid forms another passage (36-31·34-35). The mobile phase passes from 36 through 31 and brings the 2μl sample in the quantitative channel into the chromatographic column through 34 and 35, and then combines and elutes. The range of sample concentration is analyzed by recording the peaks obtained in the detection window.

[0073] In this embodiment, the simultaneous detection of the patient's plasma osimertinib concentration and cerebrospinal fluid osimertinib concentration is taken as an example.

[0074] Since the effective range of osimertinib blood concentration in clinical use is 50ng / ml to 500ng / ml, this experiment uses a comparison method to determine whether the concentration of osimertinib is within the effective drug concentration range. 41 adds a mixture of 2μl 100ng / ml osimertinib (dissolved in mobile phase) and 2μl 500ng / ml osimertinib (dissolved in mobile phase); 42 adds a mixture of 2μl sample and 2μl 500ng / ml osimertinib (dissolved in mobile phase); 43 adds 3μl 500ng / ml osimertinib (dissolved in mobile phase). After passing through the microfluidic HPLC device, when detected by the UV spectrophotometer, if the area under the peak of 42 is between 41 and 43, it means that the plasma drug concentration is within the effective range. The effective concentration of osimertinib in cerebrospinal fluid is 2-5 ng / ml. Add a mixture of 2 μl of sample and 2 μl of 2 ng / ml osimertinib (dissolved in mobile phase) to 44, and add a mixture of 3 ul of 2 ng / ml osimertinib (dissolved in mobile phase) to 45. If the area under the peak of 44 is greater than that of 45, it means that the cerebrospinal fluid concentration is effective.

[0075] Elution of osimertinib with a mobile phase may include fixed component elution and gradient elution. Based on the design of the present device, preferably, a mixture of 31% potassium dihydrogen phosphate at pH = 3.6 and 69% acetonitrile is used as a mobile phase of a fixed component to elute the detected osimertinib, the column temperature is maintained at 30°C, the UV detection wavelength is 251nm, and the elution peak of osimertinib can be obtained in about 6 minutes. Preferably, if the metabolites of osimertinib are to be detected at the same time, the absorption wavelength can be selected at 265nm, and the elution peak of its metabolite AZ5104 can be obtained before the elution peak of osimertinib. If this operation is to be performed, the corresponding concentration of AZ5104 is added to 41 and 43 to make a concentration judgment.

[0076] Example 3

[0077] like Figure 3 As shown, the main body of the protein lysis device is an elliptical oblate spherical container 20, with two openings 21 and 23 near the long axis, 21 is the inlet of the specimen and reagent, and 23 is the outlet of the lysis product. Preferably, the container is made of polytetrafluoroethylene (PTFE) material, and the thickness of the container is preferably soft and extrudable.

[0078] Extract ctDNA from plasma or cerebrospinal fluid according to the following steps. Taking the sample volume of 1 ml as an example, commercially available reagents can be used for the extraction reagents, taking QIAGEN QIAamp Circulating Nucleic Acid Kit as an example: open the sealing cover 24 of 23, open the sealing cover 22 of 21, stand the container upright so that the two openings face upward, and after leaving the sample volume used in Example 2, inject all the pretreated samples in Example 1 into container 20 through 21 (1 ml), add 1 ml of lysis solution (bufferACL), and then add 20 μl of proteinase K. Preferably, heat at 56°C for 20 min with a heating mixer matched with the container, and the mixing speed is 30 times / min. If there is no matching heating mixer, water bath for 30 minutes, invert and mix during this period, and obtain a sample that releases free ctDNA. Add 1 ml of anhydrous ethanol and vortex mix thoroughly so that DNA precipitates from the solution and can be adsorbed by the silica gel membrane on the chip. 25 is a valve-shaped one-way valve, preferably the one-way valve is made of silicone material, and the processed specimen can be directly squeezed into the subsequent operation hole.

[0079] Example 4

[0080] The microfluidic micro-cavity PCR device 101 is used to detect whether the ctDNA in the blood or cerebrospinal fluid has mutations that cause reduced drug efficacy. Figure 4 The microcavity PCR device is composed of ctDNA purification devices 71-76, sample injection and mixing devices 80-89 with a rotating disk, and microcavity PCR reaction devices 91-95.

[0081] The sample after protein lysis obtained in Example 3 is directly squeezed into the conical injection groove 71. At the bottom of the conical pool, there is a silica adsorption membrane 76 integrated in the chip. Preferably, the silica gel membrane has a diameter of 1.5 mm. 72 is an elliptical cylindrical valve with a hole perpendicular to the outflow channel of the conical pool. When it is pushed to coincide with the flow channel 74, the sample is filtered when the outlet 73 is connected to a negative pressure suction device. Preferably, the negative pressure suction device is connected to 73. If there is no negative pressure suction device, a large-volume syringe can also be connected to it.

[0082] When the specimen is completely filtered through 76, close the column valve 72, add 300μl BufferAW1 to the conical pool, open the column valve 72 and remove the liquid by negative pressure. Close the column valve 72, add 300μl bufferAW2 to the conical pool, open the column valve 72 and remove the liquid by negative pressure. Repeat this step, then let it dry for 5-10 minutes. Close the column valve 72, add 2ul BufferAVE to the silica gel membrane, leave it at room temperature for 5 minutes, and prepare to proceed to the next step.

[0083] The sample injection and mixing device consists of a quantitative sample injection turntable 80 and a sample mixing structure 88. There are two microchannels on the turntable, namely a quantitative channel 81 with a volume of 1μl and a common sample injection channel 83. Turn the turntable to the 75-81-86a-84 passage, and use negative pressure to attract at 84 so that the ctDNA sample enters the quantitative channel 81 through the microchannel 75 below the conical pool. When the microchannel is full of samples, rotate the turntable 180° to connect 82-81-86b-85, add 5μl of premixed PCR reaction solution at 82, and push it into the mixing pool 85 through the injector. The volume of the mixing pool 85 is 6μl. After the injector is filled with air, the PCR reaction system in the mixing pool 85 is pushed into the mixing structure 88 again. The preferred mixing structure volume is 1μl. After mixing, the reaction system flows into the brush-shaped coating device 91 through the microchannel 89. The brush-shaped coating device is connected to the microchannel above, and the brush head is a silicone strip device with a horizontal opening. The digital PCR chip 92 is installed on the base 96. The chip has 10,000 reaction pools, and the volume of each microcavity is preferably 500 pl. A pull rope 95 is provided at one end of the base, and the process of evenly coating the specimen in the reaction pool can be completed by pulling the rope. Then paraffin oil is dripped on the digital chip 92, and the glass cover device 94 is used to seal the chip. The pull ring 95 is continuously pulled to slide the prepared microcavity PCR chip out of the microfluidic device 101 through the track 97.

[0084] The microcavity PCR chip was placed in a PCR instrument capable of amplifying the chip for amplification, and the chip was scanned using a fluorescence detection device and analyzed using software. The judgment standard was that more than 3 points were positive: if a FAM signal was read, it indicated that there was an EGFR 19-del mutation; if there was a CY5 signal, it indicated that there was an L858R mutation; if there was a VIC signal, it indicated that there was a T790M mutation, provided that there was a signal in the internal control channel.

[0085] The reaction conditions of fluorescent PCR were pre-denaturation at 95°C for 10 min, 45 cycles of denaturation at 94°C for 30 s, annealing and extension at 56°C for 60 s, terminal extension at 72°C for 2 min, and incubation at 4°C.

[0086] The reaction solution preparation system is: 3.0 μl TaqMan dPCR Master Mix (2x), 1.0 μl of each primer mixture component (10 uM), 1.0 μl of each probe (10 uM), and 1.0 μl of sample ctDNA.

[0087] The mixed components of the primers and probes are 19-del (FAM), L858R (CY5), T790M (VIC), and internal control (ROX). The primer designs for these three mutations and the internal control primer design are prior art. For details, reference may be made to the invention patents with publication numbers CN101608240A and CN104818318A. The probe self-designed sequence is as follows:

[0088] 19del: 5'-FAM-A-CCTCGATGTGAGTTTCTGCTTTGCTGTG-CG-BHQ1-3' (SEQ ID NO. 1);

[0089] 21L858R: 5'-CY5-A-CCTCCCTTACTTTGCCTCCTTCTGCA-CG-BHQ3-3' (SEQ ID NO. 2);

[0090] 20T790M: 5'-VIC-T-CTTCGGCTGCCTCCTGGACTATGT-GG-BHQ1-3' (SEQ ID NO.3);

[0091] Internal control: 5′-ROX-A-CACCAGCAAGCTTGCGACCT-GC-BHQ2-3′ (SEQ ID NO. 4).

[0092] The preferred embodiments of the present invention have been specifically described above, but the present invention is not limited to the embodiments. Those skilled in the art may make various equivalent modifications or substitutions without violating the spirit of the present invention. These equivalent modifications or substitutions are all included in the scope defined by the claims of this application.

Claims

1. A microfluidic system capable of detecting tumor drug concentration and tumor gene mutation, characterized in that: It includes sample pretreatment filtration device, microfluidic HPLC detection device, protein lysis device, microfluidic micro-chamber PCR device, The specimen pretreatment filtering device is shaped like an injection barrel, the inner part of the barrel is filled with multiple filter particle layers with gradually decreasing filter particles from top to bottom, and a filter membrane is provided at the connection part between the barrel and the head; The microfluidic HPLC detection device is used to detect drug concentration, and is a microfluidic HPLC chip, comprising a stator area, a rotor arranged above the stator, an injection area connected to the stator, a mobile phase injection port, a chromatographic column and a waste liquid collection pool, and a detection window connected to the chromatographic column outlet. The stator region is provided with 6 longitudinal microchannels arranged in a regular hexagon, which are connected to the microchannels of related functions on the chip; the rotor can be turned 60 degrees, and the rotor has 6 microholes at positions corresponding to the 6 longitudinal microchannels of the stator, and every two adjacent microholes are connected to form 3 microchannels; the injection area is provided with 5 injection ports, which are connected to a longitudinal microchannel of the stator through microchannels, and the 5 injection ports are respectively a plasma low-concentration standard sample injection port, a sample injection port for a sample to be tested, a high-concentration standard sample injection port, a cerebrospinal fluid sample injection port, and a cerebrospinal fluid high-concentration standard sample injection port; The protein lysis device is an elliptical dish-shaped container made of inert material, and a sample and reagent inlet and a lysis product outlet are arranged at the port thereof, and the lysis product outlet matches the sample inlet of the microfluidic micro-cavity PCR device; The microfluidic microcavity PCR device comprises a ctDNA purification module, a sample injection and mixing module, and a microcavity PCR reaction module. The ctDNA purification module comprises a conical sample injection pool and a ctDNA adsorption membrane integrated at the bottom of the pool. A bifurcated pipeline is provided below the adsorption membrane. The outlet at one end of the pipeline is connected to a negative pressure suction device, and the outlet at the other end is connected to the sample injection and mixing module. The sample injection and mixing module includes a quantitative sample injection turntable and a sample mixing structure. The quantitative sample injection turntable is a turntable that can rotate 180 degrees. The turntable has two parallel microchannels. One of the microchannels is used as a quantitative microchannel to connect the ctDNA purification module, and the second microchannel is connected to the reaction premix injection channel; the reaction premix injection channel is connected to the sample mixing structure. The sample mixing structure is an equilateral parallelogram, and the two diagonals are respectively provided with a premix injection port and a mixed liquid outlet. The microcavity PCR reaction module comprises a brush-shaped coating device connected to the mixed liquid outlet and a microcavity PCR chip slidably arranged thereunder. The micropores of the microcavity PCR chip are modified with positively charged groups. The reaction liquid is coated by sliding under the flat brush. After coating, a glass cover is placed on the cover and the pool is filled with paraffin oil. Subsequent PCR reactions and scanning and analysis of the PCR chip are then performed.

2. The microfluidic system for detecting tumor drug concentration and tumor gene mutation according to claim 1, characterized in that: in, The total volume of the specimen pretreatment filtration device was 9 mL; The filter particle layer is filled in the lower half of the cylinder, and there are three filter layers in total, which are filled with filter particles of φ 10μm, filter particles of φ 5μm, and particles of φ 0.5μm respectively; The pore size of the filter membrane is 0.22μm, and the head part below the filter membrane is also filled with particles with a diameter of 0.5μm.

3. The microfluidic system for detecting tumor drug concentration and tumor gene mutation according to claim 2, characterized in that: in, The filter particles are polyvinylidene fluoride particles; the filter particles of the three filter layers are filled in the column in a parabolic shape.

4. The microfluidic system for detecting tumor drug concentration and tumor gene mutation according to claim 2, characterized in that: in, In the microfluidic HPLC detection device, among the six longitudinal microchannels in the stator region, the channel between the two longitudinal microchannels at the 0 o'clock and 6 o'clock positions is a quantitative microchannel, and the remaining four microchannels are respectively connected to the injection port, the waste liquid pool, the chromatographic column and the mobile phase injection port. The rotor is provided with a handle, by which the rotor can be rotated clockwise or counterclockwise within a range of 60 degrees; the position liquid corresponding to the rotor and the stator area has 6 vertical microchannels, and the 6 vertical microchannels are connected in pairs by 3 arc-shaped microchannels. When the vertical channel of the rotor is connected with the vertical channel on the microfluidic plate, the liquid can form a certain passage.

5. The microfluidic system for detecting tumor drug concentration and tumor gene mutation according to claim 4, characterized in that: in, Each end of the five injection microchannels in the injection area is provided with a silica gel conical one-way valve device; The mobile phase in the mobile phase injection port is a mixed liquid of an inorganic phase and an organic phase in a constant ratio, and a liquid flow maintenance microchannel is located downstream thereof, connected to a micro high-pressure constant-flow plunger pump, and finally connected to one of the longitudinal microchannels in the stator region; The filling channel of the chromatographic column is connected to one of the longitudinal microchannels in the stator region upstream. The volume of the chromatographic column is about 20 μl and is filled with 1.7 μm C18. The end of the microchannel connected to the chromatographic column channel has a detection window for an ultraviolet spectrophotometer, which can be used for detection.

6. The microfluidic system for detecting tumor drug concentration and tumor gene mutation according to claim 1, characterized in that: in, The oval dish-shaped container is made of polytetrafluoroethylene material; the capsule bag can be used in conjunction with a heating mixer, and the heating mixer can adjust the temperature to 37°C and 60°C, and can adjust the speed of oscillation mixing.

7. The microfluidic system for detecting tumor drug concentration and tumor gene mutation according to claim 1, characterized in that: in, In the ctDNA purification module, the ctDNA adsorption membrane is a silica gel adsorption membrane, and a control valve is provided in the bifurcated pipeline; The sample mixing structure is an equilateral parallelogram with two acute angles and two obtuse angles. The two acute angles are respectively provided with a premixed liquid inlet and a mixed liquid outlet. The mixed liquid outlet is in the shape of a flat brush, and the flat brush is suspended above the microcavity reaction plate of the microcavity PCR reaction module in virtual contact.

8. The microfluidic system for detecting tumor drug concentration and tumor gene mutation according to claim 7, characterized in that: in, The brush head of the brush-shaped applicator is a silicone strip device with a horizontal opening; The micro-cavity PCR chip is a digital PCR chip, which is mounted on a base and is provided with 10,000 reaction pools; the base is mounted above the micro-cavity reaction plate through a track, and one end is provided with a pull rope, and the process of evenly coating the specimen in the reaction pool is completed by the pull rope; When performing PCR, the PCR amplifier, microcavity PCR chip scanner and ultraviolet spectrophotometer are integrated into a small all-in-one machine, and the PCR reaction products are detected using four fluorescence channels: fam, cy5, vic and internal control rox.

9. A method for detecting tumor drug concentration and tumor gene mutation using the microfluidic system according to any one of claims 1 to 8, characterized in that: The steps include: A. Specimen pretreatment Take a whole blood or cerebrospinal fluid specimen with a volume of 30% to 40% of the volume of the specimen pretreatment filter device, add it to the filter device, remove the sealing accessories at the head of the device, and provide pressure to the piston by pushing the handle. The air in the cylinder, the specimen to be tested with white blood cells, red blood cells, platelets and cell fragments removed will slowly flow out, including the free drugs to be tested, the drugs bound to proteins and ctDNA; B. Microfluidic HPLC Detection The rotor is adjusted so that the device is in the injection state, and the plasma low-concentration standard, the sample to be tested, the high-concentration standard or the cerebrospinal fluid sample, the cerebrospinal fluid high-concentration standard enters the corresponding longitudinal microchannel of the stator area through the injection port, a part of it enters the quantitative microchannel, and the excess sample enters the waste liquid pool; the mobile phase liquid reaches the chromatographic column through another stator microchannel, and the excess liquid enters the waste liquid pool; After the rotor rotates 60° counterclockwise, the liquid forms another passage, and the mobile phase brings the sample in the quantitative microchannel into the chromatographic column through the stator connected to the chromatographic column, and then performs binding and elution. The range of tumor drug concentration in the sample is analyzed by recording the obtained peaks in the detection window; C. Protein cleavage After setting aside the amount of the sample used in step B from the pretreated sample obtained in step A, the remaining portion is injected into the oval disc-shaped bag of the protein lysis device, and a lysis solution with the same volume as the sample and a small amount of proteinase K are added; after heating at 56° C. for a certain period of time, external force is applied to mix the sample to obtain a sample that releases free ctDNA; D. PCR detection D-1 ctDNA purification The sample after protein lysis is directly squeezed into the conical injection pool, anhydrous ethanol is added, and it is fully vortexed to precipitate DNA from the solution and can be adsorbed by the silica gel membrane on the chip; the negative pressure suction device is connected to complete the filtration of the sample under the action of negative pressure attraction; when the sample is completely filtered, close the valve and add BufferAW1 to the conical pool, open the valve to absorb the liquid by negative pressure, then close the valve and add bufferAW2 to the conical pool, open the valve and absorb the liquid by negative pressure; repeat this step, then dry it for 5-10 minutes, close the valve, add BufferAVE to the ctDNA adsorption membrane, leave it at room temperature for 5 minutes, and prepare to proceed to the next step; D-2 Pre-reaction solution mixing The turntable is rotated to connect the quantitative channel. Under the action of negative pressure suction, the ctDNA sample enters the quantitative microchannel through the microchannel below the conical pool. When the quantitative microchannel is filled with the sample, the turntable is rotated 180° to connect another microchannel. The premixed PCR reaction solution is added and pushed into the mixing pool through the injector, and the PCR reaction system in the mixing pool is pushed into the mixing structure again. Preparation of D-3 micro-chamber PCR chip After the reaction system is mixed, it flows into the brush-shaped sample applicator through the microchannel, and the sample is evenly applied to the reaction pool by pulling the rope on the base; then paraffin oil is dripped on the microcavity PCR chip, and the chip is sealed with a glass cover, and the rope is pulled continuously to slide the prepared microcavity PCR chip out from above the microcavity reaction plate; D-4PCR detection The microcavity PCR chip is placed in a PCR instrument capable of amplifying the chip for amplification, and the chip is scanned using a fluorescence detection device and analyzed using software; more than 3 points are considered positive as the judgment standard, and the corresponding gene mutation is determined based on the premise that there is a signal in the internal control channel.

10. A method for detecting osimertinib and lung cancer gene mutation using the method of claim 9, for detecting whether the blood concentration of osimertinib is within the effective range of 50 ng / ml to 500 ng / ml and / or whether the cerebrospinal fluid is within the effective concentration range of 2 to 5 ng / ml, characterized in that: In step B, when the test specimen is a blood sample, a mixture of 2 μl of 100 ng / ml osimertinib and 2 μl of 500 ng / ml osimertinib is added to the plasma low concentration standard injection port, and both are dissolved in the mobile phase; a mixture of 2 μl of sample and 2 μl of 500 ng / ml osimertinib is added to the test specimen injection port; 3 μl of 500 ng / ml osimertinib is added to the high concentration standard injection port and dissolved in the mobile phase. When the test specimen is a cerebrospinal fluid sample, a mixture of 2 μl of sample and 2 μl of 2 ng / ml osimertinib is added to the cerebrospinal fluid sample injection port; a mixture of 3 ul of 2 ng / ml osimertinib is added to the cerebrospinal fluid high concentration standard injection port and dissolved with the mobile phase. When performing HPLC detection, a mixture of 31% potassium dihydrogen phosphate (pH=3.6) and 69% acetonitrile is used as a mobile phase of a fixed component to elute the detected osimertinib, the column temperature is maintained at 30°C, the ultraviolet detection wavelength is 251nm, and the elution peak of osimertinib can be obtained in about 6 minutes; if the metabolites of osimertinib are to be detected at the same time, the absorption wavelength can be selected at 265nm, and the elution peak of its metabolite AZ5104 can be obtained before the elution peak of osimertinib, and the corresponding concentration of AZ5104 is added to the plasma low concentration standard sample injection port and the high concentration standard sample injection port for concentration judgment. After passing through the microfluidic HPLC device, when detected by the UV spectrophotometer, if the area under the peak of the sample injection port to be tested is between the area under the peak of the plasma low-concentration standard injection port and the high-concentration standard injection port, it means that the plasma drug concentration is within the effective range; if the area under the peak of the cerebrospinal fluid sample injection port is larger than that of the cerebrospinal fluid high-concentration standard injection port, it means that the cerebrospinal fluid concentration is effective; In step D, the reaction conditions of fluorescent PCR were as follows: pre-denaturation at 95°C for 10 min, 45 cycles of denaturation at 94°C for 30 s, annealing and extension at 56°C for 60 s: terminal extension at 72°C for 2 min, and incubation at 4°C; The reaction solution preparation system is as follows: 3.0 μl, 2× TaqMan dPCR Master Mix, 1.0 μl, 10 uM of each primer mixture component, 1.0 μl 10 uM of each probe, 1.0 μl sample ctDNA; If a FAM signal is read, it indicates the presence of EGFR 19-del mutation; if a CY5 signal is read, it indicates the presence of L858R mutation; if a VIC signal is read, it indicates the presence of T790M mutation, all on the premise that there is a signal in the internal control channel.

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