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

Abstract

The invention discloses a resonant QCM biosensor-based CREPT protein quantitative detection device and method, a circular recessed area is constructed on a base, a silica gel circular ring is embedded at the bottom of the recessed area of the base, and a resonant QCM biosensor is arranged on the silica gel circular ring. On the basis, a hollow cylinder is placed on the base, and the circular through hole is matched with the circular sunken area, so that the high-sensitivity resonant QCM biosensor can be used for quantitatively detecting the CREPT protein. During quantitative detection, firstly, a pressure-frequency sensitivity coefficient KPf and a pressure-frequency coefficient CPf are calibrated, then an obtained HA-3HA tag protein liquid and an obtained HA-3HA-CREPT protein liquid are added to the surface of an upper electrode of a QCM biosensor respectively, and after the surface of the upper electrode is completely covered, according to the resonant frequency variation difference and the adding amount, the HA-3HA tag protein liquid and the HA-3HA-CREPT protein liquid are detected according to the resonant frequency variation difference and the adding amount of the HA-3HA tag protein liquid and the HA-3HA-CREPT protein liquid. And calculating to obtain the content of the CREPT protein. Therefore, low-cost and real-time on-line quantitative detection of early tumor cell biomarker-tumor cell regulation and evaluation protein is realized, and quantitative detection of early tumor canceration degree is further realized.

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, the number of early tumor cancer cells is extremely small, which brings great difficulties to the quantitative diagnosis of the degree of early tumor carcinogenesis. Tumor highly expressed 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 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 tiny 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 based on the detected change in 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 quantitatively detect CREPT protein using a highly sensitive resonant QCM (quartz crystal microbalance) biosensor, 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 located on the upper and lower surfaces of the quartz crystal respectively;

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

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

[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 has no contact with the base. The upper and lower electrodes are respectively in contact with their respective adapted metal contact sites, and signals are transmitted through the electrode wires;

[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 ΔV 1 、ΔV 2 , solve the following system of 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 resonant frequency before and after adding pure water ΔV 1 for the first and second times respectively, Δf 22 and Δf 21 are the changes in resonant frequency before and after adding pure water ΔV 2 for the fourth and third times respectively, and f 0 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 surface of the upper electrode is completely covered, record the change in resonant frequency Δf 1 , then continue to increase the volume ΔV of the HA-3HA tagged protein liquid, and then record the change in resonant frequency Δf 2 . According to the following formula, obtain the density ρ HA-3HA of the HA-3HA tagged protein liquid:

[0019]

[0020] Calculate the content m 1 of the HA-3HA tagged protein liquid at a certain volume;

[0021] Using the same method, obtain the content m 2 of HA-3HA-CREPT at a certain volume;

[0022] Calculate the content of CREPT protein obtained after removing the HA-3HA tagged protein, that is, m 1 and then obtain the content m 2 -m 1 , thereby quantifying 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. A circular recessed area is constructed on the base, and a silica gel ring is inlaid 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 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 signals are transmitted 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 obtained liquid of the HA-3HA tagged protein and the liquid of the HA-3HA-CREPT protein are respectively added to the surface of the upper electrode of the QCM biosensor. When 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 implementation manner 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 implementation manner of the base, the silica gel ring and the 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 implementation manner 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 implementation manner 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 measuring pure water;

[0030] Figure 6 This is a specific example diagram of the resonant frequency change 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 with reference to 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 obscure the main content of the present invention, these descriptions will be omitted here.

[0032] In this embodiment, as Figure 1 , 2 , and shown in FIG. 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, the base 1 has a circular recessed area 101 adapted to the resonant QCM biosensor 2. As Figure 2 shown, the base 1 has metal contact sites 103 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 recessed area 101 of the base. The resonant QCM biosensor 2 is placed on the silica gel ring 3 and within the recessed area 101 of the base, 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 the metal contact sites 103 adapted to them, and the signal is transmitted out 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 silicone 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 silicone ring 3. In this way, when the resonant QCM biosensor 2 is placed on the silicone 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, and it is placed on Figure 1 、 2 the base 1 as shown. The circular through-hole 401 is adapted to the circular recessed area 101, and is used to flow the HA-3HA tagged protein liquid and the HA-3HA-CREPT protein liquid to be measured 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 materials. 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 electrode surface of the QCM biosensor 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 resonance 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 its resonance 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, the viscosity and density of an unknown liquid 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 are 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 upper electrode surface of the resonant QCM biosensor. When the pure water completely covers it, continue to increase the amount of pure water. According to the amount of pure water increased twice, that is, the volume ΔV 1 、ΔV 2 , 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 resonance frequency change amounts before and after the first and second increases in pure water by ΔV 1 respectively, Δf 22 and Δf 21 are the resonance frequency change amounts before and after the fourth and third increases in pure water by ΔV 2 respectively, and f 0 is the resonance frequency of the resonant QCM biosensor.

[0047] Step S2: Obtain the HA-3HA tagged protein liquid and the 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 the 3HA lentivirus to the DLD-1 colorectal cancer cell line to construct a stable colorectal cancer cell line DLD-1 3HA overexpressing the CREPT protein, that is, 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 then formed 3HA-CREPT protein.

[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 antibody was added to form the HA-3HA cell lysate and the HA-3HA-CREPT cell lysate.

[0053] In this example, lysing 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 the refrigerator at 4°C for lysis for 30 min, shake the culture dish every 10 min, transfer the lysed cells to a 1.5 ml centrifuge tube, and place it on the rotor in the refrigerator at 4°C 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 12000 rpm and 4°C for 10 min.

[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 at 4°C with rotation 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] Add 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 at 4°C with rotation for 2 - 4 hours.

[0061] Wash the beads: Centrifuge at 3000 rpm and 4°C for 2 min, carefully aspirate 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 the supernatant, add 30 μl of double-distilled water, mix well, and 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, continue to increase the amount of the HA-3HA-tagged protein liquid. 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 m of a certain volume of the HA-3HA-tagged protein liquid can be calculated 1 Specifically:

[0065] Drop the HA-3HA tagged 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 Δf. 1 Then continue to increase the volume ΔV of the HA-3HA tagged protein liquid and record the change in resonance frequency Δf. 2 Based on the following formula, obtain the density ρ of the HA-3HA tagged protein liquid. HA-3HA :

[0066]

[0067] Calculate the content m of the HA-3HA tagged protein liquid at a certain volume. 1 ;

[0068] Using the same method, obtain the content m of HA-3HA-CREPT at a certain volume. 2 ;

[0069] Calculate the content of the HA-3HA tagged protein removed at a certain volume, i.e., m 1 After that, obtain the content m of the CREPT protein 2 -m 1 Thereby quantifying the content of the CREPT protein.

[0070] Although the above describes the illustrative specific embodiments of the present invention for the convenience of those skilled in the art to understand the present invention, 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, 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 CREPT protein quantitative detection device based on a resonant QCM biosensor, comprising: A resonant QCM biosensor is composed of a circular quartz crystal and an upper electrode and a lower electrode respectively located on the upper and lower surfaces of the quartz crystal; It is characterized by further comprising: A base having a circular concave area adapted to the resonant QCM biosensor and metal contact sites adapted to the upper electrode and the lower electrode of the resonant QCM biosensor, respectively, and the metal contact sites are connected to the electrode wires on the base; A silicone ring, which is adapted to the concave area of ​​the base and embedded in the bottom of the concave area of ​​the base, the resonant QCM biosensor is placed on the silicone ring and in the concave area of ​​the base, and the lower electrode has no contact with the base, and the upper electrode and the lower electrode are respectively in contact with the metal contact sites adapted to them, and the signal is transmitted through the electrode wire; 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 HA-3HA tag protein liquid to be tested and the HA-3HA-CREPT protein liquid into the upper electrode of the resonant QCM biosensor.

2. The CREPT protein quantitative detection device based on the resonant QCM biosensor according to claim 1 is characterized in that: There is a protruding cone at the bottom center of the circular recessed area, so that it forms an annular groove with the wall of the recessed area of ​​the base, and the silicone ring is embedded in the annular groove. At the same time, the height of the protruding cone is lower than the ring width of the silicone ring.

3. The CREPT protein quantitative detection device based on the resonant QCM biosensor according to claim 1, characterized in that: The utility model also comprises a cover. The hollow cylinder and the cover are both made of plastic material. The cover is adapted to the hollow cylinder and is used to cover the upper end of the hollow cylinder.

4. A method for quantitative detection of CREPT protein based on a resonant QCM biosensor, characterized in that: The following steps are involved: (1) Calibration of the pressure-frequency sensitivity coefficient K of the resonant QCM biosensor Pf and the pressure-frequency coefficient C Pf The electrodes of the resonant QCM biosensor were cleaned using an ultrasonic cleaning instrument. Then, pure water was dropped on the surface of the electrodes of the resonant QCM biosensor. When the pure water was completely covered, the amount of pure water was continued to be increased. According to the two increases in the amount of pure water, i.e., the volumes ΔV1 and ΔV2, the following equations were solved: 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 They are the resonant frequency change before and after the first addition of pure water and the frequency change after the second addition of pure water ΔV1, Δf 22 and Δf 21 They are the changes of the resonance frequency before and after adding pure water ΔV2 for the fourth and third time, respectively. f0 is the resonance frequency of the resonant QCM biosensor; (2) Obtaining HA-3HA tagged protein liquid and HA-3HA-CREPT protein liquid; (3) Drop the HA-3HA tagged protein liquid on the upper electrode surface of the QCM biosensor at a rate of 1 μL / time. When the upper electrode surface is completely covered, record the change in the resonant frequency Δf1. Then continue to increase the amount of HA-3HA tagged protein liquid, i.e., the volume ΔV. Then record the change in the resonant frequency Δf2. According to the following formula, the density ρ of the HA-3HA tagged protein liquid is obtained: HA-3HA : Calculate the content m1 of HA-3HA tagged protein liquid under a certain volume; Using the same method, the content m2 of HA-3HA-CREPT in a certain volume was obtained; The content of CREPT protein m2-m1 was obtained by calculating the content of HA-3HA tag protein m1 in a certain volume after removing the HA-3HA tag protein, thereby quantifying the content of CREPT protein.

5. The CREPT protein quantitative detection method based on the resonant QCM biosensor according to claim 4, characterized in that: The step (2) of obtaining the HA-3HA tag protein liquid and the HA-3HA-CREPT protein liquid comprises the following steps: Step 2.1) Construction of a CREPT overexpression stable cell line: The colorectal cancer cell line DLD-1 was cultured in culture medium, and then 3HA lentivirus was added to the DLD-1 colorectal cancer cell line to construct a stable colorectal cancer cell line DLD-1 3HA for CREPT protein overexpression control, i.e., a negative control group. At the same time, the gene sequence controlling the transcription and synthesis of CREPT was loaded onto the 3HA lentivirus to form the 3HA-CREPT lentivirus. The amount of the 3HA lentivirus was consistent with that added to the negative control group. The 3HA-CREPT lentivirus (which can control the transcription and synthesis of CREPT protein in DLD-1 cells) was added to the colon cancer cell line DLD-1 to form the colon cancer cell line DLD-13HA-CREPT in which CREPT is overexpressed. Then, under the stimulation of the 3HA-CREPT lentivirus, the colon cancer cell line DLD-1 3HA-CREPT transcribed and synthesized CREPT protein, thereby forming the 3HA-CREPT protein. Step 2.2), lysing the stable colorectal cancer cell line DLD-1 3HA and the colon cancer cell line DLD-13HA-CREPT overexpressing CREPT, and adding 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.

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