A CREPT protein quantitative detection device and method based on a resonant QCM biosensor

Abstract

The present invention discloses a device and method for quantitatively detecting CREPT protein based on a resonant QCM biosensor. By constructing a circular concave area on the base, embedding a silica gel ring at the bottom of the concave area of the base, and placing the resonant QCM biosensor on the silica gel ring. On this basis, a hollow cylinder is placed on the base, and the circular through-hole is adapted to the circular concave area, so that the highly sensitive resonant QCM biosensor can be used to quantitatively detect CREPT protein. During quantitative detection, first, the pressure-frequency sensitivity coefficient K<subgt;Pf< / subgt; and the pressure-frequency coefficient C<subgt;Pf< / subgt> are calibrated, and then the obtained HA-3HA tag protein liquid and HA-3HA-CREPT protein liquid are respectively added to the surface of the upper electrode of the QCM biosensor. After the surface of the upper electrode is completely covered, according to the difference in the change in resonant frequency and the added amount, the content of CREPT protein is calculated. In this way, the low-cost and real-time online quantitative detection of the biomarker of early tumor cells - the protein for tumor cell regulation and evaluation is realized, and then the quantitative detection of the degree of early tumor carcinogenesis is achieved.

Description

Technical Field

[0001] The present invention belongs to the technical field of quantitative detection of CREPT protein. More specifically, it relates to a device and method for quantitative detection of CREPT protein based on a resonant QCM biosensor. Background Art

[0002] The diagnosis of early tumor carcinogenesis in human tissue cells is crucial for the life health and survival period of potential patients. However, there are extremely few early tumor cancer cells, which brings great difficulties to the quantitative diagnosis of the degree of early tumor carcinogenesis. Tumor high-expression cell cycle-related protein (abbreviated as CREPT protein) is a protein for tumor cell regulation and evaluation and is a biomarker for early tumor cells. At present, there is no effective means to quickly, low-costly and quantitatively analyze CREPT protein to understand the degree of early tumor carcinogenesis in human tissue cells, that is, the quantitative detection of the early Tumor carcinogenesis degree has not been realized.

[0003] The quartz crystal microbalance (QCM) biosensor is a highly sensitive mass-type sensor developed based on the piezoelectric effect principle of crystals, and it has an extremely small mass detection ability at the picogram level. The specific working principle is that when a trace amount of substance adheres to the sensitive electrode of the QCM biosensor (mass signal), its mass brings a small pressure change (pressure signal), which will cause a change in the resonant frequency of the QCM biosensor (electric signal). Therefore, we can invert the mass information according to the detected change in the resonant frequency (electric signal). Since the resonant frequency of the resonant QCM biosensor reaches dozens or even hundreds of megahertz (MHz), the lower limit of the extremely small mass that can be detected can be as low as picograms or even femtograms. It can be seen that its mass sensitivity is extremely high, which is very suitable for the medical detection field that requires extremely high mass resolution. In addition, the resonant QCM biosensor also has the advantages of simple structure, low cost, real-time online detection and high degree of digitalization.

[0004] How to use the highly sensitive resonant QCM (quartz crystal microbalance) biosensor to quantitatively detect CREPT protein, realize the low-cost and real-time online quantitative detection of the biomarker for early tumor cells - the protein for tumor cell regulation and evaluation, and further achieve the quantitative detection of the degree of early tumor carcinogenesis is the problem to be solved currently. Summary of the Invention

[0005] The object of the present invention is to overcome the deficiencies of the prior art and provide a CREPT protein quantitative detection device and method based on a resonant QCM biosensor, so as to quantitatively detect CREPT protein using a highly sensitive resonant QCM biosensor, realize low-cost and real-time online quantitative detection of early tumor cell biomarkers - tumor cell regulation and evaluation proteins, and further achieve quantitative detection of the degree of early tumor carcinogenesis.

[0006] To achieve the above object of the invention, the CREPT protein quantitative detection device based on a resonant QCM biosensor of the present invention includes:

[0007] A resonant QCM biosensor, which is composed of a circular quartz crystal and upper and lower electrodes respectively located on the upper and lower surfaces of the quartz crystal;

[0008] It is characterized in that it further includes:

[0009] A base, on which there is a circular recessed area adapted to the resonant QCM biosensor, and metal contact sites respectively adapted to the upper and lower electrodes of the resonant QCM biosensor, and the metal contact sites are connected to the electrodes on the base by wire;

[0010] A silica gel ring, which is adapted to the recessed area of the base and is embedded at the bottom of the recessed area of the base. The resonant QCM biosensor is placed on the silica gel ring and within the recessed area of the base, and the lower electrode is not in contact with the base, while the upper and lower electrodes are respectively in contact with their respective adapted metal contact sites, and the signal is transmitted through the electrode wire;

[0011] A hollow cylinder, with a circular through-hole in the middle, is placed on the base. The circular through-hole is adapted to the circular recessed area and is used to flow the liquid of the HA-3HA tagged protein to be measured and the liquid of the HA-3HA-CREPT protein to the upper electrode of the resonant QCM biosensor.

[0012] The CREPT protein quantitative detection method based on a resonant QCM biosensor of the present invention is characterized by including the following steps:

[0013] (1), Calibrate the pressure-frequency sensitivity coefficient K Pf and the pressure-frequency coefficient C Pf

[0014] Use an ultrasonic cleaning instrument to clean the electrodes of the resonant QCM biosensor. Then, drop pure water on the surface of the upper electrode of the resonant QCM biosensor. When the pure water completely covers it, continue to increase the amount of pure water. According to the four times of increasing the amount of pure water, that is, the volume ΔV1, ΔV2, solve the following equations:

[0015]

[0016] Calculate the pressure-frequency sensitivity coefficient K Pf and the pressure-frequency coefficient C Pf , where ρ L is the density of pure water, n is the overtone number of the resonant QCM biosensor, D is the diameter of the upper and lower electrodes, Δf 11 and Δf 12 are the changes in the resonant frequency before and after adding pure water ΔV1 for the first and second times respectively, Δf 22 and Δf 21 are the changes in the resonant frequency before and after adding pure water ΔV2 for the fourth and third times respectively, and f0 is the resonant frequency of the resonant QCM biosensor;

[0017] (2) Obtain the HA-3HA tagged protein liquid and the HA-3HA-CREPT protein liquid;

[0018] (3) Drop the HA-3HA tagged protein liquid onto the surface of the upper electrode of the QCM biosensor in an amount of 1 μL each time. After the upper electrode surface is completely covered, record the change in the resonant frequency Δf1. Then continue to increase the volume ΔV of the HA-3HA tagged protein liquid and record the change in the resonant frequency Δf2. According to the following formula, obtain the density ρ of the HA-3HA tagged protein liquid HA-3HA :

[0019]

[0020] Calculate the content m1 of the HA-3HA tagged protein liquid at a certain volume;

[0021] Using the same method, obtain the content m2 of HA-3HA-CREPT at a certain volume;

[0022] Calculate the content of CREPT protein, which is m2 - m1, after removing the content of HA-3HA tagged protein, i.e., m1, at a certain volume, so as to quantify the content of CREPT protein.

[0023] The object of the present invention is achieved in this way.

[0024] The CREPT protein quantitative detection device and method based on a resonant QCM biosensor. By constructing a circular recessed area on the base, inlaying a silica gel ring at the bottom of the recessed area of the base, placing the resonant QCM biosensor on the silica gel ring and within the recessed area of the base, with the lower electrode not in contact with the base, and the upper electrode and the lower electrode respectively contacting their respective adapted metal contact sites, and transmitting signals through electrode wires. On this basis, a hollow cylinder is placed on the base, and the circular through-hole is adapted to the circular recessed area, so that the liquid of the HA-3HA-tagged protein to be measured and the liquid of the HA-3HA-CREPT protein can flow into the upper electrode of the resonant QCM biosensor, and the highly sensitive resonant QCM biosensor is used to quantitatively detect the CREPT protein. During quantitative detection, first, the pressure-frequency sensitivity coefficient K Pf and the pressure-frequency coefficient C Pf of the resonant QCM biosensor are calibrated. Then, the liquid of the HA-3HA-tagged protein and the liquid of the HA-3HA-CREPT protein obtained are respectively added to the surface of the upper electrode of the QCM biosensor. After the surface of the upper electrode is completely covered, according to the difference in the change amount of the resonant frequency and the added amount, the densities of the liquid of the HA-3HA-tagged protein and the liquid of the HA-3HA-CREPT protein are calculated, and the difference between the two under a certain volume is calculated to obtain the content of the CREPT protein. In this way, the low-cost and real-time online quantitative detection of the biomarker of early tumor cells - the protein for tumor cell regulation and evaluation is realized, and further the quantitative detection of the degree of early tumor carcinogenesis is achieved. Description of the Drawings

[0025] Figure 1 is a schematic structural diagram of a specific embodiment of the base in the CREPT protein quantitative detection device based on a resonant QCM biosensor of the present invention;

[0026] Figure 2 is a schematic structural diagram of a specific embodiment of the base, silica gel ring and QCM biosensor in the CREPT protein quantitative detection device based on a resonant QCM biosensor of the present invention;

[0027] Figure 3 is a schematic structural diagram of a specific embodiment of the hollow cylinder and the lid in the CREPT protein quantitative detection device based on a resonant QCM biosensor of the present invention;

[0028] Figure 4 is a flowchart of a specific embodiment of the CREPT protein quantitative detection method based on a resonant QCM biosensor of the present invention;

[0029] Figure 5 is a specific example diagram of the change in the resonant frequency of the QCM biosensor for measuring pure water;

[0030] Figure 6 It is a specific example diagram of the change in resonance frequency when the QCM biosensor in the present invention detects the density of HA-3HA tag protein liquid and HA-3HA-CREPT protein liquid. Specific embodiments

[0031] The following describes the specific embodiments of the present invention in conjunction with the accompanying drawings, so that those skilled in the art can better understand the present invention. It should be particularly noted that in the following description, when the detailed description of known functions and designs may dilute the main content of the present invention, these descriptions will be omitted here.

[0032] In this embodiment, as Figure 1 、 2 、and shown in Figure 3, the CREPT protein quantitative detection device based on the resonant QCM biosensor of the present invention includes a base 1, a resonant QCM biosensor 2, a silica gel ring 3, a hollow cylinder 4, and a lid 5.

[0033] As Figure 1 shown, there is a circular recessed area 101 on the base 1 that is adapted to the resonant QCM biosensor 2. As Figure 2 shown, the base 1 has metal contact sites 103 that are respectively adapted to the upper electrode and the lower electrode of the resonant QCM biosensor 2, and the metal contact sites 103 are connected to the electrode wires 102 on the base. In this embodiment, the base 1 is a plastic base.

[0034] As Figure 2 shown, the resonant QCM biosensor 2 is composed of a circular quartz crystal 201 and an upper electrode 202 and a lower electrode (obscured, not shown) respectively located on the upper and lower surfaces of the quartz crystal 201.

[0035] As Figure 2 shown, the silica gel ring 3 is adapted to the recessed area of the base 1 and is embedded at the bottom of the base recessed area 101. The resonant QCM biosensor 2 is placed on the silica gel ring 3 and within the base recessed area 101, and the lower electrode is not in contact with the base 1. The upper electrode 202 and the lower electrode are respectively in contact with their respective adapted metal contact sites 103, and the signal is transmitted through the electrode wire 101. In this embodiment, as Figure 2 shown, the upper electrode 202 is a circular structure as a whole, with a diameter smaller than that of the quartz crystal 201, and there is a lead on its right side for contact with the metal contact site 103.

[0036] In this embodiment, as Figure 2As shown, there is a protruding frustum 104 at the center of the bottom inside the circular recessed area 101, so that an annular groove 105 is formed between it and the wall of the base recessed area. The silica gel ring 3 is embedded in the annular groove 105. At the same time, the height of the protruding frustum 104 is lower than the ring width of the silica gel ring 3. In this way, when the resonant QCM biosensor 2 is placed on the silica gel ring 3, the lower electrode has no contact with the base 1.

[0037] As Figure 3 shown, there is a circular through-hole 401 in the middle of the hollow cylinder 4, which is placed Figure 1 、 2 on the base 1 as shown. The circular through-hole 401 is adapted to the circular recessed area 101 and is used to flow the liquid of the HA-3HA tagged protein to be detected and the HA-3HA-CREPT protein liquid to the upper electrode 202 of the resonant QCM biosensor 2.

[0038] In this embodiment, as Figure 3 shown, it further includes a lid 5. The hollow cylinder 4 and the lid 5 are both made of plastic material. The lid 5 is adapted to the hollow cylinder 4 and is used to cover the upper end of the hollow cylinder 4.

[0039] Based on the above structure, the present invention also provides a method for quantitatively detecting CREPT protein based on a resonant QCM biosensor. As Figure 4 shown, it includes the following steps:

[0040] Step S1: Calibrate the pressure-frequency sensitivity coefficient K Pf and the pressure-frequency coefficient C Pf

[0041] According to the relationship between the liquid density and the QCM biosensor:

[0042]

[0043] It can be known that K Pf and C Pf are the pressure-frequency sensitivity coefficient and the pressure-frequency coefficient respectively. The two are parameters related to the QCM biosensor itself and have nothing to do with the type of liquid. In order to determine the above two parameters, the QCM biosensor can be ultrasonically cleaned; then, pure water is dropped on the surface of the QCM biosensor electrode successively in an amount of 1 microliter each time, and the resonant frequency value of the QCM sensor is recorded each time. Since the viscosity and density of pure water are known, it can be seen that the change in the resonant frequency after successively increasing the pure water on the electrode surface is as Figure 5As shown, when pure water does not completely cover the electrode, the change in its resonant frequency is mainly caused by the weight of the liquid (pressure - frequency coefficient) and the stress change between the liquid and the electrode surface (pressure - frequency sensitivity coefficient). When the pure water completely covers the electrode and then the amount of pure water is continuously increased, the change in the resonant frequency is mainly caused by the increase in the mass of the liquid. At this time, the stress between the pure water and the electrode no longer changes with the increase in pure water. Based on the above analysis, the density and viscosity parameters of the QCM biosensor under liquid phase operation can be decoupled respectively, that is, an unknown liquid's viscosity and density can be measured simultaneously with one sensor; and the two parameters of the QCM biosensor in liquid phase application - the pressure - frequency sensitivity coefficient and the pressure - frequency coefficient can be calibrated. Specifically, it includes the following steps:

[0044] Use an ultrasonic cleaning instrument to clean the electrode of the resonant QCM biosensor. Then, drop pure water on the surface of the upper electrode of the resonant QCM biosensor. When the pure water completely covers it, continue to increase the amount of pure water. According to the amounts of pure water added twice, namely the volumes ΔV1 and ΔV2, solve the following system of equations:

[0045]

[0046] Calculate the pressure - frequency sensitivity coefficient K Pf and the pressure - frequency coefficient C Pf , where ρ L is the density of pure water, n is the overtone number of the resonant QCM biosensor, D is the diameter of the upper and lower electrodes, Δf 11 and Δf 12 are the changes in resonant frequency before and after adding pure water ΔV1 for the first time and the second time respectively, Δf 22 and Δf 21 are the changes in resonant frequency before and after adding pure water ΔV2 for the fourth time and the third time respectively, and f0 is the resonant frequency of the resonant QCM biosensor.

[0047] Step S2: Obtain HA - 3HA tagged protein liquid and HA - 3HA - CREPT protein liquid

[0048] In this embodiment, it includes the following steps:

[0049] Step S2.1: Construct a stable CREPT - overexpressing cell line: Culture the colorectal cancer cell line DLD - 1 in a medium. Then, add 3HA lentivirus to the DLD - 1 colorectal cancer cell line to construct a stable colorectal cancer cell line DLD - 1 3HA with overexpressed CREPT protein as the negative control group.

[0050] In this example, the colorectal cancer cell line DLD-1 purchased from the American Type Culture Collection was cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum and penicillin / streptomycin.

[0051] Meanwhile, the gene sequence controlling CREPT transcription synthesis was loaded onto the 3HA lentivirus to form the 3HA-CREPT lentivirus. The amount of the 3HA lentivirus was the same as that added to the negative control group. Then, the 3HA-CREPT lentivirus (which can control the transcription synthesis of CREPT protein in DLD-1 cells) was added to the colorectal cancer cell line DLD-1 to form the colorectal cancer cell line DLD-1 3HA-CREPT with overexpressed CREPT. Then, under the stimulation of the 3HA-CREPT lentivirus, the colorectal cancer cell line DLD-1 3HA-CREPT transcribed and synthesized CREPT protein, and thus the 3HA-CREPT protein was formed.

[0052] Step S2.2: The stable colorectal cancer cell line DLD-1 3HA and the colorectal cancer cell line DLD-1 3HA-CREPT with overexpressed CREPT were respectively lysed, and HA antibodies were added to form the HA-3HA cell lysate and the HA-3HA-CREPT cell lysate.

[0053] In this example, lysing the cells is equivalent to exogenous co-Immunoprecipitation (co-IP). The specific steps are as follows:

[0054] a) Discard the medium and wash twice with pre-cooled PBS (phosphate buffered saline).

[0055] b) Add 1 ml of RIPA Lysis buffer (cell lysate) to each 10-cm dish, transfer it to a 4°C refrigerator for lysis for 30 minutes, shake the culture dish every 10 minutes, transfer the lysed cells to a 1.5-ml centrifuge tube, and place it on the rotor in the 4°C refrigerator in Room 303 for rotary lysis for 2 hours.

[0056] c) Ultrasonic treatment: Place the EP tube on ice and perform ultrasonic treatment for 10 s, pause for 10 s, for a total of 5 cycles.

[0057] d) After the lysis is completed, centrifuge at 12,000 rpm at 4°C for 10 minutes.

[0058] Adding HA antibody for exogenous IP (Immunoprecipitation): Transfer the remaining cell lysate into a new 1.5 ml EP tube, directly add 2 μg of HA antibody, and incubate with rotation at 4°C overnight. Ensure that the HA antibody binds fully to the 3HA-tagged protein (in the negative control group) and the 3HA-CREPT protein (in the target group), and form the HA-3HA-tagged protein cell lysate (control group) and the HA-3HA-CREPT protein cell lysate (target group) through co-immunoprecipitation co-IP.

[0059] Step S2.3: Extract the HA-3HA-tagged protein cell lysate and the HA-3HA-CREPT protein cell lysate respectively to obtain the HA-3HA-tagged protein liquid and the HA-3HA-CREPT protein liquid. Specifically:

[0060] Adding beads: The next day, add 30 μl of protein G / A beads that specifically recognize the heavy chain of the antibody to the cell lysate after adding the antibody, and continue to incubate with rotation at 4°C for 2 - 4 hours.

[0061] Washing beads: Centrifuge at 3000 rpm and 4°C for 2 min, carefully aspirate and discard the supernatant, avoiding aspirating the beads. Resuspend the beads with 1 ml of lysis buffer and rotate at 4°C for 10 min. Repeat the washing 4 times.

[0062] Finally, aspirate and discard the supernatant, add 30 ul of double-distilled water, mix well, and then perform on-machine detection to obtain the HA-3HA-tagged protein liquid and the HA-3HA-CREPT protein liquid.

[0063] Step S3: Quantitatively detect the content of CREPT protein

[0064] Drop the HA-3HA-tagged protein liquid onto the surface of the upper electrode of the QCM biosensor drop by drop at a rate of 1 μl each time, and record the value of the resonance frequency of the sensor each time. The change in the resonance frequency after successive addition of the HA-3HA-tagged protein liquid onto the upper electrode surface is as Figure 5 shown. After the HA-3HA-tagged protein liquid completely covers the surface, if the amount of the HA-3HA-tagged protein liquid is further increased, the change in its resonance frequency is mainly caused by the increase in the mass of the 3HA-tagged protein liquid. At this time, the stress between the HA-3HA-tagged protein liquid and the upper electrode no longer changes with the increase in the HA-3HA-tagged protein liquid. In this way, the content m1 of a certain volume of the HA-3HA-tagged protein liquid can be calculated. Specifically:

[0065] Drop the HA-3HA tag protein liquid onto the surface of the upper electrode of the QCM biosensor in an amount of 1 μL each time. After the surface of the upper electrode is completely covered, record the change in resonance frequency Δf1. Then continue to increase the amount (i.e., volume ΔV) of the HA-3HA tag protein liquid and record the change in resonance frequency Δf2. According to the following formula, obtain the density ρ of the HA-3HA tag protein liquid HA-3HA :

[0066]

[0067] Calculate the content m1 of the HA-3HA tag protein liquid at a certain volume;

[0068] Using the same method, obtain the content m2 of HA-3HA-CREPT at a certain volume;

[0069] Calculate the content of CREPT protein, which is m2 - m1, after removing the content of HA-3HA tag protein (i.e., m1) at a certain volume, so as to quantitatively determine the content of CREPT protein.

[0070] Although the above describes the illustrative specific embodiments of the present invention for the understanding of those skilled in the art of the present technology, it should be clear that the present invention is not limited to the scope of the specific embodiments. For those of ordinary skill in the art of the present technology, as long as various changes are within the spirit and scope of the present invention defined and determined by the appended claims, these changes are obvious, and all inventions and creations using the concept of the present invention are within the scope of protection.

Claims

1. A method for quantitatively detecting CREPT protein based on a resonant QCM biosensor, characterized in that, Comprising the following steps: (1) Calibrate the pressure-frequency sensitivity coefficient K of the resonant QCM biosensor Pf and the pressure-frequency coefficient C Pf Clean the electrodes of the resonant QCM biosensor using an ultrasonic cleaning instrument. Then, drop pure water onto the surface of the upper electrode of the resonant QCM biosensor. When the pure water completely covers the surface, continue to increase the amount of pure water. According to the amounts of pure water added twice, namely the volumes ΔV1 and ΔV2, solve the following system of equations: Calculate the pressure-frequency sensitivity coefficient K Pf and the pressure-frequency coefficient C Pf , where ρ L is the density of pure water, n is the overtone number of the resonant QCM biosensor, D is the diameter of the upper and lower electrodes, Δf 11 and Δf 12 are the changes in resonant frequency before and after the first addition of pure water and the change in frequency after the second addition of pure water ΔV1, respectively. Δf 22 and Δf 21 are the changes in resonant frequency before and after the fourth and third additions of pure water ΔV2, respectively. f0 is the resonant frequency of the resonant QCM biosensor; (2) Obtain HA-3HA tagged protein liquid and HA-3HA-CREPT protein liquid; (3) Drop the HA-3HA tag protein liquid onto the surface of the upper electrode of the QCM biosensor in aliquots of 1 μL each. After the upper electrode surface is completely covered, record the change in resonance frequency Δf1. Then continue to increase the volume of the HA-3HA tag protein liquid by an amount ΔV, and record the change in resonance frequency Δf2. According to the following formula, obtain the density ρ of the HA-3HA tag protein liquid HA-3HA : Calculate the content m1 of HA-3HA tagged protein liquid at a certain volume; Using the same method, obtain the content m2 of HA-3HA-CREPT at a certain volume; Calculate the content of CREPT protein, which is m2 - m1 after removing the content of HA-3HA tagged protein, i.e., m1, at a certain volume, thereby quantifying the content of CREPT protein.

2. The CREPT protein quantitative detection method based on a resonant QCM biosensor according to claim 1, wherein The obtaining of HA-3HA tagged protein liquid and HA-3HA-CREPT protein liquid described in step (2) comprises the following steps: Step 2.1): Construct a stable CREPT overexpressing cell line: Culture the colorectal cancer cell line DLD-1 in a medium. Then, add 3HA lentivirus to the DLD-1 colorectal cancer cell line to construct a stable colorectal cancer cell line DLD-1 3HA with overexpressed CREPT protein as the negative control group; Meanwhile, load the gene sequence controlling CREPT transcription synthesis onto the 3HA lentivirus to form 3HA-CREPT lentivirus. The amount of 3HA lentivirus is the same as that added to the negative control group. The 3HA-CREPT lentivirus can control the DLD-1 cells to transcribe and synthesize CREPT protein. Add it to the colorectal cancer cell line DLD-1 to form a colorectal cancer cell line DLD-1 3HA-CREPT with overexpressed CREPT. Then, under the stimulation of the 3HA-CREPT lentivirus, the colorectal cancer cell line DLD-1 3HA-CREPT transcribes and synthesizes CREPT protein, and then forms 3HA-CREPT protein; Step 2.2): Lyse the cells of the stable colorectal cancer cell line DLD-1 3HA and the colorectal cancer cell line DLD-1 3HA-CREPT with overexpressed CREPT respectively, and add HA antibody to form HA-3HA cell lysate and HA-3HA-CREPT cell lysate; Step 2.3): Extract the HA-3HA tagged protein cell lysate and the HA-3HA-CREPT protein cell lysate respectively to obtain HA-3HA tagged protein liquid and HA-3HA-CREPT protein liquid.

3. An apparatus for the method for quantitatively detecting CREPT protein based on the resonant QCM biosensor according to claim 1, comprising: A resonant QCM biosensor, which is composed of a circular quartz crystal and upper and lower electrodes located on the upper and lower surfaces of the quartz crystal respectively; Characterized in that it further comprises: A base, on which there is a circular recessed area adapted to the resonant QCM biosensor, and there are metal contact sites respectively adapted to the upper electrode and the lower electrode of the resonant QCM biosensor, and the metal contact sites are connected to the electrode wires on the base; A silicone rubber ring, which is adapted to the recessed area of the base and is embedded at the bottom of the recessed area of the base. The resonant QCM biosensor is placed on the silicone rubber ring and within the recessed area of the base, and the lower electrode has no contact with the base. The upper electrode and the lower electrode are respectively in contact with their respective adapted metal contact sites, and the signal is transmitted out through the electrode wires; A hollow cylinder, with a circular through-hole in the middle, is placed on the base. The circular through-hole is adapted to the circular recessed area and is used to flow the liquid of the HA-3HA tagged protein to be measured and the HA-3HA-CREPT protein liquid to the upper electrode of the resonant QCM biosensor.

4. The device for the quantitative detection method of CREPT protein based on a resonant QCM biosensor according to claim 3, characterized in that, There is a protruding round platform at the center of the inner bottom of the circular recessed area, so that an annular groove is formed between it and the wall of the recessed area of the base. The silicone rubber ring is embedded in the annular groove. At the same time, the height of the protruding round platform is lower than the ring width of the silicone rubber ring.

5. The device for the CREPT protein quantitative detection method based on a resonant QCM biosensor according to claim 3, wherein It further includes a lid. Both the hollow cylinder and the lid are made of plastic. The lid is adapted to the hollow cylinder and is used to cover the upper end of the hollow cylinder.

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