Microfluidic system and method for detecting tumor drug concentration and tumor gene mutations

Abstract

This invention discloses a microfluidic system and method for detecting tumor drug concentration and tumor gene mutations, including a sample pretreatment and filtration device, a microfluidic HPLC detection device, a protein lysis device, and a microfluidic microcavity PCR device. The microfluidic HPLC detection device is used for detecting tumor drug concentration, and the microfluidic microcavity PCR device is used for detecting tumor gene mutations. Using the same sample for pretreatment, processing each sample with 3 μL allows for the detection of drug concentration and common tumor mutations in patients, overcoming the technical problem of insufficient detection capability for small amounts of tumor cells in samples from special organs such as cerebrospinal fluid.

Description

Technical Field

[0001] This invention belongs to the field of medical device technology and relates to a microfluidic system and corresponding detection method for detecting tumor drug concentration and tumor gene mutation. Background Technology

[0002] EGFR-TKI therapy, or epidermal growth factor receptor tyrosine kinase inhibitor therapy, has opened the door to precision medicine for advanced lung cancer. By inhibiting the activity of EGFR tyrosine kinase, it blocks the EGFR signaling pathway, thereby inhibiting the proliferation, growth, and metastasis of tumor cells, achieving the goal of treating advanced lung cancer with EGFR mutations.

[0003] Classic EGFR mutations include: 1) Exon 19 deletion mutation (19del), one of the most common types of EGFR gene mutations, accounting for approximately 45% of all EGFR mutations. This mutation alters the structure of the tyrosine kinase domain of the EGFR receptor protein, keeping it in a persistently activated state, thereby promoting tumor cell proliferation and survival. 2) Exon 21 L858R point mutation, accounting for approximately 40-45% of all EGFR mutations. This mutation also leads to persistent activation of the tyrosine kinase domain of the EGFR receptor protein, further promoting tumor cell growth and spread. EGFR mutations are particularly common in Asian populations. EGFR-TKIs are the first-line treatment for classic EGFR mutations. Currently, three generations of EGFR-TKIs are available. Third-generation EGFR-TKIs, represented by osimertinib, are not only effective for patients with advanced lung cancer caused by primary EGFR mutations, but also for those resistant to first- and second-generation drugs due to EGFR exon 20 T790 mutations. Currently, third-generation EGFR-TKIs are widely used in clinical practice.

[0004] Currently, third-generation EGFR-TKIs are usually administered orally at a fixed dose, but several factors can affect the final drug concentration, including: 1) differences in drug absorption and metabolism, 2) drug interactions, 3) individual patient differences (patient pharmacogenomic polymorphism), and 5) special target organ structures.

[0005] In summary, when third-generation EGFR-TKIs, such as osimertinib, encounter problems with insignificant efficacy and disease progression during use, it may be due to insufficient EGFR-TKI concentration or the development of EGFR-TKI resistance mutations in the tumor. For patients with classic mutations who do not respond well to clinical treatment, the following situations generally apply: 1) If the drug concentration is below the effective range but the original classic mutation is still detectable in EGFR, the treatment effect can be observed by increasing the drug concentration; 2) If the drug concentration is within the effective range but the original classic mutation is still detectable in EGFR, other third-generation EGFR-TKIs or escalating doses of the same drug can be used; 3) If the drug concentration is within the effective range and the original classic EGFR mutation disappears, it suggests the existence of other resistance mechanisms, and further molecular resistance mechanism testing should be conducted to provide a basis for subsequent treatment adjustments. Therefore, simultaneously monitoring blood drug concentration and tumor mutation sites has important clinical roles in individualized treatment, including monitoring the disease, assessing efficacy, and optimizing treatment plans. Adjusting drug dosage according to individual differences ensures optimal treatment results while reducing unnecessary drug waste and adverse reactions.

[0006] Currently, the equipment used clinically to detect osimertinib concentration is HPLC or HPLC-MS. These devices are large, complex to operate, and standards are difficult to standardize, resulting in long experimental cycles and large quantities of samples and reagents. Especially for samples such as cerebrospinal fluid, which are difficult to obtain or have small quantities, the sample volume required for a single test may only be sufficient for drug concentration, preventing the testing of other parameters. For peripheral blood EGFR mutation detection, although many kits are available, a 10ml whole blood sample is generally required to collect a sufficient amount of ctDNA, and the detection capability for small amounts of tumor cells in special organs such as cerebrospinal fluid is insufficient.

[0007] The above only describes the application of osimertinib in non-small cell lung cancer. Similar problems and needs exist in targeted therapy of other tumor drugs. Based on the above reasons, this invention is proposed. Summary of the Invention

[0008] This invention addresses the aforementioned problems by providing a microfluidic system and method for detecting tumor drug concentration and tumor gene mutations, aiming to simultaneously detect patient drug concentration and common tumor mutations using a small amount (3 μL) and the same sample, and to achieve the possibility of point-of-care testing (POCT).

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

[0010] In a first aspect, this invention provides a microfluidic system for detecting tumor drug concentration and tumor gene mutations, comprising a sample pretreatment filtration device, a microfluidic HPLC detection device, a protein lysis device, and a microfluidic microcavity PCR device. The preferred configuration of the four devices is as follows:

[0011] I. Specimen Pretreatment Filtration Device

[0012] The specimen pretreatment filtration device is shaped like a syringe barrel with a total volume of 9 mL. The interior of the barrel is filled with multiple layers of filter particles of gradually decreasing size from top to bottom, and a filter membrane is installed at the connection between the barrel and the head. It removes white blood cells, red blood cells, platelets and other particulate impurities 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 with a diameter of 10 μm, filter particles with a diameter of 5 μm, and particles with a diameter of 0.5 μm; the pore size of the filter membrane is 0.22 μm, and the head part below the filter membrane is also filled with particles with a diameter of 0.5 μm.

[0014] Further optimization involves using polyvinylidene fluoride (PVDF) particles as the filter particles; the filter particles in all three filter layers are filled in the column in a parabolic shape, which can achieve a larger contact area and filtration efficiency.

[0015] II. Microfluidic HPLC Detection Device

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

[0017] The stator region is equipped with six vertical microchannels arranged in a regular hexagonal pattern, which are connected to the microchannels of related functions on the chip. For example, the channel between the two vertical microchannels at the 0 and 6 o'clock positions is the quantitative microchannel, and the other four microchannels are connected to the injection port, waste liquid pool, chromatographic column and mobile phase injection port, respectively. They can also be adjusted according to the actual structure.

[0018] The rotor is equipped with a handle, which can be used to rotate clockwise or counterclockwise within a range of 60°; there are 6 micro-holes at positions corresponding to the 6 longitudinal microchannels of the rotor and the stator, and every two adjacent micro-holes 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 sample injection area is equipped with 5 injection ports, which are connected to a longitudinal microchannel of the stator through microchannels. The 5 injection ports are respectively the plasma low concentration standard injection port, the sample to be tested injection port, the high concentration standard injection port, the cerebrospinal fluid sample injection port, and the cerebrospinal fluid high concentration standard injection port. Each of the 5 injection microchannels is equipped with a silicone conical one-way valve device at the end.

[0020] The mobile phase in the injection port is a mixture of inorganic and organic phases in a constant ratio. Downstream of this is a flow-maintaining microchannel, which is connected to a micro high-pressure constant flow plunger pump, and finally connected to one of the longitudinal microchannels in the stator region.

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

[0022] III. Protein lysis apparatus

[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 equipped with a 50 mm diameter sealable specimen and reagent inlet and a lysis product outlet, which is matched with the sample inlet of the microfluidic microcavity PCR device.

[0024] Preferably, the elliptical disc container is made of polytetrafluoroethylene, which has good heat resistance and does not adsorb DNA; the capsule can be used with a heating mixer for nuclease treatment. The heating mixer can be adjusted to 37°C and 60°C and can be adjusted to shake and mix at 2500 rpm.

[0025] IV. Microfluidic Microcavity PCR Device

[0026] It consists of three parts: ctDNA purification module, sample mixing module, and microcavity PCR reaction module.

[0027] The ctDNA purification module includes a conical sample inlet and an integrated ctDNA adsorption membrane (preferably a silica gel adsorption membrane) at the bottom of the inlet. Below the adsorption membrane is a branched pipe with a control valve inside. One end of the pipe is connected to a negative pressure suction device, and the other end is connected to the sample mixing module.

[0028] The sample mixing module includes a quantitative sample loading turntable and a mixing structure. The quantitative sample loading turntable is a turntable capable of rotating 180°. This turntable has two parallel microchannels. One of the microchannels serves as a quantitative microchannel connected to the ctDNA purification module, and the other microchannel connects to the reaction premix sample loading channel. The reaction premix sample loading channel is connected to the mixing structure, which is an equilateral parallelogram containing two acute angles and two obtuse angles. The premix sample inlet and the sample outlet are respectively located opposite the two acute angles. The sample outlet is a flat brush-shaped structure that is suspended in contact with the microcavity reaction plate of the microcavity PCR reaction module.

[0029] The microcavity PCR reaction module includes a brush applicator connected to the sample outlet and a microcavity PCR chip slidably positioned beneath it. The brush applicator has a silicone strip-shaped device with a horizontal opening at the brush head. The microcavity PCR chip is a digital PCR chip, mounted on a base and equipped with 10,000 reaction cells. The base is mounted above the microcavity reaction plate via a track, with a pull cord at one end. Pulling the cord allows for the uniform spreading of the sample into the reaction cells. After spreading, a glass cover is placed, and the cells are filled with paraffin oil before subsequent PCR reactions and PCR chip scanning analysis.

[0030] During PCR, the PCR amplification instrument, microcavity PCR chip scanner, and ultraviolet spectrophotometer are integrated into a small all-in-one machine. The PCR reaction products are detected using four fluorescence channels: fam, cy5, vic, and internally controlled rox.

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

[0032] A. Specimen Pretreatment

[0033] Take a whole blood or cerebrospinal fluid sample with a volume of 30% to 40% of the volume of the sample pretreatment filter device and add it to the filter device. Remove the sealing accessories at the head of the device and apply pressure to the piston by pushing the handle. The air in the cylinder will be removed, and the sample to be tested, which has been depleted of white blood cells, red blood cells, platelets and cell debris, will slowly flow out. It contains the free drug to be tested, the drug bound to the protein and ctDNA.

[0034] B. Microfluidic HPLC detection

[0035] Adjusting the rotor puts the device into the sample injection state. Low-concentration plasma standards, test samples, high-concentration standards, or cerebrospinal fluid samples and high-concentration cerebrospinal fluid standards enter the corresponding longitudinal microchannels in the stator region through the injection port. A portion enters the quantitative microchannel, and excess samples enter the waste liquid pool. The mobile phase liquid reaches the chromatographic column through another stator microchannel, and excess liquid enters the waste liquid pool.

[0036] After the rotor rotates counterclockwise by 60°, the liquid forms another pathway. The mobile phase carries the sample in the quantitative microchannel into the chromatographic column through the stator connected to the chromatographic column, where it binds and elutes. 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 reserving the amount of sample used in step B from the pretreated sample obtained in step A, the remaining portion is injected into the oval disc-shaped capsule of the protein lysis device by injection. The same volume of lysis buffer as the sample and a small amount of proteinase K are added. After heating at 56°C for a certain time, the sample is mixed with external force to obtain a sample that releases free ctDNA.

[0039] D. PCR detection

[0040] D-1ctDNA purification

[0041] After protein lysis, the sample is directly squeezed into the conical injection chamber, anhydrous ethanol is added, and the mixture is thoroughly vortexed to precipitate DNA from the solution, which can then be adsorbed onto the silica membrane on the chip. The negative pressure suction device is then connected, and the sample is filtered under negative pressure. After the sample has been completely filtered, the valve is closed, BufferAW1 is added to the conical chamber, the valve is opened, and the liquid is aspirated under negative pressure. The valve is then closed again, BufferAW2 is added to the conical chamber, the valve is opened, and the liquid is aspirated under negative pressure. This step is repeated, and the sample is then air-dried for 5-10 minutes. The valve is then closed, BufferAVE is added to the ctDNA adsorption membrane, and the sample is incubated at room temperature for 5 minutes before proceeding to the next step.

[0042] D-2 pre-reaction solution mixture

[0043] Rotate the turntable to connect the quantitative channel pathway. Under the negative pressure suction, the ctDNA sample enters the quantitative microchannel through the microchannel below the cone-shaped pool. When the quantitative microchannel is full of sample, rotate the turntable 180° to connect the other microchannel pathway. Add the premixed PCR reaction solution and push it into the mixing pool through the sample injector. This pushes the PCR reaction system in the mixing pool into the mixing structure once again.

[0044] D-3 microcavity PCR chip preparation

[0045] After the reaction system is mixed, it flows into the brush applicator through the microchannel. The sample is evenly spread in the reaction chamber by pulling the pull rope on the base. Then, paraffin oil is dropped onto the microcavity PCR chip and sealed with a glass cover. Pulling the pull rope again causes the prepared microcavity PCR chip to slide out from above the microcavity reaction plate.

[0046] D-4PCR detection

[0047] The microcavity PCR chip was placed in a PCR instrument with amplifiable chip for amplification, and the chip was scanned using a fluorescence detection device and analyzed by software. A positive result of more than 3 points was used as the interpretation criterion. Based on the premise that there is a signal in the internal control channel, the corresponding gene mutation was determined.

[0048] In a third aspect, this invention provides a method for detecting osimertinib and lung cancer gene mutations using this method, 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 concentration is within the effective range of 2 to 5 ng / ml, mainly reflected in steps B and D, as follows:

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

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

[0051] For HPLC detection, a mobile phase containing 31% potassium dihydrogen phosphate (pH 3.6) and 69% acetonitrile was used as the stationary phase to elute osimertinib. The column temperature was maintained at 30℃, and the UV detection wavelength was 251 nm. The osimertinib elution peak was obtained in about 6 minutes. If the metabolite of osimertinib is to be detected simultaneously, the absorption wavelength can be selected at 265 nm. The elution peak of its metabolite AZ5104 can be obtained before the osimertinib elution peak. At the same time, the corresponding concentrations of AZ5104 were added to the injection ports of low-concentration and high-concentration plasma standards for concentration determination.

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

[0053] In step D, the reaction conditions for fluorescent PCR are as follows: 95℃ pre-denaturation for 10 minutes, 45 cycles of 94℃ denaturation for 30 seconds, 56℃ annealing and extension for 60 seconds: 72℃ final extension for 2 minutes, and incubation at 4℃.

[0054] The reaction solution was prepared as follows: 3.0 μl of 2×TaqMan dPCR Master Mix, 1.0 μl of 10 μM of each primer mixture, 1.0 μl of 10 μM of each probe, and 1.0 μl of sample ctDNA;

[0055] If a FAM signal is read, it indicates an EGFR 19-del mutation; if a CY5 signal is read, it indicates an L858R mutation; and if a VIC signal is read, it indicates a T790M mutation, provided 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] This invention integrates a specimen pretreatment and filtration device, a microfluidic HPLC detection device, a protein lysis device, and a microfluidic microcavity PCR device. Using the same specimen for pretreatment, each sample treated with 3 μL can be used to detect drug concentrations and common tumor mutations in patients, overcoming the technical problem of insufficient detection capability for small amounts of tumor cells in specimens from special organs, such as cerebrospinal fluid. Attached Figure Description

[0058] Figure 1 This is a schematic diagram of the specimen pretreatment filtration device;

[0059] Figure 2 The diagram shows the structure of a microfluidic HPLC detection device: a) front view; b) cross-sectional view along the AA direction; c) stator structure.

[0060] Figure 3 This is a schematic diagram of a protein lysis device;

[0061] Figure 4 This is a schematic diagram of a microfluidic microcavity PCR device. Detailed Implementation

[0062] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the described embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0063] The microfluidic system of the present invention, which can detect tumor drug concentration and tumor gene mutation, integrates a sample pretreatment filtration device, a microfluidic HPLC detection device, a protein lysis 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 filled with particles 16 of varying sizes, serving as the physical support for the filter membrane 15. The filter membrane 15 has a diameter of 0.22 μm, and three different sizes of microparticles are filled on top of it. These three types 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). The particles 13 and 14 are filled into the column with a parabolic surface shape to obtain a larger contact area and filtration efficiency.

[0066] The reason for designing it as a parabola is that during specimen flow, the flow essentially follows a laminar state, with the velocity distribution exhibiting 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 composition, etc., and the pressure difference is provided by an external driving system, the shape of the parabola will vary, but it is far more efficient at filling than a horizontal line.

[0067] During specimen pretreatment, 3 ml of whole blood or cerebrospinal fluid can be added to the filter device. Remove the sealing fittings from the device head, and apply pressure to the piston by pushing the handle, compressing the air inside the device. The sample, now free of white blood cells, red blood cells, platelets, and cell debris, will slowly flow out, containing the target free drug, protein-bound drug, and ctDNA.

[0068] Example 2

[0069] Microfluidic HPLC detection devices such as Figure 2 As shown, the structures are all based on the microfluidic plate 100. The HPLC microfluidic plate includes a mobile phase injection port 51 for detection, a pump 52 for maintaining flow, five injection ports 41-45, a stator 31-36 and a rotor 30 that make up the 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 collection tank 60.

[0070] Mobile phase inlet 51 is used for the entry of the chromatographic column mobile phase; pump 52 uses a miniature high-pressure constant flow plunger pump. Inlets 41-45 are fitted with 0.22 μm filter membranes; 41 is for low-concentration plasma standards, 42 is for the test sample, 43 is for high-concentration standards, 44 is for cerebrospinal fluid samples, and 45 is for high-concentration cerebrospinal fluid standards. Preferably, the injection microchannel 40 has a diameter of 80 μm. All samples pass through their corresponding microchannels and are collected at the stator 33 of the injector via a single-hole valve 46. The single-hole valve 46 is an elliptical cylinder, perpendicular to the injection microchannel above the channel of 41, and has a diameter of 80 μm. By pulling 46 outward, each microchannel of 41-45 can become a unique pathway. When injecting samples, use channels 44, 45, 41, 42, and 43 in order of increasing concentration. If only cerebrospinal fluid or only plasma is available, channels 41-43 can be used.

[0071] The injector consists of six vertically oriented microchannels 31-36 and a rotor 30. Channels 31-36 are connected to functional channels on the HPLC microfluidic plate, and rotor 30 has six vertically oriented microchannels corresponding to the positions of channels 31-36. The rotor can be rotated clockwise or counterclockwise within a 60° range using handle 37. Figure 2 As shown in Figure c, the six vertical channels on the rotor are connected in pairs by three arc-shaped microchannels; as... Figure 2 As shown in b, when the vertical channel of the rotor is connected to the vertical channel on the microfluidic plate, the liquid can form a certain pathway. A quantitative microchannel is located between stators 31 and 34; preferably, this channel has a diameter of 80 μm, a length of 40 cm, and a total volume of 2 μl. Stator 32 is connected to the waste liquid reservoir 60 via a microchannel. Downstream of stator 35 is a reverse-phase chromatography column packed with octadecyl silica gel; preferably, the packed column has a microchannel φ of 100 μm, a total volume of 20 μl, and C18 particles with a φ of 1.7 μm. Downstream, a microchannel connects to the waste liquid reservoir; preferably, the microchannel φ is 80 μm.

[0072] like Figure 2 As shown in diagram a, when handle 37 is in the vertical direction (6 o'clock position), the liquid forms a pathway (33-34-36-35), which is the injection state. At this time, the sample reaches 33 through the injection port, passes through the rotor's microchannel, reaches 34, and enters the quantitative microchannel. Excess sample can enter the waste liquid pool. The constant flow micropump causes the mobile phase liquid to pass through 36 and 35 before reaching the chromatographic column and entering the waste liquid pool. When handle 37 is rotated counterclockwise by 60°, the liquid forms another pathway (36-31·34-35). The mobile phase from 36 through 31 carries the 2 μl sample from the quantitative channel into the chromatographic column through 34 and 35, where it binds and elutes. The sample concentration range is analyzed by recording the obtained peaks at the detection window.

[0073] In this embodiment, the simultaneous detection of osimertinib concentrations in both patient plasma and cerebrospinal fluid is taken as an example.

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

[0075] Elution of osimertinib with the mobile phase can be performed using either stationary elution or gradient elution. Based on the design of this apparatus, preferably, a mixture of 31% potassium dihydrogen phosphate (pH 3.6) and 69% acetonitrile is used as the stationary component for eluting the detected osimertinib. The column temperature is maintained at 30°C, and the UV detection wavelength is 251 nm. The osimertinib elution peak is obtained in approximately 6 minutes. Preferably, if the metabolite of osimertinib is to be detected simultaneously, an absorption wavelength of 265 nm can be selected. This allows the elution peak of its metabolite AZ5104 to be obtained before the osimertinib elution peak. To perform this operation, the appropriate concentration of AZ5104 is added to 41 and 43 for concentration determination.

[0076] Example 3

[0077] like Figure 3 As shown, the main body of the protein lysis device is an elliptical oblate spheroidal container 20, with two openings, 21 and 23, near the long axis. 21 is the inlet for the sample and reagents, and 23 is the outlet for the lysis products. Preferably, the container is made of polytetrafluoroethylene (PTFE) material, and the container thickness is suitable for being flexible and compressible.

[0078] Extract ctDNA from plasma or cerebrospinal fluid according to the following steps. Taking a sample volume of 1 ml as an example, commercially available reagents can be used, such as the QIAGEN QIAamp Circulating Nucleic Acid Kit: Open the sealing cap 24 of 23 and the sealing cap 22 of 21. Stand the container upright with both openings facing upwards. After reserving the sample volume used in Example 2, inject all of the pretreated sample from Example 1 into container 20 (1 ml) through 21, add 1 ml of lysis buffer (buffer ACL), and then add 20 μl of proteinase K. Preferably, heat at 56°C for 20 min using a matching heating mixer, mixing at a speed of 30 times / min. If a matching heating mixer is not available, incubate in a water bath for 30 minutes, inverting and mixing during this time to obtain a sample containing free ctDNA. Add 1 ml of anhydrous ethanol and vortex thoroughly to precipitate the DNA from the solution, which can then be adsorbed onto the silica membrane on the chip. 25 is a valve-shaped one-way valve, preferably made of silicone, which can directly squeeze the processed specimen into the subsequent operation hole.

[0079] Example 4

[0080] The microfluidic microcavity PCR device 101 was used to detect whether ctDNA in blood or cerebrospinal fluid contained mutations that could reduce drug efficacy. The microfluidic microcavity PCR device, such as... Figure 4 As shown. The microcavity PCR device consists of ctDNA purification devices 71-76, sample mixing devices with turntables 80-89, and microcavity PCR reaction devices 91-95.

[0081] The lysed protein sample obtained in Example 3 is directly injected into the conical sample inlet 71. At the bottom of the conical inlet is a silica gel adsorption membrane 76 integrated into 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 outlet channel of the conical inlet. When it is pushed to coincide with the flow channel 74, and with a negative pressure suction device connected to the outlet 73, the sample is filtered. Preferably, a negative pressure suction device is connected to 73. If a negative pressure suction device is unavailable, a large-volume syringe can also be connected to it.

[0082] After the specimen has been completely filtered through 76, close the column valve 72, add 300 μl of BufferAW1 to the conical cisterns, open the column valve 72, and aspirate the liquid under negative pressure. Close the column valve 72, add 300 μl of bufferAW2 to the conical cisterns, open the column valve 72, and aspirate the liquid under negative pressure. Repeat this step, then allow it to air dry for 5-10 minutes. Close the column valve 72, add 2 μl of BufferAVE to the silica gel membrane, and incubate at room temperature for 5 minutes, ready to proceed to the next step.

[0083] The sample mixing device consists of a quantitative sample loading turntable 80 and a mixing structure 88. The turntable has two microchannels: a 1 μl quantitative channel 81 and a regular sample loading channel 83. The turntable is rotated to the 75-81-86a-84 pathway. Negative pressure is applied at 84 to draw the ctDNA sample through the microchannel 75 below the cone-shaped chamber into the quantitative channel 81. Once the microchannel is full of sample, the turntable is rotated 180° to connect 82-81-86b-85. 5 μl of premixed PCR reaction solution is added at 82 and pushed into the mixing chamber 85 via the syringe. The mixing chamber 85 has a volume of 6 μl. After the syringe is filled with air, the PCR reaction system in the mixing chamber 85 is pushed again into the mixing structure 88, preferably with a volume of 1 μl. After the reaction system is mixed, it flows into a brush applicator 91 through a microchannel 89. The brush applicator is connected to the microchannel at the top, and the brush head is a silicone strip-shaped device with a horizontal opening. A digital PCR chip 92 is mounted on a base 96. The chip has 10,000 reaction chambers, preferably each with a volume of 500 pL. One end of the base has a pull cord 95, which allows the sample to be evenly spread in the reaction chambers. Then, paraffin oil is dropped onto the digital chip 92, and it is sealed with a glass cover plate 94. Pulling the pull ring 95 allows the prepared microcavity PCR chip to slide out of the microfluidic device 101 via the track 97.

[0084] The microcavity PCR chip was amplified using a PCR instrument with amplifiable chips, and the chip was scanned using a fluorescence detection device and analyzed by software. A positive result was defined as more than three dots: the presence of a FAM signal indicated an EGFR 19-del mutation; the presence of a CY5 signal indicated an L858R mutation; and the presence of a VIC signal indicated a T790M mutation, provided that the internal control channel also showed a signal.

[0085] The reaction conditions for fluorescent PCR were: 95℃ pre-denaturation for 10 minutes, 45 cycles of 94℃ denaturation for 30 seconds, 56℃ annealing and extension for 60 seconds, 72℃ final extension for 2 minutes, and incubation at 4℃.

[0086] The reaction solution was prepared as follows: 3.0 μl TaqMan dPCR Master Mix (2x), 1.0 μl primer mixture (10 μM), 1.0 μl probe (10 μM), and 1.0 μl sample ctDNA.

[0087] The primer and probe mixture consists of 19-del (FAM), L858R (CY5), T790M (VIC), and internal control (ROX). The primer design for these three mutations and the internal control primer design are existing technologies; for details, please refer to the invention patents published under CN101608240A and CN104818318A. The probe sequence is designed 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 described in detail above, but the present invention is not limited to the embodiments described. Those skilled in the art can make various equivalent modifications or substitutions without departing from the spirit of the present invention, and these equivalent modifications or substitutions are all included within the scope defined by the claims of this application.

Claims

1. A microfluidic system capable of detecting tumor drug concentration and tumor gene mutations, characterized in that, This includes a specimen pretreatment and filtration device, a microfluidic HPLC detection device, a protein lysis device, and a microfluidic microcavity PCR device. The specimen pretreatment filtration device is shaped like an injection cylinder. The inside of the cylinder is filled with multiple layers of filter particles with gradually decreasing size from top to bottom. A filter membrane is provided at the connection between the cylinder and the head. The microfluidic HPLC detection device, used for detecting drug concentration, is a microfluidic HPLC chip comprising a stator region, a rotor positioned above the stator, an injection region connected to the stator, a mobile phase injection port, a chromatographic column, a waste collection tank, and a detection window connected to the column outlet. The stator region has six longitudinal microchannels arranged in a regular hexagonal pattern, which are connected to the microchannels of related functions on the chip. The rotor can rotate 60°. The rotor has six microholes at positions corresponding to the six longitudinal microchannels of the stator. Every two adjacent microholes are connected to form three microchannels. The sample injection region has five sample injection ports, which are connected to one of the longitudinal microchannels of the stator through microchannels. These five sample injection ports are for low-concentration plasma standard, test sample, high-concentration standard, cerebrospinal fluid sample, and high-concentration cerebrospinal fluid standard. The protein lysis device is an elliptical dish-shaped container made of inert material, with a sample and reagent inlet and a lysis product outlet at its port. The lysis product outlet is matched with the sample inlet of the microfluidic microcavity PCR device. The microfluidic cavity PCR device includes a ctDNA purification module, a sample mixing module, and a cavity PCR reaction module. The ctDNA purification module includes a conical sample inlet and a ctDNA adsorption membrane integrated at the bottom of the inlet. Below the adsorption membrane is a branched channel, one end of which is connected to a negative pressure suction device, and the other end is connected to the sample mixing module. The sample mixing module includes a quantitative sample loading turntable and a mixing structure. The quantitative sample loading turntable is a turntable capable of rotating 180° and has two parallel microchannels. One microchannel serves as a quantitative microchannel connected to the ctDNA purification module, and the other microchannel connects to the reaction premixed solution injection channel. The reaction premixed solution injection channel is connected to the mixing structure, which is an equilateral parallelogram with a premixed solution inlet and a mixture outlet located at two opposite corners. The microcavity PCR reaction module includes a brush-shaped applicator connected to the mixed sample outlet and a microcavity PCR chip slidably disposed below it. The micropores of the microcavity PCR chip are modified with positively charged groups. The reaction solution is applied by sliding the brush under it. After application, a glass cover is placed on top and the cell is filled with paraffin oil. Then, the subsequent PCR reaction and PCR chip scanning analysis are performed.

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

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

4. The microfluidic system for detecting tumor drug concentration and tumor gene mutations 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 located at the 0 and 6 o'clock positions is the quantitative microchannel. The other four microchannels are respectively connected to the injection port, waste liquid reservoir, chromatographic column, and mobile phase injection port. The rotor is equipped with a handle, which can be used to rotate clockwise or counterclockwise within a range of 60°. The rotor and the stator region have six vertical microchannels in their corresponding positions. These six vertical microchannels are connected in pairs by three arc-shaped microchannels. When the vertical channels of the rotor are connected to the vertical channels on the microfluidic plate, the liquid can form a certain pathway.

5. The microfluidic system for detecting tumor drug concentration and tumor gene mutations according to claim 4, Its features are: in, Each of the five microchannels in the injection area is equipped with a silicone conical one-way valve at its end. The mobile phase in the injection port is a mixture of inorganic and organic phases in a constant ratio. Downstream of the mobile phase is a liquid flow maintenance microchannel, which is connected to a micro high-pressure constant flow plunger pump and finally connected to one of the longitudinal microchannels in the stator region. The upstream of the filling channel of the chromatographic column is connected to one of the longitudinal microchannels in the stator region. The chromatographic column has a volume of 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 a UV spectrophotometer, which can be used for detection.

6. The microfluidic system for detecting tumor drug concentration and tumor gene mutations according to claim 1, characterized in that: in, The elliptical disc-shaped container is made of polytetrafluoroethylene; the bag can be used with a heating mixer, which is temperature adjustable at 37°C and 60°C and speed adjustable for oscillating and mixing.

7. The microfluidic system for detecting tumor drug concentration and tumor gene mutations 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 installed in the branched pipe. The 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 a flat brush, which is suspended above the microcavity reaction plate of the microcavity PCR reaction module.

8. The microfluidic system for detecting tumor drug concentration and tumor gene mutations according to claim 7, characterized in that: in, The brush head of the brush applicator is a silicone strip-shaped device with a horizontal opening; The microcavity PCR chip is a digital PCR chip, which is installed on a base and has 10,000 reaction cells. The base is installed above the microcavity reaction plate via a track, and one end is equipped with a pull rope. The process of uniformly spreading the sample in the reaction cells is completed by pulling the rope. During PCR, the PCR amplification instrument, microcavity PCR chip scanner, and ultraviolet spectrophotometer are integrated into a small all-in-one machine. The PCR reaction products are detected using four fluorescence channels: fam, cy5, vic, and internally controlled rox.

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